Novel interventions in type 2 diabetes and vascular disease. Effects

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NOVEL INTERVENTIONS IN TYPE 2 DIABETES AND VASCULAR DISEASE
BETA
BETA
FUNCTION
DYSFUNCTION
CELL
CELL
ENDOTHELIAL
ENDOTHELIAL
INSULIN
INSULIN
FUNCTION
SENSITIVE
DYSFUNCTION
RESISTANT
Pleun van Poppel
NOVEL INTERVENTIONS IN TYPE 2
DIABETES AND VASCULAR DISEASE
Effects on insulin sensitivity, insulin secretion and vascular function
Pleun van Poppel
NOVEL INTERVENTIONS IN TYPE 2 DIABETES
AND VASCULAR DISEASE
Effects on insulin sensitivity, insulin secretion and vascular function
Pleun van Poppel
Author: Pleun van Poppel
Cover design and lay-out: Ontwerpbureau STUDIO 0404
Printing: Ipskamp drukkers
ISBN: 978-90-9029-355-4
© Pleun van Poppel, 2015
No part of this thesis may be produced in any form or by any means without written permission
of the author or of the publisher holding the copyright of the published articles.
Printing of this thesis was financially supported by Novo Nordisk
NOVEL INTERVENTIONS IN TYPE 2 DIABETES
AND VASCULAR DISEASE
Effects on insulin sensitivity, insulin secretion and vascular function
Proefschrift
ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen
op gezag van de rector magnificus
volgens besluit van het college van decanen
in het openbaar te verdedigen op donderdag 10 december 2015
om 10.30 uur precies
door
Pleuntje Catrien Maria van Poppel
geboren 03-08-1980
te Boxtel
Promotoren:
Prof. dr. C.J.J. Tack
Prof. dr. M.G. Netea
Manuscriptcommissie: Prof. dr. N.P. Riksen
Prof. dr. G.J. Adema
Prof. dr. S.J.L. Bakker
Table of contents
Chapter 1
General introduction and outline of the thesis 9
Chapter 2Vildagliptin improves endothelium-dependent vasodilatation in
type 2 diabetes mellitus
Diabetes Care 2011 Sept;34(9):2072-2077 31
Chapter 3The dipeptidyl peptidase-4 inhibitor vildagliptin does not affect
ex vivo cytokine response and lymphocyte function in patients
with type 2 diabetes mellitus
Diabetes Res Clin Pract 2014 Mar;103(3):395-401 49
Chapter 4 One week treatment with the IL-1 receptor antagonist anakinra
leads to a sutained improvement in insulin sensitivity in insulin
resistant patients with type 1 diabetes mellitus
Clin Immunol 2015 Oct; 160 (2):155-16265
Chapter 5The interleukin-1 receptor antagonist anakinra improves first
phase insulin secretion and insulinogenic index in subjects with
impaired glucose tolerance
Diabetes Obes Metab 2014 Dec;16(12):1269-1273 83
Chapter 6Inflammation in the subcutaneous adipose tissue is not related
to endothelial function in subjects with diabetes mellitus and
subjects with dyslipidemia and hypertension
Submitted 101
Chapter 7Salvia Miltiorrhiza water-extract (danshen) has no beneficial
effect on cardiovascular risk factors
Plos One 2015 Jul 20;10(7):e0128695
119
Chapter 8Summary and general discussion 141
Chapter 9Nederlandse samenvatting, List of publications, Curriculum Vitae,
Dankwoord 155
BETA
BETA
FUNCTION
DYSFUNCTION
CELL
CELL
ENDOTHELIAL
ENDOTHELIAL
INSULIN
INSULIN
FUNCTION
SENSITIVE
DYSFUNCTION
RESISTANT
CHAPTER 1
GENERAL INTRODUCTION AND OUTLINE OF THE THESIS
 General introduction and outline of the thesis
Introduction
Type 2 diabetes is a major global health care problem and the number of adults affected by
diabetes is currently estimated around 380 million and is expected to rise to 590 million by the
year 2035 1. This increase is due to several factors, including population growth, aging, earlier
detection and increasing prevalence of obesity and physical inactivity 2,3.
Type 2 diabetes mellitus is characterized by chronically elevated blood glucose levels and
occurs when insulin secretion by beta cells fails to compensate for insulin resistance. Chronic
hyperglycemia can lead to serious microvascular and macrovascular complications 4,5.
For once, type 2 diabetes causes a twofold excess risk for cardiovascular disease, partly
independent from other risk factors 6,7. Cardiovascular disease is globally the most frequent
cause of death with an estimated number of people dying from cardiovascular disease of 20
million annually 8. Clearly, developing better treatments and prevention strategies for type 2
diabetes mellitus and cardiovascular disease is a matter of great urgency.
Type 2 diabetes
As stated, type 2 diabetes mellitus is characterized by chronically elevated blood glucose
levels (hyperglycemia). Insulin is the key hormone for regulation of blood glucose levels and
is produced by pancreatic beta cells. Insulin lowers blood glucose levels by suppressing
gluconeogenesis and glycogenolysis in the liver and by increasing glucose uptake in the
skeletal muscle. Beta cells can adapt the secretion of insulin to changes in insulin action:
a reduction in insulin action is accompanied by an increment in insulin secretion and vice
versa. In healthy subjects, normal glucose levels are maintained by the continuous interaction
between insulin action and secretion. Hyperglycemia occurs when the beta cells can no longer
compensate insulin resistance with increased insulin secretion 9. (Figure 1)
Although it is well established that genetic and environmental factors play an important role in
the pathogenesis of type 2 diabetes mellitus 10,11, whether the major genetic component in type
2 diabetes is represented by insulin resistance (impaired insulin action) or beta cell dysfunction
(impaired insulin secretion) is still debated 12. While type 2 diabetes constitutes the vast
majority, patients with type 1 diabetes represent a sizeble subset. In type 1 diabetes, life style
factors are not involved in the pathogenesis. Patients with type 1 diabetes mellitus develop an
absolute deficiency of insulin due to auto-immune beta cell destruction 13. At diagnosis there is
some residual insulin production but as the disease progresses a complete destruction of beta
cells occurs.
10
General introduction and outline of the thesis 
1
Insulin resistance with
beta cell compensation
beta cell function
Insulin resistance without
beta cell compensation
NGT
T2DM
IGT
Insulin sensitivity
Figure 1. Relation between insulin secretion (beta cell function, Y-axis) and insulin sensitivity (X-axis).
(source : M.Stumvoll et al 9). NGT = normal glucose tolerance. IGT= impaired glucose tolerance.
T2DM= type 2 diabetes mellitus.
Insulin resistance
Insulin resistance is defined as a decreased biological response to insulin and is associated
with a cluster of metabolic disorders, such as hypertension and dyslipidemia, that contribute
to the increased cardiovascular risk 11,14. The characteristics of insulin resistance are reduced
suppression of gluconeogenesis and glycogenolysis in the liver, reduced ability of insulin
to inhibit lipolysis in the adipose tissue and decreased insulin-stimulated glucose uptake in
skeletal muscle 15,16.
Prospective and longitudinal studies have shown that insulin resistance is present before the
onset of type 2 diabetes 17.
Insulin resistance can be genetic or acquired by environmental factors such as obesity, aging
and a sedentary lifestyle. It is not completely clear how obesity causes insulin resistance.
Proposed mechanisms include increased fatty acid levels, biochemical adaptations induced
by nutrient overload, microhypoxia in adipose tissue, endoplasmatic reticulum stress, secretion
11
 General introduction and outline of the thesis
of adipocyte-derived cytokines, chronic tissue inflammation, fat distribution and variations in
central nervous system inputs 10. Obesity-associated inflammation is a central theme in this
thesis and is discussed in further detail below.
Insulin secretion
If beta cells are unable to increase insulin production (“beta cell dysfunction”) to compensate
for insulin resistance, hyperglycemia and type 2 diabetes mellitus occurs 18. The United
Kingdom Prospective Diabetes Study (UKPDS) has demonstrated that beta cell function
in patients with type 2 diabetes mellitus progressively deteriorates over time 19. At the time
of diagnosis the remaining islet function is estimated to be about 50% with the reduction in
function commencing 10-12 years before diagnosis 20.
In rodent models of obesity, an adaptive increase in beta cell mass in response to insulin
resistance has been documented 21,22. Because to date there are no methods to quantify
beta cell mass in vivo, human data are limited. Butler et al studied human pancreatic tissue
from autopsies and showed that obese subjects with type 2 diabetes mellitus had a 63%
deficit and lean subjects with type 2 diabetes mellitus had a 41% deficit in relative β-cell
volume, compared to non-diabetic obese and lean cases 23. The underlying mechanism for
the decrease in beta cell mass was postulated to be increased apoptosis. Another study
demonstrated that after matching for BMI, the beta cell mass was approximately 39% lower in
type 2 diabetes compared to non-diabetic subjects 24. Thus, available evidence suggests that
beta cell mass in patients with type 2 diabetes is diminished.
Apart from genetic factors that may determine initial beta cell mass and function, the major
factors for progressive loss of beta cell function and mass are glucotoxicity, lipotoxicity, proinflammatory cytokines, leptin and islet cell amyloid 25. This thesis investigates specifically the
role of inflammation.
12
General introduction and outline of the thesis 
Vascular complications
Patients with type 2 diabetes have an increased risk to develop cardiovascular disease 26,
with a twofold excess risk for coronary heart disease and stroke, partly independent from
1
other conventional risk factors 6,7. Type 2 diabetes affects small (microvascular disease) and/
or large (macrovascular disease) blood vessels resulting in microvascular and macrovascular
complications of the disease. Microvascular complications consists of retinopathy, neuropathy
and nefropathy while macrovascular complications, manifested by accelerated atherosclerosis,
comprise of coronary heart disease, peripheral vascular disease and stroke.
Endothelial dysfunction is regarded as an important marker of vascular complications in
type 2 diabetes mellitus 27,28. The endothelium is the biological active inner layer of the blood
vessels. It regulates vascular tone and permeability, adhesion and migration of leukocytes and
it controls coagulation 28,29. Hyperglycemia, hypercholesterolemia, obesity, hypertension and
low grade inflammation all negatively affect endothelial function 27,30,31. Endothelial dysfunction
actually contains an endothelial switch from a quiescent phenotype to an activated phenotype
which is a crucial and physiological response during acute inflammation 32. A key player in
endothelial function is endothelial nitric oxide synthase (eNOS). By generating its effector
molecule nitric oxide (NO), eNOS normally exerts a silencing influence on the endothelium. NO
causes vasodilatation, inhibits platelet aggregation, inhibits proliferation of vascular smooth
muscle cells and inhibits leukocyte adhesion 28,29. Endothelial dysfunction is the result of a
decreased availability of NO due to an increased production of superoxide by eNOS and
oxidative inactivation of NO 29,32,33. A decrease in NO availability leads to vasoconstriction,
increases endothelial adhesion of leukocytes and stimulates coagulation. Thus, endothelial
dysfunction comprises of a number of functional alternations in the vascular endothelium, such
as impaired endothelium-dependent vasodilatation, impaired barrier function, inflammatory
activation and stimulated coagulation.
Endothelium-dependent vasodilatation, which is impaired in endothelial dysfunction, can be
assessed by measuring responses to pharmacological endothelium-dependent vasodilators
such as acetylcholine. In obese subjects with and without diabetes, the endothelium
dependent vasodilatation is impaired compared to lean subjects 34. Endothelial function is
further decreased in subjects with type 2 diabetes even after adjustment for obesity 35. Ideally,
pharmacotherapy for type 2 diabetes would not only lower blood glucose levels but would also
have beneficial vascular effects independent of glucose.
13
 General introduction and outline of the thesis
Role of inflammation
Insulin resistance
As noted, obesity is an important risk factor for the development of insulin resistance and type
2 diabetes mellitus and there is evidence to suggest that inflammation may be the mechanistic
link between obesity and insulin resistance 36. First, epidemiological studies have reported
an association between inflammation and type 2 diabetes mellitus. In a prospective casecontrol study it was shown that elevated levels of CRP and IL-6 predict the development of
type 2 diabetes mellitus 37. Also, a high white blood count value predicted diabetes and was
associated with a decline in insulin sensitivity 38. A mechanistic link between inflammation and
insulin resistance was established by the demonstration of increased TNFα expression in
adipose tissue (and systemically) of obese insulin resistant animals and that neutralisation of
TNFα partially improved insulin sensitivity 39.
When obesity emerges, hypertrophy of the adipocytes due to storage of increasing amounts
of lipids develops and subsequently macrophage infiltration and increased secretion of
proinflammatory cytokines occurs 40,41. Many proinflammatory cytokines have been linked to
the development of insulin resistance and type 2 diabetes mellitus including TNFα and IL-6
. More recently, interest has grown in the proinflammatory cytokine IL-1β, a member of the
41,42
IL-1 family. In order to be released, IL-1β needs to be processed from its inactive precursor
(pro IL-1β) by an intracellular cysteine protease called caspase-1 (Figure 2) 43. Activation of
caspase-1 itself takes place by activation of a protein complex, termed the inflammasome 43.
Caspase-1 and IL-1β activity is increased in adipose tissue of obese animals 44. Furthermore,
both caspase-1 deficient and IL-1β deficient mice are more insulin sensitive than wild type
animals. Both in obesity and type 2 diabetes, circulating IL-1Ra levels are increased, possibly
as a response to an increase in IL-1β activity 45,46. These findings suggest a role for IL-1β in
metabolic function of adipose tissue and suggest that inhibition of IL-1β may improve insulin
sensitivity.
14
General introduction and outline of the thesis 
Anti-IL-1β antibodies
Y
IL-1 producing cell
IL-1 β
1
Pro IL-1 β
Caspase-1
IL-1Ra
Interleukin-1
receptor antagonist
IL-1R1
IL-1RAcP
Signal transduction
IL-1 responsive cell
Figure 2.IL-1β is produced by stimulated monocytes, macrophages, endothelial cells and smooth muscle
cells. IL-1β secretion is induced by microbial products that stimulate toll-like receptors and by
endogenous triggers. Both triggers stimulate the inflammasome which activates caspase-1.
The inactive IL-1β precursor (pro IL-1β) is cleaved by caspase-1 to the active cytokine. Active
IL-1β binds to the IL-1 receptor type 1 (IL-1R1) and thereby recruits the IL-1 receptor accessory
protein (IL-1RAcP). This forms a high-affinity heterodimeric signalling complex. When the
receptor antagonist (IL-1Ra) occupies the IL-1R1, the IL-1RAcP is not recruited, no heterodimer
is formed and no signal occurs.
Insulin secretion
In type 2 diabetes mellitus, beta cell mass is diminished and beta cell function progressively
deteriorates over time. Inflammation may play a role in beta cell destruction in type 2
diabetes mellitus.
It has been shown in type 1 diabetes mellitus that the proinflammatory cytokine interleukin1β (IL-1β) is an important mediator in the destruction of beta cells 47. Since also in type 2
diabetes beta cell mass and function is diminished, interest has grown in the role of IL-1β
in the inflammatory process in the beta cell leading to the development of type 2 diabetes.
Increased glucose concentrations stimulate beta cell proliferation on the short term but induce
cell death after prolonged exposure (concept of glucotoxicity)48. In vitro, high glucose levels
15
 General introduction and outline of the thesis
increase beta cell production of IL-1β followed by functional impairment and apoptosis of beta
cells, and the interleukin-1 receptor antagonist (IL-1Ra), a naturally occurring inhibitor of IL-1β,
protects beta cells from glucose-induced apoptosis in vitro 49. Furthermore, islets of patients
with type 2 diabetes stained positive for IL-1β while control islets were negative 50. Human beta
cells also express IL-1Ra and IL-1Ra expression is decreased in the pancreas of patients with
poorly controlled type 2 diabetes as compared to non-diabetic controls 51. These data suggest
that an imbalance between IL-1β and IL-1Ra in the pancreas can lead to beta cell destruction
and thus that beta cell destruction occurs when high levels of IL-1β are not compensated by
an increment in IL-1Ra in the pancreas. Altogether, these findings suggest that IL-1β is involved
in the destruction of beta cells and that blocking the effects of IL-1β may prevent beta cell
destruction and thereby the development of type 2 diabetes mellitus.
Vascular disease
The prevalence of obesity is rising globally and paralleled by an increasing incidence of type 2
diabetes mellitus and cardiovascular disease 2,52. Traditional risk factors for atherosclerosis are
hyperlipidemia, hypertension, diabetes mellitus, and smoking 53. More recently, inflammation
has gained much attention and atherosclerosis is now considered an inflammatory disease
. The best known inflammatory marker linked to cardiovascular disease is C-reactive protein
54,55
(CRP). Elevated plasma levels of CRP have been shown to predict cardiovascular events 56
and are viewed as an independent risk marker for cardiovascular disease 57,58. Also IL-6 and
fibrinogen are related to cardiovascular events 59,60. Additional evidence for a significant role
of CRP is the fact that statin therapy leads to a greater clinical benefit when CRP levels are
elevated 61 and statins lower CRP levels independent of LDL cholesterol levels 62,63. Finally, it
has been shown that in patients with acute coronary syndrome treated with a statin, low CRP
levels are associated with an improvement in event-free survival, an effect that was observed
at all LDL levels 64.
Similarly, there is also evidence for the involvement of the proinflammatory cytokine IL-1β in
the development of atherosclerosis. Animal studies have demonstrated less atherosclerosis
in IL-1β knockout mice 65, while in humans, IL-1β has been found in greater concentrations in
atherosclerotic coronary arteries 66.
Several mechanisms may explain the association between IL-1β levels and cardiovascular
disease. First, elevated IL-1β levels may result in secretion of other cytokines and chemokines,
increased expression of adhesion molecules, endothelial and smoothe muscle cell
proliferation, macrophage activation and increased vascular permeability. Together these
effects promote atherosclerosis. Second, IL-1 may contribute to endothelial dysfunction by
16
General introduction and outline of the thesis 
releasing endothelin-1, a vasoconstrictor, and by stimulating inducible nitric oxide synthase
which increases the formation of reactive oxygen species 67.
These data provide evidence to support the hypothesis that inflammation is linked to vascular
1
disease and that reducing inflammation might have beneficial cardiovascular effects.
LEAN
Type 2 diabetes mellitus
Insulin resistance
Beta cell dysfunction
Proinflammatory mediators
OBESE
Atherosclerosis
Endothelial dysfunction
Vascular disease
Figure 3. P
otential mechanism for obesity induced inflammation and development of type 2 diabetes
mellitus and vascular disease. The expansion of the adipose tissue leads to a proinflammatory
state. Proinflammatory mediators might contribute to the development of insulin resistance
and beta cell dysfunction and these two conditions ultimately lead to the development of type
2 diabete mellitus. Type 2 diabetes mellitus is accompanied by an increased risk of vascular
disease and endothelial dysfunction is an early marker of vascular complicatios. Finally
the proinflammatory mediators released from the adipose tissue might increase the risk of
atherosclerosis.
17
 General introduction and outline of the thesis
Interventions investigated in this thesis
Dipeptidyl peptidase-4 inhibitors
 Glycemic control
The observation that oral glucose provided a more potent insulinotropic stimulus compared to
intravenous glucose led to the postulation of hormonal factors in the gut that enhance insulin
secretion of the pancreas 68. The name incretin system was given to describe these hormonal
factors, predominantly glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic
polypeptide (GIP) 69 GLP-1 is a potent glucose-dependent insulinotropic hormone, which
inhibits gastric motility, suppresses glucagon secretion and promotes satiety. GLP-1 is
produced in enteroendocrine L cells in the gastrointestinal tract.
The in vivo half-life of GLP-1 is very short. Circulating levels of GLP-1 are decreased rapidly by
degradation of GLP-1 by dipeptidyl peptidase-4(DPP-4), resulting in GLP-1 (9-37) and GLP-1
(9-36) amide 69,70. Therefore, long acting GLP-1 receptor agonists and inhibitors of DPP- 4 have
been developed for the treatment of diabetes.
Inhibition of DPP-4 prevents degradation of the incretin hormones, GLP-1 and GIP, which
results in stimulation of insulin secretion and inhibition of glucagon release, both in a glucosedependent manner. Together, these effects improve glycemic control in type 2 diabetes 70.
 Vascular effects
Apart from glycemic actions, DPP-4 inhibitors might have beneficial vascular effects since GLP1 has been shown to ameliorate endothelial function in animals and humans 71-74. These effects
are believed to be mediated through the GLP-1 receptor, which is expressed in blood vessels
and several other tissues including pancreatic islets, lungs, kidneys, heart, stomach, intestines
and several regions of the central nervous system 69 In addition, GLP-1 may have GLP-1
receptor independent vascular effects, which appear to be mediated by metabolites of GLP-175.
It is not known whether a pharmacological approach of increasing GLP-1 levels by inhibiting
DPP-4, which changes the ratio between GLP-1 and its degradation product, exhibits the same
vascular profile. Meta-analyses assessing the effect of DPP-4 inhibitors on cardiovascular
events occurring in phase 2 and 3 studies reported a lower relative risk of DPP-4 inhibitors
compared to placebo or active control treatment 76,77.
 Effect on immune system
The enzyme DPP-4 is expressed on epithelial and endothelial cells in liver, lung, kidney,
intestinal brush-border membranes and exists as a soluble form 78. DPP-4 is also expressed
on circulating T-, B-lymphocytes and natural killer (NK) cells, and is synonymous with the cellsurface antigen CD26 and has been suggested to act as a costimulator of T-cell activation 79.
18
General introduction and outline of the thesis 
DPP-4 cleaves dipeptides from oligopeptides and besides incretin hormones, also cytokines
and chemokines might serve as substrates 80. Theoretically, DPP-4 inhibition may thus affect
immune responses, and this could translate into an altered susceptibility to infections. Some
1
meta-analyses focusing on side effects of DPP-4 inhibitors, have reported a slight increase in
upper respiratory infections, although these reports are not consistent 81-83.
Antagonizing IL-1β
While IL-1 is a proinflammatory cytokine, IL-1 mediated actions are normally counterbalanced
by the naturally occurring IL-1 receptor antagonist IL-1Ra, which competitively binds to the
IL-1 receptor-1 (IL-1R1) but does not attract the IL-1 receptor accessory protein (IL-1RAcP)
and thereby inhibits further signal transduction 84. Thus, one way to antagonize the action of
IL-1 is treatment with the IL-1Ra anakinra (Figure 2) 85. Anakinra blocks not only IL-1β but also
IL-1α since IL-1α also binds to the IL-1R1. Anakinra has a relatively short half-life of 4-6 hours
and daily subcutaneous injections are required. The most frequently observed adverse events
during the use of anakinra are injection site reactions 86. A second approach is treatment with
antibodies that specifically neutralize IL-1β. Several antibodies have been developed, including
canakinumab en gevokizumab. Other alternative approaches are inhibition of caspase-1,
administration of soluble IL-1 receptors that bind circulatory IL-1 and targeting the intracellular
pathway of IL-1 by inhibiting NF-κB.
 Glycemic control
Based on the documented in vitro role of IL-1β in beta cell failure, a proof of concept study with
anakinra was performed in 70 patients with type 2 diabetes mellitus. This study indeed showed
that anakinra treatment (100 mg once daily) for 13 weeks improved glycemic control compared
to placebo 87. The observed beneficial effects of IL-1 blockade on glycemic control were
correlated with an improvement in beta cell function. No effect of anakinra on insulin sensitivity
was observed. However, in patients with diabetes, improvement of beta cell function can
be direct, but also indirect due to improvement in glucose levels, since hyperglycemia itself
impairs both insulin secretion and insulin sensitivity (concept of glucotoxicity) 48. In a follow up
study the same authors showed that 39 weeks after the cessation of anakinra treatment beta
cell function and systemic markers of inflammation were still enhanced in the former anakinra
treated group 88. While the findings in this study were very promising, the results, so far, have
not been reproduced by others.
19
 General introduction and outline of the thesis
 Vascular effects
The in vitro findings concerning the role of IL-1β in the pathogenesis of atherosclerosis, suggest
that inhibition of IL-1β may reduce vascular disease. In animals with diabetes and in patients
with rheumatoid arthritis disease, anakinra improved endothelium-dependent vasodilatation
. A large cardiovascular outcome trial with canakinumab ( Canakinumab Anti-inflammatory
89,90
Thrombosis Outcomes Study (CANTOS)) has been started to test whether reducing
inflammation by blocking the IL-1β activity in patients with a prior acute coronary syndrome can
reduce future cardiovascular events 91. Results will not be available until 2017.
In obesity, inflammation of the adipose tissue might be the link to vascular disease. In the
development of obesity, nutrient excess leads to hypertrophy of adipocytes which initiates the
production of cytokines and chemokines by adipocytes. Due to the increased production of the
cytokines and chemokines the endothelial cells respond by increased expression of adhesion
molecules. The chemokines and increased expression of adhesion molecules recruite immune
cells, including macrophages to the adipose tissue. The proinflammatory mediators that enter
the circulation increase the risk for atherosclerosis 92. A study in obese and lean subjects
showed that obese subjects with inflamed fat had impaired flow-mediated vasodilatation
compared to lean subjects 93, while obese subjects without inflamed fat had similar vascular
function as lean subjects.
Danshen
Traditional Chinese Medicine (TCM) has been playing an important role in healthcare in Asia
for hundreds of years. Recently, there is an upsurge in the popularity of TCM products among
consumers in the West. Danshen is the dried root extract of the plant Salvia Miltiorrhiza and
is widely used in Asia to treat coronary artery disease, hyperlipidemia and cerebrovascular
disease 94. According to TCM theory, danshen is used in patients to treat “blood stasis” as it
“moves blood” 95. According to TCM, the diagnosis of “blood stasis” is made by identification
of certain changes in the tongue and pulse.
Two active components of danshen, salvianolic acid A and magnesium tanshinoate B, inhibit
LDL oxidation 95. A mixture of Chinese herbs, including danshen, has been shown to exert
beneficial effects on high-fat diet induced metabolic syndrome in rats. 96. Also in humans,
danshen may reduce LDL-levels and blood pressure 95. Clinical trials on danshen components
for the treatment of angina pectoris claim that it is as effective as sublingual nitroglycerin 94.
These data suggest that danshen has beneficial effects on cardiovascular risk factors and
potentially affects inflammation.
20
General introduction and outline of the thesis 
1
21
 General introduction and outline of the thesis
Outline of the thesis
The objective of this thesis is to evaluate the effect of different novel treatments on insulin
sensitivity, insulin secretion and vascular function.
First, DPP-4 inhibitors, a relative new class of drugs for the treatment of type 2 diabetes, were
evaluated. Evidence suggests a beneficial role for DPP-4 inhibitors on vascular function. In
chapter 2 we investigated whether inhibition of DPP-4 by vildagliptin improves endothelial
function in patients with type 2 diabetes. Endothelial function was assessed by measuring
forearm vascular responses to endothelium-dependent and endothelium-independent
vasodilators 97. As DPP-4 is involved in T-cell activation, potential side effects of DPP-4 inhibitors
are an increased risk of infection by altering immune response. In chapter 3 we analysed the
effect of inhibiting DPP-4 by vildagliptin on immune function.
Second, inhibition of the actions of IL-1β is a new potential treatment of type 2 diabetes
mellitus. Inflammation is suggested to play a role in the pathogenesis of type 2 diabetes
mellitus, both at the level of insulin secretion and at the level of insulin action, and seems to
be involved in the development of atherosclerosis. Antagonizing IL-1β by anakinra, a human
IL-1 receptor antagonist, has been shown to improve glycemic control in patients with type 2
diabetes but the study needs to be reproduced and the mechanism involved need to be further
unravelled. In chapter 4 we studied the effect of anakinra on insulin sensitivity in a population
of subjects with longstanding type 1 diabetes mellitus who were insulin resistant ( BMI > 25 kg/
m2, insulin requirement > 0.5 U/kg bodyweight per day). Given the fact that beta cell function
is absent in these patients, changes in insulin sensitivity will translate into changes in glucose
levels and insulin need. Insulin sensitivity was assessed by a euglycemic hyperinsulinemic
clamp 98. In chapter 5 we investigated the effect of blocking IL-1 by anakinra on beta cell
function. We studied subjects with impaired glucose tolerance and therefore we could study
the effects on beta cell function without confounding effects of changes in glycemic control.
Beta cell function was assessed by a hyperglycemic clamp 98,99. In chapter 6 we examined the
relationship between inflammation in the adipose tissue and endothelial function. We assessed
adipocyte size, presence of macrophages and of crown-like structures (CLS) in adipose tissue
obtained by small needle biopsy. Macrophages are present in the adipose tissue in different
forms, but the arrangement of macrophages surrounding adipocytes as CLS is considered
a strong indicator of local inflammation 100. We determined whether the level of inflammation
in adipose tissue was associated with in vivo vascular function as measured by responses to
endothelium dependent and independent vasodilators.
Finally, Traditional Chinese Medicine is becoming more popular in the west. In Asia the
herbal product danshen is used to prevent and treat atherosclerosis. The estimated number
of patients using danshen is approximately 5 million worldwide 101. However, the quality of
22
General introduction and outline of the thesis 
randomized clinical trials of danshen is poor 102 and not easily accessible to Western physicians
since most are published in Chinese. In chapter 7 we investigated the effect of danshen waterextract on traditional cardiovascular risk factors, namely hyperlipidemia and hypertension.
1
Furthermore, we studied the effect on endothelial function, markers of inflammation, markers of
oxidative stress, markers of glucose metabolism, hemostatic markers and rheological markers.
In chapter 8 a summary of our findings is provided and results of our studies are put in
perspective.
23
 General introduction and outline of the thesis
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General introduction and outline of the thesis 
1
29
BETA
CELL
FUNCTION
ENDOTHELIAL
FUNCTION
INSULIN
SENSITIVE
BETA
CELL
DYSFUNCTION
ENDOTHELIAL
DYSFUNCTION
INSULIN
RESISTANT
CHAPTER 2
VILDAGLIPTIN IMPROVES ENDOTHELIUM-DEPENDENT
VASODILATATION IN TYPE 2 DIABETES MELLITUS
P.C.M. van Poppel, M.G. Netea, P. Smits, C.J. Tack
Diabetes Care 2011 Sept;34(9):2072-2077
 Vildagliptin improves endothelium-dependent vasodilatation in type 2 diabetes mellitus
Abstract
Objective
To investigate whether the dipeptidyl peptidase-4 inhibitor vildagliptin improves endotheliumdependent vasodilatation in patients with type 2 diabetes mellitus.
Research design and methods
Sixteen subjects with type 2 diabetes (age 59.8±6.8 yr, BMI 29.1±4.8 kg/m2, HbA1c
6.97±0.61) on oral blood glucose lowering treatment were included. Participants received
vildagliptin 50 mg bid or acarbose 100 mg tid for 4 consecutive weeks in a randomised, double
blind, cross-over design. At the end of each treatment period we measured forearm vasodilator
responses to intra-arterially administered acetylcholine (endothelium-dependent vasodilator)
and sodium nitroprusside (endothelium-independent vasodilator).
Results
Infusion of acetylcholine induced a dose-dependent increase in forearm blood flow (FBF) in the
experimental arm, which was higher during vildagliptin (3.1±0.7, 7.9±1.1 and 12.6±1.4 ml.dl⁻¹.
min⁻¹ in response to 3 increasing dosages of acetylcholine) than during acarbose (2.0±0.7,
5.0± 1.2 and 11.7± 1.6 ml.dl⁻¹.min⁻¹ respectively, P = 0.01 by two-way ANOVA). Treatment
with vildagliptin did not significantly change the vascular responses to sodium nitroprusside.
Conclusions
Four weeks treatment with vildagliptin improves endothelium-dependent vasodilatation in
subjects with type 2 diabetes mellitus. This observation might have favorable cardiovascular
implications.
32
Vildagliptin improves endothelium-dependent vasodilatation in type 2 diabetes mellitus 
Introduction
Worldwide, almost 200 million people suffer from type 2 diabetes mellitus and the prevalence
is rapidly increasing 1. Type 2 diabetes causes a twofold excess risk for cardiovascular disease,
partly independent from other risk factors 2. Ideally, pharmacotherapy for type 2 diabetes not
only lowers blood glucose levels but also has glucose-independent beneficial cardiovascular
effects.
2
Endothelial dysfunction is considered an early marker of vascular complications, also in type
2 diabetes 3. Endothelial dysfunction comprises of a number of functional alternations in the
vascular endothelium, such as impaired endothelium-dependent vasodilatation, impaired
barrier function, inflammatory activation en stimulated coagulation 4. Endothelium-dependent
vasodilatation, which is impaired in endothelial dysfunction, can be assessed by measuring
responses to endothelium-dependent vasodilators such as acetylcholine 5.
Recently, incretin based therapy has been approved for the treatment of type 2 diabetes
mellitus. Incretins are a group of gastrointestinal hormones, predominantly glucagon-like
peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), that are secreted
in response to food ingestion and stimulate insulin secretion 6. Dipeptidyl peptidase-4 (DPP4) rapidly degrades the incretin hormones to inactive metabolites 7. In addition to incretins,
DPP-4 also inactivates chemokines, cytokines and neuropeptides 8. In type 2 diabetes DPP-4
inhibitors reduce the breakdown of GLP-1 and improve metabolic control by increasing insulin
secretion, improving beta cell function and decreasing glucagon secretion 9.
Apart from glycemic actions, GLP-1 has beneficial cardiovascular effects. GLP-1 has
vasodilatory actions, which are believed to be mediated through a specific GLP-1 receptor in
the vascular endothelium, and improves endothelial function in animals and humans 10,11.
In addition, GLP-1 may have GLP-1 receptor independent cardiovascular effects, which
appear to be mediated by metabolites of GLP-1 12. It is not known whether a pharmacological
approach of increasing GLP-1 levels by inhibiting DPP-4, which changes the ratio between
GLP-1 and its degradation product, exhibits the same vascular profile.
DPP-4 inhibition affects not only GLP-1 levels, but also GIP breakdown, and perhaps also
other substrates, such as chemokines and cytokines. Whether intervening in the breakdown of
these other potential substrates affects endothelial function is unknown. Indirect evidence for
a potential beneficial effect on endothelial function comes from a study demonstrating that the
DPP-4 inhibitor sitagliptin increases endothelial progenitor cells in type 2 diabetes mellitus by
inhibiting degradation of the chemokine stromal-derived factor 1 α (SDF-1α) 13.
Fact is that meta-analyses summarizing the effects of DPP-4 inhibitors on major cardiovascular
events in phase 2 and 3 studies, all suggest a lower relative risk compared to placebo or other
medication 14,15. These observations need to be confirmed in large cardiovascular outcome
33
 Vildagliptin improves endothelium-dependent vasodilatation in type 2 diabetes mellitus
studies. The present study therefore aims to determine whether the DPP-4 inhibitor vildagliptin
can improve endothelial function in patients with type 2 diabetes mellitus.
Research design and methods
Study Population
This study was conducted in accordance with the principles outlined in the Declaration of
Helsinki. The local ethics committee (Radboud University Nijmegen Medical Centre, Nijmegen,
the Netherlands) approved the study and all subjects gave written informed consent before
participation. The trial was registered at clinicaltrials.gov (number NCT01000688).
The study population consisted of 16 patients with type 2 diabetes mellitus, recruited through
advertisements in a local newspaper and through the outpatient clinic of the Radboud
University Nijmegen Medical Centre.
Included were subjects with type 2 diabetes mellitus who were treated with metformin with or
without sulphonylurea or thiazolidinediones, aged 35-75 years old and had an HbA1c < 8.0%.
Exclusion criteria were use of acetylsalicylic acid or vitamin K antagonists, heart failure (NYHA
class III or IV), smoking, pregnancy or breast feeding, a serum alanine aminotransferase or
aspartate aminotransferase level of more than 3 times the upper limit of the normal range, a
serum creatinine level higher than 130 μmol/L and inability to give informed consent.
Protocol
After inclusion in the study, subjects received either vildagliptin 50 mg bid for 28 days or
acarbose 50 mg tid for 7 days followed by acarbose 100 mg tid for the remaining 21 days.
Participants were randomized into treatment with vildagliptin in the first treatment period and
acarbose in the second treatment period or vice versa. Investigational medication was given on
top of their other medication in a randomized, double blind, cross-over design. Blinding of the
study medication was performed by a double dummy technique.
Between the two treatment periods there was a wash-out period of one week.
Participants were contacted by telephone halfway each treatment period to check for
compliance and possible side effects. At the end of each treatment period (day 25-28)
endothelial function was measured, body weight and potential side effects were determined
and arterial blood was collected for serum hematological and chemical safety profiles. A
schedule of the study protocol is shown in figure 1A. Compliance was monitored by pill counts.
34
Vildagliptin improves endothelium-dependent vasodilatation in type 2 diabetes mellitus 
A
Plethysmography and
blood samples
Randomization
Plethysmography and
blood samples
Control by phone
Control by phone
Screening phase
2
Vildagliptin
Vildagliptin
Acarbose
Acarbose
Treatment period 1
Treatment period 2
0
5
9
weeks
Arterial
catheter
Time (minutes)
0
Glucose 5%
Plethysmography
Saline
B
4
30
50
80
100
Figure 1.
The study protocol (A) in which four weeks treatment with vildagliptin and acarbose are
administered in a crossover design. The protocol of venous occlusion plethysmography (B)
started with insertion of arterial catheter at t=0 after which a 30 minute equilibration period
followed. Baseline FBF measurements and subsequently FBF measurements during the
infusion of acetylcholine (dark grey; 0.5, 2.0 and 8.0 µg.dl⁻¹.min⁻¹) are performed. Again, after
a 30 minute equilibration period baseline FBF and FBF during infusion of sodium nitroprusside
(light grey; 0.06, 0.20 and 0.60 µg.dl⁻¹.min⁻¹) is assessed.
35
 Vildagliptin improves endothelium-dependent vasodilatation in type 2 diabetes mellitus
Experimental procedures
 Endothelial function
The experiments started at 8.30 a.m. after an overnight fast in a quiet, temperature controlled
room (23˚C-24˚C). Study medication was ingested the day of the experiments. The subjects
abstained from caffeine for 24 hours prior to the experiment. The brachial artery of the non
dominant arm (experimental arm) was cannulated (Angiocath, 20-gauge, Becton Dickinson,
Sandy, UTAH, USA) under local anesthesia (Xylocaine 2%) for infusion of vasodilators and
collection of blood samples. The brachial artery catheter was connected with an arterial
pressure-monitoring line for continuous blood pressure monitoring (Hewlett Packard,
Böblingen, Germany).
Forearm blood flow (FBF) was measured using mercury-in-silastic strain gauge, venous
occlusion plethysmography as previously described 16. Forearm volume was measured with
the water displacement method and all drugs were dosed per 100 ml forearm tissue with an
infusion rate of 100 ml.dl-1.min-1.
After complete instrumentation, a 30 minute equilibration period was included, after which
baseline measurements were performed with infusion of saline. Subsequently, three increasing
doses of acetylcholine (0.5, 2.0 and 8.0 µg.dl-1.min-1, 10 mg/ml dry powder, dissolved to its final
concentration with saline, Novartis, Greece) were infused into the brachial artery. Acetylcholine
(ACH) stimulates endothelial muscarinic receptors thereby activating nitric oxide synthase. This
results in the endothelial release of nitric oxide (NO) causing vasodilatation 17. Each dose was
infused for 5 minutes and FBF was measured during the last 3 minutes of the 5 minute period.
After the infusion all three doses of acetylcholine a 30 minutes equilibration period followed.
Subsequently, baseline measurements were performed with the infusion of glucose 5%
solution. Subsequently, three increasing doses of sodium nitroprusside (0.06, 0.20 and 0.60
µg.dl-1.min-1, 25 mg/ml, dissolved to its final concentration with glucose 5% solution, Clinical
Pharmacy, Radboud University Medical Centre), an endothelium-independent vasodilator 18,
were infused in the brachial artery. Again, each dose was infused for 5 minutes and FBF was
measured during the last 3 minutes of the 5 minute period. FBF registrations of the last 2
minutes of each dosage of vasodilator were averaged to a single value for data analysis. An
overview of the measurements is shown in figure 1B.
36
Vildagliptin improves endothelium-dependent vasodilatation in type 2 diabetes mellitus 
Calculations and statistical analysis
 Sample size calculation.
We considered a 30% change in primary endpoint between treatment groups, acetylcholineinduced vasodilatation, as clinically relevant. Assuming a test-to-test correlation coefficient
of 0,5 and a mean forearm blood flow (FBF) in response to acetylcholine of 19 ml.dl⁻¹.min⁻¹,
would require a total of 15 subjects to detect a 30 % change in forearm blood flow with a
power of 80 % at a significance level of 0.05 (Zα = 1.96).
2
Before each set of drug infusion, baseline FBF measurements were performed. Repeated
measures analysis of variances (ANOVA) was used to assess differences in increments in FBF
between groups. We used the factors treatment (vildagliptin versus acarbose) and time (three
increasing dosage of vasodilator) en subjects were included as matched variables. Differences
in means of laboratory results and haemodynamic parameters were tested by paired Student’s
t-test or Wilcoxon signed rank test for non-normally distributed data. Statistical analyses were
performed using Graphpad 5.0. Results in tables and figures are expressed as mean ±SEM,
unless otherwise indicated. Significance was set at a p-value of less than 0.05.
Results
Seventeen of the twenty initially screened subjects underwent randomization and were enrolled
in the study. One participant withdrew due to nausea and upper abdominal discomfort
during the first week of vildagliptin treatment. The mean age of the remaining 16 participants
was 59.8±6.8 years. The mean duration of diabetes was 7.9±5.3 years with a HbA1c of
6.97±0.61%. Ten participants (62%) were treated with metformin and 6 (38%) with metformin in
combination with a sulfonylurea. Of these 6 participants 3 used glimepiride, 2 participants used
tolbutamide and 1 used glibenclamide.
Table 1 shows the baseline characteristics of all subjects that completed the study.
After completion of the trial but before deblinding one subject was excluded from statistical
analyses because of unreliable measurement results, probably the result of a technical
problem with one of the strain gauges.
37
 Vildagliptin improves endothelium-dependent vasodilatation in type 2 diabetes mellitus
Characteristic
Age (years)
59.8±6.8
Sex (male:female)
12:4
Weight (kg)
88.5±14.6
BMI (kg/m²)
29.1±4.8
Blood pressure systolic (mmHg)
141±8
Blood pressure diastolic (mmHg)
83±7
Non-fasting glucose (mmol/l)
9.55±3.91
HbA1c (%)
6.97±0.61
Duration diabetes (years)
7.9±5.3
Antihypertensives
44%
Statins
68%
Diabetes medication
62% metformin monotherapy
38% metformin+ sulfonylurea
Table 1. Baseline characteristics (mean±SD)
Vascular responses to acetylcholine
Baseline FBF in the experimental arm in the vildagliptin group was higher than in the acarbose
group. Baseline values for FBF in the experimental arm were 3.3±0.3 ml.dl⁻¹.min⁻¹ in the
vildagliptin group and 2.5±0.2 ml.dl⁻¹.min⁻¹ in the acarbose group (P = 0.02). Corresponding
values in the non experimental arm were 2.7±0.3 and 2.2±0.3 ml.dl⁻¹.min⁻¹ (P =0.07).
To compensate for differences in baseline FBF, responses to drugs were expressed as
absolute change in FBF (Δ FBF) above baseline. Infusion of acetylcholine induced a dosedependent increase in FBF in the experimental arm, while FBF did not change in the non
experimental arm. Vildagliptin treatment augmented the absolute increase in FBF in response
to acetylcholine from baseline compared to acarbose (P = 0.01 by two-way ANOVA). Changes
in FBF from baseline with vildagliptin were 3.1±0.7, 7.9±1.1 and 12.6±1.4 ml.dl⁻¹.min⁻¹ in
response to acetylcholine 0.5, 2.0 and 8.0 µg.dl⁻¹.min⁻¹ respectively compared to 2.0±0.7,
5.0± 1.2 and 11.7± 1.6 ml.dl⁻¹.min⁻¹ with acarbose. Results of the repeated measures
two-way ANOVA for acetylcholine were: p < 0.0001 for changes over time, p = 0.002 for
differences between treatment and p = 0.01 for their interaction. Figure 2 shows the change
in FBF from baseline in response to the different vasodilators. Comparable results were
obtained when data were expressed as relative changes in FBF from baseline. When data were
expressed as mean FBF (without correction for basleine differences) the repeated measures
two way ANOVA was still borderline significant. We did not find a carry-over effect.
38
Vildagliptin improves endothelium-dependent vasodilatation in type 2 diabetes mellitus 
Δ FBF (ml.dl-1.min-1)
Vildagliptin
Acarbose
p = 0.01
2
Δ FBF (ml.dl-1.min-1)
Saline
Ach 0.5
Ach 2.0
Ach 8.0
p = ns
Glucose
SNP 0.06 SNP 0.20 SNP 0.60
Figure 2.Change in forearm blood flow in response to acetylcholine in the experimental arm in response
to acetylcholine (top panel; dosage 0.5, 2.0, 8.0 µg.dl⁻¹.min⁻¹) and sodium nitroprusside (lower
panel; dosage 0.06, 0.20 and 0.60 µg.dl⁻¹.min⁻¹), during vildagliptin (filled circles, solid lines) or
acarbose (open squares, dashed lines). P = 0.01 by two-way repeated measures ANOVA.
39
 Vildagliptin improves endothelium-dependent vasodilatation in type 2 diabetes mellitus
Vascular responses to sodium nitroprusside
Infusion of sodium nitroprusside induced a dose-dependent increase in FBF in the
experimental arm. FBF did not change in the non experimental arm. Treatment with vildagliptin
did not affect vascular responses to sodium nitroprusside. Changes in FBF from baseline
when treated with vildagliptin were 3.6± 0.5, 7.4± 0.9 and 11.9± 1.0 ml.dl⁻¹.min⁻¹ in response
to sodium nitroprusside 0.06, 0.20 and 0.60 µg.dl⁻¹.min⁻¹ respectively, compared to 3.5±
0.4 , 6.4± 0.8 and 10.7± 1.0 ml.dl⁻¹.min⁻¹ with acarbose. Results of the repeated measures
two-way ANOVA were: p < 0.0001 for changes over time, p = 0.06 for differences between
treatment and p = 0.36 for their interaction. Again, we did not find a carry-over effect.
Haemodynamic and metabolic parameters
Both systolic and diastolic blood pressure (measured intra-arterially during the venous
occlusion plethysmography) tended to be lower when treated with vildagliptin compared to
acarbose (143.1±4.7 vs 145.2±3.6 mmHg (P = 0.37) and 73.3±2.4 vs 74.9±2.3 mmHg (P
= 0.22)). Mean arterial pressure was 100.7±3.2 during vildagliptin compared to 102.4±2.8
mmHg during acarbose treatment. (P = 0.27) Heart rate was 67.9±2.1 bpm when treated with
vildagliptin and 66.3±2.5 bpm when treated acarbose. (P = 0.41)
Fasting plasma glucose (measured intra-arterially) was lower during vildagliptin than during
acarbose treatment (6.62±0.27 vs 7.59±0.28 mmol/l, P < 0.0001). Fasting insulin levels were
14.5±2.0 mE/l after vildagliptin and 15.6±2.4 mE/l after acarbose treatment (P=NS). HbA1c
levels tended to be lower during with vildagliptin compared to acarbose treatment (6.73±0.12
versus 6.84±0.12% respectively, P = 0.07).
There was no change in body weight and lipid profile (data not shown).
Side effects and safety
Most reported side effects during vildagliptin treatment were flatulence (n=4) and nausea
(n=3). One subject withdrew during the first week of vildagliptin treatment because of upper
abdominal pain and nausea. Flatulence (n=15) and diarrhea (n=5) were most frequent side
effects during acarbose treatment. Fortunately, no hypoglycemic events occurred.
No changes in blood count, renal function and liver function were observed during vildagliptin
treatment.
40
Vildagliptin improves endothelium-dependent vasodilatation in type 2 diabetes mellitus 
Conclusions
The main finding of the present study is that four week treatment with vildagliptin improves
endothelial function in patients with type 2 diabetes. This conclusion is based on the
observation that vildagliptin augments the vasodilator response in the forearm vascular bed to
acetylcholine, an endothelium-dependent vasodilator, while the vascular response to sodium
nitroprusside, an endothelium-independent vasodilator, remains unaffected. Baseline blood
2
flow tended to be higher during vildagliptin, accompanied by slightly lower blood pressure
values, but these changes did not attain statistical significance.
A number of mechanisms may underlie these results. Given that GLP-1 is a physiological
substrate of DPP-4, DPP-4 inhibition by vildagliptin may be expected to increase circulating
levels of GLP-1 19. Several studies have reported beneficial effects of GLP-1 on the
cardiovascular system. In humans, Nikolaidis et al have shown that 72 hour infusion of GLP1 improved left ventricular function in patients with acute myocardial infarction and systolic
dysfunction after successful reperfusion therapy, an effect that was observed in both diabetic
and non-diabetic patients 20. They discussed that this observation might be explained by the
insulinotropic and insulinomimetic properties of GLP-1, but alternatively GLP-1 might also
improve endothelial function. Studies have shown that GLP-1 improves endothelium-dependent
vascular responses in the brachial artery while leaving endothelium-independent responses
unaffected in healthy humans and patients with type 2 diabetes 11,21.The cardiovascular actions
of GLP-1 may occur either directly through the GLP-1 receptor or alternatively by a GLP-1
receptor independent effect of the degradation product of GLP-1, GLP-1 (9-36) 12.
Apart from GLP-1, DPP-4 also degrades GIP, and perhaps cytokines and certain chemokines
(including SDF-1α), thus other substrates of DPP-4 may be responsible for the improvement in
endothelial function. Alternatively, vildagliptin might improve endothelial function by influencing
insulin levels and glucose level. Insulin causes vasodilatation by increasing endothelial
production of NO 22. However, we did not find changes in fasting insulin levels when treated
with vildagliptin. This is in correspondence with literature, in which DPP-4 inhibitors do not
influence fasting insulin levels 19.
Although we aimed for similar glucose levels during both treatments, this was not completely
accomplished as the fasting glucose levels were slightly higher in the acarbose group
(6.62±0.27 for vildagliptin vs 7.59±0.28 mmol/l for acarbose). It seems unlikely that this
small difference of 1.0 mmol/l causes the observed difference in vascular response. This is
supported by absence of any correlation between change in glucose and change in increase in
FBF from baseline in response to acetylcholine. (r² = 0.05, data not shown).
Finally, vildagliptin might have a direct pharmacological, yet unidentified, effect on the
endothelium.
41
 Vildagliptin improves endothelium-dependent vasodilatation in type 2 diabetes mellitus
The improvement in endothelium-dependent vasodilatation in the forearm observed after
four week treatment with vildagliptin might translate into a decrease in cardiovascular events.
Endothelial dysfunction is a marker of cardiovascular complications and a close relation exists
between endothelial function in the human coronary circulation and peripheral circulation 23,24.
This possibility is supported by a recent meta-analysis showing that treatment with vildagliptin
50 mg bid may decrease the relative risk of cardio-cerebrovascular events(RR 0.84,CI 0.621.14) 14. Of course these findings need to be confirmed by a long-term cardiovascular
outcome study.
As secondary findings we found slightly lower diastolic and systolic blood pressure with a
slightly higher heart rate after vildagliptin treatment. Furthermore, baseline measurement of FBF
were also higher after vildagliptin than after acarbose treatment (3.3±0.3 vs 2.5±0.2 ml.dl⁻¹.
min⁻ ¹ in the experimental arm and 2.7±0.3 vs 2.2±0.3 ml.dl⁻¹.min⁻ ¹ in the non experimental
arm). Basal vascular tone can be affected by the NO pathway as well as by the sympathetic
nervous system 24. The lower blood pressure and increased basal FBF might implicate that
vildagliptin is a systemic vasodilator. This observation can be due to an enhanced availability
of NO as a result of improved endothelial function or alteration in the activity of the sympathetic
nervous system. However, 26 week treatment with DPP-4 inhibitor alogliptin did not cause
clinical meaningful changes in either systolic or diastolic blood pressure 25.
Our study has limitations. First of all, the duration of treatment with vildagliptin was relatively
short. This four week period was enough to show improved endothelial function but larger
outcome trials are needed to evaluate potential beneficial effect on cardiovascular outcome.
Second, although this study was double blinded, more subjects experienced flatulence during
acarbose (15 vs 4) which might compromise this. However, the investigator analyzed the FBF
results of plethysmography of each treatment period separately and the flow measurements
are objective. Third, the forearm vasodilator response to acetylcholine is only one of the
methods to study endothelial function. Nevertheless, these functional measurements are
one of the most representative indices of endothelial function. Fourth, acarbose in an
alpha-glucosidase inhibitor that might increase GLP-1 levels. The reason for using an active
comparator drug in our study was to aim for equal glucose levels. Our study design does not
allow us to conclude that the effect of vildagliptin on endothelial function is GLP-1 dependent.
But if this were true, the observed difference in FBF would have been diminished by acarbose
and larger differences would have been found using placebo. However, when using placebo
our results might have been confounded by a larger change in fasting glucose. Furthermore,
acarbose is the optimal active comparator drug in this trial, since it is not absorbed into the
circulation and thus cannot influence endothelial function directly.
The study has also strengths, the effects of vildagliptin were studied in a relevant population
– metformin-treated patients with type 2 diabetes – and thus applicable to clinical practice. In
42
Vildagliptin improves endothelium-dependent vasodilatation in type 2 diabetes mellitus 
addition, an appropriate control treatment arm was used to avoid major differences in glycemic
control.
In conclusion, four weeks treatment with vildagliptin improves endothelium-dependent
vasodilatation. These findings may be relevant in the context of future cardiovascular
complications and provide a basis for further cardiovascular outcome studies.
2
43
 Vildagliptin improves endothelium-dependent vasodilatation in type 2 diabetes mellitus
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45
 Vildagliptin improves endothelium-dependent vasodilatation in type 2 diabetes mellitus
Online appendix
Vildagliptin
Acarbose
P-value
Diastolic BP (mmHg)
73.3±2.4
74.9±2.3
0.22
Mean arterial pressure (mmHg)
100.7±3.2
102.4±2.8
0.27
Systolic BP (mmHg)
143.1±4.7
145.2±3.6
0.37
Heart rate
67.9±2.1
66.3±2.5
0.41
HbA1c(%)
6.73±0.12
6.84±0.12
0.07
Fasting glucose (mmol/l)
6.62±0.27
7.59±0.28
<0.0001
Fasting insulin (mE/l)
14.5±2.0
15.6±2.4
0.22
Body weight
88.2±3.9
88.1±3.8
0.68
Total cholesterol (mmol/l)
4.10±0.23
4.33±0.25
0.09
Triglycerides (mmol/l)
1.52±0.14
1.61±0.20
0.66
HDL (mmol/l)
1.09±0.05
1.1±0.05
0.24
LDL (mmol/l)
2.32±0.21
2.37±0.24
0.82
Table 1. Effect of vildagliptin on various vascular and metabolic parameters. Statistical analysis by paired
Student’s t-test.
46
Vildagliptin improves endothelium-dependent vasodilatation in type 2 diabetes mellitus 
2
47
BETA
BETA
FUNCTION
DYSFUNCTION
CELL
CELL
ENDOTHELIAL
ENDOTHELIAL
INSULIN
INSULIN
FUNCTION
SENSITIVE
DYSFUNCTION
RESISTANT
CHAPTER 3
THE DIPEPTIDYL PEPTIDASE-4 INHIBITOR VILDAGLIPTIN
DOES NOT AFFECT EX VIVO CYTOKINE RESPONSE AND
LYMPHOCYTE FUNCTION IN PATIENTS WITH TYPE 2 DIABETES
MELLITUS
P.C.M. van Poppel, M.S. Gresgnigt, P. Smits, M.G. Netea, C.J. Tack
Diabetes Res Clin Pract 2014 Mar;103(3):395-401
 Vildagliptin does not affect cytokine response
Abstract
Aims
The enzyme dipeptidyl peptidase-4 (DPP-4) is a key player in the degradation of incretin
hormones that are involved in glucose metabolism. DPP-4 is also expressed on immune
cells and is associated with several immunological functions. Some studies have reported
increased rates of infections in patients treated with DPP-4 inhibitors. We therefore assessed
whether treatment with the DPP-4 inhibitor vildagliptin affected cytokine production and T-cell
differentiation.
Methods
Patients with type 2 diabetes were treated with vildagliptin or an active comparator, acarbose,
for four weeks, in a randomized cross-over trial. Blood was sampled at the end of each
treatment period and peripheral blood mononuclear cells were isolated and stimulated with a
broad spectrum of pattern recognition receptor agonists.
Results
Serum cytokine concentrations and ex-vivo cytokine production (both monocyte and T-cell
derived) did not differ during treatment with vildagliptin compared to acarbose. Similarly, ex-vivo
relative upregulation of mRNA transcription of T-cell lineage specific transcription factors was
unaffected by vildagliptin treatment.
Conclusions
These data show that a four-week treatment with vildagliptin in patients with type 2 diabetes
mellitus does not result in a significant modulation of cytokine responses. This observation
suggests that inhibition of DDP-4 does not lead to an increased risk of infection by diminishing
cytokine production.
50
Vildagliptin does not affect cytokine response 
Introduction
Dipeptidyl peptidase-4 (DPP-4) inhibitors are approved for the treatment of type 2 diabetes
mellitus. Inhibition of DPP-4 prevents degradation of the incretin hormones, glucagon-like
peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), which results in
stimulation of insulin secretion and inhibition of glucagon release, both in a glucose-dependent
manner. Together, these effects improve glycaemic control in type 2 diabetes 1. The enzyme
DPP-4 is expressed on epithelial and endothelial cells in liver, lung, kidney, intestinal brushborder membranes and exists as a soluble form 2.
DPP-4 is also expressed on circulating T-, B-lymphocytes and natural killer (NK) cells, and is
synonymous with the cell-surface antigen CD26 3. DPP-4 functions as a peptidase, acts as
3
adenosine deaminase binding site and interacts with the extracellular matrix. DPP-4/CD26
has been suggested to be a co-stimulator of T-cell activation 2,3. DPP-4 cleaves N-terminal
dipeptides from oligopeptides. Besides incretin hormones, also chemokines and cytokines
might serve as substrates 4. Theoretically, DPP-4 inhibition may thus affect immune responses,
and this could translate into an altered susceptibility to infections. Some meta-analyses
focusing on side effects of DPP-4 inhibitors, have reported a slight increase in upper respiratory
infections 5,6, although these reports are not consistent 7.
Recently, we completed a randomized, double-blind, active treatment controlled cross-over
trial in which endothelial function after treatment with the DPP-4 inhibitor vildagliptin was
assessed 8. Here we present the results of experiments assessing the effect of DPP-4 inhibitors
on the plasma concentrations of inflammatory markers, and on the ex-vivo peripheral blood
mononuclear cells (PBMC) responses to various pattern recognition receptors (PRR) stimuli.
Materials and methods
Study Population
This study was combined with a trial assessing endothelial function 8. Included were
subjects with type 2 diabetes mellitus treated with metformin with or without sulphonylurea or
thiazolidinediones, aged 35-75 years and a HbA1c < 8.0% (< 64 mmol/mol). This study was
conducted in accordance with the principles outlined in the Declaration of Helsinki. The local
ethics committee (Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands)
approved the study and all subjects gave written informed consent before participation.
51
 Vildagliptin does not affect cytokine response
Protocol
Details of the study are described elsewhere 8. In short: participants were randomized to
receive either vildagliptin 50 mg bid for 28 days or acarbose 50 mg tid for 7 days followed by
100 mg tid for the remaining 21 days on top of their medication in a cross-over double dummy
design. There was a wash-out period of 1 week. At the end of each treatment period, arterial
blood was collected for PBMC isolation and stimulation.
PBMC isolation & stimulation
Blood was diluted with PBS (1:1) and fractions were separated by Ficoll-Hypaque (Pharmacia
Biotech, Uppsala, Sweden) centrifugation. PBMCs were washed twice in PBS, resuspended in
RPMI (ICN Biomedicals, Costa Mesa, CA) (supplemented with 10 µg/ml gentamycin, 10 mM
L-glutamine and 10 mM pyruvate), counted using a particle counter (Beckmann Coulter) and
plated in 96 well round bottom plates at a concentration of 2.5*106/ml. Cells were stimulated
for 24 hours with: lipopolysaccharide (LPS) (10 ng/ml) (Escherichia coli serotype O55:B5)
(Sigma Chemical Co, St Louis, MO), Pam3Cys (10 µg/ml) (EMC microcollections, Tübingen,
Germany), β-glucan (10 µg/ml, kindly provided by Prof. David Wiliams, Tennessee University),
a combination of Pam3Cys (10 µg/ml) + β-glucan (10 µg/ml) , heat killed (HK) S. aureus
(1*107/ml) and HK C. albicans yeasts (ATCC MYA-3573 (UC820) strain, 1*105/ml). A four day
stimulation assay in the presence of 10% human serum was performed with RPMI or HK C.
albicans (1*105/ml), and a seven day stimulation assay was performed with the stimuli: RPMI,
LPS (10 ng/ml), HK S. aureus (1*107/ml), HK C. albicans (1*105/ml) and beads coated with
anti-CD3/anti-CD28 antibodies (1.25*106/ml) (Miltenyi Biotec, Germany), for the stimulation
of T-cells. After the 24h and 7 days incubation at 37°C and 5% CO2, the supernatants were
collected and stored at -20°C until cytokines were measured. After four days incubation at 37°C
at 5% CO2, cells were resuspended in 200 µl TRIzol (Sigma Chemical Co, St Louis, MO) and
stored at -20°C until RNA isolation was performed.
Quantative RT-PCR (qPCR)
RNA was isolated according to the protocol supplied with the TRIzol reagent, mRNA (1 µg) was
reverse transcribed into cDNA using iScript cDNA synthesis kit (BIORAD, Hercules, CA). qPCR
was performed using Power SYBR-Green master mix (Applied Biosystems, Foster City, CA)
and primers: 5’-ATG-AGT-ATG-CCT-GCC-GTG-TG-3’ (Beta2M FW) and 5’-CCA-AAT-GCG-GCATCT-TCA-AAC-3’ (Beta2M REV); 5’-CAA-GGG-GGC-GTC-CAA-CAA-T-3’ (T-BET FW) and 5’-TCT-
52
Vildagliptin does not affect cytokine response 
GGC-TCT-CCG-TCG-TTC-A-3’ (T-BET REV); 5’-TCA-CAA-AAT-GAA-CGG-ACA-GAA-CC-3’
(GATA3 FW) and 5’-GGT-GGT-CTG-ACA-GTT-CGC-AC-3’ (GATA3 REV); and 5’-CTG-CCCCTA-GTC-ATG-GTG-G-3’ (FOXP3 FW) and 5’-CTG-GAG-GAG-TGC-CTG-TAA-GTG-3’ (FOXP3
REV). All primers were synthesized by Biolegio (Malden, The Netherlands). PCR was performed
using Applied Biosystems 7300 real-time PCR system with PCR conditions 2 min 50°C, 10 min
95°C followed by 40 cycles at 96°C for 15 sec and 60°C for 1 min. The RNA levels of the T-cell
transcription factors T-bet, GATA-3 and FoxP3 were corrected using the signal of housekeeping
protein β2microglobulin.
3
Cytokine measurements
Cytokines were measured using ELISA-kits (Sanquin, Amsterdam, the Netherlands and R&D
Systems, Minneapolis, MN), according to the protocols supplied by the manufacturers. IL-6
and CRP were measured in serum from the participants, IL-1β, TNF-α, IL-6 and IL-10 cytokine
levels were measured in the culture supernatants from 24h stimulation and IFN-γ and IL-17 in
the supernatant from 7 day stimulations.
Ex-vivo effects of the DPP-4 inhibitor on cytokine production
To investigate the potential direct effect of inhibiting DPP-4 on cytokine production, we
performed an in-vitro experiment with the DPP-4 inhibitor K579 (R&D Systems). We chose
this DPP-4 inhibitor since it has a similar IC50 in humans as vildagliptin 9. Blood was collected
from 4 healthy controls for 24 hr stimulation and from 2 healthy controls for 7 days stimulation.
PBMCs were isolated, counted and plated as described earlier. The DPP-4 inhibitor was
dissolved according to the protocol supplied by the manufacturer and added in the following
concentrations: 2450 ng/ml, 245 ng/ml and 24.5 ng/ml. The Cmax of vildagliptin is 245 ng/ml
after oral administration 10. After an incubation period of 15 minutes, cells were stimulated for
24 hours with: RPMI, LPS (10 ng/ml), Pam3Cys (10 µg/ml), β-glucan (10 µg/ml), a combination
of Pam3Cys (10 µg/ml) + β-glucan (10 µg/ml), heat killed (HK) S. aureus (1*107/ml) and HK C.
albicans yeasts. 7 day stimulation was performed in the presence of 10% human serum with:
RPMI, HK S. aureus and HK C. albicans. After the 24h and 7 days incubation at 37°C and 5%
CO2, the supernatants were collected and stored at -20°C until cytokines were measured. IL-1β
and TNFα were measured in the culture supernatants of 24h stimulation, whereas IL-17 and
IFNγ were measured in the supernatant of 7 day stimulation.
53
 Vildagliptin does not affect cytokine response
Statistical analysis
Differences in immune response between the two treatments were analyzed using the Wilcoxon
signed rank test (a p- value of <0.05 was considered statistically significant). The data
represents cumulative results of all participants and are presented as mean ± SEM, unless
otherwise indicated. Data were analysed using GraphPad Prism version 5.0.
Characteristic
Age (years)
59.8±6.8
Sex (male:female)
12:4
Weight (kg)
88.5±14.6
BMI (kg/m²)
29.1±4.8
Blood pressure systolic (mmHg)
141±8
Blood pressure diastolic (mmHg)
83±7
Non-fasting glucose (mmol/l)
9.55±3.91
HbA1c (%)
6.97±0.61
Duration diabetes (years)
7.9±5.3
Antihypertensives
44%
Statins
68%
Diabetes medication
62% metformin monotherapy
38% metformin+ sulfonylurea
Table 1. Baseline characteristics (mean±SD)
54
Vildagliptin does not affect cytokine response 
Results
A total of 16 participants completed the trial ( age 59.8±6.8 yrs, BMI 29.1±4.8 kg/m2, HbA1c
7.0±0.6% (53±-0.7mmol/mol), blood pressure 141±8/83±7 mmHg). Ten participants were
treated with metformin, 6 with metformin in combination with a sulfonylurea. Most used statins
and anti-hypertensive drugs. No changes in blood count, lipid profile, renal function and liver
function were observed during vildagliptin treatment. (data not shown).
Plasma concentrations of IL-6 and CRP did not differ between the two treatment periods (data
not shown). The ex vivo production of the proinflammatory cytokines IL-1β, TNF-α and IL-6 by
monocytes was completely similar during vildagliptin treatment when compared to acarbose
treatment after all 24h stimulations: with LPS for TLR4 stimulation, with Pam3Cys for TLR2
3
stimulation, with β-glucan for dectin-1 stimulation, with Pam3Cys and β-glucan for induction
of TLR2/dectin-1 synergism as well as for HK S. aureus or HK C. albicans to trigger a broad
immune response (Figure 1).
Also the production of the T-cell cytokines IL-10, IFN-γ and IL-17 in response to stimulation
with several microbial ligands, and direct T-cell activation with anti-CD3/CD28 antibody-coated
beads, was completely similar during treatment with vildagliptin compared to acarbose
(Figure 2A).
Finally, the relative increase of mRNA levels of the T-cell lineage specific transcription factors
T-bet, GATA-3 and FoxP3, did not differ between PBMC’s isolated after the vildagliptin and
acarbose treatment period (Figure 2B). There were no treatment-sequence effects, which
renders carry over effects unlikely.
These data were supported by in-vitro studies, in which addition of the DPP-4 inhibitor in
different concentrations to PBMCs of healthy controls did not alter the stimulation of the
proinflammatory cytokines IL-1β, TNFα, IL-17, and IFNγ by the various PRR ligands or
microorganisms. (Figure 3)
55
 Vildagliptin does not affect cytokine response
ns
s
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IL-1β (pg/ml)
IL-1β
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TNF-α
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IL-6 (pg/ml)
IL-6
Vildagliptin
Acarbose
Figure 1.Levels of the monocyte derived cytokines IL-1β, TNF-α and IL-6 (pg/ml) in the culture
supernatants of PBMCs which were isolated and stimulated after either vildagliptin treatment
(grey bars) or acarbose treatment (black bars). The PBMCs were stimulated for 24h with LPS
(10ng/ml), Pam3Cys (10µg/ml), β-glucan (10µg/ml), Pam3Cys + β-glucan (both 10µg/ml),
HK S. aureus (1*107/ml) or HK C. albicans (1*105/ml). The bars represent the mean ± SEM.
The cytokine responses to all stimuli are of the expected magnitude as with cells isolated from
untreated healthy individuals (data not shown).
56
Vildagliptin does not affect cytokine response 
A
Gata-3
B
D3
/C
ca
bi
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Acarbose
T-Bet
3
8
s
D2
Acarbose
Vildagliptin
Acarbose
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D3
FoxP3
Acarbose
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IL-10 (pg/ml)
Transcription Change
after stimulation
IL-10
αC
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S.
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IL-17 (pg/ml)
Transcription Change
after stimulation
αC
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IL-17
Vildagliptin
D2
8
ns
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RP
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S
IFN-γ (pg/ml)
Transcription Change
after stimulation
IFN-γ
Figure 2. ( A) Levels of the T-cell derived cytokines IFN-γ, IL17, and IL-10 (pg/ml) in the culture supernatants
of PBMCs which were isolated and stimulated after either vildagliptin treatment (grey bars) or
acarbose treatment (black bars). The PBMCs were stimulated for 7 days with LPS (10ng/ml), HK
S. aureus (1*107/ml), HK C. albicans (1*105/ml) or αCD3/CD28 antibody coated beads (1.25*106/
ml) for IFN-γ and IL-17 production. For IL-10 production the PBMCs were stimulated for 24h with
LPS (10ng/ml), Pam3Cys (10µg/ml), β-glucan (10µg/ml), Pam3Cys + β-glucan (both 10µg/ml), HK
S. aureus (1*107/ml) or HK C. albicans (1*105/ml. The cytokine responses to all stimuli are of the
expected magnitude as with cells isolated from untreated healthy individuals (data not shown).
The bars represent the mean ± SEM. (B) Relative upregulation of mRNA transcription of T-cell
lineage specific transcription factors after three days of stimulation with HK C. albicans (1*105/
ml) compared to unstimulated PBMCs. No significant differences in upregulation of transcription
were observed between vildagliptin and acarbose treatment.
57
 Vildagliptin does not affect cytokine response
Discussion
The present study demonstrates that treatment with the DPP-4 inhibitor vildagliptin has neither
an effect on the innate ex vivo immune response nor on adaptive T-cell responses when
compared to acarbose treatment. We found that the production of the innate proinflammatory
cytokines IL-1β, IL-6 and TNF-α in response to a wide panel of stimuli was completely similar
during both treatments. Similarly, the T-cell derived cytokines IFN-γ, IL-10 and IL-17, respectively
Th1, Th2/Treg and Th17 cytokines, were unaffected by vildagliptin treatment, as were direct
T-cell activation using CD3 and CD28 stimulation. Finally, the expression of T-cell lineage
specific transcription factors T-bet, GATA-3 and Foxp3 was not affected either by vildagliptin
treatment.
Our observations differ from some earlier results of in vitro experiments with other DPP-4
inhibitors obtained in mice. Reinhold et al found that DPP-4 inhibitors inhibited IL-2, IL-6
and IL-10 production by mouse splenocytes and thymocytes, while TGF-β1 production was
increased 11. Subsequently, they showed that human PBMCs and T-cells stimulated in vitro with
mitogens display a suppressed IL-2, IL-12, IL-10 and IFN-γ and increased TGF-β1 production,
in the presence of certain DPP-4 inhibitors 12.
Several reasons may explain this apparent discrepancy. Firstly, some reports that ascribed the
effects on immune function to DPP-4 inhibition have probably been confounded by the use
of non-selective DPP inhibitors 13. Lankas et al showed that the use of non-selective DPP-4
inhibitors resulted in impaired T-cell activation in a human in vitro model, while selective DPP4 inhibitors had no effects on T-cell activation. By using a specific DPP-4 inhibitor (sitagliptin)
in mice, Vora et al could not reproduce the immunomodulatory effects of DPP-4 inhibition 14,
which is in line with our findings with vildagliptin treatment. Our findings are also in agreement
with a report showing that treatment with DPP-4 inhibitor sitagliptin in patients with type 2
diabetes does not affect T-cell activation 15. Secondly, in vitro and animal studies often used
very high concentrations of DPP-4 inhibitors, while in our study the immune cells were exposed
to pharmacological concentrations of the vildagliptin as obtained during treatment of patients,
making the results physiologically more relevant.
58
Vildagliptin does not affect cytokine response 
bi
s
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IL-17 (pg/ml)
IFN-γ (pg/ml)
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IL-1β (pg/ml)
DMSO
DPP4i 2450 ng/ml
DPP4i 245 ng/ml
DPP4i 24.5 ng/ml
Figure 3.
IL-1β and TNFα levels in the culture supernatants of PBMCS of 4 healthy controls which were
isolated and stimulated in the presence of DMSO (vehicle control, black bars), DPP-4 inhibitor
2450 ng/ml (white bar), DPP-4 inhibitor 245 ng/ml (light grey bar) or DPP-4 inhibitor 24.5 ng/
ml (dark grey bar). Cells were stimulated for 24 hours with LPS (10ng/ml), Pam3Cys (10µg/ml),
β-glucan (10µg/ml), Pam3Cys + β-glucan (both 10µg/ml), HK C. albicans (1*105/ml) and HK
S. aureus (1*107/ml). IL-17 and IFNγ (pg/ml) levels in the culture supernatants of PBMCS of 2
healthy controls which were isolated and stimulated in the presence of 10% human serum. Cells
were stimulated for 7 days with HK C. albicans (1*105/ml) and HK S. aureus (1*107/ml).
The bars represent the mean ± SEM.
59
 Vildagliptin does not affect cytokine response
Thirdly, experiments have often been performed with DPP-4 inhibitors added ex-vivo, while
our patients were exposed to in vivo DPP-4 inhibition. The advantage of our approach is that
we have applied a wide array of PRR ligands to investigate different aspects of the immune
response during vildagliptin treatment in a clinically relevant population. This means that the
results can be directly generalized to the relevant population: patients with type 2 diabetes
mellitus on oral treatment. Moreover, when we stimulated PBMCs in vitro with different stimuli, a
specific DPP-4 inhibitor did not alter cytokine production, supporting the in-vivo data.
We did not find differences in two markers of systemic inflammation, i.e. hsCRP and IL-6,
between vildagliptin and acarbose treatment. These results differ from other studies evaluating
the effect of DPP-4 inhibition on inflammation. Three months of treatment with the DPP-4
inhibitor sitagliptin reduces systemic levels of inflammation markers hsCRP and TNFα, but not
IL-6, compared to placebo 16. Moreover, also acarbose was shown to reduce hsCRP and IL-6
levels compared to placebo 17.
So, the potential improvement in inflammation markers by inhibition of DPP-4 could have been
overcome by choosing acarbose as control group. However, with these two markers we cannot
completely rule out the possibility that vildagliptin exerts ant-inflammatory effects on fat tissue
or vascular wall. In the current trial we mainly focus on the immune response when treated with
vildagliptin.
The relative short treatment period with vildagliptin in this study cannot completely rule out the
possibility that longer duration of treatment would affect immune responses. It also remains
possible that vildagliptin might affect other components of the host immune response, since
DPP-4 has several immunological functions. DPP-4 is expressed on many cells of the immune
system including T- lymphocytes, B-lymphocytes and NK cells 2,3. Being a peptidase, DPP-4
cleaves incretin hormones, but also chemokines and cytokines 4. In the present study we did
not test the function of all the cell types expressing DPP-4, nor did we investigate the function
of chemokines and cytokines after DPP-4 inhibition. Therefore, we cannot completely rule
out the possibility that treatment with DPP-4 inhibitors increases the risk of infections by other
mechanisms.
The expression of DPP-4/CD26 on T lymphocytes has been found to be decreased in patients
with type 2 diabetes as compared to healthy controls 18. Our study population consisted only
of subjects with type 2 diabetes who received vildagliptin and acarbose in a cross over study
design. Therefore, a potential decreased expression of CD26 in type 2 diabetes does not affect
our results.
60
Vildagliptin does not affect cytokine response 
To conclude, this study shows that a four-week treatment with vildagliptin in patients with type
2 diabetes mellitus neither attenuates ex-vivo cytokine production, nor inhibits T-cell activation
and differentiation compared to acarbose. These findings suggest that inhibition of DDP-4 does
not lead to an increased risk of infection by diminishing cytokine production.
3
61
 Vildagliptin does not affect cytokine response
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12.Reinhold D, Bank U, Buhling F, et al. Inhibitors of
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dipeptidyl peptidase-4 inhibitors in type 2 diabetes.
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4.Mentlein R. Dipeptidyl-peptidase IV (CD26)--role in
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the inactivation of regulatory peptides. Regulatory
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5.Amori RE, Lau J, Pittas AG. Efficacy and safety
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6.Richter B, Bandeira-Echtler E, Bergerhoff K, Lerch
C. Emerging role of dipeptidyl peptidase-4 inhibitors
in the management of type 2 diabetes. Vascular
health and risk management 2008;4:753-68.
7.Williams-Herman D, Engel SS, Round E, et al. Safety and tolerability of sitagliptin in clinical studies:
a pooled analysis of data from 10,246 patients
with type 2 diabetes. BMC endocrine disorders
2010;10:7.
8.van Poppel PC, Netea MG, Smits P, Tack CJ. Vildag-
diabetes and its complications;24:209-13.
16.Satoh-Asahara N, Sasaki Y, Wada H, et al. A
dipeptidyl peptidase-4 inhibitor, sitagliptin, exerts
anti-inflammatory effects in type 2 diabetic patients.
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17.Derosa G, Maffioli P, Ferrari I, et al. Acarbose
actions on insulin resistance and inflammatory parameters during an oral fat load. European journal of
pharmacology 2011;651:240-50.
18.Belle LP, Bitencourt PE, de Bona KS, Zanette RA,
Moresco RN, Moretto MB. Expression of CD26 and
its association with dipeptidyl peptidase IV activity
liptin Improves Endothelium-Dependent Vasodilata-
in lymphocytes of type 2 diabetes patients. Cell
tion in Type 2 Diabetes. Diabetes Care 2011.
Biochem Biophys 2011;61:297-302.
9.He YL, Serra D, Wang Y, et al. Pharmacokinetics
and pharmacodynamics of vildagliptin in patients
with type 2 diabetes mellitus. Clinical pharmacokinetics 2007;46:577-88.
10.He YL. Clinical pharmacokinetics and pharmacodynamics of vildagliptin. Clinical pharmacokinetics
2012;51:147-62.
11.Reinhold D, Bank U, Buhling F, et al. Inhibitors
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62
dipeptidyl peptidase IV induce secretion of transfor-
Vildagliptin does not affect cytokine response 
3
63
BETA
CELL
FUNCTION
ENDOTHELIAL
FUNCTION
INSULIN
SENSITIVE
BETA
CELL
DYSFUNCTION
ENDOTHELIAL
DYSFUNCTION
INSULIN
RESISTANT
CHAPTER 4
ONE WEEK TREATMENT WITH THE IL-1 RECEPTOR
ANTAGONIST ANAKINRA LEADS TO A SUSTAINED
IMPROVEMENT IN INSULIN SENSITIVITY IN INSULIN
RESISTANT PATIENTS WITH TYPE 1 DIABETES MELLITUS
E.J.P. van Asseldonk, P.C.M. van Poppel, D.B. Ballak, R. Stienstra, M.G. Netea, C.J. Tack
Clin Immunol 2015 Oct; 160 (2):155-162
 Anakinra improves insulin sensitivity
Abstract
Context
Inflammation associated with obesity is involved in the development of insulin resistance.
Elevated glucose levels further propel insulin resistance.
Objective
We hypothesized that treatment with the Interleukin-1 receptor antagonist, anakrina, improves
insulin sensitivity.
Study design
Open label proof-of-concept study.
Overweight patients with type 1 diabetes for more than 5 years and an HbA1c level of more
than 7·5 % (58 mmol/mol) Selecting insulin resistant patients with longstanding type 1 diabetes,
and hence absent beta-cell function, allowed us to specifically study the effect of anakinra on
insulin sensitivity.
Intervention
Patients were treated with anakinra 100 mg once daily for one week.
Main outcome measures
Insulin sensitivity, insulin need and blood glucose profiles were measured before, after one
week of treatment, and after a further four weeks of follow-up.
Results
Fourteen patients completed the study. Anakinra treatment reduced systemic inflammation.
After one week of anakinra treatment, insulin sensitivity improved, an effect that was sustained
for four weeks (glucose-infusion rate improvement after one week 4·6 (95 % CI -2·5 - 11·8)
μmol kg-1 min-1 and after five weeks 4.7 (95 % CI 1.1 – 8·3) μmol kg-1 min-1). Similarly, glucose
profiles, HbA1c levels, and insulin needs improved.
Conclusions
One week treatment with anakinra improves insulin sensitivity in patients with type 1 diabetes.
These results suggest that IL-1 plays a role in mediating the insulin resistance associated with
obesity and chronic hyperglycemia, and that anakinra leads to a sustained improvement of
insulin sensitivity.
66
Anakinra improves insulin sensitivity 
Introduction
The upsurge in the prevalence of obesity has fuelled a rapid increase in the prevalence of
type 2 diabetes mellitus worldwide. This is explained by the fact that insulin resistance
associated with obesity leads to a more rapid exhaustion of beta cell function. There is strong
evidence that low-grade inflammation is involved in the pathogenesis of obesity-induced
insulin resistance.
One of the key mechanisms involved in obesity-associated inflammation, at least at the level
of the adipose tissue, is activation of the inflammasome and release of the potent cytokine
IL-1β1-4. High levels of glucose are known to promote a pro-inflammatory state 5-7. Since it is
a well-known fact that chronically elevated glucose levels induce insulin resistance, that is
reversible upon installation of normoglycemia, an enhanced inflammatory state may underlie
this phenomenon 8,9.
Low grade, systemic inflammation also appears to play a pivotal role in the pathophysiology
of beta-cell toxicity and progressive beta-cell failure associated with diabetes 10. Similarly,
chronically elevated glucose levels further deteriorate beta cell function 11,12.
4
These lines of evidence suggest that interventions aimed at reducing inflammation in general
and IL-1-activation in particular may both improve insulin sensitivity and insulin secretion.
Indeed, blocking IL-1 in patients with type 2 diabetes mellitus using recombinant human IL-1
receptor antagonist (IL-1Ra) anakinra has been shown to improve glycemic control 13.
A study using an anti-IL-1β antibody to block IL-1β action reported an improvement in glycemic
control in animals, paralleled by an enhancement in beta cell function and insulin sensitivity 14.
However, other intervention trials using specific anti-IL1b antibodies were not consistently
reporting positive effects on glycemic control 15-17. In obese, insulin-resistant individuals, we
found evidence for an improved beta-cell function after anakinra treatment, but could not
confirm an effect on insulin sensitivity after blockade of IL-1 signaling.
It should be noted that any change in glycemic control in patients with type 2 diabetes will
affect glucose toxicity and hence both insulin resistance and beta cell function. In this regard,
any change in insulin sensitivity leading to lower glucose levels could result in improved
beta cell function.
We hypothesized that the involvement of IL-1β in obesity-associated insulin resistance may
be mostly pronounced under conditions of chronic hyperglycemia. To assess this hypothesis,
we studied the effect of the interleukin-1 receptor antagonist anakinra on insulin sensitivity in
subjects with longstanding type 1 diabetes that displayed a “type 2 phenotype”. Given the
fact that beta cell function is absent in these patients, this experimental approach has the
advantage that any change in insulin resistance will immediately translate to lower glucose
levels and lower insulin need.
67
 Anakinra improves insulin sensitivity
Our results provide in vivo evidence in humans for an insulin sensitizing effect of IL-1 blockade
in the presence of hyperglycemia, inflammation and insulin resistance.
Materials and methods
Study design
Overweight patients with type 1 diabetes mellitus were included in this open-label proof-ofconcept study. Patients were recruited from the outpatient clinic of the Radboud University
Nijmegen Medical Centre. We treated patients with anakinra at 100 mg subcutaneous for eight
consecutive days. Study medication was self-administered once daily. Anakinra was purchased
from the regular manufacturer (Swedish Orphan Biovitrum). Notably, the study was performed
completely independent from any company.
Participants
The institutional ethics review board approved the study. Written informed consent was
obtained from all subjects.
Inclusion criteria were type 1 diabetes mellitus for more than five years, body mass index
higher than 25 kg/m2, insulin requirement > 0·5 U/kg bodyweight per day, HbA1c > 7·5% (58
mmol/mol), and age between 18 and 65 years.
Exclusion criteria were immunodeficiency or immunosuppressive treatment, current use
of anti-inflammatory medication (100 mg or less of aspirin per day was allowed), signs of
current infection or treatment with antibiotics, a history of recurrent infections or tuberculosis,
pregnancy or breast feeding, a serum alanine aminotransferase or aspartate aminotransferase
level of more than three times the upper limit of the normal range, a serum creatinine level
higher than 130 μmol/l, neutropenia (a leukocyte count of less than 2x109/l), or the presence
of any other medical condition that might interfere with the current study protocol. Participants
unable to give informed consent were also excluded.
68
Anakinra improves insulin sensitivity 
Study procedures
Immediately before the first injection, directly after the last injection and four weeks after the
last injection of anakinra we performed a euglycemic hyperinsulinemic clamp (insulin infusion
rate 360 pmol/m2/min)18 and obtained a subcutaneous fat biopsy. All study procedures were
performed after an overnight fast. Patients were requested to record glucose levels and insulin
use as usual, but as a minimum the two days before start of study medication, during the
intervention, and four weeks after the last treatment with anakinra.
Subcutaneous adipose tissue biopsy
A subcutaneous adipose tissue biopsy was taken from the abdominal region just before the
clamp, about 30 minutes after a subcutaneous injection with anakinra. Biopsies were taken
under local anesthesia (2% Lidocaine hcl), from an area about 10 cm lateral of the umbilicus
using a Hepafix Luer lock syringe (Braun, Melsungen, Germany) and a 2·10 x 80 mm Braun
medical Sterican needle (Braun). Adipose tissue was washed using a 0·9% normal saline
4
solution. The adipose tissue was snap frozen and stored at -80 ºC until further analysis, or it
was fixed in 4 % paraformaldehyde for embedding in paraffin.
Immunohistochemistry
An antibody against CD68 (AbD Serotec, Kidlington, UK) was used to stain macrophages.
Immunohistochemistry was performed as described earlier 19. Macrophage influx was
quantified by counting the number of adipocytes and macrophages in ten representative fields
of adipose tissue. A mean number of 205 ± 6 adipocytes were counted per subject for each
biopsy.
Biochemical analysis
Glucose concentrations were measured using the oxidation method (Biosen C-Line; EKFdiagnostic GmbH, Barleden, Germany). Concentrations of IL-1Ra, IL-8, serum amyloid A
and adiponectin were determined using ELISAs (R&D Systems, Minneapolis, MN and serum
amyloid A: Hycult Biotech, Plymouth Meeting, PA). HsCRP was measured by in-house
developed ELISA by University Hospital Maastricht 20. IL-18 was determined using magpix (BioRad laboratories Inc, Hercules, Ca). Insulin levels were determined by a radioimmunoassay.
69
 Anakinra improves insulin sensitivity
Inter- and intra-assay CV’s were respectively: IL1-ra 5·0% and 8.0%; IL-8 7·4 % and 5·6%;
hsCRP 4·7% and 3·8%, adiponectin 4·3% and 2·4%; IL-18 7% and 5%; insulin 9·7% and 4·7%.
Measurements of other parameters were performed in the clinical laboratory unit of the
Radboud University Nijmegen Medical Centre.
Statistical analysis
We considered a 15 % improvement of the primary endpoint, insulin sensitivity, as determined
by euglycemic hyperinsulinemic clamp, as clinically relevant. Assuming a test-to-test
correlation coefficient of 0·5 (paired tests) and a mean glucose infusion rate of 45·7 ± 8·5
µmol/kg/min9, would require a total of 12 subjects to detect a 15 % change in insulin sensitivity
with a power of 80 % at a significance level of 0·05. Taking into account the technical difficulties
that are frequently experienced when performing euglycemic clamps, we included 14 subjects
in our study. Dropouts were replaced. Differences were analysed by the Student’s t-test.
Correlation analysis was performed by regression analysis. Two-tailed P ≤ 0·05 was considered
to denote significance. Data are presented as mean ± SEM.
Results
A total of 14 patients were recruited from the outpatient clinic and enrolled in the trial. All
patients completed the trial. Clinical characteristics of the study population are provided
in Table 1. Patients had longstanding type 1 diabetes mellitus, were mostly obese, had
suboptimal diabetes control, and relatively high insulin needs. Fasting C-peptide levels were
below the detection limit of the assay (0·03 nmol/l) in all patients except for one patient who
had a C-peptide level of 0·04 nmol/l, both before and after the intervention. Medication, other
than insulin, was continued during the study.
No serious adverse events were observed during the trial. One patient reported painful
injections of the study medication. Adipose tissue IL-1Ra concentrations were significantly
higher directly after the intervention, and had returned to baseline values 4 weeks after the
intervention (baseline 1339 ± 347 μg/l vs. directly after 3980 ± 416 μg/l, P < 0·01 and vs. four
weeks after the intervention 1942 ± 476 μg/l, P = 0·54) (Figure 1).
70
Anakinra improves insulin sensitivity 
Characteristic
Age (years)
47 ± 9
Sex (male:female)
9:5
Diabetes duration (years)
30.9 ± 13.1
Body mass index (kg/m )
31.7 ± 4.6
Blood pressure (mmHg)
133 ± 13 / 76 ± 9
Fasting glucose (mmol/L)
9.45 ± 2.81
HbA1c (% (mmol/mol))
8.61 ± 0.70 (70)
Insulin regimen (basal-bolus:continuous subcutaneous insulin infusion)
6:8
Insulin dose (U/day)
78.6 ± 32.9
C-reactive protein (mg/L)
1.97 ± 2.85
Leucocytes (x10 /L)
5.96 ± 2.20
Neutrophiles (x109/L)
3.76 ± 1.72
Interleukin-1Ra (ng/L)
359 ± 221
2
9
Cholesterol (mmol/)
Total
4.37 ± 0.65
HDL
1.25 ± 0.37
LDL
2.69 ± 0.53
Triglycerides
0.96 ± 0.10
4
Diabetes complications
Macrovascular
1
Nephropathy
2
Polyneuropathy
7
Retinopathy
9
Medication with potential anti-inflammatory effects (number of patients)
Acetylsalicylic acid 38 mg/day
1
Acetylsalicylic acid 100 mg/day
2
Perindopril 8 mg/day
1
Enalapril 20 mg/day
2
Enalapril 40 mg/day
1
Fosinopril 20 mg/day
1
Irbesartan 300 mg/day
1
Atorvastatin 40 mg/day
1
Rosuvastatin 5 mg/day
1
Simvastatin 20 mg/day
1
Simvastatin 40 mg/day
1
Table 1. Baseline characteristics (mean±SD)
71
IL-1RA adipose tissue (mg/l)
 Anakinra improves insulin sensitivity
before
during
after
Figure 1. IL-1Ra levels adipose tissue before (white), during (black) and 5 weeks after (grey) initiation
of study medication (mean±SEM).
Systemic inflammation
As the insulin sensitizing action of anakinra is mediated by a reduction of the inflammatory
status, several inflammation markers were measured. Leukocyte and neutrophil counts were
significantly reduced during the intervention, an effect that was sustained for up to 4 weeks
(Figure 2A and B). No differences were found in systemic levels of C-reactive protein
(Figure 2C), IL-1α, IL-6, IL-8, IL-18, TNFα and serum amyloid A. IL-1β was not detectable
systemically. Serum adiponectin levels did not change during anakinra treatment.
Fat biopsies
To quantify local inflammation induced by anakinra injection (a well known side effect of one of
the solvents) fat biopsies were taken. Macrophage numbers in fat tissue were counted using
an immuno-histochemical approach. The number of macrophages did not significantly change
during or after the intervention. (Figure 2D and E).
72
Anakinra improves insulin sensitivity 
B
Leukocytes (109/I)
Neutrophils (109/I)
A
before
during
after
during
after
D
4
HsCRP (g/I)
macrophages/100 adipocytes
C
before
before
during
after
before
during
after
E
before
during
after
Figure 2. Inflammation. Serum levels of leukocytes (A), neutrophils (B) and HsCRP (C), before (white),
during (black) and 5 weeks after (grey) initiation of study medication. Number of macrophages
per adipocyte in subcutaneous adipose tissue biopsy before (white), during (black) and 5 weeks
after (grey) initiation of study medication (D+E).
73
 Anakinra improves insulin sensitivity
Insulin sensitivity and glycemic control
At baseline, subjects were moderately insulin resistant. Insulin sensitivity as determined by the
glucose infusion rate (euglycemic hyperinsulinemic clamp), was 34·5 ± 3·7 μmol kg-1 min-1
(reference value for lean individuals is 53·9 μmol kg-1 min-1). 21 During treatment with anakinra,
insulin sensitivity numerically increased to 39·2 ± 4.0 μmol kg-1 min-1, although this change
did not attain statistical significance, but the improvement was persistent and significant four
weeks after the last administration of anakinra (insulin sensitivity 39·2 ± 3·2 μmol kg-1 min-1,
P = 0·02) (Figure 3A).
HbA1c levels four weeks after termination of anakinra treatment were in accordance with the
results of the clamps and dropped from baseline 8·61 ± 0·19% (71 mmol/mol) to 8·34 ± 0·16%
(68 mmol/mol), P < 0·01) (Figure 3B).
Improved glycemic control was also shown by a decrease in mean glucose levels from 9·71 ±
0·45 mmol/l at baseline, to 8·33 ± 0·40 mmol/l during anakinra usage (P < 0·01) and to 8·34 ±
0·55 mmol/l after termination of the anakinra intervention (P = 0·04) (Figure 3C). The improved
glycemic control was achieved with a lower requirement of insulin. At baseline, average insulin
usage was 78·6 ± 8·8 U/day, during anakinra treatment the patients used 66·9 ± 6·5 U/day
and remained lower four weeks after the last injection of anakinra (69·7 ± 7·2 U/day) (Figure 3D).
The improvement in insulin sensitivity appeared to be associated with the anti-inflammatory
efficacy of anakinra treatment since the enhancement in insulin sensitivity levels correlated with
the reduction in leukocyte (R2 = 0·39, P = 0·02) (Figure 3E) and neutrophil counts (R2 = 0·47,
P < 0·01) (Figure 3F) directly after the treatment.
Cholesterol
Lipid levels (total cholesterol, HDL-cholesterol, LDL-cholesterol and triglycerides) were not
significantly different after the intervention compared to baseline.
74
Anakinra improves insulin sensitivity 
B
Insulin use
(U/day/kg)
Insulin sensitivity
µmol/kg/m2
A
before during
after
before during
after
D
after
F
before during
after
Leukocytes (%)
Neutrophils (%)
before during
E
4
HbA1c (%)
Glucose (mmol/I)
C
Insulin sensitivity (%)
Insulin sensitivity (%)
Figure 3. Insulin sensitivity and glycemic control. Insulin sensitivity as determined by euglycemic
hyperinsulinemic clamp before (white), during (black) and 5 weeks after (grey) initiation of
study medication (A). Insulin use (B), HbA1c (C) and glucose levels during the day as reported
by patients (D) before, during and 5 weeks after initiation of study medication. Change in
systemic leukocyte counts (E) and systemic neutrophil counts (F) correlated with change in
insulin sensitivity, black lines represent linear regression analysis.
75
 Anakinra improves insulin sensitivity
Discussion
The main finding of our study is that a one week treatment with the IL-1 receptor anakinra
decreases systemic inflammation and improves insulin sensitivity in insulin resistant patients
with type 1 diabetes. This change in insulin sensitivity is mirrored by better glucose control and
decreased insulin needs.
The demonstration of improved glycemic control with anakinra treatment is in line with an
earlier study demonstrating a beneficial effect of anakinra in patients with type 2 diabetes
after 13 weeks of treatment 13. However, there are also important differences between the two
studies. Larsen et al. studied patients with type 2 diabetes and found a beneficial effect on
beta cell function, explaining the improved glucose control. However, this direct effect on the
beta cell cannot be the explanation in our study performed in patients with type 1 diabetes, as
by definition these patients have no residual beta cell function.
Earlier trials using anakinra in humans did not detect improvements in insulin sensitivity. In
the trial of Larsen et al. that used euglycemic-hyperinsulinemic clamp studies performed in a
subgroup of patients and an analysis of the insulin-sensitivity index by the homeostasis model
assessment (HOMA), did not reveal any difference after anakinra as compared to placebo
treatment 13. The same holds true for our previous trial in which glucose disposal rates were
not significantly different between anakinra and placebo treatment 22. The lack of effect of
anakinra on insulin sensitivity in these earlier studies may partially be explained by injection
site reactions that are a well-known adverse event affecting a large percentage of treated
patients. The reaction is transient and is caused by one of the solvents for solution for injection
of anakinra 23. Indeed, in our previous trial that included four weeks of treatment with anakinra,
an inverse correlation between the number of macrophages in the subcutaneous adipose
tissue caused by injection site reactions and the improvement in systemic insulin sensitivity
levels was observed 22. A recent study in mice showed a direct link between adipose tissue
macrophages and bone marrow myeloid progenitor proliferation. Treatment with anakinra
resulted in decreased leukocyte production, fewer adipose tissue macrophages and improved
glucose tolerance 24.
Because of the design of the current trial, in which the dose of anakinra was reduced by 33%
and the treatment period was reduced by 75% in comparison to our earlier trial, results were
less likely to be influenced by a pro-inflammatory reaction in the subcutaneous adipose tissue.
Indeed, patients in this trial did not suffer from an enhancement in local inflammatory status, as
the number of infiltrating macrophages in the subcutaneous abdominal adipose tissue did not
significantly change after the intervention period. Furthermore no injection site reactions were
recorded. Although we are not informed about the effect of anakinra on the inflammatory status
76
Anakinra improves insulin sensitivity 
of the visceral adipose tissue it appears reasonable to expect that an increase in inflammatory
status of the subcutaneous abdominal adipose tissue blurs a potential effect of long term
(more than one week) anakinra treatment on insulin sensitivity, as 50% of patients treated with
anakinra reported such an adverse event 13.
A second reason for the difference in findings is that our previous trial consisted of nondiabetic subjects, and hence hyperglycemia was not involved. Since glucose toxicity induces
insulin resistance, anakinra may especially reduce the insulin resistance associated with high
glucose levels, which is in agreement with earlier results showing that glucose toxicity is partly
mediated by IL-1β 25-27.
Finally, our study population existed of patients diagnosed with type 1 diabetes mellitus.
Endogenous levels of IL-1Ra in patients with type 1 diabetes mellitus are reported to be lower
in comparison to patients with type 2 diabetes mellitus 28. Hence, treatment with anakinra may
be more effective in patients that suffer from low circulating levels as compared to patients with
higher basal plasma concentrations of IL-1ra.
Studies testing IL-1β antibodies in humans report positive effects on glycemic control 15,17.
4
The clinical trial using Gevokizumab does show improvement in beta-cell function, but no
effects on insulin sensitivity measured by the insulin sensitivity index were seen. Canakinumab
did not result in improvement in homeostatic model of assessment of insulin resistance.
These antibodies are specifically blocking IL-1β, while anakinra blocks both IL-1α and IL-1β.
Furthermore, the bioavailability of these antibodies in the adipose tissue is unknown. In the
present study we show a clearly increased level of anakinra in adipose tissue during treatment.
Besides, the studies testing IL-1β antibodies did not focus on insulin sensitivity. The outcome
measures used are less sensitive in recording changes in insulin sensitivity, as compared to
the clamp technique used in this study.
The therapeutic efficacy of anakinra treatment observed in our study may largely be the
result of a decrease in systemic inflammatory levels, as represented by lower leukocyte and
neutrophil counts. This is supported by our observation that a decrease in leukocyte counts
correlates with an improvement in insulin sensitivity. One would expect reduced levels in other
inflammation parameters like hsCRP, serum amyloid A and IL-6 as well. However this was not
seen in the current trial. An explanation could be the already low inflammatory levels before
initiation of the trial. Other explanations are the short treatment interval, or an assay failure.
Our study has important potential clinical implications. While the current, once daily treatment
with anakinra is inconvenient and might be complicated with injection site reactions, a short
period of treatment with IL-1ra, either subcutaneously or i.v., may be effective in patients with
extreme insulin resistance due to longstanding high glucose levels. Here, optimal treatment
duration and dose need to be determined.
77
 Anakinra improves insulin sensitivity
Our study has limitations. First of all, this was an open trial and hence cannot exclude a study
effect. The relatively strong and sustained effect and the correlation between improvements in
insulin sensitivity and reduction in systemic inflammation argue against this possibility. Another
possibility that cannot be excluded is that patients have changed their dietary or exercise
habits that may promote improvements in insulin sensitivity. Noticeably, no significant changes
in bodyweight were reported in any of the study participants during the intervention.
The strength of our study lies in the precisely characterized treatment in a carefully selected
group of patients. By testing the effects of anakinra in patients without residual beta-cell
function we were able to rule out secondary effects on insulin sensitivity mediated by reduced
glucose toxicity.
In conclusion, this study proves that one week of treatment with the IL-1 receptor antagonist
anakinra improves insulin sensitivity, an effect that is sustained for at least 4 weeks. The change
in insulin sensitivity is mirrored by lower insulin needs and better glucose control. This study
strongly suggests involvement of IL-1 in the insulin resistance associated with the combination
of obesity and hyperglycemia. Whether this translates to a chronic treatment approach in some
subsets of patients (those with pronounced hyperglycemia-associated insulin resistance)
remains to be determined.
78
Anakinra improves insulin sensitivity 
4
79
 Anakinra improves insulin sensitivity
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81
BETA
CELL
FUNCTION
ENDOTHELIAL
FUNCTION
INSULIN
SENSITIVE
BETA
CELL
DYSFUNCTION
ENDOTHELIAL
DYSFUNCTION
INSULIN
RESISTANT
CHAPTER 5
THE INTERLEUKIN-1 RECEPTOR ANTAGONIST ANAKINRA
IMPROVES FIRST PHASE INSULIN SECRETION AND
INSULINOGENIC INDEX IN SUBJECTS WITH IMPAIRED
GLUCOSE TOLERANCE
P.C.M. van Poppel, E.J.P. van Asseldonk, J.J. Holst, T. Vilsbøll, M.G. Netea, C.J. Tack
Accepted in adapted form in Diabetes Obes Metab 2014 Dec;16(12):1269-1273
 Anakinra imroves beta cell function
Abstract
Aims
Beta cell function progressively deteriorates during the course of type 2 diabetes mellitus.
Inflammation at the level of the beta cell seems to be involved in beta cell dysfunction with a
prominent role for interleukin (IL)-1 β in this process. Blocking the effects of IL-1 in patients with
type 2 diabetes by anakinra, a recombinant human IL-1 receptor antagonist, has been shown
to improve glycemic control. There is limited information regarding the effect of blocking IL-1 on
beta cell function. In the present study we assessed the effect of anakinra on beta cell function.
Materials and methods
Sixteen participants with impaired glucose tolerance (IGT) were treated with anakinra 150 mg
daily for 4 weeks in a double blind, randomized, placebo controlled, cross-over study. At the
end of both treatment periods, an oral glucose tolerance test (OGTT) and hyperglycemic clamp
were performed to assess beta cell function.
Results
Anakinra treatment reduced C-reactive protein (CRP) levels, leukocyte and neutrophil counts.
First phase insulin secretion improved after anakinra treatment compared to placebo, 148±20
vs 123±14 mU/l respectively (p = 0.03). In line with these results, the insulinogenic index
derived from the OGTT was higher after anakinra treatment. Anakinra had no effect on second
phase insulin secretion, insulin response to arginine, insulin sensitivity index or incretin levels.
Conclusions
These results support the concept of involvement of IL-1β in the (progressive) decrease of
insulin secretion capacity associated with type 2 diabetes.
84
Anakinra imroves beta cell function 
Introduction
Type 2 diabetes mellitus occurs when beta cells fail to appropriately increase insulin secretion
in response to insulin resistance 1. Beta cell function is partly under genetic control 2,3, and
progressively deteriorates over time4,5.
Inflammation contributes to both insulin resistance and beta cell dysfunction 6-9. The
proinflammatory cytokine interleukin-1 (IL-1) is an important mediator in the inflammatory
process in the beta cell leading to the development of type 2 diabetes mellitus. In vitro, high
glucose levels increase beta cell production of IL-1β followed by functional impairment and
apoptosis of beta cells10. Beta cells can be rescued from hyperglycemia-induced apoptosis by
addition of the interleukin-1 receptor antagonist (IL-1Ra), a naturally occurring inhibitor of IL-1β
which competitively binds to the IL-1 receptor without inducing a cellular response 10,11.
Blocking the effects of IL-1 in patients with type 2 diabetes mellitus by anakinra, a recombinant
human IL-1Ra, has been shown to improve glycemic control through enhanced beta cell
function 12, However, in patients with diabetes, improvement of beta cell function can be either
direct or indirect due to improvement in glucose levels, since hyperglycemia itself impairs both
insulin secretion and insulin sensitivity (concept of glucotoxicity) 13.
Recently, the beneficial effects of anakinra were not confirmed; canakinumab, a human
monoclonal anti-IL1β antibody, did not improve glycemia or insulin secretion rates in patients
with type 2 diabetes mellitus. However, canakinumab did enhance the peak insulin levels in
5
subjects with impaired glucose tolerance (IGT) compared to placebo 14.
Thus, the potential of IL-1β blockade to attenuate the progression towards type 2 diabetes
mellitus may be mainly at the early stage of impaired glucose tolerance. We hypothesized
that treatment with the IL-1Ra anakinra will improve beta cell function in first-degree relatives
of patients with type 2 diabetes mellitus with IGT. We also investigated whether the antiinflammatory treatment of anakinra could affect the incretin system.
85
 Anakinra imroves beta cell function
Materials and methods
Study population
The study was conducted in accordance with the principles outlined in the declaration of
Helsinki. The local ethics committee (Radboud University Nijmegen Medical Centre, Nijmegen,
The Netherlands) approved the study and all subjects gave written informed consent before
participation. The trial was registered at clinicaltrials.gov (number NCT01285232).
The study population consisted of 16 first-degree relatives of patients with type 2 diabetes
mellitus. Subjects were recruited through advertisements in local newspapers and through the
outpatient clinic of the Radboud University Nijmegen Medical Centre.
Included were participants with impaired fasting glucose (IFG) and/or impaired glucose
tolerance (IGT) as assessed by 75 g oral glucose tolerance test (OGTT) and/ or hemoglobin
A1c (HbA1c) levels of 5.7-6.4%. IFG and IGT were defined by American Diabetes Association:
fasting glucose 5.6-6.9 mmol/l and 2 hour plasma glucose during OGTT of 7.8-11.0 mmol/l
respectively. Other inclusion criteria were body mass index (BMI) > 25 kg/m² and age 40-70
years. We excluded participants with known diabetes mellitus, fasting glucose ≥ 7 mmol/l or
HbA1c ≥ 6.5 %, immunodeficiency or immunosuppressive treatment, use of anti-inflammatory
drugs (aspirin in a dosage of 100 mg or less was allowed), signs of current infection or
treatment with antibiotics, a history of recurrent infections, neutropenia (defined as neutrophils
< 2 x 109/l), a serum alanine aminotransferase or aspartate aminotransferase level of more
than 3 times the upper limit of the normal range, renal disease defined as the modification of
diet in renal disease (MDRD) < 60 ml/min/1.73 m2 and inability to give informed consent.
Protocol
This was a randomized, double-blind, placebo controlled crossover study. Participants were
randomly assigned to treatment with anakinra 150 mg subcutaneous (sc) once daily for 4
consecutive weeks or placebo sc once daily for 4 consecutive weeks. The earlier anakinra
study applied 100 mg daily, the dose normally used in clinical practice in rheumatoid arthritis 15,
but noted less effect in obese subjects. As we expected our population to be obese, a higher
anakinra dose was chosen.
After a wash-out period of 4 weeks, participants crossed over to the other treatment arm.
Participants were contacted by phone halfway each treatment period to check for compliance
and side effects. At the end of each treatment period beta cell function was assessed by
hyperglycemic clamp. On a separate day a 75 g oral glucose tolerance test (OGTT) was
performed. The Pharmacy Department of Radboud University Nijmegen Medical Centre, The
86
Anakinra imroves beta cell function 
Netherlands, provided the anakinra and its placebo and was responsible for the blinding and
randomization procedure. After inclusion the subjects were given a randomization number by
the investigator in order of study entry. The Pharmacy Department randomized the treatment
order (anakinra or placebo) in blocks of four and stored the randomization key untill all data
had been collected.
Experimental procedures
 Hyperglycemic clamp
The hyperglycemic clamp procedure was performed after an overnight fast. On the morning of
the test, subjects injected their study medication at the research unit under supervision. Two
intravenous cannulas were inserted. One was positioned retrogradely into a dorsal vein of the
hand that was placed in a plexiglas box, ventilated with heated air, for sampling of arterialized
venous blood. The second cannula was inserted in an antecubital vein of the contralateral arm
for infusion of the glucose solution.
After a 30 minute equilibration period, the hyperglycemic clamp was started (t=0) by an
intravenous bolus of 0.8 ml glucose 20% solution per kg body weight followed by a constant
glucose 20 % infusion in order to maintain a blood glucose level of 10 mmol/l for a total
duration of 120 minutes. At 120 minutes an arginine bolus of 5 grams was administered to
5
measure maximum insulin secretory capacity at a steady-state glucose concentration of 10
mmol/l.
Blood glucose levels were measured in whole blood using the oxidation method (Biosen
C-line, EKF diagnostics, GmbH) every 5 minutes to allow precise adjustment of the glucose
infusion rate. Blood samples were taken at t= 0, 2.5, 5, 7.5, 10, 20, 40, 60, 80, 100, 120, 122.5,
125, 127.5, 130 and 150 minutes and stored on ice for assessment of insulin and C-peptide
concentration. After the clamp the blood samples were centrifuged and plasma was stored
at -80⁰C until insulin and C-peptide measurements were performed. After 150 minutes of
hyperglycemia glucose infusion was discontinued.
 Oral glucose tolerance test (OGTT)
The OGTT was performed in the morning after an overnight fast. After collection of the fasting
blood samples, the subject drank 75 g of anhydrous glucose in 250-300 ml water over the
course of 5 minutes. Blood samples were collected every 30 minutes for 2 hours after the test
load. The blood samples for the measurement of insulin and C-peptide were temporarily stored
on ice. After the OGTT the blood samples were centrifugated and plasma was stored at -80⁰C
until insulin and C-peptide measurements were performed (Magpix, Luminex). Plasma samples
87
 Anakinra imroves beta cell function
were assayed for total glucagon-like peptide-1 (GLP-1) immunoreactivity using antiserum
89390, which is specific for the C-terminal of the GLP-1 molecule and reacts equally with intact
GLP-1 and the primary (N-terminally truncated) metabolite 16. Glucagon was measured with
an assay directed against the C-terminal of the glucagon molecule (antibody code no. 4305-8)
and, therefore, measures glucagon of mainly pancreatic origin 16.
Calculations
 OGTT
The insulinogenic index was calculated as the increase in insulin levels from 0 to 30 minutes
divided by the increase in plasma glucose levels from 0 to 30 minutes.
 Hyperglycemic clamp
The adequacy of the clamp was assessed as mean of the blood glucose levels and the
variability as the coefficient of variance (CV) of glucose levels. First phase insulin (C-peptide)
secretion was calculated as the sum of increments of plasma insulin levels from 2.5 to 10
minutes during the hyperglycemic clamp. Second phase insulin (C-peptide) secretion was
taken as the average increment in plasma insulin levels from 80 to 120 minutes of the clamp.
Insulin sensitivity was assessed as an insulin sensitivity index (ISI), defined as glucose infusion
rate (GIR) to maintain hyperglycemia from 80 to 120 minutes divided by the mean plasma
insulin level during the same interval. The acute insulin (C-peptide) response to arginine
(∆I arginine) was calculated as the mean insulin levels of 122.5 and 125 minutes minus the
insulin levels of 120 minutes. The maximal insulin (C-peptide) concentration was the single
highest post arginine insulin level.
Statistical analysis
The sample size is based on the primary aim to detect an association between blocking
IL-1β by anakinra and insulin secretion in relatives of patients with type 2 diabetes mellitus.
Estimates of first and second phase insulin secretion in first-degree relatives of patients with
type 2 diabetes mellitus were based on literature 2. As these are paired measurements, a testtest correlation should be estimated. Assuming a test-test correlation coefficient of 0.5 and a
second phase insulin secretion of 322 pmol/l with an SD of 133 pmol/l 2, we would require a
total of 15 subjects to detect a 30% change in second phase insulin with a power of 80% at
a significance level of 0,05 (Zα=1,96). A study including 16 subjects should be sufficient to
88
Anakinra imroves beta cell function 
detect effects of anakinra on insulin secretion. Dropouts were replaced.
Statistical analyses were performed using Graphpad 5.0. Results in tables and figures are
expressed as mean±SEM, unless otherwise indicated. Differences in means were tested by
paired Student’s t-test for normally distributed data and Wilcoxon signed rank test for nonnormally distributed data. Significance was set at a p-value of less than 0.05.
Assessed for eligibility (n=37)
Excluded (n=19)

Did not meet inclusion criteria (n=16)

Diagnosed with type 2 diabetesmellitus (n=3)
Randomized (n=18)
Withdrawn before start of study
medication (n=1)
5
Received intervention (n=17)
Discontinued intervention due to
injection site reaction (n=1)
Analysed (n=16)
Figure 1. Design, enrollment, withdrawal and completion of the trial
Results
Eighteen of the 37 initially screened subjects fulfilled the inclusion criteria and and were
enrolled in the study. Of these 18 individuals, one subject withdrew before start of the study
medication. One participant withdrew in the first treatment period due injection site reaction
during the use of anakinra (see Figure 1.). A total of 16 participants (7 females and 9 males)
completed the trial. Of these 16 subjects, 6 were classified as IFG (2 isolated IFG, 4 IFG with
89
 Anakinra imroves beta cell function
IGT and 5 IFG with increased HbA1c level), 7 as IGT (3 isolated IGT, 4 IGT with IFG and 4 IGT
with increased HbA1c level), and 13 fulfilled HbA1c criteria of 5.7-6.4% (7 isolated, 5 HbA1c
with IFG, 4 HBa1c with IGT). Baseline characteristics of the participants were (mean±SD): age
55±9 years, BMI 32±5 kg/m², HbA1c 5.8±0.5 % and fasting glucose level 5.3±0.71 mmol/l.
(table 1) Concomitant diseases were hypertension (n=5), depression (n=1), hypothyreoidism
(n=1), angina pectoris (n=1), history of CABG (n=1) and asthma (n=1).
Characteristic
Age (years)
54.6±8.5
Sex (male:female)
9:7
Weight (kg)
99.0±24.1
BMI (kg/m²)
31.7±4.8
Blood pressure systolic (mmHg)
131±15
Blood pressure diastolic (mmHg)
79±9
Fasting glucose (mmol/l)
5.3±0.7
HbA1c (%)
5.8±0.5
Hemoglobin (mmol/l)
8.6±0.8
Leukocytes (x109/l)
6.4±1.4
CRP (ug/ml)
3.1±3.7
IL-1Ra (pg/ml)
774±445
Use of antihypertensives
31%
Use of statins
19%
Table 1. Baseline characteristics (mean±SD)
Systemic inflammation
Leukocyte (anakinra 5.4±0.3 x109/l vs placebo 6.2±0.4 x109/l, p = 0.017) and neutrophil
counts (anakinra 2.9±0.3 x109/l vs placebo 3.7±0.3 x109/l, p = 0.001) were significantly
reduced after anakinra treatment, as were CRP levels (anakinra 0.9±0.2 ug/ml versus placebo
2.7±0.6 ug/ml, p < 0.001).
90
Anakinra imroves beta cell function 
A
Anakinra
Placebo
Time(min)
D
Time(min)
p=0.03
Anakinra
Placebo
F
Maximum insulin
concentration (mU/I)
Second phase insulin
secretion (mU/I)
E
Time(min)
First phase insulin
secretion (mU/I)
First phase insulin
secretion (mU/I)
C
Insulin (mU/I)
Glucose infusion
rate (ml/hr)
Glucose (mmol/I)
B
Anakinra
Placebo
Anakinra
Placebo
H
∆I arginine (mU/I)
Insulin sensitivity
index
G
5
Anakinra
Placebo
Anakinra
Placebo
Figure 2.Hyperglycemic clamp.
Glucose (A) levels and glucose infusion rate (GIR) during the hyperglycemic clamp after
treatment with anakinra (dark grey) and placebo treatment (light grey). The glucose levels
are depicted as symbols with connecting line and the GIR as symbols only. Insulin (B) levels
during the hyperglycemic clamp after treatment with anakinra and placebo. First phase insulin
secretion after treatment with anakinra (dark grey) and placebo treatment (light grey) are shown
as insulin levels (C) and as the mean sum of increments (D). Second phase insulin secretion
(E), the maximal insulin concentration (F), the acute insulin response to arginine (∆I arginine)
(G) and insulin sensitivity index (ISI) (H) are shown. Data are expressed as mean±SEM.
91
 Anakinra imroves beta cell function
Beta cell function and insulin sensitivity
During the hyperglycemic clamp, glucose levels were nearly identical in both treatment periods
(anakinra 9.98± 0.04 mmol/l vs placebo 10.02±0.04 mmol/l respectively, p = 0.43). The mean
CV of the hyperglycemic clamp was below 4 % in both treatment periods. First phase insulin
secretion improved after anakinra treatment compared to placebo (148±20 vs 123±14 mU/l
respectively, p=0.03). Second phase insulin secretion, insulin response after arginine stimulus
and maximal insulin secretion, did not differ between the two treatment arms (Figure 2).
Anakinra had no effect on the insulin sensitivity index (2.0±0.3 mmol/mU for anakinra and
2.4±0.43 mmol/mU for placebo treatment, p= 0.29)
In line with the first phase clamp results, the insulinogenic index derived from the oral glucose
tolerance test improved after anakinra compared to placebo treatment (14.5±1.7 vs 11.4±1.1
mU/mmol respectively, p= 0.036). The area under the curve (AUC) for glucose during the
OGTT did not differ between anakinra and placebo treatment, while the AUC for insulin tended
to be higher after anakinra treatment (321±40 vs 286±41mU*120 min/l, p=0.09). (Figure 3)
Anakinra treatment had no effect on GLP-1 and glucagon levels during OGTT (Figure 4).
Similarly, area under the curve (AUC) for GLP-1 (anakinra 61.0±6.1 vs. placebo 59.8±6.9
pmol*120 min/l, P = 0.86) and glucagon (39.5±5.5 and 36.8±5.4 pmol*120 min/l respectively,
P = 0.78) did not differ.
Anakinra treatment did not change fasting glucose levels (5.5±0.2 vs 5.5±0.3 mmol/l) or
HbA1c levels compared to placebo (5.6±0.1 % vs 5.7±0.1%, p=0.068).
There were no carry-over effects.
Side effects and safety
The vehicle of anakinra is known to induce an injection site reaction during the first weeks of
treatment in a majority of patients17. In our study, 14 out of the 17 participants experienced
injection site reactions during anakinra treatment. One subject withdrew from the study
because of the local reaction. All reactions disappeared after the cessation of anakinra
treatment. During anakinra treatment, leukocyte and neutrophil count were lower compared to
placebo treatment. One participant developed erysipelas two days after cessation of anakinra
treatment and one participant developed a thrombophlebitis of the antecubital vein, which was
used for infusion of glucose 20% during the clamp, in the anakinra treatment period. There
were no other infections or serious adverse events.
92
Anakinra imroves beta cell function 
Anakinra
Placebo
Glucose (mmol/I)
A
Time(min)
p=0.03
C
Insulin (mU/I)
Insulinogeric index
(mU/mmol)
B
Anakinra
Time(min)
E
GLP-1 (pmol/I)
AUC GLP-1
(pmol*120min/I)
D
Placebo
Time(min)
Anakinra
Placebo
Anakinra
Placebo
G
AUC Glucagon
(pmol*120min/I)
Glucagon (pmol/I)
F
5
Time(min)
Figure 3.Oral glucose tolerance test
Glucose (A) and insulin (B) levels during OGTT for anakinra (dark grey) and placebo (light grey)
treatment. The insulinogenic index (C) was higher after treatment with anakinra compared to
placebo treatment. The GLP-levels during OGTT (D) and area under the curve for GLP-1 (E) are
shown for both anakinra (dark grey) and placebo treatment (light grey). Glucagon levels during
OGTT (F) and area under the curve for glucagon (G) are also depicted.
Data are expressed as mean±SEM.
93
 Anakinra imroves beta cell function
Discussion
The main finding of the present study is that four weeks of treatment with the IL-1 receptor
antagonist anakinra can improve beta cell function in subjects with impaired glucose
tolerance. This conclusion is based on the observation that anakinra improved the
insulinogenic index and augmented the first phase insulin secretion during a hyperglycaemic
clamp. However, second phase insulin secretion, insulin response after arginine, and the
maximal insulin concentration were not influenced by anakinra treatment. The improved first
phase insulin response was not mediated by an enhanced incretin response.
The present results are in line with the study of Larsen et al performed in patients with type
2 diabetes mellitus, treated with anakinra 100 mg once daily for 13 weeks or placebo 12.
This study showed an improvement in HbA1c, which was attributed to an enhanced beta
cell function (assessed by proinsulin to insulin ratios and glucose tolerance tests), but the
improvement could be indirect and explained by improvement in glycemic control (decreased
glucotoxicity). As we studied subjects with IGT, we could analyse the effects of anakinra
treatment on beta cell function without confounding effects of changes in glycemic control. In
our study, glucose levels did not change, so the observed enhancement in beta cell function
probably reflects a direct effect of anakinra on the beta cell. The present findings are also in
agreement with our earlier study in non-diabetic subjects with the metabolic syndrome, where
the IL-1Ra anakinra had no effect on insulin sensitivity, but improved the disposition index 18.
A recent study using canakinumab, a human monoclonal anti-IL-1β antibody, in patients with
type 2 diabetes and subjects with IGT found a trend towards improving insulin secretion rates
(derives from liquid mixed meal test) relative to glucose in patients with type 2 diabetes mellitus
and an improved insulin secretion (insulin AUC0-4hr and peak insulin levels) in subjects with IGT 14 .
Other studies applying anti-IL-1β antibodies, have yielded conflicting results regarding the
effect on glycemic control in patients with type 2 diabetes mellitus. A dose escalation study
with gevokizumab, showed moderately improved HbA1c levels after a single injection19,
but the HbA1c responded in a U-shaped manner with the high-dose group showing no
improvement in HbA1c levels. 12 Weekly injections of the anti-IL1β antibody LY2189102
slightly reduced HbA1c levels in patients with type 2 diabetes mellitus 20, while canakinumab
treatment in patients with type 2 diabetes mellitus was associated with minor non-significant
effects on HbA1c 21.
As such, the effects of various anti-IL1 biological therapies on glucose control vary, with one
showing a clear effect of anakinra and most studies with anti-IL1β antibodies, showing minor
or nonsignificant effects. Part of these differences may relate to the fact that IL-1Ra blocks both
IL-1α and IL-1β, while the specific anti-IL-1β antibodies only blocks IL-1β action 22. Nevertheless,
while anti-IL1β treatment has proven effects on glycemia, the most appropriate treatment
94
Anakinra imroves beta cell function 
mode and the most suitable population to be treated remains to be determined. Combined
with findings so far, our results suggest that beneficial effects of anti-IL1β therapy are most
consistent in patients with impaired glucose tolerance.
Although our study was not specifically designed to determine an effect of anakinra on insulin
sensitivity, the insulin sensitivity index as derived from the hyperglycemic clamp did not show
an effect of anakinra, which is in agreement with Larsen et al. 12 and with our previous study
findings 18.
The observed improvement in first-phase insulin secretion and insulinogenic index after
anakinra treatment might implicate that IL-1Ra has disease-modifying potential.
Insulin secretion in response to a glucose challenge is biphasic, with a first phase response
composed of an early burst of insulin release within 10 minutes, and a second phase response
consisting of a progressive increase in insulin secretion lasting several hours 23,24. The first
phase insulin response represents exocytosis of insulin secretory granules that are readily
releasable and the second phase secretion is composed of new insulin secretory granules
that occur in response to a glucose stimulus 25. The loss of first phase insulin secretion is a
characteristic feature of (early) type 2 diabetes mellitus 26,27.
A number of mechanisms may underlie these results. First, anakinra might inhibit intra-islet
inflammation and thereby preserve beta cell function. Beta cells expressing IL-1β have been
observed in pancreatic sections from patients with type 2 diabetes mellitus 10. Furthermore, in
5
vitro studies show that high glucose levels induce IL-1β production by beta cells followed by
beta cell dysfunction and apoptosis 10,28,29. In vitro, the deleterious effects of high glucose and
IL-1β levels on beta cells can be prevented by IL-1Ra 10. Thus, the beneficial effects of anakinra
may be a direct anti-inflammatory effect on beta cells. Second, anakinra may improve beta
cell function by decreasing systemic inflammation. Cross-sectional studies have described
elevated levels of acute phase proteins and cytokines in patients with type 2 diabetes
mellitus 30,31. Moreover, elevated levels of IL-1β, CRP and IL-6 are predictive of type 2 diabetes
mellitus 32. The reduction in inflammatory markers including leukocytes and CRP in our
study suggest that anakinra treatment induces a systemic anti-inflammatory effect. Third,
an improvement in beta cell function could theoretically be explained by a compensatory
response to a decline in insulin sensitivity. However, we did not find significant changes in
the insulin sensitivity index as derived from the hyperglycemic clamp, nor did we find any
correlation between the change in insulin sensitivity index and changes in various measures of
beta cell function. (data not shown)
Since the observed improvement of insulinogenic index reflects “early” insulin secretion
after a glucose stimulus and the first phase insulin secretion improved, we postulate that
95
 Anakinra imroves beta cell function
anakinra might influence incretin hormones. The incretin effect is reduced in subjects with
impaired glucose tolerance 33 and most likely is a result of the prediabetic state rather than
a consequence of genetic predisposition 34. One week of treatment with liraglutide (GLP-1
receptor agonist) once daily in patients with type 2 diabetes mellitus improved both first- and
second phase insulin secretion as well as the response to arginine during a hyperglycemic
clamp 35. These findings suggest that incretin based therapy can improve first-phase insulin
secretion in type 2 diabetes mellitus compared to healthy controls. Furthermore, IL-6 has been
shown to promote beta cell function by increasing GLP-1 secretion and the authors postulate
crosstalk between insulin sensitive tissues and pancreatic islets through GLP-1 36. We were not
able to identify any effects of anakinra on plasma concentrations of GLP-1 and glucagon, and
these findings suggest that the effect of anakinra on early insulin secretion cannot be explained
by modulation of GLP-1 secretion. However, we measured systemic levels of GLP-1 and
therefore cannot exclude possible changes in local levels of GLP-1.
In obese subjects and in individuals prone to develop type 2 diabetes mellitus, increased
circulating IL-Ra levels have been reported 37,38 and it has been suggested that IL-Ra levels at
baseline predict the effects of anakinra treatment on glycemic control, with subjects with the
lowest IL-Ra levels showing the highest response 39. Conversely, it has also been suggested
that elevated anti-inflammatory IL-1Ra levels are an attempt to counteract the pro-inflammatory
effects of IL-1β and to preserve beta cell function 38. In our study we did not find any association
between baseline IL-1Ra levels and improvement in insulinogenic index and first phase insulin
secretion.
Our study provides a proof-of-concept that treatment with the IL-1Ra anakinra improves insulin
secretion, but the current once daily treatment with anakinra is inconvenient and associated
with injection site reactions. Future anti-IL1 therapies, especially when effects beyond glucose
metabolism have been documented (Cankinumab Anti-inflammatory Thrombosis Outcomes
Study (CANTOS trial)) may be more convenient and suited for clinical application.
In summary, 4 weeks of treatment with anakinra can improve beta cell function in subjects with
IGT, supporting the concept of involvement of IL-1β in the (progressive) decrease of insulin
secretion capacity associated with type 2 diabetes mellitus. These findings are relevant since
improvement in beta cell function can delay and/or prevent progression to frank diabetes.
96
Anakinra imroves beta cell function 
5
97
 Anakinra imroves beta cell function
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99
BETA
BETA
FUNCTION
DYSFUNCTION
CELL
CELL
ENDOTHELIAL
ENDOTHELIAL
INSULIN
INSULIN
FUNCTION
SENSITIVE
DYSFUNCTION
RESISTANT
CHAPTER 6
INFLAMMATION IN THE SUBCUTANEOUS ADIPOSE TISSUE
IS NOT RELATED TO ENDOTHELIAL FUNCTION IN SUBJECTS
WITH DIABETES MELLITUS AND SUBJECTS WITH DYLIPIDEMIA
AND HYPERTENSION
P.C.M. van Poppel, E.J. Abbink, R. Stienstra, M.G. Netea, C.J. Tack
Submitted
 Inflammation in the subcutaneous adipose tissue is not related to endothelial function
Abstract
Background
Obesity is associated with low grade inflammation that may be related to vascular disease. We
hypothesized that inflammation in the subcutaneous adipose tissue is associated with impaired
endothelium-dependent vasodilatation.
Methods
We assessed endothelial function by measuring forearm vascular response to acetylcholine
and determined inflammation in subcutaneous fat biopsies in two groups of subjects; 15
patients with type 2 diabetes mellitus (T2DM) and 19 subjects with dyslipidemia combined with
hypertension (DcH).The adipose tissue inflammation score was based on adipocyte size, influx
of macrophages and presence of crown-like structures. We compared the vascular response
to acetylcholine between subjects with and without adipose tissue inflammation.
Results
Patients with diabetes had clearly decreased endothelium-dependent vasodilatation compared
to patients with DcH (forearm vasodilator response from baseline in T2DM: +2.0±0.7,
+5.0±1.2 and +11.7±1.6 ml.dl⁻¹.min⁻¹ [in response to acetylcholine 0.5, 2.0 and 8.0 µg.dl⁻¹.
min⁻¹ resp.] versus DcH +5.6±0.8, +10.1± 1.1 and +14.3± 1.3 ml.dl⁻¹.min, P = 0.009). 23
out of the 34 study participants had subcutaneous adipose tissue inflammation. However, there
was no difference in vascular response to acetylcholine between the group with and without
inflammation (forearm vasodilator responses to the three acetylcholine doses from baseline
in those with inflammation +3.9±0.8, +7.8±1.0 and +13.6±1.0 ml.dl⁻¹.min⁻¹ compared to
+4.3±1.0, +7.9±2.1 and +12.2±2.4 ml.dl⁻¹.min⁻¹ in in those without inflammation, P=NS) ,
nor was there a relationship between circulating hsCRP levels and endothelial function.
Conclusions
We confirm that subjects with T2DM have impaired endothelial function compared to age
and BMI matched subjects with DcH. However, endothelial function did not differ between
participants with or without inflammation in the subcutaneous adipose tissue. These results
suggest that fat tissue inflammation, at least in the subcutaneous compartment, does not
affect vascular function.
102
Inflammation in the subcutaneous adipose tissue is not related to endothelial function 
Background
Obesity is characterized by low grade inflammation which has been linked to type 2 diabetes
and atherosclerosis 1. The enhanced inflammatory state in obese individuals has been
suggested to originate from the expanded adipose tissue mass.
Obesity induced inflammation is accompanied by several morphological changes in the
adipose tissue. For instance, hypertrophy of adipocytes initiates the production of cytokines
and chemokines by the adipocytes 2. In response to the raised production of the cytokines
and chemokines, endothelial cells increase the expression of adhesion molecules. The release
of chemokines and the increased expression of adhesion molecules recruite immune cells,
including macrophages, to the adipose tissue 3. Indeed, obesity is associated with adipose
tissue macrophage infiltration in mice and human subjects 4,5. In addition, enlargement of
adipocytes leads to local hypoxia, which may result in adipocyte death that subsequently
attracts macrophages to the adipose tissue 6 that cluster in crown-like structures (CLS)
surrounding adipocytes, that are dying. Noticebly, macrophages part of CLSs are known to
display a more inflammatory profile as compared to individually dispersed macrophages in
adipose tissue 7.
Endothelial dysfunction is considered an early marker of vascular complications 8,9. Endothelial
dysfunction consists of a number of functional alternations in the vascular endothelium,
including impaired endothelium-dependent vasodilatation which can be quantified by
measuring responses to endothelium-dependent vasodilators 10,11.
We hypothesized that inflammation in the subcutaneous adipose tissue impairs endotheliumdependent vasodilatation. To test this hypothesis we assessed associations between
inflammation in human adipose fat tissue and endothelium-dependent vasodilatation.
6
Methods
Study population
This study represents a pathophysiological study embedded in two clinical trials in which
subcutaneous fat biopsies were taken and endothelial function was assessed at the same
time. Both studies were conducted in accordance with the principles outlined in the Declaration
of Helsinki. The local ethics committee (Radboud university medical centre, Nijmegen, the
Netherlands) approved each study and all subjects gave written informed consent before
participation. The study population consisted of 36 subjects; 16 patients with type 2 diabetes
mellitus (T2DM) and 20 subjects with dyslipidemia combined with hypertension (DcH)
103
 Inflammation in the subcutaneous adipose tissue is not related to endothelial function
Inclusion criteria for subjects with type 2 diabetes mellitus were treatment with metformin
with or without sulphonylurea or thiazolidinediones, age 35-75 years and HbA1c < 8.0% (64
mmol/mol) [12]. Inclusion criteria for subjects with dyslipidemia combined with hypertension
were: age 40-70 years, dyslipidemia defined as a fasting LDL cholesterol > 3.5 mmol/l and/or
triglycerides > 1.7 mmol/l (no statins or other lipid lowering drugs allowed) and hypertension
defined as systolic blood pressure > 140 mmHg and/or diastolic blood pressure > 90 mmHg
(average of three office blood pressure recordings with maximal one antihypertensive drug
was allowed. When no antihypertensives were used, the elevated blood pressure needed to
be confirmed in a second office visit). Exclusion criteria were triglycerides > 8 mmol/l, LDLcholesterol > 5 mmol/l, systolic blood pressure > 180 mmHg and/or diastolic blood pressure
> 110 mmHg respectively (for details see clinicaltrials.gov NCT01563770).
Protocol
The two studies were double-blind, randomized, controlled, cross-over trials. None of the two
studies showed carry-over effects. In the present study we used the data of the control arm of
these trials. At the end of each treatment period, endothelial function was measured by venous
occlusion plethysmography, a subcutaneous fat biopsy was taken and blood was collected for
biochemical analysis.
Experimental procedures
 Demographic and clinical characteristics
Patient demographics and use of medication were recorded. Body weight, height and blood
pressure were measured.
 Biochemical analysis
Fasting blood samples were drawn to determine blood glucose levels, HbA1c, lipids and high
sensitive C- reactive protein (hsCRP) levels.
 Plethysmography
The experiments started at 8.30 a.m. after an overnight fast in a quiet, temperature controlled
room (23˚C-24˚C). The subjects abstained from caffeine for 24 hours prior to the experiment.
The brachial artery of the non dominant arm (experimental arm) was cannulated for infusion of
vasodilators and collection of blood samples.
104
Inflammation in the subcutaneous adipose tissue is not related to endothelial function 
Forearm blood flow (FBF) was measured using mercury-in-silastic strain gauge, venous
occlusion plethysmography as previously described 12. Forearm volume was measured with
the water displacement method and all drugs were dosed per 100 ml forearm tissue with an
infusion rate of 100 ml.dl-1.min-1.
After complete instrumentation, a 30 minute equilibration period was included, after which
baseline measurements were performed with infusion of saline. Subsequently, three increasing
doses of acetylcholine (0.5, 2.0 and 8.0 µg.dl-1.min-1, 10 mg/ml dry powder, dissolved to
its final concentration with saline, Novartis, Greece) were infused into the brachial artery.
Acetylcholine (Ach) stimulates endothelial muscarinic receptors thereby activating nitric oxide
synthase. This results in the endothelial release of nitric oxide (NO) causing vasodilatation 13.
Each dose was infused for 5 minutes and FBF was measured during the last 3 minutes of the
5 minute period. After the infusion all three doses of acetylcholine a 30 minutes equilibration
period followed. Subsequently, baseline measurements were performed with the infusion of
glucose 5% solution. Subsequently, three increasing doses of sodium nitroprusside (0.06,
0.20 and 0.60 µg.dl-1.min-1, 25 mg/ml, dissolved to its final concentration with glucose 5%
solution, Clinical Pharmacy, Radboud university medical centre), an endothelium-independent
vasodilator 14, were infused in the brachial artery. Again, each dose was infused for 5 minutes
and FBF was measured during the last 3 minutes of the 5 minute period.FBF registrations
of the last 2 minutes of each dosage of vasodilator were averaged to a single value for data
analysis.
 Subcutaneous adipose tissue biopsy
A subcutaneous adipose tissue biopsy was taken from the abdominal region after completion
of the plethysmography. Biopsies were obtained under local anesthesia (2% lidocaine HCl),
6
from an area about 10 cm lateral of the umbilicus using a Hepafix Luer lock syringe (Braun,
Melsungen, Germany) and a 2.10 x 80 mm Braun medical Sterican needle (Braun). Adipose
tissue was washed using a 0.9% normal saline solution. The adipose tissue was snap
frozen and stored at -80 ºC until further analysis, or it was fixed in 4% paraformaldehyde and
embedded in paraffin for morphometric analysis.
Morphometry of individual fat cells was assessed using digital image analyses as described
previously 15. For each subject, the adipocyte cell size of all fat cells in ten microscopic fields
of view (magnification 10x) were counted and measured. Adipocyte size was expressed as
area (μm2), perimeter (μm), feretmin (μm) which is the smallest diameter and feretmax (μm)
which is the largest diameter.
An antibody against CD68 (AbD Serotec, Kidlington, UK) was used to stain macrophages.
Macrophage influx was quantified by counting the number of adipocytes and macrophages in
ten representative microscopic fields of adipose tissue (magnification 40x).
105
 Inflammation in the subcutaneous adipose tissue is not related to endothelial function
Adipose tissue inflammation was based on a composite score consisting of fat cell size,
number of CD68+ cells/adipocyte and the presence of crown like structures (CLS). The
score uses means as cut-off points within the population as a whole and of the respective
subpopulations. Cut-off levels for fat cell size (smallest diameter = Feretmin) > 70 um, number
of CD68+ cells/adipocyte > 0.12 and presence of CLS. For subjects with T2DM the score
consisted of Feretmin > 74 um, CD68+ cells/adipocyte > 0.12 and the presence of CLS. For
subjects DcH the score consists of Feretmin > 67 um, CD68+ cells/adipocyte > 0.12 and the
presence of CLS.The inflammation score was positive if a participant fulfilled at least 1 out of
the 3 criteria of inflammation. We included a group of healthy controls to document increased
inflammatory levels in adipose tissue in the presence of T2DM or DcH.
Statistical analysis
All data are represented as mean ± SEM unless otherwise indicated. Differences in means
of laboratory results were tested by Student’s t-test or Mann- Whitney test for non-normally
distributed data. Correlations were calculated by Pearson correlation coefficient. Two-tailed P <
0.05 was considered significant. NS indicates non-significant.
FBF was expressed as absolute change in FBF (Δ FBF) above baseline. Analysis of variances
(ANOVA) was used to assess differences in increments in FBF between groups (T2DM
versus DcH, inflammation versus no inflammation). Statistical analyses were performed using
Graphpad 5.0.
Results
A total of 36 participants who completed both trials, were included in the study, resulting in 34
paired subcutaneous fat biopsies and endothelial function measurements. One participant
excluded from statistical analysis because of unreliable results of the plethysmography,
probably due to a technical problem with one of the strain gauges 16, and one participant did
not provide informed consent to perform a subcutaneous fat biopsy. Baseline characteristics of
the 34 participants included in the analyses are shown in table 1.
106
Inflammation in the subcutaneous adipose tissue is not related to endothelial function 
Characteristic
T2DM
(n =15)
DcH
(n = 19)
Age (years)
59.8±7.0
57.9±7.9
Sex (male:female)
11:4
13:6
Weight (kg)
88.5±15.5
84.5±17.4
BMI (kg/m²)
29.0±5.2
28.3±5.6
Blood pressure systolic (mmHg)
141±8
156±9***
Blood pressure diastolic (mmHg)
82±7
94±5***
HbA1c (%)
6.9±0.6
5.7±0.3***
Total cholesterol (mmol/l)
4.4±1.0
5.6±0.8***
Triglycerides (mmol/l)
1.6±0.8
1.8±1.1
HDL-cholesterol (mmol/l)
1.0±0.2
1.2±.3
LDL-cholesterol (mmol/l)
2.4±1.0
3.5±0.7***
Table 1. B
aseline characteristics (mean±SD).
T2DM = type 2 diabetes mellitus. DcH= dyslipidemia combined with hypertension.
*** P < 0.001 compared to subjects with type 2 diabetes mellitus
T2DM vs DcH
The two groups were well matched regarding age, gender and BMI. Based on different
inclusion criteria the subjects differed in HbA1c levels, which was higher in the T2DM group,
while blood pressure and lipids were higher in DcH (table 1).
6
In subjects with T2DM, endothelium-dependent vasodilatation was clearly decreased as
compared to DcH. Infusion of acetylcholine induced a dose-dependent increase in FBF in
the experimental arm, while FBF did not change in the non experimental arm. Changes in
FBF from baseline in T2DM were 2.0±0.7, 5.0±1.2 and 11.7±1.6ml.dl⁻¹.min⁻¹ in response to
acetylcholine dosage 0.5, 2.0 and 8.0 µg.dl⁻¹.min⁻¹ respectively compared to 5.6±0.8, 10.1±
1.1 and 14.3± 1.3 ml.dl⁻¹.min⁻¹ in DcH (P = 0.009 by two-way ANOVA). (Figure 1- top)
There was no difference in response to sodium nitroprusside between subjects with T2DM
and DcH. Changes in FBF from baseline in T2DM were 3.5± 0.4, 6.4± 0.8 and 10.9± 1.0
ml.dl⁻¹.min⁻¹ in response to sodium nitroprusside dosage 0.06, 0.20 and 0.60 µg.dl⁻¹.min⁻¹
respectively, compared to 2.4± 0.3 , 4.8± 0.5 and 8.5± 0.9 ml.dl⁻¹.min⁻¹ in DcH (P = NS).
(Figure 1)
107
 Inflammation in the subcutaneous adipose tissue is not related to endothelial function
DM
DcH
∆ FBF (ml.dl-1.min-1)
20
15
10
5
0
Saline
Ach 0.5
Ach 2.0
Ach 8.0
∆ FBF (ml.dl-1.min-1)
15
10
5
0
Glucose
SNP 0.06 SNP 0.20 SNP 0.60
Figure 1.
Vascular response to the endothelium-dependent vasodilator acetylcholine (Ach) (top) and
endothelium-independent vasodilator sodium nitroprusside (SNP) (bottom) in subjects with type
2 diabetes mellitus (T2DM) compared to subjects with dyslipidemia and hypertension (DcH).
** P = 0.009 by two-way ANOVA.
108
Inflammation in the subcutaneous adipose tissue is not related to endothelial function 
Subcutaneous adipose tissue inflammation
Compared to BMI-matched controls, the influx of macrophages in the subcutaneous adipose
tissue, as measured by CD68+cells/adipocyte, was clearly increased in both T2DM and DcH
(0.13 in T2DM, 0.12 in DcH versus 0.03 in healthy controls).
Out of the 34 participants, 23 scored positive on the inflammation score. There was no
difference in vascular response to acetylcholine between the group with and without
inflammation. Changes in FBF from baseline in the inflammation group were 3.9±0.8,
7.8±1.0 and 13.6±1.0 ml.dl⁻¹.min⁻¹ in response to acetylcholine 0.5, 2.0 and 8.0 µg.dl⁻¹.min⁻¹
respectively compared to 4.3±1.0, 7.9±2.1 and 12.2±2.4 ml.dl⁻¹.min⁻¹ in the group without
inflammation (P= NS). (Figure 2A).
 Subgroup analyses
Of the 15 subjects with T2DM, 10 fulfilled the criteria for inflammation in the subcutaneous
adipose tissue. No difference in vascular response to acetylcholine between the group with
and without inflammation was observed. Changes in FBF from baseline in the inflammation
group were 2.5±0.9, 6.3±1.6 and 13.1±1.9 ml.dl⁻¹.min⁻¹ in response to acetylcholine 0.5, 2.0
and 8.0 µg.dl⁻¹.min⁻¹ respectively compared to 1.1±0.8, 2.3±1.2 and 8.8±2.3 ml.dl⁻¹.min⁻¹
without inflammation (P= NS). (Figure 2B).
Likewise, inflammation score was positive in 13 out of the 16 subjects with DcH, again with
no difference in endothelium-dependent vasodilatation between the group with and without
inflammation. Changes in FBF from baseline in the inflammation group were 5.6±1.0,
10.0±1.1 and 14.5±1.2 ml.dl⁻¹.min⁻¹ in response to acetylcholine 0.5, 2.0 and 8.0 µg.dl⁻¹.min⁻¹
respectively compared to 5.6±1.2, 10.3±2.6 and 13.9±3.5 ml.dl⁻¹.min⁻¹ without inflammation
(P = NS). (Figure 2C)
6
Adipose tissue macrophages can cluster in crown like structures (CLS) that possess a high
inflammatory status. Out of the 34, 7 participants displayed CLS in the subcutaneous adipose
tissue biopsy material. There was no difference in endothelium-dependent vasodilatation
between the group with and without the presence of CLS. Changes in FBF from baseline were
4.1±1.7, 7.5±2.0 and 10.9±2.2 ml.dl⁻¹.min⁻¹ in response to acetylcholine 0.5, 2.0 and 8.0
µg.dl⁻¹.min⁻¹ respectively in the group with CLS compared to 4.0±0.6, 7.9±1.0 and 13.7±1.1
ml.dl⁻¹.min⁻¹ in the group without CLS (P = NS). ( Figure 2D)
109
 Inflammation in the subcutaneous adipose tissue is not related to endothelial function
Inflammation
No Inflammation
Inflammation
No Inflammation
B
∆ FBF (ml.dl-1.min-1)
∆ FBF (ml.dl-1.min-1)
A
Saline
C
Ach 0.5 Ach 2.0
Ach 8.0
Saline
D
Ach 8.0
CLS+
CLS-
∆ FBF (ml.dl-1.min-1)
∆ FBF (ml.dl-1.min-1)
Inflammation
No Inflammation
Ach 0.5 Ach 2.0
Saline
Ach 0.5 Ach 2.0
Ach 8.0
Saline
Ach 0.5 Ach 2.0
Ach 8.0
Figure 2.Relation between inflammation in subcutaneous adipose tissue and endothelium-dependent
vasodilatation.
Ach denotes acetylcholine. Change in forearm blood flow in the experimental arm in response
to acetylcholine (dosage 0.5, 2.0, 8.0 µg.dl⁻¹.min⁻¹) in all subject (A) with inflammation (n = 23,
black) and without inflammation ( n= 11, grey) (B) in subjects with type 2 diabetes mellitus with
inflammation (n = 10, black) and without inflammation ( n= 5, grey) and (C) in subjects with
both dyslipidemia and hypertension with inflammation (n = 13, black) and without inflammation
( n= 6, grey). Finally in graph D, the change in forearm blood flow in the experimental arm in
response to acetylcholine is shown for subjects with crown-like structures (CLS) in adipose
tissue biopsy (n= 7, black) and those without (n=27, grey).
110
Inflammation in the subcutaneous adipose tissue is not related to endothelial function 
Mean circulating hs-CRP level, a marker of systemic inflammation, was 1.98±0.39 μg/ml
(n=34). Hs-CRP level did neither correlate with the influx of macrophages in the subcutaneous
fat tissue (CD68 positive cells) nor with endothelial function (area under the curve for the
response to acetylcholine and area. (Figure 3) As expected, hs-CRP levels were significantly
correlated with measures of obesity (BMI and the area of adipocytes), and with HOMA-IR.(data
not shown) .
A
P=NS
hsCRP (ug/ml)
15
10
5
0
0.0
0.1
0.2
0.3
CD68+cells/adipocyte
B
hsCRP (ug/ml)
15
P=NS
6
10
5
0
0
10
20
30
40
AUC FBF Ach
Figure 3.
Relation between hsCRP levels and inflammation of subcutaneous adipose tissue (A) and
endothelium-dependent vasodilatation (B) expressed as AUC= area under the curve in response
to three sequential acetylcholine (Ach) doses. FBF= forearm blood flow.
111
 Inflammation in the subcutaneous adipose tissue is not related to endothelial function
Discussion
The present study confirms that subjects with type 2 diabetes mellitus have impaired
endothelial function compared to age-, gender and BMI-matched subjects with dyslipidemia
combined with hypertension. Both T2DM and DcH have increased inflammation in the
subcutaneous adipose tissue compared to controls. However, there was no relationship
between adipose tissue inflammation and endothelial (dys)function. This conclusion is based
on the observation that endothelium-dependent vasodilatation did not differ between the
subjects with inflammation and without inflammation in adipose tissue. Furthermore, there was
no association between hs-CRP levels and vascular function.
Endothelial dysfunction is regarded as an important marker of vascular complications in
type 2 diabetes mellitus 8. Hyperglycemia, hypercholesterolemia, obesity and hypertension
all negatively affect endothelial function 17. In the present study, we confirm that subjects
with type 2 diabetes mellitus have impaired endothelium-dependent vasodilatation
compared to subjects with both dyslipidemia and hypertension, two well known risk factors
for cardiovascular disease. This is in line with the well-known increased cardiovascular
disease risk associated with type 2 diabetes and underlines that this increased risk is partly
independent from other risk factors 18,19.
In this study, we were able to associate detailed vascular responses with adipose tissue
inflammation in a fairly large group of subjects with diabetes and subjects with cardiometabolic
risk factors. The study did not reveal an impaired endothelial-dependent vasodilatation in
subjects with inflammation in subcutaneous adipose tissue as compared to those without
inflammation whatsoever. These results seem to be in contrast with two previous studies
showing that the presence of CLS in adipose tissue of obese subjects is associated with
endothelial dysfunction 20,21. There are important differences in population and methodology
that may explain these diverging findings. One study in obese subjects shows that the
presence of CLS in the subcutaneous fat compartment is associated with impaired endothelial
function, as measured by flow-mediated vasodilatation (FMD) 20, but the population in this
study consisted mainly of morbidly obese individuals. Farb et al demonstrate that obese
subjects with inflamed fat, defined as the presence of CLS, showed lower FMD compared
to lean subjects and obese subjects without inflamed fat 21. Again, obese subjects in this
study consisted of morbidly obese subjects many of whom subsequently underwent bariatric
surgery. The lean control group was also younger. Interestingly, the percentage of patients with
diabetes did not differ between the obese subjects with or without adipose tissue inflammation
in both studies.
112
Inflammation in the subcutaneous adipose tissue is not related to endothelial function 
In our study, the population investigated was moderately overweight/obese, with a mean BMI
of approximately 29 kg/m2. A second relevant difference with the earlier studies is that we used
vascular responses to acetylcholine to assess endothelial function, while in the other studies FMD
was used. These different measures might have led to different results. Zeiher et al demonstrated
that three methods of measuring endothelial function in the coronary system, i.e. flow-dependent
dilation, cold pressor test and infusion of acetylcholine, produced different results in patients with
different stages of atherosclerosis 22. This implies that the degree of atherosclerosis influences
the results of testing endothelial function. Intra-arterial acetylcholine infusion measures vascular
responses in the forearm resistence vessels while FMD measures the response of conduit artery
(a. brachialis). Although it is tempting to conclude that intra-arterial infusion of endotheliumdependent vasodilators represents a more direct and reproducible parameter of endothelial
function,ultimately, both response to acetylcholine and FMD are negatively associated with
cardiovascular outcomes 10.
Finally in the present study, we used an overall inflammation score while the other studies only
used the presence of CLS. There is no clear definition of adipose tissue inflammation. We used a
scoring system including adipocyte size, macrophages influx and presence of CLS to categorize
inflammation. However, if we analysed subjects with and without CLS, there still was no difference
in endothelium-dependent vasodilatation. Percentage wise, the number of subjects with CLS was
clearly lower than those in the earlier studies, perhaps related to the less extreme level of obesity.
Taken together, adipose tissue inflammation may be relevant for vascular dysfunction in the
extreme end of obesity, but of relatively minor importance in the intermediate obesity range and in
the context of other cardiometabolic risk factors such as diabetes, dyslipidemia and hypertension.
As fat tissue inflammation is more pronounced in the visceral fat compartment 23, our findings do
not exclude a potential relationship between visceral fat inflammation and endothelial function. So
6
far, no studies have reported on the relationship between visceral fat inflammation and functional
vascular measurements.
CRP is a marker of low-grade inflammation and a risk marker for cardiovascular disease
since it has been shown to predict cardiovascular events 24,25. In vitro studies have shown
that CRP decreases NO bioactivity in endothelial cells by down regulation of endothelial nitric
oxide synthase (eNOS) 26,27. Given the effect that CRP has on NO biology one would expect
that high CRP levels are associated with impaired endothelial function. Our study shows no
relation between CRP levels and vascular response to acetylcholine, which is partly in line with
other studies in healthy subjects, which did not show consistent results with regard to the
association between CRP levels and endothelial function 28,29. In subjects with coronary artery
disease Fichtlscherer et al showed that elevated CRP levels were associated with endothelial
dysfunction 30.
113
 Inflammation in the subcutaneous adipose tissue is not related to endothelial function
It is important to underline that our study also has limitations. First, the cross-sectional design
provides only of associations between inflammation and endothelial function, but provides
no information on the sequence of events, therefore causality cannot be assessed. Second,
we used data from the control arm of two different studies, which enabled us to compare
endothelial function between T2DM and DcH. The two groups were comparable with regards
to age, BMI and gender, but differed in lipid levels, blood pressure and use of medication
(T2DM 44% used antihypertensives and 68% used statins, DcH 47% used antihypertensives
and no statins were used). Despite lower blood pressure and lower lipid levels, T2DM still had
impaired endothelial function.
In summary, we confirm that subjects with T2DM have impaired endothelial function compared
to age and BMI matched subjects with DcH, despite lower lipid and blood pressure levels in
the diabetes group. This finding emphasizes the increased risk of cardiovascular complications
inherent to type 2 diabetes mellitus. The present study did not find a difference in endothelial
function between the groups of participants with and without inflammation in the subcutaneous
adipose tissue. These results suggest that fat tissue inflammation in patients with moderate
overweight/obesity, at least in the subcutaneous compartment, does not affect vascular
function.
114
Inflammation in the subcutaneous adipose tissue is not related to endothelial function 
6
115
 Inflammation in the subcutaneous adipose tissue is not related to endothelial function
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11.Bakker W, Eringa EC, Sipkema P, van Hinsbergh
diate cardiometabolic phenotype in obesity. Journal
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interpreting the responses to drugs and mediators.
7.
22.Zeiher AM, Drexler H, Wollschlager H, Just H. Endo-
Inflammation in the subcutaneous adipose tissue is not related to endothelial function 
thelial dysfunction of the coronary microvasculature
is associated with coronary blood flow regulation
in patients with early atherosclerosis. Circulation
1991;84:1984-92.
23.Koenen TB, Stienstra R, van Tits LJ, et al. The
inflammasome and caspase-1 activation: a new
mechanism underlying increased inflammatory activity in human visceral adipose tissue. Endocrinology
2011;152:3769-78.
24.Ridker PM, Cook N. Clinical usefulness of very high
and very low levels of C-reactive protein across the
full range of Framingham Risk Scores. Circulation
2004;109:1955-9.
25.Ridker PM. Clinical application of C-reactive protein
for cardiovascular disease detection and prevention. Circulation 2003;107:363-9.
26.Verma S, Wang CH, Li SH, et al. A self-fulfilling
prophecy: C-reactive protein attenuates nitric oxide
production and inhibits angiogenesis. Circulation
2002;106:913-9.
27.Venugopal SK, Devaraj S, Yuhanna I, Shaul P, Jialal
I. Demonstration that C-reactive protein decreases
eNOS expression and bioactivity in human aortic
endothelial cells. Circulation 2002;106:1439-41.
28.Cleland SJ, Sattar N, Petrie JR, Forouhi NG, Elliott
HL, Connell JM. Endothelial dysfunction as a possible link between C-reactive protein levels and cardiovascular disease. Clinical science 2000;98:531-
6
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117
BETA
BETA
FUNCTION
DYSFUNCTION
CELL
CELL
ENDOTHELIAL
ENDOTHELIAL
INSULIN
INSULIN
FUNCTION
SENSITIVE
DYSFUNCTION
RESISTANT
CHAPTER 7
SALVIA MILTIORRHIZA WATER-EXTRACT (DANSHEN) HAS NO
BENEFICIAL EFFECT ON CARDIOVASCULAR RISK FACTORS
P.C.M. van Poppel, P. Breedveld, E.J. Abbink, H. Roelofs, W. van Heerde, P. Smits, W. Lin, A. Tan,
F. Russel, R. Donders, C.J. Tack, G.A. Rongen
Plos One 2015 Jul 20;10(7):e0128695
 Salvia Miltiorrhiza water-extract (danshen) has no beneficial effect on cardiovascular risk factors
Abstract
Purpose
Danshen is the dried root extract of the plant Salvia Miltiorrhiza and is widely used as traditional
Chinese medicinal herbal product to prevent and treat atherosclerosis. However, its efficacy to
prevent and treat atherosclerosis has not been thoroughly investigated. This study was initiated
to evaluate the effect of danshen on hyperlipidemia and hypertension, two well known risk
factors for the development of atherosclerosis.
Methods
This was a randomized, placebo-controlled, crossover study. Participants were randomized
to treatment with danshen (water-extract) or placebo for 4 consecutive weeks. There was
a wash out period of 4 weeks. Inclusion criteria were: age 40-70 years, hyperlipidemia and
hypertension. At the end of each treatment period, plasma lipids were determined (primary
outcome), 24 hours ambulant blood pressure measurement (ABPM) was performed, and
vasodilator endothelial function was assessed in the forearm.
Results
LDL cholesterol levels were 3.82±0.14 mmol/l after danshen and 3.52±0.16 mmol/l after
placebo treatment (p<0.05). danshen treatment had no effect on blood pressure (ABPM
138/84 after danshen and 136/87 after placebo treatment). These results were further
substantiated by the observation that danshen had neither an effect on endothelial function
nor on markers of inflammation, oxidative stress, glucose metabolism, hemostasis and blood
viscosity.
Conclusion
Four weeks of treatment with danshen (water-extract) slightly increased LDL-cholesterol without
affecting a wide variety of other risk markers. These observations do not support the use of
danshen to prevent or treat atherosclerosis.
The trial was registered at clinicaltrials.gov ( NCT01563770).
120
Salvia Miltiorrhiza water-extract (danshen) has no beneficial effect on cardiovascular risk factors 
Introduction
Worldwide, cardiovascular disease (atherosclerosis) is the leading cause of mortality 1.
Traditional risk factors for atherosclerosis are hyperlipidemia, hypertension, diabetes mellitus
and smoking and inflammation which has gained much attention more recently 2,3. In both
primary and secondary prevention, treatment of hypertension and hyperlipidemia reduces the
risk of cardiovascular events 4-6.
Traditional Chinese Medicine (TCM) has been playing an important role in healthcare in Asia
for hundreds of years. Recently, there is an upsurge in the popularity of TCM products among
consumers in outside China. In 2010, the United States spent $7.6 billion on TCM products
and Europe $2 billion 7. Factors that contribute to the increased use of TCM products in the
West are the increased prevalence of obesity and related chronic disorders, increasing costs
of conventional medicines and the belief that TCM products are safer and more effective than
prescription drugs 8.
Danshen is the dried root extract of the plant Salvia Miltiorrhiza and is widely used in Asia to
treat coronary artery disease, hyperlipidemia and cerebrovascular disease 9. The estimated
number of patients using danshen is approximately 5 million worldwide 10. According to TCM
theory, danshen is used in patients to treat “blood stasis” as it “moves blood” 11. The TCMquantification of “blood stasis” is made by identification of certain changes in the tongue
and pulse. The major components of danshen are hydrophilic salvianolic acids and lipophilic
tanshinones 12. In vitro treatment of human monocyte derived macrophages with a combination
of danshen and Gegen resulted in a suppression of acetylated Low Density Lipoprotein (LDL)
uptake 13. Two active components of danshen, salvianolic acid A and magnesium tanshinoate
B, inhibit LDL oxidation 11. A mixture of Chinese herbs, including danshen, has been shown to
exert beneficial effects on high-fat diet induced metabolic syndrome in rats 14. Also in humans,
danshen may reduce LDL-levels and blood pressure 11. However, the quality of randomized
clinical trials of danshen is poor 15 and not easily accessible to Western physicians since most
7
are published in Chinese.
Therefore, this study was designed to evaluate the effect of danshen on hyperlipidemia and
hypertension, two well validated risk factors for cardiovascular disease according to evidencebased medicine.
121
 Salvia Miltiorrhiza water-extract (danshen) has no beneficial effect on cardiovascular risk factors
Patients and methods
Study Population
The study population consisted of 20 subjects aged 40-70 years with hyperlipidemia and
hypertension, who were recruited through advertisements in local newspapers. (Figure 1)
Included were subjects with a fasting LDL cholesterol > 3.5 mmol/l and/or triglycerides > 1.7
mmol/l. Patients treated with antihypertensive drugs were eligible when the average of three
office blood pressure recordings (sphygmomanometer, sitting position, at least 5 minutes
interval) revealed a systolic blood pressure > 140 mmHg and/or diastolic blood pressure > 90
mmHg. For subjects that were not treated with antihypertensives, blood pressure was recorded
three times on two separate visits (in total 6 recordings, in sitting position, sphygmomanometer,
5 minute intervals between recordings on each visit) and again average blood pressure had
to fulfill the criterium of > 140 mmHg for systolic and/or > 90 mmHg for diastolic blood
pressure. Excluded were subjects with triglyderides > 8 mmol/l and/or LDL-cholesterol > 5
mmol/l or systolic blood pressure > 180 mmHg and/or diastolic blood pressure > 110 mmHg.
Further exclusion criteria were treatment with statins, use of more than 1 antihypertensive
drug, use of angiotensin-converting-enzyme inhibitors, angiotensin II receptor antagonists
, calcium antagonists or vitamin and/or herbal supplements, diabetes mellitus treated with
insulin, a history of cardiovascular disease, use of anticoagulant drugs, a serum alanine
aminotransferase or aspartate aminotransferase level of more than 3 times the upper limit of
normal and abnormal creatinin clearance defined as MDRD< 60 ml/min/1.73m2.
Protocol
This was a randomized, double-blind, placebo controlled crossover study. After inclusion in
the study, subjects were randomly assigned to treatment with danshen capsules, 4 capsules
of 500 mg tid, for 28 days or placebo capsules, 4 capsules tid, for 28 days. After a washout
period of 4 weeks participants crossed over to the other treatment arm. Investigational
medication was given on top of their other medication.
Participants were contacted by telephone halfway each treatment period to check for
compliance and possible side effects.
At the end of each treatment period blood was collected, blood pressure, body weight and
endothelium-mediated vasodilation were measured, and potential side effects were recorded.
Compliance was monitored by pill counts. Prescribed medication was not altered during the
trial and participants did not change their dietary habits. (Figure 2)
122
Salvia Miltiorrhiza water-extract (danshen) has no beneficial effect on cardiovascular risk factors 
Enrollment
Assessed for eligibility (n=36)
Excluded (n=13)
..
Not meeting inclusion criteria (n=13)
Randomized (n=23)
Allocation
Allocated to placebo first
Allocated to danshen first
..
Received allocated intervention (n=12)
..
Received allocated intervention (n=11)
..
Did not receive allocated intervention (n=0)
..
Did not receive allocated intervention (n=1);
consent withdrawal
Follow-Up
Lost to follow-up (n=0)
Lost to follow-up (n=0)
Discontinued intervention (n=1); SAE
Discontinued intervention (n=0)
Analysis
Analysed (n=11)
Analysed (n=9)
..
..
Excluded from analysis (n=0)
Excluded from analysis (n=1): protocol
violation
Figure 1.CONSORT flow diagram.
SAE: Serious Adverse Event.
7
Salvia miltiorrhiza extract (danshen)
Salvia miltiorrhiza extract was produced by Kaiser Pharmaceuticals Co. in Taiwan. This product
was obtained from a specialized pharmacy NatuurApotheek. Using this extract, Basic Pharma
Manufacturing BV produced Danshen and placebo capsules. Each capsule contained 500
mg granulate consisting of 50% water-extract of danshen and 50% starch. Therefore, a
dosage of 4 capsules tid with 500 mg granulate is equal to 4 capsules with 250 mg extract tid
(3 g daily). Since the root – extract ratio is 5:1, 3 gram extract originates from 15 g root. The
Pharmacopoeia of the People’s Republic of China recommends a dosage of 9-15 g root
123
 Salvia Miltiorrhiza water-extract (danshen) has no beneficial effect on cardiovascular risk factors
daily 10. So, we used in this study the highest dose of danshen that is regularly used in TCM. In
this way, we wanted to reduce any doubts about insufficient dosing in case of lack of efficacy
or overdosing in case of unexpected adverse events.
Briefly, the production process of danshen is as follows: firsta water decoction of Salvia root
is made. Subsequently, the water-extract is concentrated 5 times via a standard distillation
procedure. After the concentration procedure, a drying and granulation procedure (with 50%
starch as carrier) followed using a fluid bed spray dryer. Then the granules are filtered, which
results in a final bulk product consisting of granules of a specified size. Finally, the bulk product
is filled into containers
Screening
phase
Randomization, TCM,
blood samples and
automatic BP
Plethysmography and
blood samples
Plethysmography and
blood samples
Blood samples and
automatic BP
ABPM
and TCM
ABPM
and TCM
Danshen 4 capsules tid
Danshen 4 capsules tid
Placebo 4 capsules tid
Placebo 4 capsules tid
weeks
0
4
8
12
Figure 2.Graphic presentation of the study protocol.
Four weeks treatment with danshen and placebo in a crossover design. BP= blood pressure.
ABPM= 24-h Ambulatory Blood Pressure Monitoring. TCM= traditional Chinese Medicine
diagnosis.
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Salvia Miltiorrhiza water-extract (danshen) has no beneficial effect on cardiovascular risk factors 
Biochemical analyses
 Lipids
Blood was drawn in fasting state before and after each treatment period to measure total
cholesterol, triglycerides, High Density Lipoprotein (HDL) -cholesterol and apolipoprotein B
(Apo-B). The LDL- cholesterol was calculated using the Friedewald equation( LDL-cholesterol=
total cholesterol – HDL-cholesterol – 0.45 x triglycerides ) 16. Total cholesterol and triglyceride
concentrations were measured using an enzymatic method. For determination of HDL-levels,
direct immunoinhibition was used. Apolipoprotein B levels were determined by turbidimetry. (all
Architect, Abbott Diagnostics Division). Coefficients of variation were for total cholesterol <3%,
triglycerides <5%, HDL-cholesterol ≤4% and for apolipoprotein B ≤6.5%.
 Metabolic and inflammatory parameters
Glucose concentrations were measured using the glucose oxidase-peroxidase
method. (Architect, Abbott Diagnostics Division). Insulin levels were determined by an
electrochemiluminescence immunoassay (Modular E170, Roche Diagnostics). The coefficients
of variation were ≤ 5% for glucose and 3.2% for insulin.
Homeostasis model assessment of insulin sensitivity (HOMA IR) was calculated using the
following formula: fasting glucose (mmol/l) x fasting insulin (mU/l)/ 22.5. For HOMA of beta cell
function (HOMA-B) the formula was: 20x fasting insulin (mU/l)/ fasting glucose (mmol/l)-3.5 17.
As marker of oxidative stress, lipid peroxidation was determined by measuring ThioBarbituric
Acid Reactive Substances (TBARS) in plasma using a fluorimetric assay 18. Furthermore,
antioxidant capacity was determined by means of Ferric Reducing Ability of Plasma assay
(FRAP) 19.
Measurements of safety parameters (hemogram, electrolytes, creatinin and liver enzymes)
were performed at the department of Laboratory Medicine,Radboud university medical center.
Blood pressure measurement
7
Before start of study medication blood pressure was measured for 20 minutes at two minute
intervals by an automated device (Nihon Kohden BSM 2301, Nihon Kohden, Japan) using the
oscillometric principle with the subject in supine position.
Furthermore, in the last week of each treatment period blood pressure was measured at home
by 24-h Ambulatory Blood Pressure Monitor (ABPM) using a fully automated device (Mobil-OGraph NG, I.E.M. GmbH, Germany). ABPM results were expressed as mean total, day (from
8.00 a.m. to 10.59 p.m.) and night (from 11.00 pm to 7.59 a.m.) blood pressure.
125
 Salvia Miltiorrhiza water-extract (danshen) has no beneficial effect on cardiovascular risk factors
Forearm vasodilator response to acetylcholine and sodium nitroprusside
Forearm blood flow (FBF) was assessed by venous occlusion plethysmography using mercuryin-silastic strain gauges (Hokanson EC4. Hokanson, Inc) as previously described 20. The
experiments started at 8.30 a.m. after an overnight fast in a quiet, temperature controlled room
(23˚C-24˚C). Study medication was ingested at home, prior to visiting our clinical research
department. The subjects were studied after 24 hours of caffeine abstinence. The brachial
artery of the non-dominant arm (experimental forearm) was cannulated (27-gauge needle,
Braun, Melsungen, Germany) for infusion of vasodilators. FBF was measured at both forearms
simultaneously. The upper arm cuffs were inflated using a rapid cuff inflator (Hokanson E-20,
DE Hokanson, Bellevue, WA). Wrist cuffs were inflated to 220 mm Hg to occlude the hand
circulation during the infusion of both vasodilators and their baselines 21. Forearm volume was
measured with the water displacement method and all drugs were dosed per 100 ml forearm
tissue with a constant infusion rate of 100 µl.dl-1.min-1 during infusion of solvent ( baseline
recordings) as well as during infusion of increasing doses of vasodilators.
After complete instrumentation, a 30 minute equilibration period followed, after which baseline
measurements were performed with infusion of saline. Subsequently, three increasing doses
of acetylcholine (0.5, 2.0 and 8.0 µg.dl-1.min-1, 10 mg/ml dry powder, dissolved to its final
concentration with saline, Novartis, Greece) were infused into the brachial artery. Acetylcholine
stimulates endothelial muscarinic receptors thereby activating nitric oxide synthase. This
results in endothelial release of nitric oxide (NO) causing vasodilation 22. Each dose was
infused for 5 minutes and FBF was measured. After the last acetylcholine dose, a 30 minutes
equilibration period followed. Subsequently, baseline measurements were performed with the
infusion of glucose 5% solution. Subsequently, three increasing doses of sodium nitroprusside
(0.06, 0.20 and 0.60 µg.dl-1.min-1, 25 mg/ml, dissolved to its final concentration with glucose 5%
solution, Clinical Pharmacy, Radboud university medical centre), a NO-donor and endotheliumindependent vasodilator 23, were infused. Again, each dose was infused for 5 minutes and
FBF was measured. FBF registrations of the last 2 minutes of each dosage of vasodilator were
averaged to a single value for data analysis.
Hemostatic and rheological parameters
Screening for primary hemostasis abnormalities was performed using the platelet function
analyzer (PFA-100, Siemens, Germany). The closure time was measured with the collagen
epinephrine and collagen-ADP cassette. Normal reference ranges for the collagen epinephrine
cassette is ≤ 170 sec with an inter assay variation of 12.4 %. Normal reference ranges for the
collagen ADP cassette is ≤ 120 sec with an inter-assay variation of 12.7 %.
126
Salvia Miltiorrhiza water-extract (danshen) has no beneficial effect on cardiovascular risk factors 
Von Willebrand Factor (vWF) antigen levels were measured using the Asserachrom VWF:Ag
ELISA from STAGO. The Lower Limit Of Quantification (LLOQ) of this assay is 12.5% with an
inter-assay variation coefficient of 4.7%. Reference intervals are blood group dependent and
ranges from 50-150% (blood group O) up to 70 – 210% (blood group AB).
The effect of danshen on coagulation and fibrinolysis was measured using the Nijmegen
Hemostasis Assay which measures thrombin and plasmin generation in time 24. The thrombin
generation curve represents the following parameters: (i) lag-time, the time to the start of
thrombin formation; (ii) thrombin peak time, i.e. the time when thrombin production reaches
maximal velocity; (iii) thrombin peak height, the maximal velocity of thrombin generation; and
(iv) the area under the curve (AUC) for the total amount of thrombin formed. Plasmin generation
is described by: (v) fibrin lysis time , the time between the initiation of thrombin generation and
the time plasmin generation reaches maximal velocity; (vi) plasmin peak-height, the maximal
velocity of plasmin production; and (vii) plasmin potential, area under the curve that represents
the total amount of plasmin generated. The inter-assay variation of thrombin generation
parameters varies from 5.9% (AUC) to 25% (lag time thrombin generation). The inter-assay
variation of plasmin generation parameters varies from 10% (plasmin peak height) to 14%
(plasmin potential).
All hemostatic parameters were measured at the Department of Laboratory Medicine of the
Radboud university medical Centre.
Blood viscosity was determined in vitro at a temperature of 37°C and at a shear rate of 3.16 sˉ¹
with the viscometer LS300 (ProRheo GmbH, Germany).
TCM qualification of “blood stasis”
Two TCM practitioners independently determined the presence of “blood stasis” by pulse and
tongue diagnosis and interrogation at baseline and after both treatment periods. The TCM
practitioners were asked to categorize patients as having definite “blood stasis” (score 1),
7
definite absence of “blood stasis” (score 3) or some characteristics of “blood stasis” but not
fulfilling the criteria for a full qualification (score 2). Kappa values and polychoric correlations
were calculated to estimate the agreement of both TCM-practitioners for the three visits
separately. The polychoric correlation was calculated in addition to kappa-value since the
distribution of the diagnostic score appeared not homogeneous with overpresentation of score
2, a situation that makes the kappa-value very sensitive to a single disagreement .
127
 Salvia Miltiorrhiza water-extract (danshen) has no beneficial effect on cardiovascular risk factors
Calculations and statistical analysis
We considered a change of 0.225 mmol/l in primary endpoint LDL-cholesterol between
treatment groups as clinically relevant. Assuming a test-to-test correlation coefficient of 0.7 we
would require a total of 20 subjects to detect a change of 0.225 mmol/l in LDL-cholesterol with
a power of 80 % at a significance level of 0.05 (Zα = 1.96). When the test-to-test correlation was
0.5 , a study including 20 participants would be able to detect a difference of 0.3 mmol/l.
Drop-outs were replaced.
Differences in means of laboratory results and blood pressure measurements were tested
by paired Student’s t-test or Wilcoxon signed rank test for non-normally distributed data. In
addition, the effect of danshen on plasma lipids and ApoB-lipoprotein was analyzed with the
CROS analysis according to Senn (Cross-over trials in clinical research. Vol 5, John Wiley &
Sons, 2002). Repeated measures analysis of variances (ANOVA) was used to assess the effect
of danshen on FBF-response to acetylcholine and nitroprusside. Statistical analyses were
performed using Graphpad 5.0. Results are expressed as mean ± Standard Error of the
Mean (SEM), unless otherwise indicated. Significance was set at a two-sided p-value of less
than 0.05.
Ethics statement
This study was conducted in accordance with the principles outlined in the Declaration of
Helsinki and Good Clinical Practice (GCP) guidelines. The local ethics committee (Radboud
university medical centre) approved the study and all subjects gave written informed consent
before participation. The trial was registered at clinicaltrials.gov ( NCT01563770).
Results
Twenty-three of the 36 initially screened subjects underwent randomization and were enrolled
in the study. One participant withdrew before start of the study medication. One participant
discontinued the intervention due to a facial nerve paralysis during danshen treatment.
Furthermore, one subject was excluded from statistical analyses because of insufficient
compliance to the trial protocol. See table 1 for baseline characteristics of the remaining 20
evaluable participants. All 20 evaluable participants were included in all analyses.
128
Salvia Miltiorrhiza water-extract (danshen) has no beneficial effect on cardiovascular risk factors 
Characteristics of 20 participating subjects
Age (years)
58.0±7.7
Sex (male:female)
14:6
Weight (kg)
86.4±18.9
BMI (kg/m²)
28.6±5.6
Blood pressure systolic (mmHg)
156±9
Blood pressure diastolic (mmHg)
94±5
Total cholesteroL (mmol/l)
6.2±0.7
Triglycerides (mmol/l)
1.9±1.3
HDL-cholesterol (mmol/l)
1.3±0.4
LDL-cholesterol (mmol/l)
4.0±0.5
Apolipoprotein B (g/l)
1.1±0.2
Fasting glucose (mmol/l)
5.5±0.6
HbA1c (%)
5.7±0.3
Antihypertensives (%)
n =10 (50)
Diuretics n =6 ( 30)
Beta- blockers n = 4 (20)
Table 1.Baseline characteristics
(mean±SD)
Lipids
Danshen treatment did not affect HDL cholesterol, TG and ApoB. However, total cholesterol
and LDL cholesterol were significantly higher after danshen treatment compared to placebo,
approximately 0.3 mmol/l (Table 2). Baseline levels of total cholesterol and LDL cholesterol
were not different for both treatment periods. Furthermore, total cholesterol and LDL
7
cholesterol were slightly but significantly higher after treatment with danshen compared to
baseline values (p < 0.01) while placebo treatment did not change baseline levels. When the
effect on danshen was expressed as change from baseline and corrected for the changes
observed during placebo treatment, LDL-cholesterol was significantly increased (Figure 3)
while other lipid fractions were not affected.
129
 Salvia Miltiorrhiza water-extract (danshen) has no beneficial effect on cardiovascular risk factors
Danshen
Placebo
Baseline
Treatment
Baseline
Treatment
Total cholesterol (mmol/l)
5.61±0.15
5.84±0.18* #
5.60±0.19
5.55±0.18
Triglycerides (mmol/l)
1.76±0.27
1.75±0.22
1.71±0.25
1.81±0.24
HDL cholesterol (mmol/l)
1.23±0.07
1.23±0.07
1.21±0.06
1.22±0.07
LDL cholesterol (mmol/l)
3.67±0.12
3.82±0.14* #
3.63±0.16
3.52±0.16
Apolipoprotein B (g/l)
1.00±0.04
1.02±0.04¶
0.99±0.05
0.97±0.04
0.5
0.0
0.0
-0.5
-0.5
-1.0
-1.0
po
pr
ot
ei
ro
l
st
e
oB
Li
C
ho
le
st
e
L
C
ho
le
LD
Ap
es
rid
L
ce
H
D
ly
Tr
ig
st
e
le
To
ta
lC
ho
n
0.5
ro
l
1.0
ro
l
1.0
ApoB lipoprotein
(g/l, mean, 95% Cl)
Serum lipids
(mmol/l, mean, 95% Cl)
Table 2.Lipid levels before and after treatment with danshen and placebo
(mean±SEM). Baseline values were not significantly different between danshen and placebo
treatment.* p< 0.01 compared to baseline. ¶ p <0.05 and # p< 0.01 compared to treatment
with placebo
Figure 3.The absolute changes in lipid levels during treatment with danshen, corrected for the changes
during treatment with placebo.
CROS analysis according to Senn. *: p<0.05
130
Salvia Miltiorrhiza water-extract (danshen) has no beneficial effect on cardiovascular risk factors 
Blood pressure
24 h ABPM did not change in response to danshen treatment (Table 3). The total number of
measurements during ABPM was 67±9 of which 17±2 (mean± standard deviation (SD))
during the night with danshen and 63±10 and 17±2 with placebo treatment, respectively.
Twenty minute automatic blood pressure measurements were performed prior to placebo and
danshen administration (‘baselines’). Again, both systolic and diastolic blood pressures were
not different at baseline (systolic blood pressures before danshen 140±3 mmHg and before
placebo 140±3 mmHg. Corresponding values for diastolic blood pressure were 85±2 mmHg
and 85±2 mmHg, respectively).
ABPM
Danshen
Placebo
total BP (mmHg)
139±4/89±3
136±4/87±2
day BP (mmHg)
142±4/91±3
139±4/90±2
night BP (mmHg)
130±4/82±2
130±4/79±2
Table 3.Blood pressure measurement before and after treatment with danshen or placebo
(mean±SEM). BP= blood pressure. ABPM= 24-h Ambulatory Blood Pressure Monitoring
Forearm vascular responses to acetylcholine
Infusion of acetylcholine induced a dose-dependent increase in FBF in the experimental
forearm while FBF did not change in the non-experimental forearm. danshen treatment did not
alter the FBF in response to acetylcholine compared to placebo. With danshen, FBF increased
from 2.3±0.2 ml.dl⁻¹.min⁻¹ during saline to 7.5±0.8, 12.1±1.0 and 16.3±1.3 ml.dl⁻¹.min⁻¹
7
during acetylcholine 0.5, 2.0 and 8.0 µg.dl⁻¹.min⁻¹ respectively compared to 2.6±0.4 during
saline and 8.2±0.9, 12.6± 1.1 and 16.6± 1.3 ml.dl⁻¹.min⁻¹ during acetylcholine with placebo
(Figure 4). Comparable results were obtained when data were expressed as absolute and
relative changes in FBF from baseline.
131
 Salvia Miltiorrhiza water-extract (danshen) has no beneficial effect on cardiovascular risk factors
Forearm vascular responses to sodium nitroprusside
Infusion of sodium nitroprusside induced a dose-dependent increase in FBF in the
experimental forearm. FBF did not change in the non-experimental forearm. Treatment
with danshen did not affect vascular responses to sodium nitroprusside. FBF was 2.6±0.3
ml.dl⁻¹.min⁻¹ during glucose and 5.0± 0.4, 7.8± 0.6 and 11.5± 0.8 ml.dl⁻¹.min⁻¹ during
sodium nitroprusside 0.06, 0.20 and 0.60 µg.dl⁻¹.min⁻¹, respectively after danshen treatment,
compared to 3.0±0.5 at baseline and 5.3± 0.5 , 7.7± 0.7 and 11.3± 1.0 ml.dl⁻¹.min⁻¹ during
nitroprusside after placebo (Figure 4).
Danshen
Placebo
FBF(ml.dl-1.min-1)
20
15
10
5
0
Saline
Ach 0.5 Ach 2.0 Ach 8.0
FBF(ml.dl-1.min-1)
15
10
5
0
Glucose SNP 0.06 SNP 0.20 SNP 0.60
Figure 4.Assessment of endothelial function.
Forearm blood flow in response to acetylcholine in the experimental arm (solid lines) and in the
non experimental arm (dashed lines) in response to acetylcholine (top panel; dosage 0.5, 2.0, 8.0
µg.dl⁻¹.min⁻¹) and sodium nitroprusside ( bottom panel; dosage 0.06, 0.20 and 0.60 µg.dl⁻¹.min⁻¹),
during danshen (dark grey circles) or placebo (light grey squares).
132
Salvia Miltiorrhiza water-extract (danshen) has no beneficial effect on cardiovascular risk factors 
Metabolic and inflammatory parameters
Treatment with danshen did not affect fasting plasma glucose levels (danshen 5.7±0.1
mmol/l vs placebo 5.8±0.2 mmol/l, p = 0.48) and HbA1c levels (5.8±0.09% and 5.7±0.08%,
respectively, p = 0.45). Also fasting insulin levels, HOMA-IR and HOMA-B did not change
during danshen treatment.
C-reactive protein (CRP) levels were not significantly different after danshen (2.9±0.7 mg/l)
compared to placebo treatment (2.7±0.7 mg/l).
The markers of oxidative stress TBARs and FRAP did not change during danshen treatment
compared to placebo (TBARs 0.09±0.01 vs 0.09±0.01 µmol/mg protein and FRAP 268±11 vs
267±11 µM/mg protein). Furthermore, there were no differences in oxidized LDL, Intercellular
Adhesion Molecule (ICAM), Vascular Cell Adhesion Molecule (VCAM) and E-selectin between
danshen and placebo treatment (data not shown). Danshen did not change body weight (data
not shown).
Hemostatic and rheological parameters
Danshen treatment did not change von Willebrand factor antigen levels compared to placebo
(112±9% vs 113±9%). The PFA with collagen and epinephrine was 132±7 s when treated with
danshen and 148±7 s when treated with placebo, both within the normal range (p = 0.048
for comparison between danshen and placebo). Furthermore, the PFA with collagen and
ADP was not different between the two treatment periods (97±4 s and 100±4 s, respectively).
Taking both PFA tests together, we interpret these data as that danshen does not relevantly
affect platelet function.
The coagulation parameters measured by thrombin generation as well as fibrinolysis
parameters measured by plasmin generation were not altered by Danshen treatment (Figure 5).
Blood viscosity at a shear rate of 3.16 sˉ¹ was not different after treatment with danshen
(14.4±0.9 mPas*s) compared to placebo (14.2±0.8 mPas*s). However, hematocrit levels were
7
significantly higher after treatment with danshen (44 l/l vs 43 l/l, p = 0.048 ). When expressing
blood viscosity per l/l of hematocrit we did not find an effect of danshen on viscosity either.
133
 Salvia Miltiorrhiza water-extract (danshen) has no beneficial effect on cardiovascular risk factors
A
2000
1000
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sh
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Thrombin generation
(nmol/l)
3000
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Fibrinolysis time
(minutes)
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Figure 5.Results of the Nijmegen Hemostasis Assay.
A. Thrombin generation. B. Plasmin generation measured by fibrinolysis time
134
Salvia Miltiorrhiza water-extract (danshen) has no beneficial effect on cardiovascular risk factors 
TCM- qualification blood stasis
Only two out of the twenty participants were qualified as having blood stasis at inclusion. In
both subjects blood stasis was not observed in the following visits. The kappa values, as a
measure of between-observer agreement for the diagnosis of blood stasis were 0.19 for TCM
visit at baseline, 0.67 for TCM visit after the first treatment period, and -0.08 for TCM visit after
the second treatment period. In addition a polychoric correlation was calculated which was
0.43, 0.99 and 0.12 for the first, second and third visit, respectively..
Side effects and safety
Most reported side effects during danshen treatment were headache (n=5), dizziness (n=3),
change in stool frequency (n=3) and flatulence (n=2). One serious adverse event occurred
during danshen treatment: a peripheral facial nerve paralysis.
Discussion
This study did not find beneficial effects of four weeks of treatment with danshen on
cardiovascular risk factors. This is based on the observation that danshen does not reduce
LDL and triglyceride levels and does not decrease blood pressure. Furthermore, danshen
had no effect on endothelial function, markers of inflammation, oxidative stress and glucose
metabolism nor on hemostatic and rheological markers.
These results are in contrast with previous studies. A number of explanations may underlie the
lack of reduction of cardiovascular risk markers in this study. First, we only used danshen and
not a combination of Chinese herbs.
Tan et al. have shown that a combination of 3 Chinese herbs, including danshen, decreased
fasting triglycerides, cholesterol and non-esterified fatty acids in rats with metabolic
7
syndrome 14. The Fufang danshen dripping pill, which is a composite of Salvia miltiorrhiza,
Panax notogingseng and Cinnamomum camphora, reduced triglycerides, total cholesterol and
LDL cholesterol levels 9.
Second, there are over 80 components of danshen of which 3 lipophilic and 3 hydrophilic
components seem to be the major active components 9,25. Our danshen product was
produced via water- extraction method. Possibly, a low concentration in the water-extract of
pharmacologically active lipophilic compounds, including tanshinones, led to a lack of efficacy
of danshen . Unfortunately, an alcohol-extract of danshen is not available in the Netherlands for
human use.
135
 Salvia Miltiorrhiza water-extract (danshen) has no beneficial effect on cardiovascular risk factors
Third, the selection of participants was based on evidence based criteria of increased
cardiovascular risk, i.e. increased LDL cholesterol or triglyceride levels and hypertension.
The TCM qualification of “blood stasis” was only made in 2 out of the 20 participants. Since
according to the TCM theory danshen is used to treat “blood stasis” , advocators of this theory
could argue that the selected population might not be suitable to detect any cardiovascular
benefit. It is not known what diagnosis corresponds with this TCM-qualification. Therefore, we
cannot exclude the possibility that danshen is effective in reducing plasma LDL-cholesterol in a
subgroup of patients who have “blood stasis” as opposed to the observed increase in plasma
LDL-cholesterol in those who are at increased cardiovascular risk according to evidencebased criteria. However, our observed low reproducibility of the observation ‘blood stasis’ and
overpresentation of intermediate test results support our decision not to include this TCMqualification in the inclusion criteria of this trial.
In our study danshen (water-extract) actually increased LDL and total cholesterol levels
compared to placebo. This increment in cholesterol levels has also been observed for another
food product, namely coffee. It has been shown that boiled, but not filtered, coffee increases total
cholesterol and LDL cholesterol 26. The hypercholesterolemic factor in boiled coffee is retained
by the filter paper and eventually this factor was identified as cafestol 27 28. Possibly, the method
of preparation of danshen produced or preserved a “hypercholesterolemic factor” that was not
produced by methods used by others.
Danshen did not reduce endothelium-dependent vasodilation which suggests that the observed
increase in LDL cholesterol of approximately 0.3 mmol/l is too small to have a significant impact
on cardiovascular risk. Indeed, endothelium-mediated vasodilation predicts cardiovascular
disease and is responsive to changes in plasma cholesterol 29,30 31,32. The Cholesterol Treatment
Trialists’ (CTT) Collaboration has shown that each 1 mmol/l reduction in LDL cholesterol
reduces the absolute risk of major cardiovascular events with 20% 33. If we intrapolate these
epidemiological observations and assume generalizability of our findings, our observed increase
of 0.3 mmol/l in LDL cholesterol would translate into a small increment of 7% in absolute risk
of cardiovascular disease. Based on the demographic data of our study population and current
cardiovascular risk tables available for this Dutch population, absolute 10-year cardiovascular
risk of our subjects to develop a cardiovascular event is 19% for male non-smokers and 35% for
male smokers. The water extract of Danshen could theoretically increase these risks to 20.3%
and 37.5% for male non-smokers and smokers respectively. However, this projected increase in
cardiovascular risk may overestimate the real impact of danshen on cardiovascular risk, since
the increase in LDL-cholesterol was not accompanied by a detectable increase in oxidized
LDL cholesterol. Therefore, the impact of danshen on lipid profile was probably too small to be
reflected by a detectable change in acetylcholine-induced forearm vasodilation.
136
Salvia Miltiorrhiza water-extract (danshen) has no beneficial effect on cardiovascular risk factors 
The strength of our study is the randomized, double-blind, placebo controlled design with
objective and reproducible endpoints. The cross-over design is potentially limited by carryover effects. However, we did not detect an order-effect and baseline measurements in blood
pressure and plasma lipids did not differ prior to the two treatment periods.
Conclusion
Four weeks of treatment with the herbal product danshen (water extract) slightly increased
LDL-cholesterol without affecting a wide variety of other risk markers or endothelium-dependent
vasodilation, an early marker of vascular injury. These results do not support the use of
danshen water-extract as a single agent to treat risk factors of atherosclerosis.
Acknowledgements
The authors thank G. Pop, MD, PhD, Department of Cardiology, Radboud university medical
centre, for his assistance in the blood viscosity measurements, T.I. Tan, MD, R.M. Veenstra,
MD and K.A. Kruithof, MD who are all trained in TCMfor their diagnostic evaluation of blood
stasis. Finally, the authors thank CL Oei-Tan, chair of NAAV for her important role in initiating the
involvement of TCM-practitioners in our study.
Conflicts of interest
7
This study was financed by Cinmar Pharma BV, ‘s Hertogenbosch, the Netherlands,
Department of Pharmacology-Toxicology, Radboud university medical centre, Nijmegen, the
Netherlands, and the Dutch Association of Acupuncture Medicine (NAAV, Amsterdam, the
Netherlands).
The study sponsor had no role in study design, analysis and interpretation of the data and in
the writing of the manuscript.
137
 Salvia Miltiorrhiza water-extract (danshen) has no beneficial effect on cardiovascular risk factors
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139
BETA
BETA
FUNCTION
DYSFUNCTION
CELL
CELL
ENDOTHELIAL
ENDOTHELIAL
INSULIN
INSULIN
FUNCTION
SENSITIVE
CHAPTER 8
SUMMARY AND GENERAL DISCUSSION
DYSFUNCTION
RESISTANT
 Summary and general discussion
Summary
In this thesis we studied the effect of different novel treatments on insulin sensitivity, insulin
secretion and vascular function.
In chapter 2 we show that the DPP-4 inhibitor vildagliptin improves the vasodilator response
to acetylcholine while it does not change the response to sodium nitroprusside in patients
with type 2 diabetes mellitus. These results demonstrate that vildagliptin improves endothelial
function in subjects with type 2 diabetes which suggests that vildagliptin may have beneficial
effects on cardiovascular disease risk.
Since DPP-4 is involved in immune responses we also analysed the effect of the DPP-4 inhibitor
vildagliptin on immune function. As described in chapter 3, serum cytokine concentrations and
ex-vivo cytokine production (both monocyte and T-cell derived) did not differ during treatment
with vildagliptin compared to acarbose. Thus, inhibition of DDP-4 does not seem to lead to a
functional defect in innate cellular immunity.
As inflammation in general, and the proinflammatory cytokine IL-1β in particular, plays a role
in the development of type 2 diabetes mellitus, we have investigated the effect of treatment
with the IL-1β receptor antagonist anakinra on insulin sensitivity and on insulin secretion. As
described in chapter 4, we found that anakinra improved insulin sensitivity in insulin resistant
patients with type 1 diabetes, as demonstrated by a numerical increase in glucose infusion rate
(GIR) during the clamp after one week of treatment and by a significant increase in GIR after 4
weeks of follow-up. This was accompanied by decreased blood glucose day profiles, reduced
HbA1c levels and lower insulin needs. Altogether these results suggest that IL-1β is involved in
the development of insulin resistance, at least in the context of chronic hyperglycemia (glucose
toxicity) and that inhibiting IL-1β by anakinra can improve insulin resistance independent of an
effect on beta cell function.
Chapter 5 describes the effect of anakinra on insulin secretion in patients with impaired
glucose tolerance. Treatment with anakinra improved first phase insulin secretion, which is the
increment in insulin levels during the first 10 minutes of hyperglycemia. In line with this finding,
also the insulinogenic index (increase in insulin levels divided by increase in glucose levels
during the first 30 minutes of the oral glucose tolerance test), a marker of beta cell function,
improved after anakinra treatment. These findings support the concept of involvement of IL-1β
in the decrease of insulin secretion capacity associated with impaired glucose tolerance and
type 2 diabetes mellitus. This is relevant since improvement in insulin secretion can delay and/
142
Summary and general discussion 
or prevent progression to frank diabetes.
Because inflammation is linked to vascular disease, we subsequently investigated the
relationship between adipose tissue inflammation and endothelial function. In chapter 6 we
did confirm that subjects with type 2 diabetes mellitus have impaired endothelial function
compared to subjects with both dyslipidemia and hypertension. Compared to control, the
groups had increased macrophage influx in subcutaneous adipose tissue, consistent with
increased inflammation. However, we found no difference in endothelial function between the
groups of participants with and without inflammation in the subcutaneous adipose tissue.
Hence, subcutaneous fat tissue inflammation is probably not directly related to vascular
dysfunction.
Finally, chapter 7 describes the effect of the herbal product danshen on traditional
cardiovascular risk factors. Treatment with danshen did not decrease, but slightly increased
LDL cholesterol levels and had no effect on blood pressure. These results were further
substantiated by the observation that danshen had neither an effect on endothelial function
nor on markers of inflammation, oxidative stress, glucose metabolism, hemostasis and blood
viscosity. These results do not support the use of danshen as a single agent to treat risk factors
of atherosclerosis.
8
143
 Summary and general discussion
General discussion
Type 2 diabetes mellitus is extremely prevalent and leads to serious microvascular and
macrovascular complications. For once, type 2 diabetes causes a twofold excess risk for
cardiovascular disease, partly independent from other risk factors. Clearly, developing better
treatments and prevention strategies for type 2 diabetes mellitus and cardiovascular disease is
urgently needed. A number of novel interventions has been studied in this thesis.
DPP-4 inhibitor vildagliptin
As stated, type 2 diabetes mellitus doubles the risk of cardiovascular complications and
reducing this risk is one of the cornerstones of treatment. There is discussion whether lowering
of blood glucose levels reduces cardiovascular disease. While in newly diagnosed patients
with type 2 diabetes, strict glycemic control reduced the probability to develop a myocardial
infarction1,2, an effect that seemed preserved and stronger during follow up (10 yr follow up) 3,
intensive glucose control in patients with advanced diabetes resulted in excess mortality 4
without reduction in cardiovascular events 4,5. When combining all outcome trials, current
thinking is that strict glucose control decreases the risk to develop myocardial infarction by
approximately 15% for every 0.88% decrease in HbA1c 6, but that the effect takes time and can
be offset by side effects as hypoglycemia and weight gain 7,8. Ideally, blood glucose lowering
pharmacotherapy in patients with type 2 diabetes has beneficial cardiovascular effects
independent of glucose lowering. In response to cardiovascular safety concerns associated
with thiazolidinediones 9 , the Food and Drug Administration and the European Medicines
Agency require a demonstration of cardiovascular safety for all new glucose lowering
therapies. This has led to a substantial increase in the number of cardiovascular outcome trials
in type 2 diabetes.
Meta-analyses evaluating the effect of DPP-4 inhibitors on cardiovascular events in phase 2
and 3 studies have suggested beneficial effects 10,11. Our trial results showing that the DPP-4
inhibitor vildagliptin improves endothelial dependent vasodilatation (chapter 2), a surrogate
marker which might translate to a beneficial cardiovascular profile, is in line with these metaanalyses. Not all studies on markers of cardiovascular complications have found similar
effects, however. A recent trial suprisingly reported an attenuated endothelial function (as
measured by flow mediated vasodilatation (FMD)) of the DPP-4 inhibitor sitagliptin compared
to voglibose in patients with type 2 diabetes mellitus 12. The authors also showed in a separate
trial that both sitagliptin and alogliptin reduced FMD, suggesting that this is a class effect
of DPP-4 inhibitors 12. Potential reasons for the discrepancy with our study are the use of
144
Summary and general discussion 
different DPP-4 inhibitors and different methods of assessing endothelial function, i.e. FMD vs
acetylcholine induced vasodilatation. The real proof of a potential beneficial cardiovascular
effect of DPP-4 inhibitors is a cardiovascular outcome trial, preferably comparing a DPP-4
inhibitor with comparator treatment.
Recently, two cardiovascular outcome trials with DPP-4 inhibitors have been reported. Both
trials were intended to show cardiovascular safety and designed to test for non-inferiority
compared to placebo regarding cardiovascular event rate. The fact that these trials do
not provide a direct comparison to existing treatments limits their clinical implications. The
Saxagliptin Assessment of Vascular Outcomes Recorded in Patients with Diabetes Mellitus
(SAVOR)- Thrombolysis in Myocardial Infarction (TIMI) 53 trial was designed to evaluate
cardiovascular safety of saxagliptin 13. A total of 16.492 patients were randomized to treatment
with either saxagliptin or placebo and similar rates of cardiovascular complications occurred
in both groups ( p < 0.001 for non-inferiority and p = ns for superiority) over a median period
of 24 months. There was a slight (0.2%) but significant difference in HbA1c levels at the end
of the trial between the two groups. Another cardiovascular outcome trial, the Examination
of Cardiovascular Outcomes with Alogliptin versus Standard of Care (EXAMINE), in 5380
patients also showed equal rates of cardiovascular events in patients treated with alogliptin as
compared to patients assigned to placebo 14.
Potential explanations for the absence of beneficial cardiovascular effects in these two trials,
as opposed to our finding of improved endothelial function, include the short intervention
period and follow-up (24 and 30 months) and the patient category to detect a beneficial
cardiovascular effect. Type 2 diabetes mellitus is a chronic progressive disease and beneficial
effects of glucose lowering on macrovascular complications in type 2 diabetes mellitus
took 10 years to occur in the UKPDS trial 3. This suggests that long term trials are needed
in type 2 diabetes mellitus to assess benefits and risks of glucose-lowering interventions.
Second, in order to obtain sufficient statistical power to prove safety, patients with a high
cardiovascular risk were included while in our study we included subjects with type 2 diabetes
without a previous history of cardiovascular events and subjects were not smoking. In
theory, the process of atherosclerosis in the outcome trials may have been too advanced
for improvements to occur while in our study population the process could be counteracted.
Furthermore including subjects with longstanding diabetes at high cardiovascular risk limits the
ability to obtain information on treatments intervening earlier in the progression of diabetes.
8
Finally, these outcome trials were performed with other DPP-4 inhibitors than what we used in
our study, namely vildagliptin.
Data from ongoing cardiovascular outcome trials using DPP-4 inhibitors are expected in the
near future (CAROLINA: linagliptin vs glimepiride 15,16 ,CARMELINA: linagliptin vs placebo,
TECOS: sitagliptin vs placebo 17,18) . Of these, the CAROLINA trial is of special interest since
145
 Summary and general discussion
it compares linagliptin with active comparator glimepiride. The GRADE trial is a comparative
effectiveness trial adding GLP-1 analogue, DPP-4 inhibitor or SU- derivative to metformin, but
this trial is not powered for cardiovascular endpoints 7.
To conclude, while our study findings would suggest that the DPP-4 inhibitor vildagliptin may
have beneficial cardiovascular effects on the long run, large cardiovascular outcome studies
with the DPP-4 inhibitors saxagliptin and alogliptin have shown cardiovascular safety but no net
cardiovascular benefit, at least not in the short term, in high risk patients.
Given the fact that DPP-4 is also involved in T-cell activation and has other substrates than
GLP-1, there was concern that DPP-4 inhibitors might increase the risk of infections. We show
that serum cytokine concentrations and ex-vivo cytokine production (both monocyte and T-cell
derived) were not altered by vildagliptin (chapter 3) which suggests that DPP-4 inhibitors do not
increase the risk of infections by altering cytokine production. Indeed, a recent meta-analysis
did not show an increased risk of infections with DPP-4 inhibitors 19.
Interleukin-1 receptor antagonist anakinra
A growing amount of evidence suggests that IL-1β is involved in the pathogenesis of both
insulin resistance and insulin secretion (see introduction). A proof of concept study in patients
with type 2 diabetes mellitus demonstrated that interleukin-1 receptor antagonist anakinra
improved glycemic control 20. The observed beneficial effects of anakinra on glycemic control
were correlated with an improvement in beta cell function without effects on insulin sensitivity.
However, the improvement in beta cell function could either be direct or indirect due to
improved glycemia. Furthermore, in this trial beta cell function was assessed by proinsulin/
insulin ratios and C-peptide levels during oral and intravenous glucose tolerance tests while
the hyperglycemic clamp is considered the gold standard to determine insulin secretion.
Finally, insulin sensitivity was only assessed in a subgroup of participants. Another study using
anakinra from our own group showed enhanced disposition index derived from OGTT without
effects on insulin sensitivity in subjects with the metabolic syndrome 21.
Anakinra has a relatively short half-life of 4-6 hours and daily subcutaneous injections are
required. The most frequently observed adverse events during the use of anakinra are injection
site reactions 22. These disadvantages do not occur with anti-IL-1β antibodies, which have
recently become available 23. Studies applying anti-IL1β antibodies have yielded conflicting
results regarding the effect on glycemic control in patients with type 2 diabetes mellitus 24-27.
Most studies have found no or a minor change in glycemic control (see Table 1)
146
Summary and general discussion 
The reason for the substantial effect of anakinra by Larsen versus the various effect of IL-1β
antibodies on glycemic control in patients with type 2 diabetes is not clear. It may be that
IL-1Ra blocks both IL-1α and IL-1β, while the specific anti-IL-1β antibodies only blocks IL-1β
action 29. So far, the study by Larsen has not been reproduced by other groups and blockade
by anakinra may not be effective in all patients. The effect, although limited, of blocking IL-1β
on glucose control may be explained by increasing insulin sensitivity, by improving insulin
secretion or both.
Our group found that in subjects with the metabolic syndrome, anakinra did not improve
insulin sensitivity 21 which is in accordance with findings of Larsen in the initial anakinra trial 20.
Also studies using anti-IL-1β antibodies in patients with 2 diabetes found no effect on insulin
sensitivity 24,26. These findings seem to be in contrast with our finding of improved insulin
sensitivity in insulin resistant hyperglycemic patients (chapter 4), which may be explained by
the fact that we focused on the combination hyperglycemia/insulin resistance. Perhaps the
pathophysiological involvement of IL-1β is more pronounced in the obese state in the context
of chronic hyperglycemia. Furthermore, the trials using IL-1β antibodies were not designed to
evaluate insulin sensitivity and the measures used to assess insulin sensitivity are less accurate
than the clamp technique in our study. Clearly, more work is needed to delineate the exact
value of IL-1β blockade on insulin sensitivity.
8
147
 Summary and general discussion
Drug
Dose
Subjects
HbA1c effects
Other effects
Ref
Anakinra
100 mg sc once
daily
70 patients T2DM
↓ HbA1c
↑ insulin secretion
20
-0.46% after 13
weeks
≈ insulin sensitivity
150 mg sc once
daily
13 subjects
metabolic
syndrome
Anakinra
100 mg sc once
daily
69 patients recent
onset T1DM
≈ HbA1c
≈ insulin secretion
28
Gevokizumab
Single dose iv
98 patients T2DM
↓ HbA1c
≈ insulin sensitivity
24
-0.11,-0.44 and
-0.85% after 1,2,3
months for single
intermediate dose
only
≈ insulin secretion
↓ HbA1c
≈ insulin sensitivity
-0.27, -0.38 and
≈ insulin secretion
Anakinra
(0.01, 0.03, 0.1, 0.3,
1 or 3 mg/kg)
Single dose sc
(0.03, 0.1 or 0.3
mg/kg)
↑ insulin secretion
21
≈ insulin sensitivity
Multiple dose sc
(0.03 or 0.3 mg/kg)
LY2189102
IL-1β antibody
0.6, 18 or 180 mg
sc weekly
109 patients
T2DM
26
-0.25% after 12
weeks
Canakinumab
150 mg single sc
injection
246 patients
T2DM and IGT
≈ HbA1c
≈ insulin secretion
T2DM
25
↑ insulin secretion
IGT
Canakinumab
5,15, 50 or 150 mg
sc monthly
556 patients
T2DM
≈ HbA1c
Canakinumab
2 mg/kg sc monthly
69 patients recent
onset T1DM
≈ HbA1c
27
≈ insulin secretion
Table 1.Clinical studies using anti-IL1β therapy. Sc= subcutaneous. iv= intravenous. T2DM= type 2
diabetes mellitus. T1DM= type 1 diabetes mellitus. IGT= impaired glucose tolerance.
148
28
Summary and general discussion 
In agreement with Larsen et al we do show that anakinra improves insulin secretion
(chapter 5). While other studies with anti-IL1β antibodies have found no effect on insulin
secretion, a study using canakinumab in patients with type 2 diabetes and subjects with
IGT found a trend towards improving insulin secretion rates in patients with type 2 diabetes
mellitus and improved insulin secretion in subjects with IGT 25. Combined with findings so far,
our results suggest that beneficial effects of anti-IL1β therapy on insulin secretion are most
consistent in patients with impaired glucose tolerance. Whether these effects can translate to a
decreased progression to diabetes remains to be proven.
To conclude, the current value of anti-IL1β treatment has not yet been determined in full detail.
While in some studies it has shown an effect on glycemia, effects vary across studies. Thus,
the most appropriate anti-IL1β treatment strategies and the most suitable population to be
treated remains to be determined.
The herbal product danshen
Globally, cardiovascular disease is the most frequent cause of death 30. Traditional risk
factors for atherosclerosis are hyperlipidemia, hypertension, diabetes mellitus, and smoking.
According to Traditional Chinese Medicine, herbal products represent an alternative treatment
strategy for hyperlipidemia and hypertension 31,32. We tested the effects of danshen, the dried
root extract of the plant Salvia Miltiorrhiza, which is widely used in Asia to treat coronary artery
disease, hyperlipidemia and cerebrovascular disease. In contrast to previous studies, we
found no beneficial effect but even an increase in LDL-cholesterol levels and no effect on blood
pressure. A number of explanations may underlie these findings. First, other studies have used
a combination of herbs while we have used monotherapy with danshen only. Second, we used
danshen produced by water-extraction which might have led to a low concentration of lipophilic
compounds. There are over 80 components of danshen of which 3 hydrophilic and 3 lipophilic
are the active components 33. Third, the selection of participants was based on Western
criteria of increased cardiovascular risk, i.e. increased LDL cholesterol or triglyceride levels
and hypertension. According to the TCM theory danshen is used to treat “blood stasis” . Since
the TCM diagnosis “blood stasis” was only made in 2 out of the 20 participants, the selected
population might not be suitable to detect any cardiovascular benefit in terms of the traditional
8
TCM diagnosis.
In our study danshen actually increased LDL- cholesterol levels with 0.3 mmol/l. The
Cholesterol Treatment Trialists’ (CTT) Collaboration has shown that each 1 mmol/l reduction
in LDL cholesterol reduces the absolute risk of major cardiovascular events with 20% 34. If we
intrapolate these epidemiological observations and assume generalizability of our findings, the
149
 Summary and general discussion
observed increase of 0.3 mmol/l in LDL cholesterol would translate into a small increment of
7% in absolute risk of cardiovascular disease. Although the impact of danshen on lipid profile
was not reflected in any detectable change in acetylcholine-induced forearm vasodilatation, the
results of our study do not support the use of danshen water-extract as a single agent to treat
risk factors of atherosclerosis.
Type 2 diabetes mellitus and associated vascular disease are a major health problem. Current
treatment includes glucose lowering agents and treatment of cardiovascular risk factors.
This thesis provides insight into the vascular effects of new oral glucose lowering agents and
complementary treatment of cardiovascular risk factors. Furthermore, it contributes to a better
understanding of the role of interleukin-1β in the development of type 2 diabetes mellitus with
the suggestion that some subjects might benefit from anti-IL1β therapy. Further work is needed
to determine the most appropriate patient category for such an intervention and to advance
towards a truly personalized diabetes treatment.
150
Summary and general discussion 
8
151
 Summary and general discussion
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8
27.Ridker PM, Howard CP, Walter V, et al. Effects of
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153
BETA
BETA
FUNCTION
DYSFUNCTION
CELL
CELL
ENDOTHELIAL
ENDOTHELIAL
INSULIN
INSULIN
FUNCTION
SENSITIVE
CHAPTER 9
NEDERLANDSE SAMENVATTING
LIST OF PUBLICATIONS
CURRICULUM VITAE
DANKWOORD
DYSFUNCTION
RESISTANT
 Nederlandse samenvatting
Nederlandse samenvatting
Diabetes mellitus type 2 (suikerziekte) is een belangrijk gezondheidsprobleem en komt veel
voor zowel in Nederland als wereldwijd. Het aantal mensen in de wereld dat lijdt aan diabetes
wordt momenteel geschat op 380 miljoen en de verwachting is dat dit aantal verder zal
stijgen naar 590 miljoen in het jaar 2035. Diabetes mellitus type 2 wordt gekenmerkt door een
chronisch verhoogde glucoseconcentratie in het bloed. Chronisch te hoge glucosewaarden
leiden tot complicaties aan de grote en kleine bloedvaten. Zo hebben mensen met type 2
diabetes mellitus twee keer zo veel kans op het ontwikkelen van hart- en vaatziekten als
mensen zonder diabetes. Complicaties van de kleine bloedvaten kunnen leiden tot ernstige
problemen met de ogen, de nieren en het zenuwstelsel.
Zoals gezegd wordt diabetes mellitus gekenmerkt verhoogde glucosewaarden in het bloed.
Voor een goede regulatie van de glucosewaarde is insuline het belangrijkste hormoon. Insuline
wordt gemaakt door betacellen in de eilandjes van Langerhans in de alvleesklier en verlaagt
de glucosewaarde op verschillende manieren: het remt de glucoseaanmaak in de lever en
stimuleert de glucoseopname in de spiercel.
Bij sommige mensen is de gevoeligheid voor insuline verminderd, we spreken dan van
insulineresistentie. De betacellen in de alvleesklier kunnen in die situatie meer insuline
aanmaken en zo compenseren voor de verminderde gevoeligheid. Bij gezonde mensen zal
de balans tussen gevoeligheid voor insuline en productie van insuline ervoor zorgen dat de
glucosewaarde in het bloed normaal blijft. Als de alvleesklier niet langer kan compenseren
voor de verminderde gevoeligheid voor insuline, ontstaan er hoge glucosewaarden en
daarmee diabetes mellitus type 2. De gevoeligheid voor insuline en de productie van insuline
worden door verschillende factoren beïnvloed, waaronder genetische aanleg maar ook door
omgevingsfactoren zoals overgewicht, oudere leeftijd en door een hogere ontstekingsactiviteit.
Wereldwijd zijn hart- en vaatziekten de meest voorkomende doodsoorzaak waarbij er per
jaar 20 miljoen mensen sterven aan deze aandoeningen. Het is daarom van groot belang om
goede behandel- en preventiestrategieën voor diabetes mellitus type 2 en hart- en vaatziekten
te ontwikkelen.
In dit proefschrift hebben we de effecten van verschillende nieuwe behandelingen op
insulinegevoeligheid, insulineafgifte en vaatfunctie onderzocht.
In hoofdstuk 2 worden de resultaten beschreven van een onderzoek waarbij het effect van
een nieuw geneesmiddel voor de behandeling van diabetes, vildagliptin, op de functie van
de bloedvaatwand wordt onderzocht. Vildagliptin remt het enzym DPP-4. DPP-4 breekt het
hormoon GLP-1 in het lichaam af, dus het remmen van DPP-4 zorgt ervoor dat er minder GLP-1
wordt afgebroken en dus de concentratie van GLP-1 in het bloed stijgt. Van het hormoon GLP-
156
Nederlandse samenvatting 
1 is in eerder onderzoek aangetoond dat het de functie van de bloedvaatwand verbetert. Om
te onderzoeken of de DPP-4 remmer vildagliptin de functie van de bloedvaatwand verbetert,
hebben 16 proefpersonen met diabetes gedurende 4 weken vildagliptin en gedurende 4
weken een controlepil geslikt. Aan het einde van beide perioden werd bloed geprikt en werd de
vaatfunctie getest. Het onderzoek toonde aan dat het geneesmiddel vildagliptin de vaatfunctie
verbetert en dat zou kunnen betekenen dat het gebruik van dit geneesmiddel de kans op harten vaatziekten bij patiënten met diabetes vermindert.
Omdat het geneesmiddel vildagliptin invloed zou kunnen hebben op het afweersysteem
en daardoor mogelijk het ontstaan van infecties zou kunnen bevorderen, hebben we in het
onderzoek dat is beschreven in hoofdstuk 3 ook onderzocht of vildagliptin effect heeft
op de productie van verschillende afweerstoffen. Hiervoor hebben we uit het bloed van
de proefpersonen de witte bloedcellen geïsoleerd en gestimuleerd om afweerstoffen te
produceren. De productie van afweerstoffen na de periode van 4 weken vildagliptin werd
vergeleken met na de periode van 4 weken controlepil. Er bleek geen verschil tussen beide
behandelingen in de productie van afweerstoffen. Vildagliptin heeft dus geen invloed op de
aanmaak van afweerstoffen en dit betekent dat het risico op het ontstaan van infecties niet
verhoogd is.
Ontsteking, en in het bijzonder de ontstekingsbevorderende stof interleukine-1β (IL-1β), speelt
een rol bij het ontstaan van type 2 diabetes mellitus. In dit proefschrift hebben we de effecten
van het remmen van IL-1β op zowel de insulinegevoeligheid als de insulineafgifte onderzocht.
In hoofdstuk 4 hebben we onderzocht of het remmen van IL-1β door het geneesmiddel
anakinra effect heeft op de insulinegevoeligheid. Hiervoor hebben we 14 proefpersonen met
lang bestaande type 1 diabetes met overgewicht geselecteerd. Type 1 diabetes ontstaat
doordat de alvleesklier geen insuline kan produceren en mensen met lang bestaande
type 1 diabetes hebben geen enkele eigen insulineproductie meer. Insulinegevoeligheid
en insulineproductie hebben invloed op elkaar: als de insulinegevoeligheid vermindert,
compenseert de alvleesklier dat door meer insuline af te geven. Door deze proefpersonen te
selecteren kunnen we het effect van anakinra op insulinegevoeligheid bestuderen zonder dat
er invloed is van een verandering in de insulineproductie. De proefpersonen werden 4 weken
behandeld met het geneesmiddel anakinra en 4 weken met nepmedicijn (placebo). Aan het
einde van elke behandelperiode werd de insulinegevoeligheid getest. Uit het onderzoek bleek
dat anakinra de insulinegevoeligheid verbetert.
Hoofdstuk 5 beschrijft de effecten van anakinra op de insulineafgifte. Hiervoor hebben 16
personen gedurende 4 weken anakinra en gedurende 4 weken placebo gebruikt. Aan het
einde van beide behandelperiodes werd de maximale insulineafgifte door de eilandjes van
Langerhans gemeten. We vonden dat na behandeling met anakinra de insulineafgifte in de
9
157
 Nederlandse samenvatting
eerste 10 minuten van de test toenam. Deze bevinding ondersteunt de hypothese dat IL-1β
betrokken is bij de afname van de insulineafgifte en dus bij het ontstaan van type 2 diabetes
mellitus.
Uit eerder onderzoek was al bekend dat er een associatie bestaat tussen een toegenomen
ontstekingsactiviteit in het bloed en het ontstaan van hart- en vaatziekten. Omdat die
ontsteking zich typisch voordoet in het vetweefsel hebben we in hoofdstuk 6 onderzocht of
er een relatie bestond tussen ontsteking in het onderhuidse vetweefsel en de functie van de
bloedvaatwand. Hiervoor hebben we hapjes die we uit het onderhuids vetweefsel genomen
(vetbiopten) van patiënten met diabetes mellitus type 2 en van patiënten met hoge bloeddruk
en een hoog cholesterol en deze onder de microscoop beoordeeld op het bestaan van
kenmerken van ontsteking. Bij dezelfde proefpersonen is de functie van de bloedvaatwand
onderzocht. We vonden echter geen verband tussen ontsteking in het onderhuidse vetweefsel
en de functie van de bloedvaatwand. Wel konden we bevestigen dat personen met type
2 diabetes mellitus een slechtere vaatwandfunctie hebben dan patiënten met een hoge
bloeddruk en hoog cholesterol.
Hoofdstuk 7 beschrijft het effect van het kruidenpreparaat danshen op risicofactoren voor
hart- en vaatziekten. Het kruidenpreparaat danshen wordt in China gebruikt bij de behandeling
van hart- en vaatziekten. We vonden dat danshen het cholesterol niet verlaagt, maar juist zelfs
een beetje verhoogt, en geen effect heeft op de bloeddruk. Daarnaast had danshen geen
effect op de functie van de bloedvaatwand en ook niet op andere kenmerken voor hart- en
vaatziekten. Dit onderzoek kan dus niet bevestigen dat het gebruik van danshen nuttig is voor
de behandeling van risicofactoren voor hart- en vaatziekten.
Type 2 diabetes mellitus en de daarmee geassocieerde hart- en vaatziekten zijn een
groot gezondheidsprobleem. De huidige behandeling bestaat uit het verlagen van de
glucosewaarden en de behandeling van de risicofactoren voor hart- en vaatziekten. Dit
proefschrift geeft inzicht in de effecten van een nieuw glucoseverlagend geneesmiddel op de
bloedvaatwand en inzicht in de behandeling van risicofactoren voor hart- en vaatziekten door
chinese kruiden. Verder draagt het proefschrift bij aan het beter begrijpen van de rol van de
ontstekingsbevorderende stof interleukine-1β in het ontstaan van diabetes mellitus type 2 en
laat het zien dat sommige personen baat kunnen hebben bij het remmen interleukine-1β. Meer
werk is nodig om te bepalen wat de meest geschikte categorie van patiënten is die voordeel
halen hebben bij deze behandeling zodat mensen met diabetes meer op maat kunnen worden
behandeld.
158
Nederlandse samenvatting 
9
159
 List of publications
160
List of publications 
List of publications
1.
Agressief non-hodgkin lymfoom bij 3 patiënten met reumatoïde artritis: eerst
methotrexaat stoppen.
P.C.M. van Poppel, H.A.M. Sinnige, R. Fijnheer, J.F. Haverman.
Nederlands tijdschrift voor geneeskunde; 2008; 152; 2351-2356
2.
Hyperchloremische metabole acidose bij een patiënt met bricker lis.
P.C.M. van Poppel, C.D. Stehouwer, J.J. Beutler, M.B. Korst, H.P. Beerlage,
E.K. Hoogeveen.
Nederlands Tijdschrift voor Geneeskunde; 2009; 153; B317
3.
Vildagliptin improves endothelium-dependent vasodilatation in type 2 diabetes mellitus. P.C.M. van Poppel, M.G. Netea, P. Smits, C.J. Tack.
Diabetes Care; 2011; 34; 2072-2077
4. The dipeptidyl peptidase-4 inhibitor vildagliptin does not affect ex vivo cytokine response and lymphocyte function in patients with type 2 diabetes mellitus.
P.C.M. van Poppel, M.S. Gresgnigt, P. Smits, M.G. Netea, C.J. Tack
Diabetes Res Clin Pract 2014 Mar;103(3):395-401
5. The interleukin-1 receptor antagonist anakinra improves first phase insulin secretion and insulinogenic index in subjects with impaired glucose tolerance.
P.C.M. van Poppel, E.J.P. van Asseldonk, J.J. Holst, T. Vilsbøll, M.G. Netea, C.J. Tack
Diabetes Obes Metab 2014 Dec;16(12):1269-1273
6. Salvia Miltiorrhiza water-extract (danshen) has no beneficial effect on cardiovascular
risk factors.
P.C.M. van Poppel, P. Breedveld, E.J. Abbink, H. Roelofs, W. van Heerde, P. Smits,
W. Lin, A. Tan, F. Russel, R. Donders, C.J. Tack, G.A. Rongen
Plos One 2015 Jul 20;10(7):e0128695
7. One week treatment with the IL-1 receptor antagonist anakinra leads to a sutained improvement in insulin sensitivity in insulin resistant patients with type 1 diabetes mellitus
E.J.P. van Asseldonk, P.C.M. van Poppel, D.B. Ballak, R. Stienstra, M.G. Netea, C.J. Tack Clin Immunol 2015 Oct; 160 (2):155-162
8. Inflammation in the subcutaneous adipose tissue is not related to endothelial function in subjects with diabetes mellitus and subjects with dyslipidemia and hypertension.
P.C.M. van Poppel, A.J. Abbink, R. Stienstra, M.G. Netea, C.J. Tack
Submitted
9
161
 Curriculum Vitae
162
Curriculum Vitae 
Curriculum Vitae
Pleun van Poppel werd geboren op 3 augustus 1980 te Boxtel. In 1998 behaalde zij cum laude
haar gymnasium diploma aan het Maurick-College te Vught. In datzelfde jaar begon ze met
haar studie Geneeskunde aan de Universiteit Maastricht. Na het behalen van haar artsexamen
(cum laude) in 2004 begon ze als arts-assistent interne geneeskunde in het Amphia Ziekenhuis
in Breda. In januari 2006 begon ze met de opleiding tot internist in het Jeroen Bosch
Ziekenhuis te ’s Hertogenbosch (voormalig opleider: dr. P.M. Netten). In 2009 vervolgde ze haar
opleiding in het RadboudUMC (opleiders:prof. dr. J. de Graaf, prof. dr. J.W.A. Smit, voormalig
opleider prof. dr. J.W.M. van der Meer). In datzelfde jaar begon ze met haar promotieonderzoek
onder begeleiding van prof. dr. C.J. Tack en prof. dr. M.G. Netea. Van augustus 2009 tot
oktober 2014 volgde ze het aandachtsgebied endocrinologie (opleider: prof. dr. A.R.M.M.
Hermus). In de periode oktober 2014 tot januari 2015 was zij staflid Algemene Interne
Geneeskunde, sectie Diabetes waarbij klinische werkzaamheden werden gecombineerd
met wetenschappelijk onderzoek. Sinds januari 2015 is werkzaam als internist in het Maxima
Medisch Centrum te Veldhoven/Eindhoven.
Zij is getrouwd met Bart Wouters. Samen hebben zij twee kinderen, Loek (2013) en Lize (2014).
9
163
 Dankwoord
Dankwoord
Met het schrijven van het laatste hoofdstuk is er een eind gekomen aan een periode van
een promotietraject van 6 jaar. Graag zou ik de vele mensen willen bedanken die hebben
bijgedragen aan het tot stand komen van dit proefschrift.
Proefpersonen, allereerst wil ik jullie natuurlijk bedanken omdat jullie bereid waren om deel te
nemen aan alle onderzoeken. Deelname betekende voor jullie dat jullie maanden bezig waren
met het innemen of zelfs injecteren van medicatie en het ondergaan van lange testochtenden
met infusen, vetbiopten of lijnen. Heel veel dank voor jullie inzet en tijd. Zonder jullie was dit
proefschrift er nooit gekomen.
Prof. Dr. C.J Tack, beste Cees, ruim 6 jaar geleden kwam ik bij je binnen met de vraag of
er een mogelijkheid was om klinisch wetenschappelijk onderzoek te doen. Op dat moment
was die mogelijkheid er niet. Een paar maanden later belde je me op dat je een idee had
voor onderzoek en of ik daar mee aan de slag zou willen gaan. Mijn enthousiasme straalde
ik niet meteen uit, maar daarna zijn we er samen vol voor gegaan. Ik wil je bedanken voor
het door jou in mij gestelde vertrouwen. Jouw enthousiasme, kritische opmerkingen en
wetenschappelijke kunde hebben me steeds weer op weg geholpen als ik even niet verder
kwam. Op jouw aanraden ga ik mijn promotie met een feestje vieren.
Prof. Dr. M.G. Netea, beste Mihai, je hebt me op besprekingen keer op keer weten te
verbazen met jouw kennis over zo veel uiteenlopende onderwerpen en jouw enthousiasme
daarover werkt motiverend. Dank voor je begeleiding bij het tot stand komen van dit
proefschrift.
Prof. Dr. G.A. Rongen, beste Gerard, dank voor het meedenken en opschrijven van het
‘danshen stuk’. Ik bewonder jouw liefde voor wetenschap en jouw vasthoudendheid. Dit kwam
zeker van pas als we te maken hadden met weerbarstige editors.
Promotieonderzoek combineren met de opleiding tot medisch specialist is niet mogelijk zonder
een werkomgeving die dat ondersteunt. De stafleden van de afdeling Endocriene Ziekten,
de stafleden van de diabetes sectie en mijn collega-fellows uit het RadboudUMC hebben
mij in die periode bijgestaan met hun adviezen en geduld. Ook hebben ze daar waar nodig
mijn klinische taken waargenomen, wat mij in staat heeft gesteld me op mijn onderzoek te
concentreren. Daarnaast wil ik alle AIOS interne geneeskunde en opleiders van zowel het
Jeroen Bosch Ziekenhuis als het RadboudUMC bedanken voor de prettige werksfeer en
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Dankwoord 
flexibiliteit. Een aantal personen wil ik hier graag nog apart noemen. Prof. Dr. A.R.M.M.
Hermus, beste Ad, nadat ik twee maanden bezig was met het aandachtsgebied endocriene
ziekten onderbrak ik voor het eerst mijn opleiding voor het verrichten van wetenschappelijk
onderzoek. De jaren die volgden bestonden steeds uit delen onderzoek en delen klinische
opleiding. Dit stelde je voor uitdagingen bij het maken van het rooster. Ik wil je danken voor
je flexibiliteit waardoor ik de afgelopen jaren beide heb kunnen combineren. Prof. Dr. J. de
Graaf, beste Jacqueline, als ik voor mijn jaargesprek weer de benodigde papieren invulde
met mijn wensen voor het komende jaar, was ik stiekem toch altijd weer een beetje bang
voor jouw reactie. Onderzoek en opleiding combineren in constructies van 30/70%, 50/50%,
0/100% en dat binnen het jaar in nog verschillende periodes was geen probleem. Jij dacht in
oplossingen en probeerde mij altijd te steunen in mijn wens om beide te kunnen voltooien.
Dr. P. Netten, beste Paetrick, mijn opleiding tot internist ben ik bij jou als opleider begonnen
in het Jeroen Bosch Ziekenhuis. Jij wist dat ik onderzoek wilde gaan doen en met een klein
duwtje in mijn rug van jou ben ik aan dit traject begonnen. Dank voor het duwtje!
Artsen in de Chinese Traditionele Geneeswijze, W. Lin, A. Tan, H. Tan, K. Kruithof. Dank voor
de samenwerking en een kijkje in elkaars keuken: East meets West.
Lieve collega’s van de buitenhoek Janna, Rinke, Edwin, Pieter, Duby, Heleen, Anneleen,
Henry, Mark S, Nico, Mark G, Theo. Veel dank voor het overleggen over statistiek en
Graphpad, (ont)spanning op congressen delen en de gezellige sfeer onderling. Succes met
jullie verdere carrières.
Beste Anneke, Heidi, Helga en Cor (laboratorium Experimentele Interne Geneeskunde).
Dank voor alle hulp en raad omtrent de laboratoriumwerkzaamheden voor de verschillende
studies. En voor de dropjes.
Beste Karin, Anja, Mariëlle, Evertine, Christel en Corina (CRCN). Bedankt voor jullie
hulp en de gezellige tijd tijdens het uitvoeren van de experimenten op de afdeling klinische
fysiologie en later op het CRCN.
Beste Dr. Pop, dank voor je hulp bij en de enthousiaste uitleg over het meten van de viscositeit
van het bloed. Tevens hartelijk dank voor het uitlenen van uw apparaat.
Beste collega internisten van het Maxima Medisch Centrum. Heel hartelijk dank voor de
prettige samenwerking en steun tijdens het afronden van mijn boekje.
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 Dankwoord
Lieve vrienden en vriendinnen, bedankt voor alle leuke, gezellige, sportieve, feestelijke,
culinaire en mooie dingen die ik met jullie mee heb mogen maken. Omdat ik niemand wil
vergeten noem ik geen namen, maar jullie weten wel dat ik jullie bedoel. Dank voor jullie
vriendschap en steun. Dat er nog vele mooie momenten mogen volgen.
Beste paranimfen. Lieve Annemarie, het kan bijna niet anders dan dat jij vandaag naast me
staat. Wij hebben hetzelfde carrièrepad gevolgd. We zijn vriendinnen geworden tijdens onze
tijd als ANIOS interne geneeskunde in Breda, hebben samen veel meegemaakt als AIOS
interne in het JBZ en hebben jaren lief en leed gedeeld al carpoolend naar het RadboudUMC.
Jij hebt je opleiding tot internist-oncoloog afgerond en volgend jaar ben jij aan de beurt! Lieve
Boukje, bij de meeste belangrijke momenten in mijn leven heb jij aan mijn zijde gestaan (van
Take That concert, tot Australië, tot aan mijn bruiloft) en ik zou je vandaag dan ook niet kunnen
missen. Dank voor jouw vriendschap.
Lieve (schoon) familie bedankt! Lieve pap en mam, nu is het eindelijk af. Hier kunnen jullie
lezen waar ik al die jaren mee bezig ben geweest. Op jullie kan ik altijd rekenen voor praktische
steun (oppassen op de kindjes, onze tuin bijhouden, voor ons koken) en gezelligheid, bij jullie
kom ik nog steeds “thuis”. Dank voor jullie onvoorwaardelijke steun en liefde. Lieve Stijn, we
zien elkaar niet zo vaak als we zouden willen maar zonder het uit te hoeven spreken weten
we beide dat we er altijd voor elkaar zullen zijn. Lieve Elly, wat is het fijn dat je altijd voor ons
klaar staat. Daarnaast dank voor alle keren dat je naar Den Bosch bent gekomen om op Loek
te passen en voor alle keren dat je nu op onze beide kindjes past en deze verwent. Ook heb
je het mogelijk gemaakt dat wij regelmatig onze batterij hebben kunnen opladen in Canyelles
Petites.
Lieve Loek en Lize, jullie maken mij gelukkig met jullie blije gezichtjes als ik na een dag
werken thuiskom. Ik geniet van alle momenten met jullie al zijn die nog zo klein: een lach, een
knuffel, een kus met een cappuccino snor en op zondag met zijn allen in bed “uitslapen”.
En dan tot slot mijn allerliefste Bart, dank voor al jouw praktische hulp en steun. Met het
afronden van dit proefschrift blijft er hopelijk weer meer tijd over voor ons samen. Ik kijk uit naar
al het moois wat we samen nog mogen gaan meemaken! Ik hou van jou.
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Dankwoord 
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167
NOVEL INTERVENTIONS IN TYPE 2 DIABETES AND VASCULAR DISEASE
BETA
BETA
FUNCTION
DYSFUNCTION
CELL
CELL
ENDOTHELIAL
ENDOTHELIAL
INSULIN
INSULIN
FUNCTION
SENSITIVE
DYSFUNCTION
RESISTANT
Pleun van Poppel
NOVEL INTERVENTIONS IN TYPE 2
DIABETES AND VASCULAR DISEASE
Effects on insulin sensitivity, insulin secretion and vascular function
Pleun van Poppel