Anti-inflammatory effects of simvastatin on adipokines in type 2 diabetic patients with carotid atherosclerosis.

Abstract

Statins are extensively used for lowering LDL-cholesterol and reducing cardiovascular events. Recent studies have shown that statins have beneficial anti-inflammatory effects. We aimed to determine whether and how adipokines are regulated during statin treatment in type 2 diabetic patients.
In this study,we investigated the changes of CRP and inflammation-related adipokines (SAA,IL-6,TNFalpha and adiponectin) in 23 type 2 diabetic patients with atherosclerosis who received statin therapy, and 20 diabetic patients with atherosclerosis and 14 diabetic patients without atherosclerosis who did not receive statin therapy for a period of three months.
By the end of the simvastatin treatment (40 mg, daily), LDL-cholesterol was decreased by 16.7% and HDL-cholesterol was increased by 31.9%. SAA, CRP, TNFalpha and IL-6 levels were decreased by 31.8%, 66.2%, 53.9% and 14%, respectively and adiponectin was increased by 59.6%, compared with the baseline levels. Interestingly, the decrease of SAA was positively correlated with that of LDL-cholesterol but negatively with HDL-cholesterol during statin treatment. Among the adipokines, the decrease of SAA was positively correlated with TNFalpha (r = 0.50, p = 0.016).
The results suggest that adipokines may be differentially regulated and independent of cholesterol changes and that adipokines may be a mediator, and the adipose tissue may be a target of statins' anti-inflammatory effect.

Full text

Page 1

http://dvr.sagepub.com/
Diabetes and Vascular Disease Research
http://dvr.sagepub.com/content/6/4/262
The online version of this article can be found at:

DOI: 10.1177/1479164109339966
2009 6: 262 Diabetes and Vascular Disease Research
Yun Hu, Guoyu Tong, Wei Xu, Jiajia Pan, Kathy Ryan, Rongze Yang, Alan R. Shuldiner, Da-Wei Gong and Dalong Zhu
atherosclerosis
Anti-inflammatory effects of simvastatin on adipokines in type 2 diabetic patients with carotid


Published by:

http://www.sagepublications.com
On behalf of:


International Society of Diabetes Vascular Disease

can be found at:
Diabetes and Vascular Disease Research

Additional services and information for





http://dvr.sagepub.com/cgi/alerts
Email Alerts:

http://dvr.sagepub.com/subscriptions
Subscriptions:



http://www.sagepub.com/journalsReprints.nav
Reprints:


http://www.sagepub.com/journalsPermissions.nav

Permissions:

http://dvr.sagepub.com/content/6/4/262.refs.html
Citations:
What is This?

- Sep 17, 2009Version of Record >>
by guest on October 14, 2011dvr.sagepub.com Downloaded from

Page 2

Introduction
Recent studies indicate a close association between chronic
inflammation and CVD in that inflammatory status is ele-
vated in patients with CVD risk factors including obesity,
insulin resistance, type 2 diabetes and dyslipidaemia, as
evidenced by the increase of inflammation marker CRP1-3
and SAA.4 Furthermore, pharmacological interventions
(e.g. statin, thiazolidinediones) that improve CVD risk fac-
tors, also decrease serum inflammatory markers.5-7 Thus, a
decrease of systemic inflammatory status may be partly
responsible for the reduction of CVD events by the treat-
ment. However, questions remain about the source of the
inflammation and the target tissue of the anti-inflammatory
action of these drugs.
Statins are a class of chemicals that inhibit 3-hydroxy-3-
methyl-glutaryl-coenzyme A reductase, a rate-limiting
enzyme in cholesterol synthesis, and are extensively used
for reduction of cardiovascular events through a well-known
mechanism of lowering LDL-cholesterol. Recent studies
have shown that statins have beneficial pleiotropic effects,
decreasing inflammation.8-10 Many studies show elevated
levels of systemic inflammatory markers such as CRP,
matrix metalloproteinase-9, TNFα and IL-611 in patients
with dyslipidaemia or atherosclerosis.10, 12-15 Fewer studies
have been carried out to examine the effect of statins on adi-
pokines, a group of inflammatory and anti-inflammatory
cytokines secreted by adipose tissue. Obesity, a risk factor
for CVD, is associated with ‘inflamed’ adipose tissue,16 with
decreased expression of the anti-inflammatory adipokine,
adiponectin, and increased secretion of a variety of inflam-
matory cytokines, e.g. TNFα, IL-6 and SAA.17-22
Anti-inflammatory effects of simvastatin
on adipokines in type 2 diabetic patients
with carotid atherosclerosis
Yun Hu1, Guoyu Tong1, Wei Xu1, Jiajia Pan1, Kathy Ryan2,
Rongze Yang2, Alan R. Shuldiner2, Da-Wei Gong2 and Dalong Zhu1
Abstract
Objective: Statins are extensively used for lowering LDL-cholesterol and reducing cardiovascular events. Recent studies
have shown that statins have beneficial anti-inflammatory effects. We aimed to determine whether and how adipokines
are regulated during statin treatment in type 2 diabetic patients.
Method: In this study, we investigated the changes of CRP and inflammation-related adipokines (SAA, IL-6, TNFα and adiponectin)
in 23 type 2 diabetic patients with atherosclerosis who received statin therapy, and 20 diabetic patients with atherosclerosis and
14 diabetic patients without atherosclerosis who did not receive statin therapy for a period of three months.
Results: By the end of the simvastatin treatment (40 mg, daily), LDL-cholesterol was decreased by 16.7% and HDL-
cholesterol was increased by 31.9%. SAA, CRP, TNFα and IL-6 levels were decreased by 31.8%, 66.2%, 53.9% and 14%,
respectively and adiponectin was increased by 59.6%, compared with the baseline levels. Interestingly, the decrease of
SAA was positively correlated with that of LDL-cholesterol but negatively with HDL-cholesterol during statin treatment.
Among the adipokines, the decrease of SAA was positively correlated with TNFα (r = 0.50, p = 0.016).
Conclusion: The results suggest that adipokines may be differentially regulated and independent of cholesterol changes
and that adipokines may be a mediator, and the adipose tissue may be a target of statins’ anti-inflammatory effect.
Keywords
statin, anti-inflammation, adipokines, atherosclerosis, type 2 diabetes
1 Division of Endocrinology, The Affiliated Drum Tower Hospital of
Nanjing University, Nanjing 210008, China
2 Division of Endocrinology, Diabetes and Nutrition Department of
Medicine, University of Maryland School of Medicine, Baltimore, MD
21201, USA
Corresponding author:
Dalong Zhu, Division of Endocrinology, The Affiliated Drum Tower
Hospital of Nanjing University, Nanjing 200021, China.
Email: zhudldr@gmail.com
Original article
Diabetes & Vascular Disease Research
6(4) 262 –268
© The Author(s) 2009
Reprints and permission: http://www.
sagepub.co.uk/journalsPermission.nav
DOI: 10.1177/1479164109339966
http://dvr.sagepub.com
by guest on October 14, 2011dvr.sagepub.com Downloaded from

Page 3

Hu et al.
263
Data on the effect of statins on adipokines in type 2 dia-
betic patients are sparse. Thus, we investigated the effect of
simvastatin on changes in the levels of adipokines in type 2
diabetes patients with atherosclerosis during three months
of statin treatment.
Methods
Study subjects
All human studies were approved by the Ethical Committee
of the Nanjing University Drum Tower Hospital. Human
participants were enrolled in the Nanjing University Drum
Tower Hospital between 2006 and 2007. All subjects had no
history of heart, liver, kidney and lung diseases, and had no
overt acute or chronic infection, trauma or surgery during
the follow-up period. Type 2 diabetes was diagnosed based
on diagnostic criteria of the American Diabetes Associa-
tion.23 Diabetic patients were consecutively recruited and
divided into groups of AS and non-AS and based on carotid
atherosclerotic plaques and IMT) (see below). The AS group
was randomised to either statin or non statin therapy.
Anthropometric measurements including weight, height
and waist measurements were obtained using standardised
techniques. BMI was calculated using the following for-
mula: weight (kg)/height (m2).Venous blood was drawn at
pretreatment baseline and every month after a 12- to 14-hour
overnight fast. HbA1c was measured from whole blood.
Plasma was promptly separated by low-speed centrifuga-
tion at 4ºC and immediately used for measurement of lipid,
lipoprotein and glucose. Aliquots of the plasma specimens
were also immediately frozen at -80ºC for cytokine assays.
Carotid IMT measurement
The carotid ultrasound examination was carried out using a
colour Doppler ultrasound system (HDI-5000, Philips,
Eindhoven, Netherlands) with a scanning frequency of
5–12 MHz in B-mode, following the same protocol through-
out, by a single trained operator blind to the study group.
The flow divider between the internal and external carotid
arteries was identified, and the common carotid arteries
were explored starting 1 cm below the flow divider. Mea-
surements were obtained by tracing the leading edge of the
lumen-intima and the media-adventitia interfaces. Maxi-
mum right and left IMTs were averaged to obtain the carotid
IMT measurement. Subjects with significant carotid plaques
were defined as carotid IMT > 1.2 mm. IMT ≥ 0.9 mm with
or without carotid plaques were defined as having AS.
Statin intervention
A total of 57 type 2 diabetic patients completed the clinical
trial and were divided into two groups of non-AS (n = 14)
and AS (n = 43) by their IMT thickness and plaque pres-
ence. The AS group was further randomly divided into the
statin group of 23 subjects who received 40 mg simvastatin
(Merck), daily for 12 weeks and the control group of 20
subjects without simvastatin treatment.
All patients with diabetes received routine medication
(e.g. insulin, metformin, sulfonylurea) for glucose control.
During the three-month study period, dose adjustments for
diabetes medication were allowed, but there were no
changes in medication.
Measurement of adipokines and inflammation
markers
Serum levels of human high-sensitive CRP were deter-
mined by a highly sensitive nephelometric assay (Kyowa,
Tokyo, Japan). Serum levels of adiponectin, IL-6 and TNFα
(eBioscience, San Diego, CA, USA) and human SAA (Bio-
Source, Camarillo, CA, USA) were measured in duplicate
with enzyme-linked immunosorbent assay kits according to
instructions of the respective manufacturers. The intra-as-
say coefficient of variations of the CRP, adiponectin, IL-6,
TNFα, and SAA measurements were 5.5%, 10%, 2%, 10%
and 7.4%, respectively.
Other laboratory analyses
Lipids in plasma and fractionated lipoproteins were analy-
sed by standard enzymatic methods. HbA1c levels were
measured by high-performance liquid chromatography.
Statistical analyses
Results are expressed as the mean 6 SEM. Non-normally
distributed variables were ln-transformed prior to analysis,
and were compared using the Student’s unpaired or paired
t-test, or analysis of covariance, as appropriate. Differences
were considered to be significant at p < 0.05. Spearman
rank order correlation analyses were used to determine the
relationships between the inflammatory factors and lipo-
protein parameters. Statistical analysis was carried out
using SPSS statistical software.
Results
Basal clinical profile
Study participants were recruited and divided into non-AS
and AS groups; the AS group was further randomised to
statin treatment and no statin treatment. Table 1 shows base-
line clinical characteristics of the study participants. IMT
was the main criterion for the diagnosis of AS and by defini-
tion, was higher in the AS group than the non-AS group.
Prior to intervention, the pooled AS group (n = 43) had no
statistically significant differences in age, sex or BMI from
the non-AS control group (n = 14), except for larger waist
conference (p = 0.03). As for lipids and adipokines, serum
triglyceride level was higher and HDL was lower in the AS
by guest on October 14, 2011dvr.sagepub.com Downloaded from

Page 4

264
Diabetes and Vascular Disease Research 6(4)
Table 1. Baseline anthropometric, metabolic and clinical characteristics of the study groups
















p-value
non-AS
versus AS
pooled (un-
adjusted)
p-value
non-AS
versus AS
pooled
AS**
p-value
AS no
statin
versus
AS+statin
AS no
statin
(n = 20)
Non-AS
(n = 14)
AS+statin
(23)
Pooled
(n = 43)
Male sex (%)
Age (y)
HbA1c (%)
IMT (cm)
BMI (kg/m2)
Waist (cm)
hsCRP (mg/L)
TNFα (pg/ml)
SAA (µg/ml)*
Adiponectin (µg/ml)
IL-6 (pg/ml)
TG (mmol/L)*
TC (mmol/L)
HDL (mmol/L)
LDL (mmol/L)
42.9 70 39.1 53.5 0.60
0.07
0.49
0.24
0.005
0.31
0.006
0.02
0.37
0.10
0.24
0.05
0.03
0.05
0.40
0.61
0.04
0.77
0.47
0.05
0.06
0.29
0.60
0.86
0.17
0.54
0.25
0.42
0.46
0.007
0.26
0.02
0.60
51.6 ± 2.6
8.8 ± 0.7
0.076 ± 0.002
22.8 ± 0.9
83.3 ± 2.7
1.1 ± 0.3
0.065 ± 0.009
16.7 ± 1.8
7.3 ± 0.7
21.8 ± 3.0
1.7 ± 0.3
4.8 ± 0.3
1.2 ± 0.1
2.8 ± 0.2
55.2 ± 2.3
10.2 ± 0.5
0.094 ± 0.005
24.1 ± 0.8
89.4 ± 2.0
3.8 ± 1.4
0.092 ± 0.018
24.1 ± 2.8
4.5 ± 0.4
39.0 ± 5.3
2.0 ± 0.5
4.5 ± 0.2
1.1 ± 0.1
2.5 ± 0.2
58.5 ± 1.6
8.6 ± 0.5
0.097 ± 0.003
24.5 ± 0.6
89.1 ± 1.6
4.3 ± 1.0
0.127 ± 0.035
26.6 ± 2.6
4.8 ± 0.6
38.0 ± 3.5
2.5 ± 0.3
4.8 ± 0.2
0.9 ± 0.0
2.6 ± 0.1
57.0 ± 1.4
9.3 ± 0.4
0.096 ± 0.003 0.0004
24.3 ± 0.5
89.2 ± 1.3
4.1 ± 0.8
0.111 ± 0.021
25.4 ± 1.9
4.7 ± 0.4
38.5 ± 3.1
2.2 ± 0.3
4.6 ± 0.1
1.0 ± 0.1
2.6 ± 0.1
0.11
0.03
0.03
0.08
0.03
0.008
0.02
0.31
0.51
0.03
0.38
*log-transformed values used for significance testing; **p-values adjusted for age, sex and BMI; age p-values adjusted for sex and BMI
only, sex p-values adjusted for age and BMI only, BMI adjusted for age and sex only
group compared with the non-AS group (Table 1). More-
over, serum levels of inflammatory adipokines CRP, SAA
and IL-6 were higher and the anti-inflammatory adipokine
adiponectin level was lower in the AS compared with the
non-AS group (Table 1). The differences in these parame-
ters between AS and non-AS groups remained statistically
significant after adjustment for age and sex, however these
differences, except for adiponectin, were attenuated after
adjustment for BMI (data not shown). After adjustment for
age, sex and BMI, SAA and IL-6 levels were higher whereas
adiponectin and HDL levels were lower in the AS group
than in the non-AS group. There was no significant differ-
ence in IMT and adipokine levels between the AS group
randomised to statin treatment (AS+statin) compared with
the AS group that did not receive statin treatment (AS no
statin), although the AS+statin group was slightly older and
had higher TG and lower HDL levels (Table 1).
Correlations among baseline variables
When data before simvastatin treatment from all three
groups (n = 57) were pooled, the result (shown in Table 2)
provides insight into the relationships among anthropo-
metric and biochemical parameters. As shown in Table 2,
the ln of SAA, lnCRP and IL-6 were correlated well with
BMI (r = 0.42, p < 0.01; r = 0.34, p < 0.01 and r = 0.46, p
< 0.001, respectively) and with lnTG (r = 0.55, p < 0.001,
r = 0.28, p < 0.05 and r = 0.37, p < 0.01, respectively).
Significantly, lnSAA and IL-6 levels are negatively HDL
levels (r = -0.58, p < 0.01 and r = -0.35, p < 0.01, respec-
tively). Interestingly, lnCRP was correlated with BMI,
lnTG and TNFα, but not with SAA; however, the
correlation became significant after adjustment for BMI (r
= 0.27, p < 0.05). No significant correlations were observed
for the level of adiponectin with those of other adipokines
and lipids.
Changes of plasma lipids during simvastatin
treatment
During the treatment period, there were no significant
changes in glucose levels and BMI in any of the groups
(data not shown). We measured changes in cholesterol and
adipokines monthly during the three-month study period.
In the simvastatin group, LDL was decreased by 15.8%
from 2.65 mmol/L to 2.22 mmol/L within the first month
whereas HDL was increased by 28.4% from 0.92 mmol/L
to 1.18 mmol/L in the same period. The LDL and HDL
levels remained at similar levels thereafter. Interestingly,
TG levels were increased by 10% in the first month but
decreased significantly by 30% by the second month and
continued to decrease by an additional 18% by the third
month (Figure 1a). In comparison, no significant changes
were observed in plasma lipids in the no-statin group during
the same period (Figure 2a).
With respect to adipokines, SAA was significantly
decreased by 20.0%, 10.1% and 5%, and CRP level was
decreased by 45.4%, 25.1% and 17.2% each month in the
statin treatment group. Likewise, TNFα and IL-6 levels
were decreased in the same group. Conversely, adiponectin
gradually increased during the treatment (Figure 1b). Over-
all, by the end of the three-month simvastatin treatment,
SAA, CRP, TNFα and IL-6 levels were decreased by 31.8%,
66.2%, 53.9% and 14%, respectively, and adiponectin was
AS no statin and AS+statin (n = 43)
by guest on October 14, 2011 dvr.sagepub.comDownloaded from

Page 5

Hu et al.
265
increased by 59.6 %, compared with the baseline levels. In
contrast, there were no significant changes in adipokine
levels during the same period in the non-statin AS patients
(Figure 2b) or in the non-AS controls (data not shown).
Correlations of changes among lipids and
adipokines during simvastatin treatment
We next analysed the correlations of percentage changes
among plasma lipids and adipokines during simvastatin
treatment in the AS patients. After the first month of simv-
astatin treatment, positive correlations were observed
between the increase of adiponectin and HDL (r = 0.45,
p = 0.031). By the end of the second month of treatment, the
decrease of SAA was correlated with that of LDL (r = 0.53,
p = 0.009) and with the increase of HDL (r = -0.54,
p = 0.007). At the end of the third month of statin treatment,
the correlation of the changes of SAA was no longer cor-
related with LDL but remained negatively correlated with
HDL. Among adipokines, the decrease of SAA was posi-
tively correlated with TNFα (r = 0.50, p = 0.016) but not
with the other adipokines.
Discussion
Diabetic patients have at least a two-fold greater risk of
developing CVDs than the general population.24 Dyslipi-
daemia and systemic inflammation in diabetes may
contribute to the increased CDV risk,25 as atherosclerosis is
considered an inflammatory vascular disease, characterised
Table 2. Spearman correlation analysis of baseline anthropometric and biochemical characteristics in all participants (n=57)
LnSAA LnCRP LnTNFα IL-6 lnAdiponectin
No Adj BMI Adj No Adj BMI Adj No Adj BMI Adj No Adj BMI Adj No Adj BMI Adj
BMI
Waist
Cholesterol
LnTG
LDL
HDL
LnSAA
LnCRP
LnTNFα
IL-6
0.42**
0.44**
−0.05
0.55**
−0.28*
−0.58**




NA
0.30*
−0.2
0.41**
−0.27*
−0.59***




0.34**
0.22
0.07
0.28*
0.05
−0.19
0.3



NA
−0.1
0.04
0.25
0.11
−0.19
0.27*



0.16
0.18
−0.02
0.05
0.07
−0.09
0.2
0.32*


NA
−0.05
−0.07
−0.01
0.01
0.1
0.03
0.29*


0.46***
0.45***
0.14
0.37**
−0.07
−0.35**
0.59**
0.25
−0.09

NA
0.18
−0.02
0.21
−0.11
−0.33*
0.53***
0.21
−0.17

0.02
0.09
−0.07
0.02
−0.01
−0.02
0.16
−0.03
0.07
0.08
NA
−0.08
0.01
0.1
−0.02
−0.04
0.09
−0.07
−0.11
−0.17
*p < 0.05; **p < 0.01; ***p < 0.001. No Adj = not adjusted; BMI adj = adjusted for BMI
0123
0
1
2
3
4
5
6
TG
TC
HDL
LDL
**
**
**
**
**
**
** ***
***
***
Simvastatin treatment group
A
Months
Concentration (mM)
B
CRP
TNFa
SAA
Adiponectin
IL-6
Months
0123
3
8
13
18
23
28
33
38
43
*
*
**
**
**
**
**
**
***
***
*
***
Simvastatin treatment group
Concentration
Figure 1. Changes of lipids and adipokines during simvastatin treatment. Levels of (A) TG (mmol/L), total cholesterol (mmol/L),
HDL and LDL (mmol/L) and (B) SAA (µg/ml), TNFα (pg/ml × 100), IL-6 (pg/ml), high-sensitive CRP (mg/L) and adiponectin (µg/
ml) were measured before, one, two and three months after simvastatin treatment (40 mg/day) in type 2 diabetic patients with
carotid atherosclerosis. Data are expressed as mean ± SEM (ln-transformed for analysis, back-transformed for presentation).
*p < 0.05, **p < 0.01, ***p < 0.001
by guest on October 14, 2011 dvr.sagepub.comDownloaded from