Vaspin Serum Concentration in Patients with Type 2 Diabetes and Carotid Plaque

Abstract

Objective:

To examine whether there is an association between the serum concentration of vaspin and the presence of carotid plaque in early stage type 2 diabetes mellitus (T2DM).

Methods:

Patients (n = 61) with T2DM within 3 years of diagnosis were divided into those with and those without carotid plaque. Fasting serum vaspin levels, measured by enzymelinked immunosorbent assay, and blood pressure were compared between these two groups and also with an age-matched, apparently healthy control group (n = 26).

Results:

Fasting serum vaspin concentrations were significantly higher in patients with T2DM without carotid plaque than in controls, but significantly lower in T2DM patients with carotid plaque than inthose without. Multivariate logistic regression analysis showed a significant positive association between the presence of carotid plaque and systolic blood pressure and a significant inverse association between the presence of carotid plaque and fasting serum vaspin concentration.

Conclusion:

A significant inverse association was found, in patients with T2DM within 3 years of diagnosis, between serum vaspin concentration and the presence of carotid plaque.

Introduction

Type 2 diabetes mellitus (T2DM) is one of a cluster of risk factors for atherosclerotic vascular disease. Patients with T2DM have markedly higher rates of mortality and morbidity due to cardiovascular disease compared with nondiabetic subjects. ¹

The many cytokines released by adipose tissue (adipokines) regulate body weight homeostasis as well as inflammation, fibrinolysis, insulin resistance and diabetes, and are involved in the pathogenesis of atherosclerosis.^2,3 Both the degree of obesity and the location of fat deposits are risk factors for atherosclerosis. ⁴ Visceral adipose tissue has been reported to be more closely associated than total body fat with the risk of insulin resistance, hypertension, dyslipidaemia and cardiovascular disease.^5,6 This may be due to increased production of adipokines, mainly from visceral fat.^7,8

Vaspin is an adipokine with insulin-sensitizing effects and is predominantly secreted by visceral adipose tissue. It was reported to decrease with worsening diabetes and body weight loss in a rat model of T2DM. ⁹ Klöting et al. ¹⁰ showed that vaspin mRNA expression in human adipose tissue is specific to fat depots and is related to parameters of obesity, insulin resistance and glucose metabolism in adults. Seeger et al. ¹¹ noted that glomerular filtration rate and C-reactive protein levels are independently associated with vaspin. Another study reported that diabetic women with good glycaemic control had a lower level of vaspin than those with poor glycaemic control, but that microvascular complications were associated with low vaspin levels. ¹² These findings demonstrate that vaspin may be involved in the development of cardiovascular disease.

To our knowledge, the level of circulating vaspin in patients with diabetes who also have subclinical macrovascular disease has not been previously investigated. The present study, therefore, investigated the level of circulating vaspin in relation to intima– media thickness of the carotid artery, anthropometric parameters and other atherosclerotic risk factors.

Patients and methods

Study Participants

Inpatients and outpatients with T2DM who presented within 3 years of first diagnosis at the diabetes clinic of the Fourth People's Hospital of Jinan City, China between November 2011 and March 2012 were recruited for this study. The diagnosis of T2DM was according to World Health Organization criteria (1999). ¹³ Patients were excluded if they had other types of diabetes, liver disease, clinical evidence of cardiovascular or peripheral vascular disease, thyroid dysfunction, complications of diabetic proliferative retinopathy or macroalbuminuria (> 300 mg/day), chronic renal diseases (> 120 μmol/l of serum creatinine), infection, malignancy, recent major surgery or illness, or if they were receiving treatment with steroids or lipid-lowering drugs. Standard lifestyle modifications (exercise and dietary changes) had been adopted by each patient about 1 month before inclusion.

The patients were divided into two groups according to whether carotid plaque was present or absent (see below). There were no differences in treatment for diabetes or hypertension between the two groups.

Also recruited were age-matched, apparently healthy controls, who were attending the Fourth People's Hospital of Jinan City, China for routine examination, showed normal glucose tolerance and had no family history of diabetes.

The study was approved by the Ethics Committee of the Fourth People's Hospital of Jinan City and written informed consent was obtained from all study participants.

Clinical and Biochemical Assessments

All patients underwent a complete physical examination and routine biochemical analyses of blood. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured with a manual sphygmo -manometer after the patient had rested for 5 min. Body mass index (BMI) was calculated as body weight in kilograms divided by the square of height in metres. Hip and waist circumferences were measured and the waist-to-hip ratio was calculated. The homeostatic model assessment for insulin resistance (HOMA-IR) was calculated by the steady-state model formula: ¹⁴ HOMA-IR = FBG (mmol/l) × FINS (mU/l) / 22.5, where FBG is the fasting blood glucose level and FINS is the fasting insulin level.

Blood was collected from each participant after an overnight fast and serum was obtained by centrifugation and kept at –80 °C until it was assayed. Fasting blood glucose, total cholesterol, triglyceride, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol were determined with an automatic biochemical analyser. Glycosylated haemoglobin (HbA_1c) was detected by cation exchange column chromatography. Serum vaspin concentrations were measured by enzyme-linked immunosorbent assay (AdipoGen, Seoul, South Korea; minimum detection level 0.08 ng/ml, intra- and interassay coefficients of variation < 5% and 14%, respectively). All samples were analysed at the Key Laboratory of Cardiovascular Remodelling and Function Research Centre, Qilu Hospital, Shandong University, Jinan, China.

Carotid artery status in all patients was examined on the same day as blood collection using the same high-resolution B-mode ultrasonograph (iU22; Philips Healthcare, Andover, MA, USA) and by the same operator. The ultrasound devices were used with linear transducers (5 – 12 MHz). Subjects were placed in a supine position with the neck hyperextended and the head turned slightly to the contralateral side. The common carotid artery, the carotid artery bifurcation and the bilateral internal carotid artery were assessed for the presence of carotid plaque. Plaque was defined as a localized intima–media thickness  1.3 mm with marked protuberance; a thickness < 0.9 mm was considered plaque-negative. All measurements were made by trained ultrasonographers.

Statistical Analyses

The mean ± SD was calculated for continuous variables. Before statistical analysis, non-normally distributed parameters were logarithmically transformed to approximate a normal distribution. Statistical analyses were performed using the χ²-test, Student's t-test or Pearson's correlation coefficient (r), as appropriate. Logistic forward regression analysis was used to calculate the odds ratios of each variable for carotid plaque. A multivariate logistic regression model (β-value), in which carotid plaque was taken as the dependent variable, was applied to all patients with T2DM. All statistical analyses were performed with the SPSS^® statistical package, version 13.0 (SPSS Inc., Chicago, IL, USA). All statistical tests were two-sided and a P-value < 0.05 was considered statistically significant.

Results

Study Participants

This study enrolled 61 consecutive patients (33 males, 28 females) and 26 age-matched, apparently healthy controls (15 males, 11 females).

Clinical and Biochemical Characteristics

Demographic, clinical and biochemical data for the patients and controls are shown in Table 1. Age and gender distribution were similar in the three subgroups (controls, diabetic patients with carotid plaque and diabetic patients without carotid plaque). BMI, SBP, and the levels of fasting blood glucose, total cholesterol, low-density lipoprotein cholesterol and triglyceride were significantly higher in the T2DM patients than in the controls (P < 0.01). Fasting serum vaspin concentration in the diabetic patients (with or without plaque) was also significantly higher than in the controls (P < 0.05). High-density lipoprotein cholesterol was significantly lower in the T2DM patients than in the controls (P < 0.01).

TABLE 1:

Demographic clinical and biochemical characteristics, including the fasting serum vaspin concentrations, in the type 2 diabetes mellitus patients, with or without carotid plaque, and control subjects included in the present study

Characteristics	Control subjects	All diabetic patients	Diabetic patients, plaque absent	Diabetic patients, plaque present
Number of patients, n	26	61	28	33
Age, years	55.38 ± 9.49	59.31 ± 9.67	57.07 ± 9.83	61.21 ± 9.26
Gender, males/females, n	15/11	33/28	15/13	18/15
Duration of diabetes, months	–	23.64 ± 8.38	24.18 ± 9.15	23.16 ± 7.80
Family history of CVD, %	15.4	31.2	21.4	39.4c,d
Current smoker, %	19.2	29.5	24.2	30.3
Hypertension, % a	19.2	62.3 c	46.4	75.8c,d
Body mass index, kg/m2	23.02 ± 3.27	26.07 ± 3.58 c	26.75 ± 3.73 c	25.50 ± 3.40 b
Waist circumference, cm	81.42 ± 9.90	92.44 ± 9.36 c	93.12 ± 9.65	91.90 ± 9.12
Waist-to-hip ratio	0.87 ± 0.08	0.90 ± 0.06	0.89 ± 0.05	0.90 ± 0.07
Systolic blood pressure, mmHg	120 ± 14	144 ± 20 c	136 ± 19 c	151 ± 18 c , e
Diastolic blood pressure, mmHg	76 ± 10	83 ± 9	85 ± 8 c	82 ± 10 b
Fasting blood glucose, mmol/l	4.94 ± 0.50	8.44 ± 2.45 c	7.83 ± 2.38 c	8.96 ± 2.42 c , d
HbA1c′ %	ND	8.13 ± 1.55	7.65 ± 1.59	8.53 ± 1.41 d
Fasting insulin, μU/ml	ND	14.06 ± 4.98	13.05 ± 4.41	14.93 ± 5.34 d
HOMA-IR	ND	5.22 ± 2.17	4.54 ± 2.05	5.79 ± 2.15 d
Total cholesterol, mmol/l	4.35 ± 0.51	5.38 ± 0.96 c	5.09 ± 0.81 c	5.62 ± 1.02 c , d
Triglycerides, mmol/l	0.99 ± 0.30	1.69 ± 0.65 c	1.61 ± 0.73 c	1.75 ± 0.58 c
HDL cholesterol, mmol/l	1.36 ± 0.25	1.04 ± 0.25 c	1.05 ± 0.26 c	1.02 ± 0.24 c
LDL cholesterol, mmol/l	2.55 ± 0.33	3.59 ± 0.92 c	3.37 ± 0.75 c	3.77 ± 1.01 c
Fasting serum vaspin, ng/ml	0.46 ± 0.12	0.59 ± 0.24 b	0.65 ± 0.24 c	0.52 ± 0.23 d

Data presented as mean ± SD unless otherwise stated.

Demographic data were analysed by the Student's t-test for numerical variables; categorical data were analysed by the χ²-test.

a

^aSystolic blood pressure  140 mmHg and/or diastolic blood pressure  90 mmHg, or using antihypertensive treatment to control blood pressure.

b

P < 0.05 versus control

c

P < 0.01 versus control

d

P < 0.05 versus patients without plaque

e

P < 0.01 versus patients without plaque.

CVD, cardiovascular disease; HbA_1c, glycosylated haemoglobin; HOMA-IR, homeostatic model assessment of insulin resistance; HDL, high-density lipoprotein; LDL, low-density lipoprotein; ND, not determined.

Amongst patients with T2DM, there were no significant differences in smoking habit, BMI, waist circumference and waist-to-hip ratio between the groups with and without plaque. The prevalences of a family history of cardiovascular disease and hypertension were higher amongst T2DM patients with plaque than in those without plaque (P < 0.05). Patients with plaque had a significantly higher systolic blood pressure (P < 0.01), and significantly higher serum levels of total cholesterol, fasting blood glucose, HbA_1c and fasting insulin than those without plaque (P < 0.05).

The fasting serum vaspin level was significantly higher in T2DM patients without plaque than in controls (P < 0.01), but was significantly lower in T2DM patients with plaque than in those without plaque (P < 0.05).

Correlation Analyses

Correlation analyses undertaken on all participants showed that the fasting serum concentration of vaspin was positively correlated with BMI (r = 0.242, P = 0.024), waist circumference (r = 0.323, P = 0.002) and triglyceride level (r = 0.263, P = 0.014). No significant inverse associations were observed. In patients with diabetes, bivariate correlation analysis showed that the fasting serum vaspin level correlated significantly with waist circumference (r = 0.371, P = 0.003) and HOMA-IR (r = –0.272, P = 0.034).

Multivariate Logistic Regression Analysis

Multivariate logistic regression analysis was applied to data from all the T2DM patients, with carotid plaque as the dependent variable. The analysis showed a significant positive association between the presence of carotid plaque and SBP (β = 0.054, P < 0.05) and a significant inverse association between the presence of carotid plaque and fasting serum vaspin concentration (β = –4.234, P < 0.05).

Discussion

Obesity, especially visceral fat, may play a crucial role in the early phase of subclinical macrovascular disease. ¹⁵ Accumulation of visceral fat is associated with early carotid atherosclerosis (also called preclinical carotid artery changes) and is assessed by measuring intima–media thickness. ¹⁶

The vascular endothelium plays a major role in maintaining cardiovascular homeostasis. Increasing evidence indicates that there is a strong interaction between diabetes, adipokines and the endothelium. The normal regulation of adipose factors is perturbed in obesity or diabetes. Adipose tissue can produce several compounds able to affect endothelial function, including leptin, resistin, adiponectin, tumour necrosis factor-α, interleukin 6, monocyte chemoattractant protein 1 and plasminogen activator inhibitor 1. ¹⁷ Adipokines may play multiple roles in the inflammatory process of atherosclerosis and may contribute to the development of endothelial dysfunction. ¹⁸ Endothelial dysfunction may precede the development of overt diabetes ¹⁹ and initiate progressive atherosclerosis.

There is debate in the literature on the role of vaspin. ²⁰ An animal study suggested that vaspin expression increases with increasing body weight and circulating insulin levels in the visceral adipose tissue of Otsuka Long– Evans Tokushima Fatty rats, an animal model of T2DM that is characterized by abdominal obesity, insulin resistance, hypertension and dyslipidaemia. ⁹ Vaspin was highly expressed when obesity, body weight and insulin levels peaked, and its serum level decreased when diabetes worsened and body weight was lost. ⁹ Youn et al. ²¹ showed that there was no difference in circulating vaspin levels between individuals with normal glucose tolerance and patients with T2DM of different durations. Kim et al. ²² reported that a 10-month lifestyle modification programme in patients with metabolic syndrome did not lead to favourable changes in vaspin concentrations. Aust et al. ²³ found that serum concentrations of vaspin were significantly lower in patients with carotid stenosis who had experienced a recent ischaemic event compared with asymptomatic patients, and that the more recent the ischaemic event the lower the vaspin level. A further two studies demonstrated that serum levels of vaspin were significantly lower in subjects with coronary artery disease (CAD) than healthy controls, and that low vaspin concentrations seemed to correlate with CAD severity.^24,25

The present study indicated that, in the early stage of T2DM, serum vaspin concentration in patients without carotid plaque was significantly higher than in healthy controls and significantly higher than in patients with carotid plaque. The serum concentration of vaspin may, therefore, be associated with the process of carotid plaque development in the early stage of T2DM. We postulate that the increased vaspin production in human adipose tissue in the early stage of T2DM may be a compensatory mechanism associated with obesity, severe insulin resistance and T2DM, and that vaspin could serve as a novel marker and a protective factor for macrovascular lesions. As the compensatory capacities of vaspin secretion gradually decline with prolonged duration of diabetes or cardiovascular disease and the aggravation of vascular sclerosis, the levels of vaspin slowly decline, as reported elsewhere.^9,23–25

There was no significant difference in the duration of diabetes between the two subgroups of patients. The difference in the extent of carotid atherosclerosis between the two subgroups may have been due to poor glycaemic, blood pressure and lipid control in the group with plaque, or to late diagnosis of diabetes in some subjects in this group. The known duration of diabetes may not reflect the real duration of the disease in some patients. ²⁶ Poor control of several risk factors for atherosclerosis might shorten the compensatory stage of vaspin upregulation, and decreased production of vaspin may contribute to accelerated progression of atherosclerosis.

In conclusion, the results of the present study demonstrated a significant association between the serum concentration of vaspin and the presence of carotid plaque in patients with T2DM. The exact mechanism of action of vaspin remains to be clarified.