Relationship of Selected Adipokines with Markers of Vascular Damage in Patients with Type 2 Diabetes

Abstract

Background:

In this study we compared levels of selected adipokines between patients with type 2 diabetes (T2D) and healthy individuals and we determined their relationship with early vascular damage markers.

Methods:

Seventy-seven subjects: 56 patients with T2D (34 men and 22 women) and 21 healthy controls (8 men and 13 women) were examined in this cross-sectional study. Selected adipokines [adiponectin, adipocyte fatty acid-binding protein (A-FABP), fibroblast growth factor 21 (FGF-21), C1q/TNF-related protein 9 (CTRP-9), and allograft inflammatory factor-1 (AIF-1)] with possible cardiovascular impact were measured in all participants. To identify markers of vascular damage von Willebrand factor (vWF), plasminogen activator inhibitor-1 (PAI-1) and arterial stiffness parameters were examined in all the subjects.

Results:

When compared with healthy controls, T2D had significantly higher levels of A-FABP [50.0 (38.1–68.6) vs. 28.6 (23.6–32.9) ng/mL, P < 0.0001] and lower levels of adiponectin [5.9 (4.3–9.0) vs. 11.3 (8.7–14.8) μg/mL, P < 0.0001]. Differences in other adipokines were not statistically significant. Adiponectin level correlated negatively with vWF levels (ρ = −0.29, P < 0.05) and PAI-1 (ρ = −0.36, P < 0.05) and A-FABP positively with vWF (ρ = 0.61, P < 0.05) and PAI-1 (ρ = 0.47, P < 0.05) and augmentation index (ρ = 0.26, P < 0.05). Multivariate regression analysis showed independent association between A-FABP and vWF (b = 0.24, P < 0.05).

Conclusions:

Patients with T2D have significantly higher levels of A-FABP and lower levels of adiponectin. These adipokines correlate with indicators of vascular damage and could contribute to cardiovascular risk in patients with T2D. A-FABP may participate in direct endothelium damage.

Introduction

Cardiovascular disease (CVD) is the primary cause of death in patients with type 1 and type 2 diabetes (T2D).¹ In addition to obesity and metabolic syndrome, diabetes is often associated with the pathological function of adipose tissue. Adipose tissue can be considered as a huge gland producing paracrine and endocrine hormones, adipokines. There is growing evidence that these adipokines may link obesity to CVD.² Increased arterial stiffness also leads to a higher risk of CVD. A number of studies have shown an escalation in arterial stiffness in patients with type 1³ and type 2 diabetes.^4,5 Increased arterial stiffness may be one important pathway linking diabetes to the reported increase in cardiovascular risk of this disease.⁶

There are a number of potential mechanisms by which diabetes might affect arterial stiffness–insulin resistance, advanced glycation end products, inflammatory process, oxidative stress, or metabolically dysfunctional adipose tissue.⁷ Adipokines have been recognized as adipocyte proteins that link obesity with metabolic and vascular disease.⁸ Adipokines are peptides that signal the functional status of adipose tissue to target organs and tissues and they are promising candidates for both novel pharmacological treatment strategies and diagnostic tools.⁹

Proinflammatory adipokines (adipocyte fatty acid-binding protein (A-FABP), plasminogen activator inhibitor-1 (PAI-1), tumor necrosis factor alfa, interleukin 6, resistin, leptin, and monocyte chemoattractant protein) exert an adverse effect on the vasculature by promoting insulin resistance and monocyte infiltration into the vessel wall,¹⁰ contributing to oxidative stress, chronic inflammation, and endothelial damage. By contrast, adiponectin and fibroblast growth factor 21 (FGF-21) improves glucose and lipid metabolism.

Selected adipokines [adiponectin, A-FABP, FGF-21, C1q/TNF-related protein 9 (CTRP-9), and allograft inflammatory factor-1 (AIF-1)] with possible cardiovascular impact were measured in our study. Main functions and characteristics of selected adipokines are summarized in the Table 1.

Table 1.

Main Characteristics of Selected Adipokines

Adipokines	Characteristics, effects and functions
Adiponectin	Plays a major role in glucose and lipid metabolism.
	Prevents development of cardiovascular changes due to its antioxidative, anti-inflammatory, endothelium-protective, and antiapoptotic effects.^11,12
	Inhibits the production of inflammatory cytokines and adhesion molecules, including ICAM-1, VCAM-1, and E-selectin.¹³
	Is a marker that can predict insulin resistance in patients with type 2 diabetes and may have an important role in the pathogenesis of diabetes.¹⁴
A-FABP	Its main function is binding of free fatty acids.
	Increases lipolysis and insulin resistance, reduces contractility of cardiomyocytes, promotes chronic inflammation and formation of vulnerable atherosclerotic plaques.¹⁵
	Has recently been identified as a biomarker for the development of metabolic syndrome based on insulin resistance.¹⁶
FGF-21	Is considered as a metabolic regulator of noninsulin-dependent glucose transport in cells.
	Improves insulin sensitivity, glucose and lipid homeostasis, and preserves beta-cell functions in diabetic animal models.^17 –19
	FGF-21 elevation may indicate a compensatory response to metabolic stress or a resistance to FGF-21.^20,21
	Is an independent factor for the protection of cardiovascular system cells.^21,22
	Is increased in metabolic diseases such as obesity, diabetes, and cardiovascular disease.^23,24
CTRP-9	Is the closest adiponectin paralog.²⁵
	Improves glucose metabolism and promotes insulin sensitivity in mouse models.^26,27
	Has beneficial cardiovascular effects by vascular relaxation²⁸ and modulation of vascular smooth muscle cell proliferation.²⁹
	Is a protective factor for coronary artery disease³⁰ and is associated with increased arterial stiffness.³¹
AIF-1	Promotes macrophages, vascular smooth muscle cells, and endothelial cells activation, proliferation, and migration.³²
AIF-1	Correlates with clinical and biochemical metabolic parameters and is associated with atherogenesis.^22,32

A-FABP, adipocyte fatty acid-binding protein; AIF-1, allograft inflammatory factor-1; CTRP-9, C1q/TNF-related protein; FGF-21, fibroblast growth factor 21; ICAM-1, intercellular adhesion molecule 1; VCAM-1, vascular cell adhesion protein 1.

The aim of this pilot study was to compare selected adipokines levels in patients with T2D and in healthy individuals and to determine their relationship to the indicators of early vascular damage such as markers of endothelial dysfunction and of arterial stiffness.

Methods

Study design

The study was undertaken as a cross-sectional study with T2D patients and healthy volunteers. T2D patients were monitored in the Diabetes Centre of the third Department of Internal Medicine, University Hospital Olomouc, Czech Republic. All participants filled in a questionnaire about their previous medical history, especially cardiovascular status, medication, diabetic complications, and duration. Body mass index (BMI), waist circumference, systolic and diastolic blood pressure (SBP and DBP) measurements, and complete physical examination were realized. In all participants, we examined the arterial stiffness by applanation manometry: augmentation index (AIx), augmentation index normalized for heart rate of 75 bpm (AIx 75/min), augmentation pressure, aortic systolic pressure (SP), aortic pulse pressure, and pulse wave velocity (PWV).

Besides adipokines, lipids, anthropological parameters, indicator of insulin resistance markers of endothelial dysfunction– von Willebrand factor (vWF), and plasminogen activator PAI-1 were tested. Exclusion criteria were as follows: type 1 diabetes, secondary or genetic type of diabetes, acute infection, or trauma. Hypertension was defined as a sitting blood pressure above 140/90 mmHg, taken as a mean of three readings or on regular antihypertensive medications. Diabetes was defined as fasting plasma glucose ≥7 mM or use of oral antidiabetic drugs and/or insulin. The study was reviewed and approved by the Institutional Ethics Committee of medical Faculty and University Hospital Olomouc and informed consent was obtained from all participants.

Subjects

Individuals (77 subjects) formed two groups: 56 patients with T2D (age 48.7 ± 10.7 years, 34 men, 22 women, average diabetes duration 9.6 ± 8.5 years) and 21 healthy controls (age 42.1 ± 10.3 years, 8 men, and 13 women). The control group consisted of healthy volunteers with no medication and without hypertension or diabetes.

According to the glycated hemoglobin (HbA1c≤64 mmol/mol) 46% of T2D patients had well-controlled diabetes. In our study there were 33% T2D patients with HbA1c≤53 mmol/mol, which is an indicator to change the antidiabetic therapy, and 12% patients with HbA1c≤45 mmol/mol (the target HbA1c).

In our study, 84% of diabetic patients met the criteria of metabolic syndrome according to the International Diabetes Federation and European Association for the Study of Diabetes. There were 63% obese patients with T2D (BMI >30 kg/m²), 9% had normal weight (BMI <25 kg/m²), 28% were overweight (BMI = 25–29.9 kg/m²), 33% had 1.class obesity (BMI = 30–34.9 kg/m²), 25% suffer from 2.class obesity (BMI = 35–39.9 kg/m²), and 5% had severe obesity (BMI >40 kg/m²). In the control group there were 62% participants with normal weight and 38% subject were overweight.

All patients with T2D were treated by insulin and/or oral antidiabetics. Seventy seven percent patients were treated by insulin, 80% were treated by oral antidiabetics. The most commonly used oral antidiabetic was metformin (84%), then incretins (both glucagon-like peptide analogs and dipeptidyl peptidase-4 inhibitors - 38%), gliflozines (11%), and only two patients used sulfonylureas (4%). In T2D group 61% subjects were treated by hypolipidemic therapy (59% used statin and 18% fenofibrates). In our study 80% of diabetics were treated for hypertension (57% used angiotensin-converting-enzyme inhibitors, 20% sartans, 41% beta-blockers, 39% calcium channel blockers, and 39% used diuretics).

There were 26% smokers in the T2D group and only 10% in the control group.

Arterial stiffness measurement

Parameters associated with arterial stiffness were estimated using the SphygmoCor System (AtCor Medical Pty Ltd Head Office, West Ryde, Australia), currently accepted as the gold standard for measuring arterial stiffness. SphygmoCor System measurements were performed on patients in a sitting position and resting their arm on a rigid surface. Pulse wave analysis was performed with the sensor in the radial artery, using mathematical transformation to estimate the aortic pulse wave. PWV was measured with the patient in the supine position. The pulse wave of carotid and femoral arteries was analyzed, its delay with respect to the electrocardiogram wave was estimated and the PWV was calculated. Distances were measured using a measuring tape from the sternal notch to the carotid and femoral artery at the sensor location.

Laboratory analyses

Venous blood samples were drawn in the morning after 12–hr fasting period. Routine serum biochemical parameters were analyzed in the day of blood collection, concentrations of adipokines were measured in the sample aliquots stored at −80°C. Total cholesterol, triglycerides (TG), and high-density lipoprotein cholesterol (HDL–C) were determined enzymatically on Cobas 8000 analyzer (Roche, Manheim, Germany) using commercially available kits (Roche). HDL-C was measured by direct method without precipitation of apoB containing lipoproteins. Low density lipoprotein cholesterol (LDL–C) levels were calculated using Friedewald formula. Non-HDL-cholesterol (non-HDL–C) was calculated as total cholesterol–HDL-C. Concentration of apoB was determined immunoturbidimetrically using Tina-Quant Apo B and Apo A1 kits (Roche).

Glycated hemoglobin levels were measured by ion exchange chromatography using Arkray Adams HA-8180 V analyzer (Arkray Corporation, Kyoto, Japan). Glycemia was measured by the enzymatic hexokinase method (Roche). C-peptide was determined by commercially available kits (Immunotech, Marseille, France) using specific antibodies by the IRMA method. High sensitive C-reactive protein (Hs-CRP) was assessed by means of an ultra-sensitive latex immunoturbidimetric method (CRP latex TInaQuant kit; Roche). In measuring of all adipocytes ELISA (enzyme-linked immunosorbent assay) kits (BioVendor laboratory Medicine, Inc., Brno, Czech Republic) were used. FGF-21 levels were determined using Human FGF-21 ELISA kits, A-FABP was determined in serum by ELISA according to the manufacturer's instructions and adiponectin was measured by Human Adiponectin ELISA kit, competitive enzymoimunoanalytical method. For quantitative measurement CTRP-9 commercial sets CTRP-9 ELISA were used. Quantitative values of AIF-1 were obtained by the ELISA method. The following endothelial/hemostatic markers were examined: vWF by immuniturbidimetric assay (Instrumentation Laboratory Spa, Milan, Italy) and PAI-1 by ELISA (Technoclone, Vienna, Austria).

Statistical analysis

All values were expressed as means ± standard deviation (SD), or as median (1st–3rd quartile of values) for parameters with non-normal distribution. The Shapiro–Wilk's test was used to test for normal distribution. Differences in variables between the groups were analyzed with ANOVA after the adjustment for age and sex. Spearman correlation analyses tested univariate correlations between parameters. Multivariate regression analyses were used for testing for an independent association between dependent and independent variables. Non-normally distributed variables were logarithmically transformed before analyses.

Results

Basic characteristic

The basic characteristics of investigated groups are summarized in the Table 2. Patients with T2D had significantly higher body weight, BMI; and waist circumference. Compared with healthy controls there were no significant differences in SBP and DBP. Hs-CRP was significantly higher in T2D. Levels of TG were significantly higher and HDL-C levels were significantly lower in diabetic group. Differences in total cholesterol, LDL-C, non-HDL-C, and apolipoprotein B levels did not reach the statistical significance due to hypolipidemic therapy in diabetics. Levels of glycated hemoglobin (HbA1c), C-peptide was significantly higher in T2D group, as expected.

Table 2.

Characteristics of Investigated Groups

	Type 2 diabetes N (female/male) = 56 (22/34)	Healthy controls N (female/male) = 21(13/8)	Statistical significance p^b
weight (kg)	94.5 ± 15.8	71.2 ± 12.1	<0.001
BMI (kg/m²)	31.3 ± 6.2	22.5 ± 5.5	<0.001
waist circumference (cm)	111 (94–131.5)	84.5 (78–89)	<0.001
SBP (mmHg)	135.0 ± 16.6	125 ± 12.8	NS
DBP (mmHg)	79.6 ± 11.3	78.2 ± 8.9	NS
hs-CRP (mg/L)^a	3.8 (1.2–7.5)	1.2 (0.6–2.3)	<0.01
total cholesterol (mM)^a	4.3 (3.9–4.9)	5.12 (4.7–5.5)	NS
TG (mM)^a	1.8 (1.3–2.7)	0.8 (0.7–1.3)	<0.05
HDL-C (mM)^a	1.0 (0.8–1.3)	1.8 (1.4–2.2)	<0.0001
LDL-C (mM)^a	2.2 (1.8–2.9)	3.1 (2.3–3.2)	NS
non-HDL-C (mM)^a	3.2 (2.7–3.9)	3.6 (2.7–4.0)	NS
apolipoprotein B (g/L)^a	1.0 (0,8–1,0)	0.9 (0,8–1,0)	NS
HbA1c (mmol/mol)^a	68.5 (49.0–92.0)	33.0 (31.0–35.0)	<0.001
C peptide (pM)^a	813 (556–1117)	387 (334–595)	<0.05
FPG (mM)^a	8.3 (6.9–11.4)	4.5 (4.3–4.7)	NS
vWF (%)^a	177.5 (142.0–202.0)	112.0 (93.0–160.0)	<0.01
PAI-1 (ng/mL)^a	78.0 (41.0–106.0)	36.9 (27.0–42.0)	<0.001
AIx (%)^a	22.5 (17.0–32.0)	15.0 (9.0–25.0)	NS
AIx 75/min (%)^a	22.0 (14.0–28.0)	11.0 (-1.0–19.0)	<0.05
AP (mmHg)^a	10.0 (6.0–14.0)	5.0 (3.0–8.0)	NS
Aortic SP (mmHg)^a	122.0 (111.0–132.0)	105 (101–122)	NS
Aortic PP (mmHg)^a	39.0 (31.0–47.0)	31.0 (26.0–37.0)	NS
PWV(m/s)^a	9.0 (7.4–10.1)	7.1 (6.4–8.6)	NS

Data are expressed as mean ± standard deviation.

Non-normal data are expressed as median (25th–75th percentile values).

Differences in variables between groups were analyzed with ANOVA after the adjustment for age and sex.

AIx, augmentation index; AIx 75/min, augmentation index normalized for heart rate of 75 bpm; AP, augmentation pressure; BMI, body mass index; DBP, diastolic blood pressure; FPG, fasting plasma glucose; HbA1c, glycated haemoglobin; HDL-C, high density lipoprotein cholesterol; hs-CRP, high sensitive C-reactive protein; LD-C, low density lipoprotein cholesterol; non-HDL-C, nonhigh density lipoprotein cholesterol; PAI, plasminogen activator inhibitor; PP, aortic pulse pressure; PWV, pulse wave velocity; SBP systolic blood pressure; SP, systolic pressure; TG, triglycerides; vWF, von Willebrand factor; NS, not statistically significant, P > 0.05.

Vascular damage markers

Compared with healthy controls, PAI and vWF levels, as the indicator of endothelial dysfunction, were significantly higher in T2D patients.

Only augmentation index normalized for heart rate of 75 bpm was significantly higher in T2D patients. Differences in other parameters of arterial stiffness (aortic SP, pulse pressure, and PWV) were not statistically significant. Results are presented in Table 2.

Adipokines

Compared with healthy controls, T2D patients had significantly higher levels of A-FABP and lower levels of adiponectin–see Table 3. Although there was a trend to find higher levels of FGF-21 and lower levels of CTRP-9 between the monitored groups, those differences were not statistically significant. There was no statistical difference in AIF-1 levels.

Table 3.

Adipokines Characteristics of Investigated Groups

	Type 2 diabetes N (female/male) = 56 (22/34)	Healthy controls N (female/male) = 21(13/8)	Statistical significance p^b
Adiponectin^a (μg/mL)	5.9 (4.3–9.0)	11.3 (8.7–14.8)	<0.0001
A-FABP^a (ng/mL)	50.0 (38.1–68.6)	28.6 (23.6–32.9)	<0.0001
FGF-21 (pg/mL)	545.5 (348.1–847.9)	267.9 (68.0–389.6)	NS
CTRP-9^a (pg/mL)	41.2 (40.4–297.1)	198.6 (43.2–1781.2)	NS
AIF-1^a (pg/mL)	773.0 (619.3–922.2)	673.1 (566.7–851.5)	NS

Data are expressed as mean ± standard deviation.

Non-normal data are expressed as median (25th–75th percentile values).

Differences in variables between groups were analyzed with ANOVA after the adjustment for age and sex.

A-FABP, adipocyte fatty acid-binding protein; AIF-1, allograft inflammatory factor-1; CTRP-9, C1q/TNF-related protein; FGF-21, fibroblast growth factor 21; NS, not statistically significant, P > 0.05.

Association between adipokines, clinical and laboratory parameters

Spearman correlation analysis showed statistically significant (P < 0.05) correlations between adipokines and anthropometric and metabolic parameters (Table 4). From the vascular damage markers, vWF correlated with adiponectin (ρ = −0.29) and A-FABP (ρ = 0.61), PAI-1 correlated with adiponectin (ρ = −0.36), A-FABP (ρ = 0.47), and FGF-21 (ρ = 0.27). Statistically significant correlation was found between A-FABP and parameters describing arterial stiffness: augmentation pressure (ρ = 0.28), AIx (ρ = 0.26), and AIx 75/min (ρ = 0.39). Detailed description of the correlation is shown in Table 4.

Table 4.

Spearman Correlation Analysis Between Adipokines and Other Clinical and Laboratory Parameters in All Participants

	Adiponectin	A-FABP	FGF-21	CTRP-9	AIF-1
Age	ρ = −0.25, P < 0.05	ρ = 0.43, P < 0.05	NS	NS	NS
Weight	ρ = −0.46, P < 0.05	ρ = 0.46, P < 0.05	NS	NS	ρ = 0.24, P < 0.05
BMI	ρ = −0.31, P < 0.05	ρ = 0.56, P < 0.05	NS	NS	NS
Waist circumference	ρ = −0.59, P < 0.05	ρ = 0.82, P < 0.05	NS	NS	NS
SBP	NS	ρ = 0.29, P < 0.05	NS	NS	NS
DBP	NS	NS	NS	NS	NS
Total cholesterol	NS	NS	NS	NS	NS
HDL cholesterol	ρ = 0.68, P < 0.05	ρ = −0.38, P < 0.05	ρ = −0.26, P < 0.05	NS	ρ = −0.35, P < 0.05
LDL cholesterol	NS	NS	NS	NS	NS
TG	ρ = −0.38, P < 0.05	NS	NS	NS	NS
Non-HDL cholesterol	NS	NS	ρ = 0.24, P < 0.05	NS	ρ = 0.27, P < 0.05
Apolipoprotein B	NS	NS	ρ = 0.26, P < 0.05	NS	ρ = 0.28, P < 0.05
HbA1c	ρ = −0,47, P < 0.05	ρ = 0.41, P < 0.05	ρ = 0.24, P < 0.05	NS	NS
C-peptide	ρ = −0,25, P < 0.05	ρ = 0.28, P < 0.05	NS	ρ = −0.26, P < 0.05	NS
FPG	NS	ρ = 0.25, P < 0.05	NS	NS	NS
Smoking	ρ = −0.25, P < 0.05	NS	NS	NS	NS
CRP	NS	NS	ρ = 0.23, P < 0.05	NS	NS
PAI-1	ρ = −0.36, P < 0.05	ρ = 0.47, P < 0.05	ρ = 0.27, P < 0.05	NS	NS
vWF	ρ = −0.29, P < 0.05	ρ = 0.61, P < 0.05	NS	NS	NS
SP aortic	NS	NS	NS	NS	NS
AIx	NS	ρ = 0.26, P < 0.05	NS	NS	NS
AIx 75/min	NS	ρ = 0.39, P < 0.05	NS	NS	NS
AP	NS	ρ = 0.28, P < 0.05	NS	NS	NS

ρ, a correlation coefficient; P, statistical significance.

Multivariate regression analysis

Multivariate regression analysis performed in all participants showed following significant results: adiponectin was independently associated with BMI (b = 0.93, P < 0.01) and HDL-C (b = 0.62, P < 0.0001); A-FABP with BMI (b = 0.64, P < 0.05), vWF (b = 0.24, P < 0.05), and HDL-C levels (b = −0.21, P < 0.01); and FGF-21 was associated with hs-CRP (b = 0.13, P < 0.05) and HbA1c (b = 0.99, P < 0.0001)–see Table 5.

Table 5.

Multivariate Regression Analysis Between Selected Adipokines, Clinical and Laboratory Parameters

	Adiponectin	A-FABP	FGF-21
	R² = 0.914	R² = 0.992	R² = 0.965
BMI	b = 0.93, P < 0.01	b = 0.64, P < 0.05	NS
TG	—	—	—
HDL-C	b = 0.62, P < 0.0001	b = −0.21, P < 0.01	NS
non-HDL-C	—	—	NS
apolipoprotein B	—	—	NS
vWF	NS	b = 0.24, P < 0.05	NS
PAI–1	NS	—	NS
hs-CRP	NS	NS	b = 0.13, P < 0.05
HbA1c	NS	NS	b = 0.99, P < 0.0001
C peptide	—	NS	—
SBP	—	NS	—
age	NS	NS	—
AIx	—	NS	—
Smoking	NS	—	—

Non-normally distributed variables were logarithmically transformed before analyses.

b, standardized coefficient beta.

R ², correlation coefficient; NS, not statistically significant, P > 0.05.

Discussion

Patients with T2D had significantly higher levels of A-FABP and lower levels of adiponectin compared with the control group. Additionally, levels of adiponectin and A-FABP significantly correlated with vWF and PAI levels. A-FABP also correlated with augmenattion pressure and AIx. Multivariate regression analysis demonstrated that vWF was independent predictor of the serum A-FABP concentration.

This study revealed highly significant differences in the levels of adiponectin and A-FABP between T2D patients and the control group. From a clinical point of view those results are important because it was previously found that decreased adiponectin and increased A-FABP are important risk factors for the development of CVD. Adiponectin protects against obesity-related CVD through its pleiotropic actions on the heart and vasculature. Hypoadiponectinemia is associated with obesity, diabetes, hypertension, mixed dyslipidemia, metabolic syndrome, nonalcoholic steatosis, coronary artery disease, and others.¹⁵

By contrast, A-FABP mediates obesity-related cardiac and vascular dysfunctions by potentiating lipid-induced inflammation and by acting as a cardiodepressant factor to suppress cardiac contractility. Therefore, adiponectin and A-FABP, represent prognostic biomarkers and promising therapeutic targets.¹⁵ In our study, we also showed a tendency to increase in FGF-21 and decrease CTRP-9 levels in diabetic group compared with the control group, but it did not reach statistical significance. CTRP-9 is reported to be a protective factor for coronary artery disease³⁰ and FGF-21 elevation may indicate a compensatory response to metabolic stress.²¹ This finding supports the association between adipokines and CV risk and disease. Understanding the mode of action for novel adipokines may facilitate their future clinical use as pharmacotherapies, drug targets, or predictors of a variety of diseases.⁹

In this study adiponectin correlated negatively with weight, levels of TG, and HbA1c levels and positively with HDL-C. Multivariate regression analysis also demonstrated independent associations of adiponectin and BMI and HDL-C. These correlations, which were also reported by others, support the findings that higher adiponectin levels prevent development of cardiovascular changes and that alterations in adiponectin secretion may contribute to obesity-related diseases.^{9,11,15,33
–35} Found negative correlation between the markers of endothelial dysfunction (vWF levels and PAI-1) and adiponectin also signals its protective effect.³⁶

There were strong correlations between A-FABP and metabolic parameters (positive with BMI, weight, SBP, non-HDL-C, and negative with HDL-C levels) and glucose metabolism parameters (HbA1c, C-peptide, and fasting plasma glucose) too. In a 10-year follow-up study on Chinese population, the plasma A-FABP level was found to be a strong predictor of T2D development independently of the traditional risk factors including obesity, insulin resistance, or glycemic indexes.³⁷ These findings support the contradictory effect of adiponectin and A-FABP for the risk of development of disease associated with metabolic syndrome parameters, T2D, coronary atherosclerosis,³⁸ and CVD.¹⁵

In Tso study, levels of A-FABP were associated with increased 3-month mortality in patients with ischemic stroke, suggesting that A-FABP may serve as a potential prognostic marker for early mortality.³⁹ Epidemiological studies on different ethnic groups have demonstrated a close association between serum levels of A-FABP and a cluster of obesity-related cardiometabolic risk factors, endothelial dysfunction, and macrovascular complications of diabetes.^15,40
–42 Clinical investigations and animal models identified A-FABP as a factor associated with insulin resistance, adverse lipid profiles, hyperglycemia, obesity, metabolic syndrome, type T2D, and also with the development of atherosclerosis.^15,37,43

Takagi et al. study showed that A-FABP concentration was an independent factor associated with future cardiovascular events. In this study, the serum A-FABP concentration was associated with prognosis in patients with stable angina undergoing percutaneous coronary intervention, suggesting that serum A-FABP concentration could be useful for risk assessment of secondary prevention.⁴⁴ Furuhashi et al. showed that FABP-4 concentration was an independent predictor of the progression of carotid atherosclerosis.⁴⁵ In patients with coronary artery disease recruited to undergo elective percutaneous coronary intervention, Miyoshi et al. showed that increased serum A-FABP levels were significantly associated with a greater coronary plaque burden as quantified by intravascular ultrasound.³⁸

Moreover in our study plasma A-FABP levels were positively correlated with endothelial/hemostatic parameters (PAI-1, vWF) and with markers of arterial stiffness (augmenattion pressure and AIx 75/min), as indicators of atherosclerosis. The Zachariah et al. study investigated whether adipokines were associated with altered small and large artery function. Higher A-FABP levels were associated with higher mean arterial pressure and higher carotid-femoral PWV.⁴⁶ A-FABP was significantly positively correlated with arterial stiffness in hypertensive patients with metabolic syndrome inn Chen et al. study.⁴⁷

Multivariate regression analysis demonstrated that vWF was an independent predictor of the serum A-FABP concentration. In our previous study with dyslipidemic nondiabetic patients A-FABP was also independently associated only with vWF.⁴⁸ There are also other factors influencing the levels of PAI-1 (endothelium, liver, adipose tissue)⁴⁹; PAI-1 is therefore a less specific marker for vascular damage than vWF. The independent association between a specific endothelial marker, vWF and A-FABP may point out A-FABP participation in direct endothelium damage.

FGF-21 levels were higher in T2D patients compared with the control group, but did not reach the statistical significance. This increase in basal FGF-21 concentrations was observed in patients with obesity and other conditions related to insulin resistance. This may be explained as a compensatory response to the underlying metabolic disturbances or tissue resistance to FGF-21 action. Furthermore, the results of clinical trials have shown that increased FGF-21 concentrations were associated with increased CV risk.⁵⁰

We also showed a relationship between FGF-21 and markers of vascular damage and endothelial dysfunction—negative correlation with aortic SP and positive correlation with PAI-1, which could be assessed as a markers of CV risk. FGF-21 correlated positively with metabolic parameters (lipid and HbA1c levels) and hs-CRP. Multiple regression analysis also found hs-CRP as an independent predictor of serum FGF-21 concentration. Numerous animal studies have demonstrated that FGF-21 improves glucose and lipid metabolism and exerts anti-inflammatory effects.^20,50,51 However, data obtained from human studies have shown contradictory results; circulating FGF-21 levels were often elevated in patients with obesity, dyslipidemia, T2D, and other conditions connected with insulin resistance.⁵⁰

Although we found lower CTRP-9 levels in diabetic group compared with control group, it did not reach the statistical significance. Recent animal studies have suggested that CTRP-9 is the closest paralog of adiponectin and may play cardio-protective role⁵² and alterations in circulating CTRP-9 have also been observed in patients with CVD and diabetes.⁵³ Although, in Wang study conversely CTRP-9 levels were positively associated with increased arterial stiffness.³¹ Jang study showed a statistically significant positive association between the serum CRTP-9 brachial PWV (after adjusting for total adiponectin) in T2D patients.⁵² Other animal studies showed that CTRP-9 have beneficial cardiovascular effects by vascular relaxation²⁸ and modulation of vascular smooth muscle cell proliferation.²⁹

In study by Wolf et al. CTRP-9 levels were significantly higher in the obese group compared with the control group and significantly decreased with weight loss after bariatric surgery. They hypothesized that elevated CTRP-9 levels in obesity is a compensatory response due to CTRP-9 effect (glucose lowering and insulin sensitizing). This could explain the difference in our study's results with other human studies; thus further validation of our results is needed.⁵³

Correlations between AIF-1 and metabolic parameters could relate to low-grade chronic inflammation that is associated with obesity-related cardiometabolic risk factors.⁵⁴ Rawhia et al. study showed a significant positive correlation between AIF-1 and diabetes duration, SBP and DBP.⁵⁵ Lack of differences between AIF-1 levels among investigated groups may be a consequence of difference sensitivity in serum and in tissues. Perhaps measuring AIF-1 in serum was not the best way for assessment of its real levels.

Conclusion

Patients with T2D had significantly higher levels of A-FABP and lower levels of adiponectin, when compared with control group. Both levels of adiponectin and A-FABP significantly correlated with vWF and PAI-1 levels and A-FABP alone correlated with augmentation pressure and augmentation index. Multivariate regression analysis demonstrated that vWF was an independent predictor of the serum A-FABP concentration and it also showed an independent association between these two adipokines and metabolic parameters (BMI and HDL cholesterol levels). Levels of these adipokines correlated with indicators of vascular damage and could thus contribute to CV risk individuals with diabetes. A-FABP could participate in direct endothelium damage.

Footnotes

Acknowledgment

This study was supported by grant no. IGA LF_2017_015 (Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech Republic) and by the Ministry of Health, Czech Republic DRO (FNOL 00098892).

Authors' Contributions

All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis; J.S.: acquisition of data and article writing; J.S., D.K.: study concept and design, literature search; V.K.: acquisition of data, laboratory analysis, D.K., O.K., D.G., J.Z., J.S., V.K.: literature search and critical reading; all authors: article revision.

Author Disclosure Statement

No conflicting financial interests exist.

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