D. Karasek, J. Gajdova, V. Kubickova, O. Krystynik, L. Cibickova, H D. Karasek, J. Gajdova, V. Kubickova, O. Krystynik, L. Cibickova, H. Vaverkova 3rd Department of Internal Medicine and Department of Clinical Biochemistry University Hospital Olomouc, Czech Republic Selected adipokines in patients with type 2 diabetes: relationship to markers of endothelial dysfunction and arterial stiffness 1

Adiponectin one of the most abundant peptide hormones derived from adipose tissue plays a major role in glucose and lipid metabolism and prevents development of cardiovascular changes due to its anti-oxidative, anti-inflammatory, endothelium-protective and anti-apoptotic effects hypoadiponectinemia is associated with obesity, diabetes, hypertension, mixed dyslipidemia, metabolic syndrome, non-alcoholic steatosis, coronary artery disease, etc. Xu A, Vanhoutte PM. Am J Physiol Heart Circ Physiol. 2012 Fisman EZ, Tenenbaum A. Cardiovascular Diabetology 2014

Adipocyte Fatty Acid Binding Protein is formed mainly in adipocytes and macrophages, partially in lymphocytes, its main function is the binding of free fatty acids increases lipolysis and insulin resistance, reduces contractility of cardiomyocytes, promotes chronic inflammation and formation of vulnerable atherosclerotic plaques its increased levels were found in obesity, metabolic syndrome, T2DM, NASH, CAD, ischemic stroke is a biomarker (maybe even the main mediator) of obesity related cardiovascular diseases Xu A, Vanhoutte PM. Am J Physiol Heart Circ Physiol. 2012 Pasterkamp G. Arterioscler Thromb Vasc Biol. 2012

Fibroblast Growth Factor 21 is produced by liver, pancreas, muscles and adipocytes participates in the regulation of metabolism of sugars (increases insulin sensitivity, gluconeogenesis, glucose uptake) and fat (increases oxidation of fatty acids, ketogenesis, HDL-C levels, decreases TG and LDL-C) stimulates thermogenesis and reduces weight (its analogs were tested as anti-obesity drugs) its elevation is found in obesity, metabolic syndrome, non-alcoholic steatosis and coronary artery disease. (maybe due to its resistance or as a compensatory mechanism) Woo YC, et al. Clin Endocrinol (Oxf). 2013, Li H, et al. Front Med. 2013, Kim KH, Lee MS. Diabetes Metab J. 2014 , Jin L et al. Diabetes Metab J. 2016

C1q/TNF-related protein 9 is the closest adiponectin paralog (↓glycemia, ↓IR, anti-steatotic effect → ↓TG accumulation) is predominantly expressed in adipose tissue besides its metabolic roles that overlap with those of adiponectin, has attracted a lot of attention due to its beneficial cardiovascular effects detected in animal models: significant endothelium-dependent vasorelaxation suppression of neointimal hyperplasia and vascular smooth muscle cells proliferation attenuation adverse cardiac remodeling In human: its low levels correlated with visceral obesity, adverse metabolic profile and presence of metabolic syndrome its circulated levels and local production protect against CAD XX its high levels correlated with obesity and decreased after bariatric surgery and weight loss its increased levels correlated with C-IMT in T2DM HC CAD Wong GW et al. FASEB J. 2009, Jung CH et al. J Clin Endocrinol Metab. 2014, Hwang YC et al. Int J Obes (Lond). 2014, Wang J et al. Biomed Res Int. 2015, Li J et al. Biochem Biophys Res Commun. 2015, Wolf RM et al. J Clin Endocrinol Metab. 2016, Asada M et al. J Diabetes Res. 2016

Allograft Inflammatory Factor–1 is not produced by adipocytes, but by macrophages, which belong to numerous cells presented in fat tissue promotes macrophages, VSMCs and endothelial cells activation, proliferation and migration correlates with clinical (BMI, waist) and biochemical (FPG, TG, HDL-C, HbA1c, uric acid) metabolic parameters and maybe associated with atherogenesis Tian Y et al. Am J Physiol Cell Physiol. 2009, Fukui M et al. Metabolism. 2012, Zhao YY et al. Cell Immunol. 2013, Wang J et al. Cell Physiol Biochem. 2013, Fukui M et al. Diabetes Res Clin Pract. 2012

Aim of study, subjects Anti-diabetic medication (%) Co-morbidities (%) To compare adipokines levels in patients with diabetes and in healthy individuals To determine their relationship to indicators of early vascular damage Subjects 54 patients with type 2 diabetes (32 men, 22 women) and 21 healthy controls (8 men, 13 women) Anti-diabetic medication (%) Co-morbidities (%)

Methods Cross-sectional study: adiponectin, A-FABP, FGF-21, CTRP-9, AIF-1 anthropological parameters (BMI, waist, SBP, DBP), smoking, diabetes duration lipids: CH, TG, HDL-C, LDL-C, apoB parameters of insulin resistance and diabetes compensation: insulin, C-peptide, FBG, HOMA, HbA1C parameters of inflammation and CKD: hs-CRP, IL-6, ACR, eGF markers of endothelial dysfunction: vWF, PAI-1, t-PA markers of arterial stiffness (SfymgoCor): augmentation index (AIx=AP/ PP*100) a pulse wave velocity (PWV)

Markers of vascular damage T2DM (n=54) nephropathy – (n=33) nephropathy + (n=21) healthy controls vWF (%) 133.1 (110.7-163.2)** 126.0 (115.0-137.1) 143.5 (110.2-173.4)* 98.5 (84.4-125.0) PAI-1 (ng/ml) 78.1 (41.2-106.5)*** 73.6 (56.0-89.7) 80.0 (31.9-112.4)** 36.9 (27.5-41.9) t-PA 2.9 (2.5-3.5) 2.9 (2.5-4.1) 2.9 (2.5-3.2) 2.7 (2.4-2.9) AIx (1) 22.5 (17.0-31.9) 18.4 (6.8-19.0) 27.1 (18.1-33.7)* 15.0 (8.9-24.8) PWV (m/s) 9.0 (7.4-10.1) 7.2 (5.8-10.3) 9.3 (7.9-10.1) 7.1 (6.4-8.6) ANOVA (after adjustment for age, sex and BMI), * p<0.05, ** p<0.01 *** p<0.001

Adiponectin (μg/ml) p<0.05 p<0.001 ANOVA (after adjustment for age, sex and BMI) Statistically significant correlations (Pearson´s correlation coefficient, p<0.05): Negative: age (r=-0.25), BMI (r=-0.31), waist (r=-0.59), smoking (r=-0.24), TG (r=-0.39), HbA1C (r=-0.47), C-peptide (r=-0.24) vWF (r=-0.29), PAI-1 (r=-0.35) Positive: HDL-C (r=0.68)

A-FABP (ng/ml) p<0.01 p<0.001 ANOVA (after adjustment for age, sex and BMI) Statistically significant correlations (Pearson´s correlation coefficient, p<0.05): Positive: age (r=0.43), BMI (r=0.56), waist (r=0.82), SBP (r=-0.29), pulse (r=0.29), HbA1C (r=0.41), C-peptide (r=0.28), FBG (r=0.24), insulin (r=0.29) vWF (r=0.61), PAI-1 (r=0.47), AIx (r=0.39) Negative: HDL-C (r=-0.38)

FGF-21 (pg/ml) n.s. ANOVA (after adjustment for age, sex and BMI) Statistically significant correlations (Pearson´s correlation coefficient, p<0.05): Positive: hs-CRP (r=0.23), HbA1C (r=0.24), non-HDL-C (r=0.24), ACR (r=0.28) PAI-1 (r=0.27) Negative: HDL-C (r=-0.38)

CTRP-9 (pg/ml) n.s. ANOVA (after adjustment for age, sex and BMI) Statistically significant correlations (Pearson´s correlation coefficient, p<0.05): Negative: C-peptide (r=-0.26)

AIF-1 (pg/ml) ANOVA (after adjustment for age, sex and BMI) n.s. Statistically significant correlations (Pearson´s correlation coefficient, p<0.05): Positive: non-HDL-C (r=0.27) Negative: HDL-C (r=-0.35)

Conclusion Patients with type 2 diabetes have significantly higher levels of A-FABP and lower levels of adiponectin. The levels of these adipokines correlate with indicators of vascular damage and could thus directly contribute to cardiovascular risk individuals with diabetes. A-FABP could participate in direct endothelium damage. The increase of FGF-21 could be a secondary result as an effort to increase low adiponectin (maybe also CTRP-9) levels and to protect vessels against the vascular injury. fat tissue endothelium A-FABP ↑ FGF-21 liver adiponectin, (CTRP-9 ?)