life Article Low Levels of Serum Fetuin-A and Retinol-Binding Protein 4 Correlate with Lipoprotein Subfractions in Morbid Obese and Lean Non-Diabetic Subjects Hajnalka L˝orincz 1, Imre Csige 2,3, Mariann Harangi 1, Anita Szentpéteri 1, Ildikó Seres 1, Zoltán Szabó 2, György Paragh 1 and Sándor Somodi 1,2,* 1 Department of Internal Medicine, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary; [email protected] (H.L.); [email protected] (M.H.); [email protected] (A.S.); [email protected] (I.S.); [email protected] (G.P.) 2 Department of Emergency Medicine, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary; [email protected] (I.C.); [email protected] (Z.S.) 3 Doctoral School of Health Sciences, University of Debrecen, 4032 Debrecen, Hungary * Correspondence: [email protected] Abstract: Background: Fetuin-A and retinol-binding protein 4 (RBP4) are secreted as both hepatokine and adipokine. These are involved in insulin resistance, obesity-related dyslipidemia, and atheroscle- rosis. To date, correlations of circulating fetuin-A and RBP4 with lipoprotein subfractions as well as high-density lipoprotein (HDL)-linked proteins have not been entirely investigated in morbid obese and lean non-diabetic subjects. Methods: One-hundred obese non-diabetic patients (body mass index, BMI: 42.5 ± 8.1 kg/m2) along with 32 gender and age-matched normal weight controls 2 Citation: L˝orincz,H.; Csige, I.; (BMI: 24.5 ± 2.5 kg/m ) were enrolled in our study. Serum fetuin-A and RBP4 were measured by Harangi, M.; Szentpéteri, A.; Seres, I.; ELISA. Lipoprotein subfractions were distributed by Lipoprint gelelectrophoresis. Results: Serum Szabó, Z.; Paragh, G.; Somodi, S. Low fetuin-A and RBP4 were unexpectedly lower in obese patients (p < 0.01 and p < 0.01, respectively) Levels of Serum Fetuin-A and compared to controls and correlated with each other (r = 0.37; p < 0.001). Fetuin-A had positive Retinol-Binding Protein 4 Correlate correlations with HDL-C (r = 0.22; p = 0.02), apolipoprotein AI (apoAI) (r = 0.33; p < 0.001), very-low with Lipoprotein Subfractions in density lipoprotein (VLDL) subfraction (r = 0.18; p = 0.05), and large HDL subfraction levels (r = 0.3; Morbid Obese and Lean Non- p = 0.001) but did not show correlation with carbohydrate parameters in all subjects. RBP4 correlated Diabetic Subjects. Life 2021, 11, 881. positively with HDL-C (r = 0.2; p = 0.025), apoAI (r = 0.23; p = 0.01), VLDL subfraction (r = 0.37; https://doi.org/10.3390/life11090881 p < 0.001), intermediate HDL subfraction (r = 0.23; p = 0.01), and small HDL subfraction (r = 0.21; p = 0.02) concentrations, as well as C-peptide levels in overall participants. Backward stepwise Academic Editor: Vasily Sukhorukov multiple regression analysis showed that serum fetuin-A concentration is best predicted by RBP4 Received: 12 July 2021 and large HDL subfraction. In model 2, VLDL subfraction was the independent predictor of serum Accepted: 23 August 2021 RBP4 level. Conclusions: Our data may indicate a potential role of fetuin-A and RBP4 in impaired Published: 27 August 2021 lipoprotein metabolism associated with obesity. Publisher’s Note: MDPI stays neutral Keywords: fetuin-A; retinol-binding protein 4; lipoprotein; obesity; insulin resistance; diabetes with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction Incidence of central obesity and its related co-morbidities have been dramatically grow- ing worldwide due to a sedentary lifestyle, fat and carbohydrate-rich nutrition, together Copyright: © 2021 by the authors. with genetic effects. White adipose tissue and liver-generated hormone-like peptides— Licensee MDPI, Basel, Switzerland. collectively termed as adipokines and hepatokines, respectively—have paracrine, en- This article is an open access article docrine, and systemic effects. The dysregulation of these adipo- and hepatokines (e.g., lep- distributed under the terms and tin, chemerin, plasminogen activator inhibitor-1, afamin, fetuin-A, retinol-binding protein 4 conditions of the Creative Commons etc.) has been associated with central obesity and obesity-related co-morbidities including Attribution (CC BY) license (https:// metabolic syndrome, type 2 diabetes, dyslipidemia, and enhanced atherogenesis. However, creativecommons.org/licenses/by/ the downregulation of favorable adipokines, such as adiponectin, vaspin, and omentin-1, 4.0/). Life 2021, 11, 881. https://doi.org/10.3390/life11090881 https://www.mdpi.com/journal/life Life 2021, 11, 881 2 of 13 has been linked to insulin resistance [1]. Therefore, investigation of obesity-associated adipokine and hepatokine production has been intensively studied. Fetuin-A or α-2-Heremans–Schmid-glycoprotein (α2-HS-glycoprotein, AHSG) is a multifactorial bioactive protein that is mostly produced by hepatocytes and partially by visceral adipose tissue. It is secreted into the circulation at relatively high concentration [2]. Being originally described as an essential developmental factor in the fetal circulation both in humans and animals [2], it serves as a potent inhibitor of vascular calcification in endothelial cells [3]. An increased level of fetuin-A is found to be involved in the development of insulin resistance through its effect on the endogenous inhibitor of insulin receptor tyrosine kinase [4]. Indeed, fetuin-A level is considered as a biomarker for obesity [5], dyslipidemia [6], and subclinical atherosclerosis [7]. In addition, a recent meta- analysis suggests that fetuin-A may prove to be an important indicator for the components of metabolic syndrome [8]. Genetic influences also impact fetuin-A concentrations and functions; AHSG single-nucleotide polymorphism was found to be susceptible for obesity and early-onset co-morbidities [9]. Similar to fetuin-A, retinol-binding protein 4 (RBP4) is synthetized and secreted as both hepatokine and adipokine. RBP4 transfers retinol (vitamin A) from the liver to the target tissues within the circulation in combination with transthyretin, which prevents renal filtration and the catabolism of RBP4 [10]. Although there has been an exponential growth of knowledge about the function of RBP4, data from physiological and pathological concentration of RBP4 are contradictory. Fan et al. described a U-shaped association between serum RBP4 concentrations and the risk of incident of type 2 diabetes in predi- abetic patients [11]. Higher serum RBP4 levels were involved in obesity, type 2 diabetes, and in patients with cardiovascular disease [10,12]. A decreased GLUT4 expression in visceral adipose tissue also leads to an increased circulating concentration of RBP4 associ- ated with insulin resistance in the hepatic and muscular cells [13]. Furthermore, current studies showed that RBP4 and retinoids are strongly involved in obesity-associated dys- lipidemia [14], but results are not fully elucidated. Obesity-related dyslipidemia is one of the well-known complications in obese pa- tients including higher concentration of very low-density lipoprotein (VLDL), small-sized low-density lipoprotein (LDL) subfractions, and decreased mean LDL size. In addition, we previously published a shift toward the small-sized high-density lipoprotein (HDL) subfraction in morbid obese patients compared to lean controls [15]. Recently, proteomic analyses on HDL described an extended list of HDL-associated proteins including fetuin-A, RBP4, and transthyretin [16]; however, some authors indicated that the data are highly controversial and strongly depend on HDL isolation as well as the purification method [17]. To date, data are highly contradictory about the direct association of fetuin-A and RBP4 molecule to HDL particle; moreover, data are lacking about the relationship between impaired circulating fetuin-A and RBP4 production and the markers of obesity-related dyslipidemia (i.e., small HDL, mean LDL size, etc.) in a special morbid obese but non- diabetic population. We hypothesized that there is a correlation between serum fetuin-A and RBP4 with lipoprotein subfractions, including HDL subfractions and other HDL-linked proteins in morbid obese, non-diabetic, and healthy lean participants. 2. Patients and Methods 2.1. Patients We enrolled one-hundred non-diabetic obese patients from our obesity outpatient clinic at the Department of Medicine, Faculty of Medicine, University of Debrecen, Hun- gary. Thirty-two aged and gender-matched healthy lean volunteers (checked with routine laboratory parameters) were also enrolled from the university. Anthropometric and lab- oratory parameters of participants are summarized in Table1. Permission to carry out this study was granted by the Regional Ethics Committee of the University of Debrecen and the Medical Research Council (registration number: DE RKEB/IKEB 5513B-2020 and IV/7989-1/2020/EKU, respectively). All participants signed a written informed consent. Life 2021, 11, 881 3 of 13 Obesity was defined as body mass index (BMI) ≥ 30 kg/m2. The main medications of obese patients are shown in Table1. The control group was free of medication. Individuals with liver, kidney, endocrine disorders (including both type 1 and 2 diabetes mellitus), pulmonary, neurological, gastrointestinal, psychiatric, acute infective, autoimmune disease, or malignancies were excluded. Additional exclusion criteria including pregnancy, breast feeding, smoking, and regular alcohol consumption were applied. 2.2. Sample Collection and Biochemical Measurements