Circulating Level of Soluble Leptin Receptor and its Association with Cardiometabolic Risk Factors among Obese Subjects in Kerala, South India

Background and Aims: Leptin, the peptide hormone secreted mainly by adipose tissue is reported to play the central role in the pathogenesis of obesity. Leptin exerts its biological effects through specific receptor molecules present in target tissues. Among the different isoforms of leptin receptor, the Soluble Leptin Receptor (SLR) is the major leptin binding protein seen in circulation which modulates the bioavailability of leptin. Our objectives were to analyse the level of circulating SLR among obese subjects and its association with biomarkers of obesity, serum leptin, insulin and cardiometabolic risk factors in comparison with healthy age and sex matched control subjects. Methods: About 173 study participants of both genders were selected and grouped as case (n=102) and control (n=71) with a cut off point of BMI 25kg/m 2 . Waist to hip ratio (WHR) and body Original Research Article Manju et al.; JPRI, 33(28B): 232-239, 2021; Article no.JPRI.68208 233 fat percentage (BF%) were calculated from anthropometric measurements. Leptin, insulin, soluble leptin receptor were estimated in fasting blood samples by sandwich ELISA method. Fasting plasma glucose and lipid profile were measured by standard enzymatic methods in autoanalyzer. Homeostasis Model Assessment of Insulin resistance (HOMA-IR) was calculated. Comparison between groups was done by independent sample ‘t’ test. P values <.05 were considered statistically significant. Results: The SLR level was found to be increased in obese group in comparison with control group(P =.001). A significant increase in serum leptin and insulin level was observed in obese group when compared to control (P =.001). Obese group showed more than two fold increase in insulin resistance expressed as HOMA-IR when compared to control subjects (P =.001). But no significant difference in the synthesis of insulin expressed as HOMA-beta between the groups. No significant difference in serum lipoprotein levels was observed between the two groups. Conclusion: Increased level of circulating soluble leptin receptor has been observed in obese subjects in comparison with control subjects and is associated with hyperleptinemia, hypertension and insulin resistance.


INTRODUCTION
Obesity is a major cause accountable for the increased incidence of diabetes, hypertension, dyslipidemia, metabolic syndrome, cardiovascular diseases etc. among the population in Kerala [1,2]. Leptin, the peptide hormone secreted by the adipose tissue in response to body fat has been reported to be elevated in obesity [3,4]. Leptin exerts it's physiological functions by binding with specific receptors in target tissues. Leptin receptor is a single transmembrane domain receptor which displays a structural similarity to class 1 cytokine receptor family [5]. The Leptin receptor is produced in several alternatively spliced forms designated as LEPRa -LEPRf, that share the common extracellular domain with more than 800 amino acids and the transmembrane domain with 34 amino acids, and have a variable intracellular domain characteristic of each of the isoforms [6]. There are three classes of leptin receptor isoforms such as short, long and secreted forms. The 'short forms' of LEPR may have roles in binding (LEPRe), transport (LEPRa), and clearance (LEPRc and LEPRd) of leptin [7] whereas only the 'long form' (LEPRb) encodes all protein motifs capable of activating the janus kinase -signal transduction and activation (JAK-STAT) pathway which in turn stimulates transcription of target genes [8]. The 'secreted forms' are formed either by the alternative splicing of mRNA species or by the proteolytic cleavage of membrane bound forms of leptin receptor. The secreted forms of leptin receptor (Ob-Re) consists entirely of the extracellular ligand-binding domain and it lacks the transmembrane residues and intracellular domain responsible for signal transduction. The extracellular domain binds the circulating leptin, thereby regulating the concentration of free leptin and is known as soluble leptin receptor (SLR) [9]. SLR is the cleaved portion of the extracellular domain of the membrane bound leptin receptor and acts as the main binding protein for leptin in human blood and inhibits leptin transfer thereby modulating the bioavailability of leptin [10].
To exert it's action, leptin must reach the brain through blood brain barrier by binding with specific leptin receptor. The increase in adiposity increases the serum leptin levels which can lead to the development of resistance at the blood brain barrier due to impaired transport of leptin across the blood-brain barrier in obesity [11]. The mechanisms involved in this effect remains unknown. Less amount of leptin reaching the brain may lead to reduced activation of the signalling pathway for body weight regulation. Studies reported that reduced brain access may be the source of leptin resistance in obesity and further increase in body weight [12,13] According to certain studies, the high levels of circulating leptin could activate the mechanisms of desensitization and downregulation, causing the degradation of leptin receptors [14]. Increased levels of SLR point out regulation of leptin signalling, but the exact mechanism of SLR formation is not yet completely elucidated. However it has been proposed that increased leptin levels, cellular stress and inflammation and reduced cellular expression of leptin receptors may in part mediate serum SLR levels. Free Leptin Index (FLI), the ratio of total leptin and SLR concentrations is the biomarker of leptin resistance and the status of leptin action. Research in rodent models reported that the SLR modulates serum leptin levels by delaying its clearance thereby determines the amount of free as well as bound leptin levels in serum [15]. In this background, the present study was designed to determine the level of serum soluble leptin receptor among obese subjects and to compare with that in age and sex matched control subjects. Adiposity measures, cardiometabolic risk biomarkers and insulin resistance was also analysed. We also measured the serum leptin level in order to analyse role of SLR in the regulation of serum leptin level.

MATERIALS AND METHODS
The study participants were selected from the obesity clinic Govt. Medical College, Trivandrum and also from the outpatient units of Sree Gokulam Medical College and Research Foundation, Trivandrum. Obese individuals with a BMI≥25, with no history of any prior medication were selected as cases. Healthy subjects with BMI≤24.9 were selected as controls. Subjects with a history of drug intake affecting the study variables (BMI, blood glucose, insulin, leptin, lipid profile etc.), those with thyroid diseases, polycystic ovarian diseases (PCOD), pregnant and post-partum women and those taking oral contraceptive pills were excluded from the study. Subjects under the age of 20years, pregnant and post-partum women, those with polycystic ovarian diseases (PCOD) and thyroid diseases and those taking oral contraceptive pills were excluded from the study.
A detailed informed consent was obtained from each subject and the data were confidential. Sampling was done after getting ethical approval from both institutions. All participants answered a life style questionnaire. Information about age, gender, race, socioeconomic factors, lifestyle habits and family history of diseases was gathered from questionnaire. Body height was recorded with the subjects wearing light clothes and no shoes. Body height measured to the nearest 0.1cm with a fixed stadiometer. Body weight determined to the nearest 0.1kg in the fasting state using a standard weighing machine. BMI was calculated using the standard formula. Waist and hip circumferences measured with a fibre reinforced plastic tape to the nearest 0.1cm at the level of umbilicus (L4-L5) and the maximum extension of the buttocks respectively at the end of expiration with the subject upright and his/her hands by the side. Body fat (%) was The data were analysed using Statistical Package for Social Sciences (SPSS) for Windows version 17.0 (SPSS Inc, Chicago, IL, USA). Study variables were expressed as mean ± SEM and the significance of study variables between groups was tested by unpaired Student's t test. The statistical significance was defined by P <0.05.

RESULTS
The mean BMI of obese group was 29.2 kg/m 2 while the control group had the mean BMI of 21.45kg/m 2 . About 51% of each group were males and 49% females. There was no significant difference in age between the groups (P =.50). Statistically significant increase (P=.001) in adiposity measures such as weight, BMI, waist and hip circumferences and body fat was observed in obese group compared to control (Fig. 1). The mean waist circumference of obese group was 100.3cm and that of control group was 82.4cm. The average height was more in control group and the difference in height was not statistically significant between the two groups (P =.50). The average body fat percentage in obese group was 37.5 while that of control group was 26.6. The increase in both systolic and diastolic blood pressure in obese group was found to be statistically significant in comparison with control group(P =.001).
Fasting Plasma Glucose and insulin levels showed a statistically significant increase (P =001) in obese group compared to control (Table1). Obese group showed more than two fold increase in HOMA-IR (P =.001) along with a significant increase in serum insulin level (P =.001) than control. But there was no statistically significant difference in the synthesis of insulin expressed as HOMA-β between the groups. The insulin sensitivity expressed as Quick Insulin Sensitivity Check Index (QUICKI) decreased significantly in obese group (0.29±0.003) in comparison with control (0.33 ± 0.005) (P =.001). There was no statistically significant difference in serum lipoprotein levels between the groups.
The mean leptin level of obese group was 60.9ng/ml with a significant difference from control group of 13.2ng/ml. The concentration of serum SLR in obese group (24.26 ± 0.98, mean± SEM) showed a statistically significant increase in comparison with that in control group (18.84 ± 0.46). The mean FLI showed approximately four fold increase in obese group when compared to control group (P =.02).    . In our study population, serum insulin level was found to be increased among obese group with two fold increase in insulin resistance in comparison with control subjects; while there was no statistically significant difference in the synthesis of insulin expressed as HOMA-β between the groups. At the same time, the insulin sensitivity expressed as QUICKI decreased significantly in obese group in comparison with control.

CONCLUSION
Increased levels of circulating soluble leptin receptor have been observed in obese subjects in comparison with age and sex matched control subjects. The increased level of circulating soluble leptin receptor was found to be associated with hypertension, visceral obesity, hyperleptinemia and insulin resistance. No significant association was observed between serum lipoproteins and soluble leptin receptor.

CONSENT AND ETHICAL APPROVAL
This study was conducted in accordance with Declaration of Helsinki. Ethical approval was obtained from the Human ethics committee of both institutions. The study subjects were explained the entire study in detail and written informed consent was obtained with due negligence.