Investigation of plasma leptin level in patients with type 2 diabetes mellitus

Objectives: Adipose tissue-derived hormones are associated with metabolic disorders including type 2 diabetes mellitus (T2DM). This study investigated clinical parameters and the levels of leptin cytokine in patients with T2DM with or without overweight/obese.

Journal of military pharmaco-medicine No7-2016 INVESTIGATION OF PLASMA LEPTIN LEVEL IN PATIENTS WITH TYPE DIABETES MELLITUS Nguyen Van Hoan*; Nguyen Thanh Binh**; Doan Van De*; Nguyen Linh Toan*** SUMMARY Objectives: Adipose tissue-derived hormones are associated with metabolic disorders including type diabetes mellitus (T2DM) This study investigated clinical parameters and the levels of leptin cytokine in patients with T2DM with or without overweight/obese Materials and methods: Based on body mass index (BMI), 130 patients with T2DM were divided either as overweight/obese or non-overweight/non-obese subjects 125 overweight/obese and nonoverweight/non-obese individuals devoid of T2DM were included as healthy controls The leptin levels were measured in the plasma samples in all study subjects by ELISA and clinical parameters Results: The leptin levels were significantly lower in patients with T2DM compared to controls ([1202.75 pg/ml median vs 1715.4 pg/ml], p = 0.05) The leptin levels were increased in overweight/obese T2DM patients and in overweight/obese healthy subjects compared to without overweight/obese T2DM patients and healthy induviduals (p < 0.05) The levels of leptin were significantly increased in female patients with T2DM and in T2DM patients with metabolic syndrome (MS) compared to those of male patients (p < 0.001) and T2DM patients with none-MS (p < 0.05) Conclusions: Leptin cytokine is associated with T2DM and may serve as a prognostic marker for overweight/obese-related T2DM * Keywords: Type diabetes mellitus; Overweight; Obesity; Leptin; Insulin INTRODUCTION Type diabetes mellitus is a chronic metabolic disorder with an exponential increase in developing countries The international diabetes federation (IDF) reported 382 million diabetes cases in 2013 with a prediction of 592 million cases by 2035, and 80% of these cases are in developing countries [1] In Vietnam, the number of patients with T2DM is growing with an estimated 3.3 million diabetes cases reported for 2014 The prevalence of diabetes in the age group of 30 - 69 years is estimated to be 5.7% across Vietnam and 7% in urban areas [2] T2DM constitutes up to 95% of all diabetes and is characterized by chronic hyperglycaemia resulting from defects in insulin secretion and/or insulin action and metabolic disorders of protein and lipids [3] The pathogenesis of T2DM consists of two major abnormalities including insulin resistance and dysfunction of insulin production, which lead to the disability to regulate blood glucose level [4] * 103 Hospital * Nghean Endocrine Hospital *** Military Medical University Corresponding author: Nguyen Linh Toan (toannl@vmmu.edu.vn) 41 Journal of military pharmaco-medicine No7-2016 The damage of pancreatic β cells to produce sufficient insulin and adiponectin as well as an increased production of pro-inflammatory cytokines due to obesity are the major contributing factors for T2DM [3] Insulin resistance appeared years before the clinical manifestation of T2DM and is significantly associated with obesity, especially with abdominal and visceral obesity with an abnormally increased waist-to-hip ratio (WHR), dyslipidemia, hypertension and other metabolic disorders Therefore, obesity largely contributes to insulin resistance in patients with T2DM [5] Adipose tissue is recently recognized as an organ for metabolism of sexual steroids and production of adipsin that significantly contributes to loss of body weight [6] Adipose tissue-derived proteins regulate metabolic functionalities including hormone activities [7] Adipose tissue is also known as an endocrine organ that can produce various peptides with bioactivities namely adipokines Increased adipose tissue due to obesity, especially deposition of visceral fat, is associated with insulin resistance, increased blood glucose levels, lipid metabolic disorders, hypertension and inflammation [7] The adipose tissuederived hormones such as adiponectin, leptin, adipsin, and resistin have been shown to be associated with metabolic disorders and are risk factors for T2DM and cardiovascular diseases This study aims to investigate the levels of leptin and their correlations with insulin resistance of overweight/obese and T2DM 42 MATERIALS AND METHODS Patients and controls One hundred thirty (n = 130) patients with T2DM and one hundred twenty five (n = 125) control individuals were included in the study The patients were classified into two subgroups based on their BMI and T2DM status The first subgroup includes the patients with both overweight/obese (BMI ≥ 23) and T2DM (patients with overweight T2DM, n = 57) The second subgroup includes patients with T2DM but without overweight (BMI < 23) (nonoverweight T2DM, n = 73) The patients were diagnosed for T2DM based on the standard criteria reported by world health organization (WHO) in 1998 and by International Diabetes Federation (IDF) in 2005 The anthropometric indicators such as height, weight, waist and hip circumference were measured for all study participants BMI and waist-hip ratio (WHR) were calculated based on their anthropometric indicators (table 1) All the individuals in the control group were clinically examined and were considered healthy during sampling None of them had any chronic infectious diseases or conditions such as hepatitis, liver cirrhosis, obstructive pulmonary disease, gout and/or any infection The control group was also further divided into two subgroups The first control subgroup include normal healthy individuals, (non-overweight control individuals, n = 57), with fasting venous blood glucose test < 5.6 mmol/L, blood pressure < 130 mmHg and < 85 mmHg, ECG in normal limits, other tests in the normal range, Journal of military pharmaco-medicine No7-2016 BMI from 18.5 to 23 The second control subgroup is healthy individuals with BMI ≥ 23 (overweight/obese control individuals, n = 68), fasting venous blood glucose test < 5.6 mmol/L, blood pressure < 130 mmHg, and < 85 mmHg, ECG in normal limits (table 1) Measurement of biochemical parameters The levels of lipid components including cholesterol (CT), triglycerides (TG), highdensity lipoprotein - cholesterol (HDL-C), low-density lipoprotein - cholesterol (LDL-C) were measured by using automatic biochemical AVADIA 1800 (Siemens, USA) Fasting glucose levels were measured in blood by measurement of the UV with Hexokinase Insulin levels were measured by the Achitech i2000SR (Abbott, USA) Blood fasting glycosylated hemoglobin (HbA1c) levels were quantified by the ionexchange method using high performance liquid chromatography (HPLC) Insulin resistance index was evaluated according to the homeostasis model assessment insulin resistance index (HOMA-IR index) and the quantitative insulin sensitivity check insulin resistance index (QUICK-IR index) In addition, the β-cell function (homeostatic model assessment- HOMA-β) and the insulin secretion of β-cell were evaluated according to Matthews’ method Measurement of leptin levels The levels of leptin were measured in the respective plasma samples of the study participants by using a commercial ELISA kit following the manufacturer’s instruction (Human leptin, Sigma, USA) * Statistical analysis: Clinical and demographic data were presented in median values with range for continuous variables The student’s t-tests or one-way ANOVA were used for comparing mean of two or more groups, respectively Chi-square or Fisher’s exact tests were used to compare categorical variables Kruskal-Wallis or Mann-Whitney U test was used to analyze the plasma levels of leptin in the patients with T2DM and in controls wherever appropriate All statistical analyses were performed using IBM Statistics SPSS v.19 (IBM Corp, Armonk, NY the USA), and the level of significance was set at a P value of less than 0.05 RESULTS Demographic, clinical and biochemical characteristics of the study subjects Table 1: Characteristics of patients with type diabetes mellitus and controls Type diabetes mellitus Characteristics Age (years) Gender (M/F) Without type diabetes mellitus NonOverweight/obese overweight p value T2DM, n = 73 T2DM, n = 57 p value (*) Overweight/obese Non-overweight individuals, individuals, p value n = 68 n = 57 57.82 ± 7.98 58.89 ± 6.99 NS 55.88 ± 8.54 57.84 ± 6.93 NS < 0.05 46/27 32/25 NS 28/40 13/44 0.02 0.03 BMI 26.03 ± 2.24 21.24 ± 1.37 < 0.0001 24.99 ± 1.7 20.69 ± 1.63 < 0.0001 NS WHR 0.93 ± 0.04 0.92 ± 0.07 0.91 ± 0.08 0.88 ± 0.06 < 0.05 0.001 < 0.05 43 Journal of military pharmaco-medicine No7-2016 Fasting glucose (mmol/L) 9.50 ± 3.07 8.63 ± 2.07 NS 5.19 ± 0.34 5.17 ± 0.35 NS < 0.0001 Total Cholesterol (mmol/L) 5.14 ± 0.99 5.17 ± 0.75 NS 5.13 ± 0.97 5.09 ± 1.02 NS NS Triglyceride (mmol/L) 2.91 ± 2.65 2.22 ± 1.59 NS 2.08 ± 2.10 1.59 ± 1.13 NS < 0.05 HDL-C (mmol/L) 1.15 ± 0.25 1.27 ± 0.52 NS 1.31 ± 0.32 1.45 ± 0.37 NS NS LDL-C (mmol/L) 2.70 ± 0.82 2.93 ± 0.74 NS 2.95 ± 0.88 2.99 ± 1.06 NS NS HbA1c (%) 7.37 ± 1.23 7.24 ± 1.3 NS 5.63 ± 0.57 5.6 ± 0.28 NS NS Insulin (pmol/L) 10.64 ± 9.87 7.30 ± 4.30 < 0.001 7.33 ± 2.42 6.32 ± 2.94 NS < 0.0001 HOMA-RI 4.90 ± 6.15 2.82 ± 1.77 < 0.05 1.69 ± 0.58 1.46 ± 0.70 < 0.05 < 0.0001 QUICKI 0.81 ± 0.10 0.85 ± 0.08 < 0.01 0.92 ± 0.07 0.97 ± 0.10 < 0.01 < 0.0001 HOMA-β 38.80 ± 35.83 31.27 ± 20.78 > 0.05 92.12 ± 47.35 78.60 ± 38.79 > 0.05 < 0.0001 (*) Comparison between type diabetes mellitus and non-diabetes mellitus The mean age of the patients with T2DM was higher than control individuals (without T2DM) (p < 0.05), while there was no difference in mean age between the patients with overweight T2DM and non-overweight T2DM The proportion of male patients with T2DM was higher than the control group (p = 0.03) The levels of fasting glucose, triglycerides were significantly higher in the patients with T2DM compared to control individuals (p < 0.05) The levels of insulin and homeostasis model assessment insulin resistance (HOMA-RI) were significantly higher in the patients with T2DM compared to control individuals (p < 0.001) In contrast, the levels of quantitative insulin sensitivity check index (QUICKI) and homeostatic model assessment β-cell function (HOMA-β) were significantly lower in the patients with T2DM compared to control individuals (p < 0.001) Leptin levels in patients with type diabetes mellitus and in controls Figure 1: Leptin level in type diabetes mellitus (T2D) patients and heathy controls (HC) (A) Leptin level was measured in the plasma of patients with T2DM and healthy controls and was compared between groups (B) Leptin level in patients with overweight/obese 44 Journal of military pharmaco-medicine No7-2016 (OV/O) T2DM and those with-without overweight/obese (WOV/O) T2DM and in OV/O and WOV/O control individuals P values were calculated by using Mann-Whitney U test The leptin levels were significantly lower in the patients with T2DM [median: 1202.75 pg/ml] compared to the controls [median: 1715.4 pg/ml] (p = 0.05) (figure 1A) Among the patients with T2DM, the leptin levels were significantly lower in the T2DM patients with WOV/O compared to those with OV/O T2DM (p < 0.05) Among the control group, the leptin levels were significantly increased in the overweight/obese individuals compared to the WOV/O individuals (p < 0.05) (figure 1B) Figure 2: Leptin level in male and female patients with T2DM, in the T2DM patients with and without MS and in overweight/obese levels of subjects The levels of leptin were significantly increased in female patients with T2DM and in T2DM patients with MS compared to those of male patients (p < 0.001) and T2DM patients with none-MS (p < 0.05) (figure 2A and 2B) The levels of leptin were significantly increased associated to BMI of subjects (p < 0.001) (figure 2C) 45 Journal of military pharmaco-medicine No7-2016 DISCUSSION Leptin, a hormone secreted by adipocytes in quantities which mainly reflect fat mass and serves as an important signal of body energy stores Leptin deficiency in mice and/or in humans is associated with neuroendocrine and metabolic abnormalities, including insulin resistance and diabetes All these abnormalities are corrected by exogenous leptin administration, suggesting that leptin plays a role in glucose homeostasis and possibly in the pathogenesis of other obesity-related metabolic complications [4] Previous studies have shown that plasma leptin levels are closely associated with adiposity [8], and its level correlated with increased MS components [9] Plasma leptin level in diabetes is still controversial; one recent study does not observe any significant difference between diabetic and non-diabetic subjects, others reported significant lower level in type diabetic subjects and type diabetics with similar adiposity [10] In line with other studies, our results revealed that leptin levels were significantly increased in OV/O patients with T2DM and in OV/O healthy individuals compared to patients with WOV/O T2DM and healthy individuals, respectively (figure 1) In addition, our investigations revealed that leptin levels were increased in female T2DM patients and in T2MS patients with MS The increasing leptin levels were associated with BMI of subjects (figure 2) These results indicate that increased leptin may potentially respond to the development of overweight/obese and T2DM and metabolic disorders 46 Recently studies shown that low leptin levels were also associated with the pathogenesis of many other diseases such as lipid metabolic disorders and obesity The leptin level was significant correlated to T2DM clinical parameters including insulin (r = 0.23, p < 0.05) and HOMA-β (r = 0.216, p < 0.05) and inversity correlated to QUICKI (r = -0.366, p < 0.0001) However, our data show that the leptin levels are not correlated with numerous clinical parameters such as triglycerides, cholesterol, HDL-C and LDL-C (data not shown) Those observations indicate that adiponectin levels involve in the development of T2DM Therefore, based on the levels of leptin, an adipose-derive cytokine could be useful for diagnosis of insulin resistance However, more studies are required to propose a new index and verify the diagnostic accuracy in clinical practice and to establish a cut-off value and reference range of insulin sensitivity for specific populations [11] CONCLUSON Our study demonstrated that the levels of leptin are significantly modulated during the development of overweight/obese and T2DM The leptin levels were significantly associated to BMI, genders and MS and with clinical parameters of obesity and T2DM Leptin may possibly modulate the pathogenesis of overweight and T2DM Acknowledgements We thank all the study subjects for their participation This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 106-YS.02-2014.36 Journal of military pharmaco-medicine No7-2016 REFERANCES IDF Annual Report 6th Edition 2013 Pham N M, Eggleston K Diabetes prevalence and risk factors among Vietnamese adults: findings from community-based screening programs Diabetes Care 2015, 38, e77-e78 Donath MY, Shoelson SE Type diabetes as an inflammatory disease Nat Rev Immunol 2011, 11, pp.98-107 Kahn SE, Hull RL, Utzschneider KM Mechanisms linking obesity to insulin resistance and type diabetes Nature 2006, 444, pp.840846 Guilherme A, Virbasius JV, Puri V et al Adipocyte dysfunctions linking obesity to insulin resistance and type diabetes Nat Rev Mol Cell Biol 2008, 9, pp.367-377 Kershaw EE, Flier JS Adipose tissue as an endocrine organ J Clin Endocrinol Metab 2004, 89, pp.2548-2556 Jung UJ, Choi MS Obesity and its metabolic complications: the role of adipokines and the relationship between obesity, inflammation, insulin resistance, dyslipidemia and nonalcoholic fatty liver disease Int J Mol Sci 2014, 15, pp.6184-6223 Matsuzawa Y, Hibi K, Kimura K Risk assessment for cardiovascular disease microvascular dysfunction Circ J 2010, 74, pp.1296-1297 Nishimura R, Sano H, Matsudaira T, Morimoto A, Miyashita Y, Shirasawa T et al Changes in body mass index, leptin and adiponectin in Japanese children during a three-year follow-up period: a population-based cohort study Cardiovasc Diabetol 2009, 8, p.30 10 Abu-Farha M, Behbehani K, Elkum N Comprehensive analysis of circulating adipokines and hs-CRP association with cardiovascular disease risk factors and metabolic syndrome in Arabs Cardiovasc Diabetol 2014, 13, p.76 11 Tatti P, Masselli L, Buonanno A, Di Mauro P, Strollo F Leptin levels in diabetic and nondiabetic subjects Endocrine 2001, 15 (3), pp.305-308 47 ... patients with type diabetes mellitus and in controls Figure 1: Leptin level in type diabetes mellitus (T2D) patients and heathy controls (HC) (A) Leptin level was measured in the plasma of patients with. .. were increased in female T2DM patients and in T2MS patients with MS The increasing leptin levels were associated with BMI of subjects (figure 2) These results indicate that increased leptin may... and without MS and in overweight/obese levels of subjects The levels of leptin were significantly increased in female patients with T2DM and in T2DM patients with MS compared to those of male patients