Leptin levels in obese women with and without type 2 diabetes mellitus.

INTRODUCTION: The role of leptin has been more clear in the endocrinology area after the discovery of its secretion from the adipose tissue. The aim of the study is to investigate the leptin levels in obese women in whom type 2 diabetes mellitus were present or absent. MATERIALS AND METHODS: Thirty-five obese women with type 2 diabetes mellitus (group 1) and 34 obese women without type 2 diabetes mellitus (group 2) were enrolled in the study. In both groups the body mass index (BMI), waist circumference, and waist-to-hip ratio were measured. Leptin, HbA1c, creatinine and the lipid profile were assessed. RESULTS: Leptin was found to be statistically significantly lower in group 1 than in group 2 (40.22 +/- 17.77 ng/ml versus 50.12 +/- 15.51 ng/ml, respectively; p = 0.019). It was well correlated with BMI in group 1 (r = 0.60, p = 0.0001). In group 1 also, correlation of leptin was moderate with creatinine and high-density lipoprotein-cholesterol (r = 0.36, p = 0.037 versus r = 0.37, p = 0.027, respectively), whereas triglyceride had a negative correlation (r = -0.34, p = 0.046). In group 2, the only significant correlation with leptin was BMI (r = 0.41, p = 0.02). Leptin was also significantly lower in 17 subjects with poorly controlled diabetes mellitus than in 18 well-controlled diabetics (33.54 +/- 15.82 ng/ml versus 44.61 +/- 17.54 ng/ml, respectively; p = 0.038). CONCLUSION: Since leptin is lower in obese women with diabetes than without diabetes and additionally it is even lower in the poorly controlled diabetes subgroup, we think that further studies a rerequired to make clear the issue for lower leptin levels, whether it is a reason or an outcome.


Introduction
Obesity that is associated with a chronic low-grade inflammation state is an increasing worldwide health problem. 1 In the pathogenesis, a genetic tendency, disturbances of thermogenesis in the central nervous system, and impairment of the signals from the adipose tissue on the way to the brain are believed to play a major role. 2,3It is gaining more and more importance since the mechanism leading to type 2 diabetes mellitus */another epidemic metabolic issue */in both adolescents and adults is more comprehensible after recent studies. 4eptin together with other molecules that are secreted from the adipose tissue does affect the insulin sensitivity, and it is accepted to play a role in the pathogenesis of obesity-related disorders 5,6 via stimulating vascular inflammation and oxidative stress that may contribute to pathogenesis of athero-sclerosis and other cardiovascular complications of obesity. 7,8It has also a link with nutritional status and energy balance */which are both important in the physiopathology of diabetes */and proinflammatory T-helper 1 immune responses. 9Strong positive correlations found between interleukin-6, tumor necrosis factor alpha, leptin and serum C-reactive protein suggest that cytokines secreted by adipose tissue in obese subjects may play a role in increased inflammatory proteins secretion by the liver. 8,10Its possible role in liver disease as a fat deposition, in inflammation and fibrosis, 11 in overexpression in intestinal inflammation, 12 by activating nuclear factor-kap-paB, 13 and inappropriate increase independent of body mass index (BMI) in acute ulcerative colitis 14 have been studied by many investigators.
Approximately 80% of the subjects with type 2 diabetes mellitus are obese.As long as the obesity continues, the risk of development of type 2 diabetes mellitus increases at that rate. 15It has been found that subjects who have obesity for over 10 years have a two times greater risk for diabetes than the subjects who have obesity for less than 5 years. 15In gestational diabetes mellitus, major changes were in the expression profile of placental genes with a prominent increase in markers and mediators of inflammation. 16Regarding this, upregulation of interleukins, leptin and tumor necrosis factor alpha receptors and their downstream molecular adaptors indicate an activation of pathway recruiting stress activated protein-kinases leading to reorganization of placenta. 16n an eastern Mediterranean area of Turkey where the prevalence of type 2 diabetes is quite high 17 we aimed to investigate leptin levels in obese female subjects with type 2 diabetes mellitus and in a small group with poor glycemic control.

Subjects
In the study, subjects were collected consecutively from the Outpatient Department of Internal Medicine, Kahramanmaras Sutcu Imam University, Faculty of Medicine.All the participants were obese women with normal creatinine levels.Subjects with type 2 diabetes mellitus (group 1) numbered 35 and those without diabetes (group 2) numbered 34.All diabetic subjects met the American Diabetes Association criteria for type 2 diabetes mellitus. 18Patients having HbA1c greater than 8.5% were accepted as poorly controlled diabetes.Seventeen subjects were poorly controlled diabetics.
The height (m), weight (kg) and BMI (kg/m 2 ) were recorded.All subjects had a BMI equal to or greater than 30 for participating in the study as the obese subject.The waist (waist circumference was measured with a soft tape on standing subjects, mid-way between the lowest rib and iliac crest), hip (hip circumference was measured over the widest part of the gluteal region) and therefore waist-to hip-ratio were all measured by the same investigator so as to minimalize interobserver variability during each measurement.
For group 2, in order to exclude new onset diabetes, subjects who were known not to have diabetes by taking history were asked to drink 75 g of glucose for the oral glucose tolerance test.
Local Research Ethics Commitee approval was taken for the study before it commenced.Patients were also asked to sign a consent form.
Physical examinations, BMI, waist and hip measurements and routine laboratory measurements except leptin were performed in Kahramanmaras Sutcu Imam University Faculty of Medicine.

Samples and measurements
Before collecting venous blood samples using standard venipuncture into plain tubes, subjects were asked to have a fasting period of 12 h.For the standardization blood was drawn in a sitting position from the antecubital vein.Leptin, total cholesterol, low-density lipoprotein-cholesterol, high-density lipoprotein (HDL)-cholesterol, triglyceride and creatinine were kept in plain tubes, whereas for HbA1c special tubes containing K 3 EDTA were provided so as not to give rise to coagulation.All measurements were performed daily except for leptin.Leptin levels were measured at the Laboratories of Department of Biochemistry, Gaziantep University, Faculty of Medicine.
Lipids and creatinine levels were measured by biochemical autoanalyzer via enzymatic-calorimetric method (Roche-Hitachi Modular Analytics, Tokyo, Japan).Low-density lipoprotein cholesterol was calculated via the Friedewald formula. 19For HbA 1 c an autoanalyzer (DCA 2000; Bayer, USA) was used via the method of inhibition of latex agglutination.
Sera that were separated immediately after centrifugation with 3000 )/g for 10 min were stored at (/208C until the assays for leptin were performed and the last subject fulfilling the inclusion criteria was admitted.Serum leptin levels were measured using the enzyme-linked immunosorbent assay kit (Diagnostic Systems Laboratories, Inc., Webster, TX, USA).Analytical sensitivity of the leptin assay was 0.05 ng/ml.The intra-assay coefficient of variation was 3.7% and the interassay coefficient of variation was 4.43%.

Statistical analysis
Results are presented as the mean9/standard deviation.Data collected were analyzed by SPSS for Windows package (version 9.05; SPSS, Chicago, IL, USA).Leptin levels of group 1 and group 2 were compared via an independent t -test.Correlation of leptin with the other parameters were evaluated via Pearson correlation analyses.For the comparison of the leptin levels between subgroups who had well and poorly controlled diabetes mellitus the Mann Á/ Whitney U-test was used since the distributions of the data were not normal.For all statistical assessments a value of p B/0.05 was accepted to be statistically significant.

Results
Baseline characteristics and biochemical parameters of subjects in the diabetic and control groups are summarized in Table When we compared group 1 and group 2 regarding weight, height, waist circumference, hip circumference, waist-to-hip ratio, creatinine and lipid profile, no difference was observed (Table 1).

Discussion
After the discovery of leptin secretion from the adipose tissue, its role has been more clear in the endocrinology area.Even though leptin limits food ingestion and increases energy expenditure, it has been found high in obesity.This can be explained by the statement that ''obesity may be the consequence of leptin resistance''. 20Decreased levels of leptin concentrations during food deprivation leading to impaired immune function gave rise to spending more time to understand role of this molecule in chronic inflammation and autoimmunity together with therapeutic implications of its modulators. 21erum leptin levels are found higher in women than in men, 20,22 Á 25 and this is probably owing to adipose tissue in women being higher than in the opposite sex, the existence of negative correlation between leptin and testosterone levels, 22,26 and the stimulation of mRNA production by 17b-estradiol, which is one of the female sexuality hormones. 27All the subjects enrolled in our study were female obese subjects and we found no correlation between the  age and serum leptin levels.Á 24,28 Á 33 In our study, also, leptin had a correlation with BMI both for subjects with diabetes and subjects who did not have diabetes.The BMI seems to be applied easily at outpatient departments; however, obesity itself cannot be defined simply by this method only.Therefore, waist and hip circumferences and the waist-to-hip ratio have all been performed.As a result, we found no relationship with leptin and waist or waist-to-hip ratio measurements in diabetic and non-diabetic groups.
Leptin correlated positively with HDL-cholesterol and negatively with triglycerides in the diabetic group, as in the study where serum leptin concentrations were investigated in a group of patients with moderate and severe obesity. 24The same correlations were found to be independent of age and gender in a Caucasion population. 30reatinine was found to have a correlation with leptin in our diabetic group.This was matching with the findings of another investigation, but none of the participants had diabetes in that study. 34Leptin is known to accumulate in a uremic state; however, leptin levels and renal creatinine clearence showed no significant correlation in peritoneal dialysis patients. 35eptin has the ability of regulation of insulin secretion from the pancreatic islet cells. 36,37After leptin was given to mice who had leptin deficiency, it has been demonstrated that there had been a decrease in hyperglycemia and hyperinsulinemia, inhibition in hepatic gluconeogenesis and insulin secretion via direct effects on beta cells. 29,38,39ome investigators have found that in more obese patients with type 2 diabetes mellitus, the leptin levels were less in patients with not well controlled diabetes than in well-controlled diabetic subjects. 40his was related to the insulin deficiency.In our study also, leptin levels were significantly lower in patients who have HbA1c above 8.5%.It has been found that untreated diabetes gave rise to a reduction in leptin levels owing to an ineffective release of insulin by the monodrug therapy. 41An absence of leptin receptor expression in human diabetic foot ulcer, which is the consequence of poor glycemic control for many years, was well documented while investigating the role of leptin for the inflammatory response in diabetes-impaired skin repair. 42This may show that leptin itself might function as a regulatory link between endocrine and immune system in the context of skin repair. 42However, contrarily to these findings, no significant relation between leptin levels and the degree of the severity of diabetes was observed in another study. 23bdominal fat tissue that can be detected by densitometry, hydrometry, dual-energy X-ray absorbtiometry, a chemical multicompartment model, bioelectrical impedance, computerized tomography and magnetic resonance imaging is also well correlated with leptin levels. 31Leptin's relationship with total body fat and insulin resistance was independent of age and gender. 24,25,43In our study we did not investigate the abdominal fat tissue percentage in the participants with the aforementioned instruments and imaging techniques.
We speculate that as a consequence of insulin deficiency and the different distribution of fat tissue throughout the body, we have found low leptin levels in diabetic subjects and even lower levels in subjects who have poorly controlled diabetes.Further large-scale studies */which should investigate the physiopathologic mechanisms */are required to make clear the issue for lower leptin levels, whether it is a reason or an outcome.

Table 1 .
Baseline characteristics and biochemical parameters of subjects with diabetes and the control group

Table 2 .
Correlations of leptin levels with biochemical parameters in diabetes and control groups