Serum glucose concentration in obese type 2 diabetic patients on metformin and glimepiride therapy. 

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... double by the year of 2030 . Type 2 DM (T2DM) is a more complex metabolic disorder characterized by obesity, impaired β -cell function, increased endogenous hepatic glucose output and insulin resistance (IR) in target tissues (2) . DM and obesity have a complex relationship where obesity may be a precursor for T2DM following IR (3) . Obesity is associated with decreased responsiveness to insulin in muscle, liver and fat. On the other hand, weight gain associated with insulin therapy need to increase insulin dose and subsequently greater weight gain and obesity (4) . Not all subjects with T2DM are obese and many obese subjects do not have DM, but most of the subjects with T2DM are overweight or obese. These are largely preventable with change in life style and avoidance of sedentary habits and over- consumption of energy (5) . Insulin is a small protein hormone, with a molecular weight of about 6000 Daltons composed of two chains held together by disulfide bonds and synthesized in significant quantities only in β -cells in the pancreas. Binding of insulin to extracellular portion of the receptor activates its kinase activity resulting in autophosphorylation of specific intracellular tyrosine residues (6) . IR and a relative deficiency in insulin secretion contribute to the pathogenesis of T2DM (7) . Glimepiride is a potent sulfonylurea and is associated with a low rate of hypoglycemia (0.9 – 1.7%). In addition to effects on pancreatic B-cell function, glimepiride also may enhance tissue sensitivity to insulin and has a favorable safety and efficacy profile with once-daily dosing of 1-8 mg/day (8) . The antidiabetic biguanide Metformin is one of the most prescribed, first- line medications in the treatment of T2DM. In contrast to (sulphonylureas and insulin), metformin does not cause weight gain and can lead to significant weight loss (9) . One of the known targets of metformin action is the intracellular signaling enzyme, adenosine monophosphate-activated protein kinase (AMPK). In the liver and muscle, AMPK activation reduces hepatic gluconeogenesis and promotes fatty acid oxidation, respectively (10) . The consequences of insulin resistance manifest at many levels in metabolic processes, including glucose intolerance, overt hyperglycemia, hyperinsulinemia, and atherogenic dyslipidemia, collectively referred to as metabolic syndrome (11) . The role of visceral or intra-abdominal accumulation of adipose tissue seems to be strongly associated with metabolic syndrome rather than upper body subcutaneous fat . Visceral adipose tissue works as an active endocrine organ able to secrete a wide variety of inflammatory cytokines and hormones with key functions in the development of DM (13) . Recent evidence has demonstrated the complex function of adipose tissue as an endocrine organ through release of hormones into the blood stream involved in physiological activities of the body with potential implication in insulin resistance, obesity and diabetes. One of the most important of these hormones is recently discovered ghrelin, which is a 28 amino acid peptide hormone, primarily produced by the stomach; it has an octanoyl group on the serine at the third position in the amino acid chain which gives the peptide hormone its biological activity (14) . Ghrelin is an appetite-stimulating hormone that increases growth hormone secretion and food intake in animals and humans (15) . The appetite stimulating effects are thought to be mediated via the arcuate nucleus of the hypothalamus and the messenger peptides neuropeptide Y and Agouti-related protein (16) . Leptin, is a single-chain proteohormone with a molecular mass of 16 kDa that is thought to play a key role in the regulation of body weight, it is produced by differentiated adipocytes, although production has been demonstrated in other tissues, such as the fundus of the stomach, skeletal muscle, liver and the placenta (17) . Leptin is a hormone that works as a mediator in the stomach – hypothalamus pathway and provides information about the body's energy storage in adipocytes in addition, its level is associated with obesity (18) . Leptin acts on the central nervous system (CNS), in particular the hypothalamus, suppressing food intake and stimulating energy expenditure (19) . Evidence suggests that circulating ghrelin may work in concert with leptin as an adiposity signal in the CNS (20) . Whether low or high concentration of such hormones is primary event in DM or secondary to anti diabetic drugs is unclear and also its relationship is ambiguous. Therefore, this study was designed to evaluate and compare the possible effects of using metformin or glimepiride on serum concentrations of ghrelin, leptin and IR in obese T2DM patients in Basra, Iraq and to predict the relationship between above-mentioned parameters in the study groups. Forty T2DM obese patients were attended the private medical clinic of Dr. Qais Ali Aljazaari in Basra, Iraq, from Sep. 2013 to Mar. 2014 during their periodic visit seeking for medical advice concerning their diet modification, weight reduction and drug prescription. In addition to 20 healthy subjects with age and sex matched group served as the control group. We divided the patients into 2 groups (20 patients in each) according to their drug used to treat DM: Group 1 includes T2DM patients on glimepiride therapy while group 2 includes T2DM patients on metformin treatment for at least 4 months in both groups. The inclusion criteria were those with BMI < 30 kg/m 2 and age range between 35-50 years old. The exclusion criteria includes those with BMI > 30 kg/m 2 , patients with chronic disease other than DM, pregnant or lactating female patients and any patient with renal or hepatic impairment, and those who are on treatment with drugs, which could interfere with the tested parameters. The study also excluded the patients with any obvious major complications of DM, including heart diseases and patients who were taking other drugs like lipid lowering medications. Blood samples were taken after at least 8 hours of fasting in all the participants and subjects must refrained from strenuous physical activity for at least 2 hours. Serum glucose was measured by the glucose-peroxidase colorimetric enzymatic assay method. Insulin concentrations were determined by the DRG Insulin ELISA Kit, it is a solid phase ELISA based on the sandwich principle. The microtiter wells are coated with a monoclonal antibody directed towards a unique antigenic site on the Insulin molecule with range of assay between 1.76-100 μIU/mL. Regarding leptin levels were measured by using The DRG Leptin ELISA Kit. The microtiter wells are coated with a monoclonal antibody directed towards a unique antigenic site on a Leptin molecule. An aliquot of specimen sample containing endogenous leptin is incubated in the coated well with a specific biotinylated monoclonal anti Leptin antibody. Serum ghrelin levels were measured by enzyme Immunoassay DRG- kit were designed to detect a specific peptide (ghrelin) based on the principle of “competitive” enzyme immunoassay. The immunoplate in this kit was pre-coated with secondary antibody and the nonspecific binding sites are blocked. The secondary antibody can bind to the Fc fragment of the primary antibody (peptide antibody) whose Fab fragment will be competitively bound by both biotinylated peptide and peptide standard or targeted peptide in sample. IR was assessed using the (HOMA-IR) according to the formula: fasting insulin (μIU/ml) x fasting glucose (mmol/l)/22.5 (21) . Values were expressed as mean ± SD; this values were statistically tested using unpaired Student's t-test and one way analysis of variance (ANOVA), supported by Bonferroni’s post hoc analysis. Values with P < 0.05 considered significantly different. Analysis was performed using GraphPad Prism software for Windows (version 5.0, GraphPad Software, Inc., San Diego, CA). Fasting serum glucose concentrations were significantly increased in both meformin and glimepiride treated groups as compared with control but these concentrations with in upper limit of normal fasting serum glucose range (Fig. 1). The highest serum insulin concentration and IR levels were clearly reported in glimepiride treated group as compared to control and metformin treated group this result clearly summarized in fig. 2 and 3. Regarding leptin our finding show no significant differences in all studied groups, Obese T2DM patients on metformin therapy had higher leptin levels compared to obese control, but not significantly so as shown in fig. 4. Ghrelin levels were significantly lower in obese T2DM patients on both metformin and glimepiride therapies as compared with control group. Ghrelin levels were significantly lower in metformin group as compared to glimepiride treated group (Fig. 5). Table 1 demonstrate the relationship between BMI, IR and studied parameters (ghrelin, leptin and insulin) the result showed there were significant correlation between IR and ghrelin, In all subjects, no correlation observed between BMI and tested parameters was observed. The relationship between ghrelin, leptin and insulin among three groups are summarized in fig. 6-8. Positive correlation was observed between ghrelin and insulin in metformin treated group in other hand our finding showed negative correlation between ghrelin and leptin in control group. leptin and insulin in metformin treated group while the correlation observed with insulin only in glimepiride and control ...