Leptin

At the end of experiment, blood was collected from the tail vein and centrifuged (4,000 rpm, 4°C, 20 min) to separate sera that were used to determine glucose, TGs, TC and HDL-C using Randox colorimetric reagent kits (Antrim, United Kingdom). LDL-C was calculated according to the Friedewald equation (Friedewald et al., 1972 (link)):
The serum levels of insulin (RayBiotech, Inc., Norcross, GA, United States; Cat #: ELR-insulin), fructosamine (MyBioSource, Inc., San Diego, CA, United States; Cat #: MBS2601586), leptin (RayBiotech, Inc., Norcross, GA, United States; Cat #: ELR-leptin), adiponectin (Invitrogen, Carlsbad, CA, United States; Cat #: KRP0041), and GLP-1 (LifeSpan BioSciences, Inc., Seattle, WA, United States; Cat #: LS-F23155) were analyzed using commercially available ELISA kits according to the manufacturers’ instructions.
The HOMA-I/R was determined using the following formula (Matthews et al., 1985 (link)):
HOMA-I/R = (serum glucose (mmol/L) × serum insulin (μIU/ml))/22.5

Plasma was used for biochemical measurements with reagents from Randox Laboratories Ltd. (County Antrim, UK). Plasma Glucose was measured by Glucose Oxidase method, Total Cholesterol by Cholesterol Oxidase method, Total Triglycerides by Lipase/GPO-PAP method. Glycated hemoglobin (HbA1c) was measured by HPLC (D10 Hemoglobin analyzer, Bio-Rad, Hercules, CA, USA). Plasma Leptin (RayBiotech, Norcross, GA, USA), Insulin (Merck Millipore, MA, USA), DPP4 (R&D Systems, MN, USA), and GLP-1(Active) (Merck Millipore, MA, USA) levels were measured by ELISA. Homeostatic model assessment (HOMA2) designed by Diabetes Trials Unit, The Oxford Center for Diabetes, Endocrinology, and Metabolism was used to estimate Insulin resistance (HOMA2 IR) from all fasting venous samples.