Ch201

To obtain HDL-C and LDL-C fractions, we used precipitating reagent to remove VLDL-C, and chylomicron fractions from plasma (HDL precipitant, catalog no. 135409990885, LDL precipitant, catalog no. 143309990885, DiaSYS respons®). For hepatic cholesterol, 2 mL of extraction reagent (chloroform: methanol 2: 1, v/v) was added to 0.5 g of tissue samples and homogenized. Ten-milliliter extracts were vacuumed-dried and concentrated. A commercial kit (CH201, Randox, UK) detected cholesterol concentration of specimens. One milliliter of working solution was added to prepared specimens; absorbance was read at 500 nm and 37 °C, VLDL-C calculated as total cholesterol-(HDL-C + LDL-C).

Plasma levels of aspartate aminotransferase (AST; AS521, Randox Laboratories, Antrim, UK), alanine aminotransferase (ALT; AL520, Randox Laboratories), total cholesterol (CH201, Randox Laboratories), and triglycerides (TR213, Randox Laboratories) were determined through enzymatic colorimetric methods using commercial kits (Randox Laboratories). In addition, levels of insulin (Mercodia AB, Uppsala, Sweden) and glycosylated hemoglobin (HbA1c; Fortress Diagnostics, UK) were determined.

Fasting blood samples (6 ml) for biochemical analyses were collected into EDTA-treated vacutainer tubes in the morning, after overnight fasting, before the commencement and one day after completion of the training program. Total antioxidant status (TAS) and total oxidative capacity (TOC) of the plasma were determined with the use of commercial diagnostic kits (DM P-4100, DM P-4200, LDN Labor Diagnostika Nord GmbH & Co., Germany). Serum 25(OH)D was assessed with an immunoenzymatic test (EIA-5810, DRG Instruments GmbH, Germany). Serum cytokine concentrations (IL-6 and IL-1β) were measured with an immunoenzymatic test (EIA-4640, EIA-4437, DRG Instruments GmbH, Germany). Total serum cholesterol (TC), HDL-cholesterol (HDL-C), and triglycerides (TG) were evaluated by enzymatic methods using commercially available diagnostic kits (CH201, CH203, and TR210, Randox, United Kingdom). LDL-cholesterol (LDL-C) concentrations were calculated with the Friedewald formula. The risk of cardiovascular disease was evaluated on the basis of lipid ratios (TC/HDL-C, LDL-C/HDL-C, and TG/HDL-C) and the atherogenic index of plasma (AIP = log₁₀ (TG/HDL), with TG and HDL-C expressed in molar concentrations) [25 (link)].

In this step, fasting blood glucose (FBG) in the tail vein blood sample was measured at the beginning of the experiment through a standard glucometer (Accu‐Chek Active) to ensure that the animals had euglycemia. Following the HFD/STZ‐induced T2D, blood glucose level was measured to assess the initiation of hyperglycemia (FBG 200 mg/dl). Moreover, the enzyme‐linked immunosorbent assay (ELISA; Cat No: 10‐1250‐01, Mercodia) was utilized to measure the plasma insulin levels in the rats that had been fasted for 4 h. In addition, total concentrations of serum cholesterol, high‐density lipoprotein cholesterol (HDL‐C), and triglycerides (TG) were assessed by enzymatic methods using commercially available diagnostic kits (CH201, CH203, and TR210, respectively), based on the company's directions (Randox Laboratories Ltd., Crumlin, Co.).