Automatic analyzer

After 12-hour overnight fast, blood samples
were collected. Serum and plasma
samples were separated using a centrifuge
(Beckman Coulter Inc., USA) at 1500 rpm
for 15 minutes. Fasting insulin levels were
measured using enzyme-linked immunosorbent
assay (ELISA) kits (Monobind Inc.,
USA). Fasting plasma Glu was measured using
enzymatic procedures by an automatic
analyzer (Abbott, USA). IR was estimated
by HOMA using the following formula: HOMA-
IR=fasting insulin (μU/ml)×fasting Glu
(mg/dl)/405 (10 (link)). The concentrations of TC
and TG were measured using enzymatic procedure
with commercial kits (Pars Azmon,
IRI), while HDL-C was measured by a direct
method using polyethylene-glycol-pretreated
enzymes by an automatic analyzer (Abbott,
USA). LDL-C was calculated using Friedewald’s
formula (11 (link)). Lipoprotein ratios (TC/
HDL-C, TG/HDL-C and LDL-C/HDL) were
then calculated.

Serum TG, total cholesterol (TC), low density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C), alanine aminotransferase (ALT), and aspartic acid transaminase (AST) levels were determined using an automatic analyzer (Abbott Diagnostics, Abbott Park, IL, United States). The mice were made to fast overnight prior to intraperitoneal injection of glucose (IPGTT) at the 18th week, as described previously (Hong et al., 2018 (link)). Tail vein blood was collected, and fasting blood glucose was measured using Accu-Chek (Roche, Mannheim, Germany).

The mice were kept in metabolic cages (Fengshi Inc., Suzhou, JS, China) and 24 h urine samples were collected every 4 weeks for urine volume and urine albumin-to-creatinine ratio (UACR) measurement. All the mice were fasted overnight before collection of blood samples and euthanasia. Serum triglycerides (TG), total cholesterol (TC), low-density lipid cholesterol (LDL-C), high-density lipid cholesterol (HDL-C), and urine creatinine were measured using an automatic analyzer (Abbott Diagnostics, Abbott Park, IL, USA). Urine albumin was measured by enzyme linked immunosorbent assay (ELISA) using an ELISA Quantitation Set kit (Bethyl Laboratories Inc., Montgomery, TX), according to the manufacturer's instructions.