80 insrt e01

Glucose (GAGO-20, Sigma-Aldrich, Gillingham, UK), triglycerides (LabAssay Trigylceride, Wako, Neuss, Germany), non-esterified fatty acids (NEFA-HR(2), Wako, Neuss, Germany), insulin (80-INSRT-E01, Alpco, Salem, NH, USA) and leptin (EZRL-83K, Merck, Darmstadt, Germany) were measured as previously described following the manufacturer’s instructions [8 (link)].

Fasting plasma was analyzed in male offspring using commercial ELISAs for leptin (Cat. no. EZRL-83K; EMD Millipore, Billerica, MA, USA) and insulin (Cat. no. 80-INSRT-E01, ALPCO, New Hampshire, USA) at birth and 17 weeks post-weaning. Post-weaning plasma leptin concentrations were adjusted for total visceral fat-pad mass (g). Fasting plasma glucose (Cat#10009582, Cayman Chemical Co., Ann Arbor, MI, USA) was measured at 17 weeks post-weaning, and the homeostatic model assessment of insulin resistance (HOMA-IR) was calculated as:
Post-weaning total plasma free fatty acids (FFA, Cat# ab65341, Abcam) and plasma triglycerides (Cat#10010303, Cayman Chemical, Ann Arbor, MI, USA) were also measured according to the manufacturers’ instructions.

Insulin was measured by a rat‐specific ELISA kit, as per the manufacturer's instructions (80‐INSRT‐E01; ALPCO, Salem, NH, USA). Levels of triglycerides (ab65336) and cholesterol (total cholesterol, high‐density lipoprotein [HDL], low‐density lipoprotein [LDL]/very low‐density lipoprotein [VLDL]; ab65390) were quantified according to the manufacturer's instructions (Abcam, Cambridge, UK). Plasma was diluted 1:20 before it was assayed for insulin, triglycerides, and total cholesterol. HDL and LDL/VLDL fractions were diluted 1:4. All dilutions were performed using the appropriate sample buffer specified in the manufacturer's protocol. Natural product studies are reported in compliance with the recommendations made by the British Journal of Pharmacology (Izzo et al., 2020 (link)).

Blood glucose was measured using glucometer, unless reading exceed glucometer capabilities, in which case, plasma glucose was measured using a glucose analyzer (Analox GM9, Stourbridge, UK). Fed and fasted plasma insulin was measured via ELISA following manufacturer’s protocol (Cat. # 80-INSRT-E01, Alpco, Salem, NH, USA). Circulating leptin at 8 week, 16 week, and 24 week timepoints was measured using multiplex assay following manufacturer’s protocol (Cat. # RECYTMAG-65K, Millipore, Burlington, MA, USA). Vena cava leptin and portal glucagon-like peptide-1 (GLP-1) from control and OFS-treated rats was determined by ELISA following manufacturer’s protocol (Cat. #EGLP-35K, Millipore; Cat. # EZRL-83K, Millipore).

Plasma was thawed gently on ice. Plasma concentrations of glucose (GAGO-20, Sigma-Aldrich, Gillingham, United Kingdom), triglycerides (GPO DAOS method, Wako, Neuss, Germany), non-esterified fatty acids [NEFA-HR(2), Wako, Neuss, Germany], insulin (80-INSRT-E01, Alpco, Salem, NH, United States), corticosterone (K014-H1, Arbor Assays, Ann Arbor, MI, United States) and leptin (EZRL-83K, Merck, Darmstadt, Germany) were measured with commercial assays, respectively, following manufacturer’s instructions.

Triglyceride levels in plasma were measured using commercial kits (AM 157S-K, ASAN, Korea). Levels of HDL, total cholesterol, and low-density lipoprotein (LDL) in plasma were measured using HDL and LDL assay kits (E2HL-100, Bio Assay Systems, Germany). Levels of insulin in plasma were measured using commercial kits (80-INSRT-E01, ALPCO, UK). Levels of C-reactive protein (CRP) in plasma were measured using commercial kits (557825, BD Biosciences, America). Levels of leptin in plasma were measured using commercial kits (ab100773, Abcam, UK). Levels of T-Bill and BUN in plasma were measured using commercial kits (77184, Arkray, Japan).