Human insulin

4′,6-diamidino-2-phenylindole (DAPI), Sigma-Aldrich, D9542; Aspirin, Sigma-Aldrich, A2093; 7,8-dihydroxyflavone (7,8-DHF), Tokyo Chemical Industry Co., Ltd., D1916; Human insulin, Eli Lilly, 00002831501; d-glucose, RPI, G32040.

For the GTT, C57BL/6 mice were overnight fasted (15 h) after five weeks of treatment and injected with 2 g/kg body weight D-glucose into the peritoneal cavity. Blood samples were obtained from the tail vein of each mouse and levels of glucose were determined at indicated time points (0, 15, 30, 60, 90, and 120 min). For the ITT, 5 h fasted mice (from 6 am to 11 am) after six weeks of treatment, the glucose concentrations were measured at 0 and 15, 30, 60, 90, and 120 min after injection of human insulin (Eli Lilly) at 1 U/kg body weight. Plasma glucose concentration was measured using a YuYue Ultra Glucose Meter (LifeScan, Inc., Milpitas, CA, USA).

Yeast α-glucosidase, acarbose, ursolic acid, p-nitrophenyl phosphate (pNPP), p-nitrophenyl α-d-glucopyranoside (pNPG), and ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma-Aldrich. PTP1B (human recombinant) was purchased from Biomol^® International LP (Plymouth Meeting, PA, USA), and dithiothreitol (DTT) was purchased from Bio-Rad Laboratories (Hercules, CA, USA). Minimum essential medium (MEM), penicillin-streptomycin, 0.25% trypsin (EDTA), fetal bovine serum (FBS), sodium pyruvate and non-essential amino acids were purchased from Gibco-BRL Life Technologies (Grand Island, NY, USA).The fluorescent d-glucose analogue and glucose tracer 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG) was purchased from Life Technologies (Carlsbad, CA, USA). Human insulin was purchased from Eli Lilly (Fegersheim, France). All other chemicals and solvents used were purchased from Merck and Sigma-Aldrich, unless otherwise stated.

Glucose tolerance tests were performed by fasting the mice overnight for 16 hr and then administering glucose (1 g/kg) intraperitoneally (Arriola Apelo et al., 2016b (link)). Insulin tolerance tests were performed by fasting mice overnight for 16 hr, and then injecting 0.75 U/kg human insulin (Eli Lilly) intraperitoneally. Blood glucose was measured periodically for 2 hr after administration of glucose or insulin using a Bayer Contour blood glucose meter and test strips. For glucose-stimulated insulin secretion, mice were fasted overnight for 16 hr, blood glucose levels were determined and plasma was collected immediately prior to and 15 min after administering glucose (1 g/kg) intraperitoneally. Plasma insulin was quantified according to the manufacturer’s protocol using an ultrasensitive mouse insulin ELISA kit (90080) from Crystal Chem. Fasting glucose and insulin levels were then used to calculate HOMA2-IR (Levy et al., 1998 (link)).

The insulin tolerance tests (ITT) were carried out with WT (n = 6) and Adipoq^−/− (n = 7) male mice. Blood glucose concentration was measured using OneTouch Ultra blood glucose meter and test strips (LifeScan). For ITT, mice were i.p. injected with human insulin (Eli Lilly) at a dose of 1.0 U/kg of body weight following a 6 h of fasting period in the morning. Blood glucose was assessed at 0, 30, 60, 90 and 120 min after injections. For glucose tolerance tests (GTT), mice were i.p. injected with glucose at a dose of 2.0 g/kg body weight following a 18 h overnight fasting. The blood glucose was measured at 0, 15, 30, 60 and 120 min after injection.

For GTT, the mice were fasted for 18 h and received an intraperitoneal (i.p.) injection of D-glucose (2 mg/g body weight). For ITT, the mice were injected intraperitoneally with human insulin (Eli Lilly; 0.8 mU/g body weight) after 6 h fasting. Blood glucose levels were measured at 0, 15, 30, 60, 90 and 120 minutes after injection (a glucometer monitor, Roche). For the GTT/ITT assays, the area of the curve (AOC) was calculated using the conventional trapezoid rule. Levels of serum alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured using the kits from Applygen, China (#E2021 and E2023, respectively) according to the manufacturer’s instructions.

After an overnight fasting, [3-³H]-glucose (HPLC purified; Perkinelmer, USA) was infused at a rate of 0.05 μCi min⁻¹ for basal 2 h to assess the basal glucose turnover. Following the basal period, hyperinsulinemic-euglycemic clamp was conducted for 150 min with a primed/continuous infusion of human insulin (21.4 mU per kg during priming and 3 mU per kg per min during infusion, Eli Lilly), while plasma glucose was maintained at basal concentrations as previously described31 (link) with slight modification. To estimate insulin-stimulated whole body glucose fluxes, [3-³H]-glucose was infused at a rate of 0.1 μCi min⁻¹ throughout the clamps.