80 insmsu e01

All metabolic analyses were conducted in conscious, restrained mice, and blood samples were collected via the saphenous vein using heparinized microhematocrit tubes. Blood glucose levels were measured using a handheld glucometer (LifeScan, Burnaby, BC, Canada).
Body weight and blood glucose were measured weekly or 2x/week following a 4-h morning fast. Saphenous blood was collected at GD15.5, P1, lactation (P14), and weaning (P21) following a 4-h morning fast to measure plasma insulin levels by ELISA (#80-INSMSU-E01, ALPCO, Salem, NH, USA). For all metabolic tests, time 0 indicates the blood samples collected prior to the administration of glucose or insulin. For glucose tolerance tests (GTTs), the mice received an i.p. bolus of glucose (2 g/kg) following a 4-h morning fast. Blood samples were collected at 0, 15, and 30 min to measure plasma insulin levels by ELISA. For insulin tolerance tests (ITTs), the mice received an i.p. bolus of insulin (0.7 IU/kg: Figure 2, Figure 4D, and Supplemental Fig. S4B; 1.0 IU/kg: Figure 4B) (#02024233, Novolin ge Toronto, Novo Nordisk Canada) following a 4-h morning fast. Cardiac blood was collected at week 11 of the metabolic challenge to measure non-fasted plasma leptin levels (#90030, Crystal Chem, Elk Grove Village, IL, USA), proinsulin levels (ALPCO, #80-PINMS-E01), and insulin levels (ALPCO, #80-INSMSU-E01) by ELISA.

Triglyceride, glycerol (TR0100, Sigma-Aldrich) and NEFA (NEFA-HR(2), Wako Chemicals GmbH) were measured in plasma and supernatant of liver explants and insulin concentration in plasma by ELISA (80-Insmsu-E01, ALPCO Diagnostics) following manufacturer's protocol.^{18 (link)}