Is130d

Plasma glucose was assessed using the glucose oxidase method via an automated analyzer (Analox GM7, Analox Instruments Ltd., Lunenburg, MA, USA). Serum insulin concentrations were determined via commercially available enzyme-linked immunosorbent assay kits (IS130D; Calbiotech, Inc., Spring Valley, CA, USA) and a spectrophotometer (Eon, BioTek Instruments, Inc., Winooski, VT, USA). Following analyses, total area under the glucose and insulin curves (AUC_glu and AUC_ins, respectively) were calculated using a standard trapezoidal method. To eliminate interassay variance, all samples were analyzed in duplicate by a single technician. The coefficient of variation for each assay was 0.87% for glucose and 8.24% for insulin.

Plasma insulin levels were measured by ELISA kit (IS130D, Calbiotech, Spring Valley, CA), by a customized Milliplex surface fluorescent-coded magnetic bead assay (HMHMAG-34K, Millipore, Billerica, MA), and/or by porcine ELISA kit (Cat #10-1200-01, Mercodia, Uppsala, Sweden). Insulin measurements using the Milliplex and porcine ELISA assays were standardized to responses of the Calbiotech kit by 12 co-measures across a range (1–50 µM/mL) of ferret insulin concentrations. Plasma cortisol was measured using an ELISA kit (CO103S, Calbiotech). Plasma glucagon, PP, GLP-1, and GIP were measured using the same Milliplex assay as used for insulin. Real-time glucose levels were measured on whole blood withdrawn from the arterial catheter using a glucose meter (One Touch, LifeScan Inc.,Milpitas, CA).