Figure 3. LOKO mice are glucose intolerant but insulin sensitive.

(A) Borderline fasting hyperglycemia and insulinopenia in LOKO mice. Mice were fasted overnight and glucose measured by glucometer, and insulin, measured by multiplexed bead assay (Luminex, Millipore) (N=5 per group). (B) Random blood glucose levels from a separate group of OB and LOKO mice, showing significant hyperglycemia. Similar results were obtained in 3 separate experiments. Glycosuria in LOKO mice is shown by chemical reaction with clinical urine test strips. (C) Glucose tolerance tests were performed by I.P. injection of 1 mg/kg glucose into OB and LOKO mice (N=5 per group) fasted for 6 h. Time course measurements of blood glucose and insulin from the same experiment are shown. Values are means +/− SEM. This experiment was repeated 3 times in 2 different cohorts of mice. (D) LOKO mice are highly insulin sensitive in response to exogenous insulin. A separate cohort of OB (N = 15) and LOKO (N = 8) was analyzed by the hyperinsulinemic-euglycemic clamp technique as described in Methods. ISGDR, insulin-stimulated glucose disposal rate; HGP, hepatic glucose production. Values are expressed as means +/− SEM. (E) Western blotting for hepatic Akt, and its phosphorylated form (pSer473) showing increased basal pAkt expression in LOKO mice. Blotting was performed on whole livers from individual animals (n=3) of each genotype (OB, LOKO) under three conditions: i) fasting for 6 hours [Fast], ii) fasting for 6 hours with a single intraperitoneal injection of insulin (12 U/kg) administered 30 minutes before sacrifice [Ins 30’], and iii) at the end of hyperinsulinemic-euglycemic clamps from Fig 3D [Clamp]. A single representative blot is shown. Band density was quantified by image analysis and expressed relative to the fasted OB animals. (F) Quantification of total beta-cell mass from pancreatic sections stained for insulin production (N = 10-15 mice per genotype). Additional sections were immunofluorescently stained for insulin and a proliferative marker, Ki-67, and hand counted for cell numbers (N = 10 -11 for each genotype; N = 277,990 individual beta-cells tabulated across all genotypes). The average number of beta-cells and proliferating (Ki-67+) beta-cells for each genotype are shown. (G) Pancreatic islets were isolated from obese mice as described in Methods for glucose-stimulated insulin perifusion studies. After overnight recovery, islets were exposed to a low basal level of glucose (4 mM) for 40 minutes and then switched into high glucose media (16 mM) for another 40 minutes. Samples of the perfusate were measured for islet-secreted insulin by ELISA (n=3 separate channels per time point). Statistical analysis by Student's t-test (A, B, D, F) or 2-way ANOVA with Bonferroni post-tests (C, G): *, P < 0.05; **, P < 0.01; ***, P < 0.001.