Loss of peripheral insulin signaling in <i>sir2</i> mutants is due to defects in insulin sensitivity.

<p>(A-C) Protein isolates from control (cont) and <i>sir2</i> mutant males (<i>sir2 –</i>) were analyzed on western blots using antibodies directed against phosphorylated AKT (P-AKT), total AKT, or Tubulin as a control. Animals were collected at one, two, or three weeks of age following a fasting-refeeding paradigm (–/+ glucose). The ratio of P-AKT levels to total AKT levels in refed controls and <i>sir2</i> mutants was quantified using data from three independent experimental replicates. The fold change between these ratios in mutants and controls is as follows, representing the mean ± SEM: (A) one week of age 0.8±0.3 (NS), (B) two weeks of age 0.2±0.06 (p = 0.005), (C) three weeks of age 0.1±0.08 (p = 0.008). (D,E) Insulin tolerance tests were performed at one (D) or two weeks (E) of age by comparing P-AKT levels after injection of bovine insulin (–/+ insulin, D; increasing insulin, E) following a fast. The ratio of P-AKT levels to total AKT levels in injected controls and <i>sir2</i> mutants was quantified using data from two biological replicates for each insulin concentration. The fold change between these ratios in mutants and controls is as follows, representing the mean ± SEM. (D) One week of age (- insulin) 3.3±1.5, (+ insulin) 0.7±0.01, two-way ANOVA between genotypes across insulin concentrations (NS). (E) Two weeks of age with increasing insulin concentrations, 1.0±0.6, 0.2±0.1, 0.3±0.2, two-way ANOVA between genotypes across insulin concentrations (p<0.05). (F,G) The amount of circulating DILP2 in controls (open circles) or <i>sir2</i> mutants (black circles) was determined using an ELISA assay following a fasting-refeeding paradigm. Each data point represents a single biological replicate (n = 10 flies/sample, n = 12–30 samples/group). Data is normalized to the fasting state for each experiment and presented as a scatter plot with the mean ±SEM indicated. Quantification of the fold-change in secreted DILP2 levels from the fasted to fed state is: one week controls: 1.0±0.09 to 1.7±0.1, two week controls: 1.0±0.2 to 2.2±0.2, one week <i>sir2</i> mutants: 1.0±0.07 to 2.5±0.2, two week <i>sir2</i> mutants: 1.0±0.08 to 2.7±0.4. In both controls and <i>sir2</i> mutants, there is a significant increase in DILP2 secretion in fed versus fasted animals (p<0.0001), but not between one and two weeks of age, as determined by two-way ANOVA. <i>sir2</i> mutants at one week of age have a stronger induction of DILP2 secretion in response to feeding than do controls (p = 0.008), while at two weeks the difference is not significant as determined by two-way ANOVA.