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Animals and Treatment Groups. Three groups of 6- to 8-week-old male muscle insulin receptor knockout (MIRKO) mice and their Lox/lox controls were maintained on a 12-h light/12-h dark cycle and fed a standard mouse diet (9F 5020, Purina). One group of each was given daily i.p. injections of sodium citrate (pH 4.3) for 3 days (controls). A second group of each was treated with an i.p. injection of streptozotocin (Sigma), 100 m g/g body weight in sodium citrate for 3 consecutive days. When these mice achieved fed glucoses of >400 mg/dl for 3 consecutive days, they were separated into two groups. Half were not treated, and the half were treated with s.c. insulin pellets (LinShin, Toronto, ON, Canada), to obtain fed glucose <200 mg/dl for at least 3 consecutive days (V.Y., unpublished data). Thus, six experimental groups each consisting of at least 6 mice were created. All mice were killed between 1:00 and 4:00 p.m., and hindlimb skeletal muscle was snap frozen in liquid nitrogen and stored at –80°C.

RNA Extraction and Microarray Hybridization. Detailed methods have been described (1). Briefly, RNA was extracted from muscle by using TRIzol (Invitrogen). Two pools consisting of equal quantities of RNA from three mice within each group were created for each of the experimental groups. This pooled RNA and RNA from five to six individual mice in each group were purified by using RNeasy (Qiagen, Valencia, CA), allowing for a total of seven to eight arrays per group. Because the animals were of mixed genetic background, this larger number of arrays minimized biological and methodological variability. Biotinylated cRNA was generated by using 25 m g of the RNA samples (Affymetrix, Santa Clara, CA) and quantitated after adjusting for carryover of residual RNA. We fragmented 15 m g of adjusted cRNA and hybridized it to MG-U74A-v2 arrays (Affymetrix) for 16 h, and it was then washed and scanned. Data were analyzed by using genechip microarray suite (version 5.0), genespring (version 4.1), and excel (Microsoft). Data analysis was performed as described by using three filters of significance (1). First, all genes were excluded for which mean expression value was below the sum of the average background and the average standard difference threshold (SDT, four times scaled noise) in both control and the diabetic groups. Genes that passed the first filter were subjected to a second filter, which selected for genes with an absolute difference between the means of the control and experimental groups that was greater than the average SDT. The third filter considered only those genes that had a significance of P £ 0.05, obtained with a two-tailed t test assuming unequal variance between groups. These genes were then labeled as being significantly changed between the control and the experimental groups. A gene was labeled as responsive to insulin treatment if the expression intensity of the gene in the insulin-treated group reverted toward the control by at least one-half of the expression difference between control and diabetic groups.

Protein Extraction and Immunoblotting. Hindlimb muscles of two wild-type and two streptozotocin (STZ) diabetic mice were homogenized with a Polytron (Beckman Coulter) in-tissue lysis buffer (25 mM Tris•HCl, pH 7.4/2 mM sodium vanadate/10 mM sodium fluoride/10 mM sodium orthophosphate/1 mM EDTA/1 mM EGTA/5 m g/ml leupeptin/5 m g/ml aprotinin/1 mM PMSF/1% Nonidet P-40). The homogenate was centrifuged at 1,500 × g for 10 min. The supernatant was then centrifuged at 30,000 × g, and the resultant supernatant was used as the cytosolic protein extract after removing the upper fat layer. The pellet was washed with tissue lysis buffer with 25% glycerol and then lysed with the nuclear extraction buffer (nuclear wash buffer with 330 mM sodium chloride) by passing it through an 18G needle five times. This lysate was rotated at 4°C for 20 min and then centrifuged at 14,000 × g; the supernatant was collected as the nuclear/mitochondrial protein extract. Protein concentration was measured by using the Lowry method. Equal amounts of protein (1 mg) were immunoprecipitated at 4°C for 12 h with anti-sir2 antibody (Zymed) and protein G Sepharose beads. After separation by SDS/PAGE, immunoprecipitates were subjected to Western blotting with the same antibody and visualized by enhanced chemiluminescence (Pierce) and quantitated by using labworks (BioImaging Systems, Upland, CA).

1. Yechoor, V. K., Patti, M. E., Saccone, R. & Kahn, C. R. (2002) Proc. Natl. Acad. Sci. USA 99, 10587–10592.