Journal: The journals of gerontology. Series A, Biological sciences and medical sciences

Article Title: Differential Development of Inflammation and Insulin Resistance in Different Adipose Tissue Depots Along Aging in Wistar Rats: Effects of Caloric Restriction.

doi: 10.1093/gerona/glv117

Figure Lengend Snippet: Figure 2. Effect of aging and caloric restriction (CR) on insulin-stimulated insulin receptor (IR) and AKT phosphorylation and on insulin signaling pathway in different adipose tissue depots. (A) Ex vivo adipose tissue explants from epididymal white adipose tissue (eWAT), perirenal WAT (pWAT), subcutaneous WAT (sWAT), and brown adipose tissue (BAT) of 3-, 8-, and 24-month-old rats fed ad libitum (AL), and 8- and 24-month-old caloric-restricted rats were stimulated with 80 nM insulin or saline for 10 minutes as described in Methods section. Whole extracts were subjected to Western blot analysis. For insulin-stimulated IR, Tyr phosphorylation membranes were blotted with anti-phospho-Tyr-IRβ (P-Tyr-IRβ) and anti-IRβ antibodies. For insulin-stimulated AKT phosphorylation, membranes were blotted with anti-phospho-AKT (P-AKT) and anti-AKT antibodies. Upper panels show representative Western blots. Bars, in the lower panels, represent quantification of P-Tyr-IRβ and P-AKT after correction for IRβ and AKT, respectively, by scanning densitometry. Results are expressed as fold increase of IR and AKT phosphorylation. (B) Insulin receptor β (IRβ), insulin receptor substrate 1 (IRS1), phospho-Ser IRS1 (P-Ser-IRS1), and PTP1B levels were analyzed in whole adipose extracts by Western blot. Membranes were blotted with anti-IRβ, anti-IRS1, anti-P-Ser-IRS1, and anti-PTP1B antibodies. Anti-β-actin antibodies were used as a loading control. Upper panels show representative Western blots. Bars, in the lower panels, represent quantifications by scanning densitometry after correction for β-actin. Results, expressed as arbitrary densitometric units (A.D.U.) relative to 3-month-old rats equaled to 100, are means ± SEM of 5–8 animals per group. The data AL were evaluated by one-way analysis of variance test followed by Tukey’s post hoc test. *p < .05, **p < .01, ***p < .001 vs 3-month- old rats. CR was compared with its AL age-mate group using nonpaired Student’s t test. #p < .05, ##p < .01 vs same age-fed AL.

Article Snippet: Antibodies directed toward phospho IRβ (Tyr 1162/1163), insulin-Rβ (C-19), insulin receptor substrate 1 (IRS1; C-20), and PTP1B (H135) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Phospho-proteomics, Ex Vivo, Saline, Western Blot, Control