Basal phosphorylation of Akt in hepatocytes and liver from young and old rats. Basal phosphorylation status of Akt at the Thr308 and Ser473 residues in (a) hepatocytes from young (Y) and old (O) rats in primary culture and (b) homogenized liver tissue from young and old rats. Western blotting was performed for phospho-Ser473, phospho-Thr308 Akt, and total Akt. Actin was used as a loading control. Bands were scanned densitometrically (arbitrary units), and phospho-to-total Akt was graphed using the mean ± SE (N = 3-4 animals for each). Statistical analyses were performed using Student’s t-test a P = 0.64 for Thr308, *P = 0.03 for Ser473; b P = 0.94 for Thr308, *P = 0.01 for Ser473)

Insulin-induced phosphorylation of Thr308 and Ser473 Akt in hepatocytes from young and old rats. Insulin was withdrawn from cultured hepatocytes from young and old rats by washing the cells and replacing the media with insulin-free media. After a 4 h acclimation, a time course of insulin repletion was performed (10 ng/ml). Representative western blots (N = 3 animals) and graphs of (a) phospho-Thr308 Akt-tototal Akt, and (b) phospho-Ser473 Akt-to-total Akt. Actin was used as a loading control. Bands were scanned and densitometry was graphed (arbitrary units) using the mean ± SE. Statistical analyses were performed by two-way ANOVA (a P = 0.04 for time, P = 0.10 for age; b P < 0.0001 for time, P = 0.12 for age), with Bonferroni’s post-hoc test for young versus old (a P > 0.05 at all time points; b *P < 0.05 at 5 and 15 min)

Increased vulnerability to oxidative stress through loss of Akt activity is ameliorated by LA. Hepatocytes from young and old rats were treated with 25 mM glucose and 100 mU/ml glucose oxidase (system designated as Gox), and/or 10 μM Akt inhibitor 1/2 (Akti1/2). Graphic representation of uncleaved procaspase-3 cleavage in cells treated with Gox and/or Akti1/2, with or without 50 μM LA pretreatment. Actin was used as a loading control. Bands were scanned and densitometry was graphed (arbitrary units) using the mean ± SE (N = 3 animals). One-way ANOVA was performed (P = 0.40 for young and P = 0.001 for old), and Tukey’s multiple comparison test was performed to determine statistical significance (P < 0.05) in treated versus untreated cells (Young: P > 0.05 for all treatments versus untreated; Old: *P < 0.05 for Akti1/2, Gox + Akti1/2, and LA + Akti1/2 versus untreated, **P < 0.01 for Gox + LA + Akti1/2 versus untreated, and ***P < 0.001 for Gox versus untreated)

LA induces phosphorylation of Akt at Ser473 and increases overall activity. (a) Ser473 Akt phosphorylation status in cultured hepatocytes from young and old rats treated with 50 μM LA. Western blotting was performed for phospho-Ser473, total Akt, and actin. Bands were scanned densitometrically; data represents the mean ± SE (N = 7 animals) of phospho-to-total Akt. Statistical analyses were performed by two-way ANOVA (P = 0.04 for time, and P < 0.0001 for age), with Bonferroni’s post-hoc test for young versus old (*P < 0.05 at 0, 30, and 60 min). (b) Akt activity was tested by the phosphorylation of a GST-GSK3α/β fusion protein, using extracts of hepatocytes from young and old rats, with and without a 90 min. 50 μM LA pretreatment. Phospho-Ser21/9 GSK3α/β was determined by western blotting, bands were scanned densitometrically, and data represents the mean ± SE (N = 3 animals). Statistical analyses were performed using Student’s t-test. Young versus old *P = 0.04, young versus young + LA: P = 0.50, young versus old + LA: P = 0.56

LA induces phosphorylation of Akt at Ser473 in liver tissue in young and old rats. Ser473 Akt phosphorylation status in liver tissue from young (Y) and old (O) rats given LA by gavage (120 mg/kg). Western blotting was performed for phospho-Ser473, total Akt, and Actin. Bands were scanned densitometrically; data represents the mean ± SE (N = 3 animals) for phospho-to-total Akt. Statistical analyses were performed by two-way ANOVA (P = 0.0003 for time, P = 0.001 for age), with Bonferroni’s post-hoc test for young versus old (*P < 0.05 at 0 min)

LA induces phosphorylation of Akt in the absence of insulin. Insulin was withdrawn from cultured hepatocytes from young and old rats by washing the cells and replacing the media with insulin-free media. After a 4 h acclimation, a time course of LA treatment was performed. Representative western blots and graphic representation (N = 3) of (a) phospho-Thr308 Akt-to-total Akt and (b) phospho-Ser473 Akt-to-total Akt. Actin was used as a loading control. Bands were scanned and densitometry was graphed (arbitrary units) using the mean ± SE. Statistical analyses were performed by two-way ANOVA (a P = 0.35 for time, P = 0.0007 for age; b P < 0.0001 for time, P < 0.0001 for age), with Bonferroni’s post-hoc test for young versus old (a P > 0.05 at all time points; b *P < 0.05 at 5, 15, 60, 90, and 120 min)

LA-induced Ser473 Akt phosphorylation is dependent upon PI3K. Hepatocytes from young and old rats were cultured in insulin-replete media, then pretreated with 50 μM LY294002 2 h prior to administration of LA. Phospho-Ser473 Akt-to-total Akt levels were measured by western blot (N = 3) and bands measured densitometrically. Actin was used as a loading control. Data was graphed (arbitrary units) using the mean ± SE. P = 0.009 for age and P < 0.0001 for time by two-way ANOVA. *P < 0.05 for young versus old at 0 min LA in the absence of inhibitor, as well as for all LY294002-treated samples, by Bonferroni’s post-hoc test

LA suppresses the activity of the phosphatases PTEN and PP2A. Graphic representation of western blot results for (a) total PTEN (P = 0.39) and phospho-Ser380 PTEN (P = 0.97), and (b) total PP2A-C (P = 0.26) and phospho-Tyr307 PP2A-C (P = 0.39) in homogenized liver tissue from young and old rats (N = 6 for each, statistical significance determined by Student’s t-test). Graphic representation of time course by western blot for (c) PTEN phosphorylation at Ser380 (P = 0.0009 for time and P = 0.03 for age), and (d) PP2A-C phosphorylation at Tyr307 (P = 0.0009 for time and P = 0.01 for age) in cultured hepatocytes from young and old rats, treated with 50 μM LA (N = 3 for each, statistical significance determined by ANOVA). Actin was used as a loading control. Data represent the mean ± SE. Bonferroni’s post-hoc test was used to determine statistical significance for treated versus untreated cells (c Young: *P < 0.05 for 0 vs. 90 min, *P < 0.01 for 0 vs. 120 min; Old: P < 0.05 for 0 vs. 90 and 120 min; d Young: *P < 0.01 for 0 vs. 30, 60, 90, and 120 min; Old: P < 0.05 for 0 vs. 90 and 120 min)