Brief calorie restriction increases Akt2 phosphorylation in insulin-stimulated rat skeletal muscle

Abstract

Skeletal muscle insulin sensitivity improves with short-term reduction in calorie intake. The goal of this study was to evaluate changes in the abundance and phosphorylation of Akt1 and Akt2 as potential mechanisms for enhanced insulin action after 20 days of moderate calorie restriction [CR; 60% of ad libitum (AL) intake] in rat skeletal muscle. We also assessed changes in the abundance of SH2 domain-containing inositol phosphatase (SHIP2), a negative regulator of insulin signaling. Fisher 344 x Brown Norway rats were assigned to an AL control group or a CR treatment group for 20 days. Epitrochlearis muscles were dissected and incubated with or without insulin (500 microU/ml). Total Akt serine and threonine phosphorylation was significantly increased by 32 (P < 0.01) and 30% (P < 0.005) in insulin-stimulated muscles from CR vs. AL. Despite an increase in total Akt phosphorylation, there was no difference in Akt1 serine or Akt1 threonine phosphorylation between CR and AL insulin-treated muscles. However, there was a 30% decrease (P < 0.05) in Akt1 abundance for CR vs. AL. In contrast, there was no change in Akt2 protein abundance, and there was a 94% increase (P < 0.05) in Akt2 serine phosphorylation and an increase of 75% (P < 0.05) in Akt2 threonine phosphorylation of insulin-stimulated CR muscles compared with AL. There was no diet effect on SHIP2 abundance in skeletal muscle. These results suggest that, with brief CR, enhanced Akt2 phosphorylation may play a role in increasing insulin sensitivity in rat skeletal muscles.

Fig. 1

Total Akt serine and threonine phosphorylation. A: immunoblot (IB) analysis of serine phosphorylation in homogenates of insulin-stimulated epitrochlearis muscle after 20 days of ad libitum (AL) and calorie-restricted (CR) feeding (40 μg/lane). Phospho-pan-Akt (Ser^473/474) antibody recognizes serine-phosphorylated Akt1 and Akt2. Data are means ± SE for 15 rats/group. B: immunoblot analysis of threonine phosphorylation in homogenates of insulin-stimulated epitrochlearis muscle (60 μg/lane). Phospho-pan-Akt (Thr^308/309) antibody recognizes threonine-phosphorylated Akt1 and Akt2. Data are means ± SE for 8 rats/group. Data are relative to the average of AL values on each blot. Data were analyzed by t-test, with *P < 0.05, CR significantly different from AL. Representative immunoblots are shown.

Fig. 2

Akt1 and Akt2 abundance. A: Akt1 abundance was measured by immunoblot assay using an Akt1-specific antibody in homogenates from insulin-stimulated epitrochlearis muscles (60 μg/lane). Data are means ± SE for 15 rats/group. B: Akt2 abundance was measured by immunoblot assay using an Akt2-specific antibody in homogenates from insulin-stimulated epitrochlearis muscle (25 μg/lane). Data are means ± SE for 8–9 rats/group. All data are relative to the average of AL values on each blot. Data were analyzed by t-test, with *P < 0.05, CR significantly different from AL. Representative immunoblots are shown.

Fig. 3

Serine phosphorylation of Akt1 and Akt2. Immunoprecipated (IP) Akt1 (200 μg; A) or immunoprecipated Akt2 (250 μg; B) from homogenates of epitrochlearis muscles, incubated without (open bars) or with (filled bars) 500 μU/ml insulin, was analyzed by immunoblot assay using phospho-pan-Akt (Ser^473/474) antibody. Data are means ± SE for 4–6 rats/group. Data were analyzed by t-test, with *P < 0.05, CR significantly different from AL. Representative immunoblots are shown.

Fig. 4

Threonine phosphorylation of Akt1 and Akt2. Immunoprecipated Akt1 (200 μg; A) or immunoprecipated Akt2 (250 μg; B) from homogenates of epitrochlearis muscles, incubated without (open bars) or with (filled bars) 500 μU/ml insulin, was analyzed by immunoblot assay with phospho-pan-Akt (Thr^308/309) antibody. Data are means ± SE for 5–9 rats/group. Data were analyzed by t-test, with *P < 0.05, CR significantly different from AL. Representative immunoblots are shown.

Fig. 5

SH2 domain-containing inositol phosphatase (SHIP2) abundance. SHIP2 abundance was measured by immunoblot assay (200 μg/lane) in homogenates from epitrochlearis (Epi) muscle (A) or plantaris muscle (B). Data are means ± SE for 8–9 rats/group. Data were analyzed by t-test, with *P < 0.05, CR significantly different from AL. Representative immunoblots are shown.