Inhibition of Akt2 phosphorylation abolishes the calorie restriction-induced improvement in insulin-stimulated glucose uptake by rat soleus muscle

Abstract

Calorie restriction (CR; ∼60%-65% of ad libitum consumption) can enhance insulin-stimulated glucose uptake (ISGU) in predominantly slow-twitch skeletal muscles (e.g., soleus) by an incompletely understood mechanism. We used an Akt inhibitor (MK-2206) to eliminate CR's effect on insulin-stimulated Akt2 phosphorylation in isolated rat soleus muscles. We found long-term CR-enhanced ISGU was abolished by eliminating the CR effect on Akt2 phosphorylation, suggesting the CR-induced benefit on ISGU in the predominantly slow-twitch soleus relies on enhanced Akt2 phosphorylation.

Keywords: dietary restriction; glucose transport; insulin signaling; muscle à secousse lente; protein kinase B; protéine kinase B; restriction calorique; signalisation de l’insuline; slow-twitch muscle; transport du glucose.

Figure 1

Immunoblot analysis of Akt phosphorylation in rat soleus muscle. (A) pan-Akt Thr308 phosphorylation (pAkt^Thr308). (B) Akt2 Thr309 phosphorylation (pAkt2^Thr309), muscle lysates were immunoprecipitated with Akt2 antibody prior to immunoblotting with phospho-Akt^Thr308 antibody, (C) pan-Akt Ser473 phosphorylation (pAkt^Ser473), and (D) Akt2 Ser474 phosphorylation (pAkt2^Ser474), muscle lysates were immunoprecipitated with Akt2 antibody prior to immunoblotting with phospho-Akt^Ser473 antibody. Filled bars are muscles from AL-fed rats, open bars are muscles from CR-fed rats, and hatched bar are muscles from CR-fed rats incubated with MK-2206. “ns” indicates no statistical difference between AL and CR groups in the absence of insulin. Insulin-stimulated groups with matching letters are not statistically different, and groups with differing letters are statistically (P < 0.05) different from each other. Values are means ± SE, n = 6–10 per treatment group. All lanes are from the same gel, however, the representative blots have a vertical line separating the final 2 lanes indicating that the original blot has been re-ordered to match the respective bar graphs.

Figure 2

AS160 Thr⁶⁴²-phosphorylation (pAS160^Thr642) and 2-deoxy-D-glucose (2-DG) uptake in rat soleus muscle. (A) pAS160^Thr642. (B) 2-DG uptake. Filled bars are muscles from AL-fed rats, open bars are muscles from CR-fed rats, and hatched bar are muscles from CR-fed rats incubated with MK-2206. “ns” indicates no statistical difference between AL and CR groups in the absence of insulin. Insulin-stimulated groups with matching letters are not statistically different, and groups with differing letters are statistically (P < 0.05) different from each other. Values are means ± SE, n = 12–18 per treatment group.