Effects of exercise on AMPK signaling and downstream components to PI3K in rat with type 2 diabetes

Abstract

Exercise can increase skeletal muscle sensitivity to insulin, improve insulin resistance and regulate glucose homeostasis in rat models of type 2 diabetes. However, the potential mechanism remains poorly understood. In this study, we established a male Sprague-Dawley rat model of type 2 diabetes, with insulin resistance and β cell dysfunction, which was induced by a high-fat diet and low-dose streptozotocin to replicate the pathogenesis and metabolic characteristics of type 2 diabetes in humans. We also investigated the possible mechanism by which chronic and acute exercise improves metabolism, and the phosphorylation and expression of components of AMP-activated protein kinase (AMPK) and downstream components of phosphatidylinositol 3-kinase (PI3K) signaling pathways in the soleus. As a result, blood glucose, triglyceride, total cholesterol, and free fatty acid were significantly increased, whereas insulin level progressively declined in diabetic rats. Interestingly, chronic and acute exercise reduced blood glucose, increased phosphorylation and expression of AMPKα1/2 and the isoforms AMPKα1 and AMPKα2, and decreased phosphorylation and expression of AMPK substrate, acetyl CoA carboxylase (ACC). Chronic exercise upregulated phosphorylation and expression of AMPK upstream kinase, LKB1. But acute exercise only increased LKB1 expression. In particular, exercise reversed the changes in protein kinase C (PKC)ζ/λ phosphorylation, and PKCζ phosphorylation and expression. Additionally, exercise also increased protein kinase B (PKB)/Akt1, Akt2 and GLUT4 expression, but AS160 protein expression was unchanged. Chronic exercise elevated Akt (Thr(308)) and (Ser(473)) and AS160 phosphorylation. Finally, we found that exercise increased peroxisome proliferator-activated receptor-γ coactivator 1 (PGC1) mRNA expression in the soleus of diabetic rats. These results indicate that both chronic and acute exercise influence the phosphorylation and expression of components of the AMPK and downstream to PIK3 (aPKC, Akt), and improve GLUT4 trafficking in skeletal muscle. These data help explain the mechanism how exercise regulates glucose homeostasis in diabetic rats.

Figure 1. Effects of chronic exercise on the protein kinases in the AMPK pathway.

A–E: relative levels of p-AMPKα1/2 (Thr¹⁷²) (A), p-AMPKα1 (Thr¹⁷²) (B), p-AMPKα2 (Thr¹⁷²) (C), p-ACC (Ser⁷⁹) (D), and p-LKB1 (Ser⁴³¹) (E). F: representative western blots for each protein of interest. Values are means ± SE. ^* P<0.05 and ^** P<0.01 vs. the control group; ^§ P<0.05 vs. the HFD group; ††P<0.01 vs. the HFD group; ^‡ P<0.05, ^‡‡ P<0.01 vs. the HFD+STZ group. CON: control; HFD: high-fat diet; CE: chronic exercise; STZ: streptozotocin. N = 7–8.

Figure 2. Effects of chronic exercise on the phosphorylation and protein expression of aPKC.

p-PKCζ/λ (Thr^410/403) (A) and p-PKCζ (Thr⁴¹⁰) (B). C: representative western blots for each protein of interest. Values are means ± SE. ^* P<0.05 vs. the control group; ^§ P<0.05 vs. the HFD group; †P<0.05 vs. the HFD group; ^‡ P<0.05 vs. the HFD+STZ group. CON: control; HFD: high-fat diet; CE: chronic exercise; STZ: streptozotocin. N = 7–8.

Figure 3. Effects of chronic exercise on components of the insulin signaling pathway.

A–F: expression levels of p-Akt1 (Ser⁴⁷³) (A), p-Akt2 (Ser⁴⁷⁴) (B), p-Akt (Thr³⁰⁸) (C), p-Akt (Ser⁴⁷³) (D), p-AS160 (Thr⁶⁴²) (E), and GLUT4 (F). G: representative western blots of the proteins of interest. Values are shown as mean ± SE. ^* P<0.05 and ^** P<0.01 vs. the control group; ^§ P<0.05 vs. the HFD group; ^‡‡ P<0.01 vs. the HFD+STZ group. CON: control; HFD: high-fat diet; CE: chronic exercise; STZ: streptozotocin. N = 7–8.

Figure 4. Effects of acute exercise on the protein kinases in the AMPK pathway.

A–E: relative expression levels of p-AMPKα1/2 (Thr¹⁷²) (A), p-AMPKα1 (Thr¹⁷²) (B), p-AMPKα2 (Thr¹⁷²) (C), p-ACC (Ser⁷⁹) (D), and p-LKB1 (Ser⁴³¹) (E). F: representative western blots for each protein of interest. Values are means ± SE. ^* P<0.05 and ^** P<0.01 vs. the control group; ^‡ P<0.05 and ^‡‡ P<0.01 vs. the HFD+STZ group. CON: control; HFD: high-fat diet; STZ: streptozotocin; AE: acute exercise. N = 7–8.

Figure 5. Effects of acute exercise on the phosphorylation and protein expression of aPKC.

p-PKCζ/λ (Thr^410/403) (A) and p-PKCζ (Thr⁴¹⁰) (B). C: representative western blots for each protein of interest. Values are means ± SE. ^* P<0.05 and ^** P<0.01 vs. the control group; ^‡ P<0.05, ^‡‡ P<0.01 vs. the HFD+STZ group. CON: control; HFD: high-fat diet; STZ: streptozotocin; AE: acute exercise. N = 7–8.

Figure 6. Effects of acute exercise on components of the insulin signaling pathway.

A–F: expression levels of p-Akt1 (Ser⁴⁷³) (A), p-Akt2 (Ser⁴⁷⁴) (B), p-AS160 (Thr⁶⁴²) (C), and GLUT4 (D). E: representative western blots of the proteins of interest. Values are means ± SE. ^** P<0.01 vs. the control group; ^‡ P<0.05 and ^‡‡ P<0.01 vs. the HFD+STZ group. CON: control; HFD: high-fat diet; STZ: streptozotocin; AE: acute exercise. N = 7–8.

Figure 7. Effects of chronic and acute exercise on PGC1α and NRF1 mRNA expression.

A,B: effects of chronic exercise. C,D: effects of acute exercise. Values are means ± SE. ^** P<0.01 vs. the control group; ^§ P<0.05 vs. the HFD+STZ group; ^‡ P<0.05 and ^‡‡ P<0.01 vs. the HFD+STZ group. PGC1α: peroxisome proliferator-activated receptor-γ coactivator; NRF1: nuclear respiratory factor 1; CON: control; HFD: high-fat diet; STZ, streptozotocin; CE: chronic exercise; AE: acute exercise. N = 8.