Acute exercise reduces hepatic glucose production through inhibition of the Foxo1/HNF-4alpha pathway in insulin resistant mice

Abstract

Protein hepatocyte nuclear factor 4alpha (HNF-4alpha) is atypically activated in the liver of diabetic rodents and contributes to hepatic glucose production. HNF-4alpha and Foxo1 can physically interact with each other and represent an important signal transduction pathway that regulates the synthesis of glucose in the liver. Foxo1 and HNF-4alpha interact with their own binding sites in the phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) promoters, and this binding is required for their effects on those promoters. However, the effect of physical activity on the HNF-4alpha/Foxo1 pathway is currently unknown. Here, we investigate the protein levels of HNF-4alpha and the HNF-4alpha/Foxo1 pathway in the liver of leptin-deficient (ob/ob) and diet-induced obese Swiss (DIO) mice after acute exercise. The ob/ob and DIO mice swam for four 30 min periods, with 5 min rest intervals for a total swimming time of 2h. Eight hours after the acute exercise protocol, the mice were submitted to an insulin tolerance test (ITT) and determination of biochemical and molecular parameters. Acute exercise improved insulin signalling, increasing insulin-stimulated Akt and Foxo1 phosphorylation and decreasing HNF-4alpha protein levels in the liver of DIO and ob/ob mice under fasting conditions. These phenomena were accompanied by a reduction in the expression of gluconeogenesis genes, such as PEPCK and G6Pase. Importantly, the PI3K inhibitor LY292004 reversed the acute effect of exercise on fasting hyperglycaemia, confirming the involvement of the PI3K pathway. The present study shows that exercise acutely improves the action of insulin in the liver of animal models of obesity and diabetes, resulting in increased phosphorylation and nuclear exclusion of Foxo1, and a reduction in the Foxo1/HNF-4alpha pathway. Since nuclear localization and the association of these proteins is involved in the activation of PEPCK and G6Pase, we believe that the regulation of Foxo1 and HNF-4alpha activities are important mechanisms involved in exercise-induced improvement of glucose homeostasis in insulin resistant states.

Figure 1. Insulin signalling in the hepatic tissue of controls, ob/ob and swiss DIO mice at rest or after acute exercise

Liver extracts from mice injected with saline or insulin were prepared as described in Methods. A and B, tissue extracts were immunoprecipitated (IP) with anti-IR antibody and blotted (IB) with anti-PY antibody (upper panels) or anti-IR antibody (lower panels). C and D, tissue extracts were IP with anti-IRS-2 antibody and IB with anti-PY (upper panels) and anti-IRS-2 antibody (lower panels). E and F, liver extracts were IB with anti-phospho Akt or anti-Akt antibody (upper and lower panels, respectively). G and H, liver extracts were IB with anti-phospho Foxo1 or anti-Foxo1 antibody (upper and lower panels, respectively). The results of scanning densitometry are expressed as arbitrary units. Bars represent means ±s.e.m. of n= 6 mice. *P < 0.05, control stimulated-insulin (+) versus no insulin (−); ^#P < 0.05 ob/ob or DIO mice versus respective control; and §P < 0.05, exercised group versus ob/ob or DIO mice at rest.

Figure 2. Isolation, immunoprecipitation, and nuclear extract expression of Foxo1 and HNF-4α in control, ob/ob and DIO mice at rest or after acute exercise

Liver extracts from mice injected with saline or insulin were prepared as described in Methods. A and B, evaluation of the efficiency of isolation. IRS-2 expression in cytoplasm and nuclear extracts in ob/ob and DIO mice (upper panel) and histone expression in cytoplasm and nuclear extracts in ob/ob and DIO mice (lower panel). C and D, liver extracts were IB with anti-HNF-4α antibody. E and F, liver extracts were IB with anti-Foxo1 antibody. G and H, nuclear extracts were immunoprecipitated (IP) with anti-HNF-4α antibody and blotted (IB) with anti-Foxo1 antibody. Bars represent means ±s.e.m. of n= 6 mice. *P < 0.05, control stimulated-insulin (+) versus no insulin (−); ^#P < 0.05 ob/ob or DIO mice versus respective control; and §P < 0.05, exercised group versus ob/ob or DIO mice at rest.

Figure 3. PEPCK and G6Pase expression, hepatic glycogen contents and clamp under fasting conditions in control, ob/ob and DIO mice at rest or after acute exercise

Liver extracts from mice were prepared as described in Methods. A and B, tissue extracts were blotted (IB) with anti-PEPCK antibody in ob/ob and DIO mice, respectively. C and D, tissue extracts were blotted (IB) with anti-G6Pase antibody in ob/ob and DIO mice, respectively. The results of scanning densitometry are expressed as arbitrary units (n= 6). E and F, glycogen contents (mg per 100 mg of tissue) (n= 8). G and H, the rate of glucose consumption (mg kg⁻¹ min⁻¹) during a euglycaemic–hyperinsulinaemic clamp is presented (n= 6). Bars represent means ±s.e.m.*P < 0.05, ob/ob or DIO mice versus respective control and ^#P < 0.05, exercised group versus ob/ob or DIO mice at rest.

Figure 4. Analysis of insulin-induced phosphorylation of the Akt and Foxo1, expression of HNF-4α and immunoprecipitation of Foxo1 in the liver of ob/ob and DIO mice submitted to the exercise protocol plus single injection of LY294002

Liver extracts from mice were prepared as described in Methods. A and B, liver extracts were IB with anti-phospho Akt or anti-Akt antibody (upper and lower panels, respectively). C and D, liver extracts were IB with anti-phospho Foxo1 or anti-Foxo1 antibody (upper and lower panels, respectively). E and F, liver extracts were IB with anti-phospho HNF-4α or anti-β-actin antibodies (upper and lower panels, respectively). G and H tissue extracts were IP with anti-Foxo1 and IB anti-HNF-4α (upper panels) or IB anti-HNF-4α antibodies (lower panels). Bars represent means ±s.e.m. of n= 6 mice. *P < 0.05, control stimulated-insulin (+) versus no insulin (−); ^#P < 0.05 ob/ob or DIO mice versus respective control; §P < 0.05, exercised group versus ob/ob or DIO mice at rest, and $P < 0.05, exercised plus LY290402 groups versus ob/ob or DIO exercised mice.

Figure 5. PEPCK and G6Pase expression in control, ob/ob and DIO mice after acute exercise plus a single injection of LY294002

Liver extracts from mice were prepared as described in Methods. A and B, tissue extracts were blotted (IB) with anti-PEPCK or anti-β-actin antibodies (upper and lower panels, respectively). C and D, tissue extracts were blotted (IB) with anti-G6Pase or anti-β-actin antibodies (upper and lower panels, respectively). The results of scanning densitometry are expressed as arbitrary units. Bars represent means ±s.e.m. of n= 6 mice. *P < 0.05, ob/ob or DIO mice versus respective control; ^#P < 0.05, exercised group versus ob/ob or DIO mice at rest; and §P < 0.05, exercised plus LY290402 groups versus ob/ob or DIO exercised mice.