Exercise training decreases mitogen-activated protein kinase phosphatase-3 expression and suppresses hepatic gluconeogenesis in obese mice

Abstract

Insulin plays an important role in the control of hepatic glucose production. Insulin resistant states are commonly associated with excessive hepatic glucose production, which contributes to both fasting hyperglycaemia and exaggerated postprandial hyperglycaemia. In this regard, increased activity of phosphatases may contribute to the dysregulation of gluconeogenesis. Mitogen-activated protein kinase phosphatase-3 (MKP-3) is a key protein involved in the control of gluconeogenesis. MKP-3-mediated dephosphorylation activates FoxO1 (a member of the forkhead family of transcription factors) and subsequently promotes its nuclear translocation and binding to the promoters of gluconeogenic genes such as phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). In this study, we investigated the effects of exercise training on the expression of MKP-3 and its interaction with FoxO1 in the livers of obese animals. We found that exercised obese mice had a lower expression of MKP-3 and FoxO1/MKP-3 association in the liver. Further, the exercise training decreased FoxO1 phosphorylation and protein levels of Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and gluconeogenic enzymes (PEPCK and G6Pase). These molecular results were accompanied by physiological changes, including increased insulin sensitivity and reduced hyperglycaemia, which were not caused by reductions in total body mass. Similar results were also observed with oligonucleotide antisense (ASO) treatment. However, our results showed that only exercise training could reduce an obesity-induced increase in HNF-4α protein levels while ASO treatment alone had no effect. These findings could explain, at least in part, why additive effects of exercise training treatment and ASO treatment were not observed. Finally, the suppressive effects of exercise training on MKP-3 protein levels appear to be related, at least in part, to the reduced phosphorylation of Extracellular signal-regulated kinases (ERK) in the livers of obese mice.

Figure 1

Schematic diagram of treatment with the antisense oligonucleotide targeting MKP-3.

Figure 2

Figure 3

A, hepatic MKP-3 protein levels in lean and diet-obesity mice (DIO, n = 5). B, MKP-3 inhibition assay was assessed in a dose–response experiment with sense and antisense oligonucleotides to MKP-3 (n = 5). Representative blots are shown. Data were expressed as the means ± SEM of five mice. *P < 0.05 vs. the lean group (chow) and #P < 0.05 vs.the obese group (DIO).

Figure 4

Lean, lean mice; DIO, obese mice; DIO-EXE, exercised obese mice; DIO-ASO, obese mice treated with the antisense oligonucleotide targeting MKP-3; DIO-EXE-ASO, obese mice exercised and treated with the antisense oligonucleotide targeting MKP-3 concomitantly. A, cumulative caloric intake (n = 8); B, body mass (n = 8); C, epididymal fat (n = 8); D, fasting glucose (n = 8); E, fasting insulin (n = 8); F, insulin tolerance test (ITT) (n = 8). Bars represent the means ± SEM of eight mice. *P < 0.05 vs. the lean group (chow) and #P < 0.05 vs. the obese group (DIO).

Figure 5

Abbreviations as in Fig. 4. A, liver samples were immunoblotted (IB) with an anti-MKP-3 antibody and an anti-β-actin antibody (n = 5, upper and lower panels, respectively); B, immunoprecipitation (IP) and immunoblot (IB) assays were performed to evaluate the FoxO1/MKP-3 association and FoxO1 protein levels in hepatic samples from various groups of mice (n = 5, upper and lower panels, respectively); C, liver samples were immunoblotted (IB) with anti-phospho-FoxO1 and anti-FoxO1 antibodies (n = 5, upper and lower panels, respectively); D, nuclear extraction assay was performed to evaluate FoxO1 protein levels in hepatic samples from various groups of animals (n = 5). Data were expressed as the means ± SEM of five mice. *P < 0.05 vs. the lean group (chow), #P < 0.05 vs. the obese group (DIO) and ¥P < 0.05 vs. the exercised obese (DIO-EXE).

Figure 6

Proteins levels of PGC-1α, HNF-4α, PEPCK and G6Pase in hepatic tissues from the different study groups were evaluated by immunoblot (IB). A, samples of livers were immunoblotted (IB) with antibodies to PGC-1α and β-actin (n = 5, upper and lower panels, respectively). B, liver samples were immunoblotted (IB) with antibodies to HNF-4α and β-actin (n = 5, upper and lower panels, respectively). C, liver samples were immunoblotted (IB) with antibodies to PEPCK and β-actin (n = 5, upper and lower panels, respectively). D, liver samples were immunoblotted (IB) with antibodies to G6Pase and β-actin (n = 5, upper and lower panels, respectively). Blood glucose levels during the pyruvate tolerance test (PTT, n = 8; E) and area under curve (AUC; F). Data were expressed as the means ± SEM of five or eight mice. *P < 0.05 vs. the lean group (chow) and #P < 0.05 vs. the obese group (DIO).

Figure 7

A, liver samples were immunoblotted (IB) with antibodies to pERK and β-actin (n = 5, upper and lower panels, respectively). B, liver samples were immunoblotted (IB) with antibodies to MKP-3 and β-actin (n = 5, upper and lower panels, respectively). C–E, confocal microscopy was performed to evaluate the localization of MKP-3 (green) in the livers of lean mice (C), obese mice (D) and exercised mice (E), with ×200 magnification (scale bar, 20 μm). Yellow arrows indicate MKP-3-positive cells. Data were expressed as the means ± SEM of five mice. *P < 0.05 vs. the lean group (chow) and #P < 0.05 vs. the obese group (DIO).