Acute regulation of metabolic genes and insulin receptor substrates in the liver of mice by one single bout of treadmill exercise

Abstract

Acute exercise performance represents a major metabolic challenge for the skeletal muscle, but also for the liver as the most important source of energy. However the molecular adaptation of the liver to one single bout of exercise is largely unknown. C57BL/6 mice performed a 60 min treadmill run at high aerobic intensity. Liver, soleus and white gastrocnemius muscle were removed immediately after exercise. The single bout of exercise resulted in a very rapid and pronounced induction of hepatic metabolic enzymes and regulators of metabolism or transcription: glucose-6-phosphatase (G6Pase; 3-fold), pyruvate dehydrogenase kinase-4 (PDK4; 4.8-fold), angiopoietin-like 4 (2.1-fold), insulin receptor substrate (IRS)-2 (5.1-fold), peroxisome proliferator activated receptor-gamma coactivator 1alpha (PGC-1alpha; 3-fold). In soleus and white gastrocnemius muscle the up-regulation of IRS-2 and PDK4 was less pronounced compared with the liver and no significant induction of PGC-1alpha could be detected at this early time point. Activation of AMPK was found in both liver and white gastrocnemius muscle as phosphorylation of Thr-172. The induction of endogenous insulin secretion by a glucose load directly after the exercise bout resulted in a significantly higher PKB/Akt phosphorylation in the liver of exercised mice. The markedly enhanced IRS-2 protein amount, and presumably reduced serine/threonine phosphorylation of the IRS proteins induced by the acute exercise could be responsible for this enhanced action of insulin. In conclusion, acute exercise induced a rapid and pronounced transcriptional adaptation in the liver, and regulated hepatic IRS proteins leading to improved cellular insulin signal transduction.

Figure 1. Parameters of metabolism in the plasma

Plasma concentrations are shown of glucose (A), insulin (B), glucagon (C), FFA (E) and lactate (F), as well as the insulin/glucagon ratio (D) of sedentary (sed) and exercised (run) mice (n= 8, mean ±s.e.m.). In A and F individual values of the same animals before and after the exercise bout are given.

Figure 2. Hepatic mRNA expression

G6Pase (A), PEPCK (B), PDK4 (C), PGC-1α (D), Angptl-4 (E), IRS-2 (F), IRS-1 (G), Fasn (H), COXI (I), and CPT1a (J) mRNA content in the liver of sedentary (sed) or exercised (run) mice. Values are shown as arbitrary units (n= 8, mean ±s.e.m.). K, 150 μg protein of liver and white gastrocnemius muscle extracts were separated by 7.5% SDS-PAGE and immunoblotted with antiphospho-Thr-172 antibodies and reprobed with mixed anti-AMPK α1 +α2 antibodies. Shown are representative blots with two animals of each group.

Figure 3. Muscle mRNA expression

IRS-2 (A and B), Angptl-4 (C and D), PDK4 (E and F), PGC-1α (G and H), Fasn (I and J), COXI (K and L) and CPT1b (M and N) mRNA content in soleus (A, C, E, G, I, K and M) and white gastrocnemius muscle (B, D, F, H, J, L and N) of sedentary (sed) and exercised (run) mice. Values are shown as arbitrary units (n= 8, mean ±s.e.m.).

Figure 4. Regulation of IRS proteins and insulin signalling in the liver by acute exercise

A, detection of IRS proteins in liver extracts of sedentary (sed) and exercised (run) mice. Liver protein (150 μg) was separated by 7.5% SDS-PAGE and immunoblotted with anti-IRS-2, -IRS-1 and -β-actin antibodies. B, C and D, plasma concentration of insulin (B), FFA (C) and glucose (D) of sedentary mice (sed), exercised mice immediately after the exercise bout (run), sedentary and exercised mice 30 min after the glucose load (+ glucose i.p.). (n= 6, mean ±s.e.m., *P < 0.05 versus mice of the same group without glucose, #P < 0.05 versus sedentary mice with glucose). E. 150 μg protein of liver extracts was separated by 7.5% SDS-PAGE and immunoblotted with anti-phospho-Ser-473 antibodies and membranes were reprobed with anti-Akt/PKB antibodies. Shown is one representative immunoblot and the densitometric quantification, phosphorylation of Akt/PKB in extracts obtained from sendentary, glucose-treated mice was set as 1 (n= 6, mean ±s.e.m., *P < 0.05 versus mice of the same group without glucose, #P < 0.05 versus sedentary mice with glucose). F and G, tyrosine phosphorylation of IRS-1 (F) and IRS-2 (G). Protein of liver extracts (1 mg) was immunoprecipitated with anti-IRS-1 or anti-IRS-2 antibodies, and immunoprecipitates were separated by 7.5% SDS-PAGE and immunoblotted with anti-phospho-tyrosine antibodies. Membranes were reprobed with anti-IRS-1 or anti-IRS-2 anitbodies. Shown are representative immunoblots and the densitometric quantification (n= 6, mean ±s.e.m., *P < 0.05 versus mice of the same group without glucose). H, 150 μg protein of liver extracts were left untreated, incubated with buffer, or incubated with buffer and λ protein phosphatase (λPPase) for 30 min at 30°C, separated by 7.5% SDS-PAGE and immunoblotted with anti-IRS-2-antibodies.