Effect of acute exercise on AMPK signaling in skeletal muscle of subjects with type 2 diabetes: a time-course and dose-response study

Abstract

Activation of AMP-activated protein kinase (AMPK) by exercise induces several cellular processes in muscle. Exercise activation of AMPK is unaffected in lean (BMI approximately 25 kg/m(2)) subjects with type 2 diabetes. However, most type 2 diabetic subjects are obese (BMI >30 kg/m(2)), and exercise stimulation of AMPK is blunted in obese rodents. We examined whether obese type 2 diabetic subjects have impaired exercise stimulation of AMPK, at different signaling levels, spanning from the upstream kinase, LKB1, to the putative AMPK targets, AS160 and peroxisome proliferator-activated receptor coactivator (PGC)-1alpha, involved in glucose transport regulation and mitochondrial biogenesis, respectively. Twelve type 2 diabetic, eight obese, and eight lean subjects exercised on a cycle ergometer for 40 min. Muscle biopsies were done before, during, and after exercise. Subjects underwent this protocol on two occasions, at low (50% Vo(2max)) and moderate (70% Vo(2max)) intensities, with a 4-6 week interval. Exercise had no effect on LKB1 activity. Exercise had a time- and intensity-dependent effect to increase AMPK activity and AS160 phosphorylation. Obese and type 2 diabetic subjects had attenuated exercise-stimulated AMPK activity and AS160 phosphorylation. Type 2 diabetic subjects had reduced basal PGC-1 gene expression but normal exercise-induced increases in PGC-1 expression. Our findings suggest that obese type 2 diabetic subjects may need to exercise at higher intensity to stimulate the AMPK-AS160 axis to the same level as lean subjects.

FIG. 1

Basal AMPK, ACC, AS160, and Akt. AMPK subunit, ACC, AS160, and Akt protein content (A) and phosphorylation (B) were measured in 8 lean (□), 8 obese (▥), and 12 type 2 diabetic (T2DM) (■) subjects. Data are means ± SE. Blots are shown for three subjects/group.

FIG. 2

Effect of exercise on AMPK and ACC phosphorylation. Biopsies were done at basal (□), after 10 (▥) and 40 min (■) of exercise, and 150 min postexercise (▨). Data are means ± SE in 8 lean, 8 obese, and 12 type 2 diabetic (T2DM) subjects. Data are expressed as arbitrary units (A and C) and as fold change (B and D). *P < 0.05 vs. basal of respective group; †P < 0.05 vs. lean group at 40 min. Blots are shown for one subject/group. B, basal; R, rest postexercise.

FIG. 2

Effect of exercise on AMPK and ACC phosphorylation. Biopsies were done at basal (□), after 10 (▥) and 40 min (■) of exercise, and 150 min postexercise (▨). Data are means ± SE in 8 lean, 8 obese, and 12 type 2 diabetic (T2DM) subjects. Data are expressed as arbitrary units (A and C) and as fold change (B and D). *P < 0.05 vs. basal of respective group; †P < 0.05 vs. lean group at 40 min. Blots are shown for one subject/group. B, basal; R, rest postexercise.

FIG. 3

AMPK activity. AMPKα1, AMPKα2, and total AMPK activities were measured before (□), after 40 min of exercise (■), and 150 min postexercise (▨). Data are means ± SE. Data are expressed as kinase activity (A, C, and E) and as fold change (B, D, and F). n = 6–12 in each time point (samples were not available for all the assays). *P < 0.05 vs. basal of respective group; †P < 0.05 vs. lean group in respective time point. T2DM, type 2 diabetes.

FIG. 4

LKB1 expression and activity. Equal amounts of protein (40 μg) were used for blotting of LKB1 and MO25α (A). Blots are shown for two subjects per group. LKB1 activity was measured as described in research design and methods (B). B, basal; T2DM, type 2 diabetes.

FIG. 5

AS160 phosphorylation. Immunoblots are shown for two subjects in the basal state and after 40 min of exercise, after immunoprecipitation with AS160 and probing with PAS antibody (A). Biopsies were done at basal (□), after 10 (▥) and 40 min (■) of exercise, and 150 min postexercise (▨). Data are means ± SE in 8 lean, 8 obese, and 12 type 2 diabetic (T2DM) subjects. Data are expressed as arbitrary units (B) and as fold change (C). *P < 0.05 vs. basal of respective group; †P < 0.05 vs. lean at 150 min postexercise. Blots are shown for one subject/group. B, basal; R, rest postexercise.

FIG. 6

Akt phosphorylation. Biopsies were done at basal (□), after 10 (▥) and 40 min (■) of exercise, and 150 min postexercise (▨). Akt-Ser⁴⁷³ (A) and Thr³⁰⁸ (B) were measured as described in research design and methods. Data are means ± SE in 8 lean, 8 obese, and 12 type 2 diabetic (T2DM) subjects. *P < 0.05 vs. basal of respective group. Blots are shown for one subject/group. B, basal; R, rest postexercise.

FIG. 7

PGC-1 and NRF-1 gene expression. Basal PGC-1α (A) and NRF-1 (C) gene expression. Effect of exercise on PGC-1 (B) and NRF-1 (D) gene expression. Gene expression was measured at baseline, after 40 min of exercise, and 150 min postexercise. Data are means ± SE in 8 lean, 8 obese, and 12 type 2 diabetic (T2DM) subjects. *P < 0.05 vs. type 2 diabetes group; †P < 0.05 vs. basal of respective group; ‡P = 0.05 vs. basal in obese group.