GLUT4 and glycogen synthase are key players in bed rest-induced insulin resistance

Abstract

To elucidate the molecular mechanisms behind physical inactivity-induced insulin resistance in skeletal muscle, 12 young, healthy male subjects completed 7 days of bed rest with vastus lateralis muscle biopsies obtained before and after. In six of the subjects, muscle biopsies were taken from both legs before and after a 3-h hyperinsulinemic euglycemic clamp performed 3 h after a 45-min, one-legged exercise. Blood samples were obtained from one femoral artery and both femoral veins before and during the clamp. Glucose infusion rate and leg glucose extraction during the clamp were lower after than before bed rest. This bed rest-induced insulin resistance occurred together with reduced muscle GLUT4, hexokinase II, protein kinase B/Akt1, and Akt2 protein level, and a tendency for reduced 3-hydroxyacyl-CoA dehydrogenase activity. The ability of insulin to phosphorylate Akt and activate glycogen synthase (GS) was reduced with normal GS site 3 but abnormal GS site 2+2a phosphorylation after bed rest. Exercise enhanced insulin-stimulated leg glucose extraction both before and after bed rest, which was accompanied by higher GS activity in the prior-exercised leg than the rested leg. The present findings demonstrate that physical inactivity-induced insulin resistance in muscle is associated with lower content/activity of key proteins in glucose transport/phosphorylation and storage.

FIG. 1.

Representative blots showing GLUT4, HKII, Akt1, Akt2, TBC1D4, and GS protein before and after 7 days of bed rest in rested (Rest) and prior-exercised (Ex) leg for the same subject. Although the average Akt2 protein content of all subjects is reduced with bed rest (Table 2), it may be noted that the abundance of Akt2 is not reduced with bed rest in the subject used for the representative blots. kD, kilodaltons.

FIG. 2.

A: GIR during 3 h of hyperinsulinemic euglycemic clamp initiated 3 h after 45 min of one-legged knee extensor exercise before and after 7 days' bed rest. B: Arterio-venous (a-v) glucose difference across rested (Rest leg) and prior-exercised (Ex leg) vastus lateralis during 3 h of hyperinsulinemic euglycemic clamp performed 3 h after 45 min of one-legged knee extensor exercise before and after bed rest. Samples were obtained before (B) as well as during (I) the clamp, presented here by the accumulated data during the last hour of the clamp (2–3 h). C: Muscle glycogen concentration (mmol/kg dry weight [dw]) in rested and prior-exercised leg before (B) and after (I) 3 h of hyperinsulinemic euglycemic clamp performed 3 h after one-legged knee extensor exercise before and after bed rest. Values are means ± SE; n = 6. *Significantly different from B, P < 0.05. †Significantly different from before bed rest, P < 0.05. #Significantly different from Rest leg at given time point, P < 0.05. (†)Tendency, 0.05 ≤ P < 0.1.

FIG. 3.

Akt Thr³⁰⁸ phosphorylation (A), GSK-3β Ser⁹ phosphorylation (B), and representative blots (C) in rested (Rest leg) and prior-exercised (Ex leg) vastus lateralis before (B) and after (I) 3-h hyperinsulinemic euglycemic clamp performed 3 h after one-legged knee extensor exercise before and after 7 days of bed rest. AU, arbitrary units; kD, kilodaltons. Values are means ± SE; n = 6. *Significantly different from B, P < 0.05. †Significantly different from before bed rest, P < 0.05. #Significantly different from Rest leg at given time point, P < 0.05. (#)Tendency, 0.05 ≤ P < 0.1.

FIG. 4.

TBC1D4 Ser⁵⁸⁸ phosphorylation (A), TBC1D4 Ser⁷⁵¹ phosphorylation (B), TBC1D4 Thr⁶⁴² phosphorylation (C), and representative blots (D) in rested (Rest leg) and prior-exercised (Ex leg) vastus lateralis before (B) and after (I) 3-h hyperinsulinemic euglycemic clamp performed 3 h after one-legged knee extensor exercise before and after 7 days of bed rest. AU, arbitrary units; kD, kilodaltons. Values are means ± SE; n = 6. *Significantly different from B, P < 0.05.

FIG. 5.

Glycogen synthase (GS) activity (%FV) (A), representative blots (B), GS site 3a phosphorylation (C), and GS site 2+2a phosphorylation (D) in rested (Rest leg) and prior-exercised (Ex leg) vastus lateralis before (B) and after (I) 3-h hyperinsulinemic euglycemic clamp performed 3 h after one-legged knee extensor exercise before and after 7 days of bed rest. AU, arbitrary units; kD, kilodaltons. Values are means ± SE; n = 6. *Significantly different from B, P < 0.05. †Significantly different from before bed rest, P < 0.05. #Significantly different from Rest leg at given time point, P < 0.05. Symbols within parentheses indicate tendency, 0.05 ≤ P < 0.1.