Impaired translocation of GLUT4 results in insulin resistance of atrophic soleus muscle

Abstract

Whether or not the atrophic skeletal muscle induces insulin resistance and its mechanisms are not resolved now. The antigravity soleus muscle showed a progressive atrophy in 1-week, 2-week, and 4-week tail-suspended rats. Hyperinsulinemic-euglycemic clamp showed that the steady-state glucose infusion rate was lower in 4-week tail-suspended rats than that in the control rats. The glucose uptake rates under insulin- or contraction-stimulation were significantly decreased in 4-week unloaded soleus muscle. The key protein expressions of IRS-1, PI3K, and Akt on the insulin-dependent pathway and of AMPK, ERK, and p38 on the insulin-independent pathway were unchanged in unloaded soleus muscle. The unchanged phosphorylation of Akt and p38 suggested that the activity of two signal pathways was not altered in unloaded soleus muscle. The AS160 and GLUT4 expression on the common downstream pathway also was not changed in unloaded soleus muscle. But the GLUT4 translocation to sarcolemma was inhibited during insulin stimulation in unloaded soleus muscle. The above results suggest that hindlimb unloading in tail-suspended rat induces atrophy in antigravity soleus muscle. The impaired GLUT4 translocation to sarcolemma under insulin stimulation may mediate insulin resistance in unloaded soleus muscle and further affect the insulin sensitivity of whole body in tail-suspended rats.

Figure 1

The insulin sensitivity of whole body during a 60-min hyperinsulinemic-euglycemic clamp experiment in the control (CON) and 4-week tail-suspended (SUS) rats. (a) Blood glucose. (b) Glucose infusion rate. (c) Average blood glucose level during 60-min clamp. (d) Average glucose infusion rate during 60-min clamp. Values are mean ± SEM, n = 6 rats for each group. ^** P < 0.01 versus CON group.

Figure 2

Effects of tetanic contraction and insulin stimulation on the glucose uptake in the control and unloaded soleus and EDL muscles. (a) Glucose uptake of soleus muscles. (b) Glucose uptake in EDL muscles. CTC, tetanic contraction of CON muscle. CIN, CON muscle treated by insulin. STC, tetanic contraction of SUS muscle. SIN, SUS muscle treated by insulin. Values are mean ± SEM, n = 6 samples for each group. ^* P < 0.05 or ^** P < 0.01 versus CON group. ^# P < 0.05 or ^## P < 0.01 versus SUS group.

Figure 3

Expression of IRS-1, PI3K, Akt, and P-Akt in the unloaded soleus of rats. (a) Representative Western blots. (b) Ratios of IRS-1 to β-actin. (c) Ratios of PI3K to β-actin. (d) Ratios of Akt to β-actin. (e) Ratios of P-Akt to total Akt. Values are mean ± SEM, n = 6 samples for each group.

Figure 4

Expression of AMPK, ERK, p38, and P-p38 in the unloaded soleus of rats. (a) Representative Western blots. (b) Ratios of AMPK to β-actin. (c) Ratios of p38 to β-actin. (d) Ratios of P-p38 to p38. (e) Ratios of ERK1/2 to β-actin. Values are mean ± SEM, n = 6 samples for each group.

Figure 5

Expression of AS160 and GLUT4 in the unloaded soleus of rats. (a) Representative Western blots. (b) Ratios of AS160 to β-actin. (c) Ratios of GLUT4 to β-actin. Values are mean ± SEM, n = 6 samples for each group.

Figure 6

Changes in GLUT4 of plasma membrane fraction (PM) under insulin stimulation in the unloaded soleus of rats. (a) Representative Western blots. (b) Ratios of total GLUT4 to β-actin. (c) Ratios of PM GLUT4 to β-actin. ^* P < 0.05 versus control with insulin stimulation group [insulin(+)], ^** P < 0.01 versus control without insulin stimulation group [insulin(−)]. Values are mean ± SEM, n = 6 samples for each group.

Figure 7

Changes in GLUT4 membrane translocation ratio under insulin stimulation in the unloaded soleus of rats. (a) Representative immunofluorescent sections. GLUT4 was stained in red fluorescent rhodamine. Nuclei were stained in blue by Hochest 33258. (b) Quantitative GLUT4 membrane translocation ratio. Insulin(+), with insulin stimulation. Insulin(−), without insulin stimulation. Scale bar, 5 μm. Values are mean ± SEM, n = 30 muscle fibers for each group. ^** P < 0.01 versus CON group.