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. 2005 Feb 1;562(Pt 3):829-38.
doi: 10.1113/jphysiol.2004.073593. Epub 2004 Nov 18.

Alterations in insulin receptor signalling in the rat epitrochlearis muscle upon cessation of voluntary exercise

David S Kump  1 , Frank W Booth
Affiliations

Alterations in insulin receptor signalling in the rat epitrochlearis muscle upon cessation of voluntary exercise

David S Kump et al. J Physiol. .

Abstract

The major purpose of this study was to elucidate mechanisms by which decreasing enhanced physical activity induces decreased insulin sensitivity in skeletal muscle. Rats with access to voluntary running wheels for 3 weeks had their wheels locked for 5 h (WL5), 29 h (WL29), or 53 h (WL53); a separate group of rats never had wheel access (sedentary, SED). Relative to WL5, submaximal insulin-stimulated 2-deoxyglucose uptake into the epitrochlearis muscle was lower in WL53 and SED. Insulin binding, insulin receptor beta-subunit (IRbeta) protein level, submaximal insulin-stimulated IRbeta tyrosine phosphorylation, and glucose transporter-4 protein level were each lower in both WL53 and SED than in WL5 and WL29. Akt/protein kinase B Ser(473) phosphorylation was lower in WL53 and SED than in WL5. Protein levels of protein tyrosine phosphatase-1B, Src homology phosphatase-2, and protein kinase C- did not vary among groups. The amount of protein tyrosine phosphatase-1B, Src homology phosphatase-2, and protein kinase C- associated with IRbeta in insulin-stimulated muscle also did not differ among the four groups. The mean of SED and WL53 had a significantly higher IRbeta-associated protein tyrosine phosphatase-1B than the mean of WL5 and WL29. The enclosure of multiple changes (decreases in insulin binding, IRbeta protein, IRbeta tyrosine phosphorylation, and glucose transporter-4 protein) in the epitrochlearis muscle within the 29th to 53rd hour after cessation of voluntary wheel running raises the possibility that a single regulatory event could be responsible for the coordinated decrease.

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Figures

Figure 1
Figure 1. Experimental design
The length of the line for each experimental group corresponds to the time line at the top of the figure. See text for description. VRW, voluntary running wheel. n = 24 animals for each experimental group.
Figure 2
Figure 2. Effect of physical inactivity on 2-DOG uptake into isolated epitrochlearis muscle under basal (no added insulin), submaximal insulin-stimulated (60 μU ml−1), and maximal insulin-stimulated (2 mU ml−1) conditions
Groups had wheels locked for 5 h (WL5), 29 h (WL29), or 53 h (WL53) after 3 weeks of voluntary wheel running, or were sedentary (SED) without running wheels. Columns are mean ± s.e.m.* Significantly different from groups without an asterisk within the same insulin concentration (ANOVA, P≤ 0.05). n = 15–16 in each group for basal and 7–8 in each group for both insulin concentrations.
Figure 3
Figure 3. Effect of decreased physical activity on descriptive indices of the insulin receptor and its activation in epitrochlearis muscle
A, submaximal insulin binding to the epitrochlearis muscle. B, IRβ protein levels relative to loading control (see Methods) with a representative immunoblot (above graph). C, tyrosine phosphorylation of IRβ in response to 40 min of submaximal insulin stimulation with a representative immunoblot (above graph). IRβ immunoprecipitates were subjected to immunoblotting for phosphotyrosine, then stripped and re-probed for IRβ (see Methods). Data were normalized to a loading control (see Methods) and are expressed relative to the normalized band intensity for IRβ protein present in the same lane. Columns are mean ± s.e.m.* Signficantly different (ANOVA, P≤ 0.05) from groups without an asterisk. n = 6–8 in each group.
Figure 4
Figure 4. Effect of decreased physical activity on negative regulators of insulin receptor activation in epitrochlearis muscle
PTP1B (A), SHP2 (B) and PKC-Θ (C) protein levels normalized to a loading control (see Methods) with representative immunoblots (above graphs). Columns are mean ± s.e.m.* Significantly different from WL53 (ANOVA, P≤ 0.05). n = 6–8 in each group.
Figure 5
Figure 5. Representative immunoblots (A) and protein levels of PTP1B (B), SHP2 (C) and PKC-Θ (D) in IRβ immunoprecipitates from epitrochlearis homogenates from muscle incubated with submaximal insulin for 40 min
IRβ immunoprecipitates were subjected to immunoblotting for IRβ, then stripped and re-probed for PTP1B, SHP2, or PKC-Θ (see Methods). Data in each panel were normalized to a loading control (see Methods) on the same blot and are expressed relative to the normalized band intensity for IRβ present in the same lane. IgG in the first lane of panel A was immunoprecipitated with rabbit immunoglobulin G and is a negative control with no detectable signal. Columns in the main graphs represent the means for each group ± s.e.m. The columns in the insets illustrate the means ± s.e.m. for the linear combination of the individual group means for WL5 and WL29 (WL5+29) and for WL53 and SED (WL53+S). See Results for more detail. IP, immunoprecipitation; IB, immunoblot. * Significantly different (ANOVA with contrast statement, P≤ 0.05). n = 6–8 in each group.
Figure 6
Figure 6. Biochemical adaptations to decreased physical activity in the epitrochlearis muscle
A, Akt Ser473 phosphorylation relative to the total amount of Akt in the same lane and normalized to a loading control (see Methods) with a representative immunoblot (above graph). B, citrate synthase activity. C, glycogen concentration. D, GLUT4 protein levels normalized to a loading control (see Methods) with a representative immunoblot (above graph). Columns are mean ± s.e.m.* Signficantly different (ANOVA, P≤ 0.05) from groups without an asterisk. ** Significantly different (ANOVA, P≤ 0.05) from WL5. n = 6–8 in each group.
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