Postexercise improvement in insulin-stimulated glucose uptake occurs concomitant with greater AS160 phosphorylation in muscle from normal and insulin-resistant rats

Abstract

Earlier research on rats with normal insulin sensitivity demonstrated that acute exercise increased insulin-stimulated glucose uptake (GU) concomitant with greater phosphorylation of Akt substrate of 160 kDa (pAS160). Because mechanisms for exercise effects on GU in insulin-resistant muscle are unknown, our primary objective was to assess insulin-stimulated GU, proximal insulin signaling (insulin receptor [IR] tyrosine phosphorylation, IR substrate 1-phosphatidylinositol-3-kinase, and Akt phosphorylation and activity), and pAS160 in muscles from acutely exercised (one session) and sedentary rats fed either chow (low-fat diet [LFD]; normal insulin sensitivity) or a high-fat diet (HFD; for 2 weeks, insulin-resistant). At 3 h postexercise (3hPEX), isolated epitrochlearis muscles were used for insulin-stimulated GU and insulin signaling measurements. Although exercise did not enhance proximal signaling in either group, insulin-stimulated GU at 3hPEX exceeded respective sedentary control subjects (Sedentary) in both diet groups. Furthermore, insulin-stimulated GU for LFD-3hPEX was greater than HFD-3hPEX values. For HFD-3hPEX muscles, pAS160 exceeded HFD-Sedentary, but in muscle from LFD-3hPEX rats, pAS160 was greater still than HFD-3hPEX values. These results implicated pAS160 as a potential determinant of the exercise-induced elevation in insulin-stimulated GU for each diet group and also revealed pAS160 as a possible mediator of greater postexercise GU of insulin-stimulated muscles from the insulin-sensitive versus insulin-resistant group.

© 2014 by the American Diabetes Association.

Figures

Figure 1

A: Plasma insulin values. *P < 0.05, HFD-Sedentary greater vs. all other groups; †P < 0.05, LFD-IPEX less vs. all other groups. B: 2-DG uptake (without insulin). *P < 0.05, IPEX vs. Sedentary within the same diet group. C: Glycogen. *P < 0.05, IPEX vs. Sedentary within the same diet group. Data were analyzed by two-way ANOVA, and Tukey post hoc analysis was performed to identify the source of significant variance. Values are means ± SEM; n = 4–17/group.

Figure 2

A: Total protein abundance for AMPK and AS160 in epitrochlearis muscles of IPEX and time-matched Sedentary rats. There were no significant differences for total abundance of either protein. B: AMPKThr172 phosphorylation. C: AS160Thr642 phosphorylation. D: AS160Ser588 phosphorylation in epitrochlearis muscles of IPEX and Sedentary rats. Data were analyzed by two-way ANOVA, and Tukey post hoc analysis was performed to identify the source of significant variance. Values are means ± SEM; n = 4–6 per group. *P < 0.05, IPEX vs. Sedentary within the same diet group.

Figure 3

A: 2-DG uptake measured in paired epitrochlearis muscles without or with insulin 3hPEX. *P < 0.05, LFD-Sedentary vs. LFD-3hPEX without insulin; †P < 0.05, LFD-3hPEX group with insulin vs. all other groups with insulin; ‡P < 0.05, GU with insulin of HFD-Sedentary vs. HFD-3hPEX rats. B: 2-DG uptake delta-insulin values (delta = value with insulin − value without insulin from paired muscles). *P < 0.05, HFD-Sedentary vs. all other delta-insulin groups; †P < 0.05, LFD-3hPEX vs. all other delta-insulin groups; ‡P < 0.05, HFD-Sedentary vs. HFD-3hPEX. Data were analyzed by two-way ANOVA within each insulin level (minus or plus insulin) or for delta values, and Tukey post hoc analysis was performed to identify the source of significant variance. Values are means ± SEM; n = 17–22/group.

Figure 4

Total protein abundance for IR, IRS-1, Akt, AS160, and GLUT4. Data were analyzed by two-way ANOVA within each insulin level (minus or plus insulin). There were no significant differences for the total abundance of any of these proteins.

Figure 5

A: IRTyr1162/1163 phosphorylation measured by multiplex analysis (with this bead-based method, there is no representative blot to display) in paired epitrochlearis muscles at 3hPEX. B: Delta IRTyr1162/1163 phosphorylation (delta = value with insulin − value without insulin from paired muscles). C: IRS-1–PI3K association measured in paired epitrochlearis muscles at 3hPEX. D: Delta IRS-1–PI3K association. Data were analyzed by two-way ANOVA within each insulin level (minus or plus insulin) or for delta values. Values are means ± SEM; n = 8–12/group.

Figure 6

A: AktThr308 phosphorylation measured in paired epitrochlearis muscles at 3hPEX. B: Delta AktThr308 phosphorylation (delta = value with insulin − value without insulin from paired muscles). C: AktSer473 phosphorylation measured by multiplex analysis (with this bead-based method, there is no representative blot to display) in paired epitrochlearis muscles at 3hPEX. D: Delta AktSer473 phosphorylation. E: Akt activity measured in paired epitrochlearis muscles at 3hPEX. F: Delta Akt activity. Data were analyzed by two-way ANOVA within each insulin level (minus or plus insulin) or for delta values, and Tukey post hoc analysis was performed to identify the source of significant variance. Values are means ± SEM; n = 19–21/group.

Figure 7

A: AS160Thr642 phosphorylation measured in paired epitrochlearis muscles at 3hPEX. *P < 0.05, LFD-Sedentary vs. LFD-3hPEX with no insulin; †P < 0.05, LFD-3hPEX vs. HFD-3hPEX with insulin. B: Delta AS160Thr642 phosphorylation (delta = value with insulin − value without insulin from paired muscles). *P < 0.05, LFD-3hPEX vs. HFD-3hPEX with no insulin. C: AS160Ser588 phosphorylation measured in paired epitrochlearis muscles 3hPEX. *P < 0.05, LFD-3hPEX with insulin is greater than all other groups with insulin; †P < 0.05, HFD-Sedentary with insulin is less than all other groups with insulin; ‡P < 0.05, LFD vs. HFD within Sedentary. D: Delta AS160Ser588 phosphorylation. *P < 0.05, for LFD-3hPEX vs. all other groups. Data were analyzed by two-way ANOVA within each insulin level (minus or plus insulin) or for delta values, and Tukey post hoc analysis was performed to identify the source of significant variance. Values are means ± SEM; n = 13–16 per group.