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We investigated if acute endurance-type exercise interacts with insulin-stimulated activation of atypical protein kinase C (aPKC) and insulin signalling to peptide chain elongation in human skeletal muscle. Four hours after acute one-legged exercise, insulin-induced glucose uptake was ∼80% higher (N= 12, P < 0.05) in previously exercised muscle, measured during a euglycaemic–hyperinsulinaemic clamp (100 μU ml−1). Insulin increased (P < 0.05) both insulin receptor substrate (IRS)-1 and IRS-2 associated phosphatidylinositol (PI)-3 kinase activity and led to increased (P < 0.001) phosphorylation of Akt on Ser473 and Thr308 in skeletal muscle. Interestingly, in response to prior exercise IRS-2-associated PI-3 kinase activity was higher (P < 0.05) both at basal and during insulin stimulation. This coincided with correspondingly altered phosphorylation of the extracellular-regulated protein kinase 1/2 (ERK 1/2), p70S6 kinase (P70S6K), eukaryotic elongation factor 2 (eEF2) kinase and eEF2. aPKC was similarly activated by insulin in rested and exercised muscle, without detectable changes in aPKC Thr410 phosphorylation. However, when adding phosphatidylinositol-3,4,5-triphosphate (PIP3), the signalling product of PI-3 kinase, to basal muscle homogenates, aPKC was more potently activated (P= 0.01) in previously exercised muscle. Collectively, this study shows that endurance-type exercise interacts with insulin signalling to peptide chain elongation. Although protein turnover was not evaluated, this suggests that capacity for protein synthesis after acute endurance-type exercise may be improved. Furthermore, endurance exercise increased the responsiveness of aPKC to PIP3 providing a possible link to improved insulin-stimulated glucose uptake after exercise.
Insulin exerts a variety of actions favouring uptake and deposition of nutrients in many tissues, including skeletal muscle. In skeletal muscle, several of these actions are improved when insulin stimulation is preceded by muscle contraction (Zorzano et al. 1985; Cartee et al. 1989; Wasserman et al. 1991; Wojtaszewski et al. 1997, 2000; Biolo et al. 1999). This phenomenon is often referred to as improved insulin sensitivity, and may depend on intensity and type of exercise, time of evaluation and availability of nutrients. Correspondingly, exercise is generally endorsed as an important element in prevention and treatment of impaired insulin action in skeletal muscle, a hallmark feature of type 2 diabetes.
Immediately after endurance exercise, increased muscle glucose uptake is partly due to a residual effect of exercise whereas at later stages (> 3 h) increased insulin sensitivity to stimulate muscle glucose uptake is the primary mechanism (Richter et al. 1982, 1989; Garetto et al. 1984; Mikines et al. 1988; Cartee et al. 1989). At the cellular level this beneficial effect of prior exercise is manifested in an increased insulin-induced membrane abundance of GLUT4 (Hansen et al. 1998) and an increased activity of glycogen synthase (GS) (Richter et al. 1982; Wojtaszewski et al. 2000), the rate-limiting enzyme in glycogen synthesis. However, insulin signalling measured in whole muscle lysates through insulin receptor tyrosine kinase (IRTK), insulin receptor substrate (IRS)-1-associated phosphatidylinositol-3 (PI-3) kinase, Akt as well as glycogen synthase kinase-3 (GSK-3) does not appear to be enhanced (Treadway et al. 1989; Goodyear et al. 1995; Wojtaszewski et al. 1997, 2000). This suggests that improved overall delivery of insulin to muscle fibres cannot explain why insulin sensitivity of muscle glucose uptake is increased more than 3 h after endurance exercise. Rather, any molecular interaction between exercise and subsequent insulin action on glucose uptake is probably occurring at levels of the insulin signalling cascade not yet investigated and/or in processes directly related to GLUT4 translocation.
One aim of this study was to investigate if in human skeletal muscle, insulin-stimulated aPKC activity, similar to glucose uptake, is enhanced in response to prior endurance exercise. Furthermore, we investigated if responsiveness of aPKC towards PIP3, the signalling product of PI-3 kinase, is altered by prior exercise.
Compared with insulin-stimulated glucose uptake, only few studies have evaluated the effect of endurance exercise on insulin regulation of muscle protein kinetics. Insulin is a potent anabolic stimulus in skeletal muscle, increasing uptake of amino acids and protein synthesis (Biolo et al. 1995). Both endurance and resistance exercise improves insulin-stimulated uptake of selected amino acids in skeletal muscle (Zorzano et al. 1985; Biolo et al. 1999) and furthermore, the anabolic efficacy of amino acids and insulin is increased after resistance exercise (Biolo et al. 1997, 1999). This has not been investigated in response to endurance exercise in humans in vivo, and results from two rodent studies in vitro are conflicting showing either increased or unaltered insulin-stimulated protein synthesis (Davis & Karl, 1986; Balon et al. 1990).
Acute insulin-stimulated protein synthesis (mRNA translation) primarily results from rapid activation of existing components of the translational apparatus. In particular, peptide elongation is stimulated by insulin through a signalling sequence involving PI-3 kinase, Akt, mammalian target of rapamycin (mTOR), eukaryotic elongation factor-2 (eEF2) kinase and ultimately eEF2 as illustrated in Fig. 1. Interestingly, interaction with this pathway has been observed at the level of eEF2 kinase in response to activation of the extracellular-regulated protein kinase 1/2 (ERK 1/2) (Wang et al. 1998, 2001; Wang & Proud, 2002b) and furthermore, ERK activation seems to be required for improved insulin-stimulated muscle protein synthesis 16 h after resistance exercise in rodents (Fluckey et al. 2006). In human skeletal muscle, insulin-stimulated ERK signalling is improved 24 h after endurance exercise, although this has only been investigated in the obese and subjects with T2DM (Cusi et al. 2000). In human skeletal muscle myotubes, ERK is a downstream target of IRS-2-associated PI-3 kinase (Bouzakri et al. 2006). Furthermore, in both rodent (Howlett et al. 2002) and human (Howlett et al. 2006) muscle, IRS-2-associated PI-3 kinase activity is markedly increased in response to insulin stimulation immediately after endurance exercise. Collectively, this suggests a possible signalling interaction between endurance exercise and insulin signalling to mRNA translation.
Figure 1. Diagram showing insulin signalling through IRS-1 and IRS-2 to stimulate peptide chain elongation Signalling through IRS-2 is suggested as an alternative pathway to signalling through IRS-1, allowing for exercise to interact with insulin signalling. Bold full circles show signalling improved by prior endurance exercise. Bold dashed circles show signalling not regulated by prior endurance exercise. Regular full circles show signalling components not investigated.
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The second aim of the present study was to evaluate if the interaction between exercise and IRS-2-associated PI-3 kinase activity is present in human skeletal muscle 4 h after acute endurance-type exercise and furthermore if this coincides with altered downstream signalling to ERK and eEF2.
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The main findings of the present study are that improved insulin-stimulated glucose uptake in human skeletal muscle after exercise coincides with improved responsiveness of aPKC towards PIP3, the endogenous signalling product of PI-3 kinase activity. However, insulin-stimulated aPKC activity measured in vitro was not significantly greater after exercise. In response to prior exercise, IRS-2 (but not IRS-1)-associated PI-3 kinase activity was increased in basal and insulin-stimulated muscle. This coincided with corresponding altered phosphorylation levels of ERK 1/2, P70S6K, eEF2K and eEF2, suggesting a signalling mechanism that may allow for an additive effect of prior endurance exercise on subsequent insulin-stimulated protein synthesis.
Exercise is a useful intervention in order to increase insulin-stimulated glucose uptake in human skeletal muscle. However, the underlying mechanisms are still poorly understood (Wojtaszewski et al. 2002). A working hypothesis is that prior exercise interacts with downstream elements of the insulin-signalling pathway allowing for a more potent response upon subsequent insulin stimulation. This is supported by findings that metabolic endpoint events of insulin action in skeletal muscle (i.e. GS activity, GLUT4 translocation and glucose uptake) are increased more by insulin in previously exercised compared with rested muscle (Richter et al. 1982; Hansen et al. 1998; Thorell et al. 1999; Wojtaszewski et al. 2000). However, work from our group has previously shown that in human skeletal muscle insulin signalling at the level of IRTK, IRS-1 tyrosine phosphorylation, IRS-1-associated PI-3 kinase, Akt and GSK-3 is not enhanced by prior exercise (Wojtaszewski et al. 1997, 2000). Data from the present study confirm that prior exercise does not influence insulin-stimulated activation of IRS-1-associated PI-3 kinase (Fig. 4A) and Akt Ser473 phosphorylation (Fig. 8A). Furthermore, this study shows for the first time that Akt Thr308 phosphorylation (Fig. 8B) is likewise not affected by prior exercise.
A primary aim of the present study was to investigate if improved insulin action on glucose uptake coincided with altered regulation of aPKC in human skeletal muscle. In response to insulin stimulation, in vitro aPKC activity increased 1.3- to 1.5-fold. This somewhat lesser response compared with previous findings in healthy humans (Kim et al. 2003; Beeson et al. 2003) should probably be ascribed to the lower dose of insulin used in the present study (∼100 μU ml−1versus 700–900 μU ml−1). The increased aPKC activity was observed in the absence of any detectable changes in aPKC Thr410 phosphorylation. This seems inconsistent with the proposed model of activation based on studies in rat adipocytes (Bandyopadhyay et al. 1999; Kanoh et al. 2001) and the finding of increased aPKC Thr410 phosphorylation (Kanoh et al. 2003) in response to supra-physiological insulin stimulation in rat skeletal muscle. However, it should be emphasized that this has not previously been investigated in response to physiological insulin stimulation in humans. In this context, two previous studies have failed to show increased Thr410 phosphorylation in response to exercise in human muscle, despite increased in vitro activity of aPKC (Rose et al. 2004; Richter et al. 2004). This shows that activation of the enzyme does not always involve changes in Thr410 phosphorylation. Clearly more research is needed in order to fully understand the pattern of activation of aPKC in human skeletal muscle. However, based on the above, the proposed model of activation of aPKC based on studies in cells and rodent muscle should be cautiously adapted to intact human muscle.
In a range of models where insulin action is impaired, in rodent (Kanoh et al. 2001; Hori et al. 2002; Standaert et al. 2004), monkey (Chen et al. 2002) and human skeletal muscle (Vollenweider et al. 2002; Beeson et al. 2003; Kim et al. 2003), both IRS-1-associated PI-3 kinase activity as well as in vitro aPKC activity is decreased. This indicates that production of PIP3 through IRS-1-associated PI-3 kinase action is probably a key upstream signalling event in regulation of aPKC activity and stimulation of glucose uptake. It has furthermore been demonstrated that responsiveness of aPKC to direct allosteric activation by PIP3 is a site of regulation as illustrated by impaired activation of aPKC in muscle samples from obese glucose-intolerant as well as type 2 diabetic subjects in response to PIP3 in vitro (Beeson et al. 2003). An interesting finding of the present study is that responsiveness of aPKC towards PIP3 is significantly greater in basal samples of previously exercised muscle. In contrast to PIP3 responsiveness, insulin-stimulated aPKC activation measured in vitro was not significantly greater in previously exercised muscle (P= 0.17). This apparent dissociation between PIP3 responsiveness and in vitro aPKC activity might seem a bit puzzling if the improved PIP3 responsiveness is of physiological relevance. To our knowledge, no absolute measures of PIP3 concentrations in skeletal muscle are available. However, it cannot be ruled out that sensitivity of the in vitro activity assay does not enable detection of improved PIP3 responsiveness within a lower physiological range of PIP3 concentrations. Nonetheless, the present study demonstrates a novel interaction between exercise and regulation of aPKC, warranting further investigation. This interaction is probably of a robust nature since it can be detected in vitro after the procedure of immuno-precipitation. This study supports previous correlative findings that the ability of aPKC to be activated in response to PIP3 may be relevant to insulin-stimulated glucose uptake (Beeson et al. 2003) and furthermore that measurement of PIP3 responsiveness in vitro may be a useful analytical tool in this regard. However, based on the existing data it can only be speculated if altered PIP3 responsiveness is directly involved in improving insulin action on glucose uptake after exercise.
Activation of PI-3 kinase is involved in many of the insulin actions in skeletal muscle (Yeh et al. 1995; Lund et al. 1995; Shepherd et al. 1997, 1998). PI3-kinase is activated during insulin stimulation through binding of the regulatory src homology 2 domain to a phosphorylated IRS protein. The present study shows that in response to physiological insulin stimulation in vivo both IRS-1- and IRS-2-associated PI-3 kinase activities are increased in human skeletal muscle. Furthermore, it is shown that prior exercise increases IRS-2-associated PI-3 kinase activity in basal and insulin-stimulated muscle. Previously, it has been shown that immediately after exercise, IRS-2-associated PI-3 kinase activity is increased (Howlett et al. 2002, 2006) in insulin-stimulated (but not basal) muscle. Together with the present findings, this suggests that in a prolonged period after exercise, enzymatic capacity for production of PIP3 is increased due to regulation at the level of IRS-2-associated PI-3 kinase.
During insulin stimulation, signalling to protein synthesis is believed to involve PI-3 kinase and Akt to increase both initiation and elongation in the process of mRNA translation (Proud, 2006). However, other stimuli of protein synthesis (i.e. phorbol ester in HEK 293 cells, and phenylephrine and endothelin-1 in cardiac myocytes) have been shown to act through ERK in a PI-3 kinase- and Akt-independent manner (Herbert et al. 2000; Wang & Proud, 2002a,b). Importantly, these stimuli result in dephosphorylation of eEF2, supposedly through phosphorylation at Ser366 and deactivation of the upstream eEF2 kinase (Wang et al. 2001; Wang & Proud, 2002b). This study shows that phosphorylation of eEF2 decreases with physiological insulin stimulation in human muscle and, interestingly, in basal and insulin-stimulated muscle, eEF2 phosphorylation is further reduced by prior exercise. This finding is supported by increased Ser366 phosphorylation (leading to deactivation) of the upstream eEF2K in exercised muscle. Furthermore, this site on eEF2K is a target of S6 kinases (Wang et al. 2001) and correspondingly we observe increased phosphorylation of P70S6K Thr389 (a marker of activity) in exercised muscle during insulin stimulation. Although correlative, this study suggests an interaction between prior exercise and insulin signalling to peptide elongation through IRS-2-associated PI3-K, ERK and P70S6K leading to deactivation of eEF2K thus relieving inhibitory phosphorylation of eEF2 (see Fig. 1). In this context it is interesting that ERK activation seems to be required for improved insulin-stimulated muscle protein synthesis 16 h after resistance exercise in rodents, supporting the proposed link (Fluckey et al. 2006). Furthermore, it is noteworthy that immediately after exercise the interaction at the level of IRS-2 is markedly greater compared with the present study (Wang et al. 2001; Howlett et al. 2006), suggesting that at earlier (< 4 h) time points after exercise perhaps capacity for protein synthesis is even greater. Whether insulin signalling to translation initiation is similarly influenced by prior exercise remains to be investigated. However, this seems questionable since signalling through Akt and glycogen synthase kinase 3 (GSK-3), required for insulin-stimulated initiation (Proud, 2007), is not regulated by prior exercise using the present protocol (this study and Wojtaszewski et al. 2000). In the present study, protein kinetics were not evaluated, and as described, results from previous rodent studies are unequivocal (Davis & Karl, 1986; Balon et al. 1990). Interestingly, in human muscle during 3 h recovery from endurance exercise of comparable duration and intensity to the present study, a positive fractional synthesis rate of protein is observed (Levenhagen et al. 2001; Bolster et al. 2005); however, a positive net protein balance is only observed when exogenous amino acids are supplemented during recovery (Levenhagen et al. 2001). This suggests that in the present investigation protein synthesis most probably occurs, particularly considering the anabolic effect of insulin whereas net protein balance may still be negative. However, future studies will be needed to test if improved capacity for peptide elongation as observed at the signalling level in this study results in improved insulin-stimulated protein synthesis.
Activity of IRS-associated PI-3 kinase is mainly regulated by tyrosine/serine phosphorylation of the IRS protein and in particular through activation of IRTK during insulin stimulation. Since IRTK activity is not altered in response to prior exercise (Wojtaszewski et al. 2000), a possible explanation for increased signalling through IRS-2 after exercise is that exercise regulates activity of other kinases/phosphatases able to interact with IRS proteins. A number of kinases including PKB, c-Jun NH2 kinase, AMPK and aPKC (Aronson et al. 1998; Fujii et al. 2000; Jakobsen et al. 2001; Sakamoto et al. 2002; Moeschel et al. 2004; Weigert et al. 2005) have these characteristics; however, most research so far has evaluated regulation in regard to IRS-1. Interestingly, impaired IRS-2 signalling seems to be part of the metabolic syndrome as demonstrated by the finding of impaired IRS-2-associated PI-3 kinase activity in insulin-stimulated muscle from obese subjects with IGT (Vollenweider et al. 2002) and type 2 diabetic subjects (Kim et al. 1999; Vollenweider et al. 2002). Data from the present study thus suggest acute exercise as a beneficial interaction in prevention or treatment of this defect in human skeletal muscle.
In conclusion, the present study shows several novel molecular sites of interaction between exercise and subsequent insulin signalling in human skeletal muscle. Four hours after acute exercise IRS-2-associated PI-3 kinase activity, ERK phosphorylation and subsequent signalling to activation of eEF2 is increased in both basal and insulin-stimulated muscle, establishing a molecular link to increased capacity for insulin-stimulated protein synthesis after exercise. Furthermore, responsiveness of aPKC towards PIP3 is improved in response to prior exercise. This should be expected to counteract the observed impairment in PIP3 responsiveness in states of insulin resistance and overt T2DM. However, in the present study no effect of prior exercise on in vitro aPKC activity was observed (P= 0.17) thus a link to improved insulin-stimulated glucose uptake remains to be established.