REVIEW Exercise training-induced improvements in insulin action

Acta Physiol 2008, 192, 127 135 REVIEW Exercise training-induced improvements in insulin action J. A. Hawley and S. J. Lessard Exercise Metabolism Group, School of Medical Sciences, RMIT University, Bundoora, Vic., Australia Received 13 July 2007, accepted 17 August 2007 Correspondence: J. A. Hawley, Exercise Metabolism Group, School of Medical Sciences, RMIT University, PO Box 71, Plenty Road, Bundoora, Vic. 3083, Australia. E-mail: john.hawley@rmit.edu.au Abstract Individuals with insulin resistance are characterized by impaired insulin action on whole-body glucose uptake, in part due to impaired insulin-stimulated glucose uptake into skeletal muscle. A single bout of exercise increases skeletal muscle glucose uptake via an insulin-independent mechanism that bypasses the typical insulin signalling defects associated with these conditions. However, this insulin sensitizing effect is short-lived and disappears after 48 h. In contrast, repeated physical activity (i.e. exercise training) results in a persistent increase in insulin action in skeletal muscle from obese and insulin-resistant individuals. The molecular mechanism(s) for the enhanced glucose uptake with exercise training have been attributed to the increased expression and/or activity of key signalling proteins involved in the regulation of glucose uptake and metabolism in skeletal muscle. Evidence now suggests that the improvements in insulin sensitivity associated with exercise training are also related to changes in the expression and/or activity of proteins involved in insulin signal transduction in skeletal muscle such as the AMP-activated protein kinase (AMPK) and the protein kinase B (Akt) substrate AS160. In addition, increased lipid oxidation and/or turnover is likely to be another mechanism by which exercise improves insulin sensitivity: exercise training results in an increase in the oxidative capacity of skeletal muscle by up-regulating lipid oxidation and the expression of proteins involved in mitochondrial biogenesis. Determination of the underlying biological mechanisms that result from exercise training is essential in order to define the precise variations in physical activity that result in the most desired effects on targeted risk factors, and to aid in the development of such interventions. Keywords AMPK, AS160, insulin sensitivity, lipid metabolism, mitochondrial biogenesis, muscle glycogen. Historical perspective In recent decades intense research effort has focused on understanding the signalling mechanisms leading to exercise-stimulated glucose transporter 4 (GLUT4) translocation and the increased skeletal muscle glucose uptake and metabolism that follow a single bout of exercise. Results from many studies undertaken by independent laboratories demonstrate that muscle contraction stimulates glucose in the complete absence of insulin; that the maximal effects of contraction and insulin are additive; and that contraction and insulin stimulate glucose transport by separate pathways (for reviews, see, Holloszy & Hansen 1996, Ivy 1987, Henriksen 2002, Sakamoto & Goodyear 2002, Zierath 2002, Holloszy 2003, 2005). Following exercise there is a prolonged and persistent increase in glucose uptake by skeletal muscle (Ivy & Holloszy 1981, Young et al. 1983, Ren et al. 1993). Reversal of this increase in muscle insulin sensitivity after exercise occurs simultaneously Journal compilation Ó 2008 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2007.01783.x 127

Exercise and insulin action Æ J A Hawley and S J Lessard Acta Physiol 2008, 192, 127 135 with muscle glycogen repletion and can be accelerated by carbohydrate feeding or attenuated by keeping muscle glycogen content low by fasting (Young et al. 1983). If adequate carbohydrate is supplied throughout the post-exercise recovery period, muscle glycogen stores can be supercompensated to levels that are twofold higher than the fed sedentary state (Bergstrom & Hultman 1966, Cartee et al. 1989). A contemporary objective of exercise biochemistry is to understand the molecular mechanisms by which the various metabolic pathways involved in substrate turnover are regulated during exercise and identify how these acute signals may initiate responses that form the basis of the adaptations to chronic exercise (Hawley et al. 2006). In this brief review, we will focus on several of the key putative signalling proteins that are likely to be responsible for some of the exercise training-induced improvements in insulin action. Exercise: an effective therapeutic intervention for enhancing insulin sensitivity Individuals with insulin resistance and type 2 diabetes are characterized by impaired insulin action on wholebody glucose uptake, in part due to impaired insulinstimulated glucose uptake in skeletal muscle (Zierath et al. 1996). However, acute exercise increases glucose uptake in skeletal muscle via an insulin-independent mechanism that bypasses the insulin signalling defects associated with these conditions (Wallberg-Henriksson & Holloszy 1984, DeFronzo et al. 1987, Ivy 1987, Cortez et al. 1991, Zierath et al. 2000, Christ-Roberts et al. 2003, Richter et al. 2004, O Gorman et al. 2006). The acute increase in glucose transport in response to a single bout of whole-body exercise is mediated by a variety of intramyocellular signalling events including increased insulin receptor signalling, activation of the AMP-activated protein kinase pathway (AMPK), Akt/ protein kinase B phosphorylation, nitric oxide production and calcium-mediated mechanisms involving Ca 2+ / calmodulin-dependent protein kinase (CaMK) and protein kinase C (PKC) (Sakamoto & Goodyear 2002, Jessen & Goodyear 2005). As the insulin-sensitizing effects of an acute exercise bout are short-lived and persist for only 48 h if another bout of exercise is not undertaken (Ivy et al. 1983, Etgen et al. 1993, Wojtaszewski et al. 2002), the pertinent question is can exercise training prevent the insulin-resistant state that precedes type 2 diabetes? The resounding answer is yes (for reviews, see, Goodyear & Kahn 1998, Albright et al. 2000, Hawley 2004, Hawley & Houmard 2004). Here we summarize evidence to show that exercise training produces metabolic adaptations that result in sustained improvements in whole-body and muscle insulin sensitivity. Effects of exercise training on insulin signalling: IRS-1, IRS-2 and PI3K Exercise training results in a rapid increase in the expression of both GLUT-4 mrna and protein in skeletal muscle (Kraniou et al. 2006) and these changes have been associated with improved glucose uptake and metabolism (Dela et al. 1993, Ren et al. 1994, Hansen et al. 1995). The role of the exercise-induced increase in GLUT protein content on glucose transport has been reviewed previously (Ivy 1997, 2004) and will not be discussed in detail here. The immediate effects of exercise on glucose action occur primarily through the level of GLUT-4 trafficking (Ploug et al. 1998; Thong et al. 2005) rather than through any enhancement of insulin signalling at the level of the insulin receptors, insulin receptor substrate (IRS)-1, IRS-2 or phosphatidylinositol-3-kinase (PI3K) (Treadway et al. 1989, Wojtaszewski et al. 2000, Howlett et al. 2002). Because the effects of exercise on insulin sensitivity persist for between 16 (Ren et al. 1994, Chibalin et al. 2000) and 48 h (Bogardus et al. 1983) after the last exercise bout, measurements made at these times in individuals who undertake regular training reflect changes in expression or activity of a variety of signalling proteins involved in the regulation of skeletal muscle glucose uptake (Zierath 2002). The results of studies of the effects of exercise training on the insulin receptor substrates IRS-1 and IRS-2 are highly variable, possibly because of differences in the training stimulus (the mode, intensity and duration of exercise), prior dietary intake, training status and the muscles and/or fibre type being assessed (Chibalin et al. 2000, Howlett et al. 2002, 2006, 2007, Frosig et al. 2007). For example, in insulin-sensitive rodents who underwent either 1 or 5 days of exhaustive swimming (6 h day )1 ), IRS-1 protein expression tended to be increased after a day of exercise, whereas it was reduced 16 h after the chronic training regimen (Chibalin et al. 2000). Yu et al. (2001) reported similar results for IRS- 1 protein levels in muscle for humans engaged in endurance-training programmes. In contrast, a single bout of resistance training results in a decrease in basal (but not insulin-stimulated) IRS-1 tyrosine phosphorylation, yet following 7 days of training, basal IRS-1 phosphorylation was similar to pre-training values (Howlett et al. 2007). With regard to IRS-2 expression, levels of this protein are increased threefold in rodent muscle 16 h after a single bout of prolonged (6 h) swimming but return to pre-training levels 16 h after 5 days of repeated training bouts (Chibalin et al. 2000). In agreement with the results from animal studies, O Gorman et al. (2006) have reported that IRS-1 and IRS-2 protein expression are unaffected by short-term (7 days) exercise training in obese diabetic subjects, while Yu et al. (2001) showed that in skeletal muscle of 128 Journal compilation Ó 2008 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2007.01783.x

Acta Physiol 2008, 192, 127 135 humans habitually involved in endurance run-training, IRS-2 was actually decreased to levels below sedentary individuals. Taken collectively, these results suggest that exercise has diverse effects on this receptor substrate that involve changes in both signal transduction and protein expression. In the final analyses it may be that IRS-2 plays only a minor role in both insulin- and exercise-stimulated glucose transport in skeletal muscle, as has been previously suggested (Higaki et al. 1999). Clearly time-course studies of the effects of different stimuli (endurance and resistance exercise) are needed to fully elucidate the role of exercise training on IRS-1 and IRS-2 in skeletal muscle. While the effects of exercise on IRS-1 and IRS-2 are inconsistent, improvements in whole-body insulinmediated glucose uptake after exercise training have been attributed to enhanced intracellular signalling via PI3K activity in both rodent (Chibalin et al. 2000) and human models (Houmard et al. 1999, Kirwan et al. 2000). Such findings are clinically relevant because PI3K activity is decreased in skeletal muscle from insulinresistant subjects and patients with type 2 diabetes (Goodyear et al. 1995, Bjornholm et al. 1997, Kim et al. 1999). Houmard et al. (1999) demonstrated that 7 days of exercise training (1 h d )1 at 75% maximal oxygen consumption) improved whole-body glucose disposal and that this increase in insulin sensitivity was accompanied by an increase in insulin-stimulated PI3K activity. Kirwan et al. (2000) reported that insulin-stimulated PI3K activity was greater in skeletal muscle from endurance-trained vs. sedentary individuals, and when these two cohorts were compared together, PI3K activation was correlated with both glucose disposal and whole-body aerobic capacity. Recently, Frosig et al. (2007) reported that 3 weeks of one-legged knee-extensor exercise in healthy subjects increased insulin-stimulated glucose uptake by 60% in the trained limb, but that training reduced IRS-1-associated PI3K activity in both basal and insulin-stimulated muscle. The physiological significance of the exercise-induced decrease in basal and insulin-stimulated IRS-1-associated PI3K activity in muscle from healthy subjects is not presently clear. However, it would appear that the major effects of repeated contractions on the insulin signalling cascade in insulin-resistant muscle are confined to a traininginduced restoration of insulin-stimulated PI3K activity and/or phosphorylation, and not to increases in the protein expression of the canonical insulin receptor substrates, IRS-1 or IRS-2. Effects of exercise training on the AMP-activated protein kinase The up-regulation of the AMP-activated protein kinase (AMPK) is another potential mechanism by which J A Hawley and S J Lessard Æ Exercise and insulin action exercise training improves insulin sensitivity. In addition to acute activation of AMPK due to muscle contraction, exercise training results in an up-regulation of AMPK protein. Lessard et al. (2007) reported that in a rodent model of insulin resistance, the high-fat fed rat, 4 weeks of endurance training (treadmill running) resulted in a significant increase in the protein expression and activity of the a1 but not the a2 isoform. In healthy individuals, 3 weeks of endurance training increases the protein content of the AMPK a1, b2 and c1 subunits (Frosig et al. 2004). Seven weeks of exercise training (treadmill running) in obese Zucker rats results in a 1.5-fold increase in AMPKa1 protein expression and restores impaired AMPK activation to the level of lean controls (Sriwijitkamol et al. 2006). Pold et al. (2005) observed that 8 weeks of treadmill running in Zucker fatty rats produced similar improvements in insulin sensitivity as daily 5-aminoimidazole-4-carboxamide-1-b-d-ribofuranoside (AICAR) administration. However, unlike leptindeficient (ob/ob mouse) and leptin receptor-deficient (fa/ fa Zucker rat) rodent models of diabetes, humans with type 2 diabetes do not exhibit decreased AMPK subunit expression or activation compared with healthy controls (Wojtaszewski et al. 2005). Wojtaszewski et al. (2005) investigated the effect of strength training on the isoform expression and heterotrimeric composition of the AMPK in human skeletal muscle from 10 patients with type 2 diabetes and seven healthy controls. Subjects undertook 6 weeks of strength training with one leg while the other leg remained untrained. Muscle biopsies were obtained before and after the training period. Basal AMPK activity and mrna and protein expression of both catalytic (a1 and a2) and regulatory (b1, b2, c2, c3) AMPK isoforms were independent of health status, whereas the protein content of a1 (+16%), b2 (+14%) and c1 (+29%) were higher with the c3 content lower ()48%) in trained compared with untrained muscle. Even so, Wojtaszewski et al. (2005) observed a comparable increase in the expression of the a1, b2 and c3 subunits of AMPK in response to 6 weeks of resistance training in patients with type 2 diabetes and healthy controls. It is also possible that exercise-induced up-regulation of AMPK mediates its effects through distal components of the insulin signalling cascade. In this regard, an Akt substrate with molecular weight of 160 kda (AS160) and a molecular signature of a Rab-GTPase-activating protein (GAP) has recently been identified as an important regulator of GLUT4 traffic (Kane et al. 2002), promoting translocation of GLUT4-containing vesicles to the plasma membrane (Sano et al. 2003). Rab-GAP domains modulate the activity of Rab proteins, which are involved in the regulation of several membrane transport steps, including vesicle budding, motility, tethering and fusion (Zerial & McBride 2001). Journal compilation Ó 2008 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2007.01783.x 129

Exercise and insulin action Æ J A Hawley and S J Lessard Acta Physiol 2008, 192, 127 135 Insulin stimulation of skeletal muscle leads to phosphorylation of AS160, a process dependent on Akt2 (Bruss et al. 2005, Bouzakri et al. 2006). AS160 is also phosphorylated in response to exercise in human skeletal muscle (Deshmukh et al. 2006, Frosig et al. 2007) and after in vitro contraction in rodent skeletal muscle (Bruss et al. 2005, Kramer et al. 2006). Lessard et al. (2007) found that 4 weeks of endurance training increased IRS1-associated PI3K activity and normalized impairments to total protein levels in the Akt/AS160/ GLUT4 signalling pathway caused by high-fat feeding. Frosig et al. (2007) reported that both basal and insulinstimulated AS160 phosphorylation were increased in human skeletal muscle after 3 weeks endurance training and attributed this to changes in AMPK activity acting upstream of AS160. Interestingly, when AS160 phosphorylation was expressed relative to total protein content, the effect of training on this protein disappeared (Frosig et al. 2007). Thus, while AS160 may be a critical point of convergence for insulin- and exercisemediated glucose uptake in skeletal muscle (Deshmukh et al. 2008), the precise role to explain the traininginduced effects on AS160 signalling on glucose transport is not known. Aside from its role in regulating both insulin-dependent and -independent glucose uptake in skeletal muscle, AMPK is also a regulator of lipid metabolism. AMPK activation results in the up-regulation of fatty acid (FA) oxidation in skeletal muscle via phosphorylation of its target protein, acetyl CoA carboxylase (ACC), the enzyme that catalyses the rate-limiting step in the conversion of acetyl CoA to malonyl CoA. AMPKinduced phosphorylation at ser-218 inhibits the action of ACC and results in decreased cellular malonyl CoA levels. As malonyl CoA is a potent inhibitor of CPT1, a reduction in malonyl CoA alleviates the inhibition of CPT1 and consequently increases the transfer of FA- CoA into the mitochondria for oxidation. Fatty acid uptake and oxidation are thought to be mismatched in type 2 diabetes and obesity, and increased capacity to oxidize lipids is associated with improved insulin sensitivity (Bruce et al. 2003, 2006, Goodpaster et al. 2003, Perdomo et al. 2004). Therefore, it seems plausible that AMPK-induced increases in FA oxidation may be an additional mechanism by which AMPK activation improves skeletal muscle insulin sensitivity. Effects of exercise training on lipid status The regulation of lipid turnover and utilization is a mechanism by which exercise training may improve insulin sensitivity (Bruce & Hawley 2004). Exercise training results in an increase in the oxidative capacity of skeletal muscle by up-regulating the expression of proteins involved in mitochondrial biogenesis such as peroxisome proliferator-activated receptor c coativator (PGC1), peroxisome proliferator-activated receptor a (PPAR-a) and nuclear respiratory factor 1 (Gollnick & Saltin 1982, Hawley 2002, Irrcher et al. 2003). Oxidative enzyme capacity is low in individuals with insulin resistance, which is thought to contribute to a state of metabolic inflexibility that does not permit the transition between fasting and postprandial states observed in healthy, insulin-sensitive individuals (Storlien et al. 2004). This inflexibility, in turn is thought to contribute to the aberrant skeletal muscle glucose and lipid metabolism that is associated with insulin resistance and type 2 diabetes. Furthermore, the maximal activities of several skeletal muscle oxidative enzymes (i.e. citrate synthase) are good predictors of whole-body insulin sensitivity, suggesting that treatments that increase oxidative capacity may also improve insulin sensitivity (Bruce et al. 2003). In support of this contention, Goodpaster et al. (2003) demonstrated that the strongest predictor of insulin sensitivity following endurance training in obese individuals was enhanced whole-body lipid oxidation. Furthermore, increased oxidative capacity following exercise training was recently associated with increased CPT1 activity and decreased ceramide and diacylglycerol content in the muscle of obese individuals (Bruce et al. 2006). The findings by Bruce et al. (2006) suggest that exercise training may improve muscle insulin sensitivity by increasing the proportion of lipids targeted for oxidation, thereby reducing the accumulation of lipid species that are known to inhibit insulin signal transduction, as has recently been proposed (Hawley & Lessard 2007). In direct support of this contention, we (Lessard et al. 2007) have shown that 4 weeks of exercise training attenuated high-fat, diet-induced increases in muscle lipid storage. Furthermore, in that study (Lessard et al. 2007) exercise training was associated with increased rates of palmitate oxidation and elevated PGC-1 expression (i.e. mitochondrial biogenesis). Finally, despite lower rates of fatty acid oxidation at rest, it is noteworthy that individuals with insulin resistance are readily able to utilize lipids during exercise. Both obese sedentary males (Goodpaster et al. 2002) and females with abdominal adiposity (Horowitz & Klein 2000) have higher rates of FA oxidation during submaximal exercise compared with their lean sedentary, fitness-matched counterparts. Thus, the acute molecular/cellular signalling events that accompany contraction act to override the metabolic constraints observed in individuals with insulin resistance at rest (i.e., metabolic inflexibility) and predispose muscle towards a preference for lipid oxidation (i.e. metabolic flexibility). It is tempting to speculate that the oxidation and turnover of muscle lipids may represent a mechanistic link between mitochondrial function, lipid 130 Journal compilation Ó 2008 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2007.01783.x

Acta Physiol 2008, 192, 127 135 accumulation, skeletal muscle insulin resistance and the impaired insulin signalling observed in obesity, type 2 diabetes and other metabolic disorders (Hawley & Lessard 2007). Contractile activity and insulin resistance: the missing link? Disease states and traits are determined by lifelong interactions among multiple genetic and environmental factors (Booth & Vyas 2001, Booth & Lees 2007). Although the precise molecular basis underpinning all chronic diseases remains elusive, an extensive body of evidence exists linking decreased physical activity and the concomitant defects in aerobic metabolism to the pathogenesis of several metabolic disorders (Booth et al. 2000, Lees & Booth 2005). Less clear is whether this breakdown in aerobic metabolism is a result of the inactive state (i.e. environmental) or is instrumental in its development (i.e. genetic). Because an individual s level of physical activity directly influences health status, and as contractile activity is known to mediate gene expression and function, the inclusion of direct or indirect measures of aerobic function in investigations of genetic effects is crucial to unravel the complex processes that ultimately determine disease risk profiles. To specifically study the mechanisms associated with several progressive metabolic disease states, a novel animal model that emulates an exercise-deficient J A Hawley and S J Lessard Æ Exercise and insulin action phenotype has been developed (Britton & Koch 2001, 2005, Koch & Britton 2001). Through twoway artificial selection, they created a rodent model of low (LCR) or high (HCR) intrinsic aerobic phenotype. In this model, 11 generations of two-way artificial selection produced rats that differed markedly in aerobic exercise capacity (374% difference between LCR and HCR) while simultaneously generating animals that differed markedly for metabolic and cardiovascular disease risk factors: LCR were insulin resistant compared with HCR, as verified by higher fasting blood glucose and insulin concentration and impaired glucose tolerance (Wisloff et al. 2005). Noland et al. (2007) recently reported that HCR animals from generation 13 are resistant to the development of high-fat diet-induced obesity and insulin resistance: upon exposure to a chronic (12 weeks) high-fat diet, LCR rats gained more weight and fat mass and their insulin-resistant condition was exacerbated despite a similar energy intake as HCR rats. These data highlight the importance of genetic factors in protecting or predisposing individuals to insulin resistance and metabolic syndrome. Summary and directions for future research Insulin resistance precedes type 2 diabetes by several decades and the complications resulting from diabetes are often irreversible. Accordingly, it would seem prudent to attempt to improve insulin resistance before Figure 1 A summary of the molecular events that contribute to exercise training-induced improvements in glucose transport into skeletal muscle. There are several ways in which exercise training may improve skeletal muscle glucose uptake. These include: (1) up-regulation of GLUT4 expression and the facilitation of insulin signal transduction, (2) the chronic activation of AMPK and (3) promoting mitochondrial biogenesis and increasing lipid oxidation and turnover thereby preventing the accumulation of deleterious lipid species. IR, insulin receptor; IRS, IR substrate; PI3K, phosphatidylinositol-3-kinase; PDK, phosphatidylinositoldependent protein kinase; Akt, protein kinase B; AS160, Akt substrate of 160 kda; GLUT, glucose transporter; AMPK, AMPactivated protein kinase; ACC, acetyl CoA carboxylase; CPT, carnitine palmitoyl transferase; PGC, peroxisome proliferatoractivated receptor c coactivator; NRF, nuclear respiratory factor; FA-CoA, fatty acyl CoA; DAG, diacylglycerol. Journal compilation Ó 2008 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2007.01783.x 131

Exercise and insulin action Æ J A Hawley and S J Lessard Acta Physiol 2008, 192, 127 135 the onset of diabetes and its secondary complications. As skeletal muscle is the major source for insulinstimulated glucose uptake, any treatment targeted to improve glucose uptake in this tissue will improve whole-body glucose homeostasis. There is irrefutable evidence that exercise training is an effective therapeutic intervention to increase insulin action in skeletal muscle from obese and insulin-resistant individuals. There are several ways in which exercise training may improve skeletal muscle glucose uptake. These include upregulation of GLUT4 expression, chronic activation of AMPK, facilitation of insulin signal transduction at the level of PI3K and AS160, as well as increases in the expression of several proteins involved in glucose and lipid utilization and turnover (Fig. 1). Determination of the underlying biological mechanisms that result from exercise training is essential in order to define the precise variations in physical activity that result in the most desired effects on targeted risk factors and to aid in the development of such interventions. Conflicts of interest The authors declared no conflicts of interest. This review was supported by a grant from the Australian Research Council (DP0663862) to the authors. References Albright, A., Franz, M., Hornsby, G., Kriska, A., Marrero, D., Ullrich, I. & Verity, L.S. 2000. American College of Sports Medicine position stand. Exercise and type 2 diabetes. Med Sci Sports Exerc 32, 1345 1360. Bergstrom, J. & Hultman, E. 1966. Muscle glycogen synthesis after exercise: an enhancing factor localized to the muscle cells in man. Nature 210, 309 310. Bjornholm, M., Kawano, Y., Lehtihet, M. & Zierath, J.R. 1997. 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