Exercise Effects on Muscle Insulin Signaling and Action 2002.pdf

8/10/2019 Exercise Effects on Muscle Insulin Signaling and Action 2002.pdf

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Exercise Effects on Muscle Insulin Signaling and ActionExercise and insulin signaling: a historical perspective

EVA TOMA S,1,2 ANTONIO ZORZANO,2 AND NEIL B. RUDERMAN11Diabetes Unit, Section of Endocrinology, Boston Medical Center and Department

of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118;

and 2Department de Bioqumica i Biologia Molecular, Facultat de Biologia,

Universitat de Barcelona, 08028 Barcelona, Spain

Tomas, Eva, Antonio Zorzano, Neil B. Ruderman. Exercise andinsulin signaling: a historical perspective.J Appl Physiol 93: 765772, 2002;

10.1152/japplphysiol.00267.2002.Over the past 30 years, a consider-able body of evidence has revealed that a prior bout of exercise canincrease the ability of insulin to stimulate glucose transport and glyco-gen synthesis in skeletal muscle. Apart from its clinical implications, thiswork has led to a considerable effort to determine at a molecular levelhow exercise causes this effect and, in particular, whether it does so byenhancing specific events in the insulin-signaling cascade. The objectiveof this review is to discuss from a historical perspective how our currentthinking in this area has evolved and the people responsible for it. Areasto be discussed include the effect or lack of effect of prior exercise on theinsulin-signaling pathway, effects of exercise on the regulation by insulinof the GLUT-4 glucose transporter in muscle, and the emerging role of

AMP-activated protein kinase as a mediator of exercise-induced signal-ing events. In addition, we will discuss briefly some of the avenues thatresearch in this area is likely to follow.

diabetes; glucose transport; 5-aminoimidazole-4-carboxamide ribofur-anoside; AMP-activated protein kinase; skeletal muscle

CHAUVEAU AND KAUFMAN(9) in 1887 reported that when ahorse chews on hay the concentration of glucose in theblood draining its masseter muscle substantially de-creases. This remarkable observation was the firstdemonstration that glucose uptake by muscle is en-hanced during exercise. In 1924, Sir Henry Dale inEngland (6) and Carl and Gerti Cori in the UnitedStates (11) demonstrated with their colleagues thatinsulin also increases glucose uptake by muscle. This

review will focus on the interaction between insulinand exercise in regulating glucose uptake and in par-ticular will focus on the question of whether a priorbout of exercise enhances the ability of insulin to stim-ulate glucose utilization by an effect on insulin signal-ing. For additional discussions of aspects of this subject

and of the signaling changes induced in muscle byexercise per se, the reader is referred to a recent paperby Richter et al. (46) and to an earlier review in thisseries by Goodyear and Sakamoto (19).

ORIGIN OF THE NOTION THAT A PRIOR BOUT

OF EXERCISE ACUTELY ENHANCES

INSULIN ACTION ON MUSCLE

The first clue that exercise might enhance the ability

of insulin to stimulate muscle glucose utilization wasprobably provided by Per Bjorntorp and his co-workersin Gothenberg, Sweden in the early 1970s. In 1972 (3),they reported that glucose tolerance was better andplasma insulin levels lower in middle-aged Swedishmen who regularly participated in competitive sportsthan in age- and weight-matched control men. Thesame investigators also reported that 6 wk of physicaltraining lowered plasma insulin levels (although it didnot affect glucose tolerance) in a group of hyperinsu-linemic, obese women (2, 4). Collectively, these resultsled to the suggestion that regular exercise can increase

Address for reprint requests and other correspondence: N. B.Ruderman, Diabetes Unit, Section of Endocrinology, Boston Medi-cal Center and Dept. of Medicine, Boston Univ. School of Medi-cine, 650 Albany St., X-825, Boston, MA 02118 (E-mail:[email protected]).

J Appl Physiol93: 765772, 2002;10.1152/japplphysiol.00267.2002.

8750-7587/02 $5.00 Copyright 2002 the American Physiological Societyhttp://www.jap.org 765


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whole body and presumably muscle insulin sensitivity,a notion subsequently confirmed in rats by Carl Mon-don, Constantine Dolkas, and Gerald Reaven at Stan-ford (37).

Bjorntorps studies did not address the question ofwhether the increase in insulin sensitivity associatedwith physical activity is an acute or a chronic effect ofexercise. The fact that adipose tissue mass, fat cell size,

and plasma lipids (cholesterol and triglycerides) werelower in the athletes whom they studied than in thecontrol men suggested the latter (3). On the otherhand, their observation that acute decreases in plasmainsulin could persist for several days after each indi-vidual exercise bout suggested the former (4). Thisquestion aside, Bjorntorps research encouraged sev-eral groups to examine the effect of several months ofregular exercise on glucose tolerance in patients withType 2 diabetes, a disorder associated with insulin resis-tance. In the first of these studies, reported in 1979,Bengt Saltin and co-workers in Sweden (49) and NeilRuderman and his colleagues at the Joslin research Lab-oratory in Boston (48) found modest improvements inglucose tolerance in patients with chemical diabetes (im-paired glucose tolerance) and diet-controlled Type 2 dia-betes, respectively. On the other hand, the relative tran-sience of the improvement (it had disappeared by 8 daysafter the last exercise bout) found by the Rudermangroup led them to question whether it was the result of along-term effect of training, such as improved fitness.This and their subsequent finding that hemoglobin A1Clevels can be markedly diminished by regular exercise insimilar patients with Type 2 diabetes (51) led them toassess the effect of a single bout of exercise on insulinaction in skeletal muscle.

In studies that they carried out at Boston University,

Erik Richter and co-workers (47) used the isolatedperfused rat hindquarter preparation to demonstratethat the ability of a physiological concentration of in-sulin (75 U/ml) to stimulate glucose uptake and gly-cogen synthesis in muscle is enhanced for severalhours after a 45-min treadmill run (Table 1). Theyshowed that this effect is restricted to muscles that hadperformed work, as judged by glycogen depletion (47),and that it was reproduced when hindquarter musclewas made to contract by electrical stimulation of thesciatic nerve. Antonio Zorzano et al. (62), working inthe same laboratory, later showed that prior exerciseenhances the ability of insulin to stimulate -amino-isobutyrate uptake by muscle, indicating that it also

acts on the Na

-dependent A system for amino acidtransport. In 1990, Greg Cartee et al. (15), in thelaboratory of John Holloszy at Washington Universityin St. Louis, reported that the period of increasedinsulin sensitivity postexercise could be greatly pro-longed if the rats were fasted or fed a low-carbohydratediet, afinding that they attributed to these nutritionalmanipulations slowing the repletion of muscle glyco-gen stores. That prior exercise enhances insulin-stim-ulated glucose utilization in human muscle was firstreported in 1987 by John Devlin and Edward Horton atthe University of Vermont (13).

Armed, in part, with the initial data from Richters

rodent studies, Stephen Schneider and co-workers atthe New Jersey College of Medicine and Boston Uni-versity School of Medicine (50) carried out studiessuggesting that the decrease in hemoglobin A1C inpatients with Type 2 diabetes caused by physical train-ing is due to the cumulative effect of the individualexercise bouts rather than improvedfitness. More spe-cifically, in patients who experienced decreases of he-moglobin A1C in excess of 1% after several months ofphysical training, Schneider et al. showed that glucosetolerance was substantially better at 12 and 17 h thanat 72 h after the last bout of exercise. Concurrentstudies in nondiabetic athletes by Heath et al. in Hol-loszys laboratory (23) and by Burstein and Posner andcolleagues at McGill (7), demonstrating that the highinsulin sensitivity of trained individuals diminishesrapidly (days) when these individuals cease exercising,strongly supported this conclusion.

In summary, these early reports established beyondquestion that a single bout of exercise enhances thesensitivity and responsiveness of skeletal muscle toinsulin in both humans and experimental animals.They suggested that both glucose and amino acidtransport are affected and that the effect of exercise ismediated in great measure by local rather than sys-temic factors. They also suggested that much of theapparent benefit of physical training on glycemic con-

trol and insulin sensitivity in patients with Type 2diabetes is attributable to a residual effect of the lastbout of exercise. As will be discussed later, however,cumulative effects of regular exercise in these patientsalmost certainly also play a major role.

EFFORTS TO EXPLAIN AT A MOLECULAR LEVEL HOW

PRIOR EXERCISE ENHANCES INSULIN-STIMULATED

GLUCOSE TRANSPORT IN MUSCLE

Insulin signaling. The demonstration that prior ex-ercise enhances certain actions of insulin in skeletalmuscle (47) took place at the same time that the early

Table 1. Glucose utilization by the isolated perfusedrat hindquarter after treadmill running:effect of insulin

Insulin,U/ml

Glucose Uptake, mol g1 h1

Control n Postexercise n

0 1.80.2 4 1.90.4 510 2.20.3 8 2.80.2 830 1.90.2 4 2.70.2* 475 3.20.2 12 6.10.3* 13

500 6.30.1 3 9.41.1* 320,000 10.20.9 8 12.60.5* 840,000 10.60.2 3 12.10.2* 2

Values are means SE; n no. of observations. Hindquarterswere placed in the perfusion system 20 min after the cessation of a30-min treadmill run or an equivalent period of rest. After 12 min ofequilibration, glucose utilization was measured over the next 45 min.Control rats were not exercised. Insulin at the indicated concentra-tions was added to the initial cell free perfusate. Between 10 and 20%of the insulin was degraded during the perfusion. *P 0.05, com-pared with control values. [Adapted from Ref. 47.]

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rat skeletal muscle by Edward Hortons group at theUniversity of Vermont and Harriet Wallberg-Henriks-son at the Karolinska Institute of Sweden (24) and byHolloszys laboratory working in conjunction withAmira Klip at the University of Toronto (14). Thelaboratories of Holloszy (34), Morris Birnbaum at theUniversity of Pennsylvania (59), and Steen Lund andOluf Pedersen (35) at Aarhus University Hospital in

Denmark later showed that such glucose transporterrecruitment could occur even when activation of PI3-kinase, which is required for the stimulation of glucosetransport by insulin, is inhibited by wortmannin.These important findings proved conclusively that ex-ercise and insulin trigger GLUT-4 translocation inmuscle by effects on different signaling mechanisms.However, they did not rule out the possibility thatinsulin and exercise stimulate some common down-stream signaling event.

Historically, one hypothesis put forth to explain thepostexercise increase in insulin-stimulated glucosetransport relates to the possibility that insulin andexercise stimulate the translocation of GLUT-4 fromdifferent pools. If this occurred, insulin could act on alarger number of transporters after exercise or it couldact on transporters with different properties. The no-tion of distinct intracellular insulin- and exercise-recruitable GLUT-4 pools in skeletal muscle was firstproposed by Amira Klip and co-workers (15) at theUniversity of Toronto, based on analyses of isolatedintracellular membranes and plasma membranes fromcontrol, exercised, and acutely insulin-treated rats.Subsequent to this, Lise Coderre (10) in Paul Pilchslaboratory at Boston University was the first to isolateand characterize biochemically intracellular insulinand exercise-recruitable GLUT-4 populations from rat

skeletal muscle by using fractions separated by discon-tinuous sucrose density-gradient centrifugation. Shefound that the two transporter populations had thesame protein composition but that they differed intheir densities and sedimentation coefficients. Themost definitive evidence for distinct insulin and exer-cise-recruitable GLUT-4 pools, however, was reportedin 1998 by Torkil Ploug at the Muscle Research Centerof the University of Copenhagen, working in collabora-tion with Samuel Cushman and Evelyn Ralston at theNational Institutes of Health (44). In an elegant set ofexperiments, they first demonstrated by immunofluo-rescence microscopy and immunogold electron micros-copy that intracellular GLUT-4 vesicles are either pos-

itive or negative for the transferrin receptor. They thenproceeded to show that only the transferrin receptor-positive vesicles were recruited by contractions. Laterstudies by Eva Tomas in Zorzanos laboratory at theUniversity of Barcelona suggested that both of thesepools (insulin and exercise) are derived from an endo-somal compartment (52). Thus the evidence for distinctexercise and insulin recruitable pools is reasonablystrong. Whether their presence will help explain thepostexercise increase in insulin-stimulated glucosetransport remains to be determined. To answer thisquestion will almost certainly require fundamental in-

formation about the distal signals by which insulin andexercise trigger GLUT-4 vesicle recruitment from in-tracellular populations, the docking of these vesicles,and their fusion with cell surface membranes.

Finally it has also been suggested that exercise couldincrease glucose uptake by enhancing the synthesis ofGLUT-4 at the level of transcription. Thus studies fromDohms laboratory (39) have shown that a single bout

of exercise can moderately increase GLUT-4 mRNA inpreviously untrained (1.4-fold) and trained rats for 3 h(1.8-fold) after a single bout of exercise. Likewise, Renet al. working in Holloszys laboratory (45) found a 50%increase in GLUT-4 protein and a twofold increase inGLUT-4 mRNA 16 h after a single bout of swimmingexercise. Thus exercise clearly also acutely inducessignals that lead to an increased expression of GLUT-4mRNA and protein. As with its effect on GLUT-4 trans-location, the precise identity of these signals is notknown. For additional information on this and othermaterial covered in this section, the reader is referred toa number of excellent recent reviews (18, 26, 30, 46, 56).

AMP-ACTIVATED PROTEIN KINASE

A major advance in understanding at a molecularlevel how contraction affects glucose transport in themuscle cell was the discovery of AMP-activated proteinkinase (AMPK). This heterotrimeric enzyme, whichcontains distinct-,-, and-subunits, is regulated bychanges in a cells energy state and possibly otherfactors. Asfirst characterized in liver by David Carlingand D. Graham Hardie (21) at the University ofDundee in Scotland, increases in the AMP-to-ATP orcreatine-to-creatine phosphate ratios of a cell, such asthose that occur in response to ischemia and other

stresses, can activate AMPK by several mechanisms.The activation of AMPK, in turn, sets in motion anumber of events that both increase ATP generation(e.g., increased fatty acid oxidation) and decrease ATPutilization for processes not required for a cells imme-diate viability (e.g., inhibition of cholesterol and fattyacid synthesis).

Thefirst indication that AMPK could be involved inthe regulation of muscle metabolism was reported byWilliam Winder at Brigham Young University in Utahin a joint effort with Hardie (55). They demonstratedthat treadmill running increased AMPK activity in redquadriceps muscle of a rat by two- to threefold within 5min. They also showed that this increase in activity

persisted for as long as the rat continued to run andthat it was associated with decreases in the activity ofacetyl-CoA carboxylase and the concentration of malo-nyl-CoA, an allosteric inhibitor of carnitine palmitoyltransferase, the enzyme that controls the transfer oflong-chain fatty acyl CoA into mitochondria where theyare oxidized. Subsequently, Demetrios Vavvas and co-workers in the Ruderman laboratory (54) and Winderet al. (55) reported similar changes in response tomuscle contraction induced by sciatic nerve stimula-tion. Vavvas et al. also showed that activation ofAMPK in this setting involved the 2-isoform that it




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was evident within seconds and that after 5 min ofintense contraction it could persist for 1 h.

A specific role for AMPK in the regulation of glucoseuptake was demonstrated in 1997 by Winder and co-workers (36). Using an isolated rat hindquarter prep-aration, they showed that perfusion with the AMPKactivator 5-aminoimidazole-4-carboxamide ribofurano-side (AICAR) increased glucose uptake by approxi-

mately twofold. Subsequent studies carried out jointlyby the Goodyear and Winder laboratories establishedthat this effect of AICAR was due to increased glucosetransport, that it involved GLUT-4 translocation (33),and that, like the increase in glucose transport inducedby muscle contraction, it was not blocked when PI3-kinase is inhibited by wortmannin (22). Likewise,chronic AICAR therapy has been shown by Winderslaboratory to increase GLUT-4 protein in muscle (asdoes exercise) in vivo (25). A similar effect, as well asan increase in GLUT-4 mRNA has been demonstratedby Holloszy et al. (41) in epitrochlearis muscle incu-bated for 24 h with AICAR. Despite its array effects onGLUT-4, the precise mechanism by which AMPK acti-vation stimulates glucose transport is still unknown.On the other hand, the molecular events activated in acell by AICAR in vitro should be easier to characterizethan those induced by muscle contraction in vivo. Forthis reason, AICAR or other pharmacological AMPKactivators may prove to be useful tools in determininghow prior exercise enhances insulin action in muscle.In this context, Fisher and Nolte (16), working inHolloszys laboratory, have recently shown that bothprior exposure to AICAR and prior tetanic contractionenhance the ability of insulin to stimulate glucosetransport in an incubated epitrochlearis muscle. Theyalso found that early and intermediate events in the

insulin-signaling pathway (e.g., PI3-kinase activation)were not involved. Thus AMPK, like exercise, appearsto work downstream or independently of PI3-kinase toimprove insulin sensitivity.

Finally, recent studies by Birnbaums laboratory (38)have shown that expression of a dominant inhibitorymutant of AMPK completely blocks the ability of hyp-oxia or AICAR to activate glucose uptake, althoughonly partially reducing contraction-stimulated glucoseuptake in skeletal muscle. These authors suggestedthatAMPK transmits a portion of the signal by whichmuscle contraction increases glucose uptake, but otherAMPK-independent pathways also contribute to theresponse.

SUBJECTS FOR FURTHER STUDY

The effects of prior exercise on insulin-resistant mus-cle. This review has focused on the effects of an acutebout of exercise on insulin action and signaling innormal skeletal muscle. However, from a clinical per-spective, the effects of prior exercise are most likely tobe relevant in muscle that is insulin resistant. Insulinresistance has been defined as an impaired ability of agiven amount of insulin to exert its usual biologicaleffect and could be related to a decrease in insulin

sensitivity or responsiveness. It has long been appre-ciated that muscle of patients with Type 2 diabetes, orat risk for developing it, are insulin resistant (12). Itwas for this reason that regular physical activity wasfirst considered for the therapy and prevention of Type2 diabetes (37, 49). Despite this, investigations of theeffect of a prior bout of exercise on insulin action ininsulin-resistant muscle have been few. A potentially

important study was carried out by Nicholas Oakes etal. (40), working in E. W. Kraegens laboratory at theGarvan Institute in Sydney, Australia. In rats madeinsulin resistant by ingestion of a eucaloric, high-fatdiet for 3 wk, they found that a single bout of exercise(24 h earlier) substantially reversed the impaired abil-ity of insulin to stimulate glucose uptake into muscleduring a euglycemic hyperinsulinemic clamp. Theyalso found that the improvement in insulin sensitivityafter exercise was associated with decreases in theconcentrations of long-chain fatty acyl CoA and malo-nyl CoA. In a subsequent study, Kim Bell et al. (1) inthe same laboratory found that prior exercise also

reversed an increase in membrane-associated proteinkinase C- in muscle of fat-fed rats. Although insulinsignaling is altered in muscle of these rats (60), theeffect of exercise on these signaling abnormalities hasnot been studied.

Effects of a single bout of exercise vs. those of physicaltraining. As already noted, the increased insulin sen-sitivity of muscle in physically trained humans disap-pears rapidly (4872 h) once they stop exercising, sug-gesting it is in large part related to the last bout ofphysical activity. On the other hand, physical trainingdiminishes adiposity, fat cell size, and plasma insulinlevels (3) and increases the expression of GLUT-4 inmuscle (61), all of which could hypothetically enhance

the ability of a given amount of insulin to stimulateglucose transport. Thus the relative importance of in-dividual exercise bouts vs. chronic effects of physicaltraining in enhancing insulin action remains unset-tled.

As already noted, improvements in glucose tolerancewere observed over 20 years ago in response to physicaltraining in patients with Type 2 diabetes. Severalyears later, complete normalization of glucose toler-ance and high plasma insulin levels were observedafter 1 yr of intense training by Holloszy and co-workers (27), and more recently increased physicalactivity has been shown to diminish progression from

impaired glucose tolerance to Type 2 diabetes by Pan etal. (42). Despite these impressive clinical findings, in-vestigations of the effects of physical training on insu-lin signaling in muscle of trained humans and experi-mental animals have yielded inconsistent results. Forfurther discussion of the effects of physical training oninsulin action in humans and experimental animals,and in particular subjects with Type 2 diabetes and/orinsulin resistance, the reader is referred to a number ofrecent reviews (18, 26, 46, 56) and several chapters inthe recently published Handbook of Exercise in Diabe-tes (47a).

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Effect of exercise on cells other than those in skeletalmuscle. Another question for future study is whetherexercise enhances insulin signaling in cells other thanthe skeletal muscle myocyte. Such cells could bepresent in the arterial wall. Thus insulin resistance, asmanifest by impaired activation of PI3-kinase and Aktby insulin, has been found in the aorta of a hyperinsu-linemic obese fa/fa (Zucker) rat by George King and

co-workers at the Joslin Research Laboratory (31). Inaddition, Yasuo Ido, David Carling, and Neil Ruder-man at Boston University and Hammersmith Hospitalin England (29) have shown that the ability of insulinto activate Akt in cultured human umbilical vein en-dothelial cells is depressed after 24 h of incubation in ahyperglycemic (30 mM glucose) medium. Relevant tothis review, they found that this impairment in insulinsignaling caused by hyperglycemia was preventedwhen the human umbilical vein endothelial cells wereconcurrently incubated with AICAR, an activator ofAMPK. Whether exercise causes a similar increase inAMPK in the endothelial cell, in vivo, and if so whetherthis contributes to its ability to diminish cardiovascu-lar disease in humans (47a) is clearly a subject forfurther study.

CONCLUDING REMARKS

The subject of exercise and insulin signaling is some-what unusual for a historical review, since the first keyobservations are barely 20 years old and papers pub-lished in the past year are also discussed. Despite this,it is increasingly clear that lifestyle changes that in-clude physical activity will very likely play an increas-ing role in the prevention and treatment of Type 2diabetes and other diseases associated with insulinresistance. In addition, it is equally clear that an un-

derstanding at a molecular level (e.g., insulin signal-ing) of how exercise diminishes insulin resistance andprevents cellular damage and/or dysfunction will becritical to the success of this effort.

This work was supported in part by National Institute of Diabetesand Digestive and Kidney Diseases Grants DK-19514 andDK-49147, a Mentor Based Fellowship award from the AmericanDiabetes Association, and a Center Grant for the Study of DiabeticComplications from the Juvenile Diabetes Research Foundation.

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highlighted topics

Exercise Effects of Muscle Insulin Signaling and ActionInvited Review: Exercise training-induced changesin insulin signaling in skeletal muscle

JULEEN R. ZIERATHDepartment of Clinical Physiology, Karolinska Hospital,

Karolinska Institutet, SE-171 77 Stockholm, Sweden

Zierath, Juleen R. Invited Review: Exercise training-inducedchanges in insulin signaling in skeletal muscle. J Appl Physiol 93:773781, 2002;10.1152/japplphysiol.00126.2002.This review will pro-

vide insight on the current understanding of the intracellular signalingmechanisms by which exercise training increases glucose metabolismand gene expression in skeletal muscle. Participation in regular exerciseprograms can have important clinical implications, leading to improvedhealth in insulin-resistant persons. Evidence is emerging that insulinsignal transduction at the level of insulin receptor substrates 1 and 2, aswell as phosphatidylinositol 3-kinase, is enhanced in skeletal muscleafter exercise training. This is clinically relevant because insulin signal-ing is impaired in skeletal muscle from insulin-resistant Type 2 diabeticand obese humans. The molecular mechanism for enhanced insulin-stimulated glucose uptake after exercise training may be partly relatedto increased expression and activity of key proteins known to regulateglucose metabolism in skeletal muscle. Exercise also leads to an insulin-independent increase in glucose transport, mediated in part by AMP-

activated protein kinase. Changes in protein expression may be relatedto increased signal transduction through the mitogen-activated proteinkinase signaling cascades, a pathway known to regulate transcriptionalactivity. Understanding the molecular mechanism for the activation ofinsulin signal transduction pathways after exercise training may providenovel entry points for new strategies to enhance glucose metabolism andfor improved health in the general population.

AMP-activated protein kinase; diabetes; gene expression; insulin recep-tor substrates; mitogen-activated protein kinase; phosphatidylinositol3-kinase

EXERCISE TRAINING: A PHYSIOLOGICAL TOOL TO

ENHANCE INSULIN ACTION

People with non-insulin-dependent Type 2 diabetesmellitus are characterized by impaired insulin actionon whole body glucose uptake, partly owing to im-paired insulin-stimulated glucose transport in skeletalmuscle (98, 99). Defects in insulin action on glucoseuptake in skeletal muscle from Type 2 diabetic patientshave been linked to impaired signal transduction (7,

43). Insulin sensitivity has been shown to be related tothe degree of physical activity (64); therefore, physicaltraining programs may offer a physiological means toimprove insulin action in some insulin-resistant peo-ple. Exercise training improves glucose tolerance andinsulin action in insulin-resistant humans (35, 59) orType 2 diabetic patients (63, 74). The molecular mech-anism for enhanced glucose uptake with exercise train-ing may be related to increased expression and/or ac-tivity of key signaling proteins involved in theregulation of glucose uptake and metabolism in skele-tal muscle (Fig. 1). For example, exercise training leadsto increased expression of glucose transporter 4(GLUT-4) content in skeletal muscle, and this has been

Address for reprint requests and other correspondence: J. R. Zier-ath, Dept. of Clinical Physiology and Integrative Physiology, Karo-linska Institutet, von Eulers vag 4, II, SE-171 77 Stockholm, Sweden(E-mail: [email protected]).

J Appl Physiol93: 773781, 2002;10.1152/japplphysiol.00126.2002.

8750-7587/02 $5.00 Copyright 2002 the American Physiological Societyhttp://www.jap.org 773


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correlated with improved insulin action on glucosemetabolism (10, 14, 29, 35, 57). However, emergingevidence suggests that these exercise-training-inducedimprovements in glucose uptake are not limited tochanges in GLUT-4 expression. The improvements ininsulin sensitivity after exercise training may be re-lated to changes in expression and/or activity of pro-teins involved in insulin signal transduction in skeletalmuscle. This review will focus on effects of exercise oninsulin signaling in skeletal muscle. Emphasis will beplaced on studies whereby insulin signaling was mea-sured several hours after an acute exercise bout orafter a period of exercise training.

EARLY STEPS IN INSULIN SIGNAL TRANSDUCTION

The insulin receptor is a heterotetrameric mem-brane glycoprotein composed of two-subunits and two-subunits, linked together by disulfide bonds (re-viewed in Ref. 77). Insulin binds to the extracellular-subunits, and this leads to activation of the trans-membrane -subunits and autophosphorylation ofthe receptor. Multiple tyrosine phosphorylation sitespresent on the -subunit of the insulin receptor playimportant functional roles in promoting receptor ki-

nase activity, mediating differential responses alongmitogenic and metabolic pathways, and facilitating theinteraction between the receptor and intracellular sub-strates. In recent years, research efforts have largelymoved from studies designed to characterize insulinbinding and receptor function to studies oriented to-ward the identification and characterization of postre-ceptor molecular targets that regulate insulin signaltransduction to different metabolic and mitogenic re-sponses. Although the picture is far from complete,some important early steps in insulin signaling haveemerged.

Insulin receptor substrates. Insulin signaling is acomplex series of events involving multiple effectorproteins that orchestrate diverse cellular responses.Importantly, insulin signaling pathways are not nec-essarily linear, as there is a high degree of cross talkbetween the signal transducers. Insulin receptor sub-strate isoforms (IRS-1 to -4) (46, 47, 69, 70), Gab-1 (30),and Cbl (58) link the initial event of insulin receptorsignaling cascade to downstream events. IRS mole-cules contain multiple tyrosine phosphorylation sitesthat become phosphorylated after insulin stimulation(reviewed in Refs. 77, 79) and bind downstream signal-ing molecules containing src homology 2 domains.IRS-1 and IRS-2 play selective roles in the regulationof metabolic and mitogenic responses in insulin-sensi-tive tissues, including skeletal muscle, adipose tissue,and liver. IRS-3 and IRS-4 are not expressed in skele-tal muscle; therefore, these substrates will not be re-viewed. Likewise, because of the paucity of data con-cerning the role of Gab-1 and Cbl in mediating insulinsignaling to glucose transport after exercise in skeletalmuscle, these substrates will not be reviewed.

Tissue-specific roles of IRS-1 and IRS-2 have beenelucidated through studies performed with different

knockout strategies in mice. IRS-1 appears to be thepredominant isoform mediating signal transduction inskeletal muscle (2, 71), whereas IRS-2 appears to beimportant in -cell development (80). Both isoformsare important for regulation of metabolism in liver(36). Although different IRS proteins clearly have se-lective roles in mediating many of metabolic and mito-genic responses, a degree of redundancy in the functionmay exist. For example, in skeletal muscle and adiposetissue from Type 2 diabetic subjects, insulin-mediatedtyrosine phosphorylation of IRS-1 is impaired, whereasIRS-2 phosphorylation is normal (7, 43, 60). Thus IRS

Fig. 1. Exercise training-induced changesin insulin signaling in skeletal muscle. In-sulin signal transduction though the insu-lin receptor, insulin receptor substrate(IRS)-1/2 and phosphatidylinositol 3-ki-nase (PI3-kinase) is enhanced in skeletalmuscle in the hours after an exercise bout.

These changes may enhance insulin sensi-tivity, as well as regulate gene expressionafter exercise. Immediately after exercise,mitogen-activated protein kinase (MAPK)signaling to downstream substrates is en-hanced, providing a possible molecularmechanism for exercise-induced transcrip-tional regulation in skeletal muscle. Acuteexercise also increases AMP-activated pro-tein kinase (AMPK) activity, leading tochanges in glucose uptake and gene ex-pression. Exercise training is associatedwith changes in mRNA of several compo-nents of insulin and MAPK signaling cas-cades. The master regulator(s) of exer-cise-responses on gene expression has notbeen completely defined.

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molecules are likely to play complementary roles in themediation of insulin action.

Phosphatidylinositol 3-kinase and downstream effec-tors. Phosphatidylinositol 3-kinase (PI3-kinase) is oneof the most characterized intermediate effector mole-cules that associate with IRSs. PI3-kinase associateswith tyrosine phosphorylated IRSs after insulin stim-ulation and catalyzes the formation of phosphatidyl-

inositol-3,4,5-trisphosphate, which serves as an allo-steric regulator of phosphoinositide-dependent kinase(1). PI3-kinase plays an important role in the acuteeffect of insulin on glucose transport and GLUT-4translocation in skeletal muscle (49, 65, 92). Becauseseveral reviews in this series will consider molecularmechanisms by which insulin or exercise mediateGLUT-4 translocation and glucose transport, this as-pect will not be considered in depth in the presentreview. The downstream effectors of PI3-kinase thatsignal to glucose transport have not been fully eluci-dated. PI3-kinase presumably mediates glucose trans-port via signaling to protein kinase B (PKB)/Akt and/orprotein kinase C (PKC)- (reviewed in Ref. 77). Tissueculture systems or animal models in which either sig-naling via AKT/PKB (11) or PKC- (4, 41) has beendisrupted suggest that these targets partly contributeto the regulation of glucose uptake, although otherintermediates are likely to participate (58). Throughcomparative genomics and pathway analysis, newdownstream components of the insulin pathway arelikely to be identified.

EFFECTS OF EXERCISE TRAINING

ON INSULIN SIGNALING

Immediately after an acute bout of exercise, glucose

transport in skeletal muscle is increased through aninsulin-independent translocation of GLUT-4 to thecell surface (18, 42, 49). Thus immediate effects ofacute exercise on glucose homeostasis occur primarilyat the level of GLUT-4 traffic rather than throughenhanced insulin signaling at the level of the insulinreceptor, IRS-1, IRS-2, or PI3-kinase (34, 48, 49, 66, 73,8688, 92, 97). Several hours after acute exercise, apersistent increase in insulin sensitivity of glucosetransport occurs in skeletal muscle. Effects of exercisecan be observed even 16 h after the last exercisesession (10, 57). Measurements made at this time mayreflect changes in protein expression (enhanced or sup-pressed) that occur in response to the exercise bout.

Exercise training increases insulin-mediated wholebody glucose disposal (15, 16, 32, 35). This effect iscorrelated with increased protein expression ofGLUT-4 (10, 15, 32, 35, 56, 57, 94), as well as withadaptive responses in expression and function of keyinsulin-signaling molecules (10, 33, 40, 94). Althoughour understanding of the signaling pathways regulat-ing glucose metabolism is limited, studies designed toexamine the effects of exercise training on known con-stitutes of the insulin signaling pathway are emerging.

Insulin receptor substrates.IRS-1 and IRS-2 are im-portant signal transducers in skeletal muscle. Exercise

training-induced effects on IRSs have been elucidated.In rodents, long-term endurance training (5 bouts/wkfor 9 wk) increased insulin receptor and IRS-1 mRNAin skeletal muscle 48 h after the last bout of exercise(37). In contrast, insulin receptor and IRS-1 mRNAwas not altered after short-term endurance training inhumans (60 min/day for 9 days) (78). However, comple-mentary studies of protein expression were not per-

formed in either of these studies (37, 78). Consistentwith thisfinding in humans, IRS-1 protein expressionis not increased 16 h after short-term endurance train-ing in rats (6 h/day for 1 or 5 days) (10). In this model,insulin-stimulated tyrosine phosphorylation of IRS-1tended to be increased after 1 day of exercise. Theincrease in IRS-1 tyrosine phosphorylation correlatedwith increased insulin receptor tyrosine phosphoryla-tion (10). Surprisingly, IRS-1 protein expression wasreduced 16 h after 5 days of exercise, despite a pro-found increase in insulin-stimulated IRS-1 tyrosinephosphorylation. The reduction in IRS-1 protein ex-pression in exercise-trained rodents is similar to the55% reduction in IRS-1 protein expression in skeletalmuscle obtained 48 h after exercise from subjects en-gaged in habitual training programs (running 50km/wk for 2 mo) (94). Major effects of exercise train-ing on insulin signaling do not include transcriptionalactivation of the IRS-1 gene. Rather, improvements ininsulin action after exercise training are likely to occurfrom more efficient signaling per molecule of IRS-1,leading to increased signal transduction to down-stream substrates.

Exercise training has differential effects on proteinexpression of IRS-1 and IRS-2. In rat epitrochlearismuscle, 16 h after an acute 6-h swim bout, IRS-2expression is increased threefold (10). In this model,

IRS-2 expression is restored to pretraining levels inmuscle studied 16 h after 5 days of repeated 6-h swimbouts. Thus increased IRS-2 protein expression partlyaccounts for increased insulin action in skeletal muscleafter exercise. In support of this hypothesis, mRNAlevels of IRS-2 in human skeletal muscle increase tran-siently 3 h after a single exercise bout, but this effect isdiminished after short-term (9 days) endurance train-ing (78). The initial observation that exercise increasesinsulin action at the level of IRS-2 was confirmed withIRS-2 knockout mice (34). In wild-type mice, insulin-mediated IRS-2 tyrosine phosphorylation was in-creased in skeletal muscle immediately after exercise,with no effect noted in IRS-2 null mice (34). Although

IRS-2 protein expression was not assessed, increasedprotein expression of IRS-2 is not likely to account forenhanced tyrosine phosphorylation after exercise.Thus exercise has multiple effects on IRS-2 that in-volve changes in signal transduction and protein ex-pression. Immediately after exercise, insulin-mediatedIRS-2 tyrosine phosphorylation is enhanced. In thehours after an acute exercise bout, IRS-2 undergoes arapid upregulation at the level of mRNA and protein.The enhanced insulin action on IRS-2 is maintained forat least 16 h after exercise. Detailed time-course stud-ies of the effects of exercise on either signal transduc-

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homeostasis associated with Type 2 diabetes and obesity(51). AMPK is a heterotrimeric protein, composed of onecatalytic () and two noncatalytic (and) subunits (84)and is activated by cellular stress associated with ATPdepletion (26). Although AMPK activity does not appearto be increased in response to insulin, some discussion ofthis kinase is warranted in the present review as it hasbeen implicated to be one of several critical regulators of

mitogenic and metabolic events in response to exercise inskeletal muscle. For example, an increase in AMPK ac-tivity in response to muscle contraction or exercise hasbeen correlated with GLUT-4 translocation and glucosetransport in skeletal muscle (5, 6, 26, 27, 45, 50). Fur-thermore, increased AMPK activity has also been corre-lated with increased free fatty acid oxidation in skeletalmuscle (6), decreased lipogenesis and lipolysis in adipo-cytes (68), and decreased free fatty acid and cholesterolsynthesis in hepatocytes (28). Thus recent evidence isconsistent with the hypothesis that AMPK plays a cen-tral role in the regulation of glucose homeostasis in re-sponse to exercise.

Exercise-mediated changes in AMPK activity. Iso-form-specific and exercise intensity-dependent changesin AMPK activity have been observed in skeletal mus-cle (21, 89). Low- to moderate-intensity aerobic exer-cise induces an isoform-specific and intensity-depen-dent increase in AMPK2but not in AMPK1activityin moderately trained subjects (21, 89). However, inresponse to anaerobic sprint exercise, activity of AMPK1and 2are both increased (9). These exercise-inten-sity differences may be related to the finding thatAMPK complexes containing the 2-isoform ratherthan the1-isoform have a greater dependence on AMP(9, 62). Although these studies do not directly linkactivation of AMPK to increased glucose uptake, direct

evidence can be acquired from studies in transgenicanimal models. Transgenic overexpression of a domi-nant inhibitory mutant of AMPK in skeletal musclecompletely blocks the ability of hypoxia to activateglucose uptake, whereas only partially reducing con-traction-stimulated glucose uptake (52). Thus AMPK-dependent and AMPK-independent pathways contrib-ute to the regulation of glucose uptake in skeletalmuscle in response to exercise. For example, in rats,glucose transport in slow-twitch muscle can be mark-edly activated in response to contraction, without mea-surable changes in AMPK activity (17). Collectively,these studies illustrate the complexity in identifyingthe precise role of the AMPK pathway in regulating

metabolic events, and they strongly suggest that addi-tional factors contribute to the regulation of exercise-mediated glucose uptake. However, the latter studiesdo not distract from the attractiveness of AMPK as atarget for exercise-induced glucose transport and acandidate for pharmacological intervention to improveglucose homeostasis.

AMPK and metabolic disease. Because AMPK ap-pears to increase glucose metabolism by insulin-inde-pendent signaling cascades (27), activation of thispathway provides an alternative strategy to increaseglucose transport in insulin-resistant skeletal muscle.

An obvious hypothesis to consider is whether pharma-cological intervention of AMPK with compounds de-signed to mimic the exercise response on glucose up-take or fatty acid oxidation may be efficacious in themanagement of metabolic abnormalities associatedwith Type 2 diabetes mellitus. One compound com-monly utilized to test this hypothesis is 5-aminoimida-zole-4-carboxamide ribonucleoside (AICAR). AICAR is

an adenosine analog that can be taken up into intacthepatocytes, adipocytes, and skeletal muscle and canbe phosphorylated to form 5-aminoimidazole-4-carbox-amide ribonucleotide, the monophosphorylated deriva-tive that mimics the effects of AMP on AMPK withoutaffecting ATP or ADP content. In isolated epitrochle-aris muscle incubated in serum, AICAR exposure leadsto an increase in insulin sensitivity that appears tomimic an exercise response (20).

AICAR effects on whole body glucose homeostasishave been determined in diabetic rodents. Treatmentof diabetic ob/ob (67) or KKAy-CETP (19) mice withAICAR lowers blood glucose and insulin concentrationand improves glucose tolerance. Furthermore, in vitroexposure of isolated skeletal muscle to AICAR elicits anormal increase in glucose transport in insulin-resis-tantob/obmice (67). This is consistent with studies inType 2 diabetic subjects whereby exercise is reported toelicit a normal increase in AMPK2activity in skeletalmuscle (53). These studies provide evidence to suggestthat exercise-induced AMPK activity and AICAR-induced AMPK activity are not impaired in insulin-resistant skeletal muscle. However, AICAR treatmentof ob/ob (67) and KKAy-CETP (19) mice is associatedwith a worsening of the blood lipid profile. BecauseAICAR is a nonspecific AMPK activator (12, 76), long-term exposure to AICAR may trigger effects other than

activation of AMPK in either liver or adipose tissueand this may influence plasma lipid mobilization. Inthis respect, the recent work from Moller and col-leagues (95) is important to emphasize, as they haveidentified AMPK as the elusive target of metformin, fur-ther highlighting the importance of AMPK in the regu-lation of glucose homeostasis and providing proof ofconcept that activation of this target can enhance insulinsensitivity, as metformin is a widely used drug for treat-ment of Type 2 diabetes mellitus. Through use of a novelAMPK inhibitor, AMPK activation was shown to be re-quired for metformins inhibitory effect on glucose pro-duction by hepatocytes. Furthermore, incubation of iso-lated epitrochlearis muscle with metformin resulted in

an increase in the activity of both catalytic subunits ofAMPK, coincident with an increase in glucose uptake.Thesefindings (95) have important clinical implicationsbecause metformin also increases insulin-stimulated glu-cose transport in skeletal muscle from Type 2 diabeticsubjects (22, 23).

MECHANISMS FOR INCREASED PROTEIN EXPRESSION

IN SKELETAL MUSCLE AFTER EXERCISE

Mitogen-activated protein kinase signaling. One fu-ture direction will be the identification of pathways

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that regulate gene expression in skeletal muscle afterexercise. Clearly, multiple mechanisms contribute tothe regulation of insulin action and protein expression.Recent evidence suggests that mitogen-activated pro-tein kinase (MAPK) signaling cascades may constituteone important cellular signaling mechanism mediatingexercise-induced adaptations in skeletal muscle.MAPK activation has been implicated as an important

mechanism governing cellular proliferation and differ-entiation in many cell types (reviewed in Ref. 55).Although the possible involvement of MAPK signaltransduction pathways in exercise-mediated regula-tion of gene expression in skeletal muscle has beenconsidered in detail (reviewed in Ref. 82), a brief re-view is warranted.

Members of the MAPK family form at least threeparallel signaling cascades that include the extracellu-lar-regulated protein kinase (ERK1/2 or p42 and p44MAPK), p38 MAPK, and c-Jun NH2 kinase. Evidenceis emerging that MAPK signaling pathways are di-rectly activated in human skeletal muscle in responseto acute, short-term exercise (3, 44, 81, 83) or endur-ance running (8, 93). Activity of several downstreamsubstrates of ERK and p38 MAPK signaling cascades,such as MAPK-activated protein kinase (MAPKAPK) 1and 2, as well as the mitogen and stress-activatedkinase (MSK) 1 and 2, are increased immediately afteracute sprint (44) or endurance exercise (93). Substratespecificity for MAPK signaling cascades has been de-termined with an ex vitro system to achieve contrac-tion (electrical stimulation) of isolated rat epitrochle-aris muscle, combined with the use of chemicalinhibitors of ERK and p38 MAPK (61). Thus contrac-tion-induced inductions of MAPKAPK1 and MAP-KAPK2 occur via separate pathways, reflecting ERK

and p38 MAPK stimulation, respectively. In contrast,induction of MSK1 and MSK2 requires simultaneousactivation of ERK and p38 MAPK (61). The direct linkbetween MAPK activation and changes in gene expres-sion in skeletal muscle after exercise has yet to beestablished, as the majority of studies to address thispoint have been correlative (reviewed in Ref. 82). Fu-ture work directed toward understanding whether ex-ercise-induced MAPK signaling directly suppresses orenhances gene expression is necessary.

AMPK signaling.AMPK has been proposed to regu-late gene expression (25). This may be partly throughdirect targeting of AMPK complexes containing the2-isoform to the nucleus (62). AMPK is involved in

transcriptional regulation by repressing genes in-volved in glucose signaling in hepatocytes (62, 90) andupregulating genes involved in glucose uptake andsubstrate metabolism in skeletal muscle (31, 54, 85).For example, activation of AMPK mimics several clas-sic exercise-mediated responses on gene expression,including increases in GLUT-4 mRNA and protein con-tent, hexokinase II mRNA and activity, uncouplingprotein-3 mRNA, mitochondrial enzymes, and glycogencontent in skeletal muscle (31, 54, 85, 96). Thesechanges can also be observed in skeletal muscle fromdiabetic rodents. Hexokinase II and GLUT-4 protein

expressions, as well as in vitro MEF2 sequence-specificbinding activity, are increased in skeletal muscle fromlean and ob/ob mice after 7 days of AICAR treatment(67), presumably through increased AMPK activity. Asimilar increase in MEF2 sequence-specific bindingactivity has also been observed in human skeletalmuscle after marathon running (93). Thus increasedMEF2 sequence-specific binding activity may confer

exercise-specific changes in gene expression. Consis-tent with this hypothesis, the MEF2 site appears to beessential for GLUT-4 expression, because deletions orpoint mutations within the MEF2 consensus bindingsequence of the human GLUT-4 promoter completelyprevent tissue-specific and hormonal/metabolic regula-tion of GLUT-4 (72).

Cross talk between MAPK and AMPK signaling path-ways.AMPK may activate other downstream effectorssuch as p38 MAPK and mitogen-activated protein ki-nase kinase 3 (91). For example in clone 9 cells, acti-vation of p38 was reported to be required for AICAR-stimulated glucose transport, because treatment of thecells with the p38 inhibitor SB-203580 or overexpres-sion of dominant-negative p38 mutant inhibited glu-cose transport (91). Thus AICAR-mediated activationof glucose transport in clone 9 cells involves AMPKsignaling to p38. Future work aimed to determinewhether there is similar cross talk between AMPKand MAPK pathways in skeletal muscle will be impor-tant to understand the nature of signals that lead tochanges in gene expression in response to exercise.Identification of AMPK and MAPK substrates thatactivate or repress specific genes should reveal impor-tant regulatory mechanisms controlling protein ex-pression in skeletal muscle.

SUMMARY AND FUTURE DIRECTIONS

Exercise training appears to enhance insulin sensi-tivity by increased postreceptor insulin signaling. In-creased insulin-mediated glucose transport appears tobe related to enhanced signal transduction at the levelof IRS proteins and PI3-kinase. These findings areclinically relevant because insulin-stimulated tyrosinephosphorylation of IRS-1 and activity of PI3-kinase arereduced in skeletal muscle from Type 2 diabetic pa-tients (7, 13, 43). Thus exercise training may be onetherapeutic strategy to restore impaired insulin signaltransduction in skeletal muscle from Type 2 diabeticpatients.

Because the insulin-signaling pathway(s) to glucosetransport has not been fully elucidated, a more com-plete mapping of the necessary and required compo-nents of this network is required. Identification ofintermediates in the insulin signaling pathway may beachieved through comparative genomics, using genet-ically modified model organisms, combined with bioin-formatic approaches to identify mammalian homo-logues for pathway analysis. Studies with ex vivomodels and chemical inhibitors may directly link in-sulin signaling and MAPK or AMPK pathways tochanges in gene expression in response to exercise

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training. Transgenic and knockout mice in which com-ponents of insulin signaling and MAPK or AMPK cas-cades have been overexpressed or ablated will revealthe requirements for these signaling intermediates inexercise-mediated responses. Knowledge of the humangenome sequence, used in concert with gene and/orprotein array technology, will provide a powerfulmeans to facilitate efforts in revealing molecular tar-

gets that regulate glucose homeostasis in response toexercise training. This will also offer quicker waysforward to identifying gene expression profiles in insu-lin-sensitive and insulin-resistant human tissue andmay by useful to identify biochemical entry points fordrug intervention to improve glucose homeostasis.

This review was supported by grants from the Swedish MedicalResearch Council, Swedish Diabetes Association, Swedish NationalCentre for Research in Sports, and Novo-Nordisk Foundation.

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