molmed.biomedcentral.comacute, muscle-type specific insulin resistance following injuryacute insulin...

INTRODUCTIONInsulin activation of the insulin recep-

tor (IR) is important for the proper regu-lation of cellular metabolism. Activationof the IR results in activation of at leasttwo major signaling pathways, the phos-phatidylinositol 3-kinase (PI3-kinase)/Akt pathway, which mediates many ofthe metabolic effects of insulin, and themitogen-activated protein kinase(MAPK)/extracellular regulated kinase(ERK) pathway, which mediates many ofthe mitogenic effects of insulin (1,2). Im-pairment of one or more of these path-ways may lead to insulin resistance (3,4).Insulin resistance is defined as a state in

which normal concentrations of insulinproduce a less than normal biological re-sponse (5). Although there are numerousstudies on the development of insulin re-sistance in chronic insulin resistantstates, including type 2 diabetes, obesity,and polycystic ovarian syndrome, theexact mechanisms resulting in insulin re-sistance have been elusive. It is likelythat there are multiple possible mecha-nisms that are disease dependent, andthe mechanisms may differ in differentinsulin target tissues. An acute form ofinsulin resistance (sometimes called“stress diabetes” or “critical illness dia-betes”) is observed following severe in-

juries, surgical trauma, hemorrhage,thermal injury (burn), and sepsis (6–16).This state of insulin resistance and hy-perglycemia can occur rapidly followingphysical injury, unlike the extended peri-ods often necessary for development ofinsulin resistance in chronic diseases. In-tensive insulin therapy, to compensatefor the development of hyperglycemiaand restore normoglycemia in criticallyill individuals, results in 34%–50% reduc-tions in septicemia, renal failure, transfu-sions, polyneuropathy, and mortality(17,18). Thus, an understanding of themechanisms of acute insulin resistanceand hyperglycemia, and the ability totreat this resistance, may be importantfor new developments to increase sur-vival after injury and critical illness. Nei-ther the causative factors nor the cellularmechanisms of the acute development ofinsulin resistance following various in-juries or critical illnesses have been eluci-dated. In the chronic diseases associated

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Acute, Muscle-Type Specific Insulin Resistance FollowingInjury

LaWanda H Thompson,1 Hyeong T Kim,2 Yuchen Ma,1 Natalia A Kokorina,1 and Joseph L Messina1,3

1Department of Pathology, Division of Molecular and Cellular Pathology, The University of Alabama at Birmingham, Birmingham,Alabama, United States of America; 2Department of Internal Medicine, College of Medicine, Pochon CHA University, Seoul, SouthKorea; 3Veterans Affairs Medical Center, Birmingham, Alabama, United States of America

Acute insulin resistance can develop following critical illness and severe injury, and the mortality of critically ill patients canbe reduced by intensive insulin therapy. Thus, compensating for the insulin resistance in the clinical care setting is important.However, the molecular mechanisms that lead to the development of acute injury/infection-associated insulin resistance areunknown, and the development of acute insulin resistance is much less studied than chronic disease-associated insulin resist-ance. An animal model of injury and blood loss was utilized to determine whether acute skeletal muscle insulin resistance de-velops following injury, and surgical trauma in the absence of hemorrhage had little effect on insulin-mediated signaling. How-ever, following hemorrhage, there was an almost complete loss of insulin-induced Akt phosphorylation in triceps, and severelydecreased tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1. The severity of insulin resistance wassimilar in triceps and extensor digitorum longus muscles, but was more modest in diaphragm, and there was little change in in-sulin signaling in cardiac muscle following hemorrhage. Since skeletal muscle is an important insulin target tissue and accountsfor much of insulin-induced glucose disposal, it is important to determine its role in injury/infection-induced hyperglycemia. Thisis the first report of an acute development of skeletal muscle insulin signaling defects. The presented data indicates that thedefects in insulin signaling occurred rapidly, were reversible and more severe in some skeletal muscles, and did not occur incardiac muscle.Online address: http://www.molmed.orgdoi: 10.2119/2008-00081.Thompson

Address correspondence and reprint requests to Joseph L Messina, Department ofPathology, Division of Molecular and Cellular Pathology, The University of Alabama at

Birmingham, Volker Hall, G019J, 1530 Third Avenue S., Birmingham, AL 35294-0019. Phone:

205-934-4921; Fax: 205-975-1126; E-mail: [email protected].

Submitted July 8, 2008; Accepted for publication September 19, 2008; Epub (www.

molmed.org) ahead of print September 25, 2008.

with insulin resistance, skeletal muscle,adipose tissue, and liver become insulinresistant. However, it is not knownwhich of these three main insulin targettissues become insulin resistant acutelyfollowing injury. Since skeletal muscle isa main insulin target tissue, and accountsfor approximately 80% of insulin-inducedglucose disposal in the human body (19),it is important to understand its role inthe acute development of insulin resist-ance. In the current study, we utilized arat model of surgical trauma and hemor-rhage to determine the development,timing, and muscle selectivity ofhemorrhage-induced skeletal muscle in-sulin resistance.

MATERIALS AND METHODS

Reagents and MaterialsAll reagents and materials were ob-

tained from Fisher Scientific (Pittsburgh,PA, USA) or Sigma-Aldrich (St. Louis,MO, USA), unless otherwise noted.

Animal Model of Surgical Trauma andHemorrhage

All procedures were carried out in ac-cordance with the guidelines set forthin the Guide for the Care and Use ofLaboratory Animals and the NationalInstitutes of Health. The experimentalprotocol was approved by the Institu-tional Animal Care and Use Committeeof the University of Alabama at Birm-ingham. A model of surgical traumaand hemorrhage in the rat, as previ-ously described (6,7), was used withmodifications. Briefly, male Sprague-Dawley rats received continuous in-halation of low levels of isoflurane(Mallinckrodt Veterinary, Mundelein,IL, USA) throughout the surgery andhemorrhage periods. A 5-cm ventralmidline laparotomy was performedrepresenting soft-tissue trauma, the ab-domen was closed in layers, and thewounds were bathed with 1% lidocaine(Elkins-Sinn, Cherry Hill, NJ, USA). Theright and left femoral arteries and theright femoral vein were catheterized forbleeding, monitoring of mean arterial

pressure and fluid resuscitation, respec-tively. The rats were bled to a mean ar-terial pressure (MAP) of 35–40 mmHgwithin 10 min. Once MAP reached40 mmHg, the timing of the hemor-rhage period began and was main-tained for up to 90 min. If the rats werenot killed during the hemorrhage pe-riod, they were resuscitated withRinger’s lactate (4 × the withdrawnblood volume) infusion over 60 min.Sham-operated rats underwent thesame surgical procedures, but withouthemorrhage. To ensure that the effectsobserved were not due to the initialbleed, following the initial bleed, theanimal’s original whole blood (he-parinized) was returned, and the ani-mal maintained for 60 min.

Experimental DesignDue to the considerable stress incurred

by anesthesia and surgical trauma, it wasimpossible to have a completely un-treated control group. Thus, trauma-alone rats (T0’) that were subjected toanesthesia, laparotomy, and catheteriza-tion, and then immediately killed wereselected as the “baseline” animals inthese experiments (6,7). Additionaltrauma-only groups were subjected tothe same procedures and then killed at30 (T30’), 60 (T60’), and 90 min (T90’),and 5 (T5h) and 24 h (T24h) after cathe-terization. Trauma plus hemorrhage (TH)groups were subjected to the same proce-dures as the T groups, but also subjectedto hemorrhage (see above) and thenkilled at 0 (TH0’), 15 (TH15′), 30 (TH30’),60 (TH60’), and 90 min (TH90’), and 5(TH5h) and 24 h (TH24h) after the initialbleed period.

Blood and Tissue HarvestingProcedures

At the appropriate time points, the ab-dominal cavity was opened again, bloodsamples were collected, and insulin (5U)or saline was injected into the portalvein. The triceps, extensor digitorumlongus, diaphragm, and heart were re-moved and frozen in liquid nitrogen 2–3min following the injection.

Measurement of Fasting Blood Insulinand Glucose Levels

Serum samples were collected andstored at –80° C until analysis. Insulin lev-els were determined using a rat insulinradioimmunoassay kit (Linco Research,St. Charles, MO, USA). Glucose levelswere measured using a GM7 Analyzer(Analox Instruments, Lunenburg, MA,USA). All analysis was performed by theUAB Clinical Nutrition Research Unit.

Preparation of Tissue LysatesTissue lysates were prepared as de-

scribed previously (20). Pulverized mus-cle tissue (50–60 mg; triceps, extensordigitorum longus, diaphragm, or heart)from each animal was homogenizedwith a pestle mounted in a motorizedhomogenizer in ice-cold lysis buffer con-taining 50 mM Tris-HCl, pH 7.4, 150 mMNaCl, 5 mM EDTA, 1% Triton X-100, 1%deoxycholate, 0.1% SDS, 0.5% Igepal,100 μM PMSF, 1 mM Na3VO4, 0.1 μMokadaic acid, and 0.5 × P2714 proteaseinhibitors. A protease/phosphatase in-hibitor cocktail containing 100 μM PMSF,1 mM Na3VO4, 0.1 μM okadaic acid, and0.5 × P2714 protease inhibitors wasadded to each tissue lysate and thelysates were placed on ice for 30 min.Tissue lysates were centrifuged twice at15,000g and supernatants were stored at–80° C until use.

Western Blot AnalysisProtein concentrations were deter-

mined (BCA, Pierce, Rockford, IL, USA)and 30 μg/lane of total protein was re-solved by SDS-PAGE and transferred tonitrocellulose membranes (6,21,22). Themembranes were immunoblotted withanti-phosphoserine-Akt (S473), anti-totalAkt, and anti-total ERK1/2 antibodies(Cell Signaling Technology, Beverly, MA,USA); anti-phosphotyrosine-IR (Y972),and anti-phosphotyrosine-IRS-1 (Y612;Invitrogen, Carlsbad, CA, USA); andanti-total IRβ (Santa Cruz Biotechnol-ogy, Santa Cruz, CA, USA), also wereused. Membranes were developed withECL (Amersham Biosciences, Piscataway,NJ, USA).

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H E M O R R H A G E A N D S K E L E T A L M U S C L E I N S U L I N R E S I S T A N C E

Densitometric and Statistical AnalysisECL images of immunoblots were

scanned and quantified using FlurochemFC digital imaging software (α Innotech,San Leandro, CA, USA; 6,22). All dataare presented as means ± SEM. Analysisof variance (ANOVA) and Student t testwere performed using GraphPad InStatversion 3 software (San Diego, CA,USA).

RESULTS

Insulin-Induced Phosphorylation ofAkt Is Abolished in Skeletal MuscleFollowing Hemorrhage

The initial experiments were to deter-mine whether hemorrhage would lead tothe acute development of skeletal muscleinsulin resistance. Based on previousstudies, immediately following surgery(T0’), 90 min following surgery (T90’), or90 min following surgery and hemor-rhage (TH90’), insulin (+) or saline (–)was injected, and serine phosphorylationof Akt (S473) in skeletal muscle (triceps)was examined first. Phosphorylation ofS473 is essential for full activation of Akt(23), and was decreased dramatically fol-lowing TH90’, compared with either T0’or T90’ (Figure 1A). In many insulin re-sistant states, skeletal muscle insulin re-sistance is an early event and precedesthe development of insulin resistance inother target tissues (24). To ensure theconsistent development of hemorrhage-induced skeletal muscle insulin resist-ance, a second time point was selected.Following trauma and hemorrhage for60 min, there was abolishment of insulin-induced phosphorylation of Akt on S473(Figure 1B) in triceps, with no change intotal Akt protein levels. Skeletal muscleAkt phosphorylation was not altered fol-lowing trauma only (T60’), indicatingthat decreased insulin signaling is the re-sult of hemorrhage. Quantified data frommultiple animals is presented as the fold-change in P-Akt (S473) in the presence(+) or absence (–) of insulin (Figure 1C).In trauma followed by immediate death(T0’) and 60 min after surgery (T60’),there were increases in P-Akt (S473) in

triceps (11.6- and 9.3-fold, respectively)following insulin injection. However,following trauma and hemorrhage for 60min (TH60’), there was a complete lossof insulin-induced triceps P-Akt (S473).Trauma and hemorrhage for 60 min didresult in a small decrease in basal (non-insulin stimulated) Akt phosphorylation,but this decrease did not reach statisticalsignificance compared with trauma-onlyanimals. Thus, the data indicates arapid, hemorrhage-induced insulin sig-naling defect in skeletal muscle. Approx-imately 60% of the total blood volume isremoved during the initial bleed. To en-sure that the decrease in P-Akt in skele-tal muscle is not the result of the stressof the initial bleed step, blood with-drawal followed by immediate replace-

ment of that blood was performed. Inthese experiments, following the initial10 min bleed necessary to decrease theMAP to 35–40 mmHg, the rat’s wholeblood, with added heparin, was returned(replaced) to that rat (blood withdrawaland replacement: BWR). Following a re-covery period of 60 min (BWR60’), therewas no change in insulin-induced phos-phorylation of Akt (S473; Figure 1D). Anadditional control (blood with control:BWC) was added to this experiment toensure that the addition of heparin itselfwas without effect. This data suggeststhat the decreases observed followinghemorrhage are the result of a sustaineddecrease in MAP and are not simply dueto the initial stress of decreased bloodvolume.

R E S E A R C H A R T I C L E


Figure 1. Decreased skeletal muscle insulin signaling via phospho-Akt (P-Akt) followingtrauma and hemorrhage. Rats were subjected to trauma alone (T), or trauma and hem-orrhage (TH). At 0’, 60’, 90’ either saline (–) or 5U insulin (+) was injected via the portalvein and the triceps were removed after 2 min. Tissue lysates were subjected to Westernblotting with antibodies specific for P-Akt serine 473 (S473) or total Akt. RepresentativeWestern blots from TH90’ (A), and TH60’ (B) are presented. Autoradiographs from TH60’were quantified by scanning densitometry (C). The data are presented as mean ± SEMfold change of P-AKT (S473) by insulin of three rats (n = 3) in each group. T0’ with no in-sulin treatment was arbitrarily set to 1. $P < 0.01 compared with T0’ plus insulin; ^P < 0.01compared with T only group at the same time point plus insulin, in this case T60’. (D) Tri-cep tissue lysates from blood withdrawal and replacement 60 min (BWR60’), or heparinonly blood withdrawal control (BWC) animals were subjected to Western blotting withantibodies specific for P-Akt serine 473 (S473). A representative Western blot is presented.

Fasting Serum Glucose Levels AreIncreased Following Trauma andHemorrhage

Changes in fasting glucose and insulinlevels following trauma and hemorrhagewere examined and trauma alone for 60min (T60’) had no effect on fasting glu-cose levels compared with fasting glucoselevels in normal rats. However, followingtrauma and hemorrhage (TH60’) therewas a significant increase in fasting glu-cose levels to 161 mg/dL (Figure 2A).This increase in fasting glucose is sugges-tive of insulin resistance and may, in part,be due to a decrease in insulin action inskeletal muscle. In the current studies, therise in insulin levels varied from animal toanimal, resulting in increased insulin lev-els, but that did not yet reach statisticalsignificance 60 min following hemorrhage(Figure 2B), as it had by 90 min followinghemorrhage (6). Hyperinsulinemia canoccur following severe injury. However,

there appears to be variability dependingupon the model used and the time pointselected (6,9,25,26). No change or de-creases in insulin levels are common earlyfollowing injury during the “ebb” phaseof the injury (27,28).

IRS-1 and IR Tyrosine Phosphorylationin Skeletal Muscle Is DecreasedFollowing Hemorrhage

A reduction in insulin signaling viaP-Akt may be due to alterations in phos-phorylation of upstream components ofthe insulin signaling pathway. To addressthis possibility, experiments were per-formed to examine the phosphorylationof a specific tyrosine residue of IRS-1, ty-rosine 612, that is important for activa-tion of the PI3-kinase/Akt pathway inskeletal muscle. There was an increase intriceps insulin-stimulated P-IRS-1 (Y612)immediately following (T0’; 29-fold) and60 min (T60’; 19-fold) after the initialtrauma (Figure 3). Unlike trauma alone,trauma and hemorrhage caused an in-crease in basal IRS-1 (Y612) phosphoryla-tion in triceps. However, this increase intriceps basal P-IRS-1 (Y612) was accom-panied by a complete lack of any furthereffect of insulin (Figure 3A,B), indicatinga resistance to exogenous insulin, withno change in total protein levels (notshown). We next questioned whetherthere were changes in phosphorylationof the insulin receptor (IR), upstream ofIRS-1, in skeletal muscle. When mea-sured, phosphorylation of tyrosine 972 ofthe IR (P-IR [Y972]), required for thebinding and phosphorylation of IRS-1,was induced by insulin 6.4-fold and 8.0-fold in skeletal muscle following traumaonly for 0 min and 60 min (T0’ and T60’),respectively (Figure 3C,D). However,phosphorylation of P-IR (Y972) was de-creased significantly to two-fold follow-ing trauma and hemorrhage (TH60’),with no change in total IR protein levels.

Time Course of Alterations in SkeletalMuscle Insulin Signaling FollowingTrauma and Hemorrhage

Similar experiments were performedto determine the timing of the develop-



Figure 2. Increase in fasting glucose levelsand no significant change in fasting in-sulin levels following trauma and hemor-rhage. Rats were subjected to traumaalone (T) or trauma and hemorrhage (TH).Fasting blood glucose (A) and fasting in-sulin (B) levels were measured in normal,T60’, and TH60’ rats. Data are presentedas mean ± SEM (n = 5–7 rats/group). #P <0.05 compared with normal group; *P <0.05 compared with T only group.

Figure 3. Decreased insulin receptor sub-strate-1 (IRS-1) and insulin receptor (IR) ty-rosine phosphorylation in skeletal musclefollowing trauma and hemorrhage. Ratswere subjected to trauma alone (T) ortrauma and hemorrhage (TH) as de-scribed in Figure 1 except tissue lysateswere subjected to Western blotting withantibodies specific for phospho-IRS-1 tyro-sine 612 (P-IRS-1 Y612), total ERK1/2 (load-ing control), phospho-IR tyrosine 972 (P-IRY972) and total IR antibodies. (A) Repre-sentative Western blots are presentedand (B) autoradiographs were quantifiedby scanning densitometry; the data arepresented as mean ± SEM fold change ofP-IRS-1 (Y612) by insulin of three rats (n =3) in each group. (C) RepresentativeWestern blots are presented and (D) au-toradiographs were quantified by scan-ning densitometry; the data are pre-sented as mean ± SEM fold change ofP-IR (Y972) by insulin (n = 3 in eachgroup). T0’ with no insulin treatment wasarbitrarily set to 1. #P < 0.05 comparedwith T0’; *P < 0.05 compared with T onlygroup at the same time point 0.

ment of triceps insulin resistance fol-lowing trauma and hemorrhage. Imme-diately following surgery and after theinitial bleed (TH0’), insulin-inducedskeletal muscle P-Akt is not altered, andinsulin-induced P-Akt signaling is stillfunctional in skeletal muscle following15 and 30 min of hemorrhage (TH15′and TH30’; Figure 4A,B,C).

The time course of changes in skele-tal muscle IRS-1 and IR phosphoryla-tion after hemorrhage also was studiedand indeed there were similar de-creases observed in tyrosine phospho-rylation of IRS-1 and IR (Figure 5) inskeletal muscle. Whereas P-IRS-1(Y612) was decreased significantly 60

and 90 min following trauma and hem-orrhage (TH60’, TH90’), P-IRS-1 (Y612)was unchanged following only 30 min(TH30’; Figure 5A,B). This also wastrue of the loss of P-IR (Y972) in skele-tal muscle observed at 60 and 90(TH60’ and TH90’), but not 30 min(TH30’; Figure 5C,D) following hemor-rhage, and was consistent with a nor-mal-fold induction of P-Akt inductionby insulin in triceps at the TH30’ timepoint. Together, these data suggest adistinct time course of the hemorrhage-induced defect of insulin signaling thatoccurred in skeletal muscle between 30and 60 min following the initiation ofhemorrhage.

Insulin-Induced Phospho-Akt IsDisrupted in Extensor Digitorum Longusafter Trauma and Hemorrhage, butLess So in Diaphragm, and Not inCardiac Muscle

To determine whether there arechanges in skeletal muscle insulin sig-naling in other skeletal muscles,changes in insulin-induced P-Akt (S473)in diaphragm and extensor digitorumlongus muscles were examined. Insulin-induced phosphorylation of P-Akt wasdecreased in diaphragm followingtrauma and hemorrhage, but not traumaalone, at the 60 and 90 min time points(only TH60’ shown; Figure 6A), al-though the decrease was much less se-vere than in triceps. In contrast, exten-sor digitorum longus muscle respondedto hemorrhage in much the same way astriceps muscle: following 60 (Figure 6B)and 90 min (not shown), there was acomplete loss in insulin-induced P-Akt(S473), but no significant change oc-curred at only 30 min (not shown) fol-lowing hemorrhage. This is suggestiveof hemorrhage inducing severe insulinresistance in a subset (triceps and exten-sor digitorum longus), but not all, skele-tal muscles. The acute development ofinsulin resistance appears to be inde-pendent of muscle fiber type since bothslow-twitch (triceps) and fast-twitch (ex-tensor digitorum longus) muscles ap-pear to be affected by hemorrhage.

It was then asked whether the modest(diaphragm) or the more severe effect oftrauma and hemorrhage on the develop-ment of insulin resistance in the tricepsand EDL also occurred in cardiac muscle.Insulin-induced phosphorylation ofP-Akt was not altered in cardiac musclefollowing trauma and hemorrhage at ei-ther the 60 or 90 min hemorrhage timepoints (TH60’ and TH90’). There alsowere no significant changes in basalP-Akt (Figure 6C), and no changes intotal Akt levels (not shown), followingtrauma alone or trauma and hemorrhagein cardiac muscle. This indicates that, un-like skeletal muscle, hemorrhage had lit-tle or no effect on insulin signaling incardiac muscle.



Figure 4. Decreased skeletal muscle insulin signaling via phospho-Akt (P-Akt) at multipletime points following trauma and hemorrhage. At the same time points and treatmentregiments described in Figure 1, either saline (–) or 5U insulin (+) was injected via theportal vein, the triceps were removed after 2 min, and tissue lysates were subjected toWestern blotting with antibodies specific for P-Akt serine 473 (S473). RepresentativeWestern blots from (A) T0’, TH0’, and TH15′ and (B) TH30’, TH60’, and TH90’ are pre-sented. (C) Autoradiographs from TH30’, TH60’, and TH90’ were quantified by scanningdensitometry. The data are presented as mean ± SEM fold change of P-AKT (S473) by in-sulin of three rats (n = 3) in each group. T0’ with no insulin treatment was arbitrarily set to1. $P < 0.01 compared with T0’; ^P < 0.01 compared with T only group at the same timepoint.

Insulin-Induced Phospho-AktSignaling Is Restored in Triceps after5 h and 24 h

Lastly, it was asked whether there wasrecovery from the trauma and hemor-rhage-induced insulin signaling defect inskeletal muscle. At the end of the 90 minhemorrhage period, the animals were re-suscitated and allowed to recover. Therewas little difference in the ability of ahigh dose insulin injection to elicit in-creased phosphorylation of P-Akt follow-ing hemorrhage and resuscitation and

then 5 (TH5h) or 24 h (TH24h) of recov-ery, when compared with rats that hadundergone trauma alone (Figure 7).Thus, there was a recovery of the re-sponse to high dose insulin 5 and 24 hfollowing resuscitation.

DISCUSSIONHyperglycemia is a hallmark of insulin

resistance. Since skeletal muscle is a pri-mary site of insulin-regulated glucosedisposal, hyperglycemia can be causedby decreased insulin action on skeletal

muscle. Skeletal muscle insulin resistanceis common in many chronic clinical syn-dromes, including Type 2 diabetes, obe-sity, and metabolic syndrome (25,29–32).Common to all of these clinical syn-dromes is the chronic nature of the syn-drome, and a slow development ofsymptoms, including insulin resistance,prior to the first clinical presentation ofthe disease. In the present work, a defectin insulin signaling in skeletal musclewas observed as early as 60 min follow-ing trauma and hemorrhage.



Figure 5. Decreased insulin-induced tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and insulin receptor (IR) in the skele-tal muscle following trauma and hemorrhage. Rats were subjected to trauma (T) or trauma and hemorrhage (TH) as described in Fig-ure 4 and tissues lysates were subjected to Western blotting with antibodies specific for phospho-IRS-1 tyrosine 612 (P-IRS-1 Y612) andfor phospho-IR tyrosine 972 (P-IR Y972). (A) Representative Western blots from TH30’, TH60’, and TH90’ are presented, and (B) autoradi-ographs were quantified by scanning densitometry; the data are presented as mean ± SEM fold change of P-IRS-1 (Y612) by insulin ofthree rats (n = 3) in each group. (C) Representative Western blots from TH30’, TH60’, and TH90’ are presented and (B) autoradiographswere quantified by scanning densitometry; the data are presented as mean ± SEM fold change of P-IR (Y972) by insulin (n = 3 in eachgroup). T0’ with no insulin treatment was arbitrarily set to 1. #P < 0.05 compared with T0’; *P < 0.05 compared with T only group at thesame time point.

In the present studies, a rat model ofsurgical trauma and hemorrhage wasused (6,7) to study the acute alterationsin insulin signaling that may be involvedin hemorrhage-induced skeletal muscleinsulin resistance. We demonstrate thatskeletal muscle insulin resistance devel-oped quickly (within 60 min) followinghemorrhage. This insulin resistance wascharacterized by a rapid decrease in Aktphosphorylation following the onset ofhemorrhage at an important serineresidue, S473, which is essential for Akt

activity (23,33). The presented data alsoindicates that the alteration in Akt phos-phorylation was due to a sustained de-crease in MAP, and not simply due to thestress of a transitory decrease in bloodvolume. Coincident with the decrease ininsulin signaling is a significant rise inblood glucose levels. Because of the de-crease in hematocrit due to blood loss,the rise in blood glucose levels might beunderestimated, but hyperglycemia isobserved clearly after only 60 min fol-lowing hemorrhage.

Skeletal muscle fibers can be classifiedbroadly into two categories, slow- andfast-twitch. Insulin is known to elicit agreater response in slow-twitch skeletalmuscle versus fast-twitch skeletal muscle(34). We observed severe inhibition of in-sulin signaling both in slow-twitch (tri-ceps) and fast-twitch (extensor digitorumlongus) muscles following trauma andhemorrhage. However, the decrease ofinsulin signaling in diaphragm (slow-twitch) was less extensive. In addition toskeletal muscle, cardiac muscle also wasexamined and little change in insulin-in-duced Akt signaling was demonstratedin the heart. Thus, the rapid develop-ment of insulin resistance followingtrauma and hemorrhage is specific to theskeletal muscle and not cardiac muscle,is independent of fiber type, but may bedependent on other factors, such as mus-cle function. Following hemorrhage, themaintenance of proper insulin signalingin a subset of muscles, such as the di-aphragm (essential for proper ventila-tion) and the heart (already severelytaxed by the decrease in blood volumeand pressure) are critical to ensure oxy-genation of the remaining blood and per-fusion of the brain and heart, respec-tively, both necessary for survival. It isnot yet known whether the maintenanceof blood flow to the heart and dia-phragm protect them from developingsevere insulin resistance, or whetherthere is some alternate explanation, suchas the differential use of IGF-1/insulinhybrid receptors. This question will re-quire further research. However, it isclear from the present studies that in-



Figure 6. Insulin-induced Phospho-Akt(P-Akt) in extensor diaphragm and digito-rum longus muscles and cardiac musclefollowing trauma and hemorrhage. Ratsare subjected to trauma alone (T) ortrauma and hemorrhage (TH) as de-scribed in Figure 1 except (A) diaphragmmuscle, (B) extensor digitorum longusmuscle, and (C) cardiac (left ventricle)muscle were analyzed at the designatedtime points following the hemorrhage. Tis-sue lysates were subjected to Westernblotting with antibodies specific for P-Aktserine 473 (S473) or total Akt as indicated.Representative Western blots of at leastthree separate experiments for each tis-sue are presented for TH60’ in (A) and (B)and identical data was obtained at TH90’(TH90’ not shown). In (C) both TH60’ andTH90’ are presented.

sulin signaling in skeletal muscles in-volved in locomotion (such as the tricepsand extensor digitorum longus) may becompromised rapidly for the overall ben-efit of the critically injured individual.

In addition to altered Akt phosphory-lation, other components of the insulinsignaling pathway were altered in skele-tal muscle within 60 min followingtrauma and hemorrhage. We present thefirst evidence that both insulin-inducedIR and IRS-1 tyrosine phosphorylationwere decreased rapidly in skeletal mus-cle following hemorrhage. Previous datafrom our laboratory suggest that in theliver, IR function (phosphorylation) isnot changed by hemorrhage, suggestingpost-receptor defects are solely responsi-ble for the hepatic alterations observed(7), but here there is clearly a defect atthe level of insulin receptor activation.Together with the current data, it sug-gests that there are tissue-specific differ-ences in the mechanism underlyinghemorrhage-induced insulin resistance.A clearer understanding of this differ-ence will require further examination.However, it is important to note thatvariability in the phosphorylation stateof the IR is not uncommon and has beenhighly variable when measured by dif-ferent laboratories (35–42).

Figure 7. The recovery of skeletal muscleinsulin signaling via phospho-Akt (P-Akt)following trauma and hemorrhage.Ratswere subjected to trauma alone (T), ortrauma and hemorrhage (TH). At 5h or24h following resuscitation, either saline(–) or 5U insulin (+) was injected via theportal vein. Tricep lysates were subjectedto Western blotting with antibodies spe-cific for P-Akt serine 473 (S473) or totalAkt (not shown). Representative Westernblots of at least three separate experi-ments from each time point, TH5h andTH24h, are presented.

In recent years, the importance of IRS-1in the molecular mechanism underlyinginsulin resistance has emerged (4). Re-duced tyrosine phosphorylation of IRS-1may contribute to insulin resistance inhuman skeletal muscle in chronic dis-eases such as obesity and Type 2 diabetes(35,39,43,44). Phosphorylation of a spe-cific tyrosine (Y612) of IRS-1, which mayserve as a docking site for PI3-kinase(45), was examined in the present studiesand there was an acute decrease of in-sulin-induced IRS-1 tyrosine 612 phos-phorylation in skeletal muscle followingtrauma and hemorrhage, which likely re-sulted in reduced association with PI3-kinase. Most relevant is that acute de-fects of insulin signaling occurred by 60min, not the weeks, months, or yearsoften necessary in chronic diseases.

There is a significant recovery of in-sulin signaling that occurs upon resusci-tation following hemorrhage. Resuscita-tion with Ringer’s lactate is similar to thetreatment of patients following surgicalor accidental blood loss. It is not knownhow rapidly recovery occurs, but thedata indicates that in response to a highdose of insulin, insulin signaling ismostly recovered within 5 h after resusci-tation. To determine whether there iscomplete recovery, more extensive time-course and dose response studies willneed to be performed. However, moreimportantly, further information on themechanisms by which skeletal musclebecomes insulin resistant is needed. Ourpreliminary data indicates a complexarray of contributing factors that are in-jury-type and tissue-specific. Once thesemechanisms are understood, and thismay be as complicated as chronic statesof insulin resistance, there will need to bean understanding of the mechanisms bywhich the insulin signaling defects arereversed.

The rapid recovery of insulin respon-siveness in rats following resuscitationindicates no permanent tissue damageand minimal or no loss of cells (apopto-sis or necrosis) following hemorrhage for90 min. However, longer bouts of re-duced blood pressure and more exten-

sive blood loss are less conducive to re-covery, but the point at which blood lossis too severe for recovery of insulin sig-naling is not yet known and may be tis-sue and species specific. In recent work,we found that less blood loss results inmore modest forms of insulin resistancein liver (8). In ICU patients, attainment ofeuglycemia, achieved either on their ownor following intensive treatment with in-sulin, greatly enhances recovery and re-duces mortality (17,18,46,47). Thereforethe ability to recover insulin responsive-ness, and/or euglycemia, may be an im-portant predictor for reduction of mor-bidity and mortality in the intensive careenvironment.

In summary, skeletal muscle insulinresistance following hemorrhage occursquickly and involves multiple alter-ations in the IR/IRS-1/Akt signalingpathway. This insulin resistance occursin both fast-twitch (extensor digitorumlongus) and slow-twitch (triceps) skele-tal muscle, but it is not uniform to allmuscles. The insulin signaling defectsare not permanent, with recovery by 5 hfollowing fluid resuscitation. There aremultiple neural, hormonal, and immune/cytokine responses to hemorrhage thatmay play a role in the acute develop-ment of insulin resistance, and in the re-covery of insulin signaling following re-suscitation. There are likely differentcausative factors for the acute develop-ment of insulin resistance in different tis-sues, and additional factors in more se-vere versus less severe degrees of injuryand/or hemorrhage, which will requirecontinued research to delineate. Thepresent work indicates that a rapid de-fect of insulin signaling can be measuredin some rat skeletal muscles, but it is notuniform, and seems to occur less in con-stantly used muscles (diaphragm) andnot at all in cardiac muscle.

ACKNOWLEDGMENTSThis work is supported by grants

from the National Institutes of Health(DK62071), the Department of Defense(W81XWH-0510387), and the VeteransAdministration Merit Review to JLM.

A University of Alabama ComprehensiveMinority Faculty and Student Develop-ment Program Fellowship provided sup-port for LHT. We would like to acknowl-edge the members of the University ofAlabama Center for Surgical Research forvital assistance in setting up the animalmodel in our lab, MM Bamman and DJKosek for additional technical assistance,and the Metabolism Core Laboratory ofthe Clinical Nutrition Research Center atUniversity of Alabama (P30-DK56336)for the serum insulin and TNF-α meas-urements. We also would like to thank JLFranklin, Drs. AB Keeton, and MGSchwacha, for their helpful and insight-ful discussions and suggestions.

DISCLOSUREThe authors have nothing to disclose.

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