Effects of the HIV Protease Inhibitor Ritonavir on GLUT4Knock-out Mice*

Received for publication, August 18, 2010, and in revised form, September 22, 2010 Published, JBC Papers in Press, September 23, 2010, DOI 10.1074/jbc.M110.176321

Arpita Kalla Vyas‡1, Joseph C. Koster§1, Anatoly Tzekov‡, and Paul W. Hruz‡§2

From the Departments of ‡Pediatrics and §Cell Biology and Physiology, Washington University School of Medicine,St. Louis, Missouri 63110

HIV protease inhibitors acutely block glucose transporters(GLUTs) in vitro, and this may contribute to altered glucosehomeostasis in vivo. However, several GLUT-independentmechanisms have been postulated. To determine the contri-bution of GLUT blockade to protease inhibitor-mediated glu-cose dysregulation, the effects of ritonavir were investigatedin mice lacking the insulin-sensitive glucose transporterGLUT4 (G4KO). G4KO and control C57BL/6J mice wereadministered ritonavir or vehicle at the start of an intraperito-neal glucose tolerance test and during hyperinsulinemic-eugly-cemic clamps. G4KOmice exhibited elevated fasting blood glu-cose comparedwith C57BL/6Jmice. Ritonavir impaired glucosetolerance in control mice but did not exacerbate glucose intol-erance in G4KO mice. Similarly, ritonavir reduced peripheralinsulin sensitivity in control mice but not in G4KOmice. Seruminsulin levels were reduced in vivo in ritonavir-treated mice.Ritonavir reduced serum leptin levels in C57BL/6Jmice but hadno effect on serum adiponectin. No change in these adipokineswas observed following ritonavir treatment of G4KO mice.These data confirm that a primary effect of ritonavir on periph-eral glucose disposal is mediated through direct inhibition ofGLUT4 activity in vivo. The ability of GLUT4 blockade to con-tribute to derangements in the other molecular pathways thatinfluence insulin sensitivity remains to be determined.

The use ofHIV protease inhibitors (PIs)3 as part of combinedantiretroviral therapy (cART) has altered the course of the HIVepidemic by greatly reducing disease-related morbidity andmortality (1). The success of PI-based therapies is tempered bythe recognition that many PIs contribute to insulin resistanceand dyslipidemia (2). As HIV-infected patients live longer,there is growing concern that these adverse metabolic effectsare contributing to a rise in cardiovascular disease (3). Thedevelopment of impaired glucose tolerance has been moststrongly linked to several first generation PIs (e.g. indinavir andritonavir) and does not appear to occur with the newer non-peptidomimetic drugs (e.g. tipranavir and darunavir) (4, 5).

Although the mechanisms that are responsible for impairedglucose homeostasis during cART have been intensively inves-tigated, fewdirectmolecular targets have been identified.Acuteand reversible inhibition of the insulin-sensitive facilitative glu-cose transporter GLUT4 in fat and muscle has provided themost direct explanation for peripheral insulin resistance inpatients taking PIs (6). In cultured adipocytes, PIs acutely andreversibly inhibit insulin-stimulated glucose uptake at pharma-cologically relevant drug levels (7). Furthermore, these in vitrofindings correlate with acute and reversible induction of insulinresistance in vivo both in rodents (8) and in HIV-negative vol-unteers (9).Despite a high degree of correlation between in vitro and in

vivo findings, questions remain regarding the contribution ofdirect GLUT blockade to changes in glucose homeostasis intreated patients. Impaired insulin signaling (10, 11), reducedinsulin secretion (12), and altered adipocytokine levels (13–15)have also been implicated. To determine the specific contribu-tion of PI-mediated GLUT inhibition to altered glucose home-ostasis, we tested the effects of ritonavir on glucose tolerance intransgenic mice lacking GLUT4 (G4KO) (16). We report evi-dence supporting our hypothesis that GLUT4 inhibition is aprimary mechanism leading to changes in glucose tolerance.

EXPERIMENTAL PROCEDURES

Materials—Homozygous GLUT4 knock-out mice (G4KO)on an isogenic C57BL/6J background were a kind gift fromMaureen Charron (16). For all experiments, age- and sex-matched control C57BL/6J mice were obtained from JacksonImmunoResearch Laboratories (Bar Harbor, ME). Ritonavirwas obtained in oil-based form (Merck) and resuspended in100% ethanol (0.79 g/ml) prior to dilution with water to an 11%(v/v) working concentration. 2-[3H]Deoxyglucose (2-[3H]DG)was purchased from Sigma.BloodGlucose and Insulin Levels—Blood glucosewas assayed

using the glucose dehydrogenase-based enzymatic assay andquantitated using an Elite XL glucometer (Bayer). Blood insulinlevels were assayed in 15 �l of mouse serum using an ELISAaccording to the manufacturer’s protocol (Crystal Chem Inc.,Downers Grove, IL). Intraperitoneal glucose tolerance tests(GTTs) were performed on 3–5-month-old mice following anovernight 16-h fast. Animals were injected intraperitoneallywith either ritonavir (10 mg/kg) or vehicle 15 min prior to theintraperitoneal administration of 25% dextrose (1 g/kg). Bloodwas isolated from the tail vein at the times indicated andassayed for glucose as described above. Intraperitoneal insulintolerance tests were performed on 3–5-month-oldmice follow-

* This work was supported, in whole or in part, by National Institutes of HealthGrants DK064572 and HL092798.

1 Both authors contributed equally to this work.2 To whom correspondence should be addressed: Dept. of Pediatrics, Wash-

ington University School of Medicine, 660 South Euclid Ave., Campus Box8208, St. Louis, MO 63110. Fax: 314-286-2892; E-mail: hruz_p@kids.wustl.edu.

3 The abbreviations used are: PI, protease inhibitor; cART, combined antiret-roviral therapy; GLUT, glucose transporter; 2-DG, 2-deoxyglucose; GTT,glucose tolerance test.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 47, pp. 36395–36400, November 19, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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ing a 6-h fast. Animalswere injected intraperitoneallywith insulin(0.5 units/kg). Blood was isolated from the tail vein at the timesindicated and assayed for glucose as described above. Data arepresented as the mean � S.E. (Student’s unpaired t test).Hyperinsulinemic-Euglycemic Clamp Experiments—Weight-

matchedG4KOandC57BL/6Jmicewere anesthetizedwith ket-amine/xylazine (87 and 13.4 mg/kg intraperitoneally), andcatheters (MRE 025, Braintree Scientific Inc., Braintree, MA)were implanted into both the right internal jugular vein andfemoral artery and allowed to recover for 5–7 days. Following a5-h fast, catheters were flushed with normal saline, and heparin(20 units/kg) was administered to maintain catheter patency.After determination of fasting blood glucose levels, a constantinfusion of ritonavir (0.35 mg/kg/min) was started through thevenous catheter at a rate of 0.5�l/min using aHarvard 11 appa-ratus pump. After 30 min of drug infusion, insulin (20 milli-units/kg/min) in normal saline containing 0.1% BSA wasinfused through the venous catheter. At 10-min intervals, 100�l of blood was removed from the arterial catheter into a syringe.Blood (5 �l) was then sampled directly from the catheter for thedetermination of blood glucose levels. Blood glucose was assayedusing a Contour TS glucometer. The dead space blood was thenreinfused into the animal.Dextrose (25%)was infused through thevenous catheter at a rate sufficient to maintain a plasma glucoselevel of 100–110 mg/dl. Peripheral glucose disposal (Rd�) wasdeterminedby the averageglucose infusion rateduring the final 30min of each 120-min clamp experiment.Skeletal Muscle and Adipose Tissue 2-DG Uptake—2-[3H]DG

(5 �Ci) was administered through the arterial catheter 30 minbefore the conclusion of hyperinsulinemic-euglycemic clampexperiments. Bloodwas collected at 0, 3, 5, 10, 15, 20, 25, and 30min for determination of the tracer-specific activity (8). Imme-diately after euthanasia by cervical dislocation, hind limb mus-cles and epididymal fat were harvested, washed with PBS, andplaced in liquid nitrogen pending subsequent analysis. Frozentissue samples were ground with a mortar and pestle, boiled in1.2 ml of water for 10 min, and spun in a microcentrifuge at15,000 � g for 10 min. Accumulated 2-DG 6-phosphate in thesupernatant was separated from 2-DG by ion exchange chro-matography using a Dowex 1-X8 (100–200 mesh) anionexchange column (8).Adipokine and Leptin ELISAs—Serum leptin and adiponec-

tin levels were determined using a commercially availablemouse ELISA kit (Millipore) according to the manufacturer’sprotocol. Serum samples for adiponectin were diluted 1000-fold prior to assay.Drug Assays—Serum PI levels were determined by the HPLC

method of Foisy and Sommadossi (17) using a Waters 626HPLC system with a Microsorb C-8 column (Waters Corp.,Milford, MA). Samples were run in duplicate in 50 �l of serum.Standard curves were generated by adding pure PI standardsdirectly to control mouse serum.

RESULTS

Glucose Tolerance in G4KO Mice Is Not Acutely Altered byRitonavir—In the acute setting, HIV PIs such as ritonavir arehypothesized to impair glucose handling by targeting andinhibiting the insulin-sensitive glucose transporter GLUT4 in

fat (6) and muscle (18), although several alternative mecha-nisms have been proposed (11, 19–21). If the adverse effects ofPIs on glucose homeostasis are mediated primarily throughGLUT4, then these effects should be ameliorated on a GLUT4-null background. To characterize physiologic responses to glu-cose load, 3–5-month-old whole body GLUT4 knock-out mice(G4KO) (16) underwent intraperitoneal GTTs in the presenceor absence of ritonavir (10mg/kg). As shown in Fig. 1A, admin-istration of ritonavir before the start of theGTT in age-matchedcontrol C57BL/6J mice (WT) significantly altered glucoseexcursion at the 15- and 30-min time points compared withvehicle treatment. Blood glucose before and after glucose loadwas significantly higher in G4KO mice compared with controlC57BL/6J mice (Fig. 1B), consistent with a loss of insulin-sen-sitive glucose uptake upon GLUT4 ablation (fasting blood glu-cose: WT, 126 � 15 mg/dl (n � 7); and G4KO, 331 � 45 mg/dl(n � 7); p � 0.01). Importantly, administration of ritonavir toG4KO mice, in contrast to control mice, did not further exac-erbate the glucose intolerance. No significant differences in theresults of GTTs were observed when the mice were separatedby sex.Qualitatively similar results between the genotypeswerealso obtained when ritonavir was administered at the start ofthe GTT (data not shown). HPLC analysis of serum samplestaken at the 30-min time point, the peak of the PI effect incontrolmice, revealed amean ritonavir concentration of 16.8�2.0 �M (n � 3), which is near the reported peak therapeuticconcentration (22). Taken together, these data support the con-

FIGURE 1. Glucose tolerance in G4KO mice is unaffected by ritonavir.Ritonavir (10 mg/kg) or vehicle (ethanol, 7.9 g/kg) was administered by intra-peritoneal injection 15 min prior to the intraperitoneal injection of glucose (1g/kg) to age-matched control C57BL/6J mice (A) or G4KO mice (B). Data rep-resent the means � S.E. (n � five to nine mice, 3–5 months old). *, p � 0.05; **,p � 0.01 (Student’s unpaired two-tailed t test).

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clusion that PIs target GLUT4 in vivo and acutely impair glu-cose handling.Glucose-stimulated Insulin Secretion Is Inhibited by Ritona-

vir in WT and G4KO Mice—To assess the contribution of invivo�-cell responsiveness to PI-induced changes in glucose tol-erance, serum insulin levels were measured 30 min followingco-injection with glucose (1 g/kg) and either ritonavir (10mg/kg) or vehicle. As shown in Fig. 2A, ritonavir caused anexpected increase in blood glucose levels inWTmice compared

with vehicle treatment (WT: vehi-cle, 251 � 15 mg/dl; and ritonavir,386 � 25 mg/dl; p � 0.01), butimportantly, this rise was notaccompanied by a compensatoryrise in serum insulin levels (WT:vehicle, 0.52 � 0.06 ng/ml; andritonavir, 0.45 � 0.09 ng/ml; p �0.5), indicating relative �-cell dys-function. As expected, blood glu-cose levels were not affected, as theG4KO mice are virtually insensitiveto changes in circulating insulin dueto loss of the GLUT4 isoform(G4KO: vehicle, 404 � 31 mg/dl;and ritonavir, 373 � 28 mg/dl; p �

0.43) (Fig. 2B). Insulin levels were significantly lower in ritona-vir-treated G4KO mice (G4KO: vehicle, 1.94 � 0.4 ng/ml; andritonavir, 0.8 � 0.18 ng/ml; p � 0.01). These data indicate thatthe �-cell does not respond appropriately with an increase inserum insulin levels during PI-induced insulin resistance.Insulin Does Not Reduce Glucose Levels in G4KO Mice—To

assess the effect of ritonavir on peripheral glucose uptake, theglucose-lowering effect of intraperitoneal insulin injection (0.5units/kg) was determined using insulin tolerance tests on 6-hfasted mice. As predicted from previous studies on the acuteeffect of the PI indinavir on rodents (8), the glucose-loweringeffect of insulin was blunted in control mice injected withritonavir (10 mg/kg) (Fig. 3A). Ablation of GLUT4 eliminatedthe insulin-sensitive component of glucose uptake, and there-fore, assaying insulin sensitivity in the G4KOmice was withouteffect even at concentrations up to 2.5 units/kg (Fig. 3B anddatanot shown). Although average glucose levels were slightlyhigher 15 and 30 min after insulin injection in the G4KOmice,the values were not statistically different between treatmentgroups. Thus, the glucose intolerance in the control mice likelyreflects both impaired �-cell function and a reduction inperipheral insulin sensitivity.Peripheral Glucose Disposal Is Not Altered by Ritonavir in

G4KO Mice—To more directly determine the extent to whichthe observed effects of ritonavir on glucose tolerance are medi-ated through effects on peripheral glucose disposal, hyperinsu-linemic-euglycemic clamps, the established gold standardmeasurement of peripheral insulin sensitivity (24), was deter-mined in wild-type and G4KO mice exposed to ritonavir bycontinuous intravenous infusion (0.35 mg/kg/min). Previousstudies have established that this infusion rate provides steady-state ritonavir blood levels of 10–15 �M (25). As shown in Fig.4A, glucose Rd� was reduced by 68% in ritonavir-treated wild-type mice (30 � 11mg/kg/min) compared with vehicle-treatedanimals (99 � 12 mg/kg/min). As shown in Fig. 4B, the trans-port of 2-DG into skeletal muscle was inhibited 70% by ritona-vir in wild-type mice. Consistent with the lack of insulin-re-sponsive glucose transport, glucose Rd� in vehicle-treatedG4KO mice was significantly lower than that in wild-type ani-mals (37 � 9 mg/kg/min). Whole body glucose disposal andsoleus muscle 2-DG uptake were not different between ritona-vir- and vehicle-treated G4KO mice. The uptake of 2-DG into

FIGURE 2. Ritonavir suppresses the �-cell response to glucose load in vivo. Shown are the serum insulin(ng/ml) and glucose (mg/dl) levels in age-matched control C57BL/6J mice (n � 13) (A) and G4KO mice (n � 12)(B) at the 30-min time point following glucose administration (1 kg/kg) in the presence or absence of ritonavir(10 mg/kg). Glucose and either vehicle (7.9 g/kg) or ritonavir (10 mg/kg) were co-injected at 0 min. Data areshown as the mean � S.E. of all animals/group. p values were determined by Student’s unpaired t test.

FIGURE 3. Ritonavir significantly decreases insulin sensitivity in age-matched control C57BL/6J mice. A, shown are intraperitoneal insulin toler-ance test results from age-matched control C57BL/6J mice 3–5 months old.Data are blood glucose measurements versus time following intraperitonealbolus insulin injection (0.5 units/kg of body weight) after 6 h of fasting.B, insulin-sensitive glucose uptake was not observed in G4KO mice (at con-centrations up to 2.5 units/kg). Data represent the mean � S.E. (n � six to ninemice/group). *, p � 0.05 (Student’s unpaired two-tailed t test.

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white adipose tissue fromWTmice (5.7 �mol/100 g/min) was�10% of skeletal muscle glucose uptake. The effect of ritonaviron 2-DG uptake was more modest (17%) in this tissue and didnot reach statistical significance (data not shown). The lowamount of white adipose tissue in G4KOmice precluded accu-rate measurement of the effect of ritonavir on 2-DG uptakefrom this tissue in vivo. Nevertheless, these data indicate thatdirect inhibition of white adipose tissue glucose transport is notprimarily responsible for the acute effect of ritonavir on glucosehomeostasis in the WT mice.Adipocytokine Levels Are Not Acutely Altered by Ritonavir in

G4KO Mice—Although skeletal muscle is directly responsiblefor the majority of peripheral glucose disposal, drug-inducedeffects on adipose tissue could still potentially impair glucosehomeostasis indirectly through changes in adipocytokinesecretion. To determine whether the acute effects of ritonaviron glucose tolerance are influenced by changes in adiponectinor leptin secretion, we first measured serum levels of these adi-pokines in WT mice before and after treatment with ritonavir(1%, w/w) in standard rodent chow for 12 h overnight. Total

food intake was not different between experimental groupsduring this interval. As shown in Fig. 5A, leptin levels weresignificantly reduced in WT mice following ritonavir treat-ment. Consistent with the reduced fat content in G4KO mice(16), leptin levels were significantly lower in G4KO mice com-pared with WT animals. Ritonavir treatment did not furtherinfluence serum leptin levels in G4KO mice. As shown in Fig.5B, ritonavir treatment did not significantly alter adiponectinlevels in either WT or G4KO mice.

DISCUSSION

Metabolic side effects and the development of viral resis-tance to HIV PIs continue to represent the most serious limita-tions to the use of these agents in antiretroviral treatment reg-imens. Knowledge of the precise molecular interactions thatcontribute to the development of insulin resistance is a key tothe rational design and development of novel PIs that maintainclinical efficacy without the induction of glucose intolerance.

FIGURE 4. Ritonavir does not acutely alter peripheral glucose disposal inG4KO mice. A, whole body glucose disposal (Rd�) during hyperinsulinemic-euglycemic clamp conditions. Data represent the mean � S.E. (n � three tofive animals/group). B, uptake of 2-[3H]DG into soleus muscle during the final30 min of hyperinsulinemic clamps. Rg�, glucose metabolism index. Data rep-resent the mean � S.E. (n � four animals/group). p values were determinedusing Student’s unpaired t test.

FIGURE 5. Ritonavir lowers leptin levels in WT but not G4KO mice. Serumadipokine levels were measured in serum from WT (C57BL/6J) and G4KO mice12 h after the addition of ritonavir (1%, w/w) to standard rodent chow. A,serum leptin levels (n � 13 and 19/group for WT/vehicle and WT/ritonavir,respectively; and n � 3/group for G4KO). B, adiponectin levels (n � 9 and15/group for WT/vehicle and WT/ritonavir, respectively; and n � 3/group forG4KO). Values are shown as the mean � S.E. p values were determined usingStudent’s unpaired t test.

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Previous studies have supported an important role of PI-in-duced GLUT4 blockade in the development of altered glucosehomeostasis during cART. Although in vitro data have estab-lished that PIs act as acute and reversible non-competitiveinhibitors of GLUT4 (7) and that these drugs act through directinteraction with the transport protein (26), the existence andinfluence of additional molecular targets have remainedunclear. Furthermore, although excellent correlation betweenPI-mediated insulin resistance in vitro and in rodent modelsand healthy human volunteers has been established (27), eachof these systems alone has been insufficient to prove that the invivo effects on peripheral glucose disposal aremediated primar-ily through the direct inhibition of facilitative glucose transport.In this study, the demonstration that glucose tolerance is notacutely affected by ritonavir in mice lacking GLUT4 providesconfirmation of a central role of GLUT4 blockade in these invivo effects.Because GLUT4 mediates the most distal step in insulin-

stimulated glucose uptake, it is difficult to assess changes inmore proximal signaling events following genetic or pharmaco-logic ablation of this transporter. Therefore, given the severebase-line insulin resistance present in GLUT4 knock-out mice,our findings cannot completely exclude the possibility thatritonavir has additional effects on glucose homeostasis in addi-tion to those mediated directly through GLUT4 blockade.Glucose transport has been previously implicated in the reg-

ulation of leptin secretion from primary adipocytes (28). Thisearlier study used several nonselective GLUT inhibitors over1–4 days. The lower serum leptin levels observed in G4KOmice, together with the failure to observe further changes inleptin levels following ritonavir treatment of the transgenic ani-mals, further support a role of GLUT4 in leptin secretion fromadipocytes. However, it remains unclear whether or to whatdegree the observed changes in serum leptin levels contributeto the observed changes in peripheral glucose disposal. Adi-ponectin does not appear to contribute to the acute changes inperipheral glucose disposal induced by ritonavir.Because these studies were all performed in the setting of

acute drug exposure, it remains possible that additional long-term effects of ritonavir also indirectly impact insulin sensitiv-ity. When administered chronically to rodents and humans,ritonavir is also known to induce hyperlipidemia (29), which inturn may lead to impaired insulin signaling (30). It is possiblethat with chronic ablation of GLUT4, up-regulation of the con-stitutively active transporter GLUT1 is able to partially com-pensate for the defect in glucose transport. If PIs that do notalter the activity of GLUT1 in vitro also do not affect this trans-porter in vivo, compensatory up-regulation of GLUT1 may,over time, diminish the acute effects of these PIs on peripheralglucose disposal. If this is true in treated patients, chroniceffects on insulin secretion and/or signaling would predomi-nate, providing an explanation for varying reports on PI-in-duced changes in insulin sensitivity (11, 31, 32). Compensatorychanges may also be responsible for alterations in hepatic glu-cose production following extended PI exposure (33).Similarly, although insulin insufficiency is necessary for the

progression to overt diabetes and such changes have been dem-onstrated in HIV-infected patients receiving long-term cART

(34, 35), the degree to which PI therapy underlies impaired�-cell function remains controversial (31, 36, 37). The identifi-cation of a direct �-cell target of PIs has the potential to eluci-date the molecular basis for observed alterations in glucose-stimulated insulin secretion. The hypothesis that GLUT2 is thetarget of PIs in�-cells is based primarily upon inference from invitro findings in which glucose transport blockade was directlycorrelatedwith impaired insulin secretion (38) and acute block-ade of GLUT2 in Xenopus oocytes heterologously expressingthis facilitative glucose transporter isoform (7). However, glu-cokinase activity and not glucose transport is understood to bethe rate-limiting step in ATP production (39). Although theprecise molecular target of PIs in �-cells remains to be identi-fied, there is evidence that perturbation of voltage-dependentK� channels and anion channels may be involved in mediatingimpaired insulin secretion (40).Consistent with our in vitro data (38), we have demonstrated

that ritonavir also acutely impairs �-cell function in mice chal-lengedwith a glucose load. This further supports a contributionof impaired �-cell function in addition to peripheral insulinresistance in the glucose intolerance observed in HIV-infectedhumans receiving PIs. Because of potential differences in therelative expression of the glucose transporter isoforms(GLUT1, GLUT2, GLUT9, and/or GLUT3) in the pancreasbetween mouse and human (23, 41, 42), the magnitude ofritonavir effects on the human �-cell in relation to the effect onperipheral glucose disposal may differ from our present study.Taken together, these studies provide definitive evidence for

the central role of GLUT4 inmediating the acute in vivo effectsof PIs. Thus, the continued development of newer PIs that donot alter GLUT4 activity is likely to reduce the risk of impairedglucose homeostasis in patients receiving cART.

Acknowledgment—We thank Dr. Maureen Charron for providingGLUT4 knock-out mice.

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Protease Inhibitors and Glucose Dysregulation

36400 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 47 • NOVEMBER 19, 2010

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Arpita Kalla Vyas, Joseph C. Koster, Anatoly Tzekov and Paul W. HruzEffects of the HIV Protease Inhibitor Ritonavir on GLUT4 Knock-out Mice

doi: 10.1074/jbc.M110.176321 originally published online September 23, 20102010, 285:36395-36400.J. Biol. Chem. 

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