skeletal muscle action of estrogen receptor a is critical for ...metabolism skeletal muscle action...

R E S EARCH ART I C L E

METABOL I SM

http://stm.sciencem

agD

ownloaded from

Skeletal muscle action of estrogen receptor a is criticalfor the maintenance of mitochondrial function andmetabolic homeostasis in femalesVicent Ribas,1* Brian G. Drew,1* Zhenqi Zhou,1* Jennifer Phun,1 Nareg Y. Kalajian,1

Teo Soleymani,1 Pedram Daraei,1 Kevin Widjaja,1 Jonathan Wanagat,2

Thomas Q. de Aguiar Vallim,3 Amy H. Fluitt,1,4 Steven Bensinger,4 Thuc Le,4,5 Caius Radu,4,5

Julian P. Whitelegge,6 Simon W. Beaven,7 Peter Tontonoz,7 Aldons J. Lusis,3,8 Brian W. Parks,8

Laurent Vergnes,8 Karen Reue,8 Harpreet Singh,9 Jean C. Bopassa,9 Ligia Toro,9 Enrico Stefani,9

Matthew J. Watt,10 Simon Schenk,11 Thorbjorn Akerstrom,12 Meghan Kelly,12†

Bente K. Pedersen,12 Sylvia C. Hewitt,13 Kenneth S. Korach,13 Andrea L. Hevener1,14‡

Impaired estrogen receptor a (ERa) action promotes obesity and metabolic dysfunction in humans and mice;however, the mechanisms underlying these phenotypes remain unknown. Considering that skeletal muscle isa primary tissue responsible for glucose disposal and oxidative metabolism, we established that reduced ERaexpression in muscle is associated with glucose intolerance and adiposity in women and female mice. To test thisrelationship, we generated muscle-specific ERa knockout (MERKO) mice. Impaired glucose homeostasis andincreased adiposity were paralleled by diminishedmuscle oxidativemetabolism and bioactive lipid accumulationin MERKOmice. Aberrant mitochondrial morphology, overproduction of reactive oxygen species, and impairment inbasal and stress-induced mitochondrial fission dynamics, driven by imbalanced protein kinase A–regulator ofcalcineurin 1–calcineurin signaling through dynamin-related protein 1, tracked with reduced oxidative metabolismin MERKO muscle. Although muscle mitochondrial DNA (mtDNA) abundance was similar between the genotypes, ERadeficiency diminished mtDNA turnover by a balanced reduction in mtDNA replication and degradation. Our findingsindicate the retention of dysfunctional mitochondria inMERKOmuscle and implicate ERa in the preservation ofmito-chondrial health and insulin sensitivity as a defense against metabolic disease in women.

.org
by guest on O
ctobe/

INTRODUCTION

Insulin resistance is a central underpinning in the pathogenesis oftype 2 diabetes (T2D) and is a defining feature of the metabolic syn-drome, a clustering of abnormalities including obesity, glucose in-tolerance, dyslipidemia, and hypertension (1). Although the incidenceof T2D is higher in men than in premenopausal women of a similar

1Division of Endocrinology, Diabetes and Hypertension, Department of Medicine, DavidGeffen School of Medicine at University of California, Los Angeles (UCLA), Los Angeles,CA 90095, USA. 2Division of Geriatrics, David Geffen School of Medicine at UCLA, LosAngeles, CA 90095, USA. 3Division of Cardiology, David Geffen School of Medicine atUCLA, Los Angeles, CA 90095, USA. 4Department of Molecular and Medical Pharmacol-ogy, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA. 5AhmansonTranslational Imaging Division, David Geffen School of Medicine at UCLA, Los Angeles,CA 90095, USA. 6Pasarow Mass Spectrometry Laboratory and Neuropsychiatric Institute–Semel Institute for Neuroscience & Human Behavior, David Geffen School of Medicine atUCLA, Los Angeles, CA 90095, USA. 7Howard Hughes Medical Institute and Departmentof Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, LosAngeles, CA 90095, USA. 8Department of Human Genetics, David Geffen School of Med-icine at UCLA, Los Angeles, CA 90095, USA. 9Department of Anesthesiology, David GeffenSchool of Medicine at UCLA, Los Angeles, CA 90095, USA. 10Department of Physiology,Monash University, Clayton, Victoria 3800, Australia. 11Department of Orthopaedic Sur-gery, University of California, San Diego, La Jolla, CA 92093, USA. 12Centre of Inflammationand Metabolism and Centre for Physical Activity Research, Rigshospitalet, University ofCopenhagen, Copenhagen 2200, Denmark. 13Receptor Biology Section, National Instituteof Environmental Health Sciences, National Institutes of Health, Research Triangle Park,Durham, NC 27709, USA. 14UCLA Iris Cantor Women’s Health Research Center, Los Angeles,CA 90095, USA.*These authors contributed equally to this work.†Present address: Department of Orthopaedics and Rehabilitation, University of RochesterSchool of Medicine and Dentistry, Rochester, NY 14642, USA.‡Corresponding author. E-mail: [email protected]

www.Scie

r 17, 2020

age and body mass index (BMI) (2), the mechanisms underlying fe-male protection against metabolic dysfunction and insulin resistanceremain unclear (3–5). Unfortunately, after menopause, the incidenceof metabolic-related chronic disease increases significantly. Similarly,inactivating mutations in the estrogen receptor a gene (ESR1/Esr1) inboth humans and mice recapitulate metabolic dysfunction and height-ened disease risk. Although strong relationships between whole-bodyestrogen action, oxidative function, and insulin sensitivity are now wellestablished (6–8), our understanding of the tissue-specific molecularactions of estrogen and its a form receptor (ERa) in metabolic tissuesrequires further interrogation.

Skeletal muscle is a primary tissue responsible for oxidative me-tabolism and insulin-stimulated glucose disposal. Women and femalerodents show enhanced insulin sensitivity in skeletal muscle comparedwith male counterparts when insulin action is normalized to musclemass (9). In addition, muscle from female human subjects exhibits ahighermitochondrial abundance with enhanced antioxidant scavengingcapacity comparedwithmuscle frommen andmale rodents (10). Undervarious stress conditions, mitochondria produce excessive amounts ofreactive oxygen species (ROS) known to attack lipid bilayers, mutateDNA, and alter the activity of specific enzymes critical for the mainte-nance of oxidative function (11–14), and female muscle is refractory tothese effects of oxidative stress. Secondary to oxidant scavenging, mito-chondrial fission-fusion events are engaged to eliminate and replacedamaged mitochondrial components as well as stimulate the constantmixing ofmitochondria retained in themitochondrial network (15, 16).In contrast to rapidly dividing cells, which are known to activate apoptosis

nceTranslationalMedicine.org 13 April 2016 Vol 8 Issue 334 334ra54 1

http://stm.sciencemag.org/


as a means of quality control, long-lived postmitotic cells, including myo-cytes, require a more sophisticated mitochondrial surveillance system topreserve cellular health. Thus, effective elimination of damagedmitochon-dria incapable of copingwith changing energy demands is thought to abateinflammation and cellular aging (11, 17).

by guest on October 17, 2020

http://stm.sciencem

ag.org/D

ownloaded from

Here, we tested the impact of skeletalmuscle–specific ERa deletion on mito-chondrial quality and function in relationto metabolic health. We found that micewith a skeletalmuscle–specific ERa knock-out (MERKO) displayed obesity and pro-found skeletal muscle insulin resistance,phenocopying the whole-body ERahomozygous null model previously de-scribed byour laboratory (6).Mitochon-drial dysfunction in MERKOmuscle wasdistinguished by reduced basal and max-imally stimulated oxygen consumption,impaired calcium buffering capacity, andexcessive production of ROS. These de-fects were paralleled by reduced mito-chondrial fission and macroautophagysignaling in part as a consequence of im-balancedproteinkinaseA(PKA)–regulatorof calcineurin 1 (RCAN1)–calcineurin sig-naling through dynamin-related protein 1(Drp1). Although mitochondrial DNA(mtDNA)content was similar betweenthe genotypes under basal conditions, acoordinated reduction inmtDNAreplica-tion and degradation was observed inERa-deficient muscle.

Collectively, our data support thenotion that ERa promotes mtDNA rep-lication and the turnover of dysfunc-tional mitochondria in skeletal muscle.In MERKOmice, the retention of dam-aged mitochondria harboring mutantDNA likely contributes to muscle oxida-tive stress and inflammation culminatingin skeletal muscle insulin resistance. Theseobservations, together with our findingsof markedly reduced ERa expressionlevels in muscle from women who dis-play the metabolic syndrome (MetSyn),underscore the clinical relevance of ourwork and the central role of muscle ERain protecting against metabolic disease.

RESULTS

Skeletal muscle ERa expressioncorrelates with metabolic healthin femalesTo establish the clinical rationale for ourstudies in rodents and cells, we first de-termined the relation between natural

www.Scie

variation in muscle ESR1 expression and metabolic phenotypic traitsof women. We found that adiposity and fasting insulin are inverselyassociated with muscle ESR1 expression in women (Fig. 1, A and B),and this relationwas phenocopied in a variety of inbred strains of female

Fig. 1. Skeletal muscle ERa expression correlates with metabolic health in females. (A and B) Inverserelationship between Esr1 (natural variation in expression) versus adiposity and fasting insulin in women
(n = 42; •, premenopausal n = 29, postmenopausal n = 13). AU, arbitrary units. (C andD) In 10 strains of femalemice (•, n = 4 mice per indicated strain; age, 16 weeks), detected by Pearson’s correlation test, *P < 0.05 (Ppia,peptidylprolyl isomerase A housekeeping gene, for comparison]. (E) Muscle ESR1 expression in pre-menopausal women with (MetSyn) and without (Healthy) the MetSyn (n = 18 to 21 per group). (F and G)Esr1 expression and representative immunoblots of ERa protein in skeletal muscle (quadriceps) from leanand LepOb female mice (n = 6 per genotype; age, 12 weeks). Values are expressed as means ± SEM. All expres-sion values were normalized to 1.0. Mean differences were detected by Student’s t test or analysis of variance(ANOVA); *P < 0.05, between-group comparison.



mice (Fig. 1, C and D). Muscle ESR1 expression was markedly reduced(P = 0.0001) in women who displayed clinical features of MetSyn (Fig.1E and table S1), and this finding was reproduced in genetically obesemice compared with lean controls (Fig. 1, F and G).

www.Scie

MERKO mice show impaired glucose homeostasis andinsulin actionTo investigate the impact of muscle Esr1 expression on insulin action,we generatedMERKOmice (Fig. 2, A and B). Esr2 (ERb) is expressed at


http://stm.sciencem

ag.org/D

ownloaded from

Fig. 2. Muscle-specific ERadeletion impairs glucose tol-
erance andmuscle insulin ac-tion. (A) ERa protein inmuscle,liver,andadiposetissuefromCon-trol f/f and MERKO female mice(representative immunoblot,n=3per genotype). (B) Esr1 ex-pression inmuscles [quadriceps,gastrocnemius, tibialis, soleus,and extensor digitorum longus(EDL)] from female MERKO andControl f/f (n=8miceper geno-type) assessed by quantitativepolymerase chain reaction(qPCR). WAT, white adiposetissue. (C) Relative comparisonof Esr2 and Gper (ER G protein–coupled receptor) expressionin quadriceps from Control f/fand MERKO mice (n = 8 pergenotype). P > 0.05. (D) Glu-cose tolerance [glucose toler-ance test (GTT), 1000 mg/kg]inControl f/f andMERKOfemalemice (n = 6 to 8 per genotype;age, 24 to 26 weeks). *P < 0.05detected by Student’s t test forAUC. (E) Skeletalmuscle andhe-patic insulin sensitivity (IS-GDR,insulin-stimulated glucose dis-posal rate; HGP, hepatic glucoseproduction) assessedbyglucoseclampforControl f/f andMERKOmice (n = 6 to 8mice per geno-type; age, 28 weeks). P < 0.05.(F) Ex vivo soleus muscle glu-cose uptake (fold change frombasal; n= 6mice per genotype).Values for insulin sensitivity areexpressed as means ± SEM;*P < 0.05 detected by ANOVA.(G and H) GLUT4 transcript andprotein levels in basal 6-hour–fasted quadriceps muscle fromControl f/f and MERKO mice(n = 5 to 8mice per genotype).(I and J) Representative immu-noblots and densitometry of in-sulin signaling (IRS-1–associatedp85 and p-Akt) in quadricepsand soleusmuscle fromglucose clamp and ex vivo glucose uptake studies, respectively, in Control f/f andMERKOmice (n = 6mice per genotype per condition). P < 0.05. IP, immuno-precipitation; IB, immunoblot. (K toM) Esr1 expression (K), ERa protein levels(L), and insulin signaling (M) (IRS-1pY– and IRS-1–associated p85) in control
(Scr) and Esr1-KD C2C12 myotubes (0 to 100 nM insulin; studies performedin triplicate).Densitometric analyses areexpressedasmeans±SEM inarbitraryunits normalized to 1.0; *P<0.05, between-groupdifferences; #P<0.05,within-group treatment comparison, detected by Student’s t test and ANOVA.





http://stm.sciencem

ag.org/D

ownloaded from

levels several hundredfold less than Esr1, and no compensatory increasein ERbmRNA (P = 0.23) or the estrogen-responsive transmembrane Gprotein (heterotrimeric guanine nucleotide–binding protein)–coupledreceptor (Gper, GPR30; P = 0.53) was detected in muscle from femaleMERKO mice (Fig. 2C). Circulating levels of insulin and leptin were ele-vated (82%,P=0.01 and200%,P=0.001, respectively),whereas circulatingadiponectin levels were reduced by 34% (P = 0.004) in 6-hour–fastedMERKO compared with Control f/f mice (table S2). The increased cir-culating leptin levels in MERKO mice, relative to Control f/f mice,were consistent with a 2.75-fold increase (P= 0.0001; table S2) in gonadaladipose tissue mass and could not be explained by altered food andwater consumption (P= 0.095 andP= 0.13, respectively) or ambulatorymovement (P = 0.54) compared with Control f/f mice.

In addition, female MERKO animals showed impaired glucose tol-erance (IGT) [area under the glucose curve (AUC) P = 0.025; Fig. 2D]and whole-body insulin resistance [glucose infusion rate (GIR); Fig. 2Eand table S2] compared to controls. Marked skeletal muscle insulin re-sistance inMERKOmice was reflected by a 45% (P = 0.003) reduction inthe insulin-stimulated glucose disposal rate (GDR) during glucose clampstudies (Fig. 2E) and a 55% (P = 0.002) reduction in ex vivo insulin-stimulated 2-deoxyglucose glucose uptake into the soleus muscle com-pared with Control f/f mice (Fig. 2F). MERKO skeletal muscle insulinresistance could not be explained by reduced expression or total proteinlevels of the insulin-responsive glucose transporter GLUT4 (Fig. 2, Gand H) as previously proposed by Barros et al. (18), but rather was aconsequence of impaired insulin signal transduction in skeletal muscle(Fig. 2, I and J). Furthermore, in the mouse myoblast C2C12 cell linedifferentiated to myotubes, Esr1 knockdown (KD) [using lentiviral-mediated Esr1-targeted short hairpin RNA (shRNA)] (Fig. 2, K and L)impaired insulin signal transduction, as evidenced by the observationsthat insulin-stimulated insulin receptor substrate 1 (IRS-1) tyrosine phos-phorylation and IRS-1–p85 association were markedly reduced com-pared to control scramble shRNA (Scr) (Fig. 2M), thus recapitulatinginsulin resistance similar to that observed in MERKO mice.

ERa regulates oxidative metabolism and muscle lipidaccumulation in skeletal muscleImpaired skeletal muscle insulin action was paralleled by heightenedinflammation inMERKOmuscle [as shown by increased inflammatorysignaling via p-c-JunN-terminal kinase 1/2 (JNK 1/2) and p–IkBkinaseb (IKKb) relative to controls; P < 0.05, respectively] (Fig. 3A, left panel)and in C2C12myotubes with Esr1-KD (P < 0.01) (Fig. 3A, right panel).As would be predicted, muscle inflammation paralleled the accumu-lation of triacylglycerol and bioactive lipid intermediates [diacylglycerol(DAG) and ceramides; Fig. 3B]. To determine whether impaired fattyacid oxidation contributed to the buildup of muscle lipids, we inves-tigated the cellular deposition of [14C]palmitate in C2C12 myotubeswith Esr1-KD. The myotube Esr1 deficiency caused a 50% reductionin fatty acid oxidation (P = 0.02) and a concomitant increase in fattyacid storage into complex lipids (P < 0.01) relative to controlmyotubes(Fig. 3C). This alteration in fatty acid handling was associated with amarked reduction in basal and maximally stimulated cellular oxygenconsumption andATP (adenosine 5′-triphosphate) synthesis (Fig. 3Dand fig. S1) as measured by real-time respirometry.

Next, we investigated the mechanism underlying diminished oxi-dativemetabolism in ERa-deficient muscle. Considering that obesityand insulin resistance are often associated with a reduction in mito-chondrial number (19), we assessed surrogatemarkers ofmitochondrial

www.Scie

mass and biogenesis inmuscle from female control versusMERKOmice.We observed no difference inmaximal activity of muscle citrate synthase(amitochondrial enzyme that is part of the Krebs cycle) (Fig. 3E), expres-sion of transcriptional regulators of mitochondrial biogenesis (Fig. 3Fand tables S3 and S4, microarray analyses), or expression or proteinabundance of select components of the mitochondrial electron trans-port complex (Fig. 3G) between the genotypes. Anunanticipated increasein peroxisome proliferator–activated receptor g coactivator 1b (Ppargc1b),acetyl–coenzyme A (CoA) carboxylase 2 (Acacb), peroxisomal acyl-CoA(Acox), and the fatty acid transporter cluster of differentiation (Cd36)expressionwas observed inMERKOmuscle comparedwith Control f/f.We found that myocytes that lacked ERa produced markedly elevatedamounts of the oxygen radicals H2O2 and O2

− (Fig. 3, H and I). In ad-dition, ROS scavenging capacity was diminished because glutathioneperoxidase 3 (Gpx3) mRNA and protein levels were significantly re-duced in MERKO muscle, relative to control (Fig. 3J), a finding in linewith previous reports showing thatGpx3 expression is regulated by ERa(20). Increased oxidative stress promoted protein carbonylation (a kindof protein oxidation) inmuscle fromMERKOmice comparedwithCon-trol f/f mice (Fig. 3K). Thus, it is likely that the marked inflammationobserved inMERKOmusclewas in largepartmediatedby the cumulativeeffects of chronic oxidative stress.

An important function of the mitochondria is calcium handling.We found a 21% reduction (P = 0.017) in the maximal calcium con-centration that initiated mitochondrial permeability transition pore(mPTP) opening in isolatedmitochondria fromMERKO versus Con-trol f/f (Fig. 3L and table S3). Recent studies have shown that openingofmPTP linksmitochondrial dysfunction to insulin resistance (21). Theimpairments in calcium handling and ATP production along with theelevation in ROS production by the mitochondria were likely factorsunderlying decrements in muscle contractile function, because singlemuscle fibers fromMERKOmice fatigued faster than fibers from Con-trol f/f muscle (fig. S2A). Moreover, we observed cytochrome oxidase–negative fiber staining with aging only in MERKO muscle (fig. S2B).Collectively, these data indicate thatmetabolism requisite to fuel repeatedmuscular contractions inMERKOmice is limiting and that reduced ERaaction accelerates the development of specific muscle phenotypes asso-ciated with aging.

In contrast to muscular endurance, peak tension and peak tetanicforce were identical between the genotypes, whereas time to peak ten-sion was improved inMERKOmuscle compared to Control f/f (fig. S2,C to E). We had initially speculated that ERa deletion might promotemuscle atrophy with aging; however, on the contrary, muscle proteinlevels, including the abundant z-line protein a actinin, weremaintained(fig. S2F and table S3). This could be a consequence of a reduction in theexpression of atrogenes (genes that influence muscle atrophy; includingFoxo1,Fbxo32, andTrim63) inMERKOversusControl f/fmice (fig. S2G).Thus, despite the impairment in muscle oxidative metabolism, musclesize andmaximum force weremaintained inMERKO, in part, by a com-pensatory down-regulation of muscle degradation pathways.

ERa regulates mtDNA replication and mitochondrialturnover in muscleAlthough we observed no difference in the expression of genes associ-ated withmitochondrial biogenesis or total mtDNA content between thegenotypes of female mice (Fig. 4A), we did detect a significant reductionin mtDNA polymerase g1 (Polg1) and mtDNA-directed RNA poly-merase (Polrmt) expression levels inMERKO versus Control f/f muscle





http://stm.sciencem

ag.org/D

ownloaded from

protein carbonylation in quadriceps muscle from Control f/f versus MERKO mice (n = 8 per gencapacity/mPTP opening in mitochondria isolated from Control f/f versus MERKO mice (n = 5 perpressed as means ± SEM. Mean differences detected by Student’s t test and ANOVA where app

www.ScienceTranslationalMedicine.org 13 April 2016

Fig. 3. Esr1 deletion pro-motes mitochondrial dys-function, ROS production,and increased muscle in-flammation. (A) Heightenedproinflammatory signaling(p-IKKb and p-JNK 1/2) (foldchange over baseline nor-malized to 1.0) in femaleMERKO muscle (n = 6 pergenotype; left panel) andmyotubes (n = 4 per condi-tion; right panel), respec-tively. P < 0.05. (B) Bioactivelipid intermediates (TAG,triacylglycerol; ceramides) inquadriceps muscle from fe-male Control f/f and MERKOmice (n=6musclespergeno-type). P < 0.05. (C and D)Palmitate oxidation and es-terification as well as basalandmaximal (stimulatedusingcarbonylcyanide p-trifluoro-methoxyphenylhydrazone,FCCP) oxygen consumptionand ATP synthesis in control(Scr)versusEsr1-KDmyotubes(n = 6). (E) Citrate synthaseactivity in muscle harvestedfromControl f/fversusMERKOmice (n = 6 per genotype).(F) Quantitative reverse tran-scription PCR (RT-PCR) analy-sesofmuscle fromControl f/fversus MERKO mice (n = 6per genotype). (G) Immuno-blots and correspondingdensitometry of mitochon-drial electron transport com-plexes versus actin loadingcontrol in muscle from Con-trol f/f versus MERKO mice(n = 6 per genotype). (H andI) H2O2 and O2

− productionin control (Scr) versus Esr1-KD C2C12 myocytes. (J) Gpx3expression and protein inControl f/f versus MERKOmuscle (n = 6 per genotype).(K) Oxidative stress–induced

otype). (L) Calcium bufferinggenotype). All values are ex-ropriate; *P < 0.05.

Vol 8 Issue 334 334ra54 5




http://stm.sciencem

ag.org/D

ownloaded from

(Fig. 4B). We confirmed regulation of Polg1 expression by ERa byshowing that selective inhibition of ERa using the ERa antagonist ICI182,780 (fulvestrant) or an ERa DNA binding mutation (ERa DBDD)reduced Polg1 levels in C2C12myotubes and quadriceps muscle, respec-tively (fig. S3, A and B). To determine whether reduced expression ofPolg1 and Polrmtmanifested a functional decrement in mtDNA replica-tion inMERKOmuscle,we assessed the incorporation of deuteratedwaterinto newly synthesizedmtDNA. Consistent with gene expression data, weobserved reduced 2H2O labeling of newly synthesized mitochondrial

www.ScienceTranslationalMedic

deoxyribonucleosides after heavy water consump-tion in MERKO versus Control f/f mice (Fig. 4C).In order for the mtDNA pool to be maintained inMERKOmuscle to a level similar to that ofControlf/f mice, it follows that a reduction inmtDNA rep-licationmust be balanced by a reduction in the rateof mtDNA degradation.

To examine this notion further, we determinedwhether mitochondrial morphology and mito-chondrial turnover were altered by ERa deficien-cy. Using transmission electron microscopy, wefound that mitochondrial morphology and cellu-lar distribution were altered by ERa inactivation(Fig. 4D).MERKOmitochondriawere dysmorphic(enlarged, elongated, and hyperfused), misalignedalong the Z-disc, and less densely distributed inthe subsarcolemmal compartment compared withmitochondria of Control f/f (Fig. 4D). The mito-chondrial area was increased twofold (P = 0.0001)inMERKOmuscle comparedwithControl f/fmus-cle (Fig. 4E). A similar mitochondrial architecturewas observed in muscle from female ERa DBDDmice, suggesting that ERa DNA binding is requi-site, whereas ERa tethering is insufficient, to altergene transcription and restore the mitochondrialmorphology seen in Control f/f muscle (fig. S3C).Although the cellular architecture of myotubes inculture differs from murine muscle, altered mor-phological features of MERKO mitochondria(that is, elongation and hyperfusion) were recapi-tulated in C2C12 Esr1-KD myotubes comparedwith C2C12 control-Scr myotubes (fig. S3D). Anelongated and fused mitochondrial phenotype isoften observed as a consequence ofmitochondrialstress and is a compensatory response to stabilizethe mtDNA and dilute damaged mitochondrialcontents across the mitochondrial network (22, 23).Because the lysosome has a cargo-size restriction,mitochondrial elongation is thought to occur as amechanism to preventmitophagy (degradation ofmitochondria via autophagy). For example, mito-chondrial elongation is engaged during starvationwhen macroautophagy is maximally activated(24–26).

Becausemuscle mtDNA abundance was iden-tical between the genotypes despite a reduction inthe rate ofmtDNA replication, a coordinate reduc-tion inmtDNA turnover is required (Fig. 4E) (27).It has been hypothesized that mtDNA replication

stress may contribute to increased mtDNA mutation susceptibility be-cause the half-life of mtDNA is increased (27). To test this notion, wedetermined whether ERa is critical for controlling mtDNA turnover.Wequantified mtDNA mutation load and mtDNA abundance as twosurrogate markers of mtDNA turnover after H2O2 treatment of controland Esr1-KD myotubes (28). Basal mtDNA mutation load was elevatedand failed to return to basal levels in Esr1-KD myotubes during the re-covery period when the genotoxic stress was removed (in contrast tocontrol cells) (Fig. 4F). Moreover, although basal mtDNA abundance

Fig. 4. Impaired mtDNA replication and altered mitochondrial morphology in MERKOmuscle. (A) Muscle mtDNA content, expressed as a ratio of mtDNA/nuclear (nuc) DNA (n = 8
per group). (B)Muscle polymerase g1 (Polg1), Polg2, and Polrmt expression. (C)mtDNA replication asmeasured by 2H2O incorporation into newly synthesized mtDNA (deoxynucleosides, dG and dC)from Control f/f and MERKO mice (n = 6 per genotype). (D) Representative electron micrographsof soleus muscle from female Control f/f and MERKO mice, low- and high-magnification inset (IM,intramyofibrillar; SS, subsarcolemmal; scale bar, 1 mm). (E) Skeletal muscle intermyofibrillar mitochon-drial area (n = 3 mice per genotype). (F) mtDNA mutation load was detected by random mutationcapture (RMC) at basal (no treatment), after H2O2 treatment, and during recovery from H2O2 treat-ment. (G) mtDNA copy number at basal, after H2O2 treatment [in the presence of chloramphenicol(Chl) plus cycloheximide (Chx), and during recovery (in the presence of bafilomycin A1 (BafA1)]. Allvalues are expressed as means ± SEM. Mean differences detected by Student’s t test and ANOVAwhere appropriate; *P < 0.05, between-genotype difference; #P < 0.05, within-genotype between-treatment difference.
ine.org 13 April 2016 Vol 8 Issue 334 334ra54 6




http://stm.sciencem

ag.org/D

ownloaded from

was similar between the genotypes, mtDNA after H2O2 treatment wasreduced from basal only in control cells, suggesting a possible impair-ment in mtDNA turnover in Esr1-KD cells. During recovery, mtDNAabundance was significantly elevated over basal in control cells, but therecovery responsewasmarkedly blunted for Esr1-KDcells, findings thatsupport our observation of a defect in mtDNA replication in MERKOmice. Thus, our findings suggest that ERa is critical for the eliminationof damagedmtDNA as well as for promoting mitochondrial biogenesisduring recovery from genotoxic insult (Fig. 4, F and G).

ERa deletion impairs mitochondrial fission-fusion dynamicsin skeletal muscleThe marked alteration in mitochondrial morphology, reduction inmtDNA replication rate, and impairment in mtDNA turnover in ERa-deficient muscle prompted us to assess effectors of mitochondrialfission-fusion dynamics. On the basis of the mitochondrial hyperfusionphenotype, which showed elongated and highly interconnected mito-chondria in muscle of MERKO mice (Fig. 4D), we hypothesized thatthat there also would be a strong suppression of mitochondrial fissionsignaling in MERKO muscle. Because mitochondrial fission is respon-sible for producing smaller organelles and is critical for mitophagy, ourfindings of altered mitochondrial morphology and impaired mtDNAturnover after genotoxic stress support this hypothesis. To test thisnotion further, we quantified mitochondrial fission signaling by im-munoblotting of MERKO muscle; we observed a threefold increase(P = 0.0001) in DRP1 phosphorylation at the inhibitory residue Ser637

(Fig. 5A) and a reduction inDRP1protein associatedwithmitochondriafrom MERKO muscle compared with Control f/f (Fig. 5B). These datawere confirmed in C2C12myotubes with Esr1-KD (fig. S4). In line withincreasedDRP1Ser637 phosphorylation, we observed diminished proteininteraction between DRP1 and the putative phosphatase calcineurin,which was previously shown to induce mitochondrial fission (Fig. 5C).Moreover, a ~50% reduction in total calcineurin activity was detectedinMERKOmuscle (Fig. 5D) and in myotubes deficient in ERa (fig. S5,A and B), compared to respective controls.

In opposition to the mitochondrial pro-fission enzyme calcineurin,activity and phosphorylation status of PKA—the putative kinase linkedwithphosphorylationofDRP1on the inhibitorySer637 residue (29)—wereelevated (Fig. 5E and fig. S5, C andD) aswas the scaffolding protein thatassociates PKA with the mitochondrion [A kinase anchoring protein1 (AKAP1)] (table S4 and fig. S4, A and B). Notably, in addition to in-creased PKA total protein and phospho-PKA in whole-cell musclelysates, a near twofold increase in PKA was detected in mitochondria-enriched fractions from Esr1-KD myotubes compared with Esr1-repletecontrol cells (fig. S4, C and D).

With respect to additional mitochondria-specific proteins involvedin fission-fusion signaling, we observed a 50% (P = 0.005) reduction inthe fission-relatedoutermitochondrialmembraneDRP1 anchoringpro-tein, FIS1 (Fig. 5F), in MERKO versus Control f/f. Conversely, the innerand outer mitochondrial membrane fusion proteins, optic atrophy 1protein (OPA1), and mitofusin 2 (MFN2), respectively, were elevated50 to 60% in MERKO muscle and Esr1-KD myotubes compared withrespective controls (Fig. 5, G and H, and fig. S4, C and F). Alterations intranscript mirrored the observed differences in protein abundance be-tween the genotypes (Fig. 5I). Collectively, these findings strongly suggestthat a loss of myocellular ERa dampens mitochondrial fission signaling,promoting unopposed mitochondrial fusion. These signaling data arestrongly supported by electron micrographs showing an elongated

www.Scie

and hyperfused mitochondrial morphology in muscle from MERKOmice (Fig. 4D).

Mitophagic signaling is impaired in the context of ERa deficiencyMitochondria are highly dynamic and constantly undergoing fission-fusion events to repair and selectively retain healthy organelles in thenetwork (30, 31). To investigatewhether impairedmitochondrial fissionis associated with decrements in mitochondrial quality control in themuscle of MERKO mice, we next assessed mitophagic signaling. Theexpression levels of Park family members Park2 (Parkin) and Park6[phosphatase and tensin homolog (PTEN)–induced putative kinase 1(PINK1)] were significantly elevated in ERa-deficient muscle (Fig. 6A).Despite elevated transcript abundance, PINK1 andParkin protein levelswere reduced by 75% (P = 0.001) and 40% (P = 0.001), respectively, inmuscle fromMERKOcomparedwithControl f/fmice (Fig. 6B). Similarto findings in skeletal muscle, Park2/6 transcripts were elevated (Fig. 6C),whereas PINK1 and Parkin protein levels in whole-cell lysates were re-duced in Esr1-KD versus control (Scr) C2C12 myotubes (Fig. 6D).

Because the accumulation of full-length PINK1 protein in the outermitochondrial membrane is critical for initiation of mitophagy, weperformed mitochondrial isolation studies in ERa-replete and ERa-deficient myotubes.We observed a reduced abundance of cytosolic andmitochondria-associated full-length 63-kD PINK1 protein and the54-kD degradation product in ERa-deficient myotubes compared withcontrol (Scr) in the basal state (Fig. 6E and fig. S4, C and G). To deter-mine whether ERa is critical for surveillance of mitochondrial health,we treated myotubes with the mitochondrial membrane depolarizingagent carbonyl cyanide m-chlorophenylhydrazone (CCCP), a well-described stimulus shown to inducemitophagy. In line with previousreports, CCCP induced an increase in PINK1 protein in ERa-repletecontrol (Scr) myotubes (Fig. 6E, open bars) (32). In contrast to control(Scr) cells, basal PINK1 protein levels were reduced and there was noinduction of PINK1 protein above the vehicle control after CCCP treat-ment in Esr1-KD myotubes (Fig. 6E, closed bars).

Our findings indicate that ERa deficiency causes a reduction inPark family protein levels and implicates increased PINK1/Parkin pro-tein turnover as a potential mechanism for reduced mitophagic signal-ing in MERKO muscle. Together, these data support the notion thatERa is critical for mitochondrial health surveillance and mitophagicsignaling. We recently linked impaired mitophagic signaling to meta-bolic dysfunction by showing that Parkin inactivation reduces oxidativemetabolism and promotes skeletal muscle insulin resistance, similar tophenotypic observations made for female MERKO mice herein (33).

Impaired autophagy in ERa-deficient muscleBecause the turnover ofmitochondria is shown to be reliant on intactmacroautophagy, we next studied the impact of ERa expression onautophagic signaling under basal and starvation conditions. Macro-autopahagy is a cellular housekeeping process that allows for theremoval of dysfunctional or unnecessary proteins or organelles. Au-tophagosomes (spherical structures with double-layer membranes)deliver the unwanted cellular components to the lysosome for deg-radation. The formation of autophagosomes is incompletely under-stood but is regulated by a well-conserved family of genes, Atg’s, andmicrotubule-associated protein light chain 3 (LC3) complexes. LC3proteins (for our purposes, LC3B) undergo posttranslational modifica-tions during autophagy, including immediate cleavage to LC3BI andsubsequent lipidation (phosphatidylethanolamine conjugation of





http://stm.sciencem

ag.org/D

ownloaded from

the carboxyl glycine) processing to yield LC3BII, thus allowing forLC3B to become associated with the autophagic vesicle to control au-tophagosome fusion. In contrast to themarked increase in LC3BII [ob-served in Control f/f muscle after nutrient deprivation (P = 0.03)], LC3Bprocessing (LC3BI to LC3BII) during starvation was impaired inMERKOmuscle; no significant increase in LC3BII protein was detected betweenthe fed to fasted state (P= 0.2; Fig. 6F and fig. S6A). Further, we detectedno difference in the expression of themicrotubule-associated protein 1B

www.Scie

light chain 3 (Maplc3b) LC3B transcript between the MERKO and con-trol genotypes during the basal or fasted conditions (Maplc3b is thegene that encodes the LC3B transcript) (fig. S6B). Although expressionof most autophagy-related genes (Atg) is unaltered by nutrient depriva-tion (34), we observed an induction of Sqstm1 (p62) andAtg7 in Controlf/f muscle after fasting, a phenotype that failed to be reproduced inMERKO mice (fig. S6, C and D); Atg7, an E1-like enzyme consideredessential in facilitating the association between Atg5 and Atg12 for LC3

Fig. 5. Altered fission-fusion dynamics signaling inmuscle fromMERKOversus Control f/fmice. (A andB)DRP1Ser637 phosphorylation (A) and DRP1 protein abundance (B) in enriched mitochondrial fractions fromfemale Control f/f andMERKOquadricepsmuscle (n= 4; representative immunoblots). Porin, control. (C) Protein-protein association between DRP1 and the phosphatase calcineurin (CnA) (above; n = 3 to 6 per genotype) inmuscle from Control f/f and MERKO mice. (D and E) Calcineurin and PKA activity in quadriceps muscle fromControl f/f and MERKO mice (n = 6 to 10 per genotype). (F to H) FIS1, OPA1, and MFN2 protein expressionin muscle from Control f/f and MERKO mice relative to glyceraldehyde-3-phosphate dehydrogenase(GAPDH) expression (control) (n = 6 per genotype). (I) Expression of Fis1, Mfn2, and Opa1 proteins in quad-ricepsmuscle from female Control f/f andMERKOmice relative to Ppia expression (housekeeping gene con-trol) (n= 6 per genotype). All values are expressed asmeans ± SEM.Mean differences detected by Student’s ttest or ANOVA. *P < 0.05, difference between genotypes.





http://stm.sciencem

ag.org/D

ownloaded from

Fig. 6. ERa deficiency impairs mitophagic signaling inmuscle. (A toD) Expression of Park familymembers (Park2and Park6) in muscle from female Control f/f and MERKOmice (n = 6 per genotype, A and B) and C2C12myotubes(n = 6 per group, C and D). (E) CCCP-induced accumulationof full-length (63-kD) PINK1 protein in control (Scr) and Esr1-KD myotubes (n = 6 observations per condition). Veh,
vehicle (control). (F) Densitometric analysis of LC3BII protein levels inmusclefrom fed and fasted (24 hours) female Control f/f versus MERKO mice rel-ative to GAPDH expression (n = 6 mice per condition). (G and H) Confocalmicroscopy and flow cytometry analyses of dually labeled RFP-GFP-LC3Bexpressed in control (Scr) and Esr1-KD muscle cells during nutrient depri-vation. (G) Reduced red punctae were observed in Esr1-KD muscle cellsversus Scr-control, indicating diminished autolysosome formation (scalebar, 1 mm). (H) To more accurately quantify fluorescence signals in musclecells, flow cytometry analyses were performed in triplicate during nutrientdeprivation in the presence and absence of BafA1 (10,000 live events
www.Scie

quantified). Bars depicting quantification of dual signal represent autopha-gosomeabundance (closedbars); a singleRFP signal represents autolysosomeabundance (gray bars); and cells showing loss of signal represent autophago-some degradation (hatched bars). (I to K) The impact of PKA inhibition (H89,50 mM)onp62 protein levels and LC3Bprocessing in ERa-repletemyotubes inthe absence or presence of CCCP (20 mM) to stimulate mitophagy (n = 3 percondition). (I) Representative immunoblots and (J and K) densitometric analy-ses. All values are expressed as means ± SEM. Mean differences detected byStudent’s t test or ANOVA. *P < 0.05, difference between genotypes; #P< 0.05,within-group, between-treatment difference.




Dow

nl

activation, was reduced in MERKO muscle even under basal fedconditions (fig. S6D). The increase in basal expression of Atg12,Atg5, and Atg4b might be a compensatory response to the decrementinAtg7 and impaired downstreamLC3 processing (fig. S6, E toG) in anattempt to restore autophagic flux.

Our findings in vivo were reproduced in C2C12 cells in culture. Amarked accumulation of p62 and reduced LC3BII/LC3BI were ob-served in Esr1-KD versus control (Scr) myotubes in the basal stateand after serum deprivation with and without the lysosomal inhibitorbafilomycin A1 (fig. S6, H and I). Because p62 is an adaptor moleculebelieved to target ubiquitinated proteins to LC3BII for selective up-take and degradation by the lysosome, increased p62 protein levels,despite its reduced transcript expression, are considered reflective ofimpaired autophagy (35). Moreover, although we observed no differ-ence in the expression of Maplc3b between the genotypes, LC3BI wasconsistently elevated in ERa-deficient muscle under basal conditions,suggesting a possible delay in processing of LC3B protein (fig. S6, A,H, and I).


http://stm.sciencem

ag.org/oaded from

To confirm our signaling findings and providean additional readout of LC3 processing and au-tophagolysosome formation, we generated myo-tubes that expressed LC3B protein with boundtandem red and green fluorescent protein (RFP-GFP-LC3B). BecauseGFP fluorescence is quenchedin the acidic pH of the lysosomal compartment,the use of GFP-LC3B is limited to the identifica-tion of autophagosomes. However, RFP is pH-resistant and continues to fluoresce at acidic pH;thus, RFP-LC3B can be used to identify both auto-phagosomes and autolysosomes (36, 37). Theapparent reduction in red fluorescencepunctae ob-served in Esr1-KDversus control (Scr)myotubes(Fig. 6G) was fully consistent with reduced au-tophagic flux, specifically reduced autolysosomeformation (36, 37). In addition, flow cytometrystudies were performed, providing a more sensi-tive and quantitative determination of the GFPand RFP signals, including loss of signal deter-mination for assessing starvation-induced LC3B-autophagosome turnover. Consistent with theprotein signaling data, functional analyses indi-cate that ERa inactivation markedly suppressedautolysosome formation and autophagosometurnover in the context of starvation-induced au-tophagy (Fig. 6H and fig. S7).

One possible explanation for impaired autoph-agic signaling and delayed autophagosome for-mation is direct phosphorylation of LC3B byPKA, an event shown to reduce LC3 recruitmentto autophagosomes (38). Considering that PKAactivity was consistently elevated twofold in bothMERKOmuscle andC2C12myotubes with Esr1-KD, we next tested the relation between PKA andLC3B maturation in control ERa-replete C2C12myotubes. Similar to findings previously reportedin SH-SY5Y human neuroblastoma cells (38), wefound that kinase inhibition by H89 enhancedbasal LC3BI-to-LC3BII conversion and p62 deg-

www.Scien

radation.Moreover, these findingswere intensified byCCCP treatment,a known inducer of mitophagy (Fig. 6, I to K). Collectively, these dataindicate that autophagic signaling is blunted in ERa-deficient muscleand that increased PKA activitymight contribute, in part, to the impair-ment in autophagosome processing during macroautophagy.

Drp1 is critical for insulin action and oxidative metabolismTo test directly whether impairment of mitochondrial fission could in-duce similar phenotypic outcomes as those observed in Esr1-KD cells,we performed chemical inhibition and gene deletion of Drp1. First, wetreated murine myotubes with the Drp1 inhibitor Mdivi-1 and foundthat insulin-stimulated phosphorylation of pyruvate dehydrogenasekinase (PDK), Akt, and glycogen synthase kinase 3b (GSK3b) was re-duced compared to vehicle-treated basal control myotubes (Fig. 7A).Next, we used shRNA to knock downDrp1 inmyotubes (Fig. 7B). Sim-ilar to findings for studies of Drp1 inhibition, Drp1-KD markedly im-paired insulin action inmyotubes (Fig. 7, C to E), and similar toMERKOmuscle, lipid levels were elevated in Drp1-KD myotubes (Fig. 7F). In

Fig. 7. Impaired mitochondrial fission signaling through DRP1impairs oxidative metabolism and induces insulin resistance.(A) Insulin signaling (p-PDK1S241, p-AktSer473, and p-GSK3b) in C2C12cells treated with the Drp1 selective inhibitor Mdivi-1 (50 mM) versusvehicle control (n = 6 per condition). (B) DRP1 protein levels in C2C12myotubes relative to GAPDH expression, with Drp1-KD achieved bylentiviral delivery of shRNA (five clones tested, A to E). (C to E) Drp1-KDimpaired insulin signaling in C2C12myotubes (1, 10, and 100 nM insulintreatment). (F) Drp1-KD promoted accumulation of lipid in myotubesas determined byOil RedO staining (n = 6 observations per genotype)(using two of the five shRNA lentiviral clones, C and D). Values are ex-pressed as means ± SEM. Mean differences detected by Student’s ttest or ANOVA. *P < 0.05, difference between genotypes.

ceTra
nslationalMedicine.org 13 April 2016 Vol 8 Issue 334 334ra54 10


http://stm.sci

Dow

nloaded from

aggregate, these data suggest that Drp1 action is critical for the main-tenance of insulin sensitivity and potentially oxidative metabolism, andthus, impaired fission dynamics might in part underlie the pathogenicphenotypes observed in MERKO muscle.

The oxidative stress–responsive Rcan1 links impaired fissionwith insulin resistanceBecause ERa is a hormone nuclear receptor involved in gene activationand repression, we screened microarrays to identify genes differentiallyexpressed in skeletal muscle between MERKO and Control f/f. Rcan1(also calledDscr1 andMcip1)—a known inhibitor of calcineurin (39–41)whose encoding gene is responsive to oxidative stress (39, 40)—was up-regulated 6.8-fold (P = 0.01) by ERa deletion (table S4); we confirmedthis finding by qPCR (Fig. 8A). Rcan1 expression was also increased inmuscle from premenopausal women with MetSyn (Fig. 8B). Similar toRCAN1 mRNA transcript abundance in muscle, the RCAN1-1 andRCAN1-4 protein levels also were significantly elevated (Fig. 8, C to E),and these findings were reproduced in C2C12 myotubes with Esr1-KD(fig. S5, C, E, and F). Although increased Rcan1mRNA transcript couldaccount for the elevated RCAN1protein levels inMERKOmuscle, PKAis also known to phosphorylate and stabilize RCAN1 protein, thus in-creasing its half-life (42). When Rcan1 was overexpressed in C2C12 orprimary myotubes, insulin action was impaired (Fig. 8, F to J). Thus,together, our findings suggest a conserved mechanism by which ERaregulates a PKA-Rcan1-calcineurin signaling axis to control mitochon-drial fission and insulin action (Fig. 8K).


encemag.org/

DISCUSSION

Reduced estrogen action is clinically tied to obesity, metabolic dys-function, and increased risk of chronic disease. However, the molecularactions of ERa and the tissues responsible for conferring these clinicaloutcomes remain inadequately understood. In the current investigation,we studied the importance of skeletalmuscle ERa action and the impactof skeletal muscle–specific receptor inactivation on themanifestation ofmetabolic dysfunction. We focused our efforts on skeletal muscle be-cause it is a predominant tissue responsible for fatty acid metabolismand whole-body insulin-stimulated glucose disposal.

First, we established the clinical rationale for our studies by showingthat ERa expression was reduced in muscle from women displayingclinical features of the MetSyn. Moreover, skeletal muscle ESR1/Esr1was highly predictive of fasting insulin concentration and adiposity inwomen and female mice. To interrogate this relationship further, weperformed a selective deletion of Esr1 from skeletal muscle in femalemice and myotubes in culture. ERa deletion caused marked insulin re-sistance in skeletal muscle and cultured myotubes compared to controlERa-replete muscle and cells. Studies in myotubes indicated that thedefects in insulin action are a direct effect of Esr1 deficiency within thatcell type and that muscle insulin resistance in MERKOmice occurs in-dependently of, but is likely exacerbated by, secondary phenotypes inadipose tissue and liver. Targeted deletion of ERa from skeletal musclefully recapitulated the insulin resistance and obesity observed in whole-body Esr1−/− mice, and the metabolic dysfunction appears more severethan other mouse harboring cell-specific deletions of Esr1 (43–46).

Paralleling insulin resistance in ERa-deficient myocytes, there was amarked reduction in mitochondrial functionality including diminishedrates of oxygen consumption, reduced fatty acid oxidation, impaired

www.Scien

calcium buffering capacity/heightened susceptibility for mPTP open-ing, and increased ROS production. It is reasonable to conclude thatthese combinatory defects in mitochondrial function contributed totissue inflammation and impaired insulin action in skeletal muscle,because each of these features of mitochondrial dysfunction has inde-pendently been shown to promote inflammatory signaling and insulinresistance (21, 47, 48).

Underlying these alterations in mitochondrial function in ERa-deficient muscle were defects in mtDNA replication and organellequality control including impairments in mitochondrial health sur-veillance, fission-fusion dynamics, and autophagy. In addition to reduc-tions in PINK1 and Parkin signaling, critical for the initiation ofmitophagy and subsequent elimination of damaged mitochondrialcontents, aberrant signaling through the PKA-Rcan1-calcineurin-Drp1 axis was linked with imbalanced fission-fusion dynamics anddelayed autophagic flux. Whether a primary defect in mitochondrialreplication was the initiating factor responsible for driving a com-pensatory reduction in mitophagy to sustain mtDNA abundance inMERKO muscle remains to be determined. However, it appears thattotalmtDNAcontent was preserved inMERKOmuscle at the expenseof organelle health.

As previously described, a marked alteration in mitochondrialmorphology characterized by enlarged, tubulated, and, in many cases,fused mitochondria extending across multiple z-lines of myofibrils isentirely consistent with heightened mitochondrial stress (22). Similarmorphological features ofmitochondriawere observed previouslywhencomparing electron micrographs of muscle from obese versus lean hu-man subjects and young versus aged mice (19, 49). A mitochondrialhyperfusion phenotype is thought to be a protective mechanism to di-lute damaged organelle contents, for example, DNA and enzymes,across the mitochondrial network (25, 50). Although the mechanismsunderlying mitochondrial targeting for turnover remain incompletelyunderstood, it is believed that in addition to PINK1 and Parkinsignaling, organelle size alone may serve as an important factor ex-cluding a mitochondrion from autophagosome incorporation andlysosomal degradation (25, 51). Thus, the morphology of MERKOmitochondria alone supports our hypothesis that ERa deletion inducesmitochondrial stress and impaired organelle turnover.

It is widely accepted that overproduction of ROS, decreased ROSscavenging capacity, and impaired DNA and protein repair mecha-nisms contribute to mtDNA damage and cellular aging (52). Con-sidering that MERKO muscle exhibited decrements in all threeprocesses, it follows that ERa inactivation is likely to accelerate mus-cle aging. Indeed, Esr1-KDmyotubes showed increased susceptibilityof mtDNA to mutations induced experimentally by exogenous H2O2.Cells are typically equipped with a sensitive mitochondrial surveillancesystem to combat the damaging effects of excessive mitochondrial ROSproduction by elimination of compromised mitochondrial componentsvia mitophagy. We show that this surveillance system, specificallyPINK1 and Parkin responsiveness to CCCP and H2O2, was markedlydepressed in the absence of ERa. Muscle from MERKO mice phe-nocopied features of loss-of-function mutations in PINK1 and Parkinin model organisms including impaired mitochondrial function, re-tention of enlarged organelles, sensitivity to oxidative stress, and de-rangements in metabolism contributing to reduced longevity (53, 54).Polymorphisms in Park family members have now been linked withT2D susceptibility and aging-related disease in humans (55). More re-cently, we confirmed this clinical relationship in mice by showing that

ceTranslationalMedicine.org 13 April 2016 Vol 8 Issue 334 334ra54 11




http://stm.sciencem

ag.org/D

ownloaded from

)

l)

,

,

l

l

muscle Parkin inactivation impairs mitochondrial function and insulinaction (33).

Mitochondrial fission is thought to be a central process required forthe autophagic degradation of damaged mitochondrial components(that is, mitophagy). In mammals, mitochondrial fission is primarilyregulated by the dynamin-related GTPase (guanosine triphosphatase)DRP1. Upon stimulation, DRP1 translocates to the mitochondria and

www.Scien

via self-assembly acts to remodel the outer mitochondrial membrane,making the organelle physically available for digestion by the lysosome,relocalization by the cytoskeleton, or fusionwith othermitochondria forretention to the network (16, 56, 57). Similar to findings in ERa-deficientmuscle, loss-of-function Drp1 mutant cells are fission-incompetent andexhibit a fused and extensively tubulatedmitochondrial morphology thatis associated with reduced ATP synthesis, increased ROS production

Fig. 8. Rcan1 isup-regulated inERa-deficient muscle and im-
pairs insulin action. (A and BRcan1geneexpression is elevatedin female MERKO muscle (n = 8per genotype) and in humanmus-cle fromwomenwithMetSyn (n=8per group) comparedwith respec-tive controls. (C to E) Rcan1-1 andRcan1-4protein levels are elevatedin MERKO muscle versus Controf/f (n = 8 per genotype). (F to JRcan1 overexpression by adeno-virus (Ad) infection of C2C12 orprimarymyotubes impairs insulinaction as shown by (G) reducedinsulin–stimulated p-AktSer473
(1, 10, and 100 nM insulin), (I) IRS-1–p85 association (50 nM insulin)and (J) 2-deoxyglucose uptake(n = 3 per condition in triplicate50 nM insulin). All values are ex-pressed as means ± SEM. Meandifferences detectedby Student’st test and ANOVA. *P < 0.05, differ-encebetweengenotypes; #P<0.05within-group, between-treatmentdifference. (K) Schematic overviewof the MERKO phenotype. Skeletamuscle–specific ERa deletion re-duced mtDNA replication andimpaired muscle oxidative me-tabolism, despite maintenance ofmtDNA copy number. ElevatedRcan1 and PKA reduced calcineur-in activity levels, promoting elon-gated, hyperfused mitochondriain femaleMERKOmuscle. Themor-phological changes coupledwithan imbalanced PKA-calcineurinaxis blunted mitochondrial fissionsignaling through DRP1 and im-paired macroautophagy, pro-cesses critical for mitochondriaturnover, mitophagy. Unopposedfusionof theouter and innermito-chondrial membranes was per-mitted by increased expressionof mitochondria-specific fusionproteins Mfn2 and OPA1. The re-tentionofdamagedmitochondriato the network was paralleled byincreased ROSproduction, inflam-mation, and insulin resistance in
skeletal muscle from female MERKO mice.



by guest on Ohttp://stm

.sciencemag.org/

Dow

nloaded from

(58), and mtDNA replication stress–linked DNA damage (59). Ourstudies in Drp1-KD muscle cells recapitulate these published find-ings and strongly implicate a link between impaired fission signalingand the development of insulin resistance. Indeed, recent findings byDrummond et al. show reducedDrp1 and Parkin expression levels inmuscle paralleled by hyperinsulinemia in inactive elderly women com-pared with younger female controls (60).

In further support of fission incompetency in the context of ERainactivation, calcineurin, the fission-promoting Ser/Thr phosphatase(61, 62) shown to dephosphorylate and activate DRP1, was reduced inexpression (regulatory subunit CnB), overall activity, and protein as-sociation with Drp1 in MERKO muscle compared with Control f/f.Impaired calcineurin action was paralleled by a marked increase intranscript and protein expression of the calcineurin inhibitor RCAN1in bothMERKOmuscle and Esr1-KDmyotubes. Notably,RCAN1 ex-pression was also significantly increased in muscle from women display-ing clinical features of the MetSyn. PKA-mediated phosphorylationof RCAN1 enhances its inhibitory effect on calcineurin, which, giventhe increased phosphorylation and total activity of PKA seen in parallelwith a marked phosphorylation of DRP1Ser637 in MERKO muscle, issuggestive of a primary role of PKA-Rcan1–mediated repression of fis-sion in Esr1-deficient muscle by calcineurin-DRP1 inactivation. Ourfindings support that ERa exerts a strong regulatory function overthe PKA-calcineurin-Rcan1-DRP1 axis to control mitochondrial dy-namics and maintain a healthy organelle network.

In overview, our studies highlight the critical importance of skeletalmuscle ERa for themaintenance ofmitochondrial health andmetabolichomeostasis. This research provides clinically relevant evidence thatnatural variation in expression of muscle ERa is predictive of adiposity,and importantly, we showed thatmuscle ESR1 expression is diminishedin women displaying clinical features of the MetSyn. Our findings haveclear implications for understanding the pathobiology ofmetabolic dys-function in women, and support that strategies to maintain or elevateERa action in muscle may be of therapeutic benefit in amelioratingcomplications associated with metabolic-related disease.

ctober 17, 2020

MATERIALS AND METHODS

Study designThe objective of this research was to understand the role of muscle ERain regulating metabolic homeostasis and insulin sensitivity. First, toestablish a clinical rationale for our studies in genetically engineeredmice, we determined the relationship betweenmuscle ERa expressionand indices of metabolic function using historical deidentified data andsample from a human study previously conducted by Pederson andcolleagues from the University of Copenhagen (63, 64) and mouse dataand samples fromprevious studies performed onmice of theUniversityof California, LosAngeles,HybridMouseDiversity Panel (UCLAHMDP)(65). Each subject provided written informed consent before the orig-inal study participation, and the study procedures were approved bythe Scientific-Ethical Committee of the Frederiksberg and Copenhagencounties and are in accordance with the principles of the Declaration ofHelsinki. All procedures in rodents were performed in accordance withthe Guide for the Care and Use of Laboratory Animals of the NationalInstitutes of Health and were approved by the Animal Subjects Com-mittee of UCLA. Because muscle ERa expression was inversely cor-related with adiposity and fasting insulin levels, we next determined

www.Scien

whether muscle ERa levels were reduced in women with MetSyn com-pared with lean healthy controls. Indeed, we observed a marked reduc-tion in muscle ERa levels in women with MetSyn.

To determinewhether themetabolic dysfunction inMetSyn subjectsmight be causally related to the reduction in muscle ERa expressionlevels, next we generatedMERKOmice using the standard Lox-Cre ap-proach. We performed an exhaustive phenotypic evaluation of at least10 cohorts of MERKO and Control f/f mice using a variety of in vivoand ex vivo approaches. The number of animals used for each studywasdetermined by Power calculations using a desired P value of 0.05 (ani-mal numbers for each study are indicated in the figure legends), and allstudies performed to assess glucose homeostasis and insulin actionwereblinded for animal genotype. In addition to studies in rodents, we gen-erated primary myotubes from muscles obtained from Control f/f andMERKOmice as well as C2C12 cells with shRNA-mediated KD of ERausing a lentiviral-mediated approach. Nearly all in vitro studies wereperformed in triplicate with aminimumof three independent replicatesas indicated in the figure legends.

AnimalsThe strategy for the generation of the floxed exon 3 ERa (ERaf/f) wasdescribed in (66). Floxed mice were crossed with a muscle-specificCre transgenic mouse (MCK-Cre) line and bred to obtain the followinggenotypes of females: MCK-Cre-ERaf/f (MERKO) and ERa f/f CRE-negative control mice (Control f/f). Animals were studied at variousages as indicated. Primers used for genotyping are in table S5. Tissuesfrom 10 strains of female inbredmice (16 weeks of age)—(i) FVB/NJ,(ii) PL/J, (iii) BXA2/PgnJ, (iv)AxB4/PgnJ, (v)CxB3/ByJ, (vi) BxD34/TyJ,(vii) BxD24/TyJ-Cep293, (viii) BxD24/TyJ-Cep293, (ix)CxB12/HiAJ, and(x) BxD1/TyJ—were obtained from the Lusis Laboratory HMDPUCLA. Lean wild-type and obese (LepOb) female mice were obtainedfrom Jackson Laboratories at 8 weeks of age and studied at 12 weeksof age. Skeletalmuscle fromERaDBDDmicewas obtained fromK.Korachas previously described (67).

Control f/f andMERKOmice weremaintained on a normal chowstandard diet and divided into two general groups: basal or insulin-stimulated. Within these categories, mice were further divided intogroups depending on the duration of fasting (6 to 24 hours) or whetherthey were used for in vivo (glucose clamps) or ex vivo (soleus muscle2-deoxyglucose uptake assays) assessment of insulin sensitivity. Ani-mals used for the deuterium studies to assess mtDNA replication wereweight-matched and administered an intraperitoneal injection of2H2O (600ml; Cambridge Isotope Laboratories) followed by ad libitumdrinking of heavy water [4% (v/v) deuterium oxide] for 30 days beforeeuthanasia and hindlimb tissue harvest for mtDNA isolation and sub-sequent mass spectrometry analyses (as described below).

Human subjectsWe recruited potential female volunteers by advertising on the Copen-hagen hospital staff Web sites and in local newspapers. The potentialvolunteers visited the laboratory for an initial screening in the morningafter an overnight fast. A health examination was conducted by a phy-sician followed by a 2-hour 75-g oral glucose tolerance test (OGTT) anda maximum working rate (Pmax) test on a cycle ergometer (Ergometric839E, Monark). Volunteers included in the study were either leanhealthy individuals (n = 18; 9 premenopausal and 9 postmenopausal)or individuals classified as MetSyn showing IGT and/or insulin resist-ance (n= 25; 21 premenopausal and 4 postmenopausal) and in addition





http://stm.sciencem

ag.org/D

ownloaded from

met the following criteria: overweight (BMI, >25 kg/m2), aged 30 to60 years, and a low VO2max for their age and sex (68). Exclusion criteriawere diabetes, impaired fasting glucose, blood pressure >140/90mmHg,smoking, and any diagnosed systemic disease. All of the premenopausalwomen were in the mid to late follicular phase of their menstrual cycle(days 10 to 14), and oral contraceptives were included as an exclusioncriteria. Subject characteristics are presented in table S1 and were, in part,previously described (64). The test day started at 8:00 a.m., after an over-night fast. Subjects were asked to refrain from drinking coffee, tea, softdrinks, or alcohol 24 hours before, and from performing any rigorousphysical exercise 72 hours before the test day. Weight and height weremeasured to the nearest 0.1 kg and 0.5 cm, respectively. Blood pressurewasmeasured twice in the supine position, with 10min of rest precedingeach measurement, using an automatic sphygmomanometer (Green-light 300, AC Cossor & Son Accoson Works) placed on the left arm.

Volunteers were then subjected to a 3-hour 75-g OGTT. A catheterwas placed in an antecubital vein.Within a 5-min period, subjects drank75 g of glucose (anhydrous glucose, Unikem) diluted in water (5 dl).Eight blood samples of 7ml (the baseline sample contained 16ml) weretaken for the determination of blood glucose and plasma concentrationsof insulin. The blood samples were collected before glucose ingestion at0min and at 10, 20, 30, 60, 90, 120, and 180min after glucose ingestion.Blood samples were immediately centrifuged for 15 min at 2400g and4°C. Plasmawas removed and pipetted into 3 × 1.5–mlEppendorf tubes(Brand), and stored at−80°C for subsequent analysis. Blood glucose andcirculatinghormone levels including insulin, estradiol, follicle-stimulatinghormone, and luteinizing hormone were measured by the Departmentof Clinical Biochemistry, Rigshospitalet.

At 11:30 a.m., the volunteers received a light lunch and then werescanned in a dual-energy x-ray absorptiometry scanner (GEMedicalSystems Lunar, Prodigy Advance) to estimate whole body fat percent-age using software version 8.8. After a ~10-min rest on a hospital bed inthe supine position, amuscle biopsy was obtained from the vastus later-alis using the Bergstrom percutaneous needle biopsy technique withsuction. Biopsy samples (100 to 200 mg) were immediately frozen inliquid nitrogen and stored at −80°C until analyzed.

Animal characteristicsBlood was drawn from 6-hour–fasted, 20-week-old female mice andanalyzed for circulating factors: glucose (HemoCue), insulin, leptin, andadiponectin (Millipore; table S2). After a 2-week recovery, GTTs (1 g/kgdextrose) were performed on 6-hour–fasted female mice as previouslydescribed (69, 70). Feeding andmovement were recorded over 48 hoursafter animalswere acclimated tometabolic chambers (Oxymax, ColumbusInstruments).

Hyperinsulinemic-euglycemic clamp studiesTwo weeks after the GTT, dual catheters were surgically placed in theright jugular vein, and glucose clamp studies were performed on femalemice 3 days after surgery as previously described (69, 70). All animalswere fasted for 6hours before the clampand studied in the conscious state.Basal glucose turnoverwas determined after a 90-min constant infusion of(5.0 mCi/hour, 0.12 ml/hour) D-[3-3H]glucose (PerkinElmer). After thebasal period, glucose (50% dextrose, Abbott Laboratories) and insulin(12 mU kg−1 min−1, Novo Nordisk Pharmaceutical Industries) plustracer (5.0 mCi/hour) infusions were initiated simultaneously, and glucoselevels clamped at euglycemia (~120mg/dl) using a variableGIR.At steadystate, the total GDR,measured by tracer dilution technique, is equal to the

www.Scien

sum of the rate of endogenous or hepatic glucose production and theexogenous (cold) GIR (69–71). The insulin-stimulated component of thetotal GDR is equal to the total GDRminus the basal glucose turnover rate.

Immunoblot analysisMouse tissue samples were pulverized in liquid nitrogen and homo-genized in radioimmunoprecipitation assay (RIPA) lysis buffer con-taining freshly added protease (cOmplete EDTA-free, Roche) andphosphatase inhibitors (Sigma). All lysates were clarified, centrifuged,and resolved by SDS–polyacrylamide gel electrophoresis. Samples weretransferred to polyvinylidene difluoride membranes and subsequentlyprobed with the following antibodies for protein and phospho-proteindetection: ERa and tubulin (Santa Cruz Biotechnology); GLUT4,Mfn2,andRCAN1-1/1-4 (Sigma-Aldrich);GAPDHanda-actinin (Millipore);pan-actin, AKAP, t-Akt/p-AktSer473, IKKb/p-IKKa/bSer180/181, JNK/p-JNKSer183/Tyr185, Parkin (4211and2132), LC3B, tDRP1/p-DRP1Ser616/637,and t-PKA/p-PKA (Cell Signaling); FIS1 (GeneTex); OPA1 (BD Bio-sciences); GPX3 and calcineurin pan anda/b (R&DSystems);MitoPro-file OXPHOS (MS604/ab110413; CI-NDUFB8 20 kD, CII-SDHB 30kD, CIII-UQCR2 Core protein 2 48 kD, CIV-MTCO1 40 kD, and CV-ATP5A 55 kD) and Porin (MitoSciences, Abcam); PINK1 (CaymanChemical); p62 (Progen Biotechnik GmbH); and GFP (Abcam). To an-alyze tyrosine phosphorylation of IRS-1 (Santa Cruz Biotechnology),lysates were immunoprecipitated overnight with IRS-1 or IRS-2 anti-body (Santa Cruz Biotechnology) and immobilized on protein A(Upstate Millipore) or G agarose beads (Santa Cruz Biotechnology).Phosphorylation was detected by immunoblotting with anti-pY20 an-tibody (Cell Signaling). Additionally, immunoprecipitates were alsoimmunoblotted for PI 3-kinase (phosphatidylinositol 3-kinase)p85 protein (Santa Cruz Biotechnology). Densitometric analysiswas performed using Bio-Rad Quantity One image software.

Muscle lipids and lipid intermediatesLipids were extracted from the quadriceps muscle by the Folch method(72). Triacylglycerol was saponified in an ethanol/KOH solution at60°C, and glycerol content was determined fluorometrically. DAG andceramidewere extracted and quantified as previously described (72–74).Lipid accumulation in Drp1KD myotubes was assessed by Oil Red Ostaining. Differentiated cells were fixed for 1 hour in 10% formalin.After washing with 60% isopropanol, cells were stained with Oil Red Ofor 10 min. Cells were rinsed with water and then imaged. Quantifica-tion was performed by eluting Oil Red O with 100% isopropanol for10 min and by measuring optical density at 500 nm for a 0.5-s reading.

Quantitative reverse transcription PCRTissues were first homogenized using TRIzol reagent, and RNA wasisolated and further cleaned using RNeasy columns (Qiagen) withDNase digestion. For RNA isolation from cells, the RNeasy Plus Kitwas used as per the manufacturer’s instructions. Complementary DNAsynthesis was performed using 1 to 3 mg of RNA with SuperScript IIReverse Transcriptase (Invitrogen). PCRswere prepared using iQ SYBRGreen Supermix (Bio-Rad). All PCRs were performed in a Bio-RadMyiQ Real-Time Detection System. Quantification of a given gene, ex-pressed as relative mRNA level compared with control, was calculatedafter normalization to a standard housekeeping gene (b-actin, Ppia). Weperformed separate control experiments to ensure that the efficienciesof target and reference amplification are equal, as described in the UserBulletin 2 from Applied Biosystems. Primer pairs were designed using





http://stm.sciencem

ag.org/D

ownloaded from

Primer Express 2.0 software (Applied Biosystems) or previously pub-lished sequences (70, 75, 76). Primer sets were selected spanning at leastone exon-exon junction when possible and were checked for specificityusing BLAST (Basic Local Alignment Search Tool; National Center forBiotechnology Information). The specificity of the PCR amplificationwas confirmed by melting curve analysis ensuring that a single productwith its characteristic melting temperature was obtained. To determinemtDNA abundance, total DNA was isolated from cells or tissues usingthe DNeasy Kit (Qiagen), and the mtCO3 oligos and SDHA (succinatedehydrogenase complex subunit A) were used for quantification ofmitochondrial and nuclear genomes. Primer sequences for the specifictarget genes analyzed are in table S5.

Illumina microarray analysesMicroarray analysis was performed using the Illumina Bead ChipSystem (MouseRef-8 v2.0) by the Southern California Genome Con-sortium on RNA isolated from quadriceps as described above. Each ofthe eight separate arrays on the chip (fourControl f/f and fourMERKO)was performed on pooled RNA (from three mice) that was subjected topurity and integrity analysis using an Agilent Bioanalyzer. Array inter-nal control and basic analysis was performed using GenomeStudio(v2010.3). Findings for differential gene expression MERKO versusControl f/f are presented at 5% false discovery rate.

Cell culture, transfections, and treatmentsC2C12 cellsweremaintained inhigh-glucoseDulbecco’smodifiedEagle’smedium (DMEM) 10% fetal bovine serumwith penicillin/streptomycin.To obtain C2C12myotubes, cells were allowed to reach confluence andthemediumwas switched for high-glucoseDMEM2%horse serumwithpenicillin/streptomycin for 5 to 7 days. Bafilomycin A1 (25 nM) and cy-cloheximide (Sigma; 20 mM)were used to block lysosomal degradationand protein synthesis, respectively. Chloramphenicol (Sigma; 100 mg/ml)was used to block mitochondrial protein synthesis, and carbonyl cyanidem-chlorophenylhydrazone (CCCP; 20 mM) is a mitochondrial uncouplerused to induce mitophagy. H89 kinase inhibitor (Tocris; 50 mM) wasused to test the impact of PKA inhibition on LC3B processing.

For the induction of mtDNA mutations with H2O2, C2C12 cellswere treated with 0.5mMH2O2 (Sigma) for 24 hours and left to recoverin growth medium for an additional 24 hours. Mitochondria wereisolated using a dounce homogenizer and the Mitochondria IsolationKit for Cultured Cells according to the manufacturers’ instructions(Pierce/Thermo Scientific), and mtDNA was extracted using DNeasyextraction kit (Qiagen). Afterward, mtDNA was digested with endo-nuclease Taq I for 6 hours with the addition of 100 U of enzyme everyhour. Then, two sets of primers were used in real-time qPCR as describedfor RMC assay (77). The first set flanks one Taq I site that is amplifiedonly when there is a pointmutation in any of the bases of the Taq I targetsite. The second set amplifies an adjacent region not containing a Taq Isite, which quantifies the total amount of mtDNA in the sample (tableS5). Point mutation frequency was calculated by the amount of mutantmolecules normalized to the amount of totalmtDNAmolecules. For tan-dem Lc3b imaging, C2C12 myocytes were transfected with tandemmRfp-Gfp-Lc3 plasmid (Addgene plasmid #21074) onto glass coverslipsusing Lipofectamine Plus reagent (Invitrogen).

Lentiviral-induced ERa KD in C2C12 myocytesTo achieve Esr1-KD, lentiviral particles (Sigma) carrying shRNA tar-geted to ERa were used to transduce C2C12 myoblasts. After selecting

www.Scien

positive transformants with puromycin (5 mg/ml), the selected cloneswere expanded and analyzed for KD efficiency as measured by qRT-PCR and immunoblotting. The resulting cultures were then used forsubsequent assays in the undifferentiated and differentiated states.

Mitochondria isolation from C2C12 myotubesMitochondria were isolated frommyotubes as instructed (MitochondriaIsolation Kit for Culture Cells, Thermo Scientific, 89874). Briefly, 2 × 107

cell pellet was resuspended with mitochondria isolation reagent A andvortexed at medium speed for 5 s and then incubated on ice for 2 min.After incubation, 10 ml of reagent B was added and vortexed at maxi-mum speed for 5 s followed by an additional incubation on ice for 5min.After adding 800 ml of reagent C, tubes were inverted and then centri-fuged (700g for 10min at 4°C). In a new tube, the supernatant was cen-trifuged again (12,000g for 15min at 4°C). Themitochondrial pellet wasspun (12,000g for 5 min) and cleaned again by adding 500 ml of reagentC before mitochondrial lysis using RIPA buffer.

Insulin signal transduction in C2C12 myotubesC2C12myotube insulin signalingwas performed in 12-well culture plates.Briefly, cellswere serum-starved for2hours in low-glucoseDMEMcontain-ing 0.1% bovine serum albumin (BSA), then placed in phosphate-bufferedsaline (PBS) containing 0.1% BSA for 30 min, and allowed to equili-brate. Cells were then stimulated with or without insulin (dose response1, 10, and 100 nM) in the same medium for 15 min. Insulin signalingwas terminated by four sequential washes with ice-cold PBS before lysisin 0.3 M NaOH.

Fatty acid oxidation and esterification in myocytesMyotube fatty acid oxidation and esterification were performed in six-well culture plates, as adapted from the method previously described(78, 79). Briefly, cells were incubated in low-glucose DMEM containing1% fatty acid–free BSA and 0.5 mMpalmitate for 1 hour. After this, themedium was replaced with the addition of [14C]palmitate (1 mCi/ml)and allowed to incubate for a further 2 hours. Complete oxidation ofpalmitate was measured by benzethonium hydroxide captured 14CO2,liberated fromacid-treated culturemedium.Cellular aqueous and ester-ified lipids were isolated from cell monolayers by sequential inorganicand organic phase separations, respectively. Calculations were normal-ized to the specific activity of [14C]palmitate in the medium.

Cellular oxygen consumption in myocytesReal-timemeasurement of oxygen consumption and proton produc-tion in cells was carried out using the XF24 Extracellular Flux Analyzer(Seahorse Bioscience). To directly assess mitochondrial metabolism,measurements of oxygen consumption were made continuously whilecells were sequentially treatedwith oligomycin (ATP synthase inhibitor,which allows determination of the levels of ATP synthesis), FCCP (anuncoupler, which allows determination of maximal mitochondrial res-piratory capacity), and rotenone/myxothiazol (inhibitors of complexI/III of the electron transport chain, which allow determination of thenonmitochondrial oxygen consumption).

ROS productionC2C12myoblasts werewashed and incubated in low-glucoseDMEMat37°C, 5% CO2 in the dark with 25 mMcarboxy-H2DCF-DA (MolecularProbes), washedwith PBS and incubated for 15minwith 5 mMmitoSOX(Molecular Probes), washed and quickly trypsinized, pelleted, and





http://stm.sciencem

ag.org/D

ownloaded from

retained on ice. Cells were resuspended in FACS (fluorescence-activated cellsorting)buffer (PBS3%BSA)withDAPI (4′,6-diamidino-2-phenylindole)(25 mg/ml) and analyzed immediately by flow cytometry on an LSRII(Becton Dickinson) with FlowJo software (Tree Star Inc.). Unstainedand single stains were used for establishing compensation and gates,and only live cells (DAPI-negative) were analyzed by the software.

Ex vivo skeletal muscle glucose uptakeWhole-muscle ex vivo glucose uptakewas assessed using 2-deoxyglucose,with minor changes to that described previously (80). Briefly, soleusmuscles were carefully excised from anesthetized animals and immedi-ately incubated for 30 min in complete Krebs-Henseleit buffer with orwithout insulin (60 mU/ml) at 35°C.Muscles were then transferred to thesamebuffer containing [3H]2-deoxyglucose (3mCi/ml) and [14C]mannitol(0.053 mCi/ml), and incubated for exactly 20min before being blotted andsnap-frozen.Muscles were homogenized in lysis buffer and counted forradioactivity or subjected toWestern blotting. Glucose uptake was stan-dardized to the nonspecific uptake of mannitol and estimated asmicro-mole of glucose uptake per gram of tissue.

Citrate synthase activityCitrate synthase enzyme activity assays were performed on soleusand gastrocnemius muscle using standard methods, as previously de-scribed (81).

Mitochondrial calcium retention capacityPreparation ofmitochondria was adapted from quick isolationmethod,previously described (82). Gastrocnemius muscles were placed in iso-lationbufferA (70mMsucrose, 210mMmannitol, 1mMEDTA, 50mMtris, pH 7.4). The tissue was finely minced with scissors and digestedwith bacterial proteinase type XXIV (0.2 mg/ml) (Sigma) for 5 min onice, washed, and quickly homogenized in a Potter-Elvehjem tissuegrinder in the same buffer supplemented with 1% albumin. The ho-mogenate was centrifuged at 1300g for 3min, and the supernatant wascentrifuged at 10,000g for 10min. Themitochondrial pellet was resus-pended in isolation buffer B (150 mM sucrose, 50 mM KCl, 2 mMKH2PO4, succinic acid, and 20 mM tris-HCl, pH 7.4). The initiationofmPTPopeningwas assessed after in vitro Ca2+ overload as previouslydescribed (82, 83). Free Ca2+ concentration outside the mitochondriawas recorded with 0.5 mM calcium green-5N (Molecular Probes) (exci-tation, 500 nm; emission, 530 nm). Isolated mitochondria (500 mg ofprotein) were suspended in 2ml of buffer B. Samples were preincubatedin continuous stirring for 90 s in the spectrofluorometer cell, and CaCl2pulses (200 pmol of CaCl2) were applied every 60 s. The calcium reten-tion capacity was defined as the amount of Ca2+ required to trigger Ca2+

release and is expressed as micromole of CaCl2 per milligram of mito-chondrial protein.

Muscular force and endurance analysesMice were anesthetized (sodium pentobarbital, 60 mg/kg), and thesoleusmuscle was dissected with both tendons intact. Themuscles weremounted in a chamber continuously perfused with an oxygenated (95%O2, 5% CO2) Tyrode’s solution (121 mM NaCl, 5 mM KCl, 1.8 mMCaCl2, 0.5 mM MgCl2, 0.4 mM NaH2PO4, 24 mM NaHCO3, 5.5 mMglucose, 0.1 mM EGTA) at room temperature to ensure no unstirredlayers around the muscle, sufficient oxygenation to the core of themuscle, and stable pH (7.4). One tendon was tied with silk thread toa force transducer and the other tendon was tied to an adjustable tube

www.Scien

at the opposite end of the chamber, allowingmuscle length to be alteredincrementally to determine optimalmuscle length (Lo) and the length atwhich maximal tetanic force is produced. Muscles were stimulated atsupramaximal voltage using platinum electrodes placed on either sideof the muscle. After 15 min of equilibration, the muscle was tested formaximal isometric tetanic force and fatigue.

Maximal isometric tetanic forcewas elicitedwith a 500-ms train dura-tion and 0.2-ms pulse duration at 80 Hz. Fatigue was measured usingisometric tetanic contractions of 500-ms train duration and 0.2-ms pulseduration at 50 Hz. Stimulation frequency was increased every 2 minin a progressivemanner (from a contraction every 8 to 4 to 3 to 2 to 1 s)and terminated when the developed force had fallen to ~50% of the ini-tialmaximal developed force.Muscle fatigue was determined by the du-ration of time to reach 60%of themaximal developed force. After testing,the cross-sectional area was calculated as previously described (84), andthe maximal isometric tetanic force (Po) was expressed as N/cm2.

Electron microscopyMuscleswere harvested and immediately immersed in 2%glutaraldehydein PBS for 2 hours at room temperature and then at 4°C overnight. Fixedtissues were washed and postfixed in a solution of 1% OsO4 for 2 hours.After furtherwashes in buffer, tissueswere dehydrated through serial im-mersions in graded ethanol solutions (50 to 100%), passed through pro-pylene oxide, and infiltrated in mixtures of Epon 812 and propyleneoxide 1:1 and then 2:1 for 2 hours each and then in pure Epon 812 over-night. Embeddingwas then performed in pure Epon 812, and curingwasdone at 60°C for 48 hours. Muscle longitudinal sections of 60-nm thick-ness were cut using an ultramicrotome (RMCMTX). The sections weredouble-stained in aqueous solutions of 8% uranyl acetate for 25 min at60°C and lead citrate for 3 min at room temperature. Thin sections weresubsequently examined with a 100CX JEOL electron microscope at theUCLA Brain Research Institute. The mitochondrial area was assessed for300 to 350 organelles per mouse (n = 3) per genotype using ImageJ.

Confocal microscopy and flow cytometryTo detect autophagy, both C2C12 control scramble (Scr) and Esr1-KDmyocytes were incubated with Rfp-Gfp-Lc3b (tandem Lc3) adenovirusgenerated using theViraPowerAdenoviral Expression System (Invitrogen)according to the manufacturer’s instructions. Briefly, the coding se-quence for tandem Lc3 with flanking BP sites was amplified from theAddgene plasmid #21074 using High-Fidelity Pfx50 DNA polymerase(Invitrogen). Entry clones for tandemLc3were obtained by recombina-tion of this purified DNA fragment with pDONR221 vector usingGateway BP Clonase II. Tandem Lc3 insert was transferred from theentry clones into the pAd/CMV/V5-DEST vector (Invitrogen) usingthe Gateway LR Clonase II enzyme mix. Recombinant adenoviral pur-ified plasmid was used to generate adenoviral particles according tovector manufacturer’s instructions (Invitrogen). Adenoviral titer wasdetermined using the Adeno-X Rapid Titer Kit (Clontech). Autophagywas induced with nutrient starvation in Hanks’ balanced salt solution(Gibco) for 4 or 8 hours followed with or without autophagy inhibitionby 25 nM bafilomycin A1 (Sigma) for another 4 hours. After treatment,cells were washed twice with PBS, fixed in 4% paraformaldehyde for15min at room temperature, and resuspended in FACS buffer for anal-ysis on anLSRII (BectonDickinson)usingFlowJo software (Tree Star Inc.).Untreated cells, cells infected with adenovirus carrying EGFP-LC3, andcells transfected with mRFP-LC3 plasmid were used for establishingcompensation and gates.





http://stm.sciencem

ag.org/D

ownloaded from

For tandem LC3B imaging by confocal microscopy, confluentC2C12 myocytes were transfected with tandem Rfp-Gfp-LC3 plasmid(Addgene plasmid #21074) onto glass coverslips using LipofectaminePlus reagent (Invitrogen). Cells then were induced to differentiate tomyotubes for 5 days. For imaging, cells were nutrient-starved withHanks’ balanced salt solution for 4 hours and then fixed for 15 minin 4% paraformaldehyde-PBS at room temperature, washed with PBStwice, and sealed before being visualized on a Leica TCS-SP2 AOBSconfocal microscope.

Newly synthesized mtDNA labeling with 2H2OA highly enriched population of mitochondria were isolated fromhindlimb muscles obtained from female Control f/f and MERKOmiceafter 30 days of heavy water (Cambridge Isotope Laboratories; deute-rium oxide) consumption with modifications to previously describedtechniques (85, 86). mtDNAwas isolated, assessed for purity (Controlf/f 91.5 ± 2.62% versus MERKO 92 ± 1.54%; P = 0.756), and digestedwith a cocktail of nuclease enzymes using a commercial kit followingthe manufacturers’ protocol (DNA Degradase Plus, Zymo Research;2.5 ml of 10× DNA Degradase Reaction buffer, 1 ml of DNA DegradasePlus, and water to make a total reaction volume of 25 ml). After incuba-tion (37°C, >1 hour), aqueous formic acid was added (25ml; 0.1%, v/v)to yield a final concentration of 40 ng of digested DNA/ml. The DNAhydrolysis samples were injected onto a reversed-phase column (Hyper-carb 2.1 × 100 mm, 3-mm particle size, Thermo Scientific) equilibratedwith buffer A (0.1% aqueous formic acid) and eluted (200 ml/min) withan increasing concentration of buffer B (methanol: min/%B; 0/5, 2/5,12/50, 14/50, 15/5, 20/5). The effluent from the column was directedto an electrospray ion source (Agilent Jet Stream) connected to a triplequadrupole mass spectrometer (Agilent 6460 QQQ) operating in thepositive ion neutral loss mode of 116, 117, 118, and 119, which corre-sponds to the loss of a neutral fragment of unlabeled deoxyribose (dR),one deuterium–labeled dR (dR+1), two deuterium–labeled dR (dR+2),and three deuterium–labeled dR (dR+3). The intensity of precursor iontransitions from either dR loss was recorded. The precursor ion or thenucleosides of interest were the following: deoxycytosine (dC) mass/charge ratio (m/z) 228.1, thymidine (dT) m/z 243.1, deoxyguanosine(dG) m/z 268.1, and deoxyadenosine (dA) m/z 252.1. The measuredpeak area for the nucleosides of a specific dR loss was measured andanalyzed (Agilent MassHunter Quantitative Analysis, version B.04.00).The measured percentage of labeled nucleosides was calculated by theratio of the labeled to the total peak area counts for each nucleoside.

StatisticsValues presented are expressed as means ± SEM. Statistical analyseswere performed using Student’s t test when comparing two groups ofsamples or one-way ANOVA with Tukey’s post hoc comparison foridentification of significance within and between groups using SPSS(Graduate Pack) or GraphPad Prism 5 (GraphPad Software). Signifi-cance was set a priori at P < 0.05.

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/8/334/334ra54/DC1Table S1. Basal metabolic characteristics in female subjects.Table S2. Animal characteristics after a 6-hour fast in the basal or clamped state.Table S3. Annotation of functionally enriched terms from Illumina microarray analyses onquadriceps muscle from female Control f/f versus MERKO mice.

www.Scien

Table S4. Microarray analyses on quadriceps muscle from Control f/f versus MERKO mice.Table S5. Primer sequences for qPCR analyses on tissues and for genotyping of Control f/f andMERKO mice.Fig. S1. Real-time respirometry in C2C12 myotubes.Fig. S2. Muscle peak tension and time to fatigue.Fig. S3. Polg1 expression is regulated by ERa in muscle.Fig. S4. Altered mitochondrial fission-fusion signaling in ERa-deficient C2C12 myotubes.Fig. S5. Calcineurin-PKA-Rcan1 axis in C2C12 myotubes with Esr1-KD.Fig. S6. Expression of autophagy-related genes in MERKO muscle.Fig. S7. Autophagic flux studies in C2C12 myocytes with Esr1-KD.

REFERENCES AND NOTES

1. R. A. DeFronzo, R. C. Bonadonna, E. Ferrannini, Pathogenesis of NIDDM: A balancedoverview. Diabetes Care 15, 318–368 (1992).

2. Y.-W. Park, S. Zhu, L. Palaniappan, S. Heshka, M. R. Carnethon, S. B. Heymsfield, The metabolicsyndrome: Prevalence and associated risk factor findings in the US population from the ThirdNational Health and Nutrition Examination Survey, 1988–1994. Arch. Intern. Med. 163, 427–436(2003).

3. A. Hevener, D. Reichart, A. Janez, J. Olefsky, Female rats do not exhibit free fatty acid–induced insulin resistance. Diabetes 51, 1907–1912 (2002).

4. Y. Gómez-Pérez, E. Amengual-Cladera, A. Català-Niell, E. Thomàs-Moyà, M. Gianotti,A. M. Proenza, I. Lladó, Gender dimorphism in high-fat-diet-induced insulin resistance inskeletal muscle of aged rats. Cell. Physiol. Biochem. 22, 539–548 (2008).

5. J. P. Frias, G. B. Macaraeg, J. Ofrecio, J. G. Yu, J. M. Olefsky, Y. T. Kruszynska, Decreasedsusceptibility to fatty acid–induced peripheral tissue insulin resistance in women. Diabetes50, 1344–1350 (2001).

6. V. Ribas, M. T. A. Nguyen, D. C. Henstridge, A.-K. Nguyen, S. W. Beaven, M. J. Watt, A. L. Hevener,Impaired oxidative metabolism and inflammation are associated with insulin resistance inERa-deficient mice. Am. J. Physiol. Endocrinol. Metab. 298, E304–E319 (2010).

7. T. M. D’Eon, S. C. Souza, M. Aronovitz, M. S. Obin, S. K. Fried, A. S. Greenberg, Estrogenregulation of adiposity and fuel partitioning. Evidence of genomic and non-genomicregulation of lipogenic and oxidative pathways. J. Biol. Chem. 280, 35983–35991(2005).

8. S. E. Campbell, K. A. Mehan, R. J. Tunstall, M. A. Febbraio, D. Cameron-Smith, 17b-Estradiolupregulates the expression of peroxisome proliferator-activated receptor a and lipid oxidativegenes in skeletal muscle. J. Mol. Endocrinol. 31, 37–45 (2003).

9. H. Yki-Järvinen, Sex and insulin sensitivity. Metabolism 33, 1011–1015 (1984).10. C. Borrás, J. Sastre, D. García-Sala, A. Lloret, F. V. Pallardó, J. Viña, Mitochondria from

females exhibit higher antioxidant gene expression and lower oxidative damage thanmales. Free Radic. Biol. Med. 34, 546–552 (2003).

11. T. Oka, S. Hikoso, O. Yamaguchi, M. Taneike, T. Takeda, T. Tamai, J. Oyabu, T. Murakawa,H. Nakayama, K. Nishida, S. Akira, A. Yamamoto, I. Komuro, K. Otsu, Mitochondrial DNAthat escapes from autophagy causes inflammation and heart failure. Nature 485, 251–255(2012).

12. A. Salminen, K. Kaarniranta, A. Kauppinen, Inflammaging: Disturbed interplay between au-tophagy and inflammasomes. Aging 4, 166–175 (2012).

13. R. Zhou, A. S. Yazdi, P. Menu, J. Tschopp, A role for mitochondria in NLRP3 inflammasomeactivation. Nature 469, 221–225 (2011).

14. J. Tschopp, Mitochondria: Sovereign of inflammation? Eur. J. Immunol. 41, 1196–1202(2011).

15. D. C. Chan, Mitochondria: Dynamic organelles in disease, aging, and development. Cell125, 1241–1252 (2006).

16. S. A. Detmer, D. C. Chan, Functions and dysfunctions of mitochondrial dynamics. Nat. Rev.Mol. Cell Biol. 8, 870–879 (2007).

17. S. Mai, B. Muster, J. Bereiter-Hahn, M. Jendrach, Autophagy proteins LC3B, ATG5 andATG12 participate in quality control after mitochondrial damage and influence lifespan.Autophagy 8, 47–62 (2012).

18. R. P. A. Barros, U. F. Machado, M. Warner, J.-A. Gustafsson, Muscle GLUT4 regulationby estrogen receptors ERb and ERa. Proc. Natl. Acad. Sci. U.S.A. 103, 1605–1608(2006).

19. V. B. Ritov, E. V. Menshikova, J. He, R. E. Ferrell, B. H. Goodpaster, D. E. Kelley, Deficiency ofsubsarcolemmal mitochondria in obesity and type 2 diabetes. Diabetes 54, 8–14(2005).

20. K. A. Baltgalvis, S. M. Greising, G. L. Warren, D. A. Lowe, Estrogen regulates estrogen re-ceptors and antioxidant gene expression in mouse skeletal muscle. PLOS One 5, e10164(2010).

21. E. P. Taddeo, R. C. Laker, D. S. Breen, Y. N. Akhtar, B. M. Kenwood, J. A. Liao, M. Zhang,D. J. Fazakerley, J. L. Tomsig, T. E. Harris, S. R. Keller, J. D. Chow, K. R. Lynch, M. Chokki,


http://www.sciencetranslationalmedicine.org/cgi/content/full/8/334/334ra54/DC1




http://stm.sciencem

ag.org/D

ownloaded from

J. D. Molkentin, N. Turner, D. E. James, Z. Yan, K. L. Hoehn, Opening of the mitochondrialpermeability transition pore links mitochondrial dysfunction to insulin resistance in skeletalmuscle. Mol. Metab. 3, 124–134 (2014).

22. A. M. van der Bliek, Fussy mitochondria fuse in response to stress. EMBO J. 28, 1533–1534(2009).

23. H. Chen, M. Vermulst, Y. E. Wang, A. Chomyn, T. A. Prolla, J. M. McCaffery, D. C. Chan,Mitochondrial fusion is required for mtDNA stability in skeletal muscle and tolerance ofmtDNA mutations. Cell 141, 280–289 (2010).

24. A. S. Rambold, B. Kostelecky, J. Lippincott-Schwartz, Fuse or die: Shaping mitochondrialfate during starvation. Commun. Integr. Biol. 4, 752–754 (2011).

25. L. C. Gomes, G. Di Benedetto, L. Scorrano, During autophagy mitochondria elongate,are spared from degradation and sustain cell viability. Nat. Cell Biol. 13, 589–598(2011).

26. A. S. Rambold, B. Kostelecky, J. Lippincott-Schwartz, Together we are stronger: Fusionprotects mitochondria from autophagosomal degradation. Autophagy 7, 1568–1569(2011).

27. Z. Y. Tam, J. Gruber, B. Halliwell, R. Gunawan, Mathematical modeling of the role of mito-chondrial fusion and fission in mitochondrial DNA maintenance. PLOS One 8, e76230(2013).

28. M. Vermulst, J. Wanagat, G. C. Kujoth, J. H. Bielas, P. S. Rabinovitch, T. A. Prolla, L. A. Loeb,DNA deletions and clonal mutations drive premature aging in mitochondrial mutatormice. Nat. Genet. 40, 392–394 (2008).

29. L. C. Gomes, L. Scorrano, Mitochondrial elongation during autophagy: A stereotypical re-sponse to survive in difficult times. Autophagy 7, 1251–1253 (2011).

30. C. S. Palmer, L. D. Osellame, D. Stojanovski, M. T. Ryan, The regulation of mitochondrialmorphology: Intricate mechanisms and dynamic machinery. Cell. Signal. 23, 1534–1545(2011).

31. A. Kowald, T. B. L. Kirkwood, Evolution of the mitochondrial fusion–fission cycle and its rolein aging. Proc. Natl. Acad. Sci. U.S.A. 108, 10237–10242 (2011).

32. D. P. Narendra, S. M. Jin, A. Tanaka, D.-F. Suen, C. A. Gautier, J. Shen, M. R. Cookson, R. J. Youle,PINK1 is selectively stabilized on impaired mitochondria to activate Parkin. PLOS Biol. 8,e1000298 (2010).

33. B. G. Drew, V. Ribas, J. A. Le, D. C. Henstridge, J. Phun, Z. Zhou, T. Soleymani, P. Daraei, D. Sitz,L. Vergnes, J. Wanagat, K. Reue, M. A. Febbraio, A. L. Hevener, HSP72 is a mitochondrial stresssensor critical for Parkin action, oxidative metabolism, and insulin sensitivity in skeletalmuscle. Diabetes 63, 1488–1505 (2014).

34. N. Mizushima, B. Levine, Autophagy in mammalian development and differentiation. Nat.Cell Biol. 12, 823–830 (2010).

35. D. J. Klionsky, H. Abeliovich, P. Agostinis, D. K. Agrawal, G. Aliev, D. S. Askew, M. Baba,E. H. Baehrecke, B. A. Bahr, A. Ballabio, B. A. Bamber, D. C. Bassham, E. Bergamini, X. Bi,M. Biard-Piechaczyk, J. S. Blum, D. E. Bredesen, J. L. Brodsky, J. H. Brumell, U. T. Brunk,W. Bursch, N. Camougrand, E. Cebollero, F. Cecconi, Y. Chen, L.-S. Chin, A. Choi, C. T. Chu,J. Chung, R. S. B. Clark, P. G. H. Clarke, S. G. Clarke, C. Clave, J. L. Cleveland, P. Codogno,M. I. Colombo, A. Coto-Montes, J. M. Cregg, A. M. Cuervo, J. Debnath, P. B. Dennis, P. A. Dennis,F. Demarchi, V. Deretic, R. J. Devenish, F. Di Sano, J. F. Dice, C. W. Distelhorst, S. P. Dinesh-Kumar,N. T. Eissa, M. DiFiglia, M. Djavaheri-Mergny, F. C. Dorsey, W. Dröge, M. Dron, W. A. Dunn Jr.,M. Duszenko, Z. Elazar, A. Esclatine, E.-L. Eskelinen, L. Fésüs, K. D. Finley, J. M. Fuentes,J. Fueyo-Margareto, K. Fujisaki, B. Galliot, F.-B. Gao, D. A. Gewirtz, S. B. Gibson, A. Gohla,A. L. Goldberg, R. Gonzalez, C. González-Estévez, S. M. Gorski, R. A. Gottlieb, D. Häussinger,Y.-W. He, K. Heidenreich, J. A. Hill, M. Høyer-Hansen, X. Hu, W.-P. Huang, A. Iwasaki,M. Jäättelä, W. T. Jackson, X. Jiang, S. V. Jin, T. Johansen, J. U. Jung, M. Kadowaki, C. Kang,A. Kelekar, D. H. Kessel, J. A. K. W. Kiel, H. P. Kim, A. Kimchi, T. J. Kinsella, K. Kiselyov, K. Kitamoto,E. Knecht, M. Komatsu, E. Kominami, S. Kondo, A. L. Kovács, G. Kroemer, C.-Y. Kuan, R. Kumar,M. Kundu, J. Landry, M. Laporte, W. Le, H.-Y. Lei, B. Levine, A. P. Lieberman, K.-L. Lim, F.-C. Lin,W. Liou, L. F. Liu, G. Lopez-Berestein, C. López-Otín, B. Lu, K. F. Macleod, W. Malorni, W. Martinet,K. Matsuoka, J. Mautner, A. J. Meijer, A. Meléndez, P. Michels, G. Miotto, W. P. Mistiaen,N. Mizushima, B. Mograbi, M. N. Moore, P. I. Moreira, Y. Moriyasu, T. Motyl, C. Münz, L. O. Murphy,N. I. Naqvi, T. P. Neufeld, I. Nishino, R. A. Nixon, T. Noda, B. Nürnberg, M. Ogawa, N. L. Oleinick,L. J. Olsen, B. Ozpolat, S. Paglin, G. E. Palmer, I. S. Papassideri, M. Parkes, D. H. Perlmutter,G. Perry, M. Piacentini, R. Pinkas-Kramarski, M. Prescott, T. Proikas-Cezanne, N. Raben, A. Rami,F. Reggiori, B. Rohrer, D. C. Rubinsztein, K. M. Ryan, J. Sadoshima, H. Sakagami, Y. Sakai,M. Sandri, C. Sasakawa, M. Sass, C. Schneider, P. O. Seglen, O. Seleverstov, J. Settleman,J. J. Shacka, I. M. Shapiro, A. A. Sibirny, E. C. M. Silva-Zacarin, H.-U. Simon, C. Simone, A. Simonsen,M. A. Smith, K. Spanel-Borowski, V. Srinivas, M. Steeves, H. Stenmark, P. E. Stromhaug,C. S. Subauste, S. Sugimoto, D. Sulzer, T. Suzuki, M. S. Swanson, I. Tabas, F. Takeshita,N. J. Talbot, Z. Tallóczy, K. Tanaka, K. Tanaka, I. Tanida, G. S. Taylor, J. P. Taylor, A. Terman,G. Tettamanti, C. B. Thompson, M. Thumm, A. M. Tolkovsky, S. A. Tooze, R. Truant, L. V. Tumanovska,Y. Uchiyama, T. Ueno, N. L. Uzcátegui, I. J. van der Klei, E. C. Vaquero, T. Vellai, M. W. Vogel,H.-G. Wang, P. Webster, Z. Xi, G. Xiao, J. Yahalom, J.-M. Yang, G. S. Yap, X.-M. Yin, T. Yoshimori,Z. Yue, M. Yuzaki, O. Zabirnyk, X. Zheng, X. Zhu, R. L. Deter, Guidelines for the use and inter-

www.Scien

pretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 4, 151–175(2008).

36. S. Kimura, T. Noda, T. Yoshimori, Dissection of the autophagosome maturation processby a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy 3, 452–460(2007).

37. D. J. Klionsky, F. C. Abdalla, H. Abeliovich, R. T. Abraham, A. Acevedo-Arozena, K. Adeli,L. Agholme, M. Agnello, P. Agostinis, J. A. Aguirre-Ghiso, H. J. Ahn, O. Ait-Mohamed, S. Ait-Si-Ali,T. Akematsu, S. Akira, H. M. Al-Younes, M. A. Al-Zeer, M. L. Albert, R. L. Albin, J. Alegre-Abarrategui,M. F. Aleo, M. Alirezaei, A. Almasan, M. Almonte-Becerril, A. Amano, R. K. Amaravadi, S. Amarnath,A. O. Amer, N. Andrieu-Abadie, V. Anantharam, D. K. Ann, S. Anoopkumar-Dukie, H. Aoki,N. Apostolova, G. Arancia, J. P. Aris, K. Asanuma, N. Y. O. Asare, H. Ashida, V. Askanas,D. S. Askew, P. Auberger, M. Baba, S. K. Backues, E. H. Baehrecke, B. A. Bahr, X.-Y. Bai,Y. Bailly, R. Baiocchi, G. Baldini, W. Balduini, A. Ballabio, B. A. Bamber, E. T. W. Bampton,G. Juhász, C. R. Bartholomew, D. C. Bassham, R. C. Bast Jr., H. Batoko, B.-H. Bay, I. Beau,D. M. Béchet, T. J. Begley, C. Behl, C. Behrends, S. Bekri, B. Bellaire, L. J. Bendall, L. Benetti,L. Berliocchi, H. Bernardi, F. Bernassola, S. Besteiro, I. Bhatia-Kissova, X. Bi, M. Biard-Piechaczyk,J. S. Blum, L. H. Boise, P. Bonaldo, D. L. Boone, B. C. Bornhauser, K. R. Bortoluci, I. Bossis, F. Bost,J.-P. Bourquin, P. Boya, M. Boyer-Guittaut, P. V. Bozhkov, N. R. Brady, C. Brancolini, A. Brech,J. E. Brenman, A. Brennand, E. H. Bresnick, P. Brest, D. Bridges, M. L. Bristol, P. S. Brookes,E. J. Brown, J. H. Brumell, N. Brunetti-Pierri, U. T. Brunk, D. E. Bulman, S. J. Bultman, G. Bultynck,L. F. Burbulla, W. Bursch, J. P. Butchar, W. Buzgariu, S. P. Bydlowski, K. Cadwell, M. Cahová,D. Cai, J. Cai, Q. Cai, B. Calabretta, J. Calvo-Garrido, N. Camougrand, M. Campanella, J. Campos-Salinas,E. Candi, L. Cao, A. B. Caplan, S. R. Carding, S. M. Cardoso, J. S. Carew, C. R. Carlin, V. Carmignac,L. A. M. Carneiro, S. Carra, R. A. Caruso, G. Casari, C. Casas, R. Castino, E. Cebollero, F. Cecconi,J. Celli, H. Chaachouay, H.-J. Chae, C.-Y. Chai, D. C. Chan, E. Y. Chan, R. C.-C. Chang, C.-M. Che,C.-C. Chen, G.-C. Chen, G.-Q. Chen, M. Chen, Q. Chen, S. S.-L. Chen, W. Chen, X. Chen, X. Chen,X. Chen, Y.-G. Chen, Y. Chen, Y. Chen, Y.-J. Chen, Z. Chen, A. Cheng, C. H. K. Cheng, Y. Cheng,H. Cheong, J.-H. Cheong, S. Cherry, R. Chess-Williams, Z. H. Cheung, E. Chevet, H.-L. Chiang,R. Chiarelli, T. Chiba, L.-S. Chin, S.-H. Chiou, F. V. Chisari, C. H. Cho, D.-H. Cho, A. M. K. Choi,D. Choi, K. S. Choi, M. E. Choi, S. Chouaib, D. Choubey, V. Choubey, C. T. Chu, T.-H. Chuang,S.-H. Chueh, T. Chun, Y.-J. Chwae, M.-L. Chye, R. Ciarcia, M. R. Ciriolo, M. J. Clague, R. S. B. Clark,P. G. H. Clarke, R. Clarke, P. Codogno, H. A. Coller, M. I. Colombo, S. Comincini, M. Condello,F. Condorelli, M. R. Cookson, G. H. Coombs, I. Coppens, R. Corbalan, P. Cossart, P. Costelli,S. Costes, A. Coto-Montes, E. Couve, F. P. Coxon, J. M. Cregg, J. L. Crespo, M. J. Cronjé,A. M. Cuervo, J. J. Cullen, M. J. Czaja, M. D’Amelio, A. Darfeuille-Michaud, L. M. Davids,F. E. Davies, M. De Felici, J. F. de Groot, C. A. M. de Haan, L. De Martino, A. De Milito, V. De Tata,J. Debnath, A. Degterev, B. Dehay, L. M. D. Delbridge, F. Demarchi, Y. Z. Deng, J. Dengjel, P. Dent,D. Denton, V. Deretic, S. D. Desai, R. J. Devenish, M. Di Gioacchino, G. Di Paolo, C. Di Pietro,G. Díaz-Araya, I. Díaz-Laviada, M. T. Diaz-Meco, J. Diaz-Nido, I. Dikic, S. P. Dinesh-Kumar,W.-X. Ding, C. W. Distelhorst, A. Diwan, M. Djavaheri-Mergny, S. Dokudovskaya, Z. Dong,F. C. Dorsey, V. Dosenko, J. J. Dowling, S. Doxsey, M. Dreux, M. E. Drew, Q. Duan, M. A. Duchosal,K. E. Duff, I. Dugail, M. Durbeej, M. Duszenko, C. L. Edelstein, A. L. Edinger, G. Egea, L. Eichinger,N. T. Eissa, S. Ekmekcioglu, W. S. El-Deiry, Z. Elazar, M. Elgendy, L. M. Ellerby, K. E. Eng,A.-M. Engelbrecht, S. Engelender, J. Erenpreisa, R. Escalante, A. Esclatine, E.-L. Eskelinen,L. Espert, V. Espina, H. Fan, J. Fan, Q.-W. Fan, Z. Fan, S. Fang, Y. Fang, M. Fanto, A. Fanzani,T. Farkas, J.-C. Farre, M. Faure, M. Fechheimer, C. G. Feng, J. Feng, Q. Feng, Y. Feng, L. Fésüs,R. Feuer, M. E. Figueiredo-Pereira, G. M. Fimia, D. C. Fingar, S. Finkbeiner, T. Finkel, K. D. Finley,F. Fiorito, E. A. Fisher, P. B. Fisher, M. Flajolet, M. L. Florez-McClure, S. Florio, E. A. Fon, F. Fornai,F. Fortunato, R. Fotedar, D. H. Fowler, H. S. Fox, R. Franco, L. B. Frankel, M. Fransen, J. M. Fuentes,J. Fueyo, J. Fujii, K. Fujisaki, E. Fujita, M. Fukuda, R. H. Furukawa, M. Gaestel, P. Gailly, M. Gajewska,B. Galliot, V. Galy, S. Ganesh, B. Ganetzky, I. G. Ganley, F.-B. Gao, G. F. Gao, J. Gao,L. Garcia, G. Garcia-Manero, M. Garcia-Marcos, M. Garmyn, A. L. Gartel, E. Gatti, M. Gautel,T. R. Gawriluk, M. E. Gegg, J. Geng, M. Germain, J. E. Gestwicki, D. A. Gewirtz, S. Ghavami,P. Ghosh, A. M. Giammarioli, A. N. Giatromanolaki, S. B. Gibson, R. W. Gilkerson, M. L. Ginger,H. N. Ginsberg, J. Golab, M. S. Goligorsky, P. Golstein, C. Gomez-Manzano, E. Goncu, C. Gongora,C. D. Gonzalez, R. Gonzalez, C. González-Estévez, R. A. González-Polo, E. Gonzalez-Rey,N. V. Gorbunov, S. Gorski, S. Goruppi, R. A. Gottlieb, D. Gozuacik, G. E. Granato, G. D. Grant,K. N. Green, A. Gregorc, F. Gros, C. Grose, T. W. Grunt, P. Gual, J.-L. Guan, K.-L. Guan,S. M. Guichard, A. S. Gukovskaya, I. Gukovsky, J. Gunst, Å. B. Gustafsson, A. J. Halayko, A. N. Hale,S. K. Halonen, M. Hamasaki, F. Han, T. Han, M. K. Hancock, M. Hansen, H. Harada, M. Harada,S. E. Hardt, J. W. Harper, A. L. Harris, J. Harris, S. D. Harris, M. Hashimoto, J. A. Haspel, S. Hayashi,L. A. Hazelhurst, C. He, Y.-W. He, M.-J. Hébert, K. A. Heidenreich, M. H. Helfrich, G. V. Helgason,E. P. Henske, B. Herman, P. K. Herman, C. Hetz, S. Hilfiker, J. A. Hill, L. J. Hocking, P. Hofman,T. G. Hofmann, J. Höhfeld, T. L. Holyoake, M.-H. Hong, D. A. Hood, G. S. Hotamisligil, E. J. Houwerzijl,M. Høyer-Hansen, B. Hu, C. A. Hu, H.-M. Hu, Y. Hua, C. Huang, J. Huang, S. Huang, W.-P. Huang,T. B. Huber, W.-K. Huh, T.-H. Hung, T. R. Hupp, G. M. Hur, J. B. Hurley, S. N. A. Hussain, P. J. Hussey,J. J. Hwang, S. Hwang, A. Ichihara, S. Ilkhanizadeh, K. Inoki, T. Into, V. Iovane, J. L. Iovanna,N. Y. Ip, Y. Isaka, H. Ishida, C. Isidoro, K. Isobe, A. Iwasaki, M. Izquierdo, Y. Izumi, P. M. Jaakkola,M. Jäättelä, G. R. Jackson, W. T. Jackson, B. Janji, M. Jendrach, J.-H. Jeon, E.-B. Jeung,H. Jiang, H. Jiang, J. X. Jiang, M. Jiang, Q. Jiang, X. Jiang, X. Jiang, A. Jiménez, M. Jin,





http://stm.sciencem

ag.org/D

ownloaded from

S. V. Jin, C. O. Joe, T. Johansen, D. E. Johnson, G. V. W. Johnson, N. L. Jones, B. Joseph, S. K. Joseph,A. M. Joubert, G. Juhász, L. Juillerat-Jeanneret, C. H. Jung, Y.-K. Jung, K. Kaarniranta, A. Kaasik,T. Kabuta, M. Kadowaki, K. Kågedal, Y. Kamada, V. O. Kaminskyy, H. H. Kampinga, H. Kanamori,C. Kang, K. B. Kang, K. I. Kang, R. Kang, Y.-A. Kang, T. Kanki, T.-D. Kanneganti, H. Kanno,A. G. Kanthasamy, A. Kanthasamy, V. Karantza, G. P. Kaushal, S. Kaushik, Y. Kawazoe,P.-Y. Ke, J. H. Kehrl, A. Kelekar, C. Kerkhoff, D. H. Kessel, H. Khalil, J. A. K. W. Kiel, A. A. Kiger,A. Kihara, D. R. Kim, D.-H. Kim, D.-H. Kim, E.-K. Kim, H.-R. Kim, J.-S. Kim, J. H. Kim, J. C. Kim,J. K. Kim, P. K. Kim, S. W. Kim, Y.-S. Kim, Y. Kim, A. Kimchi, A. C. Kimmelman, J. S. King, T. J. Kinsella,V. Kirkin, L. A. Kirshenbaum, K. Kitamoto, K. Kitazato, L. Klein, W. T. Klimecki, J. Klucken, E. Knecht,B. C. B. Ko, J. C. Koch, H. Koga, J.-Y. Koh, Y. H. Koh, M. Koike, M. Komatsu, E. Kominami, H. J. Kong,W.-J. Kong, V. I. Korolchuk, Y. Kotake, M. I. Koukourakis, J. B. Kouri Flores, A. L. Kovács, C. Kraft,D. Krainc, H. Krämer, C. Kretz-Remy, A. M. Krichevsky, G. Kroemer, R. Krüger, O. Krut, N. T. Ktistakis,C.-Y. Kuan, R. Kucharczyk, A. Kumar, R. Kumar, S. Kumar, M. Kundu, H.-J. Kung, T. Kurz, H. J. Kwon,A. R. La Spada, F. Lafont, T. Lamark, J. Landry, J. D. Lane, P. Lapaquette, J. F. Laporte, L. László,S. Lavandero, J. N. Lavoie, R. Layfield, P. A. Lazo, W. Le, L. Le Cam, D. J. Ledbetter, A. J. X. Lee,B.-W. Lee, G. M. Lee, J. Lee, J. Lee, M. Lee, M.-S. Lee, S. H. Lee, C. Leeuwenburgh, P. Legembre,R. Legouis, M. Lehmann, H.-Y. Lei, Q.-Y. Lei, D. A. Leib, J. Leiro, J. J. Lemasters, A. Lemoine,M. S. Lesniak, D. Lev, V. V. Levenson, B. Levine, E. Levy, F. Li, J.-L. Li, L. Li, S. Li, W. Li, X.-J. Li,Y.-B. Li, Y.-P. Li, C. Liang, Q. Liang, Y.-F. Liao, P. P. Liberski, A. Lieberman, H. J. Lim, K.-L. Lim,K. Lim, C.-F. Lin, F.-C. Lin, J. Lin, J. D. Lin, K. Lin, W.-W. Lin, W.-C. Lin, Y.-L. Lin, R. Linden, P. Lingor,J. Lippincott-Schwartz, M. P. Lisanti, P. B. Liton, B. Liu, C.-F. Liu, K. Liu, L. Liu, Q. A. Liu, W. Liu,Y.-C. Liu, Y. Liu, R. A. Lockshin, C.-N. Lok, S. Lonial, B. Loos, G. Lopez-Berestein, C. López-Otín,L. Lossi, M. T. Lotze, P. Low, B. Lu, Z. Lu, F. Luciano, N. W. Lukacs, A. H. Lund, M. A. Lynch-Day,Y. Ma, F. Macian, J. P. MacKeigan, K. F. Macleod, F. Madeo, L. Maiuri, M. C. Maiuri, D. Malagoli,M. C. V. Malicdan, W. Malorni, N. Man, E.-M. Mandelkow, S. Manon, I. Manov, K. Mao, X. Mao,Z. Mao, P. Marambaud, D. Marazziti, Y. L. Marcel, K. Marchbank, P. Marchetti, S. J. Marciniak,M. Marcondes, M. Mardi, G. Marfe, G. Mariño, M. Markaki, M. R. Marten, S. J. Martin, C. Martinand-Mari,W. Martinet, M. Martinez-Vicente, M. Masini, P. Matarrese, S. Matsuo, R. Matteoni, A. Mayer,N. M. Mazure, D. J. McConkey, M. J. McConnell, C. McDermott, C. McDonald, G. M. McInerney,S. L. McKenna, B. McLaughlin, P. J. McLean, C. R. McMaster, G. A. McQuibban, A. J. Meijer,M. H. Meisler, A. Meléndez, T. J. Melia, G. Melino, M. A. Mena, J. A. Menendez, R. F. S. Menna-Barreto,M. B. Menon, F. M. Menzies, C. A. Mercer, A. Merighi, D. E. Merry, S. Meschini, C. G. Meyer, T. F. Meyer,C.-Y. Miao, J.-Y. Miao, P. A. M. Michels, C. Michiels, D. Mijaljica, A. Milojkovic, S. Minucci,C. Miracco, C. K. Miranti, I. Mitroulis, K. Miyazawa, N. Mizushima, B. Mograbi, S. Mohseni,X. Molero, B. Mollereau, F. Mollinedo, T. Momoi, I. Monastyrska, M. M. Monick, M. J. Monteiro,M. N. Moore, R. Mora, K. Moreau, P. I. Moreira, Y. Moriyasu, J. Moscat, S. Mostowy, J. C. Mottram,T. Motyl, C. E.-H. Moussa, S. Müller, S. Muller, K. Münger, C. Münz, L. O. Murphy, M. E. Murphy,A. Musarò, I. Mysorekar, E. Nagata, K. Nagata, A. Nahimana, U. Nair, T. Nakagawa, K. Nakahira,H. Nakano, H. Nakatogawa, M. Nanjundan, N. I. Naqvi, D. P. Narendra, M. Narita, M. Navarro,S. T. Nawrocki, T. Y. Nazarko, A. Nemchenko, M. G. Netea, T. P. Neufeld, P. A. Ney, I. P. Nezis,H. P. Nguyen, D. Nie, I. Nishino, C. Nislow, R. A. Nixon, T. Noda, A. A. Noegel, A. Nogalska,S. Noguchi, L. Notterpek, I. Novak, T. Nozaki, N. Nukina, T. Nürnberger, B. Nyfeler, K. Obara,T. D. Oberley, S. Oddo, M. Ogawa, T. Ohashi, K. Okamoto, N. L. Oleinick, F. J. Oliver, L. J. Olsen,S. Olsson, O. Opota, T. F. Osborne, G. K. Ostrander, K. Otsu, J.-J. Ou, M. Ouimet, M. Overholtzer,B. Ozpolat, P. Paganetti, U. Pagnini, N. Pallet, G. E. Palmer, C. Palumbo, T. Pan, T. Panaretakis,U. B. Pandey, Z. Papackova, I. Papassideri, I. Paris, J. Park, O. K. Park, J. B. Parys, K. R. Parzych,S. Patschan, C. Patterson, S. Pattingre, J. M. Pawelek, J. Peng, D. H. Perlmutter, I. Perrotta, G. Perry,S. Pervaiz, M. Peter, G. J. Peters, M. Petersen, G. Petrovski, J. M. Phang, M. Piacentini, P. Pierre,V. Pierrefite-Carle, G. Pierron, R. Pinkas-Kramarski, A. Piras, N. Piri, L. C. Platanias, S. Pöggeler,M. Poirot, A. Poletti, C. Poüs, M. Pozuelo-Rubio, M. Prætorius-Ibba, A. Prasad, M. Prescott,M. Priault, N. Produit-Zengaffinen, A. Progulske-Fox, T. Proikas-Cezanne, S. Przedborski,K. Przyklenk, R. Puertollano, J. Puyal, S.-B. Qian, L. Qin, Z.-H. Qin, S. E. Quaggin, N. Raben,H. Rabinowich, S. W. Rabkin, I. Rahman, A. Rami, G. Ramm, G. Randall, F. Randow, V. A. Rao,J. C. Rathmell, B. Ravikumar, S. K. Ray, B. H. Reed, J. C. Reed, F. Reggiori, A. Régnier-Vigouroux,A. S. Reichert, J. J. Reiners Jr., R. J. Reiter, J. Ren, J. L. Revuelta, C. J. Rhodes, K. Ritis, E. Rizzo,J. Robbins, M. Roberge, H. Roca, M. C. Roccheri, S. Rocchi, H. P. Rodemann, S. Rodríguez de Córdoba,B. Rohrer, I. B. Roninson, K. Rosen, M. M. Rost-Roszkowska, M. Rouis, K. M. A. Rouschop,F. Rovetta, B. P. Rubin, D. C. Rubinsztein, K. Ruckdeschel, E. B. Rucker III, A. Rudich, E. Rudolf,N. Ruiz-Opazo, R. Russo, T. E. Rusten, K. M. Ryan, S. W. Ryter, D. M. Sabatini, J. Sadoshima,T. Saha, T. Saitoh, H. Sakagami, Y. Sakai, G. H. Salekdeh, P. Salomoni, P. M. Salvaterra, G. Salvesen,R. Salvioli, A. M. J. Sanchez, J. A. Sánchez-Alcázar, R. Sánchez-Prieto, M. Sandri, U. Sankar,P. Sansanwal, L. Santambrogio, S. Saran, S. Sarkar, M. Sarwal, C. Sasakawa, A. Sasnauskiene,M. Sass, K. Sato, M. Sato, A. H. V. Schapira, M. Scharl, H. M. Schätzl, W. Scheper, S. Schiaffino,C. Schneider, M. E. Schneider, R. Schneider-Stock, P. V. Schoenlein, D. F. Schorderet, C. Schüller,G. K. Schwartz, L. Scorrano, L. Sealy, P. O. Seglen, J. Segura-Aguilar, I. Seiliez, O. Seleverstov,C. Sell, J. B. Seo, D. Separovic, V. Setaluri, T. Setoguchi, C. Settembre, J. J. Shacka, M. Shanmugam,I. M. Shapiro, E. Shaulian, R. J. Shaw, J. H. Shelhamer, H.-M. Shen, W.-C. Shen, Z.-H. Sheng, Y. Shi,K. Shibuya, Y. Shidoji, J.-J. Shieh, C.-M. Shih, Y. Shimada, S. Shimizu, T. Shintani, O. S. Shirihai,G. C. Shore, A. A. Sibirny, S. B. Sidhu, B. Sikorska, E. C. M. Silva-Zacarin, A. Simmons, A. K. Simon,H.-U. Simon, C. Simone, A. Simonsen, D. A. Sinclair, R. Singh, D. Sinha, F. A. Sinicrope, A. Sirko,

www.Scien

P. M. Siu, E. Sivridis, V. Skop, V. P. Skulachev, R. S. Slack, S. S. Smaili, D. R. Smith, M. S. Soengas,T. Soldati, X. Song, A. K. Sood, T. W. Soong, F. Sotgia, S. A. Spector, C. D. Spies, W. Springer,S. M. Srinivasula, L. Stefanis, J. S. Steffan, R. Stendel, H. Stenmark, A. Stephanou, S. T. Stern,C. Sternberg, B. Stork, P. Strålfors, C. S. Subauste, X. Sui, D. Sulzer, J. Sun, S.-Y. Sun, Z.-J. Sun,J. J. Y. Sung, K. Suzuki, T. Suzuki, M. S. Swanson, C. Swanton, S. T. Sweeney, L.-K. Sy, G. Szabadkai,I. Tabas, H. Taegtmeyer, M. Tafani, K. Takács-Vellai, Y. Takano, K. Takegawa, G. Takemura,F. Takeshita, N. J. Talbot, K. S. W. Tan, K. Tanaka, D. Tang, D. Tang, I. Tanida, B. A. Tannous,N. Tavernarakis, G. S. Taylor, G. A. Taylor, J. P. Taylor, L. S. Terada, A. Terman, G. Tettamanti,K. Thevissen, C. B. Thompson, A. Thorburn, M. Thumm, F. Tian, Y. Tian, G. Tocchini-Valentini,A. M. Tolkovsky, Y. Tomino, L. Tönges, S. A. Tooze, C. Tournier, J. Tower, R. Towns, V. Trajkovic,L. H. Travassos, T.-F. Tsai, M. P. Tschan, T. Tsubata, A. Tsung, B. Turk, L. S. Turner, S. C. Tyagi,Y. Uchiyama, T. Ueno, M. Umekawa, R. Umemiya-Shirafuji, V. K. Unni, M. I. Vaccaro, E. M. Valente,G. Van den Berghe, I. J. van der Klei, W. G. van Doorn, L. F. van Dyk, M. van Egmond,L. A. van Grunsven, P. Vandenabeele, W. P. Vandenberghe, I. Vanhorebeek, E. C. Vaquero,G. Velasco, T. Vellai, J. M. Vicencio, R. D. Vierstra, M. Vila, C. Vindis, G. Viola, M. T. Viscomi,O. V. Voitsekhovskaja, C. von Haefen, M. Votruba, K. Wada, R. Wade-Martins, C. L. Walker,C. M. Walsh, J. Walter, X.-B. Wan, A. Wang, C. Wang, D. Wang, F. Wang, F. Wang, G. Wang,H. Wang, H.-G. Wang, H.-D. Wang, J. Wang, K. Wang, M. Wang, R. C. Wang, X. Wang, X. J. Wang,Y.-J. Wang, Y. Wang, Z.-B. Wang, Z. C. Wang, Z. Wang, D. G. Wansink, D. M. Ward, H. Watada,S. L. Waters, P. Webster, L. Wei, C. C. Weihl, W. A. Weiss, S. M. Welford, L.-P. Wen, C. A. Whitehouse,J. L. Whitton, A. J. Whitworth, T. Wileman, J. W. Wiley, S. Wilkinson, D. Willbold, R. L. Williams,P. R. Williamson, B. G. Wouters, C. Wu, D.-C. Wu, W. K. K. Wu, A. Wyttenbach, R. J. Xavier, Z. Xi,P. Xia, G. Xiao, Z. Xie, Z. Xie, D. Xu, J. Xu, L. Xu, X. Xu, A. Yamamoto, A. Yamamoto, S. Yamashina,M. Yamashita, X. Yan, M. Yanagida, D.-S. Yang, E. Yang, J.-M. Yang, S. Y. Yang, W. Yang, W. Y. Yang,Z. Yang, M.-C. Yao, T.-P. Yao, B. Yeganeh, W.-L. Yen, J.-J. Yin, X.-M. Yin, O.-J. Yoo, G. Yoon, S.-Y. Yoon,T. Yorimitsu, Y. Yoshikawa, T. Yoshimori, K. Yoshimoto, H. J. You, R. J. Youle, A. Younes, L. Yu, L. Yu,S.-W. Yu, W. H. Yu, Z.-M. Yuan, Z. Yue, C.-H. Yun, M. Yuzaki, O. Zabirnyk, E. Silva-Zacarin, D. Zacks,E. Zacksenhaus, N. Zaffaroni, Z. Zakeri, H. J. Zeh III, S. O. Zeitlin, H. Zhang, H.-L. Zhang, J. Zhang,J.-P. Zhang, L. Zhang, L. Zhang, M.-Y. Zhang, X. D. Zhang, M. Zhao, Y.-F. Zhao, Y. Zhao, Z. J. Zhao,X. Zheng, B. Zhivotovsky, Q. Zhong, C.-Z. Zhou, C. Zhu, W.-G. Zhu, X.-F. Zhu, X. Zhu, Y. Zhu,T. Zoladek, W.-X. Zong, A. Zorzano, J. Zschocke, B. Zuckerbraun, Guidelines for the use andinterpretation of assays for monitoring autophagy. Autophagy 8, 445–544 (2012).

38. S. J. Cherra III, S. M. Kulich, G. Uechi, M. Balasubramani, J. Mountzouris, B. W. Day, C. T. Chu,Regulation of the autophagy protein LC3 by phosphorylation. J. Cell Biol. 190, 533–539 (2010).

39. H. Peiris, D. Dubach, C. F. Jessup, P. Unterweger, R. Raghupathi, H. Muyderman, M. P. Zanin,K. Mackenzie, M. A. Pritchard, D. J. Keating, RCAN1 regulates mitochondrial function andincreases susceptibility to oxidative stress in mammalian cells. Oxid. Med. Cell. Longev.2014, 520316 (2014).

40. G. Ermak, C. D. Harris, K. J. A. Davies, The DSCR1 (Adapt78) isoform 1 protein calcipressin 1 in-hibits calcineurin and protects against acute calcium-mediated stress damage, including tran-sient oxidative stress. FASEB J. 16, 814–824 (2002).

41. B. Rothermel, R. B. Vega, J. Yang, H. Wu, R. Bassel-Duby, R. S. Williams, A protein encodedwithin the Down syndrome critical region is enriched in striated muscles and inhibits cal-cineurin signaling. J. Biol. Chem. 275, 8719–8725 (2000).

42. S. S. Kim, Y. Oh, K. C. Chung, S. R. Seo, Protein kinase A phosphorylates Down syndromecritical region 1 (RCAN1). Biochem. Biophys. Res. Commun. 418, 657–661 (2012).

43. K. E. Davis, M. D. Neinast, K. Sun, W. M. Skiles, J. D. Bills, J. A. Zehr, D. Zeve, L. D. Hahner,D. W. Cox, L. M. Gent, Y. Xu, Z. V. Wang, S. A. Khan, D. J. Clegg, The sexually dimorphicrole of adipose and adipocyte estrogen receptors in modulating adipose tissue expansion,inflammation, and fibrosis. Mol. Metab. 2, 227–242 (2013).

44. Y. Xu, T. P. Nedungadi, L. Zhu, N. Sobhani, B. G. Irani, K. E. Davis, X. Zhang, F. Zou, L. M. Gent,L. D. Hahner, S. A. Khan, C. F. Elias, J. K. Elmquist, D. J. Clegg, Distinct hypothalamic neurons mediateestrogenic effects on energy homeostasis and reproduction. Cell Metab. 14, 453–465 (2011).

45. G. Kilic, A. I. Alvarez-Mercado, B. Zarrouki, D. Opland, C. W. Liew, L. C. Alonso, M. G. Myers Jr.,J.-C. Jonas, V. Poitout, R. N. Kulkarni, F. Mauvais-Jarvis, The islet estrogen receptor-a is in-duced by hyperglycemia and protects against oxidative stress-induced insulin-deficient di-abetes. PLOS One 9, e87941 (2014).

46. L. Zhu, W. C. Brown, Q. Cai, A. Krust, P. Chambon, O. P. McGuinness, J. M. Stafford, Estrogentreatment after ovariectomy protects against fatty liver and may improve pathway-selectiveinsulin resistance. Diabetes 62, 424–434 (2013).

47. S. D. Martin, S. L. McGee, The role of mitochondria in the aetiology of insulin resistance andtype 2 diabetes. Biochim. Biophys. Acta 1840, 1303–1312 (2014).

48. J. Kim, Y. Wei, J. R. Sowers, Role of mitochondrial dysfunction in insulin resistance. Circ. Res.102, 401–414 (2008).

49. J.-P. Leduc-Gaudet, M. Picard, F. St-Jean Pelletier, N. Sgarioto, M.-J. Auger, J. Vallée, R. Robitaille,D. H. St-Pierre, G. Gouspillou, Mitochondrial morphology is altered in atrophied skeletal muscleof aged mice. Oncotarget 6, 17923–17937 (2015).

50. A. S. Rambold, B. Kostelecky, N. Elia, J. Lippincott-Schwartz, Tubular network formationprotects mitochondria from autophagosomal degradation during nutrient starvation. Proc.Natl. Acad. Sci. U.S.A. 108, 10190–10195 (2011).





http://stm.sciencem

ag.org/D

ownloaded from

51. J. Nunnari, A. Suomalainen, Mitochondria: In sickness and in health. Cell 148, 1145–1159(2012).

52. D. C. Wallace, Mitochondrial DNA mutations in diseases of energy metabolism. J. Bioenerg.Biomembr. 26, 241–250 (1994).

53. A. C. Poole, R. E. Thomas, L. A. Andrews, H. M. McBride, A. J. Whitworth, L. J. Pallanck, ThePINK1/Parkin pathway regulates mitochondrial morphology. Proc. Natl. Acad. Sci. U.S.A.105, 1638–1643 (2008).

54. C. A. Gautier, T. Kitada, J. Shen, Loss of PINK1 causes mitochondrial functional defects andincreased sensitivity to oxidative stress. Proc. Natl. Acad. Sci. U.S.A. 105, 11364–11369 (2008).

55. C. Scheele, A. R. Nielsen, T. B. Walden, D. A. Sewell, C. P. Fischer, R. J. Brogan, N. Petrovic,O. Larsson, P. A. Tesch, K. Wennmalm, D. S. Hutchinson, B. Cannon, C. Wahlestedt, B. K. Pedersen,J. A. Timmons, Altered regulation of the PINK1 locus: A link between type 2 diabetes andneurodegeneration? FASEB J. 21, 3653–3665 (2007).

56. S. Hoppins, L. Lackner, J. Nunnari, The machines that divide and fuse mitochondria. Annu. Rev.Biochem. 76, 751–780 (2007).

57. A. Santel, S. Frank, Shaping mitochondria: The complex posttranslational regulation of themitochondrial fission protein DRP1. IUBMB Life 60, 448–455 (2008).

58. E. Smirnova, D.-L. Shurland, S. N. Ryazantsev, A. M. van der Bliek, A human dynamin-related protein controls the distribution of mitochondria. J. Cell Biol. 143, 351–358 (1998).

59. W. Qian, S. Choi, G. A. Gibson, S. C. Watkins, C. J. Bakkenist, B. Van Houten, Mitochondrialhyperfusion induced by loss of the fission protein Drp1 causes ATM-dependent G2/Marrest and aneuploidy through DNA replication stress. J. Cell Sci. 125, 5745–5757(2012).

60. M. J. Drummond, O. Addison, L. Brunker, P. N. Hopkins, D. A. McClain, P. C. LaStayo, R. L. Marcus,Downregulation of E3 ubiquitin ligases and mitophagy-related genes in skeletal muscle ofphysically inactive, frail older women: A cross-sectional comparison. J. Gerontol. A Biol. Sci.Med. Sci. 69, 1040–1048 (2014).

61. K. Elgass, J. Pakay, M. T. Ryan, C. S. Palmer, Recent advances into the understanding ofmitochondrial fission. Biochim. Biophys. Acta 1833, 150–161 (2013).

62. G. M. Cereghetti, A. Stangherlin, O. Martins de Brito, C. R. Chang, C. Blackstone, P. Bernardi,L. Scorrano, Dephosphorylation by calcineurin regulates translocation of Drp1 to mitochon-dria. Proc. Natl. Acad. Sci. U.S.A. 105, 15803–15808 (2008).

63. S. Nielsen, C. Scheele, C. Yfanti, T. Åkerström, A. R. Nielsen, B. K. Pedersen, M. Laye, Musclespecific microRNAs are regulated by endurance exercise in human skeletal muscle. J. Physiol.588, 4029–4037 (2010).

64. S. Nielsen, T. Hvid, M. Kelly, B. Lindegaard, C. Dethlefsen, K. Winding, N. Mathur, C. Scheele,B. K. Pedersen, M. J. Laye, Muscle specific miRNAs are induced by testosterone and indepen-dently upregulated by age. Front. Physiol. 4, 394 (2014).

65. B. W. Parks, T. Sallam, M. Mehrabian, N. Psychogios, S. T. Hui, F. Norheim, L. W. Castellani,C. D. Rau, C. Pan, J. Phun, Z. Zhou, W.-P. Yang, I. Neuhaus, P. S. Gargalovic, T. G. Kirchgessner,M. Graham, R. Lee, P. Tontonoz, R. E. Gerszten, A. L. Hevener, A. J. Lusis, Genetic architectureof insulin resistance in the mouse. Cell Metab. 21, 334–346 (2015).

66. S. C. Hewitt, G. E. Kissling, K. E. Fieselman, F. L. Jayes, K. E. Gerrish, K. S. Korach, Biologicaland biochemical consequences of global deletion of exon 3 from the ERa gene. FASEB J. 24,4660–4667 (2010).

67. D. L. Ahlbory-Dieker, B. D. Stride, G. Leder, J. Schkoldow, S. Trölenberg, H. Seidel, C. Otto,A. Sommer, M. G. Parker, G. Schütz, T. M. Wintermantel, DNA binding by estrogen receptor-a isessential for the transcriptional response to estrogen in the liver and the uterus.Mol. Endocrinol.23, 1544–1555 (2009).

68. P.-O. Aåstrand, Human physical fitness with special reference to sex and age. Physiol. Rev.36, 307–335 (1956).

69. A. L. Hevener, W. He, Y. Barak, J. Le, G. Bandyopadhyay, P. Olson, J. Wilkes, R. M. Evans,J. Olefsky, Muscle-specific Pparg deletion causes insulin resistance. Nat. Med. 9, 1491–1497(2003).

70. A. L. Hevener, J. M. Olefsky, D. Reichart, M. T. A. Nguyen, G. Bandyopadyhay, H.-Y. Leung,M. J. Watt, C. Benner, M. A. Febbraio, A.-K. Nguyen, B. Folian, S. Subramaniam, F. J. Gonzalez,C. K. Glass, M. Ricote, Macrophage PPARg is required for normal skeletal muscle and hepaticinsulin sensitivity and full antidiabetic effects of thiazolidinediones. J. Clin. Invest. 117,1658–1669 (2007).

71. R. Steele, Influences of glucose loading and of injected insulin on hepatic glucose output.Ann. N.Y. Acad. Sci. 82, 420–430 (1959).

72. K. N. Frayn, P. F. Maycock, Skeletal muscle triacylglycerol in the rat: Methods forsampling and measurement, and studies of biological variability. J. Lipid Res. 21,139–144 (1980).

73. J. Preiss, C. R. Loomis, W. R. Bishop, R. Stein, J. E. Niedel, R. M. Bell, Quantitative measurement ofsn-1,2-diacylglycerols present in platelets, hepatocytes, and ras- and sis-transformed normalrat kidney cells. J. Biol. Chem. 261, 8597–8600 (1986).

74. J. B. Allred, D. G. Guy, Determination of coenzyme A and acetyl CoA in tissue extracts. Anal.Biochem. 29, 293–299 (1969).

75. M. T. A. Nguyen, S. Favelyukis, A.-K. Nguyen, D. Reichart, P. A. Scott, A. Jenn, R. Liu-Bryan,C. K. Glass, J. G. Neels, J. M. Olefsky, A subpopulation of macrophages infiltrates hyper-

www.Scien

trophic adipose tissue and is activated by free fatty acids via Toll-like receptors 2 and 4and JNK-dependent pathways. J. Biol. Chem. 282, 35279–35292 (2007).

76. X. Yang, M. Downes, R. T. Yu, A. L. Bookout, W. He, M. Straume, D. J. Mangelsdorf, R. M. Evans,Nuclear receptor expression links the circadian clock to metabolism. Cell 126, 801–810 (2006).

77. M. Vermulst, J. H. Bielas, L. A. Loeb, Quantification of random mutations in the mitochon-drial genome. Methods 46, 263–268 (2008).

78. C. R. Bruce, A. J. Hoy, N. Turner, M. J. Watt, T. L. Allen, K. Carpenter, G. J. Cooney, M. A. Febbraio,E. W. Kraegen, Overexpression of carnitine palmitoyltransferase-1 in skeletal muscle is suffi-cient to enhance fatty acid oxidation and improve high-fat diet-induced insulin resistance.Diabetes 58, 550–558 (2009).

79. M. B. Chen, A. J. McAinch, S. L. Macaulay, L. A. Castelli, P. E. O’Brien, J. B. Dixon, D. Cameron-Smith,B. E. Kemp, G. R. Steinberg, Impaired activation of AMP-kinase and fatty acid oxidation byglobular adiponectin in cultured human skeletal muscle of obese type 2 diabetics. J. Clin.Endocrinol. Metab. 90, 3665–3672 (2005).

80. C. E. McCurdy, G. D. Cartee, Akt2 is essential for the full effect of calorie restriction oninsulin-stimulated glucose uptake in skeletal muscle. Diabetes 54, 1349–1356 (2005).

81. K. Vercauteren, R. A. Pasko, N. Gleyzer, V. M. Marino, R. C. Scarpulla, PGC-1-related coacti-vator: Immediate early expression and characterization of a CREB/NRF-1 binding domainassociated with cytochrome c promoter occupancy and respiratory growth. Mol. Cell. Biol.26, 7409–7419 (2006).

82. J. C. Bopassa, M. Eghbali, L. Toro, E. Stefani, A novel estrogen receptor GPER inhibits mitochon-dria permeability transition pore opening and protects the heart against ischemia-reperfusioninjury. Am. J. Physiol. Heart Circ. Physiol. 298, H16–H23 (2010).

83. L. Gomez, M. Paillard, H. Thibault, G. Derumeaux, M. Ovize, Inhibition of GSK3b by post-conditioning is required to prevent opening of the mitochondrial permeability transitionpore during reperfusion. Circulation 117, 2761–2768 (2008).

84. R. I. Close, The relations between sarcomere length and characteristics of isometric twitchcontractions of frog sartorius muscle. J. Physiol. 220, 745–762 (1972).

85. R. H. J. Bandsma, J.-P. Rake, G. Visser, R. A. Neese, M. K. Hellerstein, W. van Duyvenvoorde,H. M. G. Princen, F. Stellaard, G. P. A. Smit, F. Kuipers, Increased lipogenesis and resistanceof lipoproteins to oxidative modification in two patients with glycogen storage diseasetype 1a. J. Pediatr. 140, 256–260 (2002).

86. E. G. Zimmerman, D. R. Akins, J. V. Planz, M. J. Schurr, A rapid procedure for isolatingmitochondrial DNA. Gene Anal. Tech. 5, 102–104 (1988).

Acknowledgments: We thank J. Olefsky and H. Cohen from the University of California, SanDiego/UCLA Diabetes Research Center for their continued support of our work; D. Chan, M. Guo,D. Shackelford, and A. van der Bliek for their helpful discussions regarding mitochondrial fusion,fission, and mitophagy; C. Pan and C. Rau for assistance with bioinformatics; D. Henstridge forassistance with the isolation of primary muscle cells; S. Kohan from the UCLA Brain ResearchInstitute for assistance with electron microscopy analyses; R. Boyadjian for assistance withmeasuring circulating factors; W. Foster for assistance with the PKA activity assay; K. Wroblewskifor assistance with metabolic chamber analyses; and L. Nielsen for assistance with qRT-PCRanalyses on human muscle. Funding: These studies were supported in part by research grantsfrom the NIH [DK060484, DK073227, and Nuclear Receptor Signaling Atlas (NURSA) Data SourceProject grant DK097748], UCLA Department of Medicine, and UCLA Iris Cantor Women’s HealthCenter–UCLA Clinical and Translational Science Institute (UL1TR000124) to A.L.H. and Z.Z. V.R.was supported by the Spanish Ministry of Health, and B.G.D. was supported by the AustralianNational Health and Medical Research Council and the UCLA Jonsson Comprehensive CancerCenter. During the study period, the Centre of Inflammation and Metabolism was supported bya grant from the Danish National Research Foundation (DNRF55) to B.K.P. A.J.L. is supported byNIH (grants HL28481 and HL30568), and B.W.P. is supported by NIH R00 grant HL123021. TheCentre for Physical Activity Research is supported by a grant from TrygFonden. A.J.L. issupported by NIH grants HL28481 and HL30568, and L.V. and K.R. are supported in part byNIH grant HL28481. The Seahorse XF24 instrument was acquired by a shared instrument grantfrom the NIHNational Center for Research Resources (S10RR026744). P.T. is an investigator of theHoward Hughes Medical Institute and is supported by NIH (DK063491). Author contributions:These authors were involved in data collection and analysis: V.R. (performed in vitro insulinaction, mitochondrial real-time respirometry, immunoblotting qPCR, RMC assays, and confocalmicroscopy studies), B.G.D. (cloning and fatty acid oxidation and insulin action assays), Z.Z. (studiesof Drp1 action and mitochondrial isolations, immunoblotting, and qPCR), J.P. (performed geno-typing, animal husbandry, immunoblotting, and qPCR), N.Y.K. (performed animal husbandry,genotyping, and immunoblotting), T.S. (performed animal husbandry and qPCR), P.D. (performedanimal husbandry, genotyping, plasma analyses, and qPCR), K.W. and J.W. (performed musclehistological analyses and assisted with the RMC assays), T.Q.d.A.V. (designed and synthesized ad-enovirus), A.H.F. and S.B. (performed flow cytometry analyses of autophagy), T.L. (performedassessment of deuterium labeling), J.P.W. (performed mitochondrial proteomics analyses),S.W.B. (performed metabolic chamber analyses), B.W.P. (performed all studies associated withUCLA HMDP), L.V. (performed mitochondrial real-time respirometry), H.S. and J.C.B. (performedmitochondrial calcium homeostasis mPTP analyses), M.J.W. (performed bioactive lipid analysesinmuscle), S.S. (muscle function testing), T.A. andM.K. (human study design and data collection),




and A.L.H. (performed all in vivo studies in mice). These authors directed the research: A.L.H.(orchestrated the entire project and directed in vivo and ex vivo studies as well as performeddata integration), E.S. and L.T. (directed mitochondrial calcium handling studies), P.T. (directedstudies of indirect calorimetry and movement), K.R. (directed the real-time respirometry stu-dies), B.K.P. (directed all human studies), and A.J.L. (directed studies associated with UCLAHMDP and gene arrays performed on f/f and MERKO mouse muscle). These authorscontributed reagents, samples, or instrumentation: S.C.H. and K.S.K. (provided the ERa floxedand ERa DBD mutant mouse lines), P.T. (provided metabolic chambers), S.S. (provided instru-mentation and reagents to assess muscle function), L.T. and E.S. (provided instrumentationand reagents to assess calcium fluxes in isolated mitochondria), K.R. (provided Seahorse Bio-sciences XF24 instrument), B.K.P. (provided reagents and tissues for human muscle analyses),A.J.L. (provided data and muscle samples from the HMDP studies), C.R. (provided mass spec-troscopy instrumentation and reagents for the deuterium studies), and J.P.W. (provided massspectroscopy instrumentation and reagents for the mitochondrial proteomics analyses).These authors contributed to drafting of the original manuscript: V.R., B.G.D., Z.Z., and A.L.H.

www.Scien

All authors contributed to the final drafting of the manuscript. Competing interests: Theauthors declare that they have no competing interests.

Submitted 14 September 2015Accepted 28 February 2016Published 13 April 201610.1126/scitranslmed.aad3815

Citation: V. Ribas, B. G. Drew, Z. Zhou, J. Phun, N. Y. Kalajian, T. Soleymani, P. Daraei, K. Widjaja,J. Wanagat, T. Q. de Aguiar Vallim, A. H. Fluitt, S. Bensinger, T. Le, C. Radu, J. P. Whitelegge,S. W. Beaven, P. Tontonoz, A. J. Lusis, B. W. Parks, L. Vergnes, K. Reue, H. Singh, J. C. Bopassa,L. Toro, E. Stefani, M. J. Watt, S. Schenk, T. Akerstrom, M. Kelly, B. K. Pedersen, S. C. Hewitt,K. S. Korach, A. L. Hevener, Skeletal muscle action of estrogen receptor a is critical for themaintenance of mitochondrial function and metabolic homeostasis in females. Sci. Transl.Med. 8, 334ra54 (2016).



http://stm.sciencem

ag.org/D

ownloaded from


mitochondrial function and metabolic homeostasis in females is critical for the maintenance ofαSkeletal muscle action of estrogen receptor

Meghan Kelly, Bente K. Pedersen, Sylvia C. Hewitt, Kenneth S. Korach and Andrea L. HevenerHarpreet Singh, Jean C. Bopassa, Ligia Toro, Enrico Stefani, Matthew J. Watt, Simon Schenk, Thorbjorn Akerstrom,P. Whitelegge, Simon W. Beaven, Peter Tontonoz, Aldons J. Lusis, Brian W. Parks, Laurent Vergnes, Karen Reue,

JulianWidjaja, Jonathan Wanagat, Thomas Q. de Aguiar Vallim, Amy H. Fluitt, Steven Bensinger, Thuc Le, Caius Radu, Vicent Ribas, Brian G. Drew, Zhenqi Zhou, Jennifer Phun, Nareg Y. Kalajian, Teo Soleymani, Pedram Daraei, Kevin

DOI: 10.1126/scitranslmed.aad3815, 334ra54334ra54.8Sci Transl Med

metabolic homeostasis in females. action in skeletal muscle helps maintain mitochondrial function andαimpaired. These findings imply that ER

mouse and found that glucose homeostasis was disrupted, fat accumulation increased, and mitochondrial function knockout (MERKO)αcorrelated with metabolic health in human females. They then created a muscle-specific ER

expression in muscleαtissue responsible for insulin-stimulated glucose disposal, the authors first showed that ERsensitivity in mice, but the precise tissue-specific mechanisms remain unclear. Because skeletal muscle is a main

The estrogen receptor (ER) is known to participate in the preservation of mitochondrial health and insulinpostmenopausal chinks in a woman's energy homeostasis armor.

investigate the mechanism underlying theet al.chronic disease associated with metabolic dysfunction. Now, Ribas a loss of protection against insulin resistance, which brings with it a constellation of consequences in the form of

Menopause ushers in a host of changes that range from unpleasant to undesirable. One undesirable shift isPostmenopausal muscle and mitochondrial mayhem

ARTICLE TOOLS http://stm.sciencemag.org/content/8/334/334ra54

MATERIALSSUPPLEMENTARY http://stm.sciencemag.org/content/suppl/2016/04/11/8.334.334ra54.DC1

CONTENTRELATED

http://stke.sciencemag.org/content/sigtrans/11/536/eaam5855.fullhttp://science.sciencemag.org/content/sci/352/6291/aad0189.fullhttp://stke.sciencemag.org/content/sigtrans/9/429/ra53.fullhttp://stke.sciencemag.org/content/sigtrans/10/489/eaao3266.fullhttp://stke.sciencemag.org/content/sigtrans/10/489/eaam6870.fullhttp://stke.sciencemag.org/content/sigtrans/9/429/pc12.fullhttp://stm.sciencemag.org/content/scitransmed/6/247/247fs29.fullhttp://stm.sciencemag.org/content/scitransmed/8/323/323ps3.fullhttp://stm.sciencemag.org/content/scitransmed/8/323/323rv2.fullhttp://stm.sciencemag.org/content/scitransmed/7/292/292ra98.fullhttp://stm.sciencemag.org/content/scitransmed/8/326/326rv3.full

REFERENCES

http://stm.sciencemag.org/content/8/334/334ra54#BIBLThis article cites 86 articles, 27 of which you can access for free

Terms of ServiceUse of this article is subject to the

registered trademark of AAAS. is aScience Translational MedicineScience, 1200 New York Avenue NW, Washington, DC 20005. The title

(ISSN 1946-6242) is published by the American Association for the Advancement ofScience Translational Medicine

Copyright © 2016, American Association for the Advancement of Science


http://stm.sciencem

ag.org/D

ownloaded from

http://stm.sciencemag.org/content/8/334/334ra54

http://stm.sciencemag.org/content/suppl/2016/04/11/8.334.334ra54.DC1

http://stm.sciencemag.org/content/scitransmed/8/326/326rv3.full

http://stm.sciencemag.org/content/scitransmed/7/292/292ra98.full

http://stm.sciencemag.org/content/scitransmed/8/323/323rv2.full

http://stm.sciencemag.org/content/scitransmed/8/323/323ps3.full

http://stm.sciencemag.org/content/scitransmed/6/247/247fs29.full

http://stke.sciencemag.org/content/sigtrans/9/429/pc12.full

http://stke.sciencemag.org/content/sigtrans/10/489/eaam6870.full

http://stke.sciencemag.org/content/sigtrans/10/489/eaao3266.full

http://stke.sciencemag.org/content/sigtrans/9/429/ra53.full

http://science.sciencemag.org/content/sci/352/6291/aad0189.full

http://stke.sciencemag.org/content/sigtrans/11/536/eaam5855.full

http://stm.sciencemag.org/content/8/334/334ra54#BIBL

http://www.sciencemag.org/about/terms-service


PERMISSIONS http://www.sciencemag.org/help/reprints-and-permissions

Terms of ServiceUse of this article is subject to the

registered trademark of AAAS. is aScience Translational MedicineScience, 1200 New York Avenue NW, Washington, DC 20005. The title

(ISSN 1946-6242) is published by the American Association for the Advancement ofScience Translational Medicine

Copyright © 2016, American Association for the Advancement of Science


http://stm.sciencem

ag.org/D

ownloaded from

http://www.sciencemag.org/help/reprints-and-permissions

http://www.sciencemag.org/about/terms-service


skeletal muscle action of estrogen receptor a is critical for ...metabolism skeletal muscle action...

Documents