Disruption of Mitochondria-Associated EndoplasmicReticulum Membrane (MAM) Integrity Contributes toMuscle Insulin Resistance in Mice and HumansEmily Tubbs,1 Stéphanie Chanon,1 Maud Robert,1,2 Nadia Bendridi,1 Gabriel Bidaux,1

Marie-Agnès Chauvin,1 Jingwei Ji-Cao,1 Christine Durand,1 Daphné Gauvrit-Ramette,1 Hubert Vidal,1,2

Etienne Lefai,1 and Jennifer Rieusset1,2

Diabetes 2018;67:636–650 | https://doi.org/10.2337/db17-0316

Modifications of the interactions between endoplasmicreticulum (ER) and mitochondria, defined as mitochondria-associated membranes (MAMs), were recently shownto be involved in the control of hepatic insulin actionand glucose homeostasis, but with conflicting results.Whereas skeletal muscle is the primary site of insulin-mediated glucose uptake and the main target for alter-ations in insulin-resistant states, the relevance of MAMintegrity in muscle insulin resistance is unknown. Deci-phering the importance of MAMs on muscle insulin sig-naling could help to clarify this controversy. Here, weshow in skeletal muscle of different mice models of obe-sity and type 2 diabetes (T2D) a marked disruption ofER-mitochondria interactions as an early event precedingmitochondrial dysfunction and insulin resistance. Further-more, in human myotubes, palmitate-induced insulin re-sistance is associated with a reduction of structural andfunctional ER-mitochondria interactions. Importantly, ex-perimental increase of ER-mitochondria contacts in hu-man myotubes prevents palmitate-induced alterations ofinsulin signaling and action, whereas disruption of MAMintegrity alters the action of the hormone. Lastly, we foundan association between altered insulin signaling andER-mitochondria interactions in human myotubes fromobese subjects with or without T2D compared with healthylean subjects. Collectively, our data reveal a new role ofMAM integrity in insulin action of skeletal muscle andhighlight MAM disruption as an essential subcellularalteration associated with muscle insulin resistance inmice and humans. Therefore, reduced ER-mitochondria

coupling could be a common alteration of several insulin-sensitive tissues playing a key role in altered glucosehomeostasis in the context of obesity and T2D.

Whereas mitochondria and endoplasmic reticulum (ER)dysfunction were largely shown to contribute independentlyto insulin resistance (1,2), it has been now demonstratedthat alterations in their physical interactions are also involved(3). Indeed, mitochondria and ER interact at contact points,called mitochondria-associated ER membranes (MAMs), toexchange calcium (Ca2+) and lipids, thus regulating cell me-tabolism and fate (4,5). A few years ago, we indentified thatMAM integrity controlled hepatic insulin signaling and thatdisruption of organelle coupling contributed to hepatic in-sulin resistance (6). This was recently confirmed by anindependent group (7). Accordingly, mice models with liver-specific invalidation of proteins located at MAM interface,such as mitofusin 2 (MFN2) (8) and mammalian target ofrapamycin complex 2 (mTORC2) (9), as well as mutation ofinositol 1,4,5-triphosphate receptor 1 (IP3R1) (10), displayhyperglycemia, glucose intolerance, and increased neogluco-genesis. Alterations of mitochondrial Ca2+ uptake from the ERcan also disrupt insulin signaling in cardiomyocytes (11). Theliterature is still equivocal regarding the exact role of MAMsin hepatic insulin resistance, however, because altered hepaticinsulin sensitivity can be associated with enrichment of ER-mitochondria contacts (12) and reduced IP3R levels can be as-sociated with decreased glucose production by the liver (13).

1Laboratoire de Recherche en Cardiovasculaire, Métabolisme, Diabétologie etNutrition (CarMeN), INSERM U1060, INRA U1397, Institut National des SciencesAppliquées-Lyon, Université Claude Bernard Lyon1, Oullins, Lyon, France2Endocrinology, Diabetology and Nutrition Service, Lyon-Sud Hospital, HospicesCivils de Lyon, Pierre Bénite, Lyon, France

Corresponding author: Jennifer Rieusset, jennifer.rieusset@univ-lyon1.fr.

Received 14 March 2017 and accepted 5 January 2018.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0316/-/DC1.

S.C., M.R., and N.B. contributed equally to this work.

© 2018 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

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Another tissue crucial for glucose homeostasis is theskeletal muscle. This tissue is indeed the primary site ofinsulin-stimulated glucose uptake and, therefore, the maintarget for alterations in insulin resistant states (14). Inter-estingly, interactions of mitochondria with the sarcoplasmic-endoplasmic reticulum (SR/ER) have been demonstrated inskeletal muscle, although the molecular components of thetethers remain to be characterized (15). Furthermore, a localtransfer of Ca2+ from SR/ER to mitochondria was illustratedupon caffein stimulation in skeletal muscle (16), which seemsimportant for the control of oxidative metabolism (17). Ac-cordingly, insulin-dependent Ca2+ mobilization participatesto the translocation of the glucose transporter GLUT4 tothe cell surface and to glucose uptake (18), suggesting a po-tential role of organelle coupling in muscle insulin action.

However, the relevance of MAMs in skeletal muscleinsulin resistance has not been investigated up to now;thus, the current study was conducted to gain more insightinto this important issue. We show here that SR/ER-mitochondria interactions in skeletal muscle are altered ininsulin-resistant states in both mice and humans and thatthe modulation of organelle coupling regulates insulinsignaling and action in human myotubes.

RESEARCH DESIGN AND METHODS

Animal StudiesAnimal studies were performed in accordance with theFrench guidelines for the care and use of animals and wereapproved by an ethics committee of the Rhône-Alpes region(France). Male C57BL/6J and ob/ob mice (12 weeks old)were purchased from Harlan and adapted to the facilityfor 1 week before study. Male C57Bl/6J mice (5 weeksold) were fed a standard diet (SD) or a high-fat and high-sucrose diet (HFHSD), prepared by the Unit of ExperimentalFood Preparation in Jouy-en-Josas, France, for 16 weeks,as previously described (6,19). Metabolic characteristics ofthese mice were reported previously (6).

For this study, gastrocnemius muscles were removed andfreshly used for MAM purification or ex vivo insulin-signalingstudies (61027 mol/L insulin, 15 min). An independentgroup of mice (5 weeks old) was fed the SD or HFHSD for1, 4, 8, 12, or 16 weeks (n = 3 mice/group), and their gas-trocnemius muscles were fixed in formaldehyde for paraffininclusion or frozen. Finally, 12-week-old male C57BL/6Jmice were used to infect gastrocnemius muscles (intramus-cular injection, 109 infection forming units/muscle) withrecombinant adenoviruses encoding for green fluorescentprotein (Ad-GFP) or FATE1 (Ad-FATE1) proteins for 7 days.

MAM PurificationIsolation of MAM fractions was performed by differentialultracentrifugation as previously described (6).

Transmission Electron Microscope AnalysisFixation and posttreatment of muscle fractions or myotubesand the observation of ultrathin sections were performed aspreviously described (6). For analysis of SR/ER-mitochondria

contacts in human myotubes, we delimitated both organellesusing ImageJ, and the fraction of mitochondria in contactwith SR/ER within a 50-nm range was calculated and nor-malized to the mitochondria perimeter.

Human MyotubesPrimary culture of human myotubes was initiated fromsatellite cells of vastus lateralis muscle biopsy tissue samplesobtained from donors during surgical procedures at EdourdHerriot Hospital (Lyon, France). Myotubes from sevendonors (one man and six women; age: 68.8 6 5.3 yearsold; BMI: 25.3 6 0.7 kg/m2) were used for palmitate/oligomycin treatments. Myotubes from 10 lean subjects,15 obese subjects without diabetes, and 12 obese patientswith type 2 diabetes (T2D) were used to investigate whetherorganelle interactions are altered during obesity and T2D(Table 1). Patients with T2D were being treated with anoral antidiabetic drug or with insulin, or both, and didnot interrupt their treatment before the operation. Allparticipants gave their written consent after being informedof the nature and purpose of the study. The experimentalprotocol (DIOMEDE, agreement number 2012-111/A13-06)was approved by the Ethical Committees Sud-EST IV andperformed according to the French legislation.

The myoblasts were purified, and differentiated myo-tubes were prepared according to the procedure previouslydescribed (20). Human myotubes were incubated with pal-mitate (500 mmol/L) for 24 h to induce insulin resistance(21) or treated with oligomycin (1 and 10 mmol/L, 16 h) toalter mitochondria function. For the analysis of insulin sig-naling, myotubes were depleted for 3 h in serum and fur-ther incubated with or without 1027 mol/L of insulin for15 min in serum-free medium.

Modulation of MAM Protein ExpressionHuman myotubes were transfected in six-well plates, for48 h, with 2 mg expression plasmids or small interfering(si)RNA (25 or 50 nmol/L; Qiagen) for specific targets, usingExGen 500 transfection reagent (Roche Diagnostics) orHigh Perfect transfection reagent (Qiagen) respectively,as previously reported (6).

FATE1 Expression Vectors and RecombinantAdenovirusExpression vectors pcDNA4/TO and pcDNA4/FATE1 (fetaland adult testis expressed 1) were a gift from Enzo Lalli

Table 1—Donor characteristics for primary myotubes fromlean subjects, obese subjects without diabetes, and obesesubjects with T2D

Subjects n

Sex (n)

Age (years) BMI (kg/m2)Men Women

Lean 10 3 7 65 6 4.9 24 6 0.8

Obese 15 8 7 44 6 3.4*** 45 6 1.8***

T2D 12 3 9 54 6 1.9 41 6 2.3***

Data are presented as mean 6 SD unless indicated otherwise.***P , 0.001 compared with lean subjects.

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(CNRS-UMR7275, Valbonne, France) (22). FATE1 wassubcloned into pcDNA-internal ribosome entry site (IRES)-GFP vector, obtained as previously described (23), to obtainpcDNA-FATE1-IRES-GFP vector, allowing the simultaneousexpression of both FATE1 and GFP proteins into cells underthe control of the same promoter. Recombinant Ad-GFP (asa control) or Ad-FATE1 were generated by homologous re-combination in the VmAdcDNA3 plasmid and amplifiedand purified as previously described (24,25).

Western Blotting and Real-time PCRProtein expression was analyzed by SDS-PAGE, and mRNAlevels were measured by real-time RT-PCR. To compareintergel analysis of insulin-stimulated protein kinase B (PKB)phosphorylation in human myotubes from lean subjects,obese subjects, and subjects with T2D, a pooled sample ofhuman myotubes was used as an internal standard on eachSDS-PAGE gels and used to normalize protein expression.For that, the intensity (in pixel) of each targeted protein(i.e., phosphorylated PKB or total PKB) is normalized by theintensity of the internal standard before determining theratio of phosphorylated PKB to total PKB.

In Situ Proximity Ligation AssayVoltage-dependant anion channel 1 (VDAC1) and IP3R1proximity were measured by in situ proximity ligationassay (PLA) (Olink Bioscience) to detect and quantify ER-mitochondria interactions, as previously described andthoroughly validated (6,26). PLA was also used here to an-alyze PKB phosphorylation by using separate primary anti-bodies (Cell Signaling) against PKB protein and the S473phosphorylation site of PKB. In muscle tissues, in situ PLAswere performed on paraffin-embedded gastrocnemius sec-tions, after an antigen retrieval at pH 6, using a bright-fieldrevelation, as previously described (27).

Wide-Field Ca2+ ImagingThe concentration of cytosolic calcium ([Ca2+]c) was mea-sured using the ratiometric dye Fura2- acetoxymethyl ester(AM) (5 mmol/L) loaded in human myotubes at 37°C for1 h, whereas that of mitochondria ([Ca2+]m) was measuredusing ratiometric 4mtD3cpv biosensor expressed after2 days of adenovirus infection of human myotubes at37°C. After three washes with a Ca2+-free Tyrode solution(140 mmol/L NaCl, 5 mmol/L KCl, 10 mmol/L HEPES,1 mmol/L MgCl2, 10 mmol/L glucose, and 100 mmol/LEGTA at pH 7.4), dishes were set on a Leica DMI6000 Bmicroscope equipped with a 320 objective, an ORCA-Flash4.0 Scientific CMOS camera (Hamamatsu), and a LambdaDG-4+ illumination system (Sutter Instrument). Myotubesthat were elongated (crossing the field of vision), branched,and polynucleated were selected. Cells were treated with200 mmol/L Na-ATP (28) and 5 mmol/L thapsigargin(Tg). Because not all myotubes responded to Tg by an in-crease in free Ca2+ in cytosol or in mitochondria, we se-lected only cells showing a significant difference in theaverage fluorescence between the 40 s before and after Tg

treatment. These cells were considered as having a goodprobe load and sufficient Ca2+ stores to enable measure-ment of variations in free [Ca2+]. Four independent experi-ments were analyzed. The fluorescence ratio was normalizedby the fluorescence at the origin (F/F0), and all cells weregathered for statistical analysis.

Glucose TransportGlucose transport was measured in human myotubes, aspreviously described (29).

Mitochondrial DNA QuantificationTotal DNA was extracted from gastrocnemius muscle ofmice, and mitochondrial and nuclear DNAs were quantifiedby real-time PCR, as previously described (30).

Cytochrome C Oxidase and Citrate Synthase ActivitiesCytochrome C oxidase (COX) and citrate synthase (CS)activities were measured spectrophotometrically in totallysates from gastrocnemius muscle of mice, as previouslydescribed (31,32).

Statistical AnalysisData are expressed as the mean 6 SEM, and statisticalsignificance was defined as a value of P, 0.05. Normal dis-tribution of the data was tested using Shapiro-Wilk. Com-parisons between more than two groups were analyzed byone-way ANOVA, followed by Turkey post hoc tests. For allother analysis, data were compared by Student t test. ForCa2+ imaging, statistical error (a), statistical power (b), andeffect size (d) were calculated in function of the numberof cells, and statistical difference was defined as a , 0.05and b . 0.8.

RESULTS

ER-Mitochondria Interactions Are Altered in SkeletalMuscle of Obese and Diabetic MiceWe performed subcellular fractionation of gastrocnemiusmuscle from genetically (ob/ob) and diet-induced insulin-resistant mice. The skeletal muscles were sampled on the micethat have been previously used to study MAM integrity inthe liver, and these mice were glucose intolerant and insulinresistant (6). The ob/ob (Fig. 1A) and HFHSD (Fig. 1B) miceboth showed altered muscle insulin signaling, as illustratedby the significant reduction of insulin-stimulated PKB andglycogen synthase kinase 3-b phosphorylations in gastroc-nemius explants. Interestingly, we found in both modelsa marked reduction of MAM amount in gastrocnemiusmuscle assessed by subcellular fractionation (Fig. 1C andD, respectively). This reduction of MAMs in diabetic musclewas also observed when MAM amounts were normalizedrelative to mitochondria amounts. The purity of MAM frac-tions was validated by Western blotting and TEM (Supple-mentary Fig. 1A and B), and the expression levels of 75-kDaglucose-regulated protein (GRP75), VDAC1, MFN2, andcyclophilin D were not modified in muscle MAM fractionsby obesity and T2D (Supplementary Fig. 2A and B). Wethen used in situ PLA as an additional strategy to quantify

638 MAM Disruption and Muscle Insulin Resistance Diabetes Volume 67, April 2018

Figure 1—Disruption of MAM integrity in skeletal muscle of genetically and diet-induced obese and diabetic mice. A and B: RepresentativeWestern blots (at top) and quantitative analysis (below) of insulin-stimulated PKB and GSK3b in gastrocnemius muscle of wild-type (WT) andob/ob mice (A) and in SD- and HFHSD-fed mice (B) (n = 3). *P , 0.0001. Basal and insulin-stimulated values are summarized in SupplementaryTables 1 and 2, respectively. Quantitative analysis of protein levels in MAM fractions after subcellular fractionation of gastrocnemius muscles of

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the number of MAM contact points in paraffin-embeddedgastrocnemius muscle by measuring the proximity betweenVDAC1 and IP3R1, as previously described (6,26). The ob/ob(Fig. 1E) and HFHSD (Fig. 1F) mice both showed a significantreduction of VDAC1/IP3R1 interactions compared with re-spective control mice, illustrating a marked disruption of or-ganelle coupling in skeletal muscles of insulin-resistant mice.

Palmitate-Induced Insulin Resistance in HumanMyotubes Is AssociatedWith Altered Organelle CouplingWe next induced insulin resistance in primary cultures ofhuman myotubes from control subjects using palmitate treat-ment (500 mmol/L, 24 h). As expected, palmitate-treatedmyotubes displayed a reduction of insulin-mediated PKBand GSK3b phosphorylations (Fig. 2A) and an inhibition ofinsulin-stimulated glucose transport (Fig. 2B). Importantly,they also showed a dramatic reduction of VDAC1-IP3R1interactions measured by in situ PLA (Fig. 2C) and a decreasein the percentage of mitochondrial membrane in contactwith SR/ER, analyzed by TEM (Fig. 2D), indicating a disrup-tion of organelle coupling.

Then, we analyzed organelle Ca2+ exchange in palmitate-treated human myotubes using Ca2+ video-imaging micro-fluorometry. Cells were infected with a 4mtD3cpv-endocingadenovirus for 48 h to measure free [Ca2+] in the mitochon-drial matrix ([Ca2+]m) or incubated with 5 mmol/L Fur-a2-AM for 1 h to measure free [Ca2+] in cytosol ([Ca2+]c).Then, 200 mmol/L ATP were added in the medium to in-duce the IP3-mediated mobilization of Ca2+ stores (28).Conversely, cells were treated with 5 mmol/L Tg to releaseCa2+ from ER stores independently of MAMs, because Ca2+

leak from ER stores could be carried by several types ofchannels that have not been described at MAMs (33). Asshown in Fig. 2E and F, ATP induced a transient increase inboth [Ca2+]c and [Ca2+]m in BSA-treated cells but only in-duced increased [Ca2+]c in palmitate-treated myotubes, sug-gesting that palmitate induced a decrease of ATP-mediatedCa2+ mobilization from ER stores or a decreased Ca2+ up-take by mitochondria. We thus compared both ATP- andTg-mediated variations in [Ca2+]m and [Ca2+]c. Tg induceda similar increase in the free [Ca2+]c in BSA- and palmitate-treated myotubes, suggesting no modification in the ER Ca2+

stores. A slight but nonsignificant (Fig. 2G and Supplemen-tary Table 5) decrease in [Ca2+]m was observed in palmitate-treated myotubes, suggesting that a part of Tg-mediatedCa2+ could be released in MAMs.

SR/ER-Mitochondria Miscommunication Is Independentof Mitochondrial Dysfunction and Insulin ResistanceBecause mitochondrial physiology is markedly altered inskeletal muscle during obesity and T2D, it is difficult to

determine whether alterations of muscle insulin actionresult from organelle miscommunication or mitochondriadysfunction, or both. To get more insight into this issue, weexamined the relationship between organelle communica-tion and mitochondria physiology in insulin-resistant statesin vivo and in vitro. Firstly, we analyzed insulin sensitivity(insulin tolerance test), SR/ER-mitochondria interactions(in situ PLA), and mitochondrial biology (mitochondrial[mt]DNA amount and COX/CS activity) in muscle of SDand HFHSD mice after 1, 4, 8, 12, or 16 weeks of the diet.Diet-induced insulin resistance appeared in HFHSD miceafter 12 weeks of the diet (Supplementary Table 6). SR/ER-mitochondria interactions are decreased in skeletal muscleof HFHSD mice as soon as 1 week of feeding and remaineddiminished during the entire feeding period (Fig. 3A). Con-versely, mtDNA amount and COX/CS activity are reducedonly after 16 weeks of HFHSD feeding (Fig. 3B and C,respectively), whereas we found a transient increase ofCOX/CS activity during the first weeks of HFHSD feeding(Fig. 3C).

Secondly, we thought to alter mitochondria function byoligomycin (an ATPase inhibitor) treatment (1 or 10 mmol/Lfor 16 h) in human myotubes and to analyze the reper-cussions on organelle contacts and insulin signaling.Oligomycin treatment markedly induced mitochondriafragmentation (Fig. 3D), without altering cell viability (datanot shown). Importantly, 1 and 10 mmol/L oligomycintreatments markedly induced VDAC1-IP3R1 interactions(Fig. 3E), whereas only 1 mmol/L oligomycin significantlyincreased insulin-stimulated PKB phosphorylation (Fig. 3F).

Palmitate-Induced Insulin Resistance and ER Stress ArePrevented by Increasing MAMs in Human MyotubesNext, we investigated whether the inhibitory action ofpalmitate on insulin signaling could be prevented byincreasing organelle coupling through Grp75 or Mfn2overexpression, as previously performed in hepatocytes(6). Using a fluorescent vector, we estimated that 46.7 62.3% of myotubes (n = 20 images) were transfected in ourconditions (data not shown) and sufficient to observe anincrease of Grp75 and Mfn2 protein expression (Fig. 4B).As expected, transient Grp75 or Mfn2 overexpression in-creased VDAC1-IP3R1 interactions in human myotubes(Fig. 4A). Interestingly, increasing organelle coupling byoverexpressing Grp75 or Mfn2 restored palmitate-inducedalterations of insulin signaling, as illustrated by the increaseof insulin-mediated PKB and GSK3b phosphorylations(Fig. 4B). To evaluate the effects of MAM reinforcementon other parameters related to insulin action, we analyzedthe effect of the hormone on the regulation of GLUT4,hexokinase II (HKII), and SREBP1c mRNA, because the

ob/ob (C) and HFHSD-fed mice (D). MAM protein levels were normalized by muscle weight or by protein in pure mitochondria (Mp) fraction.(n = 5). *P , 0.05. Representative images (at top) and quantitative analysis (below) of the VDAC1-IP3R1 interactions (brown dots) measured byin situ PLA in paraffin-embedded gastrocnemius muscle of ob/ob mice (E) and of HFHSD-fed mice at 16 weeks (16W) (F) (n = 3–5/group,5–10 pictures/mice). ***P , 0.00001. a.u., arbitrary unit.

640 MAM Disruption and Muscle Insulin Resistance Diabetes Volume 67, April 2018

Figure 2—Palmitate-induced alterations of insulin signaling and action are associated with disruption of MAM integrity and function in humanmyotubes. Human myotubes were treated with BSA or palmitate (500 mmol/L) for 24 h. A: Representative Western blots (at top) and quantitativeanalysis (below) of insulin-stimulated PKB and GSK3b after BSA or palmitate treatments (n = 3). *P, 0.0001. Basal and insulin-stimulated valuesare summarized in Supplementary Table 3. B: Effect of palmitate treatment on insulin-mediated glucose uptake in human myotubes (n = 5).

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regulation of these genes by insulin is altered in muscle ofpatients with T2D (34) and on glucose transport. We foundthat insulin induced expression of GLUT4, HKII, andSREBP1c in BSA-treated myotubes transfected with anempty vector, whereas palmitate treatment completelyabolished these regulations (Fig. 4C). Importantly, Grp75and Mfn2 overexpression both restored insulin-mediatedregulation of these three genes in palmitate-treated myo-tubes (Fig. 4C). Similarly, palmitate treatment altered insulin-mediated glucose uptake in human myotubes transfectedwith an empty vector, whereas insulin-mediated glucoseuptake was restored in palmitate-treated myotubes overex-pressing Grp75 or Mfn2 (Fig. 4D). Altogether, these datademonstrate that increasing ER-mitochondria tethering inmuscle cells is sufficient to restore metabolic insulin signal-ing and metabolic action.

In hepatocytes, reduction of MAM integrity has beenassociated with ER stress (35), which may contribute toinsulin resistance. To determine whether the same phenom-enon also occurs in human myotubes, we evaluated theeffects of different treatments on the unfolded proteinresponse (UPR) markers. Palmitate treatment increasedmRNA levels of the 78-kDa GRP (GRP78), the splicedX-box-binding protein 1 (Xbp1-s), and CHOP (Fig. 4E), con-firming palmitate-mediated ER stress in human myotubes(21). Interestingly, overexpression of Grp75 or Mfn2 coun-teracted palmitate-induced ER stress, as illustrated by thesignificant reduction of all UPR markers (Fig. 4E), indicatingthat reinforcement of MAM can improve palmitate-inducedER stress in muscle cells.

Disruption of Organelle Coupling Alters Insulin Signalingand Action in Human MyotubesWe next explored whether the reduction of organellecoupling, by reducing GRP75 or MFN2 protein levels byspecific siRNA, can alter insulin action in muscle cells asobserved previously in hepatocytes (6). We validated theefficiency of siRNA targeting in human myotubes and foundthat 51 6 4% of human myotubes (n = 20 images) weretargeted by fluorescent siRNA (data not shown). Silencingof Grp75 and Mfn2 using specific siRNA (Fig. 5A) reducedVDAC1-IP3R1 interactions in human myotubes (Fig. 5B).Importantly, in both conditions of disrupted ER-mitochondriainteractions, insulin action was altered in muscle cells, asillustrated 1) by the reduction of insulin-mediated PKB

phosphorylation (Fig. 5C), 2) by a reduction of Glut4mRNA expression (Fig. 5D), and 3) by an inhibition of in-sulin-mediated glucose uptake (Fig. 5E). Lastly, cotreatmentof human myotubes with siRNA (Grp75 or Mfn2) and pal-mitate did not lead to additive effects on insulin-stimulatedPKB phosphorylation (Supplementary Fig. 3), suggestingthat the two treatments may affect the same regulatoryprocess.

FATE1-Mediated Disruption of Organelle Coupling AltersInsulin SignalingEven if genetic manipulation of MAM proteins is a goodapproach to modulate SR/ER mitochondria interactions,none of these proteins are exclusively expressed at MAMs,and their manipulation may result in alterations of cellu-lar functions outside of MAMs. Therefore, we sought tooverexpress FATE1, a cancer-testis antigen recently identi-fied as an uncoupler of MAMs (22) and normally notexpressed in skeletal muscle. For that, we constructeda pcDNA-FATE1-IRES-GFP vector, which simultaneouslyoverexpresses FATE1 and GFP proteins and allows us toanalyze organelle interactions by in situ PLA only in trans-fected cells (green cells), compared with untransfected cells(unfluorescent cells). Firstly, we confirmed that FATE1 isnot expressed in human myotubes and that its overexpres-sion increased its protein levels (Fig. 6A). More importantly,transient FATE1 overexpression reduced VDAC1-IP3R1interactions in FATE1-transfected myotubes comparedwith cells transfected with an empty vector or with untrans-fected cells in FATE1-transfected myotubes (Fig. 6B), vali-dating the uncloupling activity of FATE1 in nontestis cells.To analyze the repercussions on insulin signaling similarlyin both transfected and untransfected myotubes, we ana-lyzed insulin-stimulated PKB phosphorylation by in situPLA by using separate primary antibodies against PKB pro-tein and the S473 phosphorylation site of PKB. As expected,insulin increased pS473 phosphorylation of PKB in myo-tubes transfected with an empty vector (Fig. 6C), validatingthe use of PLA for the analysis of insulin signaling. Impor-tantly, insulin-stimulated PKB phosphorylation is markedlyand specifically reduced in FATE1-overexpressing myotubes(Fig. 6C), indicating that disrupting organelle interactionsindependently of endogenous MAM proteins also altersinsulin action.

Next, we constructed an adenovirus starting from thepcDNA-FATE1-IRES-GFP vector and we used an Ad-GFP

*P , 0.05. Basal and insulin-stimulated values are summarized in Supplementary Table 4. C: Representative PLA images (left) (original mag-nification 363 and scale bar = 20 mm) and quantitative analysis (right) of VDAC1-IP3R1 interactions in human myotubes treated with BSA orpalmitate (500 mmol/L) for 24 h (n = 3). *P , 0.05. D: Representative images (at top) and quantitative analysis (below) of the percentage ofmitochondria (M) membrane in contact with ER in human myotubes treated with BSA or palmitate (500 mmol/L) for 24 h (n = 30 images percondition in 3 independent experiments). *P, 0.05. Scale bars, 500 nm (at left) and 200 nm (at right); arrows point ER-mitochondria contacts.Average time traces of the normalized fluorescence (F/F0) reporting the [Ca2+]m (E) or [Ca2+]c (F) in human myotubes incubated with 500 mmol/LBSA or 500 mmol/L palmitate (Palm) for 24 h. [Ca2+]c was measured with Fura2-AM, and [Ca2+]m was measured with 4mtD3cpv biosensor, asexplained in the RESEARCHDESIGNANDMETHODS. Experimentswere initiated in the absence of extracellular Ca2+. ER Ca2+ release was induced by applying200 mmol/L ATP for 5 min before 5 mmol/L Tg. Values from four independent human samples were pooled and are presented as mean 6 95%CI. Statistics and cell numbers are presented in the Supplementary Table 5. G: Histogram reports the average variation in F/F0 over 40 s afterdrug treatment (values are mean6 SD). ***P, 0.00001. More detailed statistics are presented in the Supplementary Table 5. a.u., arbitrary unit.

642 MAM Disruption and Muscle Insulin Resistance Diabetes Volume 67, April 2018

Figure 3—MAM alterations in insulin-resistance states are independent of mitochondrial dysfunction in skeletal muscle. A: Quantitative analysisof VDAC1-IP3R1 interactions in gastrocnemius muscles of SD and HFHSD mice after 1, 4, 8, 12, and 16 weeks of feeding (n = 30 images in3 mice/group). *P , 0.05 vs. SD. B: Quantification of mtDNA amount measured by real-time PCR in gastrocnemius muscle of SD and HFHSDmice after 1, 4, 8, 12, and 16 weeks of feeding (n = 3 mice/group). *P , 0.05 vs. SD. C: COX/CS activity measured by spectrophotometry ingastrocnemius muscle of SD and HFHSDmice after 1, 4, 8, 12, and 16 weeks of feeding (n = 3–15 mice/group). *P, 0.05; **P, 0.01 vs. SD. D:Geometrical analysis and representative images of MitoTracker-labeled mitochondria after a 16-h oligomycin treatment (1 and 10 mmol/L)compared with ethanol (EtOH) (n = 30 images in 3 independent experiments). *P , 0.05; **P , 0.01, ***P , 0.001 vs. ethanol. Originalmagnification 363 and scale bar = 20 mm. E: Representative PLA images (at top, original magnification 363 and scale bar = 20 mm) andquantitative analysis (below) of VDAC1-IP3R1 interactions in human myotubes incubated with ethanol or oligomycin (10 and 10 mmol/L) for 16 h

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as a control. We validated that the use of Ad-FATE1 inhuman myotubes reduced ER-mitochondria interactions(Supplementary Fig. 4A) and altered insulin-stimulated PKBphosphorylation (Supplementary Fig. 4B), as in transienttransfection experiments. Interestingly, the same experi-ment performed in the HuH7 hepatic cell line also showeda clear reduction of both organelle interactions (Supple-mentary Fig. 5A) and insulin-stimulated IRS2 and PKBphosphorylations (Supplementary Fig. 5B), validating thatFATE1-mediated organelle uncoupling also alters insulinsignaling in hepatocytes, in agreement with our previousobservations when we modulated endogenous MAM pro-teins (6). Therefore, we tested in vivo the injection ofAd-FATE1 in gastrocnemius muscle of mice. We validatedthat infected muscle fibers of both Ad-GFP and Ad-FATE1mice express the GFP protein in muscle fibers (Supplemen-tary Fig. 6). Importantly, FATE1 overexpression reducedVDAC1-IP3R1 interactions (Fig. 6D) and altered the effectof insulin on PKB phosphorylation (Fig. 6E) in gastrocne-mius muscle of infected mice.

ER-Mitochondria Interactions Are Altered in HumanMyotubes of Obese Subjects and Patients With T2DBecause myotubes in primary culture are known tomaintain insulin sensitivity of the donor (36,37), we inves-tigated whether ER-mitochondria interactions are altered inprimary myotubes established from obese subjects anddonors with T2D compared with cells from healthy leancontrol subjects. The patients with T2D were matched forBMI and age with the obese subjects and for age with thelean donors (Table 1). We observed a reduction of insulin-mediated PKB phosphorylation in myotubes from obesedonors and patients with T2D compared with lean donors(Fig. 7A), whereas no significant difference was observedbetween obese donors without diabetes and obese patientswith T2D, indicating a similar degree of insulin resistance.Importantly, VDAC1-IP3R1 interactions, measured byin situ PLA, were decreased in myotubes from obese sub-jects without diabetes and those with diabetes comparedwith healthy donors (Fig. 7B). No significant difference wasfound between myotubes from obese donors without andwith diabetes. Lastly, VDAC1-IP3R1 interactions were sig-nificantly and positively correlated with the effect of in-sulin on PKB phosphorylation (Fig. 7C) (P = 0.33, R2 = 0.11;P = 0.04) when all the subjects were analyzed.

DISCUSSION

We recently showed that MAM integrity contributes toinsulin action in the liver and is altered in situations ofhepatic insulin resistance (6). Here, we investigated whetherthis mechanism is restricted to the liver or could be gener-alized to other insulin-sensitive tissues, particularly skeletal

muscles. This study in muscle cells is particularly importantbecause the few reported studies on this issue in the hep-atocytes are highly controversial (6,12). We demonstratein vitro and in vivo, from mice to humans, that the disrup-tion of organelle coupling may contribute to muscle insulinresistance and that reinforcing MAM increases insulin ac-tion, at least in human myotubes. Therefore, targetingMAM could be a novel strategy to improve insulin sensitiv-ity and restore glucose homeostasis.

As previously observed in the liver (6,27), we found thatER-mitochondria interactions are reduced in skeletal muscleof genetically and diet-induced obese and diabetic mice, inpalmitate-treated human myotubes, and in myotubes fromobese patients and patients with T2D, indicating a closerelationship between MAM integrity and muscle insulinsensitivity. These structural analyses were systematicallyperformed by in situ PLA in vivo and in vitro, whereassubcellular fractionation or TEM analysis confirms PLAresults in vivo and in vitro, respectively. We tried to analyzeER-mitochondria interactions in skeletal muscle by TEMbut were not able to perform a reliable analysis due tothe architecture of muscle fibers. We believe that TEM isnot really able to reveal fine structural details because of theoverlapping density/noisy background in muscle tissues.This issue notwithstanding, cumulative evidence supportsthe use of in situ PLA as a reliable method to study in situER-mitochondria interactions in skeletal muscle, as previ-ously validated in the liver (6,26,27). Furthermore, thefunctionality of MAMs is also altered in insulin-resistantstates, because Ca2+ transfer from ER to mitochondria isreduced in palmitate-treated myotubes, supporting a poten-tial implication of organelle miscommunication in muscleinsulin resistance.

Next, we found that MAM integrity is required for anefficient insulin action in skeletal muscle, because theexperimental disruption of MAM dampens insulin signalingand action in human myotubes, whereas the experimentalinduction of organelle interactions prevents palmitate-induced insulin resistance. These genetic experiments areinteresting but present two weaknesses. Firstly, all myo-tubes are not transfected, and therefore, we analyzedorganelle tethering on a pool of untransfected and trans-fected cells, reducing the robustness of the measurements.Secondly, none of MAM proteins is exclusively expressed atMAMs; therefore, their modifications may result in alter-ations of cellular function outside of MAMs. To get aroundthese two problems, we sought to overexpress FATE1, anorganelle uncoupler not expressed in skeletal muscle (22),by using the pCDNA-FATE1-IRES-GFP vector allowing thesimultaneous expression of both FATE1 and GFP proteins.Using this strategy, we are able to analyze by in situ PLAVDAC1-IP3R1 interactions and PKB phosphorylation in

(n = 30 images in 3 independent experiments). ***P , 0.0005 vs. ethanol. F: Representative Western blots (at top) and quantitative analysis(below) of basal and insulin-stimulated PKB phosphorylation in human myotubes treated with ethanol or 1 mmol/L oligomycin for 16 h (n = 3).**P , 0.01 vs. control (Co); $P , 0.05 vs. respective ethanol. AR, aspect ratio; a.u., arbitrary unit; FF, form factor; w, week.

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Figure 4—Experimental reinforcement of MAM prevents palmitate-induced alterations of insulin signaling and action in human myotubes.Human myotubes were transfected with empty (Co), Grp75-, or Mfn2-expressing vectors and treated after 24 h with BSA or palmitate(200 mmol/L) for an additional 24 h. When required, cells were depleted for 3 h in serum at the end of the treatment and incubated with insulin(1027 mol/L, 15 min for insulin signaling, 1 h for glucose transport). A: Representative PLA images (left, original magnification 363 and scalebar = 20 mm) and quantitative analysis (right) of VDAC1-IP3R1 interactions in transfected human myotubes treated with BSA or palmitate (Palm;200 mmol/L) during 24 h (n = 3). *P , 0.01 vs. BSA pcDNA3 Co; #P , 0.05 vs. palmitate pcDNA3 Co. B: Representative Western blots (at top)and quantitative analysis (below) of insulin-stimulated PKB and GSK3b after transfection and BSA or palmitate treatments. Basal and insulin-stimulated values are summarized in Supplementary Table 7. Validations of Grp75 andMfn2 overexpression are also shown (n = 3). *P, 0.05 vs.BSA pcDNA3 Co; #P, 0.05 vs. palmitate pcDNA3 Co.C: Effect of 6-h insulin treatment on Glut4, HKII, and SREBP1cmRNA levels measured byreal-time PCR in transfected myotubes treated with BSA or palmitate for 24 h (n = 3). *P , 0.05 vs. BSA pcDNA3 Co; #P , 0.05 vs. palmitatepcDNA3 Co. Basal and insulin-stimulated values are summarized in Supplementary Table 8. D: Measurement of insulin-mediated glucoseuptake in MFN2-overexpressing human myotubes treated with BSA or palmitate for 24 h (n = 4). *P , 0.01 vs. BSA pcDNA3 Co; #P , 0.05 vs.palmitate pcDNA3 Co. Basal and insulin-stimulated values are summarized in Supplementary Table 9. E: mRNA levels of ER stress markersmeasured by real-time PCR in transfected myotubes treated with BSA or palmitate for 24 h (n = 3). *P , 0.05 vs. BSA pcDNA3 Co; #P ,0.01 vs. palmitate pcDNA3 Co. a.u., arbitrary unit.

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transfected and untransfected cells as a negative control.We validated that FATE1-mediated reduction in organellecoupling is specific to transfected myotubes, indicating thatFATE1 overexpression is a good strategy to reduce ER-mitochondria interactions in nontestis cells. Furthermore,we found that overexpression of FATE1 specifically alteredinsulin-stimulated PKB phosphorylation in transfected

or Ad-FATE1–infected myotubes and in gastrocnemiusmuscle of mice. Altogether, these data demonstrate thatFATE1-mediated organelle uncoupling is sufficient to alterinsulin signaling in vitro and in vivo.

Because mitochondria physiology was previously reportedto be altered in skeletal muscle of obese and diabetic mice(30,38) and in palmitate-induced insulin-resistant myotubes

Figure 5—Experimental disruption of MAM integrity alters insulin action in human myotubes. Human myotubes were transfected with specificsiRNA for Grp75 or Mfn2 for 48 h. When required, cells were depleted for 3 h in serum and treated with insulin (1027 mol/L, 15 min for insulinsignaling and 1 h for glucose transport). A: Validation of the silencing of Grp75 or Mfn2 in human myotubes. B: Representative PLA images (attop, original magnification 363 and scale bar = 20 mm) and quantitative analysis (below) of VDAC1-IP3R1 interactions in human myotubessilenced for Grp75 or Mfn2 (n = 3). *P, 0.0001 vs. siRNA control (Co). C: Representative Western blots (at top) and quantitative analysis (below)of insulin-stimulated PKB in human myotubes silenced for Grp75 or Mfn2 (n = 3). Basal and insulin-stimulated values are summarized inSupplementary Table 10. *P , 0.0001 vs. siRNA Co. D: Effect of 6-h insulin treatment on Glut4 mRNA levels measured by real-time PCR inmyotubes silenced for Grp75 or Mfn2 (n = 3). **P, 0.01 vs. siRNA Co. E: Measurement of insulin-mediated glucose uptake in human myotubessilenced for Mfn2 (n = 4). Basal and insulin-stimulated values are summarized in Supplementary Table 11. a.u., arbitrary unit.

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Figure 6—FATE1-induced disruption of MAMs is sufficient to alter insulin action in skeletal muscle. A: Validation by Western blot that FATE1is not expressed in human myotubes and that its overexpression increases its protein levels. B: Representative PLA images (at top,original magnification 363 and scale bar = 20 mm) and quantitative analysis (below) of VDAC1-IP3R1 interactions in human myotubes trans-fected with pcDNA-T0 and pcDNA-FATE1-IRES-GFP vectors (n = 30 images in 3 independent experiments). **P , 0.005; ***P , 0.0001.C: Analysis of insulin-stimulated PKB phosphorylation by in situ PLA (pS473 PKB-PKB proximity) in human myotubes infected with Ad-GFP orAd-FATE1 for 48 h and stimulated or not with insulin (1027 mol/L, 15 min) (n = 30 images in 3 independent experiments). ***P , 0.0001 vs.control (Co); ##P , 0.001 vs. T0 insulin. Original magnification 363 and scale bar = 20 mm. D: Representative PLA images (at top) andquantitative analysis (below) of VDAC1-IP3R1 interactions in gastrocnemius muscle of Ad-GFP– or Ad-FATE1–infected mice 7 days post-infection (n = 30 images in 3 independent mice). ***P, 0.0005. E: Representative Western blots (top) and quantitative analysis (below) of insulin-stimulated PKB phosphorylation in gastrocnemius muscle of Ad-GFP–or Ad-FATE1–infected mice 7 days postinfection (n = 5/group). Basal andinsulin-stimulated values are summarized in Supplementary Table 12. *P , 0.05.

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(21), the reduction of ER-mitochondria interactions in all ofthese models could be related to mitochondrial alterations.Nevertheless, we firstly confirmed that the reduction ofMAM amount persists after normalization by mitochondriaamount in the subcellular fractionation of mouse muscle.Furthermore, we demonstrated that organelle miscommu-nication is an early event in diet-induced insulin resistancepreceding mitochondrial dysfunction and insulin resistance.Lastly, oligomycin-induced mitochondrial dysfunction inhuman myotubes rather increased SR/ER-mitochondriainteractions and potentiated insulin signaling, probably asan adaptive mechanism to improve mitochondrial functionand cell homeostasis. Altogether, these results suggest thatmuscle organelle miscommunication in the diabetic state isnot a consequence of mitochondrial alterations or of theinsulin-resistance state, supporting its participation inthe development of muscle insulin resistance. Nevertheless,future experiments are required to demonstrate that pre-venting MAM disruption could improve muscle insulinsensitivity in vivo.

The mechanisms linking organelle miscommunication tomuscle insulin resistance are currently unknown. Modula-tion of Ca2+ signaling and exchange between organellescould participate, as previously found in the liver (35). Ac-tivation of the UPR into the ER could also play a role,because we found here that the reinforcement of MAMs

through Mfn2 or Grp75 overexpression prevented the reg-ulation of UPR markers by palmitate. Nevertheless, becauseMfn2 was shown to directly modulate UPR (39), we cannotexclude an effect independent from MAMs. Furthermore,the involvement of ER stress in muscle insulin resistance ismore controversial than in the liver (38), and we previouslyfound that reducing ER stress by genetic or pharmacologicalapproaches is not sufficient to improve palmitate-inducedinsulin resistance in myotubes (21), suggesting that ERstress is probably not the link between MAM disruptionand muscle insulin resistance. Alternatively, intramyocellu-lar lipid accumulation has been associated to reducedmuscle insulin sensitivity (40). In this context, mitochondriadysfunction and the subsequent impaired ability to oxidizefatty acids could play an important role in muscle insulinresistance, although the causality of this association isstill controversial (1). We previously demonstrated (30)and confirmed here that mitochondrial alterations werenot an early event in the diet-induced development of in-sulin resistance, contrary to MAM disruption. Therefore,organelle miscommunication could participate to mitochon-drial dysfunction in skeletal muscle, subsequently leading toinsulin resistance. Finally, boosting mitochondrial functionmight be beneficial to muscle insulin sensitivity and patienthealth (41). Because organelle coupling is known to controlmitochondrial bioenergetics (42), MAM-mediated improve-ment of insulin action in human myotubes may be relatedto improved mitochondria function. However, furtherinvestigations are required to clarify this hypothesis.

Whereas organelle miscommunication recently emergedas a new mechanism of insulin resistance, the few reportedstudies on this issue are highly controversial. Indeed, weand others found that insulin resistance is associated withreduced ER-mitochondria contacts in the liver (6,7) and inadipose tissue (43), whereas another group found excessiveorganelle coupling in the liver of obese and diabetic mice(12). Of course, MAMs are dynamic structures strongly de-pendent on nutritional and metabolic status (27) as well ason environmental conditions and stress factors (44). Inaddition, their precise quantitative measurement is partic-ularly tricky, potentially depending on the methodology,and most importantly whether the measurement takesinto account the number, length, or thickness of the organ-elle coupling (45). That several types of mitochondria-ERcontact sites exist is likely, with potentially different proteincomplexes and functions, and it is plausible that these dif-ferent tethers could be differentially regulated, as recentlysuggested (46). Consequently, all of these factors probablyparticipate to the contrasting conclusions in this excitingtopic. Lastly, there are currently no good in vivo models ofMAM disruption or reinforcement and no optimal reporterto dynamically follow ER-mitochondria interactions, mak-ing studies more difficult. Further studies are thus requiredto solve this controversy, and the future use of the veryrecent split-GFP-based contact site sensor (SPLICS) tool tofollow narrow and wide organelle coupling (46) could bea relevant strategy. In the meantime and as a first step in

Figure 7—MAM integrity is altered in myotubes from insulin-resistantsubjects. Myotubes from lean healthy subjects (lean), obese subjectswithout diabetes (obese), and obese patients with T2D were analyzedfor ER-mitochondria interactions (A) and insulin-stimulated PKB phos-phorylation (B). *P , 0.05; **P , 0.01; ***P , 0.001 compared withlean subjects. C: Correlation between VDAC1-IP3R1 interactions andinsulin effect on PKB phosphorylation in myotubes from lean subjects,obese subjects, and subjects with T2D (n = 37).

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this quest, we confirmed here that FATE1-mediated organ-elle uncoupling reduced insulin-stimulated IRS2 and PKB phos-phorylation in HuH7 cells, as we previously observed when wemodulated endogenousMAM proteins (6). In this context, thecurrent study in skeletal muscle confirms that disruption oforganelle interactions is also associated with insulin resistance,in another insulin-sensitive tissue, and further extends theresults to humans, an aspect not investigated until now.

In summary, we demonstrated in vivo and in vitro thatdefective ER-mitochondria coupling is closely associatedwith impaired muscle insulin sensitivity in mice and humans.Furthermore, organelle miscommunication is an early eventin diet-induced insulin resistance preceding mitochon-drial dysfunction. Lastly, disruption of MAM integrityalters insulin signaling in vitro and in vivo, whereas reinforc-ing organelle coupling improves palmitate-induced insulinresistance in human myotubes. Taking into account thatsimilar observations were done previously in the liver (6),and although additional studies are required, especially todemonstrate that MAM reinforcement in muscle of insulin-resistant patients improves insulin action, the presenteddata pave the way for considering targeting the MAMinterface in insulin-sensitive tissues as a potential attractivestrategy to improve glucose homeostasis.

Acknowledgments. The authors thank Enzo Lalli (CNRS UMR7275,Valbonne, France) for the gift of pcDNA-T0 and pcDNA-FATE1 vectors, ElisabethErrazuriz for her technical help at the CIQLE Imaging Center (Lyon, France),Kassem Makki (INSERM U1060, Lyon, France) for his help with statisticalanalyses, Elsa Hoibian (INSERM U1060, Lyon, France) for her help with mice,Aurélie Vieille Marchiset (INSERM U1060, Lyon, France) for her help with culture ofhuman myotubes, and Mélanie Paillard (INSERM U1060, Lyon, France) for her helpfuldiscussion on FATE1.Funding. E.T. was supported by a research fellowship from French governmentof higher education and research and by a scholarship from Lund University. Thiswork was supported by INSERM, the Agence Nationale de la Recherche (ANR-09-JCJC-0116 to J.R.), and the “Fondation pour la Recherche Médicale”(DRM20101220461).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. E.T. and J.R. designed the experiments, researcheddata, contributed to discussion, and wrote the manuscript. S.C., N.B., G.B., M.-A.C.,J.J.-C., C.D., and D.G.-R. researched data. M.R. and E.L. constituted the DIOMEDEbiobank. G.B., H.V., and E.L., contributed to discussion and reviewed and edited themanuscript. J.R. is the guarantor of this work and, as such, had full access to all thedata in the study and takes responsibility for the integrity of the data and the accuracyof the data analysis.Prior Presentation. Parts of this study were presented in abstract form atthe Congress of the French Society of Diabetes, Lyon, France, 22–25 March2016; at the 2016 Nutrition Congress of the French Nutrition Society, Montpellier,France, 30 November–2 December 2016; and at the Research and Health Day ofINSERM, Paris, France, 22 November 2016.

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