Hypoxia in Combination With Muscle ContractionImproves Insulin Action and Glucose Metabolism inHuman Skeletal Muscle via the HIF-1a PathwaySven W. Görgens,1 Tim Benninghoff,2 Kristin Eckardt,1,3 Christian Springer,2 Alexandra Chadt,2

Anita Melior,1 Jakob Wefers,1 Andrea Cramer,1 Jørgen Jensen,4 Kåre I. Birkeland,5 Christian A. Drevon,3

Hadi Al-Hasani,2,6 and Jürgen Eckel1,6

Diabetes 2017;66:2800–2807 | https://doi.org/10.2337/db16-1488

Skeletal muscle insulin resistance is the hallmark oftype 2 diabetes and develops long before the onset of thedisease. It is well accepted that physical activity improvesglycemic control, but the knowledge on underlying mech-anismsmediating the beneficial effects remains incomplete.Exercise is accompanied by a decrease in intramuscularoxygen levels, resulting in induction of HIF-1a. HIF-1a is amaster regulator of gene expression and might play animportant role in skeletal muscle function and metab-olism. Here we show that HIF-1a is important for glucosemetabolism and insulin action in skeletal muscle. By usinga genome-wide gene expression profiling approach, weidentified RAB20 and TXNIP as two novel exercise/HIF-1a–regulated genes in skeletal muscle. Loss of Rab20impairs insulin-stimulated glucose uptake in human andmouse skeletal muscle by blocking the translocation ofGLUT4 to the cell surface. In addition, exercise/HIF-1a down-regulates the expression of TXNIP, a well-known negativeregulator of insulin action. In conclusion, we are the firstto demonstrate that HIF-1a is a key regulator of glucosemetabolism in skeletal muscle by directly controlling the tran-scription of RAB20 and TXNIP. These results hint toward anovel function of HIF-1a as a potential pharmacologicaltarget to improve skeletal muscle insulin sensitivity.

Skeletal muscle is one of the largest organs in the body andplays a major role during whole-body glucose homeostasis.Insulin resistance (IR) of skeletal muscle is one early hallmark

in the development of type 2 diabetes (T2D) (1). Therefore,the identification of novel targets for improving skeletalmuscle IR represents a fundamental challenge and plays akey role in developing novel therapeutic strategies for T2D.In contrast to the very limited nonexercise-based strategiesto combat IR, physical activity is well known to exert mul-tiple beneficial effects on the progression of IR and T2D(2,3). However, our knowledge on the cellular mechanismsmediating these adjuvant actions of physical activity re-mains incomplete. Exercise is accompanied by a reduction inintramuscular oxygen tension, resulting in enhanced HIF-1aprotein expression (4). In response to cellular hypoxia,HIF-1a is activated to regulate the transcription of .100target genes, e.g., glycolytic enzymes and glucose transporters(5). Furthermore, it could be shown that loss of HIF-1a inmurine skeletal muscle cells impairs GLUT4 translocationand glucose uptake, indicating a possible role for HIF-1a inthe regulation of skeletal muscle glucose metabolism (6).One particular aspect of skeletal muscle is that HIF-1a pro-tein is highly expressed in this tissue even in normoxicconditions, suggesting that HIF-1a function on muscle ho-meostasis is not exclusively dependent on the oxygen stateof the muscle cell (7). Importantly, endurance exercise un-der hypoxia is known to be even more efficient to improveglycemic control in individuals with T2D (8), potentiallyreflecting the synergistic or additive regulation of HIF-1adownstream target genes. Therefore, we hypothesized that the

1Paul-Langerhans-Group for Integrative Physiology, German Diabetes Center(DDZ), Düsseldorf, Germany2Institute for Clinical Biochemistry and Pathobiochemistry, German DiabetesCenter (DDZ), Düsseldorf, Germany3Department of Nutrition, Institute of Basic Medical Sciences, Faculty of Medi-cine, University of Oslo, Oslo, Norway4Department of Physical Performance, Norwegian School of Sport Sciences, Oslo, Norway5Department of Endocrinology, Morbid Obesity, and Preventive Medicine, OsloUniversity Hospital and University of Oslo, Oslo, Norway6German Center for Diabetes Research (DZD e.V.), Düsseldorf, Germany

Corresponding author: Jürgen Eckel, eckel@uni-duesseldorf.de.

Received 1 December 2016 and accepted 8 August 2017.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db16-1488/-/DC1.

© 2017 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.

2800 Diabetes Volume 66, November 2017

METABOLISM

molecular analysis of hypoxic muscle contraction may pave theway to identify novel targets for improving skeletal muscle IR.

RESEARCH DESIGN AND METHODS

Culture of Human Skeletal Muscle CellsPrimary human skeletal muscle cells isolated from eighthealthy Caucasian donors (four males, 16, 21, 41, and 47 yearsof age; four females, 16, 25, 33, and 37 years of age) weresupplied as proliferating myoblasts and cultured as de-scribed previously (9).

Electrical Pulse StimulationDifferentiated myotubes were subjected to electrical pulsestimulation (EPS) as recently described (9).

Combination of EPS and Reduced Oxygen TensionMyotubes were cultured for 24 h with or without EPS in anatmosphere containing 21, 7, or 2% O2 supplemented with5% CO2 and respective concentrations of nitrogen in anXvivo hypoxia chamber system (Biospherix, Parish, NY).

Glucose Uptake and OxidationGlucose uptake and oxidation assays were performed asdescribed previously (9).

Real-time PCR and Western BlottingWe used predesigned primers (Qiagen) in a SYBR Green–based quantitative real-time PCR. Thioredoxin-interactingprotein (TXNIP), HIF-1a, anti–phospho AktSer473, anti–phospho AMPKaThr172, anti–phospho AktThr308, anti–phospho AS160Thr642, anti–phospho GSK3a/bSer21/9, totalAkt, AMPKa, GSK3a/b, and total AS160 antibodies weresupplied by Cell Signaling Technology (Frankfurt, Germany).Polyclonal Rab20 antibody was supplied by Abcam (Cambridge,U.K.). Polyclonal antiserum against GLUT4 was describedpreviously (10).

Silencing ExperimentsSilencing experiments were performed by using FlexiTubesmall interfering RNA (siRNA) and HiPerfect (Qiagen) accord-ing to the manufacturer’s instructions. Control cells weretreated with AllStars Negative Control siRNA (Qiagen).

Immunofluorescence StainingImmunofluorescence staining was performed as describedpreviously (9).

Lactate AssayL-(+)-lactate was detected in the supernatant fraction with akit (Lactate Assay Kit II; BioVision).

GLUT4 Translocation AssayGLUT-myc translocation assay was performed as describedpreviously (11).

Cell Membrane FractionationThe protocol is described in detail by Zhao et al. (12).

In Vivo Muscle Electroporation and Glucose Uptake inIsolated Skeletal MuscleIn vivo muscle electroporation and ex vivo glucose uptakewere adapted from previous reports (13).

Human Study: 12-Week Exercise Intervention StudyThis human exercise intervention study is in detail describedby Langleite et al. (14).

Mouse Exercise StudyC57BL/6J mice were trained 5 days per week for 6 weeksduring their active dark phase. Each training sessionconsisted of alternating 5-min intervals with moderate tohigh (15–18 m/min) or low running intensity (10 m/min),respectively. Mice were sacrificed at least 24 h after theirlast exercise bout. Gastrocnemius muscle was used for theanalysis of TXNIP and Rab20 protein or mRNA expression.

High-Throughput mRNA Sequencing and DifferentialExpression AnalysisHuman skeletal muscle cells were treated with 300 mmol/Lcobalt chloride (CoCl2) for 6 h, and RNA sequencing anddata analysis were performed by the GATC Biotech GmbH(Konstanz, Germany).

RESULTS

7% O2 and Human Myotube Contraction Improve InsulinActionWe cultured human myotubes at different oxygen levels (21,14, 7, and 2%) with or without EPS for 24 h and analyzedbasal and insulin-stimulated glucose uptake (Fig. 1D andSupplementary Fig. 1A and B). Only EPS in combinationwith 7% O2 significantly increased insulin-stimulated glu-cose uptake. In accordance, we observed increased insulinsignaling (Fig. 1A–C). However, EPS in combination with7% O2 enhanced the EPS-induced lactate release, whereasglucose oxidation was not affected (Supplementary Fig. 2Aand B). In addition, we observed a significant increase ofHIF-1a protein levels at 7% O2 and in combination withEPS (Fig. 1A). The well-known HIF-1a target GLUT1 wasnot regulated at these conditions (Supplementary Fig. 3B).However, GLUT4 mRNA expression was significantly upre-gulated in the combined setting of 7% O2 and EPS (Supple-mentary Fig. 3C).

Loss of HIF-1a Inhibits Insulin Action and Contraction-Stimulated Glucose UptakeSilencing of HIF-1a (siHIF1A) in human myotubes led toa downregulation of insulin-mediated AktSer473 phosphory-lation and substantially reduced the phosphorylation ofAS160Thr642, whereas insulin receptor bTyr1150/1151 phos-phorylation was not affected (Fig. 1E). In line, in vivo elec-troporation of siHif1a into murine soleus muscle abrogatedinsulin-mediated AS160Thr642 phosphorylation (Supplemen-tary Fig. 4B–D). Insulin- and EPS-stimulated glucose uptakewere strongly inhibited by HIF-1a silencing in human myo-tubes (Fig. 1F). In addition, insulin-stimulated ex vivo glu-cose uptake was inhibited after in vivo electroporation ofsiHif1a into murine soleus muscle (Supplementary Fig. 4A).

Upregulation of HIF-1a Improves Insulin ActionCoCl2 substantially augments HIF-1a protein levels (Fig.1G). In addition, we observed a strong increase in insulin-induced AktSer473 as well as basal and insulin-mediated

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Figure 1—HIF-1a regulates glucose and insulin action and the transcription of Rab20 and TXNIP. A–D: Differentiated human myotubes werecultured at 21 or 7% O2 with or without EPS for 24 h followed by insulin stimulation (100 nmol/L) for 30 min. A: Representative Western blotimages show phosphorylation of AktSer473 and AS160Thr642 and total HIF-1a and Akt protein levels. B and C: Quantification of AktSer473 andAS160Thr642 phosphorylation. D: Glucose uptake was assessed for 2 h. E: Representative Western blot images show phosphorylation of insulinreceptor b-subunit IRbTyr1150/1151, AktSer473, and AS160Thr642 and total HIF-1a, Akt, and AS160 protein levels of untreated cells (control) or aftertreatment with 40 nmol/L AllStars Negative Control siRNA (NT) or HIF-1a siRNA (siHIF1A) for 24 h. F: siRNA-transfected cells were cultured at7% O2 with or without EPS for 24 h followed by insulin stimulation (100 nmol/L) for 30 min. Glucose uptake was assessed for 2 h. G: Humanmyotubes were treated with 300 mmol/L CoCl2 for 6 h followed by insulin stimulation (100 nmol/L) for 10 min. Representative Western blotimages show phosphorylation of AktSer473 and AS160Thr642 and total HIF-1a, GLUT1, and GLUT4 protein levels. H: Glucose uptake after CoCl2treatment. I: Heat map of the top-regulated genes after 6 h of CoCl2 (300 mmol/L) treatment. J and K: RAB20mRNA expression was analyzed byreal-time PCR after indicated treatment, and data were normalized to ACTB mRNA expression. L: Quantification of Rab20 protein abundanceafter CoCl2 and hypoxic treatment. M: Representative Western blot images show Rab20 and TXNIP protein levels after indicated treatment. Nand O: TXNIP mRNA expression was assessed by real-time PCR. P: Quantification of TXNIP protein abundance after CoCl2 and hypoxictreatment. Data are mean values 6 SEM, n = 6. *P < 0.05, **P < 0.005, and ***P < 0.0001 vs. corresponding control or as indicated (ANOVA).#P < 0.05, as indicated for single comparisons (Student t test). All experiments were performed with cells from at least six different donors.2-DOG, 2-Deoxy-D-glucose; AU, arbitrary units.

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AS160Thr642 phosphorylation (Fig. 1G). Both basal andinsulin-regulated glucose uptake were increased (Fig. 1H).Pathway analysis from RNA sequencing data indicatedthat CoCl2 treatment specifically induced HIF-1a signaling(Supplementary Fig. 5A–C). The top 15 upregulated and5 downregulated genes are displayed as a heat map (Fig.1I). RAB20 was one of the most upregulated genes, and real-time PCR and Western blot data confirmed that Rab20 isregulated by CoCl2 and hypoxia (Fig. 1J–M). Compared withall other detectable Rab genes in human myotubes, Rab20showed by far the strongest increase after CoCl2 treatment(Supplementary Fig. 6). In contrast, TXNIP was the mostdownregulated gene after CoCl2 treatment (Fig. 1I). Valida-tion by real-time PCR and Western blot confirmed thatTXNIP is regulated by hypoxia in human myotubes (Fig.1N–P).

Rab20 Is Upregulated During Myogenesis and RegulatesGlucose UptakeRab20 expression was upregulated during human skeletalmuscle cell differentiation (Fig. 2A and B). Knockdown ofRab20 inhibited the insulin- and contraction-stimulatedglucose uptake (Fig. 2C). In line, in vivo electroporation ofsiRab20 in murine soleus muscle inhibited insulin-mediatedglucose uptake (Fig. 2D). Abrogation of insulin-stimulatedglucose uptake was associated with impaired mobilizationof GLUT4 to the plasma membrane in human myotubes(Fig. 2K and L). In addition, siRab20 treatment reducedinsulin-mediated GLUT4-myc translocation to the cell sur-face in L6 myoblast overexpressing AS160 and GLUT4-myc(Fig. 2H and I). Importantly, Rab20 was not colocalized withGLUT4 after insulin stimulation (Fig. 2J). Insulin signalingat the level of Akt and AS160 phosphorylation was notaltered by Rab20 silencing (Fig. 2E–G).

Rab20 Is Regulated by ExerciseRAB20 mRNA expression was upregulated after acute andlong-term exercise (Fig. 3A and D). Only RAB20 was en-hanced after acute exercise compared with all other detect-able Rab genes in human skeletal muscle biopsies (Fig. 3G).Also HIF1A mRNA expression was significantly upregulatedby acute and chronic exercise (Supplementary Fig. 7A–D),and HIF1A mRNA expression was positively correlated withRAB20 mRNA expression (Fig. 3B). The increase of RAB20mRNA expression was positively correlated with the increaseof HIF1A mRNA expression after acute exercise (Fig. 3C).Furthermore, skeletal muscle RAB20mRNA expression inthe control group was positively correlated with glucoseinfusion rate (GIR) (Fig. 3E and F). In addition, exercisetraining increased Rab20 in murine gastrocnemius muscle(Fig. 3H–J).

TXNIP Is Downregulated by Exercise TrainingAcute exercise induced TXNIP in both groups, wherebythe control group showed a significantly higher induction(Fig. 4A and B). However, long-term exercise tended to re-duce the expression of TXNIP mRNA in skeletal muscle(control; P = 0.055) (Fig. 4C and D). Moreover, TXNIP

mRNA expression was negatively correlated with the GIR(Fig. 4E). In addition, endurance training reduced TxnipmRNA expression as well as protein levels in murine muscle(Fig. 4I and J). Importantly, the loss of TXNIP in humanmyotubes by siRNA enhanced insulin-stimulated Akt as wellas As160 phosphorylation (Fig. 4F–H).

DISCUSSION

The observation that endurance exercise under hypoxic con-ditions is more efficient to improve glycemic control (8)potentially reflects additive regulation of HIF-1a downstreamtarget genes. We show that HIF-1a protein levels are sig-nificantly increased in the combined setting of muscle con-traction and hypoxia, indicating that reduced oxygentension and muscle contraction exert an additive effect onHIF-1a stabilization. Furthermore, we show that HIF-1aimproves glucose metabolism and insulin action in humanskeletal muscle, which is in line with previous findingsshowing that HIF-1a regulates insulin-stimulated glucoseuptake in murine skeletal muscle cells (6). Collectively, thesedata identify HIF-1a as an important regulator of insulin-mediated glucose metabolism.

To analyze the underlying mechanism, we used a genome-wide gene expression profiling approach of cells treated withCoCl2. We show that Rab20 is expressed in skeletal muscle,and the expression is induced by HIF-1a. In accordance,Hackenbeck et al. (15) reported that RAB20 is a direct targetof HIF-1a. The fact that RAB20 expression is substantiallyupregulated by exercise in skeletal muscle underpins thebiological significance. Furthermore, skeletal muscle RAB20mRNA expression was positively correlated with whole-body insulin sensitivity, indicating a potential role of Rab20for skeletal muscle glucose metabolism. In line, loss of Rab20inhibited insulin- and contraction-stimulated glucose uptake,which was associated with a reduction of GLUT4 transloca-tion. A change in the proportion of GLUT4 at the cell surfacemight be caused by alterations in the endocytosis/exocytosisrate. Rab8A and Rab10 are involved in vesicle tethering viainteraction with the exocyst, and Rab5 is involved in sortingof GLUT4 from the recycling endosome to the insulin-sensitivecompartment (16–18). Rab20 is colocalized with Rab5 onendosomal membranes (19). Furthermore, Rab20 was shownto interact with Rabex-5, a guanine nucleotide exchangefactor for Rab5 (20). Thus, we can speculate that high levelsof Rab20 during exercise activate Rab5, which in turnincreases recycling of endosomes to the insulin-sensitivecompartment.

The mechanism underlying HIF-1a–mediated regulationof Akt or AS160 phosphorylation is still unclear. The well-known recognized function of HIF-1a consists of transcrip-tional regulation, and therefore it is most unlikely thatHIF-1a directly affects the phosphorylation process of Aktor downstream targets. Our data indicate that TXNIP isstrongly downregulated by HIF-1a. Furthermore, downre-gulation of TXNIP in human myotubes results in enhancedinsulin-stimulated Akt and downstream signaling. In line,whole-body knockout of TXNIP in mice resulted in enhanced

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Figure 2—Rab20 regulates insulin- and contraction-stimulated glucose uptake. A: Representative Western blot images show Rab20, GLUT1,GLUT4, and IRb during myogenesis. B: RAB20, SLC2A1, and SLC2A4 mRNA expression were analyzed by real-time PCR, and data werenormalized to ACTBmRNA expression. C: Glucose uptake after treatment with 40 nmol/L AllStars Negative Control siRNA (NT) or RAB20 siRNA(siRAB20) was assessed for 2 h. D: Insulin-stimulated glucose uptake in isolated mouse soleus muscle after in vivo electroporation with siRNAagainst Rab20 (siRab20). Data are mean values 6 SEM, n = 4–6. *P < 0.05 (ANOVA). E: Representative Western blot images show phosphor-ylation of AktSer473 and AS160Thr642 and total Rab20 protein levels after indicated treatment. F and G: Quantification of AS160Thr642 and AktSer473

phosphorylation. H: Fluorescent on-cell Western image of GLUT4-myc from siRab20-treated L6 myoblasts overexpressing GLUT4-myc andAS160 stimulated with or without insulin (50 nmol/L) for 1 h. I: Quantification of GLUT4-myc intensity. J: Immunofluorescence staining of Rab20(red), GLUT4 (green), DAPI (blue), andmerged images of differentiated humanmyotubes under basal and insulin-stimulated conditions (100 nmol/L for30min). Detection of GLUT4 trafficking to the plasmamembrane in human skeletal muscle cells. Cells were treated with 100 nmol/L insulin for 30min,and GLUT4 levels in the plasma membrane fraction and total cell lysate were detected by Western blot. Boxes indicate picture detail. K: Repre-sentative Western blot images show Rab20 and GLUT4 protein levels. The contents of IRb and b-actin were confirmed as a marker for plasmamembrane fraction or total cell lysate, respectively. L: Quantification of GLUT4 protein levels in the plasma membrane or in total cell lysate. M:RAB20, SLC2A1, and SLC2A4 mRNA expression after siRAB20 treatment. Data are mean values6 SEM, n = 6–8. *P < 0.05, **P < 0.005, and***P < 0.0001 vs. corresponding control or NT (ANOVA). #P < 0.05, as indicated for single comparisons (Student t test). All experiments wereperformed with cells from at least six different donors. 2-DOG, 2-Deoxy-D-glucose; AU, arbitrary units; FC, fold change.

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Figure 3—Rab20 is regulated by exercise in a HIF-1a–dependent manner. Changes in skeletal muscle RAB20 mRNA expression in healthysedentary men (control) and in participants with abnormal glucose metabolism (pT2D) in response to exercise. A: Skeletal muscle biopsies wereobtained before (pre-ex) and immediately after (post-ex) exercise of 45 min ergometer cycling (70% VO2max). B: Linear regression analysisof RAB20 and HIF1A mRNA expression. Data are collected from baseline and after the intervention (12 weeks). C: Linear regression analysis ofexercise-induced RAB20 and HIF1A mRNA expression. D: Muscle biopsies were taken at baseline and after the long-term training period of12 weeks (control, n = 13; pT2D, n = 11). Samples were processed for mRNA sequencing, and normalized gene expression levels are expressedin fragments per kB mapped reads (FPKM). Bars depict means 6 SEM. Correlation between RAB20 mRNA expression and GIR (E), and linearregression analysis of the increase of RAB20 and the improvements in GIR after the 12-week exercise intervention (F). G: Quantification of themRNA expression of all detectable RAB molecules in human skeletal muscle biopsies in response to acute exercise (n = 24). Data are meanvalues 6 SEM. H: Representative Western blot images show Rab20 protein levels in murine gastrocnemius muscle from sedentary and trained(6-week endurance exercises) mice. I: Quantification of Rab20 protein levels (sedentary, 14 animals; trained, 10 animals). J: Rab20 mRNAexpression in murine gastrocnemius muscle (sedentary, 4 animals; trained, 4 animals). *P < 0.05, as indicated; **P < 0.005 and ***P < 0.0001between pre-exercise values and after exercise. AU, arbitrary units.

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Akt signaling and insulin sensitivity (21). Furthermore, weshow that exercise training is able to reduce TXNIP expres-sion in muscle and that muscle TXNIP mRNA expressioncorrelates negatively with the GIR. Johnson et al. (22) dem-onstrated that caloric restriction improves insulin sensitivity,which was also associated with downregulation of TXNIPin the muscle. Therefore, downregulation of TXNIP in

muscle could improve insulin signaling as well as glucosemetabolism in humans.

In conclusion, this study provides further insights intothe molecular regulation of glucose metabolism in skeletalmuscle during exercise, implicating a key role for thetranscription factor HIF-1a in this process. Therefore, wesuggest that the skeletal muscle HIF-1a signaling pathway

Figure 4—TXNIP is downregulated by exercise training. Changes in skeletal muscle TXNIPmRNA expression in healthy sedentary men (control)and in participants with abnormal glucose metabolism (pT2D) in response to exercise. A and B: Skeletal muscle biopsies were obtained before(pre-ex) and immediately after (post-ex) exercise of 45 min ergometer cycling (70% VO2max). C and D: Muscle biopsies were taken at baselineand after the long-term training period of 12 weeks (control, n = 13; pT2D, n = 11). Bars depict means 6 SEM. **P < 0.01 and ***P < 0.001between pre-exercise values and after exercise. E: Correlation between TXNIPmRNA expression and GIR. Data are collected from baseline andafter the intervention (12 weeks). F–G: Quantification of insulin-stimulated AktSer473 and AS160Thr642 phosphorylation in human skeletal musclecells after TXNIP silencing (siTXNIP). H: Representative Western blot images show phosphorylation of AktSer473 and AS160Thr642 and total TXNIP,Akt, and AS160 protein levels. Data are mean values6 SEM, n = 6. *P< 0.05, as indicated (ANOVA). All experiments were performed with cellsfrom at least six different donors. I: Representative Western blot images show TXNIP protein levels in murine gastrocnemius muscle fromsedentary and trained (6-week endurance exercise) mice. J: Quantification of TXNIP protein levels (sedentary, 14 animals; trained, 10 animals).***P< 0.001 between pre-exercise values and after exercise. K: TxnipmRNA expression in murine gastrocnemius muscle (sedentary, 4 animals;trained, 4 animals). Bars depict means6 SEM. *P< 0.05, as indicated (ANOVA). AU, arbitrary units; FPKM, fragments per kB mapped reads; NT,AllStars Negative Control siRNA.

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may represent a novel therapeutic target to improve insulinsensitivity in humans.

Acknowledgments. The authors thank A. Heck and B. Nellemann (Departmentof Physical Performance, Norwegian School of Sport Sciences) for taking the biopsiesand A.R. Enget, A. Kielland, K.J. Kolnes, D.S. Tangen, T.I. Gloppen, T. Dalen,H. Moen, M.A. Dahl, G. Grøthe, E. Johansen, K.A. Krog, and O. Skattebo (Departmentof Physical Performance, Norwegian School of Sport Sciences and Department ofNutrition, University of Oslo) for being responsible for and helping out on differentaspects of the human strength and endurance intervention. The authors thankN. Tennagels and C.W. Jung (Sanofi Deutschland GmbH) for providing the GLUT4-myc L6 myoblasts. The technical assistance of M. Koenen and the secretarialassistance of B. Hurow (Paul-Langerhans-Group for Integrative Physiology, GermanDiabetes Center [DDZ]) are gratefully acknowledged.Funding. This work was supported by the Ministry of Science and Research ofthe State of North Rhine-Westphalia, the Federal Ministry of Health, and the GermanAcademic Exchange Service (project no. 57068553). K.E. is supported by theDeutsche Forschungsgemeinschaft (EC 440/1-1 and EC 440/2-1).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. S.W.G. and J.E. contributed to the concept; designedand performed the experiments; acquired, analyzed, and interpreted data; and wrotethe manuscript. T.B., K.E., C.S., A.Ch., A.M., J.W., A.Cr., and K.I.B. performed theresearch and contributed to analysis and interpretation of data. J.J., C.A.D., and H.A.-H.performed the research, contributed to analysis and interpretation of data, andreviewed and edited the manuscript. All authors approved the final version of themanuscript. S.W.G. is the guarantor of this work and, as such, had full access to allthe data in the study and takes responsibility for the integrity of the data and theaccuracy of the data analysis.

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