Elisabetta Trevellin,1 Michele Scorzeto,2 Massimiliano Olivieri,1 Marnie Granzotto,1 Alessandra Valerio,3

Laura Tedesco,4 Roberto Fabris,1 Roberto Serra,1 Marco Quarta,2 Carlo Reggiani,2 Enzo Nisoli,4

and Roberto Vettor1

Exercise Training InducesMitochondrial Biogenesis andGlucose Uptake inSubcutaneous Adipose TissueThrough eNOS-DependentMechanismsDiabetes 2014;63:2800–2811 | DOI: 10.2337/db13-1234

Insulin resistance and obesity are associated witha reduction of mitochondrial content in various tissues ofmammals. Moreover, a reduced nitric oxide (NO) bioavail-ability impairs several cellular functions, including mito-chondrial biogenesis and insulin-stimulated glucose up-take, two important mechanisms of body adaptation inresponse tophysical exercise.Although thesemechanismshave been thoroughly investigated in skeletal muscle andheart, fewstudieshavefocusedontheeffectsofexerciseonmitochondria and glucosemetabolism in adipose tissue. Inthis study, we compared the in vivo effects of chronicexercise in subcutaneous adipose tissue of wild-type (WT)and endothelial NO synthase (eNOS) knockout (eNOS2/2)mice after a swim training period. We then investigated thein vitro effects of NO on mouse 3T3-L1 and human subcu-taneous adipose tissue–derived adipocytes after a chronictreatmentwithanNOdonor:diethylenetriamine-NO(DETA-NO). We observed that swim training increases mitochon-drial biogenesis, mitochondrial DNA content, and glucoseuptake in subcutaneous adipose tissue of WT but noteNOS2/2 mice. Furthermore, we observed that DETA-NOpromotes mitochondrial biogenesis and elongation, glu-cose uptake, and GLUT4 translocation in cultured murineand human adipocytes. These results point to the crucial

role of the eNOS-derived NO in the metabolic adaptationof subcutaneous adipose tissue to exercise training.

Reduced mitochondrial content and/or activity is associ-ated with impaired cell function in several diseases (1,2).In particular, it has been hypothesized that mitochondrialimpairment may be involved in the pathogenesis of obe-sity and insulin resistance and their progression towardtype 2 diabetes (3,4), even though the role of mitochon-dria in health and disease is still under discussion (5,6). Atthe same time, metabolic disorders are also associatedwith a reduction of endothelial nitric oxide synthase(eNOS) enzymatic activity (7,8); in fact, mice lacking theeNOS gene are considered a useful murine model for met-abolic syndrome because they display typical features, in-cluding hypertension, hypertriglyceridemia, endothelialdysfunction, insulin resistance, and visceral obesity (9).

It is well known that physical exercise inducesprofound physiological adaptations in several tissues asa response to increased metabolic requirements. One ofthe major events induced by physical activity is theupregulation of eNOS gene expression and the consequent

1Internal Medicine 3, Endocrine-Metabolic Laboratory, Department of MedicineDIMED, University of Padua, Padua, Italy2Department of Biomedical Sciences, University of Padua, Padua, Italy3Department of Molecular and Translational Medicine, University of Brescia,Brescia, Italy4Center for Study and Research on Obesity, Department of Medical Biotechnologyand Translational Medicine, University of Milan, Milan, Italy

Corresponding author: Roberto Vettor, roberto.vettor@unipd.it.

Received 12 August 2013 and accepted 9 March 2014.

E.N. and R.V. contributed equally to this work.

M.Q. is currently affiliated with the Department of Neurology and NeurologicalSciences, School of Medicine, Stanford University, Stanford, CA.

© 2014 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, andthe work is not altered.

See accompanying article, p. 2606.

2800 Diabetes Volume 63, August 2014

PATHOPHYSIO

LOGY

increase of tissue nitric oxide (NO) production, which inturn induces mitochondrial biogenesis and cell glucoseuptake in skeletal and cardiac muscle (10–12). Moreover,a selective depletion of peroxisome proliferator–activatedreceptor-g (PPAR-g) coactivator 1a (PGC-1a), a key reg-ulator of mitochondrial biogenesis, leads to a blunting ofexercise-induced increases in mitochondrial respiratorychain proteins in muscle (13,14). Thus, a lack of re-sponse to physical exercise in mitochondrial biogenesiscould be related to reduced or impaired NO metabolism.

Insulin sensitivity is also increased after a trainingperiod, and a single bout of exercise could enhance basalglucose uptake by increasing GLUT4 translocation to thecell membrane of skeletal (15) and cardiac (16) myocytes.Similar results have been observed in mouse and humantissues not directly involved in mechanical work and ox-idative processes (17), but few studies have focused onsubcutaneous adipose tissue. The capacity to oxidize fuelsubstrates to meet the energy demand is increased intissues involved in contractile activity during exercise,such as heart and skeletal muscles; therefore, lipid andglucose uptake and oxidation need to be increased.

Emerging evidence suggests that these adaptationsoccur not only in working tissues, such as skeletal muscleand heart, but also in white adipose tissue (WAT) andbrown adipose tissue, liver, brain, and kidney. Severalstudies show that exercise induces a “browning effect” inWAT by increasing mitochondrial protein activity (18) orbrown adipocyte–specific gene expression (19). A recentstudy hypothesized that this effect might be mediated byirisin (FNDC5), a PGC-1a–dependent myokine secretedduring physical activity that promotes thermogenesisand uncoupling processes in white adipocytes, whereasleptin and other key regulator genes of “white” develop-ment are downregulated (20). Despite numerous resultssupporting this hypothesis, the role of irisin is still con-troversial (21) and requires further investigations.

The aim of the current study was to investigate themechanism underlying the response of subcutaneous adi-pose tissue to physical exercise in terms of mitochondrialbiogenesis and glucose uptake. In particular, we exploredwhether NO can play a role in such metabolic adaptations.

RESEARCH DESIGN AND METHODS

Mice and Exercise ProtocolThirty-six adult (8 weeks old) male wild-type (WT; C57BL/6J) and eNOS2/2 (B6.129P2-Nos3tm1Unc/J) mice (all fromThe Jackson Laboratory) were treated according to theEuropean Union guidelines and with the approval of theinstitutional ethical committee. Body weight and foodconsumption were monitored throughout the experimen-tal period. WT and eNOS2/2 mice (n = 18 mice per group)were assigned randomly to swim training (12) or to haveno lifestyle modifications. Mice swam once a day for5 days/week in a graduated protocol starting at 10 mindaily, with a 10-min increase each day until 90 min dailyat the end of the second week. Then, mice swam 30 days

on the full training regimen (90 min daily, 5 days/week).Swim sessions were supervised, water temperature wasmaintained between 30°C and 35°C, and mice were care-fully towel-dried after each training session.

Muscle Contractile Performance In VivoMuscle strength developed by WT and eNOS2/2mice duringinstinctive grasp was measured with the protocol indicatedas grip test (22). Briefly, the mouse was held by the tail neara trapeze bar connected to the shaft of a force transducer.Once the mouse had firmly grabbed the trapeze, a gentle pullwas exerted on the tail. The measurement of the peak forcegenerated by the mouse was repeated several times, withappropriate intervals to avoid fatigue, and average peak forcevalues were expressed relative to body mass. Endurance wasmeasured with a test to exhaustion on a treadmill. Initialspeed (5 cm/s) was increased after 2 min at 10 cm/s. Thespeed was then increased by 2 cm/s every minute up to 50cm/s, and time to exhaustion was recorded.

Tissue Glucose Utilization IndexAt the end of the training period and 2 days before theclamp studies, a catheter was inserted into the right femoralvein under general anesthesia with sodium pentobarbital.Tissue glucose uptake studies were performed on mice underconscious and unstressed conditions after an 8-h fast. Aspreviously described, with minor modifications (23), 10 mCiof the nonmetabolizable glucose analog 2-deoxy-D-[1-3H]glucose ([3H]-DG) (Amersham Biosciences) was injected asan intravenous bolus in the basal condition or after a hyper-insulinemic euglycemic clamp. Animals were killed 120 minafter the tracer injection, and subcutaneous adipose tissuefrom the inguinal fat pad was quickly collected in liquidnitrogen and kept at 280°C for subsequent analysis. Theglucose utilization index was derived from the amount of[3H]-DG-6-phosphate ([3H]-DGP) measured in adipose tis-sue as previously described (24), thus using the accumula-tion of [3H]-DGP as an index of the glucose metabolic rate.

Norepinephrine TreatmentA group of sedentary mice (n = 10 WT and n = 10 eNOS2/2)of the same strains as those used for the training experi-ments received a single intraperitoneal injection of nor-epinephrine hydrogen tartrate (5 mg/kg; Galenica Senese)or vehicle (0.9% saline solution). Animals were killed 24 hafter the norepinephrine injection, and the subcutaneousinguinal fat pad was quickly removed and kept at 280°Cfor subsequent mRNA expression analysis. An additionalgroup of sedentary mice (n = 10 WT and n = 10 eNOS2/2)received the same norepinephrine treatment and werekilled 30 min after the intraperitoneal injection. Theinguinal fat pad was quickly removed and kept at 280°Cto quantify protein expression and phosphorylation.

Immunoblot AnalysisProteins were isolated from inguinal subcutaneous adi-pose tissues using T-PER Mammalian Protein ExtractionReagent (Pierce), as indicated by the manufacturer, inthe presence of 1 mmol/L NaVO4, 10 mmol/L NaF, and

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a cocktail of protease inhibitors (Sigma-Aldrich). Proteincontent was determined by the bicinchoninic acid pro-tein assay (Pierce), and 70 mg proteins were run on SDS-PAGE under reducing conditions. The separated proteinswere then electrophoretically transferred to a nitrocel-lulose membrane (Pierce). Proteins of interest wererevealed with specific antibodies: anti-GLUT4, anti-AKT,anti–phospho-AKT, anti-p44/42 mitogen-activated proteinkinase (MAPK) extracellular signal-regulated kinases 1and 2 (ERK1/2), anti–phospho-ERK1/2, anti–hormone-sensitive lipase (HSL), anti–phospho-HSL (all from CellSignaling), and anti–b-actin (Sigma-Aldrich). The immu-nostaining was detected using horseradish peroxidase-conjugated anti-rabbit or anti-mouse Ig for 1 h at roomtemperature. Bands were revealed by the SuperSignal Sub-strate (Pierce) and quantified by densitometry usingImageJ software (National Institutes of Health).

Cell Cultures and TreatmentStromal vascular fraction from subcutaneous adipose tissueof five healthy patients undergoing bariatric surgery wasisolated as previously described (25). Human-derived and3T3-L1 (ATCC CL-173) preadipocytes were plated in Dulbecco’smodified Eagle’s medium, supplemented with 10% FBS, 150units/mL streptomycin, 200 units/mL penicillin, 2 mmol/Lglutamine, and 1 mmol/L HEPES (all from Life Technolo-gies). At confluence, adipogenic differentiation was inducedby adding 1 mmol/L dexamethasone, 0.5 mmol/L 3-isobutyl-1-methyl-xantine (IBMX, Sigma-Aldrich), and 70 nmol/L in-sulin (Novo Nordisk). IBMX was removed frommedium after3 days of culture. Cells were cultured at 37°C in a humidifiedatmosphere of 5% CO2 until fully differentiated. Cells wereexposed to 100 mmol/L diethylenetriamine-NO (DETA-NO;Sigma-Aldrich), a potent NO donor (26), or vehicle for 72 hand then harvested for further analysis.

Cell Glucose UptakeHuman-derived and 3T3-L1 mature adipocytes wereserum-starved for 8 h, incubated in the presence or absenceof 2 mmol/L insulin (Novo Nordisk) for 30 min, and thenwith 1.5 mCi/mL [3H]-DG (Amersham Biosciences) for15 min. Cells were washed with ice-cold PBS and lysed in0.5 mol/L NaOH. Radioactivity was measured by liquidscintillation counting (Wallac). Each condition was assayedin three independent experiments in triplicate.

RNA Isolation and Real-Time Quantitative PCRTotal RNA was isolated from cultured adipocytes andfrozen subcutaneous adipose tissues of mice using RNeasyMini kit or RNeasy Lipid Tissue Mini Kit (Qiagen),respectively, treated with DNase (TURBO-DNase-free,Ambion), and reverse-transcribed using random primers(Promega) and M-MLV reverse transcriptase (Promega).mRNA levels were measured by real-time quantitative PCR(qPCR) (DNA Engine Opticon2, MJ Research) using SYBRGreen PCR Master Mix (Applied Biosystems) and specificintron-spanning primers according to the manufacturer’s

instructions. All data were collected in triplicate and nor-malized to 18S gene expression.

Mitochondrial DNAMitochondrial DNA (mtDNA) copy number was measuredby means of qPCR from the cytochrome B mtDNA genecompared with the large ribosomal protein p0 (36B4)nuclear gene, as previously described (27).

Mitochondrial Morphology and Membrane PotentialAfter DETA-NO treatment, cells were incubated with 100nmol/L of MitoTracker Green FM (MTG) dye (MolecularProbes) or with 5 mmol/L JC-1 (Molecular Probes) for 30min at 37°C and 5% of CO2. After a 37°C medium wash,cells were observed using a Nikon Ti-E equipped withDS-2M cooled camera (Nikon) and a top-stage incubatorTokai Hit INU (Tokai Hit) to maintain optimal culturingconditions. MTG staining was evaluated by fluorescenceimaging (490/516 nm) with a 360 1.45 numerical aper-ture objective, and JC-1 staining was evaluated (514/529nm for the monomeric state to 585/590 nm as dimericstate) with a 3100 0.75 numerical aperture objective.

Digital Imaging ProcessingAfter acquisition, images were processed with NIS-ElementsAR software (Nikon) to evaluate the shape and area ofMTG-stained cells. Briefly, binary objects were obtained byimage segmentation; then, an automatic measurement toolwas used to calculate the area and elongation (intendedas the maximum Feret-to-minimum Feret diameter ratio,where Feret diameter is the distance between the twoparallel planes restricting the object perpendicular to thatdirection) of each mitochondria.

GLUT4 StainingAfter 2 mmol/L insulin stimulation for 30 min, cells werefixed on coverslips with 4% paraformaldehyde and perme-abilized with 0.25% Triton X-100. GLUT4 was detected byincubation with a polyclonal primary antibody (Santa CruzBiotechnology) using standard procedures. After washingwith PBS, binding of primary antibodies was detected withAlexa-488–conjugated secondary antibodies (Life Technolo-gies). Coverslips were mounted with ProLong Gold antifademedium (Life Technologies) and analyzed with the confocalLeica DMI6000 CS SP8 laser scanning microscope (LeicaMicrosystem). Image analysis was performed with ImageJsoftware.

Statistical AnalysisResults are expressed as means 6 SEM. Data were ana-lyzed by GraphPad Prism 5.0 software using unpaired Stu-dent t test, one-way ANOVA, or two-way ANOVA withNewman-Keuls post hoc test, as appropriate.

RESULTS

eNOS Is Required for Mitochondrial Biogenesis inResponse to Exercise in Subcutaneous Adipose TissueSwim training lasting 6 weeks was selected to inducemitochondrial biogenesis in WT mice and in eNOS2/2

mice. No significant differences in muscle strength, as

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measured with grip tests, were present between sedentaryWT and eNOS2/2 mice, although slightly higher valueswere observed in trained animals (Table 1). Importantly,the training was able to significantly improve endurancein WT but not in eNOS2/2 mice. To determine if swimtraining induces an increase in mitochondrial biogenesisand/or function in adipose tissue and whether such anincrease is NO-dependent, we measured the mRNA levelof multiple members of mitochondrial transcriptional ma-chinery as well as mtDNA content (an index of mitochon-drial mass) in the subcutaneous adipose tissue of WT and

eNOS2/2 mice. The expression of PGC-1a, nuclear respi-ratory factor 1 (NRF-1), mitochondrial transcription fac-tor A (Tfam), and cytochrome c oxidase IV (COX IV)mRNA was increased in subcutaneous adipose tissueof WT trained mice compared with sedentary controllittermates (Fig. 1A).

In contrast, eNOS2/2 mice, which showed reducedmRNA content of PGC-1a and COX IV in untrained con-ditions compared with WT mice, failed to display the mi-tochondrial biogenic response to exercise after the swimtraining period (Fig. 1A). The “browning” effect of exercise

Table 1—Effect of swim training on mice performance evaluated in vivo

WT eNOS2/2

Sedentary (n = 8) Trained (n = 8) Sedentary (n = 8) Trained (n = 8)

Grip test force (mN/g) 207.4 6 6.6 228.3 6 20.9 208.5 6 5.5 221.8 6 11.3

Treadmill exhaustion time (%) 100.0 6 6.9 133.8 6 14.7* 100.3 6 16.7 100.4 6 9.7

Grip test performance is expressed as generated force (mN) relative to mouse body mass (g). Endurance was evaluated by time toexhaustion in treadmill running at increasing speeds and is expressed relative to the exhaustion time of WT sedentary mice (37.6 6 2.6min). *P , 0.05 relative to sedentary.

Figure 1—Mitochondrial biogenesis in mouse subcutaneous adipose tissue. WT and eNOS2/2 mice (n = 8 per group) were swim-trainedfor 6 weeks, killed, and subcutaneous adipose tissue was collected from the inguinal fat pad. A: Relative mRNA levels were measured bycombined reverse transcription (RT) and qPCR techniques (qRT-PCR) using 18s rRNA as the internal control and expressed as fold change.B: mtDNA content was measured by means of real-time PCR and expressed as % of mtDNA copy number per nuclear DNA copy number.C: Representative Western blots show PGC-1a, COX IV, and b-actin immunodetected signals in WAT lysates of mice. SED, sedentary; D:Protein expression levels were measured by Western blot analysis using b-actin as the internal control and are expressed as fold change.All graphs depict mean 6 SEM. Two-way ANOVA, *P < 0.05 and **P < 0.01 relative to sedentary mice; †P < 0.05 and ††P < 0.01 relativeto WT mice.

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training on WAT was confirmed by the increase of UCP-1mRNA content in trained WT animals, whereas the basalcontent of UCP-1 in eNOS2/2 mice was very low and didnot increase after exercise training. Interestingly, we alsomeasured a basal mRNA expression of FNDC5, which is theprecursor of the newly discovered hormone irisin (20), insubcutaneous adipose tissue of WT mice. FNDC5 washigher in WT compared with eNOS2/2 sedentary miceand did not change after exercise training. Furthermore,swim training increased mtDNA content of subcutaneousadipose tissue of WT but not eNOS2/2 mice (Fig. 1B).

We observed a significant increase in PGC-1a and COXIV protein expression in subcutaneous adipose tissue ofWT but not eNOS2/2 mice after the swim training pro-tocol. Moreover, eNOS2/2 mice displayed a significantlylower PGC-1a protein content in trained conditions com-pared with WT trained animals (Fig. 1C and D).

To ascertain if modifications in b-adrenergic sensitivitycould have influenced our findings, the effect of one in-traperitoneal injection of norepinephrine (5 mg/kg) wasassessed in subcutaneous adipose tissue of WT and

eNOS2/2 mice. We observed a significant increase inPGC-1a and COX IV mRNA levels in WT mice 24 h afterthe norepinephrine treatment (Fig. 2A). This effect waspresent also in eNOS2/2 mice but to a remarkably lesserextent. Moreover, a slight increase in Tfam and NRF-1mRNA levels was observed after b-adrenergic stimulationin WT but not in eNOS2/2 mice (Fig. 2A). To furtherdemonstrate that the attenuated norepinephrine-inducedexpression of PGC-1a and other mitochondrial biogenesisfactors in adipose tissue of eNOS2/2 mice is due to a lackof eNOS and not to an impaired b-adrenergic signaling,we evaluated the phosphorylation state of two proteinkinase A (PKA) substrates, ERK1/2 and HSL, 30 min afterthe adrenergic stimulation. We observed that phospho-ERK1, phospho-ERK2, and phospho-HSL protein levelswere significantly higher after norepinephrine injectionin WAT of WT and eNOS2/2 mice compared with un-treated control animals (Fig. 2B and C). Notably, we didnot observe any significant difference in basal proteinlevels of ERK1/2 or HSL between WT and eNOS2/2

mice (Fig. 2B and C). These results suggest the functional

Figure 2—Norepinephrine-induced mitochondrial biogenesis and b-adrenergic signaling in mouse subcutaneous adipose tissue. WT andeNOS2/2 mice (n = 10 per group) received one intraperitoneal injection of 0.9% saline solution (control [CTRL]) or norepinephrine (5 mg/kg),were killed, and subcutaneous adipose tissue was collected from inguinal fat pad. A: Relative mRNA levels were measured by qRT-PCR inWAT lysates of mice killed 24 h after injection, using 18s rRNA as the internal control and expressed as fold change. B: RepresentativeWestern blots show HSL, phospho (P)-HSL, ERK1/2, phospho (P)-ERK1/2, and b-actin immunodetected signals in WAT lysates of micekilled 30 min after the injection with saline (CTRL) or norepinephrine (TREATED) solution. C: Protein expression was measured by Westernblot analysis using b-actin as the internal control. The signals obtained from phosphorylated proteins were normalized, each one to thecorresponding total protein level. All graphs depict mean6 SEM. Two-way ANOVA, *P< 0.05 relative to control mice; †P< 0.05 relative toWT mice.

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integrity of the b-adrenergic signaling pathway in eNOS-null mutant mice.

To rule out a possible nonspecific effect caused by thestress elicited by the intraperitoneal injection, we comparedthe previous results with those obtained in a control groupof WT and eNOS2/2 mice that did not receive any injection.We did not observe any statistically significant differencebetween treated and untreated mice (data not shown).

eNOS Is Required for Basal and Insulin-StimulatedGlucose Uptake in Subcutaneous Adipose Tissue inResponse to ExerciseTo explore if swim training improves glucose metabolismor insulin sensitivity in subcutaneous adipose tissue ofeNOS2/2 mice, we measured the tissue glucose utilizationindex as the amount of [3H]-DG uptake in the basal con-dition and after a hyperinsulinemic euglycemic clamp.Swim training increased basal and insulin-stimulated glu-cose uptake of subcutaneous adipose tissue in WT butnot eNOS2/2 mice (Fig. 3A). Moreover, eNOS2/2 micedisplayed a lower glucose uptake capacity, even in thesedentary condition, compared with WT control mice(Fig. 3A). We observed a significant increase in pAKT-to-AKT protein content in subcutaneous adipose tissue ofWT trained mice compared with sedentary littermates(Fig. 3B and C). The pAKT-to-AKT ratio did not increasein the subcutaneous adipose tissue in eNOS2/2 after theswim protocol, and they showed a significantly lower pro-tein expression in trained conditions than trained WTanimals. We also measured the GLUT4 total protein

content in subcutaneous adipose tissue of sedentary andtrained animals, but no significant changes were observedin WT or eNOS2/2 mice (Fig. 3B and C).

NO Promotes Mitochondrial Biogenesis andElongation, Glucose Uptake, and GLUT4 Translocationin 3T3-L1 AdipocytesTo determine if an increased NO bioavailability could im-prove mitochondrial biogenesis and/or glucose uptake inmouse adipocytes, we first measured mRNA content of PGC-1a, NRF1, Tfam, and COX IV in fully differentiated 3T3-L1cells after a 72-h treatment with 100 mmol/L DETA-NO.Gene expression of mitochondrial biogenesis markers wasupregulated in DETA-NO–treated cells compared with un-treated control cells (Fig. 4A). We then evaluated the mito-chondrial morphometric parameters by MTG labeling,followed by a digital image analysis able to measure thearea and the elongation of each mitochondrion (see DIGITAL

IMAGINGPROCESSING for details). We observed a higher presenceof larger (area .1.5 mm2) and longer (length .3 mm) mi-tochondria in treated cells compared with those observed inuntreated control cells (Fig. 4B–D). Glucose uptake was mea-sured after DETA-NO treatment by performing an in vitro[3H]-DG uptake assay. We observed that DETA-NO in-creased glucose transport of 3T3-L1 cells in basal and ininsulin-stimulated (2 mmol/L) conditions (Fig. 5A). MoreoverDETA-NO treatment induced an increase in GLUT4 trans-location to the cell membrane (measured as cytoplasm-to-membrane fluorescence ratio) in basal and insulin-stimulatedconditions compared with untreated control cells (Fig. 5B).

Figure 3—Glucose uptake and insulin signaling in mouse subcutaneous adipose tissue. A: For glucose uptake measurement, mice (n = 8 pergroup) were fasted 8 h and injected with PBS (left) or insulin (0.5 units/kg body weight, right). B: Representative Western blots show AKT,phospho (P)-AKT, GLUT4, and b-actin immunodetected signals in protein lysates obtained from WAT of sedentary (SED) and trained WT andeNOS2/2 mice. C: Protein expression levels were measured by Western blot analysis using b-actin as the internal control and expressed asfold change. All graphs depict mean 6 SEM. Two-way ANOVA, *P < 0.05 relative to sedentary mice; †P < 0.05 relative to WT mice.

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NO Promotes Mitochondrial Biogenesis andPolarization in Human Subcutaneous AdipocytesTo further investigate the role of NO in adipose tissue,DETA-NO effects were also studied in subcutaneousadipocytes obtained from abdominal biopsy specimensof subjects undergoing bariatric surgery. The stromalvascular fraction of adipose tissue was isolated andadipocytes cultured to full differentiation in vitro. Weobserved that PGC-1a, Tfam, COX IV, and ATP-synthasesubunit-b (ATPS) gene expression was upregulated in hu-man mature adipocytes treated with DETA-NO comparedwith untreated cells (Fig. 6A). Moreover, DETA-NO treat-ment increased mtDNA content in human mature adipo-cytes (Fig. 6B). We then labeled the adipocyte mitochondriawith the vital staining dye JC-1 and observed that mito-chondrial membrane potential was higher in cells afterDETA-NO treatment compared with mitochondrial mem-brane potential of untreated cells (Fig. 6C).

NO Promotes Glucose Uptake and GLUT4Translocation in Human Subcutaneous AdipocytesGlucose uptake and GLUT4 translocation were measuredin fully differentiated human adipocytes as describedfor 3T3-L1 cells. We observed an increase in basal andinsulin-stimulated glucose uptake capacity in DETA-NO–treated cells compared with untreated cells (Fig. 7A).

Moreover, the fraction of GLUT4 translocated on cellsmembrane was significantly higher after DETA-NO treat-ment, even in the basal condition, and slightly increasedin insulin-stimulated conditions (Fig. 7B and C).

DISCUSSION

The current study provides strong evidence that NOgenerated by eNOS plays a crucial role in the mitochon-drial biogenesis and metabolic activation taking place inadipocytes in response to physical activity. A reduction ofmitochondrial abundance and activity has been linkedwith insulin resistance in obesity and type 2 diabetes (28).One of the most important physiological conditionswidely recognized to increase insulin sensitivity and mi-tochondrial biogenesis is physical activity: the commonthought that exercise increases mitochondrial biogenesismostly, if not exclusively, in skeletal muscles has recentlybeen replaced by the notion that exercise training caninduce mitochondrial biogenesis in a wide range of tissuesnot normally associated with the metabolic demand ofexercise (29). Much emphasis has been placed on therole of mitochondrial biogenesis in the adipose organ inrelation to its potential shift from the white to brownphenotype or to the activation of the beige phenotype.This phenomenon has been shown to be associated withincreased energy expenditure and whole-body insulin

Figure 4—Mitochondrial biogenesis in 3T3-L1 adipocytes. Fully differentiated 3T3-L1 adipocytes were treated for 72 h with 100 mmol/Lvehicle (CTRL) or 100 mmol/L DETA-NO. A: mRNA levels were analyzed by means of qRT-PCR using 18s rRNA as the internal control andexpressed as fold change (n = 3 independent experiments). B: MTG dye was used as an indicator of mitochondrial mass in live cells.Mitochondrial area (C) and elongation (D) were analyzed as the percentages of larger (>1.5 mm2) and longer (>3 mm) mitochondria in DETA-NO–treated cells compared with untreated cells (n = 3 independent experiments). Data are expressed as mean 6 SEM. Student t tests,*P < 0.05 and **P < 0.01 relative to untreated cells (CTRL).

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sensitivity (30,31). In this study, we report that physicaltraining in mice was able to induce an increase in sub-cutaneous adipose tissue mRNA and protein levels ofkey transcriptional regulators of mitochondrial biogen-esis, along with an increase in mtDNA content. Inter-estingly, at the end of the training period, an increasedWAT mRNA expression of UCP1, the specific marker ofbrown adipose tissue, was also observed in subcutane-ous fat.

These results confirm and extend what was previouslyobserved by Sutherland et al. (32), who reported thatexercise training increased PGC-1a and Tfam mRNA ex-pression, as well as COX IV protein and citrate synthaseactivity in rat visceral fat depots, further supporting theevidence that exercise training is an effective stimulus formitochondrial biogenesis in WAT. This phenomenon hasbeen explained by the sympathetic stimulation duringexercise, which has been shown to induce PGC-1a in sev-eral tissues, including brown and WAT. Adrenaline di-rectly stimulates PGC-1a in WAT, and the b-blockerpropranolol is able to reduce the exercise-induced rise inPGC-1a by ;40% (32). Among the mechanisms respon-sible for the remaining ;60% of the exercise effect on

WAT mitochondria biogenesis, an increased production ofmyokines, hormones (i.e., thyroid hormones, glucocorti-coids), signaling molecules coming from the exercisingmuscles (i.e., irisin, NO), or other still unknown factorscould be taken into consideration. The results collected inthis study add a novel and important piece of evidenceshowing that a significant portion of the induction inmitochondrial biogenesis in WAT after training is dueto activation of eNOS system. We observed a reducedbasal expression of genes regulating mitochondrial bio-genesis in adipose tissue of eNOS2/2 mice and, moreimportantly, the physical training period failed to in-duce any increase in these transcriptional regulators orin mtDNA content. The sympathetic stimulation ofbrown adipose tissue in vivo and in vitro significantlyincreased eNOS expression and activity (33), and weobserved that the norepinephrine-induced upregulationof mitochondrial biogenesis factors was reduced in WATof eNOS2/2 mice.

To evaluate any changes of the sympathetic activity inthe knockout mice, we investigated the noradrenergicsignaling in these animals. In adipose tissue, HSL enzymeactivity is strongly induced by b-adrenergic stimulation.

Figure 5—Glucose uptake and GLUT4 translocation in 3T3-L1 adipocytes. Fully differentiated 3T3-L1 adipocytes were treated for 72 h withvehicle (CTRL) or 100 mmol/L DETA-NO. Cells were serum-starved for 8 h, treated with PBS (basal) or 2 mmol/L insulin for 30 min andsubjected to a [3H]-DG uptake assay (A) or immunostained with anti-GLUT4 antibody (B). Fluorescence was detected by confocalmicroscopy, and ratio between the membrane and cytoplasmic signal was measured by means of image analysis. All data are expressedas mean6 SEM (n = 3 independent experiments). Two-way ANOVA, *P< 0.05 and **P< 0.01 relative to control cells; †P< 0.05 relative tocells in basal conditions.

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HSL and ERK are major targets for PKA-mediated phos-phorylation, the first step downstream of the b-adrenergicreceptor. ERK also phosphorylates HSL to modulate theactivity of the enzyme. We observed that the norepinephrine-induced phosphorylation of ERK1/2 and HSL were notimpaired in WAT of eNOS2/2 mice. These results suggestthat the eNOS system may play a role in mediating theadrenergic effect on mitochondrial biogenesis activationalso in WAT.

Apparently, an increase in UCP1 expression in WATmay be in contrast with the known effects of exercise onmitochondrial coupling efficiency in skeletal muscle (thatincreases to produce more ATP), but this phenomenoncould be explained by considering the different metabolicfunctions of skeletal muscle and adipose tissue, whichimply different regulatory mechanisms of mitochondrialrespiration and energy expenditure (34). Moreover, it hasbeen observed that physical exercise could induce an in-crease of uncoupling activity also in skeletal muscle(35,36); therefore, the balance between substrates oxida-tion, membrane potential, and ATP production is a com-plex phenomenon that may have controversial aspects.

The role of NO in the activation of mitochondrialbiogenesis and the regulation of skeletal muscle glucoseuptake during exercise have been extensively studied inhumans and in rodents (37), but so far, little data havebeen available regarding the effect of exercise on adiposetissue insulin sensitivity and the role of NO in mediatingthis effect (38,39). The data presented here clearly showthat the significant increase in the exercise-induced adi-pose tissue glucose uptake in basal conditions and afterinsulin infusion in WT mice is abolished in eNOS2/2

mice. Therefore, NO mediates, at least partially, theinsulin-sensitizing effect of exercise on subcutaneous ad-ipose tissue. To further clarify whether the observed ef-fect of exercise on adipose tissue mitochondrial biogenesisand insulin sensitivity could be a genuine consequence ofeNOS activation, irrespective of the stimulation of fat cellb-adrenoceptors (40,41), we studied the in vitro effect ofan NO donor on murine and human fully differentiatedadipocytes. The obtained results allowed us to confirm therole of NO in inducing the master genes regulating mito-chondrial biogenesis and function, along with a parallel in-crease of basal and insulin-stimulated GLUT4 translocation

Figure 6—Mitochondrial biogenesis and membrane potential in human adipocytes. Fully differentiated human adipocytes isolated fromsubcutaneous adipose tissue were treated for 72 h with control (CTRL) vehicle or 100 mmol/L DETA-NO. A: mRNA levels were analyzed byqRT-PCR using 18s rRNA as the internal control and are expressed as fold change (n = 3 independent experiments). B: mtDNA content wasmeasured by means of qPCR and expressed as % of mtDNA copy number per nuclear DNA copy number (n = 3 independent experiments).Data are expressed as mean 6 SEM. Student t tests, *P < 0.05 and **P < 0.01 relative to control cells. C: JC-1 assay was used as anindicator of mitochondrial membrane polarization in live cells. A representative image from three independent experiments is shown.

2808 Exercise, Adipose Tissue, and Energy Metabolism Diabetes Volume 63, August 2014

and glucose uptake in mouse and human white adipocytes.Our findings highlight the important role of mitochon-dria in the regulation of adipocyte glucose homeostasisand support recent observations obtained after induc-tion of mitochondrial dysfunction by knocking downTfam, which led to a downregulation of GLUT4 expres-sion and to an attenuation of insulin-stimulated glucoseuptake in 3T3-L1 adipocytes (42). Accordingly, the treat-ment with PPAR-g agonists improves the dysfunctionaladipose organ by increasing white adipocyte insulin sen-sitivity and mitochondrial biogenesis in WAT of insulin-resistant mice (43).

Morphological and structural changes of mitochondria—from small fragmented units to larger networks of elon-gated organelles—play a role in several cellular processesaccording to the functional status of the cell. It has beenrecently reported that mitochondrial elongation is criticalto sustain cell viability during macroautophagy induced bynutrient restriction (44). Longer mitochondria are protectedfrom being degraded and possess more cristae where activityof the ATP synthase is increased, optimizing ATP productionin times of nutrient restriction (44). Mitochondria unable toelongate during nutrient deprivation consume cellular ATP,

leading to cell death. On the contrary, if elongation is blocked,mitochondria become dysfunctional and “cannibalize”cytoplasmic ATP to maintain their membrane potential,precipitating cell death (45). Whenever energy is needed,as in prolonged exercise (which mimics what happensduring nutrient restriction), morphological changes ofmitochondrial shape occur (46,47).

We aimed to determine if an increased NO availabilitycould influence mitochondrial remodeling in adipocytes andfound that chronic treatment with DETA-NO was able toinduce a significant increase in mitochondrial area andpromoted mitochondrial elongation in 3T3-L1 cells, possiblycontributing to improved bioenergetics. We also observedthat the transcriptional machinery of mitochondrial bio-genesis was activated in DETA-NO–treated cells, along withan increased COX IV and ATP-synthase mRNA expressionand increased mitochondrial potential. In contrast with thein vivo studies, we observed only a slight, not significantincrease in UCP1 mRNA expression in white adipocytestreated with the NO donor. This could be explained con-sidering the in vivo situation, where we observed theresults of a complexity of synergistic phenomena occurringduring exercise, including the prolonged sympathetic

Figure 7—Glucose uptake and GLUT4 translocation in human subcutaneous adipocytes. Fully differentiated human adipocytes isolatedfrom subcutaneous adipose tissue were treated for 72 h with control (CTRL) vehicle or 100 mmol/L DETA-NO. Cells were serum-starved for8 h, treated with PBS (basal) or 2 mmol/L insulin for 30 min, and subjected to a [3H]-DG uptake assay (A) or immunostained with anti-GLUT4antibody (B). C: Fluorescence was detected by confocal microscopy, and the ratio between the membrane and cytoplasmic signal wasmeasured by means of image analysis. All data are expressed as mean 6 SEM (n = 3 independent experiments). Two-way ANOVA,*P < 0.05 and **P < 0.01 relative to control cells; †P < 0.05 relative to cells in basal conditions.

diabetes.diabetesjournals.org Trevellin and Associates 2809

overdrive. On the contrary, the in vitro environment prob-ably excludes some major determinants that play a role inmediating the exercise effect on adipose tissue in vivo, evenif it represents a useful model to assess the importance ofNO per se in influencing mitochondrial biogenesis andglucose handling by fat cells. The role of exercise in pre-venting obesity-related metabolic disorders by acting onadipose tissue has been recently highlighted (48,49). Theeffect of exercise is not limited to the exercising muscles,increasing fatty acids mobilization to match the peripheralenergy requirement, but deeply influences the wholeadipocyte energy metabolism by activating mitochondrialbiogenesis and promoting insulin sensitivity (50). Our datashow that these two phenomena are tightly associated andpoint to a crucial role of the eNOS system in mediating theeffect of exercise training on the bioenergetic adaptationin subcutaneous white fat.

Funding. This study was supported by Ministero dell’Università e dellaRicerca (grants 20075HJTHM_003 to A.V., 2007HJTHM_001 to E.N., and20112010329EKE_005 to R.V.), Ministero della Salute (grant RF-2009-1526404 to R.V.), and the Cariparo Foundation.Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. E.T., M.S., M.O., M.G., L.T., R.F., R.S., and M.Q.performed research. C.R. and E.N. contributed new reagents and analytic tools.E.T., M.S., and A.V. analyzed data. E.T., A.V., C.R., E.N., and R.V. wrote themanuscript. E.N. and R.V. designed the research. E.N. and R.V. are the guaran-tors of this work and, as such, had full access to all the data in the study and takeresponsibility for the integrity of the data and the accuracy of the data analysis.

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