Exercise training induces mitochondrial biogenesis and glucose uptake in subcutaneous adipose tissue through eNOS-dependent mechanisms Running title: Exercise, adipose tissue and energy metabolism. Elisabetta Trevellin1, Michele Scorzeto2, Massimiliano Olivieri1, Marnie Granzotto1, Alessandra Valerio 3, Laura Tedesco4, Roberto Fabris1, Roberto Serra1, Marco Quarta2*, Carlo Reggiani2, Enzo Nisoli4, Roberto Vettor1 1
Internal Medicine 3, Endocrine-Metabolic Laboratory, Department of Medicine DIMED, University of Padua, 35121 Padua, Italy 2
3
Department of Biomedical Sciences, University of Padua, 35121 Padua, Italy
Department of Molecular and Translational Medicine, University of Brescia, 25121 Brescia, Italy
4
Center for Study and Research on Obesity, Department of Medical Biotechnology and Translational Medicine, University of Milan, 20121 Milan, Italy; Corresponding author: Roberto Vettor, [email protected] R.V. and E.N. contributed equally to this work Prof. Roberto Vettor Department of Medicine-DIMED Head of Internal Medicine 3 Endocrine-Metabolic Unit University of Padua via Ospedale 105 35128 Padua Italy Phone: + 39 (0) 49 821 2648 Fax: + 39 (0) 49 821 3332 Mobile Phone +39 3457125856
*Dr. Quarta is currently affiliated with the Dept. of Nephrology and Neurological Sciences, School of Medicine, Stanford University, USA.
Diabetes Publish Ahead of Print,©published online March 19, 2014
Total word count in main text: 4466; word count in the abstract: 200; tables: 1; figures: 7. Abstract
Insulin resistance and obesity are associated with a reduction of mitochondrial content in various tissues of mammals. Moreover, a reduced nitric oxide (NO) bioavailability impairs several cellular functions including mitochondrial biogenesis and insulin-stimulated glucose uptake, two important mechanisms of body adaptation in response to physical exercise. Although these mechanisms have been thoroughly investigated in skeletal muscle and heart, few studies have focused on the effects of exercise on mitochondria and glucose metabolism in adipose tissue. In this study we compared the in vivo effects of chronic exercise in subcutaneous adipose tissue of wild type (WT) and eNOS knockout (eNOS-/-) mice after a swim training period. Then we investigated the in vitro effects of NO on mouse 3T3-L1 and human subcutaneous adipose tissue-derived adipocytes, after a chronic treatment with a NO donor (DETA-NO). We
observed
that
swim
training
increases
mitochondrial
biogenesis,
mitochondrial DNA (mtDNA) content and glucose uptake in subcutaneous adipose tissue of WT but not eNOS-/- mice. Furthermore we observed that DETA-NO promotes mitochondrial biogenesis and elongation, glucose uptake and GLUT4 translocation in cultured murine and human adipocytes. These results point to the crucial role of the eNOS-derived NO in the metabolic adaptation of subcutaneous adipose tissue to exercise training.
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A reduced mitochondrial content and/or activity is associated with impaired cell function in several diseases (1; 2). In particular, it has been hypothesized that mitochondrial impairment may be involved in the pathogenesis of obesity, insulin resistance and their progression towards type 2 diabetes (3; 4), even though the role of mitochondria in health and disease is still under discussion (5; 6). At the same time, metabolic disorders are also associated with a reduction of endothelial nitric oxide synthase (eNOS) enzymatic activity (7; 8), in fact mice lacking eNOS gene are considered a useful murine model for metabolic syndrome because they display typical
features
including
hypertension,
hypertriglyceridemia,
endothelial
dysfunction, insulin resistance and visceral obesity (9). It is well known that physical exercise induces profound physiological adaptations in several tissues as a response to increased metabolic requirements. One of the major event induced by physical activity is the upregulation of eNOS gene expression and the consequent increase of tissue nitric oxide (NO) production which in turn induces mitochondrial biogenesis and cell glucose uptake in skeletal and cardiac muscle (10-12) . Moreover, a selective depletion of peroxisome proliferator-activated receptor γ (PPARγ) co-activator 1 (PGC-1), a key regulator of mitochondrial biogenesis, leads to a blunting of exercise-induced increases in mitochondrial respiratory chain proteins in muscle (13) (14). Thus, a lack of response to physical exercise in terms of mitochondrial biogenesis could be related to a reduced or impaired NO metabolism. Insulin sensitivity is also increased after a training period and a single bout of exercise could enhance basal glucose uptake by increasing GLUT4 translocation to the cell membrane of skeletal (15) and cardiac (16) myocytes. Similar results have been observed in mouse and human tissues not directly involved in mechanical work and oxidative processes (17), but few studies have focused on subcutaneous adipose
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tissue. Tissues involved in the contractile activity during exercise, such as heart and skeletal muscles, increase their capacity to oxidize fuel substrates in order to meet the energy demand. Therefore they need to increase both lipid and glucose uptake and their oxidation. Emerging evidence suggests that these adaptations occur not only in working tissues such as skeletal muscle and heart but also in white and brown adipose tissue, liver, brain and kidney. Several studies show that exercise induces a “browning effect” in white adipose tissue (WAT) by increasing mitochondrial protein activity (18) or brown adipocyte-specific gene expression (19). A recent study hypothesized that this effect might be mediated by irisin (FNDC5), a PGC-1α-dependent myokine secreted during physical activity which promotes thermogenesis and uncoupling processes in white adipocytes, while leptin and other key regulator genes of “white” development are downregulated (20). Despite numerous results supporting this hypothesis, the role of irisin is still controversial (21) and requires further investigations. The aim of the present study was to investigate the mechanism underlying the response of subcutaneous adipose tissue to physical exercise, in terms of mitochondrial biogenesis and glucose uptake. In particular we explored whether NO can play a role in such metabolic adaptations.
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Research Design and Methods
Mice and exercise protocol. Thirty-six adult (8-weeks-old) male wild-type (C57BL/6J) and eNOS-/- (B6.129P2-Nos3tm1Unc/J) mice (all from Jackson Laboratory) were treated according to the EU guidelines and with the approval of the Institutional Ethical Committee. Body weight and food consumption were monitored throughout the experimental period. Both WT and eNOS-/- mice (n = 18 mice per group) were assigned randomly to either swim training (12) or to have no lifestyle modifications. Mice swam once a day for 5 days/week in a graduated protocol starting at 10 min daily, with 10 min increase each day until 90 min daily at 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 was maintained between 30°C and 35°C and mice were carefully towel-dried after each training session.
Muscle contractile performance in vivo. Muscle strength developed by WT and eNOS-/- mice during instinctive grasp was measured with the protocol indicated as grip test (22). Briefly, the mouse was held by the tail in proximity to a trapeze bar connected with the shaft of a force transducer. Once the mouse had firmly grabbed the trapeze, a gentle pull was exerted on the tail. The measurement of the peak force generated by the mouse was repeated several times with appropriate intervals to avoid fatigue and average peak force values were expressed relative to body mass. Endurance was measured with a test to exhaustion on treadmill. Initial speed (5 cm/s) was increased after 2 minutes at 10 cm/s. The speed was then increased by 2 cm/s every minute up to 50 cm/s and time to exhaustion was recorded.
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Tissue glucose utilization index. At the end of the training period and two days before the clamp studies, a catheter was inserted into the right femoral vein under general anesthesia with sodium pentobarbital. Tissue glucose uptake studies were performed on mice under conscious and unstressed conditions after a 8-h fast. 10 μCi of the nonmetabolizable glucose analog 2-deoxy-D-[1-3H]glucose ([3H]-DG) (Amersham Biosciences) were injected as a i.v. bolus in basal condition or after hyperinsulinemic
euglycaemic
clamp
as
previously
described
with
minor
modifications (23). Animals were killed 120 minutes after tracer injection, subcutaneous adipose tissue from inguinal fat pad was quickly collected in liquid nitrogen and kept at −80°C for subsequent analysis. The glucose utilization index was derived from the amount of [3H]-DG-6-phosphate ([3H]-DGP) measured in adipose tissue as previously described (24) thus using the accumulation of [3H]-DGP as an index of the glucose metabolic rate.
Norepinephrine treatment. A group of sedentary mice (n = 10 WT and n = 10 eNOS-/-) of the same strains as those utilized for the training experiments received a single i.p. injection of Norepinephrine hydrogentartrate 5mg/kg (Galenica Senese) or vehicle (0.9% saline solution). Animals were killed 24 hours after norepinephrine injection and the subcutaneous inguinal fat pad was quickly removed and kept at −80°C for subsequent mRNA expression analysis. An additional group of sedentary mice (n = 10 WT and n = 10 eNOS-/-) received the same norepinephrine treatment and were killed 30 minutes after the i.p. injection. Inguinal fat pad was quickly removed and kept at −80°C in order to quantify the protein expression and phosphorylation.
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Immunoblot analysis. Proteins were isolated from inguinal subcutaneous adipose tissues using T-PER Mammalian Protein Extraction Reagent (Pierce) as indicated by the manufacturer, in the presence of 1 mM NaVO4, 10 mM NaF and a cocktail of protease inhibitors (Sigma Aldrich). Protein content was determined by the bicinchoninic acid protein assay (Pierce) and 70 µg of proteins were run on SDSPAGE
under
reducing
conditions.
The
separated
proteins
were
then
electrophoretically transferred to a nitrocellulose membrane (Pierce). Proteins of interest were revealed with specific antibodies: anti-GLUT4, anti-AKT, anti-PhosphoAKT, anti-p44/42 MAPK (Erk1/2), anti-Phospho-p44/42 MAPK (Erk1/2), anti-HSL, anti-Phospho-HSL (all from Cell Signaling) and anti-β actin (Sigma-Aldrich). The immunostaining was detected using horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin for 1 h at room temperature. Bands were revealed by the SuperSignal Substrate (Pierce), and quantified by densitometry using NIH ImageJ software.
Cell cultures and treatment. Stromal vascular fraction from subcutaneous adipose tissue of 5 healthy patients undergoing bariatric surgery was isolated as previously described (25). Human-derived and 3T3-L1 (ATCC CL-173) preadipocytes were plated in Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10% fetal bovine serum, 150U/ml streptomycin, 200U/ml penicillin, 2mM glutamine and 1mM HEPES (all from Life Technologies). At confluence, adipogenic differentiation was induced by adding 1μM dexamethasone, 0.5mM 3-isobutyl-1-methyl-xantine (IBMX, Sigma-Aldrich) and 70nM insulin (Novo Nordisk). IBMX was removed from medium after 3 days of culture. Cells were cultured at 37˚C in a humidified atmosphere of 5% CO2 until fully differentiated. Cells were exposed to 100M
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diethylentriamine-NO (DETA-NO, Sigma-Aldrich) a potent NO donor (26), or vehicle for 72 h and then harvested for further analysis.
Cell glucose uptake. Human-derived and 3T3-L1 mature adipocytes were serumstarved for 8 h, incubated in the presence or absence of 2μM insulin (Novo Nordisk) for 30 min, then with 1.5 μCi/ml [3H]-DG (Amersham Biosciences) for 15 min. Cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed in 0.5 M NaOH. Radioactivity was measured by liquid scintillation counting (Wallac). Each condition was assayed in three independent experiments in triplicate.
RNA isolation and Real-Time quantitative PCR. Total RNA was isolated from cultured adipocytes and frozen subcutaneous adipose tissues of mice using RNeasy Mini 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 PCR (DNA Engine Opticon2, MJ Research) using SYBR Green PCR Master Mix (Applied Biosystems) and specific intron-spanning primers according to manufacturer’s instructions. All data were collected in triplicate and normalized to 18S gene expression.
Mitochondrial DNA. Mitochondrial DNA copy number was measured by means of quantitative PCR (qPCR) from the cytochrome B mtDNA gene compared with the large ribosomal protein p0 (36B4) nuclear gene as previously described (27).
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Mitochondrial morphology and membrane potential. After DETA-NO treatment, cells were incubated with 100nM of Mitotracker Green FM (MTG) dye (Molecular Probes) or with 5μM JC-1 (Molecular Probes) for 30 minutes at 37°C and 5% of CO2. After a 37°C medium wash, cells were observed using a Nikon Ti-E equipped with DS-2M cooled camera (Nikon) and a top-stage incubator Tokai Hit INU (Tokai Hit) in order to maintain optimal culturing conditions. MTG staining was evaluated by fluorescence imaging (490/516nm) with a 60X 1.45 NA objective and JC-1 staining was evaluated (514/529nm for the monomeric state to 585/590nm as dimeric state) with a 100x 0.75 NA objective.
Digital imaging processing. After acquisition, images were processed with NISElements AR software (Nikon) to evaluate shape and area of MTG stained cells. Briefly, binary objects were obtained by image segmentation, then using automatic measurement tool, area and elongation (intended as the ratio of Max Feret over Min Feret diameter, where Feret diameter is distance between the two parallel planes restricting the object perpendicular to that direction) of each mitochondria were calculated.
GLUT4 staining. After 2μM insulin stimulation for 30 min cells were fixed on coverslips with 4% paraformaldehyde and permeabilized with 0.25% Triton X-100. GLUT4 was detected by incubation with a polyclonal primary antibody (Santa Cruz Biotechnology) using standard procedures. After washing with PBS, binding of primary antibodies was detected with Alexa-488-conjugated secondary antibodies (Life Technologies). Coverslips were mounted with ProLong Gold antifade medium (Life Technologies) and analyzed with the confocal laser scanning microscope Leica
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DMI6000 CS SP8 (Leica Microsystem). Image analysis was performed with the NIH ImageJ software.
Statistical analysis. Results were expressed as means ± SEM. Data were analyzed by GraphPad Prism 5.0 software using either unpaired Student's t test, one-way ANOVA or two way ANOVA with Newman-Keuls’ post hoc test as appropriate.
Results
eNOS is required for mitochondrial biogenesis in response to exercise in subcutaneous adipose tissue. A swim training lasting 6 weeks was selected to induce mitochondrial biogenesis in wild type (WT) mice and in eNOS-/- mice. No significant differences in muscle strength, as measured with grip tests, were present between sedentary WT and eNOS-/- mice, although slightly higher values were observed in trained animals. Importantly, the training was able to significantly improve endurance in WT but not in eNOS-/- mice (Table 1). To determine if swim training induces an increase in mitochondrial biogenesis and/or function in adipose tissue and whether such increase is NO-dependent, we measured the mRNA level of multiple members of mitochondrial transcriptional machinery as well as mitochondrial DNA (mtDNA) content (an index of mitochondrial mass) in the subcutaneous adipose tissue of WT and eNOS-/- mice. The expression of PGC-1, Nuclear respiratory factor 1 (NRF-1), mitochondrial transcription factor A (Tfam) and cytochrome c oxidase IV (COX IV) mRNA was increased in subcutaneous adipose tissue of WT trained mice, compared to sedentary control littermates (Fig. 1A).
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In contrast, eNOS-/- mice, which showed reduced mRNA content of PGC-1 and COX IV in untrained conditions compared to WT mice, failed to display the mitochondrial biogenic response to exercise after the swim traninig period (Fig. 1A). The "browning" effect of exercise training on WAT was confirmed by the increase of UCP-1 mRNA content in trained WT animals, while in eNOS-/- mice the basal content of UCP-1 was very low an did not increase after exercise training. Interestingly, we also measured a basal mRNA expression of FNDC5, which is the precursor of the newly discovered hormone irisin (20), in subcutaneous adipose tissue of WT mice. FNDC5 was higher in WT in comparison with eNOS
-/-
sedentary mice
and did not change after exercise training. Furthermore, swim training increased mtDNA content of subcutaneous adipose tissue of WT but not eNOS-/- mice (Fig. 1B). We observed a significant increase in PGC-1α and COX IV protein expression in subcutaneous adipose tissue of WT but not eNOS-/- mice after the swim training protocol. Moreover eNOS-/- mice displayed a significantly lower PGC-1α protein content in trained conditions, compared to WT trained animals (Fig. 1C and 1D). In order to ascertain if modifications in β-adrenergic sensitivity could have influenced our findings, the effect of a single i.p. injection of 5 mg/kg norepinephrine was assessed in subcutaneous adipose tissue of WT and eNOS-/- mice. We observed a significant increase in PGC-1α and COX IV mRNA levels in WT mice 24 hours after the norepinephrine treatment (Fig. 2A). This effect was present also in eNOS-/- mice but to a remarkable lesser extent. Moreover, a slight increase in Tfam and NRF-1 mRNA levels was observed after β-adrenergic stimulation in WT but not in eNOS-/mice (Fig. 2A). To further demonstrate that the attenuated norepinephrine-induced expression of PGC-1α and other mitochondrial biogenesis factors in adipose tissue of eNOS-/- mice is due to a lack of eNOS and not to an impaired β-adrenergic signaling,
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we evaluated the phosphorylation state of two protein kinase A (PKA) substrates, e.g. the extracellular signal-regulated kinases 1/2 (ERK1/2) and the hormone sensitive lipase (HSL), 30 minutes after the adrenergic stimulation. We observed that phosphoERK1, phospho-ERK2 and phospho-HSL protein levels were significantly higher after norepinephrine injection in WAT of both WT and eNOS-/- mice, compared to untreated control animals (Fig. 2B and 2C). Notably, we did not observe any significant difference in basal protein levels of ERK1/2 or HSL between WT and eNOS-/- mice (Fig. 2B and 2C). These results suggest the functional integrity of the βadrenergic signaling pathway in eNOS null mutant mice. In order to rule out a possible non-specific effect due to the stress elicited by the i.p. injection, we compared the previous results with those obtained in a control group of both WT and eNOS-/- mice that did not receive any injection. We did not observe any statistically significant difference between treated and untreated mice (data not shown).
eNOS is required for basal and insulin stimulated glucose uptake in subcutaneous adipose tissue in response to exercise. To explore if swim training improves glucose metabolism or insulin sensitivity in subcutaneous adipose tissue of eNOS-/- mice, we measured tissue glucose utilization index as the amount of 2-deoxy[3H]glucose uptake in basal condition and after hyperinsulinemic euglycaemic clamp. Swim training increased both basal and insulin stimulated glucose uptake of subcutaneous adipose tissue in WT but not eNOS-/- mice (Fig. 3A). Moreover, eNOS-/mice displayed a lower glucose uptake capacity even in sedentary condition, compared to WT control mice (Fig. 3A). We observed a significant increase in pAKT/AKT protein content in subcutaneous adipose tissue of WT trained mice
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compared to sedentary littermates (Fig. 3B and 3C). eNOS-/- mice didn't increased their pAKT/AKT ratio in subcutaneous adipose tissue after swim protocol and showed a significant lower protein expression in trained conditions compared to trained WT animals. We measured also the GLUT4 total protein content in subcutaneous adipose tissue of both sedentary and trained animals, but no significant changes were observed in WT or eNOS-/- mice (Fig. 3B and 3C).
Nitric oxide promotes mitochondrial biogenesis and elongation, glucose uptake and GLUT4 translocation in 3T3-L1 adipocytes. To determine if an increased NO bioavailability could improve mitochondrial biogenesis and/or glucose uptake in mouse adipocytes we first measured mRNA content of PGC-1, NRF1, Tfam and COX IV in fully differentiated 3T3-L1 cells after a 72 hours treatment with 100μM DETA-NO. Gene expression of mitochondrial biogenesis markers was upregulated in DETA-NO treated cells compared to untreated control cells (Fig. 4A). Then, we evaluated the mitochondrial morphometric parameters by MitoTracker Green FM labeling, followed by a digital image analysis able to measure the area and the elongation of each mitochondrion (see digital imaging processing for details). We observed a higher presence of larger (area > 1.5μm2 ) and longer (length >3μm) mitochondria in treated cells, compared to those observed in untreated control cells (Fig. 4 B-D). Glucose uptake was measured after DETA-NO treatment by performing an in vitro 2-deoxy-[3H]glucose uptake assay. We observed that DETA-NO increased glucose transport of 3T3-L1 cells in both basal and insulin-stimulated (2μM) conditions (Fig. 5A). Moreover DETA-NO treatment induced an increase in GLUT4 translocation to cell membrane (measured as cytoplasm/membrane fluorescence ratio)
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in both basal and insulin-stimulated conditions, in comparison with untreated control cells (Fig. 5B).
Nitric oxide promotes mitochondrial biogenesis and polarization in human subcutaneous adipocytes. To further investigate the role of NO in adipose tissue, DETA-NO effects were studied also in subcutaneous adipocytes obtained from abdominal biopsies of subjects undergoing bariatric surgery. Stromal vascular fraction of adipose tissue was isolated and adipocytes cultured to full differentiation in vitro. We observed that PGC-1, Tfam, COX IV and ATP-synthase subunit β (ATPS) gene expression was upregulated in human mature adipocytes treated with DETA-NO compared to untreated cells (Fig. 6A). Moreover DETA-NO treatment increased mtDNA content in human mature adipocytes (Fig. 6B). Then we labeled the adipocytes mitochondria with the vital staining dye JC-1 and we observed that mitochondrial membrane potential was higher in cells after DETA-NO treatment, compared to mitochondrial membrane potential of untreated cells (Fig. 6C).
Nitric oxide promotes glucose uptake and GLUT4 translocation in human subcutaneous adipocytes. Glucose uptake and GLUT4 translocation were measured in fully differentiated human adipocytes as described for 3T3-L1 cells. We observed an increase in both basal and insulin-stimulated glucose uptake capacity in DETA-NO treated cells compared to untreated cells (Fig. 7A). Moreover the fraction of GLUT4 translocated on cells membrane was significantly higher after DETA-NO treatment even in basal condition, and slightly increased in insulin-stimulated conditions (Fig. 7B-C).
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Discussion The present study provides strong evidence that NO generated by eNOS plays a crucial role in the mitochondrial biogenesis and metabolic activation taking place in adipocytes, in response to physical activity. A reduction of mitochondrial abundance and activity has been linked with insulin resistance in obesity and type 2 diabetes (28). One of the most important physiological conditions widely recognized to increase both insulin sensitivity and mitochondrial biogenesis is physical activity: the common thought that exercise increases mitochondrial biogenesis mostly, if not exclusively, in skeletal muscles has recently been replaced by the notion that exercise training can induce mitochondrial biogenesis in a wide range of tissues not normally associated with the metabolic demand of exercise (29). Much emphasis has been placed on the role of mitochondrial biogenesis in the adipose organ in relation to its potential shift from white to brown phenotype or to the activation of beige phenotype. This phenomenon has been shown to be associated with increased energy expenditure and whole body insulin sensitivity (30; 31). In this study we report that physical training in mice was able to induce an increase in subcutaneous adipose tissue mRNA and protein levels of key transcriptional regulators of mitochondrial biogenesis, along with an increase in mtDNA content. Interestingly, at the end of the training period an increased in WAT mRNA expression of UCP1, the specific marker of brown adipose tissue, was also observed in subcutaneous fat. These results confirm and extend what was previously observed by Sutherland and colleagues who reported that exercise training increased PGC-1α and Tfam mRNA expression, as well as COX IV protein and citrate synthase activity in rat visceral fat depots (32), further supporting the evidence that exercise training is an effective stimulus for mitochondrial biogenesis in WAT. This phenomenon has been explained
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by the sympathetic stimulation during exercise, which has been shown to induce PCG-1α in several tissues including brown and white adipose tissue. Adrenaline directly stimulates PGC-1α in WAT and the β-blocker propranolol is able to reduce the exercise-induced rise in PGC-1α by responsible for the remaining
40% (32). Among the mechanisms
60% of the exercise effect on WAT mitochondria
biogenesis an increased production of myokines, hormones (i.e. thyroid hormones, glucocorticoids), signaling molecules coming from the exercising muscles (i.e. irisin, nitric oxide) or other still unknown factors could be taken into consideration. The results collected in this study add a novel and important piece of evidence showing that a significant portion of the induction in mitochondrial biogenesis in WAT after training is due to activation of eNOS system. We have observed a reduced basal expression of genes regulating mitochondrial biogenesis in adipose tissue of eNOS-/mice and, more importantly, the physical training period failed to induce any increase in these transcriptional regulators or in mtDNA content. It has been shown that the sympathetic stimulation of brown adipose tissue in vivo and in vitro significantly increased eNOS expression and activity (33) and we observed that the norepinephrine-induced up-regulation of mitochondrial biogenesis factors was reduced in WAT of eNOS-/- mice. To evaluate any changes of the sympathetic activity in the knockout mice, we investigated the noradrenergic signaling in these animals. In adipose tissue, HSL enzyme activity is strongly induced by β-adrenergic stimulation. HSL and ERK are major targets for PKA-mediated phosphorylation, the first step downstream the βadrenergic receptor. ERK also phosphorylates HSL to modulate the activity of the enzyme. We observed that the norepinephrine-induced phosphorylation of ERK1/2 and HSL were not impaired in WAT of eNOS-/- mice.
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These results suggest that eNOS system may play a role in mediating the adrenergic effect on mitochondrial biogenesis activation also in WAT. Apparently, an increase in UCP1 expression in WAT may be in contrast with the known effects of exercise on mitochondrial coupling efficiency in skeletal muscle (that increases, in order to produce more ATP) but this phenomenon could be explained by considering the different metabolic functions of skeletal muscle and adipose tissue, which imply different regulatory mechanisms of mitochondrial respiration and energy expenditure (34). Moreover it has been observed that physical exercise could induce an increase of uncoupling activity also in skeletal muscle (35; 36), therefore the balance between substrates oxidation, membrane potential and ATP production is a complex phenomenon that may have controversial aspects. The role of NO in the activation of mitochondrial biogenesis and the regulation of skeletal muscle glucose uptake during exercise have been extensively studied both in humans and in rodents (37), but so far little data has been available regarding the effect of exercise on adipose tissue insulin sensitivity and the role of NO in mediating this effect (38; 39). The data presented in this paper clearly show that the significant increase in the exercise-induced adipose tissue glucose uptake both in basal conditions and after insulin infusion in WT mice, is abolished in eNOS-/- mice. Therefore NO mediates, at least partially, the insulin sensitizing effect of exercise on subcutaneous adipose tissue. In order to further clarify whether the observed effect of exercise on adipose tissue mitochondrial biogenesis and insulin sensitivity could be a genuine consequence of eNOS activation, irrespective of the stimulation of fat cell βadrenoceptors (40; 41), we studied the in vitro effect of a NO donor on murine and human fully differentiated adipocytes. The obtained results allowed us to confirm the
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role of NO in inducing the master genes regulating mitochondrial biogenesis and function, along with a parallel increase of both basal and insulin stimulated GLUT4 traslocation and glucose uptake in mouse and human white adipocytes. Our findings highlight the important role of mitochondria in the regulation of adipocyte glucose homeostasis and support recent observations obtained after induction of mitochondrial dysfunction by knocking down Tfam, which led to a downregulation of GLUT4 expression and to an attenuation of insulin stimulated glucose uptake in 3T3-L1 adipocytes (42). Accordingly, the treatment with PPARγ agonists improves the dysfunctional adipose organ by increasing white adipocyte insulin sensitivity and mitochondrial biogenesis in WAT of insulin resistant mice (43). Morphological and structural changes of mitochondria -from small, fragmented units to larger networks of elongated organelles- play a role in several cellular processes according to the functional status of the cell. It has been recently reported that mitochondrial elongation is critical to sustain cell viability during macroautophagy induced by nutrient restriction (44). Longer mitochondria are protected from being degraded and possess more cristae where activity of the ATP synthase is increased, optimizing ATP production in times of nutrient restriction (44). Mitochondria unable to elongate 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 happens during nutrient restriction) morphological changes of mitochondrial shape occur (46; 47). We aimed to determine if an increased NO availability could influence mitochondrial remodeling in adipocytes and we found that chronic treatment with DETA-NO is able to induce a significant increase in mitochondrial
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area and promoted mitochondrial elongation in 3T3-L1 cells, possibly contributing to improved bioenergetics. We have also observed that the transcriptional machinery of mitochondrial biogenesis was activated, along with an increased COX IV and ATPsynthase mRNA expression and increased mitochondrial potential, in DETA-NO treated cells. In contrast with the in vivo studies we have observed only a slight, not significant increase in UCP1 mRNA expression in white adipocytes treated with the NO donor. This could be explained considering the in vivo situation, where we observe the results of a complexity of synergistic phenomena occurring during exercise, including the prolonged sympathetic overdrive. On the contrary, the in vitro environment probably excludes some major determinants which play a role in mediating the exercise effect on adipose tissue in vivo, even if it represents a useful model to assess the importance of NO per se in influencing mitochondrial biogenesis and glucose handling by fat cells. The role of exercise in preventing obesity-related metabolic disorders by acting on adipose tissue has been recently highlighted (48) (49). The impact of exercise is not limited to the exercising muscles, increasing fatty acids mobilization to match the peripheral energy requirement, but deeply influences the whole adipocyte energy metabolism by activating mitochondrial biogenesis and promoting insulin sensitivity (50). Our data show that these two phenomena are tightly associated and point to a crucial role of the eNOS system in mediating the effect of exercise training on the bioenergetic adaptation in subcutaneous white fat.
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Acknowledgments Author contributions: E.N. and R.V. designed research; E.T., M.S., M.O., M.G., R.F., R.S., M.Q and L.T. performed research; E.N., C.R., contributed new reagents/analytic tools; E.T., M.S., A.V. analyzed data; E.T., C.R., E.N., A.V., and R.V. wrote the paper. R.V. and E.N. are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. The authors declare no conflict of interest. This study was supported by Ministero dell'Università e della Ricerca (grants 20112010329EKE_005 to R.V., 2007HJTHM_001 to E.N. and 20075HJTHM_003 to A.V.), Ministero della Salute (grant RF-2009-1526404 to R.V.) and Cariparo Foundation.
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31. Szendroedi J, Phielix E, Roden M: The role of mitochondria in insulin resistance and type 2 diabetes mellitus. Nat Rev Endocrinol 2012;8:92-103 32. Sutherland LN, Bomhof MR, Capozzi LC, Basaraba SA, Wright DC: Exercise and adrenaline increase PGC-1{alpha} mRNA expression in rat adipose tissue. J Physiol 2009;587:1607-1617 33. Giordano A, Tonello C, Bulbarelli A, Cozzi V, Cinti S, Carruba MO, Nisoli E: Evidence for a functional nitric oxide synthase system in brown adipocyte nucleus. Febs Lett 2002;514:135-140 34. Ricquier D: Respiration uncoupling and metabolism in the control of energy expenditure. Proc Nutr Soc 2005;64:47-52 35. Befroy DE, Petersen KF, Dufour S, Mason GF, Rothman DL, Shulman GI: Increased substrate oxidation and mitochondrial uncoupling in skeletal muscle of endurance-trained individuals. Proc Natl Acad Sci U S A 2008;105:16701-16706 36. Jiang N, Zhang G, Bo H, Qu J, Ma G, Cao D, Wen L, Liu S, Ji LL, Zhang Y: Upregulation of uncoupling protein-3 in skeletal muscle during exercise: a potential antioxidant function. Free Radic Biol Med 2009;46:138-145 37. McConell GK, Wadley GD: Potential role of nitric oxide in contraction-stimulated glucose uptake and mitochondrial biogenesis in skeletal muscle. Clin Exp Pharmacol Physiol 2008;35:1488-1492 38. Gollisch KS, Brandauer J, Jessen N, Toyoda T, Nayer A, Hirshman MF, Goodyear LJ: Effects of exercise training on subcutaneous and visceral adipose tissue in normal- and high-fat diet-fed rats. Am J Physiol Endocrinol Metab 2009;297:E495504 39. Hussey SE, McGee SL, Garnham A, Wentworth JM, Jeukendrup AE, Hargreaves M: Exercise training increases adipose tissue GLUT4 expression in patients with type 2 diabetes. Diabetes Obes Metab 2011;13:959-962 40. Arner P, Kriegholm E, Engfeldt P: In situ studies of catecholamine-induced lipolysis in human adipose tissue using microdialysis. J Pharmacol Exp Ther 1990;254:284-288 41. Wahrenberg H, Bolinder J, Arner P: Adrenergic regulation of lipolysis in human fat cells during exercise. Eur J Clin Invest 1991;21:534-541 42. Wang CH, Wang CC, Huang HC, Wei YH: Mitochondrial dysfunction leads to impairment of insulin sensitivity and adiponectin secretion in adipocytes. Febs J 2013;280:1039-1050 43. Rong JX, Qiu Y, Hansen MK, Zhu L, Zhang V, Xie M, Okamoto Y, Mattie MD, Higashiyama H, Asano S, Strum JC, Ryan TE: Adipose mitochondrial biogenesis is suppressed in db/db and high-fat diet-fed mice and improved by rosiglitazone. Diabetes 2007;56:1751-1760 44. Rambold AS, Kostelecky B, Elia N, Lippincott-Schwartz J: Tubular network formation protects mitochondria from autophagosomal degradation during nutrient starvation. Proc Natl Acad Sci U S A 2011;108:10190-10195 45. Gomes LC, Di Benedetto G, Scorrano L: During autophagy mitochondria elongate, are spared from degradation and sustain cell viability. Nat Cell Biol 2011;13:589-598 46. Bo H, Zhang Y, Ji LL: Redefining the role of mitochondria in exercise: a dynamic remodeling. Ann N Y Acad Sci 2010;1201:121-128 47. Garnier A, Fortin D, Zoll J, N'Guessan B, Mettauer B, Lampert E, Veksler V, Ventura-Clapier R: Coordinated changes in mitochondrial function and biogenesis in healthy and diseased human skeletal muscle. Faseb J 2005;19:43-52
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48. Vissers D, Hens W, Taeymans J, Baeyens JP, Poortmans J, Van Gaal L: The effect of exercise on visceral adipose tissue in overweight adults: a systematic review and meta-analysis. Plos One 2013;8:e56415 49. Vieira VJ, Valentine RJ, Wilund KR, Antao N, Baynard T, Woods JA: Effects of exercise and low-fat diet on adipose tissue inflammation and metabolic complications in obese mice. Am J Physiol Endocrinol Metab 2009;296:E1164-1171 50. Ruschke K, Fishbein L, Dietrich A, Kloting N, Tonjes A, Oberbach A, Fasshauer M, Jenkner J, Schon MR, Stumvoll M, Bluher M, Mantzoros CS: Gene expression of PPARgamma and PGC-1alpha in human omental and subcutaneous adipose tissues is related to insulin resistance markers and mediates beneficial effects of physical training. Eur J Endocrinol 2010;162:515-523
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Grip test force (mN/g) Treadmill exhaustion time (%)
WT sedentary (n=8)
WT trained (n=8)
eNOS-/sedentary (n=8)
eNOS-/trained (n=8)
207.4 ± 6.6
228.3 ± 20.9
208.5 ± 5.5
221.8 ± 11.3
100.0 ± 6.9
133.8 ± 14.7* 100.3 ± 16.7 100.4 ± 9.7
Table 1. Effect of swim training on mice performance evaluated in vivo. Grip test performance was expressed as generated force (mN) relative to mouse body mass (g). Endurance was evaluated by time to exhaustion in treadmill running at increasing speeds and expressed relative to the exhaustion time of WT sedentary mice (37,6 +/2,6 min). *P< 0.05 relative to sedentary.
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Figure legends Fig 1. Mitochondrial biogenesis in mouse subcutaneous adipose tissue. Wild type and eNOS-/- mice (n=8 per group) were swim-trained for 6 weeks, then killed and subcutaneous adipose tissue was collected from inguinal fat pad. (A) Relative mRNA levels were measured by qRT-PCR using 18s rRNA as internal control and expressed as fold change. (B) mtDNA content was measured by means of qPCR and expressed as mtDNA copy number per nuclear DNA copy number. (C) Representative Western blots showing PGC-1α, COX IV and β-actin immunodetected signals in WAT lysates of mice. (D) Protein expression levels was measured by Western blot analysis using β-actin as internal control and expressed as fold change. All graphs depict mean ± SEM. Two-way ANOVA, *P< 0.05 and **P
Internal Medicine 3, Endocrine-Metabolic Laboratory, Department of Medicine DIMED, University of Padua, 35121 Padua, Italy 2
3
Department of Biomedical Sciences, University of Padua, 35121 Padua, Italy
Department of Molecular and Translational Medicine, University of Brescia, 25121 Brescia, Italy
4
Center for Study and Research on Obesity, Department of Medical Biotechnology and Translational Medicine, University of Milan, 20121 Milan, Italy; Corresponding author: Roberto Vettor, [email protected] R.V. and E.N. contributed equally to this work Prof. Roberto Vettor Department of Medicine-DIMED Head of Internal Medicine 3 Endocrine-Metabolic Unit University of Padua via Ospedale 105 35128 Padua Italy Phone: + 39 (0) 49 821 2648 Fax: + 39 (0) 49 821 3332 Mobile Phone +39 3457125856
*Dr. Quarta is currently affiliated with the Dept. of Nephrology and Neurological Sciences, School of Medicine, Stanford University, USA.
Diabetes Publish Ahead of Print,©published online March 19, 2014
Total word count in main text: 4466; word count in the abstract: 200; tables: 1; figures: 7. Abstract
Insulin resistance and obesity are associated with a reduction of mitochondrial content in various tissues of mammals. Moreover, a reduced nitric oxide (NO) bioavailability impairs several cellular functions including mitochondrial biogenesis and insulin-stimulated glucose uptake, two important mechanisms of body adaptation in response to physical exercise. Although these mechanisms have been thoroughly investigated in skeletal muscle and heart, few studies have focused on the effects of exercise on mitochondria and glucose metabolism in adipose tissue. In this study we compared the in vivo effects of chronic exercise in subcutaneous adipose tissue of wild type (WT) and eNOS knockout (eNOS-/-) mice after a swim training period. Then we investigated the in vitro effects of NO on mouse 3T3-L1 and human subcutaneous adipose tissue-derived adipocytes, after a chronic treatment with a NO donor (DETA-NO). We
observed
that
swim
training
increases
mitochondrial
biogenesis,
mitochondrial DNA (mtDNA) content and glucose uptake in subcutaneous adipose tissue of WT but not eNOS-/- mice. Furthermore we observed that DETA-NO promotes mitochondrial biogenesis and elongation, glucose uptake and GLUT4 translocation in cultured murine and human adipocytes. These results point to the crucial role of the eNOS-derived NO in the metabolic adaptation of subcutaneous adipose tissue to exercise training.
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A reduced mitochondrial content and/or activity is associated with impaired cell function in several diseases (1; 2). In particular, it has been hypothesized that mitochondrial impairment may be involved in the pathogenesis of obesity, insulin resistance and their progression towards type 2 diabetes (3; 4), even though the role of mitochondria in health and disease is still under discussion (5; 6). At the same time, metabolic disorders are also associated with a reduction of endothelial nitric oxide synthase (eNOS) enzymatic activity (7; 8), in fact mice lacking eNOS gene are considered a useful murine model for metabolic syndrome because they display typical
features
including
hypertension,
hypertriglyceridemia,
endothelial
dysfunction, insulin resistance and visceral obesity (9). It is well known that physical exercise induces profound physiological adaptations in several tissues as a response to increased metabolic requirements. One of the major event induced by physical activity is the upregulation of eNOS gene expression and the consequent increase of tissue nitric oxide (NO) production which in turn induces mitochondrial biogenesis and cell glucose uptake in skeletal and cardiac muscle (10-12) . Moreover, a selective depletion of peroxisome proliferator-activated receptor γ (PPARγ) co-activator 1 (PGC-1), a key regulator of mitochondrial biogenesis, leads to a blunting of exercise-induced increases in mitochondrial respiratory chain proteins in muscle (13) (14). Thus, a lack of response to physical exercise in terms of mitochondrial biogenesis could be related to a reduced or impaired NO metabolism. Insulin sensitivity is also increased after a training period and a single bout of exercise could enhance basal glucose uptake by increasing GLUT4 translocation to the cell membrane of skeletal (15) and cardiac (16) myocytes. Similar results have been observed in mouse and human tissues not directly involved in mechanical work and oxidative processes (17), but few studies have focused on subcutaneous adipose
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tissue. Tissues involved in the contractile activity during exercise, such as heart and skeletal muscles, increase their capacity to oxidize fuel substrates in order to meet the energy demand. Therefore they need to increase both lipid and glucose uptake and their oxidation. Emerging evidence suggests that these adaptations occur not only in working tissues such as skeletal muscle and heart but also in white and brown adipose tissue, liver, brain and kidney. Several studies show that exercise induces a “browning effect” in white adipose tissue (WAT) by increasing mitochondrial protein activity (18) or brown adipocyte-specific gene expression (19). A recent study hypothesized that this effect might be mediated by irisin (FNDC5), a PGC-1α-dependent myokine secreted during physical activity which promotes thermogenesis and uncoupling processes in white adipocytes, while leptin and other key regulator genes of “white” development are downregulated (20). Despite numerous results supporting this hypothesis, the role of irisin is still controversial (21) and requires further investigations. The aim of the present study was to investigate the mechanism underlying the response of subcutaneous adipose tissue to physical exercise, in terms of mitochondrial biogenesis and glucose uptake. In particular we explored whether NO can play a role in such metabolic adaptations.
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Research Design and Methods
Mice and exercise protocol. Thirty-six adult (8-weeks-old) male wild-type (C57BL/6J) and eNOS-/- (B6.129P2-Nos3tm1Unc/J) mice (all from Jackson Laboratory) were treated according to the EU guidelines and with the approval of the Institutional Ethical Committee. Body weight and food consumption were monitored throughout the experimental period. Both WT and eNOS-/- mice (n = 18 mice per group) were assigned randomly to either swim training (12) or to have no lifestyle modifications. Mice swam once a day for 5 days/week in a graduated protocol starting at 10 min daily, with 10 min increase each day until 90 min daily at 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 was maintained between 30°C and 35°C and mice were carefully towel-dried after each training session.
Muscle contractile performance in vivo. Muscle strength developed by WT and eNOS-/- mice during instinctive grasp was measured with the protocol indicated as grip test (22). Briefly, the mouse was held by the tail in proximity to a trapeze bar connected with the shaft of a force transducer. Once the mouse had firmly grabbed the trapeze, a gentle pull was exerted on the tail. The measurement of the peak force generated by the mouse was repeated several times with appropriate intervals to avoid fatigue and average peak force values were expressed relative to body mass. Endurance was measured with a test to exhaustion on treadmill. Initial speed (5 cm/s) was increased after 2 minutes at 10 cm/s. The speed was then increased by 2 cm/s every minute up to 50 cm/s and time to exhaustion was recorded.
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Tissue glucose utilization index. At the end of the training period and two days before the clamp studies, a catheter was inserted into the right femoral vein under general anesthesia with sodium pentobarbital. Tissue glucose uptake studies were performed on mice under conscious and unstressed conditions after a 8-h fast. 10 μCi of the nonmetabolizable glucose analog 2-deoxy-D-[1-3H]glucose ([3H]-DG) (Amersham Biosciences) were injected as a i.v. bolus in basal condition or after hyperinsulinemic
euglycaemic
clamp
as
previously
described
with
minor
modifications (23). Animals were killed 120 minutes after tracer injection, subcutaneous adipose tissue from inguinal fat pad was quickly collected in liquid nitrogen and kept at −80°C for subsequent analysis. The glucose utilization index was derived from the amount of [3H]-DG-6-phosphate ([3H]-DGP) measured in adipose tissue as previously described (24) thus using the accumulation of [3H]-DGP as an index of the glucose metabolic rate.
Norepinephrine treatment. A group of sedentary mice (n = 10 WT and n = 10 eNOS-/-) of the same strains as those utilized for the training experiments received a single i.p. injection of Norepinephrine hydrogentartrate 5mg/kg (Galenica Senese) or vehicle (0.9% saline solution). Animals were killed 24 hours after norepinephrine injection and the subcutaneous inguinal fat pad was quickly removed and kept at −80°C for subsequent mRNA expression analysis. An additional group of sedentary mice (n = 10 WT and n = 10 eNOS-/-) received the same norepinephrine treatment and were killed 30 minutes after the i.p. injection. Inguinal fat pad was quickly removed and kept at −80°C in order to quantify the protein expression and phosphorylation.
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Immunoblot analysis. Proteins were isolated from inguinal subcutaneous adipose tissues using T-PER Mammalian Protein Extraction Reagent (Pierce) as indicated by the manufacturer, in the presence of 1 mM NaVO4, 10 mM NaF and a cocktail of protease inhibitors (Sigma Aldrich). Protein content was determined by the bicinchoninic acid protein assay (Pierce) and 70 µg of proteins were run on SDSPAGE
under
reducing
conditions.
The
separated
proteins
were
then
electrophoretically transferred to a nitrocellulose membrane (Pierce). Proteins of interest were revealed with specific antibodies: anti-GLUT4, anti-AKT, anti-PhosphoAKT, anti-p44/42 MAPK (Erk1/2), anti-Phospho-p44/42 MAPK (Erk1/2), anti-HSL, anti-Phospho-HSL (all from Cell Signaling) and anti-β actin (Sigma-Aldrich). The immunostaining was detected using horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin for 1 h at room temperature. Bands were revealed by the SuperSignal Substrate (Pierce), and quantified by densitometry using NIH ImageJ software.
Cell cultures and treatment. Stromal vascular fraction from subcutaneous adipose tissue of 5 healthy patients undergoing bariatric surgery was isolated as previously described (25). Human-derived and 3T3-L1 (ATCC CL-173) preadipocytes were plated in Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10% fetal bovine serum, 150U/ml streptomycin, 200U/ml penicillin, 2mM glutamine and 1mM HEPES (all from Life Technologies). At confluence, adipogenic differentiation was induced by adding 1μM dexamethasone, 0.5mM 3-isobutyl-1-methyl-xantine (IBMX, Sigma-Aldrich) and 70nM insulin (Novo Nordisk). IBMX was removed from medium after 3 days of culture. Cells were cultured at 37˚C in a humidified atmosphere of 5% CO2 until fully differentiated. Cells were exposed to 100M
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diethylentriamine-NO (DETA-NO, Sigma-Aldrich) a potent NO donor (26), or vehicle for 72 h and then harvested for further analysis.
Cell glucose uptake. Human-derived and 3T3-L1 mature adipocytes were serumstarved for 8 h, incubated in the presence or absence of 2μM insulin (Novo Nordisk) for 30 min, then with 1.5 μCi/ml [3H]-DG (Amersham Biosciences) for 15 min. Cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed in 0.5 M NaOH. Radioactivity was measured by liquid scintillation counting (Wallac). Each condition was assayed in three independent experiments in triplicate.
RNA isolation and Real-Time quantitative PCR. Total RNA was isolated from cultured adipocytes and frozen subcutaneous adipose tissues of mice using RNeasy Mini 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 PCR (DNA Engine Opticon2, MJ Research) using SYBR Green PCR Master Mix (Applied Biosystems) and specific intron-spanning primers according to manufacturer’s instructions. All data were collected in triplicate and normalized to 18S gene expression.
Mitochondrial DNA. Mitochondrial DNA copy number was measured by means of quantitative PCR (qPCR) from the cytochrome B mtDNA gene compared with the large ribosomal protein p0 (36B4) nuclear gene as previously described (27).
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Mitochondrial morphology and membrane potential. After DETA-NO treatment, cells were incubated with 100nM of Mitotracker Green FM (MTG) dye (Molecular Probes) or with 5μM JC-1 (Molecular Probes) for 30 minutes at 37°C and 5% of CO2. After a 37°C medium wash, cells were observed using a Nikon Ti-E equipped with DS-2M cooled camera (Nikon) and a top-stage incubator Tokai Hit INU (Tokai Hit) in order to maintain optimal culturing conditions. MTG staining was evaluated by fluorescence imaging (490/516nm) with a 60X 1.45 NA objective and JC-1 staining was evaluated (514/529nm for the monomeric state to 585/590nm as dimeric state) with a 100x 0.75 NA objective.
Digital imaging processing. After acquisition, images were processed with NISElements AR software (Nikon) to evaluate shape and area of MTG stained cells. Briefly, binary objects were obtained by image segmentation, then using automatic measurement tool, area and elongation (intended as the ratio of Max Feret over Min Feret diameter, where Feret diameter is distance between the two parallel planes restricting the object perpendicular to that direction) of each mitochondria were calculated.
GLUT4 staining. After 2μM insulin stimulation for 30 min cells were fixed on coverslips with 4% paraformaldehyde and permeabilized with 0.25% Triton X-100. GLUT4 was detected by incubation with a polyclonal primary antibody (Santa Cruz Biotechnology) using standard procedures. After washing with PBS, binding of primary antibodies was detected with Alexa-488-conjugated secondary antibodies (Life Technologies). Coverslips were mounted with ProLong Gold antifade medium (Life Technologies) and analyzed with the confocal laser scanning microscope Leica
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DMI6000 CS SP8 (Leica Microsystem). Image analysis was performed with the NIH ImageJ software.
Statistical analysis. Results were expressed as means ± SEM. Data were analyzed by GraphPad Prism 5.0 software using either unpaired Student's t test, one-way ANOVA or two way ANOVA with Newman-Keuls’ post hoc test as appropriate.
Results
eNOS is required for mitochondrial biogenesis in response to exercise in subcutaneous adipose tissue. A swim training lasting 6 weeks was selected to induce mitochondrial biogenesis in wild type (WT) mice and in eNOS-/- mice. No significant differences in muscle strength, as measured with grip tests, were present between sedentary WT and eNOS-/- mice, although slightly higher values were observed in trained animals. Importantly, the training was able to significantly improve endurance in WT but not in eNOS-/- mice (Table 1). To determine if swim training induces an increase in mitochondrial biogenesis and/or function in adipose tissue and whether such increase is NO-dependent, we measured the mRNA level of multiple members of mitochondrial transcriptional machinery as well as mitochondrial DNA (mtDNA) content (an index of mitochondrial mass) in the subcutaneous adipose tissue of WT and eNOS-/- mice. The expression of PGC-1, Nuclear respiratory factor 1 (NRF-1), mitochondrial transcription factor A (Tfam) and cytochrome c oxidase IV (COX IV) mRNA was increased in subcutaneous adipose tissue of WT trained mice, compared to sedentary control littermates (Fig. 1A).
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In contrast, eNOS-/- mice, which showed reduced mRNA content of PGC-1 and COX IV in untrained conditions compared to WT mice, failed to display the mitochondrial biogenic response to exercise after the swim traninig period (Fig. 1A). The "browning" effect of exercise training on WAT was confirmed by the increase of UCP-1 mRNA content in trained WT animals, while in eNOS-/- mice the basal content of UCP-1 was very low an did not increase after exercise training. Interestingly, we also measured a basal mRNA expression of FNDC5, which is the precursor of the newly discovered hormone irisin (20), in subcutaneous adipose tissue of WT mice. FNDC5 was higher in WT in comparison with eNOS
-/-
sedentary mice
and did not change after exercise training. Furthermore, swim training increased mtDNA content of subcutaneous adipose tissue of WT but not eNOS-/- mice (Fig. 1B). We observed a significant increase in PGC-1α and COX IV protein expression in subcutaneous adipose tissue of WT but not eNOS-/- mice after the swim training protocol. Moreover eNOS-/- mice displayed a significantly lower PGC-1α protein content in trained conditions, compared to WT trained animals (Fig. 1C and 1D). In order to ascertain if modifications in β-adrenergic sensitivity could have influenced our findings, the effect of a single i.p. injection of 5 mg/kg norepinephrine was assessed in subcutaneous adipose tissue of WT and eNOS-/- mice. We observed a significant increase in PGC-1α and COX IV mRNA levels in WT mice 24 hours after the norepinephrine treatment (Fig. 2A). This effect was present also in eNOS-/- mice but to a remarkable lesser extent. Moreover, a slight increase in Tfam and NRF-1 mRNA levels was observed after β-adrenergic stimulation in WT but not in eNOS-/mice (Fig. 2A). To further demonstrate that the attenuated norepinephrine-induced expression of PGC-1α and other mitochondrial biogenesis factors in adipose tissue of eNOS-/- mice is due to a lack of eNOS and not to an impaired β-adrenergic signaling,
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we evaluated the phosphorylation state of two protein kinase A (PKA) substrates, e.g. the extracellular signal-regulated kinases 1/2 (ERK1/2) and the hormone sensitive lipase (HSL), 30 minutes after the adrenergic stimulation. We observed that phosphoERK1, phospho-ERK2 and phospho-HSL protein levels were significantly higher after norepinephrine injection in WAT of both WT and eNOS-/- mice, compared to untreated control animals (Fig. 2B and 2C). Notably, we did not observe any significant difference in basal protein levels of ERK1/2 or HSL between WT and eNOS-/- mice (Fig. 2B and 2C). These results suggest the functional integrity of the βadrenergic signaling pathway in eNOS null mutant mice. In order to rule out a possible non-specific effect due to the stress elicited by the i.p. injection, we compared the previous results with those obtained in a control group of both WT and eNOS-/- mice that did not receive any injection. We did not observe any statistically significant difference between treated and untreated mice (data not shown).
eNOS is required for basal and insulin stimulated glucose uptake in subcutaneous adipose tissue in response to exercise. To explore if swim training improves glucose metabolism or insulin sensitivity in subcutaneous adipose tissue of eNOS-/- mice, we measured tissue glucose utilization index as the amount of 2-deoxy[3H]glucose uptake in basal condition and after hyperinsulinemic euglycaemic clamp. Swim training increased both basal and insulin stimulated glucose uptake of subcutaneous adipose tissue in WT but not eNOS-/- mice (Fig. 3A). Moreover, eNOS-/mice displayed a lower glucose uptake capacity even in sedentary condition, compared to WT control mice (Fig. 3A). We observed a significant increase in pAKT/AKT protein content in subcutaneous adipose tissue of WT trained mice
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compared to sedentary littermates (Fig. 3B and 3C). eNOS-/- mice didn't increased their pAKT/AKT ratio in subcutaneous adipose tissue after swim protocol and showed a significant lower protein expression in trained conditions compared to trained WT animals. We measured also the GLUT4 total protein content in subcutaneous adipose tissue of both sedentary and trained animals, but no significant changes were observed in WT or eNOS-/- mice (Fig. 3B and 3C).
Nitric oxide promotes mitochondrial biogenesis and elongation, glucose uptake and GLUT4 translocation in 3T3-L1 adipocytes. To determine if an increased NO bioavailability could improve mitochondrial biogenesis and/or glucose uptake in mouse adipocytes we first measured mRNA content of PGC-1, NRF1, Tfam and COX IV in fully differentiated 3T3-L1 cells after a 72 hours treatment with 100μM DETA-NO. Gene expression of mitochondrial biogenesis markers was upregulated in DETA-NO treated cells compared to untreated control cells (Fig. 4A). Then, we evaluated the mitochondrial morphometric parameters by MitoTracker Green FM labeling, followed by a digital image analysis able to measure the area and the elongation of each mitochondrion (see digital imaging processing for details). We observed a higher presence of larger (area > 1.5μm2 ) and longer (length >3μm) mitochondria in treated cells, compared to those observed in untreated control cells (Fig. 4 B-D). Glucose uptake was measured after DETA-NO treatment by performing an in vitro 2-deoxy-[3H]glucose uptake assay. We observed that DETA-NO increased glucose transport of 3T3-L1 cells in both basal and insulin-stimulated (2μM) conditions (Fig. 5A). Moreover DETA-NO treatment induced an increase in GLUT4 translocation to cell membrane (measured as cytoplasm/membrane fluorescence ratio)
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in both basal and insulin-stimulated conditions, in comparison with untreated control cells (Fig. 5B).
Nitric oxide promotes mitochondrial biogenesis and polarization in human subcutaneous adipocytes. To further investigate the role of NO in adipose tissue, DETA-NO effects were studied also in subcutaneous adipocytes obtained from abdominal biopsies of subjects undergoing bariatric surgery. Stromal vascular fraction of adipose tissue was isolated and adipocytes cultured to full differentiation in vitro. We observed that PGC-1, Tfam, COX IV and ATP-synthase subunit β (ATPS) gene expression was upregulated in human mature adipocytes treated with DETA-NO compared to untreated cells (Fig. 6A). Moreover DETA-NO treatment increased mtDNA content in human mature adipocytes (Fig. 6B). Then we labeled the adipocytes mitochondria with the vital staining dye JC-1 and we observed that mitochondrial membrane potential was higher in cells after DETA-NO treatment, compared to mitochondrial membrane potential of untreated cells (Fig. 6C).
Nitric oxide promotes glucose uptake and GLUT4 translocation in human subcutaneous adipocytes. Glucose uptake and GLUT4 translocation were measured in fully differentiated human adipocytes as described for 3T3-L1 cells. We observed an increase in both basal and insulin-stimulated glucose uptake capacity in DETA-NO treated cells compared to untreated cells (Fig. 7A). Moreover the fraction of GLUT4 translocated on cells membrane was significantly higher after DETA-NO treatment even in basal condition, and slightly increased in insulin-stimulated conditions (Fig. 7B-C).
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Discussion The present study provides strong evidence that NO generated by eNOS plays a crucial role in the mitochondrial biogenesis and metabolic activation taking place in adipocytes, in response to physical activity. A reduction of mitochondrial abundance and activity has been linked with insulin resistance in obesity and type 2 diabetes (28). One of the most important physiological conditions widely recognized to increase both insulin sensitivity and mitochondrial biogenesis is physical activity: the common thought that exercise increases mitochondrial biogenesis mostly, if not exclusively, in skeletal muscles has recently been replaced by the notion that exercise training can induce mitochondrial biogenesis in a wide range of tissues not normally associated with the metabolic demand of exercise (29). Much emphasis has been placed on the role of mitochondrial biogenesis in the adipose organ in relation to its potential shift from white to brown phenotype or to the activation of beige phenotype. This phenomenon has been shown to be associated with increased energy expenditure and whole body insulin sensitivity (30; 31). In this study we report that physical training in mice was able to induce an increase in subcutaneous adipose tissue mRNA and protein levels of key transcriptional regulators of mitochondrial biogenesis, along with an increase in mtDNA content. Interestingly, at the end of the training period an increased in WAT mRNA expression of UCP1, the specific marker of brown adipose tissue, was also observed in subcutaneous fat. These results confirm and extend what was previously observed by Sutherland and colleagues who reported that exercise training increased PGC-1α and Tfam mRNA expression, as well as COX IV protein and citrate synthase activity in rat visceral fat depots (32), further supporting the evidence that exercise training is an effective stimulus for mitochondrial biogenesis in WAT. This phenomenon has been explained
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by the sympathetic stimulation during exercise, which has been shown to induce PCG-1α in several tissues including brown and white adipose tissue. Adrenaline directly stimulates PGC-1α in WAT and the β-blocker propranolol is able to reduce the exercise-induced rise in PGC-1α by responsible for the remaining
40% (32). Among the mechanisms
60% of the exercise effect on WAT mitochondria
biogenesis an increased production of myokines, hormones (i.e. thyroid hormones, glucocorticoids), signaling molecules coming from the exercising muscles (i.e. irisin, nitric oxide) or other still unknown factors could be taken into consideration. The results collected in this study add a novel and important piece of evidence showing that a significant portion of the induction in mitochondrial biogenesis in WAT after training is due to activation of eNOS system. We have observed a reduced basal expression of genes regulating mitochondrial biogenesis in adipose tissue of eNOS-/mice and, more importantly, the physical training period failed to induce any increase in these transcriptional regulators or in mtDNA content. It has been shown that the sympathetic stimulation of brown adipose tissue in vivo and in vitro significantly increased eNOS expression and activity (33) and we observed that the norepinephrine-induced up-regulation of mitochondrial biogenesis factors was reduced in WAT of eNOS-/- mice. To evaluate any changes of the sympathetic activity in the knockout mice, we investigated the noradrenergic signaling in these animals. In adipose tissue, HSL enzyme activity is strongly induced by β-adrenergic stimulation. HSL and ERK are major targets for PKA-mediated phosphorylation, the first step downstream the βadrenergic receptor. ERK also phosphorylates HSL to modulate the activity of the enzyme. We observed that the norepinephrine-induced phosphorylation of ERK1/2 and HSL were not impaired in WAT of eNOS-/- mice.
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These results suggest that eNOS system may play a role in mediating the adrenergic effect on mitochondrial biogenesis activation also in WAT. Apparently, an increase in UCP1 expression in WAT may be in contrast with the known effects of exercise on mitochondrial coupling efficiency in skeletal muscle (that increases, in order to produce more ATP) but this phenomenon could be explained by considering the different metabolic functions of skeletal muscle and adipose tissue, which imply different regulatory mechanisms of mitochondrial respiration and energy expenditure (34). Moreover it has been observed that physical exercise could induce an increase of uncoupling activity also in skeletal muscle (35; 36), therefore the balance between substrates oxidation, membrane potential and ATP production is a complex phenomenon that may have controversial aspects. The role of NO in the activation of mitochondrial biogenesis and the regulation of skeletal muscle glucose uptake during exercise have been extensively studied both in humans and in rodents (37), but so far little data has been available regarding the effect of exercise on adipose tissue insulin sensitivity and the role of NO in mediating this effect (38; 39). The data presented in this paper clearly show that the significant increase in the exercise-induced adipose tissue glucose uptake both in basal conditions and after insulin infusion in WT mice, is abolished in eNOS-/- mice. Therefore NO mediates, at least partially, the insulin sensitizing effect of exercise on subcutaneous adipose tissue. In order to further clarify whether the observed effect of exercise on adipose tissue mitochondrial biogenesis and insulin sensitivity could be a genuine consequence of eNOS activation, irrespective of the stimulation of fat cell βadrenoceptors (40; 41), we studied the in vitro effect of a NO donor on murine and human fully differentiated adipocytes. The obtained results allowed us to confirm the
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role of NO in inducing the master genes regulating mitochondrial biogenesis and function, along with a parallel increase of both basal and insulin stimulated GLUT4 traslocation and glucose uptake in mouse and human white adipocytes. Our findings highlight the important role of mitochondria in the regulation of adipocyte glucose homeostasis and support recent observations obtained after induction of mitochondrial dysfunction by knocking down Tfam, which led to a downregulation of GLUT4 expression and to an attenuation of insulin stimulated glucose uptake in 3T3-L1 adipocytes (42). Accordingly, the treatment with PPARγ agonists improves the dysfunctional adipose organ by increasing white adipocyte insulin sensitivity and mitochondrial biogenesis in WAT of insulin resistant mice (43). Morphological and structural changes of mitochondria -from small, fragmented units to larger networks of elongated organelles- play a role in several cellular processes according to the functional status of the cell. It has been recently reported that mitochondrial elongation is critical to sustain cell viability during macroautophagy induced by nutrient restriction (44). Longer mitochondria are protected from being degraded and possess more cristae where activity of the ATP synthase is increased, optimizing ATP production in times of nutrient restriction (44). Mitochondria unable to elongate 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 happens during nutrient restriction) morphological changes of mitochondrial shape occur (46; 47). We aimed to determine if an increased NO availability could influence mitochondrial remodeling in adipocytes and we found that chronic treatment with DETA-NO is able to induce a significant increase in mitochondrial
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area and promoted mitochondrial elongation in 3T3-L1 cells, possibly contributing to improved bioenergetics. We have also observed that the transcriptional machinery of mitochondrial biogenesis was activated, along with an increased COX IV and ATPsynthase mRNA expression and increased mitochondrial potential, in DETA-NO treated cells. In contrast with the in vivo studies we have observed only a slight, not significant increase in UCP1 mRNA expression in white adipocytes treated with the NO donor. This could be explained considering the in vivo situation, where we observe the results of a complexity of synergistic phenomena occurring during exercise, including the prolonged sympathetic overdrive. On the contrary, the in vitro environment probably excludes some major determinants which play a role in mediating the exercise effect on adipose tissue in vivo, even if it represents a useful model to assess the importance of NO per se in influencing mitochondrial biogenesis and glucose handling by fat cells. The role of exercise in preventing obesity-related metabolic disorders by acting on adipose tissue has been recently highlighted (48) (49). The impact of exercise is not limited to the exercising muscles, increasing fatty acids mobilization to match the peripheral energy requirement, but deeply influences the whole adipocyte energy metabolism by activating mitochondrial biogenesis and promoting insulin sensitivity (50). Our data show that these two phenomena are tightly associated and point to a crucial role of the eNOS system in mediating the effect of exercise training on the bioenergetic adaptation in subcutaneous white fat.
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Acknowledgments Author contributions: E.N. and R.V. designed research; E.T., M.S., M.O., M.G., R.F., R.S., M.Q and L.T. performed research; E.N., C.R., contributed new reagents/analytic tools; E.T., M.S., A.V. analyzed data; E.T., C.R., E.N., A.V., and R.V. wrote the paper. R.V. and E.N. are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. The authors declare no conflict of interest. This study was supported by Ministero dell'Università e della Ricerca (grants 20112010329EKE_005 to R.V., 2007HJTHM_001 to E.N. and 20075HJTHM_003 to A.V.), Ministero della Salute (grant RF-2009-1526404 to R.V.) and Cariparo Foundation.
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Grip test force (mN/g) Treadmill exhaustion time (%)
WT sedentary (n=8)
WT trained (n=8)
eNOS-/sedentary (n=8)
eNOS-/trained (n=8)
207.4 ± 6.6
228.3 ± 20.9
208.5 ± 5.5
221.8 ± 11.3
100.0 ± 6.9
133.8 ± 14.7* 100.3 ± 16.7 100.4 ± 9.7
Table 1. Effect of swim training on mice performance evaluated in vivo. Grip test performance was expressed as generated force (mN) relative to mouse body mass (g). Endurance was evaluated by time to exhaustion in treadmill running at increasing speeds and expressed relative to the exhaustion time of WT sedentary mice (37,6 +/2,6 min). *P< 0.05 relative to sedentary.
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Figure legends Fig 1. Mitochondrial biogenesis in mouse subcutaneous adipose tissue. Wild type and eNOS-/- mice (n=8 per group) were swim-trained for 6 weeks, then killed and subcutaneous adipose tissue was collected from inguinal fat pad. (A) Relative mRNA levels were measured by qRT-PCR using 18s rRNA as internal control and expressed as fold change. (B) mtDNA content was measured by means of qPCR and expressed as mtDNA copy number per nuclear DNA copy number. (C) Representative Western blots showing PGC-1α, COX IV and β-actin immunodetected signals in WAT lysates of mice. (D) Protein expression levels was measured by Western blot analysis using β-actin as internal control and expressed as fold change. All graphs depict mean ± SEM. Two-way ANOVA, *P< 0.05 and **P
