- Email: [email protected]
Effects of physical exercise on myokines expression and brown adipose-like phenotype modulation in rats fed a high-fat diet
Life Sciences 165 (2016) 100–108
Contents lists available at ScienceDirect
Life Sciences journal homepage: www.elsevier.com/locate/lifescie
Effects of physical exercise on myokines expression and brown adipose-like phenotype modulation in rats fed a high-fat diet Sílvia Rocha-Rodrigues a,⁎, Amaia Rodríguez b,e,f, Alexandra M. Gouveia c,g, Inês O. Gonçalves a, Sara Becerril b,e,f, Beatriz Ramírez b,e,f, Jorge Beleza a, Gema Frühbeck b,d,e,f, António Ascensão a, José Magalhães a a
CIAFEL - Research Centre in Physical Activity, Health and Leisure, Faculty of Sport, University of Porto, Porto, Portugal Metabolic Research Laboratory, Clínica Universidad de Navarra, Pamplona, Spain Department of Experimental Biology, Faculty of Medicine, University of Porto, Porto, Portugal d Department of Endocrinology & Nutrition, Clínica Universidad de Navarra, Pamplona, Spain e Obesity & Adipobiology Group, Instituto de Investigación Sanitario de Navarra (IdiSNA), Pamplona, Spain f CIBEROBN, Instituto de Salud Carlos III, Pamplona, Spain g Instituto de Investigação e Inovação em Saúde, Institute for Molecular and Cell Biology (IBMC), University of Porto, Porto, Portugal b c
a r t i c l e
i n f o
Article history: Received 26 July 2016 Received in revised form 16 September 2016 Accepted 26 September 2016 Available online 28 September 2016 Keywords: Endurance training Skeletal muscle Adiposity FNDC5/irisin IL-6 Browning UCP1
a b s t r a c t Aims: Exercise-stimulated myokine secretion into circulation may be related with browning in white adipose tissue (WAT), representing a positive metabolic effect on whole-body fat mass. However, limited information is yet available regarding the impact of exercise on myokine-related modulation of adipocyte phenotype in WAT from obese rats. Main methods: Sprague-Dawley rats (n = 60) were divided into sedentary and voluntary physical activity (VPA) groups and fed with standard (35 kcal% fat) or high-fat (HFD, 71 kcal% fat)-isoenergetic diets. The VPA-groups had unrestricted access to wheel running throughout the protocol. After-9 weeks, half of sedentary standard (SS) and sedentary HFD (HS)-fed animals were exercised on treadmill (endurance training, ET) for 8-weeks while maintaining the dietary treatments. Key findings: The adipocyte hypertrophy induced by HFD were attenuated by VPA and ET. HFD decreased 5′ AMPactivated protein kinase (AMPK) activity in muscle as well as peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) and uncoupling protein 1 (UCP1) proteins in eWAT, while not affecting circulating irisin. VPA increased eWAT Tmem26 mRNA levels in the standard diet-fed group, whereas ET increased AMPK, interleukin 6 (IL-6) and fibronectin type III domain-containing protein 5 (FNDC5) protein expression in muscle, but had no impact on circulating irisin protein content. In eWAT, ET increased bone morphogenetic protein 7 (Bmp7), Cidea and PGC-1α in both diet-fed animals, whereas BMP7, Prdm16, UCP1 and FNDC5 only in standard diet-fed group. Significance: Data suggest that ET-induced myokine production seems to contribute, at least in part, to the “brown-like” phenotype in WAT from rats fed a HFD. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Overweight and obesity are highly prevalent conditions in industrialized countries. Although genetic factors contribute to the variance of adiposity, in most cases, obesity results from an imbalance between energy intake and expenditure due to a high consumption of hypercaloric food and/or reduced levels of physical activity. The white adipose tissue (WAT) constitutes the main store of lipids, buffering daily dietary fat entering from the circulation [1]. Nonetheless, obesity-induced WAT ⁎ Corresponding author at: Research Centre in Physical Activity, Health and Leisure, Faculty of Sport Sciences, University of Porto, Rua Dr. Plácido Costa, 91, 4200-450 Porto, Portugal. E-mail address: [email protected] (S. Rocha-Rodrigues).
http://dx.doi.org/10.1016/j.lfs.2016.09.023 0024-3205/© 2016 Elsevier Inc. All rights reserved.
dysfunction can lead to ectopic fat accumulation in other tissues, which have been associated with obesity-related metabolic diseases [1–3]. White and brown adipocytes are two distinct types of fat cells with opposite functions. White adipocytes are highly adapted to store excess energy, whereas brown adipocytes utilize fatty acids for generating heat via mitochondrial uncoupling protein 1 (UCP1) [4], thereby dropping the availability of substrates for storage in WAT. Moreover, two populations of brown-like fat cells with thermogenic properties have been identified, the “classical brown” and “beige” adipocytes [4]. Interestingly, the abundance of these two brown cell types can be induced in response to appropriate physiological stimuli, such as chronic cold exposure, irisin, peroxisome proliferator-activated receptor γ (PPARγ) agonists or β-adrenergic stimulation [5–8], increasing whole-body
S. Rocha-Rodrigues et al. / Life Sciences 165 (2016) 100–108
metabolic rate, and therefore preventing diet-induced obesity (DIO) and related diseases [4,6]. The metabolic alterations induced by physical exercise have pleiotropic effects. Previous studies have shown that regular exercise affects not only the exercised skeletal muscle, but also other non-contractile organs over time [5,6,9]. One potential mechanism underlying this crosstalk is the secretion of proteins that mediate communication between muscle and other tissues through endocrine mechanisms [10, 11], including liver and visceral adipose tissue (VAT). Accordingly, several studies have focused on myokines released from muscle during and immediately after exercise, such as interleukins (IL)-6, -8 and -15, leukemia inhibitory factor (LIF) and fibroblast growth factor 21 (FGF21) [for a review see ref. 10, 11]. In addition, the discovery of fibronectin type III-domain containing 5 (FNDC5) as a PGC-1α-dependent myokine showed that its derived product secreted into circulation (irisin) has the ability to drive a brown adipocyte-like phenotype in WAT [5]. In vitro undifferentiated white adipocytes treated with FNDC5 overexpressed UCP1-positive cells [5] and increased mitochondrial density and gene expression, which led to increased oxygen consumption, heat loss and greater energy expenditure [6]. In this context, a new role for myokines has emerged in the field of exercise-related adaptations; however little is still known regarding the physiological impact of myokines on the adaptive response to chronic exercise, and particularly on the brown adipocyte-like phenotype stimulation of VAT in a rat model of DIO. Thus, using this nutritional model of obesity in rats, the main goal of the present study was to analyze the role of voluntary physical activity (VPA) and endurance training (ET) as hypothetical modulators of an increase in brown-like phenotype in WAT. 2. Material and methods 2.1. Reagents Bradford reagent, Laemmli sample buffer and block reagent were purchased from Bio-Rad (Hercules, CA, USA). Chemiluminescent reagent ECL-Plus™ from GE Healthcare, Amersham Biosciences (Buckinghamshire, U.K.). Polyvinylidene difluoride membranes (Immobilon-N) were purchased from Merck Millipore Corporation (Bedford, MA, USA) and nitrocellulose membranes were obtained from Whatman, Protan (Pittsburgh, PA, USA). Protease inhibitor and phosphatase inhibitor cocktails were purchased from Sigma-Aldrich (Barcelona, Spain). Goat anti-BMP7 (sc9305), goat anti-β-actin (sc1616) and secondary peroxidase-conjugated antibodies (anti-rabbit, sc-2317, anti-mouse, sc-2317 and anti-goat, sc-2020) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Rabbit anti-phospho [Thr172] AMPKα (2531) and rabbit anti-SIRT3 (C73E3) were obtained from Cell Signalling Tecnhology, Inc. (Danvers, MA, USA). Goat anti-PGC-1α (ab106814), rabbit anti-IL-6 (ab6672), mouse anti-AMPK (ab80039), rabbit monoclonal anti-FNDC5 (ab174833), rabbit anti-UCP1 (ab23841) were acquired from Abcam (Cambridge, UK). Goat antiUCP2 (Sab2501087) was obtained from Sigma Aldrich and rabbit antiSIRT1 (13161-1-AP) from Proteintech Group, Inc. (Chicago, IL, USA). TRIzol® reagent and SYBR® Green PCR Master Mix were purchased from Life Technologies (Carlsbad, USA), NZY First-strand cDNA Synthesis Kit (NZYTech, Lisbon, Portugal). All other chemicals were purchased from Sigma-Aldrich (Barcelona, Spain). 2.2. Animals and diets Six-week-old male Sprague-Dawley rats (n = 60, Charles River, L'Arbresle, France) were kept in a pathogen-free room, constant temperature (21–22 °C) and humidity (50–60%) with a fixed 12-h light/ 12-h dark cycle. Rats (initial body weight 233.9 ± 2.6 g) were fed ad libitum during 17 weeks a nutritionally adequate isoenergetic, isoproteic standard (composed by 35% kcal fat, 47% kcal carbohydrates,
101
and 18% kcal protein) or a high-fat (HFD, composed by 71% kcal fat, 11% kcal carbohydrate, and 18% kcal protein)-liquid diet purchased from Dyets Inc. (catalog no. 710027 and 712031, respectively). This DIO model has been commonly used to induce visceral adipose tissue accumulation and related metabolic disorders in experimental animals [2, 12]. All animals had free access to water and food (provided in the liquid state) throughout the entire protocol. During the first week, the standard diet was given to all animals as an adaptation to the liquid feeding. Afterwards, animals were divided into six groups (n = 10/group): standard-diet sedentary (SS), standard-diet voluntary physical activity (SVPA), standard-diet endurance-trained (SET), high-fat diet sedentary (HS), high-fat diet voluntary physical activity (HVPA) and high-fat diet endurance-trained (HET), as previously described [2]. Energy intake (kilocalories per day) was measured daily while body weights were monitored once weekly during the period of the present study. The study was approved by the Institutional Ethics Committee of the Faculty of Sport, University of Porto and followed the guidelines for the care and use of laboratory animals in research advised by the Federation of European Laboratory Animal Science Association (FELASA). 2.3. Voluntary physical activity and endurance training interventions VPA intervention: voluntary-exercised animals (SVPA and HVPA) were assigned in cages equipped with 365-mm (diameter) running wheels. Based on the evidence that VPA may be an effective strategy to prevent the progress of, at least, some obesity-associated metabolic abnormalities [2], animals had unrestricted access to the running wheels during the 17 weeks of the feeding protocol. Wheel revolutions were daily recorded from a digital counter between 08.00 and 10.00 h. SVPA and HVPA animals were moved to standard cages for one night before sacrifice to avoid interference of exercise-related acute effects. ET program: Eight weeks after the diet intervention, SET and HET animals were submitted to a chronic ET program. Initially, animals were progressively acclimated to the motor-driven treadmill (Le8700, Panlab Harvard Apparatus) for 5 days per week at 15 m min−1 and 0% grade during 30 min. Then, animals performed a moderate intensity ET program (corresponding to 50–60% of VO2 max) consisted of 5 sessions per week and 60 min per day at a starting speed of 15 m min−1, which was progressively increased over the training program until 25 m min−1 was reached [3,13]. In order to understand the therapeutic effects of ET, animals started the ET program 8 weeks after the diet treatment since at this time point HFD-fed animals already exhibited obesity-related metabolic features. Sedentary animals (SS and HS groups) were placed on a non-moving treadmill during the training sessions to be exposed to the same environmental conditions, but without promoting any physical training adaptation. In order to eliminate acute effects of exercise, SET and HET animals were sacrificed 48 h after the last training session. 2.4. Visceral adipose tissue and blood collection All animals were fasted overnight for 12 h with access to drinking water before sacrifice. Animals were weighed at the day of sacrifice and anesthetized with ketamine (90 mg·kg− 1) and xylazine (10 mg·kg−1). Blood was collected and plasma separated by centrifugation 3000g for 15 min at 4 °C. Afterwards, visceral WAT depots (mesenteric, epididymal and retroperitoneal) and skeletal muscle (gastrocnemius and soleus) were excised and weighted. Epididymal fat pad and both gastrocnemius and soleus were processed for analysis as described below. 2.5. Adipocyte-size profiling Morphological analyses were performed using light microscopy as previously described [14]. Briefly, visceral WAT was fixed in 4%
102
S. Rocha-Rodrigues et al. / Life Sciences 165 (2016) 100–108
formaldehyde solution (Sigma Aldrich) embedded in paraffin blocks, sectioned, and stained with hematoxylin and eosin. Stained samples were visualized using a microscope (Zeiss AX10 imager A.1, Oberkochen, Germany) under ×40 magnification. Average of adipocyte area was determined, removing any objects below an area of 350 μm2, as these cells may be a mixture of adipocytes and stromal vascular cells [14]. The average of adipocyte area and the frequency of distribution were quantified from 4 sections per rat and 4 rats per group (N1500 adipocytes counted per group). 2.6. Citrate synthase activity in skeletal muscle Citrate synthase (CS) activity was measured in soleus homogenate using the method proposed by Coore et al. [15]. Briefly, CoA-SH released from the reaction of acetyl-CoA with oxaloacetate was measured by its reaction with a colorimetric agent [5, 5-dithiobis (2-nitrobenzoate)]. The change in color was monitored spectrophotometrically at 412 nm. 2.7. Circulating irisin content Plasma samples were diluted (1:20) in Tris buffered saline (TBS; 100 mmol Tris, 1.5 mmol NaCl, pH 8.00) and 100 μL was slot-blotted into a nitrocellulose membrane (Whatman, Protan). The slot-blot membranes were blocked with 5% (w/v) dry nonfat milk in TBS with 0.05% Tween 20 (TBS-T) and then incubated for 30 min with rabbit antimonoclonal FNDC5 diluted 1:1000 in 5% (w/v) nonfat dry milk in TBST. Afterwards, membranes were incubated with a solution of horseradish-conjugated anti-rabbit antibody diluted at 1:10,000. The blots were detected by ChemiDoc™ XRS + System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and band densities were quantified using Image Lab™ software 262 5.2.1 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Control for protein loading was confirmed by Ponceau S staining. 2.8. Western Blot studies The gastrocnemius muscle and epididymal white adipose tissue (eWAT) were homogenized in ice-cold RIPA buffer containing 50 mmol/L Tris-HCl (pH 7.40), 1 mmol/L EDTA, 0.2% sodium dodecyl sulfate, 0.2% deoxycholate and 1% Triton X-100 supplemented with protease and phosphatase inhibitors using a Polytron homogenizer for 30 s. Samples homogenates were centrifuged (13,000g for 10 min at 4 °C) and the supernatant was harvested. An aliquot of the tissue lysates was used to determine the concentration of protein in each sample by Bradford method. Samples were prepared in 2 × Laemmli buffer containing 710 mmol/L β-mercaptoethanol and heated in a boiling water bath for 5 min. To determine the total-AMPK, pAMPK (62 kDa), IL-6 (28 kDa) and FNDC5 (25 kDa) contents in gastrocnemius, aliquots containing 25 μg were subjected to SDS-PAGE and then transferred to polyvinyldifluoride membranes (Millipore). To determine PGC-1α (91 kDa), SIRT1 (75 kDa), SIRT3 (28 kDa), FNDC5 (25 kDa), UCP2 (33 kDa), UCP1 (33 kDa), BMP7 (55 kDa) and β-actin (42 kDa) contents in eWAT, 50 μg protein were loaded onto the gels. All primary antibodies were at 1:1000 dilution. The blots were detected by ChemiDoc™ XRS + System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and band densities were quantified using Image Lab™ software 262 5.2.1 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Membranes were stripped and re-probed with an anti-β-actin antibody, as an internal control. The values were obtained by dividing the density of the band of interest by that of either β-actin (as indicated in the figure legends) from the same blot. 2.9. RT-qPCR analyses Total RNA of eWAT was isolated using TRIzol® reagent as described by the manufacturer. The concentration and purity of RNA were assessed using NanoDrop™ 1000 spectrophotometer by reading
absorbance at 230 nm, 260 nm and 280 nm (Thermo Scientific, CA, USA). One point five micrograms of total RNA worked as template for cDNA production using the NZY First-strand cDNA Synthesis Kit (MB12501), which included a combination of random hexamers and oligo(dT) primers. Quantitative Real-time PCR (RT-qPCR) was conducted using SYBR® Green PCR Master Mix on a Step One Plus thermocycler (Applied Biosystems) using paired reverse and forward primers as shown in Table 1. Each sample was assayed in a 12 μL reaction in duplicate. If the duplicate contained a cycle threshold (CT) standard deviation of N0.5, it was reassayed. All reactions were performed under the same conditions 95 °C for 3 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The data were analyzed using GAPDH as the internal control with the cycle threshold (2−ΔΔCT) method as recommended by Applied Biosystems. 2.10. Statistical analysis Values are presented as mean ± standard error of the mean (SEM). The effect of diet and exercise was analyzed by a two-way analysis of variance (ANOVA). The Bonferroni post hoc test was applied for comparisons between groups and differences were considered significant at p ≤ 0.05. Pearson's correlation coefficients were used to analyze possible associations between variables. Statistical analysis was performed using SPSS 15.0 for Windows (SPSS Inc., Chicago IL, USA). 3. Results 3.1. Effects of exercise and diet on body weight, energy intake and visceral adiposity As illustrated in Fig. 1A, body weight of HS animals remained similar to that of SS group. Moreover, VPA had no impact on body weight while ET for 8 weeks significantly reduced body weight in both exercised groups compared with sedentary counterparts. No significant alterations were observed in cumulative energy intake (kcal) between studied groups (Fig. 1B), as expected in an isoenergetic pair-feeding diet. Nevertheless, HFD induced an increase in the relative weights of mesenteric, epididymal and retroperitoneal fat depots, which were reverted by VPA in mesenteric fat depot, epididymal and retroperitoneal fat pads (Fig. 1C–E). In addition, ET reduced all WAT depots in SET and HET groups compared with their sedentary counterparts. 3.2. Effects of diet and exercise on adipocyte-area profiling visceral WAT Animals from HS group presented a higher percentage of larger adipocytes (≥ 5000 μm2) when compared to SS group (Fig. 2). VPA did not alter the percentage of adipocytes below 5000 μm2 in both SVPA and HVPA groups; however, VPA reduced the percentage of larger adipocytes in HVPA group compared to sedentary counterpart. Moreover, ET significantly increased the percentage of smaller adipocytes (b2000 μm2) and reduced the number of larger adipocytes (≥5000 μm2) in both endurance-trained groups (Fig. 2C–D).
Table 1 Primer sequences used for RT-qPCR. GenBank
Accession no.
Primer sequence (5′–3′)
Tmem26
NM_001107623
Bmp7
NM_001191856
Cidea
NM_001170467
Prdm16
XM_008764418
Gapdh
NM_017008
F-CCGAGGCTACAAATGGCTTTC R-ACTGGTTTCCATGGTGCATTTC F-CCTGCAAGAAACACGAGCTGTAT R-AGGCACACTCTCCCTCACAGTAGT F-TGACATTCATGGGGTTGCAGA R-GGCCAGTTGTGATGACCAAGA F-CTCCGAGATCCGAAACTT R-CTCAGGCCGTTTGTCCAT F-GGTGAAGGTCGGAGTCAACG R-CAAAGTTGTCATGGATGACC
S. Rocha-Rodrigues et al. / Life Sciences 165 (2016) 100–108
103
Fig. 1. Effects of diet and exercise on body weight, energy intake and relative fat depot weights. The mean body weight gain (A) and cumulative energy intake, expressed as kcal per day (B), and the relatives weights of mesenteric (C), epididymal (D) and retroperitoneal (E) fat depots. Data are expressed as the mean ± SEM. SS, Standard diet sedentary; SVPA, Standard diet voluntary physical activity; SET, Standard diet endurance trained; HS, High-fat diet sedentary; HVPA, High-fat diet voluntary physical activity; HET, High-fat diet endurance training (p b 0.05). a vs. SS; b vs. HS; c vs. SVPA; d vs. SET; e vs. HVPA.
3.3. Effects of diet and physical exercise on skeletal muscle characteristics and on circulating irisin Long-term HFD did not affect neither soleus, gastrocnemius nor total muscle mass/body weight ratio (Fig. 3A–C). On the other hand, VPA increased the relative gastrocnemius weight and total
muscle mass in SVPA group. Neither HFD nor VPA interventions have impact on muscle CS activity, a validated marker of oxidative adaptation to ET program (Fig. 3D). In contrast, 8-week ET increased the relative soleus, gastrocnemius and muscle mass weights, as well as the CS activity in SET and HET groups when compared with their sedentary counterparts.
Fig. 2. Effects of diet and exercise on (A) adipocyte-size profiling of adipocytes from WAT. (C) Bar graphs representing the percentage (%) of adipocytes with area below 2000 and higher or equal 2000 μm2 and, (D) below 5000 and higher or equal 5000 μm2. Adipocytes areas were determined from 4 sections per rat. Representative images of hematoxylin and eosin staining of visceral WAT (B). Data are expressed as the mean ± SEM. SS, Standard diet sedentary; SVPA, Standard diet voluntary physical activity; SET, Standard diet endurance trained; HS, High-fat diet sedentary; HVPA, High-fat diet voluntary physical activity; HET, High-fat diet endurance training (p b 0.05). a vs. SS; b vs. HS; c vs. SVPA; d vs. SET; e vs. HVPA; *significant between areas in the same group.
104
S. Rocha-Rodrigues et al. / Life Sciences 165 (2016) 100–108
Fig. 3. Effects of diet and exercise on soleus (A) and gastrocnemius (B) muscle weight and the total muscle mass-body weight ratio (C), and citrate synthase (CS) activity (D). Data are expressed as the mean ± SEM. SS, Standard diet sedentary; SVPA, Standard diet voluntary physical activity; SET, Standard diet endurance trained; HS, High-fat diet sedentary; HVPA, High-fat diet voluntary physical activity; HET, High-fat diet endurance training (p b 0.05). a vs. SS; b vs. HS; c vs. SVPA; d vs. SET; e vs. HVPA.
Fig. 4. (A–E): Effect of diet and exercise on the activation/phosphorylation of AMPK (A), IL-6 (B), FNDC5 (C) on skeletal muscle and circulating irisin content (D). Representative blots of each protein (E). Values of protein levels are expressed as % AU (arbitrary units), where SS group was set as 100%. Data are expressed as the mean ± SEM. SS- Standard diet sedentary; SVPA- Standard diet voluntary physical activity; SET, Standard diet endurance trained; HS, High-fat diet sedentary; HVPA, High-fat diet voluntary physical activity; HET, High-fat diet endurance training (p b 0.05). a vs. SS; b vs. HS; c vs. SVPA; d vs. SET; e vs. HVPA.
S. Rocha-Rodrigues et al. / Life Sciences 165 (2016) 100–108
As shown in Fig. 4, HS animals exhibited diminished skeletal muscle 5′ AMP-activated protein kinase (AMPK) activation (pAMPK/ tAMPK ratio), as quantification of AMPK Thr(172) phosphorylation by immunoblotting is a recognized surrogate for AMPK activity [16], compared to SS group. Concerning exercise interventions, VPA had no effect in any parameter, but ET significantly increased pAMPK/tAMPK ratio in both endurance-trained groups compared to their sedentary and voluntary-exercised counterparts. The HFD regimen per se had no impact on skeletal muscle IL-6 expression, while VPA tended to increase and ET significantly increased its expression in both standard and HFD-fed animals compared to sedentary and voluntary-exercised counterparts. Moreover, skeletal muscle IL-6 positively correlated with muscle mass (r = 0.71, p b 0.001) and negatively correlated with body weight (r = − 0.65, p = 0.001) and total VAT depots (r = − 0.79, p b 0.001). Although without statistical significance, HFD intervention tended to reduce skeletal muscle FNDC5 protein expression in HS group. Despite the absence of any effect prompt by VPA, ET for 8 weeks increased FNDC5 protein content in both endurance-trained groups. Skeletal muscle FNDC5 correlated positively with muscle mass (r = 0.48, p = 0.02) and negatively with body weight (r = − 0.70, p b 0.001), total VAT depots (r = − 0.64, p = 0.001). Circulating irisin protein content remained similar within groups. A negative correlation between circulating irisin and total VAT depots (r = − 0.54, p = 0.01) and eWAT (r = − 0.49, p = 0.023) was observed. 3.4. Effects of diet and physical exercise on eWAT beige and brown-specific markers Long-term HFD per se and VPA interventions did not affect eWAT bone morphogenetic protein (BMP7) gene or protein expression, whereas 8-week-ET significantly increased both BMP7 mRNA and protein expression in SET group and gene expression in HET group. BMP7 protein negatively correlated with body weight (r = − 0.61, p = 0.002) and total VAT depots (r = − 0.64, p = 0.001). Regarding eWAT, Tmem26, mRNA levels remained unaltered with both HFD and ET interventions, but were significantly increased in SVPA animals compared to sedentary counterparts. HFD feeding had no impact on either Cidea or Prdm16 mRNA levels in eWAT. Moreover, no significant alterations were observed in the
105
expression of both genes after VPA intervention with standard or HFDfed animals. In contrast, ET for 8 weeks increased Cidea mRNA levels in both endurance-trained groups and Prdm16 mRNA levels in SET animals. A negative correlation was observed between Cidea (r = −0.79, p b 0.001) and Prdm16 (r = −0.67, p = 0.001) and total VAT depots. The HFD regimen significantly decreased eWAT UCP1 protein content compared to standard-fed counterpart, while VPA did not induce any significant alteration in both diet regimens. Moreover, ET increased UCP1 protein levels in standard-fed animals and tended to attenuate the decreased expression in HFD-fed animals (Fig. 5). A strong correlation between UCP1 and body weight (r = −0.77, p b 0.001) and total VAT depots (r = 0.74, p b 0.001). A positive correlation between skeletal muscle FNDC5 and brown-specific markers, including BMP7 (r = 0.74, p b 0.001), Cidea (r = 0.70, p = 0.001) and UCP1 (r = 0.63, p = 0.001) was observed. Also, skeletal muscle IL-6 positively correlated with brown-specific markers, including BMP7 (r = 0.72, p b 0.001), Cidea (r = 0.67, p = 0.002), Prdm16 (r = 0.67, p = 0.002) and UCP1 (r = 0.75, p b 0.001). As illustrated in Fig. 6A, animals fed a HFD reduced eWAT PGC-1α protein expression compared to standard diet counterparts. The VPA intervention was unable to revert the down-regulation induced by HFD; however, ET increased PGC-1α protein content in both endurancetrained groups compared to sedentary counterparts. Concerning sirtuin 1 (SIRT1) levels (Fig. 6B), the HFD had no impact per se, but the ET increased SIRT1 protein expression in HET group compared to SET, HS and HVPA groups. A significant negative correlation between SIRT1 expression and body weight (r = − 0.55, p = 0.01) and total VAT depots (r = − 0.53, p = 0.01) was observed. No significant alterations were observed on eWAT sirtuin 3 (SIRT3) protein content after the HFD regimen or both the exercise interventions, although a tendency in endurance-trained animals was noted (Fig. 6C). A negative correlation was found between SIRT3 and total VAT depots (r = − 0.47, p = 0.02). Regarding UCP2 protein content, both HFD and exercise interventions failed to induce any significant alterations (Fig. 6D). With exception of the increased eWAT FNDC5 protein expression observed in SET group (vs. SS and SVPA), while no other significant alterations were found between groups (Fig. 6E). Strong negative correlations between FNDC5 protein content and body weight (r = − 0.71, p b 0.001) and total VAT depots (r = − 0.72, p b 0.001) were observed.
Fig. 5. (A–G) Effects of diet and exercise on the expression of beige and brown adipose-selective markers. Semi quantitative RT-qPCR analysis of Bmp7 mRNA (A) and expression of BMP7 protein (B). Quantitative RT-qPCR analysis for Tmem26 (C), Cidea (D), Prdm16 (E) and Western Blot for UCP1 (F) on eWAT. Representative images of Western Blot (G). Values of protein and gene levels are expressed as % AU (arbitrary units), where SS group was set as 100%. Data are expressed as the mean ± SEM. SS, Standard diet sedentary; SVPA, Standard diet voluntary physical activity; SET, Standard diet endurance trained; HS, High-fat diet sedentary; HVPA, High-fat diet voluntary physical activity; HET, High-fat diet endurance training (p b 0.05). a vs. SS; b vs. HS; c vs. SVPA; d vs. SET; e vs. HVPA.
106
S. Rocha-Rodrigues et al. / Life Sciences 165 (2016) 100–108
Fig. 6. (A–F) Effects of diet and exercise on PGC-1α (A), SIRT1 (B), SIRT3 (C), UCP2 (D) and FNDC5 (E) protein expression on eWAT and the representative Western Blot images (F). Values of protein levels are expressed as % AU (arbitrary units), where SS group was set as 100%. Data are expressed as the mean ± SEM. SS, Standard diet sedentary; SVPA, Standard diet voluntary physical activity; SET, Standard diet endurance trained; HS, High-fat diet sedentary; HVPA, High-fat diet voluntary physical activity; HET, High-fat diet endurance training (p b 0.05). a vs. SS; b vs. HS; c vs. SVPA; d vs. SET; e vs. HVPA.
4. Discussion The main findings of the present study suggest that rats submitted to a Lieber DeCarli-isoenergetic diet developed several VAT morphological and metabolic alterations, which strongly resemble the pathological features observed under obesity-related scenarios [6,7,12]. Importantly, ET for 8 weeks was able to antagonize some of the VAT alterations induced by the HFD by stimulating IL-6 and FNDC5 production by skeletal muscle which may be involved in the “brown-like” phenotype of VAT from rats fed a HFD. Skeletal muscle has been considered a major endocrine organ in the human body. In fact, besides its local paracrine-mediated impact on several signaling pathways involved in muscle structure and metabolism, it has been postulated that skeletal muscle also produces and releases myokines, which work in an endocrine fashion, acting on other organs and tissues [17]. Therefore, myokines likely provide a conceptual basis to understand how skeletal muscle “talks” with other organs and tissues, such as WAT, which is of particular interest in context of obesity. However, to our best knowledge, the role of exercise-induced myokines release and its potential signaling influence on WAT metabolism is still a matter of debate. During chronic HFD feeding adipocytes expand in size and in number altering whole VAT distribution and function, accordingly with our findings, which was prevented and attenuated (VPA and ET, respectively) after physical activity interventions, also reported by others [3,6]. A small-sized adipocyte may positively influence the secretion of appropriated levels of adipokines, thus affecting the whole-body metabolism and neuroendocrine control of behavior that are related to feeding (for ref. see [1]). The IL-6 is considered the prototype myokine [11,18,19], with its circulating levels being acutely elevated in response to exercise. IL-6 has the ability to promote metabolic alterations in skeletal muscle itself and in other organs, including WAT, in an endocrine manner [10]. Due to the pleiotropic properties of IL-6 [11], data demonstrated that whole-body Il6-knockout mice are more prone to develop obesity [18] and that endogenous IL-6 seems to play an important role preventing HFD-induced insulin resistance in mice [19]. The beneficial endocrine
effects of increased IL-6 levels in response to exercise on distinct organs, such as WAT may be related to the increases in AMPK activity [19–21]. In fact, AMPK is a fuel-sensing enzyme that among other actions increases its activity in cellular depressed energy states. Considering that the increase in IL-6 concentration correlates temporally with increases in AMPK activity, some studies suggest that IL-6 is involved in AMPK activation during the later stage of exercise when the energetic state of skeletal muscle is down [20,21]. In accordance with [6], data from the present study show that HFD feeding decreased AMPK activity. However, the phosphorylation/activation of AMPK was higher after ET in animals fed with both diet type conditions but not after VPA, which is in accordance with others [22], reporting that AMPK is activated by exercise in an intensity-dependent manner. It is particularly noteworthy that an 8-week-program of ET is able to significantly increase AMPK and IL-6 content even in obesity context as both factors have been related with reduced body weight, VAT accumulation and smallsized adipocyte in HFD-induced obese animals [19]. Recently, the role of the myokine FNDC5/irisin has also been a matter of intense debate. This myokine production and secretion seems to induce exercise-based adaptations in muscle metabolism apparently through AMPK–PGC-1α signaling cascade [23]. In accordance with our findings, some studies [6,23] have demonstrated that physical activity increased muscle FNDC5 expression. Moreover, chronic VPA did not alter FNDC5 protein expression in both diets, which suggests that similarly to AMPK modulation, exercise distinctly modulated FNDC5 expression. Our findings are also consistent with other studies [9,24–26] reporting an increase in muscle FNDC5 expression although no alterations in circulating irisin levels under exercising conditions, which seems to be related to the acutely increase of irisin levels during exercise. In fact, Norheim and coworkers [9] found a peak concentration of irisin immediately after 45 min ergometer cycling (~ 1.2-fold). This acute increase was independent of an increase in FNDC5 mRNA, suggesting that the observed increase of irisin in plasma is caused by increased translation of FNDC5 mRNA in skeletal muscle. Thus, it seems that the irisin generated by endurance exercise training might be used to initiate exercise-based adaptations in skeletal muscle.
S. Rocha-Rodrigues et al. / Life Sciences 165 (2016) 100–108
The myokine FNDC5/irisin has received particular interest due its potential to mediate the browning effects in WAT [5,6]. Emerging evidence suggests that a specific beige and/or brown adipocytes accumulation within WAT might be triggered by skeletal muscle-derived FNDC5 in response to exercise, which produces subsequently positive effects on systemic metabolism and whole-body fat mass [27]. Therefore, an important goal of the present study was to understand whether or not two distinct programs of physical activity (VPA and ET) were able to modulate eWAT structure and key metabolic genes and proteins, particularly inducing a “brown-like” phenotype in WAT from animals fed a HFD. In this regard, a new role for BMP7 in brown adipogenesis and consequent increased energy expenditure has been suggested [28]. Altogether, data suggest that BMP7 activates a full program of brown adipogenesis including the induction of early regulators of brown fat fate PRDM16 and PGC-1α, increased expression of the brown-specific marker UCP1 and adipogenic transcription factors PPARγ and CCAAT/ enhancer-binding proteins, and induction of mitochondrial biogenesis via p38 mitogen-activated protein kinase and PGC-1α-dependent pathways [28]. However, data regarding the role of exercise in the modulation of BMP7 gene and protein expression in VAT is still scarce. In accordance with others [5–9], data from the present study revealed that ET increased the transcript levels of Bmp7 gene and others brown adipocyte-specific markers (Cidea, Prdm16 and UCP1), but did not modulate the native beige adipocyte-specific marker Tmem26. As state above, SIRT1 may also play a role on the induction of brown-like phenotype in WAT by deacetylating two critical lysine residues on PPARγ [29]. In accordance with Chalkiadaki and coworkers [30], the full-length SIRT1 protein was overexpressed in our HET animals, which suggest that ET plays an important role in the modulation of eWAT phenotype via SIRT1, thus contributing to improve the browning-adipocyte like phenotype in VAT. Given the pleiotropic effects of IL-6, the participation of this myokine in the browning process has also been suggested [31,32]. In fact, the overexpression of the Il6 gene seems to increase the expression of thermogenic gene and elevate the protein levels of UCP1 in rat BAT [32] and WAT [31]. Accordingly, ET-induced skeletal muscle IL-6 and FNDC5 expression was associated with the increased transcript levels of brown genes in non-obese and obese conditions. This ET-induced browning effect was consistent with the amelioration of several obesity-related outcomes, such as reductions on body weight, VAT accumulation, adipocyte size, and hepatic fat accumulation in HFD-induced NASH, as previously demonstrated by our group [2]. Previous studies shown that WAT is also able to produce FNDC5 in human and animals [6,27,33], supporting the cross-talk between skeletal muscle and adipose tissue. This cross-talk is of particular interest on obesity conditions as a dysregulation of secretion and production of adipokines and myokines might contribute to increased visceral adiposity and consequent obesity-related diseases [33]. In fact, reduced VAT FNDC5 gene levels were observed in obese patients with or without type 2 diabetes [27], suggesting that this adipokine could underlie the obesity-associated lower amounts of brown or beige adipocytes in human adipose tissue [34]. Interestingly, few studies have shown that FNDC5 in adipose tissue was upregulated after exercise [6,33], which are in agreement with our findings. However, other reports shown that FNDC5 seems to be differentially regulated in skeletal muscle and WAT in response to leptin and fasting [33,35].
5. Conclusions Taken together, data suggest that both VPA and ET induced improvements in obesity-related features, such as reductions in body weight, visceral WAT depots and adipocyte size, whereas only ET was able to induce myokine production as well as brown-like morphology and function in eWAT.
107
Statement of authorship All the authors contributed significantly to this work, providing the concept, as well as participating in the experimental design of the study and the writing of the manuscript. Moreover, all authors performed the functional and biochemical experiments and contributed to the interpretation of the results and the critical revision of the manuscript draft. Conflicts of interest The authors declare that there are no conflicts of interest. Funding sources This work was supported by a grant of Portuguese Foundation for Science and Technology (FCT) to the Research Center in Physical Activity, Health and Leisure (CIAFEL) (UID/DTP/00617/2013), to JM (PTDC/ DTP-DES/7087/2014) (POCI-01-0145-FEDER-016690) and to S.R-R (SFRH/BD/89807/2012). This work was also supported by Fondo de Investigación Sanitaria-FEDER (FIS PI10/01677, PI12/00515 and PI13/ 01430) from the Spanish Instituto de Salud Carlos III, the Department of Health of the Gobierno de Navarra (61/2014). CIBER de Fisiopatología de la Obesidad y Nutrición (CIBERobn) is an initiative of the Instituto de Salud Carlos III, Spain. References [1] A. Guilherme, et al., Adipocyte dysfunctions linking obesity to insulin resistance and type 2 diabetes, Nat. Rev. Mol. Cell Biol. 9 (5) (2008) 367–377. [2] I.O. Goncalves, et al., Physical exercise antagonizes clinical and anatomical features characterizing Lieber-DeCarli diet-induced obesity and related metabolic disorders, Clin. Nutr. (2014). [3] R.A. Sertie, et al., Cessation of physical exercise changes metabolism and modifies the adipocyte cellularity of the periepididymal white adipose tissue in rats, J. Appl. Physiol. 115 (3) (2013) 394–402 (1985). [4] M. Harms, P. Seale, Brown and beige fat: development, function and therapeutic potential, Nat. Med. 19 (10) (2013) 1252–1263. [5] P. Bostrom, et al., A PGC1-alpha-dependent myokine that drives brown-fat-like development of white fat and thermogenesis, Nature 481 (7382) (2012) 463–468. [6] M.V. Wu, et al., Thermogenic capacity is antagonistically regulated in classical brown and white subcutaneous fat depots by high fat diet and endurance training in rats: impact on whole-body energy expenditure, J. Biol. Chem. 289 (49) (2014) 34129–34140. [7] J.P. Tiano, D.A. Springer, S.G. Rane, SMAD3 negatively regulates serum irisin and skeletal muscle FNDC5 and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1alpha) during exercise, J. Biol. Chem. 290 (12) (2015) 7671–7684. [8] T.L. Morton, et al., Exercise increases and browns muscle lipid in high-fat diet-fed mice, Front. Endocrinol. (Lausanne) 7 (2016) 80. [9] F. Norheim, et al., The effects of acute and chronic exercise on PGC-1alpha, irisin and browning of subcutaneous adipose tissue in humans, FEBS J. 281 (3) (2014) 739–749. [10] B.K. Pedersen, M.A. Febbraio, Muscles, exercise and obesity: skeletal muscle as a secretory organ, Nat. Rev. Endocrinol. 8 (8) (2012) 457–465. [11] C. Weigert, et al., The secretome of the working human skeletal muscle—a promising opportunity to combat the metabolic disaster? Proteomics Clin. Appl. 8 (1–2) (2014) 5–18. [12] C.S. Lieber, et al., Model of nonalcoholic steatohepatitis, Am. J. Clin. Nutr. 79 (3) (2004) 502–509. [13] J. Magalhaes, et al., Modulation of cardiac mitochondrial permeability transition and apoptotic signaling by endurance training and intermittent hypobaric hypoxia, Int. J. Cardiol. 173 (1) (2014) 40–45. [14] S.D. Parlee, et al., Quantifying size and number of adipocytes in adipose tissue, Methods Enzymol. 537 (2014) 93–122. [15] H.G. Coore, et al., Regulation of adipose tissue pyruvate dehydrogenase by insulin and other hormones, Biochem. J. 125 (1) (1971) 115–127. [16] C.T. Lim, et al., Measurement of AMP-activated protein kinase activity and expression in response to ghrelin, Methods Enzymol. 514 (2012) 271–287. [17] B.K. Pedersen, Muscles and their myokines, J. Exp. Biol. 214 (Pt 2) (2011) 337–346. [18] V. Wallenius, et al., Interleukin-6-deficient mice develop mature-onset obesity, Nat. Med. 8 (1) (2002) 75–79. [19] J.G. Knudsen, et al., Skeletal muscle interleukin-6 regulates metabolic factors in iWAT during HFD and exercise training, Obesity (Silver Spring) 23 (8) (2015) 1616–1624. [20] S. Bijland, S.J. Mancini, I.P. Salt, Role of AMP-activated protein kinase in adipose tissue metabolism and inflammation, Clin. Sci. (Lond.) 124 (8) (2013) 491–507.
108
S. Rocha-Rodrigues et al. / Life Sciences 165 (2016) 100–108
[21] N.B. Ruderman, et al., Interleukin-6 regulation of AMP-activated protein kinase. Potential role in the systemic response to exercise and prevention of the metabolic syndrome, Diabetes 55 (Suppl. 2) (2006) S48–S54. [22] J.F. Horowitz, Fatty acid mobilization from adipose tissue during exercise, Trends Endocrinol. Metab. 14 (8) (2003) 386–392. [23] J.Y. Huh, et al., Exercise-induced irisin secretion is independent of age or fitness level and increased irisin may directly modulate muscle metabolism through AMPK activation, J. Clin. Endocrinol. Metab. (2014), jc20141437. [24] Q. Zhou, et al., Dihydromyricetin stimulates irisin secretion partially via the PGC1alpha pathway, Mol. Cell. Endocrinol. 412 (2015) 349–357. [25] J. Liu, et al., Effects of high-intensity treadmill training on timeliness and plasticity expression of irisin in mice, Eur. Rev. Med. Pharmacol. Sci. 19 (12) (2015) 2168–2173. [26] M.J. Vosselman, et al., Low brown adipose tissue activity in endurance-trained compared with lean sedentary men, Int. J. Obes. (2015). [27] J.M. Moreno-Navarrete, et al., Irisin is expressed and produced by human muscle and adipose tissue in association with obesity and insulin resistance, J. Clin. Endocrinol. Metab. 98 (4) (2013) E769–E778. [28] Y.H. Tseng, et al., New role of bone morphogenetic protein 7 in brown adipogenesis and energy expenditure, Nature 454 (7207) (2008) 1000–1004.
[29] H.C. Chang, L. Guarente, SIRT1 and other sirtuins in metabolism, Trends Endocrinol. Metab. 25 (3) (2014) 138–145. [30] A. Chalkiadaki, L. Guarente, High-fat diet triggers inflammation-induced cleavage of SIRT1 in adipose tissue to promote metabolic dysfunction, Cell Metab. 16 (2) (2012) 180–188. [31] J.G. Knudsen, et al., Role of IL-6 in exercise training- and cold-induced UCP1 expression in subcutaneous white adipose tissue, PLoS One 9 (1) (2014), e84910. [32] G. Li, et al., Induction of uncoupling protein 1 by central interleukin-6 gene delivery is dependent on sympathetic innervation of brown adipose tissue and underlies one mechanism of body weight reduction in rats, Neuroscience 115 (3) (2002) 879–889. [33] A. Roca-Rivada, et al., FNDC5/irisin is not only a myokine but also an adipokine, PLoS One 8 (4) (2013), e60563. [34] W.D. van Marken Lichtenbelt, et al., Cold-activated brown adipose tissue in healthy men, N. Engl. J. Med. 360 (15) (2009) 1500–1508. [35] A. Rodríguez, et al., Leptin administration activates irisin-induced myogenesis via nitric oxide-dependent mechanisms, but reduces its effect on subcutaneous fat browning in mice, Int. J. Obes. 39 (3) (2015) 397–407.
Contents lists available at ScienceDirect
Life Sciences journal homepage: www.elsevier.com/locate/lifescie
Effects of physical exercise on myokines expression and brown adipose-like phenotype modulation in rats fed a high-fat diet Sílvia Rocha-Rodrigues a,⁎, Amaia Rodríguez b,e,f, Alexandra M. Gouveia c,g, Inês O. Gonçalves a, Sara Becerril b,e,f, Beatriz Ramírez b,e,f, Jorge Beleza a, Gema Frühbeck b,d,e,f, António Ascensão a, José Magalhães a a
CIAFEL - Research Centre in Physical Activity, Health and Leisure, Faculty of Sport, University of Porto, Porto, Portugal Metabolic Research Laboratory, Clínica Universidad de Navarra, Pamplona, Spain Department of Experimental Biology, Faculty of Medicine, University of Porto, Porto, Portugal d Department of Endocrinology & Nutrition, Clínica Universidad de Navarra, Pamplona, Spain e Obesity & Adipobiology Group, Instituto de Investigación Sanitario de Navarra (IdiSNA), Pamplona, Spain f CIBEROBN, Instituto de Salud Carlos III, Pamplona, Spain g Instituto de Investigação e Inovação em Saúde, Institute for Molecular and Cell Biology (IBMC), University of Porto, Porto, Portugal b c
a r t i c l e
i n f o
Article history: Received 26 July 2016 Received in revised form 16 September 2016 Accepted 26 September 2016 Available online 28 September 2016 Keywords: Endurance training Skeletal muscle Adiposity FNDC5/irisin IL-6 Browning UCP1
a b s t r a c t Aims: Exercise-stimulated myokine secretion into circulation may be related with browning in white adipose tissue (WAT), representing a positive metabolic effect on whole-body fat mass. However, limited information is yet available regarding the impact of exercise on myokine-related modulation of adipocyte phenotype in WAT from obese rats. Main methods: Sprague-Dawley rats (n = 60) were divided into sedentary and voluntary physical activity (VPA) groups and fed with standard (35 kcal% fat) or high-fat (HFD, 71 kcal% fat)-isoenergetic diets. The VPA-groups had unrestricted access to wheel running throughout the protocol. After-9 weeks, half of sedentary standard (SS) and sedentary HFD (HS)-fed animals were exercised on treadmill (endurance training, ET) for 8-weeks while maintaining the dietary treatments. Key findings: The adipocyte hypertrophy induced by HFD were attenuated by VPA and ET. HFD decreased 5′ AMPactivated protein kinase (AMPK) activity in muscle as well as peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) and uncoupling protein 1 (UCP1) proteins in eWAT, while not affecting circulating irisin. VPA increased eWAT Tmem26 mRNA levels in the standard diet-fed group, whereas ET increased AMPK, interleukin 6 (IL-6) and fibronectin type III domain-containing protein 5 (FNDC5) protein expression in muscle, but had no impact on circulating irisin protein content. In eWAT, ET increased bone morphogenetic protein 7 (Bmp7), Cidea and PGC-1α in both diet-fed animals, whereas BMP7, Prdm16, UCP1 and FNDC5 only in standard diet-fed group. Significance: Data suggest that ET-induced myokine production seems to contribute, at least in part, to the “brown-like” phenotype in WAT from rats fed a HFD. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Overweight and obesity are highly prevalent conditions in industrialized countries. Although genetic factors contribute to the variance of adiposity, in most cases, obesity results from an imbalance between energy intake and expenditure due to a high consumption of hypercaloric food and/or reduced levels of physical activity. The white adipose tissue (WAT) constitutes the main store of lipids, buffering daily dietary fat entering from the circulation [1]. Nonetheless, obesity-induced WAT ⁎ Corresponding author at: Research Centre in Physical Activity, Health and Leisure, Faculty of Sport Sciences, University of Porto, Rua Dr. Plácido Costa, 91, 4200-450 Porto, Portugal. E-mail address: [email protected] (S. Rocha-Rodrigues).
http://dx.doi.org/10.1016/j.lfs.2016.09.023 0024-3205/© 2016 Elsevier Inc. All rights reserved.
dysfunction can lead to ectopic fat accumulation in other tissues, which have been associated with obesity-related metabolic diseases [1–3]. White and brown adipocytes are two distinct types of fat cells with opposite functions. White adipocytes are highly adapted to store excess energy, whereas brown adipocytes utilize fatty acids for generating heat via mitochondrial uncoupling protein 1 (UCP1) [4], thereby dropping the availability of substrates for storage in WAT. Moreover, two populations of brown-like fat cells with thermogenic properties have been identified, the “classical brown” and “beige” adipocytes [4]. Interestingly, the abundance of these two brown cell types can be induced in response to appropriate physiological stimuli, such as chronic cold exposure, irisin, peroxisome proliferator-activated receptor γ (PPARγ) agonists or β-adrenergic stimulation [5–8], increasing whole-body
S. Rocha-Rodrigues et al. / Life Sciences 165 (2016) 100–108
metabolic rate, and therefore preventing diet-induced obesity (DIO) and related diseases [4,6]. The metabolic alterations induced by physical exercise have pleiotropic effects. Previous studies have shown that regular exercise affects not only the exercised skeletal muscle, but also other non-contractile organs over time [5,6,9]. One potential mechanism underlying this crosstalk is the secretion of proteins that mediate communication between muscle and other tissues through endocrine mechanisms [10, 11], including liver and visceral adipose tissue (VAT). Accordingly, several studies have focused on myokines released from muscle during and immediately after exercise, such as interleukins (IL)-6, -8 and -15, leukemia inhibitory factor (LIF) and fibroblast growth factor 21 (FGF21) [for a review see ref. 10, 11]. In addition, the discovery of fibronectin type III-domain containing 5 (FNDC5) as a PGC-1α-dependent myokine showed that its derived product secreted into circulation (irisin) has the ability to drive a brown adipocyte-like phenotype in WAT [5]. In vitro undifferentiated white adipocytes treated with FNDC5 overexpressed UCP1-positive cells [5] and increased mitochondrial density and gene expression, which led to increased oxygen consumption, heat loss and greater energy expenditure [6]. In this context, a new role for myokines has emerged in the field of exercise-related adaptations; however little is still known regarding the physiological impact of myokines on the adaptive response to chronic exercise, and particularly on the brown adipocyte-like phenotype stimulation of VAT in a rat model of DIO. Thus, using this nutritional model of obesity in rats, the main goal of the present study was to analyze the role of voluntary physical activity (VPA) and endurance training (ET) as hypothetical modulators of an increase in brown-like phenotype in WAT. 2. Material and methods 2.1. Reagents Bradford reagent, Laemmli sample buffer and block reagent were purchased from Bio-Rad (Hercules, CA, USA). Chemiluminescent reagent ECL-Plus™ from GE Healthcare, Amersham Biosciences (Buckinghamshire, U.K.). Polyvinylidene difluoride membranes (Immobilon-N) were purchased from Merck Millipore Corporation (Bedford, MA, USA) and nitrocellulose membranes were obtained from Whatman, Protan (Pittsburgh, PA, USA). Protease inhibitor and phosphatase inhibitor cocktails were purchased from Sigma-Aldrich (Barcelona, Spain). Goat anti-BMP7 (sc9305), goat anti-β-actin (sc1616) and secondary peroxidase-conjugated antibodies (anti-rabbit, sc-2317, anti-mouse, sc-2317 and anti-goat, sc-2020) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Rabbit anti-phospho [Thr172] AMPKα (2531) and rabbit anti-SIRT3 (C73E3) were obtained from Cell Signalling Tecnhology, Inc. (Danvers, MA, USA). Goat anti-PGC-1α (ab106814), rabbit anti-IL-6 (ab6672), mouse anti-AMPK (ab80039), rabbit monoclonal anti-FNDC5 (ab174833), rabbit anti-UCP1 (ab23841) were acquired from Abcam (Cambridge, UK). Goat antiUCP2 (Sab2501087) was obtained from Sigma Aldrich and rabbit antiSIRT1 (13161-1-AP) from Proteintech Group, Inc. (Chicago, IL, USA). TRIzol® reagent and SYBR® Green PCR Master Mix were purchased from Life Technologies (Carlsbad, USA), NZY First-strand cDNA Synthesis Kit (NZYTech, Lisbon, Portugal). All other chemicals were purchased from Sigma-Aldrich (Barcelona, Spain). 2.2. Animals and diets Six-week-old male Sprague-Dawley rats (n = 60, Charles River, L'Arbresle, France) were kept in a pathogen-free room, constant temperature (21–22 °C) and humidity (50–60%) with a fixed 12-h light/ 12-h dark cycle. Rats (initial body weight 233.9 ± 2.6 g) were fed ad libitum during 17 weeks a nutritionally adequate isoenergetic, isoproteic standard (composed by 35% kcal fat, 47% kcal carbohydrates,
101
and 18% kcal protein) or a high-fat (HFD, composed by 71% kcal fat, 11% kcal carbohydrate, and 18% kcal protein)-liquid diet purchased from Dyets Inc. (catalog no. 710027 and 712031, respectively). This DIO model has been commonly used to induce visceral adipose tissue accumulation and related metabolic disorders in experimental animals [2, 12]. All animals had free access to water and food (provided in the liquid state) throughout the entire protocol. During the first week, the standard diet was given to all animals as an adaptation to the liquid feeding. Afterwards, animals were divided into six groups (n = 10/group): standard-diet sedentary (SS), standard-diet voluntary physical activity (SVPA), standard-diet endurance-trained (SET), high-fat diet sedentary (HS), high-fat diet voluntary physical activity (HVPA) and high-fat diet endurance-trained (HET), as previously described [2]. Energy intake (kilocalories per day) was measured daily while body weights were monitored once weekly during the period of the present study. The study was approved by the Institutional Ethics Committee of the Faculty of Sport, University of Porto and followed the guidelines for the care and use of laboratory animals in research advised by the Federation of European Laboratory Animal Science Association (FELASA). 2.3. Voluntary physical activity and endurance training interventions VPA intervention: voluntary-exercised animals (SVPA and HVPA) were assigned in cages equipped with 365-mm (diameter) running wheels. Based on the evidence that VPA may be an effective strategy to prevent the progress of, at least, some obesity-associated metabolic abnormalities [2], animals had unrestricted access to the running wheels during the 17 weeks of the feeding protocol. Wheel revolutions were daily recorded from a digital counter between 08.00 and 10.00 h. SVPA and HVPA animals were moved to standard cages for one night before sacrifice to avoid interference of exercise-related acute effects. ET program: Eight weeks after the diet intervention, SET and HET animals were submitted to a chronic ET program. Initially, animals were progressively acclimated to the motor-driven treadmill (Le8700, Panlab Harvard Apparatus) for 5 days per week at 15 m min−1 and 0% grade during 30 min. Then, animals performed a moderate intensity ET program (corresponding to 50–60% of VO2 max) consisted of 5 sessions per week and 60 min per day at a starting speed of 15 m min−1, which was progressively increased over the training program until 25 m min−1 was reached [3,13]. In order to understand the therapeutic effects of ET, animals started the ET program 8 weeks after the diet treatment since at this time point HFD-fed animals already exhibited obesity-related metabolic features. Sedentary animals (SS and HS groups) were placed on a non-moving treadmill during the training sessions to be exposed to the same environmental conditions, but without promoting any physical training adaptation. In order to eliminate acute effects of exercise, SET and HET animals were sacrificed 48 h after the last training session. 2.4. Visceral adipose tissue and blood collection All animals were fasted overnight for 12 h with access to drinking water before sacrifice. Animals were weighed at the day of sacrifice and anesthetized with ketamine (90 mg·kg− 1) and xylazine (10 mg·kg−1). Blood was collected and plasma separated by centrifugation 3000g for 15 min at 4 °C. Afterwards, visceral WAT depots (mesenteric, epididymal and retroperitoneal) and skeletal muscle (gastrocnemius and soleus) were excised and weighted. Epididymal fat pad and both gastrocnemius and soleus were processed for analysis as described below. 2.5. Adipocyte-size profiling Morphological analyses were performed using light microscopy as previously described [14]. Briefly, visceral WAT was fixed in 4%
102
S. Rocha-Rodrigues et al. / Life Sciences 165 (2016) 100–108
formaldehyde solution (Sigma Aldrich) embedded in paraffin blocks, sectioned, and stained with hematoxylin and eosin. Stained samples were visualized using a microscope (Zeiss AX10 imager A.1, Oberkochen, Germany) under ×40 magnification. Average of adipocyte area was determined, removing any objects below an area of 350 μm2, as these cells may be a mixture of adipocytes and stromal vascular cells [14]. The average of adipocyte area and the frequency of distribution were quantified from 4 sections per rat and 4 rats per group (N1500 adipocytes counted per group). 2.6. Citrate synthase activity in skeletal muscle Citrate synthase (CS) activity was measured in soleus homogenate using the method proposed by Coore et al. [15]. Briefly, CoA-SH released from the reaction of acetyl-CoA with oxaloacetate was measured by its reaction with a colorimetric agent [5, 5-dithiobis (2-nitrobenzoate)]. The change in color was monitored spectrophotometrically at 412 nm. 2.7. Circulating irisin content Plasma samples were diluted (1:20) in Tris buffered saline (TBS; 100 mmol Tris, 1.5 mmol NaCl, pH 8.00) and 100 μL was slot-blotted into a nitrocellulose membrane (Whatman, Protan). The slot-blot membranes were blocked with 5% (w/v) dry nonfat milk in TBS with 0.05% Tween 20 (TBS-T) and then incubated for 30 min with rabbit antimonoclonal FNDC5 diluted 1:1000 in 5% (w/v) nonfat dry milk in TBST. Afterwards, membranes were incubated with a solution of horseradish-conjugated anti-rabbit antibody diluted at 1:10,000. The blots were detected by ChemiDoc™ XRS + System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and band densities were quantified using Image Lab™ software 262 5.2.1 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Control for protein loading was confirmed by Ponceau S staining. 2.8. Western Blot studies The gastrocnemius muscle and epididymal white adipose tissue (eWAT) were homogenized in ice-cold RIPA buffer containing 50 mmol/L Tris-HCl (pH 7.40), 1 mmol/L EDTA, 0.2% sodium dodecyl sulfate, 0.2% deoxycholate and 1% Triton X-100 supplemented with protease and phosphatase inhibitors using a Polytron homogenizer for 30 s. Samples homogenates were centrifuged (13,000g for 10 min at 4 °C) and the supernatant was harvested. An aliquot of the tissue lysates was used to determine the concentration of protein in each sample by Bradford method. Samples were prepared in 2 × Laemmli buffer containing 710 mmol/L β-mercaptoethanol and heated in a boiling water bath for 5 min. To determine the total-AMPK, pAMPK (62 kDa), IL-6 (28 kDa) and FNDC5 (25 kDa) contents in gastrocnemius, aliquots containing 25 μg were subjected to SDS-PAGE and then transferred to polyvinyldifluoride membranes (Millipore). To determine PGC-1α (91 kDa), SIRT1 (75 kDa), SIRT3 (28 kDa), FNDC5 (25 kDa), UCP2 (33 kDa), UCP1 (33 kDa), BMP7 (55 kDa) and β-actin (42 kDa) contents in eWAT, 50 μg protein were loaded onto the gels. All primary antibodies were at 1:1000 dilution. The blots were detected by ChemiDoc™ XRS + System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and band densities were quantified using Image Lab™ software 262 5.2.1 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Membranes were stripped and re-probed with an anti-β-actin antibody, as an internal control. The values were obtained by dividing the density of the band of interest by that of either β-actin (as indicated in the figure legends) from the same blot. 2.9. RT-qPCR analyses Total RNA of eWAT was isolated using TRIzol® reagent as described by the manufacturer. The concentration and purity of RNA were assessed using NanoDrop™ 1000 spectrophotometer by reading
absorbance at 230 nm, 260 nm and 280 nm (Thermo Scientific, CA, USA). One point five micrograms of total RNA worked as template for cDNA production using the NZY First-strand cDNA Synthesis Kit (MB12501), which included a combination of random hexamers and oligo(dT) primers. Quantitative Real-time PCR (RT-qPCR) was conducted using SYBR® Green PCR Master Mix on a Step One Plus thermocycler (Applied Biosystems) using paired reverse and forward primers as shown in Table 1. Each sample was assayed in a 12 μL reaction in duplicate. If the duplicate contained a cycle threshold (CT) standard deviation of N0.5, it was reassayed. All reactions were performed under the same conditions 95 °C for 3 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The data were analyzed using GAPDH as the internal control with the cycle threshold (2−ΔΔCT) method as recommended by Applied Biosystems. 2.10. Statistical analysis Values are presented as mean ± standard error of the mean (SEM). The effect of diet and exercise was analyzed by a two-way analysis of variance (ANOVA). The Bonferroni post hoc test was applied for comparisons between groups and differences were considered significant at p ≤ 0.05. Pearson's correlation coefficients were used to analyze possible associations between variables. Statistical analysis was performed using SPSS 15.0 for Windows (SPSS Inc., Chicago IL, USA). 3. Results 3.1. Effects of exercise and diet on body weight, energy intake and visceral adiposity As illustrated in Fig. 1A, body weight of HS animals remained similar to that of SS group. Moreover, VPA had no impact on body weight while ET for 8 weeks significantly reduced body weight in both exercised groups compared with sedentary counterparts. No significant alterations were observed in cumulative energy intake (kcal) between studied groups (Fig. 1B), as expected in an isoenergetic pair-feeding diet. Nevertheless, HFD induced an increase in the relative weights of mesenteric, epididymal and retroperitoneal fat depots, which were reverted by VPA in mesenteric fat depot, epididymal and retroperitoneal fat pads (Fig. 1C–E). In addition, ET reduced all WAT depots in SET and HET groups compared with their sedentary counterparts. 3.2. Effects of diet and exercise on adipocyte-area profiling visceral WAT Animals from HS group presented a higher percentage of larger adipocytes (≥ 5000 μm2) when compared to SS group (Fig. 2). VPA did not alter the percentage of adipocytes below 5000 μm2 in both SVPA and HVPA groups; however, VPA reduced the percentage of larger adipocytes in HVPA group compared to sedentary counterpart. Moreover, ET significantly increased the percentage of smaller adipocytes (b2000 μm2) and reduced the number of larger adipocytes (≥5000 μm2) in both endurance-trained groups (Fig. 2C–D).
Table 1 Primer sequences used for RT-qPCR. GenBank
Accession no.
Primer sequence (5′–3′)
Tmem26
NM_001107623
Bmp7
NM_001191856
Cidea
NM_001170467
Prdm16
XM_008764418
Gapdh
NM_017008
F-CCGAGGCTACAAATGGCTTTC R-ACTGGTTTCCATGGTGCATTTC F-CCTGCAAGAAACACGAGCTGTAT R-AGGCACACTCTCCCTCACAGTAGT F-TGACATTCATGGGGTTGCAGA R-GGCCAGTTGTGATGACCAAGA F-CTCCGAGATCCGAAACTT R-CTCAGGCCGTTTGTCCAT F-GGTGAAGGTCGGAGTCAACG R-CAAAGTTGTCATGGATGACC
S. Rocha-Rodrigues et al. / Life Sciences 165 (2016) 100–108
103
Fig. 1. Effects of diet and exercise on body weight, energy intake and relative fat depot weights. The mean body weight gain (A) and cumulative energy intake, expressed as kcal per day (B), and the relatives weights of mesenteric (C), epididymal (D) and retroperitoneal (E) fat depots. Data are expressed as the mean ± SEM. SS, Standard diet sedentary; SVPA, Standard diet voluntary physical activity; SET, Standard diet endurance trained; HS, High-fat diet sedentary; HVPA, High-fat diet voluntary physical activity; HET, High-fat diet endurance training (p b 0.05). a vs. SS; b vs. HS; c vs. SVPA; d vs. SET; e vs. HVPA.
3.3. Effects of diet and physical exercise on skeletal muscle characteristics and on circulating irisin Long-term HFD did not affect neither soleus, gastrocnemius nor total muscle mass/body weight ratio (Fig. 3A–C). On the other hand, VPA increased the relative gastrocnemius weight and total
muscle mass in SVPA group. Neither HFD nor VPA interventions have impact on muscle CS activity, a validated marker of oxidative adaptation to ET program (Fig. 3D). In contrast, 8-week ET increased the relative soleus, gastrocnemius and muscle mass weights, as well as the CS activity in SET and HET groups when compared with their sedentary counterparts.
Fig. 2. Effects of diet and exercise on (A) adipocyte-size profiling of adipocytes from WAT. (C) Bar graphs representing the percentage (%) of adipocytes with area below 2000 and higher or equal 2000 μm2 and, (D) below 5000 and higher or equal 5000 μm2. Adipocytes areas were determined from 4 sections per rat. Representative images of hematoxylin and eosin staining of visceral WAT (B). Data are expressed as the mean ± SEM. SS, Standard diet sedentary; SVPA, Standard diet voluntary physical activity; SET, Standard diet endurance trained; HS, High-fat diet sedentary; HVPA, High-fat diet voluntary physical activity; HET, High-fat diet endurance training (p b 0.05). a vs. SS; b vs. HS; c vs. SVPA; d vs. SET; e vs. HVPA; *significant between areas in the same group.
104
S. Rocha-Rodrigues et al. / Life Sciences 165 (2016) 100–108
Fig. 3. Effects of diet and exercise on soleus (A) and gastrocnemius (B) muscle weight and the total muscle mass-body weight ratio (C), and citrate synthase (CS) activity (D). Data are expressed as the mean ± SEM. SS, Standard diet sedentary; SVPA, Standard diet voluntary physical activity; SET, Standard diet endurance trained; HS, High-fat diet sedentary; HVPA, High-fat diet voluntary physical activity; HET, High-fat diet endurance training (p b 0.05). a vs. SS; b vs. HS; c vs. SVPA; d vs. SET; e vs. HVPA.
Fig. 4. (A–E): Effect of diet and exercise on the activation/phosphorylation of AMPK (A), IL-6 (B), FNDC5 (C) on skeletal muscle and circulating irisin content (D). Representative blots of each protein (E). Values of protein levels are expressed as % AU (arbitrary units), where SS group was set as 100%. Data are expressed as the mean ± SEM. SS- Standard diet sedentary; SVPA- Standard diet voluntary physical activity; SET, Standard diet endurance trained; HS, High-fat diet sedentary; HVPA, High-fat diet voluntary physical activity; HET, High-fat diet endurance training (p b 0.05). a vs. SS; b vs. HS; c vs. SVPA; d vs. SET; e vs. HVPA.
S. Rocha-Rodrigues et al. / Life Sciences 165 (2016) 100–108
As shown in Fig. 4, HS animals exhibited diminished skeletal muscle 5′ AMP-activated protein kinase (AMPK) activation (pAMPK/ tAMPK ratio), as quantification of AMPK Thr(172) phosphorylation by immunoblotting is a recognized surrogate for AMPK activity [16], compared to SS group. Concerning exercise interventions, VPA had no effect in any parameter, but ET significantly increased pAMPK/tAMPK ratio in both endurance-trained groups compared to their sedentary and voluntary-exercised counterparts. The HFD regimen per se had no impact on skeletal muscle IL-6 expression, while VPA tended to increase and ET significantly increased its expression in both standard and HFD-fed animals compared to sedentary and voluntary-exercised counterparts. Moreover, skeletal muscle IL-6 positively correlated with muscle mass (r = 0.71, p b 0.001) and negatively correlated with body weight (r = − 0.65, p = 0.001) and total VAT depots (r = − 0.79, p b 0.001). Although without statistical significance, HFD intervention tended to reduce skeletal muscle FNDC5 protein expression in HS group. Despite the absence of any effect prompt by VPA, ET for 8 weeks increased FNDC5 protein content in both endurance-trained groups. Skeletal muscle FNDC5 correlated positively with muscle mass (r = 0.48, p = 0.02) and negatively with body weight (r = − 0.70, p b 0.001), total VAT depots (r = − 0.64, p = 0.001). Circulating irisin protein content remained similar within groups. A negative correlation between circulating irisin and total VAT depots (r = − 0.54, p = 0.01) and eWAT (r = − 0.49, p = 0.023) was observed. 3.4. Effects of diet and physical exercise on eWAT beige and brown-specific markers Long-term HFD per se and VPA interventions did not affect eWAT bone morphogenetic protein (BMP7) gene or protein expression, whereas 8-week-ET significantly increased both BMP7 mRNA and protein expression in SET group and gene expression in HET group. BMP7 protein negatively correlated with body weight (r = − 0.61, p = 0.002) and total VAT depots (r = − 0.64, p = 0.001). Regarding eWAT, Tmem26, mRNA levels remained unaltered with both HFD and ET interventions, but were significantly increased in SVPA animals compared to sedentary counterparts. HFD feeding had no impact on either Cidea or Prdm16 mRNA levels in eWAT. Moreover, no significant alterations were observed in the
105
expression of both genes after VPA intervention with standard or HFDfed animals. In contrast, ET for 8 weeks increased Cidea mRNA levels in both endurance-trained groups and Prdm16 mRNA levels in SET animals. A negative correlation was observed between Cidea (r = −0.79, p b 0.001) and Prdm16 (r = −0.67, p = 0.001) and total VAT depots. The HFD regimen significantly decreased eWAT UCP1 protein content compared to standard-fed counterpart, while VPA did not induce any significant alteration in both diet regimens. Moreover, ET increased UCP1 protein levels in standard-fed animals and tended to attenuate the decreased expression in HFD-fed animals (Fig. 5). A strong correlation between UCP1 and body weight (r = −0.77, p b 0.001) and total VAT depots (r = 0.74, p b 0.001). A positive correlation between skeletal muscle FNDC5 and brown-specific markers, including BMP7 (r = 0.74, p b 0.001), Cidea (r = 0.70, p = 0.001) and UCP1 (r = 0.63, p = 0.001) was observed. Also, skeletal muscle IL-6 positively correlated with brown-specific markers, including BMP7 (r = 0.72, p b 0.001), Cidea (r = 0.67, p = 0.002), Prdm16 (r = 0.67, p = 0.002) and UCP1 (r = 0.75, p b 0.001). As illustrated in Fig. 6A, animals fed a HFD reduced eWAT PGC-1α protein expression compared to standard diet counterparts. The VPA intervention was unable to revert the down-regulation induced by HFD; however, ET increased PGC-1α protein content in both endurancetrained groups compared to sedentary counterparts. Concerning sirtuin 1 (SIRT1) levels (Fig. 6B), the HFD had no impact per se, but the ET increased SIRT1 protein expression in HET group compared to SET, HS and HVPA groups. A significant negative correlation between SIRT1 expression and body weight (r = − 0.55, p = 0.01) and total VAT depots (r = − 0.53, p = 0.01) was observed. No significant alterations were observed on eWAT sirtuin 3 (SIRT3) protein content after the HFD regimen or both the exercise interventions, although a tendency in endurance-trained animals was noted (Fig. 6C). A negative correlation was found between SIRT3 and total VAT depots (r = − 0.47, p = 0.02). Regarding UCP2 protein content, both HFD and exercise interventions failed to induce any significant alterations (Fig. 6D). With exception of the increased eWAT FNDC5 protein expression observed in SET group (vs. SS and SVPA), while no other significant alterations were found between groups (Fig. 6E). Strong negative correlations between FNDC5 protein content and body weight (r = − 0.71, p b 0.001) and total VAT depots (r = − 0.72, p b 0.001) were observed.
Fig. 5. (A–G) Effects of diet and exercise on the expression of beige and brown adipose-selective markers. Semi quantitative RT-qPCR analysis of Bmp7 mRNA (A) and expression of BMP7 protein (B). Quantitative RT-qPCR analysis for Tmem26 (C), Cidea (D), Prdm16 (E) and Western Blot for UCP1 (F) on eWAT. Representative images of Western Blot (G). Values of protein and gene levels are expressed as % AU (arbitrary units), where SS group was set as 100%. Data are expressed as the mean ± SEM. SS, Standard diet sedentary; SVPA, Standard diet voluntary physical activity; SET, Standard diet endurance trained; HS, High-fat diet sedentary; HVPA, High-fat diet voluntary physical activity; HET, High-fat diet endurance training (p b 0.05). a vs. SS; b vs. HS; c vs. SVPA; d vs. SET; e vs. HVPA.
106
S. Rocha-Rodrigues et al. / Life Sciences 165 (2016) 100–108
Fig. 6. (A–F) Effects of diet and exercise on PGC-1α (A), SIRT1 (B), SIRT3 (C), UCP2 (D) and FNDC5 (E) protein expression on eWAT and the representative Western Blot images (F). Values of protein levels are expressed as % AU (arbitrary units), where SS group was set as 100%. Data are expressed as the mean ± SEM. SS, Standard diet sedentary; SVPA, Standard diet voluntary physical activity; SET, Standard diet endurance trained; HS, High-fat diet sedentary; HVPA, High-fat diet voluntary physical activity; HET, High-fat diet endurance training (p b 0.05). a vs. SS; b vs. HS; c vs. SVPA; d vs. SET; e vs. HVPA.
4. Discussion The main findings of the present study suggest that rats submitted to a Lieber DeCarli-isoenergetic diet developed several VAT morphological and metabolic alterations, which strongly resemble the pathological features observed under obesity-related scenarios [6,7,12]. Importantly, ET for 8 weeks was able to antagonize some of the VAT alterations induced by the HFD by stimulating IL-6 and FNDC5 production by skeletal muscle which may be involved in the “brown-like” phenotype of VAT from rats fed a HFD. Skeletal muscle has been considered a major endocrine organ in the human body. In fact, besides its local paracrine-mediated impact on several signaling pathways involved in muscle structure and metabolism, it has been postulated that skeletal muscle also produces and releases myokines, which work in an endocrine fashion, acting on other organs and tissues [17]. Therefore, myokines likely provide a conceptual basis to understand how skeletal muscle “talks” with other organs and tissues, such as WAT, which is of particular interest in context of obesity. However, to our best knowledge, the role of exercise-induced myokines release and its potential signaling influence on WAT metabolism is still a matter of debate. During chronic HFD feeding adipocytes expand in size and in number altering whole VAT distribution and function, accordingly with our findings, which was prevented and attenuated (VPA and ET, respectively) after physical activity interventions, also reported by others [3,6]. A small-sized adipocyte may positively influence the secretion of appropriated levels of adipokines, thus affecting the whole-body metabolism and neuroendocrine control of behavior that are related to feeding (for ref. see [1]). The IL-6 is considered the prototype myokine [11,18,19], with its circulating levels being acutely elevated in response to exercise. IL-6 has the ability to promote metabolic alterations in skeletal muscle itself and in other organs, including WAT, in an endocrine manner [10]. Due to the pleiotropic properties of IL-6 [11], data demonstrated that whole-body Il6-knockout mice are more prone to develop obesity [18] and that endogenous IL-6 seems to play an important role preventing HFD-induced insulin resistance in mice [19]. The beneficial endocrine
effects of increased IL-6 levels in response to exercise on distinct organs, such as WAT may be related to the increases in AMPK activity [19–21]. In fact, AMPK is a fuel-sensing enzyme that among other actions increases its activity in cellular depressed energy states. Considering that the increase in IL-6 concentration correlates temporally with increases in AMPK activity, some studies suggest that IL-6 is involved in AMPK activation during the later stage of exercise when the energetic state of skeletal muscle is down [20,21]. In accordance with [6], data from the present study show that HFD feeding decreased AMPK activity. However, the phosphorylation/activation of AMPK was higher after ET in animals fed with both diet type conditions but not after VPA, which is in accordance with others [22], reporting that AMPK is activated by exercise in an intensity-dependent manner. It is particularly noteworthy that an 8-week-program of ET is able to significantly increase AMPK and IL-6 content even in obesity context as both factors have been related with reduced body weight, VAT accumulation and smallsized adipocyte in HFD-induced obese animals [19]. Recently, the role of the myokine FNDC5/irisin has also been a matter of intense debate. This myokine production and secretion seems to induce exercise-based adaptations in muscle metabolism apparently through AMPK–PGC-1α signaling cascade [23]. In accordance with our findings, some studies [6,23] have demonstrated that physical activity increased muscle FNDC5 expression. Moreover, chronic VPA did not alter FNDC5 protein expression in both diets, which suggests that similarly to AMPK modulation, exercise distinctly modulated FNDC5 expression. Our findings are also consistent with other studies [9,24–26] reporting an increase in muscle FNDC5 expression although no alterations in circulating irisin levels under exercising conditions, which seems to be related to the acutely increase of irisin levels during exercise. In fact, Norheim and coworkers [9] found a peak concentration of irisin immediately after 45 min ergometer cycling (~ 1.2-fold). This acute increase was independent of an increase in FNDC5 mRNA, suggesting that the observed increase of irisin in plasma is caused by increased translation of FNDC5 mRNA in skeletal muscle. Thus, it seems that the irisin generated by endurance exercise training might be used to initiate exercise-based adaptations in skeletal muscle.
S. Rocha-Rodrigues et al. / Life Sciences 165 (2016) 100–108
The myokine FNDC5/irisin has received particular interest due its potential to mediate the browning effects in WAT [5,6]. Emerging evidence suggests that a specific beige and/or brown adipocytes accumulation within WAT might be triggered by skeletal muscle-derived FNDC5 in response to exercise, which produces subsequently positive effects on systemic metabolism and whole-body fat mass [27]. Therefore, an important goal of the present study was to understand whether or not two distinct programs of physical activity (VPA and ET) were able to modulate eWAT structure and key metabolic genes and proteins, particularly inducing a “brown-like” phenotype in WAT from animals fed a HFD. In this regard, a new role for BMP7 in brown adipogenesis and consequent increased energy expenditure has been suggested [28]. Altogether, data suggest that BMP7 activates a full program of brown adipogenesis including the induction of early regulators of brown fat fate PRDM16 and PGC-1α, increased expression of the brown-specific marker UCP1 and adipogenic transcription factors PPARγ and CCAAT/ enhancer-binding proteins, and induction of mitochondrial biogenesis via p38 mitogen-activated protein kinase and PGC-1α-dependent pathways [28]. However, data regarding the role of exercise in the modulation of BMP7 gene and protein expression in VAT is still scarce. In accordance with others [5–9], data from the present study revealed that ET increased the transcript levels of Bmp7 gene and others brown adipocyte-specific markers (Cidea, Prdm16 and UCP1), but did not modulate the native beige adipocyte-specific marker Tmem26. As state above, SIRT1 may also play a role on the induction of brown-like phenotype in WAT by deacetylating two critical lysine residues on PPARγ [29]. In accordance with Chalkiadaki and coworkers [30], the full-length SIRT1 protein was overexpressed in our HET animals, which suggest that ET plays an important role in the modulation of eWAT phenotype via SIRT1, thus contributing to improve the browning-adipocyte like phenotype in VAT. Given the pleiotropic effects of IL-6, the participation of this myokine in the browning process has also been suggested [31,32]. In fact, the overexpression of the Il6 gene seems to increase the expression of thermogenic gene and elevate the protein levels of UCP1 in rat BAT [32] and WAT [31]. Accordingly, ET-induced skeletal muscle IL-6 and FNDC5 expression was associated with the increased transcript levels of brown genes in non-obese and obese conditions. This ET-induced browning effect was consistent with the amelioration of several obesity-related outcomes, such as reductions on body weight, VAT accumulation, adipocyte size, and hepatic fat accumulation in HFD-induced NASH, as previously demonstrated by our group [2]. Previous studies shown that WAT is also able to produce FNDC5 in human and animals [6,27,33], supporting the cross-talk between skeletal muscle and adipose tissue. This cross-talk is of particular interest on obesity conditions as a dysregulation of secretion and production of adipokines and myokines might contribute to increased visceral adiposity and consequent obesity-related diseases [33]. In fact, reduced VAT FNDC5 gene levels were observed in obese patients with or without type 2 diabetes [27], suggesting that this adipokine could underlie the obesity-associated lower amounts of brown or beige adipocytes in human adipose tissue [34]. Interestingly, few studies have shown that FNDC5 in adipose tissue was upregulated after exercise [6,33], which are in agreement with our findings. However, other reports shown that FNDC5 seems to be differentially regulated in skeletal muscle and WAT in response to leptin and fasting [33,35].
5. Conclusions Taken together, data suggest that both VPA and ET induced improvements in obesity-related features, such as reductions in body weight, visceral WAT depots and adipocyte size, whereas only ET was able to induce myokine production as well as brown-like morphology and function in eWAT.
107
Statement of authorship All the authors contributed significantly to this work, providing the concept, as well as participating in the experimental design of the study and the writing of the manuscript. Moreover, all authors performed the functional and biochemical experiments and contributed to the interpretation of the results and the critical revision of the manuscript draft. Conflicts of interest The authors declare that there are no conflicts of interest. Funding sources This work was supported by a grant of Portuguese Foundation for Science and Technology (FCT) to the Research Center in Physical Activity, Health and Leisure (CIAFEL) (UID/DTP/00617/2013), to JM (PTDC/ DTP-DES/7087/2014) (POCI-01-0145-FEDER-016690) and to S.R-R (SFRH/BD/89807/2012). This work was also supported by Fondo de Investigación Sanitaria-FEDER (FIS PI10/01677, PI12/00515 and PI13/ 01430) from the Spanish Instituto de Salud Carlos III, the Department of Health of the Gobierno de Navarra (61/2014). CIBER de Fisiopatología de la Obesidad y Nutrición (CIBERobn) is an initiative of the Instituto de Salud Carlos III, Spain. References [1] A. Guilherme, et al., Adipocyte dysfunctions linking obesity to insulin resistance and type 2 diabetes, Nat. Rev. Mol. Cell Biol. 9 (5) (2008) 367–377. [2] I.O. Goncalves, et al., Physical exercise antagonizes clinical and anatomical features characterizing Lieber-DeCarli diet-induced obesity and related metabolic disorders, Clin. Nutr. (2014). [3] R.A. Sertie, et al., Cessation of physical exercise changes metabolism and modifies the adipocyte cellularity of the periepididymal white adipose tissue in rats, J. Appl. Physiol. 115 (3) (2013) 394–402 (1985). [4] M. Harms, P. Seale, Brown and beige fat: development, function and therapeutic potential, Nat. Med. 19 (10) (2013) 1252–1263. [5] P. Bostrom, et al., A PGC1-alpha-dependent myokine that drives brown-fat-like development of white fat and thermogenesis, Nature 481 (7382) (2012) 463–468. [6] M.V. Wu, et al., Thermogenic capacity is antagonistically regulated in classical brown and white subcutaneous fat depots by high fat diet and endurance training in rats: impact on whole-body energy expenditure, J. Biol. Chem. 289 (49) (2014) 34129–34140. [7] J.P. Tiano, D.A. Springer, S.G. Rane, SMAD3 negatively regulates serum irisin and skeletal muscle FNDC5 and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1alpha) during exercise, J. Biol. Chem. 290 (12) (2015) 7671–7684. [8] T.L. Morton, et al., Exercise increases and browns muscle lipid in high-fat diet-fed mice, Front. Endocrinol. (Lausanne) 7 (2016) 80. [9] F. Norheim, et al., The effects of acute and chronic exercise on PGC-1alpha, irisin and browning of subcutaneous adipose tissue in humans, FEBS J. 281 (3) (2014) 739–749. [10] B.K. Pedersen, M.A. Febbraio, Muscles, exercise and obesity: skeletal muscle as a secretory organ, Nat. Rev. Endocrinol. 8 (8) (2012) 457–465. [11] C. Weigert, et al., The secretome of the working human skeletal muscle—a promising opportunity to combat the metabolic disaster? Proteomics Clin. Appl. 8 (1–2) (2014) 5–18. [12] C.S. Lieber, et al., Model of nonalcoholic steatohepatitis, Am. J. Clin. Nutr. 79 (3) (2004) 502–509. [13] J. Magalhaes, et al., Modulation of cardiac mitochondrial permeability transition and apoptotic signaling by endurance training and intermittent hypobaric hypoxia, Int. J. Cardiol. 173 (1) (2014) 40–45. [14] S.D. Parlee, et al., Quantifying size and number of adipocytes in adipose tissue, Methods Enzymol. 537 (2014) 93–122. [15] H.G. Coore, et al., Regulation of adipose tissue pyruvate dehydrogenase by insulin and other hormones, Biochem. J. 125 (1) (1971) 115–127. [16] C.T. Lim, et al., Measurement of AMP-activated protein kinase activity and expression in response to ghrelin, Methods Enzymol. 514 (2012) 271–287. [17] B.K. Pedersen, Muscles and their myokines, J. Exp. Biol. 214 (Pt 2) (2011) 337–346. [18] V. Wallenius, et al., Interleukin-6-deficient mice develop mature-onset obesity, Nat. Med. 8 (1) (2002) 75–79. [19] J.G. Knudsen, et al., Skeletal muscle interleukin-6 regulates metabolic factors in iWAT during HFD and exercise training, Obesity (Silver Spring) 23 (8) (2015) 1616–1624. [20] S. Bijland, S.J. Mancini, I.P. Salt, Role of AMP-activated protein kinase in adipose tissue metabolism and inflammation, Clin. Sci. (Lond.) 124 (8) (2013) 491–507.
108
S. Rocha-Rodrigues et al. / Life Sciences 165 (2016) 100–108
[21] N.B. Ruderman, et al., Interleukin-6 regulation of AMP-activated protein kinase. Potential role in the systemic response to exercise and prevention of the metabolic syndrome, Diabetes 55 (Suppl. 2) (2006) S48–S54. [22] J.F. Horowitz, Fatty acid mobilization from adipose tissue during exercise, Trends Endocrinol. Metab. 14 (8) (2003) 386–392. [23] J.Y. Huh, et al., Exercise-induced irisin secretion is independent of age or fitness level and increased irisin may directly modulate muscle metabolism through AMPK activation, J. Clin. Endocrinol. Metab. (2014), jc20141437. [24] Q. Zhou, et al., Dihydromyricetin stimulates irisin secretion partially via the PGC1alpha pathway, Mol. Cell. Endocrinol. 412 (2015) 349–357. [25] J. Liu, et al., Effects of high-intensity treadmill training on timeliness and plasticity expression of irisin in mice, Eur. Rev. Med. Pharmacol. Sci. 19 (12) (2015) 2168–2173. [26] M.J. Vosselman, et al., Low brown adipose tissue activity in endurance-trained compared with lean sedentary men, Int. J. Obes. (2015). [27] J.M. Moreno-Navarrete, et al., Irisin is expressed and produced by human muscle and adipose tissue in association with obesity and insulin resistance, J. Clin. Endocrinol. Metab. 98 (4) (2013) E769–E778. [28] Y.H. Tseng, et al., New role of bone morphogenetic protein 7 in brown adipogenesis and energy expenditure, Nature 454 (7207) (2008) 1000–1004.
[29] H.C. Chang, L. Guarente, SIRT1 and other sirtuins in metabolism, Trends Endocrinol. Metab. 25 (3) (2014) 138–145. [30] A. Chalkiadaki, L. Guarente, High-fat diet triggers inflammation-induced cleavage of SIRT1 in adipose tissue to promote metabolic dysfunction, Cell Metab. 16 (2) (2012) 180–188. [31] J.G. Knudsen, et al., Role of IL-6 in exercise training- and cold-induced UCP1 expression in subcutaneous white adipose tissue, PLoS One 9 (1) (2014), e84910. [32] G. Li, et al., Induction of uncoupling protein 1 by central interleukin-6 gene delivery is dependent on sympathetic innervation of brown adipose tissue and underlies one mechanism of body weight reduction in rats, Neuroscience 115 (3) (2002) 879–889. [33] A. Roca-Rivada, et al., FNDC5/irisin is not only a myokine but also an adipokine, PLoS One 8 (4) (2013), e60563. [34] W.D. van Marken Lichtenbelt, et al., Cold-activated brown adipose tissue in healthy men, N. Engl. J. Med. 360 (15) (2009) 1500–1508. [35] A. Rodríguez, et al., Leptin administration activates irisin-induced myogenesis via nitric oxide-dependent mechanisms, but reduces its effect on subcutaneous fat browning in mice, Int. J. Obes. 39 (3) (2015) 397–407.