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Table of Contents
ORIGINAL ARTICLE
Year : 2020  |  Volume : 63  |  Issue : 3  |  Page : 101-112

Combination of exercise training and resveratrol attenuates obese sarcopenia in skeletal muscle atrophy


1 Department of Public Health, College of Medicine, Taipei Medical University, Taipei, Taiwan
2 Research Center for Healthcare Industry Innovation, National Taipei University of Nursing and Health Sciences, Taipei, Taiwan
3 Department of Gerontological Health Care, College of Nursing, National Taipei University of Nursing and Health Sciences, Taipei, Taiwan

Date of Submission06-Dec-2019
Date of Decision31-Mar-2020
Date of Acceptance14-Apr-2020
Date of Web Publication23-Jun-2020

Correspondence Address:
Dr. Jia-Ping Wu
Research Center for Healthcare Industry Innovation, National Taipei University of Nursing and Health Sciences, No. 365, Mingde Road, Beitou, Taipei 11219
Taiwan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/CJP.CJP_95_19

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  Abstract 

Obese sarcopenia is a progressive loss of skeletal muscle mass and strength with increases in adipocytes. The aim of this study was to investigate the effects of combination of exercise training and resveratrol on the pathological pathway from obesity to sarcopenia, and potential therapy for skeletal muscle declines in physical function. Two animal models were experimented: (1) C57BL/6J male mice were fed either a high-fat diet (HFD) for 8 weeks to induce obesity and resveratrol (low-, middle-, and high-dose) for 4 weeks. (2) senescence-accelerated mouse prone 8 (SAMP8) mice with sarcopenia were used. Skeletal muscle function of SAMP8 mice expressed an age-associated decline. In SAMP8 mice, resveratrol (150 mg/Kg BW, daily) was administered by oral gavage two times a week for 1 month of the experimental period. Exercise training based on adaptations in the muscle is training twice a week for 4 weeks. SAMP8 mouse skeletal muscle in each group was analyzed by H and E staining, transferase dUTP nick end labeling, and Western blot analysis. Mitochondrial function expression, apoptosis and relative hypertrophy signaling in HFD-induced obesity mice and SAMP8 mice were determined by Western blot analysis. Results of the present study indicate that effect of resveratrol on skeletal muscles of HFD-induced obesity mice is linked to an increase in Bcl-2 and phosphatidylinositol 3 kinase/AKT expressions. On the other hand, resveratrol, and its combination with exercise training, attenuate the aging-related mitochondrial dysfunction involving Bad, caspase 3, and interleukin-6 expressions in SAMP8 mice. Combination of exercise training and resveratrol induced hypertrophy in skeletal muscles of sarcopenia SAMP8 mice. Therefore, we suggest combination of exercise training and resveratrol as a therapeutic potential in obese sarcopenia.

Keywords: Exercise training, obese sarcopenia, obesity, resveratrol, sarcopenia


How to cite this article:
Bai CH, Alizargar J, Peng CY, Wu JP. Combination of exercise training and resveratrol attenuates obese sarcopenia in skeletal muscle atrophy. Chin J Physiol 2020;63:101-12

How to cite this URL:
Bai CH, Alizargar J, Peng CY, Wu JP. Combination of exercise training and resveratrol attenuates obese sarcopenia in skeletal muscle atrophy. Chin J Physiol [serial online] 2020 [cited 2020 Jul 9];63:101-12. Available from: http://www.cjphysiology.org/text.asp?2020/63/3/101/287455


  Introduction Top


Obese sarcopenia and sarcopenic obesity are the combination of age and obesity. Some key age- and obesity-mediated factors and pathways may aggravate sarcopenia. Indeed, sarcopenia is an age-associated skeletal muscle mass loss coupled with functional deterioration exacerbated by obesity.[1] Obesity in adulthood is a medical condition. Excess body fat has accumulated and thus reduced skeletal muscle mass with potential implications for developing sarcopenia and increasing health problems.[2],[3] Overfat in skeletal muscle tissue is leading to premature tissue aging.[4] The prevalence of obesity worldwide is increasing in all age groups. Because sarcopenia is directly attributed to obesity or age, there is no guarantee that a person will be able to prevent it.[5],[6] We determined whether resveratrol treatment would prevent skeletal muscle atrophy in high-fat diet-induced obesity mice and muscle mass decreases in aging-associated sarcopenia mice. The pathological pathway involved in obesity to sarcopenia was studied. Sarcopenia, most common in older people, is now recognized as a disease. Resveratrol is a phytoalexin polyphenolic compound derived from naturally obtained grapes with anti-aging properties. Resveratrol has beneficial effects on blood circulation, which reduces the risk of cardiovascular disease and inhibits inflammation.[7],[8] Exercise training improves oxygen consumption and transport throughout the body to improve vascular function.[9] Resveratrol with exercise training is a highly effective strategy to offset sarcopenia.[10],[11] Habitual exercise training alone did not improve mitochondrial dysfunction in healthy aged subjects with aging-associated functional decline, which may have to be combined with resveratrol intake.


  Materials and Methods Top


Obesity animal model

We purchased 49 male C57BL/6J mice at age 6 weeks from BioLASCO Taiwan Co., Ltd. C57BL/6J mice (n = 39) which were fed a high-fat diet (HFD) for 5 weeks to induce obesity, and then were assigned to three groups containing low (50 mg/kg BW, n = 10)-, middle (100 mg/kg BW, n = 10)- and high (200 mg/kg BW, n = 9)-doses of resveratrol, and 0.2% placebo (n = 10) for 4 weeks. The HFD group received a HFD, and the HFD + low-dose resveratrol (LR) group received a LR-containing HFD, the HFD + middle-dose resveratrol (MR) group received a MR-containing HFD, and the HFD + high-dose resveratrol (HR) group received a HR-containing HFD, and 0.2% placebo-containing HFD. After additional feeding of the experimental diet for 4 weeks, mice in the HFD group were highly obese compared with the mice in the standard diet-fed mice group. Adult mice were housed five per cage in a temperature-controlled room (24°C ± 1°C with a 12-h light/dark cycle 06:30 a.m.–18:30 p.m.). Mice were fed commercial laboratory chow and water ad libitum. Animals were killed after 4-week experimental period. The skeletal muscles were collected for quantifying skeletal muscle function proteins by Western blotting. We also dissected and weighed skeletal muscle and adipose tissue to analyze body drying weight, skeletal muscle drying grinding, fat weight, and body fat. All experimental procedures were approved by the Animal Care and Use Committee of Taipei Medical University (LAC-2019-0264), and all animal experiments were performed in accordance with the ARRIVE guidelines and use of laboratory animals.

Experimental design in senescence-accelerated mouse prone 8 animal model

Male adult senescence-accelerated mouse prone 8 (SAMP8) mice of 3 months of age, weighing 35 g were used in this study (total n = 31). We examined the effect of exercise training, resveratrol, and their combination on age-associated sarcopenia using a SAMP8 model. The animals were divided into four experimental groups: non-treated SAMP8 control groups (SAMP8, n = 8), exercise training SAMP8 groups (SAMP8+Ex, n = 8), resveratrol intake SAMP8 groups (SAMP8+Re, n = 7), and combination of exercise training and resveratrol intake SAMP8 groups (SAMP8+Ex+Re, n = 8). Mice were housed in two cages of one group for all experimental process before sacrifice. Mice were maintained in a room at 22°C ± 2°C, with automatic light cycles (12-h light/dark) and standard diet ad libitum. All the animals received animal care according to the Guidelines for Ethical Care of the Animal Care and Use Committee of Taipei Medical University (LAC-2019-0264). All animal studies complied with the ARRIVE guidelines. In SAMP8 mice, resveratrol (150 mg/kg BW, daily) was administered by oral gavage two times a week for 1 month of the experimental period. Exercise training based on adaptations in the muscle is training twice a week for 4 weeks. Mouse physical exercise training may assist in recovering from aging-related sarcopenia in SAMP8 mice.

H and E stain and terminal deoxynucleotidyl transferase dUTP nick end labeling positive nuclei stain

After skeletal muscle perfusion fixation with buffered formalin for 15 min, the skeletal muscle was sectioned into six equal transverse slices starting from the apex to base. Two of these sections were embedded in paraffin. The remaining sections were cut at 6 μm thickness and stained with hematoxylin and eosin (H and E) stain. Slides were deparaffinized and dehydrated. Samples were passed through a series of graded alcohols from 100% to 90% and to 70%, 5 min each. The skeletal muscle sections were processed for fibrosis quantification. H and E staining was prepared for one of the two sections and incubated for 5 min at room temperature. The other two sections were treated with transferase dUTP nick end labeling (TUNEL) assay (apoptosis detection kit, TA300, R and D Systems, Minneapolis, MN, USA). After rinsing with phosphate-buffered saline (PBS), each slide was then soaked with 85% alcohol and 100% alcohol for 15 min. Stained sections were rinsed with PBS and air dried before mounting. Cross-sections were stained by H and E stain and TUNEL assay. Color images of cross-sections were made at ×200 total magnification using a Nikon E600. Microscope lighting was optimized which increased the probability of being visualized in appropriate cross-sections and not tangential. The number of TUNEL-positive cells per muscle area was counted in twenty visual fields (magnification, ×400) for each mouse.

Western blotting analysis

The skeletal muscle was cut into small pieces in PBS buffer, homogenized in a homogenizer and then centrifuged at 12,000 × rpm for 30 min at 4°C. Supernatants were considered total cellular cytosol lysates protein. The protein samples were separated using gel electrophoresis. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was carried out with 10%–12.5% polyacrylamide gels. The proteins were electrophoresed at 100 V for 1.5 h. Electrophoresis proteins were transferred to polyvinylidene difluoride (PVDF) membranes (0.45 μm; MILLIPORE, USA) using a Bio-Red Laboratories Instruments Mini Trans-Blot Electrophoretic Transfer Cell unit (Alfred Nobel Drive, Hercules, CA, USA) at 150 mA for 2 h in transfer buffer (25 mM Tris-HCl, 192 mM glycine, and 20% methanol, pH 8.3). We blocked PVDF membranes in 5% nonfat milk buffer (diluted in Tris-buffered saline and 0.1% Tween 20) for 1 h at room temperature and then in blocking buffer containing 100 mM Tris-HCl, pH 7.5, 0.9% NaCl, and 0.1% fetal bovine serum (FBS) for 2 h at room temperature. For transfer buffers without methanol, it is essential that complete equilibration of the resolving gel is achieved to prevent distortions within the gel which would cause band smearing. Only a brief rinse is required to achieve equilibration if the transfer buffer contains methanol. Monoclonal antibodies (Santa Cruz, CA, USA) were diluted 1:250 in antibody-binding buffer containing 100 mM Tris-HCl, pH 7.5, 0.9% NaCl, 0.1% Tween-20, and 1% FBS. Incubations were performed at 4°C for 2 h. Western blot analysis was performed as previously described with antibodies detecting atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), fibroblast growth factor (FGF)-2, urokinase-type plasminogen activator (uPA), phosphatidylinositol 3 kinase (PI3K), AKT, interleukin (IL)-6, signal transducer and activator of transcription 3 (STAT3), phosphorylated c-Jun NH2-terminal kinase (p-JNK), p-P38, and tubulin (Santa Cruz, CA, USA). Bcl-2, Bad, Cytochrome c, caspase 3, matrix metalloproteinases (MMP2), MMP9 (Cell Signaling, Danvers, MA, USA), NFATc3 and extracellular signal-regulated kinase 1 (ERK1) (Abcam, Cambridge, CB, UK) were also measured. The immunoblots were washed three times in 5 ml blotting buffer for 5 min and afterward PVDF was incubated with secondary antibody solution containing horseradish peroxidase-conjugated anti-rabbit (1:1000), anti-goat (1:500), or anti-mouse (1:1000) immunoglobulin G secondary antibodies (Santa Cruz Lab, CA, USA) for 1 h at room temperature. Secondary antibodies were used for enhanced chemiluminescence (Sigma-Aldrich, Bornem, Belgium) detection of proteins. When required, the blots were reprobed after stripping in 62.5 mM Tris-HCl (pH 6.8), 2% SDS, and 100 mM β-mercaptoethanol at room temperature for 30 min, blocked, and reprobed with polyclonal or monoclonal antibody. The membranes were scanned and quantified with ImageJ image program software.

Statistical analysis

Quantitation was carried out by analyzing the intensity of the hybridization signals using ImageJ image program (Wayne Rasband, NIH, Bethesda, MD, USA) for Western blot analysis. Obesity (HFD-fed mice) data were obtained from independent experiments. Statistical analysis of the data was the values of the group of the same batch expressed as a mean ± standard error of the means (SEMs) using GraphPad Prism 6 software (GraphPad Software Inc., La Jolla, CA, USA). *P < 0.05, **P < 0.01 significant differences compared with control group,#P < 0.05,##P < 0.01 significant differences compared with HFD-fed mice without treatment group. Two-group comparisons were carried out with the one-way ANOVA followed by Turkey's post hoc tests. SAMP8 mice data among multiple groups were obtained from independent experiments. Statistical analysis of the data was performed for the values of the control group of the same batch using SigmaStat 11.0 software (Systat Software Inc., San Jose, CA, USA). Results were expressed as mean ± SEM. Two-group comparisons were carried out with the Student's t-test. Differences were considered statistically significant when P < 0.05.


  Results Top


Resveratrol prevents high-fat diet-induced skeletal muscle atrophy and body fat increases

HFD-induced obesity is associated with lower muscle mass in sarcopenic obesity. C57BL/6J mice were fed a HFD to induce obesity. Intriguingly, obesity may not coexist with sarcopenia, and skeletal muscle and fat percentage is the key role. Therefore, body drying weight, muscle drying grinding, fat weight, and body fat were investigated. The results showed that body drying weight in HFD groups increased compared to control without treatment. Body drying weight is skeletal muscle and fat couple together. Thus, we found that body drying weight increases in all HFD groups. No significant difference was observed in HFD with all doses of resveratrol intake groups, compared to HFD group [Figure 1]a. Hence, only HFD + HR (200 mg/kg) group showed significant increases as compared to control group (P < 0.05). Same result was found in skeletal muscle drying grinding weight in HFD + HR group [Figure 1]b. Skeletal muscle drying grinding weight in HFD group did not decrease to ascertain skeletal muscle loss. In turn, fat weight and body fat were detected. Fat weight and body fat in HFD group increased, but no significant difference was shown [Figure 1]c and d]. When compared with control, HFD + MR (100 mg/kg) had good effects on lowering fat weight and body fat. Indeed, body fat was significantly decreased in HFD + MR group (P < 0.05). Taken together, we suggest resveratrol can increase skeletal muscle mass and lowers fat weight during sarcopenia in adults with obesity development.
Figure 1: Resveratrol reduced fat weight of the skeletal muscle in HFD-fed C57BL/6J mice. (a) Body drying weight. (b) Skeletal muscle dring grinding. (c) Fat weight. (d) Body fat. Results are expressed mean ± standard error of the mean. Groups comparison to control or HFD were carried out with the Student's t-test. Differences were expressed at *P < 0.05. HFD: High-fat diet, LR: Low-dose resveratrol, MR: Middle-dose resveratrol, HR: High-dose resveratrol. Statistical analysis of the data was performed using SigmaStat 11.0 software.

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Exercise training, resveratrol intake, and combination of exercise training and resveratrol intake increased skeletal muscle weight and improved muscle cell apoptosis in senescence-accelerated mouse prone 8 mice

We use SAMP8 mice to investigate aging-related skeletal muscle atrophy (sarcopenia). Whether exercise training, resveratrol intake, and combination of exercise training and resveratrol intake can increase skeletal muscle weight and improve muscle cell apoptosis in SAMP8 mice were investigated. The results showed that the age-related loss of skeletal muscle mass was increased or maintained by exercise training, resveratrol intake, and their combination in SAMP8 mice [Figure 2]a. Age-related sarcopenia induced skeletal muscle fiber disorder in SAMP8 mice. Exercise training, resveratrol intake, and their combination alleviated the increase of skeletal muscle fiber space in SAMP8 mice [Figure 2]b and c]. Muscle-wasting condition in sarcopenia is affected by cell apoptosis. Cell apoptosis was observed using TUNEL staining. Exercise training, resveratrol intake, and their combination ameliorated cell apoptosis in skeletal muscle of sarcopenic SAMP8 mice [Figure 2]d and [Figure 2]e. We demonstrate that resveratrol has a therapeutic effect against skeletal muscle sarcopenia.
Figure 2: Sarcopenia is the age-related loss of muscle mass and function in senescence-accelerated mouse prone 8 (SAMP8) mice. (a) Skeletal muscle weight in exercise training, resveratrol, and combination of exercise training and resveratrol in SAMP8 mice. (b) Skeletal muscle space in exercise training, resveratrol, and combination of exercise training and resveratrol in SAMP8 mice. (c) Histology of skeletal muscle in exercise training, resveratrol, and combination of exercise training and resveratrol in SAMP8 mice using H and E staining. (d) Apoptosis cells of skeletal muscle in exercise training, resveratrol, and combination of exercise training and resveratrol intake in SAMP8 mice using TUNEL staining. (e) Statistical analysis results of skeletal muscle apoptosis cells (%). All data are expressed mean ± standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001, compared with SAMP8 mice without treatment. SAMP8+Ex: Exercise training SAMP8 groups, SAMP8+Re: Resveratrol intake SAMP8 groups, SAMP8+Ex+Re: Combination of exercise training and resveratrol SAMP8 groups.

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High-dose resveratrol counteracts obesity-induced decline of mitochondrial function in skeletal muscle of high-fat diet-fed mice, but aging-associated decline was reversed by resveratrol and its combination with exercise in senescence-accelerated mouse prone 8 mice

HFD-induced obesity is related with increased dysfunctional mitochondria in skeletal muscles. Thus, mitochondrial protein expressions were detected in HFD-induced obesity mice. The results showed that HFD-induced obesity augments skeletal muscle atrophy by induction of protein degradation in mitochondria [Figure 3]. High dose of resveratrol preserved mitochondrial activity in skeletal muscle of HFD-induced obesity mice [Figure 3]a. HFD-diet groups show a significant difference compared to control by downregulation of Bcl 2 and upregulation of Bad protein expression (vs. control, P < 0.05). Bcl2 expression was significantly increased in all doses of resveratrol and placebo groups (LR + HFD, HR + HFD vs. control and vs. HFD, P < 0.05; MR + HFD vs. control and vs. HFD, P < 0.01). Bad expression was significantly decreased in HR + HFD and placebo groups, compared with control and HFD (P < 0.05). Mitochondrial function proteins were detected in aging-related skeletal muscle sarcopenia of SAMP8 mice including Bcl 2, Bad, cytochrome and caspase 3 [Figure 3]b. There was no significant change in Bcl 2, Bad, Cytochrome c, and Caspase 3 expressions in SAMP8 mice with exercise training. Bcl 2 increase and Bad decrease were observed in mice treated with resveratrol (P < 0.05), and combination of exercise training and resveratrol intake showed significant differences compared with SAMP8 mice (P < 0.01). Resveratrol intake and combination of exercise training and resveratrol significantly decreased cytochrome c expression (P < 0.01). Especially, combination of exercise training and resveratrol intake showed a significant decrease in Caspase 3 (P < 0.05) in sarcopenic skeletal muscle of SAMP8 mice.
Figure 3: Effects of exercise, resveratrol, and combination of exercise and resveratrol on the mitochondrial dysfunction and apoptosis in skeletal muscles of obesity-induced sarcopenia mice and age-related sarcopenia senescence-accelerated mouse prone 8 (SAMP8) mice. (a) Representative western blotting analysis for Bcl 2 and Bad protein levels in HFD-induced obesity mice groups. All data are expressed mean ± standard error of the mean (SEM). *P < 0.05, **P < 0.01, compared with control.#P < 0.05,##P < 0.01, compared with HFD mice without treatment. a-tubulin was used as a loading control. HFD: High-fat diet, LR: Low-dose resveratrol, MR: Middle-dose resveratrol, HR: High-dose resveratrol. (b) Representative Western blots using antibodies against Bcl 2, Bad, Cytochome c, Caspase 3, and α-tubulin in SAMP8 mice groups. After quantification, Bcl 2/α-tubulin, Bad/α-tubulin, Cytochrome c/α-tubulin, and Caspase 3/α-tubulin ratios were calculated. α-tubulin was used as a loading control. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, compared with SAMP8 control group. NS: No significant difference. SAMP8+Ex: Exercise training SAMP8 groups, SAMP8+Re: Resveratrol SAMP8 groups, SAMP8+Ex+Re: Combination of exercise training and resveratrol SAMP8 groups.

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Resveratrol revealed dramatic decreases in sarcopenia extracellular matrix remodeling in obesity-induced sarcopenia mice and aging-associated sarcopenia mice

MMP2 and MMP9 play a role in adipose and muscle hypertrophy to atrophy and could be involved in extracellular matrix (ECM) remodeling in response to HFD fed mice. ECM remodeling of skeletal muscle occurs in HFD-induced obesity mice with skeletal muscle mass loss. MMP2 and MMP9 upregulation enhanced obesity-induced skeletal muscle atrophy. MMP2 expression was significantly decreased in all doses of resveratrol and placebo groups (vs. HFD, P < 0.05). HR + HFD and placebo groups showed significant decreases in MMP9 expression when compared with HFD control group (P < 0.05) [Figure 4]a. In skeletal muscle tissue, HFD showed slight increase in ANP and BNP expressions, compared to control. The physiological effects of BNP are identical to those of ANP. Resveratrol intake lowers ANP and BNP protein synthase, and it was found to be a predictor in sarcopenia skeletal muscle of HFD-induced obesity [Figure 4]b. BNP expression was slightly altered by resveratrol. MMP2 and MMP9 are involved in physiological processes of aging, such as skeletal muscle development and maturation. Expressions of skeletal muscle MMP2 and MMP9 decrease with age, which contributed to functional decline in sarcopenia SAMP8 mice. Exercise training did not reverse these changes. In order for exercise training to be effective, proper nutrition must be in place. Results showed resveratrol intake and combination of exercise training and resveratrol contributed to increases in MMP2 and MMP9 expression (P < 0.05 and P < 0.01, respectively) [Figure 4]c. Furthermore, the expressions of FGF2 and UPA in skeletal muscles growth were measured. Activities of both growth factors of FGF2 and UPA enhance the reinnervation of muscle. Thus, exercise training, resveratrol intake, and combination of exercise training and resveratrol intake showed decreases in activities of FGF2 and UPA in skeletal muscles [Figure 4]d.
Figure 4: Resveratrol reduces matrix metalloproteinases (MMPs) in skeletal muscles of obesity-induced sarcopenia mice, but elevates MMPs in age-related sarcopenia senescence-accelerated mouse prone 8 (SAMP8) mice. (a and b) The levels of MMP2, MMP9, ANP, and BNP protein in HFD-induced obesity mice were measured by Western blotting. All data were expressed as mean ± standard error of the mean (SEM). *P < 0.05, **P < 0.01, compared with control.#P < 0.05, compared with HFD control mice without treatment. α-tubulin was used as a loading control. (c and d) Western blot analysis of MMP2, MMP9, FGF-2, and UPA in SAMP8 mice. α-tubulin was used as a loading control. Quantification of Western blot bands was made by densitometry. All data were expressed as mean ± SEM. *P < 0.05, **P < 0.01, compared with SAMP8 control group. HFD: High-fat diet, LR: Low-dose resveratrol, MR: Middle-dose resveratrol, HR: High-dose resveratrol, ANP: Atrial natriuretic peptide, BNP: Brain natriuretic peptide, FGF: Fibroblast growth factor, UPA: Urokinase-type plasminogen activator, NS: No significant difference, SAMP8+Ex; Exercise training SAMP8 groups, SAMP8+Re: Resveratrol SAMP8 groups, SAMP8+Ex+Re: Combination of exercise training and resveratrol intake SAMP8 groups.

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Resveratrol and its combination with exercise training protected against skeletal muscle atrophy and inflammatory induced by obesity and sarcopenia

HFD-induced obesity causes skeletal muscle atrophy and is associated with low-grade chronic inflammation. HFD-induced obesity inhibited PI3K/Akt activities in skeletal muscles of HFD-fed mice (P < 0.05). Resveratrol intake of all doses (LR, MR, HR) activated PI3K/Akt activities in skeletal muscles of HFD-fed mice [Figure 5]a. There was no significant difference in STAT3 expression in HR + HFD group. Resveratrol was to be a promising material to alleviate HFD-induced muscle atrophy. Obesity is associated with chronic inflammation. Thus, IL-6 and STAT3 expressions were significantly increased in skeletal muscles of HFD-fed mice [Figure 5]b (P < 0.05). HR + HFD and placebo groups significantly lowered the expression in IL-6, compared with control and HFD (P < 0.05). STAT3 was slightly increased in skeletal muscle inflammation. Taken together, resveratrol alleviated skeletal muscle atrophy and suppressed inflammation in skeletal muscles of HFD-fed mice. Furthermore, we determined whether resveratrol could alleviate age-related sarcopenia in skeletal muscle atrophy of SAMP8 mice. Age-associated sarcopenia is a disease of muscles with conditions of skeletal muscle atrophy and inflammatory myopathies. Intriguingly, exercise training had no effect on SAMP8 mice with slight sarcopenia [Figure 5]c and d]. In this study, we only find resveratrol has good effects against skeletal muscle atrophy and inflammatory myopathies. We found PI3K/Akt expression was increased by resveratrol (P < 0.05) and combination of exercise training and resveratrol (PI3K, P < 0.05; Akt, P < 0.01) in SAMP8 mice [Figure 5]c. Interestingly, expression of IL-6 and STAT3 was decreased by resveratrol and combination of exercise training and resveratrol. IL-6 protein expression was decreased by resveratrol (P < 0.05) and combination of exercise training and resveratrol (P < 0.01). STAT3 was slightly decreased by combination of exercise training and resveratrol in skeletal muscles of sarcopenia SAMP8 mice [Figure 5]d. Obesity related inflammation reportedly is associated with pathogenesis of sarcopenia. Obese skeletal muscle pro-inflammation promotes obesity by activating the p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). JNK and p38 MAPK is upregulated in HFD-fed mice which resolved their paradoxical activation during diseased conditions [Figure 6]a. Resveratrol did not decrease activities of p38 MAPK and JNK in skeletal muscles, compared with the control group, and was resistant to the development of HFD-induced obesity. In turn, in age-related sarcopenia SAMP8 mice, p38 MAPK/JNK protein expression was decreased by exercise training and resveratrol [Figure 6]b. Resveratrol stimulated an increase in p-JNK expression, which prevented pro-inflammation in skeletal muscle (P < 0.05). Thus, low-grade systemic pro-inflammation is a component of “obese to sarcopenia.” Skeletal muscle pathologies can be prevented by regular physical exercise and resveratrol.
Figure 5: Effects of resveratrol, exercise, and their combination on expressions of PI3K, Akt, IL-6, and STAT3 in skeletal muscles of obesity-induced sarcopenia mice and age-related sarcopenia senescence-accelerated mouse prone 8 (SAMP8) mice. (a and b) Western blotting for IL-6, STAT3, PI3K, and Akt protein levels in HFD-induced obesity mice. α-tubulin was used as a loading control. Quantitative analysis of PI3K, Akt, IL-6, and STAT3 levels was shown. All data are expressed mean ± standard error of the mean (SEM). *P < 0.05, compared with control.#P < 0.05, compared with HFD mice without treatment. (c and d) Western blotting of PI3K, Akt, IL-6, and STAT3 protein levels in sarcopenia SAMP8 mice. α-tubulin was used as a loading control. Quantitative analysis of PI3K, Akt, IL-6, and STAT3 levels was shown. Data were expressed as mean ± SEM. *P < 0.05, **P < 0.01, compared with SAMP8 control group. HFD: High-fat diet, LR: Low-dose resveratrol, MR: Middle-dose resveratrol, HR: High-dose resveratrol, PI3K: Phosphatidylinositol 3 kinase, IL-6: Interleukin-6, NS: No significant difference, SAMP8+Ex: Exercise training SAMP8 groups, SAMP8+Re: Resveratrol SAMP8 groups, SAMP8+Ex+Re: Combination of exercise training and resveratrol SAMP8 groups

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Figure 6: Resveratrol induces skeletal muscle p38 MAPK and JNK activities in the skeletal muscles of obesity-induced sarcopenia mice and reduced JNK in aging-related sarcopenia senescence-accelerated mouse prone 8 (SAMP8) mice. (a) Western blotting analysis of skeletal muscle pro-inflammation showed increased levels of p-JNK and p-38 in HFD-induced obesity. α-tubulin was used as a loading control. All data are expressed mean ± standard error of the mean (SEM). *P < 0.05, compared with control. (b) Representative Western blotting analysis of p-JNK and p-P38 proteins in skeletal muscle of SAMP8 mice after exercise training, resveratrol intake or their combination was shown. α-tubulin was used as a loading control. Data are expressed as mean ± SEM. *P < 0.05, compared with SAMP8 control group. HFD: High-fat diet, LR: Low-dose resveratrol, MR: Middle-dose resveratrol, HR: High-dose resveratrol, MAPK: Mitogen-activated protein kinase, JNK: c-Jun N-terminal kinase, NS: No significant difference, SAMP8+Ex: Exercise training SAMP8 groups, SAMP8+Re: Resveratrol SAMP8 groups, SAMP8+Ex+Re: Combination of exercise training and resveratrol SAMP8 groups.

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Resveratrol in high-fat diet-fed mice promoted muscle hypertrophic process from atrophy involved NFATc3, but not ERK1/2

Nuclear factor of activated T-cells (NFATc3) causes pathological hypertrophy to prevent atrophy, but ERK1/2 causes physiological hypertrophy. Physiological hypertrophy was suppressed by pathological hypertrophy. Therefore, intake in spite of low-, middle-, and high-dose of resveratrol, NFATc3 expression was slightly increased in groups with all doses of resveratrol (LR, MR, HR), but ERK1/2 was decreased in HFD-fed induced obesity mice [Figure 7]a and b]. Sarcopenia is caused by aging, but the development of it can be attributed to skeletal muscle mass loss, resulting in skeletal mass atrophy. Results showed exercise training increased NFATc3 expression in SAMP8 mice, and did not reverse muscle atrophy (no significant difference) [Figure 7]c. There was no significant difference observed in ERK1 expression by exercise training, but resveratrol and its combination with exercise training showed significant decreases in ERK1 expression (P < 0.05) [Figure 7]d. Resveratrol prevented the exacerbation of skeletal muscle atrophy.
Figure 7: Resveratrol causes skeletal muscle hypertrophy in obesity-induced sarcopenia mice and age-related sarcopenia senescence-accelerated mouse prone 8 (SAMP8) mice. (a and b) Western blotting of NFATc3 and ERK1/2 protein levels in HFD-induced obesity mice groups. All data were expressed as mean ± standard error of the mean (SEM). *P < 0.05, **P < 0.01, compared with control. α-tubulin was used as a loading control. (c and d) Protein levels of NFATc3 and ERK1 by Western blotting in SAMP8 mice groups. α-tubulin was used as a loading control. Data are expressed as mean ± SEM. *P < 0.05, compared with SAMP8 control group. HFD: High-fat diet, LR: Low-dose resveratrol, MR: Middle-dose resveratrol, HR: High-dose resveratrol, NS: No significant difference, SAMP8+Ex: Exercise training SAMP8 groups, SAMP8+Re: Resveratrol SAMP8 groups, SAMP8+Ex+Re: Combination of exercise training and resveratrol SAMP8 groups.

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  Discussion Top


The aging of the population represents one of the largest healthcare challenges around the world today. Some papers have reported to lower age-mediated inflammation has been shown to increase the life span which slows development of skeletal muscle disorders.[11],[12] We examined the effects of resveratrol, a natural polyphenolic compound present in grapes, on sarcopenia in HFD-induced obesity mice and aging-associated sarcopenia in SAMP8 mice. The resveratrol theory of aging provides reasonable explanations for age-associated sarcopenia.[11] In addition, studies strongly suggest that resveratrol plays a significant role in the deterioration of skeletal muscle with obesity and sarcopenia.[13],[14] In this study, HFD-fed obesity mice with resveratrol showed an increase in skeletal muscle mass and inhibited fat weight [Figure 1]. Extra lipids cannot accumulate in the adipose tissue much longer, and infiltrate into peripheral organs such as the skeletal muscle, causing dysfunction of the organs.[15] Deleterious effects of aging probably reflect on skeletal muscle mass, space, and cell apoptosis [Figure 2]. Furthermore, mitochondrial protein expression levels, muscle hypertrophic process following atrophy, and inflammatory myopathies in skeletal muscle tissues from two different mice models of HFD-induced obesity mice and aging-associated sarcopenia SAMP8 mice were detected by Western blot. Resveratrol has been reported to target mitochondrial-related pathways in two different animal models. Adaptation exercise training does not play a role in regulating skeletal muscle in aging-related sarcopenia. In general, resveratrol has anti-inflammatory, anti-proliferative properties, and exhibits antioxidant activities in skeletal muscle atrophy of the two animal models. Therefore, resveratrol is currently advised as supplement in the diet of elderly combined with exercise training.[16],[17] Obese sarcopenia results in replacement of skeletal muscle and fat, and resveratrol prevents skeletal muscles atrophy from sarcopenia [Figure 3]. Resveratrol induces anti-inflammatory effects and inhibits skeletal muscles atrophy [Figure 5]. Moreover, MMPs enhanced immunoreactivity near atrophic myofibers.[18] MMP-9 was strongly expressed in atrophic myofibers in all inflammatory myopathies.[19] MMP-2 immunoreactivity is only slightly elevated in inflammatory skeletal muscle [Figure 4]. ANP and BNP binding to the natriuretic peptide receptor A, rising intracellular cGMP levels induce expression of downstream genes in skeletal muscle.[20],[21],[22] Free fatty acids from adipocyte lipolysis serve as additional ligands for the transcriptional regulator of mitochondrial biogenesis.[23],[24],[25] Thus, resveratrol increases skeletal muscle mitochondrial content (Bcl2, PI3K/Akt) which reverses skeletal muscle atrophy. p-JNK/p-p38-mediated intrinsic pathway signaling is involved in age-related muscle inflammation.[26],[27],[28],[29] Exercise training and resveratrol suppressed p-JNK mediated age-related muscle inflammation [Figure 6]b. Function of NFATc3 is regulating skeletal muscle mass through enlargement of muscle fiber size, while exercise training and resveratrol protect muscle from atrophy [Figure 7]. Therefore, protective effect of resveratrol against inflammation on progressive skeletal muscle atrophy was observed in obesity-induced sarcopenia mice and age-related sarcopenia SAMP8 mice [Figure 8].
Figure 8: Schematic representation of the inhibition effects of resveratrol on obesity and aging sarcopenia. The mechanisms underlying the pathophysiology of sarcopenic obesity. In obesity skeletal muscle, adipocytes undergo hypertrophy activation resulting in skeletal muscle atrophy due to accumulation of pro-inflammation as well as mitochondrial dysfunction together with senescent muscle cells. Skeletal muscle inflammation triggers and supports sarcopenic obesity development. ECM: Extracellular matrix, ANP: Atrial natriuretic peptide, BNP: Brain natriuretic peptide, MMP: Matrix metalloproteinases, PI3K: Phosphatidylinositol 3 kinase, UPA: Urokinase-type plasminogen activator, FGF: Fibroblast growth factor.

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Acknowledgements

Funding of this study was provided by the Ministry of Science and Technology (MOST 106-2811-B-650-003).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]



 

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