Resveratrol prevents sarcopenic obesity by reversing mitochondrial dysfunction and oxidative stress via the PKA/LKB1/AMPK pathway

RSV protected against HFD-induced muscle atrophy and dysfunction in aged rats

Six groups of rats were formed: young rats fed the normal chow diet (CD) (Y), old rats fed the normal CD (O), old rats fed the HFD (HO), old rats fed the HFD for 10 weeks and then the HFD supplemented with RSV for 10 weeks (HO+R10), old rats fed the HFD supplemented with RSV starting from the time of study initiation and throughout the 20-week experimental period (R20+HO), and old rats fed a normal CD supplemented with RSV starting from the time of study initiation and throughout the 20-week experimental period (R20+O). Prior to the experimental period, no apparent difference in baseline body weight was observed between the groups of aged rats (Figure S1A). After 20 weeks of feeding, the HFD-fed aged rats exhibited a higher body weight than the CD-fed aged rats (Figure 1A). The weights of the gastrocnemius (GAS) and tibialis anterior (TA) muscles were reduced during aging and further decreased in the aged rats fed the HFD (Figure 1B and 1C). Although the absolute weights of the soleus (SOL) muscles showed no overt differences among the six groups, the relative SOL muscle weights of the HO group were lower than those of the O group (Figure 1B and 1C). RSV supplementation reversed the HFD-induced increase in body weight and decreases in muscle weights (Figure 1A-C). A histological analysis showed that the mean fiber cross-sectional area (CSA) of the GAS muscles was decreased during aging, and a greater reduction was observed in the HFD-fed aged rats. Moreover, the rats in the HO group developed a large number of lipid droplets in their GAS muscles. The addition of RSV to the HFD attenuated these histological changes (Figure 1D and 1E). To monitor muscle function, we conducted a grip strength test. Concomitant with the changes in muscle weights, the grip strength was decreased by aging and further decreased in the aged rats fed the HFD. RSV administration prevented the HFD-induced reduction in grip strength (Figure 1F). Moreover, the weights and fiber CSA of the GAS muscles and grip strength were higher in the R20+HO group than in the HO+R10 group, and no significant differences in body weight, muscle weights, muscle fiber CSA and grip strength were observed between the O group and the R20+O groups (Figure 1). In addition, the addition of RSV to the diet increased the serum concentrations of RSV in aged rats (Figure S1B). Together, these results suggested that RSV supplementation effectively protected against HFD-induced muscle wasting and dysfunction in aged rats.

Figure 1. RSV protects against HFD-induced muscle atrophy in aged rats. (A) Final body weights of the different groups. (B) Weights of GAS, TA and SOL muscles in each group. (C) The relative muscle weights in each group. (D) Representative images of H&E staining of the GAS muscles. Scale bar, 100 μm. (E) Average muscle fiber CSA of the GAS muscles. (F) Grip strength in each group. Data are expressed as the mean ± SD. ^aP < 0.05 vs. the O-group; ^bP < 0.05 vs. the HO-group; ^cP < 0.05 vs. the Y-group; *P < 0.05 for the comparison between the marked groups.

RSV inhibited fat accumulation and ameliorated adverse changes in serum biochemical parameters

The abdominal fat tissues were analyzed through micro-computed tomography (microCT). As shown in Figure 2A-C, the volumes of visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) were increased during aging and further increased in aged rats fed the HFD. Paralleling the changes in the fat mass volumes, the weights of subcutaneous, epididymal and perirenal fat tissues were elevated by HFD feeding (Figure 2D). We also measured the TG levels in the GAS muscles and found that the intramuscular fat content was greater with aging and further elevated in the HFD-fed aged rats (Figure 2E). The serum levels of free fatty acid (FFA), total triglyceride (TG), total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) of the HO group were higher than those of the O group, and these increases were concomitant with a decrease in the serum high-density lipoprotein cholesterol (HDL-C) levels (Figure 2F and 2G). RSV supplementation significantly reduced the fat masses and ameliorated the adverse changes in the serum lipid levels observed in the HFD-fed aged rats (Figure 2). Moreover, the VAT volumes, perirenal adipose tissue weights, intramuscular TG content, and serum TG and LDL-C levels were lower in the R20+HO group than in the HO+R10 group. In addition, RSV administration reduced the weights of perirenal adipose tissues and increased the serum HDL-C levels in aged rats fed the CD (Figure 2D and 2G).

Figure 2. Effects of RSV on adipose tissue volumes and masses, intramuscular TG content and serum lipid profile. (A) Representative three-dimensional images of abdominal fat tissues (red, visceral fat; green, subcutaneous fat). (B) VAT volumes. (C) SAT volumes. (D) Fat masses. (E) TG content of the GAS muscles. (F) Serum FFA levels. (G) Serum TG, TC, HDL-C and LDL-C levels. Data are expressed as the mean ± SD. ^aP < 0.05 vs. the O group; ^bP < 0.05 vs. the HO group; ^cP < 0.05 vs. the Y group; *P < 0.05 for the comparison between the marked groups.

RSV ameliorated the HFD-induced mitochondrial morphological abnormalities in aged rats

We subsequently analyzed the mitochondrial morphology changes in the intermyofibrillar mitochondria (IFM) and subsarcolemmal mitochondria (SSM) through transmission electron microscopy (TEM). The images revealed fewer mitochondrial cristae and the presence of some lipid droplets closely adherent to SSM during aging, and these alterations were more extreme in the HFD-fed aged rats. Moreover, the aged rats fed the HFD showed many swollen mitochondria with distorted cristae and broken membranes (Figure 3A and 3B). The addition of RSV to the HFD ameliorated these morphological abnormalities, resulting in decreased amounts of aberrant mitochondria and smaller lipid droplets (Figure 3A and 3B).

Figure 3. Effects of RSV on mitochondrial morphology and related protein expression in the GAS muscles. (A and B) Representative TEM images of the IFM and SSM at an original magnification of 25,000×. The short arrows indicate damaged mitochondria; the long arrows indicate lipid droplets. (C) Representative images of the western blotting results for PGC-1α, TFAM, mfn2 and drp1 in whole muscle lysates; GAPDH was used as a loading control. (D) The bar graphs show quantification of the indicated proteins. (E) Representative images of the western blotting results for TFAM, mfn2 and drp1 in the mitochondrial subfractions; VDAC1 was used as a loading control. (F) The bar graphs show quantification of the indicated proteins. (G) Relative mtDNA content. Data are expressed as the mean ± SD. ^aP < 0.05 vs. the O group; ^bP < 0.05 vs. the HO group.

We further evaluated the protein expression of key regulators involved in mitochondrial biogenesis and dynamics. As shown in Figure 3C and 3D, the decreases in the protein levels of PGC-1α, TFAM and mfn2, as well as the increased drp1 expression in HFD-fed aged rats, were all reversed by RSV supplementation. Similar alterations were observed for the protein levels of TFAM, mfn2 and drp1 in the mitochondrial subfractions (Figure 3E and 3F). In addition, RSV administration inhibited the HFD-mediated reductions in mtDNA copy number (Figure 3G).

RSV improved mitochondrial function and antioxidant capacity in aged rats

To investigate the effects of RSV on mitochondrial function, we analyzed the mitochondrial membrane potential (Δψm), mitochondrial respiratory chain complex activities and ATP production in the GAS muscles. The data showed that RSV supplementation prevented the HFD-induced loss of Δψm (Figure 4A). Furthermore, the activities of complexes I, II, and IV and the ATP content were decreased by HFD feeding but restored by RSV administration (Figure 4B and 4C).

Figure 4. Effects of RSV on mitochondrial function and oxidative stress in the GAS muscles. (A) Δψm. (B) Activities of complexes I, II, and IV. (C) ATP content. (D and E) Activities of SOD and GPx. (F) T-AOC. (G) MDA levels. (H) Carbonyl protein content. Data are expressed as the mean ± SD. ^aP < 0.05 vs. the O group; ^bP < 0.05 vs. the HO group; *P < 0.05 for the comparison between the marked groups.

To examine the effects of RSV on oxidative status, we measured the antioxidant enzyme activities and the levels of several oxidative damage markers in skeletal muscle tissues. We found that HFD feeding resulted in decreased total antioxidative capability (T-AOC) and reduced activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx) accompanied by elevated malondialdehyde (MDA) and carbonyl protein contents. These changes were reversed by RSV supplementation (Figure 4D-F). Moreover, the T-AOC and activities of complexes I, II, and IV and superoxide dismutase (SOD) of the R20+HO group were higher than those of the HO+R10 group, and the MDA and carbonyl protein levels of the R20+HO group were lower than those of the HO+R10 group. In addition, RSV administration enhanced the antioxidant capacity and decreased the MDA levels in aged rats fed the CD (Figure 4D-H).

RSV prevented PA-induced muscle atrophy in vitro

To mimic HFD-induced muscle atrophy in aged rats, we used PA, the most abundant circulating saturated fatty acid [4], to produce a cell model of muscle atrophy. As shown in Figure 5A, no significant changes in cell viability were observed in myotubes treated with PA at concentrations of less than 0.5 mM. In addition, 0.75 mM PA notably reduced the myosin heavy chain (MHC) expression in myotubes (Figure 5B and 5C), and this concentration was therefore selected for the in vitro induction of the muscle atrophy model in subsequent experiments. Next, cells were treated with different concentrations of RSV to test its in vitro effects. RSV (1, 5, 10 and 25 μM) itself had no significant effects on cell viability in myotubes (Figure 5D) and dose-dependently inhibited the PA-induced decrease in MHC content (Figure 5E and 5F). RSV at the dose of 25 μM exerted the greatest protective effects; therefore, we chose this dose in subsequent experiments. Furthermore, the decreases in cell viability and myotube diameter, as well as the increased TG content in PA-treated myotubes, were all attenuated by RSV (Figure 5G, 5H and S2A). In addition, RSV alone exerted no significant effects on MHC expression, myotube diameter or TG content (Fig 5E, 5F, 5H and S2A). Together, these data indicated that RSV efficiently alleviated PA-induced cell death and muscle atrophy in vitro.

Figure 5. RSV inhibits PA-induced muscle atrophy in L6 myotubes. (A) Myotubes were treated with different doses (0.25, 0.5, 0.75, or 1 mM) of PA for 24 h, and cell viability was measured using a CCK-8 assay. (B) Representative image of the western blotting results for MHC; GAPDH was used as a loading control. (C) The bar chart shows the quantification of MHC. (D) Myotubes were treated with RSV at a series of concentrations (1, 5, 10, 25 or 50 μM) for 24 h, and cell viability was detected by CCK-8 assay. (E) Myotubes were treated with 0.75 mM PA in the presence or absence of different concentrations (1, 5, 10 and 25 μM) of RSV for 24 h. The protein expression of MHC was measured by western blotting. (F) The bar graph shows the quantification of MHC. (G) Myotubes were exposed to 0.75 mM PA in the presence or absence of 25 μM RSV for 24 h, and cell viability was detected by a CCK-8 assay. (H) Quantification of the myotube diameter, as described in the Materials and Methods section. Data are expressed as the mean ± SD. ^aP < 0.05 vs. the control group; ^bP < 0.05 vs. the PA-treated group.

RSV activated the PKA/LKB1/AMPK signaling pathway

We further explored the possible mechanism underlying the protective effects of RSV in muscle atrophy models. As shown in Figure 6A-F, RSV restored the PA-mediated reductions in p-PKA, p-LKB1 and p-AMPK protein levels. Moreover, LKB1 siRNA inhibited RSV-induced LKB1 activation accompanied by decreased expression of p-AMPK (Figure 6A and 6C). Furthermore, PKA siRNA abolished the effects of RSV on the p-LKB1 and p-AMPK levels (Figure 6D and 6F). RSV supplementation also increased the expression of p-PKA, p-LKB1 and p-AMPK in the GAS muscles of HFD-fed rats (Figure 6G and 6H). These results suggested that RSV activated the PKA/LKB1/AMPK signaling pathway in the HFD- and PA-induced muscle atrophy models.

Figure 6. RSV activates the PKA/LKB1/AMPK signaling pathway. (A) Representative images of the western blotting results for p-LKB1, LKB1, p-AMPK and AMPK in myotubes; GAPDH was used as a loading control. (B) The bar charts show quantification of the indicated proteins. (C) Quantitative graph shows the ratios of p-AMPK/AMPK. (D) Representative images of the western blotting results for p-PKA, PKA, p-LKB1, LKB1, p-AMPK and AMPK in myotubes; GAPDH was used as a loading control. (E) The bar charts show quantification of the indicated proteins. (F) Quantitative graph shows the ratios of p-LKB1/LKB1 and p-AMPK/AMPK. Data are expressed as the mean ± SD. ^aP < 0.05 vs. the control group; ^bP < 0.05 vs. the PA-treated group; ^cP < 0.05 vs. the RSV (25 μM) and PA (0.75 mM) cotreated group with control siRNA transfection. (G) Representative images of the western blotting results for p-PKA, PKA, p-LKB1, LKB1, p-AMPK and AMPK in the GAS muscles; GAPDH was used as a loading control. (H) The bar charts show the relative protein levels. Data are expressed as the mean ± SD. ^aP < 0.05 vs. the O group; ^bP < 0.05 vs. the HO group.

RSV improved mitochondrial morphology, inhibited mtDNA depletion and restored mitochondrial-related protein expression in PA-treated L6 myotubes

For the morphological analysis of mitochondria, L6 myotubes were labeled with MitoTracker probes. As shown in Figure 7A, the mitochondria of the control group were filamentous and exhibited a thread-like appearance. PA treatment resulted in punctate and highly fragmented mitochondria, whereas RSV administration attenuated these abnormal morphological changes. A further analysis of the fluorescence intensity showed that RSV prevented the PA-induced reduction in mitochondrial mass (Figure 7B). However, PKA siRNA, LKB1 siRNA and AMPK siRNA transfection abrogated these effects of RSV (Figure 7A and 7B). Similar alterations were observed for the mtDNA content (Figure 7C).

Figure 7. Effects of RSV on mitochondrial morphology, mtDNA content and related protein expression in L6 myotubes. (A) Representative images for visualization of the mitochondrial morphology in vitro. Scale bar, 10 μm. (B) The mitochondrial mass was quantified by calculating the fluorescent intensity of MitoTracker Red. (C) Relative mtDNA content. (D) The levels of PGC-1α, TFAM, mfn2 and drp1 in the whole muscle lysates were measured by western blotting; GAPDH was used as a loading control. (E) The bar charts show quantification of the indicated proteins. (F) Representative images of the western blotting results for TFAM, mfn2 and drp1 in the mitochondrial subfractions; VDAC1 was used as a loading control. (G) The bar charts show quantification of the indicated proteins. Data are expressed as the mean ± SD. ^aP < 0.05 vs. the control group; ^bP < 0.05 vs. the PA-treated group; ^cP < 0.05 vs. the RSV (25 μM) and PA (0.75 mM) cotreated group with control siRNA transfection.

We subsequently evaluated the effects of RSV on several mitochondrial-related protein levels. The results showed that both the decreases in the PGC-1α, TFAM and mfn2 protein levels and the increase in drp1 expression in PA-treated myotubes were rescued by RSV treatment (Figure 7D and 7E). Similar results were found for TFAM, mfn2 and drp1 protein expression in the mitochondrial subfractions (Figure 7F and 7G). However, PKA siRNA, LKB1 siRNA and AMPK siRNA transfection abolished these effects of RSV (Fig 7D-G). Together, these results indicated that RSV improved the mitochondrial morphology, restored the mtDNA copy number and reversed mitochondrial-related protein expression via the PKA/LKB1/AMPK signaling pathway in vitro.

RSV reversed PA-induced mitochondrial dysfunction in vitro

The Δψm was measured by JC-1 staining. As shown in Figure 8A and 8B, the ratio of red to green fluorescence was notably decreased after PA treatment, which suggested that the Δψm was depolarized. However, RSV administration prevented the PA-induced loss of Δψm. In addition, the protective effects of RSV on Δψm were abrogated by PKA siRNA, LKB1 siRNA and AMPK siRNA.

Figure 8. Effects of RSV on mitochondrial function in L6 myotubes. (A) Representative images used for determination of the Δψm in vitro. Scale bar, 20 μm. (B) Ratio of red to green fluorescence. (C) Respiration curve of each group. (D) The bar charts show the quantification of ATP production and maximal respiration. (E) Activities of complexes I, II, and IV. Data are expressed as the mean ± SD. ^aP < 0.05 vs. the control group; ^bP < 0.05 vs. the PA-treated group; ^cP < 0.05 vs. the RSV (25 μM) and PA (0.75 mM) cotreated group with control siRNA transfection.

We then analyzed the oxygen consumption rate (OCR) in myotubes. The data showed that RSV treatment prevented the PA-induced declines in the mitochondrial respiration capacity, as indicated by the restoration of ATP production and maximal respiration (Figure 8C and 8D). However, PKA siRNA, LKB1 siRNA and AMPK siRNA transfection abolished these effects of RSV (Figure 8C and 8D). We subsequently measured the activities of mitochondrial respiratory chain complexes and found that RSV treatment restored the reduced activities of complexes I, II, and IV (Figure 8E). These effects of RSV were also reversed by PKA siRNA, LKB1 siRNA and AMPK siRNA (Figure 8E). Together, these results suggested that RSV attenuated PA-induced mitochondrial dysfunction via the PKA/LKB1/AMPK signaling pathway in vitro.

RSV alleviated PA-induced oxidative stress in vitro

We then investigated the effects of RSV on oxidative stress. As shown in Figure 9A and 9B, PA significantly elevated the cellular total reactive oxygen species (ROS) and mitochondrial ROS (mtROS) levels, which were inhibited by RSV treatment. Moreover, the decreases in T-AOC and the activities of SOD and GPx, as well as the increases in MDA and carbonyl protein levels in PA-treated myotubes, were all sufficiently normalized by RSV treatment (Fig 9C-G). However, PKA siRNA, LKB1 siRNA and AMPK siRNA transfection abrogated these effects of RSV (Figure 9). These results suggested that the protective effects of RSV on oxidative stress were exerted via the PKA/LKB1/AMPK signaling pathway in vitro.

Figure 9. Effects of RSV on oxidative stress in L6 myotubes. (A) Cellular total ROS levels. (B) mtROS levels. (C and D) Activities of SOD and GPx in vitro. (E) T-AOC in vitro. (F) MDA levels. (G) Carbonyl protein content. Data are expressed as the mean ± SD. ^aP < 0.05 vs. the control group; ^bP < 0.05 vs. the PA-treated group; ^cP < 0.05 vs. the RSV (25 μM) and PA (0.75 mM) cotreated group with control siRNA transfection.

RSV improved protein metabolism via the PKA/LKB1/AMPK signaling pathway

Muscle atrophy occurs when the rate of protein degradation exceeds that of protein synthesis [33]. Thus, we further evaluated the expression of several key factors involved in protein metabolism. As shown in Fig 10A and 10C, the protein levels of FoxO3a, atrogin-1 and MuRF1, all of which are well-known markers of protein degradation and muscle atrophy, were robustly elevated by PA treatment but significantly inhibited by the administration of RSV in vitro. RSV also prevented the decline in protein synthesis, as evidenced by the restoration of p-mTOR and p-S6K expression in PA-treated myotubes (Figure 10A and 10B). Similar results were found in the GAS muscles of HFD-fed aged rats after RSV supplementation (Figure 10D and 10E). However, PKA siRNA, LKB1 siRNA and AMPK siRNA transfection abolished the effects of RSV on these protein metabolism-related factors in vitro (Figure 10A-C). In addition, the protective effects of RSV on the MHC content were also blocked by PKA siRNA, LKB1 siRNA and AMPK siRNA (Figure 10A and 10B). These results indicated that RSV improved protein metabolism and attenuated skeletal muscle atrophy through the PKA/LKB1/AMPK signaling pathway.

Figure 10. RSV improves protein metabolism via the PKA/LKB1/AMPK signaling pathway. (A) Representative images of the western blotting results for p-mTOR, mTOR, p-S6K, S6K, FoxO3a, atrogin-1, MuRF1 and MHC in L6 myotubes; GAPDH was used as a loading control. (B and C) The bar graphs show quantification of the indicated proteins. Data are expressed as the mean ± SD. ^aP < 0.05 vs. the control group; ^bP < 0.05 vs. the PA-treated group; ^cP < 0.05 vs. the RSV (25 μM) and PA (0.75 mM) cotreated group with control siRNA transfection. (D) Representative images of the western blotting results for p-mTOR, mTOR, p-S6K, S6K, FoxO3a, atrogin-1 and MuRF1 in the GAS muscles; GAPDH was used as a loading control. (E) The bar charts show the relative protein levels. Data are expressed as the mean ± SD. ^aP < 0.05 vs. the O group; ^bP < 0.05 vs. the HO group.

RSV improved protein metabolism by reversing mitochondrial dysfunction and oxidative stress

Previous studies have demonstrated that mitochondrial dysfunction decreases protein synthesis and increases protein degradation [10]. We therefore hypothesized that the RSV-mediated improvement in muscle protein metabolism was linked to its role in reversing mitochondrial dysfunction and oxidative stress through the PKA/LKB1/AMPK signaling pathway. To verify this hypothesis, we transfected L6 myotubes with TFAM siRNA, mfn2 siRNA and drp1 siRNA. As shown in Fig. S2B and S2C, TFAM siRNA and mfn2 siRNA transfection inhibited RSV-induced mitochondrial respiration recovery, and the transfection of these siRNAs also diminished the effects of RSV on the FoxO3a, atrogin-1, MuRF1, p-mTOR, p-S6K and MHC levels in myotubes (Figure 11A-C). In addition, drp1 siRNA transfection improved the mitochondrial respiration capacity and protein metabolism in PA-treated myotubes, and these effects were similar to those obtained with RSV (Figure 11A-C, S2B and S2C).

Figure 11. RSV improves protein metabolism by improving mitochondrial function and oxidative stress. (A) Representative images of the western blotting results for p-mTOR, mTOR, p-S6K, S6K, FoxO3a, atrogin-1, MuRF1 and MHC in L6 myotubes; GAPDH was used as a loading control. (B and C) The bar charts show quantification of the indicated proteins. Data are expressed as the mean ± SD. ^aP < 0.05 vs. the control group; ^bP < 0.05 vs. the PA-treated group; ^cP < 0.05 vs. the RSV (25 μM) and PA (0.75 mM) cotreated group with control siRNA transfection. (D) Myotubes were incubated with PA for 24 h in the presence or absence of NAC (5 mM) or MitoTEMPOL (5 μM). Protein levels were measured by western blotting. (E) The bar charts show the relative protein levels. Data are expressed as the mean ± SD. ^aP < 0.05 vs. the control group; ^bP < 0.05 vs. the PA-treated group.

Oxidative stress has also been shown to inhibit protein synthesis and promote proteolysis in skeletal muscle [34]. To investigate the role of oxidative stress in muscle protein metabolism, the ROS scavenger N-acetyl-L-cysteine (NAC) and mitochondria-targeted antioxidant MitoTEMPO were used to suppress oxidative stress in myotubes. The results showed that both NAC and MitoTEMPO markedly decreased cellular ROS levels in PA-treated myotubes (Figure S2D). Furthermore, NAC and MitoTEMPO mimicked the effects of RSV on protein metabolism-related factors and MHC content (Figure 11D-F). Additionally, NAC and MitoTEMPO alone exerted no significant effects on the protein levels of FoxO3a, atrogin-1, MuRF1, p-mTOR, p-S6K and MHC (Figure 11D-F).

Based on these findings, we concluded that RSV improved protein metabolism and prevented HFD- and PA-induced muscle atrophy by reversing mitochondrial dysfunction and oxidative stress.

Reagents and antibodies

RSV, PA, Protein Carbonyl Content Assay Kit, NAC and MitoTEMPO were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Laboratories (Kumamoto, Japan), and glass-bottom cell culture dishes were obtained from Nest Biotechnology (Wuxi, China). MitoTracker Deep Red and MitoSOX Red were obtained from Invitrogen (Carlsbad, CA, USA). Antibodies against MHC (ab 24642), PGC-1α (ab 106814), TFAM (ab 131607), mfn2 (ab 50838), FoxO3a (ab 23683) and VDAC1 (ab 15895) were purchased from Abcam (Cambridge, UK). Antibodies against LKB1 (3047), p-LKB1 (3482), AMPK (2532), p-AMPK (2531), p-mTOR (2971), mTOR (2972), p-S6K (9205), S6K (9202) and GAPDH (5174) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against PKA (sc-903), p-PKA (sc-12905), atrogin-1 (sc-33782) and MuRF1 (sc-27642) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibody against drp1 (BD611113) was obtained from BD Biosciences (San Jose, CA, USA).

Animals

Young (aged 3 months) and old (aged 18 months) male Sprague-Dawley (SD) rats were obtained from the animal center of the Third Military Medical University (Chongqing, China). The rats were maintained in collective cages and housed under pathogen-free conditions with constant temperature and humidity, a regular 12-h light:12-h dark cycle and free access to water and food. All the animal experiments were performed in accordance with the institutional guidelines on animal experimentation set by the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee of the Third Military Medical University.

Diets and experimental design

The rats were fed either a CD or a HFD for 20 weeks. The CD contained 10% kcal from fat, whereas the HFD contained 45% kcal from fat. RSV was added to the diet as described previously [83]. First, 50 g powdered CD or HFD was weighed and well mixed with 55 mL double-distilled H2O. Next, 15 mL ethanol containing 0.2 g RSV (for 0.4% in the diet) was thoroughly mixed with the diet, and the pellets were then reconstituted. The same volume of ethanol was added to the control diet. For ethanol evaporation, the diet was placed in a vacuum oven at 50 °C overnight. All the diets were irradiated by gamma irradiation and stored in light-protected and airtight containers at 4 °C.

The rats were randomly divided into the following six groups (n=10 per group): young rats fed the CD (Y), old rats fed the CD (O), old rats fed the HFD (HO), old rats fed the HFD for 10 weeks and then the HFD supplemented with RSV for 10 weeks (HO+R10), old rats fed the HFD supplemented with RSV starting from the time of study initiation and throughout the experimental period (R20+HO), and old rats fed the CD supplemented with RSV starting from the time of study initiation and throughout the experimental period (R20+O). Body weight was measured weekly, and food intake was recorded every 2–3 days. Grip strength was determined using a grip strength meter (YuYan Instruments, Shanghai, China).

At the end of the 20-week study period, the rats were fasted overnight and then anesthetized with sodium pentobarbital anesthesia. All efforts were made to alleviate suffering. Fresh blood samples, muscle tissues and adipose tissues were harvested for further analyses.

Serum parameter analysis

Serum was separated by solidification and centrifugation (4 °C, 3,000×g, 10 min). The levels of FFA, TG, TC, HDL-C, and LDL-C in serum were measured using an automatic analyzer (Olympus AU5400, Japan). The concentrations of RSV in serum were measured by liquid chromatography-mass spectrometry according to our previous studies [84].

Histological analysis

Fresh muscle tissues were fixed in 4% paraformaldehyde, embedded in paraffin wax, transversely sectioned into 5-μm slices, and stained with hematoxylin and eosin (H&E) following standard protocols. Photomicrographs were captured using an Olympus VS120 microscope. The myofiber CSA was measured using ImageJ software (NIH) by a researcher blinded to the experimental groups.

Body fat measurement

The body fat volume was analyzed using a Quantum FX MicroCT Imaging System (Perkin Elmer Inc., Waltham, MA, USA). The rats were anesthetized with 5% chloral hydrate (4 ml/kg body weight) and then subjected to the microCT scans. The images were acquired from the abdominal region (between vertebrae L1 and L6) of the rats. To vividly observe adipose tissues, two-dimensional grayscale image slices were reconstructed into three-dimensional images. The fat volume was quantitatively analyzed using Analyze 12.0 software (Analyze Direct, Overland Park, KS, USA) in a blinded manner.

Cell culture and treatments

The rat L6 myogenic cell line (obtained from ATCC-CRL-1458, Manassas, VA, USA) was grown in Dulbecco’s-modified Eagle’s medium (DMEM; Gibco-Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; HyClone, USA) and 1% penicillin/streptomycin in a humidified incubator enriched with 5% CO₂ at 37 °C. The medium was then replaced with DMEM containing 2% horse serum (Gibco, New Zealand) for the induction of myotube formation. After differentiation, the cells were treated with PA in the presence or absence of RSV for 24 h.

Cell viability measurement

Cell viability was analyzed through the CCK-8 assay according to the manufacturer’s instructions. Briefly, the cells were seeded into 96-well microplates at a density of 8,000 cells/well and differentiated into myotubes. After exposure to the indicated treatments, CCK-8 solution (20 μL/well) was added to each well, and the microplate was incubated at 37 °C for 2 h. Cell viability was determined through absorbance measurements using an Infinite™ M200 Microplate Reader (Tecan, Mannedorf, Switzerland) at 450 nm. The results are expressed as percentages relative to the control group values.

siRNA transfection

siRNAs targeting PKA, LKB1, AMPK, TFAM, mfn2, and drp1, as well as control siRNA, were obtained from Santa Cruz Biotechnology. Myotubes were transfected with 100 nM siRNA for 6 h according to the manufacturer’s directions. The cells were then transferred to fresh medium and incubated for an additional 24 h. Where indicated, the cells were treated with RSV and PA for 24 h. Thereafter, cells were harvested for subsequent experiments.

Measurement of the myotube diameter

To analyze the myotube diameter, images were captured under a phase contrast microscope at 100× magnification. The diameters of a total of 60 myotubes from at least 10 random fields were measured using ImageJ software (NIH) by a researcher blinded to the group designations. The results are expressed as percentages relative to the control group value.

Measurement of the TG content and ATP levels

The TG levels of the GAS muscles and myotubes were determined using a TG assay kit (Nanjing Jiancheng Biotechnology, China) following the manufacturer’s protocol. The protein concentrations were measured with a BCA Protein Assay Kit (Beyotime, China). The TG content is expressed as mmol/g protein. The ATP levels in the GAS muscles were measured using an ATP Assay Kit (Beyotime, China) according to the manufacturer’s instructions.

Mitochondrial morphology analysis

The changes in the mitochondrial morphology of the GAS muscles were observed by TEM. Immediately after the rats were sacrificed, the GAS muscles were dissected, trimmed to approximately 1.0-mm³ pieces, fixed in 2.5% glutaraldehyde solution and subsequently in 1% osmium tetroxide, dehydrated, and then embedded in epoxy resin. The tissues were then cut using an RMC/MTX ultramicrotome (Elexience). Ultrathin sections (60 nm) stained with 2% uranyl acetate and lead citrate were observed under a JEM-1400 microscope (JEOL, Japan) at 25,000× magnification.

For visualization of the mitochondrial network in L6 myotubes, the cells were cultured in a cell culture dish, subjected to the indicated treatment, loaded with 250 nM MitoTracker Deep Red probe and incubated for 30 min at 37 °C. The cells were then gently washed with prewarmed PBS and observed under a Zeiss confocal laser scanning microscope (Carl Zeiss, LSM 800). The mitochondrial fluorescence intensity was quantified using Zeiss LSM Image Examiner software. The results were obtained from at least 30 cells in each group from three experiments.

Determination of the Δψm

The Δψm in the GAS muscles and myotubes was measured using a JC-1 probe (Beyotime, China). For muscles, the freshly isolated mitochondria were suspended in JC-1 staining solution, and the fluorescence intensity was detected using an Infinite™ M200 Microplate Reader. For myotubes, the cells were grown in a cell culture dish, subjected to the indicated treatment, incubated with JC-1 staining solution for 20 min, gently washed with PBS and resuspended in serum-free medium. Images were acquired using a Zeiss confocal laser scanning microscope, and further analysis of the fluorescence intensity was performed using Zeiss LSM Image Examiner software. The Δψm is represented by the ratio of red to green fluorescence.

mtDNA quantification

mtDNA was isolated from GAS muscles and myotubes using a Mito DNA Extraction Kit (Genmed Scientifics, Inc., USA) following the manufacturer’s protocol. DNA was quantified spectrophotometrically (260 nm) and subjected to quantitative real-time PCR using a qTower 2.2 real-time PCR system (Analytik Jena, German). The relative mtDNA content was obtained by comparing the amplification products to those of ACTB (forward: 5'- CCACCATGTACCCAGGCATT-3'; reverse: 5'-CGGACTCATCGTACTCCTGC-3') and NADH-CoQ oxidoreductase 1 (forward: 5'-TTAATTGCCATGGC CTTCCTCACC-3'; reverse: 5'-TGGTTAGAGGGCG TATGGGTTCTT-3').

Measurement of the cell mitochondrial OCR

L6 myoblasts were seeded into XF Cell Culture Microplates and differentiated into myotubes. After the indicated treatment, the medium was replaced with unbuffered XF Base Medium containing 1 mM pyruvate, 2 mM glutamine and 10 mM glucose. The microplates were then placed into a non-CO₂ incubator for 1 h at 37 °C. Thereafter, oxygen consumption was measured using an Extracellular Flux Analyzer (Seahorse Bioscience, MA, USA) as previously described. Every point represents average data from five different wells, and the parameter was analyzed using Wave and Report Generator software.

Mitochondrial complex activity assay

Mitochondria were isolated from GAS muscles or myotubes using Mitochondria Isolation Kits (Beyotime, China) according to the manufacturer’s instructions. The activities of complexes I, II, and IV were determined in freshly isolated mitochondrial homogenates using commercial assay kits from Abcam (Cambridge, MA, USA) following the manufacturer’s protocol.

Antioxidant activity assay

The antioxidant capacity in the GAS muscles and myotubes was determined by measuring the T-AOC and activity of SOD and GPx using corresponding assay kits (Beyotime, China) according to the manufacturer’s instructions.

Assessment of the ROS levels and mitochondrial superoxide (O₂^•−) levels in L6 myotubes

The total ROS levels in myotubes were measured using an ROS Assay Kit (Beyotime, China) following the manufacturer’s protocol. The mtROS levels were determined using MitoSOX Red, a highly selective fluorescent probe for the detection of mitochondrial O2•−. Briefly, the cells were seeded into a 24-well microplate. After differentiation and treatment, the myotubes were incubated with 5 μM MitoSOX Red reagent working solution in the dark for 10 min at 37 °C. After gentle washing, the fluorescence intensity was analyzed using an Infinite™ M200 Microplate Reader.

Measurement of the MDA levels and carbonyl protein content

The MDA content was determined as a marker of lipid peroxidation in the GAS muscles and myotubes using an MDA Detection Kit (Beyotime, China) following the manufacturer’s instructions. The carbonyl protein level was measured as an indicator of protein oxidative damage in the GAS muscles and myotubes using a Protein Carbonyl Content Assay Kit according to the manufacturer’s protocol. The MDA levels and carbonyl protein content are expressed as nmol/mg protein.

Western blotting

Total proteins from the GAS muscles were extracted using a Tissue Protein Extraction Solution (Invitrogen, USA) containing a protease inhibitor (Roche Applied Science, Germany) and a PhosSTOP Phosphatase inhibitor cocktail tablet (Roche Applied Science, Germany). Total cell lysates from L6 myotubes were obtained by homogenizing the cells in RIPA lysis buffer (Beyotime, China) supplemented with phenylmethanesulfonyl fluoride (PMSF; Beyotime, China) and a PhosSTOP phosphatase inhibitor cocktail tablet. For the preparation of mitochondrial proteins, the mitochondria isolated from the muscle tissues and myotubes were homogenized in mitochondrial lysis buffer containing PMSF. All the lysates were centrifuged at 14,000×g and 4 °C for 20 min. The supernatants were collected, and the protein concentrations were determined with a BCA Protein Assay Kit. The protein samples were then applied to SDS-PAGE gels, electrotransferred to PVDF membranes (Bio-Rad), blocked with 5% nonfat milk or 5% BSA in TBST for 2 h, incubated with primary antibodies at 4 °C overnight and then incubated with anti-rabbit or anti-mouse secondary antibodies for 1 h. The blots were visualized using an enhanced chemiluminescence system (Millipore, Billerica, MA, USA), and the densitometric analysis was performed using Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).

Statistical analysis

The data analyses were performed using SPSS 19.0 software (Chicago, IL, USA). The quantitative data are presented as the mean ± standard deviations (SD). The statistical analyses were conducted using one-way analysis of variance (ANOVA) followed by Tukey–Kramer post hoc tests for multiple comparisons. The results were considered to be statistically significant if P < 0.05. Each experiment was performed a minimum of three times.