In this investigation, the potential of garcinol to stimulate the slow oxidation process of skeletal muscle was evaluated. We found that the expression of slow MyHC and PGC-1α activity could be upregulated by garcinol treatment in vitro and in vivo. Mechanistically, we proved that the expression of slow MyHC was upregulated by garcinol through the p300/PGC-1α pathway.
In the 1980s, garcinol was first extracted from Garcinia indica (G. indica, also known as kokum) of the Western Ghats in India . Many studies have indicated the anti-obesity, anti-inflammatory, antioxidant, and antiglycation effects of garcinol [27, 28]. Similar to findings in previous studies, the body weight and food intake were not affected by treatment with 500 ppm garcinol for 12 weeks in the current study. Generally, slow muscles have higher endurance and greater resistance to fatigue than fast muscles. Our data showed that dietary garcinol supplementation improved the endurance exercise performance and attenuated skeletal muscle fatigability, accompanied by upregulated slow MyHC expression. Moreover, the quantity of fast MyHC-positive cells was decreased, while the quantity of slow MyHC-positive cells was increased, as indicated by the immunofluorescence assay results. Therefore, the results show that the transformation of muscle fibers from the fast-twitch type to the slow-twitch type was accelerated by garcinol. The type of skeletal muscle fiber can be classified on the basis of the characteristics of energy metabolism, and the transformation of skeletal muscle fiber types can be indirectly affected by enzyme activity in energy metabolism [21, 29]. For example, glycolytic enzyme (LDH) has a higher activity in glycolytic fibers, while oxidases (MDH and SDH) have higher activity in oxidized fibers. Our results indicate that the activity of LDH was decreased and the activities of MDH and SDH were increased in mice, which support the supposition that the transformation of muscle fiber types is influenced by garcinol.
According to previous research, the transformation of muscle fiber types and the energy metabolism of muscle tissues are related to the PGC-1α signaling pathway [30, 31]. Several previous studies on transgenic mice concluded that the conversion of skeletal muscle fibers from the fast MyHC type to the slow MyHC type is caused by specific overexpression of PGC-1α . Additional studies found that PGC-1α transgenic animals have a higher proportion of slow MyHC muscle fibers, and the transformation of muscle fiber types to slow MyHC was promoted . In addition, the mRNA expression of glycolytic fast muscle-related intermediates MyHC-IIx and MHC-IIb was decreased and the mRNA expression of oxidized slow muscle-associated MyHC-Ⅰ was increased by the overexpression of PGC-1α . Our results showed that the expression and activity of p300 in the TA and soleus muscles were decreased by garcinol supplementation. Moreover, the muscle fiber type results indicated that the transcription level of PGC-1α was upregulated by garcinol in vivo and in vitro, which may have modulated slow MyHC-type fiber transcriptional activity. Thus, we hypothesized that garcinol can regulate the PGC-1α signaling pathway to regulate skeletal muscle fiber transformation.
To confirm whether garcinol induced changes in molecules were related to increased slow MyHC muscle fibers, we assessed PGC-1α activity in C2C12 myotubes. Garcinol can inhibit acetylase, which seemingly regulates myofiber transformation. According to previous research, garcinol could induce apoptosis cell lines of pancreatic cancer, prostate cancer and leukemia, and it can quickly inactivate p300 by inhibiting the NFκB-DNA interaction [35, 36].
In the present study, garcinol increased PGC-1α activity and increased slow-twitch muscle fibers in mice and C2C12 myotubes, which was demonstrated by the reduction in PGC-1α acetylation induced by garcinol treatment, so we propose that the p300-PGC-1α pathway may participate in the improvement of myofiber transformation in mice fed a diet supplemented with garcinol. Specifically, p300 could increase PGC-1α acetylation when p300 was overexpressed, which was observed to be inactivated after garcinol intervention in vitro. Overexpression of siRNAs inhibits p300 activity in cultured C2C12 myotubes, indicating that nongarcinol-mediated inhibition of p300 has similar effects. In addition, the expression of fast muscle fibers and biomarkers increased under PGC-1α knockdown conditions. However, the treatment of garcinol has no effect on weakening the expression of fast-twitch muscle fibers in C2C12 myotubes with knockdown PGC-1α. Therefore, we found that the change from fast-twitch to slow-twitch skeletal muscle fibers was related to higher PGC-1α activity, which fit the garcinol working model. Our results revealed that garcinol may increase slow-twitch muscle fibers via inhibiting p300-induced PGC-1α acetylation in mice and C2C12 myotubes. All of these results show that p300 is related to an increase in slow muscle fibers, but the precise regulatory mechanism needs to be studied further.