Resveratrol Induces Myotube Development by Altering Circadian Metabolism via the SIRT1-AMPK-PP2A Axis

The mammalian circadian clock is located in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus. Light perceived by the retina synchronizes SCN neurons that relay the information to peripheral tissues via neural and humoral signals [1]. The core clock consists of CLOCK (circadian locomotor output cycles protein kaput) and BMAL1 (brain and muscle Arnt-like protein-1) that activate transcription of many tissue-specific genes by binding to enhancer elements. PERIODs (PERs) and CRYPTOCHROMEs (CRYs) form the negative feedback loop that inhibits CLOCK:BMAL1-mediated transcription [2]. The synchronized expression of circadian clock genes orchestrates the expression of hormones, enzymes, nuclear receptors and transporters involved in metabolism [3]. Disturbances in circadian rhythms have been shown to disrupt metabolism [4,5].

Resveratrol is a polyphenolic compound found in grape skin, peanuts and berries [6,7]. It has been shown to have diverse benefits including anti-inflammatory, anti-oxidant, anti-diabetic and anti-carcinogenic powers [6,7]. Circadian rhythms have been shown to be affected by resveratrol [8,9]. This is achieved by activating Sirtuin 1 (SIRT1) [10,11], an important metabolic factor that alters circadian rhythms by deacetylating histones and activating the CLOCK:BMAL1-mediated transcription [12].

SIRT1 activation is usually accompanied by AMP-activated protein kinase (AMPK) activation [13]. This activation leads to the inhibition of the mTOR signaling pathway [14]. Indeed, some studies report that resveratrol leads in addition to SIRT1 activation and also to AMPK activation [15]. In addition, resveratrol has been shown to activate protein phosphatase 2A (PP2A) [8,16]. However, PP2A has been reported to dephosphorylate AMPK, leading to inactivation of the latter [17,18]. As the activation of AMPK, SIRT1 and PP2A are influenced by the cellular metabolic state, we studied the effect of resveratrol in the presence of low, normal and high glucose concentrations. These analyses were performed for 24 h to elucidate the signaling pathways involved in myotube development.

In this study, we investigated the circadian metabolic effect of resveratrol on C2C12 myotubes under increasing concentrations of glucose. We show that myotubes treated with increasing concentrations of glucose have increased mTOR and reduced AMPK signaling. Resveratrol treatment in combination with increasing concentrations of glucose affected these signaling pathways, by activating the SIRT1-AMPK-PP2A axis and inhibiting the mTOR pathway. In addition, resveratrol led to a phase advance in circadian rhythms and increased myogenin expression.

Activation of the mTOR signaling pathway and inhibition of AMPK signaling under increasing concentrations of glucose is expected as high energy levels lead to the induction of anabolic pathways and suppressed catabolic pathways [20]. The reduced activation of AMPK under increasing glucose concentrations was mirrored by increased activation of its target ACC, the rate-limiting enzyme in fatty acid synthesis. ACC is expected to be activated under increasing glucose concentrations as it generates malonyl CoA for the synthesis of fatty acids under conditions of ample energy [21]. However, resveratrol modified the cellular metabolic state. Resveratrol has been reported to activate SIRT1 [10]. SIRT1 activation is usually accompanied by AMPK activation [13]. Indeed, studies report that resveratrol leads to AMPK activation by phosphorylation [15]. PP2A has been reported to dephosphorylate AMPK leading to inactivation of the latter [17,18]. However, herein, we show that resveratrol treatment resulted in reduced levels of phosphorylated Tyr307 on PP2A, the inhibitory site, thereby increasing the activity of PP2A. This effect of resveratrol on PP2A has also been documented in several recent publications [16,22,23]. However, these studies did not measure AMPK activation in parallel. In studies in which both AMPK and PP2A were measured, the results depended on the cell type. In hepatocytes, resveratrol treatment led to PP2A activation and AMPK inhibition [24], which emphasizes the reported role of PP2A in the inactivation of AMPK [25]. We recently also reported that resveratrol shifts hepatocytes to the fasting state by increasing the activity of PP2A, reducing AMPK activity and inducing the expression of the key gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) [8]. In contrast, but similarly to our findings herein, in a rat brain, it was found that both AMPK and PP2A were activated by resveratrol leading to Tau dephosphorylation [26]. In addition, numerous studies have shown that PP2A activity can be increased by AMPK-mediated phosphorylation [27,28]. Thus, in the brain and muscle cells, resveratrol leads to both activation of PP2A and AMPK, which emphasizes the fact that AMPK may not be the main target of PP2A, but rather vice versa, in these cell types.

Resveratrol did not affect mTOR at any glucose concentration and led to AKT activation only at very high glucose concentrations. These findings are consistent with previous publications that showed that, upon PP2A activation, AKT was inhibited [29,30]. Interestingly, resveratrol led to P70S6K and S6 activation even at physiological glucose concentrations. These findings are supported in the literature, as it has been shown that P70S6K and S6 can be activated in an mTOR-independent manner in C2C12 myotubes [31,32,33,34]. Activation of P70S6K has been shown to mediate muscle differentiation and hypertrophy [35]. Indeed, our results show that resveratrol increases myogenin expression compared to glucose treatment alone. Although a change in morphology could not be detected, presumably because the effect is for the long run, it was shown that myogenin is crucial for myotube differentiation and development [36]. More specifically, the absence of myogenin has been shown to lead to loss of skeletal muscles, despite the presence of normal committed myoblasts [37]. These findings imply that there is no compensation for myogenin absence, and it has an indispensable role in terminal differentiation of myoblasts [38].

Resveratrol treatment led to advanced circadian rhythms and reduced levels of pBMAL1 and CRY1. These changes are congruent with other reports that show that altered AMPK and mTOR activity leads to modified circadian rhythms [39,40]. It has been shown that activation of the mTOR pathway leads to a rhythmic phosphorylation of BMAL1, allowing it to associate with the translational machinery as well as stimulate circadian oscillations of protein synthesis [19]. Conversely, as a transcription factor and a core component of the circadian clock, BMAL1 has the capacity to function as a negative regulator of mTORC1 signaling [39]. The diminished mTOR signaling pathway, and consequently the reduced levels of phosphorylated BMAL1 observed in this study, align with previous reports, indicating that BMAL1 remains a transcriptional activator of the core clock mechanism, leading to phase advances rather than functioning as a translational factor. These findings are consistent with our earlier observations in hepatocytes [8]. In addition, SIRT1 activation has been shown to deacetylate histones and activate the CLOCK:BMAL1-mediated transcription [12]. In addition, the levels of CRY1, a member of the core clock negative feedback loop, were reduced, most probably as a result of the activation of AMPK. AMPK has been shown to phosphorylate and, as a result, lead to the degradation of CRY1 [41]. Thus, both the reduced levels of pBMAL1 and CRY1 may promote the activity of the positive loop of the clock, leading to advanced rhythms.

Studies have shown that the expression of myogenin is under circadian control [42,43,44,45]. Myogenin expression peaks at specific times of the day, which align with periods of increased muscle repair and regeneration activity [42,45]. Our results suggest that resveratrol led to increased clock functionality and phase advances. As the clock controls myogenin expression and myogenin is required for muscle development, we surmise that resveratrol leads to muscle development via clock induction and, as a result, myogenin induced expression (Figure 5).

Herein we show that resveratrol leads to the activation of the SIRT1-AMPK-PP2A signaling pathway (Figure 5). AMPK activation decreases the activity of mTOR phosphorylation, which, in turn, leads to reduced pBMAL1, allowing BMAL1 to work as a transcription factor, rather than a translation factor. In addition, AMPK activation leads to CRY1 degradation, which relieves the inhibition from the CLOCK:BMAL1-mediated expression. BMAL1 activity, as a transcription factor, as well as reduced levels of negative feedback loop member CRY1, lead to phase advances in circadian expression (Figure 5). Activation of P70S6K, independent of the mTOR signaling pathway, leads to myogenin expression and muscle development. The mechanism by which resveratrol affects metabolic and circadian rhythms in different cell types merits further investigation.

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cells13121069/s1↗, Table S1: RT primers.

Conceptualization, N.A.-C., N.C. and O.F.; methodology, N.A.-C. and N.C.; validation, N.A.-C., N.C. and O.F.; formal analysis, N.A.-C. and N.C.; investigation, N.A.-C., N.C. and O.F.; resources, O.F.; data curation, N.A.-C. and N.C.; writing—original draft preparation, N.A.-C., N.C. and O.F.; writing—review and editing, N.A.-C., N.C. and O.F.; supervision, O.F. All authors have read and agreed to the published version of the manuscript.

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

The authors declare no conflicts of interest.

This research received no external funding.