PART2 A Cistanches Herba Fraction/𝛽-Sitosterol Causes A Redox-Sensitive Induction Of Mitochondrial Uncoupling And Activation Of Adenosine Monophosphate-Dependent Protein Kinase/Peroxisome Proliferator-Activated Receptor 𝛾 Coactivator-1 in C2C12 Myotubes: A Possible Mechanism Underlying The Weight Reduction Effect
Mar 05, 2022
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4. Discussion
HCF1, a semipurified fraction of Cistanches Herba, was shown to produce weight reduction in HFD-induced obese
mice. The weight reduction was associated with the induction of mitochondrial uncoupling and changes in energy metabolic enzyme activities in mouse skeletal muscle [4]. HCF1 and its active component, BSS, have been shown to decrease mitochondrial coupling efficiency secondary to the production of mitochondrial reactive oxygen species (ROS) in H9c2 cardiomyocytes and rat hearts [17]. In the present study, to elucidate the biochemical mechanism underlying the HCF1-induced mitochondrial uncoupling in mouse skeletal muscle, the effects of HCF1 and BSS on MMP in C2C12 myotubes were investigated. MMP is generated in the IMM as a consequence of proton pumping by the mitochondrial electron transport chain. Changes in MMP reflect an interplay of mitochondrial electron transport and the dissipation of the proton gradient by various processes, such as ATP synthase-mediated ATP Cistanche
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generation, proton leak via IMM, or an UCP-mediated mitochondrial uncoupling [18, 19]. HCF1/BSS incubation caused a transient elevation in MMP in C2C12 myotubes. The ability of rotenone, a mitochondrial respiratory complex I inhibitor, to suppress the HCF1/BSS-induced elevation in MMP suggests that this process was likely mediated by an increase in mitochondrial electron transport. This is consistent with our previous finding that HCF1/BSS increased mitochondrial state 3 respiration in H9c2 cardiomyocytes and rat hearts [17]. In addition, Shi et al. (2013) [20] reported that the incorporation of BSS into mitochondrial membranes specifically fluidizes the IMM, with consequent increases in mitochondrial
membrane potential and mitochondrial ATP content. Our finding that the co-incubation with cholesterol, and Cistanche enhancer of membrane rigidity [21], abolished the HCF1/BSS-induced increase in MMP suggests the involvement of an increase in mitochondrial membrane fluidity in the HCF1/BSS-induced increase in MMP. The ability of rotenone and GDP to abrogate the HCF1/BSS-induced decrease in MMP observable at the later period of HCF1/BSS incubation suggests the induction of Cistanche an UCP3-mediated mitochondrial uncoupling secondary to the stimulation in mitochondrial electron transport. DMTU, an antioxidant, prevented HCF1/BSS-induced mitochondrial uncoupling, suggesting a redox-sensitive activation of UCP3 in C2C12 myotubes, which is likely mediated by the glutaredoxin 2 (GRx2) induced S-deglutathionylation of UCP3. GRx2 is a matrix oxidoreductase, which modulates UCPs activities via reversible S-glutathionylation in response to the changes in the cellular redox environment [18, 22, 23].
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Taken together, our findings suggest that HCF1/BSS may fluidize the IMM, resulting in the stimulation of mitochondrial electron transport, with a resultant increase in mitochondrial ROS production. The elevated mitochondrial ROS, in turn, would trigger a redox-sensitive activation of mitochondrial uncoupling in C2C12 myotubes. In view of the HCF1-induced changes in energy metabolic enzyme activities in the skeletal muscle of HFD-fed mice, the effects of HCF1/BSS on the AMPK/PGC-1 signaling pathway, which participates actively in the control of cellular energy homeostasis [7, 8], was examined. The finding that HCF1/BSS induced a DMTU-sensitive activation of AMPK indicated the ability of HCF1/BSS to trigger a redox-sensitive AMPK activation in C2C12 myotubes. Recently, AMPK has been reported to be activated by ROS as well as reactive nitrogen species (RNS), with a resulting enhancement of cell Cistanche viability under conditions of oxidative stress. The exposure of AMPK to oxidative stress leads to the oxidation of cysteine residues of the 𝛼- and 𝛽-subunit, with resultant allosteric rearrangement of the heterotrimeric complex and thereby the activation of kinase activity [24, 25]. In addition, the S-glutathionylation of AMPK, as catalyzed by glutathione transferase (GST) and GRx, following H2O2 exposure was found to stimulate its kinase activity [26]. Taken together, our findings support the notion that HCF1/BSS can activate AMPK by way of mitochondrial ROS production. The HCF1/BSS-induced activation of AMPK was also found to be associated with an increased nuclear translocation of PGC-1. In this regard, AMPK can posttranslationally activate PGC-1 by phosphorylation of the Thr177/Ser538 of PGC-1 and thereby facilitate the deacetylation of PGC-1 by sirtuin 1 (SIRT1), which is a critical step for PGC-1𝛼 activation [7]. The activated PGC-1𝛼, in turn, interacts with nuclear receptors such as peroxisome proliferator-activated receptor 𝛾 (PPAR𝛾), nuclear respiratory factors (NRFs), and myocyte enhancer factor 2C (MEF2C), with resultant nuclear translocation and coactivation of gene expression [27–29]. The inhibition of HCF1/BSS-induced PGC-1 nuclear translocation by DMTU and the AMPK inhibitor, CC, indicates that the PGC- 1 activation is an event secondary to ROS-induced AMPK activation. By coactivating multiple transcription factors, AMPK/ PGC-1 exerts a wide spectrum of actions in controlling evidence-Based Complimentary and Alternative Medicine 11 gene expression related to muscle fiber type switching, uptake and utilization of fuel molecules, and mitochondrial metabolism, all of which provide an orchestrated network to regulate cellular energy metabolism and thereby improve overall metabolic fitness [30–32]. In this connection, our findings suggest the possible involvement of AMPK/PGC- 1 in the beneficial effect produced by HCF1 on glucose and fatty acid metabolism in both ND-fed and HFD-fed mice [4]. HCF1/BSS incubation also increased UCP3 expression and stimulated mitochondrial biogenesis via the redox-sensitive activation of AMPK/PGC-1 in C2C12 myotubes. The increased expression of UCP3 in response to HCF1/BSS, which was also observed in mouse skeletal muscle of ND- and HFD-fed mice, suggests the involvement of the AMPK/PGC-1 pathway in the upregulation of UCP3 expression in mouse skeletal muscle. Furthermore, the HCF1/BSS-induced increase in mitochondrial biogenesis suggests an enhanced mitochondrial oxidative capacity with a resultant improvement in metabolic fitness. Given that the redox-sensitive activation of the AMPK/PGC-1 signaling pathway is crucial in defending against oxidant injury in various tissues [24, 33, 34], HCF1/BSS may offer an alternative approach to ablating obesity-related oxidative stress. The exploration of pharmacological interventions aimed at ameliorating obesity has been an area of intensive research. Currently, most of the slimming agents are mainly food substitutes, appetite suppressants, and functional compounds that stimulate the sympathetic nervous system (SNS) [35]. The finding that HCF1 produced weight reduction in HFDinduced obesity via the redox-sensitive induction of mitochondrial uncoupling offers an alternative approach to weight control. Mitochondrial uncoupling has long been proven to be effective in inducing weight loss in animals and humans [36]. The findings from our previous and present studies unequivocally attest to the ability of HCF1 to produce weight reduction via the induction of mitochondrial uncoupling.
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5. Conclusion
A Cistanches Herba fraction (HCF1)/BSS causes the redox-sensitive induction of mitochondrial uncoupling and activation of AMPK/PGC-1 in C2C12 myotubes. HCF1 treatment may increase bodily energy consumption, particularly in skeletal muscle, with resultant reductions in body weight and adiposity in HFD-fed mice.
Conflict of Interests
The authors declare that there is no conflict of interest regarding the publication of this paper.
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