Timekeepers of metabolism: how PER2 and RORα flex their muscles in glucose control and metabolism

Circadian rhythms (CRs) define the intrinsic oscillations of mammalian gene and protein expression, driven by the activity of a collection of transcription factors on a daily basis. The rhythmic expression of downstream proteins is mediated through the activity of both a primary loop (comprising the proteins BMAL1, CLOCK and the families of inhibitory proteins PER1–3 and CRY1–2) and a secondary loop (comprising the families of proteins NR1D1 and RORα) in both the brain and peripheral tissues. While light is the primary entrainment factor of CRs, other behaviourally mediated factors such as diet and physical activity help maintain rhythmic regularity. Disruption of these factors can become a cause for concern due to inherent association with specific disease states in a tissue-specific manner. Desynchronization of skeletal muscle CRs results in impaired glucose homeostasis and skeletal muscle regeneration; however, our understanding of these mechanisms is limited and warrants further investigation. To add to this growing body of knowledge, Mansingh et al. (2024) performed multi-omic profiling on PER2 and RORα muscle-specific knockout (MKO) mice at multiple time points. Both models underwent a bout of acute exercise consisting of uphill running. By utilizing models which dysregulated either the positive or negative limbs of the clock, this group was able to elucidate CR limb-specific alterations including phenotype, overall muscle gene expression, amino acid metabolism and glucose homeostasis. The authors provide commentary on the findings of their study and the aim of this Journal Club article is to highlight (1) the alignment between the data for glucose tolerance and other multi-omic work provided by Mansingh et al. (2024) with other work in the extant literature and (2) alternative factors which may contribute to the reduced glucose tolerance observed in these mouse models. The study by Mansingh et al. (2024) provided critical insights into circadian rhythms and glucose metabolism. Mansingh et al. utilized PER2 and RORα MKO mice, which have reduced amplitude of core clock genes without inducing a phase shift. This effective experimental design allowed the authors to look at specific perturbations of the molecular clock's primary and secondary feedback loops. Notably, this knockout model revealed a time-of-day-related shift in glucose metabolism, ultimately linked to RORα depletion. Specifically, these mice showed altered glucose tolerance (i.e. decreased tolerance during the day vs. at night) (Mansingh et al., 2024). Consistent with the findings of Mansingh et al. (2024), a recently published meta-analysis − with a robust sample size (n = 2760) – also demonstrated the link between circadian disruptions and impaired glucose homeostasis in non-diabetic human adults (Xu et al., 2022). Although nutritional intake is a primary entrainment factor of circadian rhythms, the exact mechanisms by which the core clock regulates these processes are yet to be fully understood. In that regard, the work done by Mansingh et al. (2024), provides a mechanistic basis and highlights the importance of specific pathways through which circadian oscillations influence glucose tolerance (Mansingh et al., 2024; Xu et al., 2022). The broader implications of these findings highlight the significance of circadian rhythms in glucose regulation. Greater incidence of metabolic syndrome in populations with chronic circadian disruption (e.g. shift workers) is well-established. Therefore, broadening our understanding of direct circadian influence on glucose homeostasis will aid in the potential for translating these findings to clinical intervention for patients potentially at risk of metabolic syndrome. An interesting finding of this study was that both PER2 and RORα MKO mice have elevated blood glucose, indicating reduced glucose tolerance. A potential reason for the elevations in blood glucose may be the effects of CR disruption on ceramides. Ceramides are a collection of sphingolipids which are found in all tissues, including skeletal muscle. Ceramide accumulation in skeletal muscle contributes to the development of insulin resistance through repression of insulin-mediated Glut4 translocation. The findings of this study are in contrast with other studies that utilized a global PER1/PER2 model to determine negative CR limb influence on ceramide content and ceramide-producing enzyme transcript levels. Jang et al. (2012) reported that in PER1/PER2 KO mice, there is a reduced rhythmic expression of transcripts for ceramide synthase 2 (CerS2) and neutral sphingomyelinase (nSMase) and reduced ceramide content in liver as compared to wild-type. Although glucose tolerance was not examined and a whole-body knockout model was employed, this demonstrates the influence of negative CR limb proteins on transcript levels of CerS2 and nSMase and ceramide content (Jang et al., 2012; Mansingh et al., 2024). Moreover, examination of the transcriptomic data from Mansingh et al. (2024) did not show differential expression in CerS2 and nSMase mRNA in their PER2 KO mice. These discrepancies between muscle-specific and global knockout models inform that future work should consider examining ceramide content when examining glucose homeostasis in muscle. Understanding the role of CRs in energy balance and exercise is crucial for improving the health of metabolically compromised individuals as CRs are innately tied to metabolism. The secondary loop of the core clock, including the nuclear receptor RORα, has significant influence on glucose metabolism, lipid oxidation and exercise performance. Using a muscle-specific ablation of RORα has allowed the authors to focus on the skeletal muscle metabolome, providing the field with a step forward in the understanding of the rhythmic underpinnings of skeletal muscle metabolism. While previous research mentioned by Mansingh et al. (2024) describes the deleterious consequences of BMAL1 knockouts in skeletal muscle, their work regarding the influence of RORα in the secondary loop presents an interesting case for its purpose. As commented on in the article, the KO model of both genes displays metabolic impairment, likely due in part to the nature of RORα promoting transcription of BMAL1 in a wild-type model. However, the RORα MKO mice saw no morphological deficits in the skeletal muscle, yet exhibited an increase in exercise capacity and energy expenditure around the clock. These findings are consistent with their omics profiling displaying broadly upregulated metabolic pathways. This work showed mildly contrasting results in comparison to a 2017 study examining the metabolic effects of a systemic RORα knockdown (Billon et al., 2017). Particularly, these mice had improved glucose tolerance in comparison to their wild-type counterparts. While the purpose and parameters of each study differed significantly, of note was the difference in relative liver expression of RORα mRNA. The muscle-specific knockout in use by Mansingh et al. displayed a significant increase in liver RORα expression, whereas the systemic knockout model used by Billon et al. (2017) demonstrated the opposite. Given the systemic influence of the liver on substrate production due to the tricarboxylic acid cycle, it may be worthwhile to explore further influence of the liver on metabolism in respect to a skeletal muscle specific RORα KO. The authors employed an innovative omics approach that combined transcriptomic, proteomic and metabolomic profiling to dissect the molecular intricacies of muscle function and metabolism in relation to circadian rhythm perturbations. This integrative strategy allowed for a comprehensive understanding of the multifaceted impact of PER2 and RORα knockouts on skeletal muscle. By correlating changes at the gene expression level with alterations in protein and metabolite profiles, the study delineated the cascading effects of circadian disruptions on muscle physiology. This approach not only identified key pathways affected by gene ablations but also elucidated their systemic repercussions, enhancing our grasp of the circadian modulation of muscle plasticity and its potential implications for metabolic health. The knowledge from the present study coupled with previous studies that have shown the rhythmic control of mitochondrial morphology and function provides valuable insight on the intricate relationship between circadian biology and mitochondrial dynamics in skeletal muscle (Mansingh et al., 2024; Sardon Puig et al., 2018). Utilizing this multi-omics approach, it would be interesting to see if changes in the circadian clock could sensitize or desensitize the mitochondria within skeletal muscle to respond to various stressors. This may have implications for understanding the broader impact of circadian rhythms on muscle resilience and recovery, potentially guiding therapeutic strategies for muscle disorders and enhancing athletic performance by optimizing recovery protocols based on circadian biology. The CR governs the rhythmic expression of genes and proteins in both the central and peripheral tissues, including skeletal muscle. CR disruption is a common phenomenon, where disruptions to either the central or the peripheral clocks are associated with development of disease. Mansingh et al. (2024) uncovered the influence of positive or negative limb CR disruption through the use of PER2 and RORα KO mice. Utilizing a multi-omics approach, it was determined that model-specific alterations in metabolism and energy homeostasis were uncovered. This Journal Club article has contextualized these findings in the fields of skeletal muscle and CR research. The findings in this paper warrant further exploration, with areas such as glucose regulation and sphingolipid metabolism, liver metabolism and mitochondrial sensitivity and morphology as potential areas of interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. There are no conflicts of interest to declare. Writing, editing, and revisions were performed by all authors. All authors have read and approved the final version of this manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. F.A.R. is supported by a NSERC Canadian Graduate Scholarship. The authors would like to thank Dr Matthew Krause for his support in reading of the final document.

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