Metabolism and exercise: the skeletal muscle clock takes centre stage

Abstract

Circadian rhythms that influence mammalian homeostasis and overall health have received increasing interest over the past two decades. The molecular clock, which is present in almost every cell, drives circadian rhythms while being a cornerstone of physiological outcomes. The skeletal muscle clock has emerged as a primary contributor to metabolic health, as the coordinated expression of the core clock factors BMAL1 and CLOCK with the muscle-specific transcription factor MYOD1 facilitates the circadian and metabolic programme that supports skeletal muscle physiology. The phase of the skeletal muscle clock is sensitive to the time of exercise, which provides a rationale for exploring the interactions between the skeletal muscle clock, exercise and metabolic health. Here, we review the underlying mechanisms of the skeletal muscle clock that drive muscle physiology, with a particular focus on metabolic health. Additionally, we highlight the interaction between exercise and the skeletal muscle clock as a means of reinforcing metabolic health and discuss the possible implications of the time of exercise as a chronotherapeutic approach.

Fig. 1 |. Regulation of the core clock.

Core clock factors of the positive arm (brain and muscle ARNT-like 1 (BMAL1) and circadian locomotor output cycles kaput (CLOCK)) heterodimerize and bind to E-box elements in the promoters of downstream target genes (PER and CRY genes), the protein products of which (PERs and CRYs) form the negative arm, which feeds back to regulate (suppress) the transcriptional activity of the BMAL1–CLOCK heterodimer. The expression of additional regulatory components (retinoic acid receptor (RAR)-related orphan receptors (RORs)) or nuclear receptor subfamily 1 group D members (NR1D1 and NR1D2) following binding of the core clock BMAL1–CLOCK heterodimer to their E-box element also functions to either promote (RORs) or repress (NR1D1 and NR1D2) BMA1 expression. The protein products of the clock output genes NFIL3 and DBP, nuclear factor, interleukin-3-regulated (NFIL3) and D-box binding protein (DBP), are also involved in the transcriptional regulation of members of the ROR gene family and NR1D1 and NR1D2, thereby providing another level of regulation. This transcription–translation feedback loop takes ~24 h and is responsible for driving the diurnal oscillatory expression of a large programme of genes that are referred to as clock output genes. RORE, ROR response element.

Fig. 2 |. Clock protein modification and turnover.

Once transcribed and translated, components of the negative arm (PER and CRY proteins) are phosphorylated by various kinases (such as casein kinase 1 (CKIδ/ε)). Nuclear translocation feeding back into the transcription–translation feedback loop or cytoplasmic degradation via the ubiquitin–proteasome pathway is dependent on the phosphorylation status of PER and CRY proteins. Specifically, hypophosphorylated PER proteins are polyubiquitylated, leading to their degradation in the cytoplasm by the proteasome. PER proteins that are extensively phosphorylated on sequential serine residues form a complex with CRY and CKIδ/ε, which translocates into the nucleus to inhibit the activity of the BMAL1–CLOCK heterodimer. Regulation of CRY proteins is mediated by the F-box E3 ubiquitin ligase FBXL21. In the cytoplasm, phosphorylation of CRY by AMPK targets it for degradation. In the nucleus, however, FBXL21 inhibits another F-box E3 ligase, FBXL3, which normally facilitates proteasomal degradation of CRYs after AMPK phosphorylation. Thus, post-translational modification of the negative arm PER and CRY proteins within either cytoplasmic or nuclear compartments provides a means of fine tuning molecular clock timing and clock output. P, phosphate; Ub, ubiquitin.

Fig. 3 |. The skeletal muscle clock regulates muscle physiology.

Myogenic determination factor 1 (MYOD1) is a muscle-specific transcription factor, which, together with brain and muscle ARNT-like 1 (BMAL1) and circadian locomotor output cycles kaput (CLOCK), binds to E-box elements; nuclear receptor subfamily 1 group D members NR1D1 and NR1D2, and retinoic acid receptor (RAR)-related orphan receptors RORA, RORB and RORC bind to RAR-related orphan receptor response elements (ROREs); and D-box binding protein (DBP) or nuclear factor, interleukin-3-regulated (NFIL3) bind to D-box elements on DNA. The rhythmic binding of these circadian transcription factors to specific regulatory DNA elements regulates the transcription of clock genes themselves as well as a large programme of clock output genes. These diurnal transcription–translation feedback loops ultimately dictate circadian physiology in skeletal muscle, including metabolic flexibility, transcription factor regulation, protein turnover and sarcomeric structure regulation. P, phosphate; Ub, ubiquitin.

Fig. 4 |. Myofibres are multinucleated muscle cells.

a, Skeletal muscle fibres are unique in that they are multinucleated. Each myonucleus occupies a specific area of cytoplasm beneath the sarcolemma along the length of the muscle fibre and is thought to retain its own clock mechanism. b, A single human myofibre labelled with the nuclear stain DAPI (blue), highlighting the multinucleated nature of these cells, and telethonin (Tcap; green), which localizes to sarcomere Z-discs. Image courtesy of L. Denes, Institute for Systems Genetics, New York, USA.

Fig. 5 |. Transcriptional and metabolomic consequences of muscle-specific Bmal1 knockout in mice.

Ablating Bmal1 in skeletal muscle leads to the absence of the brain and muscle ARNT-like 1–circadian locomotor output cycles kaput (BMAL1–CLOCK) heterodimer, which functions to regulate genes containing E-boxes. Whether CLOCK still binds to E-box sites or whether the myogenic determination factor 1 (MYOD1)–CLOCK transcription factor complex still forms or is functional without BMAL1 is unknown. Additionally, the transcription–translation feedback loop ceases, leading to the chronic upregulation or downregulation of other circadian core factors. Blue coloured genes indicate core clock genes that are chronically upregulated with loss of Bmal1, red coloured genes indicate those genes that are downregulated following Bmal1 knockout. Consequently, skeletal muscle-specific Bmal1-knockout mice display a complete loss of the diurnal rhythmicity of gene transcription as well as exhibiting disrupted glucose uptake, glycogen breakdown, hyperinsulinaemia and promotion of lipid metabolism. This metabolic inefficiency leads to an increase in the number of circadian metabolites, decreased glycolytic flux and tricarboxylic acid (TCA) cycle intermediates, and therefore muscles show an increased rate of protein breakdown to support the TCA cycle.

Fig. 6 |. The effects of time-of-day exercise on muscle clock phase.

Acute exercise (in vivo) or electrically stimulated muscle contractions (in vitro) have been shown to induce phase shifts, primarily in the genes (such as PER2) involved in the negative arm of the skeletal muscle clock, in the direction of exercise timing. The representative graph shows how exercise can induce a phase shift in the normal rhythm of expression (dashed line) for the core clock gene PER2, delaying (blue) or advancing (red) in phase according to the exercise timing.