The endogenous molecular clock orchestrates the temporal separation of substrate metabolism in skeletal muscle

Abstract

Background: Skeletal muscle is a major contributor to whole-body metabolism as it serves as a depot for both glucose and amino acids, and is a highly metabolically active tissue. Within skeletal muscle exists an intrinsic molecular clock mechanism that regulates the timing of physiological processes. A key function of the clock is to regulate the timing of metabolic processes to anticipate time of day changes in environmental conditions. The purpose of this study was to identify metabolic genes that are expressed in a circadian manner and determine if these genes are regulated downstream of the intrinsic molecular clock by assaying gene expression in an inducible skeletal muscle-specific Bmal1 knockout mouse model (iMS-Bmal1 (-/-) ).

Methods: We used circadian statistics to analyze a publicly available, high-resolution time-course skeletal muscle expression dataset. Gene ontology analysis was utilized to identify enriched biological processes in the skeletal muscle circadian transcriptome. We generated a tamoxifen-inducible skeletal muscle-specific Bmal1 knockout mouse model and performed a time-course microarray experiment to identify gene expression changes downstream of the molecular clock. Wheel activity monitoring was used to assess circadian behavioral rhythms in iMS-Bmal1 (-/-) and control iMS-Bmal1 (+/+) mice.

Results: The skeletal muscle circadian transcriptome was highly enriched for metabolic processes. Acrophase analysis of circadian metabolic genes revealed a temporal separation of genes involved in substrate utilization and storage over a 24-h period. A number of circadian metabolic genes were differentially expressed in the skeletal muscle of the iMS-Bmal1 (-/-) mice. The iMS-Bmal1 (-/-) mice displayed circadian behavioral rhythms indistinguishable from iMS-Bmal1 (+/+) mice. We also observed a gene signature indicative of a fast to slow fiber-type shift and a more oxidative skeletal muscle in the iMS-Bmal1 (-/-) model.

Conclusions: These data provide evidence that the intrinsic molecular clock in skeletal muscle temporally regulates genes involved in the utilization and storage of substrates independent of circadian activity. Disruption of this mechanism caused by phase shifts (that is, social jetlag) or night eating may ultimately diminish skeletal muscle's ability to efficiently maintain metabolic homeostasis over a 24-h period.

Keywords: Anabolic; Bmal1; Catabolic; Circadian; Metabolism; Molecular clock; Rev-erbα; Skeletal muscle; Temporal separation.

Figure 1

Gene ontology analysis of the skeletal muscle circadian transcriptome. Top 15 enriched GO processes listed from left to right in order of significance.

Figure 2

Schematic acrophase diagram of circadian genes involved in lipid metabolic processes. The relative location of the circadian genes (italicized) in respect to the x-axis indicates acrophase or time of peak expression calculated by the JTK_CYCLE algorithm. Location of substrates and pathways does not represent peak substrate concentrations and/or rates of individual pathways as these were not measured in our analysis. White/grey shading is representative of the inactive and active phases, respectively.

Figure 3

Schematic acrophase diagram of circadian genes involved in carbohydrate metabolic processes. The relative location of the circadian genes (italicized) in respect to the x-axis indicates acrophase or time of peak expression calculated by the JTK_CYCLE algorithm. Location of substrates and pathways does not represent peak substrate concentrations and/or rates of individual pathways as these were not measured in our analysis. White/grey shading is representative of the inactive and active phases, respectively.

Figure 4

Characterization of iMS-Bmal1 ^−/− mice. Recombination assay (A) of genomic DNA isolated from muscle and non-muscle tissues from tamoxifen-treated (iMS-Bmal1 ^−/−) and vehicle-treated (iMS-Bmal1 ^+/+) mice at 17 to 18 weeks of age (5 weeks postinjection). Recombination of the Bmal1 gene (exon 8) yields a 572-bp PCR product. The non-recombined allele is detected at 431 bp. Western blot (B) analysis of BMAL1 expression in iMS-Bmal1 ^−/− and iMS-Bmal1 ^+/+ liver and gastrocnemius samples. Note that the original blot containing both muscle and liver samples was cut, and brightness/contrast was altered to enhance the visibility of Bmal1 in the muscle samples. (C) Real-time PCR results of time-course expression values for Bmal1, Rev-erbα, and Dbp in the iMS-Bmal1 ^+/+ (black) and iMS-Bmal1 ^−/− (red). Representative wheel running rhythms (D) of iMS-Bmal1 ^−/− and iMS-Bmal1 ^+/+ mice. White and black bars (top) indicate light and dark phases. 12 L/12D represents the 12-h light/12-h dark cycle. 12D/12D represents constant darkness conditions. Tick marks indicate wheel running activity. Representative chi-squared periodograms (E) of iMS-Bmal1 ^−/− and iMS-Bmal1 ^+/+ mice indicating approximate 24-h period lengths in both mice.

Figure 5

Differentially expressed circadian, metabolic genes in iMS-Bmal1−/− skeletal muscle. Average expression changes of the circadian carbohydrate (A) and lipid (B) genes in iMS-Bmal1−/− gastrocnemius averaged over circadian times 18, 22, 26, 30, 34, and 38. Tibialis anterior and soleus gene expression changes (Dyar et al.) averaged over circadian times 0, 4, 8, 12, 16, and 20. The red line denotes control (iMS-Bmal1 ^+/+) gene expression values. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Figure 6

Increase in slow type sarcomeric genes in iMS-Bmal1 ^−/−. Average gene expression changes of slow (A) and fast (B) type sarcomeric genes in iMS-Bmal1 ^−/− compared to control values (red line). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.