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

Hodge BA, Wen Y, Riley LA, Zhang X, England JH, Harfmann BD, Schroder EA, Esser KA - Skelet Muscle (2015)

Bottom Line: Within skeletal muscle exists an intrinsic molecular clock mechanism that regulates the timing of physiological processes.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, College of Medicine, University of Kentucky, MS 508, 800 Rose Street, Lexington, KY 40536 USA ; Center for Muscle Biology, University of Kentucky, 800 Rose Street, Lexington, KY 40536 USA.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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Fig4: 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.

Mentions: Use of the high-resolution microarray data set allowed for the identification of mRNAs expressed in a circadian pattern, but this could be due to the intrinsic molecular clock or could be a response to external behavioral (feeding/activity) or neural/humoral cues [24,128,129]. To determine the role of the intrinsic skeletal muscle molecular clock in the temporal regulation of metabolic gene expression, we generated an inducible mouse model to inactivate Bmal1 specifically in adult skeletal muscles. Upon treatment with tamoxifen in 12-week-old adult mice, we detect recombination of exon-8 (that is, DNA binding region) of the Bmal1 gene specifically in skeletal muscle (Figure 4A), confirming the tissue specificity of the mouse model. We waited until 12 weeks of age to limit possible developmental effects as BMAL1 has been shown to promote myogenesis [20,130]. As seen in Figure 4A, recombination was not detected in the skeletal muscle or non-muscle tissues of vehicle-treated mice (iMS-Bmal1+/+). Western blot analysis confirmed the depletion of BMAL1 protein in the skeletal muscle of the iMS-Bmal1−/− mice with no effect on the liver (Figure 4B). Tamoxifen-induced loss of Bmal1 in adult skeletal muscle resulted in significant and expected gene expression changes of genes involved in the core clock mechanism. In particular, genes directly activated by the BMAL1/CLOCK heterodimer, such as Rev-erbα and Dbp, are markedly downregulated in iMS-Bmal1−/− but not in the iMS-Bmal1+/+ samples (Figure 4C). Collectively, these results demonstrate the effective loss of BMAL1 protein and disruption of core-clock gene expression in the iMS-Bmal1−/− muscle tissue.Figure 4


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

Hodge BA, Wen Y, Riley LA, Zhang X, England JH, Harfmann BD, Schroder EA, Esser KA - Skelet Muscle (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig4: 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.
Mentions: Use of the high-resolution microarray data set allowed for the identification of mRNAs expressed in a circadian pattern, but this could be due to the intrinsic molecular clock or could be a response to external behavioral (feeding/activity) or neural/humoral cues [24,128,129]. To determine the role of the intrinsic skeletal muscle molecular clock in the temporal regulation of metabolic gene expression, we generated an inducible mouse model to inactivate Bmal1 specifically in adult skeletal muscles. Upon treatment with tamoxifen in 12-week-old adult mice, we detect recombination of exon-8 (that is, DNA binding region) of the Bmal1 gene specifically in skeletal muscle (Figure 4A), confirming the tissue specificity of the mouse model. We waited until 12 weeks of age to limit possible developmental effects as BMAL1 has been shown to promote myogenesis [20,130]. As seen in Figure 4A, recombination was not detected in the skeletal muscle or non-muscle tissues of vehicle-treated mice (iMS-Bmal1+/+). Western blot analysis confirmed the depletion of BMAL1 protein in the skeletal muscle of the iMS-Bmal1−/− mice with no effect on the liver (Figure 4B). Tamoxifen-induced loss of Bmal1 in adult skeletal muscle resulted in significant and expected gene expression changes of genes involved in the core clock mechanism. In particular, genes directly activated by the BMAL1/CLOCK heterodimer, such as Rev-erbα and Dbp, are markedly downregulated in iMS-Bmal1−/− but not in the iMS-Bmal1+/+ samples (Figure 4C). Collectively, these results demonstrate the effective loss of BMAL1 protein and disruption of core-clock gene expression in the iMS-Bmal1−/− muscle tissue.Figure 4

Bottom Line: Within skeletal muscle exists an intrinsic molecular clock mechanism that regulates the timing of physiological processes.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, College of Medicine, University of Kentucky, MS 508, 800 Rose Street, Lexington, KY 40536 USA ; Center for Muscle Biology, University of Kentucky, 800 Rose Street, Lexington, KY 40536 USA.

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.

No MeSH data available.


Related in: MedlinePlus