Temperature as a Circadian Marker in Older Human Subjects: Relationship to Metabolic Syndrome and Diabetes.

BACKGROUND: Circadian rhythms are characterized by approximate 24-hour oscillations in physiological and behavioral processes. Disruptions in these endogenous rhythms, most commonly associated with shift work and/or lifestyle, are recognized to be detrimental to health. Several studies have demonstrated a high correlation between disrupted circadian rhythms and metabolic disease. The aim of this study was to determine which metabolic parameters correlate with physiological measures of circadian temperature amplitude (TempAmp) and stability (TempStab). METHODS: Wrist skin temperature was measured in 34 subjects (ages 50 to 70, including lean, obese, and diabetic subjects) every 10 minutes for 7 consecutive days. Anthropometric measures and fasting blood draws were conducted to obtain data on metabolic parameters: body mass index, hemoglobin A1C, triglycerides, cholesterol, high-density lipoprotein, and low-density lipoprotein. A history of hypertension and current blood pressure was noted. RESULTS: Analysis of the data indicated a substantial reduction in TempAmp and TempStab in subjects with metabolic syndrome (three or more risk factors). To determine the impact of individual interdependent metabolic factors on temperature rhythms, stepwise multilinear regression analysis was conducted using metabolic syndrome measurements. Interestingly, only triglyceride level was consistently correlated by the analysis. Triglyceride level was shown to contribute to 33% of the variability in TempAmp and 23% of the variability in TempStab. CONCLUSION: Our results demonstrate that elevated triglycerides are associated with diminished TempAmp and TempStab in human subjects, and triglycerides may serve as a primary metabolic predictor of circadian parameters.

Intrinsic muscle clock is necessary for musculoskeletal health.

KEY POINTS: The endogenous molecular clock in skeletal muscle is necessary for maintenance of phenotype and function. Loss of Bmal1 solely from adult skeletal muscle (iMSBmal1(-/-) ) results in reductions in specific tension, increased oxidative fibre type and increased muscle fibrosis with no change in feeding or activity. Disruption of the molecular clock in adult skeletal muscle is sufficient to induce changes in skeletal muscle similar to those seen in the Bmal1 knockout mouse (Bmal1(-/-) ), a model of advanced ageing. iMSBmal1(-/-) mice develop increased bone calcification and decreased joint collagen, which in combination with the functional changes in skeletal muscle results in altered gait. This study uncovers a fundamental role for the skeletal muscle clock in musculoskeletal homeostasis with potential implications for ageing. ABSTRACT: Disruption of circadian rhythms in humans and rodents has implicated a fundamental role for circadian rhythms in ageing and the development of many chronic diseases including diabetes, cardiovascular disease, depression and cancer. The molecular clock mechanism underlies circadian rhythms and is defined by a transcription-translation feedback loop with Bmal1 encoding a core molecular clock transcription factor. Germline Bmal1 knockout (Bmal1 KO) mice have a shortened lifespan, show features of advanced ageing and exhibit significant weakness with decreased maximum specific tension at the whole muscle and single fibre levels. We tested the role of the molecular clock in adult skeletal muscle by generating mice that allow for the inducible skeletal muscle-specific deletion of Bmal1 (iMSBmal1). Here we show that disruption of the molecular clock, specifically in adult skeletal muscle, is associated with a muscle phenotype including reductions in specific tension, increased oxidative fibre type, and increased muscle fibrosis similar to that seen in the Bmal1 KO mouse. Remarkably, the phenotype observed in the iMSBmal1(-/-) mice was not limited to changes in muscle. Similar to the germline Bmal1 KO mice, we observed significant bone and cartilage changes throughout the body suggesting a role for the skeletal muscle molecular clock in both the skeletal muscle niche and the systemic milieu. This emerging area of circadian rhythms and the molecular clock in skeletal muscle holds the potential to provide significant insight into intrinsic mechanisms of the maintenance of muscle quality and function as well as identifying a novel crosstalk between skeletal muscle, cartilage and bone.

Blunted hypertrophic response in aged skeletal muscle is associated with decreased ribosome biogenesis.

The ability of skeletal muscle to hypertrophy in response to a growth stimulus is known to be compromised in older individuals. We hypothesized that a change in the expression of protein-encoding genes in response to a hypertrophic stimulus contributes to the blunted hypertrophy observed with aging. To test this hypothesis, we determined gene expression by microarray analysis of plantaris muscle from 5- and 25-mo-old mice subjected to 1, 3, 5, 7, 10, and 14 days of synergist ablation to induce hypertrophy. Overall, 1,607 genes were identified as being differentially expressed across the time course between young and old groups; however, the difference in gene expression was modest, with cluster analysis showing a similar pattern of expression between the two groups. Despite ribosome protein gene expression being higher in the aged group, ribosome biogenesis was significantly blunted in the skeletal muscle of aged mice compared with mice young in response to the hypertrophic stimulus (50% vs. 2.5-fold, respectively). The failure to upregulate pre-47S ribosomal RNA (rRNA) expression in muscle undergoing hypertrophy of old mice indicated that rDNA transcription by RNA polymerase I was impaired. Contrary to our hypothesis, the findings of the study suggest that impaired ribosome biogenesis was a primary factor underlying the blunted hypertrophic response observed in skeletal muscle of old mice rather than dramatic differences in the expression of protein-encoding genes. The diminished increase in total RNA, pre-47S rRNA, and 28S rRNA expression in aged muscle suggest that the primary dysfunction in ribosome biogenesis occurs at the level of rRNA transcription and processing.

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

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.

Identification of a conserved set of upregulated genes in mouse skeletal muscle hypertrophy and regrowth.

The purpose of this study was to compare the gene expression profile of mouse skeletal muscle undergoing two forms of growth (hypertrophy and regrowth) with the goal of identifying a conserved set of differentially expressed genes. Expression profiling by microarray was performed on the plantaris muscle subjected to 1, 3, 5, 7, 10, and 14 days of hypertrophy or regrowth following 2 wk of hind-limb suspension. We identified 97 differentially expressed genes (≥2-fold increase or ≥50% decrease compared with control muscle) that were conserved during the two forms of muscle growth. The vast majority (∼90%) of the differentially expressed genes was upregulated and occurred at a single time point (64 out of 86 genes), which most often was on the first day of the time course. Microarray analysis from the conserved upregulated genes showed a set of genes related to contractile apparatus and stress response at day 1, including three genes involved in mechanotransduction and four genes encoding heat shock proteins. Our analysis further identified three cell cycle-related genes at day and several genes associated with extracellular matrix (ECM) at both days 3 and 10. In conclusion, we have identified a core set of genes commonly upregulated in two forms of muscle growth that could play a role in the maintenance of sarcomere stability, ECM remodeling, cell proliferation, fast-to-slow fiber type transition, and the regulation of skeletal muscle growth. These findings suggest conserved regulatory mechanisms involved in the adaptation of skeletal muscle to increased mechanical loading.

Time course of gene expression during mouse skeletal muscle hypertrophy.

The purpose of this study was to perform a comprehensive transcriptome analysis during skeletal muscle hypertrophy to identify signaling pathways that are operative throughout the hypertrophic response. Global gene expression patterns were determined from microarray results on days 1, 3, 5, 7, 10, and 14 during plantaris muscle hypertrophy induced by synergist ablation in adult mice. Principal component analysis and the number of differentially expressed genes (cutoffs ≥2-fold increase or ≥50% decrease compared with control muscle) revealed three gene expression patterns during overload-induced hypertrophy: early (1 day), intermediate (3, 5, and 7 days), and late (10 and 14 days) patterns. Based on the robust changes in total RNA content and in the number of differentially expressed genes, we focused our attention on the intermediate gene expression pattern. Ingenuity Pathway Analysis revealed a downregulation of genes encoding components of the branched-chain amino acid degradation pathway during hypertrophy. Among these genes, five were predicted by Ingenuity Pathway Analysis or previously shown to be regulated by the transcription factor Kruppel-like factor-15, which was also downregulated during hypertrophy. Moreover, the integrin-linked kinase signaling pathway was activated during hypertrophy, and the downregulation of muscle-specific micro-RNA-1 correlated with the upregulation of five predicted targets associated with the integrin-linked kinase pathway. In conclusion, we identified two novel pathways that may be involved in muscle hypertrophy, as well as two upstream regulators (Kruppel-like factor-15 and micro-RNA-1) that provide targets for future studies investigating the importance of these pathways in muscle hypertrophy.