Nutrient-regulated skeletal muscle metabolism and circadian clock

Abstract: The prevalence of obesity has tripled in the last four decades, becoming a major risk for the development of cardiometabolic diseases such as type 2 diabetes. Obesity is associated with pathological storage of lipids in skeletal muscle, causing a major disturbance in metabolism. Furthermore, metabolic homeostasis is tightly regulated by the circadian clock. This thesis aims at gaining further insight in skeletal muscle metabolic regulation by lipid accumulation and the circadian clock. Study I investigated the role of oxidative stress and accumulation of the lipid aldehyde 4-hydroxy-2-hexenal (4-HHE) in the development of skeletal muscle insulin resistance. The results unveil elevated circulating levels of 4-HHE in type 2 diabetic humans and Zucker diabetic fatty rats. Acute intravenous injection of 4-HHE in rats, significantly altered whole-body insulin sensitivity and decreased glucose infusion rate. In vitro, 4-HHE impaired insulin-stimulated glucose uptake and signaling and induced carbonylation of cell proteins. Increasing intracellular glutathione pools prevented 4-HHE-induced carbonyl stress and insulin resistance. 4-HHE plays a causal role in the pathophysiology of type 2 diabetes and might constitute a potential therapeutic target to taper oxidative stress-induced insulin resistance. Study II investigated the role of obesity and systemic factors associated with insulin resistance in the regulation of skeletal muscle clock gene expression. We determined that skeletal muscle clock gene expression was affected by obesity and weight loss. When correlating clock gene expression with clinical characteristics of the participants, we observed invers correlated with plasma lipids. Circadian time-course studies revealed that core clock genes oscillate over time, and expression profiles were altered by palmitate treatment. In conclusion, skeletal muscle clock gene expression and function is altered by obesity, coincident with changes in plasma lipid levels. Study III explored the effect of the saturated fatty acid palmitate on circadian transcriptomics and examined the impact on histone H3 lysine K27 acetylation (H3K27ac) in primary human skeletal muscle myotubes. Palmitate disrupted transcriptomic rhythmicity in myotubes. Genes that lost or gained rhythmicity after palmitate treatment were involved in metabolic processes, protein translation and transport, and transcriptional regulation. Additionally, histone H3K27ac, a marker of active gene enhancers, was modified by palmitate treatment in myotubes. Our results indicate that dietary saturated fatty acids impart post-transcriptional modifications to histone proteins and regulate circadian transcriptomics. Study IV elucidated whether altered circadian rhythmicity of clock genes is associated with metabolic dysfunction in T2D. Transcriptional cycling of core clock genes BMAL1, CLOCK, and PER3 was altered in skeletal muscle from individuals with T2D. This was coupled with reduced number and amplitude of cycling genes and disturbed circadian oxygen consumption in T2D myotubes. Mitochondrial associated genes were enriched for rhythmic peaks in NGT, but not T2D, and positively correlated with insulin sensitivity. Mitochondrial disruption altered core-clock gene expression and free-radical production, phenomena that were restored by resveratrol treatment. The results identify bi-directional communication between mitochondrial function and rhythmic gene expression, processes which are disturbed in diabetes.

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