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. 2016 Jul 1;5(8):635-645.
doi: 10.1016/j.molmet.2016.06.012. eCollection 2016 Aug.

Demonstration of a day-night rhythm in human skeletal muscle oxidative capacity

Dirk van Moorsel  1 , Jan Hansen  2 , Bas Havekes  1 , Frank A J L Scheer  3 , Johanna A Jörgensen  2 , Joris Hoeks  2 , Vera B Schrauwen-Hinderling  4 , Helene Duez  5 , Philippe Lefebvre  5 , Nicolaas C Schaper  6 , Matthijs K C Hesselink  2 , Bart Staels  5 , Patrick Schrauwen  7
Affiliations

Demonstration of a day-night rhythm in human skeletal muscle oxidative capacity

Dirk van Moorsel et al. Mol Metab. .

Abstract

Objective: A disturbed day-night rhythm is associated with metabolic perturbations that can lead to obesity and type 2 diabetes mellitus (T2DM). In skeletal muscle, a reduced oxidative capacity is also associated with the development of T2DM. However, whether oxidative capacity in skeletal muscle displays a day-night rhythm in humans has so far not been investigated.

Methods: Lean, healthy subjects were enrolled in a standardized living protocol with regular meals, physical activity and sleep to reflect our everyday lifestyle. Mitochondrial oxidative capacity was examined in skeletal muscle biopsies taken at five time points within a 24-hour period.

Results: Core-body temperature was lower during the early night, confirming a normal day-night rhythm. Skeletal muscle oxidative capacity demonstrated a robust day-night rhythm, with a significant time effect in ADP-stimulated respiration (state 3 MO, state 3 MOG and state 3 MOGS, p < 0.05). Respiration was lowest at 1 PM and highest at 11 PM (state 3 MOGS: 80.6 ± 4.0 vs. 95.8 ± 4.7 pmol/mg/s). Interestingly, the fluctuation in mitochondrial function was also observed in whole-body energy expenditure, with peak energy expenditure at 11 PM and lowest energy expenditure at 4 AM (p < 0.001). In addition, we demonstrate rhythmicity in mRNA expression of molecular clock genes in human skeletal muscle.

Conclusions: Our results suggest that the biological clock drives robust rhythms in human skeletal muscle oxidative metabolism. It is tempting to speculate that disruption of these rhythms contribute to the deterioration of metabolic health associated with circadian misalignment.

Keywords: BMAL1, brain and muscle ARNT-like 1; BMI, body mass index; Biological rhythm; CLOCK, circadian locomotor output cycles kaput; CRY, cryptochrome; Energy metabolism; FCCP, carbonyl cyanide-4-trifluoromethoxyphenylhydrazone; Mitochondria; Molecular clock; NADH, reduced nicotinamide adenine dinucleotide; Oxidative capacity; PER, period; RER, respiratory exchange ratio; RT-QPCR, Real-Time Quantitative Polymerase Chain Reaction; Skeletal muscle; T2DM, type 2 diabetes mellitus; TCA cycle, tricarboxylic acid cycle.

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Figures

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Graphical abstract
Figure 1
Figure 1
Study design. Participants stayed in the research facility for 44 h starting at noon on day 1. During the second study-day we performed all measurements. B, breakfast; L, lunch; D, dinner; M, measurement (indirect calorimetry and muscle biopsy).
Figure 2
Figure 2
Time-dependent expression of transcripts in human skeletal muscle. (A) Time-dependent gene expression was assessed by microarray analysis of RNA extracted from a single donor (N = 1). Protein-encoding transcript expression patterns were visualized as a self-organizing map (Euclidean distance metrics). The color scale indicates upregulated genes (red) and downregulated genes (green) relative to the global median signal of each array. The arrow indicates clusters of genes showing the most prominent peak of expression at the indicated time point (4 AM, cluster 1 and 11 PM, cluster 2). (B and C) Gene annotation enrichment analysis. The genes extracted from cluster 1 and 2 were searched using multiple databases (GeneOntology Biological Processes, KEGG pathways and Reactome) and statistically enriched terms were determined using the Metascape tool. The most significantly enriched terms are indicated for cluster 1 (GO biological processes, panel B, peak expression 4 AM) and cluster 2 (Reactome, panel C, peak expression 11 PM). Statistically significant terms were hierarchically clustered and converted into a network. Each term is represented by a circle node, of which the size is proportional to the number of genes in the term. The color of the node indicates the statistical significance of the term belonging to the cluster (see color scale). The most significant term characterizing each cluster is indicated. Similarities between terms are indicated by connecting lines.
Figure 3
Figure 3
Rhythmicity of core molecular clock genes in human skeletal muscle. Diurnal mRNA expression of the arrhythmic muscle filament gene TTN (A), the core clock genes CLOCK and BMAL1 (B), PER2 and CRY1 (C), measured by RT-QPCR. Data are normalized to the geometric mean of 3 housekeeping genes and presented as mean ± SEM. *p < 0.05 for the effect of time in all depicted genes.
Figure 4
Figure 4
Mitochondrial oxidative capacity in human skeletal muscle displays a day-night rhythm. (A) ADP-stimulated respiration of permeabilized muscle fibers upon a lipid substrate (state 3 MO); fueled by complex I-linked substrates (state 3 MOG) and upon parallel electron input into complex I and II (state 3 MOGS). (B) Maximally uncoupled respiration upon FCCP (State U). M, malate; O, octanoylcarnitine; G, glutamate; S, succinate. Data represents oxygen consumption per mg wet weight per second and is depicted as mean ± SEM. *p < 0.05 for the effect of time in all states. #p < 0.05 vs 1 PM for Bonferroni-adjusted post-hoc analysis.
Figure 5
Figure 5
Mitochondrial content and mitochondrial marker proteins do not show rhythmicity. (A) Measurement of mitochondrial DNA copy number (DNA copy numbers of the mitochondrial-encoded gene ND1 divided over the nuclear encoded gene LPL). (B) Representative western blot depicting the oxidative phosphorylation complexes of all time-points (C–G) Protein levels of specific subunits of complex I–V of the mitochondrial electron transport chain, measured by western blotting. (H–J) Protein levels of other mitochondrial (VDAC, TOMM-20) and non-mitochondrial (SR-Actin) proteins measured by western blotting. Representative western blot images are displayed below their respective graphs. Data expressed as mean ± SEM. p > 0.05 for the effect of time in all panels.
Figure 6
Figure 6
Mitochondrial dynamics rather than mitochondrial biogenesis display a significant time-effect. Protein levels of mitochondrial biogenesis regulator PGC-1α (A), mitochondrial fission protein FIS-1 (B), mitophagy protein PINK-1 (C) and mitochondrial fusion protein OPA-1 (D). Representative western blot images are displayed below their respective graphs. Data presented as mean ± SEM. *p < 0.05 for the effect of time.
Figure 7
Figure 7
Whole-body resting energy expenditure peaks at the same time as skeletal muscle oxidative capacity whereas substrate oxidation exhibits a clear feeding and fasting pattern. Whole-body resting energy expenditure (A), respiratory exchange ratio (B), carbohydrate oxidation (C) and fat oxidation (D) during the second study-day, calculated from oxygen consumption and carbon-dioxide production measured by indirect calorimetry. Data presented as mean ± SEM. *p < 0.01 for the effect of time. #p < 0.05 for Bonferroni-adjusted post-hoc analysis.
Figure 8
Figure 8
Plasma metabolites and insulin display marked variations over 24 h, mostly associated with feeding-fasting. Plasma levels of glucose (A), insulin (B), free fatty acids (C) and triglycerides (D) throughout the second-study day. Data depicted as mean ± SEM. *p < 0.001 for the effect of time.
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