Day-night rhythm of skeletal muscle metabolism is disturbed in older, metabolically compromised individuals

Abstract

Objective: Skeletal muscle mitochondrial function and energy metabolism displays day-night rhythmicity in healthy, young individuals. Twenty-four-hour rhythmicity of metabolism has been implicated in the etiology of age-related metabolic disorders. Whether day-night rhythmicity in skeletal muscle mitochondrial function and energy metabolism is altered in older, metabolically comprised humans remains unknown.

Methods: Twelve male overweight volunteers with impaired glucose tolerance and insulin sensitivity stayed in a metabolic research unit for 2 days under free living conditions with regular meals. Indirect calorimetry was performed at 5 time points (8 AM, 1 PM, 6 PM, 11 PM, 4 AM), followed by a muscle biopsy. Mitochondrial oxidative capacity was measured in permeabilized muscle fibers using high-resolution respirometry.

Results: Mitochondrial oxidative capacity did not display rhythmicity. The expression of circadian core clock genes BMAL1 and REV-ERBα showed a clear day-night rhythm (p < 0.001), peaking at the end of the waking period. Remarkably, the repressor clock gene PER2 did not show rhythmicity, whereas PER1 and PER3 were strongly rhythmic (p < 0.001). On the whole-body level, resting energy expenditure was highest in the late evening (p < 0.001). Respiratory exchange ratio did not decrease during the night, indicating metabolic inflexibility.

Conclusions: Mitochondrial oxidative capacity does not show a day-night rhythm in older, overweight participants with impaired glucose tolerance and insulin sensitivity. In addition, gene expression of PER2 in skeletal muscle indicates that rhythmicity of the negative feedback loop of the molecular clock is disturbed. CLINICALTRIALS.

Gov id: NCT03733743.

Keywords: Day-night rhythm; Insulin resistance; Mitochondria; Skeletal muscle.

Copyright © 2020 The Authors. Published by Elsevier GmbH.. All rights reserved.

Figures

Figure 1

Mitochondrial oxidative capacity in skeletal muscle does not have a day-night rhythm. ADP-stimulated respiration of permeabilized muscle fibers fueled with (A) the lipid substrate octanoylcarinitine (state 3 MO); (B) addition of complex I substrates (state 3 MOG); (C) addition of substrates for parallel electron input into complex I and II (state 3 MOGS). Maximal uncoupled respiration after FCCP (State U) titration (D). For reference, we depicted the respiration states from our previous study in young, healthy, lean subjects [9] using dotted lines. M, malate; O, octanoylcarnitine; G, glutamate; S, succinate. The dark gray area represents the sleeping period (12AM–7AM). Data depicts oxygen consumption per mg wet weight per second and is shown as mean ± SEM. ∗p 

Figure 2

Mitochondrial respiratory chain proteins are not rhythmic. Proteins levels of oxidative phosphorylation complexes I – V (A–E). Representative western blot of one subject depicting the oxidative phosphorylation complexes of all time points (F). Protein levels of the two mitochondrial membrane proteins TOMM-20 and VDAC (G–H). Jointly, these data indicate that mitochondrial content does not possess 24-h rhythmicity. Representative western blot images are displayed below the quantification graphs. Proteins of interest were normalized to total protein content using stain-free technology. The dark gray area represents the sleeping period (12 AM–7 AM). Data is presented as mean ± SEM.

Figure 3

Regulators of mitochondrial dynamics. Protein levels of markers for mitochondrial fusion: OPA-1 (A); mitochondrial fission: FIS-1 (B); and mitophogy: PINK-1 (C). Representative western blots are depicted below the graphs. Marker proteins indicate no mitochondrial network remodeling over the 24-h period. Proteins of interest were normalized to total protein content using stain-free technology. The dark gray area represents the sleeping period (12 AM–7 AM). Data is presented as mean ± SEM. ∗p 

Figure 4

Respiratory exchange ratio shows increase in carbohydrate oxidation over the day. Whole-body resting energy expenditure (A), respiratory exchange ratio (B), carbohydrate oxidation (C), fat oxidation (D). For reference, we depicted the measurements from our earlier study in young, healthy, lean subjects [9] using dotted lines. The dark gray areas represent the sleeping periods (12 AM–7 AM) before and at the end of the test day. Data is presented as mean ± SEM. ∗p 

Figure 5

Daily variations in glucose, insulin, FFA, and triglycerides are predominantly influenced by feeding. Plasma levels of glucose (A), insulin (B), free fatty acids (C), triglycerides (D). For reference, we depicted the measurements from our earlier study in young, healthy, lean subjects [9] using dotted lines. The dark gray area represents the sleeping period (12 AM–7 AM). Data is presented as mean ± SEM. ∗p 

Figure 6

Core molecular clock gene expression in skeletal muscle. mRNA expression of BMAL1 (A), CLOCK (B), PER2 (C), CRY1 (D), REV-ERBα (E), PER1 (F), PER3 (G), and combined expression patterns of PER1, PER2, and PER3 in one graph (H). For reference, we depicted the mRNA expression of the representative gene from our earlier study in young, healthy, lean subjects [9] using dotted lines. Data are normalized to the geometric of 3 housekeeping genes. The dark gray area represents the sleeping period (12 AM–7 AM). Data is presented as mean ± SEM. ∗p < 0.05 for effect of time.