Circadian misalignment induces fatty acid metabolism gene profiles and compromises insulin sensitivity in human skeletal muscle

Abstract

Circadian misalignment, such as in shift work, has been associated with obesity and type 2 diabetes. However, direct effects of circadian misalignment on skeletal muscle insulin sensitivity and the muscle molecular circadian clock have never been studied in humans. Here, we investigated insulin sensitivity and muscle metabolism in 14 healthy young lean men [age 22.4 ± 2.8 years; body mass index (BMI) 22.3 ± 2.1 kg/m² (mean ± SD)] after a 3-d control protocol and a 3.5-d misalignment protocol induced by a 12-h rapid shift of the behavioral cycle. We show that short-term circadian misalignment results in a significant decrease in muscle insulin sensitivity due to a reduced skeletal muscle nonoxidative glucose disposal (rate of disappearance: 23.7 ± 2.4 vs. 18.4 ± 1.4 mg/kg per minute; control vs. misalignment; P = 0.024). Fasting glucose and free fatty acid levels as well as sleeping metabolic rate were higher during circadian misalignment. Molecular analysis of skeletal muscle biopsies revealed that the molecular circadian clock was not aligned to the inverted behavioral cycle, and transcriptome analysis revealed the human PPAR pathway as a key player in the disturbed energy metabolism upon circadian misalignment. Our findings may provide a mechanism underlying the increased risk of type 2 diabetes among shift workers.

Keywords: circadian misalignment; diabetes; insulin sensitivity; shift work; skeletal muscle.

Fig. 1.

Study design. Participants underwent a control and a misalignment condition in a randomized crossover design. B, breakfast; Clamp, hyperinsulinemic euglycemic clamp; D, dinner; F, measurement of fasting blood substrates and energy metabolism; L, lunch; M, muscle biopsy; RMR, resting metabolic rate; S, snack.

Fig. 2.

Plasma metabolites are altered in circadian misalignment. Plasma glucose (A), insulin (B), free fatty acids (C), and triglycerides (D) were measured in the evening before dinner (nonfasted) and in the overnight fasted state 15 min after awaking, 7 AM and 7 PM in the control and misalignment condition, respectively. Data are mean ± SEM. *P < 0.05 biological morning and evening; #P < 0.05 behavioral morning and evening.

Fig. 3.

Insulin sensitivity is decreased in circadian misalignment. EGP suppression during low and high insulin infusion steady state is depicted in A. Insulin-stimulated glucose disposal is expressed as R_d low insulin − R_d basal (A) and R_d high insulin − R_d basal (B). Oxidative glucose disposal (C) and nonoxidative glucose disposal (NOGD) were corrected for basal values. All values were calculated for the last 30 min of the basal, low, and high insulin steady states. Data are mean ± SEM. R_d, rate of disappearance. *P < 0.05.

Fig. 4.

Skeletal muscle core molecular clock genes are not aligned to behavioral rhythm upon circadian misalignment. mRNA expression levels of the core molecular clock genes BMAL1 (A), CLOCK (B), PER2 (C), and CRY1 (D) in skeletal muscle measured by RT-QPCR. Data are normalized to the geometric mean of three housekeeping genes and presented as mean ± SEM. *P < 0.05 comparing 7 AM and 7 PM in control and misalignment condition. #P < 0.05 comparing behavioral mornings in control vs. circadian misalignment condition (overnight fasted state before the clamp). Because only two time points per arm were investigated, the lack of difference in mRNA expression levels between 7 AM and 7 PM does not provide evidence that the measured parameter lacks 24 h rhythmicity.

Fig. 5.

Global gene expression analysis indicates up-regulated PPAR target genes. (A) Multilevel PLS-DA of the transcriptome data indicates similar expression changes between participants. Sample prediction area plots from a PLS-DA model applied on the transcriptome data set with the expression levels of all 29,526 genes in 47 samples. Almost all samples could be correctly classified into four classes that represented the four conditions for each individual subject [control condition (7 AM and 7 PM) and misaligned condition (7 AM and 7 PM)]. Each point represents one array of a participant at one time point. Background color indicates the two-dimensional representation of the predicted classification space of the four classes (conditions), and show that only 3 of the 47 samples were misclassified. (B) Positively enriched genes in the geneset “human PPAR targets” that were significantly up-regulated at 7 PM in the misaligned condition compared with 7 AM in the control condition (representing the timepoints of the clamp) (P < 0.05) were positioned in a biochemical map of cellular lipid metabolism.