Background
24h whole-body substrate metabolism and the endogenous circadian clock operative in human skeletal muscle are compromised in the insulin-resistant state. Skeletal muscle metabolism is an important determinant of insulin resistance but its dynamics over 24h are poorly understood. 

Methods
Therefore, we compared the skeletal muscle metabolome between young healthy (YH) vs. older insulin-resistant (OIR) men by means of serial muscle sampling performed under 24h real-life conditions. Five sequential skeletal muscle biopsies were obtained from 12 YH (22±2 years, BMI: 22.4±2.2 kg/m²) and 12 OIR men (65±9 years, BMI: 30.3±2.7 kg/m²) at 8AM, 1PM, 6PM, 11PM and 4AM. Under controlled real-life conditions, volunteers were provided with three meals, and biopsies from the vastus lateralis muscle were taken directly before meals to prevent direct postprandial effects. Muscle metabolites were determined using UPLC/HRMS-based semi-targeted metabolomics. Using the circacompare R-package, nonlinear mixed-effects models were used to assess metabolites for 24h rhythmicity. Their abundance, amplitude and acrophase were compared between groups.

Results
The overall levels of metabolites involved in the initial steps of glycolysis and the hexosamine biosynthesis were higher in OIR, whereas metabolites involved in the urea cycle, glutamine-alpha-ketoglutarate, ketone and redox metabolism were lower in OIR around the clock. The largest number of metabolites that proved to be different between groups was detected at 4AM. Both groups demonstrated 24h-rhythmicity in ~40% of metabolites but rhythmic metabolites only partially overlapped between groups. 14 metabolites were only rhythmic in YH and 22 metabolites only in OIR, with adenosine (only rhythmic in YH) showing the most pronounced difference. Acetylglutamate, cis-aconitate, FAD, hydroxybutyrate and UDP were phase delayed in OIR.

Conclusion
We observed large differences in the muscle metabolome between YH and OIR around the clock. Altered 24h-rhythmicity within the metabolome and phase delays of specific metabolites could be linked to the compromised 24h substrate metabolism and disturbance of the circadian clock of insulin-resistant skeletal muscle; however, this causal relationship remains to be investigated.