Lipidomics reveals diurnal lipid oscillations in human skeletal muscle persisting in cellular myotubes cultured in vitro

Abstract

Circadian clocks play an important role in lipid homeostasis, with impact on various metabolic diseases. Due to the central role of skeletal muscle in whole-body metabolism, we aimed at studying muscle lipid profiles in a temporal manner. Moreover, it has not been shown whether lipid oscillations in peripheral tissues are driven by diurnal cycles of rest-activity and food intake or are able to persist in vitro in a cell-autonomous manner. To address this, we investigated lipid profiles over 24 h in human skeletal muscle in vivo and in primary human myotubes cultured in vitro. Glycerolipids, glycerophospholipids, and sphingolipids exhibited diurnal oscillations, suggesting a widespread circadian impact on muscle lipid metabolism. Notably, peak levels of lipid accumulation were in phase coherence with core clock gene expression in vivo and in vitro. The percentage of oscillating lipid metabolites was comparable between muscle tissue and cultured myotubes, and temporal lipid profiles correlated with transcript profiles of genes implicated in their biosynthesis. Lipids enriched in the outer leaflet of the plasma membrane oscillated in a highly coordinated manner in vivo and in vitro. Lipid metabolite oscillations were strongly attenuated upon siRNA-mediated clock disruption in human primary myotubes. Taken together, our data suggest an essential role for endogenous cell-autonomous human skeletal muscle oscillators in regulating lipid metabolism independent of external synchronizers, such as physical activity or food intake.lipid metabolism | circadian clock | human skeletal muscle | human primary myotubes | lipidomics C ircadian oscillations are daily cycles in behavior and physiology that are driven by the existence of underlying intrinsic biological clocks with near 24-h periods. This anticipatory mechanism has evolved to ensure that all aspects of behavior and physiology, including metabolic pathways, are temporally coordinated with daily cycles of rest-activity and feeding to provide the organism with an adaptive advantage (1). In mammals, circadian oscillations are driven by a master pacemaker, located in the suprachiasmatic nucleus (SCN) of the hypothalamus, which orchestrates subsidiary oscillators in peripheral organs via neuronal, endocrine, and metabolic signaling pathways (2, 3).Large-scale gene expression datasets suggest that, in mammals, the vast majority of circadian-gene expression is highly organ-specific (4-6). Key metabolic functions in peripheral organs are subject to daily oscillations, such as carbohydrate and lipid metabolism by the liver, skeletal muscle, and endocrine pancreas (7).Skeletal muscle is a major contributor of whole-body metabolism and is the main site of glucose uptake in the postprandial state (8). Therefore, perturbations in glucose sensing and metabolism in skeletal muscle are strongly associated with insulin resistance in type 2 diabetes (T2D) (9). Recent data support a fundamental role for the circadian muscle clock in the regulation of glucose uptake, with a significant re...

Time zones of pancreatic islet metabolism

“…Accumulating evidence suggests that altogether peripheral clocks play a critical role in regulating glucose and lipid homeostasis . Of note, mutations in the essential clock genes BMAL1 and CLOCK cause various metabolic disorders, while perturbations of metabolic pathways in mice, fed with a high‐fat diet, dampen the amplitude of circadian oscillations and lengthen their period .…”

The importance of being rhythmic: Living in harmony with your body clocks

“…121 In the liver, REV-ERBα couples glucocorticoid signalling to energy metabolism via binding of the hepatocyte nuclear transcription factors HNF4A/HNF6. 40,53,[129][130][131][132][133][134][135] Integrative metabolomic profiling of 8 mouse tissues, conducted around the clock upon physiological conditions or high-fat diet, further exemplifies this tight temporal orchestration of whole-body metabolism by nutrient intake. In turn, knockdown of the m6A methyltransferase | 5 of 14 DIBNER METTL3 conducted in vitro reduced PPARα methylation and increased its mRNA stability, thus leading to the altered hepatic accumulation of lipids.…”

“…Concomitant with massive changes in gene expression, immune responses mediated by natural killer cells, notably those involving Jun/AP1 and STAT pathways were reduced. 129,133,135 Strikingly, rhythmic metabolic patterns of human serum and urine were altered by sleep deprivation. 150 Human plasma proteomic conducted across 24 hours revealed strong rhythmicity for 30 proteins, mostly associated with tyrosine kinase signalling and extracellular matrix organization.…”

“…39,[184][185][186][187] Application of this approach to primary cells established from tissue biopsies derived from different organs allowed dissecting the molecular characteristics of peripheral clocks operative in human pancreatic islets, primary skeletal myotubes, adipocytes and thyrocytes synchronized in vitro. 41,78,129,189 A comparison between circadian transcriptomic and lipidomic analyses from serial skeletal muscle tissue biopsies collected in vivo and primary myotubes synchronized in vitro suggest an important effect of the cell-autonomous muscle clock on 8 of 14 | DIBNER the temporal regulation of the transcriptional and metabolic landscape. 41,78,129,189 A comparison between circadian transcriptomic and lipidomic analyses from serial skeletal muscle tissue biopsies collected in vivo and primary myotubes synchronized in vitro suggest an important effect of the cell-autonomous muscle clock on 8 of 14 | DIBNER the temporal regulation of the transcriptional and metabolic landscape.…”

“…41,78,129,189 A comparison between circadian transcriptomic and lipidomic analyses from serial skeletal muscle tissue biopsies collected in vivo and primary myotubes synchronized in vitro suggest an important effect of the cell-autonomous muscle clock on 8 of 14 | DIBNER the temporal regulation of the transcriptional and metabolic landscape. 40,78,79,129,190 In summary, the in vitro approach for studying molecular human clocks in cultured explants and primary cells ( Figure 3) allows to assess inter-individual differences in circadian characteristics, 187 and to unravel functional inputs and outputs of the oscillators operative in nearly every human tissue, in a highly reliable and minimally invasive manner. 40,78,79,129,190 In summary, the in vitro approach for studying molecular human clocks in cultured explants and primary cells ( Figure 3) allows to assess inter-individual differences in circadian characteristics, 187 and to unravel functional inputs and outputs of the oscillators operative in nearly every human tissue, in a highly reliable and minimally invasive manner.…”