Muscle insulin sensitivity and glucose metabolism are controlled by the intrinsic muscle clock

“jsp) [27] and gene sets from the KEGG database.”

“Expression values were generated from fluorescence signals using the robust multi-array average procedure (RMA) [23].”

“Global inactivation of different clock genes leads to altered glucose metabolism: Clock mutant mice display hyperglycemia, obesity and metabolic syndrome [9]; Per1/Per2 double knockout mice and Cry1/Cr2 double knockout mice exhibit glucose”

“In addition to the core clock genes, all tissues contain a large number of oscillating genes, most of which are tissue-specific and regulated either directly by the core oscillator or by extrinsic circadian signals [7].”

“Speci fi cally, intensity levels have been background adjusted, normalized using quantile normalization, and log 2 expression values calculated using median polish summarization and Entrez custom chip de fi nition fi les for mouse arrays (version 14.1.0) [24].”

“Skeletal muscle is a major site of insulin-stimulated glucose disposal [56] and muscle insulin resistance has been considered to be one of the earliest factors in the pathogenesis of the metabolic syndrome [57].”

“Global Bmal1 knockout causes not only loss of circadian rhythm of locomotor activity but also reduced total activity levels [42] and rescue experiments in which BMAL1 was constitutively expressed in skeletal muscle of Bmal1 null mice showed that musclespecific rescue restored activity levels, though not circadian rhyt…”

“Selective knockout of Bmal1 in the liver causes hypoglycemia [10], while ablation of the same gene in the endocrine pancreas leads to fasting hyperglycemia and severe glucose intolerance [15].”

“What is the cause of the altered response to insulin in muscles from Bmal1 mKO mice? The canonical insulin signaling pathway is apparently not responsible for this change, since AKT phosphorylation, which is reduced in obesity and diabetes [60], was unchanged, and was similarly increased by insulin in muscles from con…”

“The method to measure glucose transport was similar to that described previously [30,31] with some modi fi cations [32].”

“Skeletal muscles with functional clocks are thus primed to choose “the right fuel at the right time“ [70], whereas muscle-specific Bmal1 inactivation and the consequent disruption of the muscle clock would cause a state of reduced metabolic flexibility [71], characterized by muscle insulin resistance and altered gluco…”

“Out of the 684 (Ctrl TA) and 1359 (Ctrl SOL) 24-h cycling genes identi fi ed by the JTK_CYCLE algorithm [26], 487 genes in TA and 666 genes in SOL lost their 24-h oscillation in the absence of Bmal1 , showing that a signi fi cant proportion of cycling genes in mouse skeletal muscles is controlled by…”

“intolerance [10,11]; Bmal1 null mice are also glucose intolerant, and show altered gluconeogenesis [10,12].”

“…altered glucose metabolism: Clock mutant mice display hyperglycemia, obesity and metabolic syndrome [9]; Per1 / Per2 double knockout mice and Cry1 / Cr2 double knockout mice exhibit glucose intolerance [10,11]; Bmal1 null mice are also glucose intolerant, and show altered gluconeogenesis [10,12].”

“Cryosections of fast and slow muscles were stained with monoclonal anti-myosin heavy chain (MyHC) antibodies produced in our lab [21], and now distributed by the Developmental Studies Hybridoma Bank (DSHB, University of Iowa): BA-D5 (IgG2b, supernatant, 1:100 dilution) speci fi c for MyHC-I, SC-71…”

“The regulation of body metabolism by normal circadian rhythms has important clinical implications, since chronic circadian misalignment, as occurs in shift work, is associated with a higher prevalence of insulin resistance, obesity, metabolic syndrome and diabetes [4 – 6].”

“The regulation of body metabolism by normal circadian rhythms has important clinical implications, since chronic circadian misalignment, as occurs in shift work, is associated with a higher prevalence of insulin resistance, obesity, metabolic syndrome and diabetes [4 – 6].”

“For example, the fact that Per2 oscillation was only mildly affected in mKO skeletal muscles likely reflects a role for oscillating systemic signals in the regulation of this gene, as was demonstrated in a liver-specific Bmal1 knockout [44,45].”

“While many behavioral and physiological processes linked to energy metabolism show daily fl uctuations under the control of an endogenous circadian clock and environmental cues, the circadian clock is itself in fl uenced by feeding and activity rhythms, and ultimately metabolic state [1 – 3].”

“While many behavioral and physiological processes linked to energy metabolism show daily fl uctuations under the control of an endogenous circadian clock and environmental cues, the circadian clock is itself in fl uenced by feeding and activity rhythms, and ultimately metabolic state [1 – 3].”

“This change is likely relevant, because skeletal muscle-specific GLUT4þ / mice, which have a 40–50% reduction in GLUT4 protein in skeletal muscles, also show a partial reduction in insulin-stimulated glucose uptake in skeletal muscles [66].”

“During the rest/fasting phase, reduced insulin and contractile activity levels decrease glucose uptake and glycolytic flux in muscle cells, while promoting fatty acid β-oxidation, further inhibiting glucose oxidation by PDK4 activation and PDH inhibition [69].”

“For example, the fact that Per2 oscillation was only mildly affected in mKO skeletal muscles likely reflects a role for oscillating systemic signals in the regulation of this gene, as was demonstrated in a liver-specific Bmal1 knockout [44,45].”

“While many behavioral and physiological processes linked to energy metabolism show daily fl uctuations under the control of an endogenous circadian clock and environmental cues, the circadian clock is itself in fl uenced by feeding and activity rhythms, and ultimately metabolic state [1 – 3].”

“intolerance [10,11]; Bmal1 null mice are also glucose intolerant, and show altered gluconeogenesis [10,12].”

“The method to measure glucose transport was similar to that described previously [30,31] with some modi fi cations [32].”

“The various responses of clock genes and clock-associated genes to Bmal1 inactivation were in agreement with previous reports for global and tissue-speci fi c Bmal1 knockouts [16].”

“Animals Muscle-specific inactivation of Bmal1 (mKO) was obtained from the cross between a C57BL/6 mouse line with floxed Bmal1 [18] and a C57BL/6 mouse line carrying a Cre recombinase transgene under control of the Mlc1f promoter (Mlc1f–Cre) [19].”

“Changes in skeletal muscle structure and function were described in a global Bmal1 knockout model [41]; however it is unclear whether these are direct consequences of muscle Bmal1 deficiency or secondary effects due to the disruption of the circadian pacemaker or other peripheral clocks.”

“Global metabolic profiling Metabolic profiling, peak identification, and curation were performed by Metabolon using described methods [28].”

“Other experimental conditions, such as lipid infusion [61– 63], and skeletal muscle-specific genetic models, such as overexpression of PPARα [64] or inactivation of carnitine acetyltransferase [65], were also shown to cause muscle insulin resistance without affecting AKT phosphorylation.”

“A similar shift in Pdk4 expression was previously observed in muscles from a global Clock mutant [55].”

“Skeletal muscles with functional clocks are thus primed to choose “the right fuel at the right time“ [70], whereas muscle-specific Bmal1 inactivation and the consequent disruption of the muscle clock would cause a state of reduced metabolic flexibility [71], characterized by muscle insulin resistance and altered gluco…”

“It is likely that other insulin-sensitive tissues, such as adipose tissue, compensate for reduced muscle insulin sensitivity at the whole body level [59].”

“Global Bmal1 knockout causes not only loss of circadian rhythm of locomotor activity but also reduced total activity levels [42] and rescue experiments in which BMAL1 was constitutively expressed in skeletal muscle of Bmal1 null mice showed that musclespecific rescue restored activity levels, though not circadian rhyt…”

“Pdp1 was also previously reported to be a circadian gene in the brain (listed as Ppm2c ) [49].”

“AKT is known to control skeletal muscle glucose transport by phosphorylating the Rab-GTPase-activating proteins TBC1D4 (AS160) and TBC1D1, thus promoting the translocation of GLUT4 to the plasma membrane [46–48].”

“AKT is known to control skeletal muscle glucose transport by phosphorylating the Rab-GTPase-activating proteins TBC1D4 (AS160) and TBC1D1, thus promoting the translocation of GLUT4 to the plasma membrane [46 – 48].”

“Other experimental conditions, such as lipid infusion [61 – 63], and skeletal muscle-speci fi c genetic models, such as overexpression of PPAR α [64] or inactivation of carnitine acetyltransferase [65], were also shown to cause muscle insulin resistance without affecting AKT phosphorylation.”

“Force measurements In vivo force measurements were performed as described previously [22].”

“Although a non-canonical E-Box, the core binding motif CACGGG was shown to efficiently bind the CLOCK:BMAL1 heterodimer in a screen for circadian transcription factor DNA binding activity [51].”

“Mice with whole body Bmal1 knockout were reported to stop growing around 16 weeks of age, display progressive arthropathy, sarcopenia and loss of fat tissue, and die between 26 and 52 weeks of age [39,40].”

“Tbc1d1 was previously identi fi ed as a circadian gene also in bone and atrial myocardium [49] and a comparative computational screen for conserved putative CLOCK:BMAL1-binding elements in the mouse and human genomes identi fi ed a potential cis -regulatory CLOCK:BMAL1 binding site within Tbc1d1 [50].”

“Skeletal muscle is a major site of insulin-stimulated glucose disposal [56] and muscle insulin resistance has been considered to be one of the earliest factors in the pathogenesis of the metabolic syndrome [57].”

“All transcriptome and metabolome data are accessible through the CircadiOmics Website (http://circadiomics.igb.uci.edu), allowing users to search and view genes of interest and detected metabolites in Ctrl and Bmal1 mKO skeletal muscles, as well as their interactions and statistics.”

“All transcriptome and metabolome data are accessible through the CircadiOmics Website (http://circadiomics.igb.uci.edu), allowing users to search and view genes of interest and detected metabolites in Ctrl and Bmal1 mKO skeletal muscles, as well as their interactions and statistics.”

“The Bmal1 paralog, Bmal2 (Arntl2/Mop9), which is downregulated in the liver of Bmal1 null mice [42] and can functionally replace Bmal1 [43], was found to be expressed at low levels without daily fluctuations in mouse skeletal muscle, and”

“All transcriptome and metabolome data are accessible through the CircadiOmics Website (http://circadiomics.igb.uci.edu), allowing users to search and view genes of interest and detected metabolites in Ctrl and Bmal1 mKO skeletal muscles, as well as their interactions and statistics.”

“AKT is known to control skeletal muscle glucose transport by phosphorylating the Rab-GTPase-activating proteins TBC1D4 (AS160) and TBC1D1, thus promoting the translocation of GLUT4 to the plasma membrane [46 – 48].”

“The method to measure glucose oxidation was similar to that previously described [34] with some modifications.”

“Networks and subnetworks can be explored and rearranged interactively, providing a comprehensive systems-level view of the circadian molecular pathways [29].”

“The hierarchical role of the central pacemaker was demonstrated by the finding that circadian rhythms of blood glucose, insulin and glucagon, as well as glucose tolerance, are all abolished by SCN lesion [14].”

“A second knockout model with inducible muscle-specific inactivation of Bmal1 (imKO) was obtained by crossing the Bmal1 floxed line with mice carrying an α-skeletal actin driven Cre recombinase fused to a mutated estrogen receptor [20], which can be activated by treatment with tamoxifen (i.”

“Other experimental conditions, such as lipid infusion [61 – 63], and skeletal muscle-speci fi c genetic models, such as overexpression of PPAR α [64] or inactivation of carnitine acetyltransferase [65], were also shown to cause muscle insulin resistance without affecting AKT phosphorylation.”

“Tbc1d1 was previously identi fi ed as a circadian gene also in bone and atrial myocardium [49] and a comparative computational screen for conserved putative CLOCK:BMAL1-binding elements in the mouse and human genomes identi fi ed a potential cis -regulatory CLOCK:BMAL1 binding site within Tbc1d1 [50].”

“Tbc1d1 was previously identi fi ed as a circadian gene also in bone and atrial myocardium [49] and a comparative computational screen for conserved putative CLOCK:BMAL1-binding elements in the mouse and human genomes identi fi ed a potential cis -regulatory CLOCK:BMAL1 binding site within Tbc1d1 [50].”

“SDS-PAGE and western blotting Muscle lysates were prepared as previously reported [38], protein concentration determined using the bicinchoninic acid assay (Pierce, Rockford, IL, USA) and SDS-PAGE performed using 10% hand-cast gels.”

“AKT is known to control skeletal muscle glucose transport by phosphorylating the Rab-GTPase-activating proteins TBC1D4 (AS160) and TBC1D1, thus promoting the translocation of GLUT4 to the plasma membrane [46 – 48].”

“The method to measure glucose transport was similar to that described previously [30,31] with some modifications [32].”

“A circadian rhythm of glucose uptake was also demonstrated in muscle cell cultures, but these daily fluctuations were observed both in basal and insulinstimulated conditions [17].”

“a site that increases in skeletal muscle after fasting [54], was significantly increased in mKO muscles (Figure 5D).”

“Muscles were quickly removed, rinsed in sterile saline, and washed 3 5 min (100 RPM) in ice-cold KHB to clear extracellular space of 2-DG [33].”

“Other experimental conditions, such as lipid infusion [61 – 63], and skeletal muscle-speci fi c genetic models, such as overexpression of PPAR α [64] or inactivation of carnitine acetyltransferase [65], were also shown to cause muscle insulin resistance without affecting AKT phosphorylation.”

“Similar diurnal fluctuations in PDHa activity were also reported in cardiac muscle [53].”