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Abstract
BackgroundDiabetes is the seventh leading cause of death in the USA, and disruption of circadian rhythms is gaining recognition as a contributing factor to disease prevalence. This disease is characterized by hyperglycemia and glucose intolerance and symptoms caused by failure to produce and/or respond to insulin. The skeletal muscle is a key insulin-sensitive metabolic tissue, taking up ~80 % of postprandial glucose. To address the role of the skeletal muscle molecular clock to insulin sensitivity and glucose tolerance, we generated an inducible skeletal muscle-specific Bmal1−/− mouse (iMSBmal1−/−).ResultsProgressive changes in body composition (decreases in percent fat) were seen in the iMSBmal1−/− mice from 3 to 12 weeks post-treatment as well as glucose intolerance and non-fasting hyperglycemia. Ex vivo analysis of glucose uptake revealed that the extensor digitorum longus (EDL) muscles did not respond to either insulin or 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) stimulation. RT-PCR and Western blot analyses demonstrated a significant decrease in mRNA expression and protein content of the muscle glucose transporter (Glut4). We also found that both mRNA expression and activity of two key rate-limiting enzymes of glycolysis, hexokinase 2 (Hk2) and phosphofructokinase 1 (Pfk1), were significantly reduced in the iMSBmal1−/− muscle. Lastly, results from metabolomics analyses provided evidence of decreased glycolytic flux and uncovered decreases in some tricarboxylic acid (TCA) intermediates with increases in amino acid levels in the iMSBmal1−/− muscle. These findings suggest that the muscle is relying predominantly on fat as a fuel with increased protein breakdown to support the TCA cycle.ConclusionsThese data support a fundamental role for Bmal1, the endogenous circadian clock, in glucose metabolism in the skeletal muscle. Our findings have implicated altered molecular clock dictating significant changes in altered substrate metabolism in the absence of feeding or activity changes. The changes in body composition in our model also highlight the important role that changes in skeletal muscle carbohydrate, and fat metabolism can play in systemic metabolism.
Highlights
Diabetes is the seventh leading cause of death in the USA, and disruption of circadian rhythms is gaining recognition as a contributing factor to disease prevalence
We focused on the levels of glucose transporter type 4 (Glut4), and we found that in the iMSBmal1−/−, mRNA expression is reduced by 47.2 % and protein content of the glucose transporter was reduced by 75.5 % relative to that measured in iMSBmal1+/+ mice (Fig. 4a, b)
Real-time PCR results confirmed that phosphofructokinase 1 (Pfk1) mRNA expression was reduced by 23.3 % in iMSBmal1−/− mice, and like HK2, we found that enzyme activity for Pfk1 was significantly reduced by 52.4 % (Fig. 5c, d)
Summary
Diabetes is the seventh leading cause of death in the USA, and disruption of circadian rhythms is gaining recognition as a contributing factor to disease prevalence. Results from metabolomics analyses provided evidence of decreased glycolytic flux and uncovered decreases in some tricarboxylic acid (TCA) intermediates with increases in amino acid levels in the iMSBmal1−/− muscle These findings suggest that the muscle is relying predominantly on fat as a fuel with increased protein breakdown to support the TCA cycle. Circadian rhythms are generated by a transcription-translation feedback loop known as the molecular clock Described, this clock has positive limb factors, brain and muscle arnt-like protein 1 (Bmal1) and circadian locomotor output cycles kaput (Clock), and negative limb genes period (period 1 and 2 (Per1/2)) and cryptochrome (Cry1/2) [17, 32]. Circadian rhythms have an important role in metabolism, and metabolism is a key function regulated by the molecular clock in a tissue-specific manner [2, 13, 19, 27, 36]
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