Abstract
Hepatic glycogen content is important for glucose homeostasis and exhibits robust circadian rhythms that peak at the end of the active phase in mammals. The activities of the rate-limiting enzymes for glycogenesis and glycogenolysis also show circadian rhythms, and the balance between them forms the circadian rhythm of the hepatic glycogen content. However, no direct evidence has yet implicated the circadian clock in the regulation of glycogen metabolism at the molecular level. We show here that a Clock gene mutation damps the circadian rhythm of the hepatic glycogen content, as well as the circadian mRNA and protein expression of Gys2 (glycogen synthase 2), which is the rate-limiting enzyme of glycogenesis in the liver. Transient reporter assays revealed that CLOCK drives the transcriptional activation of Gys2 via two tandemly located E-boxes. Chromatin immunoprecipitation assays of liver tissues revealed that CLOCK binds to these E-box elements in vivo, and real time reporter assays showed that these elements are sufficient for circadian Gys2 expression in vitro. Thus, CLOCK regulates the circadian rhythms of hepatic glycogen synthesis through transcriptional activation of Gys2.
Highlights
Many organisms display physiological and behavioral rhythms that are entrained to the 24-h cycle of light and darkness on Earth
Clock Mutation Affects Temporal Expression of Genes Associated with Glucose Metabolism—We examined the temporal expression profile of genes associated with glucose metabolism in the liver of wild-type and Clock mutant mice to determine the relationship between the circadian clock and glucose metabolism (Fig. 1)
The circadian expression of Glycogen synthase 2 (Gys2) was remarkably damped, whereas the expression of Pepck and Glucose transporter 2 (Glut2) was slightly damped in the livers of Clock mutant mice
Summary
Animals—Male Jcl:ICR (Clea Japan Inc., Tokyo, Japan) and homozygous Clock mutant mice on a Jcl:ICR background [20] at 7–10 weeks of age were maintained under a 12 h of light/12 h of dark cycle (lights on at 0:00 and lights off at 12:00) for at least 2 weeks before the day of the experiments. Hepatic Glycogen Content—Frozen liver tissues (ϳ200 mg) were homogenized in 10% trichloroacetic acid, and 1 ml of homogenate was separated by centrifugation at 17,800 ϫ g for 10 min at 4 °C. The hydrolyzed free glucose concentration was evaluated using a glucose C-test Wako (Wako Pure Chemical Industries, Osaka, Japan) and corrected by liver weight. Blood Metabolic Parameters—Plasma glucose was measured using a glucose C-test Wako (Wako Pure Chemical Industries) according to the manufacturer’s protocol. Glycogen accumulation was analyzed in 10-m cryosections of frozen liver using the periodic acid-Schiff’s reaction kit (Muto Pure Chemicals Co. Ltd., Tokyo, Japan). Isolation of RNA and Real Time Quantitative Reverse Transcription-PCR—Total RNA was isolated from liver tissues using RNAiso (TAKARA Bio Inc., Shiga, Japan) and reverse-transcribed using the PrimeScript RT reagent kit (TAKARA Bio) according to the manufacturer’s protocol. Western Blotting—Frozen livers were homogenized in icecold lysis buffer
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