Brain and muscle Arnt-like protein-1 (BMAL1; also known as MOP3 or Arnt3) is a transcription factor known to regulate circadian rhythm. Here, we established its involvement in the control of adipogenesis and lipid metabolism activity in mature adipocytes. During adipose differentiation in 3T3-L1 cells, the level of BMAL1 mRNA began to increase 4 days after induction and was highly expressed in differentiated cells. In white adipose tissues isolated from C57BL͞6J mice, BMAL1 was predominantly expressed in a fraction containing adipocytes, as compared with the stromalvascular fraction. BMAL1 knockout mice embryonic fibroblast cells failed to be differentiated into adipocytes. Importantly, adding BMAL1 back by adenovirus gene transfer restored the ability of BMAL1 knockout mice embryonic fibroblast cells to differentiate. Knock-down of BMAL1 expression in 3T3-L1 cells by an RNA interference technique allowed the cells to accumulate only minimum amounts of lipid droplets in the cells. Adenovirus-mediated expression of BMAL1 in 3T3-L1 adipocytes resulted in induction of several factors involved in lipogenesis. The promoter activity of these genes was stimulated in a BMAL1-dependent manner. Interestingly, expression of these factors showed clear circadian rhythm in mice adipose tissue. Furthermore, overexpression of BMAL1 in adipocytes increased lipid synthesis activity. These results indicate that BMAL1, a master regulator of circadian rhythm, also plays important roles in the regulation of adipose differentiation and lipogenesis in mature adipocytes.circadian rhythm A dipocytes play essential metabolic roles not only serving as massive energy reserves but also secreting hormones and cytokines that regulate metabolic activities (1, 2). The link between metabolic activity in adipocytes and circadian rhythm has long been studied; e.g., glucose and lipid homeostasis are well known to exhibit circadian variation (3-6). More recently, circadian expression of adiponectin receptors in adipocytes was reported (7). Therefore, molecular clock may play important roles in the regulation of metabolic activity in adipocytes. In a previous study, we reported that white adipose tissue contains functional molecular clock and that expression of several adipocytokines, including leptin, and plasminogen activator inhibitor-1 display circadian rhythm (8). The diurnal rhythm in the level of these molecules suggests that the molecular clock is at least partly associated with the onset of metabolic syndrome.The molecular clock is composed of transcriptional feedback loops in organisms ranging from cyanobacteria to humans. Brain and muscle Arnt-like protein-1 [BMAL1; also referred to as MOP3 (9) or Arnt3 (10)] is a transcription factor playing central roles in the regulation of circadian rhythms (11). BMAL1 forms heterodimers with another basic helix-loop-helix͞PAS protein, CLOCK, which drives transcription from E-box elements found in the promoter of circadian responsive genes, including period (Per)1 and cryptochrome (Cry). After translati...
A link between circadian rhythm and metabolism has long been discussed. Circadian rhythm is controlled by positive and negative transcriptional and translational feedback loops composed of several clock genes. Among clock genes, the brain and muscle Arnt-like protein-1 (BMAL1) and circadian locomotor output cycles kaput (CLOCK) play important roles in the regulation of the positive rhythmic transcription. In addition to control of circadian rhythm, we have previously shown that BMAL1 regulates adipogenesis. In metabolic syndrome patients, the function of BMAL1 is dysregulated in visceral adipose tissue. In addition, analysis of SNPs has revealed that BMAL1 is associated with susceptibility to hypertension and type II diabetes. Furthermore, the significant roles of BMAL1 in pancreatic β cells proliferation and maturation were recently reported. These results suggest that BMAL1 regulates energy homeostasis. Therefore, in this study, we examined whether loss of BMAL1 function is capable of inducing metabolic syndrome. Deficient of the Bmal1 gene in mice resulted in elevation of the respiratory quotient value, indicating that BMAL1 is involved in the utilization of fat as an energy source. Indeed, lack of Bmal1 reduced the capacity of fat storage in adipose tissue, resulting in an increase in the levels of circulating fatty acids, including triglycerides, free fatty acids, and cholesterol. Elevation of the circulating fatty acids level induced the formation of ectopic fat in the liver and skeletal muscle in Bmal1 -/- mice. Interestingly, ectopic fat formation was not observed in tissue-specific (liver or skeletal muscle) Bmal1 -/- mice even under high fat diet feeding condition. Therefore, we were led to conclude that BMAL1 is a crucial factor in the regulation of energy homeostasis, and disorders of the functions of BMAL1 lead to the development of metabolic syndrome.
Circadian rhythm is observed in most, if not all, of physiological functions, including metabolism, cell growth, etc. 1-6)The master pacemaker of circadian rhythm resides in the suprachiasmatic nucleus (SCN) of the hypothalamus and coordinates autonomous peripheral clocks located in organs such as the liver and kidney. [7][8][9][10][11][12] The molecular clock is composed of transcriptional feedback loops in organisms ranging from cyanobacteria to humans. Two transcription factors, the brain and muscle Arnt-like protein-1 (BMAL1; also referred to as MOP3 or Arnt3) and CLOCK, play central roles in the regulation of circadian rhythms.13-16) BMAL1 and CLOCK form a heterodimer and drive transcription from E-box elements found in the promoter of circadian-responsive genes, including period (Per)1 and cryptochrome (Cry). After translation of the Per and Cry proteins, the Per/Cry complex translocates to the nucleus, where it inhibits gene expression driven by BMAL1 and CLOCK. [17][18][19][20] There is a growing body of evidence that circadian rhythms also govern immunoreactions such as antigen presentation, lymphocyte proliferation, and cytokine expression. [21][22][23] More recently, diurnal expression of granzyme B and perforin in natural killer (NK) cells was observed in vivo.12) These circadian variations in the immune system are likely to be regulated by molecular clocks for the following reasons. First, RNAi-mediated Per2 knockdown caused a significant decrease of granzyme B and perforin levels in the rat-derived NK cell line RNK16.24) Second, Per2-deficient mice were more resistant to lipopolysaccharide (LPS)-induced endotoxic shock than wild-type mice.25) Also, the levels of the proinflammatory cytokines gamma interferon (IFN) and interleukin (IL)-1beta were dramatically decreased in Per2 Ϫ/Ϫ mice following LPS challenge, while the productions of tumor necrosis factor alpha (TNFa), IL-6, and IL-10 were approximately equal to those in wild-type mice.25) Furthermore, studies using BMAL1-deficient mice revealed that BMAL1 is required for B cell development. 26)Although several studies have shown that the immunoreactions and onset of the inflammatory diseases exhibit circadian variation, 27-30) the molecular details of the circadian rhythms in macrophages are still poorly understood. Therefore, in this study, we attempted to characterize circadian gene expression in mice peritoneal macrophages. We found that the expression of several clock genes such as BMAL1 exhibited daily oscillations in resident peritoneal macrophages. The expression of inflammatory factors such as monocyte chemoattractant protein-1 (MCP-1/JE) exhibited robust circadian rhythms. Suppression of BMAL1 expression by an RNAi technique lowered the nuclear factor-kappa B (NF-kB) activity followed by down-regulation of MCP-1/JE mRNA expression in RAW264.7 macrophage cells. Macrophages are known to play essential roles in immunoreactions and pathogenesis of atherosclerosis and arthropathy. 31,32) Consequently, these results may provide further insight ...
Water samples from the Nishitakase River in Kyoto, Japan, especially taken at sites below sewage plants, show significantly high mutagenicity in the Ames test. In the present study, mutagens in the river water were adsorbed to 24 g of blue rayon, extracted, and separated by HPLC on ODS columns. Five mutagenic compounds (I-V) were isolated, and they accounted for 21%, 17%, 11%, 12%, and 6%, respectively, of the total mutagenicity of the blue rayon-adsorbed materials. With compound I obtained from adsorbate to 24 g of blue rayon as a marker, a large quantity (1.1 mg) of mutagenic compound I was isolated by Sephadex LH-20 column chromatography and HPLC on ODS columns from material adsorbed to 27 kg of blue cotton. X-ray crystal analysis was carried out with the debrominated derivative of compound I. Based on this X-ray crystallography data and the UV, mass, and 1H-NMR spectra of both the derivative and compound I, the structure of compound I was determined to be 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-amino - 7-bromo-4-chloro-2H-benzotriazole (PBTA-1). PBTA-1 is a newly identified potent mutagen, inducing 1,200,000 revertants of Salmonella typhimurium YG1024 per microgram in the presence of S9 mix.
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