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...

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 ...

The maize Hm1 gene encoding the NADPH-dependent HC-toxin reductase is capable of detoxifying HC-toxin of fungus Cochliobolus carbonum. Here, we conducted the metabolic and biochemical analysis in transgenic rice plants overexpressing an HC-toxin reductase-like gene in rice (YK1 gene). Methods employing NADPH oxidation and capillary electrophoresis mass spectrometry analysis confirmed that YK1 possessed dihydroflavonol-4-reductase activity in vitro and in vivo. The overexpression of YK1 in both suspension-cultured cells and rice plants increased NAD(H) and NADP(H) levels by causing an increase in NAD synthetase and NAD kinase activities. Activity changes in enzymes that require NAD(P) as coenzymes were also noted in rice cells ectopically expressing YK1, where the cell death caused by hydrogen peroxide and bacterial disease was down-regulated. Thus, a strategy was proposed that the combination of dihydroflavonol-4-reductase activity and the elevated level of NAD(P)H pool may confer the prevention of induced cell death in planta. NADPH-dependent HC-toxin reductaseI n maize plants, the NADPH-dependent HC-toxin reductase (HCTR) detoxifies the HC-toxin produced by the fungus Cochliobolus carbonum (1). HCTR is encoded by the maize Hm1 gene (2). A similar gene is conserved in monocots such as rice (3) and barley (4). The molecular mechanism of the maize defense system against C. carbonum invasion remains largely unknown (5). The fungus C. carbonum is specialized for growth on maize, and HC-toxin is only biosynthesized in race 1. Therefore, it is uncertain that the role of Hm1 homologues in other monocots is detoxification (6). To determine the biological function of the Hm1 homologue in rice (the YK1 gene), we established transgenic rice plants with ectopic expression of YK1. The YK1-overexpressing rice plants showed enhanced resistance to rice blast disease and to abiotic stresses like UV radiation, increased salinity, and submergence (7). Here, we demonstrate that YK1 possesses dihydroflavonol-4-reductase (DFR) activity; to our knowledge, this is the first confirmation of such biochemical evidences for HCTR-like protein. Furthermore, the overexpression of YK1 in rice altered the activities of enzymes engaged in NAD biosynthesis, and of enzymes requiring NAD(P) as coenzymes. Moreover, overexpression of YK1 in rice conferred the prevention of cell death caused by the hydrogen peroxide as well as the bacterial disease. Materials and MethodsPlant Materials. Transgenic rice plants (Oryza sativa L. cv. Nipponbare) possessing either vector alone (the control line) or the YK1 gene (L-1 and L-2) were grown in a growth-cabinet (7). L-1 and L-2 are independent lines overexpressing the YK1 gene under the maize ubiquitin promoter. Suspension-cultured cells of rice were transferred weekly into liquid MS medium supplemented with sucrose [3% (wt/wt)], KH 2 PO 4 (340 mg/liter), thiamine (1 mg/liter), and 2,4-dichlorophenoxyacetic acid (0.2 mg/liter). Seedlings were maintained in the same medium devoid of 2,4-dichlorophenoxyacetic acid.