White adipose tissue and liver are important angiotensinogen (AGT) production sites. Until now, plasma AGT was considered to be a reflection of hepatic production. Because plasma AGT concentration has been reported to correlate with blood pressure, and to be associated with body mass index, we investigated whether adipose AGT is released locally and into the blood stream. For this purpose, we have generated transgenic mice either in which adipose AGT is overexpressed or in which AGT expression is restricted to adipose tissue. This was achieved by the use of the aP2 adipocyte-specific promoter driving the expression of rat agt cDNA in both wild-type and hypotensive AGT-deficient mice. Our results show that in both genotypes, targeted expression of AGT in adipose tissue increases fat mass. Mice whose AGT expression is restricted to adipose tissue have AGT circulating in the blood stream, are normotensive, and exhibit restored renal function compared with AGT-deficient mice. Moreover, mice that overexpress adipose AGT have increased levels of circulating AGT, compared with wild-type mice, and are hypertensive. These animal models demonstrate that AGT produced by adipose tissue plays a role in both local adipose tissue development and in the endocrine system, which supports a role of adipose AGT in hypertensive obese patients.

No abstract

Daily synchronous rhythms of cell division at the tissue or organism level are observed in many species and suggest that the circadian clock and cell cycle oscillators are coupled. For mammals, despite known mechanistic interactions, the effect of such coupling on clock and cell cycle progression, and hence its biological relevance, is not understood. In particular, we do not know how the temporal organization of cell division at the singlecell level produces this daily rhythm at the tissue level. Here we use multispectral imaging of single live cells, computational methods, and mathematical modeling to address this question in proliferating mouse fibroblasts. We show that in unsynchronized cells the cell cycle and circadian clock robustly phase lock each other in a 1:1 fashion so that in an expanding cell population the two oscillators oscillate in a synchronized way with a common frequency. Dexamethasone-induced synchronization reveals additional clock states. As well as the low-period phase-locked state there are distinct coexisting states with a significantly higher period clock. Cells transition to these states after dexamethasone synchronization. The temporal coordination of cell division by phase locking to the clock at a single-cell level has significant implications because disordered circadian function is increasingly being linked to the pathogenesis of many diseases, including cancer.coupled oscillators | oscillations | circadian rhythms | gating

Most living organisms show circadian (ϳ24 h) rhythms in physiology and behavior. These oscillations are generated by endogenous circadian clocks, present in virtually all cells where they control key biological processes. Although circadian gating of mitosis has been reported for many years in some peripheral tissues, the underlying molecular mechanisms have remained poorly understood. Here we show that the cell cycle inhibitor p21 WAF1/CIP1 is rhythmically expressed in mouse peripheral organs. This rhythmic pattern of mRNA and protein expression was recapitulated in vitro in serum-shocked differentiated skeletal muscle cells. p21 WAF1/CIP1 circadian expression is dramatically increased and no longer rhythmic in clock-deficient Bmal1 ؊/؊ knock-out mice. Biochemical and genetic data show that oscillation of p21 WAF1/CIP1 gene transcription is regulated by the antagonistic activities of the orphan nuclear receptors REV-ERB␣/␤ and ROR␣4/␥, which are core clock regulators. Importantly, p21 WAF1/CIP1 overexpressing Bmal1 ؊/؊ primary hepatocytes exhibit a decreased proliferation rate. This phenotype could be reversed using small interfering RNA-mediated knockdown of p21 WAF1/CIP1 . These data establish a novel molecular link between clock and cell cycle genes and suggest that the G 1 progression phase is a target of the circadian clock during liver cell proliferation.Many physiological and behavioral processes display daynight oscillations in most organisms including mammals. These biological rhythms are controlled by endogenous selfsustained circadian (ϳ24 h) oscillators that operate not only in the central clock located in the suprachiasmatic nuclei of the hypothalamus but also in virtually all peripheral cells. Light is the main synchronizer of the central clock, which in turn coordinates the phases of peripheral oscillators regulating specific physiological outputs. Forward genetics and biochemical approaches have established the molecular basis underlying circadian oscillations in mammalian tissues (1-4). This mechanism involves complex interlocked positive and negative transcriptional/posttranslational feedback loops between the clock genes Clock, Bmal1, Per1, Per2, Cry1, and Cry2 and their protein products. The robustness of the oscillations is ensured by additional regulators such as the REV-ERB␣ (NR1D1) and ROR␣ (NR1F1) orphan nuclear receptors (5, 6). Furthermore, extensive multilevel posttranslational regulation of various clock components has also been shown to play an important role in the molecular clock mechanism (7).There are substantial evidences that progression through the cell cycle occurs at specific times of the day/night cycle, suggesting that a function of the circadian clock system is to control this fundamental process. Notably, after the initial observation almost 40 years ago that cell division of the unicellular algae Euglena was controlled by an endogenous clock (8), numerous studies have shown a circadian variation of the proliferative activity in mammalian tissues such as the epithelia ...

The rat peroxisome-proliferator-activated receptor (PPAR) was expressed in insect cells and was shown to bind to a cognate PPAR response element (PPRE) from the acyl-CoA oxidase gene. Upon purification, PPAR was no longer able to bind DNA, although binding could be restored by addition of insect cell extracts. We investigated whether the retinoid X receptor (RXR) could supplement for this accessory activity. The rat RXRa cDNA was cloned and it was found that addition of in vitro-translated RXRa to purified PPAR facilitated binding of PPAR to a PPRE. Furthermore, an additional activity, which appeared to be distinct from rRXRa, was found in COS cell nuclear extracts that enabled binding of PPAR to a PPRE. Transient expression ofRXRa in CHO cells was found to be essential for the response of a chloramphenicol acetyltransferase reporter construct containing PPREs to activators of PPAR. These results raise the possibility of convergence of the PPAR and retinoid-dependent signaling pathways on promoters containing PPRE-like responsive elements.

Mutations of clock genes can lead to diabetes and obesity. REV-ERBα, a nuclear receptor involved in the circadian clockwork, has been shown to control lipid metabolism. To gain insight into the role of REV-ERBα in energy homeostasis in vivo, we explored daily metabolism of carbohydrates and lipids in chow-fed, unfed, or high-fat-fed Rev-erbα(-/-) mice and their wild-type littermates. Chow-fed Rev-erbα(-/-) mice displayed increased adiposity (2.5-fold) and mild hyperglycemia (∼10%) without insulin resistance. Indirect calorimetry indicates that chow-fed Rev-erbα(-/-) mice utilize more fatty acids during daytime. A 24-h nonfeeding period in Rev-erbα(-/-) animals favors further fatty acid mobilization at the expense of glycogen utilization and gluconeogenesis, without triggering hypoglycemia and hypothermia. High-fat feeding in Rev-erbα(-/-) mice amplified metabolic disturbances, including expression of lipogenic factors. Lipoprotein lipase (Lpl) gene, critical in lipid utilization/storage, is triggered in liver at night and constitutively up-regulated (∼2-fold) in muscle and adipose tissue of Rev-erbα(-/-) mice. We show that CLOCK, up-regulated (2-fold) at night in Rev-erbα(-/-) mice, can transactivate Lpl. Thus, overexpression of Lpl facilitates muscle fatty acid utilization and contributes to fat overload. This study demonstrates the importance of clock-driven Lpl expression in energy balance and highlights circadian disruption as a potential cause for the metabolic syndrome.

We have cloned a member of the nuclear receptor superfamily. The cDNA was isolated from a rat liver library and encodes a protein of 446 aa with a predicted mass of 50 kDa. This clone (OR-1) shows no striking homology to any known member of the steroid/thyroid hormone receptor superfamily. The most related receptor is the ecdysone receptor and the highest homologies represent <10%v in the amino-terminal domain, between 15-37% in the carboxyl-terminal domain and 50-62% in the DNA binding domain. The expression of OR-1 appears to be widespread in both fetal and adult rat tissues. Potential DNA response elements composed of a direct repeat of the hexameric motifAGGTCA spaced by 0-6 ntwere tested in gel shift experiments. OR-1 was shown to interact with the 9-cisretinoic acid receptor (retinoid X receptor, RXR) and the OR-

The circadian timing system coordinates many aspects of mammalian physiology and behavior in synchrony with the external light/dark cycle. These rhythms are driven by endogenous molecular clocks present in most body cells. Many clock outputs are transcriptional regulators, suggesting that clock genes primarily control physiology through indirect pathways. Here, we show that Krüppel-like factor 10 (KLF10) displays a robust circadian expression pattern in wild-type mouse liver but not in clock-deficient Bmal1 knockout mice. Consistently, the Klf10 promoter recruited the BMAL1 core clock protein and was transactivated by the CLOCK-BMAL1 heterodimer through a conserved E-box response element. Profiling the liver transcriptome from Klf10 ؊/؊ mice identified 158 regulated genes with significant enrichment for transcripts involved in lipid and carbohydrate metabolism. Importantly, approximately 56% of these metabolic genes are clock controlled. Male Klf10 ؊/؊ mice displayed postprandial and fasting hyperglycemia, a phenotype accompanied by a significant time-of-day-dependent upregulation of the gluconeogenic gene Pepck and increased hepatic glucose production. Consistently, functional data showed that the proximal Pepck promoter is repressed directly by KLF10. Klf10؊/؊ females were normoglycemic but displayed higher plasma triglycerides. Correspondingly, rhythmic gene expression of components of the lipogenic pathway, including Srebp1c, Fas, and Elovl6, was altered in females. Collectively, these data establish KLF10 as a required circadian transcriptional regulator that links the molecular clock to energy metabolism in the liver.In mammals, including humans, many aspects of behavior and physiology display daily oscillations. Twenty-four-hour rhythms of sleep, feeding behavior, core body temperature, hormone secretion, lipid and carbohydrate metabolism, and blood pressure are well-documented examples. These rhythms are driven by self-sustained endogenous clocks located in virtually all cells of the body and forming an integrated system (44, 48). The circadian (ϳ24-h) system is organized hierarchically with, at the top, a master clock located in the suprachiasmatic nuclei (SCN) of the hypothalamus and oscillating with an approximately 24-h period in the absence of external time cues. Every day, this central clock is reset by light through the retinohypothalamic tract to keep the physiology synchronized to the external light/dark (LD) cycle. Circadian clocks present in the periphery are entrained by the SCN through internal synchronizers that have yet to be identified, and although they display self-sustained oscillations at the single-cell level, at the organ and systemic levels they require an intact SCN clock to remain in phase (63). Forward genetics and biochemical approaches have identified a set of core clock genes that interact through complex positive-and negative-feedback loops to form a molecular clock generating ϳ24-h oscillations (29). In this mechanism, the two bHLH-PAS transcriptional activators CLOCK (or N...