Fibroblast growth factor 21 (FGF21) is an effective metabolic regulator of glucose and lipid homeostasis in the context of insulin resistance, glucose intolerance and dyslipidemia in diabetic rodents and monkeys, and peroxisome proliferator-activated receptor a a (PPARa a) directly induces FGF21 expression in the rodent liver. Recent findings suggest that the effects and regulation of FGF21 qualitatively differ between rodents and humans. Here, we examined the effects of PPARa a and PPARg g agonists on FGF21 mRNA expression in the mouse liver and in cultured hepatocytes. Intraperitoneal injection of both bezafibrate and pioglitazone induced FGF21 mRNA expression in the mouse liver. Rosiglitazone and pioglitazone as well as bezafibrate significantly induced FGF21 mRNA expression in cultured mouse hepatocytes. On the other hand, both rosiglitazone and pioglitazone significantly induced, whereas bezafibrate did not affect FGF21 mRNA expression in the human liver carcinoma cell line HepG2. Bezafibrate significantly induced pyruvate dehydrogenase kinase 4 mRNA expression, suggesting that HepG2 cells are sensitive to bezafibrate like the mouse liver. Our findings suggest that PPARg g-activating antidiabetic drugs such as rosiglitazone and pioglitazone induce FGF21 expression in mouse and human hepatocytes, and that PPARg g rather than PPARa a might play an important role in human FGF21 production.
We have been investigating transcriptional regulation of the BMAL1 gene, a critical component of the mammalian clock system including DNA methylation. Here, a more detailed analysis of the regulation of DNA methylation of BMAL1 proceeded in RPMI8402 lymphoma cells. We found that CpG islands in the BMAL1 and the PER2 promoters were hyper- and hypomethylated, respectively and that 5-aza-2′-deoxycytidine (aza-dC) not only enhanced PER2 gene expression but also PER2 oscillation within 24 h in RPMI8402 cells. That is, such hypermethylation of CpG islands in the BMAL1 promoter restricted PER2 expression which was recovered by aza-dC within 1 day in these cells. These results suggest that the circadian clock system can be recovered through BMAL1 expression induced by aza-dC within a day. The RPIB9 promoter of RPMI8402 cells, which is a methylation hotspot in lymphoblastic leukemia, was also hypermethylated and aza-dC gradually recovered RPIB9 expression in 3 days. In addition, methylation-specific PCR revealed a different degree of aza-dC-induced methylation release between BMAL1 and RPIB9. These results suggest that the aza-dC-induced recovery of gene expression from DNA methylation is dependent on a gene, for example the rapid response to demethylation by the circadian system, and thus, is of importance to clinical strategies for treating cancer.
Withanolide derivatives have anticancer,
anti-inflammatory, and
other functions and are components of Indian traditional Ayurvedic
medicine. Here, we found that 2,3-dihydro-3β-methoxy withaferin-A
(3βmWi-A), a derivative of withaferin-A (Wi-A) belonging to
a class of withanolides that are abundant in Ashwagandha (Withania somnifera), lengthened the period of the circadian
clock. This compound dose-dependently elongated circadian rhythms
in Sarcoma 180 cancer cells and in normal fibroblasts including NIH3T3
and spontaneously immortalized mouse embryonic fibroblasts (MEF).
Furthermore, 3βmWi-A dose-dependently upregulated the mRNA expression
and promoter activities of Bmal1 after dexamethasone
stimulation and of the nuclear orphan receptors, Rora and Nr1d1, that comprise the stabilization loop
for Bmal1 oscillatory expression. We showed that
3βmWi-A functions as an inverse agonist for RORa with an IC50
of 11.3 μM and that 3βmWi-A directly, but weakly, interacts
with RORa (estimated dissociation constant [K
d], 5.9 μM). We propose that 3βmWi-A is a novel
modulator of circadian rhythms.
The circadian clock is a cell-autonomous endogenous system that generates circadian rhythms in the behavior and physiology of most organisms. We previously reported that the harmala alkaloid, harmine, lengthens the circadian period of Bmal1 transcription in NIH 3T3 fibroblasts. Clock protein dynamics were examined using real-time reporter assays of PER2::LUC to determine the effects of harmine on the central Key words carboline alkaloid; circadian clock; PER2::LUC protein; real-time reporter assay; suprachiasmatic nucleus The circadian clock is an endogenous cell-autonomous system that generates circadian rhythms in the behavior and physiology of most organisms. The master clock in mammals is located in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus, 1,2) which controls most aspects of physiology such as sleep-wake cycles, body temperature, hormone secretion, blood pressure and metabolism.3,4) The circadian clock mechanism consists of a network of autoregulatory transcription-based feedback loops that drive the rhythmic expression of clock genes such as Per1, Per2 and Bmal1, 5) and these cell-autonomous and self-sustained oscillators are found not only in the SCN but also in peripheral tissues as well as dissociated cells. Acute induction of the mRNA expression of clock genes plays a critical role in phase shifting the circadian clock both in cells cultured in vitro and in vivo. In addition to transcriptional feedback regulations, some essential roles of post-translational modifications such as phosphorylation and ubiquitination have been reported. [6][7][8] The harmala alkaloid, harmine, is a β-carboline alkaloid that was originally isolated from seeds of Peganum harmala and Banisteropsis caapi, both of which have traditionally been used in ritual and medicinal preparations of the Middle East, Central Asia and South America. 9) Harmine is also found in common plant-derived foods and animal tissues, 10) and it has antiplasmodial, antioxidative, antimutagenic and antigenotoxic activities, as well as antidepressant properties.11-13) Several psychiatric conditions, including depression, are closely associated with the circadian clock.14) Although we previously reported that harmine dose-dependently lengthens the circadian period of Bmal1 expression in NIH 3T3 cells, 15) the effects of harmine on the master clock in the SCN remain unknown. We therefore evaluated clock protein dynamics in neuronal cells and in SCN slices by monitoring PER2::LUC bioluminescence in real-time to determine the effects of harmine on the master clock in the SCN.
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