Calorie restriction (CR) extends lifespans in a wide variety of species. CR induces an increase in the NAD(+)/NADH ratio in cells and results in activation of SIRT1, an NAD(+)-dependent protein deacetylase that is thought to be a metabolic master switch linked to the modulation of lifespans. CR also affects the expression of peroxisome proliferator-activated receptors (PPARs). The three subtypes, PPARalpha, PPARgamma, and PPARbeta/delta, are expressed in multiple organs. They regulate different physiological functions such as energy metabolism, insulin action and inflammation, and apparently act as important regulators of longevity and aging. SIRT1 has been reported to repress the PPARgamma by docking with its co-factors and to promote fat mobilization. However, the correlation between SIRT1 and other PPARs is not fully understood. CR initially induces a fasting-like response. In this study, we investigated how SIRT1 and PPARalpha correlate in the fasting-induced anti-aging pathways. A 24-h fasting in mice increased mRNA and protein expression of both SIRT1 and PPARalpha in the livers, where the NAD(+) levels increased with increasing nicotinamide phosphoribosyltransferase (NAMPT) activity in the NAD(+) salvage pathway. Treatment of Hepa1-6 cells in a low glucose medium conditions with NAD(+) or NADH showed that the mRNA expression of both SIRT1 and PPARalpha can be enhanced by addition of NAD(+), and decreased by increasing NADH levels. The cell experiments using SIRT1 antagonists and a PPARalpha agonist suggested that PPARalpha is a key molecule located upstream from SIRT1, and has a role in regulating SIRT1 gene expression in fasting-induced anti-aging pathways.

Although altered homeostatic regulation, including disturbance of 24-h rhythms, is often observed in the patients undergoing glucocorticoid therapy, the mechanisms underlying the disturbance remains poorly understood. We report here that chronic treatment with a synthetic glucocorticoid, prednisolone (PSL), can cause alteration of circadian clock function at molecular level. Treatment of cultured hepatic cells (HepG2) with PSL induced expression of Period1 (Per1), and the PSL treatment also attenuated the serum-induced oscillations in the expression of Period2 (Per2), Rev-erbalpha, and Bmal1 mRNA in HepG2 cells. Because the attenuation of clock gene oscillations was blocked by pretreating the cells with a Per1 antisense phosphothioate oligodeoxynucleotide, the extensive expression of Per1 induced by PSL may have resulted in the reduced amplitude of other clock gene oscillations. Continuous administration of PSL into mice constitutively increased the Per1 mRNA levels in liver and skeletal muscle, which seems to attenuate the oscillation in the expressions of Per2, Rev-erbalpha, and Bmal1. However, a single daily administration of PSL at the time of day corresponding to acrophase of endogenous glucocorticoid levels had little effect on the rhythmic expression of clock genes. These results suggest a possible pharmacological action by PSL on the core circadian oscillation mechanism and indicate the possibility that the alteration of clock function induced by PSL can be avoided by optimizing the dosing schedule.

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Methionine aminopeptidase2 (MetAP2) plays an important role in the growth of endothelial cells during the tumor angiogenesis stage. Recently, we have clarified that mouse methionine aminopeptidases (mMetAPs) show a 24-hour rhythm in implanted tumor masses. In the present study, we investigated the mechanism underlying the 24-hour rhythm of mMetAP2 activity in tumor-bearing mice under a light-dark (lights on from 7 a.m. to 7 p.m.) cycle. The 5 flanking region of mMetAP2 included eight E-boxes. The transcription of the mMetAP2 promoter was enhanced by the mCLOCK:mBMAL1 heterodimer, and its activation was inhibited by mPER2 or mCRY1. Deletion and mutation of the E-boxes in the region indicated that the E-box nearest to the initiation start site played an important role in the transcriptional regulation by clock genes. In sarcoma180-bearing mice, the pattern of binding of mCLOCK and mBMAL1 to the E-box and transcription of the mMetAP2 promoter showed a 24-hour rhythm with higher levels from the mid-light to early dark phase. The pattern of mMetAP2 transcription was closely associated with that of mMetAP2 mRNA expression in three types of tumor-bearing mice. mMetAP2 protein expression varied with higher levels from the late-dark to early light phase. The rhythmicity of the protein expression was synchronous with that of the activity of mMetAPs but out of phase with that of the mMetAP2 mRNA expression. These results suggest that the 24-hour rhythm of mMetAP2 activity is regulated by the transcription of clock genes within the clock feedback loops.