Circadian mRNA expression of coagulation and fibrinolytic factors is organ-dependently disrupted in aged mice

To evaluate the effects of aging on the circadian gene expression of coagulation and fibrinolytic factors in the mouse tissues, we examined temporal mRNA expression profiles of plasminogen activator inhibitor-1 (PAI-1), tissue-type plasminogen activator (tPA), tissue factor (TF), and thrombomodulin (TM) genes together with circadian clock genes in the brains, hearts and livers of young (5weeks old) and aged (15months old) mice. Cardiac mRNA expression of β-myosin heavy chain (β-MHC), a molecular marker of cardiac hypertrophy, was obviously increased in the aged mice. Rhythmic expression of the clock genes mPer2 and BMAL1 in these organs was almost identical between young and aged mice, whereas that of PAI-1, TF and TM mRNAs and of clock-controlled genes such as DBP and Dec1 were damped to low levels in the livers of aged mice. Expression levels of tPA mRNA were significantly decreased and those of TF were significantly elevated throughout the day in the brain of aged mice. Expression levels of PAI-1 in the heart of aged mice were continuously elevated over 2-fold the peak levels of young mice throughout the day. However, day/night fluctuations in plasma PAI-1 levels were unaffected by aging. Aging tissue- and time-dependently affects the mRNA expression of coagulation and fibrinolytic factors. Aging-dependent constitutive PAI-1 induction in the heart might be a risk factor for cardiovascular diseases that is independent of plasma PAI-1 levels.     

Immunocytochemical demonstration of day/night changes of clock gene protein levels in the murine adrenal gland: differences between melatonin-proficient (C3H) and melatonin-deficient (C57BL) mice

The circadian system comprises several peripheral oscillators and a central rhythm generator that, in mammals, is located in the suprachiasmatic nucleus of the hypothalamus. Expression of clock genes is a characteristic feature of the central rhythm generator and the peripheral oscillators. With regard to the rhythmic production of glucocorticoids, the adrenal gland can be considered as peripheral oscillator, but little is known about clock gene expression in this tissue. Therefore, the present study investigates the levels of three clock gene proteins PER1, BMAL1 and CRY2 in the murine adrenal cortex and medulla at seven different time points of a 12-hr light/12-hr dark cycle. To determine a potential role of melatonin we compared the patterns of clock gene proteins in the adrenal gland of melatonin-proficient mice (C3H) with those of melatonin-deficient mice (C57BL). In C3H mice, both, the adrenal cortex and medulla displayed day/night variation in PER1-, CRY2- and BMAL1-protein levels. PER1 and CRY2 peaked in the middle of the light phase, whereas BMAL1 peaked in the dark phase. This pattern was also observed in the adrenal medulla of C57BL, but in the adrenal cortex of C57BL clock gene protein levels did not change with time and were consistently lower than in C3H mice. These results support the hypothesis that the adrenal gland is a peripheral oscillator and raise the possibility that melatonin may be involved in the control of clock gene protein levels in the adrenal cortex of mice.     

Melatonin modulates dysregulated circadian clocks in mice with diethylnitrosamine‐induced hepatocellular carcinoma

Disruption of circadian rhythms, which are regulated by the circadian clock machinery, plays an important role in different long‐term diseases including hepatocellular carcinoma (HCC ). Melatonin has been reported to alleviate promotion and progression of HCC , but the potential contribution of circadian clock modulation is unknown. We investigated the effects of melatonin in mice which received diethylnitrosamine (DEN ) (35 mg/kg body weight ip) once a week for 8 weeks. Melatonin was given at 5 or 10 mg kg^?1d^?1 ip beginning 4 weeks after the onset of DEN administration and ending at the sacrifice time (10, 20, 30, or 40 weeks). Liver expression of Bmal1, Clock, Npas2, Rorα, and Sirt1 increased, whereas Cry1, Per1, Per2, Per3, CK 1ε, Rev‐erbα, and Rev‐erbβ decreased following DEN administration. Melatonin treatment prevented changes in the expression of clock genes, and this effect was accompanied by an upregulation of the MT 1 receptor and reduced levels of the hypoxia‐inducible factors Hif‐1α and Hif‐2α. An increased expression of p21, p53, and PARP 1/2, a higher Bax/Bcl‐2 ratio, and a lower expression of Cyclin D1, CDK 6, HSP 70, HSP 90, and GRP 78 proteins were also observed in melatonin‐treated mice. Melatonin significantly potentiated the suppression of proliferation and cell cycle arrest induced by the synthetic REV ‐ERB agonist SR 9009 in human Hep3B cells, and BMAL 1 knocking down attenuated the pro‐apoptotic and antiproliferative effect of melatonin. Results support a contribution of changes in the circadian clock components to the beneficial effects of melatonin in HCC and highlight the usefulness of strategies modulating the circadian machinery in hepatocarcinogenesis.     

Metformin opposes impaired AMPK and SIRT1 function and deleterious changes in core clock protein expression in white adipose tissue of genetically-obese db/db mice

Aim: AMPK activates SIRT1 in liver and skeletal muscle. Impaired circadian function is associated with development of obesity. SIRT1 regulates circadian function and is suppressed in white adipose tissue (WAT) of obese patients. We examined the potential role of AMPK and SIRT1 in regulation of circadian components in WAT of obese db/db mice and in mice fed a high‐fat diet (HFD), and investigated whether metformin‐mediated activation of AMPK opposed any deleterious changes in the WAT clock mechanism. Methods: db/+ and db/db mice were administered metformin (250 mg/kg/day; 7 days). Separately, mice were fed HFD for 16‐weeks. 3T3‐L1 adipocytes were incubated with metformin, EX527 or FK866, inhibitors of SIRT1 and NAMPT, respectively. Gene and protein expression were measured by qRT‐PCR and immunoblotting. Results: AMPK activity, NAMPT expression and SIRT1 expression were decreased in WAT of db/db and HFD mice, in association with suppressed expression of the core circadian components CLOCK and BMAL1. Expression of Pparγ and the adipogenic repressors Irf3 and Irf4 were also suppressed. Metformin increased AMPK activity in WAT of db/db mice and in metformin‐treated adipocytes, with increased NAMPT, SIRT1 and circadian component expression. Metformin‐mediated induction of Clock mRNA in adipocytes was blocked by inhibition of NAMPT and SIRT1. Conclusions: Decreased AMPK‐SIRT1 signalling in db/db and HFD mice impacts WAT circadian function causing dysregulated lipid regulation, favouring an obese phenotype. Metformin mediates a phenotypic shift away from lipid accretion through AMPK‐NAMPT‐SIRT1 mediated changes in clock components, supporting chronotherapeutic treatment approaches for obesity.     

Clock gene expression in the murine gastrointestinal tract: endogenous rhythmicity and effects of a feeding regimen

BACKGROUND & AIMS: Based on observations that the gastrointestinal tract is subject to various 24-hour rhythmic processes, it is conceivable that some of these rhythms are under circadian clock gene control. We hypothesized that clock genes are present in the gastrointestinal tract and that they are part of a functional molecular clock that coordinates rhythmic physiologic functions. METHODS: The effects of timed feeding and vagotomy on temporal clock gene expression (clock, bmal1, per1-3, cry1-2) in the gastrointestinal tract and suprachiasmatic nucleus (bmal, per2) of C57BL/6J mice were examined using real-time polymerase chain reaction and Western blotting (BMAL, PER2). Colonic clock gene localization was examined using immunohistochemistry (BMAL, PER1-2). RESULTS: Clock immunoreactivity was observed in the myenteric plexus and epithelial crypt cells. Clock genes were expressed rhythmically throughout the gastrointestinal tract. Timed feeding shifted clock gene expression at the RNA and protein level but did not shift clock gene expression in the central clock. Vagotomy did not alter gastric clock gene expression compared with sham-treated controls. CONCLUSIONS: The murine gastrointestinal tract contains functional clock genes, which are molecular core components of the circadian clock. Daytime feeding in nocturnal rodents is a strong synchronizer of gastrointestinal clock genes. This synchronization occurs independently of the central clock. Gastric clock gene expression is not mediated through the vagal nerve. The presence of clock genes in the myenteric plexus and epithelial cells suggests a role for clock genes in circadian coordination of gastrointestinal functions such as motility, cell proliferation, and migration.