Acute inhibition of casein kinase 1δ/ε rapidly delays peripheral clock gene rhythms.

Circadian rhythms are generated through a transcription-translation feedback loop involving clock genes and the casein kinases CSNK1D and CSNK1E. In this study, we investigated the effects of the casein kinase inhibitor PF-670462 (50 mg/kg) on rhythmic expression of clock genes in the liver, pancreas and suprachiasmatic nucleus (SCN) as well as plasma corticosterone, melatonin and running behaviour in rats and compared them to the responses to a 4 h extension of the light phase. PF-670462 acutely phase delayed the rhythmic transcription of Bmal1, Per1, Per2 and Nr1d1 in both liver and pancreas by 4.5 ± 1.3 and 4.5 ± 1.2 h, respectively, 1 day after administration. In the SCN, the rhythm of Nr1d1 and Dbp mRNA expression was delayed by 4.2 and 4 h, respectively. Despite these changes, the time of peak plasma melatonin secretion was not delayed, although the plasma corticosterone rhythm and onset of wheel-running activity were delayed by 2.1 and 1.1 h, respectively. These changes are in contrast to the effects of the 4 h light extension, which resulted in delays in peak expression of the clock genes of less than 1 h and no change in the melatonin or corticosterone rhythms. The ability of the casein kinase inhibitor to bring about large phase shifts in the rhythms of major metabolic target tissues may lead to new drugs being developed to rapidly phase adjust circadian rhythms to alleviate the metabolic impact of shift work.

Effect of N-proopiomelanocortin (1-77) and (1-49) infusions on adrenal expression of cyclin D1 in the fetal sheep.

In the sheep, there is a rapid increase in fetal adrenal growth and steroidogenesis during the last 10-15 days gestation. Recently, we have shown that infusion of POMC 1-77 increases fetal adrenal growth but does not significantly alter fetal plasma cortisol concentrations. Phosphorylation and inactivation of the pRB protein, which is required for progression into the DNA synthetic phase of the cell-cycle is conducted by a holoenzyme, for which cyclin D1 gene encodes the rate-limiting regulatory subunit. To further elucidate the mechanisms by which POMC 1-77 regulates adrenal growth, we therefore examined adrenal expression of the rate-limiting cell cycle protein, cyclin D1, from fetuses infused for 48 hr with POMC 1-77 (n = 6), POMC 1-49 or Saline (n = 6). There was no significant difference in the adrenal expression of cyclin D1 mRNA levels between POMC 1-77, 1-49 and saline infused fetuses. There was no significant correlation between cyclin D1 (4.0 Kb) and adrenal weight. In summary, these data do not demonstrate that the rate-limiting cell cycle protein, cyclin D1, is activated to stimulate adrenal growth following infusion of POMC 1-77 in the fetal sheep in late gestation.

N-proopiomelanocortin (1-77) suppresses expression of steroidogenic acute regulatory protein (StAR) mRNA in the adrenal gland of the fetal sheep.

In the sheep, there is a rapid increase in fetal adrenal growth and steroidogenesis during the last 10-15 days gestation. Recently, we have shown that infusion of POMC (1-77) increases fetal adrenal growth and expression of CYP17 mRNA but does not significantly alter fetal plasma cortisol concentrations [1]. We therefore investigated the effects of infusion of bovine POMC (1-77) and its biosynthetic derivative POMC (1-49) on adrenal StAR mRNA expression. At 136d gestation, POMC (1-77) (n=5 fetuses; 2microg/ml/h), POMC (1-49) (n=5 fetuses, 2microg/ml/h) or Saline (n=5 fetuses, 1ml/h) was infused for 48h. At 138d, fetal adrenal glands were collected and frozen in liquid N2 until RNA was extracted. Northern blot analyses demonstrated a major transcript for StAR mRNA at 3.0kb in fetal adrenal glands from all treatments. The membrane was stripped and re-probed with a P-labelled rat 18S rRNA oligo-probe to verify equal RNA loading. Infusion of POMC (1-77), but not POMC (1-49), resulted in a suppression of fetal adrenal StAR mRNA:18S rRNA when compared to adrenal StAR mRNA:18S rRNA from saline-infused controls. Our data suggest POMC (1-77) may act via separate mechanisms to increase fetal adrenal growth and to limit adrenal steroidogenesis through suppression of StAR mRNA.