Synchronous activation of gonadotropin-releasing hormone gene transcription and secretion by pulsatile kisspeptin stimulation.

Pulsatile release of hypothalamic gonadotropin-releasing hormone (GnRH) is essential for pituitary gonadotrope function. Although the importance of pulsatile GnRH secretion has been recognized for several decades, the mechanisms underlying GnRH pulse generation in hypothalamic neural networks remain elusive. Here, we demonstrate the ultradian rhythm of GnRH gene transcription in single GnRH neurons using cultured hypothalamic slices prepared from transgenic mice expressing a GnRH promoter-driven destabilized luciferase reporter. Although GnRH promoter activity in each GnRH neuron exhibited an ultradian pattern of oscillations with a period of ∼10 h, GnRH neuronal cultures exhibited partially synchronized bursts of GnRH transcriptional activity at ∼2-h intervals. Surprisingly, pulsatile administration of kisspeptin, a potent GnRH secretagogue, evoked dramatic synchronous activation of GnRH gene transcription with robust stimulation of pulsatile GnRH secretion. We also addressed the issue of hierarchical interaction between the circadian and ultradian rhythms by using Bmal1-deficient mice with defective circadian clocks. The circadian molecular oscillator barely affected basal ultradian oscillation of GnRH transcription but was heavily involved in kisspeptin-evoked responses of GnRH neurons. In conclusion, we have clearly shown synchronous bursts of GnRH gene transcription in the hypothalamic GnRH neuronal population in association with episodic neurohormone secretion, thereby providing insight into GnRH pulse generation.

Obesity in mice with adipocyte-specific deletion of clock component Arntl.

Adipocytes store excess energy in the form of triglycerides and signal the levels of stored energy to the brain. Here we show that adipocyte-specific deletion of Arntl (also known as Bmal1), a gene encoding a core molecular clock component, results in obesity in mice with a shift in the diurnal rhythm of food intake, a result that is not seen when the gene is disrupted in hepatocytes or pancreatic islets. Changes in the expression of hypothalamic neuropeptides that regulate appetite are consistent with feedback from the adipocyte to the central nervous system to time feeding behavior. Ablation of the adipocyte clock is associated with a reduced number of polyunsaturated fatty acids in adipocyte triglycerides. This difference between mutant and wild-type mice is reflected in the circulating concentrations of polyunsaturated fatty acids and nonesterified polyunsaturated fatty acids in hypothalamic neurons that regulate food intake. Thus, this study reveals a role for the adipocyte clock in the temporal organization of energy regulation, highlights timing as a modulator of the adipocyte-hypothalamic axis and shows the impact of timing of food intake on body weight.

Increased superoxide and endothelial NO synthase uncoupling in blood vessels of Bmal1-knockout mice.

RATIONALE: Disruption of the circadian clock in mice produces vascular dysfunction as evidenced by impairments in endothelium-dependent signaling, vasomotion, and blood vessel remodeling. Although the altered function of endothelial NO synthase and the overproduction of reactive oxygen species are central to dysfunction of the endothelium, to date, the impact of the circadian clock on endothelial NO synthase coupling and vascular reactive oxygen species production is not known. OBJECTIVE: The goals of the present study were to determine whether deletion of a critical component of the circadian clock, Bmal1, can influence endothelial NO synthase coupling and reactive oxygen species levels in arteries from Bmal1-knockout (KO) mice. METHODS AND RESULTS: Endothelial function was reduced in aortae from Bmal1-KO mice and improved by scavenging reactive oxygen species with polyethylene glycol-superoxide dismutase and nonselectively inhibiting cyclooxygenase isoforms with indomethacin. Aortae from Bmal1-KO mice exhibited enhanced superoxide levels as determined by electron paramagnetic resonance spectroscopy and dihydroethidium fluorescence, an elevation that was abrogated by administration of nitro-l-arginine methyl ester. High-performance liquid chromatography analysis revealed a reduction in tetrahydrobiopterin and an increase in dihydrobiopterin levels in the lung and aorta of Bmal1-KO mice, whereas supplementation with tetrahydrobiopterin improved endothelial function in the circadian clock KO mice. Furthermore, levels of tetrahydrobiopterin, dihydrobiopterin, and the key enzymes that regulate biopterin bioavailability, GTP cyclohydrolase and dihydrofolate reductase exhibited a circadian expression pattern. CONCLUSIONS: Having an established influence in the metabolic control of glucose and lipids, herein, we describe a novel role for the circadian clock in metabolism of biopterins, with a significant impact in the vasculature, to regulate coupling of endothelial NO synthase, production of superoxide, and maintenance of endothelial function.