Rhythmic clock and neuropeptide gene expression in hypothalamic mHypoE-44 neurons

The rhythmic expression of specific clock genes: Bmal1, Per2, Clock and Rev-Erbα; and specific hypothalamic neuropeptides: NPY, Crh, AgRP, neurotensin and preproghrelin, expressed in clonal hypothalamic neuronal cell lines, was assayed and analyzed using a novel non-linear least squares statistical analysis to determine rhythmicity in an in vitro milieu. In silico analysis of the neuropeptide promoter regions identified putative E-box motifs and a motif in the NPY promoter is bound to in an oscillatory fashion. Within the mHypoE-44 neurons, we demonstrate that mRNA of four core circadian components: Bmal1, Clock, Per2 and Rev-Erbα, oscillate with approximate 24 h rhythms. NPY and NT demonstrated significant 24 h gene expression. However, CRH and preproghrelin mRNA cycled significantly in an ultradian fashion, oscillating approximately every 18 h. AgRP mRNA did not show a significant rhythm. We speculate that endogenous rhythmic neuropeptide expression contributes to neuroendocrine homeostasis, which may include energy balance.