• Post-translational Regulation of Glucokinase in Hypothalamic Neurons

      McFarland, Jennifer; Rizzo, Megan A. (2020)
      Glucose-sensing tissues utilize glucokinase (GCK), the activity of which is rate-limiting for glucose metabolism, to sense and, consequently, counteract deviations from glucose homeostasis. Post-translational regulation of GCK is well defined in the liver and the pancreas, and is critical for the maintenance of glucose homeostasis; yet, post- translational regulation of GCK in hypothalamic neurons, which play a central role in maintaining glucose homeostasis, remains relatively unexplored. Here, we use a hypothalamically-derived, glucose-sensing GT1-7 neuronal cell line to provide evidence of a receptor-driven, ER Ca2+-mediated S-nitrosylation and activation of GCK. Strategic pharmacological manipulations were paired with the assessment of GCK activity, done by either measuring NAD(P)H autofluorescence while raising extracellular glucose, or through expression of a homotransfer FRET GCK biosensor. Further, a biotin-switch assay was used to confirm the presence of GCK S-nitrosylation. This work illustrates a central mechanism of post-translational GCK regulation, which may underlie metabolic signal integration in the hypothalamus and may contribute to the pathology of diabetes.
    • Prolactin receptor gene expression in the hypothalamus

      Chiu, Sufen; Wise, Phyllis M. (1993)
      Changes in behavior and neuroendocrine secretions in response to prolactin treatment may be mediated by prolactin receptors in the brain. Autoradiographic binding studies using iodinated prolactin suggest that these receptors may be located in the rat choroid plexus and hypothalamus. It remains unclear if the prolactin binding sites represent classical prolactin receptors or other proteins from the prolactin/growth hormone receptor family that have retained an affinity for prolactin. Two prolactin receptors, a short and long form resulting from alternative splicing of one gene, were cloned recently. The objectives of this dissertation were to (1) examine if the prolactin receptor gene is expressed in brain areas that are believed to bind and/or respond to prolactin by reverse-transcription polymerase chain reaction (RT-PCR), (2) determine the cellular distribution of prolactin receptor mRNA by in situ hybridization, and (3) assess the influence of physiological conditions on prolactin receptor gene expression at the individual cell level by in situ hybridization. We demonstrated that both forms of the prolactin receptor mRNA are expressed in the pituitary gland and hypothalamus but not in cortical brain tissue or skeletal muscle by RT-PCR. Using in situ hybridization, prolactin receptor gene expression was detectable in medial preoptic nucleus, supraoptic nucleus, arcuate nucleus, lateral ventromedial nucleus and choroid plexus. Prolactin receptor gene expression in the periventricular area of the preoptic nucleus, arcuate nucleus, and choroid plexus is increased during aging {dollar}(p<0.05){dollar}. Therefore, we conclude that the classical prolactin receptors appear to mediate the binding and action of prolactin in the brain and are influenced by age-related changes.