Browsing School, Graduate by Subject "S-nitrosylation"
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Post-translational Regulation of Glucokinase in Hypothalamic NeuronsGlucose-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.
Regulation of Glucokinase in Pancreatic Beta CellsGlucose homeostasis is a tightly coordinated process that ensures an adequate source of cellular energy. Blood glucose concentrations are coupled to changes in activity of glucokinase (GCK) in β-cells of the pancreas. GCK acts as the glucose sensor and is responsible for the phosphorylation of glucose that leads to insulin secretion from the pancreas to constrain glucose concentrations within physiologic levels. However, the underlying mechanistic details explaining how GCK maintains this tight control over glucose metabolism are unclear. First we elucidated the role of intracellular Ca2+, particularly Ca2+ mobilized from the endoplasmic reticulum, in GCK activation. We then investigated the cellular regulation of GCK through post-translational S-nitrosylation. Finally, we assessed changes in GCK activity that occur during the development of diabetes. To quantitatively assess this GCK activity in vitro, we used Förster resonance energy transfer (FRET) spectroscopy, fluorescence microscopy and GCK biosensors expressed in cultured pancreatic β-cells and primary mouse islets. This work describes our homotransfer FRET-GCK biosensor and improved data analysis methodology to understand the cellular regulation of GCK activity. To create the homotransfer biosensor, we attached two mVenus fluorescent proteins to GCK and transfected this single-color biosensor into cultured β-cells. We used the inherent polarization of light in combination with FRET principles to precisely measure GCK activation in living cells under various conditions.