Membrane lipids and their transfer proteins in β-cells
Sammanfattning: Insulin secretion from β-cells is essential for glucose homeostasis and is often dysregulated in diabetes. Intracellular Ca2+ and membrane lipids cooperate to control insulin secretion with high spatial and temporal precision. Phosphoinositide lipids (PIs) and products from their hydrolysis regulate processes such as ion channel conductance and protein localization and activation, but the role of these lipids in insulin secretion from β-cells remains poorly understood. In the present study, live cell Ca2+ imaging combined with molecular tools for acute depletion or synthesis of the major PI in the plasma membrane (PM), PI(4,5)P2, revealed that this lipid positively regulates Ca2+ influx. As a consequence of reduced PI(4,5)P2 and impaired Ca2+ influx, β-cells failed to secrete appropriate amounts of insulin in response to glucose stimulation. In stimulated β-cells, ATP is co-released with insulin, which leads to autocrine purinergic receptor signaling with resulting phospholipase C activation, PI(4,5)P2 hydrolysis and local formation of diacylglycerol (DAG) in the PM. The ER-anchored protein extended synaptotagmin-1 (E-Syt1) binds to PI(4,5)P2 in the PM and transfers DAG from the site of production to the ER in a Ca2+-dependent manner. It was now found that DAG forms locally in microdomains around exocytotic sites and that E-Syt1 was selectively recruited to these sites, where it removed DAG by a mechanism that required an intact lipid transport domain. The DAG removal was part of a negative feedback mechanism, and loss of this feedback as a consequence of reduced E-Syt1 expression resulted in increased glucose-stimulated insulin secretion, likely via enhanced protein kinase C activity. TMEM24, an ER-anchored protein structurally similar to E-Syt1, dynamically localizes to ER-PM contact sites in a Ca2+-dependent manner, where it is responsible for transporting a PI(4,5)P2 precursor to the PM. TMEM24 was now shown to be spatially and temporally regulated by both Ca2+ and DAG. Ca2+ induced TMEM24 dissociation from the PM and this process was counteracted by E-Syt1-mediated DAG transport and subsequent suppression of PKC activity. Although TMEM24 was involved in maintaining the ER Ca2+ stores and in membrane reuptake following insulin granule exocytosis, the protein was dispensable for glucose-stimulated insulin secretion. Together, the work presented in this thesis defines new and important roles of PIs and lipid transfer proteins for normal β-cell function.
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