| 1. |
- Nguyen, Phuoc My
(författare)
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Phosphoinositides control insulin secretion through multiple routes
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Glucose-stimulated insulin secretion from pancreatic beta cells is the sequence of events that starts with glucose uptake and ends with the fusion of insulin granules with the plasma membrane through Ca2+-triggered exocytosis. Phosphoinositides are minor components of all cellular membranes, yet play fundamental roles as regulators of many cellular processes. PI(4,5)P2 is the most abundant phosphoinositide in the plasma membrane, where it controls the activity of ion channels, endo- and exocytosis and cytoskeletal rearrangements. However, its role in the regulation of insulin secretion is unclear and there are support for both direct stimulatory and inhibitory effects. Using an optogenetic approach to acutely recruit a PI(4,5)P2 phosphatase to deplete the plasma membrane of PI(4,5)P2 in living beta cells, we found that this lipid was required to support voltage-dependent Ca2+-influx and glucose-stimulated insulin secretion. Consistently, depolarization-induced Ca2+-influx was instead augmented when the plasma membrane PI(4,5)P2 concentration was increased by light-dependent recruitment of a PI(4,5)P2-synthesizing enzyme. PI(4)P is another phosphoinositide residing in the plasma membrane and other intracellular membranes. In addition to serving as a precursor for PI(4,5)P2, PI(4)P is used to fuel lipid exchange reactions at membrane contacts sites, such as the PI(4)P/cholesterol exchange at the ER-Golgi interface catalyzed by OSBP. Sac2 is a PI(4)P phosphatase that is highly expressed in neuronal tissues and the pancreas, where it localizes to endosomes and participates in endosome maturation. We found that Sac2 additionally binds to insulin granules through interactions with granule PI(4)P and Rab3. Loss of Sac2 resulted in accumulation of both PI(4)P and cholesterol on the granule surface, impaired insulin granule docking to the plasma membrane and reduced insulin secretion. The cholesterol levels on insulin granules were normalized in cells with reduced OSBP expression, indicating that Sac2 and OSBP cooperate at insulin granules. Acute inhibition of OSBP by OSW-1 resulted in the redistribution of OSBP, and its ER localized receptor VAP-A, from the ER-Golgi interface to insulin granules. Similar to loss of Sac2, both siRNA-mediated knockdown and pharmacological inhibition of OSBP resulted in decreased insulin secretion. Together, these results show that Sac2, by negatively regulating granule PI(4)P, limits OSBP-mediated cholesterol transfer to insulin granules at ER–insulin granule contact sites. Type-2 diabetes is associated with impaired insulin granule docking and exocytosis as well as altered cholesterol homeostasis. We found that Sac2 expression was reduced in patients with type-2 diabetes, which may help to explain some of the hallmarks of this disease at the level of the beta cell and also form the basis for future interventions.
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| 2. |
- Nguyen, Phuoc My, 1983-, et al.
(författare)
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PI(4)P-cholesterol exchange at endoplasmic reticulum-secretory granule contact sites controls insulin secretion
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Insulin secretion is the process whereby insulin-containing granules fuse with the plasma membrane of β-cells. Exocytosis is preceded by cargo loading, maturation and transport of the secretory granules; processes that require modification of both the protein and lipid composition of the granules. We recently identified phosphatidylinositol-4 phosphate (PI4P) dephosphorylation by Sac2 on the surface of insulin granules as a key step that precedes stable granule docking at the plasma membrane and that is required for normal insulin secretion. Here, we show that granule PI4P is used to target the lipid exchange protein OSBP to the granule surface where it is involved in PI4P/cholesterol exchange. Loss of Sac2 resulted in excess accumulation of cholesterol on insulin granules that was normalized when OSBP expression was reduced. Acute inhibition of OSBP by OSW-1 resulted in dramatic cellular redistribution of OSBP from the Golgi to insulin granules where it colocalized with the ER-resident protein VAP-A and formed ER-granule contacts. Similar to Sac2 knockdown, inhibition of OSBP suppressed insulin secretion without affecting insulin production.
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| 3. |
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| 5. |
- Nguyen, Phuoc My, et al.
(författare)
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The PI(4)P phosphatase Sac2 controls insulin granule docking and release
- 2019
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Ingår i: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 218:11, s. 3714-3729
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Tidskriftsartikel (refereegranskat)abstract
- Insulin granule biogenesis involves transport to, and stable docking at, the plasma membrane before priming and fusion. Defects in this pathway result in impaired insulin secretion and are a hallmark of type 2 diabetes. We now show that the phosphatidylinositol 4-phosphate phosphatase Sac2 localizes to insulin granules in a substrate-dependent manner and that loss of Sac2 results in impaired insulin secretion. Sac2 operates upstream of granule docking, since loss of Sac2 prevented granule tethering to the plasma membrane and resulted in both reduced granule density and number of exocytic events. Sac2 levels correlated positively with the number of docked granules and exocytic events in clonal beta cells and with insulin secretion in human pancreatic islets, and Sac2 expression was reduced in islets from type 2 diabetic subjects. Taken together, we identified a phosphoinositide switch on the surface on insulin granules that is required for stable granule docking at the plasma membrane and impaired in human type 2 diabetes.
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| 6. |
- Panagiotou, Styliani, et al.
(författare)
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OSBP-mediated PI(4)P-cholesterol exchange at endoplasmic reticulum-secretory granule contact sites controls insulin secretion
- 2024
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Ingår i: Cell Reports. - : Cell Press. - 2211-1247. ; 43:4
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Tidskriftsartikel (refereegranskat)abstract
- Insulin is packaged into secretory granules that depart the Golgi and undergo a maturation process that involves changes in the protein and lipid composition of the granules. Here, we show that insulin secretory granules form physical contacts with the endoplasmic reticulum and that the lipid exchange protein oxysterol-binding protein (OSBP) is recruited to these sites in a Ca2+-dependent manner. OSBP binding to insulin granules is positively regulated by phosphatidylinositol-4 (PI4)-kinases and negatively regulated by the PI4 phosphate (PI(4)P) phosphatase Sac2. Loss of Sac2 results in excess accumulation of cholesterol on insulin granules that is normalized when OSBP expression is reduced, and both acute inhibition and small interfering RNA (siRNA)-mediated knockdown of OSBP suppress glucose-stimulated insulin secretion without affecting insulin production or intracellular Ca2+ signaling. In conclusion, we show that lipid exchange at endoplasmic reticulum (ER)-granule contact sites is involved in the exocytic process and propose that these contacts act as reaction centers with multimodal functions during insulin granule maturation.
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| 7. |
- Sanchez, Gonzalo, et al.
(författare)
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The β-cell primary cilium is an autonomous Ca2+ compartment for paracrine GABA signaling
- 2023
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Ingår i: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 222:1
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Tidskriftsartikel (refereegranskat)abstract
- The primary cilium is an organelle present in most adult mammalian cells that is considered as an antenna for sensing the local microenvironment. Here, we use intact mouse pancreatic islets of Langerhans to investigate signaling properties of the primary cilium in insulin-secreting β-cells. We find that GABAB1 receptors are strongly enriched at the base of the cilium, but are mobilized to more distal locations upon agonist binding. Using cilia-targeted Ca2+ indicators, we find that activation of GABAB1 receptors induces selective Ca2+ influx into primary cilia through a mechanism that requires voltage-dependent Ca2+ channel activation. Islet β-cells utilize cytosolic Ca2+ increases as the main trigger for insulin secretion, yet we find that increases in cytosolic Ca2+ fail to propagate into the cilium, and that this isolation is largely due to enhanced Ca2+ extrusion in the cilium. Our work reveals local GABA action on primary cilia that involves Ca2+ influx and depends on restricted Ca2+ diffusion between the cilium and cytosol.
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| 8. |
- Xie, Beichen, et al.
(författare)
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Feedback regulation of insulin secretion by extended synaptotagmin-1
- 2019
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Ingår i: The FASEB Journal. - 0892-6638 .- 1530-6860. ; 33:4, s. 4716-4728
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Tidskriftsartikel (refereegranskat)abstract
- Endoplasmic reticulum (ER)-plasma membrane (PM) contacts are dynamic structures with important roles in the regulation of calcium (Ca2+) and lipid homeostasis. The extended synaptotagmins (E-Syts) are ER-localized lipid transport proteins that interact with PM phosphatidylinositol 4,5-bisphosphate in a Ca2+-dependent manner. E-Syts bidirectionally transfer glycerolipids, including diacylglycerol (DAG), between the 2 juxtaposed membranes, but the biologic significance of this transport is still unclear. Using insulin-secreting cells and live-cell imaging, we now show that Ca2+-triggered exocytosis of insulin granules is followed, in sequence, by PM DAG formation and E-Syt1 recruitment. E-Syt1 counteracted the depolarization-induced DAG formation through a mechanism that required both voltage-dependent Ca2+ influx and Ca2+ release from the ER. E-Syt1 knockdown resulted in prolonged accumulation of DAG in the PM, resulting in increased glucose-stimulated insulin secretion. We conclude that Ca2+-triggered exocytosis is temporally coupled to Ca2+-triggered E-Syt1 PM recruitment and removal of DAG to negatively regulate the same process.
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| 9. |
- Xie, Beichen, et al.
(författare)
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Plasma Membrane Phosphatidylinositol 4,5-Bisphosphate Regulates Ca2+-Influx and Insulin Secretion from Pancreatic beta Cells
- 2016
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Ingår i: CELL CHEMICAL BIOLOGY. - : Elsevier BV. - 2451-9448 .- 2451-9456. ; 23:7, s. 816-826
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Tidskriftsartikel (refereegranskat)abstract
- Insulin secretion from pancreatic beta cells is regulated by the blood glucose concentration and occurs through Ca2+-triggered exocytosis. The activities of multiple ion channels in the beta cell plasma membrane are required to fine-tune insulin secretion in order to maintain normoglycemia. Phosphoinositide lipids in the plasma membrane often gate ion channels, and variations in the concentration of these lipids affect ion-channel open probability and conductance. Using light-regulated synthesis or depletion of plasma membrane phosphatidylinositol 4,5-bisphosphate (PI[4,5]P-2), we found that this lipid positively regulated both depolarization- and glucose-triggered Ca2+ influx in a dose-dependent manner. Small reductions of PI(4,5)P-2 caused by brief illumination resulted in partial suppression of Ca2+ influx that followed the kinetics of the lipid, whereas depletion resulted in marked inhibition of both Ca2+ influx and insulin secretion.
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