| 1. |
- Alenkvist, Ida, et al.
(författare)
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Recruitment of Epac2A to Insulin Granule Docking Sites Regulates Priming for Exocytosis
- 2017
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Ingår i: Diabetes. - : American Diabetes Association. - 0012-1797 .- 1939-327X. ; 66:10, s. 2610-2622
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Tidskriftsartikel (refereegranskat)abstract
- Epac is a cAMP-activated guanine nucleotide exchange factor that mediates cAMP signaling in various types of cells, including -cells, where it is involved in the control of insulin secretion. Upon activation, the protein redistributes to the plasma membrane, but the underlying molecular mechanisms and functional consequences are unclear. Using quantitative high-resolution microscopy, we found that cAMP elevation caused rapid binding of Epac2A to the -cell plasma membrane, where it accumulated specifically at secretory granules and rendered them more prone to undergo exocytosis. cAMP-dependent membrane binding required the high-affinity cyclic nucleotide-binding (CNB) and Ras association domains, but not the disheveled-Egl-10-pleckstrin domain. Although the N-terminal low-affinity CNB domain (CNB-A) was dispensable for the translocation to the membrane, it was critical for directing Epac2A to the granule sites. Epac1, which lacks the CNB-A domain, was recruited to the plasma membrane but did not accumulate at granules. We conclude that Epac2A controls secretory granule release by binding to the exocytosis machinery, an effect that is enhanced by prior cAMP-dependent accumulation of the protein at the plasma membrane.
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| 2. |
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| 3. |
- Balboa, Diego, et al.
(författare)
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Functional, metabolic and transcriptional maturation of human pancreatic islets derived from stem cells.
- 2022
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Ingår i: Nature Biotechnology. - : Springer Nature. - 1087-0156 .- 1546-1696. ; 40:7, s. 1042-1055
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Tidskriftsartikel (refereegranskat)abstract
- Transplantation of pancreatic islet cells derived from human pluripotent stem cells is a promising treatment for diabetes. Despite progress in the generation of stem-cell-derived islets (SC-islets), no detailed characterization of their functional properties has been conducted. Here, we generated functionally mature SC-islets using an optimized protocol and benchmarked them comprehensively against primary adult islets. Biphasic glucose-stimulated insulin secretion developed during in vitro maturation, associated with cytoarchitectural reorganization and the increasing presence of alpha cells. Electrophysiology, signaling and exocytosis of SC-islets were similar to those of adult islets. Glucose-responsive insulin secretion was achieved despite differences in glycolytic and mitochondrial glucose metabolism. Single-cell transcriptomics of SC-islets in vitro and throughout 6 months of engraftment in mice revealed a continuous maturation trajectory culminating in a transcriptional landscape closely resembling that of primary islets. Our thorough evaluation of SC-islet maturation highlights their advanced degree of functionality and supports their use in further efforts to understand and combat diabetes.
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| 4. |
- Gucek, Alenka, et al.
(författare)
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Fusion pore regulation by cAMP/Epac2 controls cargo release during insulin exocytosis
- 2019
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Ingår i: eLIFE. - : ELIFE SCIENCES PUBLICATIONS LTD. - 2050-084X. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Regulated exocytosis establishes a narrow fusion pore as initial aqueous connection to the extracellular space, through which small transmitter molecules such as ATP can exit. Co-release of polypeptides and hormones like insulin requires further expansion of the pore. There is evidence that pore expansion is regulated and can fail in diabetes and neurodegenerative disease. Here, we report that the cAMP-sensor Epac2 (Rap-GEF4) controls fusion pore behavior by acutely recruiting two pore-restricting proteins, amisyn and dynamin-1, to the exocytosis site in insulin-secreting beta-cells. cAMP elevation restricts and slows fusion pore expansion and peptide release, but not when Epac2 is inactivated pharmacologically or in Epac2(-/-) (Rapgef4(-/-)) mice. Consistently, overexpression of Epac2 impedes pore expansion. Widely used antidiabetic drugs (GLP-1 receptor agonists and sulfonylureas) activate this pathway and thereby paradoxically restrict hormone release. We conclude that Epac2/cAMP controls fusion pore expansion and thus the balance of hormone and transmitter release during insulin granule exocytosis.
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| 5. |
- Idevall Hagren, Olof, 1980-, et al.
(författare)
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cAMP Mediators of Pulsatile Insulin Secretion from Glucose-stimulated Single β-Cells
- 2010
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 285:30, s. 23005-23016
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Tidskriftsartikel (refereegranskat)abstract
- Pulsatile insulin release from glucose-stimulated beta-cells is driven by oscillations of the Ca2+ and cAMP concentrations in the subplasma membrane space ([Ca2+](pm) and [cAMP](pm)). To clarify mechanisms by which cAMP regulates insulin secretion, we performed parallel evanescent wave fluorescence imaging of [cAMP](pm), [Ca2+](pm), and phosphatidylinositol 3,4,5-trisphosphate (PIP3) in the plasma membrane. This lipid is formed by autocrine insulin receptor activation and was used to monitor insulin release kinetics from single MIN6 beta-cells. Elevation of the glucose concentration from 3 to 11 mM induced, after a 2.7-min delay, coordinated oscillations of [Ca2+](pm), [cAMP](pm), and PIP3. Inhibitors of protein kinase A (PKA) markedly diminished the PIP3 response when applied before glucose stimulation, but did not affect already manifested PIP3 oscillations. The reduced PIP3 response could be attributed to accelerated depolarization causing early rise of [Ca2+](pm) that preceded the elevation of [cAMP](pm). However, the amplitude of the PIP3 response after PKA inhibition was restored by a specific agonist to the cAMP-dependent guanine nucleotide exchange factor Epac. Suppression of cAMP formation with adenylyl cyclase inhibitors reduced already established PIP3 oscillations in glucose-stimulated cells, and this effect was almost completely counteracted by the Epac agonist. In cells treated with small interfering RNA targeting Epac2, the amplitudes of the glucose-induced PIP3 oscillations were reduced, and the Epac agonist was without effect. The data indicate that temporal coordination of the triggering [Ca2+](pm) and amplifying [cAMP](pm) signals is important for glucose-induced pulsatile insulin release. Although both PKA and Epac2 partake in initiating insulin secretion, the cAMP dependence of established pulsatility is mediated by Epac2.
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| 6. |
- Korol, Sergiy V, et al.
(författare)
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Functional Characterization of Native, High-Affinity GABAA Receptors in Human Pancreatic β Cells
- 2018
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Ingår i: EBioMedicine. - : Elsevier BV. - 2352-3964. ; 30
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Tidskriftsartikel (refereegranskat)abstract
- In human pancreatic islets, the neurotransmitter γ-aminobutyric acid (GABA) is an extracellular signaling molecule synthesized by and released from the insulin-secreting β cells. The effective, physiological GABA concentration range within human islets is unknown. Here we use native GABAA receptors in human islet β cells as biological sensors and reveal that 100-1000nM GABA elicit the maximal opening frequency of the single-channels. In saturating GABA, the channels desensitized and stopped working. GABA modulated insulin exocytosis and glucose-stimulated insulin secretion. GABAA receptor currents were enhanced by the benzodiazepine diazepam, the anesthetic propofol and the incretin glucagon-like peptide-1 (GLP-1) but not affected by the hypnotic zolpidem. In type 2 diabetes (T2D) islets, single-channel analysis revealed higher GABA affinity of the receptors. The findings reveal unique GABAA receptors signaling in human islets β cells that is GABA concentration-dependent, differentially regulated by drugs, modulates insulin secretion and is altered in T2D.
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| 7. |
- Mojtaba Ghiasi, Seyed, et al.
(författare)
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The Endoplasmic Reticulum Chaperone Glucose-Regulated Protein 94 is Essential for Proinsulin Handling
- 2019
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Ingår i: Diabetes. - : American Diabetes Association. - 0012-1797 .- 1939-327X. ; 68:4, s. 747-760
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Tidskriftsartikel (refereegranskat)abstract
- Although endoplasmic reticulum (ER) chaperone binding to mutant proinsulin has been reported, the role of protein chaperones in the handling of wild-type proinsulin is under-investigated. Here, we have explored the importance of glucose regulated protein 94 (GRP94), a prominent ER chaperone known to fold insulin-like growth factors, in proinsulin handling within β-cells. We found that GRP94 co-immunoprecipitated with proinsulin and that inhibition of GRP94 function and/or expression reduced glucose-dependent insulin secretion, shortened proinsulin half-life and lowered intracellular proinsulin and insulin levels. This phenotype was accompanied by post-ER proinsulin misprocessing and higher numbers of enlarged insulin granules that contained amorphic material with reduced immunogold staining for mature insulin. Insulin granule exocytosis was two-fold accelerated but the secreted insulin had diminished bioactivity. Moreover, GRP94 knockdown or knockout in β-cells selectively activated Protein Kinase R-like Endoplasmic Reticulum Kinase (PERK), without increasing apoptosis levels. Finally, GRP94 mRNA was overexpressed in islets from T2D patients. We conclude that GRP94 is a chaperone crucial for proinsulin handling and insulin secretion.
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| 8. |
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| 9. |
- Omar-Hmeadi, Muhmmad, et al.
(författare)
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Paracrine control of α-cell glucagon exocytosis is compromised in human type-2 diabetes.
- 2020
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- Glucagon is released from pancreatic α-cells to activate pathways that raise blood glucose. Its secretion is regulated by α-cell-intrinsic glucose sensing and paracrine control through insulin and somatostatin. To understand the inadequately high glucagon levels that contribute to hyperglycemia in type-2 diabetes (T2D), we analyzed granule behavior, exocytosis and membrane excitability in α-cells of 68 non-diabetic and 21 T2D human donors. We report that exocytosis is moderately reduced in α-cells of T2D donors, without changes in voltage-dependent ion currents or granule trafficking. Dispersed α-cells have a non-physiological V-shaped dose response to glucose, with maximal exocytosis at hyperglycemia. Within intact islets, hyperglycemia instead inhibits α-cell exocytosis, but not in T2D or when paracrine inhibition by insulin or somatostatin is blocked. Surface expression of somatostatin-receptor-2 is reduced in T2D, suggesting a mechanism for the observed somatostatin resistance. Thus, elevated glucagon in human T2D may reflect α-cell insensitivity to paracrine inhibition at hyperglycemia.
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| 10. |
- Petrenko, V., et al.
(författare)
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In pancreatic islets from type 2 diabetes patients, the dampened circadian oscillators lead to reduced insulin and glucagon exocytosis
- 2020
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 117:5, s. 2484-2495
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Tidskriftsartikel (refereegranskat)abstract
- Circadian clocks operative in pancreatic islets participate in the regulation of insulin secretion in humans and, if compromised, in the development of type 2 diabetes (T2D) in rodents. Here we demonstrate that human islet alpha- and beta-cells that bear attenuated clocks exhibit strongly disrupted insulin and glucagon granule docking and exocytosis. To examine whether compromised clocks play a role in the pathogenesis of T2D in humans, we quantified parameters of molecular clocks operative in human T2D islets at population, single islet, and single islet cell levels. Strikingly, our experiments reveal that islets from T2D patients contain clocks with diminished circadian amplitudes and reduced in vitro synchronization capacity compared to their nondiabetic counterparts. Moreover, our data suggest that islet clocks orchestrate temporal profiles of insulin and glucagon secretion in a physiological context. This regulation was disrupted in T2D subjects, implying a role for the islet cell-autonomous clocks in T2D progression. Finally, Nobiletin, an agonist of the core-clock proteins ROR alpha/gamma, boosted both circadian amplitude of T2D islet clocks and insulin secretion by these islets. Our study emphasizes a link between the circadian clockwork and T2D and proposes that clock modulators hold promise as putative therapeutic agents for this frequent disorder.
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