| 1. |
- Balboa, Diego, et al.
(författare)
-
Functional, metabolic and transcriptional maturation of human pancreatic islets derived from stem cells.
- 2022
-
Ingår i: Nature Biotechnology. - : Springer Nature. - 1087-0156 .- 1546-1696. ; 40:7, s. 1042-1055
-
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.
|
|
| 2. |
- Idevall Hagren, Olof, 1980-, et al.
(författare)
-
Metabolic regulation of calcium signaling in beta cells
- 2020
-
Ingår i: Seminars in Cell and Developmental Biology. - : ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD. - 1084-9521 .- 1096-3634. ; 103, s. 20-30
-
Forskningsöversikt (refereegranskat)abstract
- The cytoplasmic Ca2+ concentration ([Ca2+](cyt)) regulates a vast number of cellular functions, including insulin secretion from beta cells. The major physiological insulin secretagogue, glucose, triggers [Ca2+](cyt) oscillations in beta cells. Synchronization of the oscillations among the beta cells within an islet underlies the generation of pulsatile insulin secretion. This review describes the mechanisms generating [Ca2+](cyt) oscillations, the interactions between [Ca2+](cyt) and cell metabolism, as well as the contribution of various organelles to the shaping of [Ca2+](cyt) signals and insulin secretion. It also discusses how Ca2+ signals are coordinated and spread throughout the islets and data indicating that altered Ca2+ signaling is associated with beta cell dysfunction and development of type 2 diabetes.
|
|
| 3. |
- Omar-Hmeadi, Muhmmad, et al.
(författare)
-
Paracrine control of α-cell glucagon exocytosis is compromised in human type-2 diabetes.
- 2020
-
Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 11:1
-
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.
|
|
| 4. |
- Panagiotou, Styliani, et al.
(författare)
-
OSBP-mediated PI(4)P-cholesterol exchange at endoplasmic reticulum-secretory granule contact sites controls insulin secretion
- 2024
-
Ingår i: Cell Reports. - 2211-1247. ; 43:4
-
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.
|
|
| 5. |
- Petrenko, V., et al.
(författare)
-
In pancreatic islets from type 2 diabetes patients, the dampened circadian oscillators lead to reduced insulin and glucagon exocytosis
- 2020
-
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
-
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.
|
|
| 6. |
- Shuai, Hongyan, et al.
(författare)
-
Glucose-induced cAMP elevation in β-cells involves amplification of constitutive and glucagon-activated GLP-1 receptor signalling
- 2021
-
Ingår i: Acta Physiologica. - : John Wiley & Sons. - 1748-1708 .- 1748-1716. ; 231:4
-
Tidskriftsartikel (refereegranskat)abstract
- AIM: cAMP typically signals downstream of Gs -coupled receptors and regulates numerous cell functions. In β-cells, cAMP amplifies Ca2+ -triggered exocytosis of insulin granules. Glucose-induced insulin secretion is associated with Ca2+ - and metabolism-dependent increases of the sub-plasma-membrane cAMP concentration ([cAMP]pm ) in β-cells, but potential links to canonical receptor signalling are unclear. The aim of this study was to clarify the role of glucagon-like peptide-1 receptors (GLP1Rs) for glucose-induced cAMP signalling in β-cells.METHODS: Total internal reflection microscopy and fluorescent reporters were used to monitor changes in cAMP, Ca2+ and ATP concentrations as well as insulin secretion in MIN6 cells and mouse and human β-cells. Insulin release from mouse and human islets was also measured with ELISA.RESULTS: The GLP1R antagonist exendin-(9-39) (ex-9) prevented both GLP1- and glucagon-induced elevations of [cAMP]pm , consistent with GLP1Rs being involved in the action of glucagon. This conclusion was supported by lack of unspecific effects of the antagonist in a reporter cell-line. Ex-9 also suppressed IBMX- and glucose-induced [cAMP]pm elevations. Depolarization with K+ triggered Ca2+ -dependent [cAMP]pm elevation, an effect that was amplified by high glucose. Ex-9 inhibited both the Ca2+ and glucose-metabolism-dependent actions on [cAMP]pm . The drug remained effective after minimizing paracrine signalling by dispersing the islets and it reduced basal [cAMP]pm in a cell-line heterologously expressing GLP1Rs, indicating that there is constitutive GLP1R signalling. The ex-9-induced reduction of [cAMP]pm in glucose-stimulated β-cells was paralleled by suppression of insulin secretion.CONCLUSION: Agonist-independent and glucagon-stimulated GLP1R signalling in β-cells contributes to basal and glucose-induced cAMP production and insulin secretion.
|
|
| 7. |
- Strage, Emma, et al.
(författare)
-
Insulin release from isolated cat islets of Langerhans
- 2024
-
Ingår i: Domestic Animal Endocrinology. - : Elsevier. - 0739-7240 .- 1879-0054. ; 87
-
Tidskriftsartikel (refereegranskat)abstract
- Feline diabetes mellitus is a common endocrine disease with increasing prevalence. It shows similarities with human type 2 diabetes and is characterized by insulin resistance and deficient insulin secretion. Moreover, cats and humans belong to the very few species that form amyloid depositions in the pancreatic islets. However, little is known about cat islet function and no studies have addressed insulin secretion from isolated islets ex vivo. The aim of this study was to establish a protocol for isolation of islets of Langerhans from pancreata of cats euthanized due to disease, and to evaluate insulin secretion responses to various physiological and pharmacological stimuli. Collagenase digestion of pancreatic tissue from 13 non-diabetic cats and two cats with diabetic ketoacidosis yielded individual islets surrounded by a layer of exocrine tissue that was reduced after two days in culture. Histological examination showed islet amyloid in pancreatic biopsies from most non-diabetic and in one diabetic cat. Islets from non-diabetic cats cultured at 5.5 mM glucose responded with increased insulin secretion to 16.7 mM glucose, 30 mM K+ and 20 µM of the sulfonylurea glipizide (2-3 times basal secretion at 3 mM glucose). The glucagon-like peptide-1 receptor agonist exendin-4 (100 nM) had no effect under basal conditions but potentiated glucose-triggered insulin release. Only one of nine islet batches from diabetic cats released detectable amounts of insulin, which was enhanced by exendin-4. Culture of islets from non-diabetic cats at 25 mM glucose impaired secretion both in response to glucose and K+ depolarization. In conclusion, we describe a procedure for isolation of islets from cat pancreas biopsies and demonstrate that isolated cat islets secrete insulin in response to glucose and antidiabetic drugs. The study provides a basis for future ex vivo studies of islet function relevant to the understanding of the pathophysiology and treatment of feline diabetes.
|
|
| 8. |
- Vishnu, N., et al.
(författare)
-
Mitochondrial clearance of calcium facilitated by MICU2 controls insulin secretion
- 2021
-
Ingår i: Molecular Metabolism. - : Elsevier. - 2212-8778. ; 51
-
Tidskriftsartikel (refereegranskat)abstract
- Objective: Transport of Ca2+ into pancreatic 13 cell mitochondria facilitates nutrient-mediated insulin secretion. However, the underlying mechanism is unclear. Recent establishment of the molecular identity of the mitochondrial Ca2+ uniporter (MCU) and associated proteins allows modification of mitochondrial Ca2+ transport in intact cells. We examined the consequences of deficiency of the accessory protein MICU2 in rat and human insulin-secreting cells and mouse islets. Methods: siRNA silencing of Micu2 in the INS-1 832/13 and EndoC-13H1 cell lines was performed; Micu2-/- mice were also studied. Insulin secretion and mechanistic analyses utilizing live confocal imaging to assess mitochondrial function and intracellular Ca2+ dynamics were performed. Results: Silencing of Micu2 abrogated GSIS in the INS-1 832/13 and EndoC-13H1 cells. The Micu2-/- mice also displayed attenuated GSIS. Mitochondrial Ca2+ uptake declined in MICU2-deficient INS-1 832/13 and EndoC-13H1 cells in response to high glucose and high K+. MICU2 silencing in INS-1 832/13 cells, presumably through its effects on mitochondrial Ca2+ uptake, perturbed mitochondrial function illustrated by absent mitochondrial membrane hyperpolarization and lowering of the ATP/ADP ratio in response to elevated glucose. Despite the loss of mitochondrial Ca2+ uptake, cytosolic Ca2+ was lower in siMICU2-treated INS-1 832/13 cells in response to high K+. It was hypothesized that Ca2+ accumulated in the submembrane compartment in MICU2-deficient cells, resulting in desensitization of voltage-dependent Ca2+ channels, lowering total cytosolic Ca2+. Upon high K+ stimulation, MICU2-silenced cells showed higher and prolonged increases in submembrane Ca2+ levels. Conclusions: MICU2 plays a critical role in 13 cell mitochondrial Ca2+ uptake. 13 cell mitochondria sequestered Ca2+ from the submembrane compartment, preventing desensitization of voltage-dependent Ca2+ channels and facilitating GSIS.
|
|
| 9. |
- Xie, Beichen, 1989-
(författare)
-
Membrane lipids and their transfer proteins in β-cells
- 2020
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
|
|
| 10. |
- Yang, Mingyu, et al.
(författare)
-
Indicator-dependent differences in detection of local intracellular Ca2+release events evoked by voltage-gated Ca2+entry in pancreatic & beta;-cells
- 2023
-
Ingår i: Cellular Signalling. - : Elsevier BV. - 0898-6568 .- 1873-3913. ; 109
-
Tidskriftsartikel (refereegranskat)abstract
- Genetically encoded Ca2+ indicators have become widely used in cell signalling studies as they offer advantages over cell-loaded dye indicators in enabling specific cellular or subcellular targeting. Comparing responses from dye and protein-based indicators may provide information about indicator properties and cell physiology, but side-by-side recordings in cells are scarce. In this study, we compared cytoplasmic Ca2+ concentration ([Ca2+]i) changes in insulin-secreting & beta;-cells recorded with commonly used dyes and indicators based on circularly permuted fluorescent proteins. Total internal reflection fluorescence (TIRF) imaging of K+ depolarizationtriggered submembrane [Ca2+]i increases showed that the dyes Fluo-4 and Fluo-5F mainly reported stable [Ca2+]i elevations, whereas the proteins R-GECO1 and GCaMP5G more often reported distinct [Ca2+]i spikes from an elevated level. [Ca2+]i spiking occurred also in glucose-stimulated cells. The spikes reflected Ca2+ release from the endoplasmic reticulum, triggered by autocrine activation of purinergic receptors after exocytotic release of ATP and/or ADP, and the spikes were consequently prevented by SERCA inhibition or P2Y1-receptor antagonism. Widefield imaging, which monitors the entire cytoplasm, increased the spike detection by the Ca2+ dyes. The indicator-dependent response patterns were unrelated to Ca2+ binding affinity, buffering and mobility, and probably reflects the much slower dissociation kinetics of protein compared to dye indicators. Ca2+ dyes thus report signalling within the submembrane space excited by TIRF illumination, whereas the protein indicators also catch Ca2+ events originating outside this volume. The study highlights that voltage-dependent Ca2+ entry in & beta;-cells is tightly linked to local intracellular Ca2+ release mediated via an autocrine route that may be more important than previously reported direct Ca2+ effects on phospholipase C or on intracellular channels mediating calcium-induced calcium release.
|
|
