Biology subjects

Barg, S.

Publications and source records attributed to Barg, S..

12 recordsLinked to original sources

bioRxiv2025

Pancreatic δ-cells are resistant to auto- and paracrine inhibition in human type-2 diabetes

Somatostatin secretion from pancreatic {delta}-cells inhibits nearby -and {beta}-cells, and tunes the bodys glycemic set-point. The role of {delta}-cells in diabetes remains unclear, in part due to the difficulty separating intrinsic regulation from intra-islet paracrine effects. Here we compared the function of isolated {delta}-cells of cadaveric non-diabetic and type-2 diabetic donors, by single cell TIRF-microscopy and electrophysiology. Elevated glucose stimulated exocytosis of somatostatin, which was further amplified by glucagon, exendin-4, or forskolin, independent of diabetic status. GABA enhanced exocytosis and electrical activity, while insulin had no effect. Adrenaline and somatostatin strongly inhibited {delta}-cell activity, leading to autocrine feedback inhibition of somatostatin exocytosis. In type-2 diabetes, {delta}-cell inhibition by somatostatin and adrenaline was lost, together with a marked reduction in somatostatin receptor (SSTR2) surface expression. We further show that resistance to somatostatin leads to hyperactive {delta}-cells in type-2 diabetes, and propose that this mechanism contributes to defective blood glucose control.

cell biology↗

bioRxiv2024

Highly adaptable deep-learning platform for automated detection and analysis of vesicle exocytosis

Vesicle exocytosis is a fundamental component of intercellular communication, in all organisms. It has been studied for decades, using various imaging tools. Nevertheless, exocytosis research is still limited by the lack of reliable automated analysis procedures. To address this, we developed the Intelligent Vesicle Exocytosis Analysis Platform (IVEA), a nearly universal solution for analyzing exocytosis acquired with live cell imaging. IVEA is applicable to a wide variety of experimental model systems, microscopes and reporter fluorophores. IVEA combines state-of-the-art deep-learning and computer vision regimes to enable fully automated analysis of large data. IVEA runs as a FIJI plugin and does not require prior training or human intervention. IVEA is 60 times faster than manual analysis and is able to detect rare events often missed by the human eye. Overall, IVEA represents a breakthrough in the analysis of cellular secretory mechanisms and has a transformative potential for the exocytosis imaging field.

bioengineering↗

bioRxiv2024

Molecular mapping and functional validation of GLP-1R cholesterol binding sites in pancreatic beta cells

G protein-coupled receptors (GPCRs) are integral membrane proteins which closely interact with their plasma membrane lipid microenvironment. Cholesterol is a lipid enriched at the plasma membrane with pivotal roles in the control of membrane fluidity and maintenance of membrane microarchitecture, directly impacting on GPCR stability, dynamics and function. Cholesterol extraction from pancreatic beta cells has previously been shown to disrupt the internalisation, clustering and cAMP responses of the glucagon-like peptide-1 receptor (GLP-1R), a class B1 GPCR with key roles in the control of blood glucose levels via the potentiation of insulin secretion in beta cells and weight reduction via the modulation of brain appetite control centres. Here, we unveil the detrimental effect of a high cholesterol diet on GLP-1R-dependent glucoregulation in vivo, and the improvement in GLP-1R function that a reduction in cholesterol synthesis using simvastatin exerts in pancreatic islets. We next identify and map sites of cholesterol high occupancy and residence time on active versus inactive GLP-1Rs using coarse-grained molecular dynamics (cgMD) simulations, followed by a screen of key residues selected from these sites and detailed analyses of the effects of mutating one of these residues, Val229, to alanine on GLP-1R interactions with cholesterol, plasma membrane behaviours, clustering, trafficking and signalling in pancreatic beta cells and primary islets, unveiling an improved insulin secretion profile for the V229A mutant receptor. This study 1) highlights the role of cholesterol in regulating GLP-1R responses in vivo; 2) provides a detailed map of GLP-1R - cholesterol binding sites in model membranes; 3) validates their functional relevance in beta cells; and 4) highlights their potential as locations for the rational design of novel allosteric modulators with the capacity to fine-tune GLP-1R responses.

cell biology↗

bioRxiv2023

RFX6 haploinsufficiency predisposes to diabetes through impaired beta cell functionality

Regulatory factor X 6 (RFX6) is indispensable for pancreatic endocrine development and differentiation. The RFX6 protein-truncating variant p.His293LeufsTer7 is significantly enriched in the Finnish population with almost 1:250 individuals as a carrier. Importantly, the FinnGen study indicates a high predisposition for heterozygous carriers to develop type 2 diabetes (T2D) and gestational diabetes. To understand the role of this variant in {beta}-cell development and function, we generated allelic series of isogenic pluripotent stem cell models and directed them into pancreatic islet lineages (SC-islets). Expectedly, in-vitro models of the homozygous RFX6-/- variant failed to generate pancreatic endocrine cells, recapitulating the phenotype in Mitchell-Riley syndrome. Notably, heterozygous RFX6+/- derived SC-islets showed reduced {beta}-cell maturation markers and calcium oscillations, resulting in defective insulin secretion, without affecting {beta}-cell number or insulin content. The reduced insulin secretion is sustained during in-vivo implantation studies, consistent with the susceptibility of the carriers to develop diabetes. TeaserModeling RFX6-assocciated neonatal and type-2 diabetes using allelic series stem cell-derived islets in-vitro and in-vivo.

cell biology↗

bioRxiv2022

Real-time TIRF imaging of exocytosis in 3T3-L1 adipocytes stably expressing mCherry fused to human adiponectin.

Adiponectin is a peptide hormone abundantly released from adipocytes, and reduced circulating levels are associated with obesity-related diseases, such as type 2 diabetes, insulin resistance and cardiovascular disease. Adiponectin is released by regulated exocytosis of secretory vesicles, but traditional molecular biology and imaging techniques lack the specificity and time resolution to adequately quantify exocytosis and trafficking of adiponectin-containing vesicles. Here we generated 3T3-L1 adipocytes that stably express mCherry-tagged human adiponectin, resulting in robust labelling of small adiponectin vesicles with a diameter of 200-300 nm, in live cells. Total internal reflection fluorescence (TIRF) microscopy was used to visualise and quantify exocytosis and adiponectin release in real-time, observed as rapid disappearance of the fluorescence of individual vesicles. Bulk adiponectin secretion measurements confirmed that the labelled adiponectin was secreted to the surrounding solution under these conditions, and expressed in the same vesicle population as endogenous adiponectin. In contrast to previous electrophysiological results, elevation of cytosolic Ca2+ alone was sufficient to induce exocytosis, although at a lower rate compared to elevated cytosolic cAMP. We conclude that the adiponectin-mCherry-labelled cells are useful for studying adiponectin exocytosis at the single vesicle level, and that an intracellular elevation of either cAMP or Ca2+ can trigger adiponectin vesicle release.

cell biology↗

bioRxiv2022

Restoration of PITPNA in Type 2 diabetic human islets reverses pancreatic beta-cell dysfunction

Defects in insulin processing and granule maturation are linked to pancreatic beta-cell failure during type 2 diabetes (T2D). Phosphatidylinositol transfer protein alpha (PITPNA) stimulates activity of phosphatidylinositol (PtdIns) 4-OH kinase to produce sufficient PtdIns-4-phosphate (PtdIns-4-P) in the trans-Golgi network to promote insulin granule maturation. PITPNA in beta-cells of T2D human subjects is markedly reduced suggesting its depletion accompanies beta-cell dysfunction. Conditional deletion of Pitpna in the beta-cells of Ins-Cre;Pitpnaflox/flox mice leads to hyperglycemia resulting from decreased glucose-stimulated insulin secretion (GSIS) and reduced pancreatic beta-cell mass. Furthermore, PITPNA silencing in human islets confirmed its role in PtdIns-4-P synthesis and led to impaired insulin granule maturation and docking, GSIS, and proinsulin processing with evidence of ER stress. Restoration of PITPNA in islets of T2D human subjects reversed these beta-cell defects and identify PITPNA as a critical target linked to beta-cell failure in T2D.

cell biology↗

bioRxiv2022

Phase Separation of a Nodulin Sec14-like protein Maintains Auxin Efflux Carrier Polarity at Arabidopsis Plasma Membranes

Protein function can be modulated by phase transitions in their material properties, which can range from liquid-to solid-like; yet the mechanisms that drive these transitions and whether they are important for physiology are still unknown. Using the model plant Arabidopsis, we show that developmental robustness is reinforced by phase transitions of the plasma membrane-bound lipid-binding protein SEC14-like. Using imaging, genetics, and in vitro reconstitution experiments, we show that SEC14-like undergoes liquid-like phase separation in the root stem cells. Outside the stem cell niche, SEC14-like associates with the caspase-like protease separase and conserved microtubule motors at unique polar plasma membrane interfaces. In these interfaces, SEC14-like undergoes abrupt processing by separase, which promotes its liquid-to-solid transition. The SEC14-like liquid-to-solid transition is important for root developmental robustness, as lines expressing an uncleavable SEC14-like variant or mutants of separase, and associated microtubule motors show similar developmental phenotypes. Furthermore, the processed and solidified but not the liquid form of SEC14-like interacts with the polar protein PINFORMED2 at the plasma membrane and perhaps other polar proteins of the PINFORMED family. This work demonstrates that robust development can involve abrupt liquid-to-solid transitions mediated by proteolysis at unique plasma membrane interfaces.

cell biology↗

bioRxiv2022

Local PI(4,5)P2 generation controls fusion pore expansion during exocytosis

Phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) is an important signaling phospholipid that is required for regulated exocytosis and some forms of endocytosis. The two processes share a topologically similar pore structure that connects the vesicle lumen with the outside. Widening of the fusion pore during exocytosis leads to cargo release, while its closure initiates kiss&run or cavicapture endocytosis. We show here, using live cell TIRF microscopy of insulin granule exocytosis, that transient accumulation of PI(4,5)P2 at the release site recruits components of the endocytic fission machinery, and stalls the late fusion pore expansion that is required for peptide release. The absence of clathrin differentiates this mechanism from clathrin-mediated endocytosis. The PI(4,5)P2 transients result from local phosphatidylinositol-phosphate-5-kinase-1c (PIP5K1c) activity, and knockdown of PIP5K1c, or optogenetic ablation of PI(4,5)P2 promotes fusion pore expansion. Thus, local phospholipid signaling controls fusion pore expansion peptide release through an unconventional endocytic mechanism.

cell biology↗

bioRxiv2021

The β-cell primary cilium is an autonomous Ca2+ compartment for paracrine GABA signalling

The primary cilium is an organelle present in most adult mammalian cells and is thought of as an antenna for detection of a variety of signals. Here we use intact mouse pancreatic islets of Langerhans to investigate signalling properties of the primary cilium in {beta}-cells. Using cilia-targeted Ca2+ indicators we find that the resting Ca2+ concentration in the cilium is lower than that of the cytosol, and we uncover a Ca2+ extrusion mechanism in the cilium that effectively insulates the cilium from changes in cytosolic Ca2+. Stimuli that give rise to pronounced cytosolic Ca2+ concentration increases, such as glucose- and depolarization-induced Ca2+ influx, and mobilization of Ca2+ from the ER, was accompanied by minor increases in cilia Ca2+ concentrations that were spatially restricted to a small compartment at the base. Conversely, we observe pronounced Ca2+ concentration changes in the primary cilia of islet {beta}-cells that do not propagate into the cytosol and show that paracrine GABA signalling via cilia-localized GABA-B1-receptors is responsible for this Ca2+ signalling. Finally, we demonstrate that the cilia response to GABA involves ligand-dependent transport of GABA-B1 receptors into the cilium.

cell biology↗

bioRxiv2021

Functional, metabolic and transcriptional maturation of stem cell derived beta cells

Transplantation of pancreatic islet cells derived from human pluripotent stem cells is a promising treatment for diabetes. Despite progress in stem cell-derived islet (SC-islet) generation, detailed characterization of their functional properties has not been conducted. Here, we generated functionally mature SC-islets using an optimized protocol and comprehensively benchmarked them against primary adult islets. Biphasic glucose stimulated insulin secretion developed during in vitro maturation, associated with cytoarchitectural reorganization and increased alpha cells. Electrophysiology and exocytosis of SC-islets were comparable to 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 murine engraftment 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.

developmental biology↗

bioRxiv2019

Paracrine control of α-cell glucagon exocytosis is compromised in human type-2 diabetes

Glucagon is secreted from pancreatic -cells to activate gluconeogenesis and other pathways that raise blood glucose during hypoglycemia. Glucose-dependent regulation of glucagon secretion involves both -cell-intrinsic mechanisms and paracrine control through insulin and somatostatin. In type-2 diabetes (T2D) inadequately high glucagon levels contribute to hyperglycemia. To understand these disease-associated changes at the cellular level, and to isolate intrinsic and paracrine effects, we analyzed glucagon granule exocytosis and membrane excitability in isolated -cells from 56 non-diabetic (ND) and 15 T2D human donors. High resolution imaging showed that glucagon granule exocytosis had a U-shaped sensitivity to glucose, with the slowest rate around 7 mM glucose, and accelerated rates at <5 and >10 mM glucose. Exocytosis was reduced in T2D -cells, but their glucose sensitivity remained intact and there were no changes in voltage-dependent ion currents or granule trafficking. Instead, -cells from T2D donors were markedly insensitive to somatostatin and insulin, which rapidly inhibited exocytosis and electrical activity in ND cells. Thus, intrinsic mechanisms do not inhibit glucagon secretion at hyperglycemia, and elevated glucagon levels in human T2D reflect an insensitivity of -cells to paracrine inhibition.

cell biology↗

bioRxiv2018

Fusion pore regulation by Epac2/cAMP controls cargo release during insulin exocytosis

Regulated exocytosis establishes a narrow fusion pore as the initial aqueous connection to the extracellular space, through which small transmitter molecules such as ATP can exit. Co-release of larger peptides and hormones like insulin requires further expansion of the pore. There is evidence that pore expansion is regulated and can fail in type-2 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 leads to pore expansion and peptide release, but not when Epac2 is inactivated pharmacologically or in Epac2-/- mice. Conversely, overexpression of Epac2 impedes pore expansion. Widely used antidiabetic drugs (GLP-1 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.

cell biology↗