Genome-wide CRISPR Screen Identifies Sec31A as a Key Regulator of Alpha Cell Survival.

Introduction and Objective: Glucagon is critical for glycemic control, yet its dysregulation contributes to type 1 (T1D) and advanced type 2 diabetes (T2D) progression. Although beta cell loss defines diabetes, alpha cells retain their mass despite stress. Mechanisms enabling alpha cell resilience in the diabetic milieu remain unclear. This study identified regulators of alpha cell survival during ER stress, integrating data from animal models and human tissues for translational insights. Methods: A genome-wide CRISPR screen in mouse alphaTC6 cells identified Sec31A, a key ER-Golgi transport regulator, as essential for alpha cell survival under ER stress. Sec31A knockdown models included C. elegans and alphaTC6 cells exposed to ER stress. Sec31A expression in human islets under cytokine stress was analyzed by immunohistochemistry (IHC). RNA sequencing (RNA-seq) was performed to investigate how SEC31A expression in live T1D islets changes with ER stress induction and mitigation. Functional studies in human alpha pseudo-islets with SEC31A knockdown examined insulin signaling pathways. Results: Sec31A deficiency improved alpha cell survival under ER stress in mouse and C. elegans models. Human islets showed higher Sec31A expression in alpha versus beta cells during cytokine stress. RNA-seq of T1D islets confirmed stress-induced SEC31A upregulation, which diminished when stress was mitigated.SEC31A knockdown in human pseudo-islets revealed alpha cell-specific pathways linked to insulin receptor interactions, highlighting mechanistic differences from beta cells. Conclusion: This study identifies Sec31A as a key regulator of alpha cell resilience during ER stress. By integrating model and human data, these findings enhance our understanding of alpha cell biology and provide a foundation for targeted therapies to preserve alpha cell function. Such interventions have the potential to prevent severe hypoglycemia and enhance glucose homeostasis in diabetes. Disclosure K. Shibue: None. S. Kahraman: Employee; Boehringer-Ingelheim. J.I. Castillo-Quan: None. D.F. De Jesus: None. J. Hu: None. K. Blackwell: None. P. Yi: None. R. Kulkarni: Advisory Panel; Novo Nordisk, Biomea Fusion, REDD Pharma, Inversago Pharma. Research Support; Inversago Pharma. Stock/Shareholder; Biomea Fusion.

Insulin secretion is a highly regulated mechanism involving a complex insulin-dependent network of communication between alpha, beta and delta cells. However, whereas the role of insulin in beta cells has been well documented, very little is known about its role in alpha and delta cells. Having recently demonstrated heterogeneity of insulin receptor (INSR) isoform expression in these three endocrine cell types, our current study aimed to characterise the expression pattern of the multiple isoforms involved in the insulin signal transduction cascade in human alpha, beta and delta cells in vitro. cDNA samples prepared from single human islet cells were subjected to nested PCRs. Of 706 cells analysed, 15% were alpha cells, 28% beta cells, 8% delta cells and 46% non-endocrine cells. Profiling of expression of the insulin signalling cascade elements showed a heterogeneity between islet cell types, although at least one member of each protein family was expressed in the three populations of endocrine cells. Thus, the mRNAs coding for INSR-B, phosphoinositide-dependent protein kinase-1 and the human homologue of v-akt murine thymoma viral oncogene homologue 1 (AKT1) could not be detected in alpha cells, but were expressed by beta and delta cells. In addition, while the insulin receptor substrates IRS1 and IRS2, phosphoinositide-3-kinase, catalytic, beta polypeptide (PIK3CB) and AKT2 were expressed with relatively low frequencies in alpha and delta cells (<17% for IRS1, IRS2, PIK3CB; <25% for AKT2), their frequencies of expression in beta cells were 50, 33, 33 and 100%, respectively. Our results suggest that insulin signalling cascade elements in human alpha, beta and delta cells have distinct expression patterns.

Both alpha and beta cells are dysfunctional in type 1 diabetes (T1D), but beta cells die while alpha cells survive the immune attack. Understanding the mechanisms underlying alpha-cell resistance could identify new approaches to protect beta cells. Herein, we analysed single-cell datasets from human alpha and beta cells under basal/unstimulated conditions and under immune-mediated stress. Alpha cells exhibit enhanced immune-like gene expression compared to beta cells. We also found that the tumour suppressor Maternally Expressed Gene 3 (MEG3), a T1D risk gene, is highly expressed in beta cells while almost undetectable in alpha cells. These observations were confirmed by analysing bulk RNA-sequencing data from fluorescence-activated cell-sorted alpha and beta cells isolated from primary human islets from non-diabetic donors. Additionally, MEG3 knockdown in human insulin-producing EndoC-βH1 cells and human islets microtissues decreased cytokine-induced damage and apoptosis, preserving beta-cell function under inflammatory conditions. The fact that alpha cells exhibit increased immune-like and anti-apoptotic activity as compared to beta cells suggests that they are better equipped to endure the autoimmune assault in T1D. In addition, the marked difference in the expression of the pro-apoptotic factor MEG3 in beta cells compared to alpha cells may explain, at least in part, why beta cells are more susceptible to damage and cell death in a diabetogenic environment than neighbour alpha cells within the same islet.

Insulin-producing beta cells are known to be highly susceptible to hypoxia, which is a major factor in their destruction after pancreatic islet transplantation. However, whether the glucagon-producing pancreatic islet alpha cells are sensitive to hypoxia is not known. Our objective was to compare the sensitivity of alpha and beta cells to hypoxia. Isolated rat pancreatic islets were exposed to hypoxia (1% oxygen, 94% N(2), 5% CO(2)) for 3 days. The viability of the alpha and beta cells, as well as the stimulus-specific secretion of glucagon and insulin, was evaluated. A quantitative analysis of the proportion of beta to alpha cells indicated that, under normoxic conditions, islet cells were composed mainly of beta cells (87 ± 3%) with only 13 ± 3% alpha cells. Instead, hypoxia treatment significantly increased the proportion of alpha cells (40 ± 13%) and decreased the proportion of beta cells to 60 ± 13%. Using the fluorescent TUNEL assay we found that only a few percent of beta cells and alpha cells were apoptotic in normoxia. In contrast, hypoxia induced an abundance of apoptotic beta cells (61 ± 22%) and had no effect on the level of apoptosis in alpha cells. In conclusion, this study demonstrates that hypoxia results in severe functional abnormality in both beta and alpha cells while alpha cells display significantly decreased rate of apoptosis compared to intensive apoptotic injury of beta cells. These findings have implications for the understanding of the possible role of hypoxia in the pathophysiology of diabetes.

Glucose metabolism by pancreatic beta and alpha cells is essential for stimulation of insulin secretion and inhibition of glucagon secretion. Studies using rodent islets have suggested that the ATP/ADP ratio serves as second messenger in beta cells. This study compared the effects of glucose on glucose oxidation ([U-14C]glucose) and adenine nucleotides (luminometric method) in purified rat alpha and beta cells. The rate of glucose oxidation at 1 mM glucose was higher in beta than alpha cells (4.5-fold, i.e. approximately 2-fold after normalization for cell size). It was more strongly stimulated by 10 mM glucose in beta cells (9-fold) than in alpha cells (5-fold). At 1 mM glucose, ATP levels were similar in both cell types, which corresponds to an approximately 2-fold higher concentration in alpha cells ( approximately 6.5 mM) than in beta cells ( approximately 3 mM). In beta cells, glucose dose-dependently increased ATP and decreased ADP levels, causing a rise in the ATP/ADP ratio from 2.4 to 11.6 at 1 and 10 mM, respectively. In alpha cells, glucose did not affect ATP and ADP levels, and the ATP/ADP ratio remained stable around 7.5. In human islets, the ATP/ADP ratio progressively increased between 1 and 10 mM glucose. In duct cells, which often contaminate human islet preparations, an increase in the ATP/ADP ratio sometimes occurred between 1 and 3 mM glucose. In conclusion, the present observations establish that the regulation of glucagon secretion by glucose does not involve changes in alpha cell adenine nucleotides and further support the role of the ATP/ADP ratio in the control of insulin secretion.

Characterization of endocrine-cell functions and associated molecular signatures in diabetes is crucial to better understand why and by which mechanisms alpha and beta cells cause and perpetuate metabolic abnormalities. The now recognized role of glucagon in diabetes control is a major incentive to have a better understanding of dysfunctional alpha cells. To characterize molecular alterations of alpha cells in diabetes, we analyzed alpha-cell transcriptome from control and diabetic mice using diet-induced obesity model. To this aim, we quantified the expression levels of total mRNAs from sorted alpha and beta cells of low-fat and high-fat diet-treated mice through RNAseq experiments, using a transgenic mouse strain allowing collections of pancreatic alpha- and beta-cells after 16 weeks of diet. We now report that pancreatic alpha cells from obese hyperglycemic mice displayed minor variations of their transcriptome compared to controls. Depending on analyses, we identified 11 to 39 differentially expressed genes including non-alpha cell markers mainly due to minor cell contamination during purification process. From these analyses, we identified three new target genes altered in diabetic alpha cells and potently involved in cellular stress and exocytosis (Upk3a, Adcy1 and Dpp6). By contrast, analysis of the beta-cell transcriptome from control and diabetic mice revealed major alterations of specific genes coding for proteins involved in proliferation and secretion. We conclude that alpha cell transcriptome is less reactive to HFD diet compared to beta cells and display adaptations to cellular stress and exocytosis.

Differential expression of glucagon and glucagon-like peptide 1 receptors in mouse pancreatic alpha and beta cells in two models of alpha cell hyperplasia

Pax4 and MafA (v-maf musculoaponeurotic fibrosarcoma oncogene homolog A) are two transcription factors crucial for normal functions of islet beta cells in the mouse. Intriguingly, recent studies indicate the existence of notable difference between human and rodent islet in terms of gene expression and functions. To better understand the biological role of human PAX4 and MAFA, we investigated their expression in normal and diseased human islets, using validated antibodies. PAX4 was detected in 43.0±5.0% and 39.1±4.0% of normal human alpha and beta cells respectively. We found that MAFA, detected in 88.3±6.3% insulin+cells as in the mouse, turned out to be also expressed in 61.2±6.4% of human glucagons+ cells with less intensity than in insulin+ cells, whereas MAFB expression was found not only in the majority of glucagon+ cells (67.2±7.6%), but also in 53.6±10.5% of human insulin+ cells. Interestingly, MAFA nuclear expression in both alpha and beta cells, and the percentage of alpha cells expressing PAX4 were found altered in a substantial proportion of patients with type 2 diabetes. Both MAFA and PAX4 display, therefore, a distinct expression pattern in human islet cells, suggesting more potential plasticity of human islets as compared with rodent islets.

The fine structure of the foetal rat pancreatic alpha and beta cells was described at term (Day 22) and during prolonged gestation (Days 23, 24, and 25). Gestation was prolonged by daily subcutaneous injections of progesterone to the mothers from Days 20 through 24 of pregnancy. Alpha and beta cells at term contained numerous secretory granules and well developed cytoplasmic organelles. During prolonged gestation (Days 24 and 25 only) the beta cells exhibited an increased number of secretory granules whereas the alpha cells revealed decreased numbers of secretory granules and increased numbers of Golgi saccules and associated vesicles. In addition a few alpha cells contained numerous secretory granules which were concentrated toward the cell surface nearest a capillary. The results were discussed with respect to what role foetal alpha and beta cells might have in foetal glucose homeostasis during prolonged gestation.

Background: The endocrine pancreas contains multiple cell types co-localized into clusters called the Islets of Langerhans. The predominant cell types include alpha and beta cells, which produce glucagon and insulin, respectively. The regulated release of these hormones maintains whole body glucose homeostasis, essential for normal metabolism and to prevent diabetes and complications from the disease. Given the heterogeneous nature of islet composition and absence of unique surface markers, many previous studies have focused on the whole islet. Sorting islet cells by intracellular hormone expression overcomes this limitation and provides pure populations of individual islet cell subsets, specifically alpha and beta cells. This technique provides the framework for characterizing human islet composition and will work towards identifying the genetic changes alpha and beta cells undergo during development, growth, and proliferation. Methods: Human islets obtained from cadaveric donors are dissociated into a single cell suspension, fixed, permeabilized, and labeled with antibodies specific to glucagon, insulin, and somatostatin. Individual alpha, beta, and delta cell populations are simultaneously isolated using fluorescence activated cell sorting. Candidate gene expression and microRNA profiles have been obtained for alpha and beta cell populations using a quantitative nuclease protection assay. Thus far, RNA has been extracted from whole islets and beta cells and subjected to next generation sequencing analysis. Results: The ratio of beta to alpha cells significantly increases with donor age and trends higher in female donors; BMI does not appear to significantly alter the ratio. Further, we have begun to investigate the unique gene expression profiles of alpha and beta cells versus whole islets, and have characterized the microRNA profiles of the two cell subsets. Conclusions: By establishing methods to profile multiple characteristics of alpha and beta cells, we hope to determine how gene, miRNA, and protein expression patterns change under environmental conditions that lead to beta cell failure or promote beta cell development, growth, and proliferation.

Aims/hypothesisDiabetes is associated with the dysfunction of glucagon-producing pancreatic islet alpha cells, although the underlying mechanisms regulating glucagon secretion and alpha cell dysfunction remain unclear. While insulin secretion from pancreatic beta cells has long been known to be controlled partly by intracellular phospholipid signalling, very little is known about the role of phospholipids in glucagon secretion. Using patch-clamp electrophysiology and single-cell RNA sequencing, we previously found that expression of PIP4P2 (encoding TMEM55A, a lipid phosphatase that dephosphorylates phosphatidylinositol-4,5-bisphosphate [PIP2] to phosphatidylinositol-5-phosphate [PI5P]) correlates with alpha cell function. We hypothesise that TMEM55A is involved in glucagon secretion and aim to validate the role of TMEM55A and its potential signalling molecules in alpha cell function and glucagon secretion.MethodsCorrelation analysis was generated from the data in www.humanislets.com. Human islets were isolated at the Alberta Diabetes Institute IsletCore. Electrical recordings were performed on dispersed human or mouse islets with scrambled siRNA or si-PIP4P2 (si-Pip4p2 for mouse) transfection. Glucagon secretion was measured using an islet perfusion system with intact mouse islets. TMEM55A activity was measured using an in vitro on-beads phosphatase assay and live-cell imaging. GTPase activity was measured using an active GTPase pull-down assay. Confocal microscopy was used to quantify F-actin intensity using primary alpha cells and alphaTC1–9 cell lines after chemical treatment.ResultsTMEM55A regulated alpha cell exocytosis and glucagon secretion. TMEM55A knockdown in both human and mouse alpha cells reduced exocytosis at low glucose levels and this was rescued by the direct reintroduction of PI5P. PI5P, instead of PIP2 increased the glucagon secretion using intact mouse islets. This did not occur through an effect on Ca2+ channel activity but through a remodelling of cortical F-actin dependent on TMEM55A lipid phosphatase activity, which occurred in response to oxidative stress. TMEM55A- and PI5P-induced F-actin remodelling depends on the inactivation of GTPase and RhoA, instead of Ras-related C3 botulinum toxin substrate 1 or CDC42.Conclusions/interpretationWe reveal a novel pathway by which TMEM55A regulates alpha cell exocytosis by controlling intracellular PI5P and the F-actin network.Graphical

Type 2 diabetes mellitus is a complex multifactorial disease of epidemic proportions. It involves genetic and lifestyle factors that lead to dysregulations in hormone secretion and metabolic homeostasis. Accumulating evidence indicates that altered mitochondrial structure, function, and particularly bioenergetics of cells in different tissues have a central role in the pathogenesis of type 2 diabetes mellitus. In the present study, we explore how mitochondrial dysfunction impairs the coupling between metabolism and exocytosis in the pancreatic alpha and beta cells. We demonstrate that reduced mitochondrial ATP production is linked with the observed defects in insulin and glucagon secretion by utilizing computational modeling approach. Specifically, a 30–40% reduction in alpha cells’ mitochondrial function leads to a pathological shift of glucagon secretion, characterized by oversecretion at high glucose concentrations and insufficient secretion in hypoglycemia. In beta cells, the impaired mitochondrial energy metabolism is accompanied by reduced insulin secretion at all glucose levels, but the differences, compared to a normal beta cell, are the most pronounced in hyperglycemia. These findings improve our understanding of metabolic pathways and mitochondrial bioenergetics in the pathology of type 2 diabetes mellitus and might help drive the development of innovative therapies to treat various metabolic diseases.

Pancreatic alpha and beta cells are derived from the same progenitors but play opposing roles in the control of glucose homeostasis. Disturbances in their function are associated with diabetes mellitus. To identify many of the proteins that define their unique pathways of differentiation and functional features, we have analyzed patterns of gene expression in alphaTC1.6 vs. MIN6 cell lines by using oligonucleotide microarrays. Approximately 9-10% of >11,000 transcripts examined showed significant differences between the two cell types. Of >700 known transcripts enriched in either cell type, transcription factors and their regulators (TFR) was one of the most significantly different categories. Ninety-six members of the basic zipper, basic helix-loop-helix, homeodomain, zinc finger, high mobility group, and other transcription factor families were enriched in alpha cells; in contrast, homeodomain proteins accounted for 51% of a total of 45 TFRs enriched in beta cells. Our analysis thus highlights fundamental differences in expression of TFR subtypes within these functionally distinct islet cell types. Interestingly, the alpha cells appear to express a large proportion of factors associated with progenitor or stem-type cells, perhaps reflecting their earlier appearance during pancreatic development. The implications of these findings for a better understanding of alpha and beta cell dysfunction in diabetes mellitus are also considered.

Diabetes mellitus (DM) is characterised by pancreatic islet failure and dysregulation. The pancreatic islet is comprised of three primary cell types: alpha, beta and delta cells. Pancreatic beta cell destruction is responsible for the onset of classical symptoms of DM consequent to loss of endogenous insulin production and has been the subject of decades of research effort. The mechanisms underlying alpha cell dysfunction are less well understood, but no less important. Alpha cell dysfunction contributes to counter regulatory failure and predisposes to severe hypoglycemia, a disabling and potentially fatal diabetic complication. Thus, it is critical to identify new targets for the treatment of alpha cell dysfunction. Using a PCR‐based screen of AlphaTC1‐9 cells, we identified G‐protein coupled receptors 183 and 19 as highly expressed in alpha cells. G‐protein coupled receptor 183 (GPR183, also known as EB12) is the receptor for the potent GPR183 agonist 7alpha 25‐dihydroxycholesterol. GPR183 expression is increased in human pancreatic beta cells and GPR183 activation by 7alpha 25‐dihydroxycholesterol promotes both insulin transcription and release in these beta cells. G‐protein coupled receptor 19 (GPR19) is the receptor for adropin, a small peptide which plays roles in metabolism and energy homeostasis. Circulating adropin acts on the liver to enhance Insulin receptor substrate (IRS) sensitivity and reduce hepatic glucose release. Adropin levels are reduced in obesity, and adropin administration to diet‐induced‐obese mice reduces insulin resistance and lowers fasting blood glucose. The effect of adropin on pancreatic alpha cell function had not been examined prior to this study. We hypothesize that GPR183 and GPR19 act in the pancreatic alpha cell to facilitate glucose homeostasis. To test this hypothesis, Alpha TC1‐9 cells were treated with GPR183 agonist, 7alpha 25‐dihydroxycholesterol, at 1nM, 10nM, and 100nM, or GPR19 agonist, adropin, at 1nM, 3nM, or 9nM. AlphaTC1‐9 were treated with agonists unstimulated, stimulated for 1 hour at 25mM glucose (HG) and treated at 5.5mM glucose (NG), or stimulated for 1 hour at 25mM glucose and treated at 2.2mM glucose (LG). Treatment with the GPR183 agonist 7alpha 25‐dihydroxycholesterol increased proglucagon transcript and reduced GPR183 and GPPR19 transcript in alphaTC1‐9 cells. Treatment with adropin had no effect on proglucagon transcript and robustly increased GPR183 transcript. These results suggest GPR183 and GPR19 as novel therapeutic targets to reduce the incidence of hypoglycemia in DM.Support or Funding InformationNIDDK

Type 2 diabetes is characterised by decreased beta cell mass and islet amyloid formation. Islet amyloid formed by aggregation of human islet amyloid polypeptide (hIAPP) is associated with beta cell apoptosis. We used human and transgenic mouse islets in culture to examine whether deletion of caspase-3 protects islets from apoptosis induced by endogenously produced and exogenously applied hIAPP and compared hIAPP toxicity in islet alpha and beta cells. Human and wild-type or caspase-3 knockout mouse islet cells were treated with hIAPP. Rat insulinoma INS-1 cells were similarly cultured with hIAPP and the amyloid inhibitor Congo Red or caspase-3 inhibitor. Human and hIAPP-expressing caspase-3 knockout mouse islets were cultured to form amyloid fibrils and assessed for beta and alpha cell apoptosis, beta cell function and caspase-3 activation. hIAPP-treated INS-1 cells had increased caspase-3 activation and apoptosis, both of which were reduced by inhibitors of amyloid or caspase-3. Similarly, hIAPP-treated human and mouse islet beta cells had elevated active caspase-3- and TUNEL-positive cells, whereas mouse islet cells lacking caspase-3 had markedly lower beta cell but comparable alpha cell apoptosis. During culture, human islets that formed amyloid had higher active caspase-3- and TUNEL-positive beta cells than those without detectable amyloid. Finally, cultured hIAPP-expressing mouse islets lacking caspase-3 had markedly lower beta cell apoptosis than those expressing caspase-3, associated with an increase in islet beta cell/alpha cell ratio, insulin content and glucose response. Prevention of caspase-3 activation protects islet beta cells from apoptosis induced by fibrillogenesis of endogenously secreted and exogenously applied hIAPP. Islet beta cells are more susceptible to hIAPP toxicity than alpha cells cultured under the same conditions.