A method for the generation of human stem cell-derived alpha cells.
ABSTRACT: The generation of pancreatic cell types from renewable cell sources holds promise for cell replacement therapies for diabetes. Although most effort has focused on generating pancreatic beta cells, considerable evidence indicates that glucagon secreting alpha cells are critically involved in disease progression and proper glucose control. Here we report on the generation of stem cell-derived human pancreatic alpha (SC-alpha) cells from pluripotent stem cells via a transient pre-alpha cell intermediate. These pre-alpha cells exhibit a transcriptional profile similar to mature alpha cells and although they produce proinsulin protein, they do not secrete significant amounts of processed insulin. Compound screening identified a protein kinase c activator that promotes maturation of pre-alpha cells into SC-alpha cells. The resulting SC-alpha cells do not express insulin, share an ultrastructure similar to cadaveric alpha cells, express and secrete glucagon in response to glucose and some glucagon secretagogues, and elevate blood glucose upon transplantation in mice.
Project description:In the pancreatic islet, serotonin is an autocrine signal increasing beta cell mass during metabolic challenges such as those associated with pregnancy or high-fat diet. It is still unclear whether serotonin is relevant for regular islet physiology and hormone secretion. Here, we show that human beta cells produce and secrete serotonin when stimulated with increases in glucose concentration. Serotonin secretion from beta cells decreases cyclic AMP (cAMP) levels in neighboring alpha cells via 5-HT1F receptors and inhibits glucagon secretion. Without serotonergic input, alpha cells lose their ability to regulate glucagon secretion in response to changes in glucose concentration, suggesting that diminished serotonergic control of alpha cells can cause glucose blindness and the uncontrolled glucagon secretion associated with diabetes. Supporting this model, pharmacological activation of 5-HT1F receptors reduces glucagon secretion and has hypoglycemic effects in diabetic mice. Thus, modulation of serotonin signaling in the islet represents a drug intervention opportunity.
Project description:The generation of insulin-producing pancreatic ? cells from stem cells in vitro would provide an unprecedented cell source for drug discovery and cell transplantation therapy in diabetes. However, insulin-producing cells previously generated from human pluripotent stem cells (hPSC) lack many functional characteristics of bona fide ? cells. Here, we report a scalable differentiation protocol that can generate hundreds of millions of glucose-responsive ? cells from hPSC in vitro. These stem-cell-derived ? cells (SC-?) express markers found in mature ? cells, flux Ca(2+) in response to glucose, package insulin into secretory granules, and secrete quantities of insulin comparable to adult ? cells in response to multiple sequential glucose challenges in vitro. Furthermore, these cells secrete human insulin into the serum of mice shortly after transplantation in a glucose-regulated manner, and transplantation of these cells ameliorates hyperglycemia in diabetic mice.
Project description:In pancreatic beta-cells, uncoupling protein 2 (UCP2) influences mitochondrial oxidative phosphorylation and insulin secretion. Here, we show that alpha-cells express significantly higher levels of UCP2 than do beta-cells. Greater mitochondrial UCP2-related uncoupling was observed in alpha-cells compared with beta-cells and was accompanied by a lower oxidative phosphorylation efficiency (ATP/O). Conversely, reducing UCP2 activity in alpha-cells was associated with higher mitochondrial membrane potential generated by glucose oxidation and with increased ATP synthesis, indicating more efficient metabolic coupling. In vitro, the suppression of UCP2 activity led to reduced glucagon secretion in response to low glucose; however, in vivo, fasting glucagon levels were normal in UCP2(-/-) mice. In addition to its effects on secretion, UCP2 played a cytoprotective role in islets, with UCP2(-/-) alpha-cells being more sensitive to specific death stimuli. In summary, we demonstrate a direct role for UCP2 in maintaining alpha-cell function at the level of glucose metabolism, glucagon secretion, and cytoprotection.
Project description:Under extreme conditions or by genetic modification, pancreatic α-cells can regenerate and be converted into β-cells. This regeneration holds substantial promise for cell replacement therapy in diabetic patients. The discovery of clinical therapeutic strategies to promote β-cell regeneration is crucial for translating these findings into clinical applications. In this study, we reported that treatment with REMD 2.59, a human glucagon receptor (GCGR) monoclonal antibody (mAb), lowered blood glucose without inducing hypoglycemia in normoglycemic, streptozotocin-induced type 1 diabetic (T1D) and non-obesity diabetic mice. Moreover, GCGR mAb treatment increased the plasma glucagon and active glucagon-like peptide-1 levels, induced pancreatic ductal ontogenic α-cell neogenesis, and promoted α-cell proliferation. Strikingly, the treatment also increased the β-cell mass in these two T1D models. Using α-cell lineage-tracing mice, we found that the neogenic β-cells were likely derived from α-cell conversion. Therefore, GCGR mAb-induced α- to β-cell conversion might represent a pre-clinical approach for improving diabetes therapy.
Project description:AIMS/HYPOTHESIS:Several forkhead box (FOX) transcription factor family members have important roles in controlling pancreatic cell fates and maintaining beta cell mass and function, including FOXA1, FOXA2 and FOXM1. In this study we have examined the importance of FOXP1, FOXP2 and FOXP4 of the FOXP subfamily in islet cell development and function. METHODS:Mice harbouring floxed alleles for Foxp1, Foxp2 and Foxp4 were crossed with pan-endocrine Pax6-Cre transgenic mice to generate single and compound Foxp mutant mice. Mice were monitored for changes in glucose tolerance by IPGTT, serum insulin and glucagon levels by radioimmunoassay, and endocrine cell development and proliferation by immunohistochemistry. Gene expression and glucose-stimulated hormone secretion experiments were performed with isolated islets. RESULTS:Only the triple-compound Foxp1/2/4 conditional knockout (cKO) mutant had an overt islet phenotype, manifested physiologically by hypoglycaemia and hypoglucagonaemia. This resulted from the reduction in glucagon-secreting alpha cell mass and function. The proliferation of alpha cells was profoundly reduced in Foxp1/2/4 cKO islets through the effects on mediators of replication (i.e. decreased Ccna2, Ccnb1 and Ccnd2 activators, and increased Cdkn1a inhibitor). Adult islet Foxp1/2/4 cKO beta cells secrete insulin normally while the remaining alpha cells have impaired glucagon secretion. CONCLUSIONS/INTERPRETATION:Collectively, these findings reveal an important role for the FOXP1, 2, and 4 proteins in governing postnatal alpha cell expansion and function.
Project description:Disruption of pancreatic clock genes impairs pancreatic beta-cell function, leading to the onset of diabetes. Despite the importance of pancreatic alpha-cells in the regulation of glucose homeostasis and in diabetes pathophysiology, nothing is known about the role of clock genes in these cells. Here, we identify the clock gene Rev-erb alpha as a new intracellular regulator of glucagon secretion. Rev-erb alpha down-regulation by siRNA (60-70% inhibition) in alphaTC1-9 cells inhibited low-glucose induced glucagon secretion (p<0.05) and led to a decrease in key genes of the exocytotic machinery. The Rev-erb alpha agonist GSK4112 increased glucagon secretion (1.6 fold) and intracellular calcium signals in alphaTC1-9 cells and mouse primary alpha-cells, whereas the Rev-erb alpha antagonist SR8278 produced the opposite effect. At 0.5 mM glucose, alphaTC1-9 cells exhibited intrinsic circadian Rev-erb alpha expression oscillations that were inhibited by 11 mM glucose. In mouse primary alpha-cells, glucose induced similar effects (p<0.001). High glucose inhibited key genes controlled by AMPK such as Nampt, Sirt1 and PGC-1 alpha in alphaTC1-9 cells (p<0.05). AMPK activation by metformin completely reversed the inhibitory effect of glucose on Nampt-Sirt1-PGC-1 alpha and Rev-erb alpha. Nampt inhibition decreased Sirt1, PGC-1 alpha and Rev-erb alpha mRNA expression (p<0.01) and glucagon release (p<0.05). These findings identify Rev-erb alpha as a new intracellular regulator of glucagon secretion via AMPK/Nampt/Sirt1 pathway.
Project description:Obesity is frequently associated with insulin resistance. To compensate for this situation and maintain normoglycaemia, pancreatic beta-cells undergo several morphofunctional adaptations, which result in insulin hypersecretion and hyperinsulinaemia. However, no information exists about pancreatic alpha-cells during this compensatory stage of obesity. Here, we studied alpha-cells in mice fed a high-fat diet (HFD) for 12 weeks. These animals exhibited hyperinsulinaemia and normoglycaemia compared with control animals in addition to hypoglucagonaemia. While the in vivo response of glucagon to hypoglycaemia was preserved in the obese mice, the suppression of glucagon secretion during hyperglycaemia was impaired. Additionally, in vitro glucagon release at low glucose levels and glucagon content in isolated islets were decreased, while alpha-cell exocytosis remained unchanged. Assessment of morphological parameters revealed that alpha-cell area was reduced in the pancreas of the obese mice in association with alpha-cell hypotrophy, increased apoptosis and decreased proliferation. HFD feeding for 24 weeks led to significant deterioration in beta-cell function and glucose homeostasis. Under these conditions, the majority of alpha-cell changes were reversed and became comparable to controls. These findings indicate that pancreatic compensatory adaptations during obesity may also involve pancreatic alpha-cells. Additionally, defects in alpha-cell function during obesity may be implicated in progression to diabetes.
Project description:Diabetes is associated with disturbances in the normal levels of both insulin and glucagon, both of which play critical roles in the regulation of glycemia. Recent studies have found lipocalin-type prostaglandin D2 synthase (l-PGDS) to be an emerging target involved in the pathogenesis of type-2 diabetes. This study focused on the effect of l-PGDS on glucagon secretion from cultured pancreatic Alpha TC-1 Clone 6 cells. When cells were treated with various concentrations of l-PGDS (0, 10, 50, and 100 ug/ml) for 2 h in 1 mM glucose; glucagon secretion decreased to 670±45, 838±38, 479±11, and 437±45 pg/ml, respectively. In addition, pancreatic islets were isolated from C57BL/6 mice and stained for prostaglandin D2 receptors, DP1 and DP2, using immunohistochemistry. Our results showed that these islets express only the DP1 receptor. Pancreatic islets were then stained for alpha and beta cells, as well as DP1, to find the primary location of the receptor within the islets using immunofluorescence. Interestingly, DP1 receptor density was found primarily in alpha cells rather than in beta cells. Our study is the first to report a correlation between l-PGDS and glucagon secretion in alpha cells. Based on our obtained results, it can be concluded that higher concentrations of l-PGDS significantly reduced the secretion of glucagon in alpha cells, which may contribute to the pathogenesis of diabetes as well as offer a novel therapeutic site for the treatment of diabetes.
Project description:Blood glucose homeostasis is mainly achieved by the coordinated function of pancreatic alpha-, beta-, and delta-cells, which secrete glucagon, insulin, and somatostatin, respectively. Each cell type responds to glucose changes with different secretion patterns. Currently, considerable information can be found about the signal transduction mechanisms that lead to glucose-mediated insulin release in the pancreatic beta-cell, mitochondrial activation being an essential step. Increases in glucose stimulate the mitochondrial metabolism, activating the tricarboxylic acid cycle and raising the source of redox electron carrier molecules needed for respiratory ATP synthesis. However, little is known about the glucose-induced mitochondrial response of non-beta-cells and its role in the stimulus-secretion coupling process. This limited information is probably a result of the scarcity of these cells in the islet, the lack of identification patterns, and the technical limitations of conventional methods. In this study, we used flavin adenine dinucleotide redox confocal microscopy as a noninvasive technique to specifically monitor mitochondrial redox responses in immunoidentified alpha-, beta-, and delta-cells in freshly isolated intact islets and in dispersed cultured cells. We have shown that glucose provokes metabolic changes in beta- and delta-cell populations in a dose-dependent manner. Conversely, no significant responses were observed in alpha-cells, despite the sensitivity of their metabolism to drugs acting on the mitochondrial function, and their intact ability to develop Ca2+ signals. Identical results were obtained in islets and in cultures of dispersed cells. Our findings indicate metabolic differences in glucose utilization among the alpha-, beta-, and delta-cell populations, which might be important in the signal transduction events that lead to hormone release.
Project description:AIMS/HYPOTHESIS:Glucagon is critical for normal glucose homeostasis and aberrant secretion of the hormone aggravates dysregulated glucose control in diabetes. However, the mechanisms by which glucose controls glucagon secretion from pancreatic alpha cells remain elusive. The aim of this study was to investigate the role of the intracellular messenger cAMP in alpha-cell-intrinsic glucose regulation of glucagon release. METHODS:Subplasmalemmal cAMP and Ca2+ concentrations were recorded in isolated and islet-located alpha cells using fluorescent reporters and total internal reflection microscopy. Glucagon secretion from mouse islets was measured using ELISA. RESULTS:Glucose induced Ca2+-independent alterations of the subplasmalemmal cAMP concentration in alpha cells that correlated with changes in glucagon release. Glucose-lowering-induced stimulation of glucagon secretion thus corresponded to an elevation in cAMP that was independent of paracrine signalling from insulin or somatostatin. Imposed cAMP elevations stimulated glucagon secretion and abolished inhibition by glucose elevation, while protein kinase A inhibition mimicked glucose suppression of glucagon release. CONCLUSIONS/INTERPRETATION:Glucose concentrations in the hypoglycaemic range control glucagon secretion by directly modulating the cAMP concentration in alpha cells independently of paracrine influences. These findings define a novel mechanism for glucose regulation of glucagon release that underlies recovery from hypoglycaemia and may be disturbed in diabetes.