Mathematical modeling clarifies the paracrine roles of insulin and glucagon on the glucose-stimulated hormonal secretion of pancreatic alpha- and beta-cells.

Introduction: Blood sugar homeostasis relies largely on the action of pancreatic islet hormones, particularly insulin and glucagon. In a prototypical fashion, glucagon is released upon hypoglycemia to elevate glucose by acting on the liver while elevated glucose induces the secretion of insulin which leads to sugar uptake by peripheral tissues. This simplified view of glucagon and insulin does not consider the paracrine roles of the two hormones modulating the response to glucose of α- and ß-cells. In particular, glucose-stimulated glucagon secretion by isolated α-cells exhibits a Hill-function pattern, while experiments with intact pancreatic islets suggest a 'U'-shaped response. Methods: To this end, a framework was developed based on first principles and coupled to experimental studies capturing the glucose-induced response of pancreatic α- and ß-cells influenced by the two hormones. The model predicts both the transient and steady-state profiles of secreted insulin and glucagon, including the typical biphasic response of normal ß-cells to hyperglycemia. Results and discussion: The results underscore insulin activity as a differentiating factor of the glucagon secretion from whole islets vs. isolated α-cells, and highlight the importance of experimental conditions in interpreting the behavior of islet cells in vitro. The model also reproduces the hyperglucagonemia, which is experienced by diabetes patients, and it is linked to a failure of insulin to inhibit α-cell activity. The framework described here is amenable to the inclusion of additional islet cell types and extrapancreatic tissue cells simulating multi-organ systems. The study expands our understanding of the interplay of insulin and glucagon for pancreas function in normal and pathological conditions.

Sex-based impact of pancreatic islet stressors in GluCreERT2/Rosa26-eYFP mice.

The present study examines differences in metabolic and pancreatic islet adaptative responses following streptozotocin (STZ) and hydrocortisone (HC) administration in male and female transgenic GluCreERT2/Rosa26-eYFP mice. Mice received five daily doses of STZ (50 mg/kg, i.p.) or 10 daily doses of HC (70 mg/kg, i.p.), with parameters assessed on day 11. STZ-induced hyperglycaemia was evident in both sexes, alongside impaired glucose tolerance and reduced insulin concentrations. HC also had similar metabolic effects in male and female mice resulting in classical increases of circulating insulin indicative of insulin resistance. Control male mice had larger pancreatic islets than females and displayed a greater reduction of islet and beta-cell area in response to STZ insult. In addition, female STZ mice had lower levels of beta-cell apoptosis than male counterparts. Following HC administration, female mouse islets contained a greater proportion of alpha cells when compared to males. All HC mice presented with relatively comparable increases in beta- and alpha-cell turnover rates, with female mice being slightly more susceptible to HC-induced beta-cell apoptosis. Interestingly, healthy control female mice had inherently increased alpha-to-beta-cell transdifferentiation rates, which was decreased by HC treatment. The number of glucagon-positive alpha cells altering their lineage to insulin-positive beta cells was increased in male, but not female, STZ mice. Taken together, although there was no obvious sex-specific alteration of metabolic profile in STZ or HC mice, subtle differences in pancreatic islet morphology emphasises the impact of sex hormones on islets and importance of taking care when interpreting observations between males and females.

Alpha cell receptor for advanced glycation end products associate with glucagon expression in type 1 diabetes.

Hypoglycemia in type 1 diabetes associates with changes in the pancreatic islet α cells, where the receptor for advanced glycation end products (RAGE) is highly expressed. This study compared islet RAGE expression in donors without diabetes, those at risk of, and those with type 1 diabetes. Laser-dissected islets were subject to RNA bioinformatics and adjacent pancreatic tissue were assessed by confocal microscopy. We found that islets from type 1 diabetes donors had differential expression of the RAGE gene (AGER) and its correlated genes, based on glucagon expression. Random forest machine learning revealed that AGER was the most important predictor for islet glucagon levels. Conversely, a generalized linear model identified that glucagon expression could be predicted by expression of RAGE signaling molecules, its ligands and enzymes that create or clear RAGE ligands. Confocal imaging co-localized RAGE, its ligands and signaling molecules to the α cells. Half of the type 1 diabetes cohort comprised of adolescents and a patient with history of hypoglycemia-all showed an inverse relationship between glucagon and RAGE. These data confirm an association between glucagon and islet RAGE, its ligands and signaling pathways in type 1 diabetes, which warrants functional investigation into a role for RAGE in hypoglycemia.

Metabolic regulation of glucagon secretion.

Since the discovery of glucagon 100 years ago, the hormone and the pancreatic islet alpha cells that produce it have remained enigmatic relative to insulin-producing beta cells. Canonically, alpha cells have been described in the context of glucagon's role in glucose metabolism in liver, with glucose as the primary nutrient signal regulating alpha cell function. However, current data reveal a more holistic model of metabolic signalling, involving glucagon-regulated metabolism of multiple nutrients by the liver and other tissues, including amino acids and lipids, providing reciprocal feedback to regulate glucagon secretion and even alpha cell mass. Here we describe how various nutrients are sensed, transported and metabolised in alpha cells, providing an integrative model for the metabolic regulation of glucagon secretion and action. Importantly, we discuss where these nutrient-sensing pathways intersect to regulate alpha cell function and highlight key areas for future research.

Glucose Controls Glucagon Secretion by Regulating Fatty Acid Oxidation in Pancreatic α-Cells.

Whole-body glucose homeostasis is coordinated through secretion of glucagon and insulin from pancreatic islets. When glucose is low, glucagon is released from α-cells to stimulate hepatic glucose production. However, the mechanisms that regulate glucagon secretion from pancreatic α-cells remain unclear. Here we show that in α-cells, the interaction between fatty acid oxidation and glucose metabolism controls glucagon secretion. The glucose-dependent inhibition of glucagon secretion relies on pyruvate dehydrogenase and carnitine palmitoyl transferase 1a activity and lowering of mitochondrial fatty acid oxidation by increases in glucose. This results in reduced intracellular ATP and leads to membrane repolarization and inhibition of glucagon secretion. These findings provide a new framework for the metabolic regulation of the α-cell, where regulation of fatty acid oxidation by glucose accounts for the stimulation and inhibition of glucagon secretion. ARTICLE HIGHLIGHTS: It has become clear that dysregulation of glucagon secretion and α-cell function plays an important role in the development of diabetes, but we do not know how glucagon secretion is regulated. Here we asked whether glucose inhibits fatty acid oxidation in α-cells to regulate glucagon secretion. We found that fatty acid oxidation is required for the inhibitory effects of glucose on glucagon secretion through reductions in ATP. These findings provide a new framework for the regulation of glucagon secretion by glucose.

Oleanolic acid and moderate drinking increase the pancreatic GLP-1R expression of the ß-cell mass deficiency induced hyperglycemia.

Background: Oleanolic acid (OA) and moderate drinking have been reported to attenuate diabetes. However, the underlying mechanism of OA and moderate drinking alone or in combination on the islet ß-cell deficiency induced diabetes is not fully elucidated. Methods: Male Sprague Dawley (SD) rats were intraperitoneally injected with 55 mg/kg streptozotocin (STZ) to induce ß-cell deficiency. OA, 5% ethanol (EtOH), or a mixture of OA in 5% ethanol (OA+EtOH) were applied to three treatment groups of hyperglycemia rats for 6 weeks. Results: STZ caused the increase of fast blood glucose (FBG) level.OA and EtOH treatment alone or in combination decreased the STZ increased FBG level during the 6 weeks of treatment. In addition, OA treatment also significantly increased the ß-cell to total islet cell ratio. Both EtOH and OA+EtOH treatments promoted the increase of total islet cell number and α-cell to ß-cell ratio when compared to OA group. STZ induced hyperglycemia dramatically reduced the glucagon-like peptide-1 receptor (GLP-1R) positive cells in islets, all the three treatments significantly increased the pancreatic GLP-1R positive cell number. In the meantime, STZ induced hyperglycemia suppressed the insulin mRNA expression and boosted the glucagon mRNA expression. EtOH and OA+EtOH treatments increased the insulin mRNA expression, but none of the 3 treatments altered the elevated glucagon level. Conclusion: GLP-1R positive cell ratio in islets is crucial for the blood glucose level of diabetes. OA and 5% ethanol alone or in combination suppresses the blood glucose level of ß-cell deficiency induced diabetes by increasing islet GLP-1R expression.

Glucagon cell hyperplasia and neoplasia: a recently recognized endocrine receptor disease.

Glucagon cell hyperplasia and neoplasia (GCHN) is the name of an endocrine receptor disease, whose morphology was first described in 2006. Three years later, this rare disease was found to be to be caused by an inactivating mutation of the glucagon receptor (GCGR) gene. Functionally, the genetic defect mainly affects glucagon signaling in the liver with changes in the metabolism of glycogen, fatty acids and amino acids. Recent results of several studies in GCGR knockout mice suggested that elevated serum amino acid levels probably stimulate glucagon cell hyperplasia with subsequent transformation into glucagon cell neoplasia. This process leads over time to numerous small and some large pancreatic neuroendocrine tumors which are potentially malignant. Despite high glucagon serum levels, the patients develop no glucagonoma syndrome. In 2015, GCHN was identified as an autosomal recessive hereditary disorder.

Blocking IL-6 signaling improves glucose tolerance via SLC39A5-mediated suppression of glucagon secretion.

BACKGROUND AND AIMS: Hyperinsulinemia, hyperglucagonemia, and low-grade inflammation are frequently presented in obesity and type 2 diabetes (T2D). The pathogenic regulation between hyperinsulinemia/insulin resistance (IR) and low-grade inflammation is well documented in the development of diabetes. However, the cross-talk of hyperglucagonemia with low-grade inflammation during diabetes progression is poorly understood. In this study, we investigated the regulatory role of proinflammatory cytokine interleukin-6 (IL-6) on glucagon secretion. METHODS: The correlations between inflammatory cytokines and glucagon or insulin were analyzed in rhesus monkeys and humans. IL-6 signaling was blocked by IL-6 receptor-neutralizing antibody tocilizumab in obese or T2D rhesus monkeys, glucose tolerance was evaluated by intravenous glucose tolerance test (IVGTT). Glucagon and insulin secretion were measured in isolated islets from wild-type mouse, primary pancreatic α-cells and non-α-cells sorted from GluCre-ROSA26EYFP (GYY) mice, in which the enhanced yellow fluorescent protein (EYFP) was expressed under the proglucagon promoter, by fluorescence-activated cell sorting (FACS). Particularly, glucagon secretion in α-TC1 cells treated with IL-6 was measured, and RNA sequencing was used to screen the mediator underlying IL-6-induced glucagon secretion. SLC39A5 was knocking-down or overexpressed in α-TC1 cells to determine its impact in glucagon secretion and cytosolic zinc density. Dual luciferase and chromatin Immunoprecipitation were applied to analyze the signal transducer and activator of transcription 3 (STAT3) in the regulation of SLC39A5 transcription. RESULTS: Plasma IL-6 correlate positively with plasma glucagon levels, but not insulin, in rhesus monkeys and humans. Tocilizumab treatment reduced plasma glucagon, blood glucose and HbA1c in spontaneously obese or T2D rhesus monkeys. Tocilizumab treatment also decreased glucagon levels during IVGTT, and improved glucose tolerance. Moreover, IL-6 significantly increased glucagon secretion in isolated islets, primary pancreatic α-cells and α-TC1 cells. Mechanistically, we found that IL-6-activated STAT3 downregulated the zinc transporter SLC39A5, which in turn reduced cytosolic zinc concentration and ATP-sensitive potassium channel activity and augmented glucagon secretion. CONCLUSIONS: This study demonstrates that IL-6 increases glucagon secretion via the downregulation of zinc transporter SLC39A5. This result revealed the molecular mechanism underlying the pathogenesis of hyperglucagonemia and a previously unidentified function of IL-6 in the pathophysiology of T2D, providing a potential new therapeutic strategy of targeting IL-6/glucagon to preventing or treating T2D.

Amino acids and the changing face of the α-cell.

Glucagon has long been defined by its glucogenic action and as a result α-cells have been characterised based largely on their interaction with glucose. Recent findings have challenged this preconception, bringing to the fore the significant role glucagon plays in amino acid breakdown and underlining the importance of amino acids in glucagon secretion. The challenge that remains is defining the mechanism that underlie these effects - understanding which amino acids are most important, how they act on the α-cell and how their actions integrate with other fuels such as glucose and fatty acids. This review will describe the current relationship between amino acids and glucagon and how we can use this knowledge to redefine the α-cell.

Comparative transcriptomic analysis reveals the underlying molecular mechanism in high-fat diet-induced islet dysfunction.

Obesity, characterized by accumulation of adipose, is usually accompanied by hyperlipidemia and abnormal glucose metabolism, which destroys the function and structure of islet ß cells. However, the exact mechanism of islet deterioration caused by obesity has not yet been fully elucidated. Here, we fed C57BL/6 mice with a high-fat diet (HFD) for 2 (2M group) and 6 months (6M group) to construct obesity mouse models. Then, RNA-based sequencing was used to identify the molecular mechanisms in HFD-induced islet dysfunction. Compared with the control diet, a total of 262 and 428 differentially expressed genes (DEGs) were identified from islets of the 2M and 6M groups, respectively. GO and KEGG enrichment analysis revealed that the DEGs up-regulated in both the 2M and 6M groups are mainly enriched in response to endoplasmic reticulum stress and the pancreatic secretion pathway. DEGs down-regulated in both the 2M and 6M groups are mainly enriched in the neuronal cell body and protein digestion and absorption pathway. Notably, along with the HFD feeding, mRNA expression of islet cell markers was significantly down-regulated, such as Ins1, Pdx1, MafA (ß cell), Gcg, Arx (α cell), Sst (δcell), and Ppy (PP cell). In contrast, mRNA expression of acinar cell markers was remarkably up-regulated, such as Amy1, Prss2, and Pnlip. Besides, a large number of collagen genes were down-regulated, such as Col1a1, Col6a6, and Col9a2. Overall, our study provides a full-scale DEG map regarding HFD-induced islet dysfunction, which was helpful to understand the underlying molecular mechanism of islet deterioration further.

α-cell electrophysiology and the regulation of glucagon secretion.

Glucagon is the principal glucose-elevating hormone that forms the first-line defence against hypoglycaemia. Along with insulin, glucagon also plays a key role in maintaining systemic glucose homeostasis. The cells that secrete glucagon, pancreatic α-cells, are electrically excitable cells and use electrical activity to couple its hormone secretion to changes in ambient glucose levels. Exactly how glucose regulates α-cells has been a topic of debate for decades but it is clear that electrical signals generated by the cells play an important role in glucagon secretory response. Decades of studies have already revealed the key players involved in the generation of these electrical signals and possible mechanisms controlling them to tune glucagon release. This has offered the opportunity to fully understand the enigmatic α-cell physiology. In this review, we describe the current knowledge on cellular electrophysiology and factors regulating excitability, glucose sensing, and glucagon secretion. We also discuss α-cell pathophysiology and the perspective of addressing glucagon secretory defects in diabetes for developing better diabetes treatment, which bears the hope of eliminating hypoglycaemia as a clinical problem in diabetes care.

FAP expression in alpha cells of Langherhans insulae-implications for FAPI radiopharmaceuticals' use.

PURPOSE: Radiopharmaceuticals targeting fibroblast activation protein (FAP) alpha are increasingly studied for diagnostic and therapeutic applications. We discovered FAP expression at immunohistochemistry (IHC) in the alpha cells of the Langerhans insulae of few patients. Therefore, we planned an investigation aimed at describing FAP expression in the pancreas and discussing the implications for radioligand applications. METHODS: We retrospectively included 40 patients from 2 institutions (20 pts each) according to the following inclusion/exclusion criteria: (i) pathology proven pancreatic ductal adenocarcinoma and neuroendocrine tumors (NET), 10 pts per each group at each center; (ii) and availability of paraffin-embedded tissue; and (iii) clinical-pathological records. We performed IHC analysis and applied a semiquantitative visual scoring system (0, negative staining; 1, present in less than 30%; 2, present in more than 30% of the area). FAP expression was assessed according to histology-NET (n = 20) vs ductal adenocarcinoma (n = 20)-and to previous treatments within the adenocarcinoma group. The local ethics committee approved the study (No. INT 21/16, 28 January 2016). RESULTS: The population consisted of 24 males and 16 females, with a median age of 68 and a range of 14-84 years; 8/20 adenocarcinoma patients received chemotherapy. In all the Langerhans insulae (40/40), pancreatic alpha cells were found to express FAP, with a score of 2. No difference was found among NET (20/20) and adenocarcinoma (20/20), nor according to neoadjuvant chemotherapy in the adenocarcinoma cohort (received or not received). CONCLUSION: Pancreatic Langerhans islet alpha cells normally express FAP. This is not expected to influence the diagnostic accuracy of FAP-targeting tracers. In the therapeutic setting, our results suggest the need to better elucidate FAPI radioligands' effects on the Langerhans insulae function.

Small molecule glucagon release inhibitors with activity in human islets.

Purpose: Type 1 diabetes (T1D) accounts for an estimated 5% of all diabetes in the United States, afflicting over 1.25 million individuals. Maintaining long-term blood glucose control is the major goal for individuals with T1D. In T1D, insulin-secreting pancreatic islet ß-cells are destroyed by the immune system, but glucagon-secreting islet α-cells survive. These remaining α-cells no longer respond properly to fluctuating blood glucose concentrations. Dysregulated α-cell function contributes to hyper- and hypoglycemia which can lead to macrovascular and microvascular complications. To this end, we sought to discover small molecules that suppress α-cell function for their potential as preclinical candidate compounds. Prior high-throughput screening identified a set of glucagon-suppressing compounds using a rodent α-cell line model, but these compounds were not validated in human systems. Results: Here, we dissociated and replated primary human islet cells and exposed them to 24 h treatment with this set of candidate glucagon-suppressing compounds. Glucagon accumulation in the medium was measured and we determined that compounds SW049164 and SW088799 exhibited significant activity. Candidate compounds were also counter-screened in our InsGLuc-MIN6 ß-cell insulin secretion reporter assay. SW049164 and SW088799 had minimal impact on insulin release after a 24 h exposure. To further validate these hits, we treated intact human islets with a selection of the top candidates for 24 h. SW049164 and SW088799 significantly inhibited glucagon release into the medium without significantly altering whole islet glucagon or insulin content. In concentration-response curves SW088799 exhibited significant inhibition of glucagon release with an IC50 of 1.26 µM. Conclusion: Given the set of tested candidates were all top hits from the primary screen in rodent α-cells, this suggests some conservation of mechanism of action between human and rodents, at least for SW088799. Future structure-activity relationship studies of SW088799 may aid in elucidating its protein target(s) or enable its use as a tool compound to suppress α-cell activity in vitro.

Not the second fiddle: α cell development, identity, and function in health and diabetes.

Historic and emerging studies provide evidence for the deterioration of pancreatic α cell function and identity in diabetes mellitus. Increased access to human tissue and the availability of more sophisticated molecular technologies have identified key insights into how α cell function and identity are preserved in healthy conditions and how they become dysfunctional in response to stress. These studies have revealed evidence of impaired glucagon secretion, shifts in α cell electrophysiology, changes in α cell mass, dysregulation of α cell transcription, and α-to-ß cell conversion prior to and during diabetes. In this review, we outline the current state of research on α cell identity in health and disease. Evidence in model organisms and humans suggests that in addition to ß cell dysfunction, diabetes is associated with a fundamental dysregulation of α cell identity. Importantly, epigenetic studies have revealed that α cells retain more poised and open chromatin at key cell-specific and diabetes-dysregulated genes, supporting the model that the inherent epigenetic plasticity of α cells makes them susceptible to the transcriptional changes that potentiate the loss of identity and function seen in diabetes. Thus, additional research into the maintenance of α cell identity and function is critical to fully understanding diabetes. Furthermore, these studies suggest α cells could represent an alternative source of new ß cells for diabetes treatment.

Brief overview: glucagon history and physiology.

Glucagon is a peptide hormone that is produced primarily by the alpha cells in the islet of Langerhans in the pancreas, but also in intestinal enteroendocrine cells and in some neurons. Approximately 100 years ago, several research groups discovered that pancreatic extracts would cause a brief rise in blood glucose before they observed the decrease in glucose attributed to insulin. An overall description of the regulation of glucagon secretion requires the inclusion of its sibling insulin because they both are made primarily by the islet and they both regulate each other in different ways. For example, glucagon stimulates insulin secretion, whereas insulin suppresses glucagon secretion. The mechanism of action of glucagon on insulin secretion has been identified as a trimeric guanine nucleotide-binding protein (G-protein)-mediated event. The manner in which insulin suppresses glucagon release from the alpha cell is thought to be highly dependent on the peri-portal circulation of the islet through which blood flows downstream from beta cells to alpha cells. In this scenario, it is via the circulation that insulin is thought to suppress the release of glucagon. However, high levels of glucose also have been shown to suppress glucagon secretion. Consequently, the glucose-lowering effect of insulin may be additive to the direct effects of insulin to suppress alpha cell function, so that in vivo both the discontinuation of the insulin signal and the condition of low glucose jointly are responsible for induction of glucagon secretion.

100 years of glucagon anniversary.

The year 2023 marks 100 years since publication of the first report of a hyperglycemic factor in pancreatic extracts which C P Kimball and John R Murlin named glucagon (from GLUCose AGONist). Glucagon has a range of profound effects on metabolism including, but not limited to, stimulation of hepatic glucose production. Dysregulation of glucagon secretion is a key feature of both major forms of diabetes, leading to the concept that diabetes is a bihormonal disorder. Still, the work to fully understand the production and biological effects of glucagon has proceeded at a slower pace compared to that of insulin. A recent resurgence of interest in the islet alpha (α) cell, the predominant site of glucagon production, has been facilitated in part by technological innovations. This work has led to significant developments in the field, from defining how alpha cells develop and how glucagon secretion from pancreatic alpha cells is regulated to determining the role of glucagon in metabolic homeostasis and the progression of both major forms of diabetes. In addition, glucagon is considered to be a promising target for diabetes therapy, with many new potential applications arising from research in this field. This collection of reviews, led by Guest Editors James Cantley, Vincent Poitout and Rebecca Hull-Meichle, is intended to capture the field's current understanding of glucagon and alpha cell biology, as well stimulate additional interest and research on this important hormone.

GABA treatment does not induce neogenesis of new endocrine cells from pancreatic ductal cells.

Previous studies indicated that ductal cells can contribute to endocrine neogenesis in adult rodents after alpha cells convert into beta cells. This can occur through Pax4 mis-expression in alpha cells or through long-term administration of gamma-aminobutyric acid (GABA) to healthy mice. GABA has also been reported to increase the number of beta cells through direct effects on their proliferation, but only in specific genetic mouse backgrounds. To test whether GABA induces neogenesis of beta cells from ductal cells or affects pancreatic cell proliferation, we administered GABA or saline over 2 or 6 months to Sox9CreER;R26RYFP mice in which 60-80% of large or small ducts were efficiently lineage labeled. We did not observe any increases in islet neogenesis from ductal cells between 1 and 2 months of age in saline treated mice, nor between 2 and 6 months of saline treatment, supporting previous studies indicating that adult ductal cells do not give rise to new endocrine cells during homeostasis. Unlike previous reports, we did not observe an increase in beta cell neogenesis after 2 or 6 months of GABA administration. Nor did we observe a significant increase in the pancreatic islet area, the number of insulin and glucagon double positive cells, or cell proliferation in the pancreas. This indicates that the effect of long term GABA administration on the pancreas is minimal or highly context dependent.

A plasma membrane-associated glycolytic metabolon is functionally coupled to KATP channels in pancreatic α and ß cells from humans and mice.

The ATP-sensitive K+ (KATP) channel is a key regulator of hormone secretion from pancreatic islet endocrine cells. Using direct measurements of KATP channel activity in pancreatic ß cells and the lesser-studied α cells, from both humans and mice, we provide evidence that a glycolytic metabolon locally controls KATP channels on the plasma membrane. The two ATP-consuming enzymes of upper glycolysis, glucokinase and phosphofructokinase, generate ADP that activates KATP. Substrate channeling of fructose 1,6-bisphosphate through the enzymes of lower glycolysis fuels pyruvate kinase, which directly consumes the ADP made by phosphofructokinase to raise ATP/ADP and close the channel. We further show the presence of a plasma membrane-associated NAD+/NADH cycle whereby lactate dehydrogenase is functionally coupled to glyceraldehyde-3-phosphate dehydrogenase. These studies provide direct electrophysiological evidence of a KATP-controlling glycolytic signaling complex and demonstrate its relevance to islet glucose sensing and excitability.

EpiCRISPR targeted methylation of Arx gene initiates transient switch of mouse pancreatic alpha to insulin-producing cells.

Introduction: Beta cell dysfunction by loss of beta cell identity, dedifferentiation, and the presence of polyhormonal cells are main characteristics of diabetes. The straightforward strategy for curing diabetes implies reestablishment of pancreatic beta cell function by beta cell replacement therapy. Aristaless-related homeobox (Arx) gene encodes protein which plays an important role in the development of pancreatic alpha cells and is a main target for changing alpha cell identity. Results: In this study we used CRISPR/dCas9-based epigenetic tools for targeted hypermethylation of Arx gene promoter and its subsequent suppression in mouse pancreatic αTC1-6 cell line. Bisulfite sequencing and methylation profiling revealed that the dCas9-Dnmt3a3L-KRAB single chain fusion constructs (EpiCRISPR) was the most efficient. Epigenetic silencing of Arx expression was accompanied by an increase in transcription of the insulin gene (Ins2) mRNA on 5th and 7th post-transfection day, quantified by both RT-qPCR and RNA-seq. Insulin production and secretion was determined by immunocytochemistry and ELISA assay, respectively. Eventually, we were able to induce switch of approximately 1% of transiently transfected cells which were able to produce 35% more insulin than Mock transfected alpha cells. Conclusion: In conclusion, we successfully triggered a direct, transient switch of pancreatic alpha to insulin-producing cells opening a future research on promising therapeutic avenue for diabetes management.

The human α cell in health and disease.

In commemoration of 100 years since the discovery of glucagon, we review current knowledge about the human α cell. Alpha cells make up 30-40% of human islet endocrine cells and play a major role in regulating whole-body glucose homeostasis, largely through the direct actions of their main secretory product - glucagon - on peripheral organs. Additionally, glucagon and other secretory products of α cells, namely acetylcholine, glutamate, and glucagon-like peptide-1, have been shown to play an indirect role in the modulation of glucose homeostasis through autocrine and paracrine interactions within the islet. Studies of glucagon's role as a counterregulatory hormone have revealed additional important functions of the α cell, including the regulation of multiple aspects of energy metabolism outside that of glucose. At the molecular level, human α cells are defined by the expression of conserved islet-enriched transcription factors and various enriched signature genes, many of which have currently unknown cellular functions. Despite these common threads, notable heterogeneity exists amongst human α cell gene expression and function. Even greater differences are noted at the inter-species level, underscoring the importance of further study of α cell physiology in the human context. Finally, studies on α cell morphology and function in type 1 and type 2 diabetes, as well as other forms of metabolic stress, reveal a key contribution of α cell dysfunction to dysregulated glucose homeostasis in disease pathogenesis, making targeting the α cell an important focus for improving treatment.