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Unfortunately, humanity is exposed to mixed plasticizers such as bisphenol-A (BPA) and dibutyl phthalate (DBP) that are leached from the daily used plastic products. Previous studies have demonstrated their potential in pancreatic beta cell injury and diabetes induction. The study hypothesized that both compounds would affect the pancreatic alpha cells in albino rats when administered at environmentally relevant doses. Heat shock protein 60 (HSP60) and caspase-3 protein expression was also investigated as potential mechanisms. Thirty-six male Wistar albino rats were separated into four equal groups: control, BPA alone, DBP alone, and BPA + DBP combined groups. BPA and DBP were given in drinking water for 45 days in a dose of 4.5 and 0.8 µg/L, respectively. Fasting blood glucose, serum insulin, pancreatic tissue levels of malondialdehyde, and superoxide dismutase were measured. Pancreatic sections were subjected to hematoxylineosin (HE) staining, glucagon, HSP60, and caspase-3 immunohistochemistry. Although all three experimental groups showed diffuse islet cell HSP60 immunoreactivity, rats exposed to BPA alone showed α-cell-only apoptosis, indicated by HE changes and caspase-3 immunoreactivity, associated with reduced glucagon immunoreaction. However, rats exposed to DBP alone showed no changes in either α or β-cells. Both combined-exposed animals displayed α and β apoptotic changes associated with islet atrophy and reduced glucagon expression. In conclusion, the study suggested HSP60/caspase-3 interaction, caspase-3 activation, and initiation of apoptosis in α-cell only for BPA-alone exposure group, meanwhile DBP alone did not progress to apoptosis. Interestingly, both α/β cell effect was observed in the mixed group implying synergetic/additive action of both chemicals when combined.

Amino acids stimulate the secretion of glucagon, and glucagon receptor signaling regulates amino acid catabolism via ureagenesis, together constituting the liver-α cell axis. Impairment of the liver-α cell axis is observed in metabolic diseases such as diabetes. It is, however, unknown whether glucose affects the liver-α cell axis. We investigated the role of glucose on the liver-α cell axis in vivo and ex vivo. The isolated perfused mouse pancreas was used to evaluate the direct effect of low (3.5 mmol/L) and high (15 mmol/L) glucose levels on amino acid (10 mmol/L arginine)-induced glucagon secretion. High glucose levels alone lowered glucagon secretion, but the amino acid-induced glucagon responses were similar in high and low glucose conditions (

Producción Científica, Aims/hypothesis Type 2 diabetes is characterised by hyperglucagonaemia and perturbed function of pancreatic glucagon secreting alpha cells but the molecular mechanisms contributing to these phenotypes are poorly understood. Insulin-degrading enzyme (IDE) is present within all islet cells, mostly in alpha cells, in both mice and humans. Furthermore, IDE can degrade glucagon as well as insulin, suggesting that IDE may play an important role in alpha cell function in vivo. Methods We have generated and characterised a novel mouse model with alpha cell-specific deletion of Ide, the A-IDE-KO mouse line. Glucose metabolism and glucagon secretion in vivo was characterised; isolated islets were tested for glucagon and insulin secretion; alpha cell mass, alpha cell proliferation and α-synuclein levels were determined in pancreas sections by immunostaining. Results Targeted deletion of Ide exclusively in alpha cells triggers hyperglucagonaemia and alpha cell hyperplasia, resulting in elevated constitutive glucagon secretion. The hyperglucagonaemia is attributable in part to dysregulation of glucagon secretion, specifically an impaired ability of IDE-deficient alpha cells to suppress glucagon release in the presence of high glucose or insulin. IDE deficiency also leads to α-synuclein aggregation in alpha cells, which may contribute to impaired glucagon secretion via cytoskeletal dysfunction. We showed further that IDE deficiency triggers impairments in cilia formation, inducing alpha cell hyperplasia and possibly also contributing to dysregulated glucagon secretion and hyperglucagonaemia. Conclusions/interpretation We propose that loss of IDE function in alpha cells contributes to hyperglucagonaemia in type 2 diabetes, Ministerio de Economía, Industria y Competitividad (SAF2016-77871-C2-1-R to IC and BFU2016-77125-R to IQ), Ministerio de Ciencia, Innovación y Universidades (PID2019-110496RB-C21 to IC and PID2019-110496RB-C22 to GP), La Caixa Foundation (grant LCF/PR/PR18/51130007 to GP), National Institutes of Health from USA (GM115617), Publicación en abierto financiada por el Consorcio de Bibliotecas Universitarias de Castilla y León (BUCLE), con cargo al Programa Operativo 2014ES16RFOP009 FEDER 2014-2020 DE CASTILLA Y LEÓN, Actuación:20007-CL - Apoyo Consorcio BUCLE

β cells have a limited capacity for regeneration, which predisposes towards diabetes. Here, we show that, of the MYC family members, Mycl plays a key role in proliferation of pancreatic endocrine cells. Genetic ablation of Mycl causes a reduction in the proliferation of pancreatic endocrine cells in neonatal mice. By contrast, the expression of Mycl in adult mice stimulates the proliferation of β and α cells, and the cells persist after withdrawal of Mycl expression. A subset of the expanded α cells give rise to insulin-producing cells after this withdrawal. Transient Mycl expression in vivo is sufficient to normalize the hyperglycaemia of diabetic mice. In vitro expression of Mycl similarly provokes active replication in islet cells, even in those from aged mice. Finally, we show that MYCL stimulates the division of human adult cadaveric islet cells. Our results demonstrate that the induction of Mycl alone expands the functional β-cell population, which may provide a regenerative strategy for β cells.

Glucagon hypersecretion from the pancreatic α-cell is a characteristic sign of diabetes, which exacerbates fasting hyperglycemia. Thus, targeting glucagon secretion from α-cells may be a promising approach for combating hyperglucagonemia. We have recently identified stathmin-2 as an α-cell protein that regulates glucagon secretion by directing glucagon toward the endolysosomal system in αTC1-6 cells. We hypothesized that disruption of Stmn2-mediated trafficking of glucagon to the endolysosomes in diabetes contributes to hyperglucagonemia. In isolated islets from male mice treated with streptozotocin (STZ), glucagon secretion and cellular content were augmented, but cellular Stmn2 levels were reduced (p

Type 1 Diabetes (T1D) is caused by insulin deficiency, due to progressive autoimmune destruction of pancreatic β cells. Glucagon-secreting α cells become dysfunctional in T1D and contribute to pathophysiology, however, the mechanisms involved are unclear. While the majority of β cells are destroyed in T1D, some β cells escape this fate and become senescent but whether α cell dysfunction involves a senescence program has not been explored. Here we addressed the question of whether α cells become senescent during the natural history of T1D in the non-obese diabetic (NOD) mouse model and humans. NOD mice had several distinct subpopulations of α cells, but none were defined by markers of senescence at the transcriptional or protein level. Similarly, α cells of human T1D donors did not express senescence markers. Despite the lack of senescence in α cells in vivo, using a human islet culture model, we observed that DNA damage-induced senescence led to alterations in islet glucagon secretion, which could be rescued by inhibiting the senescence-associated secretory phenotype (SASP). Together our results suggest that α cell dysfunction in T1D is not due to activation of a senescence program, however, senescent β cell accumulation in the islet microenvironment may have a negative effect on α cell function.

Cell culture of different pancreatic islet cell lines, like the murine α-cell line αTC1.9, the rat β-cell lines INS-1 and INS-1 832/13, and the human δ-cell line QGP-1, can serve as valuable cell models for the analysis of melatonin-dependent modulation of hormone secretion. The paper summarizes in detail the requirements of culture for each cell line and includes batch protocols to stimulate hormone secretion and to treat cells with several melatonin concentrations as previously published. We here describe the processing of collected cell pellets or cell culture supernatants as well as different methods to analyze cell experiments after melatonin treatment on the basis of our own experience. Finally, we outlined for each cell line under which conditions the melatonin treatment should be performed to gain reproducible results.

Thioredoxin-interacting protein (TXNIP) has emerged as a key factor in pancreatic beta cell biology, and its upregulation by glucose and diabetes contributes to the impairment in functional beta cell mass and glucose homeostasis. In addition, beta cell deletion of TXNIP protects against diabetes in different mouse models. However, while TXNIP is ubiquitously expressed, its role in pancreatic alpha cells has remained elusive. We generated an alpha cell TXNIP knockout (aTKO) mouse and assessed the effects on glucose homeostasis. While no significant changes were observed on regular chow, after a 30-week high-fat diet, aTKO animals showed improvement in glucose tolerance and lower blood glucose levels compared to their control littermates. Moreover, in the context of streptozotocin (STZ)-induced diabetes, aTKO mice showed significantly lower blood glucose levels compared to controls. While serum insulin levels were reduced in both control and aTKO mice, STZ-induced diabetes significantly increased glucagon levels in control mice, but this effect was blunted in aTKO mice. Moreover, glucagon secretion from aTKO islets was >2-fold lower than from control islets, while insulin secretion was unchanged in aTKO islets. At the same time, no change in alpha cell or beta cell numbers or mass was observed, and glucagon and insulin expression and content were comparable in isolated islets from aTKO and control mice. Thus together the current studies suggest that downregulation of alpha cell TXNIP is associated with reduced glucagon secretion and that this may contribute to the glucose-lowering effects observed in diabetic aTKO mice.

Abnormal microRNA functions are closely associated with pancreatic β-cell loss and dysfunction in type 2 diabetes. Dysregulation of miR-30d has been reported in the individuals with diabetes. To study how miR-30d affects pancreatic β-cell functions, we generated two transgenic mouse lines that specifically overexpressed miR-30d in β-cells at distinct low and high levels. Transgenic overexpressed miR-30d systemically affected β-cell function. Elevated miR-30d at low-level (TgL, 2-fold) had mild effects on signaling pathways and displayed no significant changes to metabolic homeostasis. In contrast, transgenic mice with high-level of miR-30d expression (TgH, 12-fold) exhibited significant diet-induced hyperglycemia and β-cell dysfunction. In addition, loss of β-cell identity was invariably accompanied with increased insulin/glucagon-double positive bihormonal cells and excess plasma glucagon levels. The transcriptomic analysis revealed that miR-30d overexpression inhibited β-cell-enriched gene expression and induced α-cell-enriched gene expression. These findings implicate that an appropriate miR-30d level is essential in maintaining normal β-cell identity and function.

In contrast to the skin and the gut, where somatic stem cells and their niche are well characterized, a definitive pancreatic multipotent cell population in the adult pancreas has yet to be revealed. Of particular interest is whether such cells may be endogenous in patients with diabetes, and if so, can they be used for therapeutic purposes? In the current study, we used two separate reporter lines to target Cre-recombinase expression to the Lgr5- or glucagon-expressing cells in the pancreas. We provide evidence for the existence of a population of cells within and in the proximity of the ducts that transiently express the stem-cell marker Lgr5 during late gestational stages. Careful timing of tamoxifen treatment in Lgr5EGFP-IRES-CreERT2;R26Tomato mice allowed us to show that these Lgr5-expressing progenitor cells can differentiate into α-cells during pregnancy. Furthermore, we report on a spontaneous lineage conversion of α- to β-cells specifically after parturition. The contribution of Lgr5 progeny to the β-cell compartment through an α-cell intermediate phase early after pregnancy appears to be part of a novel mechanism that would counterbalance against excessive β-cell mass reduction during β-cell involution.

BACKGROUNDPathophysiology of type 1 diabetes (T1D) is illustrated by pancreatic islet infiltration of inflammatory lymphocytes, including CD8+ T cells; however, the molecular factors mediating their recruitment remain unknown. We hypothesized that single-cell RNA-sequencing (scRNA-Seq) analysis of immune cell populations isolated from islets of NOD mice captured gene expression dynamics providing critical insight into autoimmune diabetes pathogenesis.METHODSPancreatic sections from human donors were investigated, including individuals with T1D, autoantibody-positive (aAb+) individuals, and individuals without diabetes who served as controls. IHC was performed to assess islet hormones and both novel and canonical immune cell markers that were identified from unbiased, state-of-the-art workflows after reanalyzing murine scRNA-Seq data sets.RESULTSComputational workflows identified cell adhesion molecule 1-mediated (Cadm1-mediated) homotypic binding among the most important intercellular interactions among all cell clusters, as well as Cadm1 enrichment in macrophages and DCs from pancreata of NOD mice. Immunostaining of human pancreata revealed an increased number of CADM1+glucagon+ cells adjacent to CD8+ T cells in sections from T1D and aAb+ donors compared with individuals without diabetes. Numbers of CADM1+CD68+ peri-islet myeloid cells adjacent to CD8+ T cells were also increased in pancreatic sections from both T1D and aAb+ donors compared with individuals without diabetes.CONCLUSIONIncreased detection of CADM1+ cells adjacent to CD8+ T cells in pancreatic sections of individuals with T1D and those who were aAb+ validated workflows and indicated CADM1-mediated intercellular contact may facilitate islet infiltration of cytotoxic T lymphocytes and serve as a potential therapeutic target for preventing T1D pathogenesis.FUNDINGThe Johns Hopkins All Children's Foundation Institutional Research Grant Program, the National Natural Science Foundation of China (grant 82071326), and the Deutsche Forschungsgemeinschaft (grants 431549029-SFB1451, EXC2030-390661388, and 411422114-GRK2550).

Dysregulated glucagon secretion from pancreatic alpha-cells is a key feature of type-1 and type-2 diabetes (T1D and T2D), yet our mechanistic understanding of alpha-cell function is underdeveloped relative to insulin-secreting beta-cells. Here we show that the enzyme acetyl-CoA-carboxylase 1 (ACC1), which couples glucose metabolism to lipogenesis, plays a key role in the regulation of glucagon secretion. Pharmacological inhibition of ACC1 in mouse islets or αTC9 cells impaired glucagon secretion at low glucose (1 mmol/l). Likewise, deletion of ACC1 in alpha-cells in mice reduced glucagon secretion at low glucose in isolated islets, and in response to fasting or insulin-induced hypoglycaemia in vivo. Electrophysiological recordings identified impaired KATP channel activity and P/Q- and L-type calcium currents in alpha-cells lacking ACC1, explaining the loss of glucose-sensing. ACC-dependent alterations in S-acylation of the KATP channel subunit, Kir6.2, were identified by acyl-biotin exchange assays. Histological analysis identified that loss of ACC1 caused a reduction in alpha-cell area of the pancreas, glucagon content and individual alpha-cell size, further impairing secretory capacity. Loss of ACC1 also reduced the release of glucagon-like peptide 1 (GLP-1) in primary gastrointestinal crypts. Together, these data reveal a role for the ACC1-coupled pathway in proglucagon-expressing nutrient-responsive endocrine cell function and systemic glucose homeostasis.

Significance Cure of autoimmune type 1 diabetes (T1D) requires both reversal of autoimmunity and regeneration or resupply of insulin-producing β cells. We have observed that combination therapy with induction of haploidentical mixed chimerism and administration of gastrin and epidermal growth factor (EGF) cures firmly established T1D. The predominant source of β cell regeneration in mice comes from reactivation of dysfunctional insulinlo β cells and transdifferentiation of α cells. These studies have provided insights into β cell regeneration mechanisms in firmly established autoimmune T1D, in particular, reactivation of the insulinlo β cells after reversal of autoimmunity by induction of haploidentical mixed chimerism. These studies also provide a preclinical scientific basis for the feasibility of cure of long-standing T1D in humans., Type 1 diabetes (T1D) results from the autoimmune destruction of β cells, so cure of firmly established T1D requires both reversal of autoimmunity and restoration of β cells. It is known that β cell regeneration in nonautoimmune diabetic mice can come from differentiation of progenitors and/or transdifferentiation of α cells. However, the source of β cell regeneration in autoimmune nonobese diabetic (NOD) mice remains unclear. Here, we show that, after reversal of autoimmunity by induction of haploidentical mixed chimerism, administration of gastrin plus epidermal growth factor augments β cell regeneration and normalizes blood glucose in the firmly established diabetic NOD mice. Using transgenic NOD mice with inducible lineage-tracing markers for insulin-producing β cells, Sox9+ ductal progenitors, Nestin+ mesenchymal stem cells, and glucagon-producing α cells, we have found that both reactivation of dysfunctional low-level insulin expression (insulinlo) β cells and neogenesis contribute to the regeneration, with the latter predominantly coming from transdifferentiation of α cells. These results indicate that, after reversal of autoimmunity, reactivation of β cells and transdifferentiation of α cells can provide sufficient new functional β cells to reach euglycemia in firmly established T1D.

The islet of Langerhans is a complex endocrine micro-organ consisting of a multitude of endocrine and non-endocrine cell types. The two most abundant and prominent endocrine cell types, the beta and the alpha cells, are essential for the maintenance of blood glucose homeostasis. While the beta cell produces insulin, the only blood glucose-lowering hormone of the body, the alpha cell releases glucagon, which elevates blood glucose. Under physiological conditions, these two cell types affect each other in a paracrine manner. While the release products of the beta cell inhibit alpha cell function, the alpha cell releases factors that are stimulatory for beta cell function and increase glucose-stimulated insulin secretion. The aim of this review is to provide a comprehensive overview of recent research into the regulation of beta cell function by alpha cells, focusing on the effect of alpha cell-secreted factors, such as glucagon and acetylcholine. The consequences of differences in islet architecture between species on the interplay between alpha and beta cells is also discussed. Finally, we give a perspective on the possibility of using an in vivo imaging approach to study the interactions between human alpha and beta cells under in vivo conditions. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s00125-020-05196-3) contains a slideset of the figures for download, which is available to authorised users.

Glucagon is a peptide hormone generated by pancreatic α cells. It is the counterpart of insulin and plays an essential role in the regulation of blood glucose level. Therefore, a tight regulation of glucagon levels is pivotal to maintain homeostasis of blood glucose. However, little is known about the mechanisms regulating glucagon biosynthesis. In this study, we demonstrate that the RNA-binding protein HuD regulates glucagon expression in pancreatic α cells. HuD was found in α cells from mouse pancreatic islet and mouse glucagonoma αTC1 cell line. Ribonucleoprotein immunoprecipitation analysis, followed by RT-qPCR showed the association of HuD with glucagon mRNA. Knockdown of HuD resulted in a reduction in both proglucagon expression and cellular glucagon level by decreasing its de novo synthesis. Reporter analysis using the EGFP reporter containing 3' untranslated region (3'UTR) of glucagon mRNA showed that HuD regulates proglucagon expression via its 3'UTR. In addition, the relative level of glucagon in the islets and plasma was lower in HuD knockout (KO) mice compared to age-matched control mice. Taken together, these results suggest that HuD is a novel factor regulating the biosynthesis of proglucagon in pancreatic α cells.

Compromised β-cell identity is emerging as an important contributor to β-cell failure in diabetes; however, the precise mechanism independent of hyperglycemia is under investigation. We have previously reported that mTORC1/Raptor regulates functional maturation in β-cells. In the present study, we find that diabetic β-cell specific Raptor-deficient mice (βRapKOGFP) show reduced β-cell mass, loss of β-cell identity and acquisition of α-cell features; which are not reversible upon glucose normalization. Deletion of Raptor directly impairs β-cell identity, mitochondrial metabolic coupling and protein synthetic activity, leading to β-cell failure. Moreover, loss of Raptor activates α-cell transcription factor MafB (via modulating C/EBPβ isoform ratio) and several α-cell enriched genes i.e. Etv1 and Tspan12, thus initiates β- to α-cell reprograming. The present findings highlight mTORC1 as a metabolic rheostat for stabilizing β-cell identity and repressing α-cell program at normoglycemic level, which might present therapeutic opportunities for treatment of diabetes., During the progression of type 2 diabetes, insulin-producing β-cells can lose their identity and become reprogrammed into other cell types. Here the authors show that murine diabetic β-cells require the protein Raptor for maintaining β-cell health and preventing them from turning into α-cells, independent of Raptor’s involvement in regulating blood sugar levels.

Significance The primary cilium is a small subcompartment of the cell but has powerful influence on pancreatic islet function. In this study, we find a critical role for cilia in regulating β-cell function and energy metabolism. Importantly, the deletion of β-cell cilia disrupts intercellular communication and leads to islet dysfunction and diabetes, as seen in a number of human ciliopathy syndromes. These results should help elucidate pathophysiology of human ciliopathy and aid the development of pharmacologic agents targeting primary cilia that may lead to a more effective treatment for human diabetes., Pancreatic islets regulate glucose homeostasis through coordinated actions of hormone-secreting cells. What underlies the function of the islet as a unit is the close approximation and communication among heterogeneous cell populations, but the structural mediators of islet cellular cross talk remain incompletely characterized. We generated mice specifically lacking β-cell primary cilia, a cellular organelle that has been implicated in regulating insulin secretion, and found that the β-cell cilia are required for glucose sensing, calcium influx, insulin secretion, and cross regulation of α- and δ-cells. Protein expression profiling in islets confirms perturbation in these cellular processes and reveals additional targets of cilia-dependent signaling. At the organism level, the deletion of β-cell cilia disrupts circulating hormone levels, impairs glucose homeostasis and fuel usage, and leads to the development of diabetes. Together, these findings demonstrate that primary cilia not only orchestrate β-cell–intrinsic activity but also mediate cross talk both within the islet and from islets to other metabolic tissues, thus providing a unique role of cilia in nutrient metabolism and insight into the pathophysiology of diabetes.

The pancreatic islet is a dense cellular network comprised of several cell types with endocrine function vital in the control of glucose homeostasis, metabolism, and feeding behavior. Within the islet, endocrine hormones also form an intricate paracrine network with supportive cells (endothelial, neuronal, immune) and secondary signaling molecules regulating cellular function and survival. Modulation of these signals has potential consequences for diabetes development, progression, and therapeutic intervention. Beta cell loss, reduced endogenous insulin secretion, and dysregulated glucagon secretion are hallmark features of both type 1 and 2 diabetes that not only impact systemic regulation of glucose, but also contribute to the function and survival of cells within the islet. Advancing research and technology have revealed new islet biology (cellular identity and transcriptomes) and identified previously unrecognized paracrine signals and mechanisms (somatostatin and ghrelin paracrine actions), while shifting prior views of intraislet communication. This review will summarize the paracrine signals regulating islet endocrine function and survival, the disruption and dysfunction that occur in diabetes, and potential therapeutic targets to preserve beta cell mass and function.

In vivo regeneration of lost or dysfunctional islet β cells can fulfill the promise of improved therapy for diabetic patients. To achieve this, many mitogenic factors have been attempted, including gamma-aminobutyric acid (GABA). GABA remarkably affects pancreatic islet cells' (α cells and β cells) function through paracrine and/or autocrine binding to its membrane receptors on these cells. GABA has also been studied for promoting the transformation of α cells to β cells. Nonetheless, the gimmickry of GABA-induced α-cell transformation to β cells has two different perspectives. On the one hand, GABA was found to induce α-cell transformation to β cells in vivo and insulin-secreting β-like cells in vitro. On the other hand, GABA treatment showed that it has no α- to β-cell transformation response. Here, we will summarize the physiological effects of GABA on pancreatic islet β cells with an emphasis on its regenerative effects for transdifferentiation of islet α cells to β cells. We will also critically discuss the controversial results about GABA-mediated transdifferentiation of α cells to β cells.

Pancreatic α-cells are important in maintaining metabolic homeostasis, but their role in regulating maternal metabolic adaptations to pregnancy has not been studied. The objective of this study was to determine whether pancreatic α-cells respond to pregnancy and their contribution to maternal metabolic adaptation. Using C57BL/6 mice, our study showed that pregnancy induced a significant increase of α-cell mass by promoting α-cell proliferation that was associated with a transitory increase of maternal serum glucagon concentration in early pregnancy. Maternal pancreatic GLP-1 content also was significantly increased during pregnancy. Using the inducible Cre/loxp technique, we ablated the α-cells (α-null) before and during pregnancy while maintaining enteroendocrine L-cells and serum GLP-1 in the normal range. In contrast to an improved glucose tolerance test (GTT) before pregnancy, significantly impaired GTT and remarkably higher serum glucose concentrations in the fed state were observed in α-null dams. Glucagon receptor antagonism treatment, however, did not affect measures of maternal glucose metabolism, indicating a dispensable role of glucagon receptor signaling in maternal glucose homeostasis. However, the GLP-1 receptor agonist improved insulin production and glucose metabolism of α-null dams. Furthermore, GLP-1 receptor antagonist Exendin (9-39) attenuated pregnancy-enhanced insulin secretion and GLP-1 restored glucose-induced insulin secretion of cultured islets from α-null dams. Together, these results demonstrate that α-cells play an essential role in controlling maternal metabolic adaptation to pregnancy by enhancing insulin secretion.

Poorly controlled diabetes is characterised by a partial or complete loss of pancreatic islet β-cells, which deprives the remaining islet cells of important β-cell-derived soluble signals, such as insulin or GABA. We aimed to dissect the role of the two signals in the development of islet α-cells, focusing specifically on α-/β-cell transdifferentiation and using the stem cell differentiation factor nicotinamide as a comparator.Streptozotocin (STZ)-treated diabetic mice expressing a fluorescent reporter in pancreatic islet α-cells were injected with GABA (10 mg/kg once daily), nicotinamide (150 mg/kg once daily) or insulin (1U/kg three times daily) for 10 days. The impact of the treatment on metabolic status of the animals as well as the morphology, proliferative potential and transdifferentiation of pancreatic islet cells was assessed using biochemical methods and immunofluorescence.Metabolic status of STZ-diabetic mice was not dramatically altered by the treatment interventions, although GABA therapy did reduce circulating glucagon and augment pancreatic insulin stores. The effects of the exogenous agents on islet β-cells ranged from the attenuation of apoptosis (insulin, nicotinamide) to enhancement of proliferation (GABA). Furthermore, insulin and GABA but not nicotinamide enhanced the differentiation of α-cells into β-cells and increased relative number of 'bihormonal' cells, expressing both insulin and glucagon.Our data suggest a role for endogenous insulin and GABA signalling in α-cell plasticity, which is likely to bypass the common nicotinamide-sensitive stem cell differentiation pathway.

OBJECTIVE: The mechanisms by which glucose stimulates insulin secretion from β-cells are well established and involve inhibition of ATP-sensitive K+ (KATP) channels, followed by a rise in [Ca2+]c that triggers exocytosis. However, the mechanisms by which glucose controls glucagon release from α-cells are much less known. In particular, it is debated whether the sugar controls glucagon secretion by changing α-cell [Ca2+]c, and whether KATP channels or paracrine factors are involved. The present study addresses these issues. METHODS: We tested the effect of a decrease or an increase of glucose concentration (Gx, with x = concentration in mM) on α-cell [Ca2+]c and glucagon secretion. α-cell [Ca2+]c was monitored using GluCreGCaMP6f mice expressing the Ca2+-sensitive fluorescent protein, GCaMP6f, specifically in α-cells. [Ca2+]c was compared between dispersed α-cells and α-cells within islets to evaluate the potential contribution of an indirect effect of glucose. The same protocols were used for experiments of glucagon secretion from whole islets and [Ca2+]c measurements to test if changes in glucagon release mirror those in α-cell [Ca2+]c. RESULTS: Blockade of KATP channels by sulfonylureas (tolbutamide 100 μM or gliclazide 25 μM) strongly increased [Ca2+]c in both dispersed α-cells and α-cells within islets. By contrast, glucose had no effect on [Ca2+]c in dispersed α-cells, whereas it affected it in α-cells within islets. The effect of glucose was however different in islets expressing (Sst+/+) or not somatostatin (SST) (Sst-/-). Decreasing glucose concentration from G7 to G1 modestly increased α-cell [Ca2+]c, but to a slightly larger extent in Sst+/+ islets than in Sst-/- islets. This G1-induced [Ca2+]c rise was also observed in the continuous presence of sulfonylureas in both Sst+/+ and Sst-/- islets. Increasing glucose concentration from G7 to G20 did not affect α-cell [Ca2+]c in Sst+/+ islets which remained low, whereas it strongly increased it in Sst-/- islets. The observations that this increase was seen only in α-cells within islets but never in dispersed α-cells and that it was abrogated by the gap junction inhibitor, carbenoxolone, point to an indirect effect of G20 and suggest that, in Sst-/- islets, G20-stimulated β-cells entrain α-cells whereas, in Sst+/+ islets, the concomitant release of SST keeps α-cell [Ca2+]c at low levels. The [Ca2+]c lowering effect of endogenous SST is also supported by the observation that SST receptor antagonists (SSTR2/3) increased [Ca2+]c in α-cells from Sst+/+ islets. All these [Ca2+]c changes induced parallel changes in glucagon release. To test if glucose also controls glucagon release independently of [Ca2+]c changes, additional experiments were performed in the continuous presence of 30 mM K+ and the KATP channel opener diazoxide (250 μM). In these conditions, α-cell [Ca2+]c within islets was elevated and its steady-state level was unaffected by glucose. However, decreasing the glucose concentration from G7 to G1 stimulated glucagon release whereas increasing it from G1 to G15 inhibited it. These effects were also evident in Sst-/- islets, and opposite to those on insulin secretion. CONCLUSIONS: We propose a model according to which glucose controls α-cell [Ca2+]c and glucagon secretion through multiple mechanisms. Increasing the glucose concentration modestly decreases [Ca2+]c in α-cells independently of their KATP channels and partly via SST. The involvement of SST increases with the glucose concentration, and one major effect of SST is to keep α-cell [Ca2+]c at low levels by counteracting the effect of an entrainment of α-cells by β-cells when β-cells become stimulated by glucose. All these [Ca2+]c changes induce parallel changes in glucagon release. Glucose also decreases the efficacy of Ca2+ on exocytosis by an attenuating pathway that is opposite to the well-established amplifying pathway controlling insulin release in β-cells.

Secretion of glucagon from the pancreatic α-cells is conventionally seen as the first and most important defense against hypoglycemia. Recent findings, however, show that α-cell signals stimulate insulin secretion from the neighboring β-cell. This article focuses on these seemingly counterintuitive local actions of α-cells and describes how they impact islet biology and glucose metabolism. It is mostly based on studies published in the last decade on the physiology of α-cells in human islets and incorporates results from rodents where appropriate. As this and the accompanying articles show, the emerging picture of α-cell function is one of increased complexity that needs to be considered when developing new therapies aimed at promoting islet function in the context of diabetes.

Cre/loxP is a site-specific recombination system extensively used to enable the conditional deletion or activation of target genes in a spatial- and/or temporal-specific manner. A number of pancreatic-specific Cre driver mouse lines have been broadly established for studying the development, function and pathology of pancreatic cells. However, only a few models are currently available for glucagon-producing α-cells. Disagreement exists over the role of the MAFB transcription factor in glucagon expression during postnatal life, which might be due to the lack of α-cell-specific Cre driver mice. In the present study, we established a novel Gcg-Cre knock-in mouse line with the Cre transgene expressed under the control of the preproglucagon (Gcg) promoter without disrupting the endogenous Gcg gene expression. Then, we applied this newly developed Gcg-Cre mouse line to generate a new α-cell-specific Mafb conditional knockout mouse model (MafbΔGcg). Not only α-cell number but also glucagon production were significantly decreased in MafbΔGcg mice compared to control littermates, suggesting an indispensable role of MAFB in both α-cell development and function. Taken together, our newly developed Gcg-Cre mouse line, which was successfully utilized to uncover the role of MAFB in α-cells, is a useful tool for genetic manipulation in pancreatic α-cells, providing a new platform for future studies in this field.

Objective Islets secrete neurotransmitters including glutamate which participate in fine regulation of islet function. The excitatory ionotropic glutamate receptor GluK2 of the kainate receptor family is widely expressed in brain and also found in islets, mainly in α and γ cells. α cells co-release glucagon and glutamate and the latter increases glucagon release via ionotropic glutamate receptors. However, neither the precise nature of the ionotropic glutamate receptor involved nor its role in glucose homeostasis is known. As isoform specific pharmacology is not available, we investigated this question in constitutive GluK2 knock-out mice (GluK2−/−) using adult and middle-aged animals to also gain insight in a potential role during aging. Methods We compared wild-type GluK2+/+ and knock-out GluK2−/− mice using adult (14–20 weeks) and middle-aged animals (40–52 weeks). Glucose (oral OGTT and intraperitoneal IPGTT) and insulin tolerance as well as pyruvate challenge tests were performed according to standard procedures. Parasympathetic activity, which stimulates hormones secretion, was measured by electrophysiology in vivo. Isolated islets were used in vitro to determine islet β-cell electrical activity on multi-electrode arrays and dynamic secretion of insulin as well as glucagon was determined by ELISA. Results Adult GluK2−/− mice exhibit an improved glucose tolerance (OGTT and IPGTT), and this was also apparent in middle-aged mice, whereas the outcome of pyruvate challenge was slightly improved only in middle-aged GluK2−/− mice. Similarly, insulin sensitivity was markedly enhanced in middle-aged GluK2−/− animals. Basal and glucose-induced insulin secretion in vivo was slightly lower in GluK2−/− mice, whereas fasting glucagonemia was strongly reduced. In vivo recordings of parasympathetic activity showed an increase in basal activity in GluK2−/− mice which represents most likely an adaptive mechanism to counteract hypoglucagonemia rather than altered neuronal mechanism. In vitro recording demonstrated an improvement of glucose-induced electrical activity of β-cells in islets obtained from GluK2−/− mice at both ages. Finally, glucose-induced insulin secretion in vitro was increased in GluK2−/− islets, whereas glucagon secretion at 2 mmol/l of glucose was considerably reduced. Conclusions These observations indicate a general role for kainate receptors in glucose homeostasis and specifically suggest a negative effect of GluK2 on glucose homeostasis and preservation of islet function during aging. Our observations raise the possibility that blockade of GluK2 may provide benefits in glucose homeostasis especially during aging., Highlights • KO of GluK2 improved glucose tolerance and lowered fasting glucagon levels in vivo. • KO of GluK2 reduced in vitro islet glucagon secretion and increased β-cell activity. • GluK2 contributes to the known impact of aging on glucose homeostasis.

Glucagon secretion from pancreatic alpha cells is crucial to prevent hypoglycemia. For reasons still unknown, people with type 1 diabetes lose this glucoregulatory mechanism and are susceptible to dangerous hypoglycemia. Here we show that alpha cells in living pancreas slices from donors with type 1 diabetes failed to secrete glucagon in response to decreases in glucose concentration, thus mirroring the in vivo unresponsiveness to hypoglycemia. Glucagon content and responses to KCl depolarization were not affected, suggesting that alpha cells retained their secretory potential. By contrast, alpha cells had severely impaired signaling via glutamate receptors of the AMPA/kainate type. Under healthy conditions, activating these receptors was required to elicit full glucagon responses to decreases in glucose levels. In type 1 diabetes, reactivating residual glutamate receptor function with the positive allosteric modulators cyclothiazide and aniracetam restored glucagon secretion in response to hypoglycemia. These positive allosteric modulators are already approved to treat other conditions and could be repurposed to prevent hypoglycemia and improve management of diabetes.

The mechanisms by which glucose stimulates insulin secretion from β-cells are well established and involve inhibition of ATP-sensitive KWe tested the effect of a decrease or an increase of glucose concentration (Gx, with x = concentration in mM) on α-cell [CaBlockade of KWe propose a model according to which glucose controls α-cell [Ca

The Ca2+ modulated pulsatile glucagon and insulin secretions by pancreatic α and β cells play a crucial role in glucose homeostasis. However, how α and β cells coordinate to produce various Ca2+ oscillation patterns is still elusive. Using a microfluidic device and transgenic mice, we recorded Ca2+ signals from islet α and β cells, and observed heterogeneous Ca2+ oscillation patterns intrinsic to each islet. After a brief period of glucose stimulation, α and β cells’ oscillations were globally phase-locked. While the activation of α cells displayed a fixed time delay of ~20 s to that of β cells, β cells activated with a tunable period. Moreover, islet α cell number correlated with oscillation frequency. We built a mathematical model of islet Ca2+ oscillation incorporating paracrine interactions, which quantitatively agreed with the experimental data. Our study highlights the importance of cell-cell interaction in generating stable but tunable islet oscillation patterns.

Recently, the antimalarial drug, artemether and the neurotransmitter GABA were identified to convert alpha cells into beta-like cells in vivo. However, some of these observations were challenged by other studies. To help address the controversy, we took advantage of zebrafish as a model to perform this study.First, we performed a small-molecule screening for artemether and its skeleton analogues. Second, we used the Cre-LoxP system for lineage tracing to indicate the conversion of alpha cells into beta cells in vivo. The stable transgenic ins2:eGFP αTC1-6-cell line was used for evaluation of alpha-cell transdifferentiation in vitro. We further used multiple zebrafish transgenic and mutation lines to demonstrate beta-cell differentiation, beta-cell ablation and alpha-cell hyperplasia in this study.We showed that artemether and another sesquiterpene, aspterric acid, induced alpha-cell transdifferentiation into beta cells, both in zebrafish as well as using αTC1-6 cells. Furthermore, these two compounds also converted alpha cells into beta cells when beta cells were lost or alpha cells were hyperplastic in zebrafish. Unlike the previous report, the conversion of alpha cells to beta cells was mediated by increasing Pax4 expression, but not suppression of Arx expression.Our data suggest that in zebrafish and αTC1-6 cells, both artemether and aspterric acid induce alpha-cell transdifferentiation. Our data, along with those of Li et al. (2017), suggested that artemether and aspterric acid were able to induce alpha-cell transdifferentiation, at least in zebrafish and αTC1-6 cells.

Glucose homeostasis is maintained by hormones secreted from different types of pancreatic islets and its dysregulation can result in diseases including diabetes mellitus. The secretion of hormones from pancreatic islets is highly complex and tightly controlled by G protein-coupled receptors (GPCRs). Moreover, GPCR signaling may play a role in enhancing islet cell replication and proliferation. Thus, targeting GPCRs offers a promising strategy for regulating the functionality of pancreatic islets. Here, available RNAseq datasets from human and mouse islets were used to identify the GPCR expression profile and the impact of GPCR signaling for normal islet functionality is discussed.

There are significant injuries of pancreatic islets due to obesity and insulin resistance. Therefore, GLP-1 receptor agonists like Semaglutide might benefit the islet structural remodeling and its endocrine function in diet-induced obese mice. One-month-old male C57BL/6 mice were allotted into two dietary groups (n = 60/group) and fed for 16 weeks a control diet (C) or a high‒fat diet (HF). Then, for an additional four weeks, the main groups were resampled to include treatment (Semaglutide, S, 40 μg/kg), or paired feed with the treated group (PF), totaling six groups (n = 20/group): C, CS, CPF, HF, HFS, HFPF. Biochemistry, stereology, immunohistochemistry/immunofluorescence, confocal microscopy, and RT-qPCR were used in the study. The mouse model reproduced metabolism and bodily changes due to diet-induced obesity. Pancreatic islet hypertrophy was observed with alpha- and beta-cell remodeling, cell disarray, and apoptosis. Semaglutide increased islet cell proliferation and recovered islet size and alpha- and beta-cell masses. The changes include recovery of glucose and hormone levels, reduction of pro-inflammatory markers, improvement of pancreatic duodenal homeobox 1 (PDX-1), glucose transporter 2 (GLUT-2), v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MAF-A), and peroxisome proliferator-activated receptors (PPAR) -gamma. In conclusion, damage to the pancreatic islet caused by insulin resistance and the attempt to adapt the islet of obese mice involved different pathways, especially the pro-inflammatory pathway, PDX1, and PPAR-alpha and gamma. Semaglutide showed beneficial effects on these pathways, reducing the lesion on the islet. However, the weight loss influence of Semaglutide was of little relevance in the pancreatic islet.

In recent years the role of altered alpha cell function and glucagon secretion in type 1 diabetes has attracted scientific attention. It is well established that glucagon responses to hypoglycemia are absent in type 1 diabetes, but more uncertain whether it is intact following other physiological and metabolic stimuli compared with nondiabetic individuals. The aim of this review is to (i) summarize current knowledge on glucagon responses during hypoglycemia in normal physiology and type 1 diabetes, and (ii) review human in vivo studies investigating glucagon responses after other stimuli in individuals with type 1 diabetes and nondiabetic individuals. Available data suggest that in type 1 diabetes the absence of glucagon secretion after hypoglycemia is irreversible. This is a scenario specific to hypoglycemia, since other stimuli, including administration of amino acids, insulin withdrawal, lipopolysaccharide exposure and exercise lead to substantial glucagon responses though attenuated compared to nondiabetic individuals in head‐to‐head studies. The derailed glucagon secretion is not confined to hypoglycemia as individuals with type 1 diabetes, as opposed to nondiabetic individuals display glucagon hypersecretion after meals, thereby potentially contributing to insulin resistance. The complexity of these phenomena may relate to activation of distinct regulatory pathways controlling glucagon secretion i.e., intra‐islet paracrine signaling, direct and autonomic nervous signaling., The absent glucagon responses to hypoglycemia in type 1 diabetes is a scenario specific to hypoglycemia, since other stimuli, including administration of amino acids, insulin withdrawal, lipopolysaccharide exposure, exercise and even meals lead to profound glucagon responses.