Pancreatic Islet Alpha Research Articles

Multiple promoter and enhancer differences likely contribute to augmented G6PC2 expression in human versus mouse pancreatic islet alpha cells.

G6PC2 encodes a glucose-6-phosphatase catalytic subunit that opposes the action of glucokinase in pancreatic islets, thereby modulating the sensitivity of insulin and glucagon secretion to glucose. In mice, G6pc2 is expressed at ~20-fold higher levels in β-cells than in α-cells, whereas in humans G6PC2 is expressed at only ~5-fold higher levels in β-cells. We therefore hypothesize that G6PC2 likely influences glucagon secretion to a greater degree in humans. With a view to generating a humanized mouse that recapitulates augmented G6PC2 expression levels in α-cells, we sought to identify the genomic regions that confer differential mouse G6pc2 expression in α-cells versus β-cells as well as the evolutionary changes that have altered this ratio in humans. Studies in islet-derived cell lines suggest that the elevated G6pc2 expression in mouse β-cells versus α-cells is mainly due to a difference in the relative activity of the proximal G6pc2 promoter in these cell types. Similarly, the smaller difference in G6PC2 expression between α-cells and β-cells in humans is potentially explained by a change in relative proximal G6PC2 promoter activity. However, we show that both glucocorticoid levels and multiple differences in the relative activity of eight transcriptional enhancers between mice and humans likely contribute to differential G6PC2 expression. Finally, we show that a mouse-specific non-coding RNA, Gm13613, whose expression is controlled by G6pc2 enhancer I, does not regulate G6pc2 expression, indicating that altered expression of Gm13613 in a humanized mouse that contains both the human promoter and enhancers should not affect G6PC2 function.

OR12-03 The Pancreatic Islet Alpha Cell Arginine Transporter SLC7A2 Regulates Arginine-induced Glucagon Secretion and Glycemia

Abstract Disclosure: J.E. Stanley: None. A. Reuter: None. K. Sellick: None. A.D. Attie: None. M.P. Keller: None. E. Dean: None. Glucagon secreted from pancreatic islet α cells stimulates both hepatic glycogenolysis and gluconeogenesis resulting in the fractional extraction of amino acids from the blood. Thus, glucagon stimulates hepatic glucose output and raises blood glucose. When liver glucagon signaling is impaired, amino acid accumulation (hyperaminoacidemia) promotes subsequent α cell hyperplasia and hyperglucagonemia via this liver-α-cell axis, an endocrine feedback loop. We show that high levels of both glutamine and recently arginine are required to stimulate α cells to proliferate. Arginine also potently stimulates glucagon secretion, but the mechanisms behind this are not well defined. We find that the cationic amino acid transporter, SLC7A2/CAT2 is the most highly expressed amino acid transporter in human pancreatic α cells and three-fold higher in α cells than it is in β-cells in both mouse and human islets. We also find two SNPs in the human SLC7A2 locus are strongly associated with HbA1c (rs142010226 (A/G) - chr8: 17,367,112; p=9.11e-18; β=0.0113 and rs2517232 - chr8: 17,367,421; p=2.553e-14; β=-0.0283). Both SNPs are in the first intron of SLC7A2 but appear to exert opposite effect on HbA1c and associated with distinct haplotype blocks. ATAC-Seq analyses of human islets show that these SNPs are within 1 kb of binding sites for MAFB and FOXA2 transcription factors, both critical for α cell gene expression. To test if α cell expression of SLC7A2 plays a role in both glycemia and arginine-stimulated glucagon secretion, we generated α cell-specific Slc7a2-/- (α7A2KO) mice. Blood glucose and glucagon levels were not significantly different after a 6-hour fast in α7A2KO and 7A2WTCre+ mice. After a 6-hour fast, mice were administered an arginine bolus (2g/kg) via intraperitoneal injections and their blood glucose and glucagon levels were measured 15 minutes post-injection. α7A2KO mice had a significantly higher blood glucose level than 7A2WTcre+ mice after stimulation with arginine (arginine-stimulated blood glucose - 7A2WTCre+ 124.3 ± 26.7 mg/dl vs. α7A2KO 159.6 ± 32.1 mg/dl n=23-25 p=0.0019). However, glucagon secretion was significantly lower after stimulation with arginine in the α7A2KO mice compared to the 7A2WTCre+ mice (arginine-stimulated glucagon - 7A2WTCre+ 321.8 ± 171.9 pM vs. α7A2KO 74.6 ± 22.8 pM n=4-6; p<0.0001). Together, these data indicate that SLC7A2 mediates arginine’s effects in α cells and plays an important role in regulating glycemia. Presentation: Friday, June 16, 2023

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.

Impact of an SLC30A8 loss-of-function variant on the pancreatic distribution of zinc and manganese: laser ablation-ICP-MS and positron emission tomography studies in mice.

Common variants in the SLC30A8 gene, encoding the secretory granule zinc transporter ZnT8 (expressed largely in pancreatic islet alpha and beta cells), are associated with altered risk of type 2 diabetes. Unexpectedly, rare loss-of-function (LoF) variants in the gene, described in heterozygous individuals only, are protective against the disease, even though knockout of the homologous SLC30A8 gene in mice leads to unchanged or impaired glucose tolerance. Here, we aimed to determine how one or two copies of the mutant R138X allele in the mouse SLC30A8 gene impacts the homeostasis of zinc at a whole-body (using non-invasive 62Zn PET imaging to assess the acute dynamics of zinc handling) and tissue/cell level [using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) to map the long-term distribution of zinc and manganese in the pancreas]. Following intravenous administration of [62Zn]Zn-citrate (~7 MBq, 150 μl) in wild-type (WT), heterozygous (R138X+/-), and homozygous (R138X+/+) mutant mice (14-15 weeks old, n = 4 per genotype), zinc dynamics were measured over 60min using PET. Histological, islet hormone immunohistochemistry, and elemental analysis with LA-ICP-MS (Zn, Mn, P) were performed on sequential pancreas sections. Bulk Zn and Mn concentration in the pancreas was determined by solution ICP-MS. Our findings reveal that whereas uptake into organs, assessed using PET imaging of 62Zn, is largely unaffected by the R138X variant, mice homozygous of the mutant allele show a substantial lowering (to 40% of WT) of total islet zinc, as anticipated. In contrast, mice heterozygous for this allele, thus mimicking human carriers of LoF alleles, show markedly increased endocrine and exocrine zinc content (1.6-fold increase for both compared to WT), as measured by LA-ICP-MS. Both endocrine and exocrine manganese contents were also sharply increased in R138X+/- mice, with smaller increases observed in R138X+/+ mice. These data challenge the view that zinc depletion from the beta cell is the likely underlying driver for protection from type 2 diabetes development in carriers of LoF alleles. Instead, they suggest that heterozygous LoF may paradoxically increase pancreatic β-cell zinc and manganese content and impact the levels of these metals in the exocrine pancreas to improve insulin secretion.

Pancreatic islet remodeling in cotadutide-treated obese mice

Obesity and type 2 diabetes mellitus (T2DM) cause morphofunctional alterations in pancreatic islet alpha and beta cells. Therefore, we hypothesize that the new GLP-1/Glucagon receptor dual agonist cotadutide may benefit islet cell arrangement and function. Twelve-week-old C57BL/6 male mice were fed a control diet (C, 10 % kJ fat) or a high-fat diet (HF, 50 % kJ fat) for ten weeks. Then, the animals were divided into four groups for an additional 30 days and daily treated with subcutaneous cotadutide (30 nmol/kg) or vehicle: C, CC (control+cotadutide), HF, and HFC (high-fat+cotadutide). Cotadutide led to weight loss and reduced insulin resistance in the HFC group, increasing insulin receptor substrate 1 and solute carrier family 2 gene expressions in isolated islets. Also, cotadutide enhanced transcriptional factors related to islet cell transdifferentiation, decreasing aristaless-related homeobox and increasing the paired box 4 and 6, pancreatic and duodenal homeobox 1, v-maf musculoaponeurotic fibrosarcoma oncogene family protein A, neurogenin 3, and neurogenic differentiation 1. In addition, cotadutide improved the proliferating cell nuclear antigen, NK6 homeobox 1, B cell leukemia/lymphoma 2, but lessening caspase 3. Furthermore, cotadutide mitigated the endoplasmic reticulum (ER) stress-responsive genes, reducing transcription factor 4, DNA-damage-inducible transcript 3, and growth arrest and DNA-damage-inducible 45. In conclusion, our data demonstrated significant beneficial actions of cotadutide in DIO mice, such as weight loss, glycemic control, and insulin resistance improvement. In addition, cotadutide counteracted the pathological adaptive cellular arrangement of the pancreatic islet in obese mice, improving the markers of the transdifferentiating pathway, proliferation, apoptosis, and ER stress.

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

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. 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). The population consisted of 24 males and 16 females, with a median age of 68 and a range of 14-84years; 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). 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.

Necrolytic migratory erythema is an important visual cutaneous clue of glucagonoma

Glucagonoma is an extremely rare neuroendocrine tumor that arises from pancreatic islet alpha cells. Although glucagonoma is usually accompanied by a variety of characteristic clinical symptoms, early diagnosis is still difficult due to the scarcity of the disease. In this study, we present the cumulative experiences, clinical characteristics and treatments of seven patients diagnosed with glucagonoma during the past 10 years at the First Affiliated Hospital of Xi’an Jiaotong University. The seven patients in our cohort consisted of six females and one male with an average diagnosis age of 40.1 years (range 23–51). The average time from onset of symptoms to diagnosis of glucagonoma was 14 months (range 2–36 months). All the patients visited dermatology first for necrolytic migratory erythema (NME) 7/7 (100%), and other presenting symptoms included diabetes mellitus (DM) 4/7 (57%), stomatitis 2/7 (28%), weight loss 4/7 (57%), anemia 4/7 (57%), diarrhea 1/7 (14%), and DVT1/7 (14%). Plasma glucagon levels were increased in all patients (range 216.92–3155 pg/mL) and declined after surgery. Imaging studies revealed that four of seven patients had liver metastasis. Six of seven patients received surgical resection, and all of them received somatostatin analog therapy. Symptoms improved significantly in 6 out of 7 patients. Three of seven patients died of this disease by the time of follow-up. Our data suggest that if persistent NME is associated with DM and high glucagon levels, timely abdominal imaging should be performed to confirm glucagonoma. Once diagnosed, surgery and somatostatin analogs are effective for symptom relief and tumor control.

A glycolytic enzyme complex and creatine kinase locally and differentially regulate ATP-sensitive potassium (KATP) channels in pancreatic islet alpha and beta cells

The ATP-sensitive potassium channel (KATP) is a key regulator of membrane potential and islet hormone secretion. Here, using the inside-out excised patch configuration, we isolated the plasma membrane compartment containing KATP on β-cells and α-cells from humans and mice. The characteristic response of KATP to inhibition by ATP and activation by ADP was used to identify the presence of a glycolytic enzyme complex that is functionally coupled to KATP regulation. We found that the phosphofructokinase product fructose-1,6-bisphosphate (FBP), which oscillates in β-cells, can induce KATP closure by its metabolism through the glycolytic chain including aldolase, GAPDH, phosphoglycerate kinase, phosphoglycerate mutase, enolase and finally pyruvate kinase.

Localization of dipeptidyl peptidase-4 (CD26) to human pancreatic ducts and islet alpha cells

AimDPP-4/CD26 degrades the incretins GLP-1 and GIP. The localization of DPP-4 within the human pancreas is not well documented but is likely to be relevant for understanding incretin function. We aimed to define the cellular localization of DPP-4 in the human pancreas from cadaveric organ donors with and without diabetes. MethodsPancreas was snap-frozen and immunoreactive DPP-4 detected in cryosections using the APAAP technique. For co-localization studies, pancreas sections were double-stained for DPP-4 and proinsulin or glucagon and scanned by confocal microscopy. Pancreata were digested and cells in islets and in islet-depleted, duct-enriched digests analyzed for expression of DPP-4 and other markers by flow cytometry. ResultsDPP-4 was expressed by pancreatic duct and islet cells. In pancreata from donors without diabetes or with type 2 diabetes, DPP-4-positive cells in islets had the same location and morphology as glucagon-positive cells, and the expression of DPP-4 and glucagon overlapped. In donors with type 1 diabetes, the majority of residual cells in islets were DPP-4-positive. ConclusionIn the human pancreas, DPP-4 expression is localized to duct and alpha cells. This finding is consistent with the view that DPP-4 regulates exposure to incretins of duct cells directly and of beta cells indirectly in a paracrine manner.

Cardiopulmonary bypass and insulin resistance of islet cells

Background Cardiopulmonary bypass (CPB) can cause peripheral insulin resistance (IR).The IR is more serious when CPB lasts longer.Currently,the studies have confirmed that IR can also exist in pancreatic islet beta(β) and alpha (α)cells.However,the studies about secretory function of islet cells during CPB are few so far.Objective To provide the basis for better control of blood glucose during CPB.Content This review will focus on the relationship of CPB and IR of islet cells.Trend Islet cells may produce their own IR,which will be paid more and more attention in blood glucose management during CPB in the future. Key words: Cardiopulmonary bypass ; Hyperglycemia; Islet cells; Insulin resistance; Signal transduction

Different susceptibility of rat pancreatic alpha and beta cells to hypoxia

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

AMP-activated protein kinase regulates glucagon secretion from mouse pancreatic alpha cells

AMP-activated protein kinase (AMPK), encoded by Prkaa genes, is emerging as a key regulator of overall energy homeostasis and the control of insulin secretion and action. We sought here to investigate the role of AMPK in controlling glucagon secretion from pancreatic islet alpha cells. AMPK activity was modulated in vitro in clonal alphaTC1-9 cells and isolated mouse pancreatic islets using pharmacological agents and adenoviruses encoding constitutively active or dominant negative forms of AMPK. Glucagon secretion was measured during static incubation by radioimmunoassay. AMPK activity was assessed by both direct phosphotransfer assay and by western (immuno-)blotting of the phosphorylated AMPK α subunits and the downstream target acetyl-CoA carboxylase 1. Intracellular free [Ca²(+)] was measured using Fura-Red. Increasing glucose concentrations strongly inhibited AMPK activity in clonal pancreatic alpha cells. Forced increases in AMPK activity in alphaTC1-9 cells, achieved through the use of pharmacological agents including metformin, phenformin and A-769662, or via adenoviral transduction, resulted in stimulation of glucagon secretion at both low and high glucose concentrations, whereas AMPK inactivation inhibited both [Ca²(+)](i) increases and glucagon secretion at low glucose. Transduction of isolated mouse islets with an adenovirus encoding AMPK-CA under the control of the preproglucagon promoter increased glucagon secretion selectively at elevated glucose concentrations. AMPK is strongly regulated by glucose in pancreatic alpha cells, and increases in AMPK activity are sufficient and necessary for the stimulation of glucagon release in vitro. Modulation of AMPK activity in alpha cells may therefore provide a novel approach to controlling blood glucose concentrations.

Not for the eyes only: PAX6 and glucose metabolism

Paired box 6 (PAX6) is a member of the PAX multigene family of transcription factors: this family regulates embryonic differentiation. Nine unlinked PAX genes dispersed throughout the genome encode proteins that each include a 128-amino-acid sequence-specific highly conserved DNA-binding domain, the Paired box, which can regulate the expression of other genes [1]. PAX6 also contains another common DNA-binding element: the homeobox that encodes a 60-amino-acid homeodomain responsible for target sequence recognition [2]. The paired domain and homeodomain interact cooperatively to recognise multiple DNA binding sites [3]. Pax6 is expressed in the developing nervous system and in developing eyes [4], and also has important functions in the development of the endocrine pancreas. Genetic studies indicate that all eyes share a similar developmental cascade: mutations in Pax6 disrupt eye development in both mammals and insects [4]. Pax6 appears to be necessary for the correct execution of beta cell differentiation [5] and is essential for the normal expression of insulin [6]. PAX6 is required for the differentiation of pancreatic islet alpha cells, and competes with PAX4 in binding to a common element in the glucagon, insulin and somatostatin promoters [7]. Mice lacking both Pax4 and Pax6 fail to develop any mature endocrine cells, suggesting that both genes are required for endocrine development of the pancreas [8]. As well as binding to a common element in the glucagon, insulin and somatostatin promoters, Pax6 also transactivates the glucagon and insulin promoters [6]. Pax6-deficient mice have reductions in both insulinand glucagon-expressing cells. Human PAX6 is transcribed as a 2.7 kb mRNA and encodes a 422-amino-acid protein. PAX6 extends to over 22 kb: it contains 14 exons and intron sequences and is highly conserved among vertebrates and lower animals. Mutations in PAX6 in humans cause aniridia type II, a bilateral panocular disorder characterised by a complete or partial absence of the iris and fovea, and malformations of the lens and anterior chamber [2]. About one-third of the cases are sporadic and two-thirds are familial, with autosomal dominant inheritance and high penetrance. There is a PAX6 dosage effect in aniridia, ranging from mild loss of visual acuity and cataracts, to severe nervous system defects and anophthalmia [7]. Using magnetic resonance imaging techniques and smell testing, Sisodiya et al. [9] showed with that reduced olfaction was present in a large proportion of aniridia patients, indicating that PAX6 haploinsufficiency causes more widespread human neurodevelopmental anomalies. Yasuda et al. [10] were the first to demonstrate that carriers of the PAX6 mutation might have abnormal glucose Diabetologia (2009) 52:381–384 DOI 10.1007/s00125-008-1251-1

Loss of insulin-induced inhibition of glucagon gene transcription in hamster pancreatic islet alpha cells by long-term insulin exposure

Diabetes mellitus type 2 is characterised by hyperglucagonaemia, resulting in hepatic glucose production and hyperglycaemia. Considering that insulin inhibits glucagon secretion and gene transcription, hyperglucagonaemia in the face of hyperinsulinaemia in diabetes mellitus type 2 suggests that there is insulin resistance also at the glucagon-producing pancreatic islet alpha cells. However, the molecular mechanism of alpha cell insulin resistance is unknown. Therefore, the effect of molecules implicated in conferring insulin resistance in some other tissues was investigated on insulin-induced inhibition of glucagon gene transcription in alpha cells. Reporter gene assays and biochemical techniques were used in the glucagon-producing hamster pancreatic islet alpha cell line InR1-G9. From among 16 agents tested, chronic insulin treatment was found to abolish insulin-induced inhibition of glucagon gene transcription. Overproduction of constitutively active protein kinase B (PKB) still inhibited glucagon gene transcription after chronic insulin treatment; together with a markedly reduced insulin-induced phosphorylation and, thus, activation of PKB, this indicates that targets upstream of PKB within the insulin signalling pathway are affected. Indeed, chronic insulin treatment markedly reduced IRS-1 phosphorylation, insulin receptor (IR) autophosphorylation and IR content. Cycloheximide and in vivo labelling experiments attributed IR downregulation to enhanced degradation. These results show that an extended exposure of alpha cells to insulin induces IR downregulation and loss of insulin-induced inhibition of glucagon gene transcription. They suggest that hyperinsulinaemia, through IR downregulation, may confer insulin resistance to pancreatic islet alpha cells in diabetes mellitus type 2.

Fly-let Biology and the High Protein/Low Carb Diet

In Drosophila, a simple network of nutrient-sensing neuroendocrine cells, analogs of pancreatic islet alpha and beta cells, regulates carbohydrate metabolism. Work presented in this issue of Cell Metabolism (Buch et al., 2008) shows that signals from these cells control expression of a glycogen-specific glucosidase in response to dietary protein and carbohydrate.

Direct visualisation of peptide hormones in cultured pancreatic islet alpha‐ and beta‐cells by intact‐cell mass spectrometry

The application of intact-cell mass spectrometry (ICM) by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry to achieve direct protein-profiling of bacterial species is now well established. However, this methodology has not to our knowledge been applied to the analysis of mammalian cells in routine culture. Here, we describe a novel application of ICM by which we have identified proteins in intact cells from two lines representative of pancreatic islet alpha- and beta-cells. Adherent alphaTC1 clone 9 and betaTC6 F7 cells were harvested into phosphate-buffered saline (PBS) using enzyme-free dissociation buffer before 1 microL of cell suspension was spotted onto MALDI plates. Cells were overlaid with sinapinic acid then washed with pure water before application of a final coat of sinapinic acid. Data in the 2000-20,000 m/z range were acquired in linear mode on a Voyager DE-Pro mass spectrometer. The proteins which ionised were composed in large part of peptide hormones (e.g. insulin and glucagon) known to be packaged into the secretory granules of the beta- and alpha-cells respectively. However, in addition to visualising the peptides expected to be associated with these cells, a mass consistent with oxyntomodulin was identified in the cultured alpha-cells, a finding not previously reported to our knowledge. In summary, this paper describes, for the first time, a rapid and direct method useful for identifying secretory products in intact endocrine cells.

A KATP Channel-Dependent Pathway within α Cells Regulates Glucagon Release from Both Rodent and Human Islets of Langerhans

Glucagon, secreted from pancreatic islet α cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring β cells, or to an intrinsic glucose sensing by the α cells themselves. We examined hormone secretion and Ca2+ responses of α and β cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn2+ signalling was blocked, but was reversed by low concentrations (1–20 μM) of the ATP-sensitive K+ (KATP) channel opener diazoxide, which had no effect on insulin release or β cell responses. This effect was prevented by the KATP channel blocker tolbutamide (100 μM). Higher diazoxide concentrations (≥30 μM) decreased glucagon and insulin secretion, and α- and β-cell Ca2+ responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (<1 μM) stimulated glucagon secretion, whereas high concentrations (>10 μM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the KATP channel, inhibition of voltage-gated Na+ (TTX) and N-type Ca2+ channels (ω-conotoxin), but not L-type Ca2+ channels (nifedipine), prevented glucagon secretion. Both the N-type Ca2+ channels and α-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an α-cell KATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.

Chronic insulin exposure with a decrease of insulin receptors results in diminished insulin sensitivity of glucagon gene transcription in pancreatic alpha cells

Objectives: Insulin resistance of peripheral tissues like liver, muscle and fat and beta cell dysfunction contribute to the pathogenesis of diabetes mellitus type 2. Considering that insulin inhibits glucagon secretion and gene transcription, hyperglucagonemia and hyperinsulinemia observed in diabetes suggest that insulin resistance also affects the glucagon-producing pancreatic alpha-cells. However, the molecular mechanisms of insulin resistance at the level of the pancreatic islet alpha-cells are unknown. In the present study the effect of chronic insulin treatment on the regulation of glucagon gene transcription in the pancreatic islet alpha cell-line and on different steps of insulin signaling was investigated.

Differentially expressed Maf family transcription factors, c-Maf and MafA, activate glucagon and insulin gene expression in pancreatic islet alpha- and beta-cells.

A basic-leucine zipper transcription factor, MafA, was recently identified as one of the most important transactivators of insulin gene expression. This protein controls the glucose-regulated and pancreatic beta-cell-specific expression of the insulin gene through a cis-regulatory element called RIPE3b/MARE (Maf-recognition element). Here, we show that MafA expression is restricted to beta-cells of pancreatic islets in vivo and in insulinoma cell lines. We also demonstrate that c-Maf, another member of the Maf family of transcription factors, is expressed in islet alpha-cells and in a glucagonoma cell line (alphaTC1), but not in gamma- and delta-cells. An insulinoma cell line, betaTC6, also expressed c-Maf, albeit at a low level. Chromatin immunoprecipitation assays demonstrated that Maf proteins associate with insulin and glucagon promoters in beta- and alpha-cell lines, respectively. c-Maf protein stimulated glucagon promoter activity in a transient luciferase assay, and activation of the glucagon promoter by c-Maf was more efficient than by the other alpha-cell-enriched transcription factors, Cdx2, Pax6, and Isl-1. Furthermore, inhibition of c-Maf expression in alphaTC1 cells by specific short hairpin RNA resulted in marked reduction of the glucagon promoter activity. Thus, c-Maf and MafA are differentially expressed in alpha- and beta-cells where they regulate glucagon and insulin gene expression, respectively.

Pancreatic α- and β-Cell Function in Pheochromocytoma

Arginine infusion tests were carried out in seven patients with pheochromocytoma before and after extirpation of the tumors in order to evaluate pancreatic islet alpha- and beta-cell function during the state of endogenous catecholamine excess. Six of the patients had glucose intolerance; one did not. Preoperatively, the pancreatic glucagon response was suppressed, while the insulin response was comparable to that in normal control subjects. Plasma glucose levels decreased rapidly after the beginning of arginine infusion in all patients. Theses changes during the infusion were evident in the one patient without glucose intolerance. Postoperatively, the glucagon response and plasma glucose changes were normalized. In addition to the obvious suppression of pancreatic alpha-cell function in our patients with pheochromocytoma, it seems likely that pancreatic beta-cell function also was suppressed; there was no enhancement of the insulin response to arginine during the period of chronic hyperglycemia, a situation in which a synergistic effect between glucose and arginine might be expected.