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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., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

It is unclear whether the secretion of glucagon is regulated by an alpha-cell-intrinsic mechanism and whether signal recognition by the mitochondrial metabolism plays a role in it. To measure changes of the cytosolic ATP/ADP ratio, single alpha-cells and beta-cells from NMRI mice were adenovirally transduced with the fluorescent indicator PercevalHR. The cytosolic Ca2+ concentration ([Ca2+]i) was measured by use of Fura2 and the mitochondrial membrane potential by use of TMRE. Perifused islets were used to measure the secretion of glucagon and insulin. At 5 mM glucose, the PercevalHR ratio in beta-cells was significantly lower than in alpha-cells. Lowering glucose to 1 mM decreased the ratio to 69% within 10 minutes in beta-cells, but only to 94% in alpha-cells. In this situation, 30 mM glucose, 10 mM alpha-ketoisocaproic acid, and 10 mM glutamine plus 10 mM BCH (a nonmetabolizable leucine analogue) markedly increased the PercevalHR ratio in beta-cells. In alpha-cells, only glucose was slightly effective. However, none of the nutrients increased the mitochondrial membrane potential in alpha-cells, whereas all did so in beta-cells. The kinetics of the PercevalHR increase were reflected by the kinetics of [Ca2+]i. increase in the beta-cells and insulin secretion. Glucagon secretion was markedly increased by washing out the nutrients with 1 mM glucose, but not by reducing glucose from 5 mM to 1 mM. This pattern was still recognizable when the insulin secretion was strongly inhibited by clonidine. It is concluded that mitochondrial energy metabolism is a signal generator in pancreatic beta-cells, but not in alpha-cells., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Historically, intracellular function and metabolic adaptation within the α-cell has been understudied, with most of the attention being placed on the insulin-producing β-cells due to their role in the pathophysiology of type 2 diabetes mellitus. However, there is a growing interest in understanding the function of other endocrine cell types within the islet and their paracrine role in regulating insulin secretion. For example, there is greater appreciation for α-cell products and their contributions to overall glucose homeostasis. Several recent studies have addressed a paracrine role for α-cell-derived glucagon-like peptide-1 (GLP-1) in regulating glucose homeostasis and responses to metabolic stress. Further, other studies have demonstrated the ability of glucagon to impact insulin secretion by acting through the GLP-1 receptor. These studies challenge the central dogma surrounding α-cell biology describing glucagon's primary role in glucose counterregulation to one where glucagon is critical in regulating both hyper- and hypoglycemic responses. Herein, this review will update the current understanding of the role of glucagon and α-cell-derived GLP-1, placing emphasis on their roles in regulating glucose homeostasis, insulin secretion, and β-cell mass., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Islets represent an important site of direct action of the hormone ghrelin, with expression of the ghrelin receptor (growth hormone secretagogue receptor; GHSR) having been localized variably to alpha cells, beta cells, and/or somatostatin (SST)-secreting delta cells. To our knowledge, GHSR expression by pancreatic polypeptide (PP)-expressing gamma cells has not been specifically investigated. Here, histochemical analyses of Ghsr-IRES-Cre × Cre-dependent ROSA26-yellow fluorescent protein (YFP) reporter mice showed 85% of GHSR-expressing islet cells coexpress PP, 50% coexpress SST, and 47% coexpress PP + SST. Analysis of single-cell transcriptomic data from mouse pancreas revealed 95% of Ghsr-expressing cells coexpress Ppy, 100% coexpress Sst, and 95% coexpress Ppy + Sst. This expression was restricted to gamma-cell and delta-cell clusters. Analysis of several single-cell human pancreatic transcriptome data sets revealed 59% of GHSR-expressing cells coexpress PPY, 95% coexpress SST, and 57% coexpress PPY + SST. This expression was prominent in delta-cell and beta-cell clusters, also occurring in other clusters including gamma cells and alpha cells. GHSR expression levels were upregulated by type 2 diabetes mellitus in beta cells. In mice, plasma PP positively correlated with fat mass and with plasma levels of the endogenous GHSR antagonist/inverse agonist LEAP2. Plasma PP also elevated on LEAP2 and synthetic GHSR antagonist administration. These data suggest that in addition to delta cells, beta cells, and alpha cells, PP-expressing pancreatic cells likely represent important direct targets for LEAP2 and/or ghrelin both in mice and humans., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Increased glucagon is a hallmark of diabetes and leads to worsening of the hyperglycemia, but the molecular mechanisms causing it are still unknown. We therefore investigated the possibility that microRNAs might be involved in the regulation of glucagon. Indeed, analysis of the glucagon 3' untranslated region (UTR) revealed potential binding sites for miR-320a, and using luciferase reporter assays we found that miR-320a directly targets the 3' UTRs of human and rodent glucagon. In addition, endogenous glucagon mRNA and protein expression as well as glucagon secretion were reduced in response to miR-320a overexpression, whereas inhibition of miR-320a upregulated glucagon expression. Interestingly, miR-320a expression was decreased by high glucose, and this was associated with an increase in glucagon expression in human islets and mouse αTC1-6 cells. Moreover, miR-320a overexpression completely blunted these effects. Importantly, miR-320a was also significantly downregulated in human islets of subjects with type 2 diabetes and this was accompanied by increased glucagon expression. Thus, our data suggest that glucose-induced downregulation of miR-320a may contribute to the paradoxical increase in glucagon observed in type 2 diabetes and reveal for the first time that glucagon expression is under the control by a microRNA providing novel insight into the abnormal regulation of glucagon in diabetes., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The role of depolarization in the inverse glucose-dependence of glucagon secretion was investigated by comparing the effects of KATP channel block and of high potassium. The secretion of glucagon and insulin by perifused mouse islets was simultaneously measured. Lowering glucose raised glucagon secretion before it decreased insulin secretion, suggesting an alpha cell-intrinsic signal recognition. Raising glucose affected glucagon and insulin secretion at the same time. However, depolarization by tolbutamide, gliclazide, or 15 mM KCl increased insulin secretion before the glucagon secretion receded. In contrast to the robust depolarizing effect of arginine and KCl (15 and 40 mM) on single alpha cells, tolbutamide was of variable efficacy. Only when applied before other depolarizing agents had tolbutamide a consistent depolarizing effect and regularly increased the cytosolic Ca2+ concentration. When tested on inside-out patches tolbutamide was as effective on alpha cells as on beta cells. In the presence of 1 µM clonidine, to separate insulinotropic from glucagonotropic effects, both 500 µM tolbutamide and 30 µM gliclazide increased glucagon secretion significantly, but transiently. The additional presence of 15 or 40 mM KCl in contrast led to a marked and lasting increase of the glucagon secretion. The glucagon secretion by SUR1 knockout islets was not increased by tolbutamide, whereas 40 mM KCl was of unchanged efficiency. In conclusion a strong and sustained depolarization is compatible with a marked and lasting glucagon secretion. KATP channel closure in alpha cells is less readily achieved than in beta cells, which may explain the moderate and transient glucagonotropic effect., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Selective inhibitors of sodium glucose cotransporter-2 (SGLT2) are widely used for the treatment of type 2 diabetes and act primarily to lower blood glucose by preventing glucose reabsorption in the kidney. However, it is controversial whether these agents also act on the pancreatic islet, specifically the α cell, to increase glucagon secretion. To determine the effects of SGLT2 on human islets, we analyzed SGLT2 expression and hormone secretion by human islets treated with the SGLT2 inhibitor dapagliflozin (DAPA) in vitro and in vivo. Compared to the human kidney, SLC5A2 transcript expression was 1600-fold lower in human islets and SGLT2 protein was not detected. In vitro, DAPA treatment had no effect on glucagon or insulin secretion by human islets at either high or low glucose concentrations. In mice bearing transplanted human islets, 1 and 4 weeks of DAPA treatment did not alter fasting blood glucose, human insulin, and total glucagon levels. Upon glucose stimulation, DAPA treatment led to lower blood glucose levels and proportionally lower human insulin levels, irrespective of treatment duration. In contrast, after glucose stimulation, total glucagon was increased after 1 week of DAPA treatment but normalized after 4 weeks of treatment. Furthermore, the human islet grafts showed no effects of DAPA treatment on hormone content, endocrine cell proliferation or apoptosis, or amyloid deposition. These data indicate that DAPA does not directly affect the human pancreatic islet, but rather suggest an indirect effect where lower blood glucose leads to reduced insulin secretion and a transient increase in glucagon secretion., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

β-Cell dysfunction in type 2 diabetes (T2D) is associated with loss of cellular identity and mis-expression of alternative islet hormones, including glucagon. The molecular basis for these cellular changes has been attributed to dysregulation of core β-cell transcription factors, which regulate β-cell identity through activating and repressive mechanisms. The TLE1 gene lies near a T2D susceptibility locus and, recently, the glucagon repressive actions of this transcriptional coregulator have been demonstrated in vitro. We investigated whether TLE1 expression is disrupted in human T2D, and whether this is associated with increased islet glucagon-expressing cells. Automated image analysis following immunofluorescence in donors with (n = 7) and without (n = 7) T2D revealed that T2D was associated with higher islet α/β cell ratio (Control: 0.7 ± 0.1 vs T2D: 1.6 ± 0.4; P < .05) and an increased frequency of bihormonal (insulin+/glucagon+) cells (Control: 0.8 ± 0.2% vs T2D: 2.0 ± 0.4%, P < .05). In nondiabetic donors, the majority of TLE1-positive cells were mono-hormonal β-cells (insulin+/glucagon-: 98.2 ± 0.5%; insulin+/glucagon+: 0.7 ± 0.2%; insulin-/glucagon+: 1.1 ± 0.4%; P < .001). TLE1 expression was reduced in T2D (Control: 36 ± 2.9% vs T2D: 24 ± 2.6%; P < .05). Reduced islet TLE1 expression was inversely correlated with α/β cell ratio (r = -0.55; P < .05). TLE1 knockdown in EndoC-βH1 cells was associated with a 2.5-fold increase in glucagon gene mRNA and mis-expression of glucagon in insulin-positive cells. These data support TLE1 as a putative regulator of human β-cell identity, with dysregulated expression in T2D associated with increased glucagon expression potentially reflecting β- to α-cell conversion., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Plasma amino acids and their transporters constitute an important part of the feedback loop between the liver and pancreatic α-cell function, and glucagon regulates hepatic amino acid turnover. Disruption of hepatic glucagon receptor action activates the loop and results in high plasma amino acids and hypersecretion of glucagon associated with α-cell hyperplasia. In the present study, we report a technique to rescue implanted human pancreatic islets from the mouse kidney capsule. Using this model, we have demonstrated that expression of the amino acid transporter SLC38A4 increases in α-cells after administration of a glucagon receptor blocking antibody. The increase in SLC38A4 expression and associated α-cell proliferation was dependent on mechanistic target of rapamycin pathway. We confirmed increased α-cell proliferation and expression of SLC38A4 in pancreas sections from patients with glucagon cell hyperplasia and neoplasia (GCHN) with loss-of-function mutations in the glucagon receptor. Collectively, using a technique to rescue implanted human islets from the kidney capsule in mice and pancreas sections from patients with GCHN, we found that expression of SLC38A4 was increased under conditions of disrupted glucagon receptor signaling. These data provide support for the existence of a liver-human α-cell endocrine feedback loop., (Copyright © 2019 Endocrine Society.)

Pancreatic α cells proliferate at a low rate, and little is known about the control of this process. Here we report the characterization of human α cells by large-scale, single-cell RNA sequencing coupled with pseudotime ordering. We identified two large subpopulations and a smaller cluster of proliferating α cells with increased expression of genes involved in cell-cycle regulation. The proliferating α cells were differentiated, had normal levels of GCG expression, and showed no signs of cellular stress. Proliferating α cells were detected in both the G1S and G2M phases of the cell cycle. Human α cells proliferate at a fivefold higher rate than human β cells and express lower levels of the cell-cycle inhibitors CDKN1A and CDKN1C. Collectively, this study provides the gene signatures of human α cells and the genes involved in their cell division. The lower expression of two cell-cycle inhibitors in human α cells could account for their higher rate of proliferation compared with their insulin-producing counterparts.

Upon lowered blood glucose occurring during fasting, glucagon is secreted from pancreatic islets, exerting various metabolic effects to normalize glucose levels. A considerable portion of these effects is mediated by glucagon-activated transcription factors (TFs) in liver. Glucagon directly activates several TFs via immediate cyclic adenosine monophosphate (cAMP)- and calcium-dependent signaling events. Among these TFs, cAMP response element-binding protein (CREB) is a major factor. CREB recruits histone-modifying enzymes and cooperates with other TFs on the chromatin template to increase the rate of gene transcription. In addition to direct signal transduction, the transcriptional effects of glucagon are also influenced by dynamic TF cross talk. Specifically, assisted loading of one TF by a companion TF leads to increased binding and activity. Lastly, transcriptional regulation by glucagon is also exerted by TF cascades by which a primary TF induces the gene expression of secondary TFs that bring about their activity a few hours after the initial glucagon signal. This mechanism of a delayed response may be instrumental in establishing the temporal organization of the fasting response by which distinct metabolic events separate early from prolonged fasting. In this mini-review, we summarize recent advances and critical discoveries in glucagon-dependent gene regulation with a focus on direct TF activation, dynamic TF cross talk, and TF cascades.

The secretion of glucagon by islet α cells is normally suppressed by high blood glucose, but this suppressibility is impaired in patients with diabetes or cystic fibrosis (CF), a disease caused by mutations in the gene encoding CF transmembrane conductance regulator (CFTR), a cyclic adenosine monophosphate-activated Cl- channel. However, precisely how glucose regulates glucagon release remains controversial. Here we report that elevated glucagon secretion, together with increased glucose-induced membrane depolarization and Ca2+ response, is found in CFTR mutant (DF508) mice/islets compared with the wild-type. Overexpression of CFTR in AlphaTC1-9 cells results in membrane hyperpolarization and reduced glucagon release, which can be reversed by CFTR inhibition. CFTR is found to potentiate the adenosine triphosphate-sensitive K+ (KATP) channel because membrane depolarization and whole-cell currents sensitive to KATP blockers are significantly greater in wild-type/CFTR-overexpressed α cells compared with that in DF508/non-overexpressed cells. KATP knockdown also reverses the suppressive effect of CFTR overexpression on glucagon secretion. The results reveal that by potentiating KATP channels, CFTR acts as a glucose-sensing negative regulator of glucagon secretion in α cells, a defect of which may contribute to glucose intolerance in CF and other types of diabetes., (Copyright © 2017 Endocrine Society.)

Polymorphisms in the SLC30A8 gene, which encodes the ZnT8 zinc transporter, are associated with altered susceptibility to type 2 diabetes (T2D), and SLC30A8 haploinsufficiency is protective against the development of T2D in obese humans. SLC30A8 is predominantly expressed in pancreatic islet β-cells, but surprisingly, multiple knockout mouse studies have shown little effect of Slc30a8 deletion on glucose tolerance or glucose-stimulated insulin secretion (GSIS). Multiple other Slc30a isoforms are expressed at low levels in pancreatic islets. We hypothesized that functional compensation by the Slc30a7 isoform, which encodes ZnT7, limits the impact of Slc30a8 deletion on islet function. We therefore analyzed the effect of Slc30a7 deletion alone or in combination with Slc30a8 on in vivo glucose metabolism and GSIS in isolated islets. Deletion of Slc30a7 alone had complex effects in vivo, impairing glucose tolerance and reducing the glucose-stimulated increase in plasma insulin levels, hepatic glycogen levels, and pancreatic insulin content. Slc30a7 deletion also affected islet morphology and increased the ratio of islet α- to β-cells. However, deletion of Slc30a7 alone had no effect on GSIS in isolated islets, whereas combined deletion of Slc30a7 and Slc30a8 abolished GSIS. These data demonstrate that the function of ZnT8 in islets can be unmasked by removal of ZnT7 and imply that ZnT8 may affect T2D susceptibility through actions in other tissues where it is expressed at low levels rather than through effects on pancreatic islet function.

Glucose homeostasis depends on the coordinated secretion of glucagon, insulin, and Glucagon-like peptide (GLP)-1 by pancreas and intestine. Obesity, which is associated with an increased risk of developing insulin resistance and type 2 diabetes, affects the function of these organs. Here, we investigate the functional and molecular adaptations of proglucagon-producing cells in obese mice to better define their involvement in type 2 diabetes development. We used GLU-Venus transgenic male mice specifically expressing Venus fluorochrome in proglucagon-producing cells. Mice were subjected to 16 weeks of low-fat diet or high-fat diet (HFD) and then subdivided by measuring glycated hemoglobin (HbA1c) in 3 groups: low-fat diet mice and I-HFD (glucose-intolerant) mice with similar HbA1c and H-HFD (hyperglycemic) mice, which exhibited higher HbA1c. At 16 weeks, both HFD groups exhibited similar weight gain, hyperinsulinemia, and insulin resistance. However, I-HFD mice exhibited better glucose tolerance compared with H-HFD mice. I-HFD mice displayed functional and molecular adaptations of enteroendocrine L-cells resulting in increased intestinal GLP-1 biosynthesis and release as well as maintained pancreatic α- and β-cell functions. By contrast, H-HFD mice exhibited dysfunctional L, α- and β-cells with increased β- and L-cell numbers. Administration of the GLP-1R antagonist Exendin9-39 in I-HFD mice led to hyperglycemia and alterations of glucagon secretion without changes in insulin secretion. Our results highlight the cross-talk between islet and intestine endocrine cells and indicate that a compensatory adaptation of L-cell function in obesity plays an important role in preserving glucose homeostasis through the control of pancreatic α-cell functions.

Diabetes results from inadequate β-cell number and/or function to control serum glucose concentrations so that replacement of lost β-cells could become a viable therapy for diabetes. In addition to embryonic stem cell sources for new β-cells, evidence for transdifferentiation/reprogramming of non-β-cells to functional β-cells is accumulating. In addition, de-differentiation of β-cells observed in diabetes and their subsequent conversion to α-cells raises the possibility that adult islet cell fate is malleable and controlled by local hormonal and/or environmental cues. We previously demonstrated that inactivation of the activin antagonist, follistatin-like 3 (FSTL3) resulted in β-cell expansion and improved glucose homeostasis in the absence of β-cell proliferation. We recently reported that activin directly suppressed expression of critical α-cell genes while increasing expression of β-cell genes, supporting the hypothesis that activin is one of the local hormones controlling islet cell fate and that increased activin signaling accelerates α- to β-cell transdifferentiation. We tested this hypothesis using Gluc-Cre/yellow fluorescent protein (YFP) α-cell lineage tracing technology combined with FSTL3 knockout (KO) mice to label α-cells with YFP. Flow cytometry was used to quantify unlabeled and labeled α- and β-cells. We found that Ins+/YFP+ cells were significantly increased in FSTL3 KO mice compared with wild type littermates. Labeled Ins+/YFP+ cells increased significantly with age in FSTL3 KO mice but not wild type littermates. Sorting results were substantiated by counting fluorescently labeled cells in pancreatic sections. Activin treatment of isolated islets significantly increased the number of YFP+/Ins+ cells. These results suggest that α- to β-cell transdifferentiation is influenced by activin signaling and may contribute substantially to β-cell mass.

Glucagon and α-cell dysfunction are critical in the development of hyperglycemia during diabetes both in humans and rodents. We hypothesized that α-cell dysfunction leading to dysregulated glucagon secretion in diabetes is due to both a lack of insulin and intrinsic defects. To characterize α-cell dysfunction in diabetes, we used glucagon-Venus transgenic male mice and induced insulinopenic hyperglycemia by streptozotocin administration leading to alterations of glucagon secretion. We investigated the in vivo impact of insulinopenic hyperglycemia on glucagon-producing cells using FACS-sorted α-cells from control and diabetic mice. We demonstrate that increased glucagonemia in diabetic mice is mainly due to increases of glucagon release and biosynthesis per cell compared with controls without changes in α-cell mass. We identified genes coding for proteins involved in glucagon biosynthesis and secretion, α-cell differentiation, and potential stress markers such as the glucagon, Arx, MafB, cMaf, Brain4, Foxa1, Foxa3, HNF4α, TCF7L2, Glut1, Sglt2, Cav2.1, Cav2.2, Nav1.7, Kir6.2/Sur1, Pten, IR, NeuroD1, GPR40, and Sumo1 genes, which were abnormally regulated in diabetic mice. Importantly, insulin treatment partially corrected α-cell function and expression of genes coding for proglucagon, or involved in glucagon secretion, glucose transport and insulin signaling but not those coding for cMAF, FOXA1, and α-cell differentiation markers as well as GPR40, NEUROD1, CAV2.1, and SUMO1. Our results indicate that insulinopenic diabetes induce marked α-cell dysfunction and molecular alteration, which are only partially corrected by in vivo insulin treatment.

TGFβ superfamily ligands, receptors, and second messengers, including activins A and B, have been identified in pancreatic islets and proposed to have important roles regulating development, proliferation, and function. We previously demonstrated that Fstl3 (an antagonist of activin activity) null mice have larger islets with β-cell hyperplasia and improved glucose tolerance and insulin sensitivity in the absence of altered β-cell proliferation. This suggested the hypothesis that increased activin signaling influences β-cell expansion by destabilizing the α-cell phenotype and promoting transdifferentiation to β-cells. We tested the first part of this hypothesis by treating α- and β-cell lines and sorted mouse islet cells with activin and related ligands. Treatment of the αTC1-6 α cell line with activins A or B suppressed critical α-cell gene expression, including Arx, glucagon, and MafB while also enhancing β-cell gene expression. In INS-1E β-cells, activin A treatment induced a significant increase in Pax4 (a fate determining β-cell gene) and insulin expression. In sorted primary islet cells, α-cell gene expression was again suppressed by activin treatment in α-cells, whereas Pax4 was enhanced in β-cells. Activin treatment in both cell lines and primary cells resulted in phosphorylated mothers against decapentaplegic-2 phosphorylation. Finally, treatment of αTC1-6 cells with activins A or B significantly inhibited proliferation. These results support the hypothesis that activin signaling destabilized the α-cell phenotype while promoting a β-cell fate. Moreover, these results support a model in which the β-cell expansion observed in Fstl3 null mice may be due, at least in part, to enhanced α- to β-cell transdifferentiation.

In rodent models of type 1 diabetes, leptin administration into brain ventricles normalizes blood glucose at doses that have no effect when given peripherally. The ventromedial nucleus of the hypothalamus (VMN) is a potential target for leptin's antidiabetic effects because leptin-sensitive neurons in this brain area are implicated in glucose homeostasis. To test this hypothesis, we injected leptin directly into the bilateral VMN of rats with streptozotocin-induced uncontrolled diabetes mellitus. This intervention completely normalized both hyperglycemia and the elevated rates of hepatic glucose production and plasma glucagon levels but had no effect on tissue glucose uptake in the skeletal muscle or brown adipose tissue as measured using tracer dilution techniques during a basal clamp. To determine whether VMN leptin signaling is required for leptin-mediated normalization of diabetic hyperglycemia, we studied mice in which the leptin receptor gene was deleted in VMN steroidogenic factor 1 neurons using cre-loxP technology. Our findings indicate leptin action within these neurons is not required for the correction of diabetic hyperglycemia by central leptin infusion. We conclude that leptin signaling in the VMN is sufficient to mediate leptin's antidiabetic action but may not be necessary for this effect. Leptin action within a distributed neuronal network may mediate its effects on glucose homeostasis.

Chronic malnutrition leads to multiple changes in β-cell function and peripheral insulin actions to adapt glucose homeostasis to these restricted conditions. However, despite glucose homeostasis also depends on glucagon effects, the role of α-cells in malnutrition is largely unknown. Here, we studied α-cell function and hepatic glucagon signaling in mice fed with low-protein (LP) or normal-protein diet for 8 wk after weaning. Using confocal microscopy, we found that inhibition of Ca²⁺ signaling by glucose was impaired in α-cells of LP mice. Consistent with these findings, the ability of glucose to inhibit glucagon release in isolated islets was also diminished in LP mice. This altered secretion was not related with changes in either glucagon gene expression or glucagon content. A morphometric analysis showed that α-cell mass was significantly increased in malnourished animals, aspect that was probably related with their enhanced plasma glucagon levels. When we analyzed the hepatic function, we observed that the phosphorylation of protein kinase A and cAMP response-binding element protein in response to fasting or exogenous glucagon was impaired in LP mice. Additionally, the up-regulated gene expression in response to fasting observed in the hepatic glucagon receptor as well as several key hepatic enzymes, such as peroxisome proliferator-activated receptor γ, glucose-6-phosphatase, and phosphoenolpyruvate carboxykinase, was altered in malnourished animals. Finally, liver glycogen mobilization in response to fasting and the ability of exogenous glucagon to raise plasma glucose levels were lower in LP mice. Therefore, chronic protein malnutrition leads to several alterations in both the α-cell function and hepatic glucagon signaling.

This study investigated in a pancreatic alpha-cell line the effects of chronic exposure to palmitate on the insulin and IGF-I receptor (IGF-IR) and intracellular insulin pathways. alpha-TC1-6 cells were cultured in the presence or absence of palmitate (0.5 mmol/liter) up to 48 h. Glucagon secretion, insulin and IGF-IR autophosphorylation, and insulin receptor substrate (IRS)-1, IRS-2, phosphatidylinositol kinase (PI3K) (p85 alpha), and serine-threonine protein kinase (Akt) phosphorylated (active) forms were measured. Erk 44/42 and p38 phosphorylation (P) (MAPK pathway markers) were also measured. Because MAPK can regulate Pax6, a transcription factor that controls glucagon expression, paired box gene 6 (Pax6) and glucagon gene and protein expression were also measured. Basal glucagon secretion was increased and the inhibitory effect of acute insulin exposure reduced in alpha-TC1 cells cultured with palmitate. Insulin-stimulated insulin receptor phosphorylation was greatly reduced by exposure to palmitate. Similar results were observed with IRS-1-P, PI3K (p85 alpha), and Akt-P. In contrast, with IGF-IR and IRS-2-P, the basal levels (i.e. in the absence of insulin stimulation) were higher in cells cultured with palmitate. Similar data were obtained with Erk 44/42-P and p-38-P. Pax6 and glucagon gene and protein expression were higher in cells cultured with palmitate. In these cells cultured, specifics MAPKs inhibitors were able to reduce both Pax6 and glucagon gene and protein expression. These results indicate that alpha-cells exposed to palmitate show insulin resistance of the IRS-1/PI3K/Akt pathway that likely controls glucagon secretion. In contrast, the IRS-2/MAPKs pathway is stimulated, through an activation of the IGF-IR, leading to increased Pax6 and glucagon expression. Our data support the hypothesis that the chronic elevation of fatty acids contribute to alpha-cell dysregulation frequently observed in type 2 diabetes.

The pancreatic alpha- and beta-cells are critical components in regulating blood glucose homeostasis via secretion of glucagon and insulin, respectively. Both cell types are typically localized in the islets of Langerhans. However, little is known about the roles of paracrine interactions that contribute to their physiological functions. The lack of suitable cell lines to study alpha- and beta-cells interactions have led us to develop an alpha-cell-specific Cre-expressing transgenic line utilizing a glucagon promoter sequence, the Glu-Cre transgenic mouse. Here, we demonstrate that the Glu-Cre could specifically and efficiently excise floxed target genes in adult islet alpha-cells. We further showed that deletion of the tumor suppressor gene, multiple endocrine neoplasia type 1 (Men1), in alpha-cells led to tumorigenesis. However, to our surprise, the lack of Men1 in alpha-cells did not result in glucagonomas but rather beta-cell insulinomas. Because deletion of the Men1 alleles was only present in alpha-cells, our data suggested that cross communication between alpha- and beta-cells contributes to tumorigenesis in the absence of Men1. Together, we believed that the new model systems described here will allow future studies to decipher cellular interactions between islet alpha- and beta-cells in a physiological context.

Recent evidence supports the idea that insulin signaling through the insulin receptor substrate/phosphatidyl-inositol 3-kinase/Akt pathway is involved in the maintenance of beta-cell mass and function. We previously identified the insulin-response element binding protein-1 (IRE-BP1) as an effector of insulin-induced Akt signaling in the liver, and showed that the 50-kDa carboxyl fragment confers the transcriptional activity of this factor. In this investigation we found that IRE-BP1 is expressed in the alpha, beta, and delta-cells of the islets of Langerhans, and is localized to the cytoplasm in beta-cells in normal rats, but is reduced and redistributed to the islet cell nuclei in obese Zucker rats. To test whether IRE-BP1 modulates beta-cell function and insulin secretion, we used the rat insulin II promoter to drive expression of the carboxyl fragment in beta-cells. Transgenic expression of IRE-BP1 in FVB mice increases nuclear IRE-BP1 expression, and produces a phenotype similar to that of type 2 diabetes, with hyperinsulinemia, hyperglycemia, and increased body weight. IRE-BP1 increased islet type I IGF receptor expression, potentially contributing to the development of islet hypertrophy. Our findings suggest that increased gene transcription mediated through IRE-BP1 may contribute to beta-cell dysfunction in insulin resistance, and allow for the hypothesis that IRE-BP1 plays a role in the pathophysiology of type 2 diabetes.

The effect of long-chain free fatty acids on glucagon secretion from islet alpha-cells has been a controversial issue. This study examined direct effects of oleic acid (OA) on glucagon release from rat pancreatic islets and on cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) in single alpha-cells by fura-2 fluorescence imaging. OA at 30 microM increased glucagon release from isolated islets in the presence of low (2.8 mM) and elevated (8.3 mM) glucose concentrations. OA at 6-10 microm concentration-dependently increased [Ca(2+)](i) in alpha-cells, irrespective of glucose concentrations (1.4, 2.8, and 8.3 mM). OA at 10 mum increased [Ca(2+)](i) in 90% of alpha-cells. OA-induced [Ca(2+)](i) increases were strongly inhibited by the endoplasmic reticulum Ca(2+)-pump inhibitors cyclopiazonic acid and thapsigargin and by 2-aminoethoxydiphenyl borate, the blocker of both inositol 1,4,5-trisphosphate receptors and store-operated Ca(2+) channels. Furthermore, the amplitude, but not incidence, of OA-induced [Ca(2+)](i) increases was reduced substantially by Ca(2+)-free conditions and mildly by an L-type Ca(2+) channel blocker, nitrendipine, and an ATP-sensitive K(+) channel activator, diazoxide. OA-induced glucagon release was also inhibited mildly by nitrendipine and strongly by 2-aminoethoxydiphenyl borate. These results indicate that OA glucose-independently stimulates glucagon release by increasing [Ca(2+)](i) in rat pancreatic alpha-cells and that the [Ca(2+)](i) increase is triggered by Ca(2+) release from endoplasmic reticulum and amplified by Ca(2+) influx possibly via store-operated channels and via voltage-dependent L-type Ca(2+) channels. The glucose-independent action of OA to stimulate glucagon release from alpha-cells may operate under hypoglycemic conditions when plasma free fatty acids levels are elevated, possibly playing a role in maintaining glucose metabolism.

Pancreatic alpha-cells secrete glucagon in response to low glucose to counter insulin actions, thereby maintaining glucose homeostasis. The molecular basis of alpha-cell stimulus-secretion coupling has not been fully elucidated. We investigated the expression of voltage-gated K(+) (K(V)) and Ca(2+) (Ca(V)) channels, and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in pancreatic alpha-cells and examined their targeting to specialized cholesterol-rich lipid rafts. In alpha-cells, we detected the expression of K(V)4.1/4.3 (A-type current), K(V)3.2/3.3 (delayed rectifier current), Ca(V)1.2 (L-type current), Ca(V)2.2 (N-type current), and the SNARE (synaptosomal-associated protein of 25 kDa, syntaxin 1A, and vesicle-associated membrane protein 2) and SNARE-associated proteins (Munc-13-1 and Munc-18a). We also detected caveolin-2, a structural protein of cholesterol-rich lipid rafts. Of these proteins, caveolin-2, K(V)4.1/4.3, Ca(V)1.2, and SNARE proteins (syntaxin 1A, synaptosomal-associated protein of 25 kDa, and vesicle-associated membrane protein 2) target to lipid raft domains on alpha-cell plasma membranes. Disruption of lipid rafts by depletion of membrane cholesterol with methyl-beta-cyclodextrin decreased the association of K(V)4.1/4.3, Ca(V)1.2, and SNARE proteins with lipid rafts. This resulted in inhibition of A-type K(V) currents and enhancement of glucagon secretion from alpha-cells. Consistently, capacitance measurements of exocytosis of single alpha-cells showed enhanced exocytosis after membrane cholesterol depletion. Taken together, our results demonstrate the association of K(V)4, Ca(V)1.2, and SNARE proteins with lipid rafts in pancreatic alpha-cells. Glucagon secretion from alpha-cells is regulated by lipid rafts, and the dissociation of SNARE proteins from cholesterol-rich lipid raft domains enhances glucagon secretion.

Glucagon is a potent counterregulatory hormone that opposes the action of insulin in controlling glycemia. The cellular mechanisms by which pancreatic alpha-cell glucagon secretion occurs in response to hypoglycemia are poorly known. SUR1/K(IR)6.2-type ATP-sensitive K(+) (K(ATP)) channels have been implicated in the glucagon counterregulatory response at central and peripheral levels, but their role is not well understood. In this study, we examined hypoglycemia-induced glucagon secretion in vitro in isolated islets and in vivo using Sur1KO mice lacking neuroendocrine-type K(ATP) channels and paired wild-type (WT) controls. Sur1KO mice fed ad libitum have normal glucagon levels and mobilize hepatic glycogen in response to exogenous glucagon but exhibit a blunted glucagon response to insulin-induced hypoglycemia. Glucagon release from Sur1KO and WT islets is increased at 2.8 mmol/liter glucose and suppressed by increasing glucose concentrations. WT islets increase glucagon secretion approximately 20-fold when challenged with 0.1 mmol/liter glucose vs. approximately 2.7-fold for Sur1KO islets. Glucagon release requires Ca(2+) and is inhibited by nifedipine. Consistent with a regulatory interaction between K(ATP) channels and intra-islet zinc-insulin, WT islets exhibit an inverse correlation between beta-cell secretion and glucagon release. Glibenclamide stimulated insulin secretion and reduced glucagon release in WT islets but was without effect on secretion from Sur1KO islets. The results indicate that loss of alpha-cell K(ATP) channels uncouples glucagon release from inhibition by beta-cells and reveals a role for K(ATP) channels in the regulation of glucagon release by low glucose.

In isolated rat pancreatic alpha-cells, glucose, arginine, and the sulfonylurea tolbutamide stimulated glucagon release. The effect of glucose was abolished by the KATP-channel opener diazoxide as well as by mannoheptulose and azide, inhibitors of glycolysis and mitochondrial metabolism. Glucose inhibited KATP-channel activity by 30% (P<0.05; n=5) and doubled the free cytoplasmic Ca2+ concentration. In cell-attached recordings, azide opened KATP channels. The N-type Ca2+-channel blocker omega-conotoxin and the Na+-channel blocker tetrodotoxin inhibited glucose-induced glucagon release whereas tetraethylammonium, a blocker of delayed rectifying K+ channels, increased secretion. Glucagon release increased monotonically with increasing K+ concentrations. omega-Conotoxin suppressed glucagon release to 15 mM K+, whereas a combination of omega-conotoxin and an L-type Ca2+-channel inhibitor was required to abrogate secretion in 50 mM K+. Recordings of cell capacitance revealed that glucose increased the exocytotic response evoked by membrane depolarization 3-fold. This correlated with a doubling of glucagon secretion by glucose in intact rat islets exposed to diazoxide and high K+. In whole-cell experiments, exocytosis was stimulated by reducing the cytoplasmic ADP concentration, whereas changes of the ATP concentration in the physiological range had little effect. We conclude that glucose stimulates glucagon release from isolated rat alpha-cells by KATP-channel closure and stimulation of Ca2+ influx through N-type Ca2+ channels. Glucose also stimulated exocytosis by an amplifying mechanism, probably involving changes in adenine nucleotides. The stimulatory action of glucose in isolated alpha-cells contrasts with the suppressive effect of the sugar in intact islets and highlights the primary importance of islet paracrine signaling in the regulation of glucagon release.