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The ability of glucose to stimulate insulin secretion from the pancreatic islets of Langerhans is enhanced by the intestinal hormone glucagon-like peptide 1 (GLP-1), which is secreted from the gut in response to nutrient ingestion. This action, called the incretin effect, accounts for as much as half of the postprandial insulin response and is exploited therapeutically for diabetes treatment through the use of incretin mimetic drugs and inhibitors of dipeptidyl peptidase 4, which degrades GLP-1. Despite a prominent role for incretin mimetics in diabetes treatment, several key questions remain about GLP-1-induced insulin secretion. Most studies have examined the effects of GLP-1 at concentrations several orders of magnitude higher than those found in vivo; therefore, one might question the physiological (and perhaps even pharmacological) relevance of pathways identified in these studies and whether other important mechanisms might have been obscured. In this issue of theJCI, Shigeto and colleagues demonstrate that physiological GLP-1 does indeed amplify the insulin secretory response. Intriguingly, while much of this response is PKA dependent, as might be expected, the use of picomolar GLP-1 reveals a new and important mechanism that contributes to GLP-1-induced insulin secretion., GLP-1 amplification of glucose-stimulated insulin secretion It is well established that the incretin hormone glucagon-like peptide 1 (GLP-1) amplifies the insulin secretory response to glucose (1, 2). Specifically, nutrient sensing [...]

A maternal diet that is low in protein increases the susceptibility of offspring to type 2 diabetes by inducing long-term alterations in β cell mass and function. Nutrients and growth factor signaling converge through mTOR, suggesting that this pathway participates in β cell programming during fetal development. Here, we revealed that newborns of dams exposed to low-protein diet (LP0.5) throughout pregnancy exhibited decreased insulin levels, a lower β cell fraction, and reduced mTOR signaling. Adult offspring of LP0.5-exposed mothers exhibited glucose intolerance as a result of an insulin secretory defect and not β cell mass reduction. The β cell insulin secretory defect was distal to glucose-dependent [Ca.sup.2+] influx and resulted from reduced proinsulin biosynthesis and insulin content. Islets from offspring of LP0.5-fed dams exhibited reduced mTOR and increased expression of a subset of microRNAs, and blockade of microRNA-199a-3p and-342 in these islets restored mTOR and insulin secretion to normal. Finally, transient β cell activation of mTORC1 signaling in offspring during the last week of pregnancy of mothers fed a LP0.5 rescued the defect in the neonatal β cell fraction and metabolic abnormalities in the adult. Together, these findings indicate that a maternal low-protein diet alters microRNA and mTOR expression in the offspring, influencing insulin secretion and glucose homeostasis., Introduction The pervasiveness of type 2 diabetes (T2D) is a major public health concern worldwide. The elevated prevalence of this disease results in part from an increased rate of obesity [...]

Dysfunctional microRNA (miRNA) networks contribute to inappropriate responses following pathological stress and are the underlying cause of several disease conditions. In pancreatic β cells, miRNAs have been largely unstudied and little is known about how specific miRNAs regulate glucose-stimulated insulin secretion (GSIS) or impact the adaptation of β cell function to metabolic stress. In this study, we determined that miR-7 is a negative regulator of GSIS in β cells. Using Mir7a2 deficient mice, we revealed that miR-7a2 regulates β cell function by directly regulating genes that control late stages of insulin granule fusion with the plasma membrane and ternary SNARE complex activity. Transgenic mice overexpressing miR-7a in β cells developed diabetes due to impaired insulin secretion and β cell dedifferentiation. Interestingly, perturbation of miR-7a expression in β cells did not affect proliferation and apoptosis, indicating that miR-7 is dispensable for the maintenance of endocrine β cell mass. Furthermore, we found that miR-7a levels are decreased in obese/ diabetic mouse models and human islets from obese and moderately diabetic individuals with compensated β cell function. Our results reveal an interconnecting miR-7 genomic circuit that regulates insulin granule exocytosis in pancreatic β cells and support a role for miR-7 in the adaptation of pancreatic β cell function in obesity and type 2 diabetes., Introduction Type 2 diabetes (T2D) results from an inability of β cells to secrete sufficient insulin to meet the increased metabolic demands associated with peripheral insulin resistance (1). Genetic and [...]

Liver glucose metabolism plays a central role in glucose homeostasis and may also regulate feeding and energy expenditure. Here we assessed the impact of glucose transporter 2 (Glut2) gene inactivation [...]

PPARβ/δ protects against obesity by reducing dyslipidemia and insulin resistance via effects in muscle, adipose tissue, and liver. However, its function in pancreas remains ill defined. To gain insight into its hypothesized role in βcell function, we specifically deleted Pparb/d in the epithelial compartment of the mouse pancreas. Mutant animals presented increased numbers of islets and, more importantly, enhanced insulin secretion, causing hyperinsulinemia. Gene expression profiling of pancreatic βcells indicated a broad repressive function of PPARβ/δ affecting the vesicular and granular compartment as well as the actin cytoskeleton. Analyses of insulin release from isolated PPARβ/δ-deficient islets revealed an accelerated second phase of glucose-stimulated insulin secretion. These effects in PPARβ/δ-deficient islets correlated with increased filamentous actin (F-actin) disassembly and an elevation in protein kinase D activity that altered Golgi organization. Taken together, these results provide evidence for a repressive role for PPARβ/δ in β cell mass and insulin exocytosis, and shed a new light on PPARβ/δ metabolic action., Introduction High plasma glucose levels due to insulin resistance and inadequate insulin production characterize type 2 diabetes. Generally considered an adult illness, it is now often diagnosed in teenagers in [...]

Type 1 diabetes develops when most insulin-producing β cells of the pancreas are killed by an autoimmune attack. The in vivo conditions modulating the sensitivity and resistance of β cells to this attack remain largely obscure. Here, we show that connexin 36 (Cx36), a trans-membrane protein that forms gap junctions between β cells in the pancreatic islets, protects mouse β cells against both cytotoxic drugs and cytokines that prevail in the islet environment at the onset of type 1 diabetes. We documented that this protection was at least partially dependent on intercellular communication, which Cx36 and other types of connexin channels establish within pancreatic islets. We further found that proinflammatory cytokines decreased expression of Cx36 and that experimental reduction or augmentation of Cx36 levels increased or decreased β cell apoptosis, respectively. Thus, we conclude that Cx36 is central to β cell protection from toxic insults., Introduction Pancreatic β cells are functionally heterogeneous (1) and interconnected by channels made of connexin 36 (Cx36) (2-4), a member of the connexin (Cx) protein family (5). Previous studies have [...]

Reduced bone morphogenetic protein receptor 2 (BMPR2) expression in patients with pulmonary arterial hypertension (PAH) can impair pulmonary arterial EC (PAEC) function. This can adversely affect EC survival and promote SMC proliferation. We hypothesized that interventions to normalize expression of genes that are targets of BMPR2 signaling could restore PAEC function and prevent or reverse PAH. Here we have characterized, in human PAECs, a BMPR2-mediated transcriptional complex between PPAR Υ and β-catenin and shown that disruption of this complex impaired BMP-mediated PAEC survival. Using whole genome-wide ChIP-Chip promoter analysis and gene expression microarrays, we delineated PPARΥ/β-catenin-dependent transcription of target genes including APLN, which encodes apelin. We documented reduced PAEC expression of apelin in PAH patients versus controls. In cell culture experiments, we showed that apelin-deficient PAECs were prone to apoptosis and promoted pulmonary arterial SMC (PASMC) proliferation. Conversely, we established that apelin, like BMPR2 ligands, suppressed proliferation and induced apoptosis of PASMCs. Consistent with these functions, administration of apelin reversed PAH in mice with reduced production of apelin resulting from deletion of PPARΥ in ECs. Taken together, our findings suggest that apelin could be effective in treating PAH by rescuing BMPR2 and PAEC dysfunction., Introduction Pulmonary arterial hypertension (PAH) is characterized by a progressive increase in pulmonary vascular resistance (PVR) that culminates in right-sided heart failure. There is no cure for this condition, and [...]

Pemphigus vulgaris (PV) is a severe autoimmune disease involving blistering of the skin and mucous mebranes. It is caused by autoantibodies against desmoglein 3 (Dsg3), an adhesion molecule critical for maintaining epithelial integrity in the skin, oral mucosa, and esophagus. Knowing the antigen targeted by the autoantibodies renders PV a valuable model of autoimmunity. Recently, a role for Dsg3-specific CD4+ T helper cells in autoantibody production was demonstrated in a mouse model of PV, but whether these cells exert cytotoxicity in the tissues is unclear. Here, we analyzed 3 Dsg3-specific TCRs using transgenic mice and retrovirus induction. Dsg3-specific transgenic (Dsg3H1) T cells underwent deletion in the presence of Dsg3 in vivo. Dsg3H1 T cells that developed in the absence of Dsg3 elicited a severe pemphigus-like phenotype when cotransferred into immunodeficient mice with B cells from Dsg3-/- mice. Strikingly, in addition to humoral responses, T cell infiltration of Dsg3-expressing tissues led to interface dermatitis, a distinct form of T cell-mediated autoimmunity that causes keratinocyte apoptosis and is seen in various inflammatory/autoimmune skin diseases, including paraneoplastic pemphigus. The use of retrovirally generated Dsg3-specific T cells revealed that interface dermatitis occurred in an IFN -γ- and TCR avidity-dependent manner. This model of autoimmunity demonstrates that T cells specific for a physiological skin-associated autoantigen are capable of inducing interface dermatitis and should provide a valuable tool for further exploring the immunopathophysiology of T cell-mediated skin diseases., Introduction Desmoglein 3 (Dsg3) is a cadherin-type glycoprotein expressed in stratified squamous epithelium, including the skin, oral mucosa, and esophagus, and it plays a critical role in cell-cell adhesion. Dsg3 [...]

Despite intense efforts over the past 30 years, human pancreatic β cell lines have not been available. Here, we describe a robust technology for producing a functional human β cell line using targeted oncogenesis in human fetal tissue. Human fetal pancreatic buds were transduced with a lentiviral vector that expressed SV40LT under the control of the insulin promoter. The transduced buds were then grafted into SCID mice so that they could develop into mature pancreatic tissue. Upon differentiation, the newly formed SV40LT-expressing β cells proliferated and formed insulinomas. The resulting β cells were then transduced with human telomerase reverse transcriptase (hTERT), grafted into other SCID mice, and finally expanded in vitro to generate cell lines. One of these cell lines, EndoC-βH1, expressed many β cell-specific markers without any substantial expression of markers of other pancreatic cell types. The cells secreted insulin when stimulated by glucose or other insulin secretagogues, and cell transplantation reversed chemically induced diabetes in mice. These cells represent a unique tool for large-scale drug discovery and provide a preclinical model for cell replacement therapy in diabetes. This technology could be generalized to generate other human cell lines when the cell type-specific promoter is available., Introduction Pancreatic β cells are highly specialized endocrine cells that produce, store, and secrete insulin, the only physiological hypoglycemic hormone in the body. Clusters of such cells, which make up [...]

The worldwide prevalence of type 2 diabetes (T2D), which is caused by a combination of environmental and genetic factors, is increasing. With regard to genetic factors, variations in the gene encoding Cdk5 regulatory associated protein 1-like 1 (Cdkal1) have been associated with an impaired insulin response and increased risk of T2D across different ethnic populations, but the molecular function of this protein has not been character-ized. Here, we show that Cdkal1 is a mammalian methylthiotransferase that biosynthesizes 2-methylthio-[N.sup.6]-threonylcarbamoyladenosine (m [s.sup.2] [t.sup.6] A) in [tRNA.sup.Lys] (UUU) and that it is required for the accurate translation of AAA and AAG codons. Mice with pancreatic β cell-specific KO of Cdkal1 (referred to herein as β cell KO mice) showed pancreatic islet hypertrophy, a decrease in insulin secretion, and impaired blood glucose control. In Cdkal1-deficient β cells, misreading of Lys codon in proinsulin occurred, resulting in a reduction of glucose-stimulated proinsulin synthesis. Moreover, expression of ER stress-related genes was upregulated in these cells, and abnormally structured ER was observed. Further, the β cell KO mice were hypersensitive to high fat diet-induced ER stress. These findings suggest that glucose-stimulated translation of proinsulin may require fully modified [tRNA.sup.Lys] (UUU), which could potentially explain the molecular pathogenesis of T2D in patients carrying cdkal1 risk alleles., Introduction Type 2 diabetes (T2D) is caused by a combination of genetic and environmental factors. Recent advances in whole-genome association studies have identified a number of genetic variations associated with [...]

In pancreatic [beta] cells, ERK1 and ERK2 participate in nutrient sensing, and their activities rise and fall as a function of glucose concentration over the physiologic range. Glucose metabolism triggers calcium influx and release of calcium from intracellular stores to activate ERK1/2. Calcium influx also activates the calcium-dependent phosphatase calcineurin, which is required for maximal ERK1/2 activation by glucose. Calcineurin controls insulin gene expression by ERK1/2-dependent and -independent mechanisms. Here, we show that, in [beta] cells, glucose activates the ERK1/2 cascade primarily through B-Raf. Glucose activation of B-Raf, like that of ERK1/2, is calcineurin-sensitive. Calcineurin binds to B-Raf in both unstimulated and stimulated cells. We show that B-Raf is a calcineurin substrate; among calcineurin target residues on B-Raf is T401, a site of negative feedback phosphorylation by ERK1/2. Blocking calcineurin activity in [beta] cells prevents dephosphorylation of B-Raf T401 and decreases B-Raf and ERK1/2 activities. We conclude that the major calcineurin-dependent event in glucose sensing by ERK1/2 is the activation of B-Raf. diabetes | dimerization | scaffold doi/ 10.1073/pnas.1016630108

Three-amino-acid-loop-extension (TALE) homeodomain proteins including Meis and Pbx families are generally recognized for their roles in growth and differentiation during vertebrate embryogenesis and tumorigenesis. Whereas genetic studies indicate that Pbx1 regulates the development and function of insulin-producing pancreatic [beta]-cells, the role of Meis family members in [beta]-cells is still unknown. Here we show that Meis3 is abundantly expressed in pancreatic islets and [beta]-cells and that it regulates [beta]-cell survival. We further identify the 3-phosphoinositide--dependent protein kinase 1 (PDK1), a well-known kinase involved in the PI3K--Akt signaling pathway, as a direct Meis3 target, which mediates its role in [beta]-cell survival. This regulatory module appears to function broadly as we also identify Meis3 regulation of cell survival and PDK1 expression in ovarian carcinoma cells, suggesting a unique function for Meis3 beyond the traditional roles for TALE homeodomain factors during embryogenesis. diabetes | transcription | pancreas | apoptosis doi/ 10.1073/pnas.1007001107

The CREB-specific coactivator TORC2 (also known as CRTC2) upregulates gluconeogenic gene expression in the liver. Salt-inducible kinase (SIK) family enzymes inactivate TORC2 through phosphorylation and localize it in the cytoplasm. [Ser.sup.171] and [Ser.sup.275] were found to be phosphorylated in pancreatic [beta]-cells. Calcineurin (Cn) is proposed as the [Ser.sup.275] phosphatase, because its inhibitor cyclosporin A (CsA) stabilizes phospho-[Ser.sup.275] and retains TORC2 in the cytoplasm. Because the regulation of dephosphorylation at [Ser.sup.171] has not been fully clarified, we performed experiments with a range of doses of okadaic acid (OA), an inhibitor of PP2A/PP1, and with overexpression of various phosphatases and found that PP1 functions as an activator for TORC2, whereas PP2A acts as an inhibitor. In further studies using TORC2 mutants, we detected a disassociation between the intracellular distribution and the transcription activity of TORC2. Additional mutant analyses suggested the presence of a third phosphorylation site, [Ser.sup.307]. The [Ser.sup.307]-disrupted TORC2 was constitutively localized in the nucleus, but its coactivator activity was normally suppressed by SIK1 in COS-7 cells. CsA, but not OA, stabilized the phosphogroup at [Ser.sup.307], suggesting that differential dephosphorylation at [Ser.sup.171] and [Ser.sup.307] cooperatively regulate TORC2 activity and that the nuclear localization of TORC2 is insufficient to function as a coactivator. Because the COS-7 cell line may not possess signaling cascades for gluconeogenic programs, we next examined the importance of [Ser.sup.307] and [Ser.sup.171] for TORC2's function in mouse liver. Levels of phosphorylation at [Ser.sup.171] and [Ser.sup.307] changed in response to fasting or fed conditions and insulin resistance of the mouse liver, which were modified by treatment with CsA/OA and by overexpression of PP1/PP2A/Cn. These results suggest that multiple phosphorylation sites and their phosphatases may play important roles in regulating TORC2/CREB-mediated gluconeogenic programs in the liver. cAMP response element-binding protein; transducer of regulated cAMP response element-binding protein activity 2; nuclear export; salt-inducible kinase doi: 10.1152/ajpendo.00525.2009.

High-content screening for small-molecule inducers of insulin expression identified the compound BRD7389, which caused a-cells to adopt several morphological and gene expression features of a [beta]-cell state. Assay-performance profile analysis suggests kinase inhibition as a mechanism of action, and we show that biochemical and cellular inhibition of the RSK kinase family by BRD7389 is likely related to its ability induce a [beta]-cell-like state. BRD7389 also increases the endocrine cell content and function of donor human pancreatic islets in culture. BRD7389 | pancreatic islets | Rsk kinase | transdifferentiation | beta cells doi/ 10.1073/pnas.1010018107

We report localization of a cytosolic protein histidine phosphatase (PHP; ~16 kDa) in INS 832/13 cells, normal rat islets, and human islets, siRNA-mediated knockdown of PHP markedly reduced glucose- or mitochondrial fuel-induced but not KCl-induced insulin secretion, siRNA-mediated knockdown of PHP also attenuated mastoparan-induced insulin secretion, suggesting its participation in G protein-sensitive signaling steps, leading to insulin secretion. Functional assays revealed that the [beta]-cell PHP catalyzes the dephosphorylation of ATP-citrate lyase (ACL). Silencing of PHP expression markedly reduced ACL activity, suggesting functional regulation of ACL by PHP in [beta]-cells. Coimmunoprecipitation studies revealed modest effects of glucose on the interaction between PHP and ACL. Confocal microscopic evidence indicated that glucose promotes association between ACL and nm23-H1, a known kinase histidine kinase, but not between PHP and ACL. Furthermore, metabolic viability of INS 832/13 cells was resistant to siRNA-PHP, suggesting no regulatory roles of PHP in cell viability. Finally, long-term exposure (24 h) of INS 832/13 cells or rat islets to high glucose (30 mM) increased the expression of PHP. Such increases in PHP expression were also seen in islets derived from the Zucker diabetic fatty rat compared with islets from the lean control animals. Together, these data implicate regulatory roles for PHP in a G protein-sensitive step involved in nutrient-induced insulin secretion. In light of the current debate on putative regulatory roles of ACL in insulin secretion, additional studies are needed to precisely identify the phosphoprotein substrate(s) for PHP in the cascade of events leading to nutrient-induced insulin secretion. nm23-H1; adenosine 5'-triphosphate-citrate lyase doi: 10.1152/ajpendo.00091.2010.

Studies in various types of cells find that, on average, each mitochondrion becomes involved in a fusion event every 15 min, depending on the cell type. As most contact events do not result in mitochondrial fusion, it is expected that properties of the individual mitochondrion determine the likelihood of a fusion event. However, apart from membrane potential, the properties that influence the likelihood of entering a fusion event are not known. Here, we tag and track individual mitochondria in H9c2, INS1, and primary [beta]-cells and determine the biophysical properties that increase the likelihood of a fusion event. We found that the probability for fusion is independent of contact duration and organelle dimensions, but it is influenced by organelle motility. Furthermore, the history of a previous fusion event of the individual mitochondrion influenced both the likelihood for a subsequent fusion event, as well as the site on the mitochondrion at which the fusion occurred. These observations unravel the specific properties that distinguish mitochondria that will enter fusion events from the ones that will not. Altogether, these properties may help to elucidate the molecular mechanisms that regulate fusion at the level of the single mitochondrion. mitochondrial fusion and fisson; mitochondrial movement; H9c2 cells; photoactivatable green fluorescent protein doi: 10.1152/ajpcell.00427.2009.

Two pathways control glucose-induced insulin secretion (IS) by [beta]-cells. The triggering pathway involves ATP-sensitive potassium (KATe) channel-dependent depolarization, [Ca.sup.2+] influx, and a rise in the cytosolic [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.c]), which triggers exocytosis of insulin granules. The metabolic amplifying pathway augments IS without further increasing [[[Ca.sup.2+]].sub.c]. The underlying mechanisms are unknown. Here, we tested the hypothesis that amplification implicates actin microfilaments. Mouse islets were treated with latrunculin B and cytochalasin B to depolymerize actin or jasplakinolide to polymerize actin. They were then perifused to measure [[[Ca.sup.2+]].sub.c] and IS. Metabolic amplification was studied during imposed steady elevation of [[[Ca.sup.2+]].sub.c] by tolbutamide or KCI or by comparing the magnitude of [[[Ca.sup.2+]].sub.c] and IS changes produced by glucose and tolbutamide. Both actin polymerization and depolymerization augmented IS triggered by all stimuli without increasing (sometimes decreasing) [[[Ca.sup.2+]].sub.c], which indicates a predominantly inhibitory function of microfilaments in exocytosis at a step distal to [[[Ca.sup.2+]].sub.c] increase. When [[[Ca.sup.2+]].sub.c] was elevated and controlled by KC1 or tolbutamide, the amplifying action of glucose was facilitated by actin depolymerization and unaffected by polymerization. Both phases of IS were larger in response to high-glucose than to tolbutamide in low-glucose, although triggering [[[Ca.sup.2+]].sub.c] was lower. This difference in IS, due to amplification, persisted when the IS rate was doubled by actin depolymerization or polymerization. In conclusion, metabolic amplification is rapid and influences the first as well as the second phase of IS. It is a late step of stimulus-secretion coupling, which does not require functional actin microfilaments and could correspond to acceleration of the priming process conferring release competence to insulin granules. biphasic insulin release; cytosolic calcium; exocytosis; insulin granules; pancreatic islets doi: 10.1152/ajpcell.00138.2010.

Insufficient insulin secretion and reduced pancreatic β cell mass are hallmarks of type 2 diabetes (T2DM). Here, we confirm that a previously identified polymorphism (rs2295490/Q84R) in exon 2 of the [...]

The tumor suppressor liver kinase B1 (LKB1), also called STK11, is a protein kinase mutated in Peutz-Jeghers syndrome. LKB1 phosphorylates AMP-activated protein kinase (AMPK) and several related protein kinases. Whereas deletion of both catalytic isoforms of AMPK from the pancreatic [beta]-cell and hypothalamic neurons using the rat insulin promoter (RIP2).Cre transgene ([beta]AMPKdKO) diminishes insulin secretion in vivo, deletion of LKB1 in the [beta]-cell with an inducible Pdx-1.CreER transgene enhances insulin secretion in mice. To determine whether the differences between these models reflect genuinely distinct roles for the two kinases in the [beta]-cell or simply differences in the timing and site(s) of deletion, we have therefore created mice deleted for LKB1 with the RIP2.Cre transgene. In marked contrast to [beta]AMPKdKO mice, [beta]LKB1KO mice showed diminished food intake and weight gain, enhanced insulin secretion, unchanged insulin sensitivity, and improved glucose tolerance. In line with the phenotype of Pdx1-CreER mice, total [beta]-cell mass and the size of individual islets and [beta]-cells were increased and islet architecture was markedly altered in [beta]LKB1KO islets. Signaling by mammalian target of rapamycin (mTOR) to eIF4-binding protein-1 and ribosomal S6 kinase was also enhanced. In contrast to Pdx1-CreER-mediated deletion, the expression of Glut2, glucose-induced changes in membrane potential and intracellular [Ca.sup.2+] were sharply reduced in [beta]LKB1KO mouse islets and the stimulation of insulin secretion was modestly inhibited. We conclude that LKB1 and AMPK play distinct roles in the control of insulin secretion and that the timing of LKB1 deletion, and/or its loss from extrapancreatic sites, influences the final impact on [beta]-cell function. AMP-activated protein kinase; [beta]-cell; insulin secretion; food intake; liver kinase B1; pancreas doi: 10.1152/ajpendo.00100.2010.

Glucose homeostasis is critically dependent on insulin release from pancreatic [beta]-cells, which is strictly regulated by glucose-induced oscillations in membrane potential ([V.sub.m]) and the cytosolic calcium level ([[[Ca.sup.2+]].sub.cyt]). We propose that TRPM5, a [Ca.sup.2+]-activated monovalent cation channel, is a positive regulator of glucose-induced insulin release. Immunofluorescence revealed expression of TRPM5 in pancreatic islets. A [Ca.sup.2+]-activated nonselective cation current with TRPM5-like properties is significantly reduced in [Trpm5.sup.-l-] cells. [Ca.sup.2+]-imaging and electrophysiological analysis show that glucose-induced oscillations of Vm and [[[Ca.sup.2+]].sub.cyt] have on average a reduced frequency in [Trpm5.sup.-/-] islets, specifically due to a lack of fast oscillations. As a consequence, glucose-induced insulin release from [Trpm5.sup.-/-] pancreatic islets is significantly reduced, resulting in an impaired glucose tolerance in [Trpm5.sup.-/-] mice. [Ca.sup.2+] signaling | insulin release | pancreatic [beta]-cells | transient receptor potential ion channels | glucose sensing doi: 10.1073/pnas.0913107107

Pioglitazone preserves pancreatic [beta]-cell morphology and function in diabetic animal models. In this study, we investigated the molecular mechanisms by which pioglitazone protects [beta]-cells in diabetic db/db mice. In addition to the morphological analysis of the islets, gene expression profiles of the pancreatic islet were analyzed using laser capture microdissection and were compared with real-time RT-PCR of db/db and nondiabetic m/m mice treated with or without pioglitazone for 2 wk or 2 days. Pioglitazone treatment (2 wk) ameliorated dysmetabolism, increased islet insulin content, restored glucose-stimulated insulin secretion, and preserved [3-cell mass in db/db mice but had no significant effects in m/m mice. Pioglitazone upregulated genes that promote cell differentiation/proliferation in diabetic and nondiabetic mice. In db/db mice, pioglitazone downregulated the apoptosis-promoting caspase-activated DNase gene and upregulated anti-apoptosis-related genes. The above-mentioned effects of pioglitazone treatment were also observed after 2 days of treatment. By contrast, the oxidative stress-promoting NADPH oxidase gene was downregulated, and antioxidative stress-related genes were upregulated, in db/db mice treated with pioglitazone for 2 wk, rather than 2 days. Morphometric results for proliferative cell number antigen and 4-hydroxy-2-noneal modified protein were consistent with the results of gene expression analysis. The present results strongly suggest that pioglitazone preserves [3-cell mass in diabetic mice mostly by two ways; directly, by acceleration of cell differentiation/ proliferation and suppression of apoptosis (acute effect); and indirectly, by deceleration of oxidative stress because of amelioration of the underlying metabolic disorder (chronic effect). [beta]-cell dysfunction; pioglitazone; oxidative stress; cell proliferation; cell apoptosis doi: 10.1152/ajpendo.00388.2009

Am J Physiol Endocrinol Metab 298: E99-E107, 2010. First published October 20, 2009; doi: 10.1152/ajpendo.00242.2009.--Amylin is the major component of pancreatic amyloid, which is implicated in the development of type 2 diabetes. It is costored with insulin in the secretory granules of pancreatic [beta]-cells and cosecreted with insulin following stimulation with glucose. Here, we investigate the effect of fatty acids (FAs) on amylin expression and secretion by [beta]-cells and explore the underlying mechanisms. Palmitate and oleate dose-dependently induced amylin mRNA accumulation in murine pancreatic [beta]-cell line MIN6 and primary pancreatic islets, the inductive effect of FAs on amylin expression is independent of glucose concentration. FAs upregulated amylin expression at the transcriptional level, and FAs must be metabolized to induce amylin expression. FAs also significantly induced human amylin promoter activation. Pretreatment of MIN6 cells with [Ca.sup.2+] chelator (EGTA, BAPTA-AM) PKC inhibitor Go-6976 or protein synthesis inhibitor cycloheximide significantly inhibited FA-induced amylin mRNA expression. Transcription factors cAMP-responsive element-binding protein, pancreatic and duodenal homeobox factor-1, and peroxisome proliferator-activated receptor were not involved in FA-induced amylin expression. Palmitate and oleate both increased amylin and insulin release from MIN6 cells and stimulated amylin expression but had no effect on insulin expression. Mice refed with Intralipid had significantly higher levels of plasma FFA, amylin, and insulin than those refed with saline. These data demonstrate that FAs differently regulate amylin and insulin expression and induce both amylin and insulin release. [Ca.sup.2+] and PKC signaling pathways and de novo-synthesized protein(s) were involved in FA-induced amylin expression. Induction of amylin production and release by FA may contribute to its biological functions under physiological conditions. pancreas; islet amyloid polypeptide; insulin; gene expression; signal transduction; high-lipid diet

Am J Physiol Endocrinol Metab 298: E17-E27, 2010. First published October 20, 2009 doi: 10.1152/ajpendo.90871.2008.--The alteration of the [beta]-cell population in the Goto-Kakizaki rat (GK/Par line), a model of spontaneous type 2 diabetes, has been ascribed to significantly decreased [beta]-cell replication and neogenesis, while [beta]-cell apoptosis is surprisingly not enhanced and remains in the normal range. To gain insight into the mechanisms by which those [beta]-cells are protected from death, we studied ex vivo the apoptotic activity and the expression of a large set of pro/antiapoptotic and pro/antioxidant genes in GK/Par islet cells. This was done in vitro in freshly isolated islets as well as in response to culture conditions and calibrated reactive oxygen species (ROS) exposure (i.e., [H.sub.2][O.sub.2]). We also investigated the intracellular mechanisms of the diabetic [beta]-cell response to ROS, the role if any of the intracellular cAMP metabolism, and finally the kinetic of ROS response, taking advantage of the GK/Par rat normoglycemia until weaning. Our results show that the peculiar GK/Par [beta]-cell phenotype was correlated with an increased expression of a large panel of antioxidant genes as well as pro/antiapoptotic genes. We demonstrate that such combination confers resistance to cytotoxic [H.sub.2][O.sub.2] exposure in vitro, raising the possibility that at least some of the activated stress/defense genes have protective effects against [H.sub.2][O.sub.2]-triggered [beta]-cell death. We also present some evidence that the GK/Par [beta]-cell resistance to [H.sub.2][O.sub.2] is at least partly cAMP dependent. Finally, we show that such a phenotype is not innate but is spontaneously acquired after diabetes onset as the result of an adaptive response to the diabetic environment. [beta]-cell mass; apoptosis; replication; reactive oxygen species; cyclic adenosine monophosphate; diabetic Goto Kakizaki rat

Kilimnik G, Kim A, Jo J, Miller K, Hara M. Quantification of pancreatic islet distribution in situ in mice. Am J Physiol Endocrinol Metab 297: E1331-E1338, 2009. First published October 6, 2009; doi: 10.1152/ajpendo.00479.2009.--Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. Precisely monitoring pancreatic [beta]-cell proliferation and islet growth is a challenging area of research. We have developed a method to capture the distribution of [beta]-cells in the intact pancreas of transgenic mice with fluorescence-tagged [beta]-cells with a macro written for ImageJ (rsb.info.nih.gov/ij/). Total [beta]-cell area and islet number and size distribution are quantified with reference to specific parameters and location for each islet and for small clusters of [beta]-cells. The entire distribution of islets can now be plotted in three dimensions, and the information from the distribution on the size and shape of each islet allows a quantitative and a qualitative comparison of changes in overall [beta]-cell area at a glance. pancreatic [beta]-cells; islets; [beta]-cell area; insulinoma; diabetes doi: 10.1152/ajpendo.00479.2009

Impaired functioning of pancreatic [beta] cells is a key hallmark of type 2 diabetes. [beta] cell function is modulated by the actions of different classes of heterotrimeric G proteins. The functional consequences of activating specific [beta] cell G protein signaling pathways in vivo are not well understood at present, primarily due to the fact that [beta] cell G protein-coupled receptors (GPCRs) are also expressed by many other tissues. To circumvent these difficulties, we developed a chemical-genetic approach that allows for the conditional and selective activation of specific [beta] cell G proteins in intact animals. Specifically, we created two lines of transgenic mice each of which expressed a specific designer GPCR in [beta] cells only. Importantly, the two designer receptors differed in their G protein-coupling properties ([G.sub.q/11] versus [G.sub.s]). They were unable to bind endogenous ligand(s), but could be efficiently activated by an otherwise pharmacologically inert compound (clozapine-N-oxide), leading to the conditional activation of either[beta] cell [G.sub.q/11] or [G.sub.s] G proteins. Here we report the findings that conditional and selective activation of [beta] cell [G.sub.q/11] signaling in vivo leads to striking increases in both first- and second-phase insulin release, greatly improved glucose tolerance in obese, insulin-resistant mice, and elevated [beta] cell mass, associated with pathway-specific alterations in islet gene expression levels. Selective stimulation of [beta] cell Gs triggered qualitatively similar in vivo metabolic effects. Thus, this developed chemical-genetic strategy represents a powerful approach to study G protein regulation of [beta] cell function in vivo. beta cells | G protein-coupled receptors | transgenic mice | type 2 diabetes www.pnas.org/cgi/doi/10.1073/pnas.0906593106

Type 2 diabetes mellitus (T2DM) results from pancreatic [beta] cell failure in the setting of insulin resistance. Heterozygous mutations in the gene encoding the [beta] cell transcription factor pancreatic duodenal homeobox 1 (Pdx1) are associated with both T2DM and maturity onset diabetes of the young (MODY4), and low levels of Pdx1 accompany [beta] cell dysfunction in experimental models of glucotoxicity and diabetes. Here, we find that Pdx1 is required for compensatory [beta] cell mass expansion in response to diet-induced insulin resistance through its roles in promoting [beta] cell survival and compensatory hypertrophy. Pdx1-deficient [beta] cells show evidence of endoplasmic reticulum (ER) stress both in the complex metabolic milieu of high-fat feeding as well as in the setting of acutely reduced Pdx1 expression in the Min6 mouse insulinoma cell line. Further, Pdx1 deficiency enhances [beta] cell susceptibility to ER stress-associated apoptosis. The results of high throughput expression microarray and chromatin occupancy analyses reveal that Pdx1 regulates a broad array of genes involved in diverse functions of the ER, including proper disulfide bond formation, protein folding, and the unfolded protein response. These findings suggest that Pdx1 deficiency leads to a failure of [beta] cell compensation for insulin resistance at least in part by impairing critical functions of the ER. chromatin occupancy | diabetes | gene regulation | islet compensation www.pnas.org/cgi/doi/10.1073/pnas.0904849106

TSC1 is a tumor suppressor that associates with TSC2 to inactivate Rheb, thereby inhibiting signaling by the mammalian target of rapamycin (mTOR) complex 1 (mTORC1). mTORC1 stimulates cell growth by promoting anabolic cellular processes, such as translation, in response to growth factors and nutrient signals. To test roles for TSC1 and mTORC1 in [beta]-cell function, we utilized Rip2/Cre to generate mice lacking Tsc1 in pancreatic [beta]-cells (Rip-Tsc1cKO mice). Although obesity developed due to hypothalamic Tsc1 excision in older Rip-Tsc1cKO animals, young animals displayed a prominent gain-of-function [beta]-cell phenotype prior to the onset of obesity. The young Rip-Tsc1cKO animals displayed improved glycemic control due to mTOR-mediated enhancement of [beta]-cell size, mass, and insulin production but not determinants of [beta]-cell number (proliferation and apoptosis), consistent with an important anabolic role for mTOR in [beta]-cell function. Furthermore, mTOR mediated these effects in the face of impaired Akt signaling in [beta]-cells. Thus, mTOR promulgates a dominant signal to promote [beta]-cell/islet size and insulin production, and this pathway is crucial for [beta]-cell function and glycemic control. tuberous sclerosis complex; mammalian target of rapamycin; pancreatic [beta]-cell; conditional knockout mice; rat insulin promoter 2 doi: 10.1152/ajpendo.00262.2009.

Mitochondrial dysfunction may be a long-term consequence of a poor nutritional environment during early life. Our aim was to investigate whether a maternal low-protein (LP) diet may program mitochondrial dysfunction in islets of adult progeny before glucose intolerance ensues. To address this, pregnant Wistar rats were fed isocaloric diets containing either 20% protein (control) or 8% protein (LP diet) throughout gestation. From birth, offspring received the control diet. The mitochondrial function was analyzed in islets of 3-mo-old offspring. Related to their basal insulin release, cultured islets from both male and female LP offspring presented a lower response to glucose challenge and a blunted ATP production compared with control offspring. The expression of malate dehydrogenase as well as the subunit 6 of the ATP synthase encoded by mitochondrial genome (mtDNA) was lower in these islets, reducing the capacity of ATP production through the Krebs cycle and oxidative phosphorylation. However, mtDNA content was unchanged in LP islets compared with control. Several consequences of protein restriction during fetal life were more marked in male offspring. Only LP males showed an increased reactive oxygen species production associated with a higher expression of mitochondrial subunits of the electron transport chain NADH-ubiquinone oxireductase subunit 4L, an overexpression of peroxisome proliferator-activated receptor-[gamma] and uncoupling protein-2, and a strongly reduced beta-cell mass. In conclusion, mitochondrial function is clearly altered in islets from LP adult offspring in a sex-specific manner. That may provide a cellular explanation for the earlier development of glucose intolerance in male than in female offspring of dams fed an LP diet. developmental programming; malnutrition; metabolic syndrome; insulin secretion doi: 10.1152/ajpregu.00280.2009.

In addition to its primary role in regulating glucose production from the liver, glucagon has many other actions, reflected by the wide tissue distribution of the glucagon receptor (Gcgr). To investigate the role of glucagon in the regulation of insulin secretion and whole body glucose homeostasis in vivo, we generated mice overexpressing the Gcgr specifically on pancreatic [beta]-cells (RIP-Gcgr). In vivo and in vitro insulin secretion in response to glucagon and glucose was increased 1.7- to 3.9-fold in RIP-Gcgr mice compared with controls. Consistent with the observed increase in insulin release in response to glucagon and glucose, the glucose excursion resulting from both a glucagon challenge and intraperitoneal glucose tolerance test (IPGTT) was significantly reduced in RIP-Gcgr mice compared with controls. However, RIP-Gcgr mice display similar glucose responses to an insulin challenge. [beta]-Cell mass and pancreatic insulin content were also increased (20 and 50%, respectively) in RIP-Gcgr mice compared with controls. When fed a high-fat diet (HFD), both control and RIP-Gcgr mice developed similar degrees of obesity and insulin resistance. However, the severity of both fasting hyperglycemia and impaired glucose tolerance (IGT) were reduced in RIP-Gcgr mice compared with controls. Furthermore, the insulin response of RIP-Gcgr mice to an IPGTT was twice that of controls when fed the HFD. These data indicate that increased pancreatic [beta]-cell expression of the Gcgr increased insulin secretion, pancreatic insulin content, [beta]-cell mass, and, when mice were fed a HFD, partially protected against hyperglycemia and IGT. glucagon receptor signaling; pancreatic islet cells; insulin secretion

Pancreatic [beta]-cells are critical regulators of glucose homeostasis, and they vary dramatically in their glucose stimulated metabolic response and levels of insulin secretion. It is unclear whether these parameters are influenced by the developmental origin of individual [beta]-cells. Using HOTcre, a Cre-based genetic switch that uses heat-induction to precisely control the temporal expression of transgenes, we labeled two populations of [beta]-cells within the developing zebrafish pancreas. These populations originate in distinct pancreatic buds and exhibit gene expression profiles suggesting distinct functions during development. We find that the dorsal bud derived [beta]-cells are quiescent and exhibit a marked decrease in insulin expression postembryonically. In contrast, ventral bud derived [beta]-cells proliferate actively, and maintain high levels of insulin expression compared with dorsal bud derived [beta]-cells. Therapeutic strategies to regulate [beta]-cell proliferation and function are required to cure pathological states that result from excessive [beta]-cell proliferation (e.g., insulinoma) or insufficient [beta]-cell mass (e.g., diabetes mellitus). Our data reveal the existence of distinct populations of [beta]-cells in vivo and should help develop better strategies to regulate [beta]-cell differentiation and proliferation. zebrafish | pancreas | islet | insulin | lineage

Maturity-onset diabetes of the young (MODY) is a subtype of diabetes defined by an autosomal pattern of inheritance and a young age at onset, often before age 25. MODY is genetically heterogeneous, with 8 distinct MODY genes identified to date and more believed to exist. We resequenced 732 kb of genomic sequence at 8p23 in 6 MODY families unlinked to known MODY genes that showed evidence of linkage at that location. Of the 410 sequence differences that we identified, 5 had a frequency < 1% in the general population and segregated with diabetes in 3 of the families, including the 2 showing the strongest support for linkage at this location. The 5 mutations were all placed within 100 kb corresponding to the BLK gene. One resulted in an Ala71Thr substitution; the other 4 were noncoding and determined decreased in vitro promoter activity in reporter gene experiments. We found that BLK--a nonreceptor tyrosine-kinase of the src family of proto-oncogenes--is expressed in [beta]-cells where it enhances insulin synthesis and secretion in response to glucose by up-regulating transcription factors Pdx1 and Nkx6.1. These actions are greatly attenuated by the Ala71Thr mutation. These findings point to BLK as a previously unrecognized modulator of [beta]-cell function, the deficit of which may lead to the development of diabetes. beta cells | genetics | MODY | tyrosine kinase

Intracellular signaling by which pancreatic [beta]-cells synthesize and secrete insulin in control of glucose homeostasis is not fully understood. Here we show that Shp2, a cytoplasmic tyrosine phosphatase possessing 2 SH2 domains, coordinates signaling events required for insulin biosynthesis in [beta]-cells. Mice with conditional ablation of the Shp2/Ptpn11 gene in the pancreas exhibited defective glucose-stimulated insulin secretion and impaired glucose tolerance. Consistently, siRNA-mediated Shp2-knockdown in rat insulinoma INS-1 832/13 cells resulted in decreased insulin production and secretion despite ah increase in cellular ATP. Shp2 modulates the strength of signals flowing through Akt/FoxO1 and Erk pathways, culminating in control of Pdx1 expression and activity on Ins1 and Ins2 promoters, and forced Pdx1 expression rescued insulin production in Shp2-knockdown [beta]-cells. Therefore, Shp2 acts asa signal coordinator in [beta]-cells, orchestrating multiple pathways controlling insulin biosynthesis to maintain glucose homeostasis. Pdx1 | signal transduction | diabetes | insulin secretion | gene expression

To test the hypothesis that somatic mitochondrial (mt)DNA mutation accumulation predisposes mice to [beta]-cell loss and diabetes development, transgenic mice expressing a proofreading-deficient mtDNA polymerase-[gamma], under the control of the rat insulin-1 promoter were generated. At 6 wk of age, mtDNA mutations reached 0.01% (1.05 mutations/10,000 bp) in islets isolated from transgenic mice. This mutational burden is associated with impaired glucose tolerance and a diabetes prevalence of 52% in male transgenic mice. Female transgenic mice maintain slightly elevated fasting glucose levels, mild glucose intolerance, and a diabetes prevalence of 14%. Diabetes in transgenic animals is associated with insulin insufficiency that results from a significant reduction in [beta]-cell mass. Importantly, apoptosis of [beta]-cells is increased 7-fold in female and 11-fold in male transgenic mice compared with littermate controls. These results are consistent with a causative role of somatic mtDNA mutation accumulation in the loss of [beta]-cell mass and diabetes development. mitochondrial deoxyribonucleic acid; insulin; islet

Defective insulin secretion in response to glucose is an important component of the [beta] cell dysfunction seen in type 2 diabetes. As mitochondrial oxidative phosphorylation plays a key role in glucose-stimulated insulin secretion (GSIS), oxygen-sensing pathways may modulate insulin release. The von Hippel--Lindau (VHL) protein controls the degradation of hypoxia-inducible factor (HIF) to coordinate cellular and organismal responses to altered oxygenation. To determine the role of this pathway in controlling glucose-stimulated insulin release from pancreatic [beta] cells, we generated mice lacking Vhl in pancreatic [beta] cells ([beta]VhlKO mice) and mice lacking Vhl in the pancreas (PVhlKO mice). Both mouse strains developed glucose intolerance with impaired insulin secretion. Furthermore, deletion of Vhl in [beta] cells or the pancreas altered expression of genes involved in [beta] cell function, including those involved in glucose transport and glycolysis, and isolated [beta]VhlKO and PVhlKO islets displayed impaired glucose uptake and defective glucose metabolism. The abnormal glucose homeostasis was dependent on upregulation of Hif-1[alpha] expression, and deletion of Hif1a in Vhl-deficient [beta] cells restored GSIS. Consistent with this, expression of activated Hif-1[alpha] in a mouse [beta] cell line impaired GSIS. These data suggest that VHL/HIF oxygen-sensing mechanisms play a critical role in glucose homeostasis and that activation of this pathway in response to decreased islet oxygenation may contribute to [beta] cell dysfunction., Introduction Blood glucose levels are normally tightly controlled by the regulation of insulin release from the pancreatic [beta] cells. Glucose-stimulated insulin secretion (GSIS) is a complex metabolic process involving the [...]

Factors that promote pancreatic [beta] cell growth and function are potential therapeutic targets for diabetes mellitus. In mice, genetic experiments suggest that signaling cascades initiated by insulin and IGFs positively regulate [beta] cell mass and insulin secretion. Akt and S6 kinase (S6K) family members are activated as part of these signaling cascades, but how the interplay between these proteins controls [beta] cell growth and function has not been determined. Here, we found that although transgenic mice overexpressing the constitutively active form of Akt1 under the rat insulin promoter (RIP-MyrAkt1 mice) had enlarged [beta] cells and high plasma insulin levels, leading to improved glucose tolerance, a substantial proportion of the mice developed insulinomas later in life, which caused decreased viability. This oncogenic transformation tightly correlated with nuclear exclusion of the tumor suppressor PTEN. To address the role of the mammalian target of rapamycin (mTOR) substrate S6K1 in the MyrAkt1-mediated phenotype, we crossed RIP-MyrAkt1 and S6K1-deficient mice. The resulting mice displayed reduced insulinemia and glycemia compared with RIP-MyrAkt1 mice due to a combined effect of improved insulin secretion and insulin sensitivity. Importantly, although the increase in [beta] cell size in RIP-MyrAkt1 mice was not affected by S6K1 deficiency, the hyperplastic transformation required S6K1. Our results therefore identify S6K1 as a critical element for MyrAkt1-induced tumor formation and suggest that it may represent a useful target for anticancer therapy downstream of mTOR., Introduction Pancreatic [beta] cells in the islets of Langerhans are major nutrient sensors in mammals, as they produce and secrete the anabolic hormone insulin in response to nutritional cues. A [...]

Regulation of pancreatic [beta] cell mass and function is a major determinant for the development of diabetes. Growth factors and nutrients are important regulators of [beta] cell mass and function. The signaling pathways by which these growth signals modulate these processes have not been completely elucidated. Tsc2 is an attractive candidate to modulate these processes, because it is a converging point for growth factor and nutrient signals. In these experiments, we generated mice with conditional deletion of Tsc2 in [beta] cells ([beta][Tsc2.sup.-/-]). These mice exhibited decreased glucose levels and hyperinsulinemia in the fasting and fed state. Improved glucose tolerance in these mice was observed as early as 4 weeks of age and was still present in 52-week-old mice. Deletion of Tsc2 in [beta] cells induced expansion of [beta] cell mass by increased proliferation and cell size. Rapamycin treatment reversed the metabolic changes in [beta][Tsc2.sup.-/-] mice by induction of insulin resistance and reduction of [beta] cell mass. The reduction of [beta] cell mass in [beta][Tsc2.sup.-/-] mice by inhibition of the mTOR/Raptor (TORC1) complex with rapamycin treatment suggests that TORC1 mediates proliferative and growth signals induced by deletion of Tsc2 in [beta] cells. These studies uncover a critical role for the Tsc2/mTOR pathway in regulation of [beta] cell mass and carbohydrate metabolism in vivo. mTOR | pancreas | islets

The study presents the new advancements in the technique of calcineurin and its substrate the Nuclear Factor of Activated T-cells (NFAT) signaling in [beta]-cells. New therapeutic approaches for the treatment of diabetes are also discussed.

Diabetes results from an inadequate mass of functional [beta] cells, due to either [beta] cell loss caused by immune assault or the lack of compensation to overcome insulin resistance. Elucidating [...]

Obesity is characterized by hyperinsulinemia, hyperleptinemia, and an increase in islet volume. While the mechanisms that hasten the onset of diabetes in obese individuals are not known, it is possible [...]

The mutant hepatocyte nuclear factor-4[alpha] (HNF-4[alpha]) gene impairs the proliferation of pancreatic [beta]-cells in maturity-onset diabetes. These findings provide evidence that the gene is responsible for the growth of [beta] cell mass on increased metabolic demand.

Pancreas development relies on a network of transcription factors belonging mainly to the Homeodomain and basic Helix--Loop--Helix families. We show in this study that, in zebrafish, sox4, a member of the SRY-like HMG-box (SOX) family, is required for proper endocrine cell differentiation. We found that two genes orthologous to mammalian Sox4 are present in zebrafish and that only one of them, sox4b, is strongly expressed in the pancreatic anlage. Transcripts of sox4b were detected in mid-trunk endoderm from the 5-somite stage, well before the onset of expression of the early pancreatic gene pdx-1. Furthermore, by fluorescent double in situ hybridization, we found that expression of sox4b is mostly restricted to precursors of the endocrine compartment. This expression is not maintained in differentiated cells although transient expression can be detected in [alpha] cells and some [beta] cells. That sox4b-expressing cells belong to the endocrine lineage is further illustrated by their absence from the pancreata of slow-muscle-omitted mutant embryos, which specifically lack all early endocrine markers while retaining expression of exocrine markers. The involvement of sox4b in cell differentiation is suggested firstly by its up-regulation in mind bomb mutant embryos displaying accelerated pancreatic cell differentiation. In addition, sox4b knock-down leads to a drastic reduction in glucagon expression, while other pancreatic markers including insulin, somatostatin, and trypsin are not significantly affected. This disruption of [alpha] cell differentiation is due to down-regulation of the homeobox arx gene specifically in the pancreas. Taken together, these data demonstrate that, in zebrafish, sox4b is expressed transiently during endocrine cell differentiation and plays a crucial role in the generation of [alpha] endocrine cells Keywords: SOX; Transcription factor; Pancreas; Zebrafish; Development; Glucagon

KLF11 (TIEG2) is a pancreas-enriched transcription factor that has elicited significant attention because of its role as negative regulator of exocrine cell growth in vitro and in vivo. However, its functional role in the endocrine pancreas remains to be established. Here, we report, for the first time, to our knowledge, the characterization of KLF11 as a glucose-inducible regulator of the insulin gene. A combination of random oligonucleotide binding, EMSA, luciferase reporter, and chromatin immunoprecipitation assays shows that KLF11 binds to the insulin promoter and regulates its activity in beta cells. Genetic analysis of the KLF11 gene revealed two rare variants (Ala347Ser and Thr220Met) that segregate with diabetes in families with early-onset type 2 diabetes, and significantly impair its transcriptional activity. In addition, analysis of 1,696 type 2 diabetes mellitus and 1,776 normoglycemic subjects show a frequent polymorphic GIn62Arg variant that significantly associates with type 2 diabetes mellitus in North European populations (OR = 1.29, P = 0.00033). Moreover, this variant alters the corepressor mSin3A-binding activity of KLF11, impairs the activation of the insulin promoter and shows lower levels of insulin expression in pancreatic beta cells. In addition, subjects carrying the Gln62Arg allele show decreased plasma insulin after an oral glucose challenge. Interestingly, all three nonsynonymous KLF11 variants show increased repression of the catalase 1 promoter, suggesting a role in free radical clearance that may render beta cells more sensitive to oxidative stress. Thus, both functional and genetic analyses reveal that KLF11 plays a role in the regulation of pancreatic beta cell physiology, and its variants may contribute to the development of diabetes. insulin | polymorphisms | TGF-[beta] | type 2 diabetes

Insulin gene transcription is limited to the beta cells within the mammalian pancreas and, like insulin secretion, is regulated by glucose. Our previous studies in primary cultured beta cells suggested the presence of a strong glucose-responsive enhancer element between base pairs -341 and -260 of the human insulin promoter, the same region in which a transcriptional repressor had been identified in beta-cell tumor lines. In an attempt to map these promoter activities and resolve these conflicting data, we designed minienhancer constructs spanning this region, and tested them in primary cultured and immortalized cells. One sequence, the Z element (base pairs -292 to -243), functions as both a potent glucose-responsive transcriptional enhancer in primary cultured islet cells and as a transcriptional repressor in immortalized beta and nonbeta cells and in primary fibroblasts. In addition, the Z element binds a novel glucose-responsive protein complex that is found in the nuclei of primary cultured islet cells, but not in the nuclei of tumor cells or primary cultured fibroblasts. These data demonstrate a critical role for the Z element in human insulin gene transcription and its regulation by glucose.