Islet mRNA Research Articles

W2476 ameliorates β-cell dysfunction and exerts therapeutic effects in mouse models of diabetes via modulation of the thioredoxin-interacting protein signaling pathway.

Recent evidence shows that high glucose levels recruit carbohydrate response element-binding protein, which binds the promoter of thioredoxin-interacting protein (txnip), thereby regulating its expression in β-cells. Overexpression of txnip not only induces β-cell apoptosis but also reduces insulin production. Thus, the discovery of compounds that either inhibit TXNIP activity or suppress its expression was the focus of the present study. INS-1E cells stably transfected with either a txnip proximal glucose response element connected to a luciferase reporter plasmid (BG73) or full-length txnip promoter connected to a luciferase reporter plasmid (CL108) were used in primary and secondary high-throughput screening campaigns, respectively. From 256 000 synthetic compounds, a small molecule compound, W2476 [9-((1-(4-acetyl-phenyloxy)-ethyl)-2-)adenine], was identified as a modulator of the TXNIP-regulated signaling pathway following the screening and characterized using a battery of bioassays. The preventive and therapeutic properties of W2476 were further examined in streptozotocin-induced diabetic and diet-induced obese mice. Treatment with W2476 (1, 5, and 15 μmol/L) dose-dependently inhibited high glucose-induced TXNIP expression at the mRNA and protein levels in INS-1E cells and rat pancreatic islets. Furthermore, W2476 treatment prevented INS-1E cells from apoptosis induced by chronic exposure of high glucose and enhanced insulin production in vitro. Oral administration of W2476 (200 mg·kg-1·d-1) rescued streptozotocin-induced diabetic mice by promoting β-cell survival and enhancing insulin secretion. This therapeutic property of W2476 was further demonstrated by its ability to improve glucose homeostasis and insulin sensitivity in diet-induced obese mice. Thus, chemical intervention of the TXNIP-regulated signaling pathway might present a viable approach to manage diabetes.

Adhesion G Protein-Coupled Receptor G1 (ADGRG1/GPR56) and Pancreatic β-Cell Function.

Adhesion G protein-coupled receptor (GPCR)-G1 (ADGRG1) is the most abundant GPCR in human pancreatic islets, but its role in islet function is unclear. Investigate how ADGRG1 expression and activation by its ligand, collagen III, impacts β-cell function in normal and type 2 diabetic (T2D) islets. Genes associated with the ADGRG1 in human islets was probed by RNA-sequencing of human pancreatic islet isolated from cadaveric donors, followed by functional studies on β-cell proliferation, apoptosis, and insulin secretion in human and mouse islets and in INS-1 cells. Changes in β-cell gene expression, proliferation, apoptosis, and insulin secretion were quantified by RNA-sequencing, qPCR, Thymidine incorporation, Western blotting, and RIA, respectively. ADGRG1 is the most abundant GPCR mRNA in both human and mouse islets, and its expression in human islets strongly correlates with genes important for β-cell function and T2D risk. The expression of ADGRG1 was reduced in islets of T2D donors, in db/db mouse islets, and in isolated human islets exposed to chronic hyperglycemia. Beneficial effects of collagen type III on β-cell function via activation of the cAMP/protein kinase A pathway, suppression of RhoA and caspase-3 activity, increased β-cell viability, and proliferation were abolished when ADGRG1 was down-regulated in β-cells. We demonstrate a mechanistic link between ADGRG1 expression and β-cell function. Pharmacological agents that promote expression or activation of the ADGRG1 receptor may represent a novel approach for the treatment of T2D.

Recovery of high-quality RNA from laser capture microdissected human and rodent pancreas

Laser capture microdissection (LCM) is a powerful method to isolate specific populations of cells for subsequent analysis such as gene expression profiling, for example, microarrays or ribonucleic (RNA)-Seq. This technique has been applied to frozen as well as formalin-fixed, paraffin-embedded (FFPE) specimens with variable outcomes regarding quality and quantity of extracted RNA. The goal of the study was to develop the methods to isolate high-quality RNA from islets of Langerhans and pancreatic duct glands (PDG) isolated by LCM. We report an optimized protocol for frozen sections to minimize RNA degradation and maximize recovery of expected transcripts from the samples using quantitative real-time polymerase chain reaction (RT-PCR) by adding RNase inhibitors at multiple steps during the experiment. This technique reproducibly delivered intact RNA (RIN values 6–7). Using quantitative RT-PCR, the expected profiles of insulin, glucagon, mucin6 (Muc6), and cytokeratin-19 (CK-19) mRNA in PDGs and pancreatic islets were detected. The described experimental protocol for frozen pancreas tissue might also be useful for other tissues with moderate to high levels of intrinsic ribonuclease (RNase) activity.

Treatment with Tacrolimus and Sirolimus Reveals No Additional Adverse Effects on Human Islets In Vitro Compared to Each Drug Alone but They Are Reduced by Adding Glucocorticoids.

Tacrolimus and sirolimus are important immunosuppressive drugs used in human islet transplantation; however, they are linked to detrimental effects on islets and reduction of long-term graft function. Few studies investigate the direct effects of these drugs combined in parallel with single drug exposure. Human islets were treated with or without tacrolimus (30 μg/L), sirolimus (30 μg/L), or a combination thereof for 24 hrs. Islet function as well as apoptosis was assessed by glucose-stimulated insulin secretion (GSIS) and Cell Death ELISA. Proinflammatory cytokines were analysed by qRT-PCR and Bio-Plex. Islets exposed to the combination of sirolimus and tacrolimus were treated with or without methylprednisolone (1000 μg/L) and the expression of the proinflammatory cytokines was investigated. We found the following: (i) No additive reduction in function and viability in islets existed when tacrolimus and sirolimus were combined compared to the single drug. (ii) Increased expression of proinflammatory cytokines mRNA and protein levels in islets took place. (iii) Methylprednisolone significantly decreased the proinflammatory response in islets induced by the drug combination. Although human islets are prone to direct toxic effect of tacrolimus and sirolimus, we found no additive effects of the drug combination. Short-term exposure of glucocorticoids could effectively reduce the proinflammatory response in human islets induced by the combination of tacrolimus and sirolimus.

Dynamics of ADAM17-Mediated Shedding of ACE2 Applied to Pancreatic Islets of Male db/db Mice.

Angiotensin-converting enzyme 2 (ACE2) gene therapy aimed at counteracting pancreatic ACE2 depletion improves glucose regulation in two diabetic mouse models: db/db mice and angiotensin II-infused mice. A disintegrin and metalloproteinase 17 (ADAM17) can cause shedding of ACE2 from the cell membrane. The aim of our studies was to determine whether ADAM17 depletes ACE2 levels in pancreatic islets and β-cells. Dynamics of ADAM17-mediated ACE2 shedding were investigated in 832/13 insulinoma cells. Within a wide range of ACE2 expression levels, including the level observed in mouse pancreatic islets, overexpression of ADAM17 increases shed ACE2 and decreases cellular ACE2 levels. We provide a mathematical description of shed and cellular ACE2 activities as a function of the ADAM17 activity. The effect of ADAM17 on the cellular ACE2 content was relatively modest with an absolute control strength value less than 0.25 and approaching 0 at low ADAM17 activities. Although we found that ADAM17 and ACE2 are both expressed in pancreatic islets, the β-cell is not the major cell type expressing ACE2 in islets. During diabetes progression in 8-, 12-, and 15-week-old db/db mice, ACE2 mRNA and ACE2 activity levels in pancreatic islets were not decreased over time nor significantly decreased compared with nondiabetic db/m mice. Levels of ADAM17 mRNA and ADAM17 activity were also not significantly changed. Inhibiting basal ADAM17 activity in mouse islets failed to affect ACE2 levels. We conclude that whereas ADAM17 has the ability to shed ACE2, ADAM17 does not deplete ACE2 from pancreatic islets in diabetic db/db mice.

Prolactin receptors and placental lactogen drive male mouse pancreatic islets to pregnancy-related mRNA changes.

Pregnancy requires a higher functional beta cell mass and this is associated with profound changes in the gene expression profile of pancreatic islets. Taking Tph1 as a sensitive marker for pregnancy-related islet mRNA expression in female mice, we previously identified prolactin receptors and placental lactogen as key signalling molecules. Since beta cells from male mice also express prolactin receptors, the question arose whether male and female islets have the same phenotypic resilience at the mRNA level during pregnancy. We addressed this question in vitro, by stimulating cultured islets with placental lactogen and in vivo, by transplanting male or female islets into female acceptor mice. Additionally, the islet mRNA expression pattern of pregnant prolactin receptor deficient mice was compared with that of their pregnant wild-type littermates. When cultured with placental lactogen, or when transplanted in female recipients that became pregnant (day 12.5), male islets induced the ‘islet pregnancy gene signature’, which we defined as the 12 highest induced genes in non-transplanted female islets at day 12.5 of pregnancy. In addition, serotonin immunoreactivity and beta cell proliferation was also induced in these male transplanted islets at day 12.5 of pregnancy. In order to further investigate the importance of prolactin receptors in these mRNA changes we used a prolactin receptor deficient mouse model. For the 12 genes of the signature, which are highly induced in control pregnant mice, no significant induction of mRNA transcripts was found at day 9.5 of pregnancy. Together, our results support the key role of placental lactogen as a circulating factor that can trigger the pregnancy mRNA profile in both male and female beta cells.

Uncoupling protein-2 mRNA expression in mice subjected to intermittent hypoxia.

Objective: To investigate the effect of intermittent hypoxia-a model of obstructive sleep apnea (OSA)-on pancreatic expression of uncoupling protein-2 (UCP2), as well as on glycemic and lipid profiles, in C57BL mice. Methods: For 8 h/day over a 35-day period, male C57BL mice were exposed to intermittent hypoxia (hypoxia group) or to a sham procedure (normoxia group). The intermittent hypoxia condition involved exposing mice to an atmosphere of 92% N and 8% CO2 for 30 s, progressively reducing the fraction of inspired oxygen to 8 ± 1%, after which they were exposed to room air for 30 s and the cycle was repeated (480 cycles over the 8-h experimental period). Pancreases were dissected to isolate the islets. Real-time PCR was performed with TaqMan assays. Results: Expression of UCP2 mRNA in pancreatic islets was 20% higher in the normoxia group than in the hypoxia group (p = 0.11). Fasting serum insulin was higher in the hypoxia group than in the normoxia group (p = 0.01). The homeostasis model assessment of insulin resistance indicated that, in comparison with the control mice, the mice exposed to intermittent hypoxia showed 15% lower insulin resistance (p = 0.09) and 21% higher pancreatic β-cell function (p = 0.01). Immunohistochemical staining of the islets showed no significant differences between the two groups in terms of the area or intensity of α- and β-cell staining for insulin and glucagon. Conclusions: To our knowledge, this is the first report of the effect of intermittent hypoxia on UCP2 expression. Our findings suggest that UCP2 regulates insulin production in OSA. Further study of the role that UCP2 plays in the glycemic control of OSA patients is warranted.

Systematic profiling of mRNA and miRNA expression in the pancreatic islets of spontaneously diabetic Goto-Kakizaki rats.

Type 2 diabetes (T2DM) is a complex multifactorial metabolic disorder that affects >100 million individuals worldwide, yet the mechanisms involved in the development and progression of the disease have not yet been fully elucidated. The present study examined the mRNA and micro (mi)RNA expression profiles by microarray analysis in the pancreas islets of spontaneously diabetic Goto-Kakizaki rats with the aim to identify regulatory mechanisms underlying the pathogenesis of T2DM. A total of 9 upregulated and 10 downregulated miRNAs were identified, including miR-150, miR-497, miR-344-3p and let-7f, which were independently validated by quantitative polymerase chain reaction assays. In addition, differential expression of 670 genes was detected by mRNA microarray analysis, including 370 upregulated and 247 downregulated genes. The differentially expressed genes were statistically associated with major cellular pathways, including the immune response pathway and the extracellular matrix (ECM)-receptor interaction pathway. Finally, a reverse regulatory association of differentially expressed miRNAs and their predicted target genes was constructed, supported by analysis of their mRNA and miRNA expression profiles. A number of key pairs of miRNA-mRNA was proposed to have significant roles in the pathogenesis of T2DM rats based on bioinformatics analysis, one example being the let-7f/collagen, type II, alpha 1 pair that may regulate ECM-receptor interactions.

Transcription of the gene encoding TNF-α is increased by IL-1β in rat and human islets and β-cell lines

Synthesis and secretion of immunomodulatory proteins, such as cytokines and chemokines, controls the inflammatory response within pancreatic islets. When this inflammation does not resolve, destruction of pancreatic islet β-cells leads to diabetes mellitus. Production of the soluble mediators of inflammation, such as TNF-α and IL-1β, from resident and invading immune cells, as well as directly from islet β-cells, is also associated with suboptimal islet transplantation outcomes. In this study, we found that IL-1β induces rapid increases in TNF-α mRNA in rat and human islets and the 832/13 clonal β-cell line. The surge in transcription of the TNF-α gene required the inhibitor of kappa B kinase beta (IκKβ), the p65 subunit of the NF-κB and a signal-specific recruitment of RNA polymerase II to the gene promoter. Of note was the increased intracellular production of TNF-α protein in a manner consistent with mRNA accumulation in response to IL-1β, but no detectable secretion of TNF-α into the media. Additionally, TNF-α specifically induces expression of CD11b, but not CD11c, on neutrophils, which could contribute to the inflammatory milieu and diabetes progression. We conclude that activation of the NF-κB pathway in pancreatic β-cells leads to rapid intracellular production of the pro-inflammatory TNF-α protein through a combination of specific histone covalent modifications and NF-κB signaling pathways.

Histological analysis of glucagon-like peptide-1 receptor expression in chicken pancreas.

Glucagon-like peptide-1 (GLP-1) released from intestinal L cells in response to nutrient ingestion inhibits both gastrointestinal emptying and gastric acid secretion and promotes satiety. The main biological effect of GLP-1 is the stimulation of insulin secretion (thereby fulfilling the criterion for an incretin hormone) in order to reduce blood glucose levels in mammalian species. Chicken GLP-1 receptor (cGLP-1R) has also been identified in various tissues by gene expression analysis. Although certain effects of GLP-1 in mammals and birds are consistent, e.g., inhibition of food intake, whether GLP-1 has the same insulinotropic activity in chickens as in mammals is debated. Moreover, the expression of cGLP-1R in chicken pancreatic B cells has not been reported. The localization of cGLP-1R and its mRNA in pancreatic islets is studied by triple-immunofluorescence microscopy and in situ hybridization. Triple-immunofluorescence microscopy with antisera against cGLP-1R, somatostatin and insulin or glucagon revealed that cGLP-1R protein was exclusively localized in D cells producing somatostatin in chicken pancreatic islets. The D cells were localized in peripheral areas of the pancreatic islets and cGLP-1R mRNA was detected in the same areas, indicating that cGLP-1R mRNA was also expressed in D cells. This is the first report to demonstrate that cGLP-1R is expressed by D cells, not B cells as in mammals. Our study suggests that chicken GLP-1 performs its insulinotropic activity by a different mode of action from that of the mammalian hormone.

CNX-011-67, a novel GPR40 agonist, enhances glucose responsiveness, insulin secretion and islet insulin content in n-STZ rats and in islets from type 2 diabetic patients

BackgroundGPR40 is a G-protein coupled receptor regulating free fatty acid induced and also glucose induced insulin secretion. We generated neonatally-streptozotocin-treated female rats (n-STZ) and treated them with CNX-011-67, a GPR40 agonist to examine the role of GPR40 in modulation of glucose metabolism, insulin secretion and content.MethodsFemale n-STZ animals were orally administered with CNX-011-67 (15 mg/kg body weight, twice daily) or with vehicle for 8 weeks (n = 8 per group). Glucose tolerance in treated animals and insulin secretion, islet insulin content and gene expression in isolated islets were determined. Islets from type 2 diabetic mellitus (T2DM) patients were treated with different concentrations of glucose in presence or absence of CNX-011-67 and insulin secretion was measured.ResultsTreatment of n-STZ rats with GPR40 agonist CNX-011-67 enhanced insulin secretion in response to oral glucose load on day 0 and this response persisted during the treatment period. The treatment also produced a ‘memory effect’ during which insulin secretion in response to oral glucose load remained enhanced, for a week, even in absence of the agonist. Activation of GPR40 enhanced responsiveness of islets to glucose and increased glucose induced insulin secretion and islet insulin content. An increase in islet mRNA expression of GCK, PDX1, insulin and PC was also observed. Acute treatment of islets from n-STZ rats with GPR40 agonist enhanced cellular ATP content. Activation of GPR40 enhanced mitochondrial calcium level in NIT-1 insulinoma cells. CNX-011-67 increased insulin secretion in islets from T2DM patients which were non-responsive to increased glucose concentrationConclusionsOur data provide evidence that activation of GPR40 with CNX-011-67 stimulates glucose metabolism, enhances glucose responsiveness, increases insulin secretion and content and that pharmacological activation of GPR40 will prove beneficial for treatment of T2DM.

The proapoptotic BH3-only proteins Bim and Puma are downstream of endoplasmic reticulum and mitochondrial oxidative stress in pancreatic islets in response to glucotoxicity

Apoptosis of pancreatic beta cells is a feature of type 2 diabetes and its prevention may have therapeutic benefit. High glucose concentrations induce apoptosis of islet cells, and this requires the proapoptotic Bcl-2 homology domain 3 (BH3)-only proteins Bim and Puma. We studied the stress pathways induced by glucotoxicity in beta cells that result in apoptosis. High concentrations of glucose or ribose increased expression of the transcription factor CHOP (C/EBP homologous protein) but not endoplasmic reticulum (ER) chaperones, indicating activation of proapoptotic ER stress signaling. Inhibition of ER stress prevented ribose-induced upregulation of Chop and Puma mRNA, and partially protected islets from glucotoxicity. Loss of Bim or Puma partially protected islets from the canonical ER stressor thapsigargin. The antioxidant N-acetyl-cysteine also partially protected islets from glucotoxicity. Islets deficient in both Bim and Puma, but not Bim or Puma alone, were significantly protected from killing induced by the mitochondrial reactive oxygen species donor rotenone. Our data demonstrate that high concentrations of glucose induce ER and oxidative stress, which causes cell death mediated by Bim and Puma. We observed significantly higher Bim and Puma mRNA in islets of human donors with type 2 diabetes. This indicates that inhibition of Bim and Puma, or their inducers, may prevent beta-cell destruction in type 2 diabetes.

Dietary Selenium Deficiency Partially Rescues Type 2 Diabetes–Like Phenotypes of Glutathione Peroxidase-1–Overexpressing Male Mice1–3

This study was conducted to determine whether dietary Se deficiency precluded overproduction of glutathione peroxidase-1 (GPX1) activity in mice overexpressing (OE) this gene and thus rescued their type 2 diabetes–like phenotypes. A total of 20 male OE and wild-type (WT) mice were fed an Se-deficient (<0.02 mg/kg) diet or an Se-supplemented (0.3 mg/kg as sodium selenite) diet from 1 to 5 mo of age. Dietary Se deficiency eliminated or attenuated (P < 0.05) genotype differences in concentrations of blood glucose, plasma insulin, and/or hepatic lipids, insulin sensitivity, and glucose-stimulated insulin secretion at the end of the study. Dietary Se deficiency decreased (P < 0.05) OE islet mRNA levels of 2 key transcriptional activators (Beta2 and Foxa2) and removed genotype differences in islet mRNA levels of 7 genes (Beta2, Cfos, Foxa2, Pregluc, Ins1, p53, and Sur1) related to insulin synthesis and secretion. Compared with those of the Se-adequate OE mice, the Se-deficient OE mice had lower (P < 0.05) hepatic mRNA levels of 2 key rate-limiting enzymes for lipogenesis (Acc1) and glycolysis (Gk1), along with lower (P < 0.05) activities of hepatic glucokinase and muscle phosphoenolpyruvate carboxykinase. Dietary Se deficiency also decreased (P < 0.05) blood glucose and hepatic lipid concentrations in the WT mice. In conclusion, dietary Se deficiency precluded the overproduction of GPX1 in full-fed OE mice and partially rescued their metabolic syndromes. This alleviation resulted from modulating the expression and/or function of proinsulin genes, lipogenesis rate-limiting enzyme genes, and key glycolysis and gluconeogenesis enzymes in islets, liver, and muscle.

GPR119 expression in normal human tissues and islet cell tumors: evidence for its islet-gastrointestinal distribution, expression in pancreatic beta and alpha cells, and involvement in islet function

ObjectiveGPR119 is reportedly involved in regulating glucose metabolism and food intake in rodents, but little is known about its expression and functional significance in humans. To begin to assess the potential clinical importance of GPR119, the distribution of GPR119 gene expression in humans was examined. Materials/MethodsExpression of GPR119 mRNA in fresh samples of normal human pancreas (n=19) and pancreatic islets (n=3) and in insulinomas (n=2) and glucagonomas (n=2), all collected at surgery, was compared with the mRNA expression of various receptors highly expressed and operative in human pancreatic islets. ResultsGPR119 mRNA was most abundant in the pancreas, followed by the duodenum, stomach, jejunum, ileum and colon. Pancreatic levels of GPR119 mRNA were similar to those of GPR40 mRNA and were higher than those of GLP1R and SUR1 mRNA, which are strongly expressed in human pancreatic islets. Moreover, levels of GPR119 mRNA in pancreatic islets were more than 10 times higher than in adjacent pancreatic tissue, as were levels of GPR40 mRNA. GPR119 mRNA was also abundant in two cases of insulinoma and two cases of glucagonoma, but was undetectable in a pancreatic acinar cell tumor. Similar results were obtained with mouse pancreatic islets, MIN6 insulinoma cells and alpha-TC glucagonoma cells. ConclusionsThe results provide evidence of an islet-gastrointestinal distribution of GPR119, its expression in pancreatic beta and alpha cells, and its possible involvement in islet function. They also provide the basis for a better understanding of the potential clinical importance of GPR119.

Inhibition of Glucose-Stimulated Insulin Secretion by KCNJ15, a Newly Identified Susceptibility Gene for Type 2 Diabetes

Potassium inwardly rectifying channel, subfamily J, member 15 (KCNJ15) is a type 2 diabetes–associated risk gene, and Kcnj15 overexpression suppresses insulin secretion in rat insulinoma (INS1) cells. The aim of the current study was to characterize the role of Kcnj15 by knockdown of this gene in vitro and in vivo. Human islet cells were used to determine the expression of KCNJ15. Expression of KCNJ15 mRNA in islets was higher in subjects with type 2 diabetes. In INS1 cells, Kcnj15 expression was induced by high glucose–containing medium. Regulation of Kcnj15 by glucose and its effect on insulin secretion were analyzed in INS1 cells and in normal mice and diabetic mice by the inactivation of Kcnj15 using small interfering RNA. Knockdown of Kcnj15 increased the insulin secretion in vitro and in vivo. KCNJ15 and Ca2+-sensing receptor (CsR) interact in the kidney. Binding of Kcnj15 with CsR was also detected in INS1 cells. In conclusion, downregulation of Kcnj15 leads to increased insulin secretion in vitro and in vivo. The mechanism to regulate insulin secretion involves KCNJ15 and CsR.

Ghrelin Inhibits Insulin Release by Regulating the Expression of Inwardly Rectifying Potassium Channel 6.2 in Islets

IntroductionThe objective is to investigate the influence of ghrelin administration on both the insulin secretion and the expression of ATP-sensitive K+ channels in islet. MethodsGhrelin and [D-Lys3] growth hormone releasing peptide-6 were administered via intraperitoneal injection in Wistar rats at the doses 10 and 10μmol/kg/d for 2weeks, respectively. Then glucose tolerance tests were performed and plasma insulin concentrations were measured. Islets were isolated for insulin release experiments. Single β cells were isolated for electrophysiological experiments. Determination of the Kir6.2 and SUR1 mRNA and protein expression levels in islets was performed by polymerase chain reaction and western blotting. ResultsIntraperitoneal administration of exogenous ghrelin significantly (P<0.05) increased blood glucose concentrations, attenuated insulin responses during glucose tolerance tests, reduced insulin release from the isolated islets induced by 11.1 and 16.7mmol/L glucose, hyperpolarized the resting membrane potential and increased the Kir6.2 mRNA and protein expression levels. In contrast, counteraction of ghrelin by intraperitoneal injection of [D-Lys3] growth hormone releasing peptide-6 significantly (P<0.05) attenuated the aforementioned changes. SUR1 expression levels were not altered in this study. ConclusionsGhrelin via pancreatic growth hormone secretagogue receptor up-regulates the Kir6.2 expression in islet by hyperpolarizing the resting membrane potential which results in the inhibition of insulin release.

Immunohistochemical assessment of Pax8 expression during pancreatic islet development and in human neuroendocrine tumors

The paired box transcription factor Pax8 is critical for development of the eye, thyroid gland as well as the urinary and reproductive organs. In adult, Pax8 overexpression is associated with kidney, ovarian and thyroid tumors and has emerged as a specific marker for these cancers. Recently, Pax8 expression was also reported in human pancreatic islets and in neuroendocrine tumors, identifying Pax8 as a novel member of the Pax family expressed in the pancreas. Herein, we sought to provide a comprehensive analysis of Pax8 expression during pancreogenesis and in adult islets. Immunohistochemical analysis using the most employed Pax8 polyclonal antibody revealed strong nuclear staining in the developing mouse pancreas and in mature human and mouse islets. Astonishingly, Pax8 mRNA in mouse islets was undetectable while human islets exhibited low levels. These discrepancies raised the possibility of antibody cross-reactivity. This premise was confirmed by demonstrating that the polyclonal Pax8 antibody also recognized the islet-enriched Pax6 protein both by Western blotting and immunohistochemistry. Thus, in islets polyclonal Pax8 staining corresponds mainly to Pax6. In order to circumvent this caveat, a novel Pax8 monoclonal antibody was used to re-evaluate whether Pax8 was indeed expressed in islets. Surprisingly, Pax8 was not detected in neither the developing pancreas or in mature islets. Reappraisal of pancreatic neuroendocrine tumors using this Pax8 monoclonal antibody exhibited no immunostaining as compared to the Pax8 polyclonal antibody. In conclusion, Pax8 is not expressed in the pancreas and cast doubts on the value of Pax8 as a pancreatic neuroendocrine tumor marker.

A common variant upstream of the PAX6 gene influences islet function in man

Impaired glucose tolerance and impaired insulin secretion have been reported in families with PAX6 mutations and it is suggested that they result from defective proinsulin processing due to lack of prohormone convertase 1/3, encoded by PCSK1. We investigated whether a common PAX6 variant would mimic these findings and explored in detail its effect on islet function in man. A PAX6 candidate single nucleotide polymorphism (rs685428) was associated with fasting insulin levels in the Diabetes Genetics Initiative genome-wide association study. We explored its potential association with glucose tolerance and insulin processing and secretion in three Scandinavian cohorts (N = 8,897 individuals). In addition, insulin secretion and the expression of PAX6 and transcriptional target genes were studied in human pancreatic islets. rs685428 G allele carriers had lower islet mRNA expression of PAX6 (p = 0.01) and PCSK1 (p = 0.001) than AA homozygotes. The G allele was associated with increased fasting insulin (p (replication) = 0.02, p (all) = 0.0008) and HOMA-insulin resistance (p (replication) = 0.02, p (all) = 0.001) as well as a lower fasting proinsulin/insulin ratio (p (all) = 0.008) and lower fasting glucagon (p = 0.04) and gastric inhibitory peptide (GIP) (p = 0.05) concentrations. Arginine-stimulated (p = 0.02) insulin secretion was reduced in vivo, which was further reflected by a reduction of glucose- and potassium-stimulated insulin secretion (p = 0.002 and p = 0.04, respectively) in human islets in vitro. A common variant in PAX6 is associated with reduced PAX6 and PCSK1 expression in human islets and reduced insulin response, as well as decreased glucagon and GIP concentrations and decreased insulin sensitivity. These findings emphasise the central role of PAX6 in the regulation of islet function and glucose metabolism in man.

Bombesin Receptor Subtype-3 (BRS-3) Regulates Glucose-Stimulated Insulin Secretion in Pancreatic Islets across Multiple Species

Bombesin receptor subtype-3 (BRS-3) regulates energy homeostasis, and BRS-3 agonism is being explored as a possible therapy for obesity. Here we study the role of BRS-3 in the regulation of glucose-stimulated insulin secretion (GSIS) and glucose homeostasis. We quantified BRS-3 mRNA in pancreatic islets from multiple species and examined the acute effects of Bag-1, a selective BRS-3 agonist, on GSIS in mouse, rat, and human islets, and on oral glucose tolerance in mice. BRS-3 is highly expressed in human, mouse, rhesus, and dog (but not rat) pancreatic islets and in rodent insulinoma cell lines (INS-1 832/3 and MIN6). Silencing BRS-3 with small interfering RNA or pharmacological blockade with a BRS-3 antagonist, Bantag-1, reduced GSIS in 832/3 cells. In contrast, the BRS-3 agonist (Bag-1) increased GSIS in 832/3 and MIN6 cells. The augmentation of GSIS by Bag-1 was completely blocked by U73122, a phospholipase C inhibitor. Bag-1 also enhanced GSIS in islets isolated from wild-type, but not Brs3 knockout mice. In vivo, Bag-1 reduced glucose levels during oral glucose tolerance test in a BRS-3-dependent manner. BRS-3 agonists also increased GSIS in human islets. These results identify a potential role for BRS-3 in islet physiology, with agonism directly promoting GSIS. Thus, in addition to its potential role in the treatment of obesity, BRS-3 may also regulate blood glucose levels and have a role in the treatment of diabetes mellitus.

MCP-1 Upregulates Amylin Expression in Murine Pancreatic β Cells through ERK/JNK-AP1 and NF-κB Related Signaling Pathways Independent of CCR2

BackgroundAmylin is the most abundant component of islet amyloid implicated in the development of type 2 diabetes. Plasma amylin levels are elevated in individuals with obesity and insulin resistance. Monocyte chemoattractant protein-1 (MCP-1, CCL2) is involved in insulin resistance of obesity and type 2 diabetes. We investigated the effect of MCP-1 on amylin expression and the underlying mechanisms with murine pancreatic β-cell line MIN6 and pancreatic islets.Methodology/Principal FindingsWe found that MCP-1 induced amylin expression at transcriptional level and increased proamylin and intermediate forms of amylin at protein level in MIN6 cells and islets. However, MCP-1 had no effect on the expressions of proinsulin 1 and 2, as well as prohormone convertase (PC) 1/3 and PC2, suggesting that MCP-1 specifically induces amylin expression in β-cells. Mechanistic studies showed that although there is no detectable CCR2 mRNA in MIN6 cells and islets, pretreatment of MIN6 cells with pertussis toxin inhibited MCP-1 induced amylin expression, suggesting that alternative Gi-coupled receptor(s) mediates the inductive effect of MCP-1. MCP-1 rapidly induced ERK1/2 and JNK phosphorylation. Inhibitors for MEK1/2 (PD98059), JNK (SP600125) or AP1 (curcumin) significantly inhibited MCP-1-induced amylin mRNA expression. MCP-1 failed to induce amylin expression in pancreatic islets isolated from Fos knockout mice. EMSA showed that JNK and ERK1/2 were involved in MCP-1-induced AP1 activation. These results suggest that MCP-1 induces murine amylin expression through AP1 activation mediated by ERK1/2 or JNK. Further studies showed that treatment of MIN6 cells with NF-κB inhibitor or overexpression of IκBα dominant-negative construct in MIN6 cells significantly inhibited MCP-1-induced amylin expression, suggesting that NF-κB related signaling also participates in MCP-1-induced murine amylin expression.Conclusions/SignificanceMCP-1 induces amylin expression through ERK1/2/JNK-AP1 and NF-κB related signaling pathways independent of CCR2. Amylin upregulation by MCP-1 may contribute to elevation of plasma amylin in obesity and insulin resistance.