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Insufficient insulin secretion is a hallmark of type 2 diabetes and has been attributed to beta cell identity loss characterized by decreased expression of several key beta cell genes. The pro-inflammatory factor BMP-2 is upregulated in islets of Langerhans from individuals with diabetes and acts as an inhibitor of beta cell function and proliferation. Exposure to BMP-2 induces expression of Id1-4, Hes-1 and Hey-1 which are transcriptional regulators associated with loss of differentiation. The aim of this study was to investigate the mechanism by which BMP-2 induces beta cell dysfunction and loss of cell maturity. Mouse islets exposed to BMP-2 for 10 days showed impaired glucose-stimulated insulin secretion and beta cell proliferation. BMP-2-induced beta cell dysfunction was associated with decreased expression of cell maturity and proliferation markers specific to the beta cell such as Ins1, Ucn3 and Ki67 and increased expression of Id1-4, Hes-1 and Hey-1. Top 30 most regulated proteins significantly correlated with corresponding mRNA expression. BMP-2-induced gene expression changes were associated with a predominant reduction in acetylation of H3K27 and a decrease in NeuroD1 chromatin binding activity. These results show that BMP-2 induces loss of beta cell maturity and suggest that remodeling of H3K27ac and decreased NeuroD1 DNA binding activity participate in the effect of BMP-2 on beta-cell dysfunction.

Cytokine-induced beta cell dysfunction is a hallmark of type 2 diabetes (T2D). Chronic exposure of beta cells to inflammatory cytokines affects gene expression and impairs insulin secretion. Thus, identification of anti-inflammatory factors that preserve beta cell function represents an opportunity to prevent or treat T2D. Butyrate is a gut microbial metabolite with anti-inflammatory properties for which we recently showed a role in preventing interleukin-1β (IL-1β)-induced beta cell dysfunction, but how prevention is accomplished is unclear. Here, we investigated the mechanisms by which butyrate exerts anti-inflammatory activity in beta cells. We exposed mouse islets and INS-1E cells to a low dose of IL-1β and/or butyrate and measured expression of inflammatory genes and nitric oxide (NO) production. Additionally, we explored the molecular mechanisms underlying butyrate activity by dissecting the activation of the nuclear factor-κB (NF-κB) pathway. We found that butyrate suppressed IL-1β-induced expression of inflammatory genes, such as Nos2, Cxcl1, and Ptgs2, and reduced NO production. Butyrate did not inhibit IκBα degradation nor NF-κB p65 nuclear translocation. Furthermore, butyrate did not affect binding of NF-κB p65 to target sequences in synthetic DNA but inhibited NF-κB p65 binding and RNA polymerase II recruitment to inflammatory gene promoters in the context of native DNA. We found this was concurrent with increased acetylation of NF-κB p65 and histone H4, suggesting butyrate affects NF-κB activity via inhibition of histone deacetylases. Together, our results show butyrate inhibits IL-1β-induced inflammatory gene expression and NO production through suppression of NF-κB activation and thereby possibly preserves beta cell function.

Butyrate produced by the gut microbiota has beneficial effects on metabolism and inflammation. Butyrate-producing bacteria are supported by diets with a high fiber content, such as high-amylose maize starch (HAMS). We investigated the effects of HAMS- and butyrylated HAMS (HAMSB)-supplemented diets on glucose metabolism and inflammation in diabetic db/db mice. Mice fed HAMSB had 8-fold higher fecal butyrate concentration compared to control diet-fed mice. Weekly analysis of fasting blood glucose showed a significant reduction in HAMSB-fed mice when the area under the curve for all five weeks was analyzed. Following treatment, fasting glucose and insulin analysis showed increased homeostatic model assessment (HOMA) insulin sensitivity in the HAMSB-fed mice. Glucose-stimulated insulin release from isolated islets did not differ between the groups, while insulin content was increased by 36% in islets of the HAMSB-fed mice. Expression of insulin 2 was also significantly increased in islets of the HAMSB-fed mice, while no difference in expression of insulin 1, pancreatic and duodenal homeobox 1, MAF bZIP transcription factor A and urocortin 3 between the groups was observed. Hepatic triglycerides in the livers of the HAMSB-fed mice were significantly reduced. Finally, mRNA markers of inflammation in liver and adipose tissue were reduced in mice fed HAMSB. These findings suggest that HAMSB-supplemented diet improves glucose metabolism in the db/db mice, and reduces inflammation in insulin-sensitive tissues.

Pancreatic beta cell dysfunction caused by metabolic and inflammatory stress contributes to the development of type 2 diabetes (T2D). Butyrate, produced by the gut microbiota, has shown beneficial effects on glucose metabolism in animals and humans and may directly affect beta cell function, but the mechanisms are poorly described. The aim of this study was to investigate the effect of butyrate on cytokine-induced beta cell dysfunction in vitro. Mouse islets, rat INS-1E, and human EndoC-βH1 beta cells were exposed long-term to non-cytotoxic concentrations of cytokines and/or butyrate to resemble the slow onset of inflammation in T2D. Beta cell function was assessed by glucose-stimulated insulin secretion (GSIS), gene expression by qPCR and RNA-sequencing, and proliferation by incorporation of EdU into newly synthesized DNA. Butyrate protected beta cells from cytokine-induced impairment of GSIS and insulin content in the three beta cell models. Beta cell proliferation was reduced by both cytokines and butyrate. Expressions of the beta cell specific genes Ins, MafA, and Ucn3 reduced by the cytokine IL-1β were not affected by butyrate. In contrast, butyrate upregulated the expression of secretion/transport-related genes and downregulated inflammatory genes induced by IL-1β in mouse islets. In summary, butyrate prevents pro-inflammatory cytokine-induced beta cell dysfunction.

A central and still open question regarding the pathogenesis of autoimmune diseases, such as type 1 diabetes, concerns the processes that underlie the generation of MHC-presented autoantigenic epitopes that become targets of autoimmune attack. Proteasomal degradation is a key step in processing of proteins for MHC class I presentation. Different types of proteasomes can be expressed in cells dictating the repertoire of peptides presented by the MHC class I complex. Of particular interest for type 1 diabetes is the proteasomal configuration of pancreatic β cells, as this might facilitate autoantigen presentation by β cells and thereby their T-cell mediated destruction. Here we investigated whether so-called inducible subunits of the proteasome are constitutively expressed in β cells, regulated by inflammatory signals and participate in the formation of active intermediate or immuno-proteasomes.We show that inducible proteasomal subunits are constitutively expressed in human and rodent islets and an insulin-secreting cell-line. Moreover, the β5i subunit is incorporated into active intermediate proteasomes that are bound to 19S or 11S regulatory particles. Finally, inducible subunit expression along with increase in total proteasome activities are further upregulated by non-toxic concentrations of IL-1β stimulating proinsulin biosynthesis. These findings suggest that the β cell proteasomal repertoire is more diverse than assumed previously and may be highly responsive to a local inflammatory islet environment.

Increased expression of bone morphogenetic proteins (BMPs) in several tissues is associated with inflammation and type 2 diabetes mellitus. BMP2 and BMP4 mRNA expression is increased in pancreatic islets from db/db mice and β-cell proliferation and function are inhibited by BMP4. The effect of BMPs on α-cells is currently unknown. Here, we investigate the effects of BMP4 on mouse and human α-cells in vitro. The effects of BMP4 on α-cell proliferation and function were investigated in islets isolated from male mice and from human donors, and in α-TC1-6 cells. The effects of BMP4 on α-cell function were assessed by determination of glucagon secretion and gene expression. Treatment with BMP4 for 24-96 hours inhibited glucagon secretion in a time-dependent manner in mouse and human islets. Glucagon content, preproglucagon and aristaless related homeobox mRNA expression were reduced after incubation with BMP4 in mouse islets, but not in human islets. The percentage of proliferating α-cells was reduced from 7.3 % to 0.2 % in mouse islets incubated with BMP4. α-cell proliferation in human islets ranged from 0 to 11.8 %, and BMP4 was found to inhibit proliferation of α-cells from all donors when proliferation was present. In agreement with the observations in primary islets, BMP4 decreased glucagon content, preproglucagon, and aristaless related homeobox mRNA expression in α-TC1-6 cells. Our findings suggest that BMP4 has an inhibitory role on glucagon secretion, α-cell growth, and expression of genes maintaining α-cell identity.

The proinflammatory cytokines interleukin-1 beta (IL-1β) and interferon gamma (IFN-γ) play important roles in the progressive loss of beta-cell mass and function during development of both type 1 and type 2 diabetes. We have recently showed that bone morphogenetic protein (BMP)-2 and -4 are expressed in pancreatic islets and inhibit beta-cell growth and function. In this study, we describe that IL-1β and IFN-γ induce the expression of BMP-2 suggesting a possible role for BMP-2 in mediating the effects of IL-1β and IFN-γ on beta-cell apoptosis and dysfunction. IL-1β increased BMP-2 mRNA levels 6- and 3-fold in isolated islets of Langerhans from neonatal rat and human. Downstream target genes of the BMP pathway were also increased by cytokine treatment and could be reversed by neutralization of endogenous BMP activity. Nuclear factor kappa B- (NFκB) binding sites were identified in the rat BMP-2 promoter, and reporter assays verified the role of NFκB in cytokine-induced BMP-2 expression. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays confirmed NFκB binding to BMP-2 promoter upon IL-1β stimulation in beta cells. In conclusion, we suggest that NFκB stimulates BMP-2 mRNA expression in rat and human beta cells upon cytokine exposure.

Signal transducer and activator of transcription (STAT)5A and -5B are latent transcription factors activated by cytokines and hormones of the cytokine family. In pancreatic insulin-secreting β-cells, STAT5A and -5B are activated primarily by prolactin and growth hormone stimulation and are important mediators of the potent stimulation of proliferation and insulin production caused by these hormones. STAT5A and -5B are both expressed in β-cells and control the expression of a number of mRNAs implicated in cell replication control, insulin biosynthesis and secretion. In addition to STAT5A and -5B being transcriptional activators, they may also repress gene transcription. By these means, STAT5 proteins increase the levels of anti-apoptotic transcripts in β-cells and repress expression of pro-apoptotic genes. This review focuses on the anti-apoptotic role of STAT5 signaling, providing a mechanism for β-cell resistance to pro-apoptotic cytokines, Type 1 diabetes mellitus and obesity-associated β-cell stress. It is clear from studies of STAT5 signaling in pancreatic β-cells that STAT5 is important for postnatal β-cell compensatory growth (as in pregnancy or obesity) and in the defense against β-cell stress factors.

Normal pancreatic islet β-cells (PBCs) abundantly secrete insulin in response to elevated blood glucose levels, in order to maintain an adequate control of energy balance and glucose homeostasis. However, the molecular mechanisms underlying the insulin secretion are unclear. Improving our understanding of glucose-stimulated insulin secretion (GSIS) mechanisms under normal conditions is a prerequisite for developing better interventions against diabetes. Here, we aimed at identifying novel signaling pathways involved in the initial release of insulin from PBCs after glucose stimulation using quantitative strategies for the assessment of phosphorylated proteins and sialylated N-linked (SA) glycoproteins. Islets of Langerhans derived from newborn rats with a subsequent 9-10 days of maturation in vitro were stimulated with 20 mM glucose for 0 min (control), 5 min, 10 min, and 15 min. The isolated islets were subjected to timeresolved quantitative phosphoproteomics and sialiomics using iTRAQ-labeling combined with enrichment of phosphorylated peptides and formerly SA glycopeptides and high-accuracy LC-MS/MS. Using bioinformatics we analyzed the functional signaling pathways during GSIS, including well-known insulin secretion pathways. Furthermore, we identified six novel activated signaling pathways (e.g. agrin interactions and prolactin signaling) at 15 min GSIS, which may increase our understanding of the molecular mechanism underlying GSIS. Moreover, we validated some of the regulated phosphosites by parallel reaction monitoring, which resulted in the validation of eleven new phosphosites significantly regulated on GSIS. Besides protein phosphorylation, alteration in SA glycosylation was observed on several surface proteins on brief GSIS. Interestingly, proteins important for cell-cell interaction, cell movement, cell-ECM interaction and Focal Adhesion (e.g. integrins, semaphorins, and plexins) were found regulated at the level of sialylation, but not in protein expression. Collectively, we believe that this comprehensive Proteomics and PTMomics survey of signaling pathways taking place during brief GSIS of primary PBCs is contributing to understanding the complex signaling underlying GSIS.

Combination treatment with exendin-4 and gastrin has proven beneficial in treatment of diabetes and preservation of beta cell mass in diabetic mice. Here, we examined the chronic effects of a GLP-1-gastrin dual agonist ZP3022 on glycemic control and beta cell dysfunction in overtly diabetic Zucker Diabetic Fatty (ZDF) rats.ZDF rats aged 11 weeks were dosed s.c., b.i.d. for 8 weeks with vehicle, ZP3022, liraglutide, exendin-4, or gastrin-17 with or without exendin-4. Glycemic control was assessed by measurements of HbA1c and blood glucose levels, as well as glucose tolerance during an oral glucose tolerance test (OGTT). Beta cell dynamics were examined by morphometric analyses of beta and alpha cell fractions.ZP3022 improved glycemic control as measured by terminal HbA1c levels (6.2±0.12 (high dose) vs. 7.9±0.07% (vehicle), P0.001), as did all treatments, except gastrin-17 monotherapy. In contrast, only ZP3022, exendin-4 and combination treatment with exendin-4 and gastrin-17 significantly improved glucose tolerance and increased insulin levels during an OGTT. Moreover, only ZP3022 significantly enhanced the beta cell fraction in ZDF rats, a difference of 41%, when compared to the vehicle group (0.31±0.03 vs. 0.22±0.02%, respectively, P0.05).These data suggest that ZP3022 may have therapeutic potential in the prevention/delay of beta cell dysfunction in type 2 diabetes.

GLP-1-gastrin dual agonist ZP3022 has been shown to increase β-cell mass with a concomitant improvement of glycemic control in diabetic mice and rats. Here we tested the in-vitro effects of ZP3022 on β-cell proliferation, islet apoptosis and glucose-stimulated insulin secretion (GSIS) in rat islets of Langerhans. Moreover, gene expression profiling in whole pancreas from Zucker Diabetic Fatty (ZDF) rats was performed to characterize genes differently regulated by short-term treatment with ZP3022. Treatments with exendin-4, gastrin-17 alone or in combination were included in the studies. ZP3022 promoted β-cell proliferation, protected from palmitate-, but not from cytokine-induced apoptosis, and induced an increase in GSIS, demonstrating a glucose dependent insulinotropic action of ZP3022 on β-cells. The combination treatment with exendin-4 and gastrin-17 showed comparable effects on proliferation, apoptosis, and GSIS as did ZP3022. Microarray analysis revealed that ZP3022 exerted specific effects on pancreatic gene expression not observed when treating ZDF rats with either exendin-4 alone or in combination with gastrin-17. In particular MAPK signaling pathway was observed among the highest affected pathways; while also pathways related to insulin signaling and secretion were regulated by ZP3022. Moreover, rats treated with ZP3022 had a higher expression of genes encoding for the specific β-cell/endocrine cell markers, such as islet amyloid polypeptide (IAPP), protein convertase 1/3 and -2 (PC 1/3 and-2), as well as transmembrane protein 27(TMEM27) compared to vehicle treated rats. We conclude that ZP3022 may have therapeutic potential in the prevention/delay of β cell dysfunction.

There is a medical need for new insulin analogues. Yet, molecular alterations to the insulin molecule can theoretically result in analogues with carcinogenic effects. Preclinical carcinogenicity risk assessment for insulin analogues rests to a large extent on mitogenicity assays in cell lines. We therefore optimized mitogenicity assay conditions for a panel of five cell lines. All cell lines expressed insulin receptors (IR), IGF-I receptors (IGF-IR) and hybrid receptors, and in all cell lines, insulin as well as the comparator compounds X10 and IGF-I caused phosphorylation of the IR as well as IGF-IR. Insulin exhibited mitogenicity EC(50) values in the single-digit nanomolar to picomolar range. We observed correlations across cell types between (i) mitogenic potency of insulin and IGF-IR/IR ratio, (ii) Akt phosphorylation and mitogenic potency and (iii) Akt phosphorylation and IR phosphorylation. Using siRNA-mediated knockdown of IR and IGF-IR, we observed that in HCT 116 cells the IR appeared dominant in driving the mitogenic response to insulin, whereas in MCF7 cells the IGF-IR appeared dominant in driving the mitogenic response to insulin. Together, our results show that the IR as well as IGF-IR may contribute to the mitogenic potency of insulin. While insulin was a more potent mitogen than IGF-I in cells expressing more IR than IGF-IR, the hyper-mitogenic insulin analogue X10 was a more potent mitogen than insulin across all cell types, supporting that the hyper-mitogenic effect of X10 involves the IR as well as the IGF-IR. These results are relevant for preclinical safety assessment of developmental insulin analogues.

Impairment of beta cell mass and function is evident in both type 1 and type 2 diabetes. In healthy physiological conditions pancreatic beta cells adapt to the body's increasing insulin requirements by proliferation and improved function. We hypothesised that during the development of diabetes, there is an increase in the expression of inhibitory factors that prevent the beta cells from adapting to the increased need for insulin. We evaluated the effects of bone morphogenetic protein (BMP) 2 and -4 on beta cells.The effects of BMP2 and -4 on beta cell proliferation, apoptosis, gene expression and insulin release were studied in isolated islets of Langerhans from rats, mice and humans. The expression of BMPs was analysed by immunocytochemistry and real-time PCR. The role of endogenous BMP was investigated using a soluble and neutralising form of the BMP receptor 1A.BMP2 and -4 were found to inhibit basal as well as growth factor-stimulated proliferation of primary beta cells from rats and mice. Bmp2 and Bmp4 mRNA and protein were expressed in islets and regulated by inflammatory cytokines. Neutralisation of endogenous BMP activity resulted in enhanced proliferation of rodent beta cells. The expression of Id mRNAs was induced by BMP4 in rat and human islets. Finally, glucose-induced insulin secretion was significantly impaired in rodent and human islets pre-treated with BMP4, and inhibition of BMP activity resulted in enhanced insulin release.These data show that BMP2 and -4 exert inhibitory actions on beta cells in vitro and suggest that BMPs exert regulatory roles of beta cell growth and function.

Objective Several studies have shown increased beta cell mass during pregnancy in both rodents and humans. Proliferation of existing beta cells seems to be the predominant mechanism in rodents, whereas the mechanism in humans is unclear. We hypothesized that neogenesis contributes to the increased beta cell mass in pregnancy and that circulating factors are involved. Samples Pancreatic tissue from mice and rat and serum from pregnant women. Method Morphometric analysis of pancreas of pregnant and nonpregnant mice was carried out by immunocytochemical staining for the neogenic marker neurogenin-3. Messenger RNA levels of neurogenin-3 and the transcription factor musculoaponeurotic fibrosarcoma oncogene family protein B in fetal rat pancreas cells, cultured with serum from pregnant women, were measured by quantitative polymerase chain reaction. Main outcome measures The number of neurogenin-3-positive cells present in pregnant mice was increased compared with nonpregnant mice. Neurogenin-3 and musculoaponeurotic fibrosarcoma oncogene family protein B mRNA was detected in fetal rat pancreas exposed to serum from pregnant women. Results In pregnant mice we found a 3.6-fold increase in beta cell volume at day 18 compared with nonpregnant mice and a 3.5-fold increase in neurogenin-3 volume at day 14, mainly located in the acinar compartment where it was eightfold higher than in nonpregnant mice. In fetal rat pancreatic cells exposed to serum from pregnant women we found a marked increase in both neurogenin-3 and musculoaponeurotic fibrosarcoma oncogene family protein B mRNA levels in fibroblast-like cells. Conclusion These results suggest that neogenesis contributes to the increased beta cell mass in pregnancy and that circulating factors are involved in beta cell formation in both the maternal and fetal pancreas during pregnancy.

Decreased insulin secretory capacity in Type 2 diabetes mellitus is associated with beta-cell dedifferentiation and inflammation. We hypothesize that prolonged exposure of beta-cells to low concentrations of IL-1β induce beta-cell dedifferentiation characterized by impaired glucose-stimulated insulin secretion, reduced expression of key beta-cell genes and changes in histone modifications at gene loci known to affect beta-cell function. Ten days exposure to IL-1β at non-cytotoxic concentrations reduced insulin secretion and beta-cell proliferation and decreased expression of key beta-cell identity genes, including MafA and Ucn3 and decreased H3K27ac at the gene loci, suggesting that inflammatory cytokines directly affects the epigenome. Following removal of IL-1β, beta-cell function was normalized and mRNA expression of beta-cell identity genes, such as insulin and Ucn3 returned to pre-stimulation levels. Our findings indicate that prolonged exposure to low concentrations of IL-1β induces epigenetic changes associated with loss of beta-cell identity as observed in Type 2 diabetes.

Maternal low-protein (LP) diet during gestation results in a reduced beta cell mass in the offspring at birth and this may hamper the ability to adapt to high-energy food and sedentary lifestyle later in life. To investigate the biology behind the LP-offspring phenotype, this study aimed to identify differentially expressed genes in the pancreas and their potential role in the fetal programming.Wistar rats were given either an LP diet or normal-chow (NC) diet during gestation and differentially expressed genes in the offspring around the time of birth were identified using RNA microarray and quantitative PCR. The role of a differentially expressed gene, growth arrest specific protein 6 (GAS6), was evaluated in vitro using neonatal rat islets.The mRNA level of Gas6, known to be mitogenic in other tissues, was reduced in LP offspring. The mRNA content of Mafa was increased in LP offspring suggesting an early maturation of beta cells. When applied in vitro, GAS6 increased proliferation of neonatal pancreatic beta cells, while reducing glucose-stimulated insulin secretion without changing the total insulin content of the islets. In addition, GAS6 decreased the mRNA content of Mafa.We propose a role for GAS6 in the regulation of pancreatic beta cells in the critical period around the time of birth. Our results support the hypothesis that the reduced beta cell mass seen in LP offspring is caused by a change in the intra-uterine environment that favours premature maturation of the beta cells.

Pannexins (Panx's) are membrane proteins involved in a variety of biological processes, including cell death signaling and immune functions. The role and functions of Panx's in pancreatic β-cells remain to be clarified. Here, we show Panx1 and Panx2 expression in isolated islets, primary β-cells, and β-cell lines. The expression of Panx2, but not Panx1, was downregulated by interleukin-1β (IL-1β) plus interferon-γ (IFNγ), two pro-inflammatory cytokines suggested to contribute to β-cell demise in type 1 diabetes (T1D). siRNA-mediated knockdown (KD) of Panx2 aggravated cytokine-induced apoptosis in rat INS-1E cells and primary rat β-cells, suggesting anti-apoptotic properties of Panx2. An anti-apoptotic function of Panx2 was confirmed in isolated islets from Panx2-/- mice and in human EndoC-βH1 cells. Panx2 KD was associated with increased cytokine-induced activation of STAT3 and higher expression of inducible nitric oxide synthase (iNOS). Glucose-stimulated insulin release was impaired in Panx2-/- islets, and Panx2-/- mice subjected to multiple low-dose Streptozotocin (MLDS) treatment, a model of T1D, developed more severe diabetes compared to wild type mice. These data suggest that Panx2 is an important regulator of the insulin secretory capacity and apoptosis in pancreatic β-cells.

The relative contributions of the JNK subtypes in inflammatoryβ-cell failure and apoptosis are unclear. The JNK protein family consists of JNK1, JNK2, and JNK3 subtypes, encompassing many different isoforms. INS-1 cells express JNK1α1, JNK1α2, JNK1β1, JNK1β2, JNK2α1, JNK2α2, JNK3α1, and JNK3α2 mRNA isoform transcripts translating into 46 and 54 kDa isoform JNK proteins. Utilizing Lentiviral mediated expression of shRNAs against JNK1, JNK2, or JNK3 in insulin-producing INS-1 cells, we investigated the role of individual JNK subtypes in IL-1β-inducedβ-cell apoptosis. JNK1 knockdown prevented IL-1β-induced INS-1 cell apoptosis associated with decreased 46 kDa isoform JNK protein phosphorylation and attenuated Myc expression. Transient knockdown of Myc also prevented IL-1β-induced apoptosis as well as caspase 3 cleavage. JNK2 shRNA potentiated IL-1β-induced apoptosis and caspase 3 cleavage, whereas JNK3 shRNA did not affect IL-1β-inducedβ-cell death compared to nonsense shRNA expressing INS-1 cells. In conclusion, JNK1 mediates INS-1 cell death associated with increased Myc expression. These findings underline the importance of differentiated targeting of JNK subtypes in the development of inflammatoryβ-cell failure and destruction.

We investigated the impact of β-cell specific overexpression of suppressor of cytokine signalling-3 (SOCS-3) on the development of multiple low dose streptozotocin (MLDSTZ) induced Type 1 diabetes and the possible mechanisms involved. MLDSTZ treatment was administered to RIP-SOCS-3 transgenic and wild-type (wt) mice and progression of hyperglycemia monitored. Isolated islets from both strains were exposed to human IL-1β (25 U/ml) or a combination of human IL-1β (25 U/ml) and murine IFN-γ (1000 U/ml) for 24 h or 48 h and we investigated the expression of IL-1 receptor antagonist (IL-1Ra) mRNA in islet cells and secretion of IL-1Ra into culture medium. MLDSTZ treatment caused gradual hyperglycemia both in the wt mice and in the transgenic mice with the latter tending to be more sensitive. In vitro experiments on wt and transgenic islets did not reveal any differences in sensitivity to damaging effects of STZ. Exposure of wt islets to IL-1β or IL-1β + IFN-γ seemed to lead to a failing IL-1Ra response from SOCS-3 transgenic islets. It could be that an increased expression of a possible protective molecule against β-cell destruction may lead to a dampered response of another putative protective molecule. This may have counteracted a protective effect against MLDSTZ in SOCS-3 transgenic mice.

Both common forms of diabetes have an inflammatory pathogenesis in which immune and metabolic factors converge on interleukin-1β as a key mediator of insulin resistance and β-cell failure. In addition to improving insulin resistance and preventing β-cell inflammatory damage, there is evidence of genetic association between diabetes and histone deacetylases (HDACs); and HDAC inhibitors (HDACi) promote β-cell development, proliferation, differentiation and function and positively affect late diabetic microvascular complications. Here we review this evidence and propose that there is a strong rationale for preclinical studies and clinical trials with the aim of testing the utility of HDACi as a novel therapy for diabetes.

Biological activity of the neural cell adhesion molecule (NCAM) depends on both adhesion and acti-vation of intra-cellular signaling. Based on in vitro experiments with truncated extra-cellulardomains, several models describing homophilic NCAM trans- and cis-interactions have been pro-posed. However, cis-dimerization in living cells has not been shown directly and the role of the cyto-plasmic part in NCAM dimerization is poorly understood. Here, we used the bioluminescenceresonance energy transfer (BRET 2 ) technique to directly demonstrate that full-length NCAM cis-homodimerizes in living cells. Based on BRET 2 50 values we suggest that the intra-cellular part ofNCAM inhibits cis-dimerization, an effect mainly dependent on the palmitoylation sites.Structured summary:MINT-8071463: NCAM140 (uniprotkb:P13591-1) physically interacts (MI:0915) with NCAM140(uniprotkb:P13591-1) by bioluminescence resonance energy transfer (MI:0012)MINT-8071506: NCAM180 (genbank_protein_gi:119587605) physically interacts (MI:0915) with NCAM-180 (genbank_protein_gi:119587605) by bioluminescence resonance energy transfer (MI:0012)MINT-8071483: NCAM140 (uniprotkb:P13591-1) physically interacts (MI:0915) with NCAM140(uniprotkb:P13591-1) by competition binding (MI:0405) 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Several studies suggest a link between increased activity of the inflammatory inhibitor-kappaB kinase/nuclear factor-kappaB (IKK/NF-kappaB) pathway in skeletal muscle and insulin resistance.We aimed to study the regulation of skeletal muscle IKK/NF-kappaB pathway activity as well as the association with glucose metabolism and skeletal muscle insulin signaling.The study population included a metabolically well-characterized cohort of young and elderly predominantly nondiabetic twins (n = 181). Inhibitor-kappaBbeta (IkappaBbeta) protein levels are negatively associated with IKK/NF-kappaB pathway activity and were used to evaluate pathway activity with p65 levels included as loading control. This indirect measure for IKK/NF-kappaB pathway activity was validated by a p65 binding assay.Evaluating the effects of heritability, age, sex, obesity, aerobic capacity, and several hormonal factors (eg insulin and TNF-alpha), only sex and age were significant predictors of IkappaBbeta to p65 ratio (28% decreased ratio in the elderly, P0.01, and 49% increased in males P0.01). IkappaBbeta to p65 ratio was unrelated to peripheral insulin sensitivity (P = 0.51) and in accordance with this also unrelated to proximal insulin signaling (P = 0.81). Although no association was seen with plasma glucose after oral glucose challenge, there was a tendency for lower IkappaBbeta to p65 ratio (adjusted for age and sex) in subjects with impaired as opposed to normal glucose tolerance (P = 0.055).Altogether the subtle elevated IKK/NF-kappaB pathway activity seen in glucose-intolerant subjects suggests that IKK/NF-kappaB pathway activation may be secondary to impaired glucose tolerance and that skeletal muscle IKK/NF-kappaB pathway activity is unlikely to play any major role in the control of skeletal muscle insulin action in nondiabetic subjects.

Tumor necrosis factor-alpha (TNFalpha) is a pro-inflammatory cytokine involved in the pathogenesis of several diseases including type 1 diabetes mellitus (T1DM). TNFalpha in combination with interleukin-1-beta (IL-1beta) and/or interferon-gamma (IFNgamma) induces specific destruction of the pancreatic insulin-producing beta cells. Suppressor of cytokine signalling-3 (SOCS-3) proteins regulate signalling induced by a number of cytokines including growth hormone, IFNgamma and IL-1beta which signals via very distinctive pathways. The objective of this study was to investigate the effect of SOCS-3 on TNFalpha-induced signalling in beta cells. We found that apoptosis induced by TNFalpha alone or in combination with IL-1beta was suppressed by expression of SOCS-3 in the beta cell line INSr3#2. SOCS-3 inhibited TNFalpha-induced phosphorylation of the mitogen activated protein kinases ERK1/2, p38 and JNK in INSr3#2 cells and in primary rat islets. Furthermore, SOCS-3 repressed TNFalpha-induced degradation of IkappaB, NFkappaB DNA binding and transcription of the NFkappaB-dependent MnSOD promoter. Finally, expression of Socs-3 mRNA was induced by TNFalpha in rat islets in a transient manner with maximum expression after 1-2h. The ability of SOCS-3 to regulate signalling induced by the three major pro-inflammatory cytokines involved in the pathogenesis of T1DM makes SOCS-3 an interesting therapeutic candidate for protection of the beta cell mass.

OBJECTIVE Proinflammatory cytokines are cytotoxic to β-cells and have been implicated in the pathogenesis of type 1 diabetes and islet graft failure. The importance of the intrinsic mitochondrial apoptotic pathway in cytokine-induced β-cell death is unclear. Here, cytokine activation of the intrinsic apoptotic pathway and the role of the two proapoptotic Bcl-2 proteins, Bad and Bax, were examined in β-cells. RESEARCH DESIGN AND METHODS Human and rat islets and INS-1 cells were exposed to a combination of proinflammatory cytokines (interleukin-1β, interferon-γ, and/or tumor necrosis factor-α). Activation of Bad was determined by Ser136 dephosphorylation, mitochondrial stress by changes in mitochondrial metabolic activity and cytochrome c release, downstream apoptotic signaling by activation of caspase-9 and -3, and DNA fragmentation. The inhibitors FK506 and V5 were used to investigate the role of Bad and Bax activation, respectively. RESULTS We found that proinflammatory cytokines induced calcineurin-dependent dephosphorylation of Bad Ser136, mitochondrial stress, cytochrome c release, activation of caspase-9 and -3, and DNA fragmentation. Inhibition of Bad Ser136 dephosphorylation or Bax was found to inhibit cytokine-induced intrinsic proapoptotic signaling. CONCLUSIONS Our findings demonstrate that the intrinsic mitochondrial apoptotic pathway contributes significantly to cytokine-induced β-cell death and suggest a functional role of calcineurin-mediated Bad Ser136 dephosphorylation and Bax activity in cytokine-induced apoptosis.

Udgivelsesdato: 2009-Mar AIMS/HYPOTHESIS: The suppressor of cytokine signalling 1 (SOCS1) is a natural inhibitor of cytokine and insulin signalling pathways and may also play a role in obesity. In addition, SOCS1 is considered a candidate gene in the pathogenesis of both type 1 diabetes (T1D) and type 2 diabetes (T2D). The objective was to perform mutation analysis of SOCS1 and to test the identified variations for association to T2D-related quantitative traits, T2D or T1D. METHODS: Mutation scanning was performed by direct sequencing in 27 white Danish subjects. Genotyping was carried out by TaqMan allelic discrimination. A total of more than 8100 individuals were genotyped. RESULTS: Eight variations were identified in the 5' untranslated region (UTR) region. Two of these had allele frequencies below 1% and were not further examined. The six other variants were analysed in groups of T1D families (n = 1461 subjects) and T2D patients (n = 1430), glucose tolerant first-degree relatives of T2D patients (n = 212) and normal glucose tolerant (NGT) subjects. The rs33977706 polymorphism (-820G > T) was associated with a lower body mass index (BMI) (p = 0.004). In a second study (n = 4625 NGT subjects), significant associations of both the rs33977706 and the rs243330 (-1656G > A) variants to obesity were found (p = 0.047 and p = 0.015) respectively. The rs33977706 affected both binding of a nuclear protein to and the transcriptional activity of the SOCS1 promoter, indicating a relationship between this polymorphism and gene regulation. CONCLUSIONS/INTERPRETATION: This study demonstrates that functional variations in the SOCS1 promoter may associate with alterations in BMI in the general white population.

Udgivelsesdato: 2009-Mar Context: Whether GH promotes IGF-I production or lipolysis depends on nutritional status, but the underlying mechanisms remain unknown. Objective: To investigate the impact of fasting on GH mediated changes in substrate metabolism, insulin sensitivity and signaling pathways. Design: randomized cross-over study. Subjects: Ten healthy men (age: 24.3+/-0.6 yrs., BMI 23.1+/-0.4 kg/m(2)). Intervention: A GH bolus administered 1) postabsorptively, and 2) in the fasting state (37(1/2) hours). Skeletal muscle and adipose tissue biopsies were taken and a hyperinsulineamic euglycemic clamp was performed on both occasions. Main outcome measures: Metabolic clearance rate (MCR) of GH, substrate metabolism and insulin sensitivity. Biopsies were subjected to western blotting for expression of signaling proteins and to RT-PCR for expression of suppressor of cytokine signaling protein 3 and IGF-I mRNA. Results: Fasting was associated with: reduced MCR of GH (p

Chemokines recruit activated immune cells to sites of inflammation and are important mediators of insulitis. Activation of the pro-apoptotic receptor Fas leads to apoptosis-mediated death of the Fas-expressing cell. The pro-inflammatory cytokines IL-1beta and IFN-gamma regulate the transcription of genes encoding the Fas receptor and several chemokines. We have previously shown that suppressor of cytokine signalling (SOCS)-3 inhibits IL-1beta- and IFN-gamma-induced nitric oxide production in a beta cell line. The aim of this study was to investigate whether SOCS-3 can influence cytokine-induced Fas and chemokine expression in beta cells.Using a beta cell line with inducible Socs3 expression or primary neonatal rat islet cells transduced with a Socs3-encoding adenovirus, we employed real-time RT-PCR analysis to investigate whether SOCS-3 affects cytokine-induced chemokine and Fas mRNA expression. The ability of SOCS-3 to influence the activity of cytokine-responsive Fas and Mcp-1 (also known as Ccl2) promoters was measured by reporter analysis.IL-1beta induced a time-dependent increase in Mcp-1 and Mip-2 (also known as Cxcl2) mRNA expression after 6 h of stimulation in insulinoma (INS)-1 and neonatal rat islet cells. This induction was inhibited when Socs3 was expressed in the cells. In INS-1 cells, IL-1beta + IFN-gamma induced a tenfold and eightfold increase of Fas mRNA expression after 6 and 24 h, respectively. This induction was inhibited at both time-points when expression of Socs3 was induced. In promoter studies SOCS-3 significantly inhibited the cytokine-induced activity of Mcp-1 and Fas promoter constructs.SOCS-3 inhibits the expression of cytokine-induced chemokine and death-receptor Fas mRNA.

The pro-inflammatory cytokines IL-1 and IFNγ are critical molecules in immune-mediated beta cell destruction leading to type 1 diabetes mellitus. Suppressor of cytokine signalling (SOCS)-3 inhibits the cytokine-mediated destruction of insulinoma-1 cells. Here we investigate the effect of SOCS3 in primary rodent beta cells and diabetic animal models. Using mice with beta cell-specific Socs3 expression and a Socs3-encoding adenovirus construct, we characterised the protective effect of SOCS3 in mouse and rat islets subjected to cytokine stimulation. In transplantation studies of NOD mice and alloxan-treated mice the survival of Socs3 transgenic islets was investigated. Socs3 transgenic islets showed significant resistance to cytokine-induced apoptosis and impaired insulin release. Neither glucose-stimulated insulin release, insulin content or glucose oxidation were affected by SOCS3. Rat islet cultures transduced with Socs3-adenovirus displayed reduced cytokine-induced nitric oxide and apoptosis associated with inhibition of the IL-1-induced nuclear factor-κB and mitogen-activated protein kinase (MAPK) pathways. Transplanted Socs3 transgenic islets were not protected in diabetic NOD mice, but showed a prolonged graft survival when transplanted into diabetic allogenic BALB/c mice. SOCS3 inhibits IL-1-induced signalling through the nuclear factor-κB and MAPK pathways and apoptosis induced by cytokines in primary beta cells. Moreover, Socs3 transgenic islets are protected in an allogenic transplantation model. SOCS3 may represent a target for pharmacological or genetic engineering in islet transplantation for treatment of type 1 diabetes mellitus.

The tale of cytokines and the beta-cell is a long story, starting with in vitro discovery in 1984, evolving via descriptive and phenomenological studies to detailed mapping of the signalling pathways, gene- and protein expression patterns, molecular and biochemical effector mechanisms to in vivo studies in spontaneously diabetic and transgenic animal models. Only very recently have steps been taken to translate the accumulating compelling preclinical data into clinical trials. The aim of this chapter is to present an overview of early and recent key observations from our own groups as well as other laboratories that serve to illuminate the road from concept to clinical translation.

The immune-mediated elimination of pancreatic beta cells in type 1 diabetes involves release of cytotoxic cytokines such as IL-1beta and IFNgamma, which induce beta cell death in vitro by mechanisms that are both dependent and independent of nitric oxide (NO). Nuclear factor kappa B (NFkappaB) is a critical signalling molecule in inflammation and is required for expression of the gene encoding inducible NO synthase (iNOS) and of pro-apoptotic genes. NFkappaB has recently been shown to associate with chromatin-modifying enzymes histone acetyltransferases and histone deacetylases (HDAC), and positive effects of HDAC inhibition have been obtained in several inflammatory diseases. Thus, the aim of this study was to investigate whether HDAC inhibition protects beta cells against cytokine-induced toxicity.The beta cell line, INS-1, or intact rat islets were precultured with HDAC inhibitors suberoylanilide hydroxamic acid or trichostatin A in the absence or presence of IL-1beta and IFNgamma. Effects on insulin secretion and NO formation were measured by ELISA and Griess reagent, respectively. iNOS levels and NFkappaB activity were measured by immunoblotting and by immunoblotting combined with electrophoretic mobility shift assay, respectively. Viability was analysed by 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl-tetrazolium bromide and apoptosis by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay and histone-DNA complex ELISA.HDAC inhibition reduced cytokine-mediated decrease in insulin secretion and increase in iNOS levels, NO formation and apoptosis. IL-1beta induced a bi-phasic phosphorylation of inhibitor protein kappa Balpha (IkappaBalpha) with the 2nd peak being sensitive to HDAC inhibition. No effect was seen on IkappaBalpha degradation and NFkappaB DNA binding.HDAC inhibition prevents cytokine-induced beta cell apoptosis and impaired beta cell function associated with a downregulation of NFkappaB transactivating activity.

Interleukin-1β (IL-1β) and interferon-γ (IFNγ) contribute to type 1 diabetes (T1D) by inducing β-cell death. Tumor necrosis factor (TNF) receptor-associated factor (TRAF) proteins are adaptors that transduce signaling from a variety of membrane receptors including cytokine receptors. We show here that IL-1β and IFNγ upregulate the expression of TRAF2 in insulin-producing INS-1E cells and isolated rat pancreatic islets. siRNA-mediated knockdown (KD) of TRAF2 in INS-1E cells reduced IL-1β-induced phosphorylation of JNK1/2, but not of p38 or ERK1/2 mitogen-activated protein kinases. TRAF2 KD did not modulate NFκB activation by cytokines, but reduced cytokine-induced inducible nitric oxide synthase (iNOS) promotor activity and expression. We further observed that IFNγ-stimulated phosphorylation of STAT3 required TRAF2. KD of TRAF2 or STAT3 reduced cytokine-induced caspase 3/7 activation, but, intriguingly, potentiated cytokine-mediated loss of plasma membrane integrity and augmented the number of propidium iodide-positive cells. Finally, we found that TRAF2 KD increased cytokine-induced production of reactive oxygen species (ROS). In summary, our data suggest that TRAF2 is an important mediator of IL-1β and IFNγ signaling in pancreatic β-cells.

The inhibitory neurotransmitter GABA is synthesized by the enzyme glutamic acid decarboxylase (GAD) in neurons and in pancreatic β-cells in islets of Langerhans where it functions as a paracrine and autocrine signaling molecule regulating the function of islet endocrine cells. The localization of the two non-allelic isoforms GAD65 and GAD67 to vesicular membranes is important for rapid delivery and accumulation of GABA for regulated secretion. While the membrane anchoring and trafficking of GAD65 are mediated by intrinsic hydrophobic modifications, GAD67 remains hydrophilic, and yet is targeted to vesicular membrane pathways and synaptic clusters in neurons by both a GAD65-dependent and a distinct GAD65-independent mechanism. Herein we have investigated the membrane association and targeting of GAD67 and GAD65 in monolayer cultures of primary rat, human, and mouse islets and in insulinoma cells. GAD65 is primarily detected in Golgi membranes and in peripheral vesicles distinct from insulin vesicles in β-cells. In the absence of GAD65, GAD67 is in contrast primarily cytosolic in β-cells; its co-expression with GAD65 is necessary for targeting to Golgi membranes and vesicular compartments. Thus, the GAD65-independent mechanism for targeting of GAD67 to synaptic vesicles in neurons is not functional in islet β-cells. Therefore, only GAD65:GAD65 homodimers and GAD67:GAD65 heterodimers, but not the GAD67:GAD67 homodimer gain access to vesicular compartments in β-cells to facilitate rapid accumulation of newly synthesized GABA for regulated secretion and fine tuning of GABA-signaling in islets of Langerhans.

Aims/hypothesis Type 2 diabetes is characterised by progressive loss of pancreatic beta cell mass and function. Therefore, it is of therapeutic interest to identify factors with the potential to improve beta cell proliferation and insulin secretion. Bone morphogenetic protein 4 (BMP4) expression is increased in diabetic animals and BMP4 reduces glucose-stimulated insulin secretion (GSIS). Here, we investigate the molecular mechanism behind this inhibition. Methods BMP4-mediated inhibition of GSIS was investigated in detail using single cell electrophysiological measurements and live cell Ca2+ imaging. BMP4-mediated gene expression changes were investigated by microarray profiling, quantitative PCR and western blotting. Results Prolonged exposure to BMP4 reduced GSIS from rodent pancreatic islets. This inhibition was associated with decreased exocytosis due to a reduced Ca2+ current through voltage-dependent Ca2+ channels. To identify proteins involved in the inhibition of GSIS, we investigated global gene expression changes induced by BMP4 in neonatal rat pancreatic islets. Expression of the Ca2+-binding protein calbindin1 was significantly induced by BMP4. Overexpression of calbindin1 in primary islet cells reduced GSIS, and the effect of BMP4 on GSIS was lost in islets from calbindin1 (Calb1) knockout mice. Conclusions/interpretation We found BMP4 treatment to markedly inhibit GSIS from rodent pancreatic islets in a calbindin1-dependent manner. Calbindin1 is suggested to mediate the effect of BMP4 by buffering Ca2+ and decreasing Ca2+ channel activity, resulting in diminished insulin exocytosis. Both BMP4 and calbindin1 are potential pharmacological targets for the treatment of beta cell dysfunction.

IL-1beta released from immune cells induces beta cell pro-apoptotic signalling via mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB). In neurons, the neural cell adhesion molecule (NCAM) signals to several elements involved in IL-1beta-induced pro-apoptotic signalling in beta cells. Pancreatic beta cells express NCAM, but its biological effects in these cells are unclear. The aim of this study was to investigate whether there is cross-talk between NCAM signalling and cytokine-induced pro-apoptotic signalling.Western blotting was used to investigate levels of NCAM and inducible nitric oxide synthase, phosphorylation of Src and MAPKs, and cleavage of caspase-3. MAPK activity was investigated with an in vitro kinase assay. Apoptosis was detected by cleaved caspase-3 and a Cell Death Detection ELISA(plus) assay. NCAM-induced fibroblast growth factor receptor (FGFR) activation was investigated in NCAM(-/-) Trex293 cells where FGFR phosphorylation was measured by Western blotting after NCAM transfection.Pre-exposure of INS-1E cells to the FGFR-inhibitor SU5402, but not to the Src-inhibitor PP2, dose-dependently inhibited IL-1beta-mediated MAPK activity. A synthetic peptide, C3d, reported to bind NCAM, did not activate MAPK or Akt as reported in neurons but inhibited IL-1beta-induced MAPK activity, thereby mimicking the effect of SU5402. Furthermore, C3d inhibited NCAM-induced FGFR phosphorylation and apoptosis induced by IL-1beta plus IFN-gamma, but did not affect IL-1beta-induced NF-kappaB signalling.We suggest that NCAM signalling through FGFR is required for efficient IL-1beta pro-apoptotic signalling by facilitating IL-1beta-induced MAPK activation downstream of the NF-kappaB-MAPK branching point. Further, these data identify a novel function of C3d as an inhibitor of NCAM-induced FGFR activity and of IL-1beta-induced MAPK activation in beta cells.

IL-1beta is a candidate mediator of apoptotic beta cell destruction, a process that leads to type 1 diabetes and progression of type 2 diabetes. IL-1beta activates beta cell c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38, all of which are members of the mitogen-activated protein kinase (MAPK) family. Inhibition of JNK prevents IL-1beta-mediated beta cell destruction. In mouse embryo fibroblasts and 3DO T cells, overexpression of the gene encoding growth arrest and DNA-damage-inducible 45beta (Gadd45b) downregulates pro-apoptotic JNK signalling. The aim of this study was to investigate if Gadd45b prevents IL-1beta-induced beta cell MAPK signalling and apoptosis.Rat insulinoma INS-1E cells and mouse beta-TC3 cells stably expressing Gadd45b were generated. The effects of Gadd45b expression on signalling by JNK, ERK and p38 were assessed by Western blotting and kinase assays. Apoptosis rate was measured by terminal deoxynucleotidyl-mediated dUTP-biotin nick end-labelling (TUNEL) and an ELISA designed to detect apoptotic nucleosomes. Expression of endogenous Gadd45b mRNA was measured by RT-PCR.In INS-1E and beta-TC3 cells, expression of Gadd45b inhibited IL-1beta-induced activation of JNK and ERK, but augmented IL-1beta-mediated p38 activity. IL-1beta-induced nitric oxide production and decreases in insulin content and secretion were reduced by GADD45beta. IL-1beta-induced apoptosis was reduced by GADD45beta by up to 77%. Although IL-1beta stimulated the time-dependent induction of endogenous Gadd45b in INS-1E cells and rat islets, expression levels were lower in these cells than in IL-1beta-exposed NIH-3T3 and 3DO T cells.Inadequate induction of Gadd45b, which encodes a novel beta cell JNK and ERK inhibitor, may in part explain the pro-apoptotic response of beta cells to IL-1beta.

The beta cell destruction and insulin deficiency that characterises type 1 diabetes mellitus is partially mediated by cytokines, such as IL-1beta, and by nitric oxide (NO)-dependent and -independent effector mechanisms. IL-1beta activates mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), p38 and c-Jun NH2-terminal kinase (JNK), and the nuclear factor kappa B (NFkappaB) pathway. Both pathways are required for expression of the gene encoding inducible nitric oxide synthase (iNOS) and for IL-1beta-mediated beta cell death. The molecular mechanisms by which these two pathways regulate beta cell Nos2 expression are currently unknown. Therefore, the aim of this study was to clarify the putative crosstalk between MAPK and NFkappaB activation in beta cells.The MAPKs ERK, p38 and JNK were inhibited by SB203580, PD98059 or Tat-JNK binding domain or by cells overexpressing the JNK binding domain. The effects of MAPK inhibition on IL-1beta-induced iNOS production and kappa B inhibitor protein (IkappaB) degradation were examined by western blotting. NFkappaB DNA binding was investigated by electrophoretic mobility shift assay, while NFkappaB-induced gene transcription was evaluated by gene reporter assays.Inhibition of the MAPKs did not affect IkappaB degradation or NFkappaB DNA binding. However, inhibition of ERK reduced NFkappaB-mediated Nos2 expression; serine 276 phosphorylation of the p65 unit of the NFkappaB complex seemed critical, as evaluated by amino acid mutation analysis.ERK activity is required for NFkappaB-mediated transcription of Nos2 in insulin-producing INS-1E cells, indicating that ERK regulates Nos2 expression by increasing the transactivating capacity of NFkappaB. This may involve phosphorylation of Ser276 on p65 by an as yet unidentified kinase.

Growth hormone and prolactin are important growth factors for pancreatic β-cells. The effects exerted by these hormones on proliferation and on insulin synthesis and secretion in β-cells are largely mediated through the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway. Suppressors of cytokine signaling (SOCS) proteins are specific inhibitors of the JAK/STAT pathway acting through a negative-feedback loop. To investigate in vivo effects of SOCS-3 in growth hormone (GH)/prolactin signaling in β-cells we generated transgenic mice with β-cell-specific overexpression of SOCS-3. The relative β-cell proliferation and volume in the mice were measured by morphometry. β-Cell volume of transgenic female mice was reduced by over 30% compared with β-cell volume in wild-type female mice. Stimulation of transgenic islets in vitro with GH showed a reduced tyrosine phosphorylation of STAT-5 when compared with wild-type islets. Transduction of primary islet cultures with adenoviruses expressing various SOCS proteins followed by stimulation with GH or glucagon-like peptide-1 (GLP-1) revealed that SOCS-3 inhibited GH- but not GLP-1-mediated islet cell proliferation, indicating that the decreased β-cell volume observed in female transgenic mice could be caused by an inhibition of GH-induced β-cell proliferation by SOCS-3. In spite of the reduced β-cell volume the transgenic female mice exhibited enhanced glucose tolerance compared with wild-type littermates following an oral glucose-tolerance test. Together these data suggest that SOCS-3 modulates cytokine signaling in pancreatic β-cells and therefore potentially could be a candidate target for development of new treatment strategies for diabetes.

The c-jun N-terminal kinase (JNK) signaling pathway mediates IL-1beta-induced apoptosis in insulin-secreting cells, a mechanism relevant to the destruction of pancreatic beta-cells in type 1 and 2 diabetes. However, the mechanisms that contribute to IL-1beta activation of JNK in beta-cells are largely unknown. In this study, we investigated whether Ca(2+) plays a role for IL-1beta-induced JNK activation. In insulin-secreting rat INS-1 cells cultured in the presence of 11 mm glucose, combined pharmacological blockade of L- and T-type Ca(2+) channels suppressed IL-1beta-induced in vitro phosphorylation of the JNK substrate c-jun and reduced IL-1beta-stimulated activation of JNK1/2 as assessed by immunoblotting. Inhibition of IL-1beta-induced in vitro kinase activity toward c-jun after collective L- and T-type Ca(2+) channel blockade was confirmed in primary rat and ob/ob mouse islets and in mouse betaTC3 cells. Ca(2+) influx, specifically via L-type but not T-type channels, contributed to IL-1beta activation of JNK. Activation of p38 and ERK in response to IL-1beta was also dependent on L-type Ca(2+) influx. Membrane depolarization by KCl, exposure to high glucose, treatment with Ca(2+) ionophore A23187, or exposure to thapsigargin, an inhibitor of sarco(endo)plasmic reticulum Ca(2+) ATPase, all caused an amplification of IL-1beta-induced JNK activation in INS-1 cells. Finally, a chelator of intracellular free Ca(2+) [bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-acetoxymethyl], an inhibitor of calmodulin (W7), and inhibitors of Ca(2+)/calmodulin-dependent kinase (KN62 and KN93) partially reduced IL-1beta-stimulated c-jun phosphorylation in INS-1 or betaTC3 cells. Our data suggest that Ca(2+) plays a permissive role in IL-1beta activation of the JNK signaling pathway in insulin-secreting cells.

Upon leptin binding, the leptin receptor is activated, leading to stimulation of the JAK/STAT signal transduction cascade. The transient character of the tyrosine phosphorylation of JAK2 and STAT3 suggests the involvement of protein tyrosine phosphatases (PTPs) as negative regulators of this signalling pathway. Specifically, recent evidence has suggested that PTP1B might be a key regulator of leptin signalling, based on the resistance to diet-induced obesity and increased leptin signalling observed in PTP1B-deficient mice. The present study was undertaken to investigate the mechanism by which PTP1B mediates the cessation of the leptin signal transduction. Leptin-induced activation of a STAT3 responsive reporter was dose-dependently inhibited by co-transfection with PTP1B. No inhibition was observed when a catalytically inactive mutant of PTP1B was used or when other PTPs were co-transfected. PTP1B was able to dephosphorylate activated JAK2 and STAT3 in vitro, whereas either no or a minimal effect was observed with cluster of differentiation 45 (CD45), PTPα and leukocyte antigen-related (LAR). By utilisation of a selective PTP1B inhibitor, the leptin-induced STAT3 activation was enhanced in cells. In conclusion, these results suggested that the negative regulatory role of PTP1B on leptin signalling is mediated through a direct and selective dephosphorylation of the two signalling molecules, JAK2 and STAT3.

Malignant insulinoma is a critical cancer form with a poor prognosis. Because cure by surgery is infrequent, effective chemotherapy is in demand. Induction of cell death in tumor cells by proteasome inhibitors is emerging as a potential strategy in cancer therapy. Here we investigated whether inhibition of the proteasome has an antitumorigenic potential in insulinoma cells. Exposure of mouse betaTC3 insulinoma cells to the proteasome inhibitor N-Acetyl-Leu-Leu-Nle-CHO (ALLN) reduced cell viability, activated caspase-3, induced apoptosis, and suppressed insulin release. Treatment with ALLN also resulted in phosphorylation of c-jun N-terminal kinase (JNK) and an increase in in vitro phosphorylation of c-jun. In insulinoma cells with impaired JNK signaling, ALLN-induced apoptosis was significantly suppressed. Another proteasome inhibitor, lactacystin, also stimulated JNK activation, caused activation of caspase-3, suppressed cell viability, and induced apoptosis in betaTC3 and rat INS-1E cells. Both ALLN and lactacystin caused a marked decrease in the cellular amount of the JNK scaffold protein JNK-interacting protein 1/islet-brain-1. In primary pancreatic rat islet cells, proteasome inhibition reduced insulin secretion but had no impact on cell viability and even partially protected against the toxic effect of proinflammatory cytokines. Our findings demonstrate that proteasome inhibitors possess antitumorigenic and antiinsulinogenic effects on insulinoma cells.

Summary objective and design Hypopituitarism, in particular GH deficiency, is prevalent in patients with clinically nonfunctioning pituitary adenomas (NFPAs) both before and after surgery. The factors regulating the growth of pituitary adenomas in general and residual tumour tissue in particular are not fully characterized, and the effect of GH and IGF-I on human pituitary cell proliferation has not previously been reported. In NFPA tissue from 14 patients we evaluated GH receptor (GHR) expression and signal transduction, and the effect of GH and IGF-I exposure on cell proliferation and hormone secretion in vitro. measurements Tissue samples from 14 NFPAs were investigated. Expression of GHR in tissue samples was assessed by reverse transcription polymerase chain reaction (RT-PCR). Six tumours were immunostained with a GHR antibody. In the cell cultures, STAT5 (signal transducer and activator of transcription 5) phosphorylation was measured by Western blot analysis as an index of GHR signalling; cell proliferation was evaluated by [H3]-thymidine incorporation and glycoprotein hormone production analysed by radioimmunoassay (RIA). results All adenomas investigated expressed the GHR, but there was no detection of STAT5 phosphorylation. Overall, GH and IGF-I administration did not significantly stimulate cell proliferation in vitro, although some individual adenomas exhibited a proliferative response to various extents. GH also did not significantly influence glycoprotein hormone secretion in vitro. conclusion GH receptors are expressed in human pituitary adenoma cells but their functional role is uncertain. GH and IGF-I do not consistently influence the proliferation of cultured pituitary adenoma cells.

Signal transducer and activator of transcription 5 (STAT5) activation plays a central role in GH- and prolactin-mediated signal transduction in the pancreatic β-cells. In previous experiments we demonstrated that STAT5 activation is necessary for human (h)GH-stimulated proliferation of INS-1 cells and hGH-induced increase of mRNA-levels of the cell cycle regulator cyclin D2. In this study we have further characterized the role of STAT5 in the regulation of cyclin D expression and β-cell proliferation by hGH. Cyclin D2 mRNA and protein levels (but not cyclin D1 and D3) were induced in a time-dependent manner by hGH in INS-1 cells. Inhibition of protein synthesis by coincubation with cycloheximide did not affect the hGH-induced increase of cyclin D2 mRNA levels at 4 h. Expression of a dominant negative STAT5 mutant, STAT5aΔ749, partially inhibited cyclin D2 protein levels. INS-1 cells transiently transfected with a cyclin D2 promoter-reporter construct revealed a 3- to 5-fold increase of transcriptional activity in response to hGH stimulation. Furthermore, coexpression of a constitutive active STAT5 mutant (either CA-STAT5a or CA-STAT5b) was sufficient to drive transactivation of the promoter. CA-STAT5b was stably expressed in INS-1 cells under the control of a doxycycline-inducible promoter. Gel retardation experiments using a probe representing a putative STAT5 binding site in the cyclin D2 promoter revealed binding of the doxycycline-induced CA-STAT5b. Furthermore, induction of CA-STAT5b stimulated transcriptional activation of the cyclin D2 promoter and induced hGH-independent proliferation in these cells. In primary β-cells, adenovirus-mediated expression of CA-STAT5b profoundly stimulated DNA-synthesis (5.3-fold over control) in the absence of hGH. Our studies indicate that STAT5 activation is sufficient to drive proliferation of the β-cells and that cyclin D2 may be a critical target gene for STAT5 in this process.

One of the long-term effects of growth hormone (GH) in adipocytes is to maintain a state of refractoriness to insulin-like effects, a refractoriness which otherwise declines within a few hours of GH starvation. Here, we examined differences in GH signaling and the possible role for the recently identified family of suppressors of cytokine signaling (SOCS) proteins in the transition between the refractory and the responsive states in rat adipocytes. The ability of GH to stimulate lipogenesis and tyrosine phosphorylation of the GH receptor (GHR), Janus kinase 2 (Jak2), insulin receptor substrate-1 (IRS-1) and -2 (IRS-2) was greatly reduced in refractory as compared to responsive primary rat adipocytes. However, phosphorylation of Signal Transducer and Activator of Transcription 5 (Stat5) was not affected. SOCS-3 and CIS mRNA levels were significantly higher in refractory compared to responsive cells and could be induced by GH, whereas the level of SOCS-2 mRNA was unchanged. With overexpression of GHR, Jak2 and IRS-1 along with each of these SOCS proteins in human A293 cells, we could demonstrate that both SOCS-1 and SOCS-3 completely inhibited the GH-stimulated tyrosine phosphorylation of IRS-1, whereas SOCS-2 and CIS did not. Our data suggest that GH induces refractoriness to the insulin-like effects in a negative-feedback manner by inhibiting GH-induced GHR/Jak2/IRS-1/IRS-2 phosphorylation through upregulation of SOCS-3, which almost completely blocks Jak2 activation.

Suppressor of cytokine signaling (SOCS)-2 is a member of a family of intracellular proteins implicated in the negative regulation of cytokine signaling. The generation of SOCS-2-deficient mice, which grow to one and a half times the size of their wild-type littermates, suggests that SOCS-2 may attenuate growth hormone (GH) signaling. In vitro studies indicate that, while SOCS-2 can inhibit GH action at low concentrations, at higher concentrations it may potentiate signaling. To determine whether a similar enhancement of signaling is observed in vivo or alternatively whether increased SOCS-2 levels repress growth in vivo, we generated and analyzed transgenic mice that overexpress SOCS-2 from a human ubiquitin C promoter. These mice are not growth-deficient and are, in fact, significantly larger than wild-type mice. The overexpressed SOCS-2 was found to bind to endogenous GH receptors in a number of mouse organs, while phosphopeptide binding studies with recombinant SOCS-2 defined phosphorylated tyrosine 595 on the GH receptor as the site of interaction. Together, the data implicate SOCS-2 as having dual effects on GH signaling in vivo.

GH is an important regulator of cell growth and metabolism. In the pancreas, GH stimulates mitogenesis as well as insulin production in β-cells. The cellular effects of GH are exerted mainly through activation of the Janus kinase-signal transducer and activator of transcription (STAT) pathway. Recently it has been found that suppressors of cytokine signaling (SOCS) proteins are able to inhibit GH-induced signal transduction. In the present study, the role of SOCS-3 in GH signaling was investigated in the pancreatic β-cell lines RIN-5AH and INS-1 by means of inducible expression systems. Via stable transfection of the β-cell lines with plasmids expressing SOCS-3 under the control of an inducible promoter, a time- and dose-dependent expression of SOCS-3 in the cells was obtained. EMSA showed that SOCS-3 is able to inhibit GH-induced DNA binding of both STAT3 and STAT5 in RIN-5AH cells. Furthermore, using Northern blot analysis it was shown that SOCS-3 can completely inhibit GH-induced insulin production in these cells. Finally, 5-bromodeoxyuridine incorporation followed by fluorescence-activated cell sorting analysis showed that SOCS-3 inhibits GH-induced proliferation of INS-1 cells. These findings support the hypothesis that SOCS-3 is a major regulator of GH signaling in insulin-producing cells.

Objective Several studies have shown an increase in beta cell mass during pregnancy. Somatolactogenic hormones are known to stimulate the proliferation of existing beta cells in rodents whereas the mechanism in humans is still unclear. We hypothesize that in addition to somatolactogenic hormones there are other circulating factors involved in beta cell adaptation to pregnancy. This study aimed at screening for potential pregnancy-associated circulating beta cell growth factors. Samples Serum samples from nonpregnant and pregnant women. Methods The effect of serum from pregnant women on the proliferation of rat beta cells was studied using [3H]thymidine incorporation and 5-ethynyl-2′-deoxyuridine proliferation assays. In addition, serum from pregnant and nonpregnant women was fractionated by gel filtration and high performance liquid chromatography. The fractionated serum was screened for mitogenic activity in INS-1E cells. Proteins and peptides in mitogenic active serum fractions were identified by amino acid sequencing and mass spectrometry. Main outcome measures Presence of circulating beta cell proliferating factors. Results Late gestational pregnancy serum significantly increased proliferation of rat beta cells compared with early pregnancy and nonpregnancy. The mitogenic active serum fractions contained proteins and peptides derived from kininogen-1, fibrinogen-α, α1-antitrypsin, apolipoprotein-A1, placental lactogen, angiotensinogen and serum albumin. Conclusion Pregnancy serum is able to stimulate proliferation of rat beta cells. We have identified several circulating factors that may contribute to beta cell adaptation to pregnancy. Further studies are needed to elucidate their possible role in glucose homeostasis in the mother and her offspring.