Pancreatic Islet Beta Cells Research Articles

Preconditioning with Associated Blocking of Ca2+ Inflow Alleviates Hypoxia-Induced Damage to Pancreatic β-Cells

ObjectiveBeta cells of pancreatic islets are susceptible to functional deficits and damage by hypoxia. Here we aimed to characterize such effects and to test for and pharmacological means to alleviate a negative impact of hypoxia.Methods and DesignRat and human pancreatic islets were subjected to 5.5 h of hypoxia after which functional and viability parameters were measured subsequent to the hypoxic period and/or following a 22 h re-oxygenation period. Preconditioning with diazoxide or other agents was usually done during a 22 h period prior to hypoxia.ResultsInsulin contents decreased by 23% after 5.5 h of hypoxia and by 61% after a re-oxygenation period. Preconditioning with diazoxide time-dependently alleviated these hypoxia effects in rat and human islets. Hypoxia reduced proinsulin biosynthesis (3H-leucine incorporation into proinsulin) by 35%. Preconditioning counteracted this decrease by 91%. Preconditioning reduced hypoxia-induced necrosis by 40%, attenuated lowering of proteins of mitochondrial complexes I–IV and enhanced stimulation of HIF-1-alpha and phosphorylated AMPK proteins. Preconditioning by diazoxide was abolished by co-exposure to tolbutamide or elevated potassium (i.e. conditions which increase Ca2+ inflow). Preconditioning with nifedipine, a calcium channel blocker, partly reproduced effects of diazoxide. Both diazoxide and nifedipine moderately reduced basal glucose oxidation whereas glucose-induced oxygen consumption (tested with diazoxide) was unaffected. Preconditioning with diaxoxide enhanced insulin contents in transplants of rat islets to non-diabetic rats and lowered hyperglycemia vs. non-preconditioned islets in streptozotocin-diabetic rats. Preconditioning of human islet transplants lowered hyperglycemia in streptozotocin-diabetic nude mice.Conclusions1) Prior blocking of Ca2+ inflow associates with lesser hypoxia-induced damage, 2) preconditioning affects basal mitochondrial metabolism and accelerates activation of hypoxia-reactive and potentially protective factors, 3) results indicate that preconditioning by K+-ATP-channel openers has therapeutic potential for islet transplantations.

Connexin 30.2 is expressed in mouse pancreatic beta cells

Nowadays, connexin (Cx) 36 is considered the sole gap junction protein expressed in pancreatic beta cells. In the present research we investigated the expression of Cx30.2 mRNA and protein in mouse pancreatic islets. Cx30.2 mRNA and protein were identified in isolated islet preparations by qRT-PCR and Western blot, respectively. Immunohistochemical analysis showed that insulin-positive cells were stained for Cx30.2. Confocal images from double-labeled pancreatic sections revealed that Cx30.2 and Cx36 fluorescence co-localize at junctional membranes in islets from most pancreases. Abundant Cx30.2 tiny reactive spots were also found in cell cytoplasms. In beta cells cultured with stimulatory glucose concentrations, Cx30.2 was localized in both cytoplasms and cell membranes. In addition, Cx30.2 reactivity was localized at junctional membranes of endothelial or cluster of differentiation 31 (CD31) positive cells. Moreover, a significant reduction of Cx30.2 mRNA was found in islets preparations incubated for 24h in 22mM as compared with 3.3mM glucose. Therefore, it is concluded that Cx30.2 is expressed in beta and vascular endothelial cells of mouse pancreatic islets.

Blockage of progesterone receptor effectively protects pancreatic islet beta cell viability

The progesterone receptor (PR), a member of nuclear receptor superfamily, is closely associated with gestational, type 1 and type 2 diabetes. However, the underlying mechanisms remain obscure. Here we found that PR activation increased the pro-inflammatory cytokines (PIC)-induced injury in Min6 cells, and PR blockage with siRNA interference protected the cells from damage. Moreover, the new discovered PR antagonist SC51089 effectively improved cell survival by reducing the PIC-stimulated cell apoptosis in Min6 cells. Immunoblotting assays indicated that either PR agonist progesterone (P4) or PR-B over-expression promoted the PIC-induced reinforces of extracellular-signal-regulated kinase 1/2 phosphorylation (p-Erk) and protein 53 (p53), and the attenuations of protein kinase B phosphorylation (p-AKT) and tumor necrosis factor receptor-associated factor 2 (TRAF2). SC51089 could reverse all the P4- or PR-B over-expression induced effects. In addition, PR siRNA inference based assay further supported that SC51089 protected pancreatic islet beta cells from the PR activation or PIC-induced injury by targeting PR and this protective action was mediated by AKT signaling pathway. To our knowledge, this current work might be the first report on the regulation of PR in pancreatic islet beta cell survival. It is expected that SC51089, as a non-steroid PR antagonist, might also find its potential in anti-diabetic research.

Fibroblast Growth Factor Receptor Like-1 (FGFRL1) Interacts with SHP-1 Phosphatase at Insulin Secretory Granules and Induces Beta-cell ERK1/2 Protein Activation

FGFRL1 is a newly identified member of the fibroblast growth factor receptor (FGFR) family expressed in adult pancreas. Unlike canonical FGFRs that initiate signaling via tyrosine kinase domains, the short intracellular sequence of FGFRL1 consists of a putative Src homology domain-2 (SH2)-binding motif adjacent to a histidine-rich C terminus. As a consequence of nonexistent kinase domains, FGFRL1 has been postulated to act as a decoy receptor to inhibit canonical FGFR ligand-induced signaling. In pancreatic islet beta-cells, canonical FGFR1 signaling affects metabolism and insulin processing. This study determined beta-cell expression of FGFRL1 as well as consequent effects on FGFR1 signaling and biological responses. We confirmed FGFRL1 expression at the plasma membrane and within distinct intracellular granules of both primary beta-cells and βTC3 cells. Fluorescent protein-tagged FGFRL1 (RL1) induced a significant ligand-independent increase in MAPK signaling. Removal of the histidine-rich domain (RL1-ΔHis) or entire intracellular sequence (RL1-ΔC) resulted in greater retention at the plasma membrane and significantly reduced ligand-independent ERK1/2 responses. The SHP-1 phosphatase was identified as an RL1-binding substrate. Point mutation of the SH2-binding motif reduced the ability of FGFRL1 to bind SHP-1 and activate ERK1/2 but did not affect receptor localization to insulin secretory granules. Finally, overexpression of RL1 increased cellular insulin content and matrix adhesion. Overall, these data suggest that FGFRL1 does not function as a decoy receptor in beta-cells, but rather it enhances ERK1/2 signaling through association of SHP-1 with the receptor's intracellular SH2-binding motif.

GABA exerts anti-inflammatory and immunosuppressive effects (P5175)

Abstract Gamma amino-butyric acid (GABA) is an inhibitory neurotransmitter in the CNS, but it also exerts important functions in the immune system and the islets of Langerhans. Thus, functional GABA receptors have been identified on immune cells and islet cells. Previously, we found that in both autoimmune (NOD) and streptozotocin (STZ)-induced diabetes GABA increases islet-cell mass, while exerting anti-apoptotic effects. GABA induced the regeneration of islet beta cells. It also suppressed inflammatory cytokine production, which is likely important in allowing survival of new islet beta cells. In this study, we examine how GABA exerts immunoprotective effects. We report that GABA suppresses both T cells and macrophages. It increases TGF-beta production and regulatory T cells (Tr or Treg). Notably, GABA inhibits NF-kB activation in both lymphocytes and pancreatic islet beta cells. It also protects beta cells from apoptosis induced by various mechanisms, and this may be related to NF-kB inhibition. The mechanisms by which GABA exerts these effects are largely unknown. However, in immune cells it stimulates GABA type A receptors, which act as ligand-gated chloride channels. This may open voltage-dependent calcium channels in the membrane and decrease intra-cellular calcium levels. Conclusion: GABA acts on both T cells and macrophages and exerts potent anti-inflammatory effects, which are protective in models of type 1 diabetes.

Mechanism of insulin production in canine bone marrow derived mesenchymal stem cells

Insulin is a critical hormone in the regulation of blood glucose levels and is produced exclusively by pancreatic islet beta-cells. Insulin deficiency due to reduced pancreatic islet beta-cell number underlies the progression of diabetes mellitus, prompting efforts to develop beta-cell replacement therapies. However, precise information on beta-cell replacement and differentiation in canines is limited. In this study, we established insulin-producing cells from bone marrow derived mesenchymal stem cells transiently expressing canine pancreatic and duodenal homeobox 1 (Pdx1), beta cell transactivator 2 (Beta2) and V-maf avian musculoaponeurotic fibrosarcoma oncogene homolog A (Mafa) using a gene transfer technique. Real-time PCR analysis revealed an increase in insulin mRNA expression of transfected cells. And ELISA revealed that insulin protein expressed was detected in cytoplasmic fraction. Insulin immunostaining analysis was performed and observed in cytoplasmic fraction. These results suggest that co-transfection of Pdx1, Beta2 and Mafa induce insulin production in canine BMSCs. Our findings provide a clue to basic research into the mechanisms underlying insulin production in the canines.

An Exploratory Study of the Association between KCNB1 rs1051295 and Type 2 Diabetes and Its Related Traits in Chinese Han Population

Since the KCNB1 encoding Kv2.1 channel accounts for the majority of Kv currents modulating insulin secretion by pancreatic islet beta-cells, we postulated that KCNB1 is a plausible candidate gene for genetic variation contributing to the variable compensatory secretory function of beta-cells in type-2 diabetes (T2D). We conducted two studies, a case-control study and a cross-section study, to investigate the association of common single-nucleotide polymorphisms (SNPs) in KCNB1 with T2D and its linking traits. In the case-control study, we first examined the association of 20 tag SNPs of KCNB1 with T2D in a population with 226 T2D patients and non-diabetic subjects (screening study). We then identified the association in an enlarged population of 412 T2D patients and non-diabetic subjects (replication study). In the cross-sectional study, we investigated the linkage between the candidate SNP rs1051295 and T2D by comparing beta-cell function and insulin sensitivity among rs1051295 genotypes in a general population of 1051 subjects at fasting and after glucose loading (oral glucose tolerance tests, OGTT) in 84 fasting glucose impaired subjects, and several T2D-related traits. We found that among the 19 available tag SNPs, only the KCNB1 rs1051295 was associated with T2D (P = 0.027), with the rs1051295 TT genotype associated with an increased risk of T2D compared with genotypes CC (P = 0.009). At fasting, rs1051295 genotype TT was associated with a 9.8% reduction in insulin sensitivity compared to CC (P = 0.008); along with increased plasma triglycerides (TG) levels (TT/CC: P = 0.046) and increased waist/hip (W/H) ratio (TT/CC: P = 0.013; TT/TC: P = 0.002). OGTT confirmed that genotype TT exhibited reduced insulin sensitivity by 16.3% (P = 0.030) compared with genotype TC+CC in a fasting glucose impaired population. The KCNB1 rs1051295 genotype TT in the Chinese Han population is associated with decreased insulin sensitivity and increased plasma TG and W/H ratio, which together contribute to an increased risk for T2D.

Pancreatic islet cell phenotype and endocrine function throughout diabetes development in non-obese diabetic mice

Type 1 diabetes (T1D) results from a T cell dependent, autoimmune destruction of insulin producing beta cells in pancreatic islets of Langerhans, which results in insulin deficiency despite attempts at beta cell replacement by the emergence of newly-differentiated beta cells throughout T1D development. The origin of these cells has been difficult to assess as these are rapidly destroyed by the underlying autoimmunity. The identification of islets of Langerhans is typically assessed by either immunochemistry or immunofluorescence using antibodies directed against the different signature hormones and surface markers of various endocrine cells . However, the limited number of markers that can be used simultaneously and the uneven spatial distribution of endocrine cells within islets, limit the use of these histological analyses. To circumvent these caveats, we developed a novel approach using multi-parametric flow cytometry to assess the phenotype and function of pancreatic islet cell populations throughout T1D development. Using such strategy, we show that while beta cells undergo autoimmune destruction, insulin-producing cells arise from trans-differentiated alpha or delta cells, an outcome that was not solely the result of beta cell self-renewal. Moreover, we show that CD4+T cell-mediated inflammation correlates with the emergence of this insulin-producing beta cell-like cell.

MicroRNA Expression in Alpha and Beta Cells of Human Pancreatic Islets

microRNAs (miRNAs) play an important role in pancreatic development and adult β-cell physiology. Our hypothesis is based on the assumption that each islet cell type has a specific pattern of miRNA expression. We sought to determine the profile of miRNA expression in α-and β-cells, the main components of pancreatic islets, because this analysis may lead to a better understanding of islet gene regulatory pathways. Highly enriched (>98%) subsets of human α-and β-cells were obtained by flow cytometric sorting after intracellular staining with c-peptide and glucagon antibody. The method of sorting based on intracellular staining is possible because miRNAs are stable after fixation. MiRNA expression levels were determined by quantitative high throughput PCR-based miRNA array platform screening. Most of the miRNAs were preferentially expressed in β-cells. From the total of 667 miRNAs screened, the Significant Analysis of Microarray identified 141 miRNAs, of which only 7 were expressed more in α-cells (α-miRNAs) and 134 were expressed more in β-cells (β-miRNAs). Bioinformatic analysis identified potential targets of β-miRNAs analyzing the Beta Cell Gene Atlas, described in the T1Dbase, the web platform, supporting the type 1 diabetes (T1D) community. cMaf, a transcription factor regulating glucagon expression expressed selectively in α-cells (TFα) is targeted by β-miRNAs; miR-200c, miR-125b and miR-182. Min6 cells treated with inhibitors of these miRNAs show an increased expression of cMaf RNA. Conversely, over expression of miR-200c, miR-125b or miR-182 in the mouse alpha cell line αTC6 decreases the level of cMAF mRNA and protein. MiR-200c also inhibits the expression of Zfpm2, a TFα that inhibits the PI3K signaling pathway, at both RNA and protein levels.In conclusion, we identified miRNAs differentially expressed in pancreatic α- and β-cells and their potential transcription factor targets that could add new insights into different aspects of islet biology and pathophysiology.

Induction of hepatic epithelial-like stem cells into insulin producing cells by reprogramming defined small molecules

Using phase induction in vitro to induce hepatic epithelial-like stem cells (WB-F344) into insulin producing cells (IPCs),the IPCs were transplanted into diabetic rats,and the blood glucose level was monitored.The results of RT-PCR and immunofluorescence showed that genes and proteins of pancreatic islet beta cell and phenotypic markers were successfully detected in the IPCs,which significantly improved hyperglycemia after being transplantated into diabetic rats.These results suggest that hepatic epithelial-like stem cells in vitro can be induced into functional insulin producing cells by small molecules and that this finding may contribute to the treatment of diabetes. Key words: Hepatic epithelial-like stem cells; Small molecules; Insulin producing cells; Reprogramming; Diabetes

ATP Regulates Sodium Channel Kinetics in Pancreatic Islet Beta Cells

Pancreatic beta cells act as glucose sensors, in which intracellular ATP ([ATP](i)) are altered with glucose concentration change. The characterization of voltage-gated sodium channels under different [ATP](i) remains unclear. Here, we demonstrated that increasing [ATP](i) within a certain range of concentrations (2-8mM) significantly enhanced the voltage-gated sodium channel currents, compared with 2mM cytosolic ATP. This enhancement was attenuated by even high intracellular ATP (12mM). Furthermore, elevated ATP modulated the sodium channel kinetics in a dose-dependent manner. Increased [ATP](i) shifted both the current-voltage curve and the voltage-dependent inactivation curve of sodium channel to the right. Finally, the sodium channel recovery from inactivation was significantly faster when the intracellular ATP level was increased, especially in 8mM [ATP](i), which is an attainable concentration by the high glucose stimulation. In summary, our data suggested that elevated cytosolic ATP enhanced the activity of Na(+) channels, which may play essential roles in modulating β cell excitability and insulin release when blood glucose concentration increases.

Association Study of MAFA and MAFB Genes Related to Organ-Specific Autoimmunity, with Susceptibility to Type-1 Diabetes in Japanese and Caucasian Populations

The transcriptional factor MAFA is specifically expressed in beta cells of pancreatic islets, and activates tissuespecific transcription of insulin. We previously reported that MAFA is also expressed in the thymus and regulates intrathymic expression of insulin in the mouse. In humans, we identified a functional polymorphism of MAFA, Gly346Cys, which was suggested to be associated with type 1 diabetes. This study aimed to validate the association of MAFA with type 1 diabetes in a larger number of subjects. In addition, molecular scanning of MAFB another member of the large MAFA transcription family, and an association study with type 1 diabetes were also performed. A total of 1733 subjects, including newly recruited Japanese (346 controls and 532 cases) and Caucasians (223 controls and 228 cases), were studied. In newly recruited Japanese subjects, the minor allele frequency of MAFAGly346Cys was lower in cases than in controls (2.9 vs. 5.1%, odds ratio [95%CI]: 0.56 [0.34-0.91], p=0.02). Meta-analysis with our previous data showed a significant association of MAFA with type 1 diabetes (summary odds ratio [95%CI]: 0.49 [0.32-0.76], p=0.0013). When cases were limited to subjects with a risk genotype of INS, the association was further strengthened (odds ratio [95%CI]: 0.47 [0.30-0.74], p=0.00097). In the Caucasian population, the difference in minor allele frequency of MAFA between cases and controls was not significant (6.4% vs. 5.4%, odds ratio [95%CI]: 1.14 [0.66-1.99], NS). When data from Japanese and Caucasians were combined, summary odds ratio was 0.68 [95%CI: 0.48-0.95] (p G and 618C>A) were identified, but neither was significantly associated with type 1 diabetes. In conclusion, MAFA Gly346Cys is associated with type 1 diabetes, especially in the Japanese population, which possesses the high-risk INS genotype.

Islet Amyloid Polypeptide and Diabetes

Islet amyloid polypeptide (IAPP, amylin) is a 37 amino acid residue hormone expressed mainly by pancreatic islet beta cells and to less extent by some gastrointestinal endocrine cells and by certain regions in central nervous system. In experimental systems a number of different effects have been ascribed to IAPP but the in vivo importance of many of them is still unknown. At least effects on the central nervous system and on endocrine pancreatic cells are likely to be physiologically relevant. In these tissues calcitonin receptors and receptor activity-modifying proteins (RAMPs) 1 and 3, creating high affinity IAPP receptors have been identified. How expression of the components of these complexes are regulated and how further signaling is conducted are more or less unknown. IAPP is most well-known for its ability to aggregate into amyloid fibrils in islets of Langerhans in association with type 2 diabetes leading to loss of beta cells. In addition, amyloid is deposited between endocrine cells and between such cells and capillaries and most likely disturbs important interactions between the cells. How IAPP receptor complexes are affected by the amyloid formation process or by amyloid itself, or vice versa, are completely unknown.

Embryoid Body Formation is Required for Differentiation of Insulin-Producing Cell Clusters from Mouse Embryonic Stem Cells

In Type 1 diabetes, insulin-producing pancreatic cells, or beta cells, are destroyed by an autoimmune response. Current clinical treatments are indefinite insulin replacement therapy or transplantation of the pancreas or beta islets. The latter two treatments are limited in available donors; a potential alternative is the use of insulin-producing cell clusters (IPCCs) differentiated from embryonic stem cells (ESCs). We hypothesize that IPCCs will reproduce the insulin-producing capacity of healthy beta cells of an adult mouse, and we are testing the efficiency of distinct IPCC culture methods to achieve this goal. Among several existing ESC-IPCC differentiation protocols, Blyszczuk et al. developed the most successful method to date in producing IPCCs that showed similarities to pancreatic beta cells. However, this method is time-intensive, requiring approximately 41 days. We attempted to streamline the protocol by bypassing the formation of embryoid bodies (EBs), reducing the differentiation timeline to 27 days. At several time points during this protocol, IPCC cultures were analyzed by RT-PCR and immunofluorescence for genes and proteins expressed in pancreatic beta islet cells. The mRNA and protein expression of insulin was not observed. Furthermore, ELISA analysis detected low intracellular insulin response after challenging IPCCs with different glucose concentrations. These results reject the hypothesis that EB formation is not required for ESC-IPCC differentiation in vitro. However, it is possible that alpha cells can be differentiated, as glucagon was detected.

Antidiabetic effect of Cephalandra indica Q in diabetic rats

Background: Diabetes mellitus is an emerging global epidemic, taking its toll in developing countries as India. Presently, the treatment for diabetes using allopathic drugs gives only a temporary relief. A combination of right medicine and potency, on other hand has given Homoeopathy a high success rate in the treatment of this disease. Objective: The aim of this study was to investigate the effects of Cephalandra indica mother tincture and potencies on blood glucose level, cholesterol level, body weight, and beta-cells of pancreatic islets of Langerhans, in streptozotocin (STZ)-induced diabetic Wistar rats. Material and Methods: Glucose uptake was monitored in mother tincture-treated mouse fibroblast cell line. Diabetes mellitus was induced by intraperitoneal injection of STZ (55 mg/kg body weight) in adult male rats. After three days of injection, diabetic rats received mother tincture orally (750 μL/kg body weight) daily for three weeks. Results: There was a significant reduction of blood glucose level, regain of body weight, and regeneration of beta-cells in the pancreas of the mother tincture-treated rats. Mother tincture-treated 3T3 cells also showed reduced uptake of glucose in comparison to normal cells. Conclusion: The present study clearly indicates a significant antidiabetic effect of Cephalandra indica and lends support for its usage as a homoeopathic medicine.

Role of Fat Feeding on the Diabetic Albino Rats

Aim of the work: The nutrition of developing countries are rapidly changed and associated with high incidence of obesity as well as the metabolic syndrome and type 2diabetes. There is an increased intake of high caloric foods rich in refined carbohydrates, fats and red animal proteins instead of low caloric foods rich in fibers and containing low fats. This study aimed at the evaluation of effect of animal fats on the diabetic rats. Material and methods: This study was performed on forty eight albino rats (their body weight was 190 ± 10 grams) divided equally into four groups. Two groups (III & IV) were given intravenous streptozotocin in a concentration of 60 mg /Kg. B.W. for three days in order to induce diabetes through the destruction of pancreatic islet beta cells. Non-diabetic rats (group I) were given the laboratory diet (LD) while the non-diabetic second group of rats received an emulsion rich in fat. Third group diabetic animals were fed on the laboratory diet (LD) while group IV diabetic rats were received high-fat (HF) emulsion in the same dosage as group II. C-peptide (ng/ml), blood glucose (mg/ dl), serum triglycerides (TG; mmol/l) and total cholesterol (TC; mmol/l) were assessed before the beginning of this experiment and also once every two weeks for four months in non-fasted diabetic and non diabetic counterparts. Moreover, kidneys and liver were examined histopathologically to detect fatty intracellular accumulation and any other changes.

Glucose induces transient increase in DNA synthesis of pancreatic islet beta cells in culture

Glucose induces transient increase in DNA synthesis of pancreatic islet beta cells in culture

Gossypin, a flavonol glucoside protects pancreatic beta-cells from glucotoxicity in streptozotocin-induced experimental diabetes in rats

Chronic oxidative stress caused by glucotoxicity plays a crucial role in the onset and progression of pancreatic beta-cell dysfunction in diabetes mellitus. Several studies have reported that flavonoids protect tissues from oxidative damage by its antioxidative potential. The present study was aimed to investigate the antioxidant activity of gossypin by DPPH and ABTS radical scavenging assays and its role on glucotoxicity mediated pancreatic beta-cell dysfunction in streptozotocin-induced experimental diabetes in rats. Gossypin effectively scavenges both 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and ABTS radicals generated in the reaction mixtures, which implies the antioxidant property of gossypin. Oral administration of gossypin to diabetic rats significantly improved the antioxidants and reduced the levels of lipid peroxides, nitric oxide, IL-1β and nuclear NF-κB p65 unit in pancreatic tissues. The improved insulin content in pancreas and plasma in the experimental diabetic rats treated with gossypin as well as histological observations demonstrate the possible role of gossypin in the protection of pancreatic beta-cells. In addition, blood glucose, HbA1c, glutathione, lipid peroxides, IL-1β levels in plasma and nitric oxide level were significantly altered upon treatment with gossypin. It is concluded that the pancreatic islet beta-cell protective role of gossypin against glucotoxicity mediated oxidative damage might be attributed by its antioxidative potential.

P162 Ciliary neurotrophic factor protects mice against type 1 diabetes and interleukin-1B-induced beta-cell apoptosis through SOCS3 expression: Role of stat1/stat3 balance in cytokine- induced iNOS-dependent beta-cell death

Introduction Type 1 Diabetes (DM1) is characterized by a cytokine-dependent loss of pancreatic islet beta-cell mass with subsequent hyperglycaemia. Ciliary Neurotrophic Factor (CNTF) protects pancreatic islets against cytokine-induced apoptosis and increases SOCS3 expression. In addition, beta-cell apoptosis seems to be induced by STAT1 and inhibited by STAT3 activation. We evaluated if CNTF protects mice against Streptozotocin-induced Type 1 Diabetes, if this protection is dependent of SOCS3 and/or iNOS expression, and what the role of STAT1/STAT3 activation in CNTF effects on cytokine-induced iNOS-dependent beta-cell apoptosis. Methods We developed SOCS3 Knockdown mice (SOCS3Kd) by Antisense technique from a C57BL6 Wild-Type background. To evaluate the role of iNOS on the CNTF protective effects in vivo we used iNOS-knockout mice. For Streptozotocin-induced Type 1 Diabetes, 4–6 weeks-old mice were treated or not with 0.1 mg/kg of CNTF for 5 days, receiving, intraperitoneally, 80 mg/kg of Streptozotocin (STZ) on the 5th day. Glycaemia from non-fasted mice was measured at days 0, 1, 7, 14, 21, and 28 after STZ. DM1 was considered as blood glycaemia >11.2 mmol/l for 2 days. We evaluated mice glycaemia, DM1 incidence, time to DM1 onset and DM1 severity. Pancreatic islets were isolated by collagenase method for mRNA and protein extraction. SOCS3 was inhibited in MIN6 cells using an SOCS3 specific siRNA (SOCS3kd cells), and iNOS inhibition was obtained by treating MIN6 cells with 1 mmol/l of the iNOS inhibitor LMEM (iNOSkd cells). Wild-type (WT), SOCS3kd, and iNOSkd cells were cultured with 10 ug/ml of IL1beta, 1 nmol/l CNTF or both. mRNA levels were assessed by Real-Time RT-PCR and protein expression and phosphorylation by Western-Blot. ( n = 4 − 6) Data = Mean ± SEM. P Results CNTF reduced DM1 incidence by 80% in WT but only by 10% in SOCS3kd mice. In WT, CNTF increased STAT3 and reduced STAT1 phosphorylation, while in SOCS3kd mice CNTF further increased STAT3 but failed to reduce STAT1. STZ reduced STAT3 and increased STAT1 phosphorylation, while in SOCS3kd mice STZ failed to reduce STAT3 but further increased STAT1. CNTF prevented STZ-induced pancreatic islets apoptosis and IL1beta-induced MIN6 cells apoptosis in WT mice and cells, but not in SOCS3kd or iNOSkd cells. The incidence of STZ-induced DM1 was lower in iNOSkd mice (20% of Control), and CNTF failed to further protect these mice. Nevertheless, CNTF effects over upstream pathways, such as increased STAT3 and SOCS3, and reduced STAT1, were similar to those of WT mice. In iNOSkd cells, CNTF partially prevented IL-1beta-induced cell-death, even though its upstream effects were similar to the WT cells, as we observed in vivo . Conclusion CNTF protects against STZ-induced DM1 and against cytokine-induced beta-cell death. These effects are dependent on the increase in SOCS3 expression. The cytokine-induced apoptosis as well as the pro-survival effect of CNTF over beta-cells depend on the balance between STAT1/STAT3 activation.

Tratamento cirúrgico dos insulinomas do pâncreas

Insulinoma is a pancreatic neuroendocrine tumor originated from pancreatic islet beta cells. Although rare, is the most common pancreatic endocrine tumor, with about four cases per million people. The preferential treatment of insulinoma is surgical. To analyze the epidemiological, pathological, clinical and surgical patients treated in the last decade in two surgical services. Were retrospectively reviewed the medical records of patients undergoing surgical treatment of insulinoma in the period of 1999 to 2011. Demographic data, type and duration of symptoms, associated or not with endocrine syndrome and diagnostic tests were obtained from medical records. Were analyzed the method of surgery, intraoperative findings and immediate and late complications. Sixteen patients with insulinoma underwent surgical treatment, 68,7% were women. The age ranged from 20 to 60 years, with a mean age of 39 years. Only one case was associated with multiple endocrine neoplasia type 1. Neuropsychiatric manifestations, mainly syncope, were the most prevalent. The average duration of clinical manifestations until the diagnosis was one year and a half. Imaging tests were used in all patients with 68.7% of preoperative tumor localization. All operations were performed in a conventional (open) manner, without use of laparoscopy. The lesions were identified in all portions of the pancreas with the majority in the pancreatic head. Relief of symptoms was not obtained only in one patient. There were no deaths among the patients. The diagnosis of insulinoma is often established after several months of the onset of clinical manifestations and surgical treatment is curative in almost all patients.