Rat Beta-cells Research Articles

Levels of Blood Glucose and Insulin Expression of Beta-cells in Streptozotocin-induced Diabetic Rats Treated with Ethanolic Extract of Artocarpus altilis Leaves and GABA.

Information about the Artocarpus altilis leaf as an antidiabetic associated with the active compounds Gamma Amino Butyric Acid (GABA) is still limited. This study was conducted to determine the effects of ethanolic extract of A. altilis leaves decoction and GABA on blood glucose levels and insulin expression of beta-cells in streptozotocin-induced diabetic rats. This study was done by using completely randomized design and male Sprague Dewley rats. The rats were devided into normal control group and diabetic rats groups. Levels of bood glucose were measured using strip rapid test. The insulin expression in beta-cells was assessed using immunohistochemistry. Quantitative data were analyzed using ANOVA at 5% confidence level. The result indicated that 50 mg k-1 b.wt., GABA, 400 and 800 mg k-1 b.wt., ethanolic extract of A. altilis leaves decreased the level of blood glucose and increased the insulin expression in pancreas beta-cells. The GABA and ethanolic extract of A. altilis leaves with a minimum dose of 400 mg k-1 b.wt., can be used as an antidiabetic. Pancreas is the target organ was affected by GABA and A. altilis leaves as antidiabetic agents. Results of this study may support the development of research on the potency of GABA in natural materials as antidiabetic particularly type 1 diabetes.

β-MSCs: successful fusion of MSCs with β-cells results in a β-cell like phenotype.

Bone marrow mesenchymal stromal cells (MSC) have anti-inflammatory, anti-apoptotic and immunosuppressive properties and are a potent source for cell therapy. Cell fusion has been proposed for rapid generation of functional new reprogrammed cells. In this study, we aimed to establish a fusion protocol of bone marrow−derived human MSCs with the rat beta-cell line (INS-1E) as well as human isolated pancreatic islets in order to generate insulin producing beta-MSCs as a cell-based treatment for diabetes.Human eGFP+ puromycin+ MSCs were co-cultured with either stably mCherry-expressing rat INS-1E cells or human dispersed islet cells and treated with phytohemagglutinin (PHA-P) and polyethylene glycol (PEG) to induce fusion. MSCs and fused cells were selected by puromycin treatment.With an improved fusion protocol, 29.8 ± 2.9% of all MSCs were β-MSC heterokaryons based on double positivity for mCherry and eGFP.After fusion and puromycin selection, human NKX6.1 and insulin as well as rat Neurod1, Nkx2.2, MafA, Pdx1 and Ins1 mRNA were highly elevated in fused human MSC/INS-1E cells, compared to the mixed control population. Such induction of beta-cell markers was confirmed in fused human MSC/human dispersed islet cells, which showed elevated NEUROD1, NKX2.2, MAFA, PDX1 and insulin mRNA compared to the mixed control. Fused cells had higher insulin content and improved insulin secretion compared to the mixed control and insulin positive beta-MSCs also expressed nuclear PDX1. We established a protocol for fusion of human MSCs and beta cells, which resulted in a beta cell like phenotype. This could be a novel tool for cell-based therapies of diabetes.

Ubiquitin D Regulates IRE1α/c-Jun N-terminal Kinase (JNK) Protein-dependent Apoptosis in Pancreatic Beta Cells

Pro-inflammatory cytokines contribute to pancreatic beta cell apoptosis in type 1 diabetes at least in part by inducing endoplasmic reticulum (ER) stress and the consequent unfolded protein response (UPR). It remains to be determined what causes the transition from "physiological" to "apoptotic" UPR, but accumulating evidence indicates that signaling by the ER transmembrane protein IRE1α is critical for this transition. IRE1α activation is regulated by both intra-ER and cytosolic cues. We evaluated the role for the presently discovered cytokine-induced and IRE1α-interacting protein ubiquitin D (UBD) on the regulation of IRE1α and its downstream targets. UBD was identified by use of a MAPPIT (mammalian protein-protein interaction trap)-based IRE1α interactome screen followed by comparison against functional genomic analysis of human and rodent beta cells exposed to pro-inflammatory cytokines. Knockdown of UBD in human and rodent beta cells and detailed signal transduction studies indicated that UBD modulates cytokine-induced UPR/IRE1α activation and apoptosis. UBD expression is induced by the pro-inflammatory cytokines interleukin (IL)-1β and interferon (IFN)-γ in rat and human pancreatic beta cells, and it is also up-regulated in beta cells of inflamed islets from non-obese diabetic mice. UBD interacts with IRE1α in human and rodent beta cells, modulating IRE1α-dependent activation of JNK and cytokine-induced apoptosis. Our data suggest that UBD provides a negative feedback on cytokine-induced activation of the IRE1α/JNK pro-apoptotic pathway in cytokine-exposed beta cells.

Transcriptome landmarks of the functional maturity of rat beta-cells, from lactation to adulthood.

Research on the postnatal development of pancreatic beta-cells has become an important subject in recent years. Understanding the mechanisms that govern beta-cell postnatal maturation could bring new opportunities to therapeutic approaches for diabetes. The weaning period consists of a critical postnatal window for structural and physiologic maturation of rat beta-cells. To investigate transcriptome changes involved in the maturation of beta-cells neighboring this period, we performed microarray analysis in fluorescence-activated cell-sorted (FACS) beta-cell-enriched populations. Our results showed a variety of gene sets including those involved in the integration of metabolism, modulation of electrical activity, and regulation of the cell cycle that play important roles in the maturation process. These observations were validated using reverse hemolytic plaque assay, electrophysiological recordings, and flow cytometry analysis. Moreover, we suggest some unexplored pathways such as sphingolipid metabolism, insulin-vesicle trafficking, regulation of transcription/transduction by miRNA-30, trafficking proteins, and cell cycle proteins that could play important roles in the process mentioned above for further investigation.

Sex effect on insulin secretion and mitochondrial function in pancreatic beta cells of elderly Wistar rats

ABSTRACTAims/hypothesis: Glucose tolerance progressively declines with age, and there is a high prevalence of type 2 diabetes in the elderly people. Previous studies have reported the sex differences in risk for type 2 diabetes, especially in the elderly people, whereas reasons for these sex differences remain poorly understood. This study aims to evaluate the effect of sex on glucose-stimulated insulin secretion and mitochondrial function in pancreatic beta cells of Wistar rats. Methods: 3-month-old and 18-month-old Wistar rats of both sexes were used. Insulin secretion of islets was analyzed by glucose-stimulated insulin secretion and islet perifusion assays; ATP content and oxygen consumption rate of islets were determined to evaluate the mitochondrial function. Results: Insulin secretion of islets under high glucose conditions declined significantly with age in both sexes. Glucose-stimulated insulin secretion of elderly female groups was markedly higher than that of male groups under high glucose conditions. Importantly, islets from elderly female groups showed higher mitochondrial function compared with male counterparts, evidenced by higher ATP content and oxygen consumption rate under high glucose conditions. It was also noted that mitochondrial biogenesis of islets from elderly female rats was significant higher compared with male rats. There were notable increases in expression of genes involved in mitochondrial biogenesis in islets from elderly female rats compared with male rats. Conclusion: This study demonstrates a sex dimorphism in the age-associated impairment of pancreatic beta cell function in elderly rats, while the potential mechanism may be related to the sexual differences in mitochondrial biogenesis and function.

Co-culture of Insulin Producing Human Endoc-Βh1 Cells with Boundary Cap Neural Crest Stem Cells Protects Partially against Cytokine-induced Cell Death

We have recently observed that co-culture of mouse and rat beta-cells with mouse boundary cap neural crest stem cells (bNCSCs) protected against inflammatory cytokine-induced beta-cell death, possibly via direct cadherinmediated cell-to-cell junctions. However, it has not been addressed whether also human beta-cells can be protected via this strategy. If possible it would be an important approach for development of new protocols for improved outcome of islet transplantation to Type-1 diabetes patients. The aim of this investigation was therefore to study the effect of bNCSC co-culture with insulin producing human EndoC-βH1 cells on cytokine-induced cell death. For this purpose GFP-positive bNCSCs were cultured together with GFP-negative human EndoC-βH1 cells in the presence of the cytokines IL-1γ (50 U/ml) and IFN-γ (1000 U/ml). Cells were then stained with propidium iodide and trypsinized for flow cytometry analysis. Analysis of propidium iodide fluorescence in GFP-positive and GFP-negative cells revealed that EndoC-βH1 cells died to a lower extent when co-cultured with bNCSCs than when cultured without bNCSCs. We also observed that EndoC-βH1 cells formed N-cadherin, but not E-cadherin junctions with the bNCSCs. The bNCSC cell population contained a large proportion of beta-tubulin expressing cells indicating neuronal differentiation. A protective function of the N-cadherin junctions was verified by the finding that a neutralizing N-cadherin antibody counteracted the effect of co-culture. We conclude that the interaction between human insulin producing cells and bNCSCs results in a lowered susceptibility of insulin producing cells to pro-inflammatory cytokines in vitro.

Metforminium Decavanadate as a Potential Metallopharmaceutical Drug for the Treatment of Diabetes Mellitus.

New potential drugs based on vanadium are being developed as possible treatments for diabetes mellitus (DM) and its complications. In this regard, our working group developed metforminium decavanadate (MetfDeca), a compound with hypoglycemic and hypolipidemic properties. MetfDeca was evaluated in models of type 1 and type 2 diabetes mellitus, on male Wistar rats. Alloxan-induction was employed to produce DM1 model, while a hypercaloric-diet was employed to generate DM2 model. Two-month treatments with 3.7 μg (2.5 μM)/300 g/twice a week for DM2 and 7.18 μg (4.8 μM)/300 g/twice a week for DM1 of MetfDeca, respectively, were administered. The resulting pharmacological data showed nontoxicological effects on liver and kidney. At the same time, MetfDeca showed an improvement of carbohydrates and lipids in tissues and serum. MetfDeca treatment was better than the monotherapies with metformin for DM2 and insulin for DM1. Additionally, MetfDeca showed a protective effect on pancreatic beta cells of DM1 rats, suggesting a possible regeneration of these cells, since they recovered their insulin levels. Therefore, MetfDeca could be considered not only as an insulin-mimetic agent, but also as an insulin-enhancing agent. Efforts to elucidate the mechanism of action of this compound are now in progress.

Antidiabetic mechanism of ethanol extract of black rice bran on diabetic rats

Background: Black rice bran contains bioactive compounds; previous studies have shown the ethanol extract of black rice bran (EEBRB) can decrease levels in diabetic rats. Aims and Objective: To elucidate the mechanism of antidiabetic effect of EEBRB. Materials and Methods: This research was carried out by setting a few parameters for antidiabetic. The effects of inhibition of the enzyme alpha-glucosidase performed with in vitro studies using the extracted enzyme of intestines of mice. A total of 5 mL alpha-glucosidase enzyme was incubated for 5 min with EEBRB 1 mL with a serial concentration in phosphate buffer pH 7.2. Enzyme inhibitory effect was determined by measuring the concentration of formed glucose with Lane–Eynon method. The insulin level and glucose-lowering activity were determined on rats induced by alloxan 150 mg/kg and treated with EEBRB. Treated rats for 28 days were sacrificed and were prepared for histopathological observation of pancreas. Result: EEBRB was able to inhibit the activity of the enzyme a-glucosidase on glucose with IC50 values of 121 ± 12.3 mg%. The EEBRB extract dose of 100 mg/kg was able to decrease blood sugar levels to 151 ± 38.58 mg/dL. The level of insulin in diabetic rats administered by EEBRB dose of 50, 100, and 200 mg/kg was obtained 6.52 ± 5.94, 11.5 ± 3.5, and 15.20 ± 9.5 ng/mL, respectively. Meanwhile, histopathological observations showed an improvement on the pancreatic beta cells. Conclusion: Antidiabetic mechanism of EEBRB is associated with increased insulin levels because of regeneration of pancreatic beta cells in diabetic rats.

The non-canonical NF-κB pathway is induced by cytokines in pancreatic beta cells and contributes to cell death and proinflammatory responses in vitro.

Activation of the transcription factor nuclear factor (NF)-κB by proinflammatory cytokines plays an important role in beta cell demise in type 1 diabetes. Two main signalling pathways are known to activate NF-κB, namely the canonical and the non-canonical pathways. Up to now, studies on the role of NF-κB activation in beta cells have focused on the canonical pathway. The aim of this study was to investigate whether cytokines activate the non-canonical pathway in beta cells, how this pathway is regulated and the consequences of its activation on beta cell fate. NF-κB signalling was analysed by immunoblotting, promoter reporter assays and real-time RT-PCR, after knockdown or overexpression of key genes/proteins. INS-1E cells, FACS-purified rat beta cells and the human beta cell line EndoC-βH1 exposed to cytokines were used as models. IL-1β plus IFN-γ induced stabilisation of NF-κB-inducing kinase and increased the expression and cleavage of p100 protein, culminating in the nuclear translocation of p52, the hallmark of the non-canonical signalling. This activation relied on different crosstalks between the canonical and non-canonical pathways, some of which were beta cell specific. Importantly, cytokine-mediated activation of the non-canonical pathway controlled the expression of 'late' NF-κB-dependent genes, regulating both pro-apoptotic and inflammatory responses, which are implicated in beta cell loss in early type 1 diabetes. The atypical activation of the non-canonical NF-κB pathway by proinflammatory cytokines constitutes a novel 'feed-forward' mechanism that contributes to the particularly pro-apoptotic effect of NF-κB in beta cells.

IL-13 improves beta-cell survival and protects against IL-1beta-induced beta-cell death.

ObjectivesIL-13 is a cytokine classically produced by anti-inflammatory T-helper-2 lymphocytes; it is decreased in the circulation of type 2 diabetic patients and impacts positively on liver and skeletal muscle. Although IL-13 can exert positive effects on beta-cell lines, its impact and mode of action on primary beta-cell function and survival remain largely unexplored.MethodsBeta-cells were cultured for 48 h in the presence of IL-13 alone or in combination with IL-1β or cytokine cocktail (IL-1β, IFNγ, TNFα).ResultsIL-13 protected human and rat beta-cells against cytokine induced death. However, IL-13 was unable to protect from IL-1β impaired glucose stimulated insulin secretion and did not influence NFκB nuclear relocalization induced by IL-1β. IL-13 induced phosphorylation of Akt, increased IRS2 protein expression and counteracted the IL-1β induced regulation of several beta-cell stress response genes.ConclusionsThe prosurvival effects of IL-13 thus appear to be mediated through IRS2/Akt signaling with NFκB independent regulation of gene expression. In addition to previously documented beneficial effects on insulin target tissues, these data suggest that IL-13 may be useful for treatment of type 2 diabetes by preserving beta-cell mass or slowing its rate of decline.

The role of noggin in regulation of high glucose-induced apoptosis and insulin secretion in INS-1 rat beta cells.

The purpose of this study was to investigate the effects of Noggin on high glucose-induced apoptosis and insulin secretion in pancreatic beta cells. Different concentrations of glucose were used to examine their effects on INS-1 rat beta cells in vitro. When specific siRNA targeting Noggin and recombinant Noggin were added, apoptosis and insulin secretion were measured, respectively to determine their effects in INS-1 cells. Glucose stimulated the expression of Noggin in a dose-dependent manner. Knockdown of Noggin further increased apoptosis and reduced insulin secretion when INS-1 cells were exposed to high glucose. Conversely, administration of recombinant Noggin significantly reduced apoptotic cell number, and promoted insulin secretion. Finally, treatment with inhibitor of Smad phosphorylation exerted similar effects on cell apoptosis and insulin production to Noggin administration in glucose-treated INS-1 cells. Our findings indicate that Noggin inhibits apoptosis and promotes insulin secretion in pancreatic beta cells through the inhibition of Smad signaling. Gene therapy of delivering Noggin may facilitate the treatment for patients with type 2 diabetes mellitus.

Inflammatory Cytokines Stimulate Bone Morphogenetic Protein-2 Expression and Release from Pancreatic Beta Cells.

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.

Cytokines induce endoplasmic reticulum stress in human, rat and mouse beta cells via different mechanisms

Proinflammatory cytokines contribute to beta cell damage in type 1 diabetes in part through activation of endoplasmic reticulum (ER) stress. In rat beta cells, cytokine-induced ER stress involves NO production and consequent inhibition of the ER Ca(2+) transporting ATPase sarco/endoplasmic reticulum Ca(2+) pump 2 (SERCA2B). However, the mechanisms by which cytokines induce ER stress and apoptosis in mouse and human pancreatic beta cells remain unclear. The purpose of this study is to elucidate the role of ER stress on cytokine-induced beta cell apoptosis in these three species and thus solve ongoing controversies in the field. Rat and mouse insulin-producing cells, human pancreatic islets and human EndoC-βH1 cells were exposed to the cytokines IL-1β, TNF-α and IFN-γ, with or without NO inhibition. A global comparison of cytokine-modulated gene expression in human, mouse and rat beta cells was also performed. The chemical chaperone tauroursodeoxycholic acid (TUDCA) and suppression of C/EBP homologous protein (CHOP) were used to assess the role of ER stress in cytokine-induced apoptosis of human beta cells. NO plays a key role in cytokine-induced ER stress in rat islets, but not in mouse or human islets. Bioinformatics analysis indicated greater similarity between human and mouse than between human and rat global gene expression after cytokine exposure. The chemical chaperone TUDCA and suppression of CHOP or c-Jun N-terminal kinase (JNK) protected human beta cells against cytokine-induced apoptosis. These observations clarify previous results that were discrepant owing to the use of islets from different species, and confirm that cytokine-induced ER stress contributes to human beta cell death, at least in part via JNK activation.

Characterization of stimulus-secretion coupling in the human pancreatic EndoC-βH1 beta cell line.

Aims/HypothesisStudies on beta cell metabolism are often conducted in rodent beta cell lines due to the lack of stable human beta cell lines. Recently, a human cell line, EndoC-βH1, was generated. Here we investigate stimulus-secretion coupling in this cell line, and compare it with that in the rat beta cell line, INS-1 832/13, and human islets.MethodsCells were exposed to glucose and pyruvate. Insulin secretion and content (radioimmunoassay), gene expression (Gene Chip array), metabolite levels (GC/MS), respiration (Seahorse XF24 Extracellular Flux Analyzer), glucose utilization (radiometric), lactate release (enzymatic colorimetric), ATP levels (enzymatic bioluminescence) and plasma membrane potential and cytoplasmic Ca2+ responses (microfluorometry) were measured. Metabolite levels, respiration and insulin secretion were examined in human islets.ResultsGlucose increased insulin release, glucose utilization, raised ATP production and respiratory rates in both lines, and pyruvate increased insulin secretion and respiration. EndoC-βH1 cells exhibited higher insulin secretion, while plasma membrane depolarization was attenuated, and neither glucose nor pyruvate induced oscillations in intracellular calcium concentration or plasma membrane potential. Metabolite profiling revealed that glycolytic and TCA-cycle intermediate levels increased in response to glucose in both cell lines, but responses were weaker in EndoC-βH1 cells, similar to those observed in human islets. Respiration in EndoC-βH1 cells was more similar to that in human islets than in INS-1 832/13 cells.Conclusions/InterpretationFunctions associated with early stimulus-secretion coupling, with the exception of plasma membrane potential and Ca2+ oscillations, were similar in the two cell lines; insulin secretion, respiration and metabolite responses were similar in EndoC-βH1 cells and human islets. While both cell lines are suitable in vitro models, with the caveat of replicating key findings in isolated islets, EndoC-βH1 cells have the advantage of carrying the human genome, allowing studies of human genetic variants, epigenetics and regulatory RNA molecules.

Expression and hypoxic regulation of the endothelin system in endocrine cells of human and rat pancreatic islets.

The success of pancreatic islet transplantation depends largely on the capacity of the islet graft to survive the initial phase immediately after transplantation until revascularization is completed. Endothelin-1 (ET-1) is a strong vasoconstrictor which has been involved in solid organ graft failure but is also known to be a potent mitogenic/anti-apoptotic factor which could also potentially enhance the survival of the transplanted islets. Characterization of the endothelin system with regard to a potential endothelin agonist/antagonist treatment. Regulated expression of the endothelin system in human and rat pancreatic islets and beta-cell lines was assessed by means of immunohistochemistry, competition binding studies, western blot, RT-PCR, real-time PCR and transplant studies. ET-1, ETA- and ETB-receptor immunoreactivity was identified in the endocrine cells of human and rat pancreatic islets. The corresponding mRNA was detectable in rat beta-cell lines and isolated rat and human pancreatic islets. Competition binding studies on rat islets revealed binding sites for both receptor types. ET-1 stimulated the phosphorylation of mitogen-activated protein kinase, which was prevented by ETA- and ETB-receptor antagonists. After exposure to hypoxia equal to post-transplant environment oxygen tension, mRNA levels of ET-1 and ETB-receptor of human islets were robustly induced whereas ETA-receptor mRNA did not show significant changes. Immunostaining signals for ET-1 and ETA-receptor of transplanted rat islets were markedly decreased when compared to native pancreatic sections. In pancreatic islets, ET-1 and its receptors are differentially expressed by hypoxia and after transplantation. Our results provide the biological basis for the study of the potential use of endothelin agonists/antagonists to improve islet transplantation outcome.

Morphological analysis of the pancreatic beta cells of diabetic female rats at different ages of life

s / Placenta 36 (2015) 469e521 490 pregnancy morbidity plus vascular thrombosis (PM/VT), pregnancy morbidity without aPL (PM/aPL-) or healthy women (NHS). Drugs were added at the same time with IgG at previous established concentrations. After 24 h, the inserts were removed and trophoblast cell migration across the Matrigel coated membrane was determined using a colorimetric assay. The relative cell invasion value was assessed by comparing the optical densities with no treatment control. STAT-3 activation was determined at same conditions of invasion assays using Western blots. Results: IgGs from patients with APS-associated pregnancy morbidity (PM, PM/VT) induce a significantly decreased invasion in comparison with IgGs from the NHS and PM/aPLgroups. Enoxaparin, aspirin and ATL restore the aPL-decreased invasion and, moreover, ATL has a receptormediated effect as demonstrated by application of its antagonist Boc-2, which reverses the ATL-restored invasion. Additionally, phosphorylation of STAT-3 is reduced by IgGs from PM patients. Conclusion: We show here that some of drugs used in APS-related pregnancy morbidity treatment are involved in the modulation of trophoblast function. They possibly restore some processes essential for normal trophoblast invasion. Moreover, ATL effects may explain the benefits of aspirin treatment observed in patients with obstetric APS. Since STAT-3 is an intracellular signalling molecule involved in migration and invasion processes, our results support the view that aPL from patients with pregnancy morbidity alter the proper function of this pathway. Financial support: CODIUniversidad de Antioquia Grant 91515; Estrategia de sostenibilidad Grupo Reproducci on 2014-2015; Programa de Cooperaci on Cientifica Internacional-Proyectos de Intercambio: Colciencias-Colombia Convocatoria 614-2013, y CONICYT-Chile PCCI 130017. AMA is a fellow of Colciencias. PA.7. MORPHOLOGICAL ANALYSIS OF THE PANCREATIC BETA CELLS OF DIABETIC FEMALE RATS AT DIFFERENT AGES OF LIFE F.Q. Gallego , Y.K. Sinzato , B. Dallaqua , I.L. Iessi , R.L. Amorim , C.E. Fonseca , I.M.P. Calderon , M.V.C. Rudge , D.C. Damasceno . 1 Laboratory of Experimental Research on Gynecology and Obstetrics, Graduate Program on Gynecology, Obstetrics and Mastology, Botucatu Medical School, Brazil; Department of Pathology, Botucatu Veterinary and Zootechnology Medical School, Univ. Estadual Paulista_Unesp, Botucatu, S~ ao Paulo, Brazil Background: Recent studies have shown regeneration of pancreatic islets in physiological and pathological circumstances. We hypothesized that diabetes disturbs insulin-producing b-cell regeneration in laboratory animals. Aim: The objective was to understand the proliferation and death mechanism of pancreatic b-cells in non-diabetic and neonatally streptozotocininduced mild diabetic rats at different stages of life and during pregnancy. Methods: The rats were randomly assigned into the following groups: Non-diabetic (Control1⁄4C, glycemia<120 mg/dL) or Mild diabetes (MD) (n1⁄415/group). The mild diabetes group was induced at birth by the b-cell cytotoxic agent e Streptozotocin (STZ 100 mg/kg b.w., sc.). On postnatal days (PND) 5, 15 and 90 and on day 18.5 of pregnancy, the different groups of rats were killed to collect blood samples for insulin and glycated hemoglobin measurements; pancreas samples were processed for histological and immunohistochemical analysis for cell proliferation, apoptosis and insulin. In addition, on day 18.5 of pregnancy, the uterine corns were exposed for evaluation of reproductive outcomes. p<0.05 was considered as the significant statistical limit. Results: On PND5, the diabetic rats showed pancreatic islet morphological alterations as decreased insulin and apoptosis immunostaining, and increased cell proliferation. On PND15, cell proliferationwas lower than on PND5 in diabetic rats. In adulthood (PND90 and pregnancy), diabetic rats presented increased glycated hemoglobin levels and glucose intolerance, and no changes in pancreatic islets morphology were observed in relation to the control group. On day 18.5 of pregnancy, MD dams presented an increased number of embryonic death and rate of preimplantation losses when compared with the control group. Conclusion: Our results showed that after administration of streptozotocin pancreatic b-cells were able to regenerate partially by an increase in proliferation and a decrease in apoptosis. However, this was insufficient to reverse the diabetic status in adulthood leading to impairment of the reproductive function in diabetic mothers. Acknowledgement: FAPESP/Brazil (Process Numbers: 2011/18519-7; 2011/15606-6). PA.8. LEPTIN IS INVOLVED IN HUMAN TROPHOBLAST MIGRATION AND INVASION A. Toro , R. Sampayo , A. P erez-P erez , B. Maskin , V. S anchezMargalet , M. Simian , C. Varone . Dpto. de Quimica Biol ogica, FCENUBA, IQUIBICEN CONICET, Buenos Aires, Argentina; 2 Area de Investigaciones, Instituto de Oncologia “ Angel H. Roffo”, Buenos Aires, Argentina; Dpto. de Bioquimica M edica y Biologia Molecular. Universidad de Sevilla, Sevilla, Spain; Hospital Nacional “A. Posadas”, Buenos Aires,

Quantitative proteomics of rat and human pancreatic beta cells.

Data set description: This data set is composed by label-free alternate-scanning LC-MS/MS proteomics analysis human and Wistar rat pancreatic islet endocrine cells.The mass spectrometry data of the human and rat pancreatic beta cells and the resulting proteome search output from ProteinLynx GlobalSERVER (PLGS) have been deposited to the ProteomeXchange Consortium [1] via the PRIDE partner repository with the dataset identifiers PXD001539 (human) and PXD001816 (rat). From these mass spectrometry data, ‘relative molar amount units’ between cell types and across species were calculated.Biological relevance: These data provide a quantitative view on the unfractionated proteomes of human and rat beta and alpha cells. It is likely biased towards the proteins with higher molar abundance, relating to core functional pathways, but also includes several proteins with an islet-enriched expression. The quality of the cell preps is state-of-the-art, and the label-free quantitation is both precise and accurate, allowing detailed quantitative analysis.

Short Term Exposure of Beta Cells to Low Concentrations of Interleukin-1β Improves Insulin Secretion through Focal Adhesion and Actin Remodeling and Regulation of Gene Expression

Type 2 diabetes involves defective insulin secretion with islet inflammation governed in part by IL-1β. Prolonged exposure of islets to high concentrations of IL-1β (>24 h, 20 ng/ml) impairs beta cell function and survival. Conversely, exposure to lower concentrations of IL-1β for >24 h improves these same parameters. The impact on insulin secretion of shorter exposure times to IL-1β and the underlying molecular mechanisms are poorly understood and were the focus of this study. Treatment of rat primary beta cells, as well as rat or human whole islets, with 0.1 ng/ml IL-1β for 2 h increased glucose-stimulated (but not basal) insulin secretion, whereas 20 ng/ml was without effect. Similar differential effects of IL-1β depending on concentration were observed after 15 min of KCl stimulation but were prevented by diazoxide. Studies on sorted rat beta cells indicated that the enhancement of stimulated secretion by 0.1 ng/ml IL-1β was mediated by the NF-κB pathway and c-JUN/JNK pathway acting in parallel to elicit focal adhesion remodeling and the phosphorylation of paxillin independently of upstream regulation by focal adhesion kinase. Because the beneficial effect of IL-1β was dependent in part upon transcription, gene expression was analyzed by RNAseq. There were 18 genes regulated uniquely by 0.1 but not 20 ng/ml IL-1β, which are mostly involved in transcription and apoptosis. These results indicate that 2 h of exposure of beta cells to a low but not a high concentration of IL-1β enhances glucose-stimulated insulin secretion through focal adhesion and actin remodeling, as well as modulation of gene expression.

Potential of UCHL1 as biomarker for destruction of pancreatic beta cells.

Our bottom-up LC-MS/MS proteomics represents a pragmatic approach to identify protein-type biomarkers of pancreatic beta cell injury. UCHL1 successfully passed sequential validation steps of beta cell-selectivity, antigenicity and toxic discharge in vitro. Whole-body dynamic planar imaging of radiolabeled recombinant UCHL1 indicated rapid clearance through the liver, spleen and urinary excretion of proteolytic fragments, likely explaining non-consistent detection in vivo. Integration of kinetic biomarker clearance studies in the a priori selection criteria is recommended before engaging in resource-intensive custom development of sensitive immunoassays for clinical translation.

Species-Related Differences in the Proteome of Rat and Human Pancreatic Beta Cells.

The core proteomes of human and rat pancreatic beta cells were compared by label-free LC-MS/MS: this resulted in quantification of relative molar abundances of 707 proteins belonging to functional pathways of intermediary metabolism, protein synthesis, and cytoskeleton. Relative molar abundances were conserved both within and between pathways enabling the selection of a housekeeping network for geometric normalization and the analysis of potentially relevant differential expressions. Human beta cells differed from rat beta cells in their lower level of enzymes involved in glucose sensing (MDH1, PC, and ACLY) and upregulation of lysosomal enzymes. Human cells also expressed more heat shock proteins and radical scavenging systems: apart from SOD2, they expressed high levels of H2O2-scavenger peroxiredoxin 3 (PRDX3), confirmed by microarray, Western blotting, and microscopy. Besides conferring lower susceptibility to oxidative stress to human cells PRDX3 might also play a role in physiological redox regulation as, in rat, its expression was restricted to a beta cell subset with higher metabolic glucose responsiveness. In conclusion, although their core proteomic architecture is conserved, human and rat beta cells differ in their molar expression of key enzymes involved in glucose sensing and redox control.