Insulin Staining Research Articles

Role of Pax4 in Pdx1-VP16-mediated liver-to-endocrine pancreas transdifferentiation

Although Pdx1-VP16 expression induces hepatic cell transdifferentiation into pancreatic precursor cells (WB-1), these incompletely reprogrammed cells fail to become glucose-sensitive insulin-producing cells in the absence of the activation of late-stage pancreatic transcription factors. As Pax4 promotes late-stage β-cell differentiation and maturation, we generated lentiviral vector (LV) containing mouse Pax4 gene and developed two hepatic cell lines expressing Pax4 in the absence (WB-2 cells) or presence (WB-1A cells) of Pdx1-VP16, via LV-mediated gene transfer. Functional Pax4 protein expression in WB-2 and WB-1A cells was confirmed by electrophoretic mobility shift assay and Pdx1-VP16 protein expression in WB-1 and WB-1A cells was confirmed by Western blotting. Activation of Pax4 resulted in the expression of the late-stage transcription factors, including Pax6, Isl-1, and MafA, and generated a gene expression profile for WB-1A cells similar to that of functional rat insulinoma INS-1 cells. Insulin abundance in WB-1A cells was demonstrated by immunostaining. WB-1A cells exhibited glucose-responsive insulin release in vitro, and caused a rapid reversal of hyperglycemia following cell transplantation into streptozotocin-induced diabetic mice. Intraperitoneal glucose tolerance test showed a normal glucose response in WB-1, and WB-1A transplanted mice similar to that of normal mice. Removal of transplanted WB-1A cells resulted in a return of hyperglycemia, confirming that they were responsible for the observed normoglycemia. The explanted WB-1A cells exhibited strong insulin staining comparable to native islet β-cells. These studies indicate that activation of Pax4 in Pdx1-VP16-expressing cells reprograms pancreatic precursor-like WB-1 cells into glucose-responsive, more mature insulin-producing cells.

A new model of insulin-deficient diabetes: male NOD mice with a single copy of Ins1 and no Ins2

We describe a novel model of insulin-deficient diabetes with a single copy of the gene encoding insulin 1 (Ins1) and no gene encoding insulin 2 (Ins2). We constructed five lines of mice: mice with two copies of Ins1 (NOD( Ins1+/+,Ins2-/-)), mice with a single copy of Ins1 (NOD( Ins1+/-,Ins2-/-)), mice with two copies of Ins2 (NOD( Ins1-/-,Ins2+/+)), mice with a single copy of Ins2 (NOD( Ins1-/-,Ins2+/-)) and NOD( Ins1+/-,Ins2-/-) mice with a transgene encoding B16:Ala proinsulin. By 10 weeks of age, all male NOD( Ins1+/-,Ins2-/-) mice were diabetic, whereas all female NOD( Ins1+/-,Ins2-/-) were not diabetic (p < 0.0001). In contrast, neither male nor female NOD( Ins1-/-,Ins2+/-) with a single copy of Ins2 (rather than single copy of Ins1) developed early diabetes and no mice with two copies of either gene developed early diabetes. Islets of the diabetic male NOD( Ins1+/-,Ins2-/-) at this early age had no lymphocyte infiltration. Instead there was heterogeneous (between islet cells) weak staining for insulin. Although only male NOD( Ins1+/-,Ins2-/-) mice developed diabetes, both male and female NOD( Ins1+/-,Ins2-/-) mice had markedly decreased insulin content. In NOD( Ins1+/+,Ins2-/-), there was also a significant decrease in insulin content, whereas NOD( Ins1-/-,Ins2+/+) mice, and even NOD( Ins1-/-,Ins2+/-) mice, were normal. Male NOD( Ins1+/-,Ins2-/-) mice were completely rescued from diabetes by introduction of a transgene encoding proinsulin. On i.p. insulin tolerance testing, male mice had insulin resistance compared with female mice. These results suggest that Ins1 is a 'defective gene' relative to Ins2, and that the mouse lines created provide a novel model of sex-dimorphic insulin-deficient diabetes.

Development of an Islet‐Stabilizing Implant Construct

Islet transplantation for the purpose of treating type-1 diabetes is severely limited by several factors, including islet stability and viability. Following the isolation of islets from pancreas tissue, the ability of the islet to secrete physiologically relevant quantities of insulin is severely diminished; therefore, a current strategy for improving islet stability is to establish vascular perfusion to and within the islet, thus preserving islet function. The strategy used in this study was to create an islet implant construct that contains microvessel fragments which had been pre-cultured in a 3-dimensional collagen matrix. The islets were then placed between the microvessel gels, and the constructs were implanted into SCID mice. At 7 and 14 days post implantation, the constructs and surrounding tissues were removed, fixed, and prepared for histological analysis. Results from these experiments showed that the islets and some isolated cells expressed detectable quantities of insulin. Moreover, this insulin staining was associated with intact islets, but also with islets which were partially dissociated. Lastly, double cytochemical staining for insulin producing cells and endothelium indicate that insulin-producing islets contained endothelial cells, while islets devoid of endothelial cells did not stain for insulin. In conclusion, implant constructs containing islets and a pre-formed vasculature have been shown to stabilize islet function following 7 and 14 days of implantation.

Dynamics of Glucose-Induced Localization of PKC Isoenzymes in Pancreatic β-Cells

Glucose metabolism affects most major signal pathways in pancreatic beta-cells. Multiple protein kinases, including protein kinase C (PKC) isoenzymes, are involved in these effects; however, their role is poorly defined. Moreover, the dynamics of kinase isoenzyme activation in reference to the biphasic insulin secretion is unknown. In perfused pancreas of Wistar rats, PKCalpha staining was strongly associated with insulin staining, jointly accumulating in the vicinity of the plasma membrane during early first-phase insulin response. The signal declined before the onset of second phase and reappeared during second-phase insulin release as foci, only weekly associated with insulin staining; this signal persisted for at least 15 min after glucose stimulation. In the GK rat, glucose had minimal effect on beta-cell PKCalpha. In control beta-cells, PKCdelta stained as granulated foci with partial association with insulin staining; however, no glucose-dependent translocation was observed. In the GK rat, only minimal staining for PKCdelta was observed, increasing exclusively during early first-phase secretion. In Wistar beta-cells, PKCepsilon concentrated near the nucleus, strongly associated with insulin staining, with dynamics resembling that of biphasic insulin response, but persisting for 15 min after cessation of stimulation. In GK rats, PKCepsilon staining lacked glucose-dependent changes or association with insulin. PKCzeta exhibited bimodal dynamics in control beta-cells: during early first phase, accumulation near the cell membrane was observed, dispersing thereafter. This was followed by a gradual accumulation near the nucleus; 15 min after glucose stimulus, clear PKCzeta staining was observed within the nucleus. In the GK rat, a similar response was only occasionally observed. In control beta-cells, glucose stimulation led to a transient recruitment of PKCtheta, associated with first-phase insulin release, not seen in GK beta-cell. Data from this and related studies support a role for PKCalpha in glucose-induced insulin granule recruitment for exocytosis; a role for PKCepsilon in activation of insulin granules for exocytosis and/or in the glucose-generated time-dependent potentiation signal for insulin release; and a dual function for PKCzeta in initiating insulin release and in a regulatory role in the transcriptional machinery. Furthermore, diminished levels and/or activation of PKCalpha, PKCepsilon, PKCtheta, and PKCzeta could be part of the defective signals downstream to glucose metabolism responsible for the deranged insulin secretion in the GK rat.

Effect of Black Cumin (Nigella sativa) on Heart Rate, Some Hematological Values, and Pancreatic β-Cell Damage in Cadmium-Treated Rats

This study was designed to investigate the effect of Nigella sativa (NS) on the heart rate, some hematological values, and pancreatic beta-cell damage in cadmium (Cd)-treated rats. The rats were randomly grouped into one of three experimental groups: Control, Cd treated, and Cd + NS treated. Each group contained 10 animals. The Cd-treated and Cd + NS-treated groups were injected subcutaneously daily with CdCl2 dissolved in isotonic NaCl in the amount of 2 mL/kg for 30 d, resulting in a dosage of 0.49 mg Cd/kg/d. The control group was injected with only isotonic NaCl (2 mL/kg/d) throughout the experiment (for 30 d). Three days prior to administration of CdCl2, the Cd + NS-treated group received the daily intraperitoneal (ip) injection of 2 mL/kg NS until the end of the study; animals in all three groups were fasted for 12 h and blood samples were taken for the determination of the glucose and insulin levels, red blood cell (RBC) and white blood cell (WBC) counts, packet cell volume (PCV), and hemoglobin (Hb) concentration. The heart rates of rats were also measured by a direct writing electrocardiograph before the blood withdrawals. It was found that NS treatment increased the lowered insulin levels, RBC and WBC counts, PCV, and neutrophil percentage in Cd-treated rats. However, the WBC count of Cd-treated rats with NS treatment was still lower than those of control values. NS treatment also decreased the elevated heart rate and glucose concentration of Cd-treated rats. Pancreatic tissues were also harvested from the sacrificed animals for morphological and immunohistochemical examinations. Cd exposure alone caused a degeneration, necrosis, and weak degranulation in the beta-cells of the pancreatic islets. In Cd + NS-treated rats, increased staining of insulin and preservation of islet cells were apparent. It is concluded that NS treatment might decrease the Cd-treated disturbances on heart rate, some hematological values, and pancreatic beta-cell.

Amelioration of Obesity and Glucose Intolerance in High-Fat-Fed C57BL/6 Mice by Anthocyanins and Ursolic Acid in Cornelian Cherry (Cornus mas)

Much attention has been focused on food that may be beneficial in preventing diet-induced body fat accumulation and possibly reduce the risk of diabetes and heart disease. Cornelian cherries (Cornus mas) are used in the preparation of beverages in Europe and also to treat diabetes-related disorders in Asia. In this study, the most abundant bioactive compounds in C. mas fruits, the anthocyanins and ursolic acid, were purified, and their ability to ameliorate obesity and insulin resistance in C57BL/6 mice fed a high-fat diet was evaluated. Mice were initially fed a high-fat diet for 4 weeks and then switched to a high-fat diet containing anthocyanins (1 g/kg of high-fat diet) and ursolic acid (500 mg/kg of high-fat diet) for an additional 8 weeks. The high-fat diet induced glucose intolerance, and this was prevented by anthocyanins and ursolic acid. The anthocyanin-treated mice showed a 24% decrease in weight gain. These mice also showed decreased lipid accumulation in the liver, including a significant decrease in liver triacylglycerol concentration. Anthocyanin and ursolic acid treated mice exhibited extremely elevated insulin levels. Both treatments, however, showed preserved islet architecture and insulin staining. Overall, these data suggest that anthocyanins and ursolic acid purified from C. mas fruits have biological activities that improve certain metabolic parameters associated with diets high in saturated fats and obesity.

Dopamine D2-like Receptors Are Expressed in Pancreatic Beta Cells and Mediate Inhibition of Insulin Secretion

Dopamine signaling is mediated by five cloned receptors, grouped into D1-like (D1 and D5) and D2-like (D2, D3 and D4) families. We identified by reverse transcription-PCR the presence of dopamine receptors from both families in INS-1E insulin-secreting cells as well as in rodent and human isolated islets. D2 receptor expression was confirmed by immunodetection revealing localization on insulin secretory granules of INS-1E and primary rodent and human beta cells. We then tested potential effects mediated by the identified receptors on beta cell function. Dopamine (10 microM) and the D2-like receptor agonist quinpirole (5 microM) inhibited glucose-stimulated insulin secretion tested in several models, i.e. INS-1E beta cells, fluorescence-activated cell-sorted primary rat beta cells, and pancreatic islets of rat, mouse, and human origin. Insulin exocytosis is controlled by metabolism coupled to cytosolic calcium changes. Measurements of glucose-induced mitochondrial hyperpolarization and ATP generation showed that dopamine and D2-like agonists did not inhibit glucose metabolism. On the other hand, dopamine decreased cell membrane depolarization as well as cytosolic calcium increases evoked by glucose stimulation in INS-1E beta cells. These results show for the first time that dopamine receptors are expressed in pancreatic beta cells. Dopamine inhibited glucose-stimulated insulin secretion, an effect that could be ascribed to D2-like receptors. Regarding the molecular mechanisms implicated in dopamine-mediated inhibition of insulin release, our results point to distal steps in metabolism-secretion coupling. Thus, the role played by dopamine in glucose homeostasis might involve dopamine receptors, expressed in pancreatic beta cells, modulating insulin release.

An Improved Method of Fluorescent Dual Insulin and Endothelial Staining Allows Visualisation of the Revascularisation of Intraportally Transplanted Islets

Background Intraportally transplanted islets are avascular at the time of transplantation and take up to 14 days to fully revascularize, during which time up to 60% of islet mass may be lost. To investigate and improve islet revascularization, a robust method for the visualization and quantification of this process is required. Methods Islets isolated from Lewis rats were transplanted intraportally into the liver of diabetic syngeneic Lewis recipients. The animals were humanely killed either on the day of transplant or at 3, 5, 7, or 14 days posttransplant. The harvested livers were sectioned and stained with Bandeiraea simplicifolia lectin (for endothelial cells) and anti-insulin antibody and counterstained with DAPI. The slides were visualized with a fluorescent microscope. Results Islets were visualized over the whole time course. Insulin and endothelial staining was clearly visualized on the day of transplantation, but by day 3 endothelial staining was scarce within the islet. By day 5, early vessel formation could be seen within the islet, but insulin staining was patchy and associated with apoptotic nuclei. By day 7, vessels could be seen throughout the islet and insulin staining had returned. Day 14 sections showed a fully revascularized islet. Conclusions The staining provided good delineation of islet endothelium and β-cell location, with clear observation of the revascularization process. This technique also suggests that days 3 through 5 may be a critical period for islet survival and provides a good model for studying the effects of manipulating the revascularization process.

Human Cord Blood–Derived Cells Generate Insulin‐Producing Cells In Vivo

Here we report the capacity of human cord blood (CB)-derived cells to generate insulin-producing cells. To investigate in vivo capacity of human CB-derived cells, T cell-depleted mononuclear cells were intravenously transplanted into nonobese diabetic/severe combined immunodeficient/beta2-microglobulinnull mice within 48 hours of birth. At 1-2 months post-transplantation, immunofluorescence staining for insulin and fluorescence in situ hybridization (FISH) analysis using a human chromosome probe indicated that human CB-derived cells generated insulin-producing cells at a frequency of 0.65%+/-0.64% in xenogeneic hosts. Reverse transcription-polymerase chain reaction analysis confirmed the transcription of human insulin in the pancreatic tissue of the recipient mice. To clarify the mechanism underlying CB-derived insulin-producing cells, double FISH analysis using species-specific probes was performed. Almost equal proportions of human chromosome+ murine chromosome- insulin+ cells and human chromosome+ murine chromosome+ insulin+ cells were present in recipient pancreatic islets. Taken together, human CB contains progenitor cells, which can generate insulin-producing cells in recipient pancreatic tissues across a xenogeneic histocompatibility barrier by fusion-dependent and -independent mechanisms.

Differences in Suppressor of Cytokine Signaling-1 (SOCS-1) Expressing Islet Allograft Destruction in Normal BALB/c and Spontaneously-Diabetic NOD Recipient Mice

The ability to block interferon signaling represents an important strategy in designing therapies to prevent beta-cell destruction during islet allograft rejection. The SOCS proteins regulate cytokine signaling by blocking activation of JAK/STAT proteins. Using islets isolated from SOCS-1 transgenic mice (SOCS-1-Tg; these mice express SOCS-1 under the control of the human insulin promoter and are on the C57BL6/J background), we investigated whether SOCS proteins can prevent the destruction pancreatic islet cells transplanted beneath the kidney capsule of major histocompatibility complex mismatched normal BALB/c and spontaneously-diabetic NOD mouse recipients. Immunohistochemical staining for insulin confirmed the presence of donor SOCS-1-Tg islets in islet allografts harvested at 22 days posttransplant, whereas grafts of control non-Tg islets were destroyed by 14 days. In contrast, SOCS-1-Tg allogeneic islets were not protected from beta-cell destruction in clinically diabetic NOD mice. The islet allografts functioned for 1 week posttransplant; however, hyperglycemia returned after 2 weeks and the grafts were destroyed. Rejection of SOCS-1-Tg and non-Tg islets in autoimmune diabetic NOD mice was associated with an infiltrate of both CD4+ and CD8+ T cells and a T2-type cytokine response (IL-4) rather than the conventional T1-type cytokine response observed during islet allograft rejection. Self-antigen upregulation in response to IFN-gamma stimulation did not appear to be a factor in rejection of the islet allografts. These results demonstrate that expression of SOCS-1 in islets delays islet allograft rejection but cannot circumvent destruction of the islets by the recurrence of the tissue-specific autoimmune process of spontaneous diabetes.

Inducing Embryonic Stem Cells to Differentiate into Pancreatic β Cells by a Novel Three‐Step Approach with Activin A and All‐ Trans Retinoic Acid

Experimental induction of embryonic stem cells (ESCs) to become pancreatic beta cells can potentially provide ample resource for cell transplantation therapy of type I diabetes mellitus. Most of the previously reported induction strategies were long and complicated, and some required genetic manipulation. Moreover, it has been indicated that the insulin staining of ESC progeny was insulin uptake from the culture medium. Here we show that a simple three-step experimental approach based on the combination induction by activin A, all-trans retinoic acid, and other mature factors is able to induce murine ESCs to differentiate into insulin-producing cells in 2 weeks, and that insulin release of these induced cells is regulated by the glucose concentration. Our insulin-enhanced green fluorescent green protein reporter system excludes the possibility of insulin uptake. Transplantation of these ESC-derived insulin-positive cells can normalize blood glucose levels and rescue the survival of streptozocin-induced diabetic mice. The findings reported here offer a novel in vitro model to study the differentiation mechanism of pancreatic beta cells and a potential source of insulin-producing cells for transplantation therapy of type I diabetes mellitus.

Prevention and Cure of Autoimmune Diabetes in Nonobese Diabetic Mice by Continuous Administration of FTY720

Treatment of nonobese diabetic (NOD) mice with FTY720 before the development of insulitis prevents the onset of diabetes. In this study, the authors investigated whether FTY720 treatment of NOD mice with established insulitis prevents the development of diabetes. FTY720 (1 mg/kg) was administered continuously to euglycemic NOD mice starting at 14 or 23 weeks of age. A group of untreated, age-matched NOD mice served as controls. Mice with more than 300 mg/dL blood glucose on three consecutive measurements were considered diabetic. Diabetes developed in control mice starting at 13 weeks of age and reached 78% by 33 weeks of age. Mice at 14 and 23 weeks of age exhibited extensive insulitis that progressed with age. Continuous oral administration of FTY720 starting at either age completely prevented the development of diabetes. However, its withdrawal at 37 weeks of age led to abrupt diabetes onset. Pancreases of FTY720-treated diabetes-free mice showed peripheral insulitis, with strong insulin staining. The protection from diabetes was also achieved by intraperitoneal injection of FTY720 or sirolimus (1.5 mg/kg). Unlike FTY720, withdrawal of sirolimus did not induce diabetes. Continuous oral FTY720 (3 mg/kg) treatment in overtly diabetic NOD mice led to complete reversal of diabetes in 6 of 11 mice. The standard adoptive transfer study in NOD-severe combined immunodeficient mice showed that peripheral lymphoid organs of FTY720-treated mice contained diabetogenic cells but not dominant immunoregulatory cells. FTY720, which does not cause generalized immunosuppression, may be a safe and benign therapeutic agent for chronic use to prevent or cure type 1 diabetes.

SOURCE OF INSULIN SECRETED BY ISLET-LIKE CELL CLUSTERS (ILCCs) DERIVED FROM MURINE EMBRYONIC STEM (mES) CELLS

This study was undertaken to clarify the source of insulin secreted by ILCCs, derived from mES cells through a five-stage process. Although previous published studies demonstrated glucose-responsive insulin secretion by ILCCs, some investigators later disputed that insulin was not synthesized by ILCCs de novo, but rather exogenous insulin supplement in media was sequestered during culture and released upon glucose stimulation. To resolve this issue, exogenous insulin in media for stages 3, 4 and 5 was (1) eliminated or (2) replaced with insulin-like growth factor-I (IGF-I) to test for insulin secretion in a condition where artifactual sequestration of exogenous insulin was not possible. Insulin is known to bind to IGF-I receptors and able to activate the IGF-receptor mediated pathway. Although IGF-I-derived ILCCs showed positive staining for intracellular insulin, they failed to secrete any detectable quantity of insulin by ELISA, upon glucose stimulation. Control ILCCs cultured with neither insulin nor IGF-I did not stain for intracellular insulin. Based on our results and those of others, we propose the following working hypotheses: Insulin or IGF-I receptor-mediated pathways are necessary to initiate insulin synthesis in ILCCs. Both mechanisms of sequestration from media and de novo synthesis are the sources of insulin secreted by ILCCs while insulin absorbed by ILCCs from the media transiently suppresses de novo insulin synthesis; production resumes when exogenous insulin is depleted.

Successful incorporation of short-interfering RNA into islet cells by in situ perfusion

Islet transplantation offers a potential cure for type I diabetes, although its success has been limited, due to loss of cells by apoptosis stimulated by the procurement, ischemia, and the isolation process itself. RNA interference (RNAi) as mediated by short interfering RNAs (siRNAs) has become a potent tool to manipulate gene expression in mammalian cells. We describe the first successful introduction of siRNA directly into pancreatic islet cells both during in situ perfusion and from intravenous tail vein injection (in vivo). Methods siRNA was targeted to the pancreatic islets of BALB/c mice by retrograde portal vein perfusion or tail vein injection. Cy3-labeled siRNA was dissolved in University of Wisconsin (UW) solution at 2 μg/mL. After delivery pancreata were placed in cold storage at 4°C in UW solution for 24 hours, followed by processing for immunofluorescent staining for insulin. Fluorescent imaging was obtained using a Nikon DIAPHOT 300 Inverted Micoscope with a Zeiss AxioCam and OpenLab image capturing software. Results In situ delivery of siRNA was demonstrated by fluorescent imaging composites of (red) siRNA in and along (green) insulin stained islets from pancreas sections as compared with untreated control sections. The siRNA was detected mainly in and along venous structures throughout the pancreatic tissue. In vivo delivery of siRNA into islets was observed by fluorescent images taken of isolated islets in culture. Conclusions We have described the successful delivery of siRNA to pancreatic islets via a novel in situ pancreas perfusion technique and in vivo delivery via tail vein injection.

Nicorandil improves diabetes and rat islet beta-cell damage induced by streptozotocin in vivo and in vitro.

N-(2-hydroxyethyl)-nicotinamide nitrate (nicorandil) is a unique anti-anginal agent, reported to act as both an ATP-sensitive K(+) channel opener (PCO) and a nitric oxide donor. It also has an anti-oxidant action. We examined the effects of nicorandil on streptozotocin (STZ)-induced islet beta-cell damage both in vivo and in vitro. STZ-induced diabetic Brown Norway rats (STZ-DM) were fed with nicorandil-containing chow from day 2 (STZ-DM-N48), 3 (STZ-DM-N72), and 4 (STZ-DM-N96) to day 30. Body weight, blood glucose, and plasma insulin were measured every week. For the in vitro assay, neonatal rat islet-rich cultures were performed and cells were treated with nicorandil from 1 h before to 2 h after exposure to STZ for 30 min. Insulin secretion from islet cells was assayed after an additional 24 h of culture. We also observed the effect of nicorandil on the generation of reactive oxygen species (ROS) from rat inslinoma cells (RINm5F). Body weight loss and blood glucose levels of STZ-DM-N48 rats were significantly lower than those of STZ-DM rats. Immunohistochemical staining of insulin showed preservation of insulin-secreting islet beta-cells in STZ-DM-N48 rats. Nicorandil also dose-dependently recovered the insulin release from neonatal rat islet cells treated with STZ in in vitro experiments. Nicorandil did not act as a PCO on neonatal rat islet beta-cells or RINm5F cells, and did not show an inhibitory effect on poly(ADP-ribose) polymerase-1. However, the drug inhibited the production of ROS stimulated by high glucose (22.0 mmol/l) in RINm5F cells. These results suggested that nicorandil improves diabetes and rat islet beta-cell damage induced by STZ in vivo and in vitro. It protects islet beta-cells, at least partly, via a radical scavenging effect.

Syzygium cumini and the regeneration of insulin positive cells from the pancreatic duct

Syzygium cumini is a plant that has been used in popular medicine for the treatment of insulin dependent diabetes mellitus (DMID). This study verified the effect of Syzygium cumini upon the regeneration of insulin producing cells in the pancreatic duct wall. The animals were divided into four groups, control (C), treated control (TC), diabetic control (DC) and treated diabetic (TD). An aqueous extract from Syzygium cumini bark was given by gavage in a daily dose of 1g/kg of body weight. After a thirty day period the animals were euthanized and the pancreas taken to immunohistochemical analysis. In this study, it was observed the positive staining for insulin on cells of the pancreatic duct and connective tissue in the pancreas of TD and TC animals. These results indicate that Syzygium cumini bark extract stimulates development of insulin positive cells from the pancreatic duct epithelial cells.

Quercetin, a flavonoid antioxidant, prevents and protects streptozotocin-induced oxidative stress and β-cell damage in rat pancreas

The aim of the present study was the evaluation of possible protective effects of quercetin (QE) against β-cell damage in experimental streptozotocin (STZ)-induced diabetes in rats. STZ was injected intraperitoneally at a single dose of 50 mg kg −1 for diabetes induction. QE (15 mg kg −1 day, intraperitoneal (i.p.) injection) was injected for 3 days prior to STZ administration; these injections were continued to the end of the study (for 4 weeks). It has been believed that oxidative stress plays a role in the pathogenesis of diabetes mellitus (DM). In order to determine the changes of cellular antioxidant defense system, antioxidant enzymes such as glutathione peroxidase (GSHPx), superoxide dismutase (SOD) and catalase (CAT) activities were measured in pancreatic homogenates. Moreover we also measured serum nitric oxide (NO) and erythrocyte and pancreatic tissue malondialdehyde (MDA) levels, a marker of lipid peroxidation, if there is an imbalance between oxidant and antioxidant status. Pancreatic β-cells were examined by immunohistochemical methods. STZ induced a significant increase lipid peroxidation, serum NO concentrations and decreased the antioxidant enzyme activity. Erythrocyte MDA, serum NO and pancreatic tissue MDA significantly increased ( P < 0.05) and also the antioxidant levels significantly decreased ( P < 0.05) in diabetic group. QE treatment significantly decreased the elevated MDA and NO ( P < 0.05), and also increased the antioxidant enzyme activities ( P < 0.05). QE treatment has shown protective effect possibly through decreasing lipid peroxidation, NO production and increasing antioxidant enzyme activity. Islet cells degeneration and weak insulin immunohistochemical staining was observed in STZ induced diabetic rats. Increased staining of insulin and preservation of islet cells were apparent in the QE-treated diabetic rats. These findings suggest that QE treatment has protective effect in diabetes by decreasing oxidative stress and preservation of pancreatic β-cell integrity.

CONTINUOUS ADMINISTRATION OF FTY720 IN NONDIABETIC NOD MICE WITH ESTABLISHED INSULITIS PREVENTS SUBSEQUENT DEVELOPMENT OF AUTOIMMUNE DIABETES

O196* Aim: Treatment of nondiabetic NOD mice with FTY720 starting at 4 weeks of age, when insulitis has not developed, prevents the onset of overt diabetes. In the present study, we investigated whether FTY720 treatment of NOD mice that harbor established insulitis but remain normoglycemic prevents subsequent development of diabetes. Methods: Beginning from either 14 or 23 weeks of age, nondiabetic female NOD mice were continously given 0.5 mg/kg FTY720 via a gavage needle once a day, five times a week (Monday through Friday). A group of untreated, age-matched female NOD mice served as controls. Mice were monitored twice a week for urine glucose levels. Once urine glucose levels became positive, mice were additionally tested for a nonfasting blood glucose level every 2 – 3 days. Mice with >300 mg/dl blood glucose in 3 consecutive measurements were considered diabetic. Results: Control untreated female NOD mice developed overt diabetes starting at 13 weeks of age and showed diabetes incidence of 65 and 70% at 30 and 40 weeks of age, respectively. Histological examination of the pancreas at 14 and 23 weeks confirmed the presence of insulitis that progressed with age. None of the FTY720-treated NOD mice (8 mice in both groups) developed diabetes regardless of the timing of treatment initiation during the period when FTY720 was given. Intraperitoneal administration of FTY720 was also effective in prevention of diabetes. Light microscopic examination of the pancreases removed from the FTY720-treated diabetes-free mice at 37 or 44 weeks of age revealed islets that were either without lymphocytic infiltrates or with varying degrees of peripheral lymphocytic infiltrates. Invading insulitis was not seen. These islets stained strongly by insulin staining. When oral administration of FTY720 was withdrawn at 37 weeks of age, the majority of mice rapidly developed diabetes. Mice remained diabetes-free when they were continuously treated with FTY720 beyond 37 weeks of age (currently over 44 weeks of age). Conclusion: As generalized immunosuppression is not associated with FTY720, FTY720 may be a safe and benign therapeutic agent that could be used chronically in diabetes prone, yet normoglycemic individuals even after development of insulitis to prevent type 1 diabetes mellitus.

IN JUVENILE PIGS, DIABETES INDUCTION BY SREPTOZOTOCIN IS NOT AN APPROPRIATE MODEL FOR ISLETS TRANSPLANTATION BUT IS AN INTERESTING “IN VIVO” MODEL FOR PANCREATIC DUCTAL TRANSDIFFERENTIATION

P1191 Aims: In order to investigate the function of pancreatic islets transplantation in allogeneic and xenogeneic large animal models, induction of diabetes mellitus is needed. In this experimental study, we searched the most appropriate tool for induction of diabetes in both pig and primate models: Streptozotocin (STZ) or total pancreatectomy (TP). Methods: In juvenile pigs (20 weeks), multiple (2-3) low doses STZ (40mg/kg iv, n=4), one high dose STZ (150mg/kg iv, n=9) and total pancreatectomy (n=3) were tested to induce diabetes. Two different intravenous STZ doses were tested in Cynomolgus monkeys (50mg/kg (n=5) vs. 135mg/kg (n=2)). Hepatic enzymes (AST, ALT), renal function (creatinine), intra-venous glucose tolerance test (IVGTT: Area under the curve, AUC), non-fasting blood glucose and animal weight were assessed prior to, one and 4 weeks after diabetes induction. Beta cell destruction was evaluated by immunohistochemistry with insulin staining and non-beta cells by somatostatin, glucagon and pancreatic polypeptide staining. Results: Single or multiple low doses of STZ did not induce diabetes or side-effects in four in juvenile pigs (normal AST, ALT and creatinine). The three pigs which underwent TP became rapidly and definitively (4-5 days) hyperglycaemic and polyuric. Total pancreatectomy provoked an irreversible and pathological IVGTT (AUC: 45,513 ± 3,021 mg/dl/min) as compared to controls (n=9) (8,094 ± 1,902 mg/dl/min) (p<0.005). Similarly, one high dose STZ induced in pigs a significantly pathological IVGTT after one week (n=5) (32,134 ±7,037 mg/dl/min) (p<0.05). Four weeks after one high dose STZ injection, however, a significant decrease of AUC calculated after IVGTT was observed in six pigs tested (20,562±7,261 mg/dl/min) (p<0.005), thereby suggesting the survival or regeneration of beta cells. In fact, all the pancreas from STZ-treated pigs analysed after 4 weeks demonstrated the presence of small beta cells located at distance of vascular structure (co-staining insulin/von Willebrand factor). Moreover, a significant beta cellular hypertrophy could be evidenced between one and 4 weeks after STZ (98±7 μm2 vs. 120±12μm2) (p<0.05). Last but not least, a clear ductal-beta cell transdifferentiation (co-staining insulin/cytokeratin 19) was broadly observed in pancreas harvested 4 weeks after STZ. Cynomolgus treated with high dose STZ (135mg/kg (n=2) rapidly demonstrated an increase of hepatic enzymes (>200 IU/l)) and creatinine (>6mg/dl) due to liver steatosis and kidney tubular necrosis. These animals eventually died within two weeks after injection. Low dose of STZ (55mg/kg) induced an increase of non-fasting blood glucose (>200mg/dl), a pathological IVGTT (AUC: 5,534±747 mg/dl/min) as compared with untreated animals (13,005±3,084 mg/dl/min) (p<0.05). This STZ regimen did not induce any hepatic or renal deterioration and no residual serum C-peptide could be detected. In these animals, pathological IVGTT was confirmed 3 months after low dose STZ injection and the insulin-deficient state could be related to massive amyloid polypeptide deposits in islets. Conclusions: Low-dose STZ successfully induces irreversible diabetes in monkeys but not in young pigs. High dose STZ induces transient hyperglycaemic state in pigs but no irreversible diabetes due to a hypertrophy of beta cells and ductal transdifferentiation. This result suggests that high dose STZ could be used as a model of “in vivo” ductal differentiation. In juvenile pigs, TP seems to be the only choice for diabetes induction model for studying pig islets transplantation.

Effects of Nigella sativa on oxidative stress and beta-cell damage in streptozotocin-induced diabetic rats.

The aim of the present study was to evaluate the possible protective effects of Nigella sativa L. (NS) against beta-cell damage from streptozotocin (STZ)-induced diabetes in rats. STZ was injected intraperitoneally at a single dose of 50 mg/kg to induce diabetes. NS (0.2 ml/kg/day, i.p.) was injected for 3 days prior to STZ administration, and these injections were continued throughout the 4-week study. Oxidative stress is believed to play a role in the pathogenesis of diabetes mellitus (DM). To assess changes in the cellular antioxidant defense system, we measured the activities of antioxidant enzymes (such as glutathione peroxidase (GSHPx), superoxide dismutase (SOD), and catalase (CAT)) in pancreatic homogenates. We also measured serum nitric oxide (NO) and erythrocyte and pancreatic tissue malondialdehyde (MDA) levels, a marker of lipid peroxidation, to determine whether there is an imbalance between oxidant and antioxidant status. Pancreatic beta-cells were examined by immunohistochemical methods. STZ induced a significant increase in lipid peroxidation and serum NO concentrations, and decreased antioxidant enzyme activity. NS treatment has been shown to provide a protective effect by decreasing lipid peroxidation and serum NO, and increasing antioxidant enzyme activity. Islet cell degeneration and weak insulin immunohistochemical staining was observed in rats with STZ-induced diabetes. Increased intensity of staining for insulin, and preservation of beta-cell numbers were apparent in the NS-treated diabetic rats. These findings suggest that NS treatment exerts a therapeutic protective effect in diabetes by decreasing oxidative stress and preserving pancreatic beta-cell integrity. Consequently, NS may be clinically useful for protecting beta-cells against oxidative stress.