Wistar Rat Islets Research Articles

A data integration approach unveils a transcriptional signature of type 2 diabetes progression in rat and human islets.

Pancreatic islet failure is a key characteristic of type 2 diabetes besides insulin resistance. To get molecular insights into the pathology of islets in type 2 diabetes, we developed a computational approach to integrating expression profiles of Goto-Kakizaki and Wistar rat islets from a designed experiment with those of the human islets from an observational study. A principal gene-eigenvector in the expression profiles characterized by up-regulated angiogenesis and down-regulated oxidative phosphorylation was identified conserved across the two species. In the case of Goto-Kakizaki versus Wistar islets, such alteration in gene expression can be verified directly by the treatment-control tests over time, and corresponds to the alteration of α/β-cell distribution obtained by quantifying the islet micrographs. Furthermore, the correspondence between the dual sample- and gene-eigenvectors unveils more delicate structures. In the case of rats, the up- and down-trend of insulin mRNA levels before and after week 8 correspond respectively to the top two principal eigenvectors. In the case of human, the top two principal eigenvectors correspond respectively to the late and early stages of diabetes. According to the aggregated expression signature, a large portion of genes involved in the hypoxia-inducible factor signaling pathway, which activates transcription of angiogenesis, were significantly up-regulated. Furthermore, top-ranked anti-angiogenic genes THBS1 and PEDF indicate the existence of a counteractive mechanism that is in line with thickened and fragmented capillaries found in the deteriorated islets. Overall, the integrative analysis unravels the principal transcriptional alterations underlying the islet deterioration of morphology and insulin secretion along type 2 diabetes progression.

Features of the planimetric organization of the endocrine part of the pancreas in hypertensive rats of the SHR line

Analysis of the endocrine structure of the pancreas and assessment of its degree of heterogeneity is key to assessing such important physiological parameters as islet endocrinocyte mass in healthy people or functional reserve of β-cells in patients with type 1 and type 2 diabetes. The aim of the study is to perform a comparative planimetric analysis of pancreatic islets of normotensive Wistar rats and hypertensive SHR rats, using the method of double immunofluorescent staining of endocrinocytes. Materials and methods. The study was performed on the pancreas of SHR and Wistar rats. Insulin and glucagon in the islets of the pancreas were determined by immunofluorescence. The immunofluorescence reaction was studied with an AxioImager-M2 fluorescent microscope. Results. Planimetric analysis showed the presence of pancreatic islets of all types in the pancreatic tissue of SHR and Wistar rats. The number of single endocrinocytes in SHR hypertensive animals was 2.3 times higher (P < 0.001), and the number of giant islets was 34.2 % higher (P < 0.001), than in normotensive Wistar rats. The presence of regions in some endocrinocytes, that show immunoreactivity to both hormones at once – insulin and glucagon. Such areas were present in islets of all types of both rat lines, however, the total rates in hypertensive rats of the SHR line were 21.8 % lower (P < 0.001), than in normotensive rats of the Wistar line. Conclusions. Planimetric analysis showed, that the pancreatic islets in normotensive Wistar rats and hypertensive SHR rats occupy 1.4 % of the cross-sectional area of the pancreas. In hypertensive rats the SHR line in the gland by 16.9 % larger total area of the material immunoreactive to insulin. In contrast to Wistar rats, the number of single α-endocrinocytes in the pancreas of hypertensive SHR rats was significantly increased.

O4 Efecto del INGAP-PP sobre la expresión de genes involucrados en la remodelación de la matriz extracelular y la angiogénesis determinados por un análisis transcriptómico de islotes de ratas

Introducción: el INGAP-PP es un péptido derivado de INGAP que, en animales normales y con diabetes, aumenta la masa celular ß, la capacidad angiogénica insular y potencia la secreción de insulina estimulada por glucosa (SIEG) y otros secretagogos. Esto le adjudica un potencial terapéutico que actuaría específicamente en la patogenia celular de la diabetes. Conocer su posible efecto sobre los cambios globales en los perfiles de expresión génica insular facilitaría la comprensión de su mecanismo de acción.Objetivos: conocer, a través de un estudio transcriptómico, el efecto del INGAP-PP sobre la expresión génica de las células insulares y la posible regulación de distintas vías de señalización implicadas.Materiales y métodos: islotes aislados (colagenasa) de rata Wistar normales fueron cultivados 4 días en medio RPMI en ausencia (C) o presencia de INGAP-PP 50 µg/ml (I) y glucosa 11 mM. A su término se utilizaron 100 islotes de cada grupo para estudiar la SIEG en presencia de glucosa 3,3 mMó 16,6 mM (RIA). Del resto de los islotes se extrajo ARN total, una alícuota fue sometida a secuenciación masiva de ARN (RNA-seq) y otra fue retrotranscripta a ADNc para utilizarlo como molde en qPCR de marcadores de desarrollo, regeneración y angiogénesis.

P35 El INGAP-PP estimula cambios a nivel transcripcional tanto en células endocrinas como no endocrinas del islote

Introducción: el INGAP-PP, un pentadecapéptido derivado del INGAP (Islet Neogenesis Associated Protein), potencia la secreción de insulina frente al estímulo de glucosa, aumenta la masa de células ß insulares y las protege de la acción citotóxica de la estreptozotocina. Estas evidencias nos permiten proponerlo como un posible recurso terapéutico para la prediabetes y diabetes tipo 2.Objetivos: estudiar los mecanismos, a nivel transcriptómico, que condicionan los efectos del INGAP-PP destacando la contribución de distintos tipos celulares en su rol terapéutico.Materiales y métodos: se aislaron islotes de rata Wistar macho normales y se cultivaron 4 días en ausencia o presencia de INGAP-PP y glucosa 11 mM. Luego de comprobar el efecto potenciador del INGAP-PP sobre la secreción de insulina inducida por glucosa 16,7 mM, se extrajo ARN de los islotes de ambos grupos y se realizó una secuenciación masiva (RNA-seq).

Early weaning induces short- and long-term effects on pancreatic islets in Wistar rats of both sexes.

The World Health Organization recommends exclusive breastfeeding until 6months of age as an important strategy to reduce child morbidity and mortality. Studies have associated early weaning with the development of obesity and type 2 diabetes in adulthood. In our model, we demonstrated that early weaning leads to increased insulin secretion in adolescent males and reduced insulin secretion in adult offspring. Early weaned males exhibit insulin resistance in skeletal muscle. Early weaning did not change insulin signalling in the muscle of female offspring. Taking into account that insulin resistance is one of the primary factors for the development of type 2 diabetes mellitus, this work demonstrates the importance of breastfeeding in the fight against this disease. Early weaning (EW) leads to short- and long-term obesity and diabetes. This phenotype is also observed in experimental models, in which early-weaned males exhibit abnormal insulinaemia in adulthood. However, studies regarding the effect of EW on pancreatic islets are rare. We investigated the mechanisms by which glycaemic homeostasis is altered in EW models through evaluations of insulin secretion and its signalling pathway in offspring. Lactating Wistar rats and their pups were divided into the following groups: non-pharmacological EW (NPEW): mothers were wrapped with an adhesive bandage on the last 3days of lactation; pharmacological EW (PEW): mothers received bromocriptine to inhibit prolactin (1mg/kg body mass/day) on the last 3days of lactation; and control (C): pups underwent standard weaning at PN21. Offspring of both sexes were euthanized at PN45 and PN180. At PN45, EW males showed higher insulin secretion (vs. C). At PN170, PEW males exhibited hyperglycaemia in an oral glucose tolerance test (vs. C and NPEW). At PN180, EW male offspring were heavier; however, both sexes showed higher visceral fat. Insulin secretion was lower in EW offspring of both sexes. Males from both EW groups had lower glucokinase in islets, but unexpectedly, PEW males showed higher GLUT2, than did C. EW males exhibited lower insulin signalling in muscle. EW females exhibited no changes in these parameters compared with C. We demonstrated distinct alterations in the insulin secretion of EW rats at different ages. Despite the sex dimorphism in insulin secretion in adolescence, both sexes showed impaired insulin secretion in adulthood due to EW.

Integrator of Stress Responses Calmodulin Binding Transcription Activator 1 (Camta1) Regulates miR-212/miR-132 Expression and Insulin Secretion

Altered microRNA profiles have been demonstrated in experimental models of type 2 diabetes, including in islets of the diabetic Goto-Kakizaki (GK) rat. Our bioinformatic analysis of conserved sequences in promoters of microRNAs, previously observed to be up-regulated in GK rat islets, revealed putative CGCG-core motifs on the promoter of the miR-212/miR-132 cluster, overexpression of which has been shown to increase insulin secretion. These motifs are possible targets of calmodulin binding transcription activators Camta1 and Camta2 that have been recognized as integrators of stress responses. We also identified putative NKE elements, possible targets of NK2 homeobox proteins like the essential islet transcription factor Nkx2-2. As Camtas can function as co-activators with NK2 proteins in other tissues, we explored the role of Camta1, Camta2, and Nkx2-2 in the regulation of the miR-212/miR-132 cluster and insulin secretion. We demonstrate that exposure of control Wistar or GK rat islets to 16.7 mm glucose increases miR-212/miR-132 expression but significantly less so in the GK rat. In addition, Camta1, Camta2, and Nkx2-2 were down-regulated in GK rat islets, and knockdown of Camta1 reduced miR-212/miR-132 promoter activity and miR-212/miR-132 expression, even under cAMP elevation. Knockdown of Camta1 decreased insulin secretion in INS-1 832/13 cells and Wistar rat islets but increased insulin content. Furthermore, knockdown of Camta1 reduced K(+)-induced insulin secretion and voltage-dependent Ca(2+) currents. We also demonstrate Camta1 and Nkx2-2 protein interaction. These results indicate that Camta1 is required not only for expression of the miR-212/miR-132 cluster but at multiple levels for regulating beta cell insulin content and secretion.

Islet Stellate Cells Isolated from Fibrotic Islet of Goto-Kakizaki Rats Affect Biological Behavior of Beta-Cell.

We previously isolated islet stellate cells (ISCs) from healthy Wistar rat islets. In the present study, we isolated “already primed by diabetic environment” ISCs from islets of Goto-Kakizaki rats, determined the gene profile of these cells, and assessed the effects of these ISCs on beta-cell function and survival. We detected gene expression of ISCs by digital gene expression. INS-1 cell proliferation, apoptosis, and insulin production were measured after being treated with ISCs supernatant (SN). We observed the similar expression pattern of ISCs and PSCs, but 1067 differentially expressed genes. Insulin production in INS-1 cells cultured with ISC-SN was significantly reduced. The 5-ethynyl-2′-deoxyuridine-positive INS-1 cells treated with ISC-SN were decreased. Propidium iodide- (PI-) positive INS-1 cells were 2.6-fold higher than those in control groups. Caspase-3 activity was increased. In conclusion, ISCs presented in fibrotic islet of GK rats might be special PSCs, which impaired beta-cell function and proliferation and increased beta-cell apoptosis.

BLX-1002 restores glucose sensitivity and enhances insulin secretion stimulated by GLP-1 and sulfonylurea in type 2 diabetic pancreatic islets.

BLX‐1002 is a novel thiazolidinedione with no peroxisome proliferator‐activated receptor (PPAR) activity that has been shown to improve glycemia in type 2 diabetes without weight gain. We previously found that BLX‐1002 selectively augments glucose‐sensitive (but not basal) insulin secretion in normal mouse β‐cells. We have now extended these observations to other insulin secretagogues and to diabetic rat islets. To this end, dynamics of insulin secretion stimulated by glucose, GLP‐1, and the sulfonylurea tolbutamide were examined in pancreatic islets from nondiabetic Wistar and type 2 diabetic Goto‐Kakizaki rats ex vivo. BLX‐1002 restored normal glucose‐sensitive insulin secretion in otherwise “glucose‐blind” islets from GK rats, but did not affect basal or glucose‐stimulated secretion in normal Wistar rat islets. The stimulatory effect of BLX‐1002 on insulin secretion at high glucose required Ca2+ and involved phosphatidylinositol 3‐kinase (PI3K) activity. Consistent with its effects on insulin secretion, BLX‐1002 also augmented insulin secretion and cytoplasmic‐free Ca2+ concentrations ([Ca2+]i) stimulated by high glucose, GLP‐1, and tolbutamide in islets from GK, but not Wistar, rats. The inactive analog BLX‐1237 had no effects. In conclusion, our findings suggest that BLX‐1002 potentiates insulin secretion by different stimuli in diabetic β‐cells only, in a Ca2+‐dependent manner and involving PI3K.

Protective Antioxidant and Antiapoptotic Effects of ZnCl2 in Rat Pancreatic Islets Cultured in Low and High Glucose Concentrations

Aim/HypothesisRat pancreatic islet cell apoptosis is minimal after prolonged culture in 10 mmol/l glucose (G10), largely increased in 5 mmol/l glucose (G5) and moderately increased in 30 mmol/l glucose (G30). This glucose-dependent asymmetric V-shaped profile is preceded by parallel changes in the mRNA levels of oxidative stress-response genes like Metallothionein 1a (Mt1a). In this study, we tested the effect of ZnCl2, a potent inducer of Mt1a, on apoptosis, mitochondrial oxidative stress and alterations of glucose-induced insulin secretion (GSIS) induced by prolonged exposure to low and high vs. intermediate glucose concentrations.MethodsMale Wistar rat islets were cultured in RPMI medium. Islet gene mRNA levels were measured by RTq-PCR. Apoptosis was quantified by measuring islet cytosolic histone-associated DNA fragments and the percentage of TUNEL-positive β-cells. Mitochondrial thiol oxidation was measured in rat islet cell clusters expressing “redox sensitive GFP” targeted to the mitochondria (mt-roGFP1). Insulin secretion was measured by RIA.ResultsAs observed for Mt1a mRNA levels, β-cell apoptosis and loss of GSIS, culture in either G5 or G30 vs. G10 significantly increased mt-roGFP1 oxidation. While TPEN decreased Mt1a/2a mRNA induction by G5, addition of 50–100 µM ZnCl2 to the culture medium strongly increased Mt1a/2a mRNA and protein levels, reduced early mt-roGFP oxidation and significantly decreased late β-cell apoptosis after prolonged culture in G5 or G30 vs. G10. It did not, however, prevent the loss of GSIS under these culture conditions.ConclusionZnCl2 reduces mitochondrial oxidative stress and improves rat β-cell survival during culture in the presence of low and high vs. intermediate glucose concentrations without improving their acute GSIS.

Different PINK-1 immunohistochemical pattern in Langerhans islets of Goto-Kakizaki and Wistar rats: Implication to mitochondrial damage in animal model of diabetes mellitus

Different PINK-1 immunohistochemical pattern in Langerhans islets of Goto-Kakizaki and Wistar rats: Implication to mitochondrial damage in animal model of diabetes mellitus

Mitochondrial oxidative stress contributes differently to rat pancreatic islet cell apoptosis and insulin secretory defects after prolonged culture in a low non-stimulating glucose concentration

Pancreatic beta cells chronically exposed to low glucose concentrations show signs of oxidative stress, loss of glucose-stimulated insulin secretion (GSIS) and increased apoptosis. Our aim was to confirm the role of mitochondrial oxidative stress in rat islet cell apoptosis under these culture conditions and to evaluate whether its reduction similarly improves survival and GSIS. Apoptosis, oxidative stress-response gene mRNA expression and glucose-induced stimulation of mitochondrial metabolism, intracellular Ca(2+) concentration and insulin secretion were measured in male Wistar rat islets cultured for 1 week in RPMI medium containing 5-10 mmol/l glucose with or without manganese(III)tetrakis(4-benzoic acid)porphyrin (MnTBAP) or N-acetyl-L-: cysteine (NAC). Oxidative stress was measured in islet cell clusters cultured under similar conditions using cytosolic and mitochondrial redox-sensitive green fluorescent protein (roGFP1/mt-roGFP1). Prolonged culture in 5 vs 10 mmol/l glucose increased mt-roGFP1 (but not roGFP1) oxidation followed by beta cell apoptosis and loss of GSIS resulting from reduced insulin content, mitochondrial metabolism, Ca(2+) influx and Ca(2+)-induced secretion. Tolbutamide-induced, but not high K(+)-induced, Ca(2+) influx was also suppressed. Under these conditions, MnTBAP, but not NAC, triggered parallel ~50-70% reductions in mt-roGFP1 oxidation and beta cell apoptosis, but failed to protect against the loss of GSIS despite significant improvement in glucose-induced and tolbutamide-induced Ca(2+) influx. Mitochondrial oxidative stress contributes differently to rat pancreatic islet cell apoptosis and insulin secretory defects during culture in a low glucose concentration. Thus, targeting beta cell survival may not be sufficient to restore insulin secretion when beta cells suffer from prolonged mitochondrial oxidative stress, e.g. in the context of reduced glucose metabolism.

Mizoribine as Sole Immunosuppressive Agent in Islet Xenotransplantation Models: A Candidate Immunosuppressant Causing no Adverse Effects on Islets

Mizoribine (MZ) inhibits the differentiation and proliferation of helper T and B cells after antigen recognition by suppressing the purine biosynthesis pathway and nucleic acid synthesis. MZ has been used in kidney transplantation, but distinct data are unavailable for islet transplantation. The present study investigated the efficacy of MZ for islet xenotransplantation. Immunosuppressive effects of MZ were determined by mixed lymphocyte reaction (MLR) assay in vitro. Toxicities for Wistar rat islets were determined by adenosine triphosphate (ATP) contents of islets during 3-day culture and stimulation index in response to glucose after culture. Immunosuppressive effects in vivo were tested in a Wistar-to-B6 islet xenotransplantation model. MZ was administered continuously for 28 days subcutaneously or intramuscularly. MZ inhibited MLR response by approximately 50% at 0.1 μg/ml. ATP contents decreased with MZ >100 μg/ml, while stimulation index was maintained. Continuous infusion of MZ at 10 mg/kg maintained blood concentrations at 0.13-0.19 μg/ml, while intramuscular injection of MZ at 100 mg/kg/day (peak 520 μg/ml at 1 h postinjection) resulted in below measurable levels (<0.03 μg/ml) within 24 h. Graft survival was significantly prolonged following continuous infusion of 10 mg/kg/day compared to controls (31.0 ± 9.5 vs. 13.2 ± 5.2 days; p = 0.002). Furthermore, animals with intramuscular injection at doses of 3.2, 10, or 100 mg/kg/day showed significantly longer graft survival (20.0 ± 7.5, 22.0 ± 7.31, and 24.5 ± 8.1 days, respectively; p < 0.05 each). Histological examination showed significant suppression of lymphocyte infiltration by MZ administration. MZ showed immunosuppressive effects in an experimental islet xenotransplantation model without adverse effects on endocrine function of islet grafts.

Immunoisolation Effect of Polyvinyl Alcohol (PVA) Macroencapsulated Islets in Type 1 Diabetes Therapy

Islet transplantation has shown great success in the treatment of type 1 diabetes since the Edmonton protocol was established. However, it still has two major problems to overcome: the lack of organ donors and the side effects of immunosuppression. Encapsulated islets have emerged as a potential option for islet transplantation because it can, at least partly, overcome these two problems. Wistar rat islets suspended in 3% polyvinyl alcohol (PVA) hydrogel were frozen-thawed to make macroencapsulated islets (MEIs). The recovery rate, insulin content, and morphological change in culture medium with/without fresh human plasma (FHP) were measured in MEIs and free islets in vitro. In vivo, MEIs of either Wistar or Lewis rats were transplanted into the peritoneal cavity of streptozotocin (STZ)-induced diabetic Lewis rats and nonfasting blood glucose (NFBG), body weight, and histological evaluations were processed. FHP destroyed rat free islets but did not affect the islet morphology, islet recovery rate, or insulin content of rat MEIs. The transplantation of MEIs decreased the NFBG level and prevented body weight loss without a significant difference between the donor strains. Insulin-positive islets were observed in PVA MEIs 24 weeks after allotransplantation. These results suggest that PVA MEIs may be used as a cure for type 1 diabetes.

Improved Islet Yield and Function by Use of a Chloride Channel Blocker During Collagenase Digestion

Protection of pancreatic islets during isolation procedures is mandatory for successful islet transplantation. Chloride channel inhibition has been reported to prevent cell death induced by various stimuli. We examined the effects of the chloride channel blocker, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt (DIDS) and extracellular Cl(-)-free conditions on islet isolation outcomes. Experimental groups were created based on the collagenase solutions used for Wistar rat islet isolation: control group, Hanks' balanced salt solution; DIDS group, 200 μM DIDS; and Cl(-)-free group, sodium gluconate substituted for sodium chloride. We determined whether collagenase digestion induced the death of islet cells through Cl(-) influx into the cells. We then assessed islet yield and the viability of isolated islets. We observed an increase in intracellular Cl(-) concentration under collagenase digestion conditions using a Cl(-)-sensitive fluorescent dye and subsequent rupture of islet cells. Consequently, islet yields were significantly higher in the DIDS and Cl(-)-free groups than in the control group, and islet morphology of the former groups was preserved. Of streptozotocin-induced diabetic severe combined immunodeficiency mice transplanted with a marginal dose of islets, all seven mice in the DIDS group and six of the seven mice in the Cl(-)-free group became normoglycemic, compared with two of seven mice in the control group (control vs. DIDS, P=0.010; control vs. Cl(-) free, P=0.051). Our results indicate that DIDS inhibition of Cl(-) influx into islets protects islets during digestion procedures, offering a new strategy for the improvement of islet isolation outcomes.

Long-term graft function of cryostored alginate encapsulated rat islets

Microencapsulation of pancreatic islets before transplantation is a promising approach to enable graft function in an immunocompetent recipient without immunosuppression. However, the insufficient availability of allogenic islet tissue is a major problem. One concept to overcome these shortcomings is the cryopreservation of encapsulated allogenic islets. Recently, we reported a gentle cryopreservation protocol for rat islets encapsulated in an alginate-based microcapsule system. Here, we report for the first time long-term transplantation data of these cryopreserved microencapsulated islets. We detected a stable graft function for more than 12 month (experiments still continuing) after transplantation of 2500 cryopreserved microencapsulated CD rat islets in streptozotocin-diabetic Wistar rats. Moreover, the glucose clearance rate during an IPGTT was well preserved up to 56 weeks after transplantation. In addition, hyperglycemic blood glucose levels after removal of rat islet grafts 12 and 56 weeks after transplantation confirmed the efficacy of the encapsulated islets. Finally, the retrieved encapsulated rat islets responded well with a 7-fold increase of insulin secretion to a glucose stimulus (12 and 56 weeks). In conclusion, our study demonstrates for the first time that cryopreservation of encapsulated rat islets is possible without substantial losses on graft function for a very long time.

Preserved insulin secretion capacity and graft function of cryostored encapsulated rat islets

Encapsulation of pancreatic islets before transplantation enables survival and function in an immunocompetent recipient without immunosuppression. However, the insufficient availability of allogenic islet tissue is a major problem. One concept to overcome these shortcomings is the cryopreservation of microencapsulated allogenic islets, to allow their unlimited collection and use on demand. Therefore, this report outlines the development of a cryopreservation protocol for CD rat islets encapsulated in an alginate-based microcapsule-system. We determined RPMI-medium plus 10% FCS as freezing medium, equilibration at 0 °C for 15 min with the cryoprotectant dimethyl sulfoxide (DMSO; final concentration 2.0 M), and a stepwise removal of DMSO by sucrose dilution after thawing, as best protocol for cryopreservation of encapsulated islets. Importantly, the cryopreserved encapsulated islets showed post thawing in vitro an insulin increase upon a glucose challenge comparable to that of non-cryopreserved encapsulated islets. Moreover, a stable graft function without the need of immunosuppression was detected after transplantation of 2500 cryopreserved encapsulated CD rat islets in streptozotocin-diabetic Wistar rats. Finally, the glucose clearance rate during an IPGTT 4 weeks after transplantation was comparable to that of rats transplanted with non-cryopreserved encapsulated islets. In conclusion, our study demonstrates for the first time that cryopreservation of encapsulated rat islets is possible without substantial losses on graft function. Future studies will now have to carry on this approach to human islets, aiming to apply such a bioartificial pancreas consisting of cryopreserved encapsulated islets in humans.

Effect of inborn pancreatic islet deficit in the Munich Wister Frömter rat

The total mass of pancreatic islet cells is a critical factor in glucose metabolic control. The aim of the present study was to investigate whether in the Munich Wistar Frömter (MWF) rat, beside a reduction in the number of nephrons, there are also alterations in the number of pancreatic islets and of β cell mass. We also examined glucose metabolism, both in normal conditions and following intravenous glucose injection. The number of islets per pancreas, estimated by morphometrical analysis, was significantly lower in MWF rats than in Wistar rats (3,501±1,285 vs. 7,259±2,330 islet/rat, respectively). Also the mean number of islets per gram of body weight was significantly lower in MWF rats than in Wistar rats (18±7 in MWF rats vs. 28±10 islets/g bw in Wistar rats). Morphometric analysis of β cell mass showed an average of 77.1±7% islet cells staining for insulin in MWF rats and 83.9±2.1% in the control Wistar rats. Despite the lower number of islets and β cells, MWF and Wistar rats had comparable fasting blood glucose levels but significant differences in blood glucose following an intraperitoneal glucose tolerance test. In summary, pancreatic islets of MWF and Wistar rats showed a marked difference in morphometrical characteristics. While this difference is not associated with blood glucose levels, glucose metabolism after IPGTT between MWF and Wistar rats is significantly different. These data suggest that an inborn deficit in β cell mass of about 60% is responsible for altered glucose metabolism and could favor the development of diabetes.

Exendin-4 Protects Pancreatic Beta Cells from the Cytotoxic Effect of Rapamycin by Inhibiting JNK and p38 Phosphorylation

It has been reported that the immunosuppressant rapamycin decreases the viability of pancreatic beta cells. In contrast, exendin-4, an analogue of glucagon-like peptide-1, has been found to inhibit beta cell death and to increase beta cell mass. We investigated the effects of exendin-4 on the cytotoxic effect of rapamycin in beta cells. Incubation with 10 nM rapamycin induced cell death in 12 h in murine beta cell line MIN6 cells and Wistar rat islets, but not when coincubated with 10 nM exendin-4. Rapamycin was found to increase phosphorylation of c-Jun amino-terminal kinase (JNK) and p38 in 30 minutes in MIN6 cells and Wistar rat islets while exendin-4 decreased their phosphorylation. Akt and extracellular signal-regulated kinase (ERK) were not involved in the cytoprotective effect of exendin-4. These results indicate that exendin-4 may exert its protective effect against rapamycin-induced cell death in pancreatic beta cells by inhibiting JNK and p38 signaling.

The cytoprotection of chitosan based hydrogels in xenogeneic islet transplantation: An in vivo study in streptozotocin-induced diabetic mouse

Immune rejection and scarcity of donor tissues are the restrictions of islets transplantation. In this study, the cytoprotection of chitosan hydrogels in xenogeneic islet transplantation was demonstrated. Wistar rat islets encapsulated in chitosan hydrogels were performed glucose challenge test and live/dead cell staining in vitro. Islets/chitosan hydrogels were transplanted into the renal subcapsular space of diabetic C57BL/6 mice. Non-fasting blood glucose level (NFBG), body weight, intraperitoneal glucose tolerance test (IPGTT), and glucose disappearance rate were determined perioperatively. The serum insulin level was analyzed, and the kidney transplanted with islets/chitosan hydrogels were retrieved for histological examination after sacrifice. The present results showed that islets encapsulated in chitosan hydrogels secreted insulin in response to the glucose stimulation as naked islets with higher cell survival. The NFBG of diabetic mice transplanted with islets/chitosan hydrogels decreased from 487 ± 46 to 148 ± 32 at one day postoperation and maintained in the range of 201 ± 36 mg/dl for four weeks with an increase in body weight. IPGTT showed the glucose disappearance rate of mice transplanted with islets/chitosan hydrogels was significant faster than that of mice transplanted with naked islets; the serum insulin level increased from 0.29 ± 0.06 to 1.69 ± 0.65 μg/dl postoperatively. Histological examination revealed that the islets successfully engrafted at renal subcapsular space with positive insulin staining. The immunostain was negative for neither the T-cell lineages nor the monocyte/macrophages. This study indicates that the chitosan hydrogels deliver and protect encapsulated islets successfully in xenotransplantation.

Orexin-1 Receptor Co-Localizes with Pancreatic Hormones in Islet Cells and Modulates the Outcome of Streptozotocin-Induced Diabetes Mellitus

Recent studies have shown that orexins play a critical role in the regulation of sleep/wake states, feeding behaviour, and reward processes. The exocrine and endocrine pancreas are involved in the regulation of food metabolism and energy balance. This function is deranged in diabetes mellitus. This study examined the pattern of distribution of orexin-1 receptor (OX1R) in the endocrine cells of the pancreas of normal and diabetic Wistar (a model of type 1 diabetes), Goto-Kakizaki (GK, a model of type 2 diabetes) rats and in orexin-deficient (OX−/−) and wild type mice. Diabetes mellitus (DM) was induced in Wistar rats and mice by streptozotocin (STZ). At different time points (12 h, 24 h, 4 weeks, 8 months and 15 months) after the induction of DM, pancreatic fragments of normal and diabetic rats were processed for immunohistochemistry and Western blotting. OX1R-immunoreactive nerves were observed in the pancreas of normal and diabetic Wistar rats. OX1R was also discernible in the pancreatic islets of normal and diabetic Wistar and GK rats, and wild type mice. OX1R co-localized with insulin (INS) and glucagon (GLU) in the pancreas of Wistar and GK rats. The number of OX1R-positive cells in the islets increased markedly (p<0.0001) after the onset of DM. The increase in the number of OX1R-positive cells is associated with a high degree of co-localization with GLU. The number of GLU- positive cells expressing OX1R was significantly (p<0.0001) higher after the onset of DM. The tissue level of OX1R protein increased with the duration of DM especially in type 1 diabetes where it co-localized with cleaved caspase 3 in islet cells. In comparison to STZ-treated wild type mice, STZ-treated OX−/− animals exhibited reduced hyperglycemia and handled glucose more efficiently in glucose tolerance test. The findings suggest an important role for the OX-OX1R pathway in STZ-induced experimental diabetes.