Islet Damage Research Articles

303. Deletion of Pdx1 in Mouse Pancreatic Ducts by CRISPR-Cas9 Mediated Gene Editing

Introduction: The homeodomain transcription factor Pdx1 is crucial for pancreas formation. Pdx1-expressing cells are first observed at embryonic day 8.5 (E8.5), prior even to the earliest indication of morphogenesis, in endodermal cells designated to give rise to the pancreas. In adult mouse, Pdx1 protein is transiently expressed when pancreas is injured, such as Langerhans islets damage in alloxan induced diabetic mice, implying Pdx1 may be necessary for the neogenetic formation of β-cells from mature ducts. In order to study the role of pdx1 in the pancreatic duct for pancreatic regeneration, most investigators use the Cre-lox system to generate duct-specific Pdx1 deletions in mice. This method is able to delete Pdx1specifically from tissue or cells with or without a tamoxifen-inducible (cre-ERT2) system; however, several caveats and limitations exist with tamoxifen-induced depletion. In addition, the cre-ERT2 system may be leaky, resulting in constitutive rather than inducible activation in some cells. To overcome these flaws from the Cre-ERT2/lox system, we decided to use a novel CRISPR-Cas9 gene editing technique to delete pdx1 in pancreatic ducts. Methods: We previously used a mouse short Sox9 promoter (468 bp) to generate a recombinant adeno-associated virus (AAV) carrying a reporter GFP (AAV6-Sox9-GFP), and this recombinant virus construct was infused into the pancreatic ducts, through the common bile/pancreatic duct, to specifically tag the duct cells. In order to address pancreatic duct specific deletion of Pdx1 after alloxan ablation of beta-cells, we first to applied the CRISPR-Cas9 gene editing technique on the HEK293-CMV-GFP cell line in vitro (Figure 2Figure 2) and ROSA26 LSL tomato reporter mice ex vivo (Figure 3Figure 3), and then employed AAV6 mediated CRISPR-SaCas9 under the control of the mouse sox9 short promoter, coupled with paired guide RNAs (gRNAs) flanking the Pdx1 exon2 (Figure 4Figure 4) through pancreatic duct infusion into the pancreas. RESULTS AND CONCLUSIONS: These CRISPR-SaCas9 and gRNAs AAV viruses were co-infused into pancreases of C57BL/6J mice 2 days after alloxan treatment. One week after AAV6-SaCas9+gRNAs infusion, it appeared that only the Pdx1 in pancreatic ductal cells were deleted. Comparatively, PBS or AAV6-CMV-GFP viral infused C57BL/6J mice and ROSA26 LSL tomato reporter mice expressed fluorescence in the entire pancreas as a result of the deleted loxP sites that removed the stop cassette, the Pdx1 is not deleted. Thus, we first report here a highly efficient and specific ablation of Pdx1 in adult mouse pancreatic ducts using the CRISPR-Cas9 technique. Figure 1Figure 1. Construction of AAV CRISPR-SaCas9 and gRNA vectors. Figure 2Figure 2. Deletion of ZsGeen in HEK293-CMV-GFP cells using SacCas9 and gRNA in vitro. Figure 3Figure 3. Valiation of SaCas9 and gRNA in vivo deletion of loxP sites in ROSA26-LSL tomato mice pancreas. Figure 4Figure 4. Deletion of Pdx1 exon2 in alloxan treated C57 pancreatic ducts.View Large Image | Download PowerPoint SlideView Large Image | Download PowerPoint SlideView Large Image | Download PowerPoint SlideView Large Image | Download PowerPoint Slide

In vivo and in vitro evaluation of the effects of Urtica dioica and swimming activity on diabetic factors and pancreatic beta cells.

BackgroundUrtica dioica (UD) has been identified as a traditional herbal medicine. This study aimed to investigate the effect of UD extract and swimming activity on diabetic parameters through in vivo and in vitro experiments.MethodsAdult WKY male rats were randomly distributed in nine groups: intact control, diabetic control, diabetic + 625 mg/kg, 1.25 g/kg UD, diabetic + 100 mg/kg Metformin, diabetic + swimming, diabetic + swimming 625 mg/kg, 1.25 g/kg UD, and diabetic +100 mg/kg Metformin + swimming. The hearts of the animals were punctured, and blood samples were collected for biochemical analysis. The entire pancreas was exposed for histologic examination. The effect of UD on insulin secretion by RIN-5F cells in 6.25 or 12.5 mM glucose dose was examined. Glucose uptake by cultured L6 myotubes was determined.ResultsThe serum glucose concentration decreased, the insulin resistance and insulin sensitivity significantly increased in treated groups. These changes were more pronounced in the group that received UD extract and swimming training. Regeneration and less beta cell damage of Langerhans islets were observed in the treated groups. UD treatment increased insulin secretion in the RIN-5F cells and glucose uptake in the L6 myotubes cells.ConclusionsSwimming exercises accompanied by consuming UD aqueous extracts effectively improved diabetic parameters, repaired pancreatic tissues in streptozotocin-induced diabetics in vivo, and increased glucose uptake or insulin in UD-treated cells in vitro.

A Nonhematopoietic Erythropoietin Analogue, ARA 290, Inhibits Macrophage Activation and Prevents Damage to Transplanted Islets.

Erythropoietin exerts anti-inflammatory, antiapoptotic, and cytoprotective effects in addition to its hematopoietic action. A nonhematopoietic erythropoietin analogue, ARA 290, has similar properties. The efficacy of pancreatic islet transplantation (PITx) is reduced due to islet damage that occurs during isolation and from the severe inflammatory reactions caused by the transplantation procedure. We investigated whether ARA 290 protects islets and ameliorates inflammatory responses following PITx thus improving engraftment. The effects of ARA 290 on pancreatic islets of C57BL/6J (H-2) mice and on murine macrophages were investigated using an in vitro culture model. As a marginal PITx, 185 islets were transplanted into the liver of streptozotocin-induced diabetic mice (H-2) via the portal vein. Recipients were given ARA 290 (120 μg/kg) intraperitoneally just before and at 0, 6, and 24 hours after PITx. Liver samples were obtained at 12 hours after PITx, and expression levels of proinflammatory cytokines were assessed. ARA 290 protected islets from cytokine-induced damage and apoptosis. Secretion of pro-inflammatory cytokines (IL-6, IL-12, and TNF-α) from macrophages was significantly inhibited by ARA 290. After the marginal PITx, ARA 290 treatment significantly improved the blood glucose levels when compared to those of control animals (P < 0.001). Upregulation of monocyte chemoattractant protein-1, macrophage inflammatory protein-1β, IL-1β, and IL-6 messenger RNA expression within the liver was suppressed by ARA 290 treatment. ARA 290 protected pancreatic islets from cytokine-induced damage and apoptosis and ameliorated the inflammatory response after PITx. ARA 290 appears to be a promising candidate for improvement of PITx.

Long-term risk of type 2 diabetes in relation to habitual iron intake in women with a history of gestational diabetes: a prospective cohort study

An iron overload may induce pancreatic islet damage and increase risk of diabetes. Women with a history of gestational diabetes mellitus (GDM) are at high risk of developing type 2 diabetes mellitus (T2DM) after pregnancy. We aimed to examine the association of habitual iron intake with long-term risk of T2DM in this high-risk population. We included 3976 women with a history of GDM from the Nurses' Health Study II cohort as part of the ongoing Diabetes & Women's Health Study. The women were followed up through 2009. Iron intake was assessed with the use of a validated food-frequency questionnaire in 1991 and every 4 y thereafter. We used Cox proportional hazards models to estimate HRs and 95% CIs. We documented 641 incident T2DM cases during 57,683 person-years of observation. Adjusted HRs for T2DM for the highest quartile compared with the lowest quartile were 1.64 (95% CI: 1.20, 2.25; P-trend = 0.02) for total iron intake and 1.80 (95% CI: 1.18, 2.74; P-trend = 0.005) for dietary heme iron intake. In addition, women who consumed ≥30.0 mg supplemental Fe/d, compared with nonusers, had an adjusted HR of 1.83 (95% CI: 1.25, 2.70; P-trend = 0.002). In women with a history of GDM, greater intakes of total iron, dietary heme iron, and supplemental iron were associated with higher risk of T2DM.

Developmental endothelial locus-1 modulates platelet-monocyte interactions and instant blood-mediated inflammatory reaction in islet transplantation.

Platelet-monocyte interactions are strongly implicated in thrombo-inflammatory injury by actively contributing to intravascular inflammation, leukocyte recruitment to inflamed sites, and the amplification of the procoagulant response. Instant blood-mediated inflammatory reaction (IBMIR) represents thrombo-inflammatory injury elicited upon pancreatic islet transplantation (islet-Tx), thereby dramatically affecting transplant survival and function. Developmental endothelial locus-1 (Del-1) is a functionally versatile endothelial cell-derived homeostatic factor with anti-inflammatory properties, but its potential role in IBMIR has not been previously addressed. Here, we establish Del-1 as a novel inhibitor of IBMIR using a whole blood-islet model and a syngeneic murine transplantation model. Indeed, Del-1 pre-treatment of blood before addition of islets diminished coagulation activation and islet damage as assessed by C-peptide release. Consistently, intraportal islet-Tx in transgenic mice with endothelial cell-specific overexpression of Del-1 resulted in a marked decrease of monocytes and platelet-monocyte aggregates in the transplanted tissues, relative to those in wild-type recipients. Mechanistically, Del-1 decreased platelet-monocyte aggregate formation, by specifically blocking the interaction between monocyte Mac-1-integrin and platelet GPIb. Our findings reveal a hitherto unknown role of Del-1 in the regulation of platelet-monocyte interplay and the subsequent heterotypic aggregate formation in the context of IBMIR. Therefore, Del-1 may represent a novel approach to prevent or mitigate the adverse reactions mediated through thrombo-inflammatory pathways in islet-Tx and perhaps other inflammatory disorders involving platelet-leukocyte aggregate formation.

In Vivo Magnetic Resonance Imaging of Small Interfering RNA Nanodelivery to Pancreatic Islets.

Pancreatic islet transplantation is a promising therapeutic approach for type 1 diabetes.However, recent advances in islet transplantation are limited by significant graft loss after transplantation. Multiple immunological and nonimmunological factors contribute to this loss. Novel therapies that could target the core reasons for the islet graft loss are desperately needed. Small interfering RNA can be used to inhibit the expression of virtually any gene with single-nucleotide specificity including genes responsible for islet damage. Applying adequate delivery of siRNA molecules to pancreatic islets prior to transplantation holds a great potential for improving the survival of islet grafts. Noninvasive imaging provides means for monitoring the survival of transplanted islets in real time. Here, we summarize the approach that has been developed to deliver siRNA to pancreatic islets in conjunction with tracking of the graft outcome by in vivo magnetic resonance imaging (MRI). We synthesize a nano-sized theranostic agent consisting of magnetic nanoparticles (MN), a reporter for MRI, labeled with Cy5.5 dye for near-infrared fluorescence (NIRF) imaging, and conjugated to siRNA molecule targeting genes that are harmful to islet grafts. Pre-labeling of islets by MN-Cy5.5-siRNA allowed us to monitor the survival of transplanted islet grafts by MRI and NIRF imaging and resulted in efficient silencing of the target genes in vivo. This novel approach combines a therapeutic effect provided by RNA interference technology with in vivo MR imaging and is expected to significantly improve the outcome of islet transplantation in patients with type 1 diabetes.

Pharmacological activation of Nrf2 pathway improves pancreatic islet isolation and transplantation.

Oxidative stress is a major cause of islet damage and loss during the islet isolation process. The Nrf2 pathway plays a critical role in protecting the cells against oxidative stress. The aim of this study was to investigate the effect of an Nrf2 activator (dh404) on islet isolation and transplantation in a rodent model. Islet isolation was conducted using Nrf2-deficient and wild-type mice and vehicle-treated and Nrf2 activator (dh404)-treated rats. Islet yield, viability, and Nrf2 pathway activity were determined. An in vivo islet potency test was done. Islet yield and viability in Nrf2-deficient mice was significantly lower compared to wild-type (p < 0.05) mice. Furthermore, administration of dh404 to normal Sprague-Dawley rats enhanced nuclear translocation of Nrf2 and elevated HO-1 expression in the pancreas. Islet yield and viability in dh404-treated rats was significantly higher compared to the vehicle-treated group (p < 0.05). The diabetes cure rate in nude mice with chemically induced diabetes was significantly greater in those transplanted with islets from the dh404-treated group (6/9) than vehicle-treated rats (2/9, p < 0.05). The Nrf2 pathway plays a significant role in protecting islets against stress caused by the isolation process. Pharmacological activation of the Nrf2 pathway significantly increased HO-1 expression, improved islet yield, viability, and function after transplantation.

Essential phospholipids prevent islet damage induced by proinflammatory cytokines and hypoxic conditions.

The pancreatic islet damage that occurs through an inflammatory response and hypoxia after infusion is a major hurdle in islet transplantation. Because essential phospholipids (EPL) have been shown to exhibit anti-inflammatory properties in liver disease, we analysed their protective effect on islets in inflammatory or hypoxic conditions. We evaluated the viability of mouse and human islets cultured with cytokines or in hypoxic conditions for 48 h and measured cytokine expression in islets by quantitative polymerase chain reaction. We then employed an in vivo mouse assay, transplanting a marginal dose of human islets treated with or without EPL into the subcapsule of the kidney in diabetic nude mice and determining the cure rate. The viability of mouse and human islets damaged by cytokines was significantly improved by supplementation of EPL in the culture (p = 0.003 and <0.001 for mouse and human islets respectively). EPL significantly inhibited intracellular expression of IL-1β and IL-6 in cytokine-damaged human islets (p < 0.001). The viability of human islets in hypoxic conditions was significantly better when treated with EPL (p < 0.001). In the in vivo mouse assay, the EPL-treated islet group had a higher cure rate than the untreated control, with marginal statistical significance (75 and 17% respectively, p = 0.07). EPL could be a potent agent to protect islets from inflammatory and hypoxic conditions after isolation procedures. Further studies to clarify the effect of EPL in islet transplantation are warranted.

Activation of imidazoline-I3 receptors ameliorates pancreatic damage.

Agmatine, an endogenous ligand of imidazoline receptors, is reported to exhibit anti-hyperglycaemic and many other effects. It has been established that the imidazoline I3 receptor is involved in insulin secretion. The current study characterizes the role of the imidazoline I3 receptor in the protection of pancreatic islets. The activity effect of agmatine against on streptozotocin (STZ)-induced (5mmol/L) rat β cell apoptosis was examined by using ApoTox-Glo triplex assay, live/dead cell double staining assay, flow cytometric analysis, and western blot. Imidazoline I3 receptors antagonist KU14R and the phospholipase C inhibitor named U73122 were treated in β cells to investigate the potential signalling pathways. The serum glucose and recovery of insulin secretion were measured in STZ-treated rats after continuously injected agmatine. The apoptosis in rat β cells was reduced by agmatine in a dose-dependent manner, cell viability was improved after treatment with agmatine and these effects were suppressed after the blockade of KU14R and U73122. Western blot analysis confirmed that agmatine could decrease caspase-3 expression and increase the p-BAD levels. In STZ-treated rats, injection of agmatine for 4weeks may significantly lower the serum glucose and recovery of insulin secretion. This improvement of pancreatic islets induced by agmatine was deleted by KU14R invivo. Agmatine can activate the imidazoline I3 receptor linked with the phospholipase C pathway to induce cell protection against apoptosis induced by a low dose of STZ. This finding provides new insight into the prevention of early stage pancreatic islet damage.

The CXCR1/2 Pathway: Involvement in Diabetes Pathophysiology and Potential Target for T1D Interventions.

Although numerous chemokine/chemokine receptor pathways have been described to be implicated inthe pathogenesis of type 1 diabetes (T1D), the CXCR1/2 axis has recently been proved to be crucial for leucocyte recruitment involved in insulitis and β cell damage. Multiple strategies blocking the CXCR1/2 pathway are available such as neutralizing antibodies, small molecules and peptide-derived inhibitors. They were firstly and widely used in cancer thanks to their anti-tumorigenic activity and only recently they were tested as a new interventional approach for T1D. As well, CXCR1/2 inhibition has been demonstrated to prevent inflammation- and autoimmunity-mediated damage of the pancreatic islets through inhibiting the migration of CXCR1/2-expressing cells. Among them, neutrophils, macrophages, and, although to a smaller extent, lymphoid cells are the main CXCR1/2-expressing cells. These results supported the active role of the innate immunity in the autoimmune process and opened new interventional approaches for the management of T1D.

Recovery effect of phenylpropanoid glycosides from Magnolia obovata fruit on alloxan-induced pancreatic islet damage in zebrafish (Danio rerio)

Investigation of phytochemicals from Magnolia obovata fruit led to the isolation of three novel phenylpropanoid glycosides: obovatoside A–C (1–3) and two known phenylpropanoids, syringin (4) and pavonisol (5). The structures of 1–5 were determined by NMR, HRMS, IR and CD spectroscopic analyses. All compounds were evaluated for their effects on recovery from alloxan-induced pancreatic islet damage in zebrafish. All compounds increased the size of the injured pancreatic islet from 0.60- to 1.14-fold. Compounds 1 and 3–5 significantly increased glucose absorption in zebrafish.

Synergistic protective effect of folic acid and vitamin B12 against nicotine-induced oxidative stress and apoptosis in pancreatic islets of the rat

Context: Nicotine is an abundant and most significant component of cigarette smoke. Epidemiological evidence strongly suggests an association between cigarette smoking and pancreatic injury, although effects of smoking on endocrine pancreas are still controversial.Objective: We examined the impact and underlying mechanisms of action of folic acid and vitamin B12 on nicotine-induced damage in pancreatic islets of rats.Materials and methods: Male Wistar rats were treated with nicotine (3 mg/kg body weight/d, intraperitonealy) with or without folic acid (36 µg/kg body weight/d, orally) and vitamin B12 (0.63 µg/kg body weight/d, orally) for 21 d. Fasting blood glucose, oral glucose tolerance test, HBA1c, insulin, oxidative stress parameters, proinflammatory cytokines, and CRP level were measured. Histological evaluation, TUNEL assay, and immunohistochemical staining of NF-κB and caspase-3 were also performed.Results: Folic acid and vitamin B12 blunted the nicotine-induced impairment in fasting blood glucose (51–56% recovery), HbA1c (64–76% recovery), oral glucose tolerance, insulin level (23–40% recovery), and islet cell counts (26–74% recovery) in rats. Moreover, folic acid in combination with vitamin B12 also attenuated the nicotine-induced changes in markers of oxidative stress (17–88% recovery), TNF-α (40–99% recovery), and IL-6 level (47–65% recovery), CRP level (59–73% recovery), expression of NF-κB and caspase-3, and apoptosis in pancreatic islet cells.Discussion and conclusion: The present study shows that folic acid and vitamin B12 supplementation can reduce nicotine-induced impairment in glucose homeostasis and apoptosis and damage of pancreatic islet cells by modulating oxidative stress, levels of proinflammatory cytokines, and expression of NF-κB.

5,7-Dihydroxy-3,4,6-Trimethoxyflavone Attenuates Ischemic Damage and Apoptosis in Mouse Islets

BackgroundThe transplantation of isolated pancreatic islets is a promising treatment for diabetes. 5,7-dihydroxy-3,4,6-trimethoxyflavone (Eupatilin), a pharmacologically active flavone derived from the Artemisia plant species, has been reported to have antioxidant and anti-inflammatory activities. This study examines the hypothesis that preoperative eupatilin treatment can attenuate ischemic damage and apoptosis before islet transplantation. MethodsIslets isolated from Balb/c mice were randomly divided into 2 groups, and cultured in medium supplemented with or without eupatilin. In vitro islet viability and function were assessed. After treatment with a cytokine cocktail consisting of tumor necrosis factor (TNF)-α, interferon (INF)-γ, and interleukin (IL)-1β, islet cell viability, function, and apoptotic status were determined. The glutathione (GSH) and nitrous oxide (NO) levels were also measured. Proteins related to apoptosis were analyzed using Western blotting. ResultsThere was no difference in cell viability between the 2 groups. Islets cultured in the medium supplemented with eupatilin showed 1.4-fold higher glucose-induced insulin secretion than the islets cultured in the medium without eupatilin. After treatment with a cytokine cocktail, glucose-induced insulin release and the total insulin content of the islets were significantly improved in eupatilin-pretreated islets compared with islets not treated with eupatilin. Apoptosis was significantly decreased, and GSH levels were elevated in the eupatilin-pretreated group. Cytokine-only treated islets produced significantly higher levels of NO, iNOS, and caspase-3 than islets pretreated with eupatilin before cytokine treatment. ConclusionsThese results suggest that preoperative eupatilin administration enhances islet function before transplantation and attenuates the cytokine-induced damage associated with NO production and apoptosis.

Developing a Microfluidic Device for Adenoviral Transfection of Pancreatic Islets

Diabetes is a global epidemic that is associated with the deterioration of pancreatic beta-cell mass and function. Beta-cell function within the islet has previously been studied ex vivo and the role of specific proteins is commonly investigated with overexpression models using viral transfection. However, poor penetration and reduced transfection efficiency limits the application of this technology in whole ex vivo islets. To improve transfection efficiency and maintain islet structure, we aim to develop a method to bring viral particles deeper into the tissue. Improving upon a previous microfluidic device design featuring hydrodynamic traps (nozzles) in series, a spiral-shaped microfluidic device with parallel nozzles was created to trap, transfect and deliver nutrients to islets. The device was engineered to provide sufficient resistance and pressure drop along the nozzles, allowing for homogeneous transfection throughout captured islets. Computer-generated models were used to optimize our design parameters to minimize pressure drop variations along with shear induced damage of islets. Through quantitative fluorescence microscopy, we have measured the media exchange rate through islets and have determined a range of operating flow-rates. We have also shown successful culturing of the loaded islets in our microfluidic device. Beta-cell-specific transfection in whole islets with desired genes will facilitate the investigation of islets in a more physiologically relevant environment. Therefore, we have developed a beta-cell specific shuttle vector in order to deliver fluorescent protein-tagged genes via adenoviral particles. Currently viral penetration efficiency is being tested in this device through fluorescence microscopy and western immunoblotting. We aim to create a multi-purpose platform that is not limited to the study of beta-cell biology in islets, but may be translated to the investigation of other tissue models and diseases.

Omega-3 Polyunsaturated Fatty Acids May Attenuate Streptozotocin-Induced Pancreatic β-Cell Death via Autophagy Activation in Fat1 Transgenic Mice.

BackgroundInflammatory factors and β-cell dysfunction due to high-fat diets aggravate chronic diseases and their complications. However, omega-3 dietary fats have anti-inflammatory effects, and the involvement of autophagy in the etiology of diabetes has been reported. Therefore, we examined the protective effects of autophagy on diabetes using fat-1 transgenic mice with omega-3 self-synthesis capability.MethodsStreptozotocin (STZ) administration induced β-cell dysfunction in mice; blood glucose levels and water consumption were subsequently measured. Using hematoxylin and eosin (H&E) and Masson's trichrome staining, we quantitatively assessed STZ-induced changes in the number, mass, and fibrosis of pancreatic islets in fat-1 and control mice. We identified the microtubule-associated protein 1A/1B light chain 3-immunoreactive puncta in β-cells and quantified p62 levels in the pancreas of fat-1 and control mice.ResultsSTZ-induced diabetic phenotypes, including hyperglycemia and polydipsia, were attenuated in fat-1 mice. Histological determination using H&E and Masson's trichrome staining revealed the protective effects of the fat-1 expression on cell death and the scarring of pancreatic islets after STZ injection. In the β-cells of control mice, autophagy was abruptly activated after STZ treatment. Basal autophagy levels were elevated in fat-1 mice β-cells, and this persisted after STZ treatment. Together with autophagosome detection, these results revealed that n-3 polyunsaturated fatty acid (PUFA) enrichment might partly prevent the STZ-related pancreatic islet damage by upregulating the basal activity of autophagy and improving autophagic flux disturbance.ConclusionFat-1 transgenic mice with a n-3 PUFA self-synthesis capability exert protective effects against STZ-induced β-cell death by activating autophagy in β-cells.

Evaluation of Anti-Diabetic and Anti-lipidemic Activities of Aqueous Leaf Extract of Millettia aboensis and Its Effect on Pancreatic Histology of Alloxan-Induced Diabetic Rats

Plants provide a potential source of anti-diabetic drugs and are widely used in several traditional systems of medicine to prevent diabetes. This study evaluated the anti-diabetic and anti-lipidemic activities of oral administration of aqueous leaf extract of Milletia aboensis in, and its effect on pancreatic histology of, alloxan-induced diabetic rats. Eighty rats were grouped into eight based on average body weights. Diabetes similar to that of type 1 in human was induced by a single intra-peritoneal injection of alloxan monohydrate a -diabetogenic agent (160 mg kg-1). After 120 hours of alloxan injection, the rats from Groups 2 – 8 were confirmed diabetic having baseline fasting blood sugar levels >8.5mmol/L. Group 1 served as normal control. The extract was administered twice daily for 21 days at doses of 500 mg kg-1 – 2500 mg kg-1 in Groups 4 – 8. Group 2 was treated with a standard diabetic drug, ‘Metformin’, while Group 3 (untreated and negative control) was diabetic with increased Total cholesterol (CHOL), low density lipoproteins (LDL), triacylglycerol (TAG) levels and reduced high density lipoproteins (HDL) level throughout the experiment. Glucose was assayed for on the 7th, 14th and 21st day while CHOL, HDL, LDL, and triglyceride were assayed on the 21st day. A significant reduction (p<0.05) in the fasting blood glucose levels of rats in Groups 4-8 was observed when compared to the normal control. Similarly, there was a significant decrease (p<0.05) in TAG and LDL and a significant increase (p<0.05) in HDL levels in groups treated with the extract when compared to Group 1 (normal control). A photomicrograph of the pancreatic cells was taken before and after treatment with aqueous leaf extract. The photomicrograph before treatment showed atrophic pancreatic islet with vacuolations which indicates significant damaged Islets of langerhans in the beta cells. After treatment the photomicrograph indicates significant cell regeneration and repair. The present investigation suggests that leaf extract of M. aboensis exhibits anti-diabetic and anti-lipidemic activities and positively affected pancreatic functions in alloxan-induced diabetes rats.

Hes3 Is Expressed in the Adult Pancreatic Islet and Regulates Gene Expression, Cell Growth, and Insulin Release

The transcription factor Hes3 is a component of a signaling pathway that supports the growth of neural stem cells with profound consequences in neurodegenerative disease models. Here we explored whether Hes3 also regulates pancreatic islet cells. We showed that Hes3 is expressed in human and rodent pancreatic islets. In mouse islets it co-localizes with alpha and beta cell markers. We employed the mouse insulinoma cell line MIN6 to perform in vitro characterization and functional studies in conditions known to modulate Hes3 based upon our previous work using neural stem cell cultures. In these conditions, cells showed elevated Hes3 expression and nuclear localization, grew efficiently, and showed higher evoked insulin release responses, compared with serum-containing conditions. They also exhibited higher expression of the transcription factor Pdx1 and insulin. Furthermore, they were responsive to pharmacological treatments with the GLP-1 analog Exendin-4, which increased nuclear Hes3 localization. We employed a transfection approach to address specific functions of Hes3. Hes3 RNA interference opposed cell growth and affected gene expression as revealed by DNA microarrays. Western blotting and PCR approaches specifically showed that Hes3 RNA interference opposes the expression of Pdx1 and insulin. Hes3 overexpression (using a Hes3-GFP fusion construct) confirmed a role of Hes3 in regulating Pdx1 expression. Hes3 RNA interference reduced evoked insulin release. Mice lacking Hes3 exhibited increased islet damage by streptozotocin. These data suggest roles of Hes3 in pancreatic islet function.

In vivo pancreatic β-cell-specific expression of antiaging gene Klotho: a novel approach for preserving β-cells in type 2 diabetes.

Protein expression of an antiaging gene, Klotho, was depleted in pancreatic islets in patients with type 2 diabetes mellitus (T2DM) and in db/db mice, an animal model of T2DM. The objective of this study was to investigate whether in vivo expression of Klotho would preserve pancreatic β-cell function in db/db mice. We report for the first time that β-cell–specific expression of Klotho attenuated the development of diabetes in db/db mice. β-Cell–specific expression of Klotho decreased hyperglycemia and enhanced glucose tolerance. The beneficial effects of Klotho were associated with significant improvements in T2DM-induced decreases in number of β-cells, insulin storage levels in pancreatic islets, and glucose-stimulated insulin secretion from pancreatic islets, which led to increased blood insulin levels in diabetic mice. In addition, β-cell–specific expression of Klotho decreased intracellular superoxide levels, oxidative damage, apoptosis, and DNAJC3 (a marker for endoplasmic reticulum stress) in pancreatic islets. Furthermore, β-cell–specific expression of Klotho increased expression levels of Pdx-1 (insulin transcription factor), PCNA (a marker of cell proliferation), and LC3 (a marker of autophagy) in pancreatic islets in db/db mice. These results reveal that β-cell–specific expression of Klotho improves β-cell function and attenuates the development of T2DM. Therefore, in vivo expression of Klotho may offer a novel strategy for protecting β-cells in T2DM.

CXCR1/2 inhibition blocks and reverses type 1 diabetes in mice.

Chemokines and their receptors have been associated with or implicated in the pathogenesis of type 1 diabetes (T1D), but the identification of a single specific chemokine/receptor pathway that may constitute a suitable target for the development of therapeutic interventions is still lacking. Here, we used multiple low-dose (MLD) streptozotocin (STZ) injections and the NOD mouse model to investigate the potency of CXCR1/2 inhibition to prevent inflammation- and autoimmunity-mediated damage of pancreatic islets. Reparixin and ladarixin, noncompetitive allosteric inhibitors, were used to pharmacologically blockade CXCR1/2. Transient blockade of said receptors was effective in preventing inflammation-mediated damage in MLD-STZ and in preventing and reversing diabetes in NOD mice. Blockade of CXCR1/2 was associated with inhibition of insulitis and modification of leukocytes distribution in blood, spleen, bone marrow, and lymph nodes. Among leukocytes, CXCR2(+) myeloid cells were the most decreased subpopulations. Together these results identify CXCR1/2 chemokine receptors as "master regulators" of diabetes pathogenesis. The demonstration that this strategy may be successful in preserving residual β-cells holds the potential to make a significant change in the approach to management of human T1D.

Addition of a Specific Na+/Ca2+ Exchanger Inhibitor Into Collagenase Solution Prevents Hypoxic Damage of Islets During Isolation, Facilitating to Improve the Efficiency of Islet Transplantation in Mice.

The low efficiency of islet transplantation (tx) requiring 2-3 donors for a recipient,mainly due to early loss of transplanted islets (TI), hampers its clinical application. Previously, we have shown in mice that the early loss of TI is caused by hypoxic damage of islets on engraftment mediated by Ca2+ influx into ß cells via Na+/Ca2+ exchanger (NCX) resulting in islet-derived HMGB1 release to trigger NKT cell-dependent innate immune rejection and that importantly, the early loss of TI is prevented by the treatment of donor islets by a specific NCX inhibitor which diminishes hypoxic damage by blocking Ca2+ influx into ß cells.Here we determined whether the NCX inhibitor in collagenase prevents hypoxic damage of mouse islets (C57BL/6) during isolation, facilitating to improve the efficiency of islet tx. A specific NCX inhibitor, SEA0400 (10mM), or vehicle (DMSO) was added to the collagenase solution (2mg/ml), and islets were isolated by the standard static digestion method. The yield of islets did not differ between two groups. At first, HMGB1 in the culture medium of islets was measured since our previous study demonstrated that HMGB1 contained abundantly in islets is released parallel to the degree of their damage. The amount of HMGB1 in the 6 hour culture medium of islets from vehicle (n=5) and SEA0400 group (n=5) was 1.6±1.2 and 0.3±0.3ng/ml/10 islets, respectively (p<0.05). When islets were cultured in a hypoxic chamber (O2; 20mmHg) for 6 hrs, abundant and few PI positive cells in vehicle and SEA0400 group, respectively, were seen, indicating that hypoxic islet cell death is prevented by the addition of SEA0400. When STZ-diabetic mice received syngenic 200 islets into the liver from a donor of vehicle (n=5) or SEA0400 (n=8) group, recipient mice remained hyperglycemic or became normoglycemic, respectively. The glucose tolerance of mice bearing SEA0400 islets was superior to that of mice with vehicle islets. Histologically, well- or de-granulated ß cells were seen in the liver of normoglycemic or hyperglycemic mice, respectively. These findings show that the addition of NCX inhibitor into collagenase prevents hypoxic damage of islets during isolation and indicate that this strategy may apply to improve the efficiency of clinical islet tx.