Untreated Islets Research Articles

Improvement effects of transplanting pancreatic islet that previously incubated with biomaterials on the diabetic nephropathy in STZ- diabetic rats

BackgroundIslet transplantation is an effective treatment for diabetes or even its complications. Aim of this study is to investigate efficacy of biomaterial treated islet transplantation on treating diabetic nephropathy.MethodsMale rats were randomly divided into 6 groups; Control, diabetic control, diabetic transplanted with untreated islets, with platelet rich plasma treated islets, with pancreatic islets homogenate treated islets, or with these biomaterials combination treated islets. Islets cultured with biomaterials and transplanted to diabetic rats. After 60 days, biochemical, oxidative stress, and stereological parameters were assessed.ResultsSerum albumin and BUN concentration, decreased and increased respectively, Oxidative stress of kidney impaired, kidney weight, volume of kidney, cortex, medulla, glomerulus, proximal and distal tubules, collecting ducts, vessels, inflammatory, necrotic and fibrotic tissue in diabetic group increased compared to control group (p < 0.001). In treated groups, especially pancreatic islets homogenate treated islets transplanting animals, there was significant changes in kidney weight, and volume of kidney, proximal and distal tubules, Henle’s loop and collecting ducts compared with diabetic group (p = 0.013 to p < 0.001). Combination treated islets animals showed significant increase in vessel volume compared to diabetic group (p < 0.001). Necrotic and fibrotic tissue significantly decreased in islets treated than untreated islet animals, it was higher in pancreatic islets homogenate, and combination treated islets groups (p = 0.001).ConclusionsBiomaterials treated islets transplanting could improve diabetic nephropathy. Improvement of oxidative stress followed by controlling glucose level, and effects of growth factors presenting in biomaterials can be considered as capable underlying mechanism of ameliorating inflammatory, necrotic and fibrotic tissue volume.

PAX4 gene delivery improves β-cell function in human islets of Type II diabetes.

Aim: Type II diabetes (T2D) stems from insulin resistance, with β-cell dysfunction as a hallmark in its progression. Studies reveal that β cells undergo apoptosis or dedifferentiation during T2D development. The transcription factor PAX4 is vital for β differentiation and survival, thus may be a potential enhancer of β-cell function in T2D islets. Materials & methods: Human PAX4 cDNA was delivered into T2D human islets with an adenoviral vector, and its effects on β cells were examined. Results: PAX4 gene delivery significantly improved β-cell survival, and increased β-cell composition in the T2D human islets. Basal insulin and glucose-stimulated insulin secretion in PAX4-expressing islets were substantially higher than untreated or control-treated T2D human islets. Conclusion: Introduced PAX4 expression in T2D human islets improves β-cell function, thus could provide therapeutic benefits for T2D treatment.

Mesenchymal stromal cell secretory molecules improve the functional survival of human islets.

Human islet transplantation as a therapy for type 1 diabetes is compromised by the loss of functional beta cells in the immediate post-transplantation period. Mesenchymal stromal cells (MSCs) and MSC-derived secretory peptides improve the outcomes of islet transplantation in rodent models of diabetes. Here, we utilized a mouse model for human islet transplantation and assessed the effects of a cocktail of MSC-secreted peptides (screened by MSC-secretome for human islet GPCRs) on the functional survival of human islets. Human islets from nine donors (Age: 36-57; BMI: 20-35) were treated with a cocktail of human recombinant annexin A1 (ANXA1), stromal cell-derived factor-1 (SDF-1/CXCL12) and complement component C3 (C3a). Glucose-stimulated insulin secretion (GSIS) was assessed in static incubation, and cytokine-induced apoptosis was assessed by measuring caspase 3/7 activity. mRNA expression levels were determined by qPCR. Human islet function in vivo was assessed using a novel model for human islet transplantation into a T1D mouse model. Human islet function in vivo was assessed using islet transplantation under the kidney capsule of immunodeficient mice prior to STZ destruction of endogenous mouse beta cells to model T1DM. Pretreatment with a cocktail of MSC-secreted peptides increased GSIS in vitro and protected against cytokine-induced apoptosis in human islets isolated from nine donors. Animals transplanted with either treated or untreated human islets remained normoglycaemic for up to 28 days after STZ-administration to ablate the endogenous mouse beta cells, whereas non-transplanted animals showed significantly increased blood glucose immediately after STZ administration. Removal of the human islet graft by nephrectomy resulted in rapid increases in blood glucose to similar levels as the non-transplanted controls. Pretreating human islets with the MSC-derived cocktail significantly improved glucose tolerance in graft recipients, consistent with enhanced functional survival of the treated islets in vivo. Pretreating human islets before transplantation with a defined cocktail of MSC-derived molecules could be employed to improve the quality of human islets for transplantation therapy for type 1 diabetes.

207.7: DAMP Proteins Released via Exosomes During Islet Isolation Affects TPIAT Outcomes

Introduction: Islets undergo significant stress and damage during isolation and in the peritransplant period which results in the release of immunogenic factors called damage-associated molecular patterns (DAMPs) that can induce immune cell activation causing post-transplant islet damage. Exosomes are released by islets in response to stress and but the functional impact of these exosomes is still not clearly understood. Here, we sought to identify the DAMP proteins released by islets via exosomes in response to islet isolation stress and also study their impact on TPIAT outcomes. Methods: Islet stress during isolation and peritransplant period was mimicked in vitro by exposing human islets to hypoxia and cytokines (Cyt+Hyp) simultaneously for 24h. Separately, human islets were also treated with thapsigargin for 24hrs to induce ER stress. Exosomes were isolated after stress treatment and the differentially expressed proteins were determined by mass spectrometry and western blotting. To validate the in vitro findings, exosomes were isolated from the transplant media of TPIAT patients before the infusion to measure DAMP protein levels in them. Patients receiving an islet dose of 4000IEQ/kg were divided into insulin-dependent (ID; n=10) and insulin-independent (IID; n=10) groups. The exosomal DAMP protein levels were correlated with 1-year TPIAT outcomes. Results: The number of exosomes released was significantly elevated in (Cyt+Hyp) treated islets compared to untreated islets. The mass spectrometry analysis has revealed about 9 proteins including DAMP protein histones (H2A, H2B, H4) and Keratins (Type I and II) were differentially expressed. Islets treated with thapsigargin or Cyt+Hyp overexpressed histones and keratins intracellularly through the activation of the PERK/IRE1α pathway. This subsequently led to the increased release of histones and keratins via exosomes during stress. Exosomes isolated from transplant media of TPIAT patients showed elevated histone (p=0.0796) and decreased keratin (p=0.072) in ID compared to IID patients. Moreover, high exosomal histone/IEQ resulted in increased insulin requirement (r=0.527, p=0.029 n=17) and HBA1c%, and lower c-peptide (r=-0.494, p=0.085 n=13) and SUITO index at 1-year POD. In contrast, the exosomal keratin/IEQ showed the opposite effect on 1-year outcomes. Conclusion: The study has identified the DAMP protein-like histones are produced by the stressed islets are released via exosomes and these have been shown to affect the graft outcomes in TPIAT patients. Future directions will focus on the mechanism by which exosomal DAMP proteins affect graft outcomes and strategies to reduce DAMP secretion via exosomes.

In Vitro Impact of Pro-Senescent Endothelial Microvesicles on Isolated Pancreatic Rat Islets Function

Ischemia-driven islet isolation procedure is one of the limiting causes of pancreatic islet transplantation. Ischemia-reperfusion process is associated with endothelium dysfunction and the release of pro-senescent microvesicles. We investigated whether pro-senescent endothelial microvesicles prompt islet senescence and dysfunction in vitro. Pancreatic islets were isolated from male young rats. Replicative endothelial senescence was induced by serial passaging of primary porcine coronary artery endothelial cells, and microvesicles were isolated either from young passage 1 (P1) or senescent passage 3 (P3) endothelial cells. Islet viability was assessed by fluorescence microscopy, apoptosis by flow cytometry, and Western blot. Function was assessed by insulin secretion and islet senescence markers p53, p21, and p16 by Western blot. Microvesicles were stained by the PKH26 lipid fluorescent probe and their islet integration assessed by microscopy and flow cytometry. Regardless of the passage, half microvesicles were integrated in target islets after 24 hours incubation. Insulin secretion significantly decreased after treatment by senescent microvesicles (P3: 1.7 ± 0.2 vs untreated islet: 2.7 ± 0.2, P < .05) without altering the islet viability (89.47% ± 1.69 vs 93.15% ± 0.97) and with no significant apoptosis. Senescent microvesicles significantly doubled the expression of p53, p21, and p16 (P < .05), whereas young microvesicles had no significant effect. Pro-senescent endothelial microvesicles specifically accelerate the senescence of islets and alter their function. These data suggest that islet isolation contributes to endothelial driven islet senescence.

Polylysine-bilirubin conjugates maintain functional islets and promote M2 macrophage polarization

Macrophage polarization is one of the main factors contributing to the proinflammatory milieu of transplanted islets. It causes significant islet loss. Bilirubin exhibits protective effects during the islet transplantation process, but the mode of delivering drugs along with the islet graft has not yet been developed. In addition, it remains unclear whether bilirubin or its derivatives can modulate macrophage polarization during islet transplantation. Therefore, this study aimed to develop an ε-polylysine-bilirubin conjugate (PLL-BR) to encapsulate the islets for protection and to explore its macrophage modulation activities. In in vitro studies, the PLL-BR was shown to tightly adhere to the islet surface. It also exhibited enhanced cytoprotective effects against oxidative and inflammatory conditions by promoting M2-type macrophage polarization. In in vivo studies, the PLL-BR-protected islets successfully prolonged the euglycemia period in diabetic mice and accelerated the blood glucose clearance rate by maintaining the insulin secretion function. Compared to the untreated islets, the PLL-BR-encapsulated islets induced anti-inflammatory responses that were characterized by elevated levels of M2 macrophage markers and local vascularization. In conclusion, PLL-BR can be used as a tool for reprograming macrophage polarization while providing a more efficient immune protection for transplanted islets. Statement of significanceMacrophage polarization is one main factor that caused significant loss of transplanted islets. Bilirubin possesses protective effects toward pancreatic islet, but how to deliver the drug along with the islet graft has not yet been harnessed. More importantly, whether bilirubin or its derivatives could modulate macrophage polarization during the host rejections has also not been answered. In this study, we developed an ε-polylysine-bilirubin conjugate (PLL-BR) to encapsulate the islets and explore its role in macrophage modulation activities. PLL-BR could attach to the surface of islets and exerted high oxidation resistance and anti-inflammatory effect. For the first time, we demonstrate that bilirubin and its derivatives effectively promoted the M2-type macrophage polarization, and optimize the immune microenvironment for islets survival and function.

Intestinal Delivery of Proinsulin and IL-10 via Lactococcus lactis Combined With Low-Dose Anti-CD3 Restores Tolerance Outside the Window of Acute Type 1 Diabetes Diagnosis.

A combination treatment (CT) of proinsulin and IL-10 orally delivered via genetically modified Lactococcus lactis bacteria combined with low-dose anti-CD3 (aCD3) therapy successfully restores glucose homeostasis in newly diagnosed non-obese diabetic (NOD) mice. Tolerance is accompanied by the accumulation of Foxp3+ regulatory T cells (Tregs) in the pancreas. To test the potential of this therapy outside the window of acute diabetes diagnosis, we substituted autoimmune diabetic mice, with disease duration varying between 4 and 53 days, with syngeneic islets at the time of therapy initiation. Untreated islet recipients consistently showed disease recurrence after 8.2 ± 0.7 days, while 32% of aCD3-treated and 48% of CT-treated mice remained normoglycemic until 6 weeks after therapy initiation (P < 0.001 vs. untreated controls for both treatments, P < 0.05 CT vs. aCD3 therapy). However, mice that were diabetic for more than 2 weeks before treatment initiation were less efficient at maintaining normoglycemia than those treated within 2 weeks of diabetes diagnosis, particularly in the aCD3-treated group. The complete elimination of endogenous beta cell mass with alloxan at the time of diabetes diagnosis pointed toward the significance of continuous feeding of the islet antigen proinsulin at the time of aCD3 therapy for treatment success. The CT providing proinsulin protected 69% of mice, compared to 33% when an irrelevant antigen (ovalbumin) was combined with aCD3 therapy, or to 27% with aCD3 therapy alone. Sustained tolerance was accompanied with a reduction of IGRP+CD8+ autoreactive T cells and an increase in insulin-reactive (InsB12–20 or InsB13–2) Foxp3+CD4+ Tregs, with a specific accumulation of Foxp3+ Tregs around the insulin-containing islet grafts after CT with proinsulin. The combination of proinsulin and IL-10 via oral Lactococcus lactis with low-dose aCD3 therapy can restore tolerance to beta cells in autoimmune diabetic mice, also when therapy is started outside the window of acute diabetes diagnosis, providing persistence of insulin-containing islets or prolonged beta cell function.

Polylysine-Bilirubin Conjugates Maintain Functional Islets and Promote M2 Macrophage Polarization

Macrophage polarization has been considered as one main factor that contributes to the pro-inflammatory milieu of the transplanted islets and caused significant islet loss. Bilirubin possesses protective effects in islet transplantation, but how to deliver the drug along with the islet graft has not yet been harnessed. More importantly, whether bilirubin or its derivates could modulate macrophage polarization during the host rejections has also not been answered. To this end, we aimed to develop an e-polylysine-bilirubin conjugate (PLL-BR) to encapsulate the islets for protection and explore its macrophage modulation activities. In vitro studies indicated that PLL-BR tightly adheres to the islet surface, and achieved enhanced cytoprotective effects against oxidative and inflammatory conditions by promoting M2 type macrophage polarization. In vivo data demonstrated that PLL-BR protected islets successfully prolonged the period of euglycemia in diabetic mice and accelerated the clearance rate of blood glucose by maximumly maintain the insulin secretion function. The PLL-BR encapsulated islets elicited anti-inflammatory response characterized by the elevated proportion of M2 macrophage markers and local vascularization compared to untreated islets. Hence PLL-BR might serve as a useful tool for reprograming the macrophage polarization and providing a more efficient immune protection for transplanted islets and improve the outcomes.

Transplantation of PEGylated islets enhances therapeutic efficacy in a diabetic nonhuman primate model.

Islet cell transplantation can lead to insulin independence, reduced hypoglycemia, and amelioration of diabetes complications in patients with type 1 diabetes. The systemic delivery of anti-inflammatory agents, while considered crucial to limit the early loss of islets associated with intrahepatic infusion, increases the burden of immunosuppression. In an effort to decrease the pharmaceutical load to the patient, we modified the pancreatic islet surface with long-chain poly(ethylene glycol) (PEG) to mitigate detrimental host-implant interactions. The effect of PEGylation on islet engraftment and long-term survival was examined in a robust nonhuman primate model via three paired transplants of dosages 4300, 8300, and 10000islet equivalents per kg body weight. A reduced immunosuppressive regimen of anti-thymocyte globulin induction plus tacrolimus in the first posttransplant month followed by maintenance with sirolimus monotherapy was employed. To limit transplant variability, two of the three pairs were closely MHC-matched recipients and received MHC-disparate PEGylated or untreated islets isolated from the same donors. Recipients of PEGylated islets exhibited significantly improved early c-peptide levels, reduced exogenous insulin requirements, and superior glycemic control, as compared to recipients of untreated islets. These results indicate that this simple islet modification procedure may improve islet engraftment and survival in the setting of reduced immunosuppression.

Necrostatin-1 supplementation enhances young porcine islet maturation and in vitro function.

Necroptosis has been demonstrated to be a primary mechanism of islet cell death. This study evaluated whether the supplementation of necrostatin-1 (Nec-1), a potent inhibitor of necroptosis, to islet culture media could improve the recovery, maturation, and function of pre-weaned porcine islets (PPIs). PPIs were isolated from pre-weaned Yorkshire piglets (8-15days old) and either cultured in control islet culture media (n=6) or supplemented with Nec-1 (100µM, n=5). On days 3 and 7 of culture, islets were assessed for recovery, insulin content, viability, cellular composition, GLUT2 expression in beta cells, differentiation of pancreatic endocrine progenitor cells, function, and oxygen consumption rate. Nec-1 supplementation induced a 2-fold increase in the insulin content of PPIs on day 7 of culture. When compared to untreated islets, Nec-1 treatment doubled the beta- and alpha-cell composition and accelerated the development of delta cells. Additionally, beta cells of Nec-1-treated islets had a significant upregulation in GLUT2 expression. The enhanced development of major endocrine cells and GLUT2 expression after Nec-1 treatment subsequently led to a significant increase in the amount of insulin secreted in response to in vitro glucose challenge. Islet recovery, viability, and oxygen consumption rate were unaffected by Nec-1. This study underlines the importance of necroptosis in islet cell death after isolation and demonstrates the novel effects of Nec-1 to increase islet insulin content, enhance pancreatic endocrine cell development, facilitate GLUT2 upregulation in beta cells, and augment insulin secretion. Nec-1 supplementation to culture media significantly improves islet quality prior to xenotransplantation.

Improving the Function and Engraftment of Transplanted Pancreatic Islets Using Pulsed Focused Ultrasound Therapy

This study demonstrates that pulsed focused ultrasound (pFUS) therapy can non-invasively enhance the function and engraftment of pancreatic islets following transplantation. In vitro, we show that islets treated with pFUS at low (peak negative pressure (PNP): 106kPa, spatial peak temporal peak intensity (Isptp): 0.71 W/cm2), medium (PNP: 150kPa, Isptp: 1.43 W/cm2) or high (PNP: 212kPa, Isptp: 2.86 W/cm2) acoustic intensities were stimulated resulting in an increase in their function (i.e. insulin secretion at low-intensity: 1.15 ± 0.17, medium-intensity: 2.02 ± 0.25, and high-intensity: 2.54 ± 0.38 fold increase when compared to control untreated islets; P < 0.05). Furthermore, we have shown that this improvement in islet function is a result of pFUS increasing the intracellular concentration of calcium (Ca2+) within islets which was also linked to pFUS increasing the resting membrane potential (Vm) of islets. Following syngeneic renal sub-capsule islet transplantation in C57/B6 mice, pFUS (PNP: 2.9 MPa, Isptp: 895 W/cm2) improved the function of transplanted islets with diabetic animals rapidly re-establishing glycemic control. In addition, pFUS was able to enhance the engraftment by facilitating islet revascularization and reducing inflammation. Given a significant number of islets are lost immediately following transplantation, pFUS has the potential to be used in humans as a novel non-invasive therapy to facilitate islet function and engraftment, thereby improving the outcome of diabetic patients undergoing islet transplantation.

28: Enhancing In Vitro Islet Function Using a Novel Necroptosis Inhibitor

Introduction: Necroptosis was previously found as a mechanism of beta-cell death in diabetes and in human islet culture. Necrostatin-1 (Nec-1), a potent inhibitor of necroptosis, could reduce human islet necroptosis. This study aims to assess whether Nec-1 could improve the survival of pre-weaned porcine islets (PPIs) during maturation culture, and whether it affects their insulin secretory function. Methods: PPIs were isolated from pancreata of 4-11-day-old, pre-weaned Yorkshire pigs and cultured in optimized islet culture media (37°C, 5% CO2) for 3 days. At day 3, the islets were divided into 2 groups, in islet culture media alone (n=6) or supplemented with Nec-1 (100µM, Sigma, n=5), and then cultured until day 7. Islet recovery was calculated as the percentage of IEQ per gram of pancreatic tissue on day 7 compared to day 3. Flow cytometry (NovoCyte 3000VYB) was used to determine islet cellular viability, composition, GLUT2 expression and differentiation. In vitro function was determined by glucose-stimulated insulin release assay. Data was expressed as mean ± SEM and evaluated using ANOVA with p<0.05 considered statistically significant. Results & Discussion: Islet recovery was significantly higher in Nec-1 treated group (control=51.3±10.2% vs. Nec-1=112.8±8.4%, p<0.01, Figure 1). Nec-1 treated islets had a two-fold increase in the percentage of β-cells compared to untreated islets (control=8.9±1.3% vs. Nec-1=17.2±1.4%, p<0.01). Additionally, GLUT2 expression in β-cells was significantly elevated in Nec-1 treated group (control=29.9±3.2% vs. Nec-1=55.1±0.7%, p<0.05). A substantial increase in insulin secretion in response to glucose challenge was observed when the islets were cultured with Nec-1 (2.8 mM glucose: control=0.9±0.2 pg/ng vs. Nec-1=13.8±2 pg/ng, p<0.01; 28 mM glucose: control=2.3±1.6 pg/ng vs. Nec-1=38.8±5.2 pg/ng, p<0.01; SI: control=2.5±0.3 vs. Nec-1=5.3±0.6, p<0.01). Conclusion: Identifying cytoprotective agents that could maintain islet quantity and quality during extended culture is critical to optimize the current pre-transplant protocol. Our data showed that Nec-1 improved islet recovery, increased proportion of endocrine cells, increased GLUT2 expression in β-cells, and improved insulin response to glucose. We believe that Nec-1 supplementation in culture media will enhance islet quality before transplantation. Future studies will be done to titrate the most effective dose of Nec-1, and evaluate these islets in rodent transplant models of Type 1 diabetes. Juvenile Diabetes Research Foundation.

RGD-containing elastin-like polypeptide improves islet transplantation outcomes in diabetic mice

Successful islet transplantation critically depends on the isolation of healthy islets. However, the islet isolation procedure itself contributes to islet death due to the destruction of intra- and peri-islet extracellular matrices (ECMs) during digestion. We investigated whether an RGD-containing elastin-like polypeptide (REP) could function as a self-assembling matrix to replenish ECMs and protects islets from cell death. Immediately following isolation, islets were coated with REP coacervate particles via isothermal adsorption of an REP solution followed by thermal gelation. REP-coated islets displayed increased viability and insulin secretory capacity in pretransplant culture compared to untreated islets. Co-transplantation of REP-treated islets and REP beneath the renal sub-capsule in streptozotocin-induced diabetic mice restored normoglycemia and serum insulin levels. Mice that received co-transplants maintained normoglycemia for a longer period of time than those receiving untreated islets without REP. Moreover, co-transplantation sites exhibited enhanced β-cell proliferation and vascularization. Thus, the REP-based coacervation strategy improve the survival, function and therapeutic potential of transplanted islets. Statement of Significance1). An artificial matrix polypeptide comprised of thermoresponsive elastin-like peptides and integrin-stimulatory RGD ligands (REP) to reconstitute damaged or lost matrices.2). Through body temperature-induced coacervation, REP reconstitutes intra-islet environment and enhances islet viability and insulin secretion by activating the pro-survival and insulin signaling pathways.3). REP-coated islets were transplanted together with the matrix polypeptide under the kidney sub-capsule of mice, it develops a new peri-insular environment, which protects the islet grafts from immune rejection thus extending islet longevity.4). Our data suggest that in situ self-assembly of biomimetic islet environments become a new platform allowing for improved islet transplantation at extrahepatic sites.

Early TLR4 Blockade Attenuates Sterile Inflammation-mediated Stress in Islets During Isolation and Promotes Successful Transplant Outcomes.

During the isolation process, pancreatic islets are exposed to an environment of sterile inflammation resulting in an upregulated inflammatory state before transplantation. Toll-like receptor 4 (TLR4) has been identified as a major mediator of sterile inflammation. Therefore, we sought to determine whether early TLR4 blockade would be effective in reducing the inflammatory burden in islets pretransplant. Islets from C57BL/6 mice were treated with a TLR4 antagonist during the pancreatic ductal perfusion and digestion steps of the isolation process. Islets were then analyzed for inflammation by RT-PCR and Western blot, and for viability and function in vitro. A syngeneic transplant model using a marginal mass of islets transplanted intraportally into mice with streptozotocin-induced diabetes was used to study transplant outcomes after early TLR4 blockade. Diabetic mice receiving 150 islets treated with early TLR4 blockade achieved euglycemia at a higher rate than mice receiving untreated islets (75% vs 29%; P < 0.05) and had improved long-term function (P < 0.05). Serum markers for islet damage and inflammation were significantly reduced posttransplant (P < 0.05). Both the expression of key inflammatory genes and the activation of mitogen-activated protein kinases were reduced by early TLR4 blockade. Islet viability was improved (P < 0.05) while preserving islet insulin secretory capacity postisolation. Early TLR4 blockade protects islets from sterile inflammation-mediated stress sustained during isolation and promotes positive transplant outcomes. Our findings support the use of early TLR4 blockade during clinical islet isolation procedures to reduce pretransplant inflammation and improve transplant outcomes.

Lupinus mutabilis Extract Exerts an Anti-Diabetic Effect by Improving Insulin Release in Type 2 Diabetic Goto-Kakizaki Rats.

Lupinus mutabilis (LM) is a legume part of Bolivian traditional diet that has a nutraceutical property reducing blood glucose levels. The prevalence of type 2 diabetes is increasing worldwide thus; the search for novel anti-diabetic drugs is needed. Based on its traditional use, we evaluated the anti-diabetic effect of LM in the spontaneously diabetic Goto-Kakizaki (GK) rat, a model of type 2 diabetes and in Wistar (W) rats as healthy control. LM seeds hydroethanolic extract, analyzed by gas chromatography-mass spectrometry and high-performance liquid chromatography-high resolution mass spectrometry, is a complex mixture of volatile and non-volatile components. A single oral administration of LM extract (2000 mg/kg b.w.) improved glucose tolerance during the oral glucose tolerance test (OGTT) (30–120 min) in GK and W rats (p < 0.0001). The long-term treatment with LM (1000 mg/kg b.w.), for 21 days, improved the area under the curve (AUC) of glucose during OGTT at day 20, in both GK (p < 0.01) and W rats (p < 0.01). The HbA1c (GK rats, p < 0.05 and W rats, p < 0.0001) and the non-fasting glucose (GK rats, p < 0.05) were also reduced. LM increased both serum insulin levels (2.4-fold in GK rats and 2.5-fold W rats), and the glucose-induced (16.7 mM glucose) insulin release in isolated islets from treated animals (6.7-fold in GK rats, and 6.6-fold in W rats). Moreover, LM (10 mg/mL) stimulated in vitro glucose induced (16.7 mM glucose) insulin release in batch incubated GK and W rat islets (p < 0.0001). In perifused GK rat islets, insulin release in 16.7 mM glucose was increased 95.3-fold compared to untreated islets (p < 0.0001), while no significant differences were found in perifused W rat islets. The LM mechanism of action, evaluated using inhibitory compounds of the insulin secretion pathway, showed that LM-dependent insulin secretion was reduced 42% by diazoxide (p < 0.001), 70% by nifedipine (p < 0.001), 86.7% by H89 (p < 0.0001), 70.8% by calphostine-C (p < 0.0001) and 93% by pertussis toxin (p < 0.0001). A similar effect was observed in W rats islets. Our findings provide evidence that LM has an anti-diabetic effect through stimulation of insulin release. The effect is-dependent on L-type calcium channel, protein kinase A and C systems, and G protein-coupled exocytosis and is partially mediated by K-ATP channels.

A Simple and Promising Strategy for Pancreatic Islet Preconditioning to Reduce Immunogenicity and Improve Islet Graft Longevity

Introduction An advantage of pancreatic islet transplantation is that the pretransplant in vitro culture period provides an opportunity for cell modification; however, no “islet preconditioning” method has been established yet. We previously reported that the pretreatment of donor islets with an antitumoral antibiotic, mitomycin-C (MMC), and subsequent culture significantly prolonged graft survival in murine renal subcapsular islet transplantations. Here, we introduce an “islet preconditioning” strategy for reducing the immunogenicity of islets and for preventing the activation of proinflammatory events by using MMC in an intraportal islet transplantation model. Methods Freshly isolated BALB/c islets, either untreated or treated for 30 min with 10 μg/mL MMC, were cultured for 20 hours. Subsequently, 600 allogenic islets were transplanted via the portal vein into the liver of C57BL/6N diabetic mice. Recipients did not receive any immunosuppressive treatment. To demonstrate the specific mechanism responsible for prolonging graft survival by islet preconditioning, immunohistochemical studies and inflammatory cytokine assays were conducted. Microarrays were employed to comprehensively analyze the genes expressed in untreated or MMC-treated islets that were subsequently cultured and to identify the biological processes that were most affected by islet preconditioning. Results On islet transplantation, the allograft was rejected by all control mice (n = 11), with a mean survival time (MST) of 13.0 ± 8.3 days. Islet conditioning with MMC significantly prolonged islet allograft survival to an MST of 41.4 ± 7.3 days (n = 14) (P < 0.005). Notably, 42.8% of the recipients accepted islet allografts pretreated with MMC for more than 60 days without any immunosuppression. As shown by immunohistochemical studies, compared with untreated grafts, MMC-treated grafts showed a mild inflammatory cell response and minimal infiltration of immunocompetent cells. In an in vitro study, the expression of IL-6, MCP-1, ICAM-1, and IL-1β was significantly decreased in MMC-treated islets compared with untreated islets. Microarray gene expression analysis identified a significant downregulation of annotated functions associated with the movement of cells and revealed significant downregulation of multiple genes encoding proinflammatory mediators with chemotactic activity. Validation studies also showed that MMC-treated islets secreted lower levels of chemoattractants that reduced the immunogenic potential of islets. Conclusion Islet preconditioning with MMC and subsequent culture can prolong graft survival in intraportal islet transplantations by inhibiting the activation of proinflammatory events and by provoking the suppression of chemoattractants that are responsible for inducing unresponsiveness to islet grafts. This simple and promising islet preconditioning method could be an effective strategy inducing a breakthrough in establishing advanced islet transplantation.

Cyanidin-3-O-Glucoside Treatment of Human Islets In Vitro Leads to an Augmentation of Autophagic Flux and a Decreased in Inflammatory Cell Markers

Introduction Islet transplantation is currently being considered as an alternative treatment for Type I Diabetes. Despite recent progress, transplant patients continue to experience a progressive loss of insulin independence for reasons that are not well understood. Cyanidin-3-O-glucoside (C3G), an extract from Chinese bayberry, is an antioxidant that has been shown to be protective in vitro against the cell damage that may lead to transplant loss. In this study, we hypothesized that C3G is protective because of its ability to regulate autophagy; therefore, human islets treated with C3G should show an increased expression of autophagy marker LC3 and a decreased expression of inflammatory markers NLRP3 and IL-1β. Thus, we determined if autophagy is a possible mechanism by which C3G protects islets from damage in vitro. Materials and Methods Human islets (n=4-6) were obtained from the Alberta Diabetes Institute Human Islet Core laboratory and cultured in growth medium with or without C3G for 24 hours. Islets were then treated with human amylin, Aβ1-42, or rapamycin for 24 hours or H2O2 for 2 hours to mimic stresses encountered in the post-transplant environment. Samples of these islets were collected and stained for LC3, NLRP3, or IL-1β in addition to assessing their viability. Marker expression was visualized by immunofluorescence microscopy, and quantification of marker expression was performed using ImageJ software. Results and Discussion Islets treated only with human amylin or Aβ1-42 showed a significant decrease in cell viability compared to untreated islets (p < 0.05). Islets treated with human amylin or Aβ1-42 that received C3G presented a significant increase in cell viability compared to human amylin or Aβ1-42 without C3G (p < 0.05) indicating that C3G can protect human islets from the toxic effect of human amylin and Aβ1-42. When islets were treated with stress-inducing human amylin, Aβ1-42, rapamycin, or H2O2, LC3 expression was decreased, whereas expression of NLRP3 and IL-1β was increased. However, when islets were treated with C3G prior to the addition of a stress-inducing agent, LC3 expression was significantly increased (p ≤ 0.01) and NLRP3 and IL-1β expression was significantly decreased (p ≤ 0.01) compared to the samples not treated with C3G. These results suggest that C3G has beneficial effect on human islets by protecting them from damage induced by stresses encountered in the post-transplant environment. Conclusion Augmentation of autophagic flux is a likely mechanism by which C3G is protective against cellular stress in vitro. Further examination of the involvement of LC3 using siRNA is needed to determine the specific mechanism whereby C3G is protective. Alberta Diabetes Institute and University Hospital Foundation MF Grant (RES0027387).

English

Several studies highlight beneficial effect of glabridin as an anti-hyperglycemic agent. However, the protective mechanisms of glabridin on improvement of pancreatic islets damage due to streptozotocin (STZ) exposure have not been adequately investigated. In the present study, we aimed to elucidate the effects of glabridin on restoration of pancreatic islet architecture, at least in part by restoring collagen I and fibronectin, through an activation of protein kinase Cs (PKCs) and transforming growth factor (TGF)-&beta;1 in STZ-induced pancreatic &beta;-cell injuries. Glabridin (40 mg/kg b.wt./day) was administrated to STZ-exposed rats for 8 weeks. The effects of glabridin were then investigated by measuring body weight, blood glucose level, pancreatic morphology, and protein expressions of PKCs, TGF-&beta;1, collagen I, and fibronectin. The results revealed that glabridin prevented weight loss and significantly decreased blood glucose level. Physical distortion and impairment with irregular outline was clearly observed in untreated STZ islets. Nevertheless, rat islets showed nearly normal architecture with a high &beta;-cell number after glabridin treatment. Glabridin also ameliorated the expression levels of PKCs, TGF-&beta;1, collagen I, and fibronectin in STZ-rat pancreas. These results provide novel evidence that glabridin significantly reduced pancreatic islets abnormalities in STZ-exposed rats and these effects could be associated with its hypoglycemic activity and with restoration of normal levels of PKCs, TGF-&beta;1, collagen I and fibronectin, which could subsequently contribute to maintaining islet structure and function. Key words: Glabridin, pancreas, diabetes mellitus, anti-hyperglycemia, anti-oxidant activity, streptozotocin.

Ex-vivo generation of drug-eluting islets improves transplant outcomes by inhibiting TLR4-Mediated NFkB upregulation

The systemic administration of immunosuppressive and anti-inflammatory drugs is routinely employed in organ transplantation to minimize graft rejection and improve graft survival. Localized drug delivery has the potential to improve transplant outcomes by providing sustained exposure to efficacious drug concentrations while avoiding systemic immunosuppression and off-target effects. Here, we describe the synthesis of a novel prodrug and its direct covalent conjugation to pancreatic islets via a cleavable linker. Post-transplant, linker hydrolysis results in the release of a potent anti-inflammatory antagonist of TLR4, localized to the site of implantation. This covalent islet modification significantly reduces the time and the minimal effective dose of islets necessary to achieve normoglycemia in a murine transplantation model. In streptozotocin-induced diabetic C57BL/6 mice a syngeneic transplant of ∼100 modified islets achieved a 100% cure rate by the end of a 4-week monitoring period, compared to a 0% cure rate for untreated control islets. Overall, this direct prodrug conjugation to islets is well tolerated and preserves their functionality while affording significantly superior transplant outcomes. The development of drug-eluting tissues that deliver sustained and localized doses of small-molecule therapeutics represents a novel pathway for enhancing success in transplantation.

Nano-Encapsulation of Bilirubin in Pluronic F127–Chitosan Improves Uptake in β Cells and Increases Islet Viability and Function after Hypoxic Stress

Pancreatic islet transplantation is the only curative, noninvasive treatment for type 1 diabetes mellitus; however, high rates of cell death in the immediate postimplantation period have limited the success of this procedure. Bilirubin, an endogenous antioxidant, can improve the survival of murine pancreatic allografts during hypoxic stress but has poor solubility in aqueous solutions. We hypothesized that nano-encapsulation of bilirubin in pluronic 127–chitosan nanoparticle bilirubin (nBR) would improve uptake by murine pancreatic islet cells and improve their viability following hypoxic stress. Nano-bilirubin was synthesized, and drug release characteristics were studied in vitro. Cellular uptake of nBR was compared to free bilirubin (fBR) in an insulinoma cell line (INS-R3) model using confocal-like structured illumination microscopy. Next, C57BL/6 mouse islets were treated with concentrations of 0 to 20 μM of nBR, fBR, or empty nanoparticle (eNP), prior to incubation under standard or hypoxic conditions. Islet viability and function were compared between treatment groups. Release of bilirubin was greatest from nBR suspended in protein-rich solution. Increased, selective uptake of nBR by INS-R3 cells was demonstrated. Cell death after hypoxic stress was significantly decreased in murine islets treated with 5 μM nBR (18.5% ± 14.1) compared to untreated islets (33.5% ± 17.5%; P = 0.019), with reduction in central necrosis. Treatment group had a significant effect on glucose stimulation index [SI], (P = 0.0137) and islets treated with 5 μM nBR had the highest SI overall. Delivery of bilirubin using pluronic F127–chitosan NP improves uptake by murine islets compared to fBR and offers dose-dependent protective effects following hypoxic stress.