Islet Recovery Research Articles

Genetic lineage tracing identifies adaptive mechanisms of pancreatic islet β cells in various mouse models of diabetes with distinct age of initiation.

During the pathogenesis of type 1 diabetes (T1D) and type 2 diabetes (T2D), pancreatic islets, especially the β cells, face significant challenges. These insulin-producing cells adopt a regeneration strategy to compensate for the shortage of insulin, but the exact mechanism needs to be defined. High-fat diet (HFD) and streptozotocin (STZ) treatment are well-established models to study islet damage in T2D and T1D respectively. Therefore, we applied these two diabetic mouse models, triggered at different ages, to pursue the cell fate transition of islet β cells. Cre-LoxP systems were used to generate islet cell type-specific (α, β, or δ) green fluorescent protein (GFP)-labeled mice for genetic lineage tracing, thereinto β-cell GFP-labeled mice were tamoxifen induced. Single-cell RNA sequencing (scRNA-seq) was used to investigate the evolutionary trajectories and molecular mechanisms of the GFP-labeled β cells in STZ-treated mice. STZ-induced diabetes caused extensive dedifferentiation of β cells and some of which transdifferentiated into a or δ cells in both youth- and adulthood-initiated mice while this phenomenon was barely observed in HFD models. β cells in HFD mice were expanded via self-replication rather than via transdifferentiation from α or δ cells, in contrast, α or δ cells were induced to transdifferentiate into β cells in STZ-treated mice (both youth- and adulthood-initiated). In addition to the re-dedifferentiation of β cells, it is also highly likely that these "α or δ" cells transdifferentiated from pre-existing β cells could also re-trans-differentiate into insulin-producing β cells and be beneficial to islet recovery. The analysis of ScRNA-seq revealed that several pathways including mitochondrial function, chromatin modification, and remodeling are crucial in the dynamic transition of β cells. Our findings shed light on how islet β cells overcome the deficit of insulin and the molecular mechanism of islet recovery in T1D and T2D pathogenesis.

203.3: Reducing neutral protease dose during islet isolation does not impair islet recovery from pancreases retrieved for autologous use

203.3: Reducing neutral protease dose during islet isolation does not impair islet recovery from pancreases retrieved for autologous use

In Vitro and In Vivo Improvement of Islet Quality and Transplantation Successes following Islet Treatment with Biomaterials in Diabetic Rats.

Loss of islet survival and function, caused by native niche disruption and oxidative stress induction during mechanical and enzymatic isolation, limits the effectiveness of islet transplantation. Reconstitution of islet microenvironment, vascularization, and decreased oxidative stress with biomaterials may improve islet quality and graft outcomes. We investigated effects of two biomaterials, platelet-rich plasma and pancreatic islets homogenate combination on islet recovery and quality by evaluating in vitro islet survival, secretory function, and oxidative stress parameters and assessing in vivo transplantation outcomes. In vitro, islet viability and secretory function of isolated islets were assessed after 24 h and 72 h incubation with biomaterials. Also, oxidative stress markers were measured once after isolation and 24 h after incubation with biomaterials. For evaluating in vivo effects, cultured islets for 24 h were transplanted into subscapular space of diabetic rat kidney, and outcomes were analyzed by measuring serum glucose and insulin concentrations, glucose tolerance test, level of oxidative parameters, and pancreatic gene expression. Treating islets with biomaterials significantly increased their viability and secretory function, reduced MDA level, and elevate SOD and CAT activity. Decreased level of glucose and MDA improved insulin level, increased SOD activity, and also enhanced pdx1 and insulin gene expression in diabetic rats after islet transplantation. Biomaterials used in the present study should be consider as beneficial materials for increasing islet transplantation outcome. These materials may hamper transplantation limitation to some extent.

Effects of a Ganoderma lucidum Proteoglycan on Type 2 Diabetic Rats and the Recovery of Rat Pancreatic Islets.

Type 2 diabetes (T2D) results from both insulin resistance and pancreatic β-cell dysfunction. A natural proteoglycan extracted from Ganoderma lucidum, namely, FYGL, has been demonstrated to be capable of ameliorating insulin resistance in previous work. In this work, a T2D rat model induced by streptozocin (STZ) and a high-fat diet was used to investigate the effects of FYGL on pancreatic functions, and the transcriptomics of the rat pancreas was used to investigate the biological processes (BP) and signal pathways influenced by FYGL on the gene basis. Furthermore, the results of transcriptomics were verified both by histopathological analyses and protein expression. The studies showed that FYGL positively regulated T2D-related BP and signaling pathways and recovered the pancreatic function, therefore ameliorating hyperglycemia and hyperlipidemia in vivo. Importantly, the recovery of the pancreatic function suggested a crucial strategy to radically treat T2D.

Ameliorative Effects of Flavonoids from Platycodon grandiflorus Aerial Parts on Alloxan-Induced Pancreatic Islet Damage in Zebrafish

Platycodon grandiflorus (balloon flower), used as a food reserve as well as in traditional herbal medicine, is known for its multiple beneficial effects. In particular, this plant is widely used as a vegetable in Republic of Korea. We examined the ameliorative effects of P. grandiflorus on alloxan-induced pancreatic islet damage in zebrafish. The aerial part treatment led to a significant recovery in pancreatic islet size and glucose uptake. The efficacy of the aerial part was more potent than that of the root. Eight flavonoids (1-8) were isolated from the aerial part. Structures of two new flavone glycosides, designated dorajiside I (1) and II (2), were elucidated to be luteolin 7-O-α-L-rhamno-pyranosyl (1 → 2)-(6-O-acetyl)-β-D-glucopyranoside and apigenin 7-O-α-L-rhamnopyranosyl (1 → 2)-(6-O-acetyl)-β-D-glucopyranoside, respectively, by spectroscopic analysis. Compounds 1, 3, 4 and 6-8 yielded the recovery of injured pancreatic islets in zebrafish. Among them, compound 7 blocked KATP channels in pancreatic β-cells. Furthermore, compounds 3, 4, 6 and 7 showed significant changes with respect to the mRNA expression of GCK, GCKR, GLIS3 and CDKN2B compared to alloxan-induced zebrafish. In conclusion, the aerial part of P. grandiflorus and its constituents conferred a regenerative effect on injured pancreatic islets.

A bovine milk-derived peptide ameliorates pancreatic β-cell dedifferentiation through PI3K/Akt/FOXO1 signaling in type 2 diabetes.

The lacto-ghrestatin derived nonapeptide (LGP9), a bioactive peptide derived from lacto-ghrestatin in bovine milk with the sequence of LIVTQTMKG, was investigated to determine its effects on islet β-cell dedifferentiation and associated mechanisms in type 2 diabetes mellitus (T2DM). On the animal level, type-2-diabetic (T2D) mice were generated by high-fat-diet (HFD) and streptozocin (STZ). LGP9 was given to T2D mice for four weeks at doses of 1 mg kg-1, 3 mg kg-1, and 9 mg kg-1. A variety of techniques (immunohistochemistry, western blot, QPCR, and ELISA) were employed to evaluate the impact of LGP9 on the diabetic injury. On the cellular level, the pancreatic cell lines, Rin-m5f cells and Min6 cells, were treated with high-glucose (HG) and high-glucose-high-lipid (HG/PA), respectively. The cell models were established to investigate the mechanism of LGP9 treatment on the islet β-cell dedifferentiation. For the mechanism study, the PI3K/Akt/FOXO1 pathway was investigated by inhibiting FOXO1 with its inhibitor and siRNA. Results showed that LGP9 improved the β-cell dedifferentiation, prevented the EMT process, and upregulated the PI3K/Akt/FOXO1 signaling in the pancreas of T2D mice. In addition, LGP9 promoted the structural and functional recovery of pancreatic islets and shielded the liver tissue in T2D mice. From the cellular level data, LGP9 prevented β-cell dedifferentiation and EMT occurrence. To a certain extent, the inhibition of FOXO1 restored PI3K/Akt/FOXO1 pathway activation and prevented β-cell dedifferentiation. In conclusion, these findings suggest that LGP9 ameliorated pancreatic β-cell dedifferentiation via PI3k/Akt/FOXO1 signaling in vivo and in vitro.

Nanowarming of vitrified pancreatic islets as a cryopreservation technology for transplantation.

Biobanking of pancreatic islets for transplantation could solve the shortage of donors, and cryopreservation of vitrified islets is a possible approach. However, a technological barrier is rewarming of large volumes both uniformly and rapidly to prevent ice formation due to devitrification. Here, we describe successful recovery of islets from the vitrified state using a volumetric rewarming technology called "nanowarming," which is inductive heating of magnetic nanoparticles under an alternating magnetic field. Convective warming using a 37°C water bath as the gold standard for rewarming of vitrified samples resulted in a decrease in the viability of mouse islets in large volumes (>1 ml) owing to devitrification caused by slow warming. Nanowarming showed uniform and rapid rewarming of vitrified islets in large volumes. The viability of nanowarmed islets was significantly improved and islets transplanted into streptozotocin-induced diabetic mice successfully lowered serum glucose. The results suggest that nanowarming will lead to a breakthrough in biobanking of islets for transplantation.

P12.06: Effect of Supplementing Taurine to the Developed Lowtemperature Islet Preservation Solution

Introduction: Pancreatic islet transplantation has recently emerged as one of the most promising therapeutic approaches for improving glycemic control in type 1 diabetes patients. However, one of the problems with islet transplantation is that it is impossible to culture the isolated islet for extended periods to while recipients are selected, tested and prepared for surgery. To address this problem, we developed the islet preservation solution, and to improve the function of the preservation solution, we added taurine and conducted a preservation experiment. Taurine is antioxidant activity and it is not a classical free radical scavenger. Therefore, its mechanism remains unclear but it controls osmotic pressure. Our hypothesis is that taurine supplementation can improve islet recovery and enhance islet function after low temperature preservation. Methods: CMRL 1066 medium (Corning) was used as a control group, and a comparison group was prepared some compounds OPTI(Optipharm presevation) solution (with taurine) and OPTI-T solution (without taurine). To confirm the preservation effect of these solutions, we isolated islet from adult Yucatan pig’s pancreas using standard enzymatic digestion (Nordmark) followed by Ficoll purification, these islets were preserved in a humidified tissue culture incubator with preservation solutions and preserved in 4℃ for up to 4 weeks. We compared islet survival in groups of islets. To islet viability and islet functionality, we utilized Glucose Stimulated Insulin Secretion (GSIS) for measuring insulin release, calculated stimulation index (SI) and AO/PI viability staining for during the preservation period. Results: Highest islet recovery was preserved in the group of pig islets cultured with Taurine as compared to the group of islets hypothermically preserved in standard tissue culture media over the 4 week preservation period (p<0.001 using ANOVA). The porcine islet viability/cell number ratio in the OPTI solution maintained a high survival rate for up to 2 weeks (94%). In contrast, porcine islet in OPTI-T solution and CMRL 1066 supplemented media significantly reduced viability after 1 weeks. OPTI solution (with taurine) was significantly higher than OPTI-T solution (without taurine) and CMRL 1066 during 4 weeks after preservation. Glucose stimulation insulin secretion test was performed for functional analysis. As a result, a level of porcine insulin was higher during 2 weeks in the OPTI solution than OPTI-T solution and CMRL 1066. But after 3 weeks preservation, insulin level in high glucose, significantly decreased. Therefore, the viability and functionality of islets were improved by adding taurine to the developed preservation solution. Conclusion: Taurine supplementation has been shown to highest islet recovery, viability and insulin secretion level for 2 weeks. Based on this data, we have performed 2 weeks cold storage of isolated islets for current clinical islet transplantation. Ministry of trade industry and energy, KEIT 20011276, NTIS 1415172664.

A novel second-stage surgical strategy for severely obese patient with pancreatic neuroendocrine tumor: a case report

BackgroundSeverely obese patients can have other diseases requiring surgical treatment. In such patients, bariatric surgeries are considered a precursor to operations targeting the original disease for the purpose of reducing severe perioperative complications. Pancreatic ectopic fat deposition increases pancreas volume (PV) and thickness, which can worsen insulin resistance and islet β cell function. To address this problem, we present a novel two-stage surgical strategy performed on a severely obese patient with pancreatic neuroendocrine tumor (PNET) consisting of laparoscopic sleeve gastrectomy (LSG) as a metabolic surgery followed by laparoscopic spleen-preserving distal pancreatectomy (LSPDP).Case presentationA 56-year-old man was referred to our hospital for further investigation of a pancreatic tumor. His initial body weight and body mass index (BMI) were 94.0 kg and 37.2 kg/m2, respectively. Contrast computed tomography revealed an enhanced tumor measuring 15 mm on the pancreatic body. The pancreas thickness and PV were 32 mm and 148 mL, respectively. An endoscopic ultrasonographic fine needle aspiration identified the tumor as PNET-G1. We first performed LSG, the patient’s body weight and BMI had decreased dramatically to 64.0 kg and 25.3 kg/m2 at 6 months after LSG. The pancreas thickness and PV had also decreased to 17 mm and 99 mL, respectively, with no tumor growth. Since LSG has been shown to reduce the perioperative risk factors of LSPDP, and to improve insulin resistance and recovery of islet β cell function, we performed LSPDP for PNET-G1 as a second-stage surgery. The postoperative course was unremarkable, and the patient was discharged on postoperative day 14 without symptomatic postoperative pancreatic fistula (POPF). He was followed without recurrence or type 2 diabetes (T2D) onset for 6 months after LSPDP.ConclusionsWe present a novel two-stage surgical strategy for a severely obese patient with PNET, consisting of LSG as a metabolic surgery for severe obesity, followed by LSPDP after confirmation of good weight loss and metabolic effects. LSG before pancreatectomy may have a potential to reduce pancreas thickness and recovery of islet β cell function in severely obese patients, thereby reducing the risk of clinically relevant POPF and post-pancreatectomy T2D onset.

Pancreatic islet cryopreservation by vitrification achieves high viability, function, recovery and clinical scalability for transplantation

Pancreatic islet transplantation can cure diabetes but requires accessible, high-quality islets in sufficient quantities. Cryopreservation could solve islet supply chain challenges by enabling quality-controlled banking and pooling of donor islets. Unfortunately, cryopreservation has not succeeded in this objective, as it must simultaneously provide high recovery, viability, function and scalability. Here, we achieve this goal in mouse, porcine, human and human stem cell (SC)-derived beta cell (SC-beta) islets by comprehensive optimization of cryoprotectant agent (CPA) composition, CPA loading and unloading conditions and methods for vitrification and rewarming (VR). Post-VR islet viability, relative to control, was 90.5% for mouse, 92.1% for SC-beta, 87.2% for porcine and 87.4% for human islets, and it remained unchanged for at least 9 months of cryogenic storage. VR islets had normal macroscopic, microscopic, and ultrastructural morphology. Mitochondrial membrane potential and adenosine triphosphate (ATP) levels were slightly reduced, but all other measures of cellular respiration, including oxygen consumption rate (OCR) to produce ATP, were unchanged. VR islets had normal glucose-stimulated insulin secretion (GSIS) function in vitro and in vivo. Porcine and SC-beta islets made insulin in xenotransplant models, and mouse islets tested in a marginal mass syngeneic transplant model cured diabetes in 92% of recipients within 24–48 h after transplant. Excellent glycemic control was seen for 150 days. Finally, our approach processed 2,500 islets with >95% islets recovery at >89% post-thaw viability and can readily be scaled up for higher throughput. These results suggest that cryopreservation can now be used to supply needed islets for improved transplantation outcomes that cure diabetes.

Assessment of Culture/Preservation Conditions of Human Islets for Transplantation

Islet culture before clinical transplantation has been adopted by various centers, but its effect on the survival and function of islets relative to the culture conditions and media needs further assessment. Human islets were cultured or preserved under four different conditions and three media options. Parameters such as recovery, viability, function, islet damage, and gene expressions for markers of hypoxia, and inflammation were assessed after 48-h culture or preservation. Preservation of islets was performed at 4°C in Connaught’s Medical Research Lab (CMRL) and University of Wisconsin (UW) media. Islets were cultured at 22°C, 37°C, and 37°C–22°C in CMRL and PRODO culture media. Islets preserved in UW solution had visually good morphology and exhibited higher recovery with less islet damage compared with the rest of the groups, whereas islets preserved in CMRL at 4°C resulted in poor morphology, recovery, viability, and function compared with the rest of the treatment conditions. Culture at 22°C and 37°C demonstrated an increase in the expression of inflammatory and hypoxia-related genes. In conclusion, islets preserved at 4°C in UW solution showed the best overall outcomes after 48 h compared with islets cultured at 22°C, 37°C, or 37°C–22°C in PRODO. Advancement in islet culture media is warranted to reduce inflammatory gene activation and improve recovery of islets for transplantation.

Necrostatin-1 Supplementation to Islet Tissue Culture Enhances the In-Vitro Development and Graft Function of Young Porcine Islets.

Pre-weaned porcine islets (PPIs) represent an unlimited source for islet transplantation but are functionally immature. We previously showed that necrostatin-1 (Nec-1) immediately after islet isolation enhanced the in vitro development of PPIs. Here, we examined the impact of Nec-1 on the in vivo function of PPIs after transplantation in diabetic mice. PPIs were isolated from pancreata of 8–15-day-old, pre-weaned pigs and cultured in media alone, or supplemented with Nec-1 (100 µM) on day 0 or on day 3 of culture (n = 5 for each group). On day 7, islet recovery, viability, oxygen consumption rate, insulin content, cellular composition, insulin secretion capacity, and transplant outcomes were evaluated. While islet viability and oxygen consumption rate remained high throughout 7-day tissue culture, Nec-1 supplementation on day 3 significantly improved islet recovery, insulin content, endocrine composition, GLUT2 expression, differentiation potential, proliferation capacity of endocrine cells, and insulin secretion. Adding Nec-1 on day 3 of tissue culture enhanced the islet recovery, proportion of delta cells, beta-cell differentiation and proliferation, and stimulation index. In vivo, this leads to shorter times to normoglycemia, better glycemic control, and higher circulating insulin. Our findings identify the novel time-dependent effects of Nec-1 supplementation on porcine islet quantity and quality prior to transplantation.

Characteristics of the New Insulin-Resistant Zebrafish Model.

Insulin resistance, which occurs when insulin levels are sufficiently high over a prolonged period, causing the cells to fail to respond normally to the hormone. As a system for insulin resistance and diabetes drug development, insulin-resistant rodent models have been clearly established, but there is a limitation to high-throughput drug screening. Recently, zebrafish have been identified as an excellent system for drug discovery and identification of therapeutic targets, but studies on insulin resistance models have not been extensively performed. Therefore, we aimed to make a rapid insulin-resistant zebrafish model that complements the existing rodent models. To establish this model, zebrafish were treated with 10 μM insulin for 48 h. This model showed characteristics of insulin-resistant disease such as damaged pancreatic islets. Then we confirmed the recovery of the pancreatic islets after pioglitazone treatment. In addition, it was found that insulin-resistant drugs have as significant an effect in zebrafish as in humans, and these results proved the value of the zebrafish insulin resistance model for drug selection. In addition, RNA sequencing was performed to elucidate the mechanism involved. KEGG pathway enrichment analysis of differentially expressed genes showed that insulin resistance altered gene expression due to the MAPK signaling and calcium signaling pathways. This model demonstrates the utility of the zebrafish model for drug testing and drug discovery in insulin resistance and diabetes.

Progress in individualizing autologous islet isolation techniques for pediatric islet autotransplantation after total pancreatectomy in children for chronic pancreatitis.

Total pancreatectomy with islet autotransplantation is performed to treat chronic pancreatitis in children. Successful islet isolation must address the challenges of severe pancreatic fibrosis and young donor age. We have progressively introduced modifications to optimize enzymatic and mechanical dissociation of the pancreas during islet isolation. We evaluated 2 islet isolation metrics in 138 children-digest islet equivalents per gram pancreas tissue (IEQ/g) and digest IEQ per kilogram body weight (IEQ/kg), using multiple regression to adjust for key disease and patient features. Islet yield at digest had an average 4569 (standard deviation 2949) islet equivalent (IEQ)/g and 4946 (4009) IEQ/kg, with 59.1% embedded in exocrine tissue. Cases with very low yield (<2000 IEQ/g or IEQ/kg) have decreased substantially over time, 6.8% and 9.1%, respectively, in the most recent tertile of time compared to 19.2% and 23.4% in the middle and 34.1% and 36.4% in the oldest tertile. IEQ/g and IEQ/kg adjusted for patient and disease factors improved in consistency and yield in the modern era. Minimal mechanical disruption during digestion, warm enzymatic digestion using enzyme collagenase:NP activity ratio<10:1, coupled with extended distension and trimming time during islet isolation of younger and fibrotic pediatric pancreases, gave increased islet yield with improved patient outcomes.

A surrogate of Roux-en-Y gastric bypass (the enterogastro anastomosis surgery) regulates multiple beta-cell pathways during resolution of diabetes in ob/ob mice.

BackgroundBariatric surgery is an effective treatment for type 2 diabetes. Early post-surgical enhancement of insulin secretion is key for diabetes remission. The full complement of mechanisms responsible for improved pancreatic beta cell functionality after bariatric surgery is still unclear. Our aim was to identify pathways, evident in the islet transcriptome, that characterize the adaptive response to bariatric surgery independently of body weight changes.MethodsWe performed entero-gastro-anastomosis (EGA) with pyloric ligature in leptin-deficient ob/ob mice as a surrogate of Roux-en-Y gastric bypass (RYGB) in humans. Multiple approaches such as determination of glucose tolerance, GLP-1 and insulin secretion, whole body insulin sensitivity, ex vivo glucose-stimulated insulin secretion (GSIS) and functional multicellular Ca2+-imaging, profiling of mRNA and of miRNA expression were utilized to identify significant biological processes involved in pancreatic islet recovery.FindingsEGA resolved diabetes, increased pancreatic insulin content and GSIS despite a persistent increase in fat mass, systemic and intra-islet inflammation, and lipotoxicity. Surgery differentially regulated 193 genes in the islet, most of which were involved in the regulation of glucose metabolism, insulin secretion, calcium signaling or beta cell viability, and these were normalized alongside changes in glucose metabolism, intracellular Ca2+ dynamics and the threshold for GSIS. Furthermore, 27 islet miRNAs were differentially regulated, four of them hubs in a miRNA-gene interaction network and four others part of a blood signature of diabetes resolution in ob/ob mice and in humans.InterpretationTaken together, our data highlight novel miRNA-gene interactions in the pancreatic islet during the resolution of diabetes after bariatric surgery that form part of a blood signature of diabetes reversal.FundingEuropean Union's Horizon 2020 research and innovation programme via the Innovative Medicines Initiative 2 Joint Undertaking (RHAPSODY), INSERM, Société Francophone du Diabète, Institut Benjamin Delessert, Wellcome Trust Investigator Award (212625/Z/18/Z), MRC Programme grants (MR/R022259/1, MR/J0003042/1, MR/L020149/1), Diabetes UK (BDA/11/0004210, BDA/15/0005275, BDA 16/0005485) project grants, National Science Foundation (310030–188447), Fondation de l'Avenir.

Optimal Time to Ship Human Islets Post Tissue Culture to Maximize Islet Recovery

Access to functional high-quality pancreatic human islets is critical to advance diabetes research. The Integrated Islet Distribution Program (IIDP), a major source for human islet distribution for over 15 years, conducted a study to evaluate the most advantageous times to ship islets postisolation to maximize islet recovery. For the evaluation, three experienced IIDP Islet Isolation Centers each provided samples from five human islet isolations, shipping 10,000 islet equivalents (IEQ) at four different time periods postislet isolation (no 37°C culture and shipped within 0 to 18 hours; or held in 37°C culture for 18 to 42, 48 to 96, or 144 to 192 hours). A central evaluation center compared samples for islet quantity, quality, and viability for each experimental condition preshipment and postshipment, as well as post 37°C culture 18 to 24 hours after shipment receipt. Additional evaluations included measures of functional potency by static glucose-stimulated insulin release (GSIR), represented as a stimulation index. Comparing the results of the four preshipment holding periods, the greatest IEQ loss postshipment occurred with the shortest preshipment times. Similar patterns emerged when comparing preshipment to postculture losses. In vitro islet function (GSIR) was not adversely impacted by increased tissue culture time. These data indicate that allowing time for islet recovery postisolation, prior to shipping, yields less islet loss during shipment without decreasing islet function.

Regulation of c-Jun NH2-Terminal Kinase for Islet Transplantation.

Islet transplantation has been demonstrated to provide superior glycemic control with reduced glucose lability and hypoglycemic events compared with standard insulin therapy. However, the insulin independence rate after islet transplantation from one donor pancreas has remained low. The low frequency of islet grafting is dependent on poor islet recovery from donors and early islet loss during the first hours following grafting. The reduction in islet mass during pancreas preservation, islet isolation, and islet transplantation leads to β-cell death by apoptosis and the prerecruitment of intracellular death signaling pathways, such as c-Jun NH2-terminal kinase (JNK), which is one of the stress groups of mitogen-activated protein kinases (MAPKs). In this review, we show some of the most recent contributions to the advancement of knowledge of the JNK pathway and several possibilities for the treatment of diabetes using JNK inhibitors.

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.

Characterization of chelator-mediated recovery of pancreatic islets from barium-stabilized alginate microcapsules.

Islet recovery from within alginate-based microcapsules is necessary for certain analytical assays like flow cytometry; however, this technology has not been widely characterized. In this study, we explore the ability of EDTA, EGTA, and sodium citrate to induce reverse alginate polymerization via chelation and assess the toxicity of each chelator on pancreatic islets. EDTA, EGTA, and sodium citrate were used to dissolve single-layered Ba2+ alginate encapsulated islets and the rate of capsule breakdown calculated from analysis of imaging data. The effect of chelator exposure on islet viability and recovery was assessed using flow cytometry, while glucose-stimulated insulin release (GSIR) assay was used to measure effects on islet function. EGTA demonstrated the most rapid microcapsule dissolving rate followed by EDTA and sodium citrate. Islet recovery was significantly better when encapsulated islets were treated with EDTA than EGTA and Na+ citrate. A decrease in viability and increase in apoptotic cells were observed when encapsulated islets were treated with Na+ citrate compared to islets treated with EDTA and EGTA. Islets treated with EDTA and EGTA demonstrated comparable stimulation index values to non-treated control. Conversely, islets treated with Na+ citrate exhibited significantly decreased SI values compared to control. All chelator groups showed significantly lower insulin secretion than non-treated islets. Islet recovery from alginate microcapsule is possible using common chelators like Na+ citrate, EDTA, and EGTA. Chelation of encapsulated islets using EDTA demonstrated the most efficient dissolving capabilities with the least toxicity toward islet recovery and health.

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.