Intercellular contacts affect secretion and biosynthesis of pancreatic islet cells.

Cell protein biosynthesis is regulated by different factors, but implication of intercellular contacts on alpha and beta cell protein biosyntheses activity has not been yet investigated. Islet cell biosynthetic activity is essential in regulating not only the hormonal reserve within cells but also in renewing all the proteins involved in the control of secretion. Here we aimed to assess whether intercellular interactions affected similarly secretion and protein biosynthesis of rat alpha and beta cells. Insulin and glucagon secretion were analyzed by ELISA or reverse hemolytic plaque assay, and protein biosynthesis evaluated at single cell level using bioorthogonal noncanonical amino acid tagging. Regarding beta cells, we showed a positive correlation between insulin secretion and protein biosynthesis. We also observed that homologous contacts increased both activities at low or moderate glucose concentrations. By contrast, at high glucose concentration, homologous contacts increased insulin secretion and not protein biosynthesis. In addition, heterogeneous contacts between beta and alpha cells had no impact on insulin secretion and protein biosynthesis. Regarding alpha cells, we showed that when they were in contact with beta cells, they increased their glucagon secretion in response to a drop of glucose concentration, but, on the other hand, they decreased their protein biosynthesis under any glucose concentrations. Altogether, these results emphasize the role of intercellular contacts on the function of islet cells, showing that intercellular contacts increased protein biosynthesis in beta cells, except at high glucose, and decreased protein biosynthesis in alpha cells even when glucagon secretion is stimulated.

Identification of newly synthetized proteins by mass spectrometry to understand palmitate-induced early cellular changes in pancreatic islets.

Obesity and lipid metabolism dysregulation are often associated with insulin resistance, and can lead to type 2 diabetes. However, mechanisms linking insulin resistance, high levels of plasma free fatty acids (FFA), and ß cell failure remain unclear. The aim of this work was to search for proteins whose synthesis was modified by a short exposure to FFA. This could help in the future to identify molecular mechanisms underlying islet dysfunction in the presence of FFA. Therefore, we assessed by mass spectrometry de novo protein synthesis of freshly isolated rat islets after palmitate short exposure. Quantitative proteome and secretome analyses were performed by combining metabolic incorporation of azidohomoalanine (AHA) and pulse labeling with stable isotope labeling by amino acids in cell culture (SILAC). We showed that pancreatic islets, in response to 4-h exposure to palmitate, increased the synthesis of ribosomal proteins and proteins of the cytoskeleton, and increased their secretion of proteins involved in insulin synthesis and insulin secretion, as well as insulin itself. First, these results show that de novo protein quantification analysis by LC-MS/MS is a useful method to investigate cellular modifications induced by FFA on pancreatic islets. Also, these results show that short exposure to palmitate increases the expression of ribosomal proteins and proteins involved in insulin secretion, and it remains to be determined if these effects are responsible or linked to the harmful effect of palmitate on ß cells.NEW & NOTEWORTHY These results show that pancreatic rat islets cultured with palmitate mainly increase synthesis of ribosomal proteins and some proteins of the cytoskeleton. They also show a significant increase of secreted proteins involved in insulin synthesis and insulin secretion, as well as insulin itself. These data provide information to understand the mechanisms of ß cell failure induced by lipotoxicity via the identification of all newly synthesized proteins in islets in response to short-term exposure to palmitate.

Failure mode and effect analysis in human islet isolation: from the theoretical to the practical risk.

This study aimed to assess the global mapping risk of human islet isolation, using a failure mode and effect analysis (FMEA), and highlight the impact of quality assurance procedures on the risk level of criticality. Risks were scored using the risk priority number (RPN) scoring method. The risk level of criticality was made based on RPN and led to risk classification (low to critical). A raw risk analysis and a risk control analysis (with control means and quality assurance performance) were undertaken. The process of human islet isolation was divided into 11 steps, and 230 risks were identified. Analysis of the highest RPN of each of the 11 steps showed that the 4 highest risks were related to the pancreas digestion and islet purification stages. After implementation of reduction measures and controls, critical and severe risks were reduced by 3-fold and by 2-fold, respectively, so that 90% of risks could be considered as low to moderate. FMEA has proven to be a powerful approach for the identification of weaknesses in the islet isolation processes. The results demonstrated the importance of staff qualification and continuous training and supported the contribution of the quality assurance system to risk reduction.

Biosynthetic Activity Differs Between Islet Cell Types and in Beta Cells Is Modulated by Glucose and Not by Secretion.

A correct biosynthetic activity is thought to be essential for the long-term function and survival of islet cells in culture and possibly also after islet transplantation. Compared to the secretory activity, biosynthetic activity has been poorly studied in pancreatic islet cells. Here we aimed to assess biosynthetic activity at the single cell level to investigate if protein synthesis is dependent on secretagogues and increased as a consequence of hormonal secretion. Biosynthetic activity in rat islet cells was studied at the single cell level using O-propargyl-puromycin (OPP) that incorporates into newly translated proteins and chemically ligates to a fluorescent dye by "click" reaction. Heterogeneous biosynthetic activity was observed between the four islet cell types, with delta cells showing the higher relative protein biosynthesis. Beta cells protein biosynthesis was increased in response to glucose while 3-isobutyl-1-methylxanthine and phorbol-12-myristate-13-acetate, 2 drugs known to stimulate insulin secretion, had no similar effect on protein biosynthesis. However, after several hours of secretion, protein biosynthesis remained high even when cells were challenged to basal conditions. These results suggest that mechanisms regulating secretion and biosynthesis in islet cells are different, with glucose directly triggering beta cells protein biosynthesis, independently of insulin secretion. Furthermore, this OPP labeling approach is a promising method to identify newly synthesized proteins under various physiological and pathological conditions.

Impact of ischemia time on islet isolation success and posttransplantation outcomes: A retrospective study of 452 pancreas isolations.

Many variables impact islet isolation, including pancreas ischemia time. The ischemia time upper limit that should be respected to avoid a negative impact on the isolation outcome is not well defined. We have performed a retrospective analysis of all islet isolations in our center between 2008 and 2018. Total ischemia time, cold ischemia time, and organ removal time were analyzed. Isolation success was defined as an islet yield ≥200 000 IEQ. Of the 452 pancreases included, 288 (64%) were successfully isolated. Probability of isolation success showed a significant decrease after 8 hours of total ischemia time, 7 hours of cold ischemia time, and 80 minutes of organ removal time. Although we observed an impact of ischemia time on islet yield, a probability of isolation success of 50% was still present even when total ischemia time exceeds 12 hours. Posttransplantation clinical outcomes were assessed in 32 recipients and no significant difference was found regardless of ischemia time. These data indicate that although shorter ischemia times are associated with better islet isolation outcomes, total ischemia time >12 hours can provide excellent results in appropriately selected donors.

Engineering of Primary Pancreatic Islet Cell Spheroids for Three-dimensional Culture or Transplantation: A Methodological Comparative Study.

Three-dimensional (3D) cell culture by engineering spheroids has gained increasing attention in recent years because of the potential advantages of such systems over conventional two-dimensional (2D) tissue culture. Benefits include the ability of 3D to provide a more physiologically relevant environment, for the generation of uniform, size-controlled spheroids with organ-like microarchitecture and morphology. In recent years, different techniques have been described for the generation of cellular spheroids. Here, we have compared the efficiency of four different methods of islet cell aggregation. Rat pancreatic islets were dissociated into single cells before reaggregation. Spheroids were generated either by (i) self-aggregation in nonadherent petri dishes, (ii) in 3D hanging drop culture, (iii) in agarose microwell plates or (iv) using the Sphericalplate 5D™. Generated spheroids consisted of 250 cells, except for the self-aggregation method, where the number of cells per spheroid cannot be controlled. Cell function and morphology were assessed by glucose stimulated insulin secretion (GSIS) test and histology, respectively. The quantity of material, labor intensity, and time necessary for spheroid production were compared between the different techniques. Results were also compared with native islets. Native islets and self-aggregated spheroids showed an important heterogeneity in terms of size and shape and were larger than spheroids generated with the other methods. Spheroids generated in hanging drops, in the Sphericalplate 5D™, and in agarose microwell plates were homogeneous, with well-defined round shape and a mean diameter of 90 µm. GSIS results showed improved insulin secretion in response to glucose in comparison with native islets and self-aggregated spheroids. Spheroids can be generated using different techniques and each of them present advantages and inconveniences. For islet cell aggregation, we recommend, based on our results, to use the hanging drop technique, the agarose microwell plates, or the Sphericalplate 5D™ depending on the experiments, the latter being the only option available for large-scale spheroids production.

NLRP3 Inflammasome is Activated in Rat Pancreatic Islets by Transplantation and Hypoxia.

Hypoxia, IL-1ß production and oxidative stress are involved in islet graft dysfunction and destruction. However, the link between these events has not yet been determined in transplanted islets. The goal of this study was to determine whether NLRP3 inflammasome is responsible for IL-1ß production and if it is activated by hypoxia-induced oxidative stress in transplanted islets. Rat islets were transplanted under the kidney capsule of immunodeficient mice. At different times post-transplantation, blood samples were collected and islet grafts harvested. Rat islets were also incubated in vitro either under normoxia or hypoxia for 24 h, in the absence or presence of inhibitors of NLRP3 inflammasome (CASP1 inhibitor) or oxidative stress (NAC). NLRP3, CASP1, IL1B, BBC3 pro-apoptotic and BCL2 anti-apoptotic genes in transplanted and in vitro incubated islets were then studied using real time PCR. IL-1ß released in the blood and in the supernatant was quantified by ELISA. Cell death was analysed by propidium iodide and Annexin-V staining. NLRP3, CASP1 and BBC3 in transplanted rat islets and IL-1ß in blood transiently increased during the first days after transplantation. In islets incubated under hypoxia, NRLP3, IL1B and CASP1 and IL-1ß released in supernatant increased compared to islets incubated under normoxia. These effects were prevented by the inhibition of NLRP3 inflammasome by CASP1 or oxidative stress by NAC. However, these inhibitors did not prevent hypoxia-induced rat islet death. These data show that NLRP3 inflammasome in rat islets is transiently activated after their transplantation and induced through oxidative stress in vitro. However, NRLP3 inflammasome inhibition does not protect islet cells against hypoxia.

Shielding islets with human amniotic epithelial cells enhances islet engraftment and revascularization in a murine diabetes model.

Hypoxia is a major cause of considerable islet loss during the early posttransplant period. Here, we investigate whether shielding islets with human amniotic epithelial cells (hAECs), which possess anti-inflammatory and regenerative properties, improves islet engraftment and survival. Shielded islets were generated on agarose microwells by mixing rat islets (RIs) or human islets (HI) and hAECs (100 hAECs/IEQ). Islet secretory function and viability were assessed after culture in hypoxia (1% O2 ) or normoxia (21% O2 ) in vitro. In vivo function was evaluated after transplant under the kidney capsule of diabetic immunodeficient mice. Graft morphology and vascularization were evaluated by immunohistochemistry. Both shielded RIs and HIs show higher viability and increased glucose-stimulated insulin secretion after exposure to hypoxia in vitro compared with control islets. Transplant of shielded islets results in considerably earlier normoglycemia and vascularization, an enhanced glucose tolerance, and a higher ß cell mass. Our results show that hAECs have a clear cytoprotective effect against hypoxic damages in vitro. This strategy improves ß cell mass engraftment and islet revascularization, leading to an improved capacity of islets to reverse hyperglycemia, and could be rapidly applicable in the clinical situation seeing that the modification to HIs are minor.

NLRP3 inflammasome is expressed and regulated in human islets.

NRLP3 inflammasome is a protein complex involved in the maturation of IL1ß. In the onset of type 1 diabetes as well as in islet transplantation, IL-1ß is one of the cytokines involved in the recruitment of immune cells in islets and eventually in islet destruction. Whether IL-1ß is produced by islet cells is still under debate and NLRP3 inflammasome-dependent IL-1ß production has not been yet determined in human islets. The aim of this study was to determine the expression and the regulation of the NRLP3 inflammasome in human islets. Human islets were stimulated with LPS and successively with ATP (LPS + ATP) in the presence or absence of the inflammasome inhibitor glyburide. Islets were also incubated in hypoxic or normoxic conditions for 24 h in the presence or absence of glyburide. Then, IL1B and NLRP3 expression was studied by real time PCR, protein expression by western blot, protein localization by immunofluorescence and protein secretion by ELISA. LPS + ATP increased gene expression of NRLP3 and IL1B. Glyburide partially prevented this effect. IL-1ß protein was localized in ß and non-ß cells. Moreover, LPS + ATP increased IL-1ß protein expression and production, which were prevented by glyburide. Hypoxia increased gene expression of NRLP3 and IL1B and induced IL-1ß and caspase-1 production. Finally, hypoxia-induced cell death which was not prevented by inhibition of NLRP3 inflammasome. NRLP3 inflammasome is expressed and plays a role in IL-1ß production by human islets. By contrast, NRLP3 inflammasome activation is not involved in islet cell death induced by hypoxia.

LRH-1 agonism favours an immune-islet dialogue which protects against diabetes mellitus.

Type 1 diabetes mellitus (T1DM) is due to the selective destruction of islet beta cells by immune cells. Current therapies focused on repressing the immune attack or stimulating beta cell regeneration still have limited clinical efficacy. Therefore, it is timely to identify innovative targets to dampen the immune process, while promoting beta cell survival and function. Liver receptor homologue-1 (LRH-1) is a nuclear receptor that represses inflammation in digestive organs, and protects pancreatic islets against apoptosis. Here, we show that BL001, a small LRH-1 agonist, impedes hyperglycemia progression and the immune-dependent inflammation of pancreas in murine models of T1DM, and beta cell apoptosis in islets of type 2 diabetic patients, while increasing beta cell mass and insulin secretion. Thus, we suggest that LRH-1 agonism favors a dialogue between immune and islet cells, which could be druggable to protect against diabetes mellitus.

Multicenter Assessment of Animal-free Collagenase AF-1 for Human Islet Isolation.

Animal-free (AF) SERVA Collagenase AF-1 and Neutral Protease (NP) AF GMP Grade have recently become available for human islet isolation. This report describes the initial experiences of 3 different islet transplant centers. Thirty-four human pancreases were digested using 1 vial of the 6 different lots of Collagenase AF-1 (2,000-2,583 PZ-U/vial) supplemented with 4 different lots of NP AF in a range of 50 to 160 DMC-U per pancreas. Isolation, culture, and quality assessment were performed using standard techniques as previously described. All data are presented as mean ± standard error of the mean (SEM). Variability of pancreas weight was associated with a wide range of collagenase and NP activities, ranging from 12.7 to 46.6 PZ-U/g (26.0 ± 1.5 PZ-U/g) and 0.4 to 3.0 DMC-U/g (1.5 ± 0.1 DMC-U/g), respectively. Postpurification islet yield was 296,494 ± 33,620 islet equivalents (IEQ) equivalent to 3,274 ± 450 IEQ/g with a purity of 55.9% ± 3.2%. Quality assessment performed after 2 to 4 d of culture demonstrated a viability of 88.1% ± 1.5% and a stimulation index of 3.7 ± 0.7. Eighteen of the 34 preparations were transplanted into type 1 diabetic patients equivalent to a transplantation rate of 52.9%. Six preparations, which were infused into patients as first transplant, could be analyzed and increased the fasting C-peptide level from 0.11 ± 0.08 pretransplant to 1.23 ± 0.24 and 2.27 ± 0.31 ng/mL 3 and 6 mo posttransplant ( P < 0.05), respectively. Insulin requirements were simultaneously reduced at the same time from 39.2 ± 3.8 IU/d before transplantation to 10.8 ± 4.1 and 4.0 ± 2.3 IU/d, after 3 and 6 mo posttransplant ( P < 0.05), respectively. This study demonstrates the efficiency of AF SERVA Collagenase AF-1 and NP AF for clinical islet isolation and transplantation. The new plant-based production process makes these products a safe new option for the islet field.

Asymmetrical distribution of δ and PP cells in human pancreatic islets.

The aim of this study was to evaluate the location of PP and δ cells in relation to the vascularization within human pancreatic islets. To this end, pancreas sections were analysed by immunofluorescence using antibodies against endocrine islet and endothelial cells. Staining in different islet areas corresponding to islet cells adjacent or not to peripheral or central vascular channels was quantified by computerized morphometry. As results, α, PP and δ cells were preferentially found adjacent to vessels. In contrast to α cells, which were evenly distributed between islet periphery and intraislet vascular channels, PP and δ cells had asymmetric and opposite distributions: PP staining was higher and somatostatin staining was lower in the islet periphery than in the area around intraislet vascular channels. Additionally, frequencies of PP and δ cells were negatively correlated in the islets. No difference was observed between islets from the head and the tail of the pancreas, and from type 2 diabetic and non-diabetic donors. In conclusion, the distribution of δ cells differs from that of PP cells in human islets, suggesting that vessels at the periphery and at the centre of islets drain different hormonal cocktails.

Cell rearrangement in transplanted human islets.

The major feature of the human pancreatic islet architecture is the organization of endocrine cells into clusters comprising central ß cells and peripheral α cells surrounded by vasculature. To have an insight into the mechanisms that govern this unique islet architecture, islet cells were isolated, and reaggregation of α and ß cells into islet-like structures (pseudoislets) after culture or transplantation into mice was studied by immunohistology. The pseudoislets formed in culture displayed an unusual cell arrangement, contrasting with the transplanted pseudoislets, which exhibited a cell arrangement similar to that observed in native pancreatic islet subunits. The pattern of revascularization and the distribution of extracellular matrix around transplanted pseudoislets were alike to those observed in native pancreatic islets. This organization of transplanted pseudoislets occurred also when revascularization was abolished by treating mice with an anti-VEGF antibody, but not when contact with extracellular matrix was prevented by encapsulation of pseudoislets within alginate hydrogel. These results indicate that the maintenance of islet cell arrangement is dependent on in vivo features such as extracellular matrix but independent of vascularization.

A Simple High Efficiency Intra-Islet Transduction Protocol Using Lentiviral Vectors.

Successful normalization of blood glucose in patients transplanted with pancreatic islets isolated from cadaveric donors established the proof-of-concept that Type 1 Diabetes Mellitus is a curable disease. Nonetheless, major caveats to the widespread use of this cell therapy approach have been the shortage of islets combined with the low viability and functional rates subsequent to transplantation. Gene therapy targeted to enhance survival and performance prior to transplantation could offer a feasible approach to circumvent these issues and sustain a durable functional ß-cell mass in vivo. However, efficient and safe delivery of nucleic acids to intact islet remains a challenging task. Here we describe a simple and easy-to-use lentiviral transduction protocol that allows the transduction of approximately 80 % of mouse and human islet cells while preserving islet architecture, metabolic function and glucose-dependent stimulation of insulin secretion. Our protocol will facilitate to fully determine the potential of gene expression modulation of therapeutically promising targets in entire pancreatic islets for xenotransplantation purposes.

Cadherin engagement improves insulin secretion of single human ß-cells.

The aim of this study was to assess whether cadherin-mediated adhesion of human islet cells was affected by insulin secretagogues and explore the role of cadherins in the secretory activity of ß-cells. Experiments were carried out with single islet cells adherent to chimeric proteins made of functional E-, N-, or P-cadherin ectodomains fused to the Fc fragment of immunoglobulin (E-cad/Fc, N-cad/Fc, and P-cad/Fc) and immobilized on an inert substrate. We observed that cadherin expression in islet cells was not affected by insulin secretagogues. Adhesion tests showed that islet cells attached to N-cad/Fc and E-cad/Fc acquired, in a time- and secretagogue-dependent manner, a spreading form that was inhibited by blocking cadherin antibodies. By reverse hemolytic plaque assay, we showed that glucose-stimulated insulin secretion of single ß-cells was increased by N-cad/Fc and E-cad/Fc adhesion compared with control. In the presence of E-cad/Fc and after glucose stimulation, we showed that total insulin secretion was six times higher in spreading ß-cells compared with round ß-cells. Furthermore, cadherin-mediated adhesion induced an asymmetric distribution of cortical actin in ß-cells. Our results demonstrate that adhesion of ß-cells to E- and N-cadherins is regulated by insulin secretagogues and that E- and N-cadherin engagement promotes stimulated insulin secretion.

Enhancement of islet engraftment and achievement of long-term islet allograft survival by Toll-like receptor 4 blockade.

BACKGROUND: Toll-like receptors are key players in sterile inflammation phenomena and can link the innate and adaptive immune systems by enhancing graft immunogenicity. They are also considered mediators of types 1 and 2 diabetes development. The aim of the present study was to assess the role of Toll-like receptor-4 (TLR4) in mediating the inflammatory and immune responses to pancreatic islets, thereby promoting inflammatory destruction and immune rejection of islet grafts. METHODS: Experiments were conducted in murine and human in vitro systems and in vivo murine islet transplant models, using species-specific anti-TLR4 monoclonal antibodies. In vitro, mixed lymphocyte-islet reaction experiments were performed to assess T-cell activation and proliferation. In vivo, both a syngeneic (B6-to-B6) marginal mass islet transplant model to assess the impact of TLR4 blockade on islet engraftment and an allogeneic (DBA1-to-B6) model were used. RESULTS: In vitro TLR4 blockade decreased lipopolysaccharide-mediated ß-cell apoptosis and T-cell activation and proliferation against allogeneic islets. In vivo, TLR4 blockade resulted in significantly better syngeneic marginal mass islet engraftment and in indefinite allogeneic islet graft survival. Tolerance was not observed because donor-specific skin graft rechallenge in nonrejecting animals resulted in rejection of both skin and islets, but without accelerated rejection as compared to naive animals. CONCLUSION: Taken together, our data indicate that TLR4 blockade leads to a significant improvement of syngeneic islet engraftment and of allogeneic islet graft survival. A mechanism of graft accommodation with concurrent inhibition of donor-specific immune memory is likely to be involved.

Survival of free and encapsulated human and rat islet xenografts transplanted into the mouse bone marrow.

Bone marrow was recently proposed as an alternative and potentially immune-privileged site for pancreatic islet transplantation. The aim of the present study was to assess the survival and rejection mechanisms of free and encapsulated xenogeneic islets transplanted into the medullary cavity of the femur, or under the kidney capsule of streptozotocin-induced diabetic C57BL/6 mice. The median survival of free rat islets transplanted into the bone marrow or under the kidney capsule was 9 and 14 days, respectively, whereas that of free human islets was shorter, 7 days (bone marrow) and 10 days (kidney capsule). Infiltrating CD8+ T cells and redistributed CD4+ T cells, and macrophages were detected around the transplanted islets in bone sections. Recipient mouse splenocytes proliferated in response to donor rat stimulator cells. One month after transplantation under both kidney capsule or into bone marrow, encapsulated rat islets had induced a similar degree of fibrotic reaction and still contained insulin positive cells. In conclusion, we successfully established a small animal model for xenogeneic islet transplantation into the bone marrow. The rejection of xenogeneic islets was associated with local and systemic T cell responses and macrophage recruitment. Although there was no evidence for immune-privilege, the bone marrow may represent a feasible site for encapsulated xenogeneic islet transplantation.

Quantification of islet loss and graft functionality during immune rejection by 3-tesla MRI in a rat model.

BACKGROUND: Because metabolic markers are not suitable for early diagnosis of islet graft dysfunction, magnetic resonance imaging (MRI) has been used to study islets that were labeled pretransplantation with superparamagnetic iron oxide nanoparticles. However, the relation between graft functionality assessed by glycemia, and MRI signal remains unclear. METHODS: We transplanted hyperglycemic rats intraportally with 2500 ferucarbotran-labeled syngeneic (n=10) or allogeneic (n=12) islet equivalents or normoglycemic rats with 5000 xenogeneic human islet equivalents. Images were acquired on a clinical 3-Tesla MRI scanner. RESULTS: When rejection occurred on days 4 and 8 in xenogeneic and allogeneic recipients, 60% (57-68) and 55% (46-73) of the initial signal remained compared to 93% (71-104) and 82% (59-90) in syngeneic controls (P=0.006 and 0.03). With a cutoff value of 84% on day 4 for the diagnosis of allogeneic rejection, sensitivity of 91% and specificity of 70% were obtained. Based on MRI signal on day 4, treatment with antilymphocytic serum from day 4 allowed graft rescue in 75% of recipients. CONCLUSIONS: In this model, MRI of pretransplantation superparamagnetic iron oxide nanoparticle-labeled islet grafts allows timely diagnosis of immune rejection.

MicroRNAs contribute to compensatory ß cell expansion during pregnancy and obesity.

Pregnancy and obesity are frequently associated with diminished insulin sensitivity, which is normally compensated for by an expansion of the functional ß cell mass that prevents chronic hyperglycemia and development of diabetes mellitus. The molecular basis underlying compensatory ß cell mass expansion is largely unknown. We found in rodents that ß cell mass expansion during pregnancy and obesity is associated with changes in the expression of several islet microRNAs, including miR-338-3p. In isolated pancreatic islets, we recapitulated the decreased miR-338-3p level observed in gestation and obesity by activating the G protein-coupled estrogen receptor GPR30 and the glucagon-like peptide 1 (GLP1) receptor. Blockade of miR-338-3p in ß cells using specific anti-miR molecules mimicked gene expression changes occurring during ß cell mass expansion and resulted in increased proliferation and improved survival both in vitro and in vivo. These findings point to a major role for miR-338-3p in compensatory ß cell mass expansion occurring under different insulin resistance states.

Comparative impact on islet isolation and transplant outcome of the preservation solutions Institut Georges Lopez-1, University of Wisconsin, and Celsior.

BACKGROUND: Institut Georges Lopez-1 (IGL-1) is a preservation solution similar to University of Wisconsin (UW) with reversed Na/K contents. In this study, we assessed the impact of IGL-1, UW, and Celsior (CS) solutions on islet isolation and transplant outcome. METHODS: We retrospectively analyzed 376 islet isolations from pancreases flushed and transported with IGL-1 (n=95), UW (n=204), or CS (n=77). We determined isolation outcome and ß-cell function in vitro. Transplanted patients were divided into three groups depending on preservation solution of pancreas, and islet graft function was assessed by decrease in daily insulin needs, C-peptide/glucose ratios, ß-scores, and transplant estimated function at 1- and 6-month follow-up. RESULTS: IGL-1, UW, and CS groups were similar according to donor age, body mass index, and pancreas weight. There was no difference in islet yields between the three groups. Success rates, transplant rates, ß-cell secretory function, and viability were similar for all three groups. We observed no difference in decreased insulin needs, C-peptide glucose ratios, ß-scores, and transplant estimated function at 1- and 6-month follow-up between IGL-1, UW, and CS groups. CONCLUSIONS: Our study shows that IGL-1 is equivalent to UW or CS solutions for pancreas perfusion and cold storage before islet isolation and transplantation.