Improve Islet Function Research Articles

307.7: 3D Bioprinting of Functional Islets With Adipose-derived Stromal Cells in an Alginate/Nanocellulose Scaffold

Islet isolation procedure destroys islet vasculature and extra cellular matrix molecules, which can negatively affect islet function post transplantation. Developing scaffold that favors islet micro-environment is one solution to preserve islet function post isolation. 3D bioprinting of islets using hydrogel-based bioinks has been reported previously. However, modifying bioinks and presence of supporting cells such as adipose-derived stromal cells (ASCs) can improve survival and reduce cellular stress in islet scaffolds. We designed an implantable multi-layered 3D bioprinted scaffold for human or mouse islets -/+ human ASCs (1.2 ×106 ASCs/scaffold). Cells were embedded in alginate/nanocellulose bioink and printed with INKREDIBLE 3D Bioprinter from CELLINK AB. Micro-CT was used to study the distribution of islets inside scaffolds. In vitro cell viability and function were assessed using FDA/PI staining and glucose stimulated insulin secretion on day 1, 8 and 14 post print. The level of secreted human- and mouse-specific cytokines, IP-10, MCP-1 and GRO-α were measured on day 1, 8 and 14 post print. Islet scaffolds -/+ ASCs were transplanted intraperitoneal (IP) in diabetic immune-compromised mouse model and followed for 60 days post transplantation. We printed 100 islets/scaffold for in vitro and 400 islets/scaffold for in vivo studies. In vitro viability analysis revealed viable islets and ASCs throughout the studies. However, scaffolds with human and mouse islets + ASCs showed improved insulin secretion in response to glucose compared to the islet alone group on day 1, 8 and 14 post print (selected data points for human islets-day 8: islet+ASC, basal insulin 2387 ± 1262, stimulated insulin 4312 ± 606.8, recovery insulin post stimulation 1637 ± 100.1 pmol/L, * p< 0.05 vs stimulated insulin. Islet alone, basal insulin 2358 ± 887.9, stimulated insulin 1149 ± 352.6, recovery insulin post stimulation 999.0 ± 267.7 pmol/L, n=4). This is followed by significant reduction in the level of both human and mouse IP-10, MCP-1 and GRO-α in islet+ASC group compared to the islet alone group on day 1, 8 and 14 post print. IP transplantation of mouse islets -/+ ASCs to diabetic mice normalizes random blood glucose in both groups starting from day 6 post transplantation for islet+ASC group and day 40 for islet alone group. We detected c-peptide in both groups on day 3 and 13 post transplantation. However, at termination of the studies, the level of c-peptide was significantly higher in islet+ASC compared to the islet alone group (islet+ASC 94.01 ± 14.47 vs. islet alone 27.08 ± 18.93, p<0.05). This study presents a successful multi-layered scaffold design for islets and ASCs. Presence of ASCs in the scaffold can create a favorable micro-environment for the islets, resulting in reduced islet loss and improved islet function.

Explore the Effect and Target of Liraglutide on Islet Function in Type 2 Diabetic Rats by miRNA Omics Technology.

PurposeTo analyze the effect and potential therapeutic targets of liraglutide in type 2 diabetes through miRNA expression profiling.MethodsTen of 30 SPF Wistar rats, males at 4 weeks old, were randomly selected as the control group and given conventional feed, the other rats adopted high-sugar and high-fat diet combined with an intraperitoneal injection of streptozotocin to establish a T2DM model. One unsuccessful rat was excluded, and the remaining rats were randomized to the model and the liraglutide group. Liraglutide group was subcutaneously injected with liraglutide 0.11 mg/kg for 8 weeks. The biochemical indicators and staining HE were detected. The expression of miRNA in pancreatic tissue was detected by miRNA sequencing. The intersection of miRNA difference was used to predict the target gene, then functional enrichment was performed to identify its possible biological functions and signal transduction paths. Finally, qRT-PCR was used to verify the results.ResultsCompared to the model group, the level of fasting blood glucose (FBG), glucagon and insulin resistance index (HOMA-IR) in the liraglutide group were significantly decreased, fasting insulin (FINS) and insulin sensitivity index (ISI) were increased. Nine differential miRNAs (miR-135a-5p, miR-144-5p, miR-21-3p, miR-215, miR-451-5p, miR-486, miR-122-5p, miR-181d-5p and miR-345-5p) were identified at the intersection through two miRNA sequencing. A total of 3359 related target gene predictions were obtained. GO and pathway analyses demonstrated that differentially expressed genes were closely related to cell proliferation, angiogenesis, and proteolysis. Significant signaling pathways included PI signaling system, autophagy, FoxO and HIF-1 signaling pathway.ConclusionLiraglutide could improve islet function by regulating nine miRNAs, and the related signaling pathways included PI signaling system, autophagy, FoxO and HIF-1 signaling pathway. Our study provided the basis and direction for further exploring the molecular mechanism of liraglutide on T2DM.

Mesenchymal stem cell-conditioned medium alleviates high fat-induced hyperglucagonemia via miR-181a-5p and its target PTEN/AKT signaling

Backgroundα-cell dysregulation gives rise to fasting and postprandial hyperglycemia in type 2 diabetes mellitus(T2DM). Administration of Mesenchymal stem cells (MSCs) or their conditioned medium can improve islet function and enhance insulin secretion. However, studies showing the direct effect of MSCs on islet α-cell dysfunction are limited. MethodsIn this study, we used high-fat diet (HFD)-induced mice and α-cell line exposure to palmitate (PA) to determine the effects of bone marrow-derived MSC-conditioned medium (bmMSC-CM) on glucagon secretion. Plasma and supernatant glucagon were detected by enzyme-linked immunosorbent assay(ELISA). To investigate the potential signaling pathways, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), AKT and phosphorylated AKT(p-AKT) were assessed by Western blotting. ResultsIn vivo, bmMSC-CM infusion improved the glucose and insulin tolerance and protected against HFD-induced hyperglycemia and hyperglucagonemia. Meanwhile, bmMSC-CM infusion ameliorated HFD-induced islet hypertrophy and decreased α- and β-cell area. Consistently, in vitro, glucagon secretion from α-cells or primary islets was inhibited by bmMSC-CM, accompanied by reduction of intracellular PTEN expression and restoration of AKT signaling. Previous studies and the TargetScan database indicate that miR-181a and its target PTEN play vital roles in ameliorating α-cell dysfunction. We observed that miR-181a-5p was highly expressed in BM-MSCs but prominently lower in αTC1-6 cells. Overexpression or downregulation of miR-181a-5p respectively alleviated or aggravated glucagon secretion in αTC1-6 cells via the PTEN/AKT signaling pathway. ConclusionsOur observations suggest that MSC-derived miR-181a-5p mitigates glucagon secretion of α-cells by regulating PTEN/AKT signaling, which provides novel evidence demonstrating the potential for MSCs in treating T2DM.

Preservation of pancreas in the University of Wisconsin solution supplemented with AP39 reduces reactive oxygen species production and improves islet graft function

Ischemia-reperfusion injury (IRI) results in increased rates of delayed graft function and early graft loss. It has recently been reported that hydrogen sulfide (H2 S) protects organ grafts against prolonged IRI. Here, we investigated whether the preservation of pancreas in University of Wisconsin (UW) solution supplemented with AP39, which is a mitochondrial-targeted H2 S donor, protected pancreatic islets against IRI and improved islet function. Porcine pancreata were preserved in the UW solution with AP39 (UW + AP39) or the vehicle (UW) for 18h, followed by islet isolation. The islet yields before and after purification were significantly higher in the UW + AP39 group than in the UW group. The islets isolated from the pancreas preserved in UW + AP39 exhibited significantly decreased levels of reactive oxygen species (ROS) production and a significantly increased mitochondrial membrane potential as compared to the islets isolated from the pancreas preserved in the vehicle. We found that the pancreas preserved in UW + AP39 improved the outcome of islet transplantation in streptozotocin-induced diabetic mice. These results suggest that the preservation of pancreas in UW + AP39 protects the islet grafts against IRI and could thus serve as a novel clinical strategy for improving islet transplantation outcomes.

Protection factors used to improve in vivo islet function

The number of preclinical studies that use drugs or endogenous factors for various targetable sites aiming to improve islet function and engraftment has recently gained more interest. Several molecules have been proposed to support and augment the functionality of islets. Here, we review five different classes of beta cell protection factors, classified as antioxidants, anti-inflammatory, anti-apoptotic, immunomodulatory, vascularization enhancers, and promoter of beta cell proliferation and function and their effect on islet grafting and function after transplantation in animal models of type 1 diabetes.

Exenatide promotes cholesterol efflux in pancreatic tissue of obese diabetic rats

To study the effect of exenatide on the expression of ABCA1 and cholesterol metabolism in the pancreas of obese diabetic rats. Twenty-four normal male SD rats and 18 obese diabetic rats (induced by high-fat feeding and STZ injection) were both divided equally into 2 groups for injections of saline or exenatide. After treatment for a week, the expression of ABCA1, cholesterol metabolism, and islet function of the rats were examined using real-time PCR, Western blotting, oil red O staining, cholesterol content determination, and HE staining. The expressions of ABCA1 at both mRNA and protein levels in pancreatic tissue were significantly lower in obese diabetic rats than in normal SD rats. The obese diabetic rats showed obvious lipid deposition and increased cholesterol content in the pancreatic tissue with significantly reduced islet volume and structural changes (P < 0.05); exenatide treatment of the diabetic rats significantly up-regulated ABCA1 expression, reduced lipid deposition and cholesterol content in pancreatic tissue, and increased number and volume of the islets, which presented with more orderly alignment (P < 0.05). Obese diabetic rats have lowered ABCA1 expression, cholesterol efflux block, and cholesterol accumulation in the pancreatic tissue. Exenatide can up-regulate ABCA1 expression and promote cholesterol efflux to reduce cholesterol content in the pancreatic tissue and improve islet function in obese diabetic rats.

Mesenchymal Stromal Cell-Mediated Immune Regulation: A Promising Remedy in the Therapy of Type 2 Diabetes Mellitus

Type 2 diabetes mellitus (T2DM) is a major threat to global public health, with increasing prevalence as well as high morbidity and mortality, to which immune dysfunction has been recognized as a crucial contributor. Mesenchymal stromal cells (MSCs), obtained from various sources and possessing potent immunomodulatory abilities, have displayed great therapeutic potential for T2DM. Interestingly, the immunomodulatory capabilities of MSCs are endowed and plastic. Among the multiple mechanisms involved in MSC-mediated immune regulation, the paracrine effects of MSCs have attracted much attention. Of note, extracellular vesicles (EVs), an important component of MSC secretome, have emerged as pivotal mediators of their immunoregulatory effects. Particularly, the necrobiology of MSCs, especially apoptosis, has recently been revealed to affect their immunomodulatory functions in vivo. In specific, a variety of preclinical studies have demonstrated the beneficial effects of MSCs on improving islet function and ameliorating insulin resistance. More importantly, clinical trials have further uncovered the therapeutic potential of MSCs for T2DM. In this review, we outline current knowledge regarding the plasticity and underlying mechanisms of MSC-mediated immune modulation, focusing on the paracrine effects. We also summarize the applications of MSC-based therapies for T2DM in both preclinical studies and clinical trials, with particular emphasis on the modulation of immune system.

Morus alba leaves ethanol extract protects pancreatic islet cells against dysfunction and death by inducing autophagy in type 2 diabetes

Protection of pancreatic islet cells against dysfunction or death by regulating autophagy is considered to be an effective method for treatment of type 2 diabetes mellitus (T2DM). Morus alba leaves (mulberry leaves), a popular herbal medicine, have been used for prevention of T2DM since ancient times. This study aimed to clarify whether Morus alba leaves ethanol extract (MLE) could protect islet cells in vivo and in vitro by regulating autophagy in T2DM, and explore the possible mechanism of action. The main chemical constituents in MLE were analyzed by HPLC. The T2DM rat model was induced via high-fat diet combined with peritoneal injection of low-dose streptozotocin, and MLE was administered by oral gavage. Fasting blood glucose (FBG) and plasma insulin were measured, and homeostatic model assessment of β cell function (HOMA-β) and insulin resistance (HOMA-IR) were determined. The histomorphology of pancreas islets was evaluated by haematoxylin and eosin staining. In palmitic acid (PA)-stressed INS-1 rat insulinoma cells, cell viability was assayed by an MTT method. Expression of the autophagy-related proteins LC3 I/II, p62, p-AMPK and p-mTOR in islet tissues and INS-1 cells was evaluated by western blotting or immunohistochemistry analysis. The four main chemical constituents in MLE were identified as chlorogenic acid, rutin, isoquercitrin and quercitrin. MLE ameliorated hyperglycemia, insulin resistance and dyslipidemia of T2DM rats with prominent therapeutic effect. Further study indicated that MLE observably improved islet function, alleviated islet injury of T2DM rats, and inhibited PA-induced INS-1 cell death. On the other hand, MLE significantly induced autophagy in islet cells both in vivo and in vitro, and autophagy inhibitors abolished its therapeutic effect on T2DM rats and protective effect on islet cells. Apart from this, MLE markedly activated the AMPK/mTOR pathway in INS-1 cells, and the AMPK inhibitor prevented the autophagy induction ability of MLE. Together, MLE could protect islet cells against dysfunction and death by inducing AMPK/mTOR-mediated autophagy in T2DM, and these findings provide a new perspective for understanding the treatment mechanism of Morus alba leaves against T2DM.

Administration of metrnl delays the onset of diabetes in non-obese diabetic mice.

Type 1 diabetes is a chronic metabolic disease characterized by hyperglycemia due to progressive destruction of pancreatic beta cells via autoimmune attack. Meteorin-like protein (metrnl) is a secreted protein homologous to the neurotrophin metrn and it is induced after exercise in the skeletal muscle. In our paper published previously, we showed that the serum level of metrnl was significantly correlated with the lipid profile, glucose profile and insulin resistance. In this experiment, we asked whether intravenous administration of metrnl could delay the onset of diabetes in non-obese diabetic (NOD) mice. 4-week-old NOD mice were injected intravenously with metrnl. Blood glucose levels were measured weekly. Insulitis scoring, intraperitoneal glucose tolerance test, adoptive T cell transfer, flow cytometry analysis and real-time PCR were performed to investigate the underlying mechanism. The results showed that intravenous administration of metrnl delayed the onset of diabetes in NOD mice. Histology of pancreas showed a decreased infiltration of leukocytes, which was in association with augmentation of regulatory T cells, suppression of autoreactive T cells and altered cytokine secretion. To sum up, the present study showed that intravenous administration of metrnl ameliorated islet lymphocyte infiltration and modulated immune cell responses, raising the possibility that it might be beneficial in improving islet function clinically.

Multi-Tissue Acceleration of the Mitochondrial Phosphoenolpyruvate Cycle Improves Whole-Body Metabolic Health

The mitochondrial GTP (mtGTP)-dependent phosphoenolpyruvate (PEP) cycle couples mitochondrial PEPCK (PCK2) to pyruvate kinase (PK) in the liver and pancreatic islets to regulate glucose homeostasis. Here, small molecule PK activators accelerated the PEP cycle to improve islet function, as well as metabolic homeostasis, in preclinical rodent models of diabetes. In contrast, treatment with a PK activator did not improve insulin secretion in pck2-/- mice. Unlike other clinical secretagogues, PK activation enhanced insulin secretion but also had higher insulin content and markers of differentiation. In addition to improving insulin secretion, acute PK activation short-circuited gluconeogenesis to reduce endogenous glucose production while accelerating red blood cell glucose turnover. Four-week delivery of a PK activator invivo remodeled PK phosphorylation, reduced liver fat, and improved hepatic and peripheral insulin sensitivity in HFD-fed rats. These data provide a preclinical rationale for PK activation to accelerate the PEP cycle to improve metabolic homeostasis and insulin sensitivity.

Exosomes derived from human mesenchymal stem cells preserve mouse islet survival and insulin secretion function.

Islet cell death and loss of function after isolation and before transplantation is considered a key barrier to successful islet transplantation outcomes. Mesenchymal stem cells (MSCs) have been used to protect isolated islets owing to their paracrine potential partially through the secretion of vascular endothelial growth factor (VEGF). The paracrine functions of MSCs are also mediated, at least in part, by the release of extracellular vesicles including exosomes. In the present study, we examined (i) the effect of exosomes from human MSCs on the survival and function of isolated mouse islets and (ii) whether exosomes contain VEGF and the potential impact of exosomal VEGF on the survival of mouse islets. Isolated mouse islets were cultured for three days with MSC-derived exosomes (MSC-Exo), MSCs, or MSC-conditioned media without exosomes (MSC-CM-without-Exo). We investigated the effects of the exosomes, MSCs, and conditioned media on islet viability, apoptosis and function. Besides the expression of apoptotic and pro-survival genes, the production of human and mouse VEGF proteins was evaluated. The MSCs and MSC-Exo, but not the MSC-CM-without-Exo, significantly decreased the percentage of apoptotic cells and increased islet viability following the downregulation of pro-apoptotic genes and the upregulation of pro-survival factors, as well as the promotion of insulin secretion. Human VEGF was observed in the isolated exosomes, and the gene expression and protein production of mouse VEGF significantly increased in islets cultured with MSC-Exo. MSC-derived exosomes are as efficient as parent MSCs for mitigating cell death and improving islet survival and function. This cytoprotective effect was probably mediated by VEGF transfer, suggesting a pivotal strategy for ameliorating islet transplantation outcomes.

Angiotensin-(1-7) Improves Islet Function in a Rat Model of Streptozotocin- Induced Diabetes Mellitus by Up-Regulating the Expression of Pdx1/Glut2.

To observe the effects of angiotensin-(1-7) (Ang-(1-7)) on glucose metabolism, islet function and insulin resistance in a rat model of streptozotocin-induced diabetes mellitus (DM) and investigate its mechanism. Thirty-four male Wistar rats were randomly divided into 3 groups: control group, which was fed a standard diet, DM group, high-fat diet and injected with streptozotocin, and Ang-(1-7) group receiving an injection of streptozotocin followed by Ang-(1-7) treatment. Blood glucose level, fasting serum Ang II and insulin levels, and homeostasis model assessment of insulin resistance (HOMA-IR) were measured. The pancreases were collected for histological examination, protein and gene expression analysis. Compared with the control group, fasting blood glucose, serum angiotensin II level, and HOMA-IR value increased, while serum insulin level decreased in the DM group. Moreover, islet structure was damaged, β cells were irregularly arranged, the cytoplasm was loose in the DM group. Expressions of Pancreatic duodenal homeobox-1 (Pdx1), glucose transporter-2 (Glut2) and glucokinase (Gk) were significantly decreased in the DM group compared with the control group. However, the DM-associated changes were dramatically reversed following Ang-(1-7) treatment. Ang-(1-7) protects against streptozotocin-induced DM through the improvement of insulin secretion, insulin resistance and islet fibrosis, which is associated with the upregulation of Pdx1, Glut2 and Gk expressions.

Addition of olive oil to diet of rats with mild pre-gestational diabetes impacts offspring β-cell development.

Maternal diabetes impairs fetal development and increases the risk of metabolic diseases in the offspring. Previously, we demonstrated that maternal dietary supplementation with 6% of olive oil prevents diabetes-induced embryo and fetal defects, in part, through the activation of peroxisome proliferator-activated receptors (PPARs). In this study, we examined the effects of this diet on neonatal and adult pancreatic development in male and female offspring of mothers affected with pre-gestational diabetes. A mild diabetic model was developed by injecting neonatal rats with streptozotocin (90 mg/kg). During pregnancy, these dams were fed a chow diet supplemented or not with 6% olive oil. Offspring pancreata was examined at day 2 and 5 months of age by immunohistochemistry followed by morphometric analysis to determine number of islets, α and β cell clusters and β-cell mass. At 5 months, male offspring of diabetic mothers had reduced β-cell mass that was prevented by maternal supplementation with olive oil. PPARα and PPARγ were localized mainly in α cells and PPARβ/δ in both α and β cells. Although Pparβ/δ and Pparγ RNA expression showed reduction in 5-month-old male offspring of diabetic rats, Pparβ/δ expression returned to control levels after olive-oil supplementation. Interestingly, in vitro exposure to oleic acid (major component of olive oil) and natural PPAR agonists such as LTB4, CPC and 15dPGJ2 also significantly increased expression of all Ppars in αTC1-6 cells. However, only oleic acid and 15dPGJ2 increased insulin and Pdx-1 expression in INS-1E cells suggesting a protective role in β-cells. Olive oil may be considered a dietary supplement to improve islet function in offspring of affected mothers with pre-gestational diabetes.

2076-P: Increased Gap Junction Coupling in the Islet of Langerhans following Caloric Restriction

Intercellular communication mediated by connexin 36 gap junction channels coordinate and regulate the dynamics of islet calcium oscillations and insulin secretion. Impaired gap junction coupling occurs in prediabetic mice under a high fat diet. Dietary interventions, such as calorie restriction, can regain weight and modulate insulin secretion. However, it is unknown whether caloric restriction can recover impaired gap junctions coupling and calcium regulation in islets of mice fed a high fat diet. Here, we tested if caloric restriction can protect against obesity-induced decreases in connexin36 coupling and altered islet function. We used C57BL6 mice under a normal diet (Envigo 2020X), or a high fat diet (HFD) (Envigo TD.03584) which causes prediabetes after three months. HFD mice were also submitted to 40% caloric restriction (2g/day) for one month. We performed intraperitoneal glucose tolerance tests, insulin tolerance tests, in vivo insulin level measurements. In isolated islets we measured glucose-stimulated insulin secretion, islet Ca2+ dynamics, metabolic activity, along with gap junction permeability. We observed that caloric restriction improved glucose tolerance (p=0.002), insulin sensitivity (p=0.001), and islet function. This included normalization glucose-induced insulin secretion (p=0.01), enhanced Ca2+ oscillation amplitude (p=0.002) and greater coordination (p=0.04). These improvements correlated with restoration in gap junction permeability (p=0.02). Caloric restriction also promoted a slight increase in NAD(P)H increase by elevated glucose (p=0.04) indicating improved metabolic activity and viability of these cells. We conclude that the decline in gap junction coupling. mediated by connexin 36 in islets that occurs in diet-induced obesity, is improved by short term caloric restriction. This improvement contributes to improved islet function. Disclosure M.C. Amaral: None. V. Kravets: None. J.M. Dwulet: None. N.L. Farnsworth: None. R.A. Piscopio: None. W. Schleicher: None. J. Miranda: None. R.K. Benninger: None. Funding JDRF (5-CDA-2014-198-A-N); National Institutes of Health (R01DK102950, R01DK106412 to R.K.B.); Fundação de Amparo à Pesquisa do Estado de São Paulo (2018/04536-6)

Artemether Regulates Metaflammation to Improve Glycolipid Metabolism in db/db Mice.

BackgroundArtemether, a commonly used artemisinin derivative, has been shown to possess potential antidiabetic activities. However, only limited information is available on the mechanisms of artemether in type 2 diabetes. Therefore, in this study, we examined some of the possible mechanisms of artemether (ATM) upon glycolipid metabolism in the db/db mouse model of diabetes.Materials and MethodsMale C57BL/KsJ-db/db and C57BL/KsJ-db/+ mice at 4 weeks of age were divided into four groups (N=6/group): (1) NC (normal control – db/+ mice, 1% methylcellulose, intragastric administration), (2) DM (diabetic model – db/db mice, 1% methylcellulose, intragastric administration), (3) ATM 100 (DM + 100 mg/kg of artemether) and (4) ATM 200 (DM + 200 mg/kg of artemether). A number of assays related to diabetes were then performed following a 4-week period of these treatments.ResultsArtemether at both doses significantly reduced rates of weight gain and fasting blood glucose levels, improved islet function and insulin resistance and reduced serum lipid levels to varying degrees in db/db mice. Artemether exerted a positive effect on islet vacuolar degeneration and hepatic steatosis, and increased expressions of AMP-activated protein kinase, glucose transporter 4 and Insulin receptor β protein in the liver of these db/db mice. With the use of liver protein chip detection, we found that artemether significantly improved the immune microenvironment, down-regulated the expression of inflammatory factors and activated the cytokine-mediated signaling pathway through cytokine–cytokine receptor interactions.ConclusionArtemether may regulate glycolipid metabolism in db/db mice by improving the immune microenvironment. The results of this study provide important new information that can serve as the foundation for future research into the use of artemether as a means to improve glycolipid metabolism.

Inhibition of the prostaglandin D2\u2013GPR44/DP2 axis improves human islet survival and function

Aims/hypothesisInflammatory signals and increased prostaglandin synthesis play a role during the development of diabetes. The prostaglandin D2 (PGD2) receptor, GPR44/DP2, is highly expressed in human islets and activation of the pathway results in impaired insulin secretion. The role of GPR44 activation on islet function and survival rate during chronic hyperglycaemic conditions is not known. In this study, we investigate GPR44 inhibition by using a selective GPR44 antagonist (AZ8154) in human islets both in vitro and in vivo in diabetic mice transplanted with human islets.MethodsHuman islets were exposed to PGD2 or proinflammatory cytokines in vitro to investigate the effect of GPR44 inhibition on islet survival rate. In addition, the molecular mechanisms of GPR44 inhibition were investigated in human islets exposed to high concentrations of glucose (HG) and to IL-1β. For the in vivo part of the study, human islets were transplanted under the kidney capsule of immunodeficient diabetic mice and treated with 6, 60 or 100 mg/kg per day of a GPR44 antagonist starting from the transplantation day until day 4 (short-term study) or day 17 (long-term study) post transplantation. IVGTT was performed on mice at day 10 and day 15 post transplantation. After termination of the study, metabolic variables, circulating human proinflammatory cytokines, and hepatocyte growth factor (HGF) were analysed in the grafted human islets.ResultsPGD2 or proinflammatory cytokines induced apoptosis in human islets whereas GPR44 inhibition reversed this effect. GPR44 inhibition antagonised the reduction in glucose-stimulated insulin secretion induced by HG and IL-1β in human islets. This was accompanied by activation of the Akt–glycogen synthase kinase 3β signalling pathway together with phosphorylation and inactivation of forkhead box O-1and upregulation of pancreatic and duodenal homeobox-1 and HGF. Administration of the GPR44 antagonist for up to 17 days to diabetic mice transplanted with a marginal number of human islets resulted in reduced fasting blood glucose and lower glucose excursions during IVGTT. Improved glucose regulation was supported by increased human C-peptide levels compared with the vehicle group at day 4 and throughout the treatment period. GPR44 inhibition reduced plasma levels of TNF-α and growth-regulated oncogene-α/chemokine (C-X-C motif) ligand 1 and increased the levels of HGF in human islets.Conclusions/interpretationInhibition of GPR44 in human islets has the potential to improve islet function and survival rate under inflammatory and hyperglycaemic stress. This may have implications for better survival rate of islets following transplantation.

A Collagen Based Cryogel Bioscaffold that Generates Oxygen for Islet Transplantation.

The aim of this work was to develop, characterize and test a novel 3D bioscaffold matrix which can accommodate pancreatic islets and provide them with a continuous, controlled and steady source of oxygen to prevent hypoxia-induced damage following transplantation. Hence, we made a collagen based cryogel bioscaffold which incorporated calcium peroxide (CPO) into its matrix. The optimal concentration of CPO integrated into bioscaffolds was 0.25wt.% and this generated oxygen at 0.21±0.02mM/day (day 1), 0.19±0.01mM/day (day 6), 0.13±0.03mM/day (day 14), and 0.14±0.02mM/day (day 21). Accordingly, islets seeded into cryogel-CPO bioscaffolds had a significantly higher viability and function compared to islets seeded into cryogel alone bioscaffolds or islets cultured alone on traditional cell culture plates; these findings were supported by data from quantitative computational modelling. When syngeneic islets were transplanted into the epididymal fat pad (EFP) of diabetic mice, our cryogel-0.25wt.%CPO bioscaffold improved islet function with diabetic animals re-establishing glycemic control. Mice transplanted with cryogel-0.25wt.%CPO bioscaffolds showed faster responses to intraperitoneal glucose injections and had a higher level of insulin content in their EFP compared to those transplanted with islets alone (P<0.05). Biodegradability studies predicted that our cryogel-CPO bioscaffolds will have long-lasting biostability for approximately 5 years (biodegradation rate: 16.00±0.65%/year). Long term implantation studies (i.e. 6 months) showed that our cryogel-CPO bioscaffold is biocompatible and integrated into the surrounding fat tissue with minimal adverse tissue reaction; this was further supported by no change in blood parameters (i.e. electrolyte, metabolic, chemistry and liver panels). Our novel oxygen-generating bioscaffold (i.e. cryogel-0.25wt.%CPO) therefore provides a biostable and biocompatible 3D microenvironment for islets which can facilitate islet survival and function at extra-hepatic sites of transplantation.

Optimizing beta cell function through mesenchymal stromal cell-mediated mitochondria transfer.

Pretransplant islet culture is associated with the loss of islet cell mass and insulin secretory function. Insulin secretion from islet β‐cells is primarily controlled by mitochondrial ATP generation in response to elevations in extracellular glucose. Coculture of islets with mesenchymal stromal cells (MSCs) improves islet insulin secretory function in vitro, which correlates with superior islet graft function in vivo. This study aimed to determine whether the improved islet function is associated with mitochondrial transfer from MSCs to cocultured islets. We have demonstrated mitochondrial transfer from human adipose MSCs to human islet β‐cells in coculture. Fluorescence imaging showed that mitochondrial transfer occurs, at least partially, through tunneling nanotube (TNT)‐like structures. The extent of mitochondrial transfer to clinically relevant human islets was greater than that to experimental mouse islets. Human islets are subjected to more extreme cellular stressors than mouse islets, which may induce “danger signals” for MSCs, initiating the donation of MSC‐derived mitochondria to human islet β‐cells. Our observations of increased MSC‐mediated mitochondria transfer to hypoxia‐exposed mouse islets are consistent with this and suggest that MSCs are most effective in supporting the secretory function of compromised β‐cells. Ensuring optimal MSC‐derived mitochondria transfer in preculture and/or cotransplantation strategies could be used to maximize the therapeutic efficacy of MSCs, thus enabling the more widespread application of clinical islet transplantation.

Novel cell-permeable p38-MAPK inhibitor efficiently prevents porcine islet apoptosis and improves islet graft function.

During islet transplantation, mitogen-activated protein kinase (MAPK) p38 is preferentially activated in response to the isolation of islets and the associated inflammation. Although therapeutic effects of p38 inhibitors are expected, the clinical application of small-molecule inhibitors of p38 is not recommended because of their serious adverse effects on the liver and central nervous system. Here we designed peptides to inhibit p38, which were derived from the sites on p38 that mediate binding to proteins such as MAPK kinases. Peptide 11R-p38I110 significantly inhibited the activation of p38. To evaluate the effects of 11R-p38I110 , porcine islets were incubated with 10µmol/L 11R-p38I110 or a mutant form designated 11R-mp38I110 . After islet transplantation, blood glucose levels reached the normoglycemic range in 58.3% and 0% of diabetic mice treated with 11R-p38I110 or 11R-mp38I110 , respectively. These data suggest that 11R-p38I110 inhibited islet apoptosis and improved islet function. Peptide p38I110 is a noncompetitive inhibitor of ATP and targets a unique docking site. Therefore, 11R-p38I110 specifically inhibits p38 activation, which may avoid the adverse effects that have discouraged the clinical use of small-molecule inhibitors of p38. Moreover, our methodology to design "peptide inhibitors" could be used to design other inhibitors derived from the binding sites of proteins.

Translational assessment of a genetic engineering methodology to improve islet function for transplantation.

BackgroundThe functional quality of insulin-secreting islet beta cells is a major factor determining the outcome of clinical transplantations for diabetes. It is therefore of importance to develop methodological strategies aiming at optimizing islet cell function prior to transplantation. In this study we propose a synthetic biology approach to genetically engineer cellular signalling pathways in islet cells.MethodsWe established a novel procedure to modify islet beta cell function by combining adenovirus-mediated transduction with reaggregation of islet cells into pseudoislets. As a proof-of-concept for the genetic engineering of islets prior to transplantation, this methodology was applied to increase the expression of the V1b receptor specifically in insulin-secreting beta cells. The functional outcomes were assessed in vitro and in vivo following transplantation into the anterior chamber of the eye.FindingsPseudoislets produced from mouse dissociated islet cells displayed basic functions similar to intact native islets in terms of glucose induced intracellular signalling and insulin release, and after transplantation were properly vascularized and contributed to blood glucose homeostasis. The synthetic amplification of the V1b receptor signalling in beta cells successfully modulated pseudoislet function in vitro. Finally, in vivo responses of these pseudoislet grafts to vasopressin allowed evaluation of the potential benefits of this approach in regenerative medicine.InterpretationThese results are promising first steps towards the generation of high-quality islets and suggest synthetic biology as an important tool in future clinical islet transplantations. Moreover, the presented methodology might serve as a useful research strategy to dissect cellular signalling mechanisms of relevance for optimal islet function.