Inhibition of the prostaglandin D2-GPR44/DP2 axis improves human islet survival and function.

AIMS/HYPOTHESIS: Inflammatory 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. METHODS: Human 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-1ß. 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. RESULTS: PGD2 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-1ß in human islets. This was accompanied by activation of the Akt-glycogen synthase kinase 3ß 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/INTERPRETATION: Inhibition 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.

Cost and clinical outcome of islet transplantation in Norway 2010-2015.

Islet transplantation is a minimally invasive ß-cell replacement strategy. Islet transplantation is a reimbursed treatment in Norway. Here, we summarize the cost and clinical outcome of 31 islet transplantations performed at Oslo University Hospital (OUS) from January 2010 to June 2015. Patients were retrospectively divided into three groups. Thirteen patients received either one or two islet transplantation alone (ITA), while five patients received islet transplantation after previous solid organ transplantation. For the group receiving 2 ITA, Kaplan-Meier estimates show an insulin independence of 20% more than 4 years after their last transplantation. An estimated 70% maintain at least partial graft function, defined as fasting C-peptide >0.1 nmol L-1 , and 47% maintain a HbA1c below 6.5% or 2 percent points lower than before ITA. For all groups combined, we estimate that 44% of the patients have a 50% reduction in insulin requirement 4 years after the initial islet transplantation. The average cost for an islet transplantation procedure was 347 297±60 588 NOK, or 35 424±6182 EUR, of which isolation expenses represent 34%. We hereby add to the common pool of growing experience with islet transplantation and also describe the cost of the treatment at our center.

Graft function 1 year after pregnancy in an islet-transplanted patient.

Pancreatic islet transplantation is a treatment option for patients with type 1 diabetes (T1D), but pregnancy has generally not been advised for women after receiving an islet allograft. We hereby describe what is to our knowledge the first successful pregnancy and persistent graft function in a woman 4 years after her initial islet transplantation. A 37-year-old woman with brittle type 1 diabetes was transplanted with two separate islet graft infusions, eventually becoming insulin independent. Ten months after her second transplantation, her immunosuppression was switched from tacrolimus and sirolimus to tacrolimus, azathioprine, and prednisolone, due to her wish to become pregnant. She became pregnant one year later, and after 38 weeks of uncomplicated pregnancy, she gave birth to a healthy child by C-section. The current report suggests that pregnancy and childbirth can be accomplished after islet transplantation without loss of islet graft function.

Proteomic Profiling Reveals the Ambivalent Character of the Mesenchymal Stem Cell Secretome: Assessing the Effect of Preconditioned Media on Isolated Human Islets.

Previous studies in rodents have indicated that function and survival of transplanted islets can be substantially improved by mesenchymal stem cells (MSC). The few human islet studies to date have confirmed these findings but have not determined whether physical contact between MSC and islets is required or whether the benefit to islets results from MSC-secreted proteins. This study aimed to investigate the protective capacity of MSC-preconditioned media for human islets. MSC were cultured for 2 or 5 days in normoxia or hypoxia before harvesting the cell-depleted media for human islet culture in normoxia or hypoxia for 6-8 or 3-4 days, respectively. To characterize MSC-preconditioned media, proteomic secretome profiling was performed to identify angiogenesis- and inflammation-related proteins. A protective effect of MSC-preconditioned media on survival and in vitro function of hypoxic human islets was observed irrespective of the atmosphere used for MSC preconditioning. Islet morphology changed markedly when media from hypoxic MSC were used for culture. However, PDX-1 and insulin gene expression did not confirm a change in the genetic phenotype of these islets. Proteomic profiling of preconditioned media revealed the heterogenicity of the secretome comprising angiogenic and antiapoptotic as well as angiostatic or proinflammatory mediators released at an identical pattern regardless whether MSC had been cultured in normoxic or hypoxic atmosphere. These findings do not allow a clear discrimination between normoxia and hypoxia as stimulus for protective MSC capabilities but indicate an ambivalent character of the MSC angiogenesis- and inflammation-related secretome. Nevertheless, culture of human islets in acellular MSC-preconditioned media resulted in improved morphological and functional islet integrity suggesting a disbalance in favor of protective factors. Further approaches should aim to eliminate potentially detrimental factors to enable the production of advanced clinical grade islet culture media with higher protective qualities.

Glial cell-line derived neurotrophic factor protects human islets from nutrient deprivation and endoplasmic reticulum stress induced apoptosis.

One of the key limitations to successful human islet transplantation is loss of islets due to stress responses pre- and post-transplantation. Nutrient deprivation and ER stress have been identified as important mechanisms leading to apoptosis. Glial Cell-line Derived Neurotrophic Factor (GDNF) has recently been found to promote islet survival after isolation. However, whether GDNF could rescue human islets from nutrient deprivation and ER stress-mediated apoptosis is unknown. Herein, by mimicking those conditions in vitro, we have shown that GDNF significantly improved glucose stimulated insulin secretion, reduced apoptosis and proinsulin:insulin ratio in nutrient deprived human islets. Furthermore, GDNF alleviated thapsigargin-induced ER stress evidenced by reduced expressions of IRE1α and BiP and consequently apoptosis. Importantly, this was associated with an increase in phosphorylation of PI3K/AKT and GSK3B signaling pathway. Transplantation of ER stressed human islets pre-treated with GDNF under kidney capsule of diabetic mice resulted in reduced expressions of IRE1α and BiP in human islet grafts with improved grafts function shown by higher levels of human C-peptide post-transplantation. We suggest that GDNF has protective and anti-apoptotic effects on nutrient deprived and ER stress activated human islets and could play a significant role in rescuing human islets from stress responses.

Human Adipose-Derived Mesenchymal Stem Cells Respond to Short-Term Hypoxia by Secreting Factors Beneficial for Human Islets In Vitro and Potentiate Antidiabetic Effect In Vivo.

Adipose-derived mesenchymal stem cells (ASCs) release factors beneficial for islets in vitro and protect against hyperglycemia in rodent models of diabetes. Oxygen tension has been shown to induce metabolic changes and alter ASCs' release of soluble factors. The effects of hypoxia on the antidiabetic properties of ASCs have not been explored. To investigate this, we incubated human ASCs for 48 h in 21% (normoxia) or 1% O2 (hypoxia) and compared viability, cell growth, surface markers, differentiation capability, and soluble factors in the conditioned media (CM). Human islets were exposed to CM from ASCs incubated in either normoxia or hypoxia, and islet function and apoptosis after culture with or without proinflammatory cytokines were measured. To test hypoxic preconditioned ASCs' islet protective effects in vivo, ASCs were incubated for 48 h in normoxia or hypoxia before being injected into Balb/c Rag 1-/- immunodeficient mice with streptozotocin-induced insulitis. Progression of diabetes and insulin content of pancreas were measured. We found that incubation in hypoxia was well tolerated by ASCs and that levels of VEGF-A, FGF-2, and bNGF were elevated in CM from ASCs incubated in hypoxia compared to normoxia, while levels of HGF, IL-8, and CXCL1 were reduced. CM from ASCs incubated in hypoxia significantly improved human islet function and reduced apoptosis after culture, and reduced cytokine-induced apoptosis. In our mouse model, pancreas insulin content was higher in both groups receiving ASCs compared to control, but the mice receiving preconditioned ASCs had lower random and fasting blood glucose, as well as improved oral glucose tolerance compared to untreated mice. In conclusion, our in vitro results indicate that the islet protective potential of ASCs improves in hypoxia, and we give insight into factors involved in this. Finally we show that hypoxic preconditioning potentiates ASCs' antidiabetic effect in vivo.