Abstract Hypoxia, IL-1β 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-1β 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-1β 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-1β in blood transiently increased during the first days after transplantation. In islets incubated under hypoxia, NRLP3, IL1B and CASP1 and IL-1β 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.
Lack of rapid revascularization and inflammatory attacks at the site of transplantation contribute to impaired islet engraftment and suboptimal metabolic control after clinical islet transplantation. In order to overcome these limitations and enhance engraftment and revascularization, we have generated and transplanted pre-vascularized insulin-secreting organoids composed of rat islet cells, human amniotic epithelial cells (hAECs), and human umbilical vein endothelial cells (HUVECs). Our study demonstrates that pre-vascularized islet organoids exhibit enhanced in vitro function compared to native islets, and, most importantly, better engraftment and improved vascularization in vivo in a murine model. This is mainly due to cross-talk between hAECs, HUVECs and islet cells, mediated by the upregulation of genes promoting angiogenesis (vegf-a) and β cell function (glp-1r, pdx1). The possibility of adding a selected source of endothelial cells for the neo-vascularization of insulin-scereting grafts may also allow implementation of β cell replacement therapies in more favourable transplantation sites than the liver.
Background: Inflammation is a primary contributor to early graft loss and poor islet engraftment. Human amniotic epithelial cells (hAEC) possess regenerative, immunomodulatory and anti-inflammatory properties. In particular, these cells express HLA-G and HLA-E, involved in immunomodulation and immune tolerance. Here, we hypothesized that hAECs could protect islets from cellular damage induced by proinflammatory cytokines and we assessed the cytokine-induced expression of HLA-G and HLA-E in hAECs. Methods: Rat islets were cultured with or without hAECs for 24 hours, followed by 48- hour exposure to IFN-γ, TNF-α and IL-1β. Controls included mono or cocultures without cytokines. For all conditions, glucose stimulated insulin secretion (GSIS), apoptosis by detection of histone-associated DNA fragments, and Th1/Th2 cytokines secreted in the culture media were evaluated by ELISA. Gene expression modifications were assessed by qPCR. hAEC surface marker expression (CD105, CD90, CD326, HLA-E, HLA-G, SSEA-4) was assessed by flow cytometry after culture in control culture medium or in medium containing various concentrations of human recombinant IFN-γ for 24–48H. Results: Exposure to a pro-inflammatory cocktail significantly increased the secretion of the anti-inflammatory cytokines IL6, IL10 and G-CSF by hAECs at both 24H and 48H. IL6, IL8 and IL10 gene expression was significantly upregulated, as well as HLA-G and HLAE. This correlated with an upregulation of STAT1, STAT3 and NF-κB1gene expression levels. RI co-cultured with hAECs maintained a normal insulin secretion after cytokine exposure compared to RI cultured alone, and a significantly lower apoptosis rate. Conclusions: In conclusion, hAECs increase their anti-inflammatory and immunomodulatory potentials when exposed to inflammation in vitro, and protect pancreatic islets against pro-inflammatory cytokines in a coculture set-up.
Abstract 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.
Background: Hypoxia is a main cause of considerable islet loss during first days after intraportal transplantation. Human amniotic epithelial cells (hAECs) possess regenerative, immunomodulatory and anti-inflammatory properties and present particular interest in the context of islet transplantation to protect transplanted islets against immune attack, hypoxic and inflammatory injury. The aim of this study was to investigate whether covering islets with a shield of human amniotic epithelial cells (hAECs) improves islets survival under hypoxic conditions in vitro, as well as islet engraftment and survival in vivo. Methods: Shielded islets were generated on microwells by mixing islets and hAECs at ratio of 1:100 (100 hAECs per islets). The ability of hAECs to adhere to islets was analyzed by confocal microscopy. Engineered rat shielded or neat islets were cultured under normoxic and hypoxic conditions for 16 h. For all conditions, cell viability and islet function were assessed by static insulin release in response to glucose in vitro. Next, function of shielded islets was tested in vivo. For this, 1200 human shielded or neat islets were transplanted under the kidney capsule of diabetic SCID mice. Blood glucose and weight were monitored regularly. Intravenous glucose tolerance test was performed 1 month after transplantation. Graft morphology and vascularisation were evaluated by immunohistochemistry. Results: Islets shielded with hAECs had greater cellular insulin content and increased glucose-stimulated insulin secretion in vitro. Transplantation of shielded islets resulted in considerably earlier normoglycemia and vascularization, improved glucose tolerance, and increased insulin content, both in rat and in human islet transplantation experiments. Conclusion: Co-transplantation of islets with hAECs had a profound impact on the remodelling process, maintaining islet organisation and improving islet revascularisation. Moreover, hAECs improved the capacity of islets to reverse hyperglycaemia.
Background information Cell–cell or cell–substrate interactions are lost when cells are dissociated in culture, or during pathophysiological breakdowns, therefore impairing their structure and polarity, and affecting their function. We show that single rat β‐cells, cultured under non‐adhesive conditions, form intracytoplasmic vacuoles increasing in number and size over time. We characterized these structures and their implication in β‐cell function. Results Ultrastructurally, the vacuoles resemble vesicular apical compartments and are delimited by a membrane, containing microvilli and expressing markers of the plasma membrane, including glucose transporter 2 and actin. When insulin secretion is stimulated, insulin accumulates in the lumen of the vacuoles. By contrast, when the cells are incubated under low calcium levels, the hormone is undetectable in vesicular compartments. Insulin release studies from single cells revealed that vacuole‐containing cells release less insulin as compared to control cells. When added to the medium, a non‐permeant fluid phase marker becomes trapped within vacuoles. Inhibition of vesicular trafficking and exocytosis as well as dynamin‐dependent endocytosis changed the percentage of vacuole‐containing cells, suggesting that both endocytic and exocytic track contribute to their formation. Conclusions These results suggest that loss of cell–cell and cell–substrate contacts in isolated β‐cells affect normal vesicular trafficking and redirects insulin secretion to intracellular vesicular compartments. Significance Our study reveals for the first time that single β‐cells develop vacuolar compartments when cultured in suspension and redirect their insulin secretion to these vacuoles. This may underlie a compensatory process for cultured cells who lost their interactions with adhesive substrates or neighbouring cells.
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 β 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 β 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 β cell failure induced by lipotoxicity via the identification of all newly synthesized proteins in islets in response to short-term exposure to palmitate.
Background: Replacing damaged organs with biological substitutes capable of protecting the islets and facilitating vascularization is a great objective in the field of islet transplantation. A decellularized placenta includes a large number of cotyledons with a conserved vessel structure of the native organ. Our goal is to obtain a perfect decellularization protocol to generate pre-vascularized organoids by recellularizing this ECM with HUVECs and pancreatic islets. Methods: After blood removal, cotyledons were dissected from the placentas and decellularized using a bioreactor. Cell removal was assessed by histology and quantification of residual DNA. Presence of structural proteins and ECM structure were analyzed using SEM, CT scan and mass spectrometry. Recellularization protocols were conducted, with HUVECs or BOECs as endothelial cell sources, and with Ins-1E cells or rat islets as insulin secreting cell sources. Function of recellularized cotyledons was assessed in vitro with glucose stimulated insulin secretion tests (GSIS). To assess in vivo biocompatibility and function of the scaffolds, we transplanted in diabetic NSG mice. Glycaemia was measured every day to monitor normalization of blood glucose levels. Results: Our protocol led to successful decellularization, as evidenced by the absence of cells and the preserved ECM structure. Moreover, DNA quantification did not reveal any residual DNA. Quantification of GAG and hydroxyproline, and mass spectrometry analysis show that structural proteins are conserved. SEM and CT scan images revealed that the ECM structure was preserved after the decellularization protocol. Cells after recellularization showed a good vascularization after 7 days. The GSIS test shows a perfect organ response in the production of insulin. Conclusions: The decellularized cotyledon is the perfect scaffold to reproduce a prevascularized insulin-producing organ, which allows transplanted cells to survive during the peri-transplantation period.
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.
Background Development of macroencapsulation, neovascularized devices and biopolymer scaffolds that could be easily loaded with islets and implanted, with the aim of constructing a bioartificial pancreas may help to reduce the risks and improve the success rate of islet transplantation. Human amniotic membrane (HAM) is inexpensive and attractive as a biomaterial due to its structural similarities to islet extracellular matrix (ECM), and its immunomodulatory, anti-inflammatory and antifibrotic properties. The aim of our study was to develop hydrogel derived from HAM and assess whether it could support islet function in vitro and in vivo. Methods/ Materials The hydrogels were generated from HAM and accessed for porosity and ECM content. The protein content in HAM derived hydrogels and native HAM lysates were measured. To assess hydrogel impact on islet viability and function isolated rat islets were incorporated into the hydrogels and cultured for one week. The cell viability was evaluated by FDA/PI staining. To demonstrate islet function the glucose stimulated insulin secretion (GSIS) tests were performed using standard ELISA. Next, we assessed whether incorporation of islets into hydrogel could enhance engraftment and lead to better glycemic control in diabetic SCID mice. For this purpose 350 rat islets (IEQ) loaded into the hydrogels or islets alone (control) were transplanted into the epididymal fat of diabetic SCID mice. Blood glucose levels were monitored daily and intraperitoneal glucose tolerance tests (IPGTTs) were carried out. Grafts and serum were harvested at 1, 2, 6 and 12 weeks after transplantation to assess outcome. Results The ECM concentration in the hydrogel affected the pore size. Insulin and glucagon expression and viability of islets incorporated into hydrogel was significantly higher than that of islets in free-floating culture. In addition, significant enhancement of GSIS was observed from islets embedded in hydrogel as compared to controls. In vivo experiments showed that, transplantation of 350 IEQ embedded in hydrogel lead to enhanced engraftment, vascularization, viability and better glycaemic control compared to control mice transplanted with islets alone. Conclusions Incorporation of pancreatic islet into amnion-derived hydrogels enhances islet engraftment and is a valuable approach to improve islet transplantation outcomes. This work was supported by a grant from the Swiss National Science Foundation (Grant #310030_173138, to TB, EB, DB) and a grant from the European Foundation for Study of Diabetes (to EB and DB).
