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.

Palmitate and oleate modify membrane fluidity and kinase activities of INS-1E ß-cells alongside altered metabolism-secretion coupling.

Chronic exposure to elevated levels of glucose and free fatty acids impairs beta-cell function, leading to insulin secretion defects and eventually beta-cell failure. Using a semi-high throughput approach applied to INS-1E beta-cells, we tested multiple conditions of chronic exposure to basal, intermediate and high glucose, combined with saturated versus mono- and polyunsaturated fatty acids in order to assess cell integrity, lipid metabolism, mitochondrial function, glucose-stimulated calcium rise and secretory kinetics. INS-1E beta-cells were cultured for 3 days at different glucose concentrations (5.5, 11.1, 25 mM) without or with BSA-complexed 0.4 mM saturated (C16:0 palmitate), monounsaturated (C18:1 oleate) or polyunsaturated (C18:2 linoleate, C18:3 linolenate) fatty acids, resulting in 0.1-0.5 µM unbound fatty acids. Accumulation of triglycerides in cells exposed to fatty acids was glucose-dependent, oleate inducing the strongest lipid storage and protecting against glucose-induced cytotoxicity. The combined chronic exposure to both high glucose and either palmitate or oleate altered mitochondrial function as well as glucose-induced calcium rise. This pattern did not directly translate at the secretory level since palmitate and oleate exhibited distinct effects on the first and the second phases of glucose-stimulated exocytosis. Both fatty acids changed the activity of kinases, such as the MODY-associated BLK. Additionally, chronic exposure to fatty acids modified membrane physicochemical properties by increasing membrane fluidity, oleate exhibiting larger effects compared to palmitate. Chronic fatty acids differentially and specifically exacerbated some of the glucotoxic effects, without promoting cytotoxicity on their own. Each of the tested fatty acids functionally modified INS-1E beta-cell, oleate inducing the strongest effects.

Insulin-producing organoids engineered from islet and amniotic epithelial cells to treat diabetes.

Maintaining long-term euglycemia after intraportal islet transplantation is hampered by the considerable islet loss in the peri-transplant period attributed to inflammation, ischemia and poor angiogenesis. Here, we show that viable and functional islet organoids can be successfully generated from dissociated islet cells (ICs) and human amniotic epithelial cells (hAECs). Incorporation of hAECs into islet organoids markedly enhances engraftment, viability and graft function in a mouse type 1 diabetes model. Our results demonstrate that the integration of hAECs into islet cell organoids has great potential in the development of cell-based therapies for type 1 diabetes. Engineering of functional mini-organs using this strategy will allow the exploration of more favorable implantation sites, and can be expanded to unlimited (stem-cell-derived or xenogeneic) sources of insulin-producing cells.

Effects of remote ischaemic preconditioning on intraportal islet transplantation in a rat model.

Remote ischaemic preconditioning (RIPC), which is the intermittent interruption of blood flow to a site distant from the target organ, is known to improve solid organ resistance to ischaemia-reperfusion injury. This procedure could be of interest in islet transplantation to mitigate hypoxia-related loss of islet mass after isolation and transplantation. Islets isolated from control or RIPC donors were analyzed for yield, metabolic activity, gene expression and high mobility group box-1 (HMGB1) content. Syngeneic marginal mass transplantation was performed in four streptozotocin-induced diabetic groups: control, RIPC in donor only, RIPC in recipient only, and RIPC in donor and recipient. Islets isolated from RIPC donors had an increased yield of 20% after 24 h of culture compared to control donors (P = 0.007), linked to less cell death (P = 0.08), decreased expression of hypoxia-related genes (Hif1a P = 0.04; IRP94 P = 0.008), and increased intra-cellular (P = 0.04) and nuclear HMGB1. The use of RIPC in recipients only did not allow for reversal of diabetes, with increased serum HMGB1 at day 1; the three other groups demonstrated significantly better outcomes. Performing RIPC in the donors increases islet yield and resistance to hypoxia. Validation is needed, but this strategy could help to decrease the number of donors per islet recipient.

Combined Pancreatic Islet-Lung-Liver Transplantation in a Pediatric Patient with Cystic Fibrosis-Related Diabetes.

BACKGROUND: Cystic fibrosis-related diabetes (CFRD) is the most frequent extrapulmonary complication of cystic fibrosis (CF). METHODS: We report the first combined pancreatic islet-lung-liver transplantation in a 14-year-old adolescent. CFTR was analyzed by Sanger sequencing. Further genes were analyzed by high-throughput sequencing. RESULTS: The patient was diagnosed with CF at the age of 14 months. Nine years later, after diagnosis of CFRD, the patient's BMI and lung function began to decline. Bilateral lung transplantation with simultaneous liver transplantation was performed at the age of 14.5 years. The first islet transplantation (IT) was carried out 10 days later. Six months later, C-peptide secretion after arginine stimulation showed peak values of 371 pmol/L (vs. 569 pmol/L before IT) and insulin doses had slightly increased (1.40 vs. 1.11 units/kg/day before IT). A second IT was performed at the age of 15 years, a third at 16 years. Two years after the first IT, arginine-stimulated C-peptide secretion increased to 2,956 pmol/L and insulin doses could be reduced to 0.82 units/kg/day. HbA1c decreased from 7.3% (57.4 mmol/mol) to 5.9% (41.0 mmol/mol). CONCLUSION: IT following lung and liver transplantation, with injection of islets into a transplanted organ, is feasible. It improves C-peptide secretion, decreases insulin needs, and lowers HbA1c.

Epigallocatechin-3-gallate (EGCG) activates AMPK through the inhibition of glutamate dehydrogenase in muscle and pancreatic ß-cells: A potential beneficial effect in the pre-diabetic state?

Glucose homeostasis is determined by insulin secretion from the ß-cells in pancreatic islets and by glucose uptake in skeletal muscle and other insulin target tissues. While glutamate dehydrogenase (GDH) senses mitochondrial energy supply and regulates insulin secretion, its role in the muscle has not been elucidated. Here we investigated the possible interplay between GDH and the cytosolic energy sensing enzyme 5'-AMP kinase (AMPK), in both isolated islets and myotubes from mice and humans. The green tea polyphenol epigallocatechin-3-gallate (EGCG) was used to inhibit GDH. Insulin secretion was reduced by EGCG upon glucose stimulation and blocked in response to glutamine combined with the allosteric GDH activator BCH (2-aminobicyclo-[2,2,1] heptane-2-carboxylic acid). Insulin secretion was similarly decreased in islets of mice with ß-cell-targeted deletion of GDH (ßGlud1-/-). EGCG did not further reduce insulin secretion in the mutant islets, validating its specificity. In human islets, EGCG attenuated both basal and nutrient-stimulated insulin secretion. Glutamine/BCH-induced lowering of AMPK phosphorylation did not operate in ßGlud1-/- islets and was similarly prevented by EGCG in control islets, while high glucose systematically inactivated AMPK. In mouse C2C12 myotubes, like in islets, the inhibition of AMPK following GDH activation with glutamine/BCH was reversed by EGCG. Stimulation of GDH in primary human myotubes caused lowering of insulin-induced 2-deoxy-glucose uptake, partially counteracted by EGCG. Thus, mitochondrial energy provision through anaplerotic input via GDH influences the activity of the cytosolic energy sensor AMPK. EGCG may be useful in obesity by resensitizing insulin-resistant muscle while blunting hypersecretion of insulin in hypermetabolic states.

Postprandial macrophage-derived IL-1ß stimulates insulin, and both synergistically promote glucose disposal and inflammation.

The deleterious effect of chronic activation of the IL-1ß system on type 2 diabetes and other metabolic diseases is well documented. However, a possible physiological role for IL-1ß in glucose metabolism has remained unexplored. Here we found that feeding induced a physiological increase in the number of peritoneal macrophages that secreted IL-1ß, in a glucose-dependent manner. Subsequently, IL-1ß contributed to the postprandial stimulation of insulin secretion. Accordingly, lack of endogenous IL-1ß signaling in mice during refeeding and obesity diminished the concentration of insulin in plasma. IL-1ß and insulin increased the uptake of glucose into macrophages, and insulin reinforced a pro-inflammatory pattern via the insulin receptor, glucose metabolism, production of reactive oxygen species, and secretion of IL-1ß mediated by the NLRP3 inflammasome. Postprandial inflammation might be limited by normalization of glycemia, since it was prevented by inhibition of the sodium-glucose cotransporter SGLT2. Our findings identify a physiological role for IL-1ß and insulin in the regulation of both metabolism and immunity.

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.

Autophagy and non-alcoholic fatty liver disease.

Autophagy, or cellular self-digestion, is a catabolic process that targets cell constituents including damaged organelles, unfolded proteins, and intracellular pathogens to lysosomes for degradation. Autophagy is crucial for development, differentiation, survival, and homeostasis. Important links between the regulation of autophagy and liver complications associated with obesity, non-alcoholic fatty liver disease (NAFLD), have been reported. The spectrum of these hepatic abnormalities extends from isolated steatosis to non-alcoholic steatohepatitis (NASH), steatofibrosis, which sometimes leads to cirrhosis, and hepatocellular carcinoma. NAFLD is one of the three main causes of cirrhosis and increases the risk of liver-related death and hepatocellular carcinoma. The pathophysiological mechanisms of the progression of a normal liver to steatosis and then more severe disease are complex and still unclear. The regulation of the autophagic flux, a dynamic response, and the knowledge of the role of autophagy in specific cells including hepatocytes, hepatic stellate cells, immune cells, and hepatic cancer cells have been extensively studied these last years. This review will provide insight into the current understanding of autophagy and its role in the evolution of the hepatic complications associated with obesity, from steatosis to hepatocellular carcinoma.

Autophagy, signaling and obesity.

Autophagy is a cellular pathway crucial for development, differentiation, survival and homeostasis. Autophagy can provide protection against aging and a number of pathologies such as cancer, neurodegeneration, cardiac disease and infection. Recent studies have reported new functions of autophagy in the regulation of cellular processes such as lipid metabolism and insulin sensitivity. Important links between the regulation of autophagy and obesity including food intake, adipose tissue development, ß cell function, insulin sensitivity and hepatic steatosis exist. This review will provide insight into the current understanding of autophagy, its regulation, and its role in the complications associated with obesity.

Serum markers of hepatocyte death and apoptosis are non invasive biomarkers of severe fibrosis in patients with alcoholic liver disease.

BACKGROUND: Quantification of hepatocyte death is useful to evaluate the progression of alcoholic liver diseases. Our aims were to quantify and correlate the circulating levels of Cytokeratin 18 (CK18) and its caspases-generated fragment to disease severity in heavy alcoholics. METHODOLOGY/PRINCIPAL FINDINGS: CK18 and CK18-fragment were evaluated in the serum of 143 heavy alcoholics. Serum levels of markers of hepatocyte death (CK18), apoptosis (CK18 fragment) and necrosis (CK18 -CK18 fragment) increased in patients with severe fibrosis compared to patients with mild fibrosis. These markers strongly correlated with Mallory-Denk bodies, hepatocyte ballooning, fibrosis and with hepatic TNFα and TGFß assessed in the liver of 24 patients. Elevated levels of serum hepatocyte death and apoptotic markers were independent risk factors in predicting severe fibrosis in a model combining alkaline phosphatase, bilirubin, prothrombin index, hyaluronate, hepatocyte death and apoptotic markers. The level of markers of hepatocyte death and apoptosis had an area under the receiving operator curve that predicted severe fibrosis of 0.84 and 0.76, respectively. CONCLUSION/SIGNIFICANCE: Death of hepatocytes can be easily evaluated with serum markers and correlated with severe fibrosis in heavy alcohol drinkers. These biomarkers could be useful to rapidly evaluate liver injuries and the efficacy of therapies.

Modulation of caspase-independent cell death leads to resensitization of imatinib mesylate-resistant cells.

Imatinib mesylate is widely used for the treatment of patients with chronic myelogenous leukemia (CML). This compound is very efficient in killing Bcr-Abl-positive cells in a caspase-dependent manner. Nevertheless, several lines of evidence indicated that caspase-mediated cell death (i.e., apoptosis) is not the only type of death induced by imatinib. The goal of our study was to evaluate the importance of the newly described caspase-independent cell death (CID) in Bcr-Abl-positive cells. We established in several CML cell lines that imatinib, in conjunction with apoptosis, also induced CID. CID was shown to be as efficient as apoptosis in preventing CML cell proliferation and survival. We next investigated the potential implication of a recently identified mechanism used by cancer cells to escape CID through overexpression of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We showed here, in several CML cell lines, that GAPDH overexpression was sufficient to induce protection from CID. Furthermore, imatinib-resistant Bcr-Abl-positive cell lines were found to spontaneously overexpress GAPDH. Finally, we showed that a GAPDH partial knockdown, using specific short hairpin RNAs, was sufficient to resensitize those resistant cells to imatinib-induced cell death. Taken together, our results indicate that CID is an important effector of imatinib-mediated cell death. We also established that GAPDH overexpression can be found in imatinib-resistant Bcr-Abl-positive cells and that its down-regulation can resensitize those resistant cells to imatinib-induced death. Therefore, drugs able to modulate GAPDH administered together with imatinib could find some therapeutic benefits in CML patients.