Beneficial Effects of Two Marine Oxygen Carriers, M101 and M201, on Human Islet Quality in Hypoxic Culture Conditions.

High pancreatic islet sensitivity to hypoxia is an important issue in the field of pancreatic islet transplantation. A promising strategy to improve islet oxygenation in hypoxic conditions is to leverage the properties of hemoglobin as a natural carrier of oxygen. Studies using human or bovine hemoglobin have failed to demonstrate efficacy, probably due to the molecule being unstable in the absence of protective erythrocytes. Recently, marine worm hemoglobins have been shown to be more stable and to possess higher oxygen carrier potential, with 156 oxygen binding sites per molecule compared to four in humans. Previous studies have shown the beneficial effects of two marine worm hemoglobins, M101 and M201, on nonhuman pancreatic islets. However, their effects on human islets have not been tested or compared. In this study, we assessed the impact of both molecules during human islet culture in vitro under hypoxic conditions. Human islets were exposed to both molecules for 24 h in high islet density-induced hypoxia [600 islet equivalents (IEQ)/cm²]. M101 and M201 reduced the release of hypoxic (VEGF) and apoptotic (cyt c) markers in the medium after 24-h culture. Human islet function or viability was improved in vitro in the presence of these oxygen carriers. Thus, the utilization of M101 or M201 could be a safe and easy way to improve human islet oxygenation and survival in hypoxic conditions as observed during islet culture prior to transplantation or islet encapsulation.

STAT3 Regulates Mitochondrial Gene Expression in Pancreatic ß-Cells and Its Deficiency Induces Glucose Intolerance in Obesity.

Most obese and insulin-resistant individuals do not develop diabetes. This is the result of the capacity of ß-cells to adapt and produce enough insulin to cover the needs of the organism. The underlying mechanism of ß-cell adaptation in obesity, however, remains unclear. Previous studies have suggested a role for STAT3 in mediating ß-cell development and human glucose homeostasis, but little is known about STAT3 in ß-cells in obesity. We observed enhanced cytoplasmic expression of STAT3 in severely obese subjects with diabetes. To address the functional role of STAT3 in adult ß-cells, we generated mice with tamoxifen-inducible partial or full deletion of STAT3 in ß-cells and fed them a high-fat diet before analysis. Interestingly, ß-cell heterozygous and homozygous STAT3-deficient mice showed glucose intolerance when fed a high-fat diet. Gene expression analysis with RNA sequencing showed that reduced expression of mitochondrial genes in STAT3 knocked down human EndoC-ß1H cells, confirmed in FACS-purified ß-cells from obese STAT3-deficient mice. Moreover, silencing of STAT3 impaired mitochondria activity in EndoC-ß1H cells and human islets, suggesting a mechanism for STAT3-modulated ß-cell function. Our study postulates STAT3 as a novel regulator of ß-cell function in obesity.

Glycaemic control in diabetic rats treated with islet transplantation using plasma combined with hydroxypropylmethyl cellulose hydrogel.

Islet transplantation is one of the most efficient cell therapies used in clinics and could treat a large proportion of patients with diabetes. However, it is limited by the high requirement of pancreas necessary to provide the sufficient surviving islet mass in the hepatic tissue and restore normoglycaemia. Reduction in organ procurement requirements could be achieved by extrahepatic transplantation using a biomaterial that enhances islet survival and function. We report a plasma-supplemented hydroxypropyl methylcellulose (HPMC) hydrogel, engineered specifically using a newly developed technique for intra-omental islet infusion, known as hOMING (h-Omental Matrix Islet filliNG). The HPMC hydrogel delivered islets with better performance than that of the classical intrahepatic infusion. After the validation of the HPMC suitability for islets in vivo and in vitro, plasma supplementation modified the rheological properties of HPMC without affecting its applicability with hOMING. The biomaterial association was proven to be more efficient both in vitro and in vivo, with better islet viability and function than that of the current clinical intrahepatic delivery technique. Indeed, when the islet mass was decreased by 25% or 35%, glycaemia control was observed in the group of plasma-supplemented hydrogels, whereas no regulation was observed in the hepatic group. Plasma gelation, observed immediately post infusion, decreased anoïkis and promoted vascularisation. To conclude, the threshold mass for islet transplantation could be decreased using HPMC-Plasma combined with the hOMING technique. The simplicity of the hOMING technique and the already validated use of its components could facilitate its transfer to clinics. STATEMENT OF SIGNIFICANCE: One of the major limitations for the broad deployment of current cell therapy for brittle type 1 diabetes is the islets' destruction during the transplantation process. Retrieved from their natural environment, the islets are grafted into a foreign tissue, which triggers massive cell loss. It is mandatory to provide the islets with an 3D environment specifically designed for promoting isletimplantation to improve cell therapy outcomes. For this aim, we combined HPMC and plasma. HPMC provides suitable rheological properties to the plasma to be injectable and be maintained in the omentum. Afterwards, the plasma polymerises around the graft in vivo, thereby allowing their optimal integration into their transplantation site. As a result, the islet mass required to obtain glycaemic control was reduced by 35%.

Beneficial effects of the novel marine oxygen carrier M101 during cold preservation of rat and human pancreas.

Ischaemia impairs organ quality during preservation in a time-dependent manner, due to a lack of oxygen supply. Its impact on pancreas and islet transplantation outcome has been demonstrated by a correlation between cold ischaemia time and poor islet isolation efficiency. Our goal in the present study was to improve pancreas and islet quality using a novel natural oxygen carrier (M101, 2 g/L), which has been proven safe and efficient in other clinical applications, including kidney transplantation, and for several pre-clinical transplantation models. When M101 was added to the preservation solution of rat pancreas during ischaemia, a decrease in oxidative stress (ROS), necrosis (HMGB1), and cellular stress pathway (p38 MAPK)activity was observed. Freshly isolated islets had improved function when M101 was injected in the pancreas. Additionally, human pancreases exposed to M101 for 3 hours had an increase in complex 1 mitochondrial activity, as well as activation of AKT activity, a cell survival marker. Insulin secretion was also up-regulated for isolated islets. In summary, these results demonstrate a positive effect of the oxygen carrier M101 on rat and human pancreas during preservation, with an overall improvement in post-isolation islet quality.

Lysosomal degradation of newly formed insulin granules contributes to ß cell failure in diabetes.

Compromised function of insulin-secreting pancreatic ß cells is central to the development and progression of Type 2 Diabetes (T2D). However, the mechanisms underlying ß cell failure remain incompletely understood. Here, we report that metabolic stress markedly enhances macroautophagy-independent lysosomal degradation of nascent insulin granules. In different model systems of diabetes including of human origin, stress-induced nascent granule degradation (SINGD) contributes to loss of insulin along with mammalian/mechanistic Target of Rapamycin (mTOR)-dependent suppression of macroautophagy. Expression of Protein Kinase D (PKD), a negative regulator of SINGD, is reduced in diabetic ß cells. Pharmacological activation of PKD counters SINGD and delays the onset of T2D. Conversely, inhibition of PKD exacerbates SINGD, mitigates insulin secretion and accelerates diabetes. Finally, reduced levels of lysosomal tetraspanin CD63 prevent SINGD, leading to increased insulin secretion. Overall, our findings implicate aberrant SINGD in the pathogenesis of diabetes and suggest new therapeutic strategies to prevent ß cell failure.

Intra-Omental Islet Transplantation Using h-Omental Matrix Islet filliNG (hOMING).

Regenerative medicine based on cell therapy represents a new hope for curing disease. Current obstacles include proper in vivo validation of the efficiency of the therapy. For transfer to the recipient body, cells often need to be combined with biomaterials, especially hydrogels. However, validation of the efficacy of such a graft requires the right environment, the right hydrogel, and the right recipient site. The omentum might be such a site. Based on the example of islet transplantation, we developed the hOMING (h-Omental Matrix Islet filliNG) technique, which consists of the injection of the graft inside the tissue, in between the omental layers, to improve islet implantation and survival. To achieve this, islets have to be embedded in a hydrogel with a viscosity that enables its injection using an atraumatic needle. Syringes are loaded with a combination of hydrogel and islets. Several injections are performed inside the omental tissue at different entry points, and the deposition of the islet/hydrogel mixture is made along a line. We tested the feasibility of this innovative approach using dextran beads. The beads were well spread throughout the omental tissue, in close proximity to blood vessels. To test the efficacy of the graft, we transplanted islets into diabetic rats and perform a metabolic follow-up over two months. The transplanted islets exhibited a high rate of re-vascularization around and inside islets, and reversed diabetes. The hOMING technique could be applicable for other types of hydrogel or cell therapy, for cells with high metabolic activity.

Extra-Hepatic Islet Transplantation: Validation of the h-Omental Matrix Islet filliNG (hOMING) Technique on a Rodent Model Using an Alginate Carrier.

Following the tremendous development of hydrogels for cell therapy, there is now a growing need for surgical techniques to validate in vivo scaffold benefits for islet transplantation. Therefore, we propose a newly designed surgical procedure involving the injection of hydrogel-embedded pancreatic islets in the omentum, which is considered a favorable environment for cell survival and function. Our technique, called h-Omental Matrix Islet filliNG (hOMING) was designed to test the benefits of hydrogel on islet survival and function in vivo. Islets were implanted in the omentum of diabetic rats using the hOMING technique and alginate as an islet carrier. Blood glucose and C-peptide levels were recorded to assess graft function. After 2 months, grafts were explanted and studied using insulin and vessel staining. All rats that underwent hOMING exhibited graft function characterized by a glycemia decrease and a C-peptidemia increase ( P < 0.001 compared with preoperative levels). Furthermore, hOMING appeared to preserve islet morphology and insulin content and allowed the proper revascularization of grafted islets. The results suggest that hOMING is a viable and promising approach to test in vivo the benefits of hydrogel administration for islet transplantation into the omental tissue.

Beneficial Effect of Jojoba Seed Extracts on Hyperglycemia-Induced Oxidative Stress in RINm5f Beta Cells.

Hyperglycemia occurs during diabetes and insulin resistance. It causes oxidative stress by increasing reactive oxygen species (ROS) levels, leading to cellular damage. Polyphenols play a central role in defense against oxidative stress. In our study, we investigated the antioxidant properties of simmondsin, a pure molecule present in jojoba seeds, and of the aqueous extract of jojoba seeds on fructose-induced oxidative stress in RINm5f beta cells. The exposure of RINm5f beta cells to fructose triggered the loss of cell viability (-48%, p < 0.001) and disruption of insulin secretion (p < 0.001) associated with of reactive oxygen species (ROS) production and a modulation of pro-oxidant and antioxidant signaling pathway. Cell pre-treatments with extracts considerably increased cell viability (+86% p < 0.001) for simmondsin and +74% (p < 0.001) for aqueous extract and insulin secretion. The extracts also markedly decreased ROS (-69% (p < 0.001) for simmondsin and -59% (p < 0.001) for aqueous extract) and caspase-3 activation and improved antioxidant defense, inhibiting p22phox and increasing nuclear factor (erythroid-derived 2)-like 2 (Nrf2) levels (+70%, p < 0.001) for aqueous extract. Simmondsin had no impact on Nrf2 levels. The richness and diversity of molecules present in jojoba seed extract makes jojoba a powerful agent to prevent the destruction of RINm5f beta cells induced by hyperglycemia.

Oral insulin delivery, the challenge to increase insulin bioavailability: Influence of surface charge in nanoparticle system.

Oral administration of insulin increases patient comfort and could improve glycemic control thanks to the hepatic first passage. However, challenges remain. The current approach uses poly (d, lactic-co-glycolic) acid (PLGA) nanoparticles (NPs), an effective drug carrier system with a long acting profile. However, this system presents a bioavailability of less than 20% for insulin encapsulation. In this context, physico-chemical parameters like surface charge could play a critical role in NP uptake by the intestinal barrier. Therefore, we developed a simple method to modulate NP surface charge to test its impact on uptake in vitro and finally on NP efficiency in vivo. Various NPs were prepared in the presence (+) or absence (-) of polyvinyl alcohol (PVA), sodium dodecyl sulfate (SDS), and/or coated with chitosan chloride. In vitro internalization was tested using epithelial culture of Caco-2 or using a co-culture (Caco-2/RevHT29MTX) by flow cytometry. NPs were then administered by oral route using a pharmaceutical complex vector (100 or 250 UI/kg) in a diabetic rat model. SDS-NPs (-42 ±â€¯2 mV) were more negatively charged than -PVA-NPs (-22 ±â€¯1 mV) and chitosan-coated NPs were highly positively charged (56 ±â€¯2 mV) compared to +PVA particles (-2 ±â€¯1 mV), which were uncharged. In the Caco-2 model, NP internalization was significantly improved by using negatively charged NPs (SDS NPs) compared to using classical NPs (+PVA NPs) and chitosan-coated NPs. Finally, the efficacy of insulin SDS-NPs was demonstrated in vivo (100 or 250 UI insulin/kg) with a reduction of blood glucose levels in diabetic rats. Formulation of negatively charged NPs represents a promising approach to improve NP uptake and insulin bioavailability for oral delivery.

Beneficial effects of a red wine polyphenol extract on high-fat diet-induced metabolic syndrome in rats.

PURPOSE: Individuals with metabolic syndrome (MS) show several metabolic abnormalities including insulin resistance, dyslipidaemia, and oxidative stress (OS). Diet is one of the factors influencing the development of MS, and current nutritional advice emphasises the benefits of fruit and vegetable consumption. Here, we assessed the effects of naturally occurring antioxidants, red wine polyphenols (RWPs), on MS and OS. METHODS: Wistar rats (n = 20) weighing 200-220 g received a high-fat diet (HFD) for 2 months before they were divided into two groups that received either HFD only or HFD plus 50 mg/kg RWPs in their drinking water for an additional 2 months. A control group (n = 10) received a normal diet (ND) for 4 months. RESULTS: Rats receiving HFD increased body weight over 20 % throughout the duration of the study. They also showed increased blood levels of C-peptide, glucose, lipid peroxides, and oxidised proteins. In addition, the HFD increased OS in hepatic, pancreatic, and vascular tissues, as well as induced pancreatic islet cell hyperplasia and hepatic steatosis. Addition of RWPs to the HFD attenuated these effects on plasma and tissue OS and on islet cell hyperplasia. However, RWPs had no effect on blood glucose levels or hepatic steatosis. CONCLUSIONS: RWPs showed an antioxidant mechanism of action against MS. This result will inform future animal studies exploring the metabolic effects of RWPs in more detail. In addition, these findings support the use of antioxidants as adjunctive nutritional treatments for patients with diabetes.

Comparison of Perfluorodecalin and HEMOXCell as Oxygen Carriers for Islet Oxygenation in an In Vitro Model of Encapsulation.

Transplantation of encapsulated islets in a bioartificial pancreas is a promising alternative to free islet cell therapy to avoid immunosuppressive regimens. However, hypoxia, which can induce a rapid loss of islets, is a major limiting factor. The efficiency of oxygen delivery in an in vitro model of bioartificial pancreas involving hypoxia and confined conditions has never been investigated. Oxygen carriers such as perfluorocarbons and hemoglobin might improve oxygenation. To verify this hypothesis, this study aimed to identify the best candidate of perfluorodecalin (PFD) or HEMOXCell® to reduce cellular hypoxia in a bioartificial pancreas in an in vitro model of encapsulation ex vivo. The survival, hypoxia, and inflammation markers and function of rat islets seeded at 600 islet equivalents (IEQ)/cm2 and under 2% pO2 were assessed in the presence of 50 µg/mL of HEMOXCell or 10% PFD with or without adenosine. Both PFD and HEMOXCell increased the cell viability and decreased markers of hypoxia (hypoxia-inducible factor mRNA and protein). In these culture conditions, adenosine had deleterious effects, including an increase in cyclooxygenase-2 and interleukin-6, in correlation with unregulated proinsulin release. Despite the effectiveness of PFD in decreasing hypoxia, no restoration of function was observed and only HEMOXCell had the capacity to restore insulin secretion to a normal level. Thus, it appeared that the decrease in cell hypoxia as well as the intrinsic superoxide dismutase activity of HEMOXCell were both mandatory to maintain islet function under hypoxia and confinement. In the context of islet encapsulation in a bioartificial pancreas, HEMOXCell is the candidate of choice for application in vivo.

A Metabolomic Approach (1H HRMAS NMR Spectroscopy) Supported by Histology to Study Early Post-transplantation Responses in Islet-transplanted Livers.

Intrahepatic transplantation of islets requires a lot of islets because more than 50% of the graft is lost during the 24 hours following transplantation. We analyzed, in a rat model, early post-transplantation inflammation using systemic inflammatory markers, or directly in islet-transplanted livers by immunohistochemistry. 1H HRMAS NMR was employed to investigate metabolic responses associated with the transplantation. Inflammatory markers (Interleukin-6, α2-macroglobulin) are not suitable to follow islet reactions as they are not islet specific. To study islet specific inflammatory events, immunohistochemistry was performed on sections of islet transplanted livers for thrombin (indicator of the instant blood-mediated inflammatory reaction (IBMIR)) and granulocytes and macrophages. We observed a specific correlation between IBMIR and granulocyte and macrophage infiltration after 12 h. In parallel, we identified a metabolic response associated with transplantation: after 12 h, glucose, alanine, aspartate, glutamate and glutathione were significantly increased. An increase of glucose is a marker of tissue degradation, and could be explained by immune cell infiltration. Alanine, aspartate and glutamate are inter-connected in a common metabolic pathway known to be activated during hypoxia. An increase of glutathione revealed the presence of antioxidant protection. In this study, IBMIR visualization combined with 1H HRMAS NMR facilitated the characterization of cellular and molecular pathways recruited following islet transplantation.

In Vitro and In Vivo Investigation of the Angiogenic Effects of Liraglutide during Islet Transplantation.

INTRODUCTION: This study investigated the angiogenic properties of liraglutide in vitro and in vivo and the mechanisms involved, with a focus on Hypoxia Inducible Factor-1α (HIF-1α) and mammalian target of rapamycin (mTOR). MATERIALS AND METHODS: Rat pancreatic islets were incubated in vitro with 10 µmol/L of liraglutide (Lira) for 12, 24 and 48 h. Islet viability was studied by fluorescein diacetate/propidium iodide staining and their function was assessed by glucose stimulation. The angiogenic effect of liraglutide was determined in vitro by the measure of vascular endothelial growth factor (VEGF) secretion using enzyme-linked immunosorbent assay and by the evaluation of VEGF and platelet-derived growth factor-α (PDGFα) expression with quantitative polymerase chain reaction technic. Then, in vitro and in vivo, angiogenic property of Lira was evaluated using immunofluorescence staining targeting the cluster of differentiation 31 (CD31). To understand angiogenic mechanisms involved by Lira, HIF-1α and mTOR activation were studied using western blotting. In vivo, islets (1000/kg body-weight) were transplanted into diabetic (streptozotocin) Lewis rats. Metabolic control was assessed for 1 month by measuring body-weight gain and fasting blood glucose. RESULTS: Islet viability and function were respectively preserved and enhanced (p<0.05) with Lira, versus control. Lira increased CD31-positive cells, expression of VEGF and PDGFα (p<0.05) after 24 h in culture. Increased VEGF secretion versus control was also observed at 48 h (p<0.05). Moreover, Lira activated mTOR (p<0.05) signalling pathway. In vivo, Lira improved vascular density (p<0.01), body-weight gain (p<0.01) and reduced fasting blood glucose in transplanted rats (p<0.001). CONCLUSION: The beneficial effects of liraglutide on islets appeared to be linked to its angiogenic properties. These findings indicated that glucagon-like peptide-1 analogues could be used to improve transplanted islet revascularisation.

High-fructose and high-fat diet-induced disorders in rats: impact on diabetes risk, hepatic and vascular complications.

BACKGROUND: As a result of the increased consumption of sugar-rich and fatty-products, and the increase in preference for such products, metabolic disorders are becoming more common at a younger age. Fructose is particularly used in prepared foods and carbonated beverages. We investigated the impact of regular consumption of fructose, in combination or not with fatty food, on the onset of metabolic syndrome and type 2 diabetes (T2D). We evaluated the metabolic, oxidative, and functional effects on the liver and blood vessels, both related to diabetes complications. METHODS: High-fat diet (HFD), high-fructose beverages (HF) or both (HFHF) were compared to rats fed with normal diet (ND) for 8 months to induce T2D and its metabolic, oxidative, and functional complications. Metabolic control was determined by measuring body weight, fasting blood glucose, C-peptide, HOMA2-IR, leptin, and cholesterol; oxidative parameters were studied by lipid peroxidation and total antioxidant capacity in plasma and the use of ROS labelling on tissue. Histological analysis was performed on the liver and endothelial function was performed in main mesenteric artery using organ-baths. RESULTS: After 2 months, HFHF and HFD increased body weight, leptin, HOMA2-IR associated to steatosis, oxidative stress in plasma and tissues, whereas HF had only a transient increase of leptin and c-peptide. Only HFHF induced fasting hyperglycaemia after 6 months and persistent hyperinsulinaemia and fasting hyperglycaemia with complicated steatosis (inflammation and fibrosis) after 8 months. HFHF and HFD induced endothelial dysfunction at 8 months of diet. CONCLUSIONS: Six months, high fat and high carbohydrate induced T2D with widespread tissues effects. We demonstrated the role of oxidative stress in pathogenesis as well as in complications (hepatic and vascular), reinforcing interest in the use of antioxidants in the prevention and treatment of metabolic diseases, including T2D.

Featured Article: Oxidative stress status and liver tissue defenses in diabetic rats during intensive subcutaneous insulin therapy.

Long-term insulin delivery can reduce blood glucose variability in diabetic patients. In this study, its impact on oxidative stress status, inflammation, and liver injury was investigated. Diabetes was induced in Wistar rats with a single dose of streptozotocin (100 mg/kg). Untreated rats and rats administered Insuplant® (2 UI/200 g/day) through a subcutaneous osmotic pump for one or four weeks were compared with non-diabetic controls. Body weight, fructosamine level, total cholesterol, Insulin Growth Factor-1 (IGF-1) level, lipid peroxidation, and total antioxidant capacity were measured. Hepatic injury was determined through the measurement of glycogen content, reactive oxygen species (ROS) production, and macrophage infiltration. Liver oxidative stress status was evaluated through the measurement of superoxide dismutase (SOD), catalase (CAT), and nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) expression, and p38 mitogen-activated protein kinase (p38MAPK) activation. Induction of diabetes led to increased plasma oxidative stress and inflammation. Moreover, ROS production and macrophage infiltration increased in addition to SOD, CAT, and NADPH oxidase expression. Intensive insulin therapy improved metabolic control in diabetic animals as seen by a restoration of hepatic glycogen, plasma IGF-1 levels, and a decrease in plasma oxidative stress. However, insulin treatment did not result in a decrease in acute inflammation in diabetic rats as seen by continued ROS production and macrophage infiltration in the liver, and a decrease of p38MAPK activation. These results suggest that the onset of diabetes induces liver oxidative stress and inflammation, and that subcutaneous insulin administration cannot completely reverse these changes. Targeting oxidative stress and/or inflammation in diabetic patients could be an interesting strategy to improve therapeutic options.

Oxidative Stress Type Influences the Properties of Antioxidants Containing Polyphenols in RINm5F Beta Cells.

The in vitro methods currently used to screen bioactive compounds focus on the use of a single model of oxidative stress. However, this simplistic view may lead to conflicting results. The aim of this study was to evaluate the antioxidant properties of two natural extracts (a mix of red wine polyphenols (RWPs) and epigallocatechin gallate (EGCG)) with three models of oxidative stress induced with hydrogen peroxide (H2O2), a mixture of hypoxanthine and xanthine oxidase (HX/XO), or streptozotocin (STZ) in RINm5F beta cells. We employed multiple approaches to validate their potential as therapeutic treatment options, including cell viability, reactive oxygen species production, and antioxidant enzymes expression. All three oxidative stresses induced a decrease in cell viability and an increase in apoptosis, whereas the level of ROS production was variable depending on the type of stress. The highest level of ROS was found for the HX/XO-induced stress, an increase that was reflected by higher expression antioxidant enzymes. Further, both antioxidant compounds presented beneficial effects during oxidative stress, but EGCG appeared to be a more efficient antioxidant. These data indicate that the efficiency of natural antioxidants is dependent on both the nature of the compound and the type of oxidative stress generated.

Portal or subcutaneous insulin infusion: efficacy and impact on liver inflammation.

Intraperitoneal insulin allows physiological portal insulin administration and first-pass hepatic insulin extraction, but the impact on liver metabolism and inflammation is unknown. Our objective was to compare the impact, on metabolic control and liver function, of the same dose of insulin administered either intraperitoneally or subcutaneously during continuous infusion in diabetic rats. Wistar rats were randomly divided into 4 groups: control (C), untreated diabetic (streptozotocin, 100 mg/kg) and diabetic rats treated by continual subcutaneous Insuplant® infusion (CSII) and continual intraperitoneal Insuplant(®) infusion (CPII) of 2 UI/200 g/day (via an osmotic mini-pump for 1-4 weeks). Insulin signalling pathways were analysed through hepatic expression of growth hormone receptor and phosphorylated insulin receptor substrate 1. Metabolic control was determined by measurement of body weight, blood glucose and fructosamine. Liver function was assessed by measuring insulin-like growth factor-1 (IGF-1), with global inflammation assessed by levels of alpha-2-macroglobulin (α2M) and lipid peroxidation in plasma. Liver inflammation was evaluated by quantification of hepatic macrophage infiltration and reactive oxygen species production. CPII induced a better improvement in metabolic control and liver function than CSII, producing a significant decrease in blood glucose and fructosamine, coupled with increased IGF-1 and hepatic glycogen storage. Moreover, liver oxidative stress and liver inflammation were reduced. Such observations indicate that the same insulin level in CPII improves glucose control and hepatic glucose metabolism and function, attenuating the hepatic inflammatory response to diabetes. These data demonstrate the importance of focusing on therapeutics to allow first-pass hepatic insulin extraction or prevent diabetic complications.

In Vitro Assessment of Human Islet Vulnerability to Instant Blood-Mediated Inflammatory Reaction (IBMIR) and Its Use to Demonstrate a Beneficial Effect of Tissue Culture.

Culture of human pancreatic islets is now routinely carried out prior to clinical islet allotransplantation, using conditions that have been developed empirically. One of the major causes of early islet destruction after transplantation is the process termed instant blood-mediated inflammatory reaction (IBMIR). The aim of this study was to develop in vitro methods to investigate IBMIR and apply them to the culture conditions used routinely in our human islet isolation laboratory. Freshly isolated or precultured (24 h, 48 h) human islets were incubated in either ABO-compatible allogeneic human blood or Hank's buffered salt solution (HBSS) for 1 h at 37°C. Tissue factor (TF) expression and leukocyte migration were assessed by light microscopy. TF was also quantified by ELISA. To assess ß-cell function, glucose-stimulated insulin secretion (GSIS) assay was carried out. The extent of islet ß-cell damage was quantified using a proinsulin assay. Islets cultured for 24 h had higher GSIS when compared to freshly isolated or 48-h precultured islets. Freshly isolated islets had significantly higher TF content than 24-h and 48-h precultured islets. Incubation of freshly isolated human islets in allogeneic human blood released 6.5-fold higher level of proinsulin in comparison to freshly isolated human islets in HBSS. The high level of proinsulin released was significantly attenuated when precultured islets (24 h or 48 h) were exposed to fresh blood. Histological examination of fresh islets in blood clot showed that some islets were fragmented, showing signs of extraislet insulin leakage and extensive neutrophil infiltration and necrosis. These features were markedly reduced when the islets were cultured for 24 h. These results suggest that our standard 24-h islet culture is markedly beneficial in attenuating IBMIR, as evidenced by increased GSIS, lower content of TF, decrease islet fragmentation, and proinsulin release.

Pro-inflammatory and pro-oxidant status of pancreatic islet in vitro is controlled by TLR-4 and HO-1 pathways.

Since their isolation until implantation, pancreatic islets suffer a major stress leading to the activation of inflammatory reactions. The maintenance of controlled inflammation is essential to preserve survival and function of the graft. Identification and targeting of pathway(s) implicated in post-transplant detrimental inflammatory events, is mandatory to improve islet transplantation success. We sought to characterize the expression of the pro-inflammatory and pro-oxidant mediators during islet culture with a focus on Heme oxygenase (HO-1) and Toll-like receptors-4 signaling pathways. Rat pancreatic islets were isolated and pro-inflammatory and pro-oxidant status were evaluated after 0, 12, 24 and 48 hours of culture through TLR-4, HO-1 and cyclooxygenase-2 (COX-2) expression, CCL-2 and IL-6 secretion, ROS (Reactive Oxygen Species) production (Dihydroethidine staining, DHE) and macrophages migration. To identify the therapeutic target, TLR4 inhibition (CLI-095) and HO-1 activation (cobalt protoporphyrin,CoPP) was performed. Activation of NFκB signaling pathway was also investigated. After isolation and during culture, pancreatic islet exhibited a proinflammatory and prooxidant status (increase levels of TLR-4, COX-2, CCL-2, IL-6, and ROS). Activation of HO-1 or inhibition of TLR-4 decreased inflammatory status and oxidative stress of islets. Moreover, the overexpression of HO-1 induced NFκB phosphorylation while the inhibition of TLR-4 had no effect NFκB activation. Finally, inhibition of pro-inflammatory pathway induced a reduction of macrophages migration. These data demonstrated that the TLR-4 signaling pathway is implicated in early inflammatory events leading to a pro-inflammatory and pro-oxidant status of islets in vitro. Moreover, these results provide the mechanism whereby the benefits of HO-1 target in TLR-4 signaling pathway. HO-1 could be then an interesting target to protect islets before transplantation.

Perfluorodecalin-enriched fibrin matrix for human islet culture.

Disruption of microenvironment and decrease in oxygen supply during isolation and culture lead to pancreatic islet injury and their poor survival after transplantation. This study aimed to create a matrix for culturing islets, using fibrin as scaffold and perfluorodecalin as oxygen diffusion enhancing medium. Human pancreatic islets were divided in four groups: control, islets cultured in fibrin, islets in fibrin containing non-emulsified perfluorodecalin, and finally islets in fibrin supplemented with emulsified perfluorodecalin. After an overnight culture, cell damage (viability, proinsulin and insulin unregulated release, apoptosis (caspase-3 activation), secretory function, and presence of hypoxia markers (HIF-1a and VEGF expression) were assessed. Islets cultured in a matrix, had similar islet viability to controls (no matrix) but decreased levels of active caspase-3 and unregulated hormone release, but high level of hypoxia markers expression. Although the supplementation of fibrin with non-emulsified perfluorodecalin improves secretory response, there was no decrease in hypoxia markers expression. In contrast, emulsified perfluorodecalin added to the matrix improved islet function, islet viability and maintained level of hypoxia markers similar to control. Fibrin matrix supplemented with emulsified perfluorodecalin can provide a beneficial physical and chemical environment for improved pancreatic human islet function and viability in vitro.