E. Seyfritz

In vitro uptake evaluation in Caco-2 cells and in vivo results in diabetic rats of insulin-loaded PLGA nanoparticles.

PLGA nanoparticles (NPs) are largely developed for biological applications but little is known about their uptake. Therefore, we focused our study on the modalities of insulin-loaded PLGA NPs transport across Caco-2 monolayers, and their hypoglycaemic effect on diabetic rats. Insulin-loaded PLGA NPs were formulated by a double emulsion solvent evaporation process. NPs mean diameter was between 130 and 180 nm. NPs were smooth and spherical with an entrapment efficiency above 80%. Fluorescently labeled NPs were incubated with Caco-2 cells to study the process of uptake and the intracellular fate by flow cytometry and confocal laser scanning microscopy. The kinetic of absorption was time-dependent and occurred by clathrin-mediated endocytosis. The intracellular traffic led to a basolateral exocytosis of NPs. In vitro studies and in vivo intraduodenal administration to diabetic rats showed that NPs were resistant in intestinal conditions long enough to allow both the intestinal absorption of NPs and the delivery of functional insulin in bloodstream. The resulting in vivo hypoglycaemic effect was similar to a long-acting insulin one. As no effect on glycaemia occurred after oral administration, further studies need to be conducted to protect NPs from the degradation occurring at the enteric level.

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.

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.

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.

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.

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.

Early effects of liver regeneration on endocrine pancreas: in vivo change in islet morphology and in vitro assessment of systemic effects on β-cell function and viability in the rat model of two-thirds hepatectomy.

Liver and pancreas share key roles in glucose homeostasis. Liver regeneration is associated with systemic modifications and depends especially on pancreatic hormones. The aim of the study was to investigate the role of systemic factors released after two-thirds hepatectomy (2/3H) on early possible consequences of liver regeneration on endocrine pancreas structure and function. The pancreas and serum were harvested 1, 2, or 3 days after 2/3H or sham operation in Lewis rats. The HGF and VEGF serum concentrations and plasma microparticles levels were measured. The fate of endocrine pancreas was examined through islets histomorphometry and function in sham and 2/3H rats. β-Cell line RIN-m5F viability was assessed after 24 h of growth in media supplemented with 10% serum from 2/3H or sham rats instead of FCS. Three days after surgery, the pancreas was heavier in 2/3H compared to sham rats (0.56 vs. 0.40% of body weight, p < 0.05) and the proportion of islets of intermediate size was lower in 2/3H rats (5 vs. 15%, p < 0.05). Compared to Sham, sera obtained 3 days after hepatectomy were more efficient to maintain the viability of RIN-m5F cells (99 vs. 67%, p < 0.01). Three days after surgery, no significant differences in serum HGF, a trend to significant increase in VEGF concentration and a significant increase in microparticles levels, were observed in 2/3H vs. sham rats (9.8 vs. 6.5 nM Phtd Ser Eq., p < 0.05). Liver regeneration is associated with early effects on islets and could influence β-cell viability and function by systemic effect.

Improvement of islet graft function using liraglutide is correlated with its anti‐inflammatory properties

Liraglutide improves the metabolic control of diabetic animals after islet transplantation. However, the mechanisms underlying this effect remain unknown. The objective of this study was to evaluate the anti‐inflammatory and anti‐oxidative properties of liraglutide on rat pancreatic islets in vitro and 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.

Improvement of islet graft function using liraglutide: anti-inflammatory properties

Liraglutide improves the metabolic control of diabetic animals after islet transplantation. However, the mechanisms underlying this effect remain unknown. The objective of this study was to evaluate the anti‐inflammatory and anti‐oxidative properties of liraglutide on rat pancreatic islets in vitro and in vivo.