C. Peronet

Design, characterisation, and bioefficiency of insulin-chitosan nanoparticles after stabilisation by freeze-drying or cross-linking.

Insulin delivery by oral route would be ideal, but has no effect, due to the harsh conditions of the gastrointestinal tract. Protection of insulin using encapsulation in self-assembled particles is a promising approach. However, the lack of stability of this kind of particles in biological environments induces a low bioavailability of encapsulated insulin after oral administration. The objective of this work was to evaluate the effect of two stabilisation strategies alone or combined, freeze-drying and cross-linking, on insulin-loaded chitosan NPs, and to determine their bioefficiency in vitro and in vivo. NPs were prepared by complex coacervation between insulin and chitosan, stabilised either by cross linking with sodium tripolyphosphate solution (TPP), by freeze-drying or both treatments. In vitro bioefficiency NP uptake was evaluated by flow cytometry on epithelial models (Caco-2/RevHT29MTX (mucus secreting cells)). In vivo, NPs were injected via catheter in the peritoneum or duodenum on insulinopenic rats. Freeze-drying increased in size and charge (+15% vs control 412 ± 7 nm; + 36 ± 0.3 mV) in comparison with cross linking which decreased NP size (-25%) without impacting the NP charge. When combined the consecutive treatments reduced NPs size and increased charges as compared to standard level. Freeze drying is necessary to prevent the destruction of NP in intestinal environment in comparison with no freeze dryed one where 60% of NP were destroyed after 2h. Additionally freeze drying combined with cross linking treatments improved bioefficiency of NP with uptake in cell increased when mucus is present. Combination of both treatment showed a protection of insulin in vivo, with a reduction of glycemia when NPs were administrated. This work showed that the combination of freeze drying and cross linking treatment is necessary to stabilize (freeze-drying) and increase bioefficiency (cross-linking) of self assembled NP in the delivery of insulin in vitro and in vivo.

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 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.

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.

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.

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.

Treatment of NASH with Antioxidant Therapy: Beneficial Effect of Red Cabbage on Type 2 Diabetic Rats

Aims Oxidative stress (OS) plays a major role in type 2 diabetes and its vascular and hepatic complications, and novel therapeutic approaches include natural antioxidants. Our previous chemical and biological studies demonstrated the antioxidant activities of red cabbage (RC), and here, we aimed to determine the in vivo effects of 2-month long RC consumption using a high-fat/high-fructose model of diabetic rats. Results This vegetable, associated with lifestyle measurement, was shown to decrease OS and increase vascular endothelial NO synthase expression, ensuring vascular homeostasis. In the liver, RC consumption decreased OS by inhibiting p22phox expression and Nrf2 degradation and increasing catalase activity. It inhibited the activation of SREBP (1c, 2), ChREBP, NF-κB, ERK1/2, PPARγ, and GS and SIRT1 decrease, as observed in diabetic rats. Conclusion/innovation RC consumption led to metabolic profile improvement, together with hepatic function improvements. Although lifestyle changes are not sufficient to prevent diabetic complications, enrichment with RC avoids progression hepatic complications. This antioxidant strategy using RC does not only able to increase antioxidant defense, such as classical antioxidant, but also able to assure a metabolic and energetic balance to reverse complications. Whereas traditional medical therapy failed to reverse NASH in diabetic patients, consumption of RC should be a natural therapy to treat it.

Dyslipidémies et diabèteP116: Apport d’un régime riche en lipides et en fructose dans la mise en place d’un modèle de diabète de type 2 chez le rat : impacts métaboliques et oxydatifs

Introduction et but de l’etude L’utilisation de modeles animaux represente un outil indispensable dans la comprehension du diabete. Notre objectif est de mettre en place un modele plus proche de la physiopathologie humaine , par une alimentation grasse et sucree, et d’evaluer les consequences metaboliques, oxydatives, systemiques et tissulaires. Materiel et methodes Les rats mâles Wistar recevant une alimentation standard ou grasse + 25 % fructose dans l’eau (HFHF) sont sacrifies apres 0, 2, 4, 8 mois (M) de regime. Nous evaluons le delai d’apparition du diabete (xM) et ses repercussions metaboliques (poids, glycemies, leptinemie), oxydatives (capacite antioxydante totale CAOT, peroxydation lipidique, carbonylation proteique), tissulaires (foie, pancreas, muscles, vaisseaux). Resultats et Analyse statistique Le regime HFHF induit une augmentation du poids (1M), de la leptine (1M), un hyperinsulinisme (2M), une intolerance au glucose (2M), des hyperglycemies a jeun (6M). Les ilots pancreatiques presentent une hypertrophie (4M) associee a une augmentation de l’insuline (immunomarquage). Apres 8M, une perte des gros/moyens ilots est associee a une diminution du contenu insulinique. Une steatose hepatique (2M) s’accompagne d’une fibrose (8M). La production du glycogene hepatique est augmentee (4M). Un stress oxydant plasmatique est observe (augmentation des proteines carbonylees et de la CAOT (2M), des lipides peroxydes (4M)). Un stress oxydant tissulaire est visible dans l’ensemble des organes etudies (2M). Conclusion L’ajout de sucre au regime gras est indispensable a l’evolution du syndrome metabolique en diabete de type 2. Dans cette etude 6 mois de cette alimentation sont necessaire pour developper un diabete de type 2, avec des repercussions tissulaires generalisees. Ce modele permettra une meilleure comprehension de la physiopathologie du diabete et son evolution, dans le but unique de developper de nouvelles therapies antidiabetiques preventives/curatives.

P1041 Apport d’un régime riche en lipides et en fructose dans la mise en place d’un modèle de diabète de type 2 chez le rat : impacts métaboliques et oxydatifs

Introduction L’utilisation de modeles animaux represente un outil indispensable dans la comprehension du diabete. Notre objectif est de mettre en place un modele plus proche de la physiopathologie humaine, par une alimentation grasse et sucree, et d’evaluer les consequences metaboliques, oxydatives, systemiques et tissulaires. Materiels et methodes Les rats mâles Wistar recevant une alimentation standard ou grasse + 25 % fructose dans l’eau (HFHF) sont sacrifies apres 0, 2, 4, 8 mois (M) de regime. Nous evaluons le delai d’apparition du diabete (xM) et ses repercussions metaboliques (poids, glycemies, leptinemie), oxydatives (capacite antioxydante totale CAOT, peroxydation lipidique, carbonylation proteique), tissulaires (foie, pancreas, muscles, vaisseaux). Resultats Le regime HFHF induit une augmentation du poids (1M), de la leptine (1M), un hyperinsulinisme (2M), une intolerance au glucose (2M), des hyperglycemies a jeun (6M). Les ilots pancreatiques presentent une hypertrophie (4M) associee a une augmentation de l’insuline (immunomarquage). Apres 8M, une perte des gros/moyens ilots est associee a une diminution du contenu insulinique. Une steatose hepatique (2M) s’accompagne d’une fibrose (8M). La production du glycogene hepatique est augmentee (4M). Un stress oxydant plasmatique est observe (augmentation des proteines carbonylees et de la CAOT (2M), des lipides peroxydes (4M)). Un stress oxydant tissulaire est visible dans l’ensemble des organes etudies (2M). Conclusion 6 mois de regime hyperlipidique/glucidique sont necessaires pour developper un diabete de type 2, avec des repercussions tissulaires generalisees. Ce modele permettra une meilleure comprehension de la physiopathologie du diabete et son evolution, dans le but unique de developper de nouvelles therapies antidiabetiques preventives/curatives.