Carole Mura

Mammalian retinal horizontal cells are unconventional GABAergic neurons

J. Neurochem. (2011) 116, 350–362.

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.

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.

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.

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

&NA; 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 ± 2 mV) were more negatively charged than −PVA‐NPs (–22 ± 1 mV) and chitosan‐coated NPs were highly positively charged (56 ± 2 mV) compared to +PVA particles (−2 ± 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.

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.

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.

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.