Improvement of rat islet viability during transplantation: validation of pharmacological approach to induce VEGF overexpression:

Delayed and insufficient revascularization during islet transplantation deprives islets of oxygen and nutrients, resulting in graft failure. Vascular endothelial growth factor (VEGF) could play a critical role in islet revascularization. We aimed to develop pharmacological strategies for VEGF overexpression in pancreatic islets using the iron chelator deferoxamine (DFO), thus avoiding obstacles or safety risks associated with gene therapy. Rat pancreatic islets were infected in vivo using an adenovirus (ADE) encoding human VEGF gene (4.10(8) pfu/pancreas) or were incubated in the presence of DFO (10 mumol/L). In vitro viability, functionality, and the secretion of VEGF were evaluated in islets 1 and 3 days after treatment. Infected islets or islets incubated with DFO were transplanted into the liver of syngenic diabetic rats and the graft efficiency was estimated in vivo by measuring body weight, glycemia, C-peptide secretion, and animal survival over a period of 2 months. DFO induced transient VEGF overexpression over 3 days, whereas infection with ADE resulted in prolonged VEGF overexpression lasting 14 days; however, this was toxic and decreased islet viability and functionality. The in vivo study showed a decrease in rat deaths after the transplantation of islets treated with DFO or ADE compared with the sham and control group. ADE treatment improved body weight and C-peptide levels. Gene therapy and DFO improved metabolic control in diabetic rats after transplantation, but this effect was limited in the presence of DFO. The pharmacological approach is an interesting strategy for improving graft efficiency during transplantation, but this approach needs to be improved with drugs that are more specific

The Protective Effect of Antioxidants Consumption on Diabetes and Vascular Complications

Obesity and diabetes is generally accompanied by a chronic state of oxidative stress, disequilibrium in the redox balance, implicated in the development and progression of complications such as micro- and macro-angiopathies. Disorders in the inner layer of blood vessels, the endothelium, play an early and critical role in the development of these complications. Blunted endothelium-dependent relaxation and/or contractions are quietly associated to oxidative stress. Thus, preserving endothelial function and oxidative stress seems to be an optimization strategy in the prevention of vascular complications associated with diabetes. Diet is a major lifestyle factor that can greatly influence the incidence and the progression of type 2 diabetes and cardiovascular complications. The notion that foods not only provide basic nutrition but can also prevent diseases and ensure good health and longevity is now attained greater prominence. Some dietary and lifestyle modifications associated to antioxidative supply could be an effective prophylactic means to fight against oxidative stress in diabesity and complications. A significant benefit of phytochemicals (polyphenols in wine, grape, teas), vitamins (ascorbate, tocopherol), minerals (selenium, magnesium), and fruits and vegetables in foods is thought to be capable of scavenging free radicals, lowering the incidence of chronic diseases. In this review, we discuss the role of oxidative stress in diabetes and complications, highlight the endothelial dysfunction, and examine the impact of antioxidant foods, plants, fruits, and vegetables, currently used medication with antioxidant properties, in relation to the development and progression of diabetes and cardiovascular complications

Administration orale d'insuline par double encapsulation (Développement du système nanoparticulaire par coacervation complexe insuline/chitosane)

Le diabète est une maladie chronique qui touche plus de 3 millions de personnes en France. Se traduisant par une perte du contrôle de la glycémie, son traitement actuel par injections pluri-quotidiennes d insuline entraîne un inconfort des patients. D autres voies d administrations ont été développées, comme la voie orale qui constitue la voie d administration la plus physiologique, la plus confortable et la mieux acceptée par le patient. Néanmoins, des contraintes physico-chimiques peuvent entraîner une perte de l activité hypoglycémiante de l insuline. Afin de la protéger et de permettre son administration orale, un vecteur pharmaceutique complexe a été élaboré par double encapsulation: un véhicule apportant une protection gastrique et des nanoparticules apportant une protection intestinale. L insuline ainsi encapsulée au sein de ces nanoparticules est protégée du milieu intestinal et peut alors franchir la barrière intestinale pour rejoindre la circulation sanguine afin d être libérée. Ces nanoparticules sont obtenues par coacervation complexeavec le chitosane, polymère naturel bioassimilable. Néanmoins, celui-ci présente une solubilité limitée à pH physiologique rendant difficile sa complexation avec l insuline. Deux dérivés du chitosane ont été utilisés: un dérivé obtenu par modifications chimiques, le N-,O-carboxyméthyl chitosane (NOCC) et un sel de chitosane, le chitosane chlorhydrate. Une caractérisation approfondie de ces deux dérivés hydrosolubles du chitosane permet d envisager la complexation de l insuline à pH physiologique. L étude de la complexation est réalisée dans le cas de ces deux dérivés. L obtention de diagrammes de turbidité permet de définir la zone de formation des nanoparticules. Celles-ci présentent une taille moyenne comprise entre 300 et 500 nm, une charge positive (+ 40 mV) et contiennent en moyenne 85 à 90 % d insuline encapsulée. Des études in vitro ont permis de montrer leur stabilité en milieu intestinal. Leurs propriétés biologiques ont été vérifiées à l aide de manipulations in vivo sur des rats diabétiques. Ces essais ont montré la conservation de l activité biologique de l insuline et la capacité des nanoparticules à induire une diminution de la glycémie pouvant conduire à l obtention de la normoglycémie, 8 heures après leur administration.Les nanoparticules obtenues à l aide des deux dérivés hydrosolubles du chitosane protègent l insuline (conservation de son activité biologique) du milieu intestinal et lui permettent de traverser la barrière intestinale. Ces résultats permettent la validation du concept d encapsulation d insuline par coacervation complexe. Les nanoparticules peuvent désormais faire partie intégrante du vecteur pharmaceutique complexe afin d être administrées par voie orale.Diabetes mellitus is a chronic disease which affects more than 3 millions people in France. Translated by a loss of glycaemia control, its current treated by daily insulin injections entailing discomfort of patients. Other administrations way have been developed and the oral route constitute the most physiological, comfortable and best accepted by patients. Nevertheless, physico-chemical constraints can lead to lose hypoglyceamiant activity of insulin. To avoid it, a pharmaceutical complex vector was developed by double encapsulation: a vehicle bringing a gastric protection and nanoparticles bringing an intestinal protection. By this way, encapsulated insulin in nanoparticles is protected from the intestinal environment and can then cross intestinal barrier to join the blood circulation to be released. These nanoparticles were obtained by complex coacervation with chitosan, a natural and biocompatible polymer. Nevertheless, its low solubility at physiological pH makes its complexation with insulin difficult. Two different water-soluble chitosan derivatives were formed: one obtained by chemical modifications, N-, O-carboxymethyl chitosan (NOCC) and one chitosan salt, chitosan chlorhydrate. A detailed characterization of these two water-soluble derivatives allows insulin complexation at physiological pH. A complexation study made for these two compounds allows the definition of a complexation area where nanoparticles were formed. They had shown average size from 300 to 500 nm, positive charge (+40 mV) and encapsulation efficiency of 85-90 %. In vitro studies allowed to show nanoparticles stability as well as intestinal resistance. Their biological properties were verified by in vivo experiments on diabetic rats, showing the conservation of insulin biological activity and ability to induce glycaemia decrease going to normoglycaemia in 8 hours.Nanoparticles obtained with two water-soluble chitosan derivatives, protect insulin (conservation of biological activity) from the intestinal environment and allow it to cross intestinal barrier. These results permit the validation of insulin encapsulation by complex coacervation concept. Hence nanoparticles can be included in the complex pharmaceutical vector to be administered by oral way.STRASBOURG-Sc. et Techniques (674822102) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Prévention du stress oxydant dans le diabète et ses complications par des antioxydants d origine naturelle

Le stress oxydant, point commun entre le diabète, l'hypertension ou l'obésité, correspond à un déséquilibre entre les défenses antioxydantes endogènes (enzymes, vitamines...) et la production de molécules pro-oxydantes (espèces réactives de l'oxygène, notamment) au profit de celles-ci. Les patients diabétiques présentent une diminution de leur capacité oxydante. En effet, les cellules b du pancréas sont vulnérables au stress oxydant mais peuvent être protégées par un apport exogène alimentaire d'antioxydants. Ceux-ci peuvent améliorer la glycémie; et la consommation modérée de vin rouge (polyphénols) réduit la mortalité liée aux maladies cardiovasculaires. Le but de ce travail a été de développer des outils de screening et d'analyses d'antioxydants d'origine naturelle et de les tester sur un modèle de rat intolérant au glucose. Deux composés aux effets protecteurs sur le stress induit sur des cellules b de rat (RINm5F) ont été retenus : les extraits de polyphénols de vin rouge Corbières (PP) et l'épigallocatéchine gallate (EGCG) du thé vert. Sur des rats rendus diabétiques par une nourriture hypercalorique et présentant un stress oxydant, ces composés et l'exercice physique ont montré des effets bénéfiques : l'EGCG réduit les marqueurs du stress oxydant alors que l'exercice et les PP diminuent la stéatose. Les trois diminuent l'hyperinsulinisme mais seul l'EGCG améliore la glycémie. La combinaison des trois traitements pourrait améliorer les différents paramètres étudiés. Ainsi des mesures hygiéno-diététiques combinées à une supplémentation antioxydante constitueraient des moyens prophylactiques efficaces pour lutter contre le stress oxydant dans le diabète de type 2.Oxidative stress, common point between endogenous antioxidative defences (enzymes, vitamins...) and free radicals especially reactive oxygen species (ROS) in favour of these. Diabetic patients present a lack in antioxidative capacity. Pancreatic b-cells are very susceptible to oxidative stress but they can be protected by exogenous antioxidative molecules from diet. These antioxidants improve glycaemia and a moderate consumption of red wine, which contains antioxidative polyphenols, is associated to a reduction of mortality due to cardiovascular diseases. Oxidative stress and diabetes are strongly involved and the aim of my thesis was to develop some screening and analysing tools for natural antioxidative molecules and to test selected molecules on rat model of glucose intolerance. Thanks to these tools, two antioxidants with protective effect against oxidative stress on b-cells line (RINm5F) were selected: red wine polyphenols from Corbières (PP) and EGCG ( EpiGalloCatechin Gallate) from green tea. On rats become diabetic by high fat diet and presenting an oxidative stress, both antioxidants and exercise show benefic effects: EGCG reduces oxidative stress markers while exercise and PP decrease steatosis. All treatment seems to decrease hyperinsulinism while only EGCG improve glycemia. Combination of three treatments could improve more important different studied parameters. Thus, some dietary and lifestyle modifications associated to oxidative supply could be an effective prophylactic means to fight against oxidative stress in type 2 diabetes.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Identification des mécanismes cellulaires et moléculaires à l'origine de la perte précoce des îlots pancréatiques au cours de la transplantation

De l isolement des îlots pancréatiques à leur implantation, l inflammation est omniprésente au cours de la transplantation d îlots pancréatiques. Le maintien d une inflammation contrôlée est essentiel pour préserver la survie et la fonctionnalité du greffon à court et long terme. L objectif de ce travail de thèse est d identifier précisément les mécanismes inflammatoires à l origine de la perte précoce des îlots et de déterminer des cibles thérapeutiques pour limiter ces réactions inflammatoires.Nous avons ainsi démontré que les conditions de culture induisent des réactions à l origine du développement d un phénotype pro-inflammatoire et pro-oxydant propre à l îlot. Cette induction se caractérise par une élévation de la sécrétion de cytokines, de chimiokines pro-inflammatoires, une activation des voies de l inflammation Toll-like récepteurs (TLRs)-dépendantes et une génération d espèces réactives de l oxygène (ROS). Toutefois, ce processus peut être prévenu par l activation de l Hème oxygénase-1 (HO-1), une enzyme anti-oxydante et anti-inflammatoire.Par l étude des réactions inflammatoires sur un modèle animal de transplantation mimant les conditions de transplantation humaine, nous avons démontré qu un changement des médiateurs plasmatiques de l inflammation et du protéome hépatique s opère 12 heures après transplantation. De plus, ces résultats sont associés à une infiltration des îlots par les cellules immunitaires qui s organise 12 heures après transplantation. Nous avons également établi le rôle anti-inflammatoire de la rapamycine (une drogue immunomodulatrice) sur les îlots et les macrophages in vitro. Nous avons ainsi démontré que l usage de la rapamycine avec la mise en place d un pré-traitement des îlots et du receveur avant la greffe serait envisageable. Ces travaux ont permis de caractériser les mécanismes inflammatoires mis en oeuvre immédiatement avant et après transplantation. Ainsi, ces données offrent de nouvelles pistes thérapeutiques susceptibles de prévenir et/ou limiter l inflammation au cours de la transplantation d îlots pancréatiques.From isolation of pancreatic islets to their implantation, the inflammation is ubiquitous in the pancreatic islet transplantation. Maintaining a controlled inflammation is essential to preserve the survival of the graft and the functionality in the short and long term. The objective of this work is to identify precisely the inflammatory mechanisms behind the early loss of islets and identify therapeutic targets to reduce these inflammatory reactions. We have demonstrated that culture conditions induce reactions causing the development of a specific proinflammatory and pro-oxydant phenotype islet. This induction is characterized by an increase in the secretion of cytokines, chemokines pro-inflammatory activation pathways of inflammation Toll-like receptors (TLRs) -dependent and generation of reactive oxygen species (ROS). However, this process can be prevented by the activation of Heme oxygenase-1 (HO-1), an antioxidant and anti-inflammatory enzyme.By studying the inflammatory responses in an animal model of transplantation mimicking the conditions of human transplantation, we demonstrated that a change of plasma mediators of inflammation and liver proteome occurs 12 hours after transplantation. Furthermore, these results are associated with infiltration of the islets by immune cells which organizes 12 hours after transplantation. We also determined the anti-inflammatory role of rapamycin (an immunomodulatory drug) on the islets and macrophages in vitro. We have thus demonstrated that the use of rapamycin with the establishment of a pre-treatment of islets and recipient before transplantation could be considered. These studies have characterized the inflammatory mechanisms implemented immediately before and after transplantation. Thus, these data provide new therapeutic approaches that can prevent and / or reduce inflammation during pancreatic islet transplantationSTRASBOURG-Bib.electronique 063 (674829902) / SudocSudocFranceF

Evaluation du pouvoir anti-oxydant des aliments (recherche de leurs effets modulateurs sur le stress oxydant dans le cas du diabète)

L obésité et le diabète de type 2 s accompagne généralement d un stress oxydant chronique, participant à l installation et la progression du diabète, mais également à l apparition de nombreuses complications dont les macro- et micro-angiopathies. Certains aliments, comme les fruits et légumes naturellement riches en molécules anti-oxydantes, représentent ainsi un réel potentiel de prévention dans ce contexte. Cette étude s est intéressée au pouvoir anti-oxydant des fruits et légumes. Nous avons démontré que la majorité des fruits et légumes doivent leur capacité anti-radicalaire a des composés polaires. Leur étude par une méthode séparative (HPLC) couplée à une détection spécifique de capacité anti-radicalaire, a permis de révéler quelles sont les molécules réellement actives. Le chou rouge a été révélé comme aliment à fort potentiel anti-oxydant, de par la présence de nombreuses cyanidines dotés de capacités anti-radicalaires. L étude in vitro sur un modèle cellulaire (RINm5F) a confirmé l action anti-radicalaire des polyphénols des fruits et légumes. L extrait de chou rouge a ainsi permis la suppression du stress oxydant induit par un hyperinsulinisme. Enfin, une étude in vivo, sur le modèle de rat diabétique, a permis de confirmer les effets anti-oxydants et anti-diabétiques du chou rouge, même lorsque sa consommation est associée à un régime hypercalorique. Cependant, le chou rouge doit être associé à une alimentation équilibrée afin d être bénéfique à long terme.Obesity and type 2 diabetes is generally accompanied by a chronic state of oxidative stress, implicated in the development of complications such as macro- and micro-angiopathies. Food, and particularly fruit and vegetables naturally rich in radical scavenging compounds, represent a real preventive potential. This study was focused on the antioxidant capacity of fruit and vegetables. We demonstrated that the majority of fruit and vegetables owe their anti-oxydant capacity to a class of polar compounds. The study by means of a separative technique (HPLC) hyphenated with specific radical scavenging capacity detection, allowed to reveal the real active compounds. Red cabbage has shown amongst the highest antioxidant potential through the presence of numerous radical scavenging cyanidins. In vitro studies, using the RINm5f cell line, confirmed the radical scavenging activity of fruit and vegetables polyphenols. The red cabbage extract suppressed the hyperinsulinemic-induced oxidative stress. Finally, an in vivo study, using a diabetic rat model, confirmed the antioxidant and antidiabetic effects of red cabbage, even when its consumption is associated to a hypercaloric diet. However, the red cabbage must be associated with an equilibrated diet in order offer beneficial effects in the long term.STRASBOURG-Bib.electronique 063 (674829902) / SudocSudocFranceF

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

Influence of the physicochemical parameters of solvents in the extraction of bioactive compounds from Parinari macrophylla Sabine (Chrysobalanaceae)

International audienceThe extraction of bioactive compounds from medicinal plants requires methods which are as diverse as the chemical nature of the compounds themselves. In this study, a 96-well microplate was used where solvent mixtures spanning wide ranges of selectivity and polarity were tested with the objective of extracting a broad range bioactive compounds from plant material. Microplate wells were filled with plant material and the solvents and their mixtures were added. The obtained extracts were assessed in terms of their total antioxidant activity, oxygen radical absorbance capacity and effects on cell viability. An aqueous extract, generally used by traditional therapists, was also included in the study. The results showed that the extracts using methanol with acetic acid (0.1%, v:v), chloroform/ethanol, butanol/DMF, butanol/acetonitrile, ethylene glycol with acetic acid (0.1%, v:v), MTBE/DMSO, ethylene glycol, pentane/ethanol (v:v), ethanol, DMF, DMF with acetic acid (0.1%, v:v), DMSO, DMSO with acetic acid (0.1%, v:v) and THF had a higher antioxidant activity than the aqueous extract. Extracts with greater antioxidant activity than the aqueous extract were obtained largely from solvent mixtures with the exception of ethanol, DMF, DMSO and THF. The antioxidant activity obtained in TEAC varied between 1474.1±4.4 and 3183.0±16.0 μmol TE/g dry extract respectively for aqueous and THF extracts; in ORAC between 1727.7±8.4 and 2683.5±11.7 μmol TE/g dry extract for aqueous and DMSO acetic acid 1%, respectively, with mean ±SEM. In TEAC the THF extract had the highest antioxidant potential with 3183.0±16.0 μmol TE / g dry extract. The DMSO acetic acid (0.1%, v:v) extract had the highest antioxidant potential in ORAC with 2683.5±11.7 μmol TE / g dry extract. Cell viability test using β-pancreatic cells showed that only the acidified methanol extract was toxic after one hour of incubation. After 24 hours, cell viability was less than 70% for extracts using butanol/acetonitrile, MTBE/DMF, acidified methanol, pentane/ethanol and acidified DMF

Validation of a major modification of the mobile phase in the European Pharmacopoeia LC-UV human insulin assay

International audienceTo prevent the buildup of salt crystals inside the chromatograph tubing and pump that occurs with the high mobile phase sodium sulfate concentration (148 mM) used in the European Pharmacopoeia method for the quantification of human insulin, the use of a lower concentration was proposed. A minimal concentration of 37 mM sodium sulfate maintained the chromatographic performance while avoiding instrumental damage. This buffer concentration modification is considered as a major change by the European Pharmacopoeia and this improved method therefore had to be validated. Using a single-point calibration (600 µg.mL−1) yielded adequate precision (the highest relative standard deviations for repeatability and intermediate precision were <5% and <8%, respectively) and accuracy (bias values between −8% and +7%) for the quantification of human insulin in solutions of known concentration and in commercial insulin preparations. The limits of detection and quantification (1.6 and 4.5 µg.mL−1, respectively) were largely sufficient for the purpose of the metho