Michel Tournier

4-Hydroxyisoleucine: experimental evidence of its insulinotropic and antidiabetic properties

We have recently shown in vitro that 4-hydroxyisoleucine (4-OH-Ile), an amino acid extracted from fenugreek seeds, potentiates insulin secretion in a glucose-dependent manner. The present study was designed to investigate whether 4-OH-Ile could exert in vivo insulinotropic and antidiabetic properties. For this purpose, intravenous or oral glucose tolerance tests (IVGTTs and OGTTs, respectively) were performed not only in normal animals but also in a type II diabetes rat model. During IVGTT in normal rats or OGTT in normal dogs, 4-OH-Ile (18 mg/kg) improved glucose tolerance. The lactonic form of 4-OH-Ile was ineffective in normal rats. In non-insulin-dependent diabetic (NIDD) rats, a single intravenous administration of 4-OH-Ile (50 mg/kg) partially restored glucose-induced insulin response without affecting glucose tolerance; a 6-day subchronic administration of 4-OH-Ile (50 mg/kg, daily) reduced basal hyperglycemia, decreased basal insulinemia, and slightly, but significantly, improved glucose tolerance. In vitro, 4-OH-Ile (200 μM) potentiated glucose (16.7 mM)-induced insulin release from NIDD rat-isolated islets. So, the antidiabetic effects of 4-OH-Ile on NIDD rats result, at least in part, from a direct pancreatic B cell stimulation.We have recently shown in vitro that 4-hydroxyisoleucine (4-OH-Ile), an amino acid extracted from fenugreek seeds, potentiates insulin secretion in a glucose-dependent manner. The present study was designed to investigate whether 4-OH-Ile could exert in vivo insulinotropic and antidiabetic properties. For this purpose, intravenous or oral glucose tolerance tests (IVGTTs and OGTTs, respectively) were performed not only in normal animals but also in a type II diabetes rat model. During IVGTT in normal rats or OGTT in normal dogs, 4-OH-Ile (18 mg/kg) improved glucose tolerance. The lactonic form of 4-OH-Ile was ineffective in normal rats. In non-insulin-dependent diabetic (NIDD) rats, a single intravenous administration of 4-OH-Ile (50 mg/kg) partially restored glucose-induced insulin response without affecting glucose tolerance; a 6-day subchronic administration of 4-OH-Ile (50 mg/kg, daily) reduced basal hyperglycemia, decreased basal insulinemia, and slightly, but significantly, improved glucose tolerance. In vitro, 4-OH-Ile (200 microM) potentiated glucose (16.7 mM)-induced insulin release from NIDD rat-isolated islets. So, the antidiabetic effects of 4-OH-Ile on NIDD rats result, at least in part, from a direct pancreatic B cell stimulation.

Antihyperglycemic effect of a natural chicoric acid extract of chicory (Cichorium intybus L.): A comparative in vitro study with the effects of caffeic and ferulic acids

ETHNOPHARMACOLOGICAL RELEVANCE In Eurasia folk medicine, roots of chicory (Cichorium intybus L.) have been reported to exert antidiabetic benefits. In vitro, a natural chicoric acid extract (NCRAE) from Cichorium intybus root has been shown to increase insulin secretion by pancreatic β-cells and glucose uptake by muscle cells. MATERIALS AND METHODS In vitro experiments were designed to compare the effects of two hydroxycinnamic acids, caffeic and ferulic acids, to those obtained with NCRAE (50 and 100 µg.mL(-1)) on the three major tissues implicated in glycemic regulation (pancreas, muscle and liver). In vivo experiments were performed in Wistar rats submitted to a daily intraperitoneal injection of NCRAE (3, 15 or 30 mg kg(-1)) for 4 days. On the fourth day, an intraperitoneal glucose tolerance test (IPGTT; 1 g kg(-1)) was carried out. RESULTS Our results show that the three compounds we used are able each to induce an original response. Caffeic acid mainly promotes a decrease in hepatic glycogenolysis. Ferulic acid elicits a clear increase of insulin release and a reduction of hepatic glycogenolysis. However, this compound induces an inhibition of muscle glucose uptake. NCRAE provokes an increase of insulin release and glucose uptake without any effect on hepatic glycogenolysis. We could also show that none of these compounds implicates hepatic glucose 6-phosphatase in contrast to chlorogenic acid, known as an inhibitor of glucose 6-phosphatase and which is able to decrease glucose output from hepatocytes. Our results point out that NCRAE is able to decrease blood glucose without any effect hepatic effect. Our in vivo experiments bring evidence that 4 daily IP administrations of NCRAE improve IP glucose tolerance in a dose-dependent manner and mainly via an insulin sensitizing effect. CONCLUSIONS We conclude that NCRAE presents an antihyperglycemic effect essentially due to a peripheral effect on muscle glucose uptake.

Chemical analysis and antihyperglycemic activity of an original extract from burdock root (Arctium lappa).

In the present study, we obtained a dried burdock root extract (DBRE) rich in caffeoylquinic acids derivatives. We performed the chemical characterization of DBRE and explored its antihyperglycemic potential in both in vitro and in vivo experiments. Chemical analysis of DBRE using LC-MS and GC-MS revealed the presence of a great majority of dicaffeoylquinic acid derivatives (75.4%) of which 1,5-di-O-caffeoyl-4-O-maloylquinic acid represents 44% of the extract. In the in vitro experiments, DBRE is able to increase glucose uptake in cultured L6 myocytes and to decrease glucagon-induced glucose output from rat isolated hepatocytes together with a reduction of hepatic glucose 6-phosphatase activity. DBRE did not increase insulin secretion in the INS-1 pancreatic β-cell line. In vivo, DBRE improves glucose tolerance both after intraperitoneal and oral subchronic administration. In conclusion, our data demonstrate that DBRE constitutes an original set of caffeoylquinic acid derivatives displaying antihyperglycemic properties.

Relaxin and atrial natriuretic peptide pathways participate in the anti-fibrotic effect of a melon concentrate in spontaneously hypertensive rats

Background In spontaneously hypertensive rats (SHR), a model of human essential hypertension, oxidative stress is involved in the development of cardiac hypertrophy and fibrosis associated with hypertension. Dietary supplementation with agents exhibiting antioxidant properties could have a beneficial effect in remodeling of the heart. We previously demonstrated a potent anti-hypertrophic effect of a specific melon (Cucumis melo L.) concentrate with antioxidant properties in spontaneously hypertensive rats. Relaxin and atrial natriuretic peptide (ANP) were reported to reduce collagen deposition and fibrosis progression in various experimental models. Objective The aim of the present investigation was to test the hypothesis that, beside reduction in oxidative stress, the melon concentrate may act through relaxin, its receptor (relaxin/insulin-like family peptide receptor 1, RXFP1), and ANP in SHR. Design and results The melon concentrate, given orally during 4 days, reduced cardiomyocyte size (by 25%) and totally reversed cardiac collagen content (Sirius red staining) in SHR but not in their normotensive controls. Treatment with the melon concentrate lowered cardiac nitrotyrosine-stained area (by 45%) and increased by 17–19% the cardiac expression (Western blot) of superoxide dismutase (SOD) and glutathione peroxidase. In addition, plasma relaxin concentration was normalized while cardiac relaxin (Western blot) was lowered in treated SHR. Cardiac relaxin receptor level determined by immunohistochemical analysis increased only in treated SHR. Similarly, the melon concentrate reversed the reduction of plasma ANP concentration and lowered its cardiac expression. Conclusions The present results demonstrate that reversal of cardiac fibrosis by the melon concentrate involves antioxidant defenses, as well as relaxin and ANP pathways restoration. It is suggested that dietary SOD supplementation could be a useful additional strategy against cardiac hypertrophy and fibrosis.

Increase in insulin sensitivity by the association of chicoric acid and chlorogenic acid contained in a natural chicoric acid extract (NCRAE) of chicory (Cichorium intybus L.) for an antidiabetic effect

ETHNOPHARMACOLOGICAL RELEVANCE Chicory (Cichorium intybus L.) is an indigenous vegetable widely cultivated in Europe, America and Asia. In ancient times, the leaves, flowers, seeds, and roots have been used as a wealth of health benefits including its tonic effects, the ability to ease digestive problems and to detoxify liver. In Indian traditional therapy, chicory was known to possess antidiabetic effect. In the traditional medicine of Bulgaria and Italy, chicory was used as hypoglycemic decoctions. AIMS OF THE STUDIES We wanted to obtain the complete chemical composition of the natural chicoric acid extract (NCRAE), a chicory root extract rich in chicoric acid, which previously showed its glucose tolerance effect in normal rats. To investigate if the whole NCRAE is required to be effective, we performed a comparative in vivo experiment on STZ diabetic rats treated either with NCRAE or a mixture composed of the two major compounds of NCRAE. MATERIALS AND METHODS LC-MS method has been used to analyze the exhaustive composition of NCRAE: we have determined that chicoric acid and chlorogenic acid represented 83.8% of NCRAE. So, we have prepared a solution mixture of chicoric acid and chlorogenic acid named SCCAM, in order to compare in vivo the antidiabetic effects of this last and NCRAE in streptozotocin diabetic rats. In vitro experiments were performed on L6 cell line both for glucose uptake and for the protective effect against H2O2 oxidative stress. Also, we have evaluated DPPH and ORAC (Oxygen Radical Absorbance Capacity) antioxidative capacities of the two compositions. RESULTS The LC-MS analysis confirmed the high abundance of chicoric acid (64.2%) in NCRAE and a second part of NCRAE is composed of caffeoylquinic acids (CQAs) at 19.6% with among them the chlorogenic acid. This result has permitted us to prepare a mixture of synthetic L-chicoric acid (70%) and synthetic chlorogenic acid (30%): the solution is designated SCCAM. Our results showed that both NCRAE and SCCAM are able to improve a glucose tolerance in STZ diabetic rats after a subchronic administration of seven days. Alone NCRAE allows to significantly decrease the basal hyperglycemia after six days of treatment. To explain these difference of effects between NCRAE and SCCAM, we have compared their in vitro effects on the L6 muscle cell line both for the insulin sensitizing effect and for their protective action in pretreatment against H2O2. We have also compared their antioxidant capacities. In conclusion, we demonstrated that NCRAE, a natural extract of chicory (Cichorium intybus) rich in CRA and CQAs improves glucose tolerance and reduces the basal hyperglycemia in STZ diabetic rats.

Sodium restriction modulates innate immunity and prevents cardiac remodeling in a rat model of metabolic syndrome

In the view of the relationships between excessive sodium intake, immunity and target organ damage, we hypothesized that reduction in dietary sodium would be beneficial in the prevention of cardiac alterations through a restrained local immunity response in a rat model of metabolic syndrome. Sprague-Dawley rats were fed a 60% fructose diet with either a normal sodium (0.64% NaCl) or a low sodium content (<0.01% NaCl) for 8weeks. After 4weeks, rats were infused or not with angiotensin II (200ng·kg-1·min-1, sc) for 4weeks. Tail-cuff blood pressure was determined in conscious rats. Heart and left ventricle weight, cardiomyocyte size, and cardiac fibrosis were evaluated. We performed a transcriptomic analysis in order to identify differentially regulated cardiac mRNAs between normal and low sodium diets. We validated those results using qPCR and immunohistochemistry. Angiotensin II-induced blood pressure rise was blunted (~50%) in the low-sodium fed rats while cardiac hypertrophy and fibrosis were prevented. Transcriptomic analysis revealed 66 differentially regulated genes including 13 downregulated genes under the low sodium diet and implicated in the innate immune response. This was confirmed by reduced cardiac macrophages infiltration under the low sodium diet. Dietary sodium restriction prevents structural alterations of the heart of rats with fructose-induced insulin resistance and angiotensin II-hypertension. The reduction of cardiac inflammation and macrophage infiltration suggests that innate immunity has an important role in the beneficial effect of sodium restriction on cardiac remodeling.

P256 Biocommunication locale entre le tissu adipeux péripancréatique et la cellule béta pabcréatique : caractérisation immunologique

Rationnel Lors de l’obesite, les facteurs secretes par le tissu adipeux influencent la fonction et la masse β-cellulaire. Lorsque l’adiposite viscerale augmente, se developpe un tissu adipeux peri-pancreatique (TAPP), qui penetre le pancreas et etablit des contacts avec les ilots de Langerhans. Rebuffat et al (Endocrinology, 2012) ont montre dans un modele d’obesite et d’insulino-resistance que des facteurs secretes par le TAPP, modulent la proliferation des cellules β. Ceci est en faveur d’une biocommunication locale entre le TAPP et la cellule β. Notre objectif est de caracteriser le TAPP en termes de cellules immunitaires et de facteurs inflammatoires secretes lors du diabete de type 2 (DT2). Materiels et methodes Notre etude est realisee chez le rat ZDF un modele animal d’obesite et de DT2. Trois groupes d’animaux sont etudies, rats pre-diabetiques (6 et 10 semaines) et rats diabetiques (12 semaines). La presence de cellules immunitaires infiltrant le TAPP est etudiee par immunohistochimie a l’aide du marqueur macrophagique ED1. Le profil d’expression des cytokines secretees par les TAPP est analyse par la technologie de puce a anticorps (Chemiarray, RD System). Resultats Une augmentation des macrophages infiltrant le PPAT est observees chez les animaux pre-diabetiques. Parmi les cytokines/chimiokines analyses, SICAM-1, IL-1α, IP-10, L-selectine, TIMP-1, TNF et VEGF sont secretees par le TAPP independamment du stade de la pathologie. En revanche, CINC- 1, 2, 3, IL-1β, LIX, MIP-1α apparaissent uniquement dans le secretome du TAPP d’animaux pre-diabetiques (10 s). Le niveau de secretion de TIMP-1 est augmente d’un facteur 4. Conclusion Nos resultats montrent que le profil et le niveau d’expression de cytokines/chimiokines secretees par le TAPP varie du stade de pre-diabete au DT2. Ces variations (i) permettraient la mise en place et le maintien de l’inflammation en attirant des cellules immunitaires dans le TAPP et (ii) contribueraient au dysfonctionnement progressif β-cellulaire survenant dans le DT2.

PO21 Dégradation accrue de la NO synthase neuronale par le système ubiquitine-protéasome dans le muscle squelettique chez le rat Zucker fa/fa insulinorésistant

Introduction Le muscle squelettique, dont la sensibilite a l’insuline est alteree dans le diabete de type 2, exprime un variant d’epissage de l’isoforme neuronale de la nitric oxyde synthase (nNOS), la nNOSμ. Etant donne le role physiologique de la nNOSμ dans la captation du glucose, notre objectif est de determiner si des anomalies de la nNOSμ musculaire pourraient intervenir dans les etats prediabetiques et les stades precoces du diabete de type 2. Dans ce but, nous avons choisi comme modele, le rat obese Zucker fa / fa , associant une forte insulinoresistance et un hyperinsulinisme caracteristiques d’un etat prediabetique. Materiels et Methodes L’expression de la nNOSμ a ete mesuree par PCR quantitative et par Western blot. L’activite catalytique de la nNOSμ a ete evaluee en mesurant la production de citrulline a partir d’arginine radiomarquee. L’agent MG132 a ete utilise pour inhiber l’activite du proteasome. Enfin, la localisation subcellulaire de la nNOSμ a ete determinee par immunofluorescence a l’aide d’un anticorps specifique. Resultats Chez le rat obese Zucker fa / fa , nous avons trouve une diminution de l’activite catalytique de la nNOSμ d’environ 41 % par rapport aux rats fa /+ controles (P fa / fa , suggerant une degradation proteasomale de la nNOSμ. L’utilisation de l’inhibiteur du proteasome MG132 sur les muscles isoles nous a permis de mettre en evidence une augmentation du taux d’ubiquitination de la nNOSμ chez les rats fa / fa . De plus, en inhibant le proteasome, nous avons pu restaurer significativement (P fa /+. Par immunofluorescence, nous avons confirme la baisse d’expression de la nNOSμ chez le rat fa / fa et observe, au niveau subcellulaire, une perturbation de sa distribution sous-membranaire. Conclusion Des alterations fonctionnelles de la nNOSμ resultant d’une degradation proteasomale accrue observee dans le muscle squelettique des rats obeses fa / fa , pourraient etre impliquees dans l’insulinoresistance musculaire dans certains stades precoces du diabete de type 2.