Beta-Cell-Specific Expression of Nicotinamide Adenine Dinucleotide Phosphate Oxidase 5 Aggravates High-Fat Diet-Induced Impairment of Islet Insulin Secretion in Mice.

Aims: Nicotinamide adenine dinucleotide phosphate oxidases (NOX-es) produce reactive oxygen species and modulate ß-cell insulin secretion. Islets of type 2 diabetic subjects present elevated expression of NOX5. Here, we sought to characterize regulation of NOX5 expression in human islets in vitro and to uncover the relevance of NOX5 in islet function in vivo using a novel mouse model expressing NOX5 in doxycycline-inducible, ß-cell-specific manner (RIP/rtTA/NOX5 mice). Results:In situ hybridization and immunohistochemistry employed on pancreatic sections demonstrated NOX5 messenger ribonucleic acid (mRNA) and protein expressions in human islets. In cultures of dispersed islets, NOX5 protein was observed in somatostatin-positive (δ) cells in basal (2.8 mM glucose) conditions. Small interfering ribonucleic acid (siRNA)-mediated knockdown of NOX5 in human islets cultured in basal glucose concentrations resulted in diminished glucose-induced insulin secretion (GIIS) in vitro. However, when islets were preincubated in high (16.7 mM) glucose media for 12 h, NOX5 appeared also in insulin-positive (ß) cells. In vivo, mice with ß-cell NOX5 expression developed aggravated impairment of GIIS compared with control mice when challenged with 14 weeks of high-fat diet. Similarly, in vitro palmitate preincubation resulted in more severe reduction of insulin release in islets of RIP/rtTA/NOX5 mice compared with their control littermates. Decreased insulin secretion was most distinct in response to theophylline stimulation, suggesting impaired cyclic adenosine monophosphate (cAMP)-mediated signaling due to increased phosphodiesterase activation. Innovation and Conclusions: Our data provide the first insight into the complex regulation and function of NOX5 in islets implying an important role for NOX5 in δ-cell-mediated intraislet crosstalk in physiological circumstances but also identifying it as an aggravating factor in ß-cell failure in diabetic conditions.

Gastrointestinal Hormones Manipulation to Counteract Metabolic Syndrome Using Duodenal Targeted Embolization.

PURPOSE: Targeted embolization of gastrointestinal (GI) arteries can modify hormonal production. We aimed to evaluate the impact of the embolization of the gastroduodenal artery (GDA) on the activity of foregut mucosa. METHODS: The GDA's duodenal branch was embolized in 12 Yucatan pigs using 100-300 µm (group A; n = 4) or 300-500 µm (group B; n = 4) microspheres, followed by coiling of the branch. In 4 animals (sham), only saline was injected. The levels of GI hormones (ghrelin, glucose-dependent insulinotropic peptide [GIP], glucagon-like peptide-1 [GLP-1], insulin, peptide YY [PYY], leptin) and the gene expression of sodium-glucose-linked transporter-1 (SGLT-1) and glucose transporter-2 (GLUT-2) were assessed before (T0), 1 hour (T1), 1 month (T2), 3 months (T3), and 6 months (T4) after embolization. RESULTS: In group A, a segmental duodenal stenosis occurred in all cases, which required balloon dilatation. There was a significant drop in the baseline glycemia in group A at T1 and T4 versus sham. Ghrelin was reduced in group A versus baseline and versus group B at T2 and T3 and versus sham at T1 and T3. Insulin was significantly lower in group A versus B at T1 and at T4 but not versus sham. SGLT-1 expression increased in B and sham at T4, while it remained stable in group A. GLUT-2 expression increased in sham at T4 but not in A or B. CONCLUSIONS: GDA embolization induced a decrease in ghrelin production and influenced expression of glucose carriers in the foregut mucosa.

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.

Beneficial effects of cherry consumption as a dietary intervention for metabolic, hepatic and vascular complications in type 2 diabetic rats.

BACKGROUND: Oxidative stress (OS) plays an important role in type 2 diabetes (T2D) pathogenesis and its complications. New therapies target natural antioxidants as an alternative and/or supplemental strategy to prevent and control them. Our previous chemical and biological studies highlighted the important antioxidant activities of cherries, among other fruits and vegetables, thus we aimed to determine in vivo effects of 2-month long cherry consumption using a high-fat/high-fructose (HFHF) model of diabetic-rats (Lozano et al. in Nutr Metab 13:15, 2016). METHODS: After 2 months of HFHF, male Wistar rats were divided into: HFHF and HFHF enriched in cherry (nutritional approach) or standard diet ND (lifestyle measures) and ND plus cherry during 2 months. Metabolic, lipidic, oxidative parameters were quantified. Tissues (liver, pancreas and vessels) OS were assessed and hepatic (steatosis, fibrosis, inflammation) and vascular (endothelial dysfunction) complications were characterized. RESULTS: T2D was induced after 2 months of HFHF diet, characterized by systemic hyperglycaemia, hyperinsulinemia, glucose intolerance, dyslipidaemia, hyperleptinemia, and oxidative stress associated with endothelial dysfunction and hepatic complications. Cherry consumption for 2 months, in addition to lifestyle measures, in T2D-rats decreased and normalized the systemic disturbances, including oxidative stress complications. Moreover, in the vessel, cherry consumption decreased oxidative stress and increased endothelial nitric oxide (NO) synthase levels, thus increasing NO bioavailability, ensuring vascular homeostasis. In the liver, cherry consumption decreased oxidative stress by inhibiting NADPH oxidase subunit p22phox expression, nuclear factor erythroid-2 related factor 2 (Nrf2) degradation and the formation of reactive oxygen species. It inhibited the activation of sterol regulatory element-binding proteins (1c and 2) and carbohydrate-responsive element-binding protein, and thus decreased steatosis as observed in T2D rats. This led to the improvement of metabolic profiles, together with endothelial and hepatic function improvements. CONCLUSION: Cherry consumption normalized vascular function and controlled hepatic complications, thus reduced the risk of diabetic metabolic disorders. These results demonstrate that a nutritional intervention with a focus on OS could prevent and/or delay the onset of vascular and hepatic complications related to T2D.

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

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 ±â€¯2 mV) were more negatively charged than -PVA-NPs (-22 ±â€¯1 mV) and chitosan-coated NPs were highly positively charged (56 ±â€¯2 mV) compared to +PVA particles (-2 ±â€¯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.

Beneficial effects of a red wine polyphenol extract on high-fat diet-induced metabolic syndrome in rats.

PURPOSE: Individuals with metabolic syndrome (MS) show several metabolic abnormalities including insulin resistance, dyslipidaemia, and oxidative stress (OS). Diet is one of the factors influencing the development of MS, and current nutritional advice emphasises the benefits of fruit and vegetable consumption. Here, we assessed the effects of naturally occurring antioxidants, red wine polyphenols (RWPs), on MS and OS. METHODS: Wistar rats (n = 20) weighing 200-220 g received a high-fat diet (HFD) for 2 months before they were divided into two groups that received either HFD only or HFD plus 50 mg/kg RWPs in their drinking water for an additional 2 months. A control group (n = 10) received a normal diet (ND) for 4 months. RESULTS: Rats receiving HFD increased body weight over 20 % throughout the duration of the study. They also showed increased blood levels of C-peptide, glucose, lipid peroxides, and oxidised proteins. In addition, the HFD increased OS in hepatic, pancreatic, and vascular tissues, as well as induced pancreatic islet cell hyperplasia and hepatic steatosis. Addition of RWPs to the HFD attenuated these effects on plasma and tissue OS and on islet cell hyperplasia. However, RWPs had no effect on blood glucose levels or hepatic steatosis. CONCLUSIONS: RWPs showed an antioxidant mechanism of action against MS. This result will inform future animal studies exploring the metabolic effects of RWPs in more detail. In addition, these findings support the use of antioxidants as adjunctive nutritional treatments for patients with diabetes.

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.

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.

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.

High-fructose and high-fat diet-induced disorders in rats: impact on diabetes risk, hepatic and vascular complications.

BACKGROUND: As a result of the increased consumption of sugar-rich and fatty-products, and the increase in preference for such products, metabolic disorders are becoming more common at a younger age. Fructose is particularly used in prepared foods and carbonated beverages. We investigated the impact of regular consumption of fructose, in combination or not with fatty food, on the onset of metabolic syndrome and type 2 diabetes (T2D). We evaluated the metabolic, oxidative, and functional effects on the liver and blood vessels, both related to diabetes complications. METHODS: High-fat diet (HFD), high-fructose beverages (HF) or both (HFHF) were compared to rats fed with normal diet (ND) for 8 months to induce T2D and its metabolic, oxidative, and functional complications. Metabolic control was determined by measuring body weight, fasting blood glucose, C-peptide, HOMA2-IR, leptin, and cholesterol; oxidative parameters were studied by lipid peroxidation and total antioxidant capacity in plasma and the use of ROS labelling on tissue. Histological analysis was performed on the liver and endothelial function was performed in main mesenteric artery using organ-baths. RESULTS: After 2 months, HFHF and HFD increased body weight, leptin, HOMA2-IR associated to steatosis, oxidative stress in plasma and tissues, whereas HF had only a transient increase of leptin and c-peptide. Only HFHF induced fasting hyperglycaemia after 6 months and persistent hyperinsulinaemia and fasting hyperglycaemia with complicated steatosis (inflammation and fibrosis) after 8 months. HFHF and HFD induced endothelial dysfunction at 8 months of diet. CONCLUSIONS: Six months, high fat and high carbohydrate induced T2D with widespread tissues effects. We demonstrated the role of oxidative stress in pathogenesis as well as in complications (hepatic and vascular), reinforcing interest in the use of antioxidants in the prevention and treatment of metabolic diseases, including T2D.

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.

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.

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.

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.

Metabolic and oxidative stress markers in Wistar rats after 2 months on a high-fat diet.

BACKGROUND: Metabolic syndrome is associated with an increased risk of cardiovascular and hepatic complications. Oxidative stress in metabolic tissues has emerged as a universal feature of metabolic syndrome and its co-morbidities. We aimed to develop a rapidly and easily induced model of metabolic syndrome in rats to evaluate its impact on plasma and tissue oxidative stress. MATERIALS AND METHODS: Metabolic syndrome was induced in rats using a high-fat diet (HFD), and these rats were compared to rats fed a normal diet (ND) for 2 months. Metabolic control was determined by measuring body weight, blood glucose, triglycerides, lipid peroxidation and protein carbonylation in plasma. Insulinemia was evaluated through the measure of C-peptide. Histological analysis was performed on the pancreas, liver and blood vessels. RESULTS: After 2 months, the HFD induced an increase in body weight, insulin and triglycerides. Liver steatosis was also observed in the HFD group, which was associated with an increase in glycogen storage. In the pancreas, the HFD induced islet hyperplasia. Tissue oxidative stress was also increased in the liver, pancreas and blood vessels, but plasma oxidative stress remained unchanged. CONCLUSION: This paper reports the development of a fast and easy model of rat metabolic syndrome associated with tissue oxidative stress. This model may be a good tool for the biological validation of drugs or antioxidants to limit or prevent the complications of metabolic syndrome.

Duodenum-specific drug delivery: in vivo assessment of a pharmaceutically developed enteric-coated capsule for a broad applicability in rat studies.

Targeting new oral drug formulations in the intestine has a broad applicability in animal studies. Enteric-coated capsules are gastroresistant and specific drug delivery systems useful for the evaluation of new pharmaceutical formulations during pre-clinical validations in rats. The purpose of this study was to develop and validate in a large-scale, reliable, reproducible capsules, to offer a safe and standardized duodenum-specific delivery system adapted for studies in rats. The reproducibility of the coating method, the coating layer uniformity and thickness, the external capsules integrity and their enteric properties after in vitro dissolution in simulated gastric and intestinal media were already evaluated and validated. This study presents the in vivo tests of the gastroresistance and of the location of the disintegration. Micro-computerized tomography and a pharmacokinetic study of acetaminophen-filled capsules showed that the enteric-capsules were resistant in the stomach with no apparent leak of the capsules, and were disintegrated in the early duodenum 1-1.5h after oral administration. A positive impact on the bioavailability of acetaminophen in coated capsules was attested. In conclusion, this work, developed with a rigorous pharmaceutical technology, presents a tool adapted for duodenum-specific delivery of new formulations in rats.

Adenoviral infection or deferoxamine? Two approaches to overexpress VEGF in beta-cell lines.

Rapid and adequate revascularization of transplanted islets is important for their survival and function during transplantation. Vascular endothelial growth factor (VEGF) could play a critical role with respect to islet revascularization. The aim of this study was to compare two strategies that are used to overexpress VEGF in beta-cells: (1) gene therapy through adenoviral infection and (2) a pharmacological approach using deferoxamine (DFO). beta-Cell lines from rat insulinoma (RINm5F) were either infected using an adenovirus encoding the gene of human VEGF 165 or incubated with DFO. One day after treatment, the viability of RINm5F cells was preserved with 10 micromol/L of DFO (103.95 +/- 5.66% toward control; n = 4). In addition, adenoviral infection maintained the viability of cells for all the concentrations used. In both treatments, overexpression of VEGF was in a comparable level. Finally, the ratio of Bax/Bcl-2 indicated that the apoptosis increased in infected beta-cells whereas treatment with DFO seems to be antiapoptotic. Our results suggest that the use of DFO could be a realistic approach to improve the vascularization of islets during transplantation.

Extracellular matrix proteins involved in pseudoislets formation.

Extracellular matrix proteins are known to mediate, through integrins, cell adhesion and are involved in a number of cellular processes, including insulin expression and secretion in pancreatic islets. We investigated whether expression of some extracellular matrix proteins were implied in islets-like structure formation, named pseudoislets. For this purpose, we cultured the ß-cell line, RINm5F, during 1, 3, 5 and 7 days of culture on treated or untreated culture plate to form adherent cells or pseudoislets and analysed insulin, collagen IV, fibronectin, laminin 5 and ß1-integrin expression. We observed that insulin expression and secretion were increased during pseudoislets formation. Moreover, we showed by immunohistochemistry an aggregation of insulin secreting cells in the centre of the pseudoislets. Peripheral ß-cells of pseudoislets did not express insulin after 7 days of culture. RT-PCR and immunohistochemistry studies showed a transient expression of type IV collagen in pseudoislets for the first 3 days of culture. Study of fibronectin expression indicated that adherent cells expressed more fibronectin than pseudoislets. In contrast, laminin 5 was more expressed in pseudoislets than in adherent cells. Finally, expression of ß1-integrin was increased in pseudoislets as compared to adherent cells. In conclusion, laminin 5 and collagen IV might be implicated in pseudoislets formation whereas fibronectin might be involved in cell adhesion. These data suggested that extracellular matrix proteins may enhance the function of pseudoislets.