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

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

Islet survival and function.

<p>(A) Fluorescein diacetate/propidium iodide staining of control islets (CTL) and of islets treated with 10 μmol/L of liraglutide (Lira) at the indicated time-points. (B) Islet viability was also represented in percentage as histograms with controls (black bars) <i>versus</i> Lira (grey bars) (C) Glucose stimulation test: Insulin secreted by islet incubated in conditioned medium containing 2.5 mmol/L (black bars) and 25 mmol/L (grey bars) of glucose. Results were expressed as mean ± SEM. *p<0.05, **p<0.01 for the indicated comparisons.</p

The angiogenic effects of liraglutide <i>in vitro</i>.

<p>(A) Insulin intensity toward islet surface in control islets (CTL; black bar) as compared to islets treated with 10 μmol/L of liraglutide (Lira; grey bar) (B) CD31 intensity toward islet surface in CTL (black bar) <i>versus</i> Lira (grey bar) group (C) Immunostaining of insulin and endothelial cells after 24 h for control islets (CTL; a, b, c, d) <i>versus</i> islets cultured with Lira10μM (Lira; e, f, g, h). Nuclear DAPI staining is shown in blue (a, e); insulin staining in green (b, f); vessels are stained red (c, g); and these are merged in d and h. Results were expressed as mean ± SEM. *p<0.05, **p<0.01 for the indicated comparisons.</p

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

International audienceOxidative 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

Angiogenic mechanisms induced in rat pancreatic islets by 10 μmol/L of liraglutide.

<p>Evaluation of mammalian target of rapamycin (mTOR) activation, determined by the ratio of phosphorylated mTOR/mTOR using western blotting at the indicated time-points. Black bars, control; grey bars, Lira10μM. Results were expressed as mean ± SEM. *p<0.05.</p

Metabolic control in diabetic rats after islet transplantation.

<p>(A) Body-weight gain <i>versus</i> t = 0 day in no transplanted diabetic rats called SHAM (filled diamond), in transplanted diabetic animals with untreated islets called control group (filled square) and in transplanted diabetic rats using treated islets with Lira called Lira group (filled triangle). (B) The mean body-weight gains in SHAM (white bar), control (CTL; black bar) and 10μmol/L of liraglutide (Lira; grey bar) groups over the entire experiment. (C) Fasting glycaemia in the SHAM (filled diamond), control (filled square) and Lira (filled triangle) groups at the indicated time-points. (D) Mean fasting glycaemia in SHAM (white bar), control (black bar) and Lira (grey bar) during the experiment. Results were expressed as means ± SEM. ***p<0.001; **p<0.01; *p<0.05.</p

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

Abstract 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

<i>In Vitro</i> and <i>In Vivo</i> Investigation of the Angiogenic Effects of Liraglutide during Islet Transplantation

<div><p>Introduction</p><p>This study investigated the angiogenic properties of liraglutide <i>in vitro</i> and <i>in vivo</i> and the mechanisms involved, with a focus on Hypoxia Inducible Factor-1α (HIF-1α) and mammalian target of rapamycin (mTOR).</p><p>Materials and Methods</p><p>Rat pancreatic islets were incubated <i>in vitro</i> 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 <i>in vitro</i> 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, <i>in vitro</i> and <i>in vivo</i>, 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. <i>In vivo</i>, 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.</p><p>Results</p><p>Islet viability and function were respectively preserved and enhanced (p<0.05) with Lira, <i>versus</i> control. Lira increased CD31-positive cells, expression of VEGF and PDGFα (p<0.05) after 24 h in culture. Increased VEGF secretion <i>versus</i> control was also observed at 48 h (p<0.05). Moreover, Lira activated mTOR (p<0.05) signalling pathway. <i>In vivo</i>, Lira improved vascular density (p<0.01), body-weight gain (p<0.01) and reduced fasting blood glucose in transplanted rats (p<0.001).</p><p>Conclusion</p><p>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.</p></div

The angiogenic effects of liraglutide <i>in vivo</i>.

<p>(A) CD31 intensity toward islet surface of rats transplanted with control islets (CTL; black bar) as compared to animals grafted with islets treated with 10 μmol/L of liraglutide (Lira; grey bar) (B) CD31 intensity surrounding transplanted islets toward analysed surface in CTL (black bars) <i>versus</i> Lira (grey bars) groups (C) Insulin intensity toward islet surface in CTL islets (black bars) as compared to Lira (grey bar) (C) Immunostaining of insulin and endothelial cells 30 days post implantation for control islets (CTL; a, b, c, d) <i>versus</i> Lira (e, f, g, h). Nuclear DAPI staining is shown in blue (a, e); insulin staining in green (b, f); vessels are stained red (c, g); and these are merged in d and h. Results were expressed as mean ± SEM. *p<0.05, **p<0.01 for the indicated comparisons.</p