intra-omental-islet-transplantation-using-h-omental-matrix-islet

Intra-Omental Islet Transplantation Using h-Omental Matrix Islet filliNG (hOMING)

Regenerative medicine based on cell therapy represents a new hope for curing disease. Current obstacles include proper in vivo validation of the ...

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. 

Pro-inflammatory and pro-oxidant status of pancreatic islet in vitro is controlled by TLR-4 and HO-1 pathways.

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.

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

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.

Portal or subcutaneous insulin infusion: efficacy and impact on liver inflammation.

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.

Featured Article: Oxidative stress status and liver tissue defenses in diabetic rats during intensive subcutaneous insulin therapy.

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. 

Oxidative Stress Type Influences the Properties of Antioxidants Containing Polyphenols in RINm5F Beta Cells.

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.

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

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.

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

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).

In Vitro and In Vivo Investigation of the Angiogenic Effects of Liraglutide during Islet Transplantation.

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. H 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 H HRMAS NMR facilitated the characterization of cellular and molecular pathways recruited following islet transplantation.

A Metabolomic Approach (H HRMAS NMR Spectroscopy) Supported by Histology to Study Early Post-transplantation Responses in Islet-transplanted Livers.

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.

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

Tissue cross-talk is emerging as a determinant way to coordinate the different organs implicated in glucose homeostasis. Among the inter-organ communication factors, muscle-secreted myokines can modulate the function and survival of pancreatic beta-cells. Using primary human myotubes from soleus, vastus lateralis and triceps brachii muscles, we report here that the impact of myokines on beta-cells depends on fiber types and their metabolic status. We show that Type I and type II primary myotubes present specific mRNA and myokine signatures as well as a different sensitivity to TNF-alpha induced insulin resistance. Finally, we show that angiogenin and osteoprotegerin are triceps specific myokines with beta-cell protective actions against proinflammatory cytokines. These results suggest that type I and type II muscles could impact insulin secretion and beta-cell mass differentially in type 2 diabetes through specific myokines secretion.

Angiogenin and Osteoprotegerin are type II muscle specific myokines protecting pancreatic beta-cells against proinflammatory cytokines.

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).

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

Type 1 diabetes is an autoimmune disease initiated by the invasion of pancreatic islets by immune cells that selectively kill the β cells. We found that rodent and human T lymphocytes release exosomes containing the microRNAs (miRNAs) miR-142-3p, miR-142-5p, and miR-155, which can be transferred in active form to β cells favoring apoptosis. Inactivation of these miRNAs in recipient β cells prevents exosome-mediated apoptosis and protects non-obese diabetic (NOD) mice from diabetes development. Islets from protected NOD mice display higher insulin levels, lower insulitis scores, and reduced inflammation. Looking at the mechanisms underlying exosome action, we found that T lymphocyte exosomes trigger apoptosis and the expression of genes involved in chemokine signaling, including Ccl2, Ccl7, and Cxcl10, exclusively in β cells. The induction of these genes may promote the recruitment of immune cells and exacerbate β cell death during the autoimmune attack. Our data point to exosomal-miRNA transfer as a communication mode between immune and insulin-secreting cells.

Lymphocyte-Derived Exosomal MicroRNAs Promote Pancreatic β Cell Death and May Contribute to Type 1 Diabetes Development.

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  effects of 2-month long RC consumption using a high-fat/high-fructose model of diabetic rats.

Article	Year
Pro-inflammatory and pro-oxidant status of pancreatic islet in vitro is controlled by TLR-4 and HO-1 pathways. PloS one\| PubMed ID: 25343247	2014
Metabolic and oxidative stress markers in Wistar rats after 2 months on a high-fat diet. Diabetology & metabolic syndrome\| PubMed ID: 25960774	2014
Portal or subcutaneous insulin infusion: efficacy and impact on liver inflammation. Fundamental & clinical pharmacology\| PubMed ID: 26095147	2015
Featured Article: Oxidative stress status and liver tissue defenses in diabetic rats during intensive subcutaneous insulin therapy. Experimental biology and medicine (Maywood, N.J.)\| PubMed ID: 26385497	2016
Oxidative Stress Type Influences the Properties of Antioxidants Containing Polyphenols in RINm5F Beta Cells. Evidence-based complementary and alternative medicine : eCAM\| PubMed ID: 26508986	2015
Beneficial effects of a red wine polyphenol extract on high-fat diet-induced metabolic syndrome in rats. European journal of nutrition\| PubMed ID: 26913853	2017
High-fructose and high-fat diet-induced disorders in rats: impact on diabetes risk, hepatic and vascular complications. Nutrition & metabolism\| PubMed ID: 26918024	2016
In Vitro and In Vivo Investigation of the Angiogenic Effects of Liraglutide during Islet Transplantation. PloS one\| PubMed ID: 26974949	2016
A Metabolomic Approach (H HRMAS NMR Spectroscopy) Supported by Histology to Study Early Post-transplantation Responses in Islet-transplanted Livers. International journal of biological sciences\| PubMed ID: 27766032	2016
Oral insulin delivery, the challenge to increase insulin bioavailability: Influence of surface charge in nanoparticle system. International journal of pharmaceutics\| PubMed ID: 29501738	2018
Angiogenin and Osteoprotegerin are type II muscle specific myokines protecting pancreatic beta-cells against proinflammatory cytokines. Scientific reports\| PubMed ID: 29968746	2018
Beneficial effects of cherry consumption as a dietary intervention for metabolic, hepatic and vascular complications in type 2 diabetic rats. Cardiovascular diabetology\| PubMed ID: 30029691	2018
Lymphocyte-Derived Exosomal MicroRNAs Promote Pancreatic β Cell Death and May Contribute to Type 1 Diabetes Development. Cell metabolism\| PubMed ID: 30318337	2019
Treatment of NASH with Antioxidant Therapy: Beneficial Effect of Red Cabbage on Type 2 Diabetic Rats. Oxidative medicine and cellular longevity\| PubMed ID: 30363947	2018

Michel Pinget

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