Christophe Noll

Islet Endothelial Activation and Oxidative Stress Gene Expression Is Reduced by IL-1Ra Treatment in the Type 2 Diabetic GK Rat

Background Inflammation followed by fibrosis is a component of islet dysfunction in both rodent and human type 2 diabetes. Because islet inflammation may originate from endothelial cells, we assessed the expression of selected genes involved in endothelial cell activation in islets from a spontaneous model of type 2 diabetes, the Goto-Kakizaki (GK) rat. We also examined islet endotheliuml/oxidative stress (OS)/inflammation-related gene expression, islet vascularization and fibrosis after treatment with the interleukin-1 (IL-1) receptor antagonist (IL-1Ra). Methodology/Principal Findings Gene expression was analyzed by quantitative RT-PCR on islets isolated from 10-week-old diabetic GK and control Wistar rats. Furthermore, GK rats were treated s.c twice daily with IL-1Ra (Kineret, Amgen, 100 mg/kg/day) or saline, from 4 weeks of age onwards (onset of diabetes). Four weeks later, islet gene analysis and pancreas immunochemistry were performed. Thirty-two genes were selected encoding molecules involved in endothelial cell activation, particularly fibrinolysis, vascular tone, OS, angiogenesis and also inflammation. All genes except those encoding angiotensinogen and epoxide hydrolase (that were decreased), and 12-lipoxygenase and vascular endothelial growth factor (that showed no change), were significantly up-regulated in GK islets. After IL-1Ra treatment of GK rats in vivo, most selected genes implied in endothelium/OS/immune cells/fibrosis were significantly down-regulated. IL-1Ra also improved islet vascularization, reduced fibrosis and ameliorated glycemia. Conclusions/Significance GK rat islets have increased mRNA expression of markers of early islet endothelial cell activation, possibly triggered by several metabolic factors, and also some defense mechanisms. The beneficial effect of IL-1Ra on most islet endothelial/OS/immune cells/fibrosis parameters analyzed highlights a major endothelial-related role for IL-1 in GK islet alterations. Thus, metabolically-altered islet endothelium might affect the β-cell microenvironment and contribute to progressive type 2 diabetic β-cell dysfunction in GK rats. Counteracting islet endothelial cell inflammation might be one way to ameliorate/prevent β-cell dysfunction in type 2 diabetes.

Effects of red wine polyphenolic compounds on paraoxonase-1 and lectin-like oxidized low-density lipoprotein receptor-1 in hyperhomocysteinemic mice

Hyperhomocysteinemia, or abnormally high plasma homocysteine (Hcy) concentration, has often been associated with vascular thrombosis and the development of premature atherosclerosis. Many studies have shown that moderate wine consumption has potential beneficial effects related to the prevention of atherosclerosis, in part attributed to the biological properties of polyphenolic components, mainly flavonoids. The aim of the present study is to determine the effects of a red wine polyphenolic extract (PE) administration on hyperhomocysteinemia due to cystathionine beta-synthase (CBS) deficiency and on the associated biochemical markers of hepatic and endothelial dysfunctions in mice. Red wine PE was added for 4 weeks to the drinking water of heterozygous CBS-deficient mice fed a high-methionine diet, a murine model of hyperhomocysteinemia. Red wine PE supplementation at low dose significantly reduced plasma Hcy levels and restored the hepatic and plasma-decreased paraoxonase-1 activity induced by chronic hyperhomocysteinemia. Moreover, aortic expression of proinflammatory cytokines and adhesion molecules and levels of soluble lectin-like oxidized low-density lipoprotein receptor-1 were reduced in hyperhomocysteinemic mice fed the red wine PE supplementation. These findings suggest that red wine PE administration in low quantities has beneficial effects on biochemical markers of endothelial dysfunction due to hyperhomocysteinemia.

DYRK1A, a Novel Determinant of the Methionine-Homocysteine Cycle in Different Mouse Models Overexpressing this Down-Syndrome-Associated Kinase

Background Hyperhomocysteinemia, characterized by increased plasma homocysteine level, is associated with an increased risk of atherosclerosis. On the contrary, patients with Down syndrome appear to be protected from the development of atherosclerosis. We previously found a deleterious effect of hyperhomocysteinemia on expression of DYRK1A, a Down-syndrome-associated kinase. As increased expression of DYRK1A and low plasma homocysteine level have been associated with Down syndrome, we aimed to analyze the effect of its over-expression on homocysteine metabolism in mice. Methodology/Principal Findings Effects of DYRK1A over-expression were examined by biochemical analysis of methionine metabolites, real-time quantitative reverse-transcription polymerase chain reaction, and enzyme activities. We found that over-expression of Dyrk1a increased the hepatic NAD(P)H:quinone oxidoreductase and S-adenosylhomocysteine hydrolase activities, concomitant with decreased level of plasma homocysteine in three mice models overexpressing Dyrk1a. Moreover, these effects were abolished by treatment with harmine, the most potent and specific inhibitor of Dyrk1a. The increased NAD(P)H:quinone oxidoreductase and S-adenosylhomocysteine hydrolase activities were also found in lymphoblastoid cell lines from patients with Down syndrome. Conclusions/Significance Our results might give clues to understand the protective effect of Down syndrome against vascular defect through a decrease of homocysteine level by DYRK1A over-expression. They reveal a link between the Dyrk1a signaling pathway and the homocysteine cycle.

Resveratrol supplementation worsen the dysregulation of genes involved in hepatic lipid homeostasis observed in hyperhomocysteinemic mice

Hyperhomocysteinemia is characterized by an increase of plasma homocysteine, a thiol-containing amino acid produced during methionine metabolism. Hyperhomocysteinemia has often been associated with coronary artery disease, vascular thrombosis and the development of premature atherosclerosis. We have recently demonstrated that the supplementation of catechin, a polyphenol found in the red wine, significantly reduced plasma homocysteine level in cystathionine beta synthase (CBS) deficient mice, a murine model of hyperhomocysteinemia. In the present study, we have investigated the influence of another well-studied polyphenol found in red wine, resveratrol, on hyperhomocysteinemia. After two months on high methionine diet, heterozygous Cbs deficient mice were administrated the resveratrol in drinking water (0.001%) for one month. High methionine diet significantly increased serum homocysteine levels, and decreased the serum activity of HDL-associated enzyme paraoxonase-1. Chronic administration of resveratrol significantly increased plasma homocysteine level, which was associated with a decreased serum paraoxonase-1 activity, in hyperhomocysteinemic mice. Then we looked at gene expression of several proteins involved in HDL stability and found a down-regulation of lecithin:cholesterol acyltransferase. In conclusion, we found a deleterious effect of resveratrol onto homocysteine and HDL metabolism in a murine model of hyperhomocysteinemia.

Effect of hyperhomocysteinemia on the protein kinase DYRK1A in liver of mice.

Hyperhomocysteinemia due to cystathionine beta synthase (CBS)-deficiency confers diverse clinical manifestations, notably liver diseases. Even if hyperhomocysteinemia in liver of CBS-deficient mice, a murine model of hyperhomocysteinemia, promotes mitochondrial oxidative stress and pro-apoptotic signals, protective signals may counteract these pro-apoptotic signals, leading to chronic inflammation. As DYRK1A, a serine/threonine kinase, has been described as a candidate antiapoptotic factor, we have analyzed the expression of DYRK1A in liver of CBS-deficient mice. We found that DYRK1A protein level was reduced in liver of CBS-deficient mice, which was not observed at the gene expression level. Moreover, the use of primary hepatocytes/Kupffer cells co-culture showed that degradation of DYRK1A induced by hyperhomocysteinemia requires calpain activation. Our results demonstrate a deleterious effect of hyperhomocysteinemia on DYRK1A protein expression, and emphasize the role of hyperhomocysteinemia on calpain activation.

Homocysteine threshold value based on cystathionine beta synthase and paraoxonase 1 activities in mice

Background  Hyperhomocysteinaemia is a metabolic disorder associated with the development of premature atherosclerosis. Among the determinants which predispose to premature thromboembolic and atherothrombotic events, serum activity of paraoxonase 1, mainly synthesized in the liver, has been shown to be a predictor of cardiovascular disease and to be negatively correlated with serum homocysteine levels in human. Even though treatments of hyperhomocysteinaemic patients ongoing cardiovascular complications are commonly used, it still remains unclear above which homocysteine level a preventive therapy should be started.

Calpain activation is required for homocysteine-mediated hepatic degradation of inhibitor Ikappa B alpha

Hepatic steatosis is a clinical feature observed in severe hyperhomocysteinemic patients. In mice, cystathionine beta synthase (CBS) deficiency, the most common cause of severe hyperhomocysteinemia, is also associated with steatosis, fibrosis and inflammation. Proinflammatory cytokines usually induce apoptosis. However, hyperhomocysteinemia does not increase apoptosis in liver of CBS-deficient mice compared to wild type mice. The aim of the study was to analyze the activation state of the NF-kappaB pathway in liver of CBS-deficient mice and to investigate its possible involvement in anti-apoptotic signals. We analyzed the level of I kappaB alpha in liver of CBS-deficient mice. A co-culture of primary hepatocytes and Kupffer cells was also used in order to investigate how I kappaB alpha degradation occurs in response to homocysteine. We found lower I kappaB alpha level not only in liver of CBS-deficient mice but also in hepatocyte/Kupffer cell co-culture. The homocysteine-mediated I kappaB alpha enhanced proteolysis occurred via calcium-dependent calpains, which was supported by an increased level of calpain activity and a reduced expression of calpastatin in liver of CBS-deficient mice. Intraperitoneal administration of the inhibitor PDTC normalized the expression of two genes induced by NF-kappaB activation, heme oxygenase-1 and cellular inhibitor of apoptosis 2. Moreover, PDTC administration induced an increase of caspase-3 activity in liver of CBS-deficient mice. Our results suggest that hyperhomocysteinemia induces calpain-mediated I kappaB alpha degradation which is responsible for anti-apoptotic signals in liver.

Myocardial fibrosis and TGFB expression in hyperhomocysteinemic rats

Hyperhomocysteinemia, characterized by an elevated plasma homocysteine concentration, leads to several clinical manifestations and particularly cardiovascular diseases. Experimental models of hyperhomocysteinemia revealed several tissue injuries including heart fibrosis and ventricular hypertrophy. In order to analyze the molecular mechanisms link to these morphological alterations, a mild hyperhomocysteinemia was induced in rats via a chronic methionine administration. Effects of methionine administration were examined by histological analysis with Sirius red staining, histomorphometric analysis, zymography, and immunoblotting. Hyperhomocysteinemia due to methionine administration produces an interstitial myocardial fibrosis and a ventricular cardiomyocyte hypertrophy, which were associated with increased expression of transforming growth factor-beta1 (TGFβ1), tissue inhibitors of metalloproteinase (TIMP) 2, and JNK activation. However, the matrix metalloproteinase 2 activity was decreased in the hearts of hyperhomocysteinemic rats. Moreover, the TIMP1 protein expression was decreased, and the TIMP1–MMP1 balance was shifted. Remodeling in cardiac tissue observed in rat model of mild hyperhomocysteinemia is associated with a dysregulation in extracellular matrix degradation which results, at least in part, from enhancement of TGFβ1 level.

Cadmium alters the biotransformation of carcinogenic aromatic amines by arylamine N-acetyltransferase xenobiotic-metabolizing enzymes: molecular, cellular, and in vivo studies.

Background Cadmium (Cd) is a carcinogenic heavy metal of environmental concern. Exposure to both Cd and carcinogenic organic compounds, such as polycyclic aromatic hydrocarbons or aromatic amines (AAs), is a common environmental problem. Human arylamine N-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes that play a key role in the biotransformation of AA carcinogens. Changes in NAT activity have long been associated with variations in susceptibility to different cancers in relation with exposure to certain AAs. Objective We explored the possible interactions between Cd and the NAT-dependent biotransformation of carcinogenic AAs. Methods We exposed purified enzymes, lung epithelial cells, and mouse models to Cd and subsequently analyzed NAT-dependent metabolism of AAs. Results We found that Cd, at biologically relevant concentrations, impairs the NAT-dependent acetylation of carcinogenic AAs such as 2-aminofluorene (2-AF) in lung epithelial cells. NAT activity was strongly impaired in the tissues of mice exposed to Cd. Accordingly, mice exposed to Cd and 2-AF displayed altered in vivo toxicokinetics with a significant decrease (~ 50%) in acetylated 2-AF in plasma. We found that human NAT1 was rapidly and irreversibly inhibited by Cd [median inhibitory concentration (IC50) ≈ 55 nM; rate inhibition constant (kinact) = 5 × 104 M−1 · sec−1], with results of acetyl coenzyme A (acetyl-CoA) protection assays indicating that Cd-mediated inhibition was due to the reaction of metal with the active-site cysteine residue of the enzyme. We found similar results for human NAT2, although this isoform was less sensitive to inactivation (IC50 ≈ 1 μM; kinact = 1 × 104 M−1 · sec−1). Conclusions Our data suggest that Cd can alter the metabolism of carcinogenic AAs through the impairment of the NAT-dependent pathway, which may have important toxicological consequences.

Cystathionine beta synthase deficiency induces catalase-mediated hydrogen peroxide detoxification in mice liver

Cystathionine beta synthase deficiency induces hyperhomocysteinemia which is considered as a risk factor for vascular diseases. Studies underlined the importance of altered cellular redox reactions in hyperhomocysteinemia-induced vascular pathologies. Nevertheless, hyperhomocysteinemia also induces hepatic dysfunction which may accelerate the development of vascular pathologies by modifying cholesterol homeostasis. The aim of the present study was to analyze the modifications of redox state in the liver of heterozygous cystathionine beta synthase-deficient mice, a murine model of hyperhomocysteinemia. In this purpose, we quantified levels of reactive oxygen and nitrogen species and we assayed activities of main antioxidant enzymes. We found that cystathionine beta synthase deficiency induced NADPH oxidase activation. However, there was no accumulation of reactive oxygen (superoxide anion, hydrogen peroxide) and nitrogen (nitrite, peroxynitrite) species. On the contrary, hepatic hydrogen peroxide level was decreased independently of an activation of glutathione-dependent mechanisms. In fact, cystathionine beta synthase deficiency had no effect on glutathione peroxidase, glutathione reductase and glutathione S-transferase activities. However, we found a 50% increase in hepatic catalase activity without any variation of expression. These findings demonstrate that cystathionine beta synthase deficiency initiates redox disequilibrium in the liver. However, the activation of catalase attenuates oxidative impairments.