The protein-bound uremic toxin p-cresyl-sulfate promotes intracellular ROS production and lipid peroxidation in 3T3-L1 adipose cells.

Patients with chronic kidney disease (CKD) often exhibit increased level of oxidative stress that contribute to the deterioration of renal function and uremic complications. White adipose tissue (WAT) has been recognized as a major site of production of radical oxygen species (ROS) in the context of metabolic diseases. This study was designed to decipher whether the protein bound uremic toxin p-cresyl-sulfate (p-CS) could contribute to ROS production in WAT and promote oxidative stress. Mouse 3T3-L1 adipocytes were incubated for 2 h in culture medium containing 212 µM p-CS, a concentration chosen to mimic levels encountered in end stage renal disease patients or KCl as a control and intracellular ROS production was measured using the fluorescent probe 5-6-carboxy-2',7'-dichlorodihydrofluorescein diacetate. Oxidative insult was estimated by the measurement of malondialdehyde (MDA) content and glutathione content. The effects of probenecid (1 mM) a potent inhibitor of organic anion transporter, apocynin (1 mM) an inhibitor of NADPH oxidase or common antioxidants such as α-tocopherol (2.5 µM), ascorbate (200 µM), and N-acetylcysteine (500 µM) were further evaluated. p-CS triggered a striking increase in ROS production (+228%, p < 0.01), in MDA content (+214%, p < 0.005) and a decrease in glutathione (-47%, P < 0.01). Pre-treatment of cells with probenecid, apocynin or antioxidants prevented the p-CS induced ROS production and oxidative insults. These results suggest that in uremic state, the intracellular accumulation of p-CS in adipose cells could contribute, through an activation of NADPH oxidase, to the redox imbalance often reported in CKD patients.

Combined Deletion of Free Fatty-Acid Receptors 1 and 4 Minimally Impacts Glucose Homeostasis in Mice.

The free fatty-acid receptors FFAR1 (GPR40) and FFAR4 (GPR120) are implicated in the regulation of insulin secretion and insulin sensitivity, respectively. Although GPR120 and GPR40 share similar ligands, few studies have addressed possible interactions between these 2 receptors in the control of glucose homeostasis. Here we generated mice deficient in gpr120 (Gpr120KO) or gpr40 (Gpr40KO), alone or in combination (Gpr120/40KO), and metabolically phenotyped male and female mice fed a normal chow or high-fat diet. We assessed insulin secretion in isolated mouse islets exposed to selective GPR120 and GPR40 agonists singly or in combination. Following normal chow feeding, body weight and energy intake were unaffected by deletion of either receptor, although fat mass increased in Gpr120KO females. Fasting blood glucose levels were mildly increased in Gpr120/40KO mice and in a sex-dependent manner in Gpr120KO and Gpr40KO animals. Oral glucose tolerance was slightly reduced in male Gpr120/40KO mice and in Gpr120KO females, whereas insulin secretion and insulin sensitivity were unaffected. In hyperglycemic clamps, the glucose infusion rate was lower in male Gpr120/40KO mice, but insulin and c-peptide levels were unaffected. No changes in glucose tolerance were observed in either single or double knock-out animals under high-fat feeding. In isolated islets from wild-type mice, the combination of selective GPR120 and GPR40 agonists additively increased insulin secretion. We conclude that while simultaneous activation of GPR120 and GPR40 enhances insulin secretion ex vivo, combined deletion of these 2 receptors only minimally affects glucose homeostasis in vivo in mice.

Free fatty acid receptor 4 inhibitory signaling in delta cells regulates islet hormone secretion in mice.

OBJECTIVE: Maintenance of glucose homeostasis requires the precise regulation of hormone secretion from the endocrine pancreas. Free fatty acid receptor 4 (FFAR4/GPR120) is a G protein-coupled receptor whose activation in islets of Langerhans promotes insulin and glucagon secretion and inhibits somatostatin secretion. However, the contribution of individual islet cell types (α, ß, and δ cells) to the insulinotropic and glucagonotropic effects of GPR120 remains unclear. As gpr120 mRNA is enriched in somatostatin-secreting δ cells, we hypothesized that GPR120 activation stimulates insulin and glucagon secretion via inhibition of somatostatin release. METHODS: Glucose tolerance tests were performed in mice after administration of selective GPR120 agonist Compound A. Insulin, glucagon, and somatostatin secretion were measured in static incubations of isolated mouse islets in response to endogenous (ω-3 polyunsaturated fatty acids) and/or pharmacological (Compound A and AZ-13581837) GPR120 agonists. The effect of Compound A on hormone secretion was tested further in islets isolated from mice with global or somatostatin cell-specific knock-out of gpr120. Gpr120 expression was assessed in pancreatic sections by RNA in situ hybridization. Cyclic AMP (cAMP) and calcium dynamics in response to pharmacological GPR120 agonists were measured specifically in α, ß, and δ cells in intact islets using cAMPER and GCaMP6 reporter mice, respectively. RESULTS: Acute exposure to Compound A increased glucose tolerance, circulating insulin, and glucagon levels in vivo. Endogenous and/or pharmacological GPR120 agonists reduced somatostatin secretion in isolated islets and concomitantly demonstrated dose-dependent potentiation of glucose-stimulated insulin secretion and arginine-stimulated glucagon secretion. Gpr120 was enriched in δ cells. Pharmacological GPR120 agonists reduced cAMP and calcium levels in δ cells but increased these signals in α and ß cells. Compound A-mediated inhibition of somatostatin secretion was insensitive to pertussis toxin. The effect of Compound A on hormone secretion was completely absent in islets from mice with either global or somatostatin cell-specific deletion of gpr120 and partially reduced upon blockade of somatostatin receptor signaling by cyclosomatostatin. CONCLUSIONS: Inhibitory GPR120 signaling in δ cells contributes to both insulin and glucagon secretion in part by mitigating somatostatin release.

Identification of novel antilipogenic agents targeting fatty acid biosynthesis through structure-based virtual screening.

An Asinex Gold Platinium chemical library subset of 12 055 compounds was screened employing docking simulations in the active site of the human FAS KS domain. Among them, 13 compounds were further evaluated for their ability to inhibit fatty acid biosynthesis. Four compounds were found to be active in particular ASN05064661 and ASN05374526 with IC50 values of 6.6 and 10.5 µm, respectively. A binding mode study was further conducted with these two compounds structurally related to benzene sulfonamide and aromatic polyamide. This study showed that they fit tightly with the active site with several interactions, notably with the key residues Cys161, His293, and His331.

Ozone Atmospheric Pollution and Alzheimer's Disease: From Epidemiological Facts to Molecular Mechanisms.

Atmospheric pollution is a well-known environmental hazard, especially in developing countries where millions of people are exposed to airborne pollutant levels above safety standards. Accordingly, several epidemiological and animal studies confirmed its role in respiratory and cardiovascular pathologies and identified a strong link between ambient air pollution exposure and adverse health outcomes such as hospitalization and mortality. More recently, the potential deleterious effect of air pollution inhalation on the central nervous system was also investigated and mounting evidence supports a link between air pollution exposure and neurodegenerative pathologies, especially Alzheimer's disease (AD). The focus of this review is to highlight the possible link between ozone air pollution exposure and AD incidence. This review's approach will go from observational and epidemiological facts to the proposal of molecular mechanisms. First, epidemiological and postmortem human study data concerning residents of ozone-severely polluted megacities will be presented and discussed. Then, the more particular role of ozone air pollution in AD pathology will be described and evidenced by toxicological studies in rat or mouse with ozone pollution exposure only. The experimental paradigms used to reproduce in rodent the human exposure to ozone air pollution will be described. Finally, current insights into the molecular mechanisms through which ozone inhalation can affect the brain and play a role in AD development or progression will be recapitulated.

p-Cresyl glucuronide is a major metabolite of p-cresol in mouse: in contrast to p-cresyl sulphate, p-cresyl glucuronide fails to promote insulin resistance.

BACKGROUND: The role of uraemic toxins in insulin resistance associated with chronic kidney disease (CKD) is gaining interest. p-Cresol has been defined as the intestinally generated precursor of the prototype protein-bound uraemic toxins p-cresyl sulphate (p-CS) as the main metabolite and, at a markedly lower concentration in humans, p-cresyl glucuronide (p-CG). The objective of the present study was to evaluate the metabolism of p-cresol in mice and to decipher the potential role of both conjugates of p-cresol on glucose metabolism. METHODS: p-CS and p-CG were measured by high performance liquid chromatography-fluorescence in serum from control, 5/6 nephrectomized mice and mice injected intraperitoneously with either p-cresol or p-CG. The insulin sensitivity in vivo was estimated by insulin tolerance test. The insulin pathway in the presence of p-cresol, p-CG and/or p-CS was further evaluated in vitro on C2C12 muscle cells by measuring insulin-stimulated glucose uptake and the insulin signalling pathway (protein kinase B, PKB/Akt) by western blot. RESULTS: In contrast to in humans, where p-CS is the main metabolite of p-cresol, in CKD mice both conjugates accumulated, and after chronic p-cresol administration with equivalent concentrations but a substantial difference in protein binding (96% for p-CS and <6% for p-CG). p-CG exhibited no effect on insulin sensitivity in vivo or in vitro and no synergistic inhibiting effect in combination with p-CS. CONCLUSIONS: The relative proportion of the two p-cresol conjugates, i.e. p-CS and p-CG, is similar in mouse, in contrast to humans, pinpointing major inter-species differences in endogenous metabolism. Biologically, the sulpho- (i.e. p-CS) but not the glucuro- (i.e. p-CG) conjugate promotes insulin resistance in CKD.

Abnormalities in myo-inositol metabolism associated with type 2 diabetes in mice fed a high-fat diet: benefits of a dietary myo-inositol supplementation.

We previously reported that a chronic supplementation with myo-inositol (MI) improved insulin sensitivity and reduced fat accretion in mice. We then tested the potency of such dietary intervention in the prevention of insulin resistance in C57BL/6 male mouse fed a high-fat diet (HFD). In addition, some abnormalities in inositol metabolism were reported to be associated with insulin resistance in several animal and human studies. We then investigated the presence of such anomalies (i.e. inosituria and an inositol intra-tissue depletion) in this diet-induced obesity (DIO) mouse model, as well as the potential benefit of a MI supplementation for inositol intra-tissue deficiency correction. HFD (60 % energy from fat) feeding was associated with inosituria and inositol intra-tissue depletion in the liver and kidneys. MI supplementation (0·58 mg/g per d) restored inositol pools in kidneys (partially) and liver (fully). HFD feeding for 4 months induced ectopic lipid redistribution to liver and muscles, fasting hyperglycaemia and hyperinsulinaemia, insulin resistance and obesity that were not prevented by MI supplementation, despite a significant improvement in insulin sensitivity parameter K insulin tolerance test and a reduction in white adipose tissue (WAT) mass ( - 17 %, P< 0·05). MI supplementation significantly reduced fatty acid synthase activity in epididymal WAT, which might explain its beneficial, but modest, effect on WAT accretion in HFD-fed mice. Finally, we found some abnormalities in inositol metabolism in association with a diabetic phenotype (i.e. insulin resistance and fasting hyperglycaemia) in a DIO mouse model. Dietary MI supplementation was efficient in the prevention of inositol intra-tissue depletion, but did not prevent insulin resistance or obesity efficiently in this mouse model.

Ozone exposure triggers insulin resistance through muscle c-Jun N-terminal kinase activation.

A growing body of evidence suggests that exposure to traffic-related air pollution is a risk factor for type 2 diabetes. Ozone, a major photochemical pollutant in urban areas, is negatively associated with fasting glucose and insulin levels, but most aspects of this association remain to be elucidated. Using an environmentally realistic concentration (0.8 parts per million), we demonstrated that exposure of rats to ozone induced whole-body insulin resistance and oxidative stress, with associated endoplasmic reticulum (ER) stress, c-Jun N-terminal kinase (JNK) activation, and disruption of insulin signaling in skeletal muscle. Bronchoalveolar lavage fluids from ozone-treated rats reproduced this effect in C2C12 myotubes, suggesting that toxic lung mediators were responsible for the phenotype. Pretreatment with the chemical chaperone 4-phenylbutyric acid, the JNK inhibitor SP600125, or the antioxidant N-acetylcysteine alleviated insulin resistance, demonstrating that ozone sequentially triggered oxidative stress, ER stress, and JNK activation to impair insulin signaling in muscle. This study is the first to report that ozone plays a causative role in the development of insulin resistance, suggesting that it could boost the development of diabetes. We therefore provide a potential mechanism linking pollutant exposure and the increased incidence of metabolic diseases.

The relationship between renal function and plasma concentration of the cachectic factor zinc-alpha2-glycoprotein (ZAG) in adult patients with chronic kidney disease.

Zinc-α2-glycoprotein (ZAG), a potent cachectic factor, is increased in patients undergoing maintenance dialysis. However, there is no data for patients before initiation of renal replacement therapy. The purpose of the present study was to assess the relationship between plasma ZAG concentration and renal function in patients with a large range of glomerular filtration rate (GFR). Plasma ZAG concentration and its relationship to GFR were investigated in 71 patients with a chronic kidney disease (CKD) stage 1 to 5, 17 chronic hemodialysis (HD), 8 peritoneal dialysis (PD) and 18 non-CKD patients. Plasma ZAG concentration was 2.3-fold higher in CKD stage 5 patients and 3-fold higher in HD and PD patients compared to non-CKD controls (P<0.01). The hemodialysis session further increased plasma ZAG concentration (+39%, P<0.01). An inverse relationship was found between ZAG levels and plasma protein (rs = -0.284; P<0.01), albumin (rs = -0.282, P<0.05), hemoglobin (rs = -0.267, P<0.05) and HDL-cholesterol (rs = -0.264, P<0.05) and a positive correlation were seen with plasma urea (rs = 0.283; P<0.01). In multiple regression analyses, plasma urea and HDL-cholesterol were the only variables associated with plasma ZAG (r2 = 0.406, P<0.001). In CKD-5 patients, plasma accumulation of ZAG was not correlated with protein energy wasting. Further prospective studies are however needed to better elucidate the potential role of ZAG in end-stage renal disease.

Indoxyl sulfate and p-cresyl sulfate in chronic kidney disease. Could these toxins modulate the antioxidant Nrf2-Keap1 pathway?

Protein-bound uremic toxins (i.e., indoxyl sulfate or p-cresyl sulfate), produced by intestinal bacteria, are accumulated in the plasma of chronic kidney disease (CKD) patients. These toxins interact negatively with biological functions, having potent oxidative stress-inducing effects and a pathological effect on cardiovascular disease. Recent research in CKD has shown that oxidative stress and inflammation can be compounded by impaired activation of the nuclear factor (erythroid-2-related factor)-2 (Nrf2)-Kelch-like ECH associating protein-1 (Keap1) pathway, a major cellular defense mechanism. However, to date, many questions arise regarding the role of this system in CKD. For example, protein-bound uremic toxins promote oxidative stress in CKD patients, but their putative effect on the Nrf2-Keap1 system has yet to be examined in these patients. This review will focus on the putative relationship among protein-bound uremic toxins, oxidative stress, and a possible decreased expression of Nrf2 in CKD.

Potential role and therapeutic interests of myo-inositol in metabolic diseases.

Several inositol isomers and in particular myo-inositol (MI) and D-chiro-inositol (DCI), were shown to possess insulin-mimetic properties and to be efficient in lowering post-prandial blood glucose. In addition, abnormalities in inositol metabolism are associated with insulin resistance and with long term microvascular complications of diabetes, supporting a role of inositol or its derivatives in glucose metabolism. The aim of this review is to focus on the potential benefits of a dietary supplement of myo-inositol, by far the most common inositol isomer in foodstuffs, in human disorders associated with insulin resistance (polycystic ovary syndrome, gestational diabetes mellitus or metabolic syndrome) or in prevention or treatment of some diabetic complications (neuropathy, nephropathy, cataract). The relevance of such a nutritional strategy will be discussed for each context on the basis of the clinical and/or animal studies. The dietary sources of myo-inositol and its metabolism from its dietary uptake to its renal excretion will be also covered in this review. Finally, the actual insights into inositol insulin-sensitizing effects will be addressed and in particular the possible role of inositol glycans as insulin second messengers.

White adipose tissue overproduces the lipid-mobilizing factor zinc α2-glycoprotein in chronic kidney disease.

Chronic kidney disease (CKD) is frequently associated with protein-energy wasting, a recognized strong predictive factor of mortality. Zinc α2-glycoprotein (ZAG) is a new adipokine involved in body weight control through its lipid-mobilizing activity. Here we tested whether the uremic environment in CKD could alter ZAG production by white adipose tissue and contribute to CKD-associated metabolic disturbances. Compared with normal plasma, uremic plasma induced a significant increase in ZAG synthesis (124%), was associated with a significant increase in basal lipolysis (31%), and significantly blunted lipogenesis (-53%) in 3T3-L1 adipocytes in vitro. In 5/6 nephrectomized rats and mice in vivo, there was a significant decrease in white adipose tissue accretion (-44% and -43%, respectively) and a significantly higher white adipose tissue content of ZAG protein than in sham-operated, pair-fed control animals (498% and 106%, respectively). Subcutaneous white adipose tissue biopsies from patients with end-stage renal disease exhibited a higher content of ZAG (573%) than age-matched controls. Thus, the ZAG content is increased in white adipose tissue from patients or animal models with CKD. Overproduction of ZAG in CKD could be a major contributor to metabolic disturbances associated with CKD.

p-Cresyl sulfate promotes insulin resistance associated with CKD.

The mechanisms underlying the insulin resistance that frequently accompanies CKD are poorly understood, but the retention of renally excreted compounds may play a role. One such compound is p-cresyl sulfate (PCS), a protein-bound uremic toxin that originates from tyrosine metabolism by intestinal microbes. Here, we sought to determine whether PCS contributes to CKD-associated insulin resistance. Administering PCS to mice with normal kidney function for 4 weeks triggered insulin resistance, loss of fat mass, and ectopic redistribution of lipid in muscle and liver, mimicking features associated with CKD. Mice treated with PCS exhibited altered insulin signaling in skeletal muscle through ERK1/2 activation. In addition, exposing C2C12 myotubes to concentrations of PCS observed in CKD caused insulin resistance through direct activation of ERK1/2. Subtotal nephrectomy led to insulin resistance and dyslipidemia in mice, and treatment with the prebiotic arabino-xylo-oligosaccharide, which reduced serum PCS by decreasing intestinal production of p-cresol, prevented these metabolic derangements. Taken together, these data suggest that PCS contributes to insulin resistance and that targeting PCS may be a therapeutic strategy in CKD.

Chronic treatment with myo-inositol reduces white adipose tissue accretion and improves insulin sensitivity in female mice.

Type 2 diabetes is a complex disease characterized by a state of insulin resistance in peripheral tissues such as skeletal muscle, adipose tissue or liver. Some inositol isomers have been reported to possess insulin-mimetic activity and to be efficient in lowering blood glucose level. The aim of the present study was to assess in mice the metabolic effects of a chronic treatment with myo-inositol, the most common stereoisomer of inositol. Mice given myo-inositol treatment (0.9 or 1.2 mg g(-1) day(-1), 15 days, orally or intraperitoneally) exhibited an improved glucose tolerance due to a greater insulin sensitivity. Mice treated with myo-inositol exhibited a decreased white adipose tissue accretion (-33%, P<.005) compared with controls. The decrease in white adipose tissue deposition was due to a decrease in adipose cell volume (-33%, P<.05), while no change was noticed in total adipocyte number. In skeletal muscle, in vivo as well as ex vivo myo-inositol treatment increased protein kinase B/Akt phosphorylation under baseline and insulin-stimulated conditions, suggesting a synergistic action of myo-inositol treatment and insulin on proteins of the insulin signalling pathway. Myo-inositol could therefore constitute a viable nutritional strategy for the prevention and/or treatment of insulin resistance and type 2 diabetes.

The lipid peroxidation by-product 4-hydroxy-2-nonenal (4-HNE) induces insulin resistance in skeletal muscle through both carbonyl and oxidative stress.

Numerous oxidants are produced as by-products of aerobic cell metabolism, and there is growing evidence that they play key roles in the pathogenesis of insulin resistance. Under conditions of oxidative stress, lipid peroxidation of ω6-polyunsaturated fatty acids leads to the production of 4-hydroxy-2-nonenal (4-HNE). Several lines of evidence suggest that 4-HNE could be involved in the pathophysiology of metabolic diseases; therefore, in this study we assessed the direct effects of 4-HNE on skeletal muscle insulin sensitivity. Gastrocnemius muscle and L6 muscle cells were treated with 4-HNE. Insulin signaling was measured by Western blotting and glucose uptake using 2-deoxy-d-[3H]glucose. Carbonyl stress, glutathione content, and oxidative stress were assessed as potential mechanisms leading to insulin resistance. Protection of cells was induced by pretreatment with 3H-1,2-dithiole-3-thione, N-acetyl-cysteine, aminoguanidine, or S-adenosyl-methionine. 4-HNE induced a time- and dose-dependent decrease in insulin signaling and insulin-induced glucose uptake in muscle. It induced a state of carbonyl stress through adduction of proteins as well as a depletion in reduced glutathione and production of radical oxygen species. A pharmacological increase in glutathione pools was achieved by 3H-1,2-dithiole-3-thione and protected the cells against all deleterious effects of 4-HNE; furthermore, N-acetylcysteine, aminoguanidine, and S-adenosylmethionine prevented 4-HNE noxious effects. 4-HNE can impair insulin action in muscle cells through oxidative stress and oxidative damage to proteins, eventually leading to insulin resistance. These deleterious effects can be prevented by pretreatment with antioxidants, scavengers, or an increase in intracellular glutathione pools. Use of such molecules could represent a novel strategy to combat insulin resistance and other oxidative stress-associated pathologies.

Quantitative structure-activity relationship for 4-hydroxy-2-alkenal induced cytotoxicity in L6 muscle cells.

Lipid peroxidation is one of the most important sources of endogenous toxic metabolites. 4-Hydroxy-2-nonenal (HNE) and 4-hydroxy-2-hexenal (HHE) are produced in several oxidative stress associated diseases from peroxidation of n-6 and n-3 polyunsaturated fatty acids, respectively. Both are able to form covalent adducts with many biomolecules. Particularly, proteins adduction can induce structural and conformational changes and impair biological function, which may be involved in the toxicity of hydroxy-alkenals. The aim of this study was to compare the effect of 4-hydroxy-2-alkenals to several chemically related derivatives in order to clarify the physico-chemical requirement of their toxicity. L6 muscle cells were treated with HHE, HNE and parent derivatives (acetal derivative, trans-alkenals and alkanals). Viability and necrosis were estimated using MTT, LDH and caspase-3 tests. LogLC50 (Lethal Concentration 50) was then tested for correlation with adducts formation (estimated using dinitrophenylhydrazine) and several molecular descriptors in order to establish quantitative structure-toxicity relationship (QSTR) models. The rank of derivatives toxicity, based on LC50 was: hydroxy-alkenals>acetal derivatives approximately 2-alkenals>alkanals and a high correlation was found between logLC50 and protein carbonylation. Moreover, logLC50 was correlated to the electrophilic descriptor LUMO (lowest unoccupied molecular orbital) as well as with electronegativity-related molecular descriptors such as number of oxygen atoms, partial negative surface area (PNSA3) and partial positive surface area (PPSA3). Together, these results point out the important role of the electrophilic structure and adduct formation in hydroxy-alkenals toxicity. Our present study demonstrates that 4-hydroxy-2-alkenals dramatic effects on cell viability are due to covalent adducts formation, particularly Michael adducts. This capacity is related to the electrophilic structure and reactive CC double bond, making it highly accessible for nucleophilic addition. The present study suggests that nucleophilic scavengers might protect cells against electrophile compounds and might be of possible therapeutic value in oxidative stress associated diseases.