Anandamide and WIN 55212-2 Afford Protection in Rat Brain Mitochondria in a Toxic Model Induced by 3-Nitropropionic Acid: an In Vitro Study.

Mitochondrial dysfunction plays a key role in the development of neurodegenerative disorders. In contrast, the regulation of the endocannabinoid system has been shown to promote neuroprotection in different neurotoxic paradigms. The existence of an active form of the cannabinoid receptor 1 (CB1R) in mitochondrial membranes (mitCB1R), which might exert its effects through the same signaling mechanisms as the cell membrane CB1R, has been shown to regulate mitochondrial activity. Although there is evidence suggesting that some cannabinoids may induce protective effects on isolated mitochondria, substantial evidence on the role of cannabinoids in mitochondria remains to be explored. In this work, we developed a toxic model of mitochondrial dysfunction induced by exposure of brain mitochondria to the succinate dehydrogenase inhibitor 3-nitropropionic acid (3-NP). Mitochondria were also pre-incubated with the endogenous agonist anandamide (AEA) and the synthetic CB1R agonist WIN 55212-2 to evaluate their protective effects. Mitochondrial reduction capacity, reactive oxygen species (ROS) formation, and mitochondrial swelling were assessed as toxic markers. While 3-NP decreased the mitochondrial reduction capacity and augmented mitochondrial ROS formation and swelling, both AEA and WIN 55212-2 ameliorated these toxic effects. To explore the possible involvement of mitCB1R activation on the protective effects of AEA and WIN 55212-2, mitochondria were also pre-incubated in the presence of the selective CB1R antagonist AM281, which completely reverted the protective effects of the cannabinoids to levels similar to those evoked by 3-NP. These results show partial protective effects of cannabinoids, suggesting that mitCB1R activation may be involved in the recovery of compromised mitochondrial activity, related to reduction of ROS formation and further prevention of mitochondrial swelling.

Chronic consumption of imbalance diets high in sucrose or fat induces abdominal obesity with different pattern of metabolic disturbances and lost in Langerhans cells population.

AIM: Obesity is a worldwide health issue, associated with development of type 2 Diabetes Mellitus. The aim of this study is to analyze the effect of consumption of two hypercaloric diets on metabolic disturbance and beta cells damage. MAIN METHODS: Male Wistar rats were subjected to twelve months consumption of three diets: a Control balanced diet (CTD, carbohydrates 58 %, proteins 29 %, lipids 13 %) and two hypercaloric diets, high in sucrose (HSD, carbohydrates 68 %, proteins 22 %, lipids 10 %) or high in fat (HFD, carbohydrates 31 %, proteins 14 %, lipids 55 %). Serum levels of glucose, triglycerides and free fatty acids were measured after zoometric parameters determination. Antioxidant enzymes activity and oxidative stress-marker were measured in pancreas tissue among histological analysis of Langerhans islets. KEY FINDINGS: Although diets were hypercaloric, the amount of food consumed by rats decreased, resulting in an equal caloric consumption. The HSD induced hypertriglyceridemia and hyperglycemia with higher levels in free fatty acids (FFA, lipotoxicity); whereas HFD did not increased neither the triglycerides nor FFA, nevertheless the loss of islets' cell was larger. Both diets induced obesity with hyperglycemia and significant reduction in Langerhans islets size. SIGNIFICANCE: Our results demonstrate that consumption of HSD induces more significant metabolic disturbances that HFD, although both generated pancreas damage; as well hypercaloric diet consumption is not indispensable to becoming obese; the chronic consumption of unbalanced diets (rich in carbohydrates or lipids) may lead to abdominal obesity with metabolic and functional disturbances, although the total amount of calories are similar.

Smooth Muscle Cells from a Rat Model of Obesity and Hyperleptinemia Are Partially Resistant to Leptin-Induced Reactive Oxygen Species Generation.

The aim of this study was to evaluate the effect of leptin on reactive oxygen species' (ROS) generation of smooth muscle cells (SMCs) from a rat model of obesity and hyperleptinemia. Obesity and hyperleptinemia were induced in rats by a sucrose-based diet for 24 weeks. ROS generation was detected by using dichloro-dihydrofluorescein (DCF), a fluorescent ROS probe in primary SMCs culture. An increase in plasma leptin and oxidative stress markers was observed in sucrose-fed (SF) rats. At baseline SMCs from SF rats showed a more than twofold increase in fluorescence intensity (FI) compared to that obtained in control (C) cells. When the C cells were treated with 20 ng leptin, the FI increased by about 250%, whereas the leptin-induced FI in the SF cells increased only by 28%. In addition, sucrose feeding increased the levels of p22phox and gp91phox, subunits of Nox as an O2•- source in SMCs. Treatment of cells with leptin significantly increased p22phox and gp91phox levels in C cells and did not affect SF cells. Regarding STAT3 phosphorylation and the content of PTP1B and SOCS3 as protein markers of leptin resistance, they were found to be significantly increased in SF cells. These results suggest that SF aortic SMCs are partially resistant to leptin-induced ROS generation.

Lipotoxicity, glucotoxicity and some strategies to protect vascular smooth muscle cell against proliferative phenotype in metabolic syndrome.

Metabolic syndrome (MetS) is a risk factor for the development of cardiovascular disease (CVD) and atherosclerosis through a mechanism that involves vascular smooth muscle cell (VSMC) proliferation, lipotoxicity and glucotoxicity. Several molecules found to be increased in MetS, including free fatty acids, fatty acid binding protein 4, leptin, resistin, oxidized lipoprotein particles, and advanced glycation end products, influence VSMC proliferation. Most of these molecules act through their receptors on VSMCs by activating several signaling pathways associated with ROS generation in various cellular compartments. ROS from NADPH-oxidase and mitochondria have been found to promote VSMC proliferation and cell cycle progression. In addition, most of the natural or synthetic substances described in this review, including pharmaceuticals with hypoglycemic and hypolipidemic properties, attenuate VSMC proliferation by their simultaneous modulation of cell signaling and their scavenging property due to the presence of a phenolic ring in their structure. This review discusses recent data in the literature on the role that several MetS-related molecules and ROS play in the change from contractile to proliferative phenotype of VSMCs. Hence the importance of proposing an appropriate strategy to prevent uncontrolled VSMC proliferation using antioxidants, hypoglycemic and hypolipidemic agents.

Therapeutic Effect of Curcumin on 5/6Nx Hypertriglyceridemia: Association with the Improvement of Renal Mitochondrial ß-Oxidation and Lipid Metabolism in Kidney and Liver.

Chronic kidney disease (CKD) prevalence is constantly increasing, and dyslipidemia in this disease is characteristic, favoring cardiovascular events. However, the mechanisms of CKD dyslipidemia are not fully understood. The use of curcumin (CUR) in CKD models such as 5/6 nephrectomy (5/6Nx) has shown multiple beneficial effects, so it has been proposed to correct dyslipidemia without side effects. This work aimed to characterize CUR's potential therapeutic effect on dyslipidemia and alterations in lipid metabolism and mitochondrial ß-oxidation in the liver and kidney in 5/6Nx. Male Wistar rats were subjected to 5/6Nx and progressed by 4 weeks; meanwhile, CUR (120 mg/kg) was administered for weeks 5 to 8. Our results showed that CUR reversed the increase in liver and kidney damage and hypertriglyceridemia induced by 5/6Nx. CUR also reversed mitochondrial membrane depolarization and ß-oxidation disorders in the kidney and the increased lipid uptake and the high levels of proteins involved in fatty acid synthesis in the liver and kidney. CUR also decreased lipogenesis and increased mitochondrial biogenesis markers in the liver. Therefore, we concluded that the therapeutic effect of curcumin on 5/6Nx hypertriglyceridemia is associated with the restoration of renal mitochondrial ß-oxidation and the reduction in lipid synthesis and uptake in the kidneys and liver.

Effect of dietary chia supplementation on glucose metabolism and adipose tissue function markers in non-alcoholic fatty liver disease subjects.

Introduction: Background: adipose tissue dysfunction is a key factor for diabetes and non-alcoholic fatty liver disease (NAFLD) development. Chia (Salvia hispanica) is an abundant source of omega-3 fatty acids, antioxidants, and fiber which could improve adipose tissue functionality. Aim: to analyze the effect of an isocaloric chia-supplemented diet on glucose metabolism, adipose tissue inflammation, and endothelial function markers in patients with NAFLD and early stages of diabetes. Methods: in 32 patients with previous NAFLD diagnosis, without known diabetes, the effect of a diet supplemented with ground chia (25 g/day/8 weeks) was evaluated. Visceral (VAF) and liver fat, plasma lipids, fatty acids, and cytokine profiles, oral glucose tolerance test (OGTT), insulinogenic index (IGI30), insulin disposition index (DIO), and endothelial progenitor cells (EPC) were analyzed. Before and after eight weeks of diet supplementation. Results: chia supplementation promoted increases in plasma alpha-linolenic acid (75 %) and fiber consumption (55 %), and a higher number of EPC (+126 %). Basal OGTT showed that nine patients had normal OGTT, 17 pre-diabetes, and six newly diagnosed diabetes. In patients with diabetes, chia favored a healthier adipose tissue (VAF -7 %, NAFLD -100 %, adiponectin +47 %, resistin -30 %, IL-6 -44 %, IL-1ß -22 %) and upturn glucose metabolism through the improvement of beta-cell function (IGI30 +50 %, DIO +66 %). Conclusions: dietary supplementation with 25 g/day of ground chia may promote a healthier adipose tissue and improve pancreatic ß-cell and endothelial function. Among patients with early metabolic abnormalities, phytochemical properties of chia may retard diabetes progression and advanced stages of liver damage.

Introducción: Antecedentes: la disfunción del tejido adiposo es un factor clave para el desarrollo de diabetes e hígado graso no alcohólico (HGNA). La chía (Salvia hispanica) es una fuente abundante de ácidos grasos omega-3, antioxidantes y fibra, que podrían mejorar la funcionalidad del tejido adiposo. Objetivo: analizar el efecto de una dieta isocalórica suplementada con chía sobre el metabolismo de glucosa y los marcadores de inflamación del tejido adiposo y de función endotelial en pacientes con HGNA y etapas tempranas de diabetes. Métodos: en 32 pacientes con diagnóstico previo de HGNA, pero sin diabetes conocida, se evaluó el efecto de una dieta suplementada con chía molida (25 g/día) sobre la grasa visceral (GAV) y hepática, el perfil de lípidos, los ácidos grasos y las citoquinas en plasma, la prueba de tolerancia oral a la glucosa (OGTT), el índice insulinogénico (IGI30), el índice de disposición de insulina (DIO) y las células progenitoras endoteliales (EPC), antes y después de ocho semanas de suplementación. Resultados: la suplementación con chía promovió aumentos en el consumo de ácido alfa-linolénico en plasma (75 %) y fibra de alta viscosidad (55 %) y un mayor número de EPC (+126 %). La OGTT basal mostró que nueve pacientes tenían curva normal, 17 tenían prediabetes y seis, diabetes de recién diagnóstico. En los pacientes con diabetes, la chía favoreció un tejido adiposo más sano (GAV -7 %, NAFLD -100 %, adiponectina +47 %, resistina -30 %, IL-6 -44 %, IL-1ß -22 %) y un aumento del metabolismo de la glucosa a través de la mejora de la función de las células beta (IGI30 +50 %, DIO +66 %). Conclusiones: la suplementación de la dieta con 25 g/día de chía molida puede promover un tejido adiposo más saludable y mejorar la función endotelial y de las células ß pancreáticas. Entre los pacientes con anomalías metabólicas tempranas, las propiedades fitoquímicas de la chía pueden retrasar la progresión de la diabetes y el desarrollo de etapas avanzadas de daño hepático.

Two alpha isopropylmalate synthase isozymes with similar kinetic properties are extant in the yeast Lachancea kluyveri.

The first committed step in the leucine biosynthetic pathway is catalyzed by α-isopropylmalate synthase (α-IPMS, EC 2.3.3.13), which in the Saccaromycotina subphylum of Ascomycete yeasts is frequently encoded by duplicated genes. Following a gene duplication event, the two copies may be preserved presumably because the encoded proteins diverge in either functional properties and/or cellular localization. The genome of the petite-negative budding yeast Lachancea kluyveri includes two SAKL0E10472 (LkLEU4) and SAKL0F05170 g (LkLEU4BIS) paralogous genes, which are homologous to other yeast α-IPMS sequences. Here, we investigate whether these paralogous genes encode functional α-IPMS isozymes and whether their functions have diverged. Molecular phylogeny suggested that the LkLeu4 isozyme is located in the mitochondria and LkLeu4BIS in the cytosol. Comparison of growth rates, leucine intracellular pools and mRNA levels, indicate that the LkLeu4 isozyme is the predominant α-IPMS enzyme during growth on glucose as carbon source. Determination of the kinetic parameters indicates that the isozymes have similar affinities for the substrates and for the feedback inhibitor leucine. Thus, the diversification of the physiological roles of the genes LkLEU4 and LkLEU4BIS involves preferential transcription of the LkLEU4 gene during growth on glucose and different subcellular localization, although ligand interactions have not diverged.

Endogenous Liver Protections Against Lipotoxicity and Oxidative Stress to Avoid the Progression of Non-alcoholic Fatty Liver to more Serious Disease.

Non-alcoholic fatty liver disease (NAFLD) is a metabolic disorder characterized by an ectopic accumulation of lipids in at least 5% of hepatocytes. The first phase of the disease, called hepatic steatosis, progresses over time to chronic conditions, such as steatohepatitis, cirrhosis, and finally, hepatic insufficiency and cancer. The accumulation of free fatty acids in hepatocytes, particularly saturated fatty acids, is a key process in the development and progression of NAFLD. Furthermore, the accumulation of oxidative stress markers in NAFLD is closely linked to lipotoxicity due to impaired lipid metabolism and increased generation of reactive oxygen species (ROS). However, endogenous mechanisms are activated early in the liver to protect against lipotoxicity and oxidative stress, thus preventing liver mass loss and disease progression. Thus, in order to develop appropriate therapies, the purpose of this review is to discuss recent data from the literature regarding the importance of intrinsic mechanisms deployed by the liver in protecting itself against the adverse effects related to chronic lipid accumulation and ROS generation.

Mitochondrial Dysfunction and Alterations in Mitochondrial Permeability Transition Pore (mPTP) Contribute to Apoptosis Resistance in Idiopathic Pulmonary Fibrosis Fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by increased activation of fibroblasts/myofibroblasts. Previous reports have shown that IPF fibroblasts are resistant to apoptosis, but the mechanisms remain unclear. Since inhibition of the mitochondrial permeability transition pore (mPTP) has been implicated in the resistance to apoptosis, in this study, we analyzed the role of mitochondrial function and the mPTP on the apoptosis resistance of IPF fibroblasts under basal conditions and after mitomycin C-induced apoptosis. We measured the release of cytochrome c, mPTP opening, mitochondrial calcium release, oxygen consumption, mitochondrial membrane potential, ADP/ATP ratio, ATP concentration, and mitochondrial morphology. We found that IPF fibroblasts were resistant to mitomycin C-induced apoptosis and that calcium, a well-established activator of mPTP, is decreased as well as the release of pro-apoptotic proteins such as cytochrome c. Likewise, IPF fibroblasts showed decreased mitochondrial function, while mPTP was less sensitive to ionomycin-induced opening. Although IPF fibroblasts did not present changes in the mitochondrial membrane potential, we found a fragmented mitochondrial network with scarce, thinned, and disordered mitochondria with reduced ATP levels. Our findings demonstrate that IPF fibroblasts are resistant to mitomycin C-induced apoptosis and that altered mPTP opening contributes to this resistance. In addition, IPF fibroblasts show mitochondrial dysfunction evidenced by a decrease in respiratory parameters.

Alanine Represses γ-Aminobutyric Acid Utilization and Induces Alanine Transaminase Required for Mitochondrial Function in Saccharomyces cerevisiae.

The γ-aminobutyric acid (GABA) shunt constitutes a conserved metabolic route generating nicotinamide adenine dinucleotide phosphate (NADPH) and regulating stress response in most organisms. Here we show that in the presence of GABA, Saccharomyces cerevisiae produces glutamate and alanine through the irreversible action of Uga1 transaminase. Alanine induces expression of alanine transaminase (ALT1) gene. In an alt1Δ mutant grown on GABA, alanine accumulation leads to repression of the GAD1, UGA1, and UGA2 genes, involved in the GABA shunt, which could result in growth impairment. Induced ALT1 expression and negative modulation of the GABA shunt by alanine constitute a novel regulatory circuit controlling both alanine biosynthesis and catabolism. Consistent with this, the GABA shunt and the production of NADPH are repressed in a wild-type strain grown in alanine, as compared to those detected in the wild-type strain grown on GABA. We also show that heat shock induces alanine biosynthesis and ALT1, UGA1, UGA2, and GAD1 gene expression, whereas an uga1Δ mutant shows heat sensitivity and reduced NADPH pools, as compared with those observed in the wild-type strain. Additionally, an alt1Δ mutant shows an unexpected alanine-independent phenotype, displaying null expression of mitochondrial COX2, COX3, and ATP6 genes and a notable decrease in mitochondrial/nuclear DNA ratio, as compared to a wild-type strain, which results in a petite phenotype. Our results uncover a new negative role of alanine in stress defense, repressing the transcription of the GABA shunt genes, and support a novel Alt1 moonlighting function related to the maintenance of mitochondrial DNA integrity and mitochondrial gene expression.

Palmitoyl-CoA effect on cytochrome c release, a key process of apoptosis, from liver mitochondria of rat with sucrose diet-induced obesity.

Cytochrome c (cyt-c) release from the mitochondria to the cytosol is a key process in the initiation of hepatocyte apoptosis involved in the progression of non-alcoholic fatty liver disease (NAFLD) to fibrosis, cirrhosis and hepatocellular carcinoma. Hepatocyte apoptosis may be related to lipotoxicity due to the accumulation of palmitic acid and palmitoyl-CoA (Pal-CoA). Therefore, the aim of this study is to examine whether Pal-CoA induces cyt-c release from liver mitochondria of sucrose-fed rat (SF). Pal-CoA-induced cyt-c release was sensitive to cyclosporine A indicating the involvement of the mitochondrial membrane permeability transition (mMPT). In addition, cyt-c release from SF mitochondria remains significantly lower than C mitochondria despite the increased rate of H2O2 generation in SF mitochondria. The decreased cyt-c release from SF may be also related to the increased proportion of the palmitic acid-enriched cardiolipin, due to the high availibilty of palmitic acid in SF liver. The enrichment of cardiolipin molecular species with palmitic acid makes cardiolipin more resistant to peroxidation, a mechanism involved in the dissociation of cyt-c from mitochondrial inner membrane. These results suggest that Pal-CoA may participate in the progression of NAFLD to more severe disease through mechanisms involving cyt-c release and mMPT, a key process of apoptosis.

Rich fatty acids diet of fish and olive oils modifies membrane properties in striatal rat synaptosomes.

Background: Essential fatty acids (EFAs) and non-essential fatty acids (nEFAs) exert experimental and clinical neuroprotection in neurodegenerative diseases. The main EFAs, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), nEFAs, and oleic acid (OA) contained in olive and fish oils are inserted into the cell membranes, but the exact mechanism through which they exert neuroprotection is still unknown. Objectives and Methods: In this study, we assessed the fatty acids content and membrane fluidity in striatal rat synaptosomes after fatty acid-rich diets (olive- or a fish-oil diet, 15% w/w). Then, we evaluated the effect of enriching striatum synaptosomes with fatty acids on the oxidative damage produced by the prooxidants ferrous sulfate (FeSO4) or quinolinic acid (QUIN). Results and Discussion: Lipid profile analysis in striatal synaptosomes showed that EPA content increased in the fish oil group in comparison with control and olive groups. Furthermore, we found that synaptosomes enriched with fatty acids and incubated with QUIN or FeSO4 showed a significant oxidative damage reduction. Results suggest that EFAs, particularly EPA, improve membrane fluidity and confer antioxidant effect.

In Kluyveromyces lactis a Pair of Paralogous Isozymes Catalyze the First Committed Step of Leucine Biosynthesis in Either the Mitochondria or the Cytosol.

Divergence of paralogous pairs, resulting from gene duplication, plays an important role in the evolution of specialized or novel gene functions. Analysis of selected duplicated pairs has elucidated some of the mechanisms underlying the functional diversification of Saccharomyces cerevisiae (S. cerevisiae) paralogous genes. Similar studies of the orthologous pairs extant in pre-whole genome duplication yeast species, such as Kluyveromyces lactis (K. lactis) remain to be addressed. The genome of K. lactis, an aerobic yeast, includes gene pairs generated by sporadic duplications. The genome of this organism comprises the KlLEU4 and KlLEU4BIS paralogous pair, annotated as putative α-isopropylmalate synthases (α-IPMSs), considered to be the orthologs of the S. cerevisiae ScLEU4/ScLEU9 paralogous genes. The enzymes encoded by the latter two genes are mitochondrially located, differing in their sensitivity to leucine allosteric inhibition resulting in ScLeu4-ScLeu4 and ScLeu4-ScLeu9 sensitive dimers and ScLeu9-ScLeu9 relatively resistant homodimers. Previous work has shown that, in a Scleu4Δ mutant, ScLEU9 expression is increased and assembly of ScLeu9-ScLeu9 leucine resistant homodimers results in loss of feedback regulation of leucine biosynthesis, leading to leucine accumulation and decreased growth rate. Here we report that: (i) K. lactis harbors a sporadic gene duplication, comprising the KlLEU4, syntenic with S. cerevisiae ScLEU4 and ScLEU9, and the non-syntenic KlLEU4BIS, arising from a pre-WGD event. (ii) That both, KlLEU4 and KlLEU4BIS encode leucine sensitive α-IPMSs isozymes, located in the mitochondria (KlLeu4) and the cytosol (KlLeu4BIS), respectively. (iii) That both, KlLEU4 or KlLEU4BIS complement the Scleu4Δ Scleu9Δ leucine auxotrophic phenotype and revert the enhanced ScLEU9 transcription observed in a Scleu4Δ ScLEU9 mutant. The Scleu4Δ ScLEU9 growth mutant phenotype is only fully complemented when transformed with the syntenic KlLEU4 mitochondrial isoform. KlLEU4 and KlLEU4BIS underwent a different diversification pathways than that leading to ScLEU4/ScLEU9. KlLEU4 could be considered as the functional ortholog of ScLEU4, since its encoded isozyme can complement both the Scleu4Δ Scleu9Δ leucine auxotrophy and the Scleu4Δ ScLEU9 complex phenotype.

Mitochondrial dysfunction in metabolic and cardiovascular diseases associated with cardiolipin remodeling.

Cardiolipin (CL) is an acidic phospholipid almost exclusively found in the inner mitochondrial membrane, that not only stabilizes the structure and function of individual components of the mitochondrial electron transport chain, but regulates relevant mitochondrial processes, like mitochondrial dynamics and cristae structure maintenance among others. Alterations in CL due to peroxidation, correlates with loss of such mitochondrial activities and disease progression. In this review it is recapitulated the current state of knowledge of the role of cardiolipin remodeling associated with mitochondrial dysfunction in metabolic and cardiovascular diseases.

Sphingolipid Effects on the Plasma Membrane Produced by Addition of Fumonisin B1 to Maize Embryos.

Fumonisin B1 is a mycotoxin produced by Fusarium verticillioides that modifies the membrane properties from animal cells and inhibits complex sphingolipids synthesis through the inhibition of ceramide synthase. The aim of this work was to determine the effect of Fumonisin B1 on the plant plasma membrane when the mycotoxin was added to germinating maize embryos. Fumonisin B1 addition to the embryos diminished plasma membrane fluidity, increased electrolyte leakage, caused a 7-fold increase of sphinganine and a small decrease in glucosylceramide in the plasma membrane, without affecting phytosphingosine levels or fatty acid composition. A 20%-30% inhibition of the plasma membrane H+-ATPase activity was observed when embryos were germinated in the presence of the mycotoxin. Such inhibition was only associated to the decrease in glucosylceramide and the addition of exogenous ceramide to the embryos relieved the inhibition of Fumonisin B1. These results indicate that exposure of the maize embryos for 24 h to Fumonisin B1 allowed the mycotoxin to target ceramide synthase at the endoplasmic reticulum, eliciting an imbalance of endogenous sphingolipids. The latter disrupted membrane properties and inhibited the plasma membrane H+-ATPase activity. Altogether, these results illustrate the mode of action of the pathogen and a plant defense strategy.

Kidney dysfunction induced by a sucrose-rich diet in rat involves mitochondria ROS generation, cardiolipin changes, and the decline of autophagy protein markers.

The mechanistic link between obesity and renal failure has been proposed to involve mitochondria reactive oxygen species generation and lipotoxicity. These pathological conditions make mitochondria of particular interest in the regulation of cell function and death by both apoptosis and autophagy. Therefore, this work was undertaken to investigate mitochondria function, autophagy, and apoptosis protein markers in the kidney from a rat model of intra-abdominal obesity and renal damage induced by a high-sucrose diet. Mitochondria from sucrose-fed (SF) kidneys in the presence of pyruvate-malate generated H2O2 at a higher rate than from control (79.81 ± 4.98 vs. 65.84 ± 1.95 pmol·min-1·mg protein-1). With succinate, the release of H2O2 was significantly higher compared with pyruvate-malate, and it remained higher in SF than in control mitochondria (146.4 ± 8.8 vs. 106.1 ± 5.9 pmol·min-1·mg protein-1). However, cytochrome c release from SF kidney mitochondria was lower than from control. In addition, cardiolipin, a mitochondria-specific phospholipid, was found increased in SF mitochondria due to the enhanced amount of both cardiolipin synthase and tafazzin. Cardiolipin was also found enriched with saturated and monounsaturated fatty acids, which are less susceptible to peroxidative stress involved in cytochrome c release. Furthermore, beclin-1 and light chain 3-B, as autophagy protein markers, and caspase-9, as apoptosis protein marker, were found decreased in SF kidneys. These results suggest that the decline of autophagy protein markers and the lack of apoptosis process could be a pathological mechanism of cell dysfunction leading to the progression of renal disease in SF rats.

Glycine Increases Insulin Sensitivity and Glutathione Biosynthesis and Protects against Oxidative Stress in a Model of Sucrose-Induced Insulin Resistance.

Oxidative stress and redox status play a central role in the link between insulin resistance (IR) and lipotoxicity in metabolic syndrome. This mechanistic link may involve alterations in the glutathione redox state. We examined the effect of glycine supplementation to diet on glutathione biosynthesis, oxidative stress, IR, and insulin cell signaling in liver from sucrose-fed (SF) rats characterized by IR and oxidative stress. Our hypothesis is that the correction of glutathione levels by glycine treatment leads to reduced oxidative stress, a mechanism associated with improved insulin signaling and IR. Glycine treatment decreases the levels of oxidative stress markers in liver from SF rats and increases the concentrations of glutathione (GSH) and γ-glutamylcysteine and the amount of γ-glutamylcysteine synthetase (γ-GCS), a key enzyme of GSH biosynthesis in liver from SF rats. In liver from SF rats, glycine also decreases the insulin-induced phosphorylation of insulin receptor substrate-1 (ISR-1) in serine residue and increases the phosphorylation of insulin receptor ß-subunit (IR-ß) in tyrosine residue. Thus, supplementing diets with glycine to correct GSH deficiency and to reduce oxidative stress provides significant metabolic benefits to SF rats by improving insulin sensitivity.

Reactive Oxygen Species from NADPH Oxidase and Mitochondria Participate in the Proliferation of Aortic Smooth Muscle Cells from a Model of Metabolic Syndrome.

In metabolic diseases, the increased reactive oxygen species (ROS) represents one of the pathogenic mechanisms for vascular disease probably by promoting vascular smooth muscle cell (SMC) proliferation that contributes to the development of arterial remodeling and stenosis, hypertension, and atherosclerosis. Therefore, this work was undertaken to evaluate the participation of ROS from NADPH oxidase and mitochondria in the proliferation of SMCs from the aorta in a model of metabolic syndrome induced by sucrose feeding in rats. After 24 weeks, sucrose-fed (SF) rats develop hypertension, intra-abdominal obesity, hyperinsulinemia, and hyperleptinemia. In addition SMCs from SF rats had a higher growth rate and produce more ROS than control cells. The treatment of SMCs with DPI and apocynin to inhibit NADPH oxidase and with tempol to scavenge superoxide anion significantly blocked the proliferation of both SF and control cells suggesting the participation of NADPH oxidase as a source of superoxide anion. MitoTEMPO, which targets mitochondria within the cell, also significantly inhibited the proliferation of SMCs having a greater effect on cells from SF than from the control aorta. The higher rate of cell growth from the SF aorta is supported by the increased content of cyclophilin A and CD147, proteins involved in the mechanism of cell proliferation. In addition, caldesmon, α-actin, and phosphorylated myosin light chain, contractile phenotype proteins, were found significantly lower in SF cells in no confluent state and increased in confluent state but without difference between both cell types. Our results suggest that ROS from NADPH oxidase and mitochondria significantly participate in the difference found in the rate of cell growth between SF and control cells.

Diversification of Transcriptional Regulation Determines Subfunctionalization of Paralogous Branched Chain Aminotransferases in the Yeast Saccharomyces cerevisiae.

Saccharomyces cerevisiae harbors BAT1 and BAT2 paralogous genes that encode branched chain aminotransferases and have opposed expression profiles and physiological roles . Accordingly, in primary nitrogen sources such as glutamine, BAT1 expression is induced, supporting Bat1-dependent valine-isoleucine-leucine (VIL) biosynthesis, while BAT2 expression is repressed. Conversely, in the presence of VIL as the sole nitrogen source, BAT1 expression is hindered while that of BAT2 is activated, resulting in Bat2-dependent VIL catabolism. The presented results confirm that BAT1 expression is determined by transcriptional activation through the action of the Leu3-α-isopropylmalate (α-IPM) active isoform, and uncovers the existence of a novel α-IPM biosynthetic pathway operating in a put3Δ mutant grown on VIL, through Bat2-Leu2-Leu1 consecutive action. The classic α-IPM biosynthetic route operates in glutamine through the action of the leucine-sensitive α-IPM synthases. The presented results also show that BAT2 repression in glutamine can be alleviated in a ure2Δ mutant or through Gcn4-dependent transcriptional activation. Thus, when S. cerevisiae is grown on glutamine, VIL biosynthesis is predominant and is preferentially achieved through BAT1; while on VIL as the sole nitrogen source, catabolism prevails and is mainly afforded by BAT2.