In vivo chronic exposure to inorganic mercury worsens hypercholesterolemia, oxidative stress and atherosclerosis in the LDL receptor knockout mice.

Heavy metal exposure leads to multiple system dysfunctions. The mechanisms are likely multifactorial and involve inflammation and oxidative stress. The aim of this study was to evaluate markers and risk factors for atherosclerosis in the LDL receptor knockout mouse model chronically exposed to inorganic mercury (Hg) in the drinking water. Results revealed that Hg exposed mice present increased plasma levels of cholesterol, without alterations in glucose. As a major source and target of oxidants, we evaluated mitochondrial function. We found that liver mitochondria from Hg treated mice show worse respiratory control, lower oxidative phosphorylation efficiency and increased H2O2 release. In addition, Hg induced mitochondrial membrane permeability transition. Erythrocytes from Hg treated mice showed a 50% reduction in their ability to take up oxygen, lower levels of reduced glutathione (GSH) and of antioxidant enzymes (SOD, catalase and GPx). The Hg treatment disturbed immune system cells counting and function. While lymphocytes were reduced, monocytes, eosinophils and neutrophils were increased. Peritoneal macrophages from Hg treated mice showed increased phagocytic activity. Hg exposed mice tissues present metal impregnation and parenchymal architecture alterations. In agreement, increased systemic markers of liver and kidney dysfunction were observed. Plasma, liver and kidney oxidative damage indicators (MDA and carbonyl) were increased while GSH and thiol groups were diminished by Hg exposure. Importantly, atherosclerotic lesion size in the aorta root of Hg exposed mice were larger than in controls. In conclusion, in vivo chronic exposure to Hg worsens the hypercholesterolemia, impairs mitochondrial bioenergetics and redox function, alters immune cells profile and function, causes several tissues oxidative damage and accelerates atherosclerosis development.

Enhanced resistance to Ca2+-induced mitochondrial permeability transition in the long-lived red-footed tortoise Chelonoidis carbonaria.

The interaction between supraphysiological cytosolic Ca2+ levels and mitochondrial redox imbalance mediates the mitochondrial permeability transition (MPT). The MPT is involved in cell death, diseases and aging. This study compared the liver mitochondrial Ca2+ retention capacity and oxygen consumption in the long-lived red-footed tortoise (Chelonoidis carbonaria) with those in the rat as a reference standard. Mitochondrial Ca2+ retention capacity, a quantitative measure of MPT sensitivity, was remarkably higher in tortoises than in rats. This difference was minimized in the presence of the MPT inhibitors ADP and cyclosporine A. However, the Ca2+ retention capacities of tortoise and rat liver mitochondria were similar when both MPT inhibitors were present simultaneously. NADH-linked phosphorylating respiration rates of tortoise liver mitochondria represented only 30% of the maximal electron transport system capacity, indicating a limitation imposed by the phosphorylation system. These results suggested underlying differences in putative MPT structural components [e.g. ATP synthase, adenine nucleotide translocase (ANT) and cyclophilin D] between tortoises and rats. Indeed, in tortoise mitochondria, titrations of inhibitors of the oxidative phosphorylation components revealed a higher limitation of ANT. Furthermore, cyclophilin D activity was approximately 70% lower in tortoises than in rats. Investigation of critical properties of mitochondrial redox control that affect MPT demonstrated that tortoise and rat liver mitochondria exhibited similar rates of H2O2 release and glutathione redox status. Overall, our findings suggest that constraints imposed by ANT and cyclophilin D, putative components or regulators of the MPT pore, are associated with the enhanced resistance to Ca2+-induced MPT in tortoises.

Lack of mitochondrial NADP(H)-transhydrogenase expression in macrophages exacerbates atherosclerosis in hypercholesterolemic mice.

The atherosclerosis prone LDL receptor knockout mice (Ldlr-/-, C57BL/6J background) carry a deletion of the NADP(H)-transhydrogenase gene (Nnt) encoding the mitochondrial enzyme that catalyzes NADPH synthesis. Here we hypothesize that both increased NADPH consumption (due to increased steroidogenesis) and decreased NADPH generation (due to Nnt deficiency) in Ldlr-/- mice contribute to establish a macrophage oxidative stress and increase atherosclerosis development. Thus, we compared peritoneal macrophages and liver mitochondria from three C57BL/6J mice lines: Ldlr and Nnt double mutant, single Nnt mutant and wild-type. We found increased oxidants production in both mitochondria and macrophages according to a gradient: double mutant > single mutant > wild-type. We also observed a parallel up-regulation of mitochondrial biogenesis (PGC1a, TFAM and respiratory complexes levels) and inflammatory (iNOS, IL6 and IL1b) markers in single and double mutant macrophages. When exposed to modified LDL, the single and double mutant cells exhibited significant increases in lipid accumulation leading to foam cell formation, the hallmark of atherosclerosis. Nnt deficiency cells showed up-regulation of CD36 and down-regulation of ABCA1 transporters what may explain lipid accumulation in macrophages. Finally, Nnt wild-type bone marrow transplantation into LDLr-/- mice resulted in reduced diet-induced atherosclerosis. Therefore, Nnt plays a critical role in the maintenance of macrophage redox, inflammatory and cholesterol homeostasis, which is relevant for delaying the atherogenesis process.

Calcium-sensitive pyruvate dehydrogenase phosphatase is required for energy metabolism, growth, differentiation, and infectivity of Trypanosoma cruzi.

In vertebrate cells, mitochondrial Ca2+ uptake by the mitochondrial calcium uniporter (MCU) leads to Ca2+-mediated stimulation of an intramitochondrial pyruvate dehydrogenase phosphatase (PDP). This enzyme dephosphorylates serine residues in the E1α subunit of pyruvate dehydrogenase (PDH), thereby activating PDH and resulting in increased ATP production. Although a phosphorylation/dephosphorylation cycle for the E1α subunit of PDH from nonvertebrate organisms has been described, the Ca2+-mediated PDP activation has not been studied. In this work, we investigated the Ca2+ sensitivity of two recombinant PDPs from the protozoan human parasites Trypanosoma cruzi (TcPDP) and T. brucei (TbPDP) and generated a TcPDP-KO cell line to establish TcPDP's role in cell bioenergetics and survival. Moreover, the mitochondrial localization of the TcPDP was studied by CRISPR/Cas9-mediated endogenous tagging. Our results indicate that TcPDP and TbPDP both are Ca2+-sensitive phosphatases. Of note, TcPDP-KO epimastigotes exhibited increased levels of phosphorylated TcPDH, slower growth and lower oxygen consumption rates than control cells, an increased AMP/ATP ratio and autophagy under starvation conditions, and reduced differentiation into infective metacyclic forms. Furthermore, TcPDP-KO trypomastigotes were impaired in infecting cultured host cells. We conclude that TcPDP is a Ca2+-stimulated mitochondrial phosphatase that dephosphorylates TcPDH and is required for normal growth, differentiation, infectivity, and energy metabolism in T. cruzi Our results support the view that one of the main roles of the MCU is linked to the regulation of intramitochondrial dehydrogenases.

Direct determination of anaerobe contributions to the energy metabolism of Trypanosoma cruzi by chip calorimetry.

We describe a chip calorimetric technique that allows the investigation of biological material under anoxic conditions in a micro-scale and in real time. Due to the fast oxygen exchange through the sample flow channel wall, the oxygen concentration inside the samples could be switched between atmospheric oxygen partial pressure to an oxygen concentration of 0.5% within less than 2 h. Using this technique, anaerobic processes in the energy metabolism of Trypanosoma cruzi could be studied directly. The comparison of the calorimetric and respirometric response of T. cruzi cells to the treatment with the mitochondrial inhibitors oligomycin and antimycin A and the uncoupler FCCP revealed that the respiration-related heat rate is superimposed by strong anaerobic contributions. Calorimetric measurements under anoxic conditions and with glycolytic inhibitors showed that anaerobic metabolic processes contribute from 30 to 40% to the overall heat production rate. Similar basal and antimycin A heat rates with cells under anoxic conditions indicated that the glycolytic rates are independent of the oxygen concentration which confirms the absence of the "Pasteur effect" in Trypanosomes. Graphical abstract.

Cisplatin-induced human peripheral blood mononuclear cells' oxidative stress and nephrotoxicity in head and neck cancer patients: the influence of hydrogen peroxide.

Cisplatin is a widely used antineoplastic agent in the treatment of head and neck cancer. However, it is highly nephrotoxic. Oxidative stress is the main mechanism responsible for cisplatin-induced nephrotoxicity. The aim of this study was to characterize cisplatin-induced nephrotoxicity, oxidative stress in peripheral blood mononuclear cells, and the relationship between them. Twenty-four patients were included in the study. Patients had their blood collected prior to cisplatin administration, and 5 and 20 days after initiating therapy, to assess renal function and to determine oxidative stress with MitoSOX™Red, H2DCF-DA, and Amplex® Red tests. Renal function was assessed by measuring serum creatinine, creatinine clearance, and blood urea nitrogen (BUN). Serum creatinine and creatinine clearance were used to grade nephrotoxicity using Common Terminology Criteria for Adverse Events (CTCAE) v4.0. Compared to baseline values, the mean BUN and serum creatinine increased 135 and 100%, respectively, 5 days after cisplatin infusion. Mean creatinine clearance showed a 43% decrease compared to baseline value. Non-statistically significant changes in superoxide anion (O 2•- ), hydrogen peroxide (H2O2), and general reactive oxygen species production occurred. A higher production of H2O2 was correlated with variation in serum creatinine, and was associated with higher grades for serum creatinine increases and creatinine clearance reductions. Linear regression analyses showed an association between H2O2 production and serum creatinine, creatinine clearance, and BUN levels. These results were observed for 5 days following cisplatin administration. In conclusion, H2O2 production was significantly related to changes in all renal parameters that were evaluated, following the cisplatin infusion.

Contribution to mitochondrial research in Brazil: 10th anniversary of the mitomeeting.

This commentary introduces the subject, the context and the history of the Brazilian annually held meeting on Mitochondrial Research by the occasion of its 10th anniversary. Mitomeetings gather people interested in all aspects of mitochondrial biology in diverse species, including protists, animals, plants, and fungi.

The mitochondrial calcium uniporter complex in trypanosomes.

The presence of a conserved mechanism for mitochondrial calcium uptake in trypanosomatids was crucial for the molecular identification of the mitochondrial calcium uniporter (MCU), a long-sought channel present in most eukaryotic organisms. Since then, research efforts to elucidate the role of MCU and its regulatory elements in different biological models have multiplied. MCU is the pore-forming subunit of a multimeric complex (the MCU complex or MCUC) and its predicted structure in trypanosomes is simpler than in mammalian cells, lacking two of its subunits and probably possessing other unidentified components. MCU protein has been characterized in Trypanosoma brucei and Trypanosoma cruzi, the causative agents of African and American trypanosomiasis, respectively. Contrary to its mammalian homolog, TbMCU was found to be essential for cell growth and survival, while its paralog MCUb is an essential protein in T. cruzi. These findings could be further exploited for chemotherapeutic purposes. The emergence of new molecular tools for the genetic manipulation of trypanosomatids has been determinant for the functional characterization of the MCUC components in these organisms. However, further research has to be done to determine the role of each component in intracellular calcium signaling and cell bioenergetics. In this mini-review we summarize the original results on mitochondrial calcium uptake in trypanosomes, how did they contribute to the molecular identification of the MCU, and the functional characterization of the MCUC subunits that has so far been studied in these peculiar eukaryotes.

Mangifera indica L. extract (Vimang®) reduces plasma and liver cholesterol and leucocyte oxidative stress in hypercholesterolemic LDL receptor deficient mice.

Cardiovascular diseases are major causes of death worldwide. Beyond the classical cholesterol risk factor, other conditions such as oxidative stress are well documented to promote atherosclerosis. The Mangifera indica L. extract (Vimang®) was reported to present antioxidant and hypocholesterolemic properties. Thus, here we evaluate the effects of Vimang treatment on risk factors of the atherosclerosis prone model of familial hypercholesterolemia, the LDL receptor knockout mice. Mice were treated with Vimang during 2 weeks and were fed a cholesterol-enriched diet during the second week. The Vimang treated mice presented significantly reduced levels of plasma (15%) and liver (20%) cholesterol, increased plasma total antioxidant capacity (10%) and decreased reactive oxygen species (ROS) production by spleen mononuclear cells (50%), P < 0.05 for all. In spite of these benefits, the average size of aortic atherosclerotic lesions stablished in this short experimental period did not change significantly in Vimang treated mice. Therefore, in this study we demonstrated that Vimang has protective effects on systemic and tissue-specific risk factors, but it is not sufficient to promote a reduction in the initial steps of atherosclerosis development. In addition, we disclosed a new antioxidant target of Vimang, the spleen mononuclear cells that might be relevant for more advanced stages of atherosclerosis.

Facilitation of Ca2+ -induced opening of the mitochondrial permeability transition pore either by nicotinamide nucleotide transhydrogenase deficiency or statins treatment.

Mitochondrial redox imbalance and high Ca2+ uptake induce the opening of the permeability transition pore (PTP) that leads to disruption of energy-linked mitochondrial functions and triggers cell death in many disease states. In this review, we discuss the major results from our studies investigating the consequences of NAD(P)-transhydrogenase (NNT) deficiency, and of statins treatment for mitochondrial functions and susceptibility to Ca2+ -induced PTP. We highlight the aggravation of high fat diet-induced fatty liver disease in the context of NNT deficiency and the role of antioxidants in the prevention of statins toxicity to mitochondria.

CRISPR/Cas9-mediated endogenous C-terminal Tagging of Trypanosoma cruzi Genes Reveals the Acidocalcisome Localization of the Inositol 1,4,5-Trisphosphate Receptor.

Methods for genetic manipulation of Trypanosoma cruzi, the etiologic agent of Chagas disease, have been highly inefficient, and no endogenous tagging of genes has been reported to date. We report here the use of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system for endogenously tagging genes in this parasite. The utility of the method was established by tagging genes encoding proteins of known localization such as TcFCaBP (flagellar calcium binding protein) and TcVP1 (vacuolar proton pyrophosphatase), and two proteins of undefined or disputed localization, the TcMCU (mitochondrial calcium uniporter) and TcIP3R (inositol 1,4,5-trisphosphate receptor). We confirmed the flagellar and acidocalcisome localization of TcFCaBP and TcVP1 by co-localization with antibodies to the flagellum and acidocalcisomes, respectively. As expected, TcMCU was co-localized with the voltage-dependent anion channel to the mitochondria. However, in contrast to previous reports and our own results using overexpressed TcIP3R, endogenously tagged TcIP3R showed co-localization with antibodies against VP1 to acidocalcisomes. These results are also in agreement with our previous reports on the localization of this channel to acidocalcisomes of Trypanosoma brucei and suggest that caution should be exercised when overexpression of tagged genes is done to localize proteins in T. cruzi.

The Cratylia mollis seed lectin induces membrane permeability transition in isolated rat liver mitochondria and a cyclosporine a-insensitive permeability transition in Trypanosoma cruzi mitochondria.

Previous results provided evidence that Cratylia mollis seed lectin (Cramoll 1,4) promotes Trypanosoma cruzi epimastigotes death by necrosis via a mechanism involving plasma membrane permeabilization to Ca(2+) and mitochondrial dysfunction due to matrix Ca(2+) overload. In order to investigate the mechanism of Ca(2+) -induced mitochondrial impairment, experiments were performed analyzing the effects of this lectin on T. cruzi mitochondrial fraction and in isolated rat liver mitochondria (RLM), as a control. Confocal microscopy of T. cruzi whole cell revealed that Cramoll 1,4 binding to the plasma membrane glycoconjugates is followed by its internalization and binding to the mitochondrion. Electrical membrane potential (∆Ψm ) of T. cruzi mitochondrial fraction suspended in a reaction medium containing 10 µM Ca(2+) was significantly decreased by 50 µg/ml Cramoll 1,4 via a mechanism insensitive to cyclosporine A (CsA, membrane permeability transition (MPT) inhibitor), but sensitive to catalase or 125 mM glucose. In RLM suspended in a medium containing 10 µM Ca(2+) this lectin, at 50 µg/ml, induced increase in the rate of hydrogen peroxide release, mitochondrial swelling, and ∆Ψm disruption. All these mitochondrial alterations were sensitive to CsA, catalase, and EGTA. These results indicate that Cramoll 1, 4 leads to inner mitochondrial membrane permeabilization through Ca(2+) dependent mechanisms in both mitochondria. The sensitivity to CsA in RLM characterizes this lectin as a MPT inducer and the lack of CsA effect identifies a CsA-insensitive MPT in T. cruzi mitochondria.

Food restriction by intermittent fasting induces diabetes and obesity and aggravates spontaneous atherosclerosis development in hypercholesterolaemic mice.

Different regimens of food restriction have been associated with protection against obesity, diabetes and CVD. In the present study, we hypothesised that food restriction would bring benefits to atherosclerosis- and diabetes-prone hypercholesterolaemic LDL-receptor knockout mice. For this purpose, 2-month-old mice were submitted to an intermittent fasting (IF) regimen (fasting every other day) over a 3-month period, which resulted in an overall 20 % reduction in food intake. Contrary to our expectation, epididymal and carcass fat depots and adipocyte size were significantly enlarged by 15, 72 and 68 %, respectively, in the IF mice compared with the ad libitum-fed mice. Accordingly, plasma levels of leptin were 50 % higher in the IF mice than in the ad libitum-fed mice. In addition, the IF mice showed increased plasma levels of total cholesterol (37 %), VLDL-cholesterol (195 %) and LDL-cholesterol (50 %). As expected, in wild-type mice, the IF regimen decreased plasma cholesterol levels and epididymal fat mass. Glucose homeostasis was also disturbed by the IF regimen in LDL-receptor knockout mice. Elevated levels of glycaemia (40 %), insulinaemia (50 %), glucose intolerance and insulin resistance were observed in the IF mice. Systemic inflammatory markers, TNF-α and C-reactive protein, were significantly increased and spontaneous atherosclerosis development were markedly increased (3-fold) in the IF mice. In conclusion, the IF regimen induced obesity and diabetes and worsened the development of spontaneous atherosclerosis in LDL-receptor knockout mice. Although being efficient in a wild-type background, this type of food restriction is not beneficial in the context of genetic hypercholesterolaemia.

Liver proteomic response to hypertriglyceridemia in human-apolipoprotein C-III transgenic mice at cellular and mitochondrial compartment levels.

BACKGROUND: Hypertriglyceridemia (HTG) is defined as a triglyceride (TG) plasma level exceeding 150 mg/dl and is tightly associated with atherosclerosis, metabolic syndrome, obesity, diabetes and acute pancreatitis. The present study was undertaken to investigate the mitochondrial, sub-mitochondrial and cellular proteomic impact of hypertriglyceridemia in the hepatocytes of hypertriglyceridemic transgenic mice (overexpressing the human apolipoproteinC-III). METHODS: Quantitative proteomics (2D-DIGE) analysis was carried out on both "low-expressor" (LE) and "high-expressor" (HE) mice, respectively exhibiting moderate and severe HTG, to characterize the effect of the TG plasma level on the proteomic response. RESULTS: The mitoproteome analysis has revealed a large-scale phenomenon in transgenic mice, i.e. a general down-regulation of matricial proteins and up-regulation of inner membrane proteins. These data also demonstrate that the magnitude of proteomic changes strongly depends on the TG plasma level. Our different analyses indicate that, in HE mice, the capacity of several metabolic pathways is altered to promote the availability of acetyl-CoA, glycerol-3-phosphate, ATP and NADPH for TG de novo biosynthesis. The up-regulation of several cytosolic ROS detoxifying enzymes has also been observed, suggesting that the cytoplasm of HTG mice is subjected to oxidative stress. Moreover, our results suggest that iron over-accumulation takes place in the cytosol of HE mice hepatocytes and may contribute to enhance oxidative stress and to promote cellular proliferation. CONCLUSIONS: These results indicate that the metabolic response to HTG in human apolipoprotein C-III overexpressing mice may support a high TG production rate and that the cytosol of hepatocytes is subjected to an important oxidative stress, probably as a result of FFA over-accumulation, iron overload and enhanced activity of some ROS-producing catabolic enzymes.

Aging-dependent mitochondrial bioenergetic impairment in the skeletal muscle of NNT-deficient mice.

Overall health relies on features of skeletal muscle that generally decline with age, partly due to mechanisms associated with mitochondrial redox imbalance and bioenergetic dysfunction. Previously, aged mice genetically devoid of the mitochondrial NAD(P)+ transhydrogenase (NNT, encoded by the nicotinamide nucleotide transhydrogenase gene), an enzyme involved in mitochondrial NADPH supply, were shown to exhibit deficits in locomotor behavior. Here, by using young, middle-aged, and older NNT-deficient (Nnt-/-) mice and age-matched controls (Nnt+/+), we aimed to investigate how muscle bioenergetic function and motor performance are affected by NNT expression and aging. Mice were subjected to the wire-hang test to assess locomotor performance, while mitochondrial bioenergetics was evaluated in fiber bundles from the soleus, vastus lateralis and plantaris muscles. An age-related decrease in the average wire-hang score was observed in middle-aged and older Nnt-/- mice compared to age-matched controls. Although respiratory rates in the soleus, vastus lateralis and plantaris muscles did not significantly differ between the genotypes in young mice, the rates of oxygen consumption did decrease in the soleus and vastus lateralis muscles of middle-aged and older Nnt-/- mice. Notably, the soleus, which exhibited the highest NNT expression level, was the muscle most affected by aging, and NNT loss. Additionally, histology of the soleus fibers revealed increased numbers of centralized nuclei in older Nnt-/- mice, indicating abnormal morphology. In summary, our findings suggest that NNT expression deficiency causes locomotor impairments and muscle dysfunction during aging in mice.

Activation of the mitochondrial ATP-sensitive K+ channel reduces apoptosis of spleen mononuclear cells induced by hyperlipidemia.

BACKGROUND: We have previously demonstrated that increased rates of superoxide generation by extra-mitochondrial enzymes induce the activation of the mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) in the livers of hypertriglyceridemic (HTG) mice. The resulting mild uncoupling mediated by mitoK(ATP) protects mitochondria against oxidative damage. In this study, we investigate whether immune cells from HTG mice also present increased mitoK(ATP) activity and evaluate the influence of this trait on cell redox state and viability. METHODS: Oxygen consumption (Clark-type electrode), reactive oxygen species production (dihydroethidium and H2-DCF-DA probes) and cell death (annexin V, cytocrome c release and Trypan blue exclusion) were determined in spleen mononuclear cells. RESULTS: HTG mice mononuclear cells displayed increased mitoK(ATP) activity, as evidenced by higher resting respiration rates that were sensitive to mitoK(ATP) antagonists. Whole cell superoxide production and apoptosis rates were increased in HTG cells. Inhibition of mitoK(ATP) further increased the production of reactive oxygen species and apoptosis in these cells. Incubation with HTG serum induced apoptosis more strongly in WT cells than in HTG mononuclear cells. Cytochrome c release into the cytosol and caspase 8 activity were both increased in HTG cells, indicating that cell death signaling starts upstream of the mitochondria but does involve this organelle. Accordingly, a reduced number of blood circulating lymphocytes was found in HTG mice. CONCLUSIONS: These results demonstrate that spleen mononuclear cells from hyperlipidemic mice have more active mitoK(ATP) channels, which downregulate mitochondrial superoxide generation. The increased apoptosis rate observed in these cells is exacerbated by closing the mitoK(ATP) channels. Thus, mitoK(ATP) opening acts as a protective mechanism that reduces cell death induced by hyperlipidemia.

Enhanced insulin secretion and glucose tolerance in rats exhibiting low plasma free fatty acid levels and hypertriglyceridaemia due to congenital albumin deficiency.

Congenitally analbuminaemic individuals and rats (NARs) exhibit several metabolic abnormalities, including hypertriglyceridaemia and plasma free fatty acid deficiency. Our aim was to study glucose homeostasis and insulin secretion in NARs. Plasma concentrations of lipids, glucose and insulin and secretion of insulin from the pancreatic islets were measured in female NARs and control animals (Sprague-Dawley rats; SDRs). Glucose homeostasis tests were also performed. Plasma glucose levels were similar between NARs and SDRs, irrespective of feeding status. However, fed insulinaemia was ∼37% higher (P 0.05) in NARs than in SDRs. The NARs displayed a markedly increased glucose tolerance, i.e. the integrated glycaemic response was one-third that of the control animals. Enhanced glucose tolerance was associated with threefold higher insulinaemia at peak glycaemia after a glucose load than in the control animals. Similar peripheral insulin sensitivity was observed between groups. Isolated pancreatic islets from NARs secreted significantly more insulin than islets from SDRs in response to a wide range of glucose concentrations (2.8-33.3 mm). Despite having similar liver glycogen contents in the fully fed state, NARs had ∼40% (P 0.05) lower glycogen contents than SDRs after 6 h fasting. The injection of a gluconeogenic substrate, pyruvate, elicited a faster rise in glycaemia in NARs compared with SDRs. Overall, NARs displayed enhanced glucose tolerance, insulin secretion and gluconeogenic flux. The higher glucose tolerance in NARs compared with SDRs is attributed to enhanced islet responsiveness to secretagogues, while peripheral insulin sensitivity seems not to be involved in this alteration. We propose that the enhanced glucose metabolism is a chronic compensatory adaptation to decreased free fatty acid availability in NARs.

Aggravation of hepatic lipidosis in red-footed tortoise Chelonoidis carbonaria with age is associated with alterations in liver mitochondria.

The occurrence of hepatic lipidosis is commonly reported in different reptilian species, especially in animals under captivity. Liver accumulation of fat is associated with disorders, better described in mammals as non-alcoholic fatty liver diseases (NAFLD), ranging from simple steatosis, to non-alcoholic steatohepatitis (NASH), and to more severe lesions of cirrhosis and hepatocellular carcinoma. Mitochondria play a central role in NAFLD pathogenesis, therefore in this study we characterized livers of ad libitum fed captive red-footed tortoise Chelonoidis carbonaria through histological and mitochondrial function evaluations of juvenile and adult individuals. Livers from adult tortoises exhibited higher levels of lipids, melanomacrophages centers and melanin than juveniles. The observed high score levels of histopathological alterations in adult tortoises, such as microvesicular steatosis, inflammation and fibrosis, indicated the progression to a NASH condition. Mitochondrial oxygen consumption at different respiratory states and with different substrates was 30 to 58% lower in adult when compared to juvenile tortoises. Despite citrate synthase activity was also lower in adults, cardiolipin content was similar to juveniles, indicating that mitochondrial mass was unaffected by age. Mitochondrial Ca2+ retention capacity was reduced by 70% in adult tortoises. Overall, we found that aggravation of NAFLD in ad libitum fed captive tortoises is associated with compromised mitochondrial function, indicating a critical role of the organelle in liver disease progression in reptiles.

Dichloroacetate reactivates pyruvate-supported peroxide removal by liver mitochondria and prevents NAFLD aggravation in NAD(P)+ transhydrogenase-null mice consuming a high-fat diet.

The mechanisms by which a high-fat diet (HFD) promotes non-alcoholic fatty liver disease (NAFLD) appear to involve liver mitochondrial dysfunction and redox imbalance. The functional loss of the enzyme NAD(P)+ transhydrogenase, a main source of mitochondrial NADPH, results in impaired mitochondrial peroxide removal, pyruvate dehydrogenase inhibition by phosphorylation, and progression of NAFLD in HFD-fed mice. The present study aimed to investigate whether pharmacological reactivation of pyruvate dehydrogenase by dichloroacetate attenuates the mitochondrial redox dysfunction and the development of NAFLD in NAD(P)+ transhydrogenase-null (Nnt-/-) mice fed an HFD (60% of total calories from fat). For this purpose, Nnt-/- mice and their congenic controls (Nnt+/+) were fed chow or an HFD for 20 weeks and received sodium dichloroacetate or NaCl in the final 12 weeks via drinking water. The results showed that HFD reduced the ability of isolated liver mitochondria from Nnt-/- mice to remove peroxide, which was prevented by the dichloroacetate treatment. HFD-fed mice of both Nnt genotypes exhibited increased body and liver mass, as well as a higher content of hepatic triglycerides, but dichloroacetate treatment attenuated these abnormalities only in Nnt-/- mice. Notably, dichloroacetate treatment decreased liver pyruvate dehydrogenase phosphorylation levels and prevented the aggravation of NAFLD in HFD-fed Nnt-/- mice. Conversely, dichloroacetate treatment elicited moderate hepatocyte ballooning in chow-fed mice, suggesting potentially toxic effects. We conclude that the protection against HFD-induced NAFLD by dichloroacetate is associated with its role in reactivating pyruvate dehydrogenase and reestablishing the pyruvate-supported liver mitochondrial capacity to handle peroxide in Nnt-/- mice.

Mild Mitochondrial Uncoupling Decreases Experimental Atherosclerosis, A Proof of Concept.

AIM: Atherosclerosis is responsible for high morbidity and mortality rates around the world. Local arterial oxidative stress is involved in all phases of atherosclerosis development. Mitochondria is a relevant source of the oxidants, particularly under certain risky conditions, such as hypercholesterolemia. The aim of this study was to test whether lowering the production of mitochondrial oxidants by induction of a mild uncoupling can reduce atherosclerosis in hypercholesterolemic LDL receptor knockout mice. METHODS: The mice were chronically treated with very low doses of DNP (2,4-dinitrophenol) and metabolic, inflammatory and redox state markers and atherosclerotic lesion sizes were determined. RESULTS: The DNP treatment did not change the classical atherosclerotic risk markers, such as plasma lipids, glucose homeostasis, and fat mass, as well as systemic inflammatory markers. However, the DNP treatment diminished the production of mitochondrial oxidants, systemic and tissue oxidative damage markers, peritoneal macrophages and aortic rings oxidants generation. Most importantly, development of spontaneous and diet-induced atherosclerosis (lipid and macrophage content) were significantly decreased in the DNP-treated mice. In vitro, DNP treated peritoneal macrophages showed decreased H2O2 production, increased anti-inflammatory cytokines gene expression and secretion, increased phagocytic activity, and decreased LDL-cholesterol uptake. CONCLUSIONS: These findings are a proof of concept that activation of mild mitochondrial uncoupling is sufficient to delay the development of atherosclerosis under the conditions of hypercholesterolemia and oxidative stress. These results promote future approaches targeting mitochondria for the prevention or treatment of atherosclerosis.