Drug-induced impairment of mitochondrial fatty acid oxidation and steatosis: assessment of causal relationship with 45 pharmaceuticals.

Drug-induced liver injury (DILI) represents a major issue for pharmaceutical companies, being a potential cause of black-box warnings on marketed pharmaceuticals, or drug withdrawal from the market. Lipid accumulation in the liver also referred to as steatosis, may be secondary to impaired mitochondrial fatty acid oxidation (mtFAO). However, an overall causal relationship between drug-induced mtFAO inhibition and the occurrence of steatosis in patients has not yet been established with a high number of pharmaceuticals. Hence, 32 steatogenic and 13 non-steatogenic drugs were tested for their ability to inhibit mtFAO in isolated mouse liver mitochondria. To this end, mitochondrial respiration was measured with palmitoyl-L-carnitine, palmitoyl-CoA + L-carnitine, or octanoyl-L-carnitine. This mtFAO tri-parametric assay was able to predict the occurrence of steatosis in patients with a sensitivity and positive predictive value above 88%. To get further information regarding the mechanism of drug-induced mtFAO impairment, mitochondrial respiration was also measured with malate/glutamate or succinate. Drugs such as diclofenac, methotrexate and troglitazone could inhibit mtFAO secondary to an impairment of the mitochondrial respiratory chain, while dexamethasone, olanzapine and zidovudine appeared to impair mtFAO directly. Mitochondrial swelling, transmembrane potential and production of reactive oxygen species were also assessed for all compounds. Only the steatogenic drugs amiodarone, ketoconazole, lovastatin and toremifene altered all these 3 mitochondrial parameters. In conclusion, our tri-parametric mtFAO assay could be useful in predicting the occurrence of steatosis in patients. The combination of this assay with other mitochondrial parameters could also help to better understand the mechanism of drug-induced mtFAO inhibition.

Knockdown of ANGPTL2 promotes left ventricular systolic dysfunction by upregulation of NOX4 in mice.

Background: Angiopoietin-like 2 (ANGPTL2) is a pro-inflammatory and pro-oxidant circulating protein that predicts and promotes chronic inflammatory diseases such as atherosclerosis in humans. Transgenic murine models demonstrated the deleterious role of ANGPTL2 in vascular diseases, while deletion of ANGPTL2 was protective. The nature of its role in cardiac tissues is, however, less clear. Indeed, in adult mice knocked down (KD) for ANGPTL2, we recently reported a mild left ventricular (LV) dysfunction originating from a congenital aortic valve stenosis, demonstrating that ANGPTL2 is essential to cardiac development and function. Hypothesis: Because we originally demonstrated that the KD of ANGPTL2 protected vascular endothelial function via an upregulation of arterial NOX4, promoting the beneficial production of dilatory H2O2, we tested the hypothesis that increased cardiac NOX4 could negatively affect cardiac redox and remodeling and contribute to LV dysfunction observed in adult Angptl2-KD mice. Methods and results: Cardiac expression and activity of NOX4 were higher in KD mice, promoting higher levels of cardiac H2O2 when compared to wild-type (WT) mice. Immunofluorescence showed that ANGPTL2 and NOX4 were co-expressed in cardiac cells from WT mice and both proteins co-immunoprecipitated in HEK293 cells, suggesting that ANGPTL2 and NOX4 physically interact. Pressure overload induced by transverse aortic constriction surgery (TAC) promoted LV systolic dysfunction in WT mice but did not further exacerbate the dysfunction in KD mice. Importantly, the severity of LV systolic dysfunction in KD mice (TAC and control SHAM) correlated with cardiac Nox4 expression. Injection of an adeno-associated virus (AAV9) delivering shRNA targeting cardiac Nox4 expression fully reversed LV systolic dysfunction in KD-SHAM mice, demonstrating the causal role of NOX4 in cardiac dysfunction in KD mice. Targeting cardiac Nox4 expression in KD mice also induced an antioxidant response characterized by increased expression of NRF2/KEAP1 and catalase. Conclusion: Together, these data reveal that the absence of ANGPTL2 induces an upregulation of cardiac NOX4 that contributes to oxidative stress and LV dysfunction. By interacting and repressing cardiac NOX4, ANGPTL2 could play a new beneficial role in the maintenance of cardiac redox homeostasis and function.

Angiopoietin-like 2 is essential to aortic valve development in mice.

Aortic valve (AoV) abnormalities during embryogenesis are a major risk for the development of aortic valve stenosis (AVS) and cardiac events later in life. Here, we identify an unexpected role for Angiopoietin-like 2 (ANGPTL2), a pro-inflammatory protein secreted by senescent cells, in valvulogenesis. At late embryonic stage, mice knocked-down for Angptl2 (Angptl2-KD) exhibit a premature thickening of AoV leaflets associated with a dysregulation of the fine balance between cell apoptosis, senescence and proliferation during AoV remodeling and a decrease in the crucial Notch signalling. These structural and molecular abnormalities lead toward spontaneous AVS with elevated trans-aortic gradient in adult mice of both sexes. Consistently, ANGPTL2 expression is detected in human fetal semilunar valves and associated with pathways involved in cell cycle and senescence. Altogether, these findings suggest that Angptl2 is essential for valvulogenesis, and identify Angptl2-KD mice as an animal model to study spontaneous AVS, a disease with unmet medical need.

The Generation of an Artificial ATP Deficit Triggers Antibiotic Production in Streptomyces lividans.

In most Streptomyces species, antibiotic production is triggered in a condition of phosphate limitation, a condition that is known to be correlated with a low intracellular ATP content compared to growth in a condition of phosphate proficiency. This observation suggests that a low ATP content might be a direct trigger of antibiotic biosynthesis. In order to test this hypothesis, we introduced into the model strain Streptomyces lividans, a functional and a non-functional ATPase cloned into the replicative vector pOSV206 and expressed under the control of the strong ErmE* promoter. The functional ATPase was constituted by the α (AtpA), ß (AtpB) and γ (AtpD) sub-units of the native F1 part of the ATP synthase of S. lividans that, when separated from the membrane-bound F0 part, bears an ATPase activity. The non-functional ATPase was a mutated version of the latter, bearing a 12 amino acids deletion encompassing the active site of the AtpD sub-unit. S. lividans was chosen to test our hypothesis since this strain hardly produces any antibiotics. However, it possesses the same biosynthetic pathways of various specialized metabolites as S. coelicolor, a phylogenetically closely related strain that produces these metabolites in abundance. Our results demonstrated that the over-expression of the functional ATPase, but not that of its mutated version, indeed correlated with the production of the bioactive metabolites of the CDA, RED and ACT clusters. These results confirmed the long known and mysterious link existing between a phosphate limitation leading to an ATP deficit and the triggering of antibiotic biosynthesis. Based on this work and the previous published results of our group, we propose an entirely novel conception of the nature of this link.

Adipose tissue senescence is mediated by increased ATP content after a short-term high-fat diet exposure.

In the context of obesity, senescent cells accumulate in white adipose tissue (WAT). The cellular underpinnings of WAT senescence leading to insulin resistance are not fully elucidated. The objective of the current study was to evaluate the presence of WAT senescence early after initiation of high-fat diet (HFD, 1-10 weeks) in 5-month-old male C57BL/6J mice and the potential role of energy metabolism. We first showed that WAT senescence occurred 2 weeks after HFD as evidenced in whole WAT by increased senescence-associated ß-galactosidase activity and cyclin-dependent kinase inhibitor 1A and 2A expression. WAT senescence affected various WAT cell populations, including preadipocytes, adipose tissue progenitors, and immune cells, together with adipocytes. WAT senescence was associated with higher glycolytic and mitochondrial activity leading to enhanced ATP content in HFD-derived preadipocytes, as compared with chow diet-derived preadipocytes. One-month daily exercise, introduced 5 weeks after HFD, was an effective senostatic strategy, since it reversed WAT cellular senescence, while reducing glycolysis and production of ATP. Interestingly, the beneficial effect of exercise was independent of body weight and fat mass loss. We demonstrated that WAT cellular senescence is one of the earliest events occurring after HFD initiation and is intimately linked to the metabolic state of the cells. Our data uncover a critical role for HFD-induced elevated ATP as a local danger signal inducing WAT senescence. Exercise exerts beneficial effects on adipose tissue bioenergetics in obesity, reversing cellular senescence, and metabolic abnormalities.

Dysregulated Phenylalanine Catabolism Plays a Key Role in the Trajectory of Cardiac Aging.

BACKGROUND: Aging myocardium undergoes progressive cardiac hypertrophy and interstitial fibrosis with diastolic and systolic dysfunction. Recent metabolomics studies shed light on amino acids in aging. The present study aimed to dissect how aging leads to elevated plasma levels of the essential amino acid phenylalanine and how it may promote age-related cardiac dysfunction. METHODS: We studied cardiac structure and function, together with phenylalanine catabolism in wild-type (WT) and p21-/- mice (male; 2-24 months), with the latter known to be protected from cellular senescence. To explore phenylalanine's effects on cellular senescence and ectopic phenylalanine catabolism, we treated cardiomyocytes (primary adult rat or human AC-16) with phenylalanine. To establish a role for phenylalanine in driving cardiac aging, WT male mice were treated twice a day with phenylalanine (200 mg/kg) for a month. We also treated aged WT mice with tetrahydrobiopterin (10 mg/kg), the essential cofactor for the phenylalanine-degrading enzyme PAH (phenylalanine hydroxylase), or restricted dietary phenylalanine intake. The impact of senescence on hepatic phenylalanine catabolism was explored in vitro in AML12 hepatocytes treated with Nutlin3a (a p53 activator), with or without p21-targeting small interfering RNA or tetrahydrobiopterin, with quantification of PAH and tyrosine levels. RESULTS: Natural aging is associated with a progressive increase in plasma phenylalanine levels concomitant with cardiac dysfunction, whereas p21 deletion delayed these changes. Phenylalanine treatment induced premature cardiac deterioration in young WT mice, strikingly akin to that occurring with aging, while triggering cellular senescence, redox, and epigenetic changes. Pharmacological restoration of phenylalanine catabolism with tetrahydrobiopterin administration or dietary phenylalanine restriction abrogated the rise in plasma phenylalanine and reversed cardiac senescent alterations in aged WT mice. Observations from aged mice and human samples implicated age-related decline in hepatic phenylalanine catabolism as a key driver of elevated plasma phenylalanine levels and showed increased myocardial PAH-mediated phenylalanine catabolism, a novel signature of cardiac aging. CONCLUSIONS: Our findings establish a pathogenic role for increased phenylalanine levels in cardiac aging, linking plasma phenylalanine levels to cardiac senescence via dysregulated phenylalanine catabolism along a hepatic-cardiac axis. They highlight phenylalanine/PAH modulation as a potential therapeutic strategy for age-associated cardiac impairment.

The Phosin PptA Plays a Negative Role in the Regulation of Antibiotic Production in Streptomyces lividans.

In Streptomyces, antibiotic biosynthesis is triggered in phosphate limitation that is usually correlated with energetic stress. Polyphosphates constitute an important reservoir of phosphate and energy and a better understanding of their role in the regulation of antibiotic biosynthesis is of crucial importance. We previously characterized a gene, SLI_4384/ppk, encoding a polyphosphate kinase, whose disruption greatly enhanced the weak antibiotic production of Streptomyces lividans. In the condition of energetic stress, Ppk utilizes polyP as phosphate and energy donor, to generate ATP from ADP. In this paper, we established that ppk is co-transcribed with its two downstream genes, SLI_4383, encoding a phosin called PptA possessing a CHAD domain constituting a polyphosphate binding module and SLI_4382 encoding a nudix hydrolase. The expression of the ppk/pptA/SLI_4382 operon was shown to be under the positive control of the two-component system PhoR/PhoP and thus mainly expressed in condition of phosphate limitation. However, pptA and SLI_4382 can also be transcribed alone from their own promoter. The deletion of pptA resulted into earlier and stronger actinorhodin production and lower lipid content than the disruption of ppk, whereas the deletion of SLI_4382 had no obvious phenotypical consequences. The disruption of ppk was shown to have a polar effect on the expression of pptA, suggesting that the phenotype of the ppk mutant might be linked, at least in part, to the weak expression of pptA in this strain. Interestingly, the expression of phoR/phoP and that of the genes of the pho regulon involved in phosphate supply or saving were strongly up-regulated in pptA and ppk mutants, revealing that both mutants suffer from phosphate stress. Considering the presence of a polyphosphate binding module in PptA, but absence of similarities between PptA and known exo-polyphosphatases, we proposed that PptA constitutes an accessory factor for exopolyphosphatases or general phosphatases involved in the degradation of polyphosphates into phosphate.

Knockdown of angiopoietin-like 2 mimics the benefits of intermittent fasting on insulin responsiveness and weight loss.

Angiopoietin-like 2 (ANGPTL2) is an inflammatory adipokine linking obesity to insulin resistance. Intermittent fasting, on the other hand, is a lifestyle intervention able to prevent obesity and diabetes but difficult to implement and maintain. Our objectives were to characterize a link between ANGPTL2 and intermittent fasting and to investigate whether the knockdown of ANGPTL2 reproduces the benefits of intermittent fasting on weight gain and insulin responsiveness in knockdown and wild-type littermates mice. Intermittent fasting, access to food ad libitum once every other day, was initiated at the age of three months and maintained for four months. Intermittent fasting decreased by 63% (p < 0.05) gene expression of angptl2 in adipose tissue of wild-type mice. As expected, intermittent fasting improved insulin sensitivity (p < 0.05) and limited weight gain (p < 0.05) in wild-type mice. Knockdown mice fed ad libitum, however, were comparable to wild-type mice following the intermittent fasting regimen: insulin sensitivity and weight gain were identical, while intermittent fasting had no additional impact on these parameters in knockdown mice. Energy intake was similar between both wild-type fed intermittent fasting and ANGPTL2 knockdown mice fed ad libitum, suggesting that intermittent fasting and knockdown of ANGPTL2 equally lower feeding efficiency. These results suggest that the reduction of ANGPTL2 could be a useful and promising strategy to prevent obesity and insulin resistance, although further investigation of the mechanisms linking ANGPTL2 and intermittent fasting is warranted. Impact statement Intermittent fasting is an efficient diet pattern to prevent weight gain and improve insulin sensitivity. It is, however, a difficult regimen to follow and compliance is expected to be very low. In this work, we demonstrate that knockdown of ANGPTL2 in mice fed ad libitum mimics the beneficial effects of intermittent fasting on weight gain and insulin sensitivity in wild-type mice. ANGPTL2 is a cytokine positively associated with fat mass in humans, which inactivation in mice improves resistance to a high-fat metabolic challenge. This study provides a novel pathway by which IF acts to limit obesity despite equivalent energy intake. The development of a pharmacological ANGPTL2 antagonist could provide an efficient tool to reduce the burden of obesity.

MK2 Deletion in Mice Prevents Diabetes-Induced Perturbations in Lipid Metabolism and Cardiac Dysfunction.

Heart disease remains a major complication of diabetes, and the identification of new therapeutic targets is essential. This study investigates the role of the protein kinase MK2, a p38 mitogen-activated protein kinase downstream target, in the development of diabetes-induced cardiomyopathy. Diabetes was induced in control (MK2(+/+)) and MK2-null (MK2(-/-)) mice using repeated injections of a low dose of streptozotocin (STZ). This protocol generated in MK2(+/+) mice a model of diabetes characterized by a 50% decrease in plasma insulin, hyperglycemia, and insulin resistance (IR), as well as major contractile dysfunction, which was associated with alterations in proteins involved in calcium handling. While MK2(-/-)-STZ mice remained hyperglycemic, they showed improved IR and none of the cardiac functional or molecular alterations. Further analyses highlighted marked lipid perturbations in MK2(+/+)-STZ mice, which encompass increased 1) circulating levels of free fatty acid, ketone bodies, and long-chain acylcarnitines and 2) cardiac triglyceride accumulation and ex vivo palmitate ß-oxidation. MK2(-/-)-STZ mice were also protected against all these diabetes-induced lipid alterations. Our results demonstrate the benefits of MK2 deletion on diabetes-induced cardiac molecular and lipid metabolic changes, as well as contractile dysfunction. As a result, MK2 represents a new potential therapeutic target to prevent diabetes-induced cardiac dysfunction.

Analysis of 51 cyclodipeptide synthases reveals the basis for substrate specificity.

Cyclodipeptide synthases (CDPSs) constitute a family of peptide bond-forming enzymes that use aminoacyl-tRNAs for the synthesis of cyclodipeptides. Here, we describe the activity of 41 new CDPSs. We also show that CDPSs can be classified into two main phylogenetically distinct subfamilies characterized by specific functional subsequence signatures, named NYH and XYP. All 11 previously characterized CDPSs belong to the NYH subfamily, suggesting that further special features may be yet to be discovered in the other subfamily. CDPSs synthesize a large diversity of cyclodipeptides made up of 17 proteinogenic amino acids. The identification of several CDPSs having the same specificity led us to determine specificity sequence motifs that, in combination with the phylogenetic distribution of CDPSs, provide a first step toward being able to predict the cyclodipeptides synthesized by newly discovered CDPSs. The determination of the activity of ten more CDPSs with predicted functions constitutes a first experimental validation of this predictive approach.

Lack of angiopoietin-like-2 expression limits the metabolic stress induced by a high-fat diet and maintains endothelial function in mice.

BACKGROUND: Angiopoietin-like-2 (angptl2) is produced by several cell types including endothelial cells, adipocytes and macrophages, and contributes to the inflammatory process in cardiovascular diseases. We hypothesized that angptl2 impairs endothelial function, and that lowering angptl2 levels protects the endothelium against high-fat diet (HFD)-induced fat accumulation and hypercholesterolemia. METHODS AND RESULTS: Acute recombinant angptl2 reduced (P<0.05) acetylcholine-mediated vasodilation of isolated wild-type (WT) mouse femoral artery, an effect reversed (P<0.05) by the antioxidant N-acetylcysteine. Accordingly, in angptl2 knockdown (KD) mice, ACh-mediated endothelium-dependent vasodilation was greater (P<0.05) than in WT mice. In arteries from KD mice, prostacyclin contributed to the overall dilation unlike in WT mice. After a 3-month HFD, overall vasodilation was not altered, but dissecting out the endothelial intrinsic pathways revealed that NO production was reduced in arteries isolated from HFD-fed WT mice (P<0.05), while NO release was maintained in KD mice. Similarly, endothelium-derived hyperpolarizing factor (EDHF) was preserved in mesenteric arteries from HFD-fed KD mice but not in those from WT mice. Finally, the HFD increased (P<0.05) total cholesterol-to-high-density lipoprotein ratios, low-density lipoprotein-to-high-density lipoprotein ratios, and leptin levels in WT mice only, while glycemia remained similar in the 2 strains. KD mice displayed less triglyceride accumulation in the liver (P<0.05 versus WT), and adipocyte diameters in mesenteric and epididymal white adipose tissues were smaller (P<0.05) in KD than in WT fed an HFD, while inflammatory gene expression increased (P<0.05) in the fat of WT mice only. CONCLUSIONS: Lack of angptl2 expression limits the metabolic stress induced by an HFD and maintains endothelial function in mice.

VDAC phosphorylation, a lipid sensor influencing the cell fate.

The voltage-dependent anion channel (VDAC) or porin is a major membrane protein integrated into the mitochondrial outer membrane in eukaryotes. It is encoded as three isoforms (VDAC1 to 3), which play differential roles in metabolism and cell death. As a channel, VDAC mediates metabolites, ions and water movements through the outer membrane in physiological conditions, but it can also participate to mitochondrial membrane permeabilization, an apoptotic checkpoint in stress and pathological conditions. Indeed, due to its subcellular location, VDAC interacts with many molecules as diverse as NAD+, lipids and cytosolic proteins such as hexokinase, tubulin, GSK3, Bax and Bcl-2 family members and mitochondrial proteins, such as the adenine nucleotide translocase (ANT). All these interactions can influence VDAC role in cell fate determination. In the recent past, major efforts focused on VDAC1 channel function and regulation by calcium and reactive oxygen species, and comparatively, fewer studies have been undertaken on VDAC2 and 3 and their pathophysiological involvement. Here, we review recent insights into the role of VDAC isoforms in cell death, and its regulation by phosphorylation or protein-lipid interactions and discuss the putative consequences of this post-translational modification on cell fate, notably in the context of lipid accumulation. This might have important implications for the understanding of basic mechanisms of mitochondrial lipid sensing and might contribute to define a novel therapeutic target for future investigation.

Angiopoietin-like 2 promotes atherogenesis in mice.

BACKGROUND: Angiopoietin like-2 (angptl2), a proinflammatory protein, is overexpressed in endothelial cells (ECs) from patients with coronary artery disease (CAD). Whether angptl2 contributes to atherogenesis is unknown. We tested the hypothesis that angptl2 promotes inflammation and leukocyte adhesion onto ECs, thereby accelerating atherogenesis in preatherosclerotic dyslipidemic mice. METHODS AND RESULTS: In ECs freshly isolated from the aorta, basal expression of TNF-α and IL-6 mRNA was higher in 3-month-old severely dyslipidemic mice (LDLr(-/-); hApoB100(+/+) [ATX]) than in control healthy wild-type (WT) mice (P<0.05) and was increased in both groups by exogenous angptl2 (100 nmol/L). Angptl2 stimulated the adhesion of leukocytes ex vivo on the native aortic endothelium of ATX, but not WT mice, in association with higher expression of ICAM-1 and P-selectin in ECs (P<0.05). Antibodies against these endothelial adhesion molecules prevented leukocyte adhesion. Intravenous administration of angptl2 for 1 month in preatherosclerotic 3-month-old ATX mice increased (P<0.05) total cholesterol and LDL-cholesterol levels, strongly induced (P<0.05) the expression of endothelial proinflammatory cytokines and adhesion molecules while accelerating atherosclerotic lesion formation by 10-fold (P<0.05). Plasma and aortic tissue levels of angptl2 increased (P<0.05) with age and were higher in 6- and 12-month-old ATX mice than in age-matched WT mice. Angptl2 accumulated to high levels in the atherosclerotic lesions (P<0.05). Finally, angptl2 was greatly expressed (P<0.05) in ECs cultured from CAD patients, and circulating angptl2 levels were 6-fold higher in CAD patients compared with age-matched healthy volunteers. CONCLUSIONS: Angptl2 contributes to the pathogenesis of atherosclerosis.

Functional and genetic characterization of two extremely rare cases of Williams-Beuren syndrome associated with chronic granulomatous disease.

Williams-Beuren syndrome (WBS) is a neurodevelopmental disorder with multi-systemic manifestations, caused by a heterozygous segmental deletion of 1.55-1.83 Mb at chromosomal band 7q11.23. The deletion can include the NCF1 gene that encodes the p47(phox) protein, a component of the leukocyte NADPH oxidase enzyme, which is essential for the defense against microbial pathogens. It has been postulated that WBS patients with two functional NCF1 genes are more susceptible to occurrence of hypertension than WBS patients with only one functional NCF1 gene. We now describe two extremely rare WBS patients without any functional NCF1 gene, because of a mutation in NCF1 on the allele not carrying the NCF1-removing WBS deletion. These two patients suffer from chronic granulomatous disease with increased microbial infections in addition to WBS. Interestingly, one of these patients did suffer from hypertension, indicating that other factors than NADPH oxidase in vascular tissue may be involved in causing hypertension.

Glycogen synthase kinase 3-mediated voltage-dependent anion channel phosphorylation controls outer mitochondrial membrane permeability during lipid accumulation.

UNLABELLED: Nonalcoholic steatosis is a liver pathology characterized by fat accumulation and severe metabolic alterations involving early mitochondrial impairment and late hepatocyte cell death. However, mitochondrial dysfunction mechanisms remain elusive. Using four models of nonalcoholic steatosis, i.e., livers from patients with fatty liver disease, ob/ob mice, mice fed a high-fat diet, and in vitro models of lipotoxicity, we show that outer mitochondrial membrane permeability is altered and identified a posttranslational modification of voltage-dependent anion channel (VDAC), a membrane channel and NADH oxidase, as a cause of early mitochondrial dysfunction. Thus, in nonalcoholic steatosis VDAC exhibits reduced threonine phosphorylation, which increases the influx of water and calcium into mitochondria, sensitizes the organelle to matrix swelling, depolarization, and cytochrome c release without inducing cell death. This also amplifies VDAC enzymatic and channel activities regulation by calcium and modifies its interaction with proteic partners. Moreover, lipid accumulation triggers a rapid lack of VDAC phosphorylation by glycogen synthase kinase 3 (GSK3). Pharmacological and genetic manipulations proved GSK3 to be responsible for VDAC phosphorylation in normal cells. Notably, VDAC phosphorylation level correlated with steatosis severity in patients. CONCLUSION: VDAC acts as an early sensor of lipid toxicity and its GSK3-mediated phosphorylation status controls outer mitochondrial membrane permeabilization in hepatosteatosis.

ANT-VDAC1 interaction is direct and depends on ANT isoform conformation in vitro.

The voltage-dependent anion channel (VDAC) and the adenine nucleotide translocase (ANT) have central roles in mitochondrial functions such as nucleotides transport and cell death. The interaction between VDAC, an outer mitochondrial membrane protein and ANT, an inner membrane protein, was studied in isolated mitochondria and in vitro. Both proteins were isolated from various mitochondrial sources and reconstituted in vitro using a biomimetic system composed of recombinant human VDAC isoform 1 (rhVDAC1) immobilized on a surface plasmon resonance (SPR) sensor chip surface. Two enriched-preparations of (H)ANT (ANT from heart, mainly ANT1) and (L)ANT (ANT from liver, mainly ANT2) isoforms interacted differently with rhVDAC1. Moreover, the pharmacological ANT inhibitors atractyloside and bongkrekic acid modulated this interaction. Thus, ANT-VDAC interaction depends both on ANT isoform identity and on the conformation of ANT.

Mitochondrial roles and cytoprotection in chronic liver injury.

The liver is one of the richest organs in terms of number and density of mitochondria. Most chronic liver diseases are associated with the accumulation of damaged mitochondria. Hepatic mitochondria have unique features compared to other organs' mitochondria, since they are the hub that integrates hepatic metabolism of carbohydrates, lipids and proteins. Mitochondria are also essential in hepatocyte survival as mediator of apoptosis and necrosis. Hepatocytes have developed different mechanisms to keep mitochondrial integrity or to prevent the effects of mitochondrial lesions, in particular regulating organelle biogenesis and degradation. In this paper, we will focus on the role of mitochondria in liver physiology, such as hepatic metabolism, reactive oxygen species homeostasis and cell survival. We will also focus on chronic liver pathologies, especially those linked to alcohol, virus, drugs or metabolic syndrome and we will discuss how mitochondria could provide a promising therapeutic target in these contexts.

Inhibition of the Mitochondrial Permeability Transition for Cytoprotection: Direct versus Indirect Mechanisms.

Mitochondria are fascinating organelles, which fulfill multiple cellular functions, as diverse as energy production, fatty acid ß oxidation, reactive oxygen species (ROS) production and detoxification, and cell death regulation. The coordination of these functions relies on autonomous mitochondrial processes as well as on sustained cross-talk with other organelles and/or the cytosol. Therefore, this implies a tight regulation of mitochondrial functions to ensure cell homeostasis. In many diseases (e.g., cancer, cardiopathies, nonalcoholic fatty liver diseases, and neurodegenerative diseases), mitochondria can receive harmful signals, dysfunction and then, participate to pathogenesis. They can undergo either a decrease of their bioenergetic function or a process called mitochondrial permeability transition (MPT) that can coordinate cell death execution. Many studies present evidence that protection of mitochondria limits disease progression and severity. Here, we will review recent strategies to preserve mitochondrial functions via direct or indirect mechanisms of MPT inhibition. Thus, several mitochondrial proteins may be considered for cytoprotective-targeted therapies.

Clinical, functional and genetic analysis of twenty-four patients with chronic granulomatous disease - identification of eight novel mutations in CYBB and NCF2 genes.

Chronic granulomatous disease is an inherited disorder in which phagocytes lack a functional NADPH oxidase and cannot produce superoxide anions. The most common form is caused by mutations in CYBB encoding gp91phox. We investigated 24 CGD patients and their families. Twenty-one mutations in CYBB were classified as X91(0), X91(+) or X91(-) variants according to cytochrome b (558) expression. Point mutations in encoding regions represented 50 % of the mutations found in CYBB, splice site mutations 27 %, deletions and insertions 23 %. Eight mutations in CYBB were novel leading to X91(0)CGD cases. Two of these were point mutations: c493G>T and a double mutation c625C>G in exon 6 and c1510C>T in exon 12 leading to a premature stop codon at Gly165 in gp91phox and missense mutations His209Arg/Thr503Ile respectively. Two novel splice mutations in 5'intronic regions of introns 1 and 6 were found. A novel deletion/insertion c1024_1026delCTG/insT results in a frameshift introducing a stop codon at position 346 in gp91phox. The last novel mutation was the insertion of a T at c1373 leading to a frameshift and a premature stop codon at position 484 in gp91phox. For the first time the precise size of two large mutations in CYBB was determined by array-comparative genomic hybridization and carriers' status were evaluated by multiplex ligation-dependent probe amplification assay. No clear correlation between clinical severity and CYBB mutations could be established. Of three mutations in CYBA, NCF1 and NCF2 leading to rare autosomal recessive CGD, one nonsense mutation c29G>A in exon 1 of NCF2 was new.