Homozygosity for alanine in the mitochondrial targeting sequence of superoxide dismutase and risk for severe alcoholic liver disease.

BACKGROUND & AIMS: For similar ethanol consumption, some subjects only develop macrovacuolar steatosis whereas others develop severe liver lesions. A genetic dimorphism encodes for either alanine or valine in the mitochondrial targeting sequence of manganese superoxide dismutase and could modulate its mitochondrial import. METHODS: The DNA of 71 white patients with alcoholic liver disease and 79 white blood donors was amplified and genotyped. RESULTS: The frequency of the alanine-encoding allele and the percentage of alanine homozygotes were higher in all patients than in controls and increased with the severity of liver lesions. The percentage of alanine homozygotes was 19% in controls, 17% in alcoholic patients with macrovacuolar steatosis, 43% in patients with microvesicular steatosis, 58% in patients with alcoholic hepatitis, and 69% in patients with cirrhosis. Alcohol consumption in alcoholics was similar whatever the genotype. Alanine homozygosity did not change the risk of developing macrovacuolar steatosis in alcoholics, but increased by 3-fold that of microvesicular steatosis, and 6- and 10-fold that of alcoholic hepatitis and cirrhosis. CONCLUSIONS: Homozygosity for alanine in the mitochondrial targeting sequence of manganese superoxide does not modify alcohol consumption and the risk of macrovacuolar steatosis in alcoholics but is a major risk factor for severe alcoholic liver disease.

The presence of modified nucleotides is required for cloverleaf folding of a human mitochondrial tRNA.

Direct sequencing of human mitochondrial tRNALysshows the absence of editing and the occurrence of six modified nucleotides (m1A9, m2G10, Psi27, Psi28 and hypermodified nucleotides at positions U34 and A37). This tRNA folds into the expected cloverleaf, as confirmed by structural probing with nucleases. The solution structure of the corresponding in vitro transcript unexpectedly does not fold into a cloverleaf but into an extended bulged hairpin. This non-canonical fold, established according to the reactivity to a large set of chemical and enzymatic probes, includes a 10 bp aminoacyl acceptor stem (the canonical 7 bp and 3 new pairs between residues 8-10 and 65-63), a 13 nt large loop and an anticodon-like domain. It is concluded that modified nucleotides have a predominant role in canonical folding of human mitochondrial tRNALys. Phylogenetic comparisons as well as structural probing of selected in vitro transcribed variants argue in favor of a major contribution of m1A9 in this process.

Isoleucylation properties of native human mitochondrial tRNAIle and tRNAIle transcripts. Implications for cardiomyopathy-related point mutations (4269, 4317) in the tRNAIle gene.

A growing number of mutated mitochondrial tRNA genes have been found associated with severe human diseases. To investigate the potential interference of such mutations with the primordial function of tRNAs, i.e. their aminoacylation by cognate aminoacyl-tRNA synthetases, a human mitochondrial in vitro aminoacylation system specific for isoleucine has been established. Both native tRNAIleand isoleucyl-tRNA synthetase activity have been recovered from human placental mitochondria and the kinetic parameters of tRNA aminoacylation determined. The effect of pathological point mutations present in the mitochondrial gene encoding tRNAIlehas been tackled by investigating the isoleucylation properties of wild-type and mutated in vitro transcripts. Data show that: (i) modified nucleotides contribute to efficient isoleucylation; (ii) point mutation A4269G in the gene (A-->G at nt 7 in the tRNA), associated with a cardiomyopathy, does not affect aminoacylation significantly; (iii) point mutation A4317G (A-->G at nt 59 in the tRNA), reported in a case of fatal infantile cardiomyopathy, induces a small but significant decrease in isoleucylation. The potential implications of these findings on the understanding of the molecular mechanisms involved in the expression of pathology are discussed.

Molecular analysis of carnitine palmitoyltransferase II deficiency with hepatocardiomuscular expression.

Carnitine palmitoyltransferase (CPT) II deficiency, an inherited disorder of mitochondrial long-chain fatty-acid (LCFA) oxidation, results in two distinct clinical phenotypes, namely, an adult (muscular) form and an infantile (hepatocardiomuscular) form. The rationale of this phenotypic heterogeneity is poorly understood. The adult form of the disease is commonly ascribed to the Ser-113-Leu substitution in CPT II. Only few data are available regarding the molecular basis of the infantile form of the disease. We report herein a homozygous A-2399-C transversion predicting a Tyr-628-Ser substitution in a CPT II-deficient infant. In vitro expression of mutant cDNA in COS-1 cells demonstrated the responsibility of this mutation for the disease. Metabolic consequences of the SER-113-Leu and Tyr-628-Ser substitutions were studied in fibroblasts. The Tyr-628-Ser substitution (infantile form) resulted in a 10% CPT II residual activity, markedly impairing LCFA oxidation, whereas the Ser-113-Leu substitution (adult form) resulted in a 20% CPT II residual activity, with out consequence on LCFA oxidation. These data show that CPT II activity has to be reduced below a critical threshold in order for LCFA oxidation in fibroblasts to be impaired. The hypothesis that this critical threshold differs among tissues could provide a basis to explain phenotypic heterogeneity of CPT II deficiency.

Infantile form of carnitine palmitoyltransferase II deficiency with hepatomuscular symptoms and sudden death. Physiopathological approach to carnitine palmitoyltransferase II deficiencies.

Reported cases of carnitine palmitoyltransferase II (CPT II) deficiency are characterized only by a muscular symptomatology in young adults although the defect is expressed in extra-muscular tissues as well as in skeletal muscle. We describe here a CPT II deficiency associating hypoketotic hypoglycemia, high plasma creatine kinase level, heart beat disorders, and sudden death in a 3-mo-old boy. CPT II defect (-90%) diagnosed in fibroblasts is qualitatively similar to that (-75%) of two "classical" CPT II-deficient patients previously studied: It resulted from a decreased amount of CPT II probably arising from its reduced biosynthesis. Consequences of CPT II deficiency studied in fibroblasts differed in both sets of patients. An impaired oxidation of long-chain fatty acids was found in the proband but not in patients with the "classical" form of the deficiency. The metabolic and clinical consequences of CPT II deficiency might depend, in part, on the magnitude of residual CPT II activity. With 25% residual activity CPT II would become rate limiting in skeletal muscle but not in liver, heart, and fibroblasts. As observed in the patient described herein, CPT II activity ought to be more reduced to induce an impaired oxidation of long-chain fatty acids in these tissues.

Immunoquantitative analysis of human carnitine palmitoyltransferase I and II defects.

Carnitine palmitoyltransferase deficiency realizes two distinct clinical forms. We previously showed and confirmed in the present work that CPTII (identified as the carnitine palmitoyltransferase activity assayable in detergent conditions) is decreased in the muscular form whereas it is unaffected and CPTI is decreased in the hepatic form. The antibody previously prepared against human liver mitochondrial CPTII recognizes the same enzyme in muscle, liver, and fibroblasts. Immunoprecipitation experiments were performed in fibroblasts from patients with the muscular and hepatic forms of the defect. As compared with controls, cell lines from two patients with the hepatic form of the defect did not exhibit any qualitative nor quantitative abnormality of cross-reacting material, whereas cell lines from two patients with the muscular form of the defect exhibited a decreased amount of cross-reacting material. These data suggest that CPTII deficiency could result from a decreased production of protein. The amount of cross-reacting material in the two sets of patients only correlates with CPTII activity, which is decreased in the muscular presentation and unaffected in the hepatic form. These results strengthen the hypothesis of distinct proteins supporting CPTI and CPTII activities.

The effect of adenosine and chloroadenosine on sex differences in the energy metabolism of rat hepatocytes.

The intensity and regulation of metabolic pathways are different depending on the sex of the source animal for hepatocytes isolated from mature rats. In cells from fed animals incubated without exogenous substrate, ATP level and ketone body production are higher in males (+25% and +100%) and lactate production is higher (+64%) in females; oleate enhances mitochondrial pyruvate oxidation in hepatocytes from fed male rats but not from fed females; in cells from starved animals oleate increases gluconeogenesis in both sexes at saturating levels of gluconeogenic substrates. However, at physiological levels (1 mM lactate and 0.1 mM pyruvate), this activation can only be detected in cells from males. In both sexes, oleate activation is abolished by adenosine which reduces in parallel the mitochondrial oxidation of pyruvate; chloroadenosine, an A2-receptor agonist, increases glycogenolysis strongly in hepatocytes from male animals (+80%) but only very slightly in female cells (+12%).

Influence of oleate oxidation on pyruvate production and utilization in hepatocytes isolated from fed rats. Effect of 2-[5-(4-chlorophenyl)pentyl]oxiran-2-carboxylate.

Oleate (0.35 and 1.5 mM) decreases, in a concentration-dependent manner, lactate and pyruvate concentrations in hepatocytes, isolated from fed rats, incubated without exogenous substrate. The glycolytic flux, estimated at 18 mM-glucose by [6-3H]-glucose detritiation and apparent production of lactate and pyruvate, is decreased by oleate. The measurement of glycolytic intermediates shows a cross-over at the phosphofructokinase level, which might result from an increased citrate concentration. All those effects are dependent on oleate oxidation in mitochondria, since they are suppressed by 1 microM-2-[5-(4-chlorophenyl)pentyl]oxiran-2-carboxylate (POCA), an inhibitor of the mitochondrial entry of oleate, but not of its uptake by hepatocytes. The decrease of lactate and pyruvate also results from an oleate-induced enhancement of pyruvate utilization by hepatocytes, as shown by the increase of 14CO2 formation from [1-14C]- and [3-14C]-pyruvate, especially at low (0.4 mM) pyruvate concentration. Those oleate effects are also suppressed by POCA. They might be due to an enhanced flux through pyruvate carboxylase and pyruvate dehydrogenase, as a result of an oleate-induced increase in the mitochondrial concentrations of pyruvate and acetyl-CoA. Thus oleate oxidation inhibits production of lactate and pyruvate in fed-rat hepatocytes, as it does in other tissues. But, in the liver, it also enhances the mitochondrial utilization of pyruvate. The physiological implications of those findings are discussed.

Comparison of the effects of 2-chloropropionate and dichloroacetate on ketogenesis and lipogenesis in isolated rat hepatocytes.

The respective effects of 2-chloropropionate and dichloroacetate on the pyruvate metabolic crossroads, lipogenesis and ketogenesis, were compared in hepatocytes isolated from fed rats. 2-Chloropropionate acts as an exclusive pyruvate dehydrogenase activator: it increases ketogenesis, lipogenesis, Krebs cycle intermediates and mitochondrial NADH/NAD+ ratio. The effects of dichloroacetate depend on experimental conditions and the intensity of its catabolization into oxalate: the resultant action of dichloroacetate on tested parameters combines the effects of pyruvate dehydrogenase activation on the one hand, and pyruvate carboxylase inhibition by oxalate on the other. A mixture of 2-chloropropionate plus oxalate mimics the effects of dichloroacetate. In hepatocytes from fed rats, endogenous lipogenesis is correlated with the mitochondrial NADH/NAD+ ratio, irrespective of the effector added.

The metabolic effects of oxalate on intact red blood cells.

The metabolic effects of oxalate on pyruvate kinase were studied in intact human red blood cells and compared to the spontaneous modifications induced by congenital pyruvate kinase deficiency. In normal cells, oxalate (2-3 . 10(-4) M) produces a large increase of the monophosphoglycerates, phosphoenolpyruvate pool and decrease of pyruvate concentrations as a result of pyruvate kinase inhibition; it does not significantly modify 2,3-diphosphoglycerate level, ATP formation or overall glycolytic activity. Those effects of oxalate are not due to Mg2+ chelation. A similar metabolite pattern is observed in vivo in erythrocytes with congenital pyruvate kinase deficiency, in which ATP concentration and glycolytic activity are described. These cells are more sensitive to oxalate than normal ones. Results are discussed with reference to the rate-limiting character of normal or congenitally deficient pyruvate kinase.