The role of malonyl-coa in the coordination of fatty acid synthesis and oxidation in isolated rat hepatocytes

Fatty acid synthesis and fatty acid oxidation were examined in rat hepatocytes under a variety of experimental conditions. In cells from fed animals, glucagon acutely switched the direction of fatty acid metabolism from synthesis to oxidation. Addition of lactate plus pyruvate had the opposite effect. The inhibitory action of glucagon on fatty acid synthesis and its stimulatory effect on fatty acid oxidation were largely, but not completely, offset by the simultaneous addition of lactate plus pyruvate. Changes in cellular citrate and malonyl-CoA levels indicated that glucagon exerted its inhibitory effect on fatty acid synthesis at two levels: (i) blockade of glycolysis; and (ii) partial inhibition of a more distal step, probably acetyl-CoA carboxylase. Under all conditions, fatty acid oxidation was related in a linear and reciprocal fashion to the rate of fatty acid synthesis and the tissue malonyl-CoA content. The latter fluctuated through a range of 1 to 6 nmol per g wet weight of cells. Since malonyl-CoA inhibits carnitine acyltransferase I of liver mitochondria with a Ki in the region of 1 to 2 micron, the present studies support the concept that this compound plays a pivotal role in the coordination of hepatic fatty acid synthesis and oxidation. The ketogenic effect of glucagon on liver appears to be manifested in large part through the ability of the hormone to reduce the tissue malonyl-CoA concentration.     

Proceedings: Rehabilitation or bust: the impact of criminal justice system referrals on the treatment of drug addicts and alcoholics in a therapeutic community (Eagleville''s experience)

Esterification of fatty acids in rat liver mitochondria was studied following portal or intraperitoneal administration of various 14C-fatty acids. Incorporation of the labelled fatty acid was most marked into phospholipids (especially, phosphatidylcholine and phosphatidylethanolamine) and triglycerides in intact mitochondria and the inner mitochondrial membranes. In contrast, in the outer membranes the injected fatty acids remained free without esterification. It is concluded that esterification of fatty acids occurs in the inner membranes of rat liver mitochondria.     

Nephrotoxicity and hepatotoxicity of 5,6-dichloro-4-thia-5-hexenoic acid: evidence for fatty acid beta-oxidation-dependent bioactivation

5,6-Dichloro-4-thia-5-hexenoic acid (DCTH) is toxic to rat liver and kidney mitochondria and is cytotoxic to isolated rat hepatocytes. The object of this investigation was to test the hypothesis that DCTH is bioactivated in vivo by the enzymes of mitochondrial fatty acid beta oxidation and that the observed mitochondrial dysfunction is a consequence of this bioactivation. DCTH was a potent nephrotoxin and hepatotoxin in Long-Evans rats, whereas the odd-chain-length analog 6,7-dichloro-5-thia-6-heptenoic acid was not toxic. DCTH produced morphological changes in renal proximal convoluted tubules and the liver. The increases in urinary protein, glucose and blood urea nitrogen concentrations were consistent with the renal lesions. Hepatic lesions were associated with an increase in plasma glutamate-pyruvate transaminase activity, a marked infiltration of lipid and depletion of glycogen concentrations. A pronounced decrease in plasma glucose concentrations was also observed. DCTH decreased fatty acid beta oxidation by 75% and 40% in liver and kidney mitochondria, respectively, isolated from DCTH-treated rats. In addition, medium-chain acyl-coenzyme A dehydrogenase activity was reduced by 25% in rat liver mitochondria incubated with DCTH. The data presented are consistent with the hypothesis that DCTH is bioactivated by the mitochondrial fatty acid beta-oxidation system and that mitochondria are a critical cellular target in DCTH-induced toxicity.     

The effects of population density and malnutrition on the dynamics of whooping cough

We measured the proton leak across the inner membrane of liver mitochondria isolated from six different vertebrate species and from obese and control Zucker rats. Proton leak at 37 degrees C was similar in rat and pigeon, and in obese and control Zucker rats. Compared to rat, it was lower in cane toad, shingleback lizard, and the Madeiran lizard Lacerta dugessi. Proton leak at 20 degrees C was similar in xenopus toad and higher in rainbow trout, compared to rat. In general, proton permeability and substrate oxidation activity were greater in liver mitochondria from endotherms than those from ectotherms. Analysis of this and previous data showed that proton leak per milligram of mitochondrial protein correlated with standard metabolic rate, and proton leak per milligram of inner membrane phospholipid correlated with 11 phospholipid fatty acid compositional parameters, including unsaturation index.     

Evaluation of birth cohort patterns in population disease rates

1. The effects of piroxicam, a nonsteroidal anti-inflammatory drug, on rat liver mitochondria were investigated in order to obtain direct evidence about a possible uncoupling effect, as suggested by a previous work with the perfused rat liver. 2. Piroxicam increased respiration in the absence of exogenous ADP and decreased respiration in the presence of exogenous ADP, the ADP/O ratios and the respiratory control ratios. 3. The ATPase activity of intact mitochondria was increased by piroxicam. With 2,4-dinitrophenol uncoupled mitochondria, inhibition was observed. The ATPase activity of freeze-thawing disrupted mitochondria was insensitive to piroxicam. 4. Swelling driven by the oxidation of several substrates and safranine uptake induced by succinate oxidation were inhibited. 5. The results of this work represent a direct evidence that piroxicam acts as an uncoupler, thus, decreasing mitochondrial ATP generation.     

Genetic-demographic characteristics of farming regions and small cities of the Tomsk district

The effect of the herbicide 4,6-dinitro-o-cresol (DNOC), a structural analogue of the classical protonophore 2,4-dinitrophenol, on the bioenergetics and inner membrane permeability of isolated rat liver mitochondria was studied. We observed that DNOC (10-50 microM) acts as a classical uncoupler of oxidative phosphorylation in rat liver mitochondria, promoting both an increase in succinate-supported mitochondrial respiration in the presence or absence of ADP and a decrease in transmembrane potential. The protonophoric activity of DNOC was evidenced by the induction of mitochondrial swelling in hyposmotic K(+)-acetate medium, in the presence of valinomycin. At higher concentrations (> 50 microM), DNOC also induces an inhibition of succinate-supported respiration, and a decrease in the activity of the succinate dehydrogenase can be observed. The addition of uncoupling concentrations of DNOC to Ca(2+)-loaded mitochondria treated with Ruthenium Red results in non-specific membrane permeabilization, as evidenced by mitochondrial swelling in isosmotic sucrose medium. Cyclosporin A, which inhibits mitochondrial permeability transition, prevented DNOC-induced mitochondrial swelling in the presence of Ca2+, which was accompanied by a decrease in mitochondrial membrane protein thiol content, owing to protein thiol oxidation. Catalase partially inhibits mitochondrial swelling and protein thiol oxidation, indicating the participation of mitochondrial-generated reactive oxygen species in this process. It is concluded that DNOC is a potent potent protonophore acting as a classical uncoupler of oxidative phosphorylation in rat liver mitochondria by dissipating the proton electrochemical gradient. Treatment of Ca(2+)-loaded mitochondria with uncoupling concentrations of DNOC results in mitochondrial permeability transition, associated with membrane protein thiol oxidation by reactive oxygen species.     

4-bromotiglic acid, a novel inhibitor of thiolases and a tool for assessing the cooperation between the membrane-bound and soluble beta-oxidation systems of rat liver mitochondria

An inhibitor of long-chain 3-ketoacyl-CoA thiolase has been developed as a tool for probing the cooperation between the two fatty acid beta-oxidation systems located in the inner mitochondrial membrane and in the mitochondrial matrix, respectively. 4-Bromotiglic acid was synthesized and found to inhibit palmitoylcarnitine-supported respiration of rat liver mitochondria in concentration-dependent and time-dependent fashions. Complete inhibition of respiration was achieved after incubating coupled mitochondria with 10 microM 4-bromotiglic acid for 2 min. Uncoupled mitochondria were resistant to the toxic effect of the inhibitor. Inhibition of octanoate-supported or octanoylcarnitine-supported respiration was partially reversed when the inhibitor was removed from the incubation medium. Such reversal was not observed with either palmitoylcarnitine or 2-methyldecanoic acid as the respiratory substrate. The severity of the irreversible inhibition declined with decreasing chain length of the acylcarnitine substrate. Of all beta-oxidation enzymes, only thiolases were inactivated by the inhibitor. Under conditions at which acetoacetyl-CoA thiolase and long-chain thiolase were completely inactivated, 3-ketoacyl-CoA thiolase retained some activity. It is concluded that the degradation of palmitic acid and longer-chain fatty acids is initiated by the beta-oxidation system of the inner membrane, whereas fatty acids shorter than palmitic acid can be oxidized to a certain degree by the matrix system alone. The effectiveness of the matrix system increases with decreasing chain length of the substrate.     

Hamster brown-adipose-tissue mitochondria. The role of fatty acids in the control of the proton conductance of the inner membrane

The specific ability of fatty acids to increase the proton conductance of the inner membrane of mitochondria from the liver and brown adipose tissue of cold-adapted hamsters was compared. The liver and brown-adipose-tissue mitochondria had their effective proton conductances increased by respectively 0.028 and 0.94 nmol H+- min-1. (mV of proton electrochemical gradient)-1 for each nmol of palmitate bound. No difference could be detected between the abilities of liver and brown-adipose-tissue mitochondria to bind fatty acids. Purine nucleotides did not displace farry acids from the brown-adipase-tissue mitochondria. The endogenous fatty acid content of hamster brown-adipose-tissue mitochondria prepared in the absence of album was found to be equivalent to 17 +/- 7 nmol of palmitate/mg protein. The fatty acid content was reduced to 1 nmol/mg after preincubation of the mitochondria with CoA, ATP and carnitine. No inert pool of fatty acids could be detected. The endogenous fatty acids of hamster liver mitochondria were less than 4 nmol of palmitate equivalent/mg protein. Some of the fatty acid associated with the brown-adipose-tissue mitochondria originates during preparation of the mitochondria. In the light of these results, the physiological role of the fatty acids in controlling the proton conductance of the brown-adipose-tissue mitochondrial inner membrane, and hence- non-shivering thermogenesis, is re-evaluated.     

Direct effects of diazoxide on mitochondria in pancreatic B-cells and on isolated liver mitochondria

1. The direct effects of diazoxide on mitochondrial membrane potential, Ca2+ transport, oxygen consumption and ATP generation were investigated in mouse pancreatic B-cells and rat liver mitochondria. 2. Diazoxide, at concentrations commonly used to open adenosine 5'-triphosphate (ATP)-dependent K+-channels (K(ATP) channels) in pancreatic B-cells (100 to 1000 microM), decreased mitochondrial membrane potential in mouse intact perifused B-cells, as evidenced by an increase of rhodamine 123 fluorescence. This reversible decrease of membrane potential occurred at non-stimulating (5 mM) and stimulating (20 mM) glucose concentrations. 3. A decrease of mitochondrial membrane potential in perifused B-cells was also caused by pinacidil, but no effect could be seen with levcromakalim (500 microM each). 4. Measurements by a tetraphenylphosphonium-sensitive electrode of the membrane potential of rat isolated liver mitochondria confirmed that diazoxide decreased mitochondrial membrane potential by a direct action. Pretreatment with glibenclamide (2 microM) did not antagonize the effects of diazoxide. 5. In Fura 2-loaded B-cells perifused with the Ca2+ channel blocker, D 600, a moderate, reversible increase of intracellular Ca2+ concentration could be seen in response to 500 microM diazoxide. This intracellular Ca2+ mobilization may be due to mitochondrial Ca2+ release, since the reduction of membrane potential of isolated liver mitochondria by diazoxide was accompanied by an accelerated release of Ca2+ stored in the mitochondria. 6. In the presence of 500 microM diazoxide, ATP content of pancreatic islets incubated in 20 mM glucose for 30 min was significantly decreased by 29%. However, insulin secretion from mouse perifused islets induced by 40 mM K+ in the presence of 10 mM glucose was not inhibited by 500 microM diazoxide, suggesting that the energy-dependent processes of insulin secretion distal to Ca2+ influx were not affected by diazoxide at this concentration. 7. The effects of diazoxide on oxygen consumption and ATP production of liver mitochondria varied depending on the respiratory substrates (5 mM succinate, 10 mM alpha-ketoisocaproic acid, 2 mM tetramethyl phenylenediamine plus 5 mM ascorbic acid), indicating an inhibition of respiratory chain complex II. Pinacidil, but not levcromakalim, inhibited alpha-ketoisocaproic acid-fuelled ATP production. 8. In conclusion, diazoxide directly affects mitochondrial energy metabolism, which may be of relevance for stimulus-secretion coupling in pancreatic B-cells.     

Implication of the midgut of the centipede Lithobius forficatus in the heavy metal detoxification process

The ability of two rat liver fatty acid binding protein (L-FABP) isoforms to influence microsomal phosphatidic acid biosynthesis, a key intermediate in glycerolipid formation, and phospholipid fatty acid remodeling was examined in vitro. Isoform I enhanced microsomal incorporation of [1-14C]-oleoyl-CoA into phosphatidic acid 7-fold while isoform II had no effect relative to basal. In contrast, isoform II enhanced microsomal incorporation of [1-14C]-palmitoyl-CoA into phosphatidic acid 4-fold while isoform I had no effect. These results suggest that each L-FABP isoform selectively utilized different acyl-CoAs for glycerol-3-phosphate esterification. Both isoforms stimulated phosphatidic acid formation by increasing glycerol-3-phosphate acyltransferase activity, not by increasing lysophosphatidic acid acyltransferase activity. Furthermore, the effects of L-FABP on phosphatidic acid biosynthesis could not be correlated with protection from acyl-CoA hydrolysis. L-FABP isoforms also influenced phospholipid fatty acid remodeling in a phospholipid-dependent manner. Isoform I preferentially enhanced oleate and palmitate esterification into phosphatidylethanol-amine, while isoform II stimulated esterification into phosphatidylcholine, phosphatidylserine and sphingomyelin. Taken together, these data demonstrated a unique role of each L-FABP isoform in modulating microsomally derived phospholipid fatty acid composition. (c) 1998 Elsevier Science B.V.     

Always single and single again women: a qualitative study

This study reports the effects of a novel polyunsaturated 3-thia fatty acid, methyl 3-thiaoctadeca-6,9,12,15-tetraenoate on serum lipids and key enzymes in hepatic fatty acid metabolism compared to a saturated 3-thia fatty acid, tetradecylthioacetic acid. Palmitic acid treated rats served as controls. Fatty acids were administered by gavage in daily doses of 150 mg/kg body weight for 10 days. The aim of the present study was: (a) To investigate the effect of a polyunsaturated 3-thia fatty acid ester, methyl 3-thiaoctadeca-6,9,12,15-tetraenoate on plasma lipids in normolipidemic rats: (b) to verify whether the lipid-lowering effect could be consistent with enhanced fatty acid oxidation: and (c) to study whether decreased activity of esterifying enzymes and diversion to phospholipid synthesis is a concerted mechanism in limiting the availability of free fatty acid as a substrate for hepatic triglyceride formation. Repeated administration of the polyunsaturated 3-thia fatty acid ester for 10 days resulted in a reduction of plasma triglycerides (40%), cholesterol (33%) and phospholipids (20%) compared to controls. Administration of polyunsaturated and saturated 3-thia fatty acids (daily doses of 150 mg/kg body weight) reduced levels of lipids to a similar extent and followed about the same time-course. Both mitochondrial and peroxisomal fatty acid oxidation increased (1.4-fold- and 4.2-fold, respectively) and significantly increased activities of carnitine palmitoyltransferase (CPT) (1.6-fold), 2,4-dienoyl-CoA reductase (1.2-fold) and fatty acyl-CoA oxidase (3.0-fold) were observed in polyunsaturated 3-thia fatty acid treated animals. This was accompanied by increased CPT-II mRNA (1.7-fold). 2,4-dienoyl-CoA reductase mRNA (2.9-fold) and fatty acyl-CoA oxidase mRNA (1.7-fold). Compared to controls, the hepatic triglyceride biosynthesis was retarded as indicated by a decrease in liver triglyceride content (40%). The activities of glycerophosphate acyltransferase, acyl-CoA: 1,2-diacylglycerol acyltransferase and CTP:phosphocholine cytidylyltransferase were increased. The cholesterol lowering effect was accompanied by a reduction in HMG-CoA reductase activity (80%) and acyl-CoA:cholesterol acyltransferase activity (33%). In hepatocytes treated with methyl 3-thiaoctadeca-6,9,12,15-tetraenoate, fatty acid oxidation was increased 1.8-fold compared to controls. The results suggest that treatment with methyl 3-thiaoctadeca-6,9,12,15-tetraenoate reduces plasma triglycerides by a decrease in the availability of fatty acid substrate for triglyceride biosynthesis via enhanced fatty acid oxidation, most likely attributed to the mitochondrial fatty acid oxidation. It is hypothesized that decreased phosphatidate phosphohydrolase activity may be an additive mechanism which contribute whereby 3-thia fatty acids reduce triglyceride formation in the liver. The cholesterol-lowering effect of the polyunsaturated 3-thia fatty acid ester may be due to changes in cholesterol/cholesterol ester synthesis as 60% of this acid was observed in the hepatic cholesterol ester fraction.     

Differential inhibitory action of nitric oxide and peroxynitrite on mitochondrial electron transport

Various authors have suggested that nitric oxide (.NO) exerts cytotoxic effects through the inhibition of cellular respiration. Indeed, in intact cells .NO inhibits glutamate-malate (complex I) as well as succinate (complex II)-supported mitochondrial electron transport, without affecting TMPD/ascorbate (complex IV)-dependent respiration. However, experiments in our lab using isolated rat heart mitochondria indicated that authentic .NO inhibited electron transport mostly by reversible binding to the terminal oxidase, cytochrome a3, having a less significant effect on complex II- and no effect on complex I-electron transport components. The inhibitory action of .NO was more profound at lower oxygen tensions and resulted in differential spectra similar to that observed in dithionite-treated mitochondria. On the other hand, continuous fluxes of .NO plus superoxide (O.(2)(-)), which lead to formation of micromolar steady-state levels of peroxynitrite anion (ONOO-), caused a strong inhibition of complex I- and complex II-dependent mitochondrial oxygen consumption and significantly inhibited the activities of succinate dehydrogenase and ATPase, without affecting complex IV-dependent respiration and cytochrome c oxidase activity. In conclusion, even though nitric oxide can directly cause a transient inhibition of electron transport, the inhibition pattern of mitochondrial respiration observed in the presence of peroxynitrite is the one that closely resembles that found secondary to .NO interactions with intact cells and strongly points to peroxynitrite as the ultimate reactive intermediate accounting for nitric oxide-dependent inactivation of electron transport components and ATPase in living cells and tissues.     

t-Butylhydroperoxide and gliotoxin stimulate Ca2+ release from rat skeletal muscle mitochondria

Rat liver mitochondria have a specific Ca2+ release pathway which operates when NAD+ is hydrolysed to nicotinamide and ADPribose. NAD+ hydrolysis is Ca(2+)-dependent and inhibited by cyclosporine A (CSA). Mitochondrial Ca2+ release can be activated by the prooxidant t-butylhydroperoxide (tbh) or by gliotoxin (GT), a fungal metabolite of the epipolythiodioxopiperazine group. Tbh oxidizes NADH to NAD+ through an enzyme cascade consisting of glutathione peroxidase, glutathione reductase, and the energy linked transhydrogenase, whereas GT oxidizes some vicinal thiols to the disulfide form, a prerequisite for NAD+ hydrolysis. We report now that rat skeletal muscle mitochondria also contain a specific Ca2+ release pathway activated by both tbh and GT. Ca2+ release increases with the mitochondrial Ca2+ load, is completely inhibited in the presence of CSA, and is paralleled by pyridine nucleotide oxidation. In the presence of tbh and GT, mitochondria do not lose their membrane potential and do not swell, provided continuous release and re-uptake of Ca2+ ('Ca2+ cycling') is prevented. These data support the notion that both tbh- and GT-induced Ca2+ release are not the consequence of an unspecific increase of the inner membrane permeability ('pore' formation). Tbh induces Ca2+ release from rat skeletal muscle less efficiently than from liver mitochondria indicating that the coupling between tbh and NADH oxidation is much weaker in skeletal muscle mitochondria. This conclusion is corroborated by a much lower glutathione peroxidase activity in skeletal muscle than in liver mitochondria. The prooxidant-dependent pathway promotes, under drastic conditions (high mitochondrial Ca2+ loads and high tbh concentrations), Ca2+ release to about the same extent and rate as the Na+/Ca2+ exchanger. This renders the prooxidant-dependent pathway relevant in the pathophysiology of mitochondrial myopathies where its activation by an increased generation of reactive oxygen species probably results in excessive Ca2+ cycling and damage to mitochondria.     

Carnitine palmitoyltransferase activities: effects of serum albumin, acyl-CoA binding protein and fatty acid binding protein

The carnitine palmitoyltransferase activity of various subcellular preparations measured with octanoyl-CoA as substrate was markedly increased by bovine serum albumin at low microM concentrations of octanoyl-CoA. However, even a large excess (500 microM) of this acyl-CoA did not inhibit the activity of the mitochondrial outer carnitine palmitoyltransferase, a carnitine palmitoyltransferase isoform that is particularly sensitive to inhibition by low microM concentrations of palmitoyl-CoA. This bovine serum albumin stimulation was independent of the salt activation of the carnitine palmitoyltransferase activity. The effects of acyl-CoA binding protein (ACBP) and the fatty acid binding protein were also examined with palmitoyl-CoA as substrate. The results were in line with the findings of stronger binding of acyl-CoA to ACBP but showed that fatty acid binding protein also binds acyl-CoA esters. Although the effects of these proteins on the outer mitochondrial carnitine palmitoyltransferase activity and its malonyl-CoA inhibition varied with the experimental conditions, they showed that the various carnitine palmitoyltransferase preparations are effectively able to use palmitoyl-CoA bound to ACBP in a near physiological molar ratio of 1:1 as well as that bound to the fatty acid binding protein. It is suggested that the three proteins mentioned above affect the carnitine palmitoyltransferase activities not only by binding of acyl-CoAs, preventing acyl-CoA inhibition, but also by facilitating the removal of the acylcarnitine product from carnitine palmitoyltransferase. These results support the possibility that the acyl-CoA binding ability of acyl-CoA binding protein and of fatty acid binding protein have a role in acyl-CoA metabolism in vivo.     

The relative utilization of different molecular species of unsaturated 1,2-diacyglycerols by the acyl-CoA:1,2-diacyl-sn-glycerol acyltransferase in rat liver microsomes

The possible selectivity of the acyl-CaA:1,2-diacyl-sn-glycerol acyltransferase in rat liver microsomes towards different molecular species of 1,2-diacyl-sn-glycerols was studied. Triacylglycerol synthesis was determined by monitoring the entry of radioactive palmitate from [1-14C]palmitoyl-CoA into triacylglycerol in the presence of various diacylglycerols. The diacylglycerol acyltransferase did not exhibit any selectivity against hexaenoic precursors under the assay conditions used (0.25 mM diacylglycerol in medium) and the rates of triacylglycerol synthesis observed with various diacylglycerols were not significantly different when the data were analyzed statistically. The results that the extremely low level of docosahexaenoic acid in triacylglycerols cannot be attributed to a discrimination of the diacylglycerol acyltransferase against 1-saturated 2-docosahexaenoyl precursors.     

Effects of the nonsteroidal anti-inflammatory drug nimesulide on energy metabolism in livers from adjuvant-induced arthritic rats

The effects of nimesulide on energy metabolism and the hepatic metabolic alterations produced by adjuvant-induced arthritis were investigated in the perfused rat liver an in isolated liver mitochondria. Nimesulide, at therapeutic levels (20-50 microM), produced: (1) stimulation of oxygen consumption in the perfused rat liver and in isolated mitochondria, (2) inhibition of gluconeogenesis; (3) reduction of ADP/O ratio and the respiratory control ratio and stimulation of glycogenolysis in the livers from healthy rats, but not in livers from arthritic rats. These results indicate that nimesulide acts as a mitochondrial uncoupler. The main alterations produced by adjuvant-induced arthritis were: higher rates of oxygen consumption in both perfused livers and isolated mitochondria, with no decrease in the efficiency of mitochondrial energy transduction; (2) decreased gluconeogenesis and lack of glycogenolytic response to uncouplers, but not to alpha 1-agonists. These data allow to conclude that nimesulide-induced impairment of energy metabolism should worsen the hepatic disturbances that are already associated with the adjuvant disease.     

Microfluorometry using fluorescein diacetate reflects the integrity of the plasma membrane in UVA-irradiated cultured skin fibroblasts

Hepatic coenzyme A (CoA) plays an important role in cellular lipid metabolism. Because mitochondria and peroxisomes represent the two major subcellular sites of lipid metabolism, the present study was designed to investigate the specific impact of hepatic CoA deficiency on peroxisomal as well as mitochondrial beta-oxidation of fatty acids. CoA deficiency (47% decrease in free CoA and 23% decrease in total CoA) was produced by maintaining weanling male Sprague-Dawley rats on a semipurified diet deficient in pantothenic acid (the precursor of CoA) for 5 weeks. Hepatic mitochondrial fatty acid oxidation of short-chain and long-chain fatty acids were not significantly different between control and CoA-deficient rats. Conversely, peroxisomal beta-oxidation was significantly diminished (38% inhibition) in livers of CoA-deficient rats compared to control animals. Peroxisomal beta-oxidation was restored to normal levels when hepatic CoA was replenished. It is postulated that since the role of hepatic mitochondrial beta-oxidation is energy production while peroxisomal beta-oxidation acts mainly as a detoxification system, the mitochondrial pathway of beta-oxidation is spared at the expense of the peroxisomal pathway when liver CoA plummets. The present study may offer an animal model to investigate mechanisms involved in peroxisomal diseases.     

Disorders of the mitochondria

Recent advances in our understanding of the structure and function of mitochondria have led to the recognition that inherited and acquired mitochondrial dysfunction may be responsible for diseases affecting the liver and other organ systems. Mitochondrial health may also determine hepatocyte survival in other hepatic disorders not directly related to the mitochondrion. Primary mitochondrial hepatopathies are conditions in which there are inherited defects in structure or function of the mitochondria, most of which involve the respiratory chain and oxidative phosphorylation, fatty acid oxidation, the urea cycle, and other pathways confined to mitochondria. Maternally inherited mutations or deletions of the mitochondrial genome, or putative nuclear gene mutations encoding electron transport proteins, cause defective electron transport, oxidative stress, impaired oxidative phosphorylation, and other metabolic derangements that lead to hepatic failure or chronic liver dysfunction in affected children. The mitochondrial DNA (mtDNA) depletion syndrome, which similarly leads to liver failure and neurologic abnormalities, is caused by a putative nuclear gene that controls mtDNA replication or stability. Other proven or suspected primary mitochondrial hepatopathies include Pearson's marrow-pancreas syndrome, Alpers disease, mitochondrial neurogastrointestinal encephalomyopathy syndrome, and Navajo neuropathy. Secondary mitochondrial hepatopathies are conditions in which the mitochondria are major targets during liver injury from another cause, such as metal overload, certain drugs and toxins, alcoholic liver injury, and conditions of oxidant stress. Diagnosis of mitochondrial dysfunction may be difficult with currently available tools, however, elevated blood lactate: pyruvate ratios or arterial ketone body ratios with characteristic liver histology are initial tests. Measuring respiratory chain enzyme activities, mtDNA levels, and searching for mtDNA mutations and deletions are more specific tests. Treatment of these disorders is currently empirical, involving agents that may improve the redox status of mitochondria, promote electron flow, or act as mitochondrial antioxidants. Liver transplantation has occasionally been successful in patients who lack other systemic involvement.     

Monoethlenic C20 and C22 fatty acids in marine oil and rapeseed oil. Studies on their oxidation and on their relative ability to inhibit palmitate oxidation in heart and liver mitochondria

1. Carnitine esters of erucic acid (22:1 n-9 cis), cetoleic acid (22:1 n-11 cis), brassidic acid (22:1 n-9 trans), gadoleic acid (20:1 n-9 cis) and oleic acid (18:1 n-9 cis) have been compared as mitochondrial substrates and as inhibitors of palmitoylcarnitine oxidation in heart and liver mitochondria. 2. Both the rate of intramitochondrial-CoA acylation and the rate of beta-oxidation decreases as the chain length increases from C18 to C22. There are no significant differences among the three C22 isomers as oxidizable substrates. 3. All the tested acylcarnitines inhibit palmitoylcarnitine oxidation. The C18 and C20 acylcarnitines inhibit by virtue of being competing substrates; i.e. the respiration is not inhibited. The C22-isomers inhibit also respiration; this shows that the inhibition of palmitolycarnitine oxidation is not compensated for by oxidation of C22-acylcarnitines. Brassidoylcarnitine inhibits the oxidation of palmitoylcarnitine and respiration less than erucoyl-and cetoleoylcarnitine. The different behaviour of the C22-isomers is probably due to the difference in their competitive properties with respect to long-chain acyl-CoA dehydrogenase. 4. All C22 acylcarnitines seem to be relatively better oxidized in the liver than in the heart mitochondria while their inhibitory effect on the usage of the radioactive palmitoylcarnitine is very similar. 5. Palmitoylcarnitine inhibits almost completely the "endogenous" formation of acetyl-CoA presumably from malate via pyruvate in the liver mitochondria while the C22-acylcarnitines cause only a partial inhibiton of this acetyl-CaO formation.     

Reversibility of hepatic mitochondrial damage in rats with long-term cholestasis

BACKGROUND/AIMS: Long-term bile duct ligation in rats is associated with secondary biliary cirrhosis and metabolic alterations, e.g. mitochondrial dysfunction. We performed the current studies to characterize the reversibility of hepatic mitochondrial dysfunction after reversing biliary obstruction by Roux-en-Y anastomosis. METHODS: Rats were studied after 4 weeks of bile duct ligation, and after 5 or 14 days of reanastomosis. Control rats were pair-fed to treated rats and all rats were studied after starvation for 24 h. Mitochondria were isolated by differential centrifugation and enzyme activities determined by spectrophotometric methods. RESULTS: In comparison to controls, plasma beta-hydroxybutyrate concentrations were decreased in bile duct ligated rats (200+/-70 vs. 790+/-200 micromol/l) and remained decreased after relief of biliary obstruction. In contrast, plasma free fatty acids were not different between controls and treated rats. Oxidative metabolism of L-glutamate, succinate and duroquinol was decreased in liver mitochondria from bile duct ligated rats. After relief of biliary obstruction, the metabolism of L-glutamate and duroquinol normalized quickly, whereas succinate metabolism remained impaired. Similar results were obtained for the mitochondrial oxidases in disrupted mitochondria. The activities of complex I, II, III and V of the respiratory chain were reduced in bile duct ligated rats. After relief of biliary obstruction, complex I and III normalized quickly, whereas complex II and V remained impaired. Oxidative metabolism of long-chain fatty acids by isolated liver mitochondria was decreased in bile duct ligated rats and did not recover after relief of biliary obstruction. CONCLUSIONS: Long-term cholestasis in the rat is associated with a decrease in specific functions of liver mitochondria which recover only partially after Roux-en-Y anastomosis. The persistence of decreased mitochondrial fatty acid metabolism cannot be explained by impaired activity of the respiratory chain, but is more likely due to alterations in mitochondrial beta-oxidation.