Dietary fatty acids modulate liver mitochondrial cardiolipin content and its fatty acid composition in rats with non alcoholic fatty liver disease

No data are reported on changes in mitochondrial membrane phospholipids in non-alcoholic fatty liver disease. We determined the content of mitochondrial membrane phospholipids from rats with non alcoholic liver steatosis, with a particular attention for cardiolipin (CL) content and its fatty acid composition, and their relation with the activity of the mitochondrial respiratory chain complexes. Different dietary fatty acid patterns leading to steatosis were explored. With high-fat diet, moderate macrosteatosis was observed and the liver mitochondrial phospholipid class distribution and CL fatty acids composition were modified. Indeed, both CL content and its C18:2n-6 content were increased with liver steatosis. Moreover, mitochondrial ATP synthase activity was positively correlated to the total CL content in liver phospholipid and to CL C18:2n-6 content while other complexes activity were negatively correlated to total CL content and/or CL C18:2n-6 content of liver mitochondria. The lard-rich diet increased liver CL synthase gene expression while the fish oil-rich diet increased the (n-3) polyunsaturated fatty acids content in CL. Thus, the diet may be a significant determinant of both the phospholipid class content and the fatty acid composition of liver mitochondrial membrane, and the activities of some of the respiratory chain complex enzymes may be influenced by dietary lipid amount in particular via modification of the CL content and fatty acid composition in phospholipid.     

Fatty acid composition of phospholipids in mitochondria and microsomes during diethylnitrosamine carcinogenesis in rat liver

Changes in lipid composition and function of subcellular organelles have been described in transplanted and primary tumours. We examine here the fatty acid composition of individual phospholipids (PL) in hyperplastic nodules and primary hepatoma induced by diethylnitrosamine (DEN), compared to that of normal liver and of transplantable Yoshida AH-130 hepatoma. Phosphatidylcholine and phosphatidylethanolamine fatty acid composition in mitochondria and microsomes from primary hepatoma were markedly different from normal liver; C18:0/C18:1 ratio was lower and the ratio between monosaturated and polyunsaturated fatty acids was higher. Linoleic acid content of mitochondrial cardiolipin, usually very high in normal rat liver, was notably lower in primary hepatoma. Cholesterol/phospholipid ratio in both microsomes and mitochondria from DEN-induced hepatoma was higher than in normal liver. Hyperplastic nodules showed no changes in cholesterol content whereas modifications in fatty acid composition were already observable. These modifications of membrane structure may be related to the functional changes found in nodular cells. Changes in fatty acid composition of membrane phospholipids, occurring in both primary hepatoma and preneoplastic nodules, might be one of the causes for decreased rate of lipid peroxidation peculiar to these tissues.     

Effects of cold adaptation and re-adaptation upon the mitochondrial phospholipids of brown adipose tissue.

1. A study of the mitochondrial phospholipids, phospholipid fatty acid patterns and enzyme activities was investigated in brown tissue (B.A.T.) from rats chronically exposed to cold and/or treated with thyroxine. 2. The total activities of the oxidative enzymes were increased after cold exposure, but not after thyroxine treatment. 3. Cold exposure increased the amount of phosphatidylethanolamine, phosphatidylcholine, cardiolipin and lysophospholipids, the effect being greatest for phosphatidylethanolamine. At the same time, there were marked alterations in the fatty acid composition of the mitochondrial phospholipids (decrease of palmitic, palmitoleic and oleic acids ; increase of stearic, linoleic and arachidonic acids). 4. All these cold-induced alterations were reversed by re-adaptation of the animal to a normal temperature range. 5. The alterations of the fatty acid composition of phospholipids could be explained by changes in the rate of individual fatty acid biosynthesis.     

Composition of the inner mitochondrial membrane of porcine corpus luteum

An inner mitochondrial membrane fraction was prepared from porcine corpus luteum. The concentrations of the respiratory cytochromes, cytochrome P-450scc, cholesterol, ubiquinone, cardiolipin and the total phospholipids were measured. The fatty acid compositions of cardiolipin and the total phospholipid fraction were determined. Comparative data from porcine heart and liver were obtained using the same methods. Differences in both the concentration and the fatty acid composition of the phospholipids were observed between the tissues. It appeared that the phospholipid bilayer was expanded relative to haem a in luteal mitochondria. It is proposed that in the ovary this expansion may be necessary to accommodate cytochrome P-450scc and its substrate, cholesterol.     

Phospholipid composition and fatty acid patterns of isolated phospholipids from rabbit reticulocyte mitochondria

The phospholipid composition and fatty acid patterns of individual phospholipid classes were determined in mitochondria from rabbit reticulocytes. Compared to mitochondria from rat liver reticulocyte, mitochondria exhibit about twice the amount of phospholipids. The phospholipid pattern of reticulocyte mitochondria (phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and cardiolipin) is comparable with other mitochondrial species. Mitochondrial fractions from reticulocytes are characterized, however, by an additional content of sphingomyelin. This sphingomyelin differs in its fatty acid composition from the sphingomyelin of the plasma membrane. The fatty acid patterns of all other phospholipids essentially correspond to those of mitochondria from other sources and to those of plasma membranes as well.     

Mitochondrial dysfunction in rat with nonalcoholic fatty liver Involvement of complex I, reactive oxygen species and cardiolipin

Mitochondrial dysfunction and oxidative stress play a central role in the pathophysiology of nonalcoholic fatty liver disease (NAFLD). This study aimed to elucidate the mechanism(s) responsible for mitochondrial dysfunction in nonalcoholic fatty liver. Fatty liver was induced in rats with a choline-deficient (CD) diet for 30 days. We examined the effect of CD diet on various parameters related to mitochondrial function such as complex I activity, oxygen consumption, reactive oxygen species (ROS) generation and cardiolipin content and oxidation. The activity of complex I was reduced by 35% in mitochondria isolated from CD livers compared with the controls. These changes in complex I activity were associated with parallel changes in state 3 respiration. Hydrogen peroxide (H(2)O(2)) generation was significantly increased in mitochondria isolated from CD livers. The mitochondrial content of cardiolipin, a phospholipid required for optimal activity of complex I, decreased by 38% as function of CD diet, while there was a significantly increase in the level of peroxidized cardiolipin. The lower complex I activity in mitochondria from CD livers could be completely restored to the level of control livers by exogenously added cardiolipin. This effect of cardiolipin could not be replaced by other phospholipids nor by peroxidized cardiolipin. It is concluded that CD diet causes mitochondrial complex I dysfunction which can be attributed to ROS-induced cardiolipin oxidation. These findings provide new insights into the alterations underlying mitochondrial dysfunction in NAFLD.     

Lipid composition of brown adipose tissue mitochondria and microsomes in hyperthyroid rats

1. The effects of triiodothyronine on the lipid composition of rat brown adipose tissue (BAT) mitochondria and microsomes was investigated by high performance liquid chromatography (HPLC). 2. An increase of about 20% was noted in mitochondrial cholesterol and phospholipids, while a decrease of about 20% for both total cholesterol and phospholipids was observed in microsomes from hyperthyroid rats. 3. The BAT phospholipid composition was altered significantly in mitochondria from T3-treated rats with an increase (41%) of cardiolipin and a decrease (18%) in phosphatidylcholine. 4. In microsomes, a decrease by 25% in phosphatidylinositol was accompanied by a similar additional percentage increase in phosphatidylethanolamine. 5. Important alterations in the fatty acid pattern were found in mitochondrial neutral lipids.     

Citrate carrier activity and cardiolipin level in eel (Anguilla anguilla) liver mitochondria

The activity of the tricarboxylate (citrate) carrier has been assayed in intact liver mitochondria from yellow eel (Anguilla anguilla) and compared to that from rat. The eel-citrate carrier specific activity was approximately 1.7-fold higher than that assayed in rat-liver mitochondria. The content of the main mitochondrial phospholipids, phosphatidylethanolamine and phosphatidylcholine, did not show a significant difference between the two species, while in eel a higher cardiolipin level was observed. Fatty acid composition of eel-liver mitochondrial phospholipids was characterised by a large amount of unsaturated fatty acids, dominated by octadecaenoic acid (C(18:1) (n-9)) and docosahexaenoic acid (C(22:6) (n-3)). The cardiolipin fatty acid pattern of eel-liver mitochondria showed, with respect to the rat, a higher C(20:5) (n-3) and C(22:6) (n-3) content and a lower amount of C(18:2) (n-6) and C(20:4) (n-6). A noticeable activity of lipogenic enzymes was also detected in eel liver cytosol. The results of this study suggest that the remarkable activity of the citrate carrier in eel-liver mitochondria can most likely be ascribed to a considerable cardiolipin level. A covariance of citrate carrier and lipogenic enzyme activities was observed.     

Mitochondrial dysfunction in rat with nonalcoholic fatty liver: Involvement of complex I, reactive oxygen species and cardiolipin

Mitochondrial dysfunction and oxidative stress play a central role in the pathophysiology of nonalcoholic fatty liver disease (NAFLD). This study aimed to elucidate the mechanism(s) responsible for mitochondrial dysfunction in nonalcoholic fatty liver. Fatty liver was induced in rats with a choline-deficient (CD) diet for 30 days. We examined the effect of CD diet on various parameters related to mitochondrial function such as complex I activity, oxygen consumption, reactive oxygen species (ROS) generation and cardiolipin content and oxidation. The activity of complex I was reduced by 35% in mitochondria isolated from CD livers compared with the controls. These changes in complex I activity were associated with parallel changes in state 3 respiration. Hydrogen peroxide (H₂O₂) generation was significantly increased in mitochondria isolated from CD livers. The mitochondrial content of cardiolipin, a phospholipid required for optimal activity of complex I, decreased by 38% as function of CD diet, while there was a significantly increase in the level of peroxidized cardiolipin. The lower complex I activity in mitochondria from CD livers could be completely restored to the level of control livers by exogenously added cardiolipin. This effect of cardiolipin could not be replaced by other phospholipids nor by peroxidized cardiolipin. It is concluded that CD diet causes mitochondrial complex I dysfunction which can be attributed to ROS-induced cardiolipin oxidation. These findings provide new insights into the alterations underlying mitochondrial dysfunction in NAFLD.     

Improved mitochondrial function with diet-induced increase in either docosahexaenoic acid or arachidonic acid in membrane phospholipids

Mitochondria can depolarize and trigger cell death through the opening of the mitochondrial permeability transition pore (MPTP). We recently showed that an increase in the long chain n3 polyunsaturated fatty acids (PUFA) docosahexaenoic acid (DHA; 22:6n3) and depletion of the n6 PUFA arachidonic acid (ARA; 20:4n6) in mitochondrial membranes is associated with a greater Ca(2+) load required to induce MPTP opening. Here we manipulated mitochondrial phospholipid composition by supplementing the diet with DHA, ARA or combined DHA+ARA in rats for 10 weeks. There were no effects on cardiac function, or respiration of isolated mitochondria. Analysis of mitochondrial phospholipids showed DHA supplementation increased DHA and displaced ARA in mitochondrial membranes, while supplementation with ARA or DHA+ARA increased ARA and depleted linoleic acid (18:2n6). Phospholipid analysis revealed a similar pattern, particularly in cardiolipin. Tetralinoleoyl cardiolipin was depleted by 80% with ARA or DHA+ARA supplementation, with linoleic acid side chains replaced by ARA. Both the DHA and ARA groups had delayed Ca(2+)-induced MPTP opening, but the DHA+ARA group was similar to the control diet. In conclusion, alterations in mitochondria membrane phospholipid fatty acid composition caused by dietary DHA or ARA was associated with a greater cumulative Ca(2+) load required to induced MPTP opening. Further, high levels of tetralinoleoyl cardiolipin were not essential for normal mitochondrial function if replaced with very-long chain n3 or n6 PUFAs.     

Copper deficiency-induced changes in the fatty acid composition of mitochondrial and microsomal membranes of rat liver

The effects of copper deficiency on the fatty acid composition of mitochondrial and microsomal phospholipids in rat liver were studied. Copper deficiency was induced by a milk powder diet. To evaluate the effect of the milk diet on the fatty acid pattern of mitochondrial and microsomal phospholipids, one group of rats was fed Cusupplemented powdered milk. A decrease in the relative proportion of linoleic acid and an increase in the level of oleic and docosahexaenoic acids in membrane phospholipids were found in this group. However, no changes in the fatty acid pattern characteristic of essential fatty acid deficiency were observed. Dietary copper deficiency produced a significant decrease in the relative amounts of linoleic and arachidonic acids, as well as an increase in the docosahexaenoic acid content in both mitochondrial and microsomal membranes compared to the nondeficient controls. The disproportionate quantities of polyunsaturated fatty acids are discussed with a view to the disturbances of membrane function in copper deficiency.     

Essential fatty acids in tissue phospholipids and triglycerides of the zinc-deficient rat

This study addressed the possibility that zinc deficiency has different effects on the fatty acid composition of triglyceride compared to total phospholipid. Male weanling Sprague-Dawley rats were maintained for 6 weeks on a semisynthetic diet deficient in zinc (3 mg/kg zinc). Control rats (40 mg/kg zinc) were pair-fed. Lipid fractionation and fatty acid analysis were by thin-layer and gas chromatography, respectively. In zinc-deficient rats, the percentage of linoleic acid was increased or that of arachidonic acid was decreased in total phospholipids of plasma, liver, and testis, and in skin total lipids. Saturated and monounsaturated fatty acids were increased in the triglyceride of liver but decreased in the triglyceride of epididymal fat of zinc deficient rats. Essential fatty acids, as a proportion of total fatty acids, were decreased in triglyceride of liver but increased in triglyceride of epididymal fat of zinc-deficient rats. Our fatty acid data from tissue total phospholipids therefore support the concept that linoleic acid desaturation is impaired in zinc deficiency.     

Enhanced activity of the tricarboxylate carrier and modification of lipids in hepatic mitochondria from hyperthyroid rats

The effect of hyperthyroidism on the activity of the mitochondrial tricarboxylate carrier has been studied. The activity of this transporting system in liver mitochondria was quantitatively determined by the rate of malate-[14C]citrate exchange using the 1,2,3-benzene-tricarboxylate inhibitor stop technique. It has been found that the rate of citrate uptake is significantly enhanced in liver mitochondria from hyperthyroid rats as compared to that obtained in mitochondria from control rats. Kinetic analysis of the malate-citrate exchange reaction indicates that only the Vmax of this transporting process is enhanced, while there is practically no change in the Km values. Inhibitor titrations with the inhibitor palmitoyl-CoA show that mitochondria from hyperthyroid rats require the same concentrations of inhibitor to produce 100% inhibition of citrate uptake as control mitochondria, suggesting that the amount of functional translocase enzyme present is unaffected. The Arrhenius plot characteristics differ for tricarboxylate carrier activity in mitochondria from hyperthyroid rats as compared with control rats in that the break point of the biphasic plot decreases from 18.1 +/- 1.4 degrees C in controls to 12.9 +/- 1.2 degrees C in hyperthyroid animals. The hepatic mitochondrial lipid composition is altered significantly in hyperthyroid rats; the total cholesterol decreases and the phospholipids increase. The liver mitochondrial phospholipid composition is altered significantly in hyperthyroid rats. In particular negatively charged phospholipid cardiolipin increases by more than 50%. Minor alterations were found in the pattern of fatty acids. The thyroid hormone induced change in the activity of the tricarboxylate carrier can be ascribed either to a general modification of membrane lipid composition which increases the membrane fluidity and in turn the mobility of the carrier or to a more localized change of lipid domain (cardiolipin content) surrounding the carrier molecule in the mitochondrial membrane.     

The influence of polyunsaturated fatty acids on spermine inhibition of lipoperoxidation. Studies on liposomes prepared with microsomal and mitochondrial phospholipids of sea bass (Dicentrarchus labrax L.) and rat liver

1. Composition of phospholipids extracted from different organelles of European sea bass liver was determined and compared with that of phospholipids extracted from the same organelles of rat liver. 2. Spermine binding to the vesicles prepared from microsomal and mitochondrial phospholipids and their aggregation was studied: these parameters indicate that only the presence of acidic phospholipids and not their unsaturation was essential for polyamine action. 3. No correlation exists between polyunsaturated fatty acid and spermine inhibition of lipid peroxidation. In fact microsomal phospholipids, which have a low content of acidic phospholipids, and a prevalent presence of phosphatidylinositol, are not protected by spermine. 4. Mitochondrial phospholipids, which have high content of cardiolipin, elicit the capability of spermine to inhibit lipid peroxidation.     

Lipid metabolism in fatty liver of lysine- and threonine-deficient rats

Rats were fed a low protein diet deficient in and supplemented with lysine and threonine. Liver lipids contained more lecithin, sphingomyelin, and free fatty acids, and less amino phospholipids in the deficient rats. No variations in fatty acid composition of choline- and ethanolamine-containing phospholipids were found; only palmitic acid was increased in the serine-containing phospholipids of the deficient animals. The incorporation of acetate-(14)C into phospholipids, but not into other liver lipids, was lower in deficient rats. In the plasma of deficient rats the concentration of esterified fatty acids and phospholipids was lower, of free fatty acids higher, than in the controls. The fatty acid composition of depot fat differed from that of liver neutral fat both in deficient and supplemented animals. The results presented establish that multiple metabolic defects resulting from lysine and threonine deficiency accompany the fatty liver. The design of the experiments does not permit conclusions to be drawn regarding the causal relationship between the various alterations in lipid metabolism and the fatty liver.     

The renal mitochondrial metabolism of 25-hydroxyvitamin D-3: a possible role for phospholipids

The effect of exogenous phospholipids on chick kidney mitochondrial 25-hydroxyvitamin D-3 metabolism was examined. Phosphatidylserine, phosphatidylcholine and phosphatidylinositol had no effect on either the 1- or 24-hydroxylation of 25-hydroxyvitamin D-3. Phosphatidylethanolamine and cardiolipin both brought about a dose-dependent decrease in the 1-hydroxylase activity in mitochondria from vitamin D-deficient chicks but not from vitamin D-replete chicks. There were no major differences in the phospholipid composition of mitochondria from vitamin D-deficient and -replete chicks nor in the fatty acid composition of these phospholipids. Preliminary kinetic studies suggest that cardiolipin acts as a noncompetitive inhibitor of the 1-hydroxylase in mitochondria isolated from vitamin D-deficient chicks. It does not appear to exert its effect by virtue of altering the distribution of substrate or products. Investigation of the effect of fatty acid methyl esters on the hydroxylase activities suggests that it may be the fatty acid moiety of the phospholipid, rather than the phosphate moiety in the polar head group, that is involved in the phospholipid effect on the hydroxylation of 25-hydroxyvitamin D-3.     

The lipid composition of ovine placental tissue homogenate and its subcellular fractions

Homogenates of the placental tissue of near term sheep were separated by differential centrifugation into mitochondrial, microsomal and cytosolic fractions. The relative proportions of the major neutral lipids and phospholipids, together with their fatty acid compositions, were determined in the homogenates and in each subcellular fraction. The cytosolic fraction contained the highest proportion of cholesteryl esters (CEs) and these possessed a fatty acid composition markedly different from the total CEs extracted from the homogenate. Both the mitochondrial and microsomal fractions contained significant proportions of solvent front phospholipid (SFP) and whereas the mitochondrial SFP displayed the relatively unsaturated fatty acid composition characteristic of diphosphatidylglycerol (cardiolipin), the fatty acids of the microsomal SFP were distinctly more saturated. These results are compared with those obtained from other mammalian tissues, both ruminant and non-ruminant, and discussed in terms of the function of the components of the subcellular fractions.     

Effect of lidocaine on phospholipid and fatty acid composition of bacterial membranes

The effects of lidocaine on chemical composition of membrane phospholipids and membrane fluidity of Streptococcus mutans have been studied. Increasing concentra-tions of lidocaine induced both an increase in cardiolipin and a decrease in the degree of unsaturation of its fatty acid composition. A lidocaine-dependent decrease of membrane fluidity was observed from an electron spin resonance spectroscopic study. It was considered thal bacteria grown with lidocaine below its minimum inhibitory concentration resisted the effect of the drug by modifying phospholipid and fatty acid composition resulting in a decreased membrane fluidity.     

Effect of different levels of thyroid hormones on the fatty acid composition of lipids from rat liver mitochondria

The abundance or deficiency of thyroid hormones in rat organism influence the unsaturation and desaturation indices of total lipid fatty acids and phospholipids in liver mitochondria. The most conspicuous changes were observed in the fatty acid composition of the phospholipid fraction. The changes in the structure and function of rat liver mitochondria are considered to be due to alterations in the fatty acid composition of mitochondrial phospholipids.     

Cell biology of cardiac mitochondrial phospholipids.

Phospholipids are important structural and functional components of all biological membranes and define the compartmentation of organelles. Mitochondrial phospholipids comprise a significant proportion of the entire phospholipid content of most eukaroytic cells. In the heart, a tissue rich in mitochondria, the mitochondrial phospholipids provide for diverse roles in the regulation of various mitochondrial processes including apoptosis, electron transport, and mitochondrial lipid and protein import. It is well documented that alteration in the content and fatty acid composition of phospholipids within the heart is linked to alterations in myocardial electrical activity. In addition, reduction in the specific mitochondrial phospholipid cardiolipin is an underlying biochemical cause of Barth Syndrome, a rare and often fatal X-linked genetic disease that is associated with cardiomyopathy. Thus, maintenance of both the content and molecular composition of phospholipids synthesized within the mitochondria is essential for normal cardiac function. This review will focus on the function and regulation of the biosynthesis and resynthesis of mitochondrial phospholipids in the mammalian heart.