Changes in rat liver mitochondrial lipids in vitamin A deficiency

The alterations in the lipid profiles of rat liver mitochondria due to vitamin A deficiency were studied. The amount of total lipids and phospholipids were decreased with a concomitant increase in triglycerides and cholesterol levels in mitochondria, isolated from vitamin A-deficient animals. Of particular significance was the observation that the content of lysolecithin, a potent cytolytic agent, was increased. An analysis of individual fatty acids showed that the percentage of polyunsaturated fatty acids was decreased significantly in vitamin A deficiency. Further, mitochondria from vitamin A-deficient animals, when incubated in 0.1 M Tris-HCl buffer (pH 7.4)in vitro, produced increased amounts of malondialdehyde and lipofuchsin pigments indicating increased susceptibility of the mitochondrial membrane to peroxidative damage. These results suggest a possible role of vitamin A in the prevention of the decomposition of structural lipids.     

A spin label ESR and saturation transfer-ESR study of archaebacteria bipolar lipids

A spin label study has been carried out on bipolar lipids extracted from Sulfolobus solfataricus, an extreme thermophilic archaebacterium growing at about 85°C and pH 3. These lipids are cyclic diisopranyl tetraether molecules, quite different from the usual fatty acid lipids. Two hydrolytic fractions of the membrane complex lipids have been studied: the symmetric lipid glycerol-dialkyl-glycerol-tetraether (GDGT) and the asymmetric lipid glyceroldialkyl-nonitol-tetraether (GDNT). The ESR spectra confirm the results previously obtained from calorimetric and X-ray diffraction experiments showing a polymorphic behaviour of these lipids and indicating the critical temperature ranges at which structural transitions occur. Moreover, the present study adds information on the dynamics of the different portions of the hydrophobic chain. ST-ESR measurements show correlation times ranging from 10^-8 s up to 10^-5 s, depending upon the lipid sample, the label position and the degree of hydration. At very high temperatures, i.e. the physiological temperatures of Sulfolobus solfataricus, the nonitol head groups of the asymmetric lipids form a strongly immobilized structure. Indeed, the molecular correlation times of the outermost hydrophobic portion of GDNT are higher, by a factor up to 10³, than those of usual monopolar lipids. Anisotropic motional behaviour is observed even at such very high temperatures. Possible biological implications are discussed.Abbreviations used are ESR electron spin resonance - St-ESR saturation transfer electron spin resonance - GDGT glyceroldialkyl-glycerol-tetracther - GDNT glycerol-dialkyl-nonitoltetraether - 5 SASL 12SASL and 16SASL, stearic acid spin labels, N-oxyl-4,4-dimethyloxazolidine derivatives of 5-ketostearic acid, 12-ketostearic acid and 16-ketostearic acid, respectively - DSC differential scanning calorimetry     

The effect of dietary lipids on the thermotropic behaviour of rat liver and heart mitochondrial membrane lipids

Diets supplemented with relatively high levels of either saturated fatty acids derived from sheep kidney fat (sheep kidney fat diet) or unsaturated fatty acids derived from sunflower seed oil (sunflower seed oil diet) were fed to rats for a period of 16 weeks and changes in the thermotropic behaviour of liver and heart mitochondrial lipids were determined by differential scanning calorimetry (DSC). The diets induced similar changes in the fatty acid composition in both liver and heart mitochondrial lipids, the major change being the omega 6 to omega 3 unsaturated fatty acid ratio, which was elevated in mitochondria from animals on the sunflower seed oil diet and lowered with the mitochondria from the sheep kidney fat dietary animals. When examined by DSC, aqueous buffer dispersions of liver and heart mitochondrial lipids exhibited two independent, reversible phase transitions and in some instances a third highly unstable transition. The dietary lipid treatments had their major effect of the temperature at which the lower phase transition occurred, there being an inverse relationship between the transition temperature and the omega 6 to omega 3 unsaturated fatty acid ratio. No significant effect was observed for the temperature of the higher phase transition. These results indicate that certain domains of mitochondrial lipids, probably containing some relatively higher melting-point lipids, independently undergo formation of the solidus or gel phase and this phenomenon is not greatly influenced by the lipid composition of the mitochondrial membranes. Conversely, other domains, representing the bulk of the membrane lipids and which probably contain the relatively lower melting point lipids, undergo solidus phase formation at temperatures which reflect changes in the membrane lipid composition which are in turn, a reflection of the nature of the dietary lipid intake. These lipid phase transitions do not appear to correlate directly with those events considered responsible for the altered Arrhenius kinetics of various mitochondrial membrane-associated enzymes.     

Effect of sex and bezafibrate on incorporation of blood borne palmitate into lipids of rat liver nuclei

The aim of the present study was to investigate whether lipid metabolism in the nuclei is affected by changes in the metabolism of free fatty acids in the liver. The experiments were carried out on 3 groups of rats: 1 - control-male, 2 - female, and 3 - male, treated with bezafibrate (a peroxisome proliferator). The rats received ¹⁴C-palmitic acid intravenously. Thirty min later liver samples and blood from the abdominal aorta were taken. The liver nuclei were isolated in sucrose gradient. Lipids were extracted from the nuclei and the liver homogenate and subsequently separated into the following fractions: phospholipids, mono-, di- and triacylglycerols, free fatty acids, cholesterol and cholesterol esters. The radioactivity of each fraction was counted. Furthermore, the content of free fatty acids and the fatty acid binding proteins was measured. It was found that radioactivity was present in each lipid fraction obtained from the liver homogenate and from the nuclei. In the female group, the total radioactivity of lipids in the liver homogenate was lower, whereas in the nuclei it was higher in comparison to the male group. The reduction in the radioactivity in the liver was mostly accounted for by decreased radioactivity in the fraction of triacylglycerols and phospholipids. In the nuclei, the radioactivity of the fraction of phospholipids, free fatty acids and diacylglycerols was elevated. Bezafibrate did not affect the total radioactivity of lipids in the liver and reduced it in the nuclei. In the liver, the drug increased radioactivity mostly in the fraction of phospholipids and reduced it mainly in the fraction of triacylglycerols. In the nuclei, the radioactivity of each lipid fraction examined was reduced. The content of the fraction of free fatty acids in the liver and in the nuclei in the female and in the bezafibrate-treated groups did not differ from the respective value in the control group. The content of fatty acid binding proteins in the nuclei of the female and bezafibrate-treated groups increased in parallel to the elevation in their content in the cytosol. It is concluded that the female sex hormones and bezafibrate influence the transport of selected lipids into the nuclei. The effects seem to be a consequence of the action of these factors directly on the nucleus.     

Heme-biosynthetic enzyme activities and porphyrin accumulation in normal liver and hepatoma cell lines of rat

The activities of four heme-biosynthetic enzymes, -aminolevulinic acid (ALA) synthase, ALA dehydratase, porphobilogen (PBG) dearninase, and ferrochelatase, were studied in five epithelial cell lines of normal rat liver origin (Re, REC-10, RLC-24, M, Culb-TC) and five cell lines derived from Yoshida ascites hepatoma (JTC-1, JTC-2, JTC-15, JTC-16, JTC-24). The JTC series of hepatoma-derived cell lines exhibited decreased ALA synthase activity and increased ALA dehydratase activity, although the activities of all four enzymes and the Km values for their respective substrates varied widely from one cell line to another, a finding suggesting that specific regulatory mechanisms for porphyrin metabolism might operate in each cell type. M cells, which were transformed by 4-dimethylaminoazobenzene in vitro, gave the most abnormal Km values of heme-biosynthetic enzymes among all the cell lines studies, and were found to accumu2ate hematoporphyrin derivative (HpD).Abbreviations ALA o-aminolevulinic acid - DAB 4-dimethyl aminoazobenzene - HpD hematoporphyrin derivative - 4NQO 4-nitroquinoline 1-oxide - PBG porphobilinogen     

Effect of D-amino acids on some mitochondrial functions in rat liver

Summary.  We studied the role of the D-amino acids (D-aa) D-serine, D-alanine, D-methionine, D-aspartate, D-tyrosine and D-arginine on rat liver mitochondria. The stability of D-amino acids, mitochondrial swelling, transmembrane potential and oxygen consumption were studied under oxidative stress conditions in rat liver mitochondria. In the presence of glutamate-malate all D-aas salts increased mitochondrial swelling, while in the presence of succinate plus rotenone only D-ala, D-arg and D-ser, induced mitochondrial swelling. The transmembrane potential (ΔΨ) was decreased in the presence of 1 μM Ca²⁺. The D-aas inhibited oxygen consumption in state 3. The D-aa studied exerted effects on mitochondria via an increase of free radicals production. Received January 15, 2002 Accepted April 14, 2002 Published online September 4, 2002 Acknowledgements The authors appreciated the partial economical support from Mexican grants of CONACYT (to A.S.-M. during its sabbatical) and CIC-UMSNH (2.5) and critical readings from Rafael álvarez-González. Authors' address: Alfredo Saavedra-Molina, Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana de San Nicolás de Hidalgo, Edificio B-3. C.U., Morelia, Mich. 58030. México, Fax: 52-443-326-5788, E-mail: saavedra@zeus.umich.mx     

Isolation and comparative studies of mitochondrial F1-ATPase from Morris hepatoma and rat liver.

A simple method of isolating mitochondrial ATPase from rat liver and Morris hepatoma cell lines by chloroform extraction and chromatography on DEAE-Sephadex is described. This method is suitable even when small amounts of starting material with relatively low specific ATPase activity (in the case of hepatoma mitochondria and submitochondrial particles) are available. The isolated enzyme from both rat liver and hepatomas had a high specific activity, was similarly activated by bicarbonate and 2,4-dinitrophenol, and had a typical five-band pattern in sodium dodecyl sulfate electrophoresis. Prior to DEAE-Sephadex chromatography, an additional protein band which migrates between the δ and ? subunits in the tumor F₁-ATPase preparation was observed. The purified enzymes were cold labile and restored oxidative phosphorylation function of F₁-ATPase depleted submitochondrial particles prepared from rat liver. The ATPase activity of the isolated enzymes was inhibited by mitochondrial ATPase inhibitor protein. The apparent stoichiometry of the inhibitor protein to the purified ATPase was extrapolated to be 2:1.     

Hepatic mitochondrial transport of glutathione: studies in isolated rat liver mitochondria and H4IIE rat hepatoma cells

Glutathione (GSH) is transported into renal mitochondria by the dicarboxylate (DIC; Slc25a10) and 2-oxoglutarate carriers (OGC; Slc25a11). To determine whether these carriers function similarly in liver mitochondria, we assessed the effect of competition with specific substrates or inhibitors on GSH uptake in isolated rat liver mitochondria. GSH uptake was uniphasic, independent of ATP hydrolysis, and exhibited K_m and V_max values of 4.08 mM and 3.06 nmol/min per mg protein, respectively. Incubation with butylmalonate and phenylsuccinate inhibited GSH uptake by 45-50%, although the individual inhibitors had no effect, suggesting in rat liver mitochondria, the DIC and OGC are only partially responsible for GSH uptake. H4IIE cells, a rat hepatoma cell line, were stably transfected with the cDNA for the OGC, and exhibited increased uptake of GSH and 2-oxoglutarate and were protected from cytotoxicity induced by H₂O₂, methyl vinyl ketone, or cisplatin, demonstrating the protective function of increased mitochondrial GSH transport in the liver.     

The nomenclature and conformational analysis of lipids and lipid analogues

Lipids form an essential part of the biomembrane and it is of paramount importance to study their conformational aspects. It is found that the present methods of nomenclature for lipids are totally inadequate for describing these diverse amphipathic molecules. Further the existing methods are incompatible in terms of assignment of the absolute configuration. A systematic method for the naming of lipids which is rationally extendible to a wide class of amphipaths is described. The conformational features of the natural glycerolipids as well as a synthetic amphipath containing a glutamic acid moiety known to undergo interesting phase transitions, have been examined in detail using the framework of the current nomenclature system. The implications of the conformational flexibility of these molecules on assemblies of these systems is touched upon.     

Immunochemical analysis of the membrane proteins of rat liver and Zajdela hepatoma mitochondria

The contents of mitochondrial inner membrane protein complexes were compared in normal liver and in Zajdela hepatoma mitochondria by the immunotransfer technique. Antibodies against core proteins 1 and 2, cytochrome c1, the iron-sulfur protein of Complex III, subunits I and II of cytochrome oxidase, and the alpha and beta subunits of the F1-ATPase were used. In addition, antibodies against a primary dehydrogenase, beta-hydroxybutyrate dehydrogenase, as well as the outer membrane pore protein were used. The results indicate that the components of the cytochrome chain and porin are greatly enriched in hepatoma mitochondria compared to normal rat liver mitochondria. This enrichment was also reflected in the rates of respiration in tumor mitochondria using a variety of substrates. Enrichment of porin may partially account for increased hexokinase binding to tumor mitochondria. In contrast to the respiratory chain components, the F1-ATPase and F0 (measured by DCCD binding) were not increased in tumor mitochondria. Thus, Zajdela hepatoma mitochondria components are nonstoichiometric, being enriched in oxidative capacity but relatively deficient in ATP synthesizing capacity. Finally, beta-hydroxybutyrate dehydrogenase, which is often decreased in hepatoma mitochondria, was shown here by immunological methods to be decreased by only 40%, whereas enzyme activity was less than 5% of that in normal rat liver.     

A targeted proteomic approach for the analysis of rat liver mitochondrial outer membrane proteins with extensive sequence coverage

Membrane proteins play an important role in cellular function. However, their analysis by mass spectrometry often is hindered by their hydrophobicity and/or low abundance. In this article, we present a method for the mass spectrometric analysis of membrane proteins based on the isolation of the resident membranes, isolation of the proteins by gel electrophoresis, and electroelution followed by enzymatic digestion by both trypsin and proteinase K. With this method, we have achieved 82-99% sequence coverage for the membrane proteins carnitine palmitoyltransferase-I (CPT-I), long-chain acyl-CoA synthetase (LCAS), and voltage-dependent anion channel (VDAC), isolated from rat liver mitochondrial outer membranes, including the transmembrane domains of these integral membrane proteins. This high sequence coverage allowed the identification of the isoforms of the proteins under study. This methodology provides a targeted approach for examining membrane proteins in detail.     

Malonaldehyde acts as a mitochondrial toxin: Inhibitory effects on respiratory function and enzyme activities in isolated rat liver mitochondria

Malonaldehyde (MDA) is a product of oxidative damage to lipids, amino acids and DNA, and accumulates with aging and diseases. MDA can possibly react with amines to modify proteins to inactivity enzymes and also modify nucleosides to cause mutagenicity. Mitochondrial dysfunction is a major contributor to aging and age-associated diseases. We hypothesize that accumulated MDA due to mitochondrial dysfunction during aging targets mitochondrial enzymes to cause further mitochondrial dysfunction and contribute to aging and age-associated diseases. We investigated the effects of MDA on mitochondrial respiration and enzymes (membrane complexes I, II, III and IV, and dehydrogenases, including alpha-ketoglutaric dehydrogenase (KGDH), pyruvate dehydrogenase (PDH), malate dehydrogenase (MDH)) in isolated rat liver mitochondria. MDA showed a dose-dependent inhibition on mitochondrial NADH-linked respiratory control ratio (RCR) and ADP/O ratio declined from the concentrations of 0.2 and 0.8 micromol/mg protein, respectively, and succinate-linked mitochondrial RCR and ADP/O ratio declined from 1.6 and 0.8 micromol/mg protein. MDA also showed dose-dependent inhibition on the activity of PDH, KGDH and MDH significantly from 0.1, 0.2 and 2 micromol/mg protein, respectively. Activity of the complexes I and II was depressed by MDA at 0.8 and 1.6 micromol/mg protein. However, MDA did not affect activity of complexes III and IV in the concentration range studied (0-6.4 micromol/mg protein). These results suggest that MDA can cause mitochondrial dysfunction by inhibiting mitochondrial respiration and enzyme activity, and the sensitivity of the enzymes examined to MDA is in the order of PDH>KGDH>complexes I and II>MDH>complexes III and IV.     

Closure of VDAC causes oxidative stress and accelerates the Ca-induced mitochondrial permeability transition in rat liver mitochondria

The electron transport chain of mitochondria is a major source of reactive oxygen species (ROS), which play a critical role in augmenting the Ca²⁺-induced mitochondrial permeability transition (MPT). Mitochondrial release of superoxide anions (O₂⁻) from the intermembrane space (IMS) to the cytosol is mediated by voltage dependent anion channels (VDAC) in the outer membrane. Here, we examined whether closure of VDAC increases intramitochondrial oxidative stress by blocking efflux of O₂⁻ from the IMS and sensitizing to the Ca²⁺-induced MPT. Treatment of isolated rat liver mitochondria with 5 μM G3139, an 18-mer phosphorothioate blocker of VDAC, accelerated onset of the MPT by 6.8 ± 1.4 min within a range of 100-250 μM Ca²⁺. G3139-mediated acceleration of the MPT was reversed by 20 μM butylated hydroxytoluene, a water soluble antioxidant. Pre-treatment of mitochondria with G3139 also increased accumulation of O₂⁻ in mitochondria, as monitored by dihydroethidium fluorescence, and permeabilization of the mitochondrial outer membrane with digitonin reversed the effect of G3139 on O₂⁻ accumulation. Mathematical modeling of generation and turnover of O₂⁻ within the IMS indicated that closure of VDAC produces a 1.55-fold increase in the steady-state level of mitochondrial O₂⁻. In conclusion, closure of VDAC appears to impede the efflux of superoxide anions from the IMS, resulting in an increased steady-state level of O₂⁻, which causes an internal oxidative stress and sensitizes mitochondria toward the Ca²⁺-induced MPT.