Studies on vinblastine-induced autophagocytosis in mouse liver

Summary The origin and the structure of the limiting membranes of autophagic vacuoles (AV) in mouse hepatocytes was studied using cytochemical techniques. Autophagocytosis was induced by an intraperitoneal injection of viblastine (50 mg/kg). Imidazole-buffered osmium tetroxide impregnation was used as a marker for unsaturated fatty acids, and uranyl-lead-copper impregnation for the, determination of possible connections of AV membranes with the other cellular membranes.AV membranes stained strongly with both techniques. The staining pattern of AV membranes differed from that of the other cellular membranes. AV's were frequently seen to fuse with vesicles containing very low density lipoprotein particles. No other connections of AV membranes with other cellular membranes were observed. The results suggest that if pre-existing cellular membranes are used in AV formation some kind of transformation must occur in these membranes during AV formation. The content of unsaturated fatty acids appears to be high in AV membranes.     

Oleate stimulation of diacylglycerol formation from phosphatidylcholine through effects on phospholipase D and phosphatidate phosphohydrolase.

Hydrolysis of exogenous phosphatidylcholine (PtdCho) to 1,2-diacylglycerol by rat liver plasma membranes was stimulated by oleate concentrations as low as 0.1 mM. In the presence of 75 mM ethanol, the fatty acid also enhanced phosphatidylethanol (PtdEtOH) formation from PtdCho. These effects were also observed with linoleate and arachidonate, but not with saturated fatty acids or detergents, and were minimal in microsomes or mitochondria. Release of [3H]choline from exogenous Ptd[3H]Cho was stimulated by oleate, whereas phosphoryl[3H]choline formation was inhibited. Oleate and other unsaturated, but not saturated, fatty acids also stimulated the conversion of exogenous [14C]phosphatidic acid to [14C]diacylglycerol. These data are consistent with stimulatory effects of these fatty acids on both phospholipase D and phosphatidate phosphohydrolase in liver plasma membranes. The stimulatory effect of guanosine 5'-O-[3-thio]triphosphate) (20 microM) on PtdEtOH and diacylglycerol formation from PtdCho was enhanced by low concentrations of oleate. Phospholipase A2 also stimulated PtdEtOH and diacylglycerol formation from exogenous PtdCho. It is proposed that unsaturated fatty acids may play a physiological role in the regulation of diacylglycerol production through activation of phospholipase D and phosphatidate phosphohydrolase.     

Increased levels of a particular phosphatidylcholine species in senescent human dermal fibroblasts in vitro

Plasma membranes are essential components of living cells, and phospholipids are major components of cellular membranes. Here, we used liquid chromatography/mass spectrometry to investigate changes in the membrane phospholipid content that occur in association with aging. Our results indicate that the levels of a particular species of phosphatidylcholine comprised of stearic acid and arachidonic acid increased with age. To determine the reason for the increased levels of this particular phosphatidylcholine, we examined the effect of highly unsaturated fatty acids, such as arachidonic acid and eicosapentaenoic acid, on cellular aging. Applied arachidonic acid was incorporated into phosphatidylcholine molecules, but neither arachidonic acid nor other related unsaturated fatty acids had any effect. We conclude that increased levels of this distinctive phosphatidylcholine are a result of in vitro senescence.     

Cytochemical studies on induced autophagocytosis in mouse exocrine pancreas

1. The origin of the limiting membranes of autophagic vacuoles (AVs) in mouse pancreatic acinar cells was studied in vinblastine-induced autophagocytosis. 2. The marker enzymes used were adenosine triphosphatase, lipase, inosine diphosphatase and thiamine pyrophosphatase. The following impregnation techniques were used: unbuffered osmium tetroxide impregnation, imidazole-buffered osmium tetroxide impregnation and uranyl-lead-copper impregnation. 3. Only a weak lipase activity was observed between the limiting membranes of a few AVs. The AV membranes were stained heavily with all impregnation techniques used. 4. The origin of AV membranes seems to be same in mouse liver and exocrine pancreas in vinblastine-induced autophagocytosis.     

Molecular species of phosphatidylcholine and phosphatidylethanolamine in subcellular membranes from rat liver.

Four subfractions of phosphatidycholine and phosphyatidylethanolamine according to the degree of unsaturation of their fatty acids have been separated from lipid extracts of microsomes, and inner and outer mitochondrial membranes. The predominant species found in the three membranes contained one saturated and one unsaturated fatty acid. In microsomes completely saturated species of both phosphatidylcholine and phosphatideylethanolamine were practically nonexistent. In outer mitochondrial membranes species with two unsaturated fatty acids were absent. In the inner mitochondrial membranes, however, disaturated species and those with two unsaturated fatty acids were found.     

The role of fatty acid unsaturation in minimizing biophysical changes on the structure and local effects of bilayer membranes

Studying the effects of saturated and unsaturated fatty acids on biological and model (liposomes) membranes could provide insight into the contribution of biophysical effects on the cytotoxicity observed with saturated fatty acids. In vitro experiments suggest that unsaturated fatty acids, such as oleate and linoleate, are less toxic, and have less impact on the membrane fluidity. To understand and assess the biophysical changes in the presence of the different fatty acids, we performed computational analyses of model liposomes with palmitate, oleate, and linoleate. The computational results indicate that the unsaturated fatty acid chain serves as a membrane stabilizer by preventing changes to the membrane fluidity. Based on a Voronoi tessellation analysis, unsaturated fatty acids have structural properties that can reduce the lipid ordering within the model membranes. In addition, hydrogen bond analysis indicates a more uniform level of membrane hydration in the presence of oleate and linoleate as compared to palmitate. Altogether, these observations from the computational studies provide a possible mechanism by which unsaturated fatty acids minimize biophysical changes and protect the cellular membrane and structure. To corroborate our findings, we also performed a liposomal leakage study to assess how the different fatty acids alter the membrane integrity of liposomes. This showed that palmitate, a saturated fatty acid, caused greater destabilization of liposomes (more “leaky”) than oleate, an unsaturated fatty acid.     

Lipid Metabolism of Manganese-deficient Algae: I. Effect of Manganese Deficiency on the Greening and the Lipid Composition of Euglena Gracilis Z 1

The growth of photoautotrophic Euglena gracilis Z is strongly inhibited by manganese deficiency, whereas chlorophyll formation is not appreciably affected. The galactosyldiglyceride content of the manganese-deficient photo-autotrophic Euglena was about 40% lower on the basis of either chlorophyll content or dry weight. When dark-grown cultures of Euglena were grown photoheterotrophically in light sufficient for the greening of the cells, or photosynthesis, manganese deficiency resulted in a reduction of the cellular content of chlorophyll and galactosyldiglycerides to 40% of control values, indicating interference with chloroplast formation. The fatty acids of the photoheterotrophic manganese-deficient cells were mainly saturated, with an unusual accumulation (about 45%) of the total fatty acids) of myristic acid. In spite of this, the galactosyldiglycerides contain mainly unsaturated fatty acids. Ninety per cent of the fatty acids of the monogalactosyldiglyceride are unsaturated, including large amounts of alpha-linolenic acid. The ratio of chlorophyll to galactosyldiglyceride content of the cells was remarkably constant at all manganese deficiency levels.     

Oleate and linoleate stimulate degradation of β-casein in prolactin-treated HC11 mouse mammary epithelial cells

Although virtually all cells store neutral lipids as cytoplasmic lipid droplets, mammary epithelial cells have developed a specialized function to secrete them as milk fat globules. We have used the mammary epithelial cell line HC11 to evaluate the potential connections between the lipid and protein synthetic pathways. We show that unsaturated fatty acids induce a pronounced proliferation of cytoplasmic lipid droplets and stimulate the synthesis of adipose differentiation-related protein. Unexpectedly, the cellular level of β-casein, accumulated under lactogenic hormone treatment, decreases following treatment of the cells with unsaturated fatty acids. In contrast, saturated fatty acids have no significant effect on either cytoplasmic lipid droplet proliferation or cellular β-casein levels. We demonstrate that the action of unsaturated fatty acids on the level of β-casein is post-translational and requires protein synthesis. We have also observed that proteasome inhibitors potentiate β-casein degradation, indicating that proteasomal activity can destroy some cytosolic protein(s) involved in the process that negatively controls β-casein levels. Finally, lysosome inhibitors block the effect of unsaturated fatty acids on the cellular level of β-casein. Our data thus suggest that the degradation of β-casein occurs via the microautophagic pathway.     

The impairment of essential fatty acid metabolism as a key factor in doxorubicin-induced damage in cultured rat cardiomyocytes.

The clinical use of the antitumoral doxorubicin (DOX) is limited by its cardiotoxicity, which is mediated through different mechanisms. The membrane lipid peroxidation induced by DOX may cause disruption of the unsaturated fatty acyl chains; in the endoplasmic reticulum, containing the system catalyzing the desaturation/elongation of fatty acids, DOX could interfere with the metabolism of linoleic and alpha-linolenic acids. Using primary cultures of neonatal rat cardiomyocytes we demonstrated that the exposure to different concentrations of DOX (10(-5) and 10(-7) M) for 24 h caused an increase in the production of conjugated dienes, an impairment in the desaturation/elongation of essential fatty acids, and a reduction in the cellular content of highly unsaturated fatty acids. Conversely, 1 h exposure to 10(-5) M DOX was sufficient to induce alterations in the desaturation/elongation of linoleic and alpha-linolenic acids, but did not cause either formation of conjugated dienes or modification of the fatty acyl pattern. Therefore, DOX has a dual negative effect, depending on its concentration and on the time of exposure, one directed against the membrane highly unsaturated fatty acids, the other against the system which is required for the synthesis of these fatty acids themselves. These two effects synergically act in causing heart cell damage.     

Effects of exogenous fatty acids on calcium uptake by brush-border membrane vesicles from rabbit small intestine

The uptake of a variety of fatty acids by isolated brush-border membranes from rabbit small intestine was studied. This uptake increased with acyl chain-length and was not diminished by washing of the lipid-treated membranes with 0.25 M CsBr. The binding of fatty acid was not accompanied by a decrease in endogenous acyl groups or of cholesterol and therefore corresponded to a net uptake accountable qualitatively and quantitatively by the fatty acid added to the membranes. The uptake of Ca2+ was stimulated by treatment of the membranes with low concentrations of unsaturated fatty acids (0.05 mM) as well as with various concentrations of caprylic acid (0.10-3.00 mM) and inhibited by treatment with higher concentrations of unsaturated fatty acids (0.20-0.60 mM). Saturated fatty acids had no marked effects on Ca2+ uptake. The stimulatory concentrations of unsaturated fatty acids did not change the Ca2+-binding characteristics of the membranes, whereas the higher concentrations decreased equilibrium binding of Ca2+ and very probably the number of high-affinity binding sites. The results of this study are assessed in terms of the effects of normal fatty acids found in the diet on the absorptive properties of the brush-border membranes.     

Effect of calcium ion on fatty acid-induced generation of superoxide in guinea pig neutrophils

When phospholipases of plasma membranes are activated by certain stimuli, unsaturated fatty acids are liberated. Because unsaturated fatty acids enhance the transmembrane movement of calcium ions, the fatty acids released may modulate intracellular calcium homeostasis in various cells, including neutrophils. To determine the physiological function of these unsaturated fatty acids, we studied the effects of various fatty acids on superoxide generation and on changes in intracellular calcium contents of guinea pig neutrophils. Some unsaturated fatty acids, arachidonate and linoleate, stimulated the rate of superoxide generation concomitant with the increase in the amount of intracellular calcium. In contrast, the saturated fatty acid, myristate, stimulated the generation of superoxide without affecting the content of intracellular calcium. The stimulating actions of arachidonate and myristate were increased dramatically by the presence of a low concentration (1 microM) of extracellular calcium ion. The rate of superoxide generation in fatty acid-treated neutrophils was inhibited by chlorpromazine, an inhibitor of such calcium-binding proteins as C-kinase. These and other observations suggest that liberated unsaturated fatty acids increase the amount of intracellular calcium and enhance C-kinase activity also that the increased activity of the enzyme is involved in the chain of events leading to the stimulation of superoxide generation in fatty acid-treated neutrophils.     

The relationship between plasma membrane lipid composition and physical-chemical properties. I. Fluorescence polarization studies of fatty acid-altered EL4 tumor cell membranes

EL4 cells were cultured with exogenous fatty acids under conditions that resulted in their incorporation into membrane phospholipids. The behavior of the fluorescent lipid probes diphenylhexatriene and perylene was monitored in intact EL4 cells and in isolated EL4 plasma membranes. In whole cells substituted with unsaturated fatty acids, there was always a marked decrease in the $\text{P}$ value of both probes compared to the $\text{P}$ value of the probes in unsubstituted cells. In whole cells substituted with saturated fatty acids, on the other hand, $\text{P}$ values for both probes were unchanged compared to unsubstituted cells. In plasma membrane isolated from EL4 cells, no difference in $\text{P}$ values for either probe was observed among membranes from unsubstituted, saturated fatty acid substituted or unsaturated fatty acid substituted cells, even when the degree of fatty acid substitution was quite substantial. Most of the fluorescent signal for both probes in whole cells appeared to come from cytoplasmic lipid droplets. The value of techniques such as fluorescent polarization for monitoring physical properties of membranes (such as ‘fluidity’) is discussed.     

The manipulation of cellular cytochrome and lipid composition in a haem mutant of Saccharomyces cerevisiae.

1. The ole-3 mutant of Saccharomyces cerevisiae has an early lesion in the pathway of porphyrin biosynthesis. 2. This results in the loss of all haem-containing enzymes, including the mitochondrial cytochromes, and prevents the synthesis of components whose formation requires haem-containing enzymes, including unsaturated fatty acids, ergosterol and methionine. 3. The pleiotropic effects of the primary lesion are reversed by growing mutant ole-3 aerobically in the presence of intermediates of the porphyrin-biosynthetic pathway, and the present work reports the degree of manipulation of lipid and respiratory-cytochrome composition. 4. Supplements of delta-aminolaevulinate in the range 0.5--500 mg/l result in a progressive increase in the cellular content of unsaturated fatty acids and respiratory cytochromes, cause the replacement of lanosterol and squalene by ergosterol, and an increase in total sterol content. 5. Haematoporphyrin and protoporphyrin IX have similar but less extensive effects on cellular composition, whereas haematin allows unsaturated fatty acid synthesis and some sterol synthesis, but has no effect on the formation of respiratory cytochromes. 6. These results suggest that growth of the organism in the presence of defined amounts of delta-aminolaevulinate will be useful in the investigation of the role of lipids and cytochromes in the function and assembly of mitochondrial membranes.     

The role of membrane fatty acids in mammalian hibernation

During mammalian hibernation, cellular membranes continue to function at temperatures approaching 0 C. The molecular mechanisms that confer this capacity to the membranes are unknown but may be related to the fluidity of the membrane and to the level of unsaturated fatty acids. The basic tenets of membrane fluidity and the contribution of cholesterol, polar head groups, and fatty acids toward maintaining a fluid membrane in a liquid-crystalline state are examined in this review. It is shown that although unsaturated fatty acids can enhance membrane fluidity at low temperatures, there does not appear to be a consistent trend toward increased levels of unsatruated fatty acids during hibernation in all tissues of hibernators. Consequently, there may be some other role for the alterations in the composition of membrane fatty acids found during the hibernating cycle other than increasing membrane fluidity to permit continued activity at reduced temperatures.     

A cytochemical study on the effects of energy deprivation on autophagocytosis in Ehrlich ascites tumor cells

The effect of energy deprivation on autophagocytosis in Ehrlich ascites tumor cells was studied using cytochemical techniques. Autophagocytosis was induced with vinblastine incubation (0.1 mM) and the cellular ATP-level was lowered with 2-deoxy-D-glucose (0.35 mM). Acid phosphatase was used as a marker for lysosomal enzymes and imidazole-buffered osmium tetroxide impregnation in order to study the effects of energy deprivation on the maturation of autophagic vacuole (AV) membranes. Control and vinblastine treated cells maintained their ATP-levels throughout the incubation period tested (120 min). 2-Deoxy-D-glucose alone and with vinblastine decreased the intracellular ATP-level significantly after only 3 min incubation. Most of the AV's in control and vinblastine treated cells contained degraded material and acid phosphatase activity. Their membranes were stained only slightly or not at all with imidazole-buffered osmium tetroxide. 2-Deoxy-D-glucose alone as well as with vinblastine induced in particular an accumulation of early stages of AV's. These vacuoles contained undegraded cytoplasmic material and no acid phosphatase activity and their membranes were stained usually partly with imidazole-buffered osmium tetroxide. The membranes of some early AV's resembled endoplasmic reticulum and still had attached ribosomes. It was concluded that the inhibition of cellular energy production used in the present study did not inhibit autophagic sequestration but retarded the maturation of AV membranes and impaired the functioning of lysosomal hydrolases.     

Microsomal preparations from plant and yeast acylate free fatty acids without prior activation to acyl-thioesters

Acylation of fatty acids to hydroxy groups in cells generally require activation to a thioester (ACP or CoA) or transacylation from another oxygen ester. We now show that microsomal membranes from Arabidopsis leaves efficiently acylate free fatty acids to long chain alcohols with no activation of the fatty acids to thioesters prior to acylation. Studies of the fatty alcohol and fatty acids specificities of the reaction in membranes from Arabidopsis leaves revealed that long chain (C18-C24) unsaturated fatty alcohols and C18-C22 unsaturated fatty acids were preferred. Microsomal preparations from Arabidopsis roots and leaves and from yeast efficiently synthesized ethyl esters from ethanol and free fatty acids. This reaction also occurred without prior activation of the fatty acid to a thioester. The results presented strongly suggest that wax ester and ethyl ester formation are carried out by separate enzymes. The physiological significance of the reactions in plants is discussed in connection to suberin and cutin synthesis. The results also have implication regarding the interpretation of lipid metabolic experiments done with microsomal fraction.     

Inhibition of gastric (H+ + K+)-ATPase by unsaturated long-chain fatty acids

Arachidonic acid and unsaturated C18 fatty acids at concentrations near 10(-5) M markedly inhibited (H+ + K+)-ATPase in hog or rat gastric membranes. Arachidonic acid was a more potent inhibitor than unsaturated C18 fatty acids, but the involvement of the metabolites of arachidonic acid cascade was ruled out. Linolenic acid inhibited the formation of phosphoenzyme and the K+ -dependent p-nitrophenylphosphatase activity of the hog ATPase. Treatment with fatty acid-free bovine serum albumin abolished only the inhibitory effect of the fatty acid on the phosphatase activity without restoring the overall ATPase action. These data suggest the existence of at least two groups of hydrophobic binding sites in the gastric ATPase for unsaturated long-chain fatty acids which affect differentially the catalytic reactions of the ATPase. (H+ + K+)-ATPase in rat gastric membranes was found more susceptible to the fatty acid inhibition and also more unstable than the ATPase in hog gastric membranes. The presence of a millimolar level of lanthanum chloride or ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid stabilized the rat ATPase probably via the inhibition of Ca2+ -dependent phospholipases in the gastric membranes.     

Comparative study of the lipid composition of rabbit and lobster sarcoplasmic reticulum.

Sarcoplasmic reticulum (SR) membranes isolated from rabbit and lobster muscles have similar phospholipid classes, but they differ in plasmalogen content. The plasmalogenic species are mostly distributed among phosphatidylethanolamines (PE's) and make up about 62% of the total in rabbit SR and about 46% in lobster membranes. Lobster SR phospholipids contain large amounts of polyunsaturated fatty acids which are present in low amounts in rabbit membranes. The total unsaturated fatty acids of phosphatidylcholines (PC's) represent about 53% and 73% of the total fatty chains for rabbit and lobster SR, respectively. The values found for PE's were about 56% and 64%, respectively. Furthermore, lobster membranes contain significant amounts of PC and PE molecular species with unsaturated fatty acids in positions 1 and 2, whereas rabbit SR contain low amounts.     

Influence of eicosapentaenoic acid (20:5, n-3) on secretion of lipoproteins in CaCo-2 cells.

CaCo-2 cells, grown on filter membranes, were used to study the effects of fatty acids on cellular metabolism of triacylglycerol and phospholipids. The rate of triacylglycerol secretion was enhanced more than 2-fold, from 1 to 2 weeks after reaching confluency, in the presence of 0.6 mM fatty acids. Triacylglycerol secretion and oxidation of oleic acid increased 2- and 9-fold, respectively, with this culture system, as compared to cells grown on conventional plastic dishes. Eicosapentaenoic acid (20:5 n-3), when compared to oleic acid, did not reduce formation of triacylglycerol or enhance phospholipid synthesis in CaCo-2 cells during short term (less than 24 h) experiments, when the cells resided on membranes, regardless of what type of radioisotopes were used as precursors in the incubation media. However, the n-3 fatty acid was preferentially incorporated into phosphatidylinositol, lysophosphatidylcholine, and sphingomyelin, as compared to oleic acid. The disappearance from the apical medium and cellular uptake of labeled eicosapentaenoic and oleic acid were similar during incubations up to 24 h, and the metabolism of these fatty acids to acid-soluble materials and CO2 was equal. Light scattering analysis indicated that secreted lipoproteins of density less than 1.006 g/ml were in the same size-range as chylomicrons derived from human plasma. Assessment of secreted apolipoprotein B showed that by incubating CaCo-2 cells with oleic acid, apolipoprotein B levels increased approximately 1.4-fold when compared to cells incubated with eicosapentaenoic acid, whereas the amount of triacylglycerol and size-range of particles were similar for the two fatty acids. Our data indicate that CaCo-2 cells grown on filter membranes exhibit enterocyte-like characteristics with the ability to synthesize and secrete chylomicrons. Eicosapentaenoic acid and oleic acid are absorbed, metabolized, and influence secretion of lipoprotein particles in a similar way, except for some differences in incorporation of the fatty acids into certain phospholipid classes and a reduced secretion of apolipoprotein B. The culture conditions, including time after confluency and cellular support, are critical for the rate of secretion in the presence of eicosapentaenoic acid and oleic acid.     

The influence of fatty acids on model cholesterol/phospholipid membranes

The aim of this work was to verify the influence of the saturated (SFA) (stearic acid) and the unsaturated (UFA) (oleic and alpha-linolenic) fatty acids on model cholesterol/phospholipid membranes. The experiments were based on the Langmuir monolayer technique. Cholesterol and phospholipid were mixed in the molar ratio that corresponds to the proportion of these lipids in the majority of natural human membranes. Into the binary cholesterol/phospholipid monolayers, various amounts of fatty acids were incorporated. Our investigations were based on the analysis of the interactions between molecules in ternary (cholesterol/phospholipids/fatty acid) mixtures, however, also binary (cholesterol/fatty acid and phospholipids/fatty acid) mixed system were examined. It was concluded that the influence of the fatty acids on model cholesterol/phospholipid membrane is closely connected with the shape of the fatty acid molecule, resulting from the saturation degree of the hydrocarbon chain. It was found that the saturated fatty acid makes the model membrane more rigid, while the presence of unsaturated fatty acid increases its fluidity. The increasing amount of stearic acid gradually destabilizes model membrane, however, this effect is the weakest at low content of SFA in the mixed monolayer. Unsaturated fatty acids in a small proportion make the membrane thermodynamically more stable, while higher content of UFA decreases membrane stability. This explains low proportion of the free fatty acids to other lipids in natural membrane.