Activation of the calcium permeability of erythrocyte membranes by perfringolysin O

Calcium ions inhibited perfringolysin O-induced hemolysis at a concentration lower than 1 mM, but not the hemolysis by digitonin at 10 mM. The introduction of calcium ions into ghosts inhibited the lysis more strongly than the addition of calcium ions outside ghosts. When erythrocytes were treated with perfringolysin O in the presence of 1 mM CaCl2 containing 45CaCl2, the radioactivities inside cells rapidly increased during incubation. On the other hand, when perfringolysin O-treated erythrocytes were incubated in a calcium-free medium, the erythrocytes released calcium ions at a 3.3-fold higher rate than untreated cells. These results suggested that perfringolysin O accelerated both the calcium influx into and efflux from erythrocytes.     

Fatty acid and glycerol labeling of glycerolipids of leukocytes in response to ionophore A23187.

The effect of divalent cation ionophore, A23187, on the incorporation of [1-14C]palmitic acid, [1-14C]linoleic acid and [U-14C]glycerol into glycerolipids of polymorphonulcear leukocytes was examined. Ionophore A23187 stimulated the labeling of phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, and diacylglycerol by both labeled fatty acids and glycerol. [1-14C]Palmitic acid and [1-14C]linoleic acid incorporation into phosphatidylcholine and triacylglycerol was reduced by the presence of the ionophore in the incubation medium, while [U-14C]glycerol labeling of these lipids was not significantly changed under identical conditions. These data reflect that the acylation of sn-glycerol 3-phosphate is activated, and the acylations of lysophosphatidyl-choline and endogenous diacylglycerol are inhibited in cells incubated with ionophore A23187. External calcium was not required for the ionophore effect on the incorporation of labeled fatty acids and glycerol. It is suggested that the ionophore alters the metabolism of the fatty acid and glycerol moieties of glycerolipids by changing the distribution of intracellular calcium of leukocytes.     

Perfringolysin O, a cholesterol-binding cytolysin, as a probe for lipid rafts

Gaining an understanding of the structural and functional roles of cholesterol in membrane lipid rafts is a critical issue in studies on cellular signaling and because of the possible involvement of lipid rafts in various diseases. We have focused on the potential of perfringolysin O (theta-toxin), a cholesterol-binding cytolysin produced by Clostridium perfringens, as a probe for studies on membrane cholesterol. We prepared a protease-nicked and biotinylated derivative of perfringolysin O (BCtheta) that binds selectively to cholesterol in cholesterol-rich microdomains of cell membranes without causing membrane lesions. Since the domains fulfill the criteria of lipid rafts, BCtheta can be used to detect cholesterol-rich lipid rafts. This is in marked contrast to filipin, another cholesterol-binding reagent, which binds indiscriminately to cell cholesterol. Using BCtheta, we are now searching for molecules that localize specifically in cholesterol-rich lipid rafts. Recently, we demonstrated that the C-terminal domain of perfringolysin O, domain 4 (D4), possesses the same binding characteristics as BCtheta. BIAcore analysis showed that D4 binds specifically to cholesterol with the same binding affinity as the full-size toxin. Cell-bound D4 is recovered predominantly from detergent-insoluble, low-density membrane fractions where raft markers, such as cholesterol, flotillin and Src family kinases, are enriched, indicating that D4 also binds selectively to lipid rafts. Furthermore, a green fluorescent protein-D4 fusion protein (GFP-D4) was revealed to be useful for real-time monitoring of cholesterol in lipid rafts in the plasma membrane. In addition, the expression of GFP-D4 in the cytoplasm might allow the investigations of intracellular trafficking of lipid rafts. The simultaneous visualization of lipid rafts in plasma membranes and inside cells might help in gaining a total understanding of the dynamic behavior of lipid rafts.     

Epidermal growth factor alters metabolism of inositol lipids and activity of protein kinase C in mouse embryo palate mesenchyme cells

Epidermal growth factor (EGF) stimulated mouse embryo palate mesenchyme (MEPM) cells (1) to incorporate [32P]O4(3-) into phosphatidylinositol (PI), phosphatidylcholine, and phosphatidic acid over a period of 60 min; 2) to incorporate [32P]O4(3-) into polyphosphoinositides as a function of time; and 3) to incorporate [32P]O4(-3) into PI, only, as a function of concentration when the period of stimulation was kept short. EGF stimulated the release of radiolabeled inositol phosphates from MEPM cells that had been radiolabeled with [3H]myoinositol. The release of inositol 1-phosphate was sustained over a period of at least 60 min, whereas the release of inositol 1,4-bisphosphate and inositol trisphosphate peaked during the first 10 min of stimulation. EGF also stimulated phosphorylation of an Mr 80,000 protein whose pI, phosphopeptide map, and phosphoamino acid pattern were identical to those of an Mr 80,000 protein phosphorylated in response to phorbol 12-myristate 13-acetate. Mobilization or metabolism of arachidonic acid was not stimulated under the same conditions that permitted EGF to alter inositol lipid metabolism. We interpret these data to mean that 1) in contrast to the findings with some cell lines, alterations in inositol lipid metabolism may be part of the signalling mechanism for EGF in embryonic cells; 2) EGF is capable of activating inositol-dependent signalling pathways leading to activation of protein kinase C in MEPM cells; and 3) mobilization and metabolism of arachidonic acid are not an inherent part of this signalling mechanism.     

Nerve Growth Factor Stimulation of Arachidonic Acid Release from PC12 Cells: Independence from Phosphoinositide Turnover

The effect of nerve growth factor on the metabolism of arachidonic acid and the hydrolysis of phosphatidylinositol in PC12 cells was examined. Addition of nerve growth factor to PC12 cells isotopically labeled with [3H]arachidonic acid caused an increased release of radioactivity. In a similar manner, treatment of PC12 cells prelabeled with [3H]inositol increased inositol monophosphate accumulation in the presence of LiCl. Stimulation of [3H]arachidonic acid release by nerve growth factor was concentration dependent, attaining a maximum at 0.5 nM. Concentrations of nerve growth factor above 0.5 nM caused less than maximal stimulation. In contrast, nerve growth factor-stimulated accumulation of [3H]inositol monophosphate exhibited a sigmoidal dose-response curve with an apparent maximum at 8 nM. Increased accumulation of [3H]inositol monophosphate could be detected as early as 60 s after nerve growth factor addition, whereas nerve growth factor-stimulated release of [3H]arachidonic acid was not observed until 5 min after nerve growth factor treatment. The nerve growth factor-stimulated release of [3H]arachidonic acid was independent of extracellular calcium concentration. Increased [3H]inositol monophosphate accumulation elicited by nerve growth factor was dependent on the presence of extracellular calcium. These results suggest that the increased metabolism of arachidonic acid and the enhanced hydrolysis of phosphatidylinositol are separately regulated by nerve growth factor.     

Effect of TNF on triacylglycerol in cultured vascular smooth muscle cells

Smooth muscle cells (SMC) isolated from bovine aorta or human saphenous vein were cultured and used to study the putative effect of recombinant human tumor necrosis factor (TNF) on lipid metabolism in vascular cells. Addition of TNF to the culture medium for 24-48 h resulted in an increase of [3H]oleic acid uptake and esterification into lipids. The effect could be seen already with 0.3 ng/ml and was maximal with 30 ng/ml. The effect of TNF was mainly on the incorporation of [3H]oleic acid into triacylglycerol which increased by 140% in the bovine cells. There was also a significant increase in [3H]cholesteryl ester. In the human SMC there was a 40% increase in [3H]oleic acid into total lipids, while the rise in [3H]triacylglycerol ranged between 60-90%. TNF did not modulate cellular triacyglycerol synthesis in cultured mouse peritoneal macrophages. Since TNF was shown to be synthesized and secreted not only by macrophages but also by smooth muscle cells, it could play an autocrine role in lipid metabolism during development of atherosclerotic lesions. The cellular population of the lesions, i.e., predominance of macrophages or smooth muscle cells, could determine the relative proportion of triacylglycerol accumulation.     

Effects of unsaturated fatty acid deprivation on neutral lipid synthesis in Saccharomyces cerevisiae

The effects of unsaturated fatty acid deprivation on lipid synthesis in Saccharomyces cerevisiae strain GL7 were determined by following the incorporation of [14C]acetate. Compared to yeast cells grown with oleic acid, unsaturated fatty acid-deprived cells contained 200 times as much 14C label in squalene, with correspondingly less label in 2,3-oxidosqualene and 2,3;22,23-dioxidosqualene. Cells deprived of either methionine or cholesterol did not accumulate squalene, demonstrating that the effect of unsaturated fatty acid starvation on squalene oxidation was not due to an inhibition of cell growth. Cells deprived of olefinic supplements displayed additional changes in lipid metabolism: (i) an increase in 14C-labeled diacylglycerides, (ii) a decrease in 14C-labeled triacylglycerides, and (iii) increased levels of 14C-labeled decanoic and dodecanoic fatty acids. The changes in squalene oxidation and acylglyceride metabolism in unsaturated fatty acid-deprived cells were readily reversed by adding oleic acid. Pulse-chase studies demonstrated that the [14C]squalene and 14C-labeled diacylglycerides which accumulated during starvation were further metabolized when cells were resupplemented with oleic acid. These results demonstrate that unsaturated fatty acids are essential for normal lipid metabolism in yeasts.     

Tissue-specific effects of rapid tumour growth on lipid metabolism in the rat during lactation and on litter removal.

1. The effect of tumour burden on lipid metabolism was examined in virgin, lactating and litter-removed rats. 2. No differences in food intake or plasma insulin concentrations were observed between control animals and those bearing the Walker-256 carcinoma (3-5% of body wt.) in any group studied. 3. In virgin tumour-bearing animals, there was a significant increase in liver mass, blood glucose and lactate, and plasma triacylglycerol; the rate of oxidation of oral [14C]lipid to 14CO2 was diminished, and parametrial white adipose tissue accumulated less [14C]lipid compared with pair-fed controls. 4. These findings were accompanied by increased accumulation of lipid in plasma and decreased white-adipose-tissue lipoprotein lipase activity. 5. In lactating animals, tumour burden had little effect on the accompanying hyperphagia or on pup weight gain; tissue lipogenesis was unaffected, as was tissue [14C]lipid accumulation, plasma [triacylglycerol] and white-adipose-tissue and mammary-gland lipoprotein lipase activity. 6. On removal (24 h) of the litter, the presence of the tumour resulted in decreased rates of lipogenesis in the carcass, liver and white and brown adipose tissue, decreased [14C]lipid accumulation in white adipose tissue, but increased accumulation in plasma and liver, increased plasma [triacylglycerol] and decreased lipoprotein lipase activity in white adipose tissue. 7. The rate of triacylglycerol/fatty acid substrate cycling was significantly decreased in white adipose tissue of virgin and litter-removed rats bearing the tumour, but not in lactating animals. 8. These results demonstrate no functional impairment of lactation, despite the presence of tumour, and the relative resistance of the lactating mammary gland to the disturbance of lipid metabolism that occurs in white adipose tissue of non-lactating rats with tumour burden.     

Effect of perfringolysin O on the lateral diffusion constant of membrane proteins of hepatocytes as revealed by fluorescence recovery after photobleaching

Perfringolysin O is a thiol-activated cytolytic exotoxin the primary receptor of which is the membrane cholesterol on the cell surface. The effect of perfringolysin O was tested in various hepatocyte preparations. (i) Smears of fresh liver exposed to a mild H2O2 (1.0 mM) injury for 10 min at 37 degrees C, develop a 'peroxide-induced autofluorescence' (PIAF) on the membrane proteins. PIAF is suitable for measuring the average lateral diffusion constant (D) of the membrane proteins by means of fluorescence recovery after photobleaching technique (FRAP). Incubation for 5 min with 600 or 2000 units/ml of the perfringolysin O resulted in a significant increase (32 and 46%, respectively) of D as compared to the controls of the same age group (13-14 months). Various tests like heat denaturation of cholesterol saturation of perfringolysin O before its application as well as thiol-activation of the smears with dithiothreitol revealed that the increase of D is a specific toxin effect due mot probably to the reaction of perfringolysin O with cholesterol. (ii) Isolated hepatocytes were exposed to perfringolysin O and their viability as well as the release of two cytosolic enzymes (lactate dehydrogenase and glutamic-pyruvic transaminase) were measured; 40-60 units/ml of perfringolysin O in 30 min reduced the viability of the hepatocytes to zero and caused a release of about 70% of both cytosolic enzymes. The significance of the results is discussed from the points of view of both the toxin-effect and the FRAP method.     

Copper(II) complexes of a nonsteroidal anti-inflammatory drug niflumic acid. Synthesis, crystal structure of tetrakis-mu-(2-[3-(trifluoromethyl) phenyl]aminonicotinato)bis(dimethylsulfoxide)-dicopper(II) complex at 190 K. Anti-inflammatory properties.

The synthesis and characterization of three complexes with a potent nonsteroidal anti-inflammatory drug niflumic acid {2-[3-(trifluoromethyl)phenyl]aminonicotinic acid} with formula [Cu(niflumato)2L] (L = H2O, DMSO = dimethylsulfoxide, DMF = N,N-dimethylformamide) were investigated. The crystal and molecular structure of the {Cu(niflumato)2(DMSO)}2 was reported. Crystallographic data are as follows: monoclinic system, space group P2(1)/n, Z = 2, a = 11.1318(8), b = 17.513(2), c = 15.336(1) A, beta = 103.316(8) degrees, V = 2909.4(4) A3. The structure was refined to R = 0.030 and wR = 0.037 for 3702 reflections with I > sigma (I). It consists of centrosymmetric binuclear units with the Cu-Cui (symmetry code i: 1-x, -y, 1-z) distance between two centrosymmetrically related ions of 2.6272(5) A. Each Cu(II) ion in [Cu2(DMSO)2(mu-niflumato)4] is coordinated to an apical dimethylsulfoxide O atom on the one hand and to the equatorial carbonyl and carboxylic O atoms of two crystallographically independent niflumate moieties and their centrosymmetric counterparts on the other hand. In spite of the low-temperature (190 K) crystal measurements, one L-CF3 grouping exhibits some disorder. The biological activities of these complexes were compared to that of niflumic acid. Niflumic acid and its various copper complexes significantly inhibited polymorphonuclear leukocyte (PMNL) oxidative metabolism, as assessed by chemiluminescence and O2- generation measurement. This effect was dose-dependent. All copper complexes exerted a similar inhibiting effect which was always significantly higher than that exerted by the parent drug.     

Lysophosphatidic acid can activate platelets without increasing 32P-labelling of phosphatidic acid

Stimulation of platelets by thrombin produced a rise in [32P]phosphatidic acid labelling of platelets which was greater in medium without added calcium than in medium with 2.5 mM calcium. A rise in [32P]lysophosphatidic acid was also seen in platelets stimulated by thrombin in the presence of 2.5 mM extracellular calcium, though it was of lesser magnitude (average 35%) than the rise in phosphatidic acid. In platelets resuspended without added calcium no change in [32P]lysophosphatidic acid was seen in response to thrombin. Lysophosphatidic acid can itself induce platelet aggregation. Similarly to the calcium ionophore A23187, lysophosphatidic acid produced minimal change (in medium with no added calcium) to no change (in medium with 2.5 mM external calcium) in [32P]lysophosphatidic acid. The endoperoxide analog U46619 produced changes in 32P-labelling of platelet phosphatidic and lysophosphatidic acid similar to those produced by thrombin but of lesser magnitude. The results of these studies show that the action of lysophosphatidic acid on platelets differs from the action of thrombin, U46619 and platelet-activating factor, which produce a rapid rise in [32P]phosphatidic acid, and suggests that lysophosphatidic acid, like A23187, largely bypasses the initial receptor-coupled breakdown of phosphoinositides leading to formation of diacylglycerols and phosphatidic acid.     

Metabolism of palmitate in perfused rat liver. Effect of low and high ethanol concentrations at various concentrations of palmitate in the perfusion medium

1. The effect of ethanol on the metabolism of [1-(14)C]palmitate in rat liver was investigated in a single-pass perfusion system at concentrations of 10mm- or 80mm-ethanol and 0.2mm- or 1mm-palmitate. 2. After the perfusion the hepatic lipid was isolated in subcellular fractions. The two major fractions contained triacylglycerol from cytoplasmic lipid droplets and from endoplasmic reticulum plus Golgi apparatus respectively. 3. In experiments with 0.2mm-palmitate perfusion with 10mm- or 80mm-ethanol did not measurably increase the esterification, and the oxidation was markedly decreased and the fatty acid uptake was not affected. 4. Perfusion with ethanol, at 1mm-palmitate, increased the fatty acid uptake, increased esterification and decreased oxidation. The effects of 10mm- and 80mm-ethanol were similar. The incorporation of [1-(14)C]palmitate into triacylglycerol in cytoplasmic lipid droplets was not affected statistically significantly by ethanol. Ethanol increased the incorporation of [1-(14)C]palmitate into di- and tri-acylglycerol in the membranous fraction. Estimated chemically, the contents of di- and tri-acylglycerol were only slightly affected by ethanol. These results suggest that the effect of ethanol was to increase the turnover of fatty acids in triacylglycerol rather than to increase its accumulation. 5. The results indicate that an increased concentration of fatty acids is more important for the formation of acute fatty liver in fed rats than are the direct effects of ethanol on hepatic fatty acid metabolism.     

Phospholipid synthesis in isolated alveolar type II cells exposed in vitro to paraquat and hyperoxia.

Isolated alveolar epithelial type II cells were exposed to paraquat and to hyperoxia by gas diffusion through the thin Teflon bottom of culture dishes. After exposure, type II cells were further incubated in the presence of labelled substrates to assess their capacity to synthesize lipids. Hyperoxia alone (90% O2; 5 h) had minor effects on lipid metabolism in the type II cells. At low paraquat concentrations (5 and 10 microM), hyperoxia enhanced the paraquat-induced decrease of [Me-14C]choline incorporation into phosphatidylcholines. The incorporation rates of [Me-14C]choline, [1-14C]palmitate, [1-14C]glucose and [1,3-3H]glycerol into various phospholipid classes and neutral lipids were decreased by paraquat, depending on the concentration and duration of the exposure. The incorporation of [1-14C]acetate into phosphatidylcholines, phosphatidylglycerols and neutral lipids appeared to be very sensitive to inactivation by paraquat. At 5 microM-paraquat the rate of [1-14C]acetate incorporation was decreased to 50% of the control values. The rate of [1-14C]palmitate incorporation into lipids was much less sensitive; it even increased at low paraquat concentrations. At 10 microM-paraquat both NADPH and ATP were significantly decreased. It is concluded that lipid synthesis in isolated alveolar type II cells is extremely sensitive to paraquat. At low concentrations of this herbicide, lipid synthesis, and particularly fatty acid synthesis, is decreased. The effects on lipid metabolism may be partly related to altered NADPH and ATP concentrations.     

Perfringolysin O expression in Clostridium perfringens is independent of the upstream pfoR gene

The pathogenesis of Clostridium perfringens-mediated gas gangrene or clostridial myonecrosis involves the extracellular toxins alpha-toxin and perfringolysin O. Previous studies (T. Shimizu, A. Okabe, J. Minami, and H. Hayashi, Infect. Immun. 59:137-142, 1991) carried out with Escherichia coli suggested that the perfringolysin O structural gene, pfoA, was positively regulated by the product of the upstream pfoR gene. In an attempt to confirm this hypothesis in C. perfringens, a pfoR-pfoA deletion mutant was complemented with isogenic pfoA(+) shuttle plasmids that varied only in their ability to encode an intact pfoR gene. No difference in the ability to produce perfringolysin O was observed for C. perfringens strains carrying these plasmids. In addition, chromosomal pfoR mutants were constructed by homologous recombination in C. perfringens. Again no difference in perfringolysin O activity was observed. Since it was not possible to alter perfringolysin O expression by mutation of pfoR, it was concluded that the pfoR gene product is unlikely to have a role in the regulation of pfoA expression in C. perfringens.     

Conjugated docosahexaenoic acid inhibits lipid accumulation in rats

Conjugated linoleic acid (CLA), which contains a conjugated double-bond system, and n-3 highly unsaturated fatty acids such as docosahexaenoic acid (DHA) are widely known to improve lipid metabolism. To examine the possibility that a fatty acid with a combination of these structural features might have stronger physiological effects, we prepared conjugated DHA (CDHA) by alkaline isomerization of DHA and examined its effects on lipid and sugar metabolism in rats. Rats were force fed with 200 mg of test oils [linoleic acid (LA), DHA, CLA or CDHA] everyday for 4 weeks. Compared with the animals from the other groups, those in the CDHA group showed a significant weight loss in white adipose tissue (57% of adipose tissue weight in the LA group) and significant decreases in the levels of liver triacylglycerol (TG; 65% of TG level in the LA group) as well as total cholesterol (TC; 88% of TC level in the LA group), indicating suppression of lipid accumulation in the liver and adipose tissue. In addition, plasma TG and TC levels significantly decreased (69% of TG level and 82% of TC level in the LA group), indicating improved lipid metabolism. In the liver, the fatty acid synthesis system was inhibited and the fatty acid beta-oxidation system was activated, whereas the free fatty acid, glucose and tumor necrosis factor alpha levels in the plasma were lowered following CDHA administration. Hence, intake of CDHA appears to suppress the accumulation of fat in the liver and epididymal adipose tissue and improves lipid and sugar metabolism in rats.     

Activation of the neutrophil by calcium-mobilizing ligands. I. A chemotactic peptide and the lectin concanavalin A stimulate superoxide anion generation but elicit different calcium movements and phosphoinositide remodeling

The relevance of phosphoinositide remodeling to calcium movements and to the physiological response of superoxide anion (O2-) generation was probed in neutrophils stimulated by the chemotactic peptide fMet-Leu-Phe and the lectin concanavalin A. fMet-Leu-Phe and concanavalin A triggered O2- generation but elicited different patterns of calcium mobilization and phosphoinositide remodeling. fMet-Leu-Phe (10(-7) M) triggered a rise in cytosolic calcium by mobilization of intracellular calcium (fura-2) and increased calcium permeability (45Ca uptake), while concanavalin A (100 micrograms/ml) elicited a rise in cytosolic calcium, primarily by uptake of extracellular calcium (45Ca uptake). fMet-Leu-Phe triggered rapid breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate and phosphatidylinositol, and generation of inositol 1,4,5-trisphosphate (IP3). In contrast concanavalin A triggered breakdown of phosphatidylinositol, but not PIP2, nor was there a significant increase in IP3. However, both fMet-Leu-Phe and concanavalin A triggered a rapid biphasic increase in levels of labeled diacylglycerol (in [3H]arachidonate or [14C]glycerol prelabeled cells) and a 3-fold increase in [32P] phosphatidic acid. These results are concordant with a role for PIP2 breakdown and generation of IP3 specifically in intracellular calcium mobilization but not for other aspects of the signaling pathway for O2- generation. Calcium permeability changes were associated with elevated diacylglycerol and [32P]phosphatidic acid, although a cause and effect relationship is not apparent. Ligands such as concanavalin A enhance cytosolic calcium and trigger generation of O2- without significant PIP2 remodeling; elevated diacylglycerol and cytosolic calcium are the common events associated with ligand-induced O2- generation.     

Corticosteroid effect on Caco-2 cell lipids depends on cell differentiation

Previous studies from our laboratory have indicated that secondary hyperaldosteronism affects phospholipids of rat colonic enterocytes. To assess whether this represents a direct effect of mineralocorticoids on enterocytes, the role of aldosterone and dexamethasone in the regulation of lipid metabolism was examined in Caco-2 cells during development of their enterocyte phenotype. Differentiation of Caco-2 cells was associated with increased levels of triglycerides (TG) and cholesteryl esters (CE), a decreased content of cholesterol and phospholipids and changes in individual phospholipid classes. The phospholipids of differentiated cells had a higher content of n-6 polyunsaturated fatty acids (PUFA) and lower amounts of monounsaturated (MUFA) and saturated fatty acids than subconfluent undifferentiated cells. Differentiated cells exhibited a higher ability to incorporate [3H]arachidonic acid (AA) into cellular phospholipids and a lower ability for incorporation into TG and CE. Incubation of subconfluent undifferentiated cells with aldosterone or dexamethasone was without effect on the content of lipids, their fatty acids and [3H]AA incorporation. In contrast, aldosterone treatment of differentiated cells diminished the content of TG, increased the content of phospholipids and modulated their fatty acid composition. The percentage of n-6 and n-3 PUFA in phospholipids was increased and that of MUFA decreased, whereas no changes in TG were observed. The incorporation of [3H]AA into phospholipids was increased and into TG decreased and these changes were blocked by spironolactone. Treatment of differentiated cells with dexamethasone increased their CE content but no effect was identified upon other lipids, their fatty acid composition and on the incorporation of [3H]AA. As expected for the involvement of corticosteroid hormones the mineralocorticoid and glucocorticoid receptors were identified in Caco-2 cells by RT-PCR. The results suggest that aldosterone had a profound influence on lipid metabolism in enterocytes and that its effect depends on the stage of differentiation. The aldosterone-dependent changes occurring in phospholipids and their fatty acid composition may reflect a physiologically important phenomenon with long-term consequences for membrane structure and function.     

Regional uptake of meal fatty acids in humans

Two protocols were performed to study meal fatty acid metabolism. In protocol 1, 14 patients scheduled for elective intra-abdominal surgery (11 undergoing bariatric surgery for severe obesity) consumed a meal containing [3H]triolein in the evening before surgery. This allowed us to measure adipose tissue lipid specific activity (SA) in mesenteric and omental, deep and superficial abdominal subcutaneous adipose tissue. Intra-abdominal adipose tissue lipid SA was greater than subcutaneous lipid SA. There were no significant differences between mesenteric and omental or between deep and superficial abdominal subcutaneous adipose tissue. In protocol 2, meal fatty acid oxidation and uptake into subcutaneous and omental adipose tissue ([3H]triolein) were measured in six normal, healthy volunteers. Meal fatty acid oxidation (3H2O generation) plus that remaining in plasma ( approximately 1%) plus uptake into upper body subcutaneous, lower body subcutaneous, and visceral fat allowed us to account for 98 +/- 6% of meal fatty acids 24 h after meal ingestion. We conclude that omental fat is a good surrogate for visceral fat and that abdominal subcutaneous fat depots are comparable with regard to meal fatty acid metabolic studies. Using [3H]triolein, we were able to account for virtually 100% of meal fatty acids 24 h after meal ingestion. These results support the meal fatty acid tracer model as a way to study the metabolic fate of dietary fat.     

The effect of cadmium on lipid and fatty acid biosynthesis in tomato leaves

This research aims to examine the effect of cadmium uptake on lipid composition and fatty acid biosynthesis, in young leaves of tomato treated seedlings (Lycopersicon esculentum cv. Ibiza F1). Results in membrane lipids investigations revealed that high cadmium concentrations affect the main lipid classes, leading to strong changes in their composition and fatty acid content. Thus, the exposure of tomato plants to cadmium caused a concentration-related decrease in the unsaturated fatty acid content, resulting in a lower degree of fatty acid unsaturation. The level of lipid peroxides was significantly enhanced at high Cd concentrations. Studies of the lipid metabolism using radioactive labelling with [1-¹⁴C]acetate as a major precursor of lipid biosynthesis, showed that levels of radioactivity incorporation in total lipids as well as in all lipid classes were lowered by Cd doses. In total lipid fatty acids, [1-¹⁴C]acetate incorporation was reduced in tri-unsaturated fatty acids (C16:3 and C18:3); While it was enhanced in the palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0) and linoleic (C18:2) acids. [1-¹⁴C]acetate incorporation into C16:3 and C18:3 of galactolipids [monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG)] and some phospholipids [phosphatidylcholine (PC) and phosphatidylglycerol (PG)] was inhibited by Cd stress. Our results showed that in tomato plants, cadmium stress provoked an inhibition of polar lipid biosynthesis and reduced fatty acid desaturation process.     

Modulation of lipid synthesis,apolipoprotein biogenesis,and lipoprotein assembly by butyrate

Short-chain fatty acids (SCFAs) are potent modulators of the growth, function, and differentiation of intestinal epithelia. In addition, high-fiber diets may protect against the development of atherosclerosis because of their cholesterol-lowering effects due, in large part, to SCFA production, liver sterol metabolism, and bile acid excretion. Although the small gut plays a major role in dietary fat transport and contributes substantially to plasma cholesterol and lipoprotein homeostasis, the impact of SCFAs on intestinal lipid handling remains unknown. In the present study, the modulation of lipid synthesis, apolipoprotein biogenesis, and lipoprotein secretion by butyrate was investigated in Caco-2 cells plated on permeable polycarbonate filters, which permit separate access to the upper and lower compartments of the monolayers. Highly differentiated and polarized cells (20 days of culture) were incubated for 20 h with 20 mM butyrate in the apical medium. In the presence of [14C]oleic acid, butyrate led to a significant reduction of secreted, labeled triglycerides (27%; P < 0.01) and phospholipids (25%; P < 0.05). Similarly, butyrate significantly decreased the incorporation of [14C]acetate into exported cholesteryl ester (49%; P < 0.005). As expected from these results, with [14C]oleic acid as a precursor, butyrate significantly (P < 0.05) diminished the delivery of radiolabeled chylomicrons and very low-density lipoproteins. In parallel, [35S]methionine pulse labeling of Caco-2 cells revealed the concomitant inhibitory effect of butyrate on the synthesis of apolipoproteins B-48 (28%; P < 0.05) and A-I (32%; P < 0.01). Collectively, our data indicate that butyrate may influence lipid metabolism in Caco-2 cells, thus suggesting a potential regulation of intestinal fat absorption and circulating lipoprotein concentrations.