Mechanism of ethanol-induced changes in lipid composition of Escherichia coli: inhibition of saturated fatty acid synthesis in vivo.

The in vivo effects of ethanol on lipid synthesis in Escherichia coli have been examined. Under conditions which uncoupled fatty acid synthesis from phospholipid synthesis, ethanol decreased the amount of saturated fatty acids synthesized but had little effect on the selectivity of their incorporation into phospholipids. In the absence of fatty acid degradation and unsaturated fatty acid synthesis, E. coli was still able to adapt its membrane lipids to ethanol, while the inhibition of total fatty acid synthesis eliminated this response. During growth in the presence of ethanol, strain K1060 (an unsaturated fatty acid auxotroph) incorporated an increased amount of exogenous heptadecanoic acid (17:0) to compensate for the reduction in palmitic acid (16:0) available from biosynthesis. Thus, our results indicate that the reduced levels of saturated fatty acids observed in the phospholipids of E. coli following growth in the presence of ethanol result primarily from a decrease in the amounts of saturated fatty acids available for phospholipid synthesis.     

Electrospray/tandem mass spectrometry for quantitative analysis of lipid remodeling in essential fatty acid deficient mice

A method utilizing electrospray ionization coupled with tandem mass spectrometry was developed as a facile and rapid method to identify and quantify lipid remodeling in vivo. Electrospray/tandem mass spectrometric analyses were performed on lipids isolated from liver tissue and resident peritoneal cells from essential fatty acid sufficient and deficient mice. Essential fatty acid deficiency was chosen as the paradigm to evaluate the methodology because it epitomizes the most extreme dietary means of altering fatty acid composition of virtually all cellular lipid species. Qualitative and quantitative changes were measured in the phospholipid and cholesterol ester species directly in the chloroform/methanol lipid extract without any prior chromatographic separation. Lipid remodeling in liver and peritoneal cells from essential fatty acid deficient mice was qualitatively similar in cholesterol ester, phosphatidylcholine, and phosphatidylethanolamine. The monoenoic fatty acids palmitoleic acid (16:1 n-7) and oleic acid (18:1 n-9) were increased markedly, whereas all n-6 and n-3 polyunsaturated fatty acids were nearly depleted in phospholipid and cholesterol ester species. The n-9 polyunsaturated fatty acid surrogate, Mead acid (20:3 n-9), substituted for arachidonic acid (20:4 n-6) and docosahexaenoic acid (22:6 n-3) in phospholipid, but not in cholesterol ester, species. Another notable difference was that adrenic acid (22:4 n-6) and docosapentaenoic acid (22:5 n-6), both metabolites of arachidonic acid, accumulated in phospholipid and cholesterol ester species of peritoneal cells, but not in liver cells, of essential fatty acid sufficient mice. The overall body of data presented illustrates the implementation of electrospray/tandem mass spectrometry as a method for facile and direct quantification of changes in lipid species during lipid metabolic studies.     

Lipid composition of isolated epiphyseal cartilage cells, membranes and matrix vesicles.

1. Intact cells, cell fragments (membranes) and matrix vesicles were isolated from the proliferating and calcifying layers of epiphyseal cartilage by sequential hyaluronidase and collagenase digestion and differential centrifugation. Lipids were extracted and analyzed for various lipid classes and their fatty acid composition by column, thin-layer, paper and gas-liquid chromatography. 2. On a protein basis the isolated matrix vesicles had more total lipid than either the membrane or cell fractions, the vesicles and membranes being richer in non-polar lipids and containing smaller quantities of phospholipids than whole cells. Expressed as a percentage of the total lipid, the cells were richer in triacylglycerols and lower in free fatty acids than in the membrane or vesicle fractions. The proportion of free cholesterol and the cholesterol/phospholipid ratio were nearly twice as high in the matrix vesicles as in the other tissue fractions. Choline and ethanolamine phosphoglycerides progressively declined in the membrane and matrix vesicle fractions, whereas serine phosphoglycerides and sphinogomyelin increased. Non-phosphorus-containing polar lipids were present in all fractions, the vesicles being richer in polyhexosyl ceramides, cerebrosides, glycosyldiacylglycerols and certain uncharacterized acidic polar lipids. 3. Fatty acid patterns of the matrix vesicles were distinctive from those of isolated cells, being generally richer in 18 : 0 and 18 : 2, and lower in 16 : 1 and 18 : 1 fatty acids. Monoacyl forms were similarly increased in 16 : 0 and/or 18 : 0, and reduced in 16 : 1, 18 : 1 or 20 : 2 fatty acids, depending on the lipid class. The fatty acid composition of diphosphatidylglycerol from cells and matrix vesicles was markedly different, providing evidence that the cardiolipin in the vesicles was not from mitochondrial components. 4. Based on the fact that the matrix vesicles were significantly enriched in free cholesterol, sphingomyelin, glycolipids and serine-phosphoglycerides, it is concluded that they are derived from the plasma membrane of the cell, supporting earlier conclusions based upon morphological and enzymological evidence.     

Lipid composition of alveolar macrophage plasma membrane during postnatal development

This study was undertaken to define the age-related alterations in lipid composition that resident rabbit alveolar macrophages (AM) undergo during postnatal development. The eventual goal is to correlate these changes with the functional maturation of these cells. The number of AM recorded from total lung lavages rose markedly during the first 14 days of life, from 4.9 X 10(5) to 1.1 X 10(7). Adult lungs yielded 1.1 X 10(8) AM. A gradual but significant increase in fluorescence polarization (P) was observed during development when purified AM plasma membranes were tagged with the probe 1,6-diphenyl-1,3,5 hexatriene trimethyl ammonium. The rise ranged from a mean P value of less than or equal to 0.22 to 0.24 (p less than 0.001) for AM plasma membranes from rabbits 1- or 7-day-old to 30- or 150-day-old rabbits, respectively. This finding suggests that the fluidity of the AM plasma membrane decreased during postnatal development. Palmitic, stearic, oleic, and linoleic acids were the most prevalent fatty acids found in the neutral lipid fraction of the AM plasma membrane throughout development. The content of stearic acid rose from 10 to 16%, arachidonic acid rose from 2.8 to 9%, myristic acid decreased from 3.2 to 1.3%, palmitic acid decreased from 42 to 36%, and oleic and linoleic acids changed relatively little during the first 30 days of life. The levels of docosatetraenoic and docosapentaenoic increased gradually during the first 14 days of life, and by 30 days of life the levels declined to that observed at birth. The sum of these changes resulted in an increase in the ratio of unsaturated to saturated fatty acids (1 to 1.15) in the neutral lipid fraction. During the first month of life, the neutral lipid fatty acid pool in the total lipid fraction of AM plasma membrane increased from 12 to 18 mole %, cholesterol increased from 7 to 14 mole %, and total phospholipids decreased from 81 to 67 mole %. These changes resulted in increasing the cholesterol to phospholipid ratio from 0.09 at birth to 0.23 by 150 days of life. The levels of all three major lipid fractions were comparable at 30 days and 150 days of life. Adult levels of choline phosphoglycerides, the predominant phospholipid, were observed by 7 days of life to have decreased from 47 to 34.5 mole %, and the levels of ethanolamine phosphoglycerides and sphingomyelin increased from 17.5 to 25 mole % and from 9 to 13 mole %, respectively. Adult levels of lyso-bis-phosphatidic acid were reached by 30 days of life increasing from 8.2 to 17.8 mole %.(ABSTRACT TRUNCATED AT 400 WORDS)     

Bile formation and hepatic plasma membrane composition in guinea-pigs and rats

We compared bile formation, and biliary and liver plasma membrane composition in guinea-pigs and rats in an attempt to explain the observation that the bile flow rate and the bile acid independent fraction of bile flow (BAIF) in guinea-pigs is about five to seven times higher than in rats. Analysis of electrolytes in bile showed that bicarbonate was significantly [acid] higher in guinea-pigs while Cl⁻, phosphate and Ca²⁺ were markedly lower than in rats. High bile independent secretion in guinea-pigs was associated with a significantly lower concentration of total bile acid, phospholipid and cholesterol than in rats. Bile acid distribution studies showed that glycine conjugated chenodeoxycholate and ketolithocholate were the main bile acids in guinea-pigs, while taurine conjugated cholate and muricholate were the predominant bile acids in rats. Total fatty acid analysis of bile indicated that in rats the major fatty acids were palmitic acid (C16:0) and linoleic acid (C18:2, n-6). In guinea-pigs, the contribution of these fatty acids was lower than in rats and compensated with a significantly higher percentage of oleic acid (C18:1, n-9). Concentrations of anionic polypeptide fraction (APF), an acidic calcium binding apoprotein closely associated with biliary phospholipid and cholesterol secretion was also significantly lower in guinea-pigs. Canalicular plasma membrane analysis showed that as compared with rats, specific activities of Na⁺,K⁺ ATPase, and cholesterol and phospholipid content were markedly lower in guinea-pigs. Total fatty acid analysis of the membrane revealed that palmitic acid (C16:0), stearic acid (C18:0) and linoleic acid (C18:2, n-6) were the predominant fatty acids in guinea-pigs, while palmitic acid (C16:0), stearic acid (C18:0) and arachidonic acid (C20:4, n-6) were the most important in rats. Thus, high bile flow rate and BAIF in the guinea-pig may be attributed to the low bile acid concentration (below the critical micellar concentration), secretion of hypercholeretic bile acids (e.g. ketolithocholate) and high bicarbonate output.     

Fatty acid desaturase activities are modulated by phytosterol incorporation in microsomes

The effect of phytosterol-rich diets (3% beta-sitosterol + 2% campesterol) on rat liver microsomal fatty acid desaturases, membrane dynamics and lipid composition was investigated. After a 21 day period, phytosterol was incorporated into microsomes and the membrane fluidity decreased. There were no changes in either the phospholipid composition or in the total sterol content. However, the phytosterol/cholesterol ratio increased. In the animals fed phytosterols, the delta 5-, delta 6- and delta 9-fatty acid desaturases were significantly more active than in control animals. The changes in the lipid fatty acid composition were consistent with those of the desaturase activities. Hence, it is suggested that: (1) dietary phytosterol modulates desaturase activities; (2) phytosterols make the membrane more rigid but do not induce changes in the relative phospholipid composition; (3) delta 9-, delta 5- and delta 6-desaturase activities increase when the membrane becomes more rigid without changes in the phospholipid composition.     

Lipid Composition of Plasma Membranes Isolated from Lactating Bovine Mammary Gland

The light and heavy plasma membranes (PM) isolated from lactating bovine mammary glands contained 38~43% lipid of which 41~44% was phospholipid and 47~52% neutral lipid. The contents of phospholipid and neutral lipid were somewhat higher in the light PM than in the heavy PM. Cholesterol was contained 55 ~60% of neutral lipid and the ratio of cholesterol to phospholipid was 0.64 to 0.69. Phospholipid was composed of sphingomyelin (Sph) 29~38%, phosphatidylcholine (PC) 27~35%, phosphatidylethanolamine (PE) 16~20%, phosphatidylserine 10%, and phosphatidylinositol 6~7%. The content of Sph was higher in the heavy PM than in the light PM, while the values of PC and PE were opposite. The major fatty acids of lipid components were palmitic acid, stearic acid, and oleic acid and those of Sph were palmitic acid, stearic acid, C23:0 and 24:0. The fatty acid composition of individual lipid classes differed significantly from each other but were similar between the light and heavy PMs. Tetracosapentaenoic acid (C24:5) was the major fatty acid of the diacylglycerol fraction. The results indicated that the lipid composition, especially phospholipid components, of bovine mammary gland PMs was different from those of milk fat globule membranes which is derived from the PM of mammary secretory cells.     

Phospholipid ester-linked fatty acid profile changes during nutrient deprivation of Vibrio cholerae: increases in the trans/cis ratio and proportions of cyclopropyl fatty acids.

The phospholipid ester-linked fatty acids of 0-day-, 7-day-, and 30-day-starved cultures of Vibrio cholerae were compared. Statistically significant trends were noted in the fatty acid profiles as the cells starved. The amount of the cis-monoenoic fatty acids declined (e.g., 16:1 omega 7c: 0 day, 39%; 7 day, 18%; 30 day, 11%). In contrast, the saturated fatty acids, the cyclopropyl derivatives of the cis-monoenoic fatty acids, and trans-monoenoic fatty acids increased during starvation. For instance, the amounts of 16:1 omega 7t were: 0 day, 1%; 7 day, 13%; 30 day, 17%; which increased the trans/cis ratio for 16:1 omega 7 from 0.02 (0 day) to 0.70 (7 day) to 1.56 (30 day). This may be due to the reported high turnover rates of cis-monoenoic fatty acids of membrane phospholipids and the availability of enzymes for the metabolism of these isomers. During starvation-induced phospholipid loss, the cis-monoenoic fatty acids would, therefore, be preferentially utilized. The ability to either synthesize trans-monoenoic acids (which are not easily metabolized by bacteria) or modify the more volatile cis-monoenoic acids to their cyclopropyl derivatives may be a survival mechanism which helps maintain a functional (although structurally altered) membrane during starvation-induced lipid utilization. In addition, a trans/cis fatty acid ratio significantly greater than that reported for most bacterial cultures and environmental samples (less than 0.1) may be used as a starvation or stress lipid index. Such a ratio could help determine the nutritional status of ultramicrobacteria and other reported dormant cells in natural aquatic environments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phospholipid ester-linked fatty acid profile changes during nutrient deprivation of Vibrio cholerae: increases in the trans/cis ratio and proportions of cyclopropyl fatty acids

The phospholipid ester-linked fatty acids of 0-day-, 7-day-, and 30-day-starved cultures of Vibrio cholerae were compared. Statistically significant trends were noted in the fatty acid profiles as the cells starved. The amount of the cis-monoenoic fatty acids declined (e.g., 16:1 omega 7c: 0 day, 39%; 7 day, 18%; 30 day, 11%). In contrast, the saturated fatty acids, the cyclopropyl derivatives of the cis-monoenoic fatty acids, and trans-monoenoic fatty acids increased during starvation. For instance, the amounts of 16:1 omega 7t were: 0 day, 1%; 7 day, 13%; 30 day, 17%; which increased the trans/cis ratio for 16:1 omega 7 from 0.02 (0 day) to 0.70 (7 day) to 1.56 (30 day). This may be due to the reported high turnover rates of cis-monoenoic fatty acids of membrane phospholipids and the availability of enzymes for the metabolism of these isomers. During starvation-induced phospholipid loss, the cis-monoenoic fatty acids would, therefore, be preferentially utilized. The ability to either synthesize trans-monoenoic acids (which are not easily metabolized by bacteria) or modify the more volatile cis-monoenoic acids to their cyclopropyl derivatives may be a survival mechanism which helps maintain a functional (although structurally altered) membrane during starvation-induced lipid utilization. In addition, a trans/cis fatty acid ratio significantly greater than that reported for most bacterial cultures and environmental samples (less than 0.1) may be used as a starvation or stress lipid index. Such a ratio could help determine the nutritional status of ultramicrobacteria and other reported dormant cells in natural aquatic environments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ethanol tolerance in the yeast Saccharomyces cerevisiae is dependent on cellular oleic acid content

In this investigation, we examined the effects of different unsaturated fatty acid compositions of Saccharomyces cerevisiae on the growth-inhibiting effects of ethanol. The unsaturated fatty acid (UFA) composition of S. cerevisiae is relatively simple, consisting almost exclusively of the mono-UFAs palmitoleic acid (Delta(9)Z-C(16:1)) and oleic acid (Delta(9)Z-C(18:1)), with the former predominating. Both UFAs are formed in S. cerevisiae by the oxygen- and NADH-dependent desaturation of palmitic acid (C(16:0)) and stearic acid (C(18:0)), respectively, catalyzed by a single integral membrane desaturase encoded by the OLE1 gene. We systematically altered the UFA composition of yeast cells in a uniform genetic background (i) by genetic complementation of a desaturase-deficient ole1 knockout strain with cDNA expression constructs encoding insect desaturases with distinct regioselectivities (i.e., Delta(9) and Delta(11)) and substrate chain-length preferences (i.e., C(16:0) and C(18:0)); and, (ii) by supplementation of the same strain with synthetic mono-UFAs. Both experimental approaches demonstrated that oleic acid is the most efficacious UFA in overcoming the toxic effects of ethanol in growing yeast cells. Furthermore, the only other UFA tested that conferred a nominal degree of ethanol tolerance is cis-vaccenic acid (Delta(11)Z-C(18:1)), whereas neither Delta(11)Z-C(16:1) nor palmitoleic acid (Delta(9)Z-C(16:1)) conferred any ethanol tolerance. We also showed that the most ethanol-tolerant transformant, which expresses the insect desaturase TniNPVE, produces twice as much oleic acid as palmitoleic acid in the absence of ethanol and undergoes a fourfold increase in the ratio of oleic acid to palmitoleic acid in response to exposure to 5% ethanol. These findings are consistent with the hypothesis that ethanol tolerance in yeast results from incorporation of oleic acid into lipid membranes, effecting a compensatory decrease in membrane fluidity that counteracts the fluidizing effects of ethanol.     

Effect of benzyl viologen on the phospholipid fatty acid composition and some properties in hepatic microsomal membrane of rats

The effect of benzyl viologen (a stimulator of free radical production in cells) on lipid composition, fluidity and enzymes involved in both polyunsaturated fatty acid biosynthesis and cholesterol metabolism was studied in liver microsomal membrane of adult rats. In viologen-treated animals, a significant decrease in the levels of free cholesterol and cholesteryl esters, accompanied to a decrease at the free cholesterol/phospholipid ratio, were observed. The levels of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and acyl-coenzyme A : cholesterol acyltransferase (ACAT) were also lower in viologen-treated rats than in controls. Linoleic and arachidonic acids were both severely lower while docosatetraenoic, docosapentaenoic and docosahexaenoic acids were significantly higher as compared with controls. Furthermore, a decrease in monounsaturated/saturated ratio was found. In addition, the treatment evoked a depression in the fatty acid desaturation complex, with a diminish of ⁹, ⁹, and ⁵ desaturase activities in microsomal membrane.It was concluded that changes in phospholipid microsomal fatty acid and cholesterol content could be directly responsible for changes in membrane fluidity and function, and that extensive yield of docosahexaenoic acid may serve to maintain the physical characteristics of particular domains against oxidative stress caused by benzyl viologen treatment.     

Formula alpha-linolenic (18:3(n - 3)) and linoleic (18:2(n - 6)) acid influence neonatal piglet liver and brain saturated fatty acids, as well as docosahexaenoic acid (22:6(n - 3)).

Saturated fatty acids can be synthesized de novo and play a role in determining properties of structural membranes. The effect of dietary essential fatty acids, linoleic acid (18:2(n - 6)) and alpha-linolenic acid (18:3(n - 3)), on the saturated fatty acid content of membrane phospholipid has not previously been considered in newborn nutrition. The studies report the effect of low (1% fatty acids) or high (4%) formula 18:3(n - 3) with low (16%) or high (30-35%) formula 18:2(n - 6) on the saturated and unsaturated fatty acid composition of liver and brain structural lipid of piglets fed formula from birth for 15 days. A significant inverse relationship between the formula % 18:3(n - 3), but not 18:2(n - 6), and the liver phospholipid palmitic acid (16:0) was found. This may indicate a possible effect of dietary 18:3(n - 3) on de novo synthesis of 16:0 and requires further investigation. Monounsaturated fatty acids in both liver and brain were significantly lower in response to high 18:3(n - 3) and to high 18:2(n - 6) plus low 18:1(n - 9) in the formula. Liver phospholipid and brain total lipid % docosahexaenoic acid (22:6(n - 3)) were significantly higher when formula containing 4% rather than 1% 18:3(n - 3) was fed, suggesting that 1% 18:3(n - 3) may limit tissue (n - 3) fatty acid accretion. These results suggest that future studies of essential fatty acid requirements, specifically 18:3(n - 3), should consider possible influences on the saturated fatty acids which also play a functional role in tissue structural lipids.     

Accumulation of cholesterol in Acholeplasma laidlawii membranes in the steady-state phase of culture growth

In early logarithmic phase of growth of the A. laidlawii cells the lipid composition of plasma membrane is changed: the total lipid, glycolipid and phospholipid contents are decreased, while that of cholesterol changes only insignificantly. In late logarithmic and steady state phases the cholesterol level in the membrane is increased in parallel with the decrease of the phospholipid content. Throughout the growth period a quantitative redistribution of membrane phospholipids in fatty acids and an increase of the molar content of saturated fatty acids are observed. Accumulation of cholesterol in the steady state phase is accompanied by an increase in the membrane viscosity which results in inhibition of membrane processes in the cell.     

Sterol carrier protein-2 expression alters plasma membrane lipid distribution and cholesterol dynamics

Although sterol carrier protein-2 (SCP-2) binds, transfers, and/or enhances the metabolism of many membrane lipid species (fatty acids, cholesterol, phospholipids), it is not known if SCP-2 expression actually alters the membrane distribution of lipids in living cells or tissues. As shown herein for the first time, expression of SCP-2 in transfected L-cell fibroblasts reduced the plasma membrane levels of lipid species known to traffic through the HDL-receptor-mediated efflux pathway: cholesterol, cholesteryl esters, and phospholipids. While the ratio of cholesterol/phospholipid in plasma membranes of intact cells was not changed by SCP-2 expression, phosphatidylinositol, a molecule important to intracellular signaling and vesicular trafficking, and anionic phospholipids were selectively retained. Only modest alterations in plasma membrane phospholipid percent fatty acid composition but no overall change in the proportion of saturated, unsaturated, monounsaturated, or polyunsaturated fatty acids were observed. The reduced plasma membrane content of cholesterol was not due to SCP-2 inhibition of sterol transfer from the lysosomes to the plasma membranes. SCP-2 dramatically enhanced sterol transfer from isolated lysosomal membranes to plasma membranes by eliciting detectable sterol transfer within 30 s, decreasing the t(1/2) for sterol transfer 364-fold from >4 days to 7-15 min, and inducing formation of rapidly transferable sterol domains. In summary, data obtained with intact transfected cells and in vitro sterol transfer assays showed that SCP-2 expression (i) selectively modulated plasma membrane lipid composition and (ii) decreased the plasma membrane content cholesterol, an effect potentially due to more rapid SCP-2-mediated cholesterol transfer from versus to the plasma membrane.     

The effect of chronic ethanol consumption on the fatty acid composition of phosphatidylinositol in rat liver microsomes as determined by gas chromatography and 1H-NMR.

Cell membranes and vesicles composed of extracted phospholipids isolated from rats chronically-fed ethanol develop a resistance to disordering by ethanol in vitro (membrane tolerance) and a decreased partitioning of ethanol into the membranes. The anionic lipid phosphatidylinositol (PtdIns) is the only microsomal phospholipid from the ethanol-fed rats that confers tolerance to vesicles of microsomal phospholipids from control rats in a paradigm where phospholipid classes are sequentially swapped. To investigate the molecular basis of this adaptation, the fatty acid content of microsomal PtdIns extracted from the livers of rats chronically fed ethanol for 5 weeks and their calorically-matched controls was analyzed by gas-liquid chromatography (GLC) and 1H-NMR spectroscopy. Chronic ethanol consumption caused an 8.4% decrease in arachidonic acid [20:4(n - 6)], a 20.0% increase in oleic acid [18: 1(n - 9)] and a 47.1% increase in the quantitatively minor fatty acid [20:3(n - 6)]. 1H-NMR was used to quantitatively assay compositional changes in the delta 5 olefinic moiety of the acyl chains in PtdIns, an approach that should be broadly applicable to other lipid systems. After chronic ethanol feeding PtdIns had decreased delta 5 unsaturates (-7.9% NMR, -8.2% GLC) and a corresponding increase in delta 5 saturates (+5.4% NMR, +5.3% GLC). In the other phospholipids, chronic ethanol feeding caused alterations in the fatty acid compositions specific for each phospholipid. PtdIns was the only microsomal phospholipid that exhibited a significant decrease in both the polyunsaturate pool and the ratio of the total olefinic content to the saturated fatty acid content. The major adaptive response in rat liver microsomal PtdIns to chronic ethanol administration involves a decrease in arachidonic acid [20:4 (n - 6)], which is partly compensated for by increases in oleic acid [18:1(n - 9)] and eicosatrienoic acid [20:3 (n - 6)], resulting in a depressed unsaturation and polyunsaturation index. The decreased unsaturation at the delta 5 position may have special functional relevance, due to the proximity of this position to the membrane surface, where ethanol is believed to reside. Whether these acyl changes are merely coincident with, or causative of, membrane tolerance requires further elucidation.     

Isolation and partial characterization of basolateral membranes from rat proximal colonic epithelial cells

The isolation of basolateral membranes from rat proximal colonic epithelial cells is described. Cells were harvested using a technique combining chelation of divalent cations with mechanical dissociation. After homogenization, differential centrifugation yielded a 'crude' membrane fraction which was further purified using sucrose density centrifugation. The final membrane fraction was enriched 10-14-fold over homogenate in ouabain-sensitive sodium-potassium dependent adenosine triphosphatase and ouabain-sensitive potassium-dependent phosphatase specific activities. SDS-polyacrylamide gel electrophoresis of this membrane revealed at least 18 protein bands with molecular weights of 14600-200000. Phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, free cholesterol and fatty acids were the major lipid components of this membrane. The predominant fatty acids were palmitic (16:0), oleic (18:1), stearic (18:0) and linoleic (18:2) acid. Membranes and their liposomes were studied, using the lipid soluble fluorophore 1,6-diphenyl-1,3,5-hexatriene (DPH), by steady-state fluorescence polarization. The fluorescence anisotropy was greater in the intact membranes compared to their liposomes, indicating greater fluidity in the liposomes. Compositional studies suggested that the high fluidity of this membrane was due to its low ratios of protein/lipid (w/w), cholesterol/phospholipid (mol/mol), and sphingomyelin/phosphatidylcholine (mol/mol).     

Effect of distal small bowel resection on ACAT activity and microsomal lipid composition in rat small intestine

1. The acyl-CoA:cholesterol acyltransferase (ACAT) activity and lipid composition of intestinal microsomal membrane were investigated 6 weeks after both 50 and 75% distal small bowel resection (DSBR). 2. No changes in both microsomal ACAT activity and cholesteryl ester levels were found, while microsomal non-esterified cholesterol content was increased after the surgical operation. 3. The total phospholipid content of the microsomes did not change as a result of DSBR. 4. The microsomal phospholipid fatty acid composition showed a significant increase in saturated fatty acids together with no changes in both total monounsaturated and total polyunsaturated fatty acids after resection. 5. An increase in the levels of linoleic acid accompanied by a decrease in arachidonic acid was found in remnant intestine of resected rats.     

Lipid composition of the isolated rat intestinal microvillus membrane

1. Rat intestinal microvillus plasma membranes were prepared from previously isolated brush borders and the lipid composition was analysed. 2. The molar ratio of cholesterol to phospholipid was greatest in the membranes and closely resembled that reported for myelin. 3. Unesterified cholesterol was the major neutral lipid. However, 30% of the neutral lipid fraction was accounted for by glycerides and fatty acid. 4. Five phospholipid components were identified and measured, including phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, sphingomyelin and lysophosphatidylcholine. Though phosphatidylethanolamine was the chief phospholipid, no plasmalogen was detected. 5. In contrast with other plasma membranes in the rat, the polar lipids of the microvillus membrane were rich in glycolipid. The cholesterol:polar lipid (phospholipid+glycolipid) ratio was about 1:3 for the microvillus membrane. Published data suggest that this ratio resembles that of the liver plasma membrane more closely than myelin or the erythrocyte membrane. 6. The fatty acid composition of membrane lipids was altered markedly by a single feeding of safflower oil. Membrane polar lipids did not contain significantly more saturated fatty acids than cellular polar lipids. Differences in the proportion of some fatty acids in membrane and cellular glycerides were noted. These differences may reflect the presence of specific membrane glycerides.     

Effects of hydrostatic pressure and temperature on growth and lipid composition of the inner membrane of barotolerant Pseudomonas sp. BT1 isolated from the deep-sea

A barotolerant member of the genus Pseudomonas was isolated from deep-sea sediment obtained from the Japan Trench, at a depth of 4418 m. The growth temperature was found to affect the hydrostatic pressure range in which the bacterium could grow; the optimum hydrostatic pressure for growth shifted to a higher pressure with increasing temperature. We examined the lipid composition of the inner membrane of cells grown at various hydrostatic pressures and temperatures. The fatty acid components of the inner membrane lipids were C16:0, C16:1, C18:0, and C18:1. The phospholipid components of the inner membrane were phosphatidylethanolamine, cardiolipin, phosphatidylglycerol, and phosphatidylserine. It is evident that the effects of elevated hydrostatic pressure are comparable to the effects of low temperature on both the fatty acid composition of the inner membrane lipids and the phospholipid composition of the inner membrane of this bacterium.     

The interaction of dietary fatty acid and cholesterol on catecholamine-stimulated adenylate cyclase activity in the rat heart

Diets supplemented with high levels of saturated or unsaturated fatty acids supplied by addition of sheep kidney fat or sunflower seed oil, respectively, were fed to rats with or without dietary cholesterol. The effects of these diets on cardiac membrane lipid composition, catecholamine-stimulated adenylate cyclase and beta-adrenergic receptor activity associated with cardiac membranes, were determined. The fatty acid-supplemented diets, either with or without cholesterol, resulted in alterations in the proportion of the (n-6) to (n-3) series of unsaturated fatty acids, with the sunflower seed oil increasing and the sheep kidney fat decreasing this ratio, but did not by themselves significantly alter the ratio of saturated to unsaturated fatty acids. However, cholesterol supplementation resulted in a decrease in the proportion of saturated and polyunsaturated fatty acids and a dramatic increase in oleic acid in cardiac membrane phospholipids irrespective of the nature of the dietary fatty acid supplement. The cholesterol/phospholipid ratio of cardiac membrane lipids was also markedly increased with dietary cholesterol supplementation. Although relatively unaffected by the nature of the dietary fatty acid supplement, catecholamine-stimulated adenylate cyclase activity was significantly increased with dietary cholesterol supplementation and was positively correlated with the value of the membrane cholesterol/phospholipid ratio. Although the dissociation constant for the beta-adrenergic receptor, determined by [125I](-)-iodocyanopindolol binding, was unaffected by the nature of the dietary lipid supplement, the number of beta-adrenergic receptors was dramatically reduced by dietary cholesterol and negatively correlated with the value of the membrane cholesterol/phospholipid ratio. These results indicate that the activity of the membrane-associated beta-adrenergic/adenylate cyclase system of the heart can be influenced by dietary lipids particularly those altering the membrane cholesterol/phospholipid ratio and presumably membrane physico-chemical properties. In the face of these dietary-induced changes, a degree of homeostasis was apparent both with regard to membrane fatty acid composition in response to an altered membrane cholesterol/phospholipid ratio, and to down regulation of the beta-adrenergic receptor in response to enhanced catecholamine-stimulated adenylate cyclase activity.