Urothelial endocytic vesicle recycling and lysosomal degradative pathway regulated by lipid membrane composition

The urothelium, a specialized epithelium that covers the mucosa cell surface of the urinary bladder, undergoes dramatic morphological changes during the micturition cycle that involve a membrane apical traffic. This traffic was first described as a lysosomal pathway, in addition to the known endocytosis/exocytosis membrane recycling. In an attempt to understand the role of membrane lipid composition in those effects, we previously described the lipid-dependent leakage of the endocytosed vesicle content. In this work, we demonstrated clear differences in the traffic of both the fluid probe and the membrane-bound probe in urothelial umbrella cells by using spectrofluorometry and/or confocal and epifluorescence microscopy. Different membrane lipid compositions were established by using three diet formulae enriched in oleic acid, linoleic acid and a commercial formula. Between three and five animals for each dietary treatment were used for each analysis. The decreased endocytosis of both fluid and membrane-bound probes (approximately 32 and 49 % lower, respectively) in oleic acid-derived umbrella cells was concomitant with an increased recycling (approximately 4.0 and 3.7 times, respectively) and diminished sorting to the lysosome (approximately 23 and 37 %, respectively) when compared with the control umbrella cells. The higher intravesicular pH and the impairment of the lysosomal pathway of oleic acid diet-derived vesicles compared to linoleic acid diet-derived vesicles and control diet-derived vesicles correlate with our findings of a lower V-ATPase activity previously reported. We integrated the results obtained in the present and previous work to determine the sorting of endocytosed material (fluid and membrane-bound probes) into the different cell compartments. Finally, the weighted average effect of the individual alterations on the intracellular distribution was evaluated. The results shown in this work add evidences for the modulatory role of the membrane lipid composition on sorting of the endocytosed material. This suggests that changes in the membrane organization can be one of the underlying mechanisms for regulating the endocytosis/exocytosis processes and membrane intracellular trafficking.     

A residential study comparing the effects of diets rich in stearic acid, oleic acid, and linoleic acid on fasting blood lipids, hemostatic variables and platelets in young healthy men

Dietary fat is known to influence the variables of blood coagulation and fibrinolysis associated with vascular disease. However, the role of fat content and/or fat composition of the diet in this regard is still not well understood. In the present study, we investigated the effects of three isoenergic diets of differing fat composition in nine healthy young men in a strictly controlled residential study. Subjects consumed the three experimental diets for periods of 2 weeks each, separated by a washout period of at least 5 weeks in a randomized crossover design. The diets provided 38% of total energy intake as fat, 45% as carbohydrate, and 17% as protein, and differed only with respect to the fatty acid composition (stearic acid-rich diet: 34.1% stearic acid, 36.6% oleic acid; oleic acid-rich diet: 65.8% oleic acid; linoleic acid-rich diet: 36.5% linoleic acid, 38% oleic acid). Blood samples were collected at the beginning and at the end of each dietary period from fasted subjects for determination of factor VII coagulant activity (FVIIc), activated factor VII (FVIIa), factor VII antigen (FVIIag), tissue plasminogen activator (tPA) activity, plasminogen activator inhibitor type 1 (PAI-1) activity, fibrinogen, prothrombin fragment 1+2 (F(1+2)), and plasma lipids. There were no significant differences between diets in fasting plasma concentrations of FVIIc, FVIIa, FVIIag, fibrinogen, F(1+2), PAI-1 activity, and tPA activity. Plasma concentrations of lipids (high density lipoproteins, low density lipoproteins, triacylglycerols, and total cholesterol) were also unaffected. Although there were no changes in platelet aggregation response and membrane fluidity observed in any of the diets, increased anti-aggregatory prostaglandin E(1) binding to platelet membranes was observed only in the case of linoleic acid-rich diet. In conclusion, diets with very different fatty acid compositions, at 38% of energy as fat intake, did not significantly influence blood coagulation, fibrinolysis, or blood lipids in the fasting state in young healthy men.     

Effect of dietary fats on linoleic acid metabolism. A radiolabel study in rats

Effects on the linoleic acid metabolism in vivo of three dietary fats, rich in either oleic acid, trans fatty acids or alpha-linolenic acid, and all with the same linoleic acid content, were investigated in male Wistar rats. After 6 weeks of feeding, the rats were intubated with [1-14C]linoleic acid and [3H]oleic acid. The incorporation of these radiolabels into liver, heart and serum was investigated 2, 4, 8, 24 and 48 h after intubation. The amount of 14C-labelled arachidonic acid incorporated into the liver phospholipid of the group fed the oleic acid-rich diet was significantly higher than that of the other groups. However, compared to the trans fatty acids-containing diet, the oleic acid-rich diet induced only a slightly higher arachidonic acid level in the phospholipid fraction of the tissues as determined by GLC. Dietary alpha-linolenic acid more than halved the arachidonic acid levels. Our results do not support the hypothesis that the delta 6-desaturase system actually determines the polyunsaturated fatty acid levels in tissue lipids by regulating the amount of polyunsaturated fatty acids (e.g., arachidonic acid) synthesized. The biosynthesis of polyunsaturated fatty acids only is not sufficient to explain the complicated changes in fatty acid compositions as observed after feeding different dietary fats.     

Responses to oleic,linoleic and α-linolenic acids in high-carbohydrate,high-fat diet-induced metabolic syndrome in rats

We investigated the changes in adiposity, cardiovascular and liver structure and function, and tissue fatty acid compositions in response to oleic acid-rich macadamia oil, linoleic acid-rich safflower oil and α-linolenic acid-rich flaxseed oil (C18 unsaturated fatty acids) in rats fed either a diet high in simple sugars and mainly saturated fats or a diet high in polysaccharides (cornstarch) and low in fat. The fatty acids induced lipid redistribution away from the abdomen, more pronounced with increasing unsaturation; only oleic acid increased whole-body adiposity. Oleic acid decreased plasma total cholesterol without changing triglycerides and nonesterified fatty acids, whereas linoleic and α-linolenic acids decreased plasma triglycerides and nonesterified fatty acids but not cholesterol. α-Linolenic acid improved left ventricular structure and function, diastolic stiffness and systolic blood pressure. Neither oleic nor linoleic acid changed the left ventricular remodeling induced by high-carbohydrate, high-fat diet, but both induced dilation of the left ventricle and functional deterioration in low fat-diet-fed rats. α-Linolenic acid improved glucose tolerance, while oleic and linoleic acids increased basal plasma glucose concentrations. Oleic and α-linolenic acids, but not linoleic acid, normalized systolic blood pressure. Only oleic acid reduced plasma markers of liver damage. The C18 unsaturated fatty acids reduced trans fatty acids in the heart, liver and skeletal muscle with lowered stearoyl-CoA desaturase-1 activity index; linoleic and α-linolenic acids increased accumulation of their C22 elongated products. These results demonstrate different physiological and biochemical responses to primary C18 unsaturated fatty acids in a rat model of human metabolic syndrome.     

Differential Response of the Urothelial V-ATPase Activity to the Lipid Environment

The vesicle population beneath the apical plasma membrane of the most superficial urothelial cells is heterogeneous and their traffic and activity seems to be dependent on their membrane composition and inversely related to their development stage. Although the uroplakins, the major proteins of the highly differentiated urinary bladder umbrella cells, can maintain the bladder permeability barrier, the role of the membrane lipid composition still remains elusive. We have recently reported the lipid induced leakage of the vesicular content as a path of diversion in the degradative pathway. To extend the knowledge on how the lipid environment can affect vesicular acidification and membrane traffic through the regulation of the V-ATPase (vacuolar ATPase), we studied the proton translocation and ATP hydrolytic capacity of endocytic vesicles having different lipid composition obtained from rats fed with 18:1n-9 and 18:2n-6 fatty acid enriched diets. The proton translocation rate decreases while the enzymatic activity increases in oleic acid-rich vesicles (OAV), revealing an uncoupled state of V-ATPase complex which was further demonstrated by Western Blotting. A decrease of the very long fatty acyl chains length (C20–C24) and increase of the C16–C18 chains length in OAV membranes was observed, concomitant with increased hydrolytic activity of the V-ATPase. This response of the urothelial V-ATPase was similar to that of the Na–K ATPase when the activity of the latter was probed in reconstituted systems with lipids bearing different lengths of fatty acid chains. The studies describe for the first time a lipid composition-dependent activity of the urothelial V-ATPase, identified by immunofluorescence microscopy which is related to an effective coupling between the channel proton flux and ATP hydrolysis.     

Melittin-induced leakage from phosphatidylcholine vesicles is modulated by cholesterol: a property used for membrane targeting

Melittin, an amphiphathic peptide, affects the permeability of vesicles. This can be demonstrated using the dye release technique. Calcein, a fluorescent marker, is trapped in large unilamellar 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) vesicles and melittin-induced leakage of the dye can be monitored directly by increasing fluorescence intensity. First, we characterized the effect of increasing cholesterol content in the membrane on melittin-induced leakage and our results reveal that cholesterol inhibits the lytic activity of the peptide. Using intrinsic fluorescence of the single tryptophan of melittin and ²H-NMR of headgroup deuterated phosphatidylcholine, we demonstrated that the affinity of melittin for phosphatidylcholine vesicles is reduced in the presence of cholesterol; this is associated with the tighter lipid packing of the cholesterol-containing bilayer. This reduced binding is responsible for the reduced melittin-induced leakage from cholesterol-containing membranes. The pathway of release was determined to be an all-or-none mechanism. Finally, we investigated the possibility of achieving specific membrane targeting with melittin, when vesicles of different lipid composition are simultaneously present. Melittin incubated together with vesicles made of pure POPC and POPC containing 30(mol)% cholesterol can empty nearly all the cholesterol-free vesicles while the cholesterol-containing vesicles remain almost intact. Owing to the preferential interaction of melittin with the pure POPC vesicles, we were able to achieve controlled release of encapsulated material from a specific vesicle population. Received: 8 May 1996 / Accepted: 12 September 1996     

Plasma lipoprotein lipid and Lp[a] changes with substitution of elaidic acid for oleic acid in the diet.

The effect of additional dietary trans fatty acids (7% energy) on plasma lipids was assessed in a double-blind comparison of four separate diets: 1, enriched with butter fat (lauric-myristic-palmitic); 2, oleic acid-rich; 3, elaidic acid-rich; 4, palmitic acid-rich. The total dietary period was 11 weeks and comprised normal foods plus specific fat supplements. In 27 mildly hypercholesterolemic men, total and LDL cholesterol were significantly lower during the 3-week oleic acid-rich diet, and were similar during the other three diets. For the four diets LDL cholesterol levels were in mg/dl: 1, 163; 2, 151; 3, 165; 4, 161. HDL cholesterol was significantly higher with the palmitic acid-rich diet, 42 mg/dl, compared with elaidic acid, 38 mg/dl, which in turn was not lower than with oleic acid, 38 mg/dl. Plasma elaidic acid concentration rose seven-fold with the trans fatty acid diet but did not increase the vulnerability of LDL to oxidative change. The elaidic acid-rich diet led to significant elevations in the level of Lp[a] compared to all the other test diets. The Lp[a] level increased to 296 +/- 220 U/l in the elaidic acid-rich period from 235 +/- 182 (mean +/- SD) in the first ("butter") period (P less than 0.001) compared with 249 +/- 204 in the palmitic acid period (P less than 0.001) and 236 +/- 201 in the oleic acid period (NS).(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane lipids and proteins as modulators of urothelial endocytic vesicles pathways

The increased studies on urinary bladder umbrella cells as an important factor for maintaining the permeability barrier have suggested new pathways for the discoidal/fusiform endocytic vesicles which is one of the main features of the umbrella cells. The biological role of these vesicles was defined, for many years, as a membrane reservoir for the umbrella cell apical plasma membrane which are subject to an increased tension during the filling phase of the micturition cycle and, therefore, the vesicles are fused with the apical membrane. Upon voiding, the added membrane is reinserted via a non-clathrin or caveolin-dependant endocytosis thereby restoring the vesicle cytoplasmic pool. However, in the last decade, new evidence appeared indicating alternative pathways of the endocytic vesicles different than the cycling process of exocytosis/endocytosis. The purpose of this review is to analyze the molecular modulators, such as membrane lipids and proteins, in the permeability of endocytic vesicles, the sorting of endocytosed material to lysosomal degradation pathway and recycling of both membrane and fluid phases.     

Changes in plasma membrane lipids, aquaporins and proton pump of broccoli roots, as an adaptation mechanism to salinity

Salinity stress is known to modify the plasma membrane lipid and protein composition of plant cells. In this work, we determined the effects of salt stress on the lipid composition of broccoli root plasma membrane vesicles and investigated how these changes could affect water transport via aquaporins. Brassica oleracea L. var. Italica plants treated with different levels of NaCl (0, 40 or 80 mM) showed significant differences in sterol and fatty acid levels. Salinity increased linoleic (18:2) and linolenic (18:3) acids and stigmasterol, but decreased palmitoleic (16:1) and oleic (18:1) acids and sitosterol. Also, the unsaturation index increased with salinity. Salinity increased the expression of aquaporins of the PIP1 and PIP2 subfamilies and the activity of the plasma membrane H⁺-ATPase. However, there was no effect of NaCl on water permeability (P_f) values of root plasma membrane vesicles, as determined by stopped-flow light scattering. The counteracting changes in lipid composition and aquaporin expression observed in NaCl-treated plants could allow to maintain the membrane permeability to water and a higher H⁺-ATPase activity, thereby helping to reduce partially the Na⁺ concentration in the cytoplasm of the cell while maintaining water uptake via cell-to-cell pathways. We propose that the modification of lipid composition could affect membrane stability and the abundance or activity of plasma membrane proteins such as aquaporins or H⁺-ATPase. This would provide a mechanism for controlling water permeability and for acclimation to salinity stress.     

The effect of diet on the fatty acid compositions of serum, brain, brain mitochondria and myelin in the rat

1. Three groups of female rats (8-12 weeks old) were maintained respectively on a linoleic acid-rich diet, a linoleic acid-poor predominantly saturated-fatty acid diet and a normal diet. Changes in the fatty acid compositions of serum, brain, brain mitochondria-rich fraction and myelin were observed. 2. Of the serum fatty acids, linoleic acid showed the greatest change in the percentage of the total acids in response to diet; the change in the proportion of oleic acid was considerable. The percentages of arachidonic acid in serum fatty acids in the groups on the linoleic acid-rich and linoleic acid-poor diets were similar, but higher than those in the normal group. 3. Changes in the proportions of linoleic acid, arachidonic acid and docosahexaenoic acid occurred in brain fatty acids that to some extent paralleled those occurring in the serum. Changes in the proportions of most other acids in the serum fatty acids were not accompanied by corresponding changes in the brain fatty acids. 4. The percentage fatty acid compositions of a mitochondria-rich fraction and myelin are given, and changes in the relative proportions of linoleic acid, arachidonic acid and possibly some docosapolyenoic acids were demonstrated to occur as a result of diet. 5. The results are discussed in relation to the possible aetiology of multiple sclerosis.     

Leakage of membrane vesicle contents: determination of mechanism using fluorescence requenching.

Agents such as antimicrobial peptides and toxins can permeabilize membrane vesicles to cause leakage of entrapped contents in either a graded or an all-or-none fashion. Determination of which mode of leakage is induced is an important step in understanding the molecular mechanism of membrane permeabilization. Wimley et al. (1994, Protein Sci. 3:1362-1378) have developed a fluorescence method for distinguishing the two modes that makes use of the dye/quencher pair 8-aminonapthalene-1,3,6 trisulfonic acid (ANTS)/p-xylene-bis-pyridinium bromide (DPX) without the usual need for the physical separation of vesicles from released contents. Their "requenching" method establishes the mode of release through the fluorescence changes that occur when DPX is added externally to a solution of vesicles that have released some fraction of their contents. However, the requenching method as originally stated ignored the possibility of preferential release of dye or quencher. Here we extend the theory of the method to take into account preferential release and the effects of graded leakage. The ratio of the rates of release of the cationic quencher DPX and anionic dye 8-aminonapthalene-1,3,6 trisulfonic acid can be estimated by means of the theory. For graded leakage, we show that the release of the markers does not coincide with the fluorescence changes observed in the standard leakage assay. This is true for self-quenching dyes as well and means that 1) the amount of released material will be overestimated and 2) the kinetics will be nonexponential and have artificially high apparent rates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Higher membrane fluidity mediates the increased subcutaneous fatty acid content in pigs fed reduced protein diets

The production of pork with moderate amounts of intramuscular fat (IMF) without an increase in subcutaneous fat is highly desirable for the meat industry. Several studies indicate that dietary protein reduction during the growing–finishing period of pigs enhances IMF content, but its consequence on carcass fat deposition is still contradictory. In this study, we hypothesized that the effects of reduced protein diets (RPD), corrected or not with the limiting amino acid lysine, on subcutaneous fat deposition from pigs with distinct genotypes are mediated by adipose membranes biophysical properties. In total, 36 crossbred (Large White×Landrace×Pietrain – a lean genotype) and purebred (Alentejana breed – a fatty genotype) male pigs were randomly assigned to the control group, the RPD group or the reduced protein diet equilibrated for lysine (RPDL) group, allowing a 2×3 factorial arrangement (n=6). Backfat thickness and total fatty acid content were higher in Alentejana relative to crossbred pigs. Although dietary treatments did not change backfat thickness, RPD and RPDL increased total fatty acids content of subcutaneous fat. In order to understand this effect, adipose tissue membranes isolated from pig’s subcutaneous fat were assayed for glycerol permeability and fluidity, using 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-(trimethylamino)-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) probes. The glycerol transport across adipose membranes was not mediated by aquaglyceroporins and remained unchanged across dietary groups. Regardless of lysine correction, RPD increased membrane fluidity at the hydrocarbon region (lower DPH fluorescence anisotropy) in both genotypes of pigs. This result was associated with a lower ratio between oleic acid and linoleic acid on membrane’s fatty acid composition. Adipose membrane’s cholesterol content was independent from genotype and diet. Taken together, the present study shows that dietary protein reduction is successful in maintaining backfat thickness, although a negative side effect was observed on total fatty acids in subcutaneous fat, which may be due to changes in the fluidity of adipose membranes.     

Molecular species analysis of 1,2-diacylglycerol released in response to progesterone binding to the amphibian oocyte plasma membrane

Progesterone, the physiological inducer of amphibian meiosis, acts within minutes at plasma membrane receptors of the Rana pipiens oocyte to release 1,2-diacylglycerol (DAG) from plasma and intracellular membranes. High-performance liquid chromatography (HPLC) analysis of lipid extracts of uninduced oocytes indicates the presence of at least three classes of DAG with a total DAG content of about 150 micromol/kg wet weight. Within 3-5 min after exposure to progesterone, there was a differential increase in all three DAG classes with a twofold increase in total DAG by 10 min. The fatty acid composition of the DAGs in uninduced and progesterone-stimulated oocytes was compared using thin layer chromatographic analysis of lipid extracts from oocytes double-labeled with [14C] or [3H]glycerol and [14C] or [3H]fatty acids. The ratio of labeled fatty acid/labeled glycerol was measured in phosphatidylcholine (PC), phosphatidylinositol (PI) and DAG. The linoleic (18:2) or arachidonic (20:4) acid/glycerol ratios in basal DAG were low compared to that in PC or PI. In contrast, the myristic (14:0), palmitic (16:0) or oleic (18:1) acid/glycerol ratios in basal DAG were relatively high compared to the ratio in PC and PI. A transient increase in both linoleic and palmitic acid labeling of DAG occurred within the first 1-2 min in progesterone-treated oocytes, followed by a return to or below the basal level. Arachidonic and myristic acid labeling of DAG fall within the first minute after progesterone treatment, followed by a sustained rise over the next 10 min. The [3H]oleic acid/[14C]glycerol ratio of DAG does not change significantly following exposure to progesterone. Pretreatment with a phospholipid N-methylation inhibitor (2-methylaminoethane) precluded the rise in linoleic and palmitic acid-rich DAG, whereas pretreatment with a diglyceride kinase inhibitor (D102) produced a sustained elevation of linoleic and palmitic acid-rich DAG. These results indicate that the DAG released in response to progesterone is composed of multiple new molecular species of DAG and that both the palmitate and linolate-rich forms are rapidly phosphorylated to form phosphatidic acid (PA). The newly formed DAG species differ from the basal DAG species and reflect sequential activation of sphingomyelin (SM) synthase, PC-specific phospholipase D (PLD) and PI-specific phospholipase C in response to progesterone, which we have described previously.     

TNF-α mediates the stimulation of sclerostin expression in an estrogen-deficient condition

Obesity consists in fat accumulation leading to increase in adipose cells number and size. Adipocyte membrane biophysical properties are critical to maintain cellular viability in metabolically healthy obesity. This study investigated the effect of the genetic background and dietary protein restriction on fat tissue lipid composition, adipocyte membrane fluidity and water permeability using the pig as experimental model. Twenty-four male pigs from distinct genotypes, lean and obese, were fed on normal and reduced protein diets within a 2 × 2 factorial arrangement (two genotypes and two diets). Backfat thickness was twofold higher in obese than in lean pigs but unrelated to dietary protein level. In contrast, total fatty acids in the subcutaneous adipose tissue were dependent on both breed and diet, with increased lipid content promoted by the fatty genotype and by the restriction of dietary protein. Adipose membranes isolated from obese pig's subcutaneous fat tissue showed higher permeability to water, in line with an increased fluidity. Moreover, the reduced content of dietary protein influenced positively the fluidity of adipose membranes. Neither genotype nor diet affected total cholesterol concentration in the adipose membranes. Membrane-saturated fatty acids' content was influenced by genotype, while membrane-polyunsaturated fatty acids, particularly from the n-6 family, was influenced by diet. The ratio of oleic (18:1c9)/linoleic (18:2n-6) acids was positively correlated with membrane fluidity. All together, these findings reinforce the genetic background as a determinant player on adipose membrane biophysical properties and point to the dietary protein level as an important factor for subcutaneous lipid deposition as well as for regulation of membrane function, factors that may have impact on human obesity and metabolic syndrome.     

Significance of non-esterified fatty acids in iron uptake by intestinal brush-border membrane vesicles

Iron uptake from Fe/ascorbate by mouse brush-border membrane vesicles is not greatly inhibited by prior treatment with a variety of protein-modification reagents or heat. Non-esterified fatty acid levels in mouse proximal small intestine brush-border membrane vesicles show a close positive correlation with initial Fe uptake rates. Loading of rabbit duodenal brush-border membrane vesicles with oleic acid increases Fe uptake. Depletion of mouse brush-border membrane vesicle fatty acids by incubation with bovine serum albumin reduces Fe uptake. Iron uptake by vesicles from Fe/ascorbate is enhanced in an O2-free atmosphere. Iron uptake from Fe/ascorbate and Fe3+-nitrilotriacetate (Fe3+-NTA) were closely correlated. Incorporation of oleic acid into phosphatidylcholine/cholesterol (4:1) liposomes leads to greatly increased permeability to Yb3+, Tb3+, Fe2+/Fe3+ and Co2+. Ca2+ and Mg2+ are also transported by oleic acid-containing liposomes, but at much lower rates than transition and lanthanide metal ions. Fe3+ transport by various non-esterified fatty acids was highest with unsaturated acids. The maximal transport rate by saturated fatty acids was noted with chain length C14-16. It is suggested that Fe transport can be mediated by formation of Fe3+ (fatty acid)3 complexes.     

Visualization of endocytosed markers in freeze-fracture studies of toad urinary bladder

Antidiuretic hormone (ADH) stimulation increases the apical membrane water permeability of granular cells in toad urinary bladder. This response correlates closely with the fusion of tubular cytoplasmic vesicles with the membrane and delivery of intramembrane particle (IMP) aggregates from the tubules (aggrephores) to the apical membrane. These aggregates are believed to be associated with the channels responsible for the water permeability increase. Removal of ADH triggers apical membrane endocytosis and disappearance of aggregates from the apical membrane. However, it has been unclear whether aggregate disappearance is due to disassembly of aggregates within the apical membrane or to their endocytic retrieval as intact structures. Using colloidal gold and horseradish peroxidase to follow endocytosis from the apical surface after ADH removal, we have directly observed in cross-fractured bladder cells the intramembrane structure of intracellular vesicles that contain these fluid-phase markers. Under these conditions, intact aggregates can be identified in the membrane of tubular endocytosed vesicles. This directly demonstrates that conditions which lower apical membrane water permeability cause the tubular aggrephores to "shuttle" intact aggregates from the apical membrane back into the cytoplasm. An additional population of vesicles with tracer are found which are spherical and display structural features of the apical membrane, as well as occasional aggregates. These vesicles may be responsible for retrieval of aggregates from the surface apical membrane.     

Comparison of cis and trans fatty acid containing phosphatidylcholines on membrane properties

The ever-increasing amount of trans fatty acids in the human diet has been linked to a variety of afflictions, most notably coronary heart disease and arteriosclerosis. The mechanism of why the replacement of cis fatty acids with their trans counterparts can be detrimental to the health of an individual remains a mystery. Here, we compare the differences in membrane physical properties including molecular dynamics, lateral lipid packing, thermotropic phase behavior, "fluidity", lateral mobility, and permeability between model membranes (lipid monolayers and bilayers) composed of cis- and trans-containing phosphatidylcholines (PCs). The PCs tested have a total of zero, one, two, or four cis (oleic or linoleic) or trans (elaidic or linoelaidic) double bonds. These experiments all confirm the basic hypothesis that trans fatty acids produce membrane properties more similar to those of saturated chains than to those of acyl chains containing cis double bonds; i.e., cis double bonds induce much larger membrane perturbations than trans double bonds.     

Effect of dietary alpha-linolenate on platelet-activating factor production in rat peritoneal polymorphonuclear leukocytes.

The effects of dietary alpha-linolenate (18:3, n-3) and linoleate (18:2, n-6) on platelet-activating factor (PAF) production were examined. Rats were fed an alpha-linolenic acid-rich (perilla oil) diet or a linoleic acid-rich (safflower oil) diet for 6 wk, and polymorphonuclear leukocytes (PMN) were elicited by peritoneal injection of casein. The overall phospholipid content and composition as well as the subclass distribution of choline and ethanolamine glycerophospholipids in PMN were not altered by these diets. However, with the perilla oil diet their content of a putative precursor of PAF, 1-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine was approximately 50% of that with safflower oil diet. On exposure to various concentrations of FMLP, PAF formation by PMN in the perilla oil group was less than 50% of that by PMN in the safflower oil group. A larger difference in PAF productions by PMN in the two dietary groups was observed on their stimulation with calcium ionophore A23187. These results demonstrate that PAF production is modulated in some as yet unknown way by changing the alpha-linolenate/linoleate balance of the diet.     

Effects on plasma lipoproteins of monounsaturated, saturated, and polyunsaturated fatty acids in the diet of African green monkeys

Work by other investigators has shown that an increase in dietary content of monounsaturated fatty acids can result in a decreased plasma low density lipoprotein (LDL) cholesterol concentration. This observation, combined with the epidemiologic evidence that monounsaturated fat-rich diets are associated with decreased rates of death from coronary heart disease, suggests that inclusion of increased amounts of mono-unsaturated fat in the diet may be beneficial. The present study was carried out in a primate model, the African green monkey, to evaluate the effects of dietary monounsaturated fat on plasma lipoprotein cholesterol endpoints. Two study periods were carried out in which the fatty acid compositions of the experimental diets were varied. All diets contained 35% of calories as fat. In the first experimental period, a mixture of fats was used to set the dietary fatty acid composition to be approximately 50-60% of the desired fatty acid, either saturated, monounsaturated, or polyunsaturated (n-6). In the second experimental period, pure fats were used (palm oil, oleic acid-rich safflower oil, and linoleic acid-rich safflower oil) to maximize the difference in fatty acid composition. The effects of the more exaggerated dietary fatty acid differences of period 2 were similar to those that have been reported in humans. For the group fed the diet enriched in monounsaturated fat compared to saturated fat, whole plasma and LDL cholesterol concentrations were significantly lower while high density lipoprotein (HDL) cholesterol concentrations were not affected. For the group fed the diet enriched in polyunsaturated fat compared to saturated fat, both LDL and HDL cholesterol concentrations were significantly lower than in the group fed saturated fat. LDL cholesterol concentrations were comparable in the monounsaturated and polyunsaturated fat groups and the percentage of cholesterol in LDL was lowest in the monounsaturated fat fed group. Trends were similar for the mixed fat diets, although no statistically significant differences in plasma lipoprotein endpoints could be attributed to monounsaturated fatty acids in this dietary comparison. Since effects on plasma lipoproteins similar to those seen in humans were identified in this primate model, relevant mechanisms for the effects of dietary fatty acids on lipoprotein endpoints related to coronary artery atherosclerosis, per se, can subsequently be examined.     

Influence of dietary partially hydrogenated fat high in trans fatty acids on lipid composition and function of intestinal brush border membrane in rats

The effect of dietary hydrogenated fat (Indian vanaspati) high in trans fatty acids (6 en%) on lipid composition, fluidity and function of rat intestinal brush border membrane was studied at 2 and 8 en% of linoleic acid. Three groups of weanling rats were fed rice-pulse based diet containing 10% fat over a ten week period: Group I (groundnut oil), Group II (vanaspati), Group III (vanaspati + safflower oil). The functionality of the brush border membrane was assessed by the activity of membrane bound enzymes and transport of D-glucose and L-leucine. The levels of total cholesterol and phospholipids were similar in all groups. The data on fatty acid composition of membrane phospholipids showed that, at 2 en% of linoleic acid in the diet, trans fatty acids lowered arachidonic acid and increased linoleic acid contents indicating altered polyunsaturated fatty acid metabolism. Alkaline phosphatase activity was increased while the activities of sucrase, gamma-glutamyl transpeptidase and transport of D-glucose and L-leucine were not altered by dietary trans fatty acids. However at higher intake of linoleic acid in the diet, trans fatty acids have no effect on polyunsaturated fatty acid composition and alkaline phosphatase activity of intestinal brush border membrane. These data suggest that feeding dietary fat high in trans fatty acids is associated with alteration in intestinal brush border membrane polyunsaturated fatty acid composition and alkaline phosphatase activity only when the dietary linoleic acid is low.