Lovastatin increases arachidonic acid levels and stimulates thromboxane synthesis in human liver and monocytic cell lines.

The effect of lovastatin (LOV), the inhibitor of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase, on linoleic acid (LA, 18:2n-6) metabolism was examined in human monocytic Mono Mac 6 (MM6) and hepatoma Hep G2 cells. The desaturation of LA was examined after LOV (72 h, 10 microM) or dimethylsulfoxide (LOV carrier, < 0.1%) and [14C]LA (last 18 h, 0.3 microCi, 5 microM). In both cell lines, LOV reduced the percentage of 14C label associated with LA and increased the percentage of label in the 20:4n-6 and the 22:5n-6 fractions. In Hep G2 but not MM6 cells, this effect was fully reversible by means of coincubation with mevalonic acid (500 microM), but not with cholesterol or lipoproteins. In both cell lines, the LOV-mediated increase in LA desaturation resulted in dose-dependent reductions of LA and elevations of AA in cellular phospholipids. The lipids secreted by LOV-treated Hep G2 cells were also enriched in arachidonic acid (AA). In the MM6 cells, LOV increased release of thromboxane upon stimulation with the calcium ionophore A23187. In summary, our findings of higher LA desaturation and AA enrichment of lipids secreted by the Hep G2 cells suggest that LOV treatment may increase the delivery of AA from the liver to extrahepatic tissues. The changes in membrane fatty acid composition can influence a variety of cellular functions, such as eicosanoid synthesis in monocytic cells. The mechanism appears to be related to the reduced availability of intermediates of cholesterogenesis.

Differential effects of polyunsaturated fatty acids on cell growth and differentiation of premonocytic U937 cells.

The effect of long chain polyunsaturated fatty acids (PUFA) on cell growth and differentiation was assessed in human premonocytic U937 cells. Addition of either 10 microM arachidonic acid (AA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3) or docosahexaenoic acid (DHA, 22:6n-3) resulted in the rapid incorporation of these fatty acids into cellular phospholipids. Their uptake was greatest in the first 2 h. AA and EPA reached steady-state levels after 8 h, while levels of DHA increased steadily over 72 h. In parallel, fatty acid metabolites derived from AA and EPA, 22:4n-6, 22:5n-6 and 22:5n-3, 22:6n-3, respectively, increased continuously indicating an active fatty acid elongation and desaturation. The effects of PUFA on monocytic differentiation were examined in cells which had been enriched with AA, EPA or DHA for 8 h and subsequently treated with retinoic acid (RA), 1,25-(OH)2-vitamin D3 (1,25-D3), interferon-gamma (IFN-gamma) or their combinations for 72 h. Growth of differentiating or non-differentiating U937 cells was not affected by enrichment with PUFA. However, in cells differentiated with 1,25-D3 plus IFN-gamma, prior enrichment with all three PUFA slightly but significantly (P < 0.05) increased the expression of the monocytic surface antigens CD11b and CD14 and generation of superoxide anion. The data indicate that although n-6 and n-3 PUFA are rapidly incorporated into phospholipids, they do not affect cell growth. However, enrichment with PUFA increases monocytic differentiation of U937 cells when induced most effectively with 1,25-D3 plus IFN-gamma.

Cholesterol modulates PAF-stimulated Ca2(+)-mobilization in monocytic U937 cells.

We investigated the effect of cellular cholesterol content on platelet activating factor (PAF)-stimulated Ca2+ mobilization in the human monocytic cell line U937. When cholesterol auxotroph U937 cells were depleted of cellular cholesterol by a 48-h incubation in delipidated medium, a 40% reduction in the PAF (100 nM)-stimulated increase in cytosolic Ca2+ concentration was seen. Ca2+ mobilization following stimulation with LTD4 (10 nM) or ATP (10 microM) was not affected. Addition of LDL (100 micrograms/ml, 24 h) to the delipidated medium completely recovered cellular cholesterol content and PAF-induced Ca2+ mobilization. These two LDL effects had very similar time- and dose-dependences. Partial recoveries of PAF-induced Ca2+ mobilization, seen after addition of pure cholesterol dissolved in ethanol (30 micrograms/ml, 24 h) or acetyl-LDL (100 micrograms/ml, 24 h), were associated with partial recoveries of cellular cholesterol content. Our results indicate that cellular cholesterol influences PAF-stimulated events in monocytic cells.

Involvement of the GTPase Rho in the cellular uptake of low density lipoprotein by human skin fibroblasts.

Members of the Rho subfamily of small GTPases have been implicated in the regulation of endocytosis of ligand/receptor complexes localised to clathrin-coated pits. In this paper, we investigated the role of Rho A in the post-receptor regulation of cellular uptake and metabolism of native low density lipoprotein (LDL) by primary human skin fibroblasts. Incubations of cells with the selective Rho GTPase inhibitor C3-transferase (C3T) upregulated the binding, lysosomal degradation and cell accumulation of labelled LDL. The rate of internalisation of surface-bound LDL was also higher in C3T-treated cells. Single cell injections with C3T and dominant active V14Rho confirmed the negative regulation of LDL uptake by Rho. While cells injected with C3T internalised more 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diI)-labelled LDL, diI-LDL internalisation was dramatically suppressed in cells injected with the constitutively active V14Rho. The negative regulation of LDL uptake by Rho appeared to be independent of changes in the actin cytoskeleton. An increasing number of naturally occurring toxins and serum factors have been shown to influence Rho GTPase signalling cascades. The herein described post-translational regulation of LDL internalisation may modulate cell events occurring subsequent to cellular lipoprotein uptake.

Linoleic acid esterified in low density lipoprotein serves as substrate for increased arachidonic acid synthesis in differentiating monocytic cells.

The cellular metabolism of albumin- and lipoprotein-bound 18:2(n - 6) during monocytic differentiation was examined in the human premonocytic U937 and Mono Mac 6 cells. Differentiation for 72 h of U937 cells with retinoic acid (RA, 1 microM) or 1,25-(OH)2-vitamin D3 (1,25-D3, 10 nM) and of Mono Mac 6 cells with RA (1 microM) or lipopolysaccharide (LPS, 10 ng/ml) increased the desaturation and elongation of [1-14C]18:2(n - 6) to [1-14C]20:4(n - 6). In undifferentiated U937 and Mono Mac 6 cells, incubations with human LDL (100 micrograms/ml, 18 h) resulted in a 2.5-fold increase in 18:2(n - 6) levels in the cellular phospholipids. Differentiation of U937 cells with RA or or of Mono Mac 6 cells with LPS prior to LDL addition. Significantly reduced 18:2(n - 6) and elevated 20:4(n - 6) levels in cellular phospholipids. This increase in 20:4(n - 6) was likely not due to an increased incorporation of preformed 20:4(n - 6) esterified in LDL, as the receptor-specific degradation of [125I]LDL was reduced in both the RA-treated U937 and LPS-treated Mono Mac 6 cells. In U937 cells incubated with [1-14C]18:2(n - 6), the synthesis of TXB2, PGE2 and HHT could be detected after differentiation with RA. suggesting the availability of [1-14C]20:4(n - 6), derived from [1-14C]18:2(n - 6), for cyclooxygenase metabolism. Our results show that the conversion of 18:2(n - 6) to 20:4(n - 6) increases during monocyte differentiation. The 18:2(n - 6) supplied to the cells via the receptor-mediated uptake of LDL was utilized as substrate for the increased 20:4(n - 6) synthesis.

Increased cellular triglyceride levels in human monocytic and rat smooth muscle cells after lovastatin.

Beta-hydroxy-beta-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors reduce plasma LDL cholesterol by upregulating hepatic LDL receptors. However, their effects on lipid metabolism in extrahepatic cells may also contribute to their therapeutic benefit. We examined the effects of lovastatin (LOV) on cellular lipid levels in the human monocytic Mono Mac 6sr and cultured rat smooth muscle cells. In both cell types, LOV produced a dose-dependent increase in cellular triglycerides. This increase was observed in cells grown in the absence of exogenous lipids in the culture medium, but was more pronounced after additions of oleic acid (50 to 200 microM) and VLDL (50 to 200 microg ml-1). In Mono Mac 6sr cells grown in medium containing 10% delipidated FCS for the last 16 h, the LOV-induced rise in triglyceride levels was completely reversed by 2 mM mevalonic acid and was associated with a decrease in cellular cholesterol. However, when cells were maintained in lipoprotein-replete medium, the LOV-induced rise in triglycerides did not correlate with cellular cholesterol. LOV also reduced cellular cholesterol esterification and increased the synthesis of fatty acids and their incorporation into triglycerides and phospholipids. Increased triglyceride levels were also seen in Mono Mac 6sr cells treated with the lanosterol demethylase inhibitor RS-21607 and the acylcoenzyme A:cholesterol acyltransferase inhibitor SaH 58035. Our findings suggest that the LOV-induced triglyceride accumulation involves changes in intracellular cholesterol pools regulating cellular fatty acid concentrations. Although decreased cholesterol levels in cells participating in plaque formation are beneficial, the impact of the herein described shift in intracellular neutral lipid metabolism on other cellular functions warrants further investigation.

Retina fatty acid composition of piglets fed from birth with a linoleic acid-rich vegetable-oil formula for infants.

The effects of a vegetable-oil-based formula containing 30% 18:2n-6 (18:2 omega-6), 0.8% 18:3n-3, and no n-6 or n-3 long-chain polyunsaturated fatty acids (LCPs) on retina total lipid, ethanolamine phosphoglyceride (EPG), and phosphatidylcholine (PC) fatty acid composition were studied in neonatal piglets. Term-gestation piglets were fed sow milk (SMF) or the formula (FF) from birth for 5, 10, 15, or 25 d. After 25 d feeding, the 22:6n-3 was reduced by 24% in total lipid, 20% in EPG, and 28% in PC of retinas of FF relative to SMF piglets. A compensatory increase in 22:4n-6 and 22:5n-6 concentrations occurred in retina total lipid, EPG, and PC of FF animals. The data suggest that the exclusive feeding of formulas devoid of LCPs and high in 18:2n-6 and/or 18:2n-6 and 18:3n-3 compromises normal accretion of 22:6n-3 in neonatal piglet retina. The potential reversibility of these changes or their effects on vision are not known.

Effect of a vegetable oil formula rich in linoleic acid on tissue fatty acid accretion in the brain, liver, plasma, and erythrocytes of infant piglets.

The effect of feeding sow-milk formula (SMF) or a vegetable-oil infant formula (FF) with minimal n-6 and n-3 long-chain polyenoic fatty acids (LCPs) but high linoleic acid (18:2n-6) and a high ratio of 18:2n-6 to linolenic acid (18:3n-3) on the fatty acids of brain lipid and liver, plasma, and red cell phospholipids was studied in piglets fed from birth for 5, 10, 15, or 25 d. Compared with SMF, FF reduced the concentrations of 18:1 and n-3 LCPs, especially 22:6n-3, in all tissues and increased 22:4n-6 in brain, liver, plasma, and red cell phosphatidylethanolamine. FF also increased 22:5n-6 in brain lipid, liver, and plasma but not in red cell phospholipids. Thus, changes in tissues capable of in situ desaturation were not completely reflected in the red cell phospholipids. The increased liver and brain n-6 LCP accretion in the FF piglets may suggest competent desaturation and possible inhibition of n-3 desaturation and/or acylation by dietary n-6 fatty acids.

Menstrual cycle effects on the metabolism of tryptophan loads.

The metabolism of tryptophan (Trp) was examined during the follicular and luteal phases of the human menstrual cycle. Eight healthy women were administered capsulated Trp (3 g) or placebo (3 g lactose) during two follicular and two luteal phases of their menstrual cycles. Trp loading resulted in increased plasma concentrations of Trp and kynurenine, in an increase in the ratio of Trp to neutral amino acids in plasma, and in an increase in urinary excretion of Trp and kynurenine at both phases of the menstrual cycle. However at 3 h after Trp ingestion, plasma kynurenine levels in the luteal phase (23.6 +/- 3.1 mumol/L) were 40% higher than in the follicular phase (16.7 +/- 1.1 mumol/L) (p less than 0.05). Urinary kynurenine excretion in the luteal phase (81.6 +/- 14.4 mumol/24 h) was 28% greater than in the follicular phase (63.9 +/- 13.0 mumol/24 h) (p less than 0.05). The results indicate that the catabolism of Trp via the kynurenine pathway is affected by the phase of the human menstrual cycle.

Brain synaptosomal, liver, plasma, and red blood cell lipids in piglets fed exclusively on a vegetable-oil-containing formula with and without fish-oil supplements.

Clinical studies showed that a decrease in red blood cell 22:6n-3 caused by feeding infants formula (F) can be prevented by supplementation with fish oil (F + O). It is not known whether fish-oil supplementation is able to support normal accretion of fatty acids with greater than or equal to 20 carbons (LCPs) in the brain. Therefore piglets were fed exclusively F + O, F, or sow milk (SM) for 15 d and their liver and brain synaptosomal fatty acids were determined. Feeding F + O corrected the low n-3 LCP in the liver phospholipid (PL) and synaptosomal phosphatidylethanolamine (PE) of piglets fed F compared with SM. An apparent compensatory increase in n-6 LCPs in liver PL and synaptosomal PE of piglets fed F compared with SM was suppressed by feeding F + O. F + O also reduced the ratio of plasma PL 20:4n-6 to 20:5n-3, important for eicosanoid metabolism. Supplementation of F with n-3 LCPs as fish oil, without n-6 LCPs, at levels giving normal brain n-3 LCP, may alter n-6 LCP accretion.

Comparison of breast-feeding and formula feeding on intestinal and hepatic cholesterol metabolism in neonatal pigs.

Infant formulas (IFs) contain reduced cholesterol concentrations compared with breast milk (SM); how neonatal cholesterol metabolism responds to this difference is largely unknown. The effect of exclusive feeding of SM vs low-cholesterol IF on intestinal and hepatic cholesterol concentrations and synthesis during early postnatal development were compared in piglets. Animals were killed at birth or on days 5, 10, 15, or 25 postpartum. Plasma cholesterol concentrations were higher in SM-fed than in IF-fed piglets on days 15 and 25. In intestine both HMG-CoA reductase activity and 3H2O incorporation rates into cholesterol were similar for both groups or reduced in the IF-fed group at days 15 and 25. In liver, HMG-CoA reductase activity was higher in IF-fed than in SM-fed piglets on days 5, 10, and 15. Results indicate that during the early postpartum period, response to lower cholesterol intakes with IF occurs by increasing hepatic sterol synthesis whereas intestinal synthesis is largely unaffected.

The expression of the lectin-like oxidized low-density lipoprotein receptor (LOX-1) on human vascular smooth muscle cells and monocytes and its down-regulation by lovastatin.

Accumulation of oxidatively modified low-density lipoprotein (oxLDL) in the vascular wall is a characteristic feature of atherosclerosis. oxLDL can be taken up into monocytes, smooth muscle cells, and endothelial cells by several known scavenger receptors such as scavenger receptor class A I and II, CD36, and CD68. A new lectin-like oxLDL receptor (LOX-1) was recently found in bovine and human endothelial cells. We studied whether LOX-1 is also expressed in other cells present in the atherosclerotic lesion and whether its expression can be modified. We found LOX-1 expression in human blood monocytes, umbilical smooth muscle and endothelial cells, and 3T3 fibroblasts. LOX-1 mRNA expression in monocytes could be significantly suppressed by lovastatin. Thus, LOX-1 expression is not restricted to endothelial cells and its down-regulation by HMG-CoA reductase inhibitors could contribute to the clinical benefits of these drugs.

Enzymatically modified low-density lipoprotein upregulates CD36 in low-differentiated monocytic cells in a peroxisome proliferator-activated receptor-gamma-dependent way.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) has been suggested to upregulate CD36. Since free oxidized polyunsaturated fatty acids are PPARgamma ligands, we studied the effects of LDL modified by the simultaneous action of sPLA2 and 15-lipoxygenase (15LO) on CD36 expression and PPARgamma activation in monocytic cells. Exposure of MM6 cells, which do not express CD36 or other scavenger receptors, to such enzymatically modified LDL (enzLDL) resulted in upregulation of CD36 surface protein and mRNA expression. Similar effects were observed with free 13-hydroperoxyoctadecadienoic acid but not its esterified counterpart. Less pronounced effects were observed with LDL modified by 15LO alone. Upregulation of CD36 was inversely correlated to the state of cell differentiation, as showed by lower response to enzLDL of the scavenger receptor-expressing MM6-sr and THP1 cells. Importantly, LDL modified by sPLA2 and 15LO did not efficiently induce upregulation CD36 in PPARgamma-deficient macrophage-differentiated embryonic stem cells confirming a role of PPARgamma in CD36 expression in cells stimulated with enzLDL. Our data show that LDL modified with physiologically relevant enzymes stimulates CD36 expression in non-differentiated monocytes and that this process involves PPARgamma activation. These effects of enzLDL can be considered pro-atherogenic in the context of early atherosclerosis.

Lipids in vascular function.

Physiological and pathological vascular responses depend on the action of numerous intercellular mediators, ranging from hormones to gases like nitric oxide, proteins, and lipids. The last group consists not only of the different types of lipoproteins, but also includes a broad array of other lipophilic signaling molecules such as fatty acids, eicosanoids, phospholipids and their derivatives, sphingolipids and isoprenoids. Due to space limitations, it is impossible to discuss all the vascular effects of lipophilic mediators or compounds. Therefore, we will focus on one of the most important lipid-mediated diseases, atherosclerosis. Lipoproteins and especially their native or oxidized lipid compounds affect vascular function in many different ways, and these effects do not only modulate atherogenesis but are of paramount physiological and pathophysiological importance in other diseases, such as inflammation, tumor metastasis, or normal wound healing.

Plasma values of polyunsaturated fatty acids in extremely low birth weight (ELBW) infants fed breast milk or formula very early in life.

The influence of very early enteral feedings on plasma fatty acid levels in 29 sick, very premature infants with gestational age < 30 weeks was assessed at age 1, 3 and 7 weeks. Eighteen infants (birthweight 963 +/- 245 g, gestational age 27 +/- 1.3 weeks) received breast milk and 11 infants (829 +/- 159 g and 26 +/- 1.3 weeks) received formula, starting with small amounts on the first day after birth. Plasma phospholipid arachidonic acid (AA) levels decreased in both groups, but only the decline at 3 weeks in the formula-fed group was statistically significant (10.6 +/- 0.5 versus 8.0 +/- 0.4% weight, P < or = 0.05). The plasma phospholipid docosahexaenoic acid (DHA) levels of the formula-fed infants also declined from Week 1 to Week 7 (2.1 +/- 0.1 to 1.7 +/- 0.2 weight %; p < or = 0.05). In contrast, human milk-fed infants maintained their plasma phospholipid DHA levels, which were significantly higher at 7 weeks than those of the formula-fed infants (1.7 +/- 0.2 vs 2.3 +/- 0.2; p < or = 0.05). The decline in plasma DHA levels of our formula-fed very premature infants was of similar magnitude to that previously reported for larger premature infants. On the other hand, it is reassuring that very premature infants are able to maintain plasma DHA levels during the first weeks of life, if they receive even small amounts of breast milk.

[Nutrition in prevention of coronary heart disease]. / Ernährung in der Prävention der koronaren Herzkrankheit.

Clinical as well as basic research in the field of atherogenesis indicates that the progression of this disease process can be slowed down or even reversed. It is well established that nutrition plays an important role in the prevention and treatment of the classical atherogenic risk factors such as obesity, diabetes mellitus and hyperlipidemia. In addition, some nutrients such as the polyunsaturated n-3-fatty-acids or antioxidative vitamins can intervene directly by influencing one or more steps of the atherogenetic and/or thrombogenetic process. A comprehensive understanding of the pathogenesis of this disease as well as of the mechanisms of nutrient action are essential to the planning of successful nutritional prevention strategies. Because most nutrients influence mainly the slow and long-standing development of the atherosclerotic lesion, their inclusion in primary nutritional prevention should be started at an early age. Few nutrients such as the n-3 fatty acids, which also reduce the thrombogenetic risk factors, have demonstrated some success in the secondary prevention of CHD. Given the complexity with which nutrients intervene in the atherosclerotic process and their interactions with each other, nutritional prevention strategies should be based on well-grounded dietary modifications rather than supplementation with individual nutrients.

Acculturation to Canadian Foods by Chinese immigrant boys: changes in the perceived flavor, health value and prestige of foods.

Acculturation changes in the perceived qualities of foods was demonstrated in a group of first and second generation Chinese adolescent immigrants. The type and degree of change in perceived flavor, health value and prestige ratings varied for individual foods. The second generation subjects and those with more accultured patterns of language use, gave higher hedonic flavor and prestige ratings to dessert, snack and fast foods. This same group exhibited better discrimination between nutrient rich and poor foods as assessed by changes in perception of health value. Food perceptions of the more accultured second generation Chinese group were also found to approach those of an age and sex matched group of Canadian Anglophones. The results suggest that on immigration diet westernization may have nutritionally undesirable effects.

Nonlinear estimation of parameters in biphasic Arrhenius plots.

This paper presents a formal procedure for the statistical analysis of data on the thermotropic behavior of membrane-bound enzymes generated using the Arrhenius equation and compares the analysis to several alternatives. Data is modeled by a bent hyperbola. Nonlinear regression is used to obtain estimates and standard errors of the intersection of line segments, defined as the transition temperature, and slopes, defined as energies of activation of the enzyme reaction. The methodology allows formal tests of the adequacy of a biphasic model rather than either a single straight line or a curvilinear model. Examples on data concerning the thermotropic behavior of pig brain synaptosomal acetylcholinesterase are given. The data support the biphasic temperature dependence of this enzyme. The methodology represents a formal procedure for statistical validation of any biphasic data and allows for calculation of all line parameters with estimates of precision.