Effects Of Long-chain Fatty Acids Research Articles

Control of Gene Expression by Fatty Acids

The last decade provided evidence that major (glucose, fatty acids, amino acids) or minor (iron, vitamin, etc.) dietary constituents regulated gene expression in an hormonal-independent manner. This review focuses on molecular mechanisms by which fatty acids control the expression genes encoding regulatory protein involved in their own metabolism. Nonesterified fatty acids or their CoA derivatives seem to be the main signals involved in the transcriptional effect of long-chain fatty acids. The effects of fatty acids are mediated either directly owing to their specific binding to various nuclear receptors (PPAR, LXR, HNF-4alpha) leading to changes in the trans-activating activity of these transcription factors, or indirectly as the result of changes in the abundance of regulatory transcription factors (SREBP-1c, ChREBP, etc.). The relative contribution of each transcription factor in fatty acid-induced positive or negative gene expression is discussed.

Effects of Long-Chain Fatty Acids on Cytosolic Triacylglycerol Accumulation and Lipid Droplet Formation in Primary Cultured Bovine Mammary Epithelial Cells

Mammary epithelial cells have recently been shown to express and secrete leptin into milk and to accumulate triacylglycerol (TAG) in cytosol. We examined the effects on the accumulation of cytosolic TAG of free fatty acid addition to the medium bathing bovine mammary epithelial cells (bMEC). Both saturated (palmitic and stearic) and unsaturated (oleic and linoleic) fatty acids stimulated the accumulation of TAG in a concentration-dependent manner from 50 to 400μM and the expression of mRNA expression for CD36, which is involved in the uptake and secretion of long-chain fatty acids. However, leptin mRNA expression and lipid droplet formation were significantly increased only by the addition of unsaturated, but not saturated, fatty acids. Interestingly, both types of fatty acids stimulated αs1-casein mRNA expression. These data suggest that the expression of leptin is related to droplet formation, whereas CD36 is related to cytosolic TAG accumulation, and that fatty acids or cytosolic TAG accumulation also have a role to accelerate differentiation of bMEC as shown by casein synthesis.

LXR-mediated activation of macrophage stearoyl-CoA desaturase generates unsaturated fatty acids that destabilize ABCA1

Abnormal HDL metabolism among patients with diabetes and insulin resistance may contribute to their increased risk of atherosclerosis. ATP binding cassette transporter A1 (ABCA1) mediates the transport of cholesterol and phospholipids from cells to HDL apolipoproteins and thus modulates HDL levels and atherogenesis. Because fatty acids are increased in diabetes, we examined their effects on ABCA1 activity in cultured macrophages. cAMP analogs and ligands for the liver X receptor/retinoid X receptor (LXR/RXR) system can induce Abca1 transcription in murine macrophages. When induced by cAMP, unsaturated but not saturated long-chain fatty acids inhibit apolipoprotein-mediated lipid efflux by destabilizing ABCA1 protein. Here, we show that the saturated fatty acids palmitate and stearate also destabilize ABCA1 when Abca1 is induced by LXR/RXR ligands instead of cAMP. This was associated with increased palmitate and stearate desaturation by stearoyl-CoA desaturase (SCD), another gene product induced by LXR/RXR ligands. The SCD inhibitors conjugated linoleic acid and troglitazone nearly abolished ABCA1 destabilization by palmitate and stearate but not by linoleate. These results suggest that LXR/RXR ligands generate ABCA1-destabilizing monounsaturated fatty acids from their saturated precursors by activating SCD. Thus, with cholesterol-loaded macrophages exposed to saturated fatty acids, activated LXR/RXR may counteract the enhanced ABCA1 transcription by reducing the ABCA1 protein content.

Inhibitory effects of long-chain fatty acids on VFA degradation and β-oxidation

The inhibitory effects of major long-chain fatty acids (LCFA), which have 16 or 18 carbons, not only on acetate degradation, but also on propionate degradation and beta-oxidation were examined in anaerobic serum bottle tests at 35 degrees C with the acclimated granular sludges. A modified Gompertz equation described cumulative methane production to assess the rates of VFA degradation and beta-oxidation, which were applied to a simplified noncompetitive model and a simplified substrate inhibition model, respectively. The specific methane production rates on acetate decreased as LCFA concentration increased, which was in good agreement with the noncompetitive inhibition model. Unsaturated oleate (C18:1) and linoleate (C18:2) were more inhibitory than saturated stearate (C18:0) and palmitate (C16:0) on acetate degradation. LCFA inhibition on propionate degradation was similar to that for acetate; however, propionate degradation was less inhibited than acetate degradation. Beta-oxidation was the rate-limiting step in LCFA degradation in most cases. As LCFA concentration increased, beta-oxidation rate reached the maximum value, and then decreased, which confirmed the substrate inhibition of LCFA. Oleate, the most abundant LCFA in wastewater, could be degraded more quickly than saturated LCFA containing the same or even less carbon in spite of relatively high toxicity on acetate degradation.

Effect of long-chain fatty acids in the culture medium on fatty acid composition of WEHI-3 and J774A.1 cells

As a first step in determining the mechanism of action of specific fatty acids on immunological function of macrophages, a comparative study of the effect of long-chain polyunsaturated fatty acids (PUFA) in the medium was conducted in two macrophage cell lines, J774A.1 and WEHI-3. The baseline fatty-acid profiles of the two cell lines differed in the % distribution of saturated (SFA) and unsaturated fatty acids (UFA). J774A.1 cells had a higher % of SFA (primarily palmitic acid) than WEHI-3 cells. Conversely, WEHI-3 cells had a higher % of UFA (primarily oleic acid) than J774A.1 cells. Neither cell line had detectable amounts of α-linolenic acid (ALA) or eicosapentaenoic acid (EPA). The most abundant polyunsaturated fatty acid in both cells lines was arachidonic acid (AA). The efficiency of transport of fatty acids from the medium to the macrophages by two delivery vehicles (BSA complexes and ethanolic suspensions) was compared. Overall, fatty acids were transported satisfactorily by both delivery systems. Alpha-linolenic acid and doscosahexenoic acid (DHA) were transported more efficiently by the ethanolic suspension system. Linoleic acid (LA) was taken up more completely than ALA, and DHA was taken up more completely than EPA by both cell cultures and delivery systems. A dose–response effect was demonstrated for LA, ALA, EPA and DHA in both J774A.1 and WEHI-3 cells. Addition of polyunsaturated fatty acids (PUFA) to the cell cultures modified the total lipid fatty acid composition of the cells. The presence of ALA in the culture medium resulted in a significant decrease in AA in both cell lines. The omega-3/omega-6 fatty acid ratio (ω-3/ω-6), polyunsaturated/saturated fatty acid ratio (P/S), and unsaturation index (UI) increased directly with the amount of PUFA and ω-3 fatty acid provided in the medium. The results indicate that the macrophage cell lines have similar, but not identical, fatty acid profiles that may be the result of differences in fatty acid metabolism. These distinctions could in turn produce differences in immunological function. The ethanol fatty-acid delivery system, when compared with the fatty acid–BSA complex system, is preferable for measurement of dose–response effects, because the cellular fatty acid content increased in proportion to the amount of fatty acid provided in the medium. Similar dose–response results were observed in a previous in vivo study using flaxseed, rich in ALA, as a source of PUFA.

Long-chain fatty acids promote opening of the reconstituted mitochondrial permeability transition pore

Adenine nucleotide translocase–porin–hexokinase complex isolated from rat brain, when reconstituted into phospholipid-cholesterol vesicles, exhibits all properties of the mitochondrial permeability transition pore [Beutner, G., Rück, A., Riede, B., Welte, W. and Brdiczka, D. (1996) FEBS Lett. 396, 189–195]. In the present work, the effect of long-chain fatty acids on such reconstituted pore was examined. Opening of the pore was measured by leakage of either malate or fluorescein sulphonate entrapped inside the vesicles. It was found that myristate and oleate in the presence of 50 or 100 μM Ca2+ produced a partial release of the probes in a dose-dependent way. A dicarboxylic fatty acid analogue, that appeared inactive as protonophore in intact mitochondria, exerted no effect on pore opening in the reconstituted system. 100 μM Ca2+ alone was without effect. Pore opening by fatty acids in the reconstituted system was partly prevented by cyclosporin A. The pore opening also occurred when the vesicles were incubated in the presence of pancreatic phospholipase A2. In this case, the opening was decreased by cyclosporin A or serum albumin. These results indicate that long-chain fatty acids elicit opening of the permeability transition pore reconstituted in phospholipid vesicles in a similar way as in intact mitochondria [Więckowski, M.R. and Wojtczak, L. (1998) FEBS Lett. 423, 339–342].

Peroxisome-proliferator-activated receptor δ mediates the effects of long-chain fatty acids on post-confluent cell proliferation

Peroxisome-proliferator-activated receptor δ mediates the effects of long-chain fatty acids on post-confluent cell proliferation

Peroxisome-proliferator-activated receptor δ mediates the effects of long-chain fatty acids on post-confluent cell proliferation

Nutritional long-chain fatty acids control adipose tissue mass by regulating the number and the size of adipocytes. It is now established that peroxisome-proliferator-activated receptors (PPARs) play crucial functions in the control of gene expression and the level of cell differentiation. PPARgamma, which is activated by specific prostanoids, is a key factor in activating terminal differentiation and adipogenesis. We have recently demonstrated that PPARdelta, once activated by fatty acids, drives the expression of a limited set of genes, including that encoding PPARgamma, thereby inducing adipose differentiation. Thus far, the mechanism of action of fatty acids in the control of preadipocyte proliferation has remained unknown. We show here that PPARdelta is directly implicated in fatty acid-induced cell proliferation. Ectopic expression of PPARdelta renders 3T3C2 cells capable of responding to treatment with long-chain fatty acids by a resumption of mitosis, and this effect is limited to a few days after confluence. This response is restricted to PPARdelta activators and, for fatty acids, takes place within the range of concentrations found to trigger differentiation of preadipocytes both in vitro and in vivo. Furthermore, the use of a mutated inactive PPARdelta demonstrated that transcriptional activity of the nuclear receptor is required to mediate fatty acid-induced proliferation. These data demonstrate that PPARdelta, as a transcription factor, is directly implicated in fatty acid-induced proliferation, and this could explain the hyperplastic development of adipose tissue that occurs in high-fat-fed animals.

Effect of long-chain fatty acids on human urothelial cells in organ culture

It has been suggested that tumour-derived cells are differentially sensitive to the anti-proliferative and cytotoxic effects of long chain n-3 and n-6 polyunsaturated fatty acids (PuFAs). We have previously shown that PuFAs are also growth suppressive to highly proliferative normal human urinary bladder uro-epithelial (NHU) cells grown in monolayer culture. To determine if the effects on NHU cells are directly related to the proliferative index, we have studied the effects of long chain fatty acids in a bladder organ culture system, where proliferation and differentiation of the urothelium is under homeostatic control. A 50 μM concentration of fatty acids was chosen as this concentration of PuFA was profoundly growth inhibitory to NHU cells in monolayer culture. In organ culture, 50 μM PuFAs had no detectable effect on the proliferation or on the preservation of urothelial differentiated histioarchitecture, as assessed using a panel of phenotypic markers. These results suggest that the effects of PuFA may be modulated by the tissue microenvironment.

Long-chain fatty acid-induced changes in gene expression in neonatal cardiac myocytes

Long-chain fatty acids are the most important substrates for the heart. In addition, they have been shown to affect signalling pathways and gene expression. To explore the effects of long-chain fatty acids on cardiac gene expression, neonatal rat ventricular myocytes were cultured for 48 h with either glucose (10 mm), fatty acids (palmitic and oleic acid, 0.25 mm each), or a combination of both as exogenous substrates. Exposure to fatty acids (both in the absence or presence of glucose) neither affected cellular morphology and protein content nor induced alterations in the expression of phenotypic marker genes like atrial natriuretic factor and the Ca-ATPase SERCA2. However, incubation with fatty acids (with or without glucose) resulted in up to 4-fold increases of the mRNA levels of fatty acid translocase (FAT/CD36), heart-type fatty acid-binding protein, acyl-CoA synthetase, and long-chain acyl-CoA dehydrogenase. In contrast, the expression of genes coding for proteins involved in glucose uptake and metabolism, i.e., glucose transporter GLUT4, hexokinase II, and glyceraldehyde 3-phosphate dehydrogenase, remained constant or even declined under these conditions. These changes corresponded with a 60% increase in cardiomyocyte fatty acid oxidation capacity. Interestingly, the peroxisome proliferator-activated receptor–α (PPARα)–ligand Wy 14,643, but not the PPARγ–ligand ciglitazone, also resulted in increased mRNA levels of genes involved in fatty acid metabolism. In conclusion, fatty acids specifically and co-ordinately up-regulate transcription of genes coding for proteins involved in cardiac fatty acid transport and metabolism, most likely through activation of PPARα. —van der Lee, K. A. J. M., M. M. Vork, J. E. De Vries, P. H. M. Willemsen, J. F. C. Glatz, R. S. Reneman, G. J. Van der Vusse, and M. Van Bilsen. Long-chain fatty acid-induced changes in gene expression in neonatal cardiac myoctyes.

Unbound rather than total concentration and saturation rather than unsaturation determine the potency of fatty acids on insulin secretion

Isolated mouse islets were used to compare the effects of three saturated (myristate, palmitate and stearate) and three unsaturated (oleate, linoleate and linolenate) long-chain fatty acids on insulin secretion. By varying the concentrations of fatty acid (250–1250 μmol/l) and albumin simultaneously or independently, we also investigated whether the insulinotropic effect is determined by the unbound or total concentration of the fatty acids. Only palmitate and stearate slightly increased basal insulin secretion (3 mmol/l glucose). All tested fatty acids potentiated glucose-induced insulin secretion (10–15 mmol/l), and the following rank order of potency was obtained when they were compared at the same total concentrations: palmitate∼stearate>myristate≥oleate≥linoleate∼linolenate. The effect of a given fatty acid varied with the fatty acid to albumin molar ratio, in a way which indicated that the unbound fraction is the important one for the stimulation of β cells. When the potentiation of insulin secretion was expressed as a function of the unbound concentrations, the following rank order emerged: palmitate>myristate>stearate∼oleate>linoleate∼linolenate. In conclusion, the acute and direct effects of long-chain fatty acids on insulin secretion are due to their unbound fraction. They are observed only at fatty acid/albumin ratios higher than those normally occurring in plasma. Saturated fatty acids are stronger insulin secretagogues than unsaturated fatty acids. Unbound palmitate is by far the most potent of the six common long-chain fatty acids.

Effect of Long-Chain Fatty Acids on Triglyceride Accumulation, Gluconeogenesis, and Ureagenesis in Bovine Hepatocytes

A relationship between increased lipid concentration in the liver and reduced hepatic function has been suggested; however, there is little direct evidence of change in specific hepatic functions. Hepatocytes were obtained from ruminating calves and were incubated as monolayers for 36h. The media contained a mixture of nonesterified fatty acids (NEFA) at 0, 0.5, 1.0, and 1.5mM NEFA with molar proportions of 0.435 oleate, 0.319 palmitate, 0.144 stearate, 0.049 linoleate, and 0.053 palmitoleate. Ureagenesis or gluconeogenesis was measured from 48 to 51h after plating using hepatocytes that had only previous (12 to 48h), only concurrent (48 to 51h), or previous and concurrent (12 to 51h) exposure to NEFA. A previous 36-h exposure to NEFA caused cell triglyceride accumulation, yielding triglyceride concentrations that corresponded with liver that is clinically described as normal to moderately fatty. Previous, prolonged exposure to NEFA reduced ureagenesis and increased gluconeogenesis. Concurrent exposure to NEFA did not significantly affect gluconeogenesis or ureagenesis and did not alter the residual effect of prolonged incubation with NEFA. Reduced ureagenesis was related to increased cell triglyceride accumulation independently of other direct NEFA effects. Decreased ureagenic capacity may play a role in the morbidity associated with periparturient fatty liver in dairy cows.

Thyroid hormone-induced expression of the ADP/ATP carrier and its effect on fatty acid-induced uncoupling of oxidative phosphorylation

Liver mitochondria from rats made hypothyroid by administration of 2-mercapto-1-methylimidazole were less sensitive to the uncoupling effect of myristic acid, as measured by the increase of resting state respiration, than mitochondria from euthyroid animals, whereas subsequent administration to the animals of triiodothyronine (`hyperthyroidism') resulted in an increased uncoupling action of myristate. `Hyperthyroidism' also resulted in doubling of the carboxyatractyloside-sensitive portion of the myristate-stimulated respiration. Parallel to this was a twofold increase of the mitochondrial content of the ADP/ATP carrier protein and an over threefold increase of its activity. The uncoupling effect of phytanic acid was less sensitive to carboxyatractyloside and was increased in the hyperthyroid state to a smaller extent than in the case of myristate. These results provide further support to the thesis [Skulachev, V.P., FEBS Lett. 294 (1991) 158–162] that the ADP/ATP carrier is involved in the mechanism of the uncoupling effect of long-chain fatty acids.

Inhibition of Carnitine Palmitoyltransferase I Augments Sphingolipid Synthesis and Palmitate-induced Apoptosis

To identify cell death-induced genes, we employed a subtractive hybridization approach and isolated a cDNA encoding a mouse homolog of carnitine palmitoyltransferase I (CPT I), an enzyme that resides at the outer mitochondrial membrane and facilitates passage of long-chain fatty acids into mitochondria for beta-oxidation. Induced expression of CPT I mRNA was observed upon programmed cell death in the murine hematopoietic cell lines LyD9 and WEHI-231. To elucidate the role of CPT I in programmed cell death, we examined the effects of long-chain fatty acids and found that the addition of palmitate or stearate to cultured cells led to activation of a death program with a morphology resembling that of apoptosis. Other naturally occurring fatty acids, including myristate and palmitoleate, had no effect. Since both palmitate and stearate are sphingolipid precursors, the effect of these fatty acids on sphingolipid metabolism was tested. Our results indicate that apoptosis induced by palmitate or stearate is correlated with de novo synthesis of ceramide. Inhibition of CPT I by etomoxir enhanced palmitate-induced cell death and led to a further increase in ceramide synthesis.

Induction of Peroxisomal .BETA.-Oxidation and Fatty Acid Content in Primary Cultures of Rat Hepatocytes.

Effect of long-chain fatty acids on peroxisomal β-oxidation in rat hepatocytes was examined. Out of four long-chain fatty acids, linoleic acid (C 18:2) increased the β-oxidation activity 1.7-2.7 fold without cell toxicity. Total fatty acid content in linoleic acid-treated hepatocytes was increased more than twofold. Addition of nafenopin to the culture medium, instead of linoleic acid, produced a reciprocal relation between the β-oxidation activity and the fatty acid content with a coefficient r=0.906. Cycloheximide inhibited the increase in peroxisomal β-oxidation activity by linoleic acid to 50% of that in cultures without cycloheximide. Results of these experiments suggest that the increase in fatty acid content in hepatocytes may have a significant role in the induction of peroxisomal β-oxidation and that the enzyme proteins in the peroxisomal β-oxidation system induced by linoleic acid were the result of de novo synthesis.

Effects of Diets Containing Tallow and Soybean Oil with and Without Cholesterol on Hepatic Metabolism of Lipids and Lipoproteins in the Preruminant Calf

The effects of long-chain fatty acids (230g/kg of dietary DM) from tallow and from soybean oil, with or without cholesterol (10g/kg of dietary DM), on hepatic lipid contents and on in vivo hepatic production rates of lipids and lipoproteins were investigated in 22 preruminant male calves fitted with chronic catheters and with electromagnetic blood flow probes implanted in the hepatic vessels. Diets containing soybean oil and soybean oil with cholesterol led to the development of triglyceride infiltration in the liver and to higher apparent hepatic secretion of very low density lipoproteins than did diets containing tallow or tallow with cholesterol. Addition of cholesterol to diets favored accumulation of low density lipoproteins in plasma and the net apparent secretion of these particles by the liver, especially for the diet containing soybean oil with cholesterol. Regardless of the diet, calf liver clearly removed large high density lipoproteins of type 1 that were rich in cholesteryl esters but secreted heavy high density lipoproteins that were rich in proteins. The intensity of removal of high density lipoproteins of type 1 by the liver depended on the plasma concentration of these particles, probably by mass action. This removal did not prevent the accumulation of high density lipoproteins of type 1 in plasma, such as it did in calves fed soybean oil.

Effect of long-chain fatty acids on low-density-lipoprotein-cholesterol metabolism

The concentration of cholesterol in the low-density-lipoprotein (LDL) fraction of plasma is one of the major risk factors for coronary heart disease. Steady-state concentrations of LDL cholesterol in the plasma are determined primarily by the production rate and the rate of removal of LDL cholesterol from the circulation by receptor-dependent transport. The magnitude of these two processes is affected by the type of fatty acid in the diet. Saturated fatty acids with 14 and 16 carbon atoms suppress receptor-dependent LDL-cholesterol transport into the liver, increase the LDL-cholesterol production rate, and raise the plasma LDL-cholesterol concentration. The 9-cis 18:1 fatty acid restores receptor activity, lowers the production rate, and decreases the plasma LDL-cholesterol concentration. In contrast with these fatty acids, the 18:0 and 9-trans 18:1 fatty acids are biologically inactive and so do not change the circulating LDL-cholesterol concentration.

In vitro activation and inhibition of rat colonic phospholipase D by fatty acids

The present study was designed to examine the effect of long-chain fatty acids on phospholipase D (PLD) of the proximal colon mucosa of the rat. These fatty acids are present in the colon, in close contact with the membranes which contain the enzyme. The results indicate that unsaturated fatty acids activate the enzyme. At 4 mM, trienes are more potent activators as compared to 18:2 and 18:1. The number of double bonds and the configuration around the double bond were found to be important factors in activation of the enzyme. The metabolism of polyunsaturated fatty acids may be involved in the activation since the presence of antioxidants and cyclooxygenase inhibitors were found to influence the activation of PLD by polyunsaturated fatty acids.

A threshold membrane potential accounts for controversial effects of fatty acids on mitochondrial oxidative phosphorylation

The uncoupling effect of free fatty acids on oxidative phosphorylation in mitochondria has been known for more than 35 years. The mechanism of action, however, remains controversial. In this report the physicochemical basis of uncoupling was elucidated by studying the effect of free fatty acids on the proton permeability and membrane potential of proteoliposomes containing reconstituted cytochrome c oxidase (COX). A threshold membrane potential of about 125 mV was identified for fatty acid-induced proton permeability. Only above this potential do free fatty acids translocate protons across the biological membrane. The data explain the controversial effects of long-chain fatty acids on oxidative phosphorylation as well as their role on non-shivering thermogenesis in larger mammals.

Identification and Biological Activity of Germination-Inhibiting Long-Chain Fatty Acids in Animal-Waste Composts

Long-chain fatty acids in germination-inhibiting animal-waste composts were identified by gas chromatography-mass spectrometry as myristic, palmitic, stearic, oleic, linoleic, and linolenic acids. These acids were found at concentrations greater than 0.25 mg (g dry compost)-1. The identified acids, together with lauric acid, and five kinds of short- and medium-chain fatty acid, were tested for their effects on the germination process of sorghum seeds. The authentic long-chain fatty acids, which were dissolved in a 1:9 (v/v) mixture of methanol and distilled water at 40 mg liter-1, significantly reduced the alpha-amylase activity, physiological water uptake, and ATP content of the germinating seeds during the first 24 h of inhibition, as well as the rate of germination of seeds. Among the tested fatty acids, myristic and palmitic acids were the most potent inhibitors of germination. The inhibitory effects of long-chain fatty acids were stronger than those of the phenolic acids. The short- and medium-chain fatty acids did not have any significant germination-inhibitory effects at 40 mg liter-1. The results indicate that the long-chain fatty acids are the dominant inhibitors of germination in animal-waste composts, and that the inhibition of the alpha-amylase activity in germinating sorghum seeds is one aspect of the mode of action of these long-chain fatty acids.