Suppression of ABCA1 by unsaturated fatty acids leads to lipid accumulation in HepG2 cells

Abnormal lipid metabolism may contribute to the pathogenesis of non-alcoholic steatohepatitis (NASH). ATP-binding cassette transporter A1 (ABCA1) mediates the transport of cholesterol and phospholipids from cells to HDL apolipoproteins. We previously reported that unsaturated fatty acids destabilise ABCA1 in murine macrophages and ABCA1-transfected baby hamster kidney cells by increasing its protein degradation. Here, we examined the correlation between ABCA1 and hepatic lipids. In HepG2 cells, unsaturated but not saturated fatty acids suppressed ABCA1 protein levels by promoting its protein degradation. Over-expression of ABCA1 resulted in a decrease of cellular fatty acids and triglycerides, while repression by ABCA1 siRNA increased both cellular fatty acids and triglycerides. Rats with NASH also showed lower ABCA1 protein levels in liver cells, compared with that of the normal rats. These data indicate that steatosis is associated with a decrease in ABCA1 protein expression leading to an increase in lipid storage in hepatocytes. And it further suggests that this effect could be due to an excess of unsaturated fatty acids.     

The effects of unsaturated fatty acids on hepatic microsomal drug metabolism and cytochrome P-450

1. The effects of unsaturated fatty acids on drug-metabolizing enzymes in vitro were measured by using rat and rabbit hepatic 9000g supernatant fractions. 2. Unsaturated fatty acids inhibited the hepatic microsomal metabolism of ;type I' drugs with inhibition increasing with unsaturation: arachidonic acid>linolenic acid>linoleic acid>oleic acid. Inhibition was independent of lipid peroxidation. Linoleic acid competitively inhibited the microsomal O-demethylation of p-nitroanisole and the N-demethylation of (+)-benzphetamine. 3. The hepatic microsomal metabolism of ;type II' substrates, aniline and (-)-amphetamine, was not affected by unsaturated fatty acids. 4. The rate of reduction of p-nitrobenzoic acid and Neoprontosil was accelerated by unsaturated fatty acids. 5. Linoleic acid up to 3.5mm did not decelerate the generation of NADPH by rat liver soluble fraction, nor the activity of NADPH-cytochrome c reductase of rat liver microsomes. Hepatic microsomal NADPH oxidase activity was slightly enhanced by added linoleic acid. 6. No measurable disappearance of exogenously added linoleic acid occurred when this fatty acid was incubated with rat liver microsomes and an NADPH source. 7. The unsaturated fatty acids used in this study produced type I spectra when added to rat liver microsomes, and affected several microsomal enzyme activities in a manner characteristic of type I ligands.     

The effects of unsaturated fatty acids on the synthesis of arachidonic acid in rat kidney cells

Cultured rat kidney cells absorbed exogenous linoleic acid (cic, cis-18:2n-6) and esterified it mostly into glycerophospholipids. As the concentration of 18:2 was increased (5-200 microM) the quantity absorbed increased linearly and the amount esterified in the triacylglycerol increased. The cells possessed active acyl delta 6-desaturase and elongase which facilely converted 18:2n-6 to 20:4n-6. At low intracellular concentrations of 18:2n-6 other unsaturated fatty acids, i.e., gamma-linolenic (18:3n-6), alpha-linolenic (18:3n-3), dihomo-gamma-linolenic (20:3n-6), and especially trans, trans-linoleic acid (trans, trans-18:2n- -6) at concentrations ranging from 25 to 200 microM depressed delta 6-desaturase activity. However, suppression of 20:4 synthesis even by trans, trans-18:2 was readily overcome by increasing the concentration of available cis, cis-18:2n-6.     

Trans fatty acids alter the lipid composition and size of apoB-100-containing lipoproteins secreted by HepG2 cells

This study was conducted to determine the secretion rate and composition of lipoproteins secreted by HepG2 cells as influenced by the type of fatty acid present in the incubation medium. Cells were preincubated for 24 h with palmitic, oleic, elaidic, linoleic or conjugated linoleic acid (CLA), and the lipoproteins secreted during a subsequent incubation period of 24 h were collected for analysis. The secretion rate of apolipoprotein B-100 (apoB) was significantly greater in HepG2 cells preincubated with elaidic acid compared with those preincubated with palmitic or oleic acid; apoB secretion was greater in cells preincubated with CLA compared with those preincubated with linoleic acid. The lipid composition of secreted lipoproteins was also influenced by fatty acid treatment, resulting in significantly smaller lipoprotein particles secreted by cells preincubated with elaidic acid and CLA compared with those secreted by cells treated with oleic acid and linoleic acid, respectively. Our results are relevant to human metabolism for the following reasons: (1) the size of plasma low-density lipoproteins (LDLs) is determined, at least in part, by the composition of apoB-containing lipoproteins secreted by the liver; (2) small plasma LDL particles are associated with an increased risk of coronary heart disease; and (3) specific dietary fatty acids can affect the composition and size of plasma LDLs, thereby imparting a relative atherogenicity to plasma LDLs independent of LDL cholesterol concentration. The present study therefore suggests that elaidic acid and CLA promote the hepatic secretion of small apoB-containing lipoproteins, which could lead to an increased production of small plasma LDL particles.     

Differential effects of unsaturated fatty acids on phospholipid synthesis in human leukemia monocytic U937 cells

The biosynthesis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in monocyte-like leukemia U937 cells was monitored by adding [³H]choline, [¹⁴C]ethanolamine or [¹⁴C]glycerol to the culture media; incorporation into phospholipid (PL) increased with time. The effect of unsaturated fatty acids (UFA) on PC and PE synthesis was investigated by pretreating U937 cells for 72h with 10 μM 18:1 (n –9), 18:2 (n –6), 18:3 (n –3), 20:4 (n –6) and 20:5 (n –3). The UFA caused no alteration in cell growth, as evidenced by light microscopy and the incorporation of [³H]thymidine and [³H]leucine. Total cellular uptake of radioactive precursors remained unaffected by all the treatments. Pretreatment with 20:5 resulted in approximately 25 per cent reduction in the incorporation of [³H]choline into PL, while no significant effect was detected with the other UFAs. 18:3, 20:4 and 20:5 depressed the incorporation of [¹⁴C]ethanolamine into PL by 34 per cent, 28 per cent and 49 per cent respectively. However, there was no redistribution of label with any of the treatments. 18:3, 20:4 and 20:5 also antagonized the stimulatory effect of endotoxin (LPS) on PC and PE synthesis. In addition, the incorporation from [¹⁴C]glycerol into PC and PE was reduced by 18:3, 20:4 and 20:5. Although the PL composition of the cells remained essentially unaffected, our study shows that chronic treatment of U937 cells with n –3 PUFA (20:5) depressed PC and PE synthesis, and 18:3 and 20:4 also caused inhibition of PE synthesis.     

Trans unsaturated fatty acids are less oxidizable than cis unsaturated fatty acids and protect endogenous lipids from oxidation in lipoproteins and lipid bilayers

Epidemiological data suggest that dietary trans unsaturated fatty acids increase the risk of heart disease; however, the underlying mechanisms are unclear. In this study, we investigated one possible mechanism, namely, their effect on LDL oxidation. Supplementation of LDL with 10% 16:1 trans-cholesteryl ester (CE) inhibited the oxidation compared to that with 16:1 cis-CE. Total replacement of core lipids with 18:2 trans,trans-CE decreased the rate of LDL oxidation by 19% compared to replacement with 18:2 cis,cis-CE. When the surface phosphoglycerides were replaced with either 16:0-18:2 cis,cis-phosphatidylcholine (PC) or 16:0-18:2 trans,trans-PC, the latter was found to inhibit the rate and increase the lag time of oxidation to a greater extent than the former. To confirm these findings, we studied the oxidation of PC liposomes by assessing the formation of conjugated dienes or the degradation of a fluorescently labeled PC. By both methods, the 16:0-18:2 trans,trans-PC exhibited greater resistance to oxidation than the 16:0-18:2 cis,cis-PC. Eliminating the fluidity differences did not completely eliminate the differences in oxidation rates, suggesting that the trans double bond is inherently resistant to oxidation. The composition of the conjugated hydroperoxy products formed after oxidation differed markedly for the two 18:2 isomers. Supplementation of 16:0-18:2 cis,cis-PC liposomes with 20 mol % di16:1 trans-PC retarded oxidation rates to a greater extent than supplementation with di16:1 cis-PC. These studies show that dietary trans unsaturated fatty acids decrease the rate of lipid peroxidation, an effect that may mitigate the atherogenic effect of these fatty acids.     

The effects of alkylthioacetic acids (3-thia fatty acids) on fatty acid metabolism in isolated hepatocytes

Long-chain alkylthioacetic acids (3-thia fatty acids) inhibit fatty acid synthesis from [1-14C]acetate in isolated hepatocytes, while fatty acid oxidation is nearly unaffected or even stimulated. Desaturation of [1-14C]stearate (delta 9-desaturase) is also unaffected. [1-14C]Dodecylthioacetic acid (a 3-thia fatty acid) is incorporated in triacylglycerol and in phospholipids more efficiently than [1-14C]palmitate in isolated hepatocytes. The metabolism of [1-14C]dodecylthioacetic acid to acid-soluble products (by omega-oxidation) is slow compared to the oxidation of [1-14C]palmitate. In hepatocytes from adapted rats (rats fed tetradecylthioacetic acid for 4 days) the rate of [1-14C]palmitate oxidation is increased and its rate of esterification is decreased. Stearate desaturation is also decreased. The rate of cyanide-insensitive peroxisomal fatty acid beta-oxidation is several-fold increased. The metabolic effects of long-chain 3-thia fatty acids are discussed and it is concluded that they behave essentially like normal fatty acids except for their slow breakdown due to the sulfur atom in the 3 position, which blocks normal beta-oxidation.     

Synthesis of unsaturated fatty acids

This article reviews synthetic routes leading to polyunsaturated fatty acids having “skipped” double bonds. Emphasis is placed on the “acetylenic approach”.The suitability of building blocks, their condensation reactions as well as the controlled reduction of triple bonds to cis double bonds are discussed. In addition, the application of the various methods to the preparation of polyunsaturated fatty acids labelled with ³H and/or ¹⁴C at distinct positions of the molecules is reviewed.     

Acute effects of unsaturated fatty acids in splanchnic artery occlusion shock

Diets enriched with omega-3 unsaturated fatty acids are associated with decreased hypercholesterolemia and decreased risk of ischemic and atherosclerotic diseases. We studied the acute intravascular effects of some of these unsaturated fatty acids (i.e., eicosapentaenoic acid, EPA; docosahexaenoic acid, DHA) along with omega-6 unsaturated fatty acids, (i.e., linoleic and linolenic acid) in splanchnic artery occlusion (SAO) shock in rats. Anesthetized rats subjected to total occlusion of the celiac and superior mesenteric arteries for 40 minutes followed by reperfusion usually resulted in a fatal outcome 90-120 minutes after releasing the clamps. SAO shock rats treated with the omega-3 unsaturated fatty acid, EPA, exhibited an improved survival time and rate (p less than 0.05 from vehicle) compared to those receiving only vehicle (i.e., 50% ethanol). EPA and DHA treated SAO rats also exhibited lower plasma activities of the lysosomal protease, cathepsin D, free amino-nitrogen compounds, and the cardiotoxic peptide, myocardial depressant factor. These results indicate that omega-3 unsaturated fatty acids, especially EPA, have some acute beneficial effects in SAO shock in rats.     

Time-dependent effects of fatty acids on skeletal muscle metabolism

Increased plasma levels of free fatty acids (FFA) occur in states of insulin resistance such as type 2 diabetes mellitus, obesity, and metabolic syndrome. These high levels of plasma FFA seem to play an important role for the development of insulin resistance but the mechanisms involved are not known. We demonstrated that acute exposure to FFA (1 h) in rat incubated skeletal muscle leads to an increase in the insulin-stimulated glycogen synthesis and glucose oxidation. In conditions of prolonged exposure to FFA, however, the insulin-stimulated glucose uptake and metabolism is impaired in skeletal muscle. In this review, we discuss the differences between the effects of acute and prolonged exposure to FFA on skeletal muscle glucose metabolism and the possible mechanisms involved in the FFA-induced insulin resistance.     

Changes in mRNA levels of intracellular fatty acid metabolism regulators in human hepatoma HepG2 cells following their treatment with non-esterified fatty acids and dehydroepiandrosterone

The effects of non-esterified fatty acids (NEFA) and hormone dehydroepiandrosterone (DHEA) on the levels of mRNAs of protein kinase C (PKC) -delta and -epsilon isoforms and those of liver fatty acid binding protein (L-FABP) were investigated in the human hepatoma HepG2 cell line. The cells were kept in low-serum, low-albumin medium during experiments. Low FA levels (100 microM) and time intervals of 4 h and 20 h were used. In these conditions, the saturated (palmitic, stearic) and monounsaturated (oleic) acids rather selectively stimulated PKC-epsilon mRNA levels. Unexpectedly, we found that these acids also suppressed liver fatty-acid binding protein (L-FABP) mRNA levels. DHEA in pharmacological doses (100 microM) produced a significant increase in PKC-delta and -epsilon mRNA levels. Although molecular mechanisms underlying the identified changes have not been investigated in this paper, our findings emphasize that NEFA-induced modulation of mRNA levels of key signalling components represent an additional mechanism for how the ambient NEFA can influence metabolic homeostasis in cells.