Metabolic switching of human myotubes is improved by n-3 fatty acids.

The aim of the present study was to examine whether pretreatment with different fatty acids, as well as the liver X receptor (LXR) agonist T0901317, could modify metabolic switching of human myotubes. The n-3 FA eicosapentaenoic acid (EPA) increased suppressibility, the ability of glucose to suppress FA oxidation. Substrate-regulated flexibility, the ability to increase FA oxidation when changing from a high glucose, low fatty acid condition ("fed") to a high fatty acid, low glucose ("fasted") condition, was increased by EPA and other n-3 FAs. Adaptability, the capacity to increase FA oxidation with increasing FA availability, was enhanced after pretreatment with EPA, linoleic acid (LA), and palmitic acid (PA). T0901317 counteracted the effect of EPA on suppressibility and adaptability, but it did not affect these parameters alone. EPA per se accumulated less, however, EPA, LA, oleic acid, and T0901317 treatment increased the number of lipid droplets (LD) in myotubes. LD volume and intensity, as well as mitochondrial mass, were independent of FA pretreatment. Microarray analysis showed that EPA regulated more genes than the other FAs and that specific pathways involved in carbohydrate metabolism were induced only by EPA. The present study suggests a favorable effect of n-3 FAs on skeletal muscle metabolic switching and glucose utilization.

LXR{beta} is the dominant LXR subtype in skeletal muscle regulating lipogenesis and cholesterol efflux.

Liver X receptors (LXRs) are important regulators of cholesterol, lipid, and glucose metabolism and have been extensively studied in liver, macrophages, and adipose tissue. However, their role in skeletal muscle is poorly studied and the functional role of each of the LXRalpha and LXRbeta subtypes in skeletal muscle is at present unknown. To study the importance of each of the receptor subtypes, myotube cultures derived from wild-type (WT) and LXRalpha and LXRbeta knockout (KO) mice were established. The present study showed that treatment with the LXR agonist T0901317 increased lipogenesis and apoA1-dependent cholesterol efflux in LXRalpha KO and WT myotubes but not in LXRbeta KO cells. The functional studies were confirmed by T0901317-induced increase in mRNA levels of LXR target genes involved in lipid and cholesterol metabolism in myotubes established from WT and LXRalpha KO mice, whereas only minor changes were observed for these genes in myotubes from LXRbeta KO mice. Gene expression analysis using microarrays showed that very few genes other than the classical, well-known LXR target genes were regulated by LXR in skeletal muscle. The present study also showed that basal glucose uptake was increased in LXRbeta KO myotubes compared with WT myotubes, suggesting a role for LXRbeta in glucose metabolism in skeletal muscle. In conclusion, LXRbeta seems to be the main LXR subtype regulating lipogenesis and cholesterol efflux in skeletal muscle.

Effect of rapeseed oil and dietary n-3 fatty acids on triacylglycerol synthesis and secretion in Atlantic salmon hepatocytes.

Fish oil (FO) has traditionally been used as the dominating lipid component in fish feed. However, FO is a limited resource and the price varies considerably, which has led to an interest in using alternative oils, such as vegetable oils (VOs), in fish diets. It is far from clear how these VOs affect liver lipid secretion and fish health. The polyunsaturated fatty acids (PUFAs), eicosapentanoic acid (EPA) and docosahexanioc acid (DHA), reduce the secretion of lipoproteins rich in triacylglycerols (TAGs) in Atlantic salmon, as they do in humans. The mechanism by which n-3 fatty acids (FAs) in the diet reduce TAG secretion is not known. We have therefore investigated the effects of rapeseed oil (RO) and n-3 rich diets on the accumulation and secretion of (3)H-glycerolipids by salmon hepatocytes. Salmon, of approximately 90 g were fed for 17 weeks on one of four diets supplemented with either 13.5% FO, RO, EPA-enriched oil or DHA-enriched oil until a final average weight of 310 g. Our results show that the dietary FA composition markedly influences the endogenous FA composition and lipid content of the hepatocytes. The intracellular lipid level in hepatocytes from fish fed RO diet and DHA diet were higher, and the expressions of the genes for microsomal transfer protein (MTP) and apolipoprotein A1 (Apo A1) were lower, than those in fish fed the two other diets. Secretion of hepatocyte glycerolipids was lower in fish fed the EPA diet and DHA diet than it was in fish fed the RO diet. Our results indicate that EPA and DHA possess different hypolipidemic properties. Both EPA and DHA inhibit TAG synthesis and secretion, but only EPA induces mitochondrial proliferation and reduce intracellular lipid. Expression of the gene for peroxisome proliferator-activated receptor alpha (PPARalpha) was higher in the DHA dietary group than it was in the other groups.

Dietary supplementation of tetradecylthioacetic acid increases feed intake but reduces body weight gain and adipose depot sizes in rats fed on high-fat diets.

AIM: The pan-peroxisome proliferator-activated receptor (PPAR) ligand and fatty acid analogue tetradecylthioacetic acid (TTA) may reduce plasma lipids and enhance hepatic lipid metabolism, as well as reduce adipose tissue sizes in rats fed on high-fat diets. This study further explores the effects of TTA on weight gain, feed intake and adipose tissue functions in rats that are fed a high-fat diet for 7 weeks. METHODS: The effects on feed intake and body weight during 7 weeks' dietary supplement with TTA ( approximately 200 mg/kg bw) were studied in male Wistar rats fed on a lard-based diet containing approximately 40% energy from fat. Adipose tissue mass, body composition and expression of relevant genes in fat depots and liver were measured at the end of the feeding. RESULTS: Despite higher feed intake during the final 2 weeks of the study, rats fed on TTA gained less body weight than lard-fed rats and had markedly decreased subcutaneous, epididymal, perirenal and mesenteric adipose depots. The effects of TTA feeding with reduced body weight gain and energy efficiency (weight gain/feed intake) started between day 10 and 13. Body contents of fat, protein and water were reduced after feeding lard plus TTA, with a stronger decrease in fat relative to protein. Plasma lipids, including Non-Esterified Fatty Acids (NEFA), were significantly reduced, whereas fatty acid beta-oxidation in liver and heart was enhanced in lard plus TTA-fed rats. Hepatic UCP3 was expressed ectopically both at protein and mRNA level (>1900-fold), whereas Ucp1 mRNA was increased approximately 30-fold in epididymal and approximately 90-fold in mesenteric fat after lard plus TTA feeding. CONCLUSION: Our data support the hypothesis that TTA feeding may increase hepatic fatty acid beta-oxidation, and thereby reduce the size of adipose tissues. The functional importance of ectopic hepatic UCP3 is unknown, but might be associated with enhanced energy expenditure and thus the reduced feed efficiency.

Tetradecylthioacetic acid attenuates dyslipidaemia in male patients with type 2 diabetes mellitus, possibly by dual PPAR-alpha/delta activation and increased mitochondrial fatty acid oxidation.

AIM: We previously demonstrated that a modified fatty acid, tetradecylthioacetic acid (TTA), improves transport and utilization of lipids and increases mitochondrial fatty acid oxidation in animal and cell studies. We conducted an exploratory study of safety and effects of this novel drug in patients with type 2 diabetes mellitus and investigated the mechanism of action in human cell lines. METHODS: Sixteen male patients with type 2 diabetes mellitus received 1 g TTA daily for 28 days in an open-labelled study, with measurement of parameters of lipid metabolism, glucose metabolism and safety (ClinicalTrials.gov NCT00605787). The mechanism of action was further investigated in a human liver cell line (HepG2) and in cultured human skeletal muscle cells (myotubes). RESULTS: Mean LDL cholesterol level declined from 4.2 to 3.7 mmol/l (p < 0.001), accompanied by increased levels of the HDL apolipoproteins A1 and A2, and a decline in LDL/HDL ratio from 4.00 to 3.66 (p = 0.008). Total fatty acid levels declined, especially the fraction of the polyunsaturated n-3 fatty acids docosahexaenoic acid (-13%, p = 0.002) and eicosapentaenoic acid (-10%, p = 0.07). Glucose metabolism was not altered and the drug was well tolerated. In cultured liver cells, TTA acted as a pan-PPAR agonist with predominant PPAR-alpha and PPAR-delta activation at low TTA concentrations. In myotubes, TTA and a PPAR-delta agonist, but not the PPAR-alpha or PPAR-gamma agonists, increased the fatty acid oxidation. CONCLUSIONS: We demonstrate for the first time that TTA attenuates dyslipidaemia in patients with type 2 diabetes mellitus. These effects may occur through mechanisms involving PPAR-alpha and PPAR-delta activation, resulting in increased mitochondrial fatty acid oxidation.

Effect of dietary n-3 and n-6 fatty acids on fatty acid desaturation in rat liver.

To study the effect of high-fat diets with varying contents of n-3 and n-6 fatty acids on the metabolism of essential fatty acids, the rat liver microsomal fatty acid desaturases were measured. The rats were fed for 3 weeks with diets high in linseed oil (18:3(n-3)), sunflower seed oil (18:2(n-6)) or fish oil (20:5(n-3) and 22:6(n-3)) (20%, w/w) using pellet fed rats as a reference. The delta 6-desaturase using 18:2(n-6) or 18:3(n-3) as substrates was stimulated 1.5-2.5-fold by linseed or sunflower seed oil, compared to the pellet reference. The delta 5-desaturase was stimulated 3.5-fold with linseed oil and 2.5-fold with sunflower seed oil, while the delta 9-desaturase was inhibited by all the high-fat diets. The delta 6-, 5- and 9-desaturase activities were in all cases considerably reduced with fish oil as compared to linseed and sunflower seed oil diets. With pellet fed rats the rates were highest for delta 9-desaturation and in decreasing order lower for delta 5-desaturation, delta 6-desaturation with 18:3 (n-3) as substrate and finally delta 6-Desaturation with 18:2(n-6) as substrate. The content of 20:4(n-6) in liver phospholipids increased with the diets rich in 18:2(n-6), and was reduced for the fish oil diet enriched in 20:5 and 22:6(n-3) fatty acids. The amount of 20:5(n-3) in phospholipids was as high with linseed oil diet as with the fish oil diet, while the 22:6(n-3) content was only increased with the fish oil diet.

Inhibition of very-low-density lipoprotein secretion by chloroquine, verapamil and monensin takes place in the Golgi complex.

The effects of chloroquine, verapamil and monensin on secretion of very-low-density lipoproteins (VLDLs) were studied in cultured rat hepatocytes. Maximum inhibition of VLDL-triacylglycerol secretion by 50-90% of control was reached at 200 microM chloroquine, 200 microM verapamil and 5 microM monensin, whereas no effect on cellular triacylglycerol synthesis was observed. The inhibition could be seen within 15 min and was reversible after washout of the drugs. Chloroquine and verapamil inhibited both cellular protein synthesis and protein secretion, whereas monensin reduced protein secretion without any effect on protein synthesis. Control experiments with cycloheximide revealed that intact protein synthesis was not necessary for secretion of VLDL-triacylglycerol during 2 h. Electron micrographs of cells treated with chloroquine, verapamil or monensin showed swollen Golgi cisternae containing VLDL-like particles. By morphometry, a more than 2-fold increase in volume fractions and size indices of Golgi complexes and secondary lysosomes was observed, except that monensin had no significant effect on these parameters of secondary lysosomes. These results suggest that the inhibition of VLDL secretion by chloroquine, verapamil and monensin which takes place in the Golgi complex might be due to disruption of trans-membrane proton gradients. An increase in pH of acidic Golgi vesicles may cause swelling and disturb sorting and membrane flow through this organelle.

Postprandial decrease in plasma unesterified fatty acids during n-3 fatty acid feeding is not caused by accumulation of fatty acids in adipose tissue.

Dietary supplementation of very long-chain n-3 fatty acids to rats reduces postprandial plasma concentrations of triacylglycerol, unesterified fatty acids and glycerol after long-term feeding by unknown mechanisms [Rustan et al., J. Lipid Res. 34 (1993) 1299-1309]. In the present study we examine the role of adipose tissues in metabolism of fatty acids. Postprandial plasma concentrations of triacylglycerol, unesterified fatty acids and glycerol were reduced by 75%, 50% and 30%, respectively, during 49 days of feeding high-fat diets containing n-3 fatty acids (6.5% n-3 fatty acid concentrate, 13% lard) as compared to lard (19.5% lard). These differences were observed already after two days of feeding. Plasma concentration of unesterified very long-chain n-3 fatty acids increased to 50 microM in n-3 fatty acid-supplemented rats, whereas these fatty acids were undetectable in lard-fed animals. The n-3 fatty acid-enriched diet limited cell volumes of perirenal and epididymal adipocytes by 40% and 30%, respectively, after 49 days, as compared to lard feeding. This reduction in cell volume was not due to reduced synthesis of glycerolipids in epididymal adipocytes. Acute incubation of perirenal and epididymal adipocytes with oleic acid or eicosapentaenoic acid, caused similar increase in synthesis of triacylglycerol. Dietary supplementation with n-3 fatty acids decreased basal and total lipolysis (isoprenalin-stimulated) in perirenal adipocytes. Basal lipolysis in epididymal adipocytes was reduced by n-3 fatty acids only after 49 days. n-3 fatty acids increased total lipolysis in mesenteric and subcutaneous fat cells compared to adipocytes derived from lard-fed animals, whereas basal lipolysis was unchanged. These results suggest that the reduced postprandial plasma concentration of unesterified fatty acids after n-3 fatty acid-supplementation is not caused by accumulation of fatty acids in adipose tissue. The reduced trophic growth of adipocytes might be due to decreased supply of unesterified fatty acids for triacylglycerol storage. (c) 1998 Elsevier Science B.V.

Fatty acid uptake and metabolism in CaCo-2 cells: eicosapentaenoic acid (20:5(n-3)) and oleic acid (18:1(n-9)) presented in association with micelles or albumin.

In vivo, long-chain fatty acids are incorporated into bile salt micelles, which solubilize the hydrophobic fatty acids before they are transported across the unstirred water layer to the intestinal brush border membrane. In the present study we have used CaCo-2 cells, cultured on filter membranes as a model of human enterocytes, and compared presentation of fatty acids bound to albumin with a micellar form. Absorption of eicosapentaenoic acid and oleic acid from micellar solutions was 4-times faster than from fatty acid-albumin complexes after 5 h incubation, and resulted in a corresponding increase in triacylglycerol synthesis and secretion. Mass determination of newly synthesized, cell-associated triacylglycerol after 5 h incubation, indicated a 5-fold increase in cells exposed to a micellar solution versus albumin-complexed fatty acids. A 2-fold larger fraction of the absorbed fatty acids was incorporated into triacylglycerol than into phospholipids when the fatty acids were presented as micelles. Analysis by resistive pulse technique showed that secreted lipoproteins of density less than 1.006 g/ml were in the same size-range as chylomicrons derived from human plasma. In spite of an increased amount of secreted triacylglycerol from cells supplemented with micellar fatty acids, there was no increase in the mean size of these particles. Synthesis and secretion of triacylglycerol in cells exposed to eicosapentaenoic acid and oleic acid were similar regardless of whether the fatty acids were presented to the cells associated with albumin or micelles, although the total amount of triacylglycerol synthesized and secreted was highest with micelles. When incubating CaCo-2 monolayers with eicosapentaenoic acid or oleic acid bound to albumin, a similar amount of radioactivity was released as CO2 and acid soluble products into the medium with less than 3% of the lipids being oxidized after 5 h of incubation. The oxidation rate of fatty acids in cells incubated with micelles was increased 40 to 100%. In conclusion, micellar fatty acids are absorbed, metabolized and influence secretion of lipoprotein particles to a higher extent than albumin-bound fatty acids in CaCo-2 cells, and there is no major difference between eicosapentaenoic acid and oleic acid.

Tetradecylthioacetic acid (a 3-thia fatty acid) impairs secretion of oleic acid-induced triacylglycerol-rich lipoproteins in CaCo-2 cells.

The fatty acid analogue tetradecylthioacetic acid (TTA) has previously been shown to decrease triacylglycerol secretion in CaCo-2 cells (Gedde-Dahl et al., J. Lipid Res. 36 (1995) 535-543). The present study was designed to further elucidate the effect of TTA on lipoprotein production in CaCo-2 cells. TTA did not affect oleic acid-induced triacylglycerol synthesis, but it significantly decreased secretion of newly synthesized triacylglycerol when compared to cells incubated with oleic acid alone or oleic acid in combination with palmitic acid. In contrast, pulse-chase experiments showed no difference in the amount of labeled triacylglycerol secreted from cells exposed to either fatty acid combination during the chase period, indicating that TTA did not affect the secretory process in general. Cells incubated with TTA alone secreted triacylglycerol present at 1.025

Inhibition by chloroquine of the secretion of very low density lipoproteins by cultured rat hepatocytes.

Cultured rat hepatocytes were incubated in medium containing 1.0 mM oleic acid. The incorporation of [3H]glycerol into cell-associated and medium triacylglycerols was measured after 2 h incubation. More than 95% of the secreted [3H]triacylglycerols were recovered in the very low density lipoprotein (VLDL) fraction (d less than 1.006). Chloroquine and other lysosomotropic amines promoted a marked decrease in [3H]triacylglycerol secretion from the hepatocytes while the synthesis was unaffected. At 50-200 microM final concentration, chloroquine inhibited secretion of triacylglycerols by 70-90% of the control. Similar results were obtained when the mass of secreted triacylglycerols was measured. Chloroquine caused decreased secretion of [3H]triacylglycerols after 15-30 min incubation and the inhibitory effect was completely reversible within 1-2 h after washout of chloroquine. The reduced triacylglycerol secretion was not due to increased reuptake of secreted lipoproteins or decreased protein synthesis caused by chloroquine. Electron microscopy of chloroquine-treated cells showed that the inhibition of VLDL secretion occurs at or prior to the level of the Golgi apparatus. These results suggest that chloroquine interferes with crucial steps in the secretory process and/or that lysosomal function could be essential for secretion of VLDL.

Eicosapentaenoic acid inhibits synthesis and secretion of triacylglycerols by cultured rat hepatocytes.

Primary cultures of rat hepatocytes were used to study the effects of eicosapentaenoic and oleic acid on synthesis and secretion of triacylglycerols associated with very low density lipoproteins. From the experiments the following was observed. Oleic acid markedly stimulates secretion as well as synthesis of triacylglycerols, whereas eicosapentaenoic acid causes very little or no increase in secretion or synthesis as compared to a fatty-acid-free medium. The effects could already be observed after 15 min incubation. The inhibitory effect of eicosapentaenoic acid is reversible within 1-2 h. Eicosapentaenoic acid inhibits much of the stimulatory effect of oleic acid on synthesis and secretion of triacylglycerols. The cellular uptake of eicosapentaenoic acid is somewhat higher than that of oleic acid and the metabolism of these fatty acids to acid-soluble materials is similar. Eicosapentaenoic acid does not affect the secretory pathway of triacylglycerols per se. From these results it may be concluded that the mechanism for the inhibitory effect of eicosapentaenoic acid on triacylglycerol secretion is probably via reduced triacylglycerol synthesis.

Effect of 3- and 4-thia-substituted fatty acids on glycerolipid metabolism and mitochondrial beta-oxidation in rat liver.

Treatment of normolipidemic rats by alkylthiopropionic acid (CETTD), resulted in a dose- and time-dependent increase in total dihydroxyacetone phosphate acyltransferase (DHAPAT) activity, in extent comparable to that of 3-thiadicarboxylic acid (BCMTD) and alkylthioacetic acid (CMTTD). Thus, in CETTD- and CMTTD-treated rats, the specific DHAPAT activity increased in the microsomal, peroxisomal and mitochondrial fractions. In contrast, repeated administration of the peroxisome proliferator, BCMTD, decreased the specific DHAPAT activity both in the peroxisomal fraction and in purified peroxisomes. A three-fold increase in specific activity was, however, revealed in the mitochondrial fraction. Whether the variation of the DHAPAT activity in the mitochondrial and microsomal fractions among the feeding groups can be explained by increased number of enlarged and small peroxisomes sedimenting in the fractions, are to be considered. Subcellular fractionation studies confirmed previous findings that rat liver glycerophosphate acyltransferase (GPAT) was located both in mitochondria and the microsomal fraction. BCMTD was considerably more potent than CMTTD in stimulating the microsomal and mitochondrial GPAT activities. Administration of CETTD marginally affected the isoenzymes of GPAT. Diacylglycerol acyltransferase (DGAT) activity was increased by 35% in BCMTD and CMTTD treated rats, but by administration of CETTD the enzyme activity was decreased by more than 80%. The acyl-CoA cholesterol acyltransferase (ACAT) activity was marginally affected in animals treated with BCMTD, CMTTD and CETTD. Thus, the results indicate that the initial steps in the synthesis of triacylglycerols and ether glycerolipids as well as the last step in triacylglycerol synthesis could not be identified as mediating the fat accumulation or the lowering of triacylglycerol content in liver of CETTD, or BCMTD and CMTTD treated rats. On the other hand, CMTTD increased the palmitoyl-CoA oxidation in mitochondria, and CETTD considerably inhibited the activity. Therefore, it is conceivable that the development of fatty liver with CETTD is mostly due to inhibition of mitochondrial beta-oxidation.

Omega-3 and omega-6 fatty acids in the insulin resistance syndrome. Lipid and lipoprotein metabolism and atherosclerosis.

Dietary fatty acids appear to be of significant importance for several of the most-common diseases in modern societies. To obtain more knowledge about the health consequences of dietary fatty acids, we depend upon a better understanding of the mechanisms of action of these fatty acids in vivo. With regard to the IRS, omega-3 PUFA may exert beneficial effects upon many of the associated pathophysiological metabolic changes. Omega-3 PUFA reduce fasting and postprandial TG, may improve insulin sensitivity (as shown in animal experiments), decrease platelet and leukocyte reactivity, alter immunological functions, and may slightly decrease blood pressure. Omega-3 PUFA may also beneficially influence vessel wall characteristics and blood rheology. Furthermore, both types of PUFA (omega-3 and omega-6) have been shown to inhibit cardiac arrhythmias in animals. The role of omega-3 PUFA in blood clotting and fibrinolysis still remains controversial, whereas omega-6 fatty acids may lead to increased oxidation of lipoproteins. Regardless of the effects on LDL oxidizability, both types of PUFA have shown beneficial effects on the development of atherosclerosis. As yet, little is known about the effect of specific omega-6 fatty acids with respect to the IRS. Potential adverse effects of dietary PUFA must not be neglected, but should be viewed in light of the beneficial effects of these agents.

Omega-3 fatty acids--nutritional aspects.

Omega-3 fatty acids contain a double bond in the third position from the methyl group. The very long-chain (20 or 22 carbon atoms) omega-3 fatty acids are mostly found in fatty fish and fish oils. The omega-3 fatty acids are essential and may act as precursors for eicosanoids, altering membrane fluidity or binding to transcription factors. Dietary intake of omega-3 fatty acids reduces plasma concentration of triglycerides, probably by decreasing hepatic secretion of very low density lipoprotein (VLDL) and by increasing catabolism of chylomicrons. In addition, lipid peroxidation of omega-3 fatty acids may take place, with good and bad consequences. As the number of double bonds is high, the omega-3 fatty acids may easily react with oxygen radicals. We performed studies where 5 g/day of very long-chain omega-3 fatty acids was given as a supplement for four months along with vitamin E, whereas control groups received similar amounts of other oils. The unsaturation index was higher in fatty acids of LDL from individuals exposed to omega-3 fatty acids, and the amounts of cholesteryl esters and total lipids were lower compared with control LDL, whereas similar electrophoretic mobility and apolipoprotein B structure were observed. There was a decrease in the melting temperature of cholesteryl esters in omega-3 fatty acid-enriched LDL, but no change in the susceptibility of LDL to Cu2+ catalyzed lipid peroxidation, as measured by changes in amounts of lipid peroxides or in the uptake of LDL in macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of non-beta-oxidizable sulfur-substituted fatty acid analogues on synthesis and secretion of triacylglycerol and cholesterol in cultured rat hepatocytes.

The mechanisms behind the hypolipidemic effect of two sulfur-substituted fatty acid analogues, 3-thiadicarboxylic acid and tetradecylthioacetic acid, have been investigated in cultured hepatocytes. There was a dose-dependent reduction in incorporation of [3H] water into triacylglycerol and diacylglycerol when tetradecylthioacetic acid was added to rat hepatocytes cultured in the presence of 200 muM oleic acid. Tetradecylthioacetic acid also increased the oxidation of [14C]palmitic acid compared to oleic acid, inhibited the incorporation of radiolabeled precursors into diacylglycerol to a greater extent than into triacylglycerol, and reduced the secretion of triacylglycerol more than its synthesis. A stimulation, rather than a reduction, in glycerolipid synthesis and secretion by oxidation of fatty acids and reduces the synthesis and secretion of glycerolipids. 3-Thiadicarboxylic acid reduces the synthesis and secretion of both glycerolipids and cholesterol to approximately the same extent without a concomitant increase in the oxidation of fatty acids.

The hypotriglyceridemic effect of eicosapentaenoic acid in rats is reflected in increased mitochondrial fatty acid oxidation followed by diminished lipogenesis.

The effect of eicosapentaenoic acid (EPA) on fatty acid oxidation and on key enzymes of triglyceride metabolism and lipogenesis was investigated in the liver of rats. Repeated administration of EPA to normolipidemic rats resulted in a time-dependent decrease in plasma triglycerides, phospholipids and cholesterol. The triglyceride-lowering effect was observed after one day of feeding whereas lowering of plasma cholesterol and phospholipids was observed after five days of treatment. The triglyceride content of liver was reduced after two-day treatment. At that time, increased mitochondrial fatty acid oxidation occurred whereas mitochondrial and microsomal glycerophosphate acyltransferase was inhibited. The phosphatidate phosphohydrolase activity was unchanged. Adenosine triphosphate:citrate lyase, acetyl-CoA carboxylase, fatty acid synthetase and glucose-6-phosphate dehydrogenase were inhibited during the 15 d of EPA treatment whereas peroxisomal beta-oxidation was increased. At one day of feeding, however, when the hypotriglyceridemic effect was established, the lipogenic enzyme activities were reduced to the same extent in palmitic acid-treated animals as in EPA-treated rats. In cultured rat hepatocytes, the oxidation of [14C]palmitic acid to carbon dioxide and acid-soluble products was stimulated in the presence of EPA. These results suggest that the instant hypolipidemia in rats given EPA could be explained at least in part by a sudden increase in mitochondrial fatty acid oxidation, thereby reducing the availability of fatty acids for lipid synthesis in the liver for export, e.g., in the form of very low density lipoproteins, even before EPA induced peroxisomal fatty acid oxidation, reduced triglyceride biosynthesis and diminished lipogenesis.

Eicosapentaenoic acid, but not docosahexaenoic acid, increases mitochondrial fatty acid oxidation and upregulates 2,4-dienoyl-CoA reductase gene expression in rats.

The aim of the present study was to investigate whether eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) was responsible for the triglyceride-lowering effect of fish oil. In rats fed a single dose of EPA as ethyl ester (EPA-EE), the plasma concentration of triglycerides was decreased at 8 h after acute administration. This was accompanied by an increased hepatic fatty acid oxidation and mitochondrial 2,4-dienoyl-CoA reductase activity. The steady-state level of 2,4-dienoyl-CoA reductase mRNA increased in parallel with the enzyme activity. An increased hepatic long-chain acyl-CoA content, but a reduced amount of hepatic malonyl-CoA, was obtained at 8 h after acute EPA-EE treatment. On EPA-EE supplementation, both EPA (20:5n-3) and docosapentaenoic acid (DPA, 22:5n-3) increased in the liver, whereas the hepatic DHA (22:6n-3) concentration was unchanged. On DHA-EE supplementation retroconversion to EPA occurred. No statistically significant differences were found, however, for mitochondrial enzyme activities, malonyl-CoA, long-chain acyl-CoA, plasma lipid levels, and the amount of cellular fatty acids between DHA-EE treated rats and their controls at any time point studied. In cultured rat hepatocytes, the oxidation of [1-14C]palmitic acid was reduced by DHA, whereas it was stimulated by EPA. In the in vivo studies, the activities of phosphatidate phosphohydrolase and acetyl-CoA carboxylase were unaffected after acute EPA-EE and DHA-EE administration, but the fatty acyl-CoA oxidase, the rate-limiting enzyme in peroxisomal fatty acid oxidation, was increased after feeding these n-3 fatty acids. The hypocholesterolemic properties of EPA-EE may be due to decreased 3-hydroxy-3-methylglutaryl-CoA reductase activity. Furthermore, replacement of the ordinary fatty acids, i.e., the monoenes (16:1n-7, 18:1n-7, and 18:1n-9) with EPA and some conversion to DPA concomitant with increased fatty acid oxidation is probably the mechanism leading to changed fatty acid composition. In contrast, DHA does not stimulate fatty acid oxidation and, consequently, no such displacement mechanism operates. In conclusion, we have obtained evidence that EPA, and not DHA, is the fatty acid primarily responsible for the triglyceride-lowering effect of fish oil in rats.

Eicosapentaenoic and docosahexaenoic acid affect mitochondrial and peroxisomal fatty acid oxidation in relation to substrate preference.

Decreased triacylglycerol synthesis within hepatocytes due to decreased diacylglycerol acyltransferase (DGAT) activity has been suggested to be an important mechanism by which diets rich in fish oil lower plasma triacylglycerol levels. New findings suggest that eicosapentaenoic acid (EPA), and not docosahexaenoic acid (DHA), lowers plasma triacylglycerol by increased mitochondrial fatty acid oxidation and decreased availability of fatty acids for triacylglycerol synthesis. To contribute to the understanding of the triacylglycerol-lowering mechanism of fish oil, the different metabolic properties of EPA and DHA were studied in rat liver parenchymal cells and isolated rat liver organelles. EPA-CoA was a poorer substrate than DHA-CoA for DGAT in isolated rat liver microsomes, and in the presence of EPA, a markedly lower value for the triacyl[3H]glycerol/diacyl[3H]glycerol ratio was observed. The distribution of [1-14C]palmitic acid was shifted from incorporation into secreted glycerolipids toward oxidation in the presence of EPA (but not DHA) in rat liver parenchymal cells. [1-14C]EPA was oxidized to a much greater extent than [1-14C]DHA in rat liver parenchymal cells, isolated peroxisomes, and especially in purified mitochondria. As the oxidation of EPA was more effective and sensitive to the CPT-I inhibitor, etomoxir, when measured in a combination of both mitochondria and peroxisomes, we hypothesized that both are involved in EPA oxidation, whereas DHA mainly is oxidized in peroxisomes. In rats, EPA treatment lowered plasma triacylglycerol and increased hepatic mitochondrial fatty acid oxidation and carnitine palmitoyltransferase (CPT)-I activity in both the presence and absence of malonyl-CoA. Whereas only EPA treatment increased the mRNA levels of CPT-I, DHA treatment increased the mRNA levels of peroxisomal fatty acyl-CoA oxidase and fatty acid binding protein more effectively than EPA treatment. In conclusion, EPA and DHA affect cellular organelles in relation to their substrate preference. The present study strongly supports the hypothesis that EPA, and not DHA, lowers plasma triacylglycerol by increased mitochondrial fatty acid oxidation.

The hypotriglyceridemic effect of dietary n-3 FA is associated with increased beta-oxidation and reduced leptin expression.

To study the mechanisms responsible for the hypotriglyceridemic effect of marine oils, we monitored the effects of high dietary intake of n-3 PUFA on hepatic and muscular beta-oxidation, plasma leptin concentration, leptin receptor gene expression, and in vivo insulin action. Two groups of male Wistar rats were fed either a high-fat diet [28% (w/w) of saturated fat] or a high-fat diet containing 10% n-3 PUFA and 18% saturated fat for 3 wk. The hypotriglyceridemic effect of n-3 PUFA was accompanied by increased hepatic oxidation of palmitoyl-CoA (125%, P < 0.005) and palmitoyl-L-carnitine (480%, P < 0.005). These findings were corroborated by raised carnitine palmitoyltransferase-2 activity (154%, P < 0.001) and mRNA levels (91%, P < 0.01) as well as by simultaneous elevation of hepatic peroxisomal acyl-CoA oxidase activity (144%, P < 0.01) and mRNA content (82%, P < 0.05). In contrast, hepatic carnitine palmitoyltransferase-1 activity remained unchanged despite a twofold increased mRNA level after n-3 PUFA feeding. Skeletal muscle FA oxidation was less affected by dietary n-3 PUFA, and the stimulatory effect was found only in peroxisomes. Dietary intake of n-3 PUFA was followed by increased acyl-CoA oxidase activity (48%, P < 0.05) and mRNA level (83%, P < 0.05) in skeletal muscle. The increased FA oxidation after n-3 PUFA supplementation of the high-fat diet was accompanied by lower plasma leptin concentration (-38%, P < 0.05) and leptin mRNA expression (-66%, P < 0.05) in retroperitoneal adipose tissue, and elevated hepatic mRNA level for the leptin receptor Ob-Ra (140%, P < 0.05). Supplementation of the high-fat diet with n-3 PUFA enhanced in vivo insulin sensitivity, as shown by normalization of the glucose infusion rate during euglycemic hyperinsulinemic clamp. Our results indicate that the hypotriglyceridemic effect of dietary n-3 PUFA is associated with stimulation of FA oxidation in the liver and to a smaller extent in skeletal muscle. This may ameliorate dyslipidemia, tissue lipid accumulation, and insulin action, in spite of decreased plasma leptin level and leptin mRNA in adipose tissue.