Arild C. Rustan

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.

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.

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.

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 β-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 (20:5 n-3) increases fatty acid and glucose uptake in cultured human skeletal muscle cells.

This study was conducted to evaluate the chronic effects of eicosapentaenoic acid (EPA) on fatty acid and glucose metabolism in human skeletal muscle cells. Uptake of [14C]oleate was increased >2-fold after preincubation of myotubes with 0.6 mM EPA for 24 h, and incorporation into various lipid classes showed that cellular triacylgycerol (TAG) and phospholipids were increased 2- to 3-fold compared with control cells. After exposure to oleic acid (OA), TAG was increased 2-fold. Insulin (100 nM) further increased the incorporation of [14C]oleate into all lipid classes for EPA-treated myotubes. Fatty acid β-oxidation was unchanged, and complete oxidation (CO2) decreased in EPA-treated cells. Basal glucose transport and oxidation (CO2) were increased 2-fold after EPA, and insulin (100 nM) stimulated glucose transport and oxidation similarly in control and EPA-treated myotubes, whereas these responses to insulin were abolished after OA treatment. Lower concentrations of EPA (0.1 mM) also increased fatty acid and glucose uptake. CD36/FAT (fatty acid transporter) mRNA expression was increased after EPA and OA treatment compared with control cells. Moreover, GLUT1 expression was increased 2.5-fold by EPA, whereas GLUT4 expression was unchanged, and activities of the mitogen-activated protein kinase p38 and extracellular signal-regulated kinase were decreased after treatment with OA compared with EPA. Together, our data show that chronic exposure of myotubes to EPA promotes increased uptake and oxidation of glucose despite a markedly increased fatty acid uptake and synthesis of complex lipids.

Proteomic identification of secreted proteins from human skeletal muscle cells and expression in response to strength training

Regular physical activity protects against several types of diseases. This may involve altered secretion of signaling proteins from skeletal muscle. Our aim was to identify the most abundantly secreted proteins in cultures of human skeletal muscle cells and to monitor their expression in muscles of strength-training individuals. A total of 236 proteins were detected by proteome analysis in medium conditioned by cultured human myotubes, which was narrowed down to identification of 18 classically secreted proteins expressed in skeletal muscle, using the SignalP 3.0 and Human Genome Expression Profile databases together with a published mRNA-based reconstruction of the human skeletal muscle secretome. For 17 of the secreted proteins, expression was confirmed at the mRNA level in cultured human myotubes as well as in biopsies of human skeletal muscles. RT-PCR analyses showed that 15 of the secreted muscle proteins had significantly enhanced mRNA expression in m. vastus lateralis and/or m. trapezius after 11 wk of strength training among healthy volunteers. For example, secreted protein acidic and rich in cysteine, a secretory protein in the membrane fraction of skeletal muscle fibers, was increased 3- and 10-fold in m. vastus lateralis and m. trapezius, respectively. Identification of proteins secreted by skeletal muscle cells in vitro facilitated the discovery of novel responses in skeletal muscles of strength-training individuals.

Altered Skeletal Muscle Lipase Expression and Activity Contribute to Insulin Resistance in Humans

OBJECTIVE Insulin resistance is associated with elevated content of skeletal muscle lipids, including triacylglycerols (TAGs) and diacylglycerols (DAGs). DAGs are by-products of lipolysis consecutive to TAG hydrolysis by adipose triglyceride lipase (ATGL) and are subsequently hydrolyzed by hormone-sensitive lipase (HSL). We hypothesized that an imbalance of ATGL relative to HSL (expression or activity) may contribute to DAG accumulation and insulin resistance. RESEARCH DESIGN AND METHODS We first measured lipase expression in vastus lateralis biopsies of young lean (n = 9), young obese (n = 9), and obese-matched type 2 diabetic (n = 8) subjects. We next investigated in vitro in human primary myotubes the impact of altered lipase expression/activity on lipid content and insulin signaling. RESULTS Muscle ATGL protein was negatively associated with whole-body insulin sensitivity in our population (r = −0.55, P = 0.005), whereas muscle HSL protein was reduced in obese subjects. We next showed that adenovirus-mediated ATGL overexpression in human primary myotubes induced DAG and ceramide accumulation. ATGL overexpression reduced insulin-stimulated glycogen synthesis (−30%, P < 0.05) and disrupted insulin signaling at Ser1101 of the insulin receptor substrate-1 and downstream Akt activation at Ser473. These defects were fully rescued by nonselective protein kinase C inhibition or concomitant HSL overexpression to restore a proper lipolytic balance. We show that selective HSL inhibition induces DAG accumulation and insulin resistance. CONCLUSIONS Altogether, the data indicate that altered ATGL and HSL expression in skeletal muscle could promote DAG accumulation and disrupt insulin signaling and action. Targeting skeletal muscle lipases may constitute an interesting strategy to improve insulin sensitivity in obesity and type 2 diabetes.

Electrical Pulse Stimulation of Cultured Human Skeletal Muscle Cells as an In Vitro Model of Exercise

Background and Aims Physical exercise leads to substantial adaptive responses in skeletal muscles and plays a central role in a healthy life style. Since exercise induces major systemic responses, underlying cellular mechanisms are difficult to study in vivo. It was therefore desirable to develop an in vitro model that would resemble training in cultured human myotubes. Methods Electrical pulse stimulation (EPS) was applied to adherent human myotubes. Cellular contents of ATP, phosphocreatine (PCr) and lactate were determined. Glucose and oleic acid metabolism were studied using radio-labeled substrates, and gene expression was analyzed using real-time RT-PCR. Mitochondrial content and function were measured by live imaging and determination of citrate synthase activity, respectively. Protein expression was assessed by electrophoresis and immunoblotting. Results High-frequency, acute EPS increased deoxyglucose uptake and lactate production, while cell contents of both ATP and PCr decreased. Chronic, low-frequency EPS increased oxidative capacity of cultured myotubes by increasing glucose metabolism (uptake and oxidation) and complete fatty acid oxidation. mRNA expression level of pyruvate dehydrogenase complex 4 (PDK4) was significantly increased in EPS-treated cells, while mRNA expressions of interleukin 6 (IL-6), cytochrome C and carnitin palmitoyl transferase b (CPT1b) also tended to increase. Intensity of MitoTracker®Red FM was doubled after 48 h of chronic, low-frequency EPS. Protein expression of a slow fiber type marker (MHCI) was increased in EPS-treated cells. Conclusions Our results imply that in vitro EPS (acute, high-frequent as well as chronic, low-frequent) of human myotubes may be used to study effects of exercise.

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 thein 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.

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.