Benfotiamine increases glucose oxidation and downregulates NADPH oxidase 4 expression in cultured human myotubes exposed to both normal and high glucose concentrations.

The aim of the present work was to study the effects of benfotiamine (S-benzoylthiamine O-monophosphate) on glucose and lipid metabolism and gene expression in differentiated human skeletal muscle cells (myotubes) incubated for 4 days under normal (5.5 mM glucose) and hyperglycemic (20 mM glucose) conditions. Myotubes established from lean, healthy volunteers were treated with benfotiamine for 4 days. Glucose and lipid metabolism were studied with labeled precursors. Gene expression was measured using real-time polymerase chain reaction (qPCR) and microarray technology. Benfotiamine significantly increased glucose oxidation under normoglycemic (35 and 49% increase at 100 and 200 µM benfotiamine, respectively) as well as hyperglycemic conditions (70% increase at 200 µM benfotiamine). Benfotiamine also increased glucose uptake. In comparison, thiamine (200 µM) increased overall glucose metabolism but did not change glucose oxidation. In contrast to glucose, mitochondrial lipid oxidation and overall lipid metabolism were unchanged by benfotiamine. The expression of NADPH oxidase 4 (NOX4) was significantly downregulated by benfotiamine treatment under both normo- and hyperglycemic conditions. Gene set enrichment analysis (GSEA) showed that befotiamine increased peroxisomal lipid oxidation and organelle (mitochondrial) membrane function. In conclusion, benfotiamine increases mitochondrial glucose oxidation in myotubes and downregulates NOX4 expression. These findings may be of relevance to type 2 diabetes where reversal of reduced glucose oxidation and mitochondrial capacity is a desirable goal.

Metabolic switching of human skeletal muscle cells in vitro.

In this review we will focus on external factors that may modify energy metabolism in human skeletal muscle cells (myotubes) and the ability of the myotubes to switch between lipid and glucose oxidation. We describe the metabolic parameters suppressibility, adaptability and substrate-regulated flexibility, and show the influence of nutrients such as fatty acids and glucose (chronic hyperglycemia), and some pharmacological agents modifying nuclear receptors (PPAR and LXR), on these parameters in human myotubes. Possible cellular mechanisms for changes in these parameters will also be highlighted.

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.

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 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.

Liver X receptor antagonist reduces lipid formation and increases glucose metabolism in myotubes from lean, obese and type 2 diabetic individuals.

AIMS/HYPOTHESIS: Liver X receptors (LXRs) play important roles in lipid and carbohydrate metabolism. The purpose of the present study was to evaluate effects of the endogenous LXR agonist 22-R-hydroxycholesterol (22-R-HC) and its stereoisomer 22-S-hydroxycholesterol (22-S-HC), in comparison with the synthetic agonist T0901317 on lipid and glucose metabolism in human skeletal muscle cells (myotubes). METHODS: Myotubes established from lean and obese control volunteers and from obese type 2 diabetic volunteers were treated with LXR ligands for 4 days. Lipid and glucose metabolisms were studied with labelled precursors, and gene expression was analysed using real-time PCR. RESULTS: Treatment with T0901317 increased lipogenesis (de novo lipid synthesis) and lipid accumulation in myotubes, this increase being more pronounced in myotubes from type 2 diabetic volunteers than from lean volunteers. Furthermore, 22-S-HC efficiently counteracted the T0901317-induced enhancement of lipid formation. Moreover, synthesis of diacylglycerol, cholesteryl ester and free cholesterol from acetate was reduced below baseline by 22-S-HC, whereas glucose uptake and oxidation were increased. Both 22-S-HC and 22-R-HC, in contrast to T0901317, decreased the expression of genes involved in cholesterol synthesis, whereas only 22-R-HC, like T0901317, increased the expression of the gene encoding the reverse cholesterol transporter ATP-binding cassette subfamily A1 (ABCA1). CONCLUSIONS/INTERPRETATION: T0901317-induced lipogenesis and lipid formation was more pronounced in myotubes from type 2 diabetic patients than from lean individuals. 22-S-HC counteracted these effects and reduced de novo lipogenesis below baseline, while glucose uptake and oxidation were increased.

Cell-based multiwell assays for the detection of substrate accumulation and oxidation.

We describe multiwell assays for detecting the accumulation as well as the subsequent oxidation of (14)C-labeled substrates in cultured cells. Accumulation is monitored in real time by an established scintillation proximity assay in which the scintillator is embedded in the plate base primarily detecting cell-associated radiolabel. The substrate oxidation assay is a novel variant of previously described experimental approaches aimed at trapping (14)CO(2) produced by isolated enzymes, organelles, or intact cells. This method uses a standard 96-well tissue culture plate and, on top, an inverted filter plate immersed with NaOH that are clamped into a sandwich sealed with a silicon gasket to obtain gas-tight compartments. (14)CO(2) is captured in the filter and quantified by conventional scintillation. We demonstrate both the accumulation and subsequent oxidation of (14)C-labeled substrates in cultured human myotubes, adipocytes, and hepatocytes. Both methods are adaptable for compound screening; at the same time, these protocols provide easy-to-use and time- saving methods for in vitro studies of cellular fuel handling.

Leukaemia inhibitory factor stimulates glucose transport in isolated cardiomyocytes and induces insulin resistance after chronic exposure.

AIMS/HYPOTHESIS: Hypertrophic and failing hearts have increased utilisation of glucose, but also develop insulin resistance and reduced ability to produce ATP. Increased levels of the IL-6-related cytokine leukaemia inhibitory factor (LIF) are found in failing hearts, and we have recently shown that LIF reduces ATP production in isolated cardiomyocytes. In the present study we investigated effects of LIF on glucose metabolism, and how LIF-treated cells respond to insulin stimulation. METHODS: Cardiomyocytes were isolated from adult Wistar rats by collagen digestion, maintained in culture for 48 h, and then treated with 1 nmol/l LIF. RESULTS: Acute LIF treatment increased deoxyglucose uptake compared with controls, but no additive effect was observed in cardiomyocytes treated with LIF and insulin. The phosphatidylinositol 3-kinase inhibitor wortmannin did not affect LIF-induced glucose uptake. LIF had no effect on AMP-activated protein kinase phosphorylation. Cardiomyocytes treated with LIF for 48 h did not respond to insulin by increasing deoxyglucose uptake and showed a reduced insulin-mediated uptake of oleic acid and formation of complex lipids compared with control cells. Chronic LIF treatment increased gene expression of the suppressor of cytokine signalling (Socs) 3 and reduced expression of solute carrier family 2, member 4 (Slc2a4, previously known as glucose transporter 4 [Glut4]). In line with these observations, chronic LIF treatment reduced insulin-mediated phosphorylation of both Akt/protein kinase B (PKB) and glycogen synthase kinase (GSK)-3. CONCLUSIONS/INTERPRETATION: Acute LIF treatment increased glucose uptake in isolated cardiomyocytes by a pathway different from that of insulin. Chronic LIF treatment induced insulin resistance, possibly mediated by altered expression of Socs3 and Slc2a4, and impaired insulin-mediated phosphorylation of GSK-3 and Akt/PKB.

Lipid metabolism in human skeletal muscle cells: effects of palmitate and chronic hyperglycaemia.

This review focuses on the effect of exogenous factors known to be of importance for the development of insulin resistance in differentiated human myotubes. Recent data from our laboratory on the effects of fatty acid pre-treatment and chronic glucose oversupply on fatty acid and glucose metabolism, without and with acute insulin are presented, and discussed in the context of other recent publications in the field. Pre-treatment of myotubes with palmitate, chronic hyperglycaemia, and acute high concentrations of insulin changed fatty acid metabolism in favour of accumulation of intracellular lipids. Acute insulin exposure increased (14)C-oleate uptake and levels of free fatty acids (FFA) and triacylglycerol (TAG). Palmitate pre-treatment further increased oleate uptake, both under basal conditions and in the presence of insulin, with a marked increase in the phospholipid (PL) fraction, with a concomitant reduction in oleate oxidation. Chronic hyperglycaemia also promoted increased lipogenesis and elevated levels of cellular lipids. Changes in fatty acid metabolism in human muscle, in particular fatty acid oxidation, are probably crucial for the molecular mechanism behind skeletal muscle insulin resistance and impaired glucose metabolism. Differentiated human skeletal muscle cells may be an ideal system to further explore the mechanisms regulating lipid metabolism.

Chronic hyperglycaemia promotes lipogenesis and triacylglycerol accumulation in human skeletal muscle cells.

AIMS/HYPOTHESIS: The present study was conducted to evaluate the effect of hyperglycaemia in itself on glucose and lipid metabolism in human skeletal muscle cells. METHODS: Satellite cells were isolated from biopsy samples from the vastus lateralis muscle and differentiated into multinucleated myotubes in cultures. Metabolism studies were performed using isotopes ([3H]deoxyglucose, [14C]glucose, [14C]oleic acid and [14C]palmitic acid), and mRNA and protein levels were analysed by real-time RT-PCR and western blotting respectively. RESULTS: Exposure of myotubes to 20 mmol/l glucose for 4 days reduced insulin-stimulated glucose uptake and glycogen synthesis to 57+/-5% (p<0.0001) and 56+/-5% (p<0.0001) of normoglycaemic (5.5 mmol/l glucose) controls respectively. Basal glucose uptake and glycogen synthesis were both reduced, whereas glucose oxidation was unaltered. Total cell content of glycogen and expression of GLUT1 and GLUT4 mRNA were not affected. There was a significant increase in the incorporation of glucose into cellular NEFA (88+/-17% increase, p=0.006), triacylglycerol (44+/-21% increase, p=0.04) and cholesterol ester (89+/-36% increase, p=0.02) in hyperglycaemic myotubes compared with controls. Diacylglycerol tended to be increased though not significantly, and phospholipid formation were unchanged. Relative to controls, total cell content of triacylglycerol was increased by 25+/-7% (p=0.02) and acyl-CoA:1,2-diacylglycerol acyltransferase 1 activity was increased by 34+/-4% (p=0.004), whereas acyl-CoA:1,2-diacylglycerol acyltransferase 1 mRNA expression was unchanged. Total cellular uptake of palmitic acid was reduced by 18+/-3% (p=0.006) in hyperglycaemic cells compared with controls, while uptake of oleic acid was unchanged. Oxidation of palmitic acid or oleic acid was not affected by hyperglycaemia. CONCLUSIONS/INTERPRETATION: Chronic hyperglycaemia increased triacylglycerol accumulation and the incorporation of carbohydrate into triacylglycerol (i.e. de novo lipogenesis) concomitantly with a reduced insulin-stimulated glucose uptake and glycogen synthesis. Enhanced acyl-CoA:1,2-diacylglycerol acyltransferase 1 activity supported the increased triacylglycerol synthesis during hyperglycaemia.

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.

Effects of long-chain monounsaturated and n-3 fatty acids on fatty acid oxidation and lipid composition in rats.

Long-chain n-3 fatty acids and fat fish are reported, among multiple physiological properties, to enhance peroxisomal beta-oxidation and effect triacylglycerol status. Long-chain n-3 and monounsaturated fatty acids are the main portion of fatty acids in fat fish. The individual effect of long-chain monounsaturated fatty acids on beta-oxidation and fatty acid composition was tested and compared to the effect of n-3 polyunsaturated and saturated fatty acids in a 3-week feeding experiment of rats. To explore the contribution from long-chain monounsaturated fatty acids in these aspects, the effect of long-chain n-3 and monounsaturated fatty acids on mitochondrial and peroxisomal beta-oxidation was compared, as well as fatty acid composition of adipose tissue, liver and serum. Fatty acid oxidase, palmitoyltransferase I and II activities, the amount of serum lipids, and the fatty acid composition of lipid fractions from the organs were analysed. The peroxisomal beta-oxidation was enhanced by the n-3 fatty acids, whereas a small, significant increase with the monounsaturated fatty acids was observed. There was a stimulation of the mitochondrial oxidation with the n-3 fatty acids, but monounsaturated fatty acids gave a small, nonsignificant decrease. With n-3 fatty acids there was a considerable decrease in the levels of serum triacylglycerol, phospholipids, free fatty acids and total cholesterol, while there were only minor effects of monounsaturated fatty acids. As judged from the fatty acid composition data, there was a mobilization on n-3 fatty acids from the adipose tissue to liver and plasma with the n-3 diet. This observation was also seen with the monounsaturated fatty acid-enriched diet. In conclusion, monounsaturated fatty acids seemed to stimulate peroxisomal beta-oxidation and to increase plasma triacylglycerol, whereas the mitochondrial oxidation was slightly decreased.

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.

Changes in plasma free fatty acid concentrations in rheumatoid arthritis patients during fasting and their effects upon T-lymphocyte proliferation.

OBJECTIVE: To measure whether changes in the concentrations of circulating free fatty acids (FFAs) after a 7 day fast in rheumatoid arthritis (RA) patients would inhibit in vitro T-lymphocyte proliferation. METHODS: The concentration and composition of plasma FFAs were measured in nine RA patients at the conclusion of a 7 day fast. A FFA mixture was made up based on these findings (20% linoleic, 43% oleic, 10% stearic, 27% palmitic acid). Mitogen-induced lymphocyte proliferative responses were measured after co-culture of peripheral blood mononuclear cells (PBMC) from healthy individuals in the presence of increasing concentrations of this FFA mixture (from 0 to 2000 microM) and in the presence of FFA mixtures where the relative proportions of fatty acids varied. RESULTS: Both the concentration of the FFA mixture and the ratio between the unsaturated and saturated fatty acids significantly influenced in vitro lymphocyte proliferation (P<0.0001). Unexpectedly, the highest concentrations of the FFA mixture increased lymphocyte proliferation. At equimolar concentrations (600 microM), manipulating the amounts of oleic and linoleic fatty acids relative to stearic and palmitic fatty acids had a potent inhibitory effect upon lymphocyte proliferation. CONCLUSION: Fasting-associated increases in total plasma FFA concentrations do not inhibit, but rather enhance, in vitro lymphocyte proliferation. An inhibitory effect could only be achieved by manipulating the balance between the unsaturated and saturated fatty acids.

In contrast with docosahexaenoic acid, eicosapentaenoic acid and hypolipidaemic derivatives decrease hepatic synthesis and secretion of triacylglycerol by decreased diacylglycerol acyltransferase activity and stimulation of fatty acid oxidation.

Hypolipidaemic fatty acid derivatives and polyunsaturated fatty acids decrease concentrations of plasma triacylglycerol by mechanisms that are not fully understood. Because poor susceptibility to beta- and/or omega-oxidation is apparently a determinant of the peroxisome proliferating and hypolipidaemic capacity of fatty acids and derivatives, the relative importance of activation of the peroxisome-proliferator-activated receptor alpha (PPARalpha), fatty acid oxidation and triacylglycerol synthesis were examined. We have compared the effects of differentially beta-oxidizable fatty acids on these parameters in primary cultures of rat hepatocytes. Tetradecylthioacetic acid (TTA), 2-methyleicosapentaenoic acid and 3-thia-octadecatetraenoic acid, which are non-beta-oxidizable fatty acid derivatives, were potent activators of a glucocorticoid receptor (GR)-PPARalpha chimaera. This activation was paradoxically reflected in an substantially increased oxidation of [1-(14)C]palmitic acid and/or oleic acid. The incorporation of [1-(14)C]palmitic acid and/or oleic acid into cell-associated and secreted triacylglycerol was decreased by 15-20% and 30% respectively with these non-beta-oxidizable fatty acid derivatives. The CoA ester of TTA inhibited the esterification of 1, 2-diacylglycerol in rat liver microsomes. Both eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) activated GR-PPARalpha. EPA increased the oxidation of [1-(14)C]palmitic acid but DHA had no effect. The CoA ester of EPA inhibited the esterification of 1, 2-diacylglycerol, whereas DHA-CoA had no effect. The ratio between synthesized triacylglycerol and diacylglycerol was lower in hepatocytes cultured with EPA in the medium compared with DHA or oleic acid, indicating a decreased conversion of diacylglycerol to triacylglycerol. Indeed, the incorporation of [1-(14)C]oleic acid into secreted triacylglycerol was decreased by 20% in the presence of EPA. In conclusion, a decreased availability of fatty acids for triacylglycerol synthesis by increased mitochondrial beta-oxidation and decreased triacylglycerol formation caused by inhibition of diacylglycerol acyltransferase might explain the hypolipidaemic effect of TTA and EPA.

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

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.