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.

Phospholipid molecular species, beta-oxidation, desaturation and elongation of fatty acids in Atlantic salmon hepatocytes: effects of temperature and 3-thia fatty acids.

We have investigated the effects of a 3-thia fatty acid (TTA) and of temperature on the fatty acid (FA) metabolism of Atlantic salmon (Salmo salar). One experiment investigated the activity of the peroxisomal beta-oxidation enzyme, acyl-CoA oxidase (ACO), and the incorporation of TTA into phospholipid (PL) molecular species. Salmon hepatocytes in culture were incubated either without TTA (control(spades)) or with 0.8 mM TTA (TTA(spades)) in a short term (48 h) temperature study at 5 degrees C and at 12 degrees C. TTA was incorporated into the four PL classes studied: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS). TTA was preferentially esterified with 18:1, 16:1, 20:4 and 22:6 in the PLs. Hepatocytes incubated with TTA had higher ACO activity at 5 degrees C than at 12 degrees C. In a second experiment salmon were fed a diet based on fish meal-fish oil without any TTA added (control) or a fish meal-fish oil diet supplemented with 0.6% TTA for 8 weeks at 12 degrees C and 20 weeks at 5 degrees C. At the end of the feeding trial, hepatocytes from fish acclimated to high or low temperatures were isolated from both dietary groups and incubated with either [1-(14)C]18:1 n-9 or [1-(14)C]20:4 n-3 at 5 degrees C or 12 degrees C. Radiolabelled 18:1 n-9 was mainly esterified into neutral lipids (NL), whereas [1-(14)C]20:4 n-3 was mainly esterified into PL at both temperatures. The rate of elongation of [1-(14)C]18:1 n-9 to 20:1 n-9 was twice as high in hepatocytes from fish fed the control diet than it was in hepatocytes from fish fed the TTA diet, at both temperatures. The amount of [1-(14)C]20:4 n-3 converted to 22:6 n-3 was approximately the same in hepatocytes from the two dietary groups, but there was a tendency to higher production of 22:6 n-3 at the lower temperature. Oxidation of [1-(14)C]18:1 n-9 to acid soluble products (ASP) and CO(2) was approximately 10-fold greater in hepatocytes kept at 5 degrees C than in those kept at 12 degrees C and the main oxidation products formed were acetate, oxaloacetate and malate.

The metabolic syndrome and the hepatic fatty acid drainage hypothesis.

Much data indicates that lowering of plasma triglyceride levels by hypolipidemic agents is caused by a shift in the liver metabolism towards activation of peroxisome proliferator activated receptor (PPAR)alpha-regulated fatty acid catabolism in mitochondria. Feeding rats with lipid lowering agents leads to hypolipidemia, possibly by increased channeling of fatty acids to mitochondrial fatty acid oxidation at the expense of triglyceride synthesis. Our hypothesis is that increased hepatic fatty acid oxidation and ketogenesis drain fatty acids from blood and extrahepatic tissues and that this contributes significantly to the beneficial effects on fat mass accumulation and improved peripheral insulin sensitivity. To investigate this theory we employ modified fatty acids that change the plasma profile from atherogenic to cardioprotective. One of these novel agents, tetradecylthioacetic acid (TTA), is of particular interest due to its beneficial effects on lipid transport and utilization. These hypolipidemic effects are associated with increased fatty acid oxidation and altered energy state parameters of the liver. Experiments in PPAR alpha-null mice have demonstrated that the effects hypolipidemic of TTA cannot be explained by altered PPAR alpha regulation alone. TTA also activates the other PPARs (e.g., PPAR delta) and this might compensate for deficiency of PPAR alpha. Altogether, TTA-mediated clearance of blood triglycerides may result from a lowered level of apo C-III, with a subsequently induction of hepatic lipoprotein lipase activity and (re)uptake of fatty acids from very low density lipoprotein (VLDL). This is associated with an increased hepatic capacity for fatty acid oxidation, causing drainage of fatty acids from the blood stream. This can ultimately be linked to hypolipidemia, anti-adiposity, and improved insulin sensitivity.

Lipid-lowering and anti-inflammatory effects of tetradecylthioacetic acid in HIV-infected patients on highly active antiretroviral therapy.

BACKGROUND: Highly active antiretroviral therapy (HAART) often leads to a dramatic improvement in clinical, viral and immunologic parameters in HIV-infected individuals. However, the emergence of long-term side-effects of HAART and in particular dylipidaemia is increasingly reported. Based on the potential lipid-lowering and immunomodulatory properties of tetradecylthioacetic acid (TTA) we examined whether TTA in combination with dietary intervention could modify lipid levels in peripheral blood in HIV-infected patients on HAART. MATERIALS AND METHODS: Ten HIV-infected patients on protease inhibitor-based HAART with hyperlipidaemia followed a cholesterol-lowering diet throughout the study period (8 weeks). During the last 4 weeks of the study all patients received TTA (1 g qd) in addition to the cholesterol-lowering diet. RESULTS: Our main and novel findings were: (i) TTA in combination with dietary intervention reduces total cholesterol, LDL cholesterol, triglycerides and LDL/HDL cholesterol in these patients, and a particularly suppressing effect was observed during the TTA phase regarding total cholesterol. (ii) During the TTA phase, the cholesterol-lowering effect was accompanied by a significant reduction in plasma levels of tumour necrosis factor alpha. (iii) Our studies in peripheral blood mononuclear cells from these patients and in the liver from wild-type mice receiving TTA suggest that the hypolipidaemic effects of TTA may involve up-regulation of scavenger and LDL-receptor expression. CONCLUSIONS: Although few patients were studied, the present pilot study suggests that TTA combined with dietary intervention could be an interesting therapeutic approach in HIV-infected patients on HAART, potentially resulting in both hypolipidaemic and anti-inflammatory effects.

Long-chain acyl-CoA esters and acyl-CoA binding protein are present in the nucleus of rat liver cells.

A detailed analysis of the subcellular distribution of acyl-CoA esters in rat liver revealed that significant amounts of long-chain acyl-CoA esters are present in highly purified nuclei. No contamination of microsomal or mitochondrial marker enzymes was detectable in the nuclear fraction. C16:1 and C18:3-CoA esters were the most abundant species, and thus, the composition of acyl-CoA esters in the nuclear fraction deviates notably from the overall composition of acyl-CoA esters in the cell. After intravenous administration of the non-beta-oxidizable [(14)C]tetradecylthioacetic acid (TTA), the TTA-CoA ester could be recovered from the nuclear fraction. Acyl-CoA esters bind with high affinity to the ubiquitously expressed acyl-CoA binding protein (ACBP), and several lines of evidence suggest that ACBP functions as a pool former and transporter of acyl-CoA esters in the cytoplasm. By using immunohistochemistry, immunofluorescence microscopy, and immunoelectron microscopy we demonstrate that ACBP localizes to the nucleus as well as the cytoplasm of rat liver cell and rat hepatoma cells, suggesting that ACBP may also be involved in regulation of acyl-CoA-dependent processes in the nucleus.

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.

Fatty acyl-CoA oxidase activity is induced before long-chain acyl-CoA hydrolase activity and acyl-CoA binding protein in liver of rat treated with peroxisome proliferating 3-thia fatty acids.

1. In this study we explored the relationship between specific acyl-CoA esters and induction of acyl-CoA binding protein (ACBP) and enzymes related to the proliferation of peroxisomes. Male Wistar rats were administered a single dose (150 mg/day/kg) of sulphur-substituted fatty acid analogues, and the effects of tetradecylthioacetic acid and 3-thiadicarboxylic acid, which both act as peroxisome proliferators, were compared with the effects of tetradecylthiopropionic acid and palmitic acid which do not induce peroxisome proliferation. 2. The hepatic level of total long-chain acyl-CoA was significantly increased within 12 h of feeding these fatty acids, except in rat fed tetradecylthioacetic acid. Hplc chromatograms of liver extracts prepared from rat fed tetradecylthioacetic acid showed that tetradecylthioacetyl-CoA ester accumulated in the liver 4 h after feeding and had disappeared after 24 h. In liver extracts of the tetradecylthiopropionic acid-treated rat tetradecylthiopropionyl-CoA was not observed, but the appearance of a new long-chain acyl-CoA ester, probably a metabolite of tetradecylthiopropionic acid, was detected. This new peak reached a maximum 4h after feeding. In rat fed tetradecylthioacetic acid and 3-thiadicarboxylic acid the hepatic level of fatty acyl-CoA oxidase mRNA increased 8 h after feeding, while the acyl-CoA oxidase activity had increased after 12 h. 3. The early accumulation of specific tetradecylthioacetyl-CoA suggests that this ester may be a possible mediator of the induction of fatty acyl-CoA oxidase. The level of hepatic acyl-CoA binding protein, long-chain acyl-CoA hydrolase activity and long-chain acyl-CoA synthetase activity did not change after a single dose of all four fatty acids. Prolonged administration of 3-thia fatty acids resulted, however, in a dose- and time-dependent increase in hepatic ACBP content and ACBP mRNA level. The amount of ACBP increased in parallel to the long-chain acyl-CoA hydrolase activity. The correlated induction of fatty acyl-CoA binding protein and long-chain acyl-CoA hydrolase seems to be dependent on a sustained accumulation of total long-chain acyl-CoA esters.

Acute modulation of rat hepatic lipid metabolism by sulphur-substituted fatty acid analogues.

A single oral dose of two 3-thia (3-thiadicarboxylic and tetradecylthioacetic acids) and of 4-thia (tetradecylthiopropionic acid) fatty acids were administered to normolipidemic rats and their effects on lipid metabolism over a 24 hr period were studied. All three thia fatty acids could be detected in plasma 2 hr after treatment. Tetradecylthioacetic and tetradecylthiopropionic acids were detected in different hepatic lipid fractions but were incorporated mainly into hepatic phospholipids. Two hours after administration hepatic mitochondrial beta-oxidation and the total liver level of long-chain fatty acyl-CoA increased with a concomitant decrease in saturated fatty acids, total hepatic malonyl-CoA and plasma triacylglycerol levels in the 3-thia fatty acid groups. Tetradecylthiopropionic acid administration caused a decrease in mitochondrial beta-oxidation and an increase in plasma triacylglycerol at 24 hr. The activities of key lipogenic enzymes were unaffected in all treatment groups. Plasma cholesterol level was reduced only at 8 hr in 3-thiadicarboxylic acid treated rats although 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase was suppressed already at 2, 4, 8 and 12 hr. The results show that thia fatty acids are rapidly absorbed and are systemically available after oral administration but the 3-thia fatty acids reached systemic circulation more slowly and less completely than the 4-thia fatty acid. Very low levels of the thia fatty acids are detected in plasma 24 hr after a single administration. They are incorporated into all hepatic lipid classes, especially phospholipids. Rapid incorporation of a non beta-oxidizable thia fatty acid into hepatic lipids may cause a diversion of other fatty acids from glycerolipid biosynthesis to mitochondrial beta-oxidation. Stimulation of mitochondrial beta-oxidation and suppression of HMG-CoA reductase are primary events, occurring within hours, after 3-thia fatty acid administration. The hypotriglyceridemic effect of the 3-thia fatty acids observed at 2-4 hr is independent of the activities of key lipogenic and triacylglycerol synthesising enzymes.

Coordinate induction of hepatic fatty acyl-CoA oxidase and P4504A1 in rat after activation of the peroxisome proliferator-activated receptor (PPAR) by sulphur-substituted fatty acid analogues.

1. In the liver of rat fed a single dose of 3-thia fatty acids, 3-dithiahexadecanedioic acid (3-thiadicarboxylic acid) and tetradecylthioacetic acid, steady-state levels of P4504A1 and fatty acyl-CoA oxidase mRNAs increased in parallel. The increases were significant 8 h after administration, reaching a maximum after 12 h and decreased from 12 to 24 h after administration. 2. The corresponding enzyme activities of P4504A1 and fatty acyl-CoA oxidase were also induced in a parallel manner by the 3-thia fatty acids. The enzyme activities were significantly increased 12 h after administration and increased further after 24 h. This may reflect a possible effect of the 3-thia fatty acids not only on mRNA levels, but also on the translation and degradation rate of the two enzymes. 3. Repeated administration of 3-thia fatty acids resulted in an increase of the specific P4504A1 protein accompanied with an increased lauric acid hydroxylase activity. The correlation between induction of P4504A1 and fatty acyl-CoA oxidase mRNAs and their enzyme activities may reflect a coordinated rather than a causative induction mechanism, and that these genes respond to a common signal. This suggests that the increased P450 activity may not be responsible or be a prerequisite for fatty acyl-CoA oxidase induction. 4. Since the peroxisome proliferator-activated receptor (PPAR) plays a role in mediating the induction of fatty acyl-CoA oxidase, we analysed the activation of PPAR by fatty acids and sulphur-substituted analogues utilizing a chimera between the N-terminal and DNA-binding domain of the glucocorticoid receptor and the putative ligand-binding domain of PPAR. Arachidonic acid activated this chimeric receptor in Chinese hamster ovary cells. Inhibitors of P450 did not affect the activation of PPAR by arachidonic acid. Furthermore, dicarboxylic acids including 1,12-dodecanedioic acid or 1,16-hexadecanedioic acid only weakly activated the chimera. 3-Thidicarboxylic acid, however, was a much more effective activator than the non-sulphur-substituted analogues. In conclusion, the data suggest that the most likely mechanism of the induction process is fatty acid-induced activation of PPAR, which then leads to a coordinated induction of P4504A1 and fatty acyl-CoA oxidase.

Effect of 3-thiadicarboxylic acid on lipid metabolism in experimental nephrosis.

The effect of the sulfur-substituted fatty acid analogue 1,10 bis(carboxymethylthio)decane, also known as 3-thiadicarboxylic acid, on puromycin aminonucleoside-induced nephrotic hyperlipidemia was studied in rats. Treatment with 3-thiadicarboxylic acid (250 mg/kg) for 5 days reduced plasma levels of triglycerides from 5.8 to 2.7 mmol/L and cholesterol from 11.0 to 7.7 mmol/L. This was accounted for by decreases in very-low-density lipoprotein triglycerides, very-low-density lipoprotein cholesterol, and low-density lipoprotein cholesterol, without any major changes in the composition of plasma lipoproteins. The activities of two enzymes involved in fatty acid synthesis (ATP:citrate lyase and fatty acid synthetase) were inhibited by 3-thiadicarboxylic acid treatment, whereas acetyl-coenzyme A carboxylase activity was unchanged. In contrast, treatment with the sulfur-substituted fatty acid analogue induced the peroxisomal beta-oxidation of fatty acids ninefold and the mitochondrial beta-oxidation by 54% to 73%, depending on the substrate used. This was accompanied by a 26% reduction in hepatic triglyceride secretion rate. The hepatic phosphatidate phosphohydrolase activity was unchanged. 3-Thiadicarboxylic acid treatment suppressed the activity of the rate-limiting enzyme in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, by 58%, whereas hepatic LDL receptor expression was unaltered. The activities of lipoprotein lipase and hepatic lipase were unchanged by treatment. These results demonstrated that treatment with 3-thiadicarboxylic acid ameliorates hyperlipidemia in experimental nephrosis primarily by decreasing the overproduction of very-low-density lipoprotein present. The data also indicate that hepatic very-low-density lipoprotein synthesis and secretion is strongly influenced by the availability of the fatty acid substrate under the same hyperlipidemic conditions.

Modulation of phosphatidylcholine biosynthesis by peroxisome proliferating fatty acid analogues.

The modulation of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) biosynthesis by sulfur-substituted fatty acid analogues has been investigated in rats. We have compared the effects of two non-beta-oxidizable fatty acid analogues, 3-thiadicarboxylic acid and tetradecylthioacetic acid, which induce proliferation of peroxisomes, with those of the analogue tetradecylthiopropionic acid, which is a weak peroxisome proliferator. Repeated administration of 3-thiadicarboxylic acid for seven days resulted in increased hepatic concentrations of both PC and PE, but the PC/PE ratio was decreased. PC synthesis was increased, as evidenced by increased incorporation of [3H]choline into PC and an increased activity of cytidinetriphosphate (CTP): phosphocholine cytidylyltransferase. This was accompanied by a reduction in the pool sizes of choline and phosphocholine. The S-adenosylmethione/S-adenosylhomocysteine ratio (AdoMet/AdoHcy) was marginally affected, indicating no increase in the rate of methylation of PE to PC. Administration of tetradecylthioacetic acid also resulted in increased hepatic phospholipid levels, increased AdoMet/AdoHcy ratios and in slightly elevated activity of CTP:phosphocholine cytidylyltransferase. The most striking effect observed after tetradecylthiopropionic acid treatment was the development of fatty liver. The activity of CTP:phosphocholine cytidylyltransferase and the incorporation of [3H]choline into PC was reduced compared to 3-thiadicarboxylic acid treatment. Although the rate of methylation of PE seemed to be increased at an elevated AdoMet/AdoHcy ratio, this resulted in only minor changes in the hepatic PC and PE levels, and the PC/PE ratio remained unchanged. Furthermore, the hepatic levels of choline and phosphocholine were reduced in these rats.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

On the mechanism of the hypolipidemic effect of sulfur-substituted hexadecanedioic acid (3-thiadicarboxylic acid) in normolipidemic rats.

The mechanism behind the hypolipidemic effect of the sulfur-substituted non-beta-oxidizable fatty acid analogue 1,10 bis(carboxymethylthio)decane, also known as 3-thiadicarboxylic acid, was studied in normolipidemic rats. Treatment with 3-thiadicarboxylic acid markedly decreased plasma levels of free fatty acids, triglycerides, and cholesterol. This was accompanied by a corresponding reduction in very low density lipoprotein (VLDL)-triglyceride and low density lipoprotein (LDL)-cholesterol levels (by 46% and 42%, respectively), whereas the decrease in high density lipoprotein (HDL)-cholesterol levels was less pronounced (16%). However, the composition of the various plasma lipoprotein fractions was essentially unchanged. Fatty acid oxidation in both mitochondria and peroxisomes was stimulated in parallel; the activities of ATP:citrate lyase and fatty acid synthase, two key enzymes in fatty acid synthesis, were inhibited. Hepatic triglyceride biosynthesis was retarded, as indicated by a decrease in the liver triglyceride content along with a 30% reduction of hepatic VLDL-triglyceride secretion. This was accompanied by a 50% inhibition of phosphatidate phosphohydrolase. The activities of plasma lipoprotein lipase as well as hepatic lipase were somewhat higher (18%) in treated animals, suggesting a slight increase in the clearance potential of triglyceride-rich lipoproteins. The cholesterol-lowering effect was accompanied by a considerable reduction (75%) in HMG-CoA reductase activity and a less pronounced inhibition of cholesterol 7 alpha-hydroxylase (52%), and acyl-CoA:cholesterol acyltransferase (25%) activities. The present data suggest that the hypotriglyceridemic and hypocholesterolemic properties of sulfur-substituted fatty acid analogues are primarily due to effects on triglyceride and cholesterol synthesis.

The hypocholesterolemic effect of sulfur-substituted fatty acid analogues in rats fed a high carbohydrate diet.

Sulfur-substituted fatty acid analogues have been administered to rats fed a high carbohydrate diet, and the effect on plasma and hepatic lipid metabolism was investigated. Two of the analogues studied, 3-thiadicarboxylic acid and tetradecylthioacetic acid, reduced the plasma cholesterol level significantly, whereas the effect on plasma triacylglycerol level was only marginal. 3-Thiadicarboxylic acid was the most potent, decreasing the cholesterol level faster and at a lower dose than tetradecylthioacetic acid. The relative effects on plasma cholesterol and triacylglycerol levels were different from what have been observed in rats fed a conventional pellet diet. Tetradecylthiopropionic acid had no hypocholesterolemic effect. The activities of three lipogenic enzymes: ATP-citrate lyase, acetyl-CoA carboxylase and fatty acid synthase was measured. The two hypocholesterolemic analogues reduced the activities of these enzymes in a coordinated manner. The enzyme activities was found to correlate with the the plasma cholesterol level, indicating a coordinated regulation of these enzymes and cholesterol synthesis or secretion. The effect on two enzymes involved in cholesterol metabolism was also studied. The activity of acyl-CoA:cholesterol acyltransferase (ACAT) was reduced by the two hypocholesterolemic analogues, in contrast to the rate-limiting enzyme in cholesterol biosynthesis, HMG-CoA reductase, which tended to increase. The cholesterol lowering effect of 3-thiadicarboxylic acid and tetradecylthioacetic acid can probably be ascribed to diminished cholesterol synthesis due to a reduced availability of acetyl-CoA. A reduction in the esterification of hepatic cholesterol may be a contributing factor.

Early effects on mitochondrial and peroxisomal beta-oxidation by the hypolipidemic 3-thia-fatty acids in rat livers.

A single administration of 3-thiadicarboxylic and tetradecylthioacetic acids stimulates both mitochondrial and peroxisomal beta-oxidation and lowers plasma triacylglycerol levels. An increased rate of mitochondrial beta-oxidation and carnitine palmitoyl-transferase activity was established after 3 h and this was accompanied by a lowering of plasma triacylglycerol. Peroxisomal beta-oxidation, however, remained unchanged up to 8 h and was significantly increased after 12 h. These results suggest that after a single administration of 3-thia fatty acids mitochondrial beta-oxidation precedes peroxisomal beta-oxidation. Furthermore, they show that the observed tricylglycerol-lowering effect, which is established early (3-4 h) after the administration of 3-thia fatty acids, is initially due to an increased mitochondrial beta-oxidation.

Docosahexaenoic acid shows no triglyceride-lowering effects but increases the peroxisomal fatty acid oxidation in liver of rats.

The effect of docosahexaenoic acid (DHA) on mitochondrial and peroxisomal fatty acid oxidation and on key enzymes in triglyceride metabolism was investigated in the liver of rats fed a standard diet, a cholesterol diet, and a pelleted chow diet. Unexpectedly, in all three rat models repeated administration of highly purified DHA (92% pure) at different doses and times, at a dose of 1000 mg/day per kg body weight, resulted in no significant decrease of hepatic and plasma concentration of triglycerides. The serum concentrations of cholesterol and phospholipids showed an increase in a time-dependent manner in rats fed the pelleted chow diet. The hepatic concentration of cholesterol was increased in rats fed the cholesterol diet and pelleted chow diet after administration of DHA compared to palmitic acid. In all rat models, treatment with DHA tended to increase the peroxisomal beta-oxidation. This was accompanied with a significant increase (1.5-fold) of fatty acyl-CoA oxidase activity. The mitochondrial fatty acid oxidation system and carnitine palmitoyl-transferase activity, however, were almost unchanged. Moreover, palmitoyl-CoA synthetase activity was increased, whereas the palmitoyl-CoA hydrolase activity was decreased. Neither microsomal phosphatidate phosphohydrolase activity nor cytosolic phosphatidate phosphohydrolase activity was affected by DHA feeding in the three rat models. Acyl-CoA:1,2-diacylglycerol acyltransferase activity was also unaffected. In contrast to docosahexanoic acid feeding, eicosapentaenoic acid (EPA) administration possessed a hypotriglyceridemic effect and resulted in an increase of mitochondrial and peroxisomal oxidation of fatty acids. Carnitine palmitoyltransferase activity was also stimulated. Phosphatidate phosphohydrolase activity was unaffected whereas diacylglycerol acyltransferase activity was increased by EPA treatment compared with palmitic acid feeding. The results indicate that docosahexaenoic acid, in contrast to eicosapentaenoic acid, does not inhibit the synthesis and secretion of triglycerides in the liver. In addition, the results emphasize the importance that stimulation of peroxisomal beta-oxidation by these n-3 fatty acids is not sufficient to decrease the serum levels of triglycerides. In addition, increased mitochondrial beta-oxidation of fatty acids and thereby decreased availability of nonesterified fatty acids may be a mechanism by which EPA inhibits triglyceride, and subsequently very low density lipoprotein-triglyceride, production. Whether DHA and EPA possess different metabolic properties should be considered.

Relationship between translocation of long-chain acyl-CoA hydrolase, phosphatidate phosphohydrolase and CTP:phosphocholine cytidylyltransferase and the synthesis of triglycerides and phosphatidylcholine in rat liver.

Translocation of long-chain acyl-coenzyme A hydrolase from the microsomal fraction to the cytosolic fraction was promoted in cell-free extracts of rat liver by palmitic acid, oleic acid, tetradecylthioacetic acid, and tetradecylthiopropionic acid, and by their CoA esters. The CoA esters were more effective than the non-esterified acids in the translocation of the enzyme. Treatment of normolipidemic rats with sulfur-substituted non-beta-oxidizable fatty acid analogues resulted in a transitory increase in hepatic concentration of long-chain acyl-CoA. Longer feeding times almost normalized the hepatic long-chain acyl-CoA content. Microsomal long-chain acyl-CoA hydrolase activity was inhibited, whereas the activity of the cytosolic form was stimulated. The rise in enzyme activity coincided with a reduction in liver content of triglyceride and an increase in hepatic phospholipid content. The results suggest that the activity of long-chain acyl-CoA hydrolase in the cytosol may control the amount of acyl-CoA thioesters in the liver. Esterified and non-esterified fatty acids caused in vitro translocation of phosphatidate phosphohydrolase and cytidine 5'-triphosphate (CTP):phosphocholine cytidylyltransferase from the cytosolic fraction to the microsomal fraction. However, the translocation of these two enzyme systems was not obtained in vivo. The activity of phosphatidate phosphohydrolase decreased in microsomal and cytosolic fractions while the activity of cytidylyltransferase in these fractions increased. The activities of soluble phosphatidate phosphohydrolase and long-chain acyl-CoA hydrolase appeared to be inversely correlated.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative effects of oxygen and sulfur-substituted fatty acids on serum lipids and mitochondrial and peroxisomal fatty acid oxidation in rat.

Feeding tetradecyloxyacetic acid (a 3-oxa fatty acid) to rats led to decreased serum cholesterol and decreased serum triacylglycerol, resembling the effects of the corresponding 3-thia fatty acid (tetradecylthioacetic acid). The 3-oxa fatty acid inhibited strongly the mitochondrial fatty acid oxidation and led to the development of fatty liver, while the 3-thia fatty acid stimulated the mitochondrial fatty acid oxidation. Feeding tetradecyloxypropionic acid (a 4-oxa fatty acid) had less effect on the serum lipids. It stimulated fatty acid oxidation in the mitochondria and lowered the hepatic level of triacylglycerol. The corresponding 4-thia fatty acid (tetradecylthiopropionic acid) inhibited mitochondrial fatty acid oxidation and induced development of fatty liver. All these compounds, both the oxa and the thia fatty acids, induced some increase in the activity of the peroxisomal acyl-CoA oxidase. Repeated administration of 3-oxadicarboxylic acid to rats resulted in no lipid lowering effects, and marginal changes of fatty acyl-CoA oxidase activity. Oxidation of the S-atom of the 3-thia fatty acid to the corresponding sulfoxide or sulfone eliminated the metabolic effects of the thia fatty acid. The study has shown that the effects of 3- and 4-oxa fatty acids are in some ways opposite to those of the 3- and 4-thia fatty acids. The possibility that the lipophilicity of the fatty acid analogues may be an important factor behind the differences observed are discussed. It is suggested that these oxa- and thia-analogues of fatty acids may be useful in studies on the regulation of fatty acid metabolism.

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.

Regulation of fatty acid oxidation and triglyceride and phospholipid metabolism by hypolipidemic sulfur-substituted fatty acid analogues.

The mechanisms behind the hypotriglyceridemic effect of 1,10-bis(carboxymethylthio)decane (3-thiadicarboxylic acid) and tetradecylthioacetic acid and the development of fatty liver caused by 3-tetradecylthiopropionic acid (Aarsland et al. 1989. J. Lipid Res. 30: 1711-1718.) were studied in the rat. Repeated administration of S-substituted non-beta-oxidizable fatty acid analogues to normolipidemic rats resulted in a time-dependent decrease in plasma triglycerides, phospholipids, and free fatty acids. This was accompanied by an acute reduction in the liver content of triglycerides and an increase in the hepatic concentration of phospholipids. Mitochondrial fatty acid oxidation was stimulated, whereas lipogenesis was inhibited. The activity of phosphatidate phosphohydrolase decreased while the activity of CTP:phosphocholine cytidylyltransferase increased. These results suggest that the observed triglyceride-lowering effect was due to increased mitochondrial fatty acid oxidation accompanied by a reduction in the availability of the substrate i.e., free fatty acid, along with an enzymatic inhibition (phosphatidate phosphohydrolase). Administration of 3-tetradecylthiopropionic acid led to a drastic increase in the hepatic triglyceride content. Levels of plasma triglyceride phospholipid and free fatty acid also increased. Phosphatidate phosphohydrolase activity was stimulated whereas CTP:phosphocholine cytidylyltransferase was inhibited. Mitochondrial fatty acid oxidation was decreased. These data indicate that the development of fatty liver as an effect of 3-tetradecylpropionic acid is probably due to accelerated triglyceride biosynthesis, which is mediated by an increase in the availability of fatty acid along with stimulation of phosphatidate phosphohydrolase. The results of the present study speak strongly in favor of the hypothesis that phosphatidate phosphohydrolase is a major rate-limiting enzyme in triglyceride biosynthesis. Furthermore, they point out that the biosynthesis of triglycerides and phospholipids might be coordinately regulated. Such regulation is possibly mediated via phosphatidate phosphohydrolase and CTP:phosphocholine cytidylyltransferase. Whether the increase in hepatic phospholipids via increased CDP-pathway accounts for an increase of lipid components for proliferation of peroxisomes (3-thiadicarboxylic acid and tetradecylacetic acid) should be considered.