3- Thia fatty acids

EFFECTS OF 3-THIA FATTY ACIDS ON P-OXIDATION AND CARNITINE PALMITOYLATRANSFERASE I ACTIVITY IN CULTURED RAT HEPATOCYTES... 

Thia fatty acids represent non-p-oxidizable fatty acid analogues in which a S atom substitutes for the P-methylene group in the chain. They are known to reduce serum tri-acylglycerol and cholesterol. The hypolipaemia is detectable within 3 hours of administration and is explained initially by an increase in mitochondrial P-oxidation and a decrease in triacylglycerol secretion, followed by an increase in activity of peroxisomal P-oxidation enzymes after 12 hours. ... 

Addition of tetradecylthioacetic acid (TTA) to isolated rat hepatocytes leads to an increase in oxidation of palmitate and a strong decrease in de novo synthesis of fatty acids and cholesterol (Risan, K.A. Spydevold, 0., impublished results). Hepatocytes isolated from rats 24 hours after administration of TTA show also an elevated activity of CPT I. However, little is known about the acute effects of 3-thia fatty acids. 

In the present work we have studied effects of 3-thia fatty acids on P-oxidation and CPT I activity in freshly cultured (4-6 hours) rat hepatocytes. By using digitonin-permeabilized cells we were able to measure CPT I activity directly in hepatocytes which are possibly a more physiologically relevant system than isolated mitochondria. 

When 3-thia fatty acids are added to isolated hepatocytes they stimulate fatty acid oxidation (Fig. 1). The strength of this effect depends on the chain length of 3-thia fatty acid used. Dodecylthioacetic acid (DTA) and tetradecylthioacetic acid (TTA) are most potent, shorter and longer 3-thia fatty acids have a reduced effect. 

When palmitate is used as substrate the addition of equimolar amounts of DTA stimulates the oxidation 3 fold (Fig. 2A). With the shorter laurate as substrate (Fig. 2B) the effect decreases and is no longer detectable with octanoate (Fig. 2C). This pattern of responsiveness suggests that 3-thia fatty acids affect the mitochondrial CPT system, since oxidation of long fatty acids, in contrast to short ones, is dependent on the transport of acylcarnitines across the mitochondrial inner membrane. 

Effects of 3-Thia Fatty Acids on -Oxidation and Carnitine Paimitoyitransferase I Activity... 

We now have shown that the main target for the short-term effect of 3-thia fatty acids is a reversible activation of CPT I which is stimulated 3-4 fold in freshly activated hepatocytes. This stimulation is a very fast process, which cannot be explained by gene transcription. Maximal activity is reached after only lOmin, a time too short for new protein synthesis to take place. Consequently 3-thia fatty acids have to affect the already... 

Asiedu, D.K., Skorve, J., Willumsen, N., Demoz, A. Berge, R.K. (19%) Biochim. Biophys. Acta, 1166, 73-76. Early effects on mitochondrial and peroxisomal heta-oxidation by the hypolipidemic 3-thia-fatty acids in rat livers. 

Skrede, S., Narce, M., Bergseth, S. Bremer, J. (1989)B/oc)h> . Biophys. Acta, WI5,2% 302. The effects of alkylthioacetic acids (3-thia fatty acids) on fatty acid metabolism in isolated hepatocytes. 

Skrede, S. Bremer, J. (1993) Biochim. Biophys. Acta, 1167, 189 196. Acylcarnitine formation and fatty acid oxidation in hepatocytes from rats treated with tetradecyithioacetie add (a 3-thia fatty acid). Guzman, M. (jeelen, M.J. (1988) Biochem. Biophys. Res. Commun., 151, 781-787, Short-term regulation of carnitine palmitoyltransferase activity in isolated rat hepatocytes. 

Sulfur-substituted fatty acids, especially the 3-thia fatty acids (TTA, CH3-(CH2>i3-S-CH2-COOH), that profoundly affect P-oxidation, have facilitated studies on the concerted regulation of fatty acid oxidation and TG-biosynthesis. We have demonstrated that stimulation of p-oxidation (Table 2) may affect both TG-formation (Table 1) and plasma lipoprotein homeostasis (Table 1) under both normolipidemic and hyperlipidemic conditions. 

TTA is a fatty acid analogue in which a sulfur atom replaces the P-mehylene groups in the alkyl-chain (a 3-thia fatty acid). TTA, therefore, cannot be P-oxidized. Paradoxically, TTA is both mitochondrial and peroxisomal proliferator and the hepatic mitochondrial and peroxisomal fatty acid oxidation capacities are increased (Table 2). In addition to its biochemical and morphological effects, TTA decrease serum TG (Table 1) very low density lipoprotein (VLDL)-TG, cholesterol and free fatty acid (NEFA) levels in rats. Thus, the observed reduction in plasma TG levels during TTA administration could be accomplished by retarded synthesis, reduced hepatic output, enhanced clearance or a combination of these factors. 3-Thia fatty acid resulted in a slight inhibition in the activities of ATP-citrate lyase and fatty acid synthase. However, the impact of... 

Figure 1. EfTect of 3-thia fatty acid on plasma triglyceride entry rate after a single intravenous injection of Triton WR 1339. The values are expressed as means SD for 6 rats in each experimental group. To convert mmol/L to mg/dL, multiply by 88.5. Data from. ...

In hepatocytes cultured in the presence of oleic acid, incorporation of pH]-water into secreted lipids and TG was lower than incorporation into synthesized lipids with the 3-thia fatty acids. ° A similar phenomenon was observed with pH]-glycerol as the radioactive precursor. This suggests that some of the hypotriglyceridemic effects of 3-thia fatty acids may arise from a reduction in biosynthesis and/or secretion of TG. 

The degree of inhibition of [ H]-water incorporation into TG and phospholipids (PL) was at approximately the same level, indicating that the 3-thia fatty acids may affect a common step in the biosynthesis of these lipids. 

The incorporation of [ H]-water into synthesized diacylglycerols was reduced even more than the incorporation into the other two lipid classes. As the sulfur-substituted fatty acids had only small effects on incorporation of pH]-water into cell monoacylgjyc-erols (data not shown), these data suggest that 3-thia fatty acids decreased TG-synfliesis by affecting the step before formation of diacylglycerols. The reduced incorporation into diacylglycerols, thus, may indicate that activity of the enzyme phosphatidate phospho-hydrolase could be affected. 

When the synthesis of lipids is reduced due to the presence of fatty acid analogs, the NEFAs will be diverted from the esterification pathway. The level of NEFAs in the hepatocytes treated with 3-thia fatty acids tended to decrease. This indicates that the mitochondrial P-oxidation was increased, as the peroxisomal P-oxidation was unchanged in these hepatocytes. Thus, it is likely that the non-P-oxidizable fatty acid analogs reduced the availability of fatty acids for TG-synthesis due to increased mitochondrial fatty oxidation. The lack of effect on the peroxisomal P-oxidation confirms the in vivo data that the hypotriglyceridemic effect of the analogs can be dissociated from the proliferation of peroxisomes." ... 

Thus, it is likely that rate of TG-synthesis is controlled by coordinate regulation of the activities of mitochondrial P-oxidation and phosphatidate phosphohydrolase. The instant hypotriglyceridemic effect observed in rats given 3-thia fatty acids can be explained by a sudden increase in mitochondrial fatty acid oxidation and a decrease in phosphatidate phosphohydrolase. This alteration is accompanied by a reduction in the... 

Skrede, S. Bremer, J. 1993. Biochim. Biophys. Acta 1167 189-196. Acylcarnitine formation and fatty acid oxidation in hepatocytes from rats treated with tetradecylthioacetic acid (a 3-thia fatty acid). 

METABOLIC EFFECTS OF 3-THIA FATTY ACID IN CANCER CELLS... 

Thia fatty acids have been used as tools in experimental models to further penetrate the mechanisms of the lipid metabolism. These fatty acids are activated to their respective CoA-esters, but, they cannot be degraded by 3-oxidation because of the sulfur substitution in 3-position (reviewed ). Tetradecylthioacetic acid (TTA), a representative member of the 3-thia family, has the chemical structure of palmitic acid (C16) in which a sulfur atom is located between the 2 and 3-carbon atoms (COOH-CH2-S-(CH2)i3-CH3). TTA is a novel hypolipidemic drug which has been shown to combine several effects of CO-3 PUFAs, such as EPA, and structurally unrelated peroxisome proliferators, such as phenylacetate and fibrates. From this perspective we wanted to investigate if TTA shares the antitumor activity found for these functionally related compounds, and furthermore to study the metabolic effects of TTA in cancer cells in relation to the effects found in hepatocytes. 

Metabolic Effects of 3-Thia Fatty Acid in Omcer Cells... 

Berge, R.K. Hvattum, E. (1994). Pharmac. Ther. 61(3) 345-383. Impact of cytochrome P450 system on lipoprotein metabolism. Effect of abnormal fatty acids (3-Thia fatty acids). 

Demoz, A., Asiedu, O.K., Lie, 0. Berge, R.K. (1994). Biochim. Biophys. Acta. 1199 238-244. Modulation of plasma and hepatic oxidative status and changes in plasma lipid profile by n-3 (EPA and DHA), n-6 (com oil) and a 3-thia fatty acid in rats. 

Abdi-Dezfuli, F., Berge, R.K., Rasmussen, M., Thorsen, T. Aakvag, A. (1994). Ann. N. Y. Acad. Sci. 744 306-309. Effects of saturated and polyunsaturated fatty acids and their 3-thia fatty acid analogues on MCF-7 breast cancer cell growth. 

Details on enzyme distribution of CPT II and fatty acyl-CoA oxidase in both rat and human glioma cells are presented in Metabolic effects of 3-thia fatty acids in cancer cells by Tronstad KJ,. et al 1998 (this issue). 

It has been shown previously that chain-shortened 3-thia fatty acids have less hypohpidemic effect than TTA (Table 2). Thus the less biologieal poteney by the P-oxidable fatty acids compared to TTA could be due to chain shortening of the administered thia fatty acids by mitochondrial P-oxidation. Another possibihty is the formation of an inhibitory metabolite during P-oxidation of these p-oxidable thia fatty acids as is the case for 4-thia fatty acids. 

In order to elucidate that, we compared the effect of treating rats with the p-oxidable thia fatty acid 5-S and the non-P-oxidable 3-thia fatty acid DTA (dodecanylthio acetic acid, HOOC-CH2-S-CH2(ll)-CH3). Theoretically DTA is formed when 5-S has undergone 1 cycle of P-oxidation. In agreement with earlier findings (Table 2),... 

Table 2. Effects of 3-thia fatty acids with different chain-length and palmitic acid (control) on plasma triacylglycerol and phospholipid levels after 7 days of treatment. Results are expressed as means SD of five different animals in each group. Significantly different from control P < 0.05. From. ...

Aarsland, A., Aarsaether, N, Bremer, J, Bage, R.K. 1989. J. Lipid Res. 30 1711-1718. Alkylthioacetic acids (3-thia fatty acids) as non-fi-oxidizable fatty acid analogues a new group of hypolipidemic drugs.III. Dissociation of cholesterol- and triglyceride-lowering effects and the induction of peroxisomal fi-oxidation. 

Beige, R.K., Aarsland, A., Kryvi, H., Bremer, A. Aarsaether, N. 1989 Biochim. Biophys. Acta 1004 345-356. Alkylthioacetic acids (3-thia fatty acids)-A new group of non-ph-oxidizable peroxisome-inducing fatty acid analogues. . A study on the structural requirements for proliferation of peroxisome s and mitochondria in rat lliver. 

The availability of TG is a major driving force in the secretion of VLDL by the hver. Evidently, factors influencing the balance between TG biosynthesis and/ or fatty acid oxidation may ultimately influence plasma hpoprotein levels and metabolism. We have demonstrated this mechanism of action with sulfur-substituted fatty acids (3-thia fatty acids). In rats, the TG-lowering effect of the 3-thia fatty acid tetradecylth-ioacetic acid (C14-S-acetic acid), was estabhshed within hours of feeding and this was mainly due to stimulated mitochondrial fatty acid oxidation, thereby reducing... 

Activation of the fatty acids to their respective CoA esters, is a necessary step prior to oxidation. DHA was converted to its CoA ester in all subcellular fractions, but was a poor substrate for oxidation in both the mitochondrial- and the peroxisomal fractions. Compared to EPA-CoA, DHA-CoA was more effectively synfliesized in the peroxisomal than the mitochondrial fraction, espedally in animals treated with 3-thia fatty acids. It has been reported that very long chain fatty acyl-CoA synthases as lignoceryl-CoA are absent in mitochondria and DHA-CoA syndiase activity measured in the mitochondrial fraction, might also be due to contamination of peroxisomes. ... 

DHA-CoA was a substrate for peroxisomal fatty acyl-CoA oxidase and the activity of DHA-CoA oxidase increased several folds after treatment with 3-thia fatty acids (Table 1). Noteworthy, in contrast to EPA-CoA, DHA-CoA was a very poor substrate, if at all, for mitochondrial carnitine acyltransferase (CAT) -1 and CAT-11 (Table 1). In addition, neither mitochondrial CAT-I nor CAT-II activity increased after 3-thia fatty acid treatment (Table 1) when DHA-CoA was used as a substrate. It might, therefore, be considered that DHA can not be oxidized by the mitochondria... 

Thia fatty acids increased oxidation of the different fatty acids, but the oxidation of palmitic-, oleic acid and EPA in the peroxisomal fraction was only 5-20% of the mitochondrial fatty acid oxidation. Thus, the capacity of the mitochondria to oxidize long-chain fatty acids is of quantitative major importance. Seen as a whole, our results suggest that EPA is preferentially oxidized by mitochondria, while DHA is preferentially oxidized by peroxisomes. This might e q)lain why the EPA level was decreased 40-80% after 3-thia fatty acid treatment whereas the DHA level was decreased only 17-24%. ... 

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