Steinar Bergseth

The effects of alkylthioacetic acids (3-thia fatty acids) on fatty acid metabolism in isolated hepatocytes.

Long-chain alkylthioacetic acids (3-thia fatty acids) inhibit fatty acid synthesis from [1-14C]acetate in isolated hepatocytes, while fatty acid oxidation is nearly unaffected or even stimulated. Desaturation of [1-14C]stearate (delta 9-desaturase) is also unaffected. [1-14C]Dodecylthioacetic acid (a 3-thia fatty acid) is incorporated in triacylglycerol and in phospholipids more efficiently than [1-14C]palmitate in isolated hepatocytes. The metabolism of [1-14C]dodecylthioacetic acid to acid-soluble products (by omega-oxidation) is slow compared to the oxidation of [1-14C]palmitate. In hepatocytes from adapted rats (rats fed tetradecylthioacetic acid for 4 days) the rate of [1-14C]palmitate oxidation is increased and its rate of esterification is decreased. Stearate desaturation is also decreased. The rate of cyanide-insensitive peroxisomal fatty acid beta-oxidation is several-fold increased. The metabolic effects of long-chain 3-thia fatty acids are discussed and it is concluded that they behave essentially like normal fatty acids except for their slow breakdown due to the sulfur atom in the 3 position, which blocks normal beta-oxidation.

The effect of feeding fish oils, vegetable oils and clofibrate on the ketogenesis from long chain fatty acids in hepatocytes

Groups of rats were fed diets containing 25% fish oil (FO), 25% soybean oil, 25% partially hydrogenated fish oil (PHFO), 25% partially hydrogenated soybean oil (PHSO), 25% partially hydrogenated coconut oil or 0.3% clofibrate for 3 wk. After the animals were fasted for 24 hr, hepatocytes were isolated and ketogenesis from added palmitate, linoleatecis andtrans, arachidonate and docosahexaenoate was measured. Ketogenesis after oil feeding was significantly stimulated (two-to threefold) only in cells from the PHFO-and PHSO-fed rats. The stimulation was most apparent with the long chain unsaturated fatty acids as substrates. These fatty acids were relatively poor ketone body precursors in control hepatocytes. Essential fatty acid deficiency did not seem to be the reason for this stimulation. Clofibrate also stimulated ketogenesis significantly (1.5- to 3-fold). The degree of stimulation increased with chain length and degree of unsaturation of the substrate. The activity of the enzyme 2,4-dienoyl-CoA reductase was also studied in the same groups. Its activity was stimulated about fourfold in the clofibrate-treated rats and to a lesser extent by the PHFO, PHSO and FO diets. The activity showed no correlation with the content of unsaturated fatty acids in the diet or their oxidation in isolated hepatocytes. The 2,4-dienoyl-CoA reductase, therefore, does not seem to be a regulatory enzyme in the metabolism of dietary polyunsaturated fatty acids. It is concluded that an induction of the peroxisomal β-oxidation system most likely is involved in the reported increases in ketogenesis from very long chain polyunsaturated fatty acids.

Microsomal oxidation of dodecylthioacetic acid (a 3-thia fatty acid) in rat liver

[1-14C]Dodecylthioacetic acid (DTA), a 3-thia fatty acid, is omega (omega-1)-hydroxylated and sulfur oxygenated at about equal rates in rat liver microsomes. In prolonged incubations DTA is converted to omega-hydroxydodecylsulfoxyacetic acid. omega-Hydroxylation of DTA is catalysed by cytochrome P450IVA1 (or a very closely related isoenzyme in the same gene family), the fatty acid omega-hydroxylating enzyme. It is absolutely dependent on NADPH and inhibited by CO, and lauric acid is a competing substrate. omega-Hydroxylation of DTA is increased by feeding tetradecylthioacetic acid (TTA), a 3-thia fatty acid, for 4 days to rats. omega-Hydroxylation of [1-14C]lauric acid is also induced by TTA and other 3-thia carboxylic acids. A close relationship was observed between induction of microsomal omega-hydroxylation of fatty acid and palmitoyl-CoA hydrolase activity. DTA is omega-hydroxylated at about the same rate as the physiological substrate lauric acid. The sulfur oxygenation of DTA is catalysed by liver microsomal flavin-containing monooxygenase (FMO) (EC 1.14.13.8). It is dependent on either NADH or NADPH. The Km value for NADH was approx. five times larger than the Km value for NADPH. It is inhibited by methimazole and not affected by CO. It is not induced by TTA.

Metabolism of dicarboxylic acids in vivo and in the perfused kidney of the rat

After intraperitoneal injection of (1-14C)-labelled suberic or dodecanedioic acid, the acids themselves and their metabolites were excreted in urine and as 14CO2. There was a striking difference in the capacity to oxidize the two dicarboxylic acids. Most of the suberic acid was excreted unchanged in the urine, and less was recovered as 14CO2. A trace was excreted as adipic acid. Dodecanedioic acid was more efficiently oxidized; 2-3-times more was expired as 14CO2, and the urine contained only a trace of the unchanged acid. Adipic acid was the main metabolite. Kidney perfusion experiments confirmed these results by showing that unmetabolized suberic acid was actively excreted by the kidneys. Dodecanedioic acid was oxidized and shorter dicarboxylic acids were excreted. The perfused hindquarter did not metabolize the dicarboxylic acids. Our results show that dodecanedioic acid can be completely oxidized both in the whole animal and in the kidneys. Dicarboxylic acids in the urine may to a significant extent be formed in the kidneys themselves.

Alkylthioacetic acids (3-thia fatty acids) are metabolized and excreted as shortened dicarboxylic acids in vivo.

The metabolism of 1-14C-labeled long-chain alkylthioacetic acids (3-thia fatty acids) which are blocked for normal beta-oxidation by a sulfur atom in the beta-position has been investigated in vivo. Most of the injected radioactivity (greater than 50%) was excreted in the urine within the first 48 h. The recovered and identified metabolites were all short sulfoxydicarboxylic acids. The main metabolite from dodecylthioacetic acid was carboxypropylsulfoxy acetic acid. Some bis(carboxymethyl)sulfoxide (dithioglycolic acid sulfoxide) was also found. The main metabolite from nonylthioacetic acid was carboxyethylsulfoxyacetic acid. No sulfones were found. Less than 1% of the 1-14C from the dodecylthioacetic acid was recovered in respiratory CO2 and about 3% of the 1-14C from nonylthioacetic acid. [1-14C]Dodecyl-sulfonylacetic acid was recovered almost quantitatively as carboxypropylsulfonylacetic acid in the urine after 3 h. A significant fraction (10% of the dodecylthioacetic acid was recovered in the phospholipids and triacylglycerols from liver and epidymal fat pad 4 h after injection. These experiments show that the alkylthioacetic acids undergo an initial omega-oxidation followed by beta-oxidation to short dicarboxylic acids.

Metabolism of dicarboxylic acids in rat hepatocytes.

[carboxyl-14C]Dodecanedioic acid (DC12) is metabolized in hepatocytes at a rate about two thirds that of [1-14C]palmitate. Shorter dicarboxylates (sebacic (DC10), suberic (DC8), and adipic (DC6) acid) are formed, mainly DC6, less DC8 and only a little DC10. In hepatocytes from clofibrate-treated rats, more polar products account for most of the breakdown products, presumably because the beta-oxidation proceeds all the way to succinate and acetyl-CoA. [carboxyl-14C]Suberic acid (DC8) is oxidized at a rate only one fifth that of dodecanedioic acid. (+)-Decanoylcarnitine inhibits palmitate oxidation but not the oxidation of dodecanedioic acid. At low concentrations of [carboxyl-14C]dodecanedioic acid or of [1-14C]palmitate, acetylsulfanilamide is more efficiently labeled by the former. High concentrations of dodecanedioic acid inhibit palmitate oxidation and the acetylation of sulfanilamide, presumably because their CoA-esters accumulate in the cytosol. These results indicate that medium-chain dicarboxylic acids are beta-oxidized mainly in the peroxisomes.

The effect of adaption on the metabolism of dodecylthioacetic acid (a 3-thia fatty acid) in rat tissues

Dodecylthioacetic acid (DTA) was both omega-hydroxylated and sulfur-oxygenated at about equal rates by the microsomal fraction from liver and kidney. Feeding tetradecylthioacetic acid (TTA) for 4 days increased omega-hydroxylation 4-fold only in the liver. The sulfur oxygenation rate was similar in liver, kidney and lung, barely detectable in heart and absent in intestinal mucosa. In isolated hepatocytes from normal rats the major metabolite from dodecylthioacetic acid was carboxypropylsulfoxyacetic acid. In hepatocytes from adapted rats, the main product was identified as bis(carboxymethyl)sulfide. In kidney perfusion experiments dodecylthioacetic acid was metabolized to carboxypropyl-sulfoxyacetic acid and preferentially excreted in the urine. In hindquarter perfusion experiments no oxidative metabolites were detected. These experiments show that only liver and kidney can metabolize dodecylthioacetic acid completely and that omega-hydroxylation in the liver is the only inducible activity, in addition to the beta-oxidation.

Regulatory Properties of Carnitine Palmitoyltransferase in the Mitochondrial Membrane of Liver

Fritz (1955) reported that carnitine stimulated the oxidation of fatty acids in liver homogenates. This phenomenon has subsequently been explained by a transport of activated fatty acids in the form of acylcarnitines through the inner mitochondrial membrane (Bremer 1962, Fritz and Yue 1963). This transport links the activation of fatty acids in the outer membrane of the mitochondria (and in the endoplasmatic reticulum) to the β-oxidation of activated fatty acids in the mitochondrial matrix (Norum et. al. 1966, Skrede and Bremer 1970, Yates and Garland 1970).