Fatty Acid Chain Elongation System Research Articles

Methylene-interrupted double bond in polyunsaturated fatty acid is an essential structure for metabolism by the fatty acid chain elongation system of rat liver

Some plant oils contain non-methylene-interrupted polyunsaturated fatty acids (NMIFAs). Pinolenic acid (all cis Δ-5,9,12/18:3) and columbinic acid ( trans,cis,cis Δ-5,9,12/18:3) are NMIFAs that exist in pine seed oil and columbine seed oil, respectively. We investigated the double bond position of fatty acid recognized by the fatty acid chain elongation system (FACES) of rat liver using NMIFAs as experimental tools. In the total elongation assay, amounts of C2 unit chain-elongated metabolites of pinolenic acid and columbinic acid were 32% and 11%, respectively, compared to that of γ-linolenic (all cis Δ-6,9,12/18:3) as the substrate. In the condensation reaction assay, the rate limiting step of FACES, the conversion rates of pinolenic acid and columbinic acid to the corresponding C20 β-keto fatty acids were 19% and 9% of that of γ-linolenic acid, respectively. The formation of elongated metabolite of podocarpic acid (all cis Δ-5,11,14/20:3) was only 7% of that of arachidonic acid (all cis Δ-5,8,11,14/20:4). From these results it was concluded that the condensing enzyme of FACES could recognize the methylene-interrupted cis double bond structure vicinal to the carboxyl group in the fatty acid molecule.

Fatty acid elongation in the biosynthesis of (Z)-10-heptadecen-2-one and 2-tridecanone in ejaculatory bulb microsomes of Drosophila buzzatii

A fatty acid chain elongation process is involved in incorporation of saturated and unsaturated fatty acyl-CoA esters into 2-tridecanone and (Z)-10-heptadecen-2-one by Drosophila buzzatii. The microsomal fraction from mature male ejaculatory bulbs is chain-length specific and requires malonyl-CoA (or acetyl-CoA, if acetyl-CoA carboxylase were present) for the chain elongation step to 2-ketones. Decarboxylation of the proposed intermediate β-ketoacid results in 2-ketone biosynthesis. Incubation of the microsomes with the acetyl-CoA carboxylase inhibitor avidin indicated that acetyl-CoA carboxylase was present in the microsomal preparations; however, washing of the microsomal preparation removed the acetyl-CoA carboxylase activity. Fatty acyl-CoA esters were also chain elongated to produce fatty acids two and four carbons longer, suggesting that the enzymes for normal fatty acid chain elongation are also present in the microsomal fraction from ejaculatory bulbs. How much, if any, of this fatty acid chain elongation system is used for 2-ketone biosynthesis is yet to be determined.

Evidence that beta-hydroxyacyl-CoA dehydrase purified from rat liver microsomes is of peroxisomal origin.

The present study provides strong evidence that the previously isolated hepatic microsomal beta-hydroxyacyl-CoA dehydrase (EC 4.2.1.17), believed to be a component of the fatty acid chain-elongation system, is derived, not from the endoplasmic reticulum, but rather from the peroxisomes. The isolated dehydrase was purified over 3000-fold and showed optimal enzymic activity toward beta-hydroxyacyl-CoAs or trans-2-enoyl-CoAs with carbon chain lengths of 8-10. The purified preparation (VDH) displayed a pH optimum at 7.5 with beta-hydroxydecanoyl-CoA, and at 6.0 with beta-hydroxystearoyl-CoA. Competitive-inhibition studies suggested that VDH contained dehydrase isoforms, and SDS/PAGE showed three major bands at 47, 71 and 78 kDa, all of which reacted to antibody raised to the purified preparation. Immunocytochemical studies with anti-rabbit IgG to VDH unequivocally demonstrated gold particles randomly distributed throughout the peroxisomal matrix of liver sections from both untreated and di-(2-ethylhexyl) phthalate-treated rats. No labelling was associated with endoplasmic reticulum or with the microsomal fraction. Substrate-specificity studies and the use of antibodies to VDH and to the peroxisomal trifunctional protein indicated that VDH and the latter are separate enzymes. On the other hand, the VDH possesses biochemical characteristics similar to those of the D-beta-hydroxyacyl-CoA dehydrase recently isolated from rat liver peroxisomes [Li, Smeland & Schulz (1990) J. Biol. Chem. 265, 13629-13634; Hiltunen, Palosaari & Kunau (1989) J. Biol. Chem. 264, 13536-13540]. Neither enzyme utilizes crotonoyl-CoA or cis-2-enoyl-CoA as substrates, but both enzymes convert trans-2-enoyl substrates into the D-isomer only. In addition, the VDH also contained beta-oxoacyl-CoA reductase (beta-hydroxyacyl-CoA dehydrogenase) activity, which co-purified with the dehydrase.

Inhibition of rat liver microsomal fatty acid chain elongation by gemfibrozil in vitro

Gemfibrozil, a hypolipidemic drug mainly used in the treatment of hypertriglyceridemic states, strongly inhibits the rat hepatic microsomal fatty acid chain elongation system in vitro. The inhibition is independent on the reducing cofactor used in the assay. Furthermore, gemfibrozil seems to act by inhibiting the rate-limiting step of the elongation process, the condensing reaction, without discriminating among the proposed three different condensing enzymes, devoted to condensation of saturated, mono-unsaturated and polyunsaturated acyl-CoA substrates.

Characterization of fatty acid elongation system in porcine neutrophil microsomes

Microsomes purified from porcine neutrophils containing the fatty acid chain-elongation system for long- and very-long-chain fatty acyl-CoAs, and several enzymatic characters for the elongation of palmitoyl-CoA (16:0-CoA) and arachidoyl-CoA (20:0-CoA) were examined. The heat-inactivation profile for the elongation of 16:0-CoA was different from that of 20:0-CoA, suggesting the presence of different enzyme systems for palmitoyl-CoA and arachidoyl-CoA. Contrary to the elongation system of brain microsomes, the successive synthesis of lignoceric acid (24:0) from 20:0-CoA at 60 μM was not prominent under normal conditions in the neutrophil microsomes. The synthesis of behenic acid (22:0) was slightly inhibited by 0.5 mM N-ethylmaleimide (NEM) present in the assay mixture, whereas the pre-treatment of microsomes with 0.5 mM NEM largely inhibited the synthesis of 22:0 from 20:0-CoA. The synthesis of 24:0, however, was enhanced by 0.5 mM NEM in the elongation of 20:0-CoA and the rate of 24:0 synthesis became dominant over the synthesis of 22:0. These results suggested that the elongation enzyme for very-long-chain fatty acyl-CoA, especially for 20:0-CoA elongation to 22:0 in the neutrophil microsomes contained NEM-sensitive sulfhydryl groups in the active center and the mechanism for the synthesis of 24:0 through successive elongation from 20:0-CoA was different from that of 22:0, as the former was enhanced by NEM whereas the latter was strongly inhibited.

Evidence for two separate β-ketoacyl CoA reductase components of the hepatic microsomal fatty acid chain elongation system in the rat

The hepatic microsomal fatty acid chain elongation system can utilize either NADPH or NADH. Elongation activity, measured as the rate of malonyl CoA incorporation into palmitoyl CoA, was enhanced by a fat-free diet and by bovine serum albumin (BSA) when either cofactor was employed. When the intermediate products were determined, it was observed that in the presence of BSA and NADPH, the predominant product was the saturated elongated fatty acid, whereas in the presence of BSA and NADH, the major intermediate was the β-ketoacyl derivative. Employing β-ketostearoyl CoA as substrate, BSA markedly inhibited NADH-supported β-ketoacyl CoA reductase activity and stimulated NADPH-supported activity. Furthermore, the sum of the NADH-dependent and NADPH-dependent β-ketoreductase activities approximated the activity obtained when both cofactors were present in the incubation medium, suggesting the existence of two β-ketoacyl CoA reductases, one using NADH and the other NADPH.

Evidence for two separate β-ketoacyl CoA reductase components of the hepatic microsomal fatty acid chain elongation system in the rat

The hepatic microsomal fatty acid chain elongation system can utilize either NADPH or NADH. Elongation activity, measured as the rate of malonyl CoA incorporation into palmitoyl CoA, was enhanced by a fat-free diet and by bovine serum albumin (BSA) when either cofactor was employed. When the intermediate products were determined, it was observed that in the presence of BSA and NADPH, the predominant product was the saturated elongated fatty acid, whereas in the presence of BSA and NADH, the major intermediate was the β-ketoacyl derivative. Employing β-ketostearoyl CoA as substrate, BSA markedly inhibited NADH-supported β-ketoacyl CoA reductase activity and stimulated NADPH-supported activity. Furthermore, the sum of the NADH-dependent and NADPH-dependent β-ketoreductase activities approximated the activity obtained when both cofactors were present in the incubation medium, suggesting the existence of two β-ketoacyl CoA reductases, one using NADH and the other, NADPH.

Spectrophotometric assay for the condensing enzyme activity of the microsomal fatty acid chain elongation system

A rapid and simple spectrophotometric method was developed to measure the activity of the condensing enzyme component of the microsomal fatty acid chain elongation system. The intermediate product of the condensation reaction is the β-ketoacyl CoA which exists in two tautomeric forms, i.e., keto and enol. The addition of bovine serum albumin (BSA) to a cuvette cell containing a β-ketoacyl CoA derivative resulted in the formation of a 303-nm absorbance peak, characteristic of enolate formation. The β-ketoacyl CoAs with carbon chain length of 6 to 18 interacted with BSA to produce the 303-nm peak; acetoacetyl CoA was the only β-keto compound tested which did not interact with BSA to produce the peak. Other compounds which were unaffected by BSA included CoA, free β-keto acid, β-hydroxyacyl CoA, acyl CoA, trans-2-enoyl CoA, and malonyl CoA. BSA could not be replaced by ovalbumin; furthermore, denatured (boiling) BSA could not induce the 303-nm peak. The specific activity of the condensing enzyme measured by the spectrophotometric method compares favorably with the activity obtained by the radioactive method. The apparent extinction coefficient (ϵ) for the absorbance peak generated by the β-keto thioester varied from 5 to 30 m m −1 cm −1 depending on the β-keto derivative. The spectrophotometric procedure can be used in the determination of the condensing enzyme activity in not only hepatic microsomes but also in kidney and brain microsomes both of which have significantly lower activity. The advantages of the novel method over the radioactive method are that (i) it does not involve the use of radioactive compounds, (ii) it is much less cumbersome and significantly less costly, and (iii) it is rapid and easy to perform.

Topography of Rat Hepatic Microsomal Enzymatic Components of the Fatty Acid Chain Elongation System

The orientation of the condensing enzyme, the beta-hydroxyacyl-CoA dehydrase, and the trans-2-enoyl CoA reductase within the rat liver microsomal membrane was investigated by the use of impermeant inhibitors of enzyme activity: trypsin, chymotrypsin, subtilisin, mercury-dextran, and anti-beta-hydroxyacyl-CoA dehydrase IgG. The activity of the condensing enzyme was inhibited more than 70% by various proteases and was completely inhibited by 80 microM mercury-dextran. Similar results were obtained for the trans-2-enoyl-CoA reductase activity. On the other hand, in the absence of detergent, proteases inhibited beta-hydroxyacyl-CoA dehydrase activity by 25-40%, while in the presence of detergent the inhibition increased to 65-90%. Furthermore, anti-beta-hydroxyacyl-CoA dehydrase IgG, which in the absence of detergent produced no inhibition, in the presence of detergent inhibited beta-hydroxyacyl-CoA dehydrase activity by more than 80%; under identical conditions, preimmune IgG caused a 13% inhibition. Microsomes used throughout this study displayed greater than 90% latency with respect to mannose-6-phosphatase activity, indicating that the microsomes were intact. Latency was not affected by the proteases, by mercury-dextran, or by the presence of the enzyme assay components. These results suggest that both the condensing enzyme and the reductase are present on the cytoplasmic surface of the membrane, whereas the beta-hydroxyacyl-CoA dehydrase is embedded in the microsomal membrane.

Do rat hepatic microsomes contain multiple NADPH-supported fatty acid chain elongation pathways or a single pathway?

[2- 14C]- trans-2-hexadecenoyl CoA (16:1) and [2- 14C]- trans -2-cis-8,11,14-eicosatetraenoyl CoA (20:4) were chemically synthesized and employed as competitive substrates for the liver microsomal trans-2-enoyl CoA reductase component of the fatty acid chain elongation system. Both 7.5 μM and 15 μM 20:4 competitively inhibited the reduction of 16:1 CoA to palmitoyl CoA. In addition, the reduction of both substrates was identically inhibited to the same extent by the acetylenic derivative, dec-2-ynoyl CoA. Furthermore, trypsin, chymotrypsin and subtilisin inhibited trans-2-enoyl CoA reductase activity when three different substrates were employed—16:1, 20:4 and trans-2-cis-11-octadecadienoyl CoA (18:2). These results are consistent with the hypothesis of multiple condensing enzymes connected to a single elongation pathway.

Site of inhibition of rat liver microsomal fatty acid chain elongation system by dec-2-ynoyl coenzyme A. Possible mechanism of inhibition.

The present study examines the effect of the acetylenic thioester dec-2-ynoyl-CoA (delta 2 10 identical to 1-CoA) on the microsomal fatty acid chain elongation pathway in rat liver. When the individual reactions of the elongation system were measured in the presence of delta 2 10 identical to 1-CoA, the trans-2-enoyl-CoA reductase activity was markedly inhibited (Ki = 2.5 microM), whereas the activities of the condensing enzyme, the beta-ketoacyl-CoA reductase, and the beta-hydroxyacyl-CoA dehydrase were not affected. The absence of inhibition of total microsomal fatty acid elongation was attributed to the significant accumulation of the intermediates, beta-hydroxyacyl-CoA and trans-2-enoyl-CoA, without formation of the saturated elongated product, indicating that the trans-2-enoyl-CoA reductase-catalyzed reaction was the only site affected by the inhibitor. The nature of the inhibition was noncompetitive. In contrast to the delta 2 10 identical to 1-CoA, delta 3 10 identical to 1-CoA did not inhibit trans-2-enoyl-CoA reductase activity, suggesting that the mode of inhibition was not via formation of the 2,3-allene derivative. Based on the observation (a) that p-chloromercuribenzoate markedly inhibits reductase activity, (b) that dithiothreitol protects the enzyme against inactivation by delta 2 10 identical to 1-CoA, (c) of the spectral manifestation of the interaction between thiol reagents and delta 2 10 identical to 1-CoA depicting an absorbance peak similar to that of the beta-ketoacyl thioester-Mg2+ enolate complex, (d) of a similar absorbance spectrum formed by the interaction between delta 2 10 identical to 1-CoA and liver microsomes, and (e) of the absence of formation of a similar spectrum by delta 3 10 identical to 1-CoA, trans-2-10:1-CoA, or delta 2 10 identical to 1 free acid with liver microsomes, we propose that delta 2 10 identical to 1-CoA inactivates trans-2-enoyl-CoA reductase by covalently binding to a critical sulfhydryl group at or in close proximity to the active site of the enzyme.

Effect of the peroxisomal proliferator di(2-ethylhexyl)phthalate on component reactions of the rat hepatic microsomal fatty acid chain elongation system and on other hepatic lipogenic enzymes

The feeding of 2% di(2-ethylhexyl)phthalate (DEHP) to rats increased the hepatic microsomal elongation of palmitoyl-CoA by about twofold, while those of palmitoleoyl-CoA and γ-linolenoyl-CoA decreased to 83 and 63%, respectively, of the control values. When component reactions of the elongation pathway were measured, it was observed that only the activity of condensing enzyme was increased by twofold, while those of β-ketostearoyl-CoA reductase, β-hydroxypalmitoyl-CoA dehydrase, and trans-2-hexadecenoyl-CoA reductases were not affected. Furthermore, the time course for induction of both condensation and elongation of palmitoyl-CoA was similar. In vitro addition of DEHP had no effect on either condensation or elongation. Thus, these results indicate that the peroxisomal proliferator induces only the condensing enzyme which is the regulatory and rate-limiting step of elongation sequence. The DEHP treatment also markedly enhanced the cytosolic NADPH-generating activities of glucose-6-PO 4 dehydrogenase (2.2-fold) and malic enzyme (7.3-fold). Unexpectedly, the activities of fatty acid synthetase and citrate cleavage enzyme were unaffected. These results are discussed in light of the fact that these lipogenic enzymes are coordinatively induced by diet or hormones.

Evidence for multiple condensing enzymes in rat hepatic microsomes catalyzing the condensation of saturated, monounsaturated, and polyunsaturated acyl coenzyme A.

The condensation of palmitoyl-CoA with malonyl-CoA by rat hepatic microsomes was competitively inhibited by myristoyl-CoA, whereas it was noncompetitively inhibited by palmitoleoyl and gamma-linolenoyl-CoA. Furthermore, the condensation of palmitoleoyl-CoA with malonyl-CoA was also noncompetitively inhibited by gamma-linolenoyl-CoA. Replacement of normal diet by a fat-free high carbohydrate diet resulted in 8-, 2.5-, and 2.3-fold increases in the condensation rates of both palmitoyl- and myristoyl-CoA, palmitoleoyl-CoA, and gamma-linolenoyl-CoA, respectively. On the other hand, administration of di-(2-ethylhexyl)phthalate (DEHP) resulted in a 2-fold stimulation of the condensation activities with myristoyl- and palmitoyl-CoA, while those with palmitoleoyl- and gamma-linolenoyl-CoA decreased to about 83 and 63%, respectively. Similar results following dietary changes or DEHP administration were obtained for total elongation activities. Finally condensation activities of 16:0, 16:1, and gamma-18:3 CoA were differently affected by the proteolytic enzyme, chymotrypsin. The competitive substrate studies, those of dietary and DEHP administration, and the differential action of chymotrypsin strongly suggest the existence of at least three discrete condensing enzymes catalyzing the condensation of saturated, monounsaturated, and polyunsaturated acyl-CoAs. These studies also indicate that the condensation reaction is the regulating and rate-limiting step of the fatty acid chain elongation system.

Hepatic microsomal short-chain β-hydroxyacyl-CoA dehydrase distinct from the fatty acid elongation component: Substrate specificity of the membrane-extracted enzyme

The ability of 0.4 m KCl to extract over 80% of a short-chain β-hydroxyacyl-CoA dehydrase from rat hepatic endoplasmic reticulum, while more than 80% of the long-chain β-hydroxyacyl-CoA dehydrase component of the fatty acid chain elongation system remains intact, confirms the existence of more than one hepatic microsomal dehydrase. Following extraction from the microsomal membrane, the short-chain dehydrase undergoes, at least, a two-fold activation. Employing even-numbered trans-2-enoyl-CoA substrates ranging in carbon chain length from 4 to 16, the highest dehydrase specific activity of 16 μmol min −1 mg protein −1 was obtained with trans-2-hexenoyl-CoA; crotonyl-CoA was the second most active substrate, followed by 8 > 10 > 12 > 14 > 16. The specific activity of the short-chain dehydrase with trans-2-hexadecenoyl-CoA (C-16) was only 3% of that observed with the trans-2-hexenoyl-CoA. With crotonyl-CoA or β-hydroxybutyryl-CoA as substrates, HPLC was employed to identify the products, β-hydroxybutyryl-CoA, of the hydration reaction, or crotonyl-CoA, of the reverse dehydration reaction. It was also observed that the short-chain dehydrase catalyzed the formation of both d(−) and l(+) stereoisomers of β-hydroxybutyryl-CoA. The equilibrium constant for the dehydrase-catalyzed reaction determined at pH 7.4 and 35 °C, was calculated to be 6.38 × 10 −2 m −1, while the standard free energy change was −775 cal/mol, results similar to those obtained with crystalline crotonase. Finally, based on membrane fraction marker enzymes, substrate specificity, and heat lability of the dehydrase, it was concluded that the microsomal membrane contains a short-chain β-hydroxyacyl-CoA dehydrase which is separate from the mitochondrial crotonase.

Rat hepatic microsomal acetoacetyl-CoA reductase. A beta-ketoacyl-CoA reductase distinct from the long chain beta-ketoacyl-CoA reductase component of the microsomal fatty acid chain elongation system.

The present study provides evidence for a new rat liver microsomal enzyme, a short chain beta-ketoacyl (acetoacetyl)-CoA reductase, which is separate from the long chain beta-ketoacyl-CoA reductase component of the microsomal fatty acid chain elongation system. This microsomal reductase converts acetoacetyl-CoA to beta-hydroxybutyryl-CoA at a rate of 70 nmol/min/mg of protein; the enzyme has a specific requirement for NADH and appears to obtain electrons directly from the reduced pyridine nucleotide without the intervention of cytochrome b5 and its flavoprotein reductase. The apparent Km of the enzyme of the acetoacetyl-CoA was 21 microM and for the cofactor, 18 microM. The pH optimum was broad, ranging from 6.5 to 8.0. The product formed is the D-isomer of beta-hydroxybutyryl-CoA. High carbohydrate fat-free diet resulted in a small but significant (35%) increase in microsomal acetoacetyl-CoA reductase activity. The cytosol also contains this enzyme activity, measuring approximately 57% of that found in the microsomes. The mitochondrial activity which is 20-25% higher than the microsomal activity appears to be due to L-beta-hydroxyacyl-CoA dehydrogenase which converts acetoacetyl-CoA to L-beta-hydroxybutyryl-CoA. The microsomal acetoacetyl-CoA reductase activity was extracted from the microsomal membrane by 0.4 M KCl, resulting in an 8- to 10-fold purification; in addition, the long chain fatty acid elongation system was unaffected by this extraction procedure. Employing beta- hydroxyhexanoyl -CoA as a substrate, evidence is also provided for a separate dehydratase which acts on short chain substrates. Lastly, the liver microsomes had no detectable acetoacetyl-CoA synthetase or acetyl-CoA acetyltransferase activities. Hence, the possible involvement of the rat hepatic microsomal short chain beta-ketoacyl-CoA reductase, short chain beta-hydroxyacyl-CoA dehydratase, and the previously reported short chain trans-2-enoyl-CoA reductase in the hepatic utilization of acetoacetyl-CoA and in the synthesis of butyryl-CoA for hepatic lipogenesis is discussed.

Evidence for a new physiological role of hepatic NADPH:ferricytochrome (P-450) oxidoreductase. Direct electron input to the microsomal fatty acid chain elongation system.

Evidence for a new physiological role of hepatic NADPH:ferricytochrome (P-450) oxidoreductase. Direct electron input to the microsomal fatty acid chain elongation system.

Membrane lipid biosynthesis in Acholeplasma laidlawii b: elongation of medium- and long-chain exogenous fatty acids in growing cells.

The chain elongation of a wide variety of exogenous fatty acids and the subsequent incorporation of the chain elongation products into the total membrane lipids of Acholeplasma laidlawii B were systematically studied. Within each chemical class of fatty acids examined, the extent of chain elongation increased with increases in chain length, reached a maximum value, and then declined with further increases in chain length. Depending on chemical structure, exogenous fatty acids containing less than 6 to 9 carbon atoms or more than 15 to 18 carbon atoms were not substrates for the chain elongation system. The substrate specificity of this fatty acid elongation system was strikingly broad, and straight-chain, methyl isobranched, and methyl anteisobranched saturated fatty acids, as well as cis- and trans-monounsaturated, cis-cyclopropane, and cis-polyunsaturated fatty acids, underwent chain elongation in vivo. The extent of chain elongation and the average chain length of the primary elongation products correlated well with the physical properties (melting temperatures) of the exogenous fatty acid substrates. The specificity of fatty acid chain elongation in A. laidlawii B maintained the fluidity and physical state of the membrane lipids within a rather wide but definitely limited range. The fatty acid chain elongation system of this organism could be markedly influenced by the presence of a second exogenous fatty acid that was not itself a substrate for the chain elongation system but was incorporated directly into the membrane lipids. The presence of a relatively low-melting exogenous fatty acid increased both the extent of chain elongation and the average chain length of the elongation products generated, whereas the presence of a relatively high-melting fatty acid had the opposite effect. The extent of chain elongation and nature of the elongation products formed were not, however, dependent on the fluidity and physical state of the membrane lipids per se. The second exogenous fatty acid appeared instead to exert its characteristic effect by competing with the chain elongation substrate and elongation products for the stereospecific acylation of positions 1 and 2 of sn-glycerol-3-phosphate. The similar effects of alterations in environmental temperature, cholesterol content, and exposure to the antibiotic cerulenin on the fatty acid chain elongation and de novo biosynthetic activities suggested that the chain elongation system of this organism may be a component of the de novo biosynthetic system.

Effect of thyroid hormones on microsomal fatty acid chain elongation synthesis in rat liver.

Evidence is presented that rat liver microsomal fatty acid chain elongation synthesis and desaturation, as well as acetyl-CoA carboxylase and fatty acid synthetase, are strongly influenced by thyroid hormone level. Conversely, the fatty acid chain elongation system in mitochondria, unlike the oxidative capacity of palmitate, NADH, succinate and malate, does not seem significantly affected by the thyrotoxic state. In triiodothyronine-induced or thyroxine-induced hyperthyroidism, rat liver acetyl-CoA carboxylase, fatty acid synthetase and microsomal chain elongation and desaturation reactions are not greatly affected after the first 10 days of treatment, while after longer intervals a respective increase in these activities is shown of up to 87, 116 and 65% after 22 days. In propylthiouracil-induced hypothyroidism, all the above synthetic activities are strongly reduced immediately after three days of drug administration and diminished no further following longer periods. Although the pattern of synthesized fatty acids in the thyrotoxic state is similar to that obtained from normal subcellular rat fractions, the esterification process of fatty acids in microsomal lipids appears to be slightly inhibited in hypothyroid rats and increased following triiodothyronine or thyroxine administration. Finally, a reduction in the hepatic cyclic AMP level of about 41% is reported after 19 days of triiodothyronine-administration to rats. On the basis of the observed insensitivity of the mitochondrial fatty acid chain elongation system to the thyrotoxic state, a tentative interpretation of its role in the hepatic cell is postulated.

The influence of dietary alterations, fasting and competitive interactions on the microsomal chain elongation of fatty acids

1. 1.When rats were raised on a fat-free diet and fasted for 24, 48 and 72 h the rates for the chain elongation of palmitic acid, octadeca-6,9-dienoic acid and octadeca-6,9,12-trienoic acid were all depressed to about the same extent. 2. 2. When rats raised on a balanced diet were fasted for 72 h and then refed either a balanced diet or a fat-free diet the rates for the chain elongation of octadeca-6,9-dienoic acid and octadeca-6,9,12-trienoic acid returned only to the level of non-fasted controls. When fasted rats were refed the balanced diet the chain elongation of palmitic acid was four times greater than the non-fasted controls. When rats were refed the fat-free diet the chain elongation of palmitic acid was 12 times greater than for the non-fasted controls. These findings suggest that rat liver microsomes may contain more than one chain elongating system. 3. 3. Competitive substrate experiments also suggest that hepatic microsomes contain more than one fatty acid chain elongation system.

Mechanisms and physiological roles of fatty acid chain elongation in microsomes and mitochondria.

1. The characterization and separation of two enoyl-CoA reductases from rat liver with different catalytic properties are described. 2. Studies on the subcellular location revealed both enzymes to be constituents of a mitochondrial and microsomal fatty acid chain elongation system, respectively. 3. The microsomal enzyme shows optimal activity with hexenoyl-CoA, the mitochondrial enoyl-CoA reductase with decenoyl-CoA. The implications of this observation regarding a preference for γ-linolenic acid and linolenic acid, respectively, as the physiological substrates of microsomal and mitochondrial enoyl-CoA reductase will be discussed. 4. The mitochondrial enoyl-CoA reductases from rat liver and heart exhibit different pyridine nucleotide specificities. The implications of these differences concerning important roles in gluconeogenesis or nitrogen metabolism and in conservation of energy, respectively, is discussed. 5. A possible relationship between mitochondrial fatty acid chain elongation in the aorta and the pathogenesis of atherosclerosis is discussed.