Action of Ebselen on rat hepatic microsomal enzyme-catalyzed fatty acid chain elongation, desaturation, and drug biotransformation

In the previous study, the organoselenium-containing anti-inflammatory agent, Ebselen, was found to disrupt both hepatic microsomal NADH- and NADPH-dependent electron transport chains. In the current investigation, we focus on the action of Ebselen on three separate metabolic reactions, namely, fatty acid chain elongation, desaturation, and drug biotransformation, which utilize reducing equivalents via these microsomal electron transport pathways. Both NADH-dependent and NADPH-dependent chain elongation reactions showed (i) that the condensation step was inhibited by Ebselen; all three substrates, palmitoyl CoA (16:0), palmitoleoyl CoA (16:1), and gamma-linolenyl CoA (18:3), were differentially affected by Ebselen; for example, the apparent Ki's of Ebselen for the condensation of 16:0, 16:1, and 18:3 in the absence of bovine serum albumin (BSA) preincubation were 7, 14, and 34 microM, and those in the presence of BSA preincubation were 35, 62, and 150 microM, respectively, supporting earlier data for multiple condensing enzymes; (ii) that the beta-ketoacyl CoA reductase-catalyzed reaction step which appears to receive electrons, at least in part, from the cytochrome b5 system, was also markedly inhibited by varying Ebselen concentrations; and (iii) that similar results were obtained with the dehydrase and the enoyl CoA reductase. Hence, each of the four component steps was significantly inhibited by Ebselen. Another important fatty acid biotransformation reaction, delta 9 desaturation of stearoyl CoA to oleoyl CoA, was significantly inhibited (90%) by 30 microM Ebselen. This effect appeared to be directly related to the NADH-dependent electron transport chain rather than to a direct action on the desaturase enzyme. Last, Ebselen also inhibited both aminopyrine and benzphetamine N-demethylations, two cytochrome P450-catalyzed reactions, in untreated rats, in rats on a high carbohydrate diet, and in phenobarbital-treated rats.     

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.     

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 beta-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 beta-ketoacyl CoA derivative resulted in the formation of a 303-nm absorbance peak, characteristic of enolate formation. The beta-ketoacyl CoAs with carbon chain length of 6 to 18 interacted with BSA to produce the 303-nm peak; acetoacetyl CoA was the only beta-keto compound tested which did not interact with BSA to produce the peak. Other compounds which were unaffected by BSA included CoA, free beta-keto acid, beta-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 (epsilon) for the absorbance peak generated by the beta-keto thioester varied from 5 to 30 mM-1 cm-1 depending on the beta-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.     

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.     

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.     

Site of participation of cytochrome b5 in hepatic microsomal fatty acid chain elongation. Electron input in the first reduction step

The present study provides strong evidence for the involvement of rat liver microsomal cytochrome b5 in the first reduction step of fatty acid chain elongation. The rate of reoxidation of NADH-reduced microsomal cytochrome b5 was markedly stimulated (up to 3-fold) by the addition of increasing concentrations of beta-ketohexadecanoyl-CoA (1-8 microM). A quantitative analysis of product formation, the effect of cyanide, and anaerobiosis completely exclude the possibility that desaturase activity accounted for the beta-ketohexadecanoyl-CoA-induced stimulation of the cytochrome b5 reoxidation rate. Using liver microsomes from untreated rats, the beta-keto substrate was found to stimulate the rate of reoxidation of cytochrome b5 by 30%. However, when liver microsomes from fat-free diet rats were employed the stimulation was more than 3-fold, suggesting that the beta-ketoacyl-CoA reductase is inducible by a high carbohydrate, fat-free diet. This study also provides evidence for the noninvolvement of cytochrome b5 in the terminal reaction step (second reduction step of chain elongation), which is catalyzed by the trans-2-enoyl-CoA reductase. Although trans-2-hexadecenoyl-CoA significantly stimulated the NADH-reduced cytochrome b5 reoxidation rate under aerobic conditions, it did not have any stimulatory effect under anaerobic conditions. One interpretation of these results is that the trans-2-hexadecenoyl-CoA is substrate for the microsomal delta 9 desaturase system. Consistent with this conclusion was the fact that the trans-2-hexadecenoyl-CoA inhibited the liver microsomal delta 9 desaturation of stearoyl-CoA to oleoyl-CoA.     

The fatty acid chain elongation system of mammalian endoplasmic reticulum.

Much has been learned about FACES of the endoplasmic reticulum since its discovery in the early 1960s. FACES consists of four component reactions, requires the fatty acid to be activated in the form of a CoA derivative, utilizes reducing equivalents in the form of NADH or NADPH, is induced by a fat-free diet, resides on the cytoplasmic surface of the endoplasmic reticulum, appears to function in concert with the desaturase system and appears to exist in multiple forms (either multiple condensing enzymes connected to a single pathway or multiple pathways). FACES has been found in all tissues investigated, namely, liver, brain, kidney, lung, adrenals, retina, testis, small intestine, blood cells (lymphocytes and neutrophils) and fibroblasts, with one exception--the heart has no measurable activity. Yet, much more needs to be learned. The critical, inducible and rate-limiting condensing enzyme has resisted solubilization and purification; the purification of the other components has met with limited success. We know nothing about the site of synthesis of each component of FACES. How is each component enzyme integrated into the endoplasmic reticulum membrane? Is there a single mRNA directing synthesis of all four components or are there four separate mRNAs? How are elongation and desaturation coordinated? What is (are) the physiological regulator(s) of FACES--ADP, AMP, IP3, G-proteins, phosphorylation, CoA, Ca2+, cAMP, none of these? The molecular biology of FACES is only in the fetal stage of development. We are only scratching the surface--it is an undiscovered country.     

Solubilization and partial purification of an enzyme involved in rat liver microsomal fatty acid chain elongation: beta-hydroxyacyl-CoA dehydrase.

The solubilization and partial purification of beta-hydroxyacyl-CoA dehydrase from rat liver microsomes has been accomplished through deoxycholate solubilization, ammonium sulfate fractionation, and ion exchange chromatography. A purification of about 90-fold based on total soluble activity was achieved, with an overall yield of 40%. However, the initial solubilization is accompanied by the loss of the secondary portion of the v/s curve observed with intact microsomes. The enzyme requires detergent during the purification procedure to remain "soluble," and is strongly activated by the inclusion of Triton-X-100 at concentrations above its critical micelle concentration in the assay mixture. In addition a preference for micelles has been inferred based on discontinuities in the v/s curves relative to the measured critical micelle concentration of the substrates in the absence of Triton X-100. Kinetic parameters calculated on the basis of micelle-specific activity indicated that beta-hydroxyacyl-CoA substrates possessing even-numbered alkyl chains from 14 to 20 carbon atoms differed little in Vm', but had progressively larger Km' as the chain length increased. The partially purified preparation was also active with beta-hydroxy-8,11-eicosadienoyl-CoA; and with 2-trans-enoyl-CoA substrates in a reverse (hydration) reaction.     

Evidence for insulin dependent hepatic microsomal gamma-linolenic acid chain elongation in spontaneously diabetic Wistar BB rats.

We studied hepatic microsomal gamma-linolenoyl-CoA elongation and fatty acid composition of liver microsomes in spontaneously diabetic Wistar BB rats. The liver microsomal gamma-linolenoyl-CoA elongation was decreased in diabetic Wistar BB rats during both normo- and hyperglycemic periods and restored during the hypoglycemic period following insulin treatment. These results are in agreement with our previously reported data on linoleic acid delta 6 and delta 5 desaturations and support the non-parallel relationship between the chain elongation system and the glycemia. The fatty acid composition of BB rat liver microsomes was only partially consistent with the gamma-linolenoyl-CoA elongation activity at the different periods of glycemia, probably because factors other than elongation impairments were involved in the evolution of fatty acid composition.     

Fatty acid chain elongation by microsomal enzymes from the bovine meibomian gland

The composition of meibomian gland lipids suggested that fatty acid chain elongation might play a major role in the synthesis of such lipids. A fatty acid synthase preparation from the bovine meibomian gland catalyzed the formation of C16 acid and the enzyme was immunologically quite similar to that in the mammary gland. The microsomal fraction from the gland, on the other hand, catalyzed elongation of endogenous fatty acids in the presence of ATP and Mg2+ and of exogenous C18-CoA using malonyl-CoA and NADPH as the preferred reductant. The elongated products, ranging up to C28 in chain length, were found mainly as CoA esters and products derived from them. With C18-CoA as the exogenous primer, the elongation rate was linear with incubation time up to 20 min but the rate changed in a sigmoidal manner with increasing protein concentration. The elongation rate was maximal at a pH around 7.0. Typical Michaelis-Menten-type substrate saturation patterns were observed with both malonyl-CoA and NADPH. From linear double-reciprocal plots, the Km values for the two substrates were calculated to be 52 and 11 microM, respectively, with a V of about 340 pmol min-1 mg protein-1 with respect to malonyl-CoA. Exogenous CoA esters of C16 to C22 fatty acids were elongated to give products up to C28 without exhibiting any preference for the primer. The present elongation system could account for the formation of most of the very long chains found in meibomian lipids.     

Mechanisms for elongation in the biosynthesis of fatty acid components of epi-cuticular waxes

It is known that branched-chain amino acids can serve as precursors to iso- and anteiso-branched components of epi-cuticular waxes. Keto acid deamination products of Val, Leu and Ile are thought to serve as primers which are elongated by fatty acid synthase. However, the origin of elongation carbons has not been studied directly. Nor has the mechanism for formation of odd-carbon-length, straight- or branched-chain, cuticular ester fatty acids or free odd-carbon-length, straight fatty acid components of waxes been characterized. It is not known that α-oxidation of even-length precursors or elongation of odd-length primers is involved in these cases. Here, we present evidence which substantiates the expectation that elongation of branched as well as straight-chain precursors to wax ester acids occurs by fatty acid synthase catalyzed by addition of two carbon units via acetate. Also, we present evidence which indicates that odd-carbon-length acids can result from elongation of odd-carbon-length primers (at least branched), rather than even-length acids shortened by α-oxidation.     

Involvement of the fatty acid oxidation complex in acetyl-CoA-dependent chain elongation of fatty acids in Escherichia coli

The activity of acetyl-CoA-dependent chain elongation of fatty acids in Escherichia coli was enhanced when the organism was grown on oleic acid as the sole carbon source, but not detected when grown on glucose. Antibodies raised against fatty acid oxidation complex of E. coli inhibited both the reaction catalyzed by crotonase and the chain elongation in a similar manner, showing that the oxidation complex participates in the chain elongation. The activities of condensation and the activities of NADH- and NADPH-dependent 3-ketoacyl reduction in the cell-free extract were precipitated by antibodies to the complex in parallel with those of 3-ketoacyl-CoA thiolase and crotonase. These results together with the presence of NADPH-dependent trans-2-enoyl-CoA reductase in E. coli (Mizugaki, et al. (1982) Chem. Pharm. Bull. 30, 2503-2511) indicate that the acetyl-CoA-dependent chain elongation of fatty acids in E. coli occurs by the reversal of fatty acid oxidation other than the step of enoyl reduction.