Feedback regulation of bile acid biosynthesis in the rat

The hepatic biosynthesis of bile salts in the rat has been shown to be controlled homeostatically by the quantity of bile salt returning to the liver via the portal circulation. The feedback mechanism was demonstrated in two kinds of experiments. In the first, rats with bile fistulas were infused intraduodenally with sodium taurocholate 12 hr after surgery. If the rate of infusion was greater than 10 mg per 100 g rat per hr, the increase in bile acid output normally observed in bile fistula rats was prevented. In the second type of experiment, the rats were infused with taurocholate 48-72 hr after biliary diversion, when bile acid output had reached a maximal value. Provided the rate of infusion exceeded 10 mg per 100 g rat per hr, bile acid secretion returned to the low levels observed in intact rats. Previous attempts to demonstrate the feedback control have been unsuccessful because too little bile salt was infused. The taurocholate pool of the experimental animals was measured as approximately 15 mg per 100 g rat; it was calculated from this and the above results that this pool circulated 10-13 times daily.     

The short-term regulation of fatty acid synthesis in the rat epididymal adipocytes

Lipogenesis and fatty acid synthetase (FAS) activity of isolated rat adipocytes that were treated with insulin or epinephrine were studied. Insulin stimulated incorporation of radioactivity from D-[U-14C]glucose into CO2, saponifiable and non-saponifiable fractions, whereas epinephrine promoted lipolysis and oxidation of glucose into CO2. Whereas insulin stimulated fatty acid synthesis, epinephrine had no effect. Changes in FAS specific activity of insulin- or epinephrine-treated adipocytes were insignificant and could not account for insulin-stimulated lipogenesis. Rat adipocyte FAS, unlike hepatic FAS, was not subject to short-term regulation by insulin, although fatty acid synthesis showed such a response.     

Biochemical site of regulation of bile acid biosynthesis in the rat

The production of bile salts by rat liver is regulated by a feedback mechanism, but it is not known which enzyme controls endogenous bile acid synthesis. In order to demonstrate the biochemical site of this control mechanism, bile fistula rats were infused intravenously with (14)C-labeled bile acid precursors, and bile acid biosynthesis was inhibited as required by intraduodenal infusion of sodium taurocholate. The infusion of taurocholate (11-14 mg/100 g of rat per hr) inhibited the incorporation of acetate-1-(14)C, mevalonolactone-2-(14)C, and cholesterol-4-(14)C into bile acids by approximately 90%. In contrast, the incorporation of 7alpha-hydroxycholesterol-4-(14)C into bile acids was reduced by less than 10% during taurocholate infusion. These results indicate that the regulation of bile acid biosynthesis is exerted via cholesterol 7alpha-hydroxylase provided that hepatic cholesterol synthesis is adequate.     

Antibacterial targets in fatty acid biosynthesis

The fatty acid biosynthesis pathway is an attractive but still largely unexploited target for the development of new antibacterial agents. The extended use of the antituberculosis drug isoniazid and the antiseptic triclosan, which are inhibitors of fatty acid biosynthesis, validates this pathway as a target for antibacterial development. Differences in subcellular organization of the bacterial and eukaryotic multienzyme fatty acid synthase systems offer the prospect of inhibitors with host versus target specificity. Platensimycin, platencin, and phomallenic acids, newly discovered natural product inhibitors of the condensation steps in fatty acid biosynthesis, represent new classes of compounds with antibiotic potential. An almost complete catalog of crystal structures for the enzymes of the type II fatty acid biosynthesis pathway can now be exploited in the rational design of new inhibitors, as well as the recently published crystal structures of type I FAS complexes.     

Rapid purification of enzymes of fatty acid biosynthesis from rat adipose tissue

Acetyl CoA carboxylase, ATP-citrate lyase and fatty acid synthetase were purified to homogeneity by a simple procedure. The purification method consists of polymerization of acetyl CoA carboxylase with citrate followed by avidin-Sepharose affinity chromatography. ATP-citrate lyase and fatty acid synthetase were isolated as by-products of acetyl CoA carboxylase purification and are separated from each other by chromatography on DE-52. ATP-citrate lyase was further purified by CoA-agarose affinity chromatography and fatty acid synthetase was purified on Bio-Gel A-1.5m. Purified ATP-citrate lyase, acetyl CoA carboxylase and fatty acid synthetase had specific activities of 9.9, 2.8 and 1.8 U/mg respectively with an over all recovery of 30, 25 and 50% respectively. Using these purified enzymes, we found that ATP-citrate lyase and acetyl CoA carboxylase were phosphorylated in vitro by both cAMP-dependent protein kinase and ATP-citrate lyase kinase whereas fatty acid synthetase was not phosphorylated by these protein kinases.     

The biosynthesis of D-ring fatty acid esters of estriol

Biological esterification with fatty acids is a feature that is now known to be common to most steroids. The esterification of estradiol in the D-ring at the 17 beta-hydroxyl leads to a family of extremely active estrogens. Similarly, esterification of the weaker estrogen, estriol (E3), has an even greater impact on its hormonal potency. We have recently shown that synthetic long chain esters of E3 at either 16 alpha- or 17 beta- are highly potent estrogens. The estrogenic activity of the synthetic E3 esters led us to determine whether E3 is biologically esterified, and if so, to characterize the resulting esters. Incubation of E3 with rat lung, a tissue which is highly active in esterifying estradiol, produces a nonpolar metabolite which upon saponification is converted back into E3. There was no evidence for the formation of a diester. Purification by high performance liquid chromatography separates the non-polar metabolite into two peaks, one the C-16 alpha- (approximately 60%) and the other the C-17 beta-ester (approximately 40%). The two fractions were further purified and characterized; each is a mixture of fatty acid esters of E3. The composition of the C-16 alpha- and the C-17 beta-fatty acid esters of E3 is identical. The predominant fatty acids are arachidonate, 34%, palmitate, 26%, followed by oleate 14%, linoleate 13%, stearate 8%, and palmitoleate 5%. The similarity of the esters at C-16 and C-17 may indicate that the fatty acid precursor for the acyltransferase is the same for both hydroxyl groups. It may also suggest that the same enzyme esterifies both positions in the D-ring. Since synthetic estriol fatty acid esters are extremely potent and long-lived estrogens, the enzymatic esterification of estriol produces powerful estrogens with considerable physiological potential.     

Role of acyl-CoAs and acyl-CoA-binding protein in regulation of carbon supply for fatty acid biosynthesis

Acyl-CoA esters inhibit the plastidial glucose 6-phosphate (Glc-6-P) transporter and the adenylate transporter; the IC(50) values for the inhibition by oleoyl-CoA (18:1-CoA) are 200-400 nM and 1-2 microM respectively. The inhibition of either of these processes significantly reduces the flux of carbon from Glc-6-P or from acetate into long-chain fatty acids. The effect is dependent on the acyl chain length, e.g. lauryl-CoA is less inhibitory than oleoyl-CoA, causing 34 and 68% inhibition respectively of Glc-6-P uptake after 30 s. The inhibition of Glc-6-P and ATP transport is alleviated by addition of an equivalent concentration of acyl-CoA-binding protein (ACBP) or BSA. Acyl-CoAs do not inhibit pyruvate or glucose transporters. The endogenous concentrations of acyl-CoAs and ACBP are similar during embryo maturation.     

Branched-chain fatty acid biosynthesis in Escherichia coli

β-Ketoacyl-acyl carrier protein (ACP) synthase III (KASIII) catalyzes the first elongation step in straight-chain fatty acid (SCFA) biosynthesis in Escherichia coli. Overproduction of the corresponding KASIII gene, or the Brassica napus KASIII gene has previously been observed to lead to an increase in the amount of shorter-chain fatty acids produced by E. coli. In this study it is shown that overexpression of the KASIII gene, which initiates branched-chain fatty acid (BCFA) in Streptomyces glaucescens, does not lead to a change in the fatty acid profiles of E. coli. E. coli produces trace levels of BCFAs when grown in the presence of isobutyric acid, but the amounts of these are not significantly altered by expression of the S. glaucescens KASIII gene. In contrast, the amounts of BCFAs produced from isobutyryl CoA in vitro by E. coli cell-free extracts can be increased at least four-fold by the presence of the S. glaucescens KASIII. These observations suggest that in vivo production of isopalmitate by E. coli expressing the S. glaucescens KASIII is limited by availability of the appropriate BCFA biosynthetic primers. Journal of Industrial Microbiology & Biotechnology (2001) 27, 246–251. Received 10 January 2001/ Accepted in revised form 13 July 2001