Michael D. Greenspan

Effect of halofenate and clofibrate on lipid synthesis in rat adipocytes

The free acids of the plasma lipid-lowering agents, halofenate and clofibrate inhibited the incorporation of radioactive glucose and pyruvate into fatty acids of isolated adipocytes prepared from rat epididymal fat pads. The concentration which inhibited fatty acid synthesis was dependent on the bovine serum albumin concentration in the incubation. The 50 per cent inhibitory concentration of the free acid of halofenate in 1 per cent, 2 percent and 4 per cent albumin was 0.9 mM, 2.3 MM and 4.4 mM, respectively. The potency of clofibrate was also lowered by increasing the albumin concentration. These compounds inhibited the uptake of both [14C]glucose and [14C]pyruvate to the same degree as the incorporation of these substrates into fatty acids. However, the drugs either had no effect on , or stimulated the uptake of palmitate by the cells. Leucine accumulation by the adipocytes was unaffected by halofenate (free acid) and inhibited by clofibrate (free acid). A comparison of these agents with (minus)-hydroxycitrate, kynurenate and cerulenin (inhibitors of ATP-citrate lyase, acetyl CoA carboxylase and fatty acid synthetase, respectively) on the oxidation of pyruvate suggested that they inhibited pyruvate metabolism at or near the enzyme, pyruvate dehydrogenase.

Separation and detection of neutral lipids and free fatty acids in a liver extract by high-performance liquid chromatography

Abstract A relatively simple method to determine hepatic neutral lipids and free fatty acids by highperformance liquid chromatography is described. This method involves the preparation of a chloroform extract of the total liver lipids, followed by removal of the phospholipids by adsorption onto silicic acid and elution of neutral lipids and free fatty acids with 50% diethyl ether in hexane. This fraction is then subjected to liquid-solid chromatography with a solvent system of 2,2,4-trimethylpentane (isooctane):tetrahydrofuran:formic acid (90:10:0.5) and is detected by refractive index. Cholesterol esters, fatty acids, cholesterol, and diglycerides each elute as single peaks, easily quantitated by comparison to appropriate standards. Baseline separation of triglycerides from cholesterol esters is also achieved.

The effect of cerulenin on sterol biosynthesis inSaccharomyces cerevisiae

Cerulenin specifically inhibited fatty acid biosynthesis inSaccharomyces cerevisiae without having an effect on sterol formation. Ergosterol was not required for cell growth in the presence of cerulenin (1 μg/ml). The addition of fatty acids to the growth medium reduced the amount of ergosterol formed by 45%; further addition of cerulenin to the media had no effect on the amount of ergosterol synthesized by the cells. The incorporation of3H from3H2O into ergosterol was not affected by cerulenin whereas incorporration into fatty acids was inhibited by 90%.

Synthesis of δ-lactone, oxetane and azetidinone analogs from the naturally occurring β-lactone L-659,699. The preparation of a novel HMG CoA synthase inhibitor.

Abstract A series of δ-lactone ( 1 and 2 ), oxetane ( 3 ) and azetidinone ( 4 and 5 ) analogs were prepared from the HMG CoA synthase inhibitor L-659,699 ((E,E)-11-[3′R-(hydroxymethyl)-4′-oxo-2′R-oxetanyl]-3,5,7R- trimethyl-2,4-undecadienoic acid 1,2 ), which maintained the trans relationship of the ring side chains. The N-tosyl azetidinone analog represents the first reported member of a new series of non β-lactone HMG CoA synthase inhibitors.

The inhibition of cytoplasmic acetoacetyl-CoA thiolase by a triyne carbonate (L-660,631)

The compound L-660, 631 (2-oxo-5-(1-hydroxy-2,4,6-heptatriynyl)-1,3-dioxolane-4 heptanoic acid), a natural product isolated from an Actinomycete culture, was found to inhibit rat liver cytosolic acetoacetyl-CoA thiolase, the first step in the cholesterol biosynthesis pathway, with an IC50 of 1.0 x 10(-8) M. The inhibitor had no effect on other sulfhydryl containing enzymes of lipid synthesis such as HMG-CoA synthase, HMG-CoA reductase, and fatty acid synthase. When tested in cultured human liver Hep G2 cells the compound inhibited the incorporation of 14C-acetate and 14C-octanoate into sterols 56% and 48% respectively at 3 x 10(-6) M with no effect on fatty acid synthesis. No noticeable effect was seen on fatty acid biosynthesis. This strongly suggests that the locus of inhibition of acetate incorporation into sterols found with this compound is the acetoacetyl-CoA thiolase step in the cholesterol biosynthesis pathway.

Chapter 2 Animal and bacterial fatty acid synthetase: structure, function and regulation

Publisher Summary This chapter presents a survey of the current state of knowledge of the bacterial and animal fatty acid synthetase systems. Fatty acid synthetase (FAS) is the designation given to the enzyme system which catalyzes the synthesis of long-chain fatty acids from a short-chain acyl-CoA primer. Acetyl-CoA forms the methyl end of the fatty acid and malonyl-CoA (or in some circumstances methylmalonyl-CoA) contributes the remaining carbon atoms of the molecule. Prior to the mid-1950s it was generally assumed that fatty acid synthesis from acetyl-CoA occurred by direct reversal of the mitochondria1 8-oxidation pathway of degradation of long fatty acids to acetyl-CoA. This view became suspect when it was found that de novo synthesis of fatty acids from [14C]acetate was catalyzed by the 1,00,000 X g supernatant fraction obtained by centrifugation of the cytoplasm prepared from homogenates of pigeon liver. The cofactors, ATP, Mn, bicarbonate and NADPH were required for the conversion of acetyl-CoA to long-chain fatty acids and were different from those to be expected for the reversal of P-oxidation. Two key observations gave great impetus to studies on the mechanism of fatty acid biosynthesis: the discovery by Wakil that acetyl-CoA was carboxylated to malonyl-CoA in a system containing a protein fraction obtained from the pigeon-liver extract as well as ATP, Mn, and bicarbonate, and the additional finding that malonyl-CoA was an intermediate in fatty acid biosynthesis. Subsequent progress was quite rapid. It was soon established that the reactions of fatty acid biosynthesis occurred with the substrates and intermediates covalently bound to the enzyme. This provided the basis for much of the research on FAS from a wide variety of sources.

Studies on the in vivo synthesis of triacylglycerol in mouse liver

The in vivo biosynthesis of hepatic glycerolipids was examined by studying the incorporation of [2-3H] glycerol into triacylglycerol in the mouse. The isotope was administered by rapid injection into the portal vein. The incorporation of glycerol was linear for about 1 min and maximal rates were seen in the presence of additional oleic acid. At 20 mumol of oleic acid bound to 1 mumol of bovine serum albumin, the highest level tested, glycerol incorporation was still increasing linearly, whereas a plateau was reached at 4 mumol of glycerol. The liver incorporated 200-300 nmol of [2-3H] glycerol into triacyglycerol per min when 20 mumol of albumin-bound oleic acid plus 4 mumol of glycerol were injected in a 0.2 ml volume. Studies on the uptake by the liver after intraportal injection revealed that at 0.5 min the liver retained 2 mumol of glycerol and 5 mumol of oleic acid, indicating that the uptake of substrate was not rate-limiting. The results suggest that the availability of substrate is a major factor in the regulation of triacyglycerol biosynthesis by the liver.

A sensitive assay for phosphatidate phosphohydrolase in mouse liver microsomes

A technique is described for the assay of phosphatidate phosphohydrolase using 1,2-[9,10-3H]dioleoyl-sn-glycero-3-phosphate as a substrate. This substrate was prepared enzymatically using mouse liver microsomes washed with 0.5 M NaCl, which synthesize minimal amounts of neutral lipids at high enzyme concentrations. Measurement of the product, 1,2-[9,10-3H]dioleoylglycerol, was 10-fold more sensitive than the usual colorimetric assay for inorganic phosphate release. In addition, the assay provides information about the relative contribution of other activities which limit the availability of diacylglycerols for further esterification to triacylglycerols and/or phospholipids.