A role of acetoacetyl-CoA synthetase in acetoacetate utilization by rat liver cell fractions

The enzyme acetoacetyl-CoA synthetase which catalyzes the synthesis of acetoacetyl-CoA from acetoacetate, CoA and ATP is shown to be present in mitochondrial and cytoplasmic fractions of rat liver. It was decreased in both specific activity and amount after starvation for 48 hours. It is proposed that the synthetase normally functions in the re-utilization of some of the acetoacetate produced within the mitochondrion as well as that reaching the cytoplasm, and that acetoacetate can function as an acetyl carrier between mitochondria and cytoplasm.     

cDNA-derived amino acid sequence of acetoacetyl-CoA synthetase from rat liver

We have previously shown that the two membrane bound enzymes leukotriene C synthase and microsomal glutathione S-transferase interact in vitro and in vivo. Rat basophilic leukemia cells and murine mastocytoma cells, two well-known sources of leukotriene C synthase, both expressed microsomal glutathione S-transferase as determined by Western blot analyses. Several human tissues were found to contain both leukotriene C synthase and microsomal glutathione S-transferase mRNA. These data suggest that the interaction may be physiologically important. To study this further, expression vectors encoding the two enzymes were cotransfected into mammalian cells and the subcellular localization of the enzymes was determined by indirect immunofluorescence using confocal laser scanning microscopy. The results showed that leukotriene C synthase and microsomal glutathione S-transferase were both localized on the nuclear envelope and adjacent parts of the endoplasmic reticulum. Image overlay demonstrated virtually identical localization. We also observed that coexpression substantially reduced the catalytic activity of each enzyme suggesting that a mechanism involving protein–protein interaction may contribute to the regulation of LTC₄ production.     

Stimulation of hepatic cholesterol biosynthesis by fatty acids. Effects of oleate on cytoplasmic acetoacetyl-CoA thiolase, acetoacetyl-CoA synthetase and hydroxymethylglutaryl-CoA synthase.

The effects of oleic acid on the activities of cytosolic HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) synthase, AcAc-CoA (acetoacetyl-CoA) thiolase and AcAc-CoA synthetase, as well as microsomal HMG-CoA reductase, all enzymes in the pathway of cholesterol biosynthesis, were studied in the isolated perfused rat liver. Oleic acid bound to bovine serum albumin, or albumin alone, was infused for 4 h at a rate sufficient to sustain an average concentration of 0.61 +/- 0.05 mM fatty acid during the perfusion. Hepatic cytosol and microsomal fractions were isolated at the termination of the perfusion. Oleic acid simultaneously increased the activities of the cytosolic cholesterol-biosynthetic enzymes 1.4-2.7-fold in livers from normal fed rats and from animals fasted for 24 h. These effects were accompanied by increased net secretion by the liver of cholesterol and triacylglycerol in the very-low-density lipoprotein (VLDL). We confirmed the observations reported previously from this laboratory of the stimulation by oleic acid of microsomal HMG-CoA reductase. In cytosols from perfused livers, the increase in AcAc-CoA thiolase activity was characterized by an increase in Vmax. without any change in the apparent Km of the enzyme for AcAc-CoA. In contrast, oleic acid decreased the Km of HMG-CoA synthase for Ac-CoA, without alteration of the Vmax. of the enzyme. The Vmax. of AcAc-CoA synthetase was increased by oleic acid, and there was a trend towards a small increase in the Km of the enzyme for acetoacetate. These data allow us to conclude that the enzymes that supply the HMG-CoA required for hepatic cholesterogenesis are stimulated, as is HMG-CoA reductase, by a physiological substrate, fatty acid, that increases rates of hepatic cholesterol synthesis and cholesterol secretion. Furthermore, we suggest that these effects of fatty acid on hepatic cholesterol metabolism result from stimulation of secretion of triacylglycerol in the VLDL by fatty acids, and the absolute requirement of cholesterol as an important structural surface component of the VLDL necessary for transport of triacylglycerol from the liver.     

Radical acetoacetate oxidation by myeloperoxidase, lactoperoxidase, prostaglandin synthetase, and prostacyclin synthetase: implications for atherosclerosis

When the myeloperoxidase-catalyzed peroxidation of acetoacetate proceeds in the presence of piperidinooxy free radical, methyl glyoxal is formed, and the nitroxide group is reduced to the secondary amine. A mechanism is advanced wherein an alpha-carbon-centered acetoacetate radical, generated by the peroxidase, forms an unstable adduct with the nitroxide group, subsequently decomposing to the observed products. Formation of methyl glyoxal, detected as its bis-2,4-dinitrophenylhydrazone by radial thin-layer chromatography, represents a method of determining free radical acetoacetate peroxidation by other peroxidases. It is shown that lactoperoxidase, prostaglandin synthetase, and prostacyclin synthetase generate methyl glyoxal with requirements identical to those of myeloperoxidase. With prostaglandin synthetase, arachidonic acid could replace the supporting peroxide. Substantiation that the catalyst for the reaction in aortic microsomes was prostacyclin synthetase was obtained by showing that 15-hydroperoxyarachidonic acid strongly inhibited the activity (5). The finding that these peroxidases catalyze radical acetoacetate oxidation could have broad implications for cellular damage via lipid peroxidation (7). Specifically, radical oxidation of acetoacetate by prostacyclin synthetase is proposed to be a link between cardiovascular risk factors and the initiation of atherosclerosis.     

Transcriptional regulation of ketone body-utilizing enzyme, acetoacetyl-CoA synthetase, by C/EBPalpha during adipocyte differentiation

Acetoacetyl-CoA synthetase (AACS), an essential enzyme for the synthesis of fatty acid and cholesterol from ketone bodies, was found to be highly expressed in mouse adipose tissue, and GC box and C/EBPs motif were crucial for AACS promoter activity in 3T3-L1 adipocytes. Moreover, we found that AACS promoter activity was controlled mainly by C/EBPalpha during adipogenesis.     

Role of prostaglandins and leukotrienes in volume regulation by Ehrlich ascites tumor cells

Summary PGE₂ and LTC₄ syntheses in Ehrlich ascites cells were measured by radioimmunoassay. Hypotonic swelling results in stimulation of the leukotriene synthesis and a concomitant reduction in the prostaglandin synthesis. If the cells have access to sufficient arachidonic acid there is a parallel increase in the synthesis of both leukotrienes and prostaglandins following hypotonic exposure. PGE₂ significantly inhibits regulatory volume decrease (RVD) following hypotonic swelling in Na-containing medium but not in Na-free media, supporting the hypothesis that the effect of PGE₂ is on the Na permeability. PGE₂ also had no effect on RVD in Na-free media in the presence of the cation ionophore gramicidin. Since the Cl permeability becomes rate limiting for RVD in the presence of gramicidin, whereas the K permeability is rate limiting in its absence, it is concluded that PGE₂ neither affects Cl nor K permeability. Addition of LTD₄ accelerates RVD and since the K permeability is rate limiting for RVD this shows that LTD₄ stimulates the K permeability. Inhibition of the leukotriene synthesis by nordihydroguaiaretic acid inhibits RVD even when a high K conductance has been ensured by the presence of gramicidin. It is, therefore, proposed that an increase in leukotriene synthesis after hypotonic swelling is involved also in the activation of the Cl transport pathway.     

Regulation of purine metabolism adenylosuccinate synthetase from novikoff ascites tumor cells

Adenylosuccinate synthetase has been partially purified from Novikoff ascites tumor cells. The properties of the protein are quite different from the enzyme from rat liver in that the K_m for aspartate is higher and the K_I for the feedback inhibitor AMP is also higher. The antibiotic hadacidin has a preferential inhibitory effect on the tumor enzyme. These results suggest that the Novikoff ascites tumor enzyme is less sensitive to normal feedback controls but may be more sensitive to specific antitumor drugs.     

Role of myeloid cells in tumor angiogenesis and growth

Cells of the innate immune system have a key role in maintaining homeostasis by providing the first line of defense against many pathogens. Innate immunity can also modulate the activity of acquired immunity by several mechanisms. However, subsets of myeloid cells can facilitate tumor growth, because these cells produce angiogenic factors and can also prevent the immune system from attacking tumor cells. Recent studies also emphasize the role of myeloid cells in mediating refractoriness to anti-VEGF treatments. This function of myeloid cells occurs through a proangiogenic pathway that is, at least in part, driven by the secreted protein Bv8. This review summarizes recent findings on the complex role of bone marrow-derived cells in tumor growth.     

Selenalysine utilization by CHO cells

CHO cells allowed to grow in a medium containing selenalysine can utilize it for protein synthesis. Selenalysine is incorporated into cell proteins in substitution of lysine: a maximum of 5% of protein lysine can be substituted. Protein lysine substitution by selenalysine can be correlated to the reduced viability of cells grown in its presence.     

Relationship between fatty acid and glucose utilization in Ehrlich ascites tumor cells

Glucose greatly increased total free fatty acid (FFA) esterification by Ehrlich ascites tumor cells. However, the FFA concentration of the cells was not altered. Less exogenous FFA was oxidized to CO(2) at any given extracellular FFA:albumin molar ratio when glucose was available, but increasing amounts of radioactive CO(2) were produced as the FFA:albumin molar ratio was raised, even in the presence of glucose. It is suggested that glucose, by providing either energy or an excess of triose acceptor for fatty acid esterification, stimulated FFA uptake only indirectly, by increasing the utilization of FFA subsequent to initial uptake from the medium, i.e., by increasing the turnover rate of the cellular FFA pool. Availability of glucose decreased the oxidation of endogenous lipid radioactivity and the depletion of endogenous lipid ester radioactivity. Most of the radioactivity utilized was derived from phospholipids, and depletion of phospholipid radio-activity was spared when glucose was available. Depletion of cellular total lipid ester also was spared in the presence of glucose. Availability of FFA did not decrease total glucose uptake or its oxidation to CO(2). Glucose utilization by these cells appears not to be regulated by FFA availability in the manner that Randle and coworkers described for muscle.