ATP Citrate Lyase Activity Research Articles

Dependence of ATP citrate lyase activity on cell density of isolated rat hepatocytes

The activities of two enzymes involved in the lipogenic process, ATP citrate lyase and NADP-linked malic enzyme were evaluated as a function of cell density in isolated rat hepatocytes. The activity of ATP citrate lyase was profoundly affected by cell density with the activity/cell being higher at low cell densities than at high cell densities. Malic enzyme was not similarly affected, nor was cellular ATP content. The effect was observed regardless of dietary state but was most dramatic with hepatocytes from fasted-refed rats. Both an activator and an inhibitor of ATP citrate lyase have been isolated from conditioned medium from cells at low density and at high density, respectively. The activator fraction was heat stable while the inhibitor fraction was heat labile, and both factors had molecular weights in excess of 10,000 daltons.

Effect of transforming growth factor beta (TGF-beta) on ATP citrate lyase in isolated hepatocytes.

Transforming growth factor beta (TGF-beta) activates ATP citrate lyase in freshly isolated rat liver hepatocytes in a time dependent manner. Maximal stimulation of the enzyme occurred with less than thirty minutes of incubation of the cells with TGF-beta. The half-maximal effect on the enzyme determined in hepatocytes incubated with TGF-beta for 10 min at 37 degrees C was elicited by TGF-beta concentrations in the 10-11 - 10-12 M range. The potential role of TGF-beta stimulation of ATP citrate lyase activity in new membrane synthesis is discussed.

Calcitonin and hepatic fatty acid synthesis: effect of thyroparathyroidectomy on the elevation of ATP citrate lyase activity by refeeding of starved rats.

The effect of thyroparathyroidectomy (TPTX) on ATP citrate lyase regulation, a rate-limiting enzyme of fatty acid synthesis in hepatic cytosol, was investigated in rats refed after a 24 h fast. ATP citrate lyase activity in the hepatic cytosol was increased 2-fold by refeeding. This increase was suppressed about 50% by TPTX. The suppression of the enzyme activity by TPTX was completely restored by administration of calcitonin (CT; 80 MRC mU/100 g body weight). This hormonal effect was also observed at 20 MRC mU/100 g dose of CT. CT administration to refeeding-TPTX rats produced a significant increase in the calcium content of the liver tissue and the cytosol. The cytosolic ATP citrate lyase activity increase with CT administration was completely blocked by treatment of cytosol with EGTA (10 microM). This inhibition was clearly reversed by addition of calcium ion (1.25-5.0 microM). In addition, CT-induced rise in enzyme activity was markedly reduced by the presence of W-7 (5 and 50 microM), a calmodulin inhibitor, in the enzyme assay system. The present results suggest that CT plays a role in the elevation of hepatic ATP citrate lyase activity brought about by refeeding of fasted rats, and that this hormonal regulation might depend on Ca2+-calmodulin.

Effects of an antitumoural rhodium complex on thioacetamide-induced liver tumour in rats: Changes in the activities of ornithine decarboxylase, tyrosine aminotransferase and of enzymes involved in fatty acid and glycerolipid synthesis

Rats were injected daily for 8 weeks with 50 mg of thioacetamide per kg to produce liver tumours. Some of these rats were given three doses of 50 mg of an antitumoural Rh(III) complex kg at 14. 9 and 5 days before the end of the thioacetamide treatment. Thioacetamide decreased the rate of weight gain of the rats and the Rh(III) complex partly restored it. The activities of ATP citrate lyase, acetyl-CoA carboxylase and fatty acid synthetase in the livers were decreased by thioacetamide treatment and the Rh(III) complex partly reversed this effect. By contrast the activity of malic enzyme was increased by both thioacetamide and the Rh(III) complex and this effect probably relates to NADPH production for detoxification rather than for lipogenesis. Treatment with thioacetamide increased the rate of synthesis of di- and triacylglycerols from glycerol phosphate by liver homogenates, the activity of phosphatidate phosphohydrolase and the incorporation of [ 3H]glycerol into liver triacylglycerol in vivo. The Rh(III) complex did not produce a significant reversal of these effects of thioacetamide on glycerolipid synthesis. The total uptake of intraportally injected [ 3H]glycerol by the livers of thioacetamide treated rats was decreased and this was associated with a lowered activity of glycerol kinase. Thioacetamide increased the activity of hepatic ornithine decarboxylase by about 40-fold, but the Rh(III) complex did not reverse this effect. However, the decrease in tyrosine aminotransferase activity that was produced by thioacetamide was partly reversed by the Rh(III) complex. These results are discussed in relation to the tumour-promoting effects of thioacetamide and the antitumoural action of the Rh(III) complex.

Comparisons of Lipogenesis and Glucose Metabolism between Ovine and Bovine Adipose Tissues

Studies were initiated to compare glucose and lipid metabolism in vitro in subcutaneous adipose tissue of mature sheep and cattle. Mean adipocyte volume was significantly less in subcutaneous adipose tissue of sheep than in adipose tissue from cattle. The presence of acetate and lactate in the incubation medium increased total glucose utilization two- to three-fold in ovine adipose tissue, but had no effect on total glucose utilization in adipose tissue from cattle. Acetate provided 72–82% of the acetyl units to lipogenesis, depending on species and substrate concentration. There were no significant (P > 0.05) differences in the contribution of the pentose cycle to the provision of reducing equivalents to fatty acid biosynthesis, based on the incorporation of label from [3-3H]glucose into fatty acids. In ovine adipose tissue, acetyl-CoA carboxylase appeared to be rate-limiting to lipogenesis, while in bovine subcutaneous adipose tissue, the activity of fatty acid synthetase may have been the limiting step in lipogenesis. In addition, the low activity of ATP-citrate lyase, especially relative to aconitate hydratase, probably limited the conversion of lactate to fatty acids in ovine adipose tissue. It is unlikely that ATP-citrate lyase activity was rate-limiting to lipogenesis from lactate in bovine adipose tissue. The data indicate that extending the results obtained from adipose tissue from one species to lipid metabolism in ruminants in general may not be valid.

Apparent Inhibition of ATP Citrate Lyase by l‐Glutamate In Vitro Is Due to the Presence of Glutamine Synthetase

Preincubation in assay mixture for 30 min at 37 degrees C of ATP citrate lyase from rat brain and liver results in 65-70% inhibition in the presence of 10 mM L-glutamate. This inhibition is specific since none of the known brain metabolites of glutamate shows this effect. ATP and ammonium sulphate-suspended, commercially purified malate dehydrogenase are both important in the generation of inhibition; citrate and NADH are not. The ATP citrate lyase activity in desalted crude extracts and 11% polyethylene glycol-precipitated fractions is inhibited but the enzyme purified by dye affinity chromatography is unaffected. Such purification reveals the presence of a factor responsible for the generation of the inhibition shown to be of Mr 380,000. These lines of evidence implicate endogenous glutamine synthetase, and the involvement of this enzyme is established by the use of its inhibitor L-methionine sulphoximine and by the addition of purified glutamine synthetase to restore the glutamate inhibition of purified ATP citrate lyase. The phenomenon probably arises from the production by glutamine synthetase of ADP, a known product inhibitor of ATP citrate lyase. Therefore contrary to previous reports elsewhere, L-glutamate has no role in the regulation of brain ATP citrate lyase and thus the supply of cytoplasmic acetyl groups for biosynthesis.

Calcitonin action on ATP citrate lyase activity in the hepatic cytosol of fed rats and its calcium-calmodulin dependency.

The effect of calcitonin (CT) on ATP citrate lyase activity in the hepatic cytosol was investigated after a single subcutaneous administration of the hormone to fed rats. Administration of CT (synthetic [Asu107] eel CT; 80 MRC mU/100 g body weight) produced significant increases in ATP citrate lyase activity and calcium content in the hepatic cytosol of intact and thyroparathyroidectomized rats. Those alterations were also observed with the dose of CT at physiological level. The increased cytosolic ATP citrate lyase activity resulting from CT administration was prevented by treatment with 10 microM EGTA. This enzyme activity was restored by addition of calcium ion (2.5-10 microM). The rise in enzyme activity of CT-treated rats was markedly reduced by the presence of W-7 (10 and 100 microM), a calmodulin inhibitor, in the enzyme assay system, while that of control rats was not significantly altered by the drug. These results suggest that CT increases ATP citrate lyase activity in the hepatic cytosol of fed rats, and that this hormonal regulation may depend on calmodulin, and be mediated through raised calcium in the cytosol.

Testicular lipogenic enzymes — Direct effects of dexamethasone [formula omitted

Specific activities of NADP-Isocitrate dehydrogenase, ATP-citrate lyase, Malate dehydrogenase and Malic enzyme were studied in dexamethasone injected and adrenalectomized prepubertal and adult rat testis. 30 days after adrenalectomy the specific activity of NADP-Isocitrate dehydrogenase increased but the specific activities of the other three enzymes decreased in both age groups. An opposite effect was observed after dexamethasone injection to intact animals. The changes observed in the specific activities of enzymes of adrenalectomized and dexamethasone treated animals reverted back to normal after dexamethasone replacement and withdrawal, respectively in adult animals. However, dexamethasone injected intact prepubertal animals did not revert back to normal after the hormone withdrawal.

Halothane-induced alterations of glucose and pyruvate metabolism in rat cerebra synaptosomes.

Synaptosomes isolated from rat cerebra were used to study the effects of the inhalational anesthetic, halothane, on cholinergic processes. To identify possible mechanisms responsible for the depression of acetylcholine synthesis, we examined the effects of halothane on precursor metabolite metabolism involved with supplying the cytosol with acetyl-CoA for acetylcholine synthesis. Three percent halothane/air (vol/vol) depressed 14CO2 evolution from labeled pyruvate and glucose. Steady-state 14CO2 evolution from [1-14C]glucose was depressed 84% by halothane, while 14CO2 evolution from [6-14C]glucose and [3,4-14C]glucose was decreased 67 and 52%, respectively, when compared with control conditions. Halothane inhibited the activities of both pyruvate dehydrogenase (14% depression) and ATP-citrate lyase (32% depression). Total synaptosomal acetyl-CoA concentrations were unaffected by halothane. Three percent halothane/air (vol/vol) caused a 77% increase in medium glucose depletion rate from 1.38 nmol (mg protein)-1 min-1 to 2.44 nmol (mg protein)-1 min-1. Production of lactate by the synaptosomes in the presence of halothane increased by 231% from a control rate of 1.44 nmol (mg protein)-1 min-1 to 4.77 nmol (mg protein)-1 min-1. Lactate production rate from pyruvate was also enhanced by 56% in the presence of halothane. These data lend support to the concept that the NAD+/NADH potential may be involved in the halothane-induced depression of acetylcholine synthesis.

Carnitine acetyltransferase activity in oleaginous yeasts

The highest activities of carnitine acetyltransferase (CAT) were found in non-oleaginous yeasts (Candida utilis and Saccharomyces cerevisiae); lower activities, ranging from 50% down to 3% of the highest values, were found in various strains of oleaginous yeasts (Candida curvata, Lipomyces starkeyi, Rhodosporidium toruloides and Trichosporon cutaneum). Supply of acetyl units into the cytosol of the latter, but not of the former yeasts, was therefore necessarily reliant on the action of ATP: citrate lyase (ACL), which was present in all oleaginous yeasts. There was no correlation, however, between the amount of lipid in the oleaginous yeasts and the specific activities of either CAT or ACL. Activity of CAT was increased up to 30-fold by growing yeasts on a triacylglycerol.

Intracellular location of ATP citrate lyase in leaves of Pisum sativum L.

The aim of this work was to discover if there is enough ATP citrate lyase (EC 4.1.3.8) in the cytosol of the leaves of Pisum sativum L. to catalyse the synthesis of the acetyl CoA needed for terpenoid synthesis. Estimates of the maximum catalytic activity of the enzyme in leaves of 7-d-old peas gave values of 113 nmol min(-1) g(-1) fresh weight. The rate of carotenoid accumulation in these leaves corresponded to a requirement for acetyl CoA of 0.7 nmol min(-1) g(-1) fresh weight. The distribution of marker enzymes during fractionation of homogenates of leaves from 7 to 10-d-old peas showed that differential centrifugation led to the isolation in reasonable yields of chloroplasts, mitochondria, peroxisomes and the endomembrane system. None of the above components of the leaf contained appreciable detectable activity of ATP citrate lyase, the distribution of which closely paralleled that of the cytosolic marker. It was concluded that in young leaves of pea most of the ATP citrate lyase is in the cytosol.

Effects of phosphorylation on the kinetic properties of rat liver ATP-citrate lyase

Homogeneous rat liver ATP-citrate lyase (EC 4.1.3.8) was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. In agreement with other workers, the maximum level of phosphorylation that we observed was approx. 2 mol/mol of tetramer. Phosphorylated and non-phosphorylated forms of ATP-citrate lyase were prepared. Their kinetic properties were examined using an assay system in which the concentrations of Mg · ATP, magnesium·citrate and CoA were varied systematically at a constant concentration of Mg 2+. The phosphorylated form had a two-fold higher K m for Mg · ATP than did the non-phosphorylated form, but no other kinetic differences between the two forms were detected. When ATP-citrate lyase was assayed at a concentration of Mg · ATP well below K m, it was found that phosphorylation of the enzyme correlated well with a decrease of approx. 50% in its activity This is the first demonstration that phosphorylation can affect the activity of ATP-citrate lyase.

Effect of swine weaning age on body fat and lipogenic activity in liver and adipose tissue.

Two experiments were conducted to evaluate the effects of weaning swine at 2 or 5 wk of age on postweaning performance and fat metabolism. In the first experiment, 52 pigs were weaned at 2 or 5 wk of age with body weights determined from birth to 8 wk. The early weaned group was fed a 20% protein corn-soybean meal-oat diet containing 25% dried whey from 2 to 5 wk while both groups were fed a 20% protein cereal grain-based diet from 5 to 8 wk of age. In a second experiment, a total of 90 pigs weaned at similar ages and fed the same diet sequences were killed at weekly intervals from 2 to 8 wk of age to evaluate body fat content and lipogenesis in liver and adipose tissue. Lipogenic capacity was measured by incorporation of acetate-1 14C into the total lipid fraction in liver slices and adipose tissue minces or by monitoring liver ATP citrate lyase activity. The results demonstrate that pigs weaned at 2 wk experience a slower postweaning growth rate with lower empty body weights than those either concurrently nursing the dam or weaned at 5 wk of age. Both groups had similar body weights from 6 to 8 wk of age. The body fat content of nursing pigs increased from 2 to 5 wk of age. Pigs weaned at 2 wk lost approximately 25% of their body fat the first week postweaning while later-weaned pigs did not lose body fat postweaning. Body fat composition of both groups was similar by 8 wk of age. Lipogenic activity was higher in liver than in adipose tissue from 2 to 5 wk of age and remained relatively constant throughout the trial. Adipose tissue lipogenic activity was lower in the nursing pig but increased dramatically at 5 wk in the early-weaned group and 7 wk of age in the late weaned group. These results suggest that weaning age can affect postweaning body fat composition and that adipose contributes a greater lipogenic capacity than liver tissue as the pig matures.

Development under the control of insulin of lipogenic enzymes, lipoprotein lipase, isoproterenol and glucagon sensitivity in differentiating rat preadipocytes in primary culture

In preadipose cellular fractions (I, II and III) isolated by density gradient centrifugation from the inguinal tissue of young rats, we followed the activity of fatty acid synthetase, ATP citrate lyase and lipoprotein lipase during differentiation in culture. 1.5 nM insulin when added at confluence markedly induced the activity of ATP citrate lyase and fatty acid synthetase in the cells derived from the lighter fractions (I and II). The magnitude of this response was 25–50-fold the initial value 15 days after plating. In the cells of the heaviest fraction (III) both enzymes exhibited low activity which was slightly stimulated by the presence of insulin, VLDL and heparin. In contrast, the activity of lipoprotein lipase appeared before confluence in cells from all three fractions and peaked at day 6 after plating. This early emergence was independent of the addition of insulin to the medium. However, insulin slightly enhanced the peak activity in post-confluent cells. The development of cAMP production in response to isoproterenol (100 μM) and to glucagon (0.3 μM) was determined in the cells of fraction II in the same culture conditions. The responsiveness to isoproterenol was present very early in these cells and rose rapidly during the exponential growth phase, reaching a peak value at day 8 after plating. In contrast, the development of glucagon sensitivity occurred only during late differentiation. The stimulatory effect of glucagon was enhanced when VLDL and heparin were added with insulin to the medium.

Relative Contributions of Acetate, Lactate and Glucose to Lipogenesis in Bovine Intramuscular and Subcutaneous Adipose Tissue

Intramuscular adipose tissue from the fifth–eighth thoracic vertebrae region of the longissimus dorsi muscle, and portions of the overlying subcutaneous adipose tissue, were obtained at 16 and 18 months of age from Angus steers fed ad libitum either a corn silage (low energy) or ground corn (high energy) diet. Carcass weight, backfat thickness, and kidney, pelvic and heart fat were significantly greater in the steers fed the high energy diet; however, there were no significant differences in marbling scores between diet groups. Additionally, feeding steers the high energy diet did not result in differences in adipocyte diameter or number of adipose cells per gram tissue in either adipose tissue depot. Intramuscular adipocytes had a peak diameter (the diameter represented by the greatest number of cells) of 104 ± 2 µm; peak diameter in subcutaneous adipose tissue was 141 ± 5 µm. The activities of ATP-citrate lyase and NADP-malate dehydrogenase increased with age in the subcutaneous adipose tissue; feeding steers the high energy diet increased enzyme activities further. Age and diet had no effect on enzyme activities in intramuscular adipose tissue. A similar pattern was observed for the incorporation of lipogenic precursors into fatty acids. Acetate provided 70–80% of the acetyl units to in vitro lipogenesis in subcutaneous adipose tissue, but only 10–25% in intramuscular adipose tissue. Conversely, glucose provided 1–10% of the acetyl units in subcutaneous adipose tissue, but 50–75% in the intramuscular depot. The contribution of lactate to lipogenesis was similar in both tissues (15–30%). The pentose cycle was estimated to contribute all of the necessary NADPH required for lipogenesis in the younger steers, and 40–85% (depending on diet and tissue site) in the 18-month-old steers. There was no effect of diet on, or difference between adipose tissue depots for, the relative contribution of the pentose cycle to lipogenesis. The data suggest that different regulatory processes control de novo fatty acid synthesis in intramuscular and subcutaneous adipose tissue.

Effect of thyroxine-induced hyperthyroidism on some testicular enzymes of the pyruvate/malate cycle

The effect of thyroxine on the specific activities of testicular enzymes of the pyruvate/malate cycle involved in lipogenesis were studied in prepubertal, pubertal and adult rats. Thyroxine (25 μg/100 g body weight) treatment for 1 month increased the specific activity of isocitrate dehydrogenase (NADP +) but the specific activities of ATP-citrate lyase, malate dehydrogenase and malic enzyme were inhibited. Withdrawal of thyroxine treatment from hyperthyroid rats brought back all enzyme activities to normal. The study reveals a direct, specific influence of thyroxine on different testicular enzymes of the pyruvate/malate cycle.

The pathway of ketogenesis in rumen epithelium of the sheep.

A method for the fractionation of sheep rumen epithelium with limited mitochondrial breakage is described. The distributions of the enzymes of the 3-hydroxy-3-methylglutaryl-CoA pathway of ketogenesis indicate that this process is exclusively mitochondrial. Enzyme activities are sufficient to account for the ketogenic rates found in vivo. The failure of (-)-hydroxycitrate to block ketogenic flux supports this view. 3-Hydroxybutyrate dehydrogenase activity is largely associated with particulate material in the mitochondrial fraction. ATP citrate lyase activity was found, with appreciable acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl-CoA synthase in the cytoplasmic fraction.

Direct influence of dexamethasone invivo on rat testicular lipogenic enzymes involved in pyruvate-malate pathway

The changes in specific activities of NADP-isocitrate dehydrogenase, ATP-citrate lyase, Malate dehydrogenase and malic enzyme were studied in adrenalectomised and dexamethasone injected pubertal rats. One month after adrenalectomy the specific activity of NADP-isocitrate dehydrogenase increased but the specific activities of the other three enzymes decreased. An opposite effect was seen after dexamethasone administration to intact animals. The changes observed in the specific activities of enzymes of adrenalectomised and dexamethasone treated animals reverted back to normal after dexamethasone replacement and withdrawal, respectively.

Lipogenesis and Lipolysis in Normal and Dwarf Chickens from Lines Selected for High and Low Body Weight

Lipogenic and lipolytic comparisons were made among normal and dwarf adult males from both high-weight (HW) and low-weight (LW) selected lines. Six nonfasted birds from each of the four populations were sacrificed, and in vitro lipogenesis and lipolysis were measured in liver, adipose, and bone tissue. Also determined were activities of acetyl CoA carboxylase (E.C.6.4.1.2), NADP-malate dehydrogenase (E.C.1.1.1.40), ATP-citrate lyase (E.C.4.1.3.8), and plasma-free fatty acids (FFA). In comparison with the HW chickens, the LW males tended to exhibit higher activities of lipogenic enzymes, a greater capacity to incorporate acetate-1-14C into liver slices, an increased mobilization of FFA from adipose tissue, and high concentrations of FFA in plasma. The results indicated that the maintenance of increased postmaturational body fat associated with selection for high body weight was primarily the result of decreased lipolysis rather than enhanced lipogenesis. Effects of the dwarf allele on lipogenesis and lipolysis were not clear. Acetate incorporation into bone tissue was substantially higher than for adipose tissue, suggesting that bone may be an important site of lipogenesis in the fowl.

The enzymes of acetyl-CoA metabolism in differentiating cholinergic (s-20) and noncholinergic (NIE-115) neuroblastoma cells.

Dibutyryl cyclic AMP and butyrate inhibited growth of S-20 (cholinergic) and NIE-115 (adrenergic) neuroblastoma clones. Both these drugs resulted in a parallel increase of choline acetyltransferase and ATP-citrate lyase activities in S-20 neuroblastoma cells. On the other hand, the increase in tyrosine hydroxylase activity in NIE-115 caused by these drugs was not accompanied by a significant change in ATP-citrate lyase activity. Both dibutyryl cyclic AMP and butyrate caused a decrease in fatty acid synthetase activity in both cell lines. The activities of pyruvate dehydrogenase, citrate synthase, choline acetyltransferase, and lactate dehydrogenase in both S-20 and NIE-115 cells were not significantly influenced by the drugs. ATP-citrate lyases from S-20 and NIE-115 had similar kinetic and immunological properties, and their subunits had the same molecular weight as the rat liver enzyme. These data indicate that the differential regulation of ATP-citrate lyase activity in cholinergic and adrenergic cells does not result from the existence of different molecular forms of the enzyme in these cell lines. They also provide further evidence to support the hypothesis that ATP-citrate lyase activity increases during maturation of normal cholinergic neurons and decreases in noncholinergic cells of the brain.