Utilization and preferred metabolic pathway of ketone bodies for lipid synthesis by isolated rat hepatoma cells

1995 ◽  
Vol 269 (1) ◽  
pp. C22-C27 ◽  
Author(s):  
L. A. Hildebrandt ◽  
T. Spennetta ◽  
C. Elson ◽  
E. Shrago
Keyword(s):  
Metabolic Pathway ◽  
Metabolic Pathways ◽  
Ketone Bodies ◽  
Malignant Tumors ◽  
Lipid Synthesis ◽  
Hepatoma Cells ◽  
Atp Citrate Lyase ◽  
Citrate Lyase ◽  
Morris Hepatoma ◽  
Distribution Studies

Morris hepatoma 7777 cells freshly isolated from highly malignant tumors grown in the hindlimb of buffalo rats actively convert ketone bodies to cholesterol and fatty acids. On the basis of results obtained with (-)-hydroxycitrate, an inhibitor of the ATP citrate lyase enzyme, the metabolic pathway for acetoacetate conversion to lipids is exclusively cytoplasmic, whereas that for 3-hydroxybutyrate involves both extra- and intramitochondrial compartments. Subcellular distribution studies indicated accumulation and compartmentation of 3-hydroxybutyryl CoA primarily in the cytoplasm of hepatoma cells incubated with either ketone body. In contrast, the compartmentation of acetoacetyl CoA is dependent on whether the substrate is acetoacetate or 3-hydroxybutyrate. With acetoacetate, the acetoacetyl CoA is entirely cytoplasmic, whereas with 3-hydroxybutyrate, it is equally divided between the intra- and extramitochondrial compartments. The results are discussed in terms of the known and proposed metabolic pathways for lipid synthesis from ketone bodies, particularly that from 3-hydroxybutyrate.

Enzyme Activities of NADPH-Forming Metabolic Pathways in Normal and Leukemic Leukocytes

1975 ◽  
Vol 21 (7) ◽  
pp. 880-883 ◽  
Author(s):  
Francesco Belfiore ◽  
Vito Borzi ◽  
Luigi Lo Vecchio ◽  
Elena Napoli ◽  
Agata M Rabuazzo
Keyword(s):  
Malate Dehydrogenase ◽  
Metabolic Pathways ◽  
Normal Subjects ◽  
Atp Citrate Lyase ◽  
Phosphogluconate Dehydrogenase ◽  
Myelocytic Leukemia ◽  
Citrate Lyase ◽  
Enzymatic Characteristic ◽  
I Cells ◽  
Low Activity

Abstract With respect to the enzymes of NADPH-forming metabolic pathways in human leukocytes: (a) Glucose-6phosphate dehydrogenase and phosphogluconate dehydrogenase (decarboxylating) were less active in leukocytes (mostly myeloblasts) from eight patients with acute myeloblastic leukemia (I) than in leukocytes (mostly granulocytes) from 16 normal subjects (II) or 16 patients with chronic myelocytic leukemia (III). (b) Of the enzymes of the citrate cleavage pathway, ATP citrate lyase and malate dehydrogenase (decarboxylating) (NADP+) were virtually absent in the cells studied. (c) Isocitrate dehydrogenase (NADP+), aspartate aminotransferase, and alanine aminotransferase, which, together with the much more active malate dehydrogenase, constitute a newly proposed NADPH-forming metabolic cycle, showed a higher activity in I than in II or III, and therefore could compensate, as concerns NADPHgeneration, for the low activity of pentose cycle dehydrogenases. We are not sure whether the enzymatic characteristic of I cells is attributable to their immaturity or to their leukemic nature.

scholarly journals Lipid biosynthesis in liver slices of the foetal guinea pig

1976 ◽  
Vol 154 (1) ◽  
pp. 149-158 ◽  
Author(s):  
C T Jones ◽  
I K Ashton
Keyword(s):  
Phosphoenolpyruvate Carboxykinase ◽  
Fatty Acid Synthetase ◽  
Lipid Synthesis ◽  
Short Chain Fatty Acids ◽  
Acetate Incorporation ◽  
Atp Citrate Lyase ◽  
Acetyl Coa ◽  
Citrate Lyase ◽  
Effect Of Glucose

Lipid synthesis as measured by the incorporation of acetate or 3H2O into slices of foetal liver, is much higher than in slices of adult liver and shows a peak at about two-thirds of gestation. At this time the synthesis from glucose was low and reached a peak 10 days later. The changes in the activity of ATP citrate lyase, which mirrored acetate incorporation, and the effect of glucose and pyruvate on acetate corporation into lipid suggests that some of the lipid synthesis occurs via intramitochondrial acetyl-CoA production from acetate. Despite this, lipid synthesis was not inhibited by (-)-hydroxycitrate. The low rate of synthesis from glucose at two-thirds of gestation is ascribed to the low activity of pyruvate carboxylase at this time and a role for a phosphoenolpyruvate carboxykinase in providing oxaloacetate for lipogenesis is proposed. The activity of fatty acid synthetase broadly agreed with the changes in lipid synthesis, whereas the activity of acetyl-CoA carboxylase was barely sufficient to account for the rates of lipid synthesis in vivo. Acetate and short-chain fatty acids are likely to be the major precursors for lipid synthesis in vivo.

NPM-ALK Mediated Tyrosine Phosphorylation of ATP Citrate Lyase Regulates Lipid Metabolism and Promotes Oncogenesis of Anaplastic Large Cell Lymphoma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 465-465
Author(s):  
Johnvesly Basappa ◽  
Mahmoud A ElAzzouny ◽  
Delphine CM Rolland ◽  
Thirunavukkarasu Velusamy ◽  
Steven Hwang ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Western Blot ◽  
Tyrosine Phosphorylation ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Lipid Synthesis ◽  
Large Cell ◽  
Atp Citrate Lyase ◽  
Citrate Lyase

Abstract Introduction: Anaplastic large cell lymphoma (ALCL) is characterized by recurrent translocations involving anaplastic lymphoma kinase (ALK) and resulting in oncogenic chimeric fusion tyrosine kinases such as NPM-ALK. We integrated mass-spectrometry-driven phosphoproteomics and metabolomics of ALK+ ALCL and discovered that ATP citrate lyase (ACLY) is a candidate substrate of NPM-ALK at residue Y682. ACLY is a critical enzyme responsible for synthesis of acetyl-CoA and connects vital biosynthetic pathways such as carbohydrate and lipid metabolism. Functional regulation of ACLY by tyrosine phosphorylation is unknown and the significance of tyrosine phosphorylation of ACLY on cancer cell growth and metabolism have never been described. Methods: To identify novel substrates of NPM-ALK, we performed phospho-proteomic analyses of 3 ALK+ALCL derived cell lines. The MS-phosphoproteomic data were subjected to custom bioinformatic algorithms to identify highly statistically correlated phosphotyrosine proteins and phosphosites regulated by ALK kinase activity. We generated a phosphospecific antibody against a highly-ranked candidate (pACLY Y682) and utilized it in western blot analyses on ALK+ and ALK- ALCL lymphoma cell lines. Immunohistochemistry (IHC) on well-characterized primary human ALK+ (n=20) and ALK-ALCL biopsies (n=28) were performed. Co-immunoprecipitations (Co-IP) with HA-tagged ACLY-WT and ACLY-Y682F and ALK antibodies were performed and analyzed by western blot using the pACLY Y682 antibody. In vitro kinase assays were performed to investigate the direct role of NPM-ALK in phosphorylation of ACLY at Y682 using GST-tagged ACLY-WT or ACLY-Y682F peptides in the presence or absence of immunocomplexes containing active NPM-ALK or the kinase-defective K210R mutant. To study the impact of Y682F mutant on lipid metabolism, we generated ALCL-derived cell lines stably transduced to express GFP-tagged ACLY-WT or ACLY-Y682F and subjected them to metabolomic analysis for flux, β-oxidation or lipid synthesis using liquid chromatography/tandem mass spectrometry or gas chromatography platforms. The Seahorse XF24 Flux analyzer was used to measure oxygen consumption rates (OCR) in DEL stably expressing ACLY-WT and ACLY-Y682F. Cells stably expressing ACLY-WT and ACLY-Y682F were evaluated for cell proliferation, colony formation and tumor formation in SCID-BEIGE mouse xenograft models. Results: Phosphoproteomic analyses yielded a dataset containing a total of 626 tyrosine phosphoproteins. Bioinformatic analysis identified pACLY Y682 as a novel candidate substrate of NPM-ALK. Western blot analysis corroborated phosphoproteomic studies and demonstrated that phosphorylation of ALCY at Y682 is regulated by NPM-ALK activity. Expression of active NPM-ALK increased the phosphorylation of ACLY at Y682 in HEK293T cells compared to the K210R mutant. In vitro kinase assays demonstrated that NPM-ALK phosphorylates ACLY at Y682. Immunoprecipitation (IP) of ALK and co-IP (HA) demonstrated interaction of NPM-ALK and ACLY in co-transfected HEK293T cell lysates. Stable expression of ACLY-Y682F or pharmacologic antagonism of ALK with crizotinib resulted in decreased ACLY activity, decreased lipid synthesis and increased fatty acid β-oxidation when compared to ACLY-WT. β-oxidation of 13 C-oleic acid-labeled fatty acid demonstrated increased labeling of +2-citrate (p<0.01) and +18-Oleyol carnitine (p<0.001) in ACLY-Y682F cells when compared to ACLY-WT. Similarly, the OCR was significantly increased in cells expressing ACLY-Y682F (p<0.001). IHC studies of ALCL biopsies (ALK+ and ALK-) demonstrated significant correlation between NPM-ALK and ACLY pY682- expression (p<0.01). Expression of ACLY-Y682F in the DEL cell line resulted in dramatically decreased cell proliferation, impaired clonogenic potential as observed by small colony size in colony formation assay and abrogated tumor growth in in vivo xenograft model when compared to ACLY-WT. Conclusion: We identify for the first time a direct tyrosine kinase-mediated mechanism for ACLY regulation. In this regard, our studies demonstrate a novel oncogenic mechanism whereby NPM-ALK controls lipid synthesis and b-oxidation by phosphorylation of ACLY at Y682 and this mechanism contributes to ALCL pathogenesis. Our findings have significant implications for novel therapies targeting tumor metabolism in ALCLs. Disclosures No relevant conflicts of interest to declare.

scholarly journals Changes in lipid synthesis in rat liver during development

1967 ◽  
Vol 102 (3) ◽  
pp. 952-958 ◽  
Author(s):  
F. J. Ballard ◽  
R. W. Hanson
Keyword(s):  
Rat Liver ◽  
Lipid Synthesis ◽  
Adult Liver ◽  
Atp Citrate Lyase ◽  
Adult Rat ◽  
Potential Source ◽  
Liver Slices ◽  
Citrate Lyase ◽  
Glucose Incorporation ◽  
Cleavage Enzyme

1. Lipogenesis, as measured by the incorporation of (14)C-labelled glucose or acetate into fatty acids in liver slices, is high in foetal and adult rat liver but is low in the liver of the suckling rat, especially with glucose as substrate. 2. The rate of synthesis of non-saponifiable lipids from glucose is about 15 times as great in the liver of the 18-day foetus as in adult liver. Activity in the newborn is negligible. 3. Glucose incorporation into fat is strongly concentration-dependent in liver slices from the adult and 2-week-old rat, but less markedly so in liver slices from the foetus. 4. Changes in the activity of hepatic citrate-cleavage enzyme (ATP-citrate lyase) occur in parallel with the changes in the extent of fatty acid formation, supporting the participation of this enzyme in lipogenesis. However, NADP-malate dehydrogenase, a potential source of reduced nucleotide coenzyme for lipogenesis in the adult, could not be detected in foetal rat liver.

scholarly journals Lipogenesis from ketone bodies in rat brain. Evidence for conversion of acetoacetate into acetyl-coenzyme A in the cytosol

1976 ◽  
Vol 156 (3) ◽  
pp. 603-607 ◽  
Author(s):  
M S Patel ◽  
O E Owen
Keyword(s):  
Rat Brain ◽  
Ketone Bodies ◽  
Brain Mitochondria ◽  
Atp Citrate Lyase ◽  
Acetyl Coa ◽  
Cellular Compartment ◽  
Brain Lipids ◽  
Acetyl Coenzyme A ◽  
Citrate Lyase ◽  
Old Rats

The metabolism of acetoacetate via a proposed cytosolic pathway in brain of 1-week-old rats was investigated. (-)-Hydroxycitrate, an inhibitor of ATP citrate lyase, markedly inhibited the incorporation of carbon from labelled glucose and 3-hydroxybutyrate into cerebral lipids, but had no effect on the incorporation of labelled acetate and acetoacetate into brain lipids. Similarly, n-butylmalonate and benzene-1,2,3-tricarboxylate inhibited the incorporation of labelled 3-hydroxybutyrate but not of acetoacetate into cerebral lipids. These inhibitors had no effect on the oxidation to 14CO2 of the labelled substrates used. (-)-Hydroxycitrate decreased the incorporation of 3H from 3H2O into cerebral lipids by slices metabolizing either glucose or 3-hydroxybutyrate, but not in the presence of acetoacetate. (-)-Hydroxycitrate also differentially inhibited the incorporation of [2-14C]-leucine and [U-14C]leucine into cerebral lipids. The data show that, although the acetyl moiety of acetyl-CoA generated in brain mitochondria is largely translocated as citrate from these organelles to the cytosol, a cytosolic pathway exists by which acetoacetate is converted directly into acetyl-COA in this cellular compartment.

scholarly journals ACSS2-mediated acetyl-CoA synthesis from acetate is necessary for human cytomegalovirus infection

2017 ◽  
Vol 114 (8) ◽  
pp. E1528-E1535 ◽  
Author(s):  
Anna Vysochan ◽  
Arjun Sengupta ◽  
Aalim M. Weljie ◽  
James C. Alwine ◽  
Yongjun Yu
Keyword(s):  
Human Cytomegalovirus ◽  
Mammalian Cells ◽  
Human Fibroblasts ◽  
Lipid Synthesis ◽  
Atp Citrate Lyase ◽  
Acetyl Coa ◽  
Glucose Carbon ◽  
Citrate Lyase ◽  
Infected Cells ◽  
Human Cytomegalovirus Infection

Recent studies have shown that human cytomegalovirus (HCMV) can induce a robust increase in lipid synthesis which is critical for the success of infection. In mammalian cells the central precursor for lipid biosynthesis, cytosolic acetyl CoA (Ac-CoA), is produced by ATP-citrate lyase (ACLY) from mitochondria-derived citrate or by acetyl-CoA synthetase short-chain family member 2 (ACSS2) from acetate. It has been reported that ACLY is the primary enzyme involved in making cytosolic Ac-CoA in cells with abundant nutrients. However, using CRISPR/Cas9 technology, we have shown that ACLY is not essential for HCMV growth and virally induced lipogenesis. Instead, we found that in HCMV-infected cells glucose carbon can be used for lipid synthesis by both ACLY and ACSS2 reactions. Further, the ACSS2 reaction can compensate for the loss of ACLY. However, in ACSS2-KO human fibroblasts both HCMV-induced lipogenesis from glucose and viral growth were sharply reduced. This reduction suggests that glucose-derived acetate is being used to synthesize cytosolic Ac-CoA by ACSS2. Previous studies have not established a mechanism for the production of acetate directly from glucose metabolism. Here we show that HCMV-infected cells produce more glucose-derived pyruvate, which can be converted to acetate through a nonenzymatic mechanism.

scholarly journals The role of ATP citrate-lyase in the metabolic regulation of plasma lipids

1998 ◽  
Vol 334 (1) ◽  
pp. 113-119 ◽  
Author(s):  
Nigel J. PEARCE ◽  
John W. YATES ◽  
Theo A. BERKHOUT ◽  
Brian JACKSON ◽  
David TEW ◽  
...  
Keyword(s):  
Plasma Lipids ◽  
De Novo ◽  
Plasma Cholesterol ◽  
Low Density Lipoprotein ◽  
Lipid Synthesis ◽  
Density Lipoprotein ◽  
Acid Synthesis ◽  
Atp Citrate Lyase ◽  
Cholesterol Levels ◽  
Citrate Lyase

ATP citrate (pro-S)-lyase (EC 4.1.3.8), a cytosolic enzyme that generates acetyl-CoA for cholesterol and fatty acid synthesis de novo, is a potential target for hypolipidaemic intervention. Here we describe the biological effects of the inhibition of ATP citrate-lyase on lipid metabolism in Hep G2 cells, and plasma lipids in rats and dogs, by using SB-204990, the cell-penetrant γ-lactone prodrug of the potent ATP citrate-lyase inhibitor SB-201076 (Ki = 1 µM). Consistent with an important role of ATP citrate-lyase in the supply of acetyl-CoA units for lipid synthesis de novo, SB-204990 inhibited cholesterol synthesis and fatty acid synthesis in Hep G2 cells (dose-related inhibition of up to 91% and 82% respectively) and rats (76% and 39% respectively). SB-204990, when administered orally to rats, was absorbed into the systemic circulation; pharmacologically relevant concentrations of SB-201076 were recovered in the liver. When administered in the diet (0.05–0.25%, w/w) for 1 week, SB-204990 caused a dose-related decrease in plasma cholesterol (by up to 46%) and triglyceride levels (by up to 80%) in rats. This hypolipidaemic effect could be explained, at least in part, by a decrease (up to 48%) in hepatic very-low-density lipoprotein (VLDL) production as measured by the accumulation of VLDL in plasma after injection of Triton WR-1339. SB-204990 (25 mg/kg per day) also decreased plasma cholesterol levels (by up to 23%) and triglyceride levels (by up to 38%) in the dog, preferentially decreasing low-density lipoprotein compared with high-density lipoprotein cholesterol levels. Overall these results are consistent with the concept that ATP citrate-lyase is an important enzyme in controlling substrate supply for lipid synthesis de novo and a potential enzyme target for hypolipidaemic intervention.

New Therapies for Primary Hyperlipidaemia

2021 ◽  
Author(s):  
Carlos A Aguilar-Salinas ◽  
Rita A Gómez-Díaz ◽  
Pablo Corral
Keyword(s):  
Metabolic Pathways ◽  
Real Life ◽  
Lipid Lowering ◽  
The Novel ◽  
Recurrent Pancreatitis ◽  
Atp Citrate Lyase ◽  
Citrate Lyase ◽  
Family Aggregation ◽  
Primary Hyperlipidemia ◽  
Primary Hyperlipidaemia

Abstract Primary hyperlipidemias include a heterogeneous set of monogenic and polygenic conditions characterized by a strong family aggregation, severe forms of hypercholesterolemia and/or hypertriglyceridemia, appearance early on life and a high risk of cardiovascular events and/or recurrent pancreatitis. In real life, a small proportion of the primary hyperlipidemia cases is recognized and treated properly. Our goal is to present an update of current and upcoming therapies for patients with primary hyperlipidemia. Recently, new lipid lowering medications have obtained FDA and/or EMA authorization. These drugs target metabolic pathways, including (ATP)-citrate lyase (bempedoic acid), PCSK9 (inclisiran), apo CIII (volanesorsen) and ANGPTL3 (volanesorsen), that have additive effects with the actions of the currently available therapies (i.e. statins, ezetimibe or fibrates). We discuss the potential clinical indications for the novel medications. To conclude, the addition of these new medications to the therapeutic options for primary hyperlipidemia patients may increase the likelihood to achieve the treatment targets. Also, it could be a safer alternative for subjects with side effects for the currently available drugs.

Die Anämie bei malignen Tumorerkrankungen

1989 ◽  
Vol 28 (06) ◽  
pp. 247-254
Author(s):  
E. Aulbert
Keyword(s):  
Malignant Tumors ◽  
Gradient Centrifugation ◽  
Tumor Resection ◽  
Hemoglobin Concentration ◽  
Limiting Factor ◽  
Novikoff Hepatoma ◽  
Tumor Mass ◽  
Lysosomal Fraction ◽  
Proliferation Activity ◽  
Morris Hepatoma

The cellular uptake and lysosomal accumulation of 67Ga-labelled transferrin within tumors of different malignancy were examined using tissue fractionation and immunological techniques. As tumor models the slowly growing Morris hepatoma 5123C, the moderately growing Novikoff hepatoma and the fast and aggressive Yoshida hepatoma AH 130 were investigated. Isolation of subcellular fractions of tumor homogenates was performed by differential centrifugation and density-gradient centrifugation. The intracellular 67Gatransferrin was found to be highly concentrated within the purified lysosomes. The transferrin within the lysosomal fraction was identified by radial immunodiffusion technique using monospecific antiserum. The accumulation of 67Gatransferrin by the tumors resulted in a faster disappearance of 67Ga-transferrin from the blood. This loss of circulating 67Ga-transferrin correlated with the proliferation activity and the spread of the tumors. Since transferrin is indispensible for the utilization of iron by the heme-synthesizing red cell precursors, transferrin concentration in the blood is the limiting factor for the utilization of iron in hemoglobin synthesis. Thus, in a further series of experiments we investigated the development of anemia in tumor-bearing rats. With increasing tumor mass a progressive fall of hemoglobin concentration was found. The anemia was more severe in the faster growing Novikoff hepatoma than in the slowly growing Morris hepatoma. The most significant reduction of hemoglobin concentration was found in the very fast growing Yoshida hepatoma. After total tumor resection hemoglobin concentration and red blood cell count normalized completely within 6-8 weeks. We conclude from these data that the uptake of transferrin by the tumor cells results in a faster disappearance of transferrin from the blood. This loss of circulating transferrin correlates with tumor mass and proliferation activity and is one of the factors responsible for the anemia seen in patients with malignant tumors.

Die Anämie bei malignen Tumorerkrankungen

1989 ◽  
Vol 28 (05) ◽  
pp. 193-200 ◽  
Author(s):  
E. Aulbert
Keyword(s):  
Cellular Uptake ◽  
Tumor Tissue ◽  
Malignant Tumors ◽  
The Other ◽  
Novikoff Hepatoma ◽  
Cellular Accumulation ◽  
Tumor Mass ◽  
Proliferation Activity ◽  
Morris Hepatoma ◽  
Cellular Incorporation

Cellular uptake of 67Ga-labelled transferrin by the tumor tissue was studied in rats with tumors of different malignancy and different tumor mass using the slowly growing Morris hepatoma 5123C, the moderately growing Novikoff hepatoma and the very fast and aggressive Yoshida hepatoma AH130. The cellular accumulation of 67Ga-transferrin was found to correlate with the proliferation activity of the tumor. The 67Ga-transferrin concentration in the very fast growing Yoshida hepatoma was 4.8 times higher than the concentration in the slowly growing Morris hepatoma. The uptake of 67Ga-transferrin by the tumors resulted in a faster disappearance of circulating 67Ga-transferrin from the blood. The rate of disappearance correlated with the proliferation activity and the spread of the tumors. Using tumors of identical size the elimination of 67Ga-transferrin from the blood was much faster in the rats with Yoshida hepatoma than in those with the slowly growing Morris hepatoma. On the other hand, using tumors of different tumor size it could be demonstrated that the rate of disappearance of 67Ga-transferrin from the blood correlated directly with tumor mass. It is concluded that cellular incorporation of transferrin within the tumor cells results in a loss of circulating transferrin, which correlates with tumor mass and proliferation of tumor. This mechanism is supposed to be the cause for the hypotransferrinemia seen in patients with malignant tumors.

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