Enhancement of docosahexaenoic acid production by overexpression of ATP-citrate lyase and acetyl-CoA carboxylase in Schizochytrium sp.

Abstract Withaferin A (WA) is a promising phytochemical exhibiting in vitro and in vivo anticancer activities against prostate and other cancers, but the mechanism of its action is not fully understood. In this study, we performed RNA-seq analysis using 22Rv1 human prostate cancer cell line to identify mechanistic targets of WA. Kyoto Encyclopedia of Genes and Genomes pathway analysis of the differentially expressed genes showed most significant enrichment of genes associated with metabolism. These results were validated using LNCaP and 22Rv1 human prostate cancer cells and Hi-Myc transgenic mice as models. The intracellular levels of acetyl-CoA, total free fatty acids and neutral lipids were decreased significantly following WA treatment in both cells, which was accompanied by downregulation of mRNA (confirmed by quantitative reverse transcription-polymerase chain reaction) and protein levels of key fatty acid synthesis enzymes, including ATP citrate lyase, acetyl-CoA carboxylase 1, fatty acid synthase and carnitine palmitoyltransferase 1A. Ectopic expression of c-Myc, but not constitutively active Akt, conferred a marked protection against WA-mediated suppression of acetyl-CoA carboxylase 1 and fatty acid synthase protein expression, and clonogenic cell survival. WA was a superior inhibitor of cell proliferation and fatty acid synthesis in comparison with known modulators of fatty acid metabolism including cerulenin and etomoxir. Intraperitoneal WA administration to Hi-Myc transgenic mice (0.1 mg/mouse, three times/week for 5 weeks) also resulted in a significant decrease in circulating levels of total free fatty acids and phospholipids, and expression of ATP citrate lyase, acetyl-CoA carboxylase 1, fatty acid synthase and carnitine palmitoyltransferase 1A proteins in the prostate in vivo.

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Characterization of the phosphorylation of rat mammary ATP-citrate lyase and acetyl-CoA carboxylase by Ca2+ and calmodulin-dependent multiprotein kinase and Ca2+ and phospholipid-dependent protein kinase

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1986.tb09702.x ◽

1986 ◽

Vol 157 (3) ◽

pp. 553-561 ◽

Cited By ~ 22

Author(s):

D. Grahame HARDIE ◽

David CARLING ◽

Stefano FERRARI ◽

Paul S. GUY ◽

Alistair AITKEN

Keyword(s):

Protein Kinase ◽

Acetyl Coa Carboxylase ◽

Atp Citrate Lyase ◽

Dependent Protein Kinase ◽

Acetyl Coa ◽

Citrate Lyase ◽

Dependent Protein

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Studies on insulin-stimulated phosphorylation of acetyl-CoA carboxylase, ATP citrate lyase and other proteins in rat epididymal adipose tissue. Evidence for activation of a cyclic AMP-independent protein kinase

Biochemical Journal ◽

10.1042/bj2180733 ◽

1984 ◽

Vol 218 (3) ◽

pp. 733-743 ◽

Cited By ~ 34

Author(s):

R W Brownsey ◽

N J Edgell ◽

T J Hopkirk ◽

R M Denton

Keyword(s):

Protein Kinase ◽

Cyclic Amp ◽

High Speed ◽

Kinase Activity ◽

Epididymal Adipose Tissue ◽

Protein Kinase Activity ◽

Acetyl Coa Carboxylase ◽

Atp Citrate Lyase ◽

Acetyl Coa ◽

Citrate Lyase

Protein kinase activity in high-speed supernatant fractions prepared from rat epididymal adipose tissue previously incubated in the absence or presence of insulin was investigated by following the incorporation of 32P from [gamma-32P]ATP into phosphoproteins separated by sodium dodecyl sulphate/polyacrylamide-gel electro-phoresis. Incorporation of 32P into several endogenous proteins in the supernatant fractions from insulin-treated tissue was significantly increased. These included acetyl-CoA carboxylase and ATP citrate lyase (which exhibit increased phosphorylation within fat-cells exposed to insulin), together with two unknown proteins of subunit Mr 78000 and 43000. The protein kinase activity increased by insulin was distinct from cyclic AMP-dependent protein kinase, was not dependent on Ca2+ and was not appreciably affected by dialysis or gel filtration. The rate of phosphorylation of added purified fat-cell acetyl-CoA carboxylase and ATP citrate lyase was also increased by 60-90% in high-speed-supernatant fractions prepared from insulin-treated tissue. No evidence for any persistent changes in phosphoprotein phosphatase activity was found. It is concluded that insulin action on acetyl-CoA carboxylase, ATP citrate lyase and other intracellular proteins exhibiting increased phosphorylation involves an increase in cyclic AMP-independent protein kinase activity in the cytoplasm. The possibility that the increase reflects translocation from the plasma membrane, perhaps after phosphorylation by the protein tyrosine kinase associated with insulin receptors, is discussed.

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Molecular biology of acetyl-CoA metabolism

Biochemical Society Transactions ◽

10.1042/bst0280591 ◽

2000 ◽

Vol 28 (6) ◽

pp. 591-593 ◽

Cited By ~ 11

Author(s):

B. J. Nikolau ◽

D. J. Oliver ◽

P. S. Schnable ◽

E. S. Wurtele

Keyword(s):

Pyruvate Dehydrogenase ◽

Pyruvate Dehydrogenase Complex ◽

Pyruvate Decarboxylase ◽

Acetyl Coa Carboxylase ◽

Atp Citrate Lyase ◽

Acetyl Coa ◽

Acetaldehyde Dehydrogenase ◽

Citrate Lyase ◽

Biochemical Genetic ◽

Dehydrogenase Complex

We have characterized the expression of potential acetyl-CoA-generating genes (acetyl-CoA synthetase, pyruvate decarboxylase, acetaldehyde dehydrogenase, plastidic pyruvate dehydrogenase complex and ATP-citrate lyase), and compared these with the expression of acetyl-CoA-metabolizing genes (heteromeric and homomeric acetyl-CoA carboxylase). These comparisons have led to the development of testable hypotheses as to how distinct pools of acetyl-CoA are generated and metabolized. These hypotheses are being tested by combined biochemical, genetic and molecular biological experiments, which is providing insights into how acetyl-CoA metabolism is regulated.

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Glucocorticoid regulation of fatty acid synthase gene expression in fetal rat lung

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1993.265.2.l140 ◽

1993 ◽

Vol 265 (2) ◽

pp. L140-L147 ◽

Cited By ~ 6

Author(s):

Z. X. Xu ◽

W. Stenzel ◽

S. M. Sasic ◽

D. A. Smart ◽

S. A. Rooney

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthase ◽

Fatty Acid Biosynthesis ◽

Fetal Lung ◽

Acetyl Coa Carboxylase ◽

Rat Lung ◽

Acid Biosynthesis ◽

Atp Citrate Lyase ◽

Acetyl Coa ◽

Citrate Lyase

There are developmental and glucocorticoid-induced increases in the rate of fatty acid biosynthesis and in the activity of fatty acid synthase in late gestation fetal lung. We have now measured mRNA levels of fatty acid synthase and of two other enzymes of fatty acid biosynthesis, ATP citrate lyase and acetyl-CoA carboxylase, in developing fetal and postnatal rat lung and in fetal lung explants cultured with and without dexamethasone. There was a developmental increase in the mRNA for fatty acid synthase with the maximum level being reached on fetal day 21 (term is fetal day 22). This profile was similar to that reported for de novo fatty acid synthesis and fatty acid synthase activity. There was a similar but less pronounced developmental increase in the mRNA for ATP citrate lyase and a corresponding increase in its activity. There was no developmental change in the mRNA for acetyl-CoA carboxylase. Dexamethasone increased the level of fatty acid synthase mRNA approximately threefold but had no effect on those for ATP citrate lyase and acetyl-CoA carboxylase. The effect of dexamethasone on fatty acid synthase mRNA was rapid, biphasic, and partly inhibited by actinomycin D and cycloheximide. We conclude that glucocorticoids increase expression of the gene for fatty acid synthase in fetal lung. The effect of the hormone appears to be due to increased transcription and post-transcriptional events and is dependent on protein synthesis.

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Variations in the activity of several enzymes in the mammary glands of non-pregnant, pregnant and lactating rabbits

Biochemical Journal ◽

10.1042/bj1160657 ◽

1970 ◽

Vol 116 (4) ◽

pp. 657-661 ◽

Cited By ~ 8

Author(s):

P. E. Hartmann ◽

E. A. Jones

Keyword(s):

Mammary Gland ◽

Small Mammals ◽

Enzyme Activities ◽

Mammary Glands ◽

Acetyl Coa Carboxylase ◽

Atp Citrate Lyase ◽

Acetyl Coa ◽

Phosphogluconate Dehydrogenase ◽

Citrate Lyase ◽

Lactation Cycle

1. The enzymes phosphofructokinase (EC 2.7.1.11), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), phosphoglucomutase (EC 2.7.5.1), ATP–citrate lyase (EC 4.1.3.8), acetyl-CoA carboxylase (EC 6.4.1.2) and acetyl-CoA synthetase (EC 6.2.1.1) were assayed in rabbit mammary glands at various stages of the pregnancy–lactation cycle. 2. The activities of all enzymes were low during pregnancy and, with the exception of phosphofructokinase, in non-pregnant animals. Two- to ten-fold increases in enzyme activities occurred over the first 20 days of lactation. Although milk yield was considerably decreased, the enzyme activities remained elevated in late lactation (45 days after parturition). 3. These findings are discussed in relation to mammary-gland metabolism and compared with similar observations previously made on ruminants and other small mammals.

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Activities of Hepatic ATP-citrate Lyase and Acetyl CoA Carboxylase in Herbivorous Voles and C57BL/6J Mice

EXPERIMENTAL ANIMALS ◽

10.1538/expanim1978.40.4_565 ◽

1991 ◽

Vol 40 (4) ◽

pp. 565-567

Author(s):

Yoshiko HASEGAWA ◽

Toshiro ARAI ◽

Mie SHIOMI ◽

Minoru SASAKI ◽

Yoshio OKI

Keyword(s):

Acetyl Coa Carboxylase ◽

Atp Citrate Lyase ◽

Acetyl Coa ◽

Citrate Lyase

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Zonation of hepatic lipogenic enzymes identified by dual-digitonin-pulse perfusion

Biochemical Journal ◽

10.1042/bj2590821 ◽

1989 ◽

Vol 259 (3) ◽

pp. 821-829 ◽

Cited By ~ 17

Author(s):

J L Evans ◽

B Quistorff ◽

L A Witters

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthesis ◽

Fatty Acid Synthase ◽

Acid Synthesis ◽

Male Rats ◽

Male Rat ◽

Acetyl Coa Carboxylase ◽

Atp Citrate Lyase ◽

Acetyl Coa ◽

Citrate Lyase

The zonal distribution within rat liver of acetyl-CoA carboxylase, ATP citrate-lyase and fatty acid synthase, the principal enzymes of fatty acid synthesis, was investigated by using dual-digitonin-pulse perfusion. Analysis of enzyme mass by immunoblotting revealed that, in normally feeding male rats, the periportal/perivenous ratio of acetyl-CoA carboxylase mass was 1.9. The periportal/perivenous ratio of ATP citrate-lyase mass was 1.4, and fatty acid synthase exhibited the largest periportal/perivenous mass gradient, having a ratio of 3.1. This pattern of enzyme distribution was observed in male rats only; in females, the periportal/perivenous ratio of enzyme mass was nearly equal. The periportal/perivenous gradients for acetyl-CoA carboxylase, ATP citrate-lyase and fatty acid synthase observed in fed (and fasted) males were abolished when animals were fasted (48 h) and refed (30 h) with a high-carbohydrate/low-fat diet. As determined by enzyme assay of eluates obtained from the livers of normally feeding male rats, there is also periportal zonation of acetyl-CoA carboxylase activity, expressed either as units per mg of eluted protein or units per mg of acetyl-CoA carboxylase protein, suggesting the existence of gradients in both enzyme mass and specific activity. From these results, we conclude that the enzymes of fatty acid synthesis are zonated periportally in the liver of the normally feeding male rat.

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The role of phosphorylation in the regulation of fatty acid synthesis by insulin and other hormones

Philosophical Transactions of the Royal Society of London Series B Biological Sciences ◽

10.1098/rstb.1983.0036 ◽

1983 ◽

Vol 302 (1108) ◽

pp. 33-45 ◽

Cited By ~ 13

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthesis ◽

Pyruvate Dehydrogenase ◽

Kinase Activity ◽

Acid Synthesis ◽

Fat Cells ◽

Acetyl Coa Carboxylase ◽

Atp Citrate Lyase ◽

Acetyl Coa ◽

Citrate Lyase

Insulin stimulates fatty acid synthesis in white and brown fat cells as well as in liver and mammary tissue. Hormones that increase cellular cyclic AMP concentrations inhibit fatty acid synthesis, at least in white adipose tissue and liver. These changes in fatty acid synthesis occur within minutes. In white fat cells, they are brought about not only by changes in glucose transport but also changes in the activities of pyruvate kinase, pyruvate dehydrogenase and acetyl-CoA carboxylase. The basis of the alterations in pyruvate kinase activity in fat cells is not understood. Unlike the liver isoenzyme, the isoenzyme present in fat cells does not appear to be phosphorylated either in the absence or presence of hormones. The changes in pyruvate dehydrogenase activity in fat cells are undoubtedly due to changes in phosphorylation of the α subunits. Insulin appears to act by causing the parallel dephosphorylation of all three sites. The persistence of the effect of insulin during the preparation and subsequent incubation of mitochondria has allowed the demonstration that insulin acts mainly by stimulating pyruvate dehydrogenase phosphatase rather than inhibiting the kinase. Acetyl-CoA carboxylase within fat cells is phosphorylated on a number of different sites. The exposure of cells to insulin leads to activation of the enzyme and this is associated with increased phosphorylation of a specific site on the enzyme. Exposure to adrenalin, which results in a marked diminution in activity, also causes a small increase in the overall level of phosphorylation, but this increase is due to an enhanced phosphorylation of different sites; probably those phosphorylated by cyclic-AMP-dependent protein kinase. Acetyl-CoA carboxylase is one of a number of proteins in fat cells that exhibit increased phosphorylation with insulin. Others include ATP-citrate lyase, the ribosomal protein S 6 , the β subunit of the insulin receptor and a heat and acid stable protein of M r 22 000. Changes in phosphorylation of ATP-citrate lyase do not appear to result in any appreciable changes in catalytic activity. A central aspect of insulin action may be the activation and perhaps release of a membrane-associated protein kinase. Plasma membranes from fat cells have been shown to contain a cyclicnucleotide-independent kinase able to phosphorylate and activate acetyl-CoA carboxylase. Furthermore, high-speed supernatant fractions from cells previously exposed to insulin contain elevated levels of the same or similar kinase activity capable of phosphorylating both ATP-citrate lyase and acetyl-CoA carboxylase.

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