Continuous fatty acid oxidation and reduced fat storage in mice lacking acetyl-CoA carboxylase 2.

Malonyl-coenzyme A (malonyl-CoA), generated by acetyl-CoA carboxylases ACC1 and ACC2, is a key metabolite in the regulation of energy homeostasis. Here, we show that Acc2-/- mutant mice have a normal life span, a higher fatty acid oxidation rate, and lower amounts of fat. In comparison to the wild type, Acc2-deficient mice had 10- and 30-fold lower levels of malonyl-CoA in heart and muscle, respectively. The fatty acid oxidation rate in the soleus muscle of the Acc2-/- mice was 30% higher than that of wild-type mice and was not affected by addition of insulin; however, addition of insulin to the wild-type muscle reduced fatty acid oxidation by 45%. The mutant mice accumulated 50% less fat in their adipose tissue than did wild-type mice. These results raise the possibility that pharmacological manipulation of ACC2 may lead to loss of body fat in the context of normal caloric intake.

The subcellular localization of acetyl-CoA carboxylase 2.

Animals, including humans, express two isoforms of acetyl-CoA carboxylase (EC ), ACC1 (M(r) = 265 kDa) and ACC2 (M(r) = 280 kDa). The predicted amino acid sequence of ACC2 contains an additional 136 aa relative to ACC1, 114 of which constitute the unique N-terminal sequence of ACC2. The hydropathic profiles of the two ACC isoforms generally are comparable, except for the unique N-terminal sequence in ACC2. The sequence of amino acid residues 1-20 of ACC2 is highly hydrophobic, suggesting that it is a leader sequence that targets ACC2 for insertion into membranes. The subcellular localization of ACC2 in mammalian cells was determined by performing immunofluorescence microscopic analysis using affinity-purified anti-ACC2-specific antibodies and transient expression of the green fluorescent protein fused to the C terminus of the N-terminal sequences of ACC1 and ACC2. These analyses demonstrated that ACC1 is a cytosolic protein and that ACC2 was associated with the mitochondria, a finding that was confirmed further by the immunocolocalization of a known human mitochondria-specific protein and the carnitine palmitoyltransferase 1. Based on analyses of the fusion proteins of ACC-green fluorescent protein, we concluded that the N-terminal sequences of ACC2 are responsible for mitochondrial targeting of ACC2. The association of ACC2 with the mitochondria is consistent with the hypothesis that ACC2 is involved in the regulation of mitochondrial fatty acid oxidation through the inhibition of carnitine palmitoyltransferase 1 by its product malonyl-CoA.

Human acetyl-CoA carboxylase 2. Molecular cloning, characterization, chromosomal mapping, and evidence for two isoforms.

cDNA encoding the 280-kDa acetyl-CoA carboxylase 2 (ACC2) isoform was isolated from human liver using the polymerase chain reaction. Sequencing the cDNA revealed an open reading frame of 7,449 base pairs (bp) that encode 2,483 amino acids (Mr 279,380). Using 5-kilobase pair cDNA clones as probes, we localized the gene encoding the 280-kDa human carboxylase to chromosome 12q23. When the cDNA of ACC2 was compared with that of ACC1, the nucleotide sequences and the predicted amino acid sequences had about 60 and 80% identity, respectively. Ser77 and Ser79, which were found to be critical for the phosphorylation and subsequent inactivation of rat ACC1 (Ser78 and Ser80 of human ACC1), are conserved in ACC2 and are represented as Ser219 and Ser221, respectively. On the other hand, Ser1200, which is also a phosphorylation site in rat ACC1 (Ser1201 of human ACC1), is not conserved in ACC2. The homology between the amino acid sequences of the two human carboxylases, however, is primarily found downstream of residues Ser78 and Ser81 in human ACC1 and their equivalents, that is Ser219 and Ser221 in ACC2, suggesting that the sequence of the first 218 amino acids at the N terminus of ACC2 represents a unique peptide that accounts, in part, for the variance between the two carboxylases. Using a cDNA probe (400 bp) that encodes the N-terminal amino acid residues of ACC2 in Northern blot analyses of different human and mouse tissues showed that ACC2 is predominantly expressed in liver, heart, and the skeletal muscles. Polyclonal antibodies raised against the N-terminal peptide (amino acid residues 1-220) reacted specifically and equally with human and rat ACC2 carboxylases, confirming the uniqueness of this N-terminal peptide and its conservation in animal ACC2. In addition, we present evidence for the presence of an isoform of ACC2 (Mr 270,000) in human liver that differs from the 280-kDa ACC2 by the absence of 303 nucleotides that encode 101 amino acids in the region between Arg1114 and Asp1215. The regulation and physiological significance of the two ACC2 isoforms remain to be determined.

Human fatty acid synthase: properties and molecular cloning.

Fatty acid synthase (FAS; EC 2.3.1.85) was purified to near homogeneity from a human hepatoma cell line, HepG2. The HepG2 FAS has a specific activity of 600 nmol of NADPH oxidized per min per mg, which is about half that of chicken liver FAS. All the partial activities of human FAS are comparable to those of other animal FASs, except for the beta-ketoacyl synthase, whose significantly lower activity is attributable to the low 4'-phosphopantetheine content of HepG2 FAS. We cloned the human brain FAS cDNA. The cDNA sequence has an open reading frame of 7512 bp that encodes 2504 amino acids (M(r), 272,516). The amino acid sequence of the human FAS has 79% and 63% identity, respectively, with the sequences of the rat and chicken enzymes. Northern analysis revealed that human FAS mRNA was about 9.3 kb in size and that its level varied among human tissues, with brain, lung, and liver tissues showing prominent expression. The nucleotide sequence of a segment of the HepG2 FAS cDNA (bases 2327-3964) was identical to that of the cDNA from normal human liver and brain tissues, except for a 53-bp sequence (bases 3892-3944) that does not alter the reading frame. This altered sequence is also present in HepG2 genomic DNA. The origin and significance of this sequence variance in the HepG2 FAS gene are unclear, but the variance apparently does not contribute to the lower activity of HepG2 FAS.

Human acetyl-CoA carboxylase: characterization, molecular cloning, and evidence for two isoforms.

We have cloned and sequenced the cDNA coding for human HepG2 acetyl-CoA carboxylase (ACC; EC 6.4.1.2). The sequence has an open reading frame of 7038 bp that encode 2346 amino acids (M(r), 264,737). The C-terminal 2.6-kb sequence is very different from that recently reported for human ACC (Ha, J., Daniel, S., Kong, I.-S., Park, C.-K., Tae, H.-J. & Kim, K.-H. [1994] Eur. J. Biochem. 219, 297-306). Northern blot analysis revealed that the ACC mRNA is approximately 10 kb in size and that its level varies among the tissues tested. Evidence is presented to show that the human ACC gene is 200-480 kbp in size and maps to chromosome 17q12. We also provide evidence for the presence of another ACC-like gene with similarly sized mRNA but tissue-specific expression different from that of the ACC gene reported herein. That this second ACC-like gene encodes the 280-kDa carboxylase is not ruled out.

Isolation and chromosomal mapping of genomic clones encoding the human fatty acid synthase gene.

We have isolated and sequenced 0.5- and 3.6-kb cDNA clones that cover the N-terminal and carboxy-terminal regions, respectively, of the human fatty acid synthase. To localize the fatty acid synthase gene and to define its genomic structure, we have also isolated overlapping genomic clones by screening two human YAC libraries with PCR primers derived from the fatty acid synthase cDNA sequences. The DNA inserts in these human fatty acid synthase YACs hybridized with human synthase-specific cDNA probes. Using biotin-labeled Alu-PCR products of the human synthase YACs as probes for fluorescence in situ hybridization, we mapped the fatty acid synthase gene to chromosome 17q25. We also screened a chromosome 17-specific cosmid library with human synthase cDNA probes and isolated 12 cosmids, all of which had EcoRI fragments in common. DNA sequencing of an amplified PCR product from the fatty acid synthase cosmids confirmed that these genomic clones contained expressed fatty acid synthase sequences. Furthermore, the results of Southern analyses suggested that a single 40-kb cosmid clone encompasses the entire coding region of the fatty acid synthase gene. The synthase gene is located on chromosome 17 near the q25 band, which is close to the telomere and could serve as an important marker in analysis of this chromosome.

Antigenic determinants of the 70-kDa subunit of the Ku autoantigen.

Autoantibodies against Ku antigen were found in subsets of sera from patients with rheumatic diseases. The Ku autoantigen was characterized as a DNA-binding protein complex composed of two subunits, 70 and 86 kDa. In this study, we report the amino acid sequences of the 70-kDa subunit that are important for interactions with a monoclonal and autoimmune antibodies. Full-length cDNA and numerous 5' and 3' deletion mutants were expressed in bacteria and the immunoreactivity of the fusion proteins was analyzed by Western blotting. The reactivity of the monoclonal antibody depended on the region between Ile321 and Phe350. Ten autoimmune sera were tested for reactivity with deletion mutants in immunoblots. The reactivity of six sera strongly depended on the C-terminal amino acids and four sera did not show such dependence; however, these C-terminal sequences did not react with the sera when expressed alone. These results strongly suggest the conformational nature of the Ku autoepitopes. Interestingly, the DNA-binding activity of this Ku protein subunit analyzed by Southwestern blot depended on the same C-terminal amino acids that were involved in interactions with autoantibodies, indicating that anti-Ku autoantibodies are directed to conformationally intact Ku antigen. Reactivities of the autoimmune sera with Met1-Arg115, Met116-Val149, and Val149-Arg586 were also observed. These results demonstrate that different amino acid regions can be involved in interactions with autoimmune antibodies.

Effect of sinefungin on macromolecular biosynthesis and cell cycle of Plasmodium falciparum.

The growth of the malarial parasite P. falciparum was arrested by the adenine containing nucleoside sinefungin at the trophozoite stage. The synthesis of DNA, of polyamines and the specific proteins of the schizont stage were completely blocked by the drug. The inhibition of DNA synthesis was not due to a decrease in the amount of DNA polymerase, but to the depletion of polyamines which are required for DNA synthesis.

Effect of polyamines on the activity of malarial alpha-like DNA polymerase.

DNA polymerase from the malarial parasite Plasmodium falciparum required Mg2+ for activity, Putrescine (1 mM) caused a twofold increase in enzyme activity in the presence of a suboptimal concentration of MgCl2 (2 mM). Spermidine (1.5-2.0 mM) or spermine (0.1-0.3 mM) increased the activity of malarial DNA polymerase, in the presence of 2 mM MgCl2, by factors of 6 and 3-5, respectively. The activity of DNA polymerase from calf thymus or from NIH 3T3 cells transformed by the ras oncogene were not stimulated by these polyamines to the same extent. These findings suggest that in malaria-infected erythrocytes, polyamines, at physiological concentrations, serve as a cofactor for the parasitic alpha-like DNA polymerase. Malarial parasites grown in cultured human erythrocytes did not synthesize DNA after treatment with alpha-difluoromethylornithine, which caused polyamine depletion in the infected cells. DNA synthesis was resumed after adding putrescine to the polyamine-depleted cultures. DNA synthesis was also initiated when actinomycin D was added along with putrescine to polyamine-depleted cells. It thus appears that polyamines are essential for the translation of the DNA polymerase mRNA and that polyamines play an important role in regulating the cell cycle of the malarial parasite.

Plasmodium falciparum: properties of an alpha-like DNA polymerase, the key enzyme in DNA synthesis.

An alpha-like DNA polymerase has been identified and characterized in the extracts from the malarial parasite Plasmodium falciparum. The enzyme is sensitive to the specific inhibitors of alpha-DNA polymerase, N-ethylmaleimide and aphidicolin, and is cell-cycle specific. High activity has been found in the schizont, is lower in trophozoites, and has only negligible activity in the ring form. The enzyme has a molecular weight of about Mr 100,000-103,000 estimated by detecting activity in SDS-polyacrylamide electrophoresis and by Bio-Gel filtration. Another active band of a molecular Mr 68,000 was detected by SDS electrophoresis when the enzyme was stored for 2 months at -20 degrees C. The catalytic activity of parasite enzyme was optimal between pH 8 and pH 9. The apparent Michaelis constant for dTTP was 4.3 microM.

Fusion-mediated microinjection of active amine and diamine oxidases into cultured cells: effect on protein and DNA synthesis in chick embryo fibroblasts and in glioma cells.

Serum amine oxidase and/or porcine kidney diamine oxidase were trapped within reconstituted Sendai virus envelopes, and retained their activity. The trapped enzymes that were detected by radioimmunoblots were microinjected into cultured cells by fusion. When diamine oxidase was microinjected into cultured fibroblasts of chick or rat embryos, a temporary arrest in protein and DNA synthesis was observed. The inhibitory effect was more significant when both serum amine oxidase and kidney diamine oxidase were microinjected into those cultured cells. Fibroblasts of either chick or rat embryos transformed by Rous sarcoma virus were more susceptible to the injected enzymes than the normal cultures, showing a complete arrest in protein and DNA synthesis within 4 hours. Similar results were obtained by microinjecting diamine oxidase into cultured glioma cells. The injected enzyme catalyzed the oxidation of intracellular polyamines. The resulting oxidation product (hydrogen peroxide and aminoaldehydes) apparently caused the arrest in the synthesis of macromolecules.

Effect of polyamine depletion on macromolecular synthesis of the malarial parasite, Plasmodium falciparum, cultured in human erythrocytes.

DL-alpha-Difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, prevented the increases in putrescine and spermidine, but not in spermine, in human erythrocytes infected with the malarial parasite Plasmodium falciparum. The addition of putrescine to these polyamine-depleted cultures restored the normal concentrations of spermidine, whereas that of putrescine even exceeded that of the control cultures. DFMO also inhibited the incorporation of radioactive amino acids into the proteins of parasitized erythrocytes. Electrophoresis on polyacrylamide gels revealed that the synthesis of some proteins was completely blocked by DFMO, but the synthesis of others was not affected. DFMO also caused a partial inhibition of RNA synthesis, and DNA synthesis was completely blocked in polyamine-depleted parasitized erythrocytes. It has been suggested that putrescine and/or spermidine are required for the synthesis of certain proteins in parasitized erythrocytes and that at least one of those proteins is related to the synthesis of DNA of the malarial parasite. It appears that polyamines regulate the schizogony process of P. falciparum.

Leishmania tropica major: effect of paromomycin and pentamidine on polyamine levels in the skin of normal and infected mice.

The polyamine content of the skin of BALB/c and C3H mice was determined at intervals, after injecting Leishmania tropica major. In BALB/c mice, putrescine and spermidine levels increased three- to seven-fold; in C3H mice, spontaneous recovery occurred after 3 weeks, accompanied by a reduction in putrescine and spermidine levels. Ornithine decarboxylase activity was negligible in normal, uninfected skin of both BALB/c and C3H mice, but increased steadily during infection. Treatment with drugs that inhibit the growth of leishmanial amastigotes in the skin of mice also reduced polyamine levels and ornithine decarboxylase activity of previously infected skin. There was a close correlation between the therapeutic activity of the drugs and their effect on polyamine content and synthesis. The aminoglycoside paromomycin, which was chemotherapeutically more effective than pentamidine, also had a greater effect on polyamine levels. S-adenosyl-L-Methionine decarboxylase activity in the skin of BALB/c and C3H mice was only slightly affected by the parasites. Polyamine levels and ornithine decarboxylase activity could possibly serve as means for measuring the growth of leishmanial parasites in skin and other tissues and as a measure of the efficacy of anti-leishmanial chemotherapeutics.

Polyamines and the growth of leishmanial parasites.

The relation between the grown of leishmanial parasites and polyamine biosynthesis was studied. Polyamines, mainly putrescine and spermidine, accumulated in macrophages infected with Leishmania tropica major promastigotes grown in vitro. Similar results were obtained, when tissues of BALB/C mice infected with L. tropica major were examined. A consistent increase in cellular putrescine and spermidine levels was observed in infected skin and spleen. With the accumulation of putrescine, a concomitant increase in ornithine decarboxylase activity was detected in growing leishmanial promastigotes and in macrophages supporting the growth of leishmanial amastigotes. An increase in the activity of ornithine decarboxylase was also observed in Leishmania-infected skin and spleen from BALB/C mice.