Biomedical subjects

Salih J Wakil

Publications and source records attributed to Salih J Wakil.

13 recordsLinked to original sources

PubMed2006

Liver-specific deletion of acetyl-CoA carboxylase 1 reduces hepatic triglyceride accumulation without affecting glucose homeostasis.

In animals, liver and white adipose are the main sites for the de novo fatty acid synthesis. Deletion of fatty acid synthase or acetyl-CoA carboxylase (ACC) 1 in mice resulted in embryonic lethality, indicating that the de novo fatty acid synthesis is essential for embryonic development. To understand the importance of de novo fatty acid synthesis and the role of ACC1-produced malonyl-CoA in adult mouse tissues, we generated liver-specific ACC1 knockout (LACC1KO) mice. LACC1KO mice have no obvious health problem under normal feeding conditions. Total ACC activity and malonyl-CoA levels were approximately 70-75% lower in liver of LACC1KO mice compared with that of the WT mice. In addition, the livers of LACC1KO mice accumulated 40-70% less triglycerides. Unexpectedly, when fed fat-free diet for 10 days, there was significant up-regulation of PPARgamma and several enzymes in the lipogenic pathway in the liver of LACC1KO mice compared with the WT mice. Despite the significant up-regulation of the lipogenic enzymes, including a >2-fold increase in fatty acid synthase mRNA, protein, and activity, there was significant decrease in the de novo fatty acid synthesis and triglyceride accumulation in the liver. However, there were no significant changes in blood glucose and fasting ketone body levels. Hence, reducing cytosolic malonyl-CoA and, therefore, the de novo fatty acid synthesis in the liver, does not affect fatty acid oxidation and glucose homeostasis under lipogenic conditions.

Acetyl-CoA Carboxylase↗

PubMed2005

Mutant mice lacking acetyl-CoA carboxylase 1 are embryonically lethal.

Acetyl-CoA carboxylases (ACC1 and ACC2) catalyze the carboxylation of acetyl-CoA to form malonyl-CoA, an intermediate metabolite that plays a pivotal role in the regulation of fatty acid metabolism. We previously reported that ACC2 null mice are viable, and that ACC2 plays an important role in the regulation of fatty acid oxidation through the inhibition of carnitine palmitoyltransferase I, a mitochondrial component of the fatty-acyl shuttle system. Herein, we used gene targeting to knock out the ACC1 gene. The heterozygous mutant mice (Acc1(+/-)) had normal fertility and lifespans and maintained a similar body weight to that of their wild-type cohorts. The mRNA level of ACC1 in the tissues of Acc1(+/-) mice was half that of the wild type; however, the protein level of ACC1 and the total malonyl-CoA level were similar. In addition, there was no difference in the acetate incorporation into fatty acids nor in the fatty acid oxidation between the hepatocytes of Acc1(+/-) mice and those of the wild type. In contrast to Acc2(-/-) mice, Acc1(-/-) mice were not detected after mating. Timed pregnancies of heterozygotes revealed that Acc(-/-) embryos are already undeveloped at embryonic day (E)7.5, they die by E8.5, and are completely resorbed at E11.5. Our previous results of the ACC2 knockout mice and current studies of ACC1 knockout mice further confirm our hypotheses that malonyl-CoA exists in two independent pools, and that ACC1 and ACC2 have distinct roles in fatty acid metabolism.

Acetyl-CoA Carboxylase↗

PubMed2005

Glucose and fat metabolism in adipose tissue of acetyl-CoA carboxylase 2 knockout mice.

Acc2-/- mutant mice, when fed a high-fat/high-carbohydrate (HF/HC) diet, were protected against diet-induced obesity and diabetes. To investigate the role of acetyl-CoA carboxylase 2 (ACC2) in the regulation of energy metabolism in adipose tissues, we studied fatty acid and glucose oxidation in primary cultures of adipocytes isolated from wild-type and Acc2-/- mutant mice fed either normal chow or a HF/HC diet. When fed normal chow, oxidation of [14C]palmitate in adipocytes of Acc2-/- mutant mice was approximately 80% higher than in adipocytes of WT mice, and it remained significantly higher in the presence of insulin. Interestingly, in addition to increased fatty acid oxidation, we also observed increased glucose oxidation in adipocytes of Acc2-/- mutant mice compared with that of WT mice. When fed a HF/HC diet for 4-5 months, adipocytes of Acc2-/- mutant mice maintained a 25% higher palmitate oxidation and a 2-fold higher glucose oxidation than WT mice. The mRNA level of glucose transporter 4 (GLUT4) decreased several fold in the adipose tissue of WT mice fed a HF/HC diet; however, in the adipose tissue of Acc2-/- mutant mice, it was 7-fold higher. Moreover, lipolysis activity was higher in adipocytes of Acc2-/- mutant mice compared with that in WT mice. These findings suggest that continuous fatty acid oxidation in the adipocytes of Acc2-/- mutant mice, combined with a higher level of glucose oxidation and a higher rate of lipolysis, are major factors leading to efficient maintenance of insulin sensitivity and leaner Acc2-/- mutant mice.

Acetyl-CoA Carboxylase↗

PubMed2004

Human fatty acid synthase: structure and substrate selectivity of the thioesterase domain.

Human fatty acid synthase is a large homodimeric multifunctional enzyme that synthesizes palmitic acid. The unique carboxyl terminal thioesterase domain of fatty acid synthase hydrolyzes the growing fatty acid chain and plays a critical role in regulating the chain length of fatty acid released. Also, the up-regulation of human fatty acid synthase in a variety of cancer makes the thioesterase a candidate target for therapeutic treatment. The 2.6-A resolution structure of human fatty acid synthase thioesterase domain reported here is comprised of two dissimilar subdomains, A and B. The smaller subdomain B is composed entirely of alpha-helices arranged in an atypical fold, whereas the A subdomain is a variation of the alpha/beta hydrolase fold. The structure revealed the presence of a hydrophobic groove with a distal pocket at the interface of the two subdomains, which constitutes the candidate substrate binding site. The length and largely hydrophobic nature of the groove and pocket are consistent with the high selectivity of the thioesterase for palmitoyl acyl substrate. The structure also set the identity of the Asp residue of the catalytic triad of Ser, His, and Asp located in subdomain A at the proximal end of the groove.

Amino Acid Sequence↗

PubMed2004

Structure and function of animal fatty acid synthase.

Fatty acid synthase (FAS; EC 2.3.1.85) of animal tissues is a complex multifunctional enzyme consisting of two identical monomers. The FAS monomer (approximately 270 kDa) contains six catalytic activities and from the N-terminus the order is beta-ketoacyl synthase (KS), acetyl/malonyl transacylase (AT/MT), beta-hydroxyacyl dehydratase (DH), enoyl reductase (ER), beta-ketoacyl reductase (KR), acyl carrier protein (ACP), and thioesterase (TE). Although the FAS monomer contains all the activities needed for palmitate synthesis, only the dimer form of the synthase is functional. Both the biochemical analyses and the small-angle neutron-scattering analysis determined that in the dimer form of the enzyme the monomers are arranged in a head-to-tail manner generating two centers for palmitate synthesis. Further, these analyses also suggested that the component activities of the monomer are organized in three domains. Domain I contains KS, AT/MT, and DH, domain II contains ER, KR, and ACP, and domain III contains TE. Approximately one fourth of the monomer protein located between domains I and II contains no catalytic activities and is called the interdomain/core region. This region plays an important role in the dimer formation. Electron cryomicrographic analyses of FAS revealed a quaternary structure at approximately 19 A resolution, containing two monomers (180 x 130 x 75 A) that are separated by about 19 A, and arranged in an antiparallel fashion, which is consistent with biochemical and neutron-scattering data. The monomers are connected at the middle by a hinge generating two clefts that may be the two active centers of fatty acid synthesis. Normal mode analysis predicted that the intersubunit hinge region and the intrasubunit hinge located between domains II and III are highly flexible. Analysis of FAS particle images by using a simultaneous multiple model single particle refinement method confirmed that FAS structure exists in various conformational states. Attempts to get higher resolution of the structure are under way.

Animals↗

PubMed2004

Experimental verification of conformational variation of human fatty acid synthase as predicted by normal mode analysis.

Fatty acid synthase (FAS) is a 550 kDa homodimeric enzyme with multiple functional and structural domains. Normal mode analysis of a previously determined 19 A structure of FAS suggested that this enzyme might assume different conformational states with several distinct hinge movements. We have used a simultaneous multiple-model refinement method to search for the presence of the structural conformers from the electron images of FAS. We have demonstrated that the resulting models observed in the electron images are consistent with the predicted conformational changes. This technique demonstrates the potential of the combination of normal mode analysis with multiple model refinement to elucidate the multiple conformations of flexible proteins. Since each of these structures is based on a more homogeneous particle set, this technique has the potential, provided that sufficient references are used, to improve the resolution of the final reconstructions of single particles from electron cryomicroscopy.

Cryoelectron Microscopy↗

PubMed2004

David Ezra Green.

Explore the source record for details and available documents.

Biochemistry↗

PubMed2003

Acetyl-CoA carboxylase 2 mutant mice are protected against obesity and diabetes induced by high-fat/high-carbohydrate diets.

Malonyl-CoA, generated by acetyl-CoA carboxylases ACC1 and ACC2, is a key metabolite in the control of fatty acid synthesis and oxidation in response to dietary changes. ACC2 is associated to the mitochondria, and Acc2-/- mice have a normal lifespan and higher fatty acid oxidation rate and accumulate less fat. Mutant mice fed high-fat/high-carbohydrate diets weighed less than their WT cohorts, accumulated less fat, and maintained normal levels of insulin and glucose, whereas the WT mice became type-2 diabetic with hyperglycemic and hyperinsulinemic status. Fatty acid oxidation rates in the soleus muscle and in hepatocytes of Acc2-/- mice were significantly higher than those of WT cohorts and were not affected by the addition of insulin. mRNA levels of uncoupling proteins (UCPs) were significantly higher in adipose, heart (UCP2), and muscle (UCP3) tissues of mutant mice compared with those of the WT. The increase in the UCP levels along with increased fatty acid oxidation may play an essential role in the regulation of energy expenditure. Lowering intracellular fatty acid accumulation in the mutant relative to that of the WT mice may thus impact glucose transport by higher GLUT4 activity and insulin sensitivity. These results suggest that ACC2 plays an essential role in controlling fatty acid oxidation and is a potential target in therapy against obesity and related diseases.

Acetyl-CoA Carboxylase↗

PubMed2003

Human acetyl-CoA carboxylase 1 gene: presence of three promoters and heterogeneity at the 5'-untranslated mRNA region.

Acetyl-CoA carboxylase 1 (ACC1) catalyzes the formation of malonyl-CoA, the C2 donor for de novo synthesis of long-chain fatty acids. We have identified 64 exons, including 7 alternatively spliced minor exons (1A, 1B, 1C, 3, 5A', 5A, and 5B) in human ACC1 gene ( approximately 330 kb). The gene is regulated by three promoters (PI, PII, and PIII), which are located upstream of exons 1, 2, and 5A, respectively. PI is a constitutive promoter and has no homology with the PI sequences of other mammalian ACC1. PII is regulated by various hormones. PIII is expressed in a tissue-specific manner. The presence of several alternatively spliced exons does not alter the translation of the 265-kDa ACC1 protein starting from an ATG present in exon 5. Translation of PIII transcripts from exon 5A generates a 259-kDa isoform in which the N-terminal 75 aa of 265-kDa ACC1 are replaced with a new sequence of 17 aa. Interestingly, the inclusion of exon 5B between 5A and 6 in PIII transcripts would yield a third 257-kDa isoform, which is translated from an ATG in exon 6. However, the presence of exon 5B in PI and PII transcripts leads to an in-frame stop codon that results in an ACC1-related 77-aa peptide. The presence of alternatively spliced exons and three isoforms of ACC1 could contribute to overall ACC1 activity either by influencing the mRNA stability and translational efficiency or by increasing the stability and specific activity of the ACC1 protein, respectively.

5' Untranslated Regions↗

PubMed2003

Fatty acid synthesis is essential in embryonic development: fatty acid synthase null mutants and most of the heterozygotes die in utero.

In animals, including humans, the source of long-chain saturated fatty acids is de novo synthesis, which is mediated by fatty acid synthase (FAS), ingested food, or both. To understand the importance of de novo fatty acid synthesis, we generated FAS knockout mice. The heterozygous FAS mutants (Fasn+/-) are ostensibly normal. In Fasn+/- mice the levels of FAS mRNA and the FAS activity are approximately 50% and 35% lower, respectively, than those of WT mice; hence, FAS levels are affected by gene dosage. When the Fasn+/- mutant mice were interbred, Fasn-/- mice were not produced; thus, FAS is essential during embryonic development. Furthermore, the number of Fasn+/- progeny obtained was 70% less than predicted by Mendelian inheritance, indicating partial haploid insufficiency. Even when one of the parents was WT, the estimated loss of heterozygous progeny was 60%. This loss of Fasn+/- pups appeared to be strain-specific and became more pronounced as the heterozygous females produced more litters. Most of the Fasn-/- mutant embryos died before implantation and the Fasn+/- embryos died at various stages of their development. Feeding the breeders a diet rich in saturated fatty acids did not prevent the loss of homoor heterozygotes. These observations are very important in considering teratogenic consequences of drugs aimed at inhibiting FAS activity, to reduce either obesity or the growth of cancerous tissues.

Animals↗

PubMed2003

A Saccharomyces cerevisiae mutant strain defective in acetyl-CoA carboxylase arrests at the G2/M phase of the cell cycle.

To elucidate the essential functions of acetyl-CoA carboxylase (ACC1FAS3) in Saccharomyces cerevisiae, a temperature-sensitive mutant (acc1(ts)) was constructed. When the acc1(ts) cells were synchronized in G(1) phase with alpha-factor at the permissive temperature of 24 degrees C and then released from the blockade and incubated at the restrictive temperature of 37 degrees C, 95% of the cell population became arrested at the G(2)M phase of the cell cycle despite the presence of fatty acids (C(14)-C(26)) in the medium. These cells developed large undivided nuclei, and the spindles of the arrested mutant cells were short. Shifting the G(2) arrested cells back to the permissive temperature resulted in a reversal of the cell-cycle arrest, with cells initiating mitosis. However, after 3 h of incubation at 37 degrees C, G(2) arrested mutant cells lost viability and displayed a uniquely altered nuclear envelope. Using [1-(14)C]acetate as a precursor for fatty acids synthesis, we identified the phospholipids and sphingolipids derived from acc1(ts) cells and wild-type cells at 24 degrees C and 37 degrees C, respectively. The levels of inositol-ceramides [IPC, MIPC, and M(IP)(2)C] and very long-chain fatty acids C(24) and C(26) declined sharply in the G(2)M arrested cells because of ACC inactivation. Shifting the acc1(ts) cells to 24 degrees C after 2 h of incubation at 37 degrees C resulted in reactivation of the ACC and elevation of the ceramides and very long-chain fatty acid syntheses with normal cell-cycle progression. In contrast, synthesis of wild-type inositol-ceramides, C(24) and C(26), fatty acids were elevated on incubation at 37 degrees C and declined when the cells shifted to the permissive temperature of 24 degrees C.

Acetyl-CoA Carboxylase↗

PubMed2002

Domain movements in human fatty acid synthase by quantized elastic deformational model.

This paper reports the results of applying a computational method called the quantized elastic deformational model, to the determination of conformational flexibility of the supermolecular complex of human fatty acid synthase. The essence of this method is the ability to model large-scale conformational changes such as domain movements by treating the protein as an elastic object without the knowledge of protein primary sequence and atomic coordinates. The calculation was based on the electron density maps of the synthase at 19 A. The results suggest that the synthase is a very flexible molecule. Two types of flexible hinges in the structure were identified. One is an intersubunit hinge formed by the intersubunit connection and the other is an intrasubunit hinge located between domains I and II. Despite the fact that the dimeric synthase has a chemically symmetric structure, large domain movements around the hinge region occur in various directions and allow the molecule to adopt a wide range of conformations. These domain movements are likely to be important in facilitating and regulating the entire palmitate synthesis by coordinating the communication between components of the molecule, for instance, adjusting the distance between various active sites inside the catalytic reaction center. Finally, the ability to describe protein motions of a supermolecular complex, without the information of protein sequence and atomic coordinates, is a major advance in computational modeling of protein dynamics. The method provides an unprecedented ability to model protein motions at such a low resolution of structure.

Elasticity↗

PubMed2001

Quaternary structure of human fatty acid synthase by electron cryomicroscopy.

We present the first three-dimensional reconstruction of human fatty acid synthase obtained by electron cryomicroscopy and single-particle image processing. The structure shows that the synthase is composed of two monomers, arranged in an antiparallel orientation, which is consistent with biochemical data. The monomers are connected to each other at their middle by a bridge of density, a site proposed to be the combination of the interdomain regions of the two monomers. Each monomer subunit appears to be subdivided into three structural domains. With this reconstruction of the synthase, we propose a location for the enzyme's two fatty acid synthesis sites.

Binding Sites↗