Salih J. Wakil

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.

Continuous fat oxidation in acetyl–CoA carboxylase 2 knockout mice increases total energy expenditure, reduces fat mass, and improves insulin sensitivity

Acetyl–CoA carboxylase 2 (ACC)2 is a key regulator of mitochondrial fat oxidation. To examine the impact of ACC2 deletion on whole-body energy metabolism, we measured changes in substrate oxidation and total energy expenditure in Acc2−/− and WT control mice fed either regular or high-fat diets. To determine insulin action in vivo, we also measured whole-body insulin-stimulated liver and muscle glucose metabolism during a hyperinsulinemic–euglycemic clamp in Acc2−/− and WT control mice fed a high-fat diet. Contrary to previous studies that have suggested that increased fat oxidation might result in lower glucose oxidation, both fat and carbohydrate oxidation were simultaneously increased in Acc2−/− mice. This increase in both fat and carbohydrate oxidation resulted in an increase in total energy expenditure, reductions in fat and lean body mass and prevention from diet-induced obesity. Furthermore, Acc2−/− mice were protected from fat-induced peripheral and hepatic insulin resistance. These improvements in insulin-stimulated glucose metabolism were associated with reduced diacylglycerol content in muscle and liver, decreased PKCθ activity in muscle and PKCε activity in liver, and increased insulin-stimulated Akt2 activity in these tissues. Taken together with previous work demonstrating that Acc2−/− mice have a normal lifespan, these data suggest that Acc2 inhibition is a viable therapeutic option for the treatment of obesity and type 2 diabetes.

Fatty acid metabolism: target for metabolic syndrome

Fatty acids are a major energy source and important constituents of membrane lipids, and they serve as cellular signaling molecules that play an important role in the etiology of the metabolic syndrome. Acetyl-CoA carboxylases 1 and 2 (ACC1 and ACC2) catalyze the synthesis of malonyl-CoA, the substrate for fatty acid synthesis and the regulator of fatty acid oxidation. They are highly regulated and play important roles in the energy metabolism of fatty acids in animals, including humans. They are presently considered as an attractive target to regulate the human diseases of obesity, diabetes, cancer, and cardiovascular complications. In this review we discuss the role of fatty acid metabolism and its key players, ACC1 and ACC2, in animal evolution and physiology, as related to health and disease.

Activation of nuclear receptor CAR ameliorates diabetes and fatty liver disease

Constitutive androstane receptor CAR (NR1I3) has been identified as a central mediator of coordinate responses to xenobiotic and endobiotic stress. Here we use leptin-deficient mice (ob/ob) and ob/ob, CAR−/− double mutant mice to identify a metabolic role of CAR in type 2 diabetes. Activation of CAR significantly reduces serum glucose levels and improves glucose tolerance and insulin sensitivity. Gene expression analyses and hyperinsulinemic euglycemic clamp results suggest that CAR activation ameliorates hyperglycemia by suppressing glucose production and stimulating glucose uptake and usage in the liver. In addition, CAR activation dramatically improves fatty liver by both inhibition of hepatic lipogenesis and induction of β-oxidation. We conclude that CAR activation improves type 2 diabetes, and that these actions of CAR suggest therapeutic approaches to the disease.

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 ≈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.

A Small Molecule That Blocks Fat Synthesis By Inhibiting the Activation of SREBP

Sterol regulatory element binding proteins (SREBPs) are transcription factors that activate transcription of the genes involved in cholesterol and fatty acid biosynthesis. In the present study, we show that a small synthetic molecule we previously discovered to block adipogenesis is an inhibitor of the SREBP activation. The diarylthiazole derivative, now called fatostatin, impairs the activation process of SREBPs, thereby decreasing the transcription of lipogenic genes in cells. Our analysis suggests that fatostatin inhibits the ER-Golgi translocation of SREBPs through binding to their escort protein, the SREBP cleavage-activating protein (SCAP), at a distinct site from the sterol-binding domain. Fatostatin blocked increases in body weight, blood glucose, and hepatic fat accumulation in obese ob/ob mice, even under uncontrolled food intake. Fatostatin may serve as a tool for gaining further insights into the regulation of SREBP.

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 (∼270 kDa) contains six catalytic activities and from the N-terminus the order is β-ketoacyl synthase (KS), acetyl/malonyl transacylase (AT/MT), β-hydroxyacyl dehydratase (DH), enoyl reductase (ER), β-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 Å resolution, containing two monomers (180×130×75 Å) that are separated by about 19 Å, 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.

Human fatty acid synthase: Role of interdomain in the formation of catalytically active synthase dimer

The human and animal fatty acid synthases are dimers of two identical multifunctional proteins (Mr 272,000) arranged in an antiparallel configuration. This arrangement generates two active centers for fatty acid synthesis separated by interdomain (ID) regions and predicts that two appropriate halves of the monomer should be able to reconstitute an active fatty acid synthesizing center. This prediction was confirmed by the reconstitution of the synthase active center by using two heterologously expressed halves of the monomer protein. Each of these recombinant halves of synthase monomer contains half of the ID regions. We show here that the fatty acid synthase activity could not be reconstituted when the ID sequences present in the two recombinant halves are deleted, suggesting that these ID sequences are essential for fatty acid synthase dimer formation. Further, we confirm that the ID sequences are the only regions of fatty acid synthase monomers that showed significant dimer formation, by using the yeast two-hybrid system. These results are consistent with the proposal that the ID region, which has no known catalytic activity, associates readily and holds together the two dynamic active centers of the fatty acid synthase dimer, therefore playing an important role in the architecture of catalytically active fatty acid synthase.

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 Å. 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.

The pathways of synthesis of fatty acids by mitochondria.

Abstract Mitochondria obtained from mammalian liver are capable of incorporating acetyl CoA into long chain fatty acids. Some of the characteristics of synthesis of fatty acids within mitochondria have been reported (1,2). The purpose of this communication is to present evidence for the existence of several pathways of synthesis within mitochondria and to indicate the important role of mitochondria in the synthesis and alteration of unsaturated fatty acids.