Role In Fatty Acid Synthesis Research Articles

Analysis of ATP-citrate lyase and malic enzyme mutants of Yarrowia lipolytica points out the importance of mannitol metabolism in fatty acid synthesis

The role of the two key enzymes of fatty acid (FA) synthesis, ATP-citrate lyase (Acl) and malic enzyme (Mae), was analyzed in the oleaginous yeast Yarrowia lipolytica. In most oleaginous yeasts, Acl and Mae are proposed to provide, respectively, acetyl-CoA and NADPH for FA synthesis. Acl was mainly studied at the biochemical level but no strain depleted for this enzyme was analyzed in oleaginous microorganisms. On the other hand the role of Mae in FA synthesis in Y. lipolytica remains unclear since it was proposed to be a mitochondrial NAD(H)-dependent enzyme and not a cytosolic NADP(H)-dependent enzyme. In this study, we analyzed for the first time strains inactivated for corresponding genes. Inactivation of ACL1 decreases FA synthesis by 60 to 80%, confirming its essential role in FA synthesis in Y. lipolytica. Conversely, inactivation of MAE1 has no effects on FA synthesis, except in a FA overaccumulating strain where it improves FA synthesis by 35%. This result definitively excludes Mae as a major key enzyme for FA synthesis in Y. lipolytica. During the analysis of both mutants, we observed a negative correlation between FA and mannitol level. As mannitol and FA pathways may compete for carbon storage, we inactivated YlSDR, encoding a mannitol dehydrogenase converting fructose and NADPH into mannitol and NADP+. The FA content of the resulting mutant was improved by 60% during growth on fructose, demonstrating that mannitol metabolism may modulate FA synthesis in Y. lipolytica.

Lipoprotein lipase, tissue expression and effects on genes related to fatty acid synthesis in goat mammary epithelial cells.

Lipoprotein lipase (LPL) serves as a central factor in hydrolysis of triacylglycerol and uptake of free fatty acids from the plasma. However, there are limited data concerning the action of LPL on the regulation of milk fat synthesis in goat mammary gland. In this investigation, we describe the cloning and sequencing of the LPL gene from Xinong Saanen dairy goat mammary gland, along with a study of its phylogenetic relationships. Sequence analysis showed that goat LPL shares similarities with other species including sheep, bovine, human and mouse. LPL mRNA expression in various tissues determined by RT-qPCR revealed the highest expression in white adipose tissue, with lower expression in heart, lung, spleen, rumen, small intestine, mammary gland, and kidney. Expression was almost undetectable in liver and muscle. The expression profiles of LPL gene in mammary gland at early, peak, mid, late lactation, and the dry period were also measured. Compared with the dry period, LPL mRNA expression was markedly greater at early lactation. However, compared with early lactation, the expression was lower at peak lactation and mid lactation. Despite those differences, LPL mRNA expression was still greater at peak, mid, and late lactation compared with the dry period. Using goat mammary epithelial cells (GMEC), the in vitro knockdown of LPL via shRNA or with Orlistat resulted in a similar degree of down-regulation of LPL (respectively). Furthermore, knockdown of LPL was associated with reduced mRNA expression of SREBF1, FASN, LIPE and PPARG but greater expression of FFAR3. There was no effect on ACACA expression. Orlistat decreased expression of LIPE, FASN, ACACA, and PPARG, and increased FFAR3 and SREBF1 expression. The pattern of LPL expression was similar to the changes in milk fat percentage in lactating goats. Taken together, results suggest that LPL may play a crucial role in fatty acid synthesis.

Sweetpotato vines hydrolysate promotes single cell oils production of Trichosporon fermentans in high-density molasses fermentation

This study investigated the co-fermentation of molasses and sweetpotato vine hydrolysate (SVH) by Trichosporon fermentans. T. fermentans showed low lipid accumulation on pure molasses; however, its lipid content increased by 35% when 10% SVH was added. The strong influence of SVH on lipid production was further demonstrated by the result of sensitivity analysis on effects of factors based on an artificial neural network model because the relative importance value of SVH dosage for lipid production was only lower than that of fermentation time. Scanning electron microscope observation and flow cytometry of yeast cells grown in culture with and without SVH showed that less deformation cells were involved in the culture with SVH. The activity of malic enzyme, which plays a key role in fatty acid synthesis, increased from 2.4U/mg to 3.7U/mg after SVH added. All results indicated SVH is a good supplement for lipid fermentation on molasses.

Mitochondrial tricarboxylate and dicarboxylate-tricarboxylate carriers: from animals to plants.

The citrate carrier (CiC), characteristic of animals, and the dicarboxylate-tricarboxylate carrier (DTC), characteristic of plants and protozoa, belong to the mitochondrial carrier protein family whose members are responsible for the exchange of metabolites, cofactors, and nucleotides between the cytoplasm and the mitochondrial matrix. Most of the functional data on these transporters are obtained from the studies performed with the protein purified from rat, eel yeast, and maize mitochondria or recombinant proteins from different sources incorporated into phospholipid vesicles (liposomes). The functional data indicate that CiC is responsible for the efflux of acetyl-CoA from the mitochondria to the cytosol in the form of citrate, the primer for fatty acid, cholesterol synthesis, and histone acetylation. Like the CiC, the citrate exported by DTC from the mitochondria to the cytosol in exchange for oxaloacetate can be cleaved by citrate lyase to acetyl-CoA and oxaloacetate and used for fatty acid elongation and isoprenoid synthesis. In addition to its role in fatty acid synthesis, CiC is involved in other processes such as gluconeogenesis, insulin secretion, inflammation, and cancer progression, whereas DTC is involved in the production of glycerate, nitrogen assimilation, ripening of fruits, ATP synthesis, and sustaining of respiratory flux in fruit cells. This review provides an assessment of the current understanding of CiC and DTC structural and biochemical characteristics, underlying the structure-function relationship of these carriers. Furthermore, a phylogenetic relationship between CiC and DTC is proposed.

Expression of fatty acid synthesis genes and fatty acid accumulation in haematococcus pluvialis under different stressors

BackgroundBiofuel has been the focus of intensive global research over the past few years. The development of 4th generation biofuel production (algae-to-biofuels) based on metabolic engineering of algae is still in its infancy, one of the main barriers is our lacking of understanding of microalgal growth, metabolism and biofuel production. Although fatty acid (FA) biosynthesis pathway genes have been all cloned and biosynthesis pathway was built up in some higher plants, the molecular mechanism for its regulation in microalgae is far away from elucidation.ResultsWe cloned main key genes for FA biosynthesis in Haematococcus pluvialis, a green microalga as a potential biodiesel feedstock, and investigated the correlations between their expression alternation and FA composition and content detected by GC-MS under different stress treatments, such as nitrogen depletion, salinity, high or low temperature. Our results showed that high temperature, high salinity, and nitrogen depletion treatments played significant roles in promoting microalgal FA synthesis, while FA qualities were not changed much. Correlation analysis showed that acyl carrier protein (ACP), 3-ketoacyl-ACP-synthase (KAS), and acyl-ACP thioesterase (FATA) gene expression had significant correlations with monounsaturated FA (MUFA) synthesis and polyunsaturated FA (PUFA) synthesis.ConclusionsWe proposed that ACP, KAS, and FATA in H. pluvialis may play an important role in FA synthesis and may be rate limiting genes, which probably could be modified for the further study of metabolic engineering to improve microalgal biofuel quality and production.

Expression and localization of myosin-1d in the developing nervous system

Myosin-1d is a monomeric actin-based motor found in a wide range of tissues, but highly expressed in the nervous system. Previous microarray studies suggest that myosin-1d is found in oligodendrocytes where transcripts are upregulated during the maturation of these cells. Myosin-1d was also identified as a component of myelin-containing subcellular fractions in proteomic studies and mutations in MYO1D have been linked to autism. Despite the potential implications of these previous studies, there is little information on the expression and localization of myosin-1d in the developing nervous system. Therefore, we analyzed myosin-1d expression patterns in the peripheral and central nervous systems during postnatal development. In mouse sciatic nerve, myosin-1d is expressed along the axon and in the ensheathing myelin compartment. Analysis of mouse cerebellum prior to myelination at day 3 reveals that myosin-1d is present in the Purkinje cell layer, granule cell layer, and region of the cerebellar nuclei. Upon the onset of myelination, myosin-1d enrichment expands along axonal tracts, while still present in the Purkinje and granule cell layers. However, myosin-1d was undetectable in oligodendrocyte progenitor cells at early and late time points. We also show that myosin-1d interacts and is co-expressed with aspartoacylase, an enzyme that plays a key role in fatty acid synthesis throughout the nervous system. Together, these studies provide a foundation for understanding the role of myosin-1d in neurodevelopment and neurological disorders.

Mammalian ACSF3 Protein Is a Malonyl-CoA Synthetase That Supplies the Chain Extender Units for Mitochondrial Fatty Acid Synthesis

The objective of this study was to identify a source of intramitochondrial malonyl-CoA that could be used for de novo fatty acid synthesis in mammalian mitochondria. Because mammalian mitochondria lack an acetyl-CoA carboxylase capable of generating malonyl-CoA inside mitochondria, the possibility that malonate could act as a precursor was investigated. Although malonyl-CoA synthetases have not been identified previously in animals, interrogation of animal protein sequence databases identified candidates that exhibited sequence similarity to known prokaryotic forms. The human candidate protein ACSF3, which has a predicted N-terminal mitochondrial targeting sequence, was cloned, expressed, and characterized as a 65-kDa acyl-CoA synthetase with extremely high specificity for malonate and methylmalonate. An arginine residue implicated in malonate binding by prokaryotic malonyl-CoA synthetases was found to be positionally conserved in animal ACSF3 enzymes and essential for activity. Subcellular fractionation experiments with HEK293T cells confirmed that human ACSF3 is located exclusively in mitochondria, and RNA interference experiments verified that this enzyme is responsible for most, if not all, of the malonyl-CoA synthetase activity in the mitochondria of these cells. In conclusion, unlike fungi, which have an intramitochondrial acetyl-CoA carboxylase, animals require an alternative source of mitochondrial malonyl-CoA; the mitochondrial ACSF3 enzyme is capable of filling this role by utilizing free malonic acid as substrate.

Compensatory increase in lipogenic gene expression in adipose tissue of transgenic mice expressing constitutively active AMP-activated protein kinase-alpha1 in liver

We previously described a line of transgenic mice selectively expressing constitutively active AMPK-α1 under the control of liver-specific human apoE promoter with the hepatic control region sequence. In the short-term activation, the CA-AMPK-α1 transgenic mice at age 10–12weeks exhibited normal hepatic triglyceride content as compared to wild-type mice due to compensatory increase in mRNA expression of genes in the cholesterol and fatty acid synthesis pathways. But it was not known whether the lipogenic gene expression in white adipose tissue also changed. Here we characterized mRNA expression profile of main lipogenic genes in the cholesterol and fatty acid biosynthesis pathway in white adipose tissue. The data show that short-term chronic activation of AMPK in liver caused marked compensatory increase in lipogenic gene expression both in liver due to induction of Srebp-2 and in white adipose tissue due to upregulation of Srebp-1c. These results support the notion that in addition to its well-recognized function for fat storage adipose tissue can play an adaptive role in fatty acid synthesis when fatty acid synthesis is severely reduced in liver, the main lipogenic organ in mammals.

Knockdown of NPY Expression in the Dorsomedial Hypothalamus Promotes Development of Brown Adipocytes and Prevents Diet-Induced Obesity

Hypothalamic neuropeptide Y (NPY) has been implicated in control of energy balance, but the physiological importance of NPY in the dorsomedial hypothalamus (DMH) remains unclear. Here we report that knockdown of NPY expression in the DMH by adeno-associated virus-mediated RNAi reduced fat depots in rats fed regular chow and ameliorated high-fat diet-induced hyperphagia and obesity. DMH NPY knockdown resulted in development of brown adipocytes in inguinal white adipose tissue through the sympathetic nervous system. This knockdown increased uncoupling protein 1 expression in both inguinal fat and interscapular brown adipose tissue (BAT). Consistent with the activation of BAT, DMH NPY knockdown increased energy expenditure and enhanced the thermogenic response to a cold environment. This knockdown also increased locomotor activity, improved glucose homeostasis, and enhanced insulin sensitivity. Together, these results demonstrate critical roles of DMH NPY in body weight regulation through affecting food intake, body adiposity, thermogenesis, energy expenditure, and physical activity.

Arginine metabolism and sensing by mTOR in T cells

Arginine has been shown to play an essential role in T cells, as arginine depletion in the immune microenvironment has a negative effect on T cell function. The goal of this study was to characterize how T cells sense and metabolize arginine.Experiments were carried out using murine T cells stimulated by CD3 and CD28 as well as the lymphoma cell line EL4. Proliferation and IL‐2 ELISA was used to assess T cell function. Western Blots were performed to determine amino acid sensing by the mTOR pathway. To establish pathways of arginine metabolism, a stable arginine isotope was used and metabolic products were analyzed using GC‐MS. Metabolomic analysis by GC‐MS was used to identify any unsuspected signaling and metabolic pathways regulated by arginine. RT‐PCR was used to analyze expression of enzymes known to be involved in arginine metabolism.T cells stimulated in the absence of arginine fail to proliferate or increase production of IL‐2, and activated T cells increase their uptake of arginine. T cells differentially sense arginine, citrulline, and ornithine through mTOR. Urea cycle enzymes are not expressed in T cells. GC‐MS analysis showed that arginine is not a substrate for the Krebs cycle and potentially plays a role in fatty acid synthesis. This research was supported by a Howard Temin Award from NCI/NIH and a New Investigator Grant from the Leukemia Research Foundation.

ACC2 gene polymorphisms, metabolic syndrome, and gene-nutrient interactions with dietary fat

Acetyl-CoA carboxylase β (ACC2) plays a key role in fatty acid synthesis and oxidation pathways. Disturbance of these pathways is associated with impaired insulin responsiveness and metabolic syndrome (MetS). Gene-nutrient interactions may affect MetS risk. This study determined the relationship between ACC2 polymorphisms (rs2075263, rs2268387, rs2284685, rs2284689, rs2300453, rs3742023, rs3742026, rs4766587, and rs6606697) and MetS risk, and whether dietary fatty acids modulate this in the LIPGENE-SU.VI.MAX study of MetS cases and matched controls (n = 1754). Minor A allele carriers of rs4766587 had increased MetS risk (OR 1.29 [CI 1.08, 1.58], P = 0.0064) compared with the GG homozygotes, which may in part be explained by their increased body mass index (BMI), abdominal obesity, and impaired insulin sensitivity (P < 0.05). MetS risk was modulated by dietary fat intake (P = 0.04 for gene-nutrient interaction), where risk conferred by the A allele was exacerbated among individuals with a high-fat intake (>35% energy) (OR 1.62 [CI 1.05, 2.50], P = 0.027), particularly a high intake (>5.5% energy) of n-6 polyunsaturated fat (PUFA) (OR 1.82 [CI 1.14, 2.94], P = 0.01; P = 0.05 for gene-nutrient interaction). Saturated and monounsaturated fat intake did not modulate MetS risk. Importantly, we replicated some of these findings in an independent cohort. In conclusion, the ACC2 rs4766587 polymorphism influences MetS risk, which was modulated by dietary fat, suggesting novel gene-nutrient interactions.

Myocardial Overexpression of Mecr, a Gene of Mitochondrial FAS II Leads to Cardiac Dysfunction in Mouse

It has been recently recognized that mammalian mitochondria contain most, if not all, of the components of fatty acid synthesis type II (FAS II). Among the components identified is 2-enoyl thioester reductase/mitochondrial enoyl-CoA reductase (Etr1/Mecr), which catalyzes the NADPH-dependent reduction of trans-2-enoyl thioesters, generating saturated acyl-groups. Although the FAS type II pathway is highly conserved, its physiological role in fatty acid synthesis, which apparently occurs simultaneously with breakdown of fatty acids in the same subcellular compartment in mammals, has remained an enigma. To study the in vivo function of the mitochondrial FAS in mammals, with special reference to Mecr, we generated mice overexpressing Mecr under control of the mouse metallothionein-1 promoter. These Mecr transgenic mice developed cardiac abnormalities as demonstrated by echocardiography in vivo, heart perfusion ex vivo, and electron microscopy in situ. Moreover, the Mecr transgenic mice showed decreased performance in endurance exercise testing. Our results showed a ventricular dilatation behind impaired heart function upon Mecr overexpression, concurrent with appearance of dysmorphic mitochondria. Furthermore, the data suggested that inappropriate expression of genes of FAS II can result in the development of hereditary cardiomyopathy.

Structure of the mitochondrial β-ketoacyl-[acyl carrier protein] synthase from Arabidopsis and its role in fatty acid synthesis

Mitochondrial fatty acid synthesis is catalyzed by a dissociated fatty acid synthase similar to those of plant plastids and bacteria. The crystal structure of a mitochondrial β-ketoacyl-[acyl carrier protein] synthase (mtKAS), namely that from Arabidopsis thaliana, has been determined for the first time. This enzyme accomplishes the vital condensation steps in constructing fatty acid carbon skeletons. The product profile of mtKAS is unusual in that C 8 and C 14–16 fatty acyl chains predominate. An enzyme architecture that likely is the basis for the observed bimodal profile of mtKAS products can be derived from the shape of the acyl binding pocket.

Fatty Acid Synthesis from Glutamate in the Adipose Tissue of Normal Subjects and Obese Patients: An Enzyme Study

In the adipose tissue, besides fatty acid synthesis (FA-S) from glucose, which includes several mitochondrial steps, FA-S from glutamate has been demonstrated. FA-S from glutamate takes place in the cytosol through the backward pathway of Krebs cycle (BPKC) and is due to the sequential action of (1) alanine aminotransferase (ALT, EC 2.6.1.2), which in presence of pyruvate converts glutamate to oxoglutarate; (2) isocitrate dehydrogenase (NADP) (ICDH, EC 1.1.1.42), which converts oxoglutarate to isocitrate; (3) aconitate hydratase (ACO, EC 4.2.1.3), which transforms isocitrate to citrate; and (4) ATP citrate-lyase (ATP-CL, EC 4.1.3.8), which splits citrate to yield the acetyl-CoA needed for FA-S. We studied the enzymes involved in BPKC in homogenates of human adipose tissue. In normal subjects, the cytosolic activity (μmol/min/g protein) was: ALT = 10.3 ± 1.1, ICDH = 29.5 ± 2.8, ACO = 2.05 ± 0.23, and ATP-CL = 1.2 ± 0.2. Mitochondria contained less or no activity, values being 20, 9, 11, and 0% of total for ALT, ICDH, ACO, and ATP-CL, respectively. BPKC enzymes are more active than the enzymes limiting FA-S from glucose, i.e., phosphofructokinase (EC 2.7.1.11), pyruvate carboxylase (EC 6.4.1.1), and pyruvate dehydrogenase (EC 1.2.4.1). In the obese patients, cytosolic ALT and ATP-CL were increased (12.9 ± 0.7, P < 0.05, and 2.28 ± 0.27, P < 0.01, respectively) compared to normal, while ICDH was not changed (ACO could not be studied). Similar changes were obtained by expressing enzyme activity per fat cell number. Besides the increased enzyme activity, the BPKC may be more active in the obese patients because of hyperinsulinemia, as insulin and glucose stimulate BPKC, perhaps by increasing the supply of pyruvate through stimulation of glycolysis. Thus BPKC may play a significant role in FA-S in human adipose tissue.

Localization of sequences for the basal and insulin-like growth factor-I inducible activity of the fatty acid synthase promoter in 3T3-L1 fibroblasts.

Fatty acid synthase (FAS) plays a central role in fatty acid synthesis and its expression is under nutritional and hormonal control. We have investigated insulin-like growth factor-I (IGF-I) regulation of FAS by transfecting into 3T3-L1 fibroblasts chimeric genes comprising the 5'-flanking region of the FAS gene linked to a luciferase (LUC) reporter gene. First, the basal promoter activity of the 5' serial deletions from nucleotides -318 to -19 of the FAS gene were compared. Deletions of the promoter sequences from -136 to -19 resulted in a step-wise decrease in the promoter activity, with the -67 LUC and -19 LUC plasmids retaining 40% and 16% of the luciferase activity of -136 LUC. Regulatory sequences important for the FAS basal promoter activity in 3T3-L1 fibroblasts are, therefore, located within the -136 to -19 region. Treatment with 10 mM IGF-I also increased luciferase activity 1.8 +/- 0.2-, 1.8 +/- 0.3- and 2.5 +/- 0.1-fold in 3T3-L1 fibroblasts transiently transfected with -136 LUC, -110 LUC and -67 LUC plasmids, respectively. Deletion of sequences from -67 to -19 resulted in the loss of responsiveness to IGF-I. Physiological doses of insulin (10 nM), however, did not increase luciferase activity in 3T3-L1 fibroblasts transfected with any of the above plasmids. Only upon treatment with pharmacological doses of insulin (1 microM), probably through IGF-I receptor, did luciferase activity increase 4.3 +/- 0.4-, 3.2 +/- 0.4- and 3.5 +/- 0.5-fold when transfected with -136 LUC, -110 LUC and -67 LUC plasmids, respectively; there was no increase with -19 LUC. The half-maximal effect of IGF-I on FAS promoter activity was observed at 3 nM and a maximal effect was reached at 10 nM. These results indicate that the increased promoter activities observed are probably mediated through the IGF-I receptor. Furthermore, sequences responsible for IGF-I regulation of the FAS gene are located within the proximal promoter between nucleotides -67 and -19 of the FAS gene.

Effect of the carbonic anhydrase inhibitor acetazolamide on lipid synthesis in the locust

The zinc metalloenzyme carbonic anhydrase (CA; EC 4.2.1.1). which catalyses the hydration of C 0 2 to HCOj, plays a number of physiologically important roles, for example in respiration, acid-base regulation and several secretory processes [for reviews see Ann. N .Y . Acad. Sci. (1984) 429). This wide diversity of important functions is reflected in its relative abundance in a broad range of tissues and cells (Tashian et al., 1984). lnterest has recently been focused on the possible function of CA in the provision of HCO, for essential carboxylation reactions in metabolism. There is evidence for its involvement in ureogenesis (Dodgson el a/., 1084; Haussinger & Gerok, 1985) and in gluconeogenesis (Herbert et a/., 1983; Dodgson et a/ . , 1984; Metcalfe et a/., 1985). Moreover, Herbert & Coulson ( 1984) have suggested that it may play a role in fatty acid synthesis in reptilian liver. We have examined the possible involvement of CA in lipogenesis in the fat body of the desert locust Schistocerca gregaria. This animal is an excellent model for studying fat metabolism. It has been known since biblical times that the locust undertakes long migratory flights (Exodus). For these it uses fat as the principal energy source. The necessary fat reserves are largely contained within a specialized organ, the fat body (Weiss-Fogh, 1952; Kilby, 1965). lncorporation of radioactive label from ['*C Jacetate into total lipid in fat body preparations from the adult locusts of different ages was monitored over a 1 h period in the presence and absence of the potent CA inhibitor acetazolamide (1.6 mM). For each experiment, the fat bodies from at least five locusts of the same age group were pooled, passed through a nylon mesh to disrupt the tissue into fragments, and suspended in incubation buffer [Hepes, 30 mM; NaCI, 10

In vitro studies of the pathways of fatty acid synthesis in the rabbit

1. 1. Pathways of lipogenesis in rabbit liver and adipose tissue were investigated utilizing tissue slice incubations coupled with activities of relevant enzymes. 2. 2. Acetate was the major carbon source for fatty acid synthesis and high rates of lipogenesis were noted for adipose tissue (perirenal and subcutaneous) and liver. 3. 3. The citrate-cleavage pathway and malate-transhydrogenation pathway play only a minor role in fatty acid synthesis in rabbit liver and adipose tissue based on limited glucose incorporation into fatty acids, very low activities of ATP-citrate lyase and NADP-malate dehydrogenase and results from studies using radioactive glutamate. 4. 4. High activities of NADP-isocitrate dehydrogenase and glucose-6-phosphate dehydrogenase were consistent with the isocitrate pathway and the pentose pathway being the major sources of reducing equivalents for lipogenesis in rabbit tissues.

Tricarboxylic Acid Activator-Induced Changes at the Active Site of Acetyl-CoA Carboxylase

Publisher Summary This chapter focuses on tricarboxylic acid activator-induced changes at the active site of acetyl-CoA carboxylase. Acetyl-CoA carboxylase catalyzes the first committed step of extramitochondrial fatty acid synthesis in animal tissues and appears to be an important control point in this biosynthetic pathway. The carboxylase appears to be regulated both by alterations of its level in tissues and by modification of its catalytic efficiency with allosteric effectors. Certain tricarboxylic acids, notably citrate and isocitrate, markedly activate the carboxylase-catalyzed reaction. Activation by these acids is rather specific in that tricarballylate, a close structural analog, is completely inactive. Citrate appears to serve the dual role in fatty acid synthesis of precursor of extramitochondrial acetyl-CoA via the citrate-cleavage reaction and of “feed-forward” activator of acetyl-CoA carboxylase. Activation by tricarboxylic acids is accomplished by the rapid polymerization of carboxylase protomers to give rise to polymeric filaments. In this chapter, effect of activator on the partial reactions of acetyl-CoA carboxylation is analyzed. Effect of activator on the environment of the prosthetic group is elaborated. Effect of activator on the reactivity of the carboxylated biotin prosthetic group is also described.

Significance of Adipose Tissue and Liver as Sites of Fatty Acid Synthesis in the Pig and the Efficiency of Utilization of Various Substrates for Lipogenesis

A combination of in vivo and in vitro techniques as well as the assay of the activity of several enzymes presumably involved in lipogenesis have been employed to study the relative importance of liver and adipose tissue in overall fatty acid synthesis in the pig. Both the in vivo and in vitro results indicate that when glucose-U-14C is used as substrate, virtually all the newly synthesized fatty acids are formed in the adipose tissue. The incorporation of acetate-1-14C into liver fatty acids was, however, much greater than that of glucose-U-14C, suggesting that, if acetate was freely available in vivo, the contribution of liver to overall lipogenesis may be appreciable. Data on the activity of citrate cleavage enzyme and of three NADPH-generating dehydrogenase enzymes in liver and adipose tissue complement the results obtained in the lipogenic studies. The hepatic capacity for the production of cytoplasmic acetyl CoA from mitochondrially derived citrate is insignificant, as is its ability to generate NADPH required in the reductive synthesis of fatty acids. Collectively the results indicate that the adipose tissue plays a major, if not a nearly exclusive role in fatty acid synthesis in the pig. Nonsaponifiable lipid synthesis in the liver requires acetate rather than glucose as a starting substrate.

Citrate Cleavage Enzyme and Fatty Acid Synthesis

A series of experiments was performed to test the proposal of others that citrate cleavage enzyme (EC 4.1.3.8) regulates the rate of fatty acid synthesis in rat liver. The results show that, in recovery from fasting, fatty acid synthesis increased markedly without change in citrate cleavage enzyme activity. After the administration of alloxan, fatty acid synthesis decreased prior to any change in citrate cleavage enzyme. Substitution of a high carbohydrate, low fat diet resulted in large increases in citrate cleavage enzyme activity without concomitant change in lipogenesis. The addition of purified citrate cleavage enzyme from rat liver to the fatty acid-synthesizing system from fasted animals did not restore fatty acid synthesis. It was concluded that citrate cleavage enzyme does not play a primary regulatory role in fatty acid synthesis.