Effectors of fatty acid synthesis in hepatoma tissue culture cells

An investigation was undertaken to better understand the process of fatty acid synthesis in hepatoma tissue culture (HTC) cells. By comparing the findings to the normal liver some of the differences between normal and cancer tissue were defined. Incubation of the HTC cells in a buffered salt-defatted albumin medium showed that fatty acid synthesis was dependent upon the addition of substrate. The order of stimulation was glucose + pyruvate ～- glucose + alanine ～- glucose + lactate ～- pyruvate > glucose > alanine ? no additions. Fatty acid synthesis in HTC cells was decreased by oleate. In these respects HTC cells are similar to the liver; however, in contrast to the normal liver, N⁶, O²-dibutyryl cyclic adenosine 3′,5′-monophosphate (dibutyryl-cAMP) did not inhibit glycolysis or fatty acid synthesis. The cytoplasmic redox potential, as reflected by the lactate to pyruvate ratio, was found to be elevated compared to normal liver but unchanged by the addition of dibutyryl cAMP. Since higher rates of fatty acid synthesis are associated with lower lactate-to-pyruvate ratios in normal liver, it was expected that by decreasing the lactate-to-pyruvate ratio in HTC cells the rate of fatty acid synthesis would increase. One way to lower the lactate to pyruvate ratio is to increase the activity of the malate-aspartate shuttle. Stimulators of the hepatic malate-aspartate shuttle in normal liver (ammonium ion, glutamine, and lysine) had mixed effects on the redox state and fatty acid synthesis in HTC cells. Both ammonium ion and glutamine decreased the redox potential and increased the rate of fatty acid synthesis. Lysine was without effect on either process. Since NH₄Cl and glutamine stimulate the movement of reducing equivalents into the mitochondria and decrease the redox potential, then the stimulation of fatty acid synthesis by NH₄Cl and glutamine may be due to an increase in the movement of reducing equivalents into the mitochondria. However, if the shuttle were rate determining for fatty acid synthesis the rate from added lactate would be the same as from glucose alone but would be lower than from pyruvate which does not require the movement of reducing equivalents. This was not the case. Lactate and pyruvate gave comparable rates which were higher than glucose alone. Other possible sites of stimulation were investigated. The possibility that NH₄⁺ and glutamine stimulated fatty acid synthesis by activating pyruvate dehydrogenase was excluded by finding that dichloroacetate, an activator of pyruvate dehydrogenase, did not stimulate fatty acid synthesis when glucose was added. Stimulation by NH₄⁺ and glutamine at steps beyond pyruvate dehydrogenase was ruled out by the observation that NH₄⁺ caused no stimulation from added pyruvate. NH₄⁺ and glutamine did not alter the pentose phosphate pathway as determined by ¹⁴CO₂ production from [1-¹⁴C]- or [6-¹⁴C]glucose. Ammonium ion and glutamine increased glucose consumption and increased lactate and pyruvate accumulation. The increased glycolysis in HTC cells appears to be the explanation for the stimulation of fatty acid synthesis by NH₄⁺ and glutamine, even though glycolysis is much more rapid than fatty acid synthesis in these cells. The following observations support this conclusion. First, the percentage increase in glycolysis caused by NH₄⁺ or glutamine is closely matched by the percentage increase in fatty acid synthesis. Second, the malate-aspartate shuttle, the pentose phosphate pathway, and the steps past pyruvate are not limiting in the absence of NH₄⁺ or glutamine.     

Increased postprandial fatty acid trapping in subcutaneous adipose tissue in obese women

The objective of this study was to test the hypothesis that increased fatty acid trapping by subcutaneous adipose tissue might contribute to the development and/or maintenance of obesity. To do so, venoarterial (V-A) gradients across subcutaneous adipose tissue for triglycerides, glycerol, nonesterified fatty acid (NEFA), and acylation-stimulating protein (ASP) were determined in eight lean females [body mass index (BMI), 22.2 +/- 0.6] and eight obese females (BMI, 34.4 +/- 3.4). Plasma insulin was also measured at intervals throughout this period. Fasting plasma triglyceride was significantly higher in the obese group and postprandial triglyceride was also significantly delayed. In contrast, both triglyceride clearance and fatty acid uptake by subcutaneous adipose tissue were significantly greater in the obese group compared with the lean group. Fasting insulin did not differ between the groups, but postprandial insulin values were significantly higher in the obese group. The pattern of ASP release from subcutaneous adipose tissue also appeared to differ in that it was significantly greater in the early postprandial period (0;-90 min) in the obese group versus the lean group and this correlated with greater triglyceride clearance during this period. Moreover, there were strong, positive correlations between BMI and the V-A gradient for fasting ASP, the 0- to 90-min area under the curve (AUC) for ASP V-A gradient fasting insulin, and the 0- to 90-min AUC for fatty acid incorporation into adipose tissue. Taken together, these data demonstrate that fatty acid trapping by adipose tissue can be increased even when overall plasma triglyceride clearance is delayed. The postprandial pattern of insulin, in particular, was altered in the obese, although it is certainly possible that differences in ASP release or response could also contribute to increased fatty acid trapping in the obese.The data, therefore, suggest that increased fatty acid trapping by adipose tissue may be a feature of some forms of obesity.     

Chronic ethanol administration depresses fatty acid synthesis in rat adipose tissue.

The temperature-dependence of both the lipid order parameter (SDPH) and acetylcholinesterase (AChE) activity from native and cholesterol-enriched human erythrocyte membranes was investigated. Cholesterol enrichment abolishes an inflection observed around 30 degrees C in the temperature-dependence of native membrane lipid order parameter, whereas the Arrhenius plot of the enzymic activity is substantially unaffected. These results support the view that the breaks in the Arrhenius plot of the enzyme activity are not related to sudden changes of bulk membrane physical state, but arise from a direct effect of temperature on enzyme conformation.     

Dietary protein and the control of fatty acid synthesis in rat adipose tissue

Fatty acid synthesis in adipose tissue normally proceeds at a high rate when fasted animals are refed a diet containing carbohydrate, protein, and low levels of fat. This study investigated the effect of omitting protein from the refeeding diet. Rats were fasted for 48 hr and refed either a protein-free diet or a balanced diet, and the rate of fatty acid synthesis from glucose, pyruvate, lactate, and aspartate was measured. Refeeding the animals a diet devoid of protein resulted in a low rate of fatty acid synthesis from each of these substrates as well as a reduction in carbon flow over the citrate cleavage pathway. The activities of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADP-malate dehydrogenase, and ATP-citrate lyase were also reduced in epididymal fat pads from these rats. On the other hand, adipose tissue phosphoenolpyruvate carboxykinase activity was five times as great as that in tissue from animals refed a balanced diet. This difference could be eliminated if actinomycin D was injected coincident with refeeding. Refeeding rats diets high in carbohydrate is not, therefore, capable of inducing high rates of fatty acid synthesis in adipose tissue in the absence of dietary proteins. Thus, liver and adipose tissue respond differently to dietary protein.     

Regulation of glycolysis and fatty acid synthesis from glucose in sheep adipose tissue

1. The following were measured in adipose-tissue pieces, obtained from 7–9 month-old sheep, before or after the tissue pieces had been maintained in tissue culture for 24 h: the rates of synthesis from glucose of fatty acids, acylglycerol glycerol, pyruvate and lactate; the rate of glucose oxidation to CO₂; the rate of glucose oxidation via the pentose phosphate pathway; the activities of hexokinase, glucose 6-phosphate dehydrogenase, phosphofructokinase, pyruvate kinase, pyruvate dehydrogenase and ATP citrate lyase; the intra- and extra-cellular water content; the concentration of various metabolites and ATP, ADP and AMP. 2. The proportion of glucose carbon converted into the various products in sheep adipose tissue differs markedly from that observed in rat adipose tissue. 3. There was a general increase in the rate of glucose utilization by the adipose-tissue pieces after maintenance in tissue culture; largest changes were seen in the rates of glycolysis and fatty acid synthesis from glucose. These increases are paralleled by an increase in pyruvate kinase activity. There was no change in the activities of the other enzymes as measured, although the net flux through all the enzymes increased. 4. Incubation of fresh adipose-tissue pieces for 2–6h led to an increase in the affinity of pyruvate kinase for phosphoenolpyruvate. 5. The rate of pyruvate production by glycolysis was greater than the activity of pyruvate dehydrogenase of the tissue. 6. The results suggest that both pyruvate kinase and pyruvate dehydrogenase have important roles in restricting the utilization of glucose carbon for fatty acid synthesis in sheep adipose tissue.     

Understanding in vivo carbon precursor supply for fatty acid synthesis in leaf tissue

The principal supply of carbon precursors for fatty acid synthesis in leaf tissue has been a much debated topic, with some experiments suggesting a direct supply from the C3 products of photosynthetic carbon fixation and colleagues suggesting the utilization of free acetate (for which concentrations in leaves in the range of 0.05-1.4 mM have been reported). To address this issue we first reassessed the in vivo rate of fatty acid synthesis using a new method, that of [13C]carbon dioxide labeling of intact Arabidopsis plants with the subsequent analysis of fatty acids by gas chromatography-mass spectrometry (GC-MS). This method gave an average value of 2.3 mmoles carbon atoms h-1 mg chlorophyll-1 for photosynthetic tissues. The method was extended by isotopic dilution analysis to measure the rate of fatty acid synthesis in the dark. There was negligible fatty acid synthesis (< 5% of the rate in the light) in the dark. In addition, the method allowed an estimate of the absolute rate of fatty acid degradation of about 4% of the total fatty acid content per day. With the in vivo rate of fatty acid synthesis in the light defined, if the bulk tissue acetate concentration available for fatty acid synthesis is 1 mM, this acetate pool can sustain fatty acid synthesis for approximately 60 min. When the leaves of Arabidopsis, barley and pea were given a 5 min pulse of [14C]carbon dioxide, the label rapidly appeared in fatty acids with a lag phase of less than 2-3 min. Continuous labeling with [14C]carbon dioxide, for up to 1 h, showed a similar result. Furthermore, 14C-label in free acetate was less than 5% of that in fatty acids. In conclusion, these data suggest that either the bulk pool of acetate is not involved in fatty acid synthesis or the concentration of acetate must be less than 0.05 mM under strong illumination.     

Hormonal regulation of fatty acid synthetase, acetyl-CoA carboxylase and fatty acid synthesis in mammalian adipose tissue and liver.

The major objectives of this study were to define the roles of adrenal glucocorticoids and glucagon in the long-term regulation of fatty acid synthetase and acetyl-CoA carboxylase of mammalian adipose tissue and liver. Particular emphasis was given to elucidation of the mechanisms whereby these hormones produce their regulatory effects on enzymatic activity. To dissociate mental manipulation, nutritional conditions were ridgidly controlled in the experiments described. Administration of glucocorticoids to adult rats led to a marked reductionin activities of fatty acid synthetase and carboxylase in adipose in adipose tissue but no change occurred in liver. Adrenalectomy produced an increase in activities of these lipogenic enzymes in adipose tissure, but, again, no change was noted in liver. The decrease in enzymatic activities in adipose tissue with glucocorticoid administration correlated well with a decrease in fatty acid synthesis, determined in vivo by the 3-H2O method. The mechanisms whereby glucocorticoids led to a decrease in fatty acid synthetase activity were elucidated by the use of immunochemical techniques. Thus, the decrease in fatty acid synthetase activity observed in adipose tissue was shown to reflect a decrease in content of enzyme, and not a change in catalytic efficiency. The mechanism underlying the decrease in enzyme content is a decrease in synthesis of the enzyme. The relation of the effects of glucocorticoids to the effects of certain other hormones involved in regulation of lipogenesis was investigated in hypophysectomized and in diabetic animals. Thus, the observation that the glucocorticoid effect on synthetase and carboxylase occurred in adipose tissue of hypophysectomized rats indicated that alterations in levels of other pituitary-regulated hormones were not necessary for the effect. That glucocorticoids play some role in regulation of synthetase and carboxylase in liver, at lease in the diabetic state, was shown by the observation that the low activities of these enzymes in diabetic animals could be restored to normal by adrenalectomy. An even more pronounced restorative effect was apparent in adipose tissue of adrenalectomized, diabetic animals. Administration of glucagon during the refeeding of starved rats resulted in a marked reduction in the induction of fatty acid synthetase, acetyl-CoA carboxylase and in the rate of incorporation of 3-H from 3-H2O into fatty acids in liver, but no change in these parameters occurred in adipose tissue. Administration of theophylline resulted in intermediate reduction in liver. The mechanisms whereby glucagon led tto a decrease in fatty acid synthetase activity were elucidated by the use of immunochemical techniques. Thus, the changes in fatty acid synthetase activity were shown to reflect reductions in content of enzyme. The mechanism underlying these reductions in content is reduced synthesis of enzyme.     

Decreased rate of fatty acid synthesis in brown adipose tissue of hypothalamic obese rats

Intranuclear coinjection of the late SV40 KpnI/BclI DNA fragment and the early promotor/enhancer HpaII/BglI DNA segment into permissive monkey and non-permissive mouse cells allows late SV40 gene expression without T-antigen synthesis and DNA replication. These conditions were chosen to analyse the effect of DNA methylation on V-antigen synthesis detached from the process of DNA replication. We found that in vitro methylation of a single cytosine nucleotide proximal to the major late mRNA cap site by the HpaII methylase does not block capsid protein synthesis. This result is in contrast to reported data obtained in Xenopus laevis oocyte injection experiments [(1982) Proc. Natl. Acad. Sci. USA 79, 5142-5146].     

Mechanism of fatty acid synthesis

Significant advances have been made in the past few years in our understanding of the mechanism of synthesis of fatty acids, the structural organization of fatty acid synthetase complexes and the mechanism of regulation of activity of these enzyme systems. Numerous fatty acid synthetase complexes have been purified to homogeneity and the mechanism of synthesis of fatty acids by these enzyme systems has been ascertained from tracer, and recently, kinetic studies. The results obtained by these methods are in complete agreement. Furthermore, the kinetic results have indicated that fatty acid synthesis proceeds by a seven-site ping-pong mechanism. Several of the fatty acid synthetases have been dissociated completely to nonidentical half-molecular weight subunit species and these have been separated by affinity chromatography. From one of these subunits acyl carrier protein has been obtained. Whether the nonidentical subunits can be dissociated into individual proteins or whether these subunits are each comprised of one peptide is still a matter of controversy. However, it appears to us that each of the half-molecular weight subunits is indeed comprised of individual proteins. Studies on the regulation of activity of fatty acid synthetase complexes of avian and mammalian liver have resulted in the separation by affinity chromatography of three species (apo, holo-$\text{a}$ and holo-$\text{b}$) of fatty acid synthetase. Since these species have radically different enzyme activities they may provide a mechanism of short-term regulation of fatty acid synthetase activity. Other studies have shown that the quantity of avian and mammalian liver fatty acid synthetases is controlled by a change in the rate of synthesis of this enzyme complex. This change in the rate of synthesis of enzyme complex is under the control of insulin and glucagon. The former hormone increases the rate of enzyme synthesis, whereas the latter decreases it. Further studies on fatty acid synthetase complexes will undoubtedly concentrate upon more refined aspects of the structural organization of these enzyme systems, including the sequencing of acyl carrier proteins, the effects of protein-protein interaction on the kinetics of the partial reactions of fatty acid synthesis catalyzed by separated enzymes of the complex, the mechanism of hormonal regulation of fatty acid synthetase activity and x-ray diffraction analysis of subunits and complex.     

Increased rates of fatty acid uptake and plasmalemmal fatty acid transporters in obese Zucker rats

Giant vesicles were used to study the rates of uptake of long-chain fatty acids by heart, skeletal muscle, and adipose tissue of obese and lean Zucker rats. With obesity there was an increase in vesicular fatty acid uptake of 1.8-fold in heart, muscle and adipose tissue. In some tissues only fatty acid translocase (FAT) mRNA (heart, +37%; adipose, +80%) and fatty acid-binding protein (FABPpm) mRNA (heart, +148%; adipose, +196%) were increased. At the protein level FABPpm expression was not changed in any tissues except muscle (+14%), and FAT/CD36 protein content was altered slightly in adipose tissue (+26%). In marked contrast, the plasma membrane FAT/CD36 protein was increased in heart (+60%), muscle (+80%), and adipose tissue (+50%). The plasma membrane FABPpm was altered only in heart (+50%) and adipose tissues (+70%). Thus, in obesity, alterations in fatty acid transport in metabolically important tissues are not associated with changes in fatty acid transporter mRNAs or altered fatty acid transport protein expression but with their increased abundance at the plasma membrane. We speculate that in obesity fatty acid transporters are relocated from an intracellular pool to the plasma membrane in heart, muscle, and adipose tissues.     

Inhibiting bacterial fatty acid synthesis

The type II fatty acid synthase consists of a series of individual enzymes, each encoded by a separate gene, that catalyze discrete steps in chain elongation. The formation of fatty acids is vital to bacteria, and each of the essential enzymes and their acyl group carriers represent a potential target for the development of novel antibacterial therapeutics. High resolution x-ray and/or NMR structures of representative members of every enzyme in the type II pathway are now available, and these structures are a valuable resource to guide antibacterial drug discovery. The role of each enzyme in regulating pathway activity and the diversity in the components of the pathway in the major human pathogens are important considerations in deciding the most suitable targets for future drug development.     

Electron microscopic and radioautographic study of nucleic acid synthesis in normal fatty tissue and lipomas

Electron microscopic radioautography was used to study nucleic acid synthesis in the normal fatty tissue and lipomas of different degree of maturity. Destroyed adipocytes were found in the investigated varieties of the fatty tissue. In a slow-growing Madelung's lipoma such adipocytes occurred in a greater amount than in the normal fatty tissue and nongrowing lipomas. 3H-Thymidine was mainly incorporated by endotheliocytes and pericytes, whereas Madelung's lipoma contained a greater number of labeled cells. Based on the data obtained it is suggested that the replenishment of adipocytes occurs at the expense of the proliferation of vascular wall cells.     

Regulation of fatty acid synthesis.

Acetyl-CoA carboxylase and fatty acid synthetase are the two major enzymes involved in the synthesis of fatty acids in animals. The activities of both enzymes are affected by nutritional manipulations. Although acetyl-CoA carboxylase is considered generally to be the rate-limiting step in lipogenesis, there is evidence that suggests that fatty acid synthetase may become rate limiting under certain conditions. The principal support for the view that acetyl-CoA carboxylase is the rate-limiting enzyme for lipogenesis is that the activity of the enzyme is controlled by allosteric effectors that change the catalytic efficiency of the enzyme. Until recently, the only known control of fatty acid synthetase was through changes in rate of enzyme synthesis. Data are reviewed that show that fatty acid synthetase can exist in forms possessing different catalytic activities. Thus fatty acid synthetase appears to be subject to the type of control necessary for an enzyme to serve as a regulator of the rate of a biological process over a short term.