Showing papers on "Fatty acid synthesis published in 1970"

Intracellular localization of enzymes in white-adipose-tissue fat-cells and permeability properties of fat-cell mitochondria. Transfer of acetyl units and reducing power between mitochondria and cytoplasm

Abstract: 1. A method is described for extracting separately mitochondrial and extramitochondrial enzymes from fat-cells prepared by collagenase digestion from rat epididymal fat-pads. The following distribution of enzymes has been observed (with the total activities of the enzymes as units/mg of fat-cell DNA at 25°C given in parenthesis). Exclusively mitochondrial enzymes: glutamate dehydrogenase (1.8), NAD–isocitrate dehydrogenase (0.5), citrate synthase (5.2), pyruvate carboxylase (3.0); exclusively extramitochondrial enzymes: glucose 6-phosphate dehydrogenase (5.8), 6-phosphogluconate dehydrogenase (5.2), NADP–malate dehydrogenase (11.0), ATP–citrate lyase (5.1); enzymes present in both mitochondrial and extramitochondrial compartments: NADP–isocitrate dehydrogenase (3.7), NAD–malate dehydrogenase (330), aconitate hydratase (1.1), carnitine acetyltransferase (0.4), acetyl-CoA synthetase (1.0), aspartate aminotransferase (1.7), alanine aminotransferase (6.1). The mean DNA content of eight preparations of fat-cells was 109μg/g dry weight of cells. 2. Mitochondria showing respiratory control ratios of 3–6 with pyruvate, about 3 with succinate and P/O ratios of approaching 3 and 2 respectively have been isolated from fat-cells. From studies of rates of oxygen uptake and of swelling in iso-osmotic solutions of ammonium salts, it is concluded that fat-cell mitochondria are permeable to the monocarboxylic acids, pyruvate and acetate; that in the presence of phosphate they are permeable to malate and succinate and to a lesser extent oxaloacetate but not fumarate; and that in the presence of both malate and phosphate they are permeable to citrate, isocitrate and 2-oxoglutarate. In addition, isolated fat-cell mitochondria have been found to oxidize acetyl l-carnitine and, slowly, l-glycerol 3-phosphate. 3. It is concluded that the major means of transport of acetyl units into the cytoplasm for fatty acid synthesis is as citrate. Extensive transport as glutamate, 2-oxoglutarate and isocitrate, as acetate and as acetyl l-carnitine appears to be ruled out by the low activities of mitochondrial aconitate hydratase, mitochondrial acetyl-CoA hydrolyase and carnitine acetyltransferase respectively. Pathways whereby oxaloacetate generated in the cytoplasm during fatty acid synthesis by ATP–citrate lyase may be returned to mitochondria for further citrate synthesis are discussed. 4. It is also concluded that fat-cells contain pathways that will allow the excess of reducing power formed in the cytoplasm when adipose tissue is incubated in glucose and insulin to be transferred to mitochondria as l-glycerol 3-phosphate or malate. When adipose tissue is incubated in pyruvate alone, reducing power for fatty acid, l-glycerol 3-phosphate and lactate formation may be transferred to the cytoplasm as citrate and malate.

The regulation of triglyceride synthesis and fatty acid synthesis in rat epididymal adipose tissue. Effects of insulin, adrenaline and some metabolites in vitro

Abstract: 1. Adipose tissues from rats fed a balanced diet were incubated in the presence of glucose (20mm) with the following additions: insulin, anti-insulin serum, insulin+acetate, insulin+pyruvate, insulin+lactate, insulin+phenazine methosulphate, insulin+oleate+albumin, insulin+adrenaline+albumin, insulin+6-N-2'-O-dibutyryl 3':5'-cyclic AMP+albumin. 2. Measurements were made of the whole tissue concentrations of adenine nucleotides, hexose phosphates, triose phosphates, glycerol 1-phosphate, 3 phosphoglycerate, 6-phosphogluconate, long-chain fatty acyl-CoA, acid-soluble CoA, citrate, isocitrate, malate and 2-oxoglutarate, and of the release into the incubation medium of lactate, pyruvate and glycerol after 1h of incubation. 3. Fluxes of [(14)C]glucose carbon through the major pathways of glucose metabolism were calculated from the yields of (14)C in various products after 2h of incubation. Fluxes of [(14)C]acetate, [(14)C]pyruvate or [(14)C]lactate carbon in the presence of glucose were also determined. 4. Measurements were also made of the whole-tissue concentrations of metabolites in tissues taken directly from Nembutal-anaesthetized rats. 5. Whole tissue mass-action ratios for phosphofructokinase, phosphoglucose isomerase and the combined (aldolasextriose phosphate isomerase) reaction were similar in vivo and in vitro. The reactants of phosphofructokinase appeared to be far from mass-action equilibrium. In vitro, the reactants of hexokinase also appeared to be far from mass-action equilibrium. 6. Correlation of observed changes in glycolytic flux with changes in fructose 6-phosphate concentration suggested that phosphofructokinase may show regulatory behaviour. The enzyme appeared to be activated in the presence of oleate or adrenaline and to be inhibited in the presence of lactate or pyruvate. 7. Evidence is presented that the reactants of lactate dehydrogenase and glycerol 1-phosphate dehydrogenase may be near to mass-action equilibrium in the cytoplasm. 8. No satisfactory correlations could be drawn between the whole-tissue concentrations of long-chain fatty acyl-CoA, citrate and glycerol 1-phosphate and the observed rates of triglyceride and fatty acid synthesis. Under the conditions employed, the concentration of glycerol 1-phosphate appeared to depend mainly on the cytoplasmic [NAD(+)]/[NADH] ratios. 9. Calculated hexose monophosphate pathway flux rates roughly correlated with fatty acid synthesis rates and with whole tissue [6-phosphogluconate]/[glucose 6-phosphate] ratios. The relative rates of production of NADPH for fatty acid synthesis by the hexose monophosphate pathway and by the ;malic enzyme' are discussed. It is suggested that all NADH produced in the cytoplasm may be used in that compartment for reductive synthesis of fatty acids, lactate or glycerol 1-phosphate.

The regulation of triglyceride synthesis and fatty acid synthesis in rat epididymal adipose tissue. Effects of altered dietary and hormonal conditions.

Abstract: 1. Epididymal adipose tissues obtained from rats that had been previously starved, starved and refed a high fat diet for 72h, starved and refed bread for 144h or fed a normal diet were incubated in the presence of insulin+glucose or insulin+glucose+acetate. 2. Measurements were made of the whole-tissue concentrations of hexose phosphates, triose phosphates, glycerol 1-phosphate, 3-phosphoglycerate, 6-phosphogluconate, adenine nucleotides, acid-soluble CoA, long-chain fatty acyl-CoA, malate and citrate after 1h of incubation. The release of lactate, pyruvate and glycerol into the incubation medium during this period was also determined. 3. The rates of metabolism of glucose in the hexose monophosphate pathway, the glycolytic pathway, the citric acid cycle and into glyceride glycerol, fatty acids and lactate+pyruvate were also determined over a 2h period in similarly treated tissues. The metabolism of acetate to CO(2) and fatty acids in the presence of glucose was also measured. 4. The activities of acetyl-CoA carboxylase, fatty acid synthetase and isocitrate dehydrogenase were determined in adipose tissues from starved, starved and fat-refed, and alloxan-diabetic animals and also in tissues from animals that had been starved and refed bread for up to 96h. Changes in these activities were compared with the ability of similar tissues to incorporate [(14)C]glucose into fatty acids in vitro. 5. The activities of acetyl-CoA carboxylase and fatty acid synthetase roughly paralleled the ability of tissues to incorporate glucose into fatty acids. 6. Rates of triglyceride synthesis and fatty acid synthesis could not be correlated with tissue concentrations of long-chain fatty acyl-CoA, citrate or glycerol 1-phosphate. In some cases changes in phosphofructokinase flux rates could be correlated with changes in citrate concentration. 7. The main lesion in fatty acid synthesis in tissues from starved, starved and fat-refed, and alloxan-diabetic rats appeared to reside at the level of pyruvate utilization and to be related to the rate of endogenous lipolysis. 8. It is suggested that pyruvate utilization by the tissue may be regulated by the metabolism of fatty acids within the tissue. The significance of this in directing glucose utilization away from fatty acid synthesis and into glyceride-glycerol synthesis is discussed.

The Effects of 2‐Ethylcitrate and Tricarballylate on Citrate Transport in Rat Liver Mitochondria and Fatty Acid Synthesis in Rat White Adipose Tissue

Abstract: 2-Ethylcitrate and propane 1,2,3 tricarboxylic acid (tricarballylate) were found to be inhibitors of the citrate transporting system as monitored by cis-aconitate and isocitrate oxidation. Kinetic data showed the inhibitions to be competitive with tricarboxylate anion. When the exchange of intramitochondrial [14C]citrate with added extramitochondrial citrate or L-malate was measured at low temperatures, 2-ethylcitrate and 2-propylcitrate were found to be good inhibitors, while tricarballylate did not inhibit and in fact was found to be a good substrate for exchange on the transporting system. Neither 2-ethylcitrate nor tricarballylate at low concentrations (< 10 mM) were found to inhibit fatty acid synthesis with pyruvate as precursor in rat white adipose tissue but both were potent inhibitors of fatty acid synthesis with glucose as precursor. The implications of these findings are discussed.

Role of acetyl coenzyme A carboxylase in the control of fatty acid synthesis.

Abstract: Publisher Summary This chapter discusses recent advances in studies on the regulatory mechanisms of a biotin enzyme, liver acetyl coenzyme A (CoA) carboxylase, which plays a critical role in the control of fatty acid synthesis. To study the control mechanisms of a metabolic sequence consisting of several steps catalyzed by different enzymes, it is important to know which step is rate-limiting in the overall process. Shortly after the discovery of the fatty acid-synthesizing pathway via malonyl-CoA, evidence was presented that indicated that the carboxylation step is rate-limiting in overall synthesis from acetyl-CoA in tissue extracts. Subsequent investigations revealed that carboxylase was not fully activated under the conditions employed in these experiments. This chapter presents evidence that indicates that the regulation of carboxylation step in vivo is achieved both by changes in the amount of acetyl-CoA carboxylase—that is, the number of enzyme molecules—and by changes in the activity of carboxylase—that is, catalytic efficiency per enzyme molecule. The former mechanisms appear to be more responsible for the long-term control, while the latter for the short-term one.

Effect of Insulin on Fatty Acid Synthesis from Pyuvate, Lactate, or Endogenous Sources in Adipose Tissue: Evidence for the Hormonal Regulation of Pyruvate Dehydrogenase1

Abstract: The incorporation of radioactivity from tritiated water into fatty acids has been used as a measure of the rate of fat synthesis in rat epididymal adipose tissue. Tissue removed from fasted-refed rats incubated in the presence or absence of glucose synthesized fatty acids at a rate comparable to that of normal tissue incubated with glucose and insulin. This high rate of synthesis was increased about 100% by the addition of insulin when glucose was present. When no substrate was added, insulin produced a 40% increase in the rate of fatty acid synthesis while decreasing glyceride-glycerol and lactic acid formation. It is concluded that insulin addition alters the metabolic disposition of carbohydrate arising from intracellular sources. Tissue from normally fed rats incorporated 5 times as much tritium from water into fatty acids when 20 mM pyruvate was provided as substrate as when 20 mM lactate was present. Insulin increased fatty acid synthesis in the presence of pyruvate by 40% and with lactate by 60%. T...

Comparative aspects of fatty acid synthesis in Bacillus subtilis and Escherichia coli.

Abstract: Fatty acid synthesis in B. subtilis and E. coli is compared. Cell free fatty acid synthesizing systems from both organisms require malonyl-CoA, NADPH and a “priming” substrate. In the case of E. coli the priming substrate is acetyl-CoA and the products of fatty acid synthesis are saturated and unsaturated straight-chain fatty acids. B. subtilis synthesizes branched-chain fatty acids predominantly and uses branched-chain acyl-CoA esters as priming substrates. The fundamental difference between the fatty acid synthesizing systems of these two organisms lies in the specificity of the initial acyl-CoA:acyl carrier protein transacylase and the role this enzyme plays in dictating the nature of the fatty acids formed. 1 The utilisation of various acyl-CoA primers by a B. subtilis cell-free fatty acid synthesizing system was studied. This system was unable to utilize acetyl-CoA. However, if acetyl acyl carrier protein was used as the priming substrate, the initial acyl-CoA:acyl carrier protein transacylase was by-passed and acetate was rapidly incorporated into straight-chain fatty acids. 2 Similar experiments using a cell-free system from E. coli indicated that the substrate specificity of the E. coli acyl-CoA:acyl carrier protein transacylase was much less critical. The branched-chain CoA esters and intermediates were, as a whole, poorly utilized by the E. coli system.

Fat Metabolism in Higher Plants XL. Synthesis of Fatty Acids in the Initial Stage of Seed Germination

Abstract: To understand more fully organelle membrane assemblage, the synthesis of the first fatty acids by the germinating pea, Pisum sativum, was studied by the incorporation of either tritiated water or acetate-1-14C into lipids by the intact, initially dry seed. After a lag phase, labeling proceeded linearly. This lag phase ended when uptake of water had increased the seed weight to 185% of its original weight. The first fatty acids synthesized were palmitic and stearic followed shortly after by long chain saturated fatty acids (C20-C26). The synthesis of very long chain acids was consistently characteristic of several other seeds in early stages of germination. The majority of the radioactive acids were present in phospholipids and were localized in particulate fractions. The acyl components of phosphatidyl glycerol were highly labeled. The very long chain acids were found predominantly in the waxes. Pulse labeling indicated little turnover of the labeled fatty acids. Evidence is presented indicating that the enzymes for fatty acid synthesis are already present in the dry seed and participate in the synthesis of fatty acids once a critical water content of the seed is achieved.

Method of treating obesity

Abstract: The inhibition of fatty acid synthesis is obtained in biological systems by utilizing a specific stereoisomer of hydroxycitric acid and derivatives thereof such as esters or lactones and the non-toxic salts of these compounds. It is believed that the present method involves the inhibition of citrate cleavage enzyme. Inhibition of fatty acid synthesis by the present method is useful in the treatment of obesity.

Effects of feeding states on lipid radioactivity in liver, muscle and adipose tissue after injection of labelled glucose in the rat.

Abstract: 0.5 g of glucose/kg b.w. and (U-14C) glucose was injected i.v. to ad libitum fed, 18 hrs fasting or 48 hrs fasting-48 hrs refed rats. After 60 min blood glucose had decreased to preinjection values. Recirculation of radioactive metabolites was found in blood in pyruvate-lactate and to a small extent in glyceride-glycerol. Rats were sacrificed at this time and radioactivity measured in chloroform: methanol extracts of homogenates from muscle, liver and different adipose tissue regions. Furthermore, radioactivity was measured in fatty acids. Fatty acid radioactivity in different regions was considered to be due to the local synthesis of fatty acids, because no recirculation was discovered at the time of analysis. Liver and adipose tissue fatty acid synthesis was most pronounced in the refed rats. Adipose tissue showed higher fatty acid synthesizing activity than the liver in ad libitum fed, but not in refed rats. Glyceride-glycerol radioactivity was rather high in muscle. In adipose tissue it was more constant in different feeding states than fatty acid radioactivity was. By additions of measured radioactive metabolites glucose uptake in adipose tissue was roughly estimated to a few per cent of total glucose uptake.

The role of the cytoplasmic redox potential in the control of fatty acid synthesis from glucose, pyruvate and lactate in white adipose tissue

Abstract: The metabolism of lactate, pyruvate and glucose was studied in epididymal adipose tissue of starved, normally fed and starved–re-fed rats. Lactate conversion into fatty acid occurred at an appreciable rate only in the adipocyte of starved–re-fed animals. NNN′N′-Tetramethyl-p-phenylenediamine, an agent that transports reducing power from the cytoplasm to the mitochondria, caused large increments of fatty acid synthesis from lactate and a smaller one from glucose but a decrease in that from pyruvate. Glucose (1.0mm) increased fatty acid synthesis from lactate 4.3-fold but only 1.67-fold from pyruvate in adipocytes from normally fed animals. 2-Deoxyglucose decreased fatty acid synthesis from lactate to a greater degree (threefold) compared to that from pyruvate in adipocytes from starved–re-fed animals. l-Glycerol 3-phosphate contents were approximately equal in epididymal fat-pads, incubated in the presence of lactate or pyruvate, from normally fed animals, whereas the addition of 1mm-glucose resulted in a tenfold increase in l-glycerol 3-phosphate content only in the presence of lactate. The l-glycerol 3-phosphate content was tenfold higher in adipose tissue from starved–re-fed animals incubated in the presence of lactate than in the presence of pyruvate. 2-Deoxyglucose caused these values to be slightly lowered in the presence of lactate. We suggest that lactate metabolism is limited by the rate of NADH removal from the cytoplasm. In the starved–re-fed state, this occurs by reduction of dihydroxyacetone phosphate formed from glycogen to produce l-glycerol 3-phosphate, thus permitting lactate conversion into fatty acid. When glucose is the substrate, and rates of transport are not limiting, the rate of removal of cytoplasmic NADH limits glucose conversion into fatty acid.

Rat mammary-gland acetyl-coenzyme A carboxylase interaction with milk fatty acids.

Abstract: 1. Highly purified rat mammary-gland acetyl-CoA carboxylase was inhibited by milk obtained from rats 12h after their young were weaned. 2. All the inhibitory activity was found in the particulate fraction (R105) obtained on centrifuging the milk. It could be extracted from milk fraction R105 with acetone and identified as a complex mixture of non-esterified fatty acids, present in high concentration (nearly 10mm) in the milk. 3. Inhibition of acetyl-CoA carboxylase was observed at low concentrations (0.2–20μm) of several of these fatty acids when fresh fully active enzyme was used. Enzyme that had been partly inactivated by aging, or by storing in the absence of citrate, was stimulated by low concentrations but inhibited by high concentrations of fatty acids. 4. Various experiments suggested that fatty acids produce irreversible inactivation of acetyl-CoA carboxylase. 5. The effects of palmitoyl-CoA on mammary-gland acetyl-CoA carboxylase were found to resemble those of fatty acids, except that palmitoyl-CoA was effective at lower concentration. 6. The effect of milk fraction R105 was tested on six other enzymes previously shown to decline to various extents after weaning. Although several of these enzymes were affected by unfractionated milk fraction R105, none was significantly inhibited by the acetone extract or by low concentrations of lauric acid. 7. The findings are consistent, both qualitatively and quantitatively, with a regulatory mechanism whereby milk fatty acids shut off fatty acid synthesis in the mammary gland after weaning by inhibiting acetyl-CoA carboxylase.

Regulation of lipogenesis. Stimulation of fatty acid synthesis in vivo and in vitro in the liver of the newly hatched chick

Abstract: 1. A single glucose meal stimulated the incorporation of acetate into fatty acids in liver slices. If the glucose was added in vitro, it had no effect. Fructose and glycerol in vitro markedly stimulated fatty acid synthesis from acetate. Fructose and glycerol probably by-passed a rate-controlling reaction between glucose and triose phosphate. This reaction may have been stimulated by glucose administered in vivo. 2. The stimulation of fatty acid synthesis caused by fructose did not require the synthesis of enzyme, thus indicating that fatty acid-synthesizing enzymes were present in a latent form in the livers from unfed chicks.