Fatty acid effects on gluconeogenesis in goat, calf and guinea pig hepatocytes

1. Regulation of hepatic gluconeogenesis by fatty acid was studied in goat, calf and guinea pig hepatocytes. 2. Fatty acid effects on gluconeogenesis were dependent upon species; fatty acid and gluconeogenic substrate. 3. Oleate and octanoate inhibited gluconeogenesis from propionate in guinea pig hepatocytes and stimulated it in goat hepatocytes. 4. Oleate and octanoate markedly inhibited gluconeogenesis from lactate in guinea pig hepatocytes whereas octanoate, but not oleate, decreased glucose production from lactate in goat hepatocytes. 5. Effects of fatty acids on gluconeogenesis in calf hepatocytes were similar to goat hepatocytes suggesting control of gluconeogenesis is similar among ruminant species but differs from guinea pigs.     

Evidence for vesicles that mediate long-chain fatty acid uptake by human microvascular endothelial cells

This study analyzes the mechanisms of long-chain fatty acid (LCFA) uptake by human microvascular endothelial cells (HMEC). The time course revealed the presence of an early, carrier-mediated uptake component and a later component mediated by clathrin-coated vesicles (CCV) and caveolae, as evidenced by three different experimental approaches: 1) significant reduction of [3H]oleate uptake over 5 min by either inhibition of CCV formation by potassium depletion or hypertonic medium, or disruption of caveolae by filipin III or cyclodextrin. 2) Co-localization of intracellular 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]octadecanoic acid with CCV and caveolae using confocal laser scanning microscopy. 3) Enrichment of [3H]oleate in a subcellular fraction containing CCV and caveolae. Within 10 min, more than 75% of intracellular [3H]oleate remained unmetabolized, suggesting that HMEC preferentially shuttle LCFA through the cell using CCV and caveolae as carriers. The uptake of albumin paralleled that of oleate within the first 10 min, suggesting internalization of at least some LCFA bound to albumin. Compared to oleate and albumin, the uptake of sucrose and dextran was low, indicating a potential minor contribution of fluid-phase endocytosis to the total vesicular LCFA uptake. The data indicate a previously unrecognized role of both CCV and caveolae for the uptake of LCFA by HMEC.     

Uptake of long-chain fatty acids in HepG2 cells involves caveolae: analysis of a novel pathway

We investigated the role of caveolae in uptake and intracellular trafficking of long chain fatty acids (LCFA) in HepG2 human hepatoma cells. The uptake of [(3)H]oleic acid and [(3)H]stearic acid into HepG2 cells was measured by radioactive assays and internalization of the non-metabolizable fluorescent fatty acid 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] (12-NBD) stearate into single HepG2 cells was semi-quantitatively assessed by laser scanning microscopy. The initial rate of [(3)H]oleic acid uptake (V(0)) in HepG2 cells exhibited saturable transport kinetics with increasing concentrations of free oleic acid (V(max) 854 +/- 46 pmol mg protein(-1) min(-1), K(m) 100 +/- 14 nmol/l). While inhibition of clathrin coated pits did not influence LCFA uptake in HepG2, inhibition of caveolae formation by filipin III, cyclodextrin, and caveolin-1 antisense oligonucleotides resulted in reduction of [(3)H]oleic acid uptake by 54%, 45%, and 23%, respectively. Furthermore, filipin III inhibited the uptake of [(3)H]stearic acid and its fluorescent derivative 12-NBD stearate by 44% and 50%, respectively. Transfection studies with alpha-caveolin-1/cyanofluorescent protein chimeras showed significant colocalization of caveolae and internalized 12-NBD stearate. In conclusion, these data suggest a significant role for caveolae mediated uptake and intracellular trafficking of LCFA in HepG2 cells.     

One-step preparation of S-nitrosated human serum albumin with high biological activities

S-Nitrosated human serum albumin (SNO-HSA) is a large molecular weight nitric oxide carrier in human plasma, and because of its many beneficial effects in different tests, it is currently under investigation as a cytoprotective agent. However, making SNO-HSA preparations is a complicated and time-consuming process. We found that binding of caprylic acid (CA) and N-acetyl-l-tryptophan (N-AcTrp) to defatted mercaptalbumin increased S-nitrosation by S-nitrosoglutathione (GS-NO) by making Cys-34 of HSA more accessible and by protecting it against oxidation, respectively. Fortunately, HSA solutions for clinical use contain high concentrations of CA and N-AcTrp as stabilizers. By making use of that fact it was possible to work-out a fast and simple procedure for producing SNO-HSA: incubation of a commercial HSA formulation with GS-NO for only 1 min results in S-nitrosation of HSA. The biological usefulness of such a preparation was tested in a rat ischemia–reperfusion liver injury model. Although our procedure for making SNO-HSA is fast and straightforward, the cytoprotective effect of the preparation was similar to, or better than, that of a preparation made in a more traditional way. The clinical development of SNO-HSA as a strong cytoprotective agent is under way using this method in collaboration with clinicians and industrial developers.     

Effect of fatty acid supplementation on cholesterol and retinol esterification in J774 macrophages

J774 macrophages exposed to medium containing cholesterol-rich phospholipid dispersions accumulate cholesteryl ester. Supplementing this medium with 100 micrograms oleate/ml increased cellular cholesteryl ester contents 3-fold. Cell retinyl ester contents increased 8-fold when medium containing retinol dispersed in dimethyl sulfoxide was supplemented with oleate. These increases were not the result of increases in total lipid uptake by the cells but rather of redistribution of cholesterol and retinol into their respective ester pools. Effective oleate concentration of 15-30 micrograms/ml increased cellular retinyl and cholesteryl ester contents. The effective oleate concentration was reduced to 5 micrograms/ml when the fatty acid/albumin molar ratio was increased. The oleate-stimulated increase in cholesterol esterification was blocked by incubating cells with Sandoz 58-035, a specific inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT), indicating that the effect of fatty acid exposure is mediated through changes in ACAT activity. When cholesterol or retinol was added to cells which had been exposed to oleate for 24 h to provide a triacylglycerol store, the cellular contents of cholesteryl or retinyl ester were also significantly increased compared to cells not previously exposed to oleate. The oleate-stimulated increase in the esterification of cholesterol and/or retinol was also observed in P388D1 macrophages, human (HepG2) and rat (Fu5AH) hepatomas, human fibroblasts, rabbit aortic smooth muscle cells and MCF-7 breast carcinoma cells. In addition to oleate, a number of other fatty acids increased retinol esterification in J774 macrophages; however, cellular cholesterol esterification in these cells was increased only by unsaturated fatty acids and was inhibited in the presence of saturated fatty acids. Although the cellular uptake of radiolabeled oleate and palmitate was similar, a significant difference in the distribution of these fatty acids among the lipid classes was observed. These data demonstrate that exogenous fatty acids are one factor that regulate cellular cholesteryl and retinyl ester contents in cultured cells.     

Long chain fatty acid binding to human plasma albumin.

The binding of six physiologically important long chain fatty acids to defatted human plasma albumin was measured at 37 degrees in a calcium-free Krebs-Ringer phosphate buffer, pH 7.4. The data were analyzed in terms of multiple stepwise equilibria. With the saturated acids, the magnitude of the equilibrium (association) constants, Ki, increased as the chain length increased: laurate smaller than myristate smaller than palmitate smaller than stearate. Oleate was bound more tightly than stearate; by contrast, linoleate was bound less tightly than stearate. The equilibrium constants, K1 through K12, ranged from 2.4 times 10-6 - 3.5 times 10-3 m-1 for laurate to 2.6 times 10-8 - 3.5 times 10-5 m-1 for oleate. Successive values of Ki decrease for each of the acids, indicating that major cooperative binding effects do not occur over the physiological range of fatty acid concentrations. In no case could the Ki be segregated into distinct classes, suggesting that any grouping of albumin binding sites is somewhat arbitrary. The results were inconclusive concerning whether premicellar association of unbound fatty acid occurs. Although corrections for premicellar association produced very little change in the Ki values for myristate, they raised the Ki for palmitate and stearate by 300 to 700 per cent. A sigmoidal relationship was obtained when the logarithm of Ki was plotted against chain length for the saturated fatty acids containing 6 to 18 carbon atoms, indicating that the binding energy is not simply a statistical process dependent only on the fatty acid chain length. This selectivity that albumin contributes to the binding process may be due to varying degrees of configurational adaptability of its binding sites as the fatty acid increases in length.     

The sensitivity of yeast mutants to oleic acid implicates the peroxisome and other processes in membrane function

The peroxisome, sole site of beta-oxidation in Saccharomyces cerevisiae, is known to be required for optimal growth in the presence of fatty acid. Screening of the haploid yeast deletion collection identified approximately 130 genes, 23 encoding peroxisomal proteins, necessary for normal growth on oleic acid. Oleate slightly enhances growth of wild-type yeast and inhibits growth of all strains identified by the screen. Nonperoxisomal processes, among them chromatin modification by H2AZ, Pol II mediator function, and cell-wall-associated activities, also prevent oleate toxicity. The most oleate-inhibited strains lack Sap190, a putative adaptor for the PP2A-type protein phosphatase Sit4 (which is also required for normal growth on oleate) and Ilm1, a protein of unknown function. Palmitoleate, the other main unsaturated fatty acid of Saccharomyces, fails to inhibit growth of the sap190delta, sit4delta, and ilm1delta strains. Data that suggest that oleate inhibition of the growth of a peroxisomal mutant is due to an increase in plasma membrane porosity are presented. We propose that yeast deficient in peroxisomal and other functions are sensitive to oleate perhaps because of an inability to effectively control the fatty acid composition of membrane phospholipids.     

Transmembrane transport of fatty acids in the heart

Summary Although fatty acid uptake by the myocardium is rapid and efficient, the mechanism of their transmembrane transport has been unclear. Fatty acids are presented to the plasma membrane of cardiomyocytes as albumin complexes within the plasma. Since albumin is not taken up by the cells, it was postulated that specific high affinity binding sites at the sarcolemma may mediate the dissociation of fatty acids from the albumin molecules, before they are transported into the cells. In studies with a representative long-chain fatty acid, oleate, it was in fact shown that fatty acids bind with high affinity to isolated plasma membranes of rat heart myocytes revealing a K_D of 42 nM. Moreover, a specific membrane fatty acid-binding protein (MFABP) was isolated from these membranes. It had a molecular weight of 40 kD, an isoelectric point of 9.0, and lacked carbohydrate or lipid components. Binding to a specific membrane protein might represent the first step of a carrier mediated uptake process. Therefore, the uptake kinetics of oleate by isolated rat heart myocytes was determined under conditions where only cellular influx and not metabolism occurred. Uptake revealed saturation kinetics and was temperature dependent which were considered as specific criteria for a facilitated transport mechanism. For evaluation whether uptake is mediated by MFABP, the effect of a monospecific antibody to this protein on cellular influx of oleate was examined. Inhibition of uptake of fatty acids but not of glucose by the antibody to MFABP indicated the physiologic significance of this protein as transmembrane carrier in the cellular uptake process of fatty acids. Such a transporter might represent an important site for the metabolic regulation of fatty acid influx into the myocardium.     

Fatty acid transport and metabolism in HepG2 cells

The mechanism(s) of fatty acid uptake by liver cells is not fully understood. We applied new approaches to address long-standing controversies of fatty acid uptake and to distinguish diffusion and protein-based mechanisms. Using HepG2 cells containing an entrapped pH-sensing fluorescence dye, we showed that the addition of oleate (unbound or bound to cyclodextrin) to the external buffer caused a rapid (seconds) and dose-dependent decrease in intracellular pH (pH(in)), indicating diffusion of fatty acids across the plasma membrane. pH(in) returned to its initial value with a time course (in min) that paralleled the metabolism of radiolabeled oleate. Preincubation of cells with the inhibitors phloretin or triacsin C had no effect on the rapid pH(in) drop after the addition of oleate but greatly suppressed pH(in) recovery. Using radiolabeled oleate, we showed that its esterification was almost completely inhibited by phloretin or triacsin C, supporting the correlation between pH(in) recovery and metabolism. We then used a dual-fluorescence assay to study the interaction between HepG2 cells and cis-parinaric acid (PA), a naturally fluorescent but slowly metabolized fatty acid. The fluorescence of PA increased rapidly upon its addition to cells, indicating rapid binding to the plasma membrane; pH(in) decreased rapidly and simultaneously but did not recover within 5 min. Phloretin had no effect on the PA-mediated pH(in) drop or its slow recovery but decreased the absolute fluorescence of membrane-bound PA. Our results show that natural fatty acids rapidly bind to, and diffuse through, the plasma membrane without hindrance by metabolic inhibitors or by an inhibitor of putative membrane-bound fatty acid transporters.     

Decreased liver fatty acid binding capacity and altered liver lipid distribution in mice lacking the liver fatty acid-binding protein gene

Although liver fatty acid-binding protein (L-FABP) is an important binding site for various hydrophobic ligands in hepatocytes, its in vivo significance is not understood. We have therefore created L-FABP null mice and report here their initial analysis, focusing on the impact of this mutation on hepatic fatty acid binding capacity, lipid composition, and expression of other lipid-binding proteins. Gel-filtered cytosol from L-FABP null liver lacked the main fatty acid binding peak in the fraction that normally comprises both L-FABP and sterol carrier protein-2 (SCP-2). The binding capacity for cis-parinaric acid was decreased >80% in this region. Molar ratios of cholesterol/cholesterol ester, cholesteryl ester/triglyceride, and cholesterol/phospholipid were 2- to 3-fold greater, reflecting up to 3-fold absolute increases in specific lipid classes in the order cholesterol > cholesterol esters > phospholipids. In contrast, the liver pool sizes of nonesterified fatty acids and triglycerides were not altered. However, hepatic deposition of a bolus of intravenously injected [14C]oleate was markedly reduced, showing altered lipid pool turnover. An increase of approximately 75% of soluble SCP-2 but little or no change of other soluble (glutathione S-transferase, albumin) and membrane (fatty acid transport protein, CD36, aspartate aminotransferase, caveolin) fatty acid transporters was measured. These results (i) provide for the first time a quantitative assessment of the contribution of L-FABP to cytosolic fatty acid binding capacity, (ii) establish L-FABP as an important determinant of hepatic lipid composition and turnover, and (iii) suggest that SCP-2 contributes to the accumulation of cholesterol in L-FABP null liver.     

Evidence that the sensitivity of carnitine palmitoyltransferase I to inhibition by malonyl-CoA is an important site of regulation of hepatic fatty acid oxidation in the fetal and newborn rabbit. Perinatal development and effects of pancreatic hormones in cultured rabbit hepatocytes.

The temporal changes in oleate oxidation, lipogenesis, malonyl-CoA concentration and sensitivity of carnitine palmitoyltransferase I (CPT 1) to malonyl-CoA inhibition were studied in isolated rabbit hepatocytes and mitochondria as a function of time after birth of the animal or time in culture after exposure to glucagon, cyclic AMP or insulin. (1) Oleate oxidation was very low during the first 6 h after birth, whereas lipogenesis rate and malonyl-CoA concentration decreased rapidly during this period to reach levels as low as those found in 24-h-old newborns that show active oleate oxidation. (2) The changes in the activity of CPT I and the IC50 (concn. causing 50% inhibition) for malonyl-CoA paralleled those of oleate oxidation. (3) In cultured fetal hepatocytes, the addition of glucagon or cyclic AMP reproduced the changes that occur spontaneously after birth. A 12 h exposure to glucagon or cyclic AMP was sufficient to inhibit lipogenesis totally and to cause a decrease in malonyl-CoA concentration, but a 24 h exposure was required to induce oleate oxidation. (4) The induction of oleate oxidation by glucagon or cyclic AMP is triggered by the fall in the malonyl-CoA sensitivity of CPT I. (5) In cultured hepatocytes from 24 h-old newborns, the addition of insulin inhibits no more than 30% of the high oleate oxidation, whereas it stimulates lipogenesis and increases malonyl-CoA concentration by 4-fold more than in fetal cells (no oleate oxidation). This poor effect of insulin on oleate oxidation seems to be due to the inability of the hormone to increase the sensitivity of CPT I sufficiently. Altogether, these results suggest that the malonyl-CoA sensitivity of CPT I is the major site of regulation during the induction of fatty acid oxidation in the fetal rabbit liver.     

油酸对THP-1巨噬细胞源性泡沫细胞三磷酸腺苷结合盒转运体A1表达和胆固醇流出的影响

以THP 1巨噬细胞源性泡沫细胞为研究对象 ,观察油酸对THP 1巨噬细胞源性泡沫细胞胆固醇流出和三磷酸腺苷结合盒转运体A1(ABCA1)表达的影响 ,以探讨油酸对动脉粥样硬化发生发展的影响。用液体闪烁计数器检测细胞内胆固醇流出 ,高效液相色谱分析细胞内总胆固醇、游离胆固醇和胆固醇酯含量 ,运用逆转录多聚酶链反应和Western印迹分别检测ABCA1mRNA与ABCA1蛋白的表达 ,采用流式细胞术检测细胞平均ABCA1荧光强度。实验显示油酸引起THP 1巨噬细胞源性泡沫细胞总胆固醇、游离胆固醇与胆固醇酯呈时间依赖性增加 ,而ABCA1蛋白水平、细胞平均ABCA1荧光强度以及apoA I介导的胆固醇流出呈时间依赖性减少 ,细胞内胆固醇增多 ,但ABCA1mRNA没有明显变化。结果表明 ,油酸减少THP 1巨噬细胞源性泡沫细胞ABCA1蛋白水平 ,降低细胞内胆固醇流出 ,增加细胞内胆固醇聚积。     

Fatty acids inhibit N-methylation of phosphatidylethanolamine in rat hepatocytes and liver microsomes

Supplementation of rat hepatocytes with various fatty acids in the culture medium reduced the conversion of [3H]phosphatidylethanolamine into phosphatidylcholine. Unsaturated fatty acids were the most effective inhibitors of phospholipid methylation. The inhibition of phosphatidylethanolamine methylation by oleate (2 mM) was reversed within 1 h after replacement with fatty acid-deficient medium. Fatty acids and their CoA derivatives (0.15-0.5 mM) produced 50% inhibition of phosphatidylethanolamine methyltransferase in rat liver microsomes. The first methylation reaction was the site of fatty acid inhibition, as methylation of phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N-dimethylethanolamine was not reduced in the presence of oleate. The inhibition by oleate was reversed by inclusion of bovine serum albumin or by addition of phospholipid liposomes. Thus, while fatty acids stimulate phosphatidylcholine biosynthesis in hepatocytes via the CDP-choline pathway, the methylation pathway is inhibited.     

Selective inhibition of long-chain fatty acid uptake in short-term cultured rat hepatocytes by an antibody to the rat liver plasma membrane fatty acid-binding protein

Uptake of long-chain fatty acids by short-term cultured hepatocytes was studied. Rat hepatocytes, which were cultured for 16 h on plastic dishes (3.6 X 10(6) cells/dish), were incubated with [3H]oleate in the presence of various concentrations of bovine serum albumin as a function of the concentration of unbound [3H]oleate in the medium. At 37 degrees C initial uptake velocity (V0) was saturable (Km = 9 X 10(-8) M; Vmax = 835 pmol/min per mg protein). V0 was temperature dependent with an optimum at 37 degrees C and markedly reduced at 4 degrees C and 70 degrees C. To evaluate the biologic significance of a previously isolated rat liver plasma membrane fatty acid-binding protein as putative carrier protein in the hepatocellular uptake of fatty acids, cultured hepatocytes were treated with a monospecific rabbit antibody (IgG-fraction) to this membrane protein or the IgG-fraction of the pre-immune serum as controls. Uptake kinetics of [3H]oleate in antibody pretreated short-term cultured hepatocytes revealed a depression of Vmax by 70%, while Km was only reduced by 16% compared to controls, indicating a predominant non-competitive type of inhibition. V0 of a variety of long-chain fatty acids (oleic acid, arachidonic acid, palmitic acid, stearic acid) was reduced by 56-69%, while V0 of [35S]sulfobromophthalein, [3H]cholic acid and [14C]taurocholic acid remained unaltered. These data support the concept that in the system of cultured hepatocytes, uptake of long-chain fatty acids is mediated by the rat liver plasma membrane fatty acid-binding protein.     

Oleate prevents palmitate-induced mitochondrial dysfunction,insulin resistance and inflammatory signaling in neuronal cells

Elevated circulating levels of saturated free fatty acids (sFFAs; e.g. palmitate) are known to provoke inflammatory responses and cause insulin resistance in peripheral tissue. By contrast, mono- or poly-unsaturated FFAs are protective against sFFAs. An excess of sFFAs in the brain circulation may also trigger neuroinflammation and insulin resistance, however the underlying signaling changes have not been clarified in neuronal cells. In the present study, we examined the effects of palmitate on mitochondrial function and viability as well as on intracellular insulin and nuclear factor-κB (NF-κB) signaling pathways in Neuro-2a and primary rat cortical neurons. We next tested whether oleate preconditioning has a protective effect against palmitate-induced toxicity. Palmitate induced both mitochondrial dysfunction and insulin resistance while promoting the phosphorylation of mitogen-activated protein kinases and nuclear translocation of NF-κB p65. Oleate pre-exposure and then removal was sufficient to completely block subsequent palmitate-induced intracellular signaling and metabolic derangements. Oleate also prevented ceramide-induced insulin resistance. Moreover, oleate stimulated ATP while decreasing mitochondrial superoxide productions. The latter were associated with increased levels of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). Inhibition of protein kinase A (PKA) attenuated the protective effect of oleate against palmitate, implicating PKA in the mechanism of oleate action. Oleate increased triglyceride and blocked palmitate-induced diacylglycerol accumulations. Oleate preconditioning was superior to docosahexaenoic acid (DHA) or linoleate in the protection of neuronal cells against palmitate- or ceramide-induced cytotoxicity. We conclude that oleate has beneficial properties against sFFA and ceramide models of insulin resistance-associated damage to neuronal cells.     

Mitochondrial and peroxisomal fatty acid oxidation in liver homogenates and isolated hepatocytes from control and clofibrate-treated rats.

Mitochondrial and peroxisomal fatty acid oxidation were compared in whole liver homogenates. Oxidation of 0.2 mM palmitoyl-CoA or oleate by mitochondria increased rapidly with increasing molar substrate:albumin ratios and became saturated at ratios below 3, while peroxisomal oxidation increased more slowly and continued to rise to reach maximal activity in the absence of albumin. Under the latter condition mitochondrial oxidation was severely depressed. In homogenates from normal liver peroxisomal oxidation was lower than mitochondrial oxidation at all ratios tested except when albumin was absent. In contrast with mitochondrial oxidation, peroxisomal oxidation did not produce ketones, was cyanide-insensitive, was not dependent on carnitine, and was not inhibited by (+)-octanoylcarnitine, malonyl-CoA and 4-pentenoate. Mitochondrial oxidation was inhibited by CoASH concentrations that were optimal for peroxisomal oxidation. In the presence of albumin, peroxisomal oxidation was stimulated by Triton X-100 but unaffected by freeze-thawing; both treatments suppressed mitochondrial oxidation. Clofibrate treatment increased mitochondrial and peroxisomal oxidation 2- and 6- to 8-fold, respectively. Peroxisomal oxidation remained unchanged in starvation and diabetes. Fatty acid oxidation was severely depressed by cyanide and (+)-octanoylcarnitine in hepatocytes from normal rats. Hepatocytes from clofibrate-treated rats, which displayed a 3- to 4-fold increase in fatty acid oxidation, were less inhibited by (+)-octanoylcarnitine. Hydrogen peroxide production was severalfold higher in hepatocytes from treated animals oxidizing fatty acids than in control hepatocytes. Assuming that all H2O2 produced during fatty acid oxidation was due to peroxisomal oxidation, it was calculated that the contribution of the peroxisomes to fatty acid oxidation was less than 10% both in cells from control and clofibrate-treated animals.     

Human serum albumin. Spectroscopic studies of binding and proximity relationships for fatty acids and bilirubin.

Binding and proximity relationships of hydrophobic ligands on human serum albumin have been studied using absorption, fluorescence, circular dichroism, and electron paramagnetic resonance spectroscopy. The ligands studied were bilirubin, two conjugated linear polyene fatty acids, cis-parinaric acid and cis-eleostearic acid, and three nitroxide derivatives of stearic acid with doxyl groups at positions 5, 10, and 12, respectively. Binding of polyene fatty acids was monitored by absorption peak shifts, induced circular dichroism, enhancement of fluorescence, and energy transfer between albumin's single tryptophanyl residue and the polyene chromophore. Induced circular dichroism studies indicate excitonic ligand-ligand interaction between bound fatty acids. Fluorescence enhancement of cis-parinaric acid was analyzed using a stepwise multiple equilibrium model, and six binding constants in the range 10(8) to 10(6) M-1 were obtained, in agreement with previous measurements for other fatty acids. The temperature dependence of the equilibrium constants indicates that the binding enthalpy is nearly zero. Fluorescence energy transfer was similarly used to quantitate bilirubin binding to albumin. Energy transfer, nitroxide quenching of fluorescence, and electron paramagnetic resonance spectroscopy were used to elucidate binding geometries which support and extend proposed structural models for albumin. It is suggested that the first two fatty acids bind side-by-side in an antiparallel fashion in domain III of human serum albumin.     

Studies on the uptake of fatty acids by Escherichia coli

Oleate uptake by Escherichia coli showed saturation kinetics with a K_m of 34 μm and an activation energy of 6.25 kcal/mole indicating that the rate limiting step in oleate uptake involves an enzyme-catalyzed step. The rate of oleate uptake was decreased by the respiratory poisons, arsenate and 4-pentenoate, which apparently is activated to pentenoyl CoA, thus reducing the intracellular concentration of free intracellular CoA. These data indicated that oleate uptake is dependent on cellular ATP and CoA. During short pulses with [1-¹⁴C]oleate, most of the radioactivity which was taken up was released as ¹⁴C0₂; cells accumulated radioactivity in phospholipids and compounds with the chromatographic mobility of Krebs cycle intermediates. Neither free fatty acid nor oleyl CoA were detectable in the cells. The results support the hypothesis that long-chain fatty acids are translocated by the long-chain fatty acyl CoA synthetase and that uptake is the rate limiting step in the utilization of exogenous fatty acid.