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.     

Nutritional and hormonal control of lung and liver fatty acid synthesis.

During starvation and in streptozotocin-induced diabetes, the total activities of rat lung acetyl CoA carboxylase and fatty acid synthetase are reduced to one-third of the normal values. Refeeding of the starved animals or administration of insulin to diabetic animals restores the levels to the original values. The insulin effect is dose and time dependent. These data contrast with those in the liver, where a 30- to 50-fold depression of these enzymes is observed in the diabetic state and administration of insulin is actually followed by doubling of the activity over normal controls. Fat-free high-fructose diet (containing 60% fructose by weight) enhances the activities of liver enzymes 3- to 6-fold over the values of controls on laboratory diet but has no effect on the lung enzymes. Long-term feeding of fructose diet also increases the activities of liver enzymes from diabetic animals to twice the value of normal controls on laboratory diet. Insulin administration to fructose-fed diabetic animals restores the enzyme activities to those obtained with fructose-fed normal controls. However, the stimulation of lung enzymes of diabetic animals can be effected either by fructose or by insulin. Antigen-antibody titrations and measurements of the rate of protein synthesis show that the increased activity of the lung and liver fatty acid synthetase is due to enhanced content rather than increased specific activity. These data suggest that insulin or fructose effects on fatty acid-synthesizing enzymes are mediated through intermediate(s) whose concentration is affected in the experimental diabetes. Furthermore, all tissues may not have stringent insulin requirements since the lung enzymes can be stimulated by fructose alone.     

Role of fatty acid-binding protein in cardiac fatty acid oxidation

Summary Although abundant in most biological tissues and chemically well characterized, the fatty acid-binding protein (FABP) was until recently in search of a function. Because of its strong affinity for long chain fatty acids and its cytoplasmic origin, this protein was repeatedly claimed in the literature to be the transcytoplasmic fatty acid carrier. However, techniques to visualize and quantify the movements of molecules in the cytoplasm are still in their infancy. Consequently the carrier function of FABP remains somewhat speculative. However, FABP binds not only fatty acids but also their CoA and carnitine derivatives, two typical molecules of mitochondrial origin. Moreover, it has been demonstrated and confirmed that FABP is not exclusively cytoplasmic, but also mitochondrial. A function for FABP in the mitochondrial metabolism of fatty acids plus CoA and carnitine derivatives would therefore be anticpated. Using spin-labelling techniques, we present here evidence that FABP is a powerful regulator of acylcarnitine flux entering the mitochondrial -oxidative system. In this perspective FABP appears to be an active link between the cytoplasm and the mitochondria, regulating the energy made available to the cell. This active participation of FABP is shown to be the consequence of its gradient-like distribution in the cardiac cell, and also of the coexistence of multispecies of this protein produced by self-aggregation.     

Role of fatty acid transport protein 4 in metabolic tissues: insights into obesity and fatty liver disease

Fatty acid (FA) metabolism is a series of processes that provide structural substances, signalling molecules and energy. Ample evidence has shown that FA uptake is mediated by plasma membrane transporters including FA transport proteins (FATPs), caveolin-1, fatty-acid translocase (FAT)/CD36, and fatty-acid binding proteins. Unlike other FA transporters, the functions of FATPs have been controversial because they contain both motifs of FA transport and fatty acyl-CoA synthetase (ACS). The widely distributed FATP4 is not a direct FA transporter but plays a predominant function as an ACS. FATP4 deficiency causes ichthyosis premature syndrome in mice and humans associated with suppression of polar lipids but an increase in neutral lipids including triglycerides (TGs). Such a shift has been extensively characterized in enterocyte-, hepatocyte-, and adipocyte-specific Fatp4-deficient mice. The mutants under obese and non-obese fatty livers induced by different diets persistently show an increase in blood non-esterified free fatty acids and glycerol indicating the lipolysis of TGs. This review also focuses on FATP4 role on regulatory networks and factors that modulate FATP4 expression in metabolic tissues including intestine, liver, muscle, and adipose tissues. Metabolic disorders especially regarding blood lipids by FATP4 deficiency in different cell types are herein discussed. Our results may be applicable to not only patients with FATP4 mutations but also represent a model of dysregulated lipid homeostasis, thus providing mechanistic insights into obesity and development of fatty liver disease.     

Growth promotion of transfected hepatoma cells by liver fatty acid binding protein

Former studies have linked hepatocyte growth with liver fatty acid binding protein (L-FABP) of rat liver cytosol. In search for the roles of L-FABP in hepatocytes, we previously stably transfected rat L-FABP sense and antisense cDNAs into rat hepatoma HTC cells that do not contain L-FABP RNA or protein, thereby providing a zero-background, homologous cell model of L-FABP-expression suitable for controlled studies of its intracellular functions in hepatocyte-derived cells. The present study demonstrates the abilities of L-FABP to promote DNA synthesis and cell growth, preserve cell morphology, extend survival, and act cooperatively with unsaturated fatty acids in the transfected hepatoma cells in the absence of serum. Following removal of serum, the three control L-FABP-nonexpressing cell lines increased in cell lines increased in cell number for 24 hr and thereafter declined, whereas the three L-FABP-expressing cell lines exhibited a 39% higher rate of DNA synthesis per cell at 24 hr and grew in cell number for 48 hr. As a result, at 72 hr there were 2.5-fold (avg.) as many L-FABP-expressing cells than L-FABP-nonexpressing cells. In addition, the L-FABP-expressing cells retained their original polygonal morphology at 48 hr, when in contrast most of the control nonexpressing cells were spherical in shape with membrane blebs. In an effort to identify the agonists that collaborate with L-FABP in the growth promotion and preservation of cell morphology, various free fatty acids were examined at 48 hr for their ability to elminate the differences in behavior of the two cell types in the serum-free medium. The unsaturated fatty acids, oleic acid (18:1 ω9), linoleic acid (18:2ω6), α-linolenic acid (18: 3ω3), and arachidonic acid (20:4ω6), at 1 μM markedly elevated the level of DNA synthesis in the more depressed control L-FABP-nonexpressing cells and moderately raised it in the less depressed L-FABP-expressing cells. In accord, the control L-FABP-nonexpressing cells needed 10^?6–10^?5 M linoleic acid to achieve the extent of DNA synthesis attained by the expressing cells in the absence of added fatty acid. At 10 μM linoleic acid, their levels of DNA synthesis were equal. In contrast, five saturated fatty acids had no detectable effect on DNA synthesis. In addition, linoleic acid at 1 μM, but not the saturated fatty acid palmitic acid (16:0), prevented the above morphological alterations in the control L-FABP-nonexpressing cells observed in the absence of serum, thereby retaining their original polygonal morphology and that of the expressing cells. The findings are consistent with the concept that L-FABP improves the efficacy of the utilization of unsaturated fatty acid ligands of L-FABP in the formation, integrity, and fluidity of cell membranes that are involved in cell growth, morphology, and survival. © 1993 Wiley-Liss, Inc.     

肝型脂肪酸结合蛋白研究进展

肝型脂肪酸结合蛋白（liver fatty acid binding protein,L-FABP）是脂肪酸结合蛋白（fatty acid binding proteins,FABPs）家族重要的成员,在肝脏、小肠、肾脏等组织中均有表达。L-FABP在不饱和脂肪酸、饱和脂肪酸、胆固醇、胆汁酸等转运过程中扮演重要角色。目前研究显示L-FABP在脂肪肝、肝硬化以及肝癌发生发展中起到重要作用,并有望作为肝损伤的早期检测指标。此外,新近研究发现尿中L-FABP水平还可以用于预测1型糖尿病患者的临床结局。在2型糖尿病中,尿中L-FABP与糖尿病性肾病的病程有密切关系。主要就L-FABP的特性、结构及其与疾病的关系做一综述。     

Binding of acyl-CoA to liver fatty acid binding protein: effect on acyl-CoA synthesis

The ability of purified rat liver and heart fatty acid binding proteins to bind oleoyl-CoA and modulate acyl-CoA synthesis by microsomal membranes was investigated. Using binding assays employing either Lipidex 1000 or multilamellar liposomes to sequester unbound ligand, rat liver but not rat heart fatty acid binding protein was shown to bind radiolabeled acyl CoA. Binding studies suggest that liver fatty acid binding protein has a single binding site acyl-CoA which is separate from the two binding sites for fatty acids. Experiments were then performed to determine how binding may influence acyl-CoA metabolism by liver microsomes or heart sarcoplasmic reticulum. Using liposomes as fatty acid donors, liver fatty acid binding protein stimulated acyl-CoA production, whereas that from heart did not stimulate production over control values. 14C-labeled fatty acid-fatty acid binding protein complexes were prepared, incubated with membranes, and acyl-CoA synthetase activity was determined. Up to 70% of the fatty acid could be converted to acyl-CoA in the presence of liver fatty acid binding protein but in the presence of heart fatty acid binding protein, only 45% of the fatty acid was converted. Liver but not heart fatty acid binding protein bound the acyl-CoA formed and removed it from the membranes. The amount of product formed was not changed by additional membrane, enzyme cofactors, or incubation time. Additional liver fatty acid binding protein was the only factor found that stimulated product formation. Acyl-CoA hydrolase activity was also shown in the absence of ATP and CoA. These studies suggest that liver fatty acid binding protein can increase the amount of acyl-CoA by binding this ligand, thereby removing it from the membrane and possibly aiding transport within the cell.     

Acute effects of ethanol in the control of protein synthesis in isolated rat liver cells

The acute effect of ethanol on hepatic protein synthesis is a rather controversial issue. In view of the conflicting reports on this subject, the effect of ethanol on protein labeling from l-[³H]valine in isolated liver cells was studied under a variety of experimental conditions. When tracer doses of the isotope were utilized, ethanol consistently decreased the rate of protein labeling, regardless of the metabolic conditions of the cells. This inhibition was not prevented by doses of 4-methylpyrazole large enough to abolish all the characteristic metabolic effects of ethanol, and it was not related to perturbations on the rates of l-valine transport and/or proteolysis. When ethanol was tested in the presence of saturating doses of l-[³H]valine no effect on protein labeling was observed. These observations suggest that the ethanol effect in decreasing protein labelling from tracer doses of the radioactive precursor does not reflect variations in the rate of protein synthesis but reflects changes in the specific activity of the precursor. These changes probably are secondary to variations in the dimensions of the amino acid pool utilized for protein synthesis. Even though it showed a lack of effect when tested alone, in the presence of saturating doses of the radioactive precursor ethanol inhibited the stimulatory effects on protein synthesis mediated by glucose and several gluconeogenic substrates. This effect of ethanol was not prevented by inhibitors of alcohol dehydrogenase, indicating that a shift of the NAD system to a more reduced state is not the mediator of its action. It is suggested that ethanol probably acted by changing the steady-state levels of some common effector(s) generated from the metabolism of all these fuels or else by preventing the inactivation of a translational repressor.     

A novel acyl-CoA-binding protein from bovine liver. Effect on fatty acid synthesis.

Bovine liver was shown to contain a hitherto undescribed medium-chain acyl-CoA-binding protein. The protein co-purifies with fatty-acid-binding proteins, but was, unlike these proteins, unable to bind fatty acids. The protein induced synthesis of medium-chain acyl-CoA esters on incubation with goat mammary-gland fatty acid synthetase. The possible function of the protein is discussed.     

Cell-free synthesis of pigeon liver fatty acid synthetase.

Pigeon liver fatty acid synthetase proteins (apo- and holo-forms) have been synthesized in a cell-free system reconstituted from polysomes and a soluble enzyme fraction. Identification of the cell-free synthesized products as fatty acid synthetase was achieved by affinity chromatography, by immuno-precipitation and by the simultaneous conversion of both the authentic carrier protein and the $\text{in vitro}$ synthesized products from the holo- to the apo-form of the synthetase. The reverse conversion was also effected.     

The control of fatty acid metabolism in liver cells from fed and starved sheep.

Isolated liver cells prepared from starved sheep converted palmitate into ketone bodies at twice the rate seen with cells from fed animals. Carnitine stimulated palmitate oxidation only in liver cells from fed sheep, and completely abolished the difference between fed and starved animals in palmitate oxidation. The rates of palmitate oxidation to CO2 and of octanoate oxidation to ketone bodies and CO2 were not affected by starvation or carnitine. Neither starvation nor carnitine altered the ratio of 3-hydroxybutyrate to acetoacetate or the rate of esterification of [1-14C]palmitate. Propionate, lactate, pyruvate and fructose inhibited ketogenesis from palmitate in cells from fed sheep. Starvation or the addition of carnitine decreased the antiketogenic effectiveness of gluconeogenic precursors. Propionate was the most potent inhibitor of ketogenesis, 0.8 mM producing 50% inhibition. Propionate, lactate, fructose and glycerol increased palmitate esterification under all conditions examined. Lactate, pyruvate and fructose stimulated oxidation of palmitate and octanoate to CO2. Starvation and the addition of gluconeogenic precursors stimulated apparent palmitate utilization by cells. Propionate, lactate and pyruvate decreased cellular long-chain acylcarnitine concentrations. Propionate decreased cell contents of CoA and acyl-CoA. It is suggested that propionate may control hepatic ketogenesis by acting at some point in the beta-oxidation sequence. The results are discussed in relation to the differences in the regulation of hepatic fatty acid metabolism between sheep and rats.     

Studies on the synthesis of rat liver fatty acid synthetase.

The synthesis of the multienzyme complex rat liver fatty acid synthetase was investigated utilizing modifications of methods developed in the laboratory of Schimke (Schimke, R. T. (1964) J. Biol. Chem. 239, 3808-3817 and Arias, I. M., Doyle, D., and Schimke, R. T. (1969) J. Biol. Chem. 244, 3303-3315). The relative amounts of radioactivity from a pulse of labeled lysine appearing in polypeptides derived from purified synthetase complex can be measured compensating for the varying amounts of lysine per polypeptide chain. The results show that labeled amino acid is incorporated into polypeptides derived from the complex at heterogeneous rates. However, 10 to 15 hours after the administration of a pulse, the amount of label per lysine residue in these polypeptides is identical. The results support the previously proposed model of this multienzyme complex (Tweto, J., Dehlinger, P., and Larrabee, A. R. (1972) Biochem. Biophys. Res. Commun. 48, 1371-1377). The previous work and that reported here suggests the existence of a pool of synthetase subunits which is an obligatory intermediate in both synthesis and turnover of the complex. The results obtained in this work are consistent with this model if the exchange of subunits into the intact complex is a relatively slow process requiring several hours to reach equilibrium.     

Light control of fatty acid synthesis and diurnal fluctuations of fatty acid composition in leaves.

1. Although isolated spinach chloroplasts were almost entirely (greater than 99%) dependent on light for fatty acid synthesis, leaf discs were capable of fatty acid synthesis in the dark (up to 500nmol of 3H/h per mg of chlorophyll equivalent to approx. 400nmol of carbon/h per mg of chlorophyll), which represented 12-20% of the corresponding 'light rates'. 2. Net fatty acid accumulation by greening maize leaves occurred largely or entirely during the light period. 3. There was a diurnal fluctuation in the proportions of C18 unsaturated fatty acids in the lipids of developing spinach leaves, where an increase in the concentration of oleate during the day and a subsequent decline at night was observed; a complementary change occurred in the concentration of alpha-linolenate. The rhythm is interpreted as reflecting the continuation of oleate and linoleate desaturation at high rates when oleate synthesis is markedly decreased at night. 4. Changes in the fatty acid composition of 3-sn-phosphatidylcholine accounted for at least 60% of the total decrease in oleate over the dark period. This result is consistent with suggestions that this lipid is the substrate for the leaf microsomal oleate desaturase and an intermediate in leaf glycerolipid biosynthesis.