Regulating effect of β-ketoacyl synthase domain of fatty acid synthase on fatty acyl chain length in de novo fatty acid synthesis

Fatty acid synthase (FAS) is a multifunctional homodimeric protein, and is the key enzyme required for the anabolic conversion of dietary carbohydrates to fatty acids. FAS synthesizes long-chain fatty acids from three substrates: acetyl-CoA as a primer, malonyl-CoA as a 2 carbon donor, and NADPH for reduction. The entire reaction is composed of numerous sequential steps, each catalyzed by a specific functional domain of the enzyme. FAS comprises seven different functional domains, among which the β-ketoacyl synthase (KS) domain carries out the key condensation reaction to elongate the length of fatty acid chain. Acyl tail length controlled fatty acid synthesis in eukaryotes is a classic example of how a chain building multienzyme works. Different hypotheses have been put forward to explain how those sub-units of FAS are orchestrated to produce fatty acids with proper molecular weight. In the present study, molecular dynamic simulation based binding free energy calculation and access tunnels analysis showed that the C16 acyl tail fatty acid, the major product of FAS, fits to the active site on KS domain better than any other substrates. These simulations supported a new hypothesis about the mechanism of fatty acid production ratio: the geometric shape of active site on KS domain might play a determinate role.     

Possible involvement of fatty acid binding protein in peroxisomal β-oxidation of fatty acids

The localization of β-oxidation of fatty acids in isolated peroxisomes from rat liver was investigated. The enzyme system is soluble in the luminal compartment and carnitine does not appear to be involved in the transfer of the CoA derivatives through the peroxisomal membrane. Experiments involving proteolysis, inhibitors and competitive inhibition suggest that a fatty acid binding protein is responsible for the carrier process. This carrier protein seems to be present in increased amounts both in the supernatant and in the peroxisomes after clofibrate induction.     

Modulation of fatty acid-binding capacity of heart fatty acid-binding protein by oxygen — derived free radicals

Summary In this study, we examined the effects of exposure of heart fatty acid-binding protein (h-FABP) to chemically generated O₂ ^– or OH · with respect to its oleate binding and to its electrophoretic properties. Purified rat h-FABP at 40 M scavenged as much as 30% O₂ ^– and 85% OH ·. On the other hand, when 2 nmol (4 M) FABP were exposed to free radicals, the maximum oleate binding capacity as measured by Scatchard analysis was reduced only by 14% and 27% for O₂ ^– and OH ·, respectively. The electrophoretic pattern of free radical-exposed FABP was not markedly different when examined either by the non-denaturing or by denaturing PAGE, suggesting the absence of any degradation or aggregation of FABP by O₂ ^– or OH ·. It is hypothesized that O₂ ^– or OH · in physiological concentration may not alter the function of FABP markedly in the ischemic-reperfused myocardium.Abbreviations h-FABP Heart Fatty Acid Binding Protein - NEFA Non-Esterified Fatty Acids - O₂ ^– Superoxide anions - OH· hydroxyl radicals - OCI hypohalite radicals - H₂O₂ hydrogen peroxide - HPLC High Pressure Liquid Chromatography     

ω-Azido fatty acids as probes to detect fatty acid biosynthesis,degradation, and modification

FAs play a central role in the metabolism of almost all known cellular life forms. Although GC-MS is regarded as a standard method for FA analysis, other methods, such as HPLC/MS, are nowadays widespread but are rarely applied to FA analysis. Here we present azido-FAs as probes that can be used to study FA biosynthesis (elongation, desaturation) or degradation (β-oxidation) upon their uptake, activation, and metabolic conversion. These azido-FAs are readily accessible by chemical synthesis and their metabolic products can be easily detected after click-chemistry based derivatization with high sensitivity by HPLC/MS, contributing a powerful tool to FA analysis, and hence, lipid analysis in general.     

Is fatty acid uptake in cardiomyocytes determined by physicochemical fatty acid partition between albumin and membranes?

Summary Palmitate uptake by isolated, calcium-resistant cardiomyocytes was measured by using a stimulation chamber in which cell contraction can be evoked electrically. Experiments were performed in a medium containing physiological interstitial concentration of albumin (2%) and palmitate/albumin (P/A) ratios ranging from 0.03 to 2.5, and were compared to experiments with fixed P/A ratio (– 1).Initial rate of uptake (V_i) was calculated from fitted uptake vs. time curves as measured by accumulation of radioactivity in the cells from ¹⁴C-labelled palmitate. V_i-vs.-concentration curves exhibited a saturable component, if albumin concentration was kept constant. Almost no change in V_i was observed in experiments performed at constant P/A. This is in contrast to the albumin receptor hypothesis.The ¹⁴C-palmitate content of the myocytes as estimated by thin-layer-chromatography did reach a plateau at 30 s and had the same value at 30 min after administration. The cellular content of labelled palmitate could be attributed to the membrane compartment as calculated from partition coefficient (Kc) of fatty acids (FA) between albumin and membranes. With electrical stimulation V_i-vs.-palmitate concentration kinetics showed a shift in apparent Km from 62 µM (P/A – 0.22) to 23 µM (P/A = 0.08), and presence of 2,4-dinitrophenol increases V_i.Our results suggest that FA-transfer across the sarcolemmal membranes is determined by a physicochemical equilibrium between the compartments of extracellular FA-albumin complex, the membrane lipid phase, intracellular FA binding proteins and the respective aqueous phases. Consequently in cell suspensions the rate of palmitate uptake is controlled by a step of fatty acid metabolism possibly the formation of Fa CoA by the enzyme FA acyl CoA synthetase which is localized in membranes of endoplasmatic reticulum and mitochondria. This step is influenced by the metabolic state of the cells and by FA concentration in membranes.     

Translocation of long chain fatty acids across the plasma membrane – lipid rafts and fatty acid transport proteins

Translocation of long chain fatty acids across the plasma membrane is achieved by a concert of co-existing mechanisms. These lipids can passively diffuse, but transport can also be accelerated by certain membrane proteins as well as lipid rafts. Lipid rafts are dynamic assemblies of proteins and lipids, that float freely within the two dimensional matrix of the membrane bilayer. They are receiving increasing attention as devices that regulate membrane function in vivo and play an important role in membrane trafficking and signal transduction. In this review we will discuss how lipid rafts might be involved in the uptake process and how the candidate proteins for fatty acid uptake FAT/CD36 and the FATP proteins interact with these domains. We will also discuss the functional role of FATPs in general. To our understanding FATPs are indirectly involved in the translocation process across the plasma membrane by providing long chain fatty acid synthetase activity.     

Role of fatty acid uptake and fatty acid β-oxidation in mediating insulin resistance in heart and skeletal muscle

Fatty acids are a major fuel source used to sustain contractile function in heart and oxidative skeletal muscle. To meet the energy demands of these muscles, the uptake and β-oxidation of fatty acids must be coordinately regulated in order to ensure an adequate, but not excessive, supply for mitochondrial β-oxidation. However, imbalance between fatty acid uptake and β-oxidation has the potential to contribute to muscle insulin resistance. The action of insulin is initiated by binding to its receptor and activation of the intrinsic protein tyrosine kinase activity of the receptor, resulting in the initiation of an intracellular signaling cascade that eventually leads to insulin-mediated alterations in a number of cellular processes, including an increase in glucose transport. Accumulation of fatty acids and lipid metabolites (such as long chain acyl CoA, diacylglycerol, triacylglycerol, and/or ceramide) can lead to alterations in this insulin signaling pathway. An imbalance between fatty acid uptake and oxidation is believed to be responsible for this lipid accumulation, and is thought to be a major cause of insulin resistance in obesity and diabetes, due to lipid accumulation and inhibition of one or more steps in the insulin-signaling cascade. As a result, decreasing muscle fatty acid uptake can improve insulin sensitivity. However, the potential role of increasing fatty acid β-oxidation in the heart or skeletal muscle in order to prevent cytoplasmic lipid accumulation and decrease insulin resistance is controversial. While increased fatty acid β-oxidation may lower cytoplasmic lipid accumulation, increasing fatty acid β-oxidation can decrease muscle glucose metabolism, and incomplete fatty acid oxidation has the potential to also contribute to insulin resistance. In this review, we discuss the proposed mechanisms by which alterations in fatty acid uptake and oxidation contribute to insulin resistance, and how targeting fatty acid uptake and oxidation is a potential therapeutic approach to treat insulin resistance.     

Heat stress deteriorates mitochondrial β-oxidation of long-chain fatty acids in cultured fibroblasts with fatty acid β-oxidation disorders

Mitochondrial fatty acids β-oxidation disorder (FAOD) has become popular with development of tandem mass spectrometry (MS/MS) and enzymatic evaluation techniques. FAOD occasionally causes acute encephalopathy or even sudden death in children. On the other hand, hyperpyrexia may also trigger severe seizures or encephalopathy, which might be caused by the defects of fatty acid β-oxidation (FAO). We investigated the effect of heat stress on FAO to determine the relationship between serious febrile episodes and defect in β-oxidation of fatty acid in children. Fibroblasts from healthy control and children with various FAODs, were cultured in the medium loaded with unlabelled palmitic acid for 96 h at 37 °C or 41 °C. Acylcarnitine (AC) profiles in the medium were determined by MS/MS, and specific ratios of ACs were calculated. Under heat stress (at 41 °C), long-chain ACs (C12, C14, or C16) were increased, while medium-chain ACs (C6, C8, or C10) were decreased in cells with carnitine palmitoyl transferase II deficiency, very-long-chain acyl-CoA dehydrogenase deficiency and mitochondrial trifunctional protein deficiency, whereas AC species from short-chain (C4) to long-chain (C16) were barely affected in medium-chain acyl-CoA dehydrogenase and control. While long-chain ACs (C12–C16) were significantly elevated, short to medium-chain ACs (C4–C10) were reduced in multiple acyl-CoA dehydrogenase deficiency. These data suggest that patients with long-chain FAODs may be more susceptible to heat stress compared to medium-chain FAOD or healthy control and that serious febrile episodes may deteriorate long-chain FAO in patients with long-chain FAODs.     

Is the fatty acid composition of Daphnia galeata determined by the fatty acid composition of the ingested diet?

1. The fatty acid (FA) composition of Daphnia galeata and their algal food was analysed and showed many similarities, however, some significant differences were also found in the relative abundance of the FA C16 : 4ω3 and docosahexaenoic acid (DHA). Their relative abundances were much lower in daphnids than in their algal diet.
2. When daphnids were fed three distinct emulsion particles with DHA : eicosapentaenoic acid (EPA) ratios of c. 0.7, 2 and 4, the final DHA : EPA ratio in the daphnids always favoured EPA. The increase of the food DHA : EPA ratio resulted in a minor increase of DHA (to c. 2%). Feeding the animals on emulsion particles with increasing ratios of DHA : EPA, caused a minor ( c. 2%) increase of DHA level but EPA levels remained high ( c. 10%).
3. When labelled with [¹⁴C]linoleic acid and [¹⁴C]linolenic acid daphnids showed low conversion of both essential FA into C20 polyunsaturated fatty acids (PUFAs). This low conversion activity might explain the importance of C20 PUFAs as dietary compounds in the food of Daphnia.
4. The results indicate the insignificance of DHA and C16 : 4ω3 for daphnids. As EPA can be derived from C18 : 3ω3 it is not strictly essential, although it might be a significant factor in food quality for Daphnia.     

A facile preparation of α-hydroxy fatty acids

A convenient, one-step synthesis of α-hydroxy long chain, very long-chain and branched chain fatty acids from non-oxygenated fatty acids is described. The procedure involves preparation of fatty acid dianion using lithium diisopropylamide and oxygenation of the dianion by molecular oxygen.     

Will fatty worms help cure human obesity?

The incidence of obesity has reached epidemic proportions within industrial societies; however, research on human obesity has been hampered by our inability to control genetic and environmental factors. The control of energy homeostasis appears to be conserved among species. Recent creative research in Caenorhabditis elegans, including the application of a genome-wide RNA interference analysis, has provided insight into the genes involved in energy balance. In this article, we discuss the results of these studies and their potential importance to humans.     

Synthesis of β-hydroxy fatty acids and β-amino fatty acids by the strains of Bacillus subtilis producing iturinic antibiotics

The iturinic antibiotics, which contain long chain β-amino acids, are produced by Bacillus subtilis. Screening these strains for the presence of a possible precursor of the iturinic antibiotics, we isolated a lipopeptide containing β-hydroxy fatty acids. The structure of this compound was studied and it appears to be identical or structurally very similar to surfactin. The carbon chain of its β-hydroxy fatty acids was n C₁₆, iso C₁₆, iso C₁₅ or anteiso C₁₅. The percentages of each β-hydroxy fatty acids varied according to the strain producing iturinic antibiotics and were influenced by addition of branched-chain α-amino acids to the culture medium. These results demonstrate for the first time that iso C₁₄ β-hydroxy fatty acid is a constituent present in such a surfactin like lipopeptide. Besides, the presence of radioactive β-hydroxy fatty acids in the phospholipids when the strains were grown in the presence of sodium [¹⁴C]acetate seems also characterize the different strains producing iturinic antibiotics.     

Probing the fatty acid binding site of β-lactoglobulins

The interactions of fatty acids with porcine and bovine β-lactoglobulins were measured using tryptophan fluorescence enhancement. In the case of bovine β-lactoglobulin, the apparent binding constants for most of the saturated and unsaturated fatty acids were in the range of 10^?7 M at neutralpH. Bovine β-lactoglobulin displays only one high affinity binding site for palmitate with an apparent dissociation constant of 1·10^?7 M. The strength of the binding was decreasing in the following way: palmitate > stearate > myristate > arachidate > laurate. Caprylic and capric acids are not bound at all. The affinity of β-lactoglobulin for palmitate decreased as thepH of the incubation medium was lowered and BLG/palmitate complex was not observed atpH's lower than 4.5. Surprisingly, chemically modified bovine β-lactoglobulin and porcine β-lactoglobulin did not bind fatty acids in the applied conditions.     

Does physical inactivity cause nonalcoholic fatty liver disease?

While physical activity represents a key element in the prevention and management of many chronic diseases, we and others believe that physical inactivity is a primary cause of obesity and associated metabolic disorders. Unfortunately, accumulating evidence suggests that we have engineered physical activity out of our normal daily living activity. One such consequence of our sedentary and excessive lifestyle is nonalcoholic fatty liver disease (NAFLD), which is now considered the most common cause of chronic liver disease in Westernized societies. In this review, we will present evidence that physical inactivity, low aerobic fitness, and overnutrition, either separately or in combination, are an underlying cause of NAFLD.     

β-Lapachone alleviates alcoholic fatty liver disease in rats

Alcohol-induced liver injury is the most common liver disease in which fatty acid metabolism is altered. It is thought that altered NAD⁺/NADH redox potential by alcohol in the liver causes fatty liver by inhibiting fatty acid oxidation and the activity of tricarboxylic acid cycle reactions. β-Lapachone (βL), a naturally occurring quinone, has been shown to stimulate fatty acid oxidation in an obese mouse model by activating adenosine monophosphate-activated protein kinase (AMPK). In this report, we clearly show that βL reduced alcohol-induced hepatic steatosis and induced fatty acid oxidizing capacity in ethanol-fed rats. βL treatment markedly decreased hepatic lipids while serum levels of lipids and lipoproteins were increased in rats fed ethanol-containing liquid diets with βL administration. Furthermore, inhibition of lipolysis, enhancement of lipid mobilization to mitochondria and upregulation of mitochondrial β-oxidation activity in the soleus muscle were observed in ethanol/βL-treated animals compared to the ethanol-fed rats. In addition, the activity of alcohol dehydrogenase, but not aldehyde dehydrogenase, was significantly increased in rats fed βL diets. βL-mediated modulation of NAD⁺/NADH ratio led to the activation of AMPK signaling in these animals. Conclusion: Our results suggest that improvement of fatty liver by βL administration is mediated by the upregulation of apoB100 synthesis and lipid mobilization from the liver as well as the direct involvement of βL on NAD⁺/NADH ratio changes, resulting in the activation of AMPK signaling and PPARα-mediated β-oxidation. Therefore, βL-mediated alteration of NAD⁺/NADH redox potential may be of potential therapeutic benefit in the clinical setting.     

Lipase-catalyzed synthesis of vitamin C fatty acid esters

Fatty acid esters of vitamin C (ascorbic acid) where synthesized in a mainly solid-phase system in the presence of small amounts of organic solvent (acetone or t-butanol) catalyzed by immobilized lipase B from Candida antarctica.Highest reaction rates and yields of isolated products were obtained using fatty acid vinyl esters, e.g., 6-O-palmitoyl-l-ascorbic acid was obtained in 91% isolated yield after 48 h. As vitamin C and its esters are very sensitive to oxidation, a mild extraction method for the isolation of reaction products was developed.     

Dietary oxidized fatty acids: an atherogenic risk?

Previous studies have suggested that heated fat that contains oxidized fatty acids in the diet might contribute to the presence of oxidized components in circulating lipoproteins. On the other hand, studies in our laboratory showed that cultured cells such as smooth muscle cells take up oxidized fatty acids poorly. Because intestinal cells are morphologically quite distinct, we studied the uptake of oxidized linoleic acid by Caco-2 and smooth muscle cells (control). When 16-day-old Caco-2 cells were incubated with oxidized linoleic acid (ox-linoleic acid), its uptake was comparable to that of unoxidized linoleic acid (unox-linoleic acid) or that of oleic acid (40;-58, 70, and 55%, respectively). In contrast, the uptake of ox-linoleate by smooth muscle cells was about 3%. To determine whether the brush border structure of Caco-2 cells was responsible for increased uptake of oxidized fatty acids, we compared uptake in 4- and 16-day-old cells. The uptake of unox-linoleate and oleic acid (18:1) was comparable for the 4- and 16-day cells. In addition, saturation and competition experiments showed that the uptake of ox-linoleate by Caco-2 cells is not saturable even at 150 microm and that this uptake is diluted in the presence of unox-linoleate. In esterification experiments utilizing rat intestinal microsomes, we show that both ox- and unox-linoleate are esterified equally well.In summary, dietary oxidized fatty acids can be absorbed by the intestine and incorporated into lipoproteins and could potentially impose an oxidative stress and exacerbate atherogenesis.