Presence Of Nonesterified Fatty Acids Research Articles

NEFA-Sensitive Orai1 Expression in Regulation of De Novo Lipogenesis

Background/Aims: Non-esterified fatty acids (NEFAs) are important inducers of inflammatory responses and hepatic lipid accumulation, which lead to non-alcoholic fatty liver disease (NAFLD). High plasma NEFA is found in NAFLD patients, and associated with metabolic syndrome and type-2 diabetes. NFκB is known to upregulate Orai1, the Ca²⁺ channel responsible for store-operated Ca²⁺ entry. The present study explored the role of NEFA-sensitive NFκB-dependent Orai1 expression in the regulation of lipid synthesis. Methods: BRL-3A rat liver hepatocyte lines were studied in the absence and presence of NEFA. Transcript and protein expression levels of factors involved in lipid synthesis were quantified by quantitative polymerase chain reaction (qPCR) and western blot analyses. Fatty acids were measured by immunofluorescence. Results: NEFA significantly increased, as indicated by the expression of sterol regulatory element-binding protein 1 (SREBP-1c), fatty acid synthase (FAS), acetyl-CoA carboxylase α (ACC1), Orai1, and NFκB p65 by qPCR and western blot analyses. These effects were reversed by the Orai1 inhibitor, 2-aminoethoxydiphenyl borate, and the NFκB inhibitor, wogonin. Furthermore, SREBP-1c, FAS, ACC1, and Orai1 were significantly decreased by Orai1 silencing. Conclusions: Taken together, these results demonstrated that NEFA-sensitive NFκB-dependent Orai1 expression regulates de novo lipogenesis.

Nonesterified Fatty Acid-Induced Endoplasmic Reticulum Stress in Cattle Cumulus Oocyte Complexes Alters Cell Metabolism and Developmental Competence.

Reduced oocyte quality has been associated with poor fertility of high-performance dairy cows during peak lactation, due to negative energy balance. We examined the role of nonesterified fatty acids (NEFAs), known to accumulate within follicular fluid during under- and overnutrition scenarios, in causing endoplasmic reticulum (ER) stress of in vitro maturated cattle cumulus-oocyte complexes (COCs). NEFA concentrations were: palmitic acid (150 μM), oleic acid (200 μM), and steric acid (75 μM). Abattoir-derived COCs were randomly matured for 24 h in the presence of NEFAs and/or an ER stress inhibitor, salubrinal. Total and hatched blastocyst yields were negatively impacted by NEFA treatment compared with controls, but this was reversed by salubrinal. ER stress markers, activating transcription factor 4 (Atf4) and heat shock protein 5 (Hspa5), but not Atf6, were significantly up-regulated by NEFA treatment within whole COCs but reversed by coincubation with salubrinal. Likewise, glucose uptake and lactate production, measured in spent medium samples, showed a similar pattern, suggesting that cumulus cell metabolism is sensitive to NEFAs via an ER stress-mediated process. In contrast, while mitochondrial DNA copy number was recovered in NEFA-treated oocytes, oocyte autofluorescence of the respiratory chain cofactor, FAD, was lower following NEFA treatment of COCs, and this was not reversed by salubrinal, suggesting the negative impact was via reduced mitochondrial function. These results reveal the significance of NEFA-induced ER stress on bovine COC developmental competence, revealing a potential therapeutic target for improving oocyte quality during peak lactation.

Evidence for Presence of Nonesterified Fatty Acids as Potential Gustatory Signaling Molecules in Humans

Gustatory fatty acid signaling termed "fatty acid taste" is initiated when nonesterified fatty acids (NEFA) bind to putative fat receptors on taste receptor cells. However, the source and quantity of NEFA in the oral cavity of humans are unresolved. Dietary fat is comprised predominantly of triacylglycerol, and human lingual lipase is of questionable functionality. The objective of this study was to characterize the species of NEFA in saliva and quantify their individual concentrations during oral processing of high-fat foods. Participants chewed fixed amounts of almonds, coconut, walnuts, almond butter, and olive oil (stimuli that vary in physical state and fatty acid composition) for 1 min at the rate of 1 bite/s and expectorated. The salivary NEFA from the expectorant were quantitatively and qualitatively analyzed by gas chromatography-mass spectrometry. Palmitic, oleic, linoleic, and stearic acids were the 4 predominant salivary NEFA, reflecting their concentrations in the foods tested. Their significantly increased concentrations ranged from 20 to 60 µM. Previous animal electrophysiological studies suggest that these NEFA concentrations are sufficient to depolarize taste receptor cells. These data indicate NEFA concentrations likely to be sufficient to initiate gustatory signaling are present in the human oral cavity when masticating high-fat foods.

Compound K protects MIN6N8 pancreatic β‐cells against palmitate‐induced apoptosis through modulating SAPK/JNK activation

Chronic elevation of NEFAs (non-esterified fatty acids) due to insulin resistance and obesity has been shown to be associated with increased beta-cell apoptosis and with the aetiology of the reduced beta-cell mass of Type 2 diabetes. SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase) have been implicated in the control of apoptosis. C-K [compound K; 20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol] is the main intestinal bacterial metabolite of protopanaxadiol ginsenosides. Currently, little is known about the effects of C-K on beta-cells with the presence of NEFAs. The aim of the present study was to investigate the in vitro protective effect of C-K on MIN6N8 mouse insulinoma beta-cells against NEFA-induced apoptosis, as well as the modulating effect on SAPK/JNK activation. Our results have shown that C-K inhibited the palmitate-induced apoptosis through modulating SAPK/JNK activation. We conclude that C-K protects against beta-cell death and that, by anti-apoptotic activity, C-K may contribute to the previously reported anti-diabetic actions of ginseng.

Effects of non-esterified fatty acids on bovine granulosa cells and developmental potential of oocytes in vitro

High yielding dairy cows experience a negative energy balance (NEB) shortly after parturition, which is accompanied by high concentrations of non-esterified fatty acids (NEFA) in blood up to approximately 3 weeks post partum. We hypothesized that the elevated plasma NEFA concentration causes lower fertility by exerting negative effects on granulosa cells and oocytes in the ovary, leading to less viable embryos and insufficient corpora lutea. In two series of experiments, we studied the effects of a realistic NEFA (C18:1) concentration on both the proliferation and the progesterone production of follicular granulosa cells in vitro (part I) and on maturation, fertilization and developmental potential of oocytes (part II). For part I, granulosa cells were added to 4 groups of dishes with four different media and cultured for nine consecutive days. After a preculture period of 42 h, the presence of NEFA had a negative effect on the proliferation of granulosa cells. No effect of NEFA on the amount of progesterone production per cell was observed. For part II, a total of 1804 cumulus-oocyte-complexes were collected from slaughterhouse ovaries. Using a subgroup of 690 COC, maturation medium with NEFA caused a delay in maturation. Using another 1114 COC, fertilization, cleavage, and embryonic development after maturation in presence of NEFA were significantly reduced. We concluded that the presence of NEFA in follicular fluid and blood of post partum cows may reduce fertility due to hampered embryonic development and subnormal CL function.

Formation of new HDL particles from lipid-free apolipoprotein A-I.

Remodelling of plasma high density lipoproteins (HDL) promotes the dissociation of lipid-free apolipoprotein (apo)A-I from the particles. In the present study, we have investigated the formation of new HDL particles from lipid-free apoA-I in a process dependent on the presence of nonesterified fatty acids (NEFA) and other lipoprotein fractions (as donors of lipid). Incubations were carried out that included lipid-free apoA-I, VLDL, and lipoprotein lipase (LPL) or lipid-free apoA-I, either VLDL or LDL, and sodium oleate. Any new HDL particles that were formed were separated from lipid-free apoA-I in the ultracentrifuge. When any one of the ingredients in the incubation was absent, the apoA-I remained lipid-free; however, when all the ingredients were present, a significant proportion of the apoA-I was recovered in the HDL density fraction. This coincided with the formation of at least three HDL-sized subpopulations; one of the subpopulations was considerably smaller than HDL3c and had pre-beta 1 mobility while two were in the size range of human HDL2b and HDL3c and had pre-beta 2 electrophoretic mobility. The new HDL were predominantly discoidal in shape and their major constituents were apoA-I, phospholipid, and unesterified cholesterol. In conclusion, these results show that lipid-free apoA-I can form new HDL particles in the presence of NEFA and other lipoprotein fractions. The formation of pre-beta 1 HDL from lipid-free apoA-I indicates that this process is potentially of great importance in terms of generating plasma acceptors of cell cholesterol in reverse cholesterol transport.

Free thyroxine assessed with three assays in sera of patients with nonthyroidal illness and of subjects with abnormal concentrations of thyroxine-binding proteins

Three methods for estimating free thyroxine (FT4) in serum were studied: equilibrium dialysis, the SPAC-ET FT4 radioimmunoassay kit, and the Amerlite MAB FT4 luminometric assay. Serum samples from 10 subjects with above-normal thyroxine-binding globulin (TBG), 6 with low TBG, 30 with familial dysalbuminemic hyperthyroxinemia (FDH), 13 with nonesterified fatty acids (NEFA) concentrations in serum > 1.0 mmol/L, and 178 patients with various degrees of nonthyroidal illness (NTI) were measured and compared with samples from 42 euthyroid blood donors. The Amerlite MAB FT4 assay compared well with equilibrium dialysis, whereas the SPAC-ET assay averaged 40% lower. All three assays were not influenced by changes in TBG and showed no or only little changes in the presence of NEFA. Mean FT4 values in the FDH samples were somewhat higher than in controls when measured with the SPAC-ET assay, about equal with equilibrium dialysis, and somewhat below the mean control value with the Amerlite MAB FT4 assay, although individual results were within the control reference range. In NTI patients, no FT4 values were below the control reference range by the Amerlite MAB FT4 assay, 4 of 178 were below this range by equilibrium dialysis, and 1 of 178 was below this range by the SPAC-ET assay. In all assays a large proportion of the NTI samples showed FT4 values above the control reference range, a result that will interfere with the efficacy of these assays for assessing thyroid function in NTI patients.

Nonesterified fatty acids and lipid peroxidation.

Oxygen free radicals damage cells through peroxidation of membrane lipids. Gastrointestinal mucosal membranes were found to be resistant to in vitro lipid peroxidation as judged by malonaldehyde and conjugated diene production and arachidonic acid depletion. The factor responsible for this in this membrane was isolated and chemically characterised as the nonesterified fatty acids (NEFA), specifically monounsaturated fatty acid, oleic acid. Authentic fatty acids when tested in vitro using liver microsomes showed similar inhibition. The possible mechanism by which NEFA inhibit peroxidation is through iron chelation and iron-fatty acid complex is incapable of inducing peroxidation. Free radicals generated independent of iron was found to induce peroxidation of mucosal membranes. Gastrointestinal mucosal membranes were found to contain unusually large amount of NEFA. Circulating albumin is known to contain NEFA which was found to inhibit iron induced peroxidation whereas fatty acid free albumin did not have any effect. Addition of individual fatty acids to this albumin restored its inhibitory capacity among which monounsaturated fatty acids were more effective. These studies have shown that iron induced lipid peroxidation damage is prevented by the presence of nonesterified fatty acids.

Effects of various non-esterified fatty acids on the transfer of cholesteryl esters from HDL to LDL induced by the cholesteryl ester transfer protein

The effects of saturated, monounsaturated and polyunsaturated non-esterified fatty acids on the rate of transfer of radiolabeled cholesteryl esters from high density lipoproteins (HDL) to low density lipoproteins (LDL), induced by the cholesteryl ester transfer protein (CETP), have been studied. Human high-density lipoproteins-subfraction 3 (HDL 3) containing radiolabeled cholesteryl esters were incubated with LDL at 37°C with or without CETP and in the absence or in the presence of non-esterified fatty acids. Less than 6% of the total radioactivity was recovered in the LDL fraction after incubation of HDL 3 and LDL for 3 h at 37°C in the absence of CETP, regardless of whether or not non-esterified fatty acids were added. The addition of CETP to the incubation mixture induced a time-dependent redistribution of radiolabeled cholesteryl esters from HDL 3 to LDL. Non-esterified fatty acids were found to alter the rate of transfer of cholesteryl esters induced by CETP. While short chain saturated non-esterified fatty acids (caprylic and capric acids) had no effect on the rate of transfer of cholesteryl esters, the medium and long chain (caprylic and capric acids) had no effect on the rate of transfer of cholesteryl esters, the medium and long chain ones (lauric, myristic, palmitic and stearic acids) significantly increased the CETP-mediated transfers from HDL 3 to LDL. At low concentrations, unsaturated fatty acids also stimulated the CETP-mediated redistribution of radiolabeled cholesteryl esters from HDL 3 to LDL. As the concentration of either oleic, linoleic or arachidonic acids increased to higher levels, a significant proportion of fatty acids remained unassociated with lipoprotein particles. Under these circumstances the transfer process was inhibited. These results show that non-esterified fatty acids can modulate the CETP-mediated transfer of cholesteryl esters from HDL to LDL and that this effect is dependent on both the length and the degree of unsaturation of their monomeric carbon chain.

Nonesterified fatty acids inhibit iron-dependent lipid peroxidation

The effect of various fatty acids on lipid peroxidation of liver microsomes induced by different methods in vitro was studied using oxygen uptake and malonaldehyde (MDA) production. It was observed that fatty acids with a single double bond are effective inhibitors of peroxidation. Stereo and positional isomers of oleic acid were equally effective as oleic acid. There was an absolute requirement for a free carboxyl group, since methyl esters of fatty acids and long-chain saturated and unsaturated hydrocarbons could not inhibit peroxidation. Saturated fatty acids with a chain length of 12-16 carbon atoms showed inhibition, whereas more than 18 carbon atoms reduced the inhibitory capacity. Fatty acids of lower chain length such as capric and caprylic acids did not show inhibition. Fatty acid inhibition was partially reversed by increasing the concentration of iron in the system. Peroxidation induced by methods which were independent of iron was not inhibited by fatty acids. It was observed that intestinal microsomes which were resistant to peroxidation due to the presence of nonesterified fatty acids in their membrane lipids were able to peroxidise by methods which do not require iron. These results suggest that certain fatty acids inhibit peroxidation by chelating available free iron. In addition, they may also be involved in competing with the esterified fatty acids in the membrane lipids which are the substrates for peroxidation.

The effect of non-esterified fatty acids on the proton-pumping cytochrome c oxidase reconstituted into liposomes.

Bovine heart cytochrome c oxidase was reconstituted in phospholipid vesicles, and the effect of different non-esterified fatty acids (NEFA) was studied on its proton pump and on the proton permeability of the vesicles. Neither parameter appeared to be affected by concentrations of NEFA known to uncouple oxidative phosphorylation (10 microM). Also the permeability for K+ was not affected by them. The fatty acids caused an increase in the rate of electron transfer in the absence, but not in the presence, of uncoupler and/or valinomycin [diminution of the respiratory-control index (RCI)]. The RCI of 8.7-7.5 was decreased to about 4.5 in the presence of 0.27-10 microM-NEFA. Oleic acid was not effective at the above concentrations. Subunit III-depleted enzyme preparations gave vesicles with an RCI of about 5.5, which was decreased to 4.5 in the presence of NEFA. With both native and subunit III-depleted oxidase the RCI was never decreased to the value of 1 by NEFA, as happens with classical protonophores.

Identification of NaK-ATPase inhibitors in human plasma as nonesterified fatty acids and lysophospholipids.

Elevated plasma levels of factors with cardiac glycoside-like activity have been implicated in the response to volume expansion in animals and in the pathogenesis of certain human diseases. We recently described four fractions (IR1, EI1, EI2, EI3) from normal human plasma that inhibit NaK-ATPase, displace ouabain from the enzyme, and exhibit digoxin-like immunoreactivity (Kelly, R. A., O'Hara, D. S., Canessa, M. L., Mitch, W. E., and Smith, T. W. (1985) J. Biol. Chem. 260, 11396-11405). In this report, we identify the active component of these plasma fractions as long-chain nonesterified fatty acids (NEFA) and lysophospholipids. These lipids were present in fractions EI1, EI2, and EI3 in quantities sufficient to account for all of the NaK-ATPase inhibitory activity. The digoxin-like immunoreactivity in fraction IR1 could be attributed to hydrocortisone and other endogenous steroids. To explore the nature of the lipid-NaK-ATPase interactions, we examined the effects of various ATP or sodium concentrations on the NaK-ATPase activity measured in the presence of NEFA. Varying sodium did not affect the inhibition of NaK-ATPase by linoleic acid. At less than 0.15 mM ATP, linoleic acid stimulated NaK-ATPase, but at higher ATP concentrations, the enzyme was progressively inhibited. In summary, NEFA and lysophospholipids, at levels similar to those occurring in human plasma, may account for all of the NaK-ATPase inhibitory activity observed in human plasma fractions. These lipids probably do not directly regulate NaK-ATPase in vivo under normal physiologic conditions, but may alter the sodium pump in disease states characterized by abnormalities in lipid metabolism or plasma protein binding.

Decrease by cycloheximide of calcium binding and nonesterified fatty acids in rat-intestinal Golgi-enriched membrane fractions

Rat intestinal Golgi-enriched membrane fractions bind more Ca 2+ than do basolateral and microvillus-enriched membrane fractions, and this uptake is reduced by vitamin D-deficiency. The effect of the protein synthesis inhibitor, cycloheximide, on this Ca 2+ binding was determined in rats fed a normal, vitamin D-sufficient diet. Cycloheximide, 1.5 mg/kg, rapidly reduced protein synthesis (measured by [ 3H]leucine incorporation) to 12% of control values within 15 min, but Ca 2+ binding diminished gradually to 50% of control values by 60 min. Ca 2+ transport across gut sacs was also decreased. The reduction in Ca 2+ binding was not due to an alteration in vesicle morphology or to a direct effect of cycloheximide. Nonesterified (free) fatty acids, the probable binding sites for Ca 2+ in these membrane fractions, were reduced by cycloheximide to 48% of control values by 60 min. There was no significant change in total lipid phosphate. Cycloheximide may affect the synthesis of proteins necessary for the presence of nonesterified fatty acids in these Golgi membranes.

Differential transplacental binding of diazepam: causes and implications.

Diazepam plasma binding was determined in 17 matched pairs of maternal and foetal plasma, collected at delivery. Diazepam % free was higher (p less than 0.001) in maternal (mean 3.24%) than in either umbilical venous (mean 1.50%) or umbilical arterial (mean 1.24%) plasma. The data from in vitro dialysis studies were consistent with the reported higher diazepam concentrations in infants than in mothers at delivery. Plasma nonesterified fatty acids (NEFA) concentrations were higher (p less than 0.001) in maternal (mean = 643 microM) than in matched umbilical venous plasma (means = 211 microM) and there was a significant correlation (p less than 0.01) between diazepam % free and corresponding plasma NEFA concentration for pooled data (r = 0.871, n = 34). Multiple and partial regression analysis indicates that transplacental differences in albumin, bilirubin and total protein concentrations made a minimal contribution to diazepam binding differences between mother and foetus and that approximately 76% of the variability in diazepam % free was accounted for by plasma NEFA concentration. The binding of diazepam to human serum albumin (HSA) was markedly perturbed by the presence of NEFA but not by bilirubin and there was no apparent cooperativity between bilirubin and NEFA on diazepam-HSA binding. Moreover, our findings provide further evidence that substantial differences in binding affinities exist between foetal and maternal plasma albumins.

The effect of physiological levels of non-esterified fatty acids on the radioimmunoassay of prostaglandins

Non-esterified fatty acids (NEFA) can significantly interfere with the radioimmunoassay of PGE and PGF 2α using commercially available anti-sera. PGB 1 antigen-antibody binding is 50% inhibited by 110 pg of PGB 1, 48 ng of PGE 1, 3.5 μg of PGF 2α, or 9.0 μg linoleic, 14 μg arachidonic, 22 μg δ-linoleic, 40 μg palmitoleic or 45 μg oleic acids. PGF 2α antigen-antibody binding is 50% inhibited by 270 pg of PGF 2α, 70 ng of PGE 1, or 4.2 μg arachidonic, 14 μg δ-linolenic, 22 μg linoleic, 70 μg palmitoleic or 110 μg oleic acids. Physiological levels of NEFA, such as the quantities found in small volumes of plasma, are sufficient to prohibit accurate prostaglandin measurements. Chromatography on small columns of silicic acid proved to be an effective technique for separation of NEFA and prostaglandin from lipid extracts, however, the results of this study suggest that the interference produced by the presence of NEFA in the measurement of prostaglandin from certain physiological fluids may be avoided if the prostaglandins are not extracted prior to radioimmunoassay.

The binding of L-tryptophan to serum albumins in the presence of non-esterified fatty acids.

Bovine, human and rat serum albumins were defatted and palmitic acid, oleic acid and lauric acid added in various molar ratios. The binding of L-tryptophan to these albumins was measured at 20 degrees C in a 0.138 M salt solution at pH 7.4, by using an ultrafiltration technique, and analysed in terms of n, the number of available tryptophan-binding sites per albumin molecule, with apparent association constant, k. 2. n and k were 0.90 and 2.3x10(-4)M(minus-1) respectively for defatted bovine serum albumin and 0.87 and 9.7x10(-3)M(-minus-1) for human albumin. Addition of palmitic acid did not decrease n until the molar ratio, fatty acid/bovine albumin, approached and exceeded 2. The decrease in k was small and progressive. In contrast, lauric caused a marked decrease in n and k at ratios as low as 0.5. A similar distinction between the effects on n of palmitic acid and oleic acid and those of lauric acid was seen for human albumin. k for human albumin was not significantly affected by fatty acids under the conditions studied. 3. It is concluded that primary long-chain fatty acid sites interact only weakly with the tryptophan site on albumin and that inhibition of tryptophan binding occurs when secondary long-chain sites are occupied. Primary medium-chain fatty acid sites are distinct from primary long-chain sites but may be grouped with secondary long-chain sites. 4. The relationship between free and bound tryptophan in samples of rat plasma (Stoner et al., 1975) is discussed in terms of a similar but limited study of rat albumin.