Chain length-dependent interaction of free fatty acids with the erythrocyte membrane

Free fatty acids protect erythrocytes against hypotonic haemolysis in a certain low concentration range and become haemolytic at higher concentrations. The chain length dependence of this biphasic behaviour was investigated using human erythrocytes. The results can be summarized as follows: (i) A critical minimum chain length is required for both effects. Octanoic acid (C8) and fatty acids with a shorter chain length do not have any effect on the osmotic resistance of erythrocytes. (ii) Decanoic acid (C10) decreases the extent of hypo-osmotic haemolysis and does not become haemolytic at higher concentrations. (iii) Dodecanoic acid (C12) represents the minimum chain length for the typical concentration-dependent biphasic behaviour with protection against hypo-osmotic haemolysis at a certain low concentration range and subsequent haemolysis at higher concentrations. (iv) Tetradecanoic acid (C14) exhibits two concentration ranges of protection against hypo-osmotic haemolysis, each followed by haemolytic concentrations. (v) The observed effects are not correlated with the critical micellar concentrations of the investigated fatty acids.     

Interaction of free fatty acids with the erythrocyte membrane as affected by hyperthermia and ionizing radiation

The interference of hyperthermia and ionizing radiation, respectively, with the effects of capric (100), lauric (120), myristic (140), oleic (cis-181) and elaidic (trans-181) acids on the osmotic resistance of human erythrocytes was investigated. The results are summarized as follows: (A) not only at 37°, but also at 42° and 47°C lauric acid (120) represents the minimum chain length for the biphasic behaviour of protecting against hypotonic hemolysis at a certain lower concentration range and hemolysis promotion at subsequent higher concentrations; (B) with increasing temperatures the protecting as well as the hemolytic effects occur at lower concentrations of the fatty acids; (C) the increase of temperature promotes the extent of hemolysis and reduces the extent of protection against hypotonic hemolysis; (D) Gamma-irradiation of erythrocytes selectively affects the concentration of oleic acid at which maximum protection against hypotonic hemolysis occurs, without altering the minimum concentration for 100% hemolysis.     

Temperature-dependent specificity of cis-trans isomeric fatty acid interaction with the erythrocyte membrane

Stabilization of red cells against hypotonic haemolysis by cis-trans isomeric free C18 fatty acids occurs with pronounced specificity which is strongly temperature-dependent, but in a distinctly different manner for the two configurational isomers. Oleic acid (cis-18:1) stabilizes very efficiently at 0 degrees C, even at the highest concentrations. Elaidic acid (trans-18:1) causes neither stabilization nor haemolysis at this temperature. At room temperature (23 degrees C), elaidic acid acquires the ability to protect, without turning haemolytic at high concentrations. At 37 degrees C elaidic acid also becomes haemolytic. The protecting effect of oleic acid at 0 degrees C is the result of a rapid reaction. The characteristic, temperature-dependent specificity of cis-trans isomeric C18 fatty acid interaction with the red cell membrane appears to be a general phenomenon, since it was observed alike with erythrocytes of different species.     

Polyunsaturated fatty acids in plasma and erythrocyte membrane lipids of diabetic children

While insulin is a potent activator of essential fatty acid metabolism, portal hypoinsulinemia is common in Type 1 diabetes. Fatty acids were determined by high-resolution capillary gas-liquid chromatography in plasma and erythrocyte membrane lipids in diabetic children (n = 40) and in age-matched healthy controls (n = 40). In plasma phospholipids, values of linoleic acid (23.00 [2.35] vs. 18.13 [2.54], % by wt, median [range from the first to the third quartile], P<0.000l) and alpha-linolenic acid (0.12 [0.06] vs. 0.07 [0.07], P<0.05) were significantly higher in diabetic children than in controls. In contrast, values of arachidonic acid (10.73 [2.34] vs. 11.53 [2.50], P<0.05) and docosahexaenoic acid (2.23 [0.63] vs. 2.77 [0.98], P<0.01) were significantly lower in diabetic children than in controls. Reduced availability of long-chain polyunsaturates in diabetic children suggests that an enhanced dietary supply of long-chain polyunsaturates may be beneficial.     

Mode of interaction of phosphofructokinase with the erythrocyte membrane

Phosphofructokinase is known to associate with the human erythrocyte membrane both in vitro and in vivo. Such association activates the enzyme in vitro by relieving the allosteric inhibition imposed by ATP (Karadsheh, N.S., and Uyeda, K. (1977) J. Biol. Chem. 252, 7418-7420). We now demonstrate that ADP, ATP, and NADH, all of which are known to bind to the enzyme's adenine nucleotide activation site, are particularly potent in eluting the enzyme from the membrane. In addition, both inside-out red cell membrane vesicles and a 23-kDa fragment containing the amino terminus of the membrane protein, band 3, cause a slow, partial, and reversible inactivation of phosphofructokinase. The dependence of the residual phosphofructokinase activity on phosphofructokinase concentration demonstrates that inactivation occurs through the dissociation of active tetramers to inactive dimers. Dimers of phosphofructokinase associate with the membrane more avidly than tetramers. The kinetics of phosphofructokinase inactivation are consistent with the dissociation of tetramers in solution followed by the binding of dimers to the membrane. There is no indication of an association equilibrium between tetramers and dimers of phosphofructokinase bound to the membrane. Taken together, these results suggest that the amino-terminal segment of band 3 binds to the adenine nucleotide activation site, which is thought to be located in a cleft between the dimeric subunits of phosphofructokinase. As a result, band 3 not only rapidly activates the phosphofructokinase tetramer but also slowly inactivates the enzyme by preferentially binding its dissociated subunits.     

A differential scanning calorimetry study of the interaction of free fatty acids with phospholipid membranes

Mixtures of stearic, arachic, oleic and linoleic acids with dimyristoylphosphatidylcholine (DMPC) and distearylphosphatidylcholine (DSPC) have been studied by means of differential scanning calorimetry (DSC). The mixtures of stearic (SA) and arachic acids (AA) with DMPC and DSPC show phase diagrams of the peritectic type, with a region of solid phase immiscibility from 0 to 28.5 mol% of fatty acid. A pure component, with a stoichiometry fatty acid/phospholipid (2:1) seems to be formed except for the system AA/DSPC. The mixtures of oleic (OA) and linoleic acids (LA) show complex phase diagrams. In the case of OA, different regions where a phase separation exists can be observed and for the mixture of OA with DMPC, a pure component seems to be formed with a stoichiometry OA/DMPC (1:2). LA shows different behaviour in the mixtures with DMPC and with DSPC. For the mixture, LA/DMPC, a fluid phase immiscibility region is observed over the same range of concentration as for the mixture with OA, however, the mixture with DSPC shows a solid phase immiscibility for the samples containing 45 mol% or more of LA. The interaction of the different free fatty acids with equimolar mixtures of DMPC and DSPC, showing monotectic behaviour, has also been analyzed. From our results it can be clearly concluded that saturated fatty acids partition preferentially into solid-like domains, while cis-unsaturated fatty acids partition preferentially into fluid-like domains.     

Dietary trans fatty acids modulate erythrocyte membrane fatty acyl composition and insulin binding in monkeys

The substitution of trans- for half of the cis-monounsaturated fatty acids in the diet of Macaca fasicularis monkeys resulted in alterations in erythrocyte fatty acid composition and insulin receptor properties but not in membrane fluidity. Both cis and trans diets contained 10% fat and similar fatty acid compositions, except that approximately 50% of the cis-octadecenoate (c-18:1) in the cis diet was replaced with trans-octadecenoate isomers (t-18:1) in the trans diet. Compared with the cis diet, the trans diet resulted in the incorporation of approximately 11% t-18:1, an approximately 50% decrease in c-18:1, an approximately 16% decrease in total saturated fatty acids, and an approximately 20% increase in 18:2(n-6) in erythrocyte membrane lipids. The increase in 18:2(n-6) may reflect on homeostatic mechanisms designed to maintain overall membrane fluidity, as no diet-related changes in fluidity were observed with diphenylhexatriene steady state fluorescence polarization. Values observed for insulin binding and insulin receptor number were higher and binding affinity was lower in monkeys fed the cis diet. In the absence of an effect on overall membrane fluidity, altered receptor activity suggests that insulin receptor activity is dynamic, requiring specific fluid membrane subdomains or highly specific fatty acid-protein interactions.     

Plasma and erythrocyte membrane long chain polyunsaturated fatty acids in endogenous depression

Patients suffering from endogenous depression had significantly greater proportions of long chain polyunsaturated fatty acids (LCP), docosahexaenoic acid (22:6ω3) and eicosapentaenoic acid (20:5ω3), and a reduced proportion of linoleic acid (18:2ω6) in their plasma choline phosphoglycerides (CPG) compared with age-sex-matched healthy controls. Patients suffering from reactive depression or from other psychiatric disorders did not differ significantly from matched controls. Changes in the fatty acid composition of the major erythrocyte membrane phospholipids were in the same direction but were less marked. The importance and possible reasons for the elevated LCP in endogenous depression are discussed.     

The interaction of calmodulin with erythrocyte membrane proteins

The method of sedimentation equilibrium in an air-driven ultracentrifuge (Airfuge) has been employed to investigate the interaction of 125I-calmodulin with the cytoskeletal components of the human red cell membrane. The results indicate significant calcium-dependent calmodulin binding activity in the low and high ionic strength extracts of the human erythrocyte membrane. The interaction of 125I-calmodulin with the low ionic strength extract proteins is analysed quantitatively. Further purification of the high ionic strength extract comprising mainly band 2.1 and band 4.1 results in the elution of calmodulin binding activity in a purified fraction of band 4.1.     

Biphasic interaction of Triton detergents with the erythrocyte membrane.

Octylphenoxy polyoxyethylene ethers (Triton detergents) interact with the erythrocyte membrane in a biphasic manner, i.e. they stabilize erythrocytes against hypo-osmotic haemolysis at low concentrations (0.0001-0.01%, v/v), but become haemolytic at higher concentrations. This biphasic behaviour was demonstrated with Triton X-114, Triton X-100 and Triton X-102. However, a critical chain length is a prerequisite for the haemolytic effect, because Triton X-45, which differs from the other Tritons only by the shorter chain of the polyoxyethylene residue, does not exhibit this biphasic behaviour, but goes on protecting against osmotic rupture up to saturating concentrations. Even a 1% solution of Triton X-45 does not cause haemolysis. This structural specificity of Triton X-45, namely the lack of haemolysis and efficient stabilization against osmolysis even at higher concentrations of the detergent, is exhibited at 0 degree and 37 degrees C as well as at room temperature. Three conclusions are reached: (i) a critical chain length of the octylphenoxy polyoxyethylene ethers is required for the haemolytic effect; (ii) the different structural requirements would suggest that different mechanisms are responsible for the haemolytic and the stabilizing effect of amphiphilic substances; (iii) the results suggest that haemolysis is not caused simply by dissolution of the membrane by the detergent but is a rather more specific process.     

游离脂肪酸受体蛋白研究进展

游离脂肪酸不仅是人和动物体的一种重要能量来源,也是一种重要的信号分子。最近研究表明,游离脂肪酸受体蛋白在维持机体内的葡萄糖稳衡、脂肪形成、白细胞功能等生理过程中都有重要的作用,对于调控人或动物的营养代谢及疾病发生具有重要生理意义。     

G protein-coupled receptors for free fatty acids

Free fatty acids (FFAs) are not only an important direct source of energy but they also play key roles in regulating a range of physiological responses. Although it was long assumed that such effects of FFAs must occur following cellular uptake, and potentially via their conversion to fatty acyl-CoAs, it is now apparent that FFAs also function directly as agonists at a number of G protein-coupled receptors (GPCRs). Tissue distribution studies and, subsequently, siRNA knock-down experiments have indicated key roles for these GPCRs in glucose homeostasis, adipogenesis, white cell recruitment and potentially in a range of other processes. Considerable interest is thus now centred on the generation of potent and selective small molecule ligands, both as tool compounds to further unravel the biology and physiological role of this group of GPCRs and as starting points for possible therapeutic intervention in a range of areas, particularly those associated with 'metabolic syndrome'.     

Effects of ethanol on the incorporation of free fatty acids into cerebral membrane phospholipids

Chronic ethanol exposure is known to affect deacylation-reacylation of membrane phospholipids (PL). In our earlier studies we have demonstrated that chronic exposure to ethanol (EtOH) leads to a progressive increase in membrane phospholipase A2 (PLA2) activity. In the current study, we investigated the effects of chronic EtOH exposure on the incorporation of different free fatty acids (FFAs) into membrane PL. The results suggest that the incorporation of fatty acids into four major PL varied from 9.6 fmol/min/mg protein for docosahexaenoic acid (DHA) into phosphatidylinositol (PI) to 795.8 fmol/min/mg protein for linoleic acid (LA) into phosphatidylcholine (PC). These results also suggest a preferential incorporation of DHA into PC; arachidonic acid (AA) into PI; oleic acid into phosphatidylethanolamine (PE) and PC; LA into PC and stearic acid into PE. Chronic EtOH exposure affected the incorporation of unsaturated fatty acid into PI, phosphatidylserine (PS) and PC. However, EtOH did not affect significantly the incorporation of any of the fatty acids (FA) studied into PE. No significant differences were observed with the stearic acid. It is suggested that acyltransferases may play an important role in the membrane adaptation to the injurious effects of EtOH.     

Interaction of amphiphiles with integral membrane proteins. I. Structural destabilization of the anion transport protein of the erythrocyte membrane by fatty acids, fatty alcohols, and fatty amines

The effect of model amphiphiles on the structural stability of the anion exchange protein (band 3) of the human erythrocyte membrane was studied by differential scanning calorimetry. The concentration of membranes, as well as the concentration, head group, alkyl chain length, degree of unsaturation, and double bond configuration of a variety of alkane derivatives were all varied in a systematic way. The depression of the denaturation temperature of band 3 per unit membrane concentration of the amphiphile was then determined in order to quantitate the potency of each drug. Saturated fatty acids of chain length C8 to C24 displayed a monotonic decrease in potency up to C20, followed by a dramatic diminution in potency at C22 and C24. Unsaturation caused only minor increases in the abilities of fatty acids to perturb the anion exchanger, and surprisingly, there was neither a trend for the number of double bonds nor a significant cis-trans distinction. Arachidonic acid, as an exception, was much more effective than any other amphiphile in destabilizing band 3. Fatty acids were about three times more potent than fatty amines and fatty alcohols; however, the enhanced partitioning of the latter into the membrane compensated at certain membrane/buffer ratios for its reduced intrinsic potency. A quantitative model interpretation of the data is presented in an accompanying paper.     

Phospholipase activation, free fatty acids and the proton permeability of a biological membrane

The rate of collapse of a proton gradient across the apical membrane of rat kidney proximal tubule increases upon treatment with calcium, mercuric chloride and mellitin, substances which activate phospholipase A2. Treatment with phospholipase A2 or oleic acid also enhances the rate of proton gradient dissipation. Membrane water permeability is not affected. This phenomenon may have implications in pathological states arising from ischemia or toxic exposure.     

Dietary free fatty acids form alkaline phosphatase-enriched microdomains in the intestinal brush border membrane

Free fatty acids released during intralumenal digestion of dietary fat must pass through the enterocyte brush border membrane before triacylglycerol reassembly and subsequent chylomicron delivery to the lymph system. In the present work fluorescent BODIPY fatty acid analogs were used to study this membrane passage in organ cultured intestinal mucosal explants. We found that in addition to a rapid uptake into the cytoplasm, a fraction of the fatty acid analogs were inserted directly into the brush border membrane. Furthermore, a brief exposure of microvillar membrane vesicles to a fat mixture mimicking a physiological solution of dietary mixed micelles, rearranged the lipid raft microdomain organization of the membranes. Thus, the fat mixture generated a low-density subpopulation of microvillar detergent resistant membranes (DRMs) highly enriched in alkaline phosphatase (AP). Since this GPI-linked enzyme is the membrane protein in the brush border with the highest affinity for lipid rafts, this implies that free fatty acids selectively insert stably into these membrane microdomains. We have previously shown that absorption of dietary lipids transiently induce a selective endocytosis of AP from the brush border, and from work by others it is known that fat absorption is accompanied by a rise in serum AP and secretion of surfactant-like particles from enterocytes. We propose that these physiological processes may be triggered by the sequestering of dietary free fatty acids in lipid raft microdomains of the brush border.     

Dietary free fatty acids form alkaline phosphatase-enriched microdomains in the intestinal brush border membrane

Abstract

Free fatty acids released during intralumenal digestion of dietary fat must pass through the enterocyte brush border membrane before triacylglycerol reassembly and subsequent chylomicron delivery to the lymph system. In the present work fluorescent BODIPY fatty acid analogs were used to study this membrane passage in organ cultured intestinal mucosal explants. We found that in addition to a rapid uptake into the cytoplasm, a fraction of the fatty acid analogs were inserted directly into the brush border membrane. Furthermore, a brief exposure of microvillar membrane vesicles to a fat mixture mimicking a physiological solution of dietary mixed micelles, rearranged the lipid raft microdomain organization of the membranes. Thus, the fat mixture generated a low-density subpopulation of microvillar detergent resistant membranes (DRMs) highly enriched in alkaline phosphatase (AP). Since this GPI-linked enzyme is the membrane protein in the brush border with the highest affinity for lipid rafts, this implies that free fatty acids selectively insert stably into these membrane microdomains. We have previously shown that absorption of dietary lipids transiently induce a selective endocytosis of AP from the brush border, and from work by others it is known that fat absorption is accompanied by a rise in serum AP and secretion of surfactant-like particles from enterocytes. We propose that these physiological processes may be triggered by the sequestering of dietary free fatty acids in lipid raft microdomains of the brush border.     

Modulation of ATPase activities of human erythrocyte membranes by free fatty acids or phospholipase A2

Summary The artificial insertion of increasing amounts of unsaturated fatty acids into human erythrocyte membranes modulated ATPase activities in a biphasic manner, depending on the number and position of double bonds, their configuration, and the chain length. Uncharged long-chain fatty acid derivatives with double bonds and short-chain fatty acids were ineffective. Stearic acid stimulated Na⁺K⁺-ATPase only. Anionic and non-ionic detergents and -lysophosphatidylcholine failed to stimulate ATPase activities at low, and inhibited them at high concentrations.Mg²⁺-ATPase activity was maximally enhanced by a factor of 2 in the presence of monoenoic fatty acids; half-maximal stimulation was achieved at a molar ratio ofcis(trans)-configurated C18 acids/membrane phopholipid of 0.16 (0.26).Na⁺K⁺-ATPase activity was maximally augmented by 20% in the presence of monoenoic C18 fatty acids at 37°C. Half-maximal effects were attained at a molar ratio oleic (elaidic) acid/phospholipid of 0.032 (0.075). Concentrations of free fatty acids which inhibited ATPase activities at 37°C were most stimulatory at reduced temperatures. AT 10°C, oleic acid increased Na⁺K⁺-ATPase activity fivefold (molar ratio 0.22).Unsaturated fatty acids simulated the effect of calmodulin on Ca²⁺-ATPase of native erythrocyte membranes (i.e., increase ofV _max from 1.6 to 5 mol PO ₄ ^3– ·phospholipid^–1·hr^–1, decrease of K _Ca from 6 m to 1.4–1.8 m). Stearic acid decreasedK _Ca (2 m) only, probably due to an increase of negative surface charges.A stimulation of Mg²⁺-ATPase, Na⁺K⁺-ATPase, and Ca²⁺-ATPase could be achieved by incubation of the membranes with phospholipase A₂.An electrostatic segregation of free fatty acids by ATPases with ensuing alterations of surface charge densities and disordering of the hydrophobic environment of the enzymes provides an explanation of the results.