Effect of calcium, insulin and growth hormone on membrane fluidity. A spin label study of rat adipocyte and human erythrocyte ghosts

ESR spectra were recorded from rat epididymal adipocyte ghosts labeled with the 5-nitroxide stearic acid spin probe, I(12,3). Polarity-corrected and approximate order parameters, that are sensitive to the flexibility of the incorporated label, were used to evaluate the membrane lipid fluidity. Addition of CaCl2 a 37 degrees C decreased the fluidity, as indicated by positive increases in the order parameters. The ordering effect of Ca2+ was concentration-dependent, reached saturation at approx. 3--4 mM, and was completely reversed by excess EGTA. Previous studies indicated that low- and high-affinity sites on adipocyte plasma membranes are able to bind 45Ca2+, and our results suggest that Ca2+-induced alterations in the lipid fluidity involve cation binding to low-affinity sites. The cellular movements of Ca2+ and, in particular, the binding of Ca2+ to the plasma membrane may play important roles in insulin's action on fat cell function. The possibility that insulin directly alters the membrane fluidity was tested by adding hormone to freshly-prepared I(12,3)-labeled adipocyte ghosts. Insulin, at concentrations (10(-6) M) that enhance glucose uptake into intact adipocytes, did not affect the fluidity of ghosts suspended in buffers with or without Ca2+. The fluidities of I(12,3)-labeled rat adipocyte ghosts or human erythrocyte ghosts were also unaffected by various forms of human growth hormone.     

Increase in membrane fluidity and opening of tight junctions have similar effects on sodium-coupled uptakes in renal epithelial cells

Apical membranes of renal epithelial cells were shown to be more rigid than other plasma membranes, due in part to the abundance of sphingomyelin among their constituent phospholipids. Tight junctions play a key role in maintaining differences between the apical and the basolateral domains of the plasma membrane with respect to their lipid composition and fluidity. To evaluate the influence of alterations of membrane fluidity on the activity of two apically located transport systems, we compared the effect of opening of tight junctions, by a preincubation period in calcium-deprived medium and of increasing fluidity, with benzyl alcohol, on Na-dependent uptakes of Pi and alpha-methyl-D-glucopyranoside (MGP) in intact, confluent LLC-PK1 cells and MDCK cells. Benzyl alcohol, at 10 mM, increased the Vmax of Pi uptake by 55 and 42% in LLC-PK1 cells and MDCK cells, respectively, but decreased the Vmax of MGP uptake in LLC -PK1 cells by 23%. Similarly to 10 mM benzyl alcohol, opening of tight junctions also increased the Vmax of Pi uptake by 45 and 46% in LLC-PK1 cells and MDCK cells, respectively, and depressed MGP uptake in LLC-PK1 cells by inducing a 15% decrease of the Vmax. None of the two maneuvers (i.e. addition of benzyl alcohol or opening of tight junctions) affected the Km values of the transport systems. From these results it is concluded that (i) the increase in membrane fluidity, achieved either by benzyl alcohol or by opening of tight junctions, affects Na-Pi and Na-glucose cotransports differently, reflecting differences in the lipid environments of the two transport systems, and (ii) membrane fluidity might play a physiological role in the modulation of the activity of transport systems.     

Effect of the anesthetics benzyl alcohol and chloroform on bilayers made from monolayers.

The neutral anesthetics chloroform and benzyl alcohol, at concentrations that block the nerve impulse, greatly modify the transport parameters of positive and negative ions in lipid bilayers made from monolayers. Both chloroform and benzyl alcohol increase the membrane permeability to these ions and increase the translocation rate for tetraphenylborate. It was found that both anesthetics increase the membrane permeability to positive ions more markedly than to negative ions. It was also found that the membrane capacitance increases lineary with the concentration of benzyl alcohol. At 51 mM benzyl alcohol, the increase in capacitance is approximately 6%. Chloroform also increases the membrane capacitance; the increase in capacitance was found to be 6% at 18 mM chloroform. An analysis of the changes in the transport parameters of the lipophilic ions, together with the changes in membrane capacitance, suggests that benzyl alcohol and chloroform modify the dipole potential and dielectric constant of the membrane. Benzyl alcohol may also increase the "fluidity" of the lipid bilayer membranes. At 36 mM benzyl alcohol, the membrane permeability to acetamide increases by 38%.     

Benzyl alcohol increases membrane fluidity and modulates cyclic AMP synthesis in intact renal epithelial cells

To evaluate a possible modulation by membrane fluidity of hormonal, cAMP-mediated effects on renal epithelial cells, we studied the effect of the neutral local anesthetic, benzyl alcohol, on membrane fluidity and on basal and stimulated intracellular cAMP content in intact MDCK cells. Benzyl alcohol induced a dose-dependent decrease of lipid order which was measured by steady-state fluorescence anisotropy using trimethylammonium-diphenylhexatriene and propionyl-diphenylhexatriene as fluorescent probes. Benzyl alcohol induced a 2-fold increase in basal cAMP content, likely as a consequence of increased prostaglandin synthesis since this effect was abolished by indomethacin. The effect of benzyl alcohol on stimulated cAMP synthesis depended on the nature of the ligand: 10 mM benzyl alcohol increased significantly the stimulatory effect of prostaglandin E2, glucagon and forskolin but not of vasopressin. At higher concentrations (40 mM), benzyl alcohol did not affect significantly the glucagon-stimulated cAMP content, while it inhibited significantly the prostaglandin E2-, forskolin- and vasopressin-stimulated cAMP synthesis. The 40 mM benzyl alcohol-induced inhibition was reversed by 1 mM Mn2+, which is known to block the inhibitory GTP-binding protein Ni. These results suggest that: (i) the various components of the adenylate cyclase-cAMP system and their coupling are affected differently by changes in membrane fluidity, which might reflect differences in their lipid environment, (ii) changes in membrane fluidity can modulate responses of renal tubular cells to hormones, and thus tubular functions.     

Lipid-Overloaded Enlarged Adipocytes Provoke Insulin Resistance Independent of Inflammation

In obesity, adipocyte hypertrophy and proinflammatory responses are closely associated with the development of insulin resistance in adipose tissue. However, it is largely unknown whether adipocyte hypertrophy per se might be sufficient to provoke insulin resistance in obese adipose tissue. Here, we demonstrate that lipid-overloaded hypertrophic adipocytes are insulin resistant independent of adipocyte inflammation. Treatment with saturated or monounsaturated fatty acids resulted in adipocyte hypertrophy, but proinflammatory responses were observed only in adipocytes treated with saturated fatty acids. Regardless of adipocyte inflammation, hypertrophic adipocytes with large and unilocular lipid droplets exhibited impaired insulin-dependent glucose uptake, associated with defects in GLUT4 trafficking to the plasma membrane. Moreover, Toll-like receptor 4 mutant mice (C3H/HeJ) with high-fat-diet-induced obesity were not protected against insulin resistance, although they were resistant to adipose tissue inflammation. Together, our in vitro and in vivo data suggest that adipocyte hypertrophy alone may be crucial in causing insulin resistance in obesity.     

Temperature optimum of insulin-stimulated 2-deoxy-D-glucose uptake in rat adipocytes. Correlation of cellular transport with membrane spin-label and fluorescence-label data.

The effects of temperature alterations between 22 degrees C and 48 degrees C on basal and insulin-stimulated 2-deoxy-D-[1-14C]glucose uptake were examined in isolated rat adipocytes. A distinct optimum was found near physiological temperature for uptake in the presence of maximally effective insulin concentrations where insulin stimulation and hexose uptake were both conducted at each given assay temperature. Basal uptake was only subtly affected. Control and maximally insulin-stimulated cells incubated at 35 degrees C subsequently exhibited minimal temperature-sensitivity of uptake measured between 30 and 43 degrees C. The data are mostly consistent with the concept that insulin-sensitive glucose transporters are, after stimulation by insulin, functionally similar to basal transporters. Adipocyte plasma membranes were labelled with various spin- and fluorescence-label probes in lipid structural studies. The temperature-dependence of the order parameter S calculated from membranes labelled with 5-nitroxide stearate indicated the presence of a lipid phase change at approx. 33 degrees C. Membranes labelled with the fluorescence label 1,6-diphenylhexa-1,3,5-triene, or the cholesterol-like spin label nitroxide cholestane, reveal sharp transitions at lower temperatures. We suggest that a thermotropic lipid phase separation occurs in the adipocyte membrane that may be correlated with the temperature-dependence of hexose transport and insulin action in the intact cells.     

Glucose oxidation,glucose transport and insulin binding in isolated rat epididymal adipocytes in the presence of anesthetics

Insulin binding and glucose oxidation were measured in isolated rat adipocytes in the presence of several anesthetics; ethanol, n-octanol, pentobarbital, chlorpromazine and tetracaine. Ethanol and chlorpromazine, at anesthetic and pentobarbitol at sub-anesthetic concentrations are inhibitory to both basal and insulin stimulated rates of glucose oxidation. At all concentrations of ethanol, pentobarbital or chlorpromazine tested binding of insulin is not affected. Since anesthetics may alter membrane fluidity, it is suggested that an anesthetic-induced increase in membrane fluidity beyond that which occurs at 37°C is detrimental to glucose oxidation. Of the 5 anesthetics examined, only chlorpromazine (10 μM or less) and tetracaine (500 μM) stimulate glucose oxidation. These two agents are known to bind to a cell's cytoskeletal system; the binding of chlorpromazine to microtubules is entropy driven. The temperature and concentration dependence of chlorpromazine stimulation of glucose oxidation (transport) are consistent with this form of binding. It is proposed that chlorpromazine binds to the cytoskeletal system of the adipocyte and that this system is normally restrictive to the motion of membrane proteins. Disruption of the cytoskeletal system by chlorpromazine or tetracaine would increase the frequency of insulin-receptor and glucose-carrier contact. Activation of glucose transport could ensue.     

Role of membrane organization in the Mg+2-mediated regulation of excitation energy transfer in barley chloroplasts

Photosystem II fluorescence of barley chloroplasts has been monitored to understand the role of membrane organization in the cation mediated regulation of excitation energy transfer from photosystem II to photosystem I. Membrane organization has been perturbed by adding 60 mM benzyl alcohol which is known to increase the membrane fluidity and decrease its thickness. An addition of 60 mM benzyl alcohol increases the fluorescence at 683 nm (excitation at 436 mn) by 43% whereas 5 mM Mg⁺² increased the fluorescence by 38%. An addition of 5 mM Mg⁺² to benzyl alcohol treated chloroplasts resulted in only a small increase in the fluorescence (6.5%). Circular dichroic measurements showed that 5 mM Mg⁺² decreased the circular dichroic signals suggesting an alteration in the orientation of the chromophores. However, the effect was insignificant on the benzyl alcohol treated chloroplast membranes. Benzyl alcohol itself had large effect on the circular dichroic signals. Based on these results, it appears that a change in the orientation of photosystem I and photosystem II, rather than their segregation, is responsible for the cation-induced increase in the photosystem II fluorescence.     

The effect of membrane composition and alcohols on the insulin-sensitive reconstituted monosaccharide-transport system of rat adipocyte plasma membranes.

The monosaccharide transporter from the plasma membranes of rat adipocytes and insulin-stimulated adipocytes has been reconstituted in sonicated liposomes. The stereospecific D-glucose uptake by liposomes made from a range of phospholipids and incorporating fatty acids has been investigated. D-Glucose uptake is correlated with an increase in lipid fluidity as a consequence of the addition of fluidizing fatty acids, changes in phospholipid acyl chain length and temperature. Benzyl alcohol and ethyl alcohol, which are generally considered to increase bilayer fluidity, decrease stereo-specific D-glucose uptake in both whole adipocytes and reconstituted liposomes. It is suggested that, although these alcohols may affect D-glucose transport by lipid-mediated fluidity changes, they also interact directly with the transporter resulting in inhibition of transport.     

Effects of benzyl alcohol on enzyme activities and D-glucose transport in kidney brush-border membranes

Addition of increasing amounts of benzyl alcohol progressively reduced the steady-state anisotropies of diphenylhexatriene and trimethylammoniumdiphenylhexatriene in brush-border membranes from rat kidney. The decrease in order of membrane lipids, equivalent for 50 mM benzyl alcohol to that produced by a rise in temperature of approx. 6 degrees C, had no effect on the activities of alkaline phosphatase or gamma-glutamyltranspeptidase. On the other hand, benzyl alcohol markedly inhibited the D-glucose uptakes measured in the presence of a 100 mM sodium gradient. For concentrations less than 30 mM, benzyl alcohol reduced the Jmax without significant effects on Km, 22Na+ uptake or the vesicular volume of brush-border preparations. Comparable results were obtained substituting octanol for benzyl alcohol. Our data strongly suggest that, at constant temperature, the D-glucose carrier present in renal brush-border membranes is extremely sensitive to variations in membrane physical state.     

Fatty acid uptake in diabetic rat adipocytes

The effect of diabetic status and insulin on adipocyte plasma membrane properties and fatty acid uptake was examined. Studies with inhibitors and isolated adipocyte ghost plasma membranes indicated 9Z, 11E, 13E, 15Z-octatetraenoic acid (cis-parinaric acid) uptake was protein mediated. Cis-parinaric acid uptake was inhibited by trypsin treatment or incubation with phloretin, and competed with stearic acid. The initial rate, but not maximal uptake, of cis-parinaric acid uptake was enhanced two-fold in adipocytes from diabetic rats. Concomitantly, the structure and lipid composition of adipocyte ghost membranes was dramatically altered. However, the increased initial rate of cis-parinaric acid uptake in the diabetic adipocytes was not explained by membrane alterations or by a two-fold decrease in cytosolic adipocyte fatty acid binding protein (ALBP), unless ALBP stimulated fatty acid efflux. Thus, diabetic status dramatically altered adipocyte fatty acid uptake, plasma membrane structu re, lipid composition, and cytosolic fatty acid binding protein. (Mol Cell Biochem 167: 51-60, 1997)     

Disruption of endothelial barrier function: relationship to fluidity of membrane extracellular lamella

• 1.1. Endothelial cells were cultured in tissue culture flasks or on microcarrier beads and labeled with a lipid specific spin-label.
• 2.2. Exposure of endothelial cells to benzyl alcohol caused a dose- and time-dependent increase in membrane fluidity using electron spin resonance (ESR). Maximum fluidity was reached after a 5-min exposure to 100 mM benzyl alcohol.
• 3.3. Albumin permeability across endothelial cells cultured on micropore filters was used as an indication of endothelial monolayer integrity.
• 4.4. A significant increase in permeability occurred with 50 mM benzyl alcohol. Maximal albumin permeability was reached after a 5-min exposure to 100 mM benzyl alcohol.

     

Effect of benzyl alcohol and ethanol on cold-shock response ofBacillus subtilis

Benzyl alcohol and ethanol were found to exhibit an opposite effect on cold adaptation ofBacillus subtilis. The growth lag induced in bacteria by temperature shift-down was reduced in the presence of benzyl alcohol from 80 to 58 min, whereas ethanol prolonged this interval up to 102 min. In parallel, the physical state of the cytoplasmic membrane in intact bacteria was examined with the help of steady-state fluorescence anisotropy of diphenylhexatriene. Benzyl alcohol decreased the anisotropy as a consequence of membrane fluidization whereas ethanol increased its value, suggesting membrane rigidization. The correlation of growth and fluorescence data indicates that the control of membrane structure represents an important part of bacterial adaptation to cold. Dedicated to Professor C. John on the occasion of the his 75th birthday     

Reconstitution of the glucose transport activity of rat adipocytes

Rat epididymal fat cell membrane proteins were extracted from adipocyte ghosts with octylglucoside and incorporated by detergent dialysis into unilamellar phosphatidylcholine vesicles approx. 200 nm in diameter. The rate of glucose transport into the vesicles under zero-trans conditions was substrate dependent, saturable and inhibited by phloretin and cytochalasin B. Their maximum specific transport activity was 35.6 mumol/min per mg protein, and their half saturation constant for glucose was 15 mM. Glucose transport into the reconstituted vesicles was inhibited by only those sugars which competitively inhibited glucose transport into intact adipocytes. A major protein component of the vesicles was a 100 kDa protein which we had previously found to react with the affinity label maltosyl isothiocyanate (Malchoff, D.M., Olansky, L., Pohl, S. and Langdon, R.G. (1981) Fed. Proc. 40, 1893). Removal of adipocyte ghost membrane extrinsic proteins with dimethylmaleic anhydride followed by extraction of the resulting membrane pellet with octylglucoside yielded a solution which contained two major proteins, of Mr 100 000 and 85 000, with very small quantities of lower Mr proteins. Vesicles into which these proteins were incorporated had average specific transport activities of 624 mumol/min per mg protein and half saturation constants of 22 mM. Our results strongly indicate that the native glucose transporter of the rat adipocyte, like that of the human erythrocyte (Shelton, R.L. and Langdon, R.G. (1983) Biochim. Biophys. Acta 733, 25-33), is a 100 kDa protein.     

Arachidonic acid and glucose uptake by freshly isolated human adipocytes

Fatty acid (FA) and glucose transport into insulin-dependent cells are impaired in insulin resistance (IR; type 2 diabetes mellitus). Studies done on the effects of FAs on glucose uptake, and the influence of insulin on FA uptake by adipocytes, have yielded contradictory results. In this study, isolated human adipocytes were exposed to arachidonic acid (AA) and to insulin, and FA uptake as well as glucose uptake was measured. AA uptake into adipocyte membranes and nuclei was also investigated. Glucose uptake was inhibited by 57 +/- 8% after 30 min of exposure to arachidonate. AA was significantly taken up into adipocyte membranes (49.6 +/- 29% and 123 +/- 74%) at 20 and 30 min of exposure, respectively, and into nuclei (147.6 +/- 19.2%) after 30 min. Insulin stimulated AA uptake (24.1 +/- 14.1%) at 30 min by adipocytes from a non-obese subject, while inhibiting it (16.6 +/- 12%) in adipocytes from an obese subject. These results suggest that: (1) AA inhibits glucose uptake by adipocytes exposed over a short period, probably by a membrane-associated mechanism, (2) insulin-dependent AA uptake is dependent on the body mass index (BMI) of the donor and the insulin sensitivity of their adipocytes.     

Benzyl alcohol inhibits <Emphasis Type="Italic">N</Emphasis>-methyl-<Emphasis Type="SmallCaps">d</Emphasis>-aspartate receptor-mediated neurotoxicity and calcium accumulation in cultured rat cortical neurons

The purpose of this study is to examine whether benzyl alcohol affects N-methyl-D-aspartate (NMDA) receptor in cortical cells. Benzyl alcohol (0.5–2 mM) inhibited NMDA-induced cytotoxicity. The protective effect of benzyl alcohol on NMDA-induced toxicity disappeared by washing cells with buffer to remove benzyl alcohol. Benzyl alcohol reduced NMDA receptor-mediated calcium accumulation, indicating that benzyl alcohol inhibits NMDA receptor activity.     

Insulin and glucose modulate protein kinase C activity in rat adipocytes

In the presence of 1 mM glucose, insulin (10 ng/ml) increases both catalytic and receptor-binding properties of adipocyte cytosolic protein kinase C (PKC). Preincubation of adipocytes with 10 mM glucose raises basal PKC catalytic activity and prevents further stimulation of this enzyme by insulin. The effect of hyperglycemia is likely to be mediated by direct conversion of glucose into diacylglycerol. Thus, an incorporation of 14C-glucose into diacylglycerol is enhanced 10-fold in the presence of 10 mM glucose. These observations indicate that, in normal adipocytes, both insulin and glucose activate PKC; hyperglycemia eliminates the ability of insulin to stimulate this enzyme, thereby interfering with insulin action.     

锌_7-与镉_7-金属硫蛋白对羟自由基损伤大鼠红细胞膜保护作用的比较

用电子自旋共振自旋标记物氮氧自由基硬脂酸和马来酰亚胺标记大鼠红细胞膜脂和膜蛋白,测定膜脂流动性和膜蛋白构象改变,以硫代巴比妥酸法测定脂质过氧化产物丙二醛含量．结果表明,锌7-与镉7-金属硫蛋白对羟自由基引起的膜脂流动性减低、脂质过氧化反应增强双膜蛋白构象改变有明显抑制作用,而且,前者的作用明显强于后者．     

Impaired PI 3-kinase activation in adipocytes from early growth-restricted male rats

Epidemiological studies have established a relationship between early growth restriction and subsequent development of type 2 diabetes. Animal studies have shown that offspring of protein-restricted rats undergo a greater age-related loss of glucose tolerance than controls. The aim of this study was to investigate the possibility that this deterioration of glucose tolerance is associated with changes in adipocyte insulin action. Adipocytes from low-protein offspring had higher basal levels of glucose uptake than controls. Insulin stimulated glucose uptake into control adipocytes but had little effect on low-protein adipocytes. Both groups had similar levels of basal and isoproterenol-stimulated lipolysis. Insulin inhibited lipolysis in control adipocytes but had a reduced effect on low-protein adipocytes. These changes in insulin action were not related to altered expression of insulin receptors or insulin receptor tyrosine phosphorylation; however, they were associated with reduced phosphatidylinositol 3-kinase and protein kinase B activation. These results demonstrate that reduced glucose tolerance observed in late adult life after early growth restriction is associated with adipocyte insulin resistance.     

Increase in membrane fluidity modulates sodium-coupled uptakes and cyclic AMP synthesis by renal proximal tubular cells in primary culture.

In order to evaluate the influence of membrane fluidization on three apical transport systems and on a basolateral enzyme, and to analyse the mechanisms involved, we studied, in cultured rabbit proximal tubular cells, the effect of increasing concentrations of the local anesthetic drug benzyl alcohol on Na(+)-dependent uptakes of phosphate (Pi), methyl alpha-D-glucopyranoside (MGP), and L-alanine, as well as on basal and stimulated cyclic AMP content. At 10 mM, benzyl alcohol increased the Vmax of Pi uptake by 31%, decreased that of MGP uptake by 24%, and did not affect alanine uptake. Km values were not affected. Benzyl alcohol, up to 40 mM, increased in a concentration-dependent manner basal, PTH-stimulated, and cholera toxin-stimulated, but not forskolin-stimulated cyclic AMP accumulation. In the presence of 40 mM benzyl alcohol, the magnitude of PTH-induced inhibition of Pi uptake was enhanced from 11% to 24%. It is concluded that: (i) fluidization of apical membranes affected differently Na+/Pi, Na+/MGP, and Na+/alanine cotransports, reflecting differences in the lipidic environments of these transport system; (ii) fluidization of basolateral membranes enhanced PTH-stimulated cyclic AMP generation through improved coupling between the receptor-GS complex and the catalytic subunit of adenylate cyclase; (iii) these variations may result in physiological and pathophysiological modulation of the renal handling of solutes and of the phosphaturic effect of PTH.