Protein-mediated palmitate uptake and expression of fatty acid transport proteins in heart giant vesicles.
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Giant sarcolemmal vesicles were isolated from rat heart and hindlimb muscles for a) characterization of long-chain fatty acid transport in the absence of metabolism and b) comparison of fatty acid transport protein expression with fatty acid transport. Giant vesicles contained cytosolic fatty acid binding protein. Palmitate uptake was completely divorced from its metabolism. All palmitate taken up was recovered in the intravesicular cytosol as unesterified FA. Palmitate uptake by heart vesicles exhibited a K m of 9.7 nm, similar to that of muscle (K m = 9.7 nm). Vmax (2.7 pmol/mg protein/s) in heart was 8-fold higher than in muscle (0.34 pmol/mg protein/s). Palmitate uptake was inhibited in heart (55-80%) and muscle (31-50%) by trypsin, phloretin, sulfo-N-succinimidyloleate (SSO), or a polyclonal antiserum against the 40 kDa plasma membrane fatty acid binding protein (FABPpm). Palmitate uptake by heart and by red and white muscle vesicles correlated well with the expression of fatty acid translocase (FAT/CD36) and fatty acid binding protein FABPpm, which may act in concert. The expression of fatty acid transport protein (FATP), was 10-fold lower in heart vesicles than in white muscle vesicles. It is concluded that long-chain fatty acid uptake by heart and muscle vesicles is largely protein-mediated, involving FAT/CD36 and FABPpm. The role of FATP in muscle and heart remains uncertain. (+info)
Phloretin-induced apoptosis in B16 melanoma 4A5 cells and HL60 human leukemia cells.
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The dihydrochalcone phloretin induced apoptosis in B16 mouse melanoma 4A5 cells and HL60 human leukemia cells. Phloretin was suggested to induce apoptosis in B16 cells mainly through the inhibition of glucose transmembrane transport. The phloretin-induced apoptosis in B16 cells was inhibited by actinomycin D, Ac-YVAD-CHO caspase-1-like inhibitor, and Ac-DEVD-CHO caspase-3-like inhibitor. During the induction of apoptosis by phloretin, the expression of Bax protein in B16 cells increased and the levels of p53, Bcl-2, and Bcl-XL proteins did not change. Our results suggested that phloretin induced apoptosis through the promotion of Bax protein expression and caspases activation. On the other hand, phloretin may induce apoptosis in HL60 cells through the inhibition of protein kinase C activity because phloretin inhibited protein kinase C activity in HL60 cells more than that in B16 cells. The phloretin induced-apoptosis in HL60 cells was not inhibited by actinomycin D and the caspase-1-like inhibitor, but slightly inhibited by the caspase-3-like inhibitor. Phloretin reduced the level of caspase 3 protein in HL60 cells, but not the level of the Bcl-2 protein. Phloretin did not increase the level of Bax protein. Phloretin was suggested to induce apoptosis in HL60 cells through the inhibition of protein kinase C activity, followed by the pathway, which is different from that in B16 cells. (+info)
Effect of gramicidin A on the dipole potential of phospholipid membranes.
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The effect of channel-forming peptide gramicidin A on the dipole potential of phospholipid monolayers and bilayers has been studied. Surface pressure and surface potential isotherms of monolayers have been measured with a Langmuir trough equipped with a Wilhelmy balance and a surface potential meter (Kelvin probe). Gramicidin has been shown to shift pressure-area isotherms of phospholipids and to reduce their monolayer surface potentials. Both effects increase with the increase in gramicidin concentration and depend on the kind of phosphatidylcholine used. Application of the dual-wavelength ratiometric fluorescence method using the potential-sensitive dye RH421 has revealed that the addition of gramicidin A to dipalmitoylphosphatidylcholine liposomes leads to a decrease in the fluorescence ratio of RH421. This is similar to the effect of phloretin, which is known to decrease the dipole potential. The comparison of the concentration dependences of the fluorescence ratio for gramicidin and phloretin shows that gramicidin is as potent as phloretin in modifying the membrane dipole potential. (+info)
Fatty acid translocase/CD36 mediates the uptake of palmitate by type II pneumocytes.
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Type II pneumocytes, which synthesize, store, and secrete pulmonary surfactant, require exogenous fatty acids, in particular palmitic acid, for maximum surfactant synthesis. The uptake of palmitate by type II pneumocytes is thought to be protein mediated, but the protein involved has not been characterized. Here we show by RT-PCR and Northern blot analysis that rat type II pneumocytes express the mRNA for fatty acid translocase (FAT/CD36), a membrane-associated protein that is known to facilitate the uptake of fatty acids into adipocytes. The deduced amino acid sequence from rat type II pneumocytes reveals 98% identity to the FAT/CD36 sequence obtained from rat adipocytes. The uptake of palmitate by type II pneumocytes follows Michaelis-Menten kinetics (Michaelis-Menten constant = 11.9 +/- 1.8 nM; maximum velocity = 62.7 +/- 5.8 pmol. min(-1). 5 x 10(5) pneumocytes(-1)) and decreases reversibly under conditions of ATP depletion to 35% of control uptake. Incubation of cells at 0 degrees C inhibited the uptake of palmitate almost completely, whereas depletion of potassium was without effect. Preincubation of the cells with bromobimane or phloretin decreases the uptake of palmitate significantly as does preincubation with sulfo-N-succinimidyl oleate, the specific inhibitor of FAT/CD36 (C. M. Harmon, P. Luce, A. H. Beth, and N. A. Abumrad. J. Membr. Biol. 121: 261-268, 1991). From these data, we conclude that FAT/CD36 is expressed in type II pneumocytes and mediates the uptake of palmitate in a saturable and energy-dependent manner. The data suggest that the uptake process is independent of the formation of coated pits and endocytotic vesicles. (+info)
Interaction of phloretin with lipid monolayers: relationship between structural changes and dipole potential change.
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Phloretin is known to adsorb to lipid surfaces and alters the dipole potential of lipid monolayers and bilayers. Its adsorption to biological and artificial membranes results in a change of the membrane permeability for a variety of charged and neutral compounds. In this respect phloretin represents a model substance to study the effect of dipole potentials on membrane permeability. In this investigation we studied the interaction of phloretin with monolayers formed of different lipids in the liquid-expanded and the condensed state. Phloretin integrated into the monolayers as a function of the aqueous concentration of its neutral form, indicated by an increase of the surface pressure in the presence of phloretin. Simultaneous recording of the surface potential of the monolayers allowed us to correlate the degree of phloretin integration and the phloretin-induced dipole potential change. Increasing the surface pressure decreased the phloretin-induced shift of the isotherms, but did not influence the phloretin-induced surface potential change. This means that phloretin adsorption to the lipid surface can occur without affecting the lipid packing. The surface potential effect of phloretin is accompanied by a change of the lipid dipole moment vector dependent on the lipid packing. This means that the relation between the surface potential change and the lipid packing cannot be described by a static model alone. Taking into account the deviations of the surface potential change versus molecular area isotherms of the experimental data to the theoretically predicted course, we propose a model that relates the area change to the dipole moment in a dynamic manner. By using this model the experimental data can be described much better than with a static model. (+info)
Contribution of glucose transport to the control of the glycolytic flux in Trypanosoma brucei.
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The rate of glucose transport across the plasma membrane of the bloodstream form of Trypanosoma brucei was modulated by titration of the hexose transporter with the inhibitor phloretin, and the effect on the glycolytic flux was measured. A rapid glucose uptake assay was developed to measure the transport activity independently of the glycolytic flux. Phloretin proved a competitive inhibitor. When the effect of the intracellular glucose concentration on the inhibition was taken into account, the flux control coefficient of the glucose transporter was between 0.3 and 0.5 at 5 mM glucose. Because the flux control coefficients of all steps in a metabolic pathway sum to 1, this result proves that glucose transport is not the rate-limiting step of trypanosome glycolysis. Under physiological conditions, transport shares the control with other steps. At glucose concentrations much lower than physiological, the glucose carrier assumed all control, in close agreement with model predictions. (+info)
Acute effect of cadmium-metallothionein on glucose and amino acid transport across the apical membrane of the rabbit proximal tubule perfused in vitro.
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Acute as well as chronic exposure of cadmium (Cd) leads to proximal tubule injury. The exact cellular mechanism of this disorder and whether there is a contribution of cadmium-metallothionein (Cd-MT), a binding protein of Cd, remain unclear. We perfused isolated S2 segments of rabbit nephron, and the deflections of transmural voltage (DeltaV(t)) and apical membrane voltage (DeltaV(a)) on elimination of glucose or alanine from the perfusate were measured for the parameters of activity of Na(+)-glucose and Na(+)-amino acid cotransporters. The effects of Cd-MT or CdCl(2) to either bath or lumen for 10 min on these parameters were examined. We also measured the lumen-to-bath [(14)C]glucose flux. Addition of Cd-MT to lumen suppressed glucose- or alanine-dependent DeltaV(t) and DeltaV(a), as well as baseline V(t) and basolateral membrane voltage (V(b)), at approximately 10 min. [(14)C]glucose flux was inhibited by Cd-MT to lumen. The effects of Cd-MT to bath and CdCl(2) to either lumen or bath were 100-fold less potent than that of Cd-MT to lumen. Luminal Cd-MT immediately suppressed the glucose-dependent DeltaV(a), whereas the baseline V(a) and V(t) were unchanged. The early effect of luminal Cd-MT was simulated by addition of 10(-4) M phloretin. Addition of 10(-4) M ouabain to the bath simulated the later effect of Cd-MT. The protection of SH group by dithiothreitol prevented the early effect of Cd-MT, but not the later effect. We concluded that Cd-MT initially acts directly on Na(+)-glucose and Na(+)-amino acid cotransporters from the lumen by attacking SH group, followed by the later inhibition of Na(+)-K(+)-ATPase after entering the cell from the apical membrane. (+info)
Parallel stimulation of glucose and Mg(2+) accumulation by insulin in rat hearts and cardiac ventricular myocytes.
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The stimulation of beta-adrenoceptors in cardiac cells results in a rapid loss of cellular Mg(2+). Because insulin physiologically counteracts several of the cellular effects mediated by the activation of beta-adrenoceptors and the elevation of cytosolic cAMP levels, we investigated whether insulin administration could prevent Mg(2+) mobilization from rat hearts and ventricular myocytes. Rat hearts were perfused in a retrograde Langendorff system, and the changes in extracellular Mg(2+) were measured by atomic absorbance spectrophotometry. Pretreatment of the hearts with 6 nmol/L insulin completely prevented the Mg(2+) extrusion induced by the beta-adrenergic agonist isoproterenol. Furthermore, the administration of insulin per se induced an accumulation of Mg(2+) by the heart. This accumulation was small but detectable in the presence of 25 to 35 micromol/L [Mg(2+)](o) and increased in proportion to [Mg(2+)](o). Insulin-mediated Mg(2+) accumulation was not observed in hearts perfused with a medium devoid of glucose or with a medium containing the inhibitors of glucose transport, cytochalasin B and phloretin. Insulin-stimulated [(3)H]2-deoxyglucose accumulation was measured in collagenase-dispersed cardiac ventricular myocytes in the presence of varying levels of [Mg(2+)](o). Glucose transport was not observed below 25 micromol/L [Mg(2+)](o), and it also increased in proportion to [Mg(2+)](o). Taken together, these results indicate the presence of a major uptake of Mg(2+) into cardiac cells that is stimulated by insulin and may require the insulin-induced operation of a glucose transporter. Hence, extracellular and/or intracellular Mg(2+) may modulate glucose transport and/or utilization. (+info)