RESUMO
The effect of 1% dietary cholesterol and 0.5% cholate on the rat liver microsomal composition and fatty acid desaturase activities was studied over various periods of time. The cholesterol content of liver microsomes increased as well as that of phosphatidylcholine. Cholesterol/phosphatidylcholine and phosphatidylcholine/phosphatidylethanolamine ratios were also elevated. Phosphatidylinositol decreased, but it recovered its original values at the end of the experimental period. Phosphatidylserine and sphingomyelin slightly decreased with time. Fatty acid composition changes were expressed by a saturated acid decrease and monounsaturated acid increase. Arachidonic acid content was also reduced. A similar pattern appeared in the main phospholipids: phosphatidylcholine and phosphatidylethanolamine. Delta 9-Desaturase activity was enhanced as early as 48 h after cholesterol administration, whereas delta 5- and delta 6-desaturases were depressed during the same period and this enzymatic behaviour remained after 21 days of diet administration. The microsomal membrane was rigidized, as demonstrated by the increase of the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene.
Assuntos
Colesterol/farmacologia , Ácidos Graxos Dessaturases/metabolismo , Microssomos Hepáticos/enzimologia , Animais , Colesterol na Dieta/farmacologia , Difenilexatrieno , Ácidos Graxos/análise , Masculino , Fosfatidilcolinas/análise , Fosfatidiletanolaminas/análise , Ratos , Ratos EndogâmicosRESUMO
Rats fed a 1% cholesterol and 0.5% cholate diet for 21 days were transferred to a sterol-free diet after variable periods of time. The effect of cholesterol removal on liver microsomal composition and fatty acid desaturases was studied. Some changes were already observed after 1 day. However, after 21 days of a sterol-free diet, the cholesterol content of liver microsomes decreased as well as that of phosphatidylcholine. So did the cholesterol/phospholipid ratio. Phosphatidylinositol, phosphatidylserine and sphingomyelin slightly increased along with time. The total fatty acid composition was altered by a decrease in monounsaturated acids and an increase in the saturated acids, palmitic and stearic acids. The arachidonic acid content rose. A similar pattern of change was found in the fatty acid composition of the main phospholipids: phosphatidylcholine and phosphatidylethanolamine. delta 9-Desaturase activity steadily decreased along with cholesterol removal, whereas delta 5- and delta 6-desaturase activities were enhanced towards the end of the removal period. The microsomal membrane became more 'fluid', according to the decrease of fluorescence anisotropy of the 1,6-diphenyl-1,3,5-hexatriene incorporated into the membrane.
Assuntos
Colesterol na Dieta/farmacologia , Colesterol/fisiologia , Ácidos Cólicos/farmacologia , Ácidos Graxos Dessaturases/metabolismo , Microssomos Hepáticos/enzimologia , Animais , Colesterol/análise , Ácido Cólico , Cinética , Masculino , Fosfolipídeos/análise , Ratos , Ratos Endogâmicos , Valores de ReferênciaRESUMO
A peripheral component of the delta 6-fatty acid-desaturase system of rat liver microsomes has been isolated from the cytosol by ultracentrifugation at a saline density of 1.26 g/ml. It exhibited lipoprotein characteristics with an approximate protein/lipid ratio of 1.22 and free fatty acids and phosphatidylcholine as its main lipid components. Linoleic acid desaturation activity diminished in washed microsomes, since they lost the adsorbed cytosolic fraction. Addition of the factor reactivated the reaction and the recovery was dependent on the concentration of the factor in the medium. Linoleic acid and linoleyl-CoA were bound by the cytosolic fraction. However, the transport of substrate to the desaturase was not apparently a main function of the cytosolic fraction, since transport occurred equally in the absence of the factor. Moreover, the solubilization of linoleyl-CoA was not enhanced and the free monomeric concentration was not altered by the presence of the cytosolic fraction. In addition, the factor did not divert delta 6-desaturase substrate to or from other metabolic pathways such as esterification to phospholipids. gamma-Linolenic acid produced by delta 6-desaturation of linoleic acid in the microsomes inhibited the desaturase, but it was removed by the factor from the membrane towards the cytosol, preventing the inhibition. The anti-inhibitory effect of the cytosolic factor was blockaded by addition of columbinic acid or gamma-linolenic acid to the factor. Moreover, the inhibitory effect of arachidonic acid was not prevented by addition of the cytosolic fraction. These results suggest that the cytosolic fraction studied would optimize the delta 6-desaturation of linoleic acid in vitro in rat liver microsomes by removal of the product, gamma-linolenic acid, as it is formed.
Assuntos
Ácidos Graxos Dessaturases/metabolismo , Fígado/enzimologia , Animais , Radioisótopos de Carbono , Fracionamento Celular , Citosol/enzimologia , Cinética , Ácido Linoleico , Ácidos Linoleicos/metabolismo , Ácidos Linolênicos/metabolismo , Linoleoil-CoA Desaturase , Microssomos Hepáticos/metabolismo , Ratos , Ratos Endogâmicos , Ácido alfa-LinolênicoRESUMO
The effect of cholesterol administration in vivo on the lipid composition, dynamic properties of the microsomal membrane of guinea pig livers and the kinetic properties of UDPglucuronyl transferase were studied. Cholesterol administration in the diet evoked an increase of microsomal cholesterol, but no significant changes in the fatty-acid composition of total lipids or of each phospholipid class. Instead, the phosphatidylethanolamine, phosphatidylcholine molar ratio of the membrane was markedly decreased from 0.57 to 0.38. This decline was not enough to counterbalance the overall 'ordering' effect of cholesterol and consequently, the fluorescence anisotropy of the membranes labeled with 1,6-diphenylhexatriene was increased. The lateral diffusion evaluated by measuring the pyrene excimer formation was decreased by the cholesterol incorporation. These physical changes were associated with changes in the kinetic properties of UDPglucuronyl transferase: Vmax increased, while the Km of the different steps of the reaction decreased in the modified microsomes. Furthermore, a shift of the non-michaelian kinetics to michaelian, equivalent to a decrease of a negative homotropic effect and apparent cooperativity of UDPglucuronic acid was observed since the Hill coefficient changed, approaching 1. A non-michaelian kinetics of this enzyme is an indication of boundary lipids in the gel phase and a shift to michaelian, a change of the surrounding lipids to a liquid-crystalline structure. In consequence, our results suggest that cholesterol incorporation in the microsomal membrane while producing a condensing effect of bulk lipids would produce an opposite effect on the UDPglucuronyl transferase boundary lipids.
Assuntos
Colesterol na Dieta/farmacologia , Glucuronosiltransferase/metabolismo , Membranas Intracelulares/metabolismo , Microssomos Hepáticos/metabolismo , Animais , Polarização de Fluorescência , Cobaias , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/enzimologia , Cinética , Lipídeos de Membrana/metabolismo , Microscopia de Fluorescência , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologiaRESUMO
The phospholipid composition of rat-liver microsomes was modified by feeding weaning rats a choline-free diet. After 21 days, the phosphatidylcholine content decreased with a concomitant increment of phosphatidylserine and cholesterol. The bulk fluidity of the membrane decreased. Under these conditions, the delta 5-desaturase activity was diminished as well as the arachidonic-acid content of the membrane lipids.
Assuntos
Ácidos Graxos Dessaturases/metabolismo , Microssomos Hepáticos/enzimologia , Fosfolipídeos/metabolismo , Animais , Colina/administração & dosagem , Dessaturase de Ácido Graxo Delta-5 , Dieta , Polarização de Fluorescência , Masculino , Lipídeos de Membrana/metabolismo , Ratos , Ratos WistarRESUMO
The effect of phytosterol-rich diets (3% beta-sitosterol + 2% campesterol) on rat liver microsomal fatty acid desaturases, membrane dynamics and lipid composition was investigated. After a 21 day period, phytosterol was incorporated into microsomes and the membrane fluidity decreased. There were no changes in either the phospholipid composition or in the total sterol content. However, the phytosterol/cholesterol ratio increased. In the animals fed phytosterols, the delta 5-, delta 6- and delta 9-fatty acid desaturases were significantly more active than in control animals. The changes in the lipid fatty acid composition were consistent with those of the desaturase activities. Hence, it is suggested that: (1) dietary phytosterol modulates desaturase activities; (2) phytosterols make the membrane more rigid but do not induce changes in the relative phospholipid composition; (3) delta 9-, delta 5- and delta 6-desaturase activities increase when the membrane becomes more rigid without changes in the phospholipid composition.
Assuntos
Ácidos Graxos Dessaturases/metabolismo , Microssomos Hepáticos/efeitos dos fármacos , Fitosteróis/farmacologia , Animais , Colesterol/análogos & derivados , Colesterol/farmacologia , Dieta , Fluorescência , Masculino , Microssomos Hepáticos/enzimologia , Ratos , Ratos Endogâmicos , Sitosteroides/farmacologiaRESUMO
The effect of environmental temperature on the activity of liver microsomes of fish (Pimelodus maculatus) to desaturate and elongate oleic, linoleic and alpha-linolenic acids was studied. It was found that: 1. Fish kept at 14-15 degrees C had higher desaturation and elongation activity than animals kept at 29-30 degrees C. The ratio of activity was the same for the three fatty acids. 2. A decrease of the environmental temperature increased the V of linoleic acid desaturation to gamma-linolenic acid, but did not modify the approximate Km of the reaction. 3. The inactivation of the delta6-desaturase of microsomes separated from fish kept at 29-30 degrees C and 14-15 degrees C was the same when heated at 40 degrees C. However, the enzyme was deactivated faster when heated at 29-30 degrees C than at 14-15 degrees C. 4. The increase of the delta6-desaturation activity of the microsomes evoked by the decrease of the temperature of the aquarium was mostly compensated for by the correlative decrease of the specific reaction rate of the reaction. For this reason it is assumed that the adaptive change of the desaturation activity of the microsomes with the environmental temperature does not greatly modify the fatty acid composition of the fish.
Assuntos
Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos/biossíntese , Microssomos Hepáticos/metabolismo , Animais , Cinética , Ácidos Linoleicos/metabolismo , Ácidos Linolênicos/metabolismo , Ácidos Oleicos/metabolismoRESUMO
The cholesterol content of rat liver microsomal membranes was modified in vitro by incubating microsomes and cytosol with liposomes prepared by sonication of microsomal lipids and cholesterol. In this way, the cholesterol to phospholipid molar ratio was increased from 0.11-0.13 in untreated microsomes to a maximal of 0.8 in treated ones. Cholesterol incorporation in microsomes produced an increase in the diphenyl-hexatriene steady-state fluorescence anisotropy and a decrease in the efficiency of pyrene-excimer formation which indicated a decrease in the rotational and translational mobility, respectively, of these probes in the membranes lipid phase. Cholesterol incorporation in microsomes did not affect significantly the glucose-6-phosphatase activity in 0.1% Triton X-100 totally disrupted microsomes, but diminished the glucose-6-phosphatase activity of 'intact' microsomes. This indicates that possibly the glucose 6-phosphate translocation across the microsomal membrane is impeded by an increase in the membrane apparent 'microviscosity'. Cholesterol incorporation in microsomes decreased NADH-cytochrome c reductase without affecting NADH-ferricyanide reductase activity. The delta 9 desaturation reaction rate was enhanced by cholesterol incorporation at low but not at high palmitic acid substrate concentration. delta 5 and delta 6 desaturase reaction-rates were increased both at low and high fatty acid substrate concentrations. These results suggest that a mechanism involving fatty acid desaturase enzymes, might exist to self-regulate the microsomal membrane lipid phase 'fluidity' in the rat liver.
Assuntos
Colesterol/fisiologia , Ácidos Graxos Dessaturases/metabolismo , Glucose-6-Fosfatase/metabolismo , Microssomos Hepáticos/fisiologia , Animais , Transporte de Elétrons , Polarização de Fluorescência , Masculino , Fluidez de Membrana , Microssomos Hepáticos/enzimologia , Ratos , Ratos Endogâmicos , Temperatura , ViscosidadeRESUMO
The effect of fat deprivation on microsomal membrane fluidity of guinea-pig livers and the kinetic cooperativity of UDP-glucuronyl transferase towards its natural substrate, the UDP-glucuronic acid, were studied. Fat deprivation in the diet of weanling guinea-pigs evoked a typical essential fatty-acid-deficient pattern in the composition of the microsomal membrane. The unsaturated:saturated fatty acid ratio progressively declined in the membrane during the 21-day period tested. This decline determined a gradual increase in the fluorescence anisotropy (rs) of the membrane labeled with diphenylhexatriene and the apparent microviscosity of the lipid bilayer calculated from these values increased from 1.1 to 1.8 poise. In addition, when the infinitely slow decaying fluorescence anisotropy ((r infinity), which is proportional to the square of the lipid order parameter, was calculated from rs data, a significant increase in these parameters was also obtained. Furthermore, this decrease of the double bond index:saturated acid ratio of the membrane was associated with a parallel increase in Hill coefficients of the UDP-glucuronyl transferase that gradually lost the negative homotropic effect and cooperativity of UDP-glucuronic acid. The Hill coefficient varied from 0.39 to 0.98 during the 21-day period studied. Our observations indicate on one side that changes in the fat composition of the diet are accompanied by modifications in the lipid composition and fluidity of the microsomal membrane, and the apparent cooperativity of the enzyme. On the other side, they suggest that the evaluation of Hill coefficients of UDP-glucuronyl transferase might be used as a sensitive test to investigate conformational changes in the microsomal membrane of the liver.
Assuntos
Gorduras na Dieta/farmacologia , Ácidos Graxos/deficiência , Glucuronosiltransferase/metabolismo , Membranas Intracelulares/metabolismo , Microssomos Hepáticos/metabolismo , Animais , Ácidos Graxos/análise , Ácidos Graxos Essenciais/farmacologia , Polarização de Fluorescência , Cobaias , Cinética , Masculino , Fluidez de Membrana , Lipídeos de Membrana/análiseRESUMO
n-Butyl and isoamyl alcohols decrease the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene and enhance the efficiency of pyrene excimer formation when these probes are incorporated in rat-liver microsomal membrane, suggesting an increase in rotational and translational mobilities. Neither alcohol modifies NADH-ferricyanide reductase activity but both increase NADH-cytochrome c reductase activity. This was interpreted as an increase in the rate of lateral diffusion of the proteins cytochrome b5 and cytochrome b5 reductase as a consequence of the enhanced membrane lipid phase fluidity. Microsomal delta 9 and delta 6 desaturase activities in the presence of isoamyl alcohol were also studied. This alcohol decreases delta 9 desaturation when it is measured at a low substrate concentration (13 microM palmitic acid), but it is not modified when it is measured at a high substrate concentration (66 microM palmitic acid). delta 6 desaturation is diminished by isoamyl alcohol when it is measured with both 13 microM and 66 microM linoleic acid. The influence of isoamyl alcohol on the glucose-6-phosphatase system activity was also studied. In non-detergent-treated microsomes, isoamyl alcohol enhances glucose-6-phosphatase activity. However, if microsomes are previously treated with 0.1% Triton X-100 isoamyl alcohol does not modify this activity. The enhancement of the glucose 6-phosphate transport rate is not due to membrane permeability barrier disruption, since isoamyl alcohol does not modify mannose-6-phosphohydrolase latency. This would suggest that an increase in membrane lipid phase fluidity specifically activates glucose 6-phosphate transport across the membrane.
Assuntos
Butanóis/farmacologia , Fluidez de Membrana , Microssomos Hepáticos/fisiologia , Pentanóis/farmacologia , Animais , Transporte de Elétrons , Ácidos Graxos Dessaturases/metabolismo , Polarização de Fluorescência , Glucose-6-Fosfatase/metabolismo , Manosefosfatos/metabolismo , Fluidez de Membrana/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Permeabilidade , RatosRESUMO
The effect of dexamethasone on the oxidative desaturation of [1-14C]palmitic to palmitoleic acid on rat liver microsomes, was studied. After 12 h of dexamethasone injection (1 mg/rat) a significant increase in delta 9-desaturase activity, was observed. This effect was also produced by a factor present in a 110,000 X g supernatant soluble fraction obtained after washing crude microsomes from dexamethasone-treated rats with a low ionic strength solution. The dexamethasone-induced factor was present not only in the liver cytosolic fraction of treated animals but also in the cytosol of isolated HTC cells previously incubated with the hormone. Dexamethasone would act via a newly synthesized modulatory factor. The effect depends on an unchanged protein structure, since its biological activity is impaired by trypsin digestion.
Assuntos
Dexametasona/farmacologia , Ácidos Graxos Dessaturases/metabolismo , Fígado/fisiologia , Microssomos Hepáticos/enzimologia , Proteínas/fisiologia , Animais , Citosol/fisiologia , Feminino , Cinética , Microssomos Hepáticos/efeitos dos fármacos , Biossíntese de Proteínas , Proteínas/isolamento & purificação , Ratos , Ratos Endogâmicos , Estearoil-CoA DessaturaseRESUMO
This report supports evidence for the existence of a dexamethasone-induced factor that modulates fatty acid desaturase activities. Dexamethasone at a dose of 1 mg/rat produced a significant decrease in microsomal delta 6 and delta 5 desaturation activity 12 h after the injection. Both desaturase activities were depressed by a soluble factor present in the cytosolic fraction of cells, since the supernatant of microsomes separated at 110,000 X g from hormonal-treated rat liver homogenates, added to crude or washed control microsomes, was able to inhibit in vitro linoleic and homo-gamma-linolenic conversion to gamma-linolenic and arachidonic acids, respectively. The inhibitory factor was loosely bound to microsomes, since it was also present in a soluble fraction obtained after washing crude microsomes from dexamethasone-treated rats with a low-ionic-strength solution. Besides, trypsin digestion deactivates the dexamethasone-induced factor. Therefore, the depressing effect of glucocorticoids on delta 6 and delta 5 desaturation capacity depends on an unchanged protein structure present in the cytosolic fraction of the cell and whose biosynthesis is brought about by hormonal induction.
Assuntos
Dexametasona/farmacologia , Ácidos Graxos Dessaturases/metabolismo , Microssomos Hepáticos/enzimologia , Animais , Citosol/metabolismo , Dessaturase de Ácido Graxo Delta-5 , Cinética , Linoleoil-CoA Desaturase , Fígado/metabolismo , Masculino , Ratos , Ratos Endogâmicos , Tripsina/farmacologiaRESUMO
Studies were carried out to relate the changes of the fatty acid and lipid composition of rat microsomes with the modification of the activity of the linoleic acid desaturation evoked by an essential fatty acid deficient diet. Two steps were shown in the progression of the essential fatty acid deficienty. In a first step shown at three days of essential fatty acid deficiency the fatty acid composition was changed by decreasing linoleic and arachidonic acids and increasing oleic and eicosatrienoic (-9) acids. No change was found in the lipid distribution and approximate V and Km of the linoleic acid desaturation. In this first step the unsaturated/saturated fatty acids ratio fell in spite of the synthesis of eicosatrienoic (n-9) acid that was produced without any change of enzyme activity. In a second step shown at 15 days of essential fatty acid deficiency the change of the fatty acid composition was greater but the unsaturated/saturated acid ratio was restored. An increase of triacylglycerols and a decrease of phospholipids was also detected together with an enhanced activity of linoleic acid desaturation (higher approximate V) and a higher approximate Km. The increase of the V of linoleic acid desaturation is considered to be evoked by an increased level of active delta-6 desaturase. The increased activity of the delta-6 desaturase in this second period is a secondary and important response of the cell to maintain the unsaturated : saturated acid ratio and fluidity of the membrane.
Assuntos
Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos Essenciais/deficiência , Ácidos Linoleicos/metabolismo , Microssomos Hepáticos/enzimologia , Animais , Colesterol/biossíntese , Ésteres do Colesterol/biossíntese , Ácidos Graxos Essenciais/farmacologia , Ácidos Graxos não Esterificados/biossíntese , Glicerídeos/biossíntese , Cinética , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Fosfolipídeos/biossíntese , RatosRESUMO
The influence of a fat-free diet on the lipid dynamics of rat liver microsomes and liposomes of microsomal lipids was studied by using different fluorescence methods. Lifetime distribution and rotational diffusion of probes with different localization in the lipid bilayer were measured using multifrequency fluorometry. Lateral mobility was studied by measuring excimer formation of pyrenedodecanoic acid. Dipolar relaxation in the interfacial region was studied using 2-dimethyl-amino-6-lauroylnaphthalene (Laurdan). In spite of large changes in the fatty acid composition of microsomal lipids, polyunsaturated fatty acid deficiency showed no effect on the lifetime distribution and rotational mobility of 1,6-diphenyl-1,3,5-hexatriene (DPH). l-(4-(trimethylamino)phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), 2- 7- and 12-(9-anthroiloxy)stearic acids. The treatment did not change the lateral diffusion of pyrenedodecanoic acid, either. However, generalized polarization of Laurdan fluorescence was higher in polyunsaturated fatty acid deficient microsomes as compared to the polyunsaturated fatty acid sufficient ones. This effect was also observed in liposomes of the total microsomal lipids, indicating that the changes in fatty acid composition resulting from polyunsaturated fatty acid deficiency produced a small but significant decrease in the rate of dipolar relaxation in the region of the lipid polar groups of the bilayer. The absence of lipid gel phase domains in rat liver microsomes was also indicated by Laurdan fluorescence features.
Assuntos
Ácidos Graxos Essenciais/deficiência , Lipídeos de Membrana/química , Microssomos Hepáticos/química , Animais , Fenômenos Biofísicos , Biofísica , Transferência de Energia , Polarização de Fluorescência , Corantes Fluorescentes , Bicamadas Lipídicas/química , Lipossomos/química , Masculino , Ratos , Ratos Wistar , Espectrometria de FluorescênciaRESUMO
Discoidal recombinant high density lipoproteins (rHDL) of apolipoprotein AI (apoAI) and palmitoyloleoylphosphatidylcholine (POPC), with or without cholesterol, were prepared by cholate dialysis. By gel filtration, rHDL containing 2-4 (Lp2, Lp3 and Lp4) apoAI molecules/particle were obtained. The ApoAI conformation in these rHDL was investigated by tryptophan fluorescence, denaturation with guanidine HCl, and immunoreactivity with two monoclonal antibodies recognizing epitopes in the N-terminal and central domains. Data show that apoAI conformation is highly dependent on particle size as well as on cholesterol. The ability of rHDL to interact with lipid bilayer was studied by measuring leakage induction on POPC and POPC/cholesterol vesicles loaded with terbium/dipicolinic acid. Among the cholesterol-free rHDL, the most efficient ones were the smallest Lp2. Leakage induction on POPC vesicles is dramatically decreased by the presence of cholesterol in Lp2 and Lp3. All the rHDL, but specially those containing cholesterol, induced more leakage on the POPC/cholesterol than on the POPC vesicles. These results suggest that in small cholesterol-poor particles, apoAI could have a conformation determining a high affinity for membranes, which could facilitate cholesterol efflux. After cholesterol enrichment, a conformational change in apoAI could decrease the affinity for membranes allowing the lipoprotein release.
Assuntos
Apolipoproteína A-I/química , Colesterol/farmacologia , Lipossomos/metabolismo , Conformação Proteica , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Apolipoproteína A-I/imunologia , Ligação Competitiva , Epitopos/química , Epitopos/imunologia , Fluorescência , Guanidina/farmacologia , Lipoproteínas HDL/química , Lipoproteínas HDL/genética , Tamanho da Partícula , Permeabilidade , Fosfatidilcolinas/metabolismo , Ácidos Picolínicos/metabolismo , Desnaturação Proteica/efeitos dos fármacos , Térbio/metabolismo , Triptofano/metabolismoRESUMO
A sucrose-rich diet generates time-dependent metabolic disorders similar to those found in diabetes type 2. After 8 month (mo) this diet evoked in the rat an increase of blood glucose, free fatty acids (FFA) and triacylycerides (TG) without insulin modification, an interruption of liver stearoyl-CoA desaturase-1 (SCD-1) mRNA and activity increase found at 6 mo, and an enhacement of Delta6 and Delta5 desaturase mRNA and Delta6 activity. We found that the administration of troglitazone (TRO), a peroxisome-proliferator-activated receptors gamma (PPAR-gamma) agonist, for 2 mo normalized plasma FFA, TG, and glucose without altering the insulinemia. It depressed liver SCD-1 mRNA in both control and sucrose-fed rats, decreasing the 18:1n-9/18:0 ratio in serum and liver lipids, and eliminated the increasing effect on mRNA and activity of Delta6 and Delta5 desaturases. These findings evidence again that desaturases are not affected through an insulin resistant effect evoked by the sucrose-rich diet and TRO recovers the altered metabolic plasma parameters as it corresponds to a PPAR-gamma agonist, but its effect on hepatic desaturases can not be attributed to a direct action on liver by PPAR-gamma, insulin, and even by an insulin sensitizing mechanism, suggesting it would be evoked indirectly through hepatic PPAR-alpha deactivation induced by the FFA decrease.
Assuntos
Cromanos/farmacologia , Carboidratos da Dieta/farmacologia , Modelos Animais de Doenças , Ácidos Graxos Dessaturases/metabolismo , Resistência à Insulina , Sacarose/farmacologia , Tiazolidinedionas/farmacologia , Animais , Carboidratos da Dieta/administração & dosagem , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Masculino , Microssomos Hepáticos/metabolismo , Plasma/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Sacarose/administração & dosagem , TroglitazonaRESUMO
This study examines the changes in cellular lipids that take place when Trypanosoma cruzi epimastigotes and metacyclic trypomastigotes are transferred from 28 to 37 degrees C. We found a rise in the sterol to phospholipid ratio, as well as in the triacylglycerol and steryl ester cellular content in T. cruzi epimastigotes. In addition, saturated to unsaturated fatty acid ratios in phospholipids increase. This latter effect appears to be due to two concurrent processes. Firstly, fatty acyl delta9 and, especially, delta12 desaturations are significantly diminished at 37 degrees C. Secondly, triacylglycerols and steryl esters undergo changes in their fatty acyl composition opposite to those simultaneously observed in phospholipids, i.e. the ratio of saturated to unsaturated fatty acids markedly decreases. Similar alterations in each of the lipid classes and in the fatty acid composition of polar and neutral lipids were found in cultured metacyclic trypomastigotes on exposure to the same shift-up. These observations suggest that a global remodeling of cellular lipids that involves extensive fatty acid exchange between neutral and polar lipid pools represents a novel and important mechanism of adaptation of the parasites to the temperature changes they encounter in their life cycle.
Assuntos
Metabolismo dos Lipídeos , Trypanosoma cruzi/metabolismo , Adaptação Fisiológica , Animais , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos/análise , Lipídeos/química , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Esteróis/química , Esteróis/metabolismo , Temperatura , Triglicerídeos/química , Triglicerídeos/metabolismo , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Acyl-CoA synthetase activity with various long-chain fatty acid substrates and its kinetic properties were measured in rat adrenal microsomes. The apparent Michaelis constants (Km) for substrate fatty acids increased in the order eicosa-8,11,14-trienoic acid less than alpha-linolenic acid less than linoleic acid less than palmitic acid. The maximum velocities with these fatty acids decreased in the order linolenic greater than eicosa-8,11,14-trienoic acid greater than palmitic acid. The synthesis of radioactivity palmitoyl-CoA, linoleyl-CoA, alpha-linolenyl-CoA and eicosa-8,11,14-trienoyl-CoA from the respective radioactive substrates decreased in the presence of all the other fatty acids mentioned above. These effects were inversely correlated with their apparent Km values. These results support the idea of a single long-chain fatty acyl-CoA synthetase in the adrenal microsomal fraction for the acid tested. After testing the influence of different hormones, it was shown that the administration of epinephrine, ACTH and dexamethasone caused a significant decrease in the activity of the long-chain fatty acid-CoA synthetase. This inhibition is independent of the one produced by the same hormones on the desaturation of linoleic to gamma-linolenic acid.
Assuntos
Glândulas Suprarrenais/enzimologia , Hormônio Adrenocorticotrópico/farmacologia , Coenzima A Ligases/metabolismo , Epinefrina/farmacologia , Microssomos/enzimologia , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Animais , Feminino , Ratos , Ratos Endogâmicos , Especificidade por SubstratoRESUMO
The in vivo and in vitro effect of epinephrine on the incorporation and desaturation of [1-14C]eicosa-8,11,14-trienoic acid was studied in isolated adrenocortical cells of rats. Control cells incubated at different substrate concentrations and for different periods of time were able to incorporate eicosatrienoic acid and to desaturate it to arachidonic acid. The ultrastructural study demonstrated that most of the cells belonged to the zona fasciculata and presented a good preservation. When the cells were isolated from rats killed 7 and 12 h after the administration of a single dose of epinephrine, a decrease in the incorporation and desaturation of 20:3n6 was observed. The effect on the desaturation was independent from the incorporation of the acid, and it was also observed in the microsomes of the decapsulated adrenal gland from rats treated with epinephrine. The fine structure of adrenocortical cells did not show changes after the treatment with epinephrine. The addition of the hormone to the incubation medium containing cells isolated from untreated rats produced no effect on arachidonic acid biosynthesis, indicating that the effect of epinephrine would be indirect, through either a metabolic or a hormone mediator.
Assuntos
Córtex Suprarrenal/metabolismo , Ácidos Araquidônicos/biossíntese , Epinefrina/farmacologia , Ácido 8,11,14-Eicosatrienoico/metabolismo , Córtex Suprarrenal/ultraestrutura , Animais , Ácido Araquidônico , Feminino , Técnicas In Vitro , Microssomos/metabolismo , Oxirredução , RatosRESUMO
The presence of a very high density lipoprotein (VHDL), an hexameric protein, was explored in different tissues of Triatoma infestans throughout the last nymphal and adult stages, and in egg extracts by Western blot assays. The VHDL was always detected in both, hemolymph and fat body, during the above mentioned stages and it was also observed in the buffer soluble fraction of testis and egg homogenates. An enzyme-linked immunosorbent assay (ELISA) was used to measure the VHDL titer in these tissues. Hemolymph VHDL reaches a maximum value before the last molt, then it abruptly declines in males and females just after emergence, but during adult life it increases again. Fat body VHDL decreases slowly and continuously during the nymph growth reaching a minimum value prior to molting, and in the first week of adult life the values were even two-fold lower; then, it shows a different cycle of accumulation and depletion in males and females. In adult testis the VHDL undergoes a cycle similar to the one observed in male fat body. This protein increases progressively during embryonic development and, at the time of larval hatching it reaches its maximum value. The hexameric protein presents homologies in its N-terminal sequence with storage hexamerins of Diptera, Lepidoptera and Hymenoptera.