Biomedical subjects

R R Brenner

Publications and source records attributed to R R Brenner.

At least 55 records · Page 3Linked to original sources

PubMed1989

The influence of fatty acid unsaturation and physical properties of microsomal membrane phospholipids on UDP-glucuronyltransferase activity.

The relationship between lipid composition, the physical properties of microsomal phospholipids and the kinetics of liver UDP-glucuronyltransferase was studied in microsomes from guinea pigs supplied with a normal or a fat-free diet for 28 days. Fatty acid deficiency did not modify either the cholesterol/phospholipid molar ratio or the polar head group composition, but exclusively redistributed the unsaturated fatty acid pattern, by partially exchanging oleic for linoleic acid. This phenomenon accounts for the decrease of both rotational and translational mobilities of the fluorescent probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and pyrene respectively. When the thermotropic behaviour of the different systems was assessed, no transition temperature (gel-liquid-crystalline) between 10 and 40 degrees C was seen as a consequence of the lower degree of unsaturation, either in the microsomal membranes or in the total lipid or total phospholipid extracts from the treated animals. In spite of this, the polarization ratio of trans-parinaric acid and the fluorescence intensity of merocyanine 540 revealed that a significant lateral phase separation occurred at 20-22 degrees C in the extracted phospholipids, which was smoother in the total lipid fractions and in the native microsomal membranes. Fatty acid deficiency caused an upward shift of the midpoint temperature of the lateral phase separation. Furthermore, the phosphatidylcholine extracted from the 'normal' microsomes showed a lateral phase separation centred at a lower temperature than that extracted from 'fat-deficient' microsomes. In contrast, the Arrhenius plot of UDP-glucuronyltransferase from 'normal' microsomes exhibited a change in slope at a higher temperature than that from treated microsomes. These results would suggest that fatty acid deficiency in guinea-pig liver microsomes, while rigidizing the bulk lipids, would segregate the most unsaturated phosphatidylcholine molecules towards the UDP-glucuronyltransferase microenvironment, in accordance with our previous results with cholesterol incorporation [Castuma & Brenner (1986) Biochemistry 25, 4733-4738].

Animals↗

PubMed1989

Fatty acid biosynthesis in the integument tissue of Triatoma infestans.

1. The biosynthesis of lipids and their distribution in several tissues were investigated by injection of 1-14C acetate in females and 5th instar nymphs of the hematophagous hemiptera T. infestans. 2. Biosynthesis of palmitic, palmitoleic, stearic, oleic and very long chain fatty acids up to 26 carbons and hydrocarbons, was shown. They were found in haemolymph, fat body, integument, epicuticle and oocytes with special distribution. 3. Epicuticular hydrocarbon labelling was shown to precede that of haemolymph hydrocarbons. 4. Radioactivity incorporation into each lipid class depends on the developmental stage and the time after injection. 5. "In vitro" incubation of integument tissue with 1-14C acetate demonstrated the biosynthesis of palmitic and stearic acids, a low desaturation to oleic and palmitoleic and an elongation to acids of up to 34 carbons. Hydrocarbons were also synthesized. 6. Haemolymph in the incubation medium has a positive effect on the release of newly-synthesized fatty acid and unsaponifiable material from the integument.

Animals↗

PubMed1989

Purification and properties of the very high density lipoprotein from the hemolymph of adult Triatoma infestans.

The very high density lipoprotein (VHDL) of Triatoma infestans hemolymph from adult males has been isolated and purified by two-step density gradient ultracentrifugation. It appears to be homogeneous as judged by native polyacrylamide gel electrophoresis. The content of VHDL in hemolymph was estimated to be 8 mg protein/ml. The purified protein has a molecular weight (Mr) of 450,000, is composed of six subunits of Mr approximately equal to 77,000, and possesses a high content of aromatic amino acids. This protein is glycosylated and contains 3% of lipids by weight with a remarkable amount of free fatty acids (25% of total lipids). The T. infestans VHDL has a different lipid and amino acid composition from lipophorin. The lipid composition and the spectroscopic studies using cis-parinaric acid indicated a high fatty acid binding affinity. It has nine binding sites per mol of VHDL. Competence studies revealed that VHDL has its highest affinity for the binding of palmitic acid followed by stearic and arachidonic acids.

Amino Acids↗

PubMed1989

Microsomal delta 5 desaturation of eicosa-8,11,14-trienoic acid is activated by a cytosolic fraction.

delta 5 Desaturation of eicosa-8,11,14-trienoic acid to arachidonic acid was studied in rat liver microsomes. It was shown that delta 5 desaturation of fatty acids in vitro requires the participation of a peripheral component of cytosolic origin. Desaturation of 20:3n-6 to 20:4n-6 decreases in washed microsomes as they lose an adsorbed cytosolic fraction (CF), but the enzymatic activity can be recovered as a function of CF concentration in the incubation medium. Albumin does not substitute for CF. delta 5 Desaturation of 20:3n-6 is inhibited by arachidonic acid by a product inhibition effect, but CF prevents retroinhibition of delta 5-desaturase by 20:4n-6. This ability of CF is eliminated by preincubation of CF with 20:4n-6, but not with gamma-18:3n-6, the product of delta 6 desaturation of 18:2n-6, thus indicating that CF impairs the retroinhibitory effect of arachidonic acid on delta 5-desaturase in a specific manner. delta 6 Desaturation of linoleic acid to gamma-18:3n-6 is also activated by CF and retroinhibited by gamma-18:3n-6. CF activity on delta 6 desaturation is retained after preincubation with 20:4n-6, but it is lost after preincubation with gamma-18:3n-6. Activation of delta 6-desaturase by CF is associated with the removal of the reaction product in a specific manner. Chromatography of CF by Sephacryl S-200 separates two major subfractions which show different efficiency in reactivating delta 5- and delta 6-desaturase activities in washed microsomes. Therefore, CF may contain subfractions that can prevent delta 5- and delta 6-desaturase retroinhibition by apparently binding their respective reaction products specifically.

8,11,14-Eicosatrienoic Acid↗

PubMed1988

Transport and utilization of free fatty acids in Triatoma infestans.

Triatoma infestans hemolymph has 0.31 mg/ml of free fatty acids and 2.8 mg/ml of diacylglycerols. Almost all the diacylglycerols are transported by lipophorin whereas free fatty acids are carried by lipophorin and a very high density lipoprotein. The binding of cis-parinaric acid to lipophorin was employed to specify the free fatty acid binding properties of lipophorin. Lipophorin has 10 binding sites of high affinity (3 x 10(7)) and approximately 45 binding sites of low affinity (1 x 10(6)). The relative rate of tissue incorporation of free fatty acids and diacylglycerols was measured by injecting insects with hemolymph previously labeled in both, free fatty acids and diacylglycerols. In this way, the half-life of the hemolymph free fatty acids was estimated to be about 4 min. Based on this result and taking into account the content of free fatty acids and diacylglycerols in hemolymph, the incorporation of free fatty acids, expressed in moles of fatty acids, seems to be 3.4 times higher than that of diacylglycerols. This finding can be applied to other insects.

Animals↗

PubMed1988

In vivo cholesterol removal from liver microsomes induces changes in fatty acid desaturase activities.

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.

Animals↗

PubMed1988

A dexamethasone-induced protein stimulates delta 9-desaturase activity in rat liver microsomes.

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.

Animals↗

PubMed1988

Effect of various steroids on the biosynthesis of arachidonic acid in isolated hepatocytes and HTC cells.

The effect of various steroids on the incorporation and desaturation of eicosa-8,11,14-trienoic acid in normal hepatocytes and HTC cells was investigated. After 3 hr incubation with 11-deoxycorticosterone, both kinds of cells showed an increase in the incorporation of eicosatrienoic acid. In contrast, progesterone, cortexolone, 17-beta-estradiol, testosterone, estriol, aldosterone, corticosterone, dexamethasone, dehydroepiandrosterone, 11-beta-hydroxyandrosterone, 11-ketoaetiocholanolone, epiaetiocholanolone and 5-beta-pregnane-3 alpha,20 alpha-diol, provoked no significant changes in the uptake of the exogenous acid. Of all the steroids tested, only 11-deoxycorticosterone, dexamethasone and 17-beta-estradiol evoked a significant inhibition on the arachidonate biosynthesis in both kinds of cells. Testosterone, estriol, aldosterone and corticosterone provoked a significant inhibition of delta 5-desaturase in HTC cells. In dexamethasone, this effect was dose-dependent (0 to 10(-4) M). Simultaneous incubation with 17-beta-estradiol or 11-deoxycorticosterone with dexamethasone led to an extent of inhibition on arachidonate biosynthesis that did not surpass the effect of each drug. Pretreatment of isolated hepatocytes with the antiglucocorticoid, cortexolone, prevented the dexamethasone-induced inhibition of arachidonate biosynthesis. Normal rat liver microsomes preincubated in vitro with dexamethasone, 11-deoxycorticosterone, 17-beta-estradiol, corticosterone or estriol (10(-6) or 10(-4) M concentration), showed no significant changes in the delta 5-desaturase activity. The results obtained suggest that the effect of the steroids on arachidonic acid biosynthesis in normal hepatocytes and HTC cells requires receptor occupancy and probably is mediated through a common biochemical mechanism.

8,11,14-Eicosatrienoic Acid↗

PubMed1988

Long-chain fatty acyl-CoA synthetase of rat adrenal microsomes. Effect of ACTH and epinephrine.

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.

Adrenal Glands↗

PubMed1988

Effect of high carbohydrate and high protein diets on microsomal fatty acid composition, "fluidity" and delta 6 desaturation activity in kidney and lung.

High carbohydrate and high protein diets administered to rats for only three days decreased and increased, respectively, the arachidonate/linoleate ratio (20:4 n6/18:2 n6) in total lipids of lung, kidney, and liver microsomes. In liver and kidney this correlated with a significant decrease in the first dietary condition, and with an increase in the second, of delta 6 desaturase activity, measured by the rate of conversion of 1-14C linoleate n6 to 1-14C linolenate n6. Such an enzymatic activity was not detectable in lung microsomes, probably because of the low capacity of this tissue to produce the coenzyme A ester of the substrate used, since in lung the effects of dietary manipulation on the 20:4/18:2 ratios were as large as in liver. Significant differences were observed in microsomes of the three tissues examined in the steady state fluorescence anisotropy (rs) of diphenylhexatriene, which correlated with their cholesterol/phospholipid ratios. Both were lower in microsomes from liver than from the other two tissues, and both remained unchanged in each tissue under the dietary regimens studied. The results indicate that the observed effects of high carbohydrate and high protein diets on fatty acid delta 6 desaturation activity do not lead to apparent alterations in the physical properties of microsomal membranes.

Animals↗

PubMed1988

Lipid thermotropic transitions in Triatoma infestans lipophorin.

The structure and lipid thermotropic transitions of highly purified lipophorin of Triatoma infestans were examined by several techniques: steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), cis-parinaric acid (cis-PnA) and trans-parinaric acid (trans-PnA), light scattering fluorescence energy transfer between the lipophorin tryptophan residues and the bound chromophores, DPH, trans-parinaric acid cis-parinaric acid, gel electrophoresis, and gel filtration. Fluorescence polarization of PnAs and DPH revealed a reversible lipid thermotropic transition in intact lipophorin at about 20 degrees C and 18 degrees C, respectively. In lipophorin, lipid dispersion fluorescence polarization of DPH detected a lipid transition approximately at 20 degrees C, while trans-PnA showed a gel phase formation at a temperature below 30 degrees C. Similar experiments in which trans-PnA was incorporated into diacylglycerols and phospholipids extracted from the lipophorin revealed gel phase formation below 30 degrees C and 24 degrees C, respectively. Light scattering measurements showed that lipophorin particles aggregate irreversibly at 45 degrees C, increasing the molecular weight, as determined by gel filtration on Sephacryl S-300, from 740,000 to values larger than 1,500,000. The particle aggregation did not change the physical properties of the lipophorin studied by fluorescence polarization, indicating that the aggregation is apparently a non-denaturing process. Energy transfer between the lipophorin tryptophans and the bound chromophores cis-PnA, trans-PnA, and DPA revealed a different location of the fluorescent probes within the lipophorin. Temperature-dependence on the energy transfer efficiency for all probes confirmed a change in the ordering of the lipophorin lipids at 24 degrees C.

Animals↗

PubMed1987

Cholesterol-induced microsomal changes modulate desaturase activities.

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.

Animals↗

PubMed1987

Effects of fatty acid deficiency on the lipid composition and physical properties of guinea pig rough endoplasmic reticulum.

Twenty-six days of fat deficiency brought about a decrease of linoleic and an increase of oleic acid in rough endoplasmic reticulum (RER) of guinea pig liver. Arachidonic acid was only slightly decreased in some phospholipids whereas eicose-5,8,11-trienoic acid was not enhanced except in phosphatidyl-inositol. All these changes were relevant specifically in phosphatidylinositol molecules and less important in phosphatidylcholine and phosphatidylethanolamine. Fat deficiency did not modify the relative proportion of phospholipids and cholesterol. Therefore, fat deficient guinea pig microsomes are a good model to study the effect of unsaturated fatty acids on membrane properties. Fluorescent anisotropy of RER membranes, lipids and phospholipids labeled with diphenylhexatriene, was increased by the fat deficiency. The most important increase was observed in liposomes of a mixture of RER phosphatidylinositol, phosphatidylserine and sphingomyelin. A small change was found in phosphatidylcholine and phosphatidylethanolamine dispersions at 37 degrees C. The modification of the lipid unsaturation evoked fluorescent anisotropy changes. Temperature-dependent fluorescent polarization curves of RER membranes labeled with trans-parinaric acid did not show inflections in the temperature range from 5 to 45 degrees C but, RER lipids and phospholipids presented a phase separation at about 20 degrees C. This inflection point was not modified by the fat deficient diet. In those liposomes prepared with a mixture of RER phosphatidylinositol, phosphatidylserine and sphingomyelin, the inflection point was produced at about 37 degrees C.

Animals↗

PubMed1987

Epinephrine effect on arachidonic acid biosynthesis in isolated adrenocortical cells.

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.

8,11,14-Eicosatrienoic Acid↗

PubMed1987

The occurrence of arachidonic acid in Triatoma infestans.

1. Gas-liquid chromatography and mass spectrometry were used to verify the presence of arachidonic acid (20:4 omega 6) in adult male and female organs of the blood-suckling bug Triatoma infestans. Fat body, gonad and head lipids were analyzed. 2. Male gonads contained the highest percentage of phospholipids and the highest percentage of arachidonic acid. 3. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and sphingomyelin were isolated from gonads and heads of sexed insects and the fatty acid composition analyzed. 4. All the phospholipids analyzed contained relevant percentages of linoleic acid and fair percentages of arachidonic acid, except for sphingomyelin where no 20:4 omega 6 acid was found. 5. However, arachidonic acid was specifically incorporated in phosphatidylinositol since this phospholipid contained very large percentages of the acid. 6. In male gonads, phosphatidylcholine also contained a similar high percentage of the acid. Arachidonic acid is apparently incorporated, selectively, into these lipids from the blood of the host.

Adipose Tissue↗

PubMed1987

ACTH depresses delta 6 and delta 5 desaturation activity in rat adrenal gland and liver.

The in vivo and in vitro effect of ACTH on the biosynthesis of polyunsaturated fatty acid of rat adrenal gland and liver was studied. The administration of ACTH to intact rats produced a significant decrease in the conversion of [1-14C]linoleic acid to gamma-linolenic acid, [1-14C]alpha-linolenic acid to octadeca-6,9,12,15-tetraenoic acid, and [1-14C]eicosa-8,11,14-trienoic acid to arachidonic acid in liver and adrenal microsomes. Isolated adrenocortical cells and hepatocytes obtained from animals treated with ACTH showed a decrease in the incorporation and desaturation of exogenous [1-14C]eicosa-8,11,14-trienoic acid. The addition of ACTH to the incubation medium of adrenocortical cells and hepatocytes isolated from untreated rats also caused a decrease in delta 5 desaturation activity. The effect of ACTH on adrenal and liver desaturases could be produced as a consequence of the release of glucocorticoids, already measured in the experiments. However, the in vitro experiments carried out with hepatocytes isolated from untreated rats, where corticosterone was absent, indicated that ACTH can depress delta 5 desaturation per se.

8,11,14-Eicosatrienoic Acid↗

PubMed1987

[Effect of zinc deficiency on the in vivo biosynthesis of fatty acids of the linoleic series in the rat].

The effect of Zn deficiency on the in vivo synthesis of fatty acids of linoleic acid (n6) series was studied in rat liver. 1-14C Linoleic acid (18:2 n 6), 1-14C acetate plus delta-linolenic acid (18:3 n 6), and 1-14C eicosatrienoic acid (20:3 n 6) were administered to control rats and to animals which had been maintained for 32 days on a Zn-deficient diet. The distribution of label among microsomal fatty acids was measured one hour after the injections. 14C 18:2 n 6 was converted to 14C labeled (n 6) linolenate, eicosatrienoate, arachidonate, and docosapentaenoate. Label from 14C acetate was found in saturated fatty acids and in the above polyenes. The label from 14C 20:3 n 6 was transferred to (n 6) eicosatetraenoate and docosapentaenoate. These results are consistent with the occurrence of active delta 6, delta 5 and delta 4 desaturases, as well as fatty acid elongases in vivo. Zn deficiency modified the fatty acid composition of rat liver microsomes and decreased the rate of conversion of 14C 18:2 to higher homologs. The 20:4 n 6/18:2 n 6 ratio was decreased, which correlated with a decreased production of 14C 20:4 n 6 from 14C 18:2 n 6. The ratio (14C-labeled) phosphatidylcholine/triacylglycerols was significantly decreased in Zn-deprived rats. Zn deficiency thus decreases the activity of fatty acid desaturases and elongases and modifies the activity of the enzymes involved in the incorporation of fatty acids into lipids.

Animals↗

PubMed1986

Modulation of delta 6 and delta 5 rat liver microsomal desaturase activities by dexamethasone-induced factor.

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.

Animals↗