Carlos A. Marra

Influence of testosterone administration on the biosynthesis of unsaturated fatty acids in male and female rats

Thein vivo effect of testosterone administration to male or female rats on the biosynthesis of unsaturated fatty acids of liver was studied. Twenty-four hours after injection of testosterone (260 μg/kg), δ9 desaturase activity increased significantly, whereas the activities of δ5 and δ6 desaturases were strongly depressed. These effects were more pronounced in female than in male animals. The fatty acid composition of plasma and liver (homogenates, crude microsomes and cytosol) showed differences between the sexes. In males, the percentage of palmitic acid and the 18∶1/18∶0 ratio were higher whereas the 20∶4(n−6)/18∶2(n−6) ratio was lower than in female rats. The administration of testosterone significantly modified the fatty acid pattern in all fractions studied. Analytical data correlated with alterations in the fatty acid desaturase activities caused by the hormone. It is suggested that the mechanism by which testosterone exerts its effect on unsaturated fatty acid biosynthesis is different than that previously demonstrated by glucocorticoid action. The effects produced by testosterone may be of biological significance in atherosclerosis pathogenesis.

Modulation of Δ6 and Δ5 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 Δ6 and Δ5 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 × g from hormonal-treated rat liver homogenates, added to crude or washed control microsomes, was able to inhibit in vitro linoleic and homo-γ-linolenic conversion to γ-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 Δ6 and Δ5 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.

Incorporation and metabolic conversion of saturated and unsaturated fatty acids in SK-Hep1 human hepatoma cells in culture

We report here a study of the incorporation and metabolism of various long chain fatty acids in SK-Hep-1 cultured hepatoma cells. Medium supplementation with radiolabelled palmitic, stearic, linoleic, α-linolenic and eicosa-8, 11,14-trienoic acids (1 µM, 24 H) resulted in an active uptake of each of these precursors by the cultures. Subsequent analysis of the cellular lipids indicated that they exhibit almost all the enzymic activities of polyunsaturated fatty acid metabolism that are characteristic of normal hepatic cells. With respect to the desaturation capacities of this cell line, although α-linolenic acid reacted more extensively than did linoleic acid and the conversion of 8,11,14-eicosatrienoic acid by the Δ 5 specific enzyme was more avid than had been previously seen in normal rat or human liver: the saturated fatty acids constituted relatively poor substrates, being preferentially chain-elongated rather than (mono) desaturated at the Δ 9 position. Analysis of the fatty acid profiles of total cellular lipids and of various lipid subclasses, however, revealed a relative paucity of essential fatty acids when compared with the abundance of endogenous monoenoic acids (particularly oleic). Of the total cellular fatty acids, 58% were present in the form of phospholipids; with 33% of the remaining 42% (i.e., the neutral lipids) being associated with triacylglycerol fraction. Within the total lipids, phosphatidyl-choline and phosphatidyl-ethanolamine were the major sites for the incorporation of all metabolic products derived from the incubated radiolabelled 16- and 18-carbon fatty acid precursors, whereas the phosphatidyl-inositol fraction was the predominat recipient of nascent arachidonic acid when the eicosatrienoate was the substrate. The express purpose of this investigation was to characterize the biochemical routes involved in the anabolism of various essential fatty acids in the human hepatocyte, through the use of cultured human hepatoma cells as an experimental model system. In view of the similarities between certain aspects of the polyunsaturated fatty acid metabolism of these cells and the corresponding properties of other mammalian hepatic or liver-derived tissues, the data presented here would thus constitute a significant beginning alone those lines. Moreover, considering the extreme difficulty in obtaining for such investigation relevant tissue samples from normal human sources, we regard these results — and the availability for use of this particular human hepatoma cell line — as important new developments in the effort to characterize a useful experimental model both for gaining immediate information and for designing future experiments.

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-β-estradiol, testosterone, estriol, aldosterone, corticosterone, dexamethasone, dehydroepiandrosterone, 11-β-hydroxyandrosterone, 11-ketoaetiocholanolone, epiaetiocholanolone and 5-β-pregnane-3α,20α-diol, provoked no significant changes in the uptake of the exogenous acid. Of all the steroids tested, only 11-dexycorticosterone, dexamethasone and 17-β-estradiol evoked a significant inhibition on the arachidonate biosynthesis in both kinds of cells. Testosterone, estriol, aldosterone and corticosterone provoked a significant inhibition of Δ5-desaturase in HTC cells. In dexamethasone, this effect was dose-dependent (0 to 10−4 M). Simultaneous incubation with 17-β- 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-β-estradiol, corticosterone or estriol (10−6 or 10−4 M concentration), showed no significant changes in the Δ5-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.

Regulatory effect of various steroid hormones on the incorporation and metabolism of [14C]stearate in rat hepatoma cells in culture

We have examined the incorporation and metabolism of [14C] stearic acid within the total lipids of HTC rat-hepatoma cells in suspension culture in presence and in absence of steroidal hormone stimulation. Both, glucocorticoids (dexamethasone, cortisol and corticosterone) and mineralocorticoids (deoxycorticosterone and aldosterone) as well as the estrogen β-estradiol and the androgen testosterone enhanced the extent of Δ9 desaturation to oleic acid of the saturated precursors, whereas only the two mineralocorticoids affected the incorporation rate of the exogenous acid into total cellular lipids, thus promoting a little stimulation. Furthermore, all the hormones tested increased the radiolabelling of the total cellular phospholipids except deoxycorticosterone and testosterone, the former having no effect and the latter exerting a moderate inhibition. On the other hand, the incorporation of14C into neutral lipids was stimulated by testosterone, in contrast to the inhibition of this parameter observed exclusively with either the mineralocorticoids or the estrogen. Within the phospholipid subclasses, the radiolabelling of phosphatidylcholine was augmented by means of all the steroids tested save deoxycorticosterone and testosterone, whereas phosphatidylethanolamine exhibited a decrease only in the presence of testosterone. In a similar fashion, within the neutral lipids, the predominating triglyceride fraction was preferentially labelled—at the expense of other subclasses of lesser abundance—upon treatment with the steroids except aldosterone, which exerted no effect. The results obtained were correlated with those changes observed in the mass distribution of the different lipid subclasses either with or without prior hormonal stimulation.

Dexamethasone blocks arachidonate biosynthesis in isolated hepatocytes and cultured hepatoma cells.

The effect of dexamethasone on the incorporation and conversion of [1-14C]eicosa-8,11,14-trienoic acid to arachidonic acid in isolated hepatocytes and in hepatoma tissue culture (HTC) cells was studied. In both kinds of cells, no changes in the exogenous acid incorporation were found when the hormone was added to the incubation media at 0.1 or 0.2 mM concentration, while the biosynthesis of arachidonic acid was significantly depressed. The effect on the biosynthesis was faster in isolated normal liver cells (60 min) than in tumoral cells (120 min) and reached an inhibition of ca. 50% after 3 hr of treatment. The addition of cycloheximide (10−6 M) also caused a marked decrease in the biosynthesis of this polyun-saturated fatty acid, but when dexamethasone was added to the media simultaneously with cycloheximide, a synergistic action was not observed. The results obtained show that protein synthesis would be involved in the modulation of the biosynthesis of arachidonic acid by glucocorticoids. The changes in the Δ5 desaturation of labeled 20∶3ω6 to arachidonic acid correlated with changes in the fatty acid composition in isolated cells.

Role of Copper and Cholesterol Association in the Neurodegenerative Process

Age is one of the main factors involved in the development of neurological illnesses, in particular, Alzheimer, and it is widely held that the rapid aging of the world population is accompanied by a rise in the prevalence and incidence of Alzheimer disease. However, evidence from recent decades indicates that Cu and Cho overload are emerging causative factors in neurodegeneration, a hypothesis that has been partially investigated in experimental models. The link between these two variables and the onset of Alzheimer disease has opened up interesting new possibilities requiring more in-depth analysis. The aim of the present study was therefore to investigate the effect of the association of Cu + Cho (CuCho) as a possible synergistic factor in the development of an Alzheimer-like pathology in Wistar rats. We measured total- and nonceruloplasmin-bound Cu and Cho (free and sterified) contents in plasma and brain zones (cortex and hippocampus), markers of oxidative stress damage, inflammation, and programmed cell death (caspase-3 and calpain isoforms). The ratio beta-amyloid (1-42)/(1-40) was determined in plasma and brain as neurodegenerative biomarker. An evaluation of visuospatial memory (Barnes maze test) was also performed. The results demonstrate the establishment of a prooxidative and proinflammatory environment after CuCho treatment, hallmarked by increased TBARS, protein carbonyls, and nitrite plus nitrate levels in plasma and brain zones (cortex and hippocampus) with a consequent increase in the activity of calpains and no significant changes in caspase-3. A simultaneous increase in the plasma Aβ1-42/Aβ1-40 ratio was found. Furthermore, a slight but noticeable change in visuospatial memory was observed in rats treated with CuCho. We conclude that our model could reflect an initial stage of neurodegeneration in which Cu and Cho interact with one another to exacerbate neurological damage.

Role of delta 9 desaturase activity in the maintenance of high levels of monoenoic fatty acids in hepatoma cultured cells.

The incorporation and Δ9 desaturation of exogenous [14C]stearic acid were studied in HTC 7288c cells in suspension. We examined the uptake of the acid over a wide range of concentrations (0–160 μM) after incubating the cells for 6 h in a chemically-defined medium. Under this experimental condition, the uptake of the labeled acid was more extensive than that obtained from static cultures or from monolayer of isolated hepatocytes of rats. At an external concentration of 160 μMca. 52 nmoles of acid per mg of cellular protein was taken up. The production of oleic acid from [14C]stearate (Δ9 desaturation) correlated well with the uptake curve between 0–80 μM concentration. For higher stearate concentrations, the biosynthesis of oleic acid declined substantially and a plateau of 22 nmoles/mg cellular protein was reached. The incorporation and desaturation of an initial exogeneous concentration of [14C]stearic acid (80 μM) was also studied from 0–6 h. The results obtained demonstrated that the uptake of the substrate into cellular lipids was fast and non saturable. Quantitative gas-liquid chromatography of total cellular lipids under the different experimental conditions demonstrated a negative correlation between the decrease in the palmitic and palmitoleic acids and the increase in the intracellular levels of stearic and oleic acids. These analytical modifications took place with no changes in the saturated/monoenoic fatty acid ratio. This work also demonstrated a significant contribution of the stearoyl-CoA desaturase system to the high levels of oleic acid present in this kind of hepatoma cells.

Uptake and metabolism of eicosa-8,11,14-trienoic acid in normal hepatocytes and HTC cells

SummaryThe incorporation and conversion of eicosa-8, 11, 14-trienoic acid to arachidonic acid were studied in HTC cells (7288 c hepatoma cells) and isolated hepatocytes of rat. The cells were incubated at different concentrations of the acid during 0 to 6 hours. Both kinds of cells were able to take up the acid. However, the HTC cells have a greater avidity for the eicosatrienoic acid than normal liver cells. The incorporation of the acid modified the fatty acid composition of the cells and caused a decrease in the amount of saturated and monoenoic acid. In both cells eicosatrienoic acid was converted to arachidonic acid. However, in HTC cells arachidonic acid level was low and not correlated with the amount of the eicosatrienoic acid incorporated. This fact is apparently not due to an impairment in Δ5 desaturation activity since this enzyme is active in both cells. It would be possible that arachidonic acid production in malignant cells would be also interrupted in another metabolic pathway after Δ6 desaturation step. The strikingly low amount of arachidonic acid biosynthesized in HTC cells compared to normal hepatocytes could be interpreted as a consequence of a lower availability of eicosatrienoic acid for the microsomal desaturation system in malignant cells, in addition to the low A6 desaturase activity.

Effects of Copper and/or Cholesterol Overload on Mitochondrial Function in a Rat Model of Incipient Neurodegeneration

Copper (Cu) and cholesterol (Cho) are both associated with neurodegenerative illnesses in humans and animals models. We studied the effect in Wistar rats of oral supplementation with trace amounts of Cu (3 ppm) and/or Cho (2%) in drinking water for 2 months. Increased amounts of nonceruloplasmin-bound Cu were observed in plasma and brain hippocampus together with a higher concentration of ceruloplasmin in plasma, cortex, and hippocampus. Cu, Cho, and the combined treatment Cu + Cho were able to induce a higher Cho/phospholipid ratio in mitochondrial membranes with a simultaneous decrease in glutathione content. The concentration of cardiolipin decreased and that of peroxidation products, conjugated dienes and lipoperoxides, increased. Treatments including Cho produced rigidization in both the outer and inner mitochondrial membranes with a simultaneous increase in permeability. No significant increase in Cyt C leakage to the cytosol was observed except in the case of cortex from rats treated with Cu and Cho nor were there any significant changes in caspase-3 activity and the Bax/Bcl2 ratio. However, the Aβ(1–42)/(1–40) ratio was higher in cortex and hippocampus. These findings suggest an incipient neurodegenerative process induced by Cu or Cho that might be potentiated by the association of the two supplements.