Effect of cyclosporine and sirolimus on fatty acid desaturase activities in cultured HEPG2 cells.

The aim of the present work was to evaluate the influence of cyclosporine (CsA) and sirolimus (SRL) on fatty acid (FA) desaturase activities. These enzymes (named Delta9, Delta6, and Delta5 desaturases) catalyze reactions leading to the biosynthesis of n-9, n-6, and n-3 FA families. n-3 FA family, derived from alpha-linolenic acid, is involved in the prevention of vascular events, which appear after successful kidney transplantation. Five groups of HepG(2) cells in culture were treated with either CsA (1 microg/microL and 2 microg/microL) or SRL (10 ng/mL and 20 ng/mL) for 3 days, including a control group without immunosuppressive treatment. We studied the incorporation and metabolic conversion of radioactive [1-(14)C]palmitic, linoleic, and eicosatrienoic acids. We also analyzed fatty acid composition. The distribution of radioactive metabolic products after incubation of these cells with [1-(14)C]palmitic acid revealed a decrease in Delta9 desaturase activity in the presence of each immunosuppressive drug: CsA = 0.61 +/- 0.01; SRL = 0.59 +/- 0.04 versus control = 0.79 +/- 0.05 (P < .01). We observed a significant increase in Delta6 and Delta5 desaturase activities under the influence of the immunosuppressive drugs: radiolabeled linoleic acid (CsA: 0.93 +/- 0.04; SRL: 1.02 +/- 0.03 vs control 0.60 +/- 0.03; P < .01) and eicosatrienoic acid (CsA: 1.12 +/- 0.02; SRL: 1.07 +/- 0.01 vs control 0.75 +/- 0.01; P < .01). In conclusion, CsA and SRL modulated the biosynthesis of polyunsaturated FAs, decreasing Delta9 desaturase and increasing Delta6 and Delta5 desaturase activities.

Influence of testosterone on polyunsaturated fatty acid biosynthesis in Sertoli cells in culture.

Sertoli cells play a central role in spermatogenesis, its development and regulation. They are target cells for androgen action in the seminiferous tubules. The Sertoli cell is considered to be the most important cell type in the testis with regard to essential fatty acid metabolism. We studied the response to testosterone of cultured Sertoli cells from immature rats by determining the fatty acid composition of total cellular lipids as well as by the biosynthesis of polyunsaturated fatty acids. Fatty acid methyl esters were analysed by gas liquid chromatography and radiochromatography. Two doses of testosterone were tested (150 and 300 ng ml(-1)). Significant differences were found in fatty acids derived from total cellular lipids after 8 days in culture in the presence of testosterone (300 ng ml(-1), for 48 h). Compared to controls, the hormone produced a significant increase of 16:1 and 18:1 n-9, and of 18:2 n-6, and a decrease of 20:4 and 22:5 n-6 in total cellular lipids. The decrease in the n-6 fatty acid ratios 20:4/20:3, 20:4/18:2 and 24:5/24:4, and the increase in 18:1n-9/18:0 and 16:1n-9/16:0 ratios were taken as an indirect signal of testosterone effects on Delta5, Delta6 and Delta9 desaturase activities. The drop in Delta5 and Delta6 desaturase activities was corroborated by analysing the transformation of [1-14C]20:3 n-6 into its higher homologues. We concluded that testosterone modifies the fatty acid pattern of cultured Sertoli cells, and this hormone is involved in polyunsaturated fatty acid biosynthesis, modulating Delta5 and Delta6 desaturases activity.

Diacylglycerol generated in CHO cell plasma membrane by phospholipase C is used for triacylglycerol synthesis.

The diacylglycerol (DAG) signal generated from membrane phospholipids by hormone-activated phospholipases is attenuated by mechanisms that include lipolysis or phospholipid resynthesis. To determine whether the DAG signal might also be terminated by incorporation of DAG into triacylglycerol (TAG), we studied the direct formation of TAG from endogenous DAG generated by bacterial phospholipase C (PLC). When Chinese hamster ovary (CHO) cells prelabeled with [(14)C]oleate were treated with PLC from Clostridium perfringens for 6 h, [(14)C]phospholipid decreased 15% and labeled TAG increased 60%. This transfer of (14)C label was even greater when the cells were simultaneously exposed to PLC and 100 microM oleic acid. PLC as well as oleate treatment concomitantly increased the TAG mass within the cell. Moreover, when phospholipids were prelabeled with [(3)H]glycerol, a subsequent increase in [(3)H]TAG indicated that an intact DAG moiety was channeled into the TAG structure. Incubating CHO cells with the diacylglycerol kinase inhibitor R59022 enhanced the formation of TAG from phospholipids hydrolyzed by PLC or by PLC in the presence of 100 microM oleate, but not by incubation with oleate alone, indicating that the DAG released from plasma membrane phospholipids does not require the formation of a phosphatidic acid precursor for TAG synthesis. Similarly, the diacylglycerol lipase inhibitor RHC 80267 did not alter TAG synthesis from plasma membrane DAG, further supporting direct incorporation of DAG into TAG. These studies indicate that DAG derived from plasma membrane phospholipid is largely used for TAG formation, and support the view that this mechanism can terminate DAG signals. The studies also suggest that a transport mechanism exists to move plasma membrane-derived DAG to the endoplasmic reticulum.-Igal, R. A., J. M. Caviglia, I. N. T. de Gómez Dumm, and R. A. Coleman. Diacylglycerol generated in CHO cell plasma membrane by phospholipase C is used for triacylglycerol synthesis. J. Lipid Res. 2001. 42: 88;-95.

Acylglycerol synthesis in liver of type II diabetic rats fed a diet supplemented with either n-6 or n-3 fatty acids.

Liver is one of the tissues most actively involved in triacylglycerol synthesis and secretion. Hypertriglyceridemia is commonly associated with the diabetic state which has been detected in very young rats after the induction of experimental diabetes. In the present work, acylglycerol synthesis in liver of streptozotocin-treated rats, fed a diet supplemented with n-3 and n-6 fatty acids, was studied. At the onset of the experiment, plasma triacylglycerol levels increased significantly in diabetic animals when compared to controls. Two weeks after the dietary treatment, the aforementioned parameter decreased in diabetic animals consuming either n-6 or n-3 fatty acids. In control rats, n-3 fatty acids depressed triacylglycerol synthesis in liver microsomes. In the diabetic group both diets increased diacylglycerol and triacylglycerol synthesis. The addition of liver cytosolic fraction from control rats to the incubation medium, stimulated the triacylglycerol synthesis in all the groups. Nevertheless, the radioactivity recovered in the neutral lipid fractions was lower in the samples from rats fed n-3 fatty acids compared to n-6. We conclude that dietary n-3 fatty acids decreased significantly triacylglycerol plasma levels in diabetic rats probably through the inhibition of liver triacylglycerol secretion. In addition, there probably is an n-3 fatty acid sensitive factor in the liver cytosolic fraction able to depress triglyceride synthesis.

Acylglycerol synthesis in liver of type II diabetic rats fed a diet supplemented with either n-6 or n-3 fatty acids.

Liver is one of the tissues most actively involved in triacylglycerol synthesis and secretion. Hypertriglyceridemia is commonly associated with the diabetic state which has been detected in very young rats after the induction of experimental diabetes. In the present work, acylglycerol synthesis in liver of streptozotocin-treated rats, fed a diet supplemented with n-3 and n-6 fatty acids, was studied. At the onset of the experiment, plasma triacylglycerol levels increased significantly in diabetic animals when compared to controls. Two weeks after the dietary treatment, the aforementioned parameter decreased in diabetic animals consuming either n-6 or n-3 fatty acids. In control rats, n-3 fatty acids depressed triacylglycerol synthesis in liver microsomes. In the diabetic group both diets increased diacylglycerol and triacylglycerol synthesis. The addition of liver cytosolic fraction from control rats to the incubation medium, stimulated the triacylglycerol synthesis in all the groups. Nevertheless, the radioactivity recovered in the neutral lipid fractions was lower in the samples from rats fed n-3 fatty acids compared to n-6. We conclude that dietary n-3 fatty acids decreased significantly triacylglycerol plasma levels in diabetic rats probably through the inhibition of liver triacylglycerol secretion. In addition, there probably is an n-3 fatty acid sensitive factor in the liver cytosolic fraction able to depress triglyceride synthesis.

Lipid dismetabolism in Leydig and Sertoli cells isolated from streptozotocin-diabetic rats.

This work evaluates how the abnormalities in fatty acid biosynthesis, already described in diabetic rats, were extended to Sertoli and Leydig cells isolated from testes of streptozotocin-treated rats. Both kinds of cells were incubated in the presence of labeled eicosa-8,11,14-trienoic acid. The uptake of the substrate and its conversion to arachidonic acid were significantly depressed in both cell types compared to the non-diabetic controls. The indirect evidence of this inhibition was observed in the total fatty acid pattern of the cells where the 20:4 n-6/18:2 n-6 ratio was significantly decreased. The addition of insulin to the incubation medium produced no changes in the uptake of the substrate by the cells. Under similar experimental conditions the synthesis of arachidonic acid was partially recovered, however, the values obtained were still below those ones of cells isolated from control animals. These results were correlated with the fatty acid profile of different lipid fractions of plasma and with the activity of enzymes involved in polyunsaturated fatty acids biosynthesis measured in the testicular microsomes of diabetic rats. We conclude that Sertoli and Leydig cells evidenced similar lipid disorders than those observed in the whole testis or in other tissues of diabetic rats, and that the biosynthesis of arachidonic acid is under insulin regulation.

Early lipid alterations in spontaneously diabetic rats.

Human and experimental diabetes mellitus extensively alters lipid metabolism. The eSS is a rat strain that develops a spontaneous diabetes of slow evolution, resembling the non-insulin-dependent diabetes mellitus of young people. We report here disturbances in lipid metabolism of 5-month old eSS rats compared to age-matched alpha-controls. Normal plasmatic glucose levels were found in the fasted state, whereas a diabetic curve was evident for eSS rats after glucose load. Triglyceride content was elevated in plasma and in liver microsomal preparations of eSS animals, when compared to the controls. The diabetic strain revealed a significant fall in the amount of linoleic acid in liver and kidney microsomes and in erythrocyte membranes. In liver, an increase in 22:6 (n-3) was also noted. A depression in the content of linoleic acid as well as an enhancement of docosahexaenoic acid were detected in phosphatidylcholine and phosphatidylethanolamine fractions from liver microsomes of eSS rats. The fatty acid pattern of eSS rat testis showed a raise in the relative percentage of arachidonic and a decrease in 22:5 (n-6), 22:5 (n-3) and 22:6 (n-3) acids compared to their controls. Diabetic rats exhibited a significant increase in microsomal cholesterol content and cholesterol/phospholipid ratio in liver and testis. In the latter tissue, higher values of fluorescence anisotropy were also observed. The current observations indicate that in early stages of the diabetes onset, when eSS rats are still normoglycemic, severe alterations of lipid metabolism may contribute to the establishment and progression of the diabetic syndrome.

Modulation of rat liver lipid metabolism by prolactin.

The effect of chronic hyperprolactinemia on the delta6- and delta5-desaturation activity, total lipid and fatty acid composition, as well as fluorescence anisotropy, was studied in liver microsomes from anterior pituitary-grafted rats. We observed a depression in delta6-desaturation activity but no changes in the delta5-desaturation activity in the grafted animals. The microsomal fraction from the hyperprolactinemic rats contained significantly less amount of linoleic acid and a higher content of 20:4 n-6, 22:5 n-6 and 22:6 n-3 acids. Lipid rotational mobility was increased in microsomes as well as in liposomes obtained from the microsomes of transplanted animals. The fluidifying effect induced by PRL was located in the deepest zone of the membrane. The results obtained indicate that high levels of prolactin induce changes in polyunsaturated fatty acid distribution in liver microsomes, which regulates the lipid rotational mobility and hence membrane fluidity.

Spontaneously hypertensive rats: eicosa-8,11,14-trienoic acid metabolism and arachidonic acid biosynthesis.

Delta 9, delta 6 and delta 5 desaturation activity and the thioesterification of eicosa-8,11,14-trienoic acid (DGLA) were measured in spontaneously hypertensive rats (SHR) compared to normotensive controls (WKY). SHR exhibited lower levels in the long-chain fatty acyl-CoA (LCFA) synthetase and in delta 6 and delta 5 desaturase activities in liver. The enzymatic activity changes were reflected on the fatty acid composition of liver microsomes. In testis, the thioesterification of DGLA and its conversion to arachidonic acid, (AA), at the delta 5 desaturation step were also depressed in SHR. We conclude that, in SHR, the alteration in polyunsaturated fatty acid (PUFA) metabolism may influence the synthesis of AA-derived eicosanoids involved in the control of blood pressure regulation.

Long-chain acyl-CoA synthetase of rat testis microsomes. Substrate specificity and hormonal regulation.

We investigated long-chain fatty-acyl-CoA synthetase activity in rat testicular microsomes. The apparent Michaelis constants (Km's) for the substrate fatty acids increased while their corresponding maximal velocities decreased in the order 18:3(n-3), 20:3(n-6), and 18:0. The reaction with 20:3 as substrate was diminished in the presence of a constant amount of either 18:0, 18:2(n-6), or 18:3(n-3) in a manner consistent with their action as simple competitive inhibitors, with the Ki values for 18:0 and 18:3(n-3) being of the same order of magnitude as their respective Km's. Adrenocorticotrophin and/or dexamethasone administration to intact rats caused a significant decrease in the thioesterification of all three substrates without producing any alteration in the fatty-acid composition of the microsomal membranes. These results indicate the presence of a broad-specificity activating enzyme in testis whose function is subject to hormonal regulation.

Arachidonic acid biosynthesis in non-stimulated and adrenocorticotropin-stimulated Sertoli and Leydig cells.

The biosynthesis of arachidonic acid in Sertoli and Leydig cells isolated from the testes of mature rats has been investigated. Both types of cells incorporated [2-14C]eicosatrienoic acid from the incubation medium and transformed it into arachidonic acid. The administration of adrenocorticotropin (ACTH) to the rats decreased the delta 5 desaturating activity in the isolated testicular cells, while ACTH produced no changes in the uptake of the substrate. Similar results were obtained when ACTH was added to the incubation medium of cells isolated from non-hormone treated rats. The total fatty acid composition of the Sertoli cells isolated from ACTH-treated rats showed a significant increase in the relative percentage of 18:2n-6 and a decrease in the C20 and C22 polyenes. This may indicate that ACTH exerts an inhibitory effect on delta 6, delta 5 and delta 4 desaturase activities. Addition of corticosterone to the incubation medium also produced a significant decrease in arachidonic acid biosynthesis. Because ACTH is known to stimulate the release of corticosterone in vivo, both hormones may act cumulatively in the regulation of arachidonic acid metabolism in Sertoli and Leydig cells.

Abnormal metabolism of polyunsaturated fatty acids in adrenal glands of diabetic rats.

Studies carried out on the adrenal glands of experimental diabetic rats have shown an important inhibition in polyenoic fatty acid biosynthesis. This effect was demonstrated by testing the activities of long-chain fatty acyl-CoA synthetase, the delta 5- and delta 6-desaturases of the (n-6) essential fatty-acid series and the delta 6-desaturase of the (n-3) series in liver and adrenal microsomes. The depression in desaturating activity in the insulin-deprived animals was independent of that produced on acyl-CoA-thioester biosynthesis. Experiments measuring the incorporation and transformation of [1-14C]eicosa-8,11,14-trienoic acid in adrenocortical cells isolated from streptozotocin-diabetic animals demonstrated a significant inhibition of arachidonic acid biosynthesis compared to controls. Insulin injections in diabetic rats partially restored the delta 5- and delta 6-desaturase activities. This effect could result from direct action by the hormone since the restoration was reproduced when arachidonic acid biosynthesis was measured after insulin was added to the incubation medium of adrenocortical cells isolated from diabetic animals. The results of the present study provide new information about the implication of this abnormal metabolism in the adrenal gland of diabetic rats.

Role of fatty acids of (n-3) and (n-6) series on alpha-linolenic acid desaturation and chain elongation in HTC cells.

The desaturation and chain elongation of [1-14C] alpha-linolenic acid were studied in HTC cells preincubated for 24 h in the presence of different unlabeled fatty acids of (n-3) and (n-6) series. After 24 h in the presence of [1-14C] alpha-18:3, cells transformed this acid into labeled 20:5 and 22:5(n-3) through the desaturation-elongation pathway and into 20:3 and 22:3(n-3) by the elongation reactions. The preincubation of HTC cells with (n-3) fatty acids (alpha-18:3, 20:5 and 22:6) produced an increase in the amount of [1-14C] alpha-18:3 that remained in the cells without being metabolized and consequently, a decrease in the last product formed, the 22:5(n-3) was observed. Simultaneously, the desaturation-elongation products decreased significantly and those of the elongation pathway were not modified, except when the cells were pre-incubated with the last fatty acid of this family (22:6) which increased this metabolic route. Fatty acids of (n-6) series (gamma-18:3, 20:3, 20:4 and 22:4) decreased the desaturation-elongation pathway and increased the elongation route from [1-14C] alpha-18:3. From these results, it can be concluded that fatty acids of (n-3) family and intermediates of (n-6) series would impair the [1-14C] alpha-18:3 metabolism at the delta 6 desaturation step. The fatty acid composition of the cells was also modified by the preincubation with (n-3) and (n-6) acids showing a decrease on delta 9 desaturation activity.

Biosynthesis of polyunsaturated fatty acids of (n-6) and (n-3) series in isolated adrenocortical cells of rats. Effect of ACTH.

Both the capacity of isolated adrenocortical cells to incorporate and transform [1-14C]linoleic and [1-14C]alpha-linolenic acids and the effect of ACTH on the biosynthesis of polyunsaturated fatty acids from [1-14C]alpha-linolenic acid were investigated. The cells were able to incorporate both labeled precursor acids and convert them into higher homologs. This transformation increases along the incubation time tested. When linoleic acid was the precursor, the biosynthesis of higher homologs was carried out following the desaturating-elongating route. Both pathways, the desaturating-elongating and the elongating, were detected when the substrate was alpha-linolenic acid. The results proved the existence of delta 6, delta 5 and delta 4-desaturases in this type of cells. Isolated adrenocortical cells obtained from rats treated with ACTH showed an increase in the amount of [1-14C]alpha-18:3 that remained in the cells without metabolization and, consequently, a decrease in the last product formed (20:5 n-3) was evident compared to the controls. Simultaneously, the desaturation-elongation products decreased significantly. Similar results were obtained when cells isolated from untreated rats were incubated for 3 h in the presence of ACTH. In this case, the values obtained returned to normal levels 6 h after incubation. These results were mimicked by dibutyryl-cyclic AMP. It can be concluded that the effect of ACTH on the biosynthesis of polyunsaturated fatty acids from alpha-linolenic acid was mediated through an enhancement of the intracellular levels of cAMP.

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.

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.

Effect of high carbohydrate and high protein diets on microsomal fatty acid composition, [quot ]fluidity[quot ] 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.

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.

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.