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.     

Immunochemical evidence for the enzymatic difference of delta 6-desaturase from delta 9- and delta 5-desaturase in rat liver microsomes

The enzymatic properties of the three types of microsomal acyl-CoA desaturases, delta 6-, delta 9- and delta 5-desaturases, were immunologically compared using a monospecific antibody raised against the purified linoleoyl-CoA desaturase (delta 6-desaturase). By the double immunodiffusion technique, the anti-delta 6-desaturase antibody showed a single precipitin line to the purified delta 6-desaturase and microsomes treated with Triton X-100, but no line was observed with the partially purified delta 9-desaturase. The antibody even inhibited definitely delta 6-desaturase activity in microsomes, but neither stearoyl-CoA (delta 9-) nor eicosatrienoic acid (delta 5-) desaturations were inhibited. By these immunological investigations it was confirmed that terminal delta 6-desaturase is different enzyme from desaturases delta 9- and delta 5.     

delta5 desaturation of fatty acids in L-M cells

L-M cells grown in a lipid-free medium containing ¹⁴C-labeled 9,12-linoleic acid incorporated most of this acid into glycerolipids as linoleic acid. Only a small amount (3%) was elongated to eicosadienoic acid. No Δ6 desaturation occurred. When the cells were incubated with ¹⁴C-labeled 8, 11, 14-eicosatrienoic acid, 22% of the activity was found in 5,8,11,14-eicosatetraenoic acid. Treatment of the cells for 24 hr with N-isopropylethanolamine, a choline analog, depressed this desaturation reaction to about 60% of control values. The identity of the tetraene product was established by two different chromatographic analyses of the fatty acid methyl esters. Location of the double bond at position C-5 was determined by ozonolysis and subsequent reduction of the ozonides to aldesters followed by gas-liquid chromatography. These results prove that L-M cells have a Δ5 desaturase and an elongation enzyme converting 18:2 to 20:2, but lack a Δ6 desaturase.     

The effect of insulin on delta5 desaturation in hepG2 human hepatoma cells and L6 rat muscle myoblasts.

In humans there is a correlation between the ratio of arachidonic acid (20:4n-6) to cis 8,11,14 eicosatrienoic acid (20:3n-6) in skeletal muscle phospholipids and insulin sensitivity. This has been interpreted as indicating a link between the activity of the delta5 desaturase enzyme and muscle insulin sensitivity. The present study addressed the possibility that insulin regulates delta5 desaturase activity using L6 rat myoblasts and hepG2 human hepatoma cells. Both cell lines responded to insulin by increasing the amount of D-[U-14C] glucose incorporated into glycogen. In L6 cells, insulin stimulated cis 8,11,14 eicosatrienoic acid uptake and arachidonic acid production but had no effect on the percentage conversion of cis 8,11,14 eicosatrienoic acid to arachidonic acid. In hepG2 cells, insulin had no effect on cis 8,11,14 eicosatrienoic acid uptake or arachidonic acid production. These results suggest that insulin has no direct effect on delta5 desaturase activity in the liver but can alter arachidonic acid production in muscle by altering substrate availability.     

Mineralocorticoids modify rat liver delta 6 desaturase activity and other parameters of lipid metabolism

The changes in linoleyl-CoA desaturase activity of rat liver microsomes were studied after a single intraperitoneal injection of 11-deoxycorticosterone or aldosterone at physiological doses. Fatty acid of plasma and different liver fractions, and physical properties of microsomal membranes were also investigated. It was found that the specific activity of delta 6 desaturase decreased 4-fold 24 hr after the injection of aldosterone or deoxycorticosterone. Pretreatment of the rats with aldosterone led to a significant decrease in the percent distribution of palmitic, arachidonic, docosapentaenoic and docosahexenoic acids, with a concomitant increase in palmitoleic, oleic and linoleic acids in plasma and liver homogenates, microsomes and cytosol fractions. A similar pattern was observed after deoxycorticosterone administration. The changes resulted in a decreased unsaturation index of all fractions studied and were well-correlated with the increase observed in fluorescence depolarization of the hydrophobic probe 1,6-diphenylhexatriene in liver microsomal membranes. The interlipid and lipid/protein ratios in microsomes remained constant after hormonal treatment. These results are consistent with the idea that the inhibition of delta 6 desaturase activity and the alterations in fatty acid composition induced by mineralocorticoids, are solely responsible for the measured decrease in liver microsomal membrane fluidity.     

Increased activity of stearoyl-CoA desaturation in liver from rat fed clofibric acid

Male rats were fed a diet containing 0.5% (w/w) p-chlorophenoxyisobutyric acid (clofibric acid), a hypolipidemic drug. Activities of stearoyl-CoA desaturation in hepatic microsomes were increased approx. 4 times following the administration of clofibric acid for 7 days. An increase in the activity of desaturation of stearic acid was also observed in the liver of clofibric acid-fed rats in vivo. The increase in the activity of microsomal stearoyl-CoA desaturation by clofibric acid-feeding was due to the increase in the activity of terminal desaturase as measured by the rate constant for cytochrome b5 reoxidation, but not due to the changes in cytochrome b5 content and NADH-cytochrome b5 reductase activity. Increases in the activity of stearoyl-CoA desaturation by clofibric acid-feeding were also observed in rats of hormonally altered state, such as diabetic rats, hyperthyroid rats and hypothyroid rats. Percentages of octadecenoic acid in total fatty acid of hepatic lipid were increased with the increase in the activity of stearoyl-CoA desaturation.     

The effect of adrenocorticotrophin on protein degradation in rat adrenal gland and liver

The rate of adrenal protein degradation appears to be slower in rats to which ACTH (adrenocorticotrophin) has been chronically administered. As measured by the exponential decay of radioactively labelled adrenal protein in vivo, the mean half-lives of total protein and of mitochondrial, microsomal and 18000g-supernatant protein were significantly longer in ACTH-treated animals. Experiments in which either [(3)H]leucine or NaH(14)CO(3) was used to label proteins showed that of the fractions studied, the effect on mitochondrial protein degradation was most pronounced. The half-lives of the same subcellular fractions in rat liver were not affected by ACTH. The possibility that the results might have been caused by changes in radioisotope reutilization and pool size is discussed.     

Effects of streptozotocin and dietary fructose on delta-6 desaturation in spontaneously hypertensive rat liver

We have investigated the effects of hypertension associated with diabetes mellitus on polyunsaturated fatty acid biosynthesis. For this purpose, two rat models for these pathologies have been established: a type 1 diabetic hypertensive model obtained by streptozotocin injection to spontaneously hypertensive rat (SHR), followed or not by insulin treatment (experiment 1); a type 2 diabetic hypertensive model by feeding SHR with a fructose enriched diet (experiment 2). Liver gene expression of delta-6 desaturase (D6D), microsomal D6D activities and fatty acid composition of total lipids were estimated. In experiment 1, an increase of linoleic acid (18:2 n-6) level was observed in the streptozotocin group. D6D gene expression appeared depressed in both experimental groups. Insulin did not reverse the streptozotocin effect in SHR, as it does in insulin-dependent diabetic rats. In experiment 2, the results showed a decrease of 18:2 n-6 and of long chain products of desaturation in rats fed on fructose diet. Delta-6 n-3 desaturase activity was significantly increased, whereas gene expression tended to decrease. Feeding fructose induced a significant increase in delta-9 desaturated products, suggesting a stimulation of stearoyl-CoA desaturase. These changes in monounsaturated fatty acids strongly differ from those observed in the streptozotocin experiment, indicating that the effects on lipogenesis of hypertension linked to diabetes differ according to the type of diabetes. Then, these results indicate that the liver steatosis observed during genetic hypertension was reinforced by fructose feeding. All together, the present results showed that hypertension associated to type 1 or type 2 diabetes exacerbated the damage caused by diabetes or hypertension alone on liver lipid metabolism. The metabolic effects induced by fructose being very similar to those found in human NIDDM, SHR fed a fructose-rich diet appears to be an appropriate model for studying the consequences of the combination of hypertension and NIDDM in the metabolic syndrome diseases.     

Nestin expression in rat adrenal gland

The constituents of the intermediate filament network of adrenal gland cells have not been deeply investigated in vivo. Adrenocortical cells have been reported to express cytokeratins and vimentin, but the intermediate filament components of the adrenomedullary cells are still unknown. Nestin is an intermediate filament protein that is mainly expressed in the developing nervous and muscle systems. It has been reported to be unable to form filaments by itself and it co-assembles with vimentin. Using immunocytochemical and biochemical approaches, the present study demonstrates that nestin is expressed in situ either in the cortex or in the medulla of adult rat adrenal glands. Nestin-negative cells prevalently form the zona glomerulosa whereas the zona fasciculata and the zona reticularis are mainly nestin-immunoreactive. Nestin-positive cells always express vimentin-like immunoreactivity but several cells apparently expressing only vimentin are detectable too. Nestin is also expressed by adrenomedullary cells that also display a faint vimentin-like immunoreactivity. We hypothesise that the inconstant detection of nestin in adrenocortical cells depends on their different functional moments. Moreover, even though our data do not allow to confirm vimentin in adrenomedullary cells, in situ detection of nestin in the adrenal medulla indirectly supports in vivo expression of vimentin in chromaffin cells.     

Immunoreactive delta sleep-inducing peptide in the rat hypothalamus, pituitary and adrenal gland: effects of adrenalectomy

Immunoreactive (IR) delta sleep-inducing peptide (DSIP) was examined by immunocytochemistry in the rat pituitary and adrenal gland and found to be colocalized with IR thyroid-stimulating hormone in the pituitary and with noradrenaline in the adrenal medulla. IR-DSIP was also detectable in nerve fibers in the posterior pituitary. By radioimmunoassay, IR-DSIP was quantified in plasma and tissue extracts after uni- or bilateral adrenalectomy. Significantly elevated plasma levels of IR-DSIP were measured 5 days after bilateral adrenalectomy (p less than 0.001). IR-DSIP was increased (p less than 0.05) in pituitary extracts from bilaterally adrenalectomized rats after 5 days, but not after 14 or 28 days. Sham- and unoperated animals did not significantly differ in plasma or tissue concentration of IR-DSIP. High-performance liquid chromatography of C18 SEP-PAK purified hypothalamus extracts revealed a single peak of IR-DSIP material of lower hydrophobicity than synthetic DSIP. The elevated concentration of IR-DSIP in the rat pituitary and plasma after bilateral adrenalectomy is consistent with the previously suggested role of DSIP to influence the activity of the hypothalamic-pituitary-adrenal axis.     

Effects of age and sex on the induction by triiodothyronine of cortisone delta4-5alpha-reductase and glucose 6-phosphate dehydrogenase of rat liver and adrenal

The effects of age and sex on the induction by 3,5,3′-triiodothyronine (T-3) of cortisone Δ⁴-5α-reductase and glucose 6-phosphate dehydrogenase (G 6-P D) in liver and the latter in adrenal have been investigated. Levels of cortisone Δ⁴-(5α, 5β)-reductase and G 6-P D were measured in homogenates of tissue from normal and T-3 injected male and female rats, $\text{1 1}\text{4}$ to 21 months of age. Increases in the levels of the reductase seen under T-3 stimulation were ascribed to induction of the 5α-reductase alone. T-3 caused induction of cortisone Δ⁴-5α-reductase only in the livers of male rats $\text{1}\text{3}\text{4}$ months of age. There was induction of total G 6-P D at most ages except in the livers of old male and young female rats and adrenals of young and old male rats. At all ages in normal animals of both sexes the maximum activity of glucose 6-phosphate dehydrogenase was much greater than that of cortisone Δ⁴-(5α, 5β)-reductase. It is concluded that the amount of G 6-P D in normal liver may be sufficient to handle an increase in cortisone reduction, and factors other than cortisone Δ⁴-reductase or G 6-P D levels alone must regulate increased reduction of the steroid.     

Comparative in vivo and in vitro study of the influence of experimental diabetes on rat liver linoleic acid delta 6- and delta 5-desaturation

Rats with experimental diabetes were administered in vivo a tracer dose of either [1-14C]-linoleic, [1-14C]-gamma-linolenic or [2-14C]-dihomo-gamma-linolenic acid and sacrificed 48 h later. With all three radioactive precursors, the radioactivity incorporated into arachidonic acid was lower in experimental diabetes, compared to nondiabetic rats similarly treated, while the weights of hepatic arachidonic acid were not significantly affected by the diabetic state. Streptozotocin-treated rats were administered moderate or excessive quantities of protamine-zinc-insulin. Streptozotocin diabetes inhibits rat liver homogenate [2-14C]-dihomo-gamma-linolenic acid delta 5-desaturation; only moderate injections of protamine-zinc-insulin restore the in vitro delta 5-desaturation. These results suggest that experimental diabetes, a reported inhibitor of delta 6-desaturation, also causes partial inhibition of delta 5-desaturation in rat liver; this suggests that dihomo-gamma-linolenic acid desaturation, a secondary regulatory step in linoleic acid metabolism, may be restored through an optimum insulin therapy.