Effect of catecholamines and β-blockers on linoleic acid desaturation activity

The effect of catecholamines and adrenergic blocking agents on the oxidative desaturation of linoleic acid in rat liver microsomes was studied. Epinephrine (1 mg/kg/body weight) produced a significant decrease on the conversion of [1-14C]linoleic acid to γ-linolenic acid. The effect of epinephrine was blocked by single injections of the β blockers propranolol (10 mg/kg body weight) or dichloroisoproterenol 30 min before the hormone treatment. Isoproterenol (100 μg/kg body weight) produced a significant decrease on the activity of the linoleyl-CoA desaturase. The effect of the catecholamines was postulated to be mediated through β receptors by an enhancement of the intracellular levels of cyclic AMP.


INTRODUCTION
Since the work of Ahlquist (1), the mechanism of action of catecholamines has been explained through the presence of a and/3receptors in the membrane of the cell at the so-called adrenergic effector site~ The description by Powell and Slatter (2) of the/3-receptor blocking properties of dichloroisoproterenol did much to confirm the adrenergic receptor classification proposed by Ahlquist.Since then, numerous compounds classified as r-receptor antagonists have been described.One of them that gained general approval was propranolol.There is general agreement to consider that glycogenolytic effects of the catecholamines are related to their ability to activate adenyl cyclase and thus increase tissue levels of cyclic AMP.~Adrenergic blocking agents, such as propranolol, competitively inhibit the effect of catecholamines on the activation of adenyl cyclase.
A 6 Desaturase is a regulatory enzyme that begins the synthesis of polyunsaturated fatty acids of the essential series (3).In a previous work, we have demonstrated that epinephrine produced a decrease of A 6 desaturation activity in rat liver microsomes (4).This effect was postulated to be mediated through an enhancement of the intracellular cyclic AMP levels.
The purpose of the experiment reported in this paper was to study the effect of epinephrine and some pharmacological drugs that stimulate or antagonize the /3-receptors on the desaturation of linoleic acid in rat liver microsomes.
1The authors are members of the Carrera del Investigador Cient|fico of the Consejo Nacional de Investigaciones Cienffficas y Tdcnicas, Argentina,

Animals and Treatment of Animals
Adult female Wistar rats, weighing 180-220 g and maintained on standard Purina chow, were used~ All rats were fasted for 24 hr and then re-fed with Purina chow for 1 hr.Water was given ad libitumo Three hr later, the rats were separated into several groups.One group was injected with epinephrine at a dose of 1 mg/kg body weight~ Another group was treated with isoproterenol (100 ~g/kg body weight) and a third group was injected with the same dose of epinephrine and propranolol (10 mg/kg body weight).Propranolol was administered 30 min before epinephrine.A fourth group that received saline solution in place of the drugs was used as control~ All compounds were injected intraperitoneallyo The animals were killed 12 hr after the injection~ In the second experiment, the rats received the same treatment as in experiment one, but an additional group was injected with dichloroisoproterenol (50 mg/kg body weight) and epinephrine~ Dichloroisoproterenol was injected 30 min before the epinephrineo All the groups were killed 3 hr after the injections.bAverages of the analysis of four rats • one standard error of the mean (SEM).

Isolation of Microsomes
The rats were killed by decapitation without anesthesia~ The blood was allowed to drain and was collected for glucose determination.Livers were rapidly excised and immediately placed in ice cold homogenizing medium (5).After homogenization, samples were taken to measure protein and glycogen content.Microsomes were separated by differential centrifugation at 100,000 x g as described previously (5).

Experimental Procedures
Desaturation of the fatty acids by liver microsomes was measured by estimation of the percentage conversion of [ 1-14C]linoleic acid to 7-linolenic acid~ Three nmoles of the labeled acid and 97 nmoles of unlabeled acid were incubated with 5 mg of microsomal protein in a Dubnoff shaker at 35 C for 20 rain in a total volume of 1o5 ml of 0.15 M KC1-0.25 M sucrose solution.The medium contained 4/amoles of ATP, 0.1 /amole of CoA, 1.25 /amoles of NADH, 5 /amoles of MgCI2, 2.25 /.tmoles of glutathione, 62.5 /~moles of NaF, 0.5/amoles of nicotinamide, and 62~ /~moles of phosphate buffer (pH 7).The reaction was stopped by addition of 2 ml of 10% KOH in methanol.The fatty acids were recovered by saponification of the incubation mixture (45 min at 85 C), acidification and extraction with petroleum ether (bp 30-40 C).The fatty acids were esterified with methanolic 3 M He1 (3 hr at 68 C), and the distribution of the radioactivity between substrate and product was measured by gas liquid radiochromatography in an apparatus equipped with a Packard Proportional counter.Percentage conversion was calculated from the distribution of radioactivity between substrate and product measured directly on the radiochromatogram (6).
Protein content in the homogenate and in the microsomal fraction was determined by the biuret method of Gornall et al. ( 7) using cystalline bovine serum albumin as a standard.Blood glucose was measured by the o-toluidine method (8) and fiver glycogen by the method of Van Handel (9).

RESULTS AND DISCUSSION
The effect of the administration of epinephrine and the blocking agent propranolol on linoleic acid desaturation activity of rat liver microsomes in shown in Table I.Epinephrine markedly depressed A 6 desaturation of linoleic acid 12 hr after the injection~ Neither glycemia nor liver glycogen showed significant differences in the epinephrine group compared to the controls~ These results are consistent with experiments published earlier (4).They showed that epinephrine evokes an ordered sequence of events in liver~ It begins with cAMP increase, followed by a glycogen breakdown that leads to a blood glucose increase, and then a decrease of A 6 fatty acid desaturation occurs.This decrease of the microsomal A 6 desaturase activity is maintained for a long time and is still shown 12 hr after epinephrine injection when the glycogenolytic effect has already faded.
Table I also shows that pretreatment of the rats with propranolol did not inhibit the epinephrine effect in A 6 desaturation activity in contrast to what was expected.However, this result can be attributed to the fact that the /S-blockade weakens with time~ In this connection, it has been published that propranolol is extensively metabolized during the first passage through the liver (10).On the other hand, Evans et alo found that, in the rat, the mean half-life of propranolol was about 40 min (11).Epinephrine has a prolonged duration of action (4) while propranolol's effect is relatively short (12).Therefore, the results shown in Table I may be explained by fast fading of propranolol blocking effect that happens before epinephrine declines~ Consequently, A 6 desaturase was inhibited by the remaining effect of epinephrine, LIPIDS, VOL. 13, NO. 10 bAverages of the analysis of four rats -+ one standard error of the mean (SEM).
Table I also shows the effect of an adrenergic agonist, isoproterenol, in rat liver microsomal desaturation of linoleic acid.Isoproterenol produced a 50% decrease in A 6 desaturation activity, an effect similar to that obtained with epinephrine~ Isoproterenol produced no significant variations in the glycemia and in liver glycogen levels~ Table II shows the results obtained 3 hr after the injection of the adrenergic agonists or antagonists in microsomal A 6 desaturation activity, glycemia and liver glycogen~ Epinephrine produces a significant decrease of the microsomal conversion of linoleic acid to 7linolenic acid in liver~ This effect is completely blocked if 30 min before the injection of epinephrine the rats received either propranolol or dichloroisoproterenol.Propranolol alone did not modify significantly the results obtained in the control group~ Besides, this experiment shows that propranolol and dichloroisoproterenol are also able to inhibit epinephrine induced hyperglycemia and glycogenolysis as was previously reported (13)(14).
Therefore, we conclude that the/3-adrenergic blocking agents, propranolol and dichloroisoproterenol, are also antagonists of epinephrine effect in the activity of linoleyl CoA desaturase in the conditions of this experiment.The effect obtained with epinephrine can be mimicked with isoproterenol which also produces a significant decrease in A 6 desaturation activity (Table II).
It has been suggested that epinephrine initiated its biological effects in several tissues by increasing the intracellular concentration of cyclic AMP (15-16)./3-Adrenergic blocking agents competitively inhibit the ability of catecholamines to activate adenyl cyclase activity (17) and the formation of cyclic AMP (18).From the results obtained in the literature, it is evident that cyclic AMP mediates the activity of the /3-receptor.Epinephrine produces a decrease in the linoleyl CoA desaturase activity, an effect that is antagonistic with propranolol or dichloroisoproterenol.Dibutyryl cyclic AMP also decreases fatty acid A 6 desaturation (19).Thus, it is reasonable to assume that the depressing effect in A 6 desaturase activity produced by epinephrine is mediated by a flreceptor through an increase of the intracellular levels of cyclic AMP.It is also substantiated by the observation that isoproterenol, a /3adrenergic agonist, produces both an increase in the intracellular cyclic AMP concentration (20-21) and a significant decrease in A 6 desaturation activity.However, isoproterenol produces no significant differences in the glycemia in spite of the slight decrease in liver glycogen levels (Table II).This apparent inconsistency would be particularly important since it has been shown by Newton and Hornbrook (22) that a high dose of isoproterenol does not stimulate glycogen phosphorylase in rat liver.In contrast, it increases the intracellular levels of cyclic AMP (20-21) and the activity of rat liver adenyl cyclase in vitro (22).
However, isoproterenol produces an elevation of the nonesterified fatty acids in blood through an activation of adipose tissue lipase (23)(24).Therefore, it is possible to speculate that the in vivo effect of isoproterenol and even at least partially of epinephrine in the activity of rat liver A 6 desaturase is evoked through an activation of adipose tissue adenyl cyclase followed by an increased lipase activity.The free fatty acids released to the blood and transported to the liver would inhibit the enzyme~ Therefore, all our experiments show that cAMP is involved in the control of fatty acid A 6 desaturase activity but we have not determined yet the mechanism of action~

TABLEI
Effect of 12 hr Adminstration of Catecholamines and Propranolol on the Hepatic Microsomal Desaturation of Linoleic Acid, Glycemia a and Liver Glycogen a

TABLE I1 Effect
of 3 hr Adminstration of Catecholamines and/3-Blockers on the Hepatic Mierosomal Desaturation of Linoleic Acid, Glycemia, and Liver Glycogen a