Glucagon and epinephrine-stimulated phospholipid methylation in hepatic microsomes

Life Sciences, Vol. 28, pp. 1483-1488 Printed in the U.S.A.

Pergamon Press

GLUCAGON AND EPINEPHRINE-STIMULATED PHOSPHOLIPID METHYLATION IN HEPATIC MICROSOMES Naomi Kraus-Friedmann and Piotr Zimniak* Department of Physiology and Cell Biology, University of Texas School of Medicine, and *Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030 (Received in final form January 23, 1981)

Summary Phospholipid methylation by hepatic microsomes was measured following glucagon or epinephrine administration either to intact rats or to the isolated perfused liver. Both hormones stimulated the methylation measured as the incorporation of S-adenosyl-L-[methyl-JH]methionine into phospholipids. The labeled products were identified by thin layer chromatography and most of the counts were found to be incorporated into phosphatidylcholine. The stimulatory effects of the hormones were evident already 5 minutes following hormone administration both in in vivo and in in vitro. The observed stimulation of the methylation process by glucagon and epinephrine might be related to the previously reported stimulatory effect of these hormones on the microsomal Caz+ATPase, and indicate that methylation process(es) might mediate some of the effects of these hormones. The microsomal fraction of the liver contains a Ca2+-ATPase which stimulates the translocation of Ca2+ (1). The activity of this enzyme i~ influenced by hormones. Thus, glucagon treatment of intact rats, or addition of glucagQn to the isolated perfused liver was shown to stimulate the activity of the CaZ+-ATPase (2). In contrast, insulin is inhibitory (3). In addition, glucocorticoids also exert an influence on the enzyme, most likely by inducing i t (4). The hormone sensitivity of the microsomal Ca2+-ATPase implied that i t might play a regulatory role in cellular calcium metabolism. We were therefore interested to elucidate themechanismby which hormonal pre-treatment results in altered microsomal CaZ+-ATPase activity. I t was recently demonstrated in red cells that changing the phospholipid composition of the cell membrane by methylating phosphoa:idylethanolamine to phosphotidylcholine results in the stimulation of the CaZ+-ATPase activity (5). I t seemed feasible that a similar mechanism can account for the increased Caz+ uptake observed in microsomes isolated from glucagon treated rats. Therefore, the hormonal effects on phospholipid methylation were examined. According to the results presented here pre-treatment of rats with either glucagon or epinephrine or addition of these hormones to perfused liver stimulated the methylation of phospholipids and thus phosphatidylcholine synthesis.

0024-3205/81/131483-06502.00/0 Copyright (c) 1981 Pergamon Press Ltd.

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Materials and Methods Animals: Male Sprague-Dawley rats weighing 130-200 g and fed ad libitum laboratory chow were used in all experiments. For the in v i t r o experiments rats were anaesthetized with i . p . i n j e c t i o n of Nembutal. Subsequently, they were injected e i t h e r with epinephrine (lO0~g), s . c . , or with glucagon (lO0~g) i n t r a venously. Control rats received saline i n j e c t i o n in an identical fashion. At the indicated times, the abdomen was opened, and a lobe of the l i v e r taken out for the preparation of the microsomal f r a c t i o n . Liver Perfusion: The l i v e r perfusion technique employed has been described in d e t a i l s (6). In short, l i v e r s were perfused in situ via the portal vein with Krebs-Ringer bicarbonate buffer containing 4% bovine albumin Cohn f r a c t i o n V (Sigma). The perfusate was kept at 37°C and was oxygenated by a disk r o t a t i n g oxygenator. Preparation of Microsomes: The excised l i v e r s were placed in ice-cold sucrose solution (250 mM) containing Tris (2 mM) and d i t h i o t h r e i t o l (I mM), pH 7.2, minced with a scissor, and homogenized. The microsomal f r a c t i o n was subsequently isolated as described by Moore et al. (1,3). Determination of Phosphatide Methyl Transferases: The a c t i v i t i e s of the two methyl transferases was assayed together according to Hirata et al. (7), with the modification that the pH of the assay medium was lowered from pH I0 to pH 8, and the concentration of S-adenosyl-L-methionine was 200 ~M. I d e n t i f i c a t i o n of Reaction Products: The methods described by Hirata et al. (7) were used. The chloroform phase containing the products was dried, redissolved in 5 0 ~ I of chloroform/methanol 2/I v/v and applied to Merck S i l i c a gel 60 t h i n layer plates. Two solvent systmes were used to develop the chromatograms: I) Chloroform/propionic acid/n-propanol/H20 in a r a t i o of 2/2/3/I by v o l . , and 2) chloroform/methanol/7M NH3 in a r a t i o 60/35/5 by vol. The plates were sprayed with 10% PPO in glacial acetic acid or in methanol and fluorography was performed at -80°C. Authentic samples of phosphatidylcholine and phosphatidylethanolamine were used as standards; Rf values of the monoand dimethylated derivatives of the l a t t e r were taken from the l i t e r a t u r e (7). Results Effect of glucagon on microsomal phospholipid methylation in i n t a c t rats: The e f f e c t of glucagon administration to intact rats on the a c t i v i t i e s of the microsomal methyl transferases is presented in Table I. The methylation proceeded at a l i n e a r rate during the 30 minutes incubation time. The rate of the reaction was comparable to the values reported in the l i t e r a t u r e (8). Five minutes a f t e r the administration of glucagon the rate of phosphol i p i d methylation was s i g n i f i c a n t l y increased above control values. At l a t e r time period the differences were not s i g n i f i c a n t up to 30 minutes when again differences were present. This might indicate a phasic response. The products of the methylation process were i d e n t i f i e d by t h i n layer chromatography. Most of the r a d i o a c t i v i t y was found in phophatidylcholine with smaller amounts in mono- and dimethyl-phosphatidylethanolamine. An uni d e n t i f i e d radioactive spot with Rf close to I appeared in some experiments. I t was unchanged in the presence of glucagon, epinephrine or in absence of added hormone. I t appears therefore u n l i k e l y that i t represents O-methylated epinephrine, although under our assay conditions the catechol O-methyltransferase might be expressed (9).

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TABLE I EFFECT OF GLUCAGON ON PHOSPHOLIPID METHYLATION IN INTACT RATS

Minutes after hormone administration Glucagon Incubation Time

10

30

20 +

-

÷

770 _+ 88

779 ± 83

853

859

1026 ± 106

1098 ± 112

1781" ± 171

1465 ± 149

1312 ± 146

1825

2238

1526 ± 161

1935 ± 203

2007 ± 196

2401"* ± 211

2112 ± 221

2067 ± 214

2895

3036

2257 ± 236

2638 ± 271

3926 ± 388

4385 ± 401

3620

3575 ± 363

4564

4165

3924 ± 402

4558 ± 462

-

+

898 ± 86

910 ± 97

10

1538 ± 160

15

30

n=5

346

n=5

n=2

n=4

Fed rats were anesthetized with barbiturate. Subsequently glucagon (lO0~g) was administered via the t a i l vein. The rats were sacrificed at the indicated times. * **

p>O.05 p>O.01

(paired Student's t - t e s t )

Methylation is expressed in pmoles of methyl groups incorporated per mg microsomal protein. The data presented in this paper are taken from experiments in which no more than 10% of total radioactivity was associated with this spot. Effect of Gluca$on in the Perfused Liver: Becausein the intact rat various factors might influence the response to glucagon~ i t s effect was examined in the isolated perfused rat l i v e r . The rates of methylation in the perfused l i v e r were comparable to the rates obtained in vivo. Again, significant d i f ferences were observed at 10 minutes incubation. Although all other experimental values were higher than control values, because of v a r i a b i l i t y in this series of experiments the other values were not significant (Table 2).

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TABLE 2 EFFECTS OF EPINEPHRINE OR GLUCAGONON PHOSPHOLIPID METHYLATION IN THE PERFUSED RAT LIVER Epinephrine Incubation time Minutes 5

Glucagon +

4-

1190 ± 123

1212 ± 131

10

2019 ± 246

2340 ± 251

15

3035 ± 397

30

3879 ± 416

Epinephrine

n = 5

1007 ± 101

1340 ± 142

**

1596 1 275

2457 ± 263

3406 ± 381

**

2746 ± 283

3172 ± 263

5190 ± 541

**

4899 ± 491

5617 ± 569

Glucagon

n =4

Livers from fed Sprague-Dawley rats were perfused f o r 30 minutes; than either epinephrine (l~g/ml) or glucagon (lug/ml) were added to the perfusate. 5 minutes after the hormone reached the l i v e r , a sample of the l i v e r was homogenized, and the microsomal fraction prepared. Units and s t a t i s t i c a l evaluation are the same as in Table 1. Effects of Epinephrine on Microsomal Phospholipid Methylation: Epinephrine administration evoked similar responses to glucagon (Table 3). Again the responses were s i g n i f i c a n t l y d i f f e r e n t at 5 minutes and then again at 30 minutes. TABLE 3 EFFECT OF EPINEPHRINE ON PHOSPHOLIPID METHYLATION IN INTACTRATS Minutes after hormone administration Glucagon Incubation Time 813 86

5

10 +

30

20 +

+

972

687

832

98

71

88

1407

1856"

1295

1614

146

183

133

165

2073

2192

1962

2194

207

210

201

236

3594

3957

3447

4172

370

400

396

436

576

952

620

1136

-

+

583

723

64

78

1071

1377"*

10 1506

1668

110

130

1436

1816

145

190

2525

3150"

260

310

15 2522

2832

30 n=6

n=6

n=2

n=4

Fed rats were injected s.c. with lOOug Epinephrine and sacrificed at the indicated times. Units and s t a t i s t i c a l evaluation are the same as in Table 1.

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In contrast to the experiments with the intact rats in the perfused l i v e r 5 minutes after the administration of epinephrine the differences were s i g n i f i cant at every time point (Table 2). The reaction products were identical to those after glucagon treatment, as shown by thin layer chromatography. Discussion The data presented here demonstrate that glucagon or epinephrine administration either to the intact animal or to the isolated perfused rat l i v e r is followed by stimulation of methyl transferase activity in the microsomal fraction subsequently isolated. The changes in phospholipid methylation and the subsequent change in the phospholipid composition of the microsomal membrane probably have an effect on the physical characteristics of the membrane. Thus, i t has been demonstrated that enzymatic methylation of phosphatidylethanolamine increases the f l u i d i t y of erythrocyte membranes (10). In subsequent studies Hi rata et al. (11) demonstrated that the B-adrenergic agonist L-isoproterenol stimulated the methylation of phospholipids in rat reticulocyte ghost, an effect which resulted in increased membrane f l u i d i t y , increased translocation of methylated lipids and stimulated the coupling between the B-adrenergic receptor and the adenylate cyclase. I t is therefore reasonable to assume that the increase in methylation which is evoked by glucagon or epinephrine will also result in changes in the characteristics of the hepatic microsomal membranes. The possible importance of phospholipid methylation as a biological signal transmission was pointed out recently (12). The consequences of the hormonally evoked phospholipid methylation described here remain to be clarified. As mentioned in the introduction, i t is l i k e l y that the altered phospholipid composition is related to the observed changes in the microsomal Ca2+-ATPase following glucagon administration (3). While this paper was in preparation, a communication appeared reporting a stimulatory effect of glucagon on methyl-transferase activity in isolated hepatocytes (13). Acknowledgements The authors are grateful for the valuable advice of Dr. W. J. Strittmatter. The s k i l l f u l technical assistance of W. Cavin is acknowledged. This work was supported by U.S.P.H. grants AM-18383 and AM-19751. References 1. 2. 3. 4. 5. 6. 7.

J. Moore, T. Chen, R.H. Knapp and E.J. Landon, J. Biol. Chem. 250: 4562-4568 (1975). A.M. Andia-Waltenbaugh and N. Friedmann, Biochem. Biophys. Res. Commun. 82:603-608, 1978. A.M. Andia-Waltenbaugh, A. Lam, L. Hummel and N. Friedmann, Biochim. Biophys. Acta 630:165-175 (1980). N. Friedmann and F.D. Johnson, Life Sciences 27:837-842 (1980). W.J. Strittmatter, F. Hirata and J. Axelrod, Biochem. Biophys. Res. Commun 88:147-153 (1979). N. Friedmann and H. Rasmussen, Biochim. Biophys. Acta 222:41-52 (1970). F. Hirata, O.H. Viveros, E.J. Diliberto, Jr. and J. Axelrod, Proc. Natl. Acad. Sci. USA 75:1718-1771 (1978).

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Y. Tanaka, O. Doi and Y. Akamatsu, Biochem. Biophys. Res. Commun. 87: 1109-1115 (1979). J. Axelrod, Methods in Enzymology (S.P. Colowick and N.O. Kaplan, eds.) vol. 5, pp. 748-751, Academic Press, New York (1962). F. Hirata and J. Axelrod, Nature 275:219-220 (1978). F. Hirata, W.J. Strittmater and J. Axelrod, Proc. Natl. Acad. Sci. USA 76:368-372 (1979). F. Hirata and J. Axelrod, Science 209, 1082-1090 (1980). J. G. Castano, S. Alemany, A. Nieto, and J.M. Mato, J. Biol. Chem. 255: 9041-9043 (1980).