The effects of glucagon and TH-glucagon on steroid metabolism in isolated rat hepatocytes

203

Moleculur and Cellular Endocrinology, 55 (1988) 203-207 Elsevier Scientific Publishers Ireland, Ltd.

MCE 01795

The effects of glucagon and TH-glucagon on steroid metabolism in isolated rat hepatocytes Abas Hj. Hussin, Claire J. Allan, Victor J. Hruby

’

and Paul Skett

Molecular Pharmacology Laboratory, Department of Pharmacology, The University, Glasgow GI2 8QQ. Scotland, U.K., and ’ Department of Chemistry University of Anzona, Tucson, AR 85721, U.S.A. (Received

Key work

Glucagon,

liver; Steroid

metabolism;

8 June 1987; accepted

8 October

1987)

Phosphatidyhnositol

Summary Glucagon decreases the activity of steroid-metabolising enzymes in isolated rat liver cells at physiological concentrations. Higher concentrations are less effective. TH-glucagon (1-N-a-trinitrophenylhistidine12-homoarginine-glucagon) also reduces enzyme activity but does not lose activity at higher concentrations. The effects of the two hormones mimic closely their reported effects on phosphatidylinositol-4,5-bisphosphate breakdown. It is, thus, likely that the effect of glucagon on steroid metabolism is mediated via breakdown of this phospholipid. The calcium ionophore, A23187, had no effect on steroid metabolism whereas the phorbol ester 4/?-phorbol-12-myristate-13-acetate (PMA) mimicked the effect of glucagon, showing that activation of protein kinase C but not Ca*+ mobilization may be involved in glucagon’s action on hepatic steroid metabolism.

Introduction Glucagon has, for many years, been accepted as the classical example of a hormone acting by increasing intracellular CAMP via an activation of adenylate cyclase (Sutherland and Rail, 1958). The action of glucagon on glycogen phosphorylase (Studer et al., 1984), phosphatidate phosphohydrolase (Pittner et al., 1986) and omithine decarboxylase (Klingensmith et al., 1980) have, for instance, been linked to CAMP production. More recently, glucagon action has also been linked to changes in intracellular free calcium ([Ca2+li). Combettes et al. (1986) showed a dose-dependent effect of glucagon on [Ca2+], in isolated hepatocytes although the action was not as marked as Address for correspondence: Dr. Paul Skett, Molecular Pharmacology Laboratory, Department of Pharmacology, The University, Glasgow G12 SQQ, Scotland, U.K. 0303-7207/88/$03.50

0 1988 Elsevier Scientific

Publishers

Ireland,

with vasopressin. Staddon and Hansford (1986) also showed evidence for a glucagon-stimulated rise in [Ca2+li but showed that forskolin (which directly activates adenylate cyclase) or dibutyrylCAMP had the same effect. This suggested that glucagon increased [Ca’+], via its action on CAMP. The question of whether glucagon may directly increase [Ca2+li was answered by Wakelam et al. (1987) who showed that an analogue of glucagon, TH-glucagon (1-N-a-trinitrophenylhistidine-12homoarginine-glucagon), which did not have any effect on CAMP production under the same conditions, could cause the turnover of phosphatidylinositol-4,5-bisphosphate (PIP,) to yield inositol-1,4,5-&phosphate (IP,), a compound known to be involved in the increase in [Ca”], (Berridge and Irvine, 1984). Glucagon was also shown to have this effect although the turnover of PIP, was not seen at higher concentrations. It was suggested that the inhibition of PIP, turnover at superphysiLtd.

204

ological concentrations of glucagon was due to excess CAMP production inhibiting the polyphosphoinositide phosphodiesterase. Glucagon seems, therefore, to be able to exert its actions via two different receptor systems (termed GR-1 and -2), GR-1 being linked to PIP, turnover and GR-2 to cAMP production. The question can then be asked: which of glucagon’s effects are linked to which receptor system? In order to begin to answer this question, we have investigated the glucagonmediated decrease in steroid metabolism in isolated liver cells. Hepatic steroid metabolism is under extensive hormonal control (Gustafsson et al., 1980; Skett, 1987) and is mediated via the same enzymes as perform drug metabolism (Gibson and Skett, 1986). It is well recognised that glucagon can influence drug metabolism in vivo (Weiner et al., 1972)eand this was thought to be by its action on CAMP production. The use of isolated hepatocytes is essential to examine the effect of one hormone alone, bearing in mind the extensive interactions of hormones within the body. We have devised a method for maintaining steroid metabolism in hepatocyte cultures in serum-free medium for a period of, at least, 24 h (Hussin and Skett, 1986) and this has been used in this study. Materiak

and methods

Chemicals Collagenase was obtained from BCL, Lewes, U.K. and Ham’s F-10 medium from Gibco BRL, Paisley, Scotland, U.K. Glucagon, 4~-phorbol-12myristate-13-acetate (PMA), the calcium ionophore (A2318’7), 4-androstene-3,17-dione and bovine serum albumin were purchased from Sigma Chemical Co., Poole, U.K. TH-glucagon was synthesised from glucagon as previously described (Bregman et al., 1980), 4-[~‘4C]androstene-3,17dione was obtained from Amersham International, Aylesbury, U.K. All other chemicals were of the highest purity available commercially. An~maZs Male Wistar rats, bred in the department, were used throughout the study. The animals weighed 200-250 g when used and were housed in light-

and temperature-controlled conditions 07.00-19.00 h and 19 ~fr1°C).

(lights

on

Preparation of liver cells Isolated hepatocytes were prepared by a modification of the collagenase perfusion technique of Seglen (1973), which involved anaesthetising the animal with halothane/nitrous oxide and inserting a cannula into the hepatic portal vein. Calcium-free Hanks’ balanced salt solution (BSS) was perfused through the liver via the portal vein for 8 min, followed by the collagenase buffer (BSS supplemented with 4 mM calcium chloride and 0.5 mg collagenase/ml) for a further 12 min. The perfusate was collected through a cut in the inferior vena cava and, in the case of the collagenase buffer, recycled. Following perfusion, the liver was removed from the animal and cells isolated by carefully dissecting away the capsule and combing out the cells into calcium-free BSS. The cells were counted using a haemocytometer and assayed for viability by the trypan blue exclusion method. The yield of cells was 10” cell/g liver at greater than 90% viability. The cells were plated out on 9 cm Petri dishes (Nunclon, Denmark) at a density of 3 x lo5 cells/cm2 in Ham’s F-10 nutrient medium supplemented with 0.1% bovine serum albumin. Additions of hormones and drugs The additions were made directly to the cell culture after the cells had been allowed to attach to the Petri dish. Glucagon and TH-glucagon were dissolved in 0.1 M hydrochloric acid. The phorbol ester, PMA, was dissolved in dimethylsulpho~de. A23187 was dissolved in distilled water. In all cases the compounds were added in the smallest volume possible and controls were treated with a similar amount of vehicle. The additions did not alter the pH of the medium used. Glucagon and TH-glucagon were added at concentrations ranging from lo-i0 to 10e6 M, PMA at 5 x lo-’ M and A23187 at 10m7 M. The cell cultures were kept in a controlled environment (temperature 37 + 0.5’ C, humidity 96 + 1% and an atmosphere of 95% oxygen/5% carbon dioxide) during the preincubation period. Assay of steroid metabolism After treatment with the hormones

for the vari-

205

ous time periods, the cells were transferred into incubation medium and incubated with 4-androstene-3,17-dione by a modification of the method of Berg and Gustafsson (1973) as described by Hussin and Skett (1987). The calculated final substrate concentration in the incubation was 0.58 mM and this represents a saturating concentration of substrate for some, but not all, of the enzymes studied (Skett, 1978). This concentration of substrate was employed as this is the limit of solubility of androst-4-ene-3,17-dione under the conditions used in the assay. Calculations and statistics Results are expressed as percentage of relevant controls. Means and standard deviations were calculated and statistical analysis performed by means of Student’s t-test. The level of significance was set at P < 0.05. Results and discussion The assay for metabolism of androst-4-ene3,17-dione allowed the measurement of the 6p-, 7n- and 16a-hydroxylase, 17-oxosteroid oxidoreductase and ScY-reductase activities. As not all of the enzymes are saturated with substrate, the activities measured do not necessarily represent the V,,,, of the enzyme but simply the activity of the enzyme under the assay conditions. As can be seen from Table 1, all of the enzymes responded to treatment with glucagon in a similar manner

TABLE

and, thus, the 6p- and 7cr-hydroxylase activities are taken as representative of all the activities studied. Glucagon, at lower concentrations (10~“’ to lo-* M) caused a dose-dependent decrease in 7aand 6/?-hydroxylase activities (down to 55% and 60% of control for the SLY-and 6/I-hydroxylase respectively) but at higher concentrations the effect of glucagon was markedly reduced (Fig. 1). Indeed, there was no significant effect of glucagon at lop6 M. The maximum effect of glucagon was seen at 10-s M for all enzymes. The U-shaped dose-response curve for glucagon suggests two possibilities; either the down-regulation of glucagon receptors at higher concentrations as is seen with insulin (Desbuquois et al., 1982) or a second effect of glucagon at higher than physiological levels of the hormone. Glucagon is known to increase intracellular CAMP levels in a dose-dependent manner up to lo-’ M (Dighe et al., 1984; Studer et al., 1984) and has more recently been shown to increase [Ca2’li in a similar dose-dependent way (Combettes et al., 1986). Neither of these changes, therefore, correlates with the alterations in steroid metabolism seen. Recently, Wakelam et al. (1987) have suggested that glucagon can act on the liver via two receptor systems (GR-1 and

%Of Control

1

THE EFFECT OF GLUCAGON (1O-9 LISM OF 4-ANDROSTENE-3,17-DIONE MALE RAT HEPATOCYTES

M) ON METABOBY ISOLATED

Cells were preincubated with hormone for 24 h. Results are expressed as pmol product/min/106 cells and as mean + SD of six values. Enzyme

a

7a-Hydroxylase 6,l?-Hydroxylase 16 cY-Hydroxylase 17-OHSD Sa-Reductase a 17-OHSD * P < 0.05.

= 17-oxosteroid

Control

Treated

40f5 64+7 84k6 91*4 109f5

27+4 46+8 68k7 56*3 66*5

oxidoreductase.

* * * * *

40

I

C

-10

-9

I

1

-8

-7

,

-6

log [Qlucagonl M

Fig. 1. The effect of glucagon (lo-” to 10eh M) on the 6p(0) and 7a-hydroxylation (0) of 4-androstene-3,17-dione by male rat hepatocytes. * P c 0.05. 100% values are 64+7 and 4Ok5 pmol/min/106 cells for the 6p- and 7a-hydroxylases respectively.

206

GR-2), the first of which is linked to the breakdown of PIP, and the second to cAMP production as the transduction mechanism. The dose-response curve for the effect of glucagon on inositol phosphate production was very similar to that seen in these studies whereas that for CAMP production was different (Corvera et al., 1984). This would suggest that the effect of glucagon on hepatic steroid metabolism may be mediated via the GR-1 receptor system (i.e. linked to PIP, breakdown). To further test this possibility the glucagon analogue, TH-glucagon, was used. This analogue has no effect on CAMP production (up to 10m6 M (Bregman et al., 1980; Wakelam et al., 1987)) but stimulates PIP, tu~over in a simpfe dose-dependent manner in hepatocytes (Wakelam et al., 1987). TH-Glucagon caused a marked, dose-dependent reduction in steroid metabolism with the maximum effect being seen at 10e9 M (40% of control for the @I- and 7a-hydroxylases) (Fig. 2). There was no di~nution of effect at higher concentrations in contrast to what was seen with glucagon. This result is very similar to that seen for the action of TH-glucagon on PIP, turnover (Wakelam et al., 1987) and provides further evidence that the effect of glucagon on hepatic steroid metabolism is mediated via the CR-1 receptor system linked to PIP, turnover. The lack of effect of glucagon at higher concentrations is thought to x

be due to the CAMP produced inhibiting the breakdown of PIP,. It is, thus, unlikely that the effects on steroid metabolism seen at lower glucagon concentrations are CAMP mediated. The breakdown of PIP, in the cell leads to two active components, DAG (diacylglycerol) and IP,. IP, is linked to the mobilization of calcium and leads to an increase in [Ca2+li and DAG to activation of protein kinase C (PKc). Which one of these second messengers (DAG and/or IP,) is related to control of steroid metabolism? This question can be approached by the use of the calcium ionophore, A23187, and a potent activator of PKc, PMA. Preincubation of the hepatocytes with PMA for 1 h led to a marked decrease in 6/S and 7ar-hydroxylase activity (55% and 54% of control for ($6 and 7a-, respectively). There was little or no effect of the inactive phorbol ester, 4cy-phorbol-12,13-didecanoate, although it was added in lo-fold excess compared to PMA. The calcium ionophore, A23187, had no effect on any enzyme activity studied (Table 2). The influx of calcium caused by A23187 is, thus, not involved in regulation of steroid metabolism but activation of PKc by PMA caused the same effect as glucagon and THgiucagon. This suggests that glucagon may be causing its effect by generating DAG from PIP, and, thus, activating PKc. Glucagon can have its effects on liver via

Of control

TABLE

2

THE EFFECT OF PHORBOL ESTERS, PMA AND 4a-PDD AND THE CALCIUM IONOPHORE, A23187 ON METABOLISM OF 4-ANDROSTENE-3,17-DIONE BY ISOLATED MALE RAT HEPATOCYT~ Cells were preincubated with compound for 1 h. Results are expressed as pm01 product/min/106 cells and as mean i SD of 12 values. Treatment

c

-10

-8 log [TH-glucagonl M -9

-7

-6

Fig. 2. The effect of TH-glucagon (lo-to to 10K6 M) on the 6p- (0) and “la-hydroxylation (e) of 4-androstene-3,17-dione by male rat hepatocytes. * P < 0.05. 100% values are 86+_ 3 and 69 i 6 pmol/min/106 cells for the b@- and 7a-hydroxylase respectively.

a

Control PMA (5x lo-” M) 4c~-PDD(5x10-‘M) A23187 (lo-’ M)

7o-Hydroxylase

6/3-Hydroxylase

79+ 3 43* 4* 755 1 90f28

62rt2 34+4 * KS+8 67k4

a PMA = 4P-phorbol-12-myristate-13-acetate; phorbol-12,13-didecanoate. * P < 0.05.

4a-PDD

= 4o-

207

increases in CAMP, diacylglycerol or inositol1,4,5_trisphosphate. The effect on steroid metabolism would seem to be mediated by diacylglycerol activating protein kinase C. The mechanism by which PKc alters steroid metabolism is under investigation but may involve phosphorylation of the cytochrome P-450 which is the terminal oxidase of the enzyme complex. Such changes in activity of cytochrome P-450 after phosphorylation are already reported (Pyerin et al., 1984). Acknowledgements This work was supported by the Scottish Hospitals Endowment Research Trust (grant No. HERT 766), the Scottish Home & Health Dept. (grant No. K/MRS/50/C864), the University of Glasgow Medical Research Funds and U.S. Public Health Service Grant AM 21085. A.H.H. is grateful to the Government of Malaysia and the University Sains Malaysia for joint sponsorship. C.J.A. is grateful to the University of Glasgow, Faculty of Medicine for a studentship. We are endebted to Dev Trivedi for the synthesis of TH-glucagon. We gratefully acknowledge Dr. M.J.O. Wakelam and Professor M.D. Houslay for helpful discussions during the course of this work. References Berridge, M.J. and Irvine, R.F. (1984) Nature 312, 3155321. Bregman, M.D., Trivedi, D. and Hruby, V.J. (1980) J. Biol. Chem. 255, 11725-11731.

Combettes, L., Berthon, B., Binet, A. and Claret, M. (1986) B&hem. J. 237,675-683. Corvera, S., Huerta-Bahena, J., Pelton, J.T., Hruby, V.J., Trivedi. D. and Garcia-Sainz, J.A. (1984) B&him. Biophys. Acta 804, 434-441. Desbuquois, B., Lopez, S. and Burlet, H. (1982) J. Biol. Chem. 257, 10852-10860. Dighe, R.R., Rojas, F.J., Birnbaumer, L. and Garber, A.J. (1984) J. Clin. Invest. 73, 1013-1023. Gibson, G.G. and Skett, P. (1986) Introduction to Drug Metabolism, pp. 27-34, Chapman & Hall, London. Gustafsson, J.-A., Mode, A., Norstedt, G., Hokfelt, T., Sonnenschein, C., Eneroth, P. and Skett, P. (1980) B&hem. Action Harm. 7, 48-91. Hussin, A.H. and Skett, P. (1986) Biochem. Sot. Trans. 14, 914-915. Hussin, A.H. and Skett, P. (1987) Biochem. Pharmacol. (in press). Khngensmith, M.R., Freifeld, A.G., Pegg, A.E. and Jefferson, L.S. (1980) Endocrinology 106, 125-132. Pittner, R.A., Fears, R. and Brindley, D.N. (1986) B&hem. J. 240, 253-257. Pyerin, W., Taniguchi, H., Stier, A., Oesch, F. and Wolf, CR. (1984) Biochem. Biophys. Res. Commun. 122, 620-626. Seglen, P.O. (1973) Exp. Cell Res. 82, 391-398. Skett, P. (1978) Biochem. J. 174, 753-760. Skett, P. (1987) Prog. Drug Metab. 10, 85-140. Staddon, J.M. and Hansford, R.G. (1986) Biochem. J. 238, 737-743. Studer, R.K., Snowdowne, K.W. and Borle, A.B. (1984) J. Biol. Chem. 259, 3598-3604. Sutherland, E.W. and Rail, T.W. (1958) J. Biol. Chem. 232, 1077-1091. Wakelam, M.J.O., Murphy, G.J., Hruby, V.J. and Houslay, M.D. (1987) Nature 323, 68-71. Weiner, M., Buterbaugh, G.G. and Blake, CA. (1972) Res. Commun. Chem. Pathol. Pharmacol. 3. 249-254.