Biochimi ca et Biophysica A cta, 1092 (1991) 94-100 © 1991 Elsevier Science Publishers B.V. 0167-4889/91/$03.50 ADONIS 016748899100127Q

94

BBAMCR 12896

al-Adrenergic regulation of ketogenesis in isolated rat hepatocytes Takahide Nomura, Yukihiko Nomura, Masakatsu Tachibana, Hiroko Nomura, Kikuko Ukai, Rie Yokoyama and Yasumichi Hagino Department of Pharmacology, Fujita Health University School of Medicine, Toyoake, Aichi (Japan)

(Received 21 September 1990)

Key words: Noradrenaline; a~-Adrenergic receptor; Ketogenesis; Oleate metabolism; Glucose release; cAMP; (Rat hepatocyte)

Studies were conducted to clarify the effects of noradrenaline on oleate metabolism in isolated hepatocytes from fed rats. Noradrenaline caused an inhibition of ketogenesis from oleate along with a stimulation of glucose release through al.adrenergic receptors. Anti-ketogenic action of noradrenaline was confirmed by the suppression of the formation of radioactive acid-soluble products from [1-t4C]oleate in response to this agent. Noradrenaline increased the conversion of [I-t4C]oleate into t4CO2 but failed to affect [l-t4C]oleate esterification. When hepatocytes were incubated in a medium containing I mM EGTA but no Ca 2+, the effects of noradrenaline on oleate oxidation were negated. On the other hand, noradrenaline-induced increase in glucose release remained unchanged even in the absence of Ca 2+ in the incubation medium. Decrease in ketogenesis and increase in glucose release produced by vasopressin was completely abolished by calcium depletion. Noradrenaline caused a significant increase in cAMP levels in both the presence and absence of Ca z +, although the effect was more marked in the latter. Vasopressin did not affect it. The noradrenalineinduced increase in cAMP and glucose release in the absence of Ca z+ was also mediated by at-adrenergic receptors. These data are discussed and it is suggested that at-adrenergic agonists may control hepatic ketogenesis and glycogenolysis through two separate signal transduction mechanisms, i.e., a calcium-mobilizing system which is common with vasopressin, and a cAMP generation system which vasopressin lacks.

Introduction The action of catecholamines in hepatocytes from normal rats is mediated by al-adrenergic receptors [1]. it is now generally accepted that oq-adrenergic agonists act in the liver through a signal transduction mechanism which is common with vasopressin, namely the hydrolysis of phosphatidylinositol. 4,5-bisphosphate (reviewed in Ref. 2). Thus, it is expected that catecholamines and vasopressin produce similar metabolic effects in isolated rat hepatocytes. Indeed, it is well known that both a~-catecholamines and vasopressin stimulate glycogenolysis through a cAMP-independent mechanism associated with an elevation of the intracellular concentration of free Ca 2+ [2-6]. However at present it is not clear whether these agents exert similar effects on ketogenesis. Vasopressin inhibits ketogenesis from long-chain

Correspondence: T. Nomura, Department of Pharmacology, Fujita Health University School of Medicine, 1-98 Dengakugakubo, Kutsukake-cho, Toyoake, Aichi 470-11, Japan.

fatty acids in isolated hepatocytes from fed rats [7-11]'. On the other hand, the effects of noradrenaline and adrenaline on ketogenesis have been variously reported ranging from stimulation [12-14] to no effect [15] or inhibition [16]. In view of these controversial results in the literature, we attempted to clarify the adrenergic regulation of ketogenesis in isolated rat hepatocytes. Metabolic effects of vasopressin have been observed to be prevented when hepatocytes are incubated in KrebsHenseleit buffer containing 1 mM EGTA but no Ca 2+ [10]. In the present study, we also investigated how calcium-depletion modulates noradrenaline actions in hepatocytes. Materials and Methods Animals Male Wistar rats (300-500 g) were used. All animals were subjected to a 12 h light/12 h dark cycle, with the light period starting at 7:00 a.m., for at least 7 days prior to the experiment. The rats were allowed free access to water and standard laboratory food (Oriental Yeast Co., Tokyo, Japan).

95

Isolation and incubation of hepatocytes and analytical methods Preparation of hepatocytes commenced between 9:00 and 10:00 a.m. using the method of Berry and Friend [17] as modified by Harris [18]. The incubation procedure in the presence or absence of Ca 2+ has been described [10]. Oleate (0.5 raM) was added as a substrate for ketogenesis. Catecholamines and other agents were added at 0 min. The incubations were continued for the times indicated. Metabolite assays were conducted spectrophotometrically on KOH-neutralized H C I O 4 extracts using the enzymatic methods of Slein [19] for glucose, Williamson et al. [20] for acetoacetate and fl-hydroxybutyrate. Ketogenesis was expressed in terms of an accumulation of total ketone bodies (acetoacetate plus fl-hydroxybutyrate). The measuremc.nts of esterification and oxidation of [1-~4C]oleate were as described previously [10]. The results are reported in terms of #mol of [1-m4C]oleate incorporated into products. Cyclic A M P was determined by radioimmunoassay using a Y A M A S A cyclic A M P assay kit (Yamasa, Tokyo, Japan) in KOH-neutralized HCIO4 extracts of cell suspensions. Materials The sources of materials used in this work were as follows: collagenase (CLS 2) from Worthington Biochemical (Freehold, N J), [1-t4C]oleate from New England Nuclear (Boston, MA), ( - ) - a d r e n a l i n e ( + ) - b i tartrate, ( - ) - n o r a d r e n a l i n e hydrochloride, ( - ) - p h e n y l ephrine hydrochloride, ( - ) - i s o p r o t e r e n o l hydrochloride, clonidine hydrochloride, glucagon and dibutyryl cyclic A M P from Sigma (St. Louis, MO), propranolol hydrochloride from Sumitomo Chemical (Osaka, Japan)~ prazosin hydrochloride from Pfizer (Tokyo, Japan) and [Arg-8]vasopressin from Peptide Institute (Minoh, Japan). Statistical analysis The results are expressed as means + S.E. Statistical evaluation of the data was made by means of Student's t-test for paired data. R e s ~

Dose response of noradrenaline on ketogenesis and glucose release We found that increasing concentrations of noradrenaline cause a progressive inhibition of ketogenesis from 0.5 m M oleate (Fig. 1). The maximal effect was seen at 10 -5 M and was equivalent to 32~b inhibition. The data presented in Fig. 1 clearly indicate that the dose-response of noradrenaline on ketogenesis correlates very closely with that on glucose release, the concentration for half-maximal effect on these two parameters being approx. 3 . 1 0 - 7 M.

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Effects of noradrenaline, adrenaline and phenylephrine on ketogenesis and glucose release in the presence or absence of adrenergic blocking agents D a t a for these effects are summarized in Table I. Like noradrenaline (10 -5 M), adrenaline (10 -5 M) and TABLE !

Effects of noradrenaline, adrenaline and phenylephrine in the absence or presence of adrenergic antagonists on ketogenesis and glucose release m isolated rat hepatocytes Incubations were conducted for 30 min in the presence of 0.5 mM oleate. Agents were added at 0 rain. The data shown are the mean + S.E. of eight hepatocyte preparations. Additions

None Prazosin Propranolol Noradrenalin : Noradrenaline + prazosin Noradrenaline + propranolol Adrenaline Adrenaline + prazosin Adrenaline + propranolol Phenylephrine Phenylephrine + prazosin Phenylephrine + propranoloi

Concentration (M)

10- 6 10- 5 10- ~ 10- ~ 10- 6 10- 5 10- 5 10- 5 10- ~ 10- 6 10- 5 10- ~ 10- 5 10- ~ 10- o 10- 5 10- s

Total ketone bodies (pmol/30 min per g wet wt.)

Glucoserelease (/~mol/30 rain per g wet wt.)

8.2 ± 1.0 8.0 ± 1.1 8.0 ± 1.1 6.2 ± 0.8 * 8.2 ± 1.1

31.2±2.2 31.2±2.8 32.9±2.6 49.3±4.0 30.8±2.5

5.7 ± 0.7 *

48.1±3.9"

6.2 + 0.7 * 8.4 ± 1.0

50.8±4.1 33.6±2.9

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6.3 ± 0.8 * 8.3 ± 1.1

41.3±2.9 29.4±26

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42.3 + 3.4 *

* P < 0.05, significantlydifferent from control incubations (i.e., none).

96 TABLE II

Effects of noradrenaline on oleate metabolism in the absence or presence of glucagon Incubations were conducted for 30 min in t.he presence of 0.5 mM [1-n4C]oleate. Agents were added at 0 rain, The data shown are the mean + S.E. of seven hepatocyte preparations for acid-,,,oluble products and CO2 production and of four or five hepatocyte preparations for esterification and total ketone bodies. Additions

Concentration (M)

None Noradrenaline Glucagon Glucagon + noradrenaline

10- 5 10 -6 10 - ~ 10 -s

Total ketone bodies ( p m o l / 3 0 min per g wet wt.)

Acid-soluble products a

CO2 production a

Esterification a

6.6 ± 0.7 4.3 + 0.4 * 14.1 + 1.9 * 10,6 4-1.3 *' * *

1.82 4. 0.14 1.48 + 0.14 * 2.51 +0.09 * 2.10 + 0.09 * *

0.88 + 0.06 1.16 + 0.07 * 0.93 +0.06 * 1.18 + 0.07 *" * *

2.40 + 2.45 + 1.82 + 1.89 +

0.20 0.22 0.28 * 0.24 *

* P < 0,05, significantly different from control incubations (i.e., none). * * P < 0,05, significantly different from the vnlue observed with glucagon. ' ~mol oleate utilized/30 min per g wet wt,

phenylephrine (10-s M) inhibited ketogenesis from 0.5 mM oleate and increased glucose release. The metabolic effect of all three agents were equally blocked by the

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Fi& 2. Comparison of the effects of noradrenaline, vasopressin, glucagon and dibutyryl cyclic A M P on ketogenesis (A) or glucose release (B) in the absence or presence of Ca 2+. Hepatocytes were incubated with 0.5 mM oleate in either a normal calcium-containing medium or a calcium-free buffer containing 1 m M EGTA for 30 rain. The concentrations of agents were: Nad, noradrenaline, 10 -5 M; Vas, vasopressin, 10 -7 M; Glu, glucagon, 10 -~ M; Bta-cAMP , dibutyryi cyclic AMP, 10 -4 M. Agents were added at 0 min. The data shown are the mean-I-S.E, of eight or nine hepatocyte preparations. Values in parenthesis are expressed as a percentage of control. *, P < 0.05, significantly different from control incubations.

97

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Data for these effects are summarized in Table II. In accordance with the inhibition of total ketone bodies, noradrenaline (10 -5 M) significantly suppressed the formation of acid-soluble products from 0.5 m M [114C]oleate. Conversion of [1-14C]oleate into 14C0 2 was stimulated by noradrenaline confirming the findings of Sugden et al. [15] and Oberhaensli et al. [13]. Noradrenaline failed to affect [1-14C]oleate esterification. As expected [21], glucagon (10 -6 M) caused a marked increase in ketogenesis from oleate as well as the formation of acid-soluble products from [1-~4C]oleate, whereas [1-~4C]oieate esterification was inhibited by this hormone. Noradrenaline produced a significant suppression of ketogenesis from oleate along with an acceleration of 14CO2 production from [1-14C]oleate even in the presence of glucagon. The effects of noradrenaline (10 -5 M) on [1-~4C]oleate oxidation were also blocked by prazosin (10 -6 M) (data not shown).

Comparison of the effects of noradrenaline, vasopressin, glucagon and dibutyryl cyclic A M P on ketogenesis or glucose release in ti~e absence or presence of Ca 2 + Fig. 2A illustrates that the anti-ketogenic action of noradrenaline (10-5 M) as well as of vasopressin (10 -v M) observed in the presence of Ca 2+ was abolished when hepatocytes were incubated in Kxebs-Henseleit buffer containing 1 m M E G T A but no Ca 2 +. We found that the effect of noradrenaline on [1-14C]oleate

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Fig, 5, Effects of prazosin and propranolol on noradrenaline-induced increase in cAMP levels in the presence (A) or absence (B) of Ca 2+. Incubations were conducted with 0.5 mM oleate for 1 rain in the presence of Ca 2+ and for 2.5 min in the absence of Ca 2+. The concentrations of agents were: Nad, noradrenaline, 10 -s M, prazosin, 10 -6 M; propranolol, 10 - s M. Agents were added at 0 rain. The data shown are the mean + $.E. of 4 - 6 hepatocyte preparations. *, P < 0.05, significantly different from control incubations.

metabolism (Table II) was also eliminated by calcium depletion (data not shown). The stimulation of ketogenesis by glucagon (10 -6 M) and dibutyryl cyclic AMP (10 -4 M) was produced even in the absence of Ca 2+, although the effects were somewhat attenuated. Fig. 2B clearly indicates that stimulation of glucose release by vasopressin (10 -~ M) was prevented by calcium depletion. By contrast, noradrenaline (10 -5 M) elicited a marked increase in glucose release even in the absence of Ca" +. The stimulation of glucose release by glucagon (10 -6 M) and dibutyryl cyclic AMP 0 0 -4 M) was produced in the absence of Ca 2+ too, although the degree of increase was somewhat enhanced. Noradrenaline-induced increase in glucose release in the absence of Ca =+ was also blocked by prazosin 0 0 -6 M) and unaffected by propranolol (10 -s M) (Fig. 3). Effects of noradrenaline on c A M P accumulation in the presence or absence of Ca z +

The data in Fig. 2 indicate that the susceptibility of noradrenaline action to calcium depletion is obviously distinct from that of vasopressin action. One of the simple interpretations about the data is that noradrenaline and vasopressin may be coupled to distinct signal transduction m~h~msms. Thus, we assessed the possible involvement of cAMP in noradrenaline actions in hepatocytes. Noradrenaline (10 -5 M) was found to cause a significant increase in cAMP levels irrespective

of the presence of Ca 2+, although the effect was more marked in the absence of Ca 2+ (Fig. 4). When hepato= cytes were incubated in the presence of Ca 2+, noradrenaline enhanced cAMP accumulation at 1 min by 27 + 6~ compared with the control value at 1 min (Fig. 4A). On the other hand, in the absence of Ca 2+, noradrenaline-induced increase in cAMP accumulation at 1 min was 94 + 29% (Fig. 4B). Vasopressin (10 -7 M) produced no alteration in cAMP (Fig. 4) confirming the previous findings [5]. Prazosin (10 -6 M) inhibited the noradrenaline-induced increase in cAMP accumulation regardless of the presence of Ca 2+, while propranolol (10 -5 M) did not affect it (Fig. 5). Discussion

Results of the present study indicate that noradrenaline affects oleate metabolism by interacting with a~adrenergic receptors in isolated rat hepatocytes. In the normal Krebs-Henseleit bicarbonate medium, noradrenaline exerted essentially the same effects as vasopressin [7,9-11,22], i.e., inhibition of ketogenesis from oleate, inhibition of the formation of acid-soluble products from [1J4C]oleate and stimulation of 14CO2 production from [1J4C]oleate. Furthermore, we found that noradrenaline suppressed the glucagon-induced increase in ketogenesis in the same manner as vasopressin [7]. The present data fit well the conception that a~-

99 adrenergic agonists and vasopressin exert their physiological effects in the liver through a common signal transduction mechanism, namely the hydrolysis of phosphatidylinositol 4,5-bisphosphate [2,23]. However, it was also found in the present study that the sensitivity of noradrenaline actions to calcium depletion evidently differs from that of vasopressin actions. Effects of calcium depletion on a-adrenergicmediated glycogenolysis have been variously reported; from severe suppression [6,24] to almost no inhibition [25,26]. The present data are in agreement with the latter. It is well established that hepatic glycogenolysis is accelerated by an elevation of cAMP levels [27] a n d / o r an increase in cytosolic Ca 2+ [2]. The data obtained in the present study suggest that the mechanism involved in a~-adrenergic-mediated stimulation of glucose release in the absence of Ca 2+ can be explained at least partially by the increase in cAMP. Chan and Exton [28] have documented that a-receptor stimulation increases hepatocyte cAMP in the absence of Ca 2+ However, this finding could not be reproduced by other workers [26,29], and hence has been considered to be of questionable significance [30]. The present data are consistent with the findings of Chan and Exton [28]. It has been demonstrated that not only a~-agonists but also vasopressin increase cytosolic free Ca 2+ by mobilizing it from internal stores even in the absence of extracellular Ca 2+ [31,32]. It is therefore unlikely that distinct effects of noradrenaline and vasopressin on glucose release ~n the absence of Ca 2+ (Fig. 2B) are attributed to the difference between the effects of these agents on calcium mobilization. We observed that 5 rain preincubation of hepatocytes with TMB-8 (10 #M or 50 #M) a putative antagonist of intracellular Ca 2+ mobilization [33] did not affect stimulation of glucose release by noradrenaline in the absence of Ca 2+ (data not shown). cAMP stimulates ketogenesis (Fig. 2 and see Refs. 34, 35). Why did noradrenaline-induced increase in cAMP fail to accelerate ketogenesis in the absence of Ca 2+ (Fig. 2A)? We observed that 10 -6 M glucagon also produced a larger increase in cAMP levels in the absence of Ca "-+ than in the presence of Ca 2+ (cAMP levels at 2.5 rain in the absence of Ca2+: control; 0.34 _+ 0.01 n m o l / g wet wt., glucagon; 6.34 + 0.92 n m o l / g wet wt., cAMP levels at 2.5 min in the presence of Ca2+: control; 0.30 + 0.03 nmol/g wet wt., glucagon; 3.88 + 0.79 nmol/g wet wt., n = 4 or 5). Nevertheless, the ketogenic action of 10 - 6 M glucagc':: was less marked in the absence of Ca 2+ (Fig. 2A). Moreover, the stimulation by dibutyryl cyclic AMP (10 -4 M) of ketogenesis was also found to be attenuated by calcium depletion (Fig. 2A). Thus, it is reasoned that the modest noradrenaline-induced increase in cAMP (Fig. 4B) was not enough to lead to the detectable stimulation of ketogenesis in the absence of Ca 2+. The anti-ketogenic action of noradrenaline which we

observed in the presence of C a 2+ may be the one blunted somewhat by the slightly enhanced cAMP accumulation (Fig. 4A). Morgan et al. [36,37] have proposed that in the livers of mature rats, the same al-adrenergic receptor population becomes simultaneously coupled to two separate signal transduction mechanisms, namely Ca 2+ mobilization and cAMP generation. It is therefore suggested that the effects of ai-adrenergic agonists on ketogenesis may swing from inhibition to stimulation depending on which of signal transduction mechanisms predominates under the condition, a-Adrenergic-mediated increase in ketogenesis from palmitate has been reported [12-14]. However, an enhancement of cAMP has never been demonstrated. Kosugi et al. [12] documented that adrenaline exerts no effects on cAMP, although it is not clear in their paper when samples were taken during the incubation. We stress that time study needs to be conducted in order to assess the possible involvement of cAMP. Oberhaensli et al. [13] and Stark and Keller [14] conducted 60 min preincubation of hepatocytes with noradrenaline before the add;fion of palmitate. Thus, under their conditions, the stimulation of ketogenesis from palmitate may reflect an indirect effect of noradrenaline, namely secendary metabolic alteration induced by the preincubation with noradrenaline rather than the direct noradrenaline action. We observed that ketogenesis from 0.5 mM palmitate was also inhibited ( P < 0.05) by noradrenaline (10 -5 M) (control; 9.1 _+ 1.1 #mol/30 min per g wet wt., noradrenaline; 7.3 + 1.0 #mol/30 min pec g wet wt., n = 5). a~-Adrenergic receptors have recently been shown to be divided into two subtypes, i.e., a~a and alb [38,39]. Furthermore, it has been proposed tha'~ the subtypes c~¢ al-adrenergic receptors couple with different signal transduction mechanisms in smooth muscle [40]. It remains to be clarified whether noradrenaline-induced Ca 2+ mobilization and cAMP accumulation in hepatocytes are mediated by a single class of al-adrenergic receptor subtypes or by two separate subtypes.

Acknowledgment This study was supported by a Grant-in-Aid from Fujita Health University.

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