Biochimica el Biophysica Acta, 1135(1992)349-352
349
© 1992ElsevierScience PublishersB.V. All rights reserved0167-4889/92/$05.00
cAMP-dependent protein kinase activation mediated
by ¢13-adrenergic receptors parallels lipolysis in rat adilx)cytes D o m i n i q u e L a n g i n a, D a g E l d a o l m a, M a r t i n R i d d e r s t r ~ l e a, M a x L a f o n t a n b and Per Belfrage a Department of Medical and Physiological Chemistry 4, Lurid UniL'ersity, Lurid (Sweden) and b lnstitut National de la Sant~ el de la Recherdze M~dicale (INSERM U-317), Institut Louis Bugnard, CHU RangueiL Toulouse (France)
(Receive,i20 March 1992)
Keywords: Adrenergicreceptor;,cyclic-AMP-dependentprotein kinase;Lipolysis;(Pat adipocyte) Catecholamine-induced lipolysis is chiefly mediated through the recently characterized fl3-adrenergic receptor (AR) in rat adipocyte~. Discrepancies between the ability of fl3-AR agonists to stimulate adenylyl cyclase anP the resulting lipolysis were recently reported, cAMP-dependent protein kinase (A-kinase) activation induced by these agonists was compared to lipolys!-. Agonist potencies were similar for ,A-kinase activity ratios and lipolysis. The same A-kinase activity ratio to iipolysis relationship was found for the fl3-AR agonists tested.
The nature of adipocyte /]-adrenergic receptors CAR) has been of longstanding debate. However, significant progress was made recently in the characterization of the /]-AR subtypes involved in lipolysis. The use of novel series of I$-AR agonists [1,2] strongly suggested that the rat adipocyte /]-AR was different from the classical/3 t- and/32-subtypes [3]. This receptor, referred te as "atypical' o r / ] 3 - A ~ predominantly mediates lipolysis in this species, while the flt-AR plays a minor role [4,5]. These pharmacological data have been considerably strengthened by the isolation of genes encoding the human, mouse, and rat ,83-ARs [6-8] and the detection of the corresponding mRNAs in rat and mouse adipose tissues [7,8]. It is generally agreed that increased cAMP accounts for the lipolytic stimulation by catecholamines. An elevation of intracellular cAMP concentration leads to the activation of cAMP-dependent proteia kinase (ACorrespondence: D. Langin, Department of Medical and Physiological Chemistry4, Land University,P.O. Box 94, S-2210DLand, Sweden. Abbreviations: A-kinase, cAMP-dependent protein kinase; AR, adrenergiereceptor;,BRI.37344or BRI., 4-[2-[(2-hydrory-243-chlorophenyl)ethyl)amino~)mpylh~lenoxyacetic acid; EC.s0,concentration of the compound which w~l elicit a half max,in'sad response; i.a., intrinsicactivity;,ISO, iso~roteranol;OPC39I!, N-cycinhesyI-N-2-hydrox'yethyl-4(6-(1,2-dihydro-2-oxoquinolyloxy))butyramide; PC'V, packed cell volume; PDE, phnsphodiesterase;rofipram, 4-(3-cydopentyloxy-4-melhoxyphenylb2-pyrmlidone;RO 20-1724, 4-(3.butyoxy-4-methoxybenzyl)-2-imidazolidone.
Kinase) which mediates the activation of hormone-sensitive lipase through phosphorylation [9,10]. However, it has recently been shown that drugs acting through the /]3-AR could exhibit striking differences between their ability to stimulate adenylyi cyclase on fat-cell ghost preparation and the resulting lipolysis, indeed, BRL37344, a full and more potent agunist on lipolysis than the standard agonist isoproterenol (ISO) [4,5], behaved as a partial agonist and was less potent than ISO when measuring the activation of adenylyl cyclase [11,12]. These results could argue in favour of cAMP compartmentali=ation. BRL37344 would more efficiently direct cAMP into a "functional lipolytic compartment' than ISO. If valid, this hypothesis is important from both a fundamental (to understand quantitative relationship between cAMP and lipolysis) and a practical (e.g., for the design of new/33-AR agunists) point of view. To clarify this issue, we decided to determine the fraction of A-kinase stimulated by endogenous cAMP, the so-called A-kinase activity ratio. The activity ratio provides an indirect measurement of the cAMP that may be relevant to physiological processes [13]. Akinase activation induced by BRL37344 and ISO were therefore compared to lipolysis. Male Sprague-Dawley rats were supplied by Alab, Stockholm, Sweden. The animals were kept in a 12-h light-dark cycle with free access to EWOS-ALAB R3 standard lab chow and tap water. They were fasted overnight and killed by cervical dislocation between
350 8.00 and 9.00 am. At the day of the experiment, the rats were 35-37 days old (140-150 g). The use of animals was authorized by Lantbruksstyrelsen, l~nkfping, Sweden. Adipocytes were prepared according to Rodbell's method [14]. The packed cell volume (PCV) was determined and the cells supplemented with 200 nM adenosine were diluted as a 1% suspension. Immediately before the experiments, 100 nM phenylisopropyladenesine and 0.5 U / m l adenosine deaminase were added to the cell media to obtain a constant inh~itory control of the adenylyl cyclase via the inh~itory A~ adenosine receptors [13,15]. l-ml aliquots of cell suspert~ion were incubated for 20 min in polyprol~lene vials with the various concentrations of drugs. The incubation was terminated by the addition of 0.2 vol. of extraction mediun~ containing 50 mM "Iris (pH 7.4), 50 mM E D r A , 0.5 mM OPC3911, and 0.5 mM rolipranL This cocktail of phosphodiesterase (PDE) inh~itors was preferred to the original medium containing Ro 20-1724 [13,15] because it efficiently blocks both types of adipocyte Iow-Km PDE (i.e. cGMP-inh~ited PDE and cAMP*specific PDE) [16,17]. The mixture was mixed and homoge,fized with 10 strokes in a 1-ml Kontes homogenizer. The homogenate was transferred to a precooled 1.5-ml Eppendorf tube and cent.-~fuged at 10000 × g for 15 rain at 4cC. 200 /zl of the infranatant was transferred to another precooled Eppendorf tube and assayed immediately for A-ldnase activity. 500 ;tl of the infranatant was frozen in a dry ice/methanol bath and stored at -80~C for the subsequent determination of glycerol content. Duplicates were performed for each concentration of BRL37344 (a generous giR from Beecham Pharmaceuticals, Epsom, UK) or 1SO, and were assayed in parallel for A-kinase activity and lipolysis. A-kinase activity was determined according to Egan et al. [18] and glycerol content according to Gar!and and Randle [19]. The data were fitted to a sigmoidal equa-
>x~....
o
"° 3o
~,
.~:/
ISO) is strictly similar in both assays.
352 References 1 Arch, J.ILS~ Ains~mth. A.T. Cawthorne. M.A.. Pie:~y, V . Sennitt. M.V. Tlu~y, V.E, W'dson, C. and Wilson S. (1984) Nature 309.163-165. 2 Manara. L. and Bianchetti, A. (1990) Trends Pharmacol. Sci. I!. 229. 3 Zaagsma, J. and N ~ i , S.R. (1990) Trends PharmacoL ScLI !, 3-7. 4 HoHenga. C and Zaagsma. J. (1989) Br. J. Pharmacol. 98.14201424. 5 Langin, D~ Fondu)to..M..~ . Sa,'~.:~,.'~r-Bl~, J~. ~-~ l.~f~t,~% M. (1991) Eur. $. Pharmm~. 199, 291-301. 6 Emorine. I ! Maru]~. S~ Briem~Sutren, M.M. Pate*j, G_ "Fate. K., Delavier-Klutchko, C. and Strosberg, A.D. (1989) Science 245. 1118-1121. 7 Nahmias, C~ Blin, N., Elalouf, J.M. Maltei, M.G_ Strosherg. A.D. ~ Fa~0rine. I I . (1991) F~MBO J. 10, 3721-3727. 8 Muzzin, P . Revelfi, J.P~ Kuhne, F~ Goca~ne, J.D. McCombie, W.R.. Venter, J.C~ Gk3cobino. J.P. and Fraser. C. M. (1991) J. Biol. Chem. 266, 24053-24058. 9 Fain, J.N. and Garcia-Sain~ J.A. (1983) J. Lipid Res. 24, 945-966. 10 Belfrage, P . Fredn~son, G . Str~Ifors, P. and Tornqvist, H. (1984) in Lipases (Borgsm3m, 13. and Brockman, H , eds.), pp. 366--416, Elsevier, Amsterdam. 11 Hotlenga, C~ Bmuwer. F. ~ Zaagsrna, J. (1991) Br. J. Pharmacol. 102, 577-5-50.
12 Holle.~ga. C . Brouwer, F a~d Zaagsm& J. (1991) Eur. J. Pharm a ~ L ~ 325-330. 13 Hotm¢~. R.C., DhiL~n, G.S. and l~ndos. C. (1985) J Biol. Chem. 2~0o 15122-15129. 14 Ro~e/l, M. (1964) J. Biol. Chem. ~ 9 . 375-380. 15 Hoam~. R.C~ Db~on, G.S. and ~ C. ( ! 985) J. Biol. Chem. 2~~, 15130-15138. 16 Degennan. E., Iklfrage. P . Newman, A.H_ Rice, ICC. and M~ga~e~o, V.C. (1987) J. Biol. Chem. 262, 5797-5807. 17 Y ~ . T . Liebennan. F . Osborne, J.C. Manganie~lo, *v'.C. Vanghan. M. aud Hikada. EL (1984) Biochemistry 23, 670-675. 18 Egan. J J ~ C]laag. M.K. and ~ C. (1988) Anal. Biochem. 175552-561. 19 Garland. P.B. and Randle, P.J. (1962) Nature 196. 987-988. 29 Corbin. J.D~ Keel)', S.E., Sodeding, T.R. and Park. C.R. (1979) Adv. CycL Nud. Res. 5, 265-279. 21 Allen, D O . (1985) Biochem. Pharmacol. 34, 843-846. 22 Allen. D O . and Ouesenheny. J.T- ',1988) J. Pharmacol. Ex~. Ther. 244. 852-858. 23 F~,'e, B~ Emorin~ LJ _ Lasnier. F, Blin, N . Baude, 13. Nahmia~ C . Suosberg, A.D. and Pairault, J. (19911 J. Biol. Chem. 266, 203_~-20336. 24 Degerman, E., Smilh, C.T. T o r m ~ t . H_ Vasta. V . Belfrage, P. and ManganieUo. V.C. (1990) Proc. Natl. Acad. Sci. USA 87, 533-5~7.
349
© 1992ElsevierScience PublishersB.V. All rights reserved0167-4889/92/$05.00
cAMP-dependent protein kinase activation mediated
by ¢13-adrenergic receptors parallels lipolysis in rat adilx)cytes D o m i n i q u e L a n g i n a, D a g E l d a o l m a, M a r t i n R i d d e r s t r ~ l e a, M a x L a f o n t a n b and Per Belfrage a Department of Medical and Physiological Chemistry 4, Lurid UniL'ersity, Lurid (Sweden) and b lnstitut National de la Sant~ el de la Recherdze M~dicale (INSERM U-317), Institut Louis Bugnard, CHU RangueiL Toulouse (France)
(Receive,i20 March 1992)
Keywords: Adrenergicreceptor;,cyclic-AMP-dependentprotein kinase;Lipolysis;(Pat adipocyte) Catecholamine-induced lipolysis is chiefly mediated through the recently characterized fl3-adrenergic receptor (AR) in rat adipocyte~. Discrepancies between the ability of fl3-AR agonists to stimulate adenylyl cyclase anP the resulting lipolysis were recently reported, cAMP-dependent protein kinase (A-kinase) activation induced by these agonists was compared to lipolys!-. Agonist potencies were similar for ,A-kinase activity ratios and lipolysis. The same A-kinase activity ratio to iipolysis relationship was found for the fl3-AR agonists tested.
The nature of adipocyte /]-adrenergic receptors CAR) has been of longstanding debate. However, significant progress was made recently in the characterization of the /]-AR subtypes involved in lipolysis. The use of novel series of I$-AR agonists [1,2] strongly suggested that the rat adipocyte /]-AR was different from the classical/3 t- and/32-subtypes [3]. This receptor, referred te as "atypical' o r / ] 3 - A ~ predominantly mediates lipolysis in this species, while the flt-AR plays a minor role [4,5]. These pharmacological data have been considerably strengthened by the isolation of genes encoding the human, mouse, and rat ,83-ARs [6-8] and the detection of the corresponding mRNAs in rat and mouse adipose tissues [7,8]. It is generally agreed that increased cAMP accounts for the lipolytic stimulation by catecholamines. An elevation of intracellular cAMP concentration leads to the activation of cAMP-dependent proteia kinase (ACorrespondence: D. Langin, Department of Medical and Physiological Chemistry4, Land University,P.O. Box 94, S-2210DLand, Sweden. Abbreviations: A-kinase, cAMP-dependent protein kinase; AR, adrenergiereceptor;,BRI.37344or BRI., 4-[2-[(2-hydrory-243-chlorophenyl)ethyl)amino~)mpylh~lenoxyacetic acid; EC.s0,concentration of the compound which w~l elicit a half max,in'sad response; i.a., intrinsicactivity;,ISO, iso~roteranol;OPC39I!, N-cycinhesyI-N-2-hydrox'yethyl-4(6-(1,2-dihydro-2-oxoquinolyloxy))butyramide; PC'V, packed cell volume; PDE, phnsphodiesterase;rofipram, 4-(3-cydopentyloxy-4-melhoxyphenylb2-pyrmlidone;RO 20-1724, 4-(3.butyoxy-4-methoxybenzyl)-2-imidazolidone.
Kinase) which mediates the activation of hormone-sensitive lipase through phosphorylation [9,10]. However, it has recently been shown that drugs acting through the /]3-AR could exhibit striking differences between their ability to stimulate adenylyi cyclase on fat-cell ghost preparation and the resulting lipolysis, indeed, BRL37344, a full and more potent agunist on lipolysis than the standard agonist isoproterenol (ISO) [4,5], behaved as a partial agonist and was less potent than ISO when measuring the activation of adenylyl cyclase [11,12]. These results could argue in favour of cAMP compartmentali=ation. BRL37344 would more efficiently direct cAMP into a "functional lipolytic compartment' than ISO. If valid, this hypothesis is important from both a fundamental (to understand quantitative relationship between cAMP and lipolysis) and a practical (e.g., for the design of new/33-AR agunists) point of view. To clarify this issue, we decided to determine the fraction of A-kinase stimulated by endogenous cAMP, the so-called A-kinase activity ratio. The activity ratio provides an indirect measurement of the cAMP that may be relevant to physiological processes [13]. Akinase activation induced by BRL37344 and ISO were therefore compared to lipolysis. Male Sprague-Dawley rats were supplied by Alab, Stockholm, Sweden. The animals were kept in a 12-h light-dark cycle with free access to EWOS-ALAB R3 standard lab chow and tap water. They were fasted overnight and killed by cervical dislocation between
350 8.00 and 9.00 am. At the day of the experiment, the rats were 35-37 days old (140-150 g). The use of animals was authorized by Lantbruksstyrelsen, l~nkfping, Sweden. Adipocytes were prepared according to Rodbell's method [14]. The packed cell volume (PCV) was determined and the cells supplemented with 200 nM adenosine were diluted as a 1% suspension. Immediately before the experiments, 100 nM phenylisopropyladenesine and 0.5 U / m l adenosine deaminase were added to the cell media to obtain a constant inh~itory control of the adenylyl cyclase via the inh~itory A~ adenosine receptors [13,15]. l-ml aliquots of cell suspert~ion were incubated for 20 min in polyprol~lene vials with the various concentrations of drugs. The incubation was terminated by the addition of 0.2 vol. of extraction mediun~ containing 50 mM "Iris (pH 7.4), 50 mM E D r A , 0.5 mM OPC3911, and 0.5 mM rolipranL This cocktail of phosphodiesterase (PDE) inh~itors was preferred to the original medium containing Ro 20-1724 [13,15] because it efficiently blocks both types of adipocyte Iow-Km PDE (i.e. cGMP-inh~ited PDE and cAMP*specific PDE) [16,17]. The mixture was mixed and homoge,fized with 10 strokes in a 1-ml Kontes homogenizer. The homogenate was transferred to a precooled 1.5-ml Eppendorf tube and cent.-~fuged at 10000 × g for 15 rain at 4cC. 200 /zl of the infranatant was transferred to another precooled Eppendorf tube and assayed immediately for A-ldnase activity. 500 ;tl of the infranatant was frozen in a dry ice/methanol bath and stored at -80~C for the subsequent determination of glycerol content. Duplicates were performed for each concentration of BRL37344 (a generous giR from Beecham Pharmaceuticals, Epsom, UK) or 1SO, and were assayed in parallel for A-kinase activity and lipolysis. A-kinase activity was determined according to Egan et al. [18] and glycerol content according to Gar!and and Randle [19]. The data were fitted to a sigmoidal equa-
>x~....
o
"° 3o
~,
.~:/
ISO) is strictly similar in both assays.
352 References 1 Arch, J.ILS~ Ains~mth. A.T. Cawthorne. M.A.. Pie:~y, V . Sennitt. M.V. Tlu~y, V.E, W'dson, C. and Wilson S. (1984) Nature 309.163-165. 2 Manara. L. and Bianchetti, A. (1990) Trends Pharmacol. Sci. I!. 229. 3 Zaagsma, J. and N ~ i , S.R. (1990) Trends PharmacoL ScLI !, 3-7. 4 HoHenga. C and Zaagsma. J. (1989) Br. J. Pharmacol. 98.14201424. 5 Langin, D~ Fondu)to..M..~ . Sa,'~.:~,.'~r-Bl~, J~. ~-~ l.~f~t,~% M. (1991) Eur. $. Pharmm~. 199, 291-301. 6 Emorine. I ! Maru]~. S~ Briem~Sutren, M.M. Pate*j, G_ "Fate. K., Delavier-Klutchko, C. and Strosberg, A.D. (1989) Science 245. 1118-1121. 7 Nahmias, C~ Blin, N., Elalouf, J.M. Maltei, M.G_ Strosherg. A.D. ~ Fa~0rine. I I . (1991) F~MBO J. 10, 3721-3727. 8 Muzzin, P . Revelfi, J.P~ Kuhne, F~ Goca~ne, J.D. McCombie, W.R.. Venter, J.C~ Gk3cobino. J.P. and Fraser. C. M. (1991) J. Biol. Chem. 266, 24053-24058. 9 Fain, J.N. and Garcia-Sain~ J.A. (1983) J. Lipid Res. 24, 945-966. 10 Belfrage, P . Fredn~son, G . Str~Ifors, P. and Tornqvist, H. (1984) in Lipases (Borgsm3m, 13. and Brockman, H , eds.), pp. 366--416, Elsevier, Amsterdam. 11 Hotlenga, C~ Bmuwer. F. ~ Zaagsrna, J. (1991) Br. J. Pharmacol. 102, 577-5-50.
12 Holle.~ga. C . Brouwer, F a~d Zaagsm& J. (1991) Eur. J. Pharm a ~ L ~ 325-330. 13 Hotm¢~. R.C., DhiL~n, G.S. and l~ndos. C. (1985) J Biol. Chem. 2~0o 15122-15129. 14 Ro~e/l, M. (1964) J. Biol. Chem. ~ 9 . 375-380. 15 Hoam~. R.C~ Db~on, G.S. and ~ C. ( ! 985) J. Biol. Chem. 2~~, 15130-15138. 16 Degennan. E., Iklfrage. P . Newman, A.H_ Rice, ICC. and M~ga~e~o, V.C. (1987) J. Biol. Chem. 262, 5797-5807. 17 Y ~ . T . Liebennan. F . Osborne, J.C. Manganie~lo, *v'.C. Vanghan. M. aud Hikada. EL (1984) Biochemistry 23, 670-675. 18 Egan. J J ~ C]laag. M.K. and ~ C. (1988) Anal. Biochem. 175552-561. 19 Garland. P.B. and Randle, P.J. (1962) Nature 196. 987-988. 29 Corbin. J.D~ Keel)', S.E., Sodeding, T.R. and Park. C.R. (1979) Adv. CycL Nud. Res. 5, 265-279. 21 Allen, D O . (1985) Biochem. Pharmacol. 34, 843-846. 22 Allen. D O . and Ouesenheny. J.T- ',1988) J. Pharmacol. Ex~. Ther. 244. 852-858. 23 F~,'e, B~ Emorin~ LJ _ Lasnier. F, Blin, N . Baude, 13. Nahmia~ C . Suosberg, A.D. and Pairault, J. (19911 J. Biol. Chem. 266, 203_~-20336. 24 Degerman, E., Smilh, C.T. T o r m ~ t . H_ Vasta. V . Belfrage, P. and ManganieUo. V.C. (1990) Proc. Natl. Acad. Sci. USA 87, 533-5~7.
