Alpha-adrenergic receptors and the regulation of lipolysis in adipose tissue

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Alpha-adrenergic receptors and the regulation of lipolysis in adiipose tissue Max Lafontan and Michel Berlan Institut de Phvsiologie, Unit,ersit~Paul Sabatier, ERA 412 CN R S - - 2 rue F. Magendie, 31400 Toulouse, France and i.aboratoire de Pharmacologie M~dicale, Universit~ Paul Sabatier, ERA 412 CNRS. 36 all~es Jules Guesde, 314(]0 Toulouse, Fr~'~ce.

The mobilization ,of fat from adipose tissue has been ~tu, lied cxtensively and reviewed several times. The mobilization of free fatty, aciffi (FFA) is due to lipolysis; the hydrolysis ef triglycerides stored in the adil~ocytes i:i followed by an outflow of FFA and glycerol from the adipose tissue. These tw,~ metabolites can be used as a meil~sure of lipolysis in in vivo or in vitro investigatior~s. The release of FFA from adipose t:~ssue is strikingly susceptible to hormone, nfluence. The best known lipolytie 17ormoaes are catecholamines, glucagon and vario~Js pituitary peptidcs (adrenocorticotrophin, beta-lipotropin, growth hormo,,:). "Faere arc species variations in the resl:~msiv,-'ness of adipose tissue to adilt~)kine tic agents. In human adipose tissue. for t:xample, catecholamines are of major importance in the stimulation of lip~lysis while peptides are practically inactive. ITtis generally agreed that the stimulation of lipolysis by catecholamines is mediated by ~ta-edrenoceptors linked to an adenylate eycla,< system in the cell membrane. The: existence of alpha-adrenoceptors involved in the control of adipose tissue lil~:llyS~shas been demonstrated in human adi~pose tissue'-=. Alpha-adrenoceptors have been described in isolated fat cells of ha~nstep?, rabbits' and dogs ~ while in rat adi~r,ocytes, there is no evidence of the presence o'.' alpha-adrenoceptors controlling lipolytic processes.

Pkm'ma,~logical characterization of Mpka-a¢lrenoceptors inhibiting lipolysis in =~iipose ;lissue. Methodological ~mprov(:ments The preliminary data concerning alphaadrenoceptors in human or hamster fat cells were obtained by investigating the lipolytic responses of adipose tissue slices or isolated fat cells to various adrenoceptor agonists or antagonists. Epinephrine or FJ,~c~l~r;Nx~rlbHollandBlon~dical I~rcra1981

norepinephrine-induced lipolysis did not reach the level of isoproterenol-stimulated lipolysis; the addition of an alphaadrenergic blocking agent (phentolamine) to the flasks containing norepinephrine enhanced lipolysis to the level reached with isoproterenu! alone. Moreover. the combination of norepMephrine with propranolol (beta-adrenergi,: blocking drug) induced a decrease of lipolysis below basal levels of activity. The results of these studies provided evidence for the existence of both alpha- and beta-adrenergic receptor sites in fat cells and suggested that alpha-adrenergic receptor stimulation is able to counteract the lipolytic effect of physiological catecholamines. In the same t)pe of experiment the changes occurring in intracellular levels of cyclic AMP were seen to be parallel to those observed in glycerol or FFA release. This son of experiment has been used by various I~avestigators to study the alpha-adren~ergic responsiveness of adipose tissue (Fig. 1). However, one of the major problems has been that the commonly used alpha-antagonist, phentolamine, when used at higher concentrations inhibited the lipolytic activity of the adipocytes by a non-specific process distal to the hormone receptor site. Since the alpha-adrenergic effect is an antilipolytic effect, the need for an accurate experimental system involving antilipolysis induced by alpha-agonists became evident. Usually, the basal rate of lipolysis of isolated fat cells is low and the antilipolytic effects induced by epinephrine or norepinephrine, combined with propranolol, are v,:ry weak. However, when ba mt lipolysis is increased, the readiness to red,pond to alpha-adrenergic effects (i.e. a~'Ailipolysis promoted by alpha-agonists) i,¢reases. This effect was observed by (~tman's group s, in the adipose tissue from otb,e:¢ patients after starvation and by us in tlll~.'adipocytes of obese patients submitted

to caloric restriction and also in obeserabbit adipocyte~L The alpha-adrenergic stimulation partially inhibited both ACTH or improterenol-induced lipolysis and the rise in cyclic AMP levels promoted by these lipolytic agents in hamster fat-cells'. In human adipocytes we have shown that the antilipolytic effects of alpha-agonists on thcophyiline-stimulated lipolysis is related to the involvement of the adrenergic alpha-receptor sites (Fig. 1) and we found that the results were better than t h o ~ obtained when isoproterenol was used for the stimulation of lipolysis. Recently we characterized the function of the alpha-receptors of human ° and hamster =° fat cells, by testing the inhibiting effect of drugs with known alphaadrenergic agonist activity, on theophylline-induced lipolysis (Fig. 2). We

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Fig. 1. Two examples o f investigation o f alpha. adrenergic responsiveress in human fat cells. (A) Comparison o f the lipolytic effects of epinephrine (Q), isoproterenol (1"3) and ineremem o f epinephrineinduced lipolysis by an alpha-antagonist :phentolamine ((3). (B ) Inhibition of theophylline-induced lipolysis by epinephrine (0) associated wit/J propranolo/ (.5 t~M). Suppression o f this effect by phentolamine (1:3). Results are mcam +- S.E.M.; standard errors are indicated by the bars; the number by each curve is the number ofexperimen¢~. '~*, p < O.02; * * *, p < 0.01: results significantly different from basal values according Student's paired t-test.

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70 Fig. 2. Effect o f variom a/pha-agonists on thtrophyUine-induced lipolyxis in human isolated fat ce,!ls. The results are expressed as the percentage inhibiti(;n o f rheophylline.induced lipolysis in the presence of corresponding concentratior~ ofthe alpha-agonist. The st,ndar d errors are indicated by the bars. Me°m" o f dose re wonae curves are given +-S. E. M, (0) clonidine, ( &] tramazoline. (0) adrenaline + propranolol 15 lau), (~) phenylephrine, (A) methoxan=ine. The number o f experiments is indicated by each curve.

have shown that the alpha-adrenergic receptor which inhibits lipolysis is of the alpha= type. The relative order of potency of the agonists is: clonidine > epinephrine > phenylephrine > methoxamine, while the relative order of antagonists in the suppression of clonidine inhibition of theophylline-induced lipolysis is: phentolamine > yohimbine > piperoxan > phenoxybenzamine > prazosin. Recent data on hamster fat celPs are in agreement with ours and demonstrate that clonidine strongly reduces the levels of cyclic A M P due to isoproterenol or ACTH and that this effect is mediated by an alpha=adrenoceptor *tt=. It is noticeable that the alpha-adrenoceptors involved in glycogen metabolism and in the regulation of phosphatidylinositol turnover are of the alphaa-type. This aspect has been recently reviewed by Fain and Garcia-Sainz 'a. Although the alpha-adrenoceptors have been identified in various tissues by direct radioligand binding studies, there are few data concerning adipose tissue. In order to obtain a better characterization of the alpha-adrenoceptors of adipocytes, two groups of authors "a* have studied the binding of [nH]dihydroergocryptine ([SH]DHEC), a potent alpha-adrenoceptor antagonist, to hamster fat cell membranes and demonstrated the usefulness of this ligand, even though it is not selective for alpha, or alpha= receptors.

The recent production of radiolabelled alpha-adrenergic agents with a subtYlC~e .selectivity and the biological characterization of the alpha=-adrenoceptor of the adipose tissue led us to try to identify this receptor using the labelled agonist ["H]clonidine. We demonstrated that ["H]clonidine appears to have a major advantage for selective labelling of the alpha~-adrenoceptors of human fat cell membranes ~=. Although this radiolabelled agonist might label only a fraction of the whole alpha~-adrenoceptor population, it is noticeable that there is an excellent correlation between alpha-udrenergic agonist or antagonist affinities for the membrane [SH]clonidine binding site and their physiological potencies (i.e. antilipolytic effects of alpha-agonists or suppression of clonidine inhibition of lipolysis by alphaantagonists)~, m.,~. Such a re suit pe emits the assertion that the [aH]clonidine binding sites described are postsynaptic sites of the fat cell membrane which mediate lipolysis inhibition. Since the fat cell membrane appears to contain roughly 90% of alpha=adrenergic receptors in the hamster =. the adipose tissue should be a useful model for the investigation of the binding affinit.~ states for agonists and antagonists and their correlation with physiological events concerning postsynaptic alpha=-receptors. The recent availability of [~H]yohimbine ~ill provide another convenient tool for further investigations on the adipocyte alpha=receptor site and will be compared ~vith [SH]clonidine. However, at present, more research is needed to complete the definition of the properties of the alphaa-sites of the adipose tissue and to permit the complete understanding of alpha=adrenoceptor binding data. ~J~TAGONISTS

The biochemical e~ents linked to the stinlulatian of the alpha~-adrenergic receptoe, ha,,e not ~,el been co,'npleteb, explained. Ho'ae,,er. the reduction t~l cyclic AMP levels in Ihe fat cells after alpha-adrenergie stimulation ~ hatcher the initiating agent (i~+pmtercnt)l. ACI+H. theophylline) supports the assertion that alpha~-elfeets are due to a direct inhibit~un ol adenylate c.~cla~ activit:, ".~. Although the alph~+-adrenergic inhibit,,n of hamster fat cell adenylale cyclase ha~ been rccentlx reported '~. the relative order of potcnc3 of agonists (adr~:naline > alphamethylnoradrenalinc = noradrenaline clonidinc > phenylephrine) differ,, lroln that obtained on intact adip~cytes ~hcn lilx~lysis and c.~clic AMP le~el~ are considered (chmidinc > adrenaline :- noradrenalinc -> phenylcphrine - methoxamine). More data arc needed to explain these discrepancies. The Ix~ssiblc interactions of adrenergic agonists and antagonists ~ith fat cell membrane are shown in Fig. 3.

Can alpha= receptors pla) a role in the physiological regulation of lipolysis? At present, the amount of information provided by ligand binding studies on the regulation of beta and alpha-adrcrcrgtc eceptor binding sites, and the absence of , t rive studies, do not permit an adequate understanding of the phenomena. Ho~,.ever. ~here is good e~:,dence that alphaadreneLcic resptmsi~enc,s i, in~ol~ed in the h,+rmonal control ,+f adipose tis~,ue lipolysis. Se,~cral results obtained in human adifx)se tissue ,,upnort thin aN~cttttul l he inxohement of increased alpha-adrenergic responsiveness has t~'en proposed it+ explain the delccl in noradrenahnc-

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I i'~g. 3. Schematic rlwresentation o[the po.~ihie interacn(u~ o / adrener~w a(on~t~ and a~mt~,,(mL~L~~Lidl a/ptn~- and beta.receptor sites in die ]ht ('ell memhran,, an d their impact on tile tethdar mehlh, dl.~tPiof the mhp,,,'~n' The abbreviations used are a~ fidh)Lt x ('l.O. chmidine; EPI, epi.wphrlne: ~,'EPI. n(Jrt,pillep/irule: P H L phenylephrine: ~IEF. metho~nnune.

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stimulated lipolysis on the adipose tissue of the data deal with biochemical investiga- the hamster fat cell. the age-induced variahypothyroid man ~'. It is noticeable that this tions of ~he mechanisms of transduction of tions of the fat cell alpha-adrenergic effect is reversible after substitution the alpha-:tdrenergic signal in the fat cell. responsiveness were closely parallel to the therapy. Differences exist between the The physiological significance of the adi- age-induced variations of the number of lipolytic responsivene~,~s to adrenaline or pose alpha-receptors and their possible [aH]dihydroergocryptine binding sites. In noradreualine of human tissue from differ- hi, moral regulation has not been studied in the young or the aged and obese rat we ent regions '*.~a(Table ]). These differences sufficient depth at present but will prob- have not obtained evidence of a noticeable could be linked to a variable alpha- ably be a fruitful area for future investiga- alpha2-adrenoceptor activity in fat ceils, adrenergic inhibiting effect rather than to a tions. We have reported that the alpha- with our experimental design. Garcia Sainz modified beta-adrenergic effect, it is also adrenergic responsiveness increases with et al. '~ recently reported a similar observademonstrated that regional differences age and/or ,at cell size in dog a, rabbit ~ and tion in rat adipocytes. exist in the lipolytic response to fasting. A hamster adipocytes (Table !). It is noticestrong increase of the basal lipol~ tic activity able that the adipose tissue of these species Future investigations and clinical studies of the adipocytes occurs after starvation e or is characterized by clear cut differences At present, the existence of adipose tisa restricted diet and concomitantly concerning epinephrine responsiveness. sue alphaa-adrenoceptors, which inhibit enhanced alpha-: drenergic receptor activ- Dog fat cell lipolysis is strongly stimulated lipolysis, is clearly demonstrated in various ity is observed. This effect is more promi- by catecholamines while that of adult rab- species. However, their significance and nent in the adipose tissu." of the femoral bit fat cells is not. The unresponsiveness of their physiological role need more research region than in tha~ of hypogastric tissue '~. adult rabbit adipose tissue is related to an for a clear and complete understanding of These results suggest that the mobilization increased alpha-adrenergic reslxm- their usefulness and their regulation. The ol lipids induced by caloric restriction siveness4" the- weakened response of obese application of binding techniques could could be reduced by an increased alpha- dog fat cells to epinephrine is linked to the allow chmfication of the physiological and/ adrenergic activity since calecholamines same effect a. Moreover. in the aged obese or pathological importance of alphaagenerally inhibit lipolysis under such condi- rabbit, after dietary restriction, large fat adrenoceptors in human fat cells. tions.. The fact that lipid:~ are less rapidly cells (with an increased alpha-adrenergic Moreover, the availability of several animobilized from femoral than abdominal or responsiveness) were reduced in size and a mal models would facilitate investigations epiploic tissues may explain why the adi- significant restoration of the lipolytic effect into the nutritional, hormonal or neural pose tissue of the femoral region is less of epinephrine was observed associated to regulation of the alpha and beta receptors affected than that of the other regions dur- a weakened alpha-adrenergic responsive- suggested by the in vitro studies: ing fasting or restrictive diets. However. ness. In conclusion these results indicate The observations reported in human the increased alpha-receptor activity can- that cell size rather than age may be an adipose tissue suggest that the increment of not be considered as the only cause; the important factor affecting epinephrine- alpha-adrenergic responsiveness in concomitant modification of the number of responsiveness in rabbit adipocytes. The femoral adipose tissue or after fasting beta-sites and of the enz3'mes involved in loss or the recovery, of the lipolytic effect of might be of clinical importance, if it is also the lipolytic pathways of the fat cell should epinephrine could be explained by a modi- found in vivo. Clinical trials or in vivo be studied to clari~, this proble~,a. fication of the alpha-receptor activity; the investigations in the animal models should Aipha-adrenoceptors ha~e been beta-receptor activity being less affected. provide confirmation of the status of the described in several speciesS*-~.~:~*:most of In a recent paper 2°, it was reported that in alphas-receptors of the adipose tissue. If their involvement in obesity or some T A B L E I Inter~pecie~ differences in the alpha-adrenergic responsiveness of the white fat cells. Modifications epinephrine-resistant states is confirmed, it o | the ~lpha-adlen~. rgJc r¢.,l~m,,ive ne,~ -~ith age and/or obesity in animals; according to anatomical Iocalizations cannot be excluded that alpha2-antagonists m human,~. could contribute to solving problems linked to lipolytic-resistant states, and Adip~v,e tt,,suc therapeutic use of these agents could be from ~armu~ species Yotmg Normal adult Older obese reamnably considered. Human Finally, it should be emphasized, that the cl'nph~tc . + isolated fat cell provides a far simpler celluatvd~)minal ++ _ lar system than cerebral presynaptic alpha2-adrenoceptors or other postsynapRat tic alphaa-receptors for further investigacptdid~ real 0 0 0 tion of alphaa-adrenoceptor characteristics. Moreover, the fat cell system appears use]1m¢.nta] 0 + ++ ful in screening ,~he alphaa-agonist or Ratvbil |~t;renal !) or 4++ +++ antagonist activity of new compounds. Hamster penrenal

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Alpha-adrenergic responsivenes.s ~ a s investigated according to two different expct-:~ental pathways; ( l ) the increment of epinephnne-~timulated lipol~sLs indueed by an alpha-anlaganist : phentolamine. (2) the inhibition of the~,phylline-induocd lipolysis by an alpha*-agonist: clonidine. Under our experimental condition,; the older rats. dogs, hamsters and rabbits were obese and possessed large fat c-ctK ~(500-900 pl) while the young animals had very. small adilxx.'y|es (,60-150 pl). Legend: ~- ) not reported; tO) undetcctable: ("+ ) ~'eak; ( + + ) medium; ( + + + ) strong (under these conditions adrenaline alone_ was able to pr(~mote a~iLipolylic effecls in rabbits fat cells).

Reading li~1 l Ostman. L and Efendic. S. (1970) Acta Mid. Scand. 1 8 7 , 4 7 1 - t 7 6 2 Bums, T. W. and Langley, P. E. (, 1970) J. Lab. Clin, Med 75, 9~3-997 3 Hittelma~, K. J and Butcher, R. W. (1973) Biochem. Biophy.L Aeta 316, 40.'~-410 4 Lafontan. M. and Agid. R, (,,1976) Comp. Bioehem, Physio~ 55c. 85-90

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5 Berlan. M. and Dang 1ran. I., (1978) J. t'hr~ud. (Paris) 74, 6111-4~tl8 6 Arner. P. and Ostman. J. ( 1~76)..~cm Ah,d .Stand 200, 273-279 7 I,afimlan. M ( 1979)J. I,qmL Re~. 20, 2(1~,.-216 8 Schimmcl. R. J, (1076) Biochim. Bioph)~. At'ta 428. 379-387 9 Lafonl;m. M. and Berlan. M. 11981)) Ei~r, 1. PharmacoL 66, 87-93 1(1 ('arpene. C.. l,afnntan, M. attd Bcrlan. M (19/~1)) Ewerientia 36, 1413-1414 I I (iarcia-Sainz. J.A.. Hoffman. B.B.. I,i. %. Y.. t.efkowitz. R. J and Fain, J. N (1980) Life Set. 27, 953-961

12 Schimmcl. R.J. ~,erto. R. tl.,uch. A Y and Firman-White. [.. (IUX(I)/hot him. lhophv~..4cla 630.71 81 13 Fain. J. N.and(iaroa+Sam~'.J A (ItJSO)l.ffe.%i 2~. 118.3-1194 14 P¢cqu¢~+. R. and [iiudicelli. Y l lq~4ll)l-EB.~ l+,,u 116. 85-90 15 Berlan, M. and I,aflmtan, M. lit)SO) I-mr. J. I'harmaod. 67.481-484 I ~ Aktori¢~,,K+.Schultz, C. and Jakt~b,,. K, I I I 1'480) Mmnyn-+S('hmw~h'h,'rg~

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Metabolic activation and inactivation of chemical mutagens and carcinogens Thomas M. Guenthner and Franz Oesch Department o f Pharmacology, University o f Afainz, Obere Zahlbacher Strafle 67. D - 6 5 0 0 tlainz. F.R.G.

Twenty years ago the word 'carcinogen" was relatively unknown to the general public. Now it seems well entrenched in the common vocabulary. We are perceived to be in the midst of a sea of chemical carcinogens, pervading the food we eat, the water we drink, and the air we breathe. This perception is probably due, in equal parts, to the increasing ingenuity of synthetic organic chemists and the everincreasing capacities of toxicologists and analytical chemists. Avoidance of the equally unreasonable attitudes of indifference and over-reaction to such potential hazards demands a rational understanding of the disposition of these chemicals by the body; how are they detoxified and eliminated, or alternatively, how is their carcinogenicity potentiated'? A look at the carcinogens illustrated in Fig. 1 reveals few common structural features. As is the case of many areas of research, the mechanism of carcinogenicity of these and other organic compounds was poorly understood until a unifying basic concept was set forth, a concept which today seems strikingly simple. Development of this concept is mainly due to work beginning in the mid 1950s by groups centered at the MeArdle Institute in Madison, Wisconsin and the Chester Beatty Institute in London. In a series of seminal reviews in the late 60s and early 70s, James and Elizabeth Miller advanced the concept that majority of organic carcinogens held in

c o m m o n the capacity to be transformed by cellular e n z y m e s to reactive electrophilest'L A s Fig. 1 shows, the "precarcinogens" on the left are converted in r i v o to the metabolites on the right. These "ultimate carcinogens' are all highly reactive electrophiles [the electrophilic (electrondeficient) center is indicated by the arro~ ]. capable of r a n d o m covalent binding to n u m e r o u s nucleophiles in the cell. W h e n the binding occurs to such nucleophilic sites as oxygen a n d nitrogen a t o m s in DN A bases, the genetic template can be altered. This can result in a somatic mutation. which through a series of subsequent events m a y lead to cell transformation and tumorigenesis a. O n the o t h e r hand, these reactive electrophiles m a y bind to small molecular weight "scavenger" nucleophiles such as glutathione. Such binding is considered a detoxification step ~hich ultimately leads to the c o m p o u n d ' s excretion. With these concepts in mind, ~ e ~sish to discuss in further detail the toxification and detoxification of two carcinogens whose metabolism proceeds by quite difh~rent pathways, but nevertheless results in the production of electrophilic intermedk~tes which covalently bind to D N A .

Benzo(a)pyrene Benzo(a)pyrene (BP) is a polycyclic aromatic h y d r o c a r b o n ( P A H ) carcinogen, naturally present in the air, water and soil as a product of incomplete combustion of

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organic matter. In 1775. a L o n d o n ,~urge~m linked the high incidence of ,,crot,tl cancer a m o n g chimney ,,~ccp• to ihcir daib. c,mh~ct ~t.ith soot. Thu',. the fir'~t example ot ;i cancer attributable to an c x o g e n o u , t h e | m eal agent ~*a~ tree caused b} PAH~. Aithougl: mdi~ iduai carcinogenic agent,, d e m e d from coal tar and ,~oot ~erc ~olated and ;(entitled in the It~30~. true understanding of the m e c h a n i s m of P A H care.mogenesls ~*a• not approached until the 196{1s, with the recognition of the formation of eposide~, as a ke) step in the carcint,genic process ~ L A look at the HP molecule (Fig. 2) re~eals no ~ites of high chemical reactivity. Sufficient chemical reactivity for covalent interaction x~ith nucleophilic site• in the cell is introduced, h o ~ e v e r , by formation of an epo,,,ide dt one of man} po,dtion~. The three path~ a} • illustrated in Fig. 2 each begin v, ith the formation of such an 'arene oxide', made possible b~ a rather remarkable micro,,omal electron transport ~ s t e m ~ht,~e terminal oxidam ix c ~ t o c h m m c P4~, [ h e interaction of molecular ox}gen and N A D P H - s u p p l i e d electrons ~ith the iron of this c}tochrome results in the formation of a highly active o \ y g e n species ~ hich can oxidize exogenous ~ubstrate~ at other~i,,: poorly reactive site,,. In the ca~,e of BP. the reactive elcctrophiles thu~ fornred tag. undergo a n u m b e r of further metabolic transformations, ,,