Adrenergic regulation of adipocyte metabolism

Adrenergic regulation of adipocyte metabolism

M.Lafontan1, P.Barbe, J.Galitzky, G.Tavernier, D.Langin, C.Carpene,A.Bousquet-Melou and M.Berlan

Unite INSERM 317, Institut Louis Bugnard, Faculte de Medecine, Universite Paul Sabatier,CHU Rangueil, 31403 Toulouse Cedex 4, France.

*To whom correspondence should be addressed

Five adrenoceptor (AR) subtypes (Pi, P2, 03> oc2

and oti), are involved in the control of white andbrown fat cell function. A number of metabolicevents are controlled by the adrenergic systemin fat cells. The stimulatory effect of catecho-lamines on lipolysis and metabolism is mainlyconnected to increments in cAMP levels, cAMPprotein kinase activation and phosphorylationof various target proteins. Norepinephrine andepinephrine operate through differentialrecruitment of o^- and p*-AR subtypes on thebasis of their relative affinity for the differentsubtypes (the relative order of affinity is (X2 >Pi 22 |32 > P3 for norepinephrine). Antagonisticactions at the level of cAMP production existbetween 0C2- and p r , p2- and p3-AR-mediatedlipolytic effects in human white fat cells. Therole of fat cell (X2-ARs, which largely outnumberp-ARs in fat cells of certain fat deposits, inhuman and primate has never been clearlyunderstood. The other AR type which is notlinked to lipolysis regulation, the ocrAR, isinvolved in the control of glycogenolysis andlactate production. Pharmacological approachesusing in-situ microdialysis and selective oc2- andP-AR agonists and antagonists have revealedsex- and tissue-specific differences in theadrenergic control of fat cell function andnutritive blood flow in the tissue surroundingthe microdialysis probe.Key words: adipose tissue/p- and a-adrenoceptors/lactate/glucose transport/lipolysis

IntroductionWhite adipose tissue (WAT) is the major energysource in animals and humans. A very large part

(95%) of total body triacylglycerols (TAG) islocated in fat stores and TAG are central metabolicsubstrates. Fat storage and fat mobilization aremainly under the co-ordinated regulation of twoenzymes: lipoprotein lipase (LPL) and hormone-sensitive lipase (HSL), so named because of itsresponsiveness to catecholamines and insulin. Thereciprocal regulation of both enzymes has beendemonstrated under physiological conditions. Inthe overnight fasted state, HSL is more activewhile after a meal HSL is suppressed and LPLactivated (Frayn et al, 1995). Adipose tissue isthe essential site of lipolysis where TAG arehydrolysed intracellularly by HSL. In the post-absorptive state, this enzyme controlling fat celllipolysis plays a determinant role in whole-bodylipid fuel availability (Lafontan and Langin, 1995).The mobilization of white fat cell TAG providesthe body with a vital supply of fuel in the form offatty acids (FA) and of gluconeogenic precursorsin the form of glycerol. Adipose tissue is the majorsite of production of non-esterified fatty acids(NEFA) and largely determines the plasma NEFAconcentrations. Plasma NEFA, because of theirhigh caloric content, represent the major source ofenergy in post-absorptive states. NEFA have beenrecognized as the main energy source during starva-tion, various stressful situations (cold exposure,burn injury, surgical trauma, etc.) as well as duringexercise (Coppack et al, 1994).

In fat cells, the metabolic processes are preciselyregulated by neural, humoral and local factorsreleased by or in the neighbourhood of the cells.The isolated fat cell and preadipocyte cell lines(3T3-L1,3T3-F442A and obl7) have been valuablesystems for the delineation of the steps of the

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lipolytic cascade and the study of the regulationof various metabolic processes such as glucoseuptake and NEFA inflow and outflow. The under-standing of the mechanisms of action of the variousadrenergic receptors which contribute to fat cellcontrol has been largely improved. Numerous dif-ferences reported in humans and animals betweenintra-abdominal and subcutaneous depots and evenwithin the various subcutaneous depots show thateach adipose tissue deposit has its own specificityin terms of its metabolic responses. These systemspermit TAG to be hydrolysed at different ratesfrom fat cells of the different regions. Regionaldifferences in LPL or HSL expression and fat cellstimulators and inhibitors of adenylyl cyclase mayexplain why fat tends to accumulate more specific-ally in particular adipose tissue sites.

The in-vitro approach allows the control of anumber of variables in the fat cell environment(albumin concentrations, presence of various hor-mones and autacoids and local pH values). Never-theless, the major difficulty with in-vitro studiesis that the influence of circulatory and nervousfactors, active in situ, is impossible to evaluate.Results obtained in vitro require in-vivo validationwhenever possible. Introduction of new analyticaltechniques such as in-situ microdialysis of fatdeposits (Lonnroth et al, 1987; Arner and Bolinder,1991; Lafontan and Arner, 1996) has recentlyimproved analytical capacities.

The present review focuses on recent advancesconcerning the nature of the adrenoceptors (ARs)and the role of the adrenoceptor-mediated pathwaysin the control of the lipolysis of TAG and of othermetabolic events in the fat cell. The mechanismsof action and the relative importance of the ARscontrolling lipolysis and ensuring the fine tuningof HSL activity and of NEFA availability willbe discussed. Finally, the application of in-situmicrodialysis methods for the study of the adre-nergic control of fat cell function will be discussed.The concept that adipocytes behave as secretorycells for other proteins and lipid metabolites hasemerged during the last 10 years (Ailhaud et al,1992; Spiegelman and Flier, 1996). The adrenergiccontrol of secretory processes, of preadipocyteproliferation and adipose tissue dynamics will notbe considered in the present review.

Pleiotropic responses initiated by thestimulation of fat cell adrenoceptors

Physiological conditions known to alter sympath-etic nervous system (SNS) activity have revealedvarious effects of catecholamines in fat cells.Moreover, in-vitro studies have permitted betterdelineation of mechanisms. They are summarizedin the Table I. In addition to the well known effecton HSL activation, various metabolic events arealso initiated by stimulation of fat cell ARs; mostof them are cAMP and cAMP-dependent proteinkinase-dependent (cAMP-PK). When activated,cAMP-PK could catalyse the phosphorylation ofmany proteins in fat cells; in addition to HSL, thebest-identified substrates for cAMP-PK in fat cellsare GLUT4 glucose transporter, type III cGMP-inhibited low Km cAMP-phosphodiesterase (cGI-PDE), phosphorylase kinase, glycogen synthase,acyl-CoA carboxylase, perilipins and the pV and(32-ARs themselves (for review see Lafontan andBerlan, 1993). Elevation of cAMP concentrationscan also result in either stimulation or repressionof specific gene expression. Activated cAMP-PKalso phosphorylates and modulates the function ofnuclear proteins that bind to DNA sequencesexisting in the promoter regions of cAMP-induciblegenes (Roesler et al, 1988; Lalli and Sassone-Corsi, 1994).

Regulation of hormone-sensitive lipase activity

HSL, the rate-limiting enzyme of the lipolyticcascade, catalyses the hydrolysis of triacylglycerolto diacylglycerol and, then to monoacylglycerol.It is present in white and brown fat cells where itis playing the same role. The hallmark whichdistinguishes HSL from all other known lipases,is the control of its activity through phosphoryl-ation. Fat cell cAMP-PK phosphorylates HSLwhen activated by elevation of intracellular cAMPconcentrations (Kawamura et al, 1981; Stralforsand Belfrage, 1983). A large part of HSL activityis under the strict control of intracellular cAMPconcentrations (Langin et al, 1996). In-vitroassays, have demonstrated that the phosphorylationof HSL parallels the activation of the enzyme.Lipolytic activation is associated with an increasein phosphorylation of HSL while antilipolyticaction is associated with a decrease. Despite the



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Table I. Impact of catecholamines on white fat cell metabolism. Major targets and effector mechanisms for adrenoceptors infat cells

Adrenoreceptor subtype White fat cells

Beta-adrenoreceptors=>P!, p2 and p3

Agonists

isoproterenol (Pi, p2 and p3)

dobutamine (Pi)

terbutaline (P2)

procaterol (P2)

zinterol (P2)

CGP1277 (p3)

CL316243 (p3)

Alpha 2-adrenoreceptors=> a2A

Agonistsclonidine

UK 14304

Stimulation of adenylyl cyclase (Gs protein) => increased cAMP production => cAMP-PKA activation => increment of PKA-dependent phosphorylation of a number of targetproteins.

Short-term metabolic effects

Stimulation of lipolysis (HSL activation)

Stimulation of glycogenolysis

Biphasic regulation of glucose transport, phosphorylation of GLUT 4

Inhibition of insulin-induced glucose transport

Stimulation of long-chain NEFA transport accross adipocyte plasma membrane

Activation of paniculate cGI-PDE (phosphorylation)

Perilipins phosphorylationP2 and Pi-adrenoceptor phosphorylation

Long-term effects

Phosphorylation of proteins (CREB) binding to cAMPresponsive elements (CRE)=> regulation of cAMP inducible genes and of CRE modulators (CREM)Reduction of leptin mRNA levels and fall in the level of serum leptin

Adenylyl cyclase inhibition (Gi protein) => counteraction of adenylyl cyclase stimulationpromoted by beta-agonists and other stimulatory agents = > decrease of cAMP levels.Decrease of lipolysis rate and other cAMP-dependent events.

Activation (Gi protein) and tyrosine phosphorylation of the ppl25 focal adhesion kinase(FAK) as well as of the p42 and p44 mitogen-activated protein kinases (MAPK).

Regulation of preadipocyte growth (proliferation and differentiation)

Alpha radrenoreceptors

Agonistphenylephrine

Stimulation of phospholipase C activity (Gq protein), phosphoinositol bis-phosphatehydrolysis=> intracellular increase in diacylglycerol and inositol 1 ,4,5-triphosphate

=> increase of cytosol Ca + + levels => PKC activation.

Stimulation of glycogenolysis

Stimulation of pyruvate dehydrogenase activity

Stimulation of lactate production and release

crucial role played by changes in cAMP concentra-tions, the physicochemical state and compositionof the lipid droplet and translocation of HSLbetween fat cell compartments also contribute toHSL activation. Questions remain open concerningthe subcellular distribution of HSL upon itsphosphorylation by cAMP-PK. A mechanisminvolving AMP-activated kinase-dependentphosphorylation of HSL to block cAMP-PK-dependent phosphorylation and activation ofthe enzyme could represent a relevant regulatorypossibility. AMP-activated kinase is activated bya 'kinase kinase' in turn activated by long chain

acyl CoA esters (Hardie et al, 1989; Yeaman,1990; Hardie and MacKintosh, 1992). Physiologi-cal relevance of this mechanism is not fully de-lineated. It is considered that the hormonal controlof phosphorylation/dephosphorylation of HSLmainly occurs at the level of cAMP concentrationalthough an additional regulation at the level ofthe control of phosphatase activities cannot beruled out (Manganiello et al, 1992).

Lipoprotein lipase activity

LPL is synthesized in the adipocytes, glycosylatedand then secreted through the endothelial cells to



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bind finally as an active homodimer to heparanstalks on the capillary wall where it has access toits substrates. Many conditions in which energybalance is altered are known to alter tissue LPLactivity. LPL activity in WAT is generally regulatedin a reciprocal fashion of HSL. LPL activity ofWAT is lowered by the P-adrenergic agonistsin vitro and in vivo (Eckel, 1987). Since catecho-lamine concentrations are increased during exer-cise, lowering of LPL activity could be the resultof a P-adrenoceptor-mediated inhibition. Althoughthis view is questionable (Paulin et al, 1991),transcriptional (Raynolds et al, 1990) and post-translational inactivation of LPL (Ashby andRobinson, 1980) were described after P-adrenergicstimulation. A recent detailed study of mechanismsin vitro has demonstrated that epinephrine exertsa modest decrease in LPL catalytic activity in ratfat cells. Various effects were seen on LPL cellularprocessing. Epinephrine inhibited LPL translationalthough this action was counterbalanced by adecrease in LPL degradation and secretion. Thelack of change in cellular immunoreactive LPLlevels suggest that the decreased LPL activitypromoted by epinephrine is related to an additionalbut still undefined post-translational event (Onget al, 1992). Conversely, adrenergic stimulationof animals (Carneheim et al., 1988), cold exposureand (3-agonist administration result in an increasein LPL activity in brown adipose tissue. Beta-adrenergic stimulation of cultured brown adipo-cytes increases the activity of LPL and this responseis primarily P-adrenergic. It is discussed whetherthe elevated value is only achieved by activationof LPL gene transcription or if translationaland/or post-translational are involved. The rate ofLPL gene transcription is regulated positively bycAMP; the increase in rate of enzyme synthesiscorresponds to the increase in activity (Carneheimet al., 1984). Stimulation of LPL gene expressionis certainly an important step, cyclic AMP responseelements will be identified in the promoter regionalof LPL gene alongside with the tissue specifictranscription factors that bind to this region.

Long chain fatty acids transport

The mechanism by which long chain fatty acidsenter cells remains controversial. It has been shown

to exhibit many of the kinetic properties of afacilitated process in fat cells (Abumrad et al,1984). Various plasma membrane fatty acid bindingproteins have been isolated from different celltypes. However, the exact nature and precise struc-ture of these transport systems is not fully deter-mined. A gene encoding for a membrane proteininvolved in the transport of long-chain fatty acidsin adipocytes has recently been cloned (Abumradet al., 1993). Catecholamines promote the activa-tion of the membrane transport of long chain fattyacids in the rat adipocyte (Abumrad et al., 1985,1986). The effect is mediated by P-AR stimulationand cAMP-PK activation. It remains to be deter-mined whether the transporter protein itself orother intermediate regulation steps involve cAMP-PK-dependent phosphorylation in fat cells.

Type III cGMP-inhibited low Km

cAMP-phosphodiesterase activity.

The phosphodiesterase which accounts for majorcAMP phosphodiesterase activity (cAMP-PDE) inparticulate fractions of rat fat cells belongs to asubtype of low Km cAMP phosphodiesterase whichis potently inhibited by cGMP (cGI-PDE), althoughthere is no evidence that inhibition of this enzymeby endogenously produced cGMP is of physio-logical consequence (Degerman et al, 1987, 1990;Smith et al, 1991). This enzyme belongs to thePDE3 family (e.g. PDE3B) which are found inboth particulate and cytosolic fractions of cells(Degerman et al, 1997). The enzyme can bephosphorylated and activated by cAMP-dependentand insulin-dependent serine kinases (Manganielloet al, 1987, 1990; Degerman et al, 1997). Theactivation state of cGI-PDE reflects its relativephosphorylation state. In rat adipose tissue, thecatecholamine-dependent cAMP-mediated activa-tion of cGI-PDE involves phosphorylation of theenzyme by cAMP-PK (Degerman et al, 1990). Aconsensus sequence (-MFRRPS302LPCISREQ-) isphosphorylated in response to isoproterenol, insulinor combination of both (Degerman et al, 1997).Phosphorylation is observed under conditions inwhich isoprenaline causes activation of the enzyme.This is consistent with a causal relationshipbetween phosphorylation and activation of theenzyme. The lipolytic effectors which increase



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cAMP via activation of adenylyl cyclase(P-agonists) or via removal of inhibitory ligandsrapidly increase particulate cGI-PDE activity. Max-imal activations of lipolysis and cGI-PDE areassociated with the same extent of cAMP-PKactivation. In adipocytes, there is apparently a veryclose relationship between isoproterenol-inducedactivation of adenylyl cyclase, cGI-PDE and lipo-lysis. It reflects a tight functional coupling viacAMP-PK activation. In addition, P-agonist-induced activation of the particulate cGI-PDEalso contribute to desensitization of P-adrenergicresponses (Bousquet-Melou et al, 1995). Appar-ently, at least in fat cells, cAMP, through anintracellular 'feed-back loop' regulates the activityof its own degrading enzyme. Evidence for acAMP-dependent regulation of cAMP-PDE geneexpression was demonstrated in some target cells(Conti et al., 1991). If it occurs in fat cells, it canbe viewed as an additional feed back mechanismby which cAMP regulates the expression of itsown degrading enzymes.

The P-adrenergic stimulation on low Km

cGI-PDE could probably explain the strongP-adrenergic dependent stimulation of adeninenucleotide catabolism described in human isolatedfat cells (Kather, 1990). The ATP-lowering actionof P-agonists described in human fat cells is almostentirely due to their cAMP-elevating property.Human fat cells have a strong potential for dissipat-ing energy and the pathway involving cAMPformation and hydrolysis constitute the major routeof adenine nucleotide catabolism in the presenceof P-adrenergic agonists. A P-AR-dependentincrease in cAMP is associated with an enhancedrelease of purines that cannot be re-utilized foradenine nucleotide synthesis and therefore leadsto an irreversible depletion of cellular energystores. The question of the relationship betweenthe effects on cGI-PDE and purine release observedafter P-adrenoceptor stimulation deserve furtherinvestigation.

Glucose transport in fat cells

In the adipocyte, glucose is required for the syn-thesis of triglycerides and glucose transport repres-ents the rate-limiting step for glucose utilization.On a physiological point of view, catecholamines

are known to reduce glucose utilization of varioustissues, thereby inhibiting insulin action. This'diabetogenic' effect of catecholamines is explain-able in part by the direct inhibitory action theyexert on insulin secretion by pancreatic P cells andby the antagonization of peripheral actions ofinsulin on its major targets. Mechanisms explainingthe development of insulin resistance are of diverseorigin and are not fully explained. The site ofcatecholamine action may be a direct inhibitoryeffect on glucose uptake processes by peripheraltissues or an indirect action involving cAMP-mediated modifications of insulin receptor func-tions. Counteraction of glucose transport processesmay also depend on alterations of insulin disposalor of insulin interaction with its receptor. Someeffects could also be promoted from metabolitesproduced by catecholamine action on fat cells.There is considerable evidence that plasma NEFAexcess may regulate insulin sensitivity of varioustarget tissues including fat cells.

The impact of catecholamines on glucosetransport activity has been extensively studied inisolated rat fat cells. Basically, activation of glucosetransport by insulin is hampered by the lipolyticagents such as glucagon or catecholamines (Czechetal, 1992). In fact, both inhibitory and stimulatoryeffects of catecholamines on glucose transporthave been observed in rat, hamster and humanadipocytes. The P-adrenergic agonist isoproterenolstimulates glucose transport at low concentrations(10 nM) but clearly inhibits transport at higherconcentrations (1000 nM). However, when analys-ing the in-vitro effects of P-agonists in detail,several points merit attention. The stimulatoryeffect of isoproterenol on basal and insulin-stimu-lated hexose transport in isolated fat cells onlyappears when adenosine exist in the incubationmedium. When adenosine was prevented fromaccumulating in the incubation medium by additionof adenosine-deaminase, an adenosine-degradingenzyme, isoproterenol completely inhibitedinsulin-stimulated glucose uptake. All these resultsdemonstrate that counter-regulation of insulinaction by adenylyl cyclase activators probablyinvolves the adenylyl cyclase/G-protein complexand cAMP generated by its activation. A recentstudy in rat adipocytes has demonstrated that,

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pVAR-mediated events play a major role in theadrenergic inhibition of glucose transport whilepVP2-mediated events have a minor contribution(Carpene et al, 1993). Conversely, 0C2-adrenergicagonists and other antilipolytic agents participatein the regulation of hexose transport by antagoniz-ing both the (3-adrenoceptor-mediated stimulationor inhibition of transport (Joost et al, 1986).

Glucose uptake into the fat cell is accomplishedby membrane associated carrier proteins, calledGLUT4 and GLUT1, that bind glucose and promoteits transfer across the plasma membrane. TheGLUT4 isoform of glucose transporters is mainlyresponsible of the insulin-stimulated hexoseuptake. Insulin action is explained by redistributionof GLUT4 transporters from a low density micro-some pool to the plasma membrane (Kahn, 1992;Pessin and Bell, 1992).

The action of catecholamines ((3- and a2-AR-mediated) is not considered to account for altera-tions of the initial translocation response inducedby insulin. The effect relies on the modulation ofthe intrinsic activity of glucose transporters whichare located in the plasma membrane. Involvementof cAMP-mediated mechanisms was investigatedto explain the mechanism of action. Treatment ofisolated fat cells with isoproterenol or incubatingmicrosomal membranes with cAMP-dependentprotein kinase increased phosphorylation ofGLUT4 which occurs on a serine residue (S488) ofthe intracellular COOH terminus of the glucosetransporter protein (James et al, 1989; Lawrenceet al, 1990; Kelada et al, 1992). Under suchconditions glucose transport activity was signific-antly altered; its inhibition occurred withoutdecreasing the transporter number. Neverthelessphosphorylation does not completely explain theinhibition of transport activity and all the covalentchanges which could occur in transporter structureand activity require deeper analysis. When referingto the more recent studies, discrepancies revealedin (3-adrenergic receptor-mediated effects could beinterpreted by reference to intracellular cAMPconcentration changes promoted by P-agonists andmimicked by dibutyryl-cAMP. Small incrementsof cAMP concentrations in fat cells (i.e. lowconcentrations of isoproterenol and the presenceof adenosine in the incubation medium) promote

translocation of GLUT4 and stimulate glucosetransport. Low concentrations of dibutyryl-cAMPmimic this action and increase hexose uptake bytranslocating GLUT4 from low density microsomalmembranes to the plasma membrane. Higher valuesof intracellular cAMP (achieved at higher iso-proterenol concentrations and in the presence ofadenosine deaminase) promote phosphorylation ofGLUT4 resulting in a decrease in the intrinsicactivity of the transporters and inhibition oftransport. Recently, dibutyryl-cAMP was shown,when applied to rat fat cells at higher doses (1 mM),to inhibit glucose transport below basal althoughit further increased translocation of GLUT4. Whencomparing the results obtained with isoproterenoland dibutyryl-cAMP, it is when cAMP levels arehigher in fat cells that a decrease in intrinsicactivity of glucose transporter GLUT4 is observed.A cAMP-induced repression of GLUT4 expressionhas been described (Flores-Riveros et al, 1993).Further studies are needed to clarify thiscAMP-dependent repression of GLUT4 expres-sion. It is also necessary to determine the natureof all the catecholamine-induced post-translationalmodifications of glucose transporters and themolecular changes modifying intrinsic activity ofglucose transporters. Moreover, interactions ofcatecholamine-mediated pathways with the signalsthat target intracellular vesicles translocationremain to be defined.

Adrenergic regulation of lactate production infat cells

Lactate production from glucose and lactate releasehave recently been recognized as a novel functionof adipose tissue (DiGirolamo etal, 1992). Lactateproduction is stimulated by norepinephrine in epid-idymal rat adipocytes (Crandall et al, 1983); thiseffect is mediated by an o^B-AR (Faintrenie andGeloen, 1996). In rat fat cells, several intracellularmetabolic effects have been reported in responseto oci-AR stimulation including stimulation ofglycogen phosphorylase, inactivation of glycogensynthase and stimulation of pyruvate dehydro-genase (Lawrence and Larner, 1977). Lactate couldoriginate from glycogen mobilization when glucoseuptake of the fat cells is reduced. Stimulation ofthe a r A R activates a Gq-protein, phospholipase C

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and phosphatidylinositol cleavage to generate thetwo second messengers, e.g. inositol 1,4,5-tri-phosphate and diacylglycerol. a r ARs have beendetected in human, rat and hamster fat cells; theocrAR belongs to the o^B-subtype in rat fat cells(Fain and Garcia-Sainz, 1983; Torres-Marquezet al, 1992). A better knowledge of the regulationof lactate production by adipose tissue is stillneeded. Little is known about the role of a r ARsin human fat cells during lifetime and diseasessuch as obesity and typell diabetes.

Differential affinity of fat cell p r , p2- andpVARs for physiological amines.

Desensitization of P-adrenoceptor-mediatedresponses in fat cells

Basically, the metabolic responses of the fat cell,initiated by the activation of sympathetic nervoussystem and catecholamine release, depend on thebalanced action of adrenoceptor-dependent stimul-atory and inhibitory pathways on cAMP produc-tion. P-adrenergic receptors have been studied quiteextensively in fat cells of various species. Thefunctional significance of expressing all threeP-adrenoceptor subtypes in fat cells is becomingrather clear. Conversely, a2-APs represent the neg-lected side of the adrenergic control fat cell functionfor a large number of biologists; their functionalsignificance is less understood.

The first step leading to adrenoceptor-mediatedactivation of metabolic pathways in fat cellinvolves the multi-regulated key enzyme, adenylylcyclase, which controls cAMP production. Detailedmechanistic considerations have recently beenreviewed (Lafontan and Berlan, 1993). A numberof stimulatory effects are strictly connected to theadrenoceptor-controlled increment of intracellularcAMP concentrations which in turn promoteactivation of cAMP-PK (Honnor et al, 1985a,b)(Figure 1). Catecholamines are sophisticated regu-lators of fat cell function since they operate throughseveral separate receptors. They are able to stimu-late three subtypes of P-adrenoceptors which arepositively coupled to adenylyl cyclase by Gsproteins (GTP binding proteins), and an oc2-ARnegatively coupled to the enzyme by a Gi-protein.An important point in the metabolic actions initi-ated catecholamines concerns the functional signi-

ficance of intracellular cAMP elevations promotedby receptor-mediated adenylyl cyclase control. Adetailed quantitative study of the relationshipsexisting between intracellular cAMP concentra-tions, cAMP-PK activity and lipolysis has beenperformed in rat fat cells. An absence of correlationbetween cAMP levels, cAMP-PK activity state andlipolytic responsiveness was shown for sustainedand submaximal activation of fat cells (Fain andGarcia-Sainz, 1983; Honnor et al, 1985a,b;Langinet al, 1992). However, this kind of study has notbeen performed for the other targets of cAMP-PK. The existence of cAMP-independent lipolyticresponses and/or of cAMP compartimentalizationhave been postulated. Nevertheless, the validity ofthe concept of the involvement of different poolsof intracellular cAMP is still questioned.

In fat cells, when focusing on lipolysis regula-tion, the stimulation of the three P-adrenoceptorsleads to elevations of intracellular cAMP concen-trations and lipolysis stimulation. This seeminglyredundant signal tranduction device has in fact afunctional significance since it is bearing opportun-ities for differential sensitivity and differentialdesensitization of P-adrenoceptor-mediated events.Differential recruitment of the various P-adreno-ceptor subtypes has clearly been established in vitroin dog (Galitzky et al, 1993c), hamster (Carpeneet al, 1992) and rat white fat cells. The order ofaffinity of the various P-adrenoceptors is PJ 5= p2

> p3 for norepinephrine and P2 > Pi > P3 forepinephrine. P3-ARS are only recruited in dog andrat fat cells when the highest concentrations ofcatecholamines are reached in vitro. Detailed studyof lipomobilization in vivo in dog has revealedthat the lipomobilization induced by exogenouslyadministered catecholamines is only due to therecruitment of p r and p2-ARs. However, endo-genous catecholamines, released after pharmaco-logically-induced sympathetic nervous systemactivation, could stimulate fat cell P3-ARS only ifa high level of sympathetic nervous system activityis realized (Galitzky etal, 1993b). Striking species-specific differences in the efficiency ofP3-AR-mediated lipolysis exist as recentlyreviewed (Lafontan, 1994). It must be remindedthat the P3-AR is the principal receptor mediatingcatecholamine-stimulated thermogenesis in brown

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Norepinephrine - epinephrine

(31-(32-P3-ARs cc2-AR

Adipocyte

plasma membrane

D A e lactats

cAMP-PK(A-kinase)

Hormone-sensitive — ^lipase (HSL) ^ -

GLUT 4, perilipins,

Pi" & P2-adrenoceptors,

Phosphorylation of other cAMP-PKA substrates(acyl CoA synthase, phosphorylase kinase...)

Activators of cAMP-inducible genes and CREM promoter

LIPOLYSIS

Figure 1. Membrane-associated multiprotein sytem (adrenoceptors, Gs, Gi and Gq-proteins, and the catalyst moieties ofadenylyl cyclase and phoslipase C) responsible for regulating catecholamine (epinephrine and norepinephrine) effects in whitefat cells. Three stimulatory P-adrenoceptors (P(, P2

a nd P3X coupled to Gs protein, and one inhibitory oc2-adrenoceptor, coupledto Gi, exert antagonistic actions on adenylyl cyclase activity, cAMP production and cAMP-PK activation (cAMP-induceddissociation of R/C subunits). cAMP-PK promotes phosphorylation of hormone-sensitive lipase (HSL) which controls lipolysis.Phosphorylation of a number of other target proteins involved in the regulation of various metabolic effects is also controlledby cAMP-PK. oti-Adrenoceptors and their transducing system (Gq-protein and phospholipase C) are also represented in thediagram. Selective al-adrenoceptor stimulation promotes glycogenolysis and lactate production and release in fat cells.

adipose tissue, a tissue which oxidizes NEFA,produces heat and could be involved in the reduc-tion of excess fat. Brown adipose tissue is scarcein normal adult humans. The physiological andevolutionary relevance of these observations is notimmediately obvious and requires more compre-hensive genetic approaches. These results showhow careful we must be in the future when animalcells are chosen as a representative model of humanadipocytes.

Concerning human white fat cells, the functionalexistence and the role of the P3-receptor subtypein man has been controversial (Langin et al, 1991;Lonnqvist et al, 1993; Rosenbaum et al, 1993;Van Liefde et al, 1994; Tavernier et al, 1996).

The presence of P3-AR mRNAs is certain (Kriefet al, 1993), but the assessement of p3-dependenteffects is only based on the lipolytic effects of asingle compound, i.e. CGP12177 (a Pi^-antagon-ist which exerts (33-effects in white and brown fatcells of various species). In-vitro studies in humanisolated fat cells have revealed important differ-ences in p3-AR-dependent responsiveness betweenomental and subcutaneous fat cells which couldaccount for the differences reported by the variousgroups. Recent microdialysis studies have revealedthe existence of lipolytic P3-adrenergic responsesin subcutaneous adipose tissue of normal healthysubjects (Enoksson et al, 1995b) and an increasedP3-adrenergic function in visceral fat cells of

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markedly obese subjects (Lonnqvist et al, 1995).As shown in dog and rat fat cells, activation ofP3-ARs in human fat cells, if occurring, shouldonly exist at the highest concentrations of catecho-lamines (Bousquet-Melou et al, 1994). Whateverthe results obtained with some P3-agonists, thestimulation of p3-ARs under physiological activa-tions of the sympathetic nervous system has notbeen demonstrated. Recently, a mutation has beendescribed in the human P3-AR gene; a cytosine isreplaced by a thymidine in the codon 64 leadingto the replacement of a tryptophane by an arginine(Trp64Arg mutation). Pima Indians, homozygousfor the Trp64Arg mutation, have an early onset ofNIDDM and tend to have lower resting metabolicrate (Walston et al, 1995). In another study onpatients from Western Finland, Trp64Arg mutationof (33-AR gene was associated with abdominalobesity and resistance to insulin and the authorsthought that it may contribute to the early onsetof NIDDM (Widen et al, 1995). It is noticeablethat the frequency of mutated allele was similar inthe diabetic and non-diabetic subjects. Finally, anincreased capacity to gain weight was associatedwith Trp64Arg mutation of P3-AR gene in morbidlyobese French subjects (Clement et al, 1995).However, there was no evidence that the mutationis more frequent among obese subjects. TheTrp64Arg P3-AR mutant is pharmacologically andfunctionally indistinguishable from the wild typeP3-AR (Candelore et al, 1996). In obesity-pronesubjects, an impaired in P3-AR function may resultin defective lipolysis and thermogenesis and con-tribute to obesity. However, it is not clear if sucha reduction in the p3-adrenergic control of fat cellfunction could be important for the developmentof insulin resistance and metabolic complications.In fact, in subjects with abdominal obesity exhibit-ing several features of insulin resistance, it is notan impaired but it is an enhanced pVadrenergicreceptor activity which has been reported in vis-ceral adipose tissue (Lonnqvist et al, 1995). Fur-ther studies are needed to establish the clinicalimportance the Trp64Arg mutation of the P3-ARgene; a recent controversial study has been reported(Gagnon et al, 1996).

As well as HSL activation, cAMP increase alsopromotes p2-/Pi-AR desensitization partly explain-

able by cAMP-PK-dependent phosphorylation ofreceptor protein residues (Hausdorff et al, 1990).Such a mechanism could limit the occurrence ofsustained pr/p2-AR driven cAMP production andlipolysis in fat cells. Although useful to preventoverstimulation of cells, this mechanism couldrepresent a major functional drawback when sus-tained stimulation of the fat cell is necessaryas required during prolonged exercise or duringsustained cold exposure. Desensitization ofp-adrenergic responses have been described afterinvitro or in-vivo stimulation of human, rat, dog andhamster fat cell adrenoceptors with norepinephrineand P-agonists (see review (Lafontan, 1994)). TheP3-AR offers a major advantage since it is com-pletely resistant to the short-term and long-termagonist-induced desensitization and/or down-regu-lation (Carpene et al, 1992; Liggett et al, 1993;Nantel et al, 1993). However, for the moment,some controversial data exist in in-vitro and in-vivostudies concerning the long-term desensitization ofP3-ARs (Carpene et al, 1992; Granneman andLahners, 1992; Revelli et al, 1992; Thomas et al,1992; Liggett et al, 1993; Nantel et al, 1993).Contradictory observations could be partlyexplained by species-specific or cell-specific differ-ences (Nantel et al, 1994). Desensitization ischaracteristic of p r and P2-ARS, it is linked inpart to intrinsic properties of the receptor proteinwhich contain serine and threonine residues in thecarboxyl-terminus and the third cytoplasmic loop.These residues serve as substrates for cAMP-PK and P-receptor-specific kinases (P-ARK). TheP3-AR lacks most of such residues existing in theother P-adrenoceptor subtypes.

Recent experiments performed in the laboratoryhave also shown that isoproterenol- and norepin-ephrine-induced activation of cGI-PDE could playa major role in short-term desensitization of norepi-nephrine responsivenes of rat fat cells in vitro.Having access to recently developed molecules,the CGI-PDE activity could be dose-dependentlyblocked by the highly specific inhibitor ofcGI-PDE, OPC 3911. Under such conditionsP-agonist-induced short-term desensitizationlargely disappears. The well-known desensitizationof norepinephrine action is only observed whencGI-PDE activity is not blocked (Bousquet-Melou

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et al, 1995). This post-receptor mechanisms alsocontributes to the diverse mechanisms involvedin heterologous desensitization of P-adrenergicresponses.

P/0C2-AR interplay in the regulation of fat cellfunction

The role of fat cell a2-ARs, which largely out-number P-adrenoceptors in fat cells of certain fatdeposits, in human, primate, hamster and rabbithas never been clearly understood. Overexpressionof a2-ARs in fat cells alters catecholamine respons-iveness in various species (Lafontan and Berlan,1993). In human fat cells, based on the in-vitromeasurement of the relative affinity of physiologi-cal amines for 0C2- and P-adrenoceptors, it isthe 0C2-AR which has the highest affinity forphysiological amines and which is stimulated atthe lowest catecholamine concentrations before theactivation of P-adrenoceptors occur. The relativeorder of affinity of the various fat cell adrenoceptorsfor norepinephrine is a2 > Pi s* P2 > pV Thea2-AR has a higher affinity for epinephrine thanfor norepinephrine. Biphasic regulation of lipolysisin vitro by catecholamines, assessing existence ofdifferential recruitment of a2- and P-adrenoceptorshas clearly been demonstrated, under optimizedassay conditions, in human (Berlan and Lafontan,1985; Mauriege et al, 1987, 1991) and rabbit fatcells (Langin et al, 1990). The interplay betweena2- and P-adrenoceptors takes a key position inthe triggering of cAMP increments in fat cells; largespecies-specific discrepancies, the significance ofwhich is unknown, exist.

Intracellular cAMP concentrations are veryimportant in modulating catecholamine sensitivityand action. The experimental protocol commonlysettled for in-vitro assay of a2-adrenergic respons-iveness is based on counteraction of drug-induced-cAMP production (isoproterenol, forskolin, iso-butylmethylxanthine or adenosine deaminase). Theantilipolytic effect of oc2-agonists, like the anti-lipolytic effect of insulin, is certainly impairedwith high levels of cAMP and when cAMP-PKactivity ratio exceeds 0.6 as previously shown forinsulin in rat adipocytes (Londos et al, 1985).Discrepancies in cc2-agonist-induced antilipolyticeffects reported by various investigators are prob-

ably linked to the nature of the assay system usedto promote increments of cAMP concentrationsbefore measuring the antilipolytic effects ofa2-agonists. Since there is a non-linear relationshipbetween lipolysis and cAMP concentrations, animpairment of the antilipolytic action appears whenoc2-agonist-induced adenylyl cyclase inhibition isnot sufficient to promote a reduction in the cAMPconcentrations, to such an extent that inhibition oflipolysis could occur. Since the high-affinityoc2-ARs are preferentially stimulated by physiologi-cal amines, before concentration requirements forP-adrenoceptors are attained, it is not appropriateto explore the efficiency of cc2-AR-mediatedmechanisms by using counteraction of P-adreno-ceptor-mediated effects. In fact, considering thehigh affinity of catecholamines for fat cell oc2-ARs,it cannot be excluded that these receptors exert apermanent and tonic inhibition on lipolysis andare responsible for a tonic inhibitory componentexerted by catecholamines on 'basal lipolysis'. Themost suitable conditions to demonstrate that sucha mechanism is operative in vivo have not beenconvincingly established although a contributionof 0C2-ARS in the modulation of lipolysis at restwas suspected (Arner et al, 1990).

Pharmacological approaches using in-situ micro-dialysis and selective oc2- and P-agonists and antag-onists have revealed sex- and tissue-specificdifferences in the adrenergic control of fat cellfunction (Arner etal, 1990; Galitzky etal, 1993a).Moreover, use of the ethanol escape techniquewhich only measures tissue blood flow in a semi-quantitative fashion in the circulation around theprobe have revealed the importance of local nutrit-ive blood flow in the control of lipid mobilization(Enoksson et al, 1995a; Galitzky et al, 1993a;Barbe et al, 1996; Lafontan and Arner, 1996).Vasodilatory P2-ARS and vasoconstrictive (X2~ARsare present in the vessels draining human subcuta-neous adipose tissue. Because of their involvementin the control of lipolysis and the increment oflocal blood flow, P2-ARs probably play a majorrole in the induction of lipid mobilization. Optimaleffects are obtained when non-selective P-agonists(isoproterenol) and selective p2-agonists (terbuta-line) are used. Conversely, a2-ARs are inhibitors oflipid mobilization by a combination of antilipolytic

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and vasoconstrictor effects. When using micro-dialysis technique it seems essential to considerboth glycerol concentrations and blood flow toobtain relevant evaluation of the effects (Lafontanand Arner, 1996).

When physiological amines are used instead ofselective agonists or antagonists in the micro-dialysis probe, the relative contribution of thevarious adrenoceptor subtypes of the fat cell and thevessels are more difficult to demonstrate. Recentstudies performed in the laboratory and basedon infusion of catecholamines and activation ofsympathetic nervous system by orthostatism inpatients bearing microdialysis probes in variousfat deposits have exhibited a differential contribu-tion of oc2- and P-adrenoceptors in the physiologicalregulation of glycerol output from subcutaneousadipose tissue (Barbe et al, unpublished results).When infused in the microdialysis probe, norepi-nephrine exerted stronger effect on glycerol outputin abdominal than in gluteal fat deposits in women;although this result fits with previous in-vitroresults (Mauriege et al, 1987) changes in nutritiveblood flow could also contribute to the effects.The increase of glycerol output during activeorthostatism seems to be mainly due to the activa-tion of the subcutaneous adipose tissue P-adreno-ceptors since it was prevented by a P-antagonist(propranolol) and unaltered by an oc2-antagonist(yohimbine). Improvement of in-situ microdialysismethods and optimization of the measurements ofchanges occurring in adipose tissue blood flowwill certainly improve our understanding of oc2- andP-adrenoceptor interplay in the physiological regu-lation of adipose tissue metabolism and lipid mobil-ization.

Conclusions and future trends

To conclude, the study of isolated fat cells andpreadipose cell lines has considerably extendedour understanding of the mechanisms of action ofcatecholamines in the control of fat cell metabol-ism. It is well accepted that the adrenergic regula-tion of the metabolic responses of the fat celldepends on the balanced action of adrenoceptor-activated stimulatory and inhibitory pathwaysinvolved in cAMP production. The delineationof the contribution of the various adrenoceptor

subtypes has considerably been extended. Whenconsidering pathological alterations of fat cellfunction, alteration of one or more of the elementsof receptor-G proteins-adenylyl cyclase complexcould occur. Moreover, post-receptor mechanisms(e.g. at Gs and Gi-proteins, cAMP-PK, HSL andcGI-PDE level) may also participate to alterationof metabolic functions. In the near future, geneticand immunological techniques should enabledetailed analyses of the enzymes and pathwaysof the signal transduction system of the fat cell.The cell-targeted 'knock out' of various genescoding for the numerous elements of the pathwayscontrolling NEFA mobilization will open newperspectives for the delineation of their physio-logical importance.

Homologous and heterologous regulation ofP- and a2-ARs and pathological alterations of theadrenergic control of the fat cell have recentlybeen reviewed (Arner, 1992; Lafontan andBerlan, 1993).

In addition to the direct effect of catecholamineson fat cell function, there are important effects ofthe sympathetic nervous system on the control oflocal blood flow in the various fat deposits whichrequire improved studies to explain some negativecorrelations reported between the results yieldedby in-vitro and in-vivo studies. Normal lipolyticprocesses and abnormalities of lipolysis whichaccompany pathological conditions such as obses-ity and non-insulin-dependent diabetes requireextended studies since they may contribute toinsulin resistance and hyperlipidaemia currentlyassociated with these diseases. Newer techniquessuch as microdialysis and canulation of veinsdraining fat deposits will offer, when optimized,important improvements for the delineation of thevarious elements of the adipose tissue contributingto lipolytic processes and lipid mobilization.

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