Role of b3-adrenergic receptors in the action of a tumour lipid mobilizing factor

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1 British Journal of Caner (22) 86, ª 22 The Caner Researh Campaign All rights reserve 7 92/2 $25. Role of b3-arenergi reeptors in the ation of a tumour lipi mobilizing fator ST Russell 1, K Hirai 2 an MJ Tisale*,1 1 Pharmaeutial Sienes Researh Institute, Aston University, Birmingham, B4 7ET, UK; 2 Department of Obstetris an Gynaeology, Osaka City University Meial Shool, Osaka , Japan Inution of lipolysis in murine white aipoytes, an stimulation of aenylate ylase in aipoyte plasma membranes, by a tumour-proue lipi mobilizing fator, was attenuate by low onentrations ( M) of the speifi b3- arenoeptor antagonist SR5923A. Lipi mobilizing fator (25 nm) proue omparable inreases in intraellular yli AMP in CHOK1 ells transfete with the human b3-arenoeptor to that obtaine with isoprenaline (1 nm). In both ases yli AMP proution was attenuate by SR5923A onfirming that the effet is meiate through a b3-arenoeptor. A non-linear regression analysis of bining of lipi mobilizing fator to the b3-arenoeptor showe a high affinity bining site with a K value nm an a B max value (282+1 fmole mg protein 71 ) omparable with that of other b3-arenoeptor agonists. These results suggest that lipi mobilizing fator inues lipolysis through bining to a b3-arenoeptor. British Journal of Caner (22) 86, DOI: 1.138/sj/bj/ ª 22 The Caner Researh Campaign Keywors: ahexia; lipi mobilizing fator; b3-arenoeptor; energy metabolism Patients with aner ahexia experiene a ramati loss of boy fat as the onition progresses. A stuy of the boy omposition of lung aner patients, who ha lost 3% of their pre-illness stable weight, showe an 85% erease in total boy fat (Fearon, 1992), refleting a prolonge ataboli state. Caner patients with weight loss have been foun to have an elevate level of a lipi mobilizing fator (LMF) in both serum an urine, whih appears to parallel the weight loss (Grounwater et al, 199). We have isolate LMF from the urine of aner patients by a ombination of ion exhange, exlusion an hyrophobi interation hromatographies, an shown it to be homologous with the plasma protein Zn-a2-glyoprotein (ZAG) in primary sequene, eletrophoreti mobility an immunoreativity (Toorov et al, 1998). In vivo stuies onfirme the ability of LMF to ause seletive loss of arass fat with no hange in boy water, an a teneny to inrease the nonfat mass. LMF was haraterize by the ability to stimulate lipolysis iretly in isolate aipoytes, as a result of stimulation of aenylate ylase in a GTP-epenent proess (Hirai et al, 1998). The reeptor for this interation has not been haraterize, but iniret eviene suggests that it may be a b3- arenergi reeptor (b3-ar). Thus treatment of ob/ob mie with LMF, not only proue a speifi epletion of the aipose mass together with an elevation of serum glyerol levels, but also an inrease oxygen uptake by intersapular brown aipose tissue (BAT) (Hirai et al, 1998). Pharmaologial stuies iniate that the b-reeptor responsible for the stimulation of oxygen onsumption in BAT is exlusively of the b3-subtype (Howe, 1993). Inution of lipolysis in epiiymal aipoytes by LMF was attenuate by the b-arenergi reeptor bloker propranolol (Khan an Tisale, 1999), while the biphasi *Corresponene: MJ Tisale; MJTisale@aston.a.uk Reeive 1 January 21; revise 2 Otober 21; aepte 15 November 21 effet of GTP on yli AMP proution by LMF in aipoyte plasma membranes suggests a reeptor assoiate with both Gs an Gi. Only b3 an not b1-ar interat with Gi in aipoyte membranes (Granneman, 1995). In the present stuy the ability of LMF to interat with the b3- AR has been stuie both in white aipoytes an in CHO ells transfete with the human b3-ar. MATERIALS AND METHODS Patients Urine was ollete over a 24 h perio from patients with unresetable panreati aner an with a weight loss between.5 an 3 kg month 71. No patient ha reeive raiotherapy or hemotherapy. Urine samples were store at 728C in the absene of preservatives prior to use. Chemials [a- 32 P]-ATP (sp. at. 2 Cimmol 71 ) an Na [ 125 I] (sp. at. 415 Cimg 71 ioie) were purhase from Amersham Pharmaia Bioteh (Buks, UK). SR5923A was kinly onate by Dr L Manara of the Researh Centre Sanofi Miy, Sanofi Winthrop S.p.A., Milan, Italy. Purifiation of LMF LMF was purifie from human urine using a ombination of bath extration on DEAE-ellulose an hyrophobi interation hromatography (Toorov et al, 1998). Urine was entrifuge at 3 g for 1 min to remove partiulate material an was then ilute with 4 vol 1 mm Tris HCl, ph 8.. DEAE-ellulose, previously ativate by washing in 1 mm Tris HCl, ph 8. for 5 min was ae to the ilute urine (1 g l 71 of original urine)

2 an the mixture was stirre for 2 h at 48C. The DEAE-ellulose was reovere by seimentation by low spee entrifugation, an the LMF was elute with.5 M NaCl in 1 mm Tris HCl, ph 8.. The eluate was equilibrate an onentrate to 1 ml by ultrafiltration, in an Amion filtration ell (Millipore (UK) Lt, Watfor, Herts, UK) ontaining a membrane filter with a moleular weight ut-off of 1 kda, against PBS. Further purifiation was ahieve using a Resoure-Iso HPLC olumn (Pharmaia Bioteh, St Albans, Herts, UK), employing a ereasing (NH 4 ) 2 SO 4 onentration from 1.5 M. Ative frations ontaining LMF elute at.6 M (NH 4 ) 2 SO 4, an were esalte before use by washing five times against PBS using an Amion filtration ell. Lipolyti assay A single ell suspension of white aipoytes was prepare from the epiiymal aipose tissue of ex-breeer male NMRI mie using ollagenase igestion (Bek an Tisale, 1987). Lipolyti ativity was etermine by measuring glyerol release (Wielan, 1974) after inubation of LMF with aipoytes for 2 h at 378C in 1 ml Krebs-Ringer biarbonate buffer, ph 7.2. Control samples ontaining aipoytes alone were analyze to etermine the spontaneous glyerol release. Lipi mobilizing ativity was expresse as mmole glyerol release 1 5 aipoytes 71 2h 71. Aenylate ylase assay Plasma membranes were isolate from epiiymal aipoytes, as previously esribe (Khan an Tisale, 1999). Briefly isolate aipoytes were homogenize in 25 mm surose, 2 mm EGTA an 1 mm Tris HCl ph 7.4, followe by entrifugation at 3 g for 1 h at 48C. The membrane pellet forme was isolate an separate from other organelle membranes on a self forming Peroll graient, an the mixture was entrifuge at 1 g for 3 min at 48C. The washe plasma membranes were ilute in 1 mm Tris HCl, ph 7.4, ontaining 25 mm surose, 2 mm EGTA an 4 mm phenylmethylsulphonylfluorie at 1 2 mg ml 71, an if not use immeiately, snap frozen in liqui nitrogen an store at 778C until use. The aenylate ylase assay was base on that evelope by Salomon et al (1973) as previously esribe (Hirai et al, 1998). Briefly LMF was inubate for 1 min at 38C together with plasma membrane in 25 mm Tris HCl, ph 7.5, 5mM MgCl 2, 1 mm GTP, 8 mm reatine phosphate, 16 units ml 71 reatine phosphokinase, 1 mm 3-isobutyl-1-methylxanthine an 1 mm [a- 32 P]-ATP (sp.at. 2 Cimmole 71 ) in a total volume of 1 ml. The reation was terminate by the aition of 2% SDS, 4 mm ATP an 1.4 mm yli AMP. The yli AMP was isolate from the mixture using a ombination of Dowex 5W8-4 an Alumina WN-3 olumns, an the raioativity was etermine using a Tri-arb 2A sintillation ounter. Cyli AMP etermination CHOK1 ells transfete with the human b3-ar, uner the ontrol of hygromyin, together with the b-gal reporter onstrut, selete for resistane to G418, were a gift from Dr Ian Waell, Astra Zenea, Malesfiel, Cheshire, UK. They were grown in Dulbeo s moifie Eagles meium (DMEM) supplemente with 2 mm glutamine, 5 mg ml 71 hygromyin B an 2 mg ml 71 G418, uner an atmosphere of 1% CO 2 in air. For yli AMP assays ells were grown in 24 multi-well plates in 1 ml DMEM. Agonists were ae to the wells an inubate for 3 min, after whih the meium was remove an.5 ml 2 mm HEPES, ph 7.5, 5 mm EDTA an.1 mm isobutylmethylxanthine was ae to eah well. The plate was plae in a boiling water bath for 5 min an oole on ie for 1 min. To 5 ml of the ell extrat was ae 2 mci of [8-3 H]-yli AMP (Amersham, UK) an 2 mg of yli AMPepenent protein kinase (Sigma Chemial Co. Lt, Dorset, UK) b3-reeptors an LMF an inubate for 2 h at 48C. Unboun yli AMP was remove by asorption onto haroal an the onentration of yli AMP in the sample etermine by omparison with stanar urves using known onentrations of yli AMP. Ioination of LMF with [ 125 I] One ioo-bea (Piere an Warriner, Chester, UK), washe an rie, was inubate with Na[ 125 I] (1 mci per 1 mg protein) for 5 min in 1 ml PBS. LMF (1 mg protein) was then ae an the reation allowe to proee for 15 min. The ioo-bea was physially remove an free Na[ 125 I] was remove using a Sephaex G25 olumn elute with.1 M NaI. The [ 125 I] LMF was onentrate using a Miroon miroonentrator with a M r 1 ut-off against PBS. Bining stuies CHOK1 ells transfete with the human b3-ar were lyse by soniation in.5 M MgCl 2, 2 mm Tris HCl, ph 7.5 an rue membranes were pellete by entrifugation (45 g, 15 min, 48C). Bining stuies were onute in 4 ml.5 mm MgCl 2 5 mm Tris HCl, ph 7.5, by inubation of membranes (5 mg protein) with various onentrations of [ 125 I] LMF for 6 min at 378C. The samples were then entrifuge at 13 g for 2 min, the supernatant was remove an the raioativity of the pellet was etermine using a Pakar Cobra Moel 55 Auto-gamma ounter. Bining was analyze using non-linear regression analysis (GraphPa Prism, Version 3. for winows, GraphPa Software (San Diego, CA, USA)). RESULTS LMF inue a iret lipolyti response in murine white aipoytes, an this effet was attenuate by low onentrations ( M) of SR5923A (Figure 1A), whih has been reporte to have a 1-fol seletivity for the b3-ar over the b1- AR (Nisoli et al, 1996). Inution of lipolysis by LMF was assoiate with a stimulation of aenylate ylase in isolate aipoyte membranes in the presene of.1 mm GTP, an this ation was almost ompletely inhibite by SR5923A at onentrations as low as 1 79 M (Figure 1B). The ifferene in sensitivity of intat aipoytes an plasma membranes may be relate to aess of SR5923A to the b3-ar. SR5923A has been shown to bin strongly to albumin (Nisoli et al, 1996) reuing the effetive onentration available in the aipoyte assay. These results suggest that LMF stimulates lipolysis through interation with a b3-ar. To investigate this possibility the effet of LMF on yli AMP proution was etermine in CHOK1 ells, whih ha been transfete with the human b3-ar. The ata presente in Figure 2 shows that both isoprenaline an LMF stimulate yli AMP proution, whih reahe a omparable maximum level of 25 pmoles per 1 6 ells with both agents. However maximal yli AMP proution was ahieve with muh lower onentrations of isoprenaline (1 nm) than LMF (25 nm), suggesting that LMF ha a lower affinity for the b3-ar than isoprenaline. The inrease in intraellular yli AMP proue by both isoprenaline an LMF in CHOK1b3 was attenuate by the non-speifi b-ar antagonist propranolol (1 mm), while the effet on LMF, although signifiant, was less than omplete. However, yli AMP proution by both isoprenaline an LMF was almost ompletely attenuate by SR5923A, onfirming that the ation of LMF was meiate through a b3-ar. To etermine the affinity of bining of LMF to the b3-ar, LMF was raioioinate with 125 I an the bining to rue plasma membranes from CHOK1b3 ells was etermine. The ata is presente in Table 1. Non-linear regression analysis of bining showe a high affinity bining site for LMF with a K value about 425 ª 22 The Caner Researh Campaign British Journal of Caner (22) 86(3),

3 b3-reeptors an LMF 426 A.12 A 3 µmole glyerol 1 5 aipoytes -1 2 h AMP (pmoles 1 6 ells -1 ) b B 3 LMF (nm) B pmole AMP mg -1 protein min SR5923A (M) AMP (pmoles 1 6 ells -1 ) Isoprenaline (nm) Figure 2 Effet of LMF (A) an isoprenaline (B) on yli AMP levels in CHOK1b3 ells in the absene (*) or presene of 1 mm propranolol (*) or 1 mm SR5923A (~) Differenes from ontrols are iniate as b, P5.1 an, P5.1 as etermine by ANOVA SR5923A (M) Figure 1 (A) Effet of the b3-ar antagonist SR5923A on lipolysis in murine white aipotes; inue by LMF. Aipoytes were preinubate with the iniate onentration of SR5923A for 3 min prior to the aition of LMF (465 nm). (B) Effet of SR5923A on the stimulation of aenylate ylase in isolate murine aipoyte plasma membranes by LMF. Membranes were preinubate with the iniate onentrations of SR5923A for 3 min prior to the aition of LMF (2.35 mm) an aenylate ylase was etermine as esribe in Materials an Methos in the presene of.1 mm GTP. The results are expresse as means +s.. an the ata is representative of three separate experiments. Differenes from inubation in the absene of SR5923A is iniate, P5.5 an, P5.1 as etermine by Stuent s t-test. 1-fol lower than that of CGP 12177, a partial agonist of b3-ar (Kubo et al, 1997) an [ 125 I] iooyanopinolol (Huthinson et al, 2), ommonly use in bining stuies with b3-ar. However, the B max value for LMF was similar to that for other b3-ar agonists. Bining of [ 125 I] LMF was signifiantly reue in the presene of non-labelle LMF, the non-speifi b-ar antagonist propranolol an the seletive b3-ar antagonist SR5923A (Table 1). These results onfirm that LMF bins to a b3-ar an stimulates aenylate ylase. DISCUSSION Resting energy expeniture (REE) has been reporte to be signifiantly inrease in weight losing patients with lung (Frerix et al, 199) an panreati aner (Faloner et al, 1994). Hyltaner et al (1991) foun that aner patients ha an elevate REE an inrease fat oxiation ompare with either weight losing or weight stable ontrols, an that this was relate to an inrease heart rate. Suh patients were also foun to exhibit an inrease ariovasular an metaboli response to arenaline infusion (Drott et al, 1987), while aministration of the non-speifi b- bloker propranolol was foun to proue a erease in the basal metaboli rate (BMR) (Gambarella et al, 1999). These results le to the hypothesis of overativity of the sympatheti nervous system (SNS) in aner patients. Classial b1 an b2-ar meiate response to norarenaline release from the SNS. In aition a thir b-ar subtype has been ientifie (reviewe in Howe, 1993), whih shares only 4 5% amino ai sequene ientity with b1 an b2-ar, an is referre to as a b3-ar. These reeptors meiate lipolysis in white aipose tissue in mie an rats (Arh et al, 1984; Arh an Wilson, British Journal of Caner (22) 86(3), ª 22 The Caner Researh Campaign

4 Table 1 b3-reeptors an LMF K an B max values for LMF an other agonists to b3-ar 427 Agent K (nm)+s.e.m. B max (fmol mg protein 71 ) +s.e.m. Referene CGP Kubo et al, 1997 [ 125 I]-CYP Huthinson et al, 2 [ 125 I]-LMF This stuy [ 125 I]-LMF+ propranolol (1 mm) This stuy [ 125 I]-LMF+SR5923A (1 mm) This stuy [ 125 I]-LMF+ol LMF This stuy 1996), an thermogenesis in BAT (Arh, 1989), an are also responsible for the unexpete negative inotropi effets of ateholamines in the heart (Gauthier et al, 1996). However, the eviene that b3-ar an meiate lipolysis in human aipoytes is ontroversial, sine b3-ar mrna is expresse at a muh lower level than in rat or mouse (Langin et al, 1991), although lipolysis has been inue in human omental fat ells by the seletive b3- AR agonist CGP (Hoffstet et al, 1995), an LMF (Hirai et al, 1998). We have previously shown that ahexia in both mie an humans is assoiate with LMF proution by the tumour an exretion in the urine (Toorov et al, 1998), an that LMF stimulate lipolysis like a lassial lipolyti hormone through inreases in intraellular yli AMP as a result of the stimulation of aenylate ylase (Hirai et al, 1997). This stuy shows that LMF exerts this effet through a b3-ar, although the affinity for this reeptor appears to be less than seen with lassial b3-ar agonists. In white aipoytes both the inution of lipolysis an the stimulation of aenylate ylase were attenuate by the b3-ar antagonist SR5923A (Nisoli et al, 1996), while in CHO ells transfete with the human b3-ar LMF stimulate yli AMP proution in a similar manner to isoprenaline, although the onentration require to proue maximal stimulation was 25-fol greater. In aition SR5923A attenuate the inrease in yli AMP onfirming the effet was meiate through a b3-ar. The effet of propranolol was less omplete than with isoprenaline, suggesting that the mehanism of stimulation by LMF may be ifferent. Previous stuies (Khan an Tisale, 1999) have shown propranolol to at as a non-ompeative inhibitor of the inution of lipolysis in murine white aipoytes by LMF. This suggests that it may at at a site istal to the b3-ar an may attenuate the ation of two b3 agonists to ifferent extents. In this stuy we have use intat ells, sine the oupling effiieny of b3-ar to aenylate ylase is highly epenent upon the integrity of the ells (Granneman, 1995). However, it is known that the oupling effiieny of b3-ar is greater than that for b1-ar, thus offsetting the low bining affinity. Also unlike b1 an b2-ar the b3-ar has fewer potential phosphorylation sites an is resistant to agonistinue esentitization (Granneman, 1995). The b3-ar meiate oupling of LMF to lipolysis woul explain the lowere maximal response of human omental aipoytes to lipolysis when ompare with murine white aipoytes (Hirai et al, 1998). However, the inrease oupling effiieny together with the inution of UCP1 in brown aipose tissue (BAT) (Russell et al, 2) woul ensure maximum fat mobilization an utilization together with a net inrease in energy expeniture. These results suggest that seletive b3-ar antagonists may be useful in ontrolling energy expeniture an fat mobilization in aner ahexia. ACKNOWLEDGEMENT We wish to thank Bayer Corporation, USA for finanial support. REFERENCES Arh JRS (1989) The brown aipoyte beta-arenoeptor. Pro Nutr So 48: Arh JRS, Ainsworth AT, Cawthorne MA, Piery V, Sennitt MV, Thoy VE, Wilson C, Wilson S (1984) Atypial beta-arenoeptor on brown aipose as target for anti-obesity rugs. 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