Lactate Inhibits Lipolysis in Fat Cells through Activation of an Orphan G-protein-coupled Receptor, GPR81

Lactic acid is a well known metabolic by-product of intense exercise, particularly under anaerobic conditions. Lactate is also a key source of energy and an important metabolic substrate, and it has also been hypothesized to be a signaling molecule directing metabolic activity. Here we show that GPR81, an orphan G-protein-coupled receptor highly expressed in fat, is in fact a sensor for lactate. Lactate activates GPR81 in its physiological concentration range of 1–20 mm and suppresses lipolysis in mouse, rat, and human adipocytes as well as in differentiated 3T3-L1 cells. Adipocytes from GPR81-deficient mice lack an antilipolytic response to lactate but are responsive to other antilipolytic agents. Lactate specifically induces internalization of GPR81 after receptor activation. Site-directed mutagenesis of GPR81 coupled with homology modeling demonstrates that classically conserved key residues in the transmembrane binding domains are responsible for interacting with lactate. Our results indicate that lactate suppresses lipolysis in adipose tissue through a direct activation of GPR81. GPR81 may thus be an attractive target for the treatment of dyslipidemia and other metabolic disorders. Lactic acid is a well known metabolic by-product of intense exercise, particularly under anaerobic conditions. Lactate is also a key source of energy and an important metabolic substrate, and it has also been hypothesized to be a signaling molecule directing metabolic activity. Here we show that GPR81, an orphan G-protein-coupled receptor highly expressed in fat, is in fact a sensor for lactate. Lactate activates GPR81 in its physiological concentration range of 1–20 mm and suppresses lipolysis in mouse, rat, and human adipocytes as well as in differentiated 3T3-L1 cells. Adipocytes from GPR81-deficient mice lack an antilipolytic response to lactate but are responsive to other antilipolytic agents. Lactate specifically induces internalization of GPR81 after receptor activation. Site-directed mutagenesis of GPR81 coupled with homology modeling demonstrates that classically conserved key residues in the transmembrane binding domains are responsible for interacting with lactate. Our results indicate that lactate suppresses lipolysis in adipose tissue through a direct activation of GPR81. GPR81 may thus be an attractive target for the treatment of dyslipidemia and other metabolic disorders. GPR81 (1Lee D.K. Nguyen T. Lynch K.R. Cheng R. Vanti W.B. Arkhitko O. Lewis T. Evans J.F. George S.R. O'Dowd B.F. Gene (Amst.).. 2001; 275: 83-91Google Scholar) is an orphan G-protein-coupled receptor that is highly homologous to GPR109a and GPR109b. GPR109a and GPR109b were recently identified as receptors for niacin (also known as nicotinic acid) (2Tunaru S. Kero J. Schaub A. Wufka C. Blaukat A. Pfeffer K. Offermanns S. Nat. Med... 2003; 9: 352-355Google Scholar, 3Wise A. Foord S.M. Fraser N.J. Barnes A.A. Elshourbagy N. Eilert M. Ignar D.M. Murdock P.R. Steplewski K. Green A. Brown A.J. Dowell S.J. Szekeres P.G. Hassall D.G. Marshall F.H. Wilson S. Pike N.B. J. Biol. Chem... 2003; 278: 9869-9874Google Scholar) and subsequently characterized as receptors for the endogenous ketone body β-hydroxybutyrate (4Taggart A.K. Kero J. Gan X. Cai T.Q. Cheng K. Ippolito M. Ren N. Kaplan R. Wu K. Wu T.J. Jin L. Liaw C. Chen R. Richman J. Connolly D. Offermanns S. Wright S.D. Waters M.G. J. Biol. Chem... 2005; 280: 26649-26652Google Scholar). Niacin has been used clinically for a half-century as an effective treatment for dyslipidemia (5Garg A. Grundy S.M. J. Am. Med. Assoc... 1990; 264: 723-726Google Scholar); however, its utility is somewhat hampered by a target-related effect on dendritic Langerhans cells, which release prostaglandin D2 in response to GPR109a stimulation, resulting in a cutaneous flushing response (6Benyó Z. Gille A. Kero J. Csiky M. Suchánková M.C. Nüsing R.M. Moers A. Pfeffer K. Offermanns S. J. Clin. Invest... 2005; 115: 3634-3640Google Scholar, 7Benyó Z. Gille A. Bennett C.L. Clausen B.E. Offermanns S. Mol. Pharmacol... 2006; 70: 1844-1849Google Scholar, 8Cheng K. Wu T.J. Wu K.K. Sturino C. Metters K. Gottesdiener K. Wright S.D. Wang Z. O'Neill G. Lai E. Waters M.G. Proc. Natl. Acad. Sci. U. S. A... 2006; 103: 6682-6687Google Scholar). GPR81 is highly expressed in fat, similar to GPR109a, but is not expressed significantly in spleen; nor is it highly detected in any other tissue, and it has thus been hypothesized to be a potential target for the treatment of dyslipidemia that would be analogous to GPR109a/niacin but without the potential side effects (9Ge H. Weiszmann J. Reagan J.D. Gupte J. Baribault H. Gyuris T. Chen J.L. Tian H. Li Y. J. Lipid Res... 2008; 49: 797-803Google Scholar). In this report, we demonstrate the initial identification of the ligand activity for GPR81 from the rat tissue extracts, the purification of l-lactate from porcine brain as the source of the ligand activity, and the pharmacological characterization of l-lactate as a ligand for GPR81. In addition, we show that in its physiological concentration range, l-lactate effectively inhibits lipolysis in adipocytes from humans, mice, and rats. Adipocytes from GPR81-deficient mice lack responses to l-lactate, indicating that the antilipolytic effect of l-lactate is mediated by GPR81. Despite a long history of being considered as waste or a by-product of metabolism, l-lactate has maintained some attention as a potential signaling molecule (10Sola-Penna M. IUBMB Life.. 2008; 49: 797-803Google Scholar). As early as the 1960s, researchers have demonstrated significant effects of lactate on adipocytes (11Björntorp P. Acta Med. Scand... 1965; 178: 253-255Google Scholar); however, the mechanism by which this occurs has remained unknown. Our finding in this report provides a molecular basis for the ability of lactate to modulate lipolysis in adipocytes and establishes a new target opportunity for the treatment of dyslipidemia. Chemicals—All chemicals as for GPR81 were from of GPR81 from rat were in a of were for were and were a for through a and the and in a with and the in a in and for activation of used as C. E. S. Chen J. C. N. H. R. J. Biol. Chem... 2003; 278: in the human GPR81. of GPR81 from the GPR81 ligand from porcine of porcine brain were under similar as the rat the and the on a to for and the a from in a and in of to with to a on a Waters mm in and were and with being in a binding to the and in of of the to with and were on a were also for a of lactate from under the conditions. and of GPR81 from from humans, mice, and were in and as the were in a and the were to the were expressed in or expressed in as C. Chen J. C. S. D. P. Mol. Pharmacol... 2005; Scholar). from and were differentiated in tissue for adipocytes were lipolysis and with of lipolysis with or without human 3T3-L1 adipocytes were in a for and lactate a lactate lactate in rat were with and for were and with lactate were the lactate of as the for for GPR81, GPR81 from were as in a binding human GPR81 were with of l-lactate and with as C. E. S. Chen J. C. N. H. R. J. Biol. Chem... 2003; 278: Scholar). with GPR81 were with the receptor activity. human GPR81 by a the of the human GPR81 to the cells. after the were in and for and with a concentration of in the for in a tissue of on cells, were and with GPR81 the with l-lactate concentration to the receptor internalization for were by in without or not for to the and with the were with and with and by with a and under a of human GPR81 were with or without were with or with l-lactate for and were to to and with for without GPR81 were used as GPR81 with a the used as the for mutagenesis by a receptors were expressed and for responses to l-lactate in a binding of the GPR81 by of GPR81 mice were by transmembrane of GPR81 is by a were in tissue and differentiated as G. J. Weiszmann J. M. K. M. S. P. R.M. Chen J.L. Li Y. J. Biol. Chem... Scholar) for adipocytes and differentiated in in tissue were from 3T3-L1 and human adipocytes were with lipolysis and in lipolysis in a tissue or after and acid in the were a or a acid lipolysis in the or were from or mice with adipocytes were and lipolysis were as (9Ge H. Weiszmann J. Reagan J.D. Gupte J. Baribault H. Gyuris T. Chen J.L. Tian H. Li Y. J. Lipid Res... 2008; 49: 797-803Google Scholar). were and and acid release were a or a acid human and rat to a concentration of to to 3T3-L1 adipocytes and adipocytes from mice, lipolysis were without of for the were used to a C. C. S. Chen J. P. Wu J. D. D. J. T. R. E. R. J. Biol. Chem... 2005; 280: Scholar). for rat, and that were used for GPR81 were from from differentiated or 3T3-L1 were in by a of GPR81 in or GPR81 a of the GPR81 acid on an a concentration of with and from from or GPR81-deficient mice or 3T3-L1 and with mm mm were for and the were an in with on a with and with a GPR81 a of the of GPR81 acid which the a concentration of were for and with by the with a a concentration of in for with and as in of from GPR81 were in the as the for the of the of GPR81 from or cells. of GPR81 from were by the tissue or the for 3T3-L1 cells. and GPR81 the D. J.D. D.G. T.J. Res... Scholar). and on the of as T. G. S. K. 2003; Scholar). used as a and on the the residues of the of the were to the acid of the GPR81 the by acid side were and in a this the were and the transmembrane used to the with a the and of the were by energy of the of the ligand by energy of the and of as a for an to the endogenous for orphan GPR81, we from rat for ligand activity in cells. the results that tissue activity to binding in but not in with the in tissue of and the ligand for GPR81, porcine brain to rat brain of the porcine brain in mm and with a in a and a the of the with that its and are to that of l-lactate is by and not of the by and of l-lactate in the tissue that lactate in the tissue is with the GPR81 ligand of as a for that l-lactate is an ligand for GPR81, l-lactate for activation of GPR81 binding and direct of results show that l-lactate binding with an of mm and in with human GPR81 not in or the niacin receptors or In a under the of the GPR81, l-lactate inhibits it has effect in the binding and of that GPR81 is coupled to is by the that inhibits l-lactate binding and GPR81 also with cells, and treatment the may a of activation of the receptor or an endogenous lactate the l-lactate metabolic from and for from of to human for long long in a new l-lactate the internalization of GPR81, we a with a to the of GPR81. to l-lactate as by stimulation, and the from the receptor the of GPR81, were with by the the results show that detected in the of detected from receptor GPR81 were with by l-lactate were and and the of receptors were detected with results show that l-lactate GPR81 in the cells. with in cells, in indicating that l-lactate GPR81 this in a we the with to the a in the of Our results show that in without l-lactate treatment the in in and is to indicating that are l-lactate also GPR81 from the and rat GPR81 were with the human GPR81, and rat GPR81 are the an the the to the human GPR81 is conserved in the and rat GPR81 for and rat GPR81 were with with the and the to the in the human GPR81 the for the and not the were used for GPR81 from and GPR81 from are highly conserved for GPR81 from are in characterization of GPR81 from demonstrated that to l-lactate through of in indicating of the in a of as for GPR81 Our results show that and are also for GPR81. In and are for GPR81, and are not to GPR109a or GPR109b In addition, we that and were of GPR81 as and of as for human for of have been for but the for are not the for not responses by to are as the of the response by have been for but the for are not the for not responses by to are as the of the response by have been for but the for are not the for not responses by to are as the of the response by have been for but the for are not the for not responses by to are as the of the response by have been for but the for are not the for not responses by to are as the of the response by activity. activity for to Niacin with the concentration of not activity. activity for to Niacin with the concentration of activity. activity for to Niacin with the concentration of activity. activity for to Niacin with the concentration of activity. activity for to Niacin with the concentration of activity. activity for to Niacin with the concentration of activity. activity for to Niacin with the concentration of activity. activity for to Niacin with the concentration of activity. activity for to Niacin with the concentration of have been for but the for are not the for not responses by to are as the of the response by activity. activity for to Niacin with the concentration of not in a new of have demonstrated that an in the transmembrane of the receptor and the receptor is in the of the of acid and of this the receptor activity in response to acid Wang Z. J. Chen J.L. Tian H. L. Scholar). of a acid acid receptor J. N. Wu X. G. Reagan J. Tian H. L. J. Biol. Chem... 2006; receptor T. Y. E. A. K. N. N. S. T. T. J. Biol. Chem... and niacin receptors GPR109a and that a highly conserved in that this in receptors be in receptor ligand through the with the of acid this of this in GPR109a the receptor activity in response to niacin S. J. Kero J. G. Offermanns S. Mol. Pharmacol... 2005; Scholar). S. J. Kero J. G. Offermanns S. Mol. Pharmacol... 2005; Scholar) demonstrated that the of GPR109a is also important for its with nicotinic and are in the binding of and molecule S. J. Kero J. G. Offermanns S. Mol. Pharmacol... 2005; Scholar, A.J. A.J. P. D. J. Biol. Chem... Scholar, T. R. C. J. J. Biol. Chem... Scholar). GPR81 to niacin receptors GPR109a and GPR109b homology and the acid that GPR81 and GPR109a have similar fact that β-hydroxybutyrate is an for GPR109a and to the as niacin (4Taggart A.K. Kero J. Gan X. Cai T.Q. Cheng K. Ippolito M. Ren N. Kaplan R. Wu K. Wu T.J. Jin L. Liaw C. Chen R. Richman J. Connolly D. Offermanns S. Wright S.D. Waters M.G. J. Biol. Chem... 2005; 280: 26649-26652Google the GPR81 as l-lactate and are similar to β-hydroxybutyrate that l-lactate to GPR81 in a similar as β-hydroxybutyrate to homology modeling of human GPR81 a l-lactate with GPR81 in the molecule binding similar to has been for of l-lactate with of in the to the of to the highly conserved in to of in and in also in the ligand and this we in the human GPR81, and results that of and the response of GPR81 to l-lactate of the GPR81 are with that of the receptor of a of the potential of l-lactate as an endogenous ligand for GPR81, we the tissue of GPR81. from rat, or as well as 3T3-L1 were for GPR81 by for rat, or GPR81, As for rat, or were used to the from the for GPR81 rat, or and rat, or were and used as for of GPR81 to the of results show that in humans, GPR81 is expressed in fat, with in is also in and other in GPR81 is highly expressed in and and in in brain and other similar of GPR81 in In addition, GPR81 is highly expressed in differentiated 3T3-L1 adipocytes is with from other A. Foord S.M. Fraser N.J. Barnes A.A. Elshourbagy N. Eilert M. Ignar D.M. Murdock P.R. Steplewski K. Green A. Brown A.J. Dowell S.J. Szekeres P.G. Hassall D.G. Marshall F.H. Wilson S. Pike N.B. J. Biol. Chem... 2003; 278: 9869-9874Google Scholar, H. Weiszmann J. Reagan J.D. Gupte J. Baribault H. Gyuris T. Chen J.L. Tian H. Li Y. J. Lipid Res... 2008; 49: 797-803Google Scholar). the GPR81 in we GPR81 from or of by GPR81 that by the GPR81 and the from to and which it to and the endogenous GPR81 expressed with the demonstrated in GPR81 but a similar of on tissue from of the and GPR81-deficient mice not and the endogenous GPR81 the and of the we a a GPR81 as the and a GPR81 as the of the the for GPR81. Our results that GPR81 detected the in and with in of GPR81 were detected in the and the In addition, we also demonstrated that differentiated 3T3-L1 also GPR81 detected from any from the GPR81-deficient mice not of that the of GPR81 is in and adipocytes and that is a and to the niacin receptor GPR109a, we l-lactate, through GPR81, also modulate lipolysis in is and in adipocytes is by as and that in adipocytes In activation of the receptor by induces and the or activation of coupled receptors inhibits Our results show that similar to niacin and H. Li X. Weiszmann J. Wang P. Baribault H. Chen J.L. Tian H. Li Y. 2008; l-lactate also inhibits and acid release in differentiated 3T3-L1 and l-lactate also of lipolysis in human adipocytes differentiated in and similar results have been in adipocytes from rat and indicate that l-lactate is a of lipolysis in adipocytes and that l-lactate its antilipolytic through GPR81. that GPR81 is in lipolysis and the l-lactate and GPR81 in we a GPR81 from mice were and and from adipocytes from the of mice were from the by with and used for the of and release as a of results that l-lactate and release in adipocytes from the mice and In the and also In adipocytes from mice were to of l-lactate but responsive to and niacin and similar results were in and adipocytes from the mice not we that the of niacin in the adipocytes is have that niacin is not of GPR81. GPR109a is the of the human is in mice or its the of the GPR81 we some of the from that the of is which is by direct of the In we also the GPR81 in and mice and that the GPR81 is mice, GPR81 is and the GPR81 is of the GPR81 of the GPR81 in the mice, the of the of the GPR81 and the of that with the and a of the in the that it is expressed in and not of the and mice, and we have not a significant the and also demonstrated that as well as which are for GPR81, lipolysis in adipocytes from mice, this effect in the that and may lipolysis through however, we have also that is of but not or and not finding in to the antilipolytic effects of and are however, we a similar to that for from GPR81-deficient mice lack antilipolytic response to inhibits and acid release in adipocytes from mice but not in were to the adipocytes to and release were from the after of for and for and for the and for release from the were as and the results from other are as a of the in and are of niacin receptor and in GPR81-deficient of and l-lactate receptor the from the mice and GPR81 and GPR81 that the of and GPR81 were results from mice and mice are were to the of the and the GPR81 and the of GPR81 in of in the from the and from mice and mice were for by used as the is to the of have that l-lactate is of a known orphan GPR81. GPR81 is highly expressed in differentiated 3T3-L1 adipocytes and expressed in adipose inhibits and release in differentiated 3T3-L1 adipocytes and adipocytes from and in is well that l-lactate is intense exercise, and it demonstrated that lactate suppresses lipolysis in S.R. M. Scholar) hypothesized that lactate intense is a direct to to lipolysis a other are to the release of physiological for this is to the of have a of In of this and J. J. Med... Scholar) demonstrated that a in lactate is highly with the in GPR81 to lactate in the physiological range intense In and J. Lipid Res... Scholar) that mm lactate to in rat but effect on E. A. R. S. D. Am. J. Scholar) the of lactate to in human but significant effect on of used of lactate the of the that we have for GPR81. G. M. G. G. M. S. R. Res... Scholar) in that lactate but not lipolysis and that this effect significant lactate not in the In to exercise, l-lactate be by particularly fat, after a metabolic E. P. U. J. Am. J. 1990; Scholar, M. J. Scholar). In the of and also release l-lactate M. J. Scholar). demonstrated that in 3T3-L1 and are release lactate l-lactate may be as an ligand for signaling the to is GPR81 to be the receptor responsible for to endogenous in lactate from intense or to a metabolic GPR81 may target in the of metabolic for the treatment of dyslipidemia and other metabolic disorders. an for is of to that the for l-lactate to GPR81 is with with G-protein-coupled which are in the is l-lactate be a physiological ligand for GPR81. be an endogenous physiological ligand for a the ligand to be the of its receptor is and effective concentration In addition, to be a of receptor the of an endogenous physiological ligand for its receptor be in the range of the ligand concentration under physiological conditions. the concentration of the ligand be by its receptor and by receptor activation. In humans, l-lactate in the are in the mm range, l-lactate after intense anaerobic of mm Y. Y. A. M. T. J. Y. S. H. J. Scholar, J. Scholar, T. Y. K. T. M. J. Scholar, S. C. J. Scholar). after a metabolic as a adipocytes l-lactate as which the lactate concentration in M. J. Scholar). the GPR81 of mm in the of the physiological range of the endogenous the other that for GPR81, were l-lactate, and of are known to in that would be to for GPR81. l-lactate the attractive as the endogenous ligand for GPR81. by which to the of a finding is to it is l-lactate is a and in we hypothesized that lactate is as a physiological ligand for GPR81 as a signaling the also be fact that GPR81 from mice, and to l-lactate the physiological l-lactate concentration that the of the GPR81 is conserved and results that l-lactate is the endogenous physiological ligand for GPR81. As we the antilipolytic response to lactate in adipocytes from GPR81-deficient it that adipocytes from GPR81-deficient mice would demonstrate a response to GPR81 and are expressed on adipocytes and are highly to is that the receptors may have direct and the niacin effect in the mice that the niacin receptor is on GPR81 In lactate not GPR109a and niacin not GPR81, and of the receptors not not direct for a is and thus to niacin is effective in the mice, the of the an of the of of l-lactate to GPR81, which is for with the activity of in GPR81, to as to l-lactate is an to modulate the activity of GPR81. specifically this we to we mutagenesis and modeling to a ligand binding for lactate in the to lactate of receptor which is a with direct receptor activation and not the binding of l-lactate to GPR81, the GPR81 and GPR109a as well as that and β-hydroxybutyrate that and GPR81 in a similar to modeling that l-lactate with the in and in and in which would be analogous to to is by mutagenesis of any of key residues in the transmembrane significantly the ability of lactate to GPR81. results that l-lactate to GPR81 in a molecule binding of as a and a of of which is the of receptor internalization be a of to a receptor with a receptor the internalization of the receptor be an that is with the cells. this we were to demonstrate that l-lactate the internalization of GPR81. with ligand binding modeling and the internalization provides that lactate is in fact as a direct for GPR81 as an is also by the that in lactate is of in the of other GPR81 for of the in of is a known of and is in of its known mechanism of of have been used clinically to J. Clin. Pharmacol... 2003; Scholar, Barnes C. P.R. M. E. 2006; Scholar, P.R. E. 2008; Scholar) lactate and it is also in for the treatment of S. J. A. C. R. L. S. G. K. Scholar, M. 2008; Scholar, S. K. Y. S. C. 2008; Scholar). In addition, has been to in J. Scholar) in the J. Scholar) and J. Clin. Invest... Scholar) after and to and concentration in the after R. A. L. 2005; which is similar to that of l-lactate R. A. L. 2005; Scholar). is important to that the concentration for is in to or used in the which that the be to GPR81. is that some of the effects of be to the activation of GPR81. GPR81 the is a that has and effects in the brain and the M. Scholar). of in the brain and In addition, the release of endogenous and has been used as an for and for A. J. 2005; and Scholar) and is as for the treatment of with In the as a and to its and as the in for and in as a by the and mechanism of by unknown. is for an with the acid and binding for in the brain to that has pharmacological molecular for receptor activity has been finding that l-lactate and other GPR81 to and the GPR109a β-hydroxybutyrate are also of GPR81. we that β-hydroxybutyrate is GPR81, is a for human GPR81 with that also activates and rat with and of in human have been in the 1–20 range C. A. J. 2006; Scholar). after of of in a of mm Lai A. J. Clin. Pharmacol... Scholar, Pharmacol... Scholar). Scholar) of and from to it is that endogenous the to GPR81. it is to that may be of GPR81 the concentration would be In we identified l-lactate as an endogenous ligand for GPR81 and a of lipolysis in adipocytes through GPR81. with recently identified ligand receptor as A.J. S.M. Barnes A.A. Eilert L. D. Fraser N.J. Pike N.B. Steplewski Murdock P.R. Marshall F.H. Szekeres P.G. Wilson S. Ignar D.M. Foord S.M. A. Dowell S.J. J. Biol. Chem... 2003; 278: and A.J. S.M. Barnes A.A. Eilert L. D. Fraser N.J. Pike N.B. Steplewski Murdock P.R. Marshall F.H. Szekeres P.G. Wilson S. Ignar D.M. Foord S.M. A. Dowell S.J. J. Biol. Chem... 2003; 278: to a similar on adipocytes through of resulting in of lipolysis in response to metabolic other recently identified as and demonstrate that that have been of as or metabolic are directing through Our identification of lactate as an endogenous ligand for GPR81 of the in to of the of lactate in signaling to that has been the it provides an attractive target for the treatment of dyslipidemia. P. and for the of the also for in the characterization of as a ligand for GPR81. with of Lactate in Lipid of the mechanism the of lactate in by that lactate suppresses lipolysis in adipose tissue through a direct activation of GPR81. the that lactate suppresses lipolysis be of have that a or in as well as in adipose tissue, which energy as the of and of to of Lactate in Lipid of is a response to the by