Biochemical and Biophysical Research Communications 377 (2008) 987991

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Biochemical and Biophysical Research Communications

Role of GPR81 in lactate-mediated reduction of adipose lipolysis

Department of Cardiovascular Diseases, Merck Research Laboratories, RY80T-A100, 126 East Lincoln Avenue, Rahway, NJ 07065, USA Arena Pharmaceuticals Inc., San Diego, California 92121, USA c Deltagen, Inc. San Carlos, California 94070, USA
b

a r t i c l e

i n f o

a b s t r a c t
Heavy exercise or oxygen decit often links with higher levels of arterial lactate and lower levels of plasma free fatty acids (FFA). Treatment with lactate reduces circulating levels of FFA in vivo and lipolysis in adipose tissues in vitro. However, the underlying mechanism has remained unclear. Here we employ pharmacological and genetic approaches to show that GPR81, an orphan G-protein-coupled receptor with relatively restricted expression in the adipose tissues, functions as a receptor for lactate and can mediate an anti-lipolytic effect of lactate. GPR81 may thus function as a sensor of lactate that can modulate the FFA pool under exercise or conditions of oxygen decit. 2008 Elsevier Inc. All rights reserved.

Article history: Received 16 October 2008 Available online 24 October 2008

Keywords: Lactic acid Orphan GPCR Exercise Oxygen decit Free fatty acids

GPR81 is an orphan G-protein-coupled receptor of unknown function [10]. Expression of GPR81 is highly restricted in the adipose tissues [9], suggesting that this orphan GPCR may play a role in energy metabolism. GPR81 is most homologous (52% amino acid sequence identity in humans) to GPR109A, the receptor for the lipid lowering agent nicotinic acid [9,11,12]. A well-studied pharmacologic effect of nicotinic acid, a small carboxylic acid, is its ability to inhibit triglyceride (TG) lipolysis in adipose tissue, which results in reduced levels of plasma FFA [11,13]. Another small carboxylic acid that can reduce FFA is the ketone body b-hydroxybutyrate [14,15], and we have recently shown that this metabolite is an endogenous ligand for GPR109A [16]. b-Hydroxybutyrate is signicantly increased during starvation [17], and we postulate that it acts in a negative feed-back loop to control lipolysis in adipose tissue via activation of GPR109A, thereby enhancing the efcient use of adipose lipid stores during starvation [14]. Lactic acid (lactate) differs from b-hydroxybutyrate by being one methylene group smaller. Like b-hydroxybutyrate [14,15], infusion of lactate reduces lipolysis in vivo [46] as does in vitro treatment of adipose tissue [7,8]. The levels of lactate in plasma increase signicantly following physical exercise or as a consequence of oxygen decit (may reach up to 30 mM) [1820], and this lactate elevations often links with a reduction of plasma FFA [13]. We thus investigated whether lactate might be an endogenous ligand

for GPR81 and whether GPR81 plays a role in lactate-induced inhibition of lipolysis. Experimental procedures GPR81-, GPR109A- and GPR109B stable cell lines. For the generation of stable cell lines, 5 106 Chinese Hamster Ovary (CHO)-K1 cells were transfected with 12 lg plasmid DNA pHM6 (Invitrogen, Carlsbad, CA) containing GPR81 or plasmid DNA pCDNA3.1 (Invitrogen) containing GPR109A or GPR109B expressed from the cytomegalovirus promoter. Two days after transfection, the growth medium was supplemented with 400 lg/ml G418 to select for antibiotic resistant cells. Clonal CHO-K1 cell lines that stably express GPR81 were selected based on immunostaining with antiHA antibodies. Clonal CHO-K1 cell lines that stably express GPR109A and GPR109B were selected based on the ability of nicotinic acid (GPR109A-specic agonist) or 1-isopropylbenzotriazole5-carboxylic acid (GPR109B-specic agonist, [26]) to inhibit forskolin-induced cAMP production, as previously described [16]. 35 S-GTPcS binding assay: Membranes prepared from CHO-K1 cells stably expressing GPR109A, GPR109B, GPR81 or vector control (7 lg/assay) were diluted in assay buffer (100 mM HEPES, 100 mM NaCl and 10 mM MgCl2, pH 7.4) in Wallac Scintistrip plates and pre-incubated with test compounds diluted in assay buffer containing 40 lM GDP (nal [GDP] was 10 lM) for 10 min before addition of 35S-GTPcS to 0.3 nM. Binding was allowed to proceed for one hour before centrifuging the plates at 2500 rpm for 20 min at room temperature and subsequent

* Corresponding author. Fax: +1 732 594 2510. E-mail address: tianquan_cai@merck.com (T.-Q. Cai). 0006-291X/$ - see front matter 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.10.088

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counting in a TopCount scintillation counter. Non-linear regression analysis of the binding curves was performed in GraphPad Prism. Calcium mobilization (FLIPR) assay: CHO-K1 cells expressing an NFAT-b-lactamase reporter and the promiscuous Ga-subunit Gqi5 were stably transfected with either empty vector (pCDNA3.1, Invitrogen) or vector expressing HA-GPR81, GPR109A or GPR109B. Cells were seeded at 10,000 cells/well in 384-well culture plates and grown overnight at 37 C, 5% CO2 in Dulbeccos modied Eagles medium containing 10% FBS, 2 mM L-glutamine, 10 mM HEPES, pH 7.4, 0.1 mM MEM non-essential amino acids solution, 1 mM sodium pyruvate, 0.6 mg/ml hygromycin B, 0.5 mg/ml zeocin and 1 mg/ml geneticin (BD Biosciences). Cells were washed four times with Hanks balanced salt solution containing 10 mM HEPES, pH 7.4 and loaded with calcium-sensitive dye by incubating with an equal volume of Molecular Devices Calcium Assay Kit Loading Buffer at 37 C for one hour. Calcium response in the FLIPR assay was measured according to the directions from Molecular Devices. cAMP assay: CHO-K1 cells stably expressing HA-GPR81, GPR109A or GPR109B were cultured at 37 C, 5% CO2 in F-12 K medium containing 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 400 lg/ml of G418. Cells were harvested using cell dissociation buffer (Invitrogen), washed twice with PBS, and resuspended in PBS buffer containing 0.1% BSA and 100 lM of RO-20-1724, a non-specic phosphodiesterase inhibitor. Twentythousands of cells/well were plated into 384-well plates and treated with sodium lactate and 1 lM of forskolin. After 45 min incubation at room temperature, levels of cAMP were determined using assay kits obtained from DiscoveryX (Fremont, CA). Generation of GPR81/ mice: GPR81/ mice were obtained from Deltagen, Inc. (San Carlos, CA). Briey, to generate GPR81 mutant mice, a section of the GPR81 gene encoding GPR81 was replaced by homologous recombination in embryonic stem cells with a cassette containing the neomycin resistance and b-galactosidase genes. Male chimeric mice were generated by injection of the targeted ES cells into C57Bl/6J blastocysts. Chimeric mice were bred with C57Bl/6J mice to produce F1 heterozygotes. Germline transmission was conrmed by PCR analysis. Mice homozygous for the targeted allele of GPR81 are viable without discernable phenotype. Mice backcrossed six times with C57BL/6J were used for the study. Mice were maintained in a temperature-controlled (23 C) barrier facility with a 12 h light/dark cycle and had ad libitum access to water and regular rodent chow diet. Mouse epididymal fat pad lipolysis assay: Epididymal fat pads from male GPR81 null mice or the wild type littermates (22 weeks of age, n = 4) were removed and placed in the Hanks balanced salt solution buffer (HBSS, Invitrogen). Adipose tissues were sliced into pieces (2040 mg/piece). Fat pads (150 mg/tube) were incubated in 1 ml HBSS buffer with 1% FFA-free bovine serum albumin (Serologicals) with test compounds at 37 C for 60 min. After incubation, medium was removed and glycerol content in the medium (released from epididymal fat pads) was determined with the glycerol assay kit purchased from Sigma.

agonist of GPR109A and GPR109B [9], was active on membranes bearing either GPR109A or GPR109B, but not on HA-GPR81-bearing membranes (Fig. 1A). Since levels of L-lactate in plasma may reach up to 30 mM [18,19], we assayed sodium L-lactate at 10 mM. Sodium L-lactate signicantly stimulated 35S-GTPcS binding with HA-GPR81-bearing membranes but not with GPR109Aor GPR109B-expressing membranes (Fig. 1A). Additional studies showed that the effect of sodium L-lactate is dose-dependent with an EC50 of 1.30 0.32 mM (Fig. 1B). A similar effect was observed with D-lactate, however, the maximal stimulation by D-lactate was signicantly less than that by L-lactate suggesting that D-lactate may serve as a partial agonist. The EC50 for D-lactate was 3.03 0.21 mM. Since normally only the L- but not the D-isomer of lactate has biological signicance in vivo [6], it is unlikely that the D-lactate would act as a GPR81 agonist in vivo. Activation of HA-GPR81 by lactate was very selective as many other short chain fatty acids were inactive (see Table 1). HA-GPR81 was activated by sodium propionate with an EC50 of 2.9 1.2 mM. However, since the maximum concentration of propionic acid not bound to albumin in plasma is estimated to be <10 lM [21], this activity is unlikely to be physiologically relevant. Lactate-induced GPR81 activation was conrmed with a uorimetric imaging plate reader (FLIPR) assay, in which lactate induced a [Ca2+] ux in CHO cells stably expressing Gqi5 and HA-GPR81 but not vector control (Fig. 1C). The EC50 of lactate in the FLIPR assay was 4.3 0.73 mM, a value well within the physiological range observed in man following exercise [1820]. To determine if GPR81, like GPR109A and GPR109B, is Gi-coupled we employed a 35S-GTPcS exchange assay with membranes prepared from cells pretreated with or without pertussis toxin (PTX), an inhibitor of Gi signaling. Similar to nicotinic acid stimulation of GPR109A [9,1112], lactate-mediated activation of HAGPR81 was completely inhibited by the pretreatment of PTX (Fig. 2A) and is thus Gi-coupled. Consistent with this conclusion, we found lactate inhibited forskolin-stimulated cAMP production in CHO cells transfected with HA-GPR81 (IC50 = 2.15 0.26 mM (Fig. 2B) but not with GPR109A (data not shown). Lactate has previously been shown to reduce lipolysis in human and rat adipose tissues [7,8]. We extended this observation by showing that lactate can also suppress lipolysis in mouse fat pads in an ex vivo assay (Fig. 3A). Inhibition of both basal and, to a greater extent, isoproterenol-stimulated lipolysis was evident. Similar to an effect of nicotinic acid, lactate-mediated suppression of lipolysis were blocked by the pretreatment of mice with pertussis toxin (Fig. 3B), indicating a role of a Gi-coupled receptor in the process. The ability of lactate to suppress lipolysis in mouse fat pads allowed us to genetically test whether GPR81 was required for this process. To this end we employed fat pads from GPR81-decient mice and wild-type littermate controls. As expected, lactate suppressed lipolysis in the fat pads from wild-type control mice but failed to do so in the GPR81-decient fat pads (Fig. 3C), indicating a critical role of GPR81 in lactate-mediated suppression of adipose lipolysis. Discussion Our results clearly show that lactate can activate the adipose-localized GPCR GPR81 at concentrations well within the physiological range observed at states of relative oxygen decit such as exercise (may reach up to 30 mM, 1819). Specically, we show using a cell based cAMP assay or a [Ca2+] ux assay that the EC50 for lactate-mediated activation of GPR81 can be as low as 2.1 mM. Our demonstration that GPR81 is Gi coupled is consistent with the fact that infusion of lactate into humans can reduce plasma FFA. This is proposed to occur in a manner similar to the mechanism by which niacin lowers plasma FFAspecically, by

Results Binding of agonists to GPCRs induces guanine nucleotide exchange on their associated Ga-subunits. Thus, to explore the potential activity of lactate we employed a 35S-GTPcS binding assay with membranes prepared from cells transfected with a control vector or vectors that express HA-tagged GPR81 (HA-GPR81), or untagged GPR109A or GPR109B-a GPCR 95% identical at the amino acid with GPR109A [9]. Consistent with previous work [9,11,12], nicotinic acid stimulated guanine nucleotide exchange with membranes from cells expressing GPR109A but not with GPR109B- or HA-GPR81-expressing membranes, whereas acifran, a known dual

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A
Relative activity (%)

100 75 50

Nicotinic acid Acifran Sodium lactate

Table 1 Ligand-induced 35S-GTPcS binding with membranes from CHO cells expressing human GPR81. Data are shown as means SE of experiments repeated at least three times. Sodium (L)-lactate and sodium propionate, two compounds that stimulated the binding of GTPcS with membranes expressing GPR81 were inactive with membranes expressing GPR109A, GPR109B or vector control (data not shown). Compound EC50 (mM) 1.30 0.32 3.03 0.21 Inactive Inactive Inactive Inactive 2.90 1.20 Inactive Inactive Inactive Inactive Inactive Inactive

25 0 -25

GPR109A GPR109B

GPR81

3000

2500

Sodium (L)-lactate Sodium (D)-lactate Sodium (D)-b-hydroxybutyrate Lithium acetoacetate Acetone Sodium acetate Sodium propionate Sodium pyruvate Sodium butyrate Pentanoic acid Sodium hexanoate Sodium octanoate Sodium decanoate

CPM

2000

1500

8000

1000 6000

10-7 10-6 10-5 10-4 10-3 10-2 10-1

CPM

500

Compound (M)

4000

C
Ca++ Release

10000

2000

7500

0 0

10-5

10 -4

10-3

10-2

10-1

5000

Lactate (M)

B
2500

500000

400000 0 0 10
-5

10

-4

10

-3

10

-2

10

-1

10

Lactate (M)
Fig. 1. Lactate induces activation of GPR81. (A and B) Lactate stimulates 35S-GTPcS binding with membranes from cells transfected with GPR81. Ligand-induced 35SGTPcS binding was studied using membranes prepared from CHO-K1 cells expressing human GPR81, GPR109A or GPR109B as indicated. Binding of 35S-GTPcS was determined in the presence of (A) nicotinic acid (10 lM, lled black bars), acifran (100 lM, hatched bars) or sodium L-lactate (10 mM, gray bars), or (B) increasing concentrations of sodium L-lactate (j) or D-lactate (d). (C) Sodium 2+ L-lactate induces an increase of [Ca ] in CHO cells transfected with GPR81 and Gqi5 (N) but not with Gqi5 and vector control (d). All data are shown as the means SD of triplicates of a representative experiment. Three to four independent experiments were performed with similar results.

RLU

300000

200000

100000

0 0

10-5

10 -4

10-3

10-2

10-1

Lactate (M)
Fig. 2. GPR81 is a Gi-coupled receptor for lactate. (A) Sodium L-lactate stimulates 35 S-GTPcS binding in membranes prepared from untreated (N) or pertussis toxin (PTX, d) treated (500 ng/ml, overnight) CHO cells expressing HA-GPR81. (B) Sodium L-lactate reduces forskolin-stimulated (1 lM, 45 min) intracellular cAMP levels in CHO cells expressing HA-GPR81.

agonizing the highly related Gi-coupled GPCR GPR109A, reduction of intracellular cAMP and thus protein kinase A activity, and in turn the reduced phosphorylation of the lipid droplet coat protein perilipin and hormone sensitive lipase, which are key components of the TG hydrolytic system that generates FFA [11,13]. Glucose and FFA are considered the major metabolic fuels for energy needs. Under conditions of oxygen decit, the working muscles can not utilize FFA as a substrate. This shift in metabolism during oxygen decit often results in a drastic elevation of plasma levels of lactate [6]. Results from the current study suggest the fol-

lowing hypothesis. Under the state of oxygen decit, while many factors, such as decreased circulating insulin levels, increased sympathetic tone and circulating norepinephrine levels [22], are signaling for an increase of FFA, an elevation of plasma lactate serves as a signal to the adipose tissues to reduce lipolysis. GPR81 on adipocytes may function as such sensor. In addition to the production of lactate by muscle contraction during exercise [1820], recent studies show that adipose tissues

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A
Glycerol release ( moles/g tissue)

2.5

2.0

Non-stimulated Isoproterenol

different physiological circumstances: GPR109A playing a role in survival of starvation and GPR81 acting during conditions of high energy expenditure leading to oxygen decit. Acknowledgments

1.5

*
1.0

*
0.5

Control

Lactate (30mM)
Control mice PTX-treated mice

Glycerol ( moles/g tissue)

2.5 2.0 1.5 1.0 0.5 0.0 Vehicle

We thank Dr Kathleen Sullivan of Merck Research Laboratories for providing the CHO-K1 cells expressing an NFAT-b-lactamase reporter and the Ga subunit Gqi5, Dr. Chen Liaw (Arena Pharmaceutical), Ms. Rebecca Kaplan, Mr. Frank Xiaodong Gan for technical assistance, Dr. Andrew Howard for comments on the manuscript, and Drs. John S. Mudgett, Kimberly L. Folander and Robert J. Driscoll for coordinating the effort between Deltagen and Merck, and Dr. Daniel Connolly for comments and support. References
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Niacin (10 M) Lactate (30 mM)

C
Glycerol release ( moles/g tissue)

2.0

NaCl: 30mM Lactate: 30mM

1.0

0.5

0.0 GPR81+/+ GPR81-/-

Fig. 3. Lactate suppresses lipolysis in fat pads isolated from wild type but not GPR81 null mice. (A) Effect of sodium L-lactate on basal (non-stimulated, open bar) and 100 nM of isoproterenol-stimulated (hatched bar) lipolysis of epididymal fat pads isolated from wild type C57BL/6J mice. (B) Effect of pertussis toxin on nicotinic acid- and lactate-mediated suppression of fat pad lipolysis. Wild-type C57BL/6J mice were treated with (hatched bar) or without (open bar) 1 lg/mice of PTX via intraperitoneal injection. Three days after injection, epididymal fat pads were removed and fat pad lipolysis was done in the presence of nicotinic acid (10 lM) or sodium L-lactate (30 mM). C). Glycerol release from epididylmal fat pads isolated from wild type or GPR81 null mice in the presence of sodium chloride (30 mM) or sodium L-lactate (30 mM). Data are shown as the means SE of quadruplicates of a representative experiment. *p < 0.05.

may also produce signicant amounts of lactate [23]. The amount of lactate produced in adipocytes appears to correlate with cell size [24]. Lactate levels rise with obesity and decline with weight loss [23,25]. The results from the work presented here suggest that lactate produced in adipocytes could potentially function as an autocrine signal to regulate energy storage in adipose via activation of GPR81. Our ndings provide a molecular mechanism for the ability of lactate to suppress lipolysisagonism of the adipose GPCR GPR81. In addition, this nding, together with the identication of b-hydroxybutyrate as an endogenous ligand for GPR109A, leads to speculation that the GPR81/GPR109A family of receptors play a key roles in regulating appropriate utilization of adipose energy stores under vastly

T.-Q. Cai et al. / Biochemical and Biophysical Research Communications 377 (2008) 987991 [22] J.F. Horowitz, Fatty acid mobilization from adipose tissue during exercise, Trends Endocrinol. Metab. 14 (2003) 386392. [23] M. DiGirolamo, F.D. Newby, J. Lovejoy, Lactate production in adipose tissue: a regulated function with extra-adipose implications, FASEB J. 6 (1992) 24052412. [24] D.L. Crandall, S.K. Fried, A.A. Francendese, M. Nickel, M. DiGirolamo, Lactate release from isolated rat adipocytes: inuence of cell size, glucose concentration, insulin and epinephrine, Horm. Metab. Res. 15 (1983) 326329.

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[25] J. Lovejoy, B. Mellen, M. Digirolamo, Lactate generation following glucose ingestion: relation to obesity, carbohydrate tolerance and insulin sensitivity, Int. J. Obes. 14 (1990) 843855. [26] G. Semple, P.J. Skinner, M.C. Cherrier, P.J. Webb, C.R. Sage, S.Y. Tamura, R. Chen, J.G. Richman, D.T. Connolly, 1-Alkyl-benzotriazole-5-carboxylic acids are highly selective agonists of the human orphan G-protein-coupled receptor GPR109b, J Med. Chem. 49 (2006) 12271230.