Calcium mobilization FLIPR assay CHO K cells expressing an N

Calcium mobilization (FLIPR) assay: CHO-K1 cells expressing an NFAT-β-lactamase reporter and the promiscuous Gα-subunit Gqi5 were stably transfected with either empty vector (pCDNA3.1, Invitrogen) or vector expressing HA-GPR81, GPR109A or GPR109B. Cells were seeded at 10,000cells/well in 384-well culture plates and grown overnight at 37°C, 5% CO2 in Dulbecco’s modified Eagle’s medium containing 10% FBS, 2mM l-glutamine, 10mM HEPES, pH 7.4, 0.1mM MEM non-essential [Ala92]-p16 (84-103) solution, 1mM sodium pyruvate, 0.6mg/ml hygromycin B, 0.5mg/ml zeocin and 1mg/ml geneticin (BD Biosciences). Cells were washed four times with Hank’s balanced salt solution containing 10mM 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-12K medium containing 10% FBS, 2mM l-glutamine, 1mM sodium pyruvate, 400μg/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 100μM of RO-20-1724, a non-specific phosphodiesterase inhibitor. Twenty-thousands of cells/well were plated into 384-well plates and treated with sodium lactate and 1μM of forskolin. After 45min 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). Briefly, 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 β-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 confirmed 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 12h 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 Hank’s balanced salt solution buffer (HBSS, Invitrogen). Adipose tissues were sliced into pieces (∼20–40mg/piece). Fat pads (∼150mg/tube) were incubated in 1ml HBSS buffer with 1% FFA-free bovine serum albumin (Serologicals) with test compounds at 37°C for 60min. 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.
Results Binding of agonists to GPCRs induces guanine nucleotide exchange on their associated Gα-subunits. Thus, to explore the potential activity of lactate we employed a S-GTPγS 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 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 30mM [18], [19], we assayed sodium l-lactate at 10mM. Sodium l-lactate significantly stimulated S-GTPγS binding with HA-GPR81-bearing membranes but not with GPR109A- or GPR109B-expressing membranes (Fig. 1A). Additional studies showed that the effect of sodium L-lactate is dose-dependent with an EC50 of ∼1.30±0.32mM (Fig. 1B). A similar effect was observed with d-lactate, however, the maximal stimulation by d-lactate was significantly less than that by l-lactate suggesting that d-lactate may serve as a partial agonist. The EC50 for d-lactate was 3.03±0.21mM. Since normally only the l- but not the d-isomer of lactate has biological significance 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±1.2mM. However, since the maximum concentration of propionic acid not bound to albumin in plasma is estimated to be <10μM [21], this activity is unlikely to be physiologically relevant.