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(1) 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) (2, 3) and subsequently characterized as receptors for the endogenous ketone body ␤-hydroxybutyrate (4). Niacin has been used clinically for a half-century as an effective treatment for dyslipidemia (5); 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 (6 -8). 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 (9).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 (10). As early as the 1960s, researchers have demonstrated significant effects of lactate on adipocytes (11); however, the mechanism by which this occurs has remained unknown. Our...

Both relaxin-3 and its receptor (GPCR135) are expressed predominantly in brain regions known to play important roles in processing sensory signals. Recent studies have shown that relaxin-3 is involved in the regulation of stress and feeding behaviors. The mechanisms underlying the involvement of relaxin-3/GPCR135 in the regulation of stress, feeding, and other potential functions remain to be studied. Because relaxin-3 also activates the relaxin receptor (LGR7), which is also expressed in the brain, selective GPCR135 agonists and antagonists are crucial to the study of the physiological functions of relaxin-3 and GPCR135 in vivo. Previously, we reported the creation of a selective GPCR135 agonist (a chimeric relaxin-3/ INSL5 peptide designated R3/I5). In this report, we describe the creation of a high affinity antagonist for GPCR135 and GPCR142 over LGR7. This GPCR135 antagonist, R3(B⌬23-27)R/I5, consists of the relaxin-3 B-chain with a replacement of Gly 23 to Arg, a truncation at the C terminus (Gly 24 -Trp 27 deleted), and the A-chain of INSL5. In vitro pharmacological studies showed that R3(B⌬23-27)R/I5 binds to human GPCR135 (IC 50 ‫؍‬ 0.67 nM) and GPCR142 (IC 50 ‫؍‬ 2.29 nM) with high affinity and is a potent functional GPCR135 antagonist (pA2 ‫؍‬ 9.15) but is not a human LGR7 ligand. Furthermore, R3(B⌬23-27)R/I5 had a similar binding profile at the rat GPCR135 receptor (IC 50 ‫؍‬ 0.25 nM, pA2 ‫؍‬ 9.6) and lacked affinity for the rat LGR7 receptor. When administered to rats intracerebroventricularly, R3(B⌬23-27)R/I5 blocked food intake induced by the GPCR135 selective agonist R3/I5. Thus, R3(B⌬23-27)R/I5 should prove a useful tool for the further delineation of the functions of the relaxin-3/GPCR135 system.Relaxin-3 (R3) 2 (1) is the most recently identified member of the insulin-relaxin peptide family. Both relaxin-3 and its receptor, GPCR135 (2), are predominantly expressed in the brain (2, 3). GPCR135, an inhibitory receptor, is expressed in many regions of the rodent brain such as the superior colliculus, sensory cortex, olfactory bulb, amygdale, and paraventricular nucleus (4 -6), suggesting potential physiological involvement in neuroendocrine and sensory processing. Recent in vivo studies have further shown that relaxin-3 and GPCR135 are involved in the stress response and in regulation of feeding. More specifically, water restraint stress or intracerebroventricular corticotrophin-releasing factor (CRF) infusion induces relaxin-3 expression in cells of the nucleus incertus, a region where CRF receptor-1 is also expressed (7), and central administration of relaxin-3 induces feeding in rat (8, 9). These findings suggest that GPCR135 and relaxin-3 may be involved in multiple physiological processes, some of which might be as yet unknown.In vitro relaxin-3 activates GPCR135 (2), GPCR142 (10), and LGR7 (11) receptors. The predominant brain expression of both relaxin-3 and GPCR135, coupled with their high affinity interaction, strongly suggests that relaxin-3 is the endogenous ligand for GPCR135 (2). Phar...

Insulin-like peptide 5 (INSL5) is a peptide that belongs to the relaxin/insulin family, and its receptor has not been identified. In this report, we demonstrate that INSL5 is a specific agonist for GPCR142. Human INSL5 displaces the binding of 125 I-relaxin-3 to GPCR142 with a high affinity (K i ‫؍‬ 1.5 nM). In a saturation binding assay, 125 The relaxin/insulin family peptides include insulin (1), IGF1 1 (2), IGF2 (3), relaxin (4, 5), INSL3 (6), INSL4 (7), INSL5 (8), INSL6 (9), and relaxin-3/INSL7 (10). Except for IGF1 and IGF2, which are single chain peptides, each member of the family consists of two peptide subunits (an A-chain and a B-chain) that are linked by three disulfide bonds (4 -14). Insulin, IGF1, and IGF2 are known to be involved in the regulation of glucose metabolism (15) and signal through tyrosine kinase/ growth factor receptors, which are single transmembrane receptors (16, 17). Relaxin plays multifunctional roles including uterus relaxation, reproductive tissue growth, and collagen remodeling in females (18). In addition, relaxin has been reported to play important roles in nonreproductive functions including wound healing, cardiac protection, and allergic responses (19). The receptor for relaxin has been identified recently as a leucine-rich repeat containing the G-protein-coupled receptor (LGR) LGR7 (20). Although relaxin also activates LGR8 in vitro (20), recent studies show that LGR8 is likely the endogenous receptor for INSL3 and is involved in testis descent (21,22). To date, the receptors for INSL4, INSL5, and INSL6 have not been identified. Relaxin-3 (also known as INSL7), the most recently identified member of the family, was reported to be an additional ligand for LGR7 (23). We recently identified relaxin-3 as a ligand for two orphan G-protein-coupled receptors GPCR135 (14) and GPCR142 (24). The predominant brain expression for both 14,25, 26) and GPCR135 (14,26), coupled with their high affinity interaction, strongly suggests that relaxin-3 is the endogenous ligand for GPCR135. The tissue expression pattern of GPCR142 (also known as GPR100), which is primarily in peripheral tissues (24), is drastically different from that of relaxin-3, suggesting that GPCR142 may have an endogenous ligand other than relaxin-3. Furthermore, despite the high conservation of relaxin-3 in different species, GPCR142 is less conserved in the mouse and is a pseudogene in the rat (26), suggesting that GPCR142 may have a diminished role in rodents and may function as a receptor for a different ligand (other than relaxin-3) in other mammals. Sequence analysis among insulin/relaxin family members indicates that INSL5 shares high homology to relaxin-3 (Fig. 1A), suggesting that it may be an additional ligand for GPCR135, GPCR142, LGR7, or LGR8. In this report, we demonstrate that INSL5 is an agonist for GPCR142 but not for GPCR135, LGR7, or LGR8.

We describe a sensor capable of detecting single DNA molecules. The sensor is based on a single nanopore prepared in a polymer film by a latent ion track-etching technique. For this purpose, a polymer foil was penetrated by a single heavy ion of total kinetic energy of 2.2 GeV, followed by preferential etching of the ion track. DNA molecules were detected as they blocked current flow during translocation through the nanopore, driven by an electric field. The nanopores are highly stable and their dimensions are adjustable by controlling etching conditions. For detecting DNA, conical nanopores with opening diameters of 2 μm and 4 nm were used. The nanopore sensor was able to discriminate between DNA fragments of different lengths.