(D)--Hydroxybutyrate Inhibits Adipocyte Lipolysis via the Nicotinic Acid Receptor PUMA-G* Received for publication, May 23, 2005 Published, JBC Papers in Press, June 1, 2005, DOI 10.1074/jbc.C500213200 Andrew K. P. Taggart‡§¶, Jukka Kero§, Xiaodong Gan‡, Tian-Quan Cai‡, Kang Cheng‡, Marc Ippolito‡, Ning Ren‡, Rebecca Kaplan‡, Kenneth Wu‡, Tsuei-Ju Wu‡, Lan Jin‡, Chen Liaw**, Ruoping Chen**, Jeremy Richman**, Daniel Connolly**, Stefan Offermanns, Samuel D. Wright‡, and M. Gerard Waters‡ From the ‡Division of Cardiovascular Diseases, Merck Research Laboratories, Rahway, New Jersey 07065, the Institute of Pharmacology, University of Heidelberg, 69120 Heidelberg, Germany, and **Arena Pharmaceuticals Inc., San Diego, California 92121 As a treatment for dyslipidemia, oral doses of 1–3 grams of nicotinic acid per day lower serum triglycer- ides, raise high density lipoprotein cholesterol, and re- duce mortality from coronary heart disease (Tavintha- ran, S., and Kashyap, M. L. (2001) Curr. Atheroscler. Rep. 3, 74 – 82). These benefits likely result from the ability of nicotinic acid to inhibit lipolysis in adipocytes and thereby reduce serum non-esterified fatty acid levels (Carlson, L. A. (1963) Acta Med. Scand. 173, 719 –722). In mice, nicotinic acid inhibits lipolysis via PUMA-G, a G i/o -coupled seven-transmembrane receptor expressed in adipocytes and activated macrophages (Tunaru, S., Kero, J., Schaub, A., Wufka, C., Blaukat, A., Pfeffer, K., and Offermanns, S. (2003) Nat. Med. 9, 352–355). The human ortholog HM74a is also a nicotinic acid receptor and likely has a similar role in anti-lipolysis. Endoge- nous levels of nicotinic acid are too low to significantly impact receptor activity, hence the natural ligands(s) of HM74a/PUMA-G remain to be elucidated. Here we show that the fatty acid-derived ketone body (D)--hydroxybu- tyrate ((D)--OHB) specifically activates PUMA-G/ HM74a at concentrations observed in serum during fast- ing. Like nicotinic acid, (D)--OHB inhibits mouse adipocyte lipolysis in a PUMA-G-dependent manner and is thus the first endogenous ligand described for this orphan receptor. These findings suggests a homeostatic mechanism for surviving starvation in which (D)--OHB negatively regulates its own production, thereby pre- venting ketoacidosis and promoting efficient use of fat stores. Ketone bodies (acetone, acetoacetate (AcAc), 1 and (D)-- OHB) are produced in the liver from acetyl-CoA derived from -oxidation of fatty acids (7). AcAc and (D)--OHB are small water-soluble carboxylic acids that are important energy sources for the brain and other tissues during prolonged fasting (10). In humans, the serum concentration of (D)--OHB is typ- ically 50 M after a meal, rises to 0.2– 0.4 mM after an overnight fast, reaches 1–2 mM after 2–3 days of fasting, and plateaus at 6–8 mM upon prolonged starvation (7–9, 11). -OHB infusion into rats (12), pancreatomized dogs (13), and humans (9, 14) lowers serum NEFAs in vivo, and -OHB in- hibits lipolysis in primary rat (15, 16) or bovine (17) adipocytes, whereas AcAc does not. The fact that these effects are similar to those of nicotinic acid and that both -OHB and nicotinic acid are small carboxylic acids led us to investigate whether ketone bodies are HM74a agonists. EXPERIMENTAL PROCEDURES Materials—With the exception of Acifran and lithium (DL)--OHB, all compounds tested were from Sigma. Acifran was synthesized by chemists at Arena Pharmaceuticals, and lithium (DL)--OHB was made by titrating free (DL)--OHB acid (Sigma) with LiOH by chemists at Merck. [ 35 S]GTPS (1160 Ci/mmol) was from Amersham Biosciences, and [5,6- 3 H]nicotinic acid (50 Ci/mmol) was from American Radiola- beled Chemical (St. Louis, MO). Molecular Cloning—HM74a and HM74 were cloned by PCR using human genomic DNA as a template and the following primers, GCTG- GAGCATTCACTAGGCGAG (sense for HM74a), AGATCCTGGTTCTT- GGTGACAATG (antisense for HM74a), GGAGAATTCACTAGGCGAG- GCGCTCCATC (sense for HM74), and GGAGGATCCAGGAAACCTT- AGGCCGAGTCC (antisense for HM74). PUMA-G was cloned using mouse genomic DNA as template, the sense primer AGATCCACTCA- TGAGCAAGTCAGACC, and the antisense primer CCTTCTTGTCA- TAGTAACTTAACGAG. For the generation of stable cell lines, 5  10 6 CHO-K1 cells were transfected with 12 g of plasmid DNA (pCDNA3.1, Invitrogen) con- taining either HM74a, HM74, or PUMA-G expressed from the cytomeg- alovirus promoter. Two days after transfection, the growth medium was supplemented with 400 g/ml G418 to select for antibiotic-resistant cells. Clonal CHO-K1 cell lines that stably express HM74, HM74a, or PUMA-G were selected based on the ability of nicotinic acid (HM74a and PUMA-G) or S711589 (an Arena HM74-specific agonist; data not shown) to inhibit forskolin-induced cAMP production. Calcium Mobilization—CHO-K1 cells expressing an NFAT--lacta- mase reporter and the promiscuous G subunit G qi5 (kind gift of K. Sullivan, Merck Research Laboratories) were stably transfected with either empty vector (pCDNA3.1, Invitrogen) or vector expressing PU- MA-G, HM74a, or HM74. Cells were seeded at 10,000 cells/well in 384-well culture plates and grown overnight at 37 °C, 5% CO 2 in Dul- becco’s modified Eagle’s medium containing 10% fetal bovine serum, 2 mML-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 1 h. Calcium response in the fluorometric imaging plate reader assay was measured according to the directions from Molecular Devices. [ 35 S]GTPS Binding Assay—Membranes from untransfected CHO-K1 cells or cells stably expressing PUMA-G, HM74a, or HM74 (20 g/assay) were diluted in assay buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM MgCl 2 ) in Wallac Scintistrip plates and preincubated with test compounds diluted in assay buffer containing 40 M GDP (final * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § These authors contributed equally to this work. ¶ To whom correspondence should be addressed: Dept. of Cardiovas- cular Diseases, Merck Research Laboratories, 126 E. Lincoln Ave., Rahway, NJ 07065. Tel.: 732-594-2358; Fax: 732-594-4620; E-mail: andy_taggart@merck.com. 1 The abbreviations used are: AcAc, acetoacetate; (D)--OHB, (D)-- hydroxybutyrate; NEFA, non-esterified fatty acid; GTPS, guanosine 5'-O-(3-thiotriphosphate). Accelerated Publication THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 29, Issue of July 22, pp. 26649 –26652, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. This paper is available on line at http://www.jbc.org 26649 by guest on June 6, 2020 http://www.jbc.org/ Downloaded from