Cloning and Characterization of an Uncoupling Protein Homolog A Potential Molecular Mediator of Human Thermogenesis Ruth E. Gimeno, Marlene Dembski, Xun Weng, Nanhua Deng, Andrew W. Shyjan, Carlos J. Gimeno, Francois Iris, Stephen J. Ellis, Elizabeth A. Woolf, and Louis A. Tartaglia We have identified a novel cDNA encoding a protein highly homologous to the mammalian brown fat uncou- pling protein (UCP). Unlike the known UCP, which is expressed specifically in brown adipose tissue, the UCP homolog (UCPH) mRNA is expressed in a variety of tis- sues, with predominant expression in human white adi- pose tissue and skeletal muscle. In the white adipose tis- sue of ob/ob and db/db mice, the UCPH transcript is induced approximately fivefold relative to lean litter- mate controls. Expression of murine UCPH in yeast results in growth inhibition under conditions that require aerobic respiration, but does not affect growth under anaerobic conditions. Furthermore, UCPH expression in yeast causes a decrease in the mitochon- drial membrane potential, as judged by staining with the potential-sensitive dye DiOC 6 . These observations sug- gest that UCPH, like UCP, uncouples oxidative phos- phorylation. The possibility that the UCPH protein is an important mediator of human thermogenesis is dis- cussed. Diabetes 46:900-906, 1997 T he regulation of body fat in mammals is a complex process involving the regulation of not only appetite but also energy expenditure. An important component of energy expenditure is nonshiver- ing thermogenesis (NST). In rodents, the majority of NST appears to occur in brown adipose tissue (BAT) via the uncoupling protein (UCP) (1,2). UCP is a proton channel located exclusively in the inner mitochondrial membrane of adipocytes of the BAT (3). By allowing protons to equilibrate across the inner mitochondrial membrane, UCP uncouples oxidative phosphorylation and thus converts stored energy into heat rather than work (4,5). UCP-mediated uncoupling not only is capable of increasing body temperature in cold- acclimatized rodents and hibernating animals, but also can dissipate surplus caloric energy (6-8). A number of studies have now implicated UCP and BAT as important regulators of body weight in rodents (8-11). From Millennium Pharmaceuticals, Cambridge, Massachusetts. Address correspondence and reprint requests to Dr. Louis A. Tartaglia, Millennium Pharmaceuticals, 640 Memorial Dr., Cambridge, MA 02139. E- mail: tartaglia@mpi.com. Received for publication 10 January 1997 and accepted in revised form 24 February 1997. BAT, brown adipose tissue; EST, expressed sequence tag; FACS, fluo- rescence-activated cell sorter; NST, nonshivering thermogenesis; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; SC, synthetic complete; UCP, uncoupling protein; UCPH, UCP homolog. In humans, regulated thermogenesis is also thought to be an important component of metabolism and body weight homeostasis (12-14). However, the importance of the UCP in adult humans is questionable because of the low levels of BAT and, consequently, the low levels of UCP expression (15—18). In adult humans and other animals that do not contain large amounts of BAT, a large portion of NST and regulated ther- mogenesis is thought to be mediated by muscle and, to a lesser extent, the white adipose tissue (14,19-23); however, the molecular mediators for this are currently unknown (24). In this report, we describe the identification and characteri- zation of mouse and human cDNAs that encode proteins sharing functional properties and sequence similarity with the known UCP. RESEARCH DESIGN AND METHODS Cloning of murine and human UCP homolog. A mouse spleen cDNA library was constructed in the Uni-ZAP vector (Stratagene, La Jolla, CA). Random cDNA clones were isolated from the library and sequenced to gen- erate a database of expressed sequence tags (ESTs) that were compared with the GenBank sequence database using the BLASTX algorithm (25). This analy- sis identified an EST that is predicted to encode a peptide that shares a high level of similarity to the brown fat UCP. A 1.6-kb cDNA containing the entire mouse UCP homolog (UCPH) open reading frame was isolated from the same library by screening with a 32 P-labeled DNA probe using standard high strin- gency conditions. A 5'-truncated human UCPH clone was isolated by screen- ing a human spleen cDNA library (Stratagene) under low stringency conditions with the mouse UCPH cDNA probe. The 5'-end of the human UCPH cDNA was identified by 5'-RACE (rapid amplification of cDNA ends) using a commercially available RACE kit and human skeletal muscle 5'-RACE-ready cDNA (Clon- tech Laboratories, Palo Alto, CA). The full-length human UCPH cDNA was con- structed by inserting the 5'-RACE product into the original 5'-truncated human UCPH clone using standard molecular biology techniques. The accession numbers for the protein and nucleotide sequences reported in this paper are U94593 (mouse) and U94592 (human). RNA preparation and Northern blotting. Mouse and human poly A + RNA blots were obtained from Clontech. Total RNA was isolated using guani- dinium thiocyanate extraction and cesium chloride centrifugation (26) from the following tissues: Human skeletal muscle and white adipose tissue were obtained from a nondiabetic man with normal body weight (BMI 22.8) who underwent surgery for coronary artery disease. Mouse white and brown adi- pose tissue was obtained from three or four C57BL/6J animals. To determine the levels of UCPH expression in genetically obese animals, white adipose tis- sue was obtained from three or four animals each of C57BL/6J ob/ob, C57BL/6J +/?, C57BL/KsJ +db/+db, or C57BL/KsJ m+/m+ (Jackson Laborato- ries, Bar Harbor, ME). There was 15 ug of total RNA electrophoresed on a 1.3% agarose/formaldehyde gel and transferred onto nylon membranes. Blots were probed with 32 P-labeled DNA probes using the Rapid-Hyb buffer (Amersham, Arlington Heights, IL) according to the manufacturer's instructions. Expression of UCP and UCPH in S. cerevisiae. Yeast media were pre- pared and yeast molecular biology techniques were performed as described (27). Proteins were expressed in either the W303 genetic background (RGY12: MATa/a ura3-l/ura3-l Ieu2-3,112/leu2-3,112 Ms3-ll,15/his3- 11,15 trpl-1/TRPl ade2-100/ADE2 canl-lOO/canl-100 GaT; constructed 900 DIABETES, VOL. 46, MAY 1997