BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 235, 79–82 (1997) ARTICLE NO. RC976740 UCP3: An Uncoupling Protein Homologue Expressed Preferentially and Abundantly in Skeletal Muscle and Brown Adipose Tissue Antonio Vidal-Puig, 1 Gemma Solanes, 1 Danica Grujic, 1 Jeffrey S. Flier, and Bradford B. Lowell 2 Division of Endocrinology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts Received May 2, 1997 in rodents (6-8). However, UCP1 may be of lesser im- Uncoupling proteins (UCPs) are inner mitochon- portance in humans in whom the mass of brown adi- drial membrane transporters which dissipate the pro- pose tissue is limited. A second uncoupling protein, ton gradient, releasing stored energy as heat. UCP1 is termed UCP2 (9) or UCPH (10) was recently identified. expressed exclusively in brown adipocytes while UCP2 In contrast to UCP1, UCP2 is expressed in many tis- is expressed widely. We now report the molecular clon- sues, including sites not thought to mediate adaptive ing of a third uncoupling protein homologue, desig- thermogenesis. We now report the molecular cloning nated UCP3. At the amino acid level, hUCP3 is 71% of a third uncoupling protein homologue, designated identical to hUCP2 and 57% identical to hUCP1. UCP3 UCP3, which is distinguished from UCP1 and UCP2 is distinguished from UCP1 and UCP2 by its abundant by its preferential expression in skeletal muscle and and preferential expression in skeletal muscle in hu- brown adipose tissue, two important sites for regulated mans, and brown adipose tissue and skeletal muscle energy expenditure in humans (11-15) and rodents (8). in rodents. Since skeletal muscle and brown adipose tissue are believed to be important sites for regulated energy expenditure in humans and rodents, respec- MATERIALS AND METHODS tively, UCP3 may be an important mediator of adap- tive thermogenesis. Since UCP3 is minimally ex- RACE (rapid amplification of cDNA ends) cloning of UCP3. Full- pressed in human heart and other critical organs, it is length UCP3 cDNA sequences were generated using the Marathon cDNA Amplification Kit, human skeletal muscle Marathon-Ready a promising target for anti-obesity drug development cDNA (both from Clontech Laboratories, Palo Alto, CA) and an anti- aimed at increasing thermogenesis. q 1997 Academic Press sense primer (5* TTC ACC ACG TCC ACC GGG GAT GCC ACC 3 * ). PCR was carried out using ExTaq Polymerase (TaKaRa), Taq Start Antibody (Clontech Laboratories) and the following conditions: 1.5 min. at 947C, 20 sec. at 987C and 4 min. at 687C for 30 cycles. Calories are expended within mitochondria in a Northern blot assays. Human Multiple Tissue Northern Blots highly regulated fashion. Oxidation of fuels generates (#7760-1, #7759-1 and #7767-1) containing approximately 2 mg of a proton electrochemical gradient across the inner mi- polyA RNA per lane were purchased from Clontech Laboratories tochondrial membrane and re-entry of these protons (Palo Alto, CA). All hybridizations, membrane washes and mem- via ATP synthase drives conversion of ADP to ATP. brane strippings were performed according to manufacturers speci- fications. The blots were first hybridized to a hUCP3 probe, washed Uncoupling proteins (UCPs) are inner mitochondrial and exposed to film for 1-18 hours, then stripped, rehybridized to a membrane transporters which dissipate the proton hUCP2 probe and exposed to film for 18 hours. The hUCP3 probe gradient, releasing stored energy as heat (1, 2), and was a 293 bp fragment corresponding to residues #211-308. The are therefore potentially important determinants of hUCP2 probe was a 1125 bp fragment spanning the entire open metabolic efficiency. UCP1, the first uncoupling protein reading frame. The specific activities of both hybridization probes were similar. Mouse Northern blots were generated using total RNA to be identified (3-5), is expressed exclusively in brown isolated from a number of tissues and equal loading of lanes was adipose tissue, an important site of energy expenditure established using ethidium bromide florescence. The mouse Northern blots were hybridized using the hUCP3 probe described above or a 1207 bp mUCP2 probe which spans the entire open reading frame. 1 These three authors contributed equally to this work. 2 To whom correspondence should be addressed at Division of En- RNase protection assays. Partial human UCP-3 and UCP-2 probes were generated by reverse transcriptase-PCR using total RNA docrinology, RN-320, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215. Fax: (617) 667-2927. E-mail: blo from human muscle as follows: two primers (5* GCA GTC TTG AAG AAC GGG ACA CC 3 * and 5* TGG CAG TAG GGG GCA CAT CT well@bidmc.harvard.edu. 0006-291X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved. 79