BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 235, 760–764 (1997) ARTICLE NO. RC976852 Kupffer Cells Are a Dominant Site of Uncoupling Protein 2 Expression in Rat Liver Dominique Larrouy,* ,1 Patrick Laharrague,* Georges Carrera,† Nathalie Viguerie-Bascands,* Corinne Levi-Meyrueis,‡ Christophe Fleury,‡ Claire Pecqueur,‡ Maryse Nibbelink,* Mireille Andre ´,* Louis Casteilla,* ,1 and Daniel Ricquier‡ ,1 *CNRS/UPRESA 5018 and †INSERM CJF 91-07, Institut Louis Bugnard, Universite ´ Paul Sabatier, Ho ˆpital Rangueil, 31054 Toulouse Cedex, France; and ‡CNRS UPR 9078/CEREMOD, 9, rue Jules Hetzel, 92190 Meudon, France Received May 15, 1997 adult rats exposed to the cold (12) was in agreement The mechanisms underlying thermogenesis in liver with this hypothesis, but was later on described as arte- are not well understood. They may involve proteins factual (13). Until now, the search for UCP1 and/or related to the mitochondrial uncoupling protein UCP1 mRNA in tissues other than brown adipose tis- (UCP1) of brown adipocytes. In this paper, it is demon- sue, was performed mainly on homogenized tissues. strated that UCP1 is not expressed in any liver cell Such experiments could give rise to misleading results type of rat while UCP2, a recently cloned homologue if UCP1 was expressed only in a particular and sparse of UCP1, is expressed at a very high level in Kupffer cell type present in a tissue. Indeed, liver is a combina- cells but not in hepatocytes. This high level of expres- tion of at least 12 different cell types (14). Hence, the sion of UCP2 in Kupffer cells allowed cross immunore- initial aim of this study was to definitively establish if activity with antibodies directed against UCP1. This UCP1 is expressed in certain liver cell types using a cross reactivity was confirmed by the detection of combination of immunological, immuno-histochemical, UCP2 with anti-UCP1 antibody, in western blotting and molecular methods. Immunological methods used analysis of transfected yeasts expressing rat UCP2. in the present study, led to contradictory results, sug- The high level expression of UCP2 in Kupffer cells sug- gesting that UCP1 antibodies cross-reacted with an gests a particular function of UCP2 in macrophages.  1997 Academic Press other mitochondrial protein expressed in non-paren- chymal cells present in liver. Recently, a new protein belonging to the family of mitochondrial anion carriers was cloned from a mouse Cold induced thermogenesis in rodents is performed muscle cDNA library (15). This protein, named uncou- in part by brown adipose tissue. The mitochondrial un- pling protein 2 or UCP2, seems to have properties simi- coupling protein UCP1, specifically expressed in brown lar to UCP1 and is putatively involved in heat produc- fat cells, provokes heat dissipation by uncoupling of tion, since it was shown to be able to uncouple the oxidative phosphorylation from ATP synthesis, during mitochondrial respiration from ATP synthesis. More- sympathetic activation of the brown fat (1, 2). The ex- over, the wide tissular expression of UCP2 agrees with clusive occurence of UCP1 in brown adipose tissue was a role in energy expenditure. We report here that the ascertained by many investigators using immunodetec- UCP1 immunoreactivity of rat liver revealed the pres- tion (3-5) or mRNA analysis (6-9). A contribution of ence of very high amount of UCP2 in non parenchymal liver to cold-induced thermogenesis, was also demon- cells and in particular in Kupffer cells. strated. Nevertheless, although it was proposed that an uncoupling of respiration of liver mitochondria MATERIALS AND METHODS might exist in cold-exposed animals, the mechanism of such an uncoupling remains unknown (10, 11). The Animals. Male Wistar rats were housed at 25°C with free access detection of UCP1 mRNA in liver of newborn rats, or to food (65% carbohydrate, 11% fat and 24% protein w/w; UAR, Vil- lemoisson France) and water. Antibodies. Sheep and rabbit polyclonal antibodies against rat 1 Corresponding authors. (D.R.) Fax: /33 1 45 07 58 90. E-mail: ricquier@infobiogen.fr. (L.C.) Fax: /33 5 62 17 09 05. E-mail: casteil@ UCP were previously described (4, 16). Donkey anti-sheep IgG alka- line phosphatase-conjugated was purchased from Sigma Chemical rangueil.inserm.fr. Abbreviation: UCP2, uncoupling protein 2. (St. Louis, MO). Pig anti-rabbit IgG alkaline phosphatase-conjugated 0006-291X/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved. 760