Molecular approach to thermogenesis in brown adipose tissue: cDNA cloning of the mitochondrial uncoupling protein.

Abstract

The uncoupling protein (UCP) of mammalian brown fat is a specialized and unique component responsi- RNA (16) and immunoprecipitation of synthesized UCP were carried out as described (9,10).cDNA Synthesis and Cloning. Poly(A+) RNA from thermogenic brown fat was enriched for UCP mRNA by sucrose gradient centrifugation. The mRNA fraction sedimenting around 15S was used to synthesize double stranded (ds) cDNA according to standard procedures (17). Both first and second strands were prepared by using avian myeloblastosis virus reverse transcriptase. After hairpin loop cleavage with S1 nuclease and deproteinization with phenol and chloroform extractions, ds cDNA was size-fractionated by sedimentation on a sucrose gradient. Ten nanograms of a ds cDNA fraction (500 base pairs long) was tailed with calf thymus terminal deoxynucleotidyl transferase and dCTP (18) and was hybridized with 100 ng of dG-tailed, 20). Chimeric vectors were used to transform CaCl2-treated E. coli C600, and transformant clones were selected on L medium agar plates containing 10 mg of tetracycline per liter. About 2000 recombinant clones were obtained and further characterized. A second library of recombinant clones bearing longer cDNA inserts were prepared from a Abbreviations: UCP, uncoupling protein; ds, double stranded; kb, kilobase.

Comment to Shinohara et al. (1991) FEBS Letters 293, 1731̄74 The uncoupling protein is not expressed in rat liver

“…These data were confirmed by investigators who were unable to detect UCP rnRNA in liver or other tissues [12--161. Surprisingly, Shinohara et al, using a rat UCP cDNA originating from our laboratory [12], recently claimed they could detect UCP mRNA in newborn rat liver as well as in the liver of cold exposed rats [I?]. We present data contradicting such a conclusion and confirming the exclusive expression of UCP in brown adipocytes.…”

The uncoupling protein homologues: UCP1, UCP2, UCP3, StUCP and AtUCP

“…Before the publication of these data, a 32-kDa protein abundant in the membranes of BAT mitochondria was described : this protein was absent from liver mitochondria, induced during exposure of rats to the cold for several days, and down-regulated after readaptation to room temperature [36]. Following these studies, the 32-kDa protein was purified from hamster and rat [37,38], sequenced [39], cloned as cDNAs [40][41][42][43], and its protontranslocating activity reconstituted in liposomes [44][45][46][47]. It was therefore accepted that this protein was responsible for the regulated loose coupling of BAT mitochondria, and it was generally referred to as the uncoupling protein, UCP (UCP1).…”

Phytanic acid, but not pristanic acid, mediates the positive effects of phytol derivatives on brown adipocyte differentiation

“…Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a methyl-branched fatty acid that originates from the phytol side of the chain of chlorophyll. Phytanic acid enters the human body in foods such as dairy products or other ruminant fats.…”