Abstract
Transcription of the uncoupling protein (UCP) gene in rat brown adipose tissue has been evaluated by a nuclear run-on transcription assay. Nuclei from euthyroid rats treated with norepinephrine (NE) exhibited a 3-4-fold increase in transcription 2 h after the injection, whereas the UCP mRNA abundance increased 2-3-fold during the same interval. In contrast, neither UCP mRNA nor the transcription rate increased in response to the same treatment in hypothyroid rats, but the transcriptional response was recovered 2 h after the injection of a receptor saturating dose of 3,5,3'-L-triiodothyronine (T3). When injected to rats given T3 4 h before, NE increased UCP gene transcription and mRNA abundance by 4-5 and 3-4-fold, respectively, by 2 h. Neither cycloheximide nor emetine prevented this effect of T3. In euthyroid rats, the UCP gene transcription rate increased within 15 min of exposure to 4 degrees C. In contrast, in hypothyroid rats exposed to 4 degrees C overnight the transcription rate remained unchanged, but it started to increase steeply 2 h after the intravenous injection of a receptor saturating dose of T3 with UCP mRNA lagging approximately 1 h behind. Unilateral denervation of interscapular brown adipose tissue in hypothyroid rats caused a 60-70% ipsilateral reduction in transcription rate and UCP mRNA. When T3 was given to these rats, it only stimulated UCP gene transcription and UCP mRNA accumulation in the intact side, but there was a symmetrical stimulation when NE was injected following the T3. We conclude that the UCP gene is under dual control by T3 and NE. The primary signal seems to be generated by NE. This signal increases UCP mRNA by stimulating the transcription rate of the UCP gene. From the denervation experiments and the lack of response of UCP to NE in hypothyroid rats, we estimate that T3 amplifies 4-5-fold the transcriptional response to NE. No stabilization of UCP mRNA needs to be invoked to explain the acute effect of T3. The T3-dependent signal amplifying the NE effect is consistent with being the T3-receptor complex or another T3-derived signal that is neither a newly synthesized protein nor a rapidly turned-over pre-existing protein, activated by T3.
Highlights
Transcription of the uncoupling protein (UCP) gene genesis
Neither cycloheximide nor emetine rats [4]which is accounted for by a proportional increment pupegoxnreespcnevhoedeastnrnutaotgrene4edsdct“,orCtihbpo4iutsv“itCoeienrt.fnIfsnietgarcahroctttftoeenTdtti3hrnt.aeocIsrntei,tnareiacsnuenrhedtshyawcysprieriotspohtthtiieindyeorpnorlyai1dtr52sar,tamhetthiasUnfreeCetxeomPfr- aineminxeeHpdtchoyhespaenodUitshCtmyoPrsomlowidRwhNaenraAeimmb(yba5il,eTs6n3)att.pneadmrhtpiuecmripaatauntersessbinecho(e7ma)t.e hypothermic if The molecular generation are the intravenousinjection of a receptor saturatingdose not fully understood [8].We have found that the mitochonof T3with UCP mRNA lagging -1 h behind
Stimulation in hypothyroid rats improved in a dose-dependent manner following T3treatment [11].We found that the responses of UCP to various thyroid hormone regimens were paralleled by changes in UCP mRNA [9]
Summary
Rats and Experimental Manipulations-Male Sprague-Dawleyrats were used throughout (Zivic-Miller,Allison Park, PA). To examine the recovery of the response of UCP gene transcription to NE in hypothyroid animals, 10 pg of T3/100 g were injected 4 h before the NE, and the animals were killed 2 h after injecting this latter This is based on the time after T3 required by hypothyroid rats to recover the responsiveness of UCP mRNA to NE [12]. In these experiments, rats were injected with 20 pg of T3/100 g the night before the day of killing (-16 h) as indicated. The pellet is resuspended in 150 plof Buffer 3 (50% glycerol, 5 mM magnesium acetate, 0.1 mM EDTA, 5 mM DTT, 0.1 mM PMSF, and 50 mM Tris, pH 8.0) An aliquot of this nuclear suspension is saved for DNA measurement by the diphenylamine method [25], and the rest is used for the transcription.
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