Bianco A C, Kieffer J D, Silva J E
Department of Medicine, Beth Israel Hospital, Boston, Massachusetts.
Endocrinology. 1992 May;130(5):2625-33. doi: 10.1210/endo.130.5.1374009.
We intend to develop in vitro model systems to study the hormonal regulation of uncoupling protein (UCP) and its role in brown adipose tissue (BAT) thermogenesis. We report here that UCP mRNA responses to adrenergic and thyroid hormone manipulations in freshly dispersed, mature brown adipocytes mimic in vivo observations. Studies were performed in brown adipocytes obtained from interscapular brown fat of euthyroid or hypothyroid rats. The tissue was dispersed with collagenase, and cells were isolated by floatation over 4% BSA. UCP mRNA in these cells is 2-3 times more abundant than that in the whole tissue, indicating a selection of cells expressing the gene. In cells from euthyroid rats, UCP mRNA is maximally elevated within 2 h of exposure to 1 microM forskolin and 50 ng T3/ml (77 nM total, 0.43 nM free). T3 significantly enhances the effect of forskolin. In the absence of stimulation, UCP mRNA rapidly disappears from euthyroid brown adipocytes, and this can be prevented with the addition of T3 by a mechanism not requiring on-going transcription. In cells from hypothyroid rats, forskolin or isoproterenol plus phenylephrine fail to stimulate UCP mRNA, but within 3 h of exposure to T3, cells recover full responsiveness. As in vivo, a high extracellular concentration of T3 is required for maximal responsiveness of UCP mRNA to cAMP, while T4 can restore responsiveness in physiological concentrations (40 pM). This effect of T4 is prevented by iopanoic acid, a compound that blocks the type II T4 5'-deiodinase. In conclusion, 1) freshly dispersed brown adipocytes retain all of the properties concerning UCP regulation by thyroid hormone and sympathetic nervous system described for brown fat in vivo; 2) the observations made in vivo, thus, represent direct action of norepinephrine and thyroid hormone on these cells; 3) as in vivo, T4 is a better source of intracellular T3 than extracellular T3 for brown adipocytes; hence, the in vivo findings are the result of the cell biology of 5'-deiodinase type II rather than dynamic factors inherent to the in vivo condition; 4) stabilization of mature UCP mRNA by T3 is an important mechanism to maintain the levels of this mRNA elevated under sustained stimulation; and 5) dispersed brown adipocytes and UCP gene products constitute a powerful model to study interactions between the sympathetic nervous system and thyroid hormone at a cellular or molecular level.
我们打算开发体外模型系统，以研究解偶联蛋白（UCP）的激素调节及其在棕色脂肪组织（BAT）产热中的作用。我们在此报告，新鲜分离的成熟棕色脂肪细胞中UCP mRNA对肾上腺素能和甲状腺激素处理的反应与体内观察结果相似。研究在取自甲状腺功能正常或甲状腺功能减退大鼠肩胛间棕色脂肪的棕色脂肪细胞中进行。组织用胶原酶分散，细胞通过在4%牛血清白蛋白上漂浮分离。这些细胞中的UCP mRNA比整个组织中的丰富2 - 3倍，表明对表达该基因的细胞有选择作用。在甲状腺功能正常大鼠的细胞中，暴露于1 microM福斯高林和50 ng T3/ml（总浓度77 nM，游离浓度0.43 nM）后2小时内，UCP mRNA最大程度升高。T3显著增强福斯高林的作用。在无刺激情况下，UCP mRNA在甲状腺功能正常的棕色脂肪细胞中迅速消失，添加T3可通过一种不需要持续转录的机制阻止这种情况发生。在甲状腺功能减退大鼠的细胞中，福斯高林或异丙肾上腺素加去氧肾上腺素不能刺激UCP mRNA，但在暴露于T3 3小时内，细胞恢复完全反应性。与体内情况一样，UCP mRNA对cAMP的最大反应需要高细胞外浓度的T3，而T4在生理浓度（40 pM）时可恢复反应性。T4的这种作用被碘番酸阻止，碘番酸是一种阻断II型T4 5'-脱碘酶的化合物。总之，1）新鲜分离的棕色脂肪细胞保留了体内棕色脂肪中描述的关于甲状腺激素和交感神经系统对UCP调节的所有特性；2）因此，体内观察结果代表去甲肾上腺素和甲状腺激素对这些细胞的直接作用；3）与体内情况一样，对于棕色脂肪细胞，T4是比细胞外T3更好的细胞内T3来源；因此，体内发现是II型5'-脱碘酶细胞生物学的结果，而不是体内条件固有的动态因素；4）T3对成熟UCP mRNA的稳定作用是在持续刺激下维持该mRNA水平升高的重要机制；5）分散的棕色脂肪细胞和UCP基因产物构成了一个强大的模型，用于在细胞或分子水平研究交感神经系统与甲状腺激素之间的相互作用。
