PubMed · 8141913
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J A Kornblatt, M J Kornblatt. 1994-02-17. Warm brown fat.. https://doi.org/10.1038/367590c0
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In order to investigate whether the positive effect of adrenergic stimulation on lipoprotein lipase (LPL) gene expression in brown adipose tissue is a direct effect on the brown adipocytes themselves, the expression of the LPL gene was investigated by measuring LPL mRNA levels in brown adipocytes, isolated as precursors from the brown adipose tissue of rats and grown in culture in a fully defined medium before experimentation. Addition of noradrenaline led to an enhancement of LPL gene expression; the mRNA levels increased as a linear function of time for at least 5 h and were finally approx. 3 times higher than in control cells, an increase commensurate with that seen in vivo in both LPL mRNA levels and LPL activity during physiological stimulation. The increase was dependent on transcription. The effect of noradrenaline showed simple Michaelis-Menten kinetics with an EC50 of approx. 11 nM. beta3-Agonists (BRL-37344 and CGP-12177) could mimic the effect of noradrenaline; the beta1-agonist dobutamine and the beta2-agonist salbutamol could not; the alpha1-agonist cirazoline had only a weak effect. The effect of noradrenaline was fully inhibited by the beta-antagonist propranolol and was halved by the alpha1-antagonist prazosin; the alpha2-antagonist yohimbine was without effect. An increase in LPL mRNA level similar to (but not significantly exceeding) that caused by noradrenaline could also be induced by the cAMP-elevating agents forskolin and cholera toxin, and 8-Br-cAMP also increased LPL mRNA levels. The increase in LPL gene expression was not mediated via an increase in the level of an intermediary proteinaceous factor. It is concluded that the physiologically induced increase in LPL gene expression is a direct effect of noradrenaline on the brown adipocytes themselves, mediated via a dominant beta3-adrenergic pathway and an auxiliary alpha1-adrenergic pathway which converge at a regulatory point in transcriptional control.
The transport inhibiting nucleotide binding to the uncoupling protein (UCP) has a unique pH dependence and has been postulated to be controlled by the dissociation state of a carboxyl group in UCP with pK 4.5 and, in addition only for the nucleoside triphosphate, by a group with pK 7.2. To prove this assumption and to identify the carboxyl group, Woodward reagent K (WRK) was applied to UCP. In mitochondria, WRK was found to inhibit binding of GTP in a noncompetitive manner using WRK in the millimolar range. In isolated UCP, GTP binding is inhibited by WRK at a 1 to 2 ratio to UCP, suggesting that WRK primarily reacts with only one carboxyl group. Prebound GTP protects against WRK reaction as monitored by GTP binding. The protection decreases from pH 5 to 7 due to better reactivity of WRK and less tight GTP binding. WRK does not inhibit H+ transport by UCP but prevents GTP inhibition of H+ transport. For elucidating the WRK target residue, the WRK derivatized group was labeled with [3H] by reduction with [3H]NaBH4. Both GTP and GDP largely protected against WRK-dependent [3H] labeling. CNBr fragmentation identified the region T121-M197 as the [3H] incorporation site. Combined CNBr and tryptophane cleavage by the reagent 3-bromo-3-methyl-2-((2-nitrophenyl) thio)-3H-indole (BNPS) allowed to further delimit the 2.8 kDa peptide W173-M197 as the [3H] label carrier which contains two acid residues E190 and D195. To further identify the residue, limited tryptic digestion in sarcosyl-treated UCP was performed, and a tryptic fragment enclosing E190 and D195 was isolated which carried most of the [3H] label. Edman degradation showed the major [3H] label at the eighth position corresponding to E190 and no peak at D195. Thus, the original postulate of the pH-sensing carboxyl group regulating both the nucleoside di- and triphosphate binding has been verified. It is identified as E190 situated in the fourth transmembrane helix. In total, now four residues close to the nucleotide binding sites in UCP have been determined.
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