Short‐term control of mitochondrial adenine nucleotide translocator by thyroid hormone

Abstract

1. An improved simple technique for measuring adenine nucleotide translocator activity at low medium substrate concentrations is described.2. Confirming previous reports, thyroidectomy was shown to lead to lowered translocator activity in rat liver mitochondria. The rapidly exchangeable portion of the matrix nucleotide also decreased in hypothyroid preparations even though the total nucleotides increased substantially.3. The apparent K,,, of translocator for ADP increased from 2.8 to 6.2pM in hypothyroid preparations: Mg2+ ions raised this to about 20 pM.4. All of these changes in adenine nucleotide translocation were entirely reversed by 15min after a single intravenous near-physiological dose of triiodothyronine.The mitochondrion has long been considered a likely subcellular target of thyroid hormone action (review [I]). Mitochondria have been shown to acquire iodine [2] and triiodothyronine [3] at very short times after radiohormone administration to hypothyroid animals and a proteolipid fraction with specific tight binding sites for the hormone has been isolated from rat liver mitochondrial inner membrane [4].Rapid effects of hormone on oxidative phosphorylation have also been observed. Hoch [2] found changes in respiratory control and uncoupler sensitivity 2 min after thyroxine injection to hypothyroid rats; Sterling and his co-workers have reported increased phosphorylation in sub-mitochondrial vesicles both using direct addition of nanomolar hormone concentrations [5] and with vesicles prepared 30 rnin after injection of a near-physiological dose of triiodothyronine [I]. We have established conditions under which liver mitochondria from thyroidectomised rats show ADPjO ratios with both NAD' and FAD-linked substrates of 50-70% of those obtained using preparations from euthyroid animals with no loss of respiratory control [3,6]. Mitochondria from thyroidectomised rats isolated 15 min after intravenous injection of a physiological dose of hormone or isolated after incubation with hormone and other cell fractions showed ADPjO ratios and respiratory control ratios indistinguishable from those obtained with euthyroid preparations although the low rates of respiration per mass of protein were unchanged [3, 6, 71.One potential component in this lowered phosphorylating ability may be the reduced rate of adenine nucleotide translocation found in hypothyroid mitochondria [8,9], since the recent elegant experiments of Tager and his co-workers [lo, 1 I] have shown that a significant measure of control of oxidative phosphorylation is exerted by the transfer. Portnay et al. [8] found that the lowered translocation rate was increased after 6 days of thyroxine replacement therapy to hypothyroid aniEnzymes. Hexokinase (EC 2.7.1.1); pyruvate kinase (EC 2.7.1.40); lactate dehydrogenase (EC 3.1.1.27); adenylate kinase (EC 2.7.4.3); glucose-6-phosphate dehydrogenase (EC 1 .I .1.49). mals: in experiments with normal animals, the same group [12] had shown that a single large dose of triiodothyronine increased translocator activi...

Evidence for ADP‐ribosylation in the mechanism of rapid thyroid hormone control of mitochondria

“…The average radioactivity incorporated was 1314 dpm in the first experiment and 1586 dpm in the second. Given that the endogenous ATP content in these mitochondria is around 9 nmol/mg [12], the resulting specific radioactivity of the ATP in these incubations was around 300 dpm/pmol. Estimates of the content of Fi-ATPase put it at about 30 pmol/mg protein and the quantity of respiratory chain-linked proton pumps at about three times this [25].…”

Direct thyroid hormone signalling via ADP‐ribosylation controls mitochondrial nucleotide transport and membrane leakiness by changing the conformation of the adenine nucleotide transporter

“…of n mitochondrial preparations. The rates were derived from the best straight lines fitted to plots of Ln(Rm~-Rm) vs. time (see Section 2 [5]) for initial ADP concentrations of either 2 /aM (3.5 Ci/mol) or 20 ~M (0.35 Q/mo0. T.~ was administered 15 min prior to sacrifice via the tongue veins (at 1 nmol/ 100 g body weight) to rats thyroidectomized 6 weeks previously.…”

“…Adenine nucleotide transport was measured using the integral form of the transport equations as set out in [5], except that the buffer used was the same as for the oxygen electrode experiments. In some experiments CaC12 was added to give a final buffered concentration of 30 nM (see [8]).…”

“…This was consistent with the reports from other laboratories of ADP-ribosylation of a 30 kDa inner membrane protein in liver sub-mitochondrial particles [11,12] although somewhat strangely this is proposed to result from NAD + hydrolysis and then very selective nonspecific labelling by the ADP-ribosyl moiety [13,12]. An inner membrane protein of this size, the adenine nucleotide transporter (ANT) [14], has long been suggested as a mitochondrial target for thyroid hormones [15] and we had reported previously that the velocity of the ANT was halved in hypothyroid mitochondria because of a 2.2-fold increase in the apparent Km and subject to restoration within 15 min by hormone in vivo [5]. However, we had found that in vitro incorporation of labelled NAD + into the 30 kDa species was abolished by T3 [10] which we initially interpreted as providing no support for this protein as a target for T3 signalling involving ADPribosylation.…”

“…To complement this evidence of a putative receptor, rapid effects of hormone on an energy-driven mitochondrial process such as respiratory control, uncoupler sensitivity [2] and calcium transport [4] have been observed. In our laboratory we have found that liver mitochondria from thyroidectomized rats show a number of altered responses to ADP stimulation that hormone addition at near-physiological concentrations both in vivo and in vitro can restore to normal within 15 min [1, [5][6][7][8].…”