Early action of injected L-thyroxine on mitochondrial oxidative phosphorylation.

In hypothyroid rats, the depressed metabolic rate (02 consumption) rises 20-70 hours after thyroid hormone is injected;' the rates of ribosomall-3 and mitochondrial3 synthesis of proteins in the liver rise 36-96 hours after injection; but the rate of nuclear RNA metabolism rises as early as 10-30 hours.2 On the reasonable assumption that first occurrences are causal, this evidence has been taken to indicate that the thyroid hormone acted on nuclei to stimulate protein synthesis. 5 However, adding thyroid hormone to isolated nuclei does not stimulate RNA metabolism-evidence that the nuclear changes observed after hormone injection are not due to a direct hormonal action.2' 6 Adding thyroid hormone to isolated mitochondria, on the other hand, produces in about 5 minutes an as yet unidentified factor that stimulates ribosomal synthesis of proteins.6' 7 Our recent studies8-" have shown that injecting hypothyroid rats with a subcalorigenic dose of L-thyroxine (5.2 mug/gm, a dose at least 50 times less than those used to stimulate protein synthesis) three hours before sacrifice partly corrected the excessive respiratory control in their liver mitochondria. The hormone arrived in the liver mitochondria very rapidly. When these rats were killed two minutes after the hormone had been injected,11 12 it was found that the abnormally low (20% of normal) total iodine content had increased to 60 per cent of normal. These findings suggested that mitochondrial function might change as early as two minutes after hormone injection. The present studies examine the mitochondria from the livers of hypothyroid rats injected with a relatively large dose of L-thyroxine (5.2 pg/gm), and demonstrate a latent period of two minutes for changes in respiratory control and sensitivity to added dinitrophenol, a progression in the changes up to three hours, and an increase in the content of butanol-extractable and butanol-insoluble iodine up to 550-fold. Further details will be presented elsewhere.'2 13 Materials and Methods.-Normal and thyroidectomized male rats were obtained from the Charles River Breeding Laboratories, North Wilmington, Massachusetts. Thyroidectomized rats, weighing about 50 gm, were injected intraperitoneally with 0.25 me of I131 and maintained on a low-iodine, vitamin-enriched diet (Nutritional Biochemical Co.). Their rates of 02 consumption 3-4 weeks after operation were 30-40% lower than those of normal rats (measured as in ref. 8). They were injected intraperitoneally with dinitrophenol, 10 lsg per gram, and the rise in 02 consumption 15-45 min later was expressed as a per cent change relative to the rate just before treatment. Group A included those hypothyroid rats in whom dinitrophenol raised the metabolic rate less than 30%, and group B included those in whom dinitrophenol raised the metabolic rate more than 30%.8 Only group A was used in the present experiments. The significance of this grouping is that only in rats of group A did 5.2 mug of L-thyroxine per gram increase the calorigenic effect of dinitrophenol in vivo8 so that it was possible to screen the rats by their responses in vivo and to eliminate insufficiently hypothyroid animals. Each animal was tested with dinitrophenol at least twice before grouping and the animals were ready for experimental use 4-8 weeks after thyroidectomy. Animals were starved 17-24 hr before being used in an experiment. L-Thyroxine (Smith, Kline and French Laboratories) was injected intraperitoneally; it was prepared daily by dissolving in a minimal volume of 0.1 N NaOH and diluting with water. At least