The efficiency of oxidative phosphorylation and the rapid control by thyroid hormone of nicotinamide nucleotide reduction and transhydrogenation in intact rat liver mitochondria

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Abstract
1 In confirmation of previous work enhancement of the fluorescence emission of reduced nicotinamide nucleotides in intact rat liver mitochondria was found to depend on incubation conditions. 2 Under standard conditions the enhancement is constant at 4.8-fold in states 3 and 4 and is not altered by thyroidectomy of the animal 6 weeks prior to experiment. 3 The ADP-induced (state 4 → state 3 → state 4) fluorescence changes are significantly different in intact mitochondria from normal and hypothyroid animals and reflect the decreased rate and efficiency of oxidative phosphorylation after thyroidectomy. 4 Incubation of liver homogenates in vitro for 15 min with 1 μM triiodothyronine before isolating mitochondria significantly restores their ADP response towards normal. Direct addition of hormone to isolated mitochondria was ineffective. 5 Enzymatic measurement of mitochondrial extracts shows that thyroidectomy leads to increases in the contents of NAD(H) by 22% and NADP(H) by 33%. 6 With glutamate as substrate ADP-induced changes in the reduced/oxidized ratio of NAD+ are not significantly altered in hypothyroid preparations. By contrast the NADP+ ratio remains substantially more reduced in state 3 than it does in normal mitochondria. 7 The hypothesis is advanced that the decreased efficiency of hypothyroid preparations in phosphorylating ADP may be the result of increased energy-linked transhydrogenase activity. This is needed to supply NADPH via the glutathione peroxidase for reducing endogenously formed peroxides. 8 Direct reduction of mitochondrial glutathione with dithiothreitol had no substantial effect on ADP/O ratios or on ADP-induced redox cycles in either normal or thyroidectomised preparations. This decisively eliminates the possibility that lowered phosphorylation efficiency is the result of a leak of reducing equivalents via glutathione peroxidase.