Evidence for the rapid direct control both in vivo and in vitro of the efficiency of oxidative phosphorylation by 3,5,3''-tri-iodo-L-thyronine in rats.

1. Examination of the distribution of L-tri-iodothyronine among rat liver tissue fractions after its intravenous injection into thyroidectomized rats focused attention on mitochondria at very short times after administration. By 15 min this fraction contained 18.5% of the tissue pool; however, the content had decreased sharply by 60 min and even further over the next 3 h. By contrast, the content in all other fractions was constant or increased over 4 h. About 60% of tissue hormone was bound to soluble protein. 2. Mitochondria isolated from thyroidectomized rats showed P/O ratios that were about 50% of those found in normal controls, with both succinate and pyruvate plus malate as substrates. There was no evidence of uncoupling; the respiratory-control ratio was about 6. 3. Mitochondria isolated 15 min after injection of tri-iodothyronine into thyroidectomized rats showed P/O ratios and respiratory-control ratios that were indistinguishable from those obtained in mitochondria from euthyroid animals. The oxidation rate was, however, not restored. 4. Incubation of homogenates of livers taken from thyroidectomized animals injected with L-tri-iodothyronine before isolation of the mitochondria restored the P/O ratio to normal; by contrast, direct addition of hormone to isolated mitochondria had no effect. The role of extramitochondrial factors in rapid tri-iodothyronine action is discussed. 5. Possible mechanisms by which tri-iodothyronine might rapidly alter phosphorylation efficiency are considered: it is concluded that control of adenine nucleotide translocase is unlikely to be involved. 6. The amounts of adenine nucleotides in liver were measured both after thyroidectomy and 15 min after intravenous tri-iodo-thyronine administration to thyroidectomized animals. The concentrations found are consistent with a decreased phosphorylation efficiency in thyroidectomized animals. Tri-iodothyronine injection resulted in very significant changes in the amounts of ATP, ADP and AMP, and in the [ATP]/[ADP] ratio, consonant with those expected from an increased efficiency of ADP phosphorylation. This suggests that the changes seen in isolated mitochondria may indeed reflect a rapid response of liver in vivo to tri-iodo-thyronine.     

Effects of iodine upon the structure and function of mitochondria

The influence of iodine in its positive and negative monovalent form upon the oxygen consumption in euthyroid and thyroidectomized rats and the oxidative phosphorylation in liver mitochondria isolated from both groups of animals, as well as the spontaneous swelling and total ATPase activity of mitochondria have been studied.It was established that the administration of ICI increased the oxygen consumption of normal and thyroidectomized rats while under the same conditions no effect was found with NaI. IBr stimulated the oxygen consumptionin vitro in liver mitochondria isolated both from normal and thyroidectomized rats and decreased the P/O ratio while NaI had no effect. I₂ and IBr increased the swelling and inhibited the ATPase activity of isolated rat liver mitochondria, while these effects were not observed when KI was used. The thyroidstatic 1-methyl-2-mercaptoimidazol decreased the stimulating effect of iodine upon the swelling of mitochondria and to a certain extent lowered its inhibiting effect upon the ATPase activity.It is concluded that iodine in its positive monovalent form has a thyroxine-like effect upon the structure and function of isolated rat liver mitochondria, as well asin vivo upon the respiration of euthyroid and thyroidectomized rats.     

Triiodothyronine binding and norepinephrine uptake in the heart and liver

Administration of norepinephrine to thyroidectomized rats activates sharply the [125I]triiodothyronine binding by heart mitochondria and liver nuclei. Epinephrine stimulates the binding by the heart mitochondria and decreases the intensity of this process in the liver and heart nuclei and liver mitochondria As compared to norepinephrine, adrenoxyl is weaker in activation of [125I] triiodothyronine binding by the heart mitochondria and stronger in intensification of binding by the liver nuclei. Physiological concentrations of thyroxine like adrenoxyl administered to intact animals 2h before investigations intensify the uptake of [3H] norepinephrine by sections of the auricles, myocardium and liver. Hyperthyroidization induces contrary changes inthe uptake of [3H] norepinephrine. Norepinephrine administration decreases sharply the uptake of [3H] norepinephrine by sections of the auricles and myocardium. The blocking of beta-adrenoreceptors weakens the uptake.     

Influence of thyroid status on the electrophoretic pattern of rat liver mitochondrial proteins]

Liver mitochondria were isolated from normal and thyroidectomized rats and their protein components analyzed by polyacrylamide gel electrophoresis. In whole mitochondria 35 protein fractions with MW ranging from 10,000 to 135,000 were characterized. In the absence of thyroid hormone secretion, the amount of a MW 54,000 fraction was always decreased. Injection of small doses of 3,5,3'-triiodo-L-thyronine to the thyroidectomized animal restored the quantity of that protein fraction to normal. Isolated outer mitochondrial membranes showed the presence of 20 protein fractions. These fractions revealed no change after thyroidectomy. The mitoplast, which contained 35 fractions, exhibited a decrease of the MW 54,000 component in thyroidectomized rats. The mitoplast was separated into several fractions. Water soluble matrix proteins presented molecular weights ranging between 40,000 and 55,000. Proteins, which were slightly bound to the inner mitochondrial membrane and could be extracted by KCl, presented molecular weights between 25,000 and 45,000. Structural proteins showed a principal specific component of MW equals 23,000. Electrophoretic patterns obtained with these submitochondrial fractions were similar in normal and thyroidectomized animals. The mitoplast fraction which contained the insoluble cytochromes (a, a3, b, c1) was isolated ; its principal constituent, of MW 54,000 was significantly decreased after thyroidectomy. Thus, the lack of thyroid hormone secretion lowered the level of a protein constituent bound to the inner membrane of liver mitochondria. The synthesis of this constituent could be controlled by mitochondrial nucleic acids.     

Biochemical and stereological analysis of rat liver mitochondria in different thyroid states

The concentrations of the inner mitochondrial membrane markers cardiolipin and cytochrome alpha have been measured in liver homogenates and in purified mitochondria after thyroxine administration to thyroidectomized and normal rats. The biochemical results have been correlated with stereological electron micrographic analyses of hepatocytes in liver sections, and of isolated mitochondrial pellets. There were progressive and parallel increases in homogenate and mitochondrial cardiolipin concentration, and in mitochondrial cytochrome alpha concentration, after administration of 20 microgram of thyroxine on alternate days to thyroidectomized rats, and of 300 microgram on alternate days to normal rats. Electron microscope measurements showed marked differences in the shape of the mitochondria and in the number of cristae in different thyroid states. Hypothyroid mitochondria were shorter and wider than controls, and hyperthyroid mitochondria longer but of similar width. Mitochondrial volume per unit cell volume was virtually unchanged in hypo- and hyperthyroid animals. The most striking changes were a decrease in the area of the inner membrane plus cristae in thyroidectomized rats, and a substantial increase in membrane area after thyroxine administration. The biochemical and electron micrographic results indicate that, in rat liver, thyroid hormone administration leads to a selective increase in the relative amount of mitochondrial inner membranes, with little or no change in the mitochondrial volume per unit cell volume, or in total mitochondrial protein per unit total cell protein.     

N-methyl oxidation in liver mitochondria of triiodothyronine-treated and thyroidectomized rats

The levels of sarcosine dehydrogenase and acid-nonextractable flavin in the inner matrix of mitochondria of rat liver are decreased in animals treated with triiodothyronine and are elevated in the mitochondria obtained from thyroidectomized animals. Administration of triiodothyronine does not affect the electron-transfer flavoprotein associated with the sarcosine dehydrogenase or the relative amounts of soluble and membrane-bound proteins of the mitochondria. In phosphate-washed mitochondria from either the controls or the triiodothyronine-treated rats, the O₂ uptake equals the total of the [¹⁴C]formaldehyde and [β-¹⁴C]serine isolated as reaction products of the sarcosine-[¹⁴C]methyl group. In contrast to its restraint of sarcosine or choline oxidation in preparations capable of oxidative phosphorylation, ADP does not inhibit the oxidation of these substrates in mitochondria of rats given triiodothyronine.     

Effects of thyroid hormone administration on the susceptibility of rat liver chromatin to digestion with micrococcal nuclease

The accessibility of rat liver chromatin to digestion with micrococcal nuclease was investigated in normal, thyroidectomized and thyroid hormone-treated animals. A significant increase in digestibility of chromatin by micrococcal nuclease was produced by thyroid hormone treatment. The DNA in the soluble fraction analyzed by electrophoresis showed identical sizes in thyroidectomized and triiodothyronine-treated animals. However, DNA in the pellet obtained from thyroidectomized animals showed a relatively high concentration of polynucleosomes which were virtually undetectable in the pellet from thyroid hormone-treated animals. Analysis of proteins in the micrococcal nuclease solubilized fraction of chromatin revealed differences between thyroidectomized and thyroid hormone-treated animals. It is suggested that thyroid hormone causes changes in nucleoproteins which alter the structure of chromatin in such a way as to expose more DNA to nuclease attack and/or increases the solubility of released nucleosomes.     

The relationship between the structure and activity of rat skeletal muscle mitochondria after thyroidectomy and thyroid hormone treatment

The fine structure of rat gastrocnemius muscle fibers has been studied after changes were induced in the basal metabolic rate (BMR) by thyroidectomy and L-thyroxine administration under anabolic conditions. Biochemical analysis of skeletal muscle mitochondrial respiration and phosphorylation from the same tissue preparations has been summarized, details having been published earlier (3). As estimated from electron micrographs, the total amount of mitochondria from thyroidectomized animals was enlarged 1.5 times over that from normal controls. The total amount of mitochondria from thyroidectomized or normal animals made hypermetabolic with thyroxine was increased 2.5 to 3.5 times over that from their corresponding controls. In all cases, there was an increase in the mitochondrial population and the profile ratio of cristae to matrix was also considerably increased, thus indicating both relative and absolute enlargements of the entire surface of the cristae per unit fiber. The major structural changes persisted for at least 3 weeks after the cessation of thyroxine treatment, by which time the elevated mitochondrial respiratory and phosphorylative activity had declined to normal values. The hypertrophy and increase in mitochondrial population was more prominent in the perinuclear and subsacrolemmic regions near blood vessels than in the interstices of the fibrils. The very long interfibrillar mitochondria found in both the hypo- and hypermetabolic tissues are more likely to be derived from outgrowths of the original mitochondria rather than from a fusion of smaller ones. These findings are compatible with the ideas expressed elsewhere (see 1, 3, 10) that, under conditions close to the physiological, thyroid hormones control mitochondrial metabolic activity by a subtle alteration in mitochondrial composition with respect to their respiratory and phosphorylative constituents. The possible application of using thyroid hormones in the study of biogenesis of mitochondria and the synthesis of mitochondrial constituents are discussed.     

Effects of thyroidectomy of the rat on the structure and functions of skeletal muscle mitochondria]

Mitochondria used in the present study were isolated from skeletal muscle of normal and thyroidectomized rats. The preparations were controlled by electron microscopy. It was not possible to find any morphological change induced by thyroidectomy, nevertheless, some difference appeared in the cytochrome contents which were slightly decreased. Oxygen consumption rates of thyroidectomized rat mitochondria were decreased when the particles were maintained in states 3 and 4 in the presence of various substrates, but the P/O ratios were not modified. The activities of mitochondrial enzymes were in general slightly affected by thyroidectomy except for glycerol-1-phosphate cytochrome c reductase and NADH rotenone sensitive cytochrome c reductase which were decreased and for glutamate dehydrogenase activity which was increased. The tRNA nucleotidyltransferase activity found in the mitochondrial matrix was not influenced by the absence of thyroid secretion. Normal rat muscle mitochondria incorporate 14C-leucine with an artificial ATP-generating system or with a respiratory substrate. The amino acid incorporation was decreased by thyroidectomy. Muscle mitochondria analyzed by polyacrylamide gel electrophoresis contained more than 30 protein components with MW ranging from 10.000 to 135.000. Thyroidectomy lowered the amount of a fraction of about 54.000 MW. It is not impossible that all the data observed in the absence of thyroid secretion are in relation with changes induced in the mitochondrial genome as previously shown in mitochondria isolated from liver or thyroidectomized rats.     

Effects of streptozotocin-diabetes and l-thyroxine treatment on plasma amino acid levels in thyroidectomized rats

1) Thirty days after surgical thyroidectomy, one group of rats were made diabetic by treatment with streptozotocin and were studied for the next 14 days. These diabetic thyroidectomized animals were similar in body weight to their thyroidectomized controls but had higher plasma concentrations of most amino acids. 2) Treatment with 0.5, 1.0 or 2.0 micrograms of L-thyroxine/100 g body wt for 7 days prior to sacrifice produced no changes in either parameter in the diabetic thyroidectomized animals. On the contrary, in thyroidectomized controls, the L-thyroxine treatment was followed by a dose-dependent increment in body weight. In these animals, the administration of 0.5 microgram of L-thyroxine per day was associated with a marked rise in the plasma level of most amino acids, while only basic amino acid levels increased with 1.0 microgram, and levels decreased with 2.0 micrograms. 3) In the diabetic thyroidectomized rats treated with insulin for the last 7 days before sacrifice, body weight gain and the biphasic change of plasma amino acid levels were restored. 4) It is proposed that treatment of thyroidectomized controls with small doses of L-thyroxine accelerate protein breakdown accompanied by minor changes in amino acid utilization, while this latter effect increases with higher doses of the hormone. 5) Present results demonstrate that a certain amount of circulating insulin is required to obtain the response to exogenous thyroxine in diabetic thyroidectomized animals. Results are discussed in terms of the role of interhormone synergism as it affects normal sensitivity of the different hormones.     

Evaluation of the role of calcitonin deficiency in ovariectomy-induced osteopenia

Studies were carried out in rats to examine the role of calcitonin deficiency in the pathogenesis of ovariectomy-induced osteopenia. The parathyroid glands of 80 female Wistar rats were autotransplanted to their thigh muscle and the animals divided into 4 groups. Group 1 rats were sham ovariectomized, and thyroidectomized to make them calcitonin deficient; Group 2 rats were thyroidectomized, and ovariectomized to make them deficient in ovarian hormones as well; Group 3 rats were sham thyroidectomized and sham ovariectomized, and Group 4 rats were sham thyroidectomized and ovariectomized. A fifth group of rats were unoperated upon and served as controls. Thyroidectomized animals were maintained on thyroxine replacement and 11 months after ovariectomy all the animals were bled, killed and their femurs dissected out. In both the thyroid intact and thyroidectomized animals, ovariectomy decreased femur density significantly (P less than 0.01). Similarly, ovariectomy resulted in a decrease in femur calcium (P less than 0.01) in both groups of animals, and in a significant decrease in serum calcitonin (P less than 0.05) in the thyroid intact animals. We conclude from these findings that ovarian hormone deficiency can cause bone loss independently of lowering circulating calcitonin levels.     

Effect of thyroid hormones on delayed type hypersensitivity reaction

The effect of long term administration of thyroid hormones and its deprivation on delayed type hypersensitivity (DTH) reaction to 2-4 dinitrochlorobenzene (DNCB) was studied. Animals were either pre-treated with thyroid hormones (T3 or T4) for 15 days and then subjected to DNCB skin test or the animals received thyroid hormones and simultaneously subjected to DNCB skin test. In both the cases DTH reaction was found to be increased significantly. When DNCB skin test was performed in the thyroidectomized animals, DNCB skin reaction was significantly decreased and the reaction was restored to normal following supplementation of thyroid hormones to the thyroidectomized animals. TLC and ALC were increased significantly following hormone treatment and thyroidectomized animals. TLC hand, induced significant depression in the count which was restored by hormone administration to the thyroidectomized animals.     

Regulation of pyruvate transport in mitochondria by thyroid hormones

During thyroidectomy, the stimulating action of the catalytic amounts of a thermostable fraction of rat liver and diaphragm cytoplasm on Ca2+ transport in mitochondria, which indicates the decrease of the activity of an insulin-dependent cytoplasmic regulator (IDR) in insulin target organs. Thyroidectomized rats also manifested a decrease in blood insulin and glucose concentrations. Administration of the physiological doses of thyroxine produced an increase in both blood glucose concentration and IDR activity in the liver and diaphragm of thyroidectomized rats. Experiments with measuring the kinetics of the swelling of deenergized mitochondria in isoosmotic solution of ammonium pyruvate demonstrated the inhibition of liver mitochondrial swelling in thyroidectomized rats.     

Endocrine control of cytosolic factors stimulating adenylate cyclase in rat lung

Endocrine control of cytoplasmic factors modulating adenylate cyclase activity in rat lung membranes was investigated. Hypophysectomy, adrenalectomy and thyroidectomy showed an adverse effect on the body and organ weights. Lung protein, glycogen and DNA contents were decreased in the endocrine ablated animals which were restored to the normal values on hormone treatment. Phosphodiesterase and phosphorylase activities were increased and decreased in adrenalectomized and thyroidectomized animals, respectively. The activities of these enzymes were restored to normal values on hormone treatment. Adrenalectomy and thyroidectomy affected ATPases differently. Basal adenylate cyclase activity in rat lung membranes was not affected by adrenalectomy and hormone treatment. However, the total enzyme activity was increased by both dexamethasone (DEX) and thyroxine (T4) treatments. The activation of the particulate adenylate cyclase by the cytoplasmic factors was markedly decreased in the lung from hypophysectomized, adrenalectomized and thyroidectomized rats. This decrease in the cytoplasmic activation of adenylate cyclase was restored to or above the control values on hormone treatment. Alteration in the activation of enzyme by cytoplasmic factors did not appear to be due to the change in the responsiveness of the enzyme. Glucocorticoids appeared to have a specific effect on the cytoplasmic factors modulating the enzyme.     

Effect of hypothyroidism on mitochondrial energy metabolism and nuclear synthesis of RNA in the liver of Cynomolgus monkeys

The effects of hypothyroidism and of replacement therapy with T4 or T3 were studied on the enzymatic activities of liver subcellular fractions isolated from Cynomolgus monkeys. Animals were sacrificed 20 days after thyroidectomy. In mitochondria, thyroidectomy decreased significantly the respiratory chain activity (succinate cytochrome c-reductase), the transfer of cytosolic reducing equivalents (glycerol-3-phosphate dehydrogenase) and the phosphorylating capacity (oligomycin-sensitive ATPase and state 3 respiratory rate). The activity of nucleolar and nucleoplasmic RNA polymerases dropped by about 50% in hypothyroid monkeys. In T4 (2.5 micrograms/kg/d) or T3 (1 microgram/kg/d) treated thyroidectomized animals, the iodothyronine concentrations and the activity of mitochondria and nuclei enzymes were halfway between normal and hypothyroid values. Thus, the mitochondrial effects of thyroidectomy in monkey are, as in rat, at least partly secondary to a decrease in nucleocytoplasmic protein synthesis.     

Studies on the activation in vitro of carnitine palmitoyltransferase I in liver mitochondria from normal, diabetic and glucagon-treated rats.

The activation of overt carnitine palmitoyltransferase activity that occurs when rat liver mitochondria are incubated at near-physiological temperatures and ionic strengths was studied for mitochondria obtained from animals in different physiological states. In all instances, it was found to be due exclusively to an increase in the catalytic capacity of the enzyme and not to an increase in affinity of the enzyme for palmitoyl-CoA. The enzyme in mitochondria from fed animals always showed a larger degree of activation than that in mitochondria from starved animals. This was the case even for mitochondria (e.g. from fed diabetic animals) in which the kinetic characteristics of carnitine palmitoyltransferase were more similar to those for the enzyme in mitochondria from starved rats. Glucagon treatment of rats before isolation of the mitochondria did not affect the characteristics either of the kinetic parameters of overt carnitine palmitoyltransferase or of its activation in vitro.     

Regulation of mitochondrial protein turnover by thyroid hormone(s)

1. The effect of thyroidectomy on turnover rates of liver, kidney and brain mitochondrial proteins was examined. 2. In the euthyroid state, liver and kidney mitochondria show a synchronous turnover with all protein components showing more or less identical half-lives compared with the whole mitochondria. The brain mitochondrial proteins show asynchronous turnover, the soluble proteins having shorter half-lives. 3. Mitochondrial DNA (m-DNA) of liver and kidney has half-lives comparable with that of whole mitochondria from these tissues. 4. Thyroidectomy results in increased half-lives of liver and kidney mitochondria, with no apparent change in the half-life of brain mitochondria. 5. A detailed investigation of the turnover rates of several protein components revealed a significant decrease in the turnover rates of mitochondrial insoluble proteins from the three tissues under study. 6. The turnover rates of m-DNA of liver and kidney show a parallel decrease. 7. Thus it is apparent that thyroid hormone(s) may have a regulatory role in maintaining the synchrony of turnover of liver and kidney mitochondria in the euthyroid state. Turnover of brain mitochondria may perhaps be regulated by some other factor(s) in addition to thyroid hormone(s). 8. It seems likely that during mitochondrial turnover m-DNA and insoluble proteins may constitute a major unit. 9. The mitochondrial protein contents of the three tissues are not affected by thyroidectomy. 10. No correlation was seen between the turnover rate of mitochondria and cathepsin activity in any of the tissues under study in normal or thyroidectomized animals. 11. On the other hand, mitochondrial proteinase activity shows good correlation with the turnover rates of mitochondria in normal animals, and a parallel decrease in activity comparable with the decreased rates of turnover is observed after thyroidectomy. 12. It is concluded that mitochondrial proteinase activity may play a significant role in their protein turnover.     

The stimulation by treatment in vivo with tri-iodothyronine of amino acid incorporation into protein by isolated rat-liver mitochondria

1. Normal and thyroidectomized rats were treated with near-physiological doses of tri-iodothyronine. Liver mitochondria were isolated and incubated with radioactive amino acids. In normal rats tri-iodothyronine caused only a slight stimulation of incorporation into mitochondrial protein, but in thyroidectomized animals the incorporation was doubled. 2. There was a lag period of about 36 hr. after injection and the maximum effect was observed after 2 days. 3. Direct addition of tri-iodothyronine to the incubation medium had no effect on mitochondrial incorporation. 4. The incorporation was not due to bacterial, nuclear, lysosomal or microsomal contamination and the labelled particles had sedimentation properties identical with those of mitochondria, as followed by suitable enzyme markers. 5. Thyroid hormone treatment did not cause any marked alterations in the pattern of labelling of submitochondrial fractions and in all cases the most radioactive protein was in an insoluble lipid-rich fraction. The amino acid compositions of the total mitochondrial protein and the more radioactive lipoprotein were also unaltered. 6. Increases in the content of RNA and various cytochromes per mg. of mitochondrial protein were observed after treatment with tri-iodothyronine. These occurred slightly later than the stimulation of amino acid incorporation. 7. No uncoupling of oxidative phosphorylation was observed and the ATP production per mg. of mitochondrial protein increased. 8. It was concluded that tri-iodothyronine stimulated amino acid incorporation into mitochondrial protein and that the result is consistent with the view that treatment with thyroid hormone results in an enhanced selective synthesis of mitochondrial respiratory units.