Regulation of thyroid hormone metabolism in rat liver fractions

The nature of the conversion of thyroxine (T₄) to triiodothyronine (T₃) and reverse triiodothyronine (rT₃) was investigated in rat liver homogenate and microsomes. A 6-fold rise of T₃ and 2.5-fold rise of rT₃ levels determined by specific radioimmunoassays was observed over 6 h after the addition of T₄. An enzymic process is suggested that converts T₄ to T₃ and rT₃. For T₃ the optimal pH is 6 and for rT₃, 9.5. The converting activity for both T₃ and rT₃ is temperature dependent and can be suppressed by heat, H₂O₂, merthiolate and by 5-propyl-2-thiouracil. rT₃ and to a lesser degree iodide, were able to inhibit the production of T₃ in a dose related fashion. Therefore the pH dependendy, rT₃ and iodide may regulate the availability of T₃ or rT₃ depending on the metabolic requirements of thyroid hormones.     

Influence of thyroid hormone on glycogen metabolism in rat liver.

The livers removed from thyroidectomized and L-T4 supplemented rats were rapidly frozen by Freon-12 chilled with liquid nitrogen, and concentrations of metabolites which affect glycogen synthetase and phosphorylase were determined. Serum and liver glycose levels were not changed in any thyroid functioning. But liver G6P and ATP were increased by thyroidectomy and decreased by L-T4 supplement, while cAMP was increased by the hormone supplement. The enzyme activity ratio of glycogen synthetase a to phosphorylase a was increased by thyroidectomy and decreased by L-T4 supplement. The most intimate correlation was observed between the enzyme activity ratio and the ratio of the energy charge ratio of cAMP among other indices calculated from changes in the metabolite concentrations in the various thyroid functioning. The change in the substrate levels brought about by thyroidectomy and L-T4 supplement appeared to modulate both the enzyme activities which in turn regulate the glycogen metabolism.     

Regulation of biosynthesis of the rat liver inner mitochondrial membrane by thyroid hormone

Regulation of mitochondrial protein synthesis by thyroid hormone has been studied in isolated rat hepatocytes and liver mitochondria. Small doses (5 micrograms/100 g body wt) of triiodothyronine (T3) injected into hypothyroid rats increased both state 3 and 4 respiration by approximately 100%, while the ADP:O ratio remained constant. This suggests that T3 increases the numbers of functional respiratory chain units. T3 also induces mitochondrial protein synthesis by 50-100%. Analysis of the mitochondrial translation products show that all of the products were induced. No differential translation of the peptides involved in the respiratory chain was found. Regulation of the cytoplasmically made inner membrane peptides was also investigated in isolated hepatocytes. The majority of these peptides were not influenced by T3, in contrast to the finding with mitochondrial translation products. Those found to be regulated by T3 belong to two subsets, which were either induced or repressed by hormone. Thus, T3 stimulated a general increase in the synthesis of mitochondrially translated inner membrane peptides, but regulates selectively those inner membrane peptides translated on cytoplasmic ribosomes. The findings suggest that hormone regulation of the respiratory chain is exerted through a few selective proteins, perhaps those which require subunits made from both nuclear and mitochondrial genes.     

Regulation by thyroid hormone of the synthesis of a cytosolic thyroid hormone binding protein during liver regeneration.

To understand the regulation by thyroid hormone, 3,3',5-triiodo-L-thyronine (T3), of the synthesis of a cytosolic thyroid hormone binding protein (p58-M2) during liver regeneration, the synthesis of p58-M2 was evaluated. The synthesis of p58-M2 was measured by metabolic labeling of primary cultures derived from the regenerating liver of euthyroid, hypo- or hyperthyroid rats. During regeneration, the increase in the liver/body weight ratio is approximately 25% higher in hyper- than in hypothyroid rats. However, T3 has no effect on the rate of overall liver regeneration observed in four days. In mature liver, T3 increased the synthesis of p58-M2 by approximately 2.5-fold. During regeneration, however, the change in the synthesis of p58-M2 varied with the thyroid status. In euthyroid rats, the synthesis of p58-M2 continued to increase up to 2-fold during liver regeneration. In hyperthyroid rats, after an initial increase by 1.5-fold on day 1, the synthesis of p58-M2 subsequently declined during regeneration. In hypothyroid rats, the synthesis of p58-M2 remained virtually unchanged during regeneration. These results indicate that T3 regulates the synthesis of p58-M2 in mature and regenerating liver.     

Regulation of enzymes of glucose metabolism and lipogenesis in diabetic rat liver by thyroid hormones

Experimental diabetes in rats is associated with a degree of hypothyroidism. Hepatic enzymes involved in carbohydrate and lipid metabolism were estimated in control (untreated), control +T3 treated, alloxan diabetic and alloxan diabetic + T3-treated rats. The key glycolytic enzymes, phosphofructokinase and pyruvate kinase, were decreased in activity in diabetes and unchanged by further treatment with T3. In contrast, certain enzymes involve in lipogenesis, ATP-citrate lyase 'malic' enzyme and 6-phosphogluconate dehydrogenase, which were decreased in activity in diabetes, were increased to, or above, control values when diabetic rats were treated with T3. It is suggested that T3 deficiency may play a role in the decrease in enzyme activities observed in experimental diabetes, in particular, some enzymes associated with lipogenesis and the provision of NADPH.     

Phenylalanyl-tRNA synthetases of rat liver: differential effects of thyroid hormone.

Thyroxine and analogues inhibit rat liver aminoacyl-tRNA synthetase activity for phenylalanine and tyrosine. A high yield purification of the major cytoplasmic form of phenylalanyl-tRNA synthetase (C1) and its characterization is reported. Polyribosome-bound and other sedimentable forms are found to be indistinguishable from soluble enzyme by immunoprecipitation. Mitochondrial phenylalanyl-tRNA synthetase (M) and cytoplasmic activity (C2) resistant to anti-C1 antibody have been partially purified and characterized. Tissue levels of the three forms are estimated at 22, 1.8, and 4.1 units/g of liver for C1, C2, and M, respectively [1 unit = 1 nmol of Phe-tRNA/min, 30 degrees C]. Charging capability toward rat liver and yeast tRNA, kinetic parameters, and physical properties are compared. Only enzyme C1 is hormone inhibited [K1 = 4 x 10(-6) M for triiodothyronine]. The data indicata that C2 and M are not structurally related to C1; C2 may represent an independent cytoplasmic pool of M. Implications of C1 inhibition in relation to effects on liver protein synthesis are discussed.     

Uptake of thyroid hormone by isolated rat liver cells

L-Triiodothyronine is taken up by isolated rat liver cells by a process which is saturable and exhibits sigmoidity. Two uptake systems make themselves evident: A system with high affinity with an apparent K_t value of 52±22 nM and a system with low affinity with an apparent K_t value of 1446±764 nM. Cells heated at 60°C or after freezing do not show saturability of uptake. KCN inhibits the uptake by the low affinity system. In the presence of L-thyroxine and L-tyrosine the uptake of L-triiodothyronine is increased. The results suggest transport of L-triiodothyronine by proteins in the plasma membrane of the liver cell.     

Immunohistochemical localization of thyroid hormone in rat thyroid gland.

Direct and indirect immunofluorescence techniques were used to localize the thyroid hormones triidothyronine (T3) and thyroxine (T4) in adult rat thyroid gland. Optimum dilutions of the antisera were established and four tissue fixatives were investigated for usefulness in this technique. Use of antibodies specific for either T3 or T4 resulted in brilliant fluorescence in the colloid pools and apical cytoplasm of follicular cells. In all cases, the adjacent parathyroid gland was devoid of fluorescence. This report demonstrates that these dipeptide hormones can be localized by using immunofluorescence techniques.     

Regulation of cholesterol metabolism in the ethionine-induced premalignant rat liver

The early premalignant liver provides a model in which to study metabolic alterations that may be permissive for the development of full malignancy. Although there are biochemical changes in this model, there are no detectable morphological ones when compared with a normal, fully differentiated liver. The maintenance of cholesterol homeostasis, essential for proper functioning of mammalian cells, is known to be altered in malignancy. We used the ethionine-induced premalignant liver model to study the effects of the premalignant state on cellular parameters involved in the maintenance of hepatic cholesterol homeostasis. Cholesterol synthesis was elevated about twofold in the livers of rats treated with ethionine as was the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, its rate limiting enzyme. There was no change in reductase activation state. Acyl coenzyme A:cholesterol acyl-transferase (ACAT) was decreased about 30%, and cholesterol 7 alpha-hydroxylase, about 50%. There was no significant change in neutral cholesteryl ester hydrolase activity, but acid hydrolase activity was decreased. There was little change in low density lipoprotein receptor protein as determined by immunoblotting. Biliary lipid secretion was in the normal range when expressed per gram liver; however, bile flow was doubled. The ethionine-fed animals were mildly hypocholesterolemic and had an altered serum lipoprotein pattern. Cholesterol synthesis and HMG-CoA reductase activity exhibited decreased sensitivities to inhibition by dietary cholesterol when compared to control livers. However, sensitivity to intragastrically administered mevalonolactone was not altered. Although ACAT activity was increased by mevalonolactone administration to levels similar to those in untreated animals, it was not increased in the ethionine-fed animals by feeding cholesterol. The ethionine-induced premalignant liver responded to ethinyl estradiol treatment in a manner similar to that of the control, i.e., profound hypolipidemia, increased low density lipoprotein receptors, decreased reductase activity, and increased cholesterol esterification. Thus, these livers retained their estrogen responsiveness. Taken together, the data demonstrate that the major elements involved in maintaining hepatic cholesterol homeostasis are present in the premalignant liver, although in some cases at levels that are different from the control. However, the susceptibility to regulation was altered in these livers to suggest markedly decreased availability of cholesterol of exogenous origin to the regulatory compartment(s). Further, coupling of the different elements involved in maintenance of hepatic cholesterol homeostasis appeared to have been changed.     

Cold adaptation and thyroid hormone metabolism.

Resting oxygen consumption and energy expenditure is sensitive to slight alterations in thyroid function. This means that timing and magnitude of cold adaptation would to some extent depend on thyroid function. Local thyroid hormone metabolism is important for energy expenditure and dissipation of heat in special tissues. Recruitment of brown adipocytes and upregulation of uncoupling protein 1 in mitochondria depends on high tissue T3 concentrations. Most of this T3 is derived from local 5' deiodination of T4. Brown fat is vital for cold exposed mice and rats, and may be important for temperature adaptation in human neonates. The role of thyroid hormone metabolism in adult human cold adaptation has not been finally clarified. Hypothetically, cold exposure may enhance T3 production by deiodination of T4 in skeletal muscle, which may enhance heat production in muscle via a change in muscle fiber type. Another hypothetical possibility is recruitment of brown adipocytes embedded in white adipose tissue in human adults. Understanding cold adaptation in human adults may lead to development of new drugs against obesity.     

The role of selenium in thyroid hormone metabolism.

In animals, decreases in selenium-containing glutathione peroxidase activity and the resultant impairment of peroxide metabolism can account for many, but not all of the biochemical and clinical changes caused by selenium deficiency. Recently, however, type I iodothyronine 5'-deiodinase has also been shown to be a selenium-containing enzyme. This explains the impairment of thyroid hormone metabolism caused by selenium deficiency in animals with a normal vitamin E status. Since iodothyronine 5'-deiodinases are essential for the production of the active thyroid hormone 3,5,3'-triiodothyronine, some of the consequences of selenium deficiency may result from thyroid changes rather than inability to metabolise peroxides. In particular, the impaired thyroid hormone metabolism may be responsible for decreased growth and resistance to cold stress in selenium-deficient animals. A further consequence of the role of selenium in thyroid hormone metabolism is the exacerbation of some of the thyroid changes in iodine deficiency by a concurrent selenium deficiency. Selenium status may therefore have a major influence on the outcome of iodine deficiency in both human and animal populations.     

Bicarbonate ion-ATPase in rat liver cell fractions.

The distribution of HCO3MINUS-ATPase activity was studied in cell fractions prepared from homogenates of rat liver. The level of mitochondrial contamination in the microsomal fraction depended on the fractionation procedure and on the method of homogenization. With proper care, microsomes with undetectable mitochondrial contamination could be prepared. These microsomes had no detectable HCO3MINUS-ATPase activity. Approximately 85% of the total HCO3minus-ATPase activity of the post 6000 times g-min supernatant was recovered in the mitochondrial fraction. The properties of this mitochondrial HCO3minus-ATPase were not distinguishable from those of the various microsomal HCO3minus-ATPases previously described by other investigators.