Influence of chronically altered thyroid status on the activity of liver mitochondrial glycerol-3-phosphate dehydrogenase in female inbred lewis rats.

The activity of liver mitochondrial flavoprotein-dependent glycerol-3-phosphate dehydrogenase (GPDH) is considered a reliable marker of thyroid status in acute and short-lasting experiments. The aim of this study was to ascertain whether GPDH activity could also be used as an index of thyroid status during chronic experiments over several months. We therefore analyzed GPDH activity in liver mitochondria of female inbred Lewis rats with thyroid status altered for 2 to 12 months. Hyperthyroid state was maintained by triiodothyronine (T (3)) or thyroxine (T (4)) administration, while methimazole was employed for inducing hypothyroidism. We found a seven- and three-fold increase of GPDH activity in female rats after T (3) or T (4) administration, respectively, compared to euthyroid females (8.9 +/- 2.3 nmol/min/mg protein), whereas administration of methimazole reduced the enzyme activity almost to one-third of the euthyroid values. These changes were not significantly influenced by the duration of hyperthyroid or hypothyroid treatment. We conclude that the level of the rat liver GPDH activity could serve as a useful marker for evaluation of hyperthyroid and hypothyroid status in chronic long-lasting experiments on female inbred Lewis rats.     

Stimulation of phosphate transport in rat-liver mitochondria by thyroid hormones

The effect of hyperthyroidism on the transport of phosphate in rat-liver mitochondria has been examined. Thyroid hormones administered in vivo increased carrier mediated (mersalyl-sensitive) phosphate transport. Kinetic analysis of the phosphate transport showed that the thyroid hormone affects the Vmax of this process, while having no effect on the Km values. The higher activity of the phosphate carrier was found not to be due to a change in the endogenous content of phosphate nor to a change in the transmembrane delta pH value. Inhibitor titrations with mersalyl showed that mitochondria from both control and hyperthyroid rats required the same concentrations of inhibitor to produce total inhibition of phosphate transport, thus suggesting that the amount of functional translocase present is unaffected. The level of cardiolipin was significantly higher in mitochondrial membranes from hyperthyroid rats as compared to the control rats. The thyroid hormone induced change in the activity of the phosphate carrier appears to be due to a more favorable lipid microenvironment (cardiolipin content) surrounding the carrier molecule in the mitochondrial membrane.     

Influence of cold exposure and thyroid hormones on regulation of adrenal catecholamines.

Since thyroid hormones influence urinary excretion of catecholamines after exposure to cold, the effects of hyper- and hypo-thyroidism on adrenal tyrosine hydroxylase (TH) (EC 1.14.16.2), phenylethanolamine-N-methyl transferase (PNMT) (EC 2.1.1.28), and serum dopamine-beta-hydroxylase (DbetaH) (EC 1.14.17.1) of rats of 23 and 4 degrees C were studied. TH changes resembled the urinary excretion pattern at 4 degrees C in being higher after 8 days than after 1 day of exposure, and in declining as acclimation occurred. At 23 degrees C, TH activity of hypothyroid rats was significantly higher than in euthyroid or hyperthyroid animals, and after 1 day at 4 degrees C the value increased even more. While in the hypothyroid animals at 4 degrees C the concentration of adrenal catecholamines was less, the epinephrine to norepinephrine ratio was higher than at 23 degrees C. Very high TH activity with a decline in catecholamine concentration suggests that the capacity of TH had been exceeded. PNMT activity was significantly elevated in this group. TH activity was not decreased in the hyperthyroid group at 23 degrees C, and was increased after 8 days at 4 degrees C, suggesting that circulating thyroid hormones have no direct inhibitory effect on TH. Serum DbetaH was elevated after exposure to 4 degrees C, regardless of thyroid hormonal status. The activation of adrenal TH in hypothyroid rats at 23 degrees C and of TH, PNMT, and serum DbetaH at 4 degrees C is probably the result of increased activity of the sympathetic nervous system.     

Sex-specific phenotypes of hyperthyroidism and hypothyroidism in mice

Background

Thyroid dysfunction is more common in the female population, however, the impact of sex on disease characteristics has rarely been addressed. Using a murine model, we asked whether sex has an influence on phenotypes, thyroid hormone status, and thyroid hormone tissue response in hyper- and hypothyroidism.

Methods

Hypo- and hyperthyroidism were induced in 5-month-old female and male wildtype C57BL/6N mice, by LoI/MMI/ClO₄ ^? or T₄ i.p. treatment over 7 weeks, and control animals underwent sham treatment (N?=?8 animals/sex/treatment). Animals were investigated for impact of sex on body weight, food and water intake, body temperature, heart rate, behaviour (locomotor activity, motor coordination, and strength), liver function, serum thyroid hormone status, and cellular TH effects on gene expression in brown adipose tissue, heart, and liver.

Results

Male and female mice showed significant differences in behavioural, functional, metabolic, biochemical, and molecular traits of hyper- and hypothyroidism. Hyperthyroidism resulted in increased locomotor activity in female mice but decreased muscle strength and motor coordination preferably in male animals. Hypothyroidism led to increased water intake in male but not female mice and significantly higher serum cholesterol in male mice. Natural sex differences in body temperature, body weight gain, food and water intake were preserved under hyperthyroid conditions. In contrast, natural sex differences in heart rate disappeared with TH excess and deprivation. The variations of hyper- or hypothyroid traits of male and female mice were not explained by classical T₃/T₄ serum state. TH serum concentrations were significantly increased in female mice under hyperthyroidism, but no sex differences were found under eu- or hypothyroid conditions. Interestingly, analysis of expression of TH target genes and TH transporters revealed little sex dependency in heart, while sex differences in target genes were present in liver and brown adipose tissue in line with altered functional and metabolic traits of hyper- and hypothyroidism.

Conclusions

These data demonstrate that the phenotypes of hypo- and hyperthyroidism differ between male and female mice and indicate that sex is an important modifier of phenotypic manifestations.

     

Postnatal development of peroxisomal and mitochondrial enzymes in rat liver.

Subcellular organellles from livers of rats three days prenatal to 50 weeks postnatal were separated on sucrose gradients. The peroxisomes had a constant density of 1.243 g/ml throughout the life of the animal. The density of the mitochondria changed from about 1.236 g/ml at birth to a constant value of 1.200 g/ml after two weeks. The peroxisomal and mitochondrial fatty acid beta-oxidation and the peroxisomal and supernatant activities of catalase and glycerol-3-phosphate dehydrogenase were measured at each age, as well as the peroxisomal core enzyme, urate oxidase, and the mitochondrial matrix enzyme, glutamate dehydrogenase. All of these activities were very low or undetectable before birth. Mitochondrial glutamate dehydrogenase and peroxisomal urate oxidase reached maximal activities per g of liver at two and five weeks of age, respectively. Fatty acid beta-oxidation in both peroxisomes and mitochondria and peroxisomal glycerol-3-phosphate dehydrogenase exhibited maximum activities per g of liver between one and two weeks of age before weaning and then decreased to steady state levels in the adult. Peroxisomal beta-oxidation accounted for at least 10% of the total beta-oxidation activity in the young rat liver, but became 30% of the total in the liver of the adult female and 20% in the adult male due to a decrease in mitochondrial beta-oxidation after two weeks of age. The greatest change in beta-oxidation was in the mitochondrial fraction rather than in the peroxisomes. At two weeks of age, four times as much beta-oxidation activity was in the mitochondria as in the peroxisomal fraction. Peroxisomal glycerol-3-phosphate dehydrogenase activity accounted for 5% to 7% of the total activity in animals younger than one week, but only 1% to 2% in animals older than one week. Up to three weeks of age, 85% to 90% of the liver catalase was recovered in the peroxisomes. The activity of peroxisomal catalase per g of rat liver remained constant after three weeks of age, but the total activity of catalase further increased 2.5- to 3-fold, and all of the increased activity was in the supernatant fraction.     

Effect of thyroid hormone on the turnover of rat liver pyruvate carboxylase and pyruvate dehydrogenase.

Immunochemical techniques have been utilized to study the effect of thyroid status on the content and rates of synthesis and degradation of pyruvate carboxylase and pyruvate dehydrogenase in rat liver. Liver from hyperthyroid rats had twice the pyruvate carboxylase activity of normal rats while thyroidectomized rats had about two-thirds of normal activity. Pyruvate dehydrogenase complex activity was unchanged in the hyperthyroid state but was significantly reduced (by a third) in hypothyroid rats. Changes in catalytic activity during altered thyroid status were by immunochemical means to be closely related to the amount of the hepatic enzymes present. Isotopic studies showed that the changes in the content of pyruvate carboxylase and pyruvate dehydrogenase reflected alterations in the rate of the synthesis of the enzymes with the degradation rates little affected by thyroid status. The half-life for pyruvate carboxylase was 4.6 days, and that for pyruvate dehydrogenase, 8.1 days. In both cases, the turnover time was slower than that of the average mitochondrial protein (t1/2 = 3.8 days) for the control animals.     

Sex-specific phenotypes of hyperthyroidism and hypothyroidism in aged mice

Background

Sex and age play a role in the prevalence of thyroid dysfunction (TD), but their interrelationship for manifestation of hyper- and hypothyroidism is still not well understood. Using a murine model, we asked whether sex impacts the phenotypes of hyper- and hypothyroidism at two life stages.

Methods

Hyper- and hypothyroidism were induced by i.p. T4 or MMI/ClO₄-/LoI treatment over 7 weeks in 12- and 20-months-old female and male C57BL/6N mice. Control animals underwent PBS treatment (n?=?7–11 animals/sex/treatment). Animals were investigated for impact of sex on body weight, food and water intake, body temperature, heart rate, behaviour (locomotor activity, motor coordination and strength) and serum thyroid hormone (TH) status.

Results

Distinct sex impact was found in eu- and hyperthyroid mice, while phenotypic traits of hypothyroidism were similar in male and female mice. No sex difference was found in TH status of euthyroid mice; however, T4 treatment resulted in twofold higher TT4, FT4 and FT3 serum concentrations in adult and old females compared to male animals. Hyperthyroid females consistently showed higher locomotor activity and better coordination but more impairment of muscle function by TH excess at adult age. Importantly and in contrast to male mice, adult and old hyperthyroid female mice showed increased body weight. Higher body temperature in female mice was confirmed in all age groups. No sex impact was found on heart rate irrespective of TH status in adult and old mice.

Conclusions

By comparison of male and female mice with TD at two life stages, we found that sex modulates TH action in an organ- and function-specific manner. Sex differences were more pronounced under hyperthyroid conditions. Importantly, sex-specific differences in features of TD in adult and old mice were not conclusively explained by serum TH status in mice.

     

Thyroid hormone regulation of flavocoenzyme biosynthesis

The means by which thyroid hormone regulates flavocoenzyme biosynthesis was studied in hyper-, eu-, and hypothyroid rats by determining the activities of flavocoenzyme-forming enzymes, viz., flavokinase and FAD synthetase, as well as those of flavocoenzyme-degrading enzymes, viz., FMN phosphatase and FAD pyrophosphatase. Flavokinase activity was increased in hyperthyroid animal and decreased in hypothyroid animals. Correspondence of flavokinase activity with the amount of a high-affinity flavin-binding protein quantitated immunologically in hypo-, eu-, and hyperthyroid rats indicated that the thyroid response is caused by an increased amount of enzyme; moreover, the concomitant decrease in a low-affinity flavin-binding protein suggests an inactive precursor form of flavokinase. FAD synthetase activity showed a similar but less pronounced trend than flavokinase. Activities of FMN phosphatase and FAD pyrophosphatase were not influenced by thyroid hormone. Overall results indicate that the mechanism of thyroid hormone regulation of flavocoenzyme level is in the steps of biosynthesis, especially at flavokinase, rather than in degradation steps.     

Sex-linked changes in immunoreactive glucose-6-phosphate dehydrogenase in rat liver

The level of hepatic immunoreactive glucose-6-phosphate dehydrogenase protein was found to correlate well with the enzyme activity in adult rats fed the stock laboratory diet in a variety of hormonal conditions. The amount of immunoreactive protein and enzyme activity was 2-fold greater in sexually mature female rats compared with aged matched male animals. However, this difference was absent in diabetic animals, and furthermore although triiodothyronine administration to the diabetic male rat could restore the level of enzyme activity to that of the normoglycaemic animal, it was much less effective in the female animal. In contrast, administration of insulin to the normoglycaemic animal increased the level of glucose-6-phosphate dehydrogenase in the female, but was without effect in the male. These results are discussed in relation to the possible role of thyroid status and steroid sex hormones in the regulation of hepatic glucose-6-phosphate dehydrogenase.     

Effects of fatty (fa) allele and high-fat diet on adipose tissue leptin and lipid metabolism.

Leptin is an adipocyte-secreted hormone that binds hypothalamic receptors and potently decreases food intake. Leptin receptor defects in homozygous mutant Zucker fatty ( fa/fa) rats lead to massive obesity, hyperphagia, decreased energy expenditure, and insulin resistance, while the phenotype of heterozygous ( Fa/fa) lean rats lies between lean ( Fa/Fa) and obese ( fa/fa) rats. Whether heterezygotes exhibit specific changes in lipid metabolism in a diet-responsive manner is not clear. Thus, the specific aim of this study was to test whether the presence of one fa allele modulates lipid metabolism and leptin, and whether these effects are exacerbated by high-fat diet. We demonstrate that the presence of one fa allele significantly increases lipogenesis in adipose tissue assessed by glycerol-3-phosphate dehydrogenase (GPDH) and fatty acid synthase (FAS) activities. FAS is more responsive to high-fat diets than GPDH in Fa/fa rats. Adipose tissue leptin levels are significantly higher in fat pads of Fa/fa compared to Fa/Fa rats. Moreover, Fa/fa rats fed high-fat diet show an additional two-fold increase in leptin levels compared to wild type rats on the same diet. Collectively, these results indicate that the presence of one fa allele increase adipocyte lipogenic enzyme activities, which results in hyperleptinemia concurrent with increased adiposity.     

Appearance of a second form of hepatic glycerol-3-phosphate dehydrogenase during neonatal development in the mouse

Livers of 4-day-old Balb/c mice contain a single form of glycerol-3-phosphate dehydrogenase (GPDH, sn-glycerol-3-phosphate:NAD 2-oxido-reductase, EC 1.1.1.8), which is designated GPDH I. The amount of hepatic GPDH I declines during maturation and levels off by about 6–7 weeks of age. During neonatal development a second form of the enzyme, GPDH II, appears. In mature Balb/c mice (6–7 weeks of age) GPDH II comprises about 30% of the total hepatic GPDH activity. Half-maximal levels of GPDH II are attained by about 18 days postpartum. The rate of appearance of GPDH II is not affected by premature weaning. Hepatic GPDH II is chromatographically distinct from hepatic GPDH I or the embryonic isozyme observed in neonatal brains. Thermal denaturation studies indicate that GPDH I and II from Balb/c mice are denatured at 50 °C with a half-time of about 2 min while the embryonic isozyme is denatured with a half-time of about 30 s. GPDH I and II isolated from C57BL6 mice are denatured at 50 °C with a half-time of 6 min while forms I and II from DDS mice are denatured with a half-time of about 12 min. Kinetic studies reveal that GPDH II and the embryonic isozyme have similar apparent affinities for NADH and dihydroxyacetone phosphate. The apparent affinity of GPDH I for NAD, NADH, dihydroxyacetone phosphate, and glycerol-3-phosphate is lower than that of GPDH II.     

Enhanced activity of the tricarboxylate carrier and modification of lipids in hepatic mitochondria from hyperthyroid rats

The effect of hyperthyroidism on the activity of the mitochondrial tricarboxylate carrier has been studied. The activity of this transporting system in liver mitochondria was quantitatively determined by the rate of malate-[14C]citrate exchange using the 1,2,3-benzene-tricarboxylate inhibitor stop technique. It has been found that the rate of citrate uptake is significantly enhanced in liver mitochondria from hyperthyroid rats as compared to that obtained in mitochondria from control rats. Kinetic analysis of the malate-citrate exchange reaction indicates that only the Vmax of this transporting process is enhanced, while there is practically no change in the Km values. Inhibitor titrations with the inhibitor palmitoyl-CoA show that mitochondria from hyperthyroid rats require the same concentrations of inhibitor to produce 100% inhibition of citrate uptake as control mitochondria, suggesting that the amount of functional translocase enzyme present is unaffected. The Arrhenius plot characteristics differ for tricarboxylate carrier activity in mitochondria from hyperthyroid rats as compared with control rats in that the break point of the biphasic plot decreases from 18.1 +/- 1.4 degrees C in controls to 12.9 +/- 1.2 degrees C in hyperthyroid animals. The hepatic mitochondrial lipid composition is altered significantly in hyperthyroid rats; the total cholesterol decreases and the phospholipids increase. The liver mitochondrial phospholipid composition is altered significantly in hyperthyroid rats. In particular negatively charged phospholipid cardiolipin increases by more than 50%. Minor alterations were found in the pattern of fatty acids. The thyroid hormone induced change in the activity of the tricarboxylate carrier can be ascribed either to a general modification of membrane lipid composition which increases the membrane fluidity and in turn the mobility of the carrier or to a more localized change of lipid domain (cardiolipin content) surrounding the carrier molecule in the mitochondrial membrane.     

The induction of mitochondrial L-3-glycerophosphate dehydrogenase by thyroid hormone

L-3-Glycerophosphate dehydrogenase was purified from porcine brain mitochondria by a shorter and simpler procedure than previously reported. Immunoblotting with antiserum to the porcine enzyme established that rat liver L-3-glycerophosphate dehydrogenase has the same Mr (76 000) by SDS-polyacrylamide gel electrophoresis. In liver mitochondria from normal and hyperthyroid rats, changes in L-3-glycerophosphate dehydrogenase activity were parallelled by changes in enzyme content assayed by immunoblotting. Similar changes were found in the amount of enzyme synthesised in vitro by reticulocyte lysate programmed with rat liver mRNA, suggesting that thyroid hormone causes specific induction of L-3-glycerophosphate dehydrogenase mRNA.     

香鱼甘油-3-磷酸脱氢酶基因的克隆与表达

GPDH(glycerol-3-phosphate dehydrogenase)是合成脂肪代谢中间产物甘油-3-磷酸的关键酶。通过设计简并引物从香鱼肝cDNA文库中克隆GPDH基因,该基因cDNA序列全长577个核苷酸,单一大的开放阅读框编码一个由351个氨基酸组成的分子量为37.9kD的蛋白。蛋白序列分析表明,香鱼GPDH(aGPDH)与亚洲胡瓜鱼的GPDH序列同源性最高。系统进化树分析表明,GPDH的物种进化关系与目前接受的物种分类关系基本一致。实时荧光定量PCR结果显示,aGPDH基因在香鱼肝、脾、肾、脑、心和肌肉组织均有表达。香鱼咸淡水适应以后,肝、脾、脑、心和肌肉的aGPDH的mRNA表达水平下调。成功构建重组表达质粒pET-32a-GPDH。SDS-PAGE试验表明,目的蛋白可以在大肠杆菌中大量表达;并制备了抗血清,能与目的蛋白起强的特异性反应,但不与细菌自身蛋白起反应。本研究有助于进一步理解鱼类盐度适应过程中的脂肪代谢调控机制。     

Molecular and physiological properties of tyrosine hydroxylase induced by reserpine in rat adrenal gland

Newly synthesized tyrosine hydroxylase (TH) induced by reserpine was compared with the enzyme in control rats in terms of the molecular and physiological properties. When repeated doses of reserpine were given at daily intervals for three days, the enzyme activity measured in homogenates of the adrenal glands was increased 3-fold. Furthermore, when TH in the adrenal glands from both control and reserpine-treated rats was purified, both total activity of the enzyme and the enzyme protein content purified from reserpine-treated rats were also about 3-fold higher than those of the control rats. The two purified enzymes revealed similar properties; a single subunit with a Mr of 60,000 was observed by SDS polyacrylamide gel electrophoresis, and the Km value for a pterin cofactor, 6-methyl-tetrahydropterin was about 300 microM. In contrast, in situ TH activity measured under physiological conditions at pH 7.2 in adrenal tissue slices was elevated 6-fold by reserpine pretreatment for 3 days, and was stimulated by carbachol (0.1 mM) and elevated K+ (52 mM) in a roughly proportional rather than additive way relative to slices from untreated rats. These results indicate that newly synthesized TH induced by reserpine in rat adrenal gland had similar properties as the enzyme in control rats and that reserpine increased not only the amount of TH molecules but also the in situ activity of TH. Since reserpine also increases the biosynthesis of tetrahydrobiopterin as demonstrated by Viveros and co-workers, this 6-fold increase in in situ TH activity may depend both upon the 3-fold increase in the amount of enzyme molecules and upon the increase of the physiologically available tetrahydrobiopterin in the adrenal gland.     

Modulation of rat liver mitochondrial antioxidant defence system by thyroid hormone

In the present study the effect of thyroid hormone (T(3)) on oxidative stress parameters of mitochondria of rat liver is reported. Hypothyroidism is induced in male adult rats by giving 0.05% propylthiouracil (PTU) in drinking water for 30 days and in order to know the effect of thyroid hormone, PTU-treated rats were injected with 20 microg T(3)/100 g body weight/day for 3 days. The results of the present study indicate that administration of T(3) to hypothyroid (PTU-treated) rats resulted in significant augmentation of oxidative stress parameters such as thiobarbituric acid reactive substances and protein carbonyl content of mitochondria in comparison to its control and euthyroid rats. The hydrogen peroxide content of the mitochondria of liver increased in hypothyroid rats and was brought to a normal level by T(3) treatment. Induction of hypothyroidism by PTU treatment to rats also resulted in the augmentation of total and CN-sensitive superoxide dismutase (SOD) activities of the mitochondria, which was reduced when hypothyroid rats were challenged with T(3). Although CN-resistant SOD activity of the mitochondria remained unaltered in response to hypothyroidism induced by PTU treatment, its activity decreased when hypothyroid rats were injected with T(3). The catalase activity of the mitochondria decreased significantly by PTU treatment and was restored to normal when PTU-treated rats were given T(3). Total, Se-independent and Se-dependent glutathione peroxidase activities of the mitochondria were increased following PTU treatment and reduced when T(3) was administered to PTU-treated rats. The reduced and oxidised glutathione contents of the mitochondria of liver increased significantly in hypothyroid rats and their level was restored to normal when hypothyroid rats were injected with T(3). The results of the present study suggest that the mitochondrial antioxidant defence system is considerably influenced by the thyroid states of the body.