The influence of hypothyroidism on the transport of phosphate and on the lipid composition in rat-liver mitochondria.

The influence of hypothyroidism on the transport of phosphate and on the lipid composition in rat-liver mitochondria was examined. It was found that the rate of phosphate transport is reduced (around 40%) in mitochondria from hypothyroid rats compared to that obtained in mitochondria from normal rats. Treatment of hypothyroid rats with thyroid hormone reverses this effect completely. Kinetic analysis of the phosphate transport indicates that only the Vmax of this process is affected, while there is no change in the Km values. The lower rate of phosphate transport in mitochondria from hypothyroid rats is also demonstrated by swelling experiments. There is no significant difference either in the respiratory control ratios or in the ADP/O ratios between these two types of mitochondria. The hepatic mitochondrial lipid composition is altered significantly in hypothyroid rats. The total cholesterol increases, the phospholipids decrease and the cholesterol/phospholipid molar ratio increases (around 40%). Among the phospholipids, cardiolipin shows the greatest alteration (30% decrease in the hypothyroid rats). The phosphatidylethanolamine/phosphatidylcholine ratio also decreases. Alterations were also found in the pattern of fatty acids. These changes in lipid composition may be responsible, at least in part, for the depression of the phosphate carrier activity in mitochondria from hypothyroid rats.     

Decreased activity of the pyruvate translocator and changes in the lipid composition in heart mitochondria from hypothyroid rats

A study of the transport of pyruvate in heart mitochondria from normal and hypothyroid rats has been carried out. Heart mitochondria from hypothyroid rats translocate pyruvate via the alpha-cyanocinnamate sensitive carrier much more slowly than do mitochondria from normal rats. Kinetic analysis of the pyruvate transport shows that the Vmax of this process is decreased while there is practically no change in the Km values. Neither a decrease in the transmembrane delta pH value nor a decrease in the total number of the pyruvate carrier molecules, titrated with labeled alpha-cyanocinnamate, account for the decreased rate of pyruvate transport. The lower activity of the pyruvate translocator in mitochondria from hypothyroid rats is associated with a parallel decrease of the rate of pyruvate supported oxygen uptake. There is, however, no difference in either the respiratory control ratios or in the ADP/O ratios between these two types of mitochondria. The heart mitochondrial lipid composition is significantly altered in hypothyroid rats. Cardiolipin, particularly, was found to decrease by around 36%. In addition the pattern of fatty acids was found to be altered in mitochondrial membranes from hypothyroid rats. It is suggested that the decreased activity of the pyruvate translocator in heart mitochondria from hypothyroid rats can be ascribed to changes in the lipid environment which surrounds the pyruvate carrier molecule in the mitochondrial membrane.     

Covariance of tricarboxylate carrier activity and lipogenesis in liver of polyunsaturated fatty acid (n-6) fed rats.

The mitochondrial tricarboxylate (citrate) carrier plays an important role in hepatic intermediary metabolism because, among other functions, it supplies the cytosol with acetyl units for fatty-acid synthesis. In this study, the effect of polyunsaturated fatty acids (PUFA, n-6) on the function of this mitochondrial transporter and on lipogenic enzyme activities was investigated by feeding rats for 4 weeks with a 15%-fat diet composed of high linoleic safflower oil. Citrate transport was strongly reduced in liver mitochondria isolated from PUFA-treated rats. A reduced transport activity was also observed when solubilized mitochondrial citrate carrier from PUFA-treated rats was reconstituted into liposomes. In the same animals, a decrease of cytosolic lipogenic enzyme activities was observed. These results indicate a coordinated modulation of citrate carrier and of lipogenic enzyme activities by PUFA feeding. Kinetic analysis of the carrier activity showed that only V(max) decreased, whereas K(m) was almost virtually unaffected. The PUFA-mediated effect is most likely due to the reduced mRNA level and lower content of the citrate carrier protein observed in the safflower oil-fed rats.     

Differential effects of coconut oil- and fish oil-enriched diets on tricarboxylate carrier in rat liver mitochondria

The mitochondrial tricarboxylate carrier (TCC) plays an important role in lipogenesis being TCC-responsible for the efflux from the mitochondria to the cytosol of acetyl-CoA, the primer for fatty acid synthesis. In this study, we investigated the effects of two high-fat diets with different fatty acid composition on the hepatic TCC activity. Rats were fed for 3 weeks on a basal diet supplemented with 15% of either coconut oil (CO), abundant in medium-chain saturated fatty acids, or fish oil (FO), rich in n-3 polyunsaturated fatty acids. Mitochondrial fatty acid composition was differently influenced by the dietary treatments, while no appreciable change in phospholipid composition and cholesterol level was observed. Compared with CO, the TCC activity was markedly decreased in liver mitochondria from FO-fed rats; kinetic analysis of the carrier revealed a decrease of the Vmax, with no change of the Km. No difference in the Arrhenius plot between the two groups was observed. Interestingly, the carrier protein level and the corresponding mRNA abundance decreased following FO treatment. These data indicate that FO administration markedly decreased the TCC activity as compared with CO. This effect is most likely due to a reduced gene expression of the carrier protein.     

A tissue-specific increase in lipogenesis in rat brown adipose tissue in hypothyroidism.

1. Rats were made hypothyroid by feeding them with propylthiouracil together with a low-iodine diet for 4 weeks. 2. [U-14C]Glucose conversion into fatty acids was substantially enhanced in brown adipocytes isolated from hypothyroid rats. Incorporation of 3H2O into fatty acids in vivo was enhanced in hypothyroidism in interscapular brown fat, but not in epididymal white fat or in liver. Hypothyroidism increased the activities of fatty acid synthase and ATP citrate lyase in brown, but not in white, adipocytes. 3. Glycolytic flux in brown adipocytes, quantified by [3-3H]glucose detritiation, was increased by hypothyroidism. This change was accompanied by increased maximum activity of phosphofructokinase. In white adipocytes a large increase in phosphofructokinase maximum activity was observed in hypothyroidism, but this change was accompanied by only small increases in the rate of glucose detritiation by incubated cells. It is suggested that in the brown adipocyte the overall conversion of glucose into fatty acids is enhanced in thyroid deficiency, but that this change is muted in the white adipocyte, possibly because of limitation of glucose transport. 4. Fatty acid synthesis in brown adipocytes from hypothyroid animals was considerably less sensitive to inhibition by exogenous fatty acids than is the process in cells from euthyroid animals. Consequently, the effect of hypothyroidism to enhance lipogenesis is amplified in the presence of physiological concentrations of fatty acid.     

Adenine nucleotide translocation in liver mitochondria of hypothyroid rats.

In measurements using a disc filtration method, liver mitochondria obtained from hypothyroid rats translocate external ADP at 0 °C via the atractyloside-sensitive carrier much more slowly than do mitochondria from normal rats, confirming the findings of Portnay et al. (Biochem. Biophys. Res. Commun. 55, 17, 1973). The hypothyroid mitochondria contain 60% more ATP + ADP than do mitochondria from normals, but the excess nucleotides are not exchangeable and so do not contribute to translocation. A decrease in the first-order rate constant accounts for the decreased velocity. Neither a decrease in the number of translocator sites nor changes in ADP phosphorylation or ATPase activity seem to account for the abnormal kinetics of translocation. Although the filtration method limits the maximal translocation rate observed in normal mitochondria at temperatures above 17 °C that induce a fluid membrane state, no such transition is seen in mitochondria from hypothyroid rats up to 35 °C, indicating that the translocator is in an altered environment in hypothyroidism. Injecting a hypothyroid rat once with l-thyroxine corrects the abnormal compartmentation and produces a temperature-rate relationship like that in normal mitochondria in 3 days, a period which would accommodate the hormone actions reported on translation, membrane phospholipid synthesis, or fatty acid desaturation.     

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.     

3,5-Diiodo-L-Thyronine increases F<Subscript>o</Subscript>F<Subscript>1</Subscript>-ATP synthase activity and cardiolipin level in liver mitochondria of hypothyroid rats

Short-term effects of 3,5-L-diiodothyronine (T₂) administration to hypothyroid rats on F_oF₁-ATP synthase activity were investigated in liver mitochondria. One hour after T₂ injection, state 4 and state 3 respiration rates were noticeably stimulated in mitochondria subsequently isolated. F_oF₁-ATP synthase activity, which was reduced in mitochondria from hypothyroid rats as compared to mitochondria from euthyroid rats, was significantly increased by T₂ administration in both the ATP-synthesis and hydrolysis direction. No change in β-subunit mRNA accumulation and protein amount of the α-β subunit of F_oF₁-ATP synthase was found, ruling out a T₂ genomic effect. In T₂-treated rats, changes in the composition of mitochondrial phospholipids were observed, cardiolipin (CL) showing the greatest alteration. In mitochondria isolated from hypothyroid rats the decrease in the amount of CL was accompanied by an increase in the level of peroxidised CL. T₂ administration to hypothyroid rats enhanced the level of CL and decreased the amount of peroxidised CL in subsequently isolated mitochondria, tending to restore the CL value to the euthyroid level. Minor T₂-induced changes in mitochondrial fatty acid composition were detected. Overall, the enhanced F_oF₁-ATP synthase activity observed following injection of T₂ to hypothyroid rats may be ascribed, at least in part, to an increased level of mitochondrial CL associated with decreased peroxidation of CL.     

Citrate carrier activity and cardiolipin level in eel (Anguilla anguilla) liver mitochondria

The activity of the tricarboxylate (citrate) carrier has been assayed in intact liver mitochondria from yellow eel (Anguilla anguilla) and compared to that from rat. The eel-citrate carrier specific activity was approximately 1.7-fold higher than that assayed in rat-liver mitochondria. The content of the main mitochondrial phospholipids, phosphatidylethanolamine and phosphatidylcholine, did not show a significant difference between the two species, while in eel a higher cardiolipin level was observed. Fatty acid composition of eel-liver mitochondrial phospholipids was characterised by a large amount of unsaturated fatty acids, dominated by octadecaenoic acid (C(18:1) (n-9)) and docosahexaenoic acid (C(22:6) (n-3)). The cardiolipin fatty acid pattern of eel-liver mitochondria showed, with respect to the rat, a higher C(20:5) (n-3) and C(22:6) (n-3) content and a lower amount of C(18:2) (n-6) and C(20:4) (n-6). A noticeable activity of lipogenic enzymes was also detected in eel liver cytosol. The results of this study suggest that the remarkable activity of the citrate carrier in eel-liver mitochondria can most likely be ascribed to a considerable cardiolipin level. A covariance of citrate carrier and lipogenic enzyme activities was observed.     

Thryoid control over biomembranes. Rat liver mitochondrial inner membranes.

The effects of hypothyroidism and one injection of l-thyroxine on oxidative phosphorylation and the composition of proteins and phospholipids were examined in vesicles prepared from rat liver mitochondria by digitonin extractions. At 30 °C, the rates of ADP phosphorylation in sites I and II were below normal, and Mg²⁺-ATPase activity was greater than normal in vesicles from hypothyroid rats. At temperatures below 20 °C and above 30 °C, the Mg²⁺-ATPase was not accelerated above normal rates, a feature of temperature dependence shared by ADP phosphorylation (Chen, Y.-D. I., and Hoch, F. L., 1976, Arch. Biochem. Biophys.172, 741–744). Respiration at 30 °C was undiminished in hypothyroid vesicles, as were the flavin and cytochrome contents, and thyroxine administration corrected the phosphorylation rate at 30 °C in 3 days without changing either respiration or electron-carrier contents. The 30 °C phosphorylation defect comprised a decreased V and K_m for ADP and a decrease in the number of phosphorylating sites (measured with oligomycin) that accounted for most of the decreased phosphorylation rates, either dependent on or independent of the adenine nucleotide carrier. Vesicles from hypothyroid rats were not detectably depleted in major protein subunits, but were abnormal in phospholipid fatty acid contents. Thyroxine injection corrected the low unsaturation index of the fatty acids and the membrane contents of linoleic acid and its fatty acyl metabolites. Hypothyroidism appears to affect oxidative phosphorylation through the altered inner membrane lipid environment, which implies that previously reported direct, reversible effects of thyroxine may mimic repletion of the membranes with unsaturated fatty acyl groups.     

Role of citrate in stimulation of the fatty acid shuttle for the transport of reducing equivalents into mitochondria

The fatty acid shuttle for the transport of reducing equivalents into mitochondria was reconstituted $\text{in vitro}$. Externally added citrate increased the activity of this shuttle 30–40%. Citrate also stimulated the activity in the absence of added fatty acid (endogenous rate). The effect of citrate was prevented neither by inhibition of citric acid cycle activity nor by blocking citrate transport into the mitochondria. Inhibition of citrate efflux from the mitochondria impaired shuttle activity. A fatty acid elongation system in the outer membrane, which is stimulated by citrate, may be rate-limiting for the fatty acid shuttle.     

Effect of aging on the activity of the phosphate carrier and on the lipid composition in rat liver mitochondria

The effect of aging on the activity of the phosphate carrier and on the lipid composition in rat liver mitochondria has been investigated. It was found that the rate of phosphate transport in mitochondria from aged rats (28 months old) is significantly reduced (around 40%) compared to that obtained in mitochondria from young control rats (5 months old). Kinetic analysis of the phosphate transport indicates that only the Vmax of this process is affected, while there is no change in the Km values. The lower activity of the phosphate carrier in mitochondria from aged rats is also documented by swelling experiments. The age-related decrement in the activity of the phosphate carrier was found not to be due neither to a change in the endogenous content of phosphate nor to a change in the transmembrane delta pH value. Inhibitor titrations with mersalyl provide no evidence for a lower content of functional phosphate translocase in mitochondria from aged rats. There is no difference either in the respiratory control ratios or in the ADP/O ratios between mitochondria from young and aged animals. The hepatic mitochondrial lipid composition is altered significantly in aged rats: the total cholesterol increases (31%), the phospholipids decrease (12%), and the cholesterol/phospholipid molar ratio increases (44%). Among the phospholipids cardiolipin shows the greatest alteration (30% decrease with age). Alterations were also found in the pattern of fatty acids. The age-related decrement in the activity of the phosphate carrier appears to be dependent on changes in the lipid domain surrounding the carrier protein molecule in the mitochondrial membrane.     

Correlation between myocardial malate/aspartate shuttle activity and EAAT1 protein expression in hyper- and hypothyroidism

In the heart, elevated thyroid hormone leads to upregulation of metabolic pathways associated with energy production and development of hypertrophy. The malate/aspartate shuttle, which transfers cytosolic-reducing equivalents into the cardiac mitochondria, is increased 33% in hyperthyroid rats. Within the shuttle, the aspartate-glutamate carrier is rate limiting. The excitatory amino acid transporter type 1 (EAAT1) functions as a glutamate carrier in the malate/aspartate shuttle. In this study, we hypothesize that EAAT1 is regulated by thyroid hormone. Adult rats were injected with triiodothyronine (T3) or saline over a period of 8-9 days or provided with propylthiouracil (PTU) in their drinking water for 2 mo. Steady-state mRNA levels of EAAT1 and aralar1 and citrin (both cardiac mitochondrial aspartate-glutamate transporters) were determined by Northern blot analysis and normalized to 18S rRNA. A spectrophotometric assay of maximal malate/aspartate shuttle activity was performed on isolated cardiac mitochondria from PTU-treated and control animals. Protein lysates from mitochondria were separated by SDS-PAGE and probed with a human anti-EAAT1 IgG. Compared with control, EAAT1 mRNA levels (arbitrary units) were increased nearly threefold in T3-treated (3.1 +/- 0.5 vs. 1.1 +/- 0.2; P < 0.05) and decreased in PTU-treated (2.0 +/- 0. 3 vs. 5.2 +/- 1; P < 0.05) rats. Aralar1 mRNA levels were unchanged in T3-treated and somewhat decreased in PTU-treated (7.1 +/- 1.0 vs. 9.3 +/- 0.1, P < 0.05) rats. Citrin mRNA levels were decreased in T3-treated and unchanged in PTU-treated rats. EAAT1 protein levels (arbitrary units) in T3-treated cardiac mitochondria were increased compared with controls (8.9 +/- 0.4 vs. 5.9 +/- 0.6; P < 0.005) and unchanged in PTU-treated mitochondria. No difference in malate/aspartate shuttle capacity was found between PTU-treated and control cardiac mitochondria. Hyperthyroidism in rats is related to an increase in cardiac expression of EAAT1 mRNA and protein. The 49% increase in EAAT1 mitochondrial protein level shows that malate/aspartate shuttle activity increased in hyperthyroid rat cardiac mitochondria. Although hypothyroidism resulted in a decrease in EAAT1 mRNA, neither the EAAT1 protein level nor shuttle activity was affected. EAAT1 regulation by thyroid hormone may facilitate increased metabolic demands of the cardiomyocyte during hyperthyroidism and impact cardiac function in hyperthyroidism.     

Changes in lipid peroxidation and free radical scavengers in kidney of hypothyroid and hyperthyroid rats

Kidney weight was significantly decreased in hypothyroidism (induced by Na131I administration) and increased in hyperthyroidism (induced by thyroxine treatment) as compared to control in female Wistar rats. The tissue lipid peroxidation level remained unchanged in hyperthyroid rats but significantly increased in hypothyroid rats. Superoxide dismutase was decreased in both experimental groups but more so in hyperthyroid rats. Catalase was reduced significantly in hyperthyroid rats but remained unaffected in hypothyroid rats. Tissue glutathione peroxidase (GPx) activity was increased while reduced glutathione levels remained unaltered in both hypothyroid and hyperthyroid rats. Plasma GPx activity was significantly low in both the hypothyroid and hyperthyroid rats. The results suggest alterations in the oxidative stress in hypothyroid and hyperthyroid rat kidneys with concomitant changes of free radical scavengers.     

Effect of thyroid dysfunction upon phospholipid composition and CDP-choline incorporation in mitochondria and microsomal fraction isolated from liver and brain of suckling rats

The phospholipid composition and the in vitro incorporation of radioactive CDP-choline into phosphatidylcholine was studied in mitochondria and microsomal fraction obtained from liver and brain of 20 day old hyperthyroid or hypothyroid rats. The chemical composition of the subcellular membranes isolated from brain differed markedly in both conditions. In hyperthyroidism the microsomal fraction was slightly affected while the mitochondria were also affected, but not as severely as in hypothyroidism, in which the microsomal fraction showed no alterations.The incorporation of the radioactive precursor into brain mitochondria isolated from hyperthyroid rats was markedly decreased, while no changes were observed in microsomes. However, incorporation into brain microsomal fraction obtained from hypothyroid rats was increased, while no changes were observed in mitochondria. Similar results were obtained in the studies performed with liver subcellular membranes from hyperthyroid animals while no changes were found in those from hypothyroid rats.Our results indicate that both experimental conditions affect in a different way the structure and function of brain mitochondria and microsomal fractions. They also give further support to our hypothesis that mitochondria have a certain degree of autonomy for the synthesis of phosphatidylcholine.Abbreviations used PS+PI phosphatidylserine+phosphatidylinositol - Sph sphingomyelin - PC phosphatidylcholine - PE phosphatidylethanolamine