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.     

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.     

Decreased activity of the phosphate carrier and modification of lipids in cardiac mitochondria from senescent rats.

1. A comparative study of the effects of aging on the transport of phosphate and on the lipid composition in cardiac mitochondria isolated from young and aged rats was carried out. 2. Mitochondria from aged rats (26 month old) translocate phosphate much more slowly than do mitochondria from young control rats (4 month old). 3. Kinetic analysis of the phosphate transport show that only the Vmax of this process is decreased while there is no change in the Km value. 4. There is no appreciable difference in either the respiratory control ratios or in the ADP/O ratios between mitochondria from young and aged rats. 5. The heart mitochondrial lipid composition is altered in aged rats; in particular, the cholesterol/phospholipid molar ratio increases and the content of cardiolipin decreases with aging.     

Hypothyroidism in rats does not lower mitochondrial ADP/O and H+/O ratios.

We investigated reports that mitochondria isolated from hypothyroid rats have decreased ADP/O and H+/O ratios. We observed no decrease in the H+/O ratio in mitochondria from hypothyroid rats, in the presence of either 2% (w/v) fatty-acid-free bovine serum albumin or 100 nM free Ca2+. The ADP/O ratio in mitochondria isolated from hypothyroid rats in the presence of 2% fatty-acid-free bovine serum albumin was measured. Under normal experimental conditions we found no decrease in the ADP/O ratio, relative to that measured for littermate controls. At the low concentrations of mitochondrial protein used in the previously reported studies, the ADP/O ratio of mitochondria from hypothyroid rats was decreased, whereas that for control rats was only slightly decreased. The difference between the ADP/O ratios measured for mitochondria form hypothyroid rats and from control rats under these conditions was eliminated by inhibition of endogenous adenylate kinase. We suggest that the lowering of the apparent ADP/O ratio in mitochondria from hypothyroid rats at low concentrations of mitochondrial protein is an experimental artefact resulting from the breakdown of ADP to AMP.     

Rapid thyroid-hormone effect on mitochondrial and cytosolic ATP/ADP ratios in the intact liver cell.

The effect of thyroid-hormone application on cytosolic and mitochondrial ATP/ADP ratio was investigated in rat liver in vivo and in the isolated perfused organ. In vivo the ATP/ADP ratio in livers from hypothyroid rats was 0.84 +/- 0.08 in the mitochondrial matrix and 5.6 +/- 0.9 in the cytosol, as was observed in euthyroid controls. In contrast, hyperthyroidism was followed by a significant decrease in the mitochondrial and by an increase in the cytosolic ATP/ADP ratio (to 0.34 +/- 0.06 and 11.3 +/- 2.8 respectively). In the perfused liver from hypothyroid animals, addition of L-3,3',5-tri-iodothyronine in the perfusate also provoked, within 2 h, a significant decrease in the mitochondrial ATP/ADP ratio, whereas the cytosolic ratio was unaffected. From these and previous data in the isolated perfused liver and in isolated mitochondria from hypothyroid and tri-iodothyronine-treated rats it is concluded that thyroid hormones increase mitochondrial respiration and ATP regeneration, which is associated with an acceleration of mitochondrial adenine nucleotide transport and significant alterations in the mitochondrial and cytosolic ATP/ADP ratios.     

Changes in specific lipids regulate BAX-induced mitochondrial permeability transition

Recent evidence suggests the existence of lipid microdomains in mitochondria, apparently coexisting as structural elements with some of the mitochondrial permeability transition pore-forming proteins and members of the Bcl-2 family. The aim of this study was to investigate the relevance of the main components of membrane microdomains (e.g. cholesterol and sphingolipids) in activation of the mitochondrial permeability transition pore (mPTP) by recombinant BAX (rBAX). For this purpose, we used chemically modified renal cortex mitochondria and renal cortex mitochondria from hypothyroid rats that show a modified mitochondrial lipid composition in vivo. Oligomeric rBAX induced an enhanced permeability conformation in the mPTP of control mitochondria. rBAX failed to induce mPTP opening when the cholesterol and ganglioside content of mitochondria were modified with the chelator methyl-beta-cyclodextrin. Accordingly, hypothyroid mitochondria, with endogenously lower cholesterol and ganglioside content, showed resistance to mPTP opening induced by rBAX. These observations suggest that enriched cholesterol and ganglioside domains in the mitochondrial membranes may determine BAX interaction with the mPTP. An intriguing observation was that chemical extraction of cholesterol and ganglioside in control mitochondria did not have an effect on rBAX insertion. Conversely, in hypothyroid mitochondria, rBAX insertion was diminished dramatically compared with control mitochondria. The membrane and protein changes associated with thyroid status and their possible role in rBAX docking into the membranes are discussed.     

Lipid Composition in Synaptic and Nonsynaptic Mitochondria from Rat Brains and Effect of Aging

The cholesterol, phospholipid, and fatty acid compositions in synaptic and nonsynaptic mitochondria from rat brains and the effect of aging were studied. Both cholesterol and phospholipid contents were found to be significantly different in synaptic compared to nonsynaptic mitochondria. In both types of brain mitochondria, aging decreases the cholesterol content by 27% and the phospholipid content by approximately 12%. The difference between these decreases observed in the organelles causes decreases in the cholesterol/phospholipid molar ratios for synaptic and nonsynaptic mitochondria of 17 and 19%, respectively. Also, the phospholipid composition is significantly different in synaptic compared to nonsynaptic mitochondria. Among phospholipids, only the cardiolipin fraction showed a significant decrease (26%) in nonsynaptic mitochondria from the brains of aged rats. Instead, the fatty acid composition was not significantly different in synaptic compared to nonsynaptic mitochondria. The 21% aging decrease in linoleic acid (18:2), observed only in nonsynaptic mitochondria, may be related to a decrease in cardiolipin, which contains a large amount of this fatty acid.     

Thyroid hormone status and membrane n-3 fatty acid content influence mitochondrial proton leak

Proton leak, as determined by the relationship between respiration rate and membrane potential, was lower in mitochondria from hypothyroid rats compared to euthyroid controls. Moreover, proton leak rates diminished even more when hypothyroid rats were fed a diet containing 5% of the lipid content as n-3 fatty acids. Similarly, proton leak was lower in euthyroid rats fed the 5% n-3 diet compared to one containing only 1% n-3 fatty acids. Lower proton leaks rates were associated with increased inner mitochondrial membrane levels of n-3 fatty acids and a decrease in the ratio of n-6/n-3 fatty acids. This trend was evident in the phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and cardiolipin phospholipid fractions. These results suggest that a significant portion of the effect of thyroid hormone status on proton leak is due to alterations in membrane fatty acid composition, primarily changes in n-3 content. Both the hypothyroid state and dietary effects appear to be mediated in part by inhibition of the Delta6- and Delta5-desaturase pathways.     

Thyroid hormones stimulate Na+–Pi transport activity in rat renal brush-border membranes: Role of membrane lipid composition and fluidity

Antecedent studies have suggested that lipid composition and fluidity of cellular membranes of various organs are altered in response to thyroid hormone status. To date, the effects of thyroid hormone status on these parameters have not been examined in rat renal apical membrane in regard to sodium-dependent phosphate transport. In the present study, we determined the potential role of alterations in cortical brush-border membrane lipid composition and fluidity in modulation of Na⁺–P_i transport activity in response to thyroid hormone status. Thyroid hormone status influences the fractional excretion of Pi, which is associated with alteration in renal brush-border membrane phosphate transport. The increment in Na⁺–P_i transport in renal BBMV isolated from Hyper-T rats is manifested as an increase in the maximal velocity (V_max) of Na⁺–P_i transport. Further, the cholesterol content was significantly increased in renal BBM of Hypo-T rats and decreased in Hyper-T rats as compared to the Eu-T rats. The molar ratio of cholesterol/phospholipids was also higher in renal BBM from hypo-T rats. Subsequently, fluorescence anisotropy of diphenyl hexatriene (rDPH) and microviscosity were significantly decreased in the renal BBM of the Hyper-T rats and increased in the Hypo-T rats as compared to Eu-T rats. The result of this study, therefore, suggest that alteration in renal BBM cholesterol, cholesterol/phospholipid molar ratio, and membrane fluidity play an important role in the modulation of renal BBM Na⁺–P_i transport in response to thyroid hormone status of animals. (Mol Cell Biochem 268: 75–82, 2005)     

Effect of propylthiouracil-induced hypothyroidism on cerebral cortex of young and aged rats: Lipid composition of synaptosomes,muscarinic receptor sites,and acetylcholinesterase activity

The effect of hypothyroidism on the lipid composition of synaptosomes, density and affinity of muscarinic receptor sites, and acetylcholinesterase activity in the cerebral cortex of young and aged rats was investigated. The animals were made hypothyroid by adding 0.05% propyl-2-thiouracil to their drinking water for four weeks. This pathological state induced an increase in the relative percentage of sphingomyelin in young rats. In aged rats hypothyroidism induced a decrease of sphingomyelin and glycerophosphocholine and an increase of cholesterol. The effect of hypothyroid state on cerebral cortex resulted in an increase of acethylcholinesterase activity both in young and aged rats and was also reflected in an increase of density of M1-AChRs but only in the former.     

Thyroid hormone may induce changes in the concentration of the mitochondrial calcium uniporter

We explored the possibility that the hormone 3,3',5-tri-iodothyronine can regulate the biosynthesis of the mitochondrial calcium uniporter. To meet this objective experiments on Ca(2+) transport, and binding of the specific inhibitor Ru(360) were carried out in mitochondria isolated from euthyroid, hyperthyroid and hypothyroid rats. It was found that V(max) for Ca(2+) transport increased from 11.67+/-0.8 in euthyroid to 14.36+/-0.44 in hyperthyroid, and decreased in hypothyroid mitochondria to 8.62+/-0.63 nmol Ca(2+)/mg/s. Furthermore, the K(i) for the specific inhibitor Ru(360), depends on the thyroid status, i.e. 18, 19 and 13 nM for control, hyper- and hypothyroid mitochondria, respectively. In addition, the binding of 103Ru(360) was increased in hyperthyroid and decreased in hypothyroid mitochondria. Scatchard analysis for the binding of 103Ru(360) showed the following values: 28, 40 and 23 pmol/mg for control, hyper- and hypothyroid mitochondria, respectively. The K(d) for 103Ru(360) was found to be 30.39, 37.03 and 35.71 nM for controls, hyper- and hypothyroid groups, respectively. When hypothyroid rats were treated with thyroid hormone, mitochondrial Ca(2+) transport, as well as 103Ru(360) binding, reached similar values to those found for euthyroid mitochondria.     

Changes in the lipid composition of erythrocytes during prolonged fasting in lizard (Tropidurus torquatos) and rat (Rattus norvegicus)

During prolonged fasting in lizard and rat, plasma levels of unesterified cholesterol (UC) and phospholipids (TPL) decreased and there were reductions and increases, respectively, in the molar ratios of lecithin (PC) to sphingomyelin (SPH) and UC to TPL. Plasma lecithin: cholesterol acyltransferase (LCATase) activity in lizard and rat plasma was reduced during prolonged fasting. Erythrocyte lipid composition for fasted animals was also characterized by a reduction in the molar ratio PC/SPH and an increase in UC/TPL, and in both species there were positive correlations between these molar ratios in red cells and those in plasma. In both species these were changes in the morphology of the erythrocytes, and those from fasted rats showed alterations in osmotic fragility and permeability which correlated with alterations in lipid composition. These results suggest that changes in plasma lipoprotein lipid composition, linked to reduced LCATase activity, may cause similar alterations in the lipid composition of red cell membranes leading to altered membrane properties.     

The influence of nanomolar calcium ions and physiological levels of thyroid hormone on oxidative phosphorylation in rat liver mitochondria. A possible signal amplification control mechanism.

Using different conditions mitochondria from hypothyroid rats can show both unchanged ADP/O ratios and lowered ADP/O ratios without evidence of uncoupling when compared with euthyroid controls. Raising the free Ca2+ concentration to around 25 nM progressively lowered the ADP/O ratio in hypothyroid but not in euthyroid mitochondria. Ruthenium Red did not alter this behaviour and further increasing the Ca2+ concentration to levels below those which stimulate State 3 respiration had no additional effect. Measurements of the free Ca2+ concentration in the mitochondrial suspending medium using a Quin 2 fluorescence assay showed that the mitochondria did not buffer the free Ca2+ at these low concentrations. At 25 nM-free Ca2+, addition of 10-13) M-T3 to hypothyroid mitochondria produced an immediate and significant increase in the ADP/O ratio without altering the free Ca2+ concentration. The hormone effect was maximal by 10(-11) M. The concentration of ATP synthetase can be estimated to lie at about 10 nM in these experiments. Hence it appears possible that a substantial amplification of the hormone signal may have taken place. Comparison with binding studies suggests that T3 may have been maximally stimulating when somewhat less than half its receptor sites had been filled. The possible mechanisms by which this receptor mediated alteration of the ADP/O ratio might be achieved are discussed.     

Radiation changes in the lipid levels in liver and thymus chromatin of gamma-irradiated rats

A study was made of the lipid content of liver and thymus chromatin of intact and gamma-irradiated (10 Gy) rats 10 and 40 min after irradiation. The composition of the chromatin-bound phospholipids was shown to differ from that of phospholipids of intact nuclei and a nuclear membrane by a much larger content of cardiolipin and sphingomyelin. A decrease in the lipid phosphorus level, increase in the amount of total cholesterol, and a 1.7-fold increase in the cholesterol/phospholipids ratio were observed after irradiation: 40 min after exposure these indices were normalized. The opposite changes were noted in the lipid content of the thymus chromatin: 10 min after irradiation no changes were detected while after 40 min more than a 1.5-fold increase in the cholesterol amount and the cholesterol/phospholipids ratio was registered. The content of cardiolipin was reliably decreased in the chromatin of both organs throughout the entire period of observation.     

Neonatal hypothyroidism and early undernutrition in the rat: Defective maturation of structural membrane components in the central nervous system

The action of neonatal hypothyroidism and early undernutrition on the lipid and protein composition, as well as, on the activity of 23 cyclic nucleotide 3 phosphohydrolase was studied in different subcellular fractions isolated from 20 day old hypothyroid and undernourished rats. Based on protein content, a marked decrease (70%) in the recovery of myelin was observed in both experimental conditions. The lipid composition of myelin in both groups was, however, different; while cholesterol, total phospholipids, and total galactolipids decreased in a similar fashion in the two situations; sulfatides and plasmalogens were much more affected in hypothyroid rats.     

Aging-dependent modification of lipid composition and lipid structural order parameter of hepatic mitochondria

The effect of aging on the lipid composition of hepatic mitochondria has been determined using a rigorously defined group of Fischer 344 rats with known survivorship data. The age groups studied included mature adults as controls (8.5-month, 100% survivorship); an intermediate aged group (17.5-month, 90% survivorship); and an aged group (29-month, 20% survivorship). Lipid extracts of mitochondria were prepared using chloroform-methanol (2:1, by volume) and total phospholipid-P_i, cholesterol (free and esterfied), and phospholipid composition were determined. In the aged animals, total phospholipid-P_i decreased significantly (P = 0.019) whereas cholesterol increased (P = 0.048) with a progressive aging-dependent increase in the molar ratio of cholesterol/phospholipid. The lower total phospholipid content of hepatic mitochondria from the aged 29-month animals was due primarily to decreases in the major phospholipids with the most notable decrease being in cardiolipin (approximately 39%). Steady-state fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene as the probe was used to estimate the lipid structural order parameter of hepatic mitochondria. There was a highly significant (P = 0.01) aging-dependent increase in the lipid structural order parameter which correlated well with the increased molar ratio of cholesterol/phospholipid in the hepatic mitochondria isolated from the aged animals. The data suggest alterations in mitochondrial membrane lipid-protein interactions in aging and are consistent with the hypothesis of impairment of membrane function in the aging process.     

Lipid composition of brown adipose tissue mitochondria and microsomes in hyperthyroid rats

1. The effects of triiodothyronine on the lipid composition of rat brown adipose tissue (BAT) mitochondria and microsomes was investigated by high performance liquid chromatography (HPLC). 2. An increase of about 20% was noted in mitochondrial cholesterol and phospholipids, while a decrease of about 20% for both total cholesterol and phospholipids was observed in microsomes from hyperthyroid rats. 3. The BAT phospholipid composition was altered significantly in mitochondria from T3-treated rats with an increase (41%) of cardiolipin and a decrease (18%) in phosphatidylcholine. 4. In microsomes, a decrease by 25% in phosphatidylinositol was accompanied by a similar additional percentage increase in phosphatidylethanolamine. 5. Important alterations in the fatty acid pattern were found in mitochondrial neutral lipids.     

Supplementation of curcumin and vitamin E enhances oxidative stress,but restores hepatic histoarchitecture in hypothyroid rats

AimsIn the present study, the effects of vitamin E and curcumin on hepatic dysfunction, mitochondrial oxygen consumption as well as hyperlipidemia in hypothyroid rats are reported.Main methodsAdult male rats were rendered hypothyroid by administration of 0.05% 6-n-propyl-2-thiouracil (PTU) in their drinking water, while vitamin E (200 mg/kg body weight) and curcumin (30 mg/kg body weight) were supplemented orally for 30 days.Key findingsHypothyroidism-induced elevation in serum aspartate aminotransferase activity was found to decline in vitamin E and curcumin treated rats. Nevertheless, distorted histoarchitecture revealed in hypothyroid rat liver was alleviated to normal by vitamin E and curcumin treatment. Regulation of hypothyroidism induced decrease in complexes I and II mediated mitochondrial respiration by vitamin E and curcumin was found to be different. Administration of curcumin to hypothyroid rats alleviates the decreased state 4 respiration and increased respiratory control ratio (RCR) level in complex I mediated mitochondrial oxygen consumption, whereas complex II mediated respiration was not influenced by exogenous antioxidants. Although, increase in serum concentration of total cholesterol was not modified by exogenous antioxidants, increased level of non-high-density lipoprotein cholesterol (non-HDL-C) in serum of hypothyroid rats was further enhanced by vitamin E and curcumin. Moreover, a significant elevation in mitochondrial lipid peroxidation and protein carbonylation was noticed in hypothyroid groups treated with vitamin E and curcumin.SignificanceThe present study suggests that supplementation of curcumin and vitamin E enhances oxidative stress parameters and hyperlipidemia; nevertheless, it protects hypothyroid-induced altered rectal temperature, serum transaminase activity and hepatic histoarchitecture.     

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.     

Increased calcium intake reduces plasma cholesterol and improves vasorelaxation in experimental renal failure

Chronic renal failure (CRF) is associated with abnormal lipid metabolism and high prevalence of vascular complications. Calcium salts are commonly used in CRF as phosphate binders. Increased calcium intake may also lower plasma cholesterol and beneficially influence vascular tone. Therefore, we investigated the influence of increasing dietary calcium from 0.3% to 3.0% for 8 wk after 5/6 nephrectomy (NTX) on plasma cholesterol and mesenteric resistance vessel tone in male Sprague-Dawley rats. The groups were Sham, Sham-Calcium, NTX, and NTX-Calcium (n = 10-11). Blood pressure was modestly elevated after NTX, whereas the plasma creatinine, urea nitrogen, phosphate, and parathyroid hormone levels were clearly increased. The high-calcium diet suppressed plasma phosphate and parathyroid hormone but was without effect on blood pressure. The NTX resulted in 1.6-fold elevation in plasma total cholesterol and 40% reduction in high density-to-low density lipoprotein ratio (HDL/LDL). However, the lipid profile in NTX rats on the high-calcium diet did not differ from sham-operated controls. The endothelium-mediated relaxations induced by acetylcholine were impaired in NTX rats, whereas the response was normalized by a high-calcium diet. No differences in vasorelaxations by the endothelium-independent vasodilator nitroprusside were detected. In conclusion, improved vasorelaxation after a high-calcium diet could be due to reduced plasma total cholesterol and ameliorated HDL/LDL ratio, although decreased plasma phosphate and parathyroid hormone may also play a significant role in the vascular effects of increased calcium intake.