Mitochondrial coenzyme Q content and aging.

The main objective of this study was to determine the nature of the relationship between aging and mitochondrial coenzyme Q (CoQ) content. Mitochondria in the heart, skeletal muscle, kidney and brain of the mouse varied in both the amount of total CoQ (CoQ9 + CoQ10) content as well as in the ratio of the CoQ9 to CoQ10. CoQ content declined with age only in the skeletal muscle. Caloric restriction (CR) resulted in an increase in the amount of CoQ9 in skeletal muscle mitochondria. This effect was partially reversible upon termination of the caloric restriction regimen. Results suggest that a decrease in mitochondrial CoQ content is an integral aspect of aging in skeletal muscle.     

Differential regulation of Na,K-ATPase alpha 1, alpha 2, and beta subunit mRNA and protein levels by thyroid hormone

The purpose of this study was to determine the effect of thyroid status on the Na,K-ATPase alpha isoforms and beta in rat heart, skeletal muscle, kidney, and brain at the levels of mRNA, protein abundance, and enzymatic activity. Northern and dot-blot analysis of RNA (euthyroid, hypothyroid, and triiodothyronine-injected hypothyroids = hyperthyroids) and immunoblot analysis of protein (euthyroid and hypothyroid) revealed isoform-specific regulation of Na,K-ATPase by thyroid status in kidney, heart, and skeletal muscle and no regulation of sodium pump subunit levels in the brain. In general, in the transition from euthyroid to hypothyroid alpha 1 mRNA and protein levels are unchanged in kidney and skeletal muscle and slightly decreased in heart, while alpha 2 mRNA and protein are decreased significantly in heart and skeletal muscle. In hypothyroid heart and skeletal muscle, the decrease in alpha 2 protein levels was much greater than the decrease in alpha 2 mRNA levels relative to euthyroid indicating translational or post-translational regulation of alpha 2 protein abundance by triiodothyronine status in these tissues. The regulation of beta subunit by thyroid status is tissue-dependent. In hypothyroid kidney beta mRNA levels do not change, but immunodetectable beta protein levels decrease relative to euthyroid, and the decrease parallels the decrease in Na,K-ATPase activity. In hypothyroid heart and skeletal muscle beta mRNA levels decrease; beta protein decreases in heart and was not detected in the skeletal muscle. These findings demonstrate that the euthyroid levels of expression of alpha 1 in heart, alpha 2 in heart and skeletal muscle, and beta in kidney, heart, and skeletal muscle are dependent on the presence of thyroid hormone.     

Distribution and redox state of ubiquinones in rat and human tissues.

The distribution and redox state of ubiquinone in rat and human tissues have been investigated. A rapid extraction procedure and direct injection onto HPLC were employed. It was found in model experiments that in postmortem tissue neither oxidation nor reduction of ubiquinone occurs. In rat the highest concentrations of ubiquinone-9 were found in the heart, kidney, and liver (130-200 micrograms/g). In brain, spleen, and intestine one-third and in other tissues 10-20% of the total ubiquinone contained 10 isoprene units. In human tissues ubiquinone-10 was also present at highest concentrations in heart, kidney, and liver (60-110 micrograms/g), and in all tissues 2-5% of the total ubiquinone contained 9 isoprene units. High levels of reduction, 70-100%, could be observed in human tissues, with the exception of brain and lung. The extent of reduction displayed a similar pattern in rat, but was generally lower.     

Preserved protein synthesis in the heart in response to acute fasting and chronic food restriction despite reductions in liver and skeletal muscle

Whole body protein synthesis is reduced during the fed-to-fasted transition and in cases of chronic dietary restriction; however, less is known about tissue-specific alterations. We have assessed the extent to which protein synthesis in cardiac muscle responds to dietary perturbations compared with liver and skeletal muscle by applying a novel (2)H(2)O tracer method to quantify tissue-specific responses of protein synthesis in vivo. We hypothesized that protein synthesis in cardiac muscle would be unaffected by acute fasting or food restriction, whereas protein synthesis in the liver and gastrocnemius muscle would be reduced when there is a protein-energy deficit. We found that, although protein synthesis in liver and gastrocnemius muscle was significantly reduced by acute fasting, there were no changes in protein synthesis in the left ventricle of the heart for either the total protein pool or in isolated mitochondrial or cytosolic compartments. Likewise, a chronic reduction in calorie intake, induced by food restriction, did not affect protein synthesis in the heart, whereas protein synthesis in skeletal muscle and liver was decreased. The later observations are supported by changes in the phosphorylation state of two critical mediators of protein synthesis (4E-BP1 and eIF2alpha) in the respective tissues. We conclude that cardiac protein synthesis is maintained in cases of nutritional perturbations, in strong contrast to liver and gastrocnemius muscle, where protein synthesis is decreased by acute fasting or chronic food restriction.     

Retinal coenzyme Q in the bovine eye

Coenzyme Q plays an integral role in oxygen metabolism and management, and there is a positive correlation between low tissue coenzyme Q concentrations and the progression of many degenerative diseases. Retinal oxidative damage plays a role in the pathogenesis of many degenerative eye diseases; nevertheless, despite the retina's high rate of oxygen metabolism, there is little data relating to retinal coenzyme Q concentrations. In this study, we quantified coenzyme Q in the model bovine eye and determined whether it could function as a retinal lipid antioxidant. We found that the neural retina's ubiquinone concentration exceeded those of the vitreous humor, lens, choroid, and extraocular muscle, but it was lower than those measured in heart, kidney, liver, and brain tissues. Ubiquinol was found to be as effective as vitamin E as a retinal lipid antioxidant. The overall relatively low levels of ubiquinone found in the retina, coupled with the retina's need for lipid antioxidants and oxidative metabolism, suggests that retinal function might be sensitive to changes in ubiquinone concentrations.     

Effects of coenzyme Q(10) administration on its tissue concentrations,mitochondrial oxidant generation,and oxidative stress in the rat

Coenzyme Q (CoQ(10)) is a component of the mitochondrial electron transport chain and also a constituent of various cellular membranes. It acts as an important in vivo antioxidant, but is also a primary source of O(2)(-*)/H(2)O(2) generation in cells. CoQ has been widely advocated to be a beneficial dietary adjuvant. However, it remains controversial whether oral administration of CoQ can significantly enhance its tissue levels and/or can modulate the level of oxidative stress in vivo. The objective of this study was to determine the effect of dietary CoQ supplementation on its content in various tissues and their mitochondria, and the resultant effect on the in vivo level of oxidative stress. Rats were administered CoQ(10) (150 mg/kg/d) in their diets for 4 and 13 weeks; thereafter, the amounts of CoQ(10) and CoQ(9) were determined by HPLC in the plasma, homogenates of the liver, kidney, heart, skeletal muscle, brain, and mitochondria of these tissues. Administration of CoQ(10) increased plasma and mitochondria levels of CoQ(10) as well as its predominant homologue CoQ(9). Generally, the magnitude of the increases was greater after 13 weeks than 4 weeks. The level of antioxidative defense enzymes in liver and skeletal muscle homogenates and the rate of hydrogen peroxide generation in heart, brain, and skeletal muscle mitochondria were not affected by CoQ supplementation. However, a reductive shift in plasma aminothiol status and a decrease in skeletal muscle mitochondrial protein carbonyls were apparent after 13 weeks of supplementation. Thus, CoQ supplementation resulted in an elevation of CoQ homologues in tissues and their mitochondria, a selective decrease in protein oxidative damage, and an increase in antioxidative potential in the rat.     

Up-regulation of orphan nuclear estrogen-related receptor alpha expression during long-term caloric restriction in mice

The estrogen-related receptor alpha (ERRα) is an orphan receptor belonging to the nuclear receptor superfamily that regulates a number of target genes encoding enzymes that participate in various metabolic pathways involved in maintaining energy balance in animals. In this study, whether long-term caloric restriction (alternate days of fasting for 3 months) in mice modulates the expression of ERRα in various tissues was investigated. Western blot analyses showed positive immunoreactive ERRα protein (53 kDa) band in various mice tissue extracts, though at varying levels. Heart, kidney, and skeletal muscles expressed significant levels of ERRα, with a comparatively lower level detected in the intestine, brain, and liver. Cardiac ERRα expression was the highest, with the least detected in the liver. Caloric restricted mice exhibited a significant increase in ERRα level in the heart (5.45-fold), kidney (3.70-fold), skeletal muscle (3.0-fold), small intestine (2.72-fold), and liver (2.44-fold) extracts as compared to ad libitum fed. However, caloric restriction could not evoke any detectable receptor level change in the brain. Notably, the highest ERRα up-regulation was detected in the heart. This up-regulation in ERRα level especially in highly oxidative tissues such as heart, kidney, small intestine, and skeletal muscle of caloric restricted mice may be helpful in modulating ERRα responsive genes that participates in maintaining energy balance. This may potentially strengthen the metabolic and biochemical adaptation in such tissues, which is necessary for animal survival under long-term caloric restriction.     

Pyridoxamine (pyridoxine) phosphate oxidase activity in mammalian tissues

1. Vitamin B6-sufficient rats had moderate pyridoxamine-P oxidase specific activities in heart, brain, kidney and liver, but no detectable activity in skeletal muscle. Vitamin B6-deficiency in rats resulted in a decreased oxidase activity in liver but no change in the activities in other tissues. 2. The pyridoxamine-P oxidase activity in vitamin B6-sufficient mice was high in liver, moderate in brain and kidney, and not measurable in skeletal muscle and heart. Vitamin B6-deficient, compared with control mice, had decreased oxidase activities in brain, kidney and liver. 3. Mouse erythrocytes took up pyridoxine more rapidly than did rat and human erythrocytes. 4. Mouse and human erythrocytes rapidly converted pyridoxine to pyridoxal-P. Rat, hamster and rabbit erythrocytes had appreciably lower pyridoxamine-P oxidase activity than did mouse and human erythrocytes.     

Aging and lifelong calorie restriction result in adaptations of skeletal muscle apoptosis repressor, apoptosis-inducing factor, X-linked inhibitor of apoptosis, caspase-3, and caspase-12

The mechanisms of apoptosis in the loss of myocytes in skeletal muscle with age and the role of mitochondrial and sarcoplasmic reticulum-mediated pathways of apoptosis are unknown. Moreover, it is unknown whether lifelong calorie restriction prevents apoptosis in skeletal muscle and reverses age-related alterations in apoptosis signaling. We investigated key apoptotic regulatory proteins in the gastrocnemius muscle of 12 and 26 month old ad libitum fed and 26 month old calorie-restricted male Fischer-344 rats. We found that apoptosis increased with age and that calorie-restricted rats showed less apoptosis compared with their age-matched cohorts. Moreover, pro- and cleaved caspase-3 levels increased significantly with age and calorie-restricted rats had significantly lower levels than the aged ad libitum group. Neither age nor calorie restriction had any effect on muscle caspase-3 enzyme activity, but the levels of X-linked inhibitor of apoptosis, particularly an inhibitor of caspase-3, increased with age and were reduced significantly in the 26 month old calorie-restricted cohort. The apoptotic inhibitor apoptosis repressor with a caspase recruitment domain (ARC), which inhibits cytochrome c release, underwent an age-associated decline in the cytosol but increased with calorie restriction. In contrast, mitochondrial ARC levels increased with age and were lower in calorie-restricted rats than in age-matched controls, suggesting a translocation of this protein to attenuate oxidative stress. The translocation of ARC may explain the reduction in cytosolic cytochrome c levels observed with age and calorie restriction. Moreover, we found a striking approximately 350% increase in the expression of procaspase-12 (caspase located at the sarcoplasmic reticulum) with age which was significantly lower in the 26 month old calorie-restricted group. The total protein level of apoptosis-inducing factor in the plantaris muscle increased with age and was reduced calorie-restricted rats compared with age-matched controls, but there were no significant changes in this pro-apoptotic protein in the isolated nuclei. Calorie restriction is able to lower the apoptotic potential in aged skeletal muscle by altering several key apoptotic proteins toward cellular survival, thereby reducing the potential for sarcopenia.     

Effects of aging and caloric restriction on mitochondrial energy production in gastrocnemius muscle and heart

Mitochondria are chronically exposed to reactive oxygen intermediates. As a result, various tissues, including skeletal muscle and heart, are characterized by an age-associated increase in reactive oxidant-induced mitochondrial DNA (mtDNA) damage. It has been postulated that these alterations may result in a decline in the content and rate of production of ATP, which may affect tissue function, contribute to the aging process, and lead to several disease states. We show that with age, ATP content and production decreased by approximately 50% in isolated rat mitochondria from the gastrocnemius muscle; however, no decline was observed in heart mitochondria. The decline observed in skeletal muscle may be a factor in the process of sarcopenia, which increases in incidence with advancing age. Lifelong caloric restriction, which prolongs maximum life span in animals, did not attenuate the age-related decline in ATP content or rate of production in skeletal muscle and had no effect on the heart. 8-Oxo-7,8-dihydro-2'-deoxyguanosine in skeletal muscle mtDNA was unaffected by aging but decreased 30% with caloric restriction, suggesting that the mechanisms that decrease oxidative stress in these tissues with caloric restriction are independent from ATP availability. The generation of reactive oxygen species, as indicated by H2O2 production in isolated mitochondria, did not change significantly with age in skeletal muscle or in the heart. Caloric restriction tended to reduce the levels of H2O2 production in the muscle but not in the heart. These data are the first to show that an age-associated decline in ATP content and rate of ATP production is tissue specific, in that it occurs in skeletal muscle but not heart, and that mitochondrial ATP production was unaltered by caloric restriction in both tissues.     

Calorie-related rapid onset of alveolar loss, regeneration, and changes in mouse lung gene expression

Calorie restriction, followed by ad libitum refeeding, results, respectively, in loss and regeneration of pulmonary alveoli. We now show 35% of alveoli are lost within 72 h of onset of calorie restriction ((2/3) decreased daily chow intake), and an additional 12% of alveoli are lost over a subsequent 12 days of calorie restriction. Tissue necrosis was not seen. Within 72 h of refeeding, after 15 days of calorie restriction, the number of alveoli returns to precalorie restriction values. Microarray lung gene profiling, in conjunction with Western and RNase protection assay, demonstrate an increase of granzyme and caspase gene expression 2-3 h after onset of calorie restriction. By 12 h, granzyme and caspase expression is no longer increased, but tumor necrosis factor death receptor expression is elevated. At 336 h, Fas death receptor expression is increased. Because granzymes are found only in cytotoxic lymphocytes (CTLs) and natural killer (NK) cells, we suggest calorie restriction activates these cells, initiating a series of molecular events that results in alveolar destruction. The evidence of involvement of CTLs and NK cells and the absence of necrosis are similar to alveolar destruction in chronic obstructive pulmonary disease.