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Lustgarten MS, Jang YC, Liu Y, Muller FL, Qi W, Steinhelper M, Brooks SV, Larkin L, Shimizu T, Shirasawa T, McManus LM, Bhattacharya A, Richardson A, Van Remmen H. Conditional knockout of Mn-SOD targeted to type IIB skeletal muscle fibers increases oxidative stress and is sufficient to alter aerobic exercise capacity. Am J Physiol Cell Physiol 297: C 1520-C1532, 2009. First published September 23, 2009; doi: 10.1152/ajpcell.00372.2009.--In vitro studies of isolated skeletal muscle have shown that oxidative stress is limiting with respect to contractile function. Mitochondria are a potential source of muscle function-limiting oxidants. To test the hypothesis that skeletal muscle-specific mitochondrial oxidative stress is sufficient to limit muscle function, we bred mice expressing Cre recombinase driven by the promoter for the inhibitory subunit of troponin (TnIFastiCre) with mice containing a floxed Sod2 (Sod[2.sup.fl/fl]) allele. Mn-SOD activity was reduced by 82% in glycolytic (mainly type II) muscle fiber homogenates from young TnIFastCreSod[2.sup.fl/fl] mice. Furthermore, Mn-SOD content was reduced by 70% only in type IIB muscle fibers. Aconitase activity was decreased by 56%, which suggests an increase in mitochondrial matrix superoxide. Mitochondrial superoxide release was elevated more than twofold by mitochondria isolated from glycolytic skeletal muscle in TnIFastCreSod[2.sup.fl/fl] mice. In contrast, the rate of mitochondrial [H.sub.2][O.sub.2] production was reduced by 33%, and only during respiration with complex II substrate. [F.sub.2]-isoprostanes were increased by 36% in tibialis anterior muscles isolated from TnIFastCreSod[2.sup.fl/fl] mice. Elevated glycolytic muscle-specific mitochondrial oxidative stress and damage in TnIFastCreSod[2.sup.fl/fl] mice were associated with a decreased ability of the extensor digitorum longus and gastrocnemius muscles to produce contractile force as a function of time, whereas force production by the soleus muscle was unaffected. TnIFastCreSod.[2.sup.fl/fl] mice ran 55% less distance on a treadmill than wild-type mice. Collectively, these data suggest that elevated mitochondrial oxidative stress and damage in glycolytic muscle fibers are sufficient to reduce contractile muscle function and aerobic exercise capacity. muscle function; contractile function; oxidative damage; free radical doi: 10.1152/ajpcell.00372.2009

The widely accepted oxidative stress theory of aging postulates that aging results from accumulation of oxidative damage. Surprisingly, data from the longest-living rodent known, naked molerats [MRs; mass 35 g; maximum lifespan (MLSP) > 28.3 years), when compared with mice (MLSP 3.5 years) exhibit higher levels of lipid peroxidation, protein carbonylation, and DNA oxidative damage even at a young age. We hypothesize that age-related changes in protein structural stability, oxidation, and degradation are abrogated over the lifespan of the MR. We performed a comprehensive study of oxidation states of protein cysteines [both reversible (sulfenic, disulfide) and indirectly irreversible (sulfinic/sulfonic acids)] in liver from young and old C57BL/6 mice (6 and 28 months) and MRs (2 and >24 years). Furthermore, we compared interspecific differences in urea-induced protein unfolding and ubiquitination and proteasomal activity. Compared with data from young mice, young MRs have 1.6 times as much free protein thiol groups and similar amounts of reversible oxidative damage to cysteine. In addition, they show less urea-induced protein unfolding, less protein ubiquitination, and higher proteasome activity. Mice show a significant age-related increase in cysteine oxidation and higher levels of ubiquitination. In contrast, none of these parameters were significantly altered over 2 decades in MRs. Clearly MRs have markedly attenuated age-related accrual of oxidation damage to thiol groups and age-associated up-regulation of homeostatic proteolytic activity. These pivotal mechanistic interspecies differences may contribute to the divergent aging profiles and strongly implicate maintenance of protein stability and integrity in successful aging. cysteine oxidation | Heterocephalus glaber | mechanisms of aging | proteasome activity | protein homeostasis

Reactive oxygen species (ROS), especially mitochondrial ROS, are postulated to play a significant role in muscle atrophy. We report a dramatic increase in mitochondrial ROS generation in three conditions associated with muscle atrophy: in aging, in mice lacking CuZn-SOD (Sod[1.sup.-/-]), and in the neurodegenerative disease, amyotrophic lateral sclerosis (ALS). ROS generation in muscle mitochondria is nearly threefold higher in 28- to 32-too-old than in 10-mo-old mice and is associated with a 30% loss in gastrocnemius mass. In Sod[1.sup.-/-] mice, muscle mitochondrial ROS production is increased > 100% in 20-mo compared with 5-mo-old mice along with a >50% loss in muscle mass. ALS G93A mutant mice show a 75% loss of muscle mass during disease progression and up to 12-fold higher muscle mitochondrial ROS generation. In a second ALS mutant model, H46RH48Q mice, ROS production is approximately fourfold higher than in control mice and is associated with a less dramatic loss (30%) in muscle mass. Thus ROS production is strongly correlated with the extent of muscle atrophy in these models. Because each of the models of muscle atrophy studied are associated to some degree with a loss of innervation, we were interested in determining whether denervation plays a role in ROS generation in muscle mitochondria isolated from hindlimb muscle following surgical sciatic nerve transection. Seven days postdenervation, muscle mitochondrial ROS production increased nearly 30-fold. We conclude that enhanced generation of mitochondrial ROS may be a common factor in the mechanism underlying denervation-induced atrophy. mitochondria; reactive oxygen species; amyotrophic lateral sclerosis; copper, zinc superoxide dismutase

Because food restriction (FR) has a profound effect on most tissues, it is plausible that the modulation of aging by FR occurs through cellular processes such as gene expression. The effect of FR in lowering plasma glucose levels has been demonstrated in mice, rats, and nonhuman primates. The consistency of this finding suggests that decreased plasma glucose may be an important consequence of FR. Indeed, lowering plasma glucose in the absence of FR would be expected to change the expression of some of the same genes as seen with FR. GLUT4 transgenic (TG) mice were particularly suited to this examination because they have low plasma glucose levels like FR mice. We investigated altered gene expression by FR and the effect of low plasma glucose levels caused by genetic manipulation by measuring mRNA expression in liver tissues of 4- to 6-mo-old mice with 2.5-4.5 mo of FR using microarrays and 4 groups: GLUT4 TG (C57BL/6 background) consumed food ad libitum (AL), GLUT4 TG FR, wild-type littermates AL, and wild-type littermates FR. The 3 statistical analysis methods commonly indicated that FR altered the expression of 1277 genes; however, none of these genes was altered by additional GLUT4 expression. In fact, the low plasma glucose level in GLUT4 TG mice did not affect gene expression. Some results were confirmed by real-time quantitative RT-PCR. We conclude that a low plasma glucose level does not contribute to or coincide with the effect of FR on gene expression in the liver. J. Nutr. 134: 2965-2974, 2004. KEY WORDS: * gene expression * food restriction * GLUT4 transgenic mouse * GeneChip array

The levels of 8-oxo-2-deoxyguanosine (oxo8dG) in DNA isolated from tissues of rodents (male F344 rats, male B6D2F1 mice, male C57BL/6 mice, and female C57BL/6 mice) of various ages were measured using sodium iodide to prevent oxidative damage to DNA during DNA isolation. Oxo8dG was measured in nuclear DNA (nDNA) isolated from liver, heart, brain, kidney, skeletal muscle, and spleen and in mitochondrial DNA (mtDNA) isolated from liver. We observed a significant increase in oxo8dG levels in nDNA with age in all tissues and strains of rodents studied. The age-related increase in oxo8dG in nDNA from old mice was shown not to the result of the tissue's reduced ability to remove the oxo8dG lesion. Rather, the increase in oxo8dG levels appears to arise from an age-related increase in the sensitivity of these tissues to oxidative stress. We also observed an age-related increase in oxo8dG in mtDNA isolated from the livers of the rats and mice. Dietary restriction, which is known to retard aging and increase the lifespan of rodents, was shown to significantly reduce the age-related accumulation of oxo8dG levels in nDNA in all tissues of male B6D23F1 mice and in most tissues of male F344 rats. Our study also showed that dietary restriction prevented the age-related increase in oxo8dG levels in mtDNA isolated from the livers of both rats and mice.

Multiple stress resistance pathways were evaluated in the liver of Ames dwarf mice before and after exposure to the oxidative toxin diquat, seeking clues to the exceptional longevity conferred by this mutation. Before diquat treatment, Ames dwarf mice, compared with nonmutant littermate controls, had 2- to 6-fold higher levels of expression of mRNAs for immediate early genes and 2- to 5-fold higher levels of mRNAs for genes dependent on the transcription factor Nrf2. Diquat led to a 2-fold increase in phosphory-lation of the stress kinase ERK in control (but not Ames dwarf) mice and to a 50% increase in phosphorylation of the kinase JNK2 in Ames dwarf (but not control) mice. Diquat induction of Nrf2 protein was higher in dwarf mice than in controls. Of 6 Nrf2- responsive genes evaluated, 4 (HMOX, NQO-1, MT-1, and MT-2) remained 2- to 10-fold lower in control than in dwarf liver after diquat, and the other 2 (GCLM and TXNRD) reached levels already seen in dwarf liver at baseline. Thus, livers of Ames dwarf mice differ systematically from controls in multiple stress resistance pathways before and after exposure to diquat, suggesting mechanisms for stress resistance and extended longevity in Ames dwarf mice. [ABSTRACT FROM AUTHOR]

This study examined the effect of housing density on the longevity-extending and disease-delaying actions of calorie restriction (CR). Singly or multiply housed (four per cage) mice were either fed ad libitum (AL) or were on CR beginning at 2 months. All CR mice were fed 40% less food than were multiply housed AL mice. CR increased median longevity by 19%, and housing density had no effect on this increase. CR also reduced neoplastic lesions in both housing groups, but lymphoma, the most common neoplasm, was reduced more in singly than in multiply housed mice. Singly housed AL mice ate 40% more food than did multiply housed AL mice, but weighed the same and lived as long as multiply housed AL mice. These results indicate that CR can extend life span as effectively in multiply as in singly housed mice, even though housing density can differentially affect the cancer-reducing effect of CR. [ABSTRACT FROM AUTHOR]

Skeletal muscle contraction during exercise leads to increased production of reactive oxygen species (ROS). Because oxidative stress is linked to muscle fatigue development, we hypothesized that diaphragms from mice overexpressing antioxidant enzymes would be more resistant to contraction induced fatigue. To test this postulate, diaphragm fiber bundles from transgenic mice overexpressing the specific antioxidant enzymes: catalase (Cat; n=6), manganese superoxide dismutase (MnSOD; n=6), and copper-zinc superoxide dismutase (CuZnSOD; n=9) were examined to determine maximal specific tension, endurance, and recovery from fatigue. Wild-type littermates (WT; n=8) served as controls. Diaphragms from antioxidant overexpressing animals demonstrated lower (p<0.05) submaximal and maximal specific tension values compared to WT controls; with diaphragms from MnSOD overexpressing animals exhibiting the greatest decrement. However, no differences in diaphragm endurance or recovery from fatigue were observed between groups (p>0.05). Moreover, protein carbonyl levels of the stimulated fiber bundles did not differ between groups (p>0.05). Collectively, these results suggest that cellular ROS are critical to normal diaphragm contractile function and chronic overexpression of antioxidant enzymes is not sufficient to alleviate the development of fatigue. [ABSTRACT FROM AUTHOR]

Cu/Zn superoxide dismutase (SOD1) is an enzyme critically important for superoxide free radical elimination. Compared with age-matched wild type littermates (Sod1+/+), ∼2, ∼10 and ∼18 month old SOD1 homozygous knockout (Sod1-/-) mice have smaller body and skeletal muscle masses. Following staining for succinate dehydrogenase, grouping of positive fibers were observed in muscles of Sod1-/- mice from all three age groups. More advanced fiber type grouping was observed in skeletal muscles of 18 month old Sod1-/- mice. While 2 month and l0 month old Sod1+/+ mice did not display gross skeletal muscle-structural abnormalities, 18 month old Sod1-/- mice had extensive denervation of gastrocnemius muscle and some denervation of other hind limb muscles. The number of denervated muscle fibers observed in gastrocnemius muscles of 18 month old Sod1-/- mice was comparable to that found in skeletal muscles of ∼28 month old Sod1+/+ mice. We conclude that lack of SOD1 enzymatic activity did not result in major muscle fiber structural abnormalities in skeletal muscles of young Sod1-/- mice, but increased dramatically the onset and severity of age-related denervation of skeletal muscles. [ABSTRACT FROM AUTHOR]

PURPOSE: Doxorubicin belongs to a family of anticancer drugs that exert cytotoxic effects by release of reactive oxygen species (ROS) and triggering apoptosis. Since antioxidant enzymes scavenge ROS and defend against apoptosis, we sought to determine whether overexpression of catalase, copper-zinc superoxide dismutase (CuZnSOD), or manganese SOD (MnSOD) is protective against doxorubicin-mediated cell death. METHODS: Primary satellite cell cultures were derived from the hindlimb muscles of wild type (WT) or transgenic mice overexpressing catalase, CuZnSOD, or MnSOD. Myoblasts were cultured in growth medium (10% FBS) until 80% confluency, then replated at equal density. Following a 24 hr treatment with or without 5µM doxorubicin, cells were fixed for DAP1 and TUNEL staining. RESULTS: Doxorubicin induced a 60% decrease in nuclear number of WT cells, and this effect was abrogated in cells overexpressing CuZnSOD or MnSOD. The %TUNEL positive nuclei was elevated six-fold in doxorubicin-treated WT cells and only the CuZnSOD overexpressing cells were protected against this effect. CONCLUSIONS: Catalase overexpression did not protect cultured satellite cells from the cytotoxic effects of doxorubicin. However, overexpression of the cytosolic antioxidant enzyme, CuZnSOD, protects against DNA fragmentation, and overexpression of CuZnSOD or the mitochondrial enzyme, MnSOD, enhances cellular survival when challenged by a dose of doxorubicin known to induce apoptosis. [ABSTRACT FROM AUTHOR]

Rapamycin, an inhibitor of mechanistic target of rapamycin complex 1 (mTORC1), extends the life spans of yeast, flies, and mice. Calorie restriction, which increases life span and insulin sensitivity, is proposed to function by inhibition of mTORC1, yet paradoxically, chronic administration of rapamycin substantially impairs glucose tolerance and insulin action. We demonstrate that rapamycin disrupted a second mTOR complex, mTORC2, in vivo and that mTORC2 was required for the insulin-mediated suppression of hepatic gluconeogenesis. Further, decreased mTORC1 signaling was sufficient to extend life span independently from changes in glucose homeostasis, as female mice heterozygous for both mTOR and mLST8 exhibited decreased mTORC1 activity and extended life span but had normal glucose tolerance and insulin sensitivity. Thus, mTORC2 disruption is an important mediator of the effects of rapamycin in vivo. [ABSTRACT FROM AUTHOR]