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The tuberous sclerosis complex 1/2 (TSC1/2) is an endogenous regulator of the mechanistic target of rapamycin (mTOR). While mTOR has been shown to play an important role in health and aging, the role of TSC1/2 in aging has not been fully investigated. In the current study, a constitutive TSC1 transgenic (Tsc1 tg ) mouse model was generated and characterized. mTORC1 signaling was reduced in majority of the tissues, except the brain. In contrast, mTORC2 signaling was enhanced in Tsc1 tg mice. Tsc1 tg mice are more tolerant to exhaustive exercises and less susceptible to isoproterenol-induced cardiac hypertrophy at both young and advanced ages. Tsc1 tg mice have less fibrosis and inflammation in aged as well as isoproterenol-challenged heart than age-matched wild type mice. The female Tsc1 tg mice exhibit a higher fat to lean mass ratio at advanced ages than age-matched wild type mice. More importantly, the lifespan increased significantly in female Tsc1 tg mice, but not in male Tsc1 tg mice. Collectively, our data demonstrated that moderate increase of TSC1 expression can enhance overall health, particularly cardiovascular health, and improve survival in a gender-specific manner.

SummaryOlder adults universally suffer from sarcopenia and approximately 60–70% are diabetic or prediabetic. Nonetheless, the mechanisms underlying these aging‐related metabolic disorders are unknown. NFκB has been implicated in the pathogenesis of several aging‐related pathologies including sarcopenia and type 2 diabetes and has been proposed as a target against them. NFκB also is thought to mediate muscle wasting seen with disuse, denervation, and some systemic diseases (e.g., cancer, sepsis). We tested the hypothesis that lifelong inhibition of the classical NFκB pathway would protect against aging‐related sarcopenia and insulin resistance. Aged mice with muscle‐specific overexpression of a super‐repressor IκBα mutant (MISR) were protected from insulin resistance. However, MISR mice were not protected from sarcopenia; to the contrary, these mice had decreases in muscle mass and strength compared to wild‐type mice. In MISR mice, NFκB suppression also led to an increase in proteasome activity and alterations in several genes and pathways involved in muscle growth and atrophy (e.g., myostatin). We conclude that the mechanism behind aging‐induced sarcopenia is NFκB independent and differs from muscle wasting due to pathologic conditions. Our findings also indicate that, while suppressing NFκB improves insulin sensitivity in aged mice, this transcription factor is important for normal muscle mass maintenance and its sustained inhibition is detrimental to muscle function.

The caseinolytic peptidase P (ClpP) is the endopeptidase component of the mitochondrial matrix ATP-dependent ClpXP protease. ClpP degrades unfolded proteins to maintain mitochondrial protein homeostasis and is involved in the initiation of the mitochondrial unfolded protein response (UPR(mt)). Outside of an integral role in the UPR(mt), the cellular function of ClpP is not well characterized in mammalian cells. To investigate the role of ClpP in mitochondrial function, we generated C2C12 muscle cells that are deficient in ClpP using siRNA or stable knockdown using lentiviral transduction. Reduction of ClpP levels by ~70% in C2C12 muscle cells resulted in a number of mitochondrial alterations including reduced mitochondrial respiration and reduced oxygen consumption rate in response to electron transport chain (ETC) complex I and II substrates. The reduction in ClpP altered mitochondrial morphology, changed the expression level of mitochondrial fission protein Drp1 and blunted UPR(mt) induction. In addition, ClpP deficient cells showed increased generation of reactive oxygen species (ROS) and decreased membrane potential. At the cellular level, reduction of ClpP impaired myoblast differentiation, cell proliferation and elevated phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) suggesting an inhibition of translation. Our study is the first to define the effects of ClpP deficiency on mitochondrial function in muscle cells in vitro. In addition, we have uncovered novel effects of ClpP on mitochondrial morphology, cell proliferation and protein translation pathways in muscle cells.

BACKGROUND: Skeletal muscle atrophy during aging, a process known as sarcopenia, is associated with muscle weakness, frailty, and the loss of independence in older adults. The mechanisms contributing to sarcopenia are not totally understood, but muscle fiber loss due to apoptosis, reduced stimulation of anabolic pathways, activation of catabolic pathways, denervation, and altered metabolism have been observed in muscle from old rodents and humans. OBJECTIVE: Recently, histone deacetylases (HDACs) have been implicated in muscle atrophy and dysfunction due to denervation, muscular dystrophy, and disuse, and HDACs play key roles in regulating metabolism in skeletal muscle. In this review, we will discuss the role of HDACs in muscle atrophy and the potential of HDAC inhibitors for the treatment of sarcopenia. CONCLUSIONS: Several HDAC isoforms are potential targets for intervention in sarcopenia. Inhibition of HDAC1 prevents muscle atrophy due to nutrient deprivation. HDAC3 regulates metabolism in skeletal muscle and may inhibit oxidative metabolism during aging. HDAC4 and HDAC5 have been implicated in muscle atrophy due to denervation, a process implicated in sarcopenia. HDAC inhibitors are already in use in the clinic, and there is promise in targeting HDACs for the treatment of sarcopenia.

Genetic ablation of CuZn-superoxide dismutase (Sod1) in mice (Sod1(-/-) mice) leads to shortened lifespan with a dramatic increase in hepatocellular carcinoma and accelerated aging phenotypes, including early onset sarcopenia. To study the tissue specific effects of oxidative stress in the Sod1(-/-) mice, we generated mice that only express the human SOD1 gene specifically in the liver of Sod1(-/-) mice (Sod1(-/-)/hSOD1(alb) mice). Expression of hSOD1 in the liver of Sod1(-/-) mice improved liver function, reduced oxidative damage in liver, and partially restored the expression of several genes involved in tumorigenesis, which are abnormally expressed in the livers of the Sod1(-/-) mice. However, liver specific expression of hSOD1 did not prevent the loss of body weight and muscle mass and alterations in the structure of neuromuscular junctions. The expression of hSOD1 in the liver of Sod1(-/-) mice significantly improved the lifespan of Sod1(-/-) mice; however, the lifespan of the Sod1(-/-)/hSOD1(alb) mice was still significantly shorter than wild type mice. Published by Elsevier Ireland Ltd

Introduction: Histone deacetylases (HDACs) have been implicated in neurogenic muscle atrophy, but the mechanisms by which HDAC inhibitors might have beneficial effects are not defined. Methods: We used sciatic nerve crush to determine the effect of butyrate on denervation-induced gene expression and oxidative stress. Results: Butyrate treatment initiated 3 weeks before injury and continued 1 week after injury increases histone acetylation and reduces muscle atrophy after nerve crush. Butyrate delivered only after nerve crush similarly prevented muscle atrophy. Butyrate had no effect on the increase in histone deacetylase 4 (HDAC4) protein levels following nerve crush but prevented the increase in expression of myogenin, MuRF1, and atrogin-1. Butyrate did not affect mitochondrial reactive oxygen species production, but it increased antioxidant enzyme activity, reduced proteasome activity, and reduced oxidative damage following nerve injury. Conclusions: These data suggest that HDAC inhibitors are promising pharmacological agents for treating neurogenic muscle atrophy. Muscle Nerve 52: 859-868, 2015

SummarySarcopenia, the loss of skeletal muscle mass and function during aging, is a major contributor to disability and frailty in the elderly. Previous studies found a protective effect of reduced histone deacetylase activity in models of neurogenic muscle atrophy. Because loss of muscle mass during aging is associated with loss of motor neuron innervation, we investigated the potential for the histone deacetylase (HDAC) inhibitor butyrate to modulate age‐related muscle loss. Consistent with previous studies, we found significant loss of hindlimb muscle mass in 26‐month‐old C57Bl/6 female mice fed a control diet. Butyrate treatment starting at 16 months of age wholly or partially protected against muscle atrophy in hindlimb muscles. Butyrate increased muscle fiber cross‐sectional area and prevented intramuscular fat accumulation in the old mice. In addition to the protective effect on muscle mass, butyrate reduced fat mass and improved glucose metabolism in 26‐month‐old mice as determined by a glucose tolerance test. Furthermore, butyrate increased markers of mitochondrial biogenesis in skeletal muscle and whole‐body oxygen consumption without affecting activity. The increase in mass in butyrate‐treated mice was not due to reduced ubiquitin‐mediated proteasomal degradation. However, butyrate reduced markers of oxidative stress and apoptosis and altered antioxidant enzyme activity. Our data is the first to show a beneficial effect of butyrate on muscle mass during aging and suggests HDACs contribute to age‐related muscle atrophy and may be effective targets for intervention in sarcopenia and age‐related metabolic disease.

Nerve conduction velocity (NCV), the speed at which electrical signals propagate along peripheral nerves, is used in the clinic to evaluate nerve function in humans. A decline in peripheral nerve function is associated with a number of age-related pathologies. While several studies have shown that NCV declines with age in humans, there is little information on the effect of age on NCV in peripheral nerves in mice. In this study, we evaluated NCV in male and female C57Bl/6 mice ranging from 4 to 32 months of age. We observed a decline in NCV in both male and female mice after 20 months of age. Sex differences were detected in sensory NCV as well as the rate of decline during aging in motor nerves; female mice had slower sensory NCV and a slower age-related decline in motor nerves compared with male mice. We also tested the effect of dietary restriction on NCV in 30-month-old female mice. Dietary restriction prevented the age-related decline in sciatic NCV but not other nerves. Because NCV is clinically relevant to the assessment of nerve function, we recommend that NCV be used to evaluate healthspan in assessing genetic and pharmacological interventions that increase the life span of mice.

Oxidative stress is observed during aging and in numerous age-related diseases. Dietary restriction (DR) is a regimen that protects against disease and extends life span in multiple species. However, it is unknown how DR mediates its protective effects. One prominent and consistent effect of DR in a number of systems is the ability to reduce oxidative stress and damage. The purpose of this review is to comprehensively examine the hypothesis that dietary restriction reduces oxidative stress in rodents by decreasing reactive oxygen species (ROS) production and increasing antioxidant enzyme activity, leading to an overall reduction of oxidative damage to macromolecules. The literature reveals that the effects of DR on oxidative stress are complex and likely influenced by a variety of factors, including sex, species, tissue examined, types of ROS and antioxidant enzymes examined, and duration of DR. Here we present a comprehensive review of the existing literature on the effect of DR on mitochondrial ROS generation, antioxidant enzymes, and oxidative damage. In a majority of studies, dietary restriction had little effect on mitochondrial ROS production or antioxidant activity. On the other hand, DR decreased oxidative damage in the majority of cases. Although the effects of DR on endogenous antioxidants are mixed, we find that glutathione levels are the most likely antioxidant to be increased by dietary restriction, which supports the emerging redox-stress hypothesis of aging.

Elevated reactive oxygen species (ROS) production and ROS-dependent protein damage is a common observation in the pathogenesis of many muscle wasting disorders, including sarcopenia. However, the contribution of elevated ROS levels to -a breakdown in neuromuscular communication and muscle atrophy remains unknown. In this study, we examined a copper zinc superoxide dismutase [CuZnSOD (Sod1)] knockout mouse (Sod1-/-), a mouse model of elevated oxidative stress that exhibits accelerated loss of muscle mass, which recapitulates many phenotypes of sarcopenia as early as 5 months of age. We found that young adult Sod1-/- mice display a considerable reduction in hind limb skeletal muscle mass and strength when compared to age-matched wild-type mice. These changes are accompanied by gross alterations in neuromuscular junction (NMJ) morphology, including reduced occupancy of the motor endplates by axons, terminal sprouting and axon thinning and irregular swelling. Surprisingly however, the average density of acetylcholine receptors in endplates is preserved. Using in vivo electromyography and ex vivo electrophysiological studies of hind limb muscles in Sod1-/- mice, we found that motor axons innervating the extensor digitorum longus (EDL) and gastrocnemius muscles release fewer synaptic vesicles upon nerve stimulation. Recordings from individually identified EDL NMJs show that reductions in neurotransmitter release are apparent in the Sod1-/- mice even when endplates are close to fully innervated. However, electrophysiological properties, such as input resistance, resting membrane potential and spontaneous neurotransmitter release kinetics (but not frequency) are similar between EDL muscles of Sod1-/- and wild-type mice. Administration of the potassium channel blocker 3,4-diaminopyridine, which broadens the presynaptic action potential, improves both neurotransmitter release and muscle strength. Together, these results suggest that ROS-associated motor nerve terminal dysfunction is a contributor to the observed muscle changes in Sod1-/- mice.

Excess nutrient uptake leads to obesity, insulin resistance, and type 2 diabetes. Mammalian target of the rapamycin (mTOR), a major component of the nutrient-sensing pathway also regulates mitochondrial oxidative function. Rapamycin, a pharmacological inhibitor of mTOR, causes glucose intolerance and inhibits mitochondrial oxidative function. While a number of studies have focused on the effect of rapamycin on control wild-type mice, ours is the first to study the effect of rapamycin on mitochondrial gene expression and insulin sensitivity in the db/db mouse, a model of diabetic dyslipidemia. Female db/+ and db/db mice were fed ad libitum a rapamycin-containing diet or a control diet for 6 months, starting at two months of age. Body weight, fat mass, lean mass and food intake were measured monthly. Effect of rapamycin or control diet on markers of adipogenesis, fatty acid oxidation and mitochondrial biogenesis in the gonadal white adipose tissue (WAT) as well as different serum parameters were assessed. Whole body insulin sensitivity was measured by insulin tolerance test. Rapamycin feeding to db/db mice decreased body weight (58%) and fat mass (33%), elevated markers of fatty acid oxidation and mitochondrial biogenesis in WAT, reduced circulating non-esterified free fatty acids (NEFA), elevated circulating adiponectin and improved insulin sensitivity, compared to control diet fed db/db mice. These data demonstrate that rapamycin exhibits an anti-obesity effect and improves whole body insulin sensitivity in db/db mice and suggest an unexpected effect of simultaneous inhibition mTOR and leptin signaling in mice.

Diabetic peripheral polyneuropathy is associated with decrements in motor/sensory neuron myelination, nerve conduction and muscle function; however, the mechanisms of reduced myelination in diabetes are poorly understood. Chronic elevation of oxidative stress may be one of the potential determinants for demyelination as lipids and proteins are important structural constituents of myelin and highly susceptible to oxidation. The goal of the current study was to determine whether there is a link between protein oxidation/misfolding and demyelination. We chose two distinct models to test our hypothesis: 1) the leptin receptor deficient mouse (dbdb) model of diabetic polyneuropathy and 2) superoxide dismutase 1 knockout (Sod1(-/-) ) mouse model of in vivo oxidative stress. Both experimental models displayed a significant decrement in nerve conduction, increase in tail distal motor latency as well as reduced myelin thickness and fiber/axon diameter. Further biochemical studies demonstrated that oxidative stress is likely to be a potential key player in the demyelination process as both models exhibited significant elevation in protein carbonylation and alterations in protein conformation. Since peripheral myelin protein 22 (PMP22) is a key component of myelin sheath and has been found mutated and aggregated in several peripheral neuropathies, we predicted that an increase in carbonylation and aggregation of PMP22 may be associated with demyelination in dbdb mice. Indeed, PMP22 was found to be carbonylated and aggregated in sciatic nerves of dbdb mice. Sequence-driven hydropathy plot analysis and in vitro oxidation-induced aggregation of purified PMP22 protein supported the premise for oxidation-dependent aggregation of PMP22 in dbdb mice. Collectively, these data strongly suggest for the first time that oxidation-mediated protein misfolding and aggregation of key myelin proteins may be linked to demyelination and reduced nerve conduction in peripheral neuropathies.

Mice lacking Surf1, a complex IV assembly protein, have ∼50-70% reduction in cytochrome c oxidase activity in all tissues yet a paradoxical increase in lifespan. Here we report that Surf1(-/-) mice have lower body (15%) and fat (20%) mass, in association with reduced lipid storage, smaller adipocytes, and elevated indicators of fatty acid oxidation in white adipose tissue (WAT) compared with control mice. The respiratory quotient in the Surf1(-/-) mice was significantly lower than in the control animals (0.83-0.93 vs. 0.90-0.98), consistent with enhanced fat utilization in Surf1(-/-) mice. Elevated fat utilization was associated with increased insulin sensitivity measured as insulin-stimulated glucose uptake, as well as an increase in insulin receptor levels (∼2-fold) and glucose transporter type 4 (GLUT4; ∼1.3-fold) levels in WAT in the Surf1(-/-) mice. The expression of peroxisome proliferator-activated receptor γ-coactivator 1-α (PGC-1α) mRNA and protein was up-regulated by 2.5- and 1.9-fold, respectively, in WAT from Surf1(-/-) mice, and the expression of PGC-1α target genes and markers of mitochondrial biogenesis was elevated. Together, these findings point to a novel and unexpected link between reduced mitochondrial complex IV activity, enhanced insulin sensitivity, and increased mitochondrial biogenesis that may contribute to the increased longevity in the Surf1(-/-) mice.

Aging is a complex process that affects every major system at the molecular, cellular and organ levels. Although the exact cause of aging is unknown, there is significant evidence that oxidative stress plays a major role in the aging process. The basis of the oxidative stress hypothesis is that aging occurs as a result of an imbalance between oxidants and antioxidants, which leads to the accrual of damaged proteins, lipids and DNA macromolecules with age. Age-dependent increases in protein oxidation and aggregates, lipofuscin, and DNA mutations contribute to age-related pathologies. Many transgenic/knockout mouse models over expressing or deficient in key antioxidant enzymes have been generated to examine the effect of oxidative stress on aging and age-related diseases. Based on currently reported lifespan studies using mice with altered antioxidant defense, there is little evidence that oxidative stress plays a role in determining lifespan. However, mice deficient in antioxidant enzymes are often more susceptible to age-related disease while mice overexpressing antioxidant enzymes often have an increase in the amount of time spent without disease, i.e., healthspan. Thus, by understanding the mechanisms that affect healthy aging, we may discover potential therapeutic targets to extend human healthspan.

Abstract The San Antonio Nathan Shock Center Conferences have attracted international speakers and participants since 1995. This annual conference, held in Bandera, Texas, USA, addresses a different topic in the biology of aging each year. The venue's intimate setting, relatively remote location, and common areas are ideal for a small conference (80–100 participants), where copious informal intellectual interchange supplements that of the formal sessions. The 2011 meeting, part of an annual series sponsored by the University of Texas Health Science Center San Antonio, TX, USA, and the Nathan Shock Center of Excellence in the Biology of Aging, addressed the causes of age-associated inflammation and its effect on age-associated diseases.