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1.  Decreased in vitro mitochondrial function is associated with enhanced brain metabolism, blood flow, and memory in Surf1-deficient mice 
Recent studies have challenged the prevailing view that reduced mitochondrial function and increased oxidative stress are correlated with reduced longevity. Mice carrying a homozygous knockout (KO) of the Surf1 gene showed a significant decrease in mitochondrial electron transport chain Complex IV activity, yet displayed increased lifespan and reduced brain damage after excitotoxic insults. In the present study, we examined brain metabolism, brain hemodynamics, and memory of Surf1 KO mice using in vitro measures of mitochondrial function, in vivo neuroimaging, and behavioral testing. We show that decreased respiration and increased generation of hydrogen peroxide in isolated Surf1 KO brain mitochondria are associated with increased brain glucose metabolism, cerebral blood flow, and lactate levels, and with enhanced memory in Surf1 KO mice. These metabolic and functional changes in Surf1 KO brains were accompanied by higher levels of hypoxia-inducible factor 1 alpha, and by increases in the activated form of cyclic AMP response element-binding factor, which is integral to memory formation. These findings suggest that Surf1 deficiency-induced metabolic alterations may have positive effects on brain function. Exploring the relationship between mitochondrial activity, oxidative stress, and brain function will enhance our understanding of cognitive aging and of age-related neurologic disorders.
PMCID: PMC3790931  PMID: 23838831
glucose metabolism; memory; mitochondrial complex IV; mitochondrial dysfunction; Surf1
2.  Chronic rapamycin restores brain vascular integrity and function through NO synthase activation and improves memory in symptomatic mice modeling Alzheimer's disease 
Vascular pathology is a major feature of Alzheimer's disease (AD) and other dementias. We recently showed that chronic administration of the target-of-rapamycin (TOR) inhibitor rapamycin, which extends lifespan and delays aging, halts the progression of AD-like disease in transgenic human (h)APP mice modeling AD when administered before disease onset. Here we demonstrate that chronic reduction of TOR activity by rapamycin treatment started after disease onset restored cerebral blood flow (CBF) and brain vascular density, reduced cerebral amyloid angiopathy and microhemorrhages, decreased amyloid burden, and improved cognitive function in symptomatic hAPP (AD) mice. Like acetylcholine (ACh), a potent vasodilator, acute rapamycin treatment induced the phosphorylation of endothelial nitric oxide (NO) synthase (eNOS) and NO release in brain endothelium. Administration of the NOS inhibitor L-NG-Nitroarginine methyl ester reversed vasodilation as well as the protective effects of rapamycin on CBF and vasculature integrity, indicating that rapamycin preserves vascular density and CBF in AD mouse brains through NOS activation. Taken together, our data suggest that chronic reduction of TOR activity by rapamycin blocked the progression of AD-like cognitive and histopathological deficits by preserving brain vascular integrity and function. Drugs that inhibit the TOR pathway may have promise as a therapy for AD and possibly for vascular dementias.
PMCID: PMC3764385  PMID: 23801246
age-associated; Alzheimer's disease; cerebral amyloid angiopathy; nitric oxide synthase; rapamycin; target-of-rapamycin (TOR); vascular dysfunction
3.  Rapamycin-induced metabolic defects are reversible in both lean and obese mice 
Aging (Albany NY)  2014;6(9):742-754.
The inhibition of mTOR (mechanistic target of rapamycin) by the macrolide rapamycin has many beneficial effects in mice, including extension of lifespan and reduction or prevention of several age-related diseases. At the same time, chronic rapamycin treatment causes impairments in glucose metabolism including hyperglycemia, glucose intolerance and insulin resistance. It is unknown whether these metabolic effects of rapamycin are permanent or whether they can be alleviated. Here, we confirmed that rapamycin causes glucose intolerance and insulin resistance in both inbred and genetically heterogeneous mice fed either low fat or high fat diets, suggesting that these effects of rapamycin are independent of genetic background. Importantly, we also found that these effects were almost completely lost within a few weeks of cessation of treatment, showing that chronic rapamycin treatment does not induce permanent impairment of glucose metabolism. Somewhat surprisingly, chronic rapamycin also promoted increased accumulation of adipose tissue in high fat fed mice. However, this effect too was lost when rapamycin treatment was ended suggesting that this effect of rapamycin is also not permanent. The reversible nature of rapamycin's alterations of metabolic function suggests that these potentially detrimental side-effects might be managed through alternative dosing strategies or concurrent treatment options.
PMCID: PMC4221917  PMID: 25324470
rapamycin; glucose; insulin; obesity; mTOR
4.  Oxidative damage associated with obesity is prevented by overexpression of CuZn- or Mn-superoxide dismutase 
The development of insulin resistance is the primary step in the etiology of type 2 diabetes mellitus. There are several risk factors associated with insulin resistance, yet the basic biological mechanisms that promote its development are still unclear. There is growing literature that suggests mitochondrial dysfunction and/or oxidative stress play prominent roles in defects in glucose metabolism. Here, we tested whether increased expression of CuZn-superoxide dismutase (Sod1) or Mn-superoxide dismutase (Sod2) prevented obesity-induced changes in oxidative stress and metabolism. Both Sod1 and Sod2 overexpressing mice were protected from high fat diet-induced glucose intolerance. Lipid oxidation (F2-isoprostanes) was significantly increased in muscle and adipose with high fat feeding. Mice with increased expression of either Sod1 or Sod2 showed a significant reduction in this oxidative damage. Surprisingly, mitochondria from the muscle of high fat diet-fed mice showed no significant alteration in function. Together, our data suggest that targeting reduced oxidative damage in general may be a more applicable therapeutic target to prevent insulin resistance than by improving mitochondrial function.
PMCID: PMC3768142  PMID: 23872067
diabetes; mitochondria; F2-isoprostane; oxidative stress
5.  Dietary restriction attenuates the accelerated aging phenotype of Sod1−/− mice 
Dietary restriction is a powerful aging intervention that extends the life span of diverse biological species ranging from yeast to invertebrates to mammals, and it has been argued that the anti-aging action of dietary restriction occurs through reduced oxidative stress/damage. Using Sod1−/− mice, which have previously been shown to have increased levels of oxidative stress associated with a shorter life span and a high incidence of neoplasia, we were able to test directly the ability of dietary restriction to reverse an aging phenotype due to increased oxidative stress/damage. We found that dietary restriction increased the life span of Sod1−/− mice 30%, returning it to that of wild type, control mice fed ad libitum. Oxidative damage in Sod1−/− mice was markedly reduced by dietary restriction, as indicated by a reduction in liver and brain F2-isoprostanes, a marker of lipid peroxidation. Analysis of end of life pathology showed that dietary restriction significantly reduced the overall incidence of pathological lesions in the Sod1−/− mice fed the dietary restricted-diet compared to Sod1−/− mice fed ad libitum, including the incidence of lymphoma (27 vs 5%) and overall liver pathology. In addition to reduced incidence of overall and liver specific pathology, the burden and severity of both neoplastic and non-neoplastic lesions was also significantly reduced in the Sod1−/− mice fed the dietary restricted-diet. These data demonstrate that dietary restriction can significantly attenuate the accelerated aging phenotype observed in Sod1−/− mice that arises from increased oxidative stress/damage.
PMCID: PMC3696984  PMID: 23459073
dietary restriction; aging oxidative stress; CuZnSOD
6.  Calorie Restriction Alleviates Age-Related Decrease in Neural Progenitor Cell Division in the Aging Brain 
The European journal of neuroscience  2013;37(12):1987-1993.
Production of new neurons from stem cells is important for cognitive function, and the reduction of neurogenesis in the aging brain may contribute to the accumulation of age-related cognitive deficits. Restriction of calorie intake and prolonged treatment with rapamycin have been shown to extend the lifespan of animals and delay the onset of age-related decline in tissue and organ function. Using a reporter line in which neural stem and progenitor cells are marked by the expression of GFP, we examined the effect of prolonged exposure to calorie restriction (CR) or rapamycin on hippocampal neural stem and progenitor cell proliferation in aging mice. We show that CR increases the number of dividing cells in the dentate gyrus (DG) of female mice. The majority of these cells corresponded to Nestin-GFP-expressing neural stem or progenitor cells; however, this increased proliferative activity of stem and progenitor cells did not result in a significant increase in the number of doublecortin-positive newborn neurons. Our results suggest that restricted calorie intake may increase the number of divisions that neural stem and progenitor cells undergo in the aging brain of females.
PMCID: PMC3717568  PMID: 23773068
adult neurogenesis; stem cells; aging; calorie restriction; rapamycin
7.  Combined treatment of rapamycin and dietary restriction has a larger effect on the transcriptome and metabolome of liver 
Aging Cell  2013;13(2):311-319.
Rapamycin (Rapa) and dietary restriction (DR) have consistently been shown to increase lifespan. To investigate whether Rapa and DR affect similar pathways in mice, we compared the effects of feeding mice ad libitum (AL), Rapa, DR, or a combination of Rapa and DR (Rapa + DR) on the transcriptome and metabolome of the liver. The principal component analysis shows that Rapa and DR are distinct groups. Over 2500 genes are significantly changed with either Rapa or DR when compared with mice fed AL; more than 80% are unique to DR or Rapa. A similar observation was made when genes were grouped into pathways; two-thirds of the pathways were uniquely changed by DR or Rapa. The metabolome shows an even greater difference between Rapa and DR; no metabolites in Rapa-treated mice were changed significantly from AL mice, whereas 173 metabolites were changed in the DR mice. Interestingly, the number of genes significantly changed by Rapa + DR when compared with AL is twice as large as the number of genes significantly altered by either DR or Rapa alone. In summary, the global effects of DR or Rapa on the liver are quite different and a combination of Rapa and DR results in alterations in a large number of genes and metabolites that are not significantly changed by either manipulation alone, suggesting that a combination of DR and Rapa would be more effective in extending longevity than either treatment alone.
PMCID: PMC3989927  PMID: 24304444
dietary restriction; metabolome; rapamycin; transcriptome
8.  Methionine sulfoxide reductase A affects insulin resistance by protecting insulin receptor function 
Oxidative stress plays a significant role in the development of insulin resistance; however, the cellular targets of oxidation that cause insulin resistance have yet to be fully elucidated. Methionine sulfoxide reductases (Msr) reduce oxidized methionine residues, thereby repairing and protecting proteins from oxidation. Recently, several genome-wide analyses have found human obesity to be strongly correlated with polymorphisms near the methionine sulfoxide reductase A (MsrA) locus. In this study, we tested whether modulation of MsrA expression significantly alters the development of obesity and/or insulin resistance in mice. We show that mice lacking MsrA (MsrA−/−) are prone to the development of high fat diet-induced insulin resistance and a reduced physiological insulin response compared to high fat-fed wild type mice. We also show that oxidative stress in C2C12 cell cultures reduces both insulin-stimulated phosphorylation and autophosphorylation of the insulin receptor. Tissues from high fat-fed mice show similar reduction in insulin receptor function and the lack of MsrA further diminishes these functions. Together, these data demonstrate for the first time that MsrA plays a role in the regulation of glucose homeostasis. In addition, these data support a novel hypothesis that obesity-induced insulin resistance is caused in part by reduced function of insulin signaling proteins arising from protein oxidation.
PMCID: PMC3578155  PMID: 23089224
oxidative stress; methionine sulfoxide; diabetes; obesity; glucose homeostasis
9.  Mice Fed Rapamycin Have an Increase in Lifespan Associated with Major Changes in the Liver Transcriptome 
PLoS ONE  2014;9(1):e83988.
Rapamycin was found to increase (11% to 16%) the lifespan of male and female C57BL/6J mice most likely by reducing the increase in the hazard for mortality (i.e., the rate of aging) term in the Gompertz mortality analysis. To identify the pathways that could be responsible for rapamycin's longevity effect, we analyzed the transcriptome of liver from 25-month-old male and female mice fed rapamycin starting at 4 months of age. Few changes (<300 transcripts) were observed in transcriptome of rapamycin-fed males; however, a large number of transcripts (>4,500) changed significantly in females. Using multidimensional scaling and heatmap analyses, the male mice fed rapamycin were found to segregate into two groups: one group that is almost identical to control males (Rapa-1) and a second group (Rapa-2) that shows a change in gene expression (>4,000 transcripts) with more than 60% of the genes shared with female mice fed Rapa. Using ingenuity pathway analysis, 13 pathways were significantly altered in both Rapa-2 males and rapamycin-fed females with mitochondrial function as the most significantly changed pathway. Our findings show that rapamycin has a major effect on the transcriptome and point to several pathways that would likely impact the longevity.
PMCID: PMC3883653  PMID: 24409289
10.  Rapamycin, anti-aging, and avoiding the fate of Tithonus 
The Journal of Clinical Investigation  2013;123(8):3204-3206.
The discovery that rapamycin increased the lifespan of mice was recognized by Science as one of the top 10 scientific breakthroughs of 2009. In addition to increasing lifespan, Neff and colleagues show that while rapamycin improves several functions/pathologies that change with age, it has little effect on the majority of the physiological and structural parameters they evaluated. What do these data tell us about the ability of rapamycin to delay aging and improve quality of life, i.e., prevent the fate of Tithonus?
PMCID: PMC3726178  PMID: 24063054
11.  Chronic inhibition of mTOR by rapamycin modulates cognitive and non-cognitive components of behavior throughout lifespan in mice 
Neuroscience  2012;223:102-113.
Aging is, by far, the greatest risk factor for most neurodegenerative diseases. In non-diseased conditions, normal aging can also be associated with declines in cognitive function that significantly affect quality of life in the elderly. It was recently shown that inhibition of mTOR activity in mice by chronic rapamycin treatment extends lifespan, possibly by delaying aging {Harrison, 2009 #4}{Miller, 2011 #168}. To explore the effect of chronic rapamycin treatment on normal brain aging we determined cognitive and non-cognitive components of behavior throughout lifespan in male and female C57BL/6 mice that were fed control- or rapamycin-supplemented chow. Our studies show that rapamycin enhances cognitive function in young adult mice and blocks age-associated cognitive decline in older animals. In addition, mice fed with rapamycin-supplemented chow showed decreased anxiety and depressive-like behavior at all ages tested. Levels of three major monoamines (norepinephrine, dopamine and 5-hydroxytryptamine) and their metabolites (3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindolacetic acid) were significantly augmented in midbrain of rapamycin-treated mice compared to controls. Our results suggest that chronic, partial inhibition of mTOR by oral rapamycin enhances learning and memory in young adults, maintains memory in old C57BL/6J mice, and has concomitant anxiolytic and antidepressant-like effects, possibly by stimulating major monoamine pathways in brain.
PMCID: PMC3454865  PMID: 22750207
mammalian target of rapamycin; memory; depression; anxiety; brain aging; monoamines
12.  Dietary restriction attenuates age-associated muscle atrophy by lowering oxidative stress in mice even in complete absence of CuZnSOD 
Aging cell  2012;11(5):770-782.
Age-related loss of muscle mass and function, sarcopenia, has a major impact on the quality of life in the elderly. Among the proposed causes of sarcopenia are mitochondrial dysfunction and accumulated oxidative damage during aging. Dietary restriction (DR), a robust dietary intervention that extends lifespan and modulates age-related pathology in a variety of species has been shown to protect from sarcopenia in rodents. Although the mechanism(s) by which DR modulates aging are still not defined, one potential mechanism is through modulation of oxidative stress and mitochondrial dysfunction. To directly test the protective effect of DR against oxidative stress induced muscle atrophy in vivo, we subjected mice lacking a key antioxidant enzyme, CuZnSOD (Sod1) to DR (40% of ad libitum fed diet). We have previously shown that the Sod1−/− mice exhibit an acceleration of sarcopenia associated with high oxidative stress, mitochondrial dysfunction, and severe neuromuscular innervation defects. Despite the dramatic atrophy phenotype in the Sod1−/− mice, DR led to a reversal or attenuation of reduced muscle function, loss of innervation and muscle atrophy in these mice. DR improves mitochondrial function as evidenced by enhanced Ca2+ regulation and reduction of mitochondrial reactive oxygen species (ROS). Furthermore, we show upregulation of SIRT3 and MnSOD in DR animals, consistent with reduced mitochondrial oxidative stress and reduced oxidative damage in muscle tissue measured as F2- isoprostanes. Collectively, our results demonstrate that DR is a powerful mediator of mitochondrial function, mitochondrial ROS production, and oxidative damage, providing a solid protection against oxidative stress induced neuromuscular defects and muscle atrophy in vivo even under conditions of high oxidative stress.
PMCID: PMC3444532  PMID: 22672615
13.  Attenuation of Liver Insoluble Protein Carbonyls: Indicator of A Longevity Determinant? 
Aging cell  2011;10(4):720-723.
Oxidative damage affects protein structure and function. Progressive accumulation of oxidized proteins is considered a putative mechanism of aging; however, empirical evidence supporting their role in aging is inconsistent. This inconsistency may reflect a failure to distinguish damage to particular cellular compartments. We found significant reduction of protein carbonyl in the insoluble, but not the soluble, fraction of liver tissues of long-lived compared to short-lived animals. Of cellular components analyzed, only nuclear protein carbonyl level was uniformly reduced in long-lived compared with short-lived animals. This observation suggests that attenuated accumulation of protein carbonyls in the nucleus, where they can affect multiple aspects of gene expression and DNA repair, might contribute to the longevity in mammalian species.
PMCID: PMC3766365  PMID: 21463461
naked-mole rat; marmoset; bats; protein carbonylation; dietary restriction; oxidative stress
14.  Proteomic Screening of Glycoproteins in Human Plasma for Frailty Biomarkers 
The application of proteomics methodology for analyzing human blood samples is of increasing importance as a noninvasive method for understanding, detecting, and monitoring disease. In particular, glycoproteomic analysis may be useful in the study of age-related diseases and syndromes, such as frailty. This study demonstrates the use of methodology for isolating plasma glycoproteins using lectins, comparing the glycoproteome by frailty status using two-dimensional polyacrylamide gel electrophoresis and identifying glycoproteins using mass spectrometry. In a pilot study, we found seven glycoproteins to differ by at least twofold in prefrail compared with nonfrail older adults, including haptoglobin, transferrin, and fibrinogen, consistent with known inflammatory and hematologic changes associated with frailty. Enzyme-linked immunosorbent assay analysis found that plasma transferrin concentration was increased in frail and prefrail older adults compared with nonfrail, confirming our proteomic findings. This work provides evidence for using a reproducible methodology for conducting clinical proteomic comparative studies of age-related diseases.
PMCID: PMC3403859  PMID: 22219522
Proteomics; Frailty
15.  IDH1 Is Downregulated during Early Skin Tumorigenesis Which Can Be Inhibited by Overexpression of MnSOD 
Cancer science  2012;103(8):1429-1433.
Isocitrate dehydrogenase 1 (IDH1), a cytosolic enzyme which converts isocitrate to alpha-ketoglutarate, has been shown to be dysregulated during tumorigenesis. However, at what stage of cancer development IDH1 is dysregulated and how IDH1 may affect cell transformation and tumor promotion during early stages of cancer development, are unclear. We utilized a skin cell transformation model, and, mouse skin epidermal tissues to study the role of IDH1 in early skin tumorigenesis. Our studies demonstrate that both the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) and UVC irradiation decreased expression and activity levels of IDH1, not IDH2, in the tumor promotable JB6 P+ cell model. Skin epidermal tissues treated with DMBA/TPA also showed decreases in IDH1 expression and activity. In non-promotable JB6 P− cells, IDH1 was upregulated upon TPA treatment, whereas IDH2 was maintained at similar levels with TPA treatment. Interestingly, IDH1 knockdown enhanced, whereas IDH1 overexpression suppressed TPA-induced cell transformation. Finally, manganese superoxide dismutase (MnSOD) overexpression suppressed tumor promoter-induced decreases in IDH1 expression and mitochondrial respiration, while intracellular alpha-ketoglutarate levels were unchanged. These results suggest that decreased IDH1 expression in early stage skin tumorigenesis is highly correlated with tumor promotion. In addition, oxidative stress may contribute to IDH1 inactivation, because MnSOD, a mitochondrial antioxidant enzyme, blocked decreases in IDH1 expression and activity.
PMCID: PMC3407305  PMID: 22533343
16.  Complex I generated, mitochondrial matrix-directed superoxide is released from the mitochondria through voltage dependent anion channels 
Mitochondrial complex I has previously been shown to release superoxide exclusively towards the mitochondrial matrix, whereas complex III releases superoxide to both the matrix and the cytosol. Superoxide produced at Complex III has been shown to exit the mitochondria through voltage dependent anion channels (VDAC). To test whether complex I-derived, mitochondrial matrix-directed superoxide can be released to the cytosol, we measured superoxide generation in mitochondria isolated from wild type and from mice genetically altered to be deficient in MnSOD activity (TnIFastCreSod2fl/fl). Under experimental conditions that produce superoxide primarily by complex I (glutamate/malate plus rotenone, GM+R), MnSOD-deficient mitochondria release ~4-fold more superoxide than mitochondria isolated from wild type mice. Exogenous CuZnSOD completely abolished the EPR-derived GM+R signal in mitochondria isolated from both genotypes, evidence that confirms mitochondrial superoxide release. Addition of the VDAC inhibitor DIDS significantly reduced mitochondrial superoxide release (~75%) in mitochondria from either genotype respiring on GM+R. Conversely, inhibition of potential inner membrane sites of superoxide exit, including the matrix face of the mitochondrial permeability transition pore and the inner membrane anion channel did not reduce mitochondrial superoxide release in the presence of GM+R in mitochondria isolated from either genotype. These data support the concept that complex I-derived mitochondrial superoxide release does indeed occur and that the majority of this release occurs through VDACs.
PMCID: PMC3400138  PMID: 22613204
mitochondria; superoxide; voltage dependent anion channels
17.  Gpx4 ablation in adult mice results in a lethal phenotype accompanied by neuronal loss in brain 
Free radical biology & medicine  2012;52(9):1820-1827.
Glutathione peroxidase 4 (Gpx4) is an antioxidant defense enzyme important in reducing hydroperoxides in membrane lipids and lipoproteins. Gpx4 is essential for survival of embryos and neonatal mice; however, whether Gpx4 is required for adult animals remains unclear. In this study, we generated a floxed Gpx4 mouse (Gpx4(f/f)), in which exons 2–4 of Gpx4 gene are flanked by loxP sites. We then cross-bred the Gpx4(f/f) mice with a tamoxifen (tam)-inducible Cre transgenic mouse (R26CreER mice) to obtain mice in which the Gpx4 gene could be ablated by tam administration (Gpx4(f/f)/Cre mice). After treatment with tam, adult Gpx4(f/f)/Cre mice (6–9 months of age) showed a significant reduction of Gpx4 levels (a 75–85 % decrease) in tissues such as brain, liver, lung and kidney. Tam-treated Gpx4(f/f)/Cre mice lost body weight and died within 2 weeks, indicating that Gpx4 is essential for survival of adult animals. Tam-treated Gpx4(f/f)/Cre mice exhibited increased mitochondrial damage, as evidenced by the elevated 4-hydroxylnonenal (4-HNE) level, decreased activities of electron transport chain complex I and IV, and reduced ATP production in liver. Tam treatment also significantly elevated apoptosis in Gpx4(f/f)/Cre mice. Moreover, tam-treated Gpx4(f/f)/Cre mice showed neuronal loss in hippocampus region and had increased astrogliosis. These data indicate that Gpx4 is essential for mitochondria integrity and survival of neurons in adult animals.
PMCID: PMC3341497  PMID: 22401858
Gpx4; knockout mice; lipid peroxidation; mitochondria; neurodegeneration; apoptosis; oxidative stress
18.  Life-long rapamycin administration ameliorates age-dependent cognitive deficits by reducing IL-1β and enhancing NMDA signaling 
Aging Cell  2012;11(2):326-335.
Understanding the factors that contribute to age-related cognitive decline is imperative, particularly as age is the major risk factor for several neurodegenerative disorders. Levels of several cytokines increase in the brain during aging, including IL-1β, whose levels positively correlate with cognitive deficits. Previous reports show that reducing the activity of the mammalian target of rapamycin (mTOR) extends lifespan in yeast, nematodes, Drosophila, and mice. It remains to be established, however, whether extending lifespan with rapamycin is accompanied by an improvement in cognitive function. In this study, we show that 18-month-old mice treated with rapamycin starting at two months of age perform significantly better on a task measuring spatial learning and memory compared to age-matched mice on the control diet. In contrast, rapamycin does not improve cognition when given to 15-month-old mice with pre-existing, age-dependent learning and memory deficits. We further show that the rapamycin-mediated improvement in learning and memory is associated with a decrease in IL-1β levels and an increase in NMDA signaling. This is the first evidence to show that a small molecule known to increase lifespan also ameliorates age-dependent learning and memory deficits.
PMCID: PMC3306461  PMID: 22212527
learning and memory; mTOR; NMDA; aging; IL-1β; cytokines
19.  Oxidative stress and diabetes: what can we learn about insulin resistance from antioxidant mutant mouse models? 
The development of metabolic dysfunctions like diabetes and insulin resistance in mammals is regulated by a myriad of factors. Oxidative stress seems to play a central role in this process as recent evidence shows a general increase in oxidative damage and a decrease in oxidative defense associated with several metabolic diseases. These changes in oxidative stress can be directly correlated with increased fat accumulation, obesity and consumption of high calorie/high fat diets. Modulation of oxidant protection through either genetic mutation or treatment with antioxidants can significantly alter oxidative stress resistance and accumulation of oxidative damage in laboratory rodents. Antioxidant mutant mice have previously been utilized to examine the role of oxidative stress in other disease models, but have been relatively unexplored as models to study the regulation of glucose metabolism. In this review, we will discuss the evidence for oxidative stress as a primary mechanism linking obesity and metabolic disorders and whether alteration of antioxidant status in laboratory rodents can significantly alter the development of insulin resistance or diabetes.
PMCID: PMC3249484  PMID: 22056908
oxidative stress; diabetes; obesity; adipose; insulin resistance
20.  Thioredoxin 1 Overexpression Extends Mainly the Earlier Part of Life Span in Mice 
We examined the effects of increased levels of thioredoxin 1 (Trx1) on resistance to oxidative stress and aging in transgenic mice overexpressing Trx1 [Tg(TRX1)+/0]. The Tg(TRX1)+/0 mice showed significantly higher Trx1 protein levels in all the tissues examined compared with the wild-type littermates. Oxidative damage to proteins and levels of lipid peroxidation were significantly lower in the livers of Tg(TRX1)+/0 mice compared with wild-type littermates. The survival study demonstrated that male Tg(TRX1)+/0 mice significantly extended the earlier part of life span compared with wild-type littermates, but no significant life extension was observed in females. Neither male nor female Tg(TRX1)+/0 mice showed changes in maximum life span. Our findings suggested that the increased levels of Trx1 in the Tg(TRX1)+/0 mice were correlated to increased resistance to oxidative stress, which could be beneficial in the earlier part of life span but not the maximum life span in the C57BL/6 mice.
PMCID: PMC3210956  PMID: 21873593
Thioredoxin; Transgenic mouse; Oxidative stress; Protein carbonylation; Aging
21.  Reduction in Reactive Oxygen Species Production by Mitochondria From Elderly Subjects With Normal and Impaired Glucose Tolerance 
Diabetes  2011;60(8):2051-2060.
Aging increases the risk of developing impaired glucose tolerance (IGT) and type 2 diabetes. It has been proposed that increased reactive oxygen species (ROS) generation by dysfunctional mitochondria could play a role in the pathogenesis of these metabolic abnormalities. We examined whether aging per se (in subjects with normal glucose tolerance [NGT]) impairs mitochondrial function and how this relates to ROS generation, whether older subjects with IGT have a further worsening of mitochondrial function (lower ATP production and elevated ROS generation), and whether exercise reverses age-related changes in mitochondrial function.
Mitochondrial ATP and ROS production were measured in muscle from younger individuals with NGT, older individuals with NGT, and older individuals with IGT. Measurements were performed before and after 16 weeks of aerobic exercise.
ATP synthesis was lower in older subjects with NGT and older subjects with IGT versus younger subjects. Notably, mitochondria from older subjects (with NGT and IGT) displayed reduced ROS production versus the younger group. ATP and ROS production were similar between older groups. Exercise increased ATP synthesis in the three groups. Mitochondrial ROS production also increased after training. Proteomic analysis revealed downregulation of several electron transport chain proteins with aging, and this was reversed by exercise.
Old mitochondria from subjects with NGT and IGT display mitochondrial dysfunction as manifested by reduced ATP production but not with respect to increased ROS production. When adjusted to age, the development of IGT in elderly individuals does not involve changes in mitochondrial ATP and ROS production. Lastly, exercise reverses the mitochondrial phenotype (proteome and function) of old mitochondria.
PMCID: PMC3142073  PMID: 21677280
22.  Overexpression of human SOD1 improves survival of mice susceptible to endotoxic shock 
Protective effects of the antioxidant enzyme Cu-Zn superoxide dismutase (SOD1) against endotoxic shock have not been demonstrated in animal models. We used a murine model to investigate whether overexpression of SOD1 protects against endotoxic shock, and whether the genetic background of SOD1 affects its effective protective effects and susceptibility to endotoxic shock.
Transgenic (tg) mice overexpressing human SOD1 and control mice were divided into four groups based on their genetic background: (1) tg mice with mixed genetic background (tg-JAX); (2) wild-type (WT) littermates of tg-JAX strain (WT-JAX); (3) tg mice with C57BL/6J background (tg-TX); (4) WT littermates of tg-TX strain (WT-TX). Activity of SOD1 in the intestine, heart, and liver of tg and control mice was confirmed using a polyacrylamide activity gel. Endotoxic shock was induced by intraperitoneal injection of lipopolysaccharide. Survival rates over 120 hours (mean, 95% confidence interval) were analyzed using Kaplan–Meier survival curves.
Human SOD1 enzymatic activities were significantly higher in the intestine, heart, and liver of both tg strains (tg-JAX and tg-TX) compared with their WT littermates (WT-JAX and WT-TX, respectively). Interestingly, the endogenous SOD1 activities in tg-JAX mice were decreased compared with their WT littermates (WT-JAX), but such aberrant changes were not observed in tg-TX mice. There was no difference in the survival time between tg-JAX and WT-JAX groups after endotoxic shock (P > 0.05). However, the survival time in the tg-TX group was more than twofold longer than that in the WT-TX group (P < 0.05). In addition, WT-JAX mice survived significantly longer than WT-TX mice (P < 0.05).
Aberrant decrease of endogenous SOD1 activities may have overshadowed the effect of overexpression of SOD1 in tg mice (tg-JAX). Mice with C57BL/6J background (tg-TX) are more susceptible to lipopolysaccharide-induced endotoxic shock than those with mixed genetic background (tg-JAX). Overexpression of SOD1 is protective only in mice with C57BL/6J background (tg-TX).
PMCID: PMC3413208  PMID: 22879777
human SOD1 enzyme; endotoxic shock; transgenic mice; protective effect
23.  MnSOD deficiency results in elevated oxidative stress and decreased mitochondrial function but does not lead to muscle atrophy during aging 
Aging cell  2011;10(3):493-505.
In a previous study, we reported that a deficiency in MnSOD activity (approximately 80% reduction) targeted to type IIB skeletal muscle fibers was sufficient to elevate oxidative stress and to reduce muscle function in young adult mice (TnIFastCreSod2fl/fl mice). In the present study, we used TnIFastCreSod2fl/fl mice to examine the effect of elevated oxidative stress on mitochondrial function and to test the hypothesis that elevated oxidative stress and decreased mitochondrial function over the lifespan of the TnIFastCreSod2fl/fl mice would be sufficient to accelerate muscle atrophy associated with aging. We found that mitochondrial function is reduced in both young and old TnIFastCreSod2fl/fl mice, when compared with control mice. Complex II activity is reduced by 47% in young and by ~90% in old TnIFastCreSod2fl/fl mice, associated with reduced levels of the catalytic subunits for complex II, SDHA and SDHB. Complex II-linked mitochondrial respiration is reduced by approximately 70% in young TnIFastCreSod2fl/fl mice. Complex II-linked mitochondrial ATP production is reduced by 39% in young and was found to be almost completely absent in old TnIFastCreSod2fl/fl mice. Furthermore, in old TnIFastCreSod2fl/fl mice, aconitase activity is almost completely abolished; mitochondrial superoxide release remains greater than 2-fold elevated; and oxidative damage (measured as F2 isoprostanes) is increased by 30% relative to age-matched controls. These data show that despite elevated skeletal muscle-specific mitochondrial oxidative stress, oxidative damage and complex II-linked mitochondrial dysfunction, age-related muscle atrophy was not accelerated in old TnIFastCreSod2fl/fl mice, suggesting mitochondrial oxidative stress may not be causal for age-related muscle atrophy.
PMCID: PMC3094473  PMID: 21385310
24.  Short-Term Treatment With Rapamycin and Dietary Restriction Have Overlapping and Distinctive Effects in Young Mice 
Because rapamycin, an inhibitor of the nutrient sensor mammalian target of rapamycin, and dietary restriction both increase life span of mice, it has been hypothesized that they act through similar mechanisms. To test this hypothesis, we compared various biological parameters in dietary restriction mice (40% food restriction) and mice fed rapamycin (14 ppm). Both treatments led to a significant reduction in mammalian target of rapamycin signaling and a corresponding increase in autophagy. However, we observed striking differences in fat mass, insulin sensitivity, and expression of cell cycle and sirtuin genes in mice fed rapamycin compared with dietary restriction. Thus, although both treatments lead to significant downregulation of mammalian target of rapamycin signaling, these two manipulations have quite different effects on other physiological functions suggesting that they might increase life span through a common pathway as well as pathways that are altered differently by dietary restriction and rapamycin.
PMCID: PMC3598360  PMID: 22570137
Rapamycin; Dietary restriction; mTOR; Autophagy; Gene expression
25.  Rapamycin-induced insulin resistance is mediated by mTORC2 loss and uncoupled from longevity 
Science (New York, N.y.)  2012;335(6076):1638-1643.
Rapamycin, an inhibitor of mechanistic target of rapamycin complex 1 (mTORC1), extends the lifespans of yeast, flies, and mice. Calorie restriction, which increases lifespan 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 lifespan independently from changes in glucose homeostasis, as female mice heterozygous for both mTOR and mLST8 exhibited decreased mTORC1 activity and extended lifespan, but had normal glucose tolerance and insulin sensitivity. Thus, mTORC2 disruption is an important mediator of the effects of rapamycin in vivo.
PMCID: PMC3324089  PMID: 22461615

Results 1-25 (51)