Lifelong reduction in complex IV induces tissue-specific metabolic effects but does not reduce lifespan or healthspan in mice.

Loss of SURF1, a Complex IV assembly protein, was reported to increase lifespan in mice despite dramatically lower cytochrome oxidase (COX) activity. Consistent with this, our previous studies found advantageous changes in metabolism (reduced adiposity, increased insulin sensitivity, and mitochondrial biogenesis) in Surf1-/- mice. The lack of deleterious phenotypes in Surf1-/- mice is contrary to the hypothesis that mitochondrial dysfunction contributes to aging. We found only a modest (nonsignificant) extension of lifespan (7% median, 16% maximum) and no change in healthspan indices in Surf1-/- vs. Surf1+/+ mice despite substantial decreases in COX activity (22%-87% across tissues). Dietary restriction (DR) increased median lifespan in both Surf1+/+ and Surf1-/- mice (36% and 19%, respectively). We measured gene expression, metabolites, and targeted expression of key metabolic proteins in adipose tissue, liver, and brain in Surf1+/+ and Surf1-/- mice. Gene expression was differentially regulated in a tissue-specific manner. Many proteins and metabolites are downregulated in Surf1-/- adipose tissue and reversed by DR, while in brain, most metabolites that changed were elevated in Surf1-/- mice. Finally, mitochondrial unfolded protein response (UPRmt )-associated proteins were not uniformly altered by age or genotype, suggesting the UPRmt is not a key player in aging or in response to reduced COX activity. While the changes in gene expression and metabolism may represent compensatory responses to mitochondrial stress, the important outcome of this study is that lifespan and healthspan are not compromised in Surf1-/- mice, suggesting that not all mitochondrial deficiencies are a critical determinant of lifespan.

Down-regulation of the mitochondrial matrix peptidase ClpP in muscle cells causes mitochondrial dysfunction and decreases cell proliferation.

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.

Gene expression in the liver of female, but not male mice treated with rapamycin resembles changes observed under dietary restriction.

It is well known that in mice the extension in lifespan by rapamycin is sexually dimorphic, in that it has a larger effect in females than males. In a previous study we showed that in male C57BL6 mice, rapamycin had less profound effects in both gene expression and liver metabolites when compared to dietary restriction (DR), but no data was available in females. Because recent studies showed that rapamycin increases longevity in a dose dependent manner and at every dose tested the effect remains larger in females than in males, we hypothesized that rapamycin should have a stronger effect on gene expression in females, and this effect could be dose dependent. To test this hypothesis, we measured the changes in liver gene expression induced by rapamycin (14 ppm) with a focus on several genes involved in pathways known to play a role in aging and that are altered by DR. To investigate whether any effects are dose dependent, we also analyzed females treated with two additional doses of rapamycin (22 and 42 ppm). We observed striking differences between male and female in gene expression at 14 ppm, where females have a larger response to rapamycin than males, and the effects of rapamycin in females resemble what we observed under DR. However, these effects were generally not dose dependent. These data support the notion that female mice respond better to rapamycin, and at least with the set of genes studied here, the effect of rapamycin in females resemble the effect of DR.

Rapamycin and dietary restriction induce metabolically distinctive changes in mouse liver.

Dietary restriction (DR) is the gold standard intervention used to delay aging, and much recent research has focused on the identification of possible DR mimetics. Energy sensing pathways, including insulin/IGF1 signaling, sirtuins, and mammalian Target of Rapamycin (mTOR), have been proposed as pathways involved in the antiaging actions of DR, and compounds that affect these pathways have been suggested to act as DR mimetics, including metformin (insulin/IGF1 signaling), resveratrol (sirtuins), and rapamycin (mTOR). Rapamycin is a promising DR mimetic because it significantly increases both health span and life span in mice. Unfortunately, rapamycin also leads to some negative effects, foremost among which is the induction of insulin resistance, potentially limiting its translation into humans. To begin clarifying the mechanism(s) involved in insulin resistance induced by rapamycin, we compared several aspects of liver metabolism in mice treated with DR or rapamycin for 6 months. Our data suggest that although both DR and rapamycin inhibit lipogenesis, activate lipolysis, and increased serum levels of nonesterified fatty acids, only DR further activates ß-oxidation of the fatty acids leading to the production of ketone bodies.

Short-term rapamycin treatment in mice has few effects on the transcriptome of white adipose tissue compared to dietary restriction.

Rapamycin, a drug that has been shown to increase lifespan in mice, inhibits the target of rapamycin (TOR) pathway, a major pathway that regulates cell growth and energy status. It has been hypothesized that rapamycin and dietary restriction (DR) extend lifespan through similar mechanisms/pathways. Using microarray analysis, we compared the transcriptome of white adipose tissue from mice fed rapamycin or DR-diet for 6 months. Multidimensional scaling and heatmap analyses showed that rapamycin had essentially no effect on the transcriptome as compared to DR. For example, only six transcripts were significantly altered by rapamycin while mice fed DR showed a significant change in over 1000 transcripts. Using ingenuity pathway analysis, we found that stearate biosynthesis and circadian rhythm signaling were significantly changed by DR. Our findings showing that DR, but not rapamycin, has an effect on the transcriptome of the adipose tissue, suggesting that these two manipulations increase lifespan through different mechanisms/pathways.

Mice fed rapamycin have an increase in lifespan associated with major changes in the liver transcriptome.

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.

Combined treatment of rapamycin and dietary restriction has a larger effect on the transcriptome and metabolome of liver.

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.

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.