Effects of Sex, Strain, and Energy Intake on Hallmarks of Aging in Mice

“Analysis of steady-state levels of LC3-II, the most commonly used marker to measure autophagic compartments (Kabeya et al., 2000), revealed that 40% CR significantly increased levels of LC3-II in B6 mice when compared to 20% CR (Figures 5E and 5F).”

“Indeed, recent work has shown in humans that slight overweight is associated with the lowest level of all-cause mortality (Flegal et al., 2013; Grabowski and Ellis, 2001).”

“Interestingly, all CR livers displayed a significant increase in lipid droplets withmorphological signatures of lipophagy (Singh et al., 2009), which was rarely observed in the AL groups (Figures S4H and S4M).”

“Ubiquitinated proteins are amenable to degradation via macroautophagy, and blockage of this pathway leads to intracellular accumulation of K48- and K63-positive aggregates (Hara et al., 2006; Olzmann and Chin, 2008; Tan et al., 2008).”

“Not surprisingly, most of these interventions have strain-, diet-, and sex-specific effects on health and survival in mice (Anisimov et al., 2008; Harrison et al., 2009, 2014; Martin-Montalvo et al., 2013; Miller et al., 2011, 2014; Pearson et al., 2008a; Strong et al., 2008, 2013).”

“Whereas all CR groups showed reducedmitochondria circularity, suggestive of reducedmitophagy, only some of them displayed enhanced lipophagy (Gomes et al., 2011; Twig et al., 2008).”

“This uncoupling of lifespan/health span benefits by CR has been reported in earlier studies in mice and non-human primates (Colman et al., 2009; Mattison et al., 2012; Rikke and Johnson, 2007; Turturro and Hart, 1991).”

“, 1917) and then McCay and colleagues reported the lifespan effects of calorie-restricted diet in rats (McCay et al., 1935).”

“It has been shown that mitochondrial shape is closely related to mitophagy in such a way that elongated mitochondria are preserved from mitophagy (Gomes et al., 2011).”

“Although PARP-1 activity was not measured, it is possible that CRinduced changes in PARP-1 expression will impact on the NAD levels, thereby affecting protein deacetylation performed by sirtuins, and ultimately shape nuclear-mitochondrial communication and the control of energy homeostasis (Cantó et al., 2015; Gomes e…”

“Although PARP-1 activity was not measured, it is possible that CRinduced changes in PARP-1 expression will impact on the NAD levels, thereby affecting protein deacetylation performed by sirtuins, and ultimately shape nuclear-mitochondrial communication and the control of energy homeostasis (Cantó et al., 2015; Gomes e…”

“Not surprisingly, most of these interventions have strain-, diet-, and sex-specific effects on health and survival in mice (Anisimov et al., 2008; Harrison et al., 2009, 2014; Martin-Montalvo et al., 2013; Miller et al., 2011, 2014; Pearson et al., 2008a; Strong et al., 2008, 2013).”

“These results are consistent with CR-mediated inhibition of SIRT3 and are at odds with previous reports (Qiu et al., 2010; Tauriainen et al., 2011).”

“…content and/or restriction of specific components of the diet such as methionine or protein, different feeding regiments, and periodic periods of fasting (Fontana and Partridge, 2015; Hine et al., 2015; Masoro, 1990; Miller and Roth, 2007; MurtaghMark et al., 1995; Solon-Biet et al., 2015).”

“This uncoupling of lifespan/health span benefits by CR has been reported in earlier studies in mice and non-human primates (Colman et al., 2009; Mattison et al., 2012; Rikke and Johnson, 2007; Turturro and Hart, 1991).”

“Attachment of ubiquitin molecules to proteins is commonly used for their degradation by the 26S proteasome complex (when using the ubiquitin K48-linkage) or by autophagy (if ubiquitin chains are attached through K63-linkage) (Ciechanover, 2005; Olzmann and Chin, 2008; Tan et al., 2008).”

“, 2013), enhanced mitochondrial function (López-Lluch et al., 2006; Nisoli et al., 2005), improved proteostasis and autophagy (Cuervo, 2008; Hansen et al.”

“All these outcomes are accompanied by increases in median and maximum lifespan (Bartke et al., 2007; Gross and Dreyfuss, 1984; Kritchevsky, 2002; Weindruch and Sohal, 1997; Weindruch et al., 1986).”

“…in lipid metabolism are a consequence of the observed increase in chaperone-mediated autophagy activity, which was recently shown to contribute to regulation of hepatic lipid metabolism through the degradation of key enzymes in these pathways (Kaushik and Cuervo, 2015; Schneider et al., 2014).”

“Not surprisingly, most of these interventions have strain-, diet-, and sex-specific effects on health and survival in mice (Anisimov et al., 2008; Harrison et al., 2009, 2014; Martin-Montalvo et al., 2013; Miller et al., 2011, 2014; Pearson et al., 2008a; Strong et al., 2008, 2013).”

“Genetic mutations in growth hormone, IGF-1 receptor, or downstream effectors of insulin/IGF-1 signaling (i.e., PI-3 kinase and FOXO) have been associated with increased lifespan (Flurkey et al., 2001, 2002; Hsieh et al., 2002; Shimokawa et al., 2015; Sun et al., 2013).”

“Interestingly, strains of mice that maintain high Tb are more likely to have extended lifespan under CR (Speakman and Mitchell, 2011).”

“Further, we have recently shown that high IGF-1 is correlated with increased risk of cancer, and men with low IGF-1 because of GHR deficiency are fully protected against cancer (Guevara-Aguirre et al., 2011).”

“All these outcomes are accompanied by increases in median and maximum lifespan (Bartke et al., 2007; Gross and Dreyfuss, 1984; Kritchevsky, 2002; Weindruch and Sohal, 1997; Weindruch et al., 1986).”

“We measured fasting serum IGF-1 and IGFBP-1 in mice (Table S4) and found a reduction in IGF-1 levels with increasing level of CR regardless of the mouse sex or strain, which is in agreement with previous reports (Anson et al., 2003; Breese et al., 1991; Mitchell et al., 2015b).”

“, 2013), enhanced mitochondrial function (López-Lluch et al., 2006; Nisoli et al., 2005), improved proteostasis and autophagy (Cuervo, 2008; Hansen et al.”

“Not surprisingly, most of these interventions have strain-, diet-, and sex-specific effects on health and survival in mice (Anisimov et al., 2008; Harrison et al., 2009, 2014; Martin-Montalvo et al., 2013; Miller et al., 2011, 2014; Pearson et al., 2008a; Strong et al., 2008, 2013).”

“HumanCR studies have shown clear effects onmetabolic and molecular health in humans, including reduced multiple risk factors implicated in the pathogenesis of type 2 diabetes, obesity, cardiovascular disease, cancer, stroke, vascular dementia, and liver and kidney diseases (Fontana et al., 2014; Longo et al., 2015).”

“The beneficial effects of H2S on cellular aging may be mediated through the transcription factor Nrf2 and Cyp2a5 detoxification (Abu-Bakar et al., 2007; Chattopadhyay et al., 2012; Yang et al., 2013).”

“Hydrogen Sulfide and the Control of Protein Handling Systems Hydrogen sulfide (H2S) production via the transsulfuration pathway is an evolutionarily conserved response to CR that may mediate multiple CR-like benefits, including stress resistance and longevity (Hine et al., 2015).”

“Optimal activity of complex I requires the interaction with prohibitin (Phb), a major mitochondrial inner membrane protein implicated in cristae morphogenesis and functional integrity of mitochondria (Artal-Sanz and Tavernarakis, 2009; Merkwirth et al., 2008).”

“…of the autophagic system, we also analyzed the effect of CR on hepatic chaperone-mediated autophagy, which has been shown to contribute to metabolic regulation in vivo (Schneider et al., 2014) and restore chaperone-mediated autophagy reverse aging phenotypes in liver (Zhang and Cuervo, 2008).”

“, 2014) and restore chaperone-mediated autophagy reverse aging phenotypes in liver (Zhang and Cuervo, 2008).”

“…were challenged recently in a number of studies in wild-derived mice (Harper et al., 2006), inbred strains (Fernandes et al., 1976; Forster et al., 2003; Goodrick et al., 1990; Harrison and Archer, 1987; Turturro et al., 2002), and recombinant inbred strains (Liao et al., 2010; Rikke et al., 2010).”

“Ubiquitinated proteins are amenable to degradation via macroautophagy, and blockage of this pathway leads to intracellular accumulation of K48- and K63-positive aggregates (Hara et al., 2006; Olzmann and Chin, 2008; Tan et al., 2008).”

“Not surprisingly, most of these interventions have strain-, diet-, and sex-specific effects on health and survival in mice (Anisimov et al., 2008; Harrison et al., 2009, 2014; Martin-Montalvo et al., 2013; Miller et al., 2011, 2014; Pearson et al., 2008a; Strong et al., 2008, 2013).”

“Indeed, low levels of IGF-1 andmusclemass have been shown to be negatively correlated with decreases in median and maximal lifespan in mice (Sharples et al., 2015; Yuan et al., 2009).”

“Although PARP-1 activity was not measured, it is possible that CRinduced changes in PARP-1 expression will impact on the NAD levels, thereby affecting protein deacetylation performed by sirtuins, and ultimately shape nuclear-mitochondrial communication and the control of energy homeostasis (Cantó et al., 2015; Gomes e…”

“Not surprisingly, most of these interventions have strain-, diet-, and sex-specific effects on health and survival in mice (Anisimov et al., 2008; Harrison et al., 2009, 2014; Martin-Montalvo et al., 2013; Miller et al., 2011, 2014; Pearson et al., 2008a; Strong et al., 2008, 2013).”

“Not surprisingly, most of these interventions have strain-, diet-, and sex-specific effects on health and survival in mice (Anisimov et al., 2008; Harrison et al., 2009, 2014; Martin-Montalvo et al., 2013; Miller et al., 2011, 2014; Pearson et al., 2008a; Strong et al., 2008, 2013).”

“…improvements in hepatic carbohydrate and lipid metabolism directly through lipophagy (Singh et al., 2009) or through selective degradation of metabolic enzymes via chaperone-mediated autophagy (Schneider et al., 2014), which have a positive impact on the overall energetic balance of the organism.”

“…profiles showed an enhancement in mitochondrial respiration driven by fatty acid degradation, which was accompanied by increased expression of respiratory complexes and which is expected from greater use of fat as fuel source (Bruss et al., 2010) and concomitant alteration in body fat composition.”

“Both macroautophagy and chaperone-mediated autophagy contribute to regulate hepatic metabolism but through different mechanisms (Madrigal-Matute and Cuervo, 2016).”

“, 2004), decrease in different pathologies (Berg and Simms, 1960; Cheney et al., 1980; Ikeno et al., 2013), enhanced mitochondrial function (López-Lluch et al.”

“(I and J) Immunoblot for the indicated proteins in homogenate (HOM), CMA+ and females.”

“, 2006), inbred strains (Fernandes et al., 1976; Forster et al., 2003; Goodrick et al., 1990; Harrison and Archer, 1987; Turturro et al., 2002), and recombinant inbred strains (Liao et al.”

“CR Attenuates Age-Associated Impairments in Glucose Homeostasis and Maintains Pancreatic Islet Morphology One of the proposed mechanisms of CR-mediated lifespan extension is the maintenance of insulin sensitivity (Barzilai et al., 1998).”

“HumanCR studies have shown clear effects onmetabolic and molecular health in humans, including reduced multiple risk factors implicated in the pathogenesis of type 2 diabetes, obesity, cardiovascular disease, cancer, stroke, vascular dementia, and liver and kidney diseases (Fontana et al., 2014; Longo et al., 2015).”

“, 2006), inbred strains (Fernandes et al., 1976; Forster et al., 2003; Goodrick et al., 1990; Harrison and Archer, 1987; Turturro et al., 2002), and recombinant inbred strains (Liao et al.”

“Indeed, recent work has shown in humans that slight overweight is associated with the lowest level of all-cause mortality (Flegal et al., 2013; Grabowski and Ellis, 2001).”

“More than a century ago, Moreschi (Moreschi, 1909) and Rous (Rous, 1914) observed the beneficial impact of a reduction of caloric intake on transplanted and induced tumors.”

“(Schwer et al., 2009), the global pattern of lysine acetylation and deacetylation in liver lysates was refractory to CR-mediated protein deacetylation (Figure 4D, Figure S3B), as evidenced by the fact that the ratio of acetylated to total p53 was significantly increased with CR (Figure 4D, Figure S3B).”

“, 1979) in rodents (Duffy et al., 1989; Mitchell et al., 2015a; Speakman andMitchell, 2011), non-human primates (Lane et al.”

“Moreover, we have shown that Nrf2 is essential for the anti-carcinogenic effects of CR in mice (Pearson et al., 2008b).”

“, 2016), but a significant decrease in rodents when compared to AL-fed controls (Breese et al., 1991).”

“D2 mice exhibited 37.3% and 29.8% increases in maximum lifespan in both sexes maintained on 40% CR, respectively (Table S1), even though D2 males on 40% CR had an 18.8% reduction in first quarter (Q1) lifespan, an observation consistent with an earlier report (Forster et al., 2003).”

“…content and/or restriction of specific components of the diet such as methionine or protein, different feeding regiments, and periodic periods of fasting (Fontana and Partridge, 2015; Hine et al., 2015; Masoro, 1990; Miller and Roth, 2007; MurtaghMark et al., 1995; Solon-Biet et al., 2015).”

“Improvement in insulin sensitivity and signaling via the insulin/ IGF-1 signaling pathway is a proposed mechanism through which CR retards aging and promotes lifespan (Milman et al., 2014).”

“It was years later when Osborne (Osborne et al., 1917) and then McCay and colleagues reported the lifespan effects of calorie-restricted diet in rats (McCay et al.”

“Genetic mutations in growth hormone, IGF-1 receptor, or downstream effectors of insulin/IGF-1 signaling (i.e., PI-3 kinase and FOXO) have been associated with increased lifespan (Flurkey et al., 2001, 2002; Hsieh et al., 2002; Shimokawa et al., 2015; Sun et al., 2013).”

“The universal effects of CR on lifespan were challenged recently in a number of studies in wild-derived mice (Harper et al., 2006), inbred strains (Fernandes et al.”

“Not surprisingly, most of these interventions have strain-, diet-, and sex-specific effects on health and survival in mice (Anisimov et al., 2008; Harrison et al., 2009, 2014; Martin-Montalvo et al., 2013; Miller et al., 2011, 2014; Pearson et al., 2008a; Strong et al., 2008, 2013).”

“…content and/or restriction of specific components of the diet such as methionine or protein, different feeding regiments, and periodic periods of fasting (Fontana and Partridge, 2015; Hine et al., 2015; Masoro, 1990; Miller and Roth, 2007; MurtaghMark et al., 1995; Solon-Biet et al., 2015).”

“Indeed, low levels of IGF-1 andmusclemass have been shown to be negatively correlated with decreases in median and maximal lifespan in mice (Sharples et al., 2015; Yuan et al., 2009).”

“Interestingly, TXNIP has also a role in stress-induced macroautophagy (Qiao et al., 2015), one of the hallmarks of aging.”

“Optimal activity of complex I requires the interaction with prohibitin (Phb), a major mitochondrial inner membrane protein implicated in cristae morphogenesis and functional integrity of mitochondria (Artal-Sanz and Tavernarakis, 2009; Merkwirth et al., 2008).”

“Ubiquitinated proteins are amenable to degradation via macroautophagy, and blockage of this pathway leads to intracellular accumulation of K48- and K63-positive aggregates (Hara et al., 2006; Olzmann and Chin, 2008; Tan et al., 2008).”

“The long-term effects of CR on circulating IGFBP-1 levels are paradoxical, with increased concentrations in humans (Fontana et al., 2016), but a significant decrease in rodents when compared to AL-fed controls (Breese et al., 1991).”

“, 2003) and to upregulate autophagy-related genes and proteins in human skeletal muscle (Yang et al., 2016).”

“…page) 1094 Cell Metabolism 23, 1093–1112, June 14, 2016 age-associated diseases, attenuation of functional decline, and tumorigenesis across a variety of species and diet formulations (Gross and Dreyfuss, 1984; Kritchevsky, 2002; Tannenbaum, 1940; Weindruch and Sohal, 1997; Weindruch et al., 1986).”

“…reduced insulin/insulin-like growth factor 1 (IGF-1) signaling, reduction in oxidative stress and/or increase in antioxidant mechanisms (De Cabo et al., 2004), decrease in different pathologies (Berg and Simms, 1960; Cheney et al., 1980; Ikeno et al., 2013), enhanced mitochondrial…”

“, PI-3 kinase and FOXO) have been associated with increased lifespan (Flurkey et al., 2001, 2002; Hsieh et al., 2002; Shimokawa et al., 2015; Sun et al., 2013).”

“CR in rodents is also known to reduce fertility, perhaps as an evolutionary response to hunger, enabling the shutdown of reproduction and the investment of all energy into repair mechanisms and survival (Kirkwood, 2002; Kirkwood and Shanley, 2005).”

“Since the increase of nuclear size is a well-established marker of aging in liver (Gregg et al., 2012), we also measured this parameter to assess if the observed structural changes in mitochondria were related to the improvement of liver preservation.”

“The reduction in body temperature (Tb) has been postulated to play a role in the CR-promoting effects on lifespan in rodents and non-human primates (Duffy et al., 1989; Lane et al., 1995; Mitchell et al., 2015a; Speakman andMitchell, 2011; Turturro andHart, 1991).”

“CR has been shown to prevent the age-dependent decline inmacroautophagy activity in rodent liver (Bergamini et al., 2003) and to upregulate autophagy-related genes and proteins in human skeletal muscle (Yang et al.”

“, 2006), inbred strains (Fernandes et al., 1976; Forster et al., 2003; Goodrick et al., 1990; Harrison and Archer, 1987; Turturro et al., 2002), and recombinant inbred strains (Liao et al.”

“NQO1 also functions as a gatekeeper protein capable of competing with the 20S proteasome for interaction with different proteins (Moscovitz et al., 2015).”

“It is possible that the preservation of fat mass in CR mice between 12 and 24 months of age plays a protective role in survival and lifespan extension, in agreement with previous findings (Liao et al., 2011).”

“…2013), enhanced mitochondrial function (López-Lluch et al., 2006; Nisoli et al., 2005), improved proteostasis and autophagy (Cuervo, 2008; Hansen et al., 2008), a reduction in body temperature (Rikke et al., 2003), and reduction in reproductive investment (Mitchell et al., 2015b), among others.”

“One of the primary physiological responses to CR is the reduction in body temperature (Weindruch et al., 1979) in rodents (Duffy et al., 1989; Mitchell et al., 2015a; Speakman andMitchell, 2011), non-human primates (Lane et al., 1995), and humans (Soare et al., 2011).”

“, PI-3 kinase and FOXO) have been associated with increased lifespan (Flurkey et al., 2001, 2002; Hsieh et al., 2002; Shimokawa et al., 2015; Sun et al., 2013).”

“The decrease in MUPs observed herein with CR is consistent with recently published data in B6 males (Mitchell et al., 2015b).”

“, 2006), inbred strains (Fernandes et al., 1976; Forster et al., 2003; Goodrick et al., 1990; Harrison and Archer, 1987; Turturro et al., 2002), and recombinant inbred strains (Liao et al.”

“Binding of NQO1 to the key metabolic regulator, PGC-1a, leads to its stabilization and protection against proteasomal degradation (Adamovich et al., 2013).”

“The reduction in age-associated pancreatic islet hypertrophy combined with enhanced insulin sensitivity and lower insulin levels is likely to represent an important—although almost certainly not exclusive—mechanism by which CR increases longevity (Masternak et al., 2009).”

“To further test this possibility, we analyzed the autophagic compartments in transmission electron microscopy (TEM) images of (D) Total liver lysates were immunoblotted with the indicated primary antibodies SIRT1, PARP-1, cellular acetylated proteins, total p53, and ratio of acetylated/to (E and F) NAD determination i…”

“, PI-3 kinase and FOXO) have been associated with increased lifespan (Flurkey et al., 2001, 2002; Hsieh et al., 2002; Shimokawa et al., 2015; Sun et al., 2013).”

“, 2004), decrease in different pathologies (Berg and Simms, 1960; Cheney et al., 1980; Ikeno et al., 2013), enhanced mitochondrial function (López-Lluch et al.”

“…were challenged recently in a number of studies in wild-derived mice (Harper et al., 2006), inbred strains (Fernandes et al., 1976; Forster et al., 2003; Goodrick et al., 1990; Harrison and Archer, 1987; Turturro et al., 2002), and recombinant inbred strains (Liao et al., 2010; Rikke et al., 2010).”

“These results are consistent with CR-mediated inhibition of SIRT3 and are at odds with previous reports (Qiu et al., 2010; Tauriainen et al., 2011).”

“Serum leptin was lower but adiponectin was higher in CR-fed mice compared to their AL-fed controls, which can be attributed to the reduction in body weight and potential reprogramming of hepatic fat metabolism in response to CR (Kuhla et al., 2014).”

“All these outcomes are accompanied by increases in median and maximum lifespan (Bartke et al., 2007; Gross and Dreyfuss, 1984; Kritchevsky, 2002; Weindruch and Sohal, 1997; Weindruch et al., 1986).”

“…content and/or restriction of specific components of the diet such as methionine or protein, different feeding regiments, and periodic periods of fasting (Fontana and Partridge, 2015; Hine et al., 2015; Masoro, 1990; Miller and Roth, 2007; MurtaghMark et al., 1995; Solon-Biet et al., 2015).”

“, 2005) and whole-body energy metabolism (DeBalsi et al., 2014).”

“The decrease in MUPs observed herein with CR is consistent with recently published data in B6 males (Mitchell et al., 2015b).”

“All these outcomes are accompanied by increases in median and maximum lifespan (Bartke et al., 2007; Gross and Dreyfuss, 1984; Kritchevsky, 2002; Weindruch and Sohal, 1997; Weindruch et al., 1986).”

“In mouse hepatocytes we have shown that aging induces a decrease in mitochondrial mass together with reduction in individual mitochondria size and changes in the expression pattern of proteins related to fusion/fission dynamics (Khraiwesh et al., 2013, 2014; López-Lluch et al., 2006).”

“The beneficial effects of H2S on cellular aging may be mediated through the transcription factor Nrf2 and Cyp2a5 detoxification (Abu-Bakar et al., 2007; Chattopadhyay et al., 2012; Yang et al., 2013).”

“All these outcomes are accompanied by increases in median and maximum lifespan (Bartke et al., 2007; Gross and Dreyfuss, 1984; Kritchevsky, 2002; Weindruch and Sohal, 1997; Weindruch et al., 1986).”

“Litters were maintained with approximately eight pups to minimize the potential confounding effect of a crowded litter (Sadagurski et al., 2014).”

“NAD(P)H:quinone oxidoreductase 1 (NQO1), a key player in the antioxidant and detoxification pathways, is a Nrf2 target protein inducible by H2S (Chattopadhyay et al., 2012).”

“Like CR, H2S increases lifespan in C. elegans (Miller and Roth, 2007) and promotes redox regulation of SIRT1 (Zee et al., 2010) to help maintain the circadian rhythm of mouse hepatocytes (Shang et al., 2012).”

“The preservation of fat mass means more insulation and lower rate of health decline as these mice age, which may have contributed in having opposing effects on the classical Tb response to CR (Rikke and Johnson, 2007).”

“In C. elegans, it has been clearly demonstrated that aging induces a decline in mitochondrial function that leads to the activation of specific pathways (Chang et al., 2015) and an increase in reactive oxygen species (ROS).”

“The ratio in the number of a/b cells was significantly lower with CR, indicating that the process of islet hypertrophy that occurs as a consequence of aging (Figueroa and Taberner, 1994) appears to be ameliorated with CR independent of mouse strain and sex.”

“Multiple studies have demonstrated that caloric restriction can retard the aging decline (Mitchell et al., 2016).”