Skip to main content
PMC home page
eLife logoLink to eLife
. 2020 Jul 28;9:e56177. doi: 10.7554/eLife.56177

Ovariectomy uncouples lifespan from metabolic health and reveals a sex-hormone-dependent role of hepatic mTORC2 in aging

Sebastian I Arriola Apelo 1,2,3, Amy Lin 1,2,3, Jacqueline A Brinkman 2,3, Emma Meyer 1,2,3, Mark Morrison 2,3, Jay L Tomasiewicz 2, Cassidy P Pumper 2,3, Emma L Baar 2,3, Nicole E Richardson 2,3,4, Mohammed Alotaibi 2,3,4, Dudley W Lamming 2,3,4,5,✉
Editors: Veronica Galvan6, Jessica K Tyler7
PMCID: PMC7386906  PMID: 32720643

Abstract

Inhibition of mTOR (mechanistic Target Of Rapamycin) signaling by rapamycin promotes healthspan and longevity more strongly in females than males, perhaps because inhibition of hepatic mTORC2 (mTOR Complex 2) specifically reduces the lifespan of males. Here, we demonstrate using gonadectomy that the sex-specific impact of reduced hepatic mTORC2 is not reversed by depletion of sex hormones. Intriguingly, we find that ovariectomy uncouples lifespan from metabolic health, with ovariectomized females having improved survival despite paradoxically having increased adiposity and decreased control of blood glucose levels. Further, ovariectomy unexpectedly promotes midlife survival of female mice lacking hepatic mTORC2, significantly increasing the survival of those mice that do not develop cancer. In addition to identifying a sex hormone-dependent role for hepatic mTORC2 in female longevity, our results demonstrate that metabolic health is not inextricably linked to lifespan in mammals, and highlight the importance of evaluating healthspan in mammalian longevity studies.

Research organism: Mouse

Introduction

The high frequency and co-morbidity of age-related disease in the aged limits the benefits that can be achieved by targeting them individually. An alternative approach, which could simultaneously treat or prevent many age-related diseases, is to target the aging process itself (Kennedy et al., 2014). However, extending lifespan without also extending healthspan is generally considered undesirable (Kaeberlein, 2018), and not all geroprotective interventions may necessarily improve healthspan. Indeed, a recent study in C. elegans found that lifespan and healthspan can be uncoupled in this organism (Bansal et al., 2015). Thus, there is a clear need to assess healthspan in mammalian longevity studies.

The mechanistic Target Of Rapamycin (mTOR) is a serine/threonine protein kinase that serves as a central regulator of metabolism and aging and is found in two distinct protein complexes, mTOR Complex 1 (mTORC1) and mTORC2 (Kennedy and Lamming, 2016). mTORC1 is acutely sensitive to the action of the FDA-approved pharmaceutical rapamycin, and inhibition of mTORC1, either genetically or by rapamycin, extends lifespan and healthspan in model organisms ranging from yeast to mice (Arriola Apelo et al., 2016b; Bitto et al., 2016; Harrison et al., 2009; Kaeberlein et al., 2005; Kapahi et al., 2004; Medvedik et al., 2007; Powers et al., 2006; Vellai et al., 2003). Pharmaceutical or genetic inhibition of mTOR signaling tends to extend female lifespan to a greater extent than that of males (Lamming, 2014; Miller et al., 2014).

However, chronic treatment of mice with rapamycin induces a variety of metabolic side effects. We and others have shown that many of these effects are mediated by ‘off-target’ inhibition of mTORC2 (Arriola Apelo et al., 2016a; Dumas and Lamming, 2020; Kleinert et al., 2017; Kleinert et al., 2014; Lamming et al., 2012; Sarbassov et al., 2006; Schreiber et al., 2019; Schreiber et al., 2015). These studies highlighted an important gap in our knowledge – namely, the contribution of decreased mTORC2 signaling to the effect of rapamycin on lifespan. Studies in C. elegans and D. melanogaster have not convincingly addressed this question, as reduced mTORC2 signaling can be beneficial or detrimental in C. elegans depending on the tissue and diet, while overexpression of Rictor, which encodes an essential protein component of mTORC2, extends the lifespan of flies (Chang et al., 2019; Mizunuma et al., 2014; Robida-Stubbs et al., 2012; Soukas et al., 2009).

Deletion of Rictor in several distinct tissues or inducibly in the whole body of adult mice negatively impacts survival (Chellappa et al., 2019; Lamming et al., 2014b; Yu et al., 2019). Intriguingly, loss of hepatic mTORC2 specifically reduces male lifespan, with no effect of liver-specific deletion of Rictor on the lifespan of female mice (Lamming et al., 2014b). This difference between males and females was not due to sex-based differences in the efficiency of Rictor deletion (Lamming et al., 2014b). This sex-specific effect of hepatic mTORC2 inhibition may help to explain why rapamycin has a stronger effect on female lifespan (Lamming, 2014; Miller et al., 2014). Many sexually dimorphic responses, including to geroprotective interventions, are mediated in part by gonadal sex hormones (Garratt et al., 2017; Garratt et al., 2018). We hypothesized that gonadectomy would either protect male mice from the loss of hepatic Rictor, or sensitize female mice to the loss of hepatic Rictor.

We therefore examined how mice lacking hepatic Rictor responded to gonadectomy. We find that the male-specific negative impact of Rictor loss on overall survival persists following gonadectomy. While pre-pubertal gonadectomy negatively impacted the metabolic health of both liver Rictor knockout (L-RKO) mice and their wild-type littermates, it did not negatively impact survival, with ovariectomized mice in particular surviving longer than sham-surgery controls. Intriguingly, while as we expected based on our prior results, the overall survival of sham-surgery L-RKO female mice was not statistically different from that of female wild type controls, there was an apparent reduction in the survival of L-RKO Sham females between 400–800 days of age that was not observed in ovariectomized L-RKO mice. An in-depth analysis suggests that pre-pubertal ovariectomy promotes the midlife survival of female mice lacking hepatic Rictor, through protection from a non-cancer related cause of death. We conclude that there is a sex-specific role for hepatic mTORC2 in survival that is dependent upon sex hormones, and that ovariectomy uncouples metabolic health and longevity.

Results

Depletion of hepatic Rictor reduces the overall lifespan of male mice independently of sex hormones

We castrated male L-RKO mice and their wild-type littermates, and ovariectomized female L-RKO mice and their wild-type littermates, between 3 and 4 weeks of age; sham surgeries were performed on mice of both sexes and genotypes to control for any effects of the surgical procedures. We examined the longevity of all 8 groups of animals, separately analyzing females and males due to the expected sexually dimorphic effects. While cause of death analysis is difficult in mice, we performed gross necropsy on as many animals as possible, noting the presence or absence of observable cancers. As we expected for C57BL/6J mice (Brayton et al., 2012), we observed cancer in approximately half of the necropsied animals. There was no statistically significant difference in the frequency of observed cancer at necropsy between groups of male mice (chi-square test, p=0.06), although cancer was identified much more frequently in L-RKO CAST mice than in any other group of male mice (Figure 1—figure supplement 1).

We initially stratified our analysis of mice by the presence or absence of observable cancers at necropsy. We performed a Cox regression analysis to identify the overall effects of genotype and surgery, and then calculated the corrected two-sided log-rank sum p-value comparing individual curves for those effects identified as significant in the regression analysis. Intriguingly, there was no effect of hepatic Rictor deletion on the overall longevity of male mice that died with observed cancer (Figure 1A). In contrast, there was a dramatic effect of Rictor loss on the survival of male mice (HR = 4.84, p=0.0014) in which cancer was not observed during our gross necropsy (Figure 1B). We observed no overall effect of gonadectomy in either group of mice.

Figure 1. Deletion of hepatic Rictor impairs male survival independently of sex hormones.

(A) Kaplan-Meier plot of the survival of male mice in which cancer was observed during gross necropsy (N = 38 male mice; WT Sham 5, L-RKO Sham 9, WT CAST 10, L-RKO CAST 14; Supplementary file 1). (B) Kaplan-Meier plot of the survival of male mice in which cancer was not observed during gross necropsy (N = 32 male mice; WT Sham 7, L-RKO Sham 11, WT CAST 11, L-RKO CAST 3; Supplementary file 1). (C) Kaplan-Meier plot of the survival of male mice lacking hepatic Rictor (L-RKO) and their wild-type (WT) littermates. All mice were subjected to gonadectomy (CAST) or Sham surgery at 3 weeks of age (N = 105 male mice; WT Sham 23, L-RKO Sham 27, WT CAST 29, L-RKO CAST 26; Supplementary file 1). (A–C) The overall effect of genotype (RKO), gonadectomy (CAST), and the interaction was determined using a Cox-proportional hazards test (HR, hazard ratio). The two-sided log-rank sum p-value was then calculated comparing individual curves for effects identified as significant in the regression analysis, and corrected for multiple comparisons (Holm-Sidak).

Figure 1.

Figure 1—figure supplement 1. Frequency of cancer observed at necropsy in male mice.

Figure 1—figure supplement 1.

Frequency at which cancer was observed (bottom) or not observed (top) in male mice. N = 70 male mice; p-value was determined by chi-square analysis.
Open in a new tab

Looking at the survival of all of the male mice (Figure 1C), in agreement with our previous study (Lamming et al., 2014b) we observed an overall negative effect of hepatic Rictor loss on the survival of male mice, with deletion of Rictor leading to an increased hazard ratio (HR (RKO) = 1.75, p=0.01), and no overall effect of castration. This effect was driven in part by a statistically significant decrease (log-rank p=0.028) in the lifespan of intact male L-RKO mice relative to intact wild-type mice, with a 13.7% decrease in median lifespan. We also observed a 10% decrease in median lifespan in castrated male L-RKO relative to castrated male wild-type mice; these lifespan curves were not distinguishable (log-rank p=0.337). The almost complete overlap of the L-RKO Sham and L-RKO CAST survival curves demonstrate that castration does not rescue the lifespan defect of male mice lacking hepatic Rictor.

Depletion of hepatic Rictor impairs midlife survival of female mice, a defect rescued by ovariectomy

There was no statistically significant difference in the frequency of observed cancer at necropsy between groups of female mice (chi-square test, p=0.78) (Figure 2—figure supplement 1), and we observed no effect of hepatic Rictor deletion on the overall longevity of female mice that died with observed cancer (Figure 2A). However, to our great surprise, there was a dramatic effect of Rictor loss on the survival of female mice (HR = 6.67, p=0.00084) in which cancer was not observed during our gross necropsy (Figure 2B). Although there was no overall effect of gonadectomy, we observed a strong interaction between the effect of Rictor loss and ovariectomy (p=0.014), and the survival curves of L-RKO OVX mice overlap the survival curves of WT Sham and WT OVX, leading us to interpret this interaction as demonstrating a strong protective effect of ovariectomy against a non-cancer related cause of death in mice lacking hepatic Rictor.

Figure 2. Ovariectomy protects female mice lacking hepatic Rictor during midlife.

(A) Kaplan-Meier plot of the survival of female mice in which cancer was observed during gross necropsy (N = 37 female mice; WT Sham 11, L-RKO Sham 9, WT OVX 10, L-RKO OVX 7; Supplementary file 1). (B) Kaplan-Meier plot of the survival of female mice in which cancer was not observed during gross necropsy (N = 36 female mice; WT Sham 11, L-RKO Sham 8, WT OVX 7, L-RKO OVX 10; Supplementary file 1). (C) Kaplan-Meier plot of the survival of female mice lacking hepatic Rictor (L-RKO) and their wild-type (WT) littermates. All mice were subjected to gonadectomy (OVX) or Sham surgery at 3 weeks of age (N = 115 female mice; WT Sham 31, L-RKO Sham 29, WT OVX 27, L-RKO OVX 28; Supplementary file 1). (A–C) The overall effect of genotype (RKO), gonadectomy (OVX), and the interaction was determined using a Cox-proportional hazards test (HR, hazard ratio). The two-sided log-rank sum p-value was then calculated comparing individual curves for effects identified as significant in the regression analysis, and corrected for multiple comparisons (Holm-Sidak).

Figure 2.

Figure 2—figure supplement 1. Frequency of cancer observed at necropsy in female mice.

Figure 2—figure supplement 1.

Frequency at which cancer was observed (bottom) or not observed (top) in female mice. N = 73 female mice; p-value was determined by chi-square analysis.
Open in a new tab

Despite this strong effect, when we looked at the total population of female mice in our study, the negative effect of Rictor loss on Sham-treated female mice was masked, and in agreement with our previous study (Lamming et al., 2014b) there was no overall negative effect of Rictor loss on survival (Figure 2C). There was also no difference between thesurvival of WT Sham and WT L-RKO female mice (p=0.153, Wilcoxon rank sum). We did however find an overall protective effect of pre-pubertal ovariectomy (HR (OVX) = 0.66, p=0.05). Inspection of the Kaplan-Meir plots and our results in Figure 2B lead us to conclude that this effect is largely driven by the protective effect of pre-pubertal ovariectomy on the midlife survival of female L-RKO mice. Finally, ovariectomized L-RKO female mice had a 17% increase in median lifespan relative to sham L-RKO females, due to a divergence of these lifespan curves between 400 and 800 days of age. This difference was statistically significant (p=0.025) by the Wilcoxon rank-sum test, which does not assume proportional hazards.

Ovariectomy impairs the metabolic health of mice

During the course of our longevity study, we tracked weight and body composition (Figure 3A–F). Gonadectomy had a profound effect on the weight and body composition of wild-type mice, with castration reducing the weight of wild-type males due to a decrease in lean mass (Figure 3A and C), and with ovariectomy increasing the weight of wild-type females due an increase in fat mass (Figure 3B and F). Intriguingly, male L-RKO Sham mice weighed substantially less than their wild-type Sham littermates due to an overall decrease in fat mass (Figure 3A and E); however, no differences in fat mass due to genotype were observed in females (Figure 3F). Castration rescued the decreased adipose mass of L-RKO male mice, while not affecting fat mass in wild-type mice (Figure 3E).

Figure 3. Gonadectomy affects weight and body composition, and rescues a male-specific effect of hepatic Rictor deletion on fat mass.

Figure 3.

Open in a new tab

(A, B) The weight of A) male and (B) female mice lacking hepatic Rictor (L-RKO) and their wild-type (WT) littermates was tracked starting at 3 months of age (n varies by month; maximum female N = 22–26 mice/group, maximum male N = 20–23 mice/group). (C–F) The lean mass (C, D) and fat mass (E, F) of mice was determined every 4 months starting at 8 months of age (n varies by month; max female N = 9–17/group; max male N = 12–17/group). p-values for the overall effect of genotype (GT) or surgical treatment (Surgery) represent the p-value from pairwise two-way ANOVA testing. Error bars represent SEM.

In order to gain insight into the alterations in body weight, we used metabolic chambers to examine energy balance and fuel source utilization. We observed that ovariectomy, but not castration, had profound effects on multiple components of energy balance and altered fuel source utilization (Figure 4A–D). In particular, ovariectomy suppressed food intake, activity, and energy expenditure in both WT and L-RKO mice (Figure 4A,C,D). Ovariectomy also altered fuel source utilization in WT females, with WT OVX mice having a lower RER indicating reduced oxidation of carbohydrates and increased utilization of lipids (Figure 4B). Interesting, L-RKO female mice had a lower RER during the light cycle that was not further suppressed by ovariectomy (Figure 4B).

Figure 4. Gonadectomy alters energy balance and fuel source utilization.

Figure 4.

Open in a new tab

(A–D) Metabolic chambers were utilized to determine (A) food consumption, (B) Respiratory exchange ratio (RER), (C) Spontaneous activity, and (D) Energy expenditure in (left) female and (right) male mice lacking hepatic Rictor (L-RKO) and their wild-type (WT) littermates of the indicated surgical treatments at 12 months of age. The overall effect of genotype (RKO), gonadectomy (OVX or CAST), and the interaction represent the p-value from a two-way ANOVA conducted separately for the light and dark cycles; *=p < 0.05 from a Holm-Sidak post-test examining the effect of parameters identified as significant in the two-way ANOVA (n = Females, 10 mice/group; Males, WT Sham = 11 mice; WT CAST = 10 mice, L-RKO Sham = 9 mice, L-RKO CAST = 8 mice). Error bars represent SEM.

We next examined how hepatic Rictor loss alters glucose tolerance and insulin sensitivity as the mice age in the presence and absence of gonadal hormones. As we expected based on our previous findings (Lamming et al., 2014a; Lamming et al., 2014b; Lamming et al., 2012), loss of hepatic Rictor had a profound negative effect on glucose tolerance in male mice (Figure 5A and B). Castration did not affect glucose tolerance in either WT or L-RKO mice. We observed no effect of hepatic Rictor deletion on the glucose tolerance of 4 month old female mice (Figure 5C and D), but 11 month old female L-RKO mice had impaired glucose tolerance relative to littermate controls. There was a clear overall negative effect of ovariectomy on glucose tolerance at both ages (Figure 5C and D).

Figure 5. Independent effects of hepatic Rictor deletion and gonadectomy on glucose tolerance and insulin resistance.

(A–D) The glucose tolerance of (A, B) male and (C, D) female mice lacking hepatic Rictor (L-RKO) and their wild-type (WT) littermates, of the indicated ages and surgical treatments, was determined following an overnight fast. Area under the curve: the overall effect of genotype (RKO), gonadectomy (OVX or CAST), and the interaction represent the p-value from a two-way ANOVA; *=p < 0.05 from a Holm-Sidak post-test examining the effect of parameters identified as significant in the two-way ANOVA (n = Males: A) 9–12 mice/group, 3–5 months of age; B) 7–12 mice/group, 10–12 months of age. Females: C) 7–11 mice/group, 3–5 months of age; D) 7–11 mice/group, 10–12 months of age). (E–F) fasting blood glucose and insulin values were used to calculate HOMA2-IR in E) male and F) female 14 month old mice (n = 4–5 mice/group; the overall effect of genotype (RKO), gonadectomy (OVX or CAST), and the interaction represent the p-value from a two-way ANOVA; *=p < 0.05 from a Holm-Sidak post-test examining the effect of parameters identified as significant in the two-way ANOVA). Error bars represent SEM.

Figure 5.

Figure 5—figure supplement 1. Effect of hepatic Rictor loss and gonadectomy on insulin tolerance.

Figure 5—figure supplement 1.

(A–D) The insulin tolerance of (A, B) male and (C, D) female mice lacking hepatic Rictor (L-RKO) and their wild-type (WT) littermates, of the indicated ages and surgical treatments was determined following an overnight fast. Area under the curve: the overall effect of genotype (RKO), gonadectomy (OVX or CAST), and the interaction represent the p-value from a two-way ANOVA; *=p < 0.05 from a Holm-Sidak post-test examining the effect of parameters identified as significant in the two-way ANOVA. (n = Male: A) 9–12 mice/group, 4–5 months of age; (B) 8–9 mice/group, 12–14 months of age. Females: (C) 8–11 mice/group, 3.5–6 months of age; (D) 7–10 mice/group, 12–14 months of age). Error bars represent SEM.
Figure 5—figure supplement 2. Effect of hepatic Rictor loss and gonadectomy on glucose stimulated insulin secretion.

Figure 5—figure supplement 2.

Glucose stimulated insulin levels were measured 15 min after I.P. administration of 1 g/kg glucose (n = 4–5 mice/group; the overall effect of genotype (RKO), gonadectomy (OVX or CAST), and the interaction represent the p-value from a two-way ANOVA). Error bars represent SEM.
Open in a new tab

We assessed insulin sensitivity by conducting insulin tolerance tests (ITTs, Figure 5—figure supplement 1) and by determining insulin resistance using homeostasis model assessment (Levy et al., 1998; Figure 5E and F). In males, consistent with impaired hepatic insulin sensitivity, deletion of hepatic Rictor resulted in fasting and glucose-stimulated hyperinsulinemia, and increased HOMA2-IR, despite negligible effects on the response to an ITT (Figure 5E, Figure 5—figure supplements 1–2); castration did not have a significant effect on any of these parameters. In contrast, in female mice genotype did not significantly affect fasting blood glucose or insulin, but ovariectomy did, promoting hyperinsulinemia and increasing HOMA2-IR (Figure 5F). We observed an effect of genotype on female ITT, with young L-RKO mice having a slight impairment of insulin sensitivity, and older ovariectomized L-RKO mice have a statistically significant impairment (Figure 5—figure supplement 1).

Discussion

Here, we tested the role of sex hormones in the sexually dimorphic response to deletion of hepatic Rictor, which we previously observed to preferentially decrease the survival of male mice (Lamming et al., 2014b). We expected that if sex hormones were involved in this response, we would observe one of two possible outcomes: that castration would protect male mice from the loss of hepatic Rictor, or that alternatively, ovariectomy would sensitize female mice to the loss of hepatic Rictor. To our surprise, we observed neither effect; instead, castration failed to protect male L-RKO mice, while ovariectomy had a statistically significant beneficial effect on the midlife survival of female L-RKO mice, and may have had a mild positive effect on wild-type mice as well, increasing the median lifespan.

In contrast to our 2014 study, which found an effect of hepatic Rictor deletion only on male lifespan (Lamming et al., 2014b), here we also observed decreased midlife survival of female L-RKO mice. It is well appreciated that the lifespan of a mouse strain can vary substantially between laboratories (Nadon et al., 2008), and the relatively short lifespan of the wild-type control mice in our 2014 study, which was conducted in a different animal facility, may have obscured this difference. The reason for the decreased overall survival of male L-RKO mice and the decreased survival of female L-RKO mice during midlife is unknown; while our study suggests that this cause of death is not related to cancer, as we performed only a gross necropsy, we cannot at this time rule out the possibility that unobserved neoplasms contribute to these deaths. The mechanism by which ovariectomy protects L-RKO female mice during midlife is also unknown. Future carefully designed studies to assess the organismal health of L-RKO mice, with detailed pathology between 400 and 800 days of age, will be needed to fully address these issues.

The role of sex hormones in the regulation of lifespan has been an active area of investigation for many years. Several studies have shown that early life ablation of the germline extends the lifespan of C. elegans, while ovariectomy extends the lifespan of R. microptera (Arantes-Oliveira et al., 2002; Berman and Kenyon, 2006; Hatle et al., 2013; Iwasa et al., 2018). Although some older studies suggest that ovariectomy decrease the lifespan of rats; at least one of these studies suffers from the lack of a sham surgery control group (Asdell et al., 1967); a more recent study found a small positive effect of ovariectomy on rat lifespan (Iwasa et al., 2018). A recent mouse study found that post-pubertal ovariectomy may shorten mouse lifespan (Benedusi et al., 2015); in contrast, mice in our study were ovariectomized prior to puberty.

Here, we find that pre-pubertal ovariectomy protects the midlife survival of L-RKO mice. Whereas Benedusi and colleagues report a reduction in female lifespan following ovariectomy, we found no statistically significant effect of pre-pubertal ovariectomy on the overall survival of wild-type mice, and in fact observed a small numerical increase (7.4%) in median lifespan. Our findings align with the results of model organism studies which suggest that early life ablation of the female germline promotes survival, and suggest that the control of aging by the female germline is conserved from nematodes and insects to mammals. Critically, the overall positive effects of ovariectomy on longevity do not promote metabolic health, with ovariectomized mice suffering from adiposity and disrupted blood glucose homeostasis, and showing reduced spontaneous activity and energy expenditure. The disruption of metabolic health we observed is in agreement many previous studies showing that ovarian hormones play a critical role in the control of glucose homeostasis in humans and mice (Bailey and Ahmed-Sorour, 1980; Pirimoglu et al., 2011; Stubbins et al., 2012; Yuan et al., 2015).

In contrast, we observed no effect of pre-pubertal castration on mouse lifespan. Our findings differ from results previously reported for rats and humans (Drori and Folman, 1976; Min et al., 2012), but these studies may have been impacted by effects of castration on rodent aggression or confounded by the social status of the castrated humans. Conversely, hormone replacement promotes the survival of men with late onset hypogonadism (Comhaire, 2016). Our results do not support a model in which ablation of the male germline is beneficial for healthspan or longevity. In agreement with recent studies by other groups (Garratt et al., 2017; Harada et al., 2016; Inoue et al., 2010), we observed an effect of castration on weight and body composition, but did not observe an effect of castration on glucose homeostasis.

A limitation of this study is that we did not perform a comprehensive assessment of healthspan (Bellantuono et al., 2020), focusing instead on metabolic parameters. Despite this, it is clear that as in the case of C. elegans, where lifespan and healthspan can be uncoupled (Bansal et al., 2015), pre-pubertal ovariectomy uncouples metabolic health and survival. Scientists around the world are now beginning to focus on geroprotective interventions as a way to prevent or treat age-related diseases. An open question surrounding such interventions has been the effect on health, as while there is a broad consensus in favor of extending healthspan, there is less support for extending lifespan without extending healthspan (Kaeberlein, 2018). Our research here highlights the need to comprehensively assess the effects of geroprotectors on healthspan, as at least some aspects of healthspan are not inextricably coupled to longevity.

Materials and methods

Key resources table.

Reagent type
(species) or
resource		 Designation Source or
reference		 Identifiers Additional
information
Genetic reagent (Mus. musculus) C57BL/6J; RictorloxP/loxP David Sabatini Lab (Whitehead Institute for Biomedical Research) N/A
Genetic reagent (Mus. musculus) B6.Cg-Speer6-
ps1Tg(Alb-cre)21Mgn/J		 The Jackson Laboratory Stock number: 003574; RRID:IMSR_JAX:003574
Genetic reagent (Mus. musculus) C57BL/6J; Albumin-Cre; RictorloxP/loxP This paper See ‘Animal use and care’; Dudley Lamming Lab (UW-Madison)
Drug Human Insulin Eli Lilly NDC 0002-8215-17 (Humulin R U-100)
Commercial assay or kit Ultra-sensitive mouse insulin ELISA Crystal Chem Cat# 90080; RRID:AB_2783626
Software, algorithm Prism 8 GraphPad Software N/A
Software, algorithm R (v. 3.6.0) survival package (v. 2.44) Therneau, 2015 https://cran.r-project.org/web/packages/survival/index.html
Software, algorithm HOMA2 Calculator Levy et al., 1998 https://www.dtu.ox.ac.uk/homacalculator/
Other Normal Chow Purina Cat# 5001
Open in a new tab

Animal use and care

All animal procedures conducted at the William S. Middleton Memorial VA Hospital were approved by the Institutional Animal Care and Use Committee of the William S. Middleton Memorial Veterans Hospital (Assurance ID: D16-00403). Mice were multiply housed in microisolator cages and maintained under 12 hr light/dark cycles, and were fed Laboratory Rodent Diet 5001 (LabDiet). Mice hemizygous for Albumin-Cre and homozygous for a floxed allele of Rictor mice were obtained by crossing RictorL/L mice on a C57BL/6J background (Lamming et al., 2012), obtained from the Whitehead Institute for Biomedical Research (Cambridge, MA) and rederived by embryo transfer at the UW-Madison Biotechnology Center, with Albumin-Cre mice (The Jackson Laboratory, Stock 003574 Postic et al., 1999).

Body composition

Body composition was measured by magnetic resonance imaging (EchoMRI, Echo Medical Systems, Houston, USA).

Metabolic chambers

To assess food intake, RER, spontaneous activity, and energy expenditure by indirect calorimetry, we utilized an Oxymax/CLAMS metabolic chamber system following the manufacturer’s instructions (Columbus Instruments).

Glucose and insulin tolerance tests, glucose-stimulated insulin secretion, and hormone measurements

Glucose tolerance tests were performed by fasting the mice overnight for 16 hr and then administering glucose (1 g/kg) intraperitoneally (Arriola Apelo et al., 2016b). Insulin tolerance tests were performed by fasting mice overnight for 16 hr, and then injecting 0.75 U/kg human insulin (Eli Lilly) intraperitoneally. Blood glucose was measured periodically for 2 hr after administration of glucose or insulin using a Bayer Contour blood glucose meter and test strips. For glucose-stimulated insulin secretion, mice were fasted overnight for 16 hr, blood glucose levels were determined and plasma was collected immediately prior to and 15 min after administering glucose (1 g/kg) intraperitoneally. Plasma insulin was quantified according to the manufacturer’s protocol using an ultrasensitive mouse insulin ELISA kit (90080) from Crystal Chem. Fasting glucose and insulin levels were then used to calculate HOMA2-IR (Levy et al., 1998).

Surgery

Mice were gonadectomized or subject to sham surgery during the third week of age. Following anesthesia with isoflurane, mice were shaved and the skin surface was sterilized with betadine and alcohol. For castration (or sham control surgery), a 1.5 cm ventral midline incision was made ending 0.5 cm cranial to the prepuce. In castrated animals, the vas deferens, located along the abdominal fat body on each side, was retrieved using a forceps or hemostat, and the testicle exteriorized. The spermatic artery was then clamped and ligated with 5–0 absorbable suture, and the testis removed. In both castrated and sham control mice, the body wall was closed with 5–0 monofilament absorbable suture, and staples used to close the skin incision. For ovariectomy (or sham control surgery), two 0.5 cm incisions were made through the skin, one on each side (1 cm ventral) to the paralumbar fossa area with its cranial terminus 1.5 cm caudal to the 13th rib, and any fascia trimmed away. Approximately 1 cm ventral to the dorsal spinous processes of the third lumbar vertebra, and immediately caudal to the last rib, the body wall was bluntly dissected through with a mosquito hemostat. For ovariectomy, each ovary was removed as follows; pressure on the abdomen was applied, causing the ovary to be extruded through one of the incisions (if the ovary did not exteriorize, a forceps was inserted to retrieve the ovary) just caudal to the kidney on that side. The ovary was then clamped for 30 s to the level of the fallopian tube, the ovary was removed, and the uterine horn was returned to the body cavity. In both ovariectomized and sham control mice, the muscles on both sides were then sutured with 5–0 absorbable suture, and the skin incisions closed with staples. For pain management, 0.1 mg/kg buprenex was administered IP to the mouse peri-operatively, and every 8–12 hr thereafter (at time of monitoring) as needed. Mice were allowed to recover on a heated pad for up to 1 hr following surgery, and then returned to the home cage. Post-surgery, female mice were provided with moistened breeder chow, and up to 1 mL per day of subcutaneous sterile saline if dehydrated. Staples were removed once healing was complete.

Lifespan and necropsy

Following surgery, mice that successfully recovered were enrolled in the lifespan study at two months of age. A total of 115 female and 105 male mice were included in the lifespan study, with 23 female and 22 male mice removed for cross sectional analysis. Mice were co-housed (grouped by sex and surgical intervention) in specific pathogen-free housing. Mice were euthanized for humane reasons if moribund, if the mice developed other specified problems (e.g. excessive tumor burden), or upon the recommendation of the facility veterinarian. Mice found dead were noted at each daily inspection and saved in a refrigerator for gross necropsy, during which the abdominal and thoracic cavities were examined for the presence of solid tumors, metastases, splenomegaly, and infection; on the basis of this inspection the presence or absence of observable cancer was recorded.

Statistical analysis

Data are expressed as mean ± s.e.m. Statistical analysis was conducted using Prism 8 (GraphPad Software), except for survival analyses were conducted in R (version 3.5.0) using the ‘survival’ package (version 2.38) (T, 2015). Cox proportional hazards analysis was performed separately for each sex using genotype and surgical treatment as covariates. Mice which were removed from the study for cross-sectional analysis, and mice for which no record of age of death was available, were censored as of the last day for which a record was available.

Acknowledgements

We thank Dr. Abigail Radcliff for advice on animal surgery and the University of Wisconsin-Madison Biotechnology Center Transgenic Animal Facility for rederivation services. We thank A Broman of the UW-Madison Department of Biostatistics and Medical Informatics for assistance with analysis of lifespan data and R. We thank Dr. Joseph Baur and Dr. Maria Mihaylova for critical reading of the manuscript, and Dr. Dena Cohen for support and encouragement. This research was supported in part by an American Federation for Aging Research Junior Faculty Research Grant to DWL. Additional research support was provided by the National Institute of Health/National Institute on Aging (AG041765, AG050135, AG051974, AG056771, AG062328), a Glenn Foundation Award for Research in the Biological Mechanisms of Aging to DWL and funds from the University of Wisconsin-Madison School of Medicine and Public Health and Department of Medicine to DWL. SIAA was supported in part by a fellowship from the American Diabetes Association (1–16-PMF-001). NER was supported in part by a training grant from the UW Institute on Aging (NIA T32 AG000213). The project was supported by the Clinical and Translational Science Award (CTSA) program, through the NIH National Center for Advancing Translational Sciences (NCATS), grant UL1TR002373. The Lamming laboratory is supported in part by the U.S. Department of Veterans Affairs (I01-BX004031), and this work was supported using facilities and resources from the William S Middleton Memorial Veterans Hospital. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. This work does not represent the views of the Department of Veterans Affairs or the United States Government.

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Contributor Information

Dudley W Lamming, Email: dlamming@medicine.wisc.edu.

Veronica Galvan, UT Health San Antonio, United States.

Jessica K Tyler, Weill Cornell Medicine, United States.

Funding Information

This paper was supported by the following grants:

  • American Federation for Aging Research to Dudley W Lamming.

  • National Institute on Aging AG041765 to Nicole E Richardson, Dudley W Lamming.

  • American Diabetes Association 1-16-PMF-001 to Sebastian I Arriola Apelo.

  • National Center for Advancing Translational Sciences UL1TR002373 to Dudley W Lamming.

  • Glenn Foundation for Medical Research to Dudley W Lamming.

  • U.S. Department of Veterans Affairs I01-BX004031 to Dudley W Lamming.

  • National Institute on Aging AG050135 to Nicole E Richardson, Dudley W Lamming.

  • National Institute on Aging AG051974 to Nicole E Richardson, Dudley W Lamming.

  • National Institute on Aging AG056771 to Nicole E Richardson, Dudley W Lamming.

  • National Institute on Aging AG062328 to Nicole E Richardson, Dudley W Lamming.

  • National Institute on Aging AG000213 to Nicole E Richardson.

  • School of Medicine and Public Health, University of Wisconsin-Madison to Dudley W Lamming.

Additional information

Competing interests

No competing interests declared.

DWL has received funding from, and is a scientific advisory board member of, Aeovian Pharmaceuticals, which seeks to develop novel, selective mTOR inhibitors for the treatment of various diseases.

Author contributions

Formal analysis, Supervision, Investigation, Writing - review and editing.

Investigation.

Investigation.

Investigation.

Investigation.

Investigation.

Investigation.

Investigation.

Investigation.

Investigation.

Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review and editing.

Ethics

Animal experimentation: All animal procedures conducted at the William S. Middleton Memorial VA Hospital were approved by the Institutional Animal Care and Use Committee of the William S. Middleton Memorial Veterans Hospital. (Assurance ID: D16-00403).

Additional files

Supplementary file 1. Supplementary Tables S1-S4.

Supplementary Table S1: Survival of mice plotted in Figures 1A and 2A.

Supplementary Table S2: Survival of mice plotted in Figures 1B and 2B. Supplementary Table S3: Survival of male mice plotted in Figure 1C. 22 animals were removed for a cross-sectional analysis and censored (‘0’). Supplementary Table S4: Survival of female mice plotted in Figure 2C. 23 animals were removed for a cross-sectional analysis and censored (‘0’); one additional animal was last recorded at 592 days and censored at that age.

elife-56177-supp1.xlsx (19.9KB, xlsx)
Transparent reporting form

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files.

References

  1. Arantes-Oliveira N, Apfeld J, Dillin A, Kenyon C. Regulation of life-span by germ-line stem cells in Caenorhabditis elegans. Science. 2002;295:502–505. doi: 10.1126/science.1065768. [DOI] [PubMed] [Google Scholar]
  2. Arriola Apelo SI, Neuman JC, Baar EL, Syed FA, Cummings NE, Brar HK, Pumper CP, Kimple ME, Lamming DW. Alternative rapamycin treatment regimens mitigate the impact of rapamycin on glucose homeostasis and the immune system. Aging Cell. 2016a;15:28–38. doi: 10.1111/acel.12405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arriola Apelo SI, Pumper CP, Baar EL, Cummings NE, Lamming DW. Intermittent administration of rapamycin extends the life span of female C57BL/6J mice. The Journals of Gerontology Series A: Biological Sciences and Medical Sciences. 2016b;71:876–881. doi: 10.1093/gerona/glw064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Asdell SA, Doornenbal H, Joshi SR, Sperling GA. The effects of sex steroid hormones upon longevity in rats. Reproduction. 1967;14:113–120. doi: 10.1530/jrf.0.0140113. [DOI] [PubMed] [Google Scholar]
  5. Bailey CJ, Ahmed-Sorour H. Role of ovarian hormones in the long-term control of glucose homeostasis effects of insulin secretion. Diabetologia. 1980;19:475–481. doi: 10.1007/BF00281829. [DOI] [PubMed] [Google Scholar]
  6. Bansal A, Zhu LJ, Yen K, Tissenbaum HA. Uncoupling lifespan and healthspan in Caenorhabditis elegans longevity mutants. PNAS. 2015;112:E277–E286. doi: 10.1073/pnas.1412192112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bellantuono I, de Cabo R, Ehninger D, Di Germanio C, Lawrie A, Miller J, Mitchell SJ, Navas-Enamorado I, Potter PK, Tchkonia T, Trejo JL, Lamming DW. A toolbox for the longitudinal assessment of healthspan in aging mice. Nature Protocols. 2020;15:540–574. doi: 10.1038/s41596-019-0256-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Benedusi V, Martini E, Kallikourdis M, Villa A, Meda C, Maggi A. Ovariectomy shortens the life span of female mice. Oncotarget. 2015;6:10801–10811. doi: 10.18632/oncotarget.2984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Berman JR, Kenyon C. Germ-cell loss extends C. elegans life span through regulation of DAF-16 by kri-1 and lipophilic-hormone signaling. Cell. 2006;124:1055–1068. doi: 10.1016/j.cell.2006.01.039. [DOI] [PubMed] [Google Scholar]
  10. Bitto A, Ito TK, Pineda VV, LeTexier NJ, Huang HZ, Sutlief E, Tung H, Vizzini N, Chen B, Smith K, Meza D, Yajima M, Beyer RP, Kerr KF, Davis DJ, Gillespie CH, Snyder JM, Treuting PM, Kaeberlein M. Transient rapamycin treatment can increase lifespan and healthspan in middle-aged mice. eLife. 2016;5:e16351. doi: 10.7554/eLife.16351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Brayton CF, Treuting PM, Ward JM. Pathobiology of aging mice and GEM: background strains and experimental design. Veterinary Pathology. 2012;49:85–105. doi: 10.1177/0300985811430696. [DOI] [PubMed] [Google Scholar]
  12. Chang K, Kang P, Liu Y, Huang K, Miao T, Sagona AP, Nezis IP, Bodmer R, Ocorr K, Bai H. TGFB-INHB/activin signaling regulates age-dependent autophagy and cardiac health through inhibition of MTORC2. Autophagy. 2019;6:1–16. doi: 10.1080/15548627.2019.1704117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chellappa K, Brinkman JA, Mukherjee S, Morrison M, Alotaibi MI, Carbajal KA, Alhadeff AL, Perron IJ, Yao R, Purdy CS, DeFelice DM, Wakai MH, Tomasiewicz J, Lin A, Meyer E, Peng Y, Arriola Apelo SI, Puglielli L, Betley JN, Paschos GK, Baur JA, Lamming DW. Hypothalamic mTORC2 is essential for metabolic health and longevity. Aging Cell. 2019;18:e13014. doi: 10.1111/acel.13014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Comhaire F. Hormone replacement therapy and longevity. Andrologia. 2016;48:65–68. doi: 10.1111/and.12419. [DOI] [PubMed] [Google Scholar]
  15. Drori D, Folman Y. Environmental effects on longevity in the male rat: exercise, mating, castration and restricted feeding. Experimental Gerontology. 1976;11:25–32. doi: 10.1016/0531-5565(76)90007-3. [DOI] [PubMed] [Google Scholar]
  16. Dumas SN, Lamming DW. Next generation strategies for geroprotection via mTORC1 inhibition. The Journals of Gerontology: Series A. 2020;75:14–23. doi: 10.1093/gerona/glz056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Garratt M, Bower B, Garcia GG, Miller RA. Sex differences in lifespan extension with acarbose and 17-α estradiol: gonadal hormones underlie male-specific improvements in glucose tolerance and mTORC2 signaling. Aging Cell. 2017;16:1256–1266. doi: 10.1111/acel.12656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Garratt M, Lagerborg KA, Tsai YM, Galecki A, Jain M, Miller RA. Male lifespan extension with 17-α estradiol is linked to a sex-specific metabolomic response modulated by gonadal hormones in mice. Aging Cell. 2018;17:e12786. doi: 10.1111/acel.12786. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Harada N, Hanaoka R, Horiuchi H, Kitakaze T, Mitani T, Inui H, Yamaji R. Castration influences intestinal microflora and induces abdominal obesity in high-fat diet-fed mice. Scientific Reports. 2016;6:23001. doi: 10.1038/srep23001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Harrison DE, Strong R, Sharp ZD, Nelson JF, Astle CM, Flurkey K, Nadon NL, Wilkinson JE, Frenkel K, Carter CS, Pahor M, Javors MA, Fernandez E, Miller RA. Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. Nature. 2009;460:392–395. doi: 10.1038/nature08221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hatle JD, Kellenberger JW, Viray E, Smith AM, Hahn DA. Life-extending ovariectomy in grasshoppers increases somatic storage, but dietary restriction with an equivalent feeding rate does not. Experimental Gerontology. 2013;48:966–972. doi: 10.1016/j.exger.2013.06.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Inoue T, Zakikhani M, David S, Algire C, Blouin MJ, Pollak M. Effects of castration on insulin levels and glucose tolerance in the mouse differ from those in man. The Prostate. 2010;70:1628–1635. doi: 10.1002/pros.21198. [DOI] [PubMed] [Google Scholar]
  23. Iwasa T, Matsuzaki T, Yano K, Irahara M. The effects of ovariectomy and lifelong high-fat diet consumption on body weight, appetite, and lifespan in female rats. Hormones and Behavior. 2018;97:25–30. doi: 10.1016/j.yhbeh.2017.10.005. [DOI] [PubMed] [Google Scholar]
  24. Kaeberlein M, Powers RW, Steffen KK, Westman EA, Hu D, Dang N, Kerr EO, Kirkland KT, Fields S, Kennedy BK. Regulation of yeast replicative life span by TOR and Sch9 in response to nutrients. Science. 2005;310:1193–1196. doi: 10.1126/science.1115535. [DOI] [PubMed] [Google Scholar]
  25. Kaeberlein M. How healthy is the healthspan concept? GeroScience. 2018;40:361–364. doi: 10.1007/s11357-018-0036-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kapahi P, Zid BM, Harper T, Koslover D, Sapin V, Benzer S. Regulation of lifespan in Drosophila by modulation of genes in the TOR signaling pathway. Current Biology. 2004;14:885–890. doi: 10.1016/j.cub.2004.03.059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kennedy BK, Berger SL, Brunet A, Campisi J, Cuervo AM, Epel ES, Franceschi C, Lithgow GJ, Morimoto RI, Pessin JE, Rando TA, Richardson A, Schadt EE, Wyss-Coray T, Sierra F. Geroscience: linking aging to chronic disease. Cell. 2014;159:709–713. doi: 10.1016/j.cell.2014.10.039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kennedy BK, Lamming DW. The mechanistic target of rapamycin: the grand ConducTOR of metabolism and aging. Cell Metabolism. 2016;23:990–1003. doi: 10.1016/j.cmet.2016.05.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kleinert M, Sylow L, Fazakerley DJ, Krycer JR, Thomas KC, Oxbøll AJ, Jordy AB, Jensen TE, Yang G, Schjerling P, Kiens B, James DE, Ruegg MA, Richter EA. Acute mTOR inhibition induces insulin resistance and alters substrate utilization in vivo. Molecular Metabolism. 2014;3:630–641. doi: 10.1016/j.molmet.2014.06.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kleinert M, Parker BL, Fritzen AM, Knudsen JR, Jensen TE, Kjøbsted R, Sylow L, Ruegg M, James DE, Richter EA. Mammalian target of rapamycin complex 2 regulates muscle glucose uptake during exercise in mice. The Journal of Physiology. 2017;595:4845–4855. doi: 10.1113/JP274203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lamming DW, Ye L, Katajisto P, Goncalves MD, Saitoh M, Stevens DM, Davis JG, Salmon AB, Richardson A, Ahima RS, Guertin DA, Sabatini DM, Baur JA. Rapamycin-induced insulin resistance is mediated by mTORC2 loss and uncoupled from longevity. Science. 2012;335:1638–1643. doi: 10.1126/science.1215135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lamming DW. Diminished mTOR signaling: a common mode of action for endocrine longevity factors. SpringerPlus. 2014;3:735. doi: 10.1186/2193-1801-3-735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lamming DW, Demirkan G, Boylan JM, Mihaylova MM, Peng T, Ferreira J, Neretti N, Salomon A, Sabatini DM, Gruppuso PA. Hepatic signaling by the mechanistic target of rapamycin complex 2 (mTORC2) The FASEB Journal. 2014a;28:300–315. doi: 10.1096/fj.13-237743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lamming DW, Mihaylova MM, Katajisto P, Baar EL, Yilmaz OH, Hutchins A, Gultekin Y, Gaither R, Sabatini DM. Depletion of rictor, an essential protein component of mTORC2, decreases male lifespan. Aging Cell. 2014b;13:911–917. doi: 10.1111/acel.12256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Levy JC, Matthews DR, Hermans MP. Correct homeostasis model assessment (HOMA) evaluation uses the computer program. Diabetes Care. 1998;21:2191–2192. doi: 10.2337/diacare.21.12.2191. [DOI] [PubMed] [Google Scholar]
  36. Medvedik O, Lamming DW, Kim KD, Sinclair DA. MSN2 and MSN4 link calorie restriction and TOR to sirtuin-mediated lifespan extension in Saccharomyces cerevisiae. PLOS Biology. 2007;5:e261. doi: 10.1371/journal.pbio.0050261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Miller RA, Harrison DE, Astle CM, Fernandez E, Flurkey K, Han M, Javors MA, Li X, Nadon NL, Nelson JF, Pletcher S, Salmon AB, Sharp ZD, Van Roekel S, Winkleman L, Strong R. Rapamycin-mediated lifespan increase in mice is dose and sex dependent and metabolically distinct from dietary restriction. Aging Cell. 2014;13:468–477. doi: 10.1111/acel.12194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Min KJ, Lee CK, Park HN. The lifespan of korean eunuchs. Current Biology. 2012;22:R792–R793. doi: 10.1016/j.cub.2012.06.036. [DOI] [PubMed] [Google Scholar]
  39. Mizunuma M, Neumann-Haefelin E, Moroz N, Li Y, Blackwell TK. mTORC2-SGK-1 acts in two environmentally responsive pathways with opposing effects on longevity. Aging Cell. 2014;13:869–878. doi: 10.1111/acel.12248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Nadon NL, Strong R, Miller RA, Nelson J, Javors M, Sharp ZD, Peralba JM, Harrison DE. Design of aging intervention studies: the NIA interventions testing program. Age. 2008;30:187–199. doi: 10.1007/s11357-008-9048-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Pirimoglu ZM, Arslan C, Buyukbayrak EE, Kars B, Karsidag YK, Unal O, Turan MC. Glucose tolerance of premenopausal women after menopause due to surgical removal of ovaries. Climacteric. 2011;14:453–457. doi: 10.3109/13697137.2010.539723. [DOI] [PubMed] [Google Scholar]
  42. Postic C, Shiota M, Niswender KD, Jetton TL, Chen Y, Moates JM, Shelton KD, Lindner J, Cherrington AD, Magnuson MA. Dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic beta cell-specific gene knock-outs using cre recombinase. Journal of Biological Chemistry. 1999;274:305–315. doi: 10.1074/jbc.274.1.305. [DOI] [PubMed] [Google Scholar]
  43. Powers RW, Kaeberlein M, Caldwell SD, Kennedy BK, Fields S. Extension of chronological life span in yeast by decreased TOR pathway signaling. Genes & Development. 2006;20:174–184. doi: 10.1101/gad.1381406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Robida-Stubbs S, Glover-Cutter K, Lamming DW, Mizunuma M, Narasimhan SD, Neumann-Haefelin E, Sabatini DM, Blackwell TK. TOR signaling and rapamycin influence longevity by regulating SKN-1/Nrf and DAF-16/FoxO. Cell Metabolism. 2012;15:713–724. doi: 10.1016/j.cmet.2012.04.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sarbassov DD, Ali SM, Sengupta S, Sheen JH, Hsu PP, Bagley AF, Markhard AL, Sabatini DM. Prolonged rapamycin treatment inhibits mTORC2 assembly and akt/PKB. Molecular Cell. 2006;22:159–168. doi: 10.1016/j.molcel.2006.03.029. [DOI] [PubMed] [Google Scholar]
  46. Schreiber KH, Ortiz D, Academia EC, Anies AC, Liao CY, Kennedy BK. Rapamycin-mediated mTORC2 inhibition is determined by the relative expression of FK506-binding proteins. Aging Cell. 2015;14:265–273. doi: 10.1111/acel.12313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Schreiber KH, Arriola Apelo SI, Yu D, Brinkman JA, Velarde MC, Syed FA, Liao CY, Baar EL, Carbajal KA, Sherman DS, Ortiz D, Brunauer R, Yang SE, Tzannis ST, Kennedy BK, Lamming DW. A novel rapamycin analog is highly selective for mTORC1 in vivo. Nature Communications. 2019;10:3194. doi: 10.1038/s41467-019-11174-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Soukas AA, Kane EA, Carr CE, Melo JA, Ruvkun G. Rictor/TORC2 regulates fat metabolism, feeding, growth, and life span in Caenorhabditis elegans. Genes & Development. 2009;23:496–511. doi: 10.1101/gad.1775409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Stubbins RE, Holcomb VB, Hong J, Núñez NP. Estrogen modulates abdominal adiposity and protects female mice from obesity and impaired glucose tolerance. European Journal of Nutrition. 2012;51:861–870. doi: 10.1007/s00394-011-0266-4. [DOI] [PubMed] [Google Scholar]
  50. Therneau TM. A Package for Survival Analysis in S. 2.38CRAN. 2015 https://CRAN.R-project.org/package=survival
  51. Vellai T, Takacs-Vellai K, Zhang Y, Kovacs AL, Orosz L, Müller F. Genetics: influence of TOR kinase on lifespan in C. elegans. Nature. 2003;426:620. doi: 10.1038/426620a. [DOI] [PubMed] [Google Scholar]
  52. Yu D, Tomasiewicz JL, Yang SE, Miller BR, Wakai MH, Sherman DS, Cummings NE, Baar EL, Brinkman JA, Syed FA, Lamming DW. Calorie-Restriction-Induced insulin sensitivity is mediated by adipose mTORC2 and not required for lifespan extension. Cell Reports. 2019;29:236–248. doi: 10.1016/j.celrep.2019.08.084. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Yuan T, Li J, Zhao WG, Fu Y, Liu SN, Liu Q, Shen ZF. Effects of estrogen on insulin sensitivity and adipokines in mice. Zhongguo Yi Xue Ke Xue Yuan Xue Bao. Acta Academiae Medicinae Sinicae. 2015;37:269–273. doi: 10.3881/j.issn.1000-503X.2015.03.004. [DOI] [PubMed] [Google Scholar]

Decision letter

Editor: Veronica Galvan1

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

Dietary and genetic manipulations in mice can produce strong modulation of organismal physiology, and many of the variables involved in behaviors related to feeding. The studies reported in your article expand on your previous work by undertaking a dissection of the role and interplay of hepatic mTORC2 in key aspects of physiology and the regulation of lifespan using a genetic model of hepatic deletion in combination with gonadectomy. These studies add to our understanding of crosstalk between different major signaling systems in the regulation of lifespan.

Decision letter after peer review:

Thank you for submitting your article "Ovariectomy uncouples lifespan from metabolic health and reveals a sex-hormone dependent role of hepatic mTORC2 in aging" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Jessica Tyler as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in eLife but that some conclusions require a modest amount of additional new data, as they do with your paper, we are asking that the manuscript be revised to either limit claims to those supported by data in hand, or to explicitly state that the relevant conclusions require additional supporting data.

Summary:

Dietary and genetic manipulations in mice can produce strong modulation of organismal physiology, and many of the variables involved in behaviors related to feeding. In this manuscript Dr Lamming's group continues to expand on their previous work and undertake a careful dissection of the role and interplay of hepatic mTORC2 using a genetic model of hepatic deletion in combination with gonadectomy. Apelo et al. examine in more detail the role of hepatic deletion of Rictor on lifespan and healthspan.

The premise of the study was that males, but not females, of this genotype were short-lived, leading to the current project involving the effects of gonadectomy. The authors build on previous reports from the lab showing that deletion of mTORC2 reduces lifespan in male mice, but not female mice. Further, the authors try to tie this to the known difference in lifespan extension with rapamycin with females benefiting more than males. In this study, authors find that castration of males does not affect the male shortened lifespan of mTORC deletion mice, but surprisingly ovarectomy shortens the lifespan of mTORC deletion female mice (but not WT females) in mice that die of non-cancer reasons (but not overall). They also show sex effects of gonadectomy on metabolic function.

While the studies performed are a needed undertake, the quality of the work presented in this manuscript is high, and measuring healthspan across the lifespan in mammalian studies is of high relevance, some major concerns remain:

1) A major complicating factor to this study is that both females and males lacking Rictor in the liver are short-lived.

2) The determination of cancer or no cancer in these mice is unclear. Background strain of C57BL/6 has been reported previously 50-75% mice at death with lymphoma which could be challenging to determine by gross necropsy. Are the "cancer" animals those that present with solid tumors? From a pathological standpoint this is very different than "cancer" vs. "no cancer". I.e., it would be unlikely so many mice of this strain are dying of "no cancer".

3) The beneficial effects of ovariectomy on lifespan in females is surprising based on previous studies (including some from the 1960s) suggesting ovariectomy shortens rodent lifespan. How do authors reconcile these findings based on previous studies?

4) Similarly, previous studies suggest castration improves glucose metabolism (unlike shown here) and ovariectomy impairs (but here no effect). The effects or lack of effect on L-RKO are interesting, but in light of differences in results in WT animals it's hard to determine interpretation.

5) Statistical analysis – authors provide rationale for determining sample sizes, though oddly provide a range (20-30) rather than a specific number per group as a result of power analysis. Regardless, those group sizes decrease when segregating between "cancer" and "non-cancer". Is there then sufficient power to determine, for example, that ovariectomy of L-RKO actually alters death in a subset of "non-cancer"?

6) It is not until the Discussion that the survival differences between females in this study and the prior one are explained. This makes it hard for the reader. It should be addressed much earlier in the manuscript for clarity.

7) The authors equate healthspan to metabolic parameters, which seems to be a narrow interpretation. They should either include other component measures of healthspan or switch to metabolic health (or some other term that more closely describes what is being measured). With the limited number of metrics analyzed, it is hard to substantiate the notion that ovariectomy improves survival but negatively impacts healthspan.

8) It would be important to show the degree of deletion that is achieved by this genetic cross, does it produce the same level of change in males and females? If not available, did the authors measured any activity changes down from MTORC2? How does the female control lifespan compare to other studies published on B6 females? Any changes in litter weights, sizes or ratios males/females in the genetic crosses?

9) It would be important to report, if you have it, food consumption. Are some of the differences in body weight linked in any way to food intake?

10) Besides cancer, any other predominant disease that the animals are dying off/with?

11) For all the reports on metabolic parameters it would be great if the authors could set the Y axes to the same max values, it would make easier to compare the age and sex related changes in Figure 3A-D and Figure 4A and B, E and F.

12) It is not clear how to do this but the manuscript is difficult to read through because of the different cohorts of mice and sexes. The authors should consider ways to improve this. One possibility would be to discuss males and females separately in the Results and then bring the differences out in the Discussion. Not sure if this would improve clarity, but it might be worth a try.

eLife. 2020 Jul 28;9:e56177. doi: 10.7554/eLife.56177.sa2

Author response

[…] While the studies performed are a needed undertake, the quality of the work presented in this manuscript is high, and measuring healthspan across the lifespan in mammalian studies is of high relevance, some major concerns remain:

1) A major complicating factor to this study is that both females and males lacking Rictor in the liver are short-lived.

We apologize for the confusion, but actually, this is not quite correct from a statistical standpoint – there is only an effect of liver Rictor loss on the overall survival of male mice. As we state, “when we looked at the total population of female mice in our study, the negative effect of Rictor loss on Sham-treated female mice was masked, and there was no overall negative effect of Rictor loss on survival” (Cox regression analysis: HR Rictor knockout = 1.02, p = 0.92), Figure 2C).

Loss of liver Rictor does impact the survival of a subset of female mice (Figure 2B), which is a major finding of our manuscript. However, while this decrease in midlife survival leads to a trend towards reduced survival of all L-RKO Sham females between 400-800 days in Figure 2C, this does not reach statistical significance as the curves converge again after 800 days. To emphasize this, we have edited the text to state more clearly that “There was also no difference between the overall survival of WT Sham and WT L-RKO female mice (p = 0.153, Wilcoxon rank sum)”.

2) The determination of cancer or no cancer in these mice is unclear. Background strain of C57BL/6 has been reported previously 50-75% mice at death with lymphoma which could be challenging to determine by gross necropsy. Are the "cancer" animals those that present with solid tumors? From a pathological standpoint this is very different than "cancer" vs. "no cancer". I.e., it would be unlikely so many mice of this strain are dying of "no cancer".

We thanks the reviewers for highlighting this issue, which we agree is quite important – in fact we label our figure panels “deaths with cancer” and “cancer not observed at necropsy” rather than “deaths from cancer” or “deaths with no cancer” for this very reason. We have edited the figure legends to make this distinction as well; and we have edited our methodology to better describe our gross necropsy.

Approximately half of the mice had observable cancer during our gross necropsy, which is consistent (but on the lower end) of the range of cancer rates reported for C57BL/6J mice in the literature. We have now edited the Results and Discussion to more clearly note that there could be unobserved cancers as well. We now highlight that an effect of ovariectomy on unobserved neoplasma could contribute to the protective effect of OVX on L-RKO female lifespan, and we now state that “Future carefully designed studies to assess the organismal health of L-RKO mice, with detailed pathology between 400 and 800 days of age, will be needed to fully address these issues.”

3) The beneficial effects of ovariectomy on lifespan in females is surprising based on previous studies (including some from the 1960s) suggesting ovariectomy shortens rodent lifespan. How do authors reconcile these findings based on previous studies?

This is an interesting question – we have revised and expanded our Discussion section to address this. In brief, at least some older studies in rats suffered from the lack of sham surgery control group; a 1967 study suffering from this flaw also observed no difference between the lifespan of OVX mice and OVX mice dosed with estrogen, suggesting the difference in lifespan of wild-type and OVX mice could have resulted from the stress of surgery rather than removal of estrogens. A recent 2018 rat study which we now site reports OVX extends the lifespan of mice.

A 2015 mouse study showed that ovariectomy at 5 months of age shortens lifespan, in contrast, we performed ovariectomy at 3 weeks of age, prior to puberty, and studies in C. elegans and grasshoppers support the idea that early life removal of germ cells/ovaries extends lifespan. Thus, timing of ovariectomy may be important as well. We thank the reviewers for urging us to address these important points.

4) Similarly, previous studies suggest castration improves glucose metabolism (unlike shown here) and ovariectomy impairs (but here no effect). The effects or lack of effect on L-RKO are interesting, but in light of differences in results in WT animals it's hard to determine interpretation.

We thank the reviewer for this question, but it is based on an incorrect premise and a misunderstanding of our results. We have expanded our discussion of glucose homeostasis substantially to ensure the reviewer and reader can better understand our results, citing additional recent findings.

First – we do observe impaired glucose metabolism in OVX mice as expected and now state in the Discussion “these overall positive effects of ovariectomy on longevity do not promote healthspan, with ovariectomized mice suffering from adiposity and disrupted blood glucose homeostasis.” You can see this specifically in panels Figure 5C and D – the blue curve for WT OVX mice is significantly higher than the curve for WT Sham mice, and the AUC is significantly higher. There is similarly an effect of OVX on fasting insulin and calculated HOMA2-IR (Figure 5F).

Second, we see no effect of castration on glucose tolerance or insulin sensitivity, but we did not expect to see a positive effect – if anything, we expected an impairment as we expected body composition to worsen. Our results, showing no effect of castration on glucose homeostasis in young or aged mice, are in agreement with recent reports showing no effect of castration on glucose homeostasis in mice eating a control diet (Garratt et al., 2017; Harada et al., 2016). This latter paper reports an impairment of glucose homeostasis in castrated mice (unlike what the reviewer mentions) upon high-fat diet, but we did not examine high fed diet fed mice here.

5) Statistical analysis – authors provide rationale for determining sample sizes, though oddly provide a range (20-30) rather than a specific number per group as a result of power analysis. Regardless, those group sizes decrease when segregating between "cancer" and "non-cancer". Is there then sufficient power to determine, for example, that ovariectomy of L-RKO actually alters death in a subset of "non-cancer"?

As now better discussed in the transparent reporting guidelines we targeted a group size of 20-30 mice per group in order to provide 84-95% power to detect a 15% change in lifespan. We selected a target range as mouse breeding of genetically engineered mice is never precisely mendelian and we then needed to perform surgery on the mice. We achieved this target range for all groups.

To address the second concern we performed a power calculation using the actual mean and standard deviations observed here for the L-RKO Sham and L-RKO OVX lifespans. We find that we had 85% power to detect the effect size we observed (α = 0.05).

6) It is not until the Discussion that the survival differences between females in this study and the prior one are explained. This makes it hard for the reader. It should be addressed much earlier in the manuscript for clarity.

We thank the reviewers for this comment, and we have edited the manuscript to highlight this result in the Introduction and Results section.

7) The authors equate healthspan to metabolic parameters, which seems to be a narrow interpretation. They should either include other component measures of healthspan or switch to metabolic health (or some other term that more closely describes what is being measured). With the limited number of metrics analyzed, it is hard to substantiate the notion that ovariectomy improves survival but negatively impacts healthspan.

We completely agree that this is a limitation, and as suggested by the reviewers we now use the term “metabolic health” in the Abstract and Discussion, and state that “A limitation of this study is that we did not perform a comprehensive assessment of healthspan, focusing on metabolic parameters.”

8) It would be important to show the degree of deletion that is achieved by this genetic cross, does it produce the same level of change in males and females? If not available, did the authors measured any activity changes down from MTORC2? How does the female control lifespan compare to other studies published on B6 females? Any changes in litter weights, sizes or ratios males/females in the genetic crosses?

We thank the reviewer for this important question. As we have shown previously, and we now include in the Introduction, the genetic cross achieves equivalent deletion of liver mTORC2 in both males and females, reducing overall expression of liver RICTOR protein by approximately 80% through the essentially complete deletion of Rictor in hepatocytes and reducing phosphorylation of mTORC2 substrates by a similar percentage (Lamming et al., 2014; and Lamming et al., 2014).

The control lifespan of our female WT Sham mice are equivalent to C57BL/6J female mice from The Jackson Laboratory (Our female WT Sham median lifespan: 853; Jackson: 866) and are consistent with the lifespans of two other B6 mouse lifespan studies not involving surgery recently published by our lab (Yu et al., 2019; Chellappa et al., 2019).

9) It would be important to report, if you have it, food consumption. Are some of the differences in body weight linked in any way to food intake?

We thank the reviewer for this important question. We have included a new figure, showing that ovariectomy leads to alterations in energy balance and fuel source utilization. At least in 12 month old animals, ovariectomy seems to promote obesity as a combination of reduced physical activity and reduced energy expenditure, and promote utilization on lipids as an energy source.

10) Besides cancer, any other predominant disease that the animals are dying off/with?

While a spectrum of other age-related diseases is undoubtedly present in C57BL/6J mice, there was no other predominant cause of death that we were able to identify during our gross necropsies.

11) For all the reports on metabolic parameters it would be great if the authors could set the Y axes to the same max values, it would make easier to compare the age and sex related changes in Figure 3A-D and Figure 4A and B, E and F.

This was a great suggestion, and we have now made all Y axes that represent the same measurements have the same range and min/max values in Figures 3, 4, and 5.

12) It is not clear how to do this but the manuscript is difficult to read through because of the different cohorts of mice and sexes. The authors should consider ways to improve this. One possibility would be to discuss males and females separately in the Results and then bring the differences out in the Discussion. Not sure if this would improve clarity, but it might be worth a try.

We thank the reviewers for this suggestion – we actually started writing the manuscript this way and also tried a few other constructions, but ultimately settled on the present order as the clearest. We hope the other clarifications and edits we have made have helped to improve flow and readability of our description of this admittedly complex study.

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    Supplementary file 1. Supplementary Tables S1-S4.

    Supplementary Table S1: Survival of mice plotted in Figures 1A and 2A.

    Supplementary Table S2: Survival of mice plotted in Figures 1B and 2B. Supplementary Table S3: Survival of male mice plotted in Figure 1C. 22 animals were removed for a cross-sectional analysis and censored (‘0’). Supplementary Table S4: Survival of female mice plotted in Figure 2C. 23 animals were removed for a cross-sectional analysis and censored (‘0’); one additional animal was last recorded at 592 days and censored at that age.

    elife-56177-supp1.xlsx (19.9KB, xlsx)
    Transparent reporting form
    elife-56177-transrepform.docx (245.9KB, docx)

    Data Availability Statement

    All data generated or analyzed during this study are included in the manuscript and supporting files.

    Articles from eLife are provided here courtesy of eLife Sciences Publications, Ltd

    RESOURCES