Calorie-Restriction-Induced Insulin Sensitivity Is Mediated by Adipose mTORC2 and Not Required for Lifespan Extension

Abstract

Calorie restriction (CR) extends the healthspan and lifespan of diverse species. In mammals, a broadly conserved metabolic effect of CR is improved insulin sensitivity, which may mediate the beneficial effects of a CR diet. This model has been challenged by the identification of interventions that extend lifespan and healthspan yet promote insulin resistance. These include rapamycin, which extends mouse lifespan yet induces insulin resistance by disrupting mTORC2 (mechanistic target of rapamycin complex 2). Here, we induce insulin resistance by genetically disrupting adipose mTORC2 via tissue-specific deletion of the mTORC2 component Rictor (AQ-RKO). Loss of adipose mTORC2 blunts the metabolic adaptation to CR and prevents whole-body sensitization to insulin. Despite this, AQ-RKO mice subject to CR experience the same increase in fitness and lifespan on a CR diet as wild-type mice. We conclude that the CR-induced improvement in insulin sensitivity is dispensable for the effects of CR on fitness and longevity.

Keywords: Rictor; adipose; calorie restriction; fitness; frailty; healthspan; insulin sensitivity; lifespan; lipogenesis; mTORC2.

Figure 1.. Mice lacking adipose mTORC2 are glucose tolerant and insulin resistant.

(A) Western blot analysis and quantification of RICTOR expression and mTORC2 activity in the iWAT, eWAT, liver, and muscle of AQ-RKO mice and their wild-type (WT) littermates (n=4/group, * = p < 0.05, t-test). (B) Weights of male and female WT and AQ-RKO mice (n = 8–12/group (males), n=4/group (female); statistics for the overall effects of age, genotype (GT), and the interactions represent the p-value from a repeated measures two-way ANOVA conducted separately for each sex). (C and D) Tissue weights of 20-week-old male (C) and female (D) WT and AQ-RKO mice (n=5–9/group, * = p < 0.05, t-test). (E) Plasma IGF-1 was determined in male and female WT and AQ-RKO mice (n=5/group, statistics for the overall effects of sex, genotype (GT), and the interaction represent the p-value from a two-way ANOVA; * = p < 0.05, from a Holm-Sidak’s post-test examining the effect of parameters identified as significant in the two-way ANOVA). (F and G) Glucose (F) and (G) insulin tolerance was determined in 10–12 week old mice (n=6–9/group; for AUC, statistics for the overall effects of sex, genotype (GT), and the interaction represent the p-value from a two-way ANOVA, * = p < 0.05, from a Holm-Sidak’s post-test examining the effect of parameters identified as significant in the two-way ANOVA). Error bars represent SEM.

Figure 2.. Adipose mTORC2 is required for the CR-induced increase in insulin sensitivity.

(A and B) Glucose tolerance test of male (A) and female (B) WT and AQ-RKO mice after 9 weeks on indicated dietary regimens (n=5–14 mice/group). (C and D) Insulin tolerance test of male (C) and female (D) WT and AQ-RKO mice after 10 weeks on indicated dietary regimens (n=9–19 mice/group). (E and F) Plasma insulin in male (E) and female (F) mice after an overnight fast followed by refeeding for 3 hours (n=5–8/group). (G) Pyruvate tolerance test of male WT and AQ-RKO mice after 11 weeks on indicated dietary regimens (n=14–19/group). (A-G) Statistics for the overall effects of diet, genotype (GT), and the interaction represent the p-value from a two-way ANOVA; * = p < 0.05, from a Holm-Sidak’s post-test examining the effect of parameters identified as significant in the two-way ANOVA. Error bars represent SEM.

Figure 3.. Loss of adipose mTORC2 does not affect the response of body composition to CR.

Fat mass, weight, lean mass, and adiposity of male (A, C, E, and G) and female (B, D, F, and H) WT and AQ-RKO mice fed either AL or CR diets starting at 10 weeks of age (n=12–16/group; Statistics for the overall effects of diet, genotype (GT), and the interaction represent the p-value from a repeated measures two-way ANOVA conducted separately for each genotype). Error bars represent SEM.

Figure 4.. Loss of adipose mTORC2 attenuates CR-mediated reprogramming of adipose lipid metabolism.

(A and B) Respiratory exchange ratio (RER) of male (A) and female (B) WT and AQ-RKO mice after 7-week AL or CR regimens (n = 4–12/group; statistics for the overall effects of genotype (GT), time and the interaction represent the p-value from a repeated measures two-way ANOVA conducted on WT CR and AQ-RKO CR mice; * = p < 0.05, from a Holm-Sidak’s post-test; dark (shaded) and light phases were analyzed separately). (C and D) Expression of Chrebp-β and related lipogenic genes in the iWAT of male (C) and female (D) WT and AQ-RKO mice after 12 weeks on the indicated diets (n = 6/group; statistics for the overall effects of diet, genotype (GT), and the interaction represent the p-value from a two-way ANOVA for each gene; * = p < 0.05, from Holm-Sidak’s post-tests conducted for the effect of diet and genotype). (E and F) Plasma triglycerides (E) and free fatty acids (F) in WT and AQ-RKO mice after 12 weeks on the indicated diets (n=5–8/group; statistics for the overall effects of diet, genotype (GT), and the interaction represent the p-value from a two-way ANOVA conducted separately for each sex; * = p < 0.05, from Holm-Sidak’s post-tests conducted for the effect of diet and genotype). Error bars represent SEM.

Figure 5.. Longitudinal assessment of body composition of WT and AQ-RKO mice fed AL or CR diet.

(A-F) Longitudinal body composition analysis of WT and AQ-RKO mice. (A and B) Fat mass of male (A) and female (B) mice. (C and D) Lean mass of male (C) and female (D) mice. (A-D) Numbers vary month-by-month; maximum n = 24–39/group. (E and F) Percentage of body fat mass and lean mass of male (E) and female (F) mice at 2, 12 and 24 months of age (n = 13–28 mice/group). Statistics for the overall effects of genotype (GT), age, and the interaction represent the p-value from a repeated measures two-way ANOVA conducted for AL fed mice; * = p < 0.05, from Holm-Sidak’s post-test comparing WT AL and AQ-RKO AL fed mice at each age. Error bars represent SEM.

Figure 6.. Deletion of adipose Rictor blocks calorie restriction from improving organismal insulin sensitivity.

Insulin tolerance tests were performed on (A and B) 9-month-old male (A) and female (B) WT and AQ-RKO mice as well as (C and D) 25 month-old male (C) and female (D) WT and AQ-RKO mice. n = 6–13/group; statistics for the overall effects of diet, genotype (GT), and the interaction represent the p-value from a two-way ANOVA for each measurement; * = p < 0.05, Tukey post-test. Error bars represent SEM.

Figure 7.. Adipose mTORC2 is dispensable for the effects of CR on healthspan and lifespan.

(A and B) Frailty was assessed longitudinally in male (A) and female (B) WT and AQ-RKO mice starting at 18 months of age (numbers vary month by month; maximum n =20–37/group. Statistics for the overall effects of diet, age, and the interaction represent the p-value from a mixed-effects model (Restricted Maximum Likelihood (REML)) conducted separately for each genotype; * (black) = p < 0.05, from Holm-Sidak’s post-test comparing WT AL and WT CR-fed mice at each age, * (green) = p < 0.05, from Holm-Sidak’s post-test comparing AQ-RKO AL and AQ-RKO CR-fed mice at each age). (C and D) Kaplan-Meier plot showing the lifespan of male (C) and female (D) WT and AQ-RKO mice. For each sex, the two-tailed stratified log-rank p-value for the effect of genotype (GT) and diet is indicated. (E) Median lifespans for the survival curves plotted in C and D, and the individual log-rank p-values for the indicated pairwise comparisons. (F) CR effects on fitness and longevity are independent from its effects on insulin sensitivity and cancer. Error bars represent SEM.