1. Volume 4, Issue 5, Pages 913-920 (September 2013)
Increased Mammalian Lifespan and a Segmental and Tissue-Specific Slowing of Aging after Genetic Reduction of mTOR Expression 
J. Julie Wu, Jie Liu, Edmund B. Chen, Jennifer J. Wang, Liu Cao, Nisha Narayan, Marie M. Fergusson, Ilsa I. Rovira, Michele Allen, Danielle A. Springer, Cory U. Lago, Shuling Zhang, Wendy DuBois, Theresa Ward, Rafael deCabo, Oksana Gavrilova, Beverly Mock, Toren Finkel 
Cell Reports 
Volume 4, Issue 5, Pages (September 2013)
DOI: /j.celrep
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2. Cell Reports 2013 4, 913-920DOI: (10.1016/j.celrep.2013.07.030)
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3. Figure 1 A Mouse Model of Reduced mTOR Expression Extends Life Span
(A) Genomic Organization of the WT Allele (+) and the Hypomorphic mTOR Allele (Δ).
(B) Representative mTOR protein expression in the liver of two WT (mTOR+/+) and two mTORΔ/Δ mice. GAPDH is used as a loading control, and the normalized expression (WT = 1) of mTOR to GAPDH is shown for each mouse.
(C) Leucine-stimulated S6 Kinase phosphorylation (pS6K) in primary mouse embryonic fibroblasts isolated from WT or mTORΔ/Δ mice.
(D) Insulin-stimulated mTOR activity in pairs of WT or mTORΔ/Δ mice.
(E) Survival of a cohort of male WT and mTORΔ/Δ mice.
(F) Survival of female members of the cohort.
(G) Survival of the overall cohort.
(H) Incidence of malignant tumors found at necropsy denoted by shaded portion of each bar. While the overall incidence of cancer was different between the two genotypes, the spectrum of tumors observed was similar. ∗∗p < 0.001, Fisher’s exact test.
See also Figure S1.
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4. Figure 2
The mTORΔ/Δ Mice Are Smaller but Have No Significant Alterations in Glucose Homeostasis and Metabolism
All measurements were performed using male mice.
(A) Representative size of a WT mouse and an mTORΔ/Δ adult mouse.
(B) Body weight of WT (n = 7) and mTORΔ/Δ (n = 7). Curves are statistically different using a one-way analysis of variance followed by two-tailed t test, p < 0.01.
(C) Daily food intake is indistinguishable between WT mice (shaded bar) and mTORΔ/Δ mice (open bar) (n = seven WT mice, and n = six mTORΔ/Δ mice; food intake is normalized to body weight).
(D) Glucose tolerance of 8- to 12-week-old WT (n = 7) and mTORΔ/Δ (n = 6) mice.
(E) Insulin tolerance test of 8- to 12-week-old WT (n = 12) and mTORΔ/Δ (n = 7) mice.
(F) Respiratory exchange ratio (RER) of WT (n = 7) and mTORΔ/Δ (n = 5) mice.
(G) Measurement of rates of total body fatty acid oxidation normalized to body weight in WT (n = 7) and mTORΔ/Δ (n = 5).
(H) Total oxygen consumption normalized to body weight (n = WT and n = 5 mTORΔ/Δ mice).
(I) Total daily energy expenditure is not altered in mTORΔ/Δ mice (n = seven WT mice and n = five mTORΔ/Δ mice). For all panels, shaded bars represent the WT mice, and the open bars represent the mTORΔ/Δ mice. Where indicated, metabolic parameters are adjusted to body weight raised to the 0.75 power, as indicated by the symbol (BW).
All pooled data are presented as mean ± SEM. See also Figure S2.
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5. Figure 3
Molecular and Biochemical Biomarkers of Aging Are Reduced in Old mTORΔ/Δ Mice
(A) Assessment of the age-dependent increase in kidney mRNA levels for the cell cycle inhibitor p16INK4a normalized to GAPDH expression (n = three mice per genotype and age, with each mouse performed in triplicate).
(B) A similar assessment in old and young liver samples (n = six young WT and n = five young mTORΔ/Δ samples; n = four old WT and n = four old mTORΔ/Δ samples, with each sample performed in triplicate).
(C) Representative brain sections stained for nitrotyrosine (red, upper panels) obtained from young WT mice, old WT mice, and old mTORΔ/Δ mice. Cell nuclei with stained concurrently with DAPI (blue, lower panels).
(D) Intensity of nitrotyrosine staining in the brains of old WT mice (n = three mice, with three to five determinations per mouse) and mTORΔ/Δ mice (n = four mice, with three to five determinations per mouse).
(E) Staining for polyubquitinated proteins in brain tissue sections obtained from young WT mice, old WT mice, and old mTORΔ/Δ mice. Upper panels (red) are stained with an antibody that recognizes proteins that are polyubiquitinated, and lower panels are analyzed by nuclear DAPI staining.
(F) Quantification of polyubiquitinated protein levels in brain sections of WT mice (n = three mice, with three to five determinations per mouse) and mTORΔ/Δ mice (n = four mice, with three to five determinations per mouse).
All pooled data are presented as mean ± SEM. ∗p < ∗∗p < See also Figure S3.
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6. Figure 4
The Effects of Reduced mTOR Expression on a Range of Tissue Specific Age-Related Parameters
(A) Escape latency times on day 3 of training for the Barnes maze test for both young female (n = six mice per genotype) and old female (n = nine WT and n = 13 mTORΔ/Δ) mice. WT mice are represented by the shaded bars, while the open bars represent mTORΔ/Δ mice. ∗p < 0.05.
(B) Learning strategy of old mice in the acquisition phase for training in the Barnes maze. Arrows indicate transition point between random to directed searching, an indicator of the speed in which new spatial learning is obtained (n = nine WT female mice, and n = 13 mTORΔ/Δ female mice).
(C) Duration on the Rotarod, a measure of coordination and balance (n = six male mice per genotype for young mice; n = four old male WT, and n = seven old male mTORΔ/Δ mice). ∗p < 0.05.
(D) Stride width variance in young (n = six young female mice per genotype) and old mice (n = six old WT male and female mice, and n = 13 old male and female mTORΔ/Δ mice). ∗p < 0.05.
(E) Grip strength, normalized to gram of body weight, in young female mice (n = six per genotype) and old female mice (n = four WT mice, and n = 11 mTORΔ/Δ mice). ∗p < 0.05.
(F) Assessment of the age-dependent decline in bone volume (BV) to tissue volume (TV) (n = four young mice per genotype, and n = six old mice per genotype). ∗p < 0.05.
(G) Age-dependent incidence of visibly apparent superficial infections of the skin, eyes, or mouth of the total cohort of WT and mTORΔ/Δ mice (n = 34 WT mice, and n = 43 mTORΔ/Δ mice; statistical analysis by Fisher’s exact test). ∗p < ∗∗p < 0.01.
All bar graph data are presented as mean ± SEM. See also Figure S4.
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7. Figure S1
Characterization of Reduced mTOR Expression in the mTORΔ/Δ Mice, Related to Figure 1
(A) Western blot analysis of mTOR expression in the heart and pancreas of two wild-type and two mTORΔ/Δ mice. Actin or GAPDH are used as loading controls.
(B) Immunoprecipitation of mTOR from MEF protein lysate. Coimmunoprecipitation of Raptor or Rictor is evidence of the level of TORC1 and TORC2 complex formation. Input western blot demonstrates reduction in overall mTOR expression without discernable effect on Raptor or Rictor expression. Tubulin is used as a loading control for the input lysates.
(C) Assessment of global protein synthesis. Rates of 35S-methionine incorporation were determined over the indicated time frame in MEFs initially obtained from WT (mTOR+/+) or mTORΔ/Δ embryos.
(D) Tumor incidence observed at the time of necropsy for WT and mTORΔ/Δ mice (WT, n = 26 and mTORΔ/Δ mice, n = 36).
(E) Tumor incidence as a function of genotype and sex. For WT mice, we observed that 10/26 mice developed tumors (6F/4M). For mTORΔ/Δ mice, we noted 8/36 developed tumors (6F/2M).
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8. Figure S2
Metabolic Effects of Reduced mTOR Expression, Related to Figure 2
(A) Body composition of mTORΔ/Δ mice is not altered when compared to wild-type mice. Various tissues were obtained at sacrifice, individually weighed and expressed as a percentage of overall body weight. Tissues assayed include liver, heart, pancreas, epididymal white adipose tissues (EWAT), and brown adipose tissue (BAT). (n = 9 male mice per genotype, no significant differences were observed).
(B) Levels of fasting serum insulin in young WT (n = 7, shaded bars) or mTORΔ/Δ mice (n = 6, open bars).
(C and D) Glucose-stimulated insulin secretion (GSIS) of islets isolated from young WT (shaded bars) or mTORΔ/Δ mice (open bars). Islets were stimulated with either (C) 3 mM or (D) 16.7 mM extracellular glucose and insulin levels determined in the supernatant and subsequently normalized to islet DNA concentration. Shown is the average of two independent experiments each performed in triplicate with ∗p < 0.05.
(E) Glucose tolerance test in old male wild-type and mTORΔ/Δ mice (n = 4 per genotype).
(F) Young mice (age 2–3 months) were analyzed for free fatty acid (FFA) levels. Shown are the values for WT mice (shaded bars, n = 7) and mTORΔ/Δ mice (open bars, n = 6).
(G and H) Serum triglyceride levels (n = 7 WT and n = 6 mTORΔ/Δ) (G) and serum adiponectin levels (n = 6 WT and n = 6 mTORΔ/Δ) (H). All metabolic experiments were performed in male mice.
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9. Figure S3
Biomarkers of Aging Are Reduced in mTORΔ/Δ Mice, Related to Figure 3
(A) Representative nitrotyrosine staining in old WT or mTORΔ/Δ myocardial tissue. Top row is assessed for nitrotyrosine levels (red) while the bottom row represents a DAPI nuclear counterstain (blue).
(B) Quantification of nitrotyrosine staining in old WT or mTORΔ/Δ heart tissues. Analysis involved four mice per genotype using the average of 3–5 random sections per mouse.
(C) Polyubiquitin staining in sections obtained from old WT or mTORΔ/Δ myocardium. Top row is a representative assessment for polyubiquitin levels (red) while the bottom row is the DAPI nuclear counterstain (blue).
(D) Quantification of polyubiquitin accumulation in old WT or mTORΔ/Δ heart tissues. Analysis involved four mice per genotype using the average of 3–5 random sections per mice. ∗p < 0.05.
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10. Figure S4
Physiological Characterization of Reduced mTOR Expression, Related to Figure 4
(A) Rotarod speed analysis for wild-type and mTORΔ/Δ male mice. Analysis of maximum Rotarod speed obtained as a function of age and genotype is shown. No differences were observed between young wild-type (shaded bars, n = 6 male mice) or mTORΔ/Δ mice (open bars, n = 6 male mice). Maximum speed obtainable was reduced in old WT mice (n = 4 male mice) and old mTORΔ/Δ mice (n = 7 male mice), although the mTORΔ/Δ mice had significantly better performance; ∗p < 0.05.
(B and C) Representative histology of the testes of WT (B) or mTORΔ/Δ (C) mice that were both 31 months of age. No clear differences were observed in these animals or in four other mice analyzed.
(D) Rates of cataract formation as analyzed by histological sections obtained from mice sacrificed at 21 months (3 WT and 3 mTORΔ/Δ female mice) or at 31 months (3 WT and 3 mTORΔ/Δ male mice).
Cell Reports 2013 4, DOI: ( /j.celrep )
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