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Science 335 (6076): 1638-1643

Copyright © 2012 by the American Association for the Advancement of Science

Rapamycin-Induced Insulin Resistance Is Mediated by mTORC2 Loss and Uncoupled from Longevity

Dudley W. Lamming1,2,3,4,5,{dagger}, Lan Ye6,{dagger}, Pekka Katajisto1,2,3,4,5, Marcus D. Goncalves7, Maki Saitoh1,2,3,4,5, Deanna M. Stevens1,2,3,4,5, James G. Davis6, Adam B. Salmon8, Arlan Richardson8, Rexford S. Ahima7, David A. Guertin1,2,3,4,5,*, David M. Sabatini1,2,3,4,5,{ddagger}, and Joseph A. Baur6,{ddagger}

1 Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA.
2 Department of Biology, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA.
3 Howard Hughes Medical Institute, MIT, Cambridge, MA 02139, USA.
4 Broad Institute of Harvard and MIT, Seven Cambridge Center, Cambridge, MA 02142, USA.
5 The David H. Koch Institute for Integrative Cancer Research at MIT, Cambridge, MA 02139, USA.
6 Department of Physiology, Institute for Diabetes, Obesity, and Metabolism, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
7 Department of Medicine, Institute for Diabetes, Obesity, and Metabolism, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
8 The Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, TX 78245, USA.


Figure 1
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Fig. 1. Rapamycin-induced insulin resistance is independent of hepatic mTORC1. (A to F) Glucose infusion rate (A), rate of disappearance of glucose from the circulation (B), hepatic gluconeogenesis (C), and insulin responsiveness (D), as well as glucose uptake by white adipose tissue (E) and skeletal muscle (F), were determined during a hyperinsulinemic-euglycemic clamp in mice treated with 2 mg/kg per day of rapamycin or vehicle control for 2 weeks. (Each symbol represents a single animal, n = 13 vehicle-treated mice, 11 rapamycin-treated mice, *P < 1 x 10–5, Student’s t test; #P < 0.045 by Brown-Mood k-sample median test.) (G) Serum glucose and insulin concentration in rapamycin-treated mice during fasting and after refeeding for 4 hours (*P < 0.02). (H) Phosphorylation of the mTORC1 substrate S6K1 T389 and subsequent phosphorylation of S6 in the livers of rapamycin-treated mice. (I and J) Glucose tolerance with or without rapamycin treatment in mice lacking hepatic Raptor (*P < 0.05, #P < 0.06 for rapamycin-treated groups versus untreated). All bars indicate means and SEM.

 

Figure 2
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Fig. 2. Disruption of mTORC2 in vivo after chronic rapamycin treatment. (A and B) Effects of rapamycin on phosphorylation of PKCα, Akt, and the SGK substrate NDRG1 in liver in response to refeeding (A) or insulin (B) after an overnight fast. (C and D) Effects of rapamycin on phosphorylation of PKCα and Akt in response to refeeding in white adipose tissue (C) and muscle (D). (E to G) Effects of rapamycin on the integrity of mTORC1 and mTORC2. mTOR was immunoprecipitated from liver (E), skeletal muscle (F), and white adipose tissue (G), followed by immunoblotting for Raptor and Rictor (subunits of mTORC1 and mTORC2, respectively).

 

Figure 3
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Fig. 3. Regulation of glucose homeostasis by mTORC2. (A) Glucose tolerance of Alb-Cre RictorLoxP/LoxP mice (*P < 0.002). (B) Pyruvate tolerance of Alb-Cre RictorloxP/loxP mice (*P < 0.03). (C) Effect of rapamycin on glucose tolerance of mice with whole-body deletion of Rictor fasted for 6 hours (*P < 0.008 for all groups versus RictorloxP/loxP). (D to I) Glucose infusion rate (D), rate of disappearance of glucose from the circulation (E), hepatic gluconeogenesis (F), and insulin responsiveness (G), as well as glucose uptake by white adipose tissue (H) and skeletal muscle (I), were determined during a hyperinsulinemic-euglycemic clamp in tamoxifen-treated ubiquitinC–CreERT2 RictorloxP/loxP mice fasted for 6 hours (n = 8 RictorloxP/loxP, 7 UBC-Cre RictorloxP/loxP, **P < 0.008; *P < 0.05). All bars indicate means and SEM.

 

Figure 4
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Fig. 4. Depletion of mTOR and mLST8 uncouples longevity from decreased glucose tolerance. (A and B) Kaplan-Meier plots showing life spans of female (A) and male (B) mice heterozygous for components of the mTOR signaling pathway. (C and D) Life-spans of female (C) and male (D) mtor+/– mlst8+/– mice. Wild-type curves are repeated for comparison. (E) Quantification of phosphorylated proteins in female wild-type and mtor+/– mlst8+/– livers after an overnight fast and 45 min of refeeding (n = 13 wild-type versus 13 mtor+/– mlst8+/– female mice, *P < 0.03). (F) Quantitative real-time polymerase chain reaction analysis of mRNA levels for PEPCK and G6Pase in the livers of young female wild-type and mtor+/– mlst8+/– mice (*P < 0.03). (G and H) Immunoprecipitation of mTOR complexes reveals preferential loss of Raptor association in mtor+/– mlst8+/– female mice (*P < 0.02). All bars indicate means and SEM.

 


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