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. 2014 Jun 27;3(6):630-41.
doi: 10.1016/j.molmet.2014.06.004. eCollection 2014 Sep.

Acute mTOR inhibition induces insulin resistance and alters substrate utilization in vivo

Maximilian Kleinert  1 , Lykke Sylow  1 , Daniel J Fazakerley  2 , James R Krycer  3 , Kristen C Thomas  2 , Anne-Julie Oxbøll  1 , Andreas B Jordy  1 , Thomas E Jensen  1 , Guang Yang  2 , Peter Schjerling  4 , Bente Kiens  1 , David E James  5 , Markus A Ruegg  6 , Erik A Richter  1
Affiliations

Acute mTOR inhibition induces insulin resistance and alters substrate utilization in vivo

Maximilian Kleinert et al. Mol Metab. .

Abstract

The effect of acute inhibition of both mTORC1 and mTORC2 on metabolism is unknown. A single injection of the mTOR kinase inhibitor, AZD8055, induced a transient, yet marked increase in fat oxidation and insulin resistance in mice, whereas the mTORC1 inhibitor rapamycin had no effect. AZD8055, but not rapamycin reduced insulin-stimulated glucose uptake into incubated muscles, despite normal GLUT4 translocation in muscle cells. AZD8055 inhibited glycolysis in MEF cells. Abrogation of mTORC2 activity by SIN1 deletion impaired glycolysis and AZD8055 had no effect in SIN1 KO MEFs. Re-expression of wildtype SIN1 rescued glycolysis. Glucose intolerance following AZD8055 administration was absent in mice lacking the mTORC2 subunit Rictor in muscle, and in vivo glucose uptake into Rictor-deficient muscle was reduced despite normal Akt activity. Taken together, acute mTOR inhibition is detrimental to glucose homeostasis in part by blocking muscle mTORC2, indicating its importance in muscle metabolism in vivo.

Keywords: Glucose uptake; Glycolysis; Metabolism; Rictor; Skeletal muscle; mTORC2.

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Figures

Supplemental Figure S1
Supplemental Figure S1
Substrate utilization is altered in mice injected with AZD8055. Overnight fasted mice were intraperitoneal-injected with vehicle or AZD8055 (10 mg kg−1); or vehicle or rapamycin (4 mg kg−1) before re-feeding was initiated. (a,c) Carbohydrate (CHO) and (b,d) fat utilizations were calculated based on the respiratory exchange ratio (Figure 1) throughout 6 h of re-feeding (n = 3–4). **p < 0.01 and *p < 0.05 different from vehicle. Data are means ± SEM.
Supplemental Figure S2
Supplemental Figure S2
Total protein expression of AS160, p70S6K1, Akt2, NDRG1, and beta-actin two hours after in vivo AZD8055 injection in insulin responsive tissues. (a) Representative western blots and (b–f) quantitative analysis of indicated total protein immunoblot detection in mouse gastrocnemius, quadriceps, liver, heart, and adipose tissue two hours after vehicle or AZD8055 injection (n = 8; for adipose tissue n = 4). Corresponding phospho-blots are in Figure 2. *p < 0.05 and (*)p < 0.1 different from vehicle within each tissue. Data are means ± SEM.
Supplemental Figure S3
Supplemental Figure S3
Total protein expression of AS160, p70S6K1, Akt2, and NDRG1 following one hour of AZD8055 or rapamycin incubations ex vivo in mouse soleus and EDL muscles. (a,f) Representative western blots for soleus and EDL muscles. (b–e, g–j) quantitative analysis of indicated total protein immunoblot detection in mouse soleus and EDL muscles that were incubated in 0.1% DMSO, 640 nM AZD8055 or 1 μM rapamycin for 60 min. During the last 30 min indicated muscles were stimulated with insulin (60 nM) (n = 7–8). Corresponding phospho-blots are in Figure 5. ¥p < 0.05 main effect of insulin. Data are means ± SEM.
Figure 1
Figure 1
AZD8055 treatment alters fuel substrate utilization in vivo. (A,B) Overnight fasted mice were intraperitoneal-injected with vehicle or AZD8055 (10 mg kg−1); or vehicle or rapamycin (4 mg kg−1) before re-feeding was initiated. Respiratory exchange ratio (RER) was measured (n = 3–4). (C,D) Food eaten throughout 6 h of re-feeding in the AZD8055 and rapamycin experiment (n = 3–4). #p < 0.05 different from vehicle at indicated time points. Data are means ± SEM.
Figure 2
Figure 2
AZD8055 induces whole-body insulin resistance and impairs mTOR signaling in insulin responsive tissues. (A) Overnight fasted mice were intraperitoneal-injected with either vehicle or AZD8055 (10 mg kg−1), re-fed and mixed tail blood obtained at indicated time-points was analyzed for plasma glucose concentration (n = 5); (B,C) 3 h after AZD8055 injection additional blood was sampled for plasma insulin and fatty acids (FA) determinations (n = 4–5). (D–G) Representative western blots and quantitative analysis of mTOR signaling in mouse quadriceps, gastrocnemius, liver, heart, and adipose tissue two hours after vehicle or AZD8055 injection (n = 8; for adipose tissue n = 4). **p < 0.01 different from vehicle at indicated time-point; #p < 0.01 from vehicle; *p < 0.05 different from vehicle within each tissue. Data are means ± SEM.
Figure 3
Figure 3
AZD8055 differentially affects p-AktThr308 and p-AS160 in vivo (A,B) Representative western blots and quantitative analysis of p-AktThr308 and pan-AS160 phosphorylation in mouse quadriceps, gastrocnemius, liver, heart, and adipose tissue two hours after vehicle or AZD8055 (10 mg kg−1) injection (n = 8, for adipose tissue n = 4). *p < 0.05 different from vehicle within each tissue. Data are means ± SEM.
Figure 4
Figure 4
AZD8055, but not rapamycin, reduces muscle glucose uptake ex vivo. (A,B) Mouse soleus and EDL muscles were incubated ex vivo in the presence of 0.1% DMSO, 640 nM AZD8055 or 1 μM rapamycin for 30 min, followed by basal or insulin (60 nM) stimulation. Glucose uptake was measured with 3H-2-deoxyglucose (n = 7–8). *p < 0.05 different from corresponding DMSO. Data are means ± SEM.
Figure 5
Figure 5
AZD8055 impairs insulin-stimulated mTOR, but not AS160 signaling ex vivo. (A) Representative Western blots in soleus and EDL muscle. (B–K) Western blotting quantification of indicated proteins in mouse soleus and EDL muscles that were incubated with either 0.1% DMSO, 640 nM AZD8055 or 1 μM rapamycin for 30 min and basal or insulin-stimulated (60 nM) ex vivo (n = 7–8). *p < 0.05 different from corresponding basal; #p < 0.05 different from corresponding DMSO; ¥p < 0.05 main effect of insulin. Data are means ± SEM.
Figure 6
Figure 6
AZD8055 impairs insulin-stimulated glucose uptake but not GLUT4 translocation in muscle cells, blocks lactate release and induces glucose intolerance by blocking muscle mTORC2. (A,B) Insulin-stimulated 3H-2-deoxyglucose uptake following 50 min of 0.1% DMSO, 500 nM AZD8055 or 100 nM rapamycin pre-incubation in L6-GLUT4myc myoblasts (n = 3–5). (C) Insulin-induced (10 and 100 nM) GLUT4 translocation in L6-GLUT4myc myoblasts following AZD8055 (500 nM) or rapamycin (100 nM) pre-treatment for 50 min (n = 4). (D) Glucose transport into sarcolemmal giant vesicles pretreated with indicated inhibitors and concentrations (n = 3–4). (E) Overnight lactate release into media from SIN1 WT, KO and KO + WT MEFs that were treated with indicated inhibitors and concentrations of serum (n = 3). (F) Glucose concentrations measured from mixed tail blood at indicated time points in Rictor WT and mKO mice that were intraperitoneal-injected with either vehicle or AZD8055 (10 mg kg−1) at t = −120 min and with glucose (2 g/kg) at t = 0 (n = 8–9). (G) Area under the curve determined from (F) (n = 8–9). #p < 0.001 different from corresponding basal; ¤p < 0.05 different from corresponding DMSO; *p < 0.05 different from corresponding vehicle; ￥p < 0.05 difference between indicated groups. Data are means ± SEM.
Figure 7
Figure 7
Rictor deficient muscles have impaired in vivo glucose uptake, elevated glucose and glycogen levels. (A,B) Plasma glucose and insulin levels in Rictor WT and mKO mice just before (0 min) and 15 and 30 min after glucose or saline injection (n = 8–9). (C,D) Glucose clearance and uptake into quadriceps muscle of Rictor WT and mKO mice 30 min after saline or glucose injection (n = 7–8). (E,F) Muscle glycogen and glucose levels from Rictor WT and mKO mice 30 min after saline or glucose injection (n = 7–9). ***p < 0.001 and **p < 0.01 effect of time compared to 0 min; #p < 0.05 effect of genotype within glucose stimulation; (#)p < 0.1 effect of genotype within saline stimulation; ¤p < 0.05 main effect of genotype; *p < 0.05 different from WT glucose stimulation. Data are means ± SEM.
Figure 8
Figure 8
Glucose-induced Akt Ser473 phosphorylation is impaired in Rictor deficient muscle, but downstream Akt signaling is intact. (A) Representative western blots for indicated proteins and phosphorylation sites and (B–J) their densitometry quantification from muscles of Rictor WT and mKO mice 30 min after saline or glucose injection (n = 8–9). *p < 0.05 effect of glucose injection compared to corresponding saline group; (¤)p < 0.1 main effect of glucose injection; #p < 0.05 main effect of genotype. Data are means ± SEM.
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References

    1. Laplante M., Sabatini D. MTOR signaling in growth control and disease. Cell. 2012;149:274–293. - PMC - PubMed
    1. Hagiwara A., Cornu M., Cybulski N., Polak P., Betz C., Trapani F. Hepatic MTORC2 activates glycolysis and lipogenesis through Akt, glucokinase, and SREBP1c. Cell Metabolism. 2012;15:725–738. - PubMed
    1. Kumar A., Lawrence J.C., Jung D.Y., Ko H.J., Keller S.R., Kim J.K. Fat cell-specific ablation of rictor in mice impairs insulin-regulated fat cell and whole-body glucose and lipid metabolism. Diabetes. 2010;59:1397–1406. - PMC - PubMed
    1. Yuan M., Pino E., Wu L., Kacergis M., Soukas A.A. Identification of Akt-independent regulation of hepatic lipogenesis by mammalian target of rapamycin (MTOR) complex 2. Journal of Biological Chemistry. 2012;287:29579–29588. - PMC - PubMed
    1. Kocalis H.E., Hagan S.L., George L., Turney M.K., Siuta M.A., Laryea G.N. Rictor/MTORC2 facilitates central regulation of energy and glucose homeostasis. Molecular Metabolism. 2014;3:394–407. - PMC - PubMed