Muscle-specific deletion of rictor impairs insulin-stimulated glucose transport and enhances Basal glycogen synthase activity

Abstract

Rictor is an essential component of mTOR (mammalian target of rapamycin) complex 2 (mTORC2), a kinase complex that phosphorylates Akt at Ser473 upon activation of phosphatidylinositol 3-kinase (PI-3 kinase). Since little is known about the role of either rictor or mTORC2 in PI-3 kinase-mediated physiological processes in adult animals, we generated muscle-specific rictor knockout mice. Muscle from male rictor knockout mice exhibited decreased insulin-stimulated glucose uptake, and the mice showed glucose intolerance. In muscle lacking rictor, the phosphorylation of Akt at Ser473 was reduced dramatically in response to insulin. Furthermore, insulin-stimulated phosphorylation of the Akt substrate AS160 at Thr642 was reduced in rictor knockout muscle, indicating a defect in insulin signaling to stimulate glucose transport. However, the phosphorylation of Akt at Thr308 was normal and sufficient to mediate the phosphorylation of glycogen synthase kinase 3 (GSK-3). Basal glycogen synthase activity in muscle lacking rictor was increased to that of insulin-stimulated controls. Consistent with this, we observed a decrease in basal levels of phosphorylated glycogen synthase at a GSK-3/protein phosphatase 1 (PP1)-regulated site in rictor knockout muscle. This change in glycogen synthase phosphorylation was associated with an increase in the catalytic activity of glycogen-associated PP1 but not increased GSK-3 inactivation. Thus, rictor in muscle tissue contributes to glucose homeostasis by positively regulating insulin-stimulated glucose uptake and negatively regulating basal glycogen synthase activity.

FIG. 1.

Analysis of rictor gene expression in MRic^−/− mice. (A) Total RNA extracted from skeletal muscle (muscle) and adipose tissue (fat) was analyzed by reverse transcriptase PCR (lanes 2 to 5). A 1-kb DNA ladder (Invitrogen) was used as the molecular mass marker (lane 1). (B) Tissue extracts prepared from skeletal muscle and adipose tissue were subjected to SDS-PAGE and were immunoblotted with anti-rictor antibodies (top). The mTOR immunoblot shown in the bottom panel served as the loading control in these experiments.

FIG. 2.

In vivo insulin-stimulated phosphorylation of Akt at Ser473 was abolished but phosphorylation of Akt at Thr308 was normal in MRic^−/− muscles. (A) Muscle extracts (25 to 100 μg total protein) prepared from mice intraperitoneally injected with saline (−) or insulin (+) were subjected to SDS-PAGE and immunoblotted with phospho-specific antibodies to sites in Akt (P-S473 and P-T308 [P-S, phospho-Ser; P-T, phospho-Thr]) and total Akt antibody. The quantification data (means ± SE) presented are for immunoblots of (B) P-S473 Akt (n = 5 per group; *, P < 0.03; **, P < 0.002) and (C) P-T308 Akt (n = 4 per group; *, P < 0.04; **, P < 0.0006). (D) Protein extracts from muscle were immunoblotted with phospho-specific antibodies to S6K1 (P-T389), FoxO1 (P-T24), and GSK-3 α/β (P-S21/9) as well as with antibodies to total S6K1, actin, and GSK-3α. (E to G) Data shown (means ± SE) reflect band intensity after normalization to the total detected protein or to the level of actin for (E) P-T389 S6K1 (n = 4; *, P < 0.01; **, P < 0.0007; N.S [not significant], P < 0.2), (F) P-T24 FoxO1 (n = 5; *, P < 0.01; **, P < 0.0009; ***, P < 0.0002), and (G) P-S9 GSK-3β (n = 4; *, P < 0.003; **, P < 0.001). arb. units, arbitrary units.

FIG. 3.

Loss of rictor expression enhances basal glycogen synthase activity in muscle. (A) In vivo insulin-stimulated glycogen synthase activity in muscles of MRic^+/+ and MRic^−/− mice. Glycogen synthase activity was measured using hind limb muscle extracts prepared from saline (−)- or insulin (+)-injected mice in the absence and presence of G-6P (10 mM). The activity ratios are the activity measured in the absence of G-6P divided by that in the presence of G-6P (total activity) (means ± SE, n = 5 to 7 per group; *, P < 0.004; **, P < 0.00003). (B) Muscle extracts used for the glycogen synthase assay were resolved by SDS-PAGE and immunoblotted with phospho-specific glycogen synthase (P-S641 GS) antibodies and total glycogen synthase (GS) antibodies (top). Quantification of P-S641 GS immunoblots is shown in the bottom panel (n = 5 per group).

FIG. 4.

Ex vivo-incubated MRic^−/− muscle shows insulin-responsive glycogen synthase activity even with increased basal glycogen synthase activity. EDL muscles were incubated in Krebs-Henseleit buffer without (−) or with (+) insulin (100 mU/ml) for 30 min. The rapamycin-treated muscles were first incubated with rapamycin (200 nM final concentration) alone and then with rapamycin and insulin for 30 min. (A) Glycogen synthase activity was measured in EDL as described in the legend of Fig. 3A (means ± SE, n = 4 per group; *, P < 0.025; **, P < 0.006). (B) Protein extracts prepared from EDL muscles were subjected to SDS-PAGE and immunoblotted with phospho-specific antibodies to Akt, S6K1, GSK-3β, and glycogen synthase and antibodies to actin and total glycogen synthase. (C) Quantification of GSK-3β Ser9 phosphorylation (means ± SE, n = 4; *, P < 0.01) after normalization to actin. arb. units, arbitrary units.

FIG. 5.

Rictor ablation in muscle decreases insulin-stimulated glucose uptake, and MRic^−/− mice exhibit impaired glucose tolerance. (A) EDL muscles were isolated and incubated with (+) or without (−) insulin (20 mU/ml) in medium containing 0.5 mM 2-deoxy-[1,2-³H]glucose for 15 min. The results presented are means ± SE (n = 4 to 6; *, P < 0.04; **, P < 0.007; ***, P < 0.0005). (B) Total membranes prepared from hind limb muscle homogenates of MRic^+/+ and MRic^−/− mice were subjected to SDS-PAGE and immunoblotted with antibodies to GLUT4 and GLUT1 proteins. (C) Glucose tolerance tests were performed with six male MRic^+/+ (•) and seven male MRic^−/− (○) mice. Blood glucose was measured at indicated times after an intraperitoneal injection of glucose (1 mg/g body weight) (*, P < 0.004). (D) For insulin tolerance tests (male MRic^+/+ [•] and male MRic^−/− [○] mice), blood glucose levels were measured in fed mice at indicated times after an intraperitoneal insulin injection (0.75U/kg body weight). The data presented are means ± SE of four to seven mice per group.

FIG. 6.

AS160 Thr642 phosphorylation in MRic^−/− muscle. Muscle extracts prepared from saline (−)- and insulin (+)-injected mice of both genotypes were subjected to SDS-PAGE and immunoblotted with phospho-specific antibody to Thr642 in AS160 and total AS160 antibody (I and II). Data (means ± SE) presented are the quantification of P-T642 AS160 immunoblots after normalizing for the level of total AS160 (right) (n = 6; *, P < 0.025; **, P < 0.008; ***, P < 0.000001). arb. units, arbitrary units.

FIG. 7.

Increased PP1 activity in muscle glycogen pellets from MRic^−/− mice. PP1 activity in muscle extract (A) and glycogen pellet (C) from MRic^+/+ and MRic^−/− mice, measured in the presence of 4 nM okadaic acid using ³²P-labeled glycogen phosphorylase as the substrate. (A) The muscle extracts (3 μg total protein) prepared from overnight-fasted mice were tested for PP1 activity. (B) The muscle extracts were immunoblotted for R_GL, PP1α, and PP1β. (C) PP1 activity determined in glycogen pellets prepared from muscles of MRic^+/+ and MRic^−/− mice injected with saline (−) or insulin (+) (means ± SE, n = 4 to 5 mice per group; *, P < 0.001). (D) Glycogen pellets were immunoblotted for R_GL, PP1α, PP1β, P-S641 GS, and GS. (E) Quantification of P-Ser641 GS immunoblots of glycogen pellet (means ± SE, n = 4 per group; *, P < 0.01; **, P < 0.005). arb. units, arbitrary units. (F) GSK-3 activity was measured in crude muscle extract in the absence and presence of 200 mM lithium chloride with a glycogen synthase peptide as the substrate (means ± SE, n = 3 per group; *, P < 0.009; **, P < 0.0004).