ULK1 inhibits mTORC1 signaling,promotes multisite Raptor phosphorylation and hinders substrate binding

Protein synthesis and autophagy work as two opposing processes to control cell growth in response to nutrient supply. The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) pathway, which acts as a master regulator to control protein synthesis, has recently been shown to inhibit autophagy by phosphorylating and inactivating ULK1, an autophagy regulatory protein. ULK1 also inhibits phosphorylation of a mTORC1 substrate, S6K1, indicating that a complex signaling interplay exists between mTORC1 and ULK1. Here, we demonstrate that ULK1 induces multisite phosphorylation of Raptor in vivo and in vitro. Using phospho-specific antibodies we identify Ser855 and Ser859 as being strongly phosphorylated by ULK1, with moderate phosphorylation of Ser792 also observed. Interestingly, ULK1 overexpression also increases phosphorylation of Raptor Ser863 and the mTOR autophosphorylation site, Ser2481 in a mTORC1-dependent manner. Despite this evidence for heightened mTORC1 kinase activity following ULK1 overexpresssion, mTORC1-mediated phosphorylation of S6K1 and 4E-BP1 is significantly inhibited. ULK1 expression has no effect on protein-protein interactions between the components of mTORC1, but does reduce the ability of Raptor to bind to the substrate 4E-BP1. Furthermore, shRNA knockdown of ULK1 leads to increased phosphorylation of mTORC1 substrates and decreased phosphorylation of Raptor at Ser859 and Ser792. We propose a new mechanism whereby ULK1 contributes to mTORC1 inhibition through hindrance of substrate docking to Raptor. This is a novel negative feedback loop that occurs upon activation of autophagy to maintain mTORC1 inhibition when nutrient supplies are limiting.     

ULK1 inhibits mTORC1 signaling, promotes multisite Raptor phosphorylation and hinders substrate binding

Protein synthesis and autophagy work as two opposing processes to control cell growth in response to nutrient supply. The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) pathway, which acts as a master regulator to control protein synthesis, has recently been shown to inhibit autophagy by phosphorylating and inactivating ULK1, an autophagy regulatory protein. ULK1 also inhibits phosphorylation of a mTORC1 substrate, S6K1, indicating that a complex signaling interplay exists between mTORC1 and ULK1. Here, we demonstrate that ULK1 induces multisite phosphorylation of Raptor in vivo and in vitro. Using phospho-specific antibodies we identify Ser855 and Ser859 as being strongly phosphorylated by ULK1, with moderate phosphorylation of Ser792 also observed. Interestingly, ULK1 overexpression also increases phosphorylation of Raptor Ser863 and the mTOR autophosphorylation site, Ser2481 in a mTORC1-dependent manner. Despite this evidence for heightened mTORC1 kinase activity following ULK1 overexpresssion, mTORC1-mediated phosphorylation of S6K1 and 4E-BP1 is significantly inhibited. ULK1 expression has no effect on protein-protein interactions between the components of mTORC1, but does reduce the ability of Raptor to bind to the substrate 4E-BP1. Furthermore, shRNA knockdown of ULK1 leads to increased phosphorylation of mTORC1 substrates and decreased phosphorylation of Raptor at Ser859 and Ser792. We propose a new mechanism whereby ULK1 contributes to mTORC1 inhibition through hindrance of substrate docking to Raptor. This is a novel negative feedback loop that occurs upon activation of autophagy to maintain mTORC1 inhibition when nutrient supplies are limiting.     

mTOR Ser-2481 Autophosphorylation Monitors mTORC-specific Catalytic Activity and Clarifies Rapamycin Mechanism of Action

The mammalian target of rapamycin (mTOR) Ser/Thr kinase signals in at least two multiprotein complexes distinguished by their different partners and sensitivities to rapamycin. Acute rapamycin inhibits signaling by mTOR complex 1 (mTORC1) but not mTOR complex 2 (mTORC2), which both promote cell growth, proliferation, and survival. Although mTORC2 regulation remains poorly defined, diverse cellular mitogens activate mTORC1 signaling in a manner that requires sufficient levels of amino acids and cellular energy. Before the identification of distinct mTOR complexes, mTOR was reported to autophosphorylate on Ser-2481 in vivo in a rapamycin- and amino acid-insensitive manner. These results suggested that modulation of mTOR intrinsic catalytic activity does not universally underlie mTOR regulation. Here we re-examine the regulation of mTOR Ser-2481 autophosphorylation (Ser(P)-2481) in vivo by studying mTORC-specific Ser(P)-2481 in mTORC1 and mTORC2, with a primary focus on mTORC1. In contrast to previous work, we find that acute rapamycin and amino acid withdrawal markedly attenuate mTORC1-associated mTOR Ser(P)-2481 in cycling cells. Although insulin stimulates both mTORC1- and mTORC2-associated mTOR Ser(P)-2481 in a phosphatidylinositol 3-kinase-dependent manner, rapamycin acutely inhibits insulin-stimulated mTOR Ser(P)-2481 in mTORC1 but not mTORC2. By interrogating diverse mTORC1 regulatory input, we find that without exception mTORC1-activating signals promote, whereas mTORC1-inhibitory signals decrease mTORC1-associated mTOR Ser(P)-2481. These data suggest that mTORC1- and likely mTORC2-associated mTOR Ser-2481 autophosphorylation directly monitors intrinsic mTORC-specific catalytic activity and reveal that rapamycin inhibits mTORC1 signaling in vivo by reducing mTORC1 catalytic activity.     

Combination of mTOR and EGFR Kinase Inhibitors Blocks mTORC1 and mTORC2 Kinase Activity and Suppresses the Progression of Colorectal Carcinoma

Mammalian target of rapamycin complex 1 and 2 (mTORC1/2) are overactive in colorectal carcinomas; however, the first generation of mTOR inhibitors such as rapamycin have failed to show clinical benefits in treating colorectal carcinoma in part due to their effects only on mTORC1. The second generation of mTOR inhibitors such as PP242 targets mTOR kinase; thus, they are capable of inhibiting both mTORC1 and mTORC2. To examine the therapeutic potential of the mTOR kinase inhibitors, we treated a panel of colorectal carcinoma cell lines with PP242. Western blotting showed that the PP242 inhibition of mTORC2-mediated AKT phosphorylation at Ser 473 (AKT^S473) was transient only in the first few hours of the PP242 treatment. Receptor tyrosine kinase arrays further revealed that PP242 treatment increased the phosphorylated epidermal growth factor receptor (EGFR) at Tyr 1068 (EGFR^T1068). The parallel increase of AKT^S473 and EGFR^T1068 in the cells following PP242 treatment raised the possibility that EGFR phosphorylation might contribute to the PP242 incomplete inhibition of mTORC2. To test this notion, we showed that the combination of PP242 with erlotinib, an EGFR small molecule inhibitor, blocked both mTORC1 and mTORC2 kinase activity. In addition, we showed that the combination treatment inhibited colony formation, blocked cell growth and induced apoptotic cell death. A systemic administration of PP242 and erlotinib resulted in the progression suppression of colorectal carcinoma xenografts in mice. This study suggests that the combination of mTOR kinase and EGFR inhibitors may provide an effective treatment of colorectal carcinoma.     

IRS1 phosphorylation does not mediate mTORC1-induced insulin resistance

Increased mammalian target of rapamycin complex 1 (mTORC1) activity has been suggested to play important roles in development of insulin resistance in obesity. mTORC1 hyperactivity also increases endoplasmic reticulum (ER) stress, which in turn contributes to development of insulin resistance and glucose intolerance. Increased IRS1 phosphorylation at Ser307 in vitro is correlated with mTORC1- and ER stress-induced insulin resistance. This phosphorylation site correlates strongly with impaired insulin receptor signaling in diabetic mice and humans. In contrast, evidence from knock-in mice suggests that phosphorylation of IRS1 at Ser307 is actually required to maintain insulin sensitivity. To study the involvement of IRS1^Ser307 phosphorylation in mTORC1-mediated glucose intolerance and insulin sensitivity in vivo, we investigated the effects of liver specific TSC1 depletion in IRS1^Ser307Ala mice and controls. Our results demonstrate that blockade of IRS1^Ser307 phosphorylation in vivo does not prevent mTORC1-mediated glucose intolerance and insulin resistance.     

In vitro antiglioma action of indomethacin is mediated via AMP-activated protein kinase/mTOR complex 1 signalling pathway

We investigated the role of the intracellular energy-sensing AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway in the in vitro antiglioma effect of the cyclooxygenase (COX) inhibitor indomethacin. Indomethacin was more potent than COX inhibitors diclofenac, naproxen, and ketoprofen in reducing the viability of U251 human glioma cells. Antiglioma effect of the drug was associated with p21 increase and G₂M cell cycle arrest, as well as with oxidative stress, mitochondrial depolarization, caspase activation, and the induction of apoptosis. Indomethacin increased the phosphorylation of AMPK and its targets Raptor and acetyl-CoA carboxylase (ACC), and reduced the phosphorylation of mTOR and mTOR complex 1 (mTORC1) substrates p70S6 kinase and PRAS40 (Ser183). AMPK knockdown by RNA interference, as well as the treatment with the mTORC1 activator leucine, prevented indomethacin-mediated mTORC1 inhibition and cytotoxic action, while AMPK activators metformin and AICAR mimicked the effects of the drug. AMPK activation by indomethacin correlated with intracellular ATP depletion and increase in AMP/ATP ratio, and was apparently independent of COX inhibition or the increase in intracellular calcium. Finally, the toxicity of indomethacin towards primary human glioma cells was associated with the activation of AMPK/Raptor/ACC and subsequent suppression of mTORC1/S6K. By demonstrating the involvement of AMPK/mTORC1 pathway in the antiglioma action of indomethacin, our results support its further exploration in glioma therapy.     

cAMP inhibits mammalian target of rapamycin complex-1 and -2 (mTORC1 and 2) by promoting complex dissociation and inhibiting mTOR kinase activity

cAMP and mTOR signalling pathways control a number of critical cellular processes including metabolism, protein synthesis, proliferation and cell survival and therefore understanding the signalling events which integrate these two signalling pathways is of particular interest. In this study, we show that the pharmacological elevation of [cAMP]_i in mouse embryonic fibroblasts (MEFs) and human embryonic kidney 293 (HEK293) cells inhibits mTORC1 activation via a PKA-dependent mechanism. Although the inhibitory effect of cAMP on mTOR could be mediated by impinging on signalling cascades (i.e. PKB, MAPK and AMPK) that inhibit TSC1/2, an upstream negative regulator of mTORC1, we show that cAMP inhibits mTORC1 in TSC2 knockout (TSC2^−/−) MEFs. We also show that cAMP inhibits insulin and amino acid-stimulated mTORC1 activation independently of Rheb, Rag GTPases, TSC2, PKB, MAPK and AMPK, indicating that cAMP may act independently of known regulatory inputs into mTOR. Moreover, we show that the prolonged elevation in [cAMP]_i can also inhibit mTORC2. We provide evidence that this cAMP-dependent inhibition of mTORC1/2 is caused by the dissociation of mTORC1 and 2 and a reduction in mTOR catalytic activity, as determined by its auto-phosphorylation on Ser2481. Taken together, these results provide an important insight into how cAMP signals to mTOR and down-regulates its activity, which may lead to the identification of novel drug targets to inhibit mTOR that could be used for the treatment and prevention of human diseases such as cancer.     

Dramatic suppression of colorectal cancer cell growth by the dual mTORC1 and mTORC2 inhibitor AZD-2014

Colorectal cancer is a major contributor of cancer-related mortality. The mammalian target or rapamycin (mTOR) signaling is frequently hyper-activated in colorectal cancers, promoting cancer progression and chemo-resistance. In the current study, we investigated the anti-colorectal cancer effect of a novel mTOR complex 1 (mTORC1) and mTORC2 dual inhibitor: AZD-2014. In cultured colorectal cancer cell lines, AZD-2014 significantly inhibited cancer cell growth without inducing significant cell apoptosis. AZD-2014 blocked activation of both mTORC1 (S6K and S6 phosphorylation) and mTORC2 (Akt Ser 473 phosphorylation), and activated autophagy in colorectal cancer cells. Meanwhile, autophagy inhibition by 3-methyaldenine (3-MA) and hydroxychloroquine, as well as by siRNA knocking down of Beclin-1 or ATG-7, inhibited AZD-2014-induced cytotoxicity, while the apoptosis inhibitor had no rescue effect. In vivo, AZD-2014 oral administration significantly inhibited the growth of HT-29 cell xenograft in SCID mice, and the mice survival was dramatically improved. At the same time, in xenografted tumors administrated with AZD-2014, the activation of mTORC1 and mTORC2 were largely inhibited, and autophagic markers were significantly increased. Thus, AZD-2014 inhibits colorectal cancer cell growth both in vivo and in vitro. Our results suggest that AZD-2014 may be further investigated for colorectal cancer therapy in clinical trials.     

Feedback loops blockade potentiates apoptosis induction and antitumor activity of a novel AKT inhibitor DC120 in human liver cancer

The serine/threonine kinase AKT is generally accepted as a promising anticancer therapeutic target. However, the relief of feedback inhibition and enhancement of other survival pathways often attenuate the anticancer effects of AKT inhibitors. These compensatory mechanisms are very complicated and remain poorly understood. In the present study, we found a novel 2-pyrimidyl-5-amidothiazole compound, DC120, as an ATP competitive AKT kinase inhibitor that suppressed proliferation and induced apoptosis in liver cancer cells both in vitro and in vivo. DC120 blocked the phosphorylation of downstream molecules in the AKT signal pathway in dose- and time-dependent manners both in vitro and in vivo. However, unexpectedly, DC120 activated mammalian target of rapamycin complex 1 (mTORC1) pathway that was suggested by increased phosphorylation of 70KD ribosomal protein S6 kinase (P70S6K) and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1). The activated mTORC1 signal was because of increase of intracellular Ca²⁺ via Ca²⁺/calmodulin (CaM)/ signaling to human vacuolar protein sorting 34 (hVps34) upon AKT inhibition. Meanwhile, DC120 attenuated the inhibitory effect of AKT on CRAF by decreasing phosphorylation of CRAF at Ser259 and thus activated the mitogen-activated protein kinase (MAPK) pathway. The activation of the mTORC1 and MAPK pathways by DC120 was not mutually dependent, and the combination of DC120 with mTORC1 inhibitor and/or MEK inhibitor induced significant apoptosis and growth inhibition both in vitro and in vivo. Taken together, the combination of AKT, mTORC1 and/or MEK inhibitors would be a promising therapeutic strategy for liver cancer treatment.     

AKAP13 couples GPCR signaling to mTORC1 inhibition

The mammalian target of rapamycin complex 1 (mTORC1) senses multiple stimuli to regulate anabolic and catabolic processes. mTORC1 is typically hyperactivated in multiple human diseases such as cancer and type 2 diabetes. Extensive research has focused on signaling pathways that can activate mTORC1 such as growth factors and amino acids. However, less is known about signaling cues that can directly inhibit mTORC1 activity. Here, we identify A-kinase anchoring protein 13 (AKAP13) as an mTORC1 binding protein, and a crucial regulator of mTORC1 inhibition by G-protein coupled receptor (GPCR) signaling. GPCRs paired to Gα_s proteins increase cyclic adenosine 3’5’ monophosphate (cAMP) to activate protein kinase A (PKA). Mechanistically, AKAP13 acts as a scaffold for PKA and mTORC1, where PKA inhibits mTORC1 through the phosphorylation of Raptor on Ser 791. Importantly, AKAP13 mediates mTORC1-induced cell proliferation, cell size, and colony formation. AKAP13 expression correlates with mTORC1 activation and overall lung adenocarcinoma patient survival, as well as lung cancer tumor growth in vivo. Our study identifies AKAP13 as an important player in mTORC1 inhibition by GPCRs, and targeting this pathway may be beneficial for human diseases with hyperactivated mTORC1.     

Mesenchymal stromal cells regulate THP-1–differentiated macrophage cytokine production by activating Akt/mammalian target of rapamycin complex 1 pathway

Background aimsThe Akt/mammalian target of rapamycin (mTOR) pathway in macrophages converges inflammatory and metabolic signals from multiple receptors to regulate a cell's survival, metabolism and activation. Although mesenchymal stromal cells (MSCs) are well known to modulate macrophage activation, the effects of MSCs on the Akt/mTOR pathway in macrophages have not been elucidated.MethodsWe herein investigated whether MSCs affect the Akt/mTOR complex 1 (mTORC1) pathway to regulate macrophage polarization.ResultsResults showed that human bone marrow–derived MSCs induced activation of Akt and its downstream mTORC1 signaling in THP-1–differentiated macrophages in a p62/sequestosome 1–independent manner. Inhibition of Akt or mTORC1 attenuated the effects of MSCs on the suppression of tumor necrosis factor-α and interleukin-12 production and the promotion of interleukin-10 and tumor growth factor-β1 in macrophages stimulated by lipopolysaccharide/ATP. Conversely, activation of Akt or mTORC1 reproduced and potentiated MSC effects on macrophage cytokine production. MSCs with cyclooxygenase-2 knockdown, however, failed to activate the Akt/mTORC1 signaling in macrophages and were less effective in the modulation of macrophage cytokine production than control MSCs.ConclusionsThese data demonstrate that MSCs control THP-1–differentiated macrophage activation at least partly through upregulation of the Akt/mTORC1 signaling in a cyclooxygenase-2–dependent manner.     

RhoA modulates signaling through the mechanistic target of rapamycin complex 1 (mTORC1) in mammalian cells

The mechanistic target of rapamycin (mTOR) in complex 1 (mTORC1) pathway integrates signals generated by hormones and nutrients to control cell growth and metabolism. The activation state of mTORC1 is regulated by a variety of GTPases including Rheb and Rags. Recently, Rho1, the yeast ortholog of RhoA, was shown to interact directly with TORC1 and repress its activation state in yeast. Thus, the purpose of the present study was to test the hypothesis that the RhoA GTPase modulates signaling through mTORC1 in mammalian cells. In support of this hypothesis, exogenous overexpression of either wild type or constitutively active (ca)RhoA repressed mTORC1 signaling as assessed by phosphorylation of p70S6K1 (Thr389), 4E-BP1 (Ser65) and ULK1 (Ser757). Additionally, RhoA·GTP repressed phosphorylation of mTORC1-associated mTOR (Ser2481). The RhoA·GTP mediated repression of mTORC1 signaling occurred independent of insulin or leucine induced stimulation. In contrast to the action of Rho1 in yeast, no evidence was found to support a direct interaction of RhoA·GTP with mTORC1. Instead, expression of caRheb, but not caRags, was able to rescue the RhoA·GTP mediated repression of mTORC1 suggesting RhoA functions upstream of Rheb to repress mTORC1 activity. Consistent with this suggestion, RhoA·GTP repressed phosphorylation of TSC2 (Ser939), PRAS40 (Thr246), Akt (Ser473), and mTORC2-associated mTOR (Ser2481). Overall, the results support a model in which RhoA·GTP represses mTORC1 signaling upstream of Akt and mTORC2.     

Advances in understanding the mechanisms of evasive and innate resistance to mTOR inhibition in cancer cells

The development of drug-resistance by neoplastic cells is recognized as a major cause of targeted therapy failure and disease progression. The mechanistic (previously mammalian) target of rapamycin (mTOR) is a highly conserved Ser/Thr kinase that acts as the catalytic subunit of two structurally and functionally distinct large multiprotein complexes, referred to as mTOR complex 1 (mTORC1) and mTORC2. Both mTORC1 and mTORC2 play key roles in a variety of healthy cell types/tissues by regulating physiological anabolic and catabolic processes in response to external cues. However, a body of evidence identified aberrant activation of mTOR signaling as a common event in many human tumors. Therefore, mTOR is an attractive target for therapeutic targeting in cancer and this fact has driven the development of numerous mTOR inhibitors, several of which have progressed to clinical trials. Nevertheless, mTOR inhibitors have met with a very limited success as anticancer therapeutics. Among other reasons, this failure was initially ascribed to the activation of several compensatory signaling pathways that dampen the efficacy of mTOR inhibitors. The discovery of these regulatory feedback mechanisms greatly contributed to a better understanding of cancer cell resistance to mTOR targeting agents. However, over the last few years, other mechanisms of resistance have emerged, including epigenetic alterations, compensatory metabolism rewiring and the occurrence of mTOR mutations. In this article, we provide the reader with an updated overview of the mechanisms that could explain resistance of cancer cells to the various classes of mTOR inhibitors.     

KIF2C: a novel link between Wnt/β-catenin and mTORC1 signaling in the pathogenesis of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and is the fourth-leading cause of cancer-related deaths worldwide. HCC is refractory to many standard cancer treatments and the prognosis is often poor, highlighting a pressing need to identify biomarkers of aggressiveness and potential targets for future treatments. Kinesin family member 2C (KIF2C) is reported to be highly expressed in several human tumors. Nevertheless, the molecular mechanisms underlying the role of KIF2C in tumor development and progression have not been investigated. In this study, we found that KIF2C expression was significantly upregulated in HCC, and that KIF2C up-regulation was associated with a poor prognosis. Utilizing both gain and loss of function assays, we showed that KIF2C promoted HCC cell proliferation, migration, invasion, and metastasis both in vitro and in vivo. Mechanistically, we identified TBC1D7 as a binding partner of KIF2C, and this interaction disrupts the formation of the TSC complex, resulting in the enhancement of mammalian target of rapamycin complex1 (mTORC1) signal transduction. Additionally, we found that KIF2C is a direct target of the Wnt/β-catenin pathway, and acts as a key factor in mediating the crosstalk between Wnt/β-catenin and mTORC1 signaling. Thus, the results of our study establish a link between Wnt/β-catenin and mTORC1 signaling, which highlights the potential of KIF2C as a therapeutic target for the treatment of HCC.Electronic supplementary materialThe online version of this article (10.1007/s13238-020-00766-y) contains supplementary material, which is available to authorized users.     

Uroguanylin modulates (Na+ + K+)ATPase in a proximal tubule cell line: Interactions among the cGMP/protein kinase G,cAMP/protein kinase A,and mTOR pathways

BackgroundThe natriuretic effect of uroguanylin (UGN) involves reduction of proximal tubule (PT) sodium reabsorption. However, the target sodium transporters as well as the molecular mechanisms involved in these processes remain poorly understood.MethodsTo address the effects of UGN on PT (Na⁺ + K⁺)ATPase and the signal transduction pathways involved in this effect, we used LLC-PK1 cells. The effects of UGN were determined through ouabain-sensitive ATP hydrolysis and immunoblotting assays during different experimental conditions.ResultsWe observed that UGN triggers cGMP/PKG and cAMP/PKA pathways in a sequential way. The activation of PKA leads to the inhibition of mTORC2 activity, PKB phosphorylation at S473, PKB activity and, consequently, a decrease in the mTORC1/S6K pathway. The final effects are decreased expression of the α1 subunit of (Na⁺ + K⁺)ATPase and inhibition of enzyme activity.ConclusionsThese results suggest that the molecular mechanism of action of UGN on sodium reabsorption in PT cells is more complex than previously thought. We propose that PKG-dependent activation of PKA leads to the inhibition of the mTORC2/PKB/mTORC1/S6K pathway, an important signaling pathway involved in the maintenance of the PT sodium pump expression and activity.General significanceThe current results expand our understanding of the signal transduction pathways involved in the overall effect of UGN on renal sodium excretion.     

Discovery of novel leucyladenylate sulfamate surrogates as leucyl-tRNA synthetase (LRS)-targeted mammalian target of rapamycin complex 1 (mTORC1) inhibitors

According to recent studies, leucyl-tRNA synthetase (LRS) acts as a leucine sensor and modulates the activation of the mammalian target of rapamycin complex 1 (mTORC1) activation. Because overactive mTORC1 is associated with several diseases, including colon cancer, LRS-targeted mTORC1 inhibitors represent a potential option for anti-cancer therapy. In this work, we developed a series of simplified leucyladenylate sulfamate analogues that contain the N-(3-chloro-4-fluorophenyl)quinazolin-4-amine moiety to replace the adenine group. We identified several compounds with comparable activity to previously reported inhibitors and exhibited selective mTORC1 inhibition and anti-cancer activity. This study further supports the hypothesis that LRS is a promising target to modulate the mTORC1 pathway.     

Rapamycin does not improve insulin sensitivity despite elevated mammalian target of rapamycin complex 1 activity in muscles of ob/ob mice

Studies of cultured cells have indicated that the mammalian target of rapamycin complex 1 (mTORC1) mediates the development of insulin resistance. Because a role for mTORC1 in the development of skeletal muscle insulin resistance has not been established, we studied mTORC1 activity in skeletal muscles of ob/ob (OB) mice and wild-type (WT) mice. In vivo insulin action was assessed in muscles of mice 15 min following an intraperitoneal injection of insulin or an equivalent volume of saline. In the basal state, the phosphorylation of S6K on Thr(389), mTOR on Ser(2448), and PRAS40 on Thr(246) were increased significantly in muscles from OB mice compared with WT mice. The increase in basal mTORC1 signaling was associated with an increase in basal PKB phosphorylation on Thr(308) and Ser(473). In the insulin-stimulated state, no differences existed in the phosphorylation of S6K on Thr(389), but PKB phosphorylation on Thr(308) and Ser(473) was significantly reduced in muscles of OB compared with WT mice. Despite elevated mTORC1 activity in OB mice, rapamycin treatment did not improve either glucose tolerance or insulin tolerance. These results indicate that the insulin resistance of OB mice is mediated, in part, by factors other than mTORC1.     

mTOR Signaling: The Interface Linking Cellular Metabolism and Hepatitis B Virus Replication

Virologica Sinica - Mammalian target of rapamycin (mTOR) is a conserved Ser/Thr kinase that includes mTOR complex (mTORC) 1 and mTORC2. The mTOR pathway is activated in viral hepatitis, including...