Amino Acids Enhance Polyubiquitination of Rheb and Its Binding to mTORC1 by Blocking Lysosomal ATXN3 Deubiquitinase Activity.

Amino-acid-induced lysosomal mechanistic target of rapamycin complex 1 (mTORC1) localization through the Rag GTPases is a critical step for its activation by Rheb GTPase. However, how the mTORC1 interacts with Rheb on the lysosome remains elusive. We report that amino acids enhance the polyubiquitination of Rheb (Ub-Rheb), which shows a strong binding preference for mTORC1 and supports its activation, while the Ub-Rheb is subjected to subsequent degradation. Mechanistically, we identified ATXN3 as a Ub-Rheb deubiquitinase whose lysosomal localization is blocked by active Rag heterodimer in response to amino acid stimulation. Consistently, cells lacking functional Rag heterodimer on the lysosome accumulate Ub-Rheb, and blockade of its degradation instigates robust lysosomal mTORC1 localization and its activation without the Ragulator-Rag system. Thus, polyubiquitination of Rheb is an important post-translational modification, which facilitates the binding of mTORC1 to Rheb on the lysosome and is another crosstalk between the amino acid and growth factor signaling for mTORC1 activation.

Regulatory T Cell Migration Is Dependent on Glucokinase-Mediated Glycolysis.

Migration of activated regulatory T (Treg) cells to inflamed tissue is crucial for their immune-modulatory function. While metabolic reprogramming during Treg cell differentiation has been extensively studied, the bioenergetics of Treg cell trafficking remains undefined. We have investigated the metabolic demands of migrating Treg cells in vitro and in vivo. We show that glycolysis was instrumental for their migration and was initiated by pro-migratory stimuli via a PI3K-mTORC2-mediated pathway culminating in induction of the enzyme glucokinase (GCK). Subsequently, GCK promoted cytoskeletal rearrangements by associating with actin. Treg cells lacking this pathway were functionally suppressive but failed to migrate to skin allografts and inhibit rejection. Similarly, human carriers of a loss-of-function GCK regulatory protein gene-leading to increased GCK activity-had reduced numbers of circulating Treg cells. These cells displayed enhanced migratory activity but similar suppressive function, while conventional T cells were unaffected. Thus, GCK-dependent glycolysis regulates Treg cell migration.

Translational Control through Differential Ribosome Pausing during Amino Acid Limitation in Mammalian Cells.

Limitation for amino acids is thought to regulate translation in mammalian cells primarily by signaling through the kinases mTORC1 and GCN2. We find that a selective loss of arginine tRNA charging during limitation for arginine regulates translation through ribosome pausing at two of six arginine codons. Surprisingly, limitation for leucine, an essential and abundant amino acid in protein, results in little or no ribosome pausing. Chemical and genetic perturbation of mTORC1 and GCN2 signaling revealed that their robust response to leucine limitation prevents ribosome pausing, while an insufficient response to arginine limitation leads to loss of tRNA charging and ribosome pausing. Ribosome pausing decreases protein production and triggers premature ribosome termination without reducing mRNA levels. Together, our results suggest that amino acids that are not optimally sensed by the mTORC1 and GCN2 pathways still regulate translation through an evolutionarily conserved mechanism based on codon-specific ribosome pausing.

Distinct signaling by insulin and IGF-1 receptors and their extra- and intracellular domains.

Insulin and insulin-like growth factor 1 (IGF-1) receptors share many downstream signaling pathways but have unique biological effects. To define the molecular signals contributing to these distinct activities, we performed global phosphoproteomics on cells expressing either insulin receptor (IR), IGF-1 receptor (IGF1R), or chimeric IR-IGF1R receptors. We show that IR preferentially stimulates phosphorylations associated with mammalian target of rapamycin complex 1 (mTORC1) and Akt pathways, whereas IGF1R preferentially stimulates phosphorylations on proteins associated with the Ras homolog family of guanosine triphosphate hydrolases (Rho GTPases), and cell cycle progression. There were also major differences in the phosphoproteome between cells expressing IR versus IGF1R in the unstimulated state, including phosphorylation of proteins involved in membrane trafficking, chromatin remodeling, and cell cycle. In cells expressing chimeric IR-IGF1R receptors, these differences in signaling could be mapped to contributions of both the extra- and intracellular domains of these receptors. Thus, despite their high homology, IR and IGF1R preferentially regulate distinct networks of phosphorylation in both the basal and stimulated states, allowing for the unique effects of these hormones on organismal function.

Targeting a cytokine checkpoint enhances the fitness of armored cord blood CAR-NK cells.

Immune checkpoint therapy has resulted in remarkable improvements in the outcome for certain cancers. To broaden the clinical impact of checkpoint targeting, we devised a strategy that couples targeting of the cytokine-inducible Src homology 2-containing (CIS) protein, a key negative regulator of interleukin 15 (IL-15) signaling, with fourth-generation "armored" chimeric antigen receptor (CAR) engineering of cord blood-derived natural killer (NK) cells. This combined strategy boosted NK cell effector function through enhancing the Akt/mTORC1 axis and c-MYC signaling, resulting in increased aerobic glycolysis. When tested in a lymphoma mouse model, this combined approach improved NK cell antitumor activity more than either alteration alone, eradicating lymphoma xenografts without signs of any measurable toxicity. We conclude that targeting a cytokine checkpoint further enhances the antitumor activity of IL-15-secreting armored CAR-NK cells by promoting their metabolic fitness and antitumor activity. This combined approach represents a promising milestone in the development of the next generation of NK cells for cancer immunotherapy.

Role of the Mammalian Target of Rapamycin Pathway in Liver Cancer: From Molecular Genetics to Targeted Therapies.

Primary liver cancers, including hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA), are highly lethal tumors, with high worldwide frequency and few effective treatment options. The mammalian target of rapamycin (mTOR) complex is a central regulator of cell growth and metabolism that integrates inputs from amino acids, nutrients, and extracellular signals. The mTOR protein is incorporated into two distinct complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Specifically, mTORC1 regulates protein synthesis, glucose and lipid metabolism, and autophagy, whereas mTORC2 promotes liver tumorigenesis through modulating the adenine/cytosine/guanine family of serine/threonine kinases, especially the protein kinase B proteins. In human HCC and iCCA samples, genomics analyses have revealed the frequent deregulation of the mTOR complexes. Both in vitro and in vivo studies have demonstrated the key role of mTORC1 and mTORC2 in liver-tumor development and progression. The first-generation mTOR inhibitors have been evaluated for effectiveness in liver-tumor treatment and have provided unsatisfactory results. Current research efforts are devoted to generating more efficacious mTOR inhibitors and identifying biomarkers for patient selection as well as for combination therapies. Here, we provide a comprehensive review of the mechanisms leading to a deregulated mTOR signaling cascade in liver cancers, the mechanisms whereby the mTOR pathway contributes to HCC and iCCA molecular pathogenesis, the therapeutic strategies, and the challenges to effectively inhibit mTOR in liver-cancer treatment. Conclusion: Deregulated mTOR signaling significantly contributes to HCC and iCCA molecular pathogenesis. mTOR inhibitors, presumably administered in association with other drugs, might be effective against subsets of human liver tumors.

Muscle follistatin gene delivery increases muscle protein synthesis independent of periodical physical inactivity and fasting.

Blocking of myostatin and activins effectively counteracts muscle atrophy. However, the potential interaction with physical inactivity and fasting in the regulation of muscle protein synthesis is poorly understood. We used blockade of myostatin and activins by recombinant adeno-associated virus (rAAV)-mediated follistatin (FS288) overexpression in mouse tibialis anterior muscle. To investigate the effects on muscle protein synthesis, muscles were collected 7 days after rAAV-injection in the nighttime or in the daytime representing high and low levels of activity and feeding, respectively, or after overnight fasting, refeeding, or ad libitum feeding. Muscle protein synthesis was increased by FS288 independent of the time of the day or the feeding status. However, the activation of mTORC1 signaling by FS288 was attenuated in the daytime and by overnight fasting. FS288 also increased the amount of mTOR colocalized with lysosomes, but did not alter their localization toward the sarcolemma. This study shows that FS288 gene delivery increases muscle protein synthesis largely independent of diurnal fluctuations in physical activity and food intake or feeding status, overriding the physiological signals. This is important for eg cachectic and sarcopenic patients with reduced physical activity and appetite. The FS288-induced increase in mTORC1 signaling and protein synthesis may be in part driven by increased amount of mTOR colocalized with lysosomes, but not by their localization toward sarcolemma.

Blockade of mTORC1-NOX signaling pathway inhibits TGF-ß1-mediated senescence-like structural alterations of the retinal pigment epithelium.

The retinal pigment epithelium (RPE) undergoes characteristic structural changes and epithelial-mesenchymal transition (EMT) during normal aging, which are exacerbated in age-related macular degeneration (AMD). Although the pathogenic mechanisms of aging and AMD remain unclear, transforming growth factor-ß1 (TGF-ß1) is known to induce oxidative stress, morphometric changes, and EMT as a senescence-promoting factor. In this study, we examined whether intravitreal injection of TGF-ß1 into the mouse eye elicits senescence-like morphological alterations in the RPE and if this can be prevented by suppressing mammalian target of rapamycin complex 1 (mTORC1) or NADPH oxidase (NOX) signaling. We verified that intravitreal TGF-ß1-induced stress fiber formation and EMT in RPE cells, along with age-associated morphometric changes, including increased variation in cell size and reduced cell density. In RPE cells, exogenous TGF-ß1 increased endogenous expression of TGF-ß1 and upregulated Smad3-ERK1/2-mTORC1 signaling, increasing reactive oxygen species (ROS) production and EMT. We demonstrated that inhibition of the mTORC1-NOX4 pathway by pretreatment with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an activator of AMP-dependent protein kinase, or GKT137831, a NOX1/4 inhibitor, decreased ROS generation, prevented stress fiber formation, attenuated EMT, and improved the regularity of the RPE structure in vitro and in vivo. These results suggest that intravitreal TGF-ß1 injection could be used as a screening model to investigate the aging-related structural and functional changes to the RPE. Furthermore, the regulation of TGF-ß-mTORC1-NOX signaling could be a potential therapeutic target for reducing pathogenic alterations in aged RPE and AMD.

IRF3 and IRF7 contribute to diesel exhaust particles-induced pulmonary inflammation by mediating mTORC1 activation and restraining autophagy in mice.

Exposure to diesel exhaust particles (DEPs) is associated with acute inflammatory responses in the lung and exacerbation of respiratory diseases. However, the mechanism by which DEPs trigger the inflammatory responses remains unclear. Here, we demonstrated that the IFN response factors IRF3 and IRF7 played pivotal roles in DEP-induced pulmonary inflammation. DEPs could not directly induce inflammatory cytokine expression in mouse cells, whereas DEPs triggered autophagy both in vitro and in vivo. The DEP-induced autophagy was augmented in the absence of IRF3 and IRF7, but not in the absence of IFNAR. The expression of Raptor was induced by IRF3 and IRF7 in response to DEPs treatment. Furthermore, administration of the mechanistic target of rapamycin (mTOR) inhibitor alleviated the inflammatory responses in the lung during DEP exposure. Our findings define an IFNAR-independent role of increased autophagy in the absence of IRF3 and IRF7 during pulmonary DEP exposure, and provide the basis to develop new therapeutic approaches to counteract the adverse effects of DEPs and possibly other ambient particulate matters.

Mitochondrial General Control of Amino Acid Synthesis 5 Like 1 Regulates Glutaminolysis, Mammalian Target of Rapamycin Complex 1 Activity, and Murine Liver Regeneration.

BACKGROUND AND AIMS: The regenerative capacity of the liver plays a protective role against hepatotoxins and impaired regeneration exacerbates liver dysfunction in nonalcoholic fatty liver disease (NAFLD). Mitochondrial bioenergetic and -synthetic functions are important contributory factors in hepatic regeneration, and the control of mitochondrial protein acetylation is implicated in the mitochondrial susceptibility to liver stressors. Here, we evaluated the role of general control of amino acid synthesis 5 like 1 (GCN5L1), a mediator of mitochondrial metabolism and acetylation, in modulating murine liver regeneration (LR) in response to acute CCl4 -induced hepatotoxicity. APPROACH AND RESULTS: Initial metabolomic screening found that liver GCN5L1 knockout (LKO) mice have augmented glutaminolysis. Absence of GCN5L1 modified enzyme activity of liver-enriched glutaminase enzyme (glutaminase 2; GLS2), and GCN5L1 levels modulated GLS2 oligomerization and acetylation. This metabolic remodeling resulted in the elevation of α-ketoglutarate levels, which are known to activate mammalian target of rapamycin complex 1 (mTORC1). This signaling pathway was induced with increased phosphorylation of S6 kinase in LKO hepatocytes, and inhibition of glutaminolysis reversed aberrant mTORC1 signaling. At the same time, glutaminolysis, activity of GLS2, and activation of mTORC1 signaling were reversed by the genetic reintroduction of the mitochondrial isoform of GCN5L1 into LKO primary hepatocytes. Finally, LKO mice had a more robust regenerative capacity in response to CCl4 hepatoxicity, and this response was blunted by both the mTORC1 inhibitor, rapamycin, and by pharmacological blunting of glutaminolysis. CONCLUSIONS: These data point to a central role of glutaminolysis in modulating the regenerative capacity in the liver. Furthermore, inhibition of mitochondrial GCN5L1 to augment LR may be a useful strategy in disease states linked to hepatotoxicity.

MTOR-initiated metabolic switch and degeneration in the retinal pigment epithelium.

The retinal pigment epithelium (RPE) is a particularly vulnerable tissue to age-dependent degeneration. Over the life span, the RPE develops an expanded endo-lysosomal compartment to maintain the high efficiency of phagocytosis and degradation of photoreceptor outer segments (POS) necessary for photoreceptor survival. As the assembly and activation of the mechanistic target of rapamycin complex 1 (mTORC1) occur on the lysosome surface, increased lysosome mass with aging leads to higher mTORC1 activity. The functional consequences of hyperactive mTORC1 in the RPE are unclear. In the current study, we used integrated high-resolution metabolomic and genomic approaches to examine mice with RPE-specific deletion of the tuberous sclerosis 1 (Tsc1) gene which encodes an upstream suppressor of mTORC1. Our data show that RPE cells with constitutively high mTORC1 activity were reprogramed to be hyperactive in glucose and lipid metabolism. Lipolysis was suppressed, mitochondrial carnitine shuttle was inhibited, while genes involved in fatty acid (FA) biosynthesis were upregulated. The metabolic changes occurred prior to structural changes of RPE and retinal degeneration. These findings have revealed cellular events and intrinsic mechanisms that contribute to lipid accumulation in the RPE cells during aging and age-related degeneration.

Melatonin pretreatment alleviates renal ischemia-reperfusion injury by promoting autophagic flux via TLR4/MyD88/MEK/ERK/mTORC1 signaling.

Autophagy is an important mechanism for cellular homeostasis and survival during pathologic stress conditions in the kidney, such as ischemia-reperfusion (IR) injury. In this study, renal IR was induced in female C57BL/6 mice after melatonin administration. Renal function, histological damage, inflammatory infiltration, cytokine production, oxidative stress, antioxidant capacity, autophagy changing, apoptosis levels, and autophagy-associated intracellular signaling pathway were assessed to evaluate the impact of antecedent melatonin treatment on IR-induced renal injury. The administration of melatonin resulted in significantly preserved renal function, and the protective effect was associated with ameliorated oxidative stress, limited pro-inflammatory cytokine production, and neutrophil and macrophage infiltration. Moreover, autophagic flux was increased after melatonin administration while the apoptosis levels were decreased in the melatonin-pretreated mice. Using TAK-242 and CRX-527, we confirmed that MyD88-dependent TLR4 and MEK/ERK/mTORC1 signaling participated in melatonin-induced autophagy in IR mice. Collectively, our results provide novel evidence that antecedent melatonin treatment provides protection for the kidney against IR injury by enhancing autophagy, as regulated by the TLR4/MyD88/MEK/ERK/mTORC1 signaling pathway. Therefore, melatonin preconditioning offers a potential therapeutic approach to prevent renal IR injury related to various clinical conditions.

Negative regulation of amino acid signaling by MAPK-regulated 4F2hc/Girdin complex.

Amino acid signaling mediated by the activation of mechanistic target of rapamycin complex 1 (mTORC1) is fundamental to cell growth and metabolism. However, how cells negatively regulate amino acid signaling remains largely unknown. Here, we show that interaction between 4F2 heavy chain (4F2hc), a subunit of multiple amino acid transporters, and the multifunctional hub protein girders of actin filaments (Girdin) down-regulates mTORC1 activity. 4F2hc interacts with Girdin in mitogen-activated protein kinase (MAPK)- and amino acid signaling-dependent manners to translocate to the lysosome. The resultant decrease in cell surface 4F2hc leads to lowered cytoplasmic glutamine (Gln) and leucine (Leu) content, which down-regulates amino acid signaling. Consistently, Girdin depletion augments amino acid-induced mTORC1 activation and inhibits amino acid deprivation-induced autophagy. These findings uncovered the mechanism underlying negative regulation of amino acid signaling, which may play a role in tightly regulated cell growth and metabolism.

Ancestral T Cells in Fish Require mTORC1-Coupled Immune Signals and Metabolic Programming for Proper Activation and Function.

T cells suddenly appeared in jawed fish ∼450 million years ago. Biological studies of fish T cells may provide helpful evidence to understand evolution of adaptive immune systems. To this end, using a Nile tilapia (Oreochromis niloticus) model, we revealed the regulatory mechanism of adaptive immunity mediated by ancestral T cells in jawed fish. Nile tilapia T cells as well as a tightly regulated mammalian/mechanistic target of rapamycin complex 1 (mTORC1) pathway participate in the cellular adaptive immune response during Streptococcus agalactiae infection. Blockade of mTORC1 signaling by rapamycin impairs T cell activation and Ag-induced proliferation in this early vertebrate. More critically, we show that signals from mTORC1 are indispensable for primordial effector T cells to eliminate infection by promoting the expression of proinflammatory cytokines, cytotoxic-related molecules, and proapoptotic genes. Mechanistically, teleost mTORC1 directs effector T cell function by coordinating multiple metabolic programs, including glycolysis, glutaminolysis, and lipogenesis through activating key transcription factors c-Myc, HIF-1α, and sterol regulatory element-binding proteins, and thus links immune signals to metabolic reprogramming in jawed fish. To our knowledge, these results represent the first description of the regulatory mechanism for T cell-mediated adaptive immunity in a fish species. From an evolutionary viewpoint, our study suggests that primordial T cells are armed with sophisticated regulatory strategies like those in modern T cells prior to the divergence of bony fish from the tetrapod lineage. Therefore, our findings fill in an important gap regarding evolution of the adaptive immune system.

The Link between Circadian Clock Genes and Autophagy in Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD), a progressive respiratory disease, is characterized by the alveolar epithelium injury and persistent airway inflammation. It is documented that oscillation and dysregulated expression of circadian clock genes, like Bmal1, Per1, and Per2, involved in COPD pathogenies, including chronic inflammation and imbalanced autophagy level, and targeting the associations of circadian rhythm and autophagy is promising strategies in the management and treatment of COPD. Herein, we reviewed the mechanisms of the circadian clock and the unbalance of the autophagic level in COPD, as well as the link between the two, so as to provide further theoretical bases for the study on the pathogenesis of COPD.

Target of Rapamycin Complex 1 (TORC1), Protein Kinase A (PKA) and Cytosolic pH Regulate a Transcriptional Circuit for Lipid Droplet Formation.

Lipid droplets (LDs) are ubiquitous organelles that fulfill essential roles in response to metabolic cues. The identification of several neutral lipid synthesizing and regulatory protein complexes have propelled significant advance on the mechanisms of LD biogenesis in the endoplasmic reticulum (ER). However, our understanding of signaling networks, especially transcriptional mechanisms, regulating membrane biogenesis is very limited. Here, we show that the nutrient-sensing Target of Rapamycin Complex 1 (TORC1) regulates LD formation at a transcriptional level, by targeting DGA1 expression, in a Sit4-, Mks1-, and Sfp1-dependent manner. We show that cytosolic pH (pHc), co-regulated by the plasma membrane H+-ATPase Pma1 and the vacuolar ATPase (V-ATPase), acts as a second messenger, upstream of protein kinase A (PKA), to adjust the localization and activity of the major transcription factor repressor Opi1, which in turn controls the metabolic switch between phospholipid metabolism and lipid storage. Together, this work delineates hitherto unknown molecular mechanisms that couple nutrient availability and pHc to LD formation through a transcriptional circuit regulated by major signaling transduction pathways.

Role of Grb10 in mTORC1-dependent regulation of insulin signaling and action in human skeletal muscle cells.

Growth factor receptor-bound protein 10 (Grb10) is an adaptor protein that binds to the insulin receptor, upon which insulin signaling and action are thought to be inhibited. Grb10 is also a substrate for the mechanistic target of rapamycin complex 1 (mTORC1) that mediates its feedback inhibition on phosphatidylinositide 3-kinase (PI3K)/Akt signaling. To characterize the function of Grb10 and its regulation by mTORC1 in human muscle, primary skeletal muscle cells were isolated from healthy lean young men and then induced to differentiate into myotubes. Knockdown of Grb10 enhanced insulin-induced PI3K/Akt signaling and glucose uptake in myotubes, reinforcing the notion underlying its function as a negative regulator of insulin action in human muscle. The increased insulin responsiveness in Grb10-silenced myotubes was associated with a higher abundance of the insulin receptor. Furthermore, insulin and amino acids independently and additively stimulated phosphorylation of Grb10 at Ser476. However, acute inhibition of mTORC1 with rapamycin blocked Grb10 Ser476 phosphorylation and repressed a negative-feedback loop on PI3K/Akt signaling that increased myotube responsiveness to insulin. Chronic rapamycin treatment reduced Grb10 protein abundance in conjunction with increased insulin receptor protein levels. Based on these findings, we propose that mTORC1 controls PI3K/Akt signaling through modulation of insulin receptor abundance by Grb10. These findings have potential implications for obesity-linked insulin resistance, as well as clinical use of mTORC1 inhibitors.

Mammalian Target of Rapamycin Complex 1 Signaling Is Required for the Dedifferentiation From Biliary Cell to Bipotential Progenitor Cell in Zebrafish Liver Regeneration.

The liver has a high regenerative capacity. Upon two-thirds partial hepatectomy, the hepatocytes proliferate and contribute to liver regeneration. After severe liver injury, when the proliferation of residual hepatocytes is blocked, the biliary epithelial cells (BECs) lose their morphology and express hepatoblast and endoderm markers, dedifferentiate into bipotential progenitor cells (BP-PCs), then proliferate and redifferentiate into mature hepatocytes. Little is known about the mechanisms involved in the formation of BP-PCs after extreme liver injury. Using a zebrafish liver extreme injury model, we found that mammalian target of rapamycin complex 1 (mTORC1) signaling regulated dedifferentiation of BECs and proliferation of BP-PCs. mTORC1 signaling was up-regulated in BECs during extreme hepatocyte ablation and continuously expressed in later liver regeneration. Inhibition of mTORC1 by early chemical treatment before hepatocyte ablation blocked the dedifferentiation from BECs into BP-PCs. Late mTORC1 inhibition after liver injury reduced the proliferation of BP-PC-derived hepatocytes and BECs but did not affect BP-PC redifferentiation. mTOR and raptor mutants exhibited defects in BEC transdifferentiation including dedifferentiation, BP-PC proliferation, and redifferentiation, similar to the chemical inhibition. Conclusion: mTORC1 signaling governs BEC-driven liver regeneration by regulating the dedifferentiation of BECs and the proliferation of BP-PC-derived hepatocytes and BECs.

New Insights Into the Role of mTOR Signaling in the Cardiovascular System.

The mTOR (mechanistic target of rapamycin) is a master regulator of several crucial cellular processes, including protein synthesis, cellular growth, proliferation, autophagy, lysosomal function, and cell metabolism. mTOR interacts with specific adaptor proteins to form 2 multiprotein complexes, called mTORC1 (mTOR complex 1) and mTORC2 (mTOR complex 2). In the cardiovascular system, the mTOR pathway regulates both physiological and pathological processes in the heart. It is needed for embryonic cardiovascular development and for maintaining cardiac homeostasis in postnatal life. Studies involving mTOR loss-of-function models revealed that mTORC1 activation is indispensable for the development of adaptive cardiac hypertrophy in response to mechanical overload. mTORC2 is also required for normal cardiac physiology and ensures cardiomyocyte survival in response to pressure overload. However, partial genetic or pharmacological inhibition of mTORC1 reduces cardiac remodeling and heart failure in response to pressure overload and chronic myocardial infarction. In addition, mTORC1 blockade reduces cardiac derangements induced by genetic and metabolic disorders and has been reported to extend life span in mice. These studies suggest that pharmacological targeting of mTOR may represent a therapeutic strategy to confer cardioprotection, although clinical evidence in support of this notion is still scarce. This review summarizes and discusses the new evidence on the pathophysiological role of mTOR signaling in the cardiovascular system.

Rheb (Ras Homolog Enriched in Brain 1) Deficiency in Mature Macrophages Prevents Atherosclerosis by Repressing Macrophage Proliferation, Inflammation, and Lipid Uptake.

OBJECTIVE: Macrophage foam cell formation is an important process in atherosclerotic plaque development. The small GTPase Rheb (Ras homolog enriched in brain 1) regulates endocytic trafficking that is critical for foam cell formation. However, it is unclear whether and how macrophage Rheb regulates atherogenesis, which are the focuses of the current study. Approach and Results: Immunofluorescence study confirmed the colocalization of Rheb in F4/80 and Mac-2 (galectin-3)-labeled lesional macrophages. Western blot and fluorescence-activated cell sorting analysis showed that Rheb expression was significantly increased in atherosclerotic lesions of atherosclerosis-prone (apoE-/- [apolipoprotein E deficient]) mice fed with Western diet. Increased Rheb expression was also observed in oxidized LDL (low-density lipoprotein)-treated macrophages. To investigate the in vivo role of macrophage Rheb, we established mature RhebmKO (macrophage-specific Rheb knockout) mice by crossing the Rheb floxed mice with F4/80-cre mice. Macrophage-specific knockout of Rheb in mice reduced Western diet-induced atherosclerotic lesion by 32%, accompanied with a decrease in macrophage content in plaque. Mechanistically, loss of Rheb in macrophages repressed oxidized LDL-induced lipid uptake, inflammation, and macrophage proliferation. On the contrary, lentivirus-mediated overexpression of Rheb in macrophages increased oxidized LDL-induced lipid uptake and inflammation, and the stimulatory effect of Rheb was suppressed by the mTOR (mammalian target of rapamycin) inhibitor rapamycin or the PKA (protein kinase A) activator forskolin. CONCLUSIONS: Macrophage Rheb plays important role in Western diet-induced atherosclerosis by promoting macrophage proliferation, inflammation, and lipid uptake. Inhibition of expression and function of Rheb in macrophages is beneficial to prevent diet-induced atherosclerosis.