ß2-Adrenergic Receptor Enhances the Alternatively Activated Macrophages and Promotes Biliary Injuries Caused by Helminth Infection.

The autonomic nervous system has been studied for its involvement in the control of macrophages; however, the mechanisms underlying the interaction between the adrenergic receptors and alternatively activated macrophages (M2) remain obscure. Using FVB wild-type and beta 2 adrenergic receptors knockout, we found that ß2-AR deficiency alleviates hepatobiliary damage in mice infected with C. sinensis. Moreover, ß2-AR-deficient mice decrease the activation and infiltration of M2 macrophages and decrease the production of type 2 cytokines, which are associated with a significant decrease in liver fibrosis in infected mice. Our in vitro results on bone marrow-derived macrophages revealed that macrophages from Adrb2-/- mice significantly decrease M2 markers and the phosphorylation of ERK/mTORC1 induced by IL-4 compared to that observed in M2 macrophages from Adrb2+/+ . This study provides a better understanding of the mechanisms by which the ß2-AR enhances type 2 immune response through the ERK/mTORC1 signaling pathway in macrophages and their role in liver fibrosis.

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.

ZFP91 disturbs metabolic fitness and antitumor activity of tumor-infiltrating T cells.

Proper metabolic activities facilitate T cell expansion and antitumor function; however, the mechanisms underlying disruption of the T cell metabolic program and function in the tumor microenvironment (TME) remain elusive. Here, we show a zinc finger protein 91-governed (ZFP91-governed) mechanism that disrupts the metabolic pathway and antitumor activity of tumor-infiltrating T cells. Single-cell RNA-Seq revealed that impairments in T cell proliferation and activation correlated with ZFP91 in tissue samples from patients with colorectal cancer. T cell-specific deletion of Zfp91 in mice led to enhanced T cell proliferation and potentiated T cell antitumor function. Loss of ZFP91 increased mammalian target of rapamycin complex 1 (mTORC1) activity to drive T cell glycolysis. Mechanistically, T cell antigen receptor-dependent (TCR-dependent) ZFP91 cytosolic translocation promoted protein phosphatase 2A (PP2A) complex assembly, thereby restricting mTORC1-mediated metabolic reprogramming. Our results demonstrate that ZFP91 perturbs T cell metabolic and functional states in the TME and suggest that targeting ZFP91 may improve the efficacy of cancer immunotherapy.

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.

[New structures of mTORC1: Focus on Rag GTPases]. / Une moisson de nouvelles structures de mTORC1 - Coup de projecteur sur les GTPases Rag.

mTORC1 is a central player in cell growth, a process that is tightly regulated by the availability of nutrients and that controls various aspects of metabolism in the normal cell and in severe diseases such as cancers. mTORC1 is a large multiprotein complex, composed of the kinase subunit mTOR, of Ragulator, which attaches mTOR to the lysosome membrane, of the atypical Rag GTPases and the small GTPase RheB, whose nucleotide states directly dictate its localization to the lysosome and its kinase activity, and of RAPTOR, an adaptor that assembles the complex. The activity of the Rag GTPases is further controlled by the GATOR1 and folliculin complexes, which regulate their GTP/GDP conversion. Here, we review recent structures of important components of the mTORC1 machinery, determined by cryo-electron microscopy for the most part, which allow to reconstitute the architecture of active mTORC1 at near atomic resolution. Notably, we discuss how these structures shed new light on the roles of Rag GTPases and their regulators in mTORC1 regulation, and the perspectives that they open towards understanding the inner workings of mTORC1 on the lysosomal membrane.

TITLE: Une moisson de nouvelles structures de mTORC1 - Coup de projecteur sur les GTPases Rag. ABSTRACT: mTORC1 est un acteur central de la croissance cellulaire, un processus étroitement régulé par la disponibilité de nutriments et qui contrôle diverses étapes du métabolisme dans la cellule normale et au cours de maladies, comme les cancers. mTORC1 est un complexe multiprotéique de grande taille constitué de nombreuses sous-unités, parmi lesquelles deux types de GTPases, Rag et RheB, contrôlent directement sa localisation membranaire et son activité kinase. Dans cette revue, nous faisons le point sur une moisson de structures récentes, déterminées pour la plupart par cryo-microscopie électronique, qui sont en passe de reconstituer le puzzle de l'architecture de mTORC1. Nous discutons ce que ces structures révèlent sur le rôle des GTPases, et ce que leur connaissance ouvre comme perspectives pour comprendre comment mTORC1 fonctionne à la membrane du lysosome.

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.

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.

Targeting Wnt/ß-catenin by anthelmintic drug niclosamide overcomes paclitaxel resistance in esophageal cancer.

Esophageal cancer is an aggressive malignancy, and its current treatment strategies are plagued with high rates of recurrence. In this work, we demonstrate that niclosamide, an anthelmintic drug, is a potential sensitizing candidate for overcoming chemoresistance in esophageal cancer. Using a panel of esophageal cancer cell lines and normal cells, we show that niclosamide has anti-esophageal cancer activity and is likely to be less effective against normal esophageal epithelial and fibroblast cells. The combination of niclosamide with paclitaxel results in much greater efficacy than paclitaxel alone, suggesting that niclosamide is active against esophageal cancer cells that are resistant to paclitaxel. This is further confirmed by our results that niclosamide is effective in inhibiting proliferation and inducing apoptosis in paclitaxel-resistant esophageal cancer cells. In line with the findings obtained from in vitro cell culture system, niclosamide augments the in vivo efficacy of paclitaxel and significantly arrests paclitaxel-resistant esophageal cancer growth without causing toxicity in mice. Mechanistically, we show that niclosamide decreases ß-catenin level and activity, and inhibits phosphorylation of STAT3 and mTORC1 substrate 70S6K. Stabilization of ß-catenin level by Wnt activator lithium chloride (LiCl) significantly abolishes the inhibitory effects of niclosamide in inhibiting proliferation and survival but not suppressing phosphorylation of STAT3 and 70S6K in paclitaxel-resistant esophageal cancer cells, suggesting that niclosamide sensitizes esophageal cancer cell to paclitaxel mainly through inhibiting Wnt/ß-catenin. Our work demonstrates the efficacy of niclosamide and its underlying mechanism in paclitaxel-resistant esophageal cancer. Our work emphasizes that Wnt/ß-catenin inhibition is a sensitizing strategy in esophageal cancer.

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.

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.

Regulation of colonic epithelial cell homeostasis by mTORC1.

Cell signaling important for homeostatic regulation of colonic epithelial cells (CECs) remains poorly understood. Mammalian target of rapamycin complex 1 (mTORC1), a protein complex that contains the serine-threonine kinase mTOR, mediates signaling that underlies the control of cellular functions such as proliferation and autophagy by various external stimuli. We here show that ablation of tuberous sclerosis complex 2 (Tsc2), a negative regulator of mTORC1, specifically in intestinal epithelial cells of mice resulted in increased activity of mTORC1 of, as well as increased proliferative activity of, CECs. Such Tsc2 ablation also reduced the population of Lgr5-positive colonic stem cells and the expression of Wnt target genes in CECs. The stimulatory phosphorylation of the kinase Akt and inhibitory phosphorylation of glycogen synthase kinase 3ß were both markedly decreased in the colon of the Tsc2 conditional knockout (CKO) mice. Development of colonic organoids with cryptlike structures was enhanced for Tsc2 CKO mice compared with control mice. Finally, Tsc2 CKO mice manifested increased susceptibility to dextran sulfate sodium-induced colitis. Our results thus suggest that mTORC1 activity promotes the proliferation of, as well as the expression of Wnt target genes in, CECs and thereby contributes to colonic organogenesis and homeostasis.

Scaffold proteins in bulk and selective autophagy.

Autophagy is a crucial cellular degradation and recycling pathway. During autophagy double-membrane vesicles, called autophagosomes, encapsulate cellular components and deliver their cargo to the lytic compartment for degradation. Formation of autophagosomes is regulated by the Atg1 kinase complex in yeast and the homologous ULK1 kinase complex in mammals. While research on Atg1 and ULK1 has advanced our understanding of how these protein kinases function in autophagy, the other Atg1/ULK1 kinase complex members have received much less attention. Here, we focus on the functions of the Atg1 kinase complex members Atg11 and Atg17 as well as the ULK1 kinase complex member FIP200 in autophagy. These three proteins act as scaffolds in their respective complexes. Recent studies have made it evident that they have similar but also distinct functions. In this article, we review our current understanding of how these scaffold proteins function from autophagosome formation to fusion and also discuss their possible roles in diseases.

NOX4/H2O2/mTORC1 Pathway in Salt-Induced Hypertension and Kidney Injury.

We have reported that a high-salt (4.0% NaCl) dietary intake activates mTORC1 and inhibition of this pathway with rapamycin blunts the chronic phase of salt-induced hypertension and renal injury in Dahl salt-sensitive (SS) rats. In SS rats, high-salt intake is known to increase the renal production of H2O2 by NOX4, the most abundant NOX isoform in the kidney, and the global knockout of NOX4 blunts salt-sensitivity in these rats. Here, we explored the hypothesis that elevations of H2O2 by NOX4 in high-salt fed SS rat stimulate mTORC1 for the full development of salt-induced hypertension and renal injury. Our in vitro studies found that H2O2 activates mTORC1 independent of PI3K/AKT and AMPK pathways. To determine the in vivo relevance of NOX4/H2O2/mTORC1 in the salt-induced hypertension, SS-Nox4 knockout (SSNox4-/-) rats were daily administrated with vehicle/rapamycin fed a high-salt diet for 21 days. Rapamycin treatment of SSNox4-/- rats had shown no augmented effect on the salt-induced hypertension nor upon indices of renal injury. Significant reductions of renal T lymphocyte and macrophage together with inhibition of cell proliferation were observed in rapamycin treated rats suggesting a role of mTORC1 independent of NOX4 in the proliferation of immune cell. Given the direct activation of mTORC1 by H2O2 and absence of any further protection from salt-induced hypertension in rapamycin-treated SSNox4-/- rats, we conclude that NOX4-H2O2 is a major upstream activator of mTORC1 that contributes importantly to salt-induced hypertension and renal injury in the SS rat model.

Cdk4 and Cdk6 Couple the Cell-Cycle Machinery to Cell Growth via mTORC1.

Cell growth is coupled to cell-cycle progression in mitotically proliferating mammalian cells, but the underlying molecular mechanisms are not well understood. CyclinD-Cdk4/6 is known to phosphorylate RB to promote S-phase entry, but recent work suggests they have additional functions. We show here that CyclinD-Cdk4/6 activates mTORC1 by binding and phosphorylating TSC2 on Ser1217 and Ser1452. Pharmacological inhibition of Cdk4/6 leads to a rapid, TSC2-dependent reduction of mTORC1 activity in multiple human and mouse cell lines, including breast cancer cells. By simultaneously driving mTORC1 and E2F, CyclinD-Cdk4/6 couples cell growth to cell-cycle progression. Consistent with this, we see that mTORC1 activity is cell cycle dependent in proliferating neural stem cells of the adult rodent brain. We find that Cdk4/6 inhibition reduces cell proliferation partly via TSC2 and mTORC1. This is of clinical relevance, because Cdk4/6 inhibitors are used for breast cancer therapy.

mTORC1 directly inhibits AMPK to promote cell proliferation under nutrient stress.

Central to cellular metabolism and cell proliferation are highly conserved signalling pathways controlled by mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK)1,2, dysregulation of which are implicated in pathogenesis of major human diseases such as cancer and type 2 diabetes. AMPK pathways leading to reduced cell proliferation are well established and, in part, act through inhibition of TOR complex-1 (TORC1) activity. Here we demonstrate reciprocal regulation, specifically that TORC1 directly down-regulates AMPK signalling by phosphorylating the evolutionarily conserved residue Ser367 in the fission yeast AMPK catalytic subunit Ssp2, and AMPK α1Ser347/α2Ser345 in the mammalian homologs, which is associated with reduced phosphorylation of activation loop Thr172. Genetic or pharmacological inhibition of TORC1 signalling led to AMPK activation in the absence of increased AMP:ATP ratios; under nutrient stress conditions this was associated with growth limitation in both yeast and human cell cultures. Our findings reveal fundamental, bi-directional regulation between two major metabolic signalling networks and uncover new opportunity for cancer treatment strategies aimed at suppressing cell proliferation in the nutrient-poor tumor microenvironment.

Nanomolar melatonin influences insulin synthesis and secretion in rat insulinoma INS-1E cells.

Pancreatic beta-cell dysfunction results in reductions of insulin synthesis/secretion, cell survival, and insulin sensitivity thereby inducing diabetes mellitus. In this study, how nanomolar melatonin regulates insulin synthesis and secretion in rat insulinoma INS-1E cells was investigated. At melatonin concentrations of 10 - 100 nM for 48 hours, melatonin significantly increased the insulin protein level in INS-1E cells above the level in control cells without melatonin or glucose treatments and decreased the insulin level in media with glucose: increases in insulin synthesis and decreases in insulin secretion occurred in dose-dependent manners. Luzindole or 4-phenyl-2-propionamidotetralin (4P-PDOT), melatonin receptor antagonists, inhibited the melatonin-induced insulin level in cells and media. Levels of membrane vesicle trafficking-related proteins including Rab5, GOPC, phospho-caveolin-1, EEA1, and clathrin proteins significantly increased with melatonin treatment above that in control cells without melatonin or glucose treatments, whereas expressions of APPL1 and syntaxin-6 proteins significantly decreased with melatonin treatment. The increases in the phosphorylation of mammalian target of rapamycin (p-mTOR), raptor protein, and mTOR complex 1 (mTORC1) levels were consistent with the increments in the expressions of p-Akt (Ser473, Thr308) and stress-induced IRE1α/p-eIF2α proteins in the endoplasmic reticulum following melatonin treatment. also, expression levels of Bcl-2 and Bcl-xl proteins were significantly increased compared to those in control cells without melatonin or glucose treatments, whereas the Bax protein level decreased. These results indicate that nanomolar melatonin regulates insulin synthesis and secretion associated with membrane vesicle trafficking-related proteins, including Rab5, GOPC, p-Caveolin-1, EEA1, and clathrin, through the Akt/mTOR pathway.

Moderate-Intensity Strength Exercise to Exhaustion Results in More Pronounced Signaling Changes in Skeletal Muscles of Strength-Trained Compared With Untrained Individuals.

Lysenko, EA, Popov, DV, Vepkhvadze, TF, Sharova, AP, and Vinogradova, OL. Moderate-intensity strength exercise to exhaustion results in more pronounced signaling changes in skeletal muscles of strength-trained compared with untrained individuals. J Strength Cond Res 34(4): 1103-1112, 2020-The aim of our investigation was to compare the response pattern of signaling proteins and genes regulating protein synthesis and degradation in skeletal muscle after strength exercise sessions performed to volitional fatigue in strength-trained and untrained males. Eight healthy recreationally active males and 8 power-lifting athletes performed 4 sets of unilateral leg presses to exhaustion (65% 1 repetition maximum). Biopsy samples of m. vastus lateralis were obtained before, 1 and 5 hours after cessation of exercise. Phosphorylation of p70S6k, 4EBP1, and ACC increased, whereas phosphorylation of eEF2 and FOXO1 decreased only in the trained group after exercise. Expression of DDIT4, MURF1, and FOXO1 mRNAs increased and expression of MSTN mRNA decreased also only in the trained group after exercise. In conclusion, moderate-intensity strength exercise performed to volitional fatigue changed the phosphorylation status of mTORC1 downstream signaling molecules and markers of ubiquitin-proteasome system activation in trained individuals, suggesting activation of protein synthesis and degradation. In contrast to the trained group, signaling responses in the untrained group were considerably less pronounced. It can be assumed that the slowdown in muscle mass gain as the athletes increase in qualification cannot be associated with a decrease in the sensitivity of systems regulating protein metabolism, but possibly with inadequate intake or assimilation of nutrients necessary for anabolism. Perhaps, the intake of highly digestible protein or protein-carbohydrate dietary supplements could contribute to the increase in muscle mass in strength athletes.

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.

mTORC1 and mTORC2 Differentially Regulate Cell Fate Programs to Coordinate Osteoblastic Differentiation in Mesenchymal Stromal Cells.

Vascular regeneration depends on intact function of progenitors of vascular smooth muscle cells such as pericytes and their circulating counterparts, mesenchymal stromal cells (MSC). Deregulated MSC differentiation and maladaptive cell fate programs associated with age and metabolic diseases may exacerbate arteriosclerosis due to excessive transformation to osteoblast-like calcifying cells. Targeting mTOR, a central controller of differentiation and cell fates, could offer novel therapeutic perspectives. In a cell culture model for osteoblastic differentiation of pluripotent human MSC we found distinct roles for mTORC1 and mTORC2 in the regulation of differentiation towards calcifying osteoblasts via cell fate programs in a temporally-controlled sequence. Activation of mTORC1 with induction of cellular senescence and apoptosis were hallmarks of transition to a calcifying phenotype. Inhibition of mTORC1 with Rapamycin elicited reciprocal activation of mTORC2, enhanced autophagy and recruited anti-apoptotic signals, conferring protection from calcification. Pharmacologic and genetic negative interference with mTORC2 function or autophagy both abolished regenerative programs but induced cellular senescence, apoptosis, and calcification. Overexpression of the mTORC2 constituent rictor revealed that enhanced mTORC2 signaling without altered mTORC1 function was sufficient to inhibit calcification. Studies in mice reproduced the in vitro effects of mTOR modulation with Rapamycin on cell fates in vascular cells in vivo. Amplification of mTORC2 signaling promotes protective cell fates including autophagy to counteract osteoblast differentiation and calcification of MSC, representing a novel mTORC2 function. Regenerative approaches aimed at modulating mTOR network activation patterns hold promise for delaying age-related vascular diseases and treatment of accelerated arteriosclerosis in chronic metabolic conditions.

Metabolic Programming of Macrophages: Implications in the Pathogenesis of Granulomatous Disease.

Metabolic reprogramming is rapidly gaining appreciation in the etiology of immune cell dysfunction in a variety of diseases. Tuberculosis, schistosomiasis, and sarcoidosis represent an important class of diseases characterized by the formation of granulomas, where macrophages are causatively implicated in disease pathogenesis. Recent studies support the incidence of macrophage metabolic reprogramming in granulomas of both infectious and non-infectious origin. These publications identify the mechanistic target of rapamycin (mTOR), as well as the major regulators of lipid metabolism and cellular energy balance, peroxisome proliferator receptor gamma (PPAR-γ) and adenosine monophosphate-activated protein kinase (AMPK), respectively, as key players in the pathological progression of granulomas. In this review, we present a comprehensive breakdown of emerging research on the link between macrophage cell metabolism and granulomas of different etiology, and how parallels can be drawn between different forms of granulomatous disease. In particular, we discuss the role of PPAR-γ signaling and lipid metabolism, which are currently the best-represented metabolic pathways in this context, and we highlight dysregulated lipid metabolism as a common denominator in granulomatous disease progression. This review therefore aims to highlight metabolic mechanisms of granuloma immune cell fate and open up research questions for the identification of potential therapeutic targets in the future.