Mario Pende

Activation of 40S ribosomal protein S6 kinases (S6Ks) is mediated by anabolic signals triggered by hormones, growth factors, and nutrients. Stimulation by any of these agents is inhibited by the bacterial macrolide rapamycin, which binds to and inactivates the mammalian target of rapamycin, an S6K kinase. In mammals, two genes encoding homologous S6Ks, S6K1 and S6K2, have been identified. Here we show that mice deficient for S6K1 or S6K2 are born at the expected Mendelian ratio. Compared to wild-type mice, S6K1(-/-) mice are significantly smaller, whereas S6K2(-/-) mice tend to be slightly larger. However, mice lacking both genes showed a sharp reduction in viability due to perinatal lethality. Analysis of S6 phosphorylation in the cytoplasm and nucleoli of cells derived from the distinct S6K genotypes suggests that both kinases are required for full S6 phosphorylation but that S6K2 may be more prevalent in contributing to this response. Despite the impairment of S6 phosphorylation in cells from S6K1(-/-)/S6K2(-/-) mice, cell cycle progression and the translation of 5'-terminal oligopyrimidine mRNAs were still modulated by mitogens in a rapamycin-dependent manner. Thus, the absence of S6K1 and S6K2 profoundly impairs animal viability but does not seem to affect the proliferative responses of these cell types. Unexpectedly, in S6K1(-/-)/S6K2(-/-) cells, S6 phosphorylation persisted at serines 235 and 236, the first two sites phosphorylated in response to mitogens. In these cells, as well as in rapamycin-treated wild-type, S6K1(-/-), and S6K2(-/-) cells, this step was catalyzed by a mitogen-activated protein kinase (MAPK)-dependent kinase, most likely p90rsk. These data reveal a redundancy between the S6K and the MAPK pathways in mediating early S6 phosphorylation in response to mitogens.

The eukaryotic translation initiation factor 4B (eIF4B) plays a critical role in recruiting the 40S ribosomal subunit to the mRNA. In response to insulin, eIF4B is phosphorylated on Ser422 by S6K in a rapamycin-sensitive manner. Here we demonstrate that the p90 ribosomal protein S6 kinase (RSK) phosphorylates eIF4B on the same residue. The relative contribution of the RSK and S6K modules to the phosphorylation of eIF4B is growth factor-dependent, and the two phosphorylation events exhibit very different kinetics. The S6K and RSK proteins are members of the AGC protein kinase family, and require PDK1 phosphorylation for activation. Consistent with this requirement, phosphorylation of eIF4B Ser422 is abrogated in PDK1 null embryonic stem cells. Phosphorylation of eIF4B on Ser422 by RSK and S6K is physiologically significant, as it increases the interaction of eIF4B with the eukaryotic translation initiation factor 3.

The mammalian target of rapamycin (mTOR) and Akt proteins regulate various steps of muscle development and growth, but the physiological relevance and the downstream effectors are under investigation. Here we show that S6 kinase 1 (S6K1), a protein kinase activated by nutrients and insulin-like growth factors (IGFs), is essential for the control of muscle cytoplasmic volume by Akt and mTOR. Deletion of S6K1 does not affect myoblast cell proliferation but reduces myoblast size to the same extent as that observed with mTOR inhibition by rapamycin. In the differentiated state, S6K1(-/-) myotubes have a normal number of nuclei but are smaller, and their hypertrophic response to IGF1, nutrients and membrane-targeted Akt is blunted. These growth defects reveal that mTOR requires distinct effectors for the control of muscle cell cycle and size, potentially opening new avenues of therapeutic intervention against neoplasia or muscle atrophy.

Mammalian target of rapamycin (mTOR) is a key regulator of cell growth that associates with raptor and rictor to form the mTOR complex 1 (mTORC1) and mTORC2, respectively. Raptor is required for oxidative muscle integrity, whereas rictor is dispensable. In this study, we show that muscle-specific inactivation of mTOR leads to severe myopathy, resulting in premature death. mTOR-deficient muscles display metabolic changes similar to those observed in muscles lacking raptor, including impaired oxidative metabolism, altered mitochondrial regulation, and glycogen accumulation associated with protein kinase B/Akt hyperactivation. In addition, mTOR-deficient muscles exhibit increased basal glucose uptake, whereas whole body glucose homeostasis is essentially maintained. Importantly, loss of mTOR exacerbates the myopathic features in both slow oxidative and fast glycolytic muscles. Moreover, mTOR but not raptor and rictor deficiency leads to reduced muscle dystrophin content. We provide evidence that mTOR controls dystrophin transcription in a cell-autonomous, rapamycin-resistant, and kinase-independent manner. Collectively, our results demonstrate that mTOR acts mainly via mTORC1, whereas regulation of dystrophin is raptor and rictor independent.

Inactivation of APC is a strongly predisposing event in the development of colorectal cancer, prompting the search for vulnerabilities specific to cells that have lost APC function. Signalling through the mTOR pathway is known to be required for epithelial cell proliferation and tumour growth, and the current paradigm suggests that a critical function of mTOR activity is to upregulate translational initiation through phosphorylation of 4EBP1 (refs 6, 7). This model predicts that the mTOR inhibitor rapamycin, which does not efficiently inhibit 4EBP1 (ref. 8), would be ineffective in limiting cancer progression in APC-deficient lesions. Here we show in mice that mTOR complex 1 (mTORC1) activity is absolutely required for the proliferation of Apc-deficient (but not wild-type) enterocytes, revealing an unexpected opportunity for therapeutic intervention. Although APC-deficient cells show the expected increases in protein synthesis, our study reveals that it is translation elongation, and not initiation, which is the rate-limiting component. Mechanistically, mTORC1-mediated inhibition of eEF2 kinase is required for the proliferation of APC-deficient cells. Importantly, treatment of established APC-deficient adenomas with rapamycin (which can target eEF2 through the mTORC1-S6K-eEF2K axis) causes tumour cells to undergo growth arrest and differentiation. Taken together, our data suggest that inhibition of translation elongation using existing, clinically approved drugs, such as the rapalogs, would provide clear therapeutic benefit for patients at high risk of developing colorectal cancer.

Genetic studies have shown that the tuberous sclerosis complex (TSC) 1-TSC2-mammalian target of Rapamycin (mTOR) and the Hippo-Yes-associated protein 1 (YAP) pathways are master regulators of organ size, which are often involved in tumorigenesis. The crosstalk between these signal transduction pathways in coordinating environmental cues, such as nutritional status and mechanical constraints, is crucial for tissue growth. Whether and how mTOR regulates YAP remains elusive. Here we describe a novel mouse model of TSC which develops renal mesenchymal lesions recapitulating human perivascular epithelioid cell tumors (PEComas) from patients with TSC. We identify that YAP is up-regulated by mTOR in mouse and human PEComas. YAP inhibition blunts abnormal proliferation and induces apoptosis of TSC1-TSC2-deficient cells, both in culture and in mosaic Tsc1 mutant mice. We further delineate that YAP accumulation in TSC1/TSC2-deficient cells is due to impaired degradation of the protein by the autophagosome/lysosome system. Thus, the regulation of YAP by mTOR and autophagy is a novel mechanism of growth control, matching YAP activity with nutrient availability under growth-permissive conditions. YAP may serve as a potential therapeutic target for TSC and other diseases with dysregulated mTOR activity.

Chronic obstructive pulmonary disease (COPD) is a highly prevalent and devastating condition for which no curative treatment is available. Exaggerated lung cell senescence may be a major pathogenic factor. Here, we investigated the potential role for mTOR signaling in lung cell senescence and alterations in COPD using lung tissue and derived cultured cells from patients with COPD and from age- and sex-matched control smokers. Cell senescence in COPD was linked to mTOR activation, and mTOR inhibition by low-dose rapamycin prevented cell senescence and inhibited the proinflammatory senescence-associated secretory phenotype. To explore whether mTOR activation was a causal pathogenic factor, we developed transgenic mice exhibiting mTOR overactivity in lung vascular cells or alveolar epithelial cells. In this model, mTOR activation was sufficient to induce lung cell senescence and to mimic COPD lung alterations, with the rapid development of lung emphysema, pulmonary hypertension, and inflammation. These findings support a causal relationship between mTOR activation, lung cell senescence, and lung alterations in COPD, thereby identifying the mTOR pathway as a potentially new therapeutic target in COPD.

The role of the gluco-incretin hormones GIP and GLP-1 in the control of β cell function was studied by analyzing mice with inactivation of each of these hormone receptor genes, or both.Our results demonstrate that glucose intolerance was additively increased during oral glucose absorption when both receptors were inactivated.After intraperitoneal injections, glucose intolerance was more severe in double-as compared to single-receptor KO mice, and euglycemic clamps revealed normal insulin sensitivity, suggesting a defect in insulin secretion.When assessed in vivo or in perfused pancreas, insulin secretion showed a lack of first phase in Glp-1R -/-but not in Gipr -/-mice.In perifusion experiments, however, first-phase insulin secretion was present in both types of islets.In double-KO islets, kinetics of insulin secretion was normal, but its amplitude was reduced by about 50% because of a defect distal to plasma membrane depolarization.Thus, gluco-incretin hormones control insulin secretion (a) by an acute insulinotropic effect on β cells after oral glucose absorption (b) through the regulation, by GLP-1, of in vivo first-phase insulin secretion, probably by an action on extra-islet glucose sensors, and (c) by preserving the function of the secretory pathway, as evidenced by a β cell autonomous secretion defect when both receptors are inactivated.

To understand how extracellular signals may produce long-term effects in neural cells, we have analyzed the mechanism by which neurotransmitters and growth factors induce phosphorylation of the transcription factor cAMP response element binding protein (CREB) in cortical oligodendrocyte progenitor (OP) cells. Activation of glutamate receptor channels by kainate, as well as stimulation of G-protein-coupled cholinergic receptors by carbachol and tyrosine kinase receptors by basic fibroblast growth factor (bFGF), rapidly leads to mitogen-activated protein kinase (MAPK) phosphorylation and ribosomal S6 kinase (RSK) activation. Kainate and carbachol activation of the MAPK pathway requires extracellular calcium influx and is accompanied by protein kinase C (PKC) induction, with no significant increase in GTP binding to Ras. Conversely, growth factor-stimulated MAPK phosphorylation is independent of extracellular calcium and is accompanied by Ras activation. Both basal and stimulated MAPK activity in OP cells are influenced by cytoplasmic calcium levels, as shown by their sensitivity to the calcium chelator bis(2-aminophenoxy)ethane- N,N,N′,N′ -tetra-acetic acid. The kinetics of CREB phosphorylation in response to the various agonists corresponds to that of MAPK activation. Moreover, CREB phosphorylation and MAPK activation are similarly affected by calcium ions. The MEK inhibitor PD 098059, which selectively prevents activation of the MAPK pathway, strongly reduces induction of CREB phosphorylation by kainate, carbachol, bFGF, and the phorbol ester TPA. We propose that in OPs the MAPK/RSK pathway mediates CREB phosphorylation in response to calcium influx, PKC activation, and growth factor stimulation.

S6 kinase (S6K) deletion in metazoans causes small cell size, insulin hypersensitivity, and metabolic adaptations; however, the underlying molecular mechanisms are unclear. Here we show that S6K-deficient skeletal muscle cells have increased AMP and inorganic phosphate levels relative to ATP and phosphocreatine, causing AMP-activated protein kinase (AMPK) upregulation. Energy stress and muscle cell atrophy are specifically triggered by the S6K1 deletion, independent of S6K2 activity. Two known AMPK-dependent functions, mitochondrial biogenesis and fatty acid beta-oxidation, are upregulated in S6K-deficient muscle cells, leading to a sharp depletion of lipid content, while glycogen stores are spared. Strikingly, AMPK inhibition in S6K-deficient cells restores cell growth and sensitivity to nutrient signals. These data indicate that S6K1 controls the energy state of the cell and the AMPK-dependent metabolic program, providing a mechanism for cell mass accumulation under high-calorie diet.

The rapamycin-insensitive companion of mammalian target of rapamycin (mTOR) (Rictor) is a key member of mTOR complex-2 (mTORC2), which phosphorylates the AGC kinases Akt/PKB, PKC and SGK1 at a C-terminal hydrophobic motif. We identified several novel sites on Rictor that are phosphorylated, including Thr1135, which is conserved across all vertebrates. Phosphorylation of this site on Rictor is stimulated by amino acids and growth factors through a rapamycin-sensitive signaling cascade. We demonstrate here that Rictor is a direct target of the ribosomal protein S6 kinase-1 (S6K1). Rictor phosphorylation at Thr1135 does not lead to major changes in mTORC2-kinase activity. However, phosphorylation of this site turns over rapidly and mediates 14-3-3 binding to Rictor and mTORC2, providing possibility for altered interactions of the complex. These findings reveal an unexpected signaling input into mTORC2, which is regulated by amino acids, growth factors and rapamycin.

Rapidly proliferating cells promote glycolysis in aerobic conditions, to increase growth rate. Expression of specific glycolytic enzymes, namely pyruvate kinase M2 and hexokinase 2, concurs to this metabolic adaptation, as their kinetics and intracellular localization favour biosynthetic processes required for cell proliferation. Intracellular factors regulating their selective expression remain largely unknown. Here we show that the peroxisome proliferator-activated receptor gamma transcription factor and nuclear hormone receptor contributes to selective pyruvate kinase M2 and hexokinase 2 gene expression in PTEN-null fatty liver. Peroxisome proliferator-activated receptor gamma expression, liver steatosis, shift to aerobic glycolysis and tumorigenesis are under the control of the Akt2 kinase in PTEN-null mouse livers. Peroxisome proliferator-activated receptor gamma binds to hexokinase 2 and pyruvate kinase M promoters to activate transcription. In vivo rescue of peroxisome proliferator-activated receptor gamma activity causes liver steatosis, hypertrophy and hyperplasia. Our data suggest that therapies with the insulin-sensitizing agents and peroxisome proliferator-activated receptor gamma agonists, thiazolidinediones, may have opposite outcomes depending on the nutritional or genetic origins of liver steatosis.

Most cancer cells exhibit increased glycolysis for generation of their energy supply. This specificity could be used to preferentially kill these cells. In this study, we identified the signaling pathway initiated by glycolysis inhibition that results in sensitization to death receptor (DR)-induced apoptosis. We showed, in several human cancer cell lines (such as Jurkat, HeLa, U937), that glucose removal or the use of nonmetabolizable form of glucose (2-deoxyglucose) dramatically enhances apoptosis induced by Fas or by tumor necrosis factor-related apoptosis-inducing ligand. This sensitization is controlled through the adenosine monophosphate (AMP)-activated protein kinase (AMPK), which is the central energy-sensing system of the cell. We established the fact that AMPK is activated upon glycolysis block resulting in mammalian target of rapamycin (mTOR) inhibition leading to Mcl-1 decrease, but no other Bcl-2 anti-apoptotic members. Interestingly, we determined that, upon glycolysis inhibition, the AMPK–mTOR pathway controlled Mcl-1 levels neither through transcriptional nor through posttranslational mechanism but rather by controlling its translation. Therefore, our results show a novel mechanism for the sensitization to DR-induced apoptosis linking glucose metabolism to Mcl-1 downexpression. In addition, this study provides a rationale for the combined use of DR ligands with AMPK activators or mTOR inhibitors in the treatment of human cancers.

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The complex of Vacuolar Protein Sorting 34 and 15 (Vps34 and Vps15) has Class III phosphatidylinositol 3-kinase activity and putative roles in nutrient sensing, mammalian Target Of Rapamycin (mTOR) activation by amino acids, cell growth, vesicular trafficking and autophagy. Contrary to expectations, here we show that Vps15-deficient mouse tissues are competent for LC3-positive autophagosome formation and maintain mTOR activation. However, an impaired lysosomal function in mutant cells is traced by accumulation of adaptor protein p62, LC3 and Lamp2 positive vesicles, which can be reverted to normal levels after ectopic overexpression of Vps15. Mice lacking Vps15 in skeletal muscles, develop a severe myopathy. Distinct from the autophagy deficient Atg7(-/-) mutants, pathognomonic morphological hallmarks of autophagic vacuolar myopathy (AVM) are observed in Vps15(-/-) mutants, including elevated creatine kinase plasma levels, accumulation of autophagosomes, glycogen and sarcolemmal features within the fibres. Importantly, Vps34/Vps15 overexpression in myoblasts of Danon AVM disease patients alleviates the glycogen accumulation. Thus, the activity of the Vps34/Vps15 complex is critical in disease conditions such as AVMs, and possibly a variety of other lysosomal storage diseases.

Loss of skeletal muscle mass and force aggravates age-related sarcopenia and numerous pathologies, such as cancer and diabetes. The AKT-mTORC1 pathway plays a major role in stimulating adult muscle growth; however, the functional role of its downstream mediators in vivo is unknown. Here, we show that simultaneous inhibition of mTOR signaling to both S6K1 and 4E-BP1 is sufficient to reduce AKT-induced muscle growth and render it insensitive to the mTORC1-inhibitor rapamycin. Surprisingly, lack of mTOR signaling to 4E-BP1 only, or deletion of S6K1 alone, is not sufficient to reduce muscle hypertrophy or alter its sensitivity to rapamycin. However, we report that, while not required for muscle growth, S6K1 is essential for maintaining muscle structure and force production. Hypertrophy in the absence of S6K1 is characterized by compromised ribosome biogenesis and the formation of p62-positive protein aggregates. These findings identify S6K1 as a crucial player for maintaining muscle function during hypertrophy.

Merlin/NF2 is a bona fide tumor suppressor whose mutations underlie inherited tumor syndrome neurofibromatosis type 2 (NF2), as well as various sporadic cancers including kidney cancer. Multiple Merlin/NF2 effector pathways including the Hippo-YAP/TAZ pathway have been identified. However, the molecular mechanisms underpinning the growth and survival of NF2-mutant tumors remain poorly understood. Using an inducible orthotopic kidney tumor model, we demonstrate that YAP/TAZ silencing is sufficient to induce regression of pre-established NF2-deficient tumors. Mechanistically, YAP/TAZ depletion diminishes glycolysis-dependent growth and increases mitochondrial respiration and reactive oxygen species (ROS) buildup, resulting in oxidative-stress-induced cell death when challenged by nutrient stress. Furthermore, we identify lysosome-mediated cAMP-PKA/EPAC-dependent activation of RAF-MEK-ERK signaling as a resistance mechanism to YAP/TAZ inhibition. Finally, unbiased analysis of TCGA primary kidney tumor transcriptomes confirms a positive correlation of a YAP/TAZ signature with glycolysis and inverse correlations with oxidative phosphorylation and lysosomal gene expression, supporting the clinical relevance of our findings.

DEP-domain containing 5 (DEPDC5), encoding a repressor of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway, has recently emerged as a major gene mutated in familial focal epilepsies and focal cortical dysplasia. Here we established a global knockout rat using TALEN technology to investigate in vivo the impact of Depdc5-deficiency. Homozygous Depdc5−/− embryos died from embryonic day 14.5 due to a global growth delay. Constitutive mTORC1 hyperactivation was evidenced in the brains and in cultured fibroblasts of Depdc5−/− embryos, as reflected by enhanced phosphorylation of its downstream effectors S6K1 and rpS6. Consistently, prenatal treatment with mTORC1 inhibitor rapamycin rescued the phenotype of Depdc5−/− embryos. Heterozygous Depdc5+/− rats developed normally and exhibited no spontaneous electroclinical seizures, but had altered cortical neuron excitability and firing patterns. Depdc5+/− rats displayed cortical cytomegalic dysmorphic neurons and balloon-like cells strongly expressing phosphorylated rpS6, indicative of mTORC1 upregulation, and not observed after prenatal rapamycin treatment. These neuropathological abnormalities are reminiscent of the hallmark brain pathology of human focal cortical dysplasia. Altogether, Depdc5 knockout rats exhibit multiple features of rodent models of mTORopathies, and thus, stand as a relevant model to study their underlying pathogenic mechanisms.

Abstract The class 3 phosphoinositide 3-kinase (PI3K) is required for lysosomal degradation by autophagy and vesicular trafficking, assuring nutrient availability. Mitochondrial lipid catabolism is another energy source. Autophagy and mitochondrial metabolism are transcriptionally controlled by nutrient sensing nuclear receptors. However, the class 3 PI3K contribution to this regulation is unknown. We show that liver-specific inactivation of Vps15 , the essential regulatory subunit of the class 3 PI3K, elicits mitochondrial depletion and failure to oxidize fatty acids. Mechanistically, transcriptional activity of Peroxisome Proliferator Activated Receptor alpha (PPARα), a nuclear receptor orchestrating lipid catabolism, is blunted in Vps15 -deficient livers. We find PPARα repressors Histone Deacetylase 3 (Hdac3) and Nuclear receptor co-repressor 1 (NCoR1) accumulated in Vps15 -deficient livers due to defective autophagy. Activation of PPARα or inhibition of Hdac3 restored mitochondrial biogenesis and lipid oxidation in Vps15 -deficient hepatocytes. These findings reveal roles for the class 3 PI3K and autophagy in transcriptional coordination of mitochondrial metabolism.

Growth hormone (GH) participates in the postnatal regulation of skeletal muscle growth, although the mechanism of action is unclear. Here we show that the mass of skeletal muscles lacking GH receptors is reduced because of a decrease in myofiber size with normal myofiber number. GH signaling controls the size of the differentiated myotubes in a cell-autonomous manner while having no effect on size, proliferation, and differentiation of the myoblast precursor cells. The GH hypertrophic action leads to an increased myonuclear number, indicating that GH facilitates fusion of myoblasts with nascent myotubes. NFATc2, a transcription factor regulating this phase of fusion, is required for GH action because GH is unable to induce hypertrophy of NFATc2-/- myotubes. Finally, we provide three lines of evidence suggesting that GH facilitates cell fusion independent of insulin-like growth factor 1 (IGF-1) up-regulation. First, GH does not regulate IGF-1 expression in myotubes; second, GH action is not mediated by a secreted factor in conditioned medium; third, GH and IGF-1 hypertrophic effects are additive and rely on different signaling pathways. Taken together, these data unravel a specific function of GH in the control of cell fusion, an essential process for muscle growth.

Ribosomal S6 kinases (S6Ks) have been depicted as critical effectors downstream of growth factor pathways, which play an important role in the regulation of protein synthesis by phosphorylating the ribosomal protein, S6. The goal of this study was to determine whether S6Ks regulate heart size, are critical for the induction of cardiac hypertrophy in response to a pathological or physiological stimulus, and whether S6Ks are critical downstream effectors of the insulin-like growth factor 1 (IGF1)-phosphoinositide 3-kinase (PI3K) pathway. For this purpose, we generated and characterized cardiac-specific S6K1 and S6K2 transgenic mice and subjected S6K1−/−, S6K2−/−, and S6K1−/− S6K2−/− mice to a pathological stress (aortic banding) or a physiological stress (exercise training). To determine the genetic relationship between S6Ks and the IGF1-PI3K pathway, S6K transgenic and knockout mice were crossed with cardiac-specific transgenic mice overexpressing the IGF1 receptor (IGF1R) or PI3K mutants. Here we show that overexpression of S6K1 induced a modest degree of hypertrophy, whereas overexpression of S6K2 resulted in no obvious cardiac phenotype. Unexpectedly, deletion of S6K1 and S6K2 had no impact on the development of pathological, physiological, or IGF1R-PI3K-induced cardiac hypertrophy. These studies suggest that S6Ks alone are not essential for the development of cardiac hypertrophy.

The FASEB JournalVolume 24, Issue 9 p. 3555-3561 Research CommunicationFree to Read Coordinated maintenance of muscle cell size control by AMP-activated protein kinase Louise Lander, Louise Lander Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France INSERM U1016, Paris, FranceThese authors contributed equally to this work.Search for more papers by this authorRémi Mourner, Rémi Mourner Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France INSERM U1016, Paris, FranceThese authors contributed equally to this work.Search for more papers by this authorJocelyne Leclerc, Jocelyne Leclerc Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France INSERM U1016, Paris, FranceSearch for more papers by this authorMario Pende, Mario Pende INSERM U845, Paris, France Université Paris Descartes, UMRS-845, Paris, FranceSearch for more papers by this authorMarc Foretz, Marc Foretz Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France INSERM U1016, Paris, FranceSearch for more papers by this authorBenoit Viollet, Corresponding Author Benoit Viollet viollet@inserm.fr Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France INSERM U1016, Paris, France Correspondence: INSERM U1016, Institut Cochin, Département Endocrinologie, Métabolisme et Cancer, 24, rue du Faubourg St.-Jacques, 75014 Paris, France. E-mail: benoit. viollet@inserm.frSearch for more papers by this author Louise Lander, Louise Lander Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France INSERM U1016, Paris, FranceThese authors contributed equally to this work.Search for more papers by this authorRémi Mourner, Rémi Mourner Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France INSERM U1016, Paris, FranceThese authors contributed equally to this work.Search for more papers by this authorJocelyne Leclerc, Jocelyne Leclerc Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France INSERM U1016, Paris, FranceSearch for more papers by this authorMario Pende, Mario Pende INSERM U845, Paris, France Université Paris Descartes, UMRS-845, Paris, FranceSearch for more papers by this authorMarc Foretz, Marc Foretz Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France INSERM U1016, Paris, FranceSearch for more papers by this authorBenoit Viollet, Corresponding Author Benoit Viollet viollet@inserm.fr Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France INSERM U1016, Paris, France Correspondence: INSERM U1016, Institut Cochin, Département Endocrinologie, Métabolisme et Cancer, 24, rue du Faubourg St.-Jacques, 75014 Paris, France. E-mail: benoit. viollet@inserm.frSearch for more papers by this author First published: 11 May 2010 https://doi.org/10.1096/fj.10-155994Citations: 15 Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat ABSTRACT Skeletal muscle mass is regulated by signaling pathways that govern protein synthesis and cell proliferation, and the mammalian target of rapamycin (mTOR) plays a key role in these processes. Recent studies suggested the crucial role of AMP-activated protein kinase (AMPK) in the inhibition of protein synthesis and cell growth. Here, we address the role of AMPK in the regulation of muscle cell size in vitro and in vivo. The size of AMPK-deficient myotubes was 1.5-fold higher than for controls. A marked increase in p70S6K Thr389 and rpS6 Ser-235/236 phosphorylation was observed concomitantly with an up-regulation of protein synthesis rate. Treatment with rapamycin prevented p70S6K phosphorylation and rescued cell size control in AMPK-deficient cells. Importantly, myotubes lacking AMPK were resistant to further cell size increase beyond AMPK deletion alone, as MyrAkt-induced hypertrophy was absent in these cells. Moreover, in skeletal muscle-specific deficient AMPKα1/α2 KO mice, soleus muscle showed a higher mass with myofibers of larger size and was associated with increased p70S6K and rpS6 phosphorylation. Our results uncover the role of AMPK in the maintenance of muscle cell size control and highlight the crosstalk between AMPK and mTOR/p70S6K signaling pathways coordinating a metabolic checkpoint on cell growth.—Lantier, L., Mounier, R., Leclerc, J., Pende, M., Foretz, M., Viollet, B. Coordinated maintenance of muscle cell size control by AMP-activated protein kinase. FASEB J. 24, 3555–3561 (2010). www.fasebj.org Citing Literature Volume24, Issue9September 2010Pages 3555-3561 RelatedInformation

A defect in protein turnover underlies multiple forms of cell atrophy. Since S6 kinase (S6K)-deficient cells are small and display a blunted response to nutrient and growth factor availability, we have hypothesized that mutant cell atrophy may be triggered by a change in global protein synthesis. By using mouse genetics and pharmacological inhibitors targeting the mammalian target of rapamycin (mTOR)/S6K pathway, here we evaluate the control of translational target phosphorylation and protein turnover by the mTOR/S6K pathway in skeletal muscle and liver tissues. The phosphorylation of ribosomal protein S6 (rpS6), eukaryotic initiation factor-4B (eIF4B), and eukaryotic elongation factor-2 (eEF2) is predominantly regulated by mTOR in muscle cells. Conversely, in liver, the MAPK and phosphatidylinositol 3-kinase pathways also play an important role, suggesting a tissue-specific control. S6K deletion in muscle mimics the effect of the mTOR inhibitor rapamycin on rpS6 and eIF4B phosphorylation without affecting eEF2 phosphorylation. To gain insight on the functional consequences of these modifications, methionine incorporation and polysomal distribution were assessed in muscle cells. Rates and rapamycin sensitivity of global translation initiation are not altered in S6K-deficient muscle cells. In addition, two major pathways of protein degradation, autophagy and expression of the muscle-specific atrophy-related E3 ubiquitin ligases, are not affected by S6K deletion. Our results do not support a role for global translational control in the growth defect due to S6K deletion, suggesting specific modes of growth control and translational target regulation downstream of mTOR.

Rapamycin is an antibiotic inhibiting eukaryotic cell growth and proliferation by acting on target of rapamycin (TOR) kinase. Mammalian TOR (mTOR) is thought to work through 2 independent complexes to regulate cell size and cell replication, and these 2 complexes show differential sensitivity to rapamycin. Here we combine functional genetics and pharmacological treatments to analyze rapamycin-sensitive mTOR substrates that are involved in cell proliferation and tissue regeneration after partial hepatectomy in mice. After hepatectomy, hepatocytes proliferated rapidly, correlating with increased S6 kinase phosphorylation, while treatment with rapamycin derivatives impaired regeneration and blocked S6 kinase activation. In addition, genetic deletion of S6 kinase 1 (S6K1) caused a delay in S phase entry in hepatocytes after hepatectomy. The proliferative defect of S6K1-deficient hepatocytes was cell autonomous, as it was also observed in primary cultures and hepatic overexpression of S6K1-rescued proliferation. We found that S6K1 controlled steady-state levels of cyclin D1 (Ccnd1) mRNA in liver, and cyclin D1 expression was required to promote hepatocyte cell cycle. Notably, in vivo overexpression of cyclin D1 was sufficient to restore the proliferative capacity of S6K-null livers. The identification of an S6K1-dependent mechanism participating in cell proliferation in vivo may be relevant for cancer cells displaying high mTOR complex 1 activity and cyclin D1 accumulation.

In recent years several reports have linked mTORC1 (mammalian target of rapamycin complex 1) to lipogenesis via the SREBPs (sterol-regulatory-element-binding proteins). SREBPs regulate the expression of genes encoding enzymes required for fatty acid and cholesterol biosynthesis. Lipid metabolism is perturbed in some diseases and SREBP target genes, such as FASN (fatty acid synthase), have been shown to be up-regulated in some cancers. We have previously shown that mTORC1 plays a role in SREBP activation and Akt/PKB (protein kinase B)-dependent de novo lipogenesis. Our findings suggest that mTORC1 plays a crucial role in the activation of SREBP and that the activation of lipid biosynthesis through the induction of SREBP could be part of a regulatory pathway that co-ordinates protein and lipid biosynthesis during cell growth. In the present paper, we discuss the increasing amount of data supporting the potential mechanisms of mTORC1-dependent activation of SREBP as well as the implications of this signalling pathway in cancer.

Worldwide epidemics of metabolic diseases, including liver steatosis, are associated with an increased frequency of malignancies, showing the highest positive correlation for liver cancer. The heterogeneity of liver cancer represents a clinical challenge. In liver, the transcription factor PPARγ promotes metabolic adaptations of lipogenesis and aerobic glycolysis under the control of Akt2 activity, but the role of PPARγ in liver tumorigenesis is unknown. Here we have combined preclinical mouse models of liver cancer and genetic studies of a human liver biopsy atlas with the aim of identifying putative therapeutic targets in the context of liver steatosis and cancer. We have revealed a protumoral interaction of Akt2 signaling with hepatocyte nuclear factor 1α (HNF1α) and PPARγ, transcription factors that are master regulators of hepatocyte and adipocyte differentiation, respectively. Akt2 phosphorylates and inhibits HNF1α, thus relieving the suppression of hepatic PPARγ expression and promoting tumorigenesis. Finally, we observed that pharmacological inhibition of PPARγ is therapeutically effective in a preclinical murine model of steatosis-associated liver cancer. Taken together, our studies in humans and mice reveal that Akt2 controls hepatic tumorigenesis through crosstalk between HNF1α and PPARγ.

Employing inducible genetically engineered and orthotopic mouse models, we demonstrate a key role for transcriptional regulator Yap in maintenance of Kras-mutant pancreatic tumors. Integrated transcriptional and metabolomics analysis reveals that Yap transcribes Myc and cooperates with Myc to maintain global transcription of metabolic genes. Yap loss triggers acute metabolic stress, which causes tumor regression while inducing epigenetic reprogramming and Sox2 upregulation in a subset of pancreatic neoplastic cells. Sox2 restores Myc expression and metabolic homeostasis in Yap-deficient neoplastic ductal cells, which gradually re-differentiate into acinar-like cells, partially restoring pancreatic parenchyma in vivo. Both the short-term and long-term effects of Yap loss in inducing cell death and re-differentiation, respectively, are blunted in advanced, poorly differentiated p53-mutant pancreatic tumors. Collectively, these findings reveal a highly dynamic and interdependent metabolic, transcriptional, and epigenetic regulatory network governed by Yap, Myc, Sox2, and p53 that dictates pancreatic tumor metabolism, growth, survival, and differentiation.

Lysosomes serve dual antagonistic functions in cells by mediating anabolic growth signaling and the catabolic turnover of macromolecules. How these janus-faced activities are regulated in response to cellular nutrient status is poorly understood. We show here that lysosome morphology and function are reversibly controlled by a nutrient-regulated signaling lipid switch that triggers the conversion between peripheral motile mTOR complex 1 (mTORC1) signaling-active and static mTORC1-inactive degradative lysosomes clustered at the cell center. Starvation-triggered relocalization of phosphatidylinositol 4-phosphate (PI(4)P)-metabolizing enzymes reshapes the lysosomal surface proteome to facilitate lysosomal proteolysis and to repress mTORC1 signaling. Concomitantly, lysosomal phosphatidylinositol 3-phosphate (PI(3)P), which marks motile signaling-active lysosomes in the cell periphery, is erased. Interference with this PI(3)P/PI(4)P lipid switch module impairs the adaptive response of cells to altering nutrient supply. Our data unravel a key function for lysosomal phosphoinositide metabolism in rewiring organellar membrane dynamics in response to cellular nutrient status.

Oligodendrocytes and their progenitors (O-2A) express functional kainate- and DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-preferring glutamate receptors. The physiological consequences of activation of these receptors were studied in purified rat cortical O-2A progenitors and in the primary oligodendrocyte cell line CG-4. Changes in the mRNA levels of a set of immediate early genes were studied and were correlated to intracellular Ca2+ concentration, as measured by fura-2 Ca2+ imaging. Both in CG-4 and in cortical O-2A progenitors, basal mRNA levels of NGFI-A were much higher than c-fos, c-jun, or jun-b. Glutamate, kainate, and AMPA greatly increased NGFI-A mRNA and protein by activation of membrane receptors in a Ca(2+)-dependent fashion. Agonists at non-N-methyl-D-aspartate receptors promoted transmembrane Ca2+ influx through voltage-dependent channels as well as kainate and/or AMPA channels. The influx of Ca2+ ions occurring through glutamate-gated channels was sufficient by itself to increase the expression of NGFI-A mRNA. AMPA receptors were found to be directly involved in intracellular Ca2+ and NGFI-A mRNA regulation, because the effects of kainate were greatly enhanced by cyclothiazide, an allosteric modulator that selectively suppresses desensitization of AMPA but not kainate receptors. Our results indicate that glutamate acting at AMPA receptors regulates immediate early gene expression in cells of the oligodendrocyte lineage by increasing intracellular calcium. Consequently, modulation of these receptor channels may have immediate effects at the genomic level and regulate oligodendrocyte development at critical stages.

Neurons are highly dependent on astrocyte survival during brain damage. To identify genes involved in astrocyte function during ischemia, we performed mRNA differential display in astrocytes after oxygen and glucose deprivation (OGD). We detected a robust down-regulation of S6 kinase 1 (S6K1) mRNA that was accompanied by a sharp decrease in protein levels and activity. OGD-induced apoptosis was increased by the combined deletion of S6K1 and S6K2 genes, as well as by treatment with rapamycin that inhibits S6K1 activity by acting on the upstream regulator mTOR (mammalian target of rapamycin). Astrocytes lacking S6K1 and S6K2 (S6K1;S6K2^−/−) displayed a defect in BAD phosphorylation and in the expression of the anti-apoptotic factors Bcl-2 and Bcl-xL. Furthermore reactive oxygen species were increased while translation recovery was impaired in S6K-deficient astrocytes following OGD. Rescue of either S6K1 or S6K2 expression by adenoviral infection revealed that protective functions were specifically mediated by S6K1, because this isoform selectively promoted resistance to OGD and reduction of ROS levels. Finally, "<i>in vivo</i>" effects of S6K suppression were analyzed in the permanent middle cerebral artery occlusion model of ischemia, in which absence of S6K expression increased mortality and infarct volume. In summary, this article uncovers a protective role for astrocyte S6K1 against brain ischemia, indicating a functional pathway that senses nutrient and oxygen levels and may be beneficial for neuronal survival.

Lipin-1 deficiency is associated with massive rhabdomyolysis episodes in humans, precipitated by febrile illnesses. Despite well-known roles of lipin-1 in lipid biosynthesis and transcriptional regulation, the pathogenic mechanisms leading to rhabdomyolysis remain unknown. Here we show that primary myoblasts from lipin-1-deficient patients exhibit a dramatic decrease in LPIN1 expression and phosphatidic acid phosphatase 1 activity, and a significant accumulation of lipid droplets (LD). The expression levels of LPIN1-target genes [peroxisome proliferator-activated receptors delta and alpha (PPARδ, PPARα), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), acyl-coenzyme A dehydrogenase, very long (ACADVL), carnitine palmitoyltransferase IB and 2 (CPT1B and CPT2)] were not affected while lipin-2 protein level, a closely related member of the family, was increased. Microarray analysis of patients' myotubes identified 19 down-regulated and 51 up-regulated genes, indicating pleiotropic effects of lipin-1 deficiency. Special attention was paid to the up-regulated ACACB (acetyl-CoA carboxylase beta), a key enzyme in the fatty acid synthesis/oxidation balance. We demonstrated that overexpression of ACACB was associated with free fatty acid accumulation in patients' myoblasts whereas malonyl-carnitine (as a measure of malonyl-CoA) and CPT1 activity were in the normal range in basal conditions accordingly to the normal daily activity reported by the patients. Remarkably ACACB invalidation in patients' myoblasts decreased LD number and size while LPIN1 invalidation in controls induced LD accumulation. Further, pro-inflammatory treatments tumor necrosis factor alpha + Interleukin-1beta(TNF1α + IL-1ß) designed to mimic febrile illness, resulted in increased malonyl-carnitine levels, reduced CPT1 activity and enhanced LD accumulation, a phenomenon reversed by dexamethasone and TNFα or IL-1ß inhibitors. Our data suggest that the pathogenic mechanism of rhabdomyolysis in lipin-1-deficient patients combines the predisposing constitutive impairment of lipid metabolism and its exacerbation by pro-inflammatory cytokines.

Myostatin inhibition by follistatin (FS) offers a new approach for muscle mass enhancement. The aim of the present study was to characterize the mediators responsible for the FS hypertrophic action on skeletal muscle in male mice. Because IGF-I and IGF-II, two crucial skeletal muscle growth factors, are induced by myostatin inhibition, we assessed their role in FS action. First, we tested whether type 1 IGF receptor (IGF-IR) is required for FS-induced hypertrophy. By using mice expressing a dominant-negative IGF-IR in skeletal muscle, we showed that IGF-IR inhibition blunted by 63% fiber hypertrophy caused by FS. Second, we showed that FS caused the same degree of fiber hypertrophy in wild-type and IGF-II knockout mice. We then tested the role of the signaling molecules stimulated by IGF-IR, in particular the Akt/mammalian target of rapamycin (mTOR)/70-kDa ribosomal protein S6 kinase (S6K) pathway. We investigated whether Akt phosphorylation is required for the FS action. By cotransfecting a dominant-negative form of Akt together with FS, we showed that Akt inhibition reduced by 65% fiber hypertrophy caused by FS. Second, we evaluated the role of mTOR in FS action. Fiber hypertrophy induced by FS was reduced by 36% in rapamycin-treated mice. Finally, because the activity of S6K is increased by FS, we tested its role in FS action. FS caused the same degree of fiber hypertrophy in wild-type and S6K1/2 knockout mice. In conclusion, the IGF-IR/Akt/mTOR pathway plays a critical role in FS-induced muscle hypertrophy. In contrast, induction of IGF-II expression and S6K activity by FS are not required for the hypertrophic action of FS.

Defective hepatic insulin receptor (IR) signalling is a pathogenic manifestation of metabolic disorders including obesity and diabetes. The endo/lysosomal trafficking system may coordinate insulin action and nutrient homeostasis by endocytosis of IR and the autophagic control of intracellular nutrient levels. Here we show that class III PI3K--a master regulator of endocytosis, endosomal sorting and autophagy--provides negative feedback on hepatic insulin signalling. The ultraviolet radiation resistance-associated gene protein (UVRAG)-associated class III PI3K complex interacts with IR and is stimulated by insulin treatment. Acute and chronic depletion of hepatic Vps15, the regulatory subunit of class III PI3K, increases insulin sensitivity and Akt signalling, an effect that requires functional IR. This is reflected by FoxO1-dependent transcriptional defects and blunted gluconeogenesis in Vps15 mutant cells. On depletion of Vps15, the metabolic syndrome in genetic and diet-induced models of insulin resistance and diabetes is alleviated. Thus, feedback regulation of IR trafficking and function by class III PI3K may be a therapeutic target in metabolic conditions of insulin resistance.

On March 10 to March 12, 2015, the National Institute of Neurological Disorders and Stroke and the Tuberous Sclerosis Alliance sponsored a workshop in Bethesda, Maryland, to assess progress and new opportunities for research in tuberous sclerosis complex with the goal of updating the 2003 Research Plan for Tuberous Sclerosis (http://www.ninds.nih.gov/about_ninds/plans/tscler_research_plan.htm). In addition to the National Institute of Neurological Disorders and Stroke and Tuberous Sclerosis Alliance, participants in the strategic planning effort and workshop included representatives from six other Institutes of the National Institutes of Health, the Department of Defense Tuberous Sclerosis Complex Research Program, and a broad cross-section of basic scientists and clinicians with expertise in tuberous sclerosis complex along with representatives from the pharmaceutical industry. Here we summarize the outcomes from the extensive premeeting deliberations and final workshop recommendations, including (1) progress in the field since publication of the initial 2003 research plan for tuberous sclerosis complex, (2) the key gaps, needs, and challenges that hinder progress in tuberous sclerosis complex research, and (3) a new set of research priorities along with specific recommendations for addressing the major challenges in each priority area. The new research plan is organized around both short-term and long-term goals with the expectation that progress toward specific objectives can be achieved within a five to ten year time frame.

The role of the gluco-incretin hormones GIP and GLP-1 in the control of beta cell function was studied by analyzing mice with inactivation of each of these hormone receptor genes, or both. Our results demonstrate that glucose intolerance was additively increased during oral glucose absorption when both receptors were inactivated. After intraperitoneal injections, glucose intolerance was more severe in double- as compared to single-receptor KO mice, and euglycemic clamps revealed normal insulin sensitivity, suggesting a defect in insulin secretion. When assessed in vivo or in perfused pancreas, insulin secretion showed a lack of first phase in Glp-1R(-/-) but not in Gipr(-/-) mice. In perifusion experiments, however, first-phase insulin secretion was present in both types of islets. In double-KO islets, kinetics of insulin secretion was normal, but its amplitude was reduced by about 50% because of a defect distal to plasma membrane depolarization. Thus, gluco-incretin hormones control insulin secretion (a) by an acute insulinotropic effect on beta cells after oral glucose absorption (b) through the regulation, by GLP-1, of in vivo first-phase insulin secretion, probably by an action on extra-islet glucose sensors, and (c) by preserving the function of the secretory pathway, as evidenced by a beta cell autonomous secretion defect when both receptors are inactivated.

Factors that promote pancreatic beta cell growth and function are potential therapeutic targets for diabetes mellitus. In mice, genetic experiments suggest that signaling cascades initiated by insulin and IGFs positively regulate beta cell mass and insulin secretion. Akt and S6 kinase (S6K) family members are activated as part of these signaling cascades, but how the interplay between these proteins controls beta cell growth and function has not been determined. Here, we found that although transgenic mice overexpressing the constitutively active form of Akt1 under the rat insulin promoter (RIP-MyrAkt1 mice) had enlarged beta cells and high plasma insulin levels, leading to improved glucose tolerance, a substantial proportion of the mice developed insulinomas later in life, which caused decreased viability. This oncogenic transformation tightly correlated with nuclear exclusion of the tumor suppressor PTEN. To address the role of the mammalian target of rapamycin (mTOR) substrate S6K1 in the MyrAkt1-mediated phenotype, we crossed RIP-MyrAkt1 and S6K1-deficient mice. The resulting mice displayed reduced insulinemia and glycemia compared with RIP-MyrAkt1 mice due to a combined effect of improved insulin secretion and insulin sensitivity. Importantly, although the increase in beta cell size in RIP-MyrAkt1 mice was not affected by S6K1 deficiency, the hyperplastic transformation required S6K1. Our results therefore identify S6K1 as a critical element for MyrAkt1-induced tumor formation and suggest that it may represent a useful target for anticancer therapy downstream of mTOR.

Through analysis of mice with spatially and temporally restricted inactivation of Lpin1, we characterized its cell autonomous function in both white (WAT) and brown (BAT) adipocyte development and maintenance. We observed that the lipin 1 inactivation in adipocytes of aP2(Cre/+)/Lp(fEx2)(-)(3/fEx2)(-)(3) mice resulted in lipodystrophy and the presence of adipocytes with multilocular lipid droplets. We further showed that time-specific loss of lipin 1 in mature adipocytes in aP2(Cre-ERT2/+)/Lp(fEx2)(-)(3/fEx2)(-)(3) mice led to their replacement by newly formed Lpin1-positive adipocytes, thus establishing a role for lipin 1 in mature adipocyte maintenance. Importantly, we observed that the presence of newly formed Lpin1-positive adipocytes in aP2(Cre-ERT2/+)/Lp(fEx2)(-)(3/fEx2)(-)(3) mice protected these animals against WAT inflammation and hepatic steatosis induced by a high-fat diet. Loss of lipin 1 also affected BAT development and function, as revealed by histological changes, defects in the expression of peroxisome proliferator-activated receptor alpha (PPARα), PGC-1α, and UCP1, and functionally by altered cold sensitivity. Finally, our data indicate that phosphatidic acid, which accumulates in WAT of animals lacking lipin 1 function, specifically inhibits differentiation of preadipocytes. Together, these observations firmly demonstrate a cell autonomous role of lipin 1 in WAT and BAT biology and indicate its potential as a therapeutical target for the treatment of obesity.

Protein synthesis is traditionally associated with specific cytoplasmic compartments. We now show that OFD1, a centrosomal/basal body protein, interacts with components of the Preinitiation complex of translation (PIC) and of the eukaryotic Initiation Factor (eIF)4F complex and modulates the translation of specific mRNA targets in the kidney. We demonstrate that OFD1 cooperates with the mRNA binding protein Bicc1 to functionally control the protein synthesis machinery at the centrosome where also the PIC and eIF4F components were shown to localize in mammalian cells. Interestingly, Ofd1 and Bicc1 are both involved in renal cystogenesis and selected targets were shown to accumulate in two models of inherited renal cystic disease. Our results suggest a possible role for the centrosome as a specialized station to modulate translation for specific functions of the nearby ciliary structures and may provide functional clues for the understanding of renal cystic disease.

As a consequence of impaired glucose or fatty acid metabolism, bioenergetic stress in skeletal muscles may trigger myopathy and rhabdomyolysis. Genetic mutations causing loss of function of the LPIN1 gene frequently lead to severe rhabdomyolysis bouts in children, though the metabolic alterations and possible therapeutic interventions remain elusive. Here, we show that lipin1 deficiency in mouse skeletal muscles is sufficient to trigger myopathy. Strikingly, muscle fibers display strong accumulation of both neutral and phospholipids. The metabolic lipid imbalance can be traced to an altered fatty acid synthesis and fatty acid oxidation, accompanied by a defect in acyl chain elongation and desaturation. As an underlying cause, we reveal a severe sarcoplasmic reticulum (SR) stress, leading to the activation of the lipogenic SREBP1c/SREBP2 factors, the accumulation of the Fgf21 cytokine, and alterations of SR-mitochondria morphology. Importantly, pharmacological treatments with the chaperone TUDCA and the fatty acid oxidation activator bezafibrate improve muscle histology and strength of lipin1 mutants. Our data reveal that SR stress and alterations in SR-mitochondria contacts are contributing factors and potential intervention targets of the myopathy associated with lipin1 deficiency.

Article27 February 2017Open Access Transparent process ZRF1 is a novel S6 kinase substrate that drives the senescence programme Manuela Barilari Manuela Barilari Institut Necker-Enfants Malades, Paris, France Inserm, U1151, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Gregory Bonfils Gregory Bonfils Institut Necker-Enfants Malades, Paris, France Inserm, U1151, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Caroline Treins Caroline Treins Institut Necker-Enfants Malades, Paris, France Inserm, U1151, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Vonda Koka Vonda Koka Institut Necker-Enfants Malades, Paris, France Inserm, U1151, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Delphine De Villeneuve Delphine De Villeneuve Institut Necker-Enfants Malades, Paris, France Inserm, U1151, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Sylvie Fabrega Sylvie Fabrega Plateforme Vecteurs Viraux et Transfert de Gènes, IFR94, Hôpital Necker Enfants-Malades, Paris, France Search for more papers by this author Mario Pende Corresponding Author Mario Pende [email protected] orcid.org/0000-0002-7864-8937 Institut Necker-Enfants Malades, Paris, France Inserm, U1151, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Manuela Barilari Manuela Barilari Institut Necker-Enfants Malades, Paris, France Inserm, U1151, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Gregory Bonfils Gregory Bonfils Institut Necker-Enfants Malades, Paris, France Inserm, U1151, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Caroline Treins Caroline Treins Institut Necker-Enfants Malades, Paris, France Inserm, U1151, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Vonda Koka Vonda Koka Institut Necker-Enfants Malades, Paris, France Inserm, U1151, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Delphine De Villeneuve Delphine De Villeneuve Institut Necker-Enfants Malades, Paris, France Inserm, U1151, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Sylvie Fabrega Sylvie Fabrega Plateforme Vecteurs Viraux et Transfert de Gènes, IFR94, Hôpital Necker Enfants-Malades, Paris, France Search for more papers by this author Mario Pende Corresponding Author Mario Pende [email protected] orcid.org/0000-0002-7864-8937 Institut Necker-Enfants Malades, Paris, France Inserm, U1151, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Author Information Manuela Barilari1,2,3,‡, Gregory Bonfils1,2,3,‡, Caroline Treins1,2,3,‡, Vonda Koka1,2,3, Delphine De Villeneuve1,2,3, Sylvie Fabrega4 and Mario Pende *,1,2,3 1Institut Necker-Enfants Malades, Paris, France 2Inserm, U1151, Paris, France 3Université Paris Descartes, Sorbonne Paris Cité, Paris, France 4Plateforme Vecteurs Viraux et Transfert de Gènes, IFR94, Hôpital Necker Enfants-Malades, Paris, France ‡These authors contributed equally to this work *Corresponding author. Tel: +33 1 72 60 63 86; Fax: +33 1 72 60 64 01; E-mail: [email protected] The EMBO Journal (2017)36:736-750https://doi.org/10.15252/embj.201694966 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The inactivation of S6 kinases mimics several aspects of caloric restriction, including small body size, increased insulin sensitivity and longevity. However, the impact of S6 kinase activity on cellular senescence remains to be established. Here, we show that the constitutive activation of mammalian target of rapamycin complex 1 (mTORC1) by tuberous sclerosis complex (TSC) mutations induces a premature senescence programme in fibroblasts that relies on S6 kinases. To determine novel molecular targets linking S6 kinase activation to the control of senescence, we set up a chemical genetic screen, leading to the identification of the nuclear epigenetic factor ZRF1 (also known as DNAJC2, MIDA1, Mpp11). S6 kinases phosphorylate ZRF1 on Ser47 in cultured cells and in mammalian tissues in vivo. Knock-down of ZRF1 or expression of a phosphorylation mutant is sufficient to blunt the S6 kinase-dependent senescence programme. This is traced by a sharp alteration in p16 levels, the cell cycle inhibitor and a master regulator of senescence. Our findings reveal a mechanism by which nutrient sensing pathways impact on cell senescence through the activation of mTORC1-S6 kinases and the phosphorylation of ZRF1. Synopsis Aberrant mTORC1 signalling induces cell senescence via S6 kinase-mediated phosphorylation of epigenetic factor ZRF1, thereby linking nutritional cues to regulation of cell fitness. S6 kinases are required for the senescence programme triggered by constitutive mTORC1 activation. S6 kinases selectively regulate the p16 cell cycle inhibitor, without affecting the p53/p21-dependent response. A chemical genetic screen uncovers the epigenetic factor ZRF1 as a novel S6 kinase substrate, whose phosphorylation is triggered by nutritional cues and mTORC1 activity. ZRF1 phosphorylation by S6 kinases participates in the senescence response. Introduction Caloric restriction has lifespan benefits across different species, from unicellular organisms such as yeast to small primates and possibly humans (Longo et al, 2015). Among the intracellular signal transduction pathways adapting growth, metabolism and ageing to nutritional cues, the target of rapamycin complex 1 (TORC1) has a central role (Laplante & Sabatini, 2012). The serine/threonine kinase activity of TOR in TORC1 is upregulated by a large variety of anabolic signals, including amino acids, glucose, lipids, growth factor peptides, mitochondrial metabolites, oxygen and energy supplies. Genetic alterations of TORC1 elements affect ageing in yeast, flies, worms and mice (Lamming et al, 2013). The allosteric TOR inhibitor rapamycin has been reported to be the first longevity drug acting in mammals, that is, in genetically heterogeneous mice (Harrison et al, 2009). However, both caloric restriction and rapamycin treatments have some adverse effects on organismal physiology (Wilkinson et al, 2012). Deciphering the molecular targets downstream of mammalian TOR (mTOR) that specifically affect age-related disorders may help the design of safer interventions. S6 kinases 1 and 2 (S6Ks) are mTORC1 substrates that in turn possess serine/threonine kinase enzymatic activity (Dann et al, 2007). S6K1 and S6K2 are homologous proteins sharing similar modes of regulation and substrate specificities, which have been mainly inferred from the study of S6K1 (Pende et al, 2004). mTOR phosphorylates Thr-389 of S6K1 in the regulatory T loop, an event required for S6K1 activation. Among the known mTORC1 substrates, S6K1 phosphorylation requires a relatively high mTOR-specific activity (Kang et al, 2013). Hence, S6K1 phosphorylation is extremely sensitive to rapamycin treatment or nutrient levels, while the phosphorylation of other mTORC1 substrates is modulated to a lower extent. The modulation of S6K1 activity is therefore a sensitive functional adaptation to nutrient availability in the cell. Interestingly, genetic studies have revealed that S6K1 deletion in mice phenocopies a number of physiological adaptations to dietary restriction. Although S6K1-deficient mice have normal food intake, their body weight is reduced with a defect in cell size being particularly evident in metabolic tissues such as fat, skeletal muscle and pancreatic beta cells (Pende et al, 2000; Um et al, 2004; Ohanna et al, 2005; Aguilar et al, 2007). The insulin levels in their blood are low, while insulin sensitivity in peripheral tissues is increased. S6K1 mutant mice do not become obese when fed a high fat diet. Finally, their lifespan is longer as compared to control mice (Selman et al, 2009). Taken together, these findings suggest a linear pathway from nutrient availability to the control of growth and ageing through the activation of mTORC1 and S6K1. Senescence is an irreversible cell cycle arrest in the G1 phase (Munoz-Espin & Serrano, 2014). Although it can also be observed during embryonic development and after oncogenic insults, senescence increases during ageing (Lopez-Otin et al, 2013). In primary cell cultures, entry into senescence usually requires proliferation in the presence of DNA damage and is controlled by the tumour suppressors p53, retinoblastoma protein (Rb) and the gene products of the Ink4a/ARF locus, p16 and Arf (Munoz-Espin & Serrano, 2014). Consistent with its known role on growth and ageing, mTORC1 activity promotes senescence. Mouse embryonic fibroblasts (MEFs) with a constitutive activation of mTORC1, due to loss-of-function mutations in upstream negative regulators TSC1, TSC2 or PTEN (phosphatase and tensin homolog), senesce faster than wild type, and this effect is blocked by rapamycin (Zhang et al, 2003; Alimonti et al, 2010). Although the sensitivity to rapamycin and to the S6K1 pharmacological inhibitor PF-4708671 suggests an involvement of S6K1 in this process (Leontieva et al, 2013), whether and how S6 kinases control senescence remain to be established. The known S6K substrates belong to four broad classes (Dann et al, 2007; Ma & Blenis, 2009). The largest class are proteins involved in the RNA metabolism and protein synthesis: ribosomal protein S6 (rpS6), eukaryotic initiation factor 4B (eIF4B), PDCD4 (programmed cell death protein 4), eukaryotic elongation factor 2 kinase (eEF2K), carbamoyl-phosphate synthetase 2, aspartate transcarbamylase and dihydroorotase (CAD) and the β-subunit of chaperonin containing TCP1 (CCTβ). Second group are proteins involved in the retrograde control of insulin and mTOR signalling: insulin receptor substrate 1 (IRS1), mTOR, rictor (rapamycin insensitive companion of mTOR), Sin1 (stress-activated map kinase-interacting protein 1). Third are proteins involved in metabolic reprogramming: peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), glycogen synthase kinase-3-α and glycogen synthase kinase-3-β (GSK3α and β), AMP-activated protein kinase catalytic subunit-α (AMPKα). The fourth class are proteins with oncogenic or tumour suppressor activities: Bcl2-associated agonist of cell death (BAD), glioma-associated oncogene 1 (Gli1) and murine double-minute 2 protein (Mdm2). The molecular programme responsible for the age-related decline and senescence is still unclear. To determine the functional contribution of the S6 kinase signalling in cellular senescence control, we evaluated the impact of S6 kinases deletion in TSC1 mutant MEFs, which undergo premature senescence. We demonstrate that S6 kinases are required for the cellular senescence programme triggered by TSC1 mutation. By performing a chemical genetic screen to search for novel S6 kinase substrates, we identify ZRF1 as a molecular target relevant to this process. Results TSC1 and TSC2 deletion in MEFs is known to cause premature senescence (Zhang et al, 2003). To dissect the functional contribution of S6K activity, we scored the effects of the combined deletion of TSC1 and S6K1/2 on MEF senescence. In primary MEFs after adenoviral Cre-mediated deletion of the TSC1-floxed allele, increased rpS6 phosphorylation was abrogated by the concomitant deletion of S6K1/2 (Fig 1A). As expected, Akt phosphorylation inversely correlated with S6K activity, due to the negative feedback loop of mTORC1/S6K on mTORC2/Akt (Um et al, 2004; Hsu et al, 2011; Yu et al, 2011). Consistent with previous studies, TSC1-deficient cells, after adenoviral Cre transduction, underwent early senescence, as assessed by senescence-associated (SA) β-galactosidase staining (Fig 1B) and population doubling time (Fig 1C). The senescence programme was accompanied by upregulation of cell cycle arrest and senescence markers. These included p53 and its transcriptional target, the cell cycle inhibitor p21, together with p53-independent responses, such as the upregulation of the cell cycle inhibitor p16 (Fig 1A). As expected, the regulation of p16 and p21 abundance was at the mRNA level, while p53 was controlled post-transcriptionally (Fig 1D). Strikingly, S6K1/2 deletion blunted the senescence response (Fig 1B and C). At the molecular level, S6K1/2 deletion selectively affected p16 levels while the p53 response was spared (Fig 1A and D). Figure 1. S6 kinases control p16 expression and the senescence response in TSC1 mutant cells Immunoblot analysis of Tsc1fl/fl and Tsc1fl/fl S6K1−/−S6K2−/− primary MEFs transduced with GFP or GFP-Cre adenovirus and harvested from 9 to 12 days post-infection (passage P6 or P7). Proteins were analysed by Western blot using the indicated antibodies. Quantification by densitometric analyses of p53, p21 and p16 protein levels is presented as a graph. Data are normalized to β-actin and expressed as a fold change relative to the control cells. Data are presented as mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, ANOVA multiple comparisons. Bright-field images of β-galactosidase staining performed on Tsc1fl/fl and Tsc1fl/fl S6K1−/−S6K2−/− primary MEFs, transduced with GFP or GFP-Cre adenovirus at passage P3 and fixed 12 days post-infection, at passage P7. Scale bar: 50 μm. Data are presented in a graph as mean ± SEM of at least three independent experiments; ****P < 0.0001, n.s. not significant, ANOVA multiple comparisons. Population doubling analysis of Tsc1fl/fl and Tsc1fl/fl S6K1−/−S6K2−/− primary MEFs transduced at P1 with GFP or GFP-Cre adenoviruses was determined for nine passages. Data are presented in the curve as mean ± SEM of at least three independent experiments; *P < 0.05, multiple t-tests. Quantitative RT–PCR detection of the level of expression of the indicated genes in Tsc1fl/fl and Tsc1fl/fl S6K1−/−S6K2−/− primary MEFs transduced at P3 with GFP or GFP-Cre adenovirus and collected at passage P7. Expression levels are corrected for expression of the control gene (β-actin) and presented in the graph as fold changes relative to control cells. The values plotted are means ± SEM of three independent experiments; *P < 0.05, multiple t-tests. Download figure Download PowerPoint Since oncogene-induced senescence may be a consequence of hyperproliferation and DNA damage (Munoz-Espin & Serrano, 2014), the levels of DNA damage response mediators, phosphorylated histone H2AX (γ-H2AX) and 53BP1 foci, were measured by immunofluorescence at late passage P7 (Fig 2A and B). The deletions of TSC1 and S6K1/2 were not associated with these markers of DNA damage. In addition, S6K deletion at early passages P1 and P2 did not inhibit cell proliferation [data not shown and (Pende et al, 2004)]. Taken together, our data demonstrate that S6Ks are specifically required for the p16 branch of the senescence programme downstream of mTORC1 activation, without causing DNA damage and hyperproliferation. Interestingly, the overexpression of p16 has been shown to induce senescence in the absence of DNA damage (Coppe et al, 2011). Figure 2. TSC1 and S6 kinase deletion does not cause DNA damage-associated foci A, B. Tsc1fl/fl and Tsc1fl/fl S6K1−/−S6K2−/− primary MEFs were transduced with GFP or GFP-Cre adenovirus. Twelve days post-infection (passage P7) cells were fixed and analysed by immunofluorescence using the indicated antibodies. The formation of γH2AX or 53BP1 foci per cell was analysed in three independent experiments. Control cells treated with etoposide (6 μg/ml) overnight were used as a positive control (n = 3 cultures). Scale bars: 5 μm. Download figure Download PowerPoint As an additional strategy to inhibit S6Ks, MEFs were treated with the selective S6K inhibitor PF-4708671 for 6 days starting at passage P5. The pharmacological treatment was sufficient to reduce senescence-associated β-galactosidase staining (Fig 3A) and p16 expression of TSC1 deleted cells both at protein and at RNA level (Fig 3B and C). The downregulation of p16 expression by lentiviral transduction of specific short hairpin RNA (shRNA) mimicked the effects of S6K inhibition on senescence-associated β-galactosidase staining (Fig 3A). Moreover, the adenoviral-mediated overexpression of p16 restored the senescence programme in PF-4708671-treated TSC1 deleted cells (Fig 3A). Taken together, these data support the positive relationship between S6K activity and p16 expression in the control of TSC1 mutant cell senescence. Figure 3. p16 expression modulates the senescence programme during TSC1 deletion or S6K inhibition Representative bright-field images of SA β-galactosidase staining performed on cells fixed at passage P7. After Cre-mediated TSC1 deletion, primary MEFs at passage P5 were treated with the S6K1 inhibitor PF-4708671 (10 μM), treated with PF-4708671 and simultaneously transduced with Adeno-GFP-p16 or transduced with LV-sh-p16 or LV-sh-Scramble, as control. PF-4708671 or DMSO was added every 24 h. Scale bar: 50 μm. Quantification of β-galactosidase-positive cells was performed on three independent experiments and is shown in the graph on the right. Data are presented as mean ± SEM; *P < 0.05, ***P < 0.001, ANOVA multiple comparisons. Representative immunoblot analyses of MEF primary cells treated as in (A) and harvested at passage P7. * indicates unspecific band. Densitometric analyses of actin-normalized signal are presented in a graph as fold changes relative to the control. Data are presented as mean ± SEM of three experiments. *P < 0.05, ***P < 0.001, two-tailed, unpaired Student's t-test. Relative transcript level of p16 of MEF primary cells treated as in (A) and harvested at passage P7. Data are presented as mean ± SEM of three experiments. *P < 0.05, **P < 0.01, ANOVA multiple comparisons. Download figure Download PowerPoint To find novel S6 kinase substrates potentially implicated in senescence, we set up a chemical genetic screen. The mutation of the ATP-binding pocket in protein kinases may open the access of the catalytic domain to ATP thiophosphate analogues harbouring substitutions with bulky residues, which cannot be used by the majority of wild-type kinases. After the kinase reaction with ATP thiophosphate analogues in digitonin-permeabilized cells, the alkylation of thiophosphorylated proteins and the detection with a thiophosphate-ester-specific antibody allows us to assess whether a protein is a direct substrate of the mutant kinase in a cellular environment (Hertz et al, 2010; Banko et al, 2011). To adapt the technique for S6K, we mutated the gatekeeper residue Lys149 of S6K1 to Gly and compared the ability of the mutant kinase vs. wild type to thiophosphorylate the well-characterized S6K substrate ribosomal protein S6 (rpS6) by using benzyl-ATP thiophosphate in human embryonic kidney HEK293 cells. As shown in Fig 4A, the mutant kinase, but not the endogenous nor the ectopically expressed wild-type kinase (WT-S6K1), was able to use the ATP analogue and thiophosphorylate HA-tag rpS6. The mutation of the Ser residues to Ala in the carboxy-terminal tail of HA-tag rpS6 was sufficient to abolish thiophosphorylation, suggesting that the mutant kinase is active and specifically recognize the S6K phosphorylation sites. The mutant kinase was therefore named analogue-specific S6K1 (AS-S6K1). Figure 4. Validation of analogue-specific S6K1 mutation A. Myc-WT-S6K1 and myc-AS-S6K1 were transfected in HEK293 cells with HA-RpS6WT or HA-RpS65A. In vivo kinase assay was performed in the presence of 6-Bn-ATP-γ-S. After immunoprecipitation using an anti-HA antibody, the thiophosphorylation of RpS6 was revealed by Western blot using an anti-thiophosphate ester antibody. Expression level of myc-WT-S6K1 and myc-AS-S6K1 or total S6K1 was revealed by Western blot on total extracts using the indicated antibody. B, C. HEK293 or U2OS cells stably expressing myc-WT-S6K1 (WT) or myc-AS-S6K1 (AS) were transfected with tagged forms of S6K1 substrates (CAD, eIF4B, eEF2K, SKAR, IRS1, RpS6, PDCD4) or PRAS40. In vivo kinase assay was performed in the presence of 6-Bn-ATP-γ-S. After immunoprecipitation, the thiophosphorylation was revealed by Western blot using an anti-thiophosphate ester antibody. * indicates unspecific band. Download figure Download PowerPoint A series of S6K substrates, including rpS6, CAD, eIF4B, PDCD4, eEF2K, SKAR, IRS1, were tested for thiophosphorylation by AS-S6K1 and WT-S6K1 in HEK293 cells (Fig 4B). Generally, the expression of AS-S6K1 resulted in a sharp increase in detection by the thiophosphate-ester-specific antibody after in situ kinase reaction and derivatization, as compared to WT-S6K1. For a minority of targets, such as IRS1 and eEF2K, a relatively high background signal was already detected in WT-S6K1 expressing cells, which in the case of IRS1 was not further increased in AS-S6K1 expressing cells. Despite these exceptions, the AS-S6K1 can promote thiophosphorylation for the large majority of reported S6K substrates. Importantly, PRAS40 protein, which contains multiple residues phosphorylated by mTOR and Akt but not S6K (Sancak et al, 2007), was not thiophosphorylated by AS-S6K1. This was particularly encouraging as Akt and S6K share a similar consensus site for phosphorylation, with Ser-Thr residues surrounded by basic residues in the −3 and −5 positions. These results were confirmed in another cell type, that is the human osteosarcoma U2OS line (Fig 4C). Therefore, although some background noise may preclude the purification of endogenous substrates in the whole proteome, the AS-S6K1 is a valuable tool to reveal direct phosphorylation of candidate proteins in the cellular environment. Quantitative proteomic analyses have recently identified hundreds of proteins that are differentially phosphorylated after pharmacological treatment with rapamycin or mTOR catalytic inhibitors, or after genetic manipulation of TORC1/TORC2 components (Moritz et al, 2010; Hsu et al, 2011; Yu et al, 2011; Robitaille et al, 2013). From these databases, we selected a list of 10 proteins harbouring a putative S6K consensus site for phosphorylation and a reported function in cell cycle control, senescence and insulin sensitivity. This short list included zuotin-related factor 1 (ZRF1), homeobox protein cut-like 1 (Cux1), N-myc downstream-regulated gene 1 protein (NDRG1), nucleophosmin (NPM1), La-related protein 1 (Larp1), lamin A, JunB, 75-kDa glucose-regulated protein (Grp75), euchromatic histone-lysine N-methyltransferase 2 (EHMT2) and enhancer of mRNA-decapping protein 3 (EDC3). Next, we asked whether a Flag-tagged version could be directly thiophosphorylated by AS-S6K1. As an arbitrary cut-off to select for bona fide substrates, the following criteria were used (i) protein thiophosphorylation is increased more than fivefold in AS-S6K1 expressing cells as compared to WT-S6K1 expressing cells, and (ii) protein thiophosphorylation is confirmed in two distinct cell types. NPM1 (Fig 5A), NDRG1, Larp1, JunB, Grp75, EHMT2 and EDC3 (data not shown) gave negative results, while ZRF1, Cux1 and lamin A matched these criteria (Fig 5A) and were therefore selected as bona fide novel substrates of S6K1. Figure 5. Analogue-specific S6K1 mutation to screen for direct in vivo substrates HEK293 cells stably expressing myc-WT-S6K1 (WT) or myc-AS-S6K1 (AS), or U2OS transduced with adeno-myc-WT-S6K1 (WT) or adeno-myc-AS-S6K1 (AS), were transfected with tagged forms of candidates (lamin A, ZRF1, CUX1, NPM1). In vivo kinase assay were performed in the presence of 6-Bn-ATP-γ-S. After immunoprecipitation, the thiophosphorylation was revealed by Western blot using an anti-thiophosphate ester antibody. * indicates unspecific band. HEK293 cells stably expressing myc-WT-S6K1 (WT) or myc-AS-S6K1 (AS) were transfected with Flag-tagged forms of ZRF1 or a mutant of ZRF1, ZRF1S47A. In vivo kinase assay was performed in the presence of 6-Bn-ATP-γ-S. After immunoprecipitation using an anti-Flag antibody, the thiophosphorylation was revealed by Western blot using an anti-thiophosphate ester antibody. HEK293 was transfected with Flag-ZRF1WT or Flag-ZRF1S47A mutant plasmids. Twenty-four hours post-transfection, cells were starved overnight and treated for 3 h with Torin 1 (100 nM). After immunoprecipitation with anti-Flag antibody, an in vitro kinase assay was performed with a recombinant active S6K1. ZRF1 phosphorylation was analysed by immunoblotting. Download figure Download PowerPoint The known biological function of ZRF1 (aka DNAJC2, MIDA1 and Mpp11) drew our attention as a particularly interesting target, as in the nucleus ZRF1 may act as an epigenetic factor or transcriptional co-regulator influencing cell fate determination and senescence (Richly et al, 2010; Ribeiro et al, 2013; Aloia et al, 2014). Cytosolic ZRF1 may act as a co-chaperon forming the ribosome-associated complex (RAC) together with heat-shock 70-kDa protein 14 (HspA14) for the proper folding of nascent polypeptides at the tunnel exit of ribosomes (Hundley et al, 2005; Jaiswal et al, 2011). We therefore decided to further validate whether ZRF1 is a novel substrate of S6K. First, we determined the phosphorylation site in the ZRF1 protein by AS-S6K1. Human ZRF1 contains a Ser47 that harbours a RXRXXS motif and has been previously shown to be differentially phosphorylated following wortmannin, rapamycin and Torin treatments (Moritz et al, 2010; Hsu et al, 2011; Robitaille et al, 2013). This residue is conserved across vertebrates (Fig EV1). Interestingly, unicellular organisms, such as yeast, have a threonine residue in the homologous region that has been shown to be differentially phosphorylated after rapamycin treatment (Claudio de Virgilio, personal communication). Mutation of Ser47 to Ala in the mouse protein sequence was sufficient to completely abrogate thiophosphorylation by AS-S6K1 (Fig 5B), indicating that this is likely to be the unique phosphorylation site. Next, we addressed whether ZRF1 was a direct substrate of recombinant S6K in in vitro kinase reactions. Unphosphorylated Flag-tag wild-type ZRF1 or the Ser47 to Ala mutant was immunopurified from protein extracts of HEK293 cells that were previously transfected with ZRF1-coding plasmids and treated with the mTOR inhibitor Torin 1. As shown in Fig 5C, recombinant S6K1 protein was able to phosphorylate wild-type ZRF1, but not the Ser47 to Ala mutant, as detected by using a ZRF1 Ser47 phospho-specific antibody. Click here to expand this figure. Figure EV1. Sequence homology of ZRF1 phosphorylation motif across speciesAlignment of amino acid sequences of ZRF1 from different species encompassing the S6K consensus phosphorylation site motif (R/K-X-R/K-X-X-S/T) underlined; the arrow indicates the phosphorylation site. Download figure Download PowerPoint Next, endogenous ZRF1 phosphorylation by endogenous S6K was analysed in vivo and in cultured cells. In liver tissue from wild-type mice, ZRF1 Ser47 phosphorylation was sensitive to mTOR inhibition by rapamycin treatment (Fig 6A). In addition, overnight starvation and 3 h-refeeding, respectively, down- and upregulated ZRF1 phosphorylation in liver, white adipose tissue (WAT) and skeletal muscle (Figs 6B and EV2A). Strikingly, the combined deletion of S6 kinases 1 and 2 abolished ZRF1 phosphorylation, similar to rpS6 phosphorylation (Figs 6B and EV2A). However, S6K1 and S6K2 appeared to have relatively distinct activities towards rpS6 and ZRF1. As expected, rpS6 was a preferential substrate for S6K2, while S6K1 deletion had more potent effects than S6K2 on phospho-ZRF1 vs. phospho-rpS6 (Fig 6C). In TSC2-deficient immortalized cells, ZRF1 and rpS6 phosphorylation was insensitive to serum and amino acid withdrawal, as opposed to wild-type Tsc2+/+ cells (Figs 6D and EV2B). Importantly, in the same experimental setting to study premature senescence, that is primary MEFs after adenoviral Cre-mediated deletion of the TSC1-floxed allele (Fig 1A), the increased ZRF1 phosphorylation was abrogated by the concomitant deletion of S6K1/2 (Figs 6E and EV2C) and by the S6K inhibitor PF-4708671 (Fig 3B). Taken together, our data delineate a pathway leading to ZRF1 phosphorylation by S6K1 and S6K2 in response to nutrient availability or as a consequence of genetic perturbations activating mTORC1. Figure 6. ZRF1 is a novel S6 kinase substrate in mouse tissues in vivo and in primary MEFs Immunoblot analysis of proteins extracted from liver of WT mice that were starved overnight and refed for 4 h. When indicated, mice were injected intraperitoneally with 5 mg/kg of the rapamycin derivate temsirolimus 1 h before refeeding (n = 3 mice for each condition). Immunoblot analysis of proteins extracted from liver and WAT of WT and S6K1−/−S6K2−/− mice that were starved overnight or starved overnight and refed for 4 h. Proteins were analysed by Western blot using the indicated antibodies (n = 3 mice for each genotype). Immunoblot analysis of proteins extracted from liver of WT, S6K1−/−, S6K2−/− and S6K1−

Unlike immature neurons and the ones from the peripheral nervous system (PNS), mature neurons from the central nervous system (CNS) cannot regenerate after injury. In the past 15 years, tremendous progress has been made to identify molecules and pathways necessary for neuroprotection and/or axon regeneration after CNS injury. In most regenerative models, phosphorylated ribosomal protein S6 (p-RPS6) is up-regulated in neurons, which is often associated with an activation of the mTOR (mammalian target of rapamycin) pathway. However, the exact contribution of posttranslational modifications of this ribosomal protein in CNS regeneration remains elusive. In this study, we demonstrate that RPS6 phosphorylation is essential for PNS and CNS regeneration in mice. We show that this phosphorylation is induced during the preconditioning effect in dorsal root ganglion (DRG) neurons and that it is controlled by the p90S6 kinase RSK2. Our results reveal that RSK2 controls the preconditioning effect and that the RSK2-RPS6 axis is key for this process, as well as for PNS regeneration. Finally, we demonstrate that RSK2 promotes CNS regeneration in the dorsal column, spinal cord synaptic plasticity, and target innervation leading to functional recovery. Our data establish the critical role of RPS6 phosphorylation controlled by RSK2 in CNS regeneration and give new insights into the mechanisms related to axon growth and circuit formation after traumatic lesion.

Abstract The axonal growth‐associated protein GAP‐43 is believed to play some role in the synaptic remodelling that takes place in the hippocampus of adult rats after certain experimental lesions. GAP‐43 mRNA is highly expressed in adult CA3 pyramidal cells but almost absent in the dentate granule cells. We analysed whether the sprouting of granule cell axons, the mossy fibres of the hippocampus, caused by kainic acid‐induced seizures in adult rats was associated with any induction of GAP‐43 mRNA in granule cells and with any changes in the immunostaining pattern of GAP‐43 in the hippocampus. Increased GAP‐43 mRNA expression was found to be induced in granule cells 18, 24 and 30 h after a systemic injection of kainic acid which induced generalized seizures in adult rats, and returned to control levels by 48 h post‐treatment. No effect was observed in other regions of the hippocampus. However, when kainic acid was injected into 15‐day‐old rats, which responded with generalized seizures but no sprouting of mossy fibres, there was no induction of GAP‐43 mRNA in the granule cells, suggesting a close relation between GAP‐43 expression and sprouting of these cells. Seven days after kainic acid injections, GAP‐43 immunostaining was decreased in the inner molecular layer of the dentate gyrus except for a thin supragranular band, whereas 30 days after treatment all animals showed increased GAP‐43 immunoreactivity in the whole inner molecular layer. Since collaterals of mossy fibres grow in the inner molecular layer after kainic acid‐induced seizures, these results support the theory that GAP‐43 plays a role in synaptic remodelling in the adult central nervous system.

Kainic acid-induced seizures, in adult rats produce neurodegeneration in the hippocampus followed by sprouting of the mossy fibres in the inner molecular layer of the dentate gyrus and changes in GAP-43 expression in the granule cells. In the present study we observed that 4 days after kainic acid injection a dense plexus of silver-impregnated degenerating terminals detected by Gallyas's method and a decrease of GAP-43 immunostaining was observed in the inner molecular layer of the dentate gyrus indicating deafferentiation of this region. This was associated with the formation of an intense GAP-43 immunostained band in the supragranular layer. MK-801, a non-competitive inhibitor of the NMDA receptor, which partially inhibited the behavioural seizures induced by KA, also protected from the inner molecular layer deafferentation and markedly reduced the expression of GAP-43 mRNA in the granule cells and the intense GAP-43 immunostained band in the supragranular layer, suggesting a relationship among these events. Two months after kainic acid injection the intense supragranular GAP-43 positive band was no longer evident but the whole inner molecular layer appeared more labelled in association with the formation of the collateral sprouting of the mossy fibres in the inner molecular layer as detected by Timm's staining. These effects were also markedly reduced by the pretreatment with MK-801. Taken together, these experiments indicate for the first time a direct relationship between the increase of GAP-43 immunostaining in the inner molecular layer of the dentate gyrus and the collateral sprouting of mossy fibres in this district in response to kainic acid induced seizures. This further supports the hypothesis that the early induction of GAP-43 in granule cells may be one of the molecular mechanisms required for the synaptic reorganization of the mossy fibres.

To delineate the roles of the lactogens and GH in the control of perinatal and postnatal growth, fat deposition, insulin production, and insulin action, we generated a novel mouse model that combines resistance to all lactogenic hormones with a severe deficiency of pituitary GH. The model was created by breeding PRL receptor (PRLR)-deficient (knockout) males with GH-deficient (little) females. In contrast to mice with isolated GH or PRLR deficiencies, double-mutant (lactogen-resistant and GH-deficient) mice on d 7 of life had growth failure and hypoglycemia. These findings suggest that lactogens and GH act in concert to facilitate weight gain and glucose homeostasis during the perinatal period. Plasma insulin and IGF-I and IGF-II concentrations were decreased in both GH-deficient and double-mutant neonates but were normal in PRLR-deficient mice. Body weights of the double mutants were reduced markedly during the first 3-4 months of age, and adults had striking reductions in femur length, plasma IGF-I and IGF binding protein-3 concentrations, and femoral bone mineral density. By age 6-12 months, however, the double-mutant mice developed obesity, hyperleptinemia, fasting hyperglycemia, relative hypoinsulinemia, insulin resistance, and glucose intolerance; males were affected to a greater degree than females. The combination of perinatal growth failure and late-onset obesity and insulin resistance suggests that the lactogen-resistant/GH-deficient mouse may serve as a model for the development of the metabolic syndrome.

The innate immune response is influenced by the nutrient status of the host. Mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase 1 (ERK1) and ERK2, are activated after the stimulation of macrophages with bacterial lipopolysaccharide (LPS) and are necessary for the optimal production of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha). We uncovered a role for the extracellular nutrient arginine in the activation of ERK1/2 in LPS-stimulated macrophages. Arginine facilitated the activation of MAPKs by preventing the dephosphorylation and inactivation of the MAPK kinase kinase tumor-promoting locus 2 (TPL-2). Starvation of mice decreased the concentration of arginine in the plasma and impaired the activation of ERK1/2 by LPS. Supplementation of starved mice with arginine promoted the subsequent activation of ERK1/2 and the production of TNF-alpha in response to LPS. Thus, arginine is critical for two aspects of the innate immune response in macrophages: It is the precursor used in the generation of the antimicrobial mediator nitric oxide, and it facilitates MAPK activation and consequently cytokine production.

To sustain increased growth, rapidly proliferating cells, such as tumour cells, undergo metabolic adaptations. In recent years, the mechanisms of glycolysis activation as a key metabolic adaptation in proliferating cells became the topic of intense research. Although this phenomenon was described more than 50 years ago by Otto Warburg, the molecular mechanisms remained elusive. Only recently, it was demonstrated that the expression of specific glycolytic enzymes, namely PKM2 (pyruvate kinase M2) and HK2 (hexokinase 2), occurs simultaneously with the glycolytic addiction of cancer cells. The PI3K (phosphoinositide 3-kinase)/mTOR [mammalian (or mechanistic) target of rapamycin] signalling pathway is a central signalling hub co-ordinating the growth in response to growth factor signalling and nutrient availability. Not surprisingly, it is found to be activated in the majority of the tumour cells. In the present article, we discuss the requirement of different PI3K/mTOR downstream effectors for the metabolic adaptation in liver cancer cells driven by this signalling pathway. We provide evidence for a selective involvement of the mTOR target Akt2 in tumoral growth. In addition, PTEN (phosphatase and tensin homologue deleted on chromosome 10)-negative human hepatocellular carcinoma cell lines display an up-regulation of PKM2 expression in an Akt2-dependent manner, providing an advantage for cell proliferation and anchorage-independent growth. Our data have implications on the link between the metabolic action of insulin signal transduction and tumorigenesis, identifying Akt2 as a potential therapeutical target in liver malignancies depending on cancer genotype.

Abstract mTOR activation is essential and sufficient to cause polycystic kidneys in Tuberous Sclerosis Complex (TSC) and other genetic disorders. In disease models, a sharp increase of proliferation and cyst formation correlates with a dramatic loss of oriented cell division (OCD). We find that OCD distortion is intrinsically due to S6 kinase 1 (S6K1) activation. The concomitant loss of S6K1 in Tsc1 -mutant mice restores OCD but does not decrease hyperproliferation, leading to non-cystic harmonious hyper growth of kidneys. Mass spectrometry-based phosphoproteomics for S6K1 substrates revealed Afadin, a known component of cell-cell junctions required to couple intercellular adhesions and cortical cues to spindle orientation. Afadin is directly phosphorylated by S6K1 and abnormally decorates the apical surface of Tsc1 -mutant cells with E-cadherin and α-catenin. Our data reveal that S6K1 hyperactivity alters centrosome positioning in mitotic cells, affecting oriented cell division and promoting kidney cysts in conditions of mTOR hyperactivity.

The depolarization-evoked release of endogenous glutamate (GLU) and -aspartate (ASP) and its modulation mediated by γ-aminobutyric acid (GABA) heteroreceptors was investigated in superfused rat cerebrocortical synaptosomes. Exposure to 12 mM K+ enhanced the release of GLU and ASP. The K+-evoked overflow of both amino acids was largely Ca2+-dependent. Exogenous GABA inhibited the K+-evoked overflow of GLU (EC50 2.8 μM) and ASP (EC50 2.7 μM). The effect of GABA was mimicked by the GABAB receptor agonist (−)-baclofen (EC50 2.0 μM for GLU and 1.3 μM for ASP release) but not by the GABAA receptor agonist muscimol, up to 100 μM. Accordingly, the GABA-induced inhibition of GLU and ASP release was not affected by the GABAA receptor antagonists, bicuculline or picrotoxin, but was antagonized by the GABAB receptor antagonist, 3-amino-propyl(diethoxymethyl)phosphinic acid (CGP 35348). The GABA effect was, however, insensitive to another GABAB receptor antagonist, phaclofen, up to 1,000 μM. It can be concluded that GABA heteroreceptors of the GABAB type regulating the depolarization-evoked release of GLU and ASP are present on cortical GLU/ASP-releasing nerve terminals. These receptors may be classified as a phaclofen-insensitive GABAB receptor subtype.

Release‐regulating α 2 ‐autoreceptors in human brain were characterized pharmacologically in cortical slices from patients undergoing neurosurgery to remove subcortical tumours; the slices were prelabelled with [ 3 H]‐noradrenaline ([ 3 H]‐NA) and stimulated electrically (3 Hz, 2 ms, 24 mA) under superfusion conditions. The stimulus‐evoked tritium overflow was almost totally Ca 2+ ‐dependent and tetrodotoxin‐sensitive. Clonidine and oxymetazoline 0.01 to 1 μ m inhibited in a concentration‐dependent manner the evoked overflow of tritium. The two drugs were equipotent (EC 50 = 0.03 μ m ) and their maximal effect was approx. 45%. Phenylephrine and methoxamine, up to 1 μ m , did not affect tritium overflow. Yohimbine (0.01–0.1 μ m ) shifted the concentration‐response curve of clonidine to the right. The calculated pA 2 value was 8.29. Prazosin and 2‐[2‐[4‐( o ‐methoxyphenyl)piperazine‐1‐yl]ethyl]‐4,4‐dimethyl‐1,3(2H,4H)‐isoquinolinedione (AR‐C 239), tested at 0.3 μ m , did not modify the concentration‐response curve of clonidine. The effect of clonidine was antagonized by (+)‐mianserin (pA 2 = 7.74), but not by up to 0.3 μ m of the (−)‐enantiomer. The concentration‐response curve of clonidine was shifted to the right by the novel α 2 ‐adrenoceptor antagonist, 5‐chloro‐4‐(1‐butyl‐1,2,5,6‐tetrahydropyridin‐3‐yl)‐thiazole‐2‐amine (Z)‐2‐butenedioate (1:1) salt (ORG 20350) (pA 2 = 7.55). Yohimbine, (+)‐mianserin and ORG 20350, but not prazosin and (−)‐mianserin, increased the electrically‐evoked tritium overflow, suggesting that autoreceptors may be tonically activated by endogenous NA. Desipramine (1 μ m ) increased evoked tritium overflow from human cortex slices. The effect of clonidine (0.01 − 1 μ m ) on the evoked overflow of tritium was reduced in presence of 1 μ m desipramine. It is proposed that autoregulation of NA release can occur in human cerebral cortex. The process involves activation of α 2 ‐adrenoceptors which may be either the α 2A or the α 2D subtype.

Sustained activation of AMP-activated protein kinase (AMPK) induces apoptosis in several cell types. In pancreatic beta cells this occurs under glucose limitation, or in the presence of the pharmacological AMPK activator 5-aminoimidazole-4-carboxamide-riboside (AICAR). It is unknown whether Akt activation can counteract AMPK-mediated apoptosis, nor whether mTOR activation downstream of Akt mediates any survival signal in these conditions. We report that expression of a constitutively active form of Akt increases mTOR activity and prevents apoptosis upon AMPK activation. Akt-mediated survival was inhibited by rapamycin. Expression of a constitutively active form of the mTOR target ribosomal protein S6 kinase (S6K) or of translation factor eIF4E reduced apoptosis by glucose limitation, and co-expression of S6K and eIF4E protected beta cells to the same extent as active Akt. The protective effects of active Akt and S6K were associated with increased cellular protein synthesis activity. It is concluded that Akt stimulation of mTOR and subsequent activation of the targets by which mTOR affects protein translation are required and sufficient mechanisms for Akt-mediated survival of beta cells undergoing sustained AMPK activation.

Insulin inhibits the expression of the hepatic insulin-like growth factor-binding protein-1 (IGFBP-1) and glucose-6-phosphatase (G6Pase) genes. The signaling pathway that mediates these events requires the activation of phosphatidylinositol 3-kinase, whereas transfection studies have suggested an involvement of Akt (protein kinase B) and FKHR, a transcription factor regulated by Akt. We now demonstrate that insulin repression of endogenous IGFBP-1 gene transcription was blocked by rapamycin or by amino acid starvation. Rapamycin inhibited the mammalian target of rapamycin (mTOR) and the subsequent activation of p70/p85 S6 protein kinase-1 (S6K1) by insulin, whereas amino acid depletion prevented insulin induction of these signaling molecules. Importantly, we demonstrate that insulin regulation of the thymine-rich insulin response element of the IGFBP-1 promoter was also inhibited by rapamycin. However, sustained activation of S6K1 did not repress this promoter. In addition, rapamycin did not affect insulin regulation of G6Pase expression or Akt activation. We propose that these observations indicate that an mTOR-dependent, but S6K-independent mechanism regulates the suppression of IGFBP-1 (but not G6Pase) gene expression by insulin. Therefore, although the insulin-responsive sequence of the G6Pase gene promoter is related to that of the IGFBP-1 promoter, the signaling pathways that mediate suppression of these genes are distinct.

Type 2 diabetes mellitus (T2DM) is a worldwide heath problem that is characterized by insulin resistance and the eventual loss of β cell function. As recent studies have shown that loss of ribosomal protein (RP) S6 kinase 1 (S6K1) increases systemic insulin sensitivity, S6K1 inhibitors are being pursued as potential agents for improving insulin resistance. Here we found that S6K1 deficiency in mice also leads to decreased β cell growth, intrauterine growth restriction (IUGR), and impaired placental development. IUGR is a common complication of human pregnancy that limits the supply of oxygen and nutrients to the developing fetus, leading to diminished embryonic β cell growth and the onset of T2DM later in life. However, restoration of placental development and the rescue of IUGR by tetraploid embryo complementation did not restore β cell size or insulin levels in S6K1–/– embryos, suggesting that loss of S6K1 leads to an intrinsic β cell lesion. Consistent with this hypothesis, reexpression of S6K1 in β cells of S6K1–/– mice restored embryonic β cell size, insulin levels, glucose tolerance, and RPS6 phosphorylation, without rescuing IUGR. Together, these data suggest that a nutrient-mediated reduction in intrinsic β cell S6K1 signaling, rather than IUGR, during fetal development may underlie reduced β cell growth and eventual development of T2DM later in life.

Signaling downstream of mechanistic target of rapamycin complexes 1 and 2 (mTORC1 and mTORC2) controls specific and distinct aspects of insulin action and nutrient homeostasis in an interconnected and as yet unclear way. Mice lacking the mTORC1 substrate S6 kinase 1 (S6K1) maintain proper glycemic control with a high-fat diet. This phenotype is accompanied by insulin hypersensitivity, Akt- and AMP-activated kinase upregulation, and increased lipolysis in adipose tissue and skeletal muscle. Here, we show that, when S6K1 inactivation is combined with the deletion of the mTORC2 substrate Akt2, glucose homeostasis is compromised due to defects in both insulin action and β-cell function. After a high-fat diet, the S6K1−/− Akt2−/− double-mutant mice do not become obese, though they are severely hyperglycemic. Our data demonstrate that S6K1 is required for pancreatic β-cell growth and function during adaptation to insulin resistance states. Strikingly, the inactivation of two targets of mTOR and phosphatidylinositol 3-kinase signaling is sufficient to reproduce major hallmarks of type 2 diabetes.

The mechanistic target of rapamycin complex 1 (mTORC1) integrates cellular nutrient signaling and hormonal cues to control metabolism. We have previously shown that constitutive nutrient signaling to mTORC1 by means of genetic activation of RagA (expression of GTP-locked RagA, or RagAGTP) in mice resulted in a fatal energetic crisis at birth. Herein, we rescue neonatal lethality in RagAGTP mice and find morphometric and metabolic alterations that span glucose, lipid, ketone, bile acid and amino acid homeostasis in adults, and a median lifespan of nine months. Proteomic and metabolomic analyses of livers from RagAGTP mice reveal a failed metabolic adaptation to fasting due to a global impairment in PPARα transcriptional program. These metabolic defects are partially recapitulated by restricting activation of RagA to hepatocytes, and revert by pharmacological inhibition of mTORC1. Constitutive hepatic nutrient signaling does not cause hepatocellular damage and carcinomas, unlike genetic activation of growth factor signaling upstream of mTORC1. In summary, RagA signaling dictates dynamic responses to feeding-fasting cycles to tune metabolism so as to match the nutritional state.

Genetic evidence in living organisms from yeast to plants and animals, including humans, unquestionably identifies the Target Of Rapamycin kinase (TOR or mTOR for mammalian/mechanistic) signal transduction pathway as a master regulator of growth through the control of cell size and cell number. Among the mTOR targets, the activation of p70 S6 kinase 1 (S6K1) is exquisitely sensitive to nutrient availability and rapamycin inhibition. Of note, in vivo analysis of mutant flies and mice reveals that S6K1 predominantly regulates cell size versus cell proliferation. Here we review the putative mechanisms of S6K1 action on cell size by considering the main functional categories of S6K1 targets: substrates involved in nucleic acid and protein synthesis, fat mass accumulation, retrograde control of insulin action, senescence program and cytoskeleton organization. We discuss how S6K1 may be involved in the observed interconnection between cell size, regenerative and ageing responses.

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Cellular senescence affects many physiological and pathological processes and is characterized by durable cell cycle arrest, an inflammatory secretory phenotype and metabolic reprogramming. Here, by using dynamic transcriptome and metabolome profiling in human fibroblasts with different subtypes of senescence, we show that a homoeostatic switch that results in glycerol-3-phosphate (G3P) and phosphoethanolamine (pEtN) accumulation links lipid metabolism to the senescence gene expression programme. Mechanistically, p53-dependent glycerol kinase activation and post-translational inactivation of phosphate cytidylyltransferase 2, ethanolamine regulate this metabolic switch, which promotes triglyceride accumulation in lipid droplets and induces the senescence gene expression programme. Conversely, G3P phosphatase and ethanolamine-phosphate phospho-lyase-based scavenging of G3P and pEtN acts in a senomorphic way by reducing G3P and pEtN accumulation. Collectively, our study ties G3P and pEtN accumulation to controlling lipid droplet biogenesis and phospholipid flux in senescent cells, providing a potential therapeutic avenue for targeting senescence and related pathophysiology.

Abstract: The ability of γ‐aminobutyric acid (GABA) and glycine (Gly) to modulate each other's release was studied in synaptosomes from rat spinal cord, cerebellum, cerebral cortex, or hippocampus, prelabeled with [ 3 H]GABA or [ 3 H]‐Gly and exposed in superfusion to Gly or to GABA, respectively. GABA increased the spontaneous outflow of [ 3 H]Gly (EC 50 , 20.8 μ M ) from spinal cord synaptosomes. Neither muscimol nor (‐)‐baclofen, up to 300 μ M , mimicked the effect of GABA, which was not antagonized by either bicuculline or picrotoxin. However, the effect of GABA was counteracted by the GABA uptake inhibitors nipecotic acid and N ‐(4,4‐diphenyl‐3‐butenyl)nipecotic acid. Moreover, the GABA‐induced [ 3 H]Gly release was Na + dependent and disappeared when the medium contained 23 m M Na + . The effect of GABA was Ca 2+ independent and tetrodotoxin insensitive. Conversely, Gly enhanced the outflow of [ 3 H]‐GABA from rat spinal cord synaptosomes (EC 50 , 100.9 μ M ). This effect was insensitive to both strychnine and 7‐chlorokynurenic acid, antagonists at Gly receptors, but it was strongly Na + dependent. Also, the Gly‐evoked [ 3 H]‐GABA release was Ca 2+ independent and tetrodotoxin insensitive. GABA increased the outflow of [ 3 H]Gly (EC 50 , 11.1 μ M ) from cerebellar synaptosomes; the effect was not mimicked by either muscimol or (—)‐baclofen nor was it prevented by bicuculline or picrotoxin. The GABA effect was, however, blocked by GABA uptake inhibitors and was Na + dependent. Gly increased [ 3 H]GABA release from cerebellar synaptosomes (EC 50 , 110.7 μ M ) in a strychnine‐ and 7‐chlorokynurenic acid‐insensitive manner. This effect was Na + dependent. The effects of GABA on [ 3 H]Gly release seen in spinal cord and cerebellum could be reproduced also with cerebrocortical synaptosomes. However, in cortex, the effect of Gly on [ 3 H]GABA release was much lower than in spinal cord or cerebellum, although it was partly Na + dependent. No changes of [ 3 H]Gly release were observed in hippocampal synaptosomes exposed to GABA. It is suggested that transporters specific for GABA or Gly are colocalized on the same nerve terminal in rat spinal cord, cerebellum, and cerebral cortex, but not in hippocampus. Moreover, GABA uptake modulates Gly release and, at least in spinal cord and cerebellum, Gly uptake modulates GABA release. These conclusions are compatible with the reported coexistence of GABA and Gly in spinal and cerebellar neurons.

PIK3CA-related overgrowth syndrome (PROS) is a genetic disorder caused by somatic mosaic gain-of-function mutations of PIK3CA. Clinical presentation of patients is diverse and associated with endocrine disruption. Adipose tissue is frequently involved, but its role in disease development and progression has not been elucidated. Here, we created a mouse model of PIK3CA-related adipose tissue overgrowth that recapitulates patient phenotype. We demonstrate that PIK3CA mutation leads to GLUT4 membrane accumulation with a negative feedback loop on insulin secretion, a burst of liver IGFBP1 synthesis with IGF-1 sequestration, and low circulating levels. Mouse phenotype was mainly driven through AKT2. We also observed that PIK3CA mutation induces metabolic reprogramming with Warburg-like effect and protein and lipid synthesis, hallmarks of cancer cells, in vitro, in vivo, and in patients. We lastly show that alpelisib is efficient at preventing and improving PIK3CA-adipose tissue overgrowth and reversing metabolomic anomalies in both animal models and patients.

Intracerebroventricularly (ICV) injected 5,7-dihydroxytryptamine (5,7-DHT), which reduced by 70-90% forebrain serotonin levels, significantly raised glial fibrillary acidic protein (GFAP) mRNA levels in the hippocampus and nucleus raphe dorsalis 5 days but not 15 days after the lesion. A significant increase of mitochondrial benzodiazepine receptors (MBR), measured by binding autoradiography of 3H-PK 11195, was found in the nucleus raphe dorsalis 5 and 15 days after the ICV 5,7-DHT and also in the hippocampus, ventral tegmental area, and substantia nigra at 15 days. No significant effect was observed in the striatum and cortex for either GFAP mRNA or MBR binding. Unlike the ICV route, bilateral injection of 5,7-DHT into the medial forebrain bundle, which caused a 65-90% reduction of serotonin levels in different forebrain regions, significantly raised GFAP mRNA and MBR binding only at the site of injection with no effect in hippocampus, striatum, and cortex. MBR binding slightly increased in the nucleus raphe dorsalis 15 days after the lesion. High doses of d-fenfluramine (10 mg/kg intraperitoneally twice daily for 4 days) caused 80-90% reduction of serotonin levels 5 days after the last injection but did not change the GFAP mRNA or the MBR binding in any of the brain regions considered. These findings suggest that the effect of 5,7-DHT on microglial and glial markers is probably related to a nonspecific interaction with other neuronal systems besides the serotonin or to direct interaction with glial cells; the use of these parameters for detecting selective degeneration of serotonin axons presents some obvious limitations.

In multicellular organisms, growth and metabolism are controlled by extracellular signals, such as insulin and insulin-like growth factors (IGFs). Depending on nutrient availability, these factors regulate cell number, cell size, storage of lipids, proteins and sugars. Here we will review recent literature on the intracellular signal transduction pathways regulating the anabolic responses in skeletal muscles. Emphasis will be put on three serine/threonine kinases, mTOR, Akt and S6 Kinase (S6K), and their role in the integration of environmental cues and the coordination of muscle growth.

Lipid homeostasis is critically dependent on the liver. Hepatic genes involved in lipid biosynthesis are controlled by combinatorial actions of multiple transcription factors that include three sterol regulatory element binding proteins (SREBPs), carbohydrate responsive element binding protein, liver X receptors, and others. SREBP-1c, a seminal regulator of de novo lipogenesis, resides in the endoplasmic reticulum as a transcriptionally inert precursor and must undergo a regulated intramembrane proteolysis (RIP) prior to its nuclear translocation as a bone fide transcription factor. The regulation of biosynthesis, turnover and actions of SREBP-1c and lipogenesis are mechanistically linked to signaling kinases, canonically induced by macronutrients and insulin. Here, we briefly review the evidence showing that phosphorylation of SREBP-1c and its interacting partners, catalyzed by phosphatidyl inositol-3-kinase, protein kinase B, mechanistic target of rapamycin complex 1 and 2, mitogen activated protein kinases, glycogen synthase kinase-3β, protein kinase A and 5′ adenosine monophosphate-activated protein kinase regulates the mechanisms of RIP and stability of SREBP-1c and de novo lipogenesis.

It is established that overnutrition is a risk factor for hepatocellular carcinoma. Il has been proposed that hepatic steatosis leads to a subinflammatory response and to the production of mitogenic cytokines. Our team is focused on the role of mammalian Target of Rapamycin (mTOR) in two pathophysiological conditions that modulate liver growth: liver regeneration after partial hepatectomy, and steatosis-associated tumorigenesis. Target kinases of mTOR seem more specifically involved in these processes: while S6K1 contributes to liver regeneration following hepatectomy, Akt2 is implicated in steatosis-associated tumorigenesis. In addition, recent data indicate that the transcription factor PPARγ, through an activation of glycolytic enzymes, could promote liver steatosis, hypertrophy and hyperplasia.

Mutations in tuberous sclerosis complex 1 (TSC1) or TSC2 predispose to angiomyolipomas and lymphangioleiomyomatosis in a mTOR-dependent manner. In these mesenchymal lesions, mTOR suppresses macroautophagy-mediated lysosomal degradation of YAP, which is a transcriptional coactivator of Hippo pathway and is required for the tumorigenesis of TSC. Therapeutic applications for TSC and other diseases with dysregulated mTOR activity can be envisaged.

Tuberous Sclerosis Complex (TSC) is a genetic disease causing uncontrolled growth of hamartomas involving different organ systems. In the last decade, dysregulation of the mTORC1 pathway was shown to be a main driver of tumor growth in TSC. Recently, a new crosstalk was detected between the mTORC1 and the Hippo-YAP pathway, another major cell signaling cascade controlling cell growth and organ size. Elucidating this connection is an important step in understanding the complexity of TSC, enabling new pharmacological targets and therapeutical options.

WE have previously shown that kainic acid-induced seizures in adult rats caused an up-regulation of GAP- 43 mRNA in the granule cells of the hippocampus, suggesting an involvement of this protein in the kainic acid-induced sprouting of mossy fibres. To determine whether this effect was dependent on the synthesis of proteins activated under these experimental conditions we examined the effect of cycloheximide, a protein synthesis inhibitor, on kainic acid-induced GAP-43 mRNA. Cycloheximide, injected s.c. 2 h but not 8 h after kainic acid, markedly reduced the increased expression of GAP-43 mRNA in granule cells. These results suggest that a rapid mechanism involving new protein synthesis is activated by kainic acid to induce GAP-43 in the granule cells and possibly trigger the structural remodeling of mossy fibres.

Chronic obstructive pulmonary disease (COPD) is a highly prevalent and devastating condition for which no curative treatment is available. Exaggerated lung-cell senescence may contribute substantially to the pathogenesis of COPD. Here, we explored the potential role for the mTOR signaling pathway in cell senescence and lung alterations in COPD, using lung specimens and derived cultured cells from patients with COPD and from age- and sex-matched control smokers. Cell senescence in COPD was linked to mTOR activation, and mTOR inhibition by low-dose rapamycin prevented cell senescence and inhibited the pro-inflammatory senescence-associated secretory phenotype. To assess whether mTOR activation was causal in lung pathology, we generated mice with constitutive or conditional deletion of the tuberous sclerosis complex heterodimer TSC1 (a negative regulator of mTOR complex 1), in smooth muscle cells (SMCs), endothelial cells (ECs), or alveolar epithelial cells (AECs). In this model, mTOR activation was sufficient to induce early-onset replicative cell senescence in cultured pulmonary artery SMCs from mice with TSC1 deletion. Mice with conditional TSC1 deletion in ECs or AECs developed lung emphysema, pulmonary hypertension, and inflammation in the absence of any other stimuli and concomitantly with the accumulation of senescent lung cells. Rapamycin prevented all these effects. These results support causal links between mTOR activation, cell senescence, and lung alterations in COPD, thereby identifying the mTOR pathway as a new therapeutic target for COPD. <b>Keywords:</b> mTOR, senescence, COPD

<h3>Introduction</h3> The loss of Apc, causing Wnt-mediated epithelial proliferation, is an early event in colorectal cancer (CRC) development. This hyperproliferative state requires signalling though the mTOR pathway, with the current paradigm suggesting that upregulation of translation initiation via phosphorylation of 4EBP1 is crucial. This model predicts that the mTOR inhibitor rapamycin, which does not efficiently inhibit 4EBP1 function, would be ineffective in limiting development and progression of intestinal adenomas. <h3>Methods</h3> The inducible <i><i>in vivo</i></i> mouse models Lgr5Cre^ER and VillinCre^ER were used to selectively flox genes from intestinal stem cells and crypts respectively. mTOR complex 1 signalling was inhibited in Apc^fl/fl mice either by rapamycin treatment or co-floxing the mTORC1 essential component Raptor. Translational status was assessed by sucrose gradient ultracentrifugation of intestinal epithelial extract from these mice and ³⁵S methionine incorporation and harringtonine chase assays on organoid cultures. The role of downstream mTORC1 effectors was established by assessing the intestinal regeneration following IR irradiation of 4EBP1/2^DKO, S6K1/2^DKO, rpS6^mut and eEF2k^-/- mice. Survival studies for Apc^fl/fl mice treated with rapamycin were performed both prior to, and on development of, symptoms <h3>Results</h3> mTORC1 activity is absolutely required for the proliferation of Apc deficient, but not wild type, intestinal crypts. Surprisingly, although protein synthesis is increased in Apc^fl/fl crypts, it is translation elongation and not initiation that is the rate limiting step. Mechanistically, the inhibition of eukaryotic elongation factor (eEF2) kinase, to increase eEF2 activity downstream of mTORC1 and S6K is required for Wnt-mediated proliferation after IR irradiation. Treatment of established Apc^fl/fl adenomas with rapamycin (which inhibits the mTORC1-S6K-eEF2k-eEF2 axis) arrests tumour growth and prolongs life. Furthermore, rapamycin treatment of mice immediately following homozygous Apc loss prevents the onset of symptoms. <h3>Conclusion</h3> These data show that intestinal adenoma formation and growth requires an mTOR mediated increase in translation elongation. Treatment of patients at high risk of developing CRC, such as those with Familial Adenomatous Polyposis, with Rapalogs may therefore be of therapeutic value. <h3>Disclosure of Interest</h3> None Declared.

Abstract Background Arrhythmogenic Cardiomyopathy (AC) is a genetic cardiac disorder, mainly caused by mutations in genes encoding desmosomal proteins, and accounts for most stress-related arrhythmic sudden cardiac deaths (SCD) in the young and athletes. The AC myocardium is hallmarked by cardiomyocyte (CM) death and fibro-fatty replacement, which generate a pro-arrhythmogenic substrate. Several pathogenetic factors in AC remain obscure and better understanding of the disease mechanisms is required to develop novel efficacious therapies to prevent SCD, which are sorely missing. The lexical analogy between desmosomes and desmosomal proteins has originally biased AC research towards CMs, the paradigmatic desmosome-bearing cells in heart. However, the myocardium is composed by different cell types, many of which express desmosomal proteins, albeit in the absence of desmosomes, including CMs, sympathetic neurons, vascular cells and fibroblasts. Notably, AC mutations are transmitted at germline, and thus may manifest in all cell types expressing desmosomal proteins. This might explain why the majority of preclinical AC models, using CM specific over-expression or deletion of the disease-causing mutation, failed to fully recapitulate the human disease phenotype. Hypothesis On these bases, we aimed to generate a knock-in (KI) AC mouse model for comprehensively studying AC pathogenesis. Methods As Desmoplakin (DSP) mutations occur in a large part of the Italian AC population, we used CRISP/Cas9 to generate a KI mouse strain harboring the Serine-to-Alanine substitution of S311, the murine homolog of human S299 [Bauce et al, 2005]. We successfully obtained DSPS311A/WT KI founders, which were viable and fertile and after backcrossing for &amp;gt;10 generations, used to expand the new mouse strain. Mouse cardiac phenotype was characterized, at different stages (1,2,4,6,9 mo.) by functional (i.e. ECHO, telemetry-ECG, chronic exercise) and structural (i.e. EM, standard histology, confocal IF, TUNEL assay) analyses. Molecular/biochemical analyses probed the state of the main pathways involved in AC. Results Our analyses showed that, starting from 4 mo., DSP homozygous KI mice display contractile dysfunction, worsening during aging, and fibrotic myocardial remodelling with focal fatty lesions, accompanied by frequent arrhythmic beats, which become sustained ventricular arrhythmias upon Noradrenaline administration. Hearts showed desmosome alterations, particularly at advanced disease stages, and lateralization of cx43, which corresponded to the phenotype of human AC hearts. Heterozygous mice showed similar alterations, which only took longer to appear. Exercise accelerated disease progression and increased the incidence of SCD (DSPS311A: SCD=63%, n=11; ctrls: SCD=8%, n=12). Conclusion Our KI mice replicate the clinical and pathological phenotype of DSP-linked biventricular AC and are thus suited for the mechanistic study of the multicellular origin of the disease. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): PRIN Miur 2015

The electrically-evoked release of [3H]dopamine ([3H]DA) from rat striatal slices was studied after a monolateral intrastriatal injection of kainic acid (KA). The release in the KA-lesioned striatum measured 4 days after the lesion was largely reduced (by 80%) with respect to the contralateral striatum. Administration of GM1 ganglioside (GM1) beginning on the day of the lesion resulted in restoration of the catecholamine release. Significant recovery was observed when GM1 was administered i.p. daily at the dose of 3 mg/kg for 6 days. The ganglioside given for 6 days at 30 mg/kg restored to near normal the electrically-evoked [3H]DA release. Similar recovery from the KA-induced injury occurred spontaneously but required 50 days.

The development of strategies which allow the inactivation of specific murine genes by homologous recombination in embryonic cells has revolutionized biological science in the last 10 years. A large number of mice carrying genetic lesions, generated by gene targeting technology, has tremendously increased our knowledge in many areas of biology, culminating in the identification of mouse models for human genetic disorders. These findings have been recently complemented by "conditional" gene targeting technology, allowing gene inactivation in a defined tissue and at a specific time point during development or adulthood, thereby extending the sophistication and potential of this technology.

Pulmonary artery (PA) smooth muscle cell (SMC) proliferation in pulmonary hypertension (PH) is associated with dysregulated mammalian target of rapamycin (mTOR) signaling. The mTOR pathway involves two independent complexes, mTORC1 and mTORC2, which phosphorylate S6 kinase (S6K) and serine/threonine kinase (Akt), respectively, and differ in their sensitivity to rapamycin. Here, we investigated whether selective mTOR activation in SMCs was sufficient to induce PH in mice. To selectively activate the mTOR-S6K pathway in SMCs, we developed mice with selective deletion in SMCs of the tuberous sclerosis complex 1 gene (TSC1), which negatively controls mTOR, by crossing TSC1LoxP/LoxP mice with mice expressing the Cre recombinase under the control of the SM22alpha promoter (SM22/TSC1-/− mice). SM22/TSC1-/− mice had increased body and heart weights with normal survival at 1 year of age. Echocardiography showed normal left ventricle (LV) size and ejection fraction but increased anterior and posterior wall thicknesses. Right ventricle (RV) and LV weights were increased and the RV/LV + septum weight ratio markedly elevated. Marked activation of the mTOR-Akt signaling pathway was found in lungs from SM22/TSC1-/− mice compared to controls, with a marked PTEN decrease and activation of mTORC1 substrates S6K and 4EBP and of mTORC2 substrates P-AktSer473 and GSK3. In situ analysis of lung specimens revealed prominent P-Akt, P-S6K, and P-GSK3 staining in PA-SMCs. SM22/TSC1-/− mice spontaneously developed PH due to pulmonary vascular remodeling, as assessed by increased RV systolic pressure, muscularization of distal pulmonary arteries, and increased number of Ki67-positive dividing pulmonary vascular cells. Treatment with 5 mg/Kg rapamycin during 3 weeks reversed PH and reduced lung P-Akt, P-S6K, and P-GSK3 proteins to control levels. Cultured PA-SMCs from SM22/TSC1-/− mice exhibited an increased growth rate and sustained activation of mTORC1 and mTORC2 substrates compared to controls. PA-SMC exposure to rapamycin inhibited the serum-induced growth and diminished the difference in growth rate between cells from SM22/TSC1-/− and WT mice. These results support a critical role in PH for mTOR signaling pathway activation, which can be suppressed by rapamycin treatment.

<b>Objective:</b> Constitutive activation of the mammalian target of rapamycin (mTOR) mTORC1 and mTORC2 is associated with pulmonary hypertension (PH) and sustained growth of pulmonary-artery (PA) smooth muscle cells (SMCs). We investigated whether selective mTORC1 activation in SMCs induced by deleting the negative mTORC1 regulator tuberous sclerosis complex 1 gene (<i>TSC1</i>) was sufficient to produce PH in mice. <b>Approach and Results:</b> Mice expressing Cre recombinase under SM22 promoter control were crossed with TSC1^LoxP/LoxP mice to generate SM22-TSC1^-/- mice. At 8 weeks of age, SM22-TSC1^-/- mice exhibited PH with marked increases in distal pulmonary-artery muscularization and Ki67-positive PA-SMC counts, without systemic hypertension or cardiac dysfunction. Marked activation of the mTORC1 substrates S6K and 4EBP and of the mTORC2 substrates p-Akt^Ser473 and GSK3 was found in lungs and pulmonary vessels from SM22-TSC1^-/- mice compared to controls. Treatment with 5 mg/Kg rapamycin for 3 weeks to inhibit mTORC1 and mTORC2 fully reversed PH in SM22-TSC1^-/- mice. In chronically hypoxic mice and SM22-5HTT+ mice exhibiting PH associated with mTORC1 and mTORC2 activation, PH was maximally attenuated by low-dose rapamycin associated with selective mTORC1 inhibition. Cultured PA-SMCs from SM22-TSC1^-/-, SM22-5HTT+, and chronically hypoxic mice exhibited similar sustained growth-rate enhancement and constitutive mTORC1 and mTORC2 activation; both effects were abolished by rapamycin. Deletion of the downstream mTORC1 effectors S6K1/2 in mice also activated mTOR signaling and induced PH. Conclusion: Activation of mTORC1 signaling leads to increased PA-SMC proliferation and subsequent PH development.

Growth is a fundamental process defined as accumulation of mass and is tightly controlled by nutrient availability. Overnutrition may lead to obesity, insulin resistance, type 2 diabetes, non-alcoholic fatty liver disease and cancer. In order to increase growth rate rapidly proliferating cells promote glycolysis in aerobic conditions. Expression of specific glycolytic enzymes, namely pyruvate kinase M2 (PKM2) and hexokinase 2 (HK2), concurs to this metabolic adaptation, as their kinetics and intracellular localization favour biosynthetic processes required for cell proliferation. Intracellular factors regulating their selective expression remain largely unknown. Using mouse model of hepatocytes specific PTEN deletion which is prone to steatosis and liver cancer we revealed that the PPARγ transcription factor and nuclear hormone receptor is a potent and selective activator of PKM2 and HK2 gene expression versus the other glycolytic genes. We discovered that PPARγ expression, liver steatosis, shift to aerobic glycolysis and tumorigenesis are under the control of the Akt2 kinase in PTEN-null mouse livers. PPARγ directly binds the promoters of HK2 and PKM to activate transcription. In vivo rescue of PPARγ activity in PTEN;Akt2 deficient mice causes liver steatosis, hypertrophy and hyperplasia accompanied by increased selective expression of PKM2 and HK2 with no effect on other glycolytic enzymes including PKL, enolase or GAPDH. Furthermore the growth promoting action of PPARγ in liver was dependent on glycolysis as treatment of PPARγ-transduced mice with the glycolytic inhibitor 3-bromopyruvate (3-BrPA) inhibited liver growth. Conversely, genetic deletion of PPARγ in PTEN-null mice rescued the steatosis and liver hypertrophy. Our data imply that therapies with the insulin-sensitizing agents and PPARγ agonists thiazolidinediones, may have opposite outcomes depending on the nutritional or genetic origins of liver steatosis. Consistently, chronic activation of PPARγ in PTEN-null mice led to acceleration of hepatic tumorigenesis. Furthermore, we have observed that substantial fraction of human hepatocellular carcinomas display elevated levels of PPARγ, PKM2 and HK2 which is inversely correlated with PTEN levels. In sum, we propose that PPARγ plays an unrecognised role in pathological liver growth and could represent a potential new therapeutic target for treatment of liver cancer.

Introduction De nombreux facteurs genetiques et environnementaux vont contribuer au developpement du diabete de type 2 qui se caracterise par une resistance a l'action de l'insuline au niveau des tissus peripheriques, et a une diminution de la secretion d'insuline par les cellules beta pancreatiques. Les voies de signalisation mTORC1 (mammalian target of rapamycin complex 1) / S6K1 (Ribosomal protein S6 kinase 1) et mTORC2 (mammalian target of rapamycin complex 2) /Akt2 ont ete decrites pour jouer un role majeur dans l'action de l'insuline. En effet, les souris S6K1 -/- presentent un defaut de secretion d'insuline compense par une augmentation de la sensibilite a l'insuline au niveau des tissus peripheriques. Une augmentation de la masse beta endocrine aboutissant a une augmentation de l'insulinemie a quant a elle ete decrite chez les souris Akt2 -/- , qui compense la resistance a l'insuline de ses animaux. Nous nous sommes proposes d'etudier le dialogue entre ces deux voies de signalisation dans le controle de l'homeostasie nutritionnelle en etudiant l'impact de la deletion de ces deux kinases. Materiels et Methodes Nous avons procede a l'analyse in vivo des souris Akt2 -/- S6K1 -/- soumises a un regime riche en graisse au niveau metabolique. Une analyse morphometrique de pancreas ainsi qu'une analyse de la signalisation insulinique au niveau des tissus peripheriques ont egalement etait effectuees. Resultats Nous avons montre que la deletion combinee d'Akt2 et de S6K1 altere l'homeostasie des nutriments affectant a la fois l'action et la secretion d'insuline. Les cellules beta pancreatiques de ces animaux presentent un defaut de croissance qui empeche la compensation de leur etat de resistance a l'insuline. Ainsi, ces animaux presentent une hyperglycemie a jeun couplee a une forte intolerance au glucose. Sous regime riche en graisse, ces souris developpent un phenotype diabetique, caracterise par une hyperglycemie marquee associee a une resistance a l'insuline. Conclusion Ces resultats montrent que la deletion des deux principaux substrats des voies de signalisation mTORC1 et mTORC2 est suffisante pour entrainer l'etablissement d'un phenotype diabetique.

Skeletal muscle mass is regulated by signaling pathways that govern protein synthesis and cell proliferation, the mammalian target of rapamycin (mTOR) playing a key role in these processes. Recent studies have suggested the crucial role of AMP‐activated protein kinase (AMPK) in the inhibition of protein synthesis and cell growth. Here, we address the role of AMPK in the regulation of muscle cell size in vitro and in vivo . The size of myotubes lacking both AMPKα1 and α2 catalytic subunits was 1.5‐fold higher than for controls. A marked increase in p70S6K Thr389 and rpS6 Ser235/236 phosphorylation was observed concomitant with an up‐regulation of protein synthesis rate. Inhibition of mTOR pathway by AMPK activators (AICAR, metformin, A‐769662) was blunted and was correlated with altered raptor phosphorylation. However, treatment with rapamycin prevented both p70S6K phosphorylation and cell hypertrophy. Moreover, in skeletal muscle‐specific deficient AMPKα1/α2 KO mice, soleus muscle showed a higher mass with myofibers of larger size and was correlated with enhanced p70S6K and rpS6 phosphorylation. Our results demonstrate the cross talk between AMPK and mTOR/p70S6K pathways in muscle cell size control. ANR‐06‐PHYSIO‐026

Backgrounds: Cellular senescence (CS) is a response to diverse forms of nonlethal cellular stress.The phenotypic transformations occurring in CS include a stable cell cycle arrest, an inflammatory response, and a complex metabolic shift.Among the most prominent intrinsic stimuli are genotoxic and oncogenic perturbations, many of which can initiate or promote aging and age-related diseases.Thus, the accumulation of senescent cells in tissues emerges as a critical driver of aging.Consistently, senescent cells can be found in the affected tissues of patients with age-related diseases such as osteoarthritis, pulmonary fibrosis, atherosclerosis, and Alzheimer's disease.Conversely, recent data show that senescent cell elimination in healthy animals prolongs lifespan.Despite the important contribution of senescence to tumor suppression and aging in animals and humans, we have only begun to define the underlying mechanisms, and many unresolved questions remain.For example, 1) What are the senescence clocks that reflect biological rather than chronological cell age 2) Is the senescence clock/program common across, or specific to, different stresses, cell types, and species?3) How do senescent cells evolve, and 5) Are there senescence biomarkers that are more specific/robust to senescent cells other than currently used "soft" biomarkers, such as senescence-associated beta-galactosidase?Objectives: Our understanding of the metabolic reprogramming that occurs during cellular senescence is still highly fragmentary.Senescent cells show a plethora of metabolic changes, including increased glucose consumption and lactate production (Warburg effect) along with a reduction in dNTPs as well as reduced NAD+/ NADH and AMP/ATP ratios.Compounds that elevate NAD+, including nicotinamide mononucleotide (NMN), have been proposed as possible therapeutics for preventing several agerelated pathologies.Given the complexities of metabolism in general, and the role of metabolism in regulating both the causes and consequences of the senescence response, the field is ripe for applications of holistic approaches.In addition, there is a growing body of evidence that metabolic changes also influence epigenetic modifications.For example, α-ketoglutarate (α-KG), and cofactors such as acetyl-coenzyme A (acetyl-CoA), S-adenosylmethionine (SAM), ATP, UDP, insights by which active telomerase prevents emphysema and preserve lung functions in old mice.

Abstract Unlike immature neurons and neurons from the peripheral nervous system (PNS), mature neurons from the central nervous system (CNS) cannot regenerate after injury. In the past 15 years, huge progress has been made to identify molecules and pathways necessary for neuroprotection and/or regeneration after CNS injury. In most regenerative models, phosphorylated ribosomal protein S6 (p-RPS6) is upregulated in neurons, which is often associated with an activation of the mTOR pathway. However, the exact contribution of post-translational modifications of this ribosomal protein in CNS regeneration remains elusive. In this study, we demonstrate that RPS6 phosphorylation is essential for PNS and CNS regeneration. We show that this phosphorylation is induced during the preconditioning effect in dorsal root ganglion (DRG) neurons, and that it is controlled by the p90S6 kinase RSK2. Our results reveal that RSK2 controls the preconditioning effect and that the RSK2-RPS6 axis is key for this process, as well as for PNS regeneration. Finally, we demonstrate that RSK2 promotes CNS regeneration in the dorsal column and allows functional recovery. Our data establish the critical role of RPS6 phosphorylation controlled by RSK2 in CNS regeneration and give new insights into the mechanism related to axon growth and circuit formation after traumatic lesion.

The mammalian brain contains a myriad of receptors localized on neuronal axon terminals (presynaptic receptors) the function of which is to modulate neurotransmitter release. Similarly to postsynaptic receptors, presynaptic receptors exist as pharmacologically distinct types and subtypes. Inasmuch as modulation of release is the functional response that follows their activation, transmitter release represents an excellent model to investigate receptors in general as well as to test potentially novel and more selective drugs. Types and subtypes of presynaptic receptors regulating the release of various transmitters will be described and their potential involvement in different pathophysiological conditions will be discussed.

The effect of -aminobutyric acid (GABA) in modulating the release of various neurotransmitters was studied using superfused synaptosomes. The results show that GABA increased the spontaneous release of several of the neurotransmitters studied. The potentiating action of GABA appeared not to be mediated by activation of GABA receptors; in fact, in order to exert its modulatory role, GABA penetrated into heterologous nerve terminals through a high affinity uptake carrier selective for the aminoacid. Conversely, some neurotransmitters could enhance the spontaneous efflux of GABA by penetrating into GABAergic nerve terminals through transporters specific for the neurotransmitters themselves. These findings suggest that transport systems selective for different neurotransmitters may coexist on the external membrane of the same nerve terminal. The possible implications of these findings are discussed.