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The mechanistic target of rapamycin (mTOR), master regulator of cellular metabolism, exists in two distinct complexes: mTOR complex 1 and mTOR complex 2 (mTORC1 and 2). MTORC1 is a master switch for most energetically onerous processes in the cell, driving cell growth and building cellular biomass in instances of nutrient sufficiency, and conversely, allowing autophagic recycling of cellular components upon nutrient limitation. The means by which the mTOR kinase blocks autophagy include direct inhibition of the early steps of the process, and the control of the lysosomal degradative capacity of the cell by inhibiting the transactivation of genes encoding structural, regulatory, and catalytic factors. Upon inhibition of mTOR, autophagic recycling of cellular components results in the reactivation of mTORC1; thus, autophagy lies both downstream and upstream of mTOR. The functional relationship between the mTOR pathway and autophagy involves complex regulatory loops that are significantly deciphered at the cellular level, but incompletely understood at the physiological level. Nevertheless, genetic evidence stemming from the use of engineered strains of mice has provided significant insight into the overlapping and complementary metabolic effects that physiological autophagy and the control of mTOR activity exert during fasting and nutrient overload.

Mice expressing a constitutively active form of RagA are unable to inhibit mTORC1 after birth and to trigger autophagy, and succumb perinatally. Rag GTPases as nutrient sensors for mTORC1 The mTOR complex 1 (mTORC1) pathway is a major regulator of growth in eukaryotes and a drug target for common diseases including cancer and neurodegeneration. It is known that mTORC1 senses amino acids through the Rag family of GTPases, but their physiological importance is unknown. David M. Sabatini and colleagues show that following birth, which stops the maternal nutrient supply, mTORC1 is inhibited in mice in a Rag-dependent fashion. This inhibition triggers autophagy, which promotes the release of amino acids needed to sustain plasma glucose levels via gluconeogenesis between birth and suckling. Thus the Rag pathway acts as a general nutrient sensor, and through its regulation of mTORC1, helps maintain nutrient homeostasis and survival in neonates. The mechanistic target of rapamycin complex 1 (mTORC1) pathway regulates organismal growth in response to many environmental cues, including nutrients and growth factors 1 . Cell-based studies showed that mTORC1 senses amino acids through the RagA–D family of GTPases 2 , 3 (also known as RRAGA, B, C and D), but their importance in mammalian physiology is unknown. Here we generate knock-in mice that express a constitutively active form of RagA (RagA GTP ) from its endogenous promoter. RagA GTP/GTP mice develop normally, but fail to survive postnatal day 1. When delivered by Caesarean section, fasted RagA GTP/GTP neonates die almost twice as rapidly as wild-type littermates. Within an hour of birth, wild-type neonates strongly inhibit mTORC1, which coincides with profound hypoglycaemia and a decrease in plasma amino-acid concentrations. In contrast, mTORC1 inhibition does not occur in RagA GTP/GTP neonates, despite identical reductions in blood nutrient amounts. With prolonged fasting, wild-type neonates recover their plasma glucose concentrations, but RagA GTP/GTP mice remain hypoglycaemic until death, despite using glycogen at a faster rate. The glucose homeostasis defect correlates with the inability of fasted RagA GTP/GTP neonates to trigger autophagy and produce amino acids for de novo glucose production. Because profound hypoglycaemia does not inhibit mTORC1 in RagA GTP/GTP neonates, we considered the possibility that the Rag pathway signals glucose as well as amino-acid sufficiency to mTORC1. Indeed, mTORC1 is resistant to glucose deprivation in RagA GTP/GTP fibroblasts, and glucose, like amino acids, controls its recruitment to the lysosomal surface, the site of mTORC1 activation. Thus, the Rag GTPases signal glucose and amino-acid concentrations to mTORC1, and have an unexpectedly key role in neonates in autophagy induction and thus nutrient homeostasis and viability.

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 RagA GTP ) in mice resulted in a fatal energetic crisis at birth. Herein, we rescue neonatal lethality in RagA GTP 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 RagA GTP 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. The mechanistic target of rapamycin complex 1 (mTORC1) integrates nutrient and hormonal signals to control metabolism. Here the authors investigate the effects of constitutive nutrient signaling through genetic activation of RagA in adult mice and show that constitutive nutrient signaling regulates the response to feeding-fasting cycles and does not drive liver cancer.

The metabolic functions of the liver are spatially organized in a phenomenon called zonation, linked to the differential exposure of portal and central hepatocytes to nutrient-rich blood. The mTORC1 signaling pathway controls cellular metabolism in response to nutrients and insulin fluctuations. Here we show that simultaneous genetic activation of nutrient and hormone signaling to mTORC1 in hepatocytes results in impaired establishment of postnatal metabolic and zonal identity of hepatocytes. Mutant hepatocytes fail to upregulate postnatally the expression of Frizzled receptors 1 and 8, and show reduced Wnt/β-catenin activation. This defect, alongside diminished paracrine Wnt2 ligand expression by endothelial cells, underlies impaired postnatal maturation. Impaired zonation is recapitulated in a model of constant supply of nutrients by parenteral nutrition to piglets. Our work shows the role of hepatocyte sensing of fluctuations in nutrients and hormones for triggering a latent metabolic zonation program. The liver is segregated into spatially organized areas that serve distinct functions, though how these zones are patterned remains unclear. Here they show that mTORC1 controls spatial segregation of liver metabolic functions via modulation of Wnt signaling, and find that impaired zonation is also observed in pigs given total parenteral nutrition.

Fasting exerts beneficial effects in mice and humans, including protection from chemotherapy toxicity. To explore the involved mechanisms, we collect blood from humans and mice before and after 36 or 24 hours of fasting, respectively, and measure lipid composition of erythrocyte membranes, circulating micro RNAs (miRNAs), and RNA expression at peripheral blood mononuclear cells (PBMCs). Fasting coordinately affects the proportion of polyunsaturated versus saturated and monounsaturated fatty acids at the erythrocyte membrane; and reduces the expression of insulin signaling-related genes in PBMCs. When fasted for 24 hours before and 24 hours after administration of oxaliplatin or doxorubicin, mice show a strong protection from toxicity in several tissues. Erythrocyte membrane lipids and PBMC gene expression define two separate groups of individuals that accurately predict a differential protection from chemotherapy toxicity, with important clinical implications. Our results reveal a mechanism of fasting associated with lipid homeostasis, and provide biomarkers of fasting to predict fasting-mediated protection from chemotherapy toxicity. Fasting has been reported to protect from chemotherapy-associated toxicity. Here, the authors show that fatty acid profiles in erythrocyte membranes and gene expression from peripheral blood mononuclear cells are associated to the fasting-mediated benefits during cancer treatment in mice and patients.

Background Overweight and obesity are defined by an anomalous or excessive fat accumulation that may compromise health. To find single-nucleotide polymorphisms (SNPs) influencing metabolic phenotypes associated with the obesity state, we analyze multiple anthropometric and clinical parameters in a cohort of 790 healthy volunteers and study potential associations with 48 manually curated SNPs, in metabolic genes functionally associated with the mechanistic target of rapamycin (mTOR) pathway. Results We identify and validate rs2291007 within a conserved region in the 3′UTR of folliculin-interacting protein FNIP2 that correlates with multiple leanness parameters. The T-to-C variant represents the major allele in Europeans and disrupts an ancestral target sequence of the miRNA miR-181b-5p, thus resulting in increased FNIP2 mRNA levels in cancer cell lines and in peripheral blood from carriers of the C allele. Because the miRNA binding site is conserved across vertebrates, we engineered the T-to-C substitution in the endogenous Fnip2 allele in mice. Primary cells derived from Fnip2 C/C mice show increased mRNA stability, and more importantly, Fnip2 C/C mice replicate the decreased adiposity and increased leanness observed in human volunteers. Finally, expression levels of FNIP2 in both human samples and mice negatively associate with leanness parameters, and moreover, are the most important contributor in a multifactorial model of body mass index prediction. Conclusions We propose that rs2291007 influences human leanness through an evolutionarily conserved modulation of FNIP2 mRNA levels. Graphical Abstract

The mTOR pathway is a critical determinant of cell persistence and growth wherein mTOR complex 1 (mTORC1) mediates a balance between growth factor stimuli and nutrient availability. Amino acids or glucose facilitates mTORC1 activation by inducing RagA GTPase recruitment of mTORC1 to the lysosomal outer surface, enabling activation of mTOR by the Ras homolog Rheb. Thereby, RagA alters mTORC1-driven growth in times of nutrient abundance or scarcity. Here, we have evaluated differential nutrient-sensing dependence through RagA and mTORC1 in hematopoietic progenitors, which dynamically drive mature cell production, and hematopoietic stem cells (HSC), which provide a quiescent cellular reserve. In nutrient-abundant conditions, RagA-deficient HSC were functionally unimpaired and upregulated mTORC1 via nutrient-insensitive mechanisms. RagA was also dispensable for HSC function under nutritional stress conditions. Similarly, hyperactivation of RagA did not affect HSC function. In contrast, RagA deficiency markedly altered progenitor population function and mature cell output. Therefore, RagA is a molecular mechanism that distinguishes the functional attributes of reactive progenitors from a reserve stem cell pool. The indifference of HSC to nutrient sensing through RagA contributes to their molecular resilience to nutritional stress, a characteristic that is relevant to organismal viability in evolution and in modern HSC transplantation approaches.

TELO2‐TTI1‐TTI2 (TTT) and R2TP are multi‐subunit chaperones that cooperate with HSP90 to assemble matured complexes of the PIKK family of kinases, including mTOR complex 1 (mTORC1). WAC, a protein previously implicated in transcription, H2B ubiquitination, and autophagy, was recently identified as a regulator of mTORC1 in response to glucose and glutamine availability, acting in concert with R2TP and TTT. However, the molecular basis of the interactions of WAC with R2TP and TTT and their role in mTORC1 regulation remains poorly defined. Here, we characterized the interactions of WAC with mTOR, R2TP, and TTT and how these are affected by nutrient conditions. Using purified proteins, we establish that WAC directly binds to mTOR‐mLST8, R2TP, and TELO2, but not TTI1 and TTI2. In cells, WAC is part of complexes containing components of mTORC1, R2TP, and TTT, and these associations are modulated by nutrient availability. Notably, WAC and TELO2 strongly associate with mTOR under glucose and glutamine deprivation, and these interactions are weakened minutes after nutrient refeeding. These dynamics correlate with changes in mTORC1 activity. Transcriptomic and proteomic analysis shows that WAC, mTOR, R2TP, and TTT are co‐expressed across several human cancers, supporting that WAC is part of a functional pathway with mTOR, R2TP, and TTT. Together, our findings reveal the formation and disassembly of a WAC complex with mTOR and TELO2 that contributes to regulate mTORC1 in response to glucose and glutamine availability. TTT and R2TP chaperone complexes are required for the assembly and activation of mTORC1. WAC directly interacts with components of TTT, R2TP, and mTORC1, and these interactions are affected by the availability of glucose and glutamine, correlating with changes in mTORC1 activity. Thus, the interaction of WAC with TTT and R2TP could contribute to regulating mTORC1 in response to nutrients.

Key Points The mammalian target of rapamycin (mTOR) is a highly conserved kinase that belongs to the phosphoinositide 3-kinase-related protein kinases (PIKK) family. mTOR participates in two distinct complexes, mTOR complex 1 (mTORC1) and mTORC2. mTORC1 integrates energy, nutrients, stress and growth factors and, in response to these stimuli, it drives the growth of cells, organs and whole organisms. mTORC2, which is activated by growth factors, promotes cell proliferation and survival. mTOR signalling maximizes energy storage and consumption. Upon chronic activation, mTORC1 drives insulin resistance by suppressing insulin receptor signalling and promoting fat accumulation. mTORC1 and mTORC2 are tightly linked with signalling pathways that lead to cancer. mTORC1 drives tumorigenesis by boosting translation of oncogenes, promoting anabolism and angiogenesis and suppressing autophagy. mTORC2 activates Akt and other AGC family kinases that promote cell proliferation and survival. Therapeutic strategies that are based on novel catalytic mTOR inhibitors have shown promising preclinical results. Our increasing knowledge of the molecular mechanisms underlying ageing is revealing a major role for mTOR in this process. Thus, understanding mTORC1 and mTORC2 biology is crucial for the development of novel drugs that can stave off ageing and age-related diseases. In eukaryotes, the target of rapamycin (TOR) protein kinase simultaneously senses energy, nutrients and stress (and growth factors in metazoans) to regulate cell growth and division. Advances in our understanding of the regulation and functions of mammalian TOR (mTOR) are revealing its involvement in diabetes, cancer and ageing. In all eukaryotes, the target of rapamycin (TOR) signalling pathway couples energy and nutrient abundance to the execution of cell growth and division, owing to the ability of TOR protein kinase to simultaneously sense energy, nutrients and stress and, in metazoans, growth factors. Mammalian TOR complex 1 (mTORC1) and mTORC2 exert their actions by regulating other important kinases, such as S6 kinase (S6K) and Akt. In the past few years, a significant advance in our understanding of the regulation and functions of mTOR has revealed the crucial involvement of this signalling pathway in the onset and progression of diabetes, cancer and ageing.

The ability to sense and respond to fluctuations in environmental nutrient levels is a requisite for life. Nutrient scarcity is a selective pressure that has shaped the evolution of most cellular processes. Different pathways that detect intracellular and extracellular levels of sugars, amino acids, lipids and surrogate metabolites are integrated and coordinated at the organismal level through hormonal signals. During food abundance, nutrient-sensing pathways engage anabolism and storage, whereas scarcity triggers homeostatic mechanisms, such as the mobilization of internal stores through autophagy. Nutrient-sensing pathways are commonly deregulated in human metabolic diseases.