Germinal Center Selection and Affinity Maturation Require Dynamic Regulation of mTORC1 Kinase.

During antibody affinity maturation, germinal center (GC) B cells cycle between affinity-driven selection in the light zone (LZ) and proliferation and somatic hypermutation in the dark zone (DZ). Although selection of GC B cells is triggered by antigen-dependent signals delivered in the LZ, DZ proliferation occurs in the absence of such signals. We show that positive selection triggered by T cell help activates the mechanistic target of rapamycin complex 1 (mTORC1), which promotes the anabolic program that supports DZ proliferation. Blocking mTORC1 prior to growth prevented clonal expansion, whereas blockade after cells reached peak size had little to no effect. Conversely, constitutively active mTORC1 led to DZ enrichment but loss of competitiveness and impaired affinity maturation. Thus, mTORC1 activation is required for fueling B cells prior to DZ proliferation rather than for allowing cell-cycle progression itself and must be regulated dynamically during cyclic re-entry to ensure efficient affinity-based selection.

Lack of p38 activation in T cells increases IL-35 and protects against obesity by promoting thermogenesis.

Obesity is characterized by low-grade inflammation, energy imbalance and impaired thermogenesis. The role of regulatory T cells (Treg) in inflammation-mediated maladaptive thermogenesis is not well established. Here, we find that the p38 pathway is a key regulator of T cell-mediated adipose tissue (AT) inflammation and browning. Mice with T cells specifically lacking the p38 activators MKK3/6 are protected against diet-induced obesity, leading to an improved metabolic profile, increased browning, and enhanced thermogenesis. We identify IL-35 as a driver of adipocyte thermogenic program through the ATF2/UCP1/FGF21 pathway. IL-35 limits CD8+ T cell infiltration and inflammation in AT. Interestingly, we find that IL-35 levels are reduced in visceral fat from obese patients. Mechanistically, we demonstrate that p38 controls the expression of IL-35 in human and mouse Treg cells through mTOR pathway activation. Our findings highlight p38 signaling as a molecular orchestrator of AT T cell accumulation and function.

Corrigendum: mTORC1-dependent AMD1 regulation sustains polyamine metabolism in prostate cancer.

This corrects the article DOI: 10.1038/nature22964.

mTOR: from growth signal integration to cancer, diabetes 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.

mTORC1-dependent AMD1 regulation sustains polyamine metabolism in prostate cancer.

Activation of the PTEN-PI3K-mTORC1 pathway consolidates metabolic programs that sustain cancer cell growth and proliferation. Here we show that mechanistic target of rapamycin complex 1 (mTORC1) regulates polyamine dynamics, a metabolic route that is essential for oncogenicity. By using integrative metabolomics in a mouse model and human biopsies of prostate cancer, we identify alterations in tumours affecting the production of decarboxylated S-adenosylmethionine (dcSAM) and polyamine synthesis. Mechanistically, this metabolic rewiring stems from mTORC1-dependent regulation of S-adenosylmethionine decarboxylase 1 (AMD1) stability. This novel molecular regulation is validated in mouse and human cancer specimens. AMD1 is upregulated in human prostate cancer with activated mTORC1. Conversely, samples from a clinical trial with the mTORC1 inhibitor everolimus exhibit a predominant decrease in AMD1 immunoreactivity that is associated with a decrease in proliferation, in line with the requirement of dcSAM production for oncogenicity. These findings provide fundamental information about the complex regulatory landscape controlled by mTORC1 to integrate and translate growth signals into an oncogenic metabolic program.

Nutrient-sensing mechanisms and pathways.

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.

Regulation of mTORC1 by the Rag GTPases is necessary for neonatal autophagy and survival.

The mechanistic target of rapamycin complex 1 (mTORC1) pathway regulates organismal growth in response to many environmental cues, including nutrients and growth factors. Cell-based studies showed that mTORC1 senses amino acids through the RagA-D family of GTPases (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.

Correction: Limited Role of Murine ATM in Oncogene-Induced Senescence and p53-Dependent Tumor Suppression.

[This corrects the article DOI: 10.1371/journal.pone.0005475.].

Pharmacological and genetic increases in liver NADPH levels ameliorate NASH progression in female mice.

Non-alcoholic steatohepatitis (NASH) is one of the fastest growing liver diseases worldwide, and oxidative stress is one of NASH main key drivers. Nicotinamide adenine dinucleotide phosphate (NADPH) is the ultimate donor of reductive power to a number of antioxidant defences. Here, we explored the potential of increasing NADPH levels to prevent NASH progression. We used nicotinamide riboside (NR) supplementation or a G6PD-tg mouse line harbouring an additional copy of the human G6PD gene. In a NASH mouse model induced by feeding mice a methionine-choline deficient (MCD) diet for three weeks, both tools increased the hepatic levels of NADPH and ameliorated the NASH phenotype induced by the MCD intervention, but only in female mice. Boosting NADPH levels in females increased the liver expression of the antioxidant genes Gsta3, Sod1 and Txnrd1 in NR-treated mice, or of Gsr for G6PD-tg mice. Both strategies significantly reduced hepatic lipid peroxidation. NR-treated female mice showed a reduction of steatosis accompanied by a drop of the hepatic triglyceride levels, that was not observed in G6PD-tg mice. NR-treated mice tended to reduce their lobular inflammation, showed a reduction of the NK cell population and diminished transcription of the damage marker Lcn2. G6PD-tg female mice exhibited a reduction of their lobular inflammation and hepatocyte ballooning induced by the MCD diet, that was related to a reduction of the monocyte-derived macrophage population and the Tnfa, Ccl2 and Lcn2 gene expression. As conclusion, boosting hepatic NADPH levels attenuated the oxidative lipid damage and the exhausted antioxidant gene expression specifically in female mice in two different models of NASH, preventing the progression of the inflammatory process and hepatic injury.

NGFR regulates stromal cell activation in germinal centers.

Nerve growth factor receptor (NGFR) is expressed by follicular dendritic cells (FDCs). However, the role of NGFR in the humoral response is not well defined. Here, we study the effect of Ngfr loss on lymph node organization and function, demonstrating that Ngfr depletion leads to spontaneous germinal center (GC) formation and an expansion of the GC B cell compartment. In accordance with this effect, stromal cells are altered in Ngfr-/- mice with a higher frequency of FDCs, characterized by CD21/35, MAdCAM-1, and VCAM-1 overexpression. GCs are located ectopically in Ngfr-/- mice, with lost polarization together with impaired high-affinity antibody production and an increase in circulating autoantibodies. We observe higher levels of autoantibodies in Bcl2 Tg/Ngfr-/- mice, concomitant with a higher incidence of autoimmunity and lower overall survival. Our work shows that NGFR is involved in maintaining GC structure and function, participating in GC activation, antibody production, and immune tolerance.

Hepatic nutrient and hormone signaling to mTORC1 instructs the postnatal metabolic zonation of the liver.

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/ß-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.

A mild increase in nutrient signaling to mTORC1 in mice leads to parenchymal damage, myeloid inflammation and shortened lifespan.

The mechanistic target of rapamycin complex 1 controls cellular anabolism in response to growth factor signaling and to nutrient sufficiency signaled through the Rag GTPases. Inhibition of mTOR reproducibly extends longevity across eukaryotes. Here we report that mice that endogenously express active mutant variants of RagC exhibit multiple features of parenchymal damage that include senescence, expression of inflammatory molecules, increased myeloid inflammation with extensive features of inflammaging and a ~30% reduction in lifespan. Through bone marrow transplantation experiments, we show that myeloid cells are abnormally activated by signals emanating from dysfunctional RagC-mutant parenchyma, causing neutrophil extravasation that inflicts additional inflammatory damage. Therapeutic suppression of myeloid inflammation in aged RagC-mutant mice attenuates parenchymal damage and extends survival. Together, our findings link mildly increased nutrient signaling to limited lifespan in mammals, and support a two-component process of parenchymal damage and myeloid inflammation that together precipitate a time-dependent organ deterioration that limits longevity.

HSDL2 links nutritional cues to bile acid and cholesterol homeostasis.

In response to energy and nutrient shortage, the liver triggers several catabolic processes to promote survival. Despite recent progress, the precise molecular mechanisms regulating the hepatic adaptation to fasting remain incompletely characterized. Here, we report the identification of hydroxysteroid dehydrogenase-like 2 (HSDL2) as a mitochondrial protein highly induced by fasting. We show that the activation of PGC1α-PPARα and the inhibition of the PI3K-mTORC1 axis stimulate HSDL2 expression in hepatocytes. We found that HSDL2 depletion decreases cholesterol conversion to bile acids (BAs) and impairs FXR activity. HSDL2 knockdown also reduces mitochondrial respiration, fatty acid oxidation, and TCA cycle activity. Bioinformatics analyses revealed that hepatic Hsdl2 expression positively associates with the postprandial excursion of various BA species in mice. We show that liver-specific HSDL2 depletion affects BA metabolism and decreases circulating cholesterol levels upon refeeding. Overall, our report identifies HSDL2 as a fasting-induced mitochondrial protein that links nutritional signals to BAs and cholesterol homeostasis.

Nutrients and growth factors in mTORC1 activation.

Growth factors and nutrients regulate the mTORC1 [mammalian (or mechanistic) target of rapamycin complex 1] by different mechanisms. The players that link growth factors and mTORC1 activation have been known for several years and mouse models have validated its relevance for human physiology and disease. In contrast with the picture for growth factor signalling, the means by which nutrient availability leads to mTORC1 activation have remained elusive until recently, with the discovery of the Rag GTPases upstream of mTORC1. The Rag GTPases recruit mTORC1 to the outer lysosomal surface, where growth factor signalling and nutrient signalling converge on mTORC1 activation. A mouse model of constitutive RagA activity has revealed qualitative differences between growth-factor- and nutrient-dependent regulation of mTORC1. Regulation of mTORC1 activity by the Rag GTPases in vivo is key for enduring early neonatal starvation, showing its importance for mammalian physiology.

3D bioprinted functional skeletal muscle models have potential applications for studies of muscle wasting in cancer cachexia.

Acquired muscle diseases such as cancer cachexia are responsible for the poor prognosis of many patients suffering from cancer. In vitro models are needed to study the underlying mechanisms of those pathologies. Extrusion bioprinting is an emerging tool to emulate the aligned architecture of fibers while implementing additive manufacturing techniques in tissue engineering. However, designing bioinks that reconcile the rheological needs of bioprinting and the biological requirements of muscle tissue is a challenging matter. Here we formulate a biomaterial with dual crosslinking to modulate the physical properties of bioprinted models. We design 3D bioprinted muscle models that resemble the mechanical properties of native tissue and show improved proliferation and high maturation of differentiated myotubes suggesting that the GelMA-AlgMA-Fibrin biomaterial possesses myogenic properties. The electrical stimulation of the 3D model confirmed the contractile capability of the tissue and enhanced the formation of sarcomeres. Regarding the functionality of the models, they served as platforms to recapitulate skeletal muscle diseases such as muscle wasting produced by cancer cachexia. The genetic expression of 3D models demonstrated a better resemblance to the muscular biopsies of cachectic mouse models. Altogether, this biomaterial is aimed to fabricate manipulable skeletal muscle in vitro models in a non-costly, fast and feasible manner.

PI3K-C2ß limits mTORC1 signaling and angiogenic growth.

Phosphoinositide 3-kinases (PI3Ks) phosphorylate intracellular inositol lipids to regulate signaling and intracellular vesicular trafficking. Mammals have eight PI3K isoforms, of which class I PI3Kα and class II PI3K-C2α are essential for vascular development. The class II PI3K-C2ß is also abundant in endothelial cells. Using in vivo and in vitro approaches, we found that PI3K-C2ß was a critical regulator of blood vessel growth by restricting endothelial mTORC1 signaling. Mice expressing a kinase-inactive form of PI3K-C2ß displayed enlarged blood vessels without corresponding changes in endothelial cell proliferation or migration. Instead, inactivation of PI3K-C2ß resulted in an increase in the size of endothelial cells, particularly in the sprouting zone of angiogenesis. Mechanistically, we showed that the aberrantly large size of PI3K-C2ß mutant endothelial cells was caused by mTORC1 activation, which sustained growth in these cells. Consistently, pharmacological inhibition of mTORC1 with rapamycin normalized vascular morphogenesis in PI3K-C2ß mutant mice. Together, these results identify PI3K-C2ß as a crucial determinant of endothelial signaling and illustrate the importance of mTORC1 regulation during angiogenic growth.

The metabolic plasticity of B cells.

The humoral response requires rapid growth, biosynthetic capacity, proliferation and differentiation of B cells. These processes involve profound B-cell phenotypic transitions that are coupled to drastic changes in metabolism so as to meet the extremely different energetic requirements as B cells switch from resting to an activated, highly proliferative state and to plasma or memory cell fates. Thus, B cells execute a multi-step, energetically dynamic process of profound metabolic rewiring from low ATP production to transient and large increments of energy expenditure that depend on high uptake and consumption of glucose and fatty acids. Such metabolic plasticity is under tight transcriptional and post-transcriptional regulation. Alterations in B-cell metabolism driven by genetic mutations or by extrinsic insults impair B-cell functions and differentiation and may underlie the anomalous behavior of pathological B cells. Herein, we review molecular switches that control B-cell metabolism and fuel utilization, as well as the emerging awareness of the impact of dynamic metabolic adaptations of B cells throughout the different phases of the humoral response.

Phosphorylation of FAM134C by CK2 controls starvation-induced ER-phagy.

Selective degradation of the endoplasmic reticulum (ER) via autophagy (ER-phagy) is initiated by ER-phagy receptors, which facilitate the incorporation of ER fragments into autophagosomes. FAM134 reticulon family proteins (FAM134A, FAM134B, and FAM134C) are ER-phagy receptors with structural similarities and nonredundant functions. Whether they respond differentially to the stimulation of ER-phagy is unknown. Here, we describe an activation mechanism unique to FAM134C during starvation. In fed conditions, FAM134C is phosphorylated by casein kinase 2 (CK2) at critical residues flanking the LIR domain. Phosphorylation of these residues negatively affects binding affinity to the autophagy proteins LC3. During starvation, mTORC1 inhibition limits FAM134C phosphorylation by CK2, hence promoting receptor activation and ER-phagy. Using a novel tool to study ER-phagy in vivo and FAM134C knockout mice, we demonstrated the physiological relevance of FAM134C phosphorylation during starvation-induced ER-phagy in liver lipid metabolism. These data provide a mechanistic insight into ER-phagy regulation and an example of autophagy selectivity during starvation.

Folliculin-interacting protein FNIP2 impacts on overweight and obesity through a polymorphism in a conserved 3' untranslated region.

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.

A YAP/TAZ-TEAD signalling module links endothelial nutrient acquisition to angiogenic growth.

Angiogenesis, the process by which endothelial cells (ECs) form new blood vessels from existing ones, is intimately linked to the tissue's metabolic milieu and often occurs at nutrient-deficient sites. However, ECs rely on sufficient metabolic resources to support growth and proliferation. How endothelial nutrient acquisition and usage are regulated is unknown. Here we show that these processes are instructed by Yes-associated protein 1 (YAP)/WW domain-containing transcription regulator 1 (WWTR1/TAZ)-transcriptional enhanced associate domain (TEAD): a transcriptional module whose function is highly responsive to changes in the tissue environment. ECs lacking YAP/TAZ or their transcriptional partners, TEAD1, 2 and 4 fail to divide, resulting in stunted vascular growth in mice. Conversely, activation of TAZ, the more abundant paralogue in ECs, boosts proliferation, leading to vascular hyperplasia. We find that YAP/TAZ promote angiogenesis by fuelling nutrient-dependent mTORC1 signalling. By orchestrating the transcription of a repertoire of cell-surface transporters, including the large neutral amino acid transporter SLC7A5, YAP/TAZ-TEAD stimulate the import of amino acids and other essential nutrients, thereby enabling mTORC1 activation. Dissociating mTORC1 from these nutrient inputs-elicited by the loss of Rag GTPases-inhibits mTORC1 activity and prevents YAP/TAZ-dependent vascular growth. Together, these findings define a pivotal role for YAP/TAZ-TEAD in controlling endothelial mTORC1 and illustrate the essentiality of coordinated nutrient fluxes in the vasculature.