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.

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.

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.

The mTOR-Autophagy Axis and the Control of Metabolism.

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.

Inhibition of Rag GTPase signaling in mice suppresses B cell responses and lymphomagenesis with minimal detrimental trade-offs.

B lymphocytes are exquisitely sensitive to fluctuations in nutrient signaling by the Rag GTPases, and 15% of follicular lymphomas (FLs) harbor activating mutations in RRAGC. Hence, a potential therapeutic approach against malignant B cells is to inhibit Rag GTPase signaling, but because such inhibitors are still to be developed, efficacy and safety remain unknown. We generated knockin mice expressing a hypomorphic variant of RagC (Q119L); RagCQ119L/+ mice are viable and show attenuated nutrient signaling. B lymphocyte activation is cell-intrinsically impaired in RagCQ119L/+ mice, which also show significant suppression of genetically induced lymphomagenesis and autoimmunity. Surprisingly, no overt systemic trade-offs or phenotypic alterations caused by partial suppression of nutrient signaling are seen in other organs, and RagCQ119L/+ mice show normal longevity and normal age-dependent health decline. These results support the efficacy and safety of moderate inhibition of nutrient signaling against pathological B cells.

Limited survival and impaired hepatic fasting metabolism in mice with constitutive Rag GTPase signaling.

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.

Oncogenic Rag GTPase signaling enhances B cell activation and drives follicular lymphoma sensitive to pharmacological inhibition of mTOR.

The humoral immune response demands that B cells undergo a sudden anabolic shift and high cellular nutrient levels which are required to sustain the subsequent proliferative burst. Follicular lymphoma (FL) originates from B cells that have participated in the humoral response, and 15% of FL samples harbor point, activating mutations in RRAGC, an essential activator of mTORC1 downstream of the sensing of cellular nutrients. The impact of recurrent RRAGC mutations in B cell function and lymphoma is unexplored. RRAGC mutations, targeted to the endogenous locus in mice, confer a partial insensitivity to nutrient deprivation, but strongly exacerbate B cell responses and accelerate lymphomagenesis, while creating a selective vulnerability to pharmacological inhibition of mTORC1. This moderate increase in nutrient signaling synergizes with paracrine cues from the supportive T cell microenvironment that activates B cells via the PI3K-Akt-mTORC1 axis. Hence, Rragc mutations sustain induced germinal centers and murine and human FL in the presence of decreased T cell help. Our results support a model in which activating mutations in the nutrient signaling pathway foster lymphomagenesis by corrupting a nutrient-dependent control over paracrine signals from the T cell microenvironment.

Wolf-Hirschhorn Syndrome Candidate 1 Is Necessary for Correct Hematopoietic and B Cell Development.

Immunodeficiency is one of the most important causes of mortality associated with Wolf-Hirschhorn syndrome (WHS), a severe rare disease originated by a deletion in chromosome 4p. The WHS candidate 1 (WHSC1) gene has been proposed as one of the main genes responsible for many of the alterations in WHS, but its mechanism of action is still unknown. Here, we present in vivo genetic evidence showing that Whsc1 plays an important role at several points of hematopoietic development. Particularly, our results demonstrate that both differentiation and function of Whsc1-deficient B cells are impaired at several key developmental stages due to profound molecular defects affecting B cell lineage specification, commitment, fitness, and proliferation, demonstrating a causal role for WHSC1 in the immunodeficiency of WHS patients.