Nutrient sensing by the mTORC1 pathway in physiology

Cellular growth and metabolism must be linked to the environmental status of the cell to maintain organismal function. This coordination is achieved by mTORC1, a master growth regulator that senses and integrates a diverse array of environmental inputs, including nutrients like glucose and the amino acids leucine and arginine, whose sensing mechanisms are beginning to be identified. However, the role and implications of nutrient sensing by mTORC1 in mammalian physiology remains poorly understood. Here, we identified a critical role of leucine sensing by mTORC1 in adapting to leucine availability in vivo. Mice lacking the leucine sensors Sestrin1 and Sestrin2 fail to inhibit mTORC1 in tissues when deprived of dietary leucine. These mice suffer from severe loss of white adipose tissue and skeletal muscle when deprived of leucine, but not other essential amino acids. We showed that their white adipose tissue loss results from mTORC1 dysregulation in the liver and is driven by aberrant production of the hepatokine FGF21. We also found that leucine sensing is compartmentalized within the liver, which is established by zonated expression of Sestrin1 and Sestrin2 in the liver lobule and demonstrates an unappreciated spatial organization of nutrient sensing in tissues. Further, we identified a functionally-important temporal shift in nutrient sensitivity of the mTORC1 pathway in pancreatic β cells. We found that this shift is required for β cells to acquire glucose-responsive insulin secretion after birth. We further demonstrated that modulating nutrient-responsive mTORC1 activity can be therapeutically exploited to improve the generation of stem cell-derived β cells. Collectively, these findings demonstrate a subset of the likely many important roles of nutrient sensing by mTORC1 in physiology and begin to unravel the spatial and temporal complexity of nutrient sensing within tissues of the body.Ph.D

A Nutrient-Sensing Transition at Birth Triggers Glucose-Responsive Insulin Secretion

A drastic transition at birth, from constant maternal nutrient supply in utero to intermittent postnatal feeding, requires changes in the metabolic system of the neonate. Despite their central role in metabolic homeostasis, little is known about how pancreatic β cells adjust to the new nutritional challenge. Here, we find that after birth β cell function shifts from amino acid- to glucose-stimulated insulin secretion in correlation with the change in the nutritional environment. This adaptation is mediated by a transition in nutrient sensitivity of the mTORC1 pathway, which leads to intermittent mTORC1 activity. Disrupting nutrient sensitivity of mTORC1 in mature β cells reverts insulin secretion to a functionally immature state. Finally, manipulating nutrient sensitivity of mTORC1 in stem cell-derived β cells in vitro strongly enhances their glucose-responsive insulin secretion. These results reveal a mechanism by which nutrients regulate β cell function, thereby enabling a metabolic adaptation for the newborn

Dihydroxyacetone phosphate signals glucose availability to mTORC1

© 2020, The Author(s), under exclusive licence to Springer Nature Limited. The mechanistic target of rapamycin complex 1 (mTORC1) kinase regulates cell growth by setting the balance between anabolic and catabolic processes. To be active, mTORC1 requires the environmental presence of amino acids and glucose. While a mechanistic understanding of amino acid sensing by mTORC1 is emerging, how glucose activates mTORC1 remains mysterious. Here, we used metabolically engineered human cells lacking the canonical energy sensor AMP-activated protein kinase to identify glucose-derived metabolites required to activate mTORC1 independent of energetic stress. We show that mTORC1 senses a metabolite downstream of the aldolase and upstream of the GAPDH-catalysed steps of glycolysis and pinpoint dihydroxyacetone phosphate (DHAP) as the key molecule. In cells expressing a triose kinase, the synthesis of DHAP from DHA is sufficient to activate mTORC1 even in the absence of glucose. DHAP is a precursor for lipid synthesis, a process under the control of mTORC1, which provides a potential rationale for the sensing of DHAP by mTORC1

MITO-Tag Mice enable rapid isolation and multimodal profiling of mitochondria from specific cell types in vivo

Mitochondria are metabolic organelles that are essential for mammalian life, but the dynamics of mitochondrial metabolism within mammalian tissues in vivo remains incompletely understood. While whole-tissue metabolite profiling has been useful for studying metabolism in vivo, such an approach lacks resolution at the cellular and subcellular level. In vivo methods for interrogating organellar metabolites in specific cell types within mammalian tissues have been limited. To address this, we built on prior work in which we exploited a mitochondrially localized 3XHA epitope tag (MITO-Tag) for the fast isolation of mitochondria from cultured cells to generate MITO-Tag Mice. Affording spatiotemporal control over MITO-Tag expression, these transgenic animals enable the rapid, cell-type-specific immunoisolation of mitochondria from tissues, which we verified using a combination of proteomic and metabolomic approaches. Using MITO-Tag Mice and targeted and untargeted metabolite profiling, we identified changes during fasted and refed conditions in a diverse array of mitochondrial metabolites in hepatocytes and found metabolites that behaved differently at the mitochondrial versus whole-tissue level. MITO-Tag Mice should have utility for studying mitochondrial physiology, and our strategy should be generally applicable for studying other mammalian organelles in specific cell types in vivo.NIH (Grant R01CA103866)NIH (Grant R01CA129105)NIH (Grant R37AI047389)Department of Defense (Grant W81XWH-15-1-0230

Semi-Automated, Occupationally Safe Immunofluorescence Microtip Sensor for Rapid Detection of <i>Mycobacterium</i> Cells in Sputum

<div><p>An occupationally safe (biosafe) sputum liquefaction protocol was developed for use with a semi-automated antibody-based microtip immunofluorescence sensor. The protocol effectively liquefied sputum and inactivated microorganisms including <i>Mycobacterium tuberculosis</i>, while preserving the antibody-binding activity of <i>Mycobacterium</i> cell surface antigens. Sputum was treated with a synergistic chemical-thermal protocol that included moderate concentrations of NaOH and detergent at 60°C for 5 to 10 min. Samples spiked with <i>M. tuberculosis</i> complex cells showed approximately 10⁶-fold inactivation of the pathogen after treatment. Antibody binding was retained post-treatment, as determined by analysis with a microtip immunosensor. The sensor correctly distinguished between <i>Mycobacterium</i> species and other cell types naturally present in biosafe-treated sputum, with a detection limit of 100 CFU/mL for <i>M. tuberculosis</i>, in a 30-minute sample-to-result process. The microtip device was also semi-automated and shown to be compatible with low-cost, LED-powered fluorescence microscopy. The device and biosafe sputum liquefaction method opens the door to rapid detection of tuberculosis in settings with limited laboratory infrastructure.</p></div

Normalized fluorescence intensity results from the microtip assay.

<p>(A) Comparison of different species of <i>Mycobacterium</i> and <i>S. epidermidis</i> at 10⁴ CFU/mL. (B) Microtip detection of treated sputum samples spiked with BCG at densities ranging from 10² to 10⁵ CFU/mL. (C) Microtip detection of treated sputum samples spiked with H37Ra at densities ranging from 10² to 10⁵ CFU/mL.</p

Preliminary comparison between Olympus and Lumin raw intensity values obtained from microtip measurement.

<p>The microtips were evaluated in treated sputum samples spiked with either PBS alone (control) or PBS containing BCG cells at 10⁴ CFU/mL.</p