Slow TCA flux and ATP production in primary solid tumours but not metastases.

Tissues derive ATP from two pathways-glycolysis and the tricarboxylic acid (TCA) cycle coupled to the electron transport chain. Most energy in mammals is produced via TCA metabolism1. In tumours, however, the absolute rates of these pathways remain unclear. Here we optimize tracer infusion approaches to measure the rates of glycolysis and the TCA cycle in healthy mouse tissues, Kras-mutant solid tumours, metastases and leukaemia. Then, given the rates of these two pathways, we calculate total ATP synthesis rates. We find that TCA cycle flux is suppressed in all five primary solid tumour models examined and is increased in lung metastases of breast cancer relative to primary orthotopic tumours. As expected, glycolysis flux is increased in tumours compared with healthy tissues (the Warburg effect2,3), but this increase is insufficient to compensate for low TCA flux in terms of ATP production. Thus, instead of being hypermetabolic, as commonly assumed, solid tumours generally produce ATP at a slower than normal rate. In mouse pancreatic cancer, this is accommodated by the downregulation of protein synthesis, one of this tissue's major energy costs. We propose that, as solid tumours develop, cancer cells shed energetically expensive tissue-specific functions, enabling uncontrolled growth despite a limited ability to produce ATP.

Effects of aging on glucose and lipid metabolism in mice.

Aging is accompanied by multiple molecular changes that contribute to aging-associated pathologies, such as accumulation of cellular damage and mitochondrial dysfunction. Tissue metabolism can also change with age, in part because mitochondria are central to cellular metabolism. Moreover, the co-factor NAD+, which is reported to decline across multiple tissue types during aging, plays a central role in metabolic pathways such as glycolysis, the tricarboxylic acid cycle, and the oxidative synthesis of nucleotides, amino acids, and lipids. To further characterize how tissue metabolism changes with age, we intravenously infused [U-13C]-glucose into young and old C57BL/6J, WSB/EiJ, and Diversity Outbred mice to trace glucose fate into downstream metabolites within plasma, liver, gastrocnemius muscle, and brain tissues. We found that glucose incorporation into central carbon and amino acid metabolism was robust during healthy aging across these different strains of mice. We also observed that levels of NAD+, NADH, and the NAD+/NADH ratio were unchanged in these tissues with healthy aging. However, aging tissues, particularly brain, exhibited evidence of up-regulated fatty acid and sphingolipid metabolism reactions that regenerate NAD+ from NADH. Because mitochondrial respiration, a major source of NAD+ regeneration, is reported to decline with age, our data supports a model where NAD+-generating lipid metabolism reactions may buffer against changes in NAD+/NADH during healthy aging.

CHP1 Regulates Compartmentalized Glycerolipid Synthesis by Activating GPAT4.

Cells require a constant supply of fatty acids to survive and proliferate. Fatty acids incorporate into membrane and storage glycerolipids through a series of endoplasmic reticulum (ER) enzymes, but how these enzymes are regulated is not well understood. Here, using a combination of CRISPR-based genetic screens and unbiased lipidomics, we identified calcineurin B homologous protein 1 (CHP1) as a major regulator of ER glycerolipid synthesis. Loss of CHP1 severely reduces fatty acid incorporation and storage in mammalian cells and invertebrates. Mechanistically, CHP1 binds and activates GPAT4, which catalyzes the initial rate-limiting step in glycerolipid synthesis. GPAT4 activity requires CHP1 to be N-myristoylated, forming a key molecular interface between the two proteins. Interestingly, upon CHP1 loss, the peroxisomal enzyme, GNPAT, partially compensates for the loss of ER lipid synthesis, enabling cell proliferation. Thus, our work identifies a conserved regulator of glycerolipid metabolism and reveals plasticity in lipid synthesis of proliferating cells.

Axin proteolysis by Iduna is required for the regulation of stem cell proliferation and intestinal homeostasis in Drosophila.

Self-renewal of intestinal stem cells is controlled by Wingless/Wnt-ß catenin signaling in both Drosophila and mammals. As Axin is a rate-limiting factor in Wingless signaling, its regulation is essential. Iduna is an evolutionarily conserved ubiquitin E3 ligase that has been identified as a crucial regulator for degradation of ADP-ribosylated Axin and, thus, of Wnt/ß-catenin signaling. However, its physiological significance remains to be demonstrated. Here, we generated loss-of-function mutants of Iduna to investigate its physiological role in Drosophila Genetic depletion of Iduna causes the accumulation of both Tankyrase and Axin. Increase of Axin protein in enterocytes non-autonomously enhanced stem cell divisions in the Drosophila midgut. Enterocytes secreted Unpaired proteins and thereby stimulated the activity of the JAK-STAT pathway in intestinal stem cells. A decrease in Axin gene expression suppressed the over-proliferation of stem cells and restored their numbers to normal levels in Iduna mutants. These findings suggest that Iduna-mediated regulation of Axin proteolysis is essential for tissue homeostasis in the Drosophila midgut.

Overexpressed NLRC3 acts as an anti-inflammatory cytosolic protein.

The novel nucleotide oligomerization domain (NOD)-like receptor (NLR) with a caspase activation and recruitment domain (CARD) 3 (NLRC3) protein belongs to the NLR family of cytosolic pathogen recognition receptors. NLRC3 has the characteristic NOD and leucine-rich repeat configuration with a less well defined CARD. T lymphocytes are known to have high NLRC3 expression, which may be involved in suppression of T cell activation. Here, we report that NLRC3 is a cytoplasmic protein that negatively regulates pro-IL-1ß maturation. Among well-known inflammasome components, NLRC3 can interact with apoptosis-associated speck-like protein containing a CARD (ASC) and caspases 1 and 5. Transient transfection of NLRC3 into stable EGFP-ASC-expressing HEK293FT cells reduces NLR family, pyrin domain-containing 3 (NLRP3)/cryopyrin-induced formation of ASC specks in a dose- and time-dependent manner. This suggests that NLRC3 can regulate ASC speck formation, caspase-1 activation and IL-1ß maturation. We show for the first time that inflammasome-like complexes assemble when caspase-1 and ASC are cotransfected together with NLRC3 in HEK293FT cells. However, overexpression of NLRC3 with NLRP3/cryopyrin inflammasome components suppresses pro-caspase-1 cleavage and IL-1ß processing. Our study suggests that NLRC3 negatively regulates inflammatory responses.

Depletion of Rictor, an essential protein component of mTORC2, decreases male lifespan.

Rapamycin, an inhibitor of the mechanistic target of rapamycin (mTOR), robustly extends the lifespan of model organisms including mice. We recently found that chronic treatment with rapamycin not only inhibits mTOR complex 1 (mTORC1), the canonical target of rapamycin, but also inhibits mTOR complex 2 (mTORC2) in vivo. While genetic evidence strongly suggests that inhibition of mTORC1 is sufficient to promote longevity, the impact of mTORC2 inhibition on mammalian longevity has not been assessed. RICTOR is a protein component of mTORC2 that is essential for its activity. We examined three different mouse models of Rictor loss: mice heterozygous for Rictor, mice lacking hepatic Rictor, and mice in which Rictor was inducibly deleted throughout the body in adult animals. Surprisingly, we find that depletion of RICTOR significantly decreases male, but not female, lifespan. While the mechanism by which RICTOR loss impairs male survival remains obscure, we find that the effect of RICTOR depletion on lifespan is independent of the role of hepatic mTORC2 in promoting glucose tolerance. Our results suggest that inhibition of mTORC2 signaling is detrimental to males, which may explain in part why interventions that decrease mTOR signaling show greater efficacy in females.

Novel NLRP3/cryopyrin mutations and pro-inflammatory cytokine profiles in Behçet's syndrome patients.

Behçet's syndrome (BS) is a systemic inflammatory disorder with unknown etiology. Features of both innate and adaptive immunity have been claimed in the pathogenesis of BS. To test the possible dysregulation of the NLRP3/cryopyrin (Nod-like receptor with a pyrin domain 3) inflammasome, as a result of mutation(s), we performed single-strand conformation polymorphism analyses and/or sequencing of all the coding regions and intron-exon boundaries of NLRP3/cryopyrin and ASC (apoptosis-associated speck-like protein containing CARD) genes from Turkish BS patients and healthy controls. At the same time, we determined pro-inflammatory cytokine secretion profiles of peripheral blood cells in response to LPS treatment using ELISA. BS patients with vascular involvement showed significantly increased levels of TNF-α release at 2-, 4- and 8-h post-treatment and significantly increased IL-1ß levels were detected at 2h (P = 0.005) and 4h (P = 0.025) (n = 10). We identified four mutations in the NLRP3/cryopyrin gene, V200M (n = 3/104) and T195M (n = 1/104), in BS patients but none in control samples. No mutations were detected in the ASC gene. The effect of these NLRP3/cryopyrin mutants on ASC speck assembly and IL-1ß secretion was tested and the V200M mutant was shown to induce IL-1ß secretion. Thus, it is likely that certain mutations in NLRP3/cryopyrin in combination with yet unknown other factors may contribute to the pro-inflammatory cytokine profiles in BS patients.

MCT1-mediated transport of a toxic molecule is an effective strategy for targeting glycolytic tumors.

There is increasing evidence that oncogenic transformation modifies the metabolic program of cells. A common alteration is the upregulation of glycolysis, and efforts to target glycolytic enzymes for anticancer therapy are under way. Here, we performed a genome-wide haploid genetic screen to identify resistance mechanisms to 3-bromopyruvate (3-BrPA), a drug candidate that inhibits glycolysis in a poorly understood fashion. We identified the SLC16A1 gene product, MCT1, as the main determinant of 3-BrPA sensitivity. MCT1 is necessary and sufficient for 3-BrPA uptake by cancer cells. Additionally, SLC16A1 mRNA levels are the best predictor of 3-BrPA sensitivity and are most elevated in glycolytic cancer cells. Furthermore, forced MCT1 expression in 3-BrPA-resistant cancer cells sensitizes tumor xenografts to 3-BrPA treatment in vivo. Our results identify a potential biomarker for 3-BrPA sensitivity and provide proof of concept that the selectivity of cancer-expressed transporters can be exploited for delivering toxic molecules to tumors.

mTORC1 in the Paneth cell niche couples intestinal stem-cell function to calorie intake.

How adult tissue stem and niche cells respond to the nutritional state of an organism is not well understood. Here we find that Paneth cells, a key constituent of the mammalian intestinal stem-cell (ISC) niche, augment stem-cell function in response to calorie restriction. Calorie restriction acts by reducing mechanistic target of rapamycin complex 1 (mTORC1) signalling in Paneth cells, and the ISC-enhancing effects of calorie restriction can be mimicked by rapamycin. Calorie intake regulates mTORC1 in Paneth cells, but not ISCs, and forced activation of mTORC1 in Paneth cells during calorie restriction abolishes the ISC-augmenting effects of the niche. Finally, increased expression of bone stromal antigen 1 (Bst1) in Paneth cells­an ectoenzyme that produces the paracrine factor cyclic ADP ribose­mediates the effects of calorie restriction and rapamycin on ISC function. Our findings establish that mTORC1 non-cell-autonomously regulates stem-cell self-renewal, and highlight a significant role of the mammalian intestinal niche in coupling stem-cell function to organismal physiology.