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Glutamate decarboxylase 1 (GAD1) is best known for its role in producing the neurotransmitter γ-amino butyric acid (GABA) as part of the “GABA shunt” metabolic pathway, an alternative mechanism of glutamine anaplerosis for TCA cycle metabolism (Yogeeswari et al., 2005). However, understanding of the metabolic function of GAD1 in non-neuronal tissues has remained limited. Here, we show that GAD1 supports cancer cell proliferation independent of the GABA shunt. Despite its elevated expression in lung cancer tissue, GAD1 is not engaged in the GABA shunt in proliferating non-small cell lung cancer (NSCLC) cells, but rather is required for regulating amino acid homeostasis. Silencing GAD1 promotes a broad deficiency in amino acid uptake, leading to reduced glutamine-dependent TCA cycle metabolism and defects in serum- and amino acid-stimulated mTORC1 activation. Mechanistically, GAD1 regulates amino acid uptake through ATF4-dependent amino acid transporter expression including SLC7A5 (LAT1), an amino acid transporter required for branched chain amino acid (BCAA) uptake. Overexpression of LAT1 rescues the proliferative and mTORC1 signalling defects of GAD1-deficient tumor cells. Our results, therefore, define a non-canonical role for GAD1, independent of its characterised role in GABA metabolism, whereby GAD1 regulates amino acid homeostasis to maintain tumor cell proliferation.

Metabolic reprogramming is an essential part of the T cell activation program. Upon activation, T cells undergo dramatic rewiring of their metabolic pathways to promote ATP production and biosynthesis sufficient to support the rapid exponential growth of antigen-specific T cells. The metabolic profile of T cells is shaped by both cell-intrinsic factors, such as genetics and receptor-mediated signaling, and environmental conditions, such as nutrient availability. However, our understanding of T cell metabolism has largely been shaped by studies in vitro, where oxygen and nutrients are in excess. Here, we combined bioenergetic profiling and 13C-glutamine infusion techniques to investigate the metabolism of CD8+ T cells responding to Listeria infection. Similar to in vitro-activated T cells, glutamine is a prominent source of fuel for the TCA cycle during early expansion (3 dpi). However, at later timepoints of infection (6 dpi), the use of glutamine is substantially decreased by Teff cells. Similar to the observation of decreased glucose usage by T cells over the course of infection (Ma et al Immunity 2019). Our data suggests a dynamic flexibly in nutrient usage by T cells in vivo to fuel the different phases of T cell functions, from the early T cell response (3 dpi) to the late (6 dpi). Our work highlights the differences in T cell metabolism in vivo compared to in vitro cultures and a new method to study T cell nutrient utilization in vivo.

During immune challenge, T lymphocytes engage pathways of anabolic metabolism to meet the demands of clonal expansion and development of effector functions. Here we report a critical role for the non-essential amino acid serine in effector T cell responses. Upon activation, T cells upregulate enzymes of the serine, glycine, one-carbon (SGOC) metabolic network, and rapidly increase processing of serine into one-carbon metabolism. We show that T cell proliferation is highly dependent on extracellular serine, and that serine is required for optimal T cell expansion even in glucose concentrations sufficient to support T cell activation, bioenergetics, and effector function. Restricting dietary serine impairs pathogen-driven expansion of T cells in vivo, without affecting overall immune cell homeostasis. Mechanistically, we demonstrate that serine supplies glycine and one-carbon units for de novo nucleotide biosynthesis in proliferating T cells, and that one-carbon units from formate can rescue T cells from serine deprivation. Our data implicate serine as a key immunometabolite that directly modulates adaptive immunity by controlling T cell proliferative capacity.

Summary Naive CD8+ T cells differentiating into effector T cells increase glucose uptake and shift from quiescent to anabolic metabolism. Although much is known about the metabolism of cultured T cells, how T cells use nutrients during immune responses in vivo is less well defined. Here, we combined bioenergetic profiling and 13C-glucose infusion techniques to investigate the metabolism of CD8+ T cells responding to Listeria infection. In contrast to in vitro-activated T cells, which display hallmarks of Warburg metabolism, physiologically activated CD8+ T cells displayed greater rates of oxidative metabolism, higher bioenergetic capacity, differential use of pyruvate, and prominent flow of 13C-glucose carbon to anabolic pathways, including nucleotide and serine biosynthesis. Glucose-dependent serine biosynthesis mediated by the enzyme Phgdh was essential for CD8+ T cell expansion in vivo. Our data highlight fundamental differences in glucose use by pathogen-specific T cells in vivo, illustrating the impact of environment on T cell metabolic phenotypes.

Summary Remodeling of the tricarboxylic acid (TCA) cycle is a metabolic adaptation accompanying inflammatory macrophage activation. During this process, endogenous metabolites can adopt regulatory roles that govern specific aspects of inflammatory response, as recently shown for succinate, which regulates the pro-inflammatory IL-1β-HIF-1α axis. Itaconate is one of the most highly induced metabolites in activated macrophages, yet its functional significance remains unknown. Here, we show that itaconate modulates macrophage metabolism and effector functions by inhibiting succinate dehydrogenase-mediated oxidation of succinate. Through this action, itaconate exerts anti-inflammatory effects when administered in vitro and in vivo during macrophage activation and ischemia-reperfusion injury. Using newly generated Irg1 −/− mice, which lack the ability to produce itaconate, we show that endogenous itaconate regulates succinate levels and function, mitochondrial respiration, and inflammatory cytokine production during macrophage activation. These studies highlight itaconate as a major physiological regulator of the global metabolic rewiring and effector functions of inflammatory macrophages.

T cell activation leads to engagement of cellular metabolic pathways necessary to support cell proliferation and function. However, our understanding of the signal transduction pathways that regulate metabolism and their impact on T cell function remains limited. The liver kinase B1 (LKB1) is a serine/threonine kinase that links cellular metabolism with cell growth and proliferation. In this study, we demonstrate that LKB1 is a critical regulator of T cell development, viability, activation, and metabolism. T cell-specific ablation of the gene that encodes LKB1 resulted in blocked thymocyte development and a reduction in peripheral T cells. LKB1-deficient T cells exhibited defects in cell proliferation and viability and altered glycolytic and lipid metabolism. Interestingly, loss of LKB1 promoted increased T cell activation and inflammatory cytokine production by both CD4+ and CD8+ T cells. Activation of the AMP-activated protein kinase (AMPK) was decreased in LKB1-deficient T cells. AMPK was found to mediate a subset of LKB1 functions in T lymphocytes, as mice lacking the α1 subunit of AMPK displayed similar defects in T cell activation, metabolism, and inflammatory cytokine production, but normal T cell development and peripheral T cell homeostasis. LKB1- and AMPKα1-deficient T cells each displayed elevated mammalian target of rapamycin complex 1 signaling and IFN-γ production that could be reversed by rapamycin treatment. Our data highlight a central role for LKB1 in T cell activation, viability, and metabolism and suggest that LKB1–AMPK signaling negatively regulates T cell effector function through regulation of mammalian target of rapamycin activity.

Metformin is a biguanide widely prescribed to treat Type II diabetes that has gained interest as an antineoplastic agent. Recent work suggests that metformin directly antagonizes cancer cell growth through its actions on complex I of the mitochondrial electron transport chain (ETC). However, the mechanisms by which metformin arrests cancer cell proliferation remain poorly defined. Here we demonstrate that the metabolic checkpoint kinases AMP-activated protein kinase (AMPK) and LKB1 are not required for the antiproliferative effects of metformin. Rather, metformin inhibits cancer cell proliferation by suppressing mitochondrial-dependent biosynthetic activity. We show that in vitro metformin decreases the flow of glucose- and glutamine-derived metabolic intermediates into the Tricarboxylic Acid (TCA) cycle, leading to reduced citrate production and de novo lipid biosynthesis. Tumor cells lacking functional mitochondria maintain lipid biosynthesis in the presence of metformin via glutamine-dependent reductive carboxylation, and display reduced sensitivity to metformin-induced proliferative arrest. Our data indicate that metformin inhibits cancer cell proliferation by suppressing the production of mitochondrial-dependent metabolic intermediates required for cell growth, and that metabolic adaptations that bypass mitochondrial-dependent biosynthesis may provide a mechanism of tumor cell resistance to biguanide activity., How does the antidiabetic drug metformin inhibit cancer? This metabolomic study shows that metformin blocks tumor cell proliferation independently of the classic metabolic checkpoints by suppressing mitochondrial-dependent biosynthesis., Author Summary Cancer is a disease characterized by unregulated proliferation of transformed cells. To meet the increased biosynthetic demands of proliferation, biosynthetic building blocks required for cellular growth must be generated in large quantities. As cancer cells increase their anabolic metabolism to promote cell growth, there is significant interest in targeting these processes for cancer therapy. Metformin is a drug prescribed to treat Type II diabetes that has gained interest as an anti-tumor agent due to its suppressive effects on cancer cell proliferation. However, how metformin works to slow cancer cell growth has remained poorly understood. Here we show that metformin arrests cancer cell proliferation by starving mitochondria of the necessary metabolic intermediates required for anabolic metabolism in tumor cells. This results in reduced proliferation in part due to decreased synthesis of lipids used for membrane biosynthesis. We also show that some cancer cells use alternative metabolic pathways to synthesize lipids independently of mitochondrial metabolism, and that these cells are resistant to the antigrowth effects of metformin. Better understanding of mechanisms of metformin resistance will be crucial for metformin to be used as an effective anticancer agent.

Cancer cells adapt metabolically to proliferate under nutrient limitation. Here we used combined transcriptional-metabolomic network analysis to identify metabolic pathways that support glucose-independent tumor cell proliferation. We found that glucose deprivation stimulated re-wiring of the tricarboxylic acid (TCA) cycle and early steps of gluconeogenesis to promote glucose-independent cell proliferation. Glucose limitation promoted the production of phosphoenolpyruvate (PEP) from glutamine via the activity of mitochondrial PEP-carboxykinase (PCK2). Under these conditions, glutamine-derived PEP was used to fuel biosynthetic pathways normally sustained by glucose, including serine and purine biosynthesis. PCK2 expression was required to maintain tumor cell proliferation under limited-glucose conditions in vitro and tumor growth in vivo. Elevated PCK2 expression is observed in several human tumor types and enriched in tumor tissue from non-small-cell lung cancer (NSCLC) patients. Our results define a role for PCK2 in cancer cell metabolic reprogramming that promotes glucose-independent cell growth and metabolic stress resistance in human tumors.

All cells must manage their energetic resources to survive. This is particularly true for cancer cells, which initiate changes in their cellular metabolism as they transition from a normal to a can...

One of the major metabolic changes associated with cellular transformation is enhanced nutrient utilization, which supports tumor progression by fueling both energy production and providing biosynthetic intermediates for growth. The liver kinase B1 (LKB1) is a serine/threonine kinase and tumor suppressor that couples bioenergetics to cell-growth control through regulation of mammalian target of rapamycin (mTOR) activity; however, the influence of LKB1 on tumor metabolism is not well defined. Here, we show that loss of LKB1 induces a progrowth metabolic program in proliferating cells. Cells lacking LKB1 display increased glucose and glutamine uptake and utilization, which support both cellular ATP levels and increased macromolecular biosynthesis. This LKB1-dependent reprogramming of cell metabolism is dependent on the hypoxia-inducible factor-1α (HIF-1α), which accumulates under normoxia in LKB1-deficient cells and is antagonized by inhibition of mTOR complex I signaling. Silencing HIF-1α reverses the metabolic advantages conferred by reduced LKB1 signaling and impairs the growth and survival of LKB1-deficient tumor cells under low-nutrient conditions. Together, our data implicate the tumor suppressor LKB1 as a central regulator of tumor metabolism and growth control through the regulation of HIF-1α–dependent metabolic reprogramming.

SummaryAMPK is a metabolic sensor that helps maintain cellular energy homeostasis. Despite evidence linking AMPK with tumor suppressor functions, the role of AMPK in tumorigenesis and tumor metabolism is unknown. Here we show that AMPK negatively regulates aerobic glycolysis (the Warburg effect) in cancer cells and suppresses tumor growth in vivo. Genetic ablation of the α1 catalytic subunit of AMPK accelerates Myc-induced lymphomagenesis. Inactivation of AMPKα in both transformed and nontransformed cells promotes a metabolic shift to aerobic glycolysis, increased allocation of glucose carbon into lipids, and biomass accumulation. These metabolic effects require normoxic stabilization of the hypoxia-inducible factor-1α (HIF-1α), as silencing HIF-1α reverses the shift to aerobic glycolysis and the biosynthetic and proliferative advantages conferred by reduced AMPKα signaling. Together our findings suggest that AMPK activity opposes tumor development and that its loss fosters tumor progression in part by regulating cellular metabolic pathways that support cell growth and proliferation.

Cancer cells must adapt metabolically to survive and proliferate when nutrients are limiting. Here we used combined transcriptional-metabolomic network analysis to identify metabolic pathways that support glucose-independent tumor cell growth. We found that glucose deprivation stimulated the re-wiring of the tricarboxylic acid (TCA) cycle and early steps of gluconeogenesis to promote cell proliferation under low glucose. Glucose limitation promoted the production of phosphoenolpyruvate (PEP) from glutamine, which was used to fuel biosynthetic pathways normally sustained by glucose, including serine and purine biosynthesis. Mitochondrial PEP-carboxykinase (PCK2) was required for this glutamine-dependent metabolic reprogramming. PCK2 expression was dependent on the hypoxia-inducible factors (HIFs), and required to maintain tumor cell proliferation under limiting glucose availability. Elevated PCK2 expression is observed in several human tumor types, and enriched in tumor tissue from non-small cell lung cancer (NSCLC) patients. Our results define a novel role for PCK2 in cancer cell metabolic reprogramming that promotes glucose-independent cell growth and metabolic stress resistance in human tumors. Citation Format: Emma E. Vincent, Alexey Sergushichev, Takla Griss, Maxime N. Artyomov, Russell G. Jones. Mitochondrial phosphoenolpyruvate carboxykinase (PCK2) regulates metabolic adaptation and enables glucose-independent tumor cell growth. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A20.