Cells must duplicate their mass in order to proliferate. Glucose and glutamine are the major nutrients consumed by proliferating mammalian cells, but the extent to which these and other nutrients contribute to cell mass is unknown. We quantified the fraction of cell mass derived from different nutrients and find that the majority of carbon mass in cells is derived from other amino acids, which are consumed at much lower rates than glucose and glutamine. While glucose carbon has diverse fates, glutamine contributes most to protein, and this suggests that glutamine’s ability to replenish TCA cycle intermediates (anaplerosis) is primarily used for amino acid biosynthesis. These findings demonstrate that rates of nutrient consumption are indirectly associated with mass accumulation and suggest that high rates of glucose and glutamine consumption support rapid cell proliferation beyond providing carbon for biosynthesis.

CD4+ effector T cells (Teff cells) and regulatory T cells (Treg cells) undergo metabolic reprogramming to support proliferation and immunological function. Although signaling via the lipid kinase PI(3)K (phosphatidylinositol-3-OH kinase), the serine-threonine kinase Akt and the metabolic checkpoint kinase complex mTORC1 induces both expression of the glucose transporter Glut1 and aerobic glycolysis for Teff cell proliferation and inflammatory function, the mechanisms that regulate Treg cell metabolism and function remain unclear. We found that Toll-like receptor (TLR) signals that promote Treg cell proliferation increased PI(3)K-Akt-mTORC1 signaling, glycolysis and expression of Glut1. However, TLR-induced mTORC1 signaling also impaired Treg cell suppressive capacity. Conversely, the transcription factor Foxp3 opposed PI(3)K-Akt-mTORC1 signaling to diminish glycolysis and anabolic metabolism while increasing oxidative and catabolic metabolism. Notably, Glut1 expression was sufficient to increase the number of Treg cells, but it reduced their suppressive capacity and Foxp3 expression. Thus, inflammatory signals and Foxp3 balance mTORC1 signaling and glucose metabolism to control the proliferation and suppressive function of Treg cells.

SUMMARY Activated T cells differentiate into functional subsets with distinct metabolic programs. Glutaminase (GLS) converts glutamine to glutamate to support the tricarboxylic acid cycle and redox and epigenetic reactions. Here, we identify a key role for GLS in T cell activation and specification. Though GLS deficiency diminished initial T cell activation and proliferation and impaired differentiation of Th17 cells, loss of GLS also increased Tbet to promote differentiation and effector function of CD4 Th1 and CD8 CTL cells. This was associated with altered chromatin accessibility and gene expression, including decreased PIK3IP1 in Th1 cells that sensitized to IL-2-mediated mTORC1 signaling. In vivo, GLS null T cells failed to drive Th17-inflammatory diseases, and Th1 cells had initially elevated function but exhausted over time. Transient GLS inhibition, however, led to increased Th1 and CTL T cell numbers. Glutamine metabolism thus has distinct roles to promote Th17 but constrain Th1 and CTL effector cell differentiation.

The unique metabolic demands of cancer cells underscore potentially fruitful opportunities for drug discovery in the era of precision medicine. However, therapeutic targeting of cancer metabolism has led to surprisingly few new drugs to date. The neutral amino acid glutamine serves as a key intermediate in numerous metabolic processes leveraged by cancer cells including biosynthesis, cell signaling, and oxidative protection. Herein, we report the preclinical development of V-9302, a competitive small molecule antagonist of transmembrane glutamine flux, that selectively and potently targets the amino acid transporter ASCT2 (SLC1A5). Pharmacological blockade of ASCT2 with V-9302 resulted in attenuated cancer cell growth and proliferation, increased cell death, and increased oxidative stress, which collectively, contributed to anti-tumor responses in vitro and in vivo. Representing a new class of targeted therapy, this is the first study to demonstrate the utility of a pharmacological inhibitor of glutamine transport in oncology, laying a framework for paradigm-shifting therapies targeting cancer cell metabolism.

Sniffing, a rhythmic inhalation and exhalation of air through the nose, is a behavior thought to play a critical role in shaping how odor information is represented and processed by the nervous system. Although the mouse has become a prominent model for studying olfaction, little is known about sniffing behavior in mice. Here, we characterized mouse sniffing behavior by measuring intranasal pressure transients in behaving mice. Sniffing was monitored during unstructured exploratory behavior and during performance of 3 commonly used olfactory paradigms: a habituation/dishabituation task, a sand digging-based discrimination task, and a nose poke-based discrimination task. We found that respiration frequencies in quiescent mice ranged from 3 to 5 Hz--higher than that reported for rats. During exploration, sniff frequency increased up to approximately 12 Hz and was highly dynamic, with rapid changes in frequency, amplitude, and waveform. Sniffing behavior varied strongly between tasks as well as for different behavioral epochs of each task. For example, mice performing the digging-based task showed little increase in sniff frequency prior to digging, whereas mice performing a nose poke-based task showed robust increases. Mice showed large increases in sniff frequency prior to reward delivery in all tasks. Mice also showed increases in sniff frequency when nose poking in a nonodor-guided task. These results show that mouse sniffing behavior is highly dynamic, varies with behavioral context, and is strongly modulated by olfactory as well as nonolfactory events.

SUMMARY Targeted cancer therapies that use genetics are successful, but principles for selectively targeting tumor metabolism that is also dependent on the environment remain unknown. We now show that differences in rate-controlling enzymes during the Warburg Effect (WE), the most prominent hallmark of cancer cell metabolism, can be used to predict a response to targeting glucose metabolism. We establish a natural product, koningic acid (KA), to be a selective inhibitor of GAPDH, an enzyme we characterize to have differential control properties over metabolism during the WE. With machine learning and integrated pharmacogenomics and metabolomics, we demonstrate that KA efficacy is not determined by the status of individual genes, but by the quantitative extent of the WE leading to a therapeutic window in vivo. Thus, the basis of targeting of the WE can be encoded by molecular principles that extend beyond the status of individual genes.

Regulators of G protein signaling (RGS) constitute a family of proteins with a conserved RGS domain of ϳ120 amino acids that accelerate the intrinsic GTP hydrolysis of activated G␣ i and G␣ q subunits. The phosphorylationdependent interaction of 14-3-3 proteins with a subset of RGS proteins inhibits their GTPase-accelerating activity in vitro. The inhibitory interaction between 14-3-3 and RGS7 requires phosphorylation of serine 434 of RGS7. We now show that phosphorylation of serine 434 is dynamically regulated by TNF-␣. Cellular stimulation by TNF-␣ transiently decreased the phosphorylation of serine 434 of RGS7, abrogating the inhibitory interaction with 14-3-3. We examined the effect of 14-3-3 on RGS-mediated deactivation kinetics of G protein-coupled inwardly rectifying K ؉ channels (GIRKs) in Xenopus oocytes. 14-3-3 inhibited the function of wild-type RGS7, but not that of either RSG7 P436R or RGS4, two proteins that do not bind 14-3-3. Our findings are the first evidence that extracellular signals can modulate the activity of RGS proteins by regulating their interaction with 14-3-3.G protein-coupled receptors activate heterotrimeric G proteins by enhancing GDP release and binding of GTP to the G␣ subunit. By accelerating the inactivation of GTP-bound G␣ i and G␣ q subunits, the "regulator of G protein signaling" (RGS) 1 proteins negatively regulate the strength and duration of G protein signaling. RGS proteins act catalytically; a single molecule binds and inactivates multiple GTP-bound G␣ subunits. Functional specificity and temporal activity of RGS proteins arises from patterns of expression, transcriptional regulation, subcellular localization, and posttranslational modification (1). Whereas some RGS proteins are ubiquitously expressed, others are temporally and spatially restricted to certain tissues and developmental stages or are induced by cellular activation (reviewed in Refs. 2 and 3). Interaction with other cytoplasmic proteins affects the stability, function, and subcellular localization of RGS proteins (reviewed in Refs. 4 and 5).We have previously shown that binding to 14-3-3 modulates the activity of certain RGS proteins, including RGS7, in vitro (6). These RGS proteins contain a conserved SYP motif with upstream basic residues located in one of the three contact sites formed between G␣ i and the RGS domain. Binding of 14-3-3 to the SYP motif of RGS7 requires the phosphorylation of the serine residue at position 434. In this study, we demonstrate that TNF-␣ inhibits the phosphorylation of serine 434 and the interaction of RGS7 with 14-3-3. RGS7 is highly expressed in the mouse brain, with a substantial fraction associated with 14-3-3. Treatment of mice with TNF-␣ completely abrogated the interaction of RGS7 with 14-3-3. We examined the effect of 14-3-3 on RGS-mediated deactivation kinetics of G proteincoupled inwardly rectifying K ϩ channels (GIRKs) in a Xenopus co-expression system (7, 8). Microinjected 14-3-3 protein slowed the deactivation of GIRKs in the presence of wild-type RGS7, but ha...

T cells have dramatic functional and proliferative shifts in the course of maintaining immune protection from pathogens and cancer. To support these changes, T cells undergo metabolic reprogramming upon stimulation and again after antigen clearance. Depending on the extrinsic cell signals, T cells can differentiate into functionally distinct subsets that utilize and require diverse metabolic programs. Effector T cells (Teff) enhance glucose and glutamine uptake, whereas regulatory T cells (Treg) do not rely on significant rates of glycolysis. The dependence of these subsets on specific metabolic programs makes T cells reliant on these signaling pathways and nutrients. Metabolic pathways, such as those regulated by mTOR and Myc, augment T cell glycolysis and glutaminolysis programs to promote T cell activity. These pathways respond to signals and control metabolism through both transcriptional or post-transcriptional mechanisms. Epigenetic modifications also play an important role by stabilizing the transcription factors that define subset specific reprogramming. In addition, circadian rhythm cycling may also influence energy use, immune surveillance, and function of T cells. In this review, we focus on the metabolic and nutrient requirements of T cells, and how canonical pathways of growth and metabolism regulate nutrients that are essential for T cell function.