Synthesis Of Non-essential Amino Acids Research Articles

Myotube growth is associated with cancer-like metabolic reprogramming and is limited by phosphoglycerate dehydrogenase and pyruvate kinase M2

Skeletal myofiber size and oxidative metabolism are inversely related. Glycolytic fast type myofibers are larger than high high oxidative, slow type myofibers. Paradoxically, despite their lower capacity for protein synthesis (lower myonucluar density and RNA content), glycolytic, low oxidative are more responsive to an anabolic stimulus. We hypothesized that glycolytic myofibers may make use of a Warburg-like metabolic reprogramming in response to an anabolic stimulus. The Warburg effect links growth and glycolysis in tumor cells to generate glycolytic intermediates for synthesis of nucleotides and amino. The aim of this study was to test whether a similar ‘glycolysis-for-anabolism’ metabolic reprogramming also occurs in post-mitotic, hypertrophying myofibers. For this purpose, we first induced C2C12 myotube hypertrophy with IGF-1. We then added 14C glucose to the differentiation medium and measured radioactivity in isolated protein and RNA to establish whether 14C had entered anabolism. We found that especially protein became radioactive, suggesting a glucose → glycolytic intermediates → non-essential amino acid(s) → protein series of reactions, the rate of which was increased by IGF-1. Next, to investigate the importance of glycolytic flux and non-essential amino acid synthesis for myotube hypertrophy, we exposed C2C12 and primary mouse myotubes to the glycolysis inhibitor 2-Deoxy-D-glucose (2DG). We found that inhibiting glycolysis lowered C2C12 and primary myotube size. Similarly, siRNA silencing of PHGDH and PKM2, key enzymes of the Warburg-like metabolic reprogramming, reduced C2C12 and primary myotube growth; whereas retroviral PHGDH overexpression increased C2C12 myotube size. Together these results suggest that glycolysis is important for hypertrophying myotubes, which reprogramme their metabolism to facilitate anabolism, similar to cancer cells. Brendan M. Gabriel was supported by fellowships from the Novo Nordisk Foundation (NNF19OC0055072) & the Wenner-Gren Foundation, an Albert Renold Travel Fellowship from the European Foundation for the Study of Diabetes, and an Eric Reid Fund for Methodology from the Biochemical Society. Abdalla D. Mohamed was funded initially by Sarcoma UK (grant number SUK09.2015), then supported by funding from Postdoctoral Fellowship Program (Helmholtz Zentrum München, Germany), and currently by Cancer Research UK. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Enteric coronavirus PDCoV evokes a non-Warburg effect by hijacking pyruvic acid as a metabolic hub

The Warburg effect, also referred as aerobic glycolysis, is a common metabolic program during viral infection. Through targeted metabolomics combined with biochemical experiments and various cell models, we investigated the central carbon metabolism (CCM) profiles of cells infected with porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus with zoonotic potential. We found that PDCoV infection required glycolysis but decreased glycolytic flux, exhibiting a non-Warburg effect characterized by pyruvic acid accumulation. Mechanistically, PDCoV enhanced pyruvate kinase activity to promote pyruvic acid anabolism, a process that generates pyruvic acid with concomitant ATP production. PDCoV also hijacked pyruvic acid catabolism to increase biosynthesis of non-essential amino acids (NEAAs), suggesting that pyruvic acid is an essential hub for PDCoV to scavenge host energy and metabolites. Furthermore, PDCoV facilitated glutaminolysis to promote the synthesis of NEAA and pyrimidines for optimal proliferation. Our work supports a novel CCM model after viral infection and provides potential anti-PDCoV drug targets.

PSII-10 Ammonium Phosphate is an Appropriate Nitrogen Source in Nitrogen-Limiting Diets for Growing Pigs

Abstract The use of low protein (LP) diets is common in swine production, but there is potential for reduced dietary N for the endogenous synthesis of non-essential amino acids (NEAA), impacting overall animal growth. It has been previously demonstrated that inclusion of non-protein nitrogen (NPN) in a diet with an increased essential amino acid-nitrogen:total nitrogen (EAA-N:TN) ratio (i.e. limiting in total N) reduced the Lys requirement and maximum NR in growing pigs. The objective of this study was to determine the impact of NPN inclusion and Lys content on growth performance of growing pigs. A total of 240 growing pigs (120 gilts and 120 barrows) with an initial body weight (BW) of 20.2 ± 2.18 kg were housed in groups of 5 pigs/pen. Pens were randomly assigned to 1 of 6 dietary treatments over 3 blocks (n = 8 pens/treatment) in a 2 × 3 factorial design, with factors of NPN inclusion (no ammonium phosphate [NAP] or ammonium phosphate inclusion at 1.7% [AP]) and dietary lysine (Lys; 1.03%, 1.15% or 1.27% standardized ileal digestible). The AP and NAP diets were formulated to have an EAA-N:TN ratio of 0.48 and 0.54, respectively. All diets were corn and soybean meal-based and formulated to meet or exceed NRC (2012) nutrient requirements. The experimental period was 28 d, with individual BW and per pen feed intake determined weekly. On d 28, backfat and lean depth were measured on 2 pigs per pen, one male and one female. Data were analyzed using a mixed model with fixed effects of N content, Lys, and their interactions, and block as a random effect. Backfat and lean depth analysis included sex as a fixed effect. Inclusion of NPN reduced feed intake and increased gain:feed compared with pigs fed NAP diets (P < 0.05). Overall ADG and d 28 BW increased with increasing Lys, but were not impacted by dietary NPN content (P > 0.05). Pigs fed AP diets had increased lean depth (P < 0.05) and reduced backfat. Overall, NPN, as ammonium phosphate, can improve feed efficiency and increase lean depth while maintaining similar ADG and final BW.

Myotube growth is associated with cancer-like metabolic reprogramming and is limited by phosphoglycerate dehydrogenase

The Warburg effect links growth and glycolysis in cancer. A key purpose of the Warburg effect is to generate glycolytic intermediates for anabolic reactions, such as nucleotides → RNA/DNA and amino acids → protein synthesis. The aim of this study was to investigate whether a similar ‘glycolysis-for-anabolism’ metabolic reprogramming also occurs in hypertrophying skeletal muscle. To interrogate this, we first induced C2C12 myotube hypertrophy with IGF-1. We then added 14C glucose to the differentiation medium and measured radioactivity in isolated protein and RNA to establish whether 14C had entered anabolism. We found that especially protein became radioactive, suggesting a glucose → glycolytic intermediates → non-essential amino acid(s) → protein series of reactions, the rate of which was increased by IGF-1. Next, to investigate the importance of glycolytic flux and non-essential amino acid synthesis for myotube hypertrophy, we exposed C2C12 and primary mouse myotubes to the glycolysis inhibitor 2-Deoxy-d-glucose (2DG). We found that inhibiting glycolysis lowered C2C12 and primary myotube size. Similarly, siRNA silencing of PHGDH, the key enzyme of the serine biosynthesis pathway, decreased C2C12 and primary myotube size; whereas retroviral PHGDH overexpression increased C2C12 myotube size. Together these results suggest that glycolysis is important for hypertrophying myotubes, which reprogram their metabolism to facilitate anabolism, similar to cancer cells.

Human acute leukemia uses branched-chain amino acid catabolism to maintain stemness through regulating PRC2 function.

Cancer-specific metabolic activities play a crucial role in the pathogenesis of human malignancies. To investigate human acute leukemia-specific metabolic properties, we comprehensively measured the cellular metabolites within the CD34+ fraction of normal hematopoietic stem progenitor cells (HSPCs), primary human acute myelogenous leukemia (AML), and acute lymphoblastic leukemia (ALL) cells. Here, we show that human leukemia cells are addicted to the branched-chain amino acid (BCAA) metabolism to maintain their stemness, irrespective of myeloid or lymphoid types. Human primary acute leukemias had BCAA transporters for BCAA uptake, cellular BCAA, α-ketoglutarate (α-KG), and cytoplasmic BCAA transaminase-1 (BCAT1) at significantly higher levels than control HSPCs. Isotope-tracing experiments showed that in primary leukemia cells, BCAT1 actively catabolizes BCAA using α-KG into branched-chain α-ketoacids, whose metabolic processes provide leukemia cells with critical substrates for the trichloroacetic acid cycle and the synthesis of nonessential amino acids, both of which reproduce α-KG to maintain its cellular level. In xenogeneic transplantation experiments, deprivation of BCAA from daily diet strongly inhibited expansion, engraftment and self-renewal of human acute leukemia cells. Inhibition of BCAA catabolism in primary AML or ALL cells specifically inactivates the function of the polycomb repressive complex 2, an epigenetic regulator for stem cell signatures, by inhibiting the transcription of PRC components, such as zeste homolog 2 and embryonic ectoderm development. Accordingly, BCAA catabolism plays an important role in the maintenance of stemness in primary human AML and ALL, and molecules related to the BCAA metabolism pathway should be critical targets for acute leukemia treatment.

Amino Acid Homeostasis in Mammalian Cells with a Focus on Amino Acid Transport

Amino acid homeostasis is maintained by import, export, oxidation, and synthesis of nonessential amino acids, and by the synthesis and breakdown of protein. These processes work in conjunction with regulatory elements that sense amino acids or their metabolites. During and after nutrient intake, amino acid homeostasis is dominated by autoregulatory processes such as transport and oxidation of excess amino acids. Amino acid deprivation triggers processes such as autophagy and the execution of broader transcriptional programs to maintain plasma amino acid concentrations. Amino acid transport plays a crucial role in the absorption of amino acids in the intestine, the distribution of amino acids across cells and organs, the recycling of amino acids in the kidney, and the recycling of amino acids after protein breakdown.

A modern baseline for the paired isotopic analysis of skin and bone in terrestrial mammals.

We present the isotopic discrimination between paired skin and bone collagen from animals of known life history, providing a modern baseline for the interpretation of archaeological isotopic data. At present, the interpretation of inter-tissue variation (Δ(skin–bone)) in mummified remains is based on comparisons with other archaeological material, which have attributed divergence to their contrasting turnover rates, with rapidly remodelling skin collagen incorporating alterations in environmental, cultural and physiological conditions in the months prior to death. While plausible, the lack of baseline data from individuals with known life histories has hindered evaluation of the explanations presented. Our analysis of a range of animals raised under a variety of management practices showed a population-wide trend for skin collagen to be depleted in 13C by –0.7‰ and enriched in 15N by +1.0‰ relative to bone collagen, even in stillborn animals. These results are intriguing and difficult to explain using current knowledge; however, on the basis of the findings reported here, we caution any results which interpret simply on differing turnover rates. We hypothesize that there may be a consistent difference in the routing of dietary protein and lipids between skin and bone, with potentially on-site synthesis of non-essential amino acids using carbon and nitrogen that have been sourced via different biochemical pathways.

INVESTIGATION OF AMINO ACID COMPOSITION OF SOME MOSSES GATHERED FROM ARMENIA

Bryophytes are rich in a variety of secondary biological active compounds, thus provide a great potential for biotechnological and biopharmaceutical applications. Bryophytes contain potentially useful natural products, including polysaccharides terpenoids, lipids, amino acids and phenylpropanoids. Bryophytes isolated compounds and extracts have cytotoxic, antimicrobial activities. The result of our research indicate the presence in the extracts of bryophytes collected in Armenia, the entire spectrum of amino acids, especially essential ones. Also revealed in some species a certain amount of nitrogen – 840 mg/g, which indicates a more intensive synthesis of nonessential amino acids. The results obtained can be used in medicine, pharmacology.

Stable isotope tracing to assess tumor metabolism in vivo.

Cancer cells undergo diverse metabolic adaptations to meet the energetic demands imposed by dysregulated growth and proliferation. Assessing metabolism in intact tumors allows the investigator to observe the combined metabolic effects of numerous cancer cell-intrinsic and -extrinsic factors that cannot be fully captured in culture models. We have developed methods to use stable isotope-labeled nutrients (e.g., [13C]glucose) to probe metabolic activity within intact tumors in vivo, in mice and humans. In these methods, the labeled nutrient is introduced to the circulation through an intravenous catheter prior to surgical resection of the tumor and adjacent nonmalignant tissue. Metabolism within these tissues during the infusion transfers the isotope label into metabolic intermediates from pathways supplied by the infused nutrient. Extracting metabolites from surgical specimens and analyzing their isotope labeling patterns provides information about metabolism in the tissue. We provide detailed information about this technique, from introduction of the labeled tracer through data analysis and interpretation, including streamlined approaches to quantify isotope labeling in informative metabolites extracted from tissue samples. We focus on infusions with [13C]glucose and the application of mass spectrometry to assess isotope labeling in intermediates from central metabolic pathways, including glycolysis, the tricarboxylic acid cycle and nonessential amino acid synthesis. We outline practical considerations to apply these methods to human subjects undergoing surgical resections of solid tumors. We also discuss the method's versatility and consider the relative advantages and limitations of alternative approaches to introduce the tracer, harvest the tissue and analyze the data.

Variation in δ13C and δ15N values of mothers and their calves across southern right whale nursery grounds: The effects of nutritional stress?

AbstractSouthern right whales (SRW) are capital breeders that use stored energy reserves to sustain themselves and their calves on nursery areas. With successful calving events declining in some SRW populations, it has been hypothesized that nutritional stress in adult females causes reproductive failure or death of calves shortly after birth. Here we compared offsets in carbon and nitrogen isotope values of mothers and their offspring (Δ13Ccalf‐cow and Δ15Ncalf‐cow) among three SRW populations. SRW from Aotearoa New Zealand, with high population growth rates and body conditions scores, have negative Δ13Ccalf‐cow suggesting calves are utilizing 13C‐depleted lipid carbon in milk to fuel the synthesis of nonessential amino acids used to build new tissues and rapidly grow. In contrast, a significantly positive Δ13Ccalf‐cow offset previously reported for SRW from Argentina during a mass die‐off event was hypothesized to be due to calves consuming milk with low lipid content. Patterns in Δ15Ncalf‐cow were more difficult to interpret and highlight the complexity in nitrogen transfer between mother and offspring. When combined with similar data collected from Brazil and during a low mortality year in Argentina, we hypothesize this approach provides a way to retrospectively compare nutritional condition of breeding adult female SRW across nursery areas.

MYC Rules: Leading Glutamine Metabolism toward a Distinct Cancer Cell Phenotype.

Simple SummaryIn the last decade, metabolic reprogramming has emerged as a driving characteristic of cancer cells. The MYC oncogene, a transcription factor, has become of growing interest as a fundamental driver of differential cancer cell metabolism. Furthermore, the non-essential amino acid glutamine is deemed to be an important nutrient for cancer cells. In fact, glutamine can integrate into a wide variety of metabolic pathways, from energy metabolism to nucleotide synthesis. This review offers a comprehensive and specific overview of recent discoveries in the regulation of MYC oncogene activation on glutamine metabolism in cancer cells.Metabolic reprogramming and deregulated cellular energetics are hallmarks of cancer. The aberrant metabolism of cancer cells is thought to be the product of differential oncogene activation and tumor suppressor gene inactivation. MYC is one of the most important oncogenic drivers, its activation being reported in a variety of cancer types and sub-types, among which are the most prevalent and aggressive of all malignancies. This review aims to offer a comprehensive overview and highlight the importance of the c-Myc transcription factor on the regulation of metabolic pathways, in particular that of glutamine and glutaminolysis. Glutamine can be extensively metabolized into a variety of substrates and be integrated in a complex metabolic network inside the cell, from energy metabolism to nucleotide and non-essential amino acid synthesis. Together, understanding metabolic reprogramming and its underlying genetic makeup, such as MYC activation, allows for a better understanding of the cancer cell phenotype and thus of the potential vulnerabilities of cancers from a metabolic standpoint.

SnapShot: Cancer metabolism

Metabolic networks support cancer cell survival, proliferation, and malignant progression. Cancer cells take up large amounts of nutrients such as glucose and glutamine whose metabolism provides the energy, reducing equivalents, and biosynthetic precursors required to meet the biosynthetic demands of proliferation. Intermediates of glycolysis and the tricarboxylic acid (TCA) cycle provide critical building blocks for synthesis of non-essential amino acids, nucleotides, and fatty acids. To view this SnapShot, open or download the PDF.

Hepatic mTORC1 signaling activates ATF4 as part of its metabolic response to feeding and insulin

ObjectiveThe mechanistic target of rapamycin complex 1 (mTORC1) is dynamically regulated by fasting and feeding cycles in the liver to promote protein and lipid synthesis while suppressing autophagy. However, beyond these functions, the metabolic response of the liver to feeding and insulin signaling orchestrated by mTORC1 remains poorly defined. Here, we determine whether ATF4, a stress responsive transcription factor recently found to be independently regulated by mTORC1 signaling in proliferating cells, is responsive to hepatic mTORC1 signaling to alter hepatocyte metabolism. MethodsATF4 protein levels and expression of canonical gene targets were analyzed in the liver following fasting and physiological feeding in the presence or absence of the mTORC1 inhibitor, rapamycin. Primary hepatocytes from wild-type or liver-specific Atf4 knockout (LAtf4KO) mice were used to characterize the effects of insulin-stimulated mTORC1-ATF4 function on hepatocyte gene expression and metabolism. Both unbiased steady-state metabolomics and stable-isotope tracing methods were employed to define mTORC1 and ATF4-dependent metabolic changes. RNA-sequencing was used to determine global changes in feeding-induced transcripts in the livers of wild-type versus LAtf4KO mice. ResultsWe demonstrate that ATF4 and its metabolic gene targets are stimulated by mTORC1 signaling in the liver, in a hepatocyte-intrinsic manner by insulin in response to feeding. While we demonstrate that de novo purine and pyrimidine synthesis is stimulated by insulin through mTORC1 signaling in primary hepatocytes, this regulation was independent of ATF4. Metabolomics and metabolite tracing studies revealed that insulin-mTORC1-ATF4 signaling stimulates pathways of nonessential amino acid synthesis in primary hepatocytes, including those of alanine, aspartate, methionine, and cysteine, but not serine. ConclusionsThe results demonstrate that ATF4 is a novel metabolic effector of mTORC1 in the liver, extending the molecular consequences of feeding and insulin-induced mTORC1 signaling in this key metabolic tissue to the control of amino acid metabolism.

Nutrient limitation driven dynamics of amino acids and fatty acids in coastal phytoplankton

AbstractCoastal seas like the North Sea have been subject to major changes in nutrient inputs over the last decades, resulting in shifts of limiting nutrients for phytoplankton communities. Here, we investigated the seasonal and spatial distribution and synthesis patterns of individual amino acids and distinct fatty acid groups and show how these were affected by different nutrient limitations in natural coastal phytoplankton communities. Nitrogen limited communities exhibited substantially slower synthesis of essential amino acids compared to synthesis of nonessential amino acids. In short‐term nutrient addition experiments, this trend was reversed immediately after N addition to levels found under not limiting conditions. On the contrary, phosphorus limited communities showed no such shift in amino acids. Both N and P limitation induced a shift from structural to storage fatty acids with a concurrent decrease in the synthesis of poly‐unsaturated fatty acids. Reversed effects in fatty acid synthesis after N or P addition were only apparent after 72 h, when they could be found in both fatty acid biosynthesis and concentrations. The different strategies of qualitative and quantitative regulation of different biomolecule synthesis under nutrient scarcity may have far‐reaching consequences for the phytoplankton's nutritional value. Higher trophic levels may have to cope with the loss of essential amino acids and poly‐unsaturated fatty acids in nutrient limited phytoplankton, which could induce changes in the structure of food webs.

Translational and HIF-1α-Dependent Metabolic Reprogramming Underpin Metabolic Plasticity and Responses to Kinase Inhibitors and Biguanides

There is increasing interest in therapeutically exploiting metabolic differences between normal and cancer cells. We show that kinase inhibitors (KIs) and biguanides synergistically and selectively target a variety of cancer cells. Synthesis of non-essential amino acids (NEAAs) aspartate, asparagine, and serine, as well as glutamine metabolism, are major determinants of the efficacy of KI/biguanide combinations. The mTORC1/4E-BP axis regulates aspartate, asparagine, and serine synthesis by modulating mRNA translation, while ablation of 4E-BP1/2 substantially decreases sensitivity of breast cancer andmelanoma cells to KI/biguanide combinations. Efficacy of the KI/biguanide combinations is also determined by HIF-1α-dependent perturbations in glutamine metabolism, which were observed in VHL-deficient renal cancer cells. This suggests that cancer cells display metabolic plasticity by engaging non-redundant adaptive mechanisms, which allows them to survive therapeutic insults that target cancer metabolism.

Myocardial fatty acid uptake through CD36 is indispensable for sufficient bioenergetic metabolism to prevent progression of pressure overload-induced heart failure

The energy metabolism of the failing heart is characterized by reduced fatty acid (FA) oxidation and an increase in glucose utilization. However, little is known about how energy metabolism-function relationship is relevant to pathophysiology of heart failure. Recent study showed that the genetic deletion of CD36 (CD36KO), which causes reduction in FA use with an increased reliance on glucose, accelerates the progression from compensated hypertrophy to heart failure. Here, we show the mechanisms by which CD36 deletion accelerates heart failure in response to pressure overload. CD36KO mice exhibited contractile dysfunction and death from heart failure with enhanced cardiac hypertrophy and interstitial fibrosis when they were subjected to transverse aortic constriction (TAC). The pool size in the TCA cycle and levels of high-energy phosphate were significantly reduced in CD36KO-TAC hearts despite an increase in glycolytic flux. De novo synthesis of non-essential amino acids was facilitated in CD36KO-TAC hearts, which could cause a further decline of the pool size. The ingestion of a diet enriched in medium-chain FA improved cardiac dysfunction in CD36KO-TAC hearts. These findings suggest that myocardial FA uptake through CD36 is indispensable for sufficient ATP production and for preventing an increased glycolytic flux-mediated structural remodeling during pressure overload-induced hypertrophy.

Translational and HIF11-Dependent Metabolic Reprograming Underpin Oncometabolome Plasticity and Synergy Between Oncogenic Kinase Inhibitors and Biguanides

There is heightened interest to devise therapies that target the oncometabolome. We show that kinase inhibitors (KIs) and biguanides synergistically target melanoma, leukemia, and breast, colon and renal cancer cells, but not non-transformed cells. Metabolic profiling confirmed opposing effects of KIs and biguanides on glycolysis, but this was insufficient to explain the observed synergy between the drugs. Rather, we define a critical role for the synthesis of non-essential amino acids (NEAA) aspartate, asparagine and serine as well as reductive glutamine metabolism, in determining the sensitivity of cancer cells to KI - biguanide combinations. The mTORC1/4E-BP axis regulates aspartate, asparagine and serine synthesis by modulating translation of mRNAs encoding PC, ASNS, PHGDH and PSAT1. Ablation of 4E-BP1 and 2 results in a dramatic increase in serine, aspartate and asparagine levels and a substantial decrease in sensitivity of breast cancer and melanoma cells to KI - biguanide combinations. In turn, efficacy of KI - biguanide combinations is impeded by HIF1α and sustained reductive glutamine metabolism. These findings identify hitherto unappreciated translational reprograming of NEAA synthesis and HIF1α-dependent stimulation of reductive glutamine metabolism as critical metabolic vulnerabilities of cancer that underpin synergy between KIs and biguanides.

Glutaminolysis: A Hallmark of Cancer Metabolism.

Glutamine is the most abundant circulating amino acid in blood and muscle and is critical for many fundamental cell functions in cancer cells, including synthesis of metabolites that maintain mitochondrial metabolism; generation of antioxidants to remove reactive oxygen species; synthesis of nonessential amino acids (NEAAs), purines, pyrimidines, and fatty acids for cellular replication; and activation of cell signaling. In light of the pleiotropic role of glutamine in cancer cells, a comprehensive understanding of glutamine metabolism is essential for the development of metabolic therapeutic strategies for targeting cancer cells. In this article, we review oncogene-, tumor suppressor-, and tumor microenvironment-mediated regulation of glutamine metabolism in cancer cells. We describe the mechanism of glutamine's regulation of tumor proliferation, metastasis, and global methylation. Furthermore, we highlight the therapeutic potential of glutamine metabolism and emphasize that clinical application of in vivo assessment of glutamine metabolism is critical for identifying new ways to treat patients through glutamine-based metabolic therapy.

Gluconeogenesis, non-essential amino acid synthesis and substrate partitioning in chicken embryos during later development

We aimed to quantify the rate of gluconeogenesis (GNG), non-essential amino-acid (NEAA) synthesis, and substrate partitioning to the Krebs cycle in embryonic (e) day e14 and e19 chicken embryos. An in ovo continuous tracer infusion approach was employed to test the hypotheses that GNG and NEAA synthesis in developing chicken embryo increases from e14 to e19. [13 C6]Glucose or [13 C3]glycerol was continuously infused (8 h) into the chorio-allantoic compartment of eggs on e14 and e19. Glucose entry rate, Cori cycling, and GNG were higher (P < 0.05) in e19 compared to e14 embryos, presumably to support higher glycogen deposition in liver and muscle. Whereas de novo synthesis of alanine, aspartate, and glutamate via glycolysis and the Krebs cycle was higher (P < 0.01) in e14 embryos, synthesis of these NEAA from glycerol was higher (P < 0.05) in e19 compared to e14 embryos. These patterns of glucose and glycerol utilization suggest a metabolic shift to conserve glucose for glycogen synthesis and an increased utilization of yolk glycerol (from triacylglyceride) after e14. Although the contribution of glycerol to GNG in e19 embryos was higher (P < 0.05) than that in e14 embryos, the contribution of glycerol to GNG (1.3 to 6.0%) was minor. Based on [13 C6]glucose tracer kinetics, the activities of both pyruvate carboxylase (PC) and pyruvate dehydrogenase (PDH) in the liver were higher (P < 0.05) in e19 embryos; whereas the higher (P < 0.01) relative activity of liver PC compared to PDH in e14 embryos suggests a greater anaplerotic flux into the Krebs cycle. In summary, the in ovo continuous tracer infusion approach allowed for a measurement of chicken embryo whole body and liver metabolism over a shorter window of development. This study provided quantitative estimates of the developmental shifts in substrate utilization, GNG, and NEAA synthesis by chicken embryos, as well as qualitative estimates of the activities of enzymes central to the Krebs cycle, glucose, and fatty acid metabolism.

Abstract 2669: Glutaminase alternative splicing controls tumor growth and metabolism

Abstract Cancer cells reprogram their metabolism to meet their energetic and biosynthetic needs, with many cancer cells showing increased aerobic glycolysis and glutamine consumption. In addition to providing carbon to the TCA cycle to compensate for the diversion of glucose to lactate, glutamine is an important carbon and nitrogen donor for the synthesis of nucleotides, amino sugars and non-essential amino acids. Glutaminase (GLS) catalyzes the first step of glutamine metabolism, the hydrolysis of glutamine to glutamate, which can then be converted to alpha-ketoglutarate, glutathione or non-essential amino acids. GLS is alternatively spliced to make the KGA or more catalytically active GAC isoforms. Analysis of publicly available TCGA RNA-seq data from primary human tumors shows that GAC is the predominant splice form of GLS in many tumor types. However, the roles played by GAC versus KGA in tumor metabolism and progression are poorly understood. To facilitate the study of GLS alternative splicing, CRISPR/Cas9 mediated deletion of the GAC specific exon was used to convert predominantly GAC-expressing parental cell lines to solely KGA-expressing cell lines (ΔGAC). Despite showing decreased glutamine-derived glutamate and α-ketoglutarate, ΔGAC cells do not show impaired growth in nutrient-replete in vitro conditions compared to parental cells. 13C-isotopic labeling and metabolomics showed that ΔGAC cells have decreased levels of proline and alanine, both of which can be derived from glutamine-derived glutamate. This led us to ask if ΔGAC cells are dependent on exogenous non-essential amino acids. We found that depletion of non-essential amino acids slowed the growth of ΔGAC cells compared with parental cells, rescuable by exogenous proline and alanine. Importantly, the growth of lung (A549) and breast cancer (MDA-MB231) ΔGAC xenograft models was diminished compared with parental tumor xenografts in vivo, which is consistent with a role for GAC in nutrient challenged conditions. In summary, the GAC form of glutaminase fuels tumor growth partly through synthesis of non-essential amino acids, particularly under nutrient limiting conditions. As GLS inhibitors enter the clinic, a more thorough understanding of the role of GLS in tumor metabolism will aid in the development of treatment strategies to best make use of these drugs. Citation Format: Zachary Stine, Zandra E. Walton, Lin Zhang, Teresa MW Fan, Andrew N. Lane, Chi V. Dang. Glutaminase alternative splicing controls tumor growth and metabolism. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2669.