Compartmentalized metabolism supports midgestation mammalian development.

Mammalian embryogenesis requires rapid growth and proper metabolic regulation1. Midgestation features increasing oxygen and nutrient availability concomitant with fetal organ development2,3. Understanding how metabolism supports development requires approaches to observe metabolism directly in model organisms in utero. Here we used isotope tracing and metabolomics to identify evolving metabolic programmes in the placenta and embryo during midgestation in mice. These tissues differ metabolically throughout midgestation, but we pinpointed gestational days (GD) 10.5-11.5 as a transition period for both placenta and embryo. Isotope tracing revealed differences in carbohydrate metabolism between the tissues and rapid glucose-dependent purine synthesis, especially in the embryo. Glucose's contribution to the tricarboxylic acid (TCA) cycle rises throughout midgestation in the embryo but not in the placenta. By GD12.5, compartmentalized metabolic programmes are apparent within the embryo, including different nutrient contributions to the TCA cycle in different organs. To contextualize developmental anomalies associated with Mendelian metabolic defects, we analysed mice deficient in LIPT1, the enzyme that activates 2-ketoacid dehydrogenases related to the TCA cycle4,5. LIPT1 deficiency suppresses TCA cycle metabolism during the GD10.5-GD11.5 transition, perturbs brain, heart and erythrocyte development and leads to embryonic demise by GD11.5. These data document individualized metabolic programmes in developing organs in utero.

Histidine is selectively required for the growth of Myc-dependent dedifferentiation tumours in the Drosophila CNS.

Rewired metabolism of glutamine in cancer has been well documented, but less is known about other amino acids such as histidine. Here, we use Drosophila cancer models to show that decreasing the concentration of histidine in the diet strongly inhibits the growth of mutant clones induced by loss of Nerfin-1 or gain of Notch activity. In contrast, changes in dietary histidine have much less effect on the growth of wildtype neural stem cells and Prospero neural tumours. The reliance of tumours on dietary histidine and also on histidine decarboxylase (Hdc) depends upon their growth requirement for Myc. We demonstrate that Myc overexpression in nerfin-1 tumours is sufficient to switch their mode of growth from histidine/Hdc sensitive to resistant. This study suggests that perturbations in histidine metabolism selectively target neural tumours that grow via a dedifferentiation process involving large cell size increases driven by Myc.

Partial N-acetyl glutamate synthase deficiency presenting as postpartum hyperammonemia: Diagnosis and subsequent pregnancy management.

N-acetyl glutamate synthase (NAGS) deficiency (OMIM #: 237310) is a rare urea cycle disorder that usually presents early in life with hyperammonemia. NAGS catalyzes the synthesis of N-acetyl glutamate (NAG) which functions as an activator of the carbamoyl phosphate synthetase-1 mediated conversion of ammonia to carbamoyl phosphate. The absence of NAG results in a proximal urea cycle disorder which can result in severe neurologic sequelae secondary to hyperammonemia and even death. Unlike the other urea cycle disorders, a specific pharmacological treatment for NAGS deficiency exists in the form of carglumic acid, an analog of NAG. Here we present a 29-year-old previously healthy female who presented with hyperammonemia and obtundation just after the birth of her first child. Exome sequencing revealed two novel variants in the NAGS gene, and plasma metabolomics revealed extremely low levels of NAG. Carglumic acid treatment led to prompt resolution of her biochemical abnormalities and symptoms. She tolerated two subsequent pregnancies, 2 years and 6 years after her initial presentation, while taking carglumic acid, and breastfed her third child, all without complications in the mother or children. This case report emphasizes the importance of considering urea cycle disorders in previously-healthy adults presenting with neurological symptoms during periods of metabolic stress, including the postpartum period. It also highlights the efficacious and safe use of carglumic acid during pregnancy and while breastfeeding.

Novel pathogenic UQCRC2 variants in a female with normal neurodevelopment.

Electron transport chain (ETC) disorders are a group of rare, multisystem diseases caused by impaired oxidative phosphorylation and energy production. Deficiencies in complex III (CIII), also known as ubiquinol-cytochrome c reductase, are particularly rare in humans. Ubiquinol-cytochrome c reductase core protein 2 (UQCRC2) encodes a subunit of CIII that plays a crucial role in dimerization. Several pathogenic UQCRC2 variants have been identified in patients presenting with metabolic abnormalities that include lactic acidosis, hyperammonemia, hypoglycemia, and organic aciduria. Almost all previously reported UQCRC2-deficient patients exhibited neurodevelopmental involvement, including developmental delays and structural brain anomalies. Here, we describe a girl who presented at 3 yr of age with lactic acidosis, hyperammonemia, and hypoglycemia but has not shown any evidence of neurodevelopmental dysfunction by age 15. Whole-exome sequencing revealed compound heterozygosity for two novel variants in UQCRC2: c.1189G>A; p.Gly397Arg and c.437T>C; p.Phe146Ser. Here, we discuss the patient's clinical presentation and the likely pathogenicity of these two missense variants.

Comprehensive isotopomer analysis of glutamate and aspartate in small tissue samples.

Stable isotopes are powerful tools to assess metabolism. 13C labeling is detected using nuclear magnetic resonance (NMR) spectroscopy or mass spectrometry (MS). MS has excellent sensitivity but generally cannot discriminate among different 13C positions (isotopomers), whereas NMR is less sensitive but reports some isotopomers. Here, we develop an MS method that reports all 16 aspartate and 32 glutamate isotopomers while requiring less than 1% of the sample used for NMR. This method discriminates between pathways that result in the same number of 13C labels in aspartate and glutamate, providing enhanced specificity over conventional MS. We demonstrate regional metabolic heterogeneity within human tumors, document the impact of fumarate hydratase (FH) deficiency in human renal cancers, and investigate the contributions of tricarboxylic acid (TCA) cycle turnover and CO2 recycling to isotope labeling in vivo. This method can accompany NMR or standard MS to provide outstanding sensitivity in isotope-labeling experiments, particularly in vivo.

In vivo isotope tracing reveals a requirement for the electron transport chain in glucose and glutamine metabolism by tumors.

In mice and humans with cancer, intravenous 13C-glucose infusion results in 13C labeling of tumor tricarboxylic acid (TCA) cycle intermediates, indicating that pyruvate oxidation in the TCA cycle occurs in tumors. The TCA cycle is usually coupled to the electron transport chain (ETC) because NADH generated by the cycle is reoxidized to NAD+ by the ETC. However, 13C labeling does not directly report ETC activity, and other pathways can oxidize NADH, so the ETC's role in these labeling patterns is unverified. We examined the impact of the ETC complex I inhibitor IACS-010759 on tumor 13C labeling. IACS-010759 suppresses TCA cycle labeling from glucose or lactate and increases labeling from glutamine. Cancer cells expressing yeast NADH dehydrogenase-1, which recycles NADH to NAD+ independently of complex I, display normalized labeling when complex I is inhibited, indicating that cancer cell ETC activity regulates TCA cycle metabolism and 13C labeling from multiple nutrients.

In vivo characterization of glutamine metabolism identifies therapeutic targets in clear cell renal cell carcinoma.

Targeting metabolic vulnerabilities has been proposed as a therapeutic strategy in renal cell carcinoma (RCC). Here, we analyzed the metabolism of patient-derived xenografts (tumorgrafts) from diverse subtypes of RCC. Tumorgrafts from VHL-mutant clear cell RCC (ccRCC) retained metabolic features of human ccRCC and engaged in oxidative and reductive glutamine metabolism. Genetic silencing of isocitrate dehydrogenase-1 or isocitrate dehydrogenase-2 impaired reductive labeling of tricarboxylic acid (TCA) cycle intermediates in vivo and suppressed growth of tumors generated from tumorgraft-derived cells. Glutaminase inhibition reduced the contribution of glutamine to the TCA cycle and resulted in modest suppression of tumorgraft growth. Infusions with [amide-15N]glutamine revealed persistent amidotransferase activity during glutaminase inhibition, and blocking these activities with the amidotransferase inhibitor JHU-083 also reduced tumor growth in both immunocompromised and immunocompetent mice. We conclude that ccRCC tumorgrafts catabolize glutamine via multiple pathways, perhaps explaining why it has been challenging to achieve therapeutic responses in patients by inhibiting glutaminase.

Isotope tracing reveals glycolysis and oxidative metabolism in childhood tumors of multiple histologies.

Background: Survival among children with high-risk solid tumors remains poor. Reprogrammed metabolism promotes tumor growth and may contain therapeutic liabilities. Tumor metabolism has been assessed in adults using intra-operative 13C-glucose infusions. Pediatric tumors differ from adult cancers in their low mutational burden and derivation from embryonic tissues. Here we used 13C infusions to examine tumor metabolism in children, comparing phenotypes among tumor types and between childhood and adult cancers. Methods: Patients recruited to study NCT03686566 received an intra-operative infusion of [U-13C]glucose during tumor resection to evaluate central carbon pathways in the tumor, with concurrent metabolomics to provide a broad overview of metabolism. Differential characteristics were determined using multiple comparison tests and mixed effect analyses. Findings: We studied 23 tumors from 22 patients. All tumors analyzed by [U-13C]glucose contained labeling in glycolytic and tricarboxylic acid (TCA) cycle intermediates. Labeling in the TCA cycle indicated activity of pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC), with PDH predominating. Neuroblastomas had high lactate labeling relative to other childhood cancers and lung cancer, and were distinguished by abundant tyrosine catabolites consistent with catecholamine synthesis. Conclusions: Intra-operative [U13C]glucose infusions are safe and informative in pediatric cancer. Tumors of various histologies use glycolysis and oxidative metabolism, with subtype-selective differences evident from this small cohort. Expanding this cohort may uncover predictive biomarkers and therapeutic targets from tumor metabolism. Funding: N.C.I grants to P.L. (R21CA220090-01A1) and R.J.D. (R35CA22044901); H.H.M.I. funding to R.J.D.; Children's Clinical Research Advisory Committee funding to K.J.

Differential RET signaling pathways drive development of the enteric lymphoid and nervous systems.

During the early development of the gastrointestinal tract, signaling through the receptor tyrosine kinase RET is required for initiation of lymphoid organ (Peyer's patch) formation and for intestinal innervation by enteric neurons. RET signaling occurs through glial cell line-derived neurotrophic factor (GDNF) family receptor α co-receptors present in the same cell (signaling in cis). It is unclear whether RET signaling in trans, which occurs in vitro through co-receptors from other cells, has a biological role. We showed that the initial aggregation of hematopoietic cells to form lymphoid clusters occurred in a RET-dependent, chemokine-independent manner through adhesion-mediated arrest of lymphoid tissue initiator (LTin) cells. Lymphoid tissue inducer cells were not necessary for this initiation phase. LTin cells responded to all RET ligands in trans, requiring factors from other cells, whereas RET was activated in enteric neurons exclusively by GDNF in cis. Furthermore, genetic and molecular approaches revealed that the versatile RET responses in LTin cells were determined by distinct patterns of expression of the genes encoding RET and its co-receptors. Our study shows that a trans RET response in LTin cells determines the initial phase of enteric lymphoid organ morphogenesis, and suggests that differential co-expression of Ret and Gfra can control the specificity of RET signaling.