An LC-MS Approach to Quantitative Measurement of Ammonia Isotopologues

Ammonia is a fundamental aspect of metabolism spanning all of phylogeny. Metabolomics, including metabolic tracing studies, are an integral part of elucidating the role of ammonia in these systems. However, current methods for measurement of ammonia are spectrophotometric, and cannot distinguish isotopologues of ammonia, significantly limiting metabolic tracing studies. Here, we describe a novel LC-MS-based method that quantitatively assesses both 14N-and 15N-isotopologues of ammonia in polar metabolite extracts. This assay (1) quantitatively measures the concentration of ammonia in polar metabolite isolates used for metabolomic studies, and (2) accurately determines the percent isotope abundance of 15N-ammonia in a cell lysate for 15N-isotope tracing studies. We apply this assay to quantitatively measure glutamine-derived ammonia in lung cancer cell lines with differential expression of glutaminase

Nuclear respiratory factor 2 induces SIRT3 expression

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/113760/1/acel12360-sup-0001-FigS1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/113760/2/acel12360.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/113760/3/acel12360-sup-0002-FigS2.pd

Succinate Dehydrogenase Is a Direct Target of Sirtuin 3 Deacetylase Activity

BACKGROUND: Sirtuins (SIRT1-7) are a family of NAD-dependent deacetylases and/or ADP-ribosyltransferases that are involved in metabolism, stress responses and longevity. SIRT3 is localized to mitochondria, where it deacetylates and activates a number of enzymes involved in fuel oxidation and energy production. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we performed a proteomic screen to identify SIRT3 interacting proteins and identified several subunits of complex II and V of the electron transport chain. Two subunits of complex II (also known as succinate dehydrogenase, or SDH), SDHA and SDHB, interacted specifically with SIRT3. Using mass spectrometry, we identified 13 acetylation sites on SDHA, including six novel acetylated residues. SDHA is hyperacetylated in SIRT3 KO mice and SIRT3 directly deacetylates SDHA in a NAD-dependent manner. Finally, we found that SIRT3 regulates SDH activity both in cells and in murine brown adipose tissue. CONCLUSIONS/SIGNIFICANCE: Our study identifies SDHA as a binding partner and substrate for SIRT3 deacetylase activity. SIRT3 loss results in decreased SDH enzyme activity, suggesting that SIRT3 may be an important physiological regulator of SDH activity

Glutamine supports pancreatic cancer growth through a Kras-regulated metabolic pathway

Cancer cells exhibit metabolic dependencies that distinguish them from their normal counterparts1. Among these addictions is an increased utilization of the amino acid glutamine (Gln) to fuel anabolic processes2. Indeed, the spectrum of Gln-dependent tumors and the mechanisms whereby Gln supports cancer metabolism remain areas of active investigation. Here we report the identification of a non-canonical pathway of Gln utilization in human pancreatic ductal adenocarcinoma (PDAC) cells that is required for tumor growth. While most cells utilize glutamate dehydrogenase (GLUD1) to convert Gln-derived glutamate (Glu) into α-ketoglutarate in the mitochondria to fuel the tricarboxylic acid (TCA) cycle, PDAC relies on a distinct pathway to fuel the TCA cycle such that Gln-derived aspartate is transported into the cytoplasm where it can be converted into oxaloacetate (OAA) by aspartate transaminase (GOT1). Subsequently, this OAA is converted into malate and then pyruvate, ostensibly increasing the NADPH/NADP+ ratio which can potentially maintain the cellular redox state. Importantly, PDAC cells are strongly dependent on this series of reactions, as Gln deprivation or genetic inhibition of any enzyme in this pathway leads to an increase in reactive oxygen species and a reduction in reduced glutathione. Moreover, knockdown of any component enzyme in this series of reactions also results in a pronounced suppression of PDAC growth in vitro and in vivo. Furthermore, we establish that the reprogramming of Gln metabolism is mediated by oncogenic Kras, the signature genetic alteration in PDAC, via the transcriptional upregulation and repression of key metabolic enzymes in this pathway. The essentiality of this pathway in PDAC and the fact that it is dispensable in normal cells may provide novel therapeutic approaches to treat these refractory tumors

Intrinsic myocardial defects underlie an Rbfox-deficient zebrafish model of hypoplastic left heart syndrome

Hypoplastic left heart syndrome (HLHS) is characterized by underdevelopment of left sided structures including the ventricle, valves, and aorta. Prevailing paradigm suggests that HLHS is a multigenic disease of co-occurring phenotypes. Here, we report that zebrafish lacking two orthologs of the RNA binding protein RBFOX2, a gene linked to HLHS in humans, display cardiovascular defects overlapping those in HLHS patients including ventricular, valve, and aortic deficiencies. In contrast to current models, we demonstrate that these structural deficits arise secondary to impaired pump function as these phenotypes are rescued when Rbfox is specifically expressed in the myocardium. Mechanistically, we find diminished expression and alternative splicing of sarcomere and mitochondrial components that compromise sarcomere assembly and mitochondrial respiration, respectively. Injection of human RBFOX2 mRNA restores cardiovascular development in rbfox mutant zebrafish, while HLHS-linked RBFOX2 variants fail to rescue. This work supports an emerging paradigm for HLHS pathogenesis that centers on myocardial intrinsic defects