Metabolic requirement for GOT2 in pancreatic cancer depends on environmental context.

Mitochondrial glutamate-oxaloacetate transaminase 2 (GOT2) is part of the malate-aspartate shuttle, a mechanism by which cells transfer reducing equivalents from the cytosol to the mitochondria. GOT2 is a key component of mutant KRAS (KRAS*)-mediated rewiring of glutamine metabolism in pancreatic ductal adenocarcinoma (PDA). Here, we demonstrate that the loss of GOT2 disturbs redox homeostasis and halts proliferation of PDA cells in vitro. GOT2 knockdown (KD) in PDA cell lines in vitro induced NADH accumulation, decreased Asp and α-ketoglutarate (αKG) production, stalled glycolysis, disrupted the TCA cycle, and impaired proliferation. Oxidizing NADH through chemical or genetic means resolved the redox imbalance induced by GOT2 KD, permitting sustained proliferation. Despite a strong in vitro inhibitory phenotype, loss of GOT2 had no effect on tumor growth in xenograft PDA or autochthonous mouse models. We show that cancer-associated fibroblasts (CAFs), a major component of the pancreatic tumor microenvironment (TME), release the redox active metabolite pyruvate, and culturing GOT2 KD cells in CAF conditioned media (CM) rescued proliferation in vitro. Furthermore, blocking pyruvate import or pyruvate-to-lactate reduction prevented rescue of GOT2 KD in vitro by exogenous pyruvate or CAF CM. However, these interventions failed to sensitize xenografts to GOT2 KD in vivo, demonstrating the remarkable plasticity and differential metabolism deployed by PDA cells in vitro and in vivo. This emphasizes how the environmental context of distinct pre-clinical models impacts both cell-intrinsic metabolic rewiring and metabolic crosstalk with the TME.

High circ-SEC31A expression predicts unfavorable prognoses in non-small cell lung cancer by regulating the miR-520a-5p/GOT-2 axis.

Dysregulation of circular RNAs (circRNAs) has recently been shown to play important regulatory roles in cancer development and progression, including non-small cell lung cancer (NSCLC). However, the roles of most circRNAs in NSCLC are still unknown. In this study, we found that hsa_circ_0001421 (circ-SEC31A) was upregulated in NSCLC tissues and cell lines. Increased circ-SEC31A expression in NSCLC was significantly correlated with malignant characteristics and served as an independent risk factor for the post-surgical overall survival of NSCLC patients. Reduced circ-SEC31A expression in NSCLC decreased tumor cell proliferation, migration, invasion, and malate-aspartate metabolism. Mechanistically, we demonstrated that silencing circ-SEC31A downregulated GOT-2 expression by relieving the sponging effect of miR-520a-5p, which resulted in significantly reduced malate-aspartate metabolism in NSCLC cells. Taken together, these results revealed the important role of circ-SEC31A in the proliferation, migration, invasion, and metabolic regulation of NSCLC cells, providing a new perspective on circRNAs in NSCLC progression.

Preventing BRCA1/ZBRK1 repressor complex binding to the GOT2 promoter results in accelerated aspartate biosynthesis and promotion of cell proliferation.

Breast cancer susceptibility gene 1 (BRCA1) has been implicated in modulating metabolism via transcriptional regulation. However, direct metabolic targets of BRCA1 and the underlying regulatory mechanisms are still unknown. Here, we identified several metabolic genes, including the gene which encodes glutamate-oxaloacetate transaminase 2 (GOT2), a key enzyme for aspartate biosynthesis, which are repressed by BRCA1. We report that BRCA1 forms a co-repressor complex with ZBRK1 that coordinately represses GOT2 expression via a ZBRK1 recognition element in the promoter of GOT2. Impairment of this complex results in upregulation of GOT2, which in turn increases aspartate and alpha ketoglutarate production, leading to rapid cell proliferation of breast cancer cells. Importantly, we found that GOT2 can serve as an independent prognostic factor for overall survival and disease-free survival of patients with breast cancer, especially triple-negative breast cancer. Interestingly, we also demonstrated that GOT2 overexpression sensitized breast cancer cells to methotrexate, suggesting a promising precision therapeutic strategy for breast cancer treatment. In summary, our findings reveal that BRCA1 modulates aspartate biosynthesis through transcriptional repression of GOT2, and provides a biological basis for treatment choices in breast cancer.

Cooperative STAT/NF-κB signaling regulates lymphoma metabolic reprogramming and aberrant GOT2 expression.

Knowledge of stromal factors that have a role in the transcriptional regulation of metabolic pathways aside from c-Myc is fundamental to improvements in lymphoma therapy. Using a MYC-inducible human B-cell line, we observed the cooperative activation of STAT3 and NF-κB by IL10 and CpG stimulation. We show that IL10 + CpG-mediated cell proliferation of MYClow cells depends on glutaminolysis. By 13C- and 15N-tracing of glutamine metabolism and metabolite rescue experiments, we demonstrate that GOT2 provides aspartate and nucleotides to cells with activated or aberrant Jak/STAT and NF-κB signaling. A model of GOT2 transcriptional regulation is proposed, in which the cooperative phosphorylation of STAT3 and direct joint binding of STAT3 and p65/NF-κB to the proximal GOT2 promoter are important. Furthermore, high aberrant GOT2 expression is prognostic in diffuse large B-cell lymphoma underscoring the current findings and importance of stromal factors in lymphoma biology.

Febuxostat ameliorates secondary progressive experimental autoimmune encephalomyelitis by restoring mitochondrial energy production in a GOT2-dependent manner.

Oxidative stress and mitochondrial dysfunction are important determinants of neurodegeneration in secondary progressive multiple sclerosis (SPMS). We previously showed that febuxostat, a xanthine oxidase inhibitor, ameliorated both relapsing-remitting and secondary progressive experimental autoimmune encephalomyelitis (EAE) by preventing neurodegeneration in mice. In this study, we investigated how febuxostat protects neuron in secondary progressive EAE. A DNA microarray analysis revealed that febuxostat treatment increased the CNS expression of several mitochondria-related genes in EAE mice, most notably including GOT2, which encodes glutamate oxaloacetate transaminase 2 (GOT2). GOT2 is a mitochondrial enzyme that oxidizes glutamate to produce α-ketoglutarate for the Krebs cycle, eventually leading to the production of adenosine triphosphate (ATP). Whereas GOT2 expression was decreased in the spinal cord during the chronic progressive phase of EAE, febuxostat-treated EAE mice showed increased GOT2 expression. Moreover, febuxostat treatment of Neuro2a cells in vitro ameliorated ATP exhaustion induced by rotenone application. The ability of febuxostat to preserve ATP production in the presence of rotenone was significantly reduced by GOT2 siRNA. GOT2-mediated ATP synthesis may be a pivotal mechanism underlying the protective effect of febuxostat against neurodegeneration in EAE. Accordingly, febuxostat may also have clinical utility as a disease-modifying drug in SPMS.

Serum aminotransferases in nonalcoholic fatty liver disease are a signature of liver metabolic perturbations at the amino acid and Krebs cycle level.

BACKGROUND: Extensive epidemiologic studies have shown that cardiovascular disease and the metabolic syndrome (MetS) are associated with serum concentrations of liver enzymes; however, fundamental characteristics of this relation are currently unknown. OBJECTIVE: We aimed to explore the role of liver aminotransferases in nonalcoholic fatty liver disease (NAFLD) and MetS. DESIGN: Liver gene- and protein-expression changes of aminotransferases, including their corresponding isoforms, were evaluated in a case-control study of patients with NAFLD (n = 42), which was proven through a biopsy (control subjects: n = 10). We also carried out a serum targeted metabolite profiling to the glycolysis, gluconeogenesis, and Krebs cycle (n = 48) and an exploration by the next-generation sequencing of aminotransferase genes (n = 96). An in vitro study to provide a biological explanation of changes in the transcriptional level and enzymatic activity of aminotransferases was included. RESULTS: Fatty liver was associated with a deregulated liver expression of aminotransferases, which was unrelated to the disease severity. Metabolite profiling showed that serum aminotransferase concentrations are a signature of liver metabolic perturbations, particularly at the amino acid metabolism and Krebs cycle level. A significant and positive association between systolic hypertension and liver expression levels of glutamic-oxaloacetic transaminase 2 (GOT2) messenger RNA (Spearman R = 0.42, P = 0.03) was observed. The rs6993 located in the 3' untranslated region of the GOT2 locus was significantly associated with features of the MetS, including arterial hypertension [P = 0.028; OR: 2.285 (95% CI: 1.024, 5.09); adjusted by NAFLD severity] and plasma lipid concentrations. CONCLUSIONS: In the context of an abnormal hepatic triglyceride accumulation, circulating aminotransferases rise as a consequence of the need for increased reactions of transamination to cope with the liver metabolic derangement that is associated with greater gluconeogenesis and insulin resistance. Hence, to maintain homeostasis, the liver upregulates these enzymes, leading to changes in the amounts of amino acids released into the circulation.

Network integration of parallel metabolic and transcriptional data reveals metabolic modules that regulate macrophage polarization.

Macrophage polarization involves a coordinated metabolic and transcriptional rewiring that is only partially understood. By using an integrated high-throughput transcriptional-metabolic profiling and analysis pipeline, we characterized systemic changes during murine macrophage M1 and M2 polarization. M2 polarization was found to activate glutamine catabolism and UDP-GlcNAc-associated modules. Correspondingly, glutamine deprivation or inhibition of N-glycosylation decreased M2 polarization and production of chemokine CCL22. In M1 macrophages, we identified a metabolic break at Idh, the enzyme that converts isocitrate to alpha-ketoglutarate, providing mechanistic explanation for TCA cycle fragmentation. (13)C-tracer studies suggested the presence of an active variant of the aspartate-arginosuccinate shunt that compensated for this break. Consistently, inhibition of aspartate-aminotransferase, a key enzyme of the shunt, inhibited nitric oxide and interleukin-6 production in M1 macrophages, while promoting mitochondrial respiration. This systems approach provides a highly integrated picture of the physiological modules supporting macrophage polarization, identifying potential pharmacologic control points for both macrophage phenotypes.

Effects of sulfur dioxide on hypoxic pulmonary vascular structural remodeling.

Hypoxic pulmonary hypertension is a pathophysiological process important in the development of various cardiopulmonary diseases. Recently, we found that sulfur dioxide could be produced endogenously by pulmonary vessels, and that it showed vascular regulatory capabilities. In this paper, we examined the role of sulfur dioxide in hypoxic pulmonary vascular structural remodeling (HPVSR). A total of 48 Wistar rats were divided into six groups. Rats in the hypoxic group, hypoxic+sulfur dioxide group, and hypoxic+hydroxamate group were left under hypoxic conditions, whereas the control group, control+sulfur dioxide group, and control+hydroxamate group rats were left in room air. For each group, we measured the pulmonary arterial pressure, sulfur dioxide content in plasma and lung tissue, glutamate oxaloacetate transaminase 1 and 2 mRNAs, micro- and ultra-structural changes in pulmonary arteries, proliferation of pulmonary smooth muscle cells, vascular collagen metabolism, pulmonary endothelial cell inflammatory response, and pulmonary vascular endothelin-1 production in the rats. In hypoxic rats, the content of sulfur dioxide in plasma and lung tissue decreased significantly in comparison with those in the control groups, and significant pulmonary hypertension, pulmonary vascular structural remodeling, and increased vascular inflammatory response were also observed in hypoxic rats. Sulfur dioxide donor significantly downregulated Raf-1, mitogen-activated protein kinase kinase-1 (MEK-1) and p-ERK/ERK, and inhibited pulmonary vascular smooth muscle cell proliferation, collagen remodeling and pulmonary vascular endothelial cell nuclear factor-kappaB (NF-kappaB), and intercellular adhesion molecule 1 (ICAM-1) expressions. It also prevented pulmonary hypertension and pulmonary vascular structural remodeling in association with the upregulated sulfur dioxide/glutamate oxaloacetate transaminase pathway. Hydroxamate, however, advanced pulmonary hypertension, pulmonary vascular structural remodeling, and inflammatory response of the pulmonary artery in association with a downregulated sulfur dioxide/glutamate oxaloacetate transaminase pathway. The results suggested that sulfur dioxide markedly inhibited Raf-1, MEK-1, and the phosphorylation of extracellular signal-regulated kinase (ERK), and then inhibited pulmonary arterial smooth muscle cell (PASMC) proliferation induced by hypoxia. The downregulated sulfur dioxide/glutamate oxaloacetate transaminase pathway may be involved in the mechanisms responsible for pulmonary hypertension and pulmonary vascular structural remodeling.

Genetic differentiation of Culex pipiens (Diptera: Culicidae) in China.

The population genetic structures of Culex pipiens Linnaeus were evaluated in China over a 2000 km transect that encompasses the two subspecies, C. p. pallens and C. p. quinquefasciatus. Four polymorphic allozyme loci were investigated in 1376 mosquitoes sampled from 20 populations across four provinces. These loci were not statistically dependent with no apparent heterozygote deficit or excess. On a regional scale (intra-province), a low (Fst=0.007-0.016) and significant genetic differentiation was found, with no clear geographical pattern. On a wider scale (inter-province), the genetic differentiation was higher (Fst=0.059), and an isolation by distance emerged. The results are compared with previous population genetic surveys of this mosquito species in different geographic areas over the world. The overall pattern suggests that Culex pipiens requires considerable distance (500-1000 km) to show isolation by distance, irrespective of the subspecies (C. p. pipiens, C. p. quinquefasciatus and C. p. pallens) or the geographic location.

Fine-scale genetic structure in a free-living ungulate population.

The fine-scale genetic structure of wild animal populations has rarely been analysed, yet is potentially important as a confounding factor in quantitative genetic and allelic association studies, as well as having implications for population dynamics, inbreeding and kin selection. In this study, we examined the extent to which the three spatial subunits, or hefts, of the Village Bay population of Soay sheep (Ovis aries) on St Kilda, Scotland, are genetically structured using data from 20 microsatellite and protein loci. Allele frequencies differed significantly among three hefts in all the study years we considered (1987-2000 inclusive). Small but significantly positive F(ST) and negative F(IS) values were observed in most years, indicating that the hefts are genetically differentiated, and that within each heft there is more observed heterozygosity than would be expected if each were an isolated breeding population. Males showed less fidelity to their natal heft, and as a consequence higher levels of relatedness within hefts were observed among females than among males. There was a significant negative relationship between geographical proximity and relatedness in pairwise comparisons involving females, and on average pairs of females located within 50 m of each other were related at the equivalent level of second cousins. Structure is therefore largely driven by incomplete postnatal dispersal by females. Mating appears to be random with respect to the spatial-genetic substructure of the hefts, and therefore genetic structure does not contribute to the overall rate of inbreeding in the population. However, genetic substructure can lead to allelic associations and generate environmental effects within lineages that have the potential to confound heritability analyses and allelic association studies.

Differential expression of the L-plastin gene in human colorectal cancer progression and metastasis.

To identify molecular alterations in the progression of colorectal carcinoma, we analyzed gene expression profiles of colon cancer cell lines derived from primary and metastatic tumors from a single patient. Of 2280 cDNAs investigated using our in-house microarray, the expression of 6 genes (tumor-associated antigen L6, L-plastin, the human homologue of yeast ribosomal protein S28, the B-cell translocation gene, mitochondrial aspartate-aminotransferase, and HLA-A) increased, while that of 2 genes (keratin 5 and phosphoglucomutase) decreased in metastatic-tumor-derived cells compared with primary-tumor-derived cells. Of these genes, we assessed the L-plastin gene, an actin-bundling protein, at the protein level using a tissue microarray consisting of 58 clinically stratified colorectal cancer specimens. Consistent with our microarray results, the expression of L-plastin was significantly correlated with the progression of cancer staging. Therefore, our results suggest that the L-plastin gene is a potential metastatic marker. In addition, combining cDNA microarrays and tissue arrays, as shown here, is thought to facilitate the rapid characterization of candidate biomarkers.