Identification of glucose transporters in Aspergillus nidulans

o characterize the mechanisms involved in glucose transport, in the filamentous fungus Aspergillus nidulans, we have identified four glucose transporter encoding genes hxtB-E. We evaluated the ability of hxtB-E to functionally complement the Saccharomyces cerevisiae EBY.VW4000 strain that is unable to grow on glucose, fructose, mannose or galactose as single carbon source. In S. cerevisiae HxtB-E were targeted to the plasma membrane. The expression of HxtB, HxtC and HxtE was able to restore growth on glucose, fructose, mannose or galactose, indicating that these transporters accept multiple sugars as a substrate through an energy dependent process. A tenfold excess of unlabeled maltose, galactose, fructose, and mannose were able to inhibit glucose uptake to different levels (50 to 80 %) in these s. cerevisiae complemented strains. Moreover, experiments with cyanide-m-chlorophenylhydrazone (CCCP), strongly suggest that hxtB, -C, and –E mediate glucose transport via active proton symport. The A. nidulans ΔhxtB, ΔhxtC or ΔhxtE null mutants showed ~2.5-fold reduction in the affinity for glucose, while ΔhxtB and -C also showed a 2-fold reduction in the capacity for glucose uptake. The ΔhxtD mutant had a 7.8-fold reduction in affinity, but a 3-fold increase in the capacity for glucose uptake. However, only the ΔhxtB mutant strain showed a detectable decreased rate of glucose consumption at low concentrations and an increased resistance to 2-deoxyglucose.The authors would like to thank the Fundacao de Amparo a Pesquisa do Estado de Sao Paulo and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Brazil for financial support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Global profiling of metabolic adaptation to hypoxic stress in human glioblastoma cells.

Oncogenetic events and unique phenomena of the tumor microenvironment together induce adaptive metabolic responses that may offer new diagnostic tools and therapeutic targets of cancer. Hypoxia, or low oxygen tension, represents a well-established and universal feature of the tumor microenvironment and has been linked to increased tumor aggressiveness as well as resistance to conventional oncological treatments. Previous studies have provided important insights into hypoxia induced changes of the transcriptome and proteome; however, how this translates into changes at the metabolite level remains to be defined. Here, we have investigated dynamic, time-dependent effects of hypoxia on the cancer cell metabolome across all families of macromolecules, i.e., carbohydrate, protein, lipid and nucleic acid, in human glioblastoma cells. Using GC/MS and LC/MS/MS, 345 and 126 metabolites were identified and quantified in cells and corresponding media, respectively, at short (6 h), intermediate (24 h), and prolonged (48 h) incubation at normoxic or hypoxic (1% O2) conditions. In conjunction, we performed gene array studies with hypoxic and normoxic cells following short and prolonged incubation. We found that levels of several key metabolites varied with the duration of hypoxic stress. In some cases, metabolic changes corresponded with hypoxic regulation of key pathways at the transcriptional level. Our results provide new insights into the metabolic response of glioblastoma cells to hypoxia, which should stimulate further work aimed at targeting cancer cell adaptive mechanisms to microenvironmental stress

Altered glucose shunting in hypoxic GBM cells.

<p>A, Schematic of the polyol pathway. B and C, Hypoxic effects on metabolites of the polyol pathway in cells (B) and media (C). D, Schematic of the pentose phosphate pathway (PPP). E, Hypoxic modulation of PPP metabolites in cells. F, Schematic of metabolic pathways involved in nucleotide sugar synthesis. G, Hypoxic modulation of nucleotide sugars and metabolites involved in their synthesis. H and I, Hypoxic induction of mRNAs encoding enzymes involved in glycogen synthesis (H) and protein and lipid glycosylation (I) at the indicated time-points. Data represent fold change in hypoxic compared with normoxic cells. * P < 0.05; # 0.05 < P < 0.1. CMAS, cytidine monophosphate N-acetylneuraminic acid synthetase; FPGT, fucose-1-phosphate guanylyltransferase; FUK, fucose kinase; GALE, UDP-galactose-4 epimerase; GALK, galactokinase; GALT, galactose-1-phosphate-uridylyltransferase; GMDS, GDP-mannose 4,6-dehydratase; GMPPB, GDP-mannose pyrophosphorylase B; GNE, glucosamine (UDP-N-Acetyl)-2-epimerase/N-acetylmannosamine kinase; GNPDA, glucosamine-6-phosphate isomerase; GNPNAT, glucosamine-phosphate N-acetyltransferase 1; G6PD, glucose-6-phosphate dehydrogenase; MPI, mannose phosphate isomerase; NAGK, N-acetylglucosamine kinase; NANP, N-acetylneuraminic acid phosphatase; NANS, N-acetylneuraminic acid synthase; 6PGD, phosphogluconate dehydrogenase; PGLS, 6-phosphogluconolactonase; PGM, phosphoglucomutase; PMM1, phosphomannomutase 1; RPE, ribulose-phosphate 3-epimerase; RPIA, ribose-5-phosphate isomerase; TALDO1, transaldolase 1; TKT, transketolase; TSTA3, GDP-L-fucose synthetase; UAP1, UDP-N-acteylglucosamine pyrophosphorylase 1; UGDH, UDP-glucose 6-dehydrogenase; UGP, UDP—lucose pyrophosphorylase.</p

Protein and amino acid metabolism in hypoxic GBM cells.

<p>A-C, Hypoxic regulation of dipeptides (A), post-translationally modified amino acids (B) and acetylated amino acids (C) expressed as fold change in hypoxic <i>vs</i>. normoxic cells. D and E, Effects of hypoxia on the gamma-glutamyl cycle in GBM cells (D) and their conditioned media (E) compared with normoxic samples. F, Hypoxic effects on transcriptional activation of amino acid transporters expressed as fold change of mRNA levels at hypoxia <i>vs</i>. normoxia. G, Hypoxic modulation of amino acid catabolism in GBM cells. H, Schematic of arginine metabolism. I, Effects of hypoxia on arginine and related metabolites. J, Schematic of methionine and serine metabolism. K and L, Hypoxic modulation of methionine and its metabolites in GBM cells (K) and their conditioned media (L). M, Hypoxic effects on mRNAs of proteins involved in cysteine and glutathione synthesis. * P < 0.05; # 0.05 < P < 0.1. ADC, arginine decarboxylase; AGMAT, agmatinase; AMD1, adenosylmethionine decarboxylase 1; ARG, arginase; BHMT, betaine-homocysteine methyltransferase; CBS, cystathionine beta synthase; CTH, cystathionine gamma-lyase; DAO, diamine oxidase; GAMT, guanidinoacetate N-methyltransferase; GATM, glycine amidinotransferase; GCLC, glutamate-cysteine ligase; GSS, glutathione synthetase; NOS, nitric oxide synthase; OAT, ornithine aminotransferase; ODC, ornithine decarboxylase; SHMT, serine hydroxymethyltransferase.</p

Hypoxic effects on TCA cycle and glutamine metabolism in GBM cells.

<p>A, Overview of TCA cycle and its role in lipid metabolism. B, Hypoxic modulation of TCA cycle metabolites in GBM cells. Data represent fold change of metabolite levels in hypoxic <i>vs</i>. normoxic GBM cells. C, Hypoxic modulation of glutamine and its metabolites in GBM cells. Data represent fold change of metabolite levels by hypoxia in GBM cells. D, Hypoxic induction of GPT2 mRNA; data represent fold change at hypoxia as compared with normoxia. E, Hypoxia dramatically increases 2-HG levels in GBM cells. * P < 0.05; # 0.05 < P < 0.1. ACAT, acetoacetyl-CoA thiolase; ACC, acetyl-CoA carboxylase; FASN, fatty acid synthase; GLS, glutaminase; HMGCS, 3-hydroxy-3-methylglutaryl-CoA synthase.</p

The metabolic phenotype of hypoxic glioblastoma cells.

<p>A, U87 MG cells were grown at normoxic (N, 21% O₂) or hypoxic (H, 1% O₂) conditions for 6, 24 or 48 h. Cells (n = 6 for each time point) and the corresponding conditioned media (n = 6 for each time point) were analyzed on three separate mass spectrometry platforms. The identification of known chemical entities was based on comparison to metabolic library entries of purified standards. B and C, Class distribution of identified metabolites in cells (B) and conditioned media (C). D and E, Summary of number of biochemicals that was significantly down- or up-regulated in hypoxic cells (D) and corresponding conditioned media (E) when compared with normoxic conditions. F, Schematic diagram of glycolysis. G, Hypoxia-driven transcriptional activation of genes encoding proteins involved in glycolysis at the indicated time-points. Data represent fold change of mRNA levels in hypoxic <i>vs</i>. normoxic cells. H and I, Hypoxic modulation of glycolysis associated metabolites. Data represent fold change of metabolite levels in hypoxic GBM cells (H) and their conditioned media (I) compared with normoxic samples. * P < 0.05; # 0.05 < P < 0.1. ALDO, aldolase; ENO2, enolase 2; GPI, glucose-6-phosphate isomerase; HK, hexokinase; LDHA, lactate dehydrogenase A; PDK3, pyruvate dehydrogenase kinase 3; PFK, phosphofructokinase; PFKFB, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase; PGAM, phosphoglycerate mutase; PGK1, phosphoglycerate kinase 1; PKM2, pyruvate kinase isoenzyme type-M2.</p

Effects of hypoxia on glycerolipid metabolism in GBM cells.

<p>A, Schematic illustration of glycerolipid metabolism. B-D, Hypoxic modulation of metabolites involved in fatty acid β-oxidation (B), fatty acids (C), and metabolites involved in the synthesis of triacylglycerols and phospholipids (D) in GBM cells. E, Hypoxic induction of mRNAs of enzymes involved in glycerolipid metabolism at the indicated time-points. Data represent fold change in hypoxic as compared with normoxic cells. F, Effects of hypoxia on phosphatidylcholine-derived lysolipids in GBM cells expressed as fold change in hypoxic compared with normoxic cells. G, Schematic graph of essential fatty acid metabolism. H, Hypoxic regulation of essential fatty acids and their metabolites in GBM cells. * P < 0.05; # 0.05 < P < 0.1. CDIPT, CDP-diacylglycerol-inositol 3-phosphatidyltransferase; CDS1, CDP-diacylglycerol synthase 1; CEPT1, choline/ethanolamine phosphotransferase 1; CHK, choline kinase; DGAT, diglyceride acyltransferase; PAP, phosphatidate phosphatase; PCYT1A, choline-phosphate cytidylyltransferase 1A; PCYT2, ethanolamine-phosphate cytidylyltransferase 2; PEMT, phosphatidylethanolamine N-methyltransferase; PTDSS1, phosphatidylserine synthase 1.</p

Hypoxic accumulation of cholesterol precursors in GBM cells.

<p>A, Schematic of cholesterol metabolism. B, Hypoxic modulation of metabolites involved in cholesterol metabolism, expressed as fold change in hypoxic compared with normoxic GBM cells. C, Hypoxic effects on INSIG-1 and -2 mRNAs in U87 MG cells. * P < 0.05; # 0.05 < P < 0.1. DHCR7, 7-dehydrocholesterol reductase; FDFT1, farnesyl-diphosphate farnesyltransferase 1; SC5D, sterol-C5-desaturase.</p

Hypoxic effects on the levels of nucleotide cofactors NAD and NADP.

<p>A, Shematic of metabolic pathways involved in the synthesis of nucleotide cofactors NAD and NADP. B-D, Hypoxic modulation of NAD, NADP and metabolites involved in their synthesis. Data represent fold change of metabolites levels in hypoxic GBM cells (B and C) and their conditioned media (D) compared with normoxic samples. * P < 0.05; # 0.05 < P < 0.1. HAAO, 3-hydroxyanthranilate 3,4-dioxygenase; IDO, indoleamine 2,3-dioxygenase; KMO, kynurenine 3-monooxygenase; KYNU, kynureninase; NADSYN1, NAD synthetase 1; NAMPT, nicotinamide phosphoribosyltransferase; NAPRT, nicotinate phosphoribosyltransferase; NMNAT, nicotinamide mononucleotide adenylyltransferase; NMRK, nicotinamide riboside kinase; QPRT, quinolinate phosphoribosyltransferase.</p

αHS counter-acts hypoxia and starvation induced cell-death in ECs. A

<p>, HUVECs were grown for 72 h in full medium (10% FBS), serum free medium, or in serum free medium supplemented with AO4B08 (αHS; 3 µg/ml), as indicated, under hypoxic conditions (1% O₂). Cell lysates were analyzed for total and cleaved caspase-3 and 7 as well as α-tubulin by immunoblotting. Upper panel shows representative immunoblots from three independent experiments. Lower panels show quantification of cleaved caspase/total caspase ratios from a representative experiment. <b>B</b>, HUVECs were grown in serum free medium in the absence (Ctrl) or presence of AO4B08 (αHS; 3 µg/ml) under hypoxic conditions for 72 h, and then stained with crystal violet. Left panel shows representative images of stained cells. Right panels shows quantitative analysis of viable cells. Data are presented as the average±S.D. *Statistically different from untreated Ctrl, P<0.05.</p