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Martin C Schmidt - One of the best experts on this subject based on the ideXlab platform.
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‘Sugarcoating’ 2-Deoxyglucose: mechanisms that suppress its toxic effects
Current Genetics, 2020Co-Authors: Martin C Schmidt, Allyson F. O’donnellAbstract:Yeast and cancer cells are metabolically similar as they use fermentation of glucose as a primary means of generating energy. Reliance on glucose fermentation makes both of these cell types highly sensitive to the toxic glucose analog, 2-Deoxyglucose. Here we review the cellular and metabolic pathways that play a role in 2-Deoxyglucose sensitivity and discuss how the modifications to these pathways result in acquisition of 2-Deoxyglucose resistance. Insights gained from genetic and proteomic studies in yeast provide new ideas for the design of combinatorial therapies for cancer treatment.
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spontaneous mutations that confer resistance to 2 Deoxyglucose act through hxk2 and snf1 pathways to regulate gene expression and hxt endocytosis
PLOS Genetics, 2020Co-Authors: Samantha R Soncini, Dakshayini G Chandrashekarappa, David A Augustine, Kenny P Callahan, Allyson F Odonnell, Martin C SchmidtAbstract:Yeast and fast-growing human tumor cells share metabolic similarities in that both cells use fermentation of glucose for energy and both are highly sensitive to the glucose analog 2-Deoxyglucose. Spontaneous mutations in S. cerevisiae that conferred resistance to 2-Deoxyglucose were identified by whole genome sequencing. Missense alleles of the HXK2, REG1, GLC7 and SNF1 genes were shown to confer significant resistance to 2-Deoxyglucose and all had the potential to alter the activity and or target selection of the Snf1 kinase signaling pathway. All three missense alleles in HXK2 resulted in significantly reduced catalytic activity. Addition of 2DG promotes endocytosis of the glucose transporter Hxt3. All but one of the 2DG-resistant strains reduced the 2DG-mediated hexose transporter endocytosis by increasing plasma membrane occupancy of the Hxt3 protein. Increased expression of the DOG (Deoxyglucose) phosphatases has been associated with resistance to 2-Deoxyglucose. Expression of both the DOG1 and DOG2 mRNA was elevated after treatment with 2-Deoxyglucose but induction of these genes is not associated with 2DG-resistance. RNAseq analysis of the transcriptional response to 2DG showed large scale, genome-wide changes in mRNA abundance that were greatly reduced in the 2DG resistant strains. These findings suggest the common adaptive response to 2DG is to limit the magnitude of the response. Genetic studies of 2DG resistance using the dominant SNF1-G53R allele in cells that are genetically compromised in both the endocytosis and DOG pathways suggest that at least one more mechanism for conferring resistance to this glucose analog remains to be discovered.
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spontaneous mutations that confer resistance to 2 Deoxyglucose act through hxk2 and snf1 pathways to regulate gene expression and hxt endocytosis
bioRxiv, 2019Co-Authors: Samantha R Soncini, Dakshayini G Chandrashekarappa, David A Augustine, Kenny P Callahan, Allyson F Odonnell, Martin C SchmidtAbstract:Abstract Yeast and fast-growing human tumor cells share metabolic similarities in that both cells use fermentation of glucose for energy and both are highly sensitive to the glucose analog 2-Deoxyglucose. Spontaneous mutations in S. cerevisiae that conferred resistance to 2-Deoxyglucose were identified by whole genome sequencing. In addition to three aneuploid strains, we detected missense alleles of the HXK2, REG1, GLC7 and SNF1 genes that were shown to confer significant resistance to 2-Deoxyglucose. All three missense alleles in HXK2 resulted in significantly reduced catalytic activity. Missense alleles affecting the Snf1 kinase pathway (REG1, GLC7 and SNF1) exhibited different capacities to affect the regulation of invertase expression. Of the seven missense alleles identified in this study, all but one affected hexose transporter endocytosis by increasing plasma membrane occupancy of the Hxt3 protein. Increased expression of the DOG (Deoxyglucose) phosphatases has been associated with resistance to 2-Deoxyglucose. Expression of both the DOG1 and DOG2 mRNA was elevated after treatment with 2-Deoxyglucose. Deletion of the HXK2 and REG1 genes confers resistance to 2-Deoxyglucose and causes increased expression of the DOG2 mRNA. We conclude that Snf1 kinase-mediated regulation of the endocytosis of the hexose transporters and regulation of DOG2 expression are important mechanisms for resistance to 2-Deoxyglucose. However, the dominant SNF1-G53R allele can confer additional 2-Deoxyglucose resistance in cells that are genetically compromised in both the endocytosis and DOG pathways. Thus at least one more mechanism for conferring resistance to this glucose analog remains to be discovered. Author Summary Yeast and fast-growing human tumor cells share metabolic similarities in that both cells use fermentation of glucose for energy and both are highly sensitive to the glucose analog 2-Deoxyglucose. Another similarity between yeast cells and human tumor cells is that both cells can acquire resistance to 2-Deoxyglucose, an outcome that can limit the usefulness of some cancer therapeutics. In this study, we used bakers’ yeast as a model organism to better understand the mechanism of toxicity and acquisition of resistance to 2-Deoxyglucose. Spontaneous mutations in S. cerevisiae that conferred resistance to 2-Deoxyglucose were isolated and identified by whole genome sequencing, a technology that was not available until recently. Our studies indicate that 2-Deoxyglucose becomes toxic after it is phosphorylated by an enzyme called hexokinase. One important route to resistance is to reduce hexokinase activity. Other parallel pathways to resistance include increased expression of a hydrolase that degrades the toxic metabolite, altered localization of glucose transporters and altered glucose signal transduction pathways.
Kenny P Callahan - One of the best experts on this subject based on the ideXlab platform.
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spontaneous mutations that confer resistance to 2 Deoxyglucose act through hxk2 and snf1 pathways to regulate gene expression and hxt endocytosis
PLOS Genetics, 2020Co-Authors: Samantha R Soncini, Dakshayini G Chandrashekarappa, David A Augustine, Kenny P Callahan, Allyson F Odonnell, Martin C SchmidtAbstract:Yeast and fast-growing human tumor cells share metabolic similarities in that both cells use fermentation of glucose for energy and both are highly sensitive to the glucose analog 2-Deoxyglucose. Spontaneous mutations in S. cerevisiae that conferred resistance to 2-Deoxyglucose were identified by whole genome sequencing. Missense alleles of the HXK2, REG1, GLC7 and SNF1 genes were shown to confer significant resistance to 2-Deoxyglucose and all had the potential to alter the activity and or target selection of the Snf1 kinase signaling pathway. All three missense alleles in HXK2 resulted in significantly reduced catalytic activity. Addition of 2DG promotes endocytosis of the glucose transporter Hxt3. All but one of the 2DG-resistant strains reduced the 2DG-mediated hexose transporter endocytosis by increasing plasma membrane occupancy of the Hxt3 protein. Increased expression of the DOG (Deoxyglucose) phosphatases has been associated with resistance to 2-Deoxyglucose. Expression of both the DOG1 and DOG2 mRNA was elevated after treatment with 2-Deoxyglucose but induction of these genes is not associated with 2DG-resistance. RNAseq analysis of the transcriptional response to 2DG showed large scale, genome-wide changes in mRNA abundance that were greatly reduced in the 2DG resistant strains. These findings suggest the common adaptive response to 2DG is to limit the magnitude of the response. Genetic studies of 2DG resistance using the dominant SNF1-G53R allele in cells that are genetically compromised in both the endocytosis and DOG pathways suggest that at least one more mechanism for conferring resistance to this glucose analog remains to be discovered.
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spontaneous mutations that confer resistance to 2 Deoxyglucose act through hxk2 and snf1 pathways to regulate gene expression and hxt endocytosis
bioRxiv, 2019Co-Authors: Samantha R Soncini, Dakshayini G Chandrashekarappa, David A Augustine, Kenny P Callahan, Allyson F Odonnell, Martin C SchmidtAbstract:Abstract Yeast and fast-growing human tumor cells share metabolic similarities in that both cells use fermentation of glucose for energy and both are highly sensitive to the glucose analog 2-Deoxyglucose. Spontaneous mutations in S. cerevisiae that conferred resistance to 2-Deoxyglucose were identified by whole genome sequencing. In addition to three aneuploid strains, we detected missense alleles of the HXK2, REG1, GLC7 and SNF1 genes that were shown to confer significant resistance to 2-Deoxyglucose. All three missense alleles in HXK2 resulted in significantly reduced catalytic activity. Missense alleles affecting the Snf1 kinase pathway (REG1, GLC7 and SNF1) exhibited different capacities to affect the regulation of invertase expression. Of the seven missense alleles identified in this study, all but one affected hexose transporter endocytosis by increasing plasma membrane occupancy of the Hxt3 protein. Increased expression of the DOG (Deoxyglucose) phosphatases has been associated with resistance to 2-Deoxyglucose. Expression of both the DOG1 and DOG2 mRNA was elevated after treatment with 2-Deoxyglucose. Deletion of the HXK2 and REG1 genes confers resistance to 2-Deoxyglucose and causes increased expression of the DOG2 mRNA. We conclude that Snf1 kinase-mediated regulation of the endocytosis of the hexose transporters and regulation of DOG2 expression are important mechanisms for resistance to 2-Deoxyglucose. However, the dominant SNF1-G53R allele can confer additional 2-Deoxyglucose resistance in cells that are genetically compromised in both the endocytosis and DOG pathways. Thus at least one more mechanism for conferring resistance to this glucose analog remains to be discovered. Author Summary Yeast and fast-growing human tumor cells share metabolic similarities in that both cells use fermentation of glucose for energy and both are highly sensitive to the glucose analog 2-Deoxyglucose. Another similarity between yeast cells and human tumor cells is that both cells can acquire resistance to 2-Deoxyglucose, an outcome that can limit the usefulness of some cancer therapeutics. In this study, we used bakers’ yeast as a model organism to better understand the mechanism of toxicity and acquisition of resistance to 2-Deoxyglucose. Spontaneous mutations in S. cerevisiae that conferred resistance to 2-Deoxyglucose were isolated and identified by whole genome sequencing, a technology that was not available until recently. Our studies indicate that 2-Deoxyglucose becomes toxic after it is phosphorylated by an enzyme called hexokinase. One important route to resistance is to reduce hexokinase activity. Other parallel pathways to resistance include increased expression of a hydrolase that degrades the toxic metabolite, altered localization of glucose transporters and altered glucose signal transduction pathways.
Jiri Turinsky - One of the best experts on this subject based on the ideXlab platform.
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Insulin-Stimulated Phosphatidylinositol 3-Kinase Activity and 2-Deoxy-D-Glucose Uptake in Rat Skeletal Muscles
Biochemical and Biophysical Research Communications, 1995Co-Authors: Jeffrey S Elmendorf, Alice Damrau-abney, T. R. Smith, T.s. David, Jiri TurinskyAbstract:Abstract To date there is suggestive evidence that phosphatidylinositol 3-kinase participates in insulin-stimulated glucose transport. However, its involvement in skeletal muscle, a major site of insulin-stimulated glucose disposal, has not been addressed. Therefore, we tested the effects of wortmannin, a known inhibitor of phosphatidylinositol 3-kinase, on insulin-stimulated 2-Deoxyglucose uptake by rat soleus muscle in vitro. Wortmannin (1 μM) reversibly inhibited insulin-induced 2-Deoxyglucose uptake in soleus muscle by 44%. Inclusion of 5 μM wortmannin in the incubation medium completely abolished the insulin-induced increment in 2-Deoxyglucose uptake. In conclusion, the results are consistent with the concept that phosphatidylinositol 3-kinase plays a role in the insulin-signaling cascade linking insulin-receptor tyrosine kinase activation to glucose uptake in skeletal muscle.
Samantha R Soncini - One of the best experts on this subject based on the ideXlab platform.
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spontaneous mutations that confer resistance to 2 Deoxyglucose act through hxk2 and snf1 pathways to regulate gene expression and hxt endocytosis
PLOS Genetics, 2020Co-Authors: Samantha R Soncini, Dakshayini G Chandrashekarappa, David A Augustine, Kenny P Callahan, Allyson F Odonnell, Martin C SchmidtAbstract:Yeast and fast-growing human tumor cells share metabolic similarities in that both cells use fermentation of glucose for energy and both are highly sensitive to the glucose analog 2-Deoxyglucose. Spontaneous mutations in S. cerevisiae that conferred resistance to 2-Deoxyglucose were identified by whole genome sequencing. Missense alleles of the HXK2, REG1, GLC7 and SNF1 genes were shown to confer significant resistance to 2-Deoxyglucose and all had the potential to alter the activity and or target selection of the Snf1 kinase signaling pathway. All three missense alleles in HXK2 resulted in significantly reduced catalytic activity. Addition of 2DG promotes endocytosis of the glucose transporter Hxt3. All but one of the 2DG-resistant strains reduced the 2DG-mediated hexose transporter endocytosis by increasing plasma membrane occupancy of the Hxt3 protein. Increased expression of the DOG (Deoxyglucose) phosphatases has been associated with resistance to 2-Deoxyglucose. Expression of both the DOG1 and DOG2 mRNA was elevated after treatment with 2-Deoxyglucose but induction of these genes is not associated with 2DG-resistance. RNAseq analysis of the transcriptional response to 2DG showed large scale, genome-wide changes in mRNA abundance that were greatly reduced in the 2DG resistant strains. These findings suggest the common adaptive response to 2DG is to limit the magnitude of the response. Genetic studies of 2DG resistance using the dominant SNF1-G53R allele in cells that are genetically compromised in both the endocytosis and DOG pathways suggest that at least one more mechanism for conferring resistance to this glucose analog remains to be discovered.
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spontaneous mutations that confer resistance to 2 Deoxyglucose act through hxk2 and snf1 pathways to regulate gene expression and hxt endocytosis
bioRxiv, 2019Co-Authors: Samantha R Soncini, Dakshayini G Chandrashekarappa, David A Augustine, Kenny P Callahan, Allyson F Odonnell, Martin C SchmidtAbstract:Abstract Yeast and fast-growing human tumor cells share metabolic similarities in that both cells use fermentation of glucose for energy and both are highly sensitive to the glucose analog 2-Deoxyglucose. Spontaneous mutations in S. cerevisiae that conferred resistance to 2-Deoxyglucose were identified by whole genome sequencing. In addition to three aneuploid strains, we detected missense alleles of the HXK2, REG1, GLC7 and SNF1 genes that were shown to confer significant resistance to 2-Deoxyglucose. All three missense alleles in HXK2 resulted in significantly reduced catalytic activity. Missense alleles affecting the Snf1 kinase pathway (REG1, GLC7 and SNF1) exhibited different capacities to affect the regulation of invertase expression. Of the seven missense alleles identified in this study, all but one affected hexose transporter endocytosis by increasing plasma membrane occupancy of the Hxt3 protein. Increased expression of the DOG (Deoxyglucose) phosphatases has been associated with resistance to 2-Deoxyglucose. Expression of both the DOG1 and DOG2 mRNA was elevated after treatment with 2-Deoxyglucose. Deletion of the HXK2 and REG1 genes confers resistance to 2-Deoxyglucose and causes increased expression of the DOG2 mRNA. We conclude that Snf1 kinase-mediated regulation of the endocytosis of the hexose transporters and regulation of DOG2 expression are important mechanisms for resistance to 2-Deoxyglucose. However, the dominant SNF1-G53R allele can confer additional 2-Deoxyglucose resistance in cells that are genetically compromised in both the endocytosis and DOG pathways. Thus at least one more mechanism for conferring resistance to this glucose analog remains to be discovered. Author Summary Yeast and fast-growing human tumor cells share metabolic similarities in that both cells use fermentation of glucose for energy and both are highly sensitive to the glucose analog 2-Deoxyglucose. Another similarity between yeast cells and human tumor cells is that both cells can acquire resistance to 2-Deoxyglucose, an outcome that can limit the usefulness of some cancer therapeutics. In this study, we used bakers’ yeast as a model organism to better understand the mechanism of toxicity and acquisition of resistance to 2-Deoxyglucose. Spontaneous mutations in S. cerevisiae that conferred resistance to 2-Deoxyglucose were isolated and identified by whole genome sequencing, a technology that was not available until recently. Our studies indicate that 2-Deoxyglucose becomes toxic after it is phosphorylated by an enzyme called hexokinase. One important route to resistance is to reduce hexokinase activity. Other parallel pathways to resistance include increased expression of a hydrolase that degrades the toxic metabolite, altered localization of glucose transporters and altered glucose signal transduction pathways.
Hitoshi Ashida - One of the best experts on this subject based on the ideXlab platform.
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UNIT 12.14 Measurement of Glucose Uptake in Cultured Cells
Current protocols in pharmacology, 2011Co-Authors: Norio Yamamoto, Manabu Ueda-wakagi, Takuya Sato, Kengo Kawasaki, Keisuke Sawada, Kyuichi Kawabata, Mitsugu Akagawa, Hitoshi AshidaAbstract:Facilitative glucose uptake transport systems are ubiquitous in animal cells and are responsible for transporting glucose across cell surface membranes. Evaluation of glucose uptake is crucial in the study of numerous diseases and metabolic disorders such as myocardial ischemia, diabetes mellitus, and cancer. Detailed in this unit are laboratory methods for assessing glucose uptake into mammalian cells. The unit is divided into five sections: (1) a brief overview of glucose uptake assays in cultured cells; (2) a method for measuring glucose uptake using radiolabeled 3-O-methylglucose; (3) a method for measuring glucose uptake using radiolabeled 2-Deoxyglucose (2DG); (4) a microplate method for measuring 2DG-uptake using an enzymatic, fluorometric assay; and (5) a microplate-based method using a fluorescent analog of 2DG. © 2015 by John Wiley & Sons, Inc. Keywords: glucose uptake; cell-based assay; 3-O-methylglucose; 2-Deoxyglucose; radioisotope; nonradioisotope; 2-NBDG,fluorescence