First characterization of glucose flux through the hexosamine biosynthesis pathway (HBP) in ex vivo mouse heart.
J Biol Chem
; 295(7): 2018-2033, 2020 02 14.
Article
em En
| MEDLINE
| ID: mdl-31915250
ABSTRACT
The hexosamine biosynthesis pathway (HBP) branches from glycolysis and forms UDP-GlcNAc, the moiety for O-linked ß-GlcNAc (O-GlcNAc) post-translational modifications. An inability to directly measure HBP flux has hindered our understanding of the factors regulating protein O-GlcNAcylation. Our goals in this study were to (i) validate a LC-MS method that assesses HBP flux as UDP-GlcNAc (13C)-molar percent enrichment (MPE) and concentration and (ii) determine whether glucose availability or workload regulate cardiac HBP flux. For (i), we perfused isolated murine working hearts with [U-13C6]glucosamine (1, 10, 50, or 100 µm), which bypasses the rate-limiting HBP enzyme. We observed a concentration-dependent increase in UDP-GlcNAc levels and MPE, with the latter reaching a plateau of 56.3 ± 2.9%. For (ii), we perfused isolated working hearts with [U-13C6]glucose (5.5 or 25 mm). Glycolytic efflux doubled with 25 mm [U-13C6]glucose; however, the calculated HBP flux was similar among the glucose concentrations at â¼2.5 nmol/g of heart protein/min, representing â¼0.003-0.006% of glycolysis. Reducing cardiac workload in beating and nonbeating Langendorff perfusions had no effect on the calculated HBP flux at â¼2.3 and 2.5 nmol/g of heart protein/min, respectively. To the best of our knowledge, this is the first direct measurement of glucose flux through the HBP in any organ. We anticipate that these methods will enable foundational analyses of the regulation of HBP flux and protein O-GlcNAcylation. Our results suggest that in the healthy ex vivo perfused heart, HBP flux does not respond to acute changes in glucose availability or cardiac workload.
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MEDLINE
Assunto principal:
Acetilglucosamina
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Processamento de Proteína Pós-Traducional
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Glucose
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Miocárdio
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article