scholarly journals First characterization of glucose flux through the hexosamine biosynthesis pathway (HBP) in ex vivo mouse heart

2020 ◽  
Vol 295 (7) ◽  
pp. 2018-2033 ◽  
Author(s):  
Aaron K. Olson ◽  
Bertrand Bouchard ◽  
Wei Zhong Zhu ◽  
John C. Chatham ◽  
Christine Des Rosiers
Keyword(s):  
Ex Vivo ◽  
Mouse Heart ◽  
Biosynthesis Pathway ◽  
Perfused Heart ◽  
Glucose Flux ◽  
Hexosamine Biosynthesis ◽  
Molar Percent ◽  
Acute Changes ◽  
Hexosamine Biosynthesis Pathway ◽  
Glucose Availability

The hexosamine biosynthesis pathway (HBP) branches from glycolysis and forms UDP-GlcNAc, the moiety for O-linked β-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 ∼2.5 nmol/g of heart protein/min, representing ∼0.003–0.006% of glycolysis. Reducing cardiac workload in beating and nonbeating Langendorff perfusions had no effect on the calculated HBP flux at ∼2.3 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.

High glucose and insulin promote O-GlcNAc modification of proteins, including α-tubulin

2003 ◽  
Vol 284 (2) ◽  
pp. E424-E434 ◽  
Author(s):  
Jennie L. E. Walgren ◽  
Timothy S. Vincent ◽  
Kevin L. Schey ◽  
Maria G. Buse
Keyword(s):  
Posttranslational Modifications ◽  
High Glucose ◽  
Biosynthesis Pathway ◽  
Two Dimensional Gel Electrophoresis ◽  
Novel Proteins ◽  
L6 Myotubes ◽  
Hsp70 Family ◽  
Glucose Flux ◽  
Hexosamine Biosynthesis ◽  
Hexosamine Biosynthesis Pathway

Increased flux through the hexosamine biosynthesis pathway has been implicated in the development of glucose-induced insulin resistance and may promote the modification of certain proteins with O-linked N-acetylglucosamine ( O-GlcNAc). L6 myotubes (a model of skeletal muscle) were incubated for 18 h in 5 or 25 mM glucose with or without 10 nM insulin. As assessed by immunoblotting with an O-GlcNAc-specific antibody, high glucose and/or insulin enhanced O-GlcNAcylation of numerous proteins, including the transcription factor Sp1, a known substrate for this modification. To identify novel proteins that may be O-GlcNAc modified in a glucose concentration/insulin-responsive manner, total cell membranes were separated by one- or two-dimensional gel electrophoresis. Selected O-GlcNAcylated proteins were identified by mass spectrometry (MS) analysis. MS sequencing of tryptic peptides identified member(s) of the heat shock protein 70 (HSP70) family and rat α-tubulin. Immunoprecipitation/immunoblot studies demonstrated several HSP70 isoforms and/or posttranslational modifications, some with selectively enhanced O-GlcNAcylation following exposure to high glucose plus insulin. In conclusion, in L6 myotubes, Sp1, membrane-associated HSP70, and α-tubulin are O-GlcNAcylated; the modification is markedly enhanced by sustained increased glucose flux.

Decreased hexosamine biosynthesis in GH-deficient dwarf rat muscle. Reversal with GH, but not IGF-I, therapy

1999 ◽  
Vol 276 (3) ◽  
pp. E435-E442 ◽  
Author(s):  
Katherine A. Robinson ◽  
Steven M. Willi ◽  
Sarah Bingel ◽  
Maria G. Buse
Keyword(s):  
Insulin Resistance ◽  
Small Difference ◽  
Biosynthesis Pathway ◽  
Factor I ◽  
Glucose Flux ◽  
Igf I ◽  
Hexosamine Biosynthesis ◽  
Rate Limiting ◽  
Hexosamine Biosynthesis Pathway ◽  
Male Dwarf

Enhanced glucose flux via the hexosamine biosynthesis pathway (HNSP) has been implicated in insulin resistance. We measuredl-glutamine:d-fructose-6-phosphate amidotransferase activity (GFAT, a rate-limiting enzyme) and concentrations of UDP- N-acetyl hexosamines (UDP-HexNAc, major products of HNSP) in muscle and liver of growth hormone (GH)-deficient male dwarf (dw) rats. All parameters measured, except body weight, were similar in 5-wk-old control and dw rats. Muscle GFAT activity declined progressively with age in controls and dw rats but was consistently 30–60% lower in 8- to 14-wk-old dw rats vs. age-matched controls; UDP-HexNAc concentrations in muscle were concomitantly 30% lower in dw rats vs. controls ( P < 0.01). Concentrations of UDP-hexoses, GDP-mannose, and UDP in muscle were similar in control and dw rats. Muscle HNSP activity was similarly diminished in fed and fasted dw rats. In liver, only a small difference in GFAT activity was evident between controls and dw rats, and no differences in UDP-HexNAc concentrations were observed. Treatment with recombinant human GH (rhGH) for 5 days restored UDP-HexNAc to control levels in dw muscles ( P < 0.01) and partially restored GFAT activity. Insulin-like growth factor I treatment was ineffective. We conclude that GH participates in HNSP regulation in muscle.

scholarly journals Regulation of Akt signaling by O-GlcNAc in euglycemia

2008 ◽  
Vol 295 (4) ◽  
pp. E974-E980 ◽  
Author(s):  
Yudi A. Soesanto ◽  
Bai Luo ◽  
Deborah Jones ◽  
Rodrick Taylor ◽  
J. Scott Gabrielsen ◽  
...  
Keyword(s):  
Posttranslational Modification ◽  
Hepg2 Cells ◽  
Wheat Germ ◽  
Akt Signaling ◽  
Biosynthesis Pathway ◽  
Glucose Flux ◽  
Hexosamine Biosynthesis ◽  
Phosphoinositide 3 Kinase ◽  
Hexosamine Biosynthesis Pathway

The hexosamine biosynthesis pathway (HBP) regulates the posttranslational modification of nuclear and cytoplasmic protein by O-linked N-acetylglucosamine ( O-GlcNAc). Numerous studies have demonstrated that, in hyperglycemic conditions, excessive glucose flux through this pathway contributes to the development of insulin resistance. The role of the HBP in euglycemia, however, remains largely unknown. Here we investigated the effect of O-GlcNAc on hepatic Akt signaling at physiological concentrations of glucose. In HepG2 cells cultured in 5 mM glucose, removal of O-GlcNAc by adenoviral-mediated overexpression of O-GlcNAcase increased Akt activity and phosphorylation. We also observed that Akt was recognized by succinylated wheat germ agglutinin (sWGA), which specifically binds O-GlcNAc. Overexpression of O-GlcNAcase in HepG2 cells reduced the levels of Akt in sWGA precipitates. The increased Akt activity was accompanied by increased phosphorylation of Akt substrates and reduced mRNA for glucose-6-phosphatase and phospho enolpyruvate carboxykinase (PEPCK). The increased Akt activity was not a result of activation of its upstream activator phosphoinositide 3-kinase (PI 3-kinase). Further demonstrating Akt regulation by O-GlcNAc, we found that overexpression of O-GlcNAcase in the livers of euglycemic mice also significantly increased Akt activity, resulting in increased phosphorylation of downstream targets and decreased mRNA for glucose-6-phosphatase. Together, these data suggest that O-GlcNAc regulates Akt signaling in hepatic models under euglycemic conditions.

266-LB: Evidence for a Hexosamine Biosynthesis Pathway-Mediated Sp1 Cholesterolgenic Response Being an Early Etiological Factor of Insulin Resistance

Diabetes ◽  
2019 ◽  
Vol 68 (Supplement 1) ◽  
pp. 266-LB
Author(s):  
BRIAN A. GRICE ◽  
JACOB D. COVERT ◽  
ALEC M. KREILACH ◽  
MATTHEW THORNBURG ◽  
LIXUAN TACKETT ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Etiological Factor ◽  
Biosynthesis Pathway ◽  
Hexosamine Biosynthesis ◽  
Hexosamine Biosynthesis Pathway

scholarly journals Acute Regulation of Cardiac Metabolism by the Hexosamine Biosynthesis Pathway and Protein O-GlcNAcylation

PLoS ONE ◽  
2011 ◽  
Vol 6 (4) ◽  
pp. e18417 ◽  
Author(s):  
Boglárka Laczy ◽  
Norbert Fülöp ◽  
Arzu Onay-Besikci ◽  
Christine Des Rosiers ◽  
John C. Chatham
Keyword(s):  
Cardiac Metabolism ◽  
Biosynthesis Pathway ◽  
Hexosamine Biosynthesis ◽  
Hexosamine Biosynthesis Pathway

scholarly journals The hexosamine biosynthesis pathway is a targetable liability in KRAS/LKB1 mutant lung cancer

2020 ◽  
Vol 2 (12) ◽  
pp. 1401-1412
Author(s):  
Jiyeon Kim ◽  
Hyun Min Lee ◽  
Feng Cai ◽  
Bookyung Ko ◽  
Chendong Yang ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Biosynthesis Pathway ◽  
Hexosamine Biosynthesis ◽  
Hexosamine Biosynthesis Pathway

scholarly journals Agm1/Pgm3-Mediated Sugar Nucleotide Synthesis Is Essential for Hematopoiesis and Development

2007 ◽  
Vol 27 (16) ◽  
pp. 5849-5859 ◽  
Author(s):  
Kylie T. Greig ◽  
Jennifer Antonchuk ◽  
Donald Metcalf ◽  
Phillip O. Morgan ◽  
Danielle L. Krebs ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Cell Types ◽  
Protein Glycosylation ◽  
Biosynthesis Pathway ◽  
Nucleotide Synthesis ◽  
Glycosylphosphatidylinositol Anchor ◽  
Sugar Nucleotide ◽  
Hexosamine Biosynthesis ◽  
Hypomorphic Alleles ◽  
Hexosamine Biosynthesis Pathway

ABSTRACT Carbohydrate modification of proteins includes N-linked and O-linked glycosylation, proteoglycan formation, glycosylphosphatidylinositol anchor synthesis, and O-GlcNAc modification. Each of these modifications requires the sugar nucleotide UDP-GlcNAc, which is produced via the hexosamine biosynthesis pathway. A key step in this pathway is the interconversion of GlcNAc-6-phosphate (GlcNAc-6-P) and GlcNAc-1-P, catalyzed by phosphoglucomutase 3 (Pgm3). In this paper, we describe two hypomorphic alleles of mouse Pgm3 and show there are specific physiological consequences of a graded reduction in Pgm3 activity and global UDP-GlcNAc levels. Whereas mice lacking Pgm3 die prior to implantation, animals with less severe reductions in enzyme activity are sterile, exhibit changes in pancreatic architecture, and are anemic, leukopenic, and thrombocytopenic. These phenotypes are accompanied by specific rather than wholesale changes in protein glycosylation, suggesting that while universally required, the functions of certain proteins and, as a consequence, certain cell types are especially sensitive to reductions in Pgm3 activity.

scholarly journals Glucose regulates expression of pro-inflammatory genes IL-1β and IL-12 through a mechanism involving hexosamine biosynthesis pathway dependent regulation of αEcatenin

2021 ◽  
Author(s):  
Waruni C Dissanayake ◽  
Jin Kyo Oh ◽  
Brie Sorrenson ◽  
Peter R Shepherd
Keyword(s):  
High Glucose ◽  
Inflammatory Responses ◽  
Macrophage Polarization ◽  
Mrna Levels ◽  
Biosynthesis Pathway ◽  
Glucose Levels ◽  
Raw264.7 Macrophages ◽  
Hexosamine Biosynthesis ◽  
Induced Changes ◽  
Hexosamine Biosynthesis Pathway

High glucose levels are associated with changes in macrophage polarization and evidence indicates that the sustained or even short-term high glucose levels modulate inflammatory responses in macrophages. However, the mechanism by which macrophages can sense the changes in glucose levels are not clearly understood. We find that high glucose levels rapidly increase the α-E catenin protein level in RAW264.7 macrophages. We also find an attenuation of glucose induced increase of α-E catenin when hexosamine biosynthesis pathway is inhibited either with glutamine depletion or with the drugs azaserine and tunicamycin. This indicates the involvement of hexosamine biosynthesis pathway in this process. Then, we investigated the potential role of α-E catenin in glucose induced macrophage polarization. We find that the reduction of α-E catenin level using siRNA attenuates the glucose induced changes of both IL-1β and IL-12 mRNA levels under LPS stimulated condition but does not affect TNF-α expression. Together this indicates that α-E catenin can sense the changes in glucose levels in macrophages via hexosamine biosynthesis pathway and also can modulate the glucose induced gene expression of inflammatory markers such as IL-1β and IL-12.  This identifies a new part of the mechanism by which macrophages are able to respond to changes in glucose levels.

Sign in / Sign up

Export Citation Format

Share Document