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The glycogen-binding domain on the AMPK beta subunit allows the kinase to act as a glycogen sensor.

McBride A, Ghilagaber S, Nikolaev A, Hardie DG - Cell Metab. (2009)

Bottom Line: We synthesized a series of branched oligosaccharides and show that those with a single alpha1-->6 branch are allosteric inhibitors that also inhibit phosphorylation by upstream kinases.Inhibition by all carbohydrates tested was dependent on the glycogen-binding domain being abolished by mutation of residues required for carbohydrate binding.Our results suggest the hypothesis that AMPK, as well as monitoring immediate energy availability by sensing AMP/ATP, may also be able to sense the status of cellular energy reserves in the form of glycogen.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Physiology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.

ABSTRACT
AMPK beta subunits contain a conserved domain that causes association with glycogen. Although glycogen availability is known to affect AMPK regulation in vivo, the molecular mechanism for this has not been clear. We now show that AMPK is inhibited by glycogen, particularly preparations with high branching content. We synthesized a series of branched oligosaccharides and show that those with a single alpha1-->6 branch are allosteric inhibitors that also inhibit phosphorylation by upstream kinases. Removal of the outer chains of glycogen using phosphorylase, thus exposing the outer branches, renders inhibition of AMPK more potent. Inhibition by all carbohydrates tested was dependent on the glycogen-binding domain being abolished by mutation of residues required for carbohydrate binding. Our results suggest the hypothesis that AMPK, as well as monitoring immediate energy availability by sensing AMP/ATP, may also be able to sense the status of cellular energy reserves in the form of glycogen.

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Isolation of Isomaltose as an Inhibitor of AMPK(A) Bovine liver glycogen was subjected to partial acid hydrolysis and the hydrolysate passed through a glutathione Sepharose column to which the GST:GBD fusion had been prebound. The column was eluted with propionic acid and bound oligosaccharides analyzed by HPAEC. The peak eluting at 5 min was the only one found to contain carbohydrate.(B) Analysis of the peak eluting at 5 min by tandem ES-MS using collision-induced dissociation. Oligosaccharide fragments are observed as adducts with Na+ ions, increasing their mass by 23.(C) Effect of maltose and isomaltose on AMPK activity. The curve for isomaltose was generated using the best-fit parameters as in Figure 2A.(D) Effect of GBD double mutation on inhibition of recombinant AMPK by 20 mM isomaltose; results are mean ± SEM (n = 3). ∗Activity different from control without isomaltose by t test (p < 0.05).
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fig5: Isolation of Isomaltose as an Inhibitor of AMPK(A) Bovine liver glycogen was subjected to partial acid hydrolysis and the hydrolysate passed through a glutathione Sepharose column to which the GST:GBD fusion had been prebound. The column was eluted with propionic acid and bound oligosaccharides analyzed by HPAEC. The peak eluting at 5 min was the only one found to contain carbohydrate.(B) Analysis of the peak eluting at 5 min by tandem ES-MS using collision-induced dissociation. Oligosaccharide fragments are observed as adducts with Na+ ions, increasing their mass by 23.(C) Effect of maltose and isomaltose on AMPK activity. The curve for isomaltose was generated using the best-fit parameters as in Figure 2A.(D) Effect of GBD double mutation on inhibition of recombinant AMPK by 20 mM isomaltose; results are mean ± SEM (n = 3). ∗Activity different from control without isomaltose by t test (p < 0.05).

Mentions: In an attempt to establish the chemical nature of the regions of glycogen that bind to the GBD and cause inhibition of the AMPK complex, we carried out partial acid hydrolysis of bovine liver glycogen and utilized the β1 GBD as an affinity matrix to purify hydrolysis products that bound to the GBD. The hydrolysis conditions were chosen to yield a range of oligosaccharides containing up to six glucose units, as detected by high-performance anion exchange chromatography (HPAEC). The hydrolysate was passed down a glutathione Sepharose column to which the GST-GBD had been prebound, and bound oligosaccharides eluted with propionic acid. The eluted oligosaccharides were analyzed by HPAEC (Figure 5A). A large peak at 1.7 min was shown to be an artifact caused by propionic acid, while three peaks at 3.5, 5.0, and 6.5 min were analyzed by electrospray ionization-mass spectrometry. The peaks at 3.5 and 6.5 min did not appear to contain carbohydrate, but the peak at 5 min yielded a mass of 365, consistent with a disaccharide. That this disaccharide contained glucose was confirmed by collision-induced dissociation and tandem mass spectrometry (Figure 5B); the product ion spectrum contained a daughter peak at 203, corresponding to the mass of glucose plus one Na+ ion. The only disaccharides expected from partial acid hydrolysis of glycogen are maltose (α1→4 linked) and isomaltose (α1→6 linked). Attempts to identify the disaccharide by chemical linkage analysis were not successful. However, an isomaltose standard was found to elute from the HPAEC column at 5.1 min, whereas maltose eluted at 10.1 min. These results suggested that the disaccharide that was derived from glycogen and that bound to the β1-GBD was isomaltose.


The glycogen-binding domain on the AMPK beta subunit allows the kinase to act as a glycogen sensor.

McBride A, Ghilagaber S, Nikolaev A, Hardie DG - Cell Metab. (2009)

Isolation of Isomaltose as an Inhibitor of AMPK(A) Bovine liver glycogen was subjected to partial acid hydrolysis and the hydrolysate passed through a glutathione Sepharose column to which the GST:GBD fusion had been prebound. The column was eluted with propionic acid and bound oligosaccharides analyzed by HPAEC. The peak eluting at 5 min was the only one found to contain carbohydrate.(B) Analysis of the peak eluting at 5 min by tandem ES-MS using collision-induced dissociation. Oligosaccharide fragments are observed as adducts with Na+ ions, increasing their mass by 23.(C) Effect of maltose and isomaltose on AMPK activity. The curve for isomaltose was generated using the best-fit parameters as in Figure 2A.(D) Effect of GBD double mutation on inhibition of recombinant AMPK by 20 mM isomaltose; results are mean ± SEM (n = 3). ∗Activity different from control without isomaltose by t test (p < 0.05).
© Copyright Policy
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getmorefigures.php?uid=PMC2642990&req=5

fig5: Isolation of Isomaltose as an Inhibitor of AMPK(A) Bovine liver glycogen was subjected to partial acid hydrolysis and the hydrolysate passed through a glutathione Sepharose column to which the GST:GBD fusion had been prebound. The column was eluted with propionic acid and bound oligosaccharides analyzed by HPAEC. The peak eluting at 5 min was the only one found to contain carbohydrate.(B) Analysis of the peak eluting at 5 min by tandem ES-MS using collision-induced dissociation. Oligosaccharide fragments are observed as adducts with Na+ ions, increasing their mass by 23.(C) Effect of maltose and isomaltose on AMPK activity. The curve for isomaltose was generated using the best-fit parameters as in Figure 2A.(D) Effect of GBD double mutation on inhibition of recombinant AMPK by 20 mM isomaltose; results are mean ± SEM (n = 3). ∗Activity different from control without isomaltose by t test (p < 0.05).
Mentions: In an attempt to establish the chemical nature of the regions of glycogen that bind to the GBD and cause inhibition of the AMPK complex, we carried out partial acid hydrolysis of bovine liver glycogen and utilized the β1 GBD as an affinity matrix to purify hydrolysis products that bound to the GBD. The hydrolysis conditions were chosen to yield a range of oligosaccharides containing up to six glucose units, as detected by high-performance anion exchange chromatography (HPAEC). The hydrolysate was passed down a glutathione Sepharose column to which the GST-GBD had been prebound, and bound oligosaccharides eluted with propionic acid. The eluted oligosaccharides were analyzed by HPAEC (Figure 5A). A large peak at 1.7 min was shown to be an artifact caused by propionic acid, while three peaks at 3.5, 5.0, and 6.5 min were analyzed by electrospray ionization-mass spectrometry. The peaks at 3.5 and 6.5 min did not appear to contain carbohydrate, but the peak at 5 min yielded a mass of 365, consistent with a disaccharide. That this disaccharide contained glucose was confirmed by collision-induced dissociation and tandem mass spectrometry (Figure 5B); the product ion spectrum contained a daughter peak at 203, corresponding to the mass of glucose plus one Na+ ion. The only disaccharides expected from partial acid hydrolysis of glycogen are maltose (α1→4 linked) and isomaltose (α1→6 linked). Attempts to identify the disaccharide by chemical linkage analysis were not successful. However, an isomaltose standard was found to elute from the HPAEC column at 5.1 min, whereas maltose eluted at 10.1 min. These results suggested that the disaccharide that was derived from glycogen and that bound to the β1-GBD was isomaltose.

Bottom Line: We synthesized a series of branched oligosaccharides and show that those with a single alpha1-->6 branch are allosteric inhibitors that also inhibit phosphorylation by upstream kinases.Inhibition by all carbohydrates tested was dependent on the glycogen-binding domain being abolished by mutation of residues required for carbohydrate binding.Our results suggest the hypothesis that AMPK, as well as monitoring immediate energy availability by sensing AMP/ATP, may also be able to sense the status of cellular energy reserves in the form of glycogen.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Physiology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.

ABSTRACT
AMPK beta subunits contain a conserved domain that causes association with glycogen. Although glycogen availability is known to affect AMPK regulation in vivo, the molecular mechanism for this has not been clear. We now show that AMPK is inhibited by glycogen, particularly preparations with high branching content. We synthesized a series of branched oligosaccharides and show that those with a single alpha1-->6 branch are allosteric inhibitors that also inhibit phosphorylation by upstream kinases. Removal of the outer chains of glycogen using phosphorylase, thus exposing the outer branches, renders inhibition of AMPK more potent. Inhibition by all carbohydrates tested was dependent on the glycogen-binding domain being abolished by mutation of residues required for carbohydrate binding. Our results suggest the hypothesis that AMPK, as well as monitoring immediate energy availability by sensing AMP/ATP, may also be able to sense the status of cellular energy reserves in the form of glycogen.

Show MeSH
Related in: MedlinePlus