Green tea polyphenols control dysregulated glutamate dehydrogenase in transgenic mice by hijacking the ADP activation site - PubMed (original) (raw)

Green tea polyphenols control dysregulated glutamate dehydrogenase in transgenic mice by hijacking the ADP activation site

Changhong Li et al. J Biol Chem. 2011.

Abstract

Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of L-glutamate and, in animals, is extensively regulated by a number of metabolites. Gain of function mutations in GDH that abrogate GTP inhibition cause the hyperinsulinism/hyperammonemia syndrome (HHS), resulting in increased pancreatic β-cell responsiveness to leucine and susceptibility to hypoglycemia following high protein meals. We have previously shown that two of the polyphenols from green tea (epigallocatechin gallate (EGCG) and epicatechin gallate (ECG)) inhibit GDH in vitro and that EGCG blocks GDH-mediated insulin secretion in wild type rat islets. Using structural and site-directed mutagenesis studies, we demonstrate that ECG binds to the same site as the allosteric regulator, ADP. Perifusion assays using pancreatic islets from transgenic mice expressing a human HHS form of GDH demonstrate that the hyperresponse to glutamine caused by dysregulated GDH is blocked by the addition of EGCG. As observed in HHS patients, these transgenic mice are hypersensitive to amino acid feeding, and this is abrogated by oral administration of EGCG prior to challenge. Finally, the low basal blood glucose level in the HHS mouse model is improved upon chronic administration of EGCG. These results suggest that this common natural product or some derivative thereof may prove useful in controlling this genetic disorder. Of broader clinical implication is that other groups have shown that restriction of glutamine catabolism via these GDH inhibitors can be useful in treating various tumors. This HHS transgenic mouse model offers a highly useful means to test these agents in vivo.

PubMed Disclaimer

Figures

FIGURE 1.

Structure of the GDH-ECG complex. A, the entire GDH hexamer shown as a ribbon diagram. Yellow and orange spheres represent the bound ECG and NADPH molecules, respectively. For comparison, the HCP binding site from previous structural studies (42) is highlighted by mauve spheres. B, an averaged omit map showing the electron density for the bound ECG molecule.

FIGURE 2.

GDH-ECG interactions. A, details of the interactions between the bound ECG molecule and GDH. Dashed lines represent the possible hydrogen bonds. Also noted are the locations of the mutations analyzed in this study; S397I, R90S, and D123A. B, stereo image showing the relationships between the ECG and GTP binding sites. C, stereo image showing the relationship between the ADP and GTP sites. The orientation is identical to B to show the overlap between the ADP and ECG binding contacts.

FIGURE 3.

Effects of ECG binding site mutations on ECG/EGCG, ADP, and GTP allosteric regulation. A and B compare sensitivity of purified WT, D123A, and R90S forms of GDH to ECG and EGCG inhibition. Both mutants partially abrogate ECG/EGCG inhibition. C and D include the partially purified S397I mutant that was not included in the polyphenol assays because contaminants reacted with EGCG/ECG. The mutation at the subunit interface, S397I, blocks GTP inhibition, whereas the other two mutations in the ECG/ADP binding site have little or no effect on GTP inhibition. The S397I and R90S mutations block ADP activation, whereas the D123A mutation appears to make ADP a more effective activator. Error bars, S.E.

FIGURE 4.

Effects of EGCG on whole cell GDH activity. A, EGCG inhibits both TG and WT GDH with nearly identical efficacy. The higher concentration of ADP appears to interfere with EGCG inhibition. B, ADP can overcome EGCG inhibition. As a sign of ADP-EGCG antagonism, the ED50 for ADP under these conditions is more than 20 times higher than in the absence of EGCG (n = 4). Error bars, S.E.

FIGURE 5.

The effect of EGCG on Gln-stimulated insulin secretion in H454Y transgenic mouse β-cell islets. A, TG tissue secretes insulin in response to a Gln ramp stimulation. This is not observed in WT islets, and glutamine-stimulated insulin secretion in TG islets is blocked by the glutaminase inhibitor, DON, and by the GDH inhibitor, EGCG. Note that EGCG, but not DON, brings the basal insulin secretion levels (T = 20 min) down to that of WT (data are mean ± S.E. (error bars), n = 3 for each group). The black line representing WT tissue may be difficult to see because it lies directly under the TG + EGCG line (green). B, Gln stimulates Ca2+ influx in response to Gln, and this is also blocked by EGCG (representative data are shown; all experiments were repeated three times, and all showed comparable results). In both A and B, the addition of KCl serves as a control to demonstrate that none of the treatments affect insulin secretion per se.

FIGURE 6.

The effects of HCP on GDH-mediated insulin secretion from wild type and HHS tissue. A, in the presence of Gln, BCH stimulates insulin secretion from wild type islets via stimulation of GDH activity. This response is completely blocked by the addition of 5 μ

HCP. This concentration is consistent with our previous in vitro studies using purified GDH (42). B, HCP inhibition is also manifested as a block in the calcium influx upon the addition of Gln in HHS islets. Error bars, S.E.

FIGURE 7.

The effects of oral administration of EGCG on the hypersecretion of insulin in HHS transgenic mice. A, plasma glucose levels in WT mice (n = 12 for water- or EGCG-treated mice) are essentially unaffected by oral administration of water or EGCG prior to the administration of the amino acid mixture. However, the plasma glucose levels rapidly drop in the HHS TG mice (n = 12) upon the administration of the amino acid mixture, but this is blocked when the animals are fed EGCG (n = 16) prior to the amino acid challenge. B, the data here are the same as in A except that they are presented as percentage of basal levels of glucose. C, the effects of chronic administration of EGCG on the basal glucose levels during fasting rather than amino acid challenge (n = 6 for each treatment). Here, administration of EGCG to the HHS mice significantly improves plasma glucose levels. Error bars, S.E.

References

1. Hudson R. C., Daniel R. M. (1993) Comp. Biochem. Physiol. B 106, 767–792 - PubMed
1. Frieden C. (1965) J. Biol. Chem. 240, 2028–2037 - PubMed
1. George A., Bell J. E. (1980) Biochemistry 19, 6057–6061 - PubMed
1. Yielding K. L., Tomkins G. M. (1961) Proc. Natl. Acad. Sci. U.S.A. 47, 983–989 - PMC - PubMed
1. Iwatsubo M., Pantaloni D. (1967) Bull. Soc. Chem. Biol. 49, 1563–1572 - PubMed

Green tea polyphenols control dysregulated glutamate dehydrogenase in transgenic mice by hijacking the ADP activation site - PubMed (original) (raw)