Green Tea Polyphenols Control Dysregulated Glutamate Dehydrogenase in Transgenic Mice by Hijacking the ADP Activation Site

Changhong Li,Franz M. Matschinsky,Pan Chen,Charles A. Stanley,Ming Li,Michael J. Bennett,Srinivas Narayan,Thomas J. Smith

doi:10.1074/jbc.m111.268599

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.

Highlights

Glutamate dehydrogenase (GDH)3 is a homohexameric enzyme found in all organisms and catalyzes the reversible oxi
We demonstrated that Epigallocatechin gallate (EGCG) inhibits BCH-stimulated insulin secretion, a process that is mediated by GDH, under conditions where GDH is no longer inhibited by high energy metabolites
Structure of epicatechin gallate (ECG) Bound to GDH— it has been clearly demonstrated that ECG/EGCG inhibits GDH in tissue culture [22, 34, 35], these polyphenols are reactive molecules (36 –39) that have been shown to affect a number of cellular systems [40, 41]

Summary

EXPERIMENTAL PROCEDURES

The protein stock solution contained 5 mg/ml GDH, 0.4 mM ECG, 3 mM NADPH, and 20 mM sodium glutamate. The artificial reservoir solutions were composed of 18% PEG 8000, 0.4 M 1,6-hexanediol, 0.4 M NaCl, saturated ECG, 5 mM reduced glutathione, 2 mM NADPH, 20 mM glutamate, and 50 mM buffer triethylammonium-HCl (pH 7.0). 12-fold noncrystallographic restraints were applied to four sections of the protein: residues 10 –208, 209 –392, 393– 444, and 445– 489 These segments correspond to the glutamate binding domain, the NAD binding domain, the antenna, and the pivot helix, respectively. GDH Enzyme Assay, Insulin Secretion, and Cytosolic Calcium Measurements in Isolated Islets—Islets were isolated by collagenase digestion and cultured for 3 days in RPMI 1640 medium containing 10 mM glucose [31]. Intracellular amino acids were measured with an UltraPerformance LC system (Waters) [33]

RESULTS

DISCUSSION

30 Ϯ 2 35 Ϯ 2 18 Ϯ 2 53 Ϯ 1