Green Tea Polyphenols Modulate Insulin Secretion by Inhibiting Glutamate Dehydrogenase
2006; Elsevier BV; Volume: 281; Issue: 15 Linguagem: Inglês
10.1074/jbc.m512792200
ISSN1083-351X
AutoresChanghong Li, Aron Allen, Jae Kwagh, Nicolai M. Doliba, Wei Qin, Habiba Najafi, Heather W. Collins, Franz M. Matschinsky, Charles A. Stanley, Thomas J. Smith,
Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoInsulin secretion by pancreatic β-cells is stimulated by glucose, amino acids, and other metabolic fuels. Glutamate dehydrogenase (GDH) has been shown to play a regulatory role in this process. The importance of GDH was underscored by features of hyperinsulinemia/hyperammonemia syndrome, where a dominant mutation causes the loss of inhibition by GTP and ATP. Here we report the effects of green tea polyphenols on GDH and insulin secretion. Of the four compounds tested, epigallocatechin gallate (EGCG) and epicatechin gallate were found to inhibit GDH with nanomolar ED50 values and were therefore found to be as potent as the physiologically important inhibitor GTP. Furthermore, we have demonstrated that 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. EGCG does not affect glucose-stimulated insulin secretion under high energy conditions where GDH is probably fully inhibited. We have further shown that these compounds act in an allosteric manner independent of their antioxidant activity and that the β-cell stimulatory effects are directly correlated with glutamine oxidation. These results demonstrate that EGCG, much like the activator of GDH (BCH), can facilitate dissecting the complex regulation of insulin secretion by pharmacologically modulating the effects of GDH. Insulin secretion by pancreatic β-cells is stimulated by glucose, amino acids, and other metabolic fuels. Glutamate dehydrogenase (GDH) has been shown to play a regulatory role in this process. The importance of GDH was underscored by features of hyperinsulinemia/hyperammonemia syndrome, where a dominant mutation causes the loss of inhibition by GTP and ATP. Here we report the effects of green tea polyphenols on GDH and insulin secretion. Of the four compounds tested, epigallocatechin gallate (EGCG) and epicatechin gallate were found to inhibit GDH with nanomolar ED50 values and were therefore found to be as potent as the physiologically important inhibitor GTP. Furthermore, we have demonstrated that 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. EGCG does not affect glucose-stimulated insulin secretion under high energy conditions where GDH is probably fully inhibited. We have further shown that these compounds act in an allosteric manner independent of their antioxidant activity and that the β-cell stimulatory effects are directly correlated with glutamine oxidation. These results demonstrate that EGCG, much like the activator of GDH (BCH), can facilitate dissecting the complex regulation of insulin secretion by pharmacologically modulating the effects of GDH. The mitochondria of the pancreatic β-cell play an integrative role in the fuel stimulation of insulin secretion. The current concept is that mitochondrial oxidation of substrates increases the cellular phosphate potential that is manifested by a rise in the ATP4-/MgADP2- ratio. This in turn closes the plasma membrane KATP channels, opens voltage-gated Ca2+ channels, causes a rise of free cytoplasmic Ca2+, and leads to insulin granule exocytosis. Mitochondrial glutamate dehydrogenase (GDH) 2The abbreviations used are: GDH, glutamate dehydrogenase; EGCG, epigallocatechin gallate; EGC, epigallocatechin; ECG, epicatechin gallate; EC, epicatechin; BCH, β-2-aminobicyclo(2.2.1)heptane-2-carboxylic acid; LSIS, leucine-stimulated insulin secretion; BCH-SIS, BCH-stimulated insulin secretion; GSIS, glucose-stimulated insulin secretion; DON, 6-diazo-5-oxo-l-norleucine; HI/HA, hyperinsulinemia/hyperammonemia. catalyzes the oxidative deamination of l-glutamate and exhibits complex regulation in mammals through inhibition by palmitoyl CoA, GTP, and ATP and activation by ADP and leucine (1Brunhuber N.M.W. Blanchard J.S. Crit. Rev. Biochem. Mol. Biol. 1994; 29: 415-567Crossref PubMed Scopus (101) Google Scholar). The connection between GDH and insulin regulation was initially established using an analog of leucine that is not able to be metabolized (2Sener A. Malaisse W.J. Nature. 1980; 288: 187-189Crossref PubMed Scopus (283) Google Scholar, 3Sener A. Malaisse-Lagae F. Malaisse W.J. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 5460-5464Crossref PubMed Scopus (109) Google Scholar), BCH (β-2-aminobicycle(2.2.1)-heptane-2-carboxylic acid). These studies demonstrated that activation of GDH was tightly correlated with increased glutaminolysis. In addition, it has also been noted that factors that regulate GDH may affect insulin secretion (4Fahien L.A. MacDonald M.J. Kmiotek E.H. Mertz R.J. Fahien C.M. J. Biol. Chem. 1988; 263: 13610-13614Abstract Full Text PDF PubMed Google Scholar). It was suggested that glutamate serves as a mitochondrial intracellular messenger when glucose is being oxidized and that the GDH participates in this process by synthesizing glutamate (5Maechler P. Wollheim C.B. Nature. 1999; 402: 685-689Crossref PubMed Scopus (440) Google Scholar). However, the hypothesis that GDH (with a very high Km for ammonium) functions in the reductive amination reaction in vivo is controversial (6MacDonald M.J. Fahien L.A. J. Biol. Chem. 2000; 275: 34025-34027Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Subsequently, we postulated that glutamine could also play a secondary messenger role and that GDH plays a role in its regulation (7Li C. Najafi H. Daikhin Y. Nissim I. Collins H.W. Yudkoff M. Matschinsky F.M. Stanley C.A. J. Biol. Chem. 2003; 278: 2853-2858Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 8Li C. Buettger C. Kwagh J. Matter A. Daihkin Y. Nissiam I. Collins H.W. Yudkoff M. Stanley C.A. Matschinsky F.M. J. Biol. Chem. 2004; 279: 13393-13401Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 9Stanley C.A. O'Rahilly S. Dunger D.B. Genetic Insights in Paediatric Endocrinology and Metabolism. BioScientifica, Ltd, Bristol, UK2000: 23-30Google Scholar). The in vivo importance of GDH in glucose homeostasis was demonstrated by the discovery that a genetic hypoglycemic disorder, the hyperinsulinemia/hyperammonemia (HI/HA) syndrome, is caused by dysregulation of GDH (10Stanley C.A. Lieu Y.K. Hsu B.Y. Burlina A.B. Greenberg C.R. Hopwood N.J. Perlman K. Rich B.H. Zammarchi E. Poncz M. N. Engl. J. Med. 1998; 338: 1352-1357Crossref PubMed Scopus (620) Google Scholar, 11Stanley C.A. Fang J. Kutyna K. Hsu B.Y. Ming J.E. Glaser B. Poncz M. Diabetes. 2000; 49: 667-673Crossref PubMed Scopus (159) Google Scholar, 12MacMullen C. Fang J. Hsu B.Y. Kelly A. DeLonlay-Debeney P. Saudubray J.M. Ganguly A. Smith T.J. Stanley C.A. J. Clin. Endocrinol. Metab. 2001; 86: 1782-1787Crossref PubMed Scopus (141) Google Scholar). Therefore, allosteric regulation of GDH is central to understanding the response of the β-cell to the cellular fuel state. Children with HI/HA have increased β-cell responsiveness to leucine and susceptibility to hypoglycemia following high protein meals (13Hsu B.Y. Kelly A. Thornton P.S. Greenberg C.R. Dilling L.A. Stanley C.A. J. Pediatr. 2001; 138: 383-389Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). During glucose-stimulated insulin secretion, we have proposed that the generation of high energy phosphates inhibits GDH and promotes conversion of glutamate to glutamine, which, alone or combined, may amplify the release of insulin (7Li C. Najafi H. Daikhin Y. Nissim I. Collins H.W. Yudkoff M. Matschinsky F.M. Stanley C.A. J. Biol. Chem. 2003; 278: 2853-2858Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 8Li C. Buettger C. Kwagh J. Matter A. Daihkin Y. Nissiam I. Collins H.W. Yudkoff M. Stanley C.A. Matschinsky F.M. J. Biol. Chem. 2004; 279: 13393-13401Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Our goal was to find a nontoxic pharmacological agent that could be used to test these models and assess whether GDH might be an appropriate target in the treatment of disorders of glucose homeostasis. To that end, naturally occurring compounds from green tea were examined. According to legend, green tea was discovered by the Chinese Emperor Shen-Nung in 2737 B.C. and for centuries has been used as a folk remedy to treat a number of ailments, including diabetes mellitus. Green tea is a significant source of a type of flavonoid called catechin, which includes epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), and epicatechin (EC) (see Fig. 1 for the structure). One 200-ml cup of green tea supplies 140, 65, 28, and 17 mg of these polyphenols, respectively (14Yang C.S. Wang Z.Y. J. Natl. Cancer Inst. 1993; 85: 1038-1049Crossref PubMed Scopus (1011) Google Scholar). Over the past few decades, there has been growing interest in EGCG, because it has been suggested to decrease cholesterol levels (15Maron D.J. Lu G.P. Cai N.S. Wu Z.G. Li Y.H. Chen H. Zhu J.Q. Jin X.J. Wouters B.C. Zhao J. Arch. Intern. Med. 2003; 163: 1448-1453Crossref PubMed Scopus (197) Google Scholar), act as an antibiotic (16Hamilton-Miller J.M. Antimicrob. Agents Chemother. 1995; 39: 2375-2377Crossref PubMed Scopus (318) Google Scholar) and anticarcinogen (17Katiyar S.K. Mukhtar H. Int. J. Oncol. 1996; 8: 221-238PubMed Google Scholar), repress hepatic glucose production (18Waltner-Law M.E. Wang X.L. Law B.K. Hall R.K. Nawano M. Granner D.K. J. Biol. Chem. 2002; 277: 34933-34940Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar), and enhance insulin action (19Anderson R.A. Polansky M.M. J. Agric. Food Chem. 2002; 50: 7182-7186Crossref PubMed Scopus (309) Google Scholar). The exact mechanism of action of EGCG with regard to these various effects is largely unknown and in many cases is assumed to be due to its antioxidant activity. In this current study, we have demonstrated that EGCG specifically and allosterically inhibits GDH and, in turn, affects insulin secretion by pancreatic β-cells. Kinetic analysis demonstrates that EGCG and ECG (but not EGC and EC) inhibit purified GDH with nanomolar ED50 values. This inhibition is dependent upon the “antenna-like” protrusion on the enzyme but EGCG is unlikely to bind to the GTP inhibitory site since it inhibits forms of GDH with nonfunctional GTP sites. Studies with pancreatic β-cells have demonstrated that EGCG specifically affects insulin secretion under conditions where GDH is known to be important for glucose homeostasis. These results demonstrate that, as has been the case with the activator of GDH (BCH), EGCG can be used as a pharmacological tool to examine the complex regulation of insulin secretion by specifically blocking GDH activity. Kinetic Analyses—The bovine GDH used in these studies was obtained as an aqueous (NH4)2SO4 suspension from Sigma. Prior to kinetic analysis, aliquots of GDH were extensively dialyzed against 0.1 m sodium phosphate buffer, pH 7.0, that contained 1 mm EDTA. Human wild-type and mutant GDH was expressed in Escherichia coli (20Fang J. Macmullen C. Smith T.J. Stanley C.A. Biochem. J. 2001; 363: 81-87Crossref Google Scholar) and tGDH was purified from Tetrahymena (21Allen A. Kwagh J. Fang J. Stanley C.A. Smith T.J. Biochemistry. 2004; 43: 14431-14443Crossref PubMed Scopus (45) Google Scholar) as previously described. The enzyme concentrations were adjusted to 1 mg/ml, and the amount of enzyme added to the reaction mixture was adjusted to yield optimal steady state velocity measurements. All solutions were made immediately prior to use. Enzyme assays were performed by monitoring reduced coenzyme absorbance at 340 nm using a Shamadzu UV-1601 spectrophotometer. The reductive amination reactions were performed in 1-ml volumes at pH 7.0 in the presence of 0.1 mm NADH, 50 mm NH4Cl, and 5 mm 2-oxoglutarate. The rate of NADH oxidation was calculated using an extinction coefficient of 6.22 mm-1 cm-1. Islet Isolation and Culture—Pancreatic islets were isolated from fed adult male Wistar rats by collagenase digestion and cultured in RPMI 1640 medium (glucose-free; Sigma). The culture medium was supplemented with 10% fetal bovine serum, 2 mm glutamine, 100 units/ml penicillin, and 50 μg/ml streptomycin, and islets were incubated at 37 °C in a 5% CO2/95% air-humidified incubator. The islets were cultured with 10 mm glucose for 3-4 days. Insulin Secretion by Perifused Islets—100 cultured rat islets were loaded onto nylon filters in a small chamber and perifused in a Krebs-Ringer bicarbonate buffer (115 mmol/liter NaCl, 24 mmol/liter NaHCO3, 5 mmol/liter KCl, 1 mmol/liter MgCl2, 2.5 mmol/liter CaCl2, in 10 mm HEPES, pH 7.4) with 0.25% bovine serum albumin at a flow rate of 2 ml/min. Perifusate solutions were gassed with 95% O2/5% CO2 and maintained at 37 °C. Samples were collected every minute for insulin assay. Insulin was measured by radioimmunoassay. Islet Perifusion and Oxygen Consumption Measurement—Approximately 1000 isolated rat islets were cultured in 10 mm glucose for 3 days and perifused in a glass chamber. The perifusion apparatus consisted of a peristaltic pump, a water bath (37 °C), a gas exchanger (artificial lung, where medium flowed through the thin-walled silastic tubing loosely coiled in a glass jar that contained 20% O2 and 5%CO2 balanced with N2), and a fraction collector (Waters Division of Millipore). All transfer lines were insulated. Oxygen partial pressure was recorded every 10 s by phosphorescence lifetimes of an oxygen-sensitive porphyrin (palladium-mesotetra; 4-carboxyphenyl porphyrin dendrimer) (22Lo L.W. Vinogradov S.A. Koch C.J. Wilson D.F. Adv. Exp. Med. Biol. 1997; 428: 651-656Crossref PubMed Scopus (60) Google Scholar). The dye molecules were excited with pulses at 524 nm from a UV lead, and emission was measured at 690 nm. The inflow oxygen tension was measured in the absence of islets in the chamber before and after each experiment. The perifusate was a Krebs-Ringer bicarbonate buffer (pH 7.4) containing 1% bovine serum albumin equilibrated with 20% O2 and 5% CO2 balanced with N2. The flow rate was ∼100 μl/min, and samples were collected every 2 min for insulin measurements. [U-14C]Glutamine Oxidation—Islets were cultured with 10 mm glucose for 3 days. Batches of 100 islets were preincubated with glucose-free Krebs-Ringer bicarbonate buffer containing EGCG, EGC, or DON (6-diazo-5-oxo-l-norleucine) for 60 min. The islets were then incubated with 2 or 3 mm glutamine in the presence of BCH and EGCG, EGC, or DON (in accordance with the preincubation protocol) for another 60 min with 2 μCi [U-14C]glutamine (PerkinElmer Life Sciences) present. A trap filter was placed in each tightly sealed glass tube to collect the 14CO2 produced by the islets, and the amount of radioactivity was determined by liquid scintillation counting. [U-14C]Glucose Oxidation—Islets were prepared as described above, and batches of 100 islets were preincubated with glucose-free Krebs-Ringer bicarbonate buffer containing varying concentrations of EGCG for 60 min. The islets were then incubated with 3 and 12 mm glucose in the presence of EGCG for another 60 min with 2 μCi [U-14C]glucose (PerkinElmer Life Sciences) present. Production of 14CO2 was monitored as described above. Because green tea was suggested as a therapeutic agent for the treatment of diabetes more than 70 years ago (23Konayagi S. Minowada M. Stud. Phys., Kyoto Univ. 1933; 10: 449-454Google Scholar) and because of the role that GDH plays in insulin secretion, the effects of green tea catechins on GDH were tested in vitro. As shown in Fig. 1, EGCG and ECG (but not EGC or EC) are potent inhibitors of GDH activity with ED50 values of ∼300 nm. Because all four polyphenols have comparable antioxidant activities (24Anderson R.F. Fisher L.J. Hara Y. Harris T. Mak W.B. Melton L.D. Packer J.E. Carcinogenesis. 2001; 22: 1189-1193Crossref PubMed Google Scholar), this strongly suggests that EGCG and ECG effects are allosteric in nature. This inhibition is also reversible, because dialysis of an EGCG/GDH mixture completely alleviated the inhibition (data not shown). To further ascertain how EGCG inhibits the reductive amination reaction, the polyphenol was added to the reaction at various concentrations of NADH and 2-oxoglutarate (Fig. 2). As summarized in Table 1, EGCG affects both the slope and the y-intercept of the curves in a manner consistent with noncompetitive inhibition. These results suggest that EGCG does not act by directly competing with either coenzyme or substrate binding to the active site and that it inhibits by binding to either the free enzyme or the enzyme-substrate complex.TABLE 1Summary of the effects of EGCG on the reductive amination reaction As shown here, EGCG has marked effects on both the y-intercept and the slope of the curves and therefore infers that EGCG inhibits in a non-competitive manner.[EGCG]NADH varied2-oxoglutarate varied0 μm0.3 μm0.6 μm0 μm0.3 μm0.45 μmSlope0.0034 ± 0.00010.0041 ± 0.00030.0065 ± 0.00041.77 ± 0.0142.19 ± 0.143.5 ± 0.39y-intercept0.120 ± 0.0060.247 ± 0.0180.322 ± 0.02624.0 ± 0.7446.0 ± 7.369.0 ± 10.8R20.98800.95040.95830.99970.97990.9640 Open table in a new tab To examine how EGCG interacts with other allosteric regulators, the ability of the activators BCH, leucine, and ADP to reverse EGCG was examined. Fig. 3 displays the data in two different ways. On the left, the raw velocities of the reactions are shown in the presence and absence of EGCG at varying concentrations of the activators. On the right, the same data is plotted as a percent of the velocity of the reaction compared with the reaction rate in the absence of activators in the presence or absence of EGCG. Under these assay conditions and in the absence of EGCG, all three activators only increase the velocity of the reactions by, at most, 30%. However, in the presence of 1 μm EGCG, all three regulators can activate the reaction by as much as 3-fold. In absolute terms, the addition of 1 μm EGCG decreased GDH activity to ∼20% that of the uninhibited enzyme. In all cases, the activators partially alleviated EGCG inhibition and increased the enzymatic activity to ∼60% that of the uninhibited enzyme. This is very similar to previous studies that have shown that ADP and leucine activate by binding to spatially different sites (25Prough R.A. Culver J.M. Fisher H.F. J. Biol. Chem. 1973; 248: 8528-8533Abstract Full Text PDF PubMed Google Scholar) and can have apparently greater activation if an allosteric inhibitor is present (e.g. GTP). Abrogation of EGCG inhibition by these allosteric activators supports the above contention that EGCG acts in an allosteric manner. HI/HA syndrome is caused by the loss of GTP inhibition of GDH (9Stanley C.A. O'Rahilly S. Dunger D.B. Genetic Insights in Paediatric Endocrinology and Metabolism. BioScientifica, Ltd, Bristol, UK2000: 23-30Google Scholar). Many of the mutations lie in the GTP binding site and likely act by sterically interfering with GTP binding (12MacMullen C. Fang J. Hsu B.Y. Kelly A. DeLonlay-Debeney P. Saudubray J.M. Ganguly A. Smith T.J. Stanley C.A. J. Clin. Endocrinol. Metab. 2001; 86: 1782-1787Crossref PubMed Scopus (141) Google Scholar, 20Fang J. Macmullen C. Smith T.J. Stanley C.A. Biochem. J. 2001; 363: 81-87Crossref Google Scholar, 26Peterson P.E. Smith T.J. Structure. 1999; 7: 769-782Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 27Smith T.J. Peterson P.E. Schmidt T. Fang J. Stanley C. J. Mol. Biol. 2001; 307: 707-720Crossref PubMed Scopus (140) Google Scholar). Therefore, EGCG (or compounds that specifically target GDH) could be therapeutically useful if it also inhibited the HI/HA mutant forms of GDH. To this end, EGCG was tested against five different HI/HA mutant forms of GDH (Fig. 4) and was found to have the same efficacy as with wild-type human GDH. In addition, EGCG inhibited GDH from Tetrahymena thermophilia (tGDH), which, similar to these HI/HA mutants, is not regulated by GTP (21Allen A. Kwagh J. Fang J. Stanley C.A. Smith T.J. Biochemistry. 2004; 43: 14431-14443Crossref PubMed Scopus (45) Google Scholar). These results suggest that EGCG acts independently of the GTP inhibitory site. From our previous structural analyses, mammalian GDH has a 48-residue antenna-like feature protruding from the top of each of its six subunits (27Smith T.J. Peterson P.E. Schmidt T. Fang J. Stanley C. J. Mol. Biol. 2001; 307: 707-720Crossref PubMed Scopus (140) Google Scholar, 28Smith T.J. Schmidt T. Fang J. Wu J. Siuzdak G. Stanley C.A. J. Mol. Biol. 2002; 318: 765-777Crossref PubMed Scopus (107) Google Scholar) that is necessary for ADP, GTP, and palmitoyl CoA regulation (21Allen A. Kwagh J. Fang J. Stanley C.A. Smith T.J. Biochemistry. 2004; 43: 14431-14443Crossref PubMed Scopus (45) Google Scholar). As shown in Fig. 4, EGCG does not inhibit the “antenna-less” form of human GDH. This demonstrates that EGCG inhibition of GDH is unrelated to the antioxidant activity of the flavonoids, does not directly affect the active site, and is dependent upon the antenna structure to exert its activity. Because EGCG was found to be a potent inhibitor of GDH in vitro, it was postulated that GDH-dependent β-cell functions should also be influenced by these catechins. For example, the phenomenon of leucine-stimulated insulin secretion (LSIS) has been recently elucidated by our studies showing that LSIS is mediated by GDH and its regulation of glutaminolysis (7Li C. Najafi H. Daikhin Y. Nissim I. Collins H.W. Yudkoff M. Matschinsky F.M. Stanley C.A. J. Biol. Chem. 2003; 278: 2853-2858Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). LSIS is only observed after a prolonged period of rundown to produce a state of energy depletion. Under these conditions, the levels of GDH inhibitors ATP and GTP are reduced, whereas the concentration of the GDH activator ADP is increased. When leucine (or its nonmetabolizable analog BCH) is then added to cells in this depleted state, the flux of glutamine through glutaminase and GDH is increased, ATP is generated, and the β-cells are stimulated to secrete insulin. As shown in Fig. 5, the BCH stimulation of insulin secretion is blocked by EGCG (but not EGC) in a dose-dependent manner with an ED50 value of <10 μm. EGCG did not affect the intrinsic ability of the cells to secrete insulin, because depolarization by KCl still resulted in release of insulin. The concentration of EGCG required to abrogate LSIS is significantly higher than what is necessary to inhibit GDH in vitro. This is likely because of the bioavailability of EGCG in the mitochondria and/or that higher levels of EGCG are required in tissue to overcome antagonism by leucine, ADP, and BCH. According to our working model, the effects of EGCG on BCH-stimulated insulin secretion (BCH-SIS) are due to a block of glutamine oxidation through GDH (7Li C. Najafi H. Daikhin Y. Nissim I. Collins H.W. Yudkoff M. Matschinsky F.M. Stanley C.A. J. Biol. Chem. 2003; 278: 2853-2858Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). To test for this, the effects of EGCG and EGC on BCH-SIS were measured while simultaneously monitoring respiration rates. As shown in Fig. 6, EGCG strongly inhibited both BCH-SIS and the BCH-induced increase in respiration rates. The addition of EGCG itself does not have any significant effect on oxygen consumption in the absence of BCH. EGC has a number of interesting effects on BCH-SIS. First, it does not cause significant inhibition of BCH-SIS nor is its effect on respiration as strong as EGCG. Second, EGC seems to cause a slight sensitization of the β-cells to BCH, as manifested in a slightly faster response to BCH-mediated insulin secretion and respiration enhancement. This clearly demonstrates that the EGCG effects are not due to the catechin antioxidant activity and suggests that EGC may have some, although quite different, effects on the pancreatic cells. According to the working model (7Li C. Najafi H. Daikhin Y. Nissim I. Collins H.W. Yudkoff M. Matschinsky F.M. Stanley C.A. J. Biol. Chem. 2003; 278: 2853-2858Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 8Li C. Buettger C. Kwagh J. Matter A. Daihkin Y. Nissiam I. Collins H.W. Yudkoff M. Stanley C.A. Matschinsky F.M. J. Biol. Chem. 2004; 279: 13393-13401Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), it was predicted that EGCG would block BCH-SIS by inhibition of GDH-mediated glutaminolysis. To directly test for this, the effects of EGCG on BCH stimulation of glutamine oxidation were measured (Fig. 7A). As shown, EGCG (but not EGC) completely blocks BCH stimulation of glutaminolysis. EGCG inhibition of glutamine oxidation occurs in a dose-dependent manner with maximal effects occurring at ∼20 μm and never decreasing glutamine oxidation below that of the cells not stimulated by BCH (Fig. 7B). As shown in Fig. 7C, this is in contrast to the effects of DON, an inhibitor of glutaminase, which blocks glutaminolysis to levels lower than the unstimulated β-cells at high concentrations. These results are summarized in Fig. 7D and demonstrate that EGCG does not have an effect on basal levels of glutamine oxidation but does block BCH enhancement of glutaminolysis. Together with the enzymatic data, it is clear that EGCG effects on BCH-SIS are due to the inhibition of GDH. Because our previous studies have suggested that glutamine is an important signaling molecule for glucose-stimulated insulin secretion (GSIS) (8Li C. Buettger C. Kwagh J. Matter A. Daihkin Y. Nissiam I. Collins H.W. Yudkoff M. Stanley C.A. Matschinsky F.M. J. Biol. Chem. 2004; 279: 13393-13401Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), it was then necessary to determine whether EGCG might also affect GSIS. Islets were incubated for 3 days in the presence of 20 mm (Fig. 8A) or 10 mm (Fig. 8B) glucose, perifused for 50 min in the absence of glucose ± catechins, and then subjected to a glucose ramp. In all cases, neither EGC nor EGCG affected GSIS under these short run-down conditions. As confirmation of this, insulin secretion and oxygen consumption were measured during glucose stimulation (Fig. 9). It is clear that EGCG does not affect respiration under these conditions. Finally, as shown in Fig. 10, EGCG does not affect glucose oxidation over a wide range of EGCG concentrations when the islets are incubated in either 3 or 12 mm glucose. Therefore, the catechins do not, in and of themselves, affect cellular respiration, glucose oxidation, or insulin secretion. Rather, these results support the contention that EGCG affects insulin secretion via modulation of GDH activity. Under the brief run-down conditions used for the GSIS analysis, the levels of high energy metabolites (GTP and ATP) were not depleted, and these effectively shut down GDH activity (7Li C. Najafi H. Daikhin Y. Nissim I. Collins H.W. Yudkoff M. Matschinsky F.M. Stanley C.A. J. Biol. Chem. 2003; 278: 2853-2858Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 8Li C. Buettger C. Kwagh J. Matter A. Daihkin Y. Nissiam I. Collins H.W. Yudkoff M. Stanley C.A. Matschinsky F.M. J. Biol. Chem. 2004; 279: 13393-13401Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 29Gao Z. Li G. Najafi H. Wolf B.A. Matschinsky F.M. Diabetes. 1999; 48: 1535-1547Crossref PubMed Scopus (93) Google Scholar). Therefore, additional inhibition by EGCG does not affect glutamate/glutamine levels nor does it, in turn, affect GSIS under these conditions.FIGURE 9Effects of EGCG on glucose-stimulated insulin secretion and oxygen consumption. Isolated rat islets were cultured with 10 mm glucose for 3 days and then perifused in an oxygen consumption measurement apparatus. Shown are the sequence and additions. A shows the insulin secretion: EGCG 0 μm (open diamonds), EGCG 20 μm (solid diamonds), and EGC 20 μm (gray circles). FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone. B shows the oxygen consumption: EGCG 0 μm (light gray), EGCG 20 μm (black), and EGC 20 μm (darker gray). However, because of overlapping data, only the gray and black lines are clearly visible. Results are presented as means ± S.E. for 1000 islets from three separate experiments; because of the high density of data, S.E. only showed in every 20 samples.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 10Effects of EGCG on [U-14C]glucose oxidation. Isolated rat islets were cultured with 10 mm glucose for 3 days, and [U-14C]glucose oxidation was measured in batches of 100 islets. The effects of EGCG in a dose-dependent manner were tested in 3 mm (solid circles) and 12 mm (solid triangles) glucose oxidation. Results are presented as means ± S.E. from four separate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The data presented here clearly demonstrate that EGCG allosterically inhibits GDH in vitro with a nanomolar ED50 value. Because this inhibition is observed with EGCG and ECG (but not EC or EGC), inhibition cannot be due to the anti-oxidant property of these catechins. EGCG inhibition is apparently noncompetitive and, similar to GTP inhibition, is abrogated by leucine, BCH, and ADP. It is unlikely that EGCG acts by binding to the GTP site, because HI/HA GDH mutants and tGDH are all inhibited by EGCG but have inactive GTP binding sites. EGCG inhibition is apparently dependent upon the antenna, because the an antenna-less mutant of GDH is also unaffected by EGCG. This was also found to be the case with ADP activation and GTP inhibition (21Allen A. Kwagh J. Fang J. Stanley C.A. Smith T.J. Biochemistry. 2004; 43: 14431-14443Crossref PubMed Scopus (45) Google Scholar). This specificity of EGCG for GDH inhibition was mirrored in studies on β-cells and demonstrates the importance of GDH in the regulation of insulin secretion. Our previous studies have demonstrated that GDH plays a major role in LSIS by controlling glutaminolysis (7Li C. Najafi H. Daikhin Y. Nissim I. Collins H.W. Yudkoff M. Matschinsky F.M. Stanley C.A. J. Biol. Chem. 2003; 278: 2853-2858Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 8Li C. Buettger C. Kwagh J. Matter A. Daihkin Y. Nissiam I. Collins H.W. Yudkoff M. Stanley C.A. Matschinsky F.M. J. Biol. Chem. 2004; 279: 13393-13401Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Therefore it was not unexpected that EGCG, by inhibiting GDH activity, abrogated BCH-SIS. The specificity of this inhibition was confirmed by demonstrating that EGCG (but not EGC) causes a concomitant blockade of glutaminolysis stimulated by BCH but not in the basal level of glutamine oxidation or cellular respiration. When EGCG is added to β-cells during glucose stimulation, under conditions where GDH is known to not play a major role in the regulation of insulin secretion, no effect is observed on insulin secretion, glucose oxidation, or cellular respiration. It is worthy of note that, to keep the involvement of the oxidation of leucine in the process of insulin secretion from complicating the interpretation of the experiments, this study was limited to using BCH to stimulate GDH. Together, the data presented here clearly demonstrate that EGCG can affect BCH-SIS by inhibiting GDH activity in a manner that is consistent with our working hypothesis as summarized in Fig. 11. The foundation of this model is that GDH can regulate insulin secretion by controlling the intracellular pool of glutamine and/or glutamate. In the case of BCH-SIS, the β-cell is depleted of glucose and provided with glutamine at low concentration levels prior to the application of BCH. Therefore, when BCH is applied, the lack of ATP/GTP inhibition combined with ADP and BCH activation facilitates glutaminolysis. This leads to the generation of ATP, and combined with the exogenously added glutamine that may act as an intracellular signal, insulin secretion is facilitated. EGCG inhibits GDH, blocks glutaminolysis, and thereby prevents the BCH effects. In the case of GSIS, the effects of EGCG are likely dependent upon the energy state of the cell. When the β-cell has been run-down for a brief period of time, glutaminolysis is not the main energy source for the cell, and high energy metabolites (GTP and ATP) are still present at high enough levels to inhibit GDH activity. Under these conditions, EGCG does not affect GSIS, because GDH is already effectively inhibited. Further, these results demonstrate that EGCG does not intrinsically affect insulin secretion. These experiments support the hypothesis that GDH, through its allosteric regulation, plays an important role in the process of amino acid stimulation of insulin release. We have recently suggested that the complex allosteric regulation of GDH started to evolve in the Ciliates in response to the changing function of cellular organelles and, through exaptation, further allostery (leucine and GTP/ATP regulation) was layered on for the regulation of additional functions for the reaction (for example, insulin secretion) (21Allen A. Kwagh J. Fang J. Stanley C.A. Smith T.J. Biochemistry. 2004; 43: 14431-14443Crossref PubMed Scopus (45) Google Scholar). The present results continue to support this by the demonstration that the antenna, which evolved in the Ciliates, is essential for EGCG inhibition. Similar to other compounds, such as DON and BCH, EGCG is a new pharmacological tool that will help elucidate the intersecting metabolic pathways that regulate insulin secretion. Finally, the present observations suggest that GDH may be a novel target for future treatment of glucose homeostasis disorders.
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