Small elevations of glucose concentration redirect and amplify the synthesis of guanosine 5'-triphosphate in rat islets.

Abstract

Recent studies suggest a permissive requirement for guanosine 5'-triphosphate (GTP) in insulin release, based on the use of GTP synthesis inhibitors (such as mycophenolic acid) acting at inosine monophosphate (IMP) dehydrogenase; herein, we examine the glucose dependency of GTP synthesis. Mycophenolic acid inhibited insulin secretion equally well after islet culture at 7.8 or 11.1 mM glucose (51% inhibition) but its effect was dramatically attenuated when provided at < 6.4 mM glucose (13% inhibition; P < 0.001). These observations were explicable by a stimulation of islet GTP synthesis derived from IMP since, at high glucose: (a) total GTP content was augmented; (b) a greater decrement in GTP (1.75 vs. 1.05 pmol/ islet) was induced by mycophenolic acid; and (c) a smaller "pool" of residual GTP persisted after drug treatment. Glucose also accelerated GTP synthesis from exogenous guanine ("salvage" pathway) and increased content of a pyrimidine, uridine 5'-triphosphate (UTP), suggesting that glucose augments production of a common regulatory intermediate (probably 5-phosphoribosyl-1-pyrophosphate). Pathway-specific radiolabeling studies confirmed that glucose tripled both salvage and de novo synthesis of nucleotides. We conclude that steep changes in the biosynthesis of cytosolic pools of GTP occur at modest changes in glucose concentrations, a finding which may have relevance to the adaptive ( patho ) physiologic responses of islets to changes in ambient glucose levels. (J. Clin. Invest.

Evidence of a role for GTP in the potentiation of Ca(2+)-induced insulin secretion by glucose in intact rat islets.

“…200 islets per condition were cultured overnight ( 18-20 h) in RPMI-1640 medium (containing 10% FBS, 100 U/ml penicillin, 100 g/rm1 of streptomycin, 5 mM Hepes, 2 Ci/ml [3H]hypoxanthine and 11.1 mM glucose) and MPA when indicated. We have previously shown that [3H] hypoxanthine can be used to label both adenine and guanine nucleotides through the salvage pathway of purine nucleotide synthesis (8). After the culture period, islets were washed five times with 1 ml ice-cold KRB (pH 7.4, gassed with 95%02/5% CO2, and containing 16.7 mM glucose and 0.2% BSA).…”

“…The NTP/NDP ratios reported in the Results section are from the TLC method unless otherwise stated. In addition, eluant trinucleotide HPLC fractions were collected every 18 s and were matched against the concomitant UV chromatograms as previously described (8) for calculation of specific activities. Samples were counted for 5 min in 4 ml of scintillation cocktail.…”

Glucose- and GTP-dependent stimulation of the carboxyl methylation of CDC42 in rodent and human pancreatic islets and pure beta cells. Evidence for an essential role of GTP-binding proteins in nutrient-induced insulin secretion.

“…Under these conditions, MPA reduces GTP levels by Ͼ 80% (and GTP/ GDP ratio by Ͼ 60%) in isolated islets; coprovision of guanine or guanosine completely reverses the reduction in GTP (or GTP/GDP ratio) elicited by MPA (2,3). After this, islets were washed twice with Krebs-Ringer medium consisting of BSA (0.1%) and glucose (11.1 mM) and were labeled with [ 3 H]methionine (50 Ci/ml; 1 ml total volume) for 2 h at 37 Њ C. The labeling was terminated by addition of ice-cold Krebs-Ringer medium.…”

“…Recently, using specific inhibitors of guanosine triphosphate (GTP) synthesis, we identified a permissive role for GTP as one of the modulators of physiologic insulin secretion (2,3). However, the exact sites of action of GTP in pancreatic ␤ cells have not been identified thus far.…”

Bridging the gap between protein carboxyl methylation and phospholipid methylation to understand glucose-stimulated insulin secretion from the pancreatic β cell

“…For example, using selective inhibitors of GTP biosynthetic pathway [e.g., mycophenolic acid], a permissive role for GTP in GSIS was first established in [10][11][12]. Although the exact molecular and cellular mechanisms underlying the regulatory role[s] of GTP remain only partially understood, available evidence indicates that it might involve activation of one [or more] G-proteins.…”