Effects of Glucose and Amino Acids on Free ADP in βHC9 Insulin-Secreting Cells

Abstract

Stimulation of insulin release by glucose is widely thought to be coupled to a decrease in the activity of ATP-sensitive K + channels (K ATP channels) that is caused by a decreased concentration of free ADP. To date, most other investigators have reported only on total cellular ADP concentrations, even though only a small fraction of all ADP is free and only the free ADP affects K ATP channels. We tested the hypothesis that amino acids elicit insulin release via a decrease in the activity of K ATP channels owing to a decrease in the level of free ADP. We estimated the concentration of free ADP in ␤HC9 hyperplastic insulin-secreting cells based on the cell diameter and on luminometric measurements of ATP, phosphocreatine, and total creatine. The concentration of free ADP fell exponentially as the concentration of glucose increased. A physiological mixture of amino acids greatly stimulated insulin release at 0-30 mmol/l glucose but affected the concentration of free ADP only to a minor degree and significantly so only at ≤2 mmol/l glucose. In the presence of 2-deoxyglucose and NaN 3 , amino acids were unable to stimulate insulin release. When K ATP channels were held open with diazoxide (and the plasma membrane partially depolarized with high extracellular KCl), amino acids still stimulated insulin release. We conclude that amino acid-induced insulin release depends on two components: a yet-unknown amino acid sensor and K ATP channels, which serve to attenuate hormone release when cellular energy stores are low. We propose that glucoseinduced insulin release may be regulated similarly by two components: glucokinase and K ATP channels. Diabetes 50:291-300, 2001A ccording to the most widely accepted hypothesis, glucose induces insulin release as follows. Glucose rapidly equilibrates across the plasma membrane and is phosphorylated with the help of glucokinase, which determines flux through glycolysis (1). Pyruvate from glycolysis enters the citric acid cycle and determines ATP production from oxidative phosphorylation. As a result, the concentrations of ATP and free ADP reflect the concentration of blood glucose. The plasma membrane contains ATP-sensitive K + channels (K ATP channels). ATP inhibits the channels, and ADP relieves this inhibition (2). In the absence of glucose, K ATP channels are quite active and dictate the membrane potential. In the presence of glucose, initially, the balance between K + flux through K ATP channels and ionic fluxes through yet unknown "leak conductances" determines the membrane potential (2). Once the membrane potential is more positive than about -40 mV, Ca 2+ channels open intermittently, allowing the influx of extracellular Ca 2+ (2). As intracellular Ca 2+ rises, the exocytotic machinery is activated, which moves secretory granules containing insulin to the plasma membrane surface. In partial support of the above hypothesis, patients with a mutant glucokinase of abnormal affinity to glucose have an abnormal set point for glucose homeostasis (3-5), and other patients with K ATP c...

Regulation of ATP/ADP in Pancreatic Islets

“…By identifying a large stable pool of nucleotides in islet secretory granules, an approximate fourfold increase in ATP/ADP was seen in response to glucose stimulation (34,35). However, even these estimates are ϳ10-fold higher than estimates of free ADP level based on calculations that assume that the creatine kinase reaction that buffers ATP and ADP levels is in equilibrium (30,31).…”

“…This provides good estimates of free ATP but not of free ADP because 95% of cellular ADP is bound or sequestered (30,31). Accordingly, only small increases in ATP/ADP have been seen in response to glucose (32,33).…”

“…By identifying a large stable pool of nucleotides in islet secretory granules, an approximate fourfold increase in ATP/ADP was seen in response to glucose stimulation (34,35). However, even these estimates are ϳ10-fold higher than estimates of free ADP level based on calculations that assume that the creatine kinase reaction that buffers ATP and ADP levels is in equilibrium (30,31). Given the importance of free ATP and ADP to islet cells and the difficulty of measuring free ADP, we have suggested a new, albeit indirect, method for a continuous noninvasive measure of these key metabolic variables.…”

Mechanisms of mitogenic and anti-apoptotic signaling by glucose-dependent insulinotropic polypeptide in beta(INS-1)-cells

“…2. are involved in the generation of anti-apoptotic signals from glucose. Whether anti-apoptotic signaling by glucose is mediated by an altered ADP:ATP ratio, by glutamine or by glucokinase signaling (Isihara & Wollheim 2000, Moley & Mueckler 2000, Ronner et al 2001 remains to be elucidated. It seems likely that the availability of ATP in cytosolic compartments may be the decisive factor for the kinase activity of anti-apoptotic signaling cascades, which may imitate a second messenger effect.…”

β-Cell Mitochondria and Insulin Secretion

“…Mitochondrial metabolism increases the ATP/ ADP ratio (17). The increase in cytosolic ATP (18,19) and/or the decrease in free ADP (5,20) promotes the closure of ATP-sensitive K ϩ channels (K ATP channels) and depolarization of the plasma membrane (21). As a consequence, cytosolic Ca 2ϩ ([Ca 2ϩ ] c ) is raised by the opening of voltage-sensitive Ca 2ϩ channels (21,22).…”