ATP mediates a negative autocrine signal on stimulus-secretion coupling in mouse pancreatic ß-cells.

PURPOSE: The role of ATP, which is secreted by pancreatic ß-cells, is still a matter of debate. It has been postulated that extracellular ATP acts as a positive auto- or paracrine signal in ß-cells amplifying insulin secretion. However, there is rising evidence that extracellular ATP may also mediate a negative signal. METHODS: We evaluated whether extracellular ATP interferes with the Ca2+-mediated negative feedback mechanism that regulates oscillatory activity of ß-cells. RESULTS: To experimentally uncover the Ca2+-induced feedback we applied a high extracellular Ca2+ concentration. Under this condition ATP (100 µM) inhibited glucose-evoked oscillations of electrical activity and hyperpolarized the membrane potential. Furthermore, ATP acutely increased the interburst phase of Ca2+ oscillations and reduced the current through L-type Ca2+ channels. Accordingly, ATP (500 µM) decreased glucose-induced insulin secretion. The ATP effect was not mimicked by AMP, ADP, or adenosine. The use of specific agonists and antagonists and mice deficient of large conductance Ca2+-dependent K+ channels revealed that P2X, but not P2Y receptors, and Ca2+-dependent K+ channels are involved in the underlying signaling cascade induced by ATP. The effectiveness of ATP to interfere with parameters of stimulus-secretion coupling is markedly reduced at low extracellular Ca2+ concentration. CONCLUSION: It is suggested that extracellular ATP which is co-secreted with insulin in a pulsatile manner during glucose-stimulated exocytosis provides a negative feedback signal driving ß-cell oscillations in co-operation with Ca2+ and other signals.

The LXR Ligand T0901317 Acutely Inhibits Insulin Secretion by Affecting Mitochondrial Metabolism.

The role of liver X receptor (LXR) in pancreatic ß-cell physiology and pathophysiology is still unclear. It has been postulated that chronic LXR activation in ß-cells induces lipotoxicity, a key step in the development of ß-cell dysfunction, which accompanies type 2 diabetes mellitus. In most of these studies, the LXR ligand T0901317 has been administered chronically in the micromolar range to study the significance of LXR activation. In the current study, we have evaluated acute effects of T0901317 on stimulus-secretion coupling of ß-cells. We found that 10 µM T0901317 completely suppressed oscillations of the cytosolic Ca2+ concentration induced by 15 mM glucose. Obviously, this effect was due to inhibition of mitochondrial metabolism. T0901317 markedly depolarized the mitochondrial membrane potential, thus inhibiting adenosine triphosphate (ATP) production and reducing the cytosolic ATP concentration. This led in turn to a huge increase in KATP current and hyperpolarization of the cell membrane potential. Eventually, T0901317 inhibited glucose-induced insulin secretion. These effects were rapid in on-set and not compatible with the activation of a nuclear receptor. In vivo, T0901317 acutely increased the blood glucose concentration after intraperitoneal application. In summary, these data clearly demonstrate that T0901317 exerts acute effects on stimulus-secretion coupling. This observation questions the chronic use of T0901317 and limits the interpretation of results obtained under these experimental conditions.

Evidence against a Ca(2+)-induced potentiation of dehydrogenase activity in pancreatic beta-cells.

Pancreatic beta-cells respond to an unchanging stimulatory glucose concentration with oscillations in membrane potential (Vm), cytosolic Ca(2+) concentration ([Ca(2+)]c), and insulin secretion. The underlying mechanisms are largely ascertained. Some particular details, however, are still in debate. Stimulus-secretion coupling (SSC) of beta-cells comprises glucose-induced Ca(2+) influx into the cytosol and thus into mitochondria. It is suggested that this activates (mitochondrial) dehydrogenases leading to an increase in reduction equivalents and ATP production. According to SSC, a glucose-induced increase in ATP production would thus further augment ATP production, i.e. induce a feed-forward loop that is hardly compatible with oscillations. Consistently, other studies favour a feedback mechanism that drives oscillatory mitochondrial ATP production. If Ca(2+) influx activates dehydrogenases, a change in [Ca(2+)]c should increase the concentration of reduction equivalents. We measured changes in flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) autofluorescence in response to changes in glucose concentration or glucose-independent changes in [Ca(2+)]c. The FAD signal was altered by glucose but not by alterations in [Ca(2+)]c. NAD(P)H was increased by glucose but even decreased by Ca(2+) influx evoked by tolbutamide. The mitochondrial membrane potential ΔΨ was hyperpolarized by 4 mM glucose. As adding tolbutamide then depolarized ΔΨ, we deduce that Ca(2+) does not activate mitochondrial activity but by contrast even inhibits it by reducing the driving force for ATP production. Inhibition of Ca(2+) influx reversed the Ca(2+)-induced changes in ΔΨ and NAD(P)H. The results are consistent with a feedback mechanism which transiently and repeatedly reduces ATP production and explain the oscillatory activity of pancreatic beta-cells at increased glucose concentrations.

Mitochondrial succinate dehydrogenase is involved in stimulus-secretion coupling and endogenous ROS formation in murine beta cells.

AIMS/HYPOTHESIS: Generation of reduction equivalents is a prerequisite for nutrient-stimulated insulin secretion. Mitochondrial succinate dehydrogenase (SDH) fulfils a dual function with respect to mitochondrial energy supply: (1) the enzyme is part of mitochondrial respiratory chains; and (2) it catalyses oxidation of succinate to fumarate in the Krebs cycle. The aim of our study was to elucidate the significance of SDH for beta cell stimulus-secretion coupling (SSC). METHODS: Mitochondrial variables, reactive oxygen species (ROS) and cytosolic Ca(2+) concentration ([Ca(2+)]c) were measured by fluorescence techniques and insulin release by radioimmunoassay in islets or islet cells of C57Bl/6N mice. RESULTS: Inhibition of SDH with 3-nitropropionic acid (3-NPA) or monoethyl fumarate (MEF) reduced glucose-stimulated insulin secretion. Inhibition of the ATP-sensitive K(+) channel (KATP channel) partly prevented this effect, whereas potentiation of antioxidant defence by superoxide dismutase mimetics (TEMPOL and mito-TEMPO) or by nuclear factor erythroid 2-related factor 2 (Nrf-2)-mediated upregulation of antioxidant enzymes (oltipraz, tert-butylhydroxyquinone) did not diminish the inhibitory influence of 3-NPA. Blocking SDH decreased glucose-stimulated increase in intracellular FADH2 concentration without alterations in NAD(P)H. In addition, 3-NPA and MEF drastically reduced glucose-induced hyperpolarisation of mitochondrial membrane potential, indicative of decreased ATP production. As a consequence, the glucose-stimulated rise in [Ca(2+)]c was significantly delayed and reduced. Acute application of 3-NPA interrupted glucose-driven oscillations of [Ca(2+)]c. 3-NPA per se did not elevate intracellular ROS, but instead prevented glucose-induced ROS accumulation. CONCLUSIONS/INTERPRETATION: SDH is an important regulator of insulin secretion and ROS production. Inhibition of SDH interrupts membrane-potential-dependent SSC, pointing to a pivotal role of mitochondrial FAD/FADH2 homeostasis for the maintenance of glycaemic control.