Signal transduction crosstalk in the endocrine system: pancreatic beta-cells and the glucose competence concept

Abstract

Crosstalk between intracellular signalling systems is recognized as the principal means by which a cell orchestrates coordinate responses to stimulation by neurotransmitters, hormones or growth factors. The functional consequences of crosstalk are evident at multiple levels within a given signalling cascade, including the regulation of receptor-ligand interactions, guanine nucleotide-binding proteins, enzyme activities, ion channel function and gene expression. Here we focus on the pancreatic beta-cells of the islets of Langerhans to illustrate the important role crosstalk plays in the regulation of glucose-induced insulin secretion. Recent studies indicating a synergistic interaction in beta-cells between the glucose-regulated ATP-dependent signalling system and the hormonally regulated cAMP-dependent signalling system are emphasized. This interaction gives beta-cells the ability to match the ambient concentration of glucose to an appropriate insulin secretory response, a process we refer to as the induction of glucose competence. The glucose competence concept may provide new insights into the etiology and treatment of non-insulin-dependent diabetes mellitus (Type II diabetes).

Figure 1

Hormonal regulation of glucose-induced insulin secretion from pancreatic β-cells. On the left are illustrated essential features of the glucose-regulated ATP-dependent signalling system. The initial uptake of glucose is facilitated by the type-2 glucose transporter, whereas the conversion of intracellular glucose to glucose 6-phosphate is catalysed by glucokinase. Stimulation of aerobic glycolysis generates multiple signals, one of which is an increased ratio of intracellular ATP relative to ADP. Binding of ATP to ATP-sensitive potassium channels (K-ATP) induces closure of the channels through a (yet to be determined) mechanism that does not require hydrolysis of the nucleotide. Closure of ATP-sensitive potassium channels results in membrane depolarization which is necessary for the opening of voltage-sensitive Ca²⁺ channels (Ca-VS). The opening of Ca²⁺ channels may also be favoured by glucose-derived signalling molecules of undetermined origin. Entry of Ca²⁺ across the plasma membrane triggers vesicular insulin secretion by Ca²⁺-dependent exocytosis. Repolarization of the membrane results from the action of intracellular Ca²⁺ to activate Ca²⁺-dependent potassium channels (K-Ca) and to inhibit voltage-sensitive Ca²⁺ channels. Each of these steps in the glucose-regulated ATP-dependent signalling system is viewed as a potential target for modulation by the hormonally regulated cAMP-dependent signalling system. In this example, GLP-1 binds to cell-surface receptors and activates G_s, a heterotrimeric G protein that stimulates adenylate cyclase. Stimulation of adenylate cyclase by GLP-1 is proposed to require the activated form of calmodulin. Since calmodulin is activated by the glucose-induced rise in intracellular Ca²⁺, the stimulation of adenylate cyclase by GLP-1 is glucose-dependent. The production of cAMP by adenylate cyclase results in the activation of protein kinase A (PKA) which catalyses the phosphorylation of multiple targets within the glucose-signalling cascade. These targets may include elements of the glucose-sensing mechanism, ion channels, gap junctions and components of the secretory apparatus that are responsible for mobilization and exocytosis of insulin-containing vesicles.

Figure 2

Glucose-induced insulin secretion from β-cells results from depolarization-induced increases in the concentration of intracellular Ca²⁺. The recordings illustrated were obtained from clusters of pancreatic β-cells using either the perforated patch configuration of the patch–clamp technique (a), or fluorescence ratio imaging with the membrane-permeant Ca²⁺ indicator dye FURA-2 AM. As illustrated in a, recordings of the resting membrane potential revealed that 10 mm glucose shifted the membrane potential in the depolarizing direction and generated action potentials (spike-like phenomena) that resulted from Ca²⁺ influx through voltage-sensitive calcium channels. In a separate experiment the depolarizing action of glucose was accompanied by an increased concentration of intracellular Ca²⁺, as indicated by the increased fluorescence ratio (b). Note that in this example the rise in intracellular Ca²⁺ was detected simultaneously in two adjacent cells (labelled 1 and 2), thereby indicating that the cells were electrically and metabolically coupled by gap junctions.

Figure 3

The induction of glucose competence by GLP-1 is observed at the single cell level, is specific for a distinct subpopulation of β-cells, and is attributable to the inhibition of ATP-sensitive potassium channels. In (a) the actions of glucose and GLP-1 were tested on solitary β-cells isolated from dispersed islets of Langerhans and maintained in short-term primary cell culture, conditions under which the cells are known to exhibit diminished glucose-induced insulin secretion. Scatter plot analysis (where each triangle represents observations obtained from a single cell) revealed that the majority of the cells (those comprising Sets 1 and 3) also showed relatively little change in resting membrane potential when exposed to 10 mm glucose (x-axis). In contrast, a subpopulation of these cells (comprising Set 3) exhibited a large depolarizing response when challenged with a combined application of 10 mm glucose and 10 nm GLP-1 (y-axis). This induction of glucose competence by GLP-1 was observed in 40% of all cells tested. (b) A graphical presentation of how the synergistic interaction of 10 mm glucose and 10 nm GLP-1 depolarizes β-cells by inhibiting ATP-sensitive potassium channels, as recorded in the perforated vesicle configuration. Channel activity is expressed as a function of ‘openness’ vs time where openness is defined as the product of N and P_o (N is the number of channels in the patch and P_o is the probability that an individual channel is open within a given time frame). In this cell neither glucose (a) nor GLP-1 (b) were effective inhibitors of channel activity when applied alone, whereas a nearly complete inhibition of channel activity was observed when both substances were applied together (c). Channel activity recovered after removal of glucose/GLP-1, and was then blocked by application of 10 nm glyburide (d), indicating that these channels do in fact correspond to the ATP-sensitive potassium channels.

Figure 4

A model in which the induction of glucose competence by GLP-1 is proposed to result from the synergistic interaction of ATP and cAMP to inhibit ATP-sensitive potassium channels. The binding of GLP-1 to its receptor leads to activation of a heterotrimeric G protein composed of α-, β- and γ-subunits. G protein activation stimulates the activity of the enzyme adenylate cyclase which catalyses the formation of intracellular cAMP. The rise in cAMP levels results in the activation of protein kinase A which catalyses the phosphorylation of regulatory sites (indicated by PO₄) on the channel. Conformational switches (indicated by the double-ended arrows) in response to phosphorylation increase the affinity of the channel for ATP, thereby favouring channel closure. In this manner, GLP-1 favours the inhibition of the channel by ATP. Neither cAMP or ATP are viewed as sufficient, alone, to inhibit channel function, and for this reason bidirectional crosstalk between the GLP-1 and glucose signalling system is required for complete inhibition of channel activity.