Above: Concentration response characteristics for the effect of VIP and PACAP-38 to increase [Ca++]i in the presence of 4 mM glucose.
Left: The effect of 100 nM wortmannin on insulin secretion (4 mM glucose) and 10 nM VIP-stimulated secretion. [control: open squares, VIP: open circles, WT alone: closed squares, WT+VIP: closed circles.
A variety of physiologic, pharmacologic, biochemical and electrophysiologic techniques are in use to explore the regulation of pancreatic b-cell function. These include perifusion of pancreatic islets and cloned cells combined with radioimmunoassay, to measure the minute to minute release of insulin under basal, stimulated or inhibited conditions; the use of fluorescent dyes to measure the concentration of intracellular Ca2+ and other second messengers in stimulus-secretion coupling and biochemical techniques for enzyme assays or intracellular substrate determinations; electrophysiologic and patch clamp techniques for the measurement of ion channel activity; and capacitance measurements to monitor exocytosis in single beta-cells. Major emphasis is currently placed on three areas.
1. The action of glucose to stimulate insulin secretion is complex and includes at least two major mechanisms. One of these actions is to depolarise the cell and stimulate Ca2+ entry through voltage dependent Ca2+ channels. Increased intracellular Ca2+ is an effective stimulus to secretion. However, the major increase in secretion is due to a second action of glucose to augment the response to the Ca2+ signal. The glucose signaling pathway responsible for augmentation is thought to enter stimulus-secretion coupling at a late stage, possibly at a point close to the final exocytotic step.
2. Several hormones and peptides inhibit insulin secretion. Among them are norepinephrine, somatostatin, prostaglandins, galanin, and amylin. They all exert at least four inhibitory effects on the b-cell. These include activation of K+ channels to hyperpolarize or repolarize the membrane potential, thus inactivating the cell; inhibition of voltage-dependent Ca2+-channels to block Ca2+ entry; inhibition of adenylyl cyclase to lower cyclic AMP levels; and inhibition of exocytosis by an as yet unidentified mechanism late in stimulus-secretion coupling.
3. Understanding exocytosis, the process whereby the insulin-containing granules fuse with the plasma membrane and allow insulin to be released, is key to understanding the regulation of both stimulation, augmentation and inhibition. Thus we are studying exocytosis by both biochemical and electrophysiological techniques.
We expect that the variety of approaches we are using will lead to rapid progress in our understanding of the control of pancreatic beta-cell function. This, in turn, should lead to greater understanding of those diseases such as diabetes that involve disorders of insulin secretion.