The exact mechanism of vesicle fusion and release of its contents is beyond the scope of this paper; an excellent review of secretory granule exocytosis is provided by Burgoyne and Morgan [4]. The insulin response has two distinct phases: an acute phase that lasts only about 2-5 minutes and the prolonged, steady-state phase in which both pre-formed and newly synthesized insulin is released [5, 6]. It is also worth noting that in addition to the pathway outlined above, many other factors play a role in modulating insulin secretion. Involvement of both the phospholipase C and adenylyl cyclase pathways and sympathetic and parasympathetic divisions of the autonomic nervous system quickly complicate the picture of glucose homeostasis. Almost all the regulatory aspects of insulin secretion ultimately influence one or more component of the electrophysiology of the β-cell so the contributions of all of these factors will be summarized by their implication in the ionic basis of bursting, a topic still heavily debated.
III. Historical View of the Development of a Theory for the Control of Insulin Secretion
The term neuroendocrine is used to describe a broad range of cell types (including both β-cells and enteroendocrine cells) which are characterized by their synthesis of molecules that are subsequently released into the blood stream in a regulated and concerted fashion[7]. As the term implies these cells have several properties in common with neurons, most notably—excitability. The theoretical framework for the role of ion transport in the excitability of cellular membranes was built around the fundamental works of Goldman (1943) and Hodgkin and Katz (1949) which used the giant axon of the squid as a model system. Grodsky and Bennett (1966) and later Milner and Hales (1967) [8] determined that the Ca2+ cation is required for secretion of insulin in the perfused pancreas, a result that is suggestive of the contraction of muscle fibers which also have a Ca2+ requirement. Thus the term “stimulus-secretion” was coined to describe the mechanism of endocrine secretion in accordance with the “excitation-contraction” of muscles. This terminology was used by Douglas in 1968 to suggest that methods of electrophysiology be used to investigate secretion in endocrine cells, methods that were thus far reserved for “aristocratic” neurons[9]. The pancreatic β-cells were among the first endocrine cell types to be studied by electrophysiology. The first intracellular recordings of membrane potentials using microelectrodes were performed by Dean and Matthews by 1968[10]. Β-cell membrane potentials in response to secretory stimuli were also confirmed by Pace and Price and a host of others including Meissner, Schmelz, Atwater, Beigelman, Ribalet, Rojas, and Henquin throughout the 1970’s and 1980’s. These experiments with stimulatory and inhibitory agents and varying intracellular and extracellular ionic concentrations sought to determine the roles of ionic fluxes in the modulation of insulin secretion[11]. The contributions of these researchers to the theory of stimulus-secretion by β-cells will be summarized in the following sections.
Resting Potential and Initial Depolarization
The resting membrane potential was hyperpolarized usually between -65 and -70 mV. This is close to the equilibrium potential for K+ and, as expected ion substitutions revealed that K+ permeability through metabolically-sensitive K channels governs the maintenance of resting membrane potential and the initiation of depolarization. Experimental evidence showed a reduced 42K+ efflux from isolated islets stimulated with glucose which corresponded to an increase in membrane resistance and increased secretion (Howell & Taylor, 1968). Three lines of evidence established the underlying role of metabolism in glucose sensitivity (presumably of K channels). First, Na azide and 2,4-dinitrophenol (known metabolic uncouplers) caused hyperpolarization and ended insulin secretion. Non-metabolized glucose derivatives could not induce depolarization. Finally, non-glucose metabolic intermediates (i.e. leucine and α-ketoisocaproate) could induce electric activity[9].
Action Potential Generation
Biphasic electrical responses were induced by a step increase in glucose concentrations from 2.8 to 11mM causing depolarization to the -50 mV threshold. The first phase was a prolonged period of action potential firing at a plateau of -35 mV. Then, steady-state activity, known as bursting, takes over in which membrane potential oscillates between a silent phase (membrane potential just below threshold) and active phase with typical magnitude of spikes between 10 to 20 mV in magnitude. Varying glucose concentrations can alter the relative lengths of the active plateaus and silent phases.
Although the mechanism underlying bursting remains controversial, it is clear that a number of coordinated interactions between β-cell ion channels are required. The electrophysiological components so far identified in the β-cell include a number of different channels that maintain the balance of outward and inward currents of the K+, Ca2+, and other physiologically relevant ions. The channels identified as having major roles in stimulus-secretion coupling already mentioned are the ATP-sensitive K channel, voltage-dependent Ca2+ channels, and Ca2+ release dependent Ca2+ channels but other channels and currents may also enter the picture. For instance, delayed-rectifying K channel (KDR), Ca2+-activated K channel (KCa), voltage-gated Na+(TTX-sensitive) channels, Ach-activated, Na+ permeable channel, and background Na+ current carried by Na+ ions are a few other concerns of electrophysiologists. The workings of several of these channels are reviewed in detail by Mears and Atwater[9]. Several models highlight how interplay of these channels may explain the oscillatory electrical activity. Modeling of ion channels and extensive use of the improved patch-clamp technique[12] for cellular recording has provided insight into some of the processes that are likely involved, including: (1) gradual Ca2+ current inactivation, (2) cyclic variations in the activity of the KATP channel, (3) intercellular association of β-cells and changes in interstitial ion conductances, and (4) activation of low-conductance Ca2+ activated K channels.
Second Messengers Involvement
By the mid 1980s attention had shifted to the involvement of other molecules (second messengers) in the extending finer control of the secretory mechanism. Acceptance of some of these other factors was better received that others. Rorsman and Trube’s 1985 suggestion, for example, that ATP acts as a second messenger contradicted previous observations that the channel is almost fully blocked at [ATP] of 0.1 mM (intracellular recordings of β-cells reported 2-5 mM concentrations of ATP with little change in this level with varying glucose concentrations). This led to adoption of the idea that other intracellular constituents modulate ATP sensitivity because KATP activity was still observed in intact cell preparations despite high ATP levels. It is now believed that changes in [ADP]i may be as important in coupling metabolism with secretion as [ATP]i. Evidence supporting this idea is as follows (1) agents that elevate cellular ATP levels (and lower ADP levels) inhibit channel activity, while those that depress ATP and increase ADP, activate channels; (2) there is a good correlation between hyperpolarization of the mitochondrial membrane potential and the block of the KATP channels; and (3) changes in KATP activity and intracellular ATP and ADP concentrations occur over the same range of glucose concentrations with a similar time course. A decrease in ADP, at a relatively constant ATP, may be the link between metabolism and channel closure[8].
Wollheim’s 1987 work using electrically permeabilized mouse β-cells was particularly insightful. He investigated whether the secretory response to GTP analogues was mediated by any of the three enzyme systems regulated by GTP-binding proteins (i.e. generation of cAMP by adenylate cyclase, DAG by phospholipase C, or arachidonic acid by phospholipase A2)[13]. Throughout the early 1990’s Berridge, Tsien, Rorsman, Ashcroft, and many others honed in on the roles of second messengers in the β-cell further implicating ADP, as discussed above, and phospholipase C in producing the second messenger IP3 (which triggers Ca2+ oscillations by releasing it from ER stores).
Summary of β-cell Stimulus-Secretion
Glucose-induced stimulation of insulin secretion represents an exquisitely-controlled system that is critically dependent on the rise in intracellular calcium concentrations to promote exocytosis. Complex interactions between physiologic and pharmacologic stimuli and the intracellular events of metabolism and second messengers influence the membrane events (ionic current flows), making electrical activity of the β-cell very telling of the stimulus-secretion mechanism. Although questions still exist as to how burst duration is controlled and to how molecular mechanisms of ion channel regulation is achieved, the electrical events of β-cells can still provide useful insight into the secretory regulation used by other cell types of tissue engineering interest.
IV. How does secretion by enteroendocrine cells diverge from that of β-cells?
While literature contains extensive research on the electrophysiology of the β-cells and many other endocrine cell types and it is anticipated that the mechanism of stimulus-secretion will follow a similar scheme in L and K enteroendocrine cells. Few attempts have been made at demonstrating this, however, and very little is known about the mechanism of stimulus-secretion coupling in L cells[14].
As with β-cells, enteroendocrine cells are polar, with microvilli on their luminal side and secretory granules located basally. Although they are the most abundant cell type in the small intestine, the diffuse distribution of L cells has prevented extensive analysis of these low-viability and heterogeneous in vitro preparations very challenging. Two cell lines have been developed for studying glucagons-like peptide secretion by L cells: the GLUTag cell line and the STC-1 cell line both derived from intestinal tumors of transgenic mice. As in β-cells studies monitoring GLP-1 secretion by radioimmunoassay (RIA) demonstrate a biphasic mechanism of release is response to nutrients, hormones, and neural inputs following ingestion of a meal[15]. Also of a similar cord to insulin secretion, Drucker, Brubaker, and others have used the GLUTag cell line to hypothesize about the intracellular signals involved in L cells. These studies gave generated the following results: (1) activation of protein kinase A stimulates GLP-1 release and synthesis; (2) activation of protein kinase C results in an increased secretion of GLP-1 but does not increase transcription of the proglucagon gene; and (3) Calcium channel blockers inhibit whereas increasing intracellular calcium, [Ca2+]i stimulates secretion of GLP-1[15].
Perhaps more telling of L cell function, however, is the recent study using GLUTag cells in perforated-patch clamp and whole cell patch clamp recordings. Quiescent and hyperpolarized in absence of glucose. The resting membrane potential was -53 mV, compared to that of the islet β-cells (-65-70 mV), this may represent a significant divergence in the underlying control of ionic flux that modulates exocytosis. Increasing concentrations between 0-20 mmol/l decreased the membrane conductance, caused membrane depolarization, and triggered the generation of action potentials[14]. Action potentials were also triggered by tolbutamide (500umol/l) and suppressed by diazoxide (340umol.l) or the metabolic inhibitor azide (3 mmol/l), suggesting the involvement of KATP channels. Development of large tolbutamide-sensitive washout currents in whole cell recordings confirms the presence of KATP channels[14]. Due to the large number of similarity, the authors suggested that the stimulus-secretion pathways in L-cells and B-cells share common glucose-sensing machinery. Although it is unlikely that glucose normally acts on L cells and so must activate the release of GLP-1 by means other than a direct effect. Fatty acids, on the other hand, may act directly on the L cells [15].
Far less is known about the enteroendocrine K cells which produce glucose-dependent insulinotropic peptide (GIP) due to difficulties in isolating large numbers of pure K cells. K cells are located primarily in the duodenum an ideal place for regulation by nutrients. Indeed GIP is released rapidly in response to ingestion of nutrients which are thought to act directly on the K cells[15]. It has been suggested that many of the factors regulating hormone production and secretion, as well as timing of the peptide release are remarkably similar to those of β-cells[16]. Glyceraldehyde and methyl-pyruvate were secretagogues, indicating cells depolarized in response to changes in intracellular metabolite level[16]. Interestingly, K channel-opening drugs and sulphonylureas had little effect on insulin secretion by K cells suggesting that secretion is independent of KATP channels[16].
Obvious promise exists in using these cell types for therapies of diabetes yet much work remains to be done in characterizing their electrical behavior and stimulus-secretion mechanism and dynamics.
References Sited
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13. Wollheim, C.B., et al., Regulation of exocytosis in electrically permeabilized insulin-secreting cells; evidence for Ca2+ dependent and independent secretion. Biosci. Rep, 1987. 7: p. 443-454.
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