Stimulus secretion coupling in pancreatic beta-cells

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Impaired insulin secretion, which generally requires in addition inefficient insulin usage (insulin resistance), is a key step in the pathogenesis of type 2 diabetes. The two main components of secretion are calcium entry into pancreatic beta cells and triggering of insulin granule exocytosis by that calcium. We have modeled both components and their contributions to diabetes. We have been developing systematically over many years a model for the regulation of the other main component of insulin secretion, calcium entry. We have focused particularly on the mechanisms of calcium oscillations over a range of periods, from seconds to minutes. The slower class of oscillations (5 - 10 minute period) is the main driver of pulsatile insulin concentration in the circulation, which has been shown to be optimal for the response of insulin-sensitive tissues, especially the liver. The initial hypothesis of the model was that the oscillations result from the partnership of semi-independent electrical and metabolic oscillators, with the combined system called the Dual Oscillator Model (DOM). The electrical oscillator (EO) is based on negative feedback of calcium onto ion channels, directly onto calcium-activated potassium (K(Ca)) channels and indirectly onto ATP-dependent potassium (K(ATP)) channels because calcium reduces the ATP/ADP ratio. The metabolic oscillator (MO) is governed by positive feedback of fructose 1,6 bisphosphate (FBP) on the enzyme in glycolysis that produces it, phosphofructokinase (PFK). The MO communicates with the EO via the K(ATP) channels, which transduce the metabolic state of the cell (ATP/ADP ratio) into electrical depolarization. K(ATP) channels are of clinical significance as they are a target of insulin-stimulating drugs, such as the sulfonylureas tolbutamide and glyburide, the first class of oral medications developed for the treatment of Type 2 Diabetes. Severe gain-of-function mutations of K(ATP) are a major cause of neo-natal diabetes mellitus, whereas moderate gain-of-function mutations have been linked in genome-wide association studies (GWAS) to the milder but more common adult-onset form of diabetes, type 2 diabetes. Conversely, loss-of-function mutations of K(ATP) are a major cause of familial hyperinsulinism, a hereditary disease found in children in which beta cells are persistently electrically active and secrete insulin in the face of normal or low glucose, causing life-threatening hypoglycemia. Another major cause of hyperinsulinism is excessive activity of the enzyme glucokinase, which also plays a key role in the DOM. The activity of the MO is controlled by the availability of the substrate of PFK, fructose-6-phosphate, which in turn depends on the external glucose concentration. This makes PFK a natural gateway for plasma glucose to control the oscillations of the beta cells and thence the pulses of insulin release. Calcium, and thereby insulin, oscillates when glucose is at an intermediate concentration, corresponding to typical post-prandial levels. In the earliest versions of the DOM, the MO drove the EO to produce the slow oscillations. However, subsequent data, described in the 2016 report, obtained by probing the oscillations of FBP with a FRET-based sensor (PKAR), required a modification of the model to include a prominent but indirect role for calcium in regulating PFK. We proposed that calcium does this by activating pyruvate kinase, an enzyme downstream of PFK, which increases the rate of utilization of FBP, resulting in acceleration of the citric acid cycle and ATP production in the mitochondria. Thus, the activity of PFK is determined by the balance of input of substrate and consumption of product. Moreover, this positive feedback of calcium on ATP production complements the negative feedback of ATP on PFK and on ATP levels that was in the original DOM. In recognition of this integration of glycolytic and calcium-dependent elements of metabolism, we named the revised model the Integrated Oscillator Model (IOM). The IOM can account in detail not only for the existence of metabolic oscillations, but also for the shape of oscillations in FBP and ATP, which is usually sawtoothed, rather than pulsatile, as predicted by the classic DOM. The features of the IOM and their significance were described in a review paper in 2018 (Bertram et al, Diabetes 67(3):351-359). Critically, the IOM also predicted that large amplitude oscillations in calcium and ATP, but not FBP, could occur in the absence of an active contribution of PFK. Experiments in the laboratory of our collaborator, Les Satin, indeed found that slow calcium oscillations persisted in mice in which PFKm, the isoform that had been proposed to mediate the positive feedback by FBP mentioned above, was knocked out. Surprisingly, oscillations in FBP persisted when PFKm was completely knocked out. Further modeling showed that a second PFK isoform, PFKp, found in platelets, could take over the role of driving glycolytic oscillations when PFKm was absent. Notably, the model shows that this does not require increased compensatory expression of PFKp but merely that PFKp increase its activity. This can happen because PFKp is normally competitively inhibited by PFKm, which has higher affinity for the common substrate, F6P, and lower affinity for the common inhibitor, ATP, but can become active when PFKm is missing. A paper is in review. Other models that do not involve glycolytic oscillations can account for calcium oscillations in the absence of autocatalytic PFK, but they would not explain the many other observations we and others have made. We have generated a set of predictions of the IOM to distinguish it from these competing models. Preliminary tests of these predictions are favorable and the results will be described in a future report when completed. We have used the mathematical models to address a related question. The ATP/ADP ratio has been shown experimentally to oscillate, but those experiments do not answer whether the metabolic oscillations drive slow calcium oscillations via the K(ATP) channels or are a passive response to the calcium oscillations: since increases in cytosolic calcium activate pumps that consume ATP to bring calcium back down, any oscillation in calcium will be reflected in a oscillations in ATP/ADP. However, the models show that when ATP/ADP is the cause, not just a consequence, of calcium oscillations, then the ATP/ADP ratio remains constant as glucose is increased within the range that supports oscillations. This happens because the increase in ATP production due to higher glucose is balanced over the course of each oscillation by the increase in ATP consumption due to pump activation. Experiments in the Satin laboratory have confirmed that the ATP/ADP ratio is nearly invariant with the oscillatory regime, which supports our hypothesis that ATP/ADP is the primary active driver of the calcium oscillations. This counter-intuitive prediction would not have been possible without the prior mathematical and modeling work. A paper is in review.