Abstract
Pulses of insulin from pancreatic beta-cells help maintain blood glucose in a narrow range, although the source of these pulses is unclear. It has been proposed that a positive feedback circuit exists within the glycolytic pathway, the autocatalytic activation of phosphofructokinase-1 (PFK1), which endows pancreatic beta-cells with the ability to generate oscillations in metabolism. Flux through PFK1 is controlled by the bifunctional enzyme PFK2/FBPase2 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase) in two ways: via (1) production/degradation of fructose-2,6-bisphosphate (Fru2,6-BP), a potent allosteric activator of PFK1, as well as (2) direct activation of glucokinase due to a protein-protein interaction. In this study, we used a combination of live-cell imaging and mathematical modeling to examine the effects of inducibly-expressed PFK2/FBPase2 mutants on glucose-induced Ca2+ pulsatility in mouse islets. Irrespective of the ability to bind glucokinase, mutants of PFK2/FBPase2 that increased the kinase:phosphatase ratio reduced the period and amplitude of Ca2+ oscillations. Mutants which reduced the kinase:phosphatase ratio had the opposite effect. These results indicate that the main effect of the bifunctional enzyme on islet pulsatility is due to Fru2,6-BP alteration of the threshold for autocatalytic activation of PFK1 by Fru1,6-BP. Using computational models based on PFK1-generated islet oscillations, we then illustrated how moderate elevation of Fru-2,6-BP can increase the frequency of glycolytic oscillations while reducing their amplitude, with sufficiently high activation resulting in termination of slow oscillations. The concordance we observed between PFK2/FBPase2-induced modulation of islet oscillations and the models of PFK1-driven oscillations furthermore suggests that metabolic oscillations, like those found in yeast and skeletal muscle, are shaped early in glycolysis.
Highlights
The plasma insulin pulses arising from pancreatic beta-cells [1] are strongest in the portal blood system, more effective than continuous administration in suppressing hepatic glucose output [2,3], and lost in diabetics and their near relatives [4,5]
Each of the four PFK2/FBPase2 mutants used in this study (Fig. 1A) was Nterminally tagged with a Degradation Domain (DD), which permits transcription and translation but prevents the accumulation of functional protein in the cytosol due to rapid proteosomal degradation; degradation is inhibited in a controlled way by the addition of a small cell-permeant molecule, Shield1 [25]
Because fulllength PFK2/FBPase2 is required for the interaction with GK, a comparison between the point mutants and truncation mutants provides a method for discerning possible effects of PFK2/ FBPase2 on PFK1 from effects on GK
Summary
The plasma insulin pulses arising from pancreatic beta-cells [1] are strongest in the portal blood system, more effective than continuous administration in suppressing hepatic glucose output [2,3], and lost in diabetics and their near relatives [4,5]. Befitting their role as metabolic sensors for the organism, beta-cells adjust their metabolic output in accordance with plasma glucose concentration, and not just their own energy requirements.
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