Figure 1.
Inducible expression of degradation-domain (DD)-tagged mutants of PFK2/FBPase2 in pancreatic islets.
(A) Schematic of DD-tagged mutants of PFK2/FBPase2. (B and C) Western blots of islets transduced with DD-PFK2 and DD-FBPase2 truncations mutants (B), or DD-tagged phosphatase-dead PFK2/FBPase2 (DD-H259A) and kinase-dead PFK2/FBPase2 (DD-T55V) point mutants (C), treated with either vehicle (0.1% DMSO) or 1 µM Shield1, and visualized with an anti-DD antibody. Representative of three independent experiments (50 islets per treatment).
Figure 2.
Effect of expressing PFK2/FBPase2 truncation mutants on islet Ca2+ oscillations.
(A) Representative recordings of islet Ca2+ in the presence of the indicated constructs and 1 µM Shield1; note the difference in scaling. (B and C) Average oscillatory period (B) and amplitude (C) as determined from >20 min of intracellular Ca2+ measurements of transduced islets treated with vehicle or Shield1. Numbers in parentheses indicate the number of islets measured in ≥4 independent experiments. 11.1 mM glucose was used in all experiments. * p<0.05, ** p<0.01, *** p<0.001.
Figure 3.
Effect of expressing PFK2/FBPase2 point mutants on islet Ca2+ oscillations.
(A) Representative recordings of islets Ca2+ in the presence of the indicated constructs and 1 µM Shield1. (B and C) Average oscillatory period (B) and amplitude (C) were determined from >20 min of intracellular Ca2+ measurements of transduced islets treated with vehicle or Shield1. Numbers in parentheses indicate the number of islets measured in ≥4 independent experiments. 11.1 mM glucose was used in all experiments. * p<0.05, ** p<0.01, *** p<0.001.
Figure 4.
Fru1,6-BP oscillations produced by the two-variable glycolytic oscillator model are modified by Fru2,6-BP.
(A) Addition of Fru2,6-BP can terminate the oscillations (red), or at an intermediate level (green), make the oscillations faster and smaller. (B) The Fru6-P nullcline (black) is unaffected by Fru2,6-BP, but Fru2,6-BP pulls together the knees of the Fru1,6-BP nullcline (shown as solid blue, then green and then red lines as Fru2,6-BP is increased as in (A)), eventually stabilizing the equilibrium that exists at the intersection of the Fru6-P and Fru1,6-BP nullclines. The orbit of the oscillation with Fru2,6-BP = 0 is shown (blue dashed line) along with an arrow indicating the orientation of the orbit.
Figure 5.
Simulations with the full Dual Oscillator Model.
(A) Mitochondrial NADH oscillations occur due to oscillations in glycolysis. These are terminated when the ratio VPFK2/VPFK2, and thus the Fru2,6-BP level, is sufficiently large. (B) Slow Ca2+ oscillations are made faster and smaller by the addition of PFK2/FBPase2, with VPFK2/VPFK2 = 0.5. They are replaced by fast Ca2+ oscillations when VPFK2/VPFK2 = 2.