AIMS Conference, 2012

1. The Dual Oscillator Model for Pancreatic IsletsRichard BertramDepartment of MathematicsandPrograms in Neuroscience and Molecular Biophysics Florida State University, Tallahassee, FL. AIMS Conference, 2012

2. Collaborators Arthur Sherman (NIH) Leslie Satin (U. Michigan) Matthew Merrins (U. Michigan) Funding: NSF-DMS0917664

3. What is an Islet of Langerhans? Cluster of electrically coupled hormone-secreting cells, located throughout the pancreas. The human pancreas has about 1 million islets. Courtesy of Rohit Kulkarni Immunostained for glucagon (green) and insulin (red)

4. Insulin Secretion is Pulsatile deconvolved Porksen et al., AJP, 273:E908, 1997 measured Peripheral insulin measurements in the blood of humans exhibits oscillations, suggesting that insulin is secreted in a pulsatile manner.

5. Central Question: What is the mechanism for oscillations in insulin secretion from pancreatic β-cells?

6. Islets are Electrically Excitable Islets are like nerve cells in that they produce electrical impulses. During an upstroke of an impulse Ca2+ enters the cells, causing insulin to be released. calcium Gilon et al., JBC, 268:22265, 1993 insulin

7. Fast Bursting Oscillations Simultaneous fast Ca2+ and voltage measurements from a mouse islet in 11.1 mM glucose. From Zhang et al., Biophys. J., 84:2852, 2003

8. …have period similar to slow insulin oscillations measured from a mouse in vivo (Nunemaker et al., Diabetes, 54:3517, 2005) Slow Bursting Oscillations Slow oscillations of Ca2+ and voltage from an islet... Smith et al., FEBS Lett., 261-187, 1990 Zhang et al., Biophys. J., 84:2852, 2003

9. Compound Bursting Oscillations Henquin et al., Eur. J. Physiol., 393:322, 1982 Bursting oscillations superimposed on a slow wave of activity

10. The Dual Oscillator Model

11. Central Hypothesis Fast, slow, and compound oscillations can all be produced by a mechanism with two coupled oscillators. Calcium oscillator controls fast bursting Ca2+ K(Ca) channels Metabolic oscillator controls slower oscillation ATP  K(ATP) channels

12. Metabolic Oscillations Can Occur Through Oscillations in Glycolysis Glycolytic oscillations in yeast Dano et al., Nature, 402:320-322, 1999

13. Modeling Approach • Cells within an islet are electrically coupled, leading • to synchronization within an islet. Our model • therefore describes a single cell within the synchronized • cell population. • A β-cell is very small and spherical (diameter • ≈10 microns). Modeled as a single compartment; • equations depend on time only.

14. Electrical Component of the DOM Voltage equation reflects Kirchoff’s current law Second equation describes dynamics of the K+ activation variable n. This depends on the voltage.

15. Electrical/Calcium Components of the DOM ER is the Endoplasmic Reticulum Ca2+ enters the cell through L-type Ca2+ channels. The free cytosolic Ca2+ activates K(Ca) channels. Thus, there is mutual feedback between the electrical and Ca2+ components.

16. Fast Oscillations with the DOM When glycolysis is non-oscillatory, the DOM produces fast bursting oscillations, due to the electrical/calcium components of the model. Bertram and Sherman, BMB, 66:1313, 2004 The ER acts as a slow Ca2+filter, setting the period of bursting through its interaction with the cytosol.

17. Glycolytic Component of the DOM substrate product Key feature: The product F1,6BP feeds back positively onto the allosteric enzyme PFK1 (phosphofructokinase 1). Leads to oscillations due to substrate depletion.

18. Glycolytic oscillations in muscle extracts (Tornheim, Diabetes, 46:1375, 1997) Glycolytic Oscillations Produced if Glucokinase Rate is in the Right Range Solid-F6P, Dashed-FBP • Intermediate JGK • (B) High JGK Bertram et al., BJ, 87:3074, 2004

19. Mitochondrial Component Includes equations for mitochondrial NADH concentration, inner membrane potential, Ca2+ concentration, and ADP/ATP concentrations. Final 3-compartment model: Bertram et al., BJ, 92:1544, 2007 ATP acts on K(ATP) channels to affect V

20. The Three Types of Activity can be Reproduced by the Model No glycolytic oscillations With glycolytic oscillations With glycolytic oscillations Experiment Model Model Bertram et al., Am. J. Physiol., 293:E890, 2007

21. A Test for Glycolytic Oscillations Idea: Modify the activity of the key enzyme PFK1 and see if islet oscillations behave as predicted by the model. Fructose-2,6-bisphosphate (F2,6BP) is a high-affinity activator of PFK1 Competes with F1,6BP for binding to PFK1, thus reducing the positive feedback

22. Model Prediction F6P F1,6BP Merrins et al., PLoS One, 2012 F2,6BP should make metabolic oscillations smaller and faster by changing the F1,6BP nullcline

23. Experimental Test of the Prediction Use an adenovirus to overexpress the Bifunctional Enzyme 2 (BIF2) into islets. Then use molecular tagging to degrade either the kinase or phosphatase action of the enzyme. Kinase makes F2,6BP from F6P Phosphatase converts F2,6BP back to F6P

24. Prediction Confirmed With kinase the oscillations in calcium are faster and smaller Merrins et al., PLoS One, 2012

25. Prediction Confirmed With phosphatase oscillations are slower and larger Merrins et al., PLoS One, 2012

26. Next Step Use a FRET sensor to directly measure oscillations in the concentration of a molecule that is in the glycolytic pathway and downstream of PFK1. Do these oscillations exist?

27. Thank You!