BB 30045 Cell membranes Transporters and trafficking Lecture

BB 30045 Cell membranes Transporters and trafficking Lecture 8 Insulin secretion

Insulin secretion • Glucose transported into cell via GLUT 2. Metabolised via glucokinase (not hexokinase) to give glucose 6 -phosphate and further metabolism • Non-metabolised sugars do not stimulate insulin release • ATP/ADP ratio is increased • Closes K-ATP channels. Membrane depolarisation • Opening of voltage-dependent Calcium channels • Calcium influx • Calcium stimulates granule/vesicle fusion and insulin release • Hormones (incretins) can influence this process via G-protein receptors and intermediates that include c. AMP, DAG and IP 2 Seino, S. et al. Physiol. Rev. 85: 1303 -1342 2005

Incretins modulate and sensitize insulin secretion via adenyl cyclase GLP 1 (Glucagon-like peptide 1) Secreted by stomach Acts on pancreas to increase insulin secretion Exendin-4 Secreted by stomach Acts on pancreas to increase insulin secretion Leptin Secreted by adipose cells Acts on pancreas, brain and taste cells Reduces appetite and insulin secretion

Molecular structure of the KATP channel Ashcroft, F. M. J. Clin. Invest. 2005; 115:

Model of octameric structure of the KATP channel Model based on low resolution EM pictures Biochem. Soc. Trans. (2006) 34, 243 -246

Model of c. AMP compartmentation associated with exocytosis c. AMP is thought to be localised in discrete compartments and does not diffuse widely (because of localisations of the phosphodiesterase) PKA is also localised via interaction with anchoring protein AKAP Phosphorylation of exocytotic machinery is thought to occur (A) Additionally (B) there is a link through a c. AMP stimulated Rap-GEF. This in turn stimulates interaction with Rim (which is a Rab 3 interacting protein). Rim may also be associated with SUR 1 Many of the components of signalling have been discovered but establishing the relevant links between the components requires further study Seino, S. et al. Physiol. Rev. 85: 1303 -1342 2005; doi: 10. 1152/physrev. 00001. 2005 Copyright © 2005 American Physiological Society

Insulin release occurs as a fast phase and a slow phase Picture High K+ induces only the rapid release phase. Glucose increases both phases Release is associated with membrane potential changes (depolarisation)

Studying insulin vesicle trafficking Increase in surface area measured as increased capacitance Microscopy across cell (confocal) or near the PM (TIRF)

Only 5% of granules are in RRP (ready releasable pool) Total granule population is ~10, 000 per cell

Different phases are associated with different populations of vesicles Separate populations behave differently (different locations and docking properties) Slowly released vesicles appear to fuse rapidly once they reach the PM (shorter docking phase as the vesicles are already primed for fusion)

Calcium control of vesicle fusion SNAREs are required for fusion: VAMP 2 Syntaxin 1 SNAP 25 The most important Rab seems to be Rab 3 a Rab 3 A interacts with the effector Rim 3 which is described as a scaffolding protein or tether Calcium activation probably occurs via synaptotagmin V and VII

SNAREs and Calcium channel interactions occur Calcium is thus released locally in the vicinity of the vesicle fusion site

Forms of Diabetes mellitus Common forms (~ 4% of population): Type 1 Autoimmune disease ~10% Diagnosed typically in teens Type 2 Insulin resistance 90% Diagnosed typically after 50 years Rare forms (for example frequency of ~ 1: 50, 000 births): MODY Glucokinase mutations HNF 1 and HNF 4 mutations Diagnosed typically before 25 Neonatal diabetes Kir 6. 2 channel mutations Kir 6. 2 channel trafficking mutations Diagnoses at birth

Relative frequency of genetic forms of MODY and Neonatal diabetes Hattersley, A. T. et

Insulin secretion rates at different glucose values for different monogenic diabetes Hattersley, A. T.

Ashcroft, F. M. J. Clin. Invest. 2005; 115: 2047 -2058

Locations of mutations in ATP binding site region (gain of K channel function) Less ATP binding more channel activity (channel normally closes with glucose stimulation of cells Ashcroft, F. M. J. Clin. Invest. 2005; 115: 2047 -2058

Mutations also occur in pore neck region and affect gating (gain of K channel function) Ashcroft, F. M. Am J Physiol Endocrinol Metab 293: E 880 -E 889 2007;

ATP sensitivity of channel correlates with disease severity DEND syndrome (developmental delay, epilepsy and neonatal diabetes). i. DEND, intermediate DEND syndrome. Ashcroft, F. M. Am J Physiol Endocrinol Metab 293: E 880 -E 889 2007;

Some mutations in patients affect trafficking of Kir 6. 2 channels Targeting motifs in Kir 6. 2: YxxØ and LL Y 330 C and F 333 I mutations have slowed endocytosis and increased surface expression of channels (gain of K channel function and Neonatal Diabetes, ND) E 282 K inability of channel to bind SUR 1 in ER resulting in ER retention and reduced surface expression (loss of K channel function and Hyperinsulinaemia, HI) R 1399 H in SUR 1 retention of channel in TGN (loss of K channel function and HI)

Therapies for pancreatic insulin secretion deficiency (Type 1 and Type 2) Glucokinase activators GLP 1 agonists Inhibitors of Glp 1 proteolysis (dipeptidyl peptidase IV inhibitors) Islet replacement via stem cells Suphonylureas

Sulfonylureas stimulate insulin secretion in neonatal diabetes due to KATP channel mutations Before therapy: KATP channel cannot close Membrane hyperpolarised even when glucose Incretins are ineffective as Ca++ is low After therapy: KATP channel inhibited Membrane depolarised and insulin released Incretins are effective as Ca++ is high Ashcroft, F. M. Am J Physiol Endocrinol Metab 293: E 880 -E 889 2007

Summary and main points Insulin release occurs in response to glucose via an ATP K+ sensitive channel Kir 6. 2 Release occurs in two phases related to separate and distinguishable vesicle pools Modulation of release occurs via G-protein and c. AMP coupled signalling from incretins (GLP 1 etc) SNARE proteins are thought to linked to calcium channels and calcium sensitive synaptotagmins Mutations in Kir 6. 2 occur in the ATP binding site and channel leading to gain of channel function and neonatal diabetes Trafficking mutations give ND or HI depending on whether they increase or decrease surface Kir 6. 2 channels

Spectrum of disease susceptibility in relation to K channel activity Human mutations at extreme ends give distinct symptoms but more minor mutations may give disease susceptibility or even protection from disease. Note: Some groups working on the pancreas think type 2 diabetes is primarily a pancreatic problem. Others think the initial causes of disease are associated with peripheral insulin resistance. Ashcroft, F. M. J. Clin. Invest. 2005; 115: 2047 -2058