Regulation of Secretion by PI3 Kinase-gamma            

 

Background:

      Diabetes is a world-wide epidemic that affects more than 177 million people.  This is expected to climb to more than 300 million by 2025.  Greater than 85% of diabetics suffer from type-2 diabetes which results both from defective insulin secretion from the b-cells of pancreatic islets of Langerhans, and from an insulin insensitivity of peripheral tissues such as muscle and liver.  As a result, blood glucose is not stored properly, leading to complications that include nerve damage, microcirculatory impairment, kidney failure, heart disease and stroke.  Despite the prevalence of diabetes, we still do not completely understand how b-cells function normally.  This is essential to understanding potential causes of b-cell defects and to the development of future treatments for type-2 diabetes.

     

Stimulus-Secretion Coupling: A rise in blood glucose stimulates the secretion of insulin from b-cells, which acts at peripheral tissues to increase glucose uptake and storage, and decrease glucose production. Increased plasma glucose leads to closure of ATP-sensitive K⁺ (K_ATP) channels by increasing the intracellular ATP to ADP ratio.  Since these channels control the b-cell membrane potential, their closure depolarises the cell thus opening voltage-dependent Ca²⁺ channels (VDCCs) which allow Ca²⁺ to flow into the cell.  The increased intracellular Ca²⁺ interacts with the machinery controlling exocytosis (SNARE proteins) to stimulate secretion of insulin containing large dense-core vecisles (LDCVs).  Repolarisation of the membrane, closure of VDCCs, and subsequent limitation of insulin secretion is mediated by the opening of voltage-dependent K+ (Kv) channels.

 

PI3 Kinase and Insulin Secretion: Phosphatidyinositol-3-OH (PI3) kinase is comprised of both catalytic and regulatory subunits and has been extensively reviewed¹.  The catalytic subunit of type 1A PI3 kinases, which are regulated by tyrosine kinase receptors, is one of three p110 isoforms (a, b and d).  These associate with one of five regulatory subunits; p50a, p55a and p85a, resulting from alternative splicing of a single gene, and p55g and p85b.  While it is not entirely clear which type 1A isoforms are expressed in pancreatic islets, p110a and b associated lipid kinase activity has been demonstrated in insulinoma cells².  PI3 kinase inhibition with reagents such as wortmannin and LY294002 enhances glucose-dependent insulin secretion^3-8.  This is most likely mediated by inhibition of the type 1A PI3 kinases, since these are responsible for the majority of the PI3 kinase activity in insulin secreting cells² and their effects are consistent with the genetic down-regulation of islet type 1A PI3 kinase activity.⁴

      The only type 1B isoform of PI3 kinase, called p110g, associates with the p101 regulatory subunit and is activated directly by GPCRs through an interaction with Gbg.  Like other PI3 kinases, p110g phosphorylates phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P) to generate PtdIns(3,4,5)P in vivo and subsequently activates numerous downstream targets including PDK1, Akt, PKCz and p70S6 kinase.  Activity of p110g has been demonstrated in insulinoma cells,² while protein expression has been shown in human, dog, rat and mouse pancreas by immunohistochemistry.⁹  The potential role of this PI3 kinase isoform in b-cell function is largely unknown.

 

p110g -/- Mice: We have recently confirmed the expression of p110g in mouse and human islets and in insulin secreting cell lines.  Also, in contrast to the effects of type 1A PI3 kinase inhibition or genetic down-regulation, we find that genetic ablation of the type 1B PI3 kinase isoform p110g results in a severe reduction in glucose-stimulated insulin secretion.  This is consistent in both the in vivo plasma insulin response to IP glucose injection and in the isolated perfused pancreas, and suggests a distinct and important role for this PI3 kinase isoform in b-cell function.  Similar divergent roles for the type 1A and 1B isoforms have been observed in heart¹⁰, where the type 1A and 1B PI3 kinases regulate cell size and contractility, respectively.

 

General Aim:

      The general aim of this project is to examine the distinct roles of the receptor tyrosine kinase coupled type 1A (a, b or d) and the G-protein coupled type 1B (g) isoforms of PI3 kinase in b-cells of pancreatic islets of Langerhans.  To accomplish this, isoform specific knockout of endogenous PI3 kinase activity will be performed in insulinoma cells and primary b-cells using siRNA technology.

 

Aim 1: Generation of siRNAs targeted at PI3 kinase isoforms

 

Rationale: While knockout of p110g¹¹ or p85a⁴, and non-selective PI3 kinase inhibition^3-8 demonstrate that isoforms of PI3 kinase are important regulators of pancreatic islet function, these studies all suffer major drawbacks.  The genetic ablation of PI3 kinase subunits in the p110g -/- or p85a -/- mice are not b-cell specific and the long term and developmental effects of reduced PI3 kinase activity are unknown, and while the pharmacologic inhibitors block PI3 kinase acutely, they are not isoform specific.  In order to properly determine the roles of the type 1A (a, b or d) and type 1B (g) isoforms in islet function, an acute and selective method targeting these proteins is required.  Small interfering RNA (siRNA) technology has been used successfully in the past to acutely down-regulate the expression of PI3 kinase isoforms¹², and this technique has been used previously in insulin secreting cells¹³.  Efficient delivery of siRNA constructs to primary cells can be accomplished using a recombinant adenovirus approach¹⁴.

 

Aim 2: Effect of isoform specific PI3 kinase ablation on b-cell function

 

Rationale: Chronic reduction of PI3 kinase isoforms, and non-selective PI3 kinase inhibition, suggest that the classic receptor tyrosine kinase coupled PI3 kinases (type 1A) and the G-protein coupled PI3 kinase (type 1B; p110g) play opposing roles in regulating islet function.  While the type 1A PI3 kinases are negative regulators of insulin secretion, p110g appears to be a positive regulator of secretion .  These studies were performed either with chronic, whole-body, gene ablation or with non-selective antagonists.  It is therefore crucial to investigate the effects of selective and acute PI3 kinase isoform knock-down.  The experiments proposed in this aim will clarify the role of the various PI3 kinase isoforms in stimulus-secretion coupling, and identify at which point they act in the stimulus-secretion cascade.

 

Aim 3: Mechanism of PI3 kinase-g effects on insulin secretion

 

Rationale:  There are a number of potential signalling molecules down-stream of p110g including phosphorylated lipids (notably PI(3,4,5)P3), PKC and small G-proteins such as Rac and Cdc42.  These are known to modulate ion channel function and/or vesicle trafficking and exocytosis.¹⁵  Of particular interest this PI3 kinase isoform was recently shown to have a tonic inhibitory role on G-protein coupled adrenergic receptors in the heart¹⁰, and knockout of this protein relieved this inhibition (likely by reducing G_i signalling), resulting in increased cAMP responses and increased contractility.  The potential involvement of cAMP, or a down-stream pathway, is suggested by the finding that the blunted insulin response of p110g -/- mice is normalised by exendin 4, a glucagon-like peptide-1 (GLP-1) receptor agonist that signals through cAMP.  Adrenergic stimulation has dual effects on insulin secretion.¹⁶ While stimulation of b-adrenoreceptors stimulates insulin secretion through cAMP, the dominant effect of adrenergic stimuli is to inhibit insulin secretion through activation of the a2-adrenoreceptor which may be mediated by activation of K_ATP channels, antagonisim of Ca²⁺ channels, inhibition of cAMP formation or direct inhibition of exocytosis.^16-20  It may therefore be possible that, analogous to what is observed in heart, altered adrenergic receptor signalling contributes to the secretory defect.  Interestingly, data from the p110g -/- suggest a defect peripheral counter-regulation, of which adrenergic-stimulated glucose mobilization is an important component.

 

Significance:     Understanding pancreatic islet function under normal conditions is essential to the identification of defects that occur in type-2 diabetes and to developing effective treatments.  This study will provide important new information about the regulation of insulin secretion.  In particular, the role of G-protein coupled PI3 kinase-g as a regulator of insulin secretion will be investigated.

 

Reference List

 

     1.    S. Koyasu, Nat.Immunol. 4, 313-319 (2003).

     2.    A. Trumper et al., Mol.Endocrinol. 15, 1559-1570 (2001).

     3.    W. El Kholy et al., FASEB J. 17, 720-722 (2003).

     4.    K. Eto et al., Diabetes 51, 87-97 (2002).

     5.    S. Hagiwara et al., Biochem.Biophys.Res.Commun. 214, 51-59 (1995).

     6.    K. Nunoi et al., Biochem.Biophys.Res.Commun. 270, 798-805 (2000).

     7.    W. S. Zawalich and K. C. Zawalich, Endocrinology 141, 3287-3295 (2000).

     8.    W. S. Zawalich, G. J. Tesz, K. C. Zawalich, J.Endocrinol. 174, 247-258 (2002).

     9.    H. G. Bernstein, G. Keilhoff, M. Reiser, S. Freese, R. Wetzker, Cell Mol.Biol.(Noisy.-le-grand) 44, 973-983 (1998).

   10.    M. A. Crackower et al., Cell 110, 737-749 (2002).

   11.    P. E. Macdonald et al., Endocrinology In Press, (2004).

   12.    F. Czauderna et al., Nucleic Acids Res. 31, 670-682 (2003).

   13.    K. J. Mitchell, T. Tsuboi, G. A. Rutter, Diabetes 53, 393-400 (2004).

   14.    C. Shen, A. K. Buck, X. Liu, M. Winkler, S. N. Reske, FEBS Lett. 539, 111-114 (2003).

   15.    C. J. Barker, I. B. Leibiger, B. Leibiger, P. O. Berggren, Am.J.Physiol Endocrinol.Metab 283, E1113-E1122 (2002).

   16.    B. Ahren, Diabetologia 43, 393-410 (2000).

   17.    A. Debuyser, G. Drews, J. C. Henquin, Mol.Cell Endocrinol. 78, 179-186 (1991).

   18.    S. Yamazaki, T. Katada, M. Ui, Mol.Pharmacol. 21, 648-653 (1982).

   19.    J. Lang et al., EMBO J. 14, 3635-3644 (1995).

   20.    A. Debuyser, G. Drews, J. C. Henquin, Pflugers Arch. 419, 131-137 (1991).

 

 

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