Investigating the Effect of Pip4k2α Overexpression in Insulin Signalling in L6 Myotubes

Investigating the effect of PIP4K2α overexpression in insulin signalling in L6 myotubes

A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health

2017

Abdulrahim K. Alabri

School of Biological Sciences

Table of Contents

Table of Contents ...... 2

List of Tables ...... 7

List of Figures ...... 7

Abstract ...... 10

Declaration ...... 11

Acknowledgements ...... 13

Abbreviations ...... 14

Chapter 1 ...... 17

1.1 Introduction ...... 18

1.1.1 Glucose uptake ...... 18

1.1.2 Glucose transporter 4 ...... 19

1.1.3 Insulin signalling ...... 19

1.1.4 Insulin Receptor substrates (IRS) ...... 21

1.1.5 PI3K downstream effectors ...... 21

1.1.5.1 AKT ...... 22

1.1.5.2 Ras-related C3 botulinum toxin substrate 1 (Rac1) ...... 23

1.1.5.2.1 Regulation of Rac1 activity ...... 24

1.1.5.2.2 Rac1-GEFs ...... 25

1.1.5.2.3 Rac1-effectors ...... 26

1.1.5.2.3.1 RalA ...... 26

1.1.5.2.3.2 p21-activated protein kinases (PAKs) ...... 26

1.1.6 Roles of Cytoskeleton rearrangements in insulin-stimulated glucose uptake ...... 27

1.2 Phosphoinositides ...... 28

1.2.1 Characteristics and Synthesis of PIs ...... 28

1.2.2 Enzymes involved in PI metabolism...... 31

1.2.3 PI Phosphatases ...... 40

1.2.3.1 PTEN ...... 40

1.2.3.2 Phosphoinositide 5 phosphatases ...... 42

1.2.3.3 SHIP2 ...... 44

1.2.3.4 SKIP ...... 45

1.3 Roles of PIs: ...... 47

1.3.1 Phosphatidylinositol 3,4,5-trisphosphate PtdIns(3,4,5)P3 ...... 47

1.3.2 Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) ...... 49

1.3.3 Phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) ...... 50

1.3.4 Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) ...... 51

1.3.5 Phosphatidylinositol 3-phosphate (PtdIns3P) ...... 51

1.3.6 Phosphatidylinositol 4-phosphate (PtdIns4P) ...... 52

1.3.7 Phosphatidylinositol 5-phosphate (PtdIns5P) ...... 53

1.4 PIP4K2α overexpression in L6 myotubes ...... 54

1.5 Hypotheses and Aims ...... 55

Chapter 2 ...... 56

2. Materials and Methods...... 57

2.1 Materials ...... 57

2.1.1 Antibodies: ...... 59

2.1.2 Buffers and solutions ...... 60

2.2 Cell culture ...... 61

2.2.1 Rat L6 skeletal muscle cells ...... 61

2.2.2 AD293 cell culture ...... 62

2.2.3 Trypsinisation ...... 62

2.2.4 Cell counting ...... 62

2.3 Molecular biology ...... 63

2.3.1 PIP4K2α Plasmid preparation ...... 63

2.3.2 Viral DNA transfection of AD293 cells with FuGENE®6 ...... 63

2.3.3 Adenovirus titration...... 64

2.3.4 Optimising the multiplicity of infection for L6 cells ...... 65

2.4 mRNA Expression of Phosphoinositide 5-phosphatases ...... 65

2.4.1 Extraction and Purification of mRNA ...... 65

2.4.2 Reverse Transcriptase-PCR ...... 66

2.5 Agarose Gel Electrophoresis ...... 67

2.6 Fluorescence microscopy ...... 67

2.6.1 Coating plates and coverslips ...... 67

2.6.2 Preparation of myotubes ...... 67

2.6.3 Immunofluorescence Microscopy ...... 68

2.6.4 Confocal microscopy ...... 68

2.6.5 Quantification of total fluorescence using ImageJ ...... 68

2.7 Biochemical assays ...... 69

2.7.1 Subcellular fractionation (GLUT4 Translocation) ...... 69

2.7.2 Colourimetric assay of surface GLUT4myc ...... 69

2.8 Rac1 assay ...... 70

2.8.1 Rac1 Pull-down Activation Assay ...... 70

2.9 SKIP siRNA Transfection ...... 71

2.9.1 SKIP Knockdown in Parental L6 cells ...... 71

2.9.2 SKIP Knockdown in L6-Glut4myc cells ...... 72

2.10 Delivery of PtdIns5P by carrier 3...... 72

2.10.1 Effects of exogenous PtdIns5P on AKT and Rac1 activity ...... 72

2.10.2 Delivery of PtdIns5P by carrier 3 to examine GLUT4myc translocation ...... 73

2.11 Protein assay...... 73

2.12 Western Blot ...... 73

2.13 Protein lipid overlay ...... 74

2.13.1 Generation & Purification of GST-GRP1 and GST-TAPP1 PH domains ...... 74

2.13.2 Phosphoinositide Binding Domain preparation ...... 74

2.13.3 Purifying the expressed protein ...... 74

2.14 Neomycin beads: production and re-generation ...... 75

2.15 Lipid extraction ...... 76

2.15.1 Method 1 ...... 76

2.15.2 Method 2 ...... 77

2.16 Protein Lipid overlay (PLO) ...... 77

2.17 Total phosphorus measurements...... 78

2.18 Data analysis ...... 78

Chapter 3 ...... 79

Comparison between parental and L6-GLUT4myc ...... 79 cell lines and optimisation of techniques ...... 79

3.1 Basic morphological comparison between L6-GLUT4myc and Parental L6 ...... 80

3.2 GLUT4-myc expression ...... 83

3.3 Expression of GLUT4myc during differentiation ...... 84

3.4 Optimisation of the detection of GLUT-myc using the colourimetric Assay ...... 85

3.5 Optimisation of FLAG- PIP4K2α expression ...... 87

3.6 Expression of mRNAs of PI5Pases ...... 88

3.7 SKIP expression during cell differentiation ...... 90

3.8 Effect of PIP4K2α on SKIP expression ...... 92

3.9 Optimisation of SKIP knockdown ...... 93

3.9.1 SKIP knockdown in Parental L6 myotubes ...... 93

3.9.2 SKIP knockdown in L6-GLUT4myc myotubes ...... 94

2.19 Validation of Protein Lipid overlay (PLO) assays ...... 97

3.10.1 Extraction methods ...... 97

3.10.2 Binding specificity of GST-TAPP1-PH ...... 98

3.10 Discussion ...... 101

Chapter 4 ...... 105

Investigating the effects of PIP4K2α overexpression ...... 105 and SKIP knockdown in myotubes ...... 105

4.1 Investigating the effect of overexpressing PIP4K2α on GLUT4 translocation ...... 106

4.1.1 Parental L6 cells ...... 106

4.1.1.1 Subcellular fractionation ...... 106

4.1.2 L6-GLUT4myc ...... 109

4.1.3 GLUT4myc translocation using colourimetric assay ...... 112

4.2 Effects of PIP4K2α on AKT phosphorylation ...... 113

4.3 Rac1 activity ...... 114

4.3.1 Insulin dependent activation of Rac1 ...... 114

4.3.2 Effect of PIP4K2α overexpression on Rac1 activity ...... 115

4.4 Insulin dependent activation of PAK ...... 116

4.4.1 Time course of PAK1/2 phosphorylation ...... 116

4.4.2 Effect of PIP4K2α overexpression on PAK1/2 phosphorylation ...... 117

4.5 Effects of PIP4K2α and SKIP knockdown on PIs ...... 119

4.5.1 Effect of PIP4K2α on basal PtdIns(4,5)P2 ...... 119

4.5.2 PtdIns(3,4,5)P3 measurements ...... 120

4.5.2.1 Time course of PtdIns(3,4,5)P3 production ...... 120

4.5.2.2 Effect of PIP4K2α overexpression on PtdIns(3,4,5)P3 ...... 122

4.5.3 PtdIns(3,4)P2 measurements ...... 124

4.5.3.1 Time course of PtdIns(3,4)P2 production ...... 124

4.5.3.2 Effect of PIP4K2α overexpression on PtdIns(3,4)P2 ...... 125

4.6 Effect of PIP4K2α overexpression and SKIP knockdown ...... 127

4.6.1 Effect of PIP4K2α expression and SKIP knockdown on PtdIns(3,4,5)P3 ...... 127

4.6.2 Effect of PIP4K2α expression and SKIP knockdown on PtdIns(3,4)P2 ...... 130

4.6.3 Effects of PIP4K2α and SKIP knockdown on GLUT4 translocation ...... 132

4.6.4 Effects of PIP4K2α and SKIP knockdown on AKT ...... 134

4.6.5 Effect of PIP4K2α expression and SKIP knockdown on Rac1 activity ...... 135

4.7 Discussion ...... 137

4.7.1 Effect of PIP4K2α overexpression on GLUT4 translocation ...... 137

Chapter 5 ...... 145

Investigating the effect of delivery of exogenous PtdIns5P in L6 myotubes ...... 145

5.1 Investigating the effect of delivery of exogenous PtdIns5P on GLUT4 translocation .... 146

5.2 Effect of exogenous PtdIns5P on Rac1 activity ...... 147

5.3 Effect of exogenous PtdIns5P on p-AKT ...... 150

5.4 Discussion ...... 151

Chapter 6 ...... 155

General discussion and conclusion ...... 155

6.1 General discussion ...... 156

6.2 Roles of PtdIns5P ...... 159

References ...... 163

Word count: 55,902

List of Tables

Table 1. 1: Relative levels and the main binding domains and localisation of PIs in mammalian cells...... 30 Table 1. 2: Summary of the genes and proteins encoded for the PI3K family members and their phospholipid substrates...... 33 Table 1. 3: Types, substrate specificity and domains of INPP5Ps ...... 43

Table 2. 1: Primary and secondary Antibodies ...... 59 Table 2. 2: Buffers and solutions ...... 60 Table 2. 3: Primer sequences and expected sizes used for RT-PCR to examine the expression of nine PI5Pases...... 66 Table 2. 4: Amount (in μl) of different components used for RT-PCR reaction...... 66

List of Figures

Figure 1. 1: Insulin signalling in muscle cells (A) and Schematic structure (B) of IRS1, PAK and AKT...... 20 Figure 1. 2: Regulation of Rac1 by GDP-GTP exchange cycle...... 24 Figure 1. 3: Structure (A) and metabolism (B) of PIs...... 29 Figure 1. 4: Schematic diagram of the catalytic subunits of PI3K classes...... 32 Figure 1. 5: Schematic representation of the major domains of PIKfyve...... 39

Figure 2. 1: Purifying recombinant plasmid DNA extracted from E.Coli used for transfecting AD293 cells...... 63

Figure 2. 2: Examples of titrated cells observed under light microscopy...... 65 Figure 2. 3: Expresson and purification of GST-GRP1-PH...... 75

Figure 3. 1: Morphological features of the parental and L6-GLUT4myc cells...... 81 Figure 3. 2: Cell viability of parental and L6-GLUT4myc cells before and after 10 days of differentiation...... 82 Figure 3. 3: Validation of the expression of myc tagged GLUT4 in L6GLUT4myc myotubes by Western blotting...... 83 Figure 3. 4: Time course of GLUT4myc-expression during cell differentiation...... 85 Figure 3. 5: Colourimetric quantification of plasma membrane GLUT4myc...... 86 Figure 3. 6: Optimisation of FLAG-PIP4K2α expression in parental L6 myotubes...... 87 Figure 3. 7: Validation of adenovirus infection derived Flag-PIP4K2α overexpression using immunofluorescence microscopy...... 88 Figure 3. 8: RT-PCR analysis of mRNA expression of PI5Pases in L6 myotubes...... 89 Figure 3. 9: Expression of SKIP during L6 myoblast differentiation...... 91 Figure 3. 10: PIP4K2α overexpression does not affect SKIP expression...... 92 Figure 3. 11: Optimisation of SKIP knockdown in parental L6 myotubes...... 94 Figure 3. 12: Optimisation of SKIP knockdown in L6-GLUT4myc myotubes...... 95 Figure 3. 13: Relative expression of SKIP in control and knocked-down L6-GLUT4myc cells. .. 96

Figure 3. 14: Failure of extracting PIs (PtdIns(3,4)P2 and PtdIns(3,4,5)P3) using Method 1...... 97

Figure 3. 15: Comparing binding specificity of GST- TAPP1 to commercial PtdIns(3,4,5)P3 and

PtdIns(3,4)P2 standards...... 99

Figure 3. 16: Comparison of PtdIns(3,4,5)P3 and PtdIns(3,4)P2 levels...... 100

Figure 4. 1: Detection of GLUT4 in subcellular fractions using an ultracentrifugation method. . 107 Figure 4. 2: PIP4K2α overexpression abolishes insulin dependent-GLUT4 translocation...... 108 Figure 4.3: Effect of PIP4K2α overexpression on GLUT4 translocation in L6-GLUT4myc myotubes...... 110 Figure 4.4: Cell surface fluorescence of GLUT4myc in L6-GLUT4myc myotubes...... 111 Figure 4.5: Colourimetric quantification of GLUT4myc on plasma membrane...... 112 Figure 4.6: PIP4K2α overexpression does not affect insulin-dependent AKT-Ser473 phosphorylation...... 113 Figure 4.7: Time course of insulin-dependent activation of Rac1 in L6 myotubes...... 114 Figure 4.8: Overexpression of PIP4K2α abolishes insulin-dependent activation of Rac1...... 115 Figure 4.9: Time course of insulin-dependent phosphorylation of PAK1/2...... 117 Figure 4.10: Phosphorylation of PAK1/2 is apparently not affected by PIP4K2α overexpression...... 118

Figure 4.11: PIP4K2α overexpression does not affect basal levels of PtdIns(4,5)P2...... 120

Figure 4.12: Time course of PtdIns(3,4,5)P3 changes following insulin stimulation...... 121

Figure 4.13: Basal and time course of insulin-stimulated changes in PtdIns(3,4,5)P3 levels in myotubes expressing LacZ or PIP4K2α...... 123

Figure 4.14: Time course of PtdIns(3,4)P2 changes following insulin stimulation...... 124

Figure 4.15: Basal and time course of insulin induced PtdIns(3,4)P2 levels in myotubes expression LacZ or PIP4K2α...... 126

Figure 4.16: SKIP knockdown restores the insulin-stimulated increase of PtdIns(3,4,5)P3 in cells overexpressing PIP4K2α at 25 min stimulation ...... 129

Figure 4.17: Effect of SKIP knockdown on the level of PtdIns(3,4)P2 in cells expressing LacZ or PIP4K2α...... 131 Figure 4.18: SKIP knockdown does not restore GLUT4 translocation in cells overexpressing PIP4K2α...... 133 Figure 4.19: SKIP knockdown does not affect insulin dependent phosphorylation of AKT-Ser473...... 134 Figure 4.20: SKIP knockdown does not restore activation of Rac1 in cells overexpressing PIP4K2α...... 136

Figure 5. 1: Exogenous PtdIns5P promotes GLUT4 translocation in a wortmannin-sensitive fashion...... 147 Figure 5. 2: Delivery of exogenous PtdIns5P in myotubes activates Rac1...... 148 Figure 5. 3: PtdIns5P-dependent Rac1 activation is wortmannin insensitive...... 149 Figure 5. 4: Exogenous PtdIns5P induces a very small phosphorylation of AKT-ser473...... 150

Figure 6. 1: Proposed models of insulin signalling in control (A) or in L6 myotubes overexpressing PIP4K2α (B)...... 157 Figure 6. 2: Proposed roles of PtdIns(5)P in the insulin/PI3K pathway...... 159

Abstract

Insulin signalling is an essential process in humans by which the level of plasma glucose is maintained within the physiologically healthy range. Insulin activates the phosphoinositide 3 kinase (PI3K) signalling pathway that generates the phospholipid messenger PtdIns(3,4,5)P3, which in turn enhances the activity of two important proteins, AKT and Rac1. This then leads to increase the presence of the glucose transporter 4 (GLUT4) at the plasma membrane that enhances the intake of glucose, particularly in skeletal muscle cells and adipocytes. Insulin signalling also triggers interconversion of several other phosphoinositides (PIs) which play pivotal roles in different steps of glucose regulation.

PtdIns5P is an important PI that is robustly increased after insulin treatment in the skeletal muscle cell line, L6 myotubes. Many of PtdIns5P’s functions are not fully understood. To gain more knowledge of the role of PtdIns5P in insulin signalling in muscle cells, the PtdIns5P kinase phosphatidylinositol-5-phosphate 4-kinase α (PIP4K2α) was over-expressed in L6 myotubes as a way of removing PtdIns5P, and the consequences in insulin signalling were studied. Although

PtdIns5P is converted by PIP4K2α to PtdIns(4,5)P2 which is a precursor of the potent PI

PtdIns(3,4,5)P3, previous studies revealed that the increase in PtdIns(3,4,5)P3 induced by insulin in control cells is diminished in cells overexpressing PIP4K2α, for unknown reasons. Additionally, although the phosphorylation of the serine/threonine protein kinase AKT was not affected in these L6 cells, glucose uptake was attenuated. The current study investigates the possible causes of attenuating glucose uptake in PIP4K overexpressing myotubes by examining the small GTPase Rac1 which plays an important role in the cytoskeleton re-arrangement that is necessary for GLUT4 translocation. Furthermore, the possible roles of PI phosphatases that may cause the disturbance on the levels of PIs in response to insulin were evaluated. Additionally, the potential role of PtdIns5P in Rac1 activation in L6 myotubes was further investigated by delivering synthetic PtdIns5P using a carrier-based delivery approach.

The results showed that the attenuation of glucose uptake documented in previous studies occurred as a result of a defect in the process of translocating GLUT4 from intracellular storage to the plasma membrane. Rac1 activity was significantly reduced in cells expressing PIP4K2α. Quantifying the level of PIs suggested that PIP4K2α expression increases the removal of

PtdIns(3,4,5)P3 by the PI 5-phosphatase, SKIP. Silencing the expression of SKIP by siRNA restored the level of PtdIns(3,4,5)P3 but Rac1 activity and the attenuation GLUT4 translocation were not rescued possibly as a result of removing PtdIns5P by PIP4K2α. On the other hand, exogenous delivery of PtdIns5P in L6 myotubes activates both Rac1 and GLUT4 translocation in the absence of insulin. However, activating GLUT4 translocation by the exogenous PtdIns5P requires PI3K activity since redistribution of GLUT4 to the plasma membrane is inhibited by the PI3K inhibitor, wortmannin. Removing PtdIns5P reduces Rac1 activity and stimulates SKIP that inhibits PtdIns(3,4,5)P3 increase which attenuates GLUT4 translocation and hence glucose uptake. These results emphasise the critical role played by PtdIns5P which seems to serve as a regulator of insulin signalling, both directly and/or by regulating other enzymes involved in the metabolism of PIs.

Declaration

No portion of the work referred to in the thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning.

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Acknowledgements

First and foremost, I would like to express my sincere gratitude to my supervisor; Dr Kath Hinchliffe for the continuous support, advice and for discussions she offered thought this project. Her guidance and comments helped me in all the time of research and writing of this thesis.

I would also like to thank my advisor Dr Jason Bruce for his advices and ideas. I would like also to acknowledge the help of all the technicians and all the members of Bioimaging facility in FBMH.

I am extremely grateful to my family who have always supported me in each stage of my PhD study.

Finally, I am grateful for the financial support from the Ministry of Higher Education, Sultanate of Oman.

Abbreviations

αMEM alpha modification of minimum essential medium AKT Protein kinase B (PKB) APS Ammonium persulphate AS160 Akt substrate of 160 kDa BSA Bovine serum albumin Cdc42 cell division control protein 42 homologue CPE Cytopathic effect CTCF corrected total cell fluorescent signal DAG diacylglycerol DAPI 4',6-diamidino-2-phenylindole DMEM Dulbecco’s modified Eagle's medium DMSO Dimethylsulphoxide ECL Enhanced chemiluminescence EDTA Ethylenediamine tetraacetic acid EGF epidermal growth factor EGTA Ethylene glycol tetraacetic acid ER endoplasmic reticulum F-actin Filamentous actin FBS Foetal Bovine Serum GAP GTPase-activating protein GDI Guanine nucleotide dissociation inhibitors GDP Guanosine diphosphate GEF Guanine nucleotide exchange factor GLUT4 Glucose transporter 4 GPCRs G protein- coupled receptors G-protein GTP-binding protein GSV Glucose transporter 4 storage vesicle GTP Guanosine triphosphate HDM high density microsomes HM hydrophobic motif HRP Horseradish peroxidase IPTG Isopropyl β-D-1-thiogalactopyranoside IR Insulin Receptor IRS Insulin receptor substrate LB Lysogeny broth LDM low density microsomes

MEFs mouse embryo fibroblasts MOI Multiplicity of Infection MTM myotubularin MTMR myotubularin-related protein mTOR Mammalian target of rapamycin mTORC2 mTOR complex 2

Na2EDTA Disodium ethylene diamine tetra acetate O-GlcNAc O-linked β-N-acetylglucosamine OGT O-linked-β-N-acetylglucosamine transferase PAGE Polyacrylamide gel electrophoresis PAKs p21-activated protein kinases PBD p21-GTPase-binding domain PBS Phosphate buffered saline

PIP2BD PtdIns(4,5)P2 binding domain PCR Polymerase chain reaction PDGF Platelet-derived growth factor PDK1 3-phosphoinositide-dependent kinase 1 PDZ postsynaptic density 95, disk large, zonula occludens PFA Paraformaldehyde PFU Plaque forming unit PH Pleckstrin Homology PHD Plant homeodomain PI(s) Phosphoinositide(s) PI3K(s) Phosphoinositide 3-kinase(s) PI5-Pases Phosphoinositide 5 phosphatases PIKfyve phosphoinositide kinase, FYVE-type zinc finger containing PIP4K(s) Phosphatidylinositol-5-phosphate 4-Kinase(s) PIP5K(s) Phosphatidylinositol-4-phosphate 5-kinase(s) PLO Protein Lipid overlay PM Plasma membrane PP2A Protein phosphatase 2A

PREX PtdIns(3,4,5)P3–dependent Rac exchange factor 2 PTB Phosphotyrosine binding PtdIns Phosphatidylinositol

PtdIns(3,4)P2 Phosphatidylinositol(3,4)-bisphosphate

PtdIns(3,4,5)P3 Phosphatidylinositol(3,4,5)-trisphosphate

PtdIns(3,5)P2 Phosphatidylinositol(3,5)-bisphosphate

PtdIns(4,5)P2 Phosphatidylinositol(4,5)-bisphosphate PtdIns3P Phosphatidylinositol 3-phosphate PtdIns4P Phosphatidylinositol 4-phosphate PtdIns5P Phosphatidylinositol 5-phosphate PTEN Phosphatase and Tensin homolog PVDF Polyvinylidene difluoride PX phox homology Rac Ras-related C3 botulinum toxin substrate RalA Ras like proto-oncogene RBD Ras binding domain RTKs Receptor tyrosine kinases RT-PCR Reverse transcriptase-polymerase chain reaction Sac3 suppressor of actin 3 SDS Sodium dodecyl sulphate SH2 Src homology 2 SHIP1 Src-homology2-containing inositol polyphosphate phosphatases-1 SHIP2 Src-homology2-containing inositol polyphosphate phosphatases-2 SHP-2 Src homology 2-containing tyrosine phosphatase siRNA Small interfering RNA SKIP Skeletal muscle and kidney enriched inositol phosphatase T2D Type 2 diabetes TBAS Tetrabutylammonium Sulphate TBS Tris-buffered saline TEMED Tetramethylethylenediamine TGN Trans-Golgi network Tiam1 T-lymphoma invasion and metastasis TK Tyrosine kinase Vps34 (PIK3C3) vacuolar protein sorting 34

Chapter 1

Introduction

1.1 Introduction

Regulation of glucose homeostasis is a pivotal process for the human body requiring various types of cellular communications. Excess circulating glucose is taken up by skeletal muscle, adipose tissue and liver and stored as glycogen or triglycerides. The body needs a rapid and an efficient process to control the level of glucose particularly after meals (post-prandial). High glucose triggers pancreatic β cells to secrete insulin to enhance regulation of glucose. Skeletal muscle is the principal tissue of disposal glucose where about 90% of insulin dependent glucose uptake is taken up (Leto and Saltiel, 2012). Failure or reduction of the ability of these cells to uptake glucose in response to insulin is an early step of developing insulin resistance and hence the growing global problem, type 2 diabetes (T2D) mellitus (Saltiel and Kahn, 2001). Consequently, hyperglycaemia is developed while the intracellular glucose is dropped, to an estimated 50 times less in T2D compared to healthy individuals (Petersen and Shulman, 2006). At the cellular level, insulin resistance is attributed to inadequate signals from the insulin receptor downstream to the final effectors of insulin action. It has been reported that defects in skeletal muscle cells is the main determinant of whole body of insulin resistance (DeFronzo and Tripathy, 2009). Several impairments in the insulin signalling mechanism have been found to contribute to the pathological progress of T2D (Rask-Madsen and Kahn, 2012). Insulin signalling is a complex pathway with various incompletely characterised interactions between many of the molecules and the modulators of this pathway. Understanding the role of these regulators becomes of upmost importance for treating and controlling the T2D disease. This thesis discusses the role of one type of phospholipid that plays an essential part in the transduction of insulin signalling, the phosphoinositides (PIs).

1.1.1 Glucose uptake

Insulin plays different roles and activates several pathways that lead to increase glucose permeability of fat and muscle cells, allowing them to uptake and metabolise glucose. Insulin initiates a cascade of multi-stepped processes that trigger insulin sensitive cells such as skeletal muscle cells and adipocytes to redistribute a specific glucose protein transporter from its intracellular storage sites to the cell surface to increase glucose uptake. Glucose uptake is an example of one of the results of the activation of the insulin signalling pathway. Cascades of phosphorylation and dephosphorylation steps are initiated by specific kinases and phosphatases in response to insulin. Induction of glucose uptake by insulin is conducted by phosphoinositide 3 kinase (PI3K) dependent and independent pathways (Watson et al., 2004; Saltiel and Pessin, 2003). In the skeletal muscle and fat tissues, glucose uptake is regulated by three main processes. Firstly, by glucose delivery through the blood flow, secondly, by glucose transport which is controlled by GLUT1 under basal conditions and by GLUT4 after

18 insulin stimulation, and thirdly by glucose metabolism where glucose is converted to glucose-6- phosphate by hexokinase II (Deshmukh, 2016).

1.1.2 Glucose transporter 4

Glucose transporters (GLUTs) comprise 14 members which are expressed in different cells (Leto and Saltiel, 2012). Not all of these transporters are sensitive to insulin. In human skeletal muscle, the mRNA of GLUT4, GLUT5, GLUT12, GLUT8, GLUT11, GLUT3 and GLUT1 have all been detected (Stuart et al., 2006). Glucose transporter 4 (GLUT4) is the primary insulin- sensitive transporter that is highly expressed in skeletal muscles and it plays a crucial role in insulin- and contraction- dependent regulation of glucose. Similar to other GLUT-isoforms, GLUT4 contains 12 transmembrane domains where the N and C termini are exposed to the cytosol (Huang and Czech, 2007). The newly synthesised GLUT4 is recycled in the intracellular membrane system where the majority of GLUT4 is distributed between endosomes, the trans- Golgi network (TGN) and GLUT4 storage vesicles (GSVs) (Leto and Saltiel, 2012) The presence of GLUT4 at the plasma membrane is regulated mainly by the net rate of exocytosis and endocytosis processes. Insulin redistributes GLUT4 to the plasma membrane by increasing exocytosis and probably by decreasing endocytosis (Leto and Saltiel, 2012). Insulin for instance enhances GSVs translocation, docking and fusion with the plasma membrane which increase the net presence of GLUT4 at the cell surface and hence increases glucose uptake (Huang and Czech, 2007).

1.1.3 Insulin signalling

The process of insulin-dependent glucose uptake in insulin sensitive cells such as skeletal muscle and adipocytes begins when insulin binds to the insulin receptor (IR) at the plasma membrane (Figure 1.1). IR is a trans-membrane protein that belongs to the receptor tyrosine kinase (RTK) superfamily. IR is a α2β2 heterotetrameric protein composed of two extracellular α subunits and two transmembrane β-subunits linked together by disulphide bonds. Insulin interacts with the α-subunits while tyrosine kinase domains are located in the intracellular part of the β-subunits. IR is encoded by the INSR gene and it is expressed as two isomers (IR-A and IR-B) as a result of alternative splicing (Zaid et al., 2008). The two isoforms are expressed in distinct cells. For example, IR-A is reported to be expressed in brain, haematopoietic cells, foetal tissue and the placenta whereas IR-B is predominantly expressed in skeletal muscle, adipose tissue and liver (Frasca et al., 2008). IR-B has an extra 12-amino acid segment in the α-subunit. In mice, muscle specific IR knockouts show normal glucose homeostasis and insulin levels in blood, however, the fat mass and insulin–stimulated glucose uptake in adipose tissue is increased in these animals (Brüning et al., 1998). The insulin–independent glucose uptake upon

19 exercise was found to be intact in muscles of these mice (Wojtaszewski et al., 1999). This shows that insulin-independent signalling and fat tissue can compensate for the loss of specific insulin- dependent glucose uptake in muscle and indicates interaction between these tissues.

Figure 1. 1: Insulin signalling in muscle cells (A) and Schematic structure (B) of IRS1, PAK and AKT.

(A) Insulin binds to the IR which interacts with IRS that in turn works as an adaptor to activate

PI3K. PI3K initiates the metabolism of PIs and generates PtdIns(3,4,5)P3. PtdIns(3,4,5)P3 then activates the AKT and Rac1 pathways that lead to translocation of GLUT4 from the intracellular storage vesicles to the plasma membrane, which enhances glucose uptake. (B) Schematic structure of the main domains of IRS and PAK1 are shown. Additionally, the structure of AKT1 and AKT2 isoforms and the positions of activating phosphorylation sites (Threonine and Serine by PDK1 and mTORC2 respectively) are represented for each isoform. Abbreviations: PH (Plekstrin Homology), PHD (Plant Homeo Domain), PTB (phosphotyrosine binding), PBD (p21-GTPase-binding domain), AID (autoinhibitory domain).

1.1.4 Insulin Receptor substrates (IRS)

Upon binding of insulin to the extracellular α subunits of the IR, conformational changes are induced leading to activation of the tyrosine kinase (TK) domain. The TK then promotes auto- phosphorylation of different tyrosine residues of the intracellular part of the IR and also phosphorylates intracellular substrates such as insulin receptor substrate (IRS) proteins. IRS proteins are cytoplasmic adaptor proteins without catalytic activity (Sun et al., 1991). Several IRS isoforms are reported but IRS-1 and IRS-2 are the main mediators of insulin-dependent signalling. IRS-1 and 2 are widely expressed and are the most abundant IR substrates in muscle cells (Kruszynska et al., 2002). They have a PH domain, and a phosphotyrosine binding (PTB) domain in addition to several effector binding sites that interact with several proteins such as PI3K, the adaptor protein Grb2 and the protein-tyrosine phosphatase SHP2 (Mardilovich et al., 2009) (Figure 1.1 B). The PH domain of IRS1 has been suggested to facilitate binding of IRS-1 to membrane phospholipids that causes the IRS to locate to the vicinity of transmembrane receptors (Voliovitch et al., 1995). The PTB domain is thought to be the major way of recruiting IRS to the IR by interacting with NPXY (Asn- Pro-X- Tyr) motifs in the activated receptors (Mardilovich et al., 2009). IRS itself is regulated by tyrosine, serine and threonine phosphorylation that serves as a mechanism for negative-feedback inhibition of insulin signalling (Jean and Kiger, 2012).

IRS proteins serve as an adaptor protein needed for activating phosphoinositide 3 kinase (PI3K) (see section 1.2.2.1 for more details of PI3K isoforms and structures). Activated IRS recruits and activates PI3K via an SRC homology 2 (SH2) domain (Felder et al., 1993) of the regulatory (p85) subunit of PI3K which activates the catalytic subunit of PI3K, p110. It has been observed that reducing IRS-1 expression causes insulin resistance in muscle cells (Kruszynska et al., 2002) and in adipocytes (Pirola et al., 2003).

1.1.5 PI3K downstream effectors

The initiation of insulin signalling activates PI3K that leads to the production of the phospholipid

PtdIns(3,4,5)P3 which in turn works as a second messenger in various pathways leading to the translocation of GLUT4 from its intracellular storage location to the cell surface. Two main pathways downstream of PI3K/PtdIns(3,4,5)P3, AKT and the small GTPase-protein Rac1, are proposed to affect and regulate the process of translocating GLUT4 and thus glucose uptake particularly in muscle cells (Figure 1.1).

1.1.5.1 AKT

AKT (also commonly known as protein kinase B (PKB)) is a serine/ threonine protein kinase and it is a crucial protein involved in different signalling pathways. More than 100 substrates of AKT have been identified (Manning and Cantley, 2007; Vanhaesebroeck et al., 2012). AKT phosphorylates and regulates various important proteins and therefore it is involved in the control of cell growth, survival, apoptosis, proliferation and angiogenesis in addition to its role in the metabolism of carbohydrates, lipids and proteins (Zheng and Cartee, 2016).

AKT has 3 main isoforms (AKT1, AKT2, AKT3) that are encoded by different genes on different chromosomes (Manning and Cantley, 2007). AKT1 and AKT2 are ubiquitously expressed whereas AKT3 is found primarily in brain, lung and testis (Konishi et al., 1995) but is barely expressed in muscle cells (Manning and Cantley, 2007; Manning and Toker, 2017). AKT has three main domains (Figure 1.1B); an N- terminal PH domain that mediates binding to

PtdIns(3,4,5)P2, a central kinase domain containing a threonine residue (T308 or T309 for AKT1 or AKT2 respectively) and a carboxyl-terminal regulatory domain that has a hydrophobic motif (HM) containing a serine residue (S473 or 474 for AKT1 or AKT2 respectively) (Manning and Toker, 2017).

AKT is recruited the plasma membrane and bound to PtdIns(3,4,5)P3 and the related lipid

PtdIns(3,4)P2 via its PH domain. Upon PtdIns(3,4,5)P3 and/or PtdIns(3,4)P2 binding to the PH domain of AKT, a conformational change is induced in AKT which is then phosphorylated by the phosphoinositide dependent protein kinase (PDK1) at Thr308/9 (Alessi et al., 1996). A second phosphorylation at Ser473/4 of the AKT by mTORC2 (Riaz et al., 2012) is required to achieve full activation of AKT (Sarbassov, 2005; Alessi et al., 1996) (Figure 1.1B).

It has been reported that in skeletal muscle cells AKT1 is found in the cytoplasm and very little of it redistributed to the plasma membrane after insulin stimulation. AKT2 is located in the cytoplasmic compartments and associated with mitochondrial and nuclear fractions but it is also detected at the plasma membrane and insulin treatment significantly increases the presence of AKT2 at the plasma membrane (Zheng and Cartee, 2016). These data indicate that AKT2 is more likely to be involved in insulin signalling as has been documented in AKT knockout mice.

Total AKT1 knockout does not alter glucose levels in mice (Cho et al., 2001b) and it has been suggested that AKT1 is required for regulating growth and controlling cell and body size rather than glucose homeostasis (Cho et al., 2001b). However, in AKT2 knockout mice a diabetic phenotype (Cho et al., 2001a) and a mild growth deficiency (Garofalo et al., 2003) has been described. Insulin signalling was impaired in the liver and in the skeletal muscle of these animals which is reflected as inability of insulin to reduce blood glucose (Cho et al., 2001b; Garofalo et al., 2003). Thus, GLUT4 translocation seems to be controlled mainly by AKT2 rather than AKT1

22 as shown in adipocytes and muscle cells (Zheng and Cartee, 2016; Jiang et al., 2003; Bae et al., 2003; Nozaki et al., 2013).

One important AKT substrate that affects GLUT4 translocation (Sano, 2003) by negative regulation of GLUT4 exocytosis is a GAP protein, AKT substrate of 160 kDa (AS160). AKT2 phosphorylates AS160 which inhibits its GAP activity. This in turn activates GTP-bound Rab proteins (GTP-Rab) (Larance et al., 2005). GTP-Rab facilitates and promotes GLUT4 translocation and fusion with the plasma membrane (Ishikura and Klip, 2008). Mutant AS160 that lacks an AKT binding site prevents GLUT4 translocation (Randhawa et al., 2008). However, GLUT4 translocation is not completely an AS160-dependent process since depleting AS160 by siRNA only partially redistributes GLUT4 to the plasma membrane, which is correlated with the increase in GLUT4 exocytosis (by 3-fold) as seen in unstimulated adipocytes (Larance et al., 2005; Eguez et al., 2005). However, under insulin stimulation of AS160 knockdown-cells, a small reduction of GLUT4 at the plasma membrane is documented (Eguez et al., 2005), which may indicate a role of AS160 in maintaining GLUT4 in the cell surface of insulin-stimulated cells.

1.1.5.2 Ras-related C3 botulinum toxin substrate 1 (Rac1)

Rac1 is a small GTPase-protein (about 21 kD) consisting of 192 residues, belonging to the Rho subfamily of the small GTPases. Rho also includes the cell division control protein 42 homolog (Cdc42) and Rho protein. The Rho family is part of the larger Ras superfamily (Hodge and Ridley, 2016).

Rac1 is one of three isoforms; Rac1, Rac2, and Rac3 which are encoded by different genes. Unlike Rac1 which is expressed in all cells, Rac2 is only detected in hematopoietic cells, while Rac3 is predominantly localized in the perinuclear region of brain cells (Hajdo-Milasinović et al., 2007). These isoforms seem to have different, non-redundant roles. Overexpression of Rac1 induces cell spreading, whereas Rac3 overexpression results in contractile, round morphology neuronal cells (Hajdo-Milasinović et al., 2007).

Rac1 is activated by various cell surface receptors including the IR. Active Rac1 can then activate and regulate a range of cellular functions including motility, morphology (via cytoskeleton arrangement) and gene expression. Thus, dysfunction of Rac is associated with numerous disorders including cancers, cardiovascular and neurological diseases and diabetes. The important role of Rac1 in regulating the insulin-dependent GLUT4 translocation has been clearly described using skeletal muscle-specific Rac1-knockout mice. In this model GLUT4 translocation is significantly suppressed in comparison to control mice (Ueda et al., 2010).

1.1.5.2.1 Regulation of Rac1 activity

Similar to other small GTPases, Rac1 proteins are cycled between the inactive guanosine 5- diphosphate (GDP)-bound and the active guanosine 5-triphosphate (GTP)-bound state (Figure 1.2). This cycle is regulated by three groups of proteins; guanine nucleotide exchange factor (GEFs), GTPase-activating proteins (GAPs) and guanine nucleotide dissociation inhibitors (GDIs) (Hodge and Ridley, 2016).

More than 30 GEFs are known to affect Rac1 (Bos et al., 2007). GEFs are proteins involved in activating GTPase proteins by promoting the exchange of bound GDP for free GTP. GEFs are activated by a wide range of signalling pathways including insulin’s. GAPs negatively regulate GTPase protein action by increasing the GTP hydrolysis activity that results in the inactive GDP- bound form of the GTPase and inorganic phosphate. On the other hand, GDIs, which also inhibit GTPase activation, act by sequestering GDP-bound GTPases in the cytosol, preventing them from reaching the membranes where their GEFs are located (Kawano et al., 2014).

Figure 1. 2: Regulation of Rac1 by GDP-GTP exchange cycle. The Rho GTPase-proteins cycle such as of Rac1 consists of three main regulators; guanine nucleotide exchange factor (GEFs), GTPase-activating proteins (GAPs) and guanine nucleotide dissociation inhibitors (GDIs). GEFs such as VAV, Tiam1, FLJ00068, PREX2 activate Rac1 by catalysing the exchange of GDP for GTP. GAPs inactivate the GTPase-proteins by enhancing the intrinsic GTPase activity of them. GDI sequester GDP-bound Rho GTPases in the cytoplasm.

1.1.5.2.2 Rac1-GEFs

VAV2, FLJ00068, T-lymphoma invasion and metastasis (Tiam1) and PtdIns(3,4,5)P3–dependent Rac exchange factor 2 (PREX2) are examples of GEFs that have been found to have roles in activating Rac1 in insulin signalling, though it seems that their role is cell type specific. Overexpressing VAV2 in fibroblasts for example, is found to activate Rac1 and enhances the formation of membrane ruffles and stress fibres (Liu and Burridge, 2000). However, although VAV2 is expressed in L6 myoblasts, it was totally incapable of enhancing insulin-dependent GLUT4 translocation (Ueda et al., 2008).

FLJ00068 is a GEF whose role in activating Rac1 has been demonstrated by Ueda et al. In their study, Rac1 activation by ectopic expression of FLJ00068 in the absence of insulin is sufficient for inducing glucose uptake in L6 cells (Ueda et al., 2008). The role of FLJ00068 was then described in skeletal muscle cells. Silencing FLJ00068 by siRNA impairs Rac1 activity and the process of GLUT4 translocation is blocked (Takenaka et al., 2016). FLJ00068 is reported to be activated downstream of AKT2 in myoblasts since its activity is prevented by inhibiting AKT2 activity (Takenaka et al., 2016; Takenaka et al., 2014).

Tiam1 is another important GEF that is widely expressed, being found in brain, testes and skeletal muscle cells. Tiam1 is found to interact and become activated by PIs such as PtdIns5P (section 1.3.7). This interaction is suggested to have a role in localising Tiam1 to specific sites, leading to Rac1 activation (Viaud et al., 2014). Tiam1 overexpression activates Rac1 and results in a net gain of GLUT4 at the cell surface of L6 myoblasts in the absence of insulin (Chiu et al., 2013).

PREX2 is one more GEF that regulates Rac1 activity. PREX2 is widely expressed and has been detected in skeletal muscle, heart and placenta (Donald et al., 2004). PREX2 is activated by

PtdIns(3,4,5)P3 and by β and γ subunits of G-proteins (Gβγ) which is released upon activation of G protein- coupled receptors (GPCRs). A negative feedback loop that involved PREX2 which prevents over-activation of Rac1 was proposed and has been tested in HEK293 cells (Barrows et al., 2015). PtdIns(3,4,5)P3 and/or Gβγ recruit and activate PREX2 which enhances the activation of Rac1. Rac1 activates the kinase PAK. PAK in turn was found to be able to phosphorylate PREX2. This phosphorylation prevents PREX2 from binding again to

PtdIns(3,4,5)P3 and hence its reactivation is blocked. This leads to Rac1 inactivation due to the decrease of PREX2 GEF activity (Barrows et al., 2015).

1.1.5.2.3 Rac1-effectors

1.1.5.2.3.1 RalA

Active Rac1 activates various proteins including GTPase proteins such as the Ras like proto- oncogene (RalA) (Nozaki et al., 2012). Expressing either mutant active Rac1 or FLJ00068 activates RalA in L6 myoblasts as well as in mouse gastrocnemius muscle cells (Takenaka et al., 2016). Active RalA enhances GLUT4 translocation downstream of Rac1 since active Rac1 in cells with inactive mutant RalA or where RalA is silenced by siRNA show attenuation of the process of distributing GLUT4 to the plasma membrane (Nozaki et al., 2012; Takenaka et al., 2016).

1.1.5.2.3.2 p21-activated protein kinases (PAKs)

PAKs are serine/threonine kinases involved in different cellular activities including glucose homeostasis (Chiang and Jin, 2014). PAKs are among the most important downstream effectors of Rac1. This family has six mammalian isoforms PAK1-6 (Wells and Jones, 2010). In skeletal muscle cells, the PAK1 isoform was found to have a significant role in insulin-stimulated GLUT4 vesicle translocation (Tunduguru et al., 2014). Using PAK-inhibitor (IPA3) that prevents PAK1 from binding to its downstream effectors, significantly inhibit insulin-stimulated GLUT4 translocation and glucose uptake in L6 cells (Tunduguru et al., 2014). In muscle cells, PAK1 is activated by the small GTP binding proteins Cdc42 and Rac1 (Taglieri et al., 2014). PAK1 contains three main domains: p21-GTPase-binding domain (PBD) where Rac1 and Cdc42 are bound, autoinhibitory domain (AID) and a serine/threonine-kinase domain (Figure 1.1 B).

It has been proposed that following the binding of Rac1 or other p21 GTPases to the PBD, PAK1 dimerization is disturbed. Under resting condition, PAK activity is masked by the auto-inhibitory region of PAK1 in trans-through the formation of heterodimers. This inactive, dimerised PAK1 undergoes conformational changes after stimulation and Rac1 binding which activates its kinase domain, followed by auto-phosphorylation events (Parrini et al., 2002). Among the known seven auto-phosphorylation sites, phosphorylation of Thr423 in the kinase domain is proposed to be a key event of PAK1 activation which has been reported after Rac1 binding (JeBailey et al., 2007). Stimulating myoblasts with insulin leads to activating PAK1 in a PI3K dependent manner (Tsakiridis et al., 1996), and PAK1 knockout mice showed insulin resistance in skeletal muscles coupled to a defect in GLUT4 translocation (Wang et al., 2011). PAK1 activation via Rac1 is also involved in cytoskeleton rearrangements, and stimulating cells by insulin re-distributes PAK1 from the cytosol to cortical actin structures (Tunduguru et al., 2014). Furthermore, PAK1 but not PAK2 inhibition completely prevents insulin-dependent GLUT4 translocation and glucose uptake

26 in L6 myoblasts (Tunduguru et al., 2014). These data suggest an important role of PAK in insulin-dependent glucose uptake.

1.1.6 Roles of Cytoskeleton rearrangements in insulin-stimulated glucose uptake

One crucial event in the insulin signalling process of glucose uptake is the rearrangement of the cytoskeleton of the cell. This rearrangement is important for transferring GLUT4 from its intracellular storage compartments as well as for the retention of GLUT4 vesicles beneath the plasma membrane. Two types of cytoskeleton rearrangement are reported to be critical for this process; formation of microtubules and rearrangement of actin filaments (Satoh, 2014). GSVs move along the microtubules by interaction with kinesin proteins such as KIF3 and KIF5b whereas myosin proteins are important for transporting GSVs along actin filaments (Satoh, 2014). Actin filaments provide a path to transport GSVs whereas cortical actin remodelling is important not only for retention of GLUT4 beneath the membrane but also for the process of fusion of GLUT4 vesicles to the plasma membrane (Randhawa et al., 2008). Several studies show that Rac1 is involved in some of these processes, particularly in actin rearrangements.

The important role of Rac1 in insulin-stimulated cytoskeleton rearrangements and GLUT4 translocation was initially proposed in cell culture (Brozinick et al., 2004; Khayat et al., 2000). This role has been then subsequently described in several studies in skeletal muscles (Chiu et al., 2011; JeBailey et al., 2004; Sylow et al., 2013b; Ueda et al., 2008; Ueda et al., 2010; Nozaki et al., 2013; Takenaka et al., 2014).

1.2 Phosphoinositides

In several places in the preceding sections on insulin-stimulated glucose uptake involvement of phospholipids of the PI family has been mentioned. In this section, a more detailed introduction to these molecules and their role in insulin signalling will be provided.

Phosphatidylinositol (PtdIns) and its phosphorylated forms, the phosphatidylinositol phosphates or phosphoinositides (PIs) represent a family of lipids that is found primarily attached to different membranes of eukaryotic cells. PIs form only a small fraction of cell membranes but play crucial functions and they are involved in numerous intracellular pathways (Bridges and Saltiel, 2015; Viaud et al., 2016; Balla, 2013; Sasaki et al., 2009). In addition to their housekeeping function, PIs act as secondary messengers in which they transduce and regulate cell signalling. Due to their high diversity and many regulatory enzymes of these lipids, the literature review chapter of this thesis will focus on the metabolism of PIs and their regulatory pathways in addition to the involvement of PIs in insulin signalling with particular focus on glucose uptake in skeletal muscle cells, the experimental system used in this research project.

1.2.1 Characteristics and Synthesis of PIs

PIs consist of a six-carbon hydrophilic polar head, the myo-inositol ring, linked by a phosphodiester bond to a tail of diacylglycerol (DAG) (Figure 1.3 A). DAG is composed of two hydrophobic non polar fatty acids chains attached to a glycerol molecule. One of the chains is usually a saturated fatty acid (often stearic acid), while the other one is predominantly an unsaturated fatty acid such as arachidonic acid (Shah et al., 2013; Viaud et al., 2016). Recent studies have identified several combinations of fatty acyl species. However, it is not yet clear if the differences in the fatty acid tails have any impact on the function or location of PIs (Shah et al., 2013). These two fatty acid chains anchor PIs in various membranes of cells including the plasma membrane, Golgi apparatus, endoplasmic reticulum (ER), and lysosomes as well as in the nucleus (Shah et al., 2013).

PIs are generated from PtdIns which is synthesized by PtdIns synthase in the endoplasmic reticulum from DAG and myo-inositol (Balla, 2013). PtdIns is then translocated to other cellular membranes by PI transfer proteins or possibly via vesicular trafficking through the cell (Balla, 2013). Specific PI-kinases and phosphatases reversibly generate and interconvert seven distinct PIs by phosphorylating or dephosphorylating three hydroxyl groups of the inositol ring, positions D3, D4, and D5, in different combinations (Figure 1.3). PIs are classified as mono- phosphoinositides (PtdIns3P, PtdIns4P, PtdIns5P), bis-phosphoinositides (PtdIns(3,4)P2,

PtdIns(3,5)P2, PtdIns(4,5)P2 (also known as PIP2) and one tris-phosphoinositide

(PtdIns(3,4,5)P3 (also known as PIP3). The contribution of each one of these PIs in insulin signalling will be discussed later (section 1.3 ).

Figure 1. 3: Structure (A) and metabolism (B) of PIs.

(A):The chemical structure of PtdIns is composed of a cytoplasmic myo-inositol polar head- group (The hydroxyl groups at D3, D4, and D5 positions are phosphorylated or dephosphorylated by specific kinases and phosphatases) attached to the non-polar DAG tail. DAG has two fatty acids inserted into the inner leaflet of lipid bilayer membranes. (B): The metabolism and interconversion pathways of PIs by the different enzymes in mammalian cells are shown. PI Kinases (solid arrows), PI Phosphatases (dashed arrow), phospholipase C (grey arrow).

The relative amounts of PIs vary significantly among cell types and among intracellular compartments. PIs are sensitive to various stimuli including hormones and growth factors and therefore the relative amounts of different PIs also changes upon cellular condition and stimulation. The precursor PtdIns is highly abundant compared to all other PIs. PtdIns4P and

PtdIns(4,5)P2 are present at the highest level while PtdIns(3,4,5)P3, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns3P and PtdIns5P exist in very low quantities (Table 1.1). Some PIs such as

PtdIns(3,4,5)P3 and PtdIns5P are in many cell types is very low and only increase robustly after stimulation with growth factors like insulin, or infection, oxidative stress and other physical stimuli (De Craene et al., 2017).

Although PIs form a minor component of membranes, comprising less than 1% of total cellular lipid (Lemmon, 2008), the seven PIs play diverse roles in membrane trafficking, regulation of ion channels, cell adhesion, endo- and exocytosis, auto-phagocytosis, transcription, RNA maturation and cell survival (Fiume et al., 2015). The multiple functions associated with PIs are attributed to the fact that they can bind to a wide range of different proteins through specific binding domains (Balla, 2013). The estimated levels of PIs (Shisheva et al., 2015) and their binding domains (Kutateladze et al., 2010; Viaud et al., 2016) and their main location (Viaud et al., 2016) in intracellular compartments are shown in Table 1.1.

Table 1. 1: Relative levels and the main binding domains and localisation of PIs in mammalian cells.

Relative level PI binding protein Main localisation PI (% PtdIns) domain

Golgi apparatus, PM, PtdIns4P 5 PH, PTB, PX, Endosomes PtdIns3P 0.25 FYVE, PX, PH Endosomes, PM PM, Golgi apparatus, PtdIns5P 0.25 PHD Nucleus PM, Endosomes, PH, PTB, PX, C2, PtdIns(4,5)P 5 Golgi apparatus, 2 ENTH, PDZ Nucleus Endosomes, PtdIns(3,5)P 0.025 PH, PROPPINs 2 Lysosomes PtdIns(3,4)P2 0.005 PH, PX PM, Endosomes PtdIns(3,4,5)P3 0.005 PH, PX, C2 PM Abbreviations: C2 (conserved region-2 of protein kinase C), PH (Plekstrin Homology), FYVE finger (Fab1p, YOTB, Vac1p and EEA1), ENTH (Epsin N-Terminal Homology), PX (Phox homology) and PHD (Plant Homeo Domain), PTB (phosphotyrosine binding) ), PDZ (postsynaptic density 95, disk large, zonula occludens), PROPPINs (β-propellers that bind PIs), PM (plasma membrane).

Although mono-phosphoinositides were initially considered only to serve as a precursor for the other PIs, it turns out that all PIs including this subgroup have specific functions on their own. For example, individual PIs act as second messengers by translocating signal transduction down to their effectors. The specific binding domains (see Table 1.1) by which the PIs interact with effectors serves as a mean of regulating the location and the activity of different proteins that play a critical role in signalling pathways (Lemmon, 2008). For example, PtdIns(3,4,5)P3 interacts and activates the PH domain containing proteins such as the kinase AKT (section 1.1.5.1) (Viaud et al., 2016).

The imbalance of PI levels affects many aspects of metabolism which are seen as diseases or abnormalities such as in cancer or metabolic disease, as seen in diabetes. Similarly; infection, inflammation, neurological diseases such as Alzheimer's, Parkinson's, epilepsy, and inherited diseases like myotubular myopathy and Lowe syndrome are all examples where the levels of PIs are disturbed (Bridges and Saltiel, 2012; Waugh, 2015).

1.2.2 Enzymes involved in PI metabolism

PIs are regulated by different kinases, phosphatases and phospholipases (Figure 1.3B) which together maintain the balance and the tight regulation between them. The role and the exact contribution of some of these enzymes are poorly understood. In many cases these enzymes share common substrates; however the specific location and presence of either the substrate (PtdIns or PIs) or the enzymes determine the impact on these lipids. In this thesis the main kinases and phosphatases that are involved in the regulation of PtdIns5P and PtdIns(3,4,5)P3 axis will be discussed in more detail, as the research project mainly focuses on these PIs.

1.2.2.1 PI3Ks PI3Ks form a family of kinases that phosphorylate the hydroxyl group at the D-3 position of the inositol ring of PtdIns and/or PIs (Jean and Kiger, 2014). PI3Ks are categorised according to their catalytic subunits into three main classes (Class: I, II and III) (Figure 1.4). Class I is further sub-divided into Class IA and class IB (see Table 1.2 for the details of catalytic and regulatory subunits and the combinations of each subunit in each class). All PI3Ks have kinase domains in the catalytic subunits (Class IA: p110α, β or δ; Class IB p110γ; Class II: PI3K-C2 (α, β or γ) and vps34 in Class III). Class I and III have additionally regulatory or adaptor subunits (p85, p55 or p101 for Class I and vps15 which is specifically for Class III) making these kinases (Classes I and III) heterodimeric enzymes (Vanhaesebroeck et al., 2012). The core function of PI3K as an enzyme depends on the catalytic subunit which contains the catalytic site and a C2 domain that is common to all three classes of PI3K (Figure 1.4). Beside the PI3K core, the catalytic subunit of Classes I and II have also a Ras binding domain (RBD) where GTPase proteins might

31 activate them (Jean and Kiger, 2014; Vanhaesebroeck et al., 2010). The different subtypes of PI3Ks have distinct substrates and products (see below).

In general, PI3Ks are activated by RTKs such as the IR, or their phosphorylated substrates (for example: IRS1) (Vanhaesebroeck et al., 2010). PI3Ks might also be activated by GPCRs and possibly by small GTPases (Vanhaesebroeck et al., 2010). PI3Ks are ubiquitously expressed in eukaryotes, regulating various essential intracellular processes such as cell signalling and membrane trafficking which affect cell growth, proliferation, differentiation, motility, survival and intracellular trafficking. Most PI3K functions are mediated by phosphoinositides (Jean and Kiger, 2014).

Figure 1. 4: Schematic diagram of the catalytic subunits of PI3K classes.

According to the catalytic structure and substrate specificity, PI3Ks are classified into three groups (for details see text). ABD Adaptor binding domain (or p85-bindng domain), RBD; Ras- binding domain, C2 protein-kinase-C homology-2, a helical domain and a catalytic domain. PRD; Proline rich domain, PX; Phox homology.

Table 1. 2: Summary of the genes and proteins encoded for the PI3K family members and their phospholipid substrates.

Catalytic subunits Regulatory subunits Lipid Class of PI3K Gene Protein Gene Protein substrate PIK3R1 P85α PIK3CA P110α PIK3R2 P85β Class IA PIK3CB P110β PIK3R1 P55α PtdIns(4,5)P PIK3R1 P50α 2 PIK3CD P110δ PIK3R3 P55γ PIK3R5 P101 Class IB PIK3CG P110γ PIK3R6 P87 PIK3C2A PI3KC2α PtdIns Class II PIK3C2B PI3KC2β PtdIns4P PIK3C2G PI3KC2γ Class III PIK3C3 VPS34 PIK3R4 P150 (VPS15) PtdIns

1.2.2.1.1 Class I PI3Ks

Class I PI3K is the class that in vivo can only generate the potent lipid PtdIns(3,4,5)P3 from

PtdIns(4,5)P2. PtdIns3P and PtdIns(3,4)P2 have also been reported as a product of class I PI3K in vitro (Vanhaesebroeck et al., 2010). Class I is made of one catalytic subunit of 110 kDa and one regulatory subunit of variable size (Table 1.2). Depending on the catalytic subunits, Class I can be subdivided into class IA (p110: α, β and δ) and class IB (p110: γ) which are encoded by the genes PIK3CA, PIK3CB, PIK3CD and PIK3CG respectively (Fougerat et al., 2009; Viaud et al., 2016). The regulatory subunits of Class IA include; p55, p85, p50 whereas p101 and p87 (also known as p84) are regulatory subunits of Class IB PI3Ks. These subunits are encoded by different genes as shown in Table 1.2.

The catalytic subunits (p110α, p110β, and p110δ) bind to any of the five regulatory subunit isoforms (p85α, p55α, p50α, p85β, and p55γ). Unlike p110 α and β which are expressed in almost all cells, p110δ and γ are mainly found in immune cells (Sasaki et al., 2009). Class IA but not IB can be activated by RTK while Ras and G-protein-coupled receptors (GPCR) are thought to regulate both sub-classes (Vanhaesebroeck et al., 2010).

The regulatory subunit of Class I PI3Ks has an important role in regulating the activity of the catalytic kinase. The regulatory subunit also participates in the localisation and translocation of

the PI3K to the plasma membrane. This translocation is a crucial step for PtdIns(3,4,5)P3 production (Vanhaesebroeck et al., 2010). Different studies have emphasised the importance of the regulatory subunit in affecting the overall role of PI3K. For example, globally deleting

PIK3R1 and hence all three isoforms of the encoded Class IA regulatory subunit (p85α, p55α, and p50α) results in a perinatal lethal effect (Fruman et al., 2000). Mice that have specific- skeletal muscle knockout of all forms of the regulatory subunits of class IA develop glucose intolerance and insulin resistance (Luo et al., 2006). However, several studies have shown improvement in the PI3K signalling as seen in the upregulation of AKT in addition to the enhancement of insulin dependent phosphorylation of IRS in mice lacking one form of the regulatory subunit such as p50α, p55α, p85β or p85α (Chen et al., 2004; Ueki et al., 2002; Mauvais-Jarvis et al., 2002). In fact some studies have also showed that p85 might be a signalling protein in its own right, independent of the catalytic subunit by interacting and activating small GTPase proteins such as Cdc42 and Rac1 (Jiménez et al., 2000; Kang et al., 2002).

1.2.2.1.2 Class II PI3Ks Class II kinases have three catalytic core isoforms (PI3K: C2α, C2β and C2γ) without a regulatory subunit (Figure 1.4). Based on Northern blot analysis of different human tissues, PI3KC2α is found to be highly expressed in heart, placenta, and ovary (Domin et al., 1997). In general, class II PI3Ks are predominantly and constitutively associated with intracellular membranes with low levels in the cytosol (Vanhaesebroeck et al., 2010). PI3KC2α is localised in the Golgi apparatus, the nucleus and the endosome. One property that distinguishes PI3KC2α from other PI3Ks is its high resistance to the inhibitors wortmannin and LY294002 ((Domin et al., 1997; Maffucci et al., 2003; Falasca and Maffucci, 2012), which both potently inhibit other classes of PI3K. PI3KC2β is also widely expressed in different cell types but it is distributed in the cytoplasm and around the nucleus. PI3KC2γ is expressed mainly in liver (Sasaki et al., 2009; Vicinanza et al., 2008). All Class II PI3Ks have in their carboxyl end a Phox homology (PX) domain by which they interact with PtdIns(4,5)P2 (Stahelin et al., 2006) and to a lesser extent with other PIs (Falasca and Maffucci, 2012).

The exact product of this class in vivo is not fully resolved. It has been reported that in vitro class

II PI3K can generate both PtdIns3P and PtdIns(3,4)P2 (MacDougall et al., 1995; Falasca and

Maffucci, 2012), whilst it is generally accepted that Class II do not produce PtdIns(3,4,5)P3 (Falasca et al., 2007; Falasca and Maffucci, 2012). Although it has been shown that the main product of PI3KC2α and PI3KC2β is PtdIns3P (Falasca et al., 2007; Maffucci, 2005), evidence from other studies indicates that class II PI3K can also synthesise PtdIns(3,4)P2 (Posor et al., 2013).

Under insulin stimulation of L6 myoblasts, production of PtdIns3P is mediated mainly by PI3KC2α since when this kinase is knocked-down, these cells show significant reduction in the synthesis of PtdIns3P (Falasca,et al., 2007). A recent study using kinase-dead class II PI3K-C2β

(to discriminate between scaffold- and kinase-dependent functions) in mice showed that class II PI3K is important in insulin dependent endosomal trafficking. These mice displayed higher insulin sensitivity and glucose tolerance compared to control mice. Insulin-dependent AKT signalling is enhanced in liver, muscle, and white adipose tissue of these animals (Alliouachene et al., 2015). The authors suggest that in hepatocytes decreasing the production of PtdIns3P at endosomes, by inhibiting PI3KC2β, increases insulin signalling via affecting IR trafficking that increases overall IR levels at the cell surface. Whether other forms of class II share same mechanism in other cell types is not clear.

1.2.2.1.3 Class III PI3K Class III PI3K (also known as Vps34) is expressed in all eukaryotic cells and it only produces PtdIns3P in vivo (Backer, 2008). In mammals, the majority of PtdIns3P is thought to be generated by the type III PI3K. The structure of class III PI3K shows that it contains a regulatory (Vps15 or p150) subunit and a catalytic (Vsp34) subunit (Backer, 2008). The catalytic unit has a C2 domain in addition to the helical and catalytic kinase domains (Figure 1.4). Depriving cells of Vsp15 by shRNA delays IR degradation and therefore enhances insulin signalling in Hepa1.6 cells (Nemazanyy et al., 2015). Therefore, class III seems to be involved in the regulation of IR trafficking process and IR degradation.

Although Class III PI3K only produces PtdIns3P (Backer, 2008), Vps34 knockout not only

decreases the steady state levels of PtdIns3P but PtdIns(3,5)P2 and PtdIns5P are also negatively affected (Ikonomov et al., 2015). Similarly, deletion of class III in mouse embryo fibroblasts (MEFs) diminishes endosomal PtdIns3P production (Jaber et al., 2012). Furthermore, using tissue specific deletion of class III PI3K inhibits endocytosis and autophagic degradation in heart and liver (Jaber et al., 2012). These data support the notion that in mammalian cells, most of the endosomal pool of PtdIns3P is made by class III PI3K.

1.2.2.2 Phosphatidylinositol phosphate kinases (PIPKs) PIPKs are a group of lipid kinases (including PIP5Ks and PIP4Ks) that use mono- phosphorylated PIs as substrates to produce bis-PIs. PIP5Ks are a family that produces

PtdIns(4,5)P2. Historically, two types were classified under this family as they both produce

PtdIns(4,5)P2 but they have different molecular size (Rameh et al., 1997); Type 1 PIP5K uses the most available mono-phosphoinositide PtdIns4P and therefore most of the bulk of

PtdIns(4,5)P2 is generated through this route (Figure 1.3). On the other hand, Type 2 PIP5Ks (now simply known as PIP4Ks) use the much less abundant lipid PtdIns5P (Rameh et al., 1997).

Since the current work focus on the effects of PIP4K overexpression, PIP5K will not be discussed further in this section.

1.2.2.2.1 Phosphatidylinositol 5-phosphate 4-kinases (PIP4Ks) The function of PIP4Ks in cells is not well understood and their defined role in cell signalling is yet to be fully elucidated. In humans and mice, PIP4K has 3 isoforms (PIP4K2: α, β and γ) encoded by three different genes (PIPK2A, B, C) (Emerling et al., 2013). PIP4Ks are widely expressed in higher eukaryotes (but not in yeast) (Sasaki et al., 2009) and almost all mammalian cells have at least one of the three isoforms of PIP4K (Shim et al., 2016). Although each of these isoforms is localised in distinct locations in the cell, they have been also reported to share some compartments. These enzymes also tend to form hetero- and homo-dimers of PIP4K isoforms (Bultsma et al., 2010). In addition to their overlapping localisation, they also localise and interact with PIP5K. This interaction is thought to have a role in modulating the localisation of PIP4K (Hinchliffe et al., 2002).

Although it is widely expected that the function of PIP4K is controlling and regulating the level of PtdIns5P (Wilcox and Hinchliffe, 2008; Grainger et al., 2011; Clarke et al., 2010; Ramel et al.,

2011) (see section 1.3.7) rather than generating PtdIns(4,5)P2, a few reports have argued that the small pool of PtdIns(4,5)P2 generated from PtdIns5P phosphorylation could have a distinct physiological role (Clarke et al., 2008; Bulley et al., 2016). This was suggested since in particular the PtdIns(4,5)P2 generated from PtdIns5P is restricted to intracellular membranes where PtdIns5P is located rather than to the general plasma membrane fraction. For example in a recent study using chicken derived B-cells (DT40), a single mutation (A381E) was introduced in PIP4K2β which changes its catalytic specificity from a PIP4K to a PIP5K. Using this approach, the authors were able to distinguish between the effect on AKT caused either by PtdIns(4,5)P2 production (via PIP5K) or PtdIns5P removal (via PIP4K). The data from this study indicate that the PtdIns(4,5)P2 pool rather than PtdIns5P itself affected and sustained the activity of AKT (Bulley et al., 2016).

PIP4K isoforms

All PIP4K forms are structurally very similar to each other and they contain one highly conserved kinase domain (Sasaki et al., 2009). Knockout of each PIP4K isoform individually results in a distinctive phenotype which indicates non-redundant functions of these isoforms. Although double knockout of PIP4K2α and PIP4K2β does not affect embryonic development in mice, it results in the death of all pups (Emerling et al., 2013). However, silencing one isoform results in different phenotypes as discussed below.

1.2.2.2.1.1 PIP4K2α PIP4K2α is expressed in many cells and it is highly expressed in spleen and peripheral blood cells (Clarke et al., 2008). PIP4K2α was detected originally in the cytosol of all cells but is also found partially in the nucleus forming a dimer with the PIP4K2β isoform (Zhang et al., 1997; Bultsma et al., 2010). PIP4K2α is the most potent PIP4K isoform. In vitro, its activity was estimated to be 2000 fold more than PIP4K2β (Bultsma et al., 2010). The activity of PIP4K2α was also found to be high in vivo and the activity tended to be higher when dimerised with PIP4K2β (Bultsma et al., 2010). Heterodimerisation has been proposed to mediate translocation of PIP4K2α to intracellular targets, the nucleus by dimerization with PIP4K2β (Bultsma et al., 2010) or the secretory/transport vesicles by dimerization with PIP4K2γ (Clarke et al., 2008) (Sasaki et al., 2009).

Although mice with a deleted PIP4K2A gene seemed normal and did not show any obvious histological, growth, or reproductive phenotypes, mice with the double gene knockout (PIP4K2A and PIP4K2B genes) exhibited neonatal lethality and died within 12 hr after birth (Emerling et al., 2013). However, since the pups (with the double knockout genes) looked normal at birth but died shortly later this indicates that PIP4Kα and β are probably not essential for normal embryonic development but are essential for surviving the stresses of birth and these enzymes contribute to growth and development after birth (Emerling et al., 2013).

1.2.2.2.1.2 PIP4K2β Using multiple tissue Northern blot, quantitative PCR and Western blot analysis revealing that PIP4K2β is expressed in different cell types but it is highly expressed in skeletal muscle cells (Clarke et al., 2008; Castellino et al., 1997; Lamia et al., 2004). PIP4K2β is mainly found in the nucleus (Bultsma et al., 2010; Wang et al., 2010) and due to its lesser activity (compared to PIP4K2α) it has been suggested that one of its roles could be recruiting and determining PIP4K2α localisation via hetrodimerisation (Bulley et al., 2015).

PIP4K2β knockout mice have been reported to be healthy but with higher insulin sensitivity compared to control mice. Silencing or diminishing PIP4K2β seems also to have a protective effect against insulin resistance since PIP4K2β knockout mice did not become obese and did not develop diabetes when kept on a high fat diet (Lamia et al., 2004). This is consistent with the report that specific PIP4K2β knockout in muscle cells shows high activity of insulin-induced AKT phosphorylation (Lamia et al., 2004). Although it is not fully clear how PIP4K affects PI3K and its downstream effectors such as AKT, several studies propose different roles via PtdIns5P including inhibiting AKT-phosphatases or inhibiting PtdIns(3,4,5)P3-phosphatases (Ramel et al., 2009; Carricaburu et al., 2003). In BT474 cells (breast cancer cell which has PIP4K2B amplified), knockdown of both PI5P4Kα and β increases basal and insulin-stimulated

PtdIns(3,4,5)P3 synthesis and subsequent AKT (Emerling et al., 2013) whereas overexpressing

PIP4K2β decreases insulin induced PtdIns(3,4,5)P3 and AKT phosphorylation in CHO-IR cells (Carricaburu et al., 2003). These data suggest a negative role of PIP4K2β on PI3K signalling.

Additionally, PIP4K2β has been recently suggested as a GTP sensor in mammalian cells. PI5P4Kβ is a GTP-dependent kinase (Sumita et al., 2016). The intracellular concentration of GTP is changed depending on the biological status but detecting this change is not easy. PIP4K2β detects changes in the level of GTP and converts these variations into lipid second messenger signalling through regulating PtdIns5P (Sumita et al., 2016). Reducing the level of GTP decreases PIP4K2β activity and thereby increases PtdIns5P levels in the cell. These changes are supposed to be enough to trigger functional signalling (Sumita et al., 2016). Thus activity of PIP4K2β as a GTP-sensor might serve as a new way to understand several metabolic diseases and cancers where GTP is used (Sumita et al., 2016).

1.2.2.2.1.3 PIP4K2γ PIP4K2γ is the least active against PtdIns5P among the PIP4Ks (Clarke and Irvine, 2013). It is mainly expressed in kidney and brain (Clarke et al., 2008). PIP4K2γ does not have any detectable activity against any other PI substrate (Clarke and Irvine, 2013; Clarke et al., 2008). Although Its localisation is not well documented, the available data suggest that PIP4K2γ co- localised with a number of Golgi apparatus and ER markers (Clarke et al., 2008; Sarkes and Rameh, 2010) and its possible dimerization with other isoforms of PIP4K has been also shown in vitro (Clarke et al., 2010) and in vivo (Bultsma et al., 2010; Wang et al., 2010). Mice with a deleted PIP4K2C gene show normal growth but unlike PIP4K2B knockouts are not protected from obesity or insulin resistance (Shim et al., 2016). This mouse also shows higher inflammation markers due to the reported hyperactivity in mTORC1 signalling (which is a nutrient/energy/redox sensor and controls protein synthesis) and therefore it has been suggested that PIP4K2γ normally has a negative impact on mTORC1 signalling and in the absence of this T-cells became hyperactive. Although PIP4K2γ knockout mice remain viable, a double knockout with PIP4K2β shows a lethal effect (Shim et al., 2016).

1.2.2.3 PIKfyve PIKfyve (Phosphoinositide kinase, FYVE-type zinc finger containing) is a large PI kinase composed of 2098 amino acids encoded by a single gene. PIKfyve has five main domains (ordered from N- to C- terminal) (Figure 1.5). First, there is a Fyve (Fab1p, YOTB, Vac1p and EEA1) domain which is responsible for membrane localisation probably by binding to PtdIns3P. Next, there is a Dishevelled, Egl-10 and Pleckstrin (DEP) domain (its function is not known); this is followed by Chaperone containing TCP1 (CCT) and conserved cysteine rich (CCR) domains, which are proposed to be involved in regulation of PIKfyve through protein-protein interactions

(Michell et al., 2006). The last domain is the C-terminus kinase domain which is the catalytic lipid kinase and which also possesses many regulatory sites. PIKfyve is localised in the early endosomes and lysosomes and this localisation is probably associated with its FYVE domain that facilitates Ptdins3P-depending binding. PIKfyve is a low abundance enzyme and has been detected in adipocyte and muscle cells.

Figure 1. 5: Schematic representation of the major domains of PIKfyve.

Abbreviations: a Fyve (Fab1p, YOTB, Vac1p and EEA1), DEP (Dishevelled, Egl-10 and Pleckstrin), CCT (Chaperone containing TCP1), and CCR (conserved cysteine rich).

PIKfyve is the only enzyme known to synthesise PtdIns(3,5)P2 by phosphorylating its precursor substrate PtdIns3P. PIKfyve also plays an important role in generating and regulating PtdIns5P either directly from PtdIns or indirectly by providing PtdIns(3,5)P2 which serves as a substrate for the 3-phosphatases of the myotubularin (MTM) and myotubularin-related protein (MTMR) families. The effect of PIKfyve in insulin signalling has been reported using 3T3L1 adipocytes. Following either expressing dominant-negative lipid kinase-defective PIKfyve or silencing

PIKfyve by siRNA in these cells, PtdIns(3,5)P2 was decreased and insulin dependent glucose uptake was reduced (Ikonomov et al., 2002; Ikonomov et al., 2007).

A muscle specific PIKfyve knockout mouse shows the important role played by PIKfyve in regulating whole body glucose homeostasis (Ikonomov et al., 2013). These mice exhibit glucose intolerance and insulin resistance phenotypes particularly in muscle. Following the insulin signalling pathway revealed that glucose transport is impaired in this model as GLUT4 translocation is attenuated in comparison to control cells (Ikonomov et al., 2013). When compared to control cells, AKT status in the muscle-PIKfyve knockout mice is weakly phosphorylated after 15 but not 5 min of insulin stimulation (Ikonomov et al., 2013). These data indicate an important role of PIKfyve in regulating insulin-dependent pathways of glucose uptake and suggest a positive role of PIKfyve in GLUT4 translocation. However the individual net contribution of the PIKfyve products, PtdIns(3,5)P2 and/or PtdIns5P, in this process is still not clear. It is worth mentioning the suggestion that the effects on insulin-dependent GLUT4 translocation of PtdIns(3,5)P2 may be through the endosomal system (Ikonomov et al., 2007) whereas the contribution of PtdIns5P is through the rearrangement and disassembly of F-actin fibres (Sbrissa et al., 2012).

1.2.3 PI Phosphatases In addition to the role of lipid kinases, phosphatases play a critical role in regulating PI signalling.

Levels of the potent PtdIns(3,4,5)P3 generated by active PI3K are tightly regulated mainly by the 3-phosphatase, Phosphatase and Tensin homolog (PTEN), and by the 5-phosphatases, Src- homology2-containing inositol polyphosphate phosphatases-2 (SHIP2) and Skeletal muscle and kidney enriched inositol phosphatase (SKIP) (Nakashima et al., 2000; Wada et al., 2001; Ijuin et al., 2008). The schematic structures of the main domains of these three enzymes are illustrated in the following figure.

Figure 1.6: Schematic representation of the major domains of PTEN, SHIP2 and SKIP

Abbreviations: PIP2BD (PtdIns(4,5)P2 binding domain), PEST (proline, glutamic acid, serine, and threonine), PDZ (postsynaptic density protein, Disc large, Zona occludens), SH2 (Src homology 2), PRD (proline-rich domain), SAM (Sterile α Motif), SKICH (SKIP carboxyl homology).

1.2.3.1 PTEN PTEN was initially identified as a tumour suppressor gene that is defective in many types of cancers. The PTEN gene encodes the protein/PI phosphatase PTEN protein which acts to antagonise PI3K, playing an important role in regulating the level of PtdIns(3,4,5)P3 at the plasma membrane by hydrolysing phosphate groups at position D-3 of the inositol ring to form

PtdIns(4,5)P2 (Maehama and Dixon, 1998). This mechanism is considered as the main pathway of removing and terminating PtdIns(3,4,5)P3 signalling. PTEN is the only member of the PI 3- phosphatases that has been shown in vivo to be able to dephosphorylate PtdIns(3,4,5)P3 (Salmena et al., 2008).

In addition to its lipid phosphatase property, PTEN also has protein phosphatase activity due to its capacity to dephosphorylate several tyrosine, serine and threonine residues of important proteins. For example, PTEN can regulate IRS-1 by dephosphorylating it. PTEN is widely expressed throughout cell types and is found throughout the cytoplasm as well as in the nucleus (Salmena et al., 2008; Chung and Eng, 2005; Liu et al., 2005). It would be anticipated that in the nucleus PTEN also acts as an antagonist of PI3K. Nevertheless, that has not been shown and therefore non-lipid activity has been suggested as a role of nuclear PTEN. Nuclear PTEN participates for example in regulating DNA-replication (Feng et al., 2015). Thus, the dephosphorylation of PtdIns(3,4,5)P3 in cells is exclusively attributed to the cytoplasmic PTEN since the nuclear pool of PtdIns(3,4,5)P3 apparently is not affected by nuclear PTEN (Lindsay et al., 2006).

Although it was accepted that PTEN does not have isoforms besides the canonical PTEN (55kDa), recent studies have designated PTENα (70kDa) and PTENβ (70kDa) as another two forms of PTEN which are encoded by the same gene but are produced by alternative translation of mRNAs (Liang et al., 2014; Liang et al., 2017). PTENα is predominantly localised in the mitochondria whereas PTENβ is found mainly in the nucleus. The phosphatase activity of all the above three PTEN isoforms were found to be the same (Liang et al., 2017).

The activity of PTEN is regulated by different mechanisms including controlling its expression, regulating its localisation and translocation, and by controlling its stability (Leslie and Downes, 2004). Structurally, PTEN is composed of 5 main domains (Hopkins et al., 2014) (Figure 1.6).

PtdIns(4,5)P2 binding domain (PIP2BD): which has been suggested to have a role in translocating PTEN from its intracellular compartments to the cell membrane. Phosphatase domain: this is the enzymatic region which also contains acetylation sites by which the phosphatase activity of PTEN is regulated. Regulatory C2 domain which is suggested to play an important role in the localisation and the interaction between PTEN and the membrane. PEST (proline, glutamic acid, serine, and threonine) sequences in the carboxyl terminal tail which has phosphorylation sites that are thought to play role in stabilising PTEN. Finally, PDZ domain: This domain is important in protein-protein interaction.

The important role of PTEN is seen from the lethal effect in PTEN-null mice which die during embryonic development. Also, different tumour types were detected in heterozygous PTEN mice (Dahia, 2000; Simpson and Parsons, 2001; Vazquez et al., 2000). A recent study also found that in mothers with gestational diabetes, an increase in the level of PtdIns(4,5)P2 activates PTEN and results in decreasing AKT–mTORC signalling which then leads to ROS production in mitochondria of the embryo (Cao et al., 2016). Overexpressing PTEN reduces basal and insulin- stimulated PtdIns(3,4,5)P3 and PtdIns(3,4)P2 production (Maehama et al., 2001) while cells

41 lacking PTEN show high levels of PtdIns(3,4,5)P3 (Sun et al., 1999) and high activity of AKT (Sun et al., 1999; Taylor et al., 2000). Over expression of PTEN in adipocytes has a negative effect on insulin signalling as insulin-stimulated AKT activity (assessed by AKT phosphorylation and AKT gel shift) and GLUT4 translocation to the plasma membrane is inhibited in these cells (Nakashima et al., 2000).

Insulin hypersensitivity develops in mice with a specific-adipose PTEN knockout (Kurlawalla- Martinez et al., 2005), unlike mice that specifically have PTEN knocked-out in muscle tissue, where no effects on insulin signalling were detected (Wijesekara et al., 2005). However in the latter model (muscle PTEN-Knockout), the mice gain a protective mechanism against developing insulin resistance when high fat diet is used to induce insulin resistance (Wijesekara et al., 2005). Inhibition of PTEN expression using an anti-sense PTEN approach (applied by intraperitoneal injection) was documented to be able to normalise glucose and reduce insulin concentration in mice with T2D phenotype (db/db diabetic dyslipidemia) (Butler et al., 2002). The reduction of PTEN expression on the previous study was observed mainly in liver and fat cells (Butler et al., 2002). Insulin- stimulated AKT phosphorylation is not affected by this inhibition in lean mice although it restores the level of AKT activity in obese mice (Butler et al., 2002). In humans, patients with several mutations in the PTEN gene were found to have less insulin resistance and in fact, some of the reported mutations decreased the risk of diabetes compared to control subjects (Pal et al., 2012). These data indicate that PTEN might play an important role in regulating AKT signalling in pathological conditions particularly in insulin resistance induced by obesity.

1.2.3.2 Phosphoinositide 5 phosphatases In the literature 10 mammalian members of PI 5-phosphatases (INPP5P or PI5pases) family are described. A summary of the main characteristic of these PI 5-phosphatases is presented in Table 1.3 (based on (Dyson et al., 2012; Sasaki et al., 2009). It is worth emphasising here that in many cases the main PI substrate(s) of the PI5pases is/are not well characterised in vivo.

Table 1. 3: Types, substrate specificity and domains of INPP5Ps (Bridges et al., 2012 and Sasaki et al., 2009)

INPP5s (gene) Substrates un-stimulated- Expression Localization 5-phosphatase-1 soluble inositol (INPP5A) phosphates only:

(43 kDa) Ins(1,4,5)P3,

Ins(1,3,4,5)P4

5-phosphatase-2 PtdIns(4,5)P2, Mitochondria ubiquitously

(INPP5B) PtdIns(3,4,5)P3 Cytosol expressed (75 kDa) Golgi apparatus

OCRL PtdIns(3,5)P2, TGN, early endosomes, ubiquitously

(INPP5F) PtdIns(4,5)P2, membrane ruffles clathrin- expressed

(105 kDa) PtdIns(3,4,5)P3 coated trafficking intermediates

Synaptojanin-1 PtdIns(3,4,5)P3, brain

(INPP5G) PtdIns(4,5)P2

(145 kDa) Ins(1,4,5)P3

Ins(1,3,4,5)P4

Synaptojanin-2 PtdIns(3,4,5)P3, cytosol testis and brain

(INPP5H) PtdIns(4,5)P2,

(170 kDa) Ins(1,4,5)P3

Ins(1,3,4,5)P4

PIPP PtdIns(3,4,5)P3 plasma membrane brain, heart, (INPP5J) kidney, stomach, (108 kDa) PtdIns(4,5)P2 small intestine , lung

SKIP PtdIns(3,4,5)P3, perinuclear region, heart, skeletal

(INPP5K) PtdIns(4,5)P2, ER muscle, kidney (51 kDa)

SHIP1 PtdIns(3,4,5)P3 cytosol Hematopoietic (INPP5D) cells (104 kDa)

SHIP2 PtdIns(3,5)P2, cytosol Haematopoietic

(INPP5L1) PtdIns(4,5)P2, cells, brain,

(142 kDa) PtdIns(3,4,5)P3 skeletal muscle, heart

INPP5E PtdIns(3,4,5)P3 Plasma membrane, cytosol, brain, testis,

(72-5ptase) PtdIns(4,5)P2 perinuclear/ Golgi breast and

(72 kDa) PtdIns(3,5)P2 haemopoietic cells

In this section the main two PI 5-phosphatases (SHIP2 and SKIP) that affect insulin signalling particularly in muscle cells are discussed in more detail (Figure 1.6).

1.2.3.3 SHIP2 In humans, SHIP2 is encoded by the INPPL1 gene. Unlike its close relative SHIP1 which is expressed, and proposed to have a critical role in hydrolysing PtdIns(3,4,5)P3 in haematopoietic cells (Miletic et al., 2010), SHIP2 is widely expressed in various cell types including cells of the brain, heart and skeletal muscle (Bridges and Saltiel, 2012).

SHIP2 is known as a PtdIns(3,4,5)P3 –phosphatase. In vitro it can dephosphorylate

PtdIns(3,4,5)P3 at position D-5 of the inositol ring in addition to PtdIns(4,5)P2 and inositol phosphates. However, in vivo, SHIP2 is only reported to use PtdIns(3,4,5)P3 and PtdIns(4,5)P2 as substrates (Taylor et al., 2000). SHIP2 is localised in the cytosol and translocated upon various stimuli, including insulin to the plasma membrane (Bridges and Saltiel, 2012).

Accumulation of PtdIns(3,4,5)P3 at the plasma membrane is accompanied by an increase in

PtdIns(3,4)P2 as a result of the activity of SHIP2 (Sly et al., 2003). SHIP2 is also detected in nuclear speckles suggesting a role of SHIP2 in regulating the nuclear pool of PtdIns(3,4,5)P3 (Déléris et al., 2006).

SHIP2 consists of 3 main domains (Figure 1.6) which are the SH2 domain, the 5-phosphatase domain, and the proline-rich domain (PRD) which followed by a motif known as Sterile α Motif (SAM). The SH2 domain binds to some proteins that have phosphorylated tyrosine (Thomas et al., 2016). PRD recognises and binds to SH3 domain containing proteins (Thomas et al., 2016). PRD has been also suggested to play an important role in localising SHIP2 at endocytic clathrin- coated pits since SHIP2 lacking PRD is no longer located at that site (Nakatsu et al., 2010).

Several studies have been carried out to investigate the involvement of SHIP2 in insulin dependent glucose uptake. Overexpressing SHIP2 in L6 myotubes or in adipocyte cells negatively affects insulin signalling by degrading PtdIns(3,4,5)P3 which results in decreasing AKT activation and reduced GLUT4 translocation (Sasaoka et al., 2001; Wada et al., 2001). However, SHIP2 knockdown in L6 cells and in adipocytes did not increase the activity of AKT in either basal or in insulin stimulated conditions (Mandl et al., 2007). Although inhibiting SHIP2 using a pharmacological agent (AS1949490) in rat L6 myotubes (Suwa et al., 2009) but not in mouse C2C12 myotubes (which express very low amounts of SHIP2 compared to L6 cells) increased glucose uptake in the absence of insulin, it turns out that the glucose influx is through GLUT1 and not via GLUT4 (Suwa et al., 2010). Moreover, silencing SHIP2 in adipocytes using RNAi approaches has no effect on insulin signalling or glucose uptake (Zhou et al., 2004).

In the literature two models of SHIP2 knockout mice are described with contradictory outcomes, and hence the role of SHIP2 in insulin signalling is not clear. The first study was reported by

Clément et al., (2001) where exons 19-29 were deleted. This region encodes the active catalytic phosphatase of SHIP2. However, it turns out, as later reported by the same group (Clément et al., 2004) that the Phox2a gene has been also deleted as an off target effect in these mice, and therefore it is uncertain whether the effect seen was due to alterations in the Phox2a gene. These SHIP2-null mice die a few hours after birth (Clément et al., 2001) whereas the heterozygous SHIP2 mice develop insulin hypersensitivity and GLUT4 translocation to the plasma membrane is enhanced (Clément et al., 2001).

The second study was conducted by deleting all of the first 18 exons of SHIP2, leaving the last non-enzymatic domains (Sleeman et al., 2005). Unlike the previous study (Clément et al., 2001), the SHIP2-null mice live beyond 1 year with some marks of a growth phenotype like less weight gain and small size, however no significant effect was detected on insulin signalling in either the homozygous or the heterozygous SHIP2 mice compared to the wild type control subjects (Sleeman et al., 2005). On the other hand, these mice became protected against developing insulin resistance and diabetes induced by a high fat diet (Sleeman et al., 2005). These data suggest that SHIP2 might be involved in obesity rather than the physiological glucose homeostasis of insulin signalling.

Although several studies reported association between some polymorphisms in the SHIP2 gene (INPPL1) and diabetes (Accardi et al., 2014; Dyson et al., 2012; Bridges et al., 2012), the precise role of SHIP2 in regulating insulin signalling remains unresolved.

1.2.3.4 SKIP SKIP is a 5-phosphatase encoded by the INPP5K gene in human which is homologous to the Pps gene in mice. SKIP dephosphorylates D-5 of the inositol ring of PIs as well as some of the inositol phosphates (Ijuin et al., 2000). PtdIns(3,4,5)P3 and PtdIns(4,5)P2 but not PtdIns(3,5)P2 are substrates of SKIP (Gurung et al., 2003). Its activity is higher toward PtdIns(3,4,5)P3 than

PtdIns(4,5)P2 while it is very weak against inositol phosphates (Ijuin and Takenawa, 2003). In human tissue, SKIP is highly expressed in heart, skeletal muscle, and kidney (Ijuin et al., 2000).

As a result of RNA variants due to an alternative splicing process, two isoforms of SKIP have been detected; the full-length and a short forms which is truncated by 76 amino acids at the N terminus (Ijuin et al., 2008; Ijuin et al., 2000). The full length SKIP has higher activity than the short form (Ijuin et al., 2008). The full-length isoform has been detected in rat brain lysate and mouse neuroblastoma cells while the short form seems to be expressed in mouse C2C12 myoblast cells, and mouse fibroblast cells (Ijuin et al., 2000). In general, insulin-sensitive cells express the full length form of SKIP though adipose cells predominantly express the short form (Ijuin et al., 2008). In unstimulated cells, SKIP is found associated with the perinuclear region and the trans-Golgi vesicles, but the majority is co-localised with the ER. SKIP is translocated

45 from its intracellular compartments to the plasma membrane in response to insulin stimulation (Gurung et al., 2003; Ijuin and Takenawa, 2003). This translocation plays an important role in localising SKIP to its substrate PtdIns(3,4,5)P3. This was anticipated since under resting conditions, PtdIns(3,4,5)P3 is not affected in cells overexpressing SKIP. In contrast, the level of

PtdIns(4,5)P2 is found to be partially reduced in unstimulated cells expressing SKIP. This has been suggested to occur as a result of co-localisation of SKIP and PtdIns(4,5)P2 in the same intracellular compartments (eg ER) in comparison to SKIP/ PtdIns(3,4,5)P3 where SKIP translocation to the plasma membrane is needed to attack PtdIns(3,4,5)P3 (Ijuin and Takenawa, 2003).

SKIP has two main domains (Figure 1.6), the catalytic phosphatase domain and the SKICH domain. SKICH is revealed as an important domain for the localisation of SKIP (Gurung et al., 2003). Deleting or introducing mutations in the SKICH domain blocks SKIP translocation from its perinuclear /ER sites to the plasma membrane in response, for instance, to insulin (Gurung et al., 2003). Involvement has been also reported of another motif in the region between amino acids 199 to 266 of the SKICH domain to play a role in the interaction between SKIP and HBV virus that lead to translocation of SKIP from the ER into the nucleus, which results in decreasing the expression of the HBV core protein (Hung et al., 2009). However this translocation and the inhibitory effect seen on the core protein expression of the virus seems to be phosphatase independent (Hung et al., 2009).

In muscle cells SKIP expression has been associated with the regulation of glucose uptake and cell differentiation (Gurung et al., 2003; Ijuin and Takenawa, 2003; Xiong et al., 2011; Ijuin and Takenawa, 2012c; Ijuin et al., 2015; Ijuin and Takenawa, 2015). A recent role of SKIP was also identified, associated with ER-stress. Exposing C2C12 cells to excessive fat that induces ER stress upregulates SKIP expression which results in the suppression of insulin signalling (Ijuin et al., 2016).

An important role of SKIP in embryonic development has also been reported, as mid-gestational lethality is seen in global homozygous SKIP knockout mice (Ijuin et al., 2008). However, heterozygous mice are viable and display an enhanced insulin-dependent PI3K/AKT signalling

(Ijuin et al., 2008), consistent with the ability of SKIP to hydrolyse PtdIns(3,4,5)P3 .

SKIP plays crucial roles in insulin signalling due to its catalytic activity against PtdIns(3,4,5)P3 and to a lesser extent PtdIns(4,5)P2. SKIP negatively regulates the level of PtdIns(3,4,5)P3 and therefore its downstream effectors like the AKT pathway, which is attenuated upon increased SKIP expression (Ijuin and Takenawa, 2003). Silencing SKIP in cultured cells and in mice results in enhancing AKT activity, and more GLUT4 is present in the cell membrane of insulin stimulated myoblasts. Similarly glucose uptake, glucose metabolism and glycogen synthesis are all

46 increased in response to decreased SKIP activity (Ijuin and Takenawa, 2003; Xiong et al., 2009). Overexpression of SKIP reduces actin stress fibre assembly and insulin-stimulated lamellipodia formation indicating a negative role of SKIP in rearrangement of the cytoskeleton, which is an essential process in many insulin-stimulated processes including GLUT4 translocation and glucose uptake (Gurung et al., 2003; Ijuin and Takenawa, 2003; Ijuin et al., 2000). Furthermore, it has been observed that the termination of insulin signalling can be also delayed by suppressing SKIP activity (Ijuin and Takenawa, 2012).

In general, sensitivity to insulin is increased in SKIP heterozygous knockout mice and insulin resistance induced by high fat food is improved in these mice (Ijuin et al., 2008). Recent studies indicate that SKIP is the primary PI 5-phosphatase that plays a central role in the regulation of insulin signalling particularly in skeletal muscle cells (Ijuin and Takenawa, 2012a). In addition to the phosphatase activity, SKIP has a critical scaffolding effect which participates in the formation of protein complexes with PAK1 and other proteins in muscle cells that results in terminating insulin signalling (Ijuin and Takenawa, 2012).

Furthermore, SKIP was reported to have an important role in cell differentiation and maturation of muscle cells. SKIP expression is increased during cell differentiation and suppressing SKIP increases the fusion rate of cells that enhances the formation of myotubes (Xiong et al., 2011; Ijuin and Takenawa, 2012c). SKIP seems to negatively attenuate transcription of Insulin-like growth factor IGF-II which is controlled by mTOR. SKIP which also prevents AKT activation that in turn reduces the mTOR-dependent IGF-II production. This feedback decreases IGF-II- PI3K- AKT-mTOR signalling and terminates myogenesis (Ijuin and Takenawa, 2012b). Several mutations have recently been reported in the INPP5K gene that result in congenital muscular dystrophy syndrome (Osborn et al., 2017).

1.3 Roles of PIs:

1.3.1 Phosphatidylinositol 3,4,5-trisphosphate PtdIns(3,4,5)P3

PtdIns(3,4,5)P3 is a prime modulator of insulin signalling. In the basal state of the normal cell,

PtdIns(3,4,5)P3 is very low but it is increased robustly upon various types of stimulation including by various growth factors and hormones such as insulin, insulin-like growth factor 1( IGF1), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF).

Due to the powerful effect of PtdIns(3,4,5)P3, it is usually dephosphorylated quickly by specific phosphatases including PTEN and the family of 5-phosphatases as mentioned previously

(section 1.2.3). PtdIns(3,4,5)P3 plays critical roles in various cellular functions such as cell cycle progression, cell survival and apoptosis, cellular growth, cytoskeletal rearrangement via the

47 monomeric G-protein Rac1, intracellular vesicle trafficking, and chemotaxis (Manna and Jain, 2015). In muscle cells SHIP2 and SKIP seem to be the main 5-phosphatases regulating

PtdIns(3,4,5)P3. Although both pathways attenuate PtdIns(3,4,5)P3, PTEN can be considered as the main removal pathway of PtdIns(3,4,5)P3 since it returns it back to its precursor

PtdIns(4,5)P2 whereas the 5-phosphatase pathway converts PtdIns(3,4,5)P3 to another signalling lipid, PtdIns(3,4)P2 (Leslie et al,. 2002)

PtdIns(3,4,5)P3 recruits and interacts with various proteins that initiate and regulate several pathways downstream of PI3K. These interactions are mediated via specific domains or adaptors that serve as a connection between this lipid and proteins. PtdIns(3,4,5)P3 mediates its effect through the interaction with proteins mainly via the Pleckstrin Homology (PH) domain.

More than 40 PH-containing domain proteins were found to interact with PtdIns(3,4,5)P3 with varying affinity and specificity (Manna and Jain, 2015). AKT, 3-phosphoinositide-dependent protein kinase 1 (PDK1), and protein kinase C ζ (PKCζ) are examples of these important proteins which are involved in insulin signalling. AKT is recruited from the cytoplasm to the plasma membrane upon production of PtdIns(3,4,5)P3.

One highly specific protein that binds to PtdIns(3,4,5)P3 is general receptor for phosphoinositides-1 (GRP1) which has been used widely to estimate the amount and the location of PtdIns(3,4,5)P3 (Guillou et al., 2007). GRP1 is a GEF for ADP-ribosylation factor 6 (ARF6) that stimulates GLUT4 vesicle formation and trafficking downstream of AKT (Li et al., 2012) probably distinct from the effects of Rac (Clodi et al., 1998). Nevertheless, several other Rac-GEFs (see section 1.1.5.2) such as PREX1 and PREX2 and others which play an important role in regulating Rac1 pathway are also recruited and their activity is increased by

PtdIns(3,4,5)P3 (Ooms et al., 2009; Manna and Jain, 2015).

Excess production of PtdIns(3,4,5)P3 has been associated with various types of cancers

(Wymann, 2012) while T2D is usually associated with limited production of PtdIns(3,4,5)P3.

Deficiency in generating PtdIns(3,4,5)P3 or fast degradation has been seen as attenuation of insulin signalling where the GLUT4 expression at cell membrane and consequently glucose uptake is impaired, resulting in diabetes (Manna and Jain, 2015). However, PtdIns(3,4,5)P3 alone is not sufficient to induce glucose metabolism and other PIs are also required in this process. Introducing exogenous PtdIns(3,4,5)P3 into cells by microinjection or by lipid carrier methods failed to mimic insulin-dependent glucose uptake (Maffucci et al., 2003; Jiang et al.,

1998), though it has been proposed that in adipocytes PtdIns(3,4,5)P3 is capable of moving GLUT4 and fusing it with the plasma membrane but in an inactive form (Ishiki et al., 2005). In addition to the role of PtdIns(3,4,5)P3 in mediating insulin signalling, PtdIns(3,4,5)P3 also plays a role in terminating this signalling, probably by recruiting O-linked-β-N-acetylglucosamine transferase (OGT) protein from the nucleus to the plasma membrane. OGT catalyses the

48 modification of proteins by the addition of O-linked β-N-acetylglucosamine (O-GlcNAc) to the insulin signalling intermediates. This process inhibits phosphorylation of AKT and promotes serine phosphorylation of IRS1 which leads to the inhibition and termination of the insulin signalling pathways (Yang et al., 2008). Abnormal O-GlcNAc modification of the insulin signalling pathway may contribute to the pathophysiology of insulin resistance, obesity and T2D (Yang et al., 2008). However, despite the enormous number of studies that focus on PtdIns(3,4,5)P3, its entire regulation is not yet fully understood.

1.3.2 Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)

PtdIns(4,5)P2 is the major substrate for class I PI3K and is the most abundant phosphatidylinositol bisphosphates (see table 1.1) (De Craene et al., 2017). Most PtdIns(4,5)P2 is localised in the plasma membrane (Di Paolo and De Camilli, 2006; Hammond et al., 2009) though it has been also detected in small amounts in the ER, Golgi and nucleus (Viaud et al.,

2016; Osborne et al., 2001). The nuclear pool of PtdIns(4,5)P2 is not currently thought to play a role in insulin-stimulated glucose uptake (Shah et al., 2013) and will not be discussed in this review.

As mentioned previously PtdIns(4,5)P2 is generated mainly from PtdIns4P by PIP5K (see figure

1.3). PIP4K is also able to produce PtdIns(4,5)P2 from PtdIns5P (section 1.2.2.2.1) but this pathway would contribute negligibly to the total amount of PtdIns(4,5)P2 in cells since the level of PtdIns5P is very low in comparison to PtdIns4P (Di Paolo and De Camilli, 2006). Specifically, it has been estimated to be 50 fold less than PtdIns4P (Rameh et al., 1997). Nevertheless and As mentioned previously (section 1.2.2.2.1), the possibility that the PtdIns(4,5)P2 pool synthesised via the PtdIns5P route has specific roles and physiological importance cannot be totally excluded or ignored, particularly as it has a distinct and specific intracellular localisation (Golgi apparatus and/or nucleus) (Clarke et al., 2008; Bulley et al., 2015). However, enhancing this pathway by PIP4K overexpression displays unexpected results. Although the physiological substrate of PI3K, PtdIns(4,5)P2 is also produced by PIP4K from PtdIns5P, the generation of

PtdIns(3,4,5)P3 was found to be attenuated in different cell types overexpressing PIP4K, and hence the downstream insulin signalling is impaired (Grainger et al., 2011; Carricaburu et al., 2003).

Another pathway of generating PtdIns(4,5)P2 is through dephosphorylating PtdIns(3,4,5)P3 as mentioned earlier (Section 1.2.3.1) via PTEN, however PtdIns(3,4,5)P3 is much less abundant in the cell (see table 1.1 for relative amounts) making the contribution of this route to the overall level of PtdIns(4,5)P2 likely to be very limited.

In cellular signalling, PtdIns(4,5)P2 was initially recognised as a substrate of phospholipase C

(PLC). PLC utilizes PtdIns(4,5)P2 to produce Ins(1,4,5)P3 and diacylglycerol (DAG) which are important in releasing calcium from intracellular stores and in activating conventional and novel

(but not atypical) members of the Protein kinase C (PKC) family (Balla, 2013). PtdIns(4,5)P2 has an important role in interacting and localising different proteins to the plasma membrane. It is now known that PtdIns(4,5)P2 also interacts directly with numerous proteins through specific domains such as PH, ENTH, and FERM domains (Viaud et al., 2016). Based on these interactions PtdIns(4,5)P2 both directly and indirectly plays important roles in controlling many different cellular functions such as rearrangement of the actin cytoskeleton, endocytosis, exocytosis, and ion channel activity (Toker, 1998; Di Paolo and De Camilli, 2006) which might affect overall insulin signalling.

1.3.3 Phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2)

PtdIns(3,4)P2 is found at low amounts in unstimulated condition in similar levels to

PtdIns(3,4,5)P3 and it transiently increases in response to stimulation (De Craene et al., 2017).

It is widely accepted that most PtdIns(3,4)P2 is generated from PtdIns(3,4,5)P3 by PI-5

phosphatases (Hawkins and Stephens, 2016). Additionally, generation of PtdIns(3,4)P2 by PI3K is also reported (Li and Marshall, 2015; Hawkins and Stephens, 2016). Two inositol polyphosphate 4-phosphatases (INPP4A and INPP4B) have been reported to have relatively

specific selectivity toward hydrolysing the D-4 phosphate of PtdIns(3,4)P2, potentially making them an important negative regulator of this lipid (Li and Marshall, 2015; Hakim et al., 2012). PTEN has also been reported to have the ability to hydrolyse the phosphate at D-3 of

PtdIns(3,4)P2 (Campbell et al., 2003).

PtdIns(3,4)P2 has been suggested to regulate insulin signalling in positive or negative ways.

Positive control of insulin signalling comes from the fact that many PtdIns(3,4,5)P3 -binding proteins also bind to PtdIns(3,4)P2, including the protein kinase AKT (Li and Marshall, 2015).

PtdIns(3,4)P2 binds probably through the PH domain and activates AKT (Scheid et al,. 2002). It has been suggested that PtdIns(3,4,5)P3 attracts AKT to the plasma membrane to be phosphorylated at Thr308 by PDK1, while PtdIns(3,4)P2 is necessary for phosphorylation of AKT at Ser473 (Scheid et al., 2002). In vitro data suggest that PtdIns(3,4)P2 has a higher affinity for

AKT than PtdIns(3,4,5)P3 (Scheid et al., 2002). However, some studies suggest that

PtdIns(3,4)P2 is important for regulating negative-feedback of insulin/PI3K signalling via the tandem PH domain-containing proteins (TAPP1 and TAPP2) which have been found to bind specifically to PtdIns(3,4)P2 (Dowler et al., 2000; Wullschleger et al., 2011). Introducing point mutations that destroy the binding site between TAPP1 or 2 and PtdIns(3,4)P2 enhances the

AKT phosphorylation in heart and skeletal muscle, indicating that these TAPP1 and 2 proteins negatively regulate insulin signalling (Wullschleger et al., 2011). The specific binding of TAPP1 and 2 makes these proteins also a good tool for visualising and quantifying PtdIns(3,4)P2 which reveals that PtdIns(3,4)P2 is localised at the plasma membrane (Irino et al., 2012; Kimber et al., 2002) and in the ER and endosomes (Watt et al., 2004). A recent study also found that

PtdIns(3,4)P2 is co-localised with actin aggregation and it has been reported that PtdIns(3,4)P2 alone is sufficient for actin aggregation in neuronal cells (Zhang et al., 2017). PtdIns(3,4)P2 has also shown involvement in some crucial cellular mechanisms such as cell adhesion, migration, and cytoskeletal regulation (Xie et al., 2013).

1.3.4 Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2)

PtdIns(3,5)P2 is generated solely from PtdIns3P by PIKfyve (section 1.2.2.3) (Zolov et al., 2012; Sbrissa et al., 1999) in a wortmannin sensitive pathway (where PtdIns3P is produced via class II and III PI3K) and dephosphorylated to PtdIns5P by myotubularins. PtdIns(3,5)P2 increases in response to stimuli such as insulin (Bridges and Saltiel, 2012; Ikonomov, 2003; Ikonomov et al., 2001). Starving and refeeding cells have been reported to stimulate PIKfyve, which results in increasing PtdIns(3,5)P2 levels (Bridges and Saltiel, 2012).PtdIns(3,5)P2 is detected mostly in the intracellular compartments rather than at the plasma membrane (Shisheva, 2008), being found in endosomes and lysosomes (Takatori et al., 2016; Takatori and Fujimoto, 2016).

Expression of dominant- negative kinase-deficient PIKfyve reduces the level of PtdIns(3,5)P2, which has been reported to reduce insulin stimulated GLUT4 translocation in adipocytes (Ikonomov et al., 2002). Similar results have been also observed when PIKfyve is knocked-down by siRNA (Ikonomov et al., 2007). However, exogenous PtdIns(3,5)P2 could not translocate GLUT4 and hence glucose uptake is not activated in adipocytes by this treatment (Sbrissa et al., 2004; Shisheva, 2008).

1.3.5 Phosphatidylinositol 3-phosphate (PtdIns3P)

PtdIns3P is one of the PIs that is found in very low levels similar to the level of PtdIns5P (Shisheva et al., 2015) and much less than PtdIns4P (De Craene et al., 2017) (see table 1.1). It is generated mainly by class II and III PI3K directly from PtdIns (see section 1.2.2.1). PtdIns3P is localised mainly in the endosomal structure but it has been also detected partially at specific sub-domains of lipid rafts at the plasma membrane particularly after insulin stimulation (Maffucci et al., 2003). Proteins with PX domain as well as proteins with FYVE domains such as the early endosomal antigen 1 (EEA1) which plays an important role in regulating endosomal membrane fusion processes can recognise PtdIns3P (Pons et al., 2008).

The identity of the PI3K classes that is responsible for PtdIns3P production at specific compartments is studied and the data suggest that at the plasma membrane and in response to insulin most PtdIns3P production appears to be generated by the class II PI3K pathway as it is produced in a wortmannin resistant manner (Maffucci et al., 2003; Falasca et al., 2007; Kong et al., 2006). On the other hand, the main source of PtdIns3P in early endosomes (in podocytes) is class III PI3K (Ikonomov et al., 2015) but PI3K-C2α is also contributed in this pool (Franco et al., 2014; Ikonomov et al., 2015).

PtdIns3P can also be produced indirectly via hydrolysing the D-5 phosphate of PtdIns(3,5)P2 by suppressor of actin 3 (Sac3) (also known as Fig4) which exhibits a 5-phosphatase activity.

However since PtdIns(3,5)P2 itself exists in very low amounts, this pathway would be very limited in providing PtdIns3P. In fact PtdIns3P production itself is an important mediator for producing

PtdIns(3,5)P2 as well as PtdIns5P.

Insulin signalling can also activate generation of PtdIns3P partially via the small GTPase TC10. Overexpressing an active form of TC10 is reported to increase the level of PtdIns3P in adipocytes and L6 cells (Maffucci et al., 2003). Furthermore expressing the 72kD PI 5- phosphatase (INPP5E), which localised at endosomes, could also increase PtdIns3P levels in the absence of insulin (Kong et al., 2006).

Delivering synthetic PtdIns3P lipid into L6 cells displays outcomes that mimic insulin action on enhancing GLUT4 translocation to the plasma membrane, although increased glucose uptake was not achieved (Ishiki et al., 2005; Maffucci et al., 2003; Sweeney et al., 2004; Kong et al., 2006). On the other hand expressing myotubularin phosphatases or depleting PI3KC2a reduces insulin stimulated glucose uptake (Chaussade et al., 2003; Falasca et al., 2007). This indicates a potential role for PtdIns3P in insulin-stimulated glucose uptake. Importantly, and as has been mentioned previously PtdIns(3,4,5)P3 alone cannot induce glucose uptake, however combining

PtdIns(3,4,5)P3 and PtdIns3P could not enhance glucose entry into the cell either (Sweeney et al., 2004).

1.3.6 Phosphatidylinositol 4-phosphate (PtdIns4P)

PtdIns4P is the most abundant monophosphate PI and is produced mainly directly from PtdIns by PI4Ks (Lemmon, 2008) (Figure 1.3). PI4Ks are categorised into type II and III (Type I PIK refers to PI3K). Type II PI4K is inhibited by adenosine while type III is wortmannin sensitive (but with much less sensitivity than PI3K) (Balla and Balla, 2006). PtdIns4P is localised at the plasma membrane, endosomes and Golgi structure (Hammond et

al., 2009). As mentioned previously, it serves as the main precursor for PtdIns(4,5)P2 as a substrate for PIP5K. PtdIns4P has been described in signalling pathways as a regulator by itself

via interacting with proteins involved in the processes of endocytosis and phagocytosis. It has also been suggested to have negative effects on GLUT4 translocation via an unknown mechanism. However, in general PtdIns4P functions in insulin signalling only as a precursor of

PtdIns(4,5)P2 particularly at the plasma membrane and as a modulator of membrane trafficking, though the exact role in trafficking is not well established. Exogenous PtdIns4P delivery by microinjection or by lipid-Carrier does not affect accumulation of GLUT4 (Sbrissa et al., 2004; Maffucci et al., 2003).

1.3.7 Phosphatidylinositol 5-phosphate (PtdIns5P)

PtdIns5P is the most recently discovered PI because of its low level and due to its poor separation from the most abundant mono-PI, PtdIns4P, in HPLC (Rameh et al., 1997). The level of PtdIns5P is very low, almost the same as PtdIns3P. Although the exact route of production of PtdIns5P in vivo is not yet known, generation of PtdIns5P is thought mainly to occur by PIKfyve. Two main pathways have been suggested. The first, which is accepted widely is through dephosphorylating PtdIns(3,5)P2 (the product of PIKfyve) by the 3-phosphatases MTM and MTMR (Shisheva, 2013).The second proposed pathway is directly by PIKfyve phosphorylating the D-5 hydroxyl group of PtdIns. In both cases PIKfyve plays an essential role, andinhibiting PIKfyve reduces PtdIns5P levels in different cell types (Zolov et al., 2012; Shisheva, 2013). PtdIns5P is robustly increased after insulin stimulation in different cell types (Grainger et al., 2011; Sbrissa et al., 2004).

PtdIns5P is also proposed to be generated via de-phosphorylating the phosphate group at the

D-4 phosphate of PtdIns(4,5)P2 by PtdIns(4,5)P2 4-phosphatases. Two mammalian

PtdIns(4,5)P2 4-phosphatases (Type I and II) have been identified which, in humans, are encoded by TMEM55B and TMEM55A genes respectively (Ungewickell et al., 2005). These 4- phosphatases specifically hydrolyse PtdIns(4,5)P2 but not PtdIns(3,4,5)P3, PtdIns(3,4)P2,

PtdIns(3,5)P2, PtdIns5P, PtdIns4P, or PtdIns3P (Ungewickell et al., 2005). The level of PtdIns5P increased in HEK293 cells when Type I but not Type II PtdIns(4,5)P2 4-phosphatase were overexpressed (Zou et al., 2007). However, Type I 4-phosphatase is thought to control nuclear levels of PtdIns5P (Zou et al., 2007). The information about these two enzymes and their role in regulating the level of PtdIns5P and/or PtdIns(4,5)P2 is very limited. On the other hand, the bacterial 4-phosphatases IpgD is frequently used by researchers in mammalian cells as a powerful enzyme to generate bulk amounts of PtdIns5P also by hydrolysing the phosphate group located at D-4 of the endogenous PtdIns(4,5)P2 (Viaud et al., 2014; Niebuhr et al., 2002).

The amount of PtdIns5P is regulated mainly by PIP4K by converting it to PtdIns(4,5)P2 (see section 1.2.2.2 for more detail). The majority of PtdIns5P in the unstimulated cell is present in the plasma membrane although it has been also detected in in the intracellular compartments,

53 such as smooth endoplasmic reticulum and Golgi apparatus in addition to the nucleus (Sarkes and Rameh, 2010; Jones et al., 2006).

Non-nuclear PtdIns5P contributes to several cellular mechanisms including apoptosis, cell migration, intracellular membrane trafficking, pathogen invasion, cytoskeleton regulation, stress responses and insulin dependent GLUT4 translocation and glucose uptake (Oppelt et al., 2013; Sbrissa et al., 2012; Sbrissa et al., 2004; Lamia et al., 2004; Shisheva, 2013) whereas the nuclear level of PtdIns5P is associated with various stresses and has been also proposed to have a role in the cell cycle and in apoptosis, through increasing p53 activation. PtdIns5P is also involved in regulating nuclear responses to DNA damage (Jones et al., 2006; Gozani et al., 2003). However, most of the functions of PtdIns5P in response to acute stimulation from hormones such as insulin are attributed to the non-nuclear pool of PtdIns5P (Shisheva, 2013) and therefore an in-depth discussion of nuclear PtdIns5P is beyond the scope of this review.

As mentioned above (section 1.1.6), one of the effects of insulin is rearrangement of cytoskeleton. F-actin stress fibres (but not increase membrane ruffling) has been reported to be broken-down upon increasing levels of PtdIns5P by insulin, or by expressing PIKfyve or directly by micro-injecting PtdIns5P into CHO or 3T3L1 cells, and this arrangement was prevented when PtdIns5P is scavenged with PHD-containing binding proteins (ING2 and ACF) (Sbrissa et al., 2004) (ING2 and ACF eliminate the availability of PtdIns5P). Furthermore, this effect seemed to be PI3K independent and affected GLUT4 translocation as this study indicates that PtdIns5P increases exocytosis rather than decreasing endocytosis of GLUT4 in adipocytes (Sbrissa et al., 2004).

PtdIns5P has been found to activate Rac1 which regulates disappearance of actin stress fibres in fibroblasts (Viaud et al., 2014). In L6 myotubes, glucose uptake was observed when these cells were exposed to exogenous PtdIns5P in the absence of insulin (Grainger et al., 2011). However, whether PtdIns5P dependent– Rac1 activation also occurs in muscle cells and whether this activation alone is able to activate glucose uptake is not known.

1.4 PIP4K2α overexpression in L6 myotubes

Different cell lines have been used beside animal models to study and elucidate insulin signalling pathways. Quantification of glucose uptake by the L6 cell line shows a direct response to insulin (Nedachi and Kanzaki, 2006). L6 myoblasts differentiate to myotubes (poly-nuclear fibre-like structures) but they do not further develop T-tubules; the main part of muscle where GLUT4 localises particularly following insulin stimulation. Nevertheless, L6 myotubes express high endogenous levels of GLUT4 and have been widely used in investigating GLUT4 translocation and glucose uptake.

The current study is a continuation of previous work carried in the host lab, where the L6 cell line and the effect of different stimuli including insulin have already been established. L6 myotubes overexpressing PIP4K2α using adenoviral vectors have been used to study and to investigate the role of PIP4K2α and its substrate PtdIns5P in the insulin signalling. Although PIP4K2α converts PtdIns5P to PtdIns(4,5)P2, it has been observed that in these cells insulin-stimulated glucose uptake is abolished (Grainger et al., 2011), but in an AKT-independent fashion, since the phosphorylation of AKT is not altered by expressing PIP4K2α (Tavelis, 2012). Investigating

PI metabolism indicates low level of PtdIns(3,4,5)P3 in these cells, which could be as a result of attenuation in the biosynthesis process of this lipid, or increasing its removal by phosphatases. The activities of PI3K and PTEN were not affected by PIP4K2α (Tavelis, 2012) and therefore, investigating PI 5 phosphatases is prioritised. This notion is proposed since the PtdIns(3,4,5)P3 level was decreased while PtdIns(3,4)P2 was elevated in these cells (Tavelis, 2012). However, inhibiting SHIP2 by AS1949490 did not restore the PtdIns(3,4,5)P3 and did not explain the discrepancy observed in the PI3K pathway.

1.5 Hypotheses and Aims

The overall purpose of this study was to explore the effect of PIP4K2α overexpression, and the role of its substrate, PtdIns5P, on insulin signalling pathway downstream of PI3K in L6 myotubes. Two hypotheses are investigated in the current project. Firstly, that the disturbances on the levels of PIs induced by PIP4K-overexpression in L6 myotubes is caused by the effect of one or more of 5-phosphatases (with a particular focus on SKIP as a likely candidate).

Secondly, as there are two pathways downstream of PtdIns(3,4,5)P3, involving AKT and Rac1 respectively, both of which play an important role in GLUT4 translocation, Rac1 activity is hypothesised to be impaired in the insulin signalling pathway in L6 myotubes overexpressing PIP4K2α.

This work aims to test the two specific hypotheses in L6 myotubes 1. If the insulin-dependent GLUT4 translocation is attenuated whereas activity of AKT is not affected, then Rac1 activity is reduced.

2. If removal of PtdIns(3,4,5)P3 in myotubes overexpressing PIP4K2α is caused by

activation of SKIP, then silencing SKIP will restore the level of PtdIns(3,4,5)P3.

Chapter 2

Materials and Methods

2. Materials and Methods

2.1 Materials

Cell culture: Alpha modification of minimum essential medium (αMEM), Dulbecco’s modified Eagle's medium (DMEM), insulin, penicillin/streptomycin, L-Glutamine and Trypsin-EDTA were obtained from Sigma. Heat-inactivated Foetal bovine serum (FBS) was from Gibco. Parental rat L6 myoblasts were purchased from American Type Culture Collection (ATCC) while the L6 myoblasts expressing c-myc epitope-tagged glucose transporter protein-4 (L6-GLUT4myc) cell line was from Kerafast. All cell culture flasks, dishes and vessels were purchased from Corning.

Molecular techniques: Absolutely RNA Miniprep kit was purchased from Stratagene. Primers were from Sigma, MyTaq One step RT-PCR kit was from Bioline. Agarose was from Lonza and SafeView Nucleic Acid Stain was from NBS Biological. Tryptone, Yeast Extract and Agar were from Melford. Kanamycin sulfate was from Sigma while Ampicillin was from Fisher. Glutathione was obtained from Sigma while Glutathione SepharoseTM was purchased from GE Healthcare. Qiaquick gel extraction kit and plasmid maxi kit were purchased from Qiagen. FuGENE®6 transfection reagent was from Promega. AdEASY XL Adenovirus system was purchased from Stratagene while the Adeno-X Rapid Titer kit was from Clontech. Pac1 restriction enzyme was from New England Biolabs. OptiMEM was from Gibco. Scrambled siRNA, INPP5K 27-mer siRNAs (SR506835 A, B, and C), transfection reagent (siTran1.0) and DNA ladder were from Origene.

Plasmids: E.Coli with plasmids which encodes the following recombinant proteins were developed in the host lab: FLAG-tag PIP4K2α, GST-TAPP1-PH and GST-GRP1-PH. The domain for measuring PtdIns(4,5)P2, GFP-tagged PLC-delta-PH, was a gift from Professor Martin Lowe, University of Manchester.

Protein techniques: Acrylamide/bis-acrylamide was from National Diagnostics. Ammonium persulphate (APS) was from Flowgen. Tetramethylethylenediamine (TEMED), Tween-20, Sodium dodecyl sulphate (SDS), Igepal CA-630, β-mercaptoethanol were from Sigma. Glycine, Ammonium Formate, Glycerol, Tris Base and HEPES were from Fisher. Protease inhibitor cocktail and wortmannin were from CalBiochem. Rac1 Activation Assay Kit was from Cytoskeleton Inc. Bradford Ultra protein assay was from Expedeon while Precision Red™ Advanced Protein Assay Reagent from Cytoskeleton Inc. Clarity Western ECL substrate was from Bio-Rad. Nitrocellulose and Hybond-C extra membranes were from Amersham Life Sciences while Polyvinylidene difluoride (PVDF) membrane was from Roche.

Microscopic techniques: Collagen was from Gibco Invitrogen while Gelatin was from Sigma. Prolong antifade gold reagent was from Invitrogen. Phalloidin-TRITC, 6-diamidino-2-

57 phenylindole (DAPI) and Bovine serum albumin (BSA) were obtained from Sigma. Paraformaldehyde (PFA) was from Fisher. The o-phenylenediamine dihydrochloride (SIGMAFAST OPD) peroxidase substrate tablet was purchased from Sigma.

Lipid techniques: Triethylamine, Tetrabutylammonium Sulphate (TBAS), Ascorbic acid, Ammonium Molybdate and 80% perchloric acid were obtained from Sigma. HCl and EDTA were from BDH. Sodium deoxycholate monohydrate was from Afa Aesar. Neomycin sulfate, Methanol and chloroform were from Fisher. Uncoated Controlled pore glass beads were from Sigma. Lipid carrier 3 (P-9C3) and PtdIns5P (P-5016) were from Echelon. PtdIns(4,5)P2 from Avanti, and

PtdIns(3,4,5)P3 (64930) and PtdIns(3,4)P2 (64922) were from Cayman Chemical.

All other chemicals used for preparing buffers and solutions were of analytical grade.

2.1.1 Antibodies: Table 2. 1: Primary and secondary Antibodies

Primary antibody Primary Source Secondary Membrane/ Application (Catalogue No.) antibody antibody Vessel Dilution Dilution Rabbit PIP4K2α 1:1000 Sigma 1:3000 Nitrocellulose WB (241-255) Mouse Monoclonal 1:1000 Sigma 1:3000 PVDF WB ANTI-FLAG M2 (F3165) Mouse Monoclonal 1:1000 Cytoskeleton 1:3000 PVDF WB RAC1 (ARC03) Rabbit polyclonal 1:200 Santa Cruz 1:2000 PVDF WB GLUT4 Biotechnology (sc-7938) Rabbit SKIP 1:1000 Proteintech 1:4000 Nitrocellulose WB (4228-1-AP) Rabbit phospho- 1:1000 Cell Signaling 1:2000 Nitrocellulose/ WB AKT-S473 PVDF (9271) Mouse pan-AKT 1:1000 Cell Signaling 1:3000 Nitrocellulose/ WB (2920) PVDF Rabbit pan-PAK 1:1000 Cell Signaling 1:2000 Nitrocellulose WB (2604 ) Rabbit phospho- 1:1000 Cell Signaling 1:2000 Nitrocellulose WB PAK1 (Thr423)/ PAK2 (Thr402) (2601) Mouse β-actin 1: 3000 Sigma 1:6000 Nitrocellulose/ WB (A2228) PVDF Mouse monoclonal 1:2000 Thermo 1:2000 PVDF WB Anti-Na/K- ATPase scientific alpha-1 (MA3-929) Rabbit Anti-c-myc 1:500 Sigma 1:3000 Nitrocellulose/ WB polyclonal antibody PVDF (C3956) Mouse Anti-GST 1:2000 Santa Cruz 1:2000 Hybond-C PLO (B-14) Biotechnology extra Sc-138 nitrocellulose Sheep anti-GFP 1:1000 Lowe Lab, 1:2000 Hybond-C PLO University of extra Manchester nitrocellulose Mouse monoclonal 1:1000 Sigma 1:2000 Coverslip IF anti-Flag (F3165) Monoclonal anti 1:500 TONBO 1:2000 Coverslip/ IF/ Human-c-myc Bioscience Plate Colourimetric (9e10) assay (SKU: 70-6784 ) Mouse anti-Hexon 1:1000 Clontech. 1:500 Plate LM (632250) Abbreviations: WB, Western Blot; IF, Immunofluorescence; LM, Light microscopy; PLO, Protein Lipid Overlay; PVDF, Polyvinylidene difluoride

Secondary antibodies Source Species Horseradish peroxidase (HRP)-conjugated Dako Goat Goat anti- Rabbit (PO448) Horseradish peroxidase (HRP)-conjugated Dako Goat Goat Anti-Mouse (PO447) Anti-Mouse Alexa Fluor® 488 Invitrogen. Goat Anti-Mouse Alexa Fluor® 568 Invitrogen. Goat Anti-Rabbit Alexa Fluor® 568 Invitrogen. Goat

2.1.2 Buffers and solutions

Table 2. 2: Buffers and solutions

Buffer/Solution Composition 0.5xTBE 45 mM Tris base, 45 mM boric acid, 1mM EDTA 10x Running buffer 246 mM Tris base, 1.9M Glycine, 10%SDS 2x Separation buffer 750 mM Tris HCl pH 8.8 % 0.2% SDS 2x Stacking buffer 250 mM Tris HCl pH 6.8, 0.2% SDS 4x SDS sample buffer 160 mM Tris pH 6.8, 20% (v/v) glycerol, 4% SDS, 100 μg/ml bromophenol blue, 10% β-mercaptoethanol Agar plate 10g/l bacto-tryptone, 5g/l yeast extract, 10g/l NaCl, 20g/l agar Blotting buffer 25 mM Tris, 190 mM glycine, 20% methanol

Column Wash buffer 50 mM Tris pH8.0, 150 mM NaCl, 5mM MgCl2 Coomassie blue stain 0.05% (w/v) Coomassie Brilliant Blue R-250, 50% methanol, 10% acetic acid Differentiation medium αMEM containing 2% FBS Elution buffer Column wash buffer containing 20 mM reduced glutathione in 50 mM Tris pH 8.0 Growing medium αMEM containing 10% FBS HBSS 250 mM sucrose, 2 mM ethylene glycol tetra acetic acid

(EGTA), 5 mM NaN3, 20 mM HEPES pH 7.4 Homogenisation buffer HBSS containing protease inhibitor cocktail LB broth 10g/l tryptone, 5g/l yeast extract, 10g/l NaCl

PBS 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4 pH 7.4

PBS+ 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8.1 mM

Na2HPO4, 0.68 mM CaCl2, 0.49 mM MgCl2, pH 7.4 PBS-T PBS containing 0.1% Tween-20

Rac1 lysis buffer 50mM Tris pH 7.5, 10mM MgCl2, 0.5M NaCl, 2% Igepal CA- 630 containing protease inhibitor cocktail

Rac1 wash buffer 25 mM Tris pH 7.5, 30 mM MgCl2, 40 mM NaCl RIPA buffer 10 mM Tris pH 7.2, 150 mM NaCl, 5mM EDTA, 1% Igepal CA- 630, 0.5% w/v deoxycholic acid sodium salt, 0.1% w/v SDS,

200 μM sodium orthovanadate [Na3VO4], 10 mM Na-Beta

glycerolphosphate, 1x Protease inhibitor cocktail Stripping buffer 2% SDS in PBS containing 0.7% v/v β-mercaptoethanol Sucrose cushion 1.12 M sucrose; 20 mM HEPES pH 7.4; 1 mM EDTA TBS 0.1 M Tris, 0.3 M NaCl pH 7.4 TBST 0.1 M Tris, 0.3 M NaCl pH 7.4 containing 0.1% Tween-20 Lipid extraction

Elution solution 1 CHCl3: CH3OH:1M HCl (250:500:200 by volume)

Elution solution 2 CHCl3: CH3OH:2 M Triethylammonium bicarbonate (TEAB) (2:6:3 by volume)

PI binding solution CHCl3: CH3OH:H2O:2 M ammonium formate (100:200:16.8:3.2 by volume)

TEAB 2 M TEAB was freshly prepared by slowly bubbling CO2 gas

into a mixture of 18 ml H2O and 7 ml triethylamine on ice for about 1hour until a homogenous solution with pH7.5 to 8.0 was obtained.

Theoretical lower phase CHCl3: CH3OH: 1 M HCl (86:14:1 by volume) solution

Theoretical upper phase CHCl3: CH3OH: 1M HCl (3:48:47 by volume) solution 1 Theoretical upper phase 0.1M HCl, 5mM Tetrabutylammonium Sulphate(TBAS), 5mM solution 2 Na2EDTA pH 8.0: CH3OH :CHCl3 (0.8:1:2 by volume)

2.2 Cell culture

The work presented in this thesis is based on the rat L6 skeletal muscle cell line, which has been used in previous work in the host lab (see Chapter 1 introduction; section 1.4 . At the beginning of the project, late passage (P>12) L6 cells were used but their fusing and differentiation were of concern and therefore new low passage (P<4) cells were purchased and used. During the course of the work a cell line derived from L6 myoblasts which express GLUT4 that is tagged with myc polypeptide (Wang et al., 1998) became commercially available. This cell line has some technical advantages particularly in investigating GLUT4 translocation. Therefore, both of these cell lines have been used. Throughout this work ‘parental L6’ refers to rat L6 skeletal muscle cells while ‘L6-GLUT4myc’ refers to rat L6 skeletal muscle cells stably expressing GLUT4 myc tagged protein. However, when ‘L6 cells’ is used alone then it refers to both cell lines unless otherwise stated.

2.2.1 Rat L6 skeletal muscle cells Rat L6 myoblasts were routinely seeded at lower than 5 passages in growth Medium (αMEM with 5.5 mM glucose, containing 10% FBS) with penicillin (100U/ml), streptomycin (100 μg/ml) and L-glutamine (2 mM). The cells were incubated at 37°C in a humidified 5% CO2 atmosphere. Medium was replaced every 2-3 days. Cells were kept as monolayers until they reached 80-90% confluency. Differentiation of L6 myoblasts to myotubes was induced by dropping the serum

61 from 10 to 2% FBS. Cells were kept in differentiation medium for at least 10 days before further analysis. The fused myoblasts (myotubes) were examined under light microscopy and the differentiation was also confirmed by fluorescence microscopy where elongated multi-nucleated cells were formed.

2.2.2 AD293 cell culture AD293 cells, derivative of embryonic human kidney HEK293 cells, have improved adherence properties. AD293 cells were used for preparing the primary and the secondary (working) stocks of adenovirus.

AD293 cells were cultured in DMEM medium containing 10% FBS with penicillin (100 U/ml)/streptomycin (100 μg/ml). Cells were passaged every 3 to 4 days.

2.2.3 Trypsinisation Cells were sub-cultured from sub-confluent myoblasts or were re-seeded from differentiated myotubes in other vessels by trypsinisation. Cells were washed with PBS and detached by trypsin-EDTA and placed in the incubator at 37oC for 1-2 min. Trypsin was deactivated by adding medium containing serum.

The L6 myoblasts and the AD293 cells were stored by freezing cells in 10% Dimethyl Sulfoxide (DMSO) growth medium (αMEM for L6 and DMEM for AD293, both media containing 10%

FBS) in a -80oC freezer, or placed into liquid nitrogen for long term storage.

2.2.4 Cell counting Parental and L6-GLUT4myc (myoblasts and myotubes) cells were seeded into dishes. Myoblasts or myotubes were detached by trypsinisation and neutralised by αMEM containing 2% FBS. 0.4% trypan blue dye solution (w/v) was added and cells were counted under light microscopy using a haemocytometer.

total viable cells (unstained) Cell viability (%) = × 100 total cells (stained and unstained)

2.3 Molecular biology

2.3.1 PIP4K2α Plasmid preparation E. coli cells (XL10-Gold) harbouring PIP4K2α recombinant plasmid (pAdEasy-1) which had been previously verified and used in the host laboratory were cultured on 2% agar plates with kanamycin sulphate antibiotics. One colony was grown overnight in LB- broth containing 40 µg/ml kanamycin at 37oC with vigorous shaking. Plasmid DNA was extracted using a Maxi-prep kit (Qiagen). The Plasmid DNA was linearized using Pac I restriction enzyme and further purified using the Qiaquik extraction kit. The results of typical restriction and purification steps are shown in Figure 2.1.

Figure 2. 1: Purifying recombinant plasmid DNA extracted from E.Coli used for transfecting AD293 cells.

DNA analysed by 0.8% Agarose gel stained with Safeview showing: Lane 1 plasmid DNA (harbouring Flag-PIP4K2α) extracted from E Coli by HiSpeed Maxi-Qiagen Kit, Lane 2 plasmid DNA digested with PacI endonuclease, Lane 3 digested plasmid purified by Qiaquik extraction kit, Lane 4; DNA size marker (Hyperladder I).

2.3.2 Viral DNA transfection of AD293 cells with FuGENE®6

2.3.2.1 Primary stock A 15 µl aliquot of FuGENE®6 was added to 185 µl serum-free DMEM and then 5 µg linearize plasmid DNA was added. The mixture was kept at room temperature for 20 min and used for transfecting 50-60% confluent AD293 cells in 60-mm dishes. The infected cells were kept for up to10 days or until cytopathic effect (CPE) was observed: cells were lysed, rounded and floated. Cells were harvested by four 5 min-cycles of freezing in a dry-ice methanol bath and thawing in a 37oC water bath. The supernatant containing the viruses was then collected after spinning the tube at 12,000xg for 10 min. The amount of virus was estimated by Adeno-x Rapid Titer Kit.

2.3.2.2 Secondary adenovirus (working stock) AD293 cells were used to generate secondary stock by amplifying the primary virus stocks of LacZ or Flag-PIP4K2α. AD293 cells were infected with the primary virus at low multiplicity of infection (MOI<0.4). Cells were then harvested as described above (section 2.3.2.1) when CPE was observed. The amount of virus was estimated using Adeno-x Rapid Titer Kit.

2.3.3 Adenovirus titration The virus titration was carried out according to the manufacturer’s instructions. Briefly, AD293 cells were seeded in 24 well-plates to about 60% confluence then different amounts of virus were added using a range of PBS-diluted virus stocks. Cells were then incubated at 37oC for 48 hr. Cells were fixed using ice-cold methanol. The titration was done by detecting viral hexon protein in cells. Primary mouse anti-Hexon antibody was applied into the plate at 37oC for 1 hr. The wells were rinsed with PBS containing 1% BSA followed by incubating the wells with secondary rat anti-mouse (HRP-conjugated) antibody at 37oC for 1 hr. Wells were then washed with PBS and 1x Diaminobenzidine (DAB) substrate was added at room temperature for 10 min. This reaction gives brownish coloured spots that were counted under a light microscope (using a 10x Objective) and then converted into infectious units (ifu)/ml using the following formula

푐푒푙푙푠 푓푖푒푙푑푠 (푖푛푓푒푐푡푒푑 )푥( ) infectious units (ifu)/ml = 푓푖푒푙푑 푤푒푙푙 푣표푙푢푚푒 푣푖푟푢푠 (푚푙)푥 (푑푖푙푢푡푖표푛 푓푎푐푡표푟)

Examples of the appearance of uninfected and adenoviral infected cells are shown in Figure 2.2.

A B C

Figure 2. 2: Examples of titrated cells observed under light microscopy.

Different dilutions of adenovirus were used to infect AD293 cells, titrated by Adeno-x Rapid Titer -2 Kit. Panel A, Control cells (no virus), Panel B: CPE (10 dilution), Panel C example of field used for estimating the virus titre. Images were taken using a 10X objective.

2.3.4 Optimising the multiplicity of infection for L6 cells Parental and Glut4myc L6 cells were transfected with MOI using the secondary viral stock to select the best dose of virus expressing FLAG-tagged-PIP4K2α and LacZ. A range of MOI from 0 up to 80 per cell was used. Cells were lysed with Radio-Immunoprecipitation Assay (RIPA) buffer and the overexpressed protein was detected by western blot using anti-FLAG antibody.

Since two cell lines have been used, optimisation for both has been done. At the beginning, the number of cells was estimated from the surface area of the dishes but it has been observed that this estimation is not reliable. As has been observed and mentioned later in this work (section 3.1), L6 GLUT4myc cells were fused more efficiently than the parental cells and therefore their individual number becomes less following fusion, as shown by cell count.

2.4 mRNA Expression of Phosphoinositide 5-phosphatases

2.4.1 Extraction and Purification of mRNA Total RNA was obtained from L6 myotubes (parental and the myc tagged cells; including cells overexpressing PIP4K2α) using the Absolutely RNA Miniprep kit following the manufacturer’s instructions. Additionally, rat brain tissue was used as a positive control. The quality and quantity of the extracted DNA/RNA were assessed by agarose gel electrophoresis and by obtaining absorbance using a UV NanoDrop Spectrophotometer. Wavelengths of 260 and 280 nm were recorded. RNAs were stored at −80oC.

2.4.2 Reverse Transcriptase-PCR Complementary deoxyribonucleic acid (cDNA) was produced by Reverse transcriptase (RT) and polymerase chain reaction (PCR) in a single step using MyTaq One step RT-PCR kit. Primers were designed using the primer-blast web site. (http://www.ncbi.nlm.nih.gov/tools/primer-blast/).

Primers and the expected size of each of the RT-PCR products for the different PI5Pases tested are listed in table 2.3.

Table 2. 3: Primer sequences and expected sizes used for RT-PCR to examine the expression of nine PI5Pases.

PI5Pases Amplicon Forward Primer Reverse Primer (gene) (bp) 5- 5’-GAGCCTGCCCAGGTTT 5’-TCCTCCAATGGGCGCG phosphatase-2 152 CGACC AAGC (INPP5B) 75-5ptase 5’-ACAGGCAGCGAGGGAG 5’-TTCCCGCCTGTCAGCA 603 (INPP5E) GGAG GC OCRL 5’-CCTGGCAATGGTGGCGG 5’-GTGGAGGGGGTGGTTC 506 (INPP5F) TT CCGA 5’-CGAGGATCCCGTCTTGC 5’-CGGTGCTCACCAGCAC PIPP (INPP5J) 611 GC GAAGT SHIP1 5’-TACGCGCTCTGCGTGCT 5’-TGCTCCTCCGGGAGCC 419 (INPP5D) GT CACT SHIP2 5’-CTCCACTCCAAGCTTCC 5’-GGTGTGCACGTGCTTTT 276 (INPP5L1) CTG GAT SKIP 5’-AGCCTGCGTGGACTGA 5’-GCTCTAGCCTCCGCCC 583 (INPP5K) CCGA TGGA SYNJ1 5’-AGGAAGAGGGCTGCCT 5’-GGCAGGCGCATTGGCT 874 (INPP5G) CTGA TCAA SYNJ2 5’-GCCACCGTTCTCCGAG 5’-GAGCCTTGGGCGGTTG 205 (INPP5H) CAG CTGA

40ng/μl of total RNA from L6 myotubes or brain tissue were used and mixed with different components as shown in Table 2.4. Table 2. 4: Amount (in μl) of different components used for RT-PCR reaction.

Tube MyTaq Forward Reverse DEPC. RNA RiboSafe Reverse Total

One- Primer Primer H2O RNase transcriptase (μl) Step mix (10 μM) (10 μM) Inhibitor (2x) (10 U/μl) Sample 12.5 1 1 7.25 2.5 0.5 0.25 25 no-RT 12.5 1 1 7.5 2.5 0.5 0 25 control DEPC, Diethylpyrocarbonate

The mixture was then put in a thermocycler under the following conditions:

Reverse transcription: 45°C for 20 min 1 cycle Polymerase chain reaction: Initial Denaturation: 95°C for 1min, 1 cycle Denaturation: 95°C for 10 sec Annealing: 58°C for 10 sec 40 cycles Extension: 72°C for 30 sec Elongation 72°C for 5 min 1 cycle After the RT-PCR reaction, the products were analysed by agarose gel electrophoresis.

2.5 Agarose Gel Electrophoresis

Agarose powder was mixed with 0.5X TBE buffer and boiled. When the solution had cooled to about 60°C, SafeView was added and the gel was cast. Samples (DNA, RNA and PCR products) and DNA ladder were loaded on the gel and electrophoresis was run at 100 volts for about 45 min. Images were taken under UV using a Biorad gel documentation system.

2.6 Fluorescence microscopy

2.6.1 Coating plates and coverslips Myotube cells (particularly L6-GLUT4myc cells) were observed to detach from the 48-well plates and coverslips, so experiments to coat the plates and glass slides with 50 μg/mL collagen or 1% gelatine were conducted. During coating, coverslips were incubated for 1hr at room temperature under sterile conditions. Coverslips were washed with PBS before cells were seeded. Myotubes were differentiated in different flasks for 8 days and then trypsinised and seeded on the coated vessels.

2.6.2 Preparation of myotubes Parental L6 cells were differentiated in culture dishes and seeded on coverslips a day or two prior to the experiment. L6 GLUT4myc myoblast cells were seeded and differentiated for 10 days on coverslips. Cells were either infected with virus driving FLAG-tagged PIP4k2α or LacZ expression, or left without infection for 2-3 days followed by starving them for 4-6 hr. Cells were stimulated or not stimulated with 100 nM of insulin and incubated for different times. For detecting surface membrane proteins, PBS+ was used for washing and for preparing all buffers and solutions. Cells were kept on ice in the cold room for 10min, then washed with ice-cold PBS+ and incubated for 1 hr on ice at 4oC in the cold room with primary antibody (anti-myc 9e10 diluted 1:500 in 5% FBS). Cells were subsequently washed carefully 4 times with ice-cold PBS+. Coverslips were blocked with 5% FBS dissolved in 1x PBS+ for 15 min, prior to mild 3%

PFA fixation for 3 min at 4°C. Cells were washed again, and 0.1 M Glycine was added and incubated for 10 min to neutralise residual PFA. Cells were again washed 4 times with PBS. When cytoplasmic proteins were to be detected, cells were permeabilized with PBS/0.2% Triton after fixation.

Cells were probed and stained as follows: fluorescent FITC anti-mouse secondary antibody was used against primary FLAG-PIP4K2α. Alexa Fluor® 488 and Alexa Fluor® 586 were used to optimise detection of the cell surface GLUT4myc by the primary anti-myc antibodies (two different primary anti-myc were evaluated). TRITC-Phalloidin (red) and DAPI (blue) stain were used to visualize the actin cytoskeleton and nuclei respectively. Coverslips were then mounted onto slides with gold antifade.

2.6.3 Immunofluorescence Microscopy Wide-field upright (Olympus) or confocal (Nikon upright) fluorescence microscopes were used to obtain the images of cells probed with different stains and secondary immunofluorescent antibodies. Images for Wide-field were captured using Coolsnap ES camera (Photometrics) through MetaVue Software (Molecular Devices).

2.6.4 Confocal microscopy Confocal microscopy was used to get a more focused plane of GLUT4myc at the cell surface in comparison to the image obtained by wide-field microscopy. The adjustable pinhole and the excitation laser light of confocal allow generation of high resolution images. The confocal microscopy was set as follows: AlexaFluor 488 fluorophores were excited with a 488 nm line of the argon laser and 500-570 nm emission. Pinhole size (1 airy unit) and other variable factors were kept constant among compared images. ImageJ (National Institutes of Health) and Las-AF Lite (Leica Microsystems, Germany) software were used to view and analyse the images.

2.6.5 Quantification of total fluorescence using ImageJ ImageJ software was used for analysing images obtained from the wide-field fluorescence microscopes to obtain the corrected total cell fluorescent signal (CTCF). Briefly, cells and background area around the selected cells, the mean fluorescence of background readings area, and the integrated density are all obtained using measurements tool of ImageJ software.

CTCF is calculated using the following formula:

CTCF= Integrated Density – (Area of selected cell x Mean fluorescence of background readings)

Additionally, the measurements were normalised to the surface area of individual cells as described in McCloy et al., (2014). The average signals in different treated cells were compared and possibly significant differences were tested (see 2.18).

2.7 Biochemical assays

2.7.1 Subcellular fractionation (GLUT4 Translocation)

Stimulating myotubes with insulin results in the translocation of GLUT4 from intracellular compartments to the plasma membrane. This process allows glucose influx into the cell. Here the effect of overexpressing PIP4K2α on GLUT4 translocation was tested.

Ultracentrifugation fractionation was used to obtain 3 sub-fractions: Plasma membrane (PM), low (LDM) and high density microsomes (HDM). The method used was adapted from (Sumitani et al., (1997). Cells were starved overnight and then stimulated with 100nM insulin for 30 minutes or kept unstimulated. All steps were conducted in cold conditions (4oC). Cells were harvested by scraping in ice-cold homogenisation solution containing Roche protease inhibitor cocktail, and then subjected to 3 bursts of a tissue-tearor homogenizer followed by passing through a 22 gauge needle 10 times. The homogenate was then centrifuged at 19,000x g for 20 min. The resulting pellet was re-suspended in homogenization buffer and placed on top of a sucrose cushion followed by ultracentrifugation at 100,000xg for 1h. The interface of this step was collected and re-suspended in homogenisation buffer and centrifuged at 40,000xg for 20min; the pellet of this step was the PM fraction (P1). The supernatant from the first step (19,000x g) was centrifuged at 41,000xg for 20 minutes; the pellet was designated HDM (P2), while the supernatant was re-centrifuged at 180,000xg for 75 minutes, the pellet of this step contains LDM (P3). All three pellets were re-suspended in 20mM HEPES pH 7.4 containing 0.1% Igepal CA- 630 and stored at -80oC. The total protein in each sample was determined by the Bradford method. The presence of GLUT4 protein was detected in each fraction using anti-GLUT4 antibody for parental L6 myotubes.

The GLUT4 translocation of the L6-GLUT4myc myotubes was also explored using anti-myc antibodies. In comparison to the 225 cm2 flask used for detecting GLUT4 extracted from parental L6 myotubes, only a 60mm dish was needed for L6-GLUT4myc cells.

2.7.2 Colourimetric assay of surface GLUT4myc

The myc-tagged GLUT4 of the L6-GLUT4myc cells allows detection and investigation of GLUT4 translocation more practically than the previous method (section 2.7.1). L6-GLUT4myc-cells were cultured and differentiated for 10 days in 48well plates. On day 7, cells were either infected with virus ( LacZ or PIP4K2α) or kept without any infection. For SKIP knockdown, siRNA transfection was carried out as described in section 2.9.2 on days 6 and 8.

On day 10, myotubes were starved for 4 hr. For some cells, 100 nM wortmannin was added during the final 30 minutes of starvation, and was also present during the subsequent stimulation step. Cells were then either stimulated or not with 100 nM insulin for the desired time points (0-

30 min). Next, cells were placed on ice in the cold room for 15 min. Cells were then washed with ice-cold PBS+. Cells were blocked with 5% FBS for 10 min. Myotubes were then incubated with primary 9E10 myc mouse antibody (1:500) on ice in the cold room for 1hr. Some wells were incubated with blocking solution but without primary antibody as a negative control and used for subtracting background absorbance. 0.1% Triton X-100 permeabilization was done in some wells after fixation, followed by addition of primary antibodies. It is important to have positive and negative controls since cells were observed to be very sensitive and easily detached and floating during washing steps. After incubation with the primary antibody, cells were washed three times with PBS+ and fixed with 3% PFA for 5 min. Cells were then washed three times with PBS and remaining PFA was quenched with 0.1M glycine for 10 min, before washing again three times with PBS. Cells were then incubated with HRP secondary Anti-mouse antibody (1:1000) for 1hr followed by 5 washes with PBS.

O-Phenylenediamine dihydrochloride (OPD) was prepared freshly according to the supplier's protocol as follows: substrate and buffer tablets were dissolved in 24 ml of distilled water. 0.5 ml of the OPD reagent was added into each well of the 48 well-plate and incubated for 25 min at room temperature in darkness. Absorbance at 450 nm was taken by a microplate reader.

2.8 Rac1 assay

For extracting the active form of Rac1 (GTP-Rac1), parental L6 myoblasts were grown and differentiated on 60mm dishes. On the other hand, two 100mm diameter dishes were used for L6-GLUT4myc cells, the lysates of which were combined during lysing steps. Cells were infected with adenoviral vectors carrying either PIP4K2α or LacZ at 40 MOI and incubated for 2-3 days (3 days when SKIP was also knocked down). Knocking down SKIP was achieved by siRNA transfection carried out on day 6 and day 8 (see section 2.9). The myotubes were serum deprived for 4-6hr prior to harvest. Cells were then incubated with inhibitor or stimulated with or without 100 nM insulin for 20 min. Cells were then washed with ice-cold PBS and were lysed with Rac1 lysing buffer. The lysates were collected and spun in a refrigerated centrifuge (4oC) at 10,000xg for 1 min. Part of the extracted protein was used to quantify total protein to equalise the amount of protein across different samples. The lysates were snap-frozen in liquid nitrogen and stored in a -80oC freezer.

2.8.1 Rac1 Pull-down Activation Assay

The Rac1 assay was done using the Cytoskeleton Inc. kit, whereby the lysate containing equal amount of total protein (from section 2.8) was incubated with beads coated with PBD protein for 1 hr on a rotator in the cold room. During this step the active GTP-Rac1 is bound to the beads. The beads were then pelleted at 3500xg for 1 min in a refrigerated centrifuge. The supernatant was collected in new tubes while the beads were washed with Rac1-washing buffer. Part of the

70 supernatant was retained for total Rac1 blotting and to detect other proteins.10 to 20 µl of 2x SDS sample buffer were added to the beads and boiled for 5 min. The samples at this stage were ready for loading in 12% Polyacrylamide gel electrophoresis (PAGE). Western blot was used to detect the active and total Rac1 (~21 kDa) using mouse anti rac1 antibody. Proteins were transferred onto PVDF membranes and probed with primary and secondary antibodies that were diluted in Tris-buffered saline (TBS) containing 1% Tween 20 (without BSA).

2.9 SKIP siRNA Transfection

Preliminary experiments were conducted to test the effect of the transfection reagent on the cells. Due to the growth properties and the sensitivity of the myotubes, which were differentiated for 10 days (section 2.2.1), various protocols and conditions were tried to silence SKIP. SiRNA Oligo-nucleotides for SKIP knockdown were purchased from Origene. Three individual siRNAs and one control were received. The sequences of siRNAs (INPP5K (rat) 3 unique 27mer) were:

SR506835A: ACA CGA ACC UAC AGC AUU ACC ACG A SR506835B: GUA UAA CAA AGA AGC GCU ACA AAG A SR506835C: GGA AAU GAA UUA UGG GAU CAU AAG C

Each SiRNA was reconstituted with OriGene Duplex buffer and heated at 94oC for 2 min. During the optimisation, siRNAs were used individually and as a pool (a mixture of the same molar amounts of the three siRNAs (A, B, C)); also referred to as siSKIP. Additionally, universal scrambled siRNA (SR30004) was used as a negative control. SiTransfection (siTran 1.0) reagent was diluted in Opti-MEM. SiRNAs were diluted in the provided buffer to stock concentration (20µM) and working stock (5µM). Working siRNAs were mixed with transfection reagent and incubated at room temperature for 10 min prior to the transfection. On day 7 of differentiation, myotubes were infected with adenovirus expressing PIP4K2α or LacZ or kept without infection as a control. Cells were ready for the experiments on day 10, when they were serum deprived for 4-6 hr before being stimulated and harvested. The details of the optimal SKIP siRNA transfection conditions for the parental and GLUT4myc L6 are described below.

2.9.1 SKIP Knockdown in Parental L6 cells Parental myoblasts were seeded and differentiated on 60mm dishes. On day 6 of differentiation, myotubes were washed with PBS, detached by trypsinisation and trypsin inactivated by 2% FBS in αMEM. Cells were pelleted and the supernatant discarded. Cells were then suspended in 2.0 ml of serum-free, antibiotic-free αMEM. Transfection reagent was prepared by mixing 2µl siTrans and 8µl Opti-MEM. Diluted transfection reagent and siRNA were mixed together and incubated for 10 min at room temperature to form a complex. The siRNA-transfection complex was added to the detached cells and seeded on new 60 mm dishes for at least 6 hr (final siRNA

71 was 50nM). The αMEM was then replaced with differentiation medium αMEM (containing 2% FBS and antibiotics).The same procedure was repeated on day 8 (but with a final siRNA concentration of 75 nM).

2.9.2 SKIP Knockdown in L6-Glut4myc cells L6 GLUT4myc myoblasts were cultured and differentiated on 100 mm dishes. On day 6, myotubes were washed with PBS. Transfection reagent was prepared by mixing 4µl siTrans and 16µl Opti-MEM. Diluted transfection reagent and siRNA were mixed together and incubated for 10 min at room temperature to form a complex. The siRNA-Transfection complex was added to 4 ml of serum-free, antibiotic-free αMEM (final siRNA was 25 nM). After 6 hr, the medium was replaced by αMEM containing 2% FBS. The same transfection procedure was repeated on day 8 but with a final siRNA concentration of 37.5 nM.

2.10 Delivery of PtdIns5P by carrier 3

Preparation and delivery of the exogenous PtdIns5P was adapted and modified from Grainger et al., (2011). Lipid Carrier 3 (1 M) was reconstituted in distilled sterilized water and 1 M of synthetic PtdIns5P-diC16 was prepared by dissolving it in a solution with the ratio of 1:2:0.8 per volume of the following components CHCl3:CHOH:H2O. Carrier 3-PtdIns5P complex was prepared by drying the desired amount of PtdIns5P down with Nitrogen gas. The dried PtdIns5P and the carrier 3 were mixed and sonicated for 30 sec. The complex was then diluted with water, agitated and kept at room temperature for 10 min. The complex was then diluted 10 times with serum free αMEM and added to the serum-starved cells for 20 min. Since for each experiment different L6 cell line was used, the details of the volume of various components are stated below.

2.10.1 Effects of exogenous PtdIns5P on AKT and Rac1 activity To examine the effects of exogenous PtdIns5P on Rac1 activity, a complex of carrier3- PtdIns5P was introduced into the parental L6 cells (section 2.10). Parental L6 myotubes were seeded and differentiated in 60mm dishes for 10 days. Cells were washed with PBS and starved for 4 hr in serum-free αMEM. 10 µl of 1mM PtdIns5P was dried by Nitrogen gas and 10 µl of 1 M carrier3 was added. The mixture was diluted 10 times with free serum αMEM and mixed. 1.5 ml of the αMEM-carrier-complex was added to 60mm dishes (final concentration of 6 µM PtdIns5P). Samples were incubated either with carrier 3 alone or with the complex for 20 min. In some dishes, wortmannin (100 nM) was added 30 min prior to and throughout the incubation with carrier-PtdIns5P complex. Cell lysate was collected and Rac1 activation determined as described above (Section 2.8)

2.10.2 Delivery of PtdIns5P by carrier 3 to examine GLUT4myc translocation L6-GLUT4myc cells were seeded and differentiated on 48-well plates. Cells were serum starved on day 10 for 4h. 5.0 µl of carrier 3 was mixed with 10µl of (1 mM) PtdIns5P at a ratio of 1:2. Additionally, a third of the amount of complex was added to the same surface area in comparison with the parental L6, due to differences in cell number (see 2.3.4). Carrier 3- PtdIns5P complex was added for 20 min (final concentration of 4 µM PtdIns5P). Samples were then fixed and GLUT4myc translocation was assessed by the OPD-colourimetric method as explained in section 2.7.2. Similarly to the previous section, when 100 nM Wortmannin was used, cells were preincubated with the inhibitor for 30 min prior to the start of the experiment and the inhibitor was also present during delivery of the exogenous PtdIns5P.

2.11 Protein assay

The amount of proteins in cell lysates was quantified either using Bradford solution or using Precision Red Advanced Protein Assay Reagent. The latter was used for samples extracted using the Rac1 activation kit. Standard curves were prepared for each method using BSA as a standard. A NanoDrop spectrophotometer was used to read the absorbance at 590 nm. For each sample at least triplicate readings were taken to get the average concentration.

2.12 Western Blot

Proteins extracted from the harvested cells were snap-frozen and stored at -80°C (for GLUT4 and Rac1 assays) or at -20°C for other proteins. Equal amounts of protein as assessed by protein assay (section 2.11) were loaded onto polyacrylamide gels after mixing with SDS- sample buffer and boiling for 5 minutes (except for GLUT4 blotting: samples were heated up to 65°C). Electrophoresis was run on a gel consisting of a 4% stacking layer and 10 or 12 % separation layer of acrylamide gel at 150 V until the blue dye front had nearly run out of the gel. PVDF membrane was soaked in methanol for 30 sec before equilibration with blotting buffer. Proteins were then transferred from the gel to PVDF or nitrocellulose membranes in blotting buffer at a constant current of 100 mA for 3 hr or at 25 mA overnight. Subsequently, the membrane was blocked by 5% BSA dissolved in TBST for 1hr at room temperature. The membrane was incubated with primary antibody for 1-2 hr at room temperature or overnight at 4oC. Thereafter, the membrane was washed 3 times with TBST followed by incubation with the appropriate HRP-conjugated secondary antibody at room temperature for 1-2 hr. Next, the membrane was washed 5 times with TBST.

Finally, chemiluminescence signal was detected by the Bio-Rad documentation system after adding ECL reagent. The bands were quantified and analysed using Image Lab5 software. If the membrane was to be re-probed with different antibodies, a stripping buffer was used at room

73 temperature for 20 min. The membrane was then washed twice with TBST followed by blocking in TBST containing 5% BSA for 30 min. the membrane was then probed with the desired antibodies.

The antibodies used for detecting proteins by Western blot are listed in Table 2.1 and Table 2.2.

2.13 Protein lipid overlay

2.13.1 Generation & Purification of GST-GRP1 and GST-TAPP1 PH domains Transformed E. coli Bacteria expressing general receptor for phosphoinositides-1 (GST-GRP1- PH) or Tandem PH domain containing Protein-1 (GST- TAPP1-PH) were used and verified previously in the host lab.

2.13.2 Phosphoinositide Binding Domain preparation Recombinant bacteria, harbouring plasmids expressing verified GST-proteins when induced by Isopropyl β-D-1-thiogalactopyranoside (IPTG), were streaked from glycerol stock (-80°C) onto Lysogeny broth (LB) agar plates containing 50μg/ml ampicillin antibiotic and incubated overnight at 37°C. A single colony was inoculated overnight in 5ml LB containing ampicillin antibiotic at 37°C with shaking. 2.5ml of the broth was then inoculated into 250ml LB and incubated at 37°C with shaking for about 6 hours or until the absorbance at 600 nm was 0.5-1 AU. IPTG was added at a final concentration of 0.1mM and incubated overnight with shaking at room temperature. Samples before and after adding IPTG were aliquoted and induced expression of the target protein was visualised by Western blot and Coomassie blue staining (an example of a membrane for visualising GST-GRP1-PH is shown in Figure 2.3). Bacteria were centrifuged at 6000xg at 4°C for 15 min and the supernatant was decanted. The pellet (bacteria) was re- suspended in 250ml ice-cold column wash buffer and centrifuged. The supernatant was decanted again. The bacteria were then re-suspended in 20 ml of column wash buffer containing 1% Triton X-100, 1% Tween-20 and protease inhibitors, and kept on ice. The bacteria were disrupted by a sonicator probe. The suspension was sonicated four times on ice, each for 30 seconds. The lysate was then spun at 10000xg for 20 min at 4°C and the supernatant was used for protein purification.

2.13.3 Purifying the expressed protein Lysate was passed three times down a column of glutathione sepharose and washed using column wash buffer. GST-tagged proteins were eluted with elution buffer containing 20 mM reduced glutathione. Fractions of 0.5 ml were collected and the desired protein was visualised using PAGE and stained up with Coomassie blue. A Western blot was conducted using anti- GST to verifying the purified protein expression (Figure 2.3). The purified and total lysates were stored as 50% glycerol at −20oC.

Figure 2. 3: Expresson and purification of GST-GRP1-PH.

GST-GRP1-PH expression was induced by 0.1mM IPTG. Total Bacterial lysates were collected and purified into different eluted fractions using a glutathione sepharose column. Samples were separated on 10% PAGE and visualised using Coomassie blue stain (A) or immunoblot with Anti-GST Antibody (B). Lane 1; samples before IPTG induction, Lane 2-4; eluted fraction after IPTG induction, Lane 5; total lysate IPTG induction, Lanes between 4-5 are different eluted fractions and M; protein marker.

2.14 Neomycin beads: production and re-generation

To isolate PIs from the extracted lipids neomycin beads were used. The beads were produced following the procedure described by Schacht, (1978). Neomycin was coupled covalently to glass beads as follows: firstly 5 g of controlled pore glyceryl glass beads (diameter 500 Å, 120-

200 mesh) were mixed with 250 ml of 6 mM Sodium periodate (NaIO4) in a glass container for an hour at room temperature with continuous agitation. This reaction oxidized the beads to aldehyde form. During the reaction degassing was frequently applied to remove gas bubbles. After allowing the beads to settle, the liquid was decanted and the beads were washed 3 times with 250 ml distilled water. The oxidised beads were then mixed with 500 ml of neomycin sulphate (40 mM, pH 9 adjusted by NaOH). A 100 mg aliquot of sodium borohydride (NaBH4) was added to the solution for 20 min and degassing was applied. Another 100 mg of NaBH4 was added again after 20 min and degassing applied for a further 20 min. The beads were then washed successively with 250 ml each of the following: distilled water, 1 mM HCl, distilled water, and finally with 1:1 (volume: volume) methanol: distilled.H2O. The beads were stored in a dark container in 50% suspension (in 1:1 methanol: H2O).

50 µl of the 50% suspension was used for extracting PIs (obtained from a 60mm dish for parental L6 or a 100mm dish for L6-GLUT4myc). The neomycin coated beads were equilibrated in a solution containing ammonium formate. The beads were first washed with 1ml (5:10:2; ratios

75 per volume) of each of the following solutions; (CHCl3:CH3OH:H2O) then with (CHCl3:CH3OH: ammonium formate 0.5 M), then (CHCl3:CH3OH: H2O). Beads were finally re-suspended in

(CHCl3:CH3OH: ammonium formate 50 mM).

2.15 Lipid extraction

Two methods adapted from Jones et al.(2013), Grainger et al., (2011) and Guillou et al. (2007) were used and compared for extracting and purifying PIs. The first method (Method 1) was used mainly for extracting PIs to measure PtdIns(4,5)P2. When the same method was used for measuring PtdIns(3,4,5)P3 and PtdIns(3,4)P2, the signals were very weak or undetectable.

2.15.1 Method 1

For PtdIns(4,5)P2, parental L6 myoblasts were cultured and differentiated in 60mm dishes and the following volumes used for this size of dish. Cells were starved for 6 hr in serum-free αMEM and stimulated with 100 nM insulin for the desired time. Dishes were then washed with ice-cold PBS and cells lysed with 450 µl 1.2 M ice cold HCl. Cells were then scraped and the dishes washed with 600 µl methanol (CH3OH), and 500 µl of chloroform (CHCl3) was added and collected in microtubes. Samples were then vigorously mixed and centrifuged at 16,000xg for 1 min at room temperature (Tube1). The lower organic phase was collected into a fresh tube (Tube 2) and washed with 900 µl theoretical upper phase solution1. Samples were mixed and centrifuged as in the previous step and the lower phase was transferred to a new tube (Tube 3). For maximum lipid extraction, the upper aqueous phase from Tube 1 was washed with 450 µl theoretical lower phase solution. Tubes were centrifuged and the lower phase was pooled with the samples collected in Tube 3. The pooled sample (Tube 3) was placed in a vacuum centrifuge for about 30 min until samples were fully dried. The extracted lipids were blown by a stream of nitrogen gas and stored at -80oC until PI purification was carried out.

PIs were purified from lipid extraction using the following method adapted from Jones et al., (2013).

Dried samples (extracted lipids) were dissolved in 20 µl CHCl3 and 50 µl of 50% neomycin beads was added. Then, 950 µl PI binding solution was added and samples were mixed by rotation for 1hr at room temperature. Tubes were then spun at 1000xg for 5 min. The upper phase was removed and part of it was collected and used for measuring total phosphate (section 2.18). The beads were then washed twice with PI-binding solution and the supernatant was decanted. PIs that bind to the beads were eluted by adding 950 µl of elution solution 1. The tubes were mixed by rotation for 1 hr. The beads were then pelleted by spinning at 1000xg for 2 min. The supernatant was collected into a new tube containing 250 µl each of H2O and CHCl3. The contents were mixed, vortexed and spun at 1000xg for 2 min. The lower phase was then

76 collected and dried in a vacuum centrifuge. The purified dried samples were used directly, or stored at -80oC after blowing with nitrogen gas.

2.15.2 Method 2

For measuring PtdIns(3,4,5)P3 and PtdIns(3,4)P2, parental L6 and L6-GLUT4myc cells were seeded and differentiated into 60mm and 100mm dishes respectively. Cells were transfected with siRNA (see section 2.9) on day 6 and 8, and infected with virus for overexpression of PIP4K2α, or LacZ as a control, on day 7. On day 10 myotubes were starved and stimulated (or left unstimulated) with 100 nM insulin. Cells were washed with ice- cold PBS and lysed with 750 µl of 1M ice-cold HCl. Cells were then scraped and collected into 15 ml tubes (Tube 1). The dishes were further rinsed with 2 ml of 1 M HCl: methanol (4.5:16, by volume) containing 5 mM TBAS and pooled with the first extraction. To separate the phases, 3 ml of chloroform was added and tubes were vortexed vigorously. Samples were then spun at room temperature at 3000xg for 10 min. The lower organic phase from tube 1 was transferred into two new microtubes (Tube 2) and 1 ml of theoretical upper phase 2 was added. Tubes 2 were vortexed and spun at 16,000xg for 1 min. The lower layer of tube 2 was collected into new tubes (Tube 3).

To maximize lipid extraction, the upper phase of Tube 2 was added to the initial upper phase of the 15 ml tube (Tube 1). The combined upper-phases were re-purified by adding 1680 µl of theoretical upper phase 2. Tube 1 was then spun at room temperature at 3000xg for 10 min. The lower layer of Tube 1 was collected and pooled with Tube 3. The pooled lipid was placed in a vacuum centrifuge until completely dried. The dried lipids were stored at -80oC after applying a stream of nitrogen gas in the tubes.

Extracted Lipids were dissolved in 500 µl of (CHCl3:CH3OH: ammonium formate 50 mM) (5:10:2 per volume) and incubated with rotation at room temperature for 1hr with neomycin beads. Beads were spun at 4000xg for 1 min. Part of the supernatant was used for measuring total phosphate in each sample (see section 2.17). The PIs at this stage were bound to the beads. Beads were then washed twice with 50 mM formate solution. The PIs were eluted from the neomycin beads twice, firstly by 500 µl, and secondly by 250 µl of elution solution 2. In each elution step, tubes were rotated at room temperature for 1hr, followed by 3000xg centrifugation for 1 min. The supernatants from the two elution steps were collected and combined in a tube for drying. The eluted PIs were dried down overnight in a vacuum centrifuge at 45oC.

2.16 Protein Lipid overlay (PLO)

Dried eluted PIs (from sections 2.15.1 and 2.15.2) were dissolved in 25 µl chloroform. A range of PI standards were also prepared and diluted using chloroform. The amount of PI standard in each 5µl-spot was in the range of 0 to 200 pmole.

Samples and standards were spotted onto Hybond-C extra nitrocellulose membrane. The spotted samples were allowed to dry for 10 min. The membranes were then blocked by 5% BSA in PBS for 1hr. Membranes were probed with the appropriate PI binding domain overnight (GST-

GRP-PH for PtdIns(3,4,5)P3, GST-TAPP1-PH for PtdIns(3,4)P2 or GFP-tagged PLC-delta-PH for

PtdIns(4,5)P2) in 0.05% Tween-20/ PBS containing 3% BSA. The membrane was then washed four times with PBS containing 0.1%Tween (PBS-T) and then probed with anti-GST (1:2000) or anti- GFP (1:2000) for 1hr followed by washing four times with PBS-T for 1hr. GST-tagged PH- domains were then detected using anti-mouse HRP antibody (1:2000) while donkey anti-goat HRP-secondary antibody was used for detecting the GFP-tagged PH-domain using a 1 hour incubation. Finally the membrane was washed four times for one hour with PBS-T. Chemiluminescence signal was then detected by Clarity Western ECL Substrate (BioRad) and Image lab 5 (Bio-Rad Laboratories) was used to visualize and quantify the spots.

2.17 Total phosphorus measurements

Total Phosphorus assays of the extracted lipid samples were used to standardise the samples. Aliquots of samples were taken before the elution step; the lipid fraction that did not bind to the neomycin beads (see 2.15). 30 µl extracted from parental cells grown in 60mm dishes or 100 µl from L6-GLUT4myc cells grown in 100mm dishes were incubated overnight at 120oC in glass tubes. Additionally, phosphate standards (0-300 pmole) were prepared from Na2HPO4. Samples and standards were digested by adding 220 µl of 70% perchloric acid and tubes were heated to 170oC for 1 hr. To develop the colour, tubes were left to cool to room temperature then 1 ml distilled water, 600 µl 0.833% ammonium molybdate and 200 µl freshly prepared 10% Ascorbic acid were added. The mixture was mixed and incubated at 100oC for 7 min. The absorbance was then read at 630 nm. Standard curves were generated and the total phosphate for samples was calculated. To normalise the results, the signals obtained from spots of the membrane from the previous section (2.16) were divided by the total phosphate value obtained from this section.

2.18 Data analysis

Data obtained from different experiments were statistically analysed using GraphPad Prism 6 (GraphPad Software Inc). Data are presented as means ± standard error of the mean (SEM). Unpaired t test or Mann-Witney U test was used to compare between two groups. Analysis of variance (ANOVA) followed by Tukey’s Multiple Comparison Test or Dunn's post-hoc was used to compare between more than two groups.

Chapter 3

Comparison between parental and L6-GLUT4myc

cell lines and optimisation of techniques

Rat L6 skeletal muscle cells have been used widely in studying insulin dependent glucose uptake (Ijuin and Takenawa, 2012a; Grainger et al., 2011; Wilson et al., 1995). However, due to the laborious and time consuming techniques needed to follow the GLUT4 translocation in parental cells, the L6-GLUT4myc cell-line was constructed (Wang et al., 1998). At the beginning of this project, these cells were not commercially available. However, part-way through the project a commercial source was found, and the cells were obtained. Since the latter cells had not been used previously in the host lab, a basic comparison and optimisation was carried out for the two cell lines to ensure that the results obtained can be generalised for both cell lines.

The first section of this chapter examines the morphological pattern of the two cell lines followed by investigating the pattern of SKIP expression during differentiation. The chapter then presents the optimisation data for expressing Flag-PIP4K2α and for SKIP knockdown. Additionally, the expression profile of PI5Pases at the RNA level is documented. Evaluation of the methods used for detecting PIs is addressed at the end of this chapter.

3.1 Basic morphological comparison between L6-GLUT4myc and Parental L6

The data in this section shows the differences and similarities between the parental and L6- GLUT4myc. The two cell lines were used to obtain a complementary picture of the effects of various treatments on myotubes.

The first difference observed when L6-GLUT4myc was used was their morphological differences to the parental myotubes, which were clearly noticeable under light microscopy. This became even clearer when optimised treatment conditions (sections: 2.3, 2.9 and 2.10) for parental cells were applied to L6-GLUT4myc cells. L6-GLUT4myc could not tolerate the same amount of reagents. For example, L6-GLUT4myc could not stand same amount per volume of siRNA reagent or the same virus dose.

Under the microscope, myoblasts of the two cell lines look very similar (Figure 3.1A), unlike the myotubes (Figure 3.1B). When myoblasts reach high confluency and the serum in the medium is reduced to 2%, cells start fusing to form long multi-nucleated myotubes (Figure 3.1B and C). The morphological differences between myotubes could be most likely attributed to different rates of fusion between the two cell lines. The average number of fused cells was higher in L6- GLUT4myc which lead to a low number of individual myotubes in comparison to parental cells. This was verified by cell count, using trypan blue, of both myoblasts and myotubes (Figure 3.2). Staining nuclei of myotubes with DAPI and examining them under fluorescence microscopy showed a greater number of nuclei per myotube in L6-GLUT4myc (average 7-15) than in the parental cells (average 3-5 nuclei). The L6-GLUT4myc cells were also generally noticeably longer than parental cells.

Parental L6 L6-GLUT4myc A

Figure 3. 1: Morphological features of the parental L6 (left panels) and L6-GLUT4myc (right panels) cells.

Myoblasts (A) were seeded and grown in dishes or glass coverslips in αMEM containing 10% FBS until about 90% confluency. Cells were differentiated to myotubes (B and C) by dropping the FBS to 2% for 10 days. Cells in dishes were visualised by light microscopy (A and B). Cells on coverslips (C) were fixed and stained with DAPI to visualise nuclei and with Phalloidin-TRITC (red) to determine cell borders. Images were captured using Fluorescence microscopy. Bar scale 20µm.

Figure 3. 2: Cell viability of parental and L6-GLUT4myc cells before and after 10 days of differentiation.

Each cell line was seeded into two 35mm-dishes; one was used for myoblasts (MB) and the other for myotubes (MT). Cells were maintained in αMEM containing 10% FBS until 90% confluence (Day 0; Cell counting and protein extraction for myoblasts were done at this point). Cells were then differentiated by keeping them in αMEM containing 2%FBS for 10 days. Cells were counted using trypan blue on day 0 and day 10. (A) The graph represents averages of cell counts of two experiments. Few and similar numbers of dead cells (<3%) were observed in the two cell lines. Data on the graph are expressed as percentage of the number of myotubes (day 10) over the number of myoblasts (day 0). The remaining cells from each dish were lysed using RIPA buffer and β-actin was detected using Western blot (B).

There was about a 50% increase in the number of parental L6 myotubes (day 10) in comparison to myoblasts (day 0). This increase could be attributed to the ability of parental cells to divide and proliferate even though they were incubated in low serum (2%) medium. This is likely, as there were some parental cells with single nuclei beside other multi-nucleated cells (Figure 3.1 C). In contrast, at day 10 the number of L6-GLUT4myc myotubes was reduced by 60% in comparison to myoblasts (Figure 3.2A). In both cell lines, the viability of cells was high (dead cells<3%) and was apparently not affected by trypsinisation since when cells were trypsinised and re-seeded on day 8 the viability of cells compared to non-trypsinised cells on Day 10 was

82 unaffected (data not shown). Comparing the starting number (day 0) of L6-GLUT4myc myoblasts to the number by day 10 shows a noticeable drop (about 60% reduction). This reduction of L6-GLUT4myc numbers was also seen as a decrease in β-actin detected in cell lysates (Figure 3.2 B). The lower number of L6-GLUT4myc myotubes was most likely due to the fusing process during the 10 days of differentiation and was not a result of cell dying since the number of dead cells counted was very few.

As the work in this project progressed, trypsinising myotubes, particularly L6-GLUT4myc, and re- seeding them at lower density for microscopical investigation, was found to be challenging. Cells did not adhere uniformly, and instead they were rather aggregated. Coating the glass coverslips with collagen or gelatin as described in section 2.6.1 did not improve the adherence (not shown). Therefore, cells were grown and differentiated on the same coverslip. It is important to note that keeping cells on a glass coverslip for a long period (at least 13 days in this case) may have some negative effects on the rate of fusion and adherence properties compared to cells grown in culture plates.

3.2 GLUT4-myc expression

L6-GLUT4myc cells were used for examining the GLUT4 translocation in response to different treatments. The expression of the c-myc tagged GLUT4 in these myotubes was first established. Parental cells were used as a negative control in the Western blotting and immunofluorescence microscopy. Figure 3.3 shows the expression of myc tagged GLUT4 from total lysate of L6 myotubes, detected by immunoblot.

L6-GLUT4myc Parental

GLUT4myc

β-actin

Figure 3. 3: Validation of the expression of myc tagged GLUT4 in L6GLUT4myc myotubes by Western blotting.

L6GLUT4myc and parental L6 (as a negative control) were differentiated for 10 days in αMEM containing 2% FBS. Cells were lysed using RIPA buffer. Total cell lysates were separated on 10% SDS-PAGE and proteins were transferred onto a nitrocellulose membrane. GLUT4myc was detected by probing the membrane with Anti-myc antibody followed by the HRP-secondary antibody. β-actin was detected after the membrane was stripped and re-probed with anti- β-actin antibody.

In this work, L6-GLUT4myc cells were initially introduced, with the intention of investigating GLUT4 translocation using Immunofluorescence assays (Bradley et al., 2014; Antonescu et al., 2008). The myc tag is expressed on the first ecto-domain of the GLUT4 which is exposed on the outer surface of the plasma membrane after it is inserted in the membrane. This makes it ideal for assessing GLUT4 translocation in intact cells (Antonescu et al., 2008; Wang et al., 1998). To validate the specific detection of GLUT4myc, two anti-myc antibodies, a polyclonal and the 9E10 monoclonal (detailed in Table 2. 1) were used. Initially, the polyclonal anti-myc antibody gave high fluorescent signals even in the negative control, the parental L6. However, switching to the 9E10 monoclonal antibody improved the results and was used in the immunofluorescence techniques for evaluating GLUT4 translocation.

3.3 Expression of GLUT4myc during differentiation

Since different treatments and experiments were planned to be carried out on myotubes throughout the 10 days of differentiation, investigating GLUT4 expression would be useful to determine how to obtain maximum responses, and as a sign of full differentiation. The increase of the endogenous GLUT4 expression in myotube in comparison to myoblasts was previously reported in the L6 cells and was used as a marker of differentiation (Mitsumoto et al., 1991; Mitsumoto and Klip, 1992; Kaliman et al., 1996). Here, the expression pattern of the exogenous GLUT4myc is presented. Figure 3.4 shows the time course of expression of GLUT4myc throughout 10 days of differentiation. GLUT4myc expression was increased up to three-fold in comparison to the basal myoblast level. The Figure also shows that the maximum expression of GLUT4myc was achieved 7 days post differentiation.

Figure 3. 4: Time course of GLUT4myc-expression during cell differentiation.

L6-GLUT4myc cells were differentiated in αMEM containing 2% serum for up to 10 days. Cells were harvested with RIPA buffer and total proteins were separated using 10% PAGE. GLUT4myc expression was evaluated by Western blot using anti-myc antibody. The membrane was stripped using stripping buffer, and re-probed with a β-actin antibody. The graph represents GLUT4myc-expression normalized to β-actin.

3.4 Optimisation of the detection of GLUT-myc using the colourimetric Assay

Expressing myc tagged GLUT4 protein in L6-GLUT4myc cells makes measuring GLUT4 at the plasma membrane possible, and this can be done directly without the need of sub-fractionation or permeabilisation of the cells. Additionally, the specificity is increased by detecting the exogenous myc sequence. This also allows quantification of GLUT4 on plasma membrane colourimetrically. The colourimetric assay was optimised using 24-well plates. At the beginning of these experiments, L6-GLUT4myc myotubes appeared to be permeabilised by 4% PFA/PBS fixation for 10 min, and therefore 2% PFA dissolved in PBS+ for 3-5 min was used. This improves the reading by reducing the background, and this is particularly seen in unstimulated cells. Additionally, using monoclonal (9e10) anti-myc antibody also improves the specificity of the assay in comparison to the primary polyclonal anti-myc antibody (data not shown).

One reading set was used from collected points ranging from 15 to 25 min after adding the substrate (OPD). Absorbances at 450 nm were obtained from L6-GLUT4myc myotubes stimulated with 100 nM insulin for different time points (0, 5, 10, 15, 20, 25, 30, 40 min). Figure 3.5 shows the insulin-stimulated increase in the GLUT4myc signal on the plasma membrane compared to the unstimulated cells. Although there is about a two-fold increase at 5 and 10 min stimulation, the difference from the basal level was not statistically significant. Significant increases (3 to 4 fold) were achieved between 20 to 40 min of insulin stimulation. It is worth mentioning here that the reading of permeabilised cells was about 5-fold greater than that from non-permeabilised cells stimulated with insulin for 30 min. This indicates that the cell membranes of non-permeabilised cells have remained intact and only a fraction of the total GLUT4 in the cell was translocated to the plasma membrane.

6 0 0 )

l * * * *

t n

a * * *

o l

d 4 0 0

e n

t *

t s

m 2 0 0

% ( 0 0 5 1 0 2 0 3 0 4 0

T im e (m in )

Figure 3. 5: Colourimetric quantification of plasma membrane GLUT4myc.

L6-GLUT4myc cells were differentiated in 24-well plates for 10 days. On day 10, cells were deprived of serum for 4 hour. Myotubes were then stimulated with 100 nM insulin for the indicated times. Cells were probed with 9e10 monoclonal anti-myc antibody for 1 hour at 4oC. Cells were then fixed in 2% PFA for 5 min. Cells were probed with HRP-secondary anti-mouse antibody for 1 hour. Cells were incubated with OPD-substrate for about 25 min. Absorbance was taken at 450 nm using plate reader. The background absorbance (obtained from wells treated exactly with same procedure except were not blotted with the primary antibody) was subtracted from all readings. The results (means ± SEM; n=6-12) are expressed as a percentage of the basal unstimulated cells. Data were analysed by one way ANOVA, using Kruskal-Wallis test followed by Dunn's post-hoc analysis. Time points are compared to the unstimulated myotubes (0 min). *p<0.05, ***p<0.001, ****p<0.0001.

3.5 Optimisation of FLAG- PIP4K2α expression

This project aims to study the effects of PIP4K2α on insulin signalling by overexpressing it in L6 myotubes. At the beginning of the work, primary and secondary stocks of virus were prepared (see methodology; section 2.3) and the optimal doses of viruses in MOI were established by infecting parental L6 myotubes with adenovirus harbouring Flag- PIP4K2α for 2 days.

Figure 3.6 shows that the maximum expression was achieved at MOI of 40-80 PFU/cell. However, cells under light microscopy were observed to be de-attached and floated from the vessel at doses higher than 60 PFU/cell. MOI of 40 PFU/cell was found to be the optimal dose as FLAG-PIP4K2α overexpression was achieved and cells looked healthy (no sign of CPE).

Figure 3. 6: Optimisation of FLAG-PIP4K2α expression in parental L6 myotubes.

Myoblasts were differentiated in αMEM medium containing 2% FBS for 10 days. On day 8, cells were transfected with adenovirus harbouring FLAG-PIP4K2α at MOI doses ranging from 0-80. Infected cells were incubated for 48 hr. Cells were then lysed with RIPA buffer and equal amounts of total proteins were loaded on 10% PAGE. FLAG-PIP4K2α was detected with Western blot using anti-FLAG antibodies.

When the same volume (similar to the volume applied for parental L6) of virus per surface area of the vessels of cell culture was applied to L6-GLUT4myc myotubes, cells were detached and deteriorated. This was then corrected after cell counting (section 3.1) and after a further optimisation process. The optimal condition of Flag-PIP4K2α expression for L6-GLUT4myc was found to be also an MOI of 40 (the volume was adjusted according to the number of cells rather than total surface area of vessels) and the expression of Flag-PIP4K2α continued to be present and was detected for at least 5 days post infection (data not shown).

As this work includes some immunofluorescence experiments, it is important to ensure high efficiency of expressing Flag-PIP4K2α after viral infection. This was therefore examined using anti-Flag antibody under fluorescence microscopy. As shown in Figure 3.7, high efficiency of overexpressing PIP4K2α per cell was achieved by infecting myotubes with 40 MOI of adenovirus. Almost all cells were expressing Flag-PIP4K2α. Flag-PIP4K2α is detected throughout the cytoplasm (Figure 3.7D). Additionally and as expected no signal was detected

87 from LacZ cells (used as a negative control; did not express Flag-protein) when probed with Flag antibody (Figure 3.7 E).

D E

Figure 3. 7: Validation of adenovirus infection derived Flag-PIP4K2α overexpression using immunofluorescence microscopy.

L6 myotubes were cultured on coverslips in differentiation medium. Cells were infected with adenovirus driving PIP4K2α (A-D) or LacZ (E) expression at an MOI of 40 for 2 days. Cells were fixed and permeabilised. (A) to (E) Flag-PIP4K2α was visualised using anti-flag antibody probed with Alexa Fluor® 488 antibody (green). (B) DAPI (blue) was used to identify nuclei. (C) Merged images. Images were visualised under fluorescence microscopy using the X10 objective (A-C) scale bar 20µm, or x60 objective (D and E) scale bar 10µM.

3.6 Expression of mRNAs of PI5Pases

One of the hypotheses tested in this work is that the abolition of insulin-dependent increases of

PtdIns(3,4,5)P3 in cells overexpressing PIP4K2α is caused by the action of one or more PI5Pases (also referred to as INPP5P). The expression of these enzymes was initially screened at the level of mRNA. Total RNA extracted from rat brain, parental L6 myotubes, L6-GLUT4myc myotubes and L6-GLUT4myc myotubes overexpressing PIP4K2α was tested for the presence of different PI5Pases using RT-PCR. The RNA extracted from brain tissue was used as a positive

88 control since it expresses wide range of PI phosphatases. Each pair of primers (Table 2.3) was designed to cover an area at the junction between at least two exons in the DNA sequence to exclude any false positives due to amplification of contaminating DNA. Additionally, a negative control that did not have the RT enzyme was included for each test. Figure 3.8 shows that all tested PI5Pases are present at the level of mRNA. All amplicons were detected at the predicted size and the expressions of all tested PI5Pases in L6-GLUT4myc are not altered by overexpressing PIP4K2α. The results of negative and positive controls are as expected.

PI5Pase (Gene) Parental L6 Rat L6-GLUT4Myc myotubes Size Myotubes Brain uninfected PIP4K2α (bp) RT: - + - + - + - + 5-phosphatase-2 152 (INPP5B)

75-5ptase 603 (INPP5E)

OCRL 506 (INPP5F)

PIPP 611 (INPP5J)

SHIP1 419 (INPP5D)

SHIP2 276 (INPP5L1)

SKIP 583 (INPP5K)

SYNAPTOJANIN1 874 (INPP5G)

SYNAPTOJANIN2 205 (INPP5H)

Figure 3. 8: RT-PCR analysis of mRNA expression of PI5Pases in L6 myotubes.

L6 cells were differentiated for 10 days. Total RNAs were extracted from rat brain and myotubes of parental L6, L6-GLUT4myc and L6-GLUT4myc overexpressing PIP4K2α. MRNAs were extracted using an RNA Miniprep kit and amplified using the MyTaq One step RT-PCR kit. The amplicons were separated and visualised on 1-2% agarose gels stained with Safeview. RT: Reverse Transcriptase.

3.7 SKIP expression during cell differentiation

Reviewing published work on myotubes points to SKIP as a potential candidate for dephosphorylating PtdIns(3,4,5)P3 in the insulin signalling pathway (see section 1.2.3.4). Although RT-PCR analysis (section 3.6) did not exclude any of the PI5Pases, the expression of SKIP at the RNA level was clearly detected, and subsequent work focused primarily on this protein. Initially, the expression of SKIP in parental and L6-GLUT4myc cells was evaluated and compared. Myoblasts were differentiated for 10 days. Cells were lysed and SKIP expression was analysed using Western blot. The immunoblots in Figure 3.9 shows expression of two isoforms of SKIP (full-length; ~50 kDa and short; ~40 kDa) in both cell lines.

The expression of the full-length form, but apparently not of the short form of SKIP, was noticeably changed as cells were differentiating. Although the expression was fluctuating, an overall increase was observed as indicated in the graphs (Figure 3.9 A, B). Compared to the 9- fold increase in the SKIP expression of the parental myotubes, L6-GLUT4myc was only increased by 4 times at the sharp peak on day 9 (Figure 3. 9 C). Likewise, the SKIP expression was markedly increased earlier by three days in parental cells (day 3) in comparison to the L6- GLUT4myc cells (day 5-6). In general, the level of SKIP expression in the myotubes relative to the myoblasts is very high. Although the shape of the graph is broadly similar for the two cell lines (L6-GLUT4 myc myotubes to the parental cell line), the main differences seem to be in the level (of expression) of SKIP. Establishing the pattern of SKIP expression during differentiation is important not only as a marker of differentiation but also to help to get an idea of the level of SKIP expression which can be used to achieve the best conditions for silencing SKIP expression (section 3. 9). It is worth mentioning that a 60-mm dish of parental cells was enough for detecting SKIP in parental L6 cells while one or two combined 100-mm dishes were needed to detect it in L6-GLUT4myc myotubes. Additionally, the number of myotubes is most likely different in the two cell lines (as seen in section 3.1) making accurate comparison difficult. However, the data present in Figure 9.3 suggests a lower level of SKIP expression in L6- GLUT4myc in comparison to parental cells.

A: Parental L6 B: L6-GLUT4myc

Figure 3. 9: Expression of SKIP during L6 myoblast differentiation.

L6 myoblasts were grown to 90% confluence in αMEM containing 10% FBS. Differentiation was induced by switching the medium to αMEM containing 2% FBS. Total cell lysate from dishes were obtained daily using RIPA buffer from day 0 (myoblast) through to day 10. SKIP expression was detected by Western blot. The upper SKIP band (~51 kDa) represents the full- length isoform and the lower SKIP-band (~42 kDa) indicates the short form. After detection of SKIP, the membranes were stripped and re-probed with anti- β-actin. SKIP expression of Parental L6 (A) and L6-GLUT4myc (B) were normalised to β-actin expression. Results are displayed as arbitrary units (A, B) or as fold increases over the myoblast expression on day 0 (C). The graphs represent the level of the full isoform of SKIP obtained from one experiment. The experiment was performed in duplicate showing similar results.

3.8 Effect of PIP4K2α on SKIP expression

With the aid of previous studies (see section 1.4 overexpressing PIP4K2α in L6 cells was hypothesised to enhance insulin dependent activation of SKIP without affecting its expression. To test this hypothesis, the expression of SKIP in cells overexpressing PIP4K2α was compared to control cells. Since PIP4K2α overexpression was achieved using adenovirus, the control cells were also infected with adenovirus harbouring LacZ. The same dose of virus was used; MOI 40. Figure 3.10 indicates that PIP4K2α overexpression does not affect SKIP expression.

A B

Figure 3. 10: PIP4K2α overexpression does not affect SKIP expression.

L6 myotubes were infected with adenovirus harbouring LacZ or PIP4K2α for 48 hours. Cells were lysed using RIPA buffer and proteins were separated on 10% PAGE. Proteins were transferred to a nitrocellulose membrane. (A) SKIP expression was verified by Western blot using anti-SKIP antibody. Flag-PIP4K2α expression was detected using anti-Flag antibody. β- actin was used as a loading control. Graph (B) presents the average (Mean ± SEM) of SKIP expression obtained from 8 experiments. Data were expressed as percentage of SKIP expression of PIP4K2α compared to LacZ. Data were analysed using the Mann-Whitney U Test. No significant difference on the expression of SKIP was observed between LacZ and PIP4K2α expressing cells.

3.9 Optimisation of SKIP knockdown

Inhibiting SKIP activity would be the ideal way to investigate its role in myotube cells. Unfortunately, no pharmaceutical inhibitors are yet available and therefore knockdown of SKIP expression using siRNA was used. The effects of protein expression and activity were conducted on myotubes after 10 days from inducing differentiation. Viral infection and siRNA transfection were carried out before day 10. SKIP expression was then analysed by Western blot on day 10. In this section the optimisation of silencing SKIP mediated by siRNA in the two cell lines is shown. The condition of the transfection reagents was adjusted so that no affect was observed between transfected and non-transfected cells (data not shown).

3.9.1 SKIP knockdown in Parental L6 myotubes

At the beginning of the knockdown experiments, transfecting cells with individual or pooled siRNAs (A, B, C) for 24, 48 or 72 hours failed to reduce SKIP levels. However, introducing reverse transfection (Balasubramanian et al., 2014; Ovcharenko et al., 2005) where the transfection complex was added to the trypsinised parental L6 myotubes in suspension improved SKIP knockdown. About 20% reduction was achieved when cells were transfected once with the pooled siRNA. Another step of double-transfection was then tried. The transfections were conducted twice, on day 6 and day 8, each for 48h. Double-transfection reduced SKIP expression to about 40-50% as seen in Figure 3.11. Although siRNA-B seems to have a higher effect on depleting SKIP, due to variations on the results obtained between the experiments (data not shown) the pool of the three siRNAs (ABC) was used in the rest of the experiments in this study.

Figure 3. 11: Optimisation of SKIP knockdown in parental L6 myotubes.

Parental myoblasts were differentiated for 10 days in αMEM containing 2% FBS. SiRNA transfections were carried out using scrambled (SC) siRNA or with individual siSKIP (A, B, C) or as a pool of the three (ABC) on days 6 and 8. On the day of transfection, myotubes were trypsinised and suspended cells were transfected with siRNA in serum-free, antibiotic-free αMEM. After 6 hours, medium containing transfection reagent was replaced with fresh differentiation medium. Cells were transfected with the indicated siRNA at the following final concentrations: first transfection 50 nM, second transfection 75 nM. On day 10, cells were harvested using RIPA buffer. SKIP expression was analysed using Western blot. β-actin was used as a loading control.

3.9.2 SKIP knockdown in L6-GLUT4myc myotubes

Dramatic toxicity was observed when the optimal transfection conditions for parental L6 were applied to L6-GLUT4myc myotubes. Visualising transfected cells under microscopy showed detached, deteriorated and dead cells (data not shown). As mentioned before, the cell count (section 3.1) showed that the number of L6-GLUT4myc cells reduced during differentiation due to their hyper-fusion properties, suggesting a smaller amount of transfection reagent would be needed. Additionally, as cells fused more spaces were created between cells which facilitate the transfection process without need of a trypsinisation step.

Cells died when kept in medium containing the transfection complex overnight. Additionally, the efficiency of knocking down SKIP was reduced when cells were transfected in medium containing serum and antibiotics. Like the parental myotubes, the best transfection condition for L6-GLUT4myc was found when double-transfection was conducted on day 6 and 8. On each transfection step, cells were incubated with the transfection reagent for 6 hours and the medium was then replaced with fresh 2% FBS-medium and kept for 48 hr. Trypsinisation was found not to be required when GLUT4myc myotubes were used.

Figure 3.12 shows SKIP expression in L6-GLUT4myc myotubes transfected with scrambled siRNA and pooled si-SKIP (A, B, C) once, on day 8 only, or twice, on days 6 and 8. The Figure indicates a reduction of SKIP expression particularly when double-transfection was used. Additionally, the short isoform of SKIP is almost totally depleted compared to the full isoform. However, the full-length form of SKIP has higher phosphatase activity (100 fold higher) and is expressed more than the short form in skeletal muscle (Ijuin et al., 2008) and therefore this study focuses more on the full isoform. The Figure also indicates that cells tolerate both double transfection of siRNA and viral infection, and that SKIP knockdown is still present in virally- transduced cells. It was necessary to establish this before carrying out the next series of experiments.

Figure 3. 12: Optimisation of SKIP knockdown in L6-GLUT4myc myotubes.

Myoblasts were differentiated for 10 days. SiRNA transfections were conducted once on day 8 (+) or twice (++) on days 6 and 8. Cells were transfected with scrambled siRNA (SC) or pooled siRNA (siSKIP; ABC) in serum-free, antibiotic-free αMEM. After 6 hours, medium containing transfection reagent was replaced with differentiation medium. Cells were transfected with the indicated siRNA at the following final concentrations: first transfection 25 nM, second transfection 37.5 nM, and kept for 2 days. Myotube cells (ABC-2) were infected on day 7 with adenovirus for expressing Flag-PIP4K2α. On day 10, cells were harvested using RIPA buffer. SKIP expression was analysed using Western blot. Anti-Flag antibody was used to detect PIP4K2α overexpression. β−actin was used as a loading control.

Analysing collective data from 5 independent experiments showed a reduction (average 50%) of SKIP expression in samples treated with siSKIP compared to cells treated with scrambled control (Figure 3.13).

Figure 3. 13: Relative expression of SKIP in control and knocked-down L6-GLUT4myc cells.

Data were collected from 5 independent experiments. SKIP expression was normalised to β- actin. Results are expressed as fold decreases (mean ± SEM) of myotubes transfected with si- SKIP (si-ABC) compared to control cells (transfected with scrambled siRNA). Data were analysed with the Mann-Whitney U Test (*p<0.05).

3.10 Validation of Protein Lipid overlay (PLO) assays

3.10.1 Extraction methods

PLO assays were used to quantify the level of PIs. Two methods (section 2.15) were evaluated.

Method 1 was successful in measuring PtdIns(4,5)P2 (see next chapter, section 4.5.1) but the signals were very weak or undetectable when the same method was used for PtdIns(3,4)P2 or

PtdIns(3,4,5)P3. Figure 3.14 shows an example of this failure to detect signals from the two 3- phosphorylated PIs. However, the signals from the standards clearly illustrate the success of the GST- domains used (TAPP1-PH and GRP-PH) in detecting and quantifying the linearity of the

PtdIns(3,4)P2 and PtdIns(3,4,5)P3 standards respectively.

Figure 3. 14: Failure of extracting PIs (PtdIns(3,4)P2 and PtdIns(3,4,5)P3) using Method 1.

Samples were extracted from unstimulated and insulin stimulated myotubes grown in 100-mm dishes using Method 1 (details in section 2.15.1). Dried PIs were dissolved in 20 µl chloroform. 5 µl of standards and 10 µl of samples were spotted onto two Hybond-C extra nitrocellulose membranes (A, B). The numbers above the blots are the amount of synthetic PI in each standard (pmol per spot). The membranes were blocked with 5% BSA in PBS for 1 hour.

Membrane (A) was probed overnight with GST-TAPP1-PH to detect PtdIns(3,4)P2 while GST-

GRP-PH was used to detect PtdIns(3,4,5)P3 in membrane (B). The membranes were then washed using PBS-T and probed with Anti-GST antibody. The membranes were then probed with HRP-conjugated secondary antibody, washed with PBS-T and ECL-substrate was added. Images were visualised using a Gel-Doc system (BioRad).

3.10.2 Binding specificity of GST-TAPP1-PH

As the work in the project progressed, some unexpected results were observed particularly on the levels of PtdIns(3,4)P2 (discussed later in Chapter 4, section 4.5.3.2). In some experiments where cells overexpressing PIP4K2α, when these cells were stimulated with insulin for 10 min, unexpectedly, PtdIns(3,4)P2 did not increase as previously reported (Tavelis, 2012). Therefore the specificity of the GST-TAPP1 PH-domain construct used to detect PtdIns(3,4)P2 was validated to assure that this domain was specific for PtdIns(3,4)P2.

The specificity of GST-TAPP1-PH and selectivity to PtdIns(3,4)P2 was documented (Dowler et al., 2000) and has been previously validated in the host lab using a PIPTM Strip membrane (Tavelis, 2012). The PIPTM Strip membrane contained 100 pmol samples of 15 different lipids, including all PIs. The GST-TAPP1-PH was found to be exclusively bound to PtdIns(3,4)P2. To re-assess possible binding of GST-TAPP1-PH to PtdIns(3,4,5)P3, different amounts of

PtdIns(3,4,5)P3 and PtdIns(3,4)P2 standards were spotted onto the same Hybond-C extra nitrocellulose membrane and a PLO assay was conducted. Successful quantification of

PtdIns(3,4)P2 and PtdIns(3,4,5)P3 was achieved using Method 2, as shown in Figure 3.15 and 3.17 in addition to the Figures in Chapter 4 (section 4.5.2-3). Figure 3.15 shows high specificity and sensitivity of GST-TAPP1-PH towards PtdIns(3,4)P2 compared to PtdIns(3,4,5)P3 standards. However, due to the high dynamic range of quantifying Chemiluminescence in the Gel-Doc system (BioRad), signals from the PtdIns(3,4,5)P3 standard can be detected when the amount was more than 50 pmole. Detection of synthetic PtdIns(3,4,5)P3 standard using GST-TAPP1-PH domain in the current study, while not reported in the Tavelis (2012) study, could be possibly attributed to different practical factors including the washing off of lipids at different rates depending on their charge, the actual amount of lipids on the PIP-strip, or additionally the purity of the lipid standard. Although in this work highly pure PtdIns(3,4,5)P3 standard was used, the possible presence of PtdIns(3,4)P2 or other lipids, or dissociation of PtdIns(3,4,5)P3 standard during the preparation or during PLO assay cannot be fully excluded. However, it is important to emphasise that detection of 100 pmol or more of PtdIns(3,4,5)P3 in Figure 3.15 was accompanied with saturation of the spots of the PtdIns(3,4)P2 standard greater than 50 pmol. The rest of the results of PLO assays presented in this thesis are obtained from non-saturated spots where the signals of PtdIns(3,4,5)P3 standards were minimal or undetected when tested with GST-TAPP1-PH.

A B

Figure 3. 15: Comparing binding specificity of GST- TAPP1 to commercial PtdIns(3,4,5)P3 and PtdIns(3,4)P2 standards.

(A) Layout of spots. Zero to 200 pmol of synthetic PtdIns(3,4,5)P3 and PtdIns(3,4)P2 standards were prepared in chloroform and spotted on a Hybond-C extra nitrocellulose membrane. PLO assay was conducted. 0.5μg/ml of GST-TAPP1-PH was used for validating its binding specificity.

Another piece of evidence that excludes a significant effect from the possible binding of GST-

TAPP1 to PtdIns(3,4,5)P3 (from samples) is shown in Figure 3.16. The Figure presents a comparison of the levels of PtdIns(3,4,5)P3 and PtdIns(3,4)P2 in cells stimulated with insulin for different intervals. The same amount of the same samples was analysed using PLO. The Figure clearly indicates that the signals detected from the two PIs were not the same. For instance, at

30 min of insulin stimulation, PtdIns(3,4,5)P3 is at its maximum level whereas the PtdIns(3,4)P2 signal is at a minimum, demonstrating that the two probes are not reporting the levels of the same target.

The results presented in this section therefore confirm the validity of using GST-TAPP1-PH for quantifying PtdIns(3,4)P2.

P PtdIns(3,4,5) 3

P PtdIns(3,4) 2

Time (min) 0 10 20 30

Figure 3. 16: Comparison of PtdIns(3,4,5)P3 and PtdIns(3,4)P2 levels.

Myotubes were stimulated with 100 nM insulin for 0, 10, 20 or 30 min. PIs were extracted using Method 2. The same amounts of extracted PIs were spotted onto two Hybond-C extra nitrocellulose membranes. PLO analysis was applied using GST-GRP-PH and TAPP1-PH to detect PtdIns(3,4,5)P3 and PtdIns(3,4)P2 respectively. Signals were detected using a Gel-Doc system (BioRad).

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3.11 Discussion

The L6 myotubes were used in this work as a model for skeletal muscle cells. L6 cells were derived from rat skeletal muscle (Yaffe, 1968) and are widely used for studying insulin dependence of glucose uptake (Ijuin and Takenawa, 2012a; Grainger et al., 2011; Wilson et al., 1995). Although L6 myoblasts differentiate to polynuclear fibre-like structures, myotubes, they do not further develop sarcolemma or transverse tubules (T-tubules); the main parts of muscle tissue that expresses GLUT4 and hence glucose uptake (Lauritzen and Schertzer, 2010; Rudich and Klip, 2003; Wang et al., 1996). Therefore, differentiated myotube cells in culture do not closely mimic skeletal muscle and GLUT4 expression (Lizunov et al., 2012; Sylow et al., 2013a). In this work two L6 cell lines; parental and L6-GLUT4myc, were used. Although both cell lines were derived from the same origin (Wang et al., 1998), some morphological differences were observed between them. As they were the principle tool of this project, basic comparisons between them were therefore conducted. Additionally, although each cell line was initially designated for a particular set of tests (for example, GLUT4 translocation assays using L6- GLUT4myc while Rac1 activation test using parental L6 cells); all experiments have been initially conducted on both cell lines at least once to ensure that the biochemical investigations were not affected by the differences observed between the two cell lines. The results from the following experiments reveal that no appreciable difference was observed between the two cell lines. These results include the expression of mRNA of the PI5Pases and the effect of expressing PIP4K2α and knocking down SKIP on GLUT4 translocation, AKT phosphorylation, Rac1 activity and the levels of the measured PIs (see chapter 4). Therefore, it is safe to assume that the results obtained from one cell line are applicable for the other one.

3.11.1 Expression of the PI5Pases

The expression of mRNA of the nine PI5Pases in the two L6 cell lines was screened using RT- PCR and all screened PI5Pases (SHIP2, SHIP1, SKIP, 75-5ptase, Synaptojanin 1, Synaptojanin 2, OCRL, 5-phosphatase-2, PIPP) were expressed at the level of RNA. However, RNA expression does not always correlate with the expression of protein (Vogel and Marcotte, 2012). Reviewing the literature indicates that the expression of the following PI5Pases at the protein level was previously reported in L6 myotubes or rat skeletal muscle cells: SHIP2 (Huard et al., 2007; Sasaoka et al., 2001; Hou et al., 2010; Mandl et al., 2007), SHIP1 (Mandl et al., 2007), SKIP (Ijuin and Takenawa, 2012), 72-5ptase and OCRL (Hou et al., 2010). PIPP was detected in rat brain, heart and kidney but not in skeletal muscle using Northern blot (Mochizuki and Takenawa, 1999). Similarly, using Northern blot, Synaptojanin 1 and Synaptojanin 2 have been detected in rat brain and testis tissue but these two phosphatases were not detected in muscle

101 tissue (Nemoto et al., 2001). The data presented in this work show expression of PIPP, Synaptojanin 1 and 2 using RT-PCR; while the expression of these phosphatases was not reported by Northern blot, this difference could be attributed to the higher sensitivity of RT-PCR compared to Northern blotting. However, this may not necessary reflect the physiological role or protein expression status of these enzymes in mature skeletal muscle in vivo. Additionally, it is worth noting that not all PI5Pases are documented to hydrolyse PtdIns(3,4,5)P3 as a substrate in vivo (as outlined in table 1.3).

One aim of this project is to investigate one candidate from the nine PI5Pases that presumably dephosphorylates PtdIns(3,4,5)P3 in PIP4K2α overexpressing cells. Previous work showed that when cells were stimulated with insulin, PIP4K2α overexpression prevents PtdIns(3,4,5)P3 elevation which is accompanied by an increase in PtdIns(3,4)P2 (Tavelis, 2012) (see section introduction ). Using a SHIP2- inhibitor, it was further revealed that SHIP2 seemed not to be involved in the breakdown of PtdIns(3,4,5)P3 to PtdIns(3,4)P2 in myotubes overexpressing PIP4K2α (Tavelis, 2012). Although none of the other PI5Pases can be excluded, Synaptojanin 1, Synaptojanin 2 and PIPP were not previously reported in L6 cells at the protein level and their possible roles in glucose uptake have not been verified. SHIP1 is expressed mainly in haematopoietic cells, while the role of OCRL, 5-phosphatase-2 (INPP5B) and 75kD 5-pase (INPP5E) in glucose uptake has not yet been explored in muscle cells. Additionally, OCRL and

INPP5B have been reported to prefer PtdIns(4,5)P2 rather than PtdIns(3,4,5)P3 as a substrate (Schmid et al., 2004). Therefore, out of the nine PI5Pases, SKIP was chosen for study as a likely candidate that is activated in response to PIP4K2α overexpression. Following identification of

SKIP as a PtdIns(3,4,5)P3 phosphatase, its effect on cytoskeleton rearrangement was strongly suggested (Ijuin et al., 2000) and the Inhibitory effect of SKIP on GLUT4 translocation in L6 cells (Ijuin and Takenawa, 2003) and in mouse tissue (Ijuin et al., 2008) was documented. Attenuation of GLUT4 translocation in cells overexpressing SKIP is associated with low AKT phosphorylation, probably as a result of decreasing PtdIns(3,4,5)P3 (Gurung et al., 2003). Additionally, SKIP has been reported to inactivate the Rac-sensitive protein kinase PAK1, and SKIP-PAK1 interaction was found to be important in the cytoskeleton rearrangements (Ijuin and Takenawa, 2012b).The importance of SKIP is clearly noticed on the lethal effect of null-mutant Pps (no SKIP) mice whereas heterozygous SKIP mice are highly insulin sensitive (Ijuin et al., 2008).

3.11.2 SKIP expression

The L6 cells used in this work expressed two isoforms of SKIP, full-length and short. The full- length isoform is known to have much higher activity than the short and is expressed in insulin sensitive organs (Ijuin et al., 2008). The expression of SKIP (Figure 3.9) in the hyper-fused L6-

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GLUT4myc cells during differentiation was apparently lower and increased later than the parental L6 cells. Whether the level of SKIP expression between the two lines has an effect on the cell differentiation or the morphological differences between the two cell lines was not addressed in this work. However, SKIP expression was reported to have a negative impact on cell differentiation of mouse C2C12 muscle cells (Ijuin and Takenawa, 2012c) and it is gradually upregulated during skeletal muscle differentiation (Xiong et al., 2011). The level of SKIP seems not to be markedly affected by the overexpression of PIP4K2α (Figure 3.10). This is important to establish, to exclude the possibility that any effect of SKIP is not due to its overexpression, but rather due to its activation or, more probably, translocation to a particular cellular compartment. It has been reported that insulin and EGF stimulate translocation of SKIP to the plasma membrane in C2C12 cells and COS-7 cells respectively (Ijuin and Takenawa, 2012b; Gurung et al, 2003). However, whether overexpressing PIP4K2α affects SKIP translocation is not addressed in this work. Evidence for activation of SKIP and the effects of SKIP knockdown in myotubes are discussed in chapter 4.

3.11.3 SKIP knockdown

As mentioned previously, the size and number of nuclei per cell of the parental and L6- GLUT4myc myotubes were different probably due to the difference of fusing rate. This might have an effect on the transfection process of the SKIP knockdown. It is known that myotubes are more difficult to transfect than myoblasts (Balci and Dinçer, 2009; Neuhuber et al., 2002; Dodds et al., 1998). It has also been noticed that the uptake of the transfection complex is inversely proportional to the rate of fusion (Helbling-Leclerc et al., 1999). Moreover, since the differentiation process might be negatively affected by transfection reagents, as has been noticed in other skeletal muscle cell lines (Owens et al., 2013), the transfections were started after 6 days from the onset of the differentiation. By this time, as seen in the current work, the morphological and biochemical characteristics of myotubes were established. In this work, siRNA mediated knockdown successfully reduces SKIP expression to about 50% compared to the control cells of both cell lines. Nevertheless, this represents the total cell lysate: the efficiency of transfection in individual cells was not assessed.

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3.11.4 PLO

It is essential to validate the methods used in this project, particularly the laboratory-prepared materials. In the beginning of this work, two methods for measuring the PIs, named in this thesis as Methods 1 and 2, were validated. The results show the viability of detecting PtdIns(3,4,5)P3 and PtdIns(3,4)P2 using Method 2. From the serial attempts, the different elution steps might make the difference between the two. In Method 2 TEAB was added in the elution step, unlike in Method 1. Although Method 1 has been proposed for detecting these PIs in zebrafish embryos (Jones et al., 2013), the failure to detect them here might be also attributed to the very small amount of PtdIns(3,4,5)P3 and PtdIns(3,4)P2 compared to PtdIns(4,5)P2 in the samples extracted from the myotubes.

As mentioned previously (section 1.5 ), the central hypothesis of this project suggests that PIP4K2α overexpression may result in activating one of the PI5Pases. This hypothesis was based on the data from the previous work conducted by Tavelis (2012), which revealed elevation of PtdIns(3,4)P2 in PIP4K2α expressing cells when compared with LacZ expressing cells as control. This is specifically in insulin-stimulated cells, and it was corresponding decrease of insulin-stimulated PtdIns(3,4,5)P3 elevation. Some unexpected results with PtdIns(3,4)P2 in this work in cells overexpressing PIP4K2α are discussed in the next chapter. These surprising results obliged the project to validate the specificity and sensitivity of using TAPP1-PH domain as a selective probe for PtdIns(3,4)P2. Similar to previous work (Tavelis, 2012; Dowler et al.,

2000) specific binding of GST- TAPP1-PH domain to PtdIns(3,4)P2 was again confirmed in this work. The signals which might be detected from PtdIns(3,4,5)P3 only appear using the highly sensitive Gel-Doc system (BioRad), and where the accompanying PtdIns(3,4)P2 standards exceed the saturation level. At ideal non-saturated conditions, PtdIns(3,4,5)P3 is barely detected by the TAPP1 domain. Thus, it can be confidently stated that the signals detected by TAPP1-PH from the spotted samples obtained from cells were of the PtdIns(3,4)P2 lipid.

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Chapter 4

Investigating the effects of PIP4K2α overexpression

and SKIP knockdown in myotubes

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In the previous chapter, the results of comparison and optimisation of the methods for the L6 myotubes were presented and discussed. Here in Chapter 4, the results from applying those techniques are presented. The effects of PIP4K2α overexpression on insulin signalling were initially established. The investigation started with GLUT4 translocation, then AKT activation, followed by Rac1 activation and PAK phosphorylation. Subsequently the amounts and levels of the PIs were also examined. SKIP knockdown was introduced to investigate the possible role of

SKIP on depleting PtdIns(3,4,5)P3 in cells overexpressing PIP4K2α. As discussed later in this chapter, SKIP knockdown restored the level of PtdIns(3,4,5)P3, and so the possible effects of SKIP knockdown were again examined in insulin signalling.

4.1 Investigating the effect of overexpressing PIP4K2α on GLUT4 translocation

A previous study using L6 myotubes has shown abolition of insulin-dependent glucose uptake in cells overexpressing PIP4K2α (Grainger et al., 2011). However, whether this abolition was caused by a defect in the translocation of glucose transporter(s), most likely GLUT4, from the intracellular storage vesicles to the plasma membrane or was due to an inactive conformation of GLUT4 in the membrane, as described in other cell models (Ishiki et al., 2005; Wang et al., 1996), was not tested. To find out the most likely mechanism that might be affected, GLUT4 translocation was initially investigated using three different methods: subcellular fractionation, colourimetric assay and immunofluorescence microscopy.

4.1.1 Parental L6 cells

4.1.1.1 Subcellular fractionation

To investigate the distribution of GLUT4 between the PM and the two intracellular compartments; HDM and LDM, membrane fractions were initially isolated using an ultracentrifugation method and the presence of GLUT4 was examined in the control myotubes using immunoblots. Two bands were detected around 50kDa that are usually interpreted as different glycosylation forms of GLUT4 (Zaarour et al., 2012). Na+/K+ -ATPase was used as a marker for the plasma membrane fraction and to assess possible cross contamination between the fractions. The GLUT4 bands in Figure 4.1 reveal the response of myotubes to insulin as the intensity of the PM band with insulin is higher than the unstimulated cells. The Figure also indicates the separation of the PM from the intracellular compartments as indicated by the higher amount of Na+/K+ -ATPase present in the PM fraction compared to the HDM and LDM.

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Figure 4. 1: Detection of GLUT4 in subcellular fractions using an ultracentrifugation method.

Parental L6 myoblasts were differentiated for 10 days in 225 cm2 flasks in αMEM containing 2% FBS. Cells were serum-starved overnight and treated with or without 100 nM insulin for 30 min. Cells were sub-fractionated using ultracentrifugation into PM, HDM and LDM. Equal amounts of protein extracted from each fraction were separated in 10% PAGE and proteins were transferred onto nitrocellulose. The membrane was immunoblotted using Anti-GLUT4 to detect GLUT4. Na+/K+ -ATPase was used as a marker to assess the isolation of PM from other fractions. PM; Plasma membrane, HDM; High Density Microsome, LDM; Low Density Microsome.

Next, the effect of PIP4K2α on GLUT4 translocation was investigated by comparing the presence of GLUT4 in the plasma membrane of cells infected with adenoviruses harbouring LacZ (as a control), or PIP4K2α (Figure 4.2.A). Average data of two experiments is presented in Figure 4.1.B. The results are expressed as percentages relative to unstimulated cells. As the graph shows, LacZ cells treated with 100 nM insulin for 30 min show 2 fold increases in the amount of GLUT4 in the PM fraction compared to the unstimulated cells. In contrast, cells overexpressing PIP4K2α do not response to insulin and GLUT4 in the PM fraction does not increase. These results suggest that overexpression of PIP4K2α abolishes or attenuates insulin dependent- GLUT4 translocation in myotubes.

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Figure 4. 2: PIP4K2α overexpression abolishes insulin dependent-GLUT4 translocation.

Parental L6 myotubes were differentiated in 225 cm2 flasks in αMEM containing 2% FBS. Myotubes were infected with 40 MOI of viruses harbouring LacZ or Flag-PIP4K2α for 48 hour. Cells were serum-starved overnight and treated with or without 100nM insulin for 30 min. Cells were fractionated using ultracentrifugation. Equal amounts of the extracted protein from PM fractions were separated on 10% PAGE and GLUT4 detected using an immunoblot. Na+/K+ - ATPase was used as a marker for the plasma membrane (A). Average data of two independent experiments are shown as a graph (mean±SD) (B). Results are expressed as a percentage of the GLUT4 present in PM compared to the unstimulated control cells.

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4.1.2 L6-GLUT4myc

4.1.2.1 Subcellular fractionation

Similar to the parental L6 cells, experiments were also conducted using L6-GLUT4myc myotubes. As these cells expressing myc tagged GLUT4 that gives them the advantage of higher specificity and sensitivity of GLUT4 detection compared to the parental cells. One 60-mm dish of L6-GLUT4myc myotubes was adequate for detecting GLUT4 in the subcellular fractions. The signals detected from these cells were clearer and easier for quantification across the fractions. Similar to parental L6 cells, insulin increases the presence of GLUT4 two fold in the PM of the control cells. Likewise, the insulin-dependent GLUT4 translocation was abolished in the L6-GLUT4myc cells expressing PIP4K2α as shown in Figure 4.3. Insulin activates the translocation of GLUT4 from the intracellular compartments (HDM and LDM) to the PM fraction in control (LacZ) cells. In contrast, insulin does not increase the presence of GLUT4 in the PM of cells expressing PIP4K2α. GLUT4 stays in the intracellular compartments and is not mobilised to the PM in cells expressing PIP4K2α (Figure 4.3 A). Additionally, it seems that in cells expressing PIP4K2α, GLUT4 is more accumulated in the LDM fractions in comparison to control LacZ expressing cells. The total quantification of GLUT4 in the three fractions (PM, HDM, LDM) is apparently equal in all samples indicating that the expression of GLUT4 was not affected by PIP4K2α overexpression (data not shown). However, due to the use of the immunofluorescence techniques (see below), the maturation and / or localisation of GLUT4 in the subcellular compartments was not further investigated.

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Figure 4. 3: Effect of PIP4K2α overexpression on GLUT4 translocation in L6-GLUT4myc myotubes.

L6-GLUT4myc myotubes were differentiated in 60 mm dishes in αMEM containing 2% FBS. On day 8 post differentiation, myotubes were infected with 40 MOI of adenoviruses harbouring LacZ or Flag-PIP4K2α for 48 hours. Cells were starved overnight and treated with or without 100 nM insulin for 30 min. Cells were fractionated using ultracentrifugation into PM, HDM and LDM. Equal amounts of the extracted protein from each fraction were separated in 10% PAGE and proteins were transferred onto nitrocellulose. (A) The membrane was immunoblotted using Anti- myc to detect GLUT4. Average data of two independent experiments are shown as a graph (mean±SD) (B). Results are expressed as a percentage of GLUT4 present in PM in relation to unstimulated LacZ cells. PM; Plasma membrane, HDM; High Density Microsome, LDM; Low Density Microsome.

4.1.2.2 Intensity quantification of immunofluorescence images

Due to the technical difficulties and the time and material consuming nature of the ultracentrifugation method, two alternative immunoassays for assessing the GLUT4 translocation were also evaluated: quantification of the total surface GLUT4 fluorescence and the Immuno-colourimetric assay.

Quantification of the total surface GLUT4 was applied to images obtained from wide field microscopy. Representative images are shown in Figure 4.4.A. The corrected fluorescent intensities were calculated as described in section 2.6.5 and the results are presented as a graph in Figure 4.4.B. The graph indicates a significant increase in the intensity of the GLUT4myc fluorescence upon insulin stimulation in control cells. However, the signal intensity which corresponds to GLUT4myc translocation is abolished in myotubes expressing PIP4K2α. The presence of GLUT4myc at the cell surface was also investigated using confocal microscopy (Figure 4.4.C). The intensity of the fluorescence at the plasma membrane, as the images show, is higher in cells treated with insulin compared to unstimulated myotubes.

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Unstimulated Insulin (100nM; 30min)

Figure 4. 4: Cell surface fluorescence of GLUT4myc in L6-GLUT4myc myotubes.

L6-GLUT4myc cells were grown and differentiated on coverslips for 10 days. On day 8, myotubes were infected with adenovirus for expressing LacZ or Flag-PIP4K2α at MOI of 40 and kept for 2 days. On day 10, cells were serum-starved for 4 hours. Cells were treated with or without 100nM insulin for 30 min. Cells were probed with anti-myc antibody followed by PFA- fixation and Anti-Mouse Alexa Fluor® 488. (A) Representative images were captured with wide- field immunofluorescence microscopy. Objective X60. Scale bar: 5µm. (B) Total GLUT4myc fluorescence intensity was calculated using ImageJ software. Data were collected from 18-30 cells per condition from 2 experiments. Results are expressed as fold increase (means ± SEM) to the unstimulated control. Data were analysed using unpaired t test. *p<0.05. (C) Representative images captured with confocal immunofluorescence microscopy (Objective X63).

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4.1.3 GLUT4myc translocation using colourimetric assay

The conditions of this method have been optimised as shown in chapter 3 (section 3.4). To examine the effect of PIP4K2α overexpression on GLUT4 translocation, the time-course of GLUT4 translocation was examined using cells infected with adenovirus to express LacZ (as a control) or PIP4K2α as shown in Figure 4.5. The presence of GLUT4 in the LacZ cells continued to increase at the cell surface of the myotubes and the elevation is significant at 30 min of insulin stimulation. On the other hand, no significant increase was observed after insulin stimulation at the tested time points in cells expressing PIP4K2α. The results confirm the inhibitory effect of PIP4K2α overexpression on GLUT4 translocation.

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L6-GLUT4myc myoblasts were differentiated in 48-well plates for 10 days. On day 8, myotubes were infected with adenoviruses expressing LacZ (as a control) or Flag-PIP4K2α and were kept for 48 hours. On day 10, cells were deprived of serum for 4 hours. Myotubes were then stimulated with 100 nM insulin for the indicated times. Cells were probed with 9e10 monoclonal anti-myc antibody for 1 hour at 4oC. Cells were then fixed in 2% PFA for 5 min. Cells were probed with HRP-secondary anti-mouse antibody for 1 hour. Cells were incubated with OPD- substrate for 25 min. Absorbance was taken at 450 nm using a plate reader. The background absorbance was subtracted from all readings. The results (means ± SEM; n=6-8) were expressed as a percentage of the unstimulated (LacZ or PIP4K2α) cells. Data were analysed by one way ANOVA using Kruskal-Wallis test followed by Dunn's post-hoc analysis **p<0.01.

The results of this section obtained from the parental and L6-GLUT4myc cells indicate a defect in the translocation process in cells expression PIP4K2α.

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4.2 Effects of PIP4K2α on AKT phosphorylation

It has been previously reported that phosphorylation of AKT at Ser473 and Thr308 were not affected in myotubes overexpressing PIP4K2α (Tavelis, 2012). Since L6-GLUT4myc cells were not used previously in the host lab, and as proof of concept, the phosphorylation status of AKT at Ser473 (p-AKT-Ser473) was re-examined. There is no noticeable difference in AKT phosphorylation was observed between un-transfected cells and cells infected with virus harbouring LacZ in unstimulated or insulin stimulated conditions (data not shown). As shown in Figure 4.6, control myotubes stimulated with 100 nM insulin for 20 min results in phosphorylation of AKT-Ser473. Moreover and as the previous study reported, overexpressing PIP4K2α did not

p-AKT-Ser473

Pan-AKT

FLAG- PIP4K2α

Insulin: - + - +

LacZ PIP4K2α affect the phosphorylation of AKT.

Figure 4. 6: PIP4K2α overexpression does not affect insulin-dependent AKT-Ser473 phosphorylation.

L6 myoblasts were differentiated for 10 days in αMEM containing 2% FBS. Myotubes were infected with adenoviruses for expressing LacZ or Flag-PIP4K2α on day 8 and were kept for 48 hours. Cells were serum starved for 6 hours. Cells were then treated with or without 100 nM insulin for 20 min. Cells were harvested with RIPA buffer and total protein lysates were separated in 10% PAGE and subjected to Western blot using nitrocellulose membranes. AKT phosphorylation was detected using anti-p-AKT-Ser473 antibody. The membrane was then stripped and probed with pan-AKT antibody. Another Western blot was prepared to assess the expression of the Flag-PIP4K2α using anti-Flag antibody.

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4.3 Rac1 activity

Data from this work and the previous study (Tavelis, 2012) revealed that AKT phosphorylation was not affected in myotubes overexpressing PIP4K2α. It has been proposed that beside activation of AKT, Rac1 is necessary for translocation of GLUT4 to plasma membrane (Sylow et al., 2014; Khayat et al., 2000; L JeBailey et al., 2004). Insulin-dependent activation of AKT and Rac1 are apparently both downstream of PI3K (Sylow et al., 2014). Rac1 is involved in cytoskeleton rearrangement (Tapon and Hall, 1997) and is necessary for GLUT4 translocation (Sylow et al., 2014; Chiu et al., 2011). Thus, one of the aims of this project was to investigate the activity status of Rac1 in PIP4K2α overexpressing cells. GTP bound Rac1 (Rac1-GTP) is the active form of Rac1 and can be separated from the total Rac1 using an active Rac1 pull-down assay.

4.3.1 Insulin dependent activation of Rac1

Initially, the time course of insulin dependent Rac1 activation was established as shown in Figure 4.7. The active Rac1 can be detected as early as 5 min after 100 nM insulin stimulation. The activation continually increases and peaks at 20 min. Activation of Rac1 lasts for at least 30 min of insulin stimulation.

Time (min) 0 5 10 20 30

Rac1-GTP

Total Rac1

Insulin (100nM) - + + + +

Figure 4. 7: Time course of insulin-dependent activation of Rac1 in L6 myotubes.

Myotubes were incubated in differentiation medium for 10 days. Cells were starved for 6 hours and stimulated with 100 nM of insulin for the indicated time periods. Cells were harvested using Rac1 lysie buffer. GTP-bound Rac1 was pulled down by beads using a Cytoskeleton kit. Equal amounts of total protein lysates were used to purify the active Rac1 (Rac1-GTP). Purified Rac1 and total lysates were separated by 12% SDS-PAGE and were transferred onto PVDF membranes. Anti-Rac1 antibody was used to detect Rac1. Total Rac1 was used to assess the amount of loading.

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4.3.2 Effect of PIP4K2α overexpression on Rac1 activity

To investigate the effect of PIP4K2α on Rac1 activity, myotubes were infected with adenoviruses harbouring LacZ or Flag-PIP4K2α genes or were kept without any infection. There is no noticeable difference in Rac1 activity was observed between un-transfected cells and cells infected with virus harbouring LacZ in unstimulated or insulin stimulated conditions (data not shown). Cells were then stimulated with or without insulin for 20 min. Figure 4.8 shows the immuno-blot and the graph of the average Rac1 activity following different treatments. The Figure indicates that PIP4K2α overexpression does not affect basal levels of active Rac1. Furthermore, in comparison to the unstimulated cells, there is a 2-3 fold increase in Rac1 activity when control myotubes (LacZ) were stimulated with 100 nM insulin. However, this significant increase in control cells was lost in cells overexpressing PIP4K2α at 10 min (data not shown) and at 20 min (Figure 4.8) of insulin stimulation. Hence, PIP4K2α abolishes the insulin dependent activation of Rac1.

Figure 4. 8: Overexpression of PIP4K2α abolishes insulin-dependent activation of Rac1.

Parental myoblasts were differentiated in αMEM containing 2% FBS for 10 days. On day 8 cells were infected with adenoviruses driving expression of LacZ or Flag-PIP4K2α. Cells were serum- starved for 6 hours. Cells were then treated with or without insulin (100nM) for 20 min. Cells were lysed and 500µg of total protein of each sample was used to isolate the active form of Rac1 (GTP-Rac1) using a Cytoskeleton Kit. Equal amounts of total protein lysates were used to purify the active Rac1 (Rac1-GTP). Purified Rac1 and total lysates were separated by 12%

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SDS-PAGE and were transferred onto PVDF membrane. Anti-Rac1 antibody was used to detect Rac1. Total Rac1 was used to normalise the Rac1 activity. The results are expressed as percentages of the unstimulated LacZ cells (means ± SEM; n=5). Data were analysed by ANOVA followed by Tukey’s post hoc test (**p<0.01)

4.4 Insulin dependent activation of PAK

P21-activated protein kinase 1 (PAK1) is one of the downstream effectors of Rac1 in muscle cells. Rac1 was suggested to have a role in the activation of PAK1 by facilitating its phosphorylation (Sylow et al., 2013a; Ijuin and Takenawa, 2012b). Sylow et al., proposed the use of PAK1/2 phosphorylation (Thr423 and Thr402) as a marker for the Rac1 status (Sylow et al., 2013b; Sylow et al., 2014). Therefore, and since Rac1 activity was estimated using a relatively expensive kit, the possibility of using PAK as an indicator of Rac1 activity and the effect of PIP4K2α expression on PAK phosphorylation were investigated. Technically, the anti- phospho-PAK antibody used in this work is not specific for PAK1. It detects, besides PAK1, PAK2 and PAK3 when phosphorylated at Thr423, Thr402 and Thr421, respectively. However PAK3 is not expressed in muscle or L6-GLUT4myc myoblasts (Tunduguru et al., 2014).

4.4.1 Time course of PAK1/2 phosphorylation

Initially, the time course of phosphorylation of PAK1/2 in response to 100 nM insulin was established (Figure 4.9) using anti-phospho-PAK1(Thr423)/PAK2(Thr402) antibody. There was about a 4 fold increase in the phosphorylation of PAK1/2 as early as 5 min. The phosphorylation status peaked at 5 to 15 min of insulin stimulation and then declined, remaining around 2 fold of basal activity for at least 60 min.

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Figure 4.9: Time course of insulin-dependent phosphorylation of PAK1/2.

L6 myoblasts were differentiated in αMEM containing 2% FBS for 10 days. Myotubes were serum-starved for 6 hours. Cells were treated with or without 100 nM insulin for the indicated time (0-60 min). Cells were harvested with RIPA buffer. Equal amounts of lysates were separated using 10% PAGE followed by Western blot analysis. Phosphorylation of PAK1/2 was evaluated using Phospho-PAK1 (Thr423)/PAK2 (Thr402) antibody. β-actin was used to normalise the signal. Results are presented as immunoblots and as a graph (means ± SD). The graph shows the mean of fold-increases relative to the unstimulated cells that were collected from two experiments.

4.4.2 Effect of PIP4K2α overexpression on PAK1/2 phosphorylation

To investigate the effect of PIP4K2α on the activity of PAK, the phosphorylation at Thr423 and Thr402 was examined using Western blot. Figure 4.10 indicates that there is no clear effect of expressing PIP4K2α compared to LacZ. The phosphorylation status at different time points is apparently the same. However, in comparison to the un-infected cells (Figure 4.9), the cells infected with adenoviruses seem to have higher PAK1/2 phosphorylation in the absence of stimulation and at 3 min post insulin. Additionally, the decline of the activity is apparently faster in the infected cells compared to the un-infected cells. The previous Figure (4.9) shows maximum phosphorylation of uninfected cells at 10 min post stimulation whereas cells expressing LacZ or PIP4K2α driven by adenovirus (Figure 4.10) indicate that the phosphorylation is close to the basal level at 10 min. The results from these experiments

117 indicate that the use of p-PAK1/2 as a marker for Rac1 activity seems not to be reliable. The phosphorylation of PAKs does not mimic Rac1 activation as seen in the insulin-time courses (Figure 4.7 and Figure 4.9). For instance, Rac1 activity peaked after 20 min of insulin stimulation while PAK1/2 phosphorylation was very low at this point and peaked earlier around 10 min in the un-infected cells and even earlier (at 5 min; Figure 4.13) in cells infected with viruses. Therefore this approach was abandoned.

Figure 4.10: Phosphorylation of PAK1/2 is apparently not affected by PIP4K2α overexpression.

Parental L6 myoblasts were differentiated in αMEM containing 2% FBS for 10 days. Myotubes were serum-starved for 6 hours. Cells were infected with adenovirus driving LacZ or Flag- PIP4K2α expression on day 8 and kept for 48 hours. Cells were serum-starved for 6 hours and were stimulated with 100 nM insulin for the indicated times. Cells were harvested with RIPA buffer. Equal amounts of lysates were separated using 10% PAGE followed by Western blot analysis. Phosphorylation of PAK1/2 were evaluated using Phospho-PAK1 (Thr423)/PAK2 (Thr402) antibody. Pan-PAK antibody was used to normalize the signal. Anti-Flag antibody was used to assess Flag-PIP4K2α expression. Results are presented as immunoblots of cells expressing LacZ or PIP4K2α (Insulin: 0-20 min) (A) and as a graph (means ± SEM; n=3) showing the fold increase compared to the unstimulated cells (Insulin: 0-30 min) (B).

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4.5 Effects of PIP4K2α and SKIP knockdown on PIs

As discussed in Chapter 1, insulin signalling is mediated through PI3K resulting in cascades of robust changes of the PIs and their downstream effectors (such as AKT and probably Rac1).

PI3K generates PtdIns(3,4,5)P3 using PtdIns(4,5)P2 as a substrate. The level of PtdIns(3,4,5)P3 is regulated by phosphoinositide 3- and 5-phosphatases converting it to PtdIns(4,5)P2 or

PtdIns(3,4)P2 respectively. In this section the effects of PIP4K2α and SKIP knockdown on the levels of PtdIns(4,5)P2, PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are presented.

4.5.1 Effect of PIP4K2α on basal PtdIns(4,5)P2

PtdIns(4,5)P2 is the principle precursor of PtdIns(3,4,5)P3 and is generated mainly by PIP5K

(see introduction section). The higher levels of PtdIns(4,5)P2 compared to PtdIns(3,4,5)P3 and

PtdIns(3,4)P2 makes it easy to be detected but probably unsuitable for evaluating minor changes using the PLO assay (Method 1). In the beginning of this work and as part of evaluating the methods of extracting PIs from myotubes, PtdIns(4,5)P2 was measured in unstimulated cells.

Although little change was expected in the level of PtdIns(4,5)P2 through activating the PIP4K2α pathway, and as a proof of principle, the possible effect of PIP4K2α on the basal level of

PtdIns(4,5)P2 was therefore examined using Method 1 (section 2.15.1). As shown in Figure 4.11, the representative blot and the graph indicate that no change on the basal level of PtdIns(4,5)P2 was detected.

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Figure 4.11: PIP4K2α overexpression does not affect basal levels of PtdIns(4,5)P2.

Parental L6 cells were differentiated on 60-mm dishes for 10 days. Cells were un-infected or infected with adenovirus expressing LacZ or PIP4K2α on day 8 and were kept for 2 days. On day 10, myotubes were serum-starved overnight and lipids were extracted using Method 1. Dried PIs were dissolved in chloroform and were spotted onto Hybond extra nitrocellulose membrane. PtdIns(4,5)P2 was detected using GFP-PLC-delta-PH domain. Results of the basal level of PtdIns(4,5)P2 are expressed as (means ± SEM) from 5 experiments. Data were analysed by ANOVA. No significant difference was observed.

4.5.2 PtdIns(3,4,5)P3 measurements

4.5.2.1 Time course of PtdIns(3,4,5)P3 production

As mentioned previously, PIs are tightly regulated by kinases and phosphatases. These enzymes play important roles particularly during stimulation. In this work, the responses of L6 myotubes toward 100 nM insulin were investigated. In the beginning, insulin time courses of

PtdIns(3,4,5)P3 changes were established using a PLO assay. PIs were extracted using Method 2 (section 2.15.2). Figure 4.12 A shows examples of the standard curves of known amounts of PIs. The standards were plotted to assess the linearity and the amounts of PI in extracted samples.

Figure 4.12 B expresses the levels of PtdIns(3,4,5)P3 corrected for the total lipid phosphate (section 2.17) in each sample. The data are expressed as percentages of the basal level in unstimulated cells. As shown in the Figure, the levels of PtdIns(3,4,5)P3 fluctuate after stimulation. An initial peak appears at 5 min followed by a second, wider peak observed at 20-30 min after insulin stimulation. The increase of these peaks was at least two fold compared to the unstimulated cells. This experiment was repeated twice and similar patterns of the peaks were obtained.

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Figure 4.12: Time course of PtdIns(3,4,5)P3 changes following insulin stimulation.

L6-GLUT4myc myoblasts were grown in 100mm-dishes and were differentiated in αMEM containing 2% FBS for 10 days. Cells were serum-starved overnight. Myotubes were stimulated with or without 100 nM insulin for the indicated times (0-60 min). Lipid extraction and PI purification were conducted using Method 2. Known amounts of synthetic standards (A) were prepared in chloroform and were spotted on Hybond-C extra nitrocellulose membranes. Dried PIs from the extracted samples were dissolved in chloroform. PLO assay was applied using GST-GRP1 PH domain. The chemiluminescence intensity of the lipid spots were quantified using Image Lab software and data were corrected for total lipid phosphate. The experiment was repeated twice. (B) The percentages of corrected results normalised to unstimulated samples (0 min) from a single experiment are shown.

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4.5.2.2 Effect of PIP4K2α overexpression on PtdIns(3,4,5)P3

The results using un-infected cells from the previous section (4.5.2.1) showed a fluctuating pattern of the levels of PtdIns(3,4,5)P3 in response to insulin. The same experiment was then repeated using cells infected with adenoviruses to express LacZ as a control or with PIP4K2α.

PIP4K2α overexpression did not have a significant effect on the basal level of PtdIns(3,4,5)P3 (Figure 4.13 A).

The early peak (at 3 or 5 min) of PtdIns(3,4,5)P3 previously observed in the presence of insulin (Figure 4.12B) was detected in some but not all experiments, both in cells expressing LacZ as well as in PIP4K2α expressing cells at 3 and 5 min of insulin treatment. Figure 4.13 B shows the individual results of 5 independent experiments conducted on cells expressing either LacZ or

PIP4K2α. The pattern of the levels of PtdIns(3,4,5)P3 seems not to be affected by PIP4K2α at least at 3 min of insulin stimulation. The transient nature of this early peak may explain why it is not detected in all experiments. This early increase of PtdIns(3,4,5)P3, particularly in PIP4K2α- cells is likely to be responsible for the observed AKT phosphorylation in cells overexpressing PIP4K2α (see section 4.2).

Figure 4.13 C presents the average levels of PtdIns(3,4,5)P3 from 3 to 7 experiments, and indicates that there is about 1.5 to 2 fold increase at 20 to 30 min of insulin stimulation in control cells whereas the level is similar to the basal level for cells expressing PIP4K2α. . When data are averaged in this way the early peak at 3 or 5 min is diminished in comparison to the individual values presented in Figure 4.13 B and no statistically significant difference is seen. This discrepancy is likely to be due to the fact that the early peaks are of short duration and vary between individual experiments, being detected at slightly different times either at 3 or 5 min, but not at both time points; this has been observed in 2 experiments where the insulin stimulations were conducted at 3 and 5 min in the same experiment. However, the later, more prolonged and statistically significant PtdIns(3,4,5)P3 increase that peaks around 20 min of stimulation is abolished in cells expressing PIP4K2α.

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0 0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 T im e (m in )

Figure 4.13: Basal and time course of insulin-stimulated changes in PtdIns(3,4,5)P3 levels in myotubes expressing LacZ or PIP4K2α.

L6-GLUT4myc myotubes were grown in 100mm-dishes and differentiated in αMEM containing 2% FBS for 10 days. On day 8 myotubes were infected with 40 MOI viruses for expressing LacZ or PIP4K2α and were kept for 48 h. Cells were serum-starved overnight. Myotubes were stimulated with or without 100 nM insulin for the indicated time intervals. Lipid extraction and PI purification were conducted using Method 2. Dried PIs were dissolved in chloroform and spotted on Hybond-C extra nitrocellulose membranes. GST-GRP1 PH domain was used to detect

PtdIns(3,4,5)P3. The chemiluminescence intensity of the lipid spots were quantified using Image Lab software and data were corrected to total lipid phosphate. (A) Comparison of the basal

(unstimulated) PtdIns(3,4,5)P3 level between cells expressing LacZ or PIP4K2α. (n=13, Mann-

Whitney U test). (B) Levels of PtdIns(3,4,5)P3 from individual experiments, at 3 and 5 min of insulin stimulation on cells expressing either LacZ or PIP4K2α (n=5). (C) Data are expressed as a percentage of the basal level of PtdIns(3,4,5)P3 seen in unstimulated cells expressing either LacZ or PIP4K2α after correction for the total lipid phosphate. Results are the means ± SEM of 3 to 7 experiments. Data were analysed by one way ANOVA using Kruskal-Wallis test followed by

Dunn's post-hoc analysis *p<0.05.( PtdIns(3,4,5)P3 levels are significantly lower in PIP4K2α expressing cells compared to LacZ expressing cells at 20-30 min).

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4.5.3 PtdIns(3,4)P2 measurements

One of the main aims of this study is to investigate the possible contribution of PI5Pases in accelerating the breakdown of PtdIns(3,4,5)P3 in cells overexpressing PIP4K2α. It has been hypothesised that PtdIns(3,4,5)P3 might be dephosphorylated to PtdIns(3,4)P2 by SKIP (or other PI5Pases) in cells overexpressing PIP4K2α (see introduction Section 1.4 ). This hypothesis was based on the observation from previous work that the reduction of the amount of PtdIns(3,4,5)P3 in PIP4K2α expressing cells was accompanied by an increase in PtdIns(3,4)P2 (Tavelis, 2012).

Section 4.5.2.2 indicates that PIP4K2α prevents the insulin-stimulated PtdIns(3,4,5)P3 increase.

Since new L6 cell lines were used in this work, the levels of PtdIns(3,4)P2 were also examined.

4.5.3.1 Time course of PtdIns(3,4)P2 production

Initially the standard curve of synthetic PtdIns(3,4)P2 and the time course of insulin action on the level of this lipid was established. As Figure 4.14 indicates, and similar to the pattern seen with

PtdIns(3,4,5)P3, PtdIns(3,4)P2 levels also fluctuated with a peak at 10 min and another around

25 min. The level of PtdIns(3,4)P2 approximately doubled at those points.

A B

2 5 0

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a l

) 1 5 0

(

n i

n 1 0 0

P o

% 5 0

0 0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0

T im e (m in )

Figure 4.14: Time course of PtdIns(3,4)P2 changes following insulin stimulation. L6-GLUT4myc myoblasts were grown in 100mm-dishes and were differentiated in αMEM containing 2% FBS for 10 days. Cells were serum-starved overnight. Myotubes were stimulated with or without 100 nM insulin for the indicated times (0-60 min). Lipid extraction and PI purification were conducted using Method 2. Known amounts of synthetic standards

PtdIns(3,4)P2 (A) were prepared in chloroform and were spotted on Hybond-C extra nitrocellulose membranes. Dried PIs from the extracted samples were dissolved in chloroform and were used for PtdIns(3,4)P2 measurement. PLO assay was applied using GST-TAPP1 PH domain. The chemiluminescence intensity of the lipid spots were quantified using Image Lab software and data were corrected for total lipid phosphate. The experiment was repeated twice giving similar results. The graph (B) shows the percentages of corrected results normalised to unstimulated samples (0 min) and is representative of a single experiment.

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4.5.3.2 Effect of PIP4K2α overexpression on PtdIns(3,4)P2

The insulin-time courses of changes in PtdIns(3,4)P2 in control cells expressing LacZ and in PIP4K2α over-expressing cells were also established. PIP4K2α overexpression does not affect the basal level of PtdIns(3,4)P2 (Figure 4.15A). As shown in Figure 4.14B, un-infected cells display fluctuating responses throughout the insulin stimulation. Similarly, cells expressing LacZ show the same pattern for the levels of PtdIns(3,4)P2. However, as was the case with

PtdIns(3,4,5)P3 measurements (Figure 4.13), the averaged responses are again less informative than individual experiments, due to the high variability between the experiments which leads to noticeable effects on the average results. Also as with the PtdIns(3,4,5)P3 measurements (Figure 4.13), there appears to be a shift in the exact time of the peaks from one experiment to another. This shift is reflected by the large error bars seen in the graphs presenting the average levels (Figure 4.15D). Similar to the observation of the early peak(s) of PtdIns(3,4,5)P3 (Figure

4.13 B), there was also an early elevation of PtdIns(3,4)P2 level at 3 min of insulin stimulation in most experiments (Figure 4.15 B). In most, but not all experiments, cells overexpressing

PIP4K2α showed higher levels of PtdIns(3,4)P2 than control cells in the first 15 min of insulin stimulation (Figure 4.15 C representative of a single experiment). However, this increase apparently is reversed after 20 min insulin stimulation: the amount of PtdIns(3,4)P2 detected 20 to 30 min after insulin treatment in cells expressing PIP4K2α was lower than the control cells (LacZ). These unexpected results lead to carry out experiments to validate the detection methods as mentioned in the previous chapter (section 3.10.2).

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)

(

u d

t 1 0 0

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B % 0 ( 0 0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 T im e (m in ) T im e (m in ) L a c Z P IP 4 K 2 

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2 5 0 L a cZ P IP 4 k 2 

l 2 0 0

)

e t

P 1 5 0

)

, s

3 1 0 0

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P 5 0

% (

0 0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 T im e (m in )

Figure 4.15: Basal and time course of insulin induced PtdIns(3,4)P2 levels in myotubes expression LacZ or PIP4K2α.

L6-GLUT4myc myotubes were grown in 100 mm-dishes. Cells were differentiated in αMEM containing 2% FBS for 10 days. On day 8 myotubes were infected with 40 MOI viruses to express LacZ or PIP4K2α and were kept for 48 h. Cells were serum-starved overnight. On day 10, cells were stimulated with or without 100 nM insulin for the indicated time interval (0-40 min). Lipid extraction and PI purification were conducted using Method 2. Dried PIs were dissolved in chloroform and spotted on Hybond-C extra nitrocellulose membranes. GST-TAPP1 PH domain was used to detect PtdIns(3,4)P2. The chemiluminescence intensity of lipid spots were quantified using Image Lab software and data were corrected for total lipid phosphate. (A) Comparison of the basal (unstimulated) PtdIns(3,4)P2 level between cells expressing LacZ or PIP4K2α. (n=15,

Mann-Whitney U test). (B) Individual levels of PtdIns(3,4)P2 at 3 and 5 min stimulation of cells expressing either LacZ or PIP4K2α (n=4-6). Data are presented as a percentage of

PtdIns(3,4)P2 of the basal level seen in unstimulated cells expressing LacZ or PIP4K2α after correction for the total lipid phosphate. Results are displayed as an individual experiment (C) or as averages of 3 to 7 experiments (means ± SEM) (D).

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4.6 Effect of PIP4K2α overexpression and SKIP knockdown

As shown in Figure 4.13, the insulin-induced levels of PtdIns(3,4,5)P3 observed in control myotubes are lost in cells overexpressing PIP4K2α particularly when stimulated with insulin for more than 5 min. This attenuation most likely would not be a direct effect of PIP4K2α. It would be rather achieved via activating phosphatase(s). One of the hypotheses examined in this work was that PIP4K2α expression over-activates the PtdIns(3,4,5)P3-5 phosphatase, SKIP. In this section, investigations of the effects of SKIP knockdown mediated by siRNA on insulin signalling are therefore presented. Initially, the work focused on the possible rescue of PtdIns(3,4,5)P3 levels, followed by investigating steps downstream of this lipid including AKT, Rac1 and GLUT4 translocation.

4.6.1 Effect of PIP4K2α expression and SKIP knockdown on PtdIns(3,4,5)P3

Since SKIP breaks down PtdIns(3,4,5)P3 to PtdIns(3,4)P2 (Ijuin et al., 2000; Gurung et al., 2003;

Ijuin et al., 2012a), silencing SKIP may restore the levels of PtdIns(3,4,5)P3 if this phosphatase is involved in the effects of PIP4K2α. Although SKIP has not been silenced completely, as shown in Figure 3.13 the level of SKIP was reduced to about 50% when cells were transfected with si-SKIP. It is important to point out that since only about 50% of SKIP knockdown was achieved, SKIP activity will not be entirely abolished, and since it is an enzyme, the impact on overall activity in cells may not be greatly compromised. Nevertheless, to investigate the possible involvement of SKIP in the effects of PIP4K2α, myotubes expressing PIP4K2α or LacZ as a control were transfected with scrambled, or with si-SKIP, siRNA. The effects of these treatments on the level of PtdIns(3,4,5)P3 were then examined using the PLO assay. The basal level of PtdIns(3,4,5)P3 is reduced (p=0.041) in unstimulated SKIP knockdown cells expressing LacZ compared to cells expressing LacZ and transfected with scrambled siRNA. However, the basal level of SKIP knockdown cells overexpressing PIP4K2α cells is apparently not affected when compared to the basal level in cells overexpressing PIP4K2α and transfected with scrambled siRNA (Figure 4.16).

Due to the multi-peaks pattern of the insulin time course of PtdIns(3,4,5)P3 levels (see section 4.5.2.2), multiple- time points (10 and 25 min) were initially examined in cells expressing LacZ or PIP4K2α to pick the best time point to assess possible SKIP knockdown influences. The 25 min point was chosen, since it has been shown previously that the variations between repeated experiments were limited at this time point, as shown by the small error bar in Figure 4.13. The early peaks (at 3 and/or 5min) were excluded since PIP4K2α overexpression does not seem to affect them, as mentioned before (Figure 4.13B).

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The statistical tests of the levels of PtdIns(3,4,5)P3 at 10 min and 25 min in LacZ cells transfected with si-SKIP siRNA showed there was not a significant difference in comparison to LacZ cells transfected with scrambled siRNA (Figure 4.16A). Similarly, the average level of

PtdIns(3,4,5)P3 in SKIP knockdown-PIP4K2α cells at 10 min of insulin stimulation was not statistically significant compared to PIP4K2α cells transfected with scrambled siRNA (Figure

4.16A). However, the amount of PtdIns(3,4,5)P3 was significantly increased at 25 min (Figure 4.16 B) after insulin stimulation in PIP4K2α cells transfected with si-SKIP.

The average levels at 25 min of insulin stimulation, also presented as percentages of unstimulated control (LacZ-expressing cells transfected with scrambled siRNA), are shown in

Figure 4.16B. As shown in the Figure, there is a significant 1.5- to 2-fold PtdIns(3,4,5)P3 increase in insulin stimulated cells expressing LacZ. This increase is abolished in cells transfected with scrambled siRNA expressing PIP4K2α. Importantly, and as hypothesised, SKIP knockdown in cells expressing PIP4K2α clearly restores the increase in the level of

PtdIns(3,4,5)P3 with about a 2-fold increase compared to unstimulated SKIP-knocked-down cells expressing PIP4K2α. This increase is significantly different (P<0.01) from the insulin stimulated, PIP4K2α cells transfected with scrambled siRNA. These results indicate the involvement of SKIP in the abolition of the insulin-stimulated PtdIns(3,4,5)P3 increase in cells overexpressing PIP4K2α as this abolition is reversed when SKIP is knocked down.

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A (10 min) B (25 min)

Figure 4.16: SKIP knockdown restores the insulin-stimulated increase of PtdIns(3,4,5)P3 in cells overexpressing PIP4K2α at 25 min stimulation

L6-GLUT4myc myotubes were differentiated in αMEM containing 2% FBS for 10 days. Myotubes were transfected on days 6 and 8 with scrambled siRNA or siSKIP. On day 7 myotubes were infected with 40 MOI viruses to express LacZ or PIP4K2α. Cells were serum-starved overnight. Cells were then stimulated with or without 100 nM insulin for 10 min (n=3) (A) or 25 min (n=4-8) (B). Lipid extraction and PI purification were conducted using Method 2. Dried PIs were dissolved in chloroform and spotted on Hybond-C extra nitrocellulose membranes. GST-GRP1 domain was used to detect PtdIns(3,4,5)P3. The chemiluminescence intensity of lipid spots was quantified using Image Lab software and data were corrected to total lipid phosphate. Results are expressed as means ± SEM. Data are presented as a percentage of the PtdIns(3,4,5)P3 content of the basal level of LacZ-expressing cells transfected with scrambled siRNA, after correction for total lipid phosphate. Data were analysed using ANOVA (Kruskal-Wallis test) followed by Dunn's multiple comparisons test. (*<0.05, **<0.01).

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4.6.2 Effect of PIP4K2α expression and SKIP knockdown on PtdIns(3,4)P2

As demonstrated above (section 4.6.1) knocking down SKIP in cells overexpressing PIP4K2α restored the lost increase of PtdIns(3,4,5)P3 after insulin stimulation. According to the tested hypothesis, it was anticipated that PtdIns(3,4)P2 would decrease in SKIP Knock-down cells since most PtdIns(3,4)P2 might be generated via this route. It is worth emphasising that the PLO assays were conducted using the same lipid samples used for measuring PtdIns(3,4,5)P3. The dried PIs extracted using Method 2 were dissolved in chloroform, half was used for

PtdIns(3,4,5)P3 and the other half was used for PtdIns(3,4)P2. This is important as the levels of the two PIs were examined simultaneously from the same dishes of cells. However, measuring

PtdIns(3,4)P2 at a single point was challenging, as the time course fluctuated markedly and therefore variation was very high between the individual experiments (Figure 4.15).

The response of cells expressing PIP4K2α was not always as expected. Although

PtdIns(3,4,5)P3 was reduced at certain time points, unexpectedly PtdIns(3,4)P2 did not increase at those same time points. These results contradict the previous work in the host lab using L6 myotubes, which clearly showed a significant increase in the level of PtdIns(3,4)P2 in cells overexpressing PIP4K2α compared to control ( LacZ expressing) cells, particularly at 10 min of insulin stimulation (Tavelis, 2012). PtdIns(3,4)P2 was therefore also measured at 10 and 25 min of insulin treatments. At 10 min, the PtdIns(3,4)P2-increase generally was very weak in most experiments. The insulin stimulated PtdIns(3,4)P2 level in LacZ-expressing cells transfected with si-SKIP is apparently unchanged or in some experiments is even lower (although the difference is not statistically significant) than the basal PtdIns(3,4)P2 level of the unstimulated myotubes (Figure 4.17 A).

On the other hand, the level of PtdIns(3,4)P2 at 25 min of insulin stimulation in LacZ cells transfected with scrambled siRNA (Figure 4.17 B) shows a significant increase of PtdIns(3,4)P2 in comparison to unstimulated control cells ( LacZ, scrambled siRNA). The significant difference between the unstimulated and insulin stimulated cells is lost in SKIP knocked-down, LacZ expressing cells. Surprisingly, the basal level of PtdIns(3,4)P2 in the SKIP knocked-down LacZ- expressing cells is notably and repeatedly higher (though is not statistically significant) than in the control cells (LacZ, scrambled siRNA) (Figure 4.17 B).

The PtdIns(3,4)P2 increase at 25 min of insulin stimulation in PIP4K2α cells was unexpectedly very minor (Figure 4.17B). There is no significant difference between the basal PtdIns(3,4)P2 level and the 25 min-insulin-stimulated PtdIns(3,4)P2 level in SKIP knocked down, PIP4K2α – expressing cells. Similar to SKIP knocked-down- LacZ-expressing cells, the basal level of

PtdIns(3,4)P2 in the unstimulated SKIP knocked-down PIP4K2α cells as apparently higher

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(though again is not statistically significant) than basal PtdIns(3,4)P2 in the scrambled siRNA- PIP4K2α cells.

A (10 min) B (25min)

2 5 0 2 5 0

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c c

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)

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0 0 In s u lin - + - + - + - + In s u lin - + - + - + - + S c r a m b le + + - - + + - - S c r a m b le + + - - + + - - s i- S K IP - - + + - - + + s i- S K IP - - + + - - + +

L a c Z P IP 4 K 2  L a c Z P IP 4 K 2 

Figure 4.17: Effect of SKIP knockdown on the level of PtdIns(3,4)P2 in cells expressing LacZ or PIP4K2α.

L6-GLUT4myc myotubes were differentiated in αMEM containing 2% FBS for 10 days. Myotubes were transfected on days 6 and 8 with scrambled siRNA or siSKIP. Myotubes were infected with 40 MOI viruses to express LacZ or PIP4K2α on day 7. Cells were serum-starved overnight. Cells were stimulated with or without 100 nM insulin for 10 min (A) and 25 min (B). Lipid extraction and PI purification were conducted using Method 2. Dried PIs were dissolved in chloroform and spotted on Hybond-C extra nitrocellulose membranes. GST- TAPP1 domain was used to detect

PtdIns(3,4)P2. The chemiluminescence intensity of lipid spots were quantified using Image Lab software and data were corrected for total lipid phosphate. Results are expressed as means ±

SEM (n=4-8). Data are presented as percentages of the basal level of PtdIns(3,4)P2 in scrambled siRNA-transfected, LacZ-expressing cells after correction for total phosphate. Data were analysed using ANOVA (Kruskal-Wallis test) followed by Dunn's multiple comparisons test (*<0.05).

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4.6.3 Effects of PIP4K2α and SKIP knockdown on GLUT4 translocation

Since PtdIns(3,4,5)P3 was restored in cells expressing PIP4K2α when SKIP was depleted (Figure 4.16 B), and since the original studies show a correlation between the attenuation of glucose uptake and PtdIns(3,4,5)P3 reduction (Grainger et al., 2011), the possible restoration of GLUT4 translocation by SKIP knockdown in PIP4K2α –expressing cells was next investigated. GLUT4 translocation of L6-GLUT4myc myotubes where SKIP is knocked-down was examined using the colourimetric assay. Figure 4.18 indicates that there is about a 2-fold increase in the amount of GLUT4myc present in the plasma membrane following 30 min of insulin stimulation in LacZ cells transfected either with scrambled siRNA or with siSKIP. The difference between the two conditions of the two siRNA transfection is not statistically significant.

On the other hand and similar to the results obtained from the subcellular fractionation and quantification of the surface intensities (section 4.1.2), overexpressing PIP4K2α blocks the GLUT4 translocation in cells transfected with scrambled siRNA. There was no statistically significant difference in cells overexpressing PIP4K2α between cells treated with or without 100 nM insulin. Importantly, SKIP knockdown in cells expressing PIP4K2α does not restore GLUT4 translocation and the amount of GLUT4myc presents at the plasma membrane is similar to the amount detected on the unstimulated cells (Figure 4.18).

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Figure 4.18: SKIP knockdown does not restore GLUT4 translocation in cells overexpressing PIP4K2α.

L6-GLUT4myc cells were differentiated in αMEM containing 2% FBS for 10 days in 48-well plates. On days 6 and 8, myotubes were transfected with siRNA for SKIP knockdown. On day 7, myotubes were infected with adenovirus driving expression of LacZ or PIP4K2α. On day 10, cells were starved for 4 hours, followed by 100 nM insulin stimulation for 30 min. Cells were probed with 9e10 anti-myc antibody, then fixed with 2% PFA followed by secondary HRP-tagged antibody. GLUT4myc on the cell surface was quantified using the colourimetric method (using OPD as a substrate). Absorbances were read at 450 nm using a microplate reader. Results are expressed as Means ± SEM (n= 14-22 *p<0.05) and presented as percentages of the signal from unstimulated cells transfected with scrambled siRNA (LacZ or PIP4K2α). Data were analysed using ANOVA followed by Tukey's multiple comparisons test.

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4.6.4 Effects of PIP4K2α and SKIP knockdown on AKT

Knocking down SKIP has been reported to have a positive effect on the activation of AKT in C2C12 myoblasts (Ijuin et al., 2012a). Therefore, phosphorylation of AKT was also re-examined in L6 myotubes expressing either LacZ or PIP4K2α. These cells were again subjected to siRNA transfection to mediate SKIP-knockdown.

Figure 4.19 presents the average percentages of phosphorylation of AKT-Ser473 in different conditions. In all cases, cells were apparently responding equally to the 100 nM insulin. The graph indicates SKIP knockdown does not affect either the basal level of AKT phosphorylation in the unstimulated cells, or the insulin-dependent phosphorylation of AKT in LacZ and PIP4K2α cells.

Figure 4.19: SKIP knockdown does not affect insulin dependent phosphorylation of AKT- Ser473.

L6-GLUT4myc myoblasts were differentiated for 10 days in αMEM containing 2% FBS. Myotubes were transfected with scrambled or si-SKIP siRNA on days 6 and 8. Cells were infected with adenoviruses (MOI of 40) driving either LacZ or PIP4K2α expression on day 7. On day 10, cells were serum-starved for 6 hours. Cells were then treated with or without 100 nM insulin for 20 min. Cells were harvested and were subjected to Western blot. The membrane was probed with anti- p-AKT-Ser473 antibody. The membrane was stripped and re-probed with pan-AKT antibody. Total AKT was used to normalise the detected signals. Anti-Flag antibody was used to confirm PIP4K2α overexpression. Data are expressed as means ± SEM, (*p< 0.05, n=5). Data were analysed by ANOVA followed by Tukey’s post-hoc test.

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4.6.5 Effect of PIP4K2α expression and SKIP knockdown on Rac1 activity

Since the insulin dependent activity of Rac1 (Figure 4.8) and the increase of PtdIns(3,4,5)P3 are abolished in cells overexpressing PIP4K2α, and, as has been revealed, SKIP knockdown restored the level of PtdIns(3,4,5)P3 (Figure 4.16 B), the possibility that Rac1 activity could be rescued by knocking down SKIP was also investigated. Rac1 activities were examined in cells expressing LacZ or PIP4K2α which were transfected with scrambled siRNA or si-SKIP. As shown in Figure 4.20, no effect is observed in the basal activity of Rac1 in knocked-down cells. The Rac1 activity is increased 2 to 3 fold in response to 100 nM insulin for 20 min in control cells expressing LacZ. Additionally, no significant difference was observed in the control cells transfected with scrambled or si-SKIP SiRNA. In contrast, and similar to the results presented in section 4.3.2, Rac1 activity in cells expressing PIP4K2α did not increase in response to insulin and remained at the basal level. Importantly, knocking down SKIP does not restore Rac1 activity in PIP4K2α cells when stimulated with insulin.

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Figure 4.20: SKIP knockdown does not restore activation of Rac1 in cells overexpressing PIP4K2α.

L6-GLUT4myc myoblasts were differentiated in αMEM containing 2% FBS for 10 days. Myotubes were transfected with scrambled or si-SKIP siRNA on days 6 and 8. Cells were infected with adenoviruses (MOI of 40) driving either LacZ or PIP4K2α expression on day 7. On day 10, cells were serum-starved for 6 hours. Cells were then treated with or without 100 nM insulin for 20 min. Cells were harvested using Rac1 lysing buffer from the Cytoskeleton Kit. Equal amounts of total protein lysates were used to purify the active Rac1 (Rac1-GTP). Purified Rac1 and total lysates were separated by 12% SDS-PAGE and were transferred onto PVDF membranes. Anti-Rac1 antibody was used to detect Rac1. Total Rac1 was used to normalise Rac1 activity measurements. Anti-Flag was used to assess Flag-PIP4K2α expression. Results (means ± SEM, n=7) are expressed as percentages of the activity in unstimulated, scrambled siRNA-transfected cells expressing either LacZ or PIP4K2α. Data were analysed by ANOVA followed by Tukey’s post hoc test (*p<0.05).

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4.7 Discussion

This project aims to explore the effect of PIP4K2α overexpression in L6 myotubes. Previous work showed that PIP4K2α abolished insulin dependent PtdIns5P increases (Grainger et al.,

2011). Insulin-stimulated PtdIns(3,4,5)P3 increases were also abolished, and this was accompanied by a significant increase in PtdIns(3,4)P2 (Tavelis, 2012). Additionally, insulin- stimulated glucose uptake was attenuated in cells overexpressing PIP4K2α (Grainger et al., 2011). Surprisingly, however, insulin-stimulated activation of the classical downstream PI3K effector, AKT, was not affected in PIP4K2α –expressing myotubes (Tavelis, 2012). Based partially on these results, two main hypotheses have been investigated in this thesis; the first one is that PIP4K2α expression activates the PtdIns(3,4,5)P3 -5 phosphatase SKIP whereas the second hypothesis is that the activity of Rac1 is affected by PIP4K2α expression. The results of the experiments in this work consistentxxz with the notion that SKIP seems to actively dephosphorylate PtdIns(3,4,5)P3 in cells expressing PIP4K2α, as the level of this lipid was restored upon silencing of SKIP. However, although Rac1 activation and GLUT4 translocation are both lost following PIP4K2α expression, they are not restored by knocking down SKIP.

4.7.1 Effect of PIP4K2α overexpression on GLUT4 translocation

Although the presence of GLUT4 at the cell surface is usually correlated with increased glucose uptake, it has been documented in some cases that this is not always true. For example, GLUT4 translocation and insertion into the plasma membrane was induced by PtdIns3P, yet glucose uptake was not achieved (Kong et al., 2006). Accordingly, an activation step for GLUT4 while it is in the plasma membrane has been proposed to be necessarily for glucose absorption (Funaki et al., 2006).

In this project three different methods were used to evaluate the effect of PIP4K2α on GLUT4 translocation. These were: subcellular fractionation, and quantifying fluorescence intensity either using microscopy or using a colourimetric method. The three techniques all show about a 2-fold increase of GLUT4 in the plasma membrane in insulin stimulated myotubes compared to the unstimulated cells. However, this increase was abolished in cells overexpressing PIP4K2α. The results from the subcellular fractionation also show different accumulations of GLUT4 in the intracellular compartments between cells expressing LacZ or PIP4K2α (Figure 4.3A). This finding is consistent with the abolition of glucose uptake reported in L6 myotubes expressing PIP4K2α (Grainger et al., 2011), and therefore the attenuation observed in glucose uptake is most likely caused by a defect in the process of GLUT4 translocation. However the data obtained do not completely exclude other possible reasons affecting the presence of GLUT4 in the plasma membrane such as the maturation of GLUT4 in the intracellular compartments.

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4.7.2 Effect of PIP4K2α overexpression on AKT and Rac1

Insulin-dependent GLUT4 translocation takes place mainly downstream of PI3K (Carnagarin et al., 2015; Leney and Tavaré, 2009). Active PI3K results in the activation of two effectors of

PtdIns(3,4,5)P3, AKT and Rac1, that have both been suggested to operate together for mediating the GLUT4 translocation in muscle cells (Sylow et al., 2014; Chiu et al., 2013; Nozaki et al., 2012). To elucidate the defective step that may block GLUT4 translocation, AKT phosphorylation was initially investigated. Phosphorylation of AKT at Ser473 by 100 nM insulin was not affected by expressing PIP4K2α. This is in agreement with a previous study (Tavelis, 2012) in L6 myotube cells. Expressing PIP4K2α in the L6 myotubes abolishes the main increase of PtdIns(3,4,5)P3 seen in control, although in most experiments an early PtdIns(3,4,5)P3-peak (3-5min) has been detected in the control cells ( LacZ) as well as in the PIP4K2α –expressing myotubes. This early peak has been previously reported in control L6 myotubes (Grainger, 2010) and at 1 min stimulation in C2C12 myoblasts (Ijuin and Takenawa, 2012a). AKT activation, as previously mentioned, was not affected by PIP4K2α in myotubes. It is likely that the fact that the initial peak

(at 3 or 5min) of PtdIns(3,4,5)P3 is not abolished in PIP4K2α expressing cells explains why AKT activation is not lost. PtdIns(3,4,5)P3 and PtdIns(3,4)P2 have both been suggested to play a role in activating AKT (Scheid et al., 2002). However, the possibility that the general slight increase in the level of PtdIns(3,4)P2 seen following insulin stimulation of PIP4K2α expressing cells may also play a role cannot be excluded.

Since AKT phosphorylation is not affected in cells overexpressing PIP4K2α, the second arm of the PI3K pathway, Rac1, which had not previously been investigated in these cells, was examined. The results obtained (section 4.3.2) indicate that insulin causes about a 2.5-fold increase in Rac1 activation compared to unstimulated control cells expressing LacZ 4.3. Importantly, the insulin-dependent activation of Rac1 is abolished in myotubes overexpressing PIP4K2α.

Rac1 induces actin remodelling by activating PAKs, and inhibiting Rac1 decreases the phosphorylation of PAK1 in myotubes (Sylow et al., 2013a; JeBailey et al., 2007). Surprisingly however, in this work the phosphorylation of PAK1/2 was not affected by expressing PIP4K2α, when compared to myotubes expressing LacZ. However, both these samples of cells were infected with adenoviruses, and it has been reported previously that adenoviruses can activate PAK1 (Van den Broeke et al., 2010). This might therefore explain the differences observed between the insulin time course of PAK phosphorylation in un-infected cells and in cells infected with adenoviruses driving LacZ or PIP4K2α expression. Moreover, PAK1 is activated by a broad spectrum of regulators (Chiang et al., 2013) and therefore measuring and determining the

138 activity of Rac1 should be conducted directly instead of using PAKs, particularly when viruses are used in the studied system.

4.7.3 Effects of SKIP knockdown on PtdIns(3,4,5)P3 in cells overexpressing PIP4K2α

Another hypothesis that has been examined in this work was whether overexpression of PIP4K2α activates PI5Pases. This was based on the ability of PI5Pases to break down

PtdIns(3,4,5)P3 to PtdIns(3,4)P2 and the previous observation that PtdIns(3,4)P2 was significantly elevated in PIP4K2α expressing L6 myotubes (Tavelis, 2012) although the current work could not observe significant increase in the level of PtdIns(3,4)P2 which might be due to differences between L6 and L6-GLUT4myc cells as discussed later (section 4.7.4).

Nevertheless, previous work in the host lab excluded SHIP2 since inhibiting SHIP2 by

AS1949490 did not rescue PtdIns(3,4,5)P3 (Tavelis, 2012). In this work the effect of another potent PI5Pase, namely SKIP, was investigated. Silencing SKIP was conducted in control myotubes (expressing LacZ) and cells overexpressing PIP4K2α. In the present study, silencing

SKIP did not affect the insulin dependent increase of PtdIns(3,4,5)P3, even though one might expect a higher level of PtdIns(3,4,5)P3 in SKIP-knocked down cells. In C2C12 myoblasts, insulin stimulation significantly raised PtdIns(3,4,5)P3 levels of cells in which SKIP had been knocked down higher than insulin-stimulated cells transfected with control siRNA (Ijuin and Takenawa, 2012a). This discrepancy may be explained by experimental differences: in the current study maximum stimulation with 100 nM insulin was used and only 50% of SKIP depletion was achieved in the L6 myotubes, whereas 10 nM insulin and 80% knockdown were used in the myoblasts of the previous study. Additionally, different techniques with different sensitivity were used for measuring the PIs.

As mentioned before, expressing PIP4K2α in L6 myotubes abolishes the PtdIns(3,4,5)P3 increase after 20-30 min of insulin stimulation (Figure 4.13 and 4.16). To test the involvement of SKIP in this process, SKIP knockdown using siRNA was evaluated. Knocking down SKIP to about 50% in cells overexpressing PIP4K2α restored the level of PtdIns(3,4,5)P3 at 25 min of insulin treatment. The accumulation of PtdIns(3,4,5)P3 at 25 min may indicate that the removal pathway of PtdIns(3,4,5)P3 by SKIP rather than the generation of this lipid was affected by PIP4K2α. This indicates that SKIP is involved in this mechanism. This is consistent with the notion that SKIP is a predominant regulator of PtdIns(3,4,5)P3 when muscle cells are stimulated with insulin (Ijuin and Takenawa, 2012a). Insulin triggers translocation of SKIP from the ER to the plasma membrane ruffles as well as to insulin receptor complexes, where it plays an important role in terminating insulin signalling (Ijuin and Takenawa, 2012b). However, the nature of the involvement of SKIP in the insulin signalling of cells overexpressing PIP4K2α and whether

139 it is due to activation of the phosphatase SKIP activity, or its localisation in addition to the role of PtdIns5P in this mechanism is yet to be studied.

4.7.4 Effects of PIP4K2α overexpression on PtdIns(3,4)P2

The individual time course of insulin dependent PtdIns(3,4)P2 production was highly variable between experiments, making the results difficult to interpret and possibly contributing to the lack of statistical significance observed in the experiments. At 25 min, insulin significantly increases PtdIns(3,4)P2 to about 150% in cells expressing LacZ and transfected with scrambled siRNA compared to the unstimulated LacZ cells whereas, and opposite to what was expected, the slight elevation was not significant in cells expressing PIP4K2α and transfected with scrambled siRNA.

In most experiments in this work, which have been carried out on the L6-GLUT4myc myotubes, the significant increase of PtdIns(3,4)P2 associated with PIP4K2α expression previously reported in parental L6 myotubes (Tavelis, 2012) is not detected. This discrepancy could be due to the differences between the cell lines. It is worth emphasising, as shown in chapter 3 (section 3.7), that SKIP expression in the parental cells was higher than that of the L6-GLUT4myc myotubes.

One might therefore expect a higher increase of PtdIns(3,4)P2 in parental cells due to the higher level of SKIP compared to L6-GLUT4myc. The level of PtdIns(3,4)P2 is also likely to depend on the amount of PtdIns(3,4,5)P3 produced. However, it is difficult to compare the level of the PIs between the two cell lines since the number of cells per dishes and hence the extracted lipids were considerably different.

Alternatively, the activation of a PtdIns(3,4)P2 specific phosphatase along with the hypothesised 5-phosphatase upon insulin stimulation might be also suggested specifically in L6-GLUT4myc cells. The discrepancy between the abolition of the increase of PtdIns(3,4,5)P3 and the modest to un-changed level of PtdIns(3,4)P2 in cells expressing PIP4K2α seen in this work have also been previously reported in some experiments which were conducted on CHO-IR cells overexpressing PIP4K2β (Carricaburu et al., 2003). A similar observation was also documented in malignant glioblastoma cells (U87-MG) overexpressing the 5-phosphatase, SHIP2 (Taylor et al., 2000). In both studies an unknown but PtdIns(3,4)P2- specific phosphatase has been also proposed to account for this discrepancy.

Although PTEN as a 3-phosphatase can dephosphorylate PtdIns(3,4)P2 (Campbell et al., 2003;

McConnachie et al., 2003), its activity would be expected to be more toward PtdIns(3,4,5)P3 since it has been reported that the catalytic efficiency of PTEN for PtdIns(3,4,5)P3 as a substrate is 200-fold greater than that for PtdIns(3,4)P2 (McConnachie et al., 2003). However as has been

140 revealed in this work, the level of PtdIns(3,4,5)P3 in SKIP-knockdown myotubes expressing PIP4K2α is restored and increased and therefore the possible contribution of PTEN in reducing

PtdIns(3,4)P2 would be unlikely.

Inositol polyphosphate 4-phosphatase type I (INPP4A) and type II (INPP4B) have been reported to dephosphorylate the D-4 position of PtdIns(3,4)P2 to produce PtdIns3P (Hakim et al., 2012).

Although the specificity of these two phosphatases toward PtdIns(3,4)P2 is not clear (Sasaki et al., 2010), INPP4B has been shown to prefer de-phosphorylating PtdIns(3,4)P2 to

PtdIns(3,4,5)P3. Overexpressing INPP4B in 3T3-L6 cells reduced the level of PtdIns(3,4)P2 but did not affect PtdIns(3,4,5)P3 (Gewinner et al., 2009). Therefore it is possible that INPP4B or a similar 4-phosphatase may be consuming the PtdIns(3,4)P2 in cells overexpressing PIP4K2α, but this remains to be tested.

In addition to the two possible differences (SKIP expression and 4-phosphatase) between the current and the Tavelis (2012) results of PtdIns(3,4)P2, the activity of PI3K might also play a role in this variation. The activity of PI3K at 10 min of insulin stimulation in parental L6 myotubes was not affected by overexpressing PIP4K2α when compared to cells expressing LacZ (Tavelis, 2012). In the current work, the PI3K activity was not measured in L6-GLUT4myc and therefore, the possible attenuation of the activity of PI3K particularly at 25 min cannot be completely discounted. However, restoring the level of PtdIns(3,4,5)P3 in myotubes overexpressing PIP4K2α and where SKIP was knockdown indicates active PI3K.

4.7.5 Effects of SKIP knockdown on PtdIns(3,4)P2 in cells overexpressing PIP4K2α

In this work, the unstimulated level of PtdIns(3,4)P2 in SKIP knocked down cells was surprisingly and repeatedly higher (152.8 ± 36.83% ) than the unstimulated cells transfected with scrambled siRNA (Figure 4.17). Although this was not statistically significant; it approached significance, (p= 0.0831, n=5) and was accompanied by a slight but statistically significant reduction (71.03 ±

8.44%; n=4, p=0.041) of the unstimulated PtdIns(3,4,5)P3 (Figure 4.16 A&B). This contradicts the data obtained following the silencing of SKIP in C2C12 myoblasts, which did not show any effect on the basal level of PtdIns(3,4,5)P3 or PtdIns(3,4)P2 (Ijuin and Takenawa, 2012a).

A possible explanation for this unexpected difference could be due to the requirement for a long period for SKIP depletion in myotubes of the current study. Double- siRNA transfections over a total of 4 days were used in this study, in comparison to the one day used in the work of Ijuin and Takenawa, (2012a). SKIP is an important protein and depleting it, even partially, might force the cell to compensate for its loss by activating or upregulating other PI5Pases. In support of this possibility, SHIP2 knockdown in rat 3T3L6 myotubes using shRNA for 48 hours also increased

PtdIns(3,4)P2 rather than decreasing it (Mandl et al., 2007). These authors suggested an increase in SKIP activity or a decrease in the activity of PtdIns(3,4)P2-phosphatases as a result

141 of SHIP2 knockdown to account for this observation. Another possible explanation for the observed differences could be due to the cell line and cell differentiation where the expression of SKIP is increased. L6 myotubes were used in the present study, compared to myoblasts in Ijuin’s study. As a support for the possible role of cell line or cell differentiation, the insulin dependent level of PtdIns(3,4,5)P3 and apparently PtdIns(3,4)P2 was not altered by SHIP2 knockdown in C2C12 myoblasts (Ijuin and Takenawa, 2012a) while it was in the rat 3T3L6 myotubes (Mandl et al., 2007). Previous work in C2C12 myoblasts indicates that silencing SKIP for 24 hours did not alter the expression of SHIP2 or PTEN (Ijuin and Takenawa, 2012a). In the current work, expression of other phosphatases in SKIP-knocked down cells was not assessed and alteration in their expression cannot be discounted. The above studies point to the need of careful assessment of changes in expression of the other phosphatases during knocking down of SKIP, particularly when cells are treated for long time.

Stimulating myotubes with insulin for 25 min significantly elevates the level of PtdIns(3,4)P2 in LacZ cells transfected with scrambled siRNA (Figure 4.17) whereas the insulin dependent level of this lipid was not affected by knocking down SKIP in comparison to control cells (i.e the level of insulin dependent PtdIns(3,4)P2 is similar in control and SKIP knockdown cells expressing LacZ. Additionally, insulin does not cause a further increase above what is already been produced in basal levels of knocked-down cells). Moreover, no change of the PtdIns(3,4)P2 level was observed between cells expressing PIP4K2α that were transfected with scrambled or si-

SKIP siRNA when stimulated with insulin (Figure 4.17). Since most PtdIns(3,4)P2 production is generated via dephosphorylation of D-5 of PtdIns(3,4,5)P3 (Hawkins and Stephens, 2016) one might expect a big decrease in the level of PtdIns(3,4)P2 in cells transfected with si-SKIP particularly as PtdIns(3,4,5)P3 was significantly increased in the same cells. The remaining SKIP expression (50%) might still be responsible for the generation of the PtdIns(3,4)P2 detected in the SKIP knockdown cells.

4.7.6 Effects of SKIP knockdown on GLUT4 translocation, AKT and Rac1

As previously discussed SKIP knockdown restored insulin-stimulated PtdIns(3,4,5)P3 production in cells overexpressing PIP4K2α. The next hypothesis that was examined in this work was the possibility that restoring levels of PtdIns(3,4,5)P3 by silencing SKIP could also restore GLUT4 translocation. The effects of SKIP knockdown on the insulin signalling were therefore examined. Silencing SKIP has no significant effect on GLUT4 translocation of LacZ cells transfected with scrambled siRNA when stimulated with 100 nM insulin. Importantly SKIP knockdown did not rescue GLUT4 translocation in PIP4K2α cells. Insulin dependent phosphorylation of AKT at Ser473 is also not altered, either by PIP4K2α as reported before or by depleting SKIP. However, since the level of insulin-induced PtdIns(3,4,5)P3 is restored in SKIP-knockdown cells expressing PIP4K2α, Rac1 activities were examined.

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Rac1 activation was found to be significantly diminished in PIP4K2α overexpressing cells compared to the control cells and again SKIP knockdown does not restore the activity of Rac1. The continued low activity of Rac1 probably explains the attenuation of GLUT4 translocation in myotubes overexpressing PIP4K2α even in SKIP knocked-down cells.

Activation of Rac1 is mediated via GEFs such as Tiam1 (Lambert et al., 2002) which is a Rac1- specific GEF in myoblasts (Chiu et al., 2013). Overexpressing Tiam1 activates both Rac1 and GLUT4 translocation (Chiu et al., 2013). Recently Rac1 activation via Tiam1 was proposed to be activated by PtdIns5P (Viaud et al., 2014). Although the level of PtdIns5P was not measured in this work, it is unlikely to be restored by SKIP depletion, since PIP4K2α remains overexpressed.

Generating PtdIns5P by expressing the potent PtdIns(4,5)P2 4-phosphatase IpgD activates Rac1 in MEF-cells. Furthermore, this Rac1 activity is significantly reduced when PIP4K2β is co- expressed with IpgD (Viaud et al., 2014). A previous study in same cell line, L6 myotubes, has revealed that PIP4K2α overexpression abolishes the insulin dependent increase of PtdIns5P and glucose uptake (Grainger et al., 2011). These findings corroborate with those obtained in this study, which might be summarised as follows: overexpressing the potent kinase PIP4K2α diminishes insulin-stimulated PtdIns5P production that thereby decreases Rac1 activation, which then causes the attenuation of GLUT4 translocation. Furthermore and as previously discussed, the results from measuring the PIs in this study are also consistent with the activation of SKIP as a result of PIP4K2α overexpression. SKIP is also suggested to inhibit Rac1-dependent PAK1 scaffolding activity in myoblasts after 30, but not 10 min of insulin stimulation in the process of terminating insulin signalling (Ijuin and Takenawa, 2012b). Therefore PIP4K2α might affect the initiation steps of insulin signalling (for example via PtdIns5P/Rac1) as well as enhancing the termination steps of insulin signalling by activating SKIP that accelerates the decline of Rac1- dependent PAK1 function.

Thus, increasing PtdIns(3,4,5)P3 alone by insulin stimulation in the absence of PtdIns5P, and hence without Rac1 activation, is not sufficient for translocation of the GLUT4 to the plasma membrane, as seen in this work.

It has been shown in various studies that activating PI3K is crucial but not sufficient for activating GLUT4 translocation and glucose uptake. For example, activating PI3K and generating

PtdIns(3,4,5)P3 with interleukin 4 (IL-4) in L6 myoblasts (overexpressing IL-4R) or with PDGF in 3T3-L1 adipocytes did not stimulate either GLUT4 translocation to the plasma membrane or glucose uptake (Isakoff et al., 1995). On the other hand, administrating exogenous

PtdIns(3,4,5)P3 shows partial GLUT4 translocation in L6 myoblasts (Maffucci et al., 2003), which is sufficient to induce GLUT4myc fusion with the plasma membrane in L6 myoblasts and adipocytes but without increasing glucose uptake (Ishiki et al., 2005; Sweeney et al., 2004). The requirement for an activation step for ‘unmasking’ GLUT4 has been proposed to mediate

143 glucose uptake, and this step probably requires PtdIns3P (Ishiki et al., 2005). GLUT4 translocation and fusion with the cell membrane in L6 muscle cells and 3T3L1 adipocytes was also seen when exogenous-PtdIns(3,4,5)P3 was delivered into cells using polyethylenimine (PEI- 25) (Kachko et al., 2015). Additionally when insulin resistance, characterised by reduced the tyrosine phosphorylation of the IR, IRS1 and the activation of PI3K, was induced in L6 myoblasts with exposure to 100 nM insulin and high glucose (25 mM) for 24 h (Huang et al., 2002), the defect in the endogenous PtdIns(3,4,5)P3 production was bypassed when the cells were treated with exogenous PtdIns(3,4,5)P3. The reduction of AKT activation seen in these insulin resistant cells was restored by the exogenous PtdIns(3,4,5)P3 (Kachko et al., 2015). However, GLUT4 translocation in L6 cells was not examined in this study.

In another study using epithelial Madin-Darby canine kidney (MDCK) cells, Rac1 activity was induced at 5 min by exogenous PtdIns(3,4,5)P3 and this phenomenon was PI3K dependent since the activity of Rac1 and AKT was inhibited by LY294002 (Gassama-Diagne et al., 2006).

The authors suggested that exogenous PtdIns(3,4,5)P3 activates PI3K which in turn generate endogenous PtdIns(3,4,5)P3 that mainly mediates the activation of AKT and Rac1. The data from the previous two studies (Kachko et al., 2015; Gassama-Diagne et al., 2006) suggested a positive feedback loop between PtdIns(3,4,5)P3 and PI3K. In the current work, PtdIns(3,4,5)P3 was noticeably elevated in SKIP-knockdown cells expressing PIP4K2α. However, these cells display a form of insulin resistance as Rac1 activity and GLUT4 translocation were impaired. This is probably due to the absence of PtdIns5P.

In summary, this chapter highlights that PIP4K2α overexpression inhibits insulin-stimulated Rac1 activation. Rac1 activation is essential for GLUT4 translocation in myotubes (Sylow et al., 2014; Chiu et al., 2011) since its inhibition as also shown in this work impaired the translocation. The data are also consistent with the notion that PIP4K2α overexpression activates SKIP which dephosphorylates PtdIns(3,4,5)P3. PtdIns5P has been shown to play a vital role in GLUT4 translocation (Grainger et al., 2011), probably as could be inferred from the current study, by activating Rac1 and/or regulating SKIP.

The results from this chapter emphasise indirectly (by PIP4K2α overexpression) the important role played by PtdIns5P in the insulin signalling pathway. Some of the effects of PtdIns5P on insulin signalling are therefore further investigated in Chapter 5.

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Chapter 5

Investigating the effect of delivery of exogenous PtdIns5P in L6 myotubes

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The results presented in chapter 4 showed on abolition of insulin-dependent GLUT4 translocation in myotubes expressing PIP4K2α that also display attenuation of Rac1 activity. This may be as a consequence of diminishing PtdIns5P by PIP4K2α. Although PtdIns5P was not measured in this study, previous work in L6 myotubes in the host lab showed abolition of insulin- stimulated PtdIns5P production in PIP4K2α overexpressing cells (Grainger et al., 2011). Delivery of exogenous PtdIns5P into myotube cells has been previously shown to elevate the presence of GLUT4 in the plasma membrane, which was also confirmed by a stimulated increase in glucose uptake in these cells, even in the absence of insulin (Grainger et al., 2011). However, the accompanying phosphorylation of AKT in the PtdIns5P-treated cells was very modest, and much lower than insulin induced AKT-phosphorylation. The effect of exogenous PtdIns5P on Rac1 activity in myotubes was not examined. In this section, the effect of exogenous PtdIns5P on translocation of GLUT4 to the plasma membrane was initially investigated, followed by examining any effects on Rac1 and AKT activity.

The method of delivering PtdIns5P was adopted from Grainger et al., (2011) and optimised as detailed in section 2.10. Since two cell lines were used, L6-GLUT4myc and parental L6, the optimum ratios between the carrier 3 and the synthetic PtdIns5P were initially established for each cell line. In the present chapter, L6-GLUT4myc myotubes were used for examining GLUT4 translocation, while parental L6 myotubes were used for assessing Rac1 activity. Since these experiments were carried-out toward the end of the study, each experiment was repeated 3 times only.

5.1 Investigating the effect of delivery of exogenous PtdIns5P on GLUT4 translocation

The effect of exogenous PtdIns5P on GLUT4 translocation in L6-GLUT4myc myotubes was investigated using the colourimetric assay as described in section 2.7.2. Additionally, 100 nM of a PI3K inhibitor, wortmannin, was applied to test the dependency of the PtdIns5P-mediated GLUT4 translocation on the PI3K pathway.

Figure 5.1 shows that delivery of the synthetic PtdIns5P-diC16 into myotubes causes a significant increase (~2.5 fold) in GLUT4 translocation in comparison to cells treated with the carrier alone. Moreover, this increase was abolished when cells were treated with 100 nM wortmannin (Figure 5.1). This finding indicates that GLUT4 translocation was induced by the synthetic PtdIns5P via activation of the PI3K pathway.

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Figure 5. 1: Exogenous PtdIns5P promotes GLUT4 translocation in a wortmannin- sensitive fashion.

L6-GLUT4myc myoblasts were grown and differentiated in αMEM containing 2% FBS on 48-well plates for 10 days. Myotubes were serum-starved for 6 hours. Cells were then incubated with or without 100 nM wortmannin for the last 30 min prior to and throughout the 20 min incubation with carrier-PtdIns5P complex (4µM). Myotubes were probed with the 9e10 anti-myc antibody and then fixed with 2% PFA followed by secondary-HRP antibody. GLUT4myc on the cell surface was quantified using the colourimetric method. Absorbances were read at 450 nm using a microplate reader. Results are expressed as Means ± SEM, (n= 6-8 *p<0.05). Data are displayed as a percentage of the untreated control cells. Data were analysed by One-way ANOVA followed by Dunn's multiple comparisons test.

5.2 Effect of exogenous PtdIns5P on Rac1 activity

As previous work showed only very weak phosphorylation of AKT in response to exogenous PtdIns5P(Grainger et al., 2011), the other arm of the signalling pathway downstream of PI3K, Rac1 (Rudich and Klip, 2013) was examined. Parental L6 myotubes were used to investigate the effect of exogenous PtdIns5P on Rac1 activity. Cells were serum-starved for 6 hours, and then incubated with carrier 3- PtdIns5P complex for 20 min. As seen in Figure 5.2, the exogenous PtdIns5P activated Rac1 to about 200% compared to control cells.

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Figure 5. 2: Delivery of exogenous PtdIns5P in myotubes activates Rac1.

Parental L6 cells were differentiated in αMEM containing 2% FBS for 10 days. Myotubes were serum starved for 6 hours. Cells were treated with 6 µM carrier-PtdIns5P complex for 20 min. Total lysates were extracted with Rac1 lysis buffer. Active Rac1 (GTP-bound) was pulled down and detected using Western blot. Blots are representative of 3 independent experiments. Results are expressed as means ± SEM (n=3). Data are normalised to total Rac1 and expressed as percentages of untreated control. Data were analysed using the unpaired t test (*p<0.05).

To test if the Rac1 activity induced by exogenous PtdIns5P was PI3K dependent, wortmannin was applied. Rac1 activation stimulated by PtdIns5P or by insulin was compared. As indicated in Figure 5.3, the insulin dependent Rac1 activation is wortmannin sensitive. The activity was reduced by about 50%. However, insulin-dependent Rac1 activation was seemingly not fully inhibited by wortmannin (Figure 5.3 A). On the other hand, the PtdIns5P-dependent Rac1 activity was apparently insensitive to wortmannin (Figure 5.3 B). These results indicate different mechanisms of activating Rac1 by insulin and partially involving PtdIns5P. It also points to the possibility that more than one mechanism for Rac1 activation in response to insulin stimulation. These mechanisms are probably not all sensitive to wortmannin. This may explain the partial inhibition of Rac1 by wortmannin in insulin-stimulated cells.

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Figure 5. 3: PtdIns5P-dependent Rac1 activation is wortmannin insensitive.

Parental L6 myoblasts were differentiated in αMEM containing 2%FBS for 10 days. Myotubes were serum starved for 6 hours. Cells were incubated with or without 100 nM wortmannin for the last 30 min prior to and throughout the 20 min incubation with carrier-PtdIns5P complex (6 µM) or 100nM insulin. Total lysates were extracted using Rac1 lysis buffer. Active Rac1 (GTP-bound) was pulled down and detected using Western blot. Rac1 immunoblot indicates incomplete inhibition of insulin dependent Rac1 activity by 100 nM wortmannin (A). Results are expressed as means ± SEM (B). Data are obtained from 3 independent experiments. Data are normalised to total Rac1 and expressed as percentages of the wortmannin-free cells independently (insulin- or PtdIns5P- treated cells). Data were analysed using Mann-Whitney U Test (*p<0.05).

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5.3 Effect of exogenous PtdIns5P on p-AKT

The effect of exogenous PtdIns5P on phosphorylation of AKT-Ser473 was then examined. Previous work showed AKT phosphorylation upon delivery of PtdIns5P, which was very modest compared with insulin induced phosphorylation of AKT (Grainger et al., 2011). Due to limitations on time and resources, and since the AKT experiment has been previously investigated in the host lab, only a single time point (20 min) was examined. Additionally, the total protein lysates used in these blots were extracted from the supernatant of cells lysed with Rac1 lysis buffer, and it is possible that these conditions might affect the detection of AKT phosphorylation. In this work, AKT phosphorylation was barely detected at 20 min (Figure 5.4 A).

Figure 5. 4: Exogenous PtdIns5P induces a very small phosphorylation of AKT-ser473.

Parental L6 myoblasts were differentiated in αMEM containing 2%FBS for 10 days. Myotubes were serum starved for 6 hours. Cells were incubated with or without carrier3-PtdIns5P (6 µM) complex for 20 min. (A). Myotubes treated with or without 100 nM wortmannin for the last 30 min (B) prior to and throughout the 20 min incubation of 100nM insulin (Left panel) or carrier3- PtdIns5P (6µM) complex (Right panel). Total lysates were extracted with Rac1 lysis buffer and AKT-Ser473 was detected using the supernatant of Rac1 lysate after GTP-bound Rac1 had been pulled down. Western blotting was carried out using anti-AKT-Ser473 antibody. The membrane was stripped and re-probed with pan AKT antibody. The blots are representative of 3 independent experiments which showed similar results.

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Additionally, some cells were also treated with insulin having been pre-incubated with or without wortmannin as shown in Figure 5.4 B. The wortmannin inhibitory effect on PI3K was confirmed by examining the AKT activity. The Figure shows, as expected, that insulin triggers AKT phosphorylation while wortmannin prevents this response (Figure 5. 4B). Unfortunately the effects of wortmannin on PtdIns5P-induced phosphorylation were inconclusive due to the very weak AKT-phosphorylation in both cells treated with or without wortmannin (Figure 5. 4B, Right panel).

5.4 Discussion

The results from previous chapters are consistent with an important role played by PtdIns5P in insulin signalling in L6 cells. The discrepancies demonstrated in both Rac1 activation and GLUT4 translocation as a result of expressing PIP4K2α, which is known to diminish PtdIns5P, were not restored by rescuing the levels of PtdIns(3,4,5)P3 by SKIP knockdown. In this chapter, the effects of delivery of exogenous PtdIns5P in L6 myotubes were examined.

5.4.1 Effect of exogenous PtdIns5P on GLUT4 translocation

The delivery of synthetic PtdIns5P was shown to significantly elevate the presence of GLUT4myc in the plasma membrane compared to addition of the carrier alone. This GLUT4 translocation is PI3K dependent since adding 100 nM wortmannin inhibited the increase at the plasma membrane. These results are consistent with previous work by Grainger et al., (2011) in L6 myotubes, which also revealed increases both of GLUT4 in plasma membrane sheets and in glucose uptake following exposure to exogenous PtdIns5P. The glucose uptake in that study was also PI3K dependent (Grainger et al., 2011). Similar results on GLUT4 translocation were also reported when PtdIns5P was injected into 3T3-L1 adipocytes (Sbrissa et al., 2004).

5.4.2 Effect of exogenous PtdIns5P on Rac1 activity

The importance of Rac1 in myotubes was seen earlier in this work (Chapter 4), as Rac1 was not activated, and cells failed to translocate GLUT4 to the plasma membrane, in insulin-stimulated cells overexpressing PIP4K2α, probably and hypothetically as a consequence of PtdIns5P removal. The effect of wortmannin on basal Rac1 and AKT activities of unstimulated cells were not investigated in this study. However, it has been reported that unlike the unstimulated basal AKT which is wortmannin sensitive, basal Rac1 activity is not affected by wortmannin (Ueda et al., 2010; JeBailey et al., 2007).

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In the current study, introducing exogenous PtdIns5P into myotubes was found to induce significant activation of Rac1.This activation was found to be insensitive to wortmannin, and hence is PI3K independent. This finding is consistent with a study using exogenous short-chain C4-PtdIns5P and endogenous PtdIns5P induced by expressing the potent 4-phosphatase, IpgD, in MEF cells. Using LY294002 in that study revealed that the Rac1 activation caused by PtdIns5P is also PI3K independent (Viaud et al., 2014).

The unexpected results that inhibiting PI3K by wortmannin prevents exogenous PtdIns5P mediated GLUT4 translocation but not the activation of Rac1 as seen in this project, has also been described in other models using L6 myoblasts expressing a constitutively active form of Rac1. For example, in a study by Ueda et al., GLUT4 translocation without the presence of insulin was induced by an activated mutant of Rac1(G12V) or by expression of a constitutively active form of the GEF, FLJ00068 (also known as FLJ68ΔN). In the absence of insulin, these two models did not show an increase in AKT phosphorylation when compared to control cells (Ueda et al., 2008). Importantly, in both cases), GLUT4 translocation but not membrane ruffle formation was inhibited by wortmannin (Ueda et al., 2008). Wortmannin completely inhibited AKT phosphorylation in the basal unstimulated, insulin stimulated and active Rac1-expressing cells (Ueda et al., 2008). The authors suggested based on these observations that AKT is probably a prerequisite for starting the process of GLUT4 translocation in the Rac1-induced as well as insulin- induced GLUT4 translocation (Ueda et al., 2008).

In another study using L6-GLUT4myc cells, super-activation of Rac1 achieved by expressing the constitutively active Rac1 mutant (Rac1(G12V) was found to result in a modest increase in

PtdIns(3,4,5)P3 that causes minor AKT phosphorylation, which collectively induced GLUT4 translocation (Chiu et al., 2013). These results indicate positive feedback between Rac1 and PI3K, which has been proposed in other cells in addition to myoblasts (Jiang et al., 2014; Pandey et al., 2009; Chiu et al., 2013; Ueda et al., 2008; Welch et al., 2003; Murga et al., 2002) Therefore, and in the light of the above studies, the ability of wortmannin to abolish GLUT4 translocation mediated by exogenous PtdIns5P, as seen in this study, is probably due to inhibiting the unstimulated basal level of AKT activity or the very small PtdIns5P-stimulated AKT activation, which as mentioned before is required in addition to Rac1 for GLUT4 translocation.

On the other hand, the insulin dependent activity of Rac1 was remarkably but not completely reduced by 100 nM wortmannin, as seen in this work (Figure 5.4 B). A noticeable activity seems to be maintained in the wortmannin-treated cells when compared to the basal level seen in unstimulated cells (Figure 5.4 A). In agreement with the data presented here, Rac1 activation in L6 myoblasts, L6 myotubes, and 3T3-L1 adipocytes was also observed to be only partially prevented by 100 nM wortmannin or LY294002 in previous studies (JeBailey et al., 2004; Nozaki et al., 2012). Furthermore, and based on in situ experiments where myotubes were treated with

152 insulin in the presence or absence of wortmannin and Rac1 activations were examined in different subcellular regions, two populations of active Rac1 were distinguished depending on their sensitivity to wortmannin. The wortmannin-sensitive form occurs mostly in the cell surface areas whereas the wortmannin-insensitive Rac1 seems to be inside the cell. The authors suggested based on this observation that insulin dependent Rac1 activation might be triggered in two pathways, one of which involves PI3K and the other does not (Nozaki et al., 2012). Therefore in agreement with Nozaki et al., (2012) the results of the current study indicate that insulin may activate Rac1 by both PI3K-dependent and -independent mechanisms.

5.4.3 Effect of exogenous PtdIns5P on AKT phosphorylation

Investigating AKT phosphorylation in response to delivery of PtdIns5P showed only a very small increase of the Ser473-phosphorylation. A modest phosphorylation of AKT following exogenous PtdIns5P delivery compared to insulin stimulation has been also reported previously in myotubes (Grainger et al. 2011) and in HeLa cells when treated with short-chain PtdIns5P (Pendaries et al., 2006). Likewise, elevating PtdIns5P via expression of IpgD in MEF cells (Viaud et al., 2014) or in HEK293, Vero, Cos-7, HeLa cells and CHO-IR cells also induced AKT phosphorylation (Carricaburu et al., 2003). In this work, investigating the effect of wortmannin on AKT was difficult since the level of AKT phosphorylation was very low and hard to detect. This may be due to the use of lysate extracted by Rac1-lysis buffer from cells incubated for 20 min with the exogenous PtdIns5P. Also, due to time pressures and limited resources only one time point was used (20 min) although it had previously been shown that AKT phosphorylation in PtdIns5P- treated cells was maximum at 30 min (Grainger, 2010). Additionally, it was difficult to load the correct amount of protein into the polyacrylamide gel with the obtained volume of the total lysate. However, and similar to the effects on GLUT4 translocation, the modest AKT activation mediated by PtdIns5P is apparently PI3K dependent which is consistent with results obtained from Hela cells expressing IpgD by Pendaries et al., (2006). It has previously been shown that PtdIns5P can induce sustained AKT signalling in HeLa cells by inhibiting protein phosphatase 2A (PP2A) which de-phosphorylates AKT (Ramel et al., 2009). However, the exact mechanism of the PtdIns5P-dependent AKT activation is not clear, although possible involvement of Rac1/PI3K is discussed in the next section.

Although Rac1 and AKT were originally thought to be both downstream of PI3K and to be activated independently of each other, inhibiting AKT2 by inhibitor IV or by knockdown resulted in the prevention of both insulin dependent Rac1 activation and GLUT4 translocation in L6 myoblasts (Nozaki et al., 2013). From these results, the authors suggested that Rac1 may act downstream of AKT2 (Nozaki et al., 2013). In contrast, using AKT / Rac1- inhibitors and knockout techniques, no such cross talk was revealed between AKT and Rac1 signalling in

153 response to insulin in mature skeletal muscle (Sylow et al., 2014; Sylow et al., 2013b). These results indicate some differences in the mechanism depending on the cell type and probably on the maturity of these cells. Hence, generalising the results obtained from these studies should be taken with extra caution.

To summarise the findings in this chapter, exogenous PtdIns5P activates Rac1, which in turn probably signals to PI3K. GLUT4 translocation, but not Rac1 activation, was inhibited by wortmannin in cells treated with PtdIns5P. This indicates a PI3K-dependent mechanism is necessary in addition to Rac1 activation for GLUT4 translocation. However, whether this mechanism requires AKT or other protein is not clear.

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Chapter 6

General discussion and conclusion

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6.1 General discussion

The overall purpose of this study was to study the effect of PIP4K2α overexpression on the insulin signalling pathway in L6 myotubes. Recent studies highlight the role of Rac1 activation as an obligatory step beside AKT downstream of PI3K in insulin-stimulated GLUT4 translocation. Rac1 and AKT together mediate GLUT4 translocation to the plasma membrane and hence increase glucose uptake (Rudich et al., 2013; Sylow et al., 2014; Ueda et al., 2010). Two main unexplained findings were previously reported in insulin-stimulated myotubes expressing

PIP4K2α (1) the abolition of the insulin dependent PtdIns(3,4,5)P3 increase and (2) the attenuation of glucose uptake even though AKT was fully phosphorylated. In an attempt to answer these questions two main hypotheses were examined; (1) SKIP is involved in hydrolysing PtdIns(3,4,5)P3 in cells overexpressing PIP4K2α and therefore SKIP knockdown would restore the level of PtdIns(3,4,5)P3. (2) If AKT activity is not affected in cells overexpressing PIP4K2α, then the alternative and second arm of GLUT4 translocation, Rac1, might be inactivated.

The current and previous work has revealed that abolishing PtdIns5P production using PIP4K2α (Grainger et al., 2011) prevents the late but not the initial transient peak of insulin-dependent increase of PtdIns(3,4,5)P3 (section 4.5.2.2). Moreover glucose uptake (Grainger et al., 2011) and GLUT4 translocation (section 4.1.3) were attenuated in a non-AKT dependent manner since the phosphorylation of AKT was apparently not altered. Importantly, the PI3K activation was not affected by PIP4K2α in parental cells (Tavelis, 2012) although the activity status of PI3K has not been assessed in the L6-GLUT4myc myotubes used in the current work (discussed in section 4.7.4). Additionally, previous work has excluded the involvement of PTEN and SHIP2 as

PtdIns(3,4,5)P3-phosphatases (Tavelis, 2012).

Figure 6.1 illustrates some of the key findings obtained from this study and proposes a model of the insulin signalling downstream of PI3K in control myotubes and in cells overexpressing PIP4K2α.

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A B

Figure 6. 1: Proposed models of insulin signalling in control (A) or in L6 myotubes overexpressing PIP4K2α (B).

(A) Insulin activates production of PtdIns5P and PtdIns(3,4,5)P3. Insulin also induces the Rac1 and AKT pathways which together mediate GLUT4 translocation and hence glucose uptake. PtdIns5P may partially contribute in activating Rac1. Additionally, PtdIns5P might have inhibitory effect on SKIP activity. (B) PIP4K2α overexpression abolishes the insulin-elevation of PtdIns5P which might increase SKIP activity. PIP4K2α overexpression indirectly inhibits the late (20-30 min)

but not the early (3-5 min) PtdIns(3,4,5)P3-increase probably via SKIP phosphatase. AKT phosphorylation is not affected in cells expressing PIP4K2α is probably caused by

the early peak of PtdIns(3,4,5)P3. SKIP converts PtdIns(3,4,5)P3 to PtdIns(3,4)P2 which is probably removed by phosphatase(s) (eg INPP4B). Silencing SKIP restores the

insulin-dependent elevation of PtdIns(3,4,5)P3 in myotubes expressing PIP4K2α. Diminishing PtdIns5P by PIP4K2α abolishes Rac1 activity that in turn attenuates GLUT4

translocation and glucose uptake even in cells where PtdIns(3,4,5)P3 is rescued by depleting SKIP.

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The evidence presented in this work is consistent with the notion of higher activity of SKIP upon

PIP4K2α overexpression, since silencing SKIP in myotubes rescued the level of PtdIns(3,4,5)P3 in these cells. This is the first study that shows a possible effect of PIP4K2α on SKIP phosphatase. SKIP expression was not affected by PIP4K2α at the protein level, as shown by Western blotting (Figure 3.10). This suggests that PIP4K2α activates SKIP either by enhancing the phosphatase property of SKIP or through mobilising SKIP to PtdIns(3,4,5)P3- enriched compartments. It is not clear whether the apparent activation of SKIP has been driven directly by PIP4K2α overexpression, or by diminishing PtdIns5P (the next section discusses the possible roles of PtdIns5P). Translocating SKIP to a particular cellular compartment is thought to play an important role in its function in intact cells (Ijuin et al., 2012b; Gurung et al., 2003).

In addition to the observed effects potentially involving SKIP, another finding of this project was the fact that Rac1 activation by insulin was also abolished in PIP4K2α over-expressing cells.

Interestingly, rescuing PtdIns(3,4,5)P3 did not reactivate Rac1 (section 4.6.5). It has been reported that the robust insulin-stimulated elevation of PtdIns5P is missing in these cells, as shown by Grainger et al., (2011), and myotubes overexpressing PIP4K2α also show a defect in GLUT4 translocation, most likely as a result of the abolition of the Rac1 pathway as indicated by the current work. This solves the unanswered question of the attenuation of insulin-stimulated glucose uptake seen in this cell model even though AKT is still phosphorylated. Recent evidence clearly indicates the major role played by Rac1 as well as by AKT in the process of translocating the glucose transporter to the cell membrane, whereby these two proteins are both required for efficient translocation and enhanced glucose uptake (JeBailey et al., 2007; Rudich et al., 2013).

Restoring PtdIns(3,4,5)P3 by SKIP knockdown did not rescue either Rac1 activity or GLUT4 translocation. This finding not only emphasises the importance of Rac1 activation for GLUT4 translocation, but it also points to the role played by the missing phospholipid in this model, PtdIns5P. Although the level of PtdIns5P was not measured in this study, PIP4K2α was previously found to abolish insulin dependent production of PtdIns5P (Grainger et al., 2011).

Additionally, and in support of the suggestion that PtdIns5P is important for Rac1 activation in L6 myotubes, it was demonstrated that carrier-mediated delivery of synthetic PtdIns5P activates Rac1. These findings, in addition to the recent published data (Viaud et al., 2014; Chiu et al., 2013), support the idea that the loss of Rac1 activation seen in myotubes expressing PIP4K2α is probably as a consequence of the loss of PtdIns5P rather than of PtdIns(3,4,5)P3. This conclusion is based on the findings that (1) PIP4K2α overexpression inhibited Rac1 activity, (2) that SKIP knockdown restored PtdIns(3,4,5)P3 but still could not activate Rac1, and finally (3) that exogenous PtdIns5P activated Rac1.

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6.2 Roles of PtdIns5P

Exogenous PtdIns5P application in the absence of insulin is able to induce both GLUT4 translocation and Rac1 activation (section 5.1 and 5.3) in addition to glucose uptake (Grainger et al., 2011) in a PI3K-dependent fashion. The important role of PtdIns5P in these processes is emphasised, as it is able not only to activate Rac1 but also to mediate modest, but apparently sufficient AKT activation via the PI3K pathway, to permit GLUT4 translocation. How PtdIns5P activates PI3K is not clear, and whether this is a direct influence of PtdIns5P or occurs via the activated Rac1 which apparently works both downstream and upstream of PI3K in the insulin and exogenous PtdIns5P experiments is also not certain (Viaud et al., 2014).

Several hypotheses (summarised in Figure 6.2) have been suggested regarding how PtdIns5P might affect the PI3K pathway to affect GLUT4 translocation and hence glucose uptake.

Figure 6. 2: Proposed roles of PtdIns(5)P in the insulin/PI3K pathway.

(A) PtdIns5P regulates AKT phosphorylation by inhibiting PP2A, thus sustaining AKT activation (Ramel et al., 2009).

(B) PtdIns5P regulates the level of PtdIns(3,4,5)P3 by inhibiting PtdIns(3,4,5)P3-phosphatases (Carricaburu et al., 2003). (C) PtdIns5P activates PI3K (Pendaries et al., 2006). (D) PtdIns5P activates Rac1 via the GEF, Tiam1 (Viaud et al., 2014; Chiu et al., 2013).

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The first proposed role of PtdIns5P is in sustaining AKT phosphorylation by inhibiting PP2A (Figure 6.2 (A) (Ramel et al., 2009). However, in the current study, insulin-stimulated AKT phosphorylation is not affected and no differences are observed between cells overexpressing PIP4K2α and control cells. This casts doubt on this hypothesis being correct in myotubes. It would be anticipated that since PtdIns5P is absent, AKT would be de-phosphorylated faster, which was not observed in myotube cells stimulated by insulin over 30 min (Tavelis, 2012), though it is seen in Hela cells when compared to cells expressing IpgD (Ramel et al., 2009).

The second proposed mechanism is that PtdIns5P inhibits the PtdIns(3,4,5)P3-phosphatases (Figure 6.2 (B)) (Carricaburu et al., 2003). Evidence supporting this hypothesis was derived from the effect seen either by depletion or addition of PtdIns5P which affected and seemed to regulate SHIP2. However, PTEN and SHIP2 activities were not affected in the parental L6 myotubes (Tavelis, 2012), although Pendaries and co-workers showed that PtdIns5P causes reduction of SHIP2 phosphatase activity in vitro (Pendaries et al., 2006). However, as suggested in the present study, and as further shown in recent published studies, SKIP is the major regulator of PtdIns(3,4,5)P3 in myotubes (Ijuin et al., 2012a). As mentioned above, addition of exogenous PtdIns5P did not affect SKIP activity in vitro (Tavelis, 2012), although that does not exclude a possible influence in vivo. Moreover, as discussed above expressing PIP4K2α, which diminishes PtdIns5P, apparently may activate the phosphatase property of SKIP.

The third suggested role is that PtdIns5P is able to increase the activity of PI3K (Class IA) via promoting tyrosine phosphorylation which ultimately increases the level of PtdIns(3,4,5)P3 (Figure 6.2 (C)) (Grainger et al., 2011; Pendaries et al., 2006). Increasing PtdIns5P by ectopic expression of IpgD (which increase PtdIns(3,4,5)P3 level) or by delivering synthetic PtdIns5P into different cell types cause AKT phosphorylation in a PI3K dependent fashion since it is inhibited by LY294002 (Pendaries et al., 2006). Additionally, the activity of AKT by IpgD is significantly inhibited in different cell lines lacking certain regulatory subunits of class IA PI3K or knockdown of catalytic subunit (p110β) of class IA PI3K (Pendaries et al., 2006). However, AKT activation is only partially and modestly prevented by knocking down of class II PI3K (C2α and C2β) (Pendaries et al., 2006). These data indicate that PtdIns5P has a role in PI3K activation (mainly in class IA) though the exact mechanism is not known.

Fourthly, the recent finding of the critical role of Rac1 in GLUT4 translocation (Rudich et al., 2013) which could be activated by PtdIns5P via Tiam1 (Viaud et al., 2014; Chiu et al., 2013) or other GEFs like FLJ00068 (Ueda et al., 2008) opens up another possibility of how PIP4K2α and subsequently PtdIns5P may affect the PI3K pathway (Figure 6.2 (D)). Moreover, as has been discussed previously, PtdIns5P can activate Rac1, which possibly also could feed back positively and so modestly activate PI3K. Such feedback, which has been reported in other cells (Jiang et al., 2014; Pandey et al., 2009; Chiu et al., 2013; Ueda et al., 2008; Welch et al., 2003;

160

Murga et al., 2002) might be inferred from the finding that wortmannin inhibits GLUT4 translocation but not Rac1 activation in cells treated with synthetic PtdIns5P, as seen in this study.

As discussed before (sections 4.2, 4.7.2 and 4.7.7), AKT phosphorylation was not affected in myotubes overexpressing PIP4K2α even though PtdIns(3,4,5)P3 was significantly reduced in these cells. It is important to note, however, that the early elevation of PtdIns(3,4,5)P3 that was often observed at 3 or 5 min of insulin stimulation in control and in PIP4K2α cells possibly occurs prior to SKIP activation. This notion of the late effect of SKIP is proposed since the localisation of SKIP to membrane ruffles and its interaction with different protein complex which mediates the phosphatase activity of SKIP against PtdIns(3,4,5)P3 was observed at late steps of the suggested model of how SKIP regulate insulin signalling (Ijuin et al., 2012b). This delay of

PtdIns(3,4,5)P3 break down might be sufficient to drive the phosphorylation of AKT. More detailed study of this early peak of PtdIns(3,4,5)P3 might resolve this issue.

A number of limitations in this study need to be considered. Firstly, the process of SKIP knockdown using siRNA techniques was challenging in myotubes and required a procedure lasting 4 days. This long term experiment might cause some changes in the expression or activity of some of other proteins. An example of this might be as discussed in chapter 4, a

PtdIns(3,4)P2-specific phosphatase. Another limitation of this project was the using of the PLO assay to determine the PI levels. This assay could have some influence on the variations of the lipid measurements that were observed between the experiments. However, PLO is used as an alternative method to the expensive and more advanced techniques such as mass spectrometry or HPLC.

The findings presented in this thesis are broadly in line with other studies attempting to understand the relationship between the different PI phosphatases and kinases and how they affect each other. In summary, and most importantly, this work demonstrates that

PtdIns(3,4,5)P3 in the absence of PtdIns5P is not sufficient to activate GLUT4 translocation, whilst PtdIns5P alone can do so, probably by activating Rac1/PI3K. PtdIns5P has been suggested as a second messenger involved in insulin-stimulated glucose uptake (Grainger et al., 2011; Sbrissa et al., 2004) and it has been proposed that PtdIns5P is necessary but in the class IA PI3K-independent remodelling of actin fibres which is critical for GLUT4 translocation to the plasma membrane in response to insulin (Sbrissa et al., 2004). In fact it has been also suggested based on the previous studies (Grainger et al., 2011; Sbrissa et al., 2004) the possible that PtdIns5P-dependent activation of glucose uptake could be upstream of the PI3K/AKT pathway (Shisheva, 2013).

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PIP4K2α seems to affect the glucose uptake in myotubes at least in two ways. Firstly, is by inhibiting Rac1 activity, probably through depleting PtdIns5P. Secondly, PIP4K2α overexpression, via an unclear mechanism but probably also involved PtdIns5P leads to activation or translocation of the 5-phosphatase property of SKIP, which thus prevents the insulin-stimulated increase of PtdIns(3,4,5)P3.

Together, the findings presented in this thesis strongly imply that PtdIns5P plays important roles in regulating insulin/PI3K signalling. However, further research needs to be carried out to establish how overexpressing PIP4K2α decreases PtdIns(3,4,5)P3 (probably by activating SKIP) and whether this occurs as a result of depleting PtdIns5P. Additionally, future work should focus on the relations between PtdIns5P, Tiam1 or other GEFs, Rac1 and PI3K to understand the mechanism and the different roles of this axis in insulin-stimulated glucose uptake.

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