Thesis for doctoral degree (Ph.D.) 2012 Thesis for doctoral degree (Ph.D.) 2012

Serine/ reonine Protein Phosphatase 5 - a Double-edged Sword - in the Progression towards Diabetes Serine/ reonine Protein Phosphatase Sword 5 - a Double-edged - in the Progression towards Diabetes

Nina Grankvist Nina Grankvist Department of Clinical Science and Education, Södersjukhuset Karolinska Institutet, Stockholm, Sweden

Serine/Threonine Protein Phosphatase 5 - a Double-edged Sword - in the Progression towards Diabetes

Nina Grankvist

Stockholm 2012

All previously published papers were reproduced with permission from the publisher.

Published by Karolinska Institutet.

© Nina Grankvist, 2012 ISBN 978-91-7457-780-8

Printed by

2012

Gårdsvägen 4, 169 70 Solna

To my beloved family

“Do your best and forget the rest” – Tony Horton

Abstract

Abstract

Type 2 diabetes mellitus is increasing at an alarming rate worldwide. In the development and progression of type 2 diabetes mellitus, the insulin-producing pancreatic β-cells are commonly exposed to a hyperglycemic and hyperlipidemic environment, in which the levels of reactive oxygen species are elevated. In turn, reactive oxygen species can trigger an apoptotic response, by activating mitogen- activated protein kinase signaling networks, leading to β-cell death. When the functional β-cell mass is reduced to a level that, can no longer maintain euglycemia, type 2 diabetes mellitus is manifested. Therefore, there is a need to find new anti- diabetic drugs that are able to protect the loss of β-cell mass and in so doing prevent and treat diabetes. This thesis aimed at investigating the possible protective roles of serine/threonine protein phosphatase 5 (PP5) in this context. We generated a PP5-deficient mouse line to evaluate the biological actions of PP5. Isolated mouse embryonic fibroblasts from mice lacking PP5 (Ppp5c-/-) and their wild-type littermates (Ppp5c+/+) were exposed to DNA damage-inducing agents to investigate the role of PP5 in response to genotoxic stress. Pancreatic islets isolated from both Ppp5c+/+ and Ppp5c-/- mice, and MIN6 cells treated with short- interfering RNA targeting PP5, were exposed to H2O2 or palmitate to test the hypothesis that PP5 acts to suppress apoptotic signaling in β-cells. Ppp5c+/+ and Ppp5c-/- mice were placed on either a standard diet or high-fat diet for ten weeks to examine the role of PP5 in a diabetic environment. Our data revealed that the PP5-deficient mice were viable and fertile, demonstrating that PP5 is not essential for survival. However, the Ppp5c-/- mice were underrepresented in offspring from heterozygous mating, indicating that PP5 provides some advantage during embryonic development. Mouse embryonic fibroblasts lacking PP5 showed increased susceptibility to UV light-induced stress, suggesting that PP5 may promote cell survival. PP5 deficiency in mice was also associated with reduced weight gain, lower fasting glycemia and improved glucose tolerance. However, the genetic disruption of PP5 did not alter insulin sensitivity or islet volume. Comparison of mitogen-activated protein kinase signaling in islets from Ppp5c-/- mice and MIN6 cells with reduced PP5 levels revealed that the lack of PP5 was associated with enhanced H2O2- and palmitate-induced JNK phosphorylation and apoptosis. This indicates that PP5 can suppress stress-induced apoptosis in β-cells by a mechanism involving the regulation of JNK phosphorylation and, thereby, contributing to a protective effect. PP5 suppression in MIN6 cells correlated with hypersecretion of insulin in response to glucose. When compared to wild-type littermate controls, Ppp5c-/- mice on a high-fat diet gained less weight. The mice lacking PP5 also had lower fasting glucose, which increased by high-fat diet feeding. A finding not observed in the Ppp5c+/+ mice.

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Abstract

These observations may be explained, in part, by the enhanced serum insulin levels in the Ppp5c+/+ mice on the high-fat diet. Increased serum insulin was not observed in the Ppp5c-/- mice, which instead had lower levels of insulinemia after they were fed high-fat diet. High-fat diet feeding resulted in impaired glucose tolerance in both genotypes without an apparent difference in insulin sensitivity or β-cell function. These data indicate that the lack of PP5 protects mice against high-fat diet-induced weight gain but it cannot sustain glucose control during high-fat diet treatment. Together, our data provide evidence that PP5 is involved in the regulation of β- cell apoptosis and glucose homeostasis. PP5 may represent a double-edged sword in the fight against diabetes, since a PP5 inhibitor useful to prevent the progression towards obesity and diabetes, could possibly also harm the β-cells exposed to a glucolipotoxic environment.

Keywords: diabetes, insulin, protein phosphatase 5, knockout mouse, pancreatic islet, apoptosis, high-fat diet, mouse embryonic fibroblasts, β-cell

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List of Papers

List of Papers

This thesis is based on the following original papers, which will be referred to in the text by their Roman numerals.

I. Amable L*, Grankvist N*, Largen JW, Ortsäter H, Sjöholm Å and Honkanen RE. (* Both authors contributed equally to this work) (2011) Disruption of serine/threonine protein phosphatase 5 (PP5:PPP5c) in mice reveals a novel role for PP5 in the regulation of ultraviolet light-induced phosphorylation of serine/threonine protein kinase Chk1 (CHEK1) J. Biol. Chem. 286(47):40413-22.

II. Grankvist N, Amable L, Honkanen RE, Sjöholm Å and Ortsäter H. (2012) Serine/threonine protein phosphatase 5 regulates glucose homeostasis in vivo and apoptosis signalling in mouse pancreatic islets and clonal MIN6 cells. Diabetologia. In press.

III. Grankvist N, Honkanen RE, Sjöholm Å and Ortsäter H. Genetic disruption of protein phosphatase 5 in mice is associated with altered glucose control during normal and high-fat feeding. Manuscript.

Other publications not included in the thesis:

Darsalia V, Mansouri S, Ortsäter H, Olverling A, Nozadze N, Kappe C, Iverfeldt K, Tracy LM, Grankvist N, Sjöholm Å and Patrone C. (2012) Glucagon-like peptide-1 receptor activation reduces ischaemic brain damage following stroke in Type 2 diabetic rats. Clin. Sci. (Lond). 122(10):473-83.

Ortsäter H, Grankvist N, Wolfram S, Kuehn N and Sjöholm Å. (2012) Diet supplementation with green tea extract epigallocatechin gallate prevents progression to glucose intolerance in db/db mice. Nutr. Metab. (Lond). 9(1):11.

Reproductions were made with permission from the publishers.

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List of Abbreviations

List of Abbreviations

ADP Adenosine diphosphate AMPK 5’-AMP-activated protein kinase ANOVA Analysis of variance APC Anaphase-promoting complex ASK1 Apoptosis signal regulating kinase 1 ATM Ataxia-telangiectasia mutated ATP Adenosine triphosphate ATR Ataxia-telangiectasia and Rad3-related BSA Bovine serum albumin CaM Calmodulin cAMP Cyclic adenosine monophosphate cAMP-GEFII cAMP regulated guanine nucleotide exchange factor II carboxy-DCF 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate CD Control diet CDC Cell division cycle cDNA Complementary DNA Chk1 Checkpoint kinase 1 Chk2 Checkpoint kinase 2 C-peptide Connecting peptide CPT-1 Carnitine palmitoyltransferase-1 DAG Diacylglycerol DDR DNA damage response DMEM Dulbecco's Modified Eagle Medium DNA-PKcs DNA-dependent protein kinase catalytic subunit DPP-4 Dipeptidyl peptidase-4 ECL Enhanced luminol-based chemiluminescene EGF Epidermal growth factor EGFP Enhanced green fluorescent protein eIF2α Eukaryotic initiation factor 2α ELISA Enzyme-linked immunosorbent assay ER Endoplasmic reticulum ES Embryonic stem cell FBS Fetal bovine serum FFA Free fatty acid GAPDH Glyceraldehyde 3-phosphate dehydrogenase GDH Glutamate dehydrogenase GIP Glucose-dependent insulinotropic polypeptide GLP-1 Glucagon-like peptide-1 GLUT2 Glucose transporter 2 GLUT4 Glucose transporter 4 GR Glucocorticoid receptor GRP40 G-related protein-40 GSIS Glucose-stimulated insulin secretion

H2O2 Hydrogen peroxide HFD High-fat diet HIF1α Hypoxia inducible factor 1α

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List of Abbreviations

Hsp90 Heat-shock protein 90 i.p. Intraperitoneal

InsP3 Inositol 1,4,5-trisphosphate InsP6 Inositol hexakisphosphate IPGTT Intraperitoneal glucose tolerance test IPInsTT Intraperitoneal insulin tolerance test IR Insulin receptor IRS Insulin receptor substrate JNK c-Jun N-terminal kinase

KATP channels ATP sensitive potassium channels Malonyl-CoA Malonyl-coenzyme A MAPK Mitogen activated protein kinase MAPKK/MAPK2K Mitogen activated protein kinase kinase MAPKKK/MAPK3K Mitogen activated protein kinase kinase kinase MEF Mouse embryonic fibroblasts MODY Maturity onset diabetes of the young mTOR Mammalian target of rapamycin NEFA Non-esterified fatty acids OA Okadaic acid p53 p53 tumor suppressor protein PBS Phosphate-buffered saline PCR Polymerase chain reaction PI3K Phosphatidylinositol 3 kinase

PIP2 Phosphatidylinositol-4,5-bisphosphate PKA Protein kinase A PKB Protein kinase B PKC Protein kinase C PLC Phospholipase C PP5 Protein phosphatase 5 PPARγ Peroxisome proliferator activated receptor γ PPP Phosphoprotein phosphatase PVDF Polyvinylidene fluoride qRT-PCR Quantitative reverse transcriptase PCR Rac Rac GTP-binding protein Raf1 Raf proto-oncogene serine/threonine protein kinase ROS Reactive oxygen species RPMI Roswell Park Memorial Institute (culture medium) S100 S100-calcium binding protein SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SEM Standard error of the mean ser/thr Serine/threonine siRNA Short-interfering RNA STIP1 Stress-induced phosphoprotein 1 SU Sulfonylurea T1DM Type 1 diabetes mellitus T2DM Type 2 diabetes mellitus TNFα Tumor necrosis factor α TPA 12-O-tetradecanoylphorbol-13-acetate TPR Tetratricopeptide repeat VDCC Voltage-dependent Ca2+ channel

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Contents

Contents

Abstract ...... 5 List of Papers ...... 7 List of Abbreviations ...... 9 Contents ...... 11 Introduction ...... 13 Diabetes Mellitus ...... 13 The Pancreatic Islet and the Insulin-producing β-cell ...... 14 Glucose Sensing by the Pancreatic β-cell ...... 15 Insulin Signaling ...... 17 Glucolipotoxicity and β-cell Dysfunction ...... 18 The DNA Damage Response ...... 18 β-cell Apoptosis Signaling ...... 20 Reversible Protein Phosphorylation ...... 21 Protein Kinases and Protein Phosphatases in the Regulation of Insulin Secretion ...... 22 Serine/Threonine Protein Phosphatase 5 (PP5) ...... 23 Aims ...... 25 Materials and Methods ...... 27 Generation of the PP5 Knockout Mouse ...... 27 Targeting Construct ...... 27 Embryonic Stem Cells ...... 27 Chimeric Mice and Mouse Genotyping ...... 27 Animals, Isolation of Islets of Langerhans and Treatment ...... 28 Primary Cells and Cell Lines ...... 29 MEF Generation, Culture and Treatment ...... 29 MIN6 Cell Culture and In Vitro Treatment ...... 29 Methodology ...... 30 Glucose and Insulin Tolerance Tests ...... 30 Insulin, Proinsulin and C-peptide Analyses ...... 30 Non-esterified Fatty Acid (NEFA) Determination ...... 30 Immunohistochemistry ...... 31 Suppression of PP5 in Cultured Cells using siRNA ...... 31 Assessment of Cell Viability and Apoptosis ...... 31

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Contents

RNA Isolation, Generation of cDNA and Quantitative PCR ...... 31 Western Blot Analyses ...... 32 Analysis of ROS Levels ...... 32 PP2A Enzymatic Activity ...... 32 Statistical Analysis ...... 32 Results and Discussion ...... 35 The PP5-deficient Mice, Phenotypes and Characteristics...... 35 The Role of PP5 in Response to Genotoxic Stress ...... 37 The Role of PP5 in Pancreatic β-cells ...... 38 The Role of PP5 in a Diabetic Setting ...... 41 Conclusions...... 45 Acknowledgements ...... 47 References ...... 51

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Introduction

Introduction

Diabetes Mellitus Diabetes mellitus refers to a group of metabolic disorders characterized by impaired glucose homeostasis, caused by absent or inadequate insulin secretion from the insulin-producing β-cells of the islets of Langerhans in the pancreas [1]. There are two main types of diabetes, type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) [2]. T1DM, formerly known as insulin-dependent diabetes mellitus, results from an autoimmune reaction in which the insulin-producing β-cells are damaged and destroyed, leading to absolute insulin deficiency to such a degree that insulin therapy becomes necessary [3]. T2DM, formerly called non-insulin dependent diabetes mellitus, is the most common form of diabetes, accounting for approximately 90 % of all diabetes cases globally. The prevalence of T2DM has increased dramatically worldwide during recent years [4] and is reaching epidemic proportions in the Western world [5]. More than 350 million people worldwide have diabetes and will suffer a substantially reduced quality of life as a consequence. There are both monogenic and polygenic forms of T2DM. The monogenic forms are different variants of maturity onset diabetes of the young (MODY), all caused by mutations in genes responsible for glucose signaling and metabolism [6]. However, almost all cases of T2DM belongs to the polygenic category, involving a complex interplay between genetic and several environmental factors [7]. Two of the major hallmarks of T2DM are impaired glucose tolerance and insulin resistance. Another characteristic is the insufficient insulin supply by the pancreatic β-cells [8, 9]. Before patients display manifest diabetes with elevated fasting blood glucose levels, hyperglycemia, there is often a preceding period with excess levels of insulin, hyperinsulinemia. This is due to an adaptive response by the insulin-producing β- cells to release more insulin [10, 11], mainly as a result of an increase in β-cell mass [12-15]. This is particularly evident in obesity-related diabetes [16, 17]. Such an adaptation may explain why most obese people do not develop T2DM. However, in some cases there is a gradual decline in β-cell function [18], which to a large extent can be explained by reduced β-cell mass [12, 13], thereby triggering T2DM outbreak. T2DM is manifested when the functional β-cell mass is compromised to a level where it no longer can maintain euglycemia. It has been calculated that approximately 50 % of the β-cells have been lost at the time of T2DM diagnosis. The reduced β-cell mass is believed to arise from an increased apoptotic rate of β- cells in a hyperglycemic and hyperlipidemic environment [19]. Today there are different strategies for the treatment of T2DM. Early therapy includes exercise and diet, followed by the sequential use of oral hypoglycemic agents and, if needed, eventually also subcutaneous insulin injections. Metformin is the first-line choice of oral agents and it primarily reduces the production of glucose

13

Introduction in the liver and increases insulin sensitivity somewhat. However, many patients stop this treatment due to side effects. Sulfonylurea (SU) drugs, another traditionally used class, exert their antidiabetic effect by binding to the β-cells and by that enhance insulin secretion. SUs increase insulin secretion regardless of the existing blood glucose concentration, which can lead to severe hypoglycemia and weight gain. Another drawback of SUs is that the chronic pressure of the β-cells to release insulin can lead to exhaustion and, perhaps, later death of these cells [20]. Glitazones are drugs that specifically target insulin resistance by increasing the insulin sensitivity in peripheral tissues. Treatment with glitazones evokes sustained control of glycemia, which might be explained also by direct and/or indirect β-cell protective effects [21]. Incretin-based drugs, such as glucagon-like peptide-1 (GLP- 1) analogues and dipeptidyl peptidase-4 (DPP-4) inhibitors, hold the advantage that the effects are glucose dependent, both stimulation of insulin release and inhibition of glucagon secretion ends when euglycemia is attained. Incretin-based drugs, have also shown to have protective effects on the β-cells in vitro and in animal models [22]. Although the conventional treatment for T2DM does not really live up to the requirements necessary for the complexity in this disease, important progress has been made [23]. Importantly, a landmark study in T2DM, namely the United Kingdom Prospective Diabetes Study (UKPDS), has provided compelling evidence that β-cell function and cell numbers are reduced despite the use of current drug treatments to control blood glucose levels, eventually leaving most patients dependent on exogenous insulin [24, 25]. Hence, it is apparent that there is a need to find new antidiabetic drugs that are capable of preventing and treating diabetes, in particularly agents that protect against the loss of β-cell mass. In this thesis, the possible protective roles of protein phosphatases will be addressed.

The Pancreatic Islet and the Insulin-producing β-cell In the pancreas, endocrine cells are grouped in the islets of Langerhans, which are distributed throughout the organ. The islets constitute less than 2 % of the pancreatic mass, which represents around 1 million islets. Each islet consists of five different hormone-producing cells. The most abundant are the β-cells, which constitute more than 70 % of the islet. β-cells, synthesize, store and release the only glucose-lowering hormone, namely insulin [26]. The α-cells produce and secrete glucagon, whose function is opposite to that of insulin. Somatostatin from the δ- cells serves in a paracrine manner to counteract secretion of insulin and glucagon. The PP-cells release a 36 amino acid pancreatic polypeptide that exerts a negative feedback mechanism by inhibiting pancreatic secretion [27, 28]. More recently, the ε-cells were identified. They produce ghrelin, which is proposed to have a role in regulating satiation and energy balance [29-31]. Together, these cells are crucial for sustaining a normal glucose homeostasis, where the main function of the β-cell is to sense the nutrient status of the body and respond accordingly, with a release of insulin, to maintain euglycemia.

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Introduction

Glucose Sensing by the Pancreatic β-cell The pancreatic β-cells are equipped to rapidly sense ambient glycemia. In order for the cells to respond appropriately with insulin secretion, glucose must be metabolized within the β-cells [32, 33]. Glucose rapidly enters the cells via the efficient glucose transporter 2 (GLUT2) that enables a balance between the extracellular and intracellular concentration of glucose [34, 35]. Following entry, glucose is phosphorylated by glucokinase, which acts as a glucose sensor by controlling the amount of glucose that goes through the metabolic pathway glycolysis [36]. During glycolysis, one molecule of glucose, by multiple steps, is converted into two molecules of pyruvate. Pyruvate, in turn, enters the citric acid cycle and the mitochondria. Glucose metabolism results, among other things, in increased production of adenosine triphosphate (ATP), leading to an increased ATP/adenosine diphosphate (ADP) ratio [37], which (like SUs drugs) closes the ATP-sensitive K+ (KATP) channels [38, 39]. This causes depolarization of the plasma membrane, opening of voltage-dependent Ca2+ channels (VDCC) and influx of extracellular Ca2+. Elevation of cytosolic Ca2+ is the main trigger of granule translocation and insulin exocytosis [40, 41]. Insulin is released in a pulsatile manner, which was first described to be caused by oscillations of the intracellular Ca2+ concentration [Ca2+]i, caused by the periodical opening of the VDCC [42]. However, pulsatile release of insulin has been shown to occur without changes of Ca2+ levels [43], indicating that the mechanism is not totally contingent upon [Ca2+]i. Instead oscillations in β-cell metabolism have been correlated with the oscillations of insulin secretion, in which [Ca2+]i seems to function more as an amplifier [44, 45]. The mechanism above describing glucose-stimulated insulin secretion (GSIS) is often referred to as the triggering pathway, or the KATP-dependent pathway, and accounts for the first or early phase of insulin secretion, specifically the release of insulin secretory granules already docked at the cell membrane [46]. The function of this rapidly released insulin is believed to primarily be to suppress hepatic glucose production. Loss of first phase insulin secretion is an early pathogenic event in T2DM [47], and it seems this loss is specific for glucose [48]. If the concentration of blood glucose remains elevated, the β-cells will continue to release insulin, leading to the second or late phase of insulin release [49]. In addition, there is another pathway known as the amplifying pathway or KATP-independent pathway, where glucose can sustain insulin release independently of the KATP channels [50]. Under conditions in which K+ concentrations are increased in the presence of the drug diazoxide, which keeps the KATP channels open, glucose retains its ability to stimulate insulin secretion in a dose-dependent manner [51]. Even so, this mechanism is dependent on Ca2+, although it does not raise Ca2+ levels further, but rather serves to amplify the response to glucose and thereby requires the triggering pathway [52]. The metabolism of glucose is essential for GSIS, regardless of which pathway that is activated. In parallel with the amplifying pathway, anaplerosis is an important occurrence [53, 54], where metabolites derived from glucose metabolism and intermediates from citric acid cycle together with other nutrient secretagogues

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Introduction provide signaling roles for insulin secretion. Citrate, produced from the citric acid cycle in the mitochondria, is a precursor that forms malonyl-coenzyme A (malonyl- CoA) in the cytosol. Malonyl-CoA acts to inhibit carnitine palmitoyltransferase-1 (CPT-1) [55, 56], which is located in the outer mitochondrial membrane and responsible for transport of long-chain fatty acids across the membrane for degradation through β-oxidation within the mitochondria [57, 58]. When CPT-1 is blocked or downregulated, fatty acid oxidation is reduced leading to a raise in cytosolic levels of long-chain acyl-CoA, which can signal directly or indirectly by means of fatty acid esterification to increase insulin release [59, 60]. High levels of glucose reduce oxidation of fatty acids, augmenting the production of esterified lipids. However, high levels of free fatty acids also acutely enhance GSIS (Figure 1). There are additional intracellular signals that link glucose sensing to insulin exocytosis. In response to glucose, the intracellular levels of cyclic adenosine monophosphate (cAMP) are increased [61, 62]. The second messenger cAMP is generated via adenylate cyclase, which is part of the G-protein signaling cascades. This pathway is activated in response to glucagon and the two incretins, GLP-1 and glucose-dependent insulinotropic polypeptide (GIP), both peptide hormones that are released during a meal from the gastrointestinal tract. cAMP can enhance GSIS by prolonged opening of the VDCC [63], but the actions of cAMP is mostly mediated through protein kinase A (PKA), either in a dependent or independent manner [64]. In a PKA independent manner, insulin exocytosis can be regulated in a cAMP-dependent way by a direct target of cAMP, namely the cAMP regulated guanine nucleotide exchange factor II (cAMP-GEFII) [65]. Free fatty acids (FFAs) can bind and activate a G-protein coupled receptor, G protein receptor-40 (GRP40), leading to increased phospholipase C (PLC) activity, which results in the hydrolysis of plasma membrane phosphatidylinositol-4,5- bisphosphate (PIP2) and the subsequent generation of inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG). DAG is a second messenger that can activate protein kinase C (PKC) [66]. PKC [67] is a signaling protein that has been extensively investigated in the regulation of insulin secretion [68-71]. Phorbol esters, including 12-O-tetradecanoylphorbol-13-acetate (TPA), have been shown to increase insulin release by activating PKC [72, 73]. PKC can modulate different signaling pathways by affecting phospholipase D [74], PLC and the VDCC [75]. However, it is not likely that PKC directly mediates the action of glucose on insulin secretion [70]; instead PKC seems to be important for the mediation of Ca2+ mobilizing neurotransmitters and hormones that potentiate fuel-induced insulin exocytosis [76]. Glutamate is formed from the citric acid cycle intermediate α-ketoglutarate by glutamate dehydrogenase (GDH) and is acting downstream of the mitochondria to potentiate insulin exocytosis by affecting the secretory granules [77]. Glutamate has also been suggested to promote insulin secretion by inhibiting protein phosphatases [78]. This hypothesis, which couples glutamate to insulin exocytosis is, however, controversial since elevated glutamate levels also have been shown not to induce

16

Introduction

insulin release [79]. Inositol hexakisphosphate (InsP6), a dominant inositol phosphate in β-cells, has been shown to inhibit protein phosphatases [80], and thereby increasing the activity of VDCC, which leads to increased cytosolic Ca2+ levels and in turn insulin release [81].

K+

Ψ

KATP- channel VDCC

Glucose Ca2+ ATP/ADP Glucokinase

Glucose-6-phosphate ATP ATP FFAs Glycolysis PKA Electron transport chain Pyruvate Malonyl-CoA cAMP

Citric 1β-oxidation CPT-1 Acetyl-CoA carboxylase acid cycle Citrate Mitochondria

Figure 1: Simplified scheme of triggering and amplifying pathways of insulin secretion. See text for details.

Insulin Signaling The β-cells are responsible for synthesizing insulin, a process beginning with the precursor, preproinsulin, that after cleavage by the proteases, prohormone convertase 2 and 3, forms proinsulin. Proinsulin, in turn, is converted by the enzyme carboxypeptidase into insulin together with connecting peptide (C-peptide) [82, 83]. Insulin is an essential hormone in the control of glucose homeostasis. A primary role of insulin is to regulate glycemia by reducing the endogenous glucose production in the liver, via gluconeogenesis and glycogenolysis, which decreases the hepatic output of glucose. At the same time insulin stimulates glucose uptake into skeletal muscle and adipose tissue, which is mediated by the glucose transporter 4 (GLUT4) [84, 85]. Insulin also affects the lipid metabolism by enhancing lipid synthesis in liver and fat cells, while inhibiting the breakdown of lipids through lipolysis [86]. The action of insulin is exerted through receptor-mediated signaling [87]. The insulin receptor (IR) is located in the plasma membrane. Upon the binding of insulin, it changes conformation, producing a state in which the tyrosine kinase domains can undergo autophosphorylation. The phosphorylated residues then recruit multiple intracellular proteins, among which the insulin receptor substrate (IRS), phosphatidylinositol 3 kinase (PI3K) and Akt/protein kinase B (PKB) are the most important components in the signaling pathway for insulin function [88, 89].

17

Introduction

Glucolipotoxicity and β-cell Dysfunction Fasting blood glucose values are around 5 mM in healthy individuals, and after a meal it rarely exceeds 10 mM [90, 91]. Preceding and during the development of T2DM, when insulin resistance unfolds, the β-cells compensate in order to maintain euglycemia by hypersecreting insulin [11]. When β-cells no longer are able to meet the demands with increased insulin secretion, hyperglycemia ensues. In late stages of T2DM, or during experimental situations, where the β-cells are chronically exposed to hyperglycemia, a reduced β-cell mass is seen [12, 14]. T2DM is often linked to obesity and, as a consequence of obesity and insulin resistance, the expanded adipose tissue releases an excessive amount of fatty acids, causing increased levels of circulating FFAs [92, 93] and also their ectopic deposition in visceral organs (steatosis). In addition, the adipose tissue releases proinflammatory cytokines, such as tumor necrosis factor α (TNFα), which has provided a link between diabetes, obesity and chronic inflammation [94]. Acutely, FFAs play an essential role to amplify insulin secretion [58, 95, 96], while prolonged exposure to FFAs is toxic and decreases GSIS [97, 98]. Chronic exposure to high FFA concentrations, hyperlipidemia, causes severe damage to the β-cells, leading to decreased function and eventually loss of β-cell mass [99-101]. Preceding and during the development of T2DM, the β-cells are often exposed to a combination of a hyperglycemic and hyperlipidemic environment, producing so-called glucolipotoxicity [102]. Elevated levels of glucose and fatty acids are associated with increased production of ROS [103-105], that cause cellular damage and initiate apoptosis in human pancreatic islets [106]. During metabolism of glucose, the electron transport chain produces and often leaks free radicals, that have the potential to induce oxidative stress. Since the β-cells have low antioxidant defense [107-109], they are conspicuously susceptible to ROS-induced decrease in function and viability [110, 111]. Hence, a hyperglycemic and hyperlipidemic environment may reduce the functional β-cell mass through both functional suppression and increased apoptosis.

The DNA Damage Response Cells are continuously challenged by different kinds of stimuli, both endogenous and exogenous, that can induce DNA damage. Accordingly, the cells have evolved complex mechanisms to maintain genomic integrity and stability. One of these important mechanisms is the DNA damage response (DDR) (Figure 2), a signal transduction network composed of a large number of proteins, that by subsequent phosphorylation of their substrates, function to regulate cell cycle progression, DNA repair and apoptosis [112, 113]. In response to genomic damage caused by genotoxic agents, radiation or oxidative stress, three central PI3K-related protein kinases becomes activated, ATR (ataxia-telangiectasia and Rad3-related), ATM (ataxia-telangiectasia mutated) and DNA-PKcs (DNA-dependent protein kinase catalytic subunit) [114-116]. Via elaborate mechanisms, these protein kinases phosphorylate proteins that help

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Introduction initiate and coordinate: 1) the recruitment of repair proteins to damaged DNA, 2) the suppression of cell cycle progression while repairs are in progress and 3) the induction of apoptosis when damage cannot be adequately repaired. ATR can respond to a wide range of DNA damage and stress, while the ATM is mainly activated in the presence of double-strand breaks [117, 118]. These kinases have both similarities and differences. Thus their substrate specificity and functions occasionally are overlapping throughout the signaling network [119]. When activated, ATR and ATM phosphorylate several key proteins, including checkpoint kinase 1 (Chk1) and checkpoint kinase 2 (Chk2) [120], which sustain and amplifies the DDR signal. ATR and ATM, also phosphorylate p53, Brca1 and hRad17 [118, 121-125]. The ATR-mediated phosphorylation of Chk1, at serines (S) S317 and S345, represents a hallmark of ATR-activation [126]. In turn, Chk1 phosphorylates the p53 (S15/18 and S20/23) tumor suppressor protein. However, ATR can also directly phosphorylate p53 at many residues, by which p53 promotes cell cycle arrest and induces progression into apoptotic cell death [114]. Activated Chk1 also phosphorylates Cdc25A on S76, leading to its ubiquitination and degradation [127, 128]. When damaged DNA has been repaired, Chk1 activity returns to basal levels and Cdc25A levels increase, permitting the cell cycle progression to continue. To allow a dynamic response, in which cells that have made sufficient repairs are able to resume DNA replication and continue cell cycle progression, ser/thr protein phosphatases are needed to counter the protein kinases activity in the DDR. Currently, the knowledge about the roles of individual protein phosphatases in this context is still rudimentary.

DNA Damage

P P Sensors Rad17 γH2AX S645 S139

Initiating kinases ATR ATM

S345 S317 P P Effector kinases Chk1 Chk2

P P P S18 Effector protein Cdc25A p53 P P

Apoptosis DNA Repair Cell-cycle arrest

Figure 2. A simplified scheme of the DNA damage response. See text for details. 19

Introduction

β-cell Apoptosis Signaling Apoptosis, also called programmed cell death, is a well-regulated process leading to cell death. It is a defense mechanism to remove damaged cells and by that is important for the maintenance of cellular homeostasis [129]. A wide variety of different environmental stressors can induce apoptosis. The mitogen-activated protein kinase (MAPK) signaling cascade (Figure 3) is evolutionary well conserved and serves as an important positive regulator of apoptotic signaling [130, 131]. Therefore, dysregulation or improper functioning of this signaling network is linked to the pathogenesis of several diseases, including cancer [132], diabetes [133], neurodegenerative diseases [134] and ischemic injury [135]. The core of MAPK signaling is typically composed of three-tiered modules of sequentially activated protein kinases, such as the MAPK kinase kinase (MAPKKK, MAP3K) which phosphorylates and thereby activates the MAPK kinase (MAPKK, MAP2K), which in turn activates downstream MAPKs in a similar manner. Activated MAPKs can then regulate a range of cellular functions [131, 136, 137]. Apoptosis signal regulating kinase 1 (ASK1), is a MAP3K that plays a key role in stress-induced cell death. ASK1 activates two MAP2Ks complexes, the MKK4/MKK7 and MKK3/MKK6. These MAP2Ks, in turn, activate the c-Jun N- terminal kinases (e.g. JNK1, JNK2) and the p38 MAPK, respectively, at sites that enhance their catalytic activity [135, 138-140]. JNK and p38 MAPK are both known to regulate cell differentiation and apoptosis, through phosphorylation of specific serine and/or threonine residues of their substrate proteins. ASK1 is activated by various types of stress, among which oxidative stress caused by ROS is the most potent activator of ASK1 [141]. The action of ASK1 is tightly regulated, in part, by an antioxidant protein, thioredoxin [142]. Under non-stressed conditions where a redox balance is properly maintained, the reduced form of thioredoxin binds to the N-terminal region of ASK1 and thereby inhibits its kinase function and helps suppress apoptosis. When cells are exposed to ROS, oxidation of thioredoxin occurs, resulting in an oxidized form which is unable to bind and thereby dissociates from ASK1, which then becomes active [143]. Hence, activation of ASK1 and the subsequent activation of JNK and p38 MAPK is responsive to changes in the levels of ROS. The catalytic activity of ASK1 is further regulated by phosphorylation. Oxidative stress induces ASK1 phosphorylation on Thr845, which correlates with an increase in both ASK1 activity and ASK1-dependent apoptosis [144, 145]. The MAPKs are activated by phosphorylation and the protein phosphatases that act to dephosphorylate these kinases represent critical elements in the control of MAPK signaling.

20

Introduction

Stimulus ROS, UV, cytokines, mechanical stress etc.

MAP3K ASK1

MAP2K MKK4/7 MKK3/6

MAPK JNK p38

Stress responses

Figure 3. Regulation of the MAPK signaling pathway. See text for details.

Reversible Protein Phosphorylation The regulation of cellular processes, by changes in the levels of protein phosphorylation, is fundamental for a wide variety of cellular events. There are two classes of enzymes, protein kinases and protein phosphatases, involved in regulating the protein phosphorylation state. Protein kinases phosphorylate the target protein by adding a phosphate group, usually on a serine, threonine or tyrosine residues. Protein phosphatases counteract the kinases by catalyzing the dephosphorylation of phosphorylated amino acids [146, 147]. Therefore, the phosphorylation level in a cell is a result of a dynamic and delicate balance between protein kinases and protein phosphatases. Protein kinases and protein phosphatases are equally important, and much is known and studied about the protein kinases; however, surprisingly little is known about the protein phosphatases involved. This is partly due to the difficulties and limitations that are present when studying protein phosphatases. Protein phosphatases are a complex group of proteins that usually are classified according to their substrate specificities, such as phosphorylated serine, threonine or tyrosine residues [148, 149]. The vast majority of protein phosphorylation events occur on serine and threonine amino acid residues. The dephosphorylation of these residues is mainly accounted for by the phosphoprotein phosphatase (PPP) family, which is comprised of PP1, PP2A, PP2B/calcineurin, PP4, PP5, PP6 and PP7 [150- 156]. To characterize the roles of serine/threonine protein phosphatases, inhibitors such as okadaic acid, calyculin A, microcystin, cantharidin and fostriecin, among others, have emerged as powerful tools [157, 158]. The most commonly used inhibitor is okadaic acid, a toxin produced from marine dinoflagellates. Okadaic acid has a potent inhibitory activity against PP1, PP2A, PP4, PP5 and PP6 [159]. Studies designed to elucidate the biological function of PP5 are hampered by the absence of small molecular inhibitors that specifically target PP5. The existing inhibitors are

21

Introduction semi-selective (e.g. okadaic acid, microcystin and calyculin A) and, therefore, they provide very limited information [160]. Conversely, studying the role of PP5 when being overexpressed, by forced upregulation, also offers limited information. This is because PP5, in contrast to other PPP-family phosphatases, consists of catalytic, regulatory and targeting TPR domains within a single polypeptide chain [153, 161]. In solution PP5 has low catalytic activity because the back side of the TPR domains fold over the catalytic site and thereby blocks substrate access [162]. Interactions of PP5 with other proteins are mediated via the TPR domains and binding triggers a conformational change in which substrates are allowed to access the catalytic site. Accordingly, protein-protein interactions determine both the substrate specificity and activity of PP5. When PP5 no longer is in a protein complex, it rapidly resumes its autoinhibitory conformation. Hence, overexpression of PP5 may only produce a pool of unbound PP5 protein that does not possess enzymatic activity [163].

Protein Kinases and Protein Phosphatases in the Regulation of Insulin Secretion Multiple families of protein kinases and protein phosphatases are involved in regulating insulin secretion. In this process, the protein kinases activate, and by that induce signals, and the protein phosphatases function in the opposite way. However, both classes of enzymes contribute to insulin secretion via different mechanisms. Some ser/thr protein kinases require the presence of Ca2+ and a Ca2+ binding protein, calmodulin (CaM), for their activation. The Ca2+/CaM dependent protein kinase (CaMK) has been found both in rat islets [164] and human islets [165]. During GSIS, when the cytosolic levels of Ca2+ increase, Ca2+ binds CaM and forms a complex. That, in turn, binds to the inactive form of CaMK, which then becomes enzymatically active [166]. When activated, CaMK phosphorylates various substrate proteins that function to stimulate the secretory response [167]. An alternative way of activating CaMK is through receptor-mediated generation of InsP3, which induces Ca2+ release from the endoplasmic reticulum (ER), thereby activating CaMK as described above. Another ser/thr protein kinase is the mammalian target of rapamycin (mTOR), which belongs to the PI3K-related kinase protein family. mTOR is distinctive in that it incorporates signals from both nutrients and mitogens (e.g amino acids and glucose) to control cellular growth. mTOR is activated upon increased ATP production [168, 169]. Inhibition of mTOR, by the inhibitor rapamycin, has been shown to result in impaired GSIS in β-cells [170]. This may be of clinical significance, as rapamycin is often part of the immunosuppressive treatment following islet transplantation into diabetic patients. 5’-AMP-activated protein kinase (AMPK) is another protein kinase that is considered by some to be the ‘master fuel gauge’. It has been shown to respond to metabolic stress by inhibiting lipogenesis and cholesterol synthesis, suggesting that

22

Introduction one function of AMPK activity is to respond to fluctuations in glycemia [171]. AMPK has been shown to inhibit different steps in the GSIS. Firstly, glucose oxidation and the synthesis of ATP [172]. Secondly, AMPK may keep the KATP channels open thereby preventing the rise in cytosolic Ca2+ [173]. Thirdly, the secretory granules are negatively affected by AMPK and by that the secretory process. A few other protein kinases that are involved in increasing insulin secretion have been mentioned in a section earlier, such as PKA, Akt/PKB and PKC. It should also be noted that there is significant ‘cross talk’ between many of these signaling pathways that may impact GSIS. For instance, PKA and PKC activation is known to synergistically potentiate GSIS. Compared to the protein kinases, relatively little attention has been paid to the role of the ser/thr protein phosphatases, which act to counter the kinases by catalyzing protein dephosphorylation [174]. It has been suggested that amino acids and glucose metabolites from glycolysis and the citric acid cycle can reduce the activity of protein phosphatases, mostly PP1 and PP2A, which may contribute to an increased insulin secretion. Use of calyculin A, a phosphatase inhibitor, revealed that when the activity of protein phosphatases is reduced the GSIS is elevated [78, 80, 175, 176]. Additionally, several known insulin secretagogues and second messengers have been shown to transiently suppress protein phosphatase activity in β-cells [78, 81, 177-179].

Serine/Threonine Protein Phosphatase 5 (PP5) PP5 is a serine/threonine protein phosphatase that belongs to the PPP family [180, 181]. Although it is highly conserved among species and expressed in most, if not all, mammalian cells, the roles of PP5 in biology and disease is only beginning to emerge [182-184], and the influence of PP5 on β-cell function is entirely unknown. The human gene encoding PP5 (PPP5C) is localized on chromosome 19 [185]. PP5 is present both in the cytosol and nucleus [186]. Studying the biological roles of PP5 has proven to be challenging, partly because, just until recently, only a few physiological substrates has been identified. Arachidonic acid, a polyunsaturated fatty acid, has been shown to activate PP5 [187]. A high-throughput screening effort identified a compound, chaulmoogric acid, which can act to activate PP5 at fairly high concentrations [188]. During normal conditions PP5 is predominantly in an inactive state [162, 189]. Therefore, the basal activity of PP5 is very low and represents less than 1 % of the total measurable phosphatase activity. For this reason, changes in PP5 activity are extremely difficult to detect in the high background of other protein phosphatases, in particular PP1, PP2A and PP4. When utilizing natural toxins, such as okadaic acid/microcystin/calyculin A, to inhibit PP5 activity for investigating its cellular functions, it is important to keep in mind that those toxins are not PP5 specific, rather they affects several protein phosphatases. Hence, it would be beneficial to identify small inhibitors specific for PP5, to define its roles [159].

23

Introduction

PP5 is distinctive in that it contains a phosphatase catalytic domain near its C- terminus and a regulatory domain at the N-terminus within a single polypeptide chain [153, 161]. An additional feature, unique for PP5 among its family members, is the extended N-terminal region containing tetratricopeptide repeat (TPR) domains by which PP5 mediates protein-protein interactions [190]. PP5 is associated with various proteins involved in different signaling networks, including heat shock protein 90 (Hsp90) in complex with the glucocorticoid receptor (GR) [191, 192], the cell division cycle (CDC16/CDC27/CDC37) subunits of the anaphase-promoting complex (APC) [193, 194], cryptochrome 2 [195], ATR [196], ATM [197], DNA- PKcs [198], ASK1 [199], Hsp90-dependet heme-regulated eIF2α kinase [200], Rac [201], the A-regulatory subunit of PP2A [202], Raf1 [203], stress-induced phosphoprotein 1 (STIP1) [204] and the Gα12/Gα13 subunits of heterotrimeric GTP-binding proteins [205]. Studies have indicated that suppression of PP5 expression leads to increased activity of GR, enhanced expression of the cyclin dependent kinase inhibitor, p21Waf1/cip1 and hyperphosphorylation of p53, suggesting that PP5 antagonize GR and p53 signaling [206]. The expression of PP5 is responsive to estrogen in human breast adenocarcinoma cells (MCF-7). When PP5 is expressed constitutively, it can convert the estrogen dependency of the MCF-7 cells into an estrogen-independent phenotype, allowing a rapid proliferation in medium lacking estrogen [207, 208]. This indicates a role where PP5 may act as a link between growth suppressing and stimulating signal transduction networks. Several studies have indicated that PP5 is acting in the regulation of signaling cascades activated by oxidative stress, hypoxia and DNA damage. PP5 expression is affected by hypoxia and ROS via activation of hypoxia inducible transcription factor 1α, HIF1α [209]. Both hypoxia and elevated levels of ROS induce the association of PP5 with ASK1, this by dephosphorylation of Thr845 on ASK1, resulting in inactivation of ASK1 [199, 209-211]. This suggests that PP5 can suppress oxidative- stress-induced apoptosis, by preventing the sustained activation of ASK1 and the subsequent activation of JNK, with PP5 acting as a negative regulator of the ASK1/JNK signaling cascade and by that protecting cells from apoptosis [199, 211, 212]. After exposure to ionizing radiation or UV light, PP5 contributes to ATM [183, 197, 213] or ATR [196] mediated checkpoint regulation of the cell cycle, respectively. Taken together, these diverse interactions suggest that PP5 may play an underappreciated role in the regulation of signal transduction cascades that allow the cell to respond appropriately to various kinds of stress. Nevertheless, the regulation mechanism of PP5 activity remains poorly understood.

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Aims

Aims

The general objective of this thesis was to get a deeper understanding, at a molecu- lar level and in biology, of the serine/threonine protein phosphatase 5.

The specific aims were to:

™ Evaluate the biological actions of PP5 by generating a PP5-deficient mouse line

™ Investigate the role of PP5 in response to genotoxic stress

™ Determine if PP5 can act to suppress stress-induced signaling in pancreatic β-cells and thereby provide protection against apoptosis

™ Characterize the glucoregulatory role of PP5 in a diabetic environment in vivo

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26

Materials and Methods

Materials and Methods

Generation of the PP5 Knockout Mouse

Targeting Construct The murine gene encoding PP5 (Ppp5c) is located on chromosome 7 and it spans a region of 23 kb and is comprised of 13 exons. To generate the targeting vector, a mouse 129/SvJ genomic bacterial artificial chromosome library was obtained from Incyte Genomics and screened for the presence of both exon 1 and exon 2 using Southern analysis. 32P-labeled DNA probes were generated using PCR. First a probe targeting exon 2 was created and plasmids containing exon 2 were screened for the presence of exon 1 using a PCR-generated 32P-labeled probe. Digestion with BamHI produced a 10 kb DNA fragment containing exon 1 flanked on both sides with over 4 kb. The targeting vector containing three loxP sites, neomycin, thymidine kinase genes, and enhanced GFP (EGFP) was constructed using conventional cloning techniques (Amable L, 2009, Generation of a knockout mouse model to determine the in vivo function of protein phosphatase 5 (PP5), Ph.D. dissertation, University of South Alabama). The final construct was sequenced in its entirety to verify integrity and correct orientation.

Embryonic Stem Cells The targeting plasmid was digested with SacII, and uptake of linear DNA by 129/X1A embryonic stem cells (ES) derived from 129/SvJ blastocysts (Yale Animal Genomics) was facilitated by electroporation (Bio-Rad Gene Pulser II at 230 V and 500 mF). Following electroporation, ES cells were plated on neomycin-transgenic mouse embryo fibroblasts inactivated by γ-irradiation and grown in ES cell medium (DMEM, 15 % FBS, 1000 units/ml leukemia inhibitory factor, 2 mM glutamine, 0.001 % β-mercaptoethanol, and 0.1 mM nonessential amino acids) containing 300 µg/ml G418 for selection. Individual G418 resistant clones were selected and cultured in 24 well plates coated with 0.1 % gelatin. Southern analysis in combination with SpeI digestion was used to identify clones containing the PP5 targeted allele. Clones with the correct orientation were detected with a 3’ external probe generated via PCR. Conformation of the correct orientation was also obtained by the generation of a 6.5 kb band following digestion with 6.5 DrdI.

Chimeric Mice and Mouse Genotyping A correctly targeted clone was injected into C57BL/6J blastocysts (Yale Animal Genomics). Chimeric male offspring were mated with C57BL/6J female mice (Jackson Laboratories) and determination of different genotypes was performed on genomic DNA. Mouse DNA (from ear punches) was isolated using the Qiagen 27

Materials and Methods

DNeasy blood and tissue kit (Qiagen, Valencia, CA, USA). The amplification parameters for the PCR reaction were as follows: 94 °C for 3 minutes, 94 °C for 45 seconds, 60 °C for 45 seconds, and 72 °C for 1 minute 40 seconds for 30 cycles with a final extension of 72 °C for 2.5 minutes. The PCR product generated by using a sense primer complementary to the PP5 promoter (P1) and an antisense primer complementary to a region in intron 1 (P2), was separated according to size by electrophoresis on a 1 % agarose (Fermentas Inc., Glen Burnie, MD, USA) gel and stained with ethidium bromide (Sigma-Aldrich Corp., St. Louis, MO, USA). The 0.4 kb band indicates the presence of wild-type allele and a 1.3 kb product represents the PP5 knockout allele (Figure 4).

0.4 kb Ppp5c+/+ Ppp5c+/- Ppp5c -/- Wild-type Allele (+) P1 P2 Exon 1

1.3 kb Total Knockout Allele (-) P1 P2 EGFP

Figure 4. Schematic representation of the wild-type and knockout alleles (left panel) and representative PCR products for genotyping (right panel)

Animals, Isolation of Islets of Langerhans and Treatment Experiments were performed on male PP5 knockout (Ppp5c-/-) animals using their male wild-type (Ppp5c+/+) littermates as controls. Mice at various ages were used depending on which experiment that were performed (the specific age of mice are indicated in each paper). Mice were killed by exposure to CO2 followed by decapitation. The pancreatic gland was excised and islets were isolated by collagenase (Roche Diagnostics, Mannheim, Germany) digestion. Islets were placed in RPMI-1640 culture medium (SVA, Uppsala, Sweden) containing 11 mM glucose and supplemented with 10 % FBS, 2 mM L-glutamine (SVA), 6 mg/ml penicillin G and 5 mg/ml streptomycin sulfate (Invitrogen Inc., Carlsbad, CA, USA) for an overnight recovery at 37 ºC in 5 % CO2. The next day, islets were transferred to fresh media (described above) without FBS and supplemented with 0.5 mM palmitate (Sigma-Aldrich) equilibrated with 0.5 % BSA (fraction V fatty acid free; Roche Diagnostics). In the high-fat diet study (paper III), the animals were housed 2 mice per cage with free access to water and food. The mice were kept in ventilated and temperature-controlled rooms with 40-60 % humidity having a 12 hour light/dark cycle. The mice were fed either a high-fat diet (HFD) (D12492, Research Diets, Inc., New Brunswick, NJ, USA) or a control diet (CD) (D12450B, Research Diets, Inc.) 28

Materials and Methods for 10 weeks. The HFD is comprised of 20 % protein, 20 % carbohydrates and 60 % fat, whereas the CD contains 20 % protein, 70 % carbohydrates and 10 % fat; the energy derived from fat is 60 % in the HFD and only 10 % in the CD. Body weight and intake of water and food were monitored weekly. Blood glucose was measured on tail vein blood after a 4 hour fast using a glucometer (OneTouch® Ultra® 2, LifeScan Inc., Milpitas, CA, USA) at three time points: study start (week 0), half- time (week 5) and as a final point during the penultimate week (week 9). At the same time points, blood samples were collected for determination of proinsulin, insulin and non-esterified fatty acid levels in the serum. At the penultimate week, mice were allocated to either an intraperitoneal (i.p.) glucose- or an i.p. insulin tolerance test. Mice were killed by exposure to CO2 followed by decapitation, where after serum and organs were weighed and/or collected. The pancreas was fixed in 4 % paraformaldehyde (PFA), while other organs -- such as heart, brain, liver, kidney, spleen, muscle and fat tissue -- were frozen directly upon removal. The studies were performed according to the guidelines of Karolinska Institutet and approved by the local animal ethics committee.

Primary Cells and Cell Lines

MEF Generation, Culture and Treatment Mouse embryonic fibroblasts (MEFs) were isolated from day 13.5 old embryos from Ppp5c+/- intercrosses using conventional methods [214]. Embryo tissues were mechanically disrupted in 100 ml of 0.25 % trypsin and placed into individual 6 cm dishes in RPMI-1640 medium with 10 % FBS (Invitrogen). After a few days, Ppp5c - /-MEFs failed to thrive in conventional MEF medium. However, the Ppp5c -/-MEFs grew well when plated on gelatin-coated (0.1 %) plates and grown in DMEM-F12 medium supplemented with 5% horse serum, 30 % conditioned medium (filtered medium from the previous passage), insulin (10 µg/ml), EGF (10 µg/ml), hydrocortisone (500 ng/ml), and cholera toxin (100 ng/ml) (Invitrogen). Littermate Ppp5c -/-MEFs and Ppp5c +/+MEFs (passages 3–6) were transformed with SV40 large T-antigen and plated onto 12 well dishes. After the cells attached and grew to around 50 % confluence, they were treated with UV light (irradiated with 0, 6, 12, 18, 24, or 36 J/m2 of shortwave UV from a 23 watt, shortwave UV light source [254 nm]), camptothecin, or hydroxyurea at the different concentrations indicated in paper I.

MIN6 Cell Culture and In Vitro Treatment MIN6 cells [215], derived from mouse pancreatic β-cells, were maintained in DMEM containing 1 mM sodium pyruvate, 25 mM glucose, and supplemented with 15 % FBS, 6 mg/ml penicillin G, 5 mg/ml streptomycin sulfate, 2 mM L-glutamine and 50 µM β-mercaptoethanol (Invitrogen) at 37 ºC and 5 % CO2. Hydrogen peroxide (H2O2) (Sigma-Aldrich) was diluted in the above media to a final

29

Materials and Methods concentration of 0.5 mM. For palmitate exposure, 0.5 mM palmitate equilibrated with 0.5 % BSA was added. The molar ratio of FFA:BSA in the culture medium was 6.7:1. The BSA molecule has 7 binding sites for fatty acids, of which 3 sites are high- affinity sites that never release the fatty acids once they are bound. The remaining 4 sites are low-affinity sites and from these the fatty acids can be released into the medium and from there be taken up by the cells. This procedure, where BSA forms complexes with fatty acids, aims to fill up as many binding sites as possible, because a low amount of fatty acids in relation to the amount of BSA would only fill up the high-affinity sites and - as a consequence - no fatty acids would be released into the medium. For that reason is the molar ratio important [216].

Methodology

Glucose and Insulin Tolerance Tests Glucose tolerance was evaluated by an i.p. glucose tolerance test (IPGTT). Mice were fasted for 4 hours before they received i.p. injections containing a 30 % glucose solution (2 g/kg body weight) (Fresenius Kabi, Uppsala, Sweden). Blood was drawn from the tail vein and glycemia was measured using a glucometer (OneTouch® Ultra® 2, LifeScan Inc.), immediately before (time 0), and at 5, 15, 30, 60 and 120 minutes after the injection. Insulin sensitivity was determined by an i.p. insulin tolerance test (IPInsTT). Random-fed mice were injected i.p. with human recombinant insulin lispro analog (Humalog®, Eli Lilly, Indianapolis, IA, USA) equivalent to 1 unit/kg body weight. Blood glucose levels were measured as described above.

Insulin, Proinsulin and C-peptide Analyses Glucose-stimulated insulin release and insulin content were measured in MIN6 cells following transfection with short-interfering RNAs (siRNA) targeting PP5 or a negative control siRNA, after treatment with or without 0.5 mM palmitate, as previously described [217]. The amount of insulin was determined using a mouse insulin ELISA (Mercodia, Uppsala, Sweden). Serum levels of proinsulin and C- peptide were determined using either a rat/mouse proinsulin ELISA (Mercodia) or by mouse C-peptide ELISA (ALPCO Diagnostics, Salem, NH, USA), respectively. Both assays were performed according to the manufacturer’s instructions.

Non-esterified Fatty Acid (NEFA) Determination Serum concentrations of NEFAs were measured using a NEFA-HR(2) reagent, an enzymatic colorimetric method assay (Wako Chemicals GmbH, Neuss, Germany), according to the manufacturer’s recommendation.

30

Materials and Methods

Immunohistochemistry The pancreatic gland was dissected and placed in 4 % phosphate buffered paraformaldehyde for 48 hours. The fixed pancreas was then embedded in paraffin and sectioned at 5 µM longitudinally through the entire gland. Sections were stained for insulin (guinea-pig anti-insulin polyclonal antibody; DAKO, Glostrup, Denmark) and counterstained with hematoxylin eosin. For quantification of islet volume, we adopted the nucleofector method [218] using a computerized setup for stereology, driven by newCASTTM software (Visiopharm, Hoersholm, Denmark). For each animal, the volume was determined from all islets identified (ranging from 87-125 per animal) in three randomly chosen sections and presented as mean islet volume.

Suppression of PP5 in Cultured Cells using siRNA siRNAs targeting three different regions of mouse PP5 (NM_011155), or corresponding scrambled siRNA (negative control, Neg.C) (Santa Cruz Biotechnology, CA, USA) were used to suppress PP5 expression in MIN6 cells. Cell transfection was aided by electroporation using a Nucleofector® (Lonza, Cologne, Germany) and 100 pM of the indicated siRNA.

Assessment of Cell Viability and Apoptosis In paper I, cell viability was measured using ViaLight Plus Cell proliferation assays (Lonza Inc., Rockland, ME, USA) or CellTiter-Glo Luminescence cell viability assay (Promega Corporation, Madison, WI, USA) according to methods provided by the manufacturer. In paper II, cell viability was assessed by a Cytotoxicity Detection KitPlus (Roche Diagnostics) that measures the amount of lactate dehydrogenase released after cell lysis, which correlates inversely to the amount of living cells. Apoptosis was detected by measuring cytoplasmic DNA-histone nucleosomes generated during apoptotic DNA fragmentation using the Cell Death Detection Kit ELISAPLUS (Roche Diagnostics), according to manufacturer’s instructions.

RNA Isolation, Generation of cDNA and Quantitative PCR In paper I, RNA was isolated from equal amounts of tissues or cells as indicated using TRIzol reagent (Invitrogen). The RNA was precipitated with ethanol and resuspended in 100 µl of nuclease-free water. Then 0.75 µg of RNA was used to synthesize first strand cDNA at 42 °C with murine leukemia virus reverse transcriptase using random hexamers as primers (GeneAmp RNA PCR kit Applied Biosystems). The cDNA generated was then used as a template for PCR. PP5 was detected using primer pairs that amplify exon 1 and exon 8. GAPDH was used as an internal control. In paper II, total mRNA was isolated from cells and islets. cDNA was produced using reverse transcriptase (iScriptTM cDNA Synthesis Kit, Bio-Rad). The

31

Materials and Methods expression levels of mRNAs were measured by SYBR green-based quantitative RT- PCR (iQTM SYBR® Green Supermix). β-actin was used as an internal standard.

Western Blot Analyses Western analysis was performed essentially as described previously [217]. Briefly, protein samples from islets or cells were washed twice with PBS and homogenized in cell lysis buffer. The extracts were collected and centrifuged at 13,000 rpm for 15 minutes at 4 °C and an aliquot of the supernatants was removed for protein determination according to Lowry [219]. Equal amounts of protein (20-30 µg) were mixed with reducing SDS-PAGE sample buffer and boiled for 5 minutes and then separated by 10-15% SDS-PAGE. After electrophoresis, proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad Laboratories). Immunoblot analyses were performed using polyclonal rabbit antibodies generated against a synthetic 15-amino acid peptide identical to unique regions contained in the near C- terminal regions of PP1, PP2A, or PP5 [206, 220] or using antibodies that recognize actin (Sigma-Aldrich), Cdc25A (Abcam, Cambridge, MA, USA), PP5, (Santa Cruz Biotechnology), phosphorylated p53 (S15/18), total p53, phosphorylated Chk1 (S317 or S345), total Chk1, phosphorylated p38 MAPK, total p38 MAPK, phosphorylated JNK, total JNK, phosphorylated c-Jun, total c-Jun and the cleaved form of caspase 3 (Cell Signaling Technology, Inc., Danvers, MA, USA). Dr. Brian Wadzinski (Vanderbilt University, Nashville, TN, USA) generously provided polyclonal antibodies to PP4 and PP6. Immunoreactive bands were detected using enhanced luminol-based chemiluminescene (ECL) (GE Healthcare, Uppsala, Sweden), imaged with a GelDoc system and quantified with Quantity One software (Bio-Rad Laboratories). To verify equal protein loading after imaging, the PVDF membranes were stained with Coomassie Blue.

Analysis of ROS Levels 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-DCF) was used as fluorogenic indicator to detect ROS. Cellular levels of ROS were measured using Image-iT LIVE Green Reactive Oxygen Species Detection Kit (Invitrogen) as described previously [221].

PP2A Enzymatic Activity PP2A activity was determined by using a PP2A specific immunoprecipitation phosphatase assay kit (Millipore, Temecula, CA, USA), which measures free phosphate with malachite green dye. The assay was performed according to the manufacturer’s instructions.

Statistical Analysis Data are presented as mean values ± standard error of the mean (mean ± SEM). Student’s t-test was used when comparing the difference between two groups. For

32

Materials and Methods multiple comparisons, differences were determined by one- or two-way ANOVA followed by Bonferroni post hoc test (GraphPad Prism version 5 software). When comparing genotypic differences regarding expected and experimental distribution of inheritance, an X2 test was applied. A value of p < 0.05 was considered statistically significant.

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34

Results and Discussion

Results and Discussion

The PP5-deficient Mice, Phenotypes and Characteristics Due to the difficulties associated with studying the biological roles of PP5 (see Introduction), we developed PP5-deficient mice (Ppp5c-/-) to help identify and evaluate the actions of PP5. In the generation of the PP5 knockout mouse, the targeting vector was constructed with a homologous recombination strategy for exon 1. By using the technique of a cre/loxP system, exon 1 could be successfully removed in combination with a proper mating scheme. The breeding generated viable offspring, and in order to distinguish between the genotypes, homozygous wild-type (Ppp5c+/+), heterozygous (Ppp5c+/-) and homozygous mice lacking exon 1 (Ppp5c-/-), genotyping was performed. Northern analysis of cells from Ppp5c+/+mice and Ppp5c-/- mice, revealed no evidence of truncated mRNA species. PCR, together with Western analysis, revealed no detectable levels of PP5 either in cells or tissues from the Ppp5c-/- mice. However, PP5 was readily detected in tissue from the Ppp5c+/+ mice. These data indicate that removal of exon 1 is indeed sufficient to produce PP5-deficient mice. Levels of other protein phosphatases, such as PP1, PP2A, PP4 and PP6, were measured in tissues, and the levels of these phosphatases were unchanged after disruption of PP5, suggesting that the viability of the mice was not due to a compensatory effect by other PPP-family phosphatases. Over time some phenotypic differences between Ppp5c+/+ mice and Ppp5c-/- mice have been observed. The mice were fertile, and mating generated viable offspring. No evident aberrant phenotypes were observed in young animals (<8 weeks). After backcrossing on the C57BL/6J background to reach the “golden standard” of N10 [222], analysis of the genotyping data from N6-N10 revealed that the Ppp5c-/- mice were underrepresented. When analyzing >1000 mice generated from Ppp5c+/-/Ppp5c+/- mating, the numbers revealed that the Ppp5c-/- mice represented approximately 19 %, contrasting to the 25 % expected by Mendelian inheritance. In addition, litters from Ppp5c-/-/Ppp5c-/- mating were smaller, with about 2-3 pups/litter. This is less than the Ppp5c+/-/Ppp5c+/- mating, which generated around 5-6 pups/litter. Even so, PP5 is not essential for normal growth, because the Ppp5c-/- mice were viable, fertile and had no detectable differences at birth. In offspring from Ppp5c+/-/Ppp5c+/- mating, which were fully backcrossed and non- stressed, some phenotypical differences were observed. Male Ppp5c-/- mice weighed less than their wild-type littermates, a difference that was significant already after 2 months of age (Figure 5). A similar trend was seen for the female Ppp5c-/- mice, which also are smaller than the wild-type littermates. That difference becomes significant after 5 months of age (data regarding the female mice, unpublished). Other features that were observed in the mice lacking PP5 was associated with glucose homeostasis. Male Ppp5c-/- mice had lower fasting glycemia compared to the

35

Results and Discussion

Ppp5c+/+ mice but there was no change in serum insulin levels. However, the C- peptide levels were higher in the Ppp5c-/- mice, and this resulted in a significant increase in the C-peptide to insulin ratio. The number of pancreatic islets and their mean volume were determined in histological sections from both strains, but no differences were observed. Glucose handling of the mice was determined by an IPGTT, which showed that the Ppp5c-/- mice had improved glucose tolerance, but there was no difference in insulin sensitivity, analyzed by an IPInsTT, between the two genotypes. Taken together, these data indicate that insulin secretion is elevated in the Ppp5c-/- mice, which could explain the lower glycemia in these mice. The PP5- deficient mice could also eliminate glucose faster than wild-type mice, as shown by the IPGTT, despite unchanged insulin sensitivity. It seems that the Ppp5c-/- mice may clear insulin more efficiently, indicating that PP5 might regulate hepatic insulin clearance. Upon autopsy an additional phenotypical attribute was observed. Splenomegaly was observed in the female Ppp5c-/- mice that were one year of age. The same was observed in the male mice at 4-5 months of age (data regarding the male mice, unpublished). How PP5 deficiency contributes to this phenotypic trait is unclear. In islets from the Ppp5c+/+ mice, the expression levels of Ppp5c were comparable to that of Slc2a2 and Gck but lower than Ins2 and the housekeeping genes Actb and Gapdh. Our data indicate that these mice are indeed useful tools when investigating the role of PP5 in glucose homeostasis and other signaling pathways.

Figure 5. Size difference between 2-month-old male wild-type (upper) and PP5- deficient (lower) mice

36

Results and Discussion

The Role of PP5 in Response to Genotoxic Stress Overexpression of PP5 in human cells has been shown to provide resistance to UV- induced apoptosis, and PP5 has been reported to be essential for ATR-mediated checkpoint signaling. Therefore, we wanted to further investigate the roles of PP5 in cellular responses to DNA damage caused by UV light. We used MEFs generated from littermate Ppp5c+/+ and Ppp5c-/- mice to study the role of PP5 in response to UV light-induced damage. The MEFs generated from Ppp5c-/- mice (Ppp5c-/-MEFs) were, in contrast to MEFs generated from Ppp5c+/+ mice (Ppp5c+/+MEFs), unable to thrive in conventional MEF medium. Within a few days they died. However, when the Ppp5c-/-MEFs were placed in enriched MEF medium following transformation with SV40 large T-antigen, they were able to proliferate appropriately in culture. Under these conditions, the MEFs were stable through passage 7. However, it is not clear at this time, whether all of the components added to the MEF medium are necessary for the survival of the Ppp5c-/-MEFs. This may indicate that deletion of PP5 makes the survival process tougher. This is also seen in mice lacking PP5, which seem to have a disadvantage during embryonic development, as evident by fewer than expected Ppp5c-/- mice in litters. These data suggest that PP5 is not necessary for survival; however, it might have an advantageous role during development. The notion that the absence of PP5 might be associated with increased sensitivity to damage was confirmed by measuring cell viability after 24 hours of treatment in Ppp5c+/+MEFs and Ppp5c-/-MEFs exposed to different amount of UV light. These studies revealed that the Ppp5c-/-MEFs were indeed more vulnerable. Exposure to UV light at levels that had no effect on the Ppp5c+/+MEFs, killed approximately 25 % of the Ppp5c-/-MEFs. This indicates that the MEFs lacking PP5 have heightened sensitivity to UV irradiation. The Ppp5c-/-MEFs were also more sensitive to the two DNA damage-inducing agents, camptothecin and hydroxyurea, compared to the Ppp5c+/+MEFs. These data show that MEFs lacking PP5 are more susceptible to all three treatments, which are all known to activate ATR by inducing DNA damage albeit in different ways. We continued to study the role of PP5 downstream of ATR by examining Chk1 phosphorylation, a hallmark of ATR activation. UV light-treated Ppp5c+/+MEFs and Ppp5c-/-MEFs, both produced a transient increase in Chk1 phosphorylation at S345. Still, a comparison of the response between the genotypes showed that S345 phosphorylation was greater and remained longer in the Ppp5c-/-MEFs. In the untreated cells from both Ppp5c+/+MEFs and Ppp5c-/-MEFs, no phosphorylated Chk1 was detected, and in the treated groups the phosphorylation returned to basal levels after 24 hours. In MEFs from both genotypes, UV light also increased phosphorylation of Chk1 at S317. Phosphorylated γ-H2AX at S139, an indicator of DNA double-strand breaks, was also increased in both MEF genotypes, although it was more prominent in the Ppp5c-/-MEFs. In the same samples, p53 was analyzed, revealing increased and more robust phosphorylation at S18, at all time points, in the Ppp5c-/-MEFs compared to Ppp5c+/+MEFs. Furthermore, phosphorylation of 37

Results and Discussion p53 was observed in the untreated Ppp5c-/-MEFs, and p53 protein levels were elevated at all time points analyzed after stress, as compared to the Ppp5c+/+MEFs. Cells from Ppp5c+/+MEFs and Ppp5c-/-MEFs were pretreated with 1 mM caffeine, a concentration sufficient to inhibit activation of both ATR and ATM, before a 30 minute exposure to UV light. The UV-induced phosphorylation of both Chk1, at S354, and p53 was decreased in the Ppp5c+/+MEFs. In the Ppp5c-/-MEFs, the caffeine treatment clearly suppressed the hyperphosphorylation of Chk1, but the basal and UV-induced hyperphosphorylation of p53 was only suppressed to a small extent. Next, Cdc25A was analyzed after UV light exposure, since degradation of Cdc25A is a characteristic for ATR-mediated Chk1 activation. In the Ppp5c+/+MEFs the UV light generated a transient decrease in the Cdc25A levels, that was evident after 1 hour and returned to baseline after 5 hours. Cdc25A degradation was more prominent in the Ppp5c-/-MEFs, and after 5 hours the rebound seen for the wild-type cells had not yet occurred. However, after 24 hours the Cdc25A levels were similar for both genotypes. This suggests that PP5 probably is not the only phosphatase that acts on Chk1 at S345. Taken together, the PP5-deficient mice were viable and fertile, indicating that PP5 alone is not crucial for ATR signaling. This can be suggested because ATR knockout in mice results in embryonic lethality [223]. Since UV light exposure to these cells could induce phosphorylation of Rad17 at S645, which is dependent on ATR, we could not find any evidence that ATR activation was prevented in the Ppp5c-/-MEFs. For that reason we examined events downstream of ATR. Our data refine the role of PP5 in the UV light-induced response that was originally proposed by Zhang et al [196] by placing PP5 downstream of ATR and upstream of Chk1. The absence of PP5 was also associated with enhanced phosphorylation of p53 and enhanced p53 protein levels. Since there are numerous complex pathways regulating p53 phosphorylation, extensive studies are needed in order to clarify in what way PP5 affects p53 signaling. It should also be mentioned again that the MEFs were transfected with SV40 large T-antigen, which functions by inactivating p53 through binding and in so doing prevents apoptosis. Even so, both genotypes of MEFs were treated the same way and the results thus cannot solely be explained due to the SV40 large T-antigen. High levels of PP5 have been reported in human cancers and might contribute to tumor development, suggesting that PP5 may be involved in promoting cell survival, possibly by a compensatory mechanism that counteracts the action of ATR.

The Role of PP5 in Pancreatic β-cells In the natural development of T2DM, there is a relentless loss of β-cells due to increased apoptosis (see Introduction), contributing to the progressive natural course of the disease. Conversely, finding ways and means to protect and preserve β-cells is a major, but formidable, task in contemporary diabetology. Our finding that PP5 might promote cell survival, together with the totally unexplored role of

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Results and Discussion

PP5 in β-cells, prompted us to investigate whether PP5 might serve to protect the pancreatic β-cells. If that were to be the case, PP5 activation becomes an attractive antidiabetic target. To this end, we utilized our generated PP5-deficient mice and isolated pancreatic islets from both Ppp5c+/+ and Ppp5c-/- mice. They were subsequently exposed in vitro to 11 mM glucose together with 0.5 mM palmitate, a saturated fatty acid, for 24 hours, thus mimicking the glucolipotoxic environment β- cells are exposed to during the progression of T2DM [105]. This treatment induced islet cell apoptosis in both genotypes but was more pronounced in the islets lacking PP5 compared to wild-type islets. This correlated with an increase in phosphorylated JNK1 levels and total JNK2, indicating that PP5 deficiency is linked to increased vulnerability of the islets towards palmitate-induced stress. The MIN6 cell line was used to further investigate the role of PP5 in β-cells, by inducing stress through exposure to 0.5 mM H2O2. This treatment increased cellular levels of ROS, which was associated with enhanced MAPK signaling. H2O2 increased the phosphorylation levels of JNK1, JNK2 and the downstream substrate c-Jun, as well as the p38 MAPK. H2O2 reduced cell viability with only 5 % after 30 minutes. However, this brief exposure was sufficient to induce irreversible cell damage, as indicated by the death of approximately 75 % of the cells when they were treated for 30 minutes and then incubated in the absence of H2O2 for 8 hours. A comparable amount of cells died in the groups that were given H2O2 continuously. Notably, the effects of H2O2 on cell viability could be fully reversed by adding the ROS scavenger catalase into the medium. Next, we addressed the role of PP5 in this context by transfecting MIN6 cells with siRNA targeting PP5, which reduced the PP5 levels successfully both on mRNA and protein level. Cells with reduced levels of PP5 displayed elevated phosphorylation levels of JNK1, JNK2 and c-Jun after exposure to H2O2, indicating increased sensitivity. However, the phosphorylation levels of p38 MAPK were not affected by the loss of PP5. The silencing of PP5 did not have any additional effect on the cellular ROS levels or the expression of scavenging enzymes. These data indicate that exposure to H2O2 in MIN6 cells promotes JNK signaling, which can be negatively regulated by PP5.

In view of the fact that H2O2 is an artificial way of inducing oxidative stress, we turned to use palmitate. Exposure of MIN6 cells to palmitate in combination with 25 mM glucose increased cellular levels of ROS and enhanced apoptosis, as previously reported [106, 224]. The same treatment also induced JNK1, JNK2 and c-Jun phosphorylation, and -- in combination with low (5.5 mM) glucose -- palmitate could again activate JNK phosphorylation, although not to the same extent as observed in high glucose. In contrast, the p38 MAPK was not activated, regardless of glucose concentration. These data suggest that palmitate is involved in inducing apoptosis particularly through the JNK pathway in these cells. The effects of palmitate were further evaluated in MIN6 cells with reduced levels of PP5. The results obtained from palmitate treatment were in concordance with data obtained following H2O2 treatment, i.e. increased susceptibility in cells in which PP5 levels

39

Results and Discussion were suppressed. In cells treated with siRNA targeting PP5, phosphorylation levels of JNK2 and c-Jun were augmented compared to cells treated with negative control siRNA. In addition, DNA fragmentation and cleaved caspase 3 levels were both augmented in PP5-silenced cells. These data indicate that palmitate-induced ROS production can activate JNK signaling, which is coupled to apoptosis, and that PP5 can act to protect these cells from the harmful effects of palmitate. The PP5-deficient mice subjected to an IPGTT showed that they can clear glucose faster than their wild-type littermates, although the insulin sensitivity was the same. This suggests that the mice lacking PP5 might release insulin more efficiently. Therefore, GSIS was measured in MIN6 cells, which revealed an insulin secretory response to 20 mM glucose, which was more than doubled in cells with reduced PP5 levels compared to control cells. The difference remained when the cells were exposed to palmitate, but there was no variation in insulin content. Taken together, our data provide additional insight into the molecular actions of PP5 in the pancreatic β-cells. MIN6 cells exposed to H2O2 showed increased cellular levels of ROS together with increased phosphorylation of JNK, c-Jun, p38 MAPK and reduced cell viability. Silencing of PP5 expression resulted in more pronounced H2O2 effect on JNK signaling, but without additional increase of ROS or changed p38 MAPK levels. Palmitate treatment generated comparable results in these cells, with increased phosphorylation of JNK signaling that was augmented in cells with reduced levels of PP5, regardless or glucose concentration, indicating that the palmitate effect on JNK signaling is not dependent of a certain glucose concentration. These findings lend support to previous studies that indicate that PP5 can suppress ROS-induced JNK signaling and apoptosis in other cell types [199, 211, 225, 226]. An important issue is to explore was the possibility that other phosphatases -- structurally related to PP5 -- were upregulated in a compensatory manner when PP5 is suppressed, because other protein phosphatases (especially PP2A) have been shown to also influence JNK phosphorylation. Therefore, we measured mRNA and protein levels of Ppp1ca, Ppp2ca and Ppp3ca (calcineurin). The results revealed no such compensatory effect. Additionally, the activity of PP2A was not affected by the treatment of siRNA targeted against PP5, showing that the siRNA treatment is indeed PP5-specific. Suppression of PP5 also resulted in enhanced GSIS in MIN6 without an effect on the total insulin content of the cells. This is consistent with findings from a previous study showing that used okadaic acid inhibition, which inhibits the catalytic activity of PP1, PP2A, PP4, PP5 and PP6, lead to enhanced insulin secretion in the β-cells [176]. Furthermore, the islets from the PP5-deficient mice were more vulnerable to palmitate-induced JNK activation and subsequent apoptosis, suggesting a direct anti-apoptotic role for PP5 in the pancreatic β-cells. A study by Yamaguchi et al [227] showed that S100-calcium binding protein (S100) could bind to the TPR domains of PP5, and in so doing inhibited the interaction between PP5 and ASK1, by inhibiting the dephosphorylation of ASK1 by PP5. These S100 proteins have also been shown to accumulate in the plasma and

40

Results and Discussion potentially contribute to the pathogenesis of T2DM [228]. A recent report by Hinds et al [182], investigating the balance between lipolysis and lipogenesis in MEFs generated from another variant of PP5 knockout mice, suggested that PP5 may play a role in lipid and glucose metabolism. More studies are required in order to assess the specific role of PP5 in β-cell function and glucose homeostasis. In their entirety, our data provides evidence that PP5 is a previously unrecognized actor in the regulation of pancreatic β-cell apoptosis and glucose homeostasis. Consequently, PP5 may represent an underappreciated player in the development and progression of diabetes.

The Role of PP5 in a Diabetic Setting Based upon the finding that PP5 might be involved in regulating glucose homeostasis and also may protect β-cells from lipotoxicity, we investigated the role of PP5 in a diabetic environment induced by a high-fat diet. Age-matched Ppp5c-/- and their control littermates, Ppp5c+/+, were placed on either standard or high-fat diet for ten weeks. The Ppp5c+/+ mice, as expected, gained weight after being fed a HFD, while mice lacking PP5 gained weight to a much lesser extent. There was a clear difference between the genotypes fed HFD, with Ppp5c+/+ mice gaining more weight compared to the Ppp5c-/- mice, suggesting that the absence of PP5 is associated with a protective effect against weight gain by HFD. Remarkably the weight of the HFD fed Ppp5c-/- mice was less compared to the Ppp5c+/+ on CD. A phenotypic trait distinguishing the Ppp5c+/+ from Ppp5c-/- mice is the difference in body weight, with PP5-deficient animals being smaller, as reported previously [184]. Therefore, comparison of the groups was done both in terms of percentage of their baseline weight (weight gain) and in absolute values. Our data indicate that PP5-deficient mice are less prone to gain weight than their wild-type littermates when placed on a HFD, suggesting a correlation between PP5 deficiency and protection against HFD-induced weight gain. The difference in weight gain could not be explained by a difference in a caloric intake. Although the mice were housed as follows, Ppp5c+/+/Ppp5c+/+, Ppp5c+/+/Ppp5c-/- and Ppp5c-/-/Ppp5c-/-, no apparent difference in either food or water intake between the genotypes were observed. However, both strains of mice ate less HFD than CD, which can be explained by the energy density being higher for the HFD than the CD. To assess the diet-induced effect on glycemia and insulin levels, fasting blood glucose and serum insulin levels were determined. During a treatment period of ten weeks, euglycemia is anticipated to be maintained by increased insulin secretion. In the Ppp5c+/+ mice on HFD, insulin secretion was increased as expected and the fasting blood glucose was not changed over this time. The opposite occurred in the Ppp5c-/- mice on HFD, which were unable to increase their insulin secretion and accordingly displayed elevated fasting blood glucose after the ten weeks. In fact, the Ppp5c-/- animals showed lower fasting insulin levels than the Ppp5c+/+ mice. On the other

41

Results and Discussion hand, the Ppp5c-/- animals had lower basal fasting blood glucose on CD which was maintained over the study period compared to Ppp5c+/+ mice. This indicates that the Ppp5c-/- mice have a reduced capacity of β-cell adaptation to obesity and insulin resistance. To evaluate their glucose handling and insulin sensitivity, the mice were subjected to IPGTT and IPInsTT, respectively. The Ppp5c-/- mice on CD had significantly lower blood glucose levels compared to the Ppp5c+/+ mice during the glucose challenge. HFD impaired glucose tolerance in both genotypes, despite unchanged insulin sensitivity. To investigate whether these metabolic differences are related to changes in β- cell function, we measured serum levels of intact proinsulin and insulin and calculated the ratio, which is often used as a surrogate marker of β-cell dysfunction [229, 230]. However, neither of the genotypes showed any indication of β-cell dysfunction after HFD. Additionally, neither the Ppp5c+/+ mice nor the Ppp5c-/- mice showed any difference in NEFA levels. Next, we weighed the fat deposits after sacrifice to examine if there might be a difference in adipogenesis. The Ppp5c+/+ mice had a clear increase in kidney-, epididymal- and subcutaneous fat after HFD compared to CD, however, this was not observed in the Ppp5c-/- animals. The fat deposits of the PP5-deficient mice were slightly increased by the HFD, but significantly smaller than the wild-type mice. These data indicate that both genotypes can accumulate fat, although the PP5-deficient mice produced smaller fat deposits after HFD treatment. This is in agreement with a recently published paper [182], in which MEFs isolated from another line of PP5 knockout mice were used. MEFs without PP5 expression showed resistance to accumulation of lipids in response to adipogenic stimuli caused by increased GR phosphorylation in combination with decreased activity of the peroxisome proliferator activated receptor γ (PPARγ), which regulates genes important for lipid metabolism. These data indicate that the PP5-deficient MEFs were less responsive to adipogenic stimuli, suggesting that PP5 is necessary for adipogenesis and could be a target for the treatment of obesity. Taken together, we found that disruption of PP5 decreases HFD-induced weight gain. In order to mimic the pathogenesis of a typical T2DM patient, we employed HFD as a tool for inducing a diabetic setting with obesity and insulin resistance [231]. The HFD consists of 60 % fat, mostly lard, with high saturated fat content. From a nutritional point of view, this diet would be considered extreme if applied on humans. However, as a tool for inducing obesity in rodents to investigate dietary effects after a short period of time, HFD is commonly used [232, 233]. Together with the reduced weight gain, mice lacking PP5 also showed better glucose tolerance than wild-type littermates when fed CD. It appears that the Ppp5c-/- mice are more susceptible to the harmful and diabetogenic property caused by HFD feeding compared to the Ppp5c+/+ mice. The difference between baseline and end point in the mice lacking PP5 is bigger, but in absolute terms the Ppp5c-/- mice show lower fasting glycemia and better glucose tolerance than the Ppp5c+/+ mice. Additional studies are needed to address the direct or indirect function of PP5 in

42

Results and Discussion glucose homeostasis and regulation of insulin secretion. When considered in combination with our previous in vitro findings, suggesting that PP5 may protect β- cells from lipoapoptosis, the current in vivo data suggest that PP5 may be a potential target for novel therapies to protect pancreatic β-cells in the progression towards obesity and diabetes.

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Conclusions

Conclusions

In summary, we present data that provide an additional understanding of the roles played by PP5 in signal transduction, and for the first time assign a biological function to this phosphatase. We confirm the involvement of PP5 in the DDR response by showing that MEFs lacking PP5 are more sensitive to UV light-induced DNA damage. We then describe the site of interaction between PP5 and this stress response. Our data suggest that PP5 can regulate cell survival and that enhancement of PP5 may be a strategy to protect cells against stress-induced cell death. This is in line with the finding that overexpression of PP5 is observed in some human cancers and may aid tumor development [208]. We also prove the first evidence that PP5 may have a biological function in regulating glucose homeostasis. The PP5-deficient mice are characterized by reduced weight gain and improved glucose control with lower fasting glycemia and enhanced glucose tolerance. Also, the lack of PP5 protects mice against HFD-induced weight gain, even though they cannot sustain glucose control throughout the HFD treatment. At a molecular level, PP5 is regulating stress-induced apoptosis in β-cells by suppressing a mechanism that involves JNK signaling. Our results may imply that enhancing PP5-mediated actions could be exploited to confer protection against lipotoxicity in β-cells, an effect which may be harnessed to advantage in attempts to preserve β-cell function and mass that deteriorate during the natural course of both T1DM and T2DM. In this context, it is interesting to note that arachidonic acid, which can activate PP5 [187], was recently shown to prevent lipotoxicity in a clonal β-cell line [234]. As PP5 is a multi-tasking regulator of a number of signaling pathways, one needs to recognize that different approaches to regulate PP5 may be necessary since one treatment may function in different ways depending on tissue or environmental settings. From a clinical perspective, the conclusion that inhibitors of PP5 may be useful to treat human disorders should be approached very cautiously. On the one hand, results obtained in vitro with islets lacking PP5, corroborated by studies using PP5- silenced MIN6 cells, indicate that PP5 might protect β-cells from ROS/palmitate- induced damage. On the other hand, our in vivo data indicate that PP5 deficiency is associated with reduced weight gain, better glucose tolerance and enhanced insulin secretion. Together our data therefore suggest that PP5 may represent a double- edged sword in the fight against diabetes, with an inhibitor useful to prevent the progression from a pre-diabetic to a diabetic state, yet possibly harming β-cells exposed to a glucolipotoxic environment.

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Acknowledgements

Acknowledgements

It is truly a bittersweet feeling to complete a work like this. My thesis is the result of the inputs from several people, who have helped me in various ways in order to make my thesis doable. Their contributions have been truly indispensable. I wish to express my sincere gratitude to all of you who made this journey possible. My supervisor Åke Sjöholm deserves a special tribute. You have helped me in countless ways. Your meticulous reading, great scientific advice and guidance have provided me with many opportunities to improve my academic writing and research quality. Thank you for your never ending encouragement, contagious enthusiasm for hard work and for always believing in my academic ability. Without you this thesis would not have been completed. Richard Honkanen, you have been a wonderful co-supervisor. Your cheerful support, perceptive reading, and valuable advice have improved my work considerably. You have been of invaluable help in making me believe in my scientific capabilities, and for that I am deeply grateful. An additional thank-you for introducing me to the extraordinary world of P90X! Henrik Ortsäter has been a great co-supervisor. Thank you for valuable and constructive comments, discussions and advice. Your ceaseless appetite for questioning has helped me to sharpen my arguments and writing. Together, you three have showed me the true spirit of supervising, what it meant to write a thesis and, more importantly, that I could do it. Thank you for all the hours you have put into reading my drafts, commenting on them and for your persistent attempts to always help me improve my work. You have shaped my thinking profoundly. I would also like to thank Qimin Zhang, who was supportive during the first years of my work, when he was my co-supervisor. The Unit of Diabetes Research led by Åke Sjöholm has been my main intellectual home. To my colleagues, thank you for an inspiring research environment with many fruitful discussions. Cesare Patrone and Vladimer Darsalia for collaboration and for generously introducing me to sterology. Special thanks to my fellow PhD-students: Linnéa Eriksson, Liselotte Fransson, Victoria Rosengren, Camilla Kappe, Özlem Erdogdu, Shiva Mansouri, Anna Olverling and Hanna Dahlin. Sharing this journey with you has indeed been fun. Our lunches, coffee breaks and after works, while constantly dwelling about our research, have been an important source of energy. A great environment results in great science, and that is indeed what our group is all about! An additional thanks to Linnéa, for sharing both highs and lows with me, for our enjoyable conversations over a glass of wine, for being my technical iPhone support but foremost for being such a great friend, and

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Acknowledgments

Liselotte for our fun times during traveling to different research meetings. An additional thank-you also to Petra Wolbert for her skilled technical assistance and for excellently helping me with the PP5 mice. As part of my project, I spent three months at the Department of Biochemistry and Molecular Biology within the College of Medicine at the University of South Alabama in Mobile, U.S.A., invited by Richard Honkanen, for this I am tremendously grateful. This was truly an experience of a lifetime in many ways, in which I learned many things. The opportunity to work in another lab gave me perspective and it was as coming up for fresh air. Thank you so much for this Rich! I also wish to thank the chairman William Gerthoffer and the staff there, in particular, Danalea Skarra, Ileana Aragon, Mark Swingle, Lauren Amable, Cathy Thompson, Betty Farley, Lanette Flagge and Howard Shell for making my stay such an enlightening experience. I value my interaction with you highly and appreciate the probing and inspiring discussions we had. You have made University of South Alabama my second intellectual home. An additional thanks to Danalea, your support, our long talks over Skype and all the fun times that we shared during my visits have all meant a lot to me. Thanks for fantastic friendship and inspiration! Special gratitude goes to the Honkanen family, Richard, Mary, Megan and Kelly, for their endless hospitality and generosity. Thanks for letting me share your day-by-day life in Mobile, Perdido Key and Crescent. I had such a great time and it was a true pleasure getting to know you and I miss you all so very much! A number of people should also be especially mentioned: Past and present chairman (Sari Ponzer, Göran Erlinder and Maaret Castrén) of the Department of Clinical Science and Education, Södersjukhuset, together with past and present head of Forskningscentrum (Hans Olivecrona, Michael Eberhardson and Jeannette Lundblad-Magnusson), for providing such excellent research facilities and creating a great scientific environment and also for all the fun events. Everyone else at Forskningscentrum, present and former colleagues, thanks for creating such a pleasant and encouraging atmosphere. All staff at KISÖS throughout the years, especially Anita Stålsäter-Pettersson, Jeanette Öhrman, Lina Rejnö, Anne Edgren, Christina Hadders Medin, Matts Jonsson, Lina Benson and Hans Pettersson, for administrative assistance and for helping me with so many things on so many occasions. Viveca Holmberg for giving me the opportunity to lecture at KI SÖS Kliniska Forskarskola and also participate in the festivities, it has been a true pleasure. Christina Häll, Lotta Larsson and Monica Nordlund for all your help and support during these years. Diana Rydholm and Richelle Fall for great animal care. A special thanks goes to some unexpected contributors; All the sweet but aggressive PP5 mice that contributed to the studies. Salty licorice, Reeses peanut butter cups and Starbucks coffee, which gilded the time during my PhD studies.

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Acknowledgements

Finally, my profound gratitude goes to my family. My parents, Jan and Ann- Christin, who always encourage me to achieve my goals in life, support me when things are difficult and celebrate me when I succeed. You are the most amazing parents one could ever wish for and I am who I am because of you. My dear sister, Hannah, you are truly a great sister and more so my best friend. I owe so much to you. You have always been my steady rock and inspiration. I am so grateful for all your help and constant support during the last chaotic stage of writing. I simply could not do without you. Björn, you have been a part of this journey from the very beginning and sharing it with you has been truly wonderful. I am so grateful for your endless patience, love and support during these years. I am looking forward to share new adventures with you. To you and my family, I dedicate this thesis.

Stockholm, May 2012

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References

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