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Oxidative and ER stresses: breaking the cycle to preserve beta cells in diabetes

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Oxidative and ER stresses:

breaking the cycle to preserve beta cells in diabetes

Thesis submitted by Ana Filipa MARTINS OLIVEIRA

with a view to obtaining the PhD Degree in Biomedical and pharmaceutical

sciences (“Docteur en Sciences biomédicales et pharmaceutiques”)

Academic year 2019-2020

Supervisor: Professor Miriam Cnop

Co-supervisor: Professor Mariana Igoillo-Esteve

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Contents

Contents ... 1

Papers constituting this thesis ... 3

Abbreviations ... 5

Abstract ... 7

Résumé ... 9

Introduction ... 11

Diabetes mellitus ... 11

Role of the pancreatic beta cell ... 12

Glucose sensing and insulin secretion ... 12

Insulin biosynthesis ... 15

Role of insulin signaling in the beta cell ... 15

Type 2 diabetes and monogenic diabetes ... 16

The role of mitochondria and endoplasmic reticulum on beta cell function and survival ... 18

Oxidative stress and mitochondrial dysfunction ... 19

ER stress and the unfolded protein response ... 23

Triggers of beta cell oxidative and ER stress ... 25

Beta cell apoptosis as consequence of oxidative and ER stress ... 28

Current therapeutic approaches in type 2 diabetes ... 31

Oxidative and ER stress as therapeutic targets for the treatment of type 2 diabetes ... 32

Cellular models for beta cell research and drug discovery ... 33

Aims ... 35

Results ... 37

PAPER I ... 37

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Papers constituting this thesis

PAPER I: Oliveira AF, Cunha DA, Ladriere L, Igoillo-Esteve M, Bugliani M, Marchetti P, Cnop M (2015). In vitro use of free fatty acids bound to albumin: A comparison of protocols. Biotechniques 58(5):228-33. doi: 10.2144/000114285

PAPER II: Oliveira AF, Cunha DA, Ladriere L, Igoillo-Esteve M, Marchetti P, Eizirik DL, Cnop M. Harmine protects pancreatic beta cells from lipotoxic endoplasmic reticulum stress and apoptosis. (In preparation for submission)

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Abbreviations

ATF Activating transcription factor

BAD BCL-2 associated agonist of cell death BAK BCL2 antagonist/killer

BAX BCL2 associated X protein BCL2 B-cell lymphoma 2

BH BCL2 homology

BID BH3 interacting domain death agonist BIM BCL2 interacting mediator of cell death BiP Immunoglobulin heavy chain binding protein cAMP Cyclic adenosine monophosphate

CHOP C/EBP homologous protein CPT Carnitine palmitoyl transferase

CReP Constitutive repressor of eIF2α phosphorylation DP5 Death protein 5

DYRK Dual-specificity tyrosine phosphorylation-regulated kinase eIF Eukaryotic initiation factor

ER Endoplasmic reticulum FFA Free fatty acid

FFAR Free fatty acid receptor FFAR FFA receptor

FRDA Friedreich’s ataxia

GAA Guanine-adenine-adenine

GADD34 Growth arrest DNA damage inducible 34 GCK Glucokinase

GCL Glutamate–cysteine ligase GDM Gestational diabetes mellitus

GIP Glucose-dependent insulinotropic polypeptide GLIS3 Gli-similar 3

GLP-1 Glucagon-like peptide 1 GLP-1R GLP-1 receptor

GLUT Glucose transporter

GSIS Glucose-stimulated insulin secretion HFN Hepatocyte nuclear factor

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MAO Monoamine oxidase MCL1 Myeloid cell leukemia 1

MIDD Maternally inherited mitochondrial diabetes and deafness MIDY Mutant INS-gene-induced Diabetes of Youth

MODY Maturity-onset diabetes of youth mtDNA mitochondrial DNA

NDM Neonatal diabetes mellitus

NFAT Nuclear factor of activated T cells NF-κB Nuclear factor κappa B

NOX NADPH oxidase

NQO1 NAD(P)H:quinone oxidoreductase 1 NRF Nuclear respiratory factor

PBA 4-Phenyl butyric acid

PDX Pancreatic/duodenal homeobox

PERK Protein kinase RNA-like endoplasmic reticulum kinase PGC PPARy co-activators

PI3K Phosphoinositide 3-kinase PK Protein kinase

PP1c Protein phosphatase 1c

PPAR Peroxisome proliferator-activated receptor PSC Pluripotent stem cell

PSDM Permanent neonatal diabetes mellitus RNS Reactive nitrogen species

ROS Reactive oxygen species

SERCA Sarco/endoplasmic reticulum Ca2+ ATPase 2b

SGLT2 Sodium-glucose co-transporter 2 SUR1 Sulfonylurea receptor 1

TFB1M Transcription factor B1 mitochondrial TG Triglycerides

TNDM Transient neonatal diabetes mellitus TRB3 Tribbles 3

TUDCA Taurine-conjugated ursodeoxycholic acid tauroursodeoxycholic acid UCP Uncoupling protein

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Abstract

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Résumé

La prévalence du diabète de type 2 augmente considérablement et constitue un problème de santé publique et socio-économique majeur. Un niveau élevé d'acides gras libres saturés (AGL), associé a un régime alimentaire hautement calorique ainsi qu’à l'obésité, contribuent à la résistance à l'insuline, au dysfonctionnement des cellules bêta et sont liés au développement futur du diabète de type 2. Le dysfonctionnement mitochondrial et le stress du réticulum endoplasmique (RE) jouent un rôle central dans la défaillance et la mort des cellules bêta causée par les AGL. La synthèse et la sécrétion de l’insuline en réponse au glucose constituent un défi pour la fonction du RE des cellules bêta. La réponse aux protéines non repliées (unfolded protein response, UPR) est déclenchée pour faire face aux perturbations de l'homéostasie du RE mais elle peut finalement conduire à l'apoptose des cellules bêta si le stress du RE n'est pas résolu. En outre, la production excessive de dérivés réactifs de l'oxygène en tant que sous-produits du métabolisme oxydatif entraîne un stress oxydatif, un dysfonctionnement mitochondrial et aggrave davantage le stress du RE. Nous avons exploré les mécanismes moléculaires sous-jacents à la perte de cellules bêta due au dysfonctionnement mitochondrial et du RE et démontré le potentiel de réduction des stress oxydatifs et du RE comme approche thérapeutique du diabète de type 2.

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neurones sensoriels et les cellules bêta déficients en frataxine en normalisant l'état oxydatif mitochondrial et en améliorant la fonction mitochondriale.

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Introduction

Diabetes mellitus

Diabetes is a group of chronic, metabolic diseases characterized by high blood glucose (blood sugar) levels resulting from inherited and/or acquired defects in insulin secretion by the pancreas, insulin action, or both. This chronic hyperglycemia is associated with long-term damage, dysfunction, and failure of different organs, especially the eyes, kidneys, nerves, heart, and blood vessels. Blurry visions, excess thirst, fatigue, frequent urination, hunger and weight loss are some of the symptoms commonly seen in diabetic patients. When left untreated, hyperglycemia can lead to diabetic ketoacidosis, an acute, life-threatening condition (1).

Based on its etiology, diabetes can be classified into the following general categories:

Type 1 diabetes is a multifactorial autoimmune disease characterized by progressive

destruction of pancreatic beta cells ultimately resulting in absolute deficiency of insulin secretion and consequent chronic hyperglycemia (2). Autoimmune destruction of beta cells has multiple genetic predispositions and is also related to environmental factors. It accounts for about 10-15% of all cases of diabetes, and remains a serious chronic disorder, usually with an early onset (during childhood or adolescence) and serious short-term and long-term implications. Since the condition causes the loss of insulin, treatment focuses on maintaining normal blood glucose levels through regular monitoring, diet, exercise and insulin therapy (3, 4).

Type 2 diabetes is a chronic disease in which hyperglycemia develops due to a

defective capacity of beta cells to secrete enough insulin to compensate the peripheral insulin resistance. It is the more prevalent form of diabetes accounting for about 80% of all cases worldwide. Type 2 diabetes usually develops in middle aged and older subjects but is increasingly occurring in younger age groups as a result of the obesity epidemic (5). Treatment of the disease is mainly focused in the management of hyperglycemia through a combination of changes in lifestyle and pharmacological treatment improving insulin sensitivity and beta cell function. Since existing therapies fail to arrest the progression of beta cell dysfunction and death, combinations of drug treatments are often required (6).

Gestational diabetes mellitus describes a state of increased blood glucose levels

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Diabetes affects global population, with an estimated 422 million adults living with the disease. In addition to the impact on health and well-being, diabetes and its complications bring about substantial economic loss to individuals and their families, and to health systems and national economies, representing a significant global economic burden. Early diagnosis and treatment of diabetes is critical for the management of the disease and reduction of the risk of serious complications that may impact quality of life. However, misclassification is common, particularly in young people. A classification into more refined subtypes could provide a powerful tool to accurately predict clinical outcomes, identify individuals with increased risk of complications at diagnosis and individualise treatment regimens that ameliorate them (11, 12). The novel cluster classification of adult-onset diabetes proposed by Ahlqvist

et al. (13) focuses on disease trajectory but does not assist in improved tailored

therapy in this heterogeneous complex disorder, leaving unaccomplished a classification of diabetes designed to support the practice of personalised medicine in diabetes. Further knowledge of the mechanisms involved in beta cell demise will help developing and shaping future classifications.

Role of the pancreatic beta cell

Insulin secreting beta cells are one of four major endocrine types of cells present in the islets of Langerhans, followed by glucagon-secreting alpha, somatostatin-releasing gamma and polypeptide P cells. The islets are distributed throughout the pancreas, comprising approximately 2% of total pancreas weight in adult humans (14, 15).

Glucose sensing and insulin secretion

The primary role of beta cells is to maintain physiological glycemic homeostasis by synthesizing and secreting insulin in response to nutrients, hormones and nervous stimuli.

Glucose is the most major physiologically regulator of beta cell function through coordinated stimulation of insulin gene transcription, proinsulin biosynthesis, and insulin secretion, which in turn drives glucose uptake in the liver, muscle and fat (16). Beta cells are poised to sense circulating glucose, mainly through the passive glucose transporters (GLUT) 1 (GLUT2 in rodents) and glucokinase (GCK). After entering the cell via GLUT, glucose is phosphorylated by GCK to form glucose-6-phosphate, a rate limiting step in insulin secretion (17). Once formed, glucose-6-phosphate enters the glycolytic pathway to form pyruvate, which is then oxidized through the mitochondrial Krebs cycle to produce ATP. The increased ATP/ADP ratio causes membrane ATP-sensitive K+ channels (KATP) channels to close leading to depolarization of the plasma

membrane and subsequent opening of voltage-dependent L-type Ca2+ channels (18).

The rise in intracellular cytosolic Ca2+ is the primary mediator of exocytosis of the

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Figure 1: Glucose-stimulated insulin secretion in the pancreatic beta cell.

Increased levels of glucose in the circulation lead to increased glucose uptake into pancreatic beta cells through GLUT2. Glucose is then phosphorylated by glucokinase and pyruvate is formed via the process of glycolysis. This leads to increased activity of the Krebs cycle and production of ATP within the mitochondria, which results in an increased ATP/ADP ratio. This causes closure of ATP-sensitive potassium channels, a wave of membrane depolarisation and the subsequent activation of voltage gated calcium channels. The entry of calcium into the cell is followed by vesicle docking and insulin granules are released into the circulation. VDCC, Voltage-dependent calcium channel; ETC, electron transport chain.

Although the accelerated production of acetyl-CoA is essential for glucose-stimulated insulin secretion (GSIS), additional coupling factors and mechanisms independent of the KATP channel contribute to the full development of the secretory response (19).

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which is highly expressed in beta cells, also enhances GSIS, at least partly, through the amplification of intracellular Ca2+ signaling via activation of the Ca2+-releasing

channel inositol trisphosphate receptor (IP3R) and the increase of cellular cyclic

adenosine monophosphate (cAMP) levels (21, 22). The different effects of FFAs on insulin secretion depend on intrinsic factors, such as chain length and saturation, and the duration of exposure. Another mechanism of nutrient-stimulated insulin secretion appears to involve a GTP-dependent step that is activated through the combined effects of protein kinase (PK) A and PKC (23). Regulated insulin release shows a characteristic biphasic pattern that consists of a transient first phase followed by a more sustained second phase (24). The first phase of release, which can be triggered by both nutrient and non-nutrient secretagogues, involves the fusion of a small, readily releasable pool of granules which are already docked to the plasma membrane, through the SNAP receptor complex of proteins. The second-phase has a longer duration as it requires the remaining granules belonging to the reserve pool to undergo the Ca2+ dependent preparatory reactions (trafficking, docking and priming of granules

to the plasma membrane) before becoming available for exocytosis (25).

In the body, beta cells are constantly exposed to stimulatory signals so that GSIS is modulated by a number of other factors. In fact, a great part of the response to the ingestion of glucose (from the breakdown of carbohydrates) is due to the potentiating effect of gut-derived incretin hormones. This “incretin effect” ensures that the response in insulin secretion after oral glucose intake is greater than the one observed after an intravenous glucose load, thereby potentiating postprandial insulin secretion (26). Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) are the two primary incretins, respectively secreted from endocrine K- and L-cells, in response to food intake. GIP and GLP-1 exert their effects by binding to their specific G-protein coupled receptors, GIP receptor (GIPR) and GLP-1 receptor (GLP-1R), which activates adenylyl cyclase and generates cAMP. This in turn leads to elevation of intracellular Ca2+ through cAMP-dependent activation of second

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Figure 2: The effects of GLP-1 in the beta cell.

Signalling via the classical GLP-1R-coupled G protein pathway mediates increases in cAMP to up-regulate PKA and Epac2. This leads to increased intracellular calcium mobilization and calcineurin/NFAT, promoting insulin biosynthesis and secretion. Activation of PI3K enhance beta cell neogenesis and proliferation, which is also facilitated in part by PKA-mediated activation of factors involved in cellular stress protection.

Insulin biosynthesis

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is initiated by binding to its cell-surface receptor, a tyrosine kinase that undergoes autophosphorylation and catalyses the phosphorylation of the members of the insulin receptor substrate (IRS), to permit the recruitment and activation of the phosphatidylinositol 3-kinase (PI3K). This allows the subsequent activation of PKB (also known as AKT), atypical PKCs, and mammalian target of rapamycin pathways that are involved in the anabolic actions of insulin (36, 37). Interaction of IRS with PI3K is required, but not sufficient, to the clearance of circulating glucose through stimulated translocation of the glucose transporter GLUT4 isoform to the cell surface in muscle and adipose tissues (38) Besides regulating glucose metabolism, insulin also stimulates cell growth and differentiation and affects lipid metabolism by increasing lipid synthesis in liver and fat cells, and attenuating FFAs release from triglycerides in fat and muscle (39) (Figure 3). Therefore, mechanisms leading to insulin resistance in the classical insulin-target tissues, namely liver, muscle and fat, affect beta cell function and survival due to increased insulin demands.

Figure 3: The insulin signaling pathway.

The binding of insulin to its receptor leads to autophosphorylation on the insulin receptor subunit and the Tyr phosphorylation of insulin receptor substrate (IRS) proteins. Phosphorylated IRS serve as docking proteins for other signaling proteins like PI3K. Activation of PIK3 initiates a downstream cascade of events leading to the phosphorylation and activation of AKT. Activation of this protein promotes protein, glycogen, and lipid synthesis and is essential for GLUT4 translocation to the cellular membrane and subsequent glucose uptake. Activation of the insulin receptor also leads to activation of signaling pathways controlling cell proliferation and growth.

Type 2 diabetes and monogenic diabetes

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However, it is now well-recognized that diabetes only develops when beta cells fail to compensate for the extra demand in insulin secretion (44). This relative deficiency results from progressive beta cell dysfunction (45) and/or death (46) and underlies the deterioration of glucose tolerance observed in patients (47). Indeed, post-mortem studies have reported a significant reduction in beta cell mass (48-50) as a result of increased rates of apoptosis (46), possibly explaining the observed functional loss (44). Although recent data suggests that neogenesis of human beta cells can occur under normal physiological and pathophysiological conditions (51, 52), beta cell dysfunction and death rather than defective regeneration seem to be the key factors in the pathogenesis of type 2 diabetes.

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affecting insulin secretion (59). Rare monogenic forms of severe insulin resistance can also cause diabetes, such as lipodystrophy syndromes caused by defects in genes involved in adipocyte development, differentiation, and death pathways (60). A range of genetic syndromes may be associated with diabetes or insulin resistance including Turner syndrome, Prader-Willi syndrome, Klinefelter syndrome, Down syndrome, and Friedreich’s ataxia (FRDA), and may either present early or later in life (61).

Findings from genome wide-association studies strongly suggest that frequent polymorphisms in or near some of the genes implicated in monogenic diabetes are also involved in susceptibility to polygenic/multifactorial forms of adult type 2 diabetes (62, 63). Specific variants of HNF1A and HNF1B and common polymorphisms in

KCNJ11 and GCK were found to contribute to the risk of type 2 diabetes in some

populations. Also, a genetic variation in WSF1, resulting in the rare Wolfram syndrome characterized by early-onset non-autoimmune diabetes, is associated with susceptibility to adult type 2 diabetes (56). Mutations in Gli-similar (GLIS) 3 protein cause neonatal diabetes and GLIS3 gene region has been identified as a susceptibility risk locus for both type 1 and type 2 diabetes (64, 65). Polymorphisms in the CDK5 regulatory associated protein 1-like 1, a tRNA modifying enzyme, have been associated with type 2 diabetes across ethnic populations (66). Deficiency of this methylthiotransferase leads to impaired GSIS in beta cells and glucose intolerance in mice (67). More recently, a mutation in tRNA methyltransferase homolog gene

TRMT10A was identified in a new syndrome of young onset diabetes and

microcephaly and TRMT10A silencing was shown to induce apoptosis in beta cells, suggesting that tRNA methyltransferases may have a broader relevance in the pathogenesis of type 2 diabetes (68, 69).

Monogenic forms of diabetes therefore represent invaluable models for identifying new targets of beta cell dysfunction and the discovery of their genetic basis has greatly advanced the understanding and management of not only these rare forms of diabetes but also of the more complex, polygenic types. As specific drug targets are identified through this increased knowledge, novel therapeutics will become available in the future.

The role of mitochondria and endoplasmic reticulum on beta cell

function and survival

ER and mitochondria play a key role in beta cell function and impaired functions of these organelles have been associated with obesity, insulin resistance and metabolic defects (70, 71). The ER is responsible for the synthesis and folding of membrane and secretory proteins, as well as other important cellular functions including cellular responses to stress and Ca2+ storage (72). In the beta cell, the ER has a crucial role

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Most of the functions of the ER and mitochondria in the beta cell are associated with changes in free Ca2+ concentrations which are also controlled by these organelles.

Under normal conditions, the concentration of Ca2+ in the ER is around three orders

of magnitude higher than in the cytosol and is maintained primarily by the balance between Ca2+ uptake via the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA)

pump, and ER Ca2+ release through the IP

3Rs and ryanodine receptor (RyRs). The

rise in intracellular cytosolic Ca2+ upon physiological stimuli is followed by the

activation of SERCA to restore cytosolic Ca2+ homeostasis. Mitochondria can also

uptake Ca2+ from the cytosol. Alterations to this homeostasis due to ER (74) or

mitochondrial Ca2+ flux (75, 76) affect beta cell function and survival (77). Reduced

SERCA activity and expression have been described in rodent and human models of diabetes (78). ER Ca2+ leak from RyR2 was also shown to reduce insulin secretion

(79). Moreover, ER stress was shown to exacerbate ER Ca2+ efflux through RyRs

(80), whereas pharmacological blockade of RyRs and IP3Rs was shown to reduce ER stress-mediated cell death induced by thapsigargin (81) and WSF1 deficiency (82). Moreover, direct ER-mitochondria communication, via multiple contact sites, have been implicated in insulin signaling and glucose metabolism (83).

Proper function of the ER, mitochondria and crosstalk between these organelles is thus critical to maintain beta cell homeostasis and impairment of any of these systems may lead to beta cell demise, contributing to the development of type 2 diabetes.

Oxidative stress and mitochondrial dysfunction

ROS and reactive nitrogen species comprise a variety of molecules that can be formed in beta cells including free radicals, such as nitric oxide, superoxide and the hydroxyl radical, non-radicals such as hydrogen peroxide, or anions such as peroxynitrite. These species can be produced by mitochondrial metabolism or in the cytoplasm by enzymes such as nicotinamide adenine dinucleotide phosphate oxidase and inducible nitric oxide synthase. Redox signaling manifested by transient ROS burst at least locally is an inherent part of beta cell physiology involved in intracellular signaling and biosynthetic and secretory functions. However, excessive ROS production as a result of dysregulated redox homeostasis leads to oxidative stress and the accumulation of oxidized proteins, lipids, and DNA with multiple deleterious effects on cellular metabolism (84, 85).

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To maintain cellular redox homeostasis and reduce oxidative damage, beta cells can enhance cellular ROS-scavenging capacity by induction of a family of antioxidant/detoxification enzymes via nuclear respiratory factor (NRF) 1 pathway. These enzymes include NAD(P)H:quinone oxidoreductase 1, heme oxygenase 1, glutamate–cysteine ligase, glutathione reductase, glutathione synthetase, glutathione peroxidase, catalase, superoxide dismutase, among others. Although rodent beta cells have a low abundance of these enzymes and are thus particularly sensitive to oxidative stress (90), human beta cells seem to have a robust antioxidant capacity (91). In addition, the mitochondrial uncoupling protein (UCP) 2 is regularly associated with the stress response and its overexpression has been shown to confer partial protection beta cells against both oxidative stress and glucotoxicity (92, 93). However, this protein has been identified as a negative regulator of insulin secretion, probably due to its proton leak activity and consequent negative effect on ATP production (94). Impaired mitochondrial function can also result from defects in genes involved in mitochondria function.

Although the vast majority of mitochondrial proteins are encoded by the nuclear genome, mitochondria maintain a genome that is essential for their respiratory function. Depletion of mtDNA in beta cells causes loss of GSIS as a result of defective respiratory-chain activation and loss of ATP production. Degradation of mtDNA, mitochondrial abnormalities and increased beta cell loss were also found in different type 2 diabetes models (95-97). The most obvious and clear support for the pathogenetic role of mitochondrial dysfunction in diabetes comes from MIDD, a disorder caused by maternally inherited mutations in the mtDNA impacting mitochondrial function, and consequently beta cell stimulus-secretion coupling (98). In the large majority of cases this associates with the A3243G mutation in the mitochondrial DNA-encoded tRNA(Leu,UUR) (99). Diabetic patients carrying this mutation exhibit reduced insulin secretion after intravenous glucose load compared to noncarriers (100). More recently, a common variant in the human transcription factor B1 mitochondrial was associated with reduced insulin secretion and increased future risk of type 2 diabetes (101), probably due to mitochondrial dysfunction and loss of beta cell function and mass (102).

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function in type 2 diabetes. PGC1β has quite similar effects to PGC1α and both exert a strong positive effect on mtDNA and nuclear and mitochondrial-encoded genes (108) in part by inducing NRF1 and 2. NRF1 mainly controls expression of genes encoding the respiratory proteins and others involved in mitochondrial function (109) while NRF2 plays a more important role in antioxidant processes possibly restoring insulin secretion and enhancing beta cell viability perturbed by oxidative stress (110, 111). NRF1 has been recently found to regulate mitochondrial metabolism and GSIS in rodent islets (112) and different haplotypes have been associated with either reduced or increased risk of type 2 diabetes (113, 114). PGC1α has been shown to also regulate UCP2 (115) which has been linked to obesity, beta cell dysfunction, and type 2 diabetes (94). More importantly, PDX1 loss of function has been shown to lead to reduced GSIS and mitochondrial function, with altered expression of several mitochondrial proteins, in rodent models (116, 117). PDX1 is essential for islet development and beta cell maintenance and mutations in this transcription factor lead to several heritable forms of diabetes, including MODY, NDM and early onset type 2 diabetes. It thus seems that dysregulation of the signaling pathways controlling mitochondrial content and function can lead to major impairments in both mitochondria and beta cells.

Some diabetic syndromes are caused by a distinct gene defect in nuclear-encoded mitochondrial proteins or factors influencing mitochondrial function, such as Wolfram syndrome and FRDA. The genetic and functional characterization of these diseases has provided further insight into the possible mechanisms contributing to beta cell failure and the development of diabetes due to mitochondria dysfunction. FRDA is an autosomal recessive neurodegenerative disorder associated with cardiomyopathy and high prevalence of diabetes. In most cases (98%), it is caused by homozygous guanine-adenine-adenine (GAA) repeat expansions in the first intron of the frataxin (FXN) gene resulting in epigenetic silencing and reduced expression of frataxin protein. Remaining individuals are compound heterozygous with a single expanded allele and a point mutation in the FXN exon in the other chromosome. These patients can have some atypical clinical features (118-121). In homozygous FRDA patients, the length of the shorter GAA expansion inversely correlates with frataxin levels, age of onset, and rate of disease progression. FRDA patients have a reduction of 70- 95% in frataxin protein expression while asymptomatic heterozygous carriers have around 50% of normal levels (120, 122).

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storage, and iron–sulphur cluster (ISC) biosynthesis. Frataxin interacts with mitochondrial aconitase, ferrochelatase, and proteins of the mitochondrial ISC synthesis pathway, which depend on iron and ISC for their assembly and/or enzymatic activity (124, 125). ISC containing proteins play crucial roles in cellular respiration and ATP production, mitochondrial and cellular iron homeostasis and oxidative stress response. Hence, frataxin deficiency in FRDA results in impaired energy metabolism, iron overload and accumulation of oxidative damage. Mitochondrial dysfunction underlies the pathological changes observed in the three major organs affected in FRDA, the nervous system, the heart and the pancreas (126-128) (Figure 4). The neurological syndrome is characterized by progressive trunk and limb ataxia, dysarthria, instability of fixation, sensory neuropathy and pyramidal weakness (129). Neurodegeneration occurs early in the large proprioceptive sensory neurons of the dorsal root ganglia (DRG) and their axons in the posterior columns. Hypertrophic cardiomyopathy is the most common cause of death amongst FRDA patients. Ten to 30% of FRDA patients develop diabetes, which contributes to early mortality in affected patients (128). Diabetes in FRDA is a consequence of beta cell dysfunction and apoptosis due to mitochondrial oxidative stress and the activation of the mitochondrial pathway of apoptosis. Interestingly, the same pathway is activated in induced pluripotent stem cells (iPSCs)-derived neurons from these patients (128, 130), suggesting a common mechanism underlying neuronal and beta cell loss in FRDA.

Figure 4: The pathophysiology of FRDA is the consequence of frataxin deficiency and mitochondrial dysfunction.

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Altogether, these data highlight the role of mitochondrial impairment in beta cell dysfunction and loss, a hallmark of type 2 diabetes. It is important to note that this impairment is rarely inherited, such as due to mtDNA mutations (accounting for approximately 1% of all diabetes cases), and instead arise from a toxic environment favoured by inappropriate dietary habits and lifestyles. Aberrant mitochondrial dynamics may lead to enhanced ROS formation, which, in turn, may deteriorate mitochondrial health and further exacerbate oxidative stress in a self-perpetuating vicious cycle, culminating with beta cell apoptosis. In addition to their acute effects, ROS may also lead to increased mutations in mtDNA, exacerbated by the limited repair capacity of these cells. Mitochondrial dysfunction and excessive oxidative stress also induce ER stress, a major contributor to beta cell dysfunction and death which will be discussed in the next section.

ER stress and the unfolded protein response

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requires translocation to the Golgi for its ER stress-mediated activation and results in the upregulation of UPR homeostatic effectors involved in protein folding, processing, and degradation, such as BiP and p58IPK, and also XBP1 to help restore ER

homeostasis (145, 146). These series of events are aimed at restoring ER homeostasis by combined upregulation of the ER folding capacity, downregulation of the biosynthetic load and increased clearance of the unfolded proteins. However, when these adaptive mechanisms fail to clear the perturbation, ER stress triggers cell death by apoptosis through JNK, CHOP and ATF3, in a process involving the activation of the canonical mitochondrial pathway, which is controlled by the B cell lymphoma (BCL)-2 protein family (147). CHOP is a fundamental factor that links protein misfolding in the ER to oxidative stress and apoptosis (148).

Figure 5: Overview of the UPR.

Under basal conditions, the three main ER stress transducers IRE1, ATF6 and PERK remain inactive due to the binding to the ER chaperone BiP. When misfolded proteins accumulate in the ER lumen, BiP dissociates the ER stress transducers, thereby activating them. Active IRE1 causes alternative splicing of XBP1 mRNA which then regulates the transcription of genes involved in protein folding and export from the ER. IRE1 also activates JNK. PERK phosphorylates eIF2α, thereby inhibiting protein translation and decreasing ER workload. In parallel, translation of specific proteins is facilitated and leads to upregulation of ATF4 and its downstream targets CHOP and ATF3. Active ATF6 induces transcription of ER chaperones, such as BiP. This response is primarily aimed at restoring ER homeostasis but, when ER stress is prolonged and excessive, apoptosis is triggered through JNK, CHOP and ATF3.

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phosphorylation is detrimental to beta cells and other secretory tissues, resulting in diabetes associated with multisystem abnormalities (149). Indeed, selective inhibition of eIF2α dephosphorylation (promoting translational repression) exacerbates lipotoxicity-induced ER stress and apoptosis, suggesting that a tight regulation of this event is crucial for beta cell function and survival (150, 151). Mutations in the gene

WFS1, encoding the protein wolframin, lead to young-onset diabetes associated with

selective beta cell loss and optic atrophy in Wolfram syndrome (152). Wolframin is an ER transmembrane protein which appears to regulate cellular Ca2+ homeostasis by

modulating the SERCA pump (153). An association with insulin secretion and type 2 diabetes susceptibility was also identified for genetic variants of ATF6. Most of the INS gene mutations, causing the syndrome MIDY, are associated with proinsulin misfolding and affect proper ER signaling (57). These data suggest that any form of beta cell ER stress is likely to impair both insulin biosynthesis and secretion. More direct evidence for a role of ER stress in type 2 diabetes comes from post-mortem histological studies. Increased staining for BiP, CHOP, DNAJC3 (154) and ATF3 (155) was observed in beta cells from pancreatic sections of type 2 diabetes patients compared with nondiabetic pancreatic tissue. Nuclear localization of CHOP (156) and expansion of ER surface (157) was also reported.

As mentioned before, oxidative stress can induce ER stress by disturbing the ER redox state and disrupting protein folding and disulphide bond formation, which itself leads to the production of ROS. In addition, both stresses interfere with insulin signaling through downregulation of key transcription factors such as MAFA, MAFB, PDX1 and NKX6.1 (158), a process involving JNK activation (159, 160). Oxidative and ER stress are thus intrinsically entwined pathological events in type 2 diabetes, contributing to diminished insulin secretory capacity and beta cell apoptosis.

Triggers of beta cell oxidative and ER stress

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mitochondria impairment and production of ROS. In addition, excessive glucose levels can swamp the glycolytic process and inhibit glyceraldehyde catabolism, causing glucose and intermediates to be shunted to other pathways that further induce oxidative stress (165).

Other proposed mechanisms leading to glucotoxicity-induced beta cell death include the induction of ER stress and increase in intracellular Ca2+ levels. Overstimulation by

high glucose leads to a persistent elevation in cytoplasmic Ca2+ levels, which may

trigger beta cell dysfunction and apoptosis (166). Chronic hyperglycemia may activate the UPR once diabetes is established. Acute stimulation by glucose modestly induced the UPR including PERK-dependent activation of ATF3, CHOP and GADD3, XBP1 splicing and expression of ER chaperones BIP, GRP94, and Edem (167, 157). Activation of the IRE1-JNK pathway was also reported (168). On the contrary, in other studies high glucose alone did not induce ER stress markers (157, 169). Whether high glucose potentiates FFA-induced beta cell ER stress and apoptosis is still under debate. A potentiating effect was observed in clonal beta cells (169, 168) but not in human islets (169), suggesting that ER stress is primarily involved in lipotoxicity and not in glucolipotoxicity.

Lipotoxicity

Fatty acids are important metabolic substrates for energy production and lipid synthesis that are also involved in signaling processes (170, 171). Two of the most common fatty acids in humans are the long-chain saturated FFA palmitate (C16:0) and the monounsaturated oleate (C18:1). Short-term exposure to FFAs potentiates GSIS, an effect occurring via independent pathways involving FFA metabolism and the activation of membrane FFAR (20, 172). This stimulatory effect is physiological in nature, occurring particularly in response to acute elevations of FFAs during the fasted to fed transition. However, chronic exposure to FFAs causes beta cell dysfunction by decreasing insulin gene expression and GSIS, ultimately leading to beta cell apoptosis (173, 174, 46, 175).

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the ER (182). Because protein folding is a Ca2+-dependent process, perturbation of

ER Ca2+ homeostasis triggers ER stress. Accordingly, it was recently shown that

palmitate increases glucose-6-phosphatase catalytic subunit 2, a negative regulator of GSIS, through downregulation of the Ca2+ sensor protein sorcin involved in

maintaining ER Ca2+ (183). Palmitate was also shown to impair GSIS due to partial

mitochondrial uncoupling and ROS generation via NADPH oxidase (NOX) (184) (185). Whereas both saturated and unsaturated FFAs promote defects in GSIS, beta cell apoptosis is much more restricted to saturated FFAs. The toxic effects of FFAs are known to depend on their chain length and degree of saturation. Long-chain saturated FFAs, such as palmitate, are reported to be less efficiently incorporated into triglycerides than monounsaturated FFAs (173), leading to increased accumulation of LC-CoA derivatives such as ceramides and diacylglycerol. Depletion of the stearoyl-CoA desaturase 1 and 2 sensitizes beta cells to palmitate-induced ER stress and apoptosis, demonstrating the importance of FFA structure in the modulation of its effects (186). Palmitate elicits a more potent ER stress owing to a greater and sustained depletion of ER Ca2+ stores (169). In keeping with this observation, palmitate

leads to phosphorylation of ER Ca2+ sensor protein PERK and eIF2α, inhibition of

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28

The release of FFAs from adipose tissue is also the most critical factor in modulating insulin sensitivity. Western diets rich in saturated fats cause obesity and changes in plasma lipid levels which are associated with insulin resistance, greater risk of cardiovascular disease (195, 45, 47, 196) and predictive of diabetes development (197, 198). The mechanisms by which obesity and saturated FFAs, like palmitate, cause insulin resistance, are still not completely understood. Excessive accumulation of lipotoxic intermediates, such as ceramides and diacylglycerol, have been shown to disrupt insulin signaling by inhibition of AKT phosphorylation (199) or by interfering with the association with PI3K via serine phosphorylation of IRS (200). The resulting downregulation of insulin signalling prevents the translocation of GLUT4 to the plasma membrane and glucose uptake into skeletal muscle (201, 202). Interestingly, oleate has been shown to blunt palmitate-induced insulin resistance by promoting GLUT4 translocation, at least in part, by activating the PI3K pathway (203). FFAs also induce cellular oxidative and ER stresses which may mediate the disruption of the insulin signal by transducing some effects of lipid metabolites into an activation of the stress kinases (204). ER stress can act directly as a negative modulator of the insulin signaling pathway by activation of eIF2α kinase PKR (encoded by EIF2AK2 gene) and JNK, but also indirectly by promoting lipid accumulation (205). FFAs and ER stress also play an important role in inflammation associated with type 2 diabetes (206). In

vitro exposure of human islets to palmitate induces ER stress, mild NF-κB activation

and a proinflammatory response, similar to the mild inflammatory gene expression found in type 2 diabetes islets (207-209).

It is important to note that FFAs circulate in the plasma bound to plasma albumin due to their reduced solubility in aqueous solutions, which represents a major limitation for

in vitro and in vivo studies. For this reason, FFAs are commonly conjugated to albumin,

allowing the preparation of solutions in the physiological concentration range. A caveat of this approach is that the fraction of unbound FFAs which is accessible for cellular uptake is determined by the binding of the FFA to the albumin (210-212). Therefore, the biological effect of FFAs will depend on the ratio of total FFAs to albumin, as well as their source and mode of preparation. Variations in these factors between protocols and failure to provide detailed information on those in publications, may render interpretation and comparison of results problematic.

Beta cell apoptosis as consequence of oxidative and ER stress

Although difficult to detect in vivo, it is now well established that pancreatic beta cell death by apoptosis contributes significantly in both autoimmune type 1 and type 2 diabetes, and can be induced by multiple stresses (175).

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and anti-apoptotic members of the BCL-2 protein family (Figure 6). The BCL2 family can be grouped into several groups, namely anti-apoptotic, pro-apoptotic and BH3-only proteins which are divided into sensitizers and activators. Upon a stress signal, BH3-only sensitizers (e.g. death protein 5 (DP5), BCL-2 associated agonist of cell death (BAD), BID, BIK, NOXA) directly bind anti-death family members (e.g. BCL-2, Bcl-extra large (BCL-XL) and myeloid cell leukemia (MCL)-1), causing the release of the activators (e.g. PUMA, BIM, and tBID. Once freed in the cytoplasm these proteins bind and activate cytosolic pro-apoptotic BCL-2-associated X protein (BAX) that translocates to the mitochondrial membrane where it oligomerizes with BCL-2 homologous antagonist/killer (BAK) and forms a pore. This leads to mitochondrial outer membrane permeabilization, allowing soluble proteins such as cytochrome c to diffuse to the cytosol where formation of the apoptosome complex leads to caspase-9 activation and caspase-3–mediated cell death.

Figure 6: Regulation of the intrinsic pathway of apoptosis by the BCL-2 protein family members.

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30

Lipotoxicity-induced beta cell death

FFA-induced ER stress leads to cell death through the mitochondrial apoptosis pathway as indicated by cytochrome c release from the mitochondria after palmitate treatment (174, 214). PERK-eIF2α pathway of the UPR plays a major role in the switch from adaptive to pro-death response. CHOP induction contributes to beta cell apoptosis in rodent models of ER stress and diabetes (215) and in FFA-treated beta cells (169). This transcription factor was shown to induce cell death by downregulating BCL-2 and disturbing cellular redox state (216). Palmitate was also shown to decrease the expression of anti-apoptotic proteins MCL-1, via eIF2α-mediated translational repression (217), BCL-2 and BCL-XL (218). PERK-induced ATF3, and not CHOP, modulates the expression of BH3-only sensitizer DP5 and activator PUMA by distinct mechanisms. While ATF3 directly regulates DP5 mRNA expression by binding to its promoter, PUMA induction occurs through tribbles (TRB) 3–regulated AKT inhibition and Forkhead box O3a activation. Activation of IRE1 pathway by palmitate also upregulates DP5 through JNK phosphorylation. Induction of BH3-only proteins concomitant with loss of anti-apoptotic ones results in BAX/BAK activation, mitochondrial permeabilization and cytochrome c release. These events then lead to caspase-9 and caspase-3 activation-mediated apoptosis (218). Oleate, on the other hand, neither decreases MCL-1 (217) or BCL-2 (214) nor induces DP5, PUMA or cytochrome c release (218), in keeping with the observation that this unsaturated FFA is much less toxic (169).

Glucotoxicity-induced beta cell death

A direct role of glucotoxicity in beta cell death is less clear. Glucose toxicity is mainly mediated by ROS-induced oxidative stress which leads to defective insulin production and secretion and increased apoptosis (165). Thioredoxin-interacting protein, which inhibits the redox enzyme thioredoxin, was shown to be upregulated by glucose and critically involved in caspase activation in mouse islets (219). Data from human islets showed an upregulation of BH3-only proteins BAD, BID and BIK and downregulation of BCL-XL expression, paralleled with increased cell death (220). BIM, PUMA and BAX were shown to be required for glucose-induced apoptosis in mouse islets, whereas loss of BID, NOXA and BAK had no impact on glucose-mediated death of the islets (221). The mechanisms and transcription factors involved are not known. It has also been suggested by some (222, 223), but not reproduced by others (224, 225), that in human islets glucose induces production of IL-1β leading to NF-κB activation, Fas upregulation and apoptosis engagement through this pathway.

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Current therapeutic approaches in type 2 diabetes

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32

(233). Despite lifestyle management and the currently available pharmacological therapies, a significant proportion of patients with type 2 diabetes do not maintain adequate glycemic goals in long-term, as insulin resistance increases and pancreatic beta cell function deteriorates (226).

Restoring beta cell mass represents the ultimate therapeutic goal with the potential to decrease or eliminate the need for insulin administration and adjunctive medications. The concept has prompted attempts in beta cell replacement through conversion of other cell types and beta cell regeneration by enhancement of beta cell replication (234). Replacement of beta cells with human iPSC-derived cells is currently under investigation in a clinical trial to assess their safety in humans (235). In addition, there has been success in reprogramming developmentally related cell types into beta cells. Manipulation of specific targets or intracellular signalling pathways regulating beta cell replication represents an alternative strategy to promote the expansion of residual beta cells in diabetic patients. However, while replication of existing beta cells has been well described in rodents, a limited proliferation response to mitogenic stimuli is observed in adult human beta cell (236). Nevertheless, small molecules/compounds have been identified, such as dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitors, like harmine and 5-IT (237, 238), or GNF7156 and GNF4877, inhibitors of both DYRK1A and glycogen synthase kinase 3 (239), that induce adult human beta cell proliferation by preventing the nuclear export of NFAT. Other studies reported successful human beta cell proliferation using WS6, an IκB kinase and EBP1 inhibitor, the RANK inhibitors osteoprotegrin and Denosmab, and the protease inhibitor SerpinB1 (240). A key remaining challenge of novel pharmacologic approaches for stimulating beta cell replication is the need for cell specificity. For example, the natural beta-carboline alkaloid harmine, is a promiscuous ATP-competitive kinase inhibitor which can act on several DYRK family members, monoamine oxidases (MAOs) and cdc-like kinases, limiting its therapeutic application due to expected pharmacological side effects (241). A strategy to overcome this challenge could pass through the isolation of specific functions by optimization of the compound chemical structure as well as to develop methods for their specific delivery to beta cells. In accordance, the development of harmine derivatives with enhanced beta cell proliferation-inducing effect has been recently described (242). The targeting to beta cell might be accomplished in the future by the use of monoclonal antibodies that react specifically with beta cells and are capable of delivering small molecule cargoes or by dual-ligand strategies (243). Nevertheless, it is conceivable that restoration of beta cells through manipulation of mitogenic or regenerative pathways will encroach upon oncogenic pathways and, therefore, safe regulatory mechanisms will need to be developed before regenerative approaches become accepted.

Oxidative and ER stress as therapeutic targets for the treatment of type 2 diabetes

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34

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Aims

We hypothesize that genetic and environmental factors affecting ER and/or mitochondrial function contribute to insulin resistance and beta cell demise in type 2 diabetes. The global aim of my work is to elucidate the molecular mechanisms underlying beta cell loss due to oxidative and ER stresses and evaluate these responses as potential therapeutic targets to preserve functional beta cell mass in diabetes. To this end, we have defined the following objectives:

1) To set up a reproducible in vitro model of lipotoxicity in pancreatic beta cells, by comparing the effects of different FFA preparations on beta cell viability and expression of ER stress markers in the clonal rat beta cell line INS-1E and human islets.

2) To evaluate the potential of the novel anti-diabetic compound harmine to target ER stress and preserve beta cell mass in the model set up in 1).

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Results

PAPER I

In vitro use of free fatty acids bound to albumin: A comparison of protocols Oliveira AF, Cunha DA, Ladriere L, Igoillo-Esteve M, Bugliani M, Marchetti P, Cnop M Biotechniques 2015, 58(5):228-33. doi: 10.2144/000114285

Contribution to the study:

• Preparation of FFA solutions • INS-1E cell line culture:

- Cell treatments - Apoptosis analysis

- mRNA extraction and reverse transcription - Data analysis

• Part of protein quantification assays

• Preparing figures and writing the manuscript

Summary

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PAPER II

Harmine protects pancreatic beta cells from lipotoxic endoplasmic reticulum stress and apoptosis

Oliveira AF, Cunha DA, Ladriere L, Igoillo-Esteve M, Marchetti P, Eizirik DL, Cnop M

(In preparation for submission)

Contribution to the study:

• Experiments in rat beta cell line INS-1E: - Cell treatments

- Cell death/apoptosis assay - Western blotting

- mRNA extraction and reverse transcription • Part of the experiments in human islets:

- Cell treatments - Cell death assay • Data analysis

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Harmine protects pancreatic beta cells from lipotoxic endoplasmic reticulum stress and apoptosis

Ana Oliveira1, Daniel Cunha1, Laurence Ladriere1, Mariana Igoillo-Esteve1, Piero

Marchetti2, Décio L. Eizirik1, Miriam Cnop1,3*

1ULB Center for Diabetes Research, Université Libre de Bruxelles, Brussels, Belgium; 2Department of Endocrinology and Metabolism, University of Pisa; Pisa Italy;

3Division of Endocrinology, ULB Erasmus Hospital, Université Libre de Bruxelles.

Brussels, Belgium.

*Correspondence to: Miriam Cnop

ULB Center for Diabetes Research, Université Libre de Bruxelles CP-618, Route de Lennik 808, 1070 Brussels, Belgium.

Tel: 32.2.555.63.05; Fax: 32.2.555.62.39 Email: mcnop@ulb.ac.be

Abbreviations: activating transcription factor (ATF), B-cell lymphoma (BCL), bovine serum albumin (BSA), C/EBP homologous protein (CHOP), cyclin-dependent kinase (CDK), death protein 5 (DP5), dual-specificity tyrosine-regulated kinase (DYRK), endoplasmic reticulum (ER), eukaryotic translation initiation (eIF2α), free fatty acids (FFAs), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycogen kinase 3 (GSK3), Hoechst (HO), inhibitor of DYRK family kinases (INDY), inositol-requiring enzyme 1 (IRE1), Jun N-terminal kinase (JNK), monoamine oxidase (MAO), myeloid cell leukemia (Mcl), nuclear factor of activated T cells (NFAT), p53-upregulated modulator of apoptosis (PUMA), PKR-like ER kinase (PERK), propidium iodide (PI), stearoyl-CoA desaturase (SCD), unfolded protein response (UPR), X-box binding protein 1 (XBP1)

Abstract

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50 These findings point to the therapeutic potential of harmine to modulate ER stress and preserve beta cell mass under lipotoxic conditions.

Introduction

Type 2 diabetes has developed into a major public health concern due to major changes in lifestyle. Western diets rich in saturated fats lead to obesity and insulin resistance (1-4). Concomitantly, increased concentrations of saturated free fatty acids (FFAs), of which palmitate is the most common in man, lead to pancreatic beta cell dysfunction and death (5-7) and are associated with increased diabetes risk (8, 9). Accumulating evidence suggests that endoplasmic reticulum (ER) stress is a critical factor contributing to the pathogenesis of type 2 diabetes mediating both insulin resistance and beta cell loss, the two main defects of the disease. ER stress is a molecular mechanism of lipotoxicity in beta cells (10) and has also been implicated in type 1 diabetes and monogenic diabetes (11). Alterations to normal ER function and environment lead to the accumulation of misfolded or unfolded proteins in the organelle and activate the unfolded protein response (UPR). This adaptive response is mediated through the ER transmembrane proteins PKR-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF) 6, and aims at restoring ER homeostasis (12). PERK phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α) leads to attenuation of global translation (13, 14), thereby reducing protein load, whereas the two other branches increase ER function by upregulating ER chaperones and the ER-associated protein degradation machinery. Prolonged and unresolved ER stress triggers beta cell death through activation of the mitochondrial pathway of apoptosis (15, 16).

The intrinsic pathway of apoptosis is regulated by the balance between pro- and anti-apoptotic members of the B-cell lymphoma (BCL)-2 protein family. The BCL2 family consists of anti-apoptotic, pro-apoptotic and BH3-only proteins, which are divided into sensitizers and activators. BH3-only sensitizers (e.g. death protein 5 (DP5), Bad and Noxa) directly bind anti-death family members (e.g. Bcl-2, Bcl-XL and myeloid cell leukemia (Mcl)-1), causing the release of the activators (e.g. p53-upregulated modulator of apoptosis (PUMA) and Bim). Once freed in the cytoplasm these proteins bind and activate cytosolic pro-apoptotic Bax that translocates to the mitochondrial membrane where it oligomerizes with Bak to form a pore. The resulting mitochondrial outer membrane permeabilization allows soluble proteins such as cytochrome c to diffuse to the cytosol where formation of the apoptosome complex leads to caspase-9 activation and caspase-3–mediated cell death.

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lower glycemia in mouse models. The inhibition of DYRK1A contributes to harmine-mediated beta cell proliferation through reduced phosphorylation of nuclear factor of activated T cells (NFAT) proteins (27-29). Besides proliferation, apoptosis and other processes regulate beta cell mass. Because ER stress contributes to beta cell loss in type 2 diabetes, we evaluated the effect of harmine on ER-stress induced apoptosis.

Results

Harmine protects beta cells from ER stress-induced cell death by preventing activation of the intrinsic pathway of apoptosis

The saturated fatty acid palmitate, synthetic ER stressors thapsigargin, tunicamycin, brefeldin A or a combination of the cytokines IL-1β and IFN- induced apoptosis in both INS-1E cells and human islets. Harmine per se was non-toxic to clonal rat beta cells and human islets (Figure 1A-B). In INS-1E cells, harmine partially protected beta cells against palmitate-, thapsigargin- and brefeldin A-induced apoptosis. Harmine had no effect on tunicamycin or cytokines-induced beta cell apoptosis (Figure 1A). In keeping with the clonal rat beta cell findings, harmine protected human islets from cell death induced by palmitate and thapsigargin, but it did not protect from brefeldin A. An increase in cell death was observed when human islets were exposed to a combination of harmine and tunicamycin or cytokines (Figure 1B).

Palmitate-induced INS-1E cell apoptosis was confirmed by Western blotting for cleavage of 3 and 9. Harmine prevented the activation of caspase-3 and caspase-9 induced by 16h exposure to palmitate (Figure 1C and D). Harmine also reduced the transcriptional upregulation of the BH3-only protein DP5 by palmitate in INS-1E cells (Figure 1E). Importantly, harmine strongly decreased the levels of palmitate-induced active caspase-3 in a preliminary experiment with human islets (Supplementary Figure S1B).

These data show that harmine decreases the activation of the mitochondrial pathway of apoptosis under lipotoxic conditions and partially prevents rat and human beta cell death induced by the physiological and chemical ER stressors palmitate and thapsigargin, respectively.

Harmine decreases signaling in the PERK and IRE1 branches of the ER stress response

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52 ER expansion. IRE1 also activates Jun N-terminal kinase (JNK). Harmine reduced JNK phosphorylation and tended to decrease XBP1 splicing in palmitate-treated INS-1E cells (Figure 2E and F and Supplementary Figure S2D).

In line with the results observed in the clonal beta cell line, harmine also prevented the activation of ATF3 and reduced JNK phosphorylation induced by palmitate in the preliminary experiment with human islets (Supplementary Figure S1C and D).

Palmitate-induced activation of JNK and PERK trigger the mitochondrial pathway of apoptosis through the induction of the BH3-only protein DP5 (10). Our current data show that harmine prevents lipotoxic beta cell death by attenuating ER stress signaling and the ER-to-mitochondria crosstalk.

Harmine protects beta cells from lipotoxic apoptosis through modulation of DYRK1A/B

Inhibition of DYRK1A promotes NFAT signaling which regulates the expression of genes involved in beta cell growth and function (30, 27, 28). To assess whether DYRK1A inhibition potentially mediates the protective effect of harmine, we used another inhibitor of DYRK family kinases (INDY), and tested whether it reduced palmitate-induced beta cell apoptosis. INDY significantly decreased beta cell apoptosis, to a similar extent as harmine (Figure 3A). Both compounds also reduced cleavage of caspase-3 and caspase-9, and JNK activation (Figure 3B-E).

Together this data suggests that harmine protects beta cells from palmitate-induced ER stress and apoptosis through DYRK1A inhibition.

Discussion

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the protein glycosylation blocker tunicamycin or cytokines, which have also been shown to induce ER stress via different mechanisms in rat and human beta cells (34, 35). The particular susceptibility of human islets to brefeldin A (36) might also explain the lack of a protective effect of harmine in these cells after exposure to this inhibitor of the ER-Golgi vesicle transport. Harmine per se had no effect on cell viability, as previously reported (28). Palmitate-induced beta cell death is mediated by a crosstalk between lipotoxic ER stress and the mitochondrial pathway of apoptosis. Activation of JNK and ATF3 in PERK and IRE1 pathways of the ER stress response, contribute to the subsequent execution of beta cell apoptosis through regulation of BCL2 family members. Palmitate induces BH3-only proteins DP5 and PUMA expression through JNK and ATF3 activation (37, 10). Harmine reduced DP5 induction and caspase-3 and caspase-9 cleavage by palmitate, the latter confirming that harmine inhibits the mitochondrial pathway of apoptosis. Upstream of this cell death process, we observed that ER stress markers of the PERK and IRE1 branches of the UPR, including CHOP, ATF4, ATF3, phospho-JNK and XBP1s, were decreased by harmine.

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54 products or cell type-specific isoforms may differ greatly. Another hypothesis is that different targets, other than DYRK1A, are involved in the effects of harmine and which cannot be easily distinguished on pharmacologic grounds since available inhibitors largely overlap. In addition to DYRK1A, harmine and INDY can also inhibit the closely related isoform DYRK1B and other DYRK family members as well as cyclin family kinases (26, 50). In neuroblastoma, harmine was shown to induce significant cytotoxicity through DYRK2 inhibition, but not DYRK1A/B (51), although DYRK1B expression is correlated with tumor development (52). Interestingly, DYRK1B was reported to be involved in adipogenesis and glucose homeostasis (53), suggesting that both DYRK1A and DYRK1B might be potential therapeutic targets for diabetes. Moreover, harmine also targets MAOs which are widely distributed throughout different human tissues (54). We cannot rule out the involvement of these other targets in its cytoprotective effects.

Several studies have focused on the pharmacological actions of harmine on the central nervous system (55), mainly the antidepressant-like effects associated with the potent inhibition of MAO-A (56, 18). It is therefore expected that harmine will have several off-target effects, thereby limiting its therapeutic utility and potential for pharmaceutical development. However, it may eventually be possible to isolate the pro-survival effects of harmine on beta cells by optimization of its chemical structure, similarly to what was recently described for enhancing human beta cell proliferation (57). Computational modeling has already shown to provide valuable knowledge for the design of beta carboline-derived DYRK1A inhibitors (58) and series of novel harmine analogs with minimal or absent MAO-A inhibitory activity have also been characterized (59). In addition, understanding and optimizing the duration and dosing of future potential harmine analogs as well as developing methods for their specific delivery to beta cells are key challenges. Such targeting might be accomplished in the future by the use of monoclonal antibodies that bind specifically to beta cells and are capable of delivering small molecule cargoes or by dual-ligand strategies (60).

In conclusion, we show that harmine protects pancreatic beta cells from lipotoxic ER stress and apoptosis, reinforcing the potential of modulating the ER stress response in the preservation of beta cell mass. Together with the previously described effects of harmine on beta cell proliferation, these observations suggest that harmine analogs hold therapeutic promise for human diabetes therapy. Future approaches should focus on the development of harmine derivatives with isolated functions and methods for their targeted deliver to beta cells.

Materials and methods

Culture of INS-1E cell line and human islets

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trauma (2)) were isolated by collagenase digestion and density gradient purification. The islets were cultured and dispersed as previously described (63). The percentage of beta cells was 45±4%, as measured by insulin immunofluorescence. The collection and handling of human islets were approved by the Ethical Committee of the University of Pisa, Pisa, Italy.

Cell treatments

Cells were exposed to harmine (10 µM), thapsigargin (1 µM,), brefeldin A (0.1 µ/ml), tunicamycin (5 µg/ml), palmitate (0.5 mM) (all from Sigma), INDY (15 µM; Santa Cruz), or a combination of human IL-1β (30 or 50 U/ml; kindly provided by Dr CW Reynolds, National Cancer Institute, Bethesda, MD) and recombinant rat IFN-γ (100 U/ml; R&D Systems). The compounds were dissolved in DMSO and diluted in culture medium to their final concentrations. The control condition contained a similar dilution of DMSO. Palmitate was dissolved in ethanol and diluted in medium containing FFA-free bovine serum albumin (BSA, Roche) to a molar ratio of FFA:BSA of 3.4 (corresponding to a concentration of ~26 nM of unbound palmitate) (66). The control condition contained a similar dilution of ethanol. Palmitate was added to serum-free medium for human islets and to medium containing 1% FBS for INS-1E cells.

Assessment of beta cell death

The percentage of viable, apoptotic and necrotic cells was determined following staining with the DNA-binding dyes propidium iodide (PI, 5 μg/ml, Sigma) and Hoechst 33342 (HO, 5 μg/ml, Sigma), as described previously (37). For rat and human islets, the percentage of dead cells was semiquantitatively estimated following HO-PI staining. The cells were examined by inverted fluorescence microscopy (excitation at 365 nm for HO and at 546 nm for PI).

Western blot

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56 AGTTCAACGGCACAGTCAAG-3′ (F) and 5′-TACTCAGCACCAGCATCACC-3′ (R) (118 bp); ATF4, 5′-GTTGGTCAGTGCCTCAGACA-3′ (F) and

5′-CATTCGAAACAGAGCATCGA-3′ (R) (109 bp); CHOP,

5′-CCAGCAGAGGTCACAAGCAC-3′ (F) and 5′-CGCACTGACCACTCTGTTTC-3′ (R) (125 bp); ATF3, 5′-CTCCTGGGTCACTGGTGTTT-3′ (F) and R 5′-AGTGCACAGGAAGCCAGTTT-3 (R) (568 bp); spliced XBP1, 5′-GAGTCCGCAGCAGGTG-3′ (F) and 5′-GCGTCAGAATCCATGGGA-3 (R) (65 bp); DP5, 5′-TCTGGAAGACACCCTCTGCT-3′ (F) and 5′-CACAGAGTCCCACCATGTTG-3′ (R) (93 bp).

Statistical analysis

Data are presented as means±SEM. Comparisons were performed by two-tailed paired Student’s t test or by one-way or two-way ANOVA followed by Bonferroni's multiple comparisons test. A p value <0.05 was considered statistically significant.

Ethical approval

The animal experiments were approved by the Commission d’Ethique du Bien-Être Animal, ULB, and are in accordance with the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council, Belgium.

Acknowledgments

We thank Isabelle Millard, Michael Pangerl, Nathalie Pachera and Anyishai Musuaya from the ULB Center for Diabetes Research for excellent technical support.

This work was supported by a Fonds National de la Recherche Scientifique-Fund for Research Training in Industry and Agriculture fellowship (to AFO).

Conflict of interest

The authors declare no conflict of interest.

References

1. Boden G. Role of fatty acids in the pathogenesis of insulin resistance and NIDDM. Diabetes. 1997;46(1):3-10.

2. Boden G. Obesity, insulin resistance and free fatty acids. Current opinion in

endocrinology, diabetes, and obesity. 2011;18(2):139-143.

3. Groop LC, Saloranta C, Shank M, Bonadonna RC, Ferrannini E, DeFronzo RA. The role of free fatty acid metabolism in the pathogenesis of insulin resistance in obesity and noninsulin-dependent diabetes mellitus. The Journal of clinical

endocrinology and metabolism. 1991;72(1):96-107.

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