THE ROLE OF AMP-ACTIVATED PROTEIN KINASE IN ALLEVIATING INSULIN RESISTANCE IN HYPOTHALAMIC NEURONS

Transcription

1 THE ROLE OF AMPACTIVATED PROTEIN KINASE IN ALLEVIATING INSULIN RESISTANCE IN HYPOTHALAMIC NEURONS by Jonathan Menchella A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Physiology University of Toronto Copyright by Jonathan Menchella 2014

2 THE ROLE OF AMPACTIVATED PROTEIN KINASE IN ALLEVIATING INSULIN RESISTANCE IN HYPOTHALAMIC NEURONS Abstract Jonathan Menchella Master of Science Department of Physiology University of Toronto 2014 Insulin signaling in the brain is vital to maintain wholebody glucose and energy homeostasis. AMPactivated protein kinase (AMPK) is a key enzyme that acts as a regulator of energy homeostasis within insulin sensitive tissues. To investigate the role of AMPK on insulin resistance within the hypothalamus, the rhypoe19 and mhypoanpy/gfp hypothalamic cell lines were used. Both of these cell lines express the insulin receptor and accompanying downstream molecules involved in normal insulin signaling. To induce cellular insulin resistance, the neuronal cell lines were exposed to chronic insulin for sustained periods. It was found that pretreatment with the AMPK activator, AICAR, had an effect on insulin action and was able to slightly improve the state of chronic insulininduced resistance within both cell lines. Therefore, these studies provide a clearer understanding of the potential role of the AMPK pathway in improving the state of cellular insulin resistance within the hypothalamus. ii

3 Table of Contents List of Tables and Figures...v List of Abbreviations...vii Chapter 1: Introduction 1.1 Introduction The Role of the Hypothalamus in Energy Homeostasis Insulin Cellular insulin signaling The role of insulin in the brain The role of obesity and insulin resistance Neuronal Hypothalamic cell models The neuronal immortalization of embryonic and adult hypothalamic cell lines: rhypoexx and mhypoanpy/gfp Chronic insulin as a model of cellular insulin resistance Hypothesis and Aims Chapter 2: Material and Methods 2.1 Cell Culture and Reagents Generation and Characterization of the rhypoe19 and mhypoanpy/gfp hypothalamic cell lines Western Blot Analysis Induction of Cellular Insulin Resistance Experimental Normalization Statistics Chapter 3: Results 3.1 Chronic insulin impairs Akt phosphorylation and hyperactivates p70 S6K1 phosphorylation Chronic insulin pretreatment induces ER stress in hypothalamic neurons Pretreatment with chronic insulin attenuates AMPK protein phosphorylation Effects of AICAR and Compound C on AMPK activation AMPK activation with AICAR appears to reverse the effects of chronic insulininduced cellular insulin resistance Compound C appears to abolish the reversal of cellular insulin resistance iii

4 with AICAR treatment in the rhypoe19 cell line Chapter 4: Discussion 4.1 Overall Conclusions of significant findings Future Directions Limitations Use of cell lines as experimental model Shortcomings of statistical analysis Conclusion Acknowledgements...62 References iv

5 List of Tables and Figures Figure 1.1 Regulation of feeding within the Arcuate Nucleus. Figure 1.2 Schematic representation of insulin receptor signaling. Figure 1.3 The generation process of hypothalamic cell lines. Figure 1.4 Characterization of rhypoe19 and mhypoanpy/gfp hypothalamic neuronal models. Figure 2.1 ICC images of rhypoe19 and mhypoa NPY/GFP hypothalamic cell lines. Figure 3.1 Chronic insulin pretreatment attenuates Akt and overactivates mtormediated S6K1 activity in the rhypoe19 and mhypoanpy/gfp hypothalamic neurons. Figure 3.2. Chronic insulin pretreatment induces ER stress in hypothalamic neurons. rhypoe19 neurons were exposed to either vehicle () or chronic insulin (+) (100 nm) for 24 hrs. Figure 3.3 Pretreatment with chronic insulin attenuates AMPK protein phosphorylation. Figure 3.4 Effects of AICAR and Compound C on AMPK activation. The rhypoe19 neurons were pretreated with various doses of AICAR for one hour. Figure 3.5 AMPK activation with AICAR appears to reverse the effects of chronic insulininduced cellular insulin resistance. Figure 3.6 Compound C appears to abolish the reversal of cellular insulin resistance with AICAR treatment in the rhypoe19 cell line. Figure 4.1. Model of the putative role of central insulin resistance in attenuating insulin signaling in AgRP/NPY expressing hypothalamic neurons. v

6 Table 1.1 Anorexigenic and Orexigenic Molecules Involved in Feeding. Table 3.1A Densitometry of pakt relative to Total Akt (rhypoe19) Table 3.1B Densitometry of pakt relative to Total Akt (mhypoanpy/gfp) Table 3.1C Densitometry of ps6k1 relative to Gbeta (rhypoe19) Table 3.1D Densitometry of ps6k1 relative to Gbeta (mhypoanpy/gfp) Table 3.2A Densitometry of XBP1S relative to GBeta Table 3.2B Densitometry of pjnk relative to GBeta Table 3.2C Densitometry of peif2a relative to GBeta Table 3.3 Densitometry of pampk relative to Total AMPK Table 3.4A Densitometry of pampk relative to Total AMPK (AICAR) Table 3.4B Densitometry of pampk relative to Total AMPK (Compound C) Table 3.5A Densitometry of pakt relative to Total Akt (rhypoe19) Table 3.5B Densitometry of pakt relative to Total Akt (mhypoanpy/gfp) Table 3.6A Densitometry of pakt relative to Total Akt Table 3.6B Densitometry of pampk relative to Total AMPK vi

7 List of Abbreviations 6[4(2Piperidin1ylethoxy)phenyl]pyridin4ylpyrazolo Compound C [1,5a] pyrimidine 5' adenosine monophosphateactivated protein kinase AMPK 5aminoimidazole4carboxamide ribonucleotide Arcuate nucleus Agoutirelated peptide Blood brain barrier Bovine serum albumin Central nervous system cjun Nterminal kinase Gammaaminobutyric acid High fat diet Insulin receptor Insulin receptor substrate Intracerebroventricular Kilodaltons Mammalian target of rapamycin Neuronspecific enolase Neuronspecific IR knockout Neuropeptide Y p70s6 kinase Paraventricular nucleus Phosphate buffered saline AICAR ARC AgRP BBB BSA CNS JNK GABA HFD IR IRS ICV kda mtor NSE NIRKO S6K1 PVN PBS vii

8 Phosphoinositide3kinase Protein kinase B Simian virus 40 Suppressor of cytokine signaling Tantigen Type 2 diabetes mellitus Urocortin 2 Ventromedial hypothalamus PI3K Akt SV40 SOCS3 TAg T2DM UCN2 VMH viii

9 Chapter 1 Introduction 1

10 2 1.1 Introduction With the prevalence of obesity and T2DM on the rise, understanding the molecular events leading to deregulation of glucose homeostasis and the onset of insulin resistance is vital, in order to identify potential therapeutic options to counteract these diseases. Most cases of T2DM are associated with insulin resistance, a condition characterized by a reduced cellular response to insulin (1). This in turn leads to an increase in blood glucose, due to an inability of insulin to inhibit glucose production within the liver (1). Insulin resistance is associated with numerous etiologies, including hyperinsulemia, inflammation, ER stress, and oxidative stress (2). Several key in vivo studies have demonstrated that chronic exposure to high levels of insulin lead to attenuated insulin signaling (3,4). In addition, in vitro studies have shown that 5 to 100 nm of insulin treatment diminished insulin signal transduction in adipocytes and myocytes (5,6). In the obese state, plasma insulin levels are elevated for sustained periods, which over time, correlates to reduced insulin sensitivity (4). The combination of hyperinsulemia and insulin resistance leads to the onset of metabolic changes that in turn, lead to the development of T2DM (1). At the cellular level, hyperinsulemia translates to dysfunctional insulin signaling and the onset of insulin resistance (7). In accordance with previous studies, the Belsham lab has demonstrated that multiple cellular mechanisms lead to insulin resistance, including serine phosphorylation of the IRS1 protein, IRS1 degradation, and insulin receptor (IR) degradation (7). In normal physiology, insulin acts on the insulin receptor (IR) to initiate its cellular signaling response. Therefore, diminished insulin signaling can lead to impaired energy homeostasis.

11 3 Insulin signaling in the hypothalamus of the brain plays a key role in regulating physiological functions, such as glucose homeostasis and food intake (8,9). Within the hypothalamus, there are various nuclei and neuronal subpopulations that are involved in sustaining wholebody energy homeostasis (10). Insulin action within the arcuate nucleus (ARC) and ventral medial nucleus (VMH) has been identified to play a prominent role in controlling these processes (11,12). Although peripheral mechanisms of insulin resistance are well studied, the mechanisms underlying hypothalamic insulin resistance are unclear. Therefore, elucidating the signaling mechanisms leading to the onset of impaired hypothalamic insulin action is critical, in order to identify effective therapeutic targets to counteract the detrimental effects of insulin resistance. 1.2 The Role of the Hypothalamus in Energy Homeostasis The hypothalamus is an essential region of the brain for maintaining whole body energy homeostasis (1316). Within the hypothalamus, there is a complex circuitry containing an intricate web of neuronal subpopulation, each expressing an array of neuronal molecules required to sustain its various functions. Additionally, the hypothalamus is multinucleated, including many nuclei that are involved with feeding behavior and energy metabolism. Of these various nuclei, the arcuate nucleus (ARC), paraventricular nucleus (PVN), ventromedial nucleus (VMH), dorsomedial nucleus (DMH) and lateral hypothalamus (LH) have been identified to play a prominent role in regulating food intake and energy balance (17). For example, it has been demonstrated that lesions within the aforementioned hypothalamic nuclei resulted in obesity and

12 4 hypophagia (16, 1921). In addition, the location of the hypothalamus in the CNS is also essential, as it resides on the lateral walls of the third ventricle below the thalamus and in close proximity to the blood brain barrier. This positioning imperatively allows the hypothalamus to respond to peripheral cues (22). Therefore, the hypothalamus and periphery undergo cross talk to ensure that energy intake and expenditure are sustained. Extensive research over the last decade has provided important advances in our understanding of various endocrine hormones, such as insulin and leptin, and their direct role in regulating hypothalamic nuclei involved in energy homeostasis (10, 2325). Importantly, these hormones direct the downstream signaling that modulates gene expression of anorexigenic and orexigenic neuropeptides. Anorexigenic neuropeptides and hormones suppress appetite, whereas orexigenic neuropetides and hormones stimulate appetite. There is an abundance of orexigenic and anorexigenic neuropeptides and hormones that have been identified to be vital in regulating food intake and body weight (26, 27) (Table 1.1). Insulin and leptin, two hormones critically involved in feeding, regulate satiety and energy expenditure in the hypothalamus through the melanocortin system (28). The central melanocortin system is crucial in the sustaining the function of the hypothalamus to control feeding, and has long been known to be present within the ARC nucleus (29). Residing within the ARC nucleus are AgRP/NPY, POMC neurons, and melanocortin receptors, which essentially make up the melanocortin system. The AgRP/NPY neurons are orexigenic, which stimulate food intake (30), whereas the POMC/CART neurons are anorexigenic, suppressing food intake (3133). There are five melanocortin receptors in the ARC that have adenylyl cyclase transmembrane domains, of which MCR3 and

13 5 MCR4 have been identified as crucial downstream effectors for sustaining energy homeostasis (34). MCR3 and MCR4 receptors respond to peptides from the POMC and AgRP/NPY neurons to regulate satiety and energy expenditure. To provide strong evidence for the role of the melanocortin receptors involved in feeding behavior, it has been demonstrated that central and peripheral administration of nonselective melanocortin receptors agonists leads to a reduction in food intake and weight loss (35). Moreover, central administration of AgRP caused an increase in food intake and decrease in energy expenditure (3639). Stimulation of the POMC neurons by various cues, including insulin and leptin, causes the release of amelanocyte stimulating hormone (a MSH), a potent anorexigen (3133). In turn, the amsh leaves the POMC axonterminal to act on MCR3/4 receptors. Subsequently this causes suppression of food intake (10). In contrast, insulin and leptin have an inhibitory effect on AgRP/NPY neurons to initiate an orexigenic effect through the release of AgRP and NPY (40). Both of these orexigenic peptides antagonize the effects of amsh on the MCR3/4 receptors and stimulate food intake (40). Moreover, the release of NPY and gammaaminobutyric acid (GABA) (an amino acid inhibitory neurotransmitter) by the AgRP/NPY neurons also directly inhibits POMC action (4143) (Figure 1.1). Overall, it is evident that there is a delicate balance within the hypothalamic neuronal circuitry that must remain intact to sustain wholebody energy homeostasis. With the emergence of studies identifying the important cross talk that occurs between the hypothalamus and periphery, important insights have been provided regarding potential therapeutic targets to reverse the detrimental effects of T2DM and obesity.

14 6 Table 1.1 Anorexigenic and Orexigenic Molecules Involved in Feeding. Anorexigenic αmelanocytestimulating hormone (αmsh) Cocaine and amphetaminerelated transcript (CART) Leptin Insulin Serotonin GABA Glucagon Glucagon like peptide1 (GLP1) Neurotensin Urocortin 2 (UCN2) Peptide YY Orexigenic Neuropeptide Y (NPY) Agoutirelated peptide (AgRP) Melaninconcentrating hormone (MCH) Orexin Ghrelin Galanin Catecholamines Figure 1.1 Regulation of feeding within the Arcuate Nucleus. Insulin and leptin act on AgRP/NPY and POMC neurons within the arcuate nucleus of the hypothalamus to regulate feeding. Insulin and leptin have a stimulatory effect on POMC neurons, which in turn causes the expression of anorexigenic neuropeptides leading to suppression of satiety. In contrast, insulin and leptin have an inhibitory effect on AgRP/NPY neurons, which causes the expression of orexigenic neuropeptides and an increase in satiety and food intake.

15 7 Additionally, the importance of insulin to act both peripherally and centrally has warranted investigation into better understanding its role in sustaining wholebody energy homeostasis. 1.3 Insulin Cellular insulin signaling Insulin signaling at the cellular level in the brain occurs via the insulin receptor substrate (IRS) phosphatidylinositol3kinase (PI3K) pathway (7,44). Under normal conditions, insulin binds to the insulin receptor (IR). In turn, this elicits the downstream secondary messenger activation of Akt (protein kinase B) and subsequently causes the serine phosphorylation of the transcription factor, Forkhead box protein O1 (FoxO1) (45). Upon its activation, the serine phosphorylation of FoxO1 prevents the transcription of orexigenic neuropeptides, such as Agoutirelated peptide (AgRP) and neuropeptide Y (NPY) (46). However, when insulin signaling is impaired, as is the case with insulin resistance, insulinmediated Akt phosphorylation is attenuated, and this leads to the deregulation of FoxO1 action. At the level of the hypothalamus, impaired insulin signaling has been strongly linked to the onset of obesity. This is due in part to uncontrolled orexigenic neuropeptide gene expression as a result of relieving FoxO1 action, and thus causing an increase in food intake (47,48). In peripheral tissue, a crucial downstream effector of the insulinsignaling cascade is the mammalian target of rapamycin (mtor). When insulin signaling is intact, insulinmediated downstream activation leads to the phosphorylation of mtor, which further

16 8 causes S6 Kinase 1 (S6K1) activation (49). As a result, a negative feedback loop of upstream insulin receptor substrate1 (IRS1) is elicited through serine phosphorylation at its 1101 site (50). However, under a state of insulin resistance resulting from various factors, such as chronic insulin exposure, mtor becomes overactivated (51). Thus, sustained overactivation of mtor causes deregulated S6K1 phosphorylation, amplification of the negative feedback loop involving IRS1, and ultimately leading to insulin desensitization (Figure 1.2). Although the overactivation of mtor leading to cellular insulin resistance is well known in the periphery, mtor overactivation within the hypothalamus is unclear. Additionally, hypothalamic cellular insulin resistance has also been attributed to lysosome degradation of the insulin receptor (IR) (7) and receptor inhibition due to SOCS3 overactivation (52,53). To further exacerbate the issue, impaired insulin signaling also evokes endoplasmic reticulum (ER) stress, which acts as a compensatory mechanism to unfolded proteins resulting from overnutrition (54). For example, chronic insulin in the periphery has been associated with induction of the inositol requiring 1 (IRE1) branch of the unfolded protein response (UPR) (54). IRE1 splices Xbox binding protein 1 (XBP1) and phosphorylates cjun Nterminal kinase (JNK) (54). JNK activation has been attributed to further aggravate the state of cellular insulin resistance through direct serine phosphorylation of IRS1 (55) and indirect activation of mtor/s6k1 (56). Thus, the complex sequence of events leading to insulin desensitization and ultimately, cellular insulin resistance, has fundamental consequences to various physiological processes associated with obesity and T2DM.

17 Figure 1.2 Schematic representation of insulin receptor signaling. Insulin binding to the IR triggers receptor autophosphorylation. Autophosphorylation permits IRS proteins to dock and become activated. In turn, tyrosine phosphorylation recruits PI3K to the cell leading to its phosphorylation. Upon its activation, PI3K phosphorylates Akt. The activated Akt can then regulate numerous downstream signaling molecules and transcriptional events, such as phosphorylation of translocation of Foxo1. 9

18 The role of insulin in the brain Although studies demonstrating the link between central insulin action and wholebody glucose homeostasis have been documented, only recently has its role gained acceptance in the scientific community. In 1978, one study showed that insulin levels in whole rat brain tissue where as much as twentyfive times higher than that of plasma insulin levels (57). Moreover, a study that was conducted in baboons a year later showed for the first time that intracerebroventricular (ICV) administration of insulin decreased their body mass and altered their feeding behavior (58). It is now well known that insulin can enter the hypothalamus from the peripheral circulation through a saturable transport system of the bloodbrain barrier (59,60). Interestingly, the permeability of the bloodbrain barrier has been shown to be impaired in diabetic mice (61). In accordance with the ability of insulin to enter the CNS via the BBB, the insulin receptor is expressed throughout the brain with the highest concentration found within the hypothalamus (62, 63). Basal hypothalamic insulin concentrations have been demonstrated to match plama insulin levels of approximately 3 to 5 nm (64). Obese and diabetic states, as shown in ob/ob mice and db/db mice, have been accompanied with elevated plasma insulin levels of approximately 20 nm and 40 nm, respectively (65). Additionally, plasma insulin levels as high as 300 nm have been reported in ob/ob mice upon feeding (65). An important study involving neuronspecific knockout of the IR receptor in mice (NIRKO mice) provided a key insight into the importance of insulin action in the brain. The knockout of the insulin receptor in these mice caused them to be obese and resistant to insulin (66). Other studies have provided significant insight into the role of insulin and glucose homeostasis. For example, another crucial study showed the impact of

19 11 hypothalamic insulin receptor inhibition and the onset of obesity. Through administration of antisense oligonucleotides through the third ventricle, selective hypothalamic insulin receptors were decreased, which exacerbated the ability of insulin to suppress hepatic glucose production, impacted food intake behavior, and caused increased fat mass (67). Overall, these studies improved the understanding of the role of central insulin action in the brain for the regulation of glucose homeostasis The role of obesity and insulin resistance Peripheral studies examining insulin sensitive tissues, such as the liver, have established the role of mtormediated p70 S6K1 activity with the onset of insulin resistance, through IRS1 inhibition. It has been demonstrated that S6K1 phosphorylation is the best indicator of mtor activity (49). In vivo studies have provided key insights into the intimate link between S6K1 with insulin resistance. For example, it has been shown that S6K1 deficient mice are protected against dietinduced obesity and display improved insulin sensitivity (51). To compliment in vivo findings, in vitro studies in hepatocytes and adipocytes have uncovered the detrimental molecular consequences of mtor/s6k1 hyperactivation on insulin signaling (68,69). Although a strong link has been identified with mtormediated S6K1 overactivation to cellular insulin resistance in the periphery, the role it has in the hypothalamus is unknown. However, recent in vivo evidence in mice has suggested that hypothalamic S6K1 activation leads to dietinduced hepatic insulin resistance and decreased hypothalamic tyrosine IRS1 phosphorylation (70). The emergence of other studies have also revealed that intact insulin signaling in AgRP expressing neurons is crucial to suppress hepatic

20 12 glucose production (11). Collectively, these studies demonstrate a key role that hypothalamic p70 S6K1 plays in the development of insulin resistance. Based on these findings, it is imperative that in vitro hypothalamic studies identify the central molecular mechanisms leading to uncontrolled mtor/s6k1 activity at the neuronal level, particularly in AgRP and NPY expressing neurons. This will provide insight into potential therapeutic cellular targeting and a better connective understanding of dysfunctional energy homeostasis. Due to its global metabolic effects, AMPactivated protein kinase (AMPK) has been identified as an appealing therapeutic target for the treatment of insulin resistance due to its interplay with the mtor/s6k1 pathway (71). AMPK is a key enzyme involved in regulating energy homeostasis in insulin sensitive tissues, such as the hypothalamus, liver and βcells of the pancreas, and is activated by an increase in the AMP/ATP ratio. AMPK consists of three subunits, α, β, and γ, respectively (71). The threonine172 site of the αsubunit constitutes AMPK activation (72). In insulin sensitive peripheral tissue, the activation of AMPK has been shown to lead to mtor inhibition, thus preventing the activation of p70 S6K1. Subsequently, this removes the inhibition of IRS1 and restores insulin signaling (73). Based on this notion, it is postulated that cellular insulin resistance leads to the suppression of AMPK, thus impairing its regulation on mtor/s6k1. Additionally, there is a growing body of evidence suggesting that 5 aminoimidazole4 carboxamide ribonucleotide (AICAR), a pharmacological activator of AMPK, can improve the state of insulin resistance within the liver (74), myocytes (75,76), adipocytes (77), and βcells (78). Activation of AMPK is accomplished after AICAR undergoes intracellular conversion into the nucleotide ZMP, mimicking AMP and increasing Thr172

21 13 phosphorylation (79). 1.4 Immortalized Neuronal Hypothalamic cell models Through the use of in vivo models, invaluable information regarding the roles of various stimuli, such as hormones and neurotransmitters, on physiological functions has been established. The use of in vivo models has been applied to the hypothalamus, and has uncovered its role in regulating wholebody energy homeostasis. However, despite providing important insights, these studies have left unanswered questions regarding the molecular mechanisms underlying their findings. Moreover, the complexity of the hypothalamus has made it difficult to create in vitro hypothalamic neuronal cell lines to allow for the study of underlying molecular events. Thus, uncertainty remains regarding the elucidation of cellular and molecular mechanisms of the various hypothalamic nuclei and neuronal subpopulations in response to various stimuli. Consequently, the ability to study the precise molecular events within specific neuronal populations is not possible in vivo, due to complex neuronal innervations and circuitry. At the same time, applying the results of in vitro hypothalamic findings to explain wholebody physiological functions must be taken with a certain restraint, due to the homogeneity of the cell lines, lack of representative neuronal connections that would occur naturally in vivo and modified metabolism of the proliferating cells. According to the prevailing view, the use of proliferating cell lines, such as those used in this study, cannot be considered a model of hypothalamic neurons since traditionally, CNS neurons are postmitotic. However, for the purpose of the experiments conducted in the current thesis, the cells were exposed to low glucose in an effort to

22 14 serum starve them and inhibit their proliferation. Moreover, certain criteria to further validate whether or not the hypothalamic cell lines used are indeed neuronal could be achieved through assessing whether or not they undergo polarization, a hallmark feature of neurons. Interestingly, some literature suggests that CNS neurons are not necessarily postmitotic. Particularly, in the hippocampus of the CNS, adult neurogenesis is a widely accepted phenomenon (80). Other evidence suggests that neurogenesis also occurs in the adult hypothalamus, as some adult cells in this region of the brain coexpress Doublecortin (a neuronal marker) and BrdU (a proliferative marker) (81). Notwithstanding these limitations, hypothalamic in vitro neuronal cell lines are vital to allow for the comprehensive delineation of underlying molecular events, including cellular signaling and gene regulation, in order to compliment and further explain in vivo findings regarding the role of the hypothalamus in maintaining wholebody energy homeostasis. In 1974, De Vitry et al. were the first group to attempt to construct a neuronal cell line, HT9 (82). Although the group was successful in creating a hypothalamic immortalized cell line, the HT9 cells lacked maturity (as they did not possess axons or axon terminals as is seen in mature neurons) and differentiation. Advancement to De Vitry et al. s work was completed years later, when in 1990 Mellon et al. were successfully able to create a mature and fully differentiated hypothalamic cell line through utilizing a process of tumorigensis (83). By targeting the SV40 Tantigen expression in GnRH neurons in a transgenic mouse containing an anterior hypothalamic tumour, the group was able to subsequently isolate and culture the GnRH tumour cells to create a homogenous neuronal population. Moreover, Melon et al. further subcloned

23 15 these cells, which they termed GT1, to generate various homogenous, immortalized populations of GnRH neurons (83). Since the advent of Melon et al. s work, many other hypothalamic cell lines have been generated using a similar immortalization process, which have enabled the elucidation of underlying molecular mechanisms (8487) The neuronal immortalization of embryonic and adult hypothalamic cell lines: rhypoe19 and NPY/GFP Due to the complexity of the hypothalamus, limited cell lines have been readily available to study the molecular events in specific neuronal populations. However, in 2004, Belsham et al. constructed various clonal, neuronal cell lines derived from embryonic mice hypothalami (88). Subsequently, in 2009, other immortalized hypothalamic cell lines derived from embryonic rat hypothalami were created (89). The hypothalamic cell lines have been well characterized, express the appropriate neuronal cell markers, and contain the proper downstream signaling molecules and genes that have been linked to energy homeostasis. By implementing an immortalization process to the rat and mouse lines, rhypoexx and mhypoexx, respectively, over 60 distinct hypothalamic neuronal cell lines have been generated. The process of immortalization for these cell lines has previously been reported (88, 90) (Figure 1.2). In short, fetal hypothalami from day 15 to 17 mice, or day 18 rats were extracted, triturated and dispersed as primary cultures. The primary cultures were retrovirally infected with the SV40 large Tantigen and neomycin resistant gene to immortalize the heterogeneous cell populations. Subsequently, geneticin was incorporated into a growth media to isolate for the transfected cells. Through a serial dilution process, numerous homogenous neuronal

24 16 cell populations were established, each containing distinct intracellular machinery and morphology. Moreover, using a similar process, in 2009, Belsham et al. reported the generation of adult mouse hypothalamic cell lines, mhypoaxx (91). This was a more complicated endeavor, as adult lines do not proliferate as readily as embryonic cells, thus making it difficult to obtain enough cells to immortalize. However, after discovering that treating the cells with CNTF stimulated proliferation, this allowed for the immortalization process to be carried out and for the generation of various adult mouse hypothalamic cell lines (91). To this end, using adult, transgenic NPYGFP mice, NPY expressing neurons were isolated using flow cytometry and an mhypoanpy/gfp cell line was created in 2011 (92). The generation of these cell lines has permitted the study of molecular events pertaining to reproduction, circadian rhythms, and energy homeostasis. To elucidate the underlying molecular events and consequences of insulin resistance in AgRP and NPY expressing neurons, two hypothalamic cell lines were used; an embryonic rat, rhypoe19 cell line and adult mouse, mhypoanpy/gfp cell line, respectively. Each of these cell lines has been characterized to possess the proper downstream signaling molecules, express the appropriate neuronal markers and contain the cellular machinery to carry out the studies (Figure 1.3). Importantly, since in vivo work has discovered the link between suppression of hepatic glucose production with insulin action in AgRP and NPY expressing neurons, evaluating the molecular events at the cellular level of these neuronal populations will allow for a more comprehensive understanding of how insulin resistance impairs glucose homeostasis.

25 Figure 1.3 Generation of Clonal, Immortalized Hypothalamic Cell Lines. Whole hypothalami from embryonic rats day 18 or day 15 to 17 mice were harvested, dispersed through trituration and plated as primary culture. Cells were then retrovirally infected with the SV40 Tantigen oncogene conferring neomycin resistance. Resistant colonies were selected for using geneticin and then serially diluted to generate clonal, hypothalamic cell lines. 17

26 Chronic insulin as a model of cellular insulin resistance A hallmark feature associated with T2DM is hyperinsulemia. As it is well established, hyperinsulemia leads to various conditions, including insulin resistance and obesity (93). An abundance of studies have demonstrated that prolonged exposure to chronic insulin levels leads to disruption to the normal insulinsignaling cascade (7,50, 94). Moreover, the Belsham lab has recently reported that prolonged exposure to insulin in the mhypoe46 hypothalamic cell line leads to cellular insulin resistance through degradation of the insulin receptor, IRS1 serine phosphorylation and IRS1 protein degradation (7). In the periphery, it is well known that serine phosphorylation of IRS1 attenuates insulin action through disrupting downstream signaling. The serine phosphorylation of IRS1 has strongly been linked to the mammalian target of rapamycin (mtor)p70 ribosomal S6 kinase (S6K1) pathway (50,95) in peripheral sensitive tissues to insulin. Studies have shown overactivation of the mtor/s6k1 pathway in peripheral tissue resulting from chronic insulin exposure. However, the events of hyperactivation within the hypothalamus, particularly involved in neuronal populations involved in feeding, have yet to be elucidated (96,97). Therefore, understanding the molecular consequences of chronic insulin within the hypothalamus will improve our understanding of how obesity and T2DM affect the molecular processes required to sustain energy metabolism.

27 Figure 1.4 Characterization of rhypoe19 and mhypoanpy/gfp hypothalamic neuronal models. Listed in the table is the presence (+) or absence () of specific genes (C). AgRP, agoutirelated peptide; NPY, neuropeptide Y; IR, insulin receptor, ObRb, leptin receptor long form; IRS1 insulin receptor substrate1; IRS2 insulin receptor substrate2; SOCS3 suppressor of cytokine 3. 19

28 Hypothesis and Aims Due to the complexity of the hypothalamus and lack of in vitro cell models, the effects of AMPK activating compounds on central insulin resistance have not been well studied. In the present studies, I aimed to delineate the effects of AMPK modulators following the induction of insulin resistance. The Belsham lab has generated novel, immortalized, hypothalamic AgRP and NPY expressing neuronal cell models, rhypoe 19, and mhypoanpy/gfp, which have permitted the investigation of the effects of AMPK modulators, such as AICAR and Compound C on cellular insulin resistance. The embryonic, rat neuronal cell line, rhypoe19, as well as the mouse adult NPY/GFP expressing neuronal cell line, mhypoanpy/gfp, were used to test the hypothesis that AMPK activation can improve the state of insulin resistance within these two hypothalamic cell models. In order to test the hypothesis, the studies were broken down into three specific aims: i. Delineate the molecular events involved in the manifestation of chronic insulin induced cellular insulin resistance in a model of hypothalamic neurons. ii. Assess the role of AICAR in alleviating cellular insulin resistance in the rhypoe 19 and mhypoanpy/gfp hypothalamic neurons. iii. Determine whether Compound C prevents the improved state of insulin signaling with AICAR. Uncovering the mechanistic pathways involved in central insulin resistance and determining how AMPK, an important energymediating enzyme, regulates these pathways, will further expand our understanding of potential therapeutic targets to control complications arising from T2DM.

29 Chapter 2 Materials & Methods 21

30 Cell Culture and Reagents rhypoe19 and mhypoanpy/gfp neurons were generated as previously described (53,89). Cells were cultured in monolayer in Dulbecco s Modified Eagle Medium (DMEM) (SigmaAldrich, Oakville, Ontario, Canada) supplemented with 5% fetal bovine serum (FBS) and 1% penicillinstreptomycin (GIBCO, Burlington, Ontario, Canada). The neurons were grown under regulated air conditions with 5% CO2 at 37 C. Human biosynthetic insulin was used (gifted by NovoNordisk Canada Inc., Mississauga, Ontario, Canada), which was diluted in phosphate buffered saline (PBS). The Gprotein beta (Gβ) and XBP1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antiakt, phosphospecificakt (Ser473), antiampkα, phosphospecificampkα (Thr172), phosphospecificp70 S6 Kinase1 (Thr389), antifoxo1, phosphospecificfoxo1 (Ser256), phosphospecific eif2α (Ser51), and phosphospecificsapk/jnk (Thr183/Tyr185) antibodies were obtained from Cell Signaling Technology Inc. (Danvers, MA). The AMPK agonist, 5aminoimidazole4carboxamide ribonucleotide (AICAR), was purchased from TOCRIS (Ellisville, MS) and used at a final concentration of 1 mm. The AMPK antagonist, 6[4(2Piperidin 1ylethoxy)phenyl]pyridin4ylpyrazolo[1,5a] pyrimidine (Compound C) was purchased from Sigma Aldridge (Oakville, ON) and reconstituted in DMSO, respectively, at a final concentration of 50 µm.

31 Generation and Characterization of the rhypoe19 and mhypoanpy/gfp hypothalamic cell lines The rhypoe19 cell line is a clonal neuronal model generated from primary cultures of hypothalamic cells derived from embryonic day 18 mice, and immortalized as previously described (53, 88). Additionally, the mhypoanpy/gfp cell line is a mixed population of NPY expressing neurons derived from twomonthold NPYGFP adult mice (Figure 2.1). Using ICC to confirm cell phenotype, mhypoanpy/gfp neurons have been probed for NPY and GFP using specific antibodies, whereas the rhypoe19 neurons have probed for neuron specific enolase (NSE) (53). Furthermore, both of these cell lines display neuronal morphology and express a wide variety of neuropeptides and receptors. It has recently been reported that these cell lines contain the appropriate machinery to carry out insulin signaling studies, assessed using RTPCR (53). Specifically, the rhypoe19 and mhypoanpy/gfp neuronal models have a robust expression of the insulin receptor (IR), as well as crucial downstream signaling molecules, such as IRS1, IRS2, SOCS3, and FoxO Western Blot Analysis rhypoe19 and mhypoanpy/gfp cells were grown to 9095% confluence, serumstarved for 24 hours with 100 nm insulin or vehicle (PBS) and then rechallenged with 10 nm insulin or vehicle (PBS). The cells were harvested at 15 minutes following treatment using a 1X lysis buffer (Cell Signaling Technology Inc.) supplemented with 1 mm phenylmethylsulfonyl fluoride, 10 µl phosphatase inhibitors, and 10 µl protease inhibitors. Total protein (30 µg) was run on a 10% SDSpolyacrylamide gel and

32 24 transferred onto ImmobilonP PVDF membrane (BioRad, Mississauga, Ontario, Canada). The membranes were incubated with 5% BSA in TBS with 0.1% Tween for 1 hour in order to prevent the nonspecific binding of antibodies. Next, the blots were incubated overnight at 4 C with primary antibody, subsequently exposed with secondary antibody conjugated to horseradish peroxidase. Visualization of the blots was carried out using the enhanced chemiluminescence (ECL) method captured using the KODAK Image Station 2000R. Protein was quantified using densitometry and normalized using the relative value of the phosphorylated samples divided by the total protein or Gβ control. 2.4 Induction of Cellular Insulin Resistance The rhypoe19 and mhypoanpy/gfp neurons were pretreated with either vehicle (PBS) or 100 nm insulin for 24 hours. Following prolonged insulin exposure, the cells were washed with PBS and placed in fresh media to allow the phosphorylation levels to return to basal. Subsequently, the neurons were then rechallenged with 10 nm insulin to assess protein phosphorylation status. Protein was isolated 15 minutes following insulin rechallenge. For the study analyzing the effects of AICAR on cellular insulin resistance in the hypothalamic neurons, the rhypoe19 and mhypoanpy/gfp neurons were pretreated with either PBS vehicle or 100 nm insulin for 24 hours. During pretreatment, the cells were treated with the AMPK activator, AICAR (1 mm), for one hour prior to wash period. Following pretreatment, the cells were washed with PBS and

33 Figure 2.1 ICC images of rhypoe19 and mhypoa NPY/GFP hypothalamic cell lines. The immortalized hypothalamic mhypoanpy/gfp line expresses GFP and NPY, as determined by ICC, confirming that the cultures only contain immortalized mhypoa NPY/GFP neurons (A), whereas the rhypoe19 cell line expresses only neuron specific enolase (NSE) (B). (Menchella et al., unpublished data, contributed by Jennifer Chalmers) 25

34 26 then placed in fresh media for one hour. The cells were then rechallenged with 10 nm insulin for 15 minutes and then protein was harvested. For the inhibitor study, the rhypoe19 neurons were treated with either PBS vehicle or 100 nm insulin for 24 hours. After the 24 hour pretreatment, the neurons were treated with the AMPK inhibitor, Compound C (50 µm) or DMSO control. Subsequently, cells treated with Compound C (50 µm) were cotreated with AICAR (1 mm) for one hour prior to wash period. The cells were then washed, placed in fresh media for one hour, and rechallenged with 10 nm insulin or water vehicle for 15 minutes. The protein was then harvested. 2.5 Experimental Normalization For the Western blot experiments, the relative densitometry values of the phosphorylated samples were divided by the densitometry values of the total protein or Gbeta loading control for the specific protein analyzed. These values were then divided by the average of the phosphoprotein sample to total protein or Gbeta for Western blot experiments. Statistical analysis was subsequently used to determine significance on the relative values. 2.6 Statistics Data are presented as the mean ± the standard error of the mean (SEM) and analyzed using GraphPad Prism (GraphPad Software Inc., San Diego, CA) software. For each of the experiments conducted, both a Oneway ANOVA and Twoway ANOVA (or Threeway ANOVA) were performed, followed by a Bonferroni or HolmSidak posthoc tests. Significance was considered with a pvalue equal to less than 0.05.

35 Chapter 3 Results 27

36 Chronic insulin pretreatment attenuates insulinmediated Akt protein phosphorylation and overactivates p70 S6K1 protein phosphorylation in the rhypoe19 and NPY/GFP cell lines Thank you to Christopher Mayer for the insulin resistance protocol. As expected in the rhypoe19 and mhypoanpy/gfp neurons, insulin activated the PI3K/Akt pathway, determined by Akt protein phosphorylation 15 minutes following insulin treatment (Fig. 3.1A and 3.1B). This accords with previous studies that have reported shortterm induction of phosphoakt following insulin treatment in hepatic and hypothalamic cell lines (98, 7). We have recently reported the minimum dose of insulin pretreatment required to induce a state of cellular insulin resistance (7). Based on this previous finding, the rhypoe19 and mhypoanpy/gfp neurons were pretreated with either PBS vehicle or 100 nm insulin for 24 hours. Following prolonged insulin exposure, the cells were washed with PBS and placed in fresh media to allow the phosphorylation levels to return to basal. Subsequently, the neurons were then rechallenged with 10 nm insulin to assess protein phosphorylation status. Protein was isolated 15 min following insulin rechallenge. Using Western blot analysis, it was determined that exposing the neurons with 100 nm insulin for 24 hours significantly attenuated insulin signaling in the rhypoe19 cell line, as demonstrated by impaired insulininduced phosphorylation of Akt (Fig. 3.1A; n=3, Raw data: Table 3.1A) Specifically, Oneway ANOVA detected a significant effect among the two treatment groups (p=0.004). Through a pairwise comparison using a posthoc test, there was a significant difference found between insulin and PBS under normal conditions (p=0.005),

37 Figure 3.1 Chronic insulin pretreatment attenuates Akt and overactivates mtormediated S6K1 activity in the rhypoe19 and mhypoanpy/gfp hypothalamic neurons. rhypoe19 and mhypoanpy/gfp neurons were pretreated with PBS vehicle () or 100 nm of insulin (+) for 24 hrs. The cells were then washed and allowed to recover in fresh media for 1 hr prior to a 10 nm insulin or vehicle rechallenge. Protein was isolated at 15 min following treatment. Western blot analysis was used to assess the phosphorylation status of Akt (A) and S6K1 (C) in the rhypoe19 neurons along with the mhypoa NPY/GFP neurons (B) and (D), respectively. Protein levels were normalized to total Akt and Gb. Data are shown with SEM (n=3 independent experiments). * p < 0.05 ** p < 0.01, *** p < as per oneway ANOVA with Bonferroni posthoc test. 29

38 30 Table 3.1A. Densitometry of pakt relative to Total Akt rhypoe19 cell line No4pre>treatment 244hr4insulin4(1004nM)4pre>treatment PBS Insulin4(104nM) Table 3.1B. Densitometry of pakt relative to Total Akt mhypoanpy/gfp cell line No5pre>treatment 245hr5insulin5(1005nM)5pre>treatment PBS Insulin5(105nM) Table 3.1C. Densitometry of pp70 S6K1 relative to Gβ rhypoe19 cell line No5pre>treatment 245hr5insulin5(1005nM)5pre>treatment PBS Insulin5(105nM) Table 3.1D. Densitometry of pp70 S6K1 relative to Gβ mhypoanpy/gfp No5pre>treatment 245hr5insulin5(1005nM)5pre>treatment PBS Insulin5(105nM)

39 31 and insulin treatment under normal conditions compared to insulin rechallenge under chronic insulin conditions (p=0.017). Moreover, Twoway ANOVA detected a significant interaction between the effects of insulin and the effects of pretreatment (p=0.004), as well as a significant difference among pairwise comparisons between 24 hour insulin pretreatment and insulin treatment under normal conditions (p=0.003). These results regarding Akt impairment due to chronic insulin were similar to the mhypoanpy/gfp cell line (Fig. 3.1B; n=3, Oneway ANOVA p=0.001 (between the two treatment groups), Twoway ANOVA p=<0.001 (Pairwise comparison: Within Insulin treatment; Normal vs. 24 hr insulin pretreatment), Raw data: Table 3.1B). Importantly, attenuation of Akt was also accompanied with hyperactivation of p70 S6K1 in the rhypoe19 cell line (Fig. 3.1C; n=3, Oneway ANOVA p= (between the two treatment groups), TwoWay ANOVA p=<0.01 (Pairwise comparison: Within Insulin treatment; Normal vs. 24 hr insulin pretreatment), Raw data: Table 3.1C) and mhypoanpy/gfp cell line (Fig. 3.1D; n=3, Oneway ANOVA p= (between the two treatment groups), Twoway ANOVA p=<0.05 (Within Insulin treatment; Normal vs. 24 hr insulin pretreatment), Raw data: Table 3.1D). Therefore, the overactivation of p70 S6K1 suggests that the mtors6k1 pathway is involved with the induction of cellular insulin resistance due to chronic insulin exposure in both the rhypoe19 and mhypoanpy/gfp hypothalamic cell lines.

40 Chronic insulin induces ER stress in hypothalamic neurons Thank you to Christopher Mayer for the insulin resistance protocol. In order to investigate whether chronic insulin induced ER stress in the neurons, I accessed various branches of the UPR. As previously described, neurons were pretreated with chronic insulin (100 nm) or PBS control for 24 hours, followed by a 15 minute insulin rechallenge (10 nm). To assess whether the IRE1 branch of the UPR was induced with chronic insulin, Western Blot analysis was conducted on XBP1 spliced (activation) and JNK phosphorylation. According to Oneway ANOVA, there was a significant difference detected for XBP1 spliced (activation) among the chronic insulin pretreatment and normal condition groups (p=0.013). Pairwise comparison using a posthoc test detected a significant difference between PBS treatment under normal conditions and PBS treatment under 24 hour insulin pretreatment (p=0.025), and between PBS treatment under chronic insulin conditions and insulin treatment under normal conditions (p=0.022). However, using a more stringent Twoway ANOVA, there were no interactions identified between the effects of insulin and pretreatment (p=0.17). This indicates that there was no selective effect of 24 hour insulin pretreatment on the insulin treated groups for XBP1S activation (Fig. 3.2A; n=3, Raw Data: Table 3.2A). In addition, both Oneway and Twoway ANOVA detected a significant difference between the 24 hour pretreatment and normal condition groups for JNK phosphorylation, indicating there was a significant effect of 24 hour insulin pretreatment to the insulin treated groups in the rhypoe19 neurons (Fig. 3.2B, n=3, Oneway ANOVA p= (between the two treatment groups), Twoway ANOVA p<0.05 (Within Insulin

41 Figure 3.2. Chronic insulin pretreatment induces ER stress in hypothalamic neurons. rhypoe19 neurons were exposed to either vehicle () or chronic insulin (+) (100 nm) for 24 hrs. The cells were then washed, allowed to recover in fresh media for 1 hr prior to a rechallenge with 10 nm insulin or vehicle control. Protein was isolated at 15 min following treatment and analyzed using Western blot analysis with phosphospecific antibodies against XBP1 (A), JNK (B), and eif2α (C). Protein levels were normalized to Gβ. Data are shown with SEM (n=3 independent experiments). * p < 0.05, ** p < 0.01, *** p < as per oneway ANOVA with Bonferroni s posthoc test. 33

42 34 Table 3.2A. Densitometry of XBP1S relative to Gβ rhypoe19 cell line No5pre>treatment 245hr5insulin5(1005nM)5pre>treatment PBS Insulin5(105nM) Table 3.2B. Densitometry of pjnk relative to Gβ rhypoe19 cell line No5pre>treatment 245hr5insulin5(1005nM)5pre>treatment PBS Insulin5(105nM) Table 3.2C. Densitometry of peif2α relative to Gβ rhypoe19 cell line No5pre>treatment 245hr5insulin5(1005nM)5pre>treatment PBS Insulin5(105nM)

43 35 treatment, Normal vs. 24 hr insulin pretreatment), Raw Data: Table 3.2B). Moreover, the PERK branch of the UPR was unaffected with chronic insulin pretreatment, as assessed by eif2α phosphorylation (Fig 3.2C, n=3, Oneway ANOVA p= (between the treatment groups), Twoway ANOVA p=>0.05 (Within Insulin treatment; Normal vs. 24 hr insulin pretreatment), Raw Data: Table 3.2C). Taken together, these results suggest that ER stress mechanisms could be evoked during periods of insulin resistance in AgRP expressing neurons. 3.3 Pretreatment with chronic insulin attenuates AMPK protein phosphorylation mtormediated p70 S6K1 protein phosphorylation is known to be regulated by the vital energy sensing molecule, AMPactivated protein kinase (AMPK) (99, 100). However, studies have failed to address whether overactivation of S6K1 levels during a state of cellular insulin resistance are accompanied with decreased AMPK activity. To determine the effects of cellular insulin resistance on AMPK, the rhypoe19 neurons were pretreated with PBS vehicle or 100 nm insulin for 24 hours to induce insulin resistance. Subsequently, these neurons were washed and placed in fresh media for one hour. Following a 10 nm insulin or PBS vehicle rechallenge, protein was isolated at 15 minutes following treatment. Using Western blot analysis, it was determined that chronic insulin exposure decreased protein phosphorylation of AMPK compared to the nonpretreatment group (Fig. 3.3, n=5, Oneway ANOVA p= (between treatment groups), Twoway ANOVA p=>0.05 (Within Insulin treatment; Normal vs. 24 hr insulin pretreatment), Raw Data: Table 3.3). Overall, the decrease in AMPK activity following

44 36 A rhypoe19 cell line Relative AMPK Phosphorylation *** ** 24 hr Insulin Pretreatment (100 nm) + + Vehicle Insulin pampk Thr172 AMPK Figure 3.3. Chronic insulin pretreatment attenuates AMPK activity. rhypoe19 neurons were pretreated with PBS vehicle () or 100 nm of insulin (+) for 24 hrs. The cells were then washed, allowed to recover in fresh media for 1 hr prior to a rechallenge with 10 nm of insulin or vehicle. Protein was isolated at 15 min following treatment and analyzed using Western blot analysis with phosphospecific antibodies against AMPK (A) Protein levels were normalized to total AMPK. Data are shown with SEM (n=5 independent experiments). * p < 0.05, ** p < 0.01, *** p < as per oneway ANOVA with Bonferroni s posthoc test. Table 3.3. Densitometry of pampk relative to Total AMPK rhypoe19 cell line No#pre2treatment 24#hr#insulin#(100#nM)#pre2treatment PBS Insulin#(10#nM)

45 37 prolonged insulin pretreatment indicates that the induction of cellular insulin resistance alters the ability of AMPK to regulate mtor and normal neuropeptide expression. 3.4 Effects of AICAR and Compound C on AMPK activation To assess the appropriate treatment for the AMPK experiments, I treated the rhypoe19 neurons with various concentrations of AICAR (PBS vehicle, 0.5, 1, or 2 mm) for one hour. Following AICAR treatment, the cells were harvested and protein was isolated. Using Western Blot analysis, I found that AICAR was able to increase AMPK phosphorylation in a dosedependent manner in these neurons (Fig. 3.4A, n=3, Oneway ANOVA p=0.0014, Raw Data: Table 3.4A). Therefore, 1 mm was deemed the appropriate minimum dose to significantly activate AMPK, as assessed by induction of protein phosphorylation. Treatment with 1 mm AICAR did not have any impact on cell viability (assessed through visual observation under the microscope). This finding correlates with other in vitro studies, which have used a minimum dose of 1 mm AICAR to activate AMPK (99101). Next, to confirm that Compound C, a putative AMPK inhibitor, was able to block the effects of AICAR, I treated the cells with this inhibitor in the presence of AICAR (Fig. 3.4B, n=3, Oneway ANOVA p=0.0025, Raw Data: Table 3.4B). It has been previously demonstrated in vitro that 50 µm of Compound C is able to inhibit AMPK phosphorylation upon treatment. Similarly, I found that treatment with this concentration was able to inhibit AMPK phosphorylation in the presence of AICAR. In addition, this corresponded to no effect on cell viability or morphology (assessed through visual observation under the microscope). Collectively, these results show that AICAR

46 38 A AMPK B Relative AMPK Phosphorylation pampk 1.5 ** ** AICAR (mm) Relative AMPK Phosphorylation AICAR (1mM) Compound C (50µM) pampk * + ** + + AMPK AMPK Figure 3.4. Effects of AICAR and Compound C on AMPK activation. The rhypoe19 neurons were pretreated with various doses of AICAR for one hour. Protein was then collected and Western blot analysis was performed using phosphospecific antibodies against AMPK (A). Inhibitor studies were then performed using Compound C. AMPK activity was assessed using the AMPK phosphospecific antibody. Protein levels were normalized to total AMPK. Data are shown with SEM (n=3 independent experiments). * p < 0.05, ** p < 0.01, *** p < as per oneway ANOVA with Bonferroni s posthoc test.

47 39 Table 3.4A. Densitometry of pampk relative to Total AMPK rhypoe19 cell line Table 3.4B. Densitometry of pampk relative to Total AMPK rhypoe19 cell line

48 40 and Compound C act on AMPK to stimulate and inhibit its action respectively in the rhypoe19 cell line. 3.5 AMPK activation with AICAR appears to reverse the effects of chronic insulininduced cellular insulin resistance. Thank you to Sean McFadden for assisting with the experimental design. With AMPK being a central regulator of energy metabolism, I next wanted to investigate the mechanism of AMPK activation on cellular insulin resistance in our neuronal models. The rhypoe19 and mhypoanpy/gfp neurons were pretreated with either PBS vehicle or 100 nm insulin for 24 hours. During pretreatment, the cells were treated with the AMPK activator, AICAR (1 mm), for one hour prior to wash period. Following pretreatment, the cells were washed with PBS and then placed in fresh media for one hour. The cells were then rechallenged with 10 nm insulin for 15 minutes and then protein was harvested. To assess the status of insulin resistance upon AMPK activation with AICAR, Western Blot analysis was conducted. Oneway ANOVA found a significant effect of AICAR treatment on Akt phosphorylation. Specifically, Oneway ANOVA posthoc analysis detected a difference between AICAR and insulin treatment versus insulin rechallenge following prolonged insulin exposure (p=0.009). Using a more stringent Threeway ANOVA, there was a significant interaction detected between insulin treatment and 24 hour chronic insulin pretreatment (p=0.002), and between insulin treatment and AICAR treatment (p=0.013). However, there was no significant interaction detected between 24 hour insulin pretreatment and AICAR treatment (p=0.092). Posthoc analysis determined a significant difference between insulin levels in

49 41 the chronic insulin pretreatment and non pretreated groups (p=0.002), as well as with insulin levels with and without AICAR treatment (p=0.002). Therefore, this indicates that AICAR did have an effect on insulin action, however, only had a slight improvement to Akt phosphorylation when used in combination with insulin treatment. (Fig. 3.5A, n=4, Raw Data: Table 3.5A). Similar trends were apparent in the mhypoanpy/gfp cell line with Akt phosphorylation (Fig 3.5B, n=3, Raw Data: Table 3.5B). Specifically, Oneway ANOVA detected a significant effect among the pretreatment groups (p=0.0003). Furthermore, there was a significant difference between AICAR and insulin treatment compared to insulin rechallenge in the 24 hour insulin pretreatment group with posthoc analysis using Oneway ANOVA (p<0.05). However, AICAR treatment did not have a significant interaction with 24 hour insulin pretreatment with Threeway ANOVA. Moreover, Oneway ANOVA detected a significant difference with p70 S6K1 phosphorylation levels between the normal conditions and 24 hour insulin pretreatment following insulin rechallenge (p=0.0045). Among specific comparison groups using the Oneway ANOVA, there was a significant difference found between AICAR and insulin compared to insulin rechallenge in the 24 hour insulin pretreatment group (p=<0.05), as well as a significant difference between insulin treatment in normal conditions compared to insulin rechallenge in the chronic insulin group (p=<0.05). Again, a similar trend as in Figure 3.5A for statistical significance was detected using the more stringent Threeway ANOVA (Fig. 3.5C, n=4, Raw Data: Table 3.5C). To determine whether the effects seen were specific to the AMPK pathway, I assessed the protein levels of AMPK. Oneway ANOVA detected a significant increase in AMPK phosphorylation when comparing

50 42 A rhypoe19 cells B mhypoanpy/gfp cells Relative Akt Phosphorylation hr Insulin Pretreatment (100 nm) pakt Akt ** ** ** Vehicle * Insulin (10 nm) AICAR (1 mm) AIC + INS Relative Akt Phosphorylation hr Insulin Pretreatment (100 nm) ** ** AICAR (1 mm) AIC+INS ** ** Vehicle Insulin (10 nm) pakt Akt C rhypoe19 cells D rhypoe19 cells Relative S6K1 Phosphorylation hr Insulin Pretreatment (100 nm) ps6k1 G ** * * Vehicle Insulin (10 nm) AICAR (1 mm) AIC+INS Relative AMPK Phosphorylation hr Insulin Pretreatment (100 nm) pampk AMPK * *** Vehicle Insulin (10 nm) ** AICAR (1 mm) AIC+INS * Figure 3.5. AMPK activation with AICAR reverses the effects of chronic insulininduced cellular insulin resistance. rhypoe19 and mhypoanpy/gfp neurons were pretreated for 24 hrs with vehicle () or 100 nm insulin (+). Cells were then treated with AICAR (1 mm) or vehicle (PBS) one hour prior to insulin (10 nm) or vehicle (PBS) rechallenge. AICAR increased Akt phosphorylation following induction of insulin resistance in both the rhypoe19 (A) and mhypoanpy/gfp (B) cell lines. 100 nm insulin pretreatment increased S6K1 protein phosphorylation, however, these effects were reversed with AICAR (C). This was accompanied by an increase in AMPK protein phosphorylation levels (D). rhypoe19 neurons were pretreated for 24 hours with PBS (vehicle) or 100 nm insulin. Cells were then treated with AICAR (1 mm) or PBS (vehicle) one hour prior to insulin (10 nm) or PBS (vehicle) rechallenge. Data are shown with SEM (n=3 to 4 independent experiments). * p < 0.05, ** p < 0.01, *** p < as per oneway ANOVA with Bonferroni s posthoc test.

51 43 Table 3.5A. Densitometry of pakt relative to Total Akt rhypoe19 cell line No*pre:treatment 24*hr*insulin*(100*nM)*pre:treatment PBS Insulin*(10*nM) AICAR*(1*mM) AICAR*+*Insulin*(1*mM*+*10*nM) Table 3.5B. Densitometry of pakt relative to Total Akt mhypoanpy/gfp cell line No5preCtreatment 245hr5insulin5(1005nM)5preCtreatment PBS Insulin5(105nM) AICAR5(15mM) AICAR5+5Insulin5(15mM5+5105nM) Table 3.5C. Densitometry of pp70 S6K1 relative to Gβ rhypoe19 cell line No5preCtreatment 245hr5insulin5(1005nM)5preCtreatment PBS Insulin5(105nM) AICAR5(15mM) AICAR5+5Insulin5(15mM5+5105nM) Table 3.5D. Densitometry of pampk1 relative to Total AMPK rhypoe19 cell line No5preCtreatment 245hr5insulin5(1005nM)5preCtreatment PBS Insulin5(105nM) AICAR5(15mM) AICAR5+5Insulin5(15mM5+5105nM)

52 44 between treatment groups (p=0.0001). Threeway ANOVA also detected an increase to AMPK phosphorylation in the rhypoe19 cell line (Fig. 3.5D, n=4, Raw Data: Table 3.5D). Collectively, these results suggest that AMPK activation with AICAR had an effect on insulin action, but only slightly improved the state of chronic insulininduced insulin resistance by inhibiting mtormediated S6K1 phosphorylation. 3.6 Compound C appears to abolish the reversal of cellular insulin resistance with AICAR treatment in the rhypoe19 cell line To further confirm that the effects seen with cellular insulin resistance were attributed to AMPK activation using AICAR, I performed inhibitor studies. I used the classic AMPK inhibitor, Compound C, to investigate the effects of AMPK inhibition on insulin resistance reversal. To ensure the efficacy and specificity of the inhibitor, rhypoe19 neurons were pretreated with either water vehicle, water + insulin (10 nm), DMSO vehicle, DMSO + insulin (10 nm), or Compound C (50 µm) + AICAR (1 mm) + insulin (10 nm) with and without insulin pretreatment. I treated the rhypoe19 neurons with either PBS vehicle or 100 nm insulin for 24 hours. After the 24 hour pretreatment, the neurons were treated with the AMPK inhibitor, Compound C (50 µm) or DMSO control. Subsequently, cells treated with Compound C (50 µm) were cotreated with AICAR (1 mm) for one hour prior to wash period. The cells were then washed, placed in fresh media for one hour, and rechallenged with 10 nm insulin or water vehicle for 15 minutes. The protein was then harvested. Using Western Blot analysis, I found that Compound C was able to improve AICAR induced Akt protein phosphorylation

53 45 A Relative Akt Phosphorylation Water DMSO Insulin (10 nm) AICAR (1 mm) Compound C (50 µm) ** ** + + * *** Pretreatment Vehicle Insulin (100 nm) pakt Akt B Relative AMPK Phosphorylation Water DMSO Insulin (10 nm) AICAR (1 mm) Compound C (50 µm) ** ** Pretreatment Vehicle Insulin (100 nm) pampk AMPK Figure 3.6. Compound C appears to abolish the reversal of cellular insulin resistance with AICAR treatment. rhypoe19 neurons were pretreated for 24 hours with PBS (vehicle) or 100 nm insulin. Cells were then treated with either Compound C (50 µm) or vehicle (PBS) one hour prior to AICAR (1 mm) treatment. Compound C prevented the induction of Akt with AICAR treatment following induction of insulin resistance (A). Compound C also decreased the activation of AMPK in both pretreatment groups (B). Protein levels were normalized to total Akt and AMPK. Data are shown with SEM (n=3 independent experiments). * p < 0.05, ** p < 0.01, *** p < as per oneway ANOVA with Bonferroni s posthoc test.

54 46 Table 3.6.A Densitometry of pakt relative to Total Akt rhypoe19 cell line Water DMSO Insulin0(100mM) DMSO0+0Insulin Compound0C0(500μM)0+0AICAR0(10mM) Insulin0(10nM) Table 3.6.B Densitometry of pampk relative to Total AMPK rhypoe19 cell line No0pre@treatment 240hr0insulin0(1000nM)0pre@treatment Water DMSO Insulin0(100mM) DMSO0+0Insulin Compound0C0(500μM)0+0AICAR0(10mM) Insulin0(10nM)

55 47 following induction of insulin resistance (Fig 3.6A, n=3, Oneway ANOVA p=< (between the two treatment groups), Twoway ANOVA p=>0.05 (Within 24 hr insulin pretreatment; Insulin rechallenge vs. Insulin + AICAR + Compound C), Raw Data: Table 3.6A). Additionally, the effect of Compound C was associated with a decrease in AMPK phosphorylation (Fig 3.6B, n=3, Oneway ANOVA p=< (between the two treatment groups), Twoway ANOVA p=<0.001 (Within 24 hr insulin pretreatment; Insulin rechallenge vs. Insulin + AICAR+ Compound C), Raw Data: Table 3.6B). Taken together, these results suggest that the effect of AICAR treatment on insulin action in the rhypoe19 and mhypoanpy/gfp neurons is specifically attributed to AMPK activation.

56 Chapter 4 Discussion 48

57 Overall Conclusions of Significant Findings Insulin action in the hypothalamus is vital to maintain wholebody energy homeostasis. In the current findings, a potential mechanism of how targeted AMPK action can restore neuronal cellular insulin resistance is presented. Although the role of insulin resistance in the periphery is well characterized, there is a lack of studies identifying the molecular consequences of cellular insulin resistance at the neuronal level. However, with the recent emergence of studies indicating the vital role that insulin signaling plays at the level of the hypothalamus, particularly within AgRP expressing neurons to suppress hepatic glucose production, identifying candidate molecules is crucial to provide further insight into potential therapeutic targets to counteract the detrimental effects of T2DM and obesity (11). The role that S6K1 plays in the induction of insulin resistance in the periphery is well known. For example, it has been shown in adipose tissue, skeletal muscle, and the liver that there is an elevation of S6K1 phosphorylation following highfat diet feeding (51). More recently, an indirect role for S6K1 has been proposed. Ono et al. demonstrated that overactivation of S6K1 in the hypothalamus was accompanied by impaired Akt and IRS1 activity and led to the onset of hepatic insulin resistance (70). Moreover, the dominantnegative form of hypothalamic S6K1 was shown to reverse these effects and improve insulin sensitivity. Importantly, in the present studies it is demonstrated that the induction of cellular insulin resistance is accompanied by overactivation of the mtors6k1 pathway, assessed by S6K1 phosphorylation in both the rhypoe19 and mhypoanpy/gfp cell lines. Thus, in line with previous studies, I

58 50 present evidence for hypothalamic S6K1 as a major contributor to central cellular insulin resistance and show how targeting S6K1 using the AMPK activator AICAR can slightly improve this resistance. Using various concentrations of AICAR, it was identified that treatment with 1 mm was the lowest sufficient dose to activate AMPK in the neurons. This finding correlates with other in vitro studies, which have used a minimum dose of 1 mm AICAR to activate AMPK (99101). It was also determined within the rhypoe19 and mhypoa NPY/GFP cell lines that AICAR treatment was able to slightly improve Akt phosphorylation following prolonged chronic insulin exposure. However, AICAR did have a significant effect on insulin action. Moreover, this was accompanied by a decrease in the overactivation of S6K1 resulting from chronic insulin exposure. Thus, the slight improvement to Akt activity following AMPK activation could be due to the inhibition of mtors6k1 hyperactivation. I further confirmed the potential role that AMPK plays on insulin action through the use of the putative inhibitor, Compound C. A blunted effect of AICAR treatment in the presence of Compound C following prolonged insulin exposure in the neurons was evident, assessed by Akt phosphorylation. Collectively, it is suggested that the mild effects of restoration seen were mediated by AMPK in both hypothalamic neuronal cell lines. Interestingly, in some cases the statistical analysis using Oneway and Twoway ANOVA yielded the same result. In other cases where there was significance using the Oneway ANOVA and no significance using Twoway ANOVA, performing more trials may have conceded the same conclusion. To my knowledge, this is the first time that it has been presented at the cellular level that targeting AMPK in the hypothalamus may protect against insulin resistance through inhibition of mtor/s6k1.

59 51 Thus, in conjunction with peripheral studies, I present evidence to potentiate hypothalamic AMPK as a potential therapeutic target of central insulin resistance. A hallmark feature of cellular insulin resistance in the periphery is attenuated Akt phosphorylation. In the current studies, the effects of chronic insulin in the mhypoa NPY/GFP and rhypoe19 neuronal lines were assessed. Indeed, impairment of Akt phosphorylation due to chronic insulin in the rhypoe19 and mhypoanpy/gfp neuronal cell lines was evident. This accords with previous studies that have reported shortterm induction of phosphoakt following insulin treatment in hepatic and hypothalamic cell lines (98, 7). It has also been recently reported that chronic insulin exposure in the rhypoe19 cell line is accompanied by a decrease in the serine phosphorylation of the transcription factor FoxO1 (53). As previously mentioned, this compliments the findings in the current study as Akt phosphorylation, which is a regulator of FoxO1 action, was also impaired in the rhypoe19 cell line. Interestingly, chronic insulin also impaired the levels of AMPK in the rhypoe19 cell model. This finding is supported by other studies, which have found basal suppression of AMPK in some regions of the brain in dietinduced obese mice (DIO) (104). As a possible mechanism of this inhibition of AMPK activity, I propose this may be attributed to SOCS3 overactivation. The overactivation of SOCS3 with prolonged insulin exposure has recently been reported (53). Given the wellestablished role of SOCS3 as a negative regulator of the insulin receptor and inhibitor of AMPK activity, future studies will need to investigate whether overactivation of SOCS3 suppresses AMPK in our neuronal models (105).

60 52 Additionally, the phosphorylation status of various ER stress markers was assessed. Chronic insulin induced the IRE1 branch of the UPR through JNK phosphorylation. However, although there was a significant difference in XBP1 spliced (activation) levels under normal and 24 hour insulin pretreatment with Oneway ANOVA, there was not a significant difference between the two groups supported by the more stringent Twoway ANOVA analysis. In addition, the PERK branch of the UPR was not activated, as there were no changes in downstream eif2α activity following prolonged insulin exposure. The lack of the PERK branch activation may be due in part to the method through which insulin resistance was induced. For example, eif2α phosphorylation as a consequence of insulin resistance has been well documented in the presence of palmitate (106). Indeed, the Belsham lab has previously reported that preexposure of the mhypoe44 neuronal cells with palmitate initiated eif2α phosphorylation (107). Additionally, prolonged insulin exposure has been reported to activate only the IRE1 branch of the UPR in hepatocytes. However, it was shown that when mtor/s6k1 signaling was blocked using rapamycin, IRE1 branch activation was no longer present (54). Thus, in the case of neuronal insulin resistance due to chronic insulin, activation of the IRE1 branch of the UPR could be due to mtor/s6k1 hyperactivation. To date, studies regarding the molecular consequences of insulin resistance are limited. At the neuronal level, other groups have provided some insight into the induction of central insulin resistance by overnutrition. Zhang et al demonstrated that the IKKβ/NFκB pathway mediates obesity and glucose intolerance induced by HFD feeding (108). Interestingly, when the IKKβ/NFκB pathway was suppressed, specifically in

61 53 AgRP expressing neurons, these mice were protected against the negative consequences of insulin resistance. Additionally, elevated ER stress markers have also been reported in models of obesity (109). Through intracerebroventricular (i.c.v) administration of chaperones that target hypothalamic ER stress, Ozcan et al. were able to improve hypothalamic insulin sensitivity (109). Moreover, PTP1B expression, a negative regulator of the insulin receptor was elevated under a state of HFD feeding (110). The elevation of PTP1B was accompanied by an increase in proinflammatory cytokines, such as TNFα and SOCS3. Taken together, these studies further emphasize the importance of identifying the cellular mechanisms involved in the pathogenesis of hypothalamic insulin resistance. Collectively, in the current studies, a potential therapeutic role of targeting AMPK in AgRP/NPY expressing neurons to mediate the effects of insulin resistance due to chronic insulin exposure was investigated (Figure 4.1). Given the putative role of insulin signaling in AgRP expressing neurons to suppress hepatic glucose production, identifying therapeutic targets to improve hypothalamic insulin resistance is essential. The overactivation of S6K1 seen in the in vitro studies suggests a parallel effect of the onset of insulin resistance both centrally and peripherally. Overall, the present findings suggest that treatments aimed to target peripheral insulin resistance may also be used to improve central insulin resistance. These findings provide some insight into potential clinical application for future treatments of T2DM and obesity.

62 Future Directions The results of the recent studies presented in this thesis raise further novel questions that warrant investigation. Most cases of obesity are also associated with lowgrade inflammation, particularly an increase in circulating TNFα and other cytokines (111). Peripheral studies have demonstrated the relevance of lowgrade inflammation in the pathogenesis of type 2 diabetes mellitus (T2DM). For example, in the liver and adipose tissue the TNFα signaling cascade has been shown to undergo crosstalk with the insulinsignaling cascade to induce cellular insulin resistance (112). The onset of this inflammatory induced resistance occurred through serine phosphorylation of IRS1 and overactivation of mtor/s6k1, two downstream effectors of the insulinsignaling cascade. At the cellular level, TNFα signaling has been associated with downstream JNK, IKKβ, and NfκB activation. NfκB is a transcription factor involved in the expression of various cytokines, including IL1β, TNFα, and SOCS3. Under normal conditions, NfκB activity is suppressed by IκBα (113). However, when TNFα is initiated, as is the case with obesity, IκBα is degraded and liberates active dimers of NfκB allowing for nuclear translocation. NfκB mediated expression of SOCS3 disrupts insulin signaling, as it is a wellestablished inhibitor of the insulin receptor (113). Moreover, JNK activation has been attributed to further exacerbate the state of cellular insulin resistance through direct serine phosphorylation of IRS1 (55) and indirect activation of mtor/s6k1 (56). Therefore, to expand on the current studies presented in this thesis, the next step would be to identify whether the inflammatory cytokine, TNFα, can evoke an inflammatory response in the hypothalamus in the rhypoe19 and mhypoanpy/gfp cell lines. Furthermore, whether the canonical TNFα inflammatory

63 Figure 4.1 Model of the putative role of central insulin resistance in attenuating insulin signaling in AgRP/NPY expressing hypothalamic neurons. Prolonged exposure of the insulin receptor to insulin leads to impaired activation of the insulinsignaling cascade. Chronic activation of the insulin receptor leads to overactivation of downstream signaling molecules, such as S6K1 and mtor that phosphorylate IRS proteins on serine residues and inhibit their action. In turn, this prevents the further activation of the insulinsignaling cascade in the presence of insulin. AMPK activation with AICAR is able to alleviate this resistance through inhibition of mtor/s6k1 activity. 55