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Role of endoplasmic reticulum calcium stores in beta-cell ER stress and lipotoxicity Gwiazda, Kamila Sabina 2009

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ROLE OF ENDOPLASMIC RETICULUM CALCIUM STORES IN BETA-CELL ER STRESS AND LIPOTOXICITY by KAMILA SABINA GWIAZDA B.Sc., The University of British Columbia, 2006  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Cell and Developmental Biology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  August 2009  © Kamila Sabina Gwiazda, 2009  ii  Abstract There are strong links between obesity, elevated free fatty acids, and type 2 diabetes. Specifically, the saturated fatty acid palmitate has pleiotropic effects on !-cell function and survival. The present study sought to determine the mechanism by which palmitate affects intracellular Ca2+ in pancreatic !-cells, and in particular the role of the endoplasmic reticulum (ER). In the MIN6 β-cell line, palmitate rapidly increased cytosolic Ca2+ through a combination of Ca2+ store release and extracellular Ca2+ influx. Palmitate caused a reversible lowering of ER Ca2+, measured directly with the fluorescent protein-based ER Ca2+ sensor, D1ER. Using another genetically encoded indicator, long-lasting oscillations of cytosolic Ca2+ in palmitate-treated cells were observed. The kinetics of pharmacological SERCA inhibition on the !-cell ER stress response were characterized, and the ER calcium sensor PERK was found to be rapidly activated in response to irreversible ER calcium depletion. ER calcium depletion in palmitate-treated cells also induced rapid phosphorylation of PERK, as well as other subsequent downstream ER stress signals. In summary, the effects of the free fatty acid palmitate on pancreatic β-cell Ca2+ homeostasis were characterized in this thesis. This study provides the first direct evidence that free fatty acids reduce ER Ca2+ and sheds light on pathways involved in β-cell ER stress, lipotoxicity and the pathogenesis of type 2 diabetes.  iii  Table of Contents Abstract.............................................................................................................................................ii
 Table of Contents .......................................................................................................................... iii
 List of Tables.................................................................................................................................... v
 List of Figures .................................................................................................................................vi
 List of Abbreviations................................................................................................................... vii
 Acknowledgements ..................................................................................................................... viii
 Chapter One: Introduction...........................................................................................................1
 Pathophysiology of Type 2 Diabetes ..................................................................................................... 1
 Obesity and lipotoxicity........................................................................................................................... 2
 Measuring plasma free fatty acid levels ..........................................................................................................3
 Fatty acids and insulin secretion........................................................................................................................4
 Mechanisms of lipotoxicty ...................................................................................................................... 5
 ER stress in the !-cell.............................................................................................................................................6
 ER stress and diabetes ...........................................................................................................................................7
 Lipotoxicty and ER stress......................................................................................................................................8
 Lipotoxicity and Ca2+ signalling ........................................................................................................................9
 Ca2+ signalling in the β -cell.................................................................................................................. 10
 Imaging intracellular Ca2+ ................................................................................................................................ 12
 Caspases and apoptosis in β -cells....................................................................................................... 15
 Chapter Two: Materials and Methods................................................................................... 17
 Reagents................................................................................................................................................... 17
 MIN6 Cell Culture................................................................................................................................. 18
 Live-cell Imaging ................................................................................................................................... 19
 Protein Extraction and Western Blotting......................................................................................... 20
 Data Analysis .......................................................................................................................................... 20
 Chapter Three: Results............................................................................................................... 21
 Acute effects of palmitate on cytosolic Ca2+ ..................................................................................... 21
 D3cpv cameleon captures acute cytosolic Ca2+ signals induced by palmitate .......................... 22
 Mechanisms of palmitate-induced Ca2+ signals .............................................................................. 23
 Acute palmitate treatment causes a decrease in ER Ca2+ ............................................................. 26
 Effects of oleate on ER Ca2+ dynamics.............................................................................................. 27
 Long-term analysis of Ca2+ signals using cameleons...................................................................... 28
 Effect of chronic treatment of palmitate on Ca2+ dynamics ......................................................... 31
 Characteristics of ER stress and caspase activation in β -cells .................................................... 33
 Effects of palmitate on ER stress........................................................................................................ 34
 Chapter Three: Discussion......................................................................................................... 37
 Palmitate-induced cytosolic Ca2+ signals .......................................................................................... 39
 Long-term palmitate-induced cytosolic Ca2+ signals...................................................................... 40
 Mechanisms behind palmitate-induced ER Ca2+ signals .............................................................. 41
 Long-term palmitate-induced ER Ca2+ depletion........................................................................... 43
  iv Oleate­stimulated
ER
Ca2+
signals .................................................................................................. 44
 Glucose-dependence of palmitate Ca2+ signals ................................................................................ 45
 Kinetics of ER stress and death in β -cells ........................................................................................ 46
 Palmitate and β -cell
ER stress ............................................................................................................ 46
 Palmitate and lipotoxicity: possible mechanisms ........................................................................... 47
 Palmitate and insulin secretion........................................................................................................... 48
 Conclusions and future directions ..................................................................................................... 50
  References...................................................................................................................................... 51
 
  v  List of Tables Table 1. List of antibodies used............................................................................................18
  vi  List of Figures Figure 1. Schematic of the ER stress signalling cascade in the beta-cell................................7
 Figure 2. Schematic of the genetically engineered cameleon constructs and Ca2+-stimulated FRET.. ............................................................................................................................15
 Figure 3. Palmitate Ca2+ dose-response in MIN6 cells.. .......................................................22
 Figure 4. FRET-based cytosolic Ca2+ imaging of palmitate signalling.................................24
 Figure 5. Mechanisms of palmitate-induced cytosolic Ca2+ signals in MIN6 cells.. ............25
 Figure 6. Blocking IP3R does not block palmitate-induced cytosolic Ca2+ signals.. ............26
 Figure 7. Direct imaging of ER luminal Ca2+ in palmitate-treated MIN6 cells.. ..................28
 Figure 8. Blocking IP3R does not block palmitate-induced ER Ca2+ release........................29
 Figure 9. 2-bromopalmitate does not initiate ER Ca2+ release.. ............................................29
 Figure 10. Oleate mobilizes ER Ca2+ stores. .........................................................................30
 Figure 11. Oleate does not block palmitate’s ability to mobilize ER Ca2+ stores. ................31
 Figure 12. Long-term imaging of palmitate-induced Ca2+ dynamics....................................32
 Figure 13. Effect of chronic palmitate treatment on ER Ca2+ dynamics...............................33
 Figure 14. Effects of IP3R activation on UPR activation evoked by SERCA inhibition. .....35
 Figure 15. Blocking ER Ca2+ release does not attenuate thapsigarin-induced caspase actiavtion.. ......................................................................................................................36
 Figure 16. Palmitate induces acute PERK phosphorylation..................................................37
 Figure 17. Blocking ER Ca2+ release does not inhibit palmitate-induced XBP-1 splicing ...38
 Figure 18. Schematic summarizing palmitate’s possible effects on !-cell Ca2+ homeostasis and ER stress.. ................................................................................................................49
  vii  List of Abbreviations AUC ATF6 BSA Ca2+ CHOP eIF2α ER FBS FFA FRET GPR40 IP3R IRE1 LC-CoA RyR MIN6 PBS PERK ER kinase UPR XBP-1  Area under the curve Activating transcription factor 6 Bovine serum albumin Calcium C/EBP-homologous protein Eukaryotic translation initiation factor 2 alpha Endoplasmic reticulum Fetal bovine serum Free fatty acid Fluorescence resonance energy transfer G-protein coupled receptor 40 Inositol 1,4,5-triphosphate receptor Inositol requiring ER-to nucleus signal kinase 1 Long-chain acyl-coenzyme A Ryanodine receptor Mouse insulinoma Phosphate-buffered saline Double-stranded RNA-activated protein kinase-like Unfolded protein response X-box binding protein-1  viii  Acknowledgements The past two years have been a roller-coaster ride, and I am very grateful to everyone who helped me to get to where I am. First and foremost, my supervisor, Jim Johnson, who has inspired and motivated me, and has shaped the scientist I have become. His sheer love of science is contagious, and I will miss his uncanny way to make everything funny. Jim, thanks for putting up with my anxiety and seemingly never-ending capacity for stress; it’s not easy to do. Thank you for showing me that I can actually do this. My committee members, Chris Loewen, Tim Kieffer, and Eric Jan: thank you for all your guidance, your suggestions and your support. To everyone in the lab, past and present, who has inspired and helped me: Emilyn, Dan, Tatyana, Yalin, Ting, Jessica, Carol, Arya, Connie, Farnaz, Betty, Grace, Erin. Em, you were always there for me, and we’ve had a lot of fun, both in the lab and out. I’ll never forget our trip to Ireland, dancing and singing in the lab, and everything in between. Dan, thanks for teaching me pretty much everything I know (about everything!) and for all your advice. And to everyone else, thanks for all your kindness and help, and most of all thanks for putting up with me in the lab and for providing such a great work environment. I’ll miss you all! And most importantly, to my family: my sister, dad, and mom. Thank you for supporting me and being there for me through the years. Special thanks to my sister, for keeping me sane and laughing, and my dad for inspiring me and pushing me to work hard. It’s not over ‘til it’s over, so you may as well enjoy the ride. KG  1  Chapter One: Introduction Type 2 diabetes has become a disease of epidemic proportions, and is no longer limited to Western countries and cultures.  The World Health Organization (WHO)  estimates that the disease now affects over 180 million people, causing approximately 2.9 million deaths each year (124). Associated complications of diabetes include retinopathy, neuropathy, limb amputations, kidney failure, and increased risk for heart disease and stroke, rendering type 2 diabetes an important medical concern and a severe health care burden. The numbers of diabetes cases are expected to double between 2000 and 2030 (156), and consequently the WHO estimates that over the next 10 years diabetes deaths will increase by more than 50% without urgent action (155). There is no current cure for diabetes, only treatment to manage it and at best slow its progression. Together, these statistics indicate that this epidemic is only going to increase and continue affecting people the world over.  Pathophysiology of Type 2 Diabetes Type 2 diabetes is caused by peripheral insulin resistance, insulin secretory defects, and programmed cell death of the pancreatic insulin-secreting β-cells (16, 35, 126, 161). The best-known risk factor for the disease is obesity, which is characterized by elevated levels of circulating plasma free fatty acids (12). The lipid levels are elevated due to expanded and highly lipolytic stores of adipose tissue (12, 116). Obesity itself is associated with insulin resistance in tissues such as muscle, liver, and adipose, but most obese and insulin-resistant individuals do not develop type 2 diabetes because their β-cells can compensate by increasing their β-cell mass and hyper-secreting insulin (67). The disease occurs when  2 peripheral insulin resistance is too great and β-cells can no longer secrete the required amounts of insulin, or when the β-cells themselves fail and undergo apoptosis (16, 115). There are many causes of β-cell dysfunction and apoptosis in type 2 diabetes: hyperglycemia, hyperlipidemia, pro-inflammatory cytokines, and secreted islet amyloid polypeptide. Whether these factors are causal or secondary to the disease state, or both, is difficult to determine, but it is clear that to further understand diabetes pathophysiology, the biology of the pancreatic islet β-cell must be better understood.  Obesity and lipotoxicity The link between obesity and diabetes is robust. The vast majority (>80%) of patients with type 2 diabetes are obese and most obese individuals have symptoms of prediabetes, including impaired glucose tolerance and insulin resistance. Lipotoxicity, the phenomenon whereby excess fatty acids cause cell death, helps explain how elevated levels of lipids, such as those seen in obesity, can be detrimental to pancreatic β-cell health. Insulin resistance, considered to be one of the first steps in the onset of type 2 diabetes, also results from toxic amounts of saturated fatty acids in insulin-sensitive tissues, including the liver, adipose and muscle (91, 158). In some in vivo models of obesity, such as the Zucker diabetic fatty (ZDF) rat that develops obesity and diabetes, β-cell apoptosis is seen in the obese pre-diabetic state, when the islets are fat-laden (132). Indeed, many in vitro experiments using human and rodent primary islets as well as cultured β-cells have shown that chronic elevation of the long-chain saturated fatty acid palmitate induces !-cell death (68, 83, 85, 110, 132). In contrast, in vitro studies using treatments with monounsaturated fatty acids such as palmitoleate (C16:1) and oleate (C18:1) found that these lipids tend to be  3 less cytotoxic (26, 49, 68, 70, 90), and in some cases even protective, against palmitate toxicity (32, 85, 86). Some investigators suggest that the toxic effects of lipids occur only concurrently with high levels of glucose, a phenomenon termed glucolipotoxicity (111-113). A recent report suggested that elevated glucose levels accentuate lipotoxicity in vitro, through the regulation of ER stress by the protein synthesis activator mTORC1 (7). However, it is conceivable that hyperlipidemia may affect !-cell survival and function in the absence of hyperglycemia and frank diabetes, suggesting that lipids can indeed have deleterious effects on !-cells on their own, as has been shown in vitro (26, 85, 86, 125, 162). Further, in vitro studies using obese pre-diabetic ZDF rats demonstrate lipotoxicity and β-cell dysfunction in the absence of elevated glucose levels (50). To add further complexity to this issue, high glucose itself can induce lipotoxicty by up-regulating lipogenic genes (150), thereby creating a cellular glucolipotoxic environment separate from that caused by elevated plasma lipid levels alone. Whether the two stimuli act additively or synergistically once diabetes has been established remains to be determined.  Measuring plasma free fatty acid levels Fatty acids are essential nutrients and physiological signalling molecules, and as such there exists a spectrum of plasma free fatty acid levels that range from normolipidemic to hyperlipidemic. Under normal conditions, the vast majority of circulating plasma fatty acids are bound to carrier proteins. The most abundant of these carrier proteins is albumin, which also has the highest affinity for free fatty acids (134). However, in states where the fatty acid to albumin ratio is high (> 4:1) other molecules such as lipoproteins and phospholipid vesicles can also bind fatty acids (21). In healthy individuals, the molar ratio of  4 fatty acids to albumin ranges from 0.5:1 to 2:1 (24), while in conditions including obesity and diabetes, the ratio can increase up to 6:1 (117). There are at least 43 different FFA species present in human plasma, but in FFA preparations from human plasma albumin, the C16 and C18 FFA's accounted for 90% of total FFA (17). Additionally, palmitate is the predominant lipid produced by de novo lipogenesis (1), and this pathway contributes 20% of the palmitate stored in adipose tissue (138). Determining the concentration of free fatty acid circulating in plasma is complicated by the FFA: albumin ratio, as the binding of one fatty acid affects the binding of subsequent fatty acids to albumin (134). The presence of lipases (hormone-sensitive lipase and adipocyte triglyceride lipase) in the β-cell also increases endogenous FFA levels (40, 109). Nevertheless, the total level of palmitate in plasma of healthy humans in one study ranged from 720 µM to 2 mM (17), and a suggested healthy range of unbound FFA is 7.5 ± 2.5 nM (121). By comparison, a solution containing 0.5 mM palmitate in a 5:1 FFA: BSA ratio, a typical amount used in in vitro studies, gave a free palmitate concentration of 200 nM (112). It should also be noted that the levels of bound free fatty acids in the obese and diabetic ZDF rat were greater than 1 mM (132). Together, these studies demonstrate that there is quite a range of plasma free fatty acid levels, and re-creating a physiological fatty acid milieu in vitro can be difficult.  Fatty acids and insulin secretion Acute administration of fatty acids, especially palmitate, is thought to cause a modest increase in insulin secretion (25, 98), while prolonged exposure to this fatty acid increases basal insulin secretion and decreases the relative response to glucose both in vitro and in vivo (9, 13, 102, 163). Additionally, circulating fatty acids are thought to be critical in  5 maintaining glucose-stimulated insulin secretion after fasting (33, 34).  However, the  mechanisms by which palmitate affects β-cell insulin secretion are not fully understood. There has been recent evidence pointing to the involvement of the long-chain fatty acid Gprotein coupled receptor GPR40 (60, 69, 76, 96, 136, 141), though the effects of acute vs. chronic palmitate treatment on GPR40 activation remain controversial (3). Additionally, fatty acid uptake via CD36 and consequent intracellular signalling is also involved (99), and the literature suggests mechanisms involving altered mitochondrial metabolism (13), signalling through esterified long-chain CoA molecules (107, 114, 123), and insulin granule exocytosis (101). Intracellular lipolysis and FFA/triglyceride cycling may also play a role in insulin secretion (40, 109). Indeed in the Zucker fatty rat, which is obese but does not develop diabetes, lipolysis has been suggested as the mechanism by which the β-cells compensate for insulin resistance by enhancing insulin secretion (97).  Mechanisms of lipotoxicty There are many proposed models that account for toxic effects of chronically elevated lipids (146). One model of lipotoxicty hypothesizes that free fatty acids, especially palmitate, cause ER stress, the state in which the endoplasmic reticulum of a cell cannot cope with its protein load and initiates the unfolded protein response (UPR). If the ER stress signals are chronically elevated, they can eventually lead to apoptosis (128). Lipotoxicity has also been examined in the context of oxidative stress, ceramide generation, and altered lipid partitioning, but these models are not mutually exclusive from the ER stress model.  6  ER stress in the β-cell Pancreatic β-cells are especially susceptible to the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress, as they are high capacity secretory cells with a extensive network of ER. The ER stress response is activated under pathological conditions, but also as a normal physiological response to secure ER integrity. The UPR has three main functions. One function is to up-regulate ER chaperone proteins to help reduce the load of misfolded proteins. A second function is to decrease the overall rate of protein synthesis, while sparing proteins that are required for the UPR. A third function involves the initiation of programmed cell death, once the cell decides that it can no longer be saved (54). The ER trans-membrane proteins IRE1, PERK, and ATF6 initiate these signals by sensing an increase in the misfolding of ER client proteins. Once activated, these proteins activate their respective targets (Fig. 1). IRE1, an endonuclease and kinase, cleaves cytosolic XBP-1 mRNA, activating the transcription factor to transcribe ER stress-inducible genes, such as chaperones, to aid in protein folding. Chronic IRE1 signalling has also been shown to activate JNK and initiate apoptosis (147). PERK phosphorylates eIF2α, causing a general attenuation of translation in order to decrease the protein load on the ER. ATF6 is a transcription factor that also up-regulates ER stress-inducible target genes. Together, these molecules are the proximal UPR signals, and if chronically activated, can initiate programmed cell death through pro-apoptotic molecules such as CHOP and JNK.  7  Figure 1. Schematic of the ER stress signalling cascade in the beta-cell. Thick arrows indicate an increase in protein levels. The diagram is not to scale and many molecules have been left out for simplicity.  ER stress and diabetes There are a number of mutations that disrupt the protein folding pathway that lead to diabetes, including those found in permanent neonatal diabetes, Wolfram syndrome, and  8 Wolcot-Rallison syndrome (128). In the Akita mouse, the Insulin 2 gene carries a mutation similar to those found a subset of patients with neonatal diabetes (137), which prevents the formation of an essential disulphide bond and causes the protein to misfold and become retained in the ER. Over time, the Akita mouse develops diabetes due to a progressive loss of its !-cell mass, a consequence of excessive ER stress-induced apoptosis (103). A loss of PERK also causes a loss in !-cell mass and diabetes in mice (55), and in humans this mutation is known as the autosomal recessive Wolcot-Rallision syndrome, which also results  in  diabetes  (30).  Wolfram  syndrome,  a  human  autosomal  recessive  neurodegenerative disorder that also presents with juvenile-onset diabetes (8), is caused by a mutation in WFS1, an ER resident protein that is likely involved in ER protein degradation (54). Additionally, polymorphisms in the transcription factor ATF6 have been associated with type 2 diabetes in two different population studies (94, 143). Other mouse models have been made to further elucidate the effects of ER stress signalling machinery, including the eIF2αSer51A mouse, which develops diabetes due to PERK’s inability to phosphorylate the mutated eIF2α (133). A number of studies also demonstrate the role of islet-amyloid polypeptide toxicity, and suggest that the excess presence of this toxic protein contributes to β-cell failure through ER stress (58, 92). Together, these conditions underline the importance of ER homeostasis in !-cells and the requirement of intact ER stress machinery for healthy !-cell function and normal glucose homeostasis (128).  Lipotoxicty and ER stress Many studies have found that fatty acids, specifically palmitate, initiate !-cell ER stress in vitro (26, 39, 68, 77), as well as in vivo (77). Studies with fatty acids oleate and palmitoleate have found these monounsaturated fats to induce less ER stress than palmitate  9 in some models (26, 70, 90), and none at all in others (31, 32, 68). How fatty acids induce ER stress remains unclear, but recent reports point to a few different mechanisms. For example, chronic treatment with palmitate, but not oleate, caused morphological distention of the ER, which could affect its function and induce the stress response (68). Saturated fats, owing to their high melting point, are more likely to precipitate once formed, while unsaturated fats can be safely stored as triglycerides. Saturated fats also affect membrane fluidity, causing an increase in rigidity and hampering membrane function (139). Palmitate has also been shown to feed into de novo ceramide synthesis (108), which can activate apoptosis in ZDF rat islets (132). A recent study also found that palmitate directly affected the degradation of an important insulin-processing enzyme, carboxypeptidase E (CPE) (62). Further, β-cells exhibited significant ER stress and apoptosis in mice lacking CPE, indicating that this enzyme is required for β-cell survival and function (62). A recently discovered effect of palmitate is its ability to elicit intracellular calcium (Ca2+) signals in rodent islets and some β-cell lines (118, 129). The ER is a major cellular Ca2+ store and ER Ca2+ homeostasis is critical for ER function (130), and importantly, insulin processing (52). Accordingly, depletion of ER Ca2+ by cytokines or inhibitors of the ER Ca2+ uptake pump (SERCA) can trigger an extensive ER stress response in β-cells (18, 81). A recent report documented that blocking Ca2+ entry through voltage-gated Ca2+ channels inhibited palmitate-induced β-cell apoptosis (62). This may provide a potential link between palmitate, ER stress, and lipotoxicity.  Lipotoxicity and Ca2+ signalling Whether palmitate directly affects ER Ca2+ stores remains unclear. Recent studies employing indirect measurements of the ER filling state in INS-1 cells (68) and mouse islets  10 (101) failed to detect any effects of palmitate on ER Ca2+ homeostasis. In other studies, palmitate was shown to increase cytosolic Ca2+ levels in HIT-T15 and INS-1 cells, as well as primary mouse β-cells (118, 129). In HIT-T15 cells, the maintenance of these signals was dependent on extracellular Ca2+ influx through voltage-gated Ca2+ channels (118). However, the observation that a Ca2+ transient remained in the absence of extracellular Ca2+ suggested that a component of these Ca2+ signals might be initiated by intracellular stores (118). It is thought that the G-protein coupled receptor for long-chain fatty acids, GPR40 (15, 60, 73), may play a role in initiating palmitate-induced Ca2+ signals. Activation of G-protein coupled fatty acid receptors would be expected to mobilize IP3-sensitive Ca2+ stores on the ER (15, 60, 73). Notwithstanding, the effects of palmitate on ER Ca2+ levels have not been measured directly using targeted ER Ca2+ sensors (106). Although these previous studies suggest that Ca2+ dynamics play an important role in the effects of palmitate, the precise mechanisms by which palmitate affects β-cell Ca2+ homeostasis remain unresolved.  Ca2+ signalling in the β-cell Ca2+ is a ubiquitous second messenger in cells, involved in a vast array of cellular processes including growth, gene regulation, proliferation, metabolism, exocytosis, and apoptosis (10, 22, 122). Persistently elevated Ca2+ is toxic to cells, and thus its levels must be carefully regulated. Cytosolic baseline levels of Ca2+ are typically in the 100 nM range, about 20,000 times lower than the extracellular environment (22). The ER serves as a major intracellular Ca2+ store, storing micromolar amounts of the molecule (105). Ca2+ signals themselves, whether initiated extrinsically or intrinsically, are coded in a variety of ways: transient rises in Ca2+ can vary in amplitude, frequency, and spatial localization; they can rise locally at a mouth of a channel, be confined to one area of a cell, or propagate in a  11 global wave that can then spread between cells (122). These Ca2+ signals can originate from channels on the ER, the IP3R- and RyR-gated channels, from the various channels and pumps on the plasma membrane, or from other organelles such as the Golgi apparatus and mitochondria. Additionally, there are many channels and pumps that buffer Ca2+, to turn signals off and return the cytosol to baseline levels, including the ER Ca2+ uptake pump, SERCA. Together, these mechanisms serve as the cellular Ca2+ signalling toolkit (11), creating a complex code of signals that can control a vast array of cellular processes. Well-studied examples of Ca2+ signalling occur in secretory cells, such as the pancreatic β-cells. The combination of cellular Ca2+ channels and pumps creates cytosolic Ca2+ oscillations that drive oscillatory insulin secretion in glucose-stimulated β-cells (142). The cascade of events leading to glucose-stimulated Ca2+ signals in β-cells starts when glucose is internalized by the transporter GLUT2 (or GLUT1 in humans) (27), undergoes glycolysis and oxidative metabolism, and increases the cell ATP/ADP ratio. ATP inhibits plasma-membrane KATP channel activity, resulting in cell depolarization and activation of voltage-dependant Ca2+ channels on the plasma membrane (5). The local increase in subplasma membrane Ca2+ regulates docking and fusion of secretory granules, resulting in insulin secretion (84, 157).  Importantly, this secretion is pulsatile in nature, and  consequently creates oscillations in plasma insulin levels, which are important for insulin action on its target tissues. This pulsatility is controlled by oscillations in cytosolic Ca2+, among other factors, and this in turn is triggered by the oscillatory nature of glucose metabolism (57). These oscillations in Ca2+ are dependent on both voltage-gated extracellular Ca2+ influx as well as the sequestration of cytosolic Ca2+ into the ER via SERCA pumps (142). There are different types of oscillations: slow, fast, spiking, and fast  12 receptor-induced, and they are controlled by different stimuli (glucose, IP3, cAMP) and result in different frequencies and durations of intracellular Ca2+ increase (142). However, all of the intricate mechanisms in insulin secretion have not been fully elucidated. Other aspects of cellular metabolism, including pyruvate cycling, NADH, cAMP, and PLCmediated agonists, may also play a part in glucose-stimulated insulin secretion (57, 63, 82, 142).  Imaging intracellular Ca2+ To measure the intricacies of intracellular Ca2+ signals, probes have been developed that allow real-time live-cell imaging of intracellular Ca2+ dynamics. These probes, which have varying degrees of sensitivity, stability, and versatility, are molecules whose light absorption or emission properties change upon calcium binding, through a change in intensity or spectral shift. Since the development of the first fluorescent Ca2+ indicators (51), a variety of probes have been developed that are either small molecule fluorescent indicators (i.e. dyes), aequorin-based chemiluminescent Ca2+ sensors, or genetically encoded Ca2+ indicators (104). The most widely used small molecule Ca2+ indicators include Fura-2, Fluo-3 and Fluo-4,  and  they  differ  with  respect  to  their  resistance  to  photobleaching,  compartmentalization, leakage, loading, and dynamic range (144). These molecules are used to measure cytosolic Ca2+ levels and have proved very useful for measuring transient rises and oscillations. The next class of indicators, which is based on the jellyfish aequorin photoprotein, generates light upon calcium binding to the aequorin in the presence its prosthetic group, coelenterazine. Among the advantages of using aequorin-based indicators is that there is a very high signal to noise ratio, given that no mammalian cell produces any  13 luminescent molecules. Importantly, aequorins can be targeted to organelles and engineered to have the correct dynamic range for the organelle (4). In contrast to dyes like Fura, they have low calcium buffering capacity, preventing them from significantly affecting intracellular Ca2+ homeostasis. However, single-cell imaging is difficult with aequorins because only one photon is released per probe, giving a low level of light emission per cell, in contrast to the strongly emitting fluorescent dyes. More importantly, the reconstitution of aequorin with its cofactor is difficult, requiring the compartment to have low Ca2+ levels. For imaging Ca2+ within organelles such as the ER where Ca2+ levels are high, this step requires lowering ER Ca2+, using ionophores and inhibitors of the SERCA uptake pumps, which causes a significant alteration of organelle physiology (14). The last class of probes is the fluorescent protein-based genetically encoded indicators. These molecules have been designed with a calcium-responsive element, such as calmodulin, present with either one or two fluorescent proteins. In the single-fluorescent protein probes, termed camgaroos and pericams, the binding of Ca2+ alters the structure of the molecule, causing a change in its spectral properties. Probes containing two fluorescent proteins, the cameleons, are engineered such that Ca2+ binding changes the degree of fluorescence resonance energy transfer (FRET) between two fluorophores (93). Like aequorins, because these probes are based on recombinant cDNA expression, they can be targeted to various cellular compartments and organelles using targeting sequences. Alternatively, transgenic lines or animals can be made that express these constructs (6, 41). In contrast to the aequorins, however, these probes are easy to work with, provided they can be transfected into the cells of interest. By comparison, the fluorescent dyes tend to have faster reaction kinetics, better dynamic range, and increased sensitivity than the current  14 genetically encoded calcium indicators. However, because cells loaded with these dyes suffer from problems like dye leakage, compartmentalization, and photobleaching, the genetically engineered sensors are uniquely suitable for long-term Ca2+ imaging experiments. This is important given that the long-term dynamics of cellular Ca2+ have not been measured in most cell types, including pancreatic β-cells. Some recently developed cameleons have been fine-tuned to perform well in specific compartments, including the ER and the cytoplasm. These ‘newer generation’ cameleons are less affected by fluctuating pH levels and have improved dynamic range over their predecessor cameleons (106). Additionally, they are not affected by nor do they affect endogenous calmodulin levels, which was a problem with earlier versions of the probes (93). Two of these probes, D3cpv and D1ER, are specifically targeted to the cellular cytoplasm (D3cpv) and ER (D1ER) (Fig. 2). Both have appropriate dynamic ranges for their compartments: the affinity of D3cpv for Ca2+ is much higher than D1ER, reflecting the lower Ca2+ levels in the cytoplasm (93). D1ER is efficiently targeted to its organelle by virtue of an ER signal sequence as well as an ER retention sequence (81), and D3cpv is limited to the cytoplasm by its lack of signalling sequence. Thus, the D1ER probe presents the best method for dynamically imaging physiologically relevant luminal Ca2+ changes within β-cells (62, 105), and D3cpv allows for accurately imaging cytosolic Ca2+ signals for extended periods of time.  15  Figure 2. . Schematic of the genetically engineered cameleon constructs and Ca2+-stimulated FRET. (A) Schematic of the genetically encoded cameleons D3cpv (cytosol) and D1ER (ER). ‘m’ indicates mutant; CaM, calmodulin; smMLCK, smooth muscle myosin light chain kinase; cvV, circularly permuated Venus; CalR SS, calreticulin signal sequence; M13, peptide from skeletal muscle myosin light chain kinase; KDEL, ER retention tag sequence. (B) Schematic representation of Ca2+-stimulated FRET between the generic CFP/YFP cameleon. The diagram is not to scale.  Caspases and apoptosis in β-cells Caspases are a family of cysteine-requiring aspartate proteases that are key in regulating the initiation and execution of apoptosis in β-cells (59). To denote their function in the apoptotic pathway, they are classified as initiator (early activation) or effector (late mediator) caspases. Caspase 9, an initiator caspase, is activated by cytochrome c, released  16 from compromised mitochondria, together with Apaf-1 in the presence of dATP (59). Caspase 9 then activates pro-caspase 3, an effector executioner caspase, pro-caspase 7 and other apoptosis factors including PARP, to induce the hallmarks of apoptosis: cell blebbing, chromatin condensation, protein cross-linking, and collapse of nuclear membrane (59). In rodents, caspase 12, which is located at the ER membrane, is activated in response to ER stress by IRE1 and cleaved by Caspase 7 or m-Calpain (54) and leads to caspase 3 activation. However, caspase-12 is thought to be a pseudogene in man, and therefore may not have an evolutionarily conserved role (42), although whether or not it is important in human ER stress signalling remains controversial (88, 140, 159). Indeed, altering ER Ca2+ homeostasis by drugs that block ER Ca2+ uptake caused ER stress-induced caspase 3 activation in β-cells (81). Thus these proteins are potent executors of apoptotic cell death, but the field of caspase biology is limited in its knowledge of caspase activation kinetics.  17  Chapter Two: Materials and Methods  Reagents All reagents were from Sigma (St. Louis, MO), unless otherwise indicated. The acetoxymethyl ester of the cytoplasmic Ca2+ dye Fura-2 (Fura-2-AM; Molecular Probes; Eugene, OR) was stored in dimethyl sulfoxide (DMSO) as a 1 mM stock solution. Thapsigargin, xestospongin C (Cayman Chemical; Ann Arbor, MI), and nifedipine (Calbiochem; San Diego, CA) were stored in DMSO as a 1 mM stock solution, and ryanodine (Tocris Biosciences; Ellisville, MO) was stored in DMSO as a 100 mM stock solution. Carbachol (Calbiochem; San Diego, CA) was dissolved in water to a 100 mM stock solution. The cameleon FRET probes D1ER and D3cpv were gifts from Dr. Amy Palmer (Department of Chemistry and Biochemistry, University of Colorado). Drug solutions were prepared immediately before each experiment. Palmitic acid (Nu-Check Prep; Elysian, MN) was dissolved in 65 mM NaOH and complexed with 20% essentially fatty-acid free bovine serum albumin (BSA). The complex was added to Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen; Burlington, ON) containing 5 mM or 25 mM glucose, 10% fetal bovine serum (Invitrogen; Burlington, ON) and 1% penicillin-streptomycin (Invitrogen; Burlington, ON) for a final molar ratio of 6:1 palmitate to BSA (21), unless otherwise indicated. For imaging experiments, sodium palmitate was prepared as described (118). Briefly, a stock solution of 100 mM was prepared by dissolving palmitate in 60 mM NaOH and added to pre-warmed Ringer’s solution (3 mM glucose) containing 0.1% essentially fatty acid free BSA in a 6:1 molar ratio. 2-bromopalmitate solutions were prepared in the same way. Oleate solutions were  18 prepared as described (129), by dissolving oleic acid in hot (95°C) 100 mM NaOH to a 100 mM stock solution, and diluted by addition to pre-warmed Ringer’s solution containing 0.1% essentially fatty acid free BSA in a 6:1 molar ratio. For imaging experiments, the final concentration of palmitate, oleate, and 2-bromopalmtiate was 100 "M unless otherwise shown. Vehicle controls contained BSA and NaOH, and did not induce cell death or Ca2+ fluxes (data not shown). Table 1. List of antibodies used Antibody (Ab) 1°Ab dilution Phospho-PERK  1:500, 5% BSA  Incubation 2°Ab source, dilution 48 hours Rabbit, 1:1000  Company  Phospho-eIF2α  1:500, 5% BSA  48 hours  Rabbit, 1:1000  Cell Signalling  eIF2α XBP-1  1:1000, I-Block 1:200, I-Block  24 hours 24 hours  Mouse, 1:2500 Rabbit, 1:2000  Abcam Santa Cruz  CHOP  1:200, I-Block  24 hours  Rabbit, 1:1000  Santa Cruz  Cleaved caspase 3 Cleaved caspase 9  1:1000, I-Block 1:1000, I-Block  24 hours 24 hours  Rabbit, 1:1000 Rabbit, 1:1000  Cell Signalling Cell Signalling  Cleaved caspase 12  1:1000, I-Block  24 hours  Rabbit, 1:1000  Cell Signalling  β-actin  1:30,000, I-Block 24 hours  Mouse, 1:10,000  Novus  Cell Signalling  MIN6 Cell Culture The mouse insulinoma-derived (MIN6) cell line (95) was cultured as described (36). MIN6 cells were grown to a confluence of 30%-60% for cameleon or fura-2 imaging, and to a confluence of 80-90% for cell treatment and protein extraction. For cameleon imaging, MIN6 cells were transfected with D1ER or D3cpv cameleon DNA one day after seeding on sterile 22 mm coverslips using 1µg Lipofectamine 2000 reagent (Invitrogen, Burlington, ON) per coverslip and 1 µg/uL midi-prepped construct DNA in 50 µL Opti-MEM (Invitrogen, Burlington, ON). Cells were left to take up DNA for 4-6 hours, then fresh  19 DMEM was added and cells were imaged within 2-3 days.  Live-cell Imaging Single-cell imaging was performed in HEPES-buffered Ringer's solution containing 144 mM NaCl, 5.5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 20 mM HEPES, and 3 mM glucose, unless otherwise indicated (64). Cytosolic Ca2+ was imaged in Fura-2-AM-loaded cells as described previously (64, 80), or in D3cpv-expressing cells (106). ER luminal Ca2+ was imaged using the FRET-based D1ER cameleon (105, 106). Pre-heated solutions were applied by stable perifusion at 1 ml/minute and complete solution changes were achieved in <30 seconds. Images were taken using a 10x objective, unless otherwise indicated, with a Zeiss 200m inverted microscope, a Sutter Lambda DG4 excitation source, a Roper CoolSnap HQ2 camera and Slidebook Software (Intelligent Imaging Innovations, Denver, CO). The CFP component of the cameleons was excited at 430 nm using a S430/25x filter (Chroma). CFP emission and YFP (FRET) emission were alternately collected at 470 and 535 nm respectively, using S470/30m and S535/30m filters mounted in a Sutter Lambda 102 emission filter wheel. The FRET signal was normalized to the CFP emission intensity, and changes in ER or cytosolic Ca2+ were expressed as the FRET/CFP ratio. There was no correlation between the apparent Ca2+ levels and the intensity of the FRET probes (D3cpv and D1ER) in the cells used for this study (data not shown). The analysis of D1ERmeasured ER Ca2+ responses was performed on a cell-by-cell basis. A response was considered to have occurred if: 1) there was an inflection of the calcium signal within the treatment period that deviated from baseline by >15%, and 2) this inflection was reversed after the treatment was removed.  20  Protein Extraction and Western Blotting MIN6 cells were grown on 6-well plates (Thermo Fischer Scientific; Rochester, NY) to a confluency of 80-90%, then lysed using Cell Signalling Lysis Buffer (Beverly, MA) for CHOP, XBP-1, cleaved caspase 3, 9, and 12 or Cell Signalling Lysis Buffer supplemented with phosphatase inhibitors (50 mM sodium fluoride, 10 mM sodium pyrophosphate decahydrate,  and  10  mM  β-glycerophosphate)  for  phosphorylated  PERK  and  phosphorylated eIF2α. The cells were then sonicated for 30 seconds and cleared by centrifugation at 10,000 rpm for 5 to 15 minutes. Protein concentrations were determined via BCA assay (Pierce; Rockford, IL) and samples were loaded onto SDS-PAGE gels. Separated proteins were transferred onto PVDF membranes via a semi-dry transfer apparatus and blocked using I-Block (Tropix; Bedford, MA), containing 0.1% Tween-20. All primary antibody incubations were done overnight at 4°C except for rabbit monoclonal phosphorylated PERK and phosphorylated eIF2α primary antibodies (Cell Signalling; Boston, MA) which were incubated over 48 hours at 4°C. Secondary antibodies were incubated at room temperature for one hour in I-Block containing 0.1% Tween-20.  Data Analysis All studies were replicated at least 3 times. Results are presented as means plus or minus standard error of the means. Statistical analysis was performed as indicated in the text. Unpaired, two-tailed t-tests and one-way ANOVA with Tukey’s post-hoc analyses were used where appropriate. A p value of <0.05 was considered significant.  21  Chapter Three: Results  Acute effects of palmitate on cytosolic Ca2+ Palmitate is known to stimulate Ca2+ signals in some β-cell models (118, 129), but these Ca2+ signals have not been fully characterized. Previous data using human islet β-cells indicated that robust and complex cytosolic Ca2+ signals occurred in response to 100 µM palmitate (53). To further characterize the mechanisms of these responses we employed the MIN6 β-cell line, which is homogenous, readily available, and can be easily transfected with cameleon DNA. Many reports use different concentrations of palmitate, so a dose-response experiment was performed using MIN6 cells loaded with the cytosolic Fura-2-AM dye. Example traces of each dose, ranging from 25 µM to 500 µM palmitate, are shown (Fig. 3). Palmitate was coupled to BSA in a 6:1 ratio in all experiments (90), which is a physiological ratio of palmitate:BSA in the obese state (117). Normal plasma free fatty acid levels, which are lower in lean individuals, exist closer to a 2:1 free fatty acid:BSA ratio (24). The average cytosolic Ca2+ response was analyzed over this concentration range (Fig. 3), indicating no significant differences between the concentrations. These experiments demonstrate that palmitate induces a significant number and amplitude of responses at the lowest concentration tested.  22  Figure 3. Palmitate Ca2+ dose-response in MIN6 cells. (A-E) Representative traces of all cells from different days are shown. Cytosolic Ca2+ concentration was measured using Fura-2-AM dye. Each does represents n=3-4. The total number of cells measured for each concentration was 44 (25 µM), 66 (50 µM), 56 (100 µM), 90 (250 µM), and 62 (500 µM). All solutions were made with a 6:1 ratio of palmitate: BSA, and the baseline solution contained 3 mM glucose and 0.1% BSA. (F) Quantification of the dose-response. The response was analyzed by the average amplitude of the Fura-2 Ca2+ signal (left axis), and the percent of cells responsive to palmitate (right axis).  23  D3cpv cameleon captures acute cytosolic Ca2+ signals induced by palmitate In addition to its ability to acutely stimulate β-cells, palmitate has potent long-term effects on β-cell function and survival. To determine the effects of sustained palmitate exposure on β-cell Ca2+ homeostasis, the recently developed FRET-based cytosolic Ca2+ indicator, D3cpv, was employed to follow cytosolic Ca2+ patterns under extended palmitate treatment. This genetically-encoded cameleon allowed for long-term Ca2+ imaging experiments, in contrast to Fura-2 dye, which performs poorly over long periods of time (>1 hour) due to dye leakage, dye compartmentalization into organelles, and photo-bleaching. First, the relative sensitivity for measuring palmitate-induced signals between the D3cpv cameleon and Fura-2-AM dye was determined. As expected, the D3cpv FRET probe recorded the characteristic cytosolic Ca2+ signals evoked by acute exposures to 20 mM glucose, as well as 100 µM palmitate and 25 mM KCl in MIN6 cells (Fig. 4). Together, these experiments indicated that D3cpv is an effective Ca2+ probe for β-cells and that it accurately captured the palmitate-induced Ca2+ signals that have been previously characterized using other methods.  Mechanisms of palmitate-induced Ca2+ signals To identify the sources of the palmitate-induced cytosolic Ca2+ signals, β-cells were perifused with Ca2+-free buffer to test whether extracellular Ca2+ was required. In MIN6 βcells, palmitate caused a transient Ca2+ signal but not a sustained Ca2+ elevation in the absence of extracellular Ca2+ (Fig. 5), similar to data obtained using human β-cells (53). Additionally, removing extracellular Ca2+ inhibited palmitate-induced Ca2+ signals that had already been initiated (Fig. 5). These results demonstrate that palmitate can initiate Ca2+  24  Figure 4. FRET-based cytosolic Ca2+ imaging of palmitate signalling. (A) The cytoplasmtargeted FRET cameleon D3cpv. Nuclei are counterstained with Hoechst 33342 dye. Scale bar is 5 µm. (B) MIN6 cells expressing D3cpv exposed to 20 mM glucose (n=13 cells). Ca2+ concentration is shown as a ratio between FRET emission and CFP fluorescence of the cytosol-targeted construct. (C) MIN6 cells expressing D3cpv sequentially exposed to 100 µM palmitate and 25 mM KCl (n=21 cells).  signals in the absence of extracellular Ca2+, but that Ca2+ influx from the extracellular space is required for continued propagation of palmitate-induced Ca2+ signals. Importantly, these findings implicate intracellular Ca2+ stores as a component of fatty acid-induced Ca2+ signalling, in agreement with studies using other !-cell models (118, 129).  25  Figure 5. Mechanisms of palmitate-induced cytosolic Ca2+ signals in MIN6 cells. (A) Effects of pre-treatment with extracellular Ca2+ free solution on palmitate-induced Ca2+ signals in MIN6 cells. (B) Removal of extracellular Ca2+ during the response to palmitate blocked the sustained oscillations in MIN6 cells. Representative traces are shown (A, n=12 cells; B, n=21 cells).  The long-chain free fatty acid receptor, GPR40, can be activated by palmitate and has been suggested to generate intracellular Ca2+ signals through PLC-mediated IP3 generation in β-cells (15, 60). To determine the extent to which IP3 receptors are involved in this intracellular Ca2+ store release, cells were treated with palmitate in the presence of xestospongin C, a drug that blocks IP3 receptors (IP3R) (66). MIN6 cells were pre-treated with 1 µM xestospongin C for 10 or 20 minutes and the area under the curve of the initial (first phase) and sustained (second phase) of the palmitate-induced Ca2+ signals was quantified (Fig. 6). The first minute of response was expected to involve intracellular stores, and xestospongin C tended to reduce the 1st phase of the palmitate-induced Ca2+ signals. However, this difference did not reach statistical significance despite a large number of cells  26 studied. This result suggests that the IP3R may not play a dominant role in palmitate-induced intracellular store Ca2+ release, or that the dose of drug, duration of treatment, was too low to fully inhibit signal transduction.  Figure 6. Blocking IP3R does not block palmitate-induced cytosolic Ca2+ signals. (A) Effects of 10 or 20 minute pre-treatment of 1 µM Xestospongin C on palmitate-induced cytosolic Ca2+ signals in Fura-2-AM loaded MIN6 cells. The first and second phases of the Ca2+ response are indicated. (B) AUC quantification of the first and second phases of the Ca2+ response. n=43 cells (palmitate alone), 42 cells (10 minute Xestospongin C treatment), 50 cells (20 minute Xestospongin C treatment).  Acute palmitate treatment causes a decrease in ER Ca2+ The ER is thought to be the main dynamic intracellular Ca2+ storage compartment in βcells (39). To dynamically measure Ca2+ levels in the ER lumen of MIN6 cells, the recently developed targeted cameleon probe was used (105, 106). This genetically engineered indicator has Ca2+-responsive elements between an enhanced cyan fluorescent protein (eCFP) and the citrine yellow fluorescent protein variant (Fig. 2). FRET between these fluorescent proteins changes upon Ca2+ binding to the probe (105). Acute palmitate treatment caused a reversible decrease in ER Ca2+, in a subset (31.3 ± 4.7%) MIN6 cells (Fig. 7; 114 responsive cells of 476 cells measured in total). These Ca2+ signals were also repeatable within individual cells (Fig. 7). To determine the extent of this depletion, the  27 decrease in ER Ca2+ caused by palmitate was compared to that caused by thapsigargin (1 µM), a drug that directly inhibits the SERCA Ca2+ uptake pumps on the ER. In the palmitate-responsive MIN6 cell subset, the fatty acid depleted ER Ca2+ to a level that was 45 ± 3.7% of that caused by thapsigargin, which affected all cells (Fig. 7). In a limited number of preliminary experiments, blocking IP3R with 1 µM xestospongin C did not block palmitate-induced ER Ca2+ release in MIN6 cells (Fig. 8), suggesting that IP3 generation is not necessary for the palmitate response. Further, treatment of MIN6 cells with the nonmetabolizable palmitate analogue 2-bromopalmitate did not consistently stimulate a robust release of ER Ca2+ (Fig. 9; n = 83 cells), suggesting that palmitate metabolism is required for this effect. Together, these data provide a direct demonstration that ER intracellular Ca2+ store mobilization plays a role in palmitate-induced Ca2+ signalling.  Effects of oleate on ER Ca2+ dynamics The monounsaturated fat oleate has been shown to affect β-cells in a number of studies, although it is generally considered to be less toxic (31, 38, 49, 68, 85, 86). Oleate is thought to activate GPR40 in β-cells (46, 129). In D1ER-transfected MIN6 cells, oleate caused a decrease in ER luminal Ca2+ that was similar to that of palmitate (Fig. 10). There was a trend that a higher percentage of cells responded to oleate (45± 8% for oleate, 25±5% for palmitate; not statistically significant; Fig. 10). This may be a dose-dependent effect, as the free concentration of oleate in equilibrium with its BSA-bound form is twice that of palmitate (120). Alternatively, if this signal is initiated by GPR40 activation, oleate has been shown to have a higher affinity for GPR40 than palmitate (60). In experiments where these fatty acids were added sequentially, there was no difference in response to either fat after  28 treatment with the other (Fig. 11). Together, these results indicate that palmitate does not block the cell’s ability to respond to oleate, and that oleate also does not seem to impair the ability of palmitate to signal  . Figure 7. Direct imaging of ER luminal Ca2+ in palmitate-treated MIN6 cells. (A) The endoplasmic reticulum-targeted Ca2+ sensor D1ER. The nuclear dye, Hoechst 33342, was added to live cells for contrast. Scale bar is 5 µm. (B) Live cell imaging with D1ER revealed that 100 µM palmitate acutely and reversibly decreased ER Ca2+. The trace is representative of 12 responding cells out of 66 total cells imaged. (C,D) Comparison of palmitate-induced ER Ca2+ release to 1 µM thapsigargin. (C) Representative average trace of cells treated with 100 µM palmitate or vehicle control then stimulated with 1 µM thapsigargin. (D) Quantification of the decrease in ER Ca2+ induced by palmitate compared to that of thapsigargin. n=3 experiments done on different days; palmitate cells = 20-23, and control cells = 54-68 each experiment. The baseline solution for all experiments contained 3 mM glucose. * denotes significance over control (p<0.05).  29  Figure 8. Blocking IP3R does not block palmitate-induced ER Ca2+ release. MIN6 cells transfected with D1ER were pre-treated with 1 µM Xestospongin C and then exposed to 100 µM palmitate. Representative traces from three cells are shown. 16 cells were imaged in total, with 10 cells responding to palmitate in the presence of the drug.  Figure 9. 2-bromopalmitate does not initiate ER Ca2+ release. MIN6 cells transfected with D1ER were exposed to 100 µM 2-bromopalmitate followed by 100 µM palmitate and 100 µM carbachol. The average trace of 10 palmitate-responding cells is shown. 88 cells were imaged in total and no cell responded to 2-bromopalmitate. The baseline solution contained 3 mM glucose and 0.1% BSA.  Long-term analysis of Ca2+ signals using cameleons A major advantage of the fluorescent protein-based Ca2+ indicators is their utility in long-term experiments. In this study, experiments were done examining cytosolic and ER Ca2+ responses to palmitate in MIN6 cells over multiple hours. Interestingly, palmitate-  30  Figure 10. Oleate mobilizes ER Ca2+ stores. (A) Treatment of D1ER-transfected MIN6 cells with 100mM oleate (6:1 BSA) evoked a similar response in ER calcium release to that caused by palmitate. Shown is a representative trace of 35 cells out of 125 cells imaged. (B) Quantification of the overall fatty acid responses in D1ER-transfected MIN6 cells (oleate compared to experiments done with palmitate).  induced cytosolic Ca2+ oscillations were sustained and did not appear to diminish appreciably with time in a large proportion of cells (Fig. 12). This indicates that in the continuous presence of palmitate, the fatty acid generates signals using components that do not desensitize within the time frame of these studies. Additionally, there were distinct groups of cellular responses to palmitate. About half of the cells responded immediately, and ~75% of these exhibited sustained cytosolic Ca2+ oscillations (Fig. 12). The other half of cells did not begin responding with sustained cytosolic Ca2+ oscillations until they had been exposed to palmitate for an average of ~50 minutes. No signals were observed under baseline conditions (3 mM glucose with vehicle control) (Fig. 12). Additionally, ER Ca2+ depletion upon long-term palmitate stimulation was also observed (Fig. 12). Typically, palmitate induced release of ER luminal Ca2+ that included a gradual and oscillatory refilling of the organelle (Fig. 12). Furthermore, these cells remained responsive to 100 µM carbachol, an IP3-generating agonist, after 100 minutes of palmitate treatment. Together,  31 these results demonstrated that SERCA pumps were still active and the ER Ca2+ store was not completely and permanently depleted in the prolonged presence of palmitate.  Figure 11. Oleate does not block palmitate’s ability to mobilize ER Ca2+ stores. (A, B) When one fatty acid was applied first, there was no different between the response rate compared to the other fat. (A) Treatment of D1ER-transfected cells with 100 µM palmitate followed by 100 µM oleate (n= 22 out of 86 cells), and the converse (B) (n=15 out of 99 cells) revealed similar signals evoked by the two fatty acids that did not cause a block of the signal when applied first. 100 µM carbachol is used as a positive control for cells with responsive IP3R activation. Representative traces are shown, n=3-4 of different days of experimentation. (C) Quantification of overall responses revealed no difference in fatty acid responses.  Effect of chronic treatment of palmitate on Ca2+ dynamics In previous studies of β-cells chronically treated with palmitate, indirect measurements of Ca2+ store loading yielded conflicting results (26, 68). To directly test the hypothesis that chronic palmitate treatment alters ER Ca2+, ER Ca2+ was measured in D1ER-  32  Figure 12. Long-term imaging of palmitate-induced Ca2+ dynamics. (A) Average of vehiclecontrol treated D3cpv-expressing MIN6 cells; all cells were KCl-responsive (n=11 cells). (B) Heterogeneous, oscillatory cytosolic Ca2+ responses to palmitate with long-term exposure. Three classes of responses were observed (transient, continuous oscillations, delayed oscillations) in D3cpv-expressing MIN6 cells treated with 100 µM palmitate. Representative traces are shown (n= 44 cells). (C) Long-term imaging of ER Ca2+ depletion in response to 100 µM palmitate and 100 µM carbachol in D1ER-expressing MIN6 cells. A representative trace is shown (n=57 cells). Nonresponding cells are shown in (B,C) to demonstrate the typical photobleaching-associated signal loss.  33 transfected MIN6 cells exposed to palmitate for 24 hours in low glucose conditions (5 mM). Baseline ER Ca2+ levels were significantly lower in the palmitate-treated cells compared to the vehicle control-treated cells (Fig. 13). The amount of ER Ca2+ that could be released upon thapsigargin treatment was not significantly different, suggesting that chronic palmitate exposure did not affect the thapsigargin-sensitive pool of ER Ca2+. These results indicate that chronically high levels of palmitate may affect the ER Ca2+ ‘set-point’ and contribute to deregulation of β-cell Ca2+ homeostasis.  Figure 13. Effect of chronic palmitate treatment on ER Ca2+ dynamics. MIN6 cells transfected with D1ER were treated with 1500 µM palmitate (6:1 BSA) or vehicle control in 5mM glucose for 22-25 hours and ER Ca2+ was measured, with the addition of 1 µM thapsigarin. The trace shown is an average of 3 coverslips each of palmitate-treated cells (111) and control cells (77).  Characteristics of ER stress and caspase activation in β-cells Changes in ER Ca2+ levels can lead to chaperone dysfunction, an increase in misfolded proteins, and subsequent initiation of the ER stress response (130). Using pharmacological agents that induce ER Ca2+ depletion, the time-course of signalling events that mediate ER stress was analyzed. Thapsigargin (1 µM) and carbachol (100 µM) together  34 resulted in a rapid and homogeneous depletion of the ER Ca2+ stores, compared to either drug alone (81). The pro-apoptotic transcription factor CHOP and the death effector Capsase 3 were both activated upon chronic thapsigargin treatment (81). To elucidate the signals that may link ER Ca2+ depletion to downstream ER stress and apoptosis signals, the PERK/eIF2α arm of the ER stress pathway was characterized. PERK was rapidly phosphorylated within 5 minutes of thapsigarin treatment (Fig. 14), and this subsequently lead to eIF2α phosphorylation. Both PERK and eIF2α remained activated for the duration of the treatment, up to 4 hours (Fig. 13). This effect was not exacerbated by the additional treatment with carbachol. These kinetics implicate that rapid perturbations of ER Ca2+ homeostasis can lead to an immediate and sustained ER stress response, which under some circumstances can then lead to caspase 3 activation and apoptosis (81). To further characterize the effect of ER Ca2+ perturbation on apoptosis signalling, the activation of caspases 7 and 9 was examined. Similar to what was seen with capsase 3 cleavage (81), both caspases 7 and 9 were strongly up-regulated after chronic thapsigargin treatment (Fig. 15). Treatment with drugs that block ER Ca2+ release from IP3R (xestospongin C) and RyR (ryanodine) channels did not significantly allay the apoptosis signals in these experiments (Fig. 15). Taken together, these results demonstrate that chronic disruption of ER Ca2+ levels leads to a sustained ER stress response, which then causes βcell death.  Effects of palmitate on ER stress Sustained elevations in intracellular Ca2+ are known to predispose β-cells to programmed cell death. Moreover, extended periods of lowered ER Ca2+ also promote the  35 induction of ER-stress and cell death (81). Given the rapid effects of palmitate on luminal  Figure 14. Effects of IP3R activation on UPR activation evoked by SERCA inhibition. (A) Phosphorylation of PERK and eIF2" in 25 mM glucose cultured MIN6 cells was examined at the time points indicated. (B) Quantification of western blots for PERK and eIF2" phosphorylation. (Similar results from experiments conducted in 5 mM and 25 mM glucose were pooled; n=6). * denotes significance over control (p<0.05). Cch, carbachol; Tg, thapsigargin.  ER Ca2+, palmitate may also activate the components of the ER-stress response rapidly. Indeed, in MIN6 cells treated with palmitate, in the presence of 5 mM glucose, there was a significant increase in the phosphorylation of luminal ER Ca2+ sensor PERK, after just 5 minutes (Fig. 16). The other arm of the ER stress signalling cascade is initiated by activation of IRE1, and results in the splicing and activation of the transcription factor XBP-1 (130). Extended palmitate treatment tended to cause a increase in the activated, spliced (54kD)  36  Figure 15. Blocking ER Ca2+ release does not attenuate thapsigarin-induced caspase actiavtion. (A) Caspase 7 and caspase 9 activation in MIN6 cells upon treatment with carbachol (Cch), thapsigargin (Tg), ryanodine (Ry) or Xestospongin C (X) at the time-points indicated. (B,C). Quantifications of the western blots for caspase cleavage.  form of XBP-1 (77) (Fig. 17), which also coincided with the activation of the pro-apoptotic transcription factor CHOP (53), consistent with previous reports (23, 68, 70, 77). This also correlated with the kinetics of palmitate-induced propidium iodide incorporation, an indicator of β-cell death (53). Blocking the release of ER IP3R- and RyR-gated Ca2+ stores using xestospongin C and ryanodine concomitantly with palmitate treatment did not decrease the activation of XBP-1 (Fig. 17). Blocking extracellular Ca2+ influx through Ltype Ca2+ channels with nifedipine also failed to block palmitate-induced XBP-1 activation  37 at 12 hours, but did mitigate palmitate’s effect at 24 hours of treatment (Fig. 17). Together, these results indicate that palmitate treatment in low glucose conditions does indeed initiate a sustained ER stress response mediated in part by ER Ca2+ store mobilization. However, this modification in Ca2+ homeostasis alone is not likely to be sufficient to maintain ERstress or initiate β-cell apoptosis.  Figure 16. Palmitate induces acute PERK phosphorylation. (A) Levels of phospo-PERK after short (5 minutes) treatment with 1500 µM palmitate (6:1 palmitate to BSA), after 6 hours of culture in low (5 mM) glucose; n=5. * denotes significance over control (p<0.05).  38  Figure 17. Blocking ER Ca2+ release does not inhibit palmitate-induced XBP-1 splicing. (A) Spliced XBP-1 protein levels after 24-48 hours of treatment with 1500 µM palmitate (6:1 palmitate to BSA) in low glucose (5 mM), n=5-6 with the indicated drugs. (B) Quantification, analyzed using a one-way ANOVA and Tukey’s post-hoc test. * denotes significance over vehicle control (p<0.05).  39  Chapter Four: Discussion The objective of this project was to elucidate the effects of palmitate on pancreatic β-cell Ca2+ homeostasis. The study has three main findings. First, palmitate caused cytosolic Ca2+ signals in MIN6 cells, in agreement with previous findings in other β-cell lines, primary mouse islets, and human β-cells (53, 118, 129). Second, these Ca2+ signals were generated by a combination of extracellular Ca2+ influx and Ca2+ release from intracellular stores: both palmitate and oleate mobilized Ca2+ release from the ER lumen. Third, both pharmacological and palmitate-induced Ca2+ depletion induced the ER-stress response with rapid kinetics. Together, these findings contribute to the understanding of the mechanisms by which the saturated fatty acid palmitate alters β-cell function.  Palmitate-induced cytosolic Ca2+ signals The nature of the palmitate-induced Ca2+ signals in MIN6 cells was characterized in this study. The Ca2+ responses were qualitatively and quantitatively similar between MIN6 βcells and human β-cells (53), indicating that the MIN6 cell line may be a suitable model for studying some aspects of β-cell Ca2+ homeostasis. A recent study also described the similarities between MIN6 cells and human β-cells in terms of their susceptibility to apoptosis in response to palmitate (74), but no study has directly compared the two cell types with respect to their palmitate-induced Ca2+ responses. In MIN6 cells, even low doses of palmitate (e.g. 25 µM) effectively stimulated cytosolic Ca2+ signals. This low dose nevertheless reflects a free (unbound) nanomolar amount of palmitate (120), which is within the normal physiological range for plasma free fatty acids (135). Indeed, experiments done  40 using an increasing amount of BSA (> 0.1%) demonstrated an inhibition of FFA-mediated signal at high BSA levels, indicating that it is the unbound form of the fatty acid that is the active signalling molecule (46, 60). There have been a wide range of palmitate concentrations used to model lipotoxicty, and indeed, increasing palmitate concentration increases levels of cell death (53, 85). However, in one study a sub-toxic concentration of palmitate did not induce death but did have effects on mitochondrial membrane potential in MIN6 cells (72). This indicates that even low doses of palmitate, which may be the more relevant (and healthy) physiological levels of the fatty acid, induce signals that may be involved in our observed cytosolic Ca2+ signals. It may also be interesting to note that a study characterizing oleate signalling found that oleate induced cytosolic Ca2+ signals at concentrations as low as 3 µM (46).  Long-term palmitate-induced cytosolic Ca2+ signals The present study provides the first long-term (> 1 hour) measurements of β-cell calcium homeostasis. We compared the acute palmitate responses measured using D3cpv to those established using Fura-2-AM dye (53, 118, 129). The similarities between the signals validate the D3cpv probe as a reliable method for measuring β-cell Ca2+, and justify its use in long-term experiments. Using the D3cpv and D1ER probes over a 3-hour time period demonstrated that palmitate generates sustained, oscillatory Ca2+ signals. It was interesting to find that a nutrient other than glucose can generate sustained cytosolic Ca2+ responses. In β-cells, one theory posits that the enzyme phosphofructokinase (PFK) drives oscillatory Ca2+ signals in response to glucose metabolism, which in turn drive pulsatile insulin release (37, 100, 154). In contrast, a recent report found that multiple glycolysis-independent metabolites can also generate β-cell Ca2+ oscillations, suggesting that PFK, and glycolysis itself, is not  41 necessary for this oscillatory behavior in β-cells (56). The experiments in our study were done using a sub-stimulatory glucose concentration (3 mM), and no Ca2+ signals were observed in these baseline conditions, indicating that palmitate is solely driving the observed Ca2+ response. Additionally, because treatment with 2-bromopalmitate, a non-metabolizable analog of palmitate, does not generate cytosolic Ca2+ signals like that of palmitate (53), these sustained Ca2+ signals may be due to direct palmitate metabolism, perhaps driven by β– oxidation-derived metabolites or by downstream LC-CoA signalling.  Mechanisms behind palmitate-induced ER Ca2+ signals This work sheds new light on the mechanisms underlying the palmitate-induced Ca2+ signals. Although some investigators have reported that fatty acids do not reduce thapsigargin-mobilizable Ca2+ stores (46, 68), other reports suggested the possible role of both extracellular Ca2+ influx and intracellular store release (20, 118). Our data, using MIN6 cells, support the latter model. Importantly, for the first time palmitate-induced ER depletion was directly imaged using the D1ER cameleon probe. Previous studies have suggested a role for GPR40 in driving the observed cytosolic Ca2+ signals (46, 60, 129). GPR40, a G-proteincoupled receptor (60), would be expected to mobilize ER Ca2+ stores via Gq trimeric Gproteins (15, 60), phospholipase C (46), and IP3R-gated ER Ca2+ release. Indeed, pharmacological small-molecule agonists of GPR40 were found to induce IP3 formation in GPR40-expressing HEK293 cells (141), and inhibition of Gqα blocked palmitate potentiation of glucose-stimulated insulin release in mouse islets (76). However, another report noted that palmitate treatment did not result in an increase in IP3 content in rat islets (2). In our experiments, we were not able to define a major role for IP3 receptors using pharmacological inhibition. This may be due to the dose or treatment protocol, or due to the fact that a  42 majority of the cells (~70%) would not have an ER Ca2+ response, but yet may have rapid cytosolic Ca2+ responses, thus masking the inhibitory effect. Nevertheless, the result leaves open the possibility that palmitate may act on ryanodine receptors (RyR) or the SERCA pumps in mobilizing ER Ca2+. Indeed, previous studies in different model systems, including pancreatic acinar cells, demonstrated that palmitoyl-CoA, the esterified metabolite of palmitate, could activate RyR channels (19, 43, 47, 48), while another study in the HIT βcell line suggested an effect of palmitoyl-CoA on SERCA activity (29). Additionally, a previous study outlined the requirement for calpain-10 in palmitate-induced β-cell apoptosis, which is also involved in RyR2-activated apoptosis (65), suggesting a possible role for RyR2 in palmitate’s lipotoxic action on β-cells. To further elucidate the mechanism of palmitate-induced Ca2+ signals, experiments were carried out using the non-metabolizable palmitate analog 2-bromopalmitate. This molecule is esterified to 2-bromopalmtiate-CoA but cannot undergo !-oxidation. It also prevents other fatty acids from doing so by blocking carnitine palmitoyl transferase-1, which transports long-chain free fatty acids into the mitochondria (119). Treatment of human islets and MIN6 cells with 2-bromopalmitate caused a significantly attenuated Ca2+ signal that, nevertheless, was not fully abolished (53). However, we did not detect a decrease in ER luminal Ca2+ upon treatment with 2-bromopalmitate in MIN6 cells. This suggests that 2-bromopalmitate cannot mobilize ER Ca2+ stores, possibly indicative of its inability to activate GPR40 or other Ca2+ channels. Indeed, the effects of 2-bromopalmitate on GPR40 activation remain controversial (107, 127). The possibility remains that the esterified metabolite of 2bromopalmitate may partially activate Ca2+ channels to create the partial cytosolic response (53). A previous report found that concurrent treatment of palmitate with 2-bromopalmtiate  43 abolished palmitate-induced Ca2+ signals (118), perhaps by blocking a receptor or channel or by preventing the action of downstream palmitate metabolites. Another report noted that treatment of mouse islets with palmitoyl-CoA did not mimic palmitate’s actions on islet capacitance and Ca2+ currents (101), although Ca2+ signals were not directly measured. Together, this set of observations indicates that the non-metabolizable analog of palmitate does not replicate the actions of palmitate on β-cell ER Ca2+, suggesting that native palmitate, with its free acidic group, is required for these signals.  Long-term palmitate-induced ER Ca2+ depletion Long-term experiments also indicated that the palmitate-induced ER Ca2+ depletion is transient and tends to have an oscillatory re-filling nature. Whether this oscillatory ER behavior occurs concurrently with the observed cytosolic oscillations is unclear, although a previous report demonstrated that ATP-mediated IP3 production generated ER Ca2+ oscillations that were anti-correlated with their corresponding cytosolic Ca2+ oscillations (105). Nevertheless, after the 3-hour palmitate treatment, stimulation by the IP3-generating agonist carbachol in the presence of palmitate indicated that SERCA pumps were active and able to refill the ER Ca2+ store. Experiments comparing SERCA activity with and without palmitate were not performed, leaving open the possibility that palmitate may affect SERCA activity kinetics, but our results do indicate that even under longer-term stimulated conditions, palmitate does not fully inhibit SERCA activity. In contrast, under chronic palmitate treatment in low glucose, the basal ER Ca2+ level was affected and was indeed lower compared to control-treated cells. This could reflect the action of chronically elevated palmitate at the level of SERCA activity or expression, similar to the effect of cytokines (18), or be a direct effect of palmitate-induced chronic ER Ca2+ depletion. In agreement,  44 chronic palmitate (56) or palmitoyl-CoA (29) treatment has been reported to affect subsequent oscillatory cytosolic Ca2+ behavior in response to glucose and other stimulants, indicating the mechanism behind this effect may be due to palmitate’s alteration of ER Ca2+ levels.  Oleate-stimulated ER Ca2+ signals The monounsaturated fat oleate also transiently mobilized ER Ca2+, in agreement with previous studies showing effects of this fatty acid on !-cell calcium homeostasis (46, 129). Oleate treatment did not block the ability of subsequent palmitate to induce ER Ca2+ release, suggesting that the interaction of each fatty acid with its target may be transient. In agreement, a study in rat α-cells, which also express GPR40 (44), demonstrated that blocking ER stores abolished oleate-induced cytosolic Ca2+ signals (45). Evidence for a GPR40-mediated mechanism is given through RNAi-mediated GPR40 knockdown studies in rat islets (46) and INS-1 cells (129): the knockdown abolished oleate-stimulated Ca2+ signals. However, oleate-stimulated cytosolic Ca2+ signals were also demonstrated in cells expressing L-type calcium channels but not GPR40 (145). Palmitate’s effects on L-type calcium channels in mouse islets have also been documented (101), suggesting that palmitate may act through these channels as well. These previous studies, in concert with the present one, suggest that fatty acids as well as their metabolites may interact with more than one Ca2+ signalling component. Importantly, these studies together with ours suggest that the observed protection offered by oleate against palmitate lipotoxicity (85, 86) does not involve acute ER Ca2+ store mobilization. A similar pattern of oleate’s acute vs. chronic effects was shown in a recent study looking at mitochondrial permeability transition (MPT) in MIN6 cells. Acute treatments  45 with palmitate and oleate both induced similar MPT, but long-term oleate treatment did not induce MPT to the extent of long-term palmitate treatment, and prevented palmitate’s induction of MPT when the two fatty acids were co-administered (72). Mitochondrial dysfunction has been documented in lipotoxicity (87), possibly due to the dysregulation in ER integrity, and this study parallels ours in demonstrating oleate’s acute, but less chronically toxic, effect.  Glucose-dependence of palmitate Ca2+ signals In the present study, we observed substantial heterogeneity in Ca2+ signals induced by palmitate. This is similar to the well-described response heterogeneity seen with glucose (71, 148, 149, 151) and other agonists (66). Notably, we observed only a subset of MIN6 cells (~30%) that responded to palmitate treatment by a reduction in ER Ca2+. It is possible that a higher percentage of MIN6 cells might have responded to palmitate treatment at increased glucose levels. Previous reports have also suggested a glucose-dependence of fatty acid-induced Ca2+ signals in mouse (118) and rat !-cells (46). Remizov et al (118) suggested that this may be related to increased mitochondrial activity and inhibition of CPT-1 at stimulatory glucose levels, creating a backlog of cytosolic esterified fatty acids which might then become available for signalling. Nevertheless, we observed both cytosolic Ca2+ signals and ER Ca2+ store mobilization at baseline glucose levels in response to palmitate, providing strong evidence that elevated glucose is not essential for the formation of at least some of these signals.  46  Kinetics of ER stress and death in β-cells As dedicated high-capacity secretory cells, pancreatic β-cells are particularly susceptible to ER-stress (89). In this study, the kinetics of one branch of the UPR signalling pathway were characterized in response to pharmacological induction of ER stress. The PERKdependent ER stress cascade, which controls translational attenuation and regulates the proapoptotic transcription factor CHOP, has been proposed to increase apoptosis when chronically sustained, in contrast to IRE1, which has recently been shown to increase proliferation (79). Chronic ER Ca2+ depletion, such as that caused by the SERCA inhibitor thapsigargin, caused CHOP induction and apoptosis in MIN6 cells within 24 hours of treatment (81). ER Ca2+ release via RyR or IP3R further participated in this thapsigargininduced death (81). Previous work has indicated that the ER transmembrane protein PERK is required to initiate Ca2+-dependent ER stress signals (78). The results of the current study indicate that ER Ca2+ depletion indeed activates PERK in a rapid manner, and causes eventual apoptosis involving both caspases 7 and 9. This suggests the kinetics of luminal Ca2+ depletion can dictate the degree of ER stress, in a PERK-dependent manner.  Palmitate and β-cell ER stress Previous studies have documented an effect of palmitate on ER-stress in β-cells (20, 68, 70, 74), and these findings are confirmed here in the context of altered Ca2+ homeostasis. ER Ca2+ levels are critical in the ER stress response (160), and a recent report demonstrated that palmitate-induced CHOP induction was partially mediated by extracellular Ca2+ influx (20). Further, a component of the palmitate-induced β-cell death was shown to be due to the rapid and Ca2+-dependent degradation of CPE (61). The present study adds direct ER Ca2+  47 depletion to the many insults caused by palmitate. The extent of this ER Ca2+ release was less than half of that caused by the ER stress-inducer thapsigargin, in the subset of cells that exhibited the palmitate-induced ER Ca2+ response. However, since high luminal Ca2+ must be maintained for proper protein folding in the ER, the palmitate-induced depletion could be expected to exacerbate ER stress, especially since ER Ca2+ levels are important in proinsulin processing and transport (52). Our study demonstrated rapid PERK phosphorylation after 5 minutes, indicating an immediate response to the palmitate-induced Ca2+ release, similar to that observed with thapsigargin. In parallel, another study observed cytokineinduced SERCA down-regulation that resulted in a decrease in ER Ca2+ levels and subsequent ER stress induction (18). The observation that chronic palmitate treatment decreased basal ER Ca2+ levels is consistent with a recent report (26) and may provide an additional link between ER Ca2+ deregulation and cell survival. IRE1 activation has been implicated in palmitate lipotoxicity (26), but we found that blocking ER Ca2+ release pharmacologically did not ameliorate this pathway of ER stress, which serves to up-regulate ER chaperone proteins. Whether this arm of the ER stress pathway contributes to β-cell lipotoxicity is unclear, given a recent report that indicated an increase in the ER chaperone protein GPR78 did not inhibit palmitate-induced apoptosis (75). Together, the present study adds another layer to the palmitate-ER stress model, and perhaps establishes the link between ER Ca2+ depletion and the onset of ER stress signals by rapid PERK phosphorylation.  Palmitate and lipotoxicity: possible mechanisms It is clear that a modest Ca2+ depletion alone cannot account for β-cell ER stress and cell death. Indeed, chronic palmitate treatment has many deleterious effects on !-cell function  48 (23, 68, 74, 77, 110) and altered Ca2+ homeostasis is an important part of the lipotoxic pathway, but is not sufficient to cause death on its own. For example, blocking extracellular Ca2+ influx protects rat islets and cultured cells from palmitate-induced cell death (20, 62), but blocking IP3R alone did not exert a protective effect (20). Exogenous palmitate may also lead to an accumulation of LC-CoA’s, lipid intermediates that can be cytotoxic when in excess via different mechanisms not involving Ca2+ signals (28, 107, 131). Further, our study has demonstrated that oleate stimulation creates similar ER Ca2+ responses to those of palmitate, and others have shown that this unsaturated fatty acid is indeed less toxic than palmitate (31, 38, 68, 86). Together, these findings indicate that palmitate has multiple deleterious effects on pancreatic !-cells. Results from this study suggest a model whereby chronic palmitate-induced perturbation of ER Ca2+ homeostasis contributes to defects in proinsulin processing (62) and eventually ER stress and apoptosis (Fig. 18). This model can help explain !-cell apoptosis in the context of hyperlipidemia, where peripheral insulin resistance or hyperglycemia put an increased demand on insulin biosynthesis. It is worth noting that all our palmitate experiments were done under low glucose conditions, indicating that the lipotoxic effects of palmitate (PERK phosphorylation, ER Ca2+ alterations) can occur in low glucose in vitro.  Palmitate and insulin secretion Fatty acids can acutely stimulate insulin secretion in several model systems (98). However, in contrast to previous reports in rodent islets and cultured mouse !-cells (2, 28, 152), palmitate was not a robust insulin secretagogue in MIN6 cells (53). Similarly, palmitate treatment stimulated only a modest acute insulin response in perifused human islets, regardless of whether they were examined in high or low glucose (61). Thus, it is  49  Figure 18. Schematic summarizing palmitate’s possible effects on β-cell Ca2+ homeostasis and ER stress. [Ca2+]cyt refers to cytosolic Ca2+ levels while [Ca2+]ER refers to ER Ca2+ levels. Many cellular organelles and other molecules have been left out for clarity.  difficult to confirm studies demonstrating either a palmitate-induced decrease in insulin content or a robust glucose-dependent increase in insulin secretion (107, 153). Together, these data imply that the Ca2+ signals generated by palmitate are only weakly coupled to the insulin secretory apparatus in human β-cells and MIN6 cells (53). Perhaps human islets are more similar to MIN6 cells than to mouse islets in this regard. Whether or not this is due to  50 the spatial location of palmitate-induced Ca2+ signals, in that they may not occur near insulin granule exocytotic machinery, remains to be established.  Conclusions and future directions In conclusion, this project has examined the effects of palmitate on Ca2+ signalling in MIN6 cells, using a combination of conventional and new live-cell imaging approaches. Importantly, Ca2+ release from the β-cell ER in response to palmitate was directly measured using genetically encoded organelle-targeted probes. The kinetics of PERK activation in response to rapid ER Ca2+ depletion were characterized, indicating that fluctuations in ER Ca2+ levels can have immediate effects on the cellular ER stress response. In parallel, we found the rapid depletion of ER Ca2+ caused by palmitate also stimulated a rapid PERK response, and chronic palmitate treatment affected the basal level of ER Ca2+. This indicates that ER Ca2+ levels may contribute to palmitate-induced ER-stress in β-cells. Taken together, these data help define the mechanisms linking hyperlipidemia and β-cell ER stress and eventual failure in type 2 diabetes. The mechanisms behind palmitate’s mobilization of ER Ca2+ stores remain to be established, as well as the function of the Ca2+ signals themselves. 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