Effects of Free Fatty Acids on Insulin and Glucagon Secretion

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1320

– with special emphasis on the role of Free fatty acid receptor 1

HJALTI KRISTINSSON

ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6206 ISBN 978-91-554-9867-2 UPPSALA urn:nbn:se:uu:diva-316991 2017 Dissertation presented at Uppsala University to be publicly examined in B22, BMC, Husargatan 3, Uppsala, Friday, 19 May 2017 at 13:15 for the degree of Doctor of Philosophy (Faculty of Medicine). The examination will be conducted in English. Faculty examiner: Professor Birgitte Holst (University of Copenhagen, Department of Neuroscience and Pharmacology).

Abstract Kristinsson, H. 2017. Effects of Free Fatty Acids on Insulin and Glucagon Secretion. – with special emphasis on the role of Free fatty acid receptor 1. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1320. 54 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-554-9867-2.

Prevalence of type 2 diabetes mellitus (T2DM) is still rising and even so in the juvenile population. Obesity is highly associated with increased risk for developing T2DM. The development has been related to elevated fasting concentrations of the pancreatic islet hormones insulin and glucagon as well as to an increase in plasma lipids that occurs during obesity. Specifically, research has indicated that chronic exposure to high levels of saturated free fatty acids cause dysfunction in islet alpha- and beta-cells. Fatty acids can affect islet cells by various mechanisms one of which is the G-protein coupled receptor FFAR1/GPR40. The role of the receptor in the effects of fatty acids on pancreatic islet-cell function is not clear. The aim of this thesis was to clarify the role of FFAR1 in how fatty acids, and more specifically the long-chain saturated fatty acid palmitate, affect insulin and glucagon secretion. In children and adolescents with obesity elevated fasting levels of insulin and glucagon were positively correlated with lipid parameters. Specifically, plasma triglycerides and free fatty acids were positively correlated with insulin and glucagon at fasting as well as with visceral adipose tissue volume. Elevated glucagon levels at fasting were associated with worsening of glucose tolerance in the same population. In in vitro studies of isolated human islets palmitate stimulated basal insulin and glucagon secretion as well as mitochondrial respiration at fasting glucose levels. The effect was mediated by FFAR1 and fatty acid beta-oxidation. At higher glucose concentrations the receptor was involved in the potentiation of insulin secretion from isolated human islets and insulin-secreting MIN6 cells. Furthermore, we found that the effects of palmitate on hormone secretion were associated with enhanced mitochondrial respiration mediated by FFAR1 Gαq signaling and PKC activity as well as increased intracellular metabolism induced by the fatty acid. When islets were exposed to palmitate for long time periods and in the presence of FFAR1 antagonist, normalized insulin and glucagon secretion during culture and insulin response to glucose after culture were observed. In MIN6 cells chronic palmitate treatment increased mitochondrial uncoupling irrespective of FFAR1 involvement. However, FFAR1 antagonism during palmitate exposure resulted in elevated respiration and reduced apoptosis. In conclusion, children and adolescents with obesity have elevated fasting concentrations of insulin and glucagon that correlate with free fatty acids and fatty acid sources. High glucagon levels are linked to worsening of glucose tolerance in these subjects. In vitro the combination or synergy of FFAR1 activation and intracellular metabolism caused by palmitate is decisive for both the short-term enhancement effects and the negative chronic effects on insulin and glucagon secretion.

Keywords: FFAR1, GPR40, palmitate, lipotoxicity, islets of Langerhans, type 2 diabetes, obesity

Hjalti Kristinsson, Department of Medical Cell Biology, Box 571, Uppsala University, SE-75123 Uppsala, Sweden.

ISSN 1651-6206 ISBN 978-91-554-9867-2 urn:nbn:se:uu:diva-316991 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-316991)

to my Family

List of Papers

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

I Kristinsson, H., Smith, DM., Bergsten, P., Sargsyan, E. (2013) FFAR1 is involved in both the acute and chronic effects of palmitate on insulin secretion. Endocrinology 154:4078-88. II Kristinsson, H., Bergsten, P. & Sargsyan, E. (2015) Free fatty acid receptor 1 (FFAR1/GPR40) signaling affects insulin secretion by enhancing mitochondrial respiration during palmitate exposure. BBA-Mol Cell Res 1853, 3248-3257. III Kristinsson, H., Sargsyan, E., Smith, DM., Göpel, SO., Berg- sten, P. (2017) Basal hypersecretion of glucagon and insulin from palmitate-exposed islets depends on FFAR1 but not decreased somatostatin secretion. Submitted manuscript. IV Manell, H., Kristinsson H., Kullberg, J., Paulmichl, K., Ca- damuro, J., Sargsyan, E., Ahlström, H., Weghuber, D., For- slund, A., Bergsten, P. Hyperglucagonemia is associated with elevated plasma triglycerides and increased visceral fat in children and adolescents. Submitted manuscript.

Reprints were made with permission from the respective publishers.

Contents

Introduction ...... 11 Obesity in humans ...... 11 Obesity-related type 2 diabetes mellitus ...... 12 Metabolism, free fatty acids and pancreatic islet hormone secretion in obese subjects ...... 13 Free fatty acids in cellular function and hormone secretion from pancreatic islets ...... 14 Fatty acid G-protein coupled receptors ...... 15 Free fatty acids and insulin and glucagon secretion ...... 15 FFAR1 (GPR40) and islet function ...... 17 Metabolism and mitochondrial function in islet-cells ...... 18 Aim ...... 20 Methods ...... 21 Cohort study ...... 21 Cohort description ...... 21 Blood sampling and analyses ...... 21 Free fatty acid analysis ...... 22 Oral glucose tolerance test ...... 22 Magnetic resonance imaging ...... 23 Cell studies ...... 23 Cell culture ...... 23 Hormone secretion ...... 25 Studies on metabolism ...... 26 Apoptosis measurements ...... 27 Analysis of results ...... 27 Results and discussion ...... 28 Elevated fasting insulin, glucagon and lipid levels in children and adolescents with obesity ...... 28 Simultaneous palmitate stimulation of insulin and glucagon secretion via FFAR1 ...... 29

FFAR1 stimulation of insulin and glucagon secretion is associated with enhanced mitochondrial respiration which is dependent on Gαq signaling and PKC activation ...... 31 FFAR1 plays a role in the long-term effects of palmitate on hormone secretion ...... 33 Conclusions ...... 36 Sammanfattning på svenska ...... 37 Ágrip á íslensku ...... 39 Acknowledgements ...... 41 References ...... 44

Abbreviations

ANOVA Analysis of variance ATP Adenosine triphosphate BMI Body mass index BSA Bovine serum albumin CoA Coenzyme A CPT1 Carnitine palmitoyl transferase 1 DAG Diacylglycerol DNA Deoxyribonucleic acid ELISA Enzyme linked immunosorbent assay ER Endoplasmic reticulum FFA Free fatty acid FFAR1 Free fatty acid receptor 1 GPCR G-protein coupled receptor GSIS Glucose stimulated insulin secretion IFG Impaired fasting glucose IGT Impaired glucose tolerance IP3 Inositol triphosphate mRNA Messenger RNA mtDNA Mitochondrial DNA NADPH Nicotinamide adenine dinucleotide phosphate NEFA Non-esterified fatty acid OCR Oxygen consumption rate OGTT Oral glucose tolerance test PCR Polymerase chain reaction PDX1 Pancreatic and duodenal homeobox 1 PIP2 Phosphatidylinositol 4,5-bisphosphate PKC Protein kinase C PKD1 Protein kinase D1 PLC Phospholipase C qPCR Quantitative PCR ROS Reactive oxygen species SAT Subcutaneous adipose tissue shRNA Short hairpin ribonucleic acid T2DM Type-2-Diabetes mellitus UCP2 Uncoupling protein 2 VAT Visceral adipose tissue

Introduction

Obesity in humans Obesity develops when energy is stored at a greater rate than expended. Through human history starvation has been a bigger health risk than obesity. Therefore, physiology is programmed and very well equipped to store energy mainly as fat depots. With excessive fat mass there are changes in adipo- cyte size and number [1]. Adipose tissue consists of different compartments that can expand, where the expansion of some are particularly associated with negative health effects [2]. For example, increase in visceral adipose tissue (VAT) is coupled with negative health effects more than subcutaneous adipose tissue [3]. In recent decades the role of adipose tissue has been clari- fied by examining hormones, adipokines or metabolites released by adipocytes, which can mediate messages to other parts of the body about the size and physiological state of adipose tissue [2]. Thus, there is constant signaling and crosstalk between the central nervous system, the intestinal tract, the pancreas and adipose tissue about energy status. The diagnosis or definition of obesity is commonly done by body mass index (BMI). The interval or span of BMI is derived from longitudinal studies that found that certain BMI spans were more correlated with worsening of health parameters [4]. Some of the negative effects of obesity and its duration on health are cardiovascular with coronary artery disease and stroke, hypertension and type 2 diabetes mellitus (T2DM) [5]. The prevalence of childhood obesity has increased during the past decades [6, 7]. Since most children with obesity remain obese as adults the increase translates into a similar rise in adult obesity rates. Obesity developing already at young age implicates longer duration of obesity exposure, which is anticipated to lead to aggravated development of co-morbidities. Obesity in children and adolescents is important to study in order to investigate the etiology and in order to define ways of intervention. Research- ing childhood obesity also provides a unique opportunity to study early obesity and its progression, in most cases in drug-naïve subjects. In contrast, adults with obesity have usually a long exposure period with co-morbidities already developed that make it difficult to distinguish causes from conse- quences of obesity [8, 9].

11 Obesity-related type 2 diabetes mellitus T2DM is caused by insufficient insulin signaling and glucose uptake leading to hyperglycemia (accompanied by hyperosmolarity), which causes dysfunction of metabolism and, if left untreated, tissue damage [10, 11]. While obesity related T2DM is a disease, where glucose control is lost, in the earlier phases of obesity the subjects have in most cases normal glucose tolerance (NGT) but altered lipid metabolism. Obesity has been related to both dysfunction in the pancreatic islets of Langerhans [12], which secrete insulin and glucagon, and other tissues that respond to those hormones, such as adipocytes, hepatocytes and myocytes [13]. In the islets of Langerhans the glucagon-secreting alpha- (20-35% of mass) and insulin-secreting beta-cells (60-80% of mass) are located together with other endocrine cells. The other cells include the pancreatic polypeptide secreting PP-cells, ghrelin-secreting epsilon-cells and the somatostatin secreting delta-cells. The delta-cells are considered important paracrine regulators of alpha- and beta-cell hormone secretion [14]. The dynamic and complex interplay of the different cell types in the islets is one of the aspects that make them important to study. Islets are spherical structures consisting of about 1000 cells each and there are about 1 million islets in the pancreas contributing to about 2% of the total mass of the organ [15, 16]. The islets have an important task of maintaining plasma glucose levels within a narrow range and signaling storage or release of energy substrates for adequate organ function. Insulin is the main hormone signaling storage and growth to other tissues and facilitates the uptake of nutrients and most importantly of glucose. In addition to aiding cells in the uptake of glucose for fuel purposes and stimulating adipogenesis, insulin has an important role in avoiding abnormal elevations in glucose concentrations, which otherwise could be detrimental for organ function. Glucagon on the other hand prevents glucose concentrations from becom- ing too low, which decreases glucose availability for cells. Glucagon therefore has a more catabolic function and is important during fasting [17]. The prohormone of glucagon is also expressed in the intestine and central nervous system, where it is cleaved to different peptide sequences that form hormones that also are involved in energy homeostasis [18]. Under normal cir- cumstances elevation of glucose has opposite effects on the secretion of insulin and glucagon from the beta- and alpha-cells. In T2DM patient elevation of glucose paradoxically stimulates both hormones [19]. When beta-cells fail to meet the increased demand for insulin during obesity with resulting hyperglycemia, manifestations of beta-cell dysfunction include elevated basal insulin secretion, loss of glucose responsiveness and eventually cell death [20]. The enhanced activity of alpha-cells in obesity elevates basal glucagon secretion with enhanced liver glucose output, which further enhances the increasing insulin demand [21]. Studies on the patho-

12 genesis of T2DM have found that many genes carrying risk for developing T2DM are genes connected with the islet-cells [12, 22]. This implies that vulnerability to the disease is related to islet-cell function, which is evident especially in obesity, where the insulin need is increased [23].

Metabolism, free fatty acids and pancreatic islet hormone secretion in obese subjects Obesity is accompanied by alterations in metabolism, plasma hormonal levels and kinetics as well as sensitivity of target tissues that all may contribute to increase the risk of developing T2DM [23-25]. Increased nutritional in- take results in many changes in human physiology. The increase in adipose tissue, which leads to rise in energy storage in the form of triglycerides, is often accompanied by a state of dyslipidemia with elevated levels in plasma free fatty acids (FFAs), triglycerides and the ratio between low and high density lipoproteins [26]. Elevated levels of insulin and glucagon are seen in individuals with obesity and elevated basal or fasting levels of insulin are associated with increased risk of developing T2DM [27, 28]. The fuel surplus signals to store the extra energy and the insulin response can be interpreted as to accommo- date this signal. The simultaneously accentuated levels of glucagon are, however, counterintuitive since the hormone is catabolic and the main positive regulator of gluconeogenesis. Adding more glucose to the already nutri- tient rich plasma enhances the demand on the beta-cells. Therefore, hyperglucagonemia is proposed to play a role in the development of obesity- related T2DM [29, 30]. Intervention of glucagon signaling by antibodies or other means has shown promising results [31, 32]. The regulation of glucagon secretion is complex and there are worries that lowering of glucagon secretion induces compensatory alpha-cell hyperplasia as has been observed in animal models [33-35]. These initial attempts to counteract glucagon secretion illustrate the necessity to have good understanding of the causality of hyperglucagonemia during obesity. Better understanding of underlying causes of hyperglucagonemia would allow us to evaluate biological targets of intervention, that are likely to successfully lower glucagon secretion without interfering with other important physiology. Measuring metabolites, that are elevated in obesity and studying how they regulate pancreatic islet hormone secretion, can be an important step in developing preventive or treatment interventions. Plasma long-chain FFA, or non-esterified fatty acid (NEFA) as they are also named, concentrations are elevated in obese subjects. FFAs have been proposed to play a role in the pathogenesis of T2DM by affecting both islet- cells functionally and thereby their hormonal output [36-40], as well as hor-

13 mone sensitivity of target tissues [41]. FFAs show diurnal rhythms and in obesity there are reports of less FFA fluctuations and suppression during insulin elevation, which could enhance the effect of the higher total levels observed in obesity [42]. FFAs have been shown to stimulate basal insulin secretion [43], which as mentioned is associated with increased risk for developing T2DM [27]. There is also evidence that long-term elevation of FFAs can negatively affect glucose responsiveness of beta-cells [36]. They have also been shown to be very important for proper insulin response to glucose during fasting [44]. Despite the fact that glucagon levels are elevated in obesity it is not clear if lipid parameters are only associated with glucagon secretion during obesity or if there is a more direct relationship [45]. A positive correlation between fat mass and both insulin and glucagon levels has been demonstrated in humans [46]. However, research on animals and humans, where FFAs have been acutely removed or added, points to inverse relationship between glucagon secretion and free fatty acids [47, 48]. Other studies, where lipid rich meals are ingested, point to a stimulatory effect of fatty acids on glucagon secretion and even suggest lipids can induce alpha-cell dysfunction similar as in T2DM [49, 50]. While most studies have not focused much on the type of fatty acid, there is evidence that saturated long-chain fatty acids are more associated with negative health effects compared with unsaturated fatty acids [51-53]. The most abundant saturated long-chain FFA in plasma is the 16-carbon palmitate and studies have suggested that palmitate has detrimental effects on cellular function [54-56]. In contrast, the monounsaturated counterpart palmitoleate is associated with improved insulin sensitivity and lipid parameters, and lowering of inflammation [57, 58]. The most compelling evidence for the difference in effects of fatty acids and their saturation and chain length has come from in vitro studies, which provide an opportunity to study those differences in more detail and depth [59].

Free fatty acids in cellular function and hormone secretion from pancreatic islets FFAs can serve as substrates for adenosine triphosphate (ATP) production, messenger moieties for signaling processes, and as important components in cellular organs such as membranes [60, 61]. During starvation or low glucose levels FFAs can both be metabolized into ketone bodies or transferred directly into mitochondria for oxidative phosphorylation as acyl-CoAs [62]. In states of fuel overload excess metabolites are turned into FFAs and stored as triglycerides and utilized as a complementary energy source to glucose. FFAs interact with cells by being cleaved from triglycerides by lipoprotein

14 lipase or directly as FFAs transported by albumin as they can enter cells by protein transporters such as Cluster of Differentiation 36 (CD36) or by diffu- sion [63]. Inside the cells FFAs are usually turned into long-chain acyl-CoAs or metabolized to other lipid species [64]. Another way that FFAs interact with cells is by binding to G-protein coupled receptors on the plasma membrane [65].

Fatty acid G-protein coupled receptors Cells are able to communicate with each other by sending and receiving signals, which include hormonal signals from distant endocrine cells, paracrine signals from neighboring cells or autocrine sensing of signals emitted from the same cell. Cell surface or transmembrane receptors on the plasma membrane provide cells with the means of sensing different ligands in their environment with relevant signaling cascades, without specific uptake of the respective ligands. There are different types and subtypes of transmembrane receptors such as G-protein coupled, receptor tyrosine kinases and ion channel receptors. G-protein coupled receptors (GPCRs), or seven transmembrane receptors as they are also named, are influential on cell function. In addition they have proven to be suitable targets for drug development. In- deed, a high percentage of marketed medicines target GPCRs [66]. In the early 2000s there was much progress in the deorphanization of orphan GPCRs. Interestingly, some of the orphan GPCRs bind fatty acids of different species. At least four different FFA receptors (GPR40, GPR41, GPR43 and GPR120) were identified and a fifth that binds fatty acid amides, lysophosphatidylcholine or acyl-glycerols (GPR119) [67-69]. GPR40 or free fatty acid receptor 1 (FFAR1) as it was named, has its highest expression profile in pancreatic islets and studies found that it specifically binds medium- to long-chain fatty acids and that its activation increased insulin secretion [70-72]. Other tissues, where it is also expressed, include the enteroendocrine region [73], central nervous system [74], certain cancer types [75], osteoclasts [76] and monocytes [70]. GPR119 is mainly expressed in the pancreas and intestine [77]. GPR41 and GPR43 or free fatty acid receptor 2 and 3 (FFAR2, FFAR3), which bind short chain fatty acids and acetate, are expressed in various organs and tissues [78, 79]. GPR120 or FFAR4 is expressed in the intestine, adipose tissue and pancreatic islets, where it reportedly binds long-chain fatty acids and has higher affinity for unsaturated species [67, 80, 81]. Interestingly, some of these receptors are expressed in the same cell-type and reported to have synergistic effects [82].

Free fatty acids and insulin and glucagon secretion Insulin and glucagon are mostly connected with regulation of carbohydrate metabolism. It is, however, possible that their importance for sensing and

15 regulating lipid metabolism is equally important. Although the islets are primarily known as responders to alterations in glucose levels, there is accumulative evidence that they are highly influenced by FFAs and lipid species. In cellular models mechanisms of action of such effects can be defined. Studies on the effects of FFAs on islet hormone secretion are important in this regard and have demonstrated the complexity of hormone secretion from the islets of Langerhans.

Acute effects of elevated FFA levels Insulin is the major regulator of fuel uptake and anabolism and secretion of the hormone from pancreatic beta-cells is mainly regulated by fuel substrates [83]. Short-term exposure to FFAs in vitro amplifies insulin secretion whereas long-term exposure leads to diminished insulin secretion, cellular insulin content and increased apoptosis [84-86]. The same phenomenon has been demonstrated in vivo, where long-term infusion of lipids decreased and short-term infusion increased insulin secretion during intravenous glucose tolerance tests in healthy subjects [87]. The effect of FFAs on glucagon secretion in humans is not as well understood [18]. In rodent cell models stimulatory effects during acute exposure followed by a decline after long-term exposure have been reported [47, 88-90]. During fasting in the presence of low glucose, FFA utilization by mitochondria maintains adequate ATP production and insulin secretion [91]. In the same manner as elevated glucose levels affect insulin secretion, FFAs can also do so by elevating the ATP/ADP ratio, which closes the plasma membrane KATP channels, depolarizes the membrane and opens voltage- dependent Ca2+ channels [92, 93]. The subsequent increase in intracellular Ca2+ results in exocytosis of insulin granules [94]. Besides direct effects on oxidative production of ATP, other indirect effects of FFA metabolism can cause increased energy expenditure and stimulate insulin secretion by accel- erating the glycerolipid/FFA cycle [95]. When glucose levels are elevated it is reported that it is accompanied by a rise in intracellular FFA species due to inhibition of fatty acid beta-oxidation by a product of the glycolytic pathway, malonyl-CoA [96]. These FFA species mainly arise from esterification of long-chain acyl-CoAs into triglycerides or diacylglycerols (DAGs) with further metabolism leading to phosphatic acid and other metabolites implicated in potentiation of insulin secretion [96-101]. Signaling via phospholipase C (PLC) and protein kinase C (PKC) is in large part mediated via lipid molecules such as phospholipids and fatty acids. There are different ways how intracellular fatty acid derived lipids can enhance insulin secretion. Such mechanisms include increasing intracellular Ca2+ concentrations by enhanced endoplasmic reticulum (ER) Ca2+ release and by activating L- type Ca2+ channels. There are also reports of fatty acids directly stimulating insulin secretion by affecting the insulin granules [102-105].

16 Effects of FFAs on glucagon secretion are less clear. Most in vitro studies have as mentioned found strong stimulatory effects of FFAs on glucagon secretion from rodent cell lines or islets [47, 88-90]. How FFAs affect basal glucagon secretion in human islets has not been defined, however. One of the proposed mechanism is that FFAs affect somatostatin secretion that normally has a paracrine control of alpha-cell secretion [14]. Additionally, studies have found increased activity of L-type Ca2+ channels and elevation of intracellular Ca2+ concentrations in the effects of FFAs on glucagon secretion [106]. With the discovery of FFA signaling via GPCRs, the role of FFA- mediated signaling in islet-cells is currently being reevaluated.

Chronic effects of elevated FFA levels - cell models of increased FFAs during obesity When fatty acids are elevated for prolonged time-periods, negative effects on insulin secretion are observed. The metabolism of especially saturated FFAs has been put forth as contributing to cellular dysfunction [107]. The most abundant saturated long-chain FFA in plasma is as mentioned palmitate [108]. Chronic high palmitate levels have repeatedly been shown to induce negative effects on beta-cell function [59, 109-111]. Investigations have identified a number of different mechanisms for how fatty acids induce those effects, which include metabolic effects of palmitate and triglyceride metabolism [84, 112], formation of toxic ceramide species [113], reactive oxygen species (ROS) [114, 115], alterations in glucose metabolism [109, 115] and effects on NADPH content [116]. There is also evidence that palmitate- induced metabolic changes not only diminish glucose responsiveness directly but also indirectly by inducing ER-stress and apoptosis [86, 112, 117, 118]. Long-term studies of the effects of FFAs on glucagon secretion and alpha-cell function have indicated altered function and hypersecretion in rodent cell lines. In one study palmitate and oleate culture was found to enhance the secretion of glucagon and reduce somatostatin secretion in response to glucose [119]. A similar study on isolated rat islets found FFAs to induce glucagon hypersecretion [88]. They also found diminished expression of pancreatic and duodenal homeobox 1 (PDX1), a transcription factor important for beta- and delta-cell differentiation [88].

FFAR1 (GPR40) and islet function Fatty acids influence alpha- and beta-cells by interacting with GPCRs on the plasma membrane [70, 71]. FFAR1 activation by medium- to long-chain fatty acids and synthetic small molecule agonists is reported to signal via Gαq protein coupled signaling, which activates phospholipase C (PLC) with subsequent hydrolysis of PIP2 into DAG and inositol triphosphate (IP3). It has been proposed that DAG and IP3 potentiate insulin secretion by activating

17 PKC, protein kinase D1 (PKD1) which affects remodeling of the actin cyto- skeleton and by triggering of endoplasmic reticulum (ER) Ca2+ release [120, 121]. The main conclusion of many of these studies was that FFAR1 signaling potentiates the insulin response to elevated glucose concentrations and that it did not influence insulin secretion at low glucose concentrations [71]. As glucose stimulates insulin secretion in a biphasic manner it was found that FFAR1 mostly affects what is called the second phase of insulin secretion [122, 123]. The reported glucose-dependency of the stimulatory effects of FFAR1 on insulin secretion promoted development of synthetic FFAR1 agonists as potential therapeutic agents for the treatment of T2DM. Indeed, some have undergone clinical trials in humans with positive effects on glucose control [124, 125]. There is evidence that FFAR1 not only influences insulin secretion but also glucagon secretion, with the receptor being involved in enhanced glucagon secretion from isolated rodent islets and alpha-cells mediated by rise in cytoplasmic Ca2+ [126, 127]. Interestingly, no effect on glucagon secretion was observed in human islets exposed to a FFAR1 synthetic agonist [128]. Animal and in vitro studies have implicated FFAR1 in the long-term negative effect of FFAs, which often are termed lipotoxicity [129-134]. Other studies have not found a common mechanistic role for FFAR1 in the negative effects of FFAs [135-137]. This maybe due to differences in culture conditions and/or species differences [136, 138]. There has been little or no research on the role of FFAR1 with this regard in human in vitro models.

Metabolism and mitochondrial function in islet-cells For accurate sensing of fuel status in blood it is especially important that the metabolic machinery in pancreatic beta-cells is functioning properly. As mentioned above ATP-production affects insulin secretion together with other metabolic signaling molecules, indicating the importance of mitochondrial function in beta-cells [139, 140]. This was illustrated in a study showing that beta-cells depleted of mitochondria were unable to respond to metabolic changes with changes in insulin secretion [141]. It has been demonstrated that beta-cells have highly uncoupled mitochondrial oxygen consumption, meaning that there is a large proportion of the oxygen consumed not coupled to ATP-synthesis (also called proton leak) [142]. This makes it possible for beta-cells to burn and sense fuel-substrates without overproduc- ing ATP and losing the ability to sense changes in ATP-levels. Alpha-cells are not as well understood with regard to influence of metabolism on hormone secretion, which maybe due to the fact that alpha-cell models are not available to the same extent as beta-cell models. Glucose metabolism with ATP-generation is reported to close KATP channels in alpha- cells, just like in beta-cells, but result in lower secretion of the hormone. This is proposed to be due to the fact that action potential generation in al-

18 pha-cells is dependent on T-type Ca2+ channels and Na+ channels, which inactivate and cause decreased electrical excitability [143, 144]. In studies on rat alpha-cells dissociated from their neighboring beta- and delta-cells glucose stimulated glucagon secretion, however [145]. Studies have also found that mitochondrial ATP-generation plays a lesser role in the induction of glucagon secretion as a large portion of glucose utilization in alpha-cells does not seem to result in glucose oxidation and mitochondrial ATP-generation [146, 147]. Investigations on alpha-cell stimulus secretion coupling have found pyruvate, amino acids and FFAs to increase secretion [18]. In the case of pyruvate the difference compared to glucose is proposed to be due to different expression of enzymes involved in pyruvate and lactate metabolism between the cells [147, 148]. Amino acids are proposed to stimulate secretion by different means including their polar properties [18]. FFAs have as mentioned been shown to stimulate glucagon secretion and have even been shown to do so when the plasma membrane is already depolarized, indicating that other or additional mechanism than ATP- generation is present [106]. Studies on animal models of T2DM with high nutrient environment and isolated human islets exposed to elevated levels of nutrients have demonstrated effects on beta-cell mitochondria structure and function [149-153]. A study on pancreatic islets from diabetic patients identified changes in mitochondrial protein expression and reduction in ATP levels. There was increased expression of mitochondrial uncoupling protein 2 (UCP2), respirato- ry chain complex 1 and ATP-synthase expression as well as increased mitochondrial volume without changes in total mitochondrial number [154]. The- se studies have prompted the question if different mitochondrial fuel substrates are more or less capable of causing mitochondrial dysfunction. Interestingly, only 4 hour exposure to elevated FFAs caused fragmentation of mitochondria in a beta-cell line but elevated glucose concentrations alone for up to 24 hours had minor effects on fragmentation [155]. Another study focusing on differences between fuel metabolites in long-term exposure found elevated levels of pyruvate not to induce the same detrimental effects as elevated glucose, which induced unfavorable effects on mitochondrial metabolism and insulin secretion [156]. Investigating how elevated lipids or FFAs influence islet-cell metabolism and hormone secretion can lead to better understanding of the cellular events leading to altered hormone secretion during obesity and T2DM development.

19 Aim

The aim of this thesis was to define mechanisms by which high concentrations of free fatty acids, as observed in obese subjects, affect secretion of insulin and glucagon using cellular models and translating them to the in vivo situation in obese children and adolescents.

Specifically, the aim was to elucidate the role of FFAR1 in both the short- and long-term effects of palmitate on insulin and glucagon secretion.

Specific aims by paper:

I: Examine the role of the FFAR1 signaling pathway in the acute and long- term effects of palmitate on GSIS and insulin content of isolated human islets.

II: Investigate the effects of fatty acid metabolism and FFAR1 signaling on mitochondrial function.

III: Examine how chronically elevated FFA levels affect glucagon, insulin and also somatostatin secretion from isolated human islets at fasting glucose concentrations with special focus on the role of FFAR1.

IV: Delineate mechanisms for fasting hyperglucagonemia by studying associations between fasting glucagon concentrations and plasma lipid parameters in children and adolescents with obesity. Investigate the ability of fatty acids to trigger glucagon secretion from isolated islets of Langerhans at fasting glucose concentrations.

20 Methods

Major aspects regarding the materials and methods used in the thesis are described below. For detailed descriptions of methods and materials used in the studies reference is made to the corresponding section in the respective papers.

Cohort study Cohort description The study design was a two-center cross-sectional study carried out at the Uppsala University Hospital in Uppsala, Sweden, and at Paracelsus Medical University Hospital in Salzburg, Austria. All study subjects were part of the Beta-cell Function in Juvenile Type 2 Diabetes and Obesity (Beta-JUDO) cohort. Children and adolescents with overweight or obesity (n=147) were recruited from the obesity clinics at the respective center and lean controls (n=43) were recruited through advertisement and local schools in Uppsala and Salzburg. All examinations and procedures were harmonized between the centers. Height was measured with a stadiometer and weight with a digital scale. The age- and sex-adjusted body mass index, i.e. the BMI standard deviation score (BMI SDS) was calculated according to the WHO 2006- 2007 growth reference. Waist and hip circumference was measured to the nearest cm by a flexible tape. Children and adolescents were classified as prepubertal, pubertal or postpubertal according to Tanner stage or, when not available, by locally derived cutoffs for testosterone and assessment of growth charts for height. Ethical permission for examinations and procedures were obtained from the Regional Ethical Review Board in Uppsala, Sweden (EPN numbers 2010/036; 2010-03-19 and 2012/318; 2012-10-24) and Salzburg, Austria (2012/1544).

Blood sampling and analyses Blood was sampled through a patent venous catheter, inserted after application of an anesthetic patch to the skin (EMLA, AstraZeneca, London, UK). Plasma glucose and triglycerides were measured at hospital laboratories at each center according to local protocols. In the Salzburg Paracelsus Medical University Hospital laboratory, plasma concentrations of triglycerides and glucose were analyzed on the COBAS Modular System (Roche Diagnostics,

21 Rotkreuz, Switzerland) using enzymatic photometric or glucose oxidase methods. In the Uppsala University Hospital laboratory, plasma concentrations of triglycerides and glucose were analyzed on the Architect system (Abbott Diagnostics, Abbott Park, IL, USA) using a photometric test or glucose dehydrogenase method. Validation of analyses between hospital labs was done by reference blood samples. Plasma insulin and glucagon was analyzed centrally by specific sandwich ELISAs (Mercodia, Uppsala, Sweden). Blood samples used for the analysis of insulin, glucagon and FFAs were immediately centrifuged at 4°C and plasma aliquoted and stored in a biobank at -80°C until analyses. All analytes were measured in fasting blood and in addition, glucose was measured during an oral glucose tolerance test (OGTT).

Free fatty acid analysis A sample of 100 µl plasma was vortexed thoroughly with a 60 µl mixture of internal standards (3.33 µg/ml of nonanoic acid (C9:0) 3.33 mg/ml, 16.66 µg/ml of heptanoic acid (C17:0), and 3.33 µg/ml of tricosylic acid (C23:0)). Plasma lipids were extracted with 2 ml of organic solvent mixture (isopropa- nol: heptane: 1 M hydrochloric acid (40:10:1, v/v/v)) by vortexing for 30 min. During the extraction, lipids need to be protected against oxidation by adding 0.05 mg/ml butylated hydroxytoluene to the organic solvent mixture. The samples were incubated at room temperature for 10 min. One ml of water was added to the samples and the plasma lipids were extracted into 2 ml of heptane. Another lipid extraction step was performed by adding 1 ml of heptane. The heptane phase was dried under nitrogen gas and the dried lipids were redissolved in 100 µl of heptane. The sample was stored at -20°C until Ultra-Performance Convergence Chromatography (UPC2) analysis. FFAs were analyzed with UPC2 (Waters ACQUITY® UPC2™, Milford, MA) coupled with tandem MS (XEVO® TQ-S, Milford, MA). The analysis was performed with an Acquity UPC2 HSS C18 column (100 mm 3.0 mm, 1.8 µm at 40 °C, Waters, Milford, MA, USA). The mobile phase A is 99.99% pure compressed CO2. Methanol containing 0.1% formic acid was used as the co-solvent.

Oral glucose tolerance test After fasting overnight, subjects underwent a standard OGTT, as previously described [157]. Briefly, subjects drank a glucose solution at a dose of 1.75 g glucose/kg body weight, maximum 75 g glucose. Blood samples were drawn at time-points -5, 5, 10, 15, 30, 60, 90 and 120 min from glucose ingestion. Impaired fasting glucose (IFG), impaired glucose tolerance (IGT) and T2D was determined by fasting and/or 2-h OGTT plasma glucose, according to WHO criteria.

22 Magnetic resonance imaging A subset of subjects from the cohort with overweight or obesity (n=90) and lean controls (n=15) underwent quantification of visceral (VAT) and subcutaneous (SAT) adipose tissue volume as well as liver (LFF) and pancreatic (PFF) fat fractions by magnetic resonance imaging as previously described [158]. In brief, 1.5 T clinical MRI systems (Philips Medical System, Am- sterdam, Netherlands) were used. Water-fat imaging techniques were used throughout. All MRI-scans were performed after a light meal and in close time proximity to the OGTT, in most cases on the same day. The volumes were determined using a fully automated segmentation method. The method uses a filtering technique to separate VAT from SAT [159].

Cell studies Cell culture Isolated human islets Isolated human islets were obtained from brain-dead otherwise healthy individuals from the Uppsala University Islet Transplantation Unit and from the Islet Prodo Lab Inc. (Irvine, CA, USA). Human islets were cultured in CMRL 1066 medium (Gibco, Thermo Fisher, Waltham, MA, USA) containing 5.6 mmol/l glucose and supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher, Waltham, MA, USA) and 0.5 % fatty acid free bovine serum albumin (BSA, fraction V, Roche Diagnostics, Basel, Switzer- land), which was defined as control conditions. Ethical permission for the use and for the procedures and protocols involved in handling of human islets isolated from healthy individuals was obtained from the Regional Ethi- cal Review Board in Uppsala, Sweden (EPN number 2010/006; 2010-02- 10). All procedures involving islets were carried out in accordance with the aforementioned approval as well as in compliance with respective local guidelines and regulations.

MIN6 cells MIN6 cells were originally developed from a mouse insulinoma by targeted expression of the SV40 T antigen gene in transgenic mice [160]. The cells are generally considered a good model for the study of beta-cell function in vitro because of their good insulin secretory response to glucose and their susceptibility to the lipotoxic effects of palmitate. Cells were cultured in Dulbecco’s Modified Eagle medium (DMEM) containing 25 mM glucose and supplemented with 10% FBS.

23 EndoC-betaH1 cells EndoC-betaH1 cells are a human beta-cell line developed from fetal pancreatic buds by SV40 T antigen transduction and further expansion in mice [161]. EndoC-betaH1 cells offer the opportunity to study human beta-cell biology, which can differ from rodent beta-cell biology [162]. Cells were cultured in DMEM in Dulbecco’s Modified Eagle medium (DMEM) containing 5.6 mM glucose and supplemented with 2 % BSA as previously described [161].

Palmitate-BSA conjugate preparation Palmitate was prepared in 100 mM 50% ethanol stock solutions and diluted in culture media to a final concentration of 0.5 mM. The media contained 0.5% (weight) fatty acid free BSA. The FFA and protein were allowed to conjugate for 30 min at 37°C.

FFAR1 antagonists and agonists The role of FFAR1 in islet cell biology was addressed by using FFAR1 antagonists ANT203, ANT825 and DC260126 [131, 163, 164] and agonists TAK-875 and GW9508 [72, 128].

Down-regulation of FFAR1 by short hairpin RNA The short hairpin RNA (shRNA) of FFAR1 (Sigma Aldrich) was used to inhibit expression of FFAR1 in MIN6 cells and human islets. Non- mammalian shRNA control plasmid DNA (Sigma Aldrich) was used as a negative control. For transfection of human islets commercially available lentiviral particles pseudotyped with an envelope G glycoprotein from Ve- sicular Stomatitis Virus (VSV-G), containing shRNA targeting FFAR1, were used to enhance transfection (SHCLNV, Mission transduction particles, Sigma Aldrich).

Measurement of FFAR1 protein and mRNA level Total mRNA was isolated from islets or cells using NucleoSpin® RNA (Macherey-Nagel, Duren, Germany) and reversely transcribed into cDNA with SuperScript™ III First-Strand Synthesis System for RT-qPCR (Invitro- gen, Carlsbad, CA, USA). The real-time PCR was performed in 10 µL volume using Dynamo Capillary SYBR Green qPCR kit (Thermo Scientific, Waltham, MA, USA). FFAR1 mRNA level was normalized to the reference gene β-actin using the following formula: target amount = 2-ΔΔCt, where ΔΔCt = [Ct (GPR40 KO) − Ct (β-actin KO)] − [Ct (GPR40 control) − Ct (β- actin control) [165]. Total FFAR1 protein amount in samples of lysed islets was measured by a commercial Human FFAR1/GPR40 Sandwich ELISA (LS-F8454, LifeSpan BioSciences, WA, USA) according to the manufactur- er´s instructions.

24 Short-term exposure of cells to palmitate The acute effects of palmitate on islet-cell function were studied by exposing human islets, EndoC-betaH1 cells or MIN6 cells to 0.5 mM palmitate in the presence or absence of FFAR1 antagonists/agonist for time periods of 0.5 - 2 hours. During this treatment insulin secretion, oxygen consumption rate and metabolic activity were measured.

Long-term exposure of human islets and MIN6 cells to palmitate The long-term effects of palmitate on cell function were studied by exposing human islets to 0.5 mM palmitate for 6 hours, 1, 2, 4 and 7 days or MIN6 cells to 0.5 mM palmitate for 48 hours. FFAR1 antagonists/agonist were included or not during culture. The fatty acid and FFAR1 antagonists/agonist were removed before measuring the effect of different culture conditions on insulin secretion, oxygen consumption rate, apoptosis and mtDNA copy.

Hormone secretion Human islets were collected and placed into either a perifusion chamber for perifusion experiments, or a culture vesicle for longer incubations where media was collected after culture for hormone measurements. In perifusion experiments buffers with different compositions were pumped into the chamber and subsequently collected in time fractions for hormone measurement yielding a kinetic representation of hormone secretion. Islets were ini- tially perifused for 1 hour at 37°C with a buffer set to pH 7.4 containing base-line conditions [86]. Subsequently, samples were collected in the same conditions the next 20 minutes in 5-minute fractions as the baseline. This was followed by another 20-60 min perifusion with the buffer now containing experimental conditions. When assessing glucose-stimulated insulin secretion (GSIS), glucose concentrations were raised from 2 to 20 mM (paper I) and from 5,5 to 11 mM (paper IV). After perifusion, islets were washed with phosphate buffer saline (PBS) and lysed for measurements of hormone and protein content. Insulin was measured by competitive ELISA as previously described [166] (paper I and II), and with commercial ELISA kits from Mercodia (paper III, IV). Glucagon (Mercodia AB, Uppsala Swe- den) and somatostatin (Peninsula labs, San Carlos, CA, USA) were also measured by ELISA. Glucagon, insulin and somatostatin content was normalized to total protein content as measured by the DC protein assay (Bio- Rad Laboratories, Hercules, CA, USA). In MIN6 cells GSIS was measured after culture by incubating the cells for 1 hour at 37°C in buffer containing 2 mM glucose. Subsequently, the buffer was changed to the same type of buffer containing either 2 or 20 mM glucose. After incubation at 37°C for 30 min, aliquots of the buffer were col-

25 lected for insulin measurements. Lysates were used for measurements of insulin and protein content.

Studies on metabolism

Oxygen consumption rate measurements Mitochondrial function was determined by measuring oxygen consumption rate (OCR) in the Extracellular Flux Analyzer XF96e, which is a static model with kinetic fluorescence measurements [167]. Inhibitor of ATP-ase oligomycin, inhibitor of complex 1 rotenone and complex 3 antimycin were used to test mitochondrial function. Whereas addition of oligomycin gives estimation of the amount of respiration that is coupled to ATP-synthase, the addition of rotenone and antimycin post oligomycin blocks all mitochondrial respiration and gives estimation of the total OCR of the mitochondria. Esti- mates of total mitochondrial OCR and the degree of uncoupling and/or the amount of ATP-coupling/proton leak can be calculated. All measurements were corrected for non-mitochondrial OCR by subtracting the non- mitochondrial portion of OCR from the total OCR. Mitochondrial function was determined in MIN6 cells, EndoC-betaH1 and human islets at pH 7.4 in XF assay media (modified DMEM). About 75 000 MIN6 cells and 60 000 EndoC-BetaH1 were seeded in each well of a Seahorse cell culture plate, with 5-10 replicate wells per treatment condition. For measurements of OCR in human islets, 10-15 islets were individually picked to avoid non-islet structures and placed into each well of the same plate, with 6-10 replicate wells per condition. To examine the contribution of glucose to OCR, glycolysis was inhibited by the addition of 100 mM 2- deoxy glucose. To examine FFA contribution to OCR, fatty acid beta- oxidation was inhibited by the addition of the carnitine palmitoyl transferase 1 (CPT1) inhibitor etomoxir or the alpha-keto thiolase inhibitor trimetazidine. To study Gαq protein-coupled receptor signaling, the M3 muscarinic acetylcholine receptor agonist carbachol or the PKC inhibitor chelerythrine were used.

Glucose utilization measurements Glucose utilization in MIN6 cells was determined by addition of 2 µCi d-[5- 3H]glucose into the treatment media. The amount of [3H]OH formed during 2 hour exposure represents the relative amount of glucose that was utilized [168], and was determined by a liquid-scintillation spectrometer.

Palmitate accumulation and oxidation measurements MIN6 cells were cultured in 24-well plates in media containing 0.5 mM palmitate and 2 µCi [3H]palmitate in the absence or presence of FFAR1 agonist/antagonist for 48 hours. Subsequently, cells were lysed with H2O and

26 collected for measurements of the level of accumulated palmitate. In a paral- lel experiment, [3H]palmitate was present during the initial 24 hours but not the following 24 hours. Media from the latter 24-hour culture were collected for estimation of the level of oxidized palmitate.

Apoptosis measurements Apoptosis in MIN6 cells was measured by the Cell Death Detection ELISAPLUS Kit (Roche, Mannheim, Germany), according to the manufactur- er's instructions. The ELISA measures cytoplasmic oligonucleosomes that increase after apoptosis-associated DNA degradation. Apoptosis, determined by optical density, was correlated to total protein determined by the DC Pro- tein Assay (BioRad, Hercules, CA, USA) in separate experiments.

Analysis of results Analysis of cohort study Differences between groups was tested by Student’s t-test in case of normally distributed data or Mann-Whitney U-test in case of non-normally distributed data. Normality was tested by Shapiro-Wilk test. Differences between the study groups in the distribution of categorical variables were tested by the chi-square test. Subjects were divided into quartiles of fasting plasma glucagon concentrations. Differences in anthropometric measurements, body fat compartments and plasma lipids among quartiles of fasting plasma glucagon concentrations were tested by one-way ANOVA with a post hoc Fish- er’s LSD test comparing quartile 1 to quartile 4. In case of non-normally distributed data, the Kruskal-Wallis test with post hoc Dunn’s test was used. Associations of fasting plasma glucagon with anthropometric measurements, body fat compartments and plasma lipids were further tested by multiple linear regressions including and thus adjusting for age, sex and family history of diabetes. In case of failure to satisfy the assumptions of normality and homogeneity of variance, analysis was performed on log-transformed data. Standardised coefficients are denoted as β and unstandardized coefficients are denotes as B throughout. Due to the study population consisting of two separate groups of BMI rather than a continuum, the groups were analysed separately.

Analysis of cell studies For comparison between groups in cell studies the paired Student’s t-test, repeated measures or ordinary one-way ANOVA (with Bonferroni´s or Fish- er´s post hoc tests) were used respectively. P < 0.05 was considered statisti- cally significant. All analysis and figure presentation was done with Graph Pad Prism software, version 6 (San Diego, CA, USA).

27 Results and discussion

Elevated fasting insulin, glucagon and lipid levels in children and adolescents with obesity Obese children and adolescents had higher fasting plasma insulin and glucagon levels compared to lean controls (paper IV). The highest quartile group of fasting plasma glucagon also had the highest plasma insulin and higher prevalence of impaired glucose tolerance than those in the lowest quartile of glucagon. The fact, that fasting glucagon levels are positively correlated with impaired glucose tolerance in the current and other studies involving pediatric obesity [169-171], supports the hypothesis that elevated glucagon levels contribute to the development of T2DM. Plasma triglycerides, and total FFAs at fasting were highest in individuals in the quartile with highest plasma glucagon who also had the highest insulin. Positive correlations between all FFA species measured and glucagon was found, except for palmitoleate. Palmitate showed the greatest difference (54%) between lean controls and those with obesity (paper IV). Since triglycerides can be cleaved locally by lipoprotein lipase yielding FFAs, including in islets, triglycerides most likely contribute to FFA exposure of islet-cells. Association between plasma triglycerides and glucagon has previously been observed in adults with obesity and varying degrees of glucose intolerance [172]. In children and adolescents with obesity there was a positive correlation between VAT and glucagon levels but no correlation between SAT and glucagon levels. In adult males correlation has been observed between VAT, triglycerides and fasting insulin, and glucagon levels [173]. Two studies on central obesity and kidney function demonstrated an association between abdominal obesity and plasma glucagon concentrations in adults, which is in line with the findings of this thesis [174, 175]. Interestingly, in the earlier study, total FFAs were measured, which were substantially higher in the group with central obesity. Rapid increase or removal of FFAs has been coupled to decreased glucagon secretion [47, 48]. This may be influenced by the fact that acute addition or removal of FFA species will have strong effects on insulin secretion and other islet paracrine signals, which then can affect glucagon secretion [18]. When lipid rich meals are ingested and plasma lipids likely increase more

28 transiently, stimulatory effects of FFAs on glucagon secretion are observed [49, 50]. Since the study is cross sectional we can only describe the association and not address causality. The association between lipids and glucagon levels in obesity could also be driven by other metabolites or parameters that stimulate glucagon secretion and are concomitantly elevated in obesity. Glucagon secretion in response to raised FFA levels can in healthy subjects be observed during long periods of fasting to secure glucose levels by enhanced gluconeogenesis [176, 177]. One can speculate that in obesity the alpha-cells chronically receive signals, such as elevated FFA levels, which are normally received only during fasting. Such a scenario would then contribute to cell dysfunction and development of T2DM.

Simultaneous palmitate stimulation of insulin and glucagon secretion via FFAR1 To further investigate associations between lipids and insulin and glucagon levels observed in vivo, we tested the effects of FFAs on alpha- and beta- cells in vitro. The relationship found in children and adolescent between fatty acid sources and the dual increase in insulin and glucagon secretion was also present in isolated human islets. In experiments at fasting glucose levels the addition of FFA palmitate to the culture induced a concomitant increase in both insulin and glucagon release (paper III, IV). After 48 hour culture, the effect on secretion was still present when islets were perifused with 5.5 mM glucose in absence of palmitate (paper IV). Further analyses of 24 hour exposure to palmitate showed that this increase in hormone secretion could not be accounted for by decreased somatostatin secretion as it was also elevated during palmitate culture (paper III). In contrast, in a previous study glucagon hypersecretion and decreased somatostatin secretion were observed from palmitate-treated mouse islets in response to increased glucose concentrations [119]. In contrast, we investigated the effect on basal secretion of islet hormones during culture including somatostatin secretion (paper III). When we studied the glucagon response to glucose stimulation (paper IV) we did not measure somatostatin secretion, however. Interestingly, a study on perifused human pancreas found glucagon capable of inducing somatostatin secretion [178] . Our studies were on intact islets and it is therefore difficult to interpret these findings with regard to mechanism of action on the separate islet cell types. By decreasing FFAR1 levels or antagonizing the receptor during culture, secretion of all three hormones was decreased demonstrating the influence of the receptor in the effects of palmitate on secretion of all three hormones whether direct or indirect. Decreased hormone secretion of the three

29 hormones was also observed when fatty acid beta-oxidation was inhibited by etomoxir, indicating an additional role for oxidative phosphorylation in the effects of FFAs. In islets GSIS caused about 4-fold increase in secretion (paper I). When palmitate was present during GSIS, it was further enhanced 3-fold. Investi- gations with FFAR1 antagonists indicated that FFAR1 was at least responsi- ble for circa 50% of the amplification by palmitate (paper I). It was mostly in the second phase or later time points of GSIS that the effect of FFAR1 antagonist was present (paper I). Insulin secretion from islets in the presence of a FFAR1 agonist alone resulted in a 1.4-fold increase in GSIS compared to the aforementioned 3-fold increase by palmitate (paper I). The results indicate that at least half of the potentiation of GSIS by palmitate was mediated by activation of FFAR1 in human islets. The metabolic or non-FFAR1 dependent effects of palmitate must have also contributed together with FFAR1 signaling since a small molecule agonist, which reportedly has more activity than FFAs at the receptor [179], had less potentiation ability. These findings are in agreement with conclusions from another study, namely that receptor activation is necessary but not sufficient for the potentiation of insulin secretion by FFAs [135]. In isolated human islets the addition of palmitate enhanced insulin secretion both at high (20 mM) and fasting (5.5 mM) glucose concentrations. The increase in secretion in both cases was mediated in large part via FFAR1 as secretion was markedly attenuated, when the receptor was antagonized or down-regulated (paper I, III). The consensus from many studies on how FFAs affect insulin secretion from beta-cells has been that FFAs only potentiate insulin secretion at elevated glucose levels [122, 180]. The same findings of dependency of high glucose concentrations were often reported with regard to effects of FFAR1 [71]. The findings of the studies included in this thesis do not fit with this view. A potential reason for this discrepancy may be that most of the research on islet-cells has been done in rodent cells or islets. There are species differences between rodents and humans when it comes to the pancreatic islets [15]. One important difference when it comes to glucose is the different fasting glucose levels in rodents (around 7-10 mM) and humans (around 5 mM) and the different expression of glucose transporters in beta-cells with predominantly GLUT2 in rodents and GLUT1 and GLUT3 in humans [181-183]. Despite the aforementioned discrepancy there are studies on rodent cells that have found FFAs to increase the basal secretion of insulin at lower glucose levels [85]. As pointed out before, the elevated basal or fasting insulin secretion tone is a strong risk factor for developing T2DM. The general view is that this is due to insulin resistance, where fatty acids may be one of the mediators of resistance. Fittingly, a direct relationship between fasting fatty acids and insulin secretion has been described in vivo [43]. In the study FFAs were measured during euglycaemic–hyperinsulinaemic clamp to estimate steady

30 state FFA levels. The results showed that FFAs were positively related to insulin secretion, when measured during OGTT and that this correlation was independent of sex, obesity, insulin sensitivity and glucose tolerance status [43]. The authors concluded that FFAs exert a tonic regulation on insulin secretion. The results of this thesis and other studies indicate that glucagon levels at fasting also correlate with circulating FFA levels and glucose tolerance [45]. From our in vitro studies we identify a mechanistic way of how FFAs could elevate the basal secretion of both hormones via FFAR1. The fact that we found FFAR1 to also influence glucagon secretion at lower or fasting glucose levels could mean that obese individuals with high FFAs have a higher glucagon tone, i.e. raised glucagon secretion, with resulting increased glucose output and therefore burden on the beta-cells. These findings are from in vitro studies and need to be further investigated in vivo, however.

FFAR1 stimulation of insulin and glucagon secretion is associated with enhanced mitochondrial respiration which is dependent on Gαq signaling and PKC activation In the presence of palmitate mitochondrial OCR in human islets, EndoC- betaH1 cells and MIN6 cells was elevated. This was true at both high (paper II) and fasting (paper III) glucose concentrations. The increase in OCR was largely mediated by FFAR1 as evident when the receptor was antagonized during palmitate exposure (paper II, III). Further investigation indicated that this increase was not mediated solely by oxidative phosphorylation of palmitate and that it was more a general effect on mitochondrial OCR at higher glucose concentrations (paper II). At fasting glucose concentrations fatty acid beta-oxidation contributed more to the increase in respiration as inhibitors of fatty acid beta-oxidation trimetazidine and etomoxir lowered respiration as well as insulin and glucagon secretion (paper III). This is not surpris- ing as elevated glucose levels are reported to inhibit beta-oxidation via malonyl-CoA and therefore other fuel substrates including FFAs are likely rela- tively more abundant and available at lower glucose concentrations. Taken together the results indicate that palmitate, at least in part via FFAR1 signaling, enhances mitochondrial oxygen consumption. Receptor activation ap- pears to increase mitochondrial activity in general as FFAR1 agonists to some extent also increase respiration, although modestly compared to palmitate (paper II). When we investigated how palmitate was more efficient compared to a FFAR1 agonist in this effect on mitochondrial metabolism, we found that palmitate but not FFAR1 agonists initiated intracellular metabolism, which acted in synergy with the enhancement of respiration by FFAR1 signaling.

31 This was evident at high glucose concentrations where palmitate enhanced glucose utilization in MIN6 cells irrespective of whether the receptor was blocked or not (paper II). At lower glucose concentrations the fatty acid it- self was a fuel source for the enhancement of mitochondrial activity in human islets and EndoC-betaH1 cells (paper III). When metabolism in islets of FFAR1 knock-out mice was measured, the conclusion in one study was that it did not affect islet-cell metabolism [184]. In another study in the INS-1E beta-cell line, the authors reported that palmitate affected FFA/glycerolipid cycling and glucose utilization via metabolic and FFAR1 pathways [122], which is in line with the findings of our study. FFAs activate FFAR1 and enhance insulin secretion via downstream Gαq signaling [120]. In addition, recent reports indicate that GPCRs can have what is called biased signaling and such a phenomenon has been described for FFAR1 [185]. To verify the involvement of Gαq signaling pathway in mitochondrial respiration we investigated if the same signaling pathway activated by another Gαq-coupled receptor could enhance OCR in cells incubated with palmitate and the antagonist to FFAR1. The agonist of the M3 muscarinic acetylcholine receptor carbachol also signals via Gαq. Indeed, carbachol increased OCR effectively in the presence of palmitate and the FFAR1 antagonist and to a lesser extent with palmitate alone (paper II). Solely adding carbachol did not have as strong an effect on oxygen consumption compared to when administered together with the combination of palmitate and the FFAR1 antagonist, confirming the previous findings of synergistic dual effects of palmitate as a metabolite and receptor ligand. Taken together this indicates a general mechanism for Gαq signaling on mitochondrial respiration, which seems to depend on metabolism and fuel sub- strate availability and merits further studies. Another downstream target of Gαq signaling is protein kinase C. In line with the previous findings inhibition of the enzyme by chelerythrine lowered OCR in palmitate-treated cells. Blocking FFAR1 did not decrease OCR to the same extent in cells exposed to chelerythrine, indicating involvement of PKC in this effect (paper II). In support of our findings with respect to the role of PKC for mitochondrial respiration, the enzyme has been reported to play an active role in respiration of other cell types than beta-cells [186, 187]. Recently another study further explored the importance of PKC for mitochondrial respiration in beta-cells. In line with our findings, PKC was found to mediate important coordinated effects on mitochondrial respiration and insulin secretion [188].

32 FFAR1 plays a role in the long-term effects of palmitate on hormone secretion In human islets long-term culture in the presence of fasting glucose and elevated palmitate levels caused time-dependent alterations in GSIS and reduction in insulin content. Co-treatment with FFAR1 antagonist improved both insulin secretion in response to glucose and cellular insulin content post culture. The same pattern was observed in MIN6 cells and confirmed by shRNA-mediated down-regulation of the receptor (paper I). Co-culture with the FFAR1 agonist TAK-875 accentuated the negative effects of palmitate on GSIS and total insulin content after 7-day exposure (paper I). Treatment of human islets with the FFAR1 agonist in the absence of extracellular palmitate showed a tendency to improve GSIS and cellular insulin content and had no negative effects on its own (paper I). Long-term culture of human islets in the presence of palmitate resulted in hypersecretion of insulin and glucagon at fasting glucose levels, where FFAR1 antagonism lowered the secretion of both hormones (paper III). Still palmitate significantly enhanced basal hormone secretion of both insulin and glucagon at later time points of the 7-day culture including when FFAR1 was blocked. Thus, there are other factors in the effects of fatty acids in addition to FFAR1 that have long-term influence on basal hormone secretion (paper III). Antagonising FFAR1 during chronic palmitate exposure decreased apoptosis in MIN6 cells (paper I). This has been observed in other in vitro studies and been reported to stem from reduced phosphorylation of the stress kinases JNK and p38 MAPK [133], NADPH oxidase activity [132], superoxide formation [189] and ER stress [131]. Others have come to opposite conclusions from in vitro studies [135] and some even found that activation of FFAR1 with synthetic agonists during palmitate exposure can protect the cells from the lipotoxic effects of palmitate [137, 190]. The discrepancy could arise from differences in cell species and/or culture conditions. One example is a study where the lipotoxic condition was in fact glucolipotoxic with concomitant elevation of glucose [135]. With regard to favourable effects of additional FFAR1 agonism to the already enhanced activation by palmitate [137, 190], we as mentioned found negative effects in our study on human islets, where addition of TAK-875 further decreased GSIS and insulin content (paper I). As receptor pharmacology is complicated and the receptor reportedly has multiple binding sites with reports of allosterism [191] and biased agonism [185], it is possible that some of the synthetic agonist compounds, which alone act as strong agonists, could serve as antagonists to palmitate or modulators of receptor signaling. One such example is rosiglitazone, which is a weak or partial agonist to FFAR1 but serves as an antgonist to palmitate at the receptor [65, 189].

33 When FFAR1 was antagonised during long-term palmitate exposure, we found evidence for altered metabolism in MIN6 cells (paper II). Cells treated with palmitate and the antagonist had more intracellular accumulated fatty acids post culture and less beta-oxidation during culture (paper I). This may be caused by enhanced oxygen consumption in palmitate-exposed cells compared to cells exposed to palmitate and FFAR1 antagonist, as we found was the case when cells were acutely exposed to the fatty acid (paper II). Even though basal mitochondrial OCR in palmitate-treated cells after 48 hour culture was similar to OCR in non-treated cells, the ATP coupling efficiency, or the ratio between ATP-coupled and proton leak OCR, was consid- erably decreased (paper II). The reduction in ATP-coupling of mitochondrial respiration as induced by long-term exposure to palmitate could affect insulin secretion. It has previously been reported that in beta-cells proton leak across the mitochondrial membrane plays an essential role in the regulation of ATP/ADP ratio and increased levels of uncoupling protein 2 (UCP2) can impair GSIS [192, 193]. In our study antagonism of FFAR1 did not improve ATP-coupling efficiency. However, it enhanced the total ATP-coupled respiration and decreased apoptosis post culture, which was associated with par- tially preserved GSIS (paper II). This indicates that part of the effects of palmitate on ATP-coupled respiration was mediated by intracellular metabolism and not by receptor activation. The effect of palmitate on FFAR1 protein showed an initial rise followed by a relative decline (paper III). The relative decline could be the result of chronic basal stimulation of the receptor and negative feedback. In one study islets from T2DM patients had decreased expression of FFAR1 [194]. Lower FFAR1 protein levels could lead to suboptimal GSIS in the same individuals even at low FFA concentrations as it was recently reported that beta-cells release FFAs to the extracellular space and especially at higher glucose levels [195]. In this way FFA release by beta-cells could serve as an autocrine signal in the lipid amplification pathway of GSIS. Many studies including the ones in this thesis (paper I-III) have demonstrated that antagonizing or silencing FFAR1 in the presence of high fatty acid levels can delay or divert the lipotoxic effects of fatty acids [129-133, 196]. Studies on animals with both knockout and antagonism models have come to different conclusions on FFAR1 with regards to its role in the development of glucose intolerance ands its feasibility as a drug target [134, 138, 197]. It is important to distinguish between antagonism of FFAR1 during obesity as a preventive mean versus treatment for T2DM. The former situation is what was tested in our study (paper I-III) and is potentially a good preventive strategy to combat T2DM development in individuals with high FFAs. This view was shared by other researchers, who have studied the role of FFAR1 in animal models of obesity [130, 198]. Since FFAR1 is expressed in other tissues and is for example important for GLP-1 secretion from the intestine, such preventive antagonism could potentially be obtained

34 by using a weak partial agonist. As with many drug targets the off target effects can be significant as indicated by the discontinuation of the development of FFAR1 agonist TAK-875 due to concerns of adverse liver effects [199]. In our studies activation of FFAR1 by synthetic non-fuel agonists did not have negative effects on cell function (paper I, II). Synthetic FFAR1 agonists have been shown to increase insulin secretion and thereby improve glycemic control in subjects with or models of T2DM [122, 199]. The reason for this difference between synthetic and the endogenous ligands of FFAR1 could be that the latter activate both receptor signaling and intracellular metabolism. Due to this the effects on secretion and mitochondrial respiration are more potent especially at lower or fasting glucose concentrations. This may have implications on ROS generation, protein synthesis and feedback mechanisms. Taken together there is evidence that although synthetic non-fuel FFAR1 agonists can stimulate insulin secretion and may be helpful for the treatment of T2DM the same receptor might be involved in the events leading up to worsening of beta-cell function in chronic high fatty acid conditions [130, 134, 164, 196]. Therefore a scenario might be present in obese subjects with chronically high FFA levels, where increased basal secretion of insulin and glucagon leads to simultaneous enhanced lipogenesis and liver glucose output. Eventually basal or fasting levels of insulin could become so high that raising them further in response to elevated glucose levels is not possible for the beta-cells and results in loss of glucose control.

The findings of this thesis indicate that FFAs are important regulators of insulin and glucagon secretion and especially during obesity, where FFAs are elevated. A dual rise in the basal or fasting secretion of both hormones is a characteristic of obese children and adolescents with worsening of glucose control. Research at the cellular level puts FFAR1 forth as one of the key mediators of the dual hormone hypersecretion with accompanied enhanced mitochondrial metabolism. With regards to chronic effects of FFAs there is evidence produced in this thesis, and from other studies, that supports a role for FFAR1 in the mechanism of action behind saturated FFA impairment of islet-cell function.

35 Conclusions

• In children and adolescents with obesity insulin and glucagon levels are elevated compared to in lean individuals and correlate with plasma triglycerides, FFAs and visceral adipose tissue.

• FFAR1 contributes significantly to the stimulation of insulin and glucagon secretion by palmitate from isolated human islets at both elevated (insulin) and fasting glucose levels (both insulin and glucagon).

• Palmitate effects on insulin and glucagon secretion are associated with elevated mitochondrial respiration. The effects are mediated by intracellular metabolism and FFAR1 Gαq signaling and downstream PKC activity.

• FFAR1 activation influences beta-cell metabolism and secretory activity and can enhance the chronic lipotoxic effects of palmitate on insulin and glucagon secretion in vitro.

36 Sammanfattning på svenska

Prevalensen av diabetes typ 2 (DT2) ökar fortfarande och även hos unga människor. Personer som lider av fetma har en högre risk att utveckla DT2. Utvecklingen har kopplats till ökade fastande plasmanivåer av de blod- sockerreglerande hormonerna insulin och glukagon samt ökade nivåer av fria fettsyror. Forskning har särskilt indikerat att kronisk exponering av höga nivåer mättade fettsyror leder till dysfunktion i de Langerhanska öarnas alfa- och betaceller. Fettsyror kan påverka cellerna genom olika mekanismer och en av dessa är fettsyrornas bindning till den G-proteinkopplade receptorn FFAR1/GPR40. Receptorns uppgift vad gäller fria fettsyrornas effekt på Langerhans cellerna är oklar. Syftet med denna avhandling var att klargöra FFAR1:s roll i den omfat- tande effekten som fria fettsyror har och specifikt klargöra effekten som den mättade fettsyran palmitat har på insulin- och glukagonfrisättning. Insulin- och glukagonfastenivåer var förhöjda och korrelerade med lipid- parametrar i barn och ungdomar med fetma. Plasma triglycerider och fria fettsyror var positivt korrelerade med insulin och glukagon vid fasta så väl som med storleken av visceral fettvävnad. Förhöjda glukagonnivåer vid fasta var associerade med nedsatt glukostolerans i samma population. In vitro-studier gjorda på isolerade mänskliga pankreasöar visade att palmitat stimulerade basal insulin- och glukagonfrisättning såväl som mitokondriell respiration vid glukosnivåer motsvarande fastenivåer. Effekten me- dierades av FFAR1 och betaoxidation av fettsyror. Vid högre glukoskoncen- trationer var receptorn involverad i potentiering av insulinfrisättning från isolerade mänskliga pankreasöar samt insulinfrisättande MIN6-celler. Des- sutom visade resultaten att effekten av palmitat på hormonfrisättningen var associerad med förhöjd mitokondriell respiration medierad av FFAR1 och signallering via Gαq och PKC-aktivitet såväl som ökad intracellulär metabolism inducerad av fettsyran. När pankreasöarna blev exponerade för palmitat under långa tidsperioder samt vid närvaro av en antagonist till FFAR1, ob- serverades normaliserad insulin- och glukagonfrisättning och likaså observ- erades insulinrespons till glukos efter behandling. I MIN6-celler ökade kronisk palmitatbehandling mitokondriell frikop- pling oberoende av FFAR1. FFAR1-antagonism under palmitatbehandling resulterade i förhöjd respiration och minskad apoptos.

37 Sammanfattningsvis har barn och ungdomar som lider av fetma förhöjda fastande insulin- och glukagonnivåer som korrelerar med fria fettsyror och källor til fria fettsyror. Höga glukagonnivåer är kopplade till försämrad glukostolerans hos dessa individer. In vitro är synergin mellan FFAR1- aktiveringen och den intracellulära metabolismen orsakad av palmitat avgörande både för den tidiga ökningen samt den negativa kroniska effekten på insulin- och glukagonfrisättningen.

38 Ágrip á íslensku

Algengi sykursýki af tegund 2 (SS2) hefur aukist síðastliðna áratugi og sérstaklega hjá ungu fólki. Offita er einn helsti áhættuþáttur fyrir myndun SS2. Þróun sjúkdómsins felur í sér há fastandi blóðgildi af brishormónunum insúlíni og glúkagoni ásamt aukningu í fríum fitusýrum. Rannsóknir hafa sýnt að langvarandi há þéttni sérstaklega mettaðra frírra fitusýra getur leitt til vanvirkni í alfa- og betafrumum Langerhans eyjanna í brisi sem seyta glúkagoni og insúlíni. Fríar fitusýrur hafa áhrif á frumur Langerhans eyjanna með ýmsum leiðum og er ein slík binding þeirra við G-próteintengda viðtakann Free fatty acid receptor 1 (FFAR1/GPR40). Hlutverk viðtakans í að miðla áhrifum frírra fitusýra á frumur í Langerhans eyjum hefur ekki verið rannsakað til hlítar. Markmið rannsókna þessarar ritgerðar voru að skilgreina hlutverk FFAR1 í hinum víðtæku áhrifum frírra fitusýra og sérstaklega áhrifa mettuðu langkeðju fitusýrunnar palmítat á seytingu insúlíns og glúkagons. Í börnum og unglingum með offitu var jákvætt samband milli blóðþéttni hormónanna insúlíns og glúkagons og blóðgilda þríglýseríða og fitusýra, en einnig jákvætt samband milli hormónanna og fitumagns í kviðarholi. Glúkagon blóðgildi sýndu einnig jákvætt samband við versnandi blóðsykurstjórn í sama þýði. Rannsóknir á mennskum Langerhans eyjum in vitro sýndu að fitusýran palmítat jók seytingu á bæði glúkagoni og insúlíni við fastandi glúkósagildi ásamt því að auka súrefnisupptöku frumnanna. Áhrifin voru tengd virkni FFAR1 viðtakans og beta-oxun fitusýrunnar. Við hærri þéttni af glúkósa var virkjun FFAR1 á bakvið stóran hluta af áhrifum palmítat á aukningu insúlín- seytingar í bæði MIN6 frumum og mennskum eyjum. Niðurstöður sýndu að áhrif palmítat á hormónaseytingu höfðu í för með sér aukningu í súrefni- supptöku sem var miðluð af Gαq boðleiðum og sérstaklega af Prótein kínasa C ensíminu, en jafnframt efnaskiptum á fitusýrunni. Þegar Langerhans eyjar voru meðhöndlaðar með palmítat í lengri tíma ásamt antagonista FFAR1 viðtakans, var insúlín og glúkagon seyting álík seytingu frá eyjum við staðalaðstæður í fjarveru fitusýrunnar. Eftir meðhöndlun sýndu eyjar og MIN6 frumur sem meðhöndlaðar voru með palmítat og antagonista FFAR1 betri insúlínsvörun við glúkósahækkun og höfðu hærra hormónamagn inni í frumum eyjanna í samanburði við þær eyjar sem eingöngu voru meðhöndlaðar með palmítat.

39 Meðhöndlun á MIN6 frumum með palmítat jók hlutfall öndunar sem ekki leiðir til ATP-myndunar í hvatberum óháð því hvort antagonisti FFAR1 var hluti af ræktunaræti eða ekki. Aftur á móti leiddi blokkering á FFAR1, á meðan palmítat meðhöndlun stóð, til aukinnar súrefnisupptöku og lægri tíðni sjálfstýrðs frumudauða eftir meðhöndlun. Ályktun þessarar ritgerðar er sú að börn og unglingar með offitu hafi að jafnaði hærri blóðgildi af hormónunum insúlíni og glúkagoni. Blóðþéttni hormónanna sýnir jafnframt jákvæða fylgni við blóðþéttni frírra fitusýra og annarra uppspretta þeirra. Einnig hafa há blóðgildi af glúkagoni jákvæða fylgni við versnandi blóðsykurstjórn í sömu einstaklingum. In vitro eru það samverkandi áhrif virkjunar FFAR1 og aukning í efnaskiptum sem standa að baki bæði þeirri skammtíma aukningu og þeim neikvæðu langtíma áhrifum sem palmítat hefur á seytingu insúlíns og glúkagons.

40 Acknowledgements

For the most part the work behind the research presented in this thesis was carried out at the department of Medical Cell Biology during the years 2012- 2017. Although there is only one person listed as author of this thesis, the work behind it was carried out, guided, inspired, assisted and made possible by many people. Therefore I want to acknowledge and thank all those people and especially my co-authors.

I would like to firstly thank my supervisor Professor Peter Bergsten for giving me this opportunity. Thank you for all the support, patience, vision and encouragement during this time and for mentoring me and for allowing me to progress in the many different aspects of research.

My co-supervisor Ernest Sargsyan thanks for your guidance and support and for sharing your expertise. Special thanks for all the good scientific and also non scientific discussions and times together.

Thanks to all the current and former members of the Bergsten research group during my time. Hannes Manell for the collaborations and the many interesting discussions that led to them. Azazul Chowdhury, many thanks for providing the electron microscopy islet-cell figure for the cover, for sharing an office with me and lots of good advice and conversations. To Jing Cen for all the help and positive energy. Levon Manukyan for all the help and good times. To Johan Staaf, for good times and advice. Anna Drzazga for the collaboration and good times. To all the master students, especially Ismael, Arne, Mahla and Dalal.

Thanks to AstraZeneca and my co-authors David M. Smith and Sven Göpel for sharing interest in palmitate and FFAR1 biology and for the important scientific discussions.

Thanks to the staff at Dept. of Women’s and Children’s Health, Uppsala University and especially Anders Forslund, Marie Dahlbom, Iris Ciba and Malte Lidström for their important work that made the in vivo studies possible.

41 Thanks to Kumari Ubhayasekera and Jonas Bergquist at the Dept. of Analytical chemistry for their great work and collaborations, discussions and advice.

Thanks to Joel Kullberg and Håkan Ahlström and the staff at Dept. of Surgical Sciences, Radiology for their excellent work and collaboration.

To all members of the Beta-Judo consortium. Especially my co-authors at the Obesity Research Unit, Paracelsus Medical University, Salzburg, Aus- tria; Daniel Weghuber, Katarina Paulmichl, Fanni Zsoldos and Waru- nika Aluthgedara. Also many thanks to Anders Alderborn, Kirsten Roomp, Karlfried Grobe, Jean-Charles Sanchez, Domitille Schvartz and Reinhard Schnei- der.

Thanks to all the staff and students at the Department of Medical Cell Bi- ology, Uppsala University. Special thanks to the current and former head of the department Professor Nils Welsh and Professor Erik Gylfe. Thanks to all senior Professors during my time especially; Per-Ola Carl- son, Gunilla Westermark, Anders Tengholm, Michael Welsh, Peter Hansell and Ulf Eriksson. Thanks to; Lina Thorvaldsson, Björn Åkerblom, Martin Blixt, Shumin Pan, Carl Lundström, Camilla Sävmarker, Farank Azarbayja- ni, Olof Idevall, Antoine Giraud, Parvin Ahooghalandari, Heléne Dansk, Oleg Dyachok, Camilla Krizhanovskii, Nadine Griesch, Kyril Turpaev, Yunjian Xu and Parekh Vishal. To all the members of Friday Fika club and to the many fellow PhD students during my time especially; Nikhil, Parham, Sara, Marie, Evelina, Gustaf, Qian, Jia, Tian, Ye, Chenxiao, Hongyan, Liza, David, Mediha, Sofia, Ida, Xuan, Jalal, Beichen, Maria, Daniel and Kailash.

Thanks to my former fellow students, teachers and professors from the years at the Faculty of Pharmacy of the University of Iceland. Especially FASI5 as well as Már Másson and Einar Mäntylä for their guidance and encouragement during my master project.

Thanks to all my former colleagues at the Icelandic Medicines Agency, especially those at the inspection-unit and those who supported and encour- aged me with regard to my decision of pursuing academic research; Bryn- hildur, Helga, Gunnar, Jóhann, Þórhallur, Kolbeinn and Rannveig.

Thanks to my father Kristinn Baldursson, Hulda Guðný Ásmundsdóttir and my grandmother Helga Kristinsdóttir for their support.

42 Thanks to my parents in law Agnes Hrafnsdóttir and Páll Pálsson for their support in many ways during the years.

I would like to acknowledge and thank my cousin Ragnar Þórisson for his friendship and for inspiring me with his courage, wisdom and positivity. He is truly missed.

I thank my dear grandparents Alma Þórarinsson and Hjalti Þórarinsson for their love, encouragement, support and wisdom.

I want to thank my mother Gunnlaug Hjaltadóttir especially, for her love and for always believing in me and supporting me in various ways. Also for sparking my interest in medical science and for showing me the value of determination and work ethic.

My daughters Alma, Agnes and Embla I thank and acknowledge for inspiring me every day and for together with their mother showering me with love that has had so many positive effects on me as well as on my work.

Last but not least I would like to thank my wife, the wonderful Eva María Pálsdóttir. Your love, positive energy, support, friendship, strength and interest in the research through these years enabled me to do the work that underlies this thesis. I am forever grateful for that and for having you in my life.

The research in this thesis would not have been possible if not for the fund- ing from;

European Union's Seventh Framework Programme (FP7/2007-2013) Swedish Diabetes Foundation EXODIAB Swedish Society for Diabetology Family Ernfors Foundation

43 References

1. Bjorntorp, P., Sjostrom L,+SJOSTROM L: Number and size of adipose tissue fat cells in relation to metabolism in human obesity. Metabolism, 1971. 20(7): p. 703-13. 2. Lee, M.J., Y. Wu, and S.K. Fried, Adipose tissue heterogeneity: implication of depot differences in adipose tissue for obesity complications. Mol Aspects Med, 2013. 34(1): p. 1-11. 3. Tchernof, A. and J.P. Despres, Pathophysiology of human visceral obesity: an update. Physiol Rev, 2013. 93(1): p. 359-404. 4. Obesity: preventing and managing the global epidemic. Report of a WHO consultation. World Health Organ Tech Rep Ser, 2000. 894: p. i-xii, 1-253. 5. Kopelman, P., Health risks associated with overweight and obesity. Obes Rev, 2007. 8 Suppl 1: p. 13-7. 6. Fazeli Farsani, S., et al., Global trends in the incidence and prevalence of type 2 diabetes in children and adolescents: a systematic review and evaluation of methodological approaches. Diabetologia, 2013. 56(7): p. 1471-88. 7. Santoro, N., Childhood obesity and type 2 diabetes: the frightening epidemic. World J Pediatr, 2013. 9(2): p. 101-2. 8. Chan, J.M., et al., Obesity, fat distribution, and weight gain as risk factors for clinical diabetes in men. Diabetes Care, 1994. 17(9): p. 961-9. 9. Lorenzo, C., et al., The metabolic syndrome as predictor of type 2 diabetes: the San Antonio heart study. Diabetes Care, 2003. 26(11): p. 3153-9. 10. Brownlee, M., The pathobiology of diabetic complications: a unifying mechanism. Diabetes, 2005. 54(6): p. 1615-25. 11. Gray, S.P. and K. Jandeleit-Dahm, The pathobiology of diabetic vascular complications--cardiovascular and kidney disease. J Mol Med (Berl), 2014. 92(5): p. 441-52. 12. Andersson, S.A., et al., Reduced insulin secretion correlates with decreased expression of exocytotic genes in pancreatic islets from patients with type 2 diabetes. Mol Cell Endocrinol, 2012. 364(1-2): p. 36-45. 13. Martin, B.C., et al., Role of glucose and insulin resistance in development of type 2 diabetes mellitus: results of a 25-year follow-up study. Lancet, 1992. 340(8825): p. 925-9. 14. Fujimoto, W.Y., J.W. Ensinck, and R.H. Williams, Somatostatin inhibits insulin and glucagon release by monolayer cell cultures of rat endocrine pancreas. Life Sci, 1974. 15(11): p. 1999-2004. 15. Cabrera, O., et al., The unique cytoarchitecture of human pancreatic islets has implications for islet cell function. Proc Natl Acad Sci U S A, 2006. 103(7): p. 2334-9. 16. Mastracci, T.L. and L. Sussel, The endocrine pancreas: insights into development, differentiation, and diabetes. Wiley Interdiscip Rev Dev Biol, 2012. 1(5): p. 609-28.

44 17. Unger, R.H., A.M. Eisentraut, and L.L. Madison, The effects of total starvation upon the levels of circulating glucagon and insulin in man. J Clin Invest, 1963. 42: p. 1031-9. 18. Gromada, J., I. Franklin, and C.B. Wollheim, alpha-Cells of the Endocrine Pancreas: 35 Years of Research but the Enigma Remains. Endocr Rev, 2007. 28(1): p. 84-116. 19. Quesada, I., et al., Physiology of the pancreatic alpha-cell and glucagon secretion: role in glucose homeostasis and diabetes. J Endocrinol, 2008. 199(1): p. 5-19. 20. Butler, A.E., et al., Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes. Diabetes, 2003. 52(1): p. 102-10. 21. Baron, A.D., et al., Role of hyperglucagonemia in maintenance of increased rates of hepatic glucose output in type II diabetics. Diabetes, 1987. 36(3): p. 274-83. 22. Sladek, R., et al., A genome-wide association study identifies novel risk loci for type 2 diabetes. Nature, 2007. 445(7130): p. 881-5. 23. Polonsky, K.S., B.D. Given, and E. Van Cauter, Twenty-four-hour profiles and pulsatile patterns of insulin secretion in normal and obese subjects. J Clin Invest, 1988. 81(2): p. 442-8. 24. Wang, Y., et al., Comparison of abdominal adiposity and overall obesity in predicting risk of type 2 diabetes among men. Am J Clin Nutr, 2005. 81(3): p. 555-63. 25. Pedersen, D.J., et al., A major role of insulin in promoting obesity-associated adipose tissue inflammation. Mol Metab, 2015. 4(7): p. 507-18. 26. Howard, B.V., G. Ruotolo, and D.C. Robbins, Obesity and dyslipidemia. Endocrinol Metab Clin North Am, 2003. 32(4): p. 855-67. 27. Dankner, R., et al., Basal state hyperinsulinemia in healthy normoglycemic adults heralds dysglycemia after more than two decades of follow up. Diabetes Metab Res Rev, 2012. 28(7): p. 618-24. 28. Starke, A.A., et al., Elevated pancreatic glucagon in obesity. Diabetes, 1984. 33(3): p. 277-80. 29. D'Alessio, D., The role of dysregulated glucagon secretion in type 2 diabetes. Diabetes Obes Metab, 2011. 13 Suppl 1: p. 126-32. 30. Dunning, B.E. and J.E. Gerich, The role of alpha-cell dysregulation in fasting and postprandial hyperglycemia in type 2 diabetes and therapeutic implications. Endocr Rev, 2007. 28(3): p. 253-83. 31. Vajda, E.G., et al., Pharmacokinetics and pharmacodynamics of single and multiple doses of the glucagon receptor antagonist LGD-6972 in healthy subjects and subjects with type 2 diabetes mellitus. Diabetes Obes Metab, 2017. 19(1): p. 24-32. 32. Okamoto, H., et al., Glucagon receptor inhibition normalizes blood glucose in severe insulin-resistant mice. Proc Natl Acad Sci U S A, 2017. 33. Larger, E., et al., Pancreatic alpha-cell hyperplasia and hyperglucagonemia due to a glucagon receptor splice mutation. Endocrinol Diabetes Metab Case Rep, 2016. 2016. 34. Gelling, R.W., et al., Lower blood glucose, hyperglucagonemia, and pancreatic alpha cell hyperplasia in glucagon receptor knockout mice. Proc Natl Acad Sci U S A, 2003. 100(3): p. 1438-43. 35. Gu, W., et al., Long-term inhibition of the glucagon receptor with a monoclonal antibody in mice causes sustained improvement in glycemic control, with reversible alpha-cell hyperplasia and hyperglucagonemia. J Pharmacol Exp Ther, 2009. 331(3): p. 871-81.

45 36. Mahendran, Y., et al., Glycerol and fatty acids in serum predict the development of hyperglycemia and type 2 diabetes in Finnish men. Diabetes Care, 2013. 36(11): p. 3732-8. 37. Pankow, J.S., et al., Fasting plasma free fatty acids and risk of type 2 diabetes: the atherosclerosis risk in communities study. Diabetes Care, 2004. 27(1): p. 77-82. 38. Santomauro, A.T., et al., Overnight lowering of free fatty acids with Acipimox improves insulin resistance and glucose tolerance in obese diabetic and nondiabetic subjects. Diabetes, 1999. 48(9): p. 1836-41. 39. Morita, S., et al., Effect of exposure to non-esterified fatty acid on progressive deterioration of insulin secretion in patients with Type 2 diabetes: a long-term follow-up study. Diabet Med, 2012. 29(8): p. 980-5. 40. Carpentier, A., et al., Acute enhancement of insulin secretion by FFA in humans is lost with prolonged FFA elevation. Am J Physiol, 1999. 276(6 Pt 1): p. E1055-66. 41. Hunnicutt, J.W., et al., Saturated fatty acid-induced insulin resistance in rat adipocytes. Diabetes, 1994. 43(4): p. 540-5. 42. Toledo-Corral, C.M., et al., Fasting, post-OGTT challenge, and nocturnal free fatty acids in prediabetic versus normal glucose tolerant overweight and obese Latino adolescents. Acta Diabetol, 2015. 52(2): p. 277-84. 43. Rebelos, E., et al., Influence of endogenous NEFA on beta cell function in humans. Diabetologia, 2015. 58(10): p. 2344-51. 44. Dobbins, R.L., et al., Circulating fatty acids are essential for efficient glucose- stimulated insulin secretion after prolonged fasting in humans. Diabetes, 1998. 47(10): p. 1613-1618. 45. Calanna, S., et al., Beta and alpha cell function in metabolically healthy but obese subjects: relationship with entero-insular axis. Obesity (Silver Spring), 2013. 21(2): p. 320-5. 46. Elahi, D., et al., Effect of Age and Obesity on Fasting Levels of Glucose, Insulin, Glucagon, and Growth-Hormone in Man. Journals of Gerontology, 1982. 37(4): p. 385-391. 47. Luyckx, A.S. and P.J. Lefebvre, Arguments for a Regulation of Pancreatic Glucagon Secretion by Circulating Plasma Free Fatty Acids. Proceedings of the Society for Experimental Biology and Medicine, 1970. 133(2): p. 524-&. 48. Gerich, J.E., et al., Effects of alternations of plasma free fatty acid levels on pancreatic glucagon secretion in man. J Clin Invest, 1974. 53(5): p. 1284-9. 49. Niederwanger, A., et al., Postprandial lipemia induces pancreatic alpha cell dysfunction characteristic of type 2 diabetes: studies in healthy subjects, mouse pancreatic islets, and cultured pancreatic alpha cells. Am J Clin Nutr, 2014. 100(5): p. 1222-31. 50. Radulescu, A., M.C. Gannon, and F.Q. Nuttall, The effect on glucagon, glucagon-like peptide-1, total and acyl-ghrelin of dietary fats ingested with and without potato. J Clin Endocrinol Metab, 2010. 95(7): p. 3385-91. 51. Boden, G., Obesity and free fatty acids. Endocrinol Metab Clin North Am, 2008. 37(3): p. 635-46, viii-ix. 52. Arner, P. and M. Ryden, Fatty Acids, Obesity and Insulin Resistance. Obes Facts, 2015. 8(2): p. 147-55. 53. Sabin, M.A., et al., Fasting nonesterified fatty acid profiles in childhood and their relationship with adiposity, insulin sensitivity, and lipid levels. Pediatrics, 2007. 120(6): p. e1426-33.

46 54. Wei, Y., et al., Saturated fatty acids induce endoplasmic reticulum stress and apoptosis independently of ceramide in liver cells. Am J Physiol Endocrinol Metab, 2006. 291(2): p. E275-81. 55. Simon, J.A., et al., Serum fatty acids and the risk of coronary heart disease. Am J Epidemiol, 1995. 142(5): p. 469-76. 56. El-Assaad, W., et al., Saturated fatty acids synergize with elevated glucose to cause pancreatic beta-cell death. Endocrinology, 2003. 144(9): p. 4154-63. 57. Stefan, N., et al., Circulating palmitoleate strongly and independently predicts insulin sensitivity in humans. Diabetes Care, 2010. 33(2): p. 405-7. 58. Bernstein, A.M., M.F. Roizen, and L. Martinez, Purified palmitoleic acid for the reduction of high-sensitivity C-reactive protein and serum lipids: a double- blinded, randomized, placebo controlled study. J Clin Lipidol, 2014. 8(6): p. 612-7. 59. Maedler, K., et al., Distinct effects of saturated and monounsaturated fatty acids on beta-cell turnover and function. Diabetes, 2001. 50(1): p. 69-76. 60. Klausner, R.D., et al., Lipid domains in membranes. Evidence derived from structural perturbations induced by free fatty acids and lifetime heterogeneity analysis. J Biol Chem, 1980. 255(4): p. 1286-95. 61. Dennis, E.A., et al., Role of phospholipase in generating lipid second messengers in signal transduction. FASEB J, 1991. 5(7): p. 2068-77. 62. McGarry, J.D. and D.W. Foster, Regulation of hepatic fatty acid oxidation and ketone body production. Annu Rev Biochem, 1980. 49: p. 395-420. 63. Hames, K.C., et al., Free fatty acid uptake in humans with CD36 deficiency. Diabetes, 2014. 63(11): p. 3606-14. 64. Mashek, D.G. and R.A. Coleman, Cellular fatty acid uptake: the contribution of metabolism. Current Opinion in Lipidology, 2006. 17(3): p. 274-278. 65. Kotarsky, K., et al., A human cell surface receptor activated by free fatty acids and thiazolidinedione drugs. Biochemical and Biophysical Research Communications, 2003. 301(2): p. 406-410. 66. Pierce, K.L., R.T. Premont, and R.J. Lefkowitz, Seven-transmembrane receptors. Nat Rev Mol Cell Biol, 2002. 3(9): p. 639-50. 67. Hirasawa, A., et al., Free fatty acids regulate gut incretin glucagon-like peptide-1 secretion through GPR120. Nat Med, 2005. 11(1): p. 90-4. 68. Swaminath, G., Fatty acid binding receptors and their physiological role in type 2 diabetes. Arch Pharm (Weinheim), 2008. 341(12): p. 753-61. 69. Brown, A.J., S. Jupe, and C.P. Briscoe, A family of fatty acid binding receptors. DNA Cell Biol, 2005. 24(1): p. 54-61. 70. Briscoe, C.P., et al., The orphan G protein-coupled receptor GPR40 is activated by medium and long chain fatty acids. J Biol Chem, 2003. 278(13): p. 11303- 11. 71. Itoh, Y., et al., Free fatty acids regulate insulin secretion from pancreatic beta cells through GPR40. Nature, 2003. 422(6928): p. 173-6. 72. Briscoe, C.P., et al., Pharmacological regulation of insulin secretion in MIN6 cells through the fatty acid receptor GPR40: identification of agonist and antagonist small molecules. Br J Pharmacol, 2006. 148(5): p. 619-28. 73. Edfalk, S., P. Steneberg, and H. Edlund, Gpr40 is expressed in enteroendocrine cells and mediates free fatty acid stimulation of incretin secretion. Diabetes, 2008. 57(9): p. 2280-7. 74. Ma, D., et al., Expression of free fatty acid receptor GPR40 in the central nervous system of adult monkeys. Neurosci Res, 2007. 58(4): p. 394-401. 75. Hardy, S., et al., Oleate promotes the proliferation of breast cancer cells via the G protein-coupled receptor GPR40. J Biol Chem, 2005. 280(14): p. 13285-91.

47 76. GPR40, a free fatty acid receptor, inhibits osteoclast differentiation and spares bone. Bonekey Rep, 2013. 2: p. 341. 77. Soga, T., et al., Lysophosphatidylcholine enhances glucose-dependent insulin secretion via an orphan G-protein-coupled receptor. Biochem Biophys Res Commun, 2005. 326(4): p. 744-51. 78. Brown, A.J., et al., The Orphan G protein-coupled receptors GPR41 and GPR43 are activated by propionate and other short chain carboxylic acids. J Biol Chem, 2003. 278(13): p. 11312-9. 79. Tang, C., et al., Loss of FFA2 and FFA3 increases insulin secretion and improves glucose tolerance in type 2 diabetes. Nat Med, 2015. 21(2): p. 173-7. 80. Miyauchi, S., et al., Distribution and regulation of protein expression of the free fatty acid receptor GPR120. Naunyn Schmiedebergs Arch Pharmacol, 2009. 379(4): p. 427-34. 81. Ichimura, A., T. Hara, and A. Hirasawa, Regulation of Energy Homeostasis via GPR120. Front Endocrinol (Lausanne), 2014. 5: p. 111. 82. Ekberg, J.H., et al., GPR119, a Major Enteroendocrine Sensor of Dietary Triglyceride Metabolites Coacting in Synergy With FFA1 (GPR40). Endocrinology, 2016. 157(12): p. 4561-4569. 83. Grodsky, G.M., et al., Effects of Carbohydrates on Secretion of Insulin from Isolated Rat Pancreas. Am J Physiol, 1963. 205: p. 638-44. 84. Segall, L., et al., Lipid rather than glucose metabolism is implicated in altered insulin secretion caused by oleate in INS-1 cells. Am J Physiol, 1999. 277(3 Pt 1): p. E521-8. 85. Bollheimer, L.C., et al., Chronic exposure to free fatty acid reduces pancreatic beta cell insulin content by increasing basal insulin secretion that is not compensated for by a corresponding increase in proinsulin biosynthesis translation. J Clin Invest, 1998. 101(5): p. 1094-101. 86. Sargsyan, E., et al., Diazoxide-induced beta-cell rest reduces endoplasmic reticulum stress in lipotoxic beta-cells. J Endocrinol, 2008. 199(1): p. 41-50. 87. Paolisso, G., et al., Opposite effects of short- and long-term fatty acid infusion on insulin secretion in healthy subjects. Diabetologia, 1995. 38(11): p. 1295-9. 88. Gremlich, S., et al., Fatty acids decrease IDX-1 expression in rat pancreatic islets and reduce GLUT2, glucokinase, insulin, and somatostatin levels. J Biol Chem, 1997. 272(48): p. 30261-9. 89. Galbo, H., J.J. Holst, and N.J. Christensen, Glucagon and Plasma Catecholamine Responses to Graded and Prolonged Exercise in Man. Journal of Applied Physiology, 1975. 38(1): p. 70-76. 90. Bollheimer, L.C., et al., Stimulatory short-term effects of free fatty acids on glucagon secretion at low to normal glucose concentrations. Metabolism, 2004. 53(11): p. 1443-8. 91. Berne, C., The metabolism of lipids in mouse pancreatic islets. The biosynthesis of triacylglycerols and phospholipids. Biochem J, 1975. 152(3): p. 667-73. 92. Cook, D.L. and C.N. Hales, Intracellular ATP directly blocks K+ channels in pancreatic B-cells. Nature, 1984. 311(5983): p. 271-3. 93. Ashcroft, F.M., D.E. Harrison, and S.J. Ashcroft, Glucose induces closure of single potassium channels in isolated rat pancreatic beta-cells. Nature, 1984. 312(5993): p. 446-8. 94. Nelson, T.Y., et al., Increased cytosolic calcium. A signal for sulfonylurea- stimulated insulin release from beta cells. J Biol Chem, 1987. 262(6): p. 2608- 12.

48 95. Prentki, M. and S.R. Madiraju, Glycerolipid/free fatty acid cycle and islet beta- cell function in health, obesity and diabetes. Mol Cell Endocrinol, 2012. 353(1- 2): p. 88-100. 96. Prentki, M., et al., Malonyl-CoA and long chain acyl-CoA esters as metabolic coupling factors in nutrient-induced insulin secretion. J Biol Chem, 1992. 267(9): p. 5802-10. 97. Nolan, C.J., et al., Fatty acid signaling in the beta-cell and insulin secretion. Diabetes, 2006. 55 Suppl 2: p. S16-23. 98. Prentki, M. and F.M. Matschinsky, Ca2+, cAMP, and phospholipid-derived messengers in coupling mechanisms of insulin secretion. Physiol Rev, 1987. 67(4): p. 1185-248. 99. Prentki, M. and S.R. Madiraju, Glycerolipid metabolism and signaling in health and disease. Endocr Rev, 2008. 29(6): p. 647-76. 100. Deeney, J.T., et al., Acute stimulation with long chain acyl-CoA enhances exocytosis in insulin-secreting cells (HIT T-15 and NMRI beta-cells). J Biol Chem, 2000. 275(13): p. 9363-8. 101. Larsson, O., et al., Activation of the ATP-sensitive K+ channel by long chain acyl-CoA. A role in modulation of pancreatic beta-cell glucose sensitivity. J Biol Chem, 1996. 271(18): p. 10623-6. 102. Olofsson, C.S., et al., Palmitate increases L-type Ca2+ currents and the size of the readily releasable granule pool in mouse pancreatic beta-cells. J Physiol, 2004. 557(Pt 3): p. 935-48. 103. McGarry, J.D. and R.L. Dobbins, Fatty acids, lipotoxicity and insulin secretion. Diabetologia, 1999. 42(2): p. 128-38. 104. Gwiazda, K.S., et al., Effects of palmitate on ER and cytosolic Ca2+ homeostasis in beta-cells. Am J Physiol Endocrinol Metab, 2009. 296(4): p. E690-701. 105. Noushmehr, H., et al., Fatty acid translocase (FAT/CD36) is localized on insulin-containing granules in human pancreatic beta-cells and mediates fatty acid effects on insulin secretion. Diabetes, 2005. 54(2): p. 472-481. 106. Olofsson, C.S., et al., Palmitate stimulation of glucagon secretion in mouse pancreatic alpha-cells results from activation of L-type calcium channels and elevation of cytoplasmic calcium. Diabetes, 2004. 53(11): p. 2836-43. 107. Opara, E.C., et al., Effect of fatty acids on insulin release: role of chain length and degree of unsaturation. Am J Physiol, 1994. 266(4 Pt 1): p. E635-9. 108. Ubhayasekera, S.J., et al., Free fatty acid determination in plasma by GC-MS after conversion to Weinreb amides. Anal Bioanal Chem, 2013. 405(6): p. 1929-35. 109. Zhou, Y.P. and V.E. Grill, Long-term exposure of rat pancreatic islets to fatty acids inhibits glucose-induced insulin secretion and biosynthesis through a glucose fatty acid cycle. J Clin Invest, 1994. 93(2): p. 870-6. 110. Zhou, Y.P. and V. Grill, Long term exposure to fatty acids and ketones inhibits B-cell functions in human pancreatic islets of Langerhans. J Clin Endocrinol Metab, 1995. 80(5): p. 1584-90. 111. Hong, J., P.B. Jeppesen, and K. Hermansen, Effects of elevated fatty acid and glucose concentrations on pancreatic islet function in vitro. Diabetes Obes Metab, 2009. 11(4): p. 397-404. 112. Cnop, M., et al., Inverse relationship between cytotoxicity of free fatty acids in pancreatic islet cells and cellular triglyceride accumulation. Diabetes, 2001. 50(8): p. 1771-7.

49 113. Manukyan, L., et al., Palmitate-Induced Impairments of beta-Cell Function Are Linked With Generation of Specific Ceramide Species via Acylation of Sphingosine. Endocrinology, 2015. 156(3): p. 802-12. 114. Cai, Y., et al., Increased oxygen radical formation and mitochondrial dysfunction mediate beta cell apoptosis under conditions of AMP-activated protein kinase stimulation. Free Radic Biol Med, 2007. 42(1): p. 64-78. 115. Carlsson, C., L.A. Borg, and N. Welsh, Sodium palmitate induces partial mitochondrial uncoupling and reactive oxygen species in rat pancreatic islets in vitro. Endocrinology, 1999. 140(8): p. 3422-8. 116. Iizuka, K., et al., Metabolic consequence of long-term exposure of pancreatic beta cells to free fatty acid with special reference to glucose insensitivity. Biochim Biophys Acta, 2002. 1586(1): p. 23-31. 117. Lupi, R., et al., Lipotoxicity in human pancreatic islets and the protective effect of metformin. Diabetes, 2002. 51 Suppl 1: p. S134-7. 118. Sol, E.M., et al., Glucolipotoxicity in INS-1E cells is counteracted by carnitine palmitoyltransferase 1 over-expression. Biochem Biophys Res Commun, 2008. 375(4): p. 517-21. 119. Collins, S.C., et al., Long-term exposure of mouse pancreatic islets to oleate or palmitate results in reduced glucose-induced somatostatin and oversecretion of glucagon. Diabetologia, 2008. 51(9): p. 1689-93. 120. Fujiwara, K., F. Maekawa, and T. Yada, Oleic acid interacts with GPR40 to induce Ca2+ signaling in rat islet beta-cells: mediation by PLC and L-type Ca2+ channel and link to insulin release. Am J Physiol Endocrinol Metab, 2005. 289(4): p. E670-7. 121. Ferdaoussi, M., et al., G protein-coupled receptor (GPR)40-dependent potentiation of insulin secretion in mouse islets is mediated by protein kinase D1. Diabetologia, 2012. 55(10): p. 2682-92. 122. Burant, C.F., Activation of GPR40 as a therapeutic target for the treatment of type 2 diabetes. Diabetes Care, 2013. 36 Suppl 2: p. S175-9. 123. Kebede, M., et al., The fatty acid receptor GPR40 plays a role in insulin secretion in vivo after high-fat feeding. Diabetes, 2008. 57(9): p. 2432-7. 124. Araki, T., et al., GPR40-induced insulin secretion by the novel agonist TAK- 875: first clinical findings in patients with type 2 diabetes. Diabetes Obes Metab, 2012. 14(3): p. 271-8. 125. Naik, H., et al., Safety, tolerability, pharmacokinetics, and pharmacodynamic properties of the GPR40 agonist TAK-875: results from a double-blind, placebo-controlled single oral dose rising study in healthy volunteers. J Clin Pharmacol, 2012. 52(7): p. 1007-16. 126. Flodgren, E., et al., GPR40 is expressed in glucagon producing cells and affects glucagon secretion. Biochem Biophys Res Commun, 2007. 354(1): p. 240-5. 127. Wang, L., et al., Acute stimulation of glucagon secretion by linoleic acid results from GPR40 activation and [Ca2+]i increase in pancreatic islet {alpha}- cells. J Endocrinol, 2011. 210(2): p. 173-9. 128. Yashiro, H., et al., The effects of TAK-875, a selective G protein-coupled receptor 40/free fatty acid 1 agonist, on insulin and glucagon secretion in isolated rat and human islets. J Pharmacol Exp Ther, 2012. 340(2): p. 483-9. 129. Abaraviciene, S.M., I. Lundquist, and A. Salehi, Rosiglitazone counteracts palmitate-induced beta-cell dysfunction by suppression of MAP kinase, inducible nitric oxide synthase and caspase 3 activities. Cell Mol Life Sci, 2008. 65(14): p. 2256-65.

50 130. Meidute Abaraviciene, S., et al., GPR40 protein levels are crucial to the regulation of stimulated hormone secretion in pancreatic islets. Lessons from spontaneous obesity-prone and non-obese type 2 diabetes in rats. Mol Cell Endocrinol, 2013. 381(1-2): p. 150-9. 131. Wu, J., et al., Inhibition of GPR40 protects MIN6 beta cells from palmitate- induced ER stress and apoptosis. J Cell Biochem, 2012. 113(4): p. 1152-8. 132. Graciano, M.F., et al., Evidence for the involvement of GPR40 and NADPH oxidase in palmitic acid-induced superoxide production and insulin secretion. Islets, 2013. 5(4): p. 139-48. 133. Natalicchio, A., et al., Exendin-4 protects pancreatic beta cells from palmitate- induced apoptosis by interfering with GPR40 and the MKK4/7 stress kinase signalling pathway. Diabetologia, 2013. 56(11): p. 2456-66. 134. Steneberg, P., et al., The FFA receptor GPR40 links hyperinsulinemia, hepatic steatosis, and impaired glucose homeostasis in mouse. Cell Metab, 2005. 1(4): p. 245-58. 135. Latour, M.G., et al., GPR40 is necessary but not sufficient for fatty acid stimulation of insulin secretion in vivo. Diabetes, 2007. 56(4): p. 1087-94. 136. Alquier, T. and V. Poitout, GPR40: good cop, bad cop? Diabetes, 2009. 58(5): p. 1035-6. 137. Wagner, R., et al., Reevaluation of fatty acid receptor 1 as a drug target for the stimulation of insulin secretion in humans. Diabetes, 2013. 62(6): p. 2106-11. 138. Brownlie, R., et al., The long-chain fatty acid receptor, GPR40, and glucolipotoxicity: investigations using GPR40-knockout mice. Biochem Soc Trans, 2008. 36(Pt 5): p. 950-4. 139. Wollheim, C.B., Beta-cell mitochondria in the regulation of insulin secretion: a new culprit in type II diabetes. Diabetologia, 2000. 43(3): p. 265-77. 140. Chowdhury, A., et al., Functional differences between aggregated and dispersed insulin-producing cells. Diabetologia, 2013. 56(7): p. 1557-68. 141. Kennedy, E.D., P. Maechler, and C.B. Wollheim, Effects of depletion of mitochondrial DNA in metabolism secretion coupling in INS-1 cells. Diabetes, 1998. 47(3): p. 374-80. 142. Wikstrom, J.D., et al., A novel high-throughput assay for islet respiration reveals uncoupling of rodent and human islets. PLoS One, 2012. 7(5): p. e33023. 143. Gopel, S.O., et al., Regulation of glucagon release in mouse -cells by KATP channels and inactivation of TTX-sensitive Na+ channels. J Physiol, 2000. 528(Pt 3): p. 509-20. 144. Gromada, J., et al., ATP-sensitive K+ channel-dependent regulation of glucagon release and electrical activity by glucose in wild-type and SUR1-/- mouse alpha-cells. Diabetes, 2004. 53 Suppl 3: p. S181-9. 145. Olsen, H.L., et al., Glucose stimulates glucagon release in single rat alpha-cells by mechanisms that mirror the stimulus-secretion coupling in beta-cells. Endocrinology, 2005. 146(11): p. 4861-70. 146. Gorus, F.K., W.J. Malaisse, and D.G. Pipeleers, Differences in glucose handling by pancreatic A- and B-cells. J Biol Chem, 1984. 259(2): p. 1196- 200. 147. Schuit, F., et al., Metabolic fate of glucose in purified islet cells. Glucose- regulated anaplerosis in beta cells. J Biol Chem, 1997. 272(30): p. 18572-9. 148. Sekine, N., et al., Low lactate dehydrogenase and high mitochondrial glycerol phosphate dehydrogenase in pancreatic beta-cells. Potential role in nutrient sensing. J Biol Chem, 1994. 269(7): p. 4895-902.

51 149. Mizukami, H., et al., Augmented beta cell loss and mitochondrial abnormalities in sucrose-fed GK rats. Virchows Arch, 2008. 452(4): p. 383-92. 150. Higa, M., et al., Troglitazone prevents mitochondrial alterations, beta cell destruction, and diabetes in obese prediabetic rats. Proc Natl Acad Sci U S A, 1999. 96(20): p. 11513-8. 151. Taniyama, H., et al., Spontaneous diabetes mellitus in young cattle: histologic, immunohistochemical, and electron microscopic studies of the islets of Langerhans. Vet Pathol, 1993. 30(1): p. 46-54. 152. Del Guerra, S., et al., Gliclazide protects human islet beta-cells from apoptosis induced by intermittent high glucose. Diabetes Metab Res Rev, 2007. 23(3): p. 234-8. 153. Fex, M., et al., Enhanced mitochondrial metabolism may account for the adaptation to insulin resistance in islets from C57BL/6J mice fed a high-fat diet. Diabetologia, 2007. 50(1): p. 74-83. 154. Anello, M., et al., Functional and morphological alterations of mitochondria in pancreatic beta cells from type 2 diabetic patients. Diabetologia, 2005. 48(2): p. 282-9. 155. Molina, A.J., et al., Mitochondrial networking protects beta-cells from nutrient- induced apoptosis. Diabetes, 2009. 58(10): p. 2303-15. 156. Gohring, I., et al., Chronic high glucose and pyruvate levels differentially affect mitochondrial bioenergetics and fuel-stimulated insulin secretion from clonal INS-1 832/13 cells. J Biol Chem, 2014. 289(6): p. 3786-98. 157. Forslund, A., et al., Uppsala Longitudinal Study of Childhood Obesity: protocol description. Pediatrics, 2014. 133(2): p. e386-93. 158. Staaf, J., et al., Pancreatic Fat Is Associated With Metabolic Syndrome and Visceral Fat but Not Beta-Cell Function or Body Mass Index in Pediatric Obesity. Pancreas, 2017. 46(3): p. 358-365. 159. Berglund, J. and J. Kullberg, Three-dimensional water/fat separation and T2* estimation based on whole-image optimization--application in breathhold liver imaging at 1.5 T. Magn Reson Med, 2012. 67(6): p. 1684-93. 160. Miyazaki, J.I., et al., Establishment of a Pancreatic Beta-Cell Line That Retains Glucose-Inducible Insulin-Secretion - Special Reference to Expression of Glucose Transporter Isoforms. Endocrinology, 1990. 127(1): p. 126-132. 161. Ravassard, P., et al., A genetically engineered human pancreatic beta cell line exhibiting glucose-inducible insulin secretion. J Clin Invest, 2011. 121(9): p. 3589-97. 162. Andersson, L.E., et al., Characterization of stimulus-secretion coupling in the human pancreatic EndoC-betaH1 beta cell line. PLoS One, 2015. 10(3): p. e0120879. 163. Davenport, M.P. and H.G. Kristinsson, Channel catfish (Ictalurus punctatus) muscle protein isolate performance processed under different acid and alkali pH values. J Food Sci, 2011. 76(3): p. E240-7. 164. Kristinsson, H., et al., FFAR1 is involved in both the acute and chronic effects of palmitate on insulin secretion. Endocrinology, 2013. 154(11): p. 4078-88. 165. Livak, K.J. and T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 2001. 25(4): p. 402-8. 166. Bergsten, P. and B. Hellman, Glucose-induced amplitude regulation of pulsatile insulin secretion from individual pancreatic islets. Diabetes, 1993. 42(5): p. 670-4.

52 167. Ferrick, D.A., A. Neilson, and C. Beeson, Advances in measuring cellular bioenergetics using extracellular flux. Drug Discov Today, 2008. 13(5-6): p. 268-74. 168. Malmgren, S., et al., Tight coupling between glucose and mitochondrial metabolism in clonal beta-cells is required for robust insulin secretion. J Biol Chem, 2009. 284(47): p. 32395-404. 169. Manell, H., et al., Altered Plasma Levels of Glucagon, GLP-1 and Glicentin During OGTT in Adolescents With Obesity and Type 2 Diabetes. J Clin Endocrinol Metab, 2016. 101(3): p. 1181-9. 170. Michaliszyn, S.F., et al., beta-cell function, incretin effect, and incretin hormones in obese youth along the span of glucose tolerance from normal to prediabetes to type 2 diabetes. Diabetes, 2014. 63(11): p. 3846-55. 171. Weiss, R., et al., Basal alpha-cell up-regulation in obese insulin-resistant adolescents. J Clin Endocrinol Metab, 2011. 96(1): p. 91-7. 172. Ortega, F.J., et al., Circulating glucagon is associated with inflammatory mediators in metabolically compromised subjects. European Journal of Endocrinology, 2011. 165(4): p. 639-645. 173. Pouliot, M.C., et al., Visceral obesity in men. Associations with glucose tolerance, plasma insulin, and lipoprotein levels. Diabetes, 1992. 41(7): p. 826- 34. 174. Solerte, S.B., et al., Hyperviscosity and microproteinuria in central obesity: relevance to cardiovascular risk. Int J Obes Relat Metab Disord, 1997. 21(6): p. 417-23. 175. Solerte, S.B., et al., Serum glucagon concentration and hyperinsulinaemia influence renal haemodynamics and urinary protein loss in normotensive patients with central obesity. Int J Obes Relat Metab Disord, 1999. 23(9): p. 997-1003. 176. Fredrickson, D.S. and R.S. Gordon, Jr., The metabolism of albumin-bound C14-labeled unesterified fatty acids in normal human subjects. J Clin Invest, 1958. 37(11): p. 1504-15. 177. Wasserman, D.H., et al., Glucagon Is a Primary Controller of Hepatic Glycogenolysis and Gluconeogenesis during Muscular Work. American Journal of Physiology, 1989. 257(1): p. E108-E117. 178. Brunicardi, F.C., et al., Immunoneutralization of somatostatin, insulin, and glucagon causes alterations in islet cell secretion in the isolated perfused human pancreas. Pancreas, 2001. 23(3): p. 302-308. 179. Tsujihata, Y., et al., TAK-875, an orally available G protein-coupled receptor 40/free fatty acid receptor 1 agonist, enhances glucose-dependent insulin secretion and improves both postprandial and fasting hyperglycemia in type 2 diabetic rats. J Pharmacol Exp Ther, 2011. 339(1): p. 228-37. 180. Itoh, Y. and S. Hinuma, GPR40, a free fatty acid receptor on pancreatic beta cells, regulates insulin secretion. Hepatol Res, 2005. 33(2): p. 171-3. 181. McCulloch, L.J., et al., GLUT2 (SLC2A2) is not the principal glucose transporter in human pancreatic beta cells: implications for understanding genetic association signals at this locus. Mol Genet Metab, 2011. 104(4): p. 648-53. 182. De Vos, A., et al., Human and rat beta cells differ in glucose transporter but not in glucokinase gene expression. J Clin Invest, 1995. 96(5): p. 2489-95. 183. Rorsman, P. and M. Braun, Regulation of insulin secretion in human pancreatic islets. Annu Rev Physiol, 2013. 75: p. 155-79.

53 184. Alquier, T., et al., Deletion of GPR40 impairs glucose-induced insulin secretion in vivo in mice without affecting intracellular fuel metabolism in islets. Diabetes, 2009. 58(11): p. 2607-15. 185. Mancini, A.D., et al., beta-Arrestin Recruitment and Biased Agonism at Free Fatty Acid Receptor 1. Journal of Biological Chemistry, 2015. 290(34): p. 21131-21140. 186. Mahato, B., et al., Regulation of mitochondrial function and cellular energy metabolism by protein kinase C-lambda/iota: a novel mode of balancing pluripotency. Stem Cells, 2014. 32(11): p. 2880-92. 187. Sugawara, K., M. Fujikawa, and M. Yoshida, Screening of protein kinase inhibitors and knockdown experiments identified four kinases that affect mitochondrial ATP synthesis activity. FEBS Lett, 2013. 587(23): p. 3843-7. 188. Santo-Domingo, J., et al., Coordinated activation of mitochondrial respiration and exocytosis mediated by PKC signaling in pancreatic beta cells. FASEB J, 2017. 31(3): p. 1028-1045. 189. Meidute Abaraviciene, S., et al., Palmitate-induced beta-cell dysfunction is associated with excessive NO production and is reversed by thiazolidinedione- mediated inhibition of GPR40 transduction mechanisms. PLoS One, 2008. 3(5): p. e2182. 190. Panse, M., et al., Activation of Extracellular Signal-Regulated Protein Kinases 1 and 2 (ERK1/2) by Free Fatty Acid Receptor 1 (FFAR1/GPR40) Protects from Palmitate-Induced Beta Cell Death, but Plays no Role in Insulin Secretion. Cellular Physiology and Biochemistry, 2015. 35(4): p. 1537-1545. 191. Lin, D.C., et al., Identification and pharmacological characterization of multiple allosteric binding sites on the free Fatty Acid 1 receptor. Mol Pharmacol, 2012. 82(5): p. 843-59. 192. Chan, C.B., et al., Increased uncoupling protein-2 levels in beta-cells are associated with impaired glucose-stimulated insulin secretion: mechanism of action. Diabetes, 2001. 50(6): p. 1302-10. 193. Fleury, C., et al., Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia. Nat Genet, 1997. 15(3): p. 269-72. 194. Del Guerra, S., et al., G-protein-coupled receptor 40 (GPR40) expression and its regulation in human pancreatic islets: the role of type 2 diabetes and fatty acids. Nutr Metab Cardiovasc Dis, 2010. 20(1): p. 22-5. 195. Mugabo, Y., et al., Metabolic fate of glucose and candidate signaling and excess-fuel detoxification pathways in pancreatic beta-cells. J Biol Chem, 2017. 196. Sun, P., et al., DC260126: a small-molecule antagonist of GPR40 that protects against pancreatic beta-Cells dysfunction in db/db mice. PLoS One, 2013. 8(6): p. e66744. 197. Lan, H., et al., Lack of FFAR1/GPR40 does not protect mice from high-fat diet-induced metabolic disease. Diabetes, 2008. 57(11): p. 2999-3006. 198. Zhang, X., et al., DC260126, a small-molecule antagonist of GPR40, improves insulin tolerance but not glucose tolerance in obese Zucker rats. Biomed Pharmacother, 2010. 64(9): p. 647-51. 199. Kaku, K., et al., Long-term safety and efficacy of fasiglifam (TAK-875), a G- protein-coupled receptor 40 agonist, as monotherapy and combination therapy in Japanese patients with type 2 diabetes: a 52-week open-label phase III study. Diabetes Obes Metab, 2016. 18(9): p. 925-9.

Acta Universitatis Upsaliensis Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1320 Editor: The Dean of the Faculty of Medicine

A doctoral dissertation from the Faculty of Medicine, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine. (Prior to January, 2005, the series was published under the title “Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine”.)

ACTA UNIVERSITATIS UPSALIENSIS Distribution: publications.uu.se UPPSALA urn:nbn:se:uu:diva-316991 2017