Muscle-Macrophage & Macrophage-Macrophage Crosstalk in the Diabetogenic Environment

Transcription

1 Muscle-Macrophage & Macrophage-Macrophage Crosstalk in the Diabetogenic Environment M. Constantine Samaan A thesis submitted in conformity with the requirements for the degree of Masters of Science Institute of Medical Sciences University of Toronto Copyright by M. Constantine Samaan, 2011

2 Muscle-Macrophage & Macrophage-Macrophage Crosstalk in the Diabetogenic Environment M. Constantine Samaan Masters of Science Institute of Medical Sciences University of Toronto 2011 Abstract Diabetes and obesity are associated with inflammation and activation of the immune system with infiltration of adipose tissue by macrophages. This is mainly studied in adipose tissue, with limited information to clarify immune-skeletal muscle interactions in these conditions. We show that exposure of L6 rat skeletal muscle cells to saturated fatty acid palmitate results in insulin resistance, activation of inflammatory pathways, upregulation of pro-inflammatory cytokine and chemokine gene expression and secretion. We identified monocyte chemoattractant protein-1 [MCP-1] as the main factor responsible for macrophage attraction, as blocking it reduced macrophage migration to muscle cells. When macrophages are exposed to palmitate, a similar response ensues with production of macrophage chemoattractants and activation of inflammatory pathways and gene expression profiles, and secretion of multiple cytokines. Our work identifies MCP-1 chemokine produced in response to palmitate treatment by both muscle cells and macrophages and provides a potential link in immune-metabolic crosstalk in diabetogenic environment. ii

3 Acknowledgments I would like to thank my supervisor Dr. Amira Klip for her mentorship during this thesis work. She is a great role model that I would like to emulate in my career. She taught me many things, but most importantly how to observe my work with a critical eye to detail, and to always think of why as the data are generated. It was my privilege to be mentored by such an excellent scientist and a wonderful human being. The other person that deserves mention is Phillip Bilan, the Senior Research Associate in the lab. Phil has such excellent insights into the field of Cell Biology, and I regularly sought his opinion in some of the most challenging junctions at the start of this work, and I will miss our train ride home together that was important in shaping my thinking about my work. I would also like to acknowledge my lab mates including Kevin Foley, Tim Chiu, Alexandra Koshkina, Shlomit Boguslovsky, Yi Sun, Jonathan Schertzer, and Zhi Liu, as my daily discussions with them helped me resolve some of the most testing parts of this project. My committee members Drs. Warren Lee and Lisa Robinson helped me tremendously in critiquing my data during my committee meetings, and their helpful suggestions shaped the final form this thesis has taken. Dr. Robinson s lab members deserve a special thank you, as they were very helpful in providing advice and reagents for some of the assays done during the course of this work. Dr. Robinson also helped me shape my vision for a career choice as a physician scientist. Finally, my wife deserves a special thank you, as her encouragement to keep going to achieve my goals was instrumental in my pursuit and completion of the thesis work. iii

4 Table of Contents Acknowledgments... iii List of Tables... viii List of Figures... ix List of abbreviations... xi 1 Chapter 1: Introduction Global burden of the obesity epidemic Type 2 diabetes [T2D] is another global epidemic Obesity and T2D are associated with chronic low-grade inflammation Mechanisms of inflammation in obesity and T2D Adipose tissue is at the epicenter of responses to diabetogenic environment The adipocyte Immune system interaction with metabolic organs in obesity and T2D Innate and adaptive immune systems contribution in obesity and T2D Toll like receptors [TLRs] Immune cells interacting with metabolic organs in obesity and T2D The macrophage Neutrophils T-lymphocytes Potential mechanisms and organelles involved in skeletal muscle insulin resistance Skeletal muscle in obesity and insulin resistance Impaired insulin delivery with elevated FFA Impaired insulin action with elevated FFA Organelles in FFA-induced insulin resistance in skeletal muscle Mitochondria...17 iv

5 Endoplasmic Reticulum stress [ERS] Intracellular pathways mediating insulin action Insulin signaling cascade Insulin signaling pathway molecules affected by insulin resistance Pathways interfering with insulin signaling and leading to insulin resistance Protein Kinase C [PKC] isoforms Inhibitor of nuclear factor-kappa B kinase-β [IKKβ] c-jun N-terminal kinase [JNK] Extracellular Regulated Kinase [ERK] p38 Mitogen Activated Protein Kinase [p38mapk] Cytokines, chemokines and adipokines play important roles in causing inflammation and insulin resistance Tumor necrosis factor-α [TNFα] Interleukin-6 [IL-6] Monocyte Chemoattractant Protein-1 [MCP-1] Interleukin-8 [IL-8] Adiponectin Leptin Adipose tissue-secreted factors and metabolic-immune interactions Effects of cytokines and lipotoxicity on skeletal muscle insulin sensitivity Macrophage interactions with skeletal muscle: Rationale for thesis work Hypotheses Chapter 2: Methods Cell culture methods L6GLUT4myc myoblasts RAW264.7 macrophages...40 v

6 2.1.3 Isolation of primary rat peritoneal macrophages Generation of conditioned medium from muscle cells and macrophages Generation of conditioned media from BSA and Palmitate treated myoblasts Generation of conditioned media from BSA and palmitate treated primary rat peritoneal macrophages Macrophage migration assay Preparation of whole cell lysates for western blot Determination of protein concentration of lysates Gel preparation for western blots Membrane blotting Inhibitors and blocking antibodies RNA generation for RT-PCR Reverse transcription polymerase chain reaction (RT-PCR) NEFA measurements Endotoxin measurement in conditioned media Cytokine estimations using arrays Glucose uptake assay Preparation of palmitate and BSA Statistics Chapter 3: Muscle-macrophage interactions in diabetogenic environment Myoblast conditioned medium [MyoCM] attracts macrophages Palmitate treatment of muscle cells results in reduced glucose uptake Palmitate treatment of muscle cells activates inflammatory pathways Palmitate treatment of muscle cells upregulate inflammatory cytokine and chemokine gene expression Palmitate treatment of muscle cells results in secretion of multiple cytokines and chemokines...57 vi

7 3.6 Monocyte Chemoattractant Protein-1 [MCP-1] production is enhanced with palmitate treatment Neutralizing MCP-1 inhibits macrophage migration Identification of pathways responsible for muscle cell MCP-1 production in response to palmitate treatment Discussion Palmitate treatment of muscle cells activates inflammatory signalling Comparison of the response to fatty acids and signalling pathways involved in adipocytes Possible contribution of other chemokines and cytokines to the response to palmitate Chapter Macrophage-macrophage interaction in diabetogenic environment Effect of diabetogenic environment on macrophage: Macrophage conditioned medium attracts macrophages Palmitate treatment of macrophages attracts neutrophils Palmitate treatment of macrophages activates inflammatory pathways Palmitate treatment of macrophages up regulates inflammatory cytokines and chemokine gene expression Palmitate treatment of macrophages results in secretion of multiple cytokines and chemokines into conditioned medium Discussion Chapter 5: Conclusions & Future Directions Conclusions Future directions: Muscle-macrophage crosstalk Macrophage-macrophage crosstalk References...96 vii

8 List of Tables Table 1.1: Myokines detected in conditioned medium from palmitate-treated cells 61 Table 1.2: Quantification of endotoxin in BSA, palmitate and muscle conditioned media.62 viii

9 List of Figures Figure 1.1: Toll-like receptor signaling pathway 10 Figure 1.2: Endoplasmic reticulum [ER] stress pathways..20 Figure 1.3: Insulin signaling cascade..22 Figure 1.4: The canonical IKK-NKκB pathway.26 Figure 1.5: Mitogen Activated Protein Kinase [MAPK] pathways...29 Figure 1.6: Obesity associated mechanisms leading to insulin resistance..37 Figure 3.1: Experimental design for macrophage migration assay...50 Figure 3.2: Muscle palmitate treatment results in production of factors that attract macrophages 51 Figure 3.3: Myoblast treatment with palmitate results in reduced glucose uptake 53 Figure 3.4: Muscle palmitate treatment activates inflammatory cytokine & chemokine gene expression 54 Figure 3.5: Myoblasts treatment with palmitate activate MAP kinases.55 Figure 3.6: Myoblast treatment with palmitate leads to degradation of IκBα...56 Figure 3.7: Palmitate treatment of myoblasts results in MCP-1 protein synthesis 59 Figure 3.8 A: Muscle palmitate treatment results in secretion of cytokines/chemokines.63 Figure 3.8 B: Concentration of non-esterified fatty acids [NEFA] in MyoCM-BSA and MyoCM-PA...63 Figure 3.9: MCP-1 neutralization in MyoCM-PA impairs macrophage migration.64 ix

10 Figure 3.10: MCP-1 production by palmitate treated myoblasts occurs through ERK 1/2 and NFκB pathways.66 Figure 4.1: Experimental design for macrophage migration assay..76 Figure 4.2: Macrophage treatment with palmitate results in macrophage attraction...77 Figure 4.3: Macrophage treatment with palmitate results in attraction of neutrophils...79 Figure 4.4 A&B: Macrophage treatment with palmitate activates JNK & p38mapk...81 Figure 4.5: Macrophage palmitate treatment activates inflammatory cytokine & chemokine gene expression.82 Figure 4.6 A&B: Treatment of primary rat peritoneal macrophage with palmitate leads to secretion of multiple cytokines/chemokines into conditioned medium...84 Figure 5.1: The model for fatty acid effects on muscle in diabetogenic environment on musclemacrophage and macrophage-macrophage crosstalk...92 x

11 List of abbreviations ACC AP-1 AMP ATP Acetyl Co-A carboxylase Adaptor Protein-1 Adenosine Monophosphate Adenosine Triphosphate AS160 Akt substrate 160 AMEM AT Alpha Modification of Eagle s Medium Adipose Tissue BSA Bovine Serum Albumin BTG Brewer's Thioglycollate CPT-1 Carnitine Palmitoyltransferase-1 CCR2 Chemokine (C-C motif) receptor -2 CD3 Cluster of Differentiation 3 CD4 Cluster of Differentiation 4 CD8 Cluster of Differentiation 8 CD14 Cluster of Differentiation 14 CD44 Cluster of Differentiation 44 CD62L Cluster of Differentiation 62 Ligand CD80 Cluster of Differentiation 80 CD86 Cluster of Differentiation 86 CNS Central Nervous System CRP C - Reactive Protein DAG Diacylglycerol DMEM Dulbecco s Modified Eagle Medium ERK Extracellular Regulated Kinase ER stress Endoplasmic Reticulum Stress FBS Fetal Bovine Serum FFA Free Fatty acid xi

12 fmlp formyl-methionine-leucine-phenylalanine GLUT4 Glucose Transporter -4 HFF High Fat Fed HIF-1 Hypoxia Inducible Factor- HMG-CoA 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase reductase HOMA Homeostatic model assessment IκBα Inhibitor of NFκB-Alpha IKK IκB Kinase INFγ Interferon gamma IL-1 α IL-1 Alpha IL-1β IL-1 Beta IL-1Ra IL-1 receptor antagonist IL-1R Interleukin-1 receptor IL-2 Interleukin-2 IL-4 Interleukin-4 IL-6 Interleukin-6 IL-7 Interleukin -7 IL-8 Interleukin-8 IL-10 Interleukin-10 IL-15 Interleukin-15 ICAM-1 Intercellular Adhesion Molecule-1 IMFD Intermyocellular Fat Depot IMTG Intramyocellular Triglycerides IR Insulin Resistance IRS Insulin Receptor Substrate JAK Janus Kinase protein JNK c-jun terminal kinase kda Kilodalton KC Keratinocyte-derived chemokine xii

13 LCACoA Long Chain Fatty Acyl CoA synthetase LPS Lipopolysaccharide LBP LPS binding protein MØ Macrophage MØCM Primary rat peritoneal macrophage conditioned medium MetS Metabolic Syndrome μm Micromolar μci Microcurie mg Milligram min Minute ml Millilitre MCP-1 Monocyte Chemoattractant Protein-1 MYD88 Myeloid Differentiation primary response protein 88 MyoCM Myoblast conditioned medium NK cells Natural Killer cells NC Normal Chow NFκB Nuclear Factor kappa-light-chain-enhancer of activated B cells NO Nitric Oxide p38mapk p38 Mitogen Activated Protein Kinase PA Palmitate PKC Protein Kinase C PTX3 Pentraxin-3 PIP3 Phosphatidylinositol 3-phosphate PI3K Phosphatidylinositol-3 Kinase PPARγ Peroxisome Proliferator Activated Receptor-γ PGC-1α Peroxisome Proliferator Activated Receptor-γ coactivator-1α PAI-1 Plasminogen Activator Inhibitor-1 RBP4 Retinol Binding Protein-4 ROS Reactive Oxygen Species SCF Stem Cell Factor SOCS1 Suppressor Of Cytokine Signaling-1 SOCS3 Suppressor Of Cytokine Signaling-3 xiii

14 STAT SVF SAT TCR T reg TIRAP TLR TRAM TRIF TNFα T2D UPR UNT VAT VCAM-1 VEGF Signal Transducer and Activator of Transcription Stromal Vascular Fraction Subcutaneous Adipose Tissue T-cell receptor Regulatory T-lymphocytes TIR domain containing adaptor protein Toll-like Receptor TRIF-related adaptor molecule TIR domain-containing adaptor protein inducing Interferon-β Tumor Necrosis Factor-Alpha Type 2 Diabetes Unfolded Protein Response Untreated Visceral Adipose Tissue Vascular Cell Adhesion Molecule-1 Vascular Endothelial Growth Factor xiv

15 1 Chapter 1: Introduction 1.1 Global burden of the obesity epidemic One billion people around the world are overweight [Body Mass Index (BMI) kg/m 2 ], and of those 300 million are obese [BMI 30 kg/m 2 and above] (1). The rising rates of obesity are not restricted to adult populations, as its prevalence in children has tripled over the past three decades, and currently stands at 17% (2). In Canada, the rates of overweight and obesity have also risen, and Canadian children and adults today are less fit and have higher BMIs than two decades ago (3, 4). There is ample evidence that obese children will more likely become obese adolescents and adults (5-8), and the main risks associated with this are its contribution to future adverse cardiometabolic outcomes, including type 2 diabetes [T2D] and cardiovascular disease. It has been also shown that if obesity is present in childhood, then the likelihood of coronary events in adulthood is increased (9). In that sense, it is constructive to think of obesity as a disease in its own right which causes and exacerbates other diseases, including cardiometabolic problems. The main instigators of the obesity epidemic are excess caloric consumption with high fat and carbohydrate intake and reduced physical activity, and these factors interact with the individual s genetic, epigenetic, hormonal, chemical and other environmental factors to cause the obese phenotype (10). Obesity is part of a constellation of features collectively called metabolic syndrome (MetS), the prevalence of which is 34% in US population and reaches close to 50% in severe obesity in children (11). The metabolic syndrome in addition to obesity includes hyperlipidemia, hypertension, and insulin resistance. These conditions increase the individual s risk for developing cardiometabolic complications, shortening the lifespan and increasing morbidities. The burden of obesity and its complications to the individual, family, community, health care systems, and society are only being realized and represent one of the biggest challenges to health care systems around the world (12, 13). In addition, interventions to treat established obesity have limited long-term success, and prevention remains the best cure. The prevention of obesity requires the identification of precipitating mechanisms in order to design effective population-based intervention strategies to combat its devastating effects.

16 1.2 Type 2 diabetes [T2D] is another global epidemic The second global epidemic the world faces today is the T2D epidemic. In the year 2000, 171,000,000 people had T2D worldwide; these numbers will more than double by 2030 to around 366,000,000. Within the same time frame in Canada, more than 2,000,000 people had diabetes in 2000, and this number will surpass 3,500,000 by 2030 (14). Most of the individuals affected are in the middle age group, which means that the burden on these individuals is tremendous as they are likely to live with diabetes for decades. The rates of T2D are also rising in adolescents, and the main contributing factor to this is obesity (4, 15, 16). Diabetes and its complications are a major cause of morbidity and mortality worldwide, and the cause of diabetes epidemic is at least in part due to the obesity epidemic. If children develop T2D, the likelihood of diabetic complications including vasculopathy, nephropathy, retinopathy, and cardiovascular disease at a younger age is high. In addition, treatment regimens for T2D are complicated, and involve frequent blood glucose checks, oral medications or insulin injections, careful dietary planning, and all this impacts the individual s well being and quality of life. None of these treatments are a cure, as they merely treat the condition on a temporary basis. The metabolic syndrome is characterized by insulin resistance (17), which is a preamble to T2D and affects metabolic organs including the liver, adipose tissue and skeletal muscle. In the liver, insulin resistance results in failure to curb hepatic glucose output; in adipose tissue, this result in reduced anti-lipolytic action of insulin and increased lipolysis. In skeletal muscle, insulin resistance is characterized by reduced glucose uptake from the circulation, and as muscle is the major postprandial glucose uptake organ taking up to 75% of absorbed glucose, its insulin resistance results in hyperglycemia. The pancreatic response to elevated blood glucose levels leads to hypersecretion of insulin from the β-cells, and when the insulin supply cannot meet the demand as the β-cell will ultimately fail to keep up insulin production, T2D develops (18). 2

17 1.3 Obesity and T2D are associated with chronic low-grade inflammation Mechanisms of inflammation in obesity and T2D There is currently ample evidence that obesity and T2D are associated with chronic low-grade inflammation (19-21). Adipose tissue is the organ most widely involved in inception and progression of the inflammatory state seen in obesity, and this inflammation impacts other metabolic organs including skeletal muscle and liver (22-24). One of the main pieces of data supporting association of obesity with inflammation came almost two decades ago, when Hotamisligil et al established that Tumor Necrosis Factor-α [TNFα], a prototypical pro-inflammatory cytokine, was elevated in adipose tissue of high fat fed [HFF] mice (25, 26). This was later confirmed in human adipose tissue (27-29), and elevated circulating levels were also noted in obese humans, and these levels fell with weight loss (30).TNFα is one of the major cytokines involved in the inflammatory response and has diverse effects, from influencing insulin signaling to enhancement of production of other cytokines, including itself, via stimulation of stress pathways in cells (31). Further studies support the role of TNFα in this response where TNFα knockout mouse models and the whole body deletion of TNFα receptor show less insulin resistance, and infusing TNFα results in reduced insulin sensitivity (25, 26). TNFα antagonist use in patients with Rheumatoid Arthritis also suggests a role in development of insulin resistance, as these drugs improve insulin sensitivity (32). However, the role of TNFα has been questioned in clinical studies, showing that its levels do not correlate with muscle insulin resistance or hepatic glucose production, glucose levels, or fat metabolism (33, 34). Other indications of existence of inflammation in obesity include the presence of elevated levels of many other inflammatory mediators including fibrinogen, Interleukin-6 [IL-6], Monocyte Chemoattractant Protein-1 [MCP-1], Interleukin-8 [IL-8], and C-reactive protein [CRP] (35-37), to name a few. As will be discussed below, several other cytokines have been implicated in inflammatory response related to obesity. How obesity triggers the inflammatory phenotype seen is still unclear, but it is apparent that the initiation and propagation of the inflammatory response is associated with immune cell activation as discussed below, and it is unclear if this is reversible. Recent evidence suggests that 3

18 inflammation may be persistent in obesity, suggested by a study that looked at neutrophil activation status in morbid obesity, and demonstrated that even with significant improvement in BMI, the chronic inflammatory state persisted (38). On the other hand, macrophages that infiltrate adipose tissue with obesity decline with weight loss, both in cases of dietary restriction regimens and bariatric surgery macrophage markers decline (39, 40). In addition, it has been recently reported that anti-inflammatory macrophages infiltrate adipose tissue with weight loss and this correlates with circulating fatty acid levels and local lipolysis, and these cells disappear from adipose tissue once weight loss is achieved (41). This may highlight an additional role for macrophage subtypes in mediation of adipose tissue metabolism and remodeling with weight loss. Our current understanding of the events leading to inflammation in obesity postulates that the increase in fatty acid and caloric supply results in adipose tissue expansion with adipocyte hypertrophy and hyperplasia, and this may lead to local tissue hypoxia that among other factors that may play a role in promoting the inflammatory response (42). Enlarged adipose cells with reduced oxygen supply then will respond by releasing inflammatory cytokines and adipokines like Plasminogen Activator Inhibitor-1 [PAI-1], Vascular Endothelial Growth Factor [VEGF], and leptin in an attempt to increase blood flow and enhance angiogenesis, a response that will ultimately trigger adipose tissue remodeling but also cause local tissue inflammation. In addition, hypoxia leads to induction of the transcription factor Hypoxia-Inducible Factor-1 [HIF-1], and this in turn will suppress adiponectin production (39, 43). Oxidative stress in adipose tissue and other organs including skeletal muscle is the result of excessive fatty acid oxidation that results in production of reactive oxygen species at higher levels and causes insulin resistance. As adipose tissue expands and caloric and fat influx persists, adipocytes and other cells in adipose tissue like resident macrophages and T-cells start secreting pro-inflammatory cytokines and chemokines, which are cytokines with ability to attract immune cell migration across a gradient (42). These cytokines and chemokines exacerbate local inflammation, causing fatty acid release from adipocytes [lipolysis] and attracting bone marrow-derived monocytes. These monocytes adhere to the endothelium, and then transmigrate to local tissues where they differentiate to macrophages. These macrophages assume an inflammatory [M1] phenotype and secrete pro-inflammatory cytokines and chemokines such as TNFα, IL-1, IL-6, and IL-8 that 4

19 worsen local inflammation, and cause further lipolysis and additional cytokine release (44, 45). This process leads to a vicious cycle whereby more inflammatory cytokine production and lipolysis results in further local tissue inflammation. Cytokines and fatty acids eventually find their way into the systemic circulation and reach distant metabolic organs including skeletal muscle and liver. In these tissues, there is also deposition of fatty acids that, in addition to the fatty acids and cytokines arriving via circulation from adipose tissue, are thought to work together to propagate inflammation and lead to insulin resistance. Many of the cytokines secreted in obesity in response to the local and systemic inflammatory stimuli act in an autocrine, paracrine, and endocrine fashion to mediate further cytokine production and lipolysis. This lipotoxicity is associated with additional roles of fatty acid themselves, acting as signalling molecules that interfere with inflammatory pathways and affect insulin signaling (46) Adipose tissue is at the epicenter of responses to diabetogenic environment Adipose tissue is composed of adipocytes and several other cell types including endothelial cells, fibroblasts, macrophages, and other immune cells like neutrophils and T-cells that are also present in adipose tissue or infiltrate this depot with its expansion in obesity. The roles of these cell types in maintaining tissue homeostasis under physiological conditions are not well studied and are emerging as a novel aspect of obesity-associated inflammation. The two main compartments of adipose tissue in the bodies of rodents and humans include the subcutaneous adipose tissue [SAT] and visceral adipose tissue [VAT]. The contribution of the latter compartment to inflammation and insulin resistance in obesity is well established from epidemiological, clinical, and experimental research (47). The VAT is more lipolytic in response to catecholamines and more responsive to glucocorticoids, less responsive to insulin, secretes more IL-6 and Plasminogen Activator Inhibitor-1 [PAI-1], and less leptin and adiponectin when compared to SAT(47), so the overall phenotype of VAT is more lipolytic and more inflammatory in obesity. The size of the adipocyte in the VAT correlates with insulin resistance more than that of cells in SAT (48). It is likely that because of its proximity and drainage to the portal circulation, fatty acids and cytokines reach the liver where they affect metabolic function including insulin action to limit hepatic glucose uptake and induce cytokine production by liver cells. 5

20 1.3.3 The adipocyte The adipocyte is one of the main cells in the adipose tissue that serves as a storage facility for triglycerides from the circulation that are ingested with meals, and has the ability to respond to different stimuli with products that have roles in adipose tissue and distant organs metabolism. The adipocyte is capable of expanding up to a degree to accommodate increased fat supply in obesity, where there is excess caloric and fat intake. Fat is stored as triglycerides in those cells and released when energy substrates are needed. In addition, the adipocyte secretes a large number of molecules, collectively termed adipokines. The adipose tissue is primarily responsible for the production of some of them like leptin and adiponectin, while others are produced by other cells in addition to the adipose tissue [e.g. TNFα, IL-6] (49, 50). One important note is that some of the cytokines and chemokines produced by adipose tissue are not the product of the adipocyte alone, but also of other non-fat cells including endothelial cells and immune cells. Some of these immune cells are resident in the adipose tissue, while others infiltrate the adipose tissue in obesity and high fat feeding [HFF], and secrete cytokines that impact local tissue homeostasis, but may spill into the circulation and reach skeletal muscle and liver impacting their functions. 1.4 Immune system interaction with metabolic organs in obesity and T2D The immune cells involved in the response to obesity include both cells of the innate immune system and cells of the adaptive immune system. Macrophages and neutrophils fall into the former group, while T-lymphocytes are part of the latter group Innate and adaptive immune systems contribution in obesity and T2D The innate immune system is an evolutionarily conserved system that defends the body against pathogenic, chemical, and physical insults. It is composed of many cell types including not only cells like macrophages, polymorphonuclear leukocytes, Natural Killer cells, mast cells, and dendritic cells but also include cells that form barriers of the body organs like skin and gut linings. The innate immune system includes pattern recognition receptors like Toll-like receptors [TLRs]. This system is present in all organisms including plants, invertebrates and vertebrates. 6

21 The cells of the innate immune system recognize self from non-self in an effective fashion that was selected for throughout evolution, and the response of these cells is of immediate nature to tackle threats to the organism. Innate immune cells of a particular category are identical, and these cells respond to conserved molecular patterns including non-self molecules like LPS, mannans and glycans. The genes for innate immunity are encoded in germline DNA and do not involve receptor re-organization that is needed in adaptive immune responses (51-53). The responses of the innate immune cells include the expression of co-stimulatory molecules [CD80, CD86] that are necessary for T-lymphocyte activation, and expression and secretion of various pro-inflammatory cytokines and chemokines including IL-1β, IL-6, and IL-8, and these responses are mediated via activation of nuclear factor kappa-light-chain-enhancer of activated B cells [NFκB] (53). The adaptive immune system is composed of T-lymphocytes and B- lymphocytes. Unlike the innate immune system, the adaptive immune system is encoded for by gene segments that require rearrangements in order to produce a repertoire of immune responses. There are multiple distinct cell lineages within this system. It has evolved to recognize specific molecules including proteins, peptides, carbohydrates, nucleic acids and pathogens; this response take days to develop and is manifested as clonal expansion or anergy, secretion of Interleukin-2 [IL-2] or effector cytokines like Interleukin-4[IL-4] and Interferon gamma [INFγ] (54, 55). The recognition of non-pathogen antigens by adaptive immune cells cause allergies, autoimmune diseases and transplant rejections Toll like receptors [TLRs] TLRs [Figure 1.1] are a family of pattern recognition receptors with 12 known members that belong to the innate immune system, and play an important role in pathogen recognition as well as recognition of physical and chemical threats to the organism (56). These receptors recognize conserved pathogen associated molecules collectively termed Pathogen-Associated Molecular Patterns [PAMPs]. For example, Lipopolysaccharide [LPS] is a typical PAMP and is a component of the Gram negative bacterial cell wall that is recognized by a member of TLR family, TLR4. Other examples include peptidoglycans from Gram positive bacteria signaling through TLR2 and recognition of a protein from bacteria with flagellae called Flagellin by TLR5 (57). 7

22 Recently, two members of this family of receptors were linked to fatty acid sensing in fat, including TLR 2 & TLR4 and both activate NFκB pathway (56, 58, 59). Of note, skeletal muscle cells express both TLR2 and TLR4 (60). TLR4 is a pattern recognition receptor that is present as a homodimer spanning the plasma membrane and acts as a sensor for Lipopolysaccharide [LPS], a component of the gram-negative bacterial cell wall. Signaling through TLR4 triggers downstream signaling events leading to transcription of pro-inflammatory genes (61, 62). TLR4 has also been implicated in FFA recognition, and there are several murine models that demonstrate that TLR4 is important in inflammatory response in obesity. Murine models of TLR4 loss of function mutations on high fat diet demonstrate less obesity and insulin resistance (63). In addition, C3H/HeJ mice with a point mutation in their TLR4 have higher oxygen consumption, and improved insulin signaling in fat, skeletal muscle and liver (64). When these mice are fed high fat diet, they show less inflammatory responses with reduce activation of JNK and NFκB (65). These mice along with the deletion of TLR4 gene C57BL/10.ScCr mice demonstrate that they are tolerant to the actions of endotoxin with less inflammation (66, 67). In macrophages, knocking out TLR4 results in reduced macrophage activation of NFκB and reduced TNFα production, along with reduction of other pro-inflammatory cytokines in response to saturated fatty acid treatment [Lauric acid] (68). In addition, a constitutively active form of TLR4 induced the expression of inflammatory cytokines in monocytes (69). Importantly, there is evidence that TLR4 mediates interaction between Peroxisome Proliferator Activator Protein-γ [PPARγ] and NFκB, whereby normally PPARγ suppresses NFκB activity. TLR4 ligand LPS stimulate NFκB and this acts to reduce PPARγ mrna synthesis. LPS has no effect on PPARγ expression in TLR4-/- macrophages, and inhibits the expression of PPARγ when activity of TLR4 is restored (70) through activation of NFκB and not MAPKs. In summary, PPARγ represses NFκB in resting macrophages but when TLR4 activation takes place, NFκB in turn will downregulate PPARγ resulting in the reduction of its anti-inflammatory effects (70). The LPS binds to LPS binding protein [LBP] in the circulation (71). When reaching a cell, LBP will help transfer the LPS monomers to cell surface protein CD14. How CD14 facilitates the 8

23 recognition of LPS by TLR4 is unknown, but CD14 deficient mice have less LPS responses. Another protein, MD2, is part of the complex that attaches to TLR4 and its deficiency leads to reduced LPS signaling. Thus, a complex of TLR4, MD2, and CD14 binds LPS at the plasma membrane (62, 72, 73). When LPS binds to the above complex, intracellular signaling ensues via signaling pathways that will lead to different actions. TLR signaling involves the recruitment of one or more adaptor proteins, which will determine localization of the TLR and its subsequent actions. For example, the adaptor protein myeloid differentiation primary response protein 88 [MYD88] is the first intracellular adaptor protein recruited to cytoplasmic domain of surface TLR4 upon LPS binding; if TLR4 is internalized, then the adaptor protein TIR domaincontaining adaptor protein inducing IFN-β [TRIF] is recruited to the endosome. The decision of which adaptor protein is recruited to which location of TLR presence is dependent on the sorting adaptors TIR domain containing adaptor protein [TIRAP] and TRIF-related adaptor molecule [TRAM], respectively (74). The activation of TLR4 leads to IKK-β degradation and the release of NFκB, which will activate pro-inflammatory cytokine production (75). TLR4 also activate type I Interferon responses. TLR2 is another receptor that recognizes multiple ligands including peptidoglycans, bacterial lipoproteins, LPS, Zymosan, and FFA and activates pro-inflammatory pathways. TLR2 forms heterodimers with TLR-1 and TLR-6 for activation, which increases ligand specificity (61, 76). In addition, TLR2 acts via additional pathways including the activation of Rac-PI3K-Akt pathway that will also trigger NFκB release (59, 77, 78), the end result of which is the activation of the same inflammatory pathways activated by TLR4. It has been shown that LPS stimulation and TNFα treatment result in TLR4 induction that leads to induction of TLR2 gene expression and synthesis in adipocytes (79). These receptors play important roles in recognition of saturated fatty acids and are involved in development of insulin resistance. Importantly, it has been shown that the saturated fatty acid [SFA] palmitate, which is abundant in western diet, and composes up to 30% of SFA levels in human circulation causes IR in mouse skeletal muscle cell line through TLR2 and 4(80, 81). This was the reason we chose it in our study as a study model for inflammation induction. 9

24 Cell membrane TLR2 TLR4 MyD88 TIRAP TRAM TRIF IRAK4 TRAF6 TRADD TRAF3 IRAK1/2 Pellino-1 TRAF6 TAB2, TAB3, TAK1 RIP-1 TBK1,IKKe IRF5 MAPK NFκB IRF3 Nucleus Inflammatory cytokines Type I INF Figure 1.1: The toll-like receptor [TLR] signaling pathways. TLR-mediated responses are mediated by MyD88 that is used by TLR2 and TLR4 and TRIF which is used by TLR4. TRAM and TIRAP are adaptor molecules. In macrophages, MyD88 recruits IRAK4, IRAK1, IRAK2 AND TRAF6. TRAF 6 acts via activation of TAK1/TAB2/TAB3 complex to trigger MAPK and NFκB activation, while TRAF6 acts directly on IRF5 which stimulate inflammatory cytokine production. TRIF assembles TRADD, TRAF3 and TRAF6. Through interaction with Pellino-1, TRADD activates RIP-1 and TAK1 leading to activation of MAPK and NFκB pathways. TRIF also activate the inflammasome with TLR4 signalling. 10

25 NFκB is silenced by binding IκBα. Several stimuli including free fatty acids and cytokines signaling through different receptors activate the IκB kinases that degrade IκBα and release p50/p65 heterodimers to enter the nucleus. In the nucleus, NFκB binds to specific sites on DNA of pro-inflammatory molecules called κb sites and this activates gene transcription. The mrna generated then is processed in cytosol; protein is synthesized and then released by these cells Immune cells interacting with metabolic organs in obesity and T2D The macrophage Macrophages are mononuclear phagocytic cells and are part of the innate immune system, which is an evolutionarily conserved defense system, with its cells placed at critical ports of entry of pathogens and other environmental threats to the body. One function of macrophages is sampling antigens as they enter through mucosal barriers and then either destroying them with no memory kept of the encounter innate response, or presenting the antigen to the T-cells to mount an adaptive immune response. Macrophages are present in almost all organs in the body, where they serve to maintain normal tissue homeostasis and physiological function by secreting growth factors, cytokines, and continually crosstalk with other cells in the various tissues to deal with any threats from pathogens and other stimuli and to maintain normal function (82-84). Macrophages present two broadly defined phenotypes; in M1 or pro-inflammatory phenotype, cells are capable of producing pro-inflammatory cytokines and nitric oxide and are responsive to INFγ; the M1 cells are capable of secreting factors that induce insulin resistance in peripheral metabolic organs including adipose tissue. Cells of the M2 or anti- inflammatory phenotype on the other hand are responsive to IL-4 and IL-13 and secrete anti-inflammatory cytokines like IL-10. In addition, the induction of arginase pathway in M2 cells reduces nitric oxide synthesis (82-88). This is a rather simplistic view of the reality of their existence, as macrophages respond to cues in their environment in diverse ways. These cells exist in multiple intermediate phenotypes depending on local tissue environment and are able to adapt to changes in this environment (89). Under normal physiological conditions, macrophages are present in adipose tissue and are mainly of the M2 or anti-inflammatory type (90, 91), in contrast to M1 or pro-inflammatory 11

26 macrophages encountered in pathogen and metabolic responses that infiltrate adipose tissue as bone marrow-derived monocytes that then differentiate to macrophages (44, 92). The detection of increased macrophage repertoire in adipose tissue with HFF was a major leap forward in our understanding of adipose tissue responses to obesity, since the assumption was that the adipocyte itself was the source of the inflammatory cytokines that were found a decade previously to be elevated in adipose tissue with HFF. Two papers changed this; the first used a microarray-based approach where obese adipose tissue was analyzed for its gene expression; many of the upregulated genes were for macrophages. This result was further confirmed with immunohistochemical staining and detected an almost 4-fold increase in adipose tissue macrophages with HFF and obesity (93). The second study looked at a selected group of inflammatory genes [ADAM8, MIP-1α, MCP-1, MAC-1, F4/80, and CD68] that were upregulated in genetic mice models of obesity [leptin deficiency ob/ob; leptin receptor deficiency db/db] and in mice with diet-induced obesity [DIO], and identified those genes as macrophage-enriched. With progressive HFF, these genes were further upregulated and histological analysis confirmed the infiltration of macrophages into the stromal vascular fraction in adipose tissue (94). Since then, there has been a flurry of research further confirming and characterizing the adipose tissue inflammation in the context of obesity and HFF. With adipose tissue expansion in obesity, bone marrow-derived monocytes are attracted to adipose tissue and arrive via circulation; these cells migrate through the endothelium and blood vessel wall to reach the local tissue (44, 95-97). There, local inflammatory milieu with increased lipolysis and elevated cytokines lead to differentiation of these cells to macrophages of the proinflammatory or M1 phenotype (44, 45, 95). There is also some evidence that this affects resident tissue macrophages and they may assume a M1-like phenotype and start secreting proinflammatory cytokines; in a myeloid-cell-only knockout mouse model of I kappa B kinase-β [IKK-β], which is essential for the activity of NF-κB inflammatory pathway, these mice have less inflammation and better insulin sensitivity on HFF (98). In another model involving c-jun N terminal kinase-1 [JNK1] knockout, non-hematopoietic knockout of JNK1 resulted in protection from the effects of HFF and reduced insulin resistance compared to wild type controls due to reduced adiposity, but hematopoietic deletion of JNK1 reduced HFF-induced insulin resistance via reduced inflammation (99). 12

27 This argues for M1-like phenotype in response to HFF of resident macrophages, but the main cohort of macrophages present in obese adipose tissue is bone marrow-derived and infiltrates adipose tissue in the course of HFF (99). The adipose tissue macrophage population that expands with HFF localizes itself around large adipocytes that are thought to be dead or dying in crown like structures [CLS], representing multinucleate giant cell structures (100); this suggests that these cells may play a role in clearing the dead cells (19); CLSs are more abundant in visceral versus subcutaneous adipose tissue (101, 102). There does not appear to be a myeloid stem cell in adipose tissue so far that produces these cells as needed to respond to inflammatory environment in obesity. While the bulk of pro-inflammatory macrophages in obese adipose tissue is thought to originate from bone marrow-derived monocytes (44), it is possible that other cells may contribute to this pool. The pre-adipocytes is one other potential source of macrophages in obese adipose tissue, and these cells share some common capabilities with macrophages in response to diabetogenic and obesogenic environments. Pre-adipocytes are adipocyte precursors that reside in stromal vascular fraction [SVF], in addition to several others in this area (103). All these cells are bathed in the same cocktail of nutrients, factors and molecules that influence their interactions. Macrophages are capable of storing lipids as seen in atherosclerotic foam cells that are present in atherosclerotic plaques ( ); pre-adipocytes injected into peritoneal cavity of mice can act like macrophages, phagocytosing microorganisms and demonstrating antimicrobial actions via generation of reactive oxygen species [ROS] (107, 108), and these abilities disappear when these cells differentiate to mature adipocytes. In addition to mimicking macrophage functions, preadipocytes can also differentiate to macrophages with expression of many macrophage markers, and this is probably due to direct physical contact between pre-adipocyte and macrophage. The transcriptional profile of pre-adipocyte in fact is closer to the macrophage than the adipocyte, and these cells share many products including cytokines, chemokines, and adhesion molecules (21, 22, 109, 110). This is an important concept that requires further study, as some of the experimental work does not look at the pre-adipocytes in the adipose tissue itself but studies its interaction with macrophages and environments that are not completely similar to the adipose tissue. 13

28 Nevertheless, it highlights another potential source of macrophages in obese adipose tissue, in addition to the bone marrow-derived monocytes, and the evaluation of the exact contribution to the adipose tissue macrophage pool by these two cell types needs to be clarified. One of the important molecules in macrophage phenotypic determination is Peroxisome Proliferator Associated Receptor-γ [PPARγ]. This ligand induces macrophage phenotypic switch of macrophages in obesity, and is needed for maturation of M2 macrophages (111). PPARγ is sensor of fatty acids and PPARs control biological actions of lipids mainly through inhibiting NF-κB (112, 113), and this role has established PPARγ as a master regulator of adipogenesis. The function of PPARγ in macrophages and maintenance of normal insulin sensitivity in muscle and liver has been studied but the evidence is inconclusive. PPARγ deletion in macrophages does not significantly affect insulin sensitivity in one study (114); in another study, the inactivation of PPARγ in macrophages lead to glucose intolerance associated with muscle and hepatic IR even with normal chow [NC] diet in lean mice, with significant insulin resistance occurs with HFF Neutrophils Neutrophils are a component of the innate immune system and have a critical role in defending the body mainly against bacterial infections. Recently, a new role has been ascribed to these cells in obesity and HFF. Mice studied in the first few weeks of HFF demonstrate that neutrophils infiltrate adipose tissue within the first week of HFF, and disappeared at the end of the second week, and this was followed by increase in macrophage content. At the end of the HFF period [16 weeks], no neutrophil markers were detected in adipose tissue but macrophages were abundant as expected (115). In cell culture, adipocytes attracted neutrophils that were seen adherent to adipocytes. In addition, patients undergoing bariatric surgery had no detectable neutrophils in skeletal muscle or adipose tissue, but had elevated Myeloperoxidase [MPO] levels in the circulation, this being a marker of neutrophil presence and activation. Two years post-operatively, MPO levels remained elevated despite significant improvement in Body Mass Index [BMI] and weight reduction. This result suggests that these cells may be in a chronic activation state of a cell normally involved in acute immune responses (38). 14

29 The long-term effect of these changes and whether they result in a signature in adipose tissue that contributes to attraction and activation of the infiltrating macrophages or other immune cells is unclear. It indeed raises questions about the interaction between cells [adipocytes, neutrophils, macrophages, T-cells, other immune cells, and endothelial cells], their secreted products, and activated inflammatory pathways in the development of inflammation and insulin resistance in obesity and T2D T-lymphocytes Obese adipose tissue contains T-lymphocytes that are detected prior to macrophage infiltration with HFF. Activated effector T-lymphocytes [CD3 + CD8 + CD62L - CD44 + ] are detected in ob/ob mice and DIO mice within two weeks of commencement of HFF, and the increased number of these cells preceded the infiltration of macrophages to the expanding adipose tissue. There were also reduced CD3 + CD4 + CD8 - cells in adipose tissue and in the circulation. Using neutralizing antibodies to CD8 resulted in reduced M1 macrophage infiltration with no effect on M2 macrophage numbers and improved insulin sensitivity (116). Macrophage recruitment to adipose tissue was found to be a collaborative effort between the adipocytes and CD8 + cells. In another study, resident CD4 + cells were detected in VAT, and adoptive transfer experiments of CD4 + cells had a positive effect on normalizing insulin sensitivity in Rag-1-deficient mice with diet induced obesity, which have lymphopenia and insulin resistance (117), and this effect was mediated mainly via anti-inflammatory T H 2 T-lymphocyte signals, and the improvement in insulin sensitivity is via its effect on macrophages. Importantly, it was found that the T-cell receptor [TCR] repertoire is quite limited, suggesting that the T-lymphocyte immune response to antigens is adipose-specific. This study also showed reduced numbers of Regulatory T-cells [T reg ] in obesity, and these cells are a T-lymphocyte subset that acts to suppress immune system and maintain tolerance of self. When numbers of T reg cells was normalized, this led to improved insulin sensitivity. The normalization of T reg number was due to IL-10 production by macrophages. Similarly, T reg with a unique signature that includes not only CD4 + Foxp3 +, but also includes several adhesion and chemokines receptors were identified only in VAT, and their numbers were reduced in DIO. 15

30 These cells also had a limited receptor repertoire, indicating a response to a local adipose tissue related antigen and supplementing these cells lead to improved insulin sensitivity in DIO mice (118). In conclusion, it seems that T-lymphocytes that are present in non-obese adipose tissue have T reg and T H 2 homeostatic and anti-inflammatory phenotypes. These cells are not only capable of secreting IL-10, but can also get local tissue macrophages to secrete IL-10 as well, thus maintaining insulin sensitivity. In obesity, pro-inflammatory T H 1 and Activated effector T-lymphocytes [CD3 + CD8 + ] predominate, and this may signal to recruit macrophages to adipose tissue in DIO. In addition, these cells probably interact directly with adipocytes influencing insulin sensitivity and adipocyte metabolism. 1.5 Potential mechanisms and organelles involved in skeletal muscle insulin resistance Skeletal muscle in obesity and insulin resistance The understanding that obesity and T2D are associated with insulin resistance raises questions as to the mechanisms and organelles involved in its development. Skeletal muscle is the most important organ in postprandial glucose uptake, and obesity interferes with its insulin signaling leading to disturbances in insulin delivery through muscle capillaries and insulin action on the skeletal muscle cells. Potential mechanisms involved in these responses are discussed below Impaired insulin delivery with elevated FFA For insulin to stimulate muscle glucose uptake, it needs to reach the interstitial space where muscle cells are located and bind to its receptor. Normally, the transport of insulin occurs across a gradient traversing the capillary endothelium in muscle to reach the interstitial space, and this happens within minutes of intravenous insulin administration. Direct administration of insulin to muscle bypasses the endothelial barrier to the interstitium and this result in almost immediate glucose uptake by muscle (119). In addition, there is evidence that insulin positively affects blood flow in muscle, and this occurs possibly via nitric oxide pathway [NO] (120). Free fatty acids reduce insulin-stimulated muscle blood flow, and increase the insulin gradient across the endothelium, which means less insulin is able to cross to the interstitium; this will reduce the amount of insulin available to bind insulin receptor, reducing glucose uptake (119, 121, 122). 16

31 Impaired insulin action with elevated FFA In skeletal muscle cells, major contributors to insulin resistance include pro-inflammatory cytokines and lipotoxicity. The excess fatty acid supply noted in obesity reaches muscle via the circulation, and muscle will take up fatty acids via dedicated membrane transporters but also through diffusion through cell wall. When these fatty acids enter the cell, they will either undergo fatty acid oxidation in mitochondria, or get stored as intramyocellular triglycerides [IMTG]. If fatty acid supply increases beyond the muscle s ability to divert them for storage in IMTG sink or to undergo oxidation, then intermediate metabolic products are generated including Diacylglycerol [DAG] and ceramides. The IMTG is not the culprit in causing insulin resistance, rather its metabolites including DAG and ceramide that interfere with insulin signaling. Importantly, IMTG can serve as a marker for lipid accumulation inside muscle cells and is a correlate of insulin resistance, but does not in itself cause insulin resistance. In addition, lowering the circulating fatty acid levels without lowering IMTG does not improve insulin sensitivity, and lowering IMTG results in improved insulin sensitivity, which fits with the idea that the effect of IMTG is presumably through generation of intermediary metabolites which impact insulin action (123). We discuss cytokines in more detail below Organelles in FFA-induced insulin resistance in skeletal muscle Mitochondria Mitochondrial content and function are important factors to consider in obesity and T2D associated insulin resistance. Mitochondria are involved in fatty acid oxidation, which is important in metabolizing lipids and energy generation. The impaired oxidative capacity of skeletal muscle in obesity has been attributed to reduced mitochondrial content, and this reduction has been reported in insulin resistant offspring of individuals with type 2 diabetes, who themselves are at risk of developing diabetes later in life (124). In addition, skeletal muscle from T2D and obese subjects have impaired mitochondrial bioenergetics and reduced number (125). In diabetic patients, there is reduced muscle expression of genes linked to fatty acid oxidation including Peroxisome Proliferator-Activated Receptor gamma co-activator-1-alpha [PGC-1α] (126). Muscle in obese and T2D individuals have impairment in oxidative phosphorylation that is worse in T2D and is impaired in obesity compared to lean individuals (127, 128). 17

32 However, whether there is a relationship between intermyocellular fat depot [IMFD] and reduction in oxidative capacity, and whether this is a cause or effect is still a matter for debate (128, 129) Endoplasmic Reticulum stress [ERS] Endoplasmic Reticulum Stress [ER stress] is a cellular response to insults that interfere with normal protein synthesis and folding (130, 131). When faced with a stressor, cells will activate a stress response termed Unfolded Protein Response, the aim of which is to halt protein synthesis and generate molecular chaperones that can assist in protein folding. If this process fails, the cell will activate apoptosis program. There are three arms for UPR: protein kinase-like ER kinase [PERK], ER membrane proteins inositol-requiring enzyme-1 [IRE-1], and activating transcription factor [ATF] and these arms link ER stress, metabolism and insulin signaling. Activation of PERK leads to inhibition of protein production via phosphorylation of Eukaryotic Translation Initiation Factor 2 alpha [eif2α], and this leads to a reduction in folding load of proteins in ER to reduce cellular stress [Figure 1.2]. This protein also leads to activation of NFκB pathway via inhibition of synthesis of IκB leading to inflammatory responses in cell (132, 133). Another effect of PERK action is the activation of ATF4 and induction of genes involved in antioxidant actions and amino acid transfer (134, 135). Activation of PERK also leads to increased expression of CCAAT/enhancer binding protein (C/EBP) homologous protein [CHOP] which is a proapoptotic transcription factor. In addition, it induces another protein called growth arrest and DNA damage inducible protein 34 [Gadd34] leading to a negative feedback of eif2α phosphorylation and attenuation of PERK signaling (134, 135). Activation of IRE-1 leads to splicing of X-box binding domain [XBP-1] and mrna translation of spliced form that translocates to the nucleus and regulates the expression of ER chaperones and proteins involved in ER associated degradation [ERAD] (136). Activity of IRE-1 can also induce apoptosis and inflammatory signaling with other proteins including TNFα receptor associated factor 2[TRAF2] to activate JNK which can be induced independently of XBP-1 splicing ( ). In addition, IRE-1 is proposed to activate ERK, p38mapk and NFκB through interactions with protein complexes including IKK-TRAF2 complex and interaction with non-catalytic region of 18

33 tyrosine kinase [Nck] protein (140). Furthermore, Bcl-2 associated proteins Bax and Bak can also bind IRE-1α subunit activating extended signaling via this pathway and triggering apoptosis. The ATF6 family of proteins includes ATF6α and ATF6β subgroups. The former is a more potent inducer of ER stress related transcription. These proteins enter the Golgi apparatus in response to cellular stress and are cleaved to an active N-terminal isoform that then enters the nucleus to regulate ER chaperones expression (141, 142). In addition, ATF6α can regulate XBP-1 and has been demonstrated to increase levels of ERAD by dimerization with spliced XBP-1(143), and it also mediates the activation of NFκB pathway (144). Recently, ER stress was linked to obesity, primarily ER stress in the fat, liver and pancreatic beta cells, but more recently in muscle as well. Importantly, ER stress results in activation of JNK and this interferes with insulin signaling by inhibiting IRS-1 phosphorylation. Activation in skeletal muscle in this case is probably through the activation of JNK and NF-κB pathways. 19

34 Figure 1.2: Endoplasmic reticulum [ER] stress pathways. ER stress is a cellular response that is activated when cell faces a stressor in environment called unfolded protein response. The three molecules involved in unfolded protein response include protein kinase-like ER kinase [PERK], ER membrane protein inositol-requiring enzyme-1 [IRE-1], and activating transcription factor [ATF]. Activation of PERK leads to inhibition of protein production and reduction in folding load of proteins in ER to reduce cellular stress. This protein also leads to activation of NFκB pathway via inhibition of synthesis of IκB. Activation of IRE-1 leads to splicing of X-box binding domain [XBP-1] that translocates to the nucleus and regulates the expression of ER chaperones and proteins involved in ER associated degradation [ERAD]. Activity of IRE-1 can also induce apoptosis and inflammatory signaling with other proteins including TNFα receptor associated factor 2[TRAF2] to activate JNK which can be induced independently of XBP-1 splicing. ATF6 can regulate XBP-1 and has been demonstrated to dimerize with spliced XBP-1, activating NFκB pathway. 20

35 1.6 Intracellular pathways mediating insulin action Insulin signaling cascade Insulin is a hormone with multiple functions in different organs. Insulin is produced by the β- pancreatic islet cells that sense glucose levels and attempts to maintain it within a tight range. Insulin signals through the insulin receptor that is a tyrosine kinase receptor, composed of two α and two β subunits. On insulin binding, one beta subunit transphosphorlyates its sister subunit on specific tyrosine residues that will under normal physiological conditions leads to the recruitment and phosphorylation of tyrosine residues on the Insulin Receptor Substrate 1-4 molecules [IRS1-4], the main ones in skeletal muscle are the IRS-1 and IRS-2. In muscle, the activation of IRS-1 leads to recruitment of the p85 regulatory subunit of the phosphatidylinositol-3 kinase [PI3K], which will then recruit the p110 catalytic subunit of the enzyme. This will result in the production of Phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P 3 ] by phosphorylation of its substrate Phosphatidylinositol 4,5-biphosphate, and this recruits the kinase Akt ( ). Inhibition of PI3K activity by either using mutant forms or PI3K inhibitors like wortmannin, leads to reduced insulin signaling and glucose transporter 4 vesicles [GLUT4] translocation to cell surface [Figure 1.3] ( ). Downstream of PI3K, insulin signaling diverges to activate the serine/threonine kinases Akt and atypical PKC λ/δ. In muscle and fat, the two isoforms of Akt including Akt1 and Akt2 are activated (152) and then will activate Akt substrate 160 [AS160]. The role of Akt was recognized by means of constitutively active and phosphorylation deficient mutants in muscle (153), SiRNA of both isoforms in adipocytes (154), and with specific inhibitors of Akt 1 and Akt 2 (155). AS160 is believed to act as a Rab-GTPase activating protein [GAP] and Akt phosphorylation of AS160 inhibits this activity and causing the activation of downstream Rab GTPases, which is important in mobilizing the GLUT4 vesicles to the cell surface ( ). Another pathway that is involved in regulating insulin mediated GLUT4 translocation is the mammalian Target Of Rapamycin [mtor] pathway. This pathway acts as a feedback loop with serine phosphorylation and inactivation of IRS-1 in muscle at a site close to where PI3K binds and this limits insulin action in cells and GLUT4 translocation (159, 160). 21

36 Once mobilized to the surface, GLUT4 vesicles fuse with the cell membrane, take up glucose from outside the cell, and then internalize it to the cytosol. As glucose is internalized, it then is either utilized to generate energy [oxidative metabolism] of stored intracellularly [non-oxidative metabolism]. GLUT4 vesicle Insulin receptor P P Insulin PKC,JNK,IKKβ IRS-1/2 P P85/P110 PI3K mtor Akt GLUT4 translocation Protein synthesis AS160 P Glycogen synthesis Hepatic Gluconeogenesis/ lipolysis GLUT4 vesicle Figure 1.3: Insulin signaling cascade. Binding of insulin to its receptor at the hepatocyte, myocyte and adipocyte leads to tyrosine phosphorylation of insulin receptor substrates 1 & 2 [IRS-1 & IRS-2]. Phosphorylation enables the recruitment of the p85 regulatory subunit of Phosphatidylinositol 3 kinase [PI3K] with its associated catalytic p110 subunit which thereby becomes activated. PI3K recruits and activates the kinase Akt. Akt regulates the insulindependent inhibition of gluconeogenesis and glucose output in the liver and inhibits lipolysis in adipose tissue. Akt is also important in protein synthesis and glycogen synthesis. In muscle and fat, Akt phosphorylates the protein AS160 and this step is essential to mobilize Glucose transporter 4 [GLUT 4] to cell membrane. Glucose is then readily taken up via GLUT4 into the cell and channeled into oxidative [energy generation] and non-oxidative [storage] pathways within cells. Mammalian Target Of Rapamycin [mtor] is a negative regulator of IRS proteins providing a negative feedback loop [Adopted from Samaan MC et al, Endocrinology Rounds, SMH, 2008]. 22

37 Insulin functions to enhance lipogenesis, glycogenesis, protein synthesis and inhibiting lipolysis and gluconeogenesis, with a net anabolic effect downstream of PI-3 kinase (161, 162). In parallel to Akt, insulin signals through the small GTPase Rac, which can also lead to GLUT4 translocation (156, ). In addition, insulin activates Mitogen Activated Protein Kinase [MAPK] pathway, which also occurs via IRS-1 and PI3K/Akt and serves additional roles in the cell including gene expression, and cellular differentiation and growth (161, 166) Insulin signaling pathway molecules affected by insulin resistance The binding of insulin to its receptor triggers the acquisition of receptor tyrosine kinase activity and this in turn will phosphorylate IRS-1 on tyrosine residues. The IRS proteins are made of N- terminal Pleckstrin homology [PH] domain that is adjacent to phosphotyrosine binding domain that is associated with a C- terminal that has a number of tyrosine and serine phosphorylation sites. The PH domain is essential for insulin receptor interaction with IRS proteins, which occur through membrane phospholipids, cytoskeleton molecules and other adaptor proteins. The phosphotyrosine binding domain interacts with juxtamembrane domain of insulin receptor (167) and if this contact is affected through serine/threonine phosphorylation then that will adversely impact insulin signaling (168). A third domain is the kinase regulatory loop binding domain, which is only present in IRS-2 and interacts with phosphorylated regulatory loop of insulin receptor and the phosphorylation of two tyrosine residues within this domain are important for this activity (169). The generation of intracellular lipid intermediaries like diacylglycerol [DAG] leads to activation of inflammatory pathways including JNK, IKK, and PKC, and will lead to serine phosphorylation of IRS-1 protein and inhibits its signaling. In addition, the generation of another lipid metabolite called ceramide from intracellular fatty acid processing will interfere directly with Akt signaling and this leads to insulin resistance (163). 23

38 1.6.3 Pathways interfering with insulin signaling and leading to insulin resistance Many of the mechanisms that interfere with insulin signaling do so by interfering with early steps of insulin signaling i.e. IRS-1 phosphorylation. For example, activation of JNK, Protein Kinase C [PKC], and Inhibitor of nuclear factor-kappa B kinase [IKK] results in interference with insulin signaling at this level by causing serine phosphorylation of IRS-1 and reducing its signaling capacity. In addition, other molecules that affect IRS-1 recruitment and action include the inflammatory molecules Suppressor Of Cytokine Signaling-1 and 3 [SOCS1 and SOCS3], whereby they lead to ubiquitinylation and breakdown of IRS proteins in different cell types. Next, we discuss what pathways are involved in inhibition of insulin signaling Protein Kinase C [PKC] isoforms Elevated circulating fatty acid levels results in increased skeletal muscle uptake of fatty acids, with the increased generation of lipid intermediary metabolites including fatty acyl Co-A, ceramide and diacylglycerol [DAG]. Fatty acyl Co-A and ceramide has the potential to activate conventional PKCs and novel PKCs. PKCθ: this isoform has been implicated in muscle and liver insulin resistance, and activation of PKCθ leads to insulin resistance in-vitro and in-vivo (20, 170). Overexpression of PKCθ in a murine muscle cell line showed degradation of IRS-1 protein, reduced insulin stimulated PI3K recruitment and binding to IRS-1, and reduced Akt phosphorylation. Treatment of cells with a non-specific PKCθ inhibitor abrogated insulin resistance (170). In liver cells, exposure to high glucose/high insulin upregulated PKCθ and reduced IRS-1 level and induced insulin resistance. Using RNA interference approach for PKCθ resulted in improved insulin signaling (170). When PKCθ is deleted, knockout mice are protected from HFF-induced insulin resistance. Activation of PKCθ interferes with insulin signaling by serine phosphorylation of IRS-1 protein thereby reducing the tyrosine phosphorylation by the insulin receptor (171). 24

39 PKCε: This isoform plays an important part in hepatic insulin resistance with high fat feeding in rats, as PKCε associates with insulin receptor directly and interferes with insulin signaling. High fat feeding of rats for three days results in hepatic insulin resistance and steatosis, which is associated with the activation of PKCε but not other isoforms of PKC. Treating rats with antisense oligonucleotide against PKCε protects rats from high fat feeding induced insulin resistance and reverses impaired insulin signaling (172) Inhibitor of nuclear factor-kappa B kinase-β [IKKβ] The transcription factor NFκB is a master regulator of several genes including those responsible for production of cytokines, inducible nitric oxide synthetase, Cyclo-oxygenase-2, and growth factors in response to binding of several ligands to immune cell receptors including T- lymphocyte receptors, B- lymphocyte receptors, TLRs, and receptor members of IL-1 family [Figure 1.4] (173, 174). The NFκB pathway is composed of several members including p50, p52, RelA (p65),c-rel, and RelB (175). the structure of these factors is similar with N-Terminus composed of 300 aminoacids, a Rel Homology Domain [RHD] through which DNA binding happens to short DNA stretches known as κb sites in the promoter and enhancer regions of the DNA. RelA, c- Rel, and RelB contain c-terminus transcriptional activation domains that enable them to activate target gene expression, while p50 and p52 do not have those domains so they will repress transcription unless they bind to a protein containing those domains like RelA, c-rel, RelB, or Bcl-3. The p50 is produced by cleavage of a larger precursor molecule p105, and p52 is the result of cleavage of p100 (175). The NFκB protein activity is inhibited by binding with IκB proteins in the cytosol, including IκB ε, α, β, γ, Bcl-3, p100, and p105. When cytokines signal through their receptors, two molecules that are part of the IKK signalosome get activated, including IKK-1 and IKK-2. These will phosphorylate the IκBα leading to its degradation, and this will lead to release of NFκB protein and then it enters into the nucleus (176). In the nucleus, this transcription factor binds to specific pro-inflammatory cytokine gene promoter sequences and activate inflammatory gene expression. 25

40 The inactivation of NFκB and the reduction in NFκB production leads to reduced production on pro-inflammatory cytokines and improved insulin sensitivity and dyslipidemia in ob/ob mice and fa/fa rats. In the liver, NFκB production causes insulin resistance, where overexpression of IKKβ induces hepatic and peripheral insulin resistance and upregulate pro-inflammatory cytokine expression (177). Selective deletion of IKKβ in liver results in retention of insulin sensitivity but development of peripheral insulin resistance, while deleting IKKβ in myeloid cells protects rodents against insulin resistance (98). Cell membrane Cytokine receptor TLR Cytokines and inflammatory proteins IκBα IκB kinases P IκBα Degradation IκBα p50 p65 p50 p65 Nucleus p50 p65 mrna Inflammatory genes Figure 1.4: The canonical IKK-NFκB pathway. NFκB is silenced by binding IκBα. Several stimuli including free fatty acids and cytokines signaling through different receptors activate the IκB kinases that degrade IκBα and release p50/p65 heterodimer to enter the nucleus. In the nucleus, NFκB binds to specific sites on DNA of pro-inflammatory molecules called κb sites and this activates gene transcription. The mrna generated then is processed in cytosol and protein is synthesized and then released by these cells. 26

41 From the above evidence, the inhibition of NFκB may represent a viable strategy to combat inflammation and insulin resistance. One of the groups of drugs that has been used to this effect are the Salicylates, which are composed of acetylated [e.g. Aspirin] and non-acetylated [e.g. Salsalate] compounds. Both groups inhibit inflammation but via distinct pathways, with aspirin inhibiting Cyclooxygenase pathway and, while Salsalate and other non-acetylated formulations act on IKK-NFκB pathway (178). These drugs have been known to reduce blood glucose for almost a century, and only recently a mechanism was proposed in which salicylates inhibit NFκB in insulin responsive tissues by inhibiting IκBα phosphorylation and degradation in heterozygous IKKβ mice (179), and has been shown to improve HbA1c in humans with type 2 diabetes (177, 178, 180) c-jun N-terminal kinase [JNK] JNK, as well as ERK and p38 MAPK, are part of a system characterized by three kinases one phosphorylating the other in a cascade-like fashion resulting in intracellular actions. JNK is a serine threonine Mitogen Activated Protein Kinase [MAPK] that is part of the c-jun N-terminal kinase [JNK]/AP-1 pathway [Figure 1.5]; it is an important factor in development of insulin resistance and inflammation in obesity and T2D. Nutritional and inflammatory stimuli trigger the activation of the JNK pathway, and it is an important regulator of insulin resistance in obesity. Obesity causes activation of this pathway is skeletal muscle, liver and fat via elevated FFA or increased TNFα. Loss of JNK prevents IR in DIO and genetic obesity, and liver JNK knockdown leads to lower blood glucose and insulin levels. JNK inhibits insulin action of IRS-1 tyrosine phosphorylation and induces the production of several pro-inflammatory molecules once activated (181). JNK is encoded by Mapk8 gene; Jnk1 knockout mice have improved insulin sensitivity on HFF, which occurs via reduced serine phosphorylation at position 307 of IRS-1 protein. These animals also are less likely to put on excessive weight with HFF and maintain a certain degree of insulin sensitivity; this indicates that JNK has actions that affect adiposity in addition to its effect on insulin signaling. JNK2 also has a role in metabolic regulation, as its knockout that is bred to heterozygous Jnk1 background [Jnk1 +/- Jnk2 -/- ] have a similar phenotype to Jnk1 -/-, showing that there is crosstalk between Jnk1 and Jnk2 in modifying insulin action (181, 182). 27

42 Extracellular Regulated Kinase [ERK] The extracellular regulated kinase pathway [Figure 1.5] has two isoforms ERK1/2, that are stimulated by many factors including cytokines, growth factors, viral infections, carcinogens and protein ligands that stimulate G-protein coupled receptors (183). The three kinase phosphorylation cascade can be activated by Ras which is a proto-oncogene. This will activate C-Raf1, B-Raf, or A-Raf. This will then activate the Mitogen activated protein kinase kinase [MAP2K] which will then phosphorylate ERK1/2. This will in turn act on transcription factors and promote intracellular signaling actions that are involved in cellular growth. There is more recent evidence that ERK1 knockout mice on ob/ob background [ob/ob-erk1-/-] are protected from hyperglycemia and have improved insulin sensitivity and less hepatic steatosis with HFF. These mice also show a reduction in inflammatory cytokine gene expression in adipose tissue of high fat fed mice (184), putting forward ERK1 as a potential therapeutic target to improve insulin sensitivity in obesity p38 Mitogen Activated Protein Kinase [p38mapk] The p38 Mitogen Activated Protein Kinase is another MAPK that like ERK can be stimulated via several ways including cytokines, growth factors, LPS, UV radiation, heat shock, and osmotic shock. There are four isoforms of p38mapk including α, β, γ and δ [Figure 1.5] (185). These isoforms are expressed in different tissues but α isoform is most significantly expressed in skeletal muscle. Different stimuli will activate a diverse group of mitogen activated protein kinase kinase kinase [MKKK] that is tissue-dependent, and this in turn will activate mitogen activated protein kinase kinase 3/6 [MKK 3/6], and this will phosphorylate p38mapk on threonine 180 and tyrosine 182 in all isoforms. This will then result in transcription factors release and gene transcription (186, 187). There is also evidence to implicate other molecules in activating these MAPK isoforms including the GTPases Rac, Rho, cdc42, and Rit ( ) along with GPCRs (191). The p38mapk regulates the synthesis of pro-inflammatory cytokines including TNFα, IL-1 in LPS-stimulated monocytes (192), regulation of production of IL-8 in response to IL-1(193), and IL-6 production in response to TNFα and this appears to happen at post-transcriptional level (194). 28

43 In addition, p38mapk deficiency in Th1 cells inhibits production of INFγ production with IL12/IL18 stimulation compared with secretion induced by TCR, which suggests that its activity is restricted to one of the pathways involved in INFγ secretion (195). Growth factors Cytokines Cellular stress Growth factors Cellular stress MEKK 1/4 MLK3 ASK1 MLK3, TAK, DLK A-Raf, B-Raf, C-Raf MKK 4/7 MKK 3/6 MEK 1/2 JNK p38mapk ERK Inflammation Growth Differentiation Apoptosis Growth differentiation Figure 1.5: Mitogen Activated Protein Kinase [MAPK] pathways. These pathways include JNK, ERK 1/2 and p38mapk. They are composed of three-kinase cascades, whereby at the top of each cascade there is a MAP kinase kinase kinase [MAP3K] that is activated by various stimuli including growth factors, cytokines and cellular stressors. These will then activate MAP kinase kinase [MAP2K] that in case of JNK is MKK 4/7, p38mapk is MKK3/6, and ERK is MEK 1/2. These MAP2K will activate their respective pathways causing cellular responses including inflammation, growth, apoptosis and differentiation. 29

44 1.7 Cytokines, chemokines and adipokines play important roles in causing inflammation and insulin resistance Cytokines are peptide molecules secreted by cells in response to other cytokines, infectious, chemical, and nutritional triggers and help modulate the cellular responses to these stimuli. The production of cytokines is complex and is context dependent, and the cytokines have different functions in different tissues depending on the stimulus, its duration, and concentration (53, 81). Cytokines exert their actions by acting on cells in an autocrine, paracrine, or even endocrine fashion depending on their stability and half-life in the circulation. They exert their function by binding to their respective receptors on cell surface which recruit the Janus kinases [JAKs] (196, 197). The JAKs will then phosphorylate the receptor that then will lead to binding to Signal Transducers and activators of transcription [STATs] that after phosphorylation by JAKs will dimerize and translocate to the nucleus and bind to specific sequences in their gene promoters triggering transcription of these genes. There are four JAKs [JAK1, JAK2, JAK3, Tyk2] and seven STATs [STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, STAT6] and the binding of JAKs to STATs occur in various combinations that are pre-determined and well documented (198, 199). This binding and activation triggers gene expression and cell differentiation, growth, division, movement and apoptosis. The signaling process is controlled via classical negative feedback loops consisting of the Suppressor Of Cytokine Signaling [SOCS] molecules which limit production of these cytokines and abrogate their actions ( ). Some cytokines have the ability to attract cells to move in a particular direction in a concentration dependant manner from low to high concentration, and these are called chemotactic cytokines or chemokines (204). These chemokines are produced by virtually all cells and bind to endothelial cells or other structures or to the extracellular matrix, and help the cell move in a directional fashion or chemotactic manner from low to high concentration of the chemokine. Non-directional or chemokinetic motion does not require chemokines ( ). The adipose tissue and skeletal muscle cells produce many cytokines and chemokines. Some molecules are strictly produced by adipose tissue and are categorized under the term adipokines [e.g. adiponectin and leptin] [Figure 1.6]. Some of these molecules are discussed below. 30

45 1.7.1 Tumor necrosis factor-α [TNFα] This cytokine was the first to link obesity with inflammation and insulin resistance. As noted above, the first evidence of TNFα involvement in obesity and insulin resistance came from Hotamisligil et al (25), where TNFα was elevated in the adipose tissue of HFF mice, and this was confirmed in ob/ob mice and subsequently noted in humans, where it is elevated in obesity and is reduced with weight loss (209). This finding was replicated in skeletal muscle in obese, insulin resistant, and T2D humans. In addition, the absence of TNFα or its receptor resulted in improved insulin sensitivity and less inflammation in adipose tissue in DIO and genetic mouse models of obesity (26). The adipocyte was considered the main source of TNFα in obese adipose tissue (93, 94). Subsequently, it became clear that infiltrating macrophages are the main source of several cytokines produced from adipose tissue, including TNFα. Regardless of its source, TNFα causes local inflammation and triggers further cytokine production and lipolysis in adipocyte, and causes enhanced macrophage cytokine production, leading to insulin resistance. TNFα causes inflammation and insulin resistance in metabolic organs through several mechanisms. It inhibits Peroxisome Proliferator Activated Receptor-γ [PPARγ], a master regulator of adipogenesis in adipose tissue. Mechanistically, this occurs through the activation of NFκB pathway that lead to PPARγ mrna degradation, and activation of caspases that lead to PPARγ protein degradation (111, 210). The net effect is increased lipolysis and reduced triglyceride storage in adipose tissue, with subsequent increased TG storage in muscle (211). TNFα also interferes with insulin signaling in skeletal muscle and adipocyte in IRS-1 dependent and independent manners. Short term TNFα treatment of adipocytes stimulates serine phosphorylation of IRS-1 protein and inhibition of insulin action (212) via activation of serine kinases including Inhibitory-κB kinase (213), JNK (214), and mtor (215), and serine phosphorylation of IRS-1 will inhibit tyrosine phosphorylation mediated by insulin (213). The activation of NFκB and JNK pathways with TNFα exposure will increase serine phosphorylation of IRS-1 and enhances its degradation, which ultimately results in reduced Akt and AS160 phosphorylations and reduced glucose uptake (214, 216). TNFα will also lead to induction of SOCS1 and SOCS3 proteins, which can bind to the insulin receptor and inhibit its association with IRS-1and subsequent tyrosine phosphorylation (217). 31

46 TNFα also suppresses AMPK, which leads to a reduction in muscle fatty acid oxidation and increased intracellular lipid intermediary metabolites including DAG, and this interferes further with insulin signaling via activation of inflammatory pathways (218). In adipocytes, TNFα can also reduce gene expression of IRS-1, insulin receptor, GLUT4, hormone sensitive lipase, adiponectin, long chain fatty acyl CoA synthetase and Pparg, thus impairing insulin sensitivity in IRS-1 independent manner (219, 220) Interleukin-6 [IL-6] This cytokine has been implicated in several inflammatory conditions including autoimmune disease (221, 222). There are two receptors for IL-6, including IL-6 receptor that is specific receptor for the cytokine, and another receptor that is common also to several other cytokines called gp130 (223, 224). The signaling from both receptors involves activation of JAK-STAT pathway and specifically STAT 1 and 3. IL-6 can act as a pro-inflammatory or anti-inflammatory cytokine in a tissue-based manner. It is produced by macrophages, liver, skeletal muscle and adipose tissue. In human studies, it has been shown that IL-6 levels correlate well with insulin resistance, and are increased in age related obesity and reduced with weight loss. Its production in adipose tissue is several folds higher in visceral adipose tissue compared to subcutaneous adipose tissue, and is likely to play a significant role in hepatic insulin resistance (225). Mouse models of IL-6 knockout have yielded conflicting results in terms of its effect on obesity. It has been shown that IL-6-/- mice develop age related obesity; lipid and carbohydrate metabolism dysregulation with insulin resistance and hyperleptinemia was noted in one model that was not replicated in another study, and this is likely that in the first study these mice had reduced fat utilization and enhanced thermogenesis. In the second study the only abnormality noted was elevated glucose levels post glucose tolerance test with normal leptin and TNFα and hyperleptinemia noted only in the high fat fed group (226, 227) Monocyte Chemoattractant Protein-1 [MCP-1] MCP-1 is an inducible chemokine that represents one of the potential links between adipose tissue and skeletal muscle in insulin resistance, and a connection by which immune-metabolic interactions occur in response to metabolic stimuli. The main sources of MCP-1 are macrophages and endothelial cells, where it is secreted in response to inflammatory and metabolic stimuli, but 32

47 is also produced by skeletal muscle and adipose tissue. MCP-1 is one of the main factors that attract macrophages to obese adipose tissue ( ). Knockout mouse models have reduced macrophages in adipose tissue and reduced IR with HFF. Over-expression of MCP-1 results in enhanced macrophage migration into adipose tissue (234, 235). However, in another MCP-1 knockout model, HFF did not result in enhanced macrophage infiltration, arguing for the presence of other factors that may compensate for MCP-1 deficiency. In addition, these mice develop glucose intolerance with increased plasma glucose levels and reduced adiponectin. Therefore, MCP-1 may have a role to play in energy metabolism (236). The notion that MCP-1 has additional roles beside macrophage chemoattraction is supported by evidence in which treatment of muscle cells in culture leads to reduced glucose uptake, and inhibition of insulin signaling at levels that are even lower than those in the systemic circulation (237). This indicates that there are additional roles for MCP-1 beside macrophage attraction and that it has direct effects on glucose metabolism. The MCP-1 receptor, called Chemokine (C-C motif) receptor-2 [CCR2], has been implicated in HFF induced IR, and has been shown to have a possible role in eating behavior, as CCR2 -/- mice have reduced caloric intake which remains unexplained (238) Interleukin-8 [IL-8] Human adipocytes secrete IL-8 in response to TNFα and IL-1β and in response to HFF. Adipocytes express IL-8 receptor [CXCR2] and IL-8 signaling through its receptor induces its own production, and this in turn will lead to reduced insulin mediated Akt phosphorylation in adipose tissue (208). Using inhibitors, this effect on insulin signaling is shown to take place via activation of MAPK pathway (239, 240). In clinical settings, the levels of circulating IL-8 are increased in obese humans and correlate with clinical parameters of adverse metabolic outcomes including body mass index [BMI], waist circumference, and Homeostasis model assessment [HOMA] score for insulin resistance (241). IL-8 may have a role in the development of skeletal muscle insulin resistance, although the isolated effect of IL-8 on skeletal muscle is not documented. IL-8 is a potent neutrophil chemoattractant, and its secretion by the adipocyte and skeletal muscle under different settings including HFF and obesity may underscore the importance of the IL-8 neutrophil axis in the 33

48 metabolic processes impairment in obesogenic environment, not only via attraction of neutrophils, but directly on metabolic organs (242). This IL-8 may come from muscle cell itself or from IMFD adipocytes, and it is possible that other immune cells including macrophages that produce IL-8 may have an impact also. Adipokines involved in metabolism, inflammation & insulin resistance Adipokines include molecules that are either mainly produced by adipose tissue like adiponectin and leptin, or expands to comprise some of the most widely secreted cytokines in body. Most of the cytokines described above e.g. TNF, IL-6, IL10 are all adipokines Adiponectin This adipokine has a wide range of biological functions, mostly to increase insulin sensitivity. Its levels are reduced with visceral obesity and IR, TNFα treatment (243), IL-6 treatment (244), and increase with weight loss and PPARγ stimulation (245). Adiponectin circulates in three forms, a trimeric [low molecular weight], hexameric [medium molecular weight], and a multimeric [high molecular weight] forms. Adiponectin is an anti-inflammatory adipokine that acts in part through inhibition of TNFα-induced adhesion molecule expression. Treating obese mice with adiponectin results in reduced hyperglycemia, lower FFA, and increased insulin sensitivity (246). While adiponectin deficiency leads to insulin resistance with high fat and high fructose feeding, the evidence of its effect on insulin resistance is inconsistent, with studies showing that it does or does not cause insulin resistance (247, 248). Adiponectin has two receptors, the deletion of both results in insulin resistance with HFF. On the other hand, over-expressing adiponectin in ob/ob mice leads to obesity, but is associated with increased levels of secretion of high molecular weight isoform, which reverses diabetic phenotype and normalizes glucose and insulin levels. The expansion of the AT results in increased levels of adiponectin but only in a modest range of 2-3 folds. Adiponectin reduces liver fat content, increases subcutaneous adipose tissue, and increases insulin sensitivity. In mice model of sepsis, adiponectin knockout mice demonstrated significant mortality and induction of inflammatory responses with increased neutrophil infiltration to peritoneal cavity post irritant injection and up-regulation of inflammatory cytokines and 34

49 chemokines in immune cells and adhesion molecules in the aorta. These effects appeared to be mediated via high molecular weight adiponectin, ascribing a role for this adipokine in improved survival and endothelial inflammation in sepsis (249) Leptin Leptin is produced mainly by adipose tissue but also by muscle, placenta, bone marrow and may be the brain. It has important effects on energy and glucose homeostasis (250). While it has a major action at the level of the central nervous system where it controls appetite, and also has important actions on peripheral tissues. Serum levels correlate with amount of energy stored in adipocytes in mice and humans (251).Overall, leptin has significant input into whole-body glucose metabolism, shown by its ability to reverse hyperglycemia in ob/ob mice even before weight loss is evident, and the improvement in glucose levels in humans with lipodystrophy and leptin deficiency. Leptin levels are elevated in obesity, party by the expansion in adipose tissue and partly by leptin resistance in the central nervous system [CNS]. Indeed, leptin treatment fails to correct hyperglycemia in obese patients, a result attributed to this central resistance to its actions (252). The mechanisms of leptin resistance involve the reduction of its uptake into the CNS and the elevation of SOCS3 protein expression; SOCS3 binds to leptin receptor and phosphorylated JAK protein, and this inhibits STAT binding to the receptor and its activation (252). Leptin affects insulin sensitivity through different mechanisms in various organs; in skeletal muscle, it reduces intramyocellular fat and activates AMPK. In the liver, it acts by reducing triacylglycerol deposition, while in pancreas leptin acts on islet cells inhibiting insulin release. All these actions argue favorably for a role of leptin in glucose homeostasis(252). Leptin induces the production of pro-inflammatory cytokines including TNFα, IL-6, and IL-12 from monocytes and macrophages; it also induces production of chemokines like CCL2 and angiogenic factors in hepatic stellate cells via activation of NFκB pathway (253). 1.8 Adipose tissue-secreted factors and metabolic-immune interactions It has been recognized for almost two decades that obesity is associated with chronic low-grade inflammation (36, 254, 255), one of the main features of which is pro-inflammatory cytokine 35

50 production (21, 25, 27). While the source of these cytokines was initially thought to be the adipocyte (25), it was more recently appreciated that one of the main sources of these cytokines are the macrophages and perhaps other immune cells that infiltrate expanding adipose tissue in obesity (93, 94, 115, 256). In addition, lipotoxicity with increased fatty acid release from adipose tissue contribute to the initiation and progression of the inflammatory state seen in obesity. Proinflammatory cytokines and lipotoxicity collude to trigger insulin resistance in metabolic organs including fat, skeletal muscle and liver. Subsequently, the compensatory increase in insulin secretion that occurs with insulin resistance may not be maintained as the pancreatic β-cells fail ultimately leading to type 2 diabetes (28, 256, 257) Effects of cytokines and lipotoxicity on skeletal muscle insulin sensitivity Circulating cytokines and fatty acids and those produced locally from intermyocellular fat depot jointly affect skeletal muscle. Macrophages infiltrate IMFD when it expands with high fat feeding and obesity; the products of both macrophages and adipocytes result in local muscle inflammation, insulin resistance and attraction of other macrophages (93). As the IMFD depot is in immediate vicinity of skeletal muscle, it is likely that direct and indirect muscle-macrophage crosstalk will impact both cells. Insulin resistance is probably caused by several mechanisms including fatty acid oxidation defects due to effects on mitochondrial biogenesis, oxidative stress, accumulation of lipid intermediates in muscle, and effects of pro-inflammatory cytokine on insulin signalling ( ). While research has focused on macrophage-adipose tissue interactions in obesity, other interactions including skeletal muscle-macrophage crosstalk are possible and less documented. There is evidence that macrophages infiltrate skeletal muscle in obesity. It has been recently noted that high fat feeding increased macrophages in muscle (265), and deletion of PPARγ in myeloid cells, a master regulator of adipogenesis and inflammatory response, also led to detection of macrophage markers in muscle (266). These macrophage markers were found rather expectedly in IMFD, and rarely between myofibrils with high fat feeding using immunohistochemistry staining with macrophage markers (93) and bone marrow transplant experiments in a conditional knockout model of CD11c, which is a marker of dendritic cell/macrophage pro-inflammatory activation (267). 36

51 Genes Epigenetics Diet Lifestyle Adiponectin Leptin Visfatin Resistin RBP4 FFA TNFα IL-1 IL-6 PAI-1 IL-8 MCP-1 Immune cells Environment Production of factors that attract immune cells Inflammation Lipotoxicity Insulin resistance Figure 1.6: Obesity-associated mechanisms leading to insulin resistance. The effect of obesity on adipose tissue is mediated via genetic, epigenetic, life style and environmental factors that lead to adipose tissue production of free fatty acids [FFA], inflammatory cytokines, chemokines and adipokines that cause adipose tissue inflammation and attraction of macrophages. When macrophages arrive at adipose tissue they become activated and produce more pro-inflammatory cytokines that lead to insulin resistance. in liver, this lead to failure of insulin to curb hepatic glucose output; in skeletal muscle this leads to less glucose uptake, and in adipose tissue to reduced anti-lipolytic action of insulin leading to lipolysis and reduced adipogenesis. There is also evidence that hypothalamic resistance to leptin and insulin may play a role in obesity and insulin resistance. In this last paper, conditional knockout of CD11c reduced macrophage presence in muscle and at muscle-fat junction. In the latter case, where macrophages were detected at the muscle-fat junction, this raises the question of whether this is a chemokinetic [non-directional cell migration] or a true chemotactic response [i.e. true directional movement of macrophage within a chemokine gradient with cells moving up the gradient from low to high concentration of chemokine] to factors produced by muscle. Regardless of why or how do the macrophages get 37

52 there, they will secrete their cytokines into muscle. In human studies, macrophages were detected in skeletal muscle from obese non-diabetic subjects and this was positively associated with BMI and negatively associated with insulin sensitivity (268). In addition, macrophages were detected in muscle from subjects with normal glucose tolerance but at much lower than in adipose tissue (269). Other studies failed to demonstrate the presence of macrophage markers using microarrays of muscle from high fat fed mice (94), and no increase of macrophage markers in muscle was noted in severely obese humans undergoing lifestyle intervention program (270). Furthermore, one study demonstrated detection of CD11c+ve cells [dendritic cell marker] in muscle from high fat fed mice; fatty acid treatment of bone marrow derived macrophages and dendritic cells induced inflammation in the latter but not the former. Furthermore, conditioned medium from Free Fatty Acid treated wild type bone marrow dendritic cells, but not bone marrow derived macrophages, lead to reduced glucose uptake in L6 myotubes (271) Macrophage interactions with skeletal muscle: Rationale for thesis work The research over the past two decades helps us derive extensive data that relates obesity to inflammation and insulin resistance, chief among them is the role of cytokines, chemokines, and lipotoxicity in causing insulin resistance in obesity. The evidence for and against the infiltration of skeletal muscle by macrophages remains controversial, but appears to be leaning towards the former conclusion. i.e. macrophages infiltrate skeletal muscle in obesity and HFF in rodents and humans. The main question that these studies do not answer is the potential pathways or molecules involved in macrophage recruitment to muscle. Macrophages have been located on several occasions in the IMFD or in skeletal muscle at the vicinity of this depot. So it is unclear if the presence of macrophages in skeletal muscle is related to true chemotaxis in response to factors produced by muscle, or is mere chemokinesis, with macrophages mobilizing within IMFD and accidentally arriving at muscle. 38

53 The other question that remains unanswered is in relation to interaction between muscle and macrophages once the latter cells infiltrate muscle and the products from both cells that mediate their crosstalk in context of elevated fatty acids. It has been shown that macrophages respond to saturated fatty acids by production of factors that cause muscle insulin resistance with reduced glucose uptake and induction of pro-inflammatory cytokine gene expression (272). The focus of this work was on providing information that fatty acid treatment of macrophages can lead to secretion of factor(s) that contribute to muscle cellmacrophage crosstalk. We take this work one step further by looking at the effect of the saturated fatty acid palmitate on muscle-macrophage and macrophage-macrophage crosstalk. 1.9 Hypotheses This thesis is guided by two hypotheses: I. That muscle treatment with saturated fatty acids results in production of factors that attract macrophages, along with the activation of inflammatory pathways within the muscle cells and reduction in muscle insulin signalling. II. That macrophage exposure to saturated fatty acids activates their inflammatory pathways and leads to production of factors that attract other immune cells. Based on the results, we also propose to determine what potential factor(s) are involved in macrophage-muscle and macrophage-macrophage crosstalk when cells are challenged with saturated fatty acids. 39

54 2.1 Cell culture methods L6GLUT4myc myoblasts 2 Chapter 2: Methods This rat muscle cell line has a myc epitope inserted to the first exofacial loop (163, 272). Cells were grown in 5% Co 2 at 37 C in Alpha modification of Eagle's medium (AMEM, Wisent Inc, Montreal, QC), supplemented with 10% fetal bovine serum [FBS] (Wisent Inc, Montreal, QC) and 1% Antibiotic/Antimycotic (Wisent Inc, Montreal, QC). Cells were passaged every two days and discarded when they reached passage number ten RAW264.7 macrophages Cells were grown in AMEM supplemented with 10% FBS for migration to myoblast conditioned medium [MyoCM] and in RPMI/10% FBS for migration to macrophage conditioned medium [MØCM]. Cells were passaged every two days by scraping at 1:10 ratio Isolation of primary rat peritoneal macrophages Sprague Dawley rats ( grams) were from Charles River labs, and peritoneal macrophage isolation was done as previously described with some modifications (273). Ten milliliters of aged 4% Brewer's Thioglycollate was injected into peritoneal cavity after skin sterilization with 70% ethanol. After five days, rats were euthanized using Co 2 and 60 ml ice cold RPMI /10% FBS was injected into the peritoneal cavity. A wide bore needle was inserted into midline and typically ml was recollected. The retrieved medium was centrifuged at 1500 RPM for 5 minutes at 4 C and PBS was used to wash cells twice and cells were then resuspended in RPMI/10% FBS. Cells were seeded at a ratio of 30 ml retrieved medium per one 15 cm dish and left in incubator for 2 hours to allow macrophages to adhere to plastic and eliminate nonmacrophage cells in the peritoneal fluid collected. 2.2 Generation of conditioned medium from muscle cells and macrophages Generation of conditioned media from BSA and Palmitate treated myoblasts L6GLUT4myc myoblasts were seeded in 10 cm tissue culture plates in AMEM medium/10%fbs and 1% Antibiotic/Antimycotic. When cells were subconfluent, they were 40

55 treated in mm Bovine Serum Albumin [BSA] or 0.2mM palmitate [PA] (see below) for 24 hours. Cells were washed several times with PBS and fresh medium added (AMEM supplemented with 2%FBS and antibiotic/antimycotic) for 24 hours. The medium collected at the end of this incubation is called myoblast conditioned medium (MyoCM). The medium was centrifuged at 1500 RPM for 5 minutes and then aliquoted and frozen immediately at -80 C or used for experiments at the time of collection. Viability of cells was tested with trypan blue and consistently found to be more than 92%.Only cells in early passages (1-4) was used in generation of conditioned media Generation of conditioned media from BSA and palmitate treated primary rat peritoneal macrophages Primary rat peritoneal macrophages were isolated as above and treated as previously reported with modifications (272). Cells were treated in 0.5 mm palmitate and BSA control for 6 hours. Palmitate and BSA were washed off with PBS and fresh medium added for 16 hours. This medium was then collected and centrifuged for 1500 RPM at 4 C for 5 minutes, and immediately aliquoted and frozen at -80 C till further use. This is called Macrophage Conditioned Medium (MØCM). 2.3 Macrophage migration assay The macrophage migration assay was carried out utilizing transwell system (Corning, 8 micron pore size membrane). Six Hundred microlitres of conditioned medium from BSA and Palmitate treated muscle cells or from macrophages was added to the lower chamber of the transwell system, and RAW264.7 macrophages were added to the upper chamber at 1x10 6 cells/100μl. Cells were allowed to migrate at 37 C for three hours. The medium containing macrophages in the upper chamber was then aspirated and cells that have not migrated were removed by gentle wiping of the upper surface of the membrane with Qtips soaked in PBS. Membranes were then immersed in 4% paraformaldehyde in PBS with 4', 6-diamidino-2-phenylindole [DAPI] for 15 minutes and protected from light at room temperature. The wells were washed six times with PBS. Membranes were then separated from the plastic frame and mounted on glass slides using fluorescence mounting medium (Dako, CA) and 18mm cover slips applied. Slides were left overnight and analyzed next day using Nikon TE2000 Long term inverted microscope with high resolution monochrome camera for fluorescent imaging at the Sick Kids 41

56 imaging facility [excitation 358nm, emission 461nm]. Cells that completed migration were defined as cells seen on the lower surface of the membrane and were counted in five representative fields at 10x magnification per slide. Quantification was done by counting the total number of cells that completed migration per condition and analysis done using Volocity software [Perkin Elmer]. This software captures cells based on cell size [70 micron] and their fluorescent intensity, and has capabilities to separate adjacent cells. Conditions were run in duplicates with appropriate controls as discussed below. 2.4 Preparation of whole cell lysates for western blot Muscle cells and macrophages were grown in 6 well plates and treated as above. At the end of treatment, cells were washed twice in ice cold PBS and 200μl of 1% triton in PBS with protease inhibitor cocktail (1mM benzamidine, 10μM E-64, 1 μm leupeptin, 200 μm phenylmethylsulphonyl fluoride [PMSF] (Sigma -Aldrich, St. Louis, MO) and phosphatase inhibitors (100nM okadaic acid, 10mM sodium fluoride, and 1mM sodium orthovanadate). Lysates were then scraped and transferred to 1.5 ml eppendorf tube on ice. Lysates were syringed five times through 21- gauge needle and then centrifuged at 13,000 RPM for 10 minutes at 4 C. Supernatants were transferred to new tubes on ice and frozen at -80 C until further use. Samples were thawed on ice, and μg of protein taken and mixed with equal volume of 2X Laemmli sample buffer (LSB), and sample heated at 97 C for 5 minutes and then put on ice for 1-2 minutes. Samples were centrifuged briefly and then loaded into 10-13% sodium dodecyl sulfate polyacrylamide gel electrophoresis [SDS-PAGE] gels and run in running buffer (274). 2.5 Determination of protein concentration of lysates Protein concentration of lysates was determined using Bicinchoninic acid [BCA] Protein Assay Reagent [Thermo Fisher Scientific, Rockford, IL]. 3μl protein per condition was added to each well of a clear 96 well plate. A standard curve was constructed using BSA standards 2mg/ml (Pierce Biotechnology, Rockford, IL). The protein concentration was then read at wavelength 562 in plate reader. Concentration was calculated and μg protein loaded to gels for western blot analysis. 42

57 2.6 Gel preparation for western blots SDS-PAGE gels were used for separating protein samples. 10% gels were used for MAPKs and 13% gels used for MCP-1 detection. Gels were run in Miniprotean Tetra cell gel box [Bio-Rad, Hercules, CA] at 100volts for 2 hours and then proteins were electrotransferred onto polyvinylidene fluoride [PVDF] membranes using Miniprotean 3 cell transfer system [Bio-Rad, Hercules, CA] in transfer buffer [components in recipes section] overnight in cold room at 40 volts/hr or at 100 volts/hr for 2 hours at room temperature. Following transfer, membrane was blocked using 3% BSA for one hour at room temperature (275). 2.7 Membrane blotting Membranes were then blotted for proteins using primary antibodies in 1%BSA/wash buffer. To measure phosphorylation of proteins, we blotted with anti-phospho JNK antibody (Thr183/Tyr185,1:1000, Cell Signal, Beverly, MA), anti-phospho ERK antibody (p44/42 MAPK, 1:1000, Cell Signal, Beverly, MA), anti-phospho p38 MAPK antibody (Thr180/Tyr182, 1:1000, Cell Signal, Beverly, MA). To measure total proteins, we used anti JNK antibody (1:1000, Cell Signal, Beverly, MA), anti ERK antibody (1:1000, Cell Signal, Beverly, MA), anti p38 MAPK antibody (1:1000, Cell Signal, Beverly, MA),anti Inhibitor of IKKb alpha (IKBα, Cell Signal, Beverly, MA). MCP-1 antibody was from Abcam [ 1:1000, Abcam, Cambridge, MA], Monoclonal anti-actn1 [1:5000, monoclonal mouse IgM, clone BM-75.2, Sigma- Aldrich]. Membranes were washed three times for 15 minutes each on shaker and then secondary antibody applied for one hour at room temperature in 1%BSA/wash buffer. All secondary antibodies were diluted at 1: After secondary antibody incubation, membranes were washed in wash buffer three times for 15 minutes each. Protein bands were detected using enhanced chemiluminescence (ECL, Bio-Rad, Hercules, CA). Membranes were scanned and quantified using Image J software (National Institute of Health, Bethesda, MD). 2.8 Inhibitors and blocking antibodies NFκB inhibitor Pyrrolidine dithiocarbamate ammonium [PDTC, 25μM], acting via inhibition of NOS pathway, was from Sigma-Aldrich. JNK inhibitor [SP600125, 10 μm], ERK inhibitor [U126, 10 μm], p38 MAPK inhibitor [SB203580, 20 μm] were from EMD4Biosciences [EMD4 43

58 Biosciences, Gibbstown, NJ]. MCP-1 neutralizing antibodies and IgG controls were from BioLegend [BioLegend, Roswell, CA]. 2.9 RNA generation for RT-PCR Muscle cells or macrophages were grown to confluence and treated as above. At the end of treatment, medium was aspirated and cells washed twice in ice cold PBS. Total RNA was isolated using RNAeasy mini kit from Qiagen according to manufacturer's instructions. Then endonuclease-free water was added and the column centrifuged at RPM for 1 minute and RNA was eluted into Eppendorf tubes that were frozen at -80 C until further use (44, 276) Reverse transcription polymerase chain reaction (RT- PCR) Reverse transcription polymerase chain reaction (RT-PCR) was used to evaluate gene expression profiles of cells using one step RT-PCR kit from Qiagen (277). RNA was added at a final concentration of 200 ng to 25 μl final volume per reaction. The reactions were run on BIO-RAD C1000 thermal cycler [Bio Rad, Hercules, CA]. A proportion of the sample (10 μl) was run in 1% agarose gel in 1% TAE buffer with 4% ethidium bromide at 100 volts for minutes. Gels were imaged using Gel Doc analysis station NEFA measurements Non-esterified fatty acid [NEFA] content of conditioned media was measured using NEFA HR kit (WAKO, Richmond, VA). Cells were grown in 6 well plates and treated with BSA or PA for 24 hours. Then, 1 ml of fresh medium added of AMEM/2%FBS per well. 24 hours later, 50μl of this freshly generated medium was used for the experiments. Samples were tested in duplicates and quantified using plate reader at wavelength 550. A standard curve was constructed with standard provided (oleic acid, 0.1mM/ml) that included 0-10nM standards Endotoxin measurement in conditioned media The conditioned media and their individual components including AMEM and FBS as well as BSA and palmitate and controls were tested for endotoxin contamination using ToxinSensor Chromogenic LAL endotoxin assay kit [Genscript, Piscataway, NJ], as per manufacturer s instructions. This assay is based on modified limulus amebocyte lysate [LAL] 44

59 whereby the lysate binds endotoxin and a Chromogenic substrate then is detected as a function of color when endotoxin is present. A standard curve was generated from 0-1 EU/ml and results are expressed as EU/ml±SD. According to the manufacturer, 0.01EU/ml is considered endotoxin free conditions Cytokine estimations using arrays The cytokine components of both MyoCM and MØCM were determined using Ray Biotech profiler arrays [Ray Biotech, Norcross, GA]. This assay measures the cytokine content of conditioned media with antibodies that are captured on the membrane. The membrane was processed as per manufacturer s instruction and signal detection done using chemilumenecent dye Glucose uptake assay To measure glucose uptake by muscle cells post BSA or Palmitate treatment, we utilized a well validated technique (272, 274). L6GLUT4myc myoblasts were seeded at 40,000 cells/ well in 24 well plates for 48 hours, and then treated with 0.066mM BSA or 0.2mM palmitate for 8 hours. Cells were serum starved for 3 hours and then either treated or not with insulin Humulin R [Eli Lilly Canada, Ontario] for 20 minutes. Cells were then washed twice with Hepes Buffered saline (HBS). The HBS was aspirated and transport solution with HEPES-buffered saline containing 10 μm 2-[ 3 H] deoxyglucose (0.5 μci/ml) [PerkinElmer Life Sciences] in the absence of insulin was added for 5 minutes. The uptake was stopped by washing cells with ice cold 0.9%NaCl with 25mM glucose. This stop solution was used with four washes per well and aspirated. The plates were then stored overnight at -20 C and then cells were lysed in 500 μl of 0.05N sodium hydroxide and cells were scraped from plates and 400 μl added to 5 ml scintillation fluid in scintillation vials. This mix was shaken for 2-3 hours and allowed to settle down, and then read on scintillation counter. Ten microlitres of the original lysate was taken to determine protein concentration in sample. Protein concentration was determined using Bio-Rad protein assay solution [Bio Rad, Hercules, CA]. 200 μl or the protein assay solution was added to each 10 μl in 96 well clear plate and sample read a using plate reader at wavelength 595 using standard curve to quantify protein. Results of glucose uptake are reported in pmol/mg protein/minute. 45

60 2.15 Preparation of palmitate and BSA Palmitate was conjugated to BSA as vehicle to prevent it from coming out of solution as previously described with some modifications (272). 10% BSA solution was prepared by dissolving 1 gm of fatty acid free low endotoxin BSA (Sigma-Aldrich) and put at 37 C water bath on shaker. Sodium palmitate (Sigma-Aldrich) was prepared by generating 5 mm stock solution in 50% ethanol using endotoxin free water (Sigma-Aldrich). 1 ml of this solution was added to 9 ml 10% BSA solution prepared in endotoxin free water and left to shake at 37C for two hours. The solution was then aliquoted and frozen at -20C till further use. The molar ratio of BSA:PA in the conjugated palmitate preparation is 3:1 which is within physiological range. Samples were thawed once and the remaining sample was discarded if not used. The final preparation included either palmitate conjugated to BSA as vehicle [PA], or BSA alone [BSA] as control. To allow equimolar concentrations of PA and BSA control, PA was used at concentration of 0.2mM which in addition contains 0.066mM BSA i.e. BSA:PA ratio of 3:1. BSA control was used at 0.066mM Statistics The statistical analysis of the results used GraphPad software (GraphPad Software, San Diego, CA). Student's paired t-test was used for comparison of two groups. One way ANOVA with Tukey post-hoc test was used to test differences in effect of different stimuli. Two-way ANOVA with Bonferroni post test used when interaction of stimuli and inhibitor testing. Data are shown as means ± SD. 46

61 3 Chapter 3: Muscle-macrophage interactions in diabetogenic environment Summary Obesity is associated with chronic low-grade inflammation that starts in adipose tissue and leads to insulin resistance. In rodents and humans, this inflammatory response is characterized by adipocyte secretion of adipokines, cytokines, chemokines, and release of free fatty acid; these factors result in local inflammation in adipose tissue and attraction of bone marrow derived monocytes that differentiate to macrophages. In addition, resident macrophages as well as recruited macrophages will be stimulated to secrete factors that attract immune cells including macrophages and cause local inflammation. Fatty acids and cytokines that act locally at the start will eventually spill into circulation and reach distant metabolic organs including liver and skeletal muscle; in skeletal muscle, fatty acids and cytokines are also produced locally by the intermyocellular fat depots that expand with obesity and attract macrophages. When muscle is exposed to these inflammatory stimuli, insulin resistance ensues. While the adipose tissue-macrophage interaction is well characterized in obesity, the question whether similar crosstalk occurs between skeletal muscle and macrophage is unclear. In particular, it is unclear if muscle produces factor(s) that attract macrophages as a response to diabetogenic environment. The question whether skeletal muscle is infiltrated by macrophages in obesity has been difficult to answer. As muscle is considered a tissue with slow turnover and the need for significant number of macrophages as resident macrophages is questioned. In addition, the method used to section muscle normally generates a cross section that looks at cells. Some of the immune cells detected can be those that are in intravascular lumen and not really migrated to muscle, but because of the nature of the section this is difficult to ascertain. While several papers have documented the presence of macrophages within muscle, this has been questioned in other studies; this is quite different from adipose tissue complement of macrophages where about 5% of cells at any given time are macrophages and this number can rise to 50% in context of high fat diet in rodents. In addition to the controversy regarding macrophage infiltration of skeletal muscle in obesity, questions remain as to chemotactic potential of muscle in response to saturated fatty acid exposure. 47

62 Here we show that conditioned medium from muscle cells treated with Palmitate increased macrophage migration. In addition, palmitate treatment of muscle cells enhanced inflammatory pathway activation in Mitogen Activated Protein Kinase [MAPK] pathway including JNK, ERK, and p38mapk and degradation of IκBα. The activation of these pathways was associated with enhanced cytokine and chemokine gene expression and pro-and anti-inflammatory cytokine and chemokine secretion into the conditioned medium. One of the factors detected in the myoblast secretome is Monocyte Chemoattractant Protein-1 [MCP-1], a known chemoattractant of monocytes/macrophages. When MCP-1 effect was blocked using neutralizing antibodies, macrophage migration was attenuated. To identify the pathways responsible for MCP-1 production in muscle cells in response to palmitate, we treated cells with chemical inhibitors of JNK, ERK, p38mapk, and NFκB. MCP-1 production was shown to be mediated by ERK1/2 and NFκB. This suggests that skeletal muscle cell exposure to palmitate results in activation of inflammatory pathways that upregulate cytokine and chemokine production, resulting in macrophage attraction. MCP-1 appears to be one of the main factors responsible for macrophage attraction, and is produced by activation of ERK1/2 and NFκB. 48

63 3.1 Myoblast conditioned medium [MyoCM] attracts macrophages Macrophages infiltrate adipose tissue in context of obesity and can compose up to 50% of adipose tissue cell content in both mice and humans. One fat depot that expands with obesity and attracts macrophages is intermyocellular fat depot (IMFD). Macrophages have been detected in this area and in skeletal muscle that is in immediate vicinity of this depot. It is unclear if these macrophages are attracted to muscle by muscle produced factors i.e. chemotactic response, of if this is part of random macrophage motion i.e. chemokinetic response. To determine if muscle cells are capable of producing factors that attract macrophages when exposed to saturated fatty acids, we generated conditioned medium from palmitate-treated rat muscle cell line L6GLUT4myc; muscle cells were treated with PA 0.2mM and 0.066mM BSA control for 24 hours. Palmitate was washed off thoroughly and fresh medium added to cells for another 24 hours. This medium was collected and is called myoblast conditioned medium (MyoCM).This medium was added to the lower chamber in transwell migration system and macrophages were added to upper chamber. Macrophages were allowed to migrate for 3 hours. AMEM/2% FBS and MCP-1 were used as background and positive controls, respectively [Figure 3.1]. Palmitate treatment of muscle cells resulted in a two fold increase in macrophage migration compared to BSA control [Figure3.2] (P <0.05). 49

64 L6GLUT4myc Myoblast treatment with BSA/palmitate 24H Wash palmitate off & add fresh medium 24H Collect myoblast conditioned medium (MyoCM) 3H Migration assay Macrophages counted using Volocity software Macrophages completing migration are stained MyoCM added to lower chamber of transwell system Macrophages added to upper chamber of transwell system Figure 3.1: Experimental design for macrophage migration assay. L6GLUT4myc myoblasts were treated with 0.066mM BSA or 0.2mM palmitate for 24 hours [AMEM/10%FBS]. The palmitate was then washed off thoroughly and fresh medium added to cells for another 24 hours [AMEM/2%FBS]. This medium was then collected and called myoblast conditioned medium (MyoCM). This was added to lower chamber of transwell system, and macrophages were added to upper chamber of transwell system with 8μm pore size membrane. Macrophages were allowed to migrate for three hours, and cells that did not migrated were removed; cell that completed migration at the lower surface of membrane were stained with DAPI. Membranes were mounted on glass slides and counted using Volocity software. 50

65 Chemotactic index 5 4 * *** 3 2 ** 1 0 2%FBS MCP-1 MyoCM-BSA MyoCM-PA 2%FBS MCP-1 CM-BSA CM-PA Figure 3.2: Myoblast treatment with palmitate results in production of factors that attract macrophages. L6GLUT4myc myoblasts were treated for 24 hours with 0.2mM palmitate in AMEM/10%FBS; palmitate was washed off and fresh medium with 2%FBS added for another 24 hours. The medium was collected, centrifuged at 1,500 RPM for 5 minutes and then aliquoted and frozen at -80 C until further use. The conditioned medium was added to lower chamber and macrophages to upper chamber of transwell system. Cells that did not migrate were removed from the upper chamber and the membranes stained with DAPI. Images were taken from five fields per membrane and images were quantified using fluorescent microscopy. Results are from duplicate wells per experiment (n=3). Values are normalized to AMEM/2% FBS and expressed as chemotactic index. * P < 0.05 MCP-1 versus 2%FBS; ** P< 0.05 MCP-1 versus MyoCM- BSA; *** p <0.05 MyoCM-BSA versus MyoCM-PA. 2% FBS= background control AMEM/2%FBS, MCP-1= MCP-1 (100μg/ml) positive control in AMEM/2%FBS, MyoCM- BSA= conditioned medium from BSA treated myoblasts; MyoCM-PA= conditioned medium from palmitate treated myoblasts. 51

66 3.2 Palmitate treatment of muscle cells results in reduced glucose uptake Saturated fatty acids including palmitate exert their effect on muscle insulin signaling either directly by interacting with cell surface receptors, or indirectly via lipid metabolites that are generated after cellular uptake of the fatty acid (264). In order to test if palmitate treatment of muscle cells results in insulin resistance, we treated muscle cells with 0.066mM BSA or 0.2mM palmitate for 5 hours and serum starved the cell with palmitate for 3 hours. Palmitate reduced muscle glucose uptake in response to insulin, indicating insulin resistance [Figure 3.3] (P <0.05). 3.3 Palmitate treatment of muscle cells activates inflammatory pathways Saturated fatty acids including palmitate have been shown to activate Toll-like receptors 2 and 4 [TLR2 and TLR4], that mediates intracellular signalling events. We tested whether the inflammatory pathways including JNK, ERK, p38mapk and NFκB are activated under our experimental conditions. For this aim, we treated muscle cells or not with 0.066mM BSA or 0.2mM palmitate for 1, 3, 8, 16, and 24 hours. The phosphorylation of JNK, ERK, and p38mapk was tested, as well as the degradation of IκBα; the latter indicates the release of the transcription factor NFκB, which enters the nucleus and activate gene expression of inflammatory cytokines. Treating muscle cells with palmitate results in some degree of phosphorylation of JNK and ERK1/2 [Figure 3.4]. This treatment also results in the degradation of IκBα [Figure 3.5], indicating the activation of NFκB pathway. 3.4 Palmitate treatment of muscle cells upregulate inflammatory cytokine and chemokine gene expression From the above experiments, we observed that palmitate treatment of muscle cells results in activation of MAPKs and degradation of IκBα. We next wanted to assess if activation of these pathways will result in enhanced pro-inflammatory cytokine and chemokine gene expression profiles. Reverse transcriptase RT-PCR was conducted as per protocol in methods section; RT- PCR showed that palmitate treatment resulted in elevated pro-inflammatory cytokines [TNFα, IL-6] and chemokines [MCP-1, KC] gene expression profiles [Figure 3.6] (P < 0.05). 52

67 2DG-glucose uptake (pmol/mg protein /min) This indicated that muscle cells react to palmitate by activating inflammatory pathways that act downstream to enhance gene expression of pro-inflammatory cytokines and chemokines * * ** Basal Insulin UNT BSA PA Figure 3.3: Myoblast treatment with palmitate results in reduced glucose uptake. L6GLUT4myc myoblasts were treated with 0.2mM palmitate for 8 hours. Serum starvation was performed for 3 hours and then cells were treated or not with 100nM insulin for 20 minutes. Results are reported in pmol/mg protein/minute. Experiments were done in triplicates per condition (n=4). *P<0.05 for basal versus insulin stimulated glucose uptake in untreated and BSA-treated myoblasts. ** P<0.05 for insulin stimulated glucose uptake in BSA versus PA treated myoblasts. UNT= untreated myoblasts, BSA= BSA treated myoblasts, PA= palmitate treated myoblasts. 53

68 BSA PA Time (h): p-jnk JNK p-erk ERK p-p38 MAPK p38 MAPK Figure 3.4: Myoblasts treatment with palmitate activates MAP kinases. Myoblasts were untreated [0] or treated with BSA or palmitate for 1, 3, 8, 16 and 24 hours. Cells were lysed and 10 μg of protein was loaded per well. phosphorylation of JNK, ERK, and p38 MAPK was tested using appropriate primary antibodies (see methods section for details). Representative membranes are shown (n=4). 54

69 BSA PA Time (h): IκBα Actinin Figure 3.5: Myoblast treatment with palmitate leads to degradation of IκBα. Myoblasts were untreated [0] or treated with palmitate for 1, 3, 8, 16 and 24 hours. Cells were processed as described in methods section and 10 μg of protein was loaded per well. Membranes were probed for IκBα. Representative membranes are shown (n=4). 55

70 IL-6/GAPDH fold change KC/GAPDH fold change TNFα/GAPDH fold change MCP-1/GAPDH fold change 4 4 * * BSA PA 0 BSA PA 3 * 3 * BSA PA 0 BSA PA Figure 3.6: Myoblast palmitate treatment activates inflammatory cytokine & chemokine gene expression. Myoblasts were treated with BSA or palmitate [PA] for 24 hours. mrna was isolated using RNAeasy minikit [Qiagen], and 200ng used for RT-PCR.The data are normalized to GAPDH and BSA is set at 1 fold.* P<0.05 BSA versus PA. 56

71 3.5 Palmitate treatment of muscle cells results in secretion of multiple cytokines and chemokines As enhanced migration of macrophages to MyoCM-PA indicated that chemokines are produced by muscle cells in response to palmitate treatment, we next wanted to characterize the cytokine and chemokine content of the muscle secretome. We used membrane-based Cytokine profiler arrays [Ray Biotech, Norcross, GA] to test the MyoCM-BSA and MyoCM-PA for cytokines and chemokines. The conditioned media were applied to the arrays and processing done as per manufacturer s protocol. Images of the arrays are shown in figure 3.7 and the categorization of the cytokines detected in MyoCM-PA is reported in table 1.1. Analysis of the myoblast secretome identified several myokines including pro-inflammatory cytokines [IL-1, IL-6, TNFα, INFγ], anti-inflammatory cytokines [IL-4, IL-10, IL-13], and chemokines [MCP-1, CINC-1, GM-CSF, MIP-3α]. In the latter group, MCP-1 is a known monocyte/macrophage chemoattractant, CINC-1 and GM-CSF are neutrophil attractants, and MIP-3α attracts T-lymphocytes. It is very likely that those myokines act on muscle to activate the same inflammatory pathways activated by palmitate in addition to other independent pathways in an autocrine and paracrine fashion, which leads to further cytokine and chemokine production. Of special importance, the MyoCM-PA contains factors that can polarize the macrophage to pro-inflammatory M1 [e.g. INFγ] or anti-inflammatory M2 [e.g. IL-4, IL-13, IL-1] phenotypes. Thus, muscle cells respond to saturated fatty acids in a complex fashion; they are capable of producing factors that not only attract monocytes/macrophages, but also can modulate the phenotype of the infiltrating monocytes/macrophages to a pro- or anti-inflammatory phenotype. The phenotype of the macrophage will lead to different responses in muscle both in terms of the products synthesized and effects on muscle insulin sensitivity, as some cytokines have negative effects on insulin signaling. Some of the cytokines detected have not previously reported to be produced by muscle cells in response to saturated fatty acid treatment as far as we know e.g. MIP-3α, GM-CSF, and IL-13, and their role in muscle metabolism and responses to fatty acids remains to be clarified. 57

72 Of note, the MyoCM-BSA and MyoCM-PA and their components were tested for the presence of endotoxin and NEFA, as these two factors may lead to the responses similar to those seen in muscle. The media were tested and found to be endotoxin free [table 1.2] and NEFA were detected at very low levels of about 1/1000 of original treatment dose with palmitate in the conditioned media [MyoCM-BSA 2.7±0.007 nm; MyoCM-PA 2.5±0.005 nm, n=3]. 3.6 Monocyte Chemoattractant Protein-1 [MCP-1] production is enhanced with palmitate treatment As noted above, palmitate treatment of muscle cells results in production of multiple cytokines and chemokines. Monocyte Chemoattractant Protein-1 [MCP-1] is one of the factors detected in the MyoCM-PA and this chemokine is a well known monocyte/macrophage attractant. As we have shown that MCP-1 gene expression is upregulated with palmitate treatment, we wanted to test the kinetics of MCP-1 protein production in muscle cells in response to palmitate treatment. For this experiment, we treated muscle cells with 0.066mM BSA or 0.2mM palmitate for 1, 3, 8, 16, and 24 hours. Figure 3.8 shows that MCP-1 is present at low levels in the basal state; when cells are treated with palmitate, the levels start rising early, peak at 8 hours post treatment and continue to hold at steady levels up to 24 hours. BSA treated myoblasts demonstrate a significantly lower response to treatment compared to palmitate treated cells. 3.7 Neutralizing MCP-1 inhibits macrophage migration Since MCP-1 gene expression and protein production were upregulated with palmitate treatment, and since the MyoCM-PA attracted more macrophages than MyoCM-BSA control, we hypothesized that MCP-1 is one of the main chemoattractants in the conditioned medium that is responsible for attracting macrophages. We tested our hypothesis by blocking MCP-1 action by neutralizing antibodies added to both MyoCM-BSA and MyoCM-PA along with isotype IgG antibodies added as controls. 58

73 Concentration [nm] A BSA MyoCM-BSA BSA A B C D E F G H I J K L PA MyoCM-PA A B C D E F G H I J K L PA B MyoCM-BSA MyoCM-PA 59

74 Figure 3.7: Myoblast palmitate treatment results in secretion of multiple cytokines/chemokines. A) MyoCM-BSA and MyoCM-PA were tested for their cytokine and chemokine content using Ray Biotech rat profiler arrays. Freshly prepared conditioned medium [1ml] was applied to the membranes and processing done as per manufacturer s instructions. Positive control= A1-2, B1-2,L7-8; negative control=c1-2, D1-2, C-K 7 and C-K 8. CINC-1=I 1-2; GM-CSF= C 3-4; INFγ=E 3-4; IL-1α= F 3-4; IL-1β=G 3-4; IL-4=J 3-4; IL-6= K 3-4; IL-10=L -3-4; IL-13=A 5-6; MCP-1= E 5-6 [box]; TNFα=A 7-8. B) Quantification of NEFA in the conditioned media demonstrate low levels with no significant difference between the two conditions [n=3]. 60

75 Pro-inflammatory cytokine name Coordinates on array INFγ E 3-4 IL-1α F 3-4 IL-1β G 3-4 IL-6 K 3-4 TNFα A 7-8 Anti-inflammatory cytokine name Coordinates on array IL-4 J 3-4 IL-10 L -3-4 IL-13 A 5-6 Chemokine name Coordinates on array CINC-1 I 1-2 CINC-2α J 1-2 GM-CSF C 3-4 MCP-1 E 5-6 MIP-3α F 5-6 CXCL5 C5-6 Table 1.1: Myokines detected in conditioned medium from palmitate-treated cells [Coordinates given are for the Ray Biotech cytokine array in figure 3.7] 61

76 Condition Endotoxin concentration EU/ml SD BSA PA FBS 2% FBS 10% AMEM/no FBS AMEM/2%FBS AMEM/10%FBS MyoCM-BSA MyoCM-PA Table 1.2: Quantification of endotoxin in media. Conditioned media were generated as described in methods and frozen at -80 C. media were thawed and tested for endotoxin content along with the BSA and palmitate used to treat the cells using ToxinSensor TM Chromogenic LAL Endotoxin Assay Kit (Genscript) as per manufacturer s instructions. Media with endotoxin levels below 0.01 EU/ml are considered endotoxin-free as per manufacturer. Data shown are mean ± SD from three independent collections of media. BSA=Bovine Serum Albumin used to treat muscle cells [0.066mM, 24 hours; PA= palmitate conjugated to BSA and used to treat muscle cells [0.2mM, 24 hours]; FBS 2%=Fetal Bovine Serum from same batch used to prepare growing media diluted in endotoxin free water to 2%; FBS 10%=Fetal Bovine Serum from same batch used to prepare growing media diluted in endotoxin free water to 10%; AMEM/no FBS= AMEM with no FBS added; AMEM/2% FBS = AMEM supplemented with 2% FBS; AMEM/10% FBS= AMEM supplemented with 10% FBS; MyoCM-BSA= conditioned medium generated from BSA-treated myoblasts; MyoCM-PA= conditioned medium generated from palmitate treated myoblasts. 62

77 MCP-1/actininfold change A Time (h): BSA PA MCP-1 Actinin B 5 4 ** ** ** ** 3 * 2 BSA PA Time (h) Figure 3.8: Palmitate treatment of myoblasts results in MCP-1 protein production. A) Western blot done using protein lysates from myoblasts untreated [0] or treated with BSA or palmitate for 1, 3, 8, 16, and 24 hours, and 20μg of lysate was loaded per well. Membranes were probed with MCP-1 primary antibody. Probing for Actinin was used as loading control. Representative membranes are shown (n=3). B) Quantification of MCP-1 production by myoblasts in response to palmitate treatment. Western blots were scanned and quantification done using Image J software. The data were normalized to actinin first and then untreated BSA was set at 1, and all other values were normalized to untreated BSA. The data reported as fold change ±SE (n=3). * P< 0.05 MCP-1 with PA treatment at 1 hour versus BSA control. ** P < MCP-1 with PA treatment at 3, 8, 16, and 24 hours versus BSA control. 63

78 Chemotactic index 5 4 * ** *** 3 2 # 1 0 MyoCM-BSA MyoCM-PA Figure 3.9: MCP-1 neutralization in MyoCM-PA impairs macrophage migration. MyoCM- BSA and MyoCM-PA were incubated with MCP-1 neutralizing antibody [ BA, 2H5, 50μg/ml, Biolegend] or IgG control antibody [IgG, 50μg/ml, Biolegend] for one hour prior to starting the migration assay. RAW macrophages were added to the top chamber of the transwell system at a concentration of 1x10 6 /0.1ml, and migration was done for 3 hours. Cells that did not migrate were removed and membranes stained with DAPI. Five images from representative parts of the membrane were taken and cells were counted using Nikon long range fluorescent microscope. Counts were normalized to AMEM/2%FBS and expressed as chemotactic index. * P < 0.05 MCP-1 versus AMEM/2%FBS. ** P < 0.05 MyoCM-PA versus MyoCM-BSA. *** P < 0.05 MyoCM-BSA IgG versus MyoCM-PA IgG. # P < 0.05 MyoCM-PA IgG versus MyoCM-PA BA. Experiments were done in duplicates (n=3). MyoCM-BSA= conditioned medium from BSA treated myoblasts; MyoCM-PA=conditioned medium from palmitate treated myoblasts; CM-BSA IgG= MyoCM-BSA treated with control IgG antibody; CM-BSA BA= MyoCM-BSA treated with MCP-1 blocking antibody; CM-PA IgG= MyoCM-PA treated with control IgG antibody; CM-PA BA= MyoCM-PA treated with MCP-1 blocking antibody. 64

79 We then tested macrophage migration to the conditioned medium one hour after adding the neutralizing antibodies. MCP-1 neutralization resulted in a significant reduction in macrophage migration to MyoCM-PA [Figure 3.9, P < 0.05]. 3.8 Identification of pathways responsible for muscle cell MCP-1 production in response to palmitate treatment In order to identify the pathways involved in MCP-1 production by muscle with palmitate treatment, we treated the cells with inhibitors of JNK [SB600125], ERK 1/2 [U0126], p38 MAPK [SB203580] and NFκB [PDTC] for one hour and then added BSA or palmitate for another 7 hours in the presence of inhibitors. Western blot analysis was done and the membranes blotted for MCP-1. Treating muscle cells with the inhibitors revealed that ERK 1/2 and NFκB are the two main pathways that mediate muscle MCP-1 production in response to palmitate. JNK did not appear to be involved in stimulating MCP-1 production. The contribution of p38mapk to muscle MCP-1 production was difficult to ascertain, as treatment of cells with BSA and p38mapk inhibitor consistently showed elevation in MCP-1 levels [Figure 3.10]. A DMSO SP DMSO U0126 DMSO SB MCP-1 BSA PA BSA PA BSA PA BSA PA BSA PA BSA PA Actinin B DMSO DPTC BSA PA BSA PA MCP-1 Actinin 65

80 Fold change c 4 DMSO BSA DMSO PA 3 BSAI PAI 2 * ** 1 0 SP U126 SB DPTC Figure 3.10: MCP-1 production by palmitate treated myoblasts occurs through ERK 1/2 and NFκB pathways. A) Myoblasts were treated with inhibitors for JNK [SP600125], ERK [U0126], p38 MAPK [SB ] and B) NFκB [Pyrrolidine dithiocarbamate (PDTC)] pathways. Cells were incubated with inhibitors and DMSO controls for one hour prior to addition of BSA or palmitate for another 7 hours. Cells were lysed and 20μg of protein was loaded per well. Membranes were probed with MCP-1 primary antibody and actinin was used as loading control. Representative membranes are shown C) Quantification of experiments (N=4). P 0.52 for comparison of PA+DMSO versus PA+JNK inhibitor; *P 0.01 for comparison of PA+DMSO versus PA+ERK inhibitor; P 0.14 for comparison of PA+DMSO versus PA+p38MAPK inhibitor; **P 0.03 for comparison of PA+DMSO versus PA+NFκB inhibitor. 66

81 3.9 Discussion Palmitate treatment of muscle cells activates inflammatory signalling Palmitate is a saturated fatty acid that is abundant in western diet, constituting up to 30% of circulating free fatty acids in humans and its circulating levels are elevated in obesity (278). Palmitate is known to trigger inflammatory responses in cells (65). In the current study, we tested the effects of palmitate treatment on the L6GLUT4myc muscle cell line. Palmitate treatment activated inflammatory pathways including MAPK pathway and NFκB. The activation of these pathways was associated with upregulation of cytokine [TNFα, IL-6] and chemokine [MCP-1, KC] gene expression, and enhanced secretion of multiple cytokines and chemokines from palmitate treated myoblasts [table 1]. Moreover, palmitate treatment resulted in production of muscle cell factors that spilled into the medium, and such medium attracted macrophages. We demonstrated by the experiments summarized here that the skeletal muscle cells response to palmitate is complex with production of multiple cytokines and chemokines. Some of the cytokines and chemokines detected have not previously reported to be produced by muscle cells in response to saturated fatty acid treatment as far as we know e.g. MIP3α, GM-CSF, IL-13, and their role in muscle metabolism remains to be determined. One of the main chemokines detected in conditioned medium from palmitate treated cells was MCP-1, a chemokine known to attract macrophages. Palmitate treatment resulted in upregulation of MCP-1 gene expression, protein amount and secretion. The signalling pathways involved in the palmitate-induced rise in MCP-1 protein were identified using selective inhibitors of the MAPK s and NFκB pathways. These experiments identified ERK and NFκB as potentially important pathways for MCP-1 production. To evaluate contribution of MCP-1 in the conditioned medium to the chemoattractant function of conditioned medium from palmitate-treated muscle cells, we neutralized MCP-1 in the medium using blocking antibodies. Neutralizing MCP-1 abrogated macrophage migration to this conditioned medium when compared with an IgG isotype control. These results do not rule out contribution by other molecules in the conditioned medium to the chemoattractant response of macrophages, which may cooperate with MCP-1 to bring about the response. 67

82 However, the positive control we use is MCP-1, serving also as proof of principle that it alone can induce macrophage migration. Nonetheless, it is likely that hierarchical and collaborative relationships exist among competing chemokines, and that MCP-1 may be a major chemokine in this hierarchy upstream of other less potent chemokine(s), so that if MCP-1 is neutralized, the net effect is diminished. Of note, the conditioned medium from BSA-treated muscle cells attracted double the number of cells compared to the negative control of medium alone. This indicates that muscle cells constitutively produce macrophage chemoattractants. Blocking one of these potential chemoattractants, MCP-1, in BSA-treated myoblasts had no effect on inhibiting macrophage migration, and MCP-1 was not detected in conditioned medium from BSA-treated muscle cells. Because blocking MCP-1 in conditioned medium from palmitate-treated cells significantly inhibited macrophage migration, distinct factor(s) in the conditioned medium from BSA-treated muscle cells are likely involved in macrophage attraction. Identifying those factors will be subject of future investigation, and they may include either factors already detected by the cytokine array or additional factors not included in this detection assay Comparison of the response to fatty acids and signalling pathways involved in adipocytes When comparing myoblasts to other metabolic cells such as adipocytes, significant differences are detected in their responses to fatty acids. 3T3-L1 adipocytes in culture treated with a mixture of fatty acids also produce MCP-1 and adipocyte-conditioned medium enhances mononuclear cell migration (279). However, neutralizing MCP-1 in adipocyte-conditioned medium reduced mononuclear cell migration by only 40% (279). In addition, in mice in vivo, absence of MCP-1 (236) or its receptor (229) in adipocytes did not inhibit macrophage infiltration upon high fat feeding. Therefore, the adipocyte response to fatty acids differs from that of muscle cells, the former producing other macrophage chemoattractants when exposed to saturated fatty acids compared to the latter, whereas MCP-1 is the more dominant chemoattractant produced by muscle cells in response to palmitate. Alternatively, the differences may lie in the fatty acids tested in each case, the species of the cells (L6 muscle cells are of rat origin whereas 3T3-L1 adipocytes are of mouse origin), and/or the type of mononuclear cells assayed for migration. 68

83 In other cells including adipocytes, macrophages, and myotubes the production of proinflammatory cytokines in response to palmitate occurs via activation of inflammatory pathways MAPKs, NFκB, TLR-2 and further include TLR-4 signalling (58, 59). In our experiments, activation of MAPKs and NFκB at the early time points is likely to be mediated by direct signalling events of palmitate through TLR. However, cytokine production is likely caused by several mechanisms, including both palmitate signaling as well as intracellular lipid metabolites like ceramide and diacylglycerol ( ) and cytokines generated from palmitate treatment. Cytokines signal through their respective receptors and JAK-STAT pathways to activate MAPKs and NFκB. These pathways stimulate further production of cytokines via production of transcription factors that enter the nucleus and bind to pro-inflammatory gene promoters, resulting in further activation of inflammatory pathway and cytokine production perpetuating inflammation (19). Reports from previous cell culture models provided evidence for palmitate-mediated MCP-1 production in MAP kinase and NFκB dependant manner in myotubes (59). However, in vivo studies did not document increased MCP-1 mrna in skeletal muscle from db/db mice and wild type mice on high fat feeding despite elevated expression in adipose tissue and elevated serum levels (235). These contrasting results may be due to the timing of sampling for MCP-1 mrna. In our study we used myoblasts in the experimental system. We first tested, whether myoblasts respond similarly to myotubes. We also investigated the temporal relationship of palmitate treatment to MCP-1 production, and found that the ERK and NFκB pathways are the main ones involved in MCP-1 production, which occurs early and is sustained with continuing exposure to palmitate Possible contribution of other chemokines and cytokines to the response to palmitate In adipocytes, MCP-1 production is enhanced by other cytokines including IL-1β, TNFα, IL-6, and IL-8 and is reduced by IL-10 (228). Most of these factors were detected in conditioned medium from palmitate-treated myoblasts, which may suggest a similar process whereby cytokines trigger MCP-1 production in addition to fatty acids as demonstrated in our experiments. 69

84 It is unusual that one chemokine seems to be a major factor responsible for steering macrophages to muscle as seen from our earlier experiment with blocking antibodies. Based on evidence from literature, we expected a less dramatic effect of MCP-1 on macrophage migration than what we have reported when MCP-1 was blocked (279). It is likely that other chemokines are at play, however their production may be insufficient in our experimental conditions to elicit such a response on migration. It is also feasible that MCP-1 levels are such that chemokines with smaller effects will not be appreciated with our detection system, which relied upon cell counts and in the event of small differences would not be able to detect them. Another explanation is that additional or higher levels of chemokines may be produced during palmitate treatment and are removed when palmitate is washed off prior to the generation of the conditioned medium. While this invariably will remove factors produced at earlier time points in response to palmitate, it is important to study the effect of factors on macrophage attraction in isolation from palmitate, which might have affected macrophage chemotactic and inflammatory responses. Our results must be interpreted with certain considerations in mind. An important issue is that we removed palmitate at the end of treatment prior to generation of conditioned medium, and subsequently found very low NEFA levels (1/1000 th of input) in conditioned media. Therefore, the production of MCP-1 is likely to be mediated by palmitate stimulation early on but then palmitate intermediates and cytokines play an important role in sustaining its production. Another consideration is that no LPS was detected in the conditioned media, the AMEM or FBS components, or in palmitate and BSA used to generate the conditioned media. This is important because LPS signals through TLRs, and has been recently found to contaminate some BSA products that are used in conjugation of fatty acids to avoid fatty acids coming out of solution (283). This may complicate the interpretation of some reported studies but allows the interpretation of our results with elimination of this confounder, and focuses the cell responses on palmitate, its metabolites, and cytokines produced in response to these factors. One of the main cytokines involved in the inflammatory response to fatty acid treatment is TNFα. While we detected low levels of TNFα by the cytokine array, the gene expression data predicted that these levels would be higher. In previously published work, it has been demonstrated that TNFα treatment of adipocytes increases MCP-1 production, and when TNFα is neutralized the response is reversed (68). It is 70

85 likely that TNFα was generated when myoblasts were treated with palmitate and then washed off when palmitate was removed, and that it acted on muscle cells prior to its removal. Another possibility is that TNFα had peaked earlier and subsequently degraded so that at the time of measurement its levels were low. In addition to MCP-1 and TNFα, analysis of the myoblast secretome pointed to a complex response by muscle to palmitate. The cytokine arrays identified several molecules including proinflammatory [IL-1, IL-6, TNFα, INFγ] and anti-inflammatory [IL-4, IL-10, IL-13] cytokines, and chemokines [MCP-1, CINC-1, GM-CSF, MIP3α, CXCL5] [table1]. It is likely that those cytokines act on muscle to activate some of the same inflammatory pathways that are activated by fatty acids and their metabolites in an autocrine and paracrine fashion, leading to further cytokine and chemokine production and interference with insulin signalling pathways, leading to insulin resistance (129, 237, 268). One of the most significant findings of this study is that the conditioned medium from palmitate treated myoblasts contain factors that can polarize the macrophage to pro-inflammatory [INFγ] or anti-inflammatory [IL-4, IL-13, IL-1] phenotypes (284). Thus, in response to palmitate muscle cells are capable of producing factors that not only attract macrophages and alter insulin sensitivity and metabolism, but may also modulate the phenotype of the infiltrating monocytes/macrophages to a pro- or anti-inflammatory phenotype. This is probably the case invivo, where cells bathed in the same environment continuously communicate to affect metabolic and immune responses of both cell types (18-20). In addition to its impact on macrophage-muscle crosstalk, palmitate treated muscle cells demonstrate reduced glucose uptake, which was not associated with increased basal glucose uptake. This indicates that palmitate may exert its effects primarily on insulin stimulated glucose uptake as the basal level remain unchanged, and this is likely to occur through the activation of inflammatory pathways within the cell including JNK, IKK, and PKC by palmitate or its intermediate metabolites. Additionally, it is possible that cytokines and chemokines secreted by muscle cells themselves [e.g. IL-1, IL-6, TNFα] may contribute to the insulin resistance. This autocrine loop will be explored in future studies. 71

86 In addition, MCP-1 itself is known to induce insulin resistance in vivo with acute and chronic administration in rodents, and its increased production by muscle cells is likely to be one important factor in reducing insulin sensitivity (285). We did not test glucose uptake in muscle after blocking MCP-1 in medium. It is important to investigate glucose uptake in muscle after blocking MCP1 in future experiments to demonstrate the contribution of MCP-1 to insulin sensitivity in vitro. It has been reported that the main source of MCP-1 production in humans and rodents are adipocytes (228, 286), and that this chemokine plays an important role in attracting macrophages to adipose tissue in obesity (234, 235, 286). In vitro, mature adipocytes constitutively produce MCP-1at low levels under physiological conditions, but TNFα treatment induces its production (239). The pathways that govern MCP-1 production in adipocytes include JNK and NFκB (231). There are some differences in muscle pathways responsible for MCP-1 production, as we link NFκB and ERK 1/2 pathways to MCP-1 production in muscle cells, as their inhibition significantly affected MCP-1 synthesis. One other important function of MCP-1 is muscle regeneration by attracting macrophages that will help with muscle repair and angiogenesis (287). Whether its production with palmitate treatment is related to muscle repair of damage caused by palmitate is unknown. In summary, we present evidence that myoblast treatment with palmitate results in production of MCP-1 that attracts macrophages, and we show that muscle cells are capable of mounting a complex response to palmitate characterized by activation of inflammatory pathways, upregulation of inflammatory cytokine and chemokine gene expression and secretion. These cytokines and chemokines can alter macrophage phenotype to pro- or anti-inflammatory cell type and change insulin sensitivity. 72

87 4 Chapter 4 Macrophage-macrophage interaction in diabetogenic environment Summary Obesity is associated with macrophage infiltration to adipose tissue. The literature has suggested that adipose tissue produces factors that attract immune cells and these cells include bone marrow-derived monocytes that infiltrate this depot with high fat feeding, and differentiate to macrophages. These newly recruited macrophages are pro-inflammatory M1 macrophages in phenotype, as opposed to the resident M2 macrophages. In addition, other immune cells like T- lymphocytes and neutrophils also appear to either be present in adipose tissue prior to the arrival of monocytes or are continuously entering and leaving adipose tissue like T- lymphocytes. Their role in recruitment of macrophages and their interaction with each other and other cells in adipose tissue are not very clear. By what mechanisms do the macrophages get to adipose tissue and what are the mechanism through which these cells communicate with other macrophages again is not well defined. We first tested if palmitate treatment of primary rat peritoneal macrophages result in production of macrophage chemoattractants, and found this to be the case. We also demonstrated that macrophage conditioned medium from palmitate treated cells attract neutrophils. Palmitate activated MAP kinases JNK and p38mapk and resulted in degradation of IκBα, indicating the activation of NFκB pathway. In addition, palmitate treatment upregulated pro-inflammatory cytokines [TNFα, IL-6] and chemokines [MCP-1, KC] gene expression. To evaluate the macrophage secretome i.e. factors produced by macrophages in the conditioned medium, the conditioned medium generated from treating primary rat peritoneal macrophages was tested using profiler arrays. This showed that macrophages produce several cytokines in basal state, and this is further enhanced with palmitate treatment for some of them including MCP-1, TNFα, and IL-6 to a lesser degree. The response is different and less stark than what was seen in the muscle cells post palmitate treatment. We conclude that palmitate treatment of macrophages leads to production of macrophage chemoattractants, that in addition to MCP-1 may include other that are tested for, as MCP-1 levels were not significantly different in the BSA and palmitate generated conditioned media. Palmitate treatment also generated neutrophil chemoattractants. 73

88 4.1 Effect of diabetogenic environment on macrophage: Macrophage conditioned medium attracts macrophages It has been proposed that macrophage infiltration of adipose tissue in the context of obesity and high fat feeding is related to increased cytokine and chemokine production by an expanding adipose tissue (19, 93, 94). The source of these chemokines is believed to be from adipocytes, resident macrophages and infiltrating monocytes that differentiate to macrophages and in turn secrete their own products into the local tissue environment. Local inflammatory signals induce endothelial cell activation and these in turn will express chemokines that will attract monocytes from circulation. These cells will then have to transmigrate through the endothelium and reach the tissue involved. As monocytes reach the adipose tissue, they are exposed to signals that promote differentiation to macrophages. These macrophages are believed to differentiate to a different, pro-inflammatory [M1] phenotype compared to the resident tissue macrophages [M2]. The differences between those cell types are manifested in their arginase activity. The M1 cells are capable of producing NO, and the M2 cells are capable of breaking down arginase and mitigating inflammatory response in tissues. Lipolysis that occurs with adipose tissue expansion in obesity exposes the macrophages to high concentrations of fatty acids in the circulation and in the local adipose tissue once these cells infiltrate it. This result in M1 differentiation and these cells will then secrete pro-inflammatory cytokines and chemokines that will lead to further monocyte attraction and local tissue inflammation. This propagates the inflammatory response in local tissues and results in further metabolic dysregulation. In skeletal muscle this dysregulation is manifested as insulin resistance. This is an early event in the course of the inflammatory responses seen in obesity and leads to reduced glucose uptake into skeletal muscle cells. In order to establish that macrophages are capable of producing factors that attract macrophages in vivo, we conducted macrophage migration assays. We generated conditioned media from Thioglycollate elicited primary rat peritoneal macrophages [MØCM] by treating them with 0.5 mm palmitate for 6 hours, washing it off and adding fresh medium for another 16 hours. This medium was collected, centrifuged, aliquoted and then frozen till further use. When experiments were done, this medium from BSA [MØCM-BSA] and palmitate treated primary rat peritoneal macrophages [MØCM-PA] was added to the lower chamber of the transwell system and RAW macrophages were added to the upper chamber of the transwell system. Migration was allowed to proceed for 3 hours and membranes were processed accordingly. 74

89 The experimental design is shown in figure 4.1. When macrophages are exposed to palmitate, they are capable of producing factors that attract other macrophages [P < 0.05] [Figure 4.2]. Of note, the MØCM-BSA did attract more macrophages that the background controls, indicating the production of chemotactic factors even at the basal state by macrophages that attract other macrophages. These factors or other factors are further stimulated when cells are exposed to palmitate. As noted from the background control, these cells do have chemotactic capacity, which is likely to be to the FBS in the medium, but this capacity is further enhanced beyond this when other chemokines are present in the medium. We attempted earlier to use the primary rat peritoneal macrophages in the migration assays, but it was difficult to maintain viability when scraped for collection having adhered to the bottom of the plastic plate at initial processing. These cells are Thioglycollate elicited and this may alter their phenotype and responses to stimuli when compared to cells collected without Thioglycollate. The only reason for using Thioglycollate is the enhanced yield with more cells acquired with this treatment, but this may affect our ability to see the full scale of the response of these cells to palmitate; as noted though, these cells are capable of further stimulation when palmitate is added and do elicit an inflammatory response when palmitate is present. 4.2 Palmitate treatment of macrophages attracts neutrophils The above results confirmed that the macrophage is capable of producing factors that attract other macrophages. The next question we wanted answered is whether this is a specific response to macrophages only, or can macrophages produce factors that crosstalk to other immune cells. This has been shown to be the case in several other immune cells that have been implicated in adipose-immune cross talk including T- lymphocytes and neutrophil (115, 118). The same conditioned medium that was used in macrophage migration assay was used in neutrophil migration assay that were conducted for 30 minutes, and membrane processing was done in a similar manner to the other migration experiments. 75

90 Macrophage treatment with 0.5mM BSA/palmitate 6H Palmitate washed off & fresh medium added 16H Collect conditioned medium (MØCM) 3H Macrophages counted using Volocity software Macrophages completing migration are stained MØCM added to lower chamber of transwell system Figure 4.1: Experimental design for macrophage migration assay. Primary rat peritoneal macrophages were treated with BSA or Palmitate 0.5mM for 6 hours. The palmitate was then washed off and fresh medium added to cells for another 16 hours. This medium was then collected and called macrophage conditioned medium (MØCM). This was added to lower chamber of transwell system with 8μm hole size membrane, and macrophages were added to upper chamber of transwell system. Macrophages were allowed to migrate for three hours, and cells that completed migration at the lower surface of membrane were stained. Cells were counted using Volocity software. 76

91 Fold change 7 6 * %FBS MCP-1 MØCM-BSA MØCM-PA Conditioned medium from primary rat peritoneal macrophages Figure 4.2: Macrophage treatment with palmitate results in production of factors that attract other macrophages. Primary rat peritoneal macrophages were treated with palmitate as described in methods. Macrophage conditioned medium MØCM was added to lower chamber and RAW macrophages were added to upper chamber. Cells were allowed to migrate for 3 hours. * P<0.05 for MØCM-BSA versus MØCM-PA and results are expressed as fold change [N=3]. 77

92 It was demonstrated that the MØCM-PA attracted more neutrophils compared to MØCM-BSA control [P<0.05] [Figure 4.3]. This indicates that the response by macrophages noted in response to palmitate treatment by macrophages is also seen in neutrophils. Importantly, neutrophil chemoattractants are not present in significant amounts in MØCM-BSA when compared to MØCM-PA; this is surprising as we had expected some basal production of these chemokines that is enhanced with palmitate treatment. It appears that at basal non-stimulated conditions, the MØCM-BSA has factors that attract macrophages only. One limitation to our experiments is that we did not quantify the cytokine KC in the MØCM, as our arrays did not include this. In gene expression experiments, KC rose with palmitate treatment. This indicates that some of the responses are inducible in response to palmitate and that the BSA control has no effect on this response. 4.3 Palmitate treatment of macrophages activates inflammatory pathways As macrophage and neutrophil migration experiments above indicated the presence of macrophage and neutrophil chemoattractants in the conditioned medium post palmitate treatment, we set out to see which inflammatory pathways are activated with palmitate treatment of macrophages. We treated primary rat peritoneal macrophages with 0.5mM palmitate for 6 hours and isolated protein, or we harvested protein at the end of period of conditioned medium generation i.e. protein was obtained from cells at the end of the palmitate treatment or at the time of collection of conditioned medium. We evaluated activation of MAPK and degradation of NF-κB in response to palmitate treatment as indicators of activation of cellular stress kinases. Treatment of macrophages with palmitate led to activation of MAPKs stress kinases JNK and p38 MAPK [Figure 4.4A]. This activation was only present when palmitate was present in the medium, and disappeared when palmitate was removed. 78

93 Fold change 9 8 * 7 6 * %DMEM fmlp MØCM-BSA MØCM-PA MØCM from primary rat peritoneal macrophages Figure 4.3: Macrophage treatment with palmitate results in attraction of neutrophils. Primary rat peritoneal macrophages were treated with palmitate as in methods. Macrophage conditioned medium from BSA[MØCM-BSA] and palmitate [MØCM-PA] treated macrophages was added to lower chamber, and formyl MLP was used as positive control. Primary bone marrow derived murine neutrophils were added to the upper chamber of the transwell system and allowed to migrate for 30 minutes. *P <0.05 neutrophil migration to fmlp versus 0.2%DMEM and to MØCM-BSA versus migration to MØCM-PA [N=4]. 79

94 When the cells were treated with palmitate for 6 hours, this also resulted in degradation of IΚBα, which indicates the release of NF-ΚB that acts as a transcription factor in the nucleus activating pro-inflammatory cytokine gene transcription [Figure 4.4B]. Again, this response disappeared when palmitate was removed and there was recovery in IκBα levels in palmitate treated cells to basal levels at 16 hours. 4.4 Palmitate treatment of macrophages up regulates inflammatory cytokines and chemokine gene expression The activation of inflammatory pathways was associated with upregulation of inflammatory gene expression when assessed by RT-PCR. When primary rat peritoneal macrophages were treated with palmitate which was then removed and then lysed at the time of generation of the conditioned medium, cells showed sustained activation of inflammatory cytokine [TNF, IL-6] and chemokine [MCP-1,KC] gene expression when tested [Figure 4.5]. This indicated that the effects of palmitate treatment of macrophages are more sustained even after the removal of the stimulus, and that this effect is likely not only due to the palmitate itself, but also to its metabolites that are generated after its uptake into the cell, although we did not quantify this in our work, there s ample evidence that fatty acid metabolites in immune cells are responsible for some of the effects that are seen with this treatment. 4.5 Palmitate treatment of macrophages results in secretion of multiple cytokines and chemokines into conditioned medium As noted above, the treatment of primary rat peritoneal macrophages with palmitate resulted in activation of stress kinases, NF-κB pathway and subsequent activation of pro-inflammatory cytokine and chemokine gene expression profiles. Whether this enhanced gene expression is mirrored by an increase in protein production by the cell was not clear. Therefore, we tested the macrophage secretome for cytokines and chemokines produced by those cells in response to BSA and palmitate treatments. Palmitate treatment resulted in secretion of several pro- and antiinflammatory cytokines and chemokines into the MØCM-PA, as reported in figure

95 Time/h 6 16 Conditions UNT BSA PA0.5 UNT BSA PA0.5 p38mapk pp38mapk JNK pjnk IκBα Figure 4.4: A) Macrophage treatment with palmitate activates JNK & p38mapk. Primary rat peritoneal macrophages were treated for 6 hours with palmitate 0.5mM. Cell lysates were generated immediately or palmitate was washed off and cells were grown for another 16 hours in fresh medium and then protein was isolated. Total JNK and p38 MAPK are shown as loading controls [N=5]. B) Macrophage treatment with palmitate leads to degradation of IκBα. Primary rat peritoneal macrophages were treated in the same way as in A. Loading controls are same as in A [N=3]. Representative membranes are shown 81

96 KC/GAPDH fold change IL-6/GAPDH fold change TNFα/GAPDH fold change MCP-1/GAPDH fold change 3 * 4 * BSA PA 0 BSA PA 3 2 * * BSA PA 0 BSA PA Figure 4.5: Macrophage palmitate treatment activates inflammatory cytokine & chemokine gene expression. Primary rat peritoneal macrophages were treated as per protocol for generation of conditioned medium. RNA was isolated and one-step RT-PCR kit used with GAPDH as control. * P< 0.05 BSA versus PA per cytokine tested. [N=4 for TNFα & IL-6; N=5 MCP-1; N= 3 KC]. 82

97 Despite the fact that there are several neutrophil chemoattractants in the conditioned medium, apart from CXCL5 what was induced with palmitate treatment, others like CINC-1, CINC-2α, and CINC-3 showed no significant difference between their levels in the MØCM-BSA or MØCM-PA, but neutrophil migration occurred mainly in the latter. This is intriguing and raises several possibilities, including that CXCL5 is the attracting chemokine for neutrophils in the palmitate-generated medium, or that there is another factor that is not one of those measured above, and that these factors are not present in sufficient amounts in MØCM-BSA to cause neutrophil chemotaxis. There is also the possibility that species incompatibility is to blame, as the neutrophils were of murine origin while the conditioned medium generated from cells of rat origin. We did not measure candidates like KC, which may be present in the medium that causes neutrophil attraction; this was not part of the array used for cytokine detection. Another important point is that MCP-1 was present in the MØCM-BSA but at lower levels than in but at lower levels than in MØCM-PA, and macrophage migration did not increase to levels seen in MØCM-PA. This differs from MyoCM-BSA that had no detectable levels when measured, yet comparable macrophage migration took place. It is likely that the MCP-1 in MØCM-BSA is contributing to macrophage migration in this case, while BSA-treated muscle is producing other factors that contribute to macrophage migration. What these factors may be is a matter of speculation and needs further evaluation. Interestingly, the arrays demonstrate that at different time points, different cytokines start appearing and our quantifications did take that into account. The signal from positive controls was always saturated and this increased at increasing time points. This is one limitation that precludes normalization of values to positive control. We did not do MCP-1 neutralization to answer the question of the role of MCP-1 in macrophagemacrophage cross talk; this is the next experiment to evaluate the relative contribution of MCP-1 under basal conditions e.g. BSA treatment to macrophage migration. 83

98 A MØCM-BSA MØCM-PA A B C D E F G H I J K L A B C D E F G H I J K L Touch BSA MØCM-BSA BSA A B C D E F G H I J K L PA MØCM-PA A B C D E F G H I J K L PA 5 sec

99 Agrin beta-ngf CINC-1 CINC-2 alpha CINC-3 Fas Ligand IL-6 IL-10 MCP-1 Thymus chemokine-1 TIMP-1 TNF-alpha VEGF PA/BSA fold change B 9 Cytokine profiler array primary rat peritoneal macrophages post palmitate 0.5mM treatment [Tx 6h, CM 16h, touch exposure] Figure 4.6: Treatment of primary rat peritoneal macrophage with palmitate leads to secretion of multiple cytokines/chemokines into conditioned medium. A) MØCM-BSA and MØCM-PA were tested for their cytokine and chemokine content and exposures done for up to 5 seconds. B) Quantification of different cytokines based on time of appearance. Positive control= A1-2, B1-2, L7-8; negative control=c1-2, D1-2, C-K 7 and C-K 8. AGRIN =F1-2; beta-ngf= H1-2; CINC-1=I 1-2; CINC-2α=J1-2; CINC-3=K1-2; FasL=A3-4; IL-10=L - 3-4; LIX=C5-6;MCP-1= E 5-6; Thymus chemokine-1=k5-6;timp-1=l5-6; TNFα=A 7-8; VEGF=B

100 4.6 Discussion In this study, we investigated the effect of palmitate on macrophage-macrophage interactions in context of palmitate treatment. Treating primary rat peritoneal macrophages with palmitate resulted in induction of inflammatory responses in and production of factors that attracted RAW macrophages. Palmitate-treated macrophages also produced factors that attracted neutrophils. Palmitate-treated macrophages showed activated inflammatory pathways including JNK, p38mapk and NFκB associated with upregulation of inflammatory cytokine and chemokine gene expression and secretion of several cytokines and chemokines into the medium. As we discussed earlier, saturated fatty acids contribute to inflammation and insulin resistance. Palmitate readily enters cells by diffusion across the plasma membrane and this process is concentration-dependent (288, 289), but it is also taken up via dedicated fatty acid membrane transporters ( ). In addition, palmitate also signals through TLR2 and TLR4 on cell surface (58). The evidence for the action of saturated fatty acids such as palmitate via TLR came from animal studies, whereby mice made deficient in TLR4 were protected from inflammation and insulin resistance despite high fat diet-induced obesity (58, 293, 294). Once fatty acids are inside the cell, they enter metabolic pathways and are processed to generate fatty acid intermediates including long chain fatty acid Co-A, DAG, and ceramide. When the latter two accumulate in the cells they affect insulin signaling and fatty acid metabolism, and lead to insulin resistance. The accumulation of DAG leads to activation of JNK, IKK, and PKC pathways and this interferes with IRS-1 signaling. Ceramides interfere with Akt phosphorylation and Rac activation, among other steps in insulin signaling (282, 295). In macrophages, fatty acid exposure leads to activation of inflammatory pathways in these cells and this triggers the production of pro-inflammatory cytokines and chemokines. Less is known about the onset of these responses, whether they are initiated by fatty acid signaling through TLRs and/or mediated by intracellular fatty acid metabolites. The macrophages involved in the interaction with metabolic organs are generally classified as pro-inflammatory or M1 and alternatively activated, anti-inflammatory M2 macrophages. However, this classification is not absolute and the markers of each are still being debated. 86

101 A question that is also highly debated is whether the gain in macrophage count in the obese adipose tissue is due to infiltrating anti-inflammatory macrophages or whether resident and infiltrating cells increase in phenotypes that include inflammatory and anti-inflammatory markers. However, in general terms the cytokine production suggests that there is a gain in new infiltrating macrophages, as their numbers far exceed the resident macrophages, and their phenotype is more pro-inflammatory (95). It is assumed that the adipocyte is the main source of the chemokines that attract macrophages while macrophages are the main source of proinflammatory cytokines produced in obese adipose tissue, but again the parameters of this complex response may be time dependent, where in early stages macrophages may not be the main contributors to chemokine production and rather the adipocyte plays an important role. As obesity persists and M1 macrophages are established in adipose tissue, they start secreting chemokines as well as cytokines to facilitate further macrophage attraction (19) (296). It is important to note that M2 macrophages are present in almost every tissue in the body as resident macrophages, and can compose 5-10% of the mass of a tissue under physiological circumstances. These cells maintain homeostatic functions within tissues by secreting factors that maintain normal tissue growth and homeostasis including cytokines and chemokines. In obesity and high fat feeding, and depending on the dietary regimen used to induce weight gain, the number of macrophages can expand to constitute a significant proportion of total tissue mass. It is likely that the differentiation status of macrophages is influenced by local tissue environment and the presence of other cells, and the mechanisms behind the evolution and differentiation of macrophages is not fully understood. Most likely, crosstalk among macrophages, adipocytes, and also muscle cells, endothelial cells and responses to local tissue environment cues leads to the differentiation of infiltrating monocytes to macrophages, mostly towards the M1 proinflammatory type. By this scenario, these cells would secrete pro-inflammatory cytokines and chemokines that would affect local tissues, including muscle. Again, why these cells would become M1 cells is dictated by the local environment, but the exact molecules and pathways involved are not clear. The information available on macrophage-macrophage cross talk in obesity is scant. It has been shown that 3T3-L1 adipocytes exposed to a fatty acid mix produce factors that attract RAW macrophages (279). 87

102 As described in chapter 3, myoblasts exposed to palmitate also attract macrophages. Our experimental work provides evidence that exposing primary rat peritoneal macrophages to palmitate leads to the production of factors that attract other macrophages. However, it is unclear how fatty acids exert their effects on immune cells, and what are the pathways involved in this process. We here show that macrophage palmitate treatment results in activation of p38mapk and degradation of IκBα, an indicator of release of NFκB which is a transcription factor that translocate to the nucleus and has binding sites at promoters of pro-inflammatory cytokine genes, leading to activation of their transcription. This is in agreement with the responses we observed upon treating muscle cells with palmitate, indicating that the activation of these pathways is likely a general response. However, while palmitate treatment causes insulin resistance in muscle cells, NF B activation was not essential for this effect (297). Whether fatty acid exposure and enhanced gene expression would translate to enhanced levels and secretion of the pro-inflammatory cytokines tested was unknown. Therefore, using profiler arrays we quantified cytokine proteins in the conditioned medium from palmitate-treated macrophages. Several pro-inflammatory cytokines and chemokines were secreted in response to both BSA and palmitate treatment of primary rat peritoneal macrophages, particularly increased levels of MCP-1 and TNFα and a smaller increase in IL-6. There were also several neutrophil chemoattractants detected in both conditioned media. It would be important to clarify which factor(s) are mainly responsible for neutrophil attraction and further experiments are needed to clarify this further. Along these lines, murine muscle cell stretching results in secretion of IL-8, a known neutrophil chemoattractant (298), indicating muscle cell ability to attract neutrophils under certain conditions. When comparing cytokine arrays from BSA versus palmitate treated macrophages, several patterns emerge. First, there are several molecules that are produced constitutively by macrophages as seen in BSA treated cells, but some are upregulated with palmitate treatment and most of these molecules are chemokines. For example, MCP-1, CINC1, CINC2, and CINC3 are constitutively expressed by macrophages, and the production of some including CINC1 and MCP-1 is upregulated with palmitate treatment. In addition, Thymocyte Chemokine-1 is upregulated with palmitate treatment. 88

103 These chemokines attract not only other macrophages [MCP-1], but also neutrophils [CINC1, CINC2, CINC3, CXCL5] and T-lymphocytes [Thymus Chemokine-1]. This points to a heterogeneous chemokinetic response whereby under basal conditions, the macrophage secrete molecules that facilitate innate immune cell communication, and this is upregulated when palmitate is applied to macrophages, but this triggers adaptive immune cell recruitment to inflamed cells as well. How does the attraction of all these cell types evolve over time to impact metabolic function is not very clear, and it is probable that the production of these chemokines serve other roles in different tissues beside chemoattraction of immune cells. Second, palmitate treatment of macrophage stimulates the production of several other molecules that are exclusive to palmitate treated cells including IL-10, FasL, CXCL5, TNF, and VEGF. Some of these molecules like TNF are pro-inflammatory, while others like IL-10 and TIMP-1 are protective against inflammation and CXCL5 is a known neutrophil chemoattractant. The balance in production of and responses to these factors dictates the final response by macrophages to their environment. When comparing macrophage and muscle cell responses to palmitate, there are several distinctions to be noted. In muscle cells, there is activation of a general inflammatory response characterized by production of multiple pro- and anti-inflammatory cytokines and chemokines. Muscle cells start producing MCP-1 and induce the production of CINC1 with palmitate treatment. The cytokines and chemokines attract immune cells but also modulate their phenotype. In macrophages, the response to palmitate is predominantly chemokine-based. MCP-1, CINC1, and others are constitutively produced by macrophages and are upregulated with palmitate treatment. This takes place in conjunction with the production of other neutrophil and T-lymphocyte chemoattractants as above. In addition, Macrophages produce TNF at higher levels than muscle cells and produce very little IL-6 under our experimental conditions. This comparison needs to take into account that these two cells were treated differently, whereby muscle cells were exposed to palmitate 0.2mM for 24 hours and then medium was generated after 24 hours of washing it off, while macrophages were treatment was with 0.5mM for 6 hours followed by incubation in fresh medium for 89

104 16 hours. Nevertheless, this comparison demonstrates the different responses noted in metabolic and immune cells in response to palmitate treatment. In summary, treating macrophages with palmitate led to an inflammatory response characterized by activation of inflammatory pathways, enhancement of gene expression of inflammatory cytokines, and secretion of multiple cytokines and chemokines. These are likely to act in an autocrine and paracrine fashion to affect macrophage and neutrophil migration. 90

105 5 Chapter 5: Conclusions & Future Directions 5.1 Conclusions Our experimental work shows that the exposure of muscle cells and macrophages to the saturated fatty acid palmitate results in activation of inflammatory pathways, upregulation of cytokine and chemokine gene expression, and secretion of multiple cytokines and chemokines. We identified MCP-1 as the main factor produced by muscle cells that is synthesized and secreted in response to fatty acid treatment in the absence of confounding factors like endotoxin or residual fatty acids in the culture. Blocking MCP-1 lead to significant reduction in macrophage migration to conditioned media from palmitate treated muscle cells. This may provide a potential link between muscle-macrophage crosstalk in diabetogenic environment. We present a model for muscle response to saturated fatty acids [Figure 5.1], in which the expansion of adipose tissue in obesity which takes place in visceral compartment and this is associated with expansion of the intermyocellular fat depot. This results in an inflammatory state characterized by production of cytokines and chemokines, activation of resident and infiltrating macrophages and lipolysis. The exposure of muscle to cytokines, chemokines and fatty acids from systemic and local sources triggers an inflammatory response characterized by activation of inflammatory pathways, upregulation of gene expression, and synthesis and secretion of inflammatory cytokines and chemokines. One of the main factors produced by muscle is MCP-1, which attract macrophages and this will in turn secrete inflammatory molecules and propagate this inflammatory response resulting in muscle inflammation and insulin resistance. In addition, MCP-1 itself may confer insulin resistance on skeletal muscle as discussed in introduction. This highlights the potential for therapeutic strategies whereby targeting MCP-1 directly or the pathways responsible for muscle MCP-1 production, which in muscle cells include ERK and NFκB, may help reduce macrophage infiltration, muscle inflammation and insulin resistance, which may have a major impact on the glucose metabolism in obese and insulin resistant individuals. 91

106 Adipose tissue Adipocyte Adipokines Cytokines Chemokines FFA Intermyocellular fat depot Macrophages Muscle systemic & local exposure: FFA +FA intermediates Cytokines/chemokines Activation of MAPKs and IKK MCP-1 Monocyte/macrophage attraction Inflammation Insulin resistance Figure 5.1: The model for fatty acid effects on muscle-macrophage and macrophagemacrophage crosstalk in diabetogenic environment. Adipose tissue expansion in obesity is characterized by production of cytokines and chemokines, activation of resident adipose tissue macrophages and free fatty acid [FFA] release via increased lipolysis. This attracts monocytes that differentiate to macrophages. In addition, the expansion of the intermyocellular fat depot also attracts macrophages and this is associated with secretion of pro-inflammatory cytokines, chemokines and FFA release that occur locally. The exposure of muscle to cytokines, chemokines and fatty acids from systemic and local sources triggers an inflammatory response characterized by activation of inflammatory pathways, upregulation of gene expression, and synthesis and secretion of inflammatory cytokines and chemokines. One of the main factors produced by muscle is MCP-1; the secretion of MCP-1 will lead to macrophage attraction to muscle. The attracted macrophages will in turn secrete inflammatory molecules, which signal through MAPKs and other pathways, propagating this inflammatory response and resulting in attraction of immune cells, resulting in muscle inflammation and insulin resistance. 92