WHY DOES IRON OVERLOAD ASSOCIATED WITH INSULIN RESISTANCE OCCUR? Thesis submitted to THE TAMIL NADU DR. M.G.R. MEDICAL UNIVERSITY CHENNAI for the degree of DOCTOR OF PHILOSOPHY By JOE VARGHESE DEPARTMENT OF BIOCHEMISTRY CHRISTIAN MEDICAL COLLEGE VELLORE – 632002, INDIA DECEMBER 2017 1 TABLE OF CONTENTS Page no. 1. Introduction ………………………………………………………………………….. 4 2. Aims and objectives …………………………………………………………………. 9 3. Review of literature ………………………………………………………………….. 10 3.1. Review of current understanding of systemic iron homeostasis ………………… 10 3.2. Hepcidin, the central regulator of iron homeostasis …………………………….. 18 3.3. Insulin, the central regulator of energy homeostasis ……………………………. 29 3.4. Diabetes mellitus ………………………………………………………………… 41 3.5. Role of insulin resistance in the pathogenesis of type 2 diabetes mellitus ……… 47 3.6. Role of beta cell dysfunction in the pathogenesis of type 2 diabetes mellitus ….. 53 3.7. Role of iron in the pathogenesis of type 2 diabetes mellitus ……………………. 55 4. Scope and plan of work ……………………………………………………………… 66 5. Materials and methods ………………………………………………………………. 69 5.1. Equipment used …………………………………………………………………. 69 5.2. Materials ………………………………………………………………………... 70 5.3. Methodology ……………………………………………………………………. 72 Study 1 …………………………………………………………………… 78 Study 2 …………………………………………………………………… 126 Study 3 …………………………………………………………………… 192 Study 4 …………………………………………………………………… 245 6. Results and discussion 6.1. Study 1 …………………………………………………………………………... 75 Abstract …………………………………………………………………… 75 Introduction ………………………………………………………………. 76 Methodology ……………………………………………………………... 78 Results ……………………………………………………………………. 92 Discussion ………………………………………………………………… 115 Summary and conclusions ………………………………………………… 121 6.2. Study 2 …………………………………………………………………………… 122 Abstract …………………………………………………………………… 122 2 Introduction ……………………………………………………………….. 123 Methodology ……………………………………………………………… 126 Results …………………………………………………………………….. 139 Discussion ………………………………………………………………… 172 Summary and conclusions ………………………………………………… 185 6.3. Study 3 …………………………………………………………………………… 187 Abstract …………………………………………………………………… 187 Introduction ……………………………………………………………….. 188 Methodology ……………………………………………………………… 192 Results …………………………………………………………………….. 207 Discussion ………………………………………………………………… 234 Summary and conclusions ………………………………………………… 240 6.4. Study 4 …………………………………………………………………………… 241 Abstract …………………………………………………………………… 241 Introduction ……………………………………………………………….. 242 Methodology ……………………………………………………………… 245 Results …………………………………………………………………….. 253 Discussion ………………………………………………………………… 273 Summary and conclusions ………………………………………………… 279 7. Summary of results ……………………………………………………………………. 280 8. Conclusions …………………………………………………………………………… 284 9. Recommendations and future directions ………………………………………………. 286 10. Bibliography …………………………………………………………………………... 288 11. Appendix Appendix I: MIQE checklist ……………………………………………………… 323 Appendix II: qPCR validation data ……………………………………………….. 327 Appendix III and IV: Composition of control diet and high-fat diet……………… 330 Appendix V and VI: Patient information sheet and consent form (English) …….. 332 Appendix VII and VIII: Patient information sheet and consent form (Tamil) …… 334 12. Publications ……………………………………………………………………………. 338 3 1. INTRODUCTION Diabetes mellitus is one of the most prevalent non-communicable diseases in India and the world over. It is estimated that there are over 60 million diabetics in India and this number is expected to increase significantly over the next few decades (1). Insulin resistance (IR) is the hallmark of type 2 diabetes mellitus (T2DM), which is the most common type of diabetes. The pathogenesis of IR is complex and multi-factorial. A number of risk factors, including obesity and genetic predisposition, have been shown to be associated with IR. However, the molecular mechanisms involved have not been fully elucidated (2). Iron is a transition metal and an essential micronutrient. It is required for the synthesis of hemoglobin and is therefore essential for erythropoiesis. Approximately 25 mg of iron is required every day for normal erythropoiesis in the bone marrow. More than 95% of this is provided by the recycling of iron derived from senescent erythrocytes. The macrophages that form the reticuloendothelial system, especially in the spleen, play a major role in this process. The rest of the daily iron requirement is derived from absorption of dietary iron, which occurs in the duodenum. Duodenal absorption of iron is a highly regulated process. Any increase in iron absorption in excess of the requirement results in iron overload, as the body has no physiologically regulated mechanisms to excrete iron. Such iron overload has been shown to be associated with tissue damage seen in conditions like hereditary hemochromatosis and thalassemia (3). A large number of epidemiological studies have shown a strong association between serum ferritin levels, an indicator of body iron stores, and IR. For example, the National Health and 4 Nutrition Education Survey (NHANES) done among 9486 adults in USA, showed that the odds ratio for newly developed T2DM was 4.94 in males and 3.61 in females who had serum ferritin that exceeded 300 µg/L (4). This and a number of other studies have shown that this association remains strong even after adjusting for age, sex, race, body mass index (BMI), smoking and C- reactive protein (CRP) levels (5–9). In addition, the “normal” range of serum ferritin (15-300 µg/L in males and 15-200 µg/L in females) is quite wide and values at either end of this range may be associated with health risks that currently remain unknown (10). Although a strong association exists between increased body iron stores and T2DM, the molecular interactions between processes involved in iron homeostasis and insulin sensitivity are not known. There is some evidence that supports the view that iron overload may play a causal role in the pathogenesis of diabetes mellitus. For example, the incidence of diabetes is higher is conditions characterized by iron overload, viz. hereditary hemochromatosis (11) and thalassemia (12,13). Decreasing body iron stores, by iron chelation or phlebotomy in these conditions, tends to improve insulin sensitivity (14–16). These findings are supported by results from research using animal models of T2DM, where dietary iron overload was associated with insulin resistance (17) and iron restriction/phlebotomy was associated with improved insulin sensitivity (18,19). Insulin acts by binding to its cell surface receptor and triggering multiple intracellular signaling cascades. The phosphorylation and activation of Akt is a key event in this pathway (20). Activated Akt phosphorylates several downstream targets, which mediate the effects of insulin on metabolism, cell growth and proliferation (21). Although iron overload is thought to be 5 associated with insulin resistance, very little is known about the effects of increased intracellular iron on the insulin signaling pathway. The mechanism(s) by which iron accumulates in the body in diabetes is not known. It has been hypothesized that inappropriately low levels of hepcidin, the chief regulator of systemic iron homeostasis, may play a key role in mediating increased iron stores in T2DM (22). Hepcidin is a small-molecular-weight peptide (25 amino acids) secreted mainly by hepatocytes (23). It binds to ferroportin (the only known transporter in mammalian cells that exports iron) and triggers its internalization and degradation (24). Since ferroportin is expressed on the basolateral surface of enterocytes and also on macrophages, hepcidin decreases duodenal iron absorption and also inhibits iron recycling by macrophages. Studies on hepcidin in patients with T2DM have reported variable findings, with some studies showing increased serum hepcidin levels in diabetics (25,26), while others show decreased levels (27–29); still others show no significant differences (30). The reasons for these varying reports are unclear. A number of factors regulate hepatic hepcidin synthesis. It is known that hepcidin expression is induced in response to increased liver iron levels, increased transferrin saturation and inflammatory mediators such as interleukin-6 (IL-6). On the other hand, hepcidin expression is decreased in response to iron-deficiency anemia, hypoxia and erythropoiesis (31). The effects of IR per se on hepatic hepcidin expression and on factors that regulate it are not known. Among the factors that regulate hepatic hepcidin expression, inhibitory signals from the bone marrow, mediated by certain regulators secreted by erythroid precursors (“erythroid regulators of hepcidin”), play a very important role (32). Although the identity of these erythroid regulators is not clearly known, growth differentiation factor 15 (GDF15) (33), twisted gastrulation factor 6 (TWSG1) (34) and erythroferrone (ERFE) (35) have been proposed to be potential candidates. It is known that T2DM is associated with hyperinsulinemia and that insulin is a growth factor for erythropoiesis (36–38). However, the effects of insulin resistance on erythropoiesis or on the expression of GDF15, TWSG1 and ERFE in erythroid precursors are not clearly known. In summary, a number of epidemiological studies have shown that raised serum ferritin, a marker of body iron stores, is associated with insulin resistance. There is evidence to suggest that iron overload may play a causal role in the pathogenesis of insulin resistance, although the molecular mechanisms are not known. Dysregulation