Relationship of Microalbuminuria in Patients of Type 2 Diabetes with Insulin Resistance and Gender Difference in this Association

Transcription

1 Relationship of Microalbuminuria in Patients of Type 2 Diabetes with Insulin Resistance and Gender Difference in this Association Research Thesis for Ph.D (Faculty of Medicine) at Baqai Medical University Karachi Research Conducted By Dr. Shahid Ahmed MBBS FCPS (Medicine) Consultant Physician CMH Malir Cantt. Karachi Under the Supervision of Professor Lt Gen (R) Dr. Syed Azhar Ahmad Hilal-e-Imtiaz (M), Sitara-e-Bisalat MBBS, Ph.D (London), FRCPath,FCPS, Vice Chancellor Baqai Medical University

2 Dedicated To My Parents 2

3 Acknowledgements I wish to thank the following people, for their support to make this work possible: 1. My supervisor, Professor Lt Gen (Retd) Dr. Syed Azhar Ahmad, for continuous supervision, support and encouragement throughout my work. 2. Lt Col Nadir, consultant haematologist and officer in charge of Combined Military Hospital Malir laboratory and his staff for providing excellent technical assistance for this research. 3. Brig Ishaq, Commandant CMH Malir, for providing technical, administrative and financial support for this research. 4. Pharmaceutical companies especially Eli Lilly and Novonordisk for provision of funds for expensive lab kits. 5. Administrative staff of Baqai Medical University for a very supportive and responsible attitude. 6. Most importantly, my wife and kids for providing me time, support and peace of mind to perform this work. 3

4 Contents a. Abstract 1 b. Introduction 3 c. Review of literature 8 1. Introduction to Insulin Resistance 8 2. Multiple Sites of Insulin Resistance 9 3. Molecular Mechanisms of Insulin Resistance Adipokines, Insulin Sensitivity and Type 2 Diabetes Inflammation and Insulin Resistance Fatty Acids and Insulin Resistance Insulin Resistance Syndrome Metabolic Syndrome Microalbuminuria and Nephropathy in Type 2 Diabetes Microalbuminuria and Insulin Resistance Measurement of Insulin Resistance Treatment of Insulin Resistance Reduces Microalbuminuria 49 d. Figures 54 e. Materials and Methods 59 f. Results 63 g. Tables 67 h. Graphs 78 i. Discussion 82 j. Summary 89 k. Conclusion 91 l. References 92 4

5 ABSTRACT Insulin resistance is considered as a fundamental component of the pathogenesis of type 2 diabetes. People have suggested the possibility that treating and modifying insulin resistance in patients of type 2 diabetes would improve urine albumin excretion. This study was planned with an objective to investigate the relationship of insulin resistance with microalbuminuria in patients of type 2 diabetes mellitus and observe any difference between men and women in this association, in Pakistani population. This study was carried out and Diabetes clinic of Combined Military Hospital, Malir Cantt, from April 2007 to August One hundred and fifty five patients of type 2 diabetes mellitus were included in the study that had either microalbuminuria or normoalbuminuria. Body mass index, waist circumference and blood pressure were recorded. Fasting venous blood sample was collected for plasma glucose (FPG), serum insulin, total and HDL cholesterol, triglycerides, creatinine and HbA1c. Urine albumin excretion was determined using urine albumin to creatinine ratio. Insulin resistance was calculated from fasting plasma glucose and serum insulin levels, using homeostatic model assessment of insulin resistance (HOMA-IR). Microalbuminuria was found to be significantly correlated with HOMA-IR (r = 0.33, p = < ), serum insulin (r = 0.28, p = < 0.001), body mass index (r = 0.18, p = 0.02) and waist circumference (r = 0.21, p = 0.008). This correlation was more significant in women (n = 85, r = 0.48, p = < ) as compared to men (n = 70, r = 0.14, p = 0.12). The correlation between HOMA-IR and urine albumin excretion remained highly significant (p = 0.001) after controlling for gender, age, duration of diabetes, waist circumference, hypertension, triglycerides and HbA1c. 5

6 HOMA-IR was also significantly correlated with waist circumference (r = 0.24, p = 0.001), BMI (r = 0.16, p = 0.02), triglycerides (r = 0.22, p = 0.003), HDL cholesterol (r = 0.18, p = 0.01), HbA1c (r = 0.35, p = < ), FPG (r = 0.44, p = < ) and metabolic syndrome (r = 0.18, p = 0.01) The study concludes that urine albumin excretion in patients of type 2 diabetes is strongly associated with insulin resistance and related cardiovascular risk factors. This association appears to be stronger in women than men, in our population. 6

7 INTRODUCTION Type 2 diabetes mellitus is a worldwide public health concern and an important cause of morbidity and mortality. Through lifelong vascular complications, diabetes leads to excessive rates of myocardial infarction, stroke, renal failure, blindness and amputations. The projections of its future impact are alarming. According to the World Health Organization, diabetes affects more than 170 million people worldwide, and this number will rise to 370 million by 2030 [1]. Type 2 diabetes results from disorders of insulin action and insulin secretion, either of which may be the predominant feature and both of which are usually present when the disease becomes clinically manifest [2]. By definition, specific causes are not known and autoimmune destruction of the pancreas does not occur. Type 2 DM is preceded by insulin resistance and impaired glucose tolerance (IGT). Once insulin resistance is pronounced, the likelihood of type 2 DM development depends on the ability of beta cells to compensate adequately by increasing insulin secretion. The disease thus, is of insidious onset and may remain asymptomatic for many years. The true duration of disease is often not known. It has been reported that duration of more than 6 years of diabetes may have existed before diagnosis [3] About one third of type 2 diabetics will eventually have progressive deterioration of renal function [4]. Diabetic nephropathy is a public health concern of increasing proportions. It has become the most common single cause of end-stage renal disease all over the world [5]. The first clinical sign of renal dysfunction in patients with diabetes is generally, microalbuminuria (a sign of endothelial dysfunction that is not necessarily confined to the kidney). The degree of microalbuminuria determines the progression of diabetic nephropathy. It may reflect the 7

8 renal manifestation of a global vascular dysfunction [6]. Microalbuminuria is also a marker of inflammation and an independent risk factor for cardiovascular mortality [7]. Microalbuminuria is often present at the time of diagnosis, either due to insidious nature and asymptomatic initial years of type 2 diabetes, or its positive association with insulin resistance, even in non diabetic people [8]. It refers to the excretion of albumin in the urine at a rate that exceeds normal limits but is less than the detection level for traditional dipstick methods [9]. The normal rate of albumin excretion is less than 20 mg/day (15 µg/min); persistent albumin excretion between 30 and 300 mg/day (20 to 200 µg/min) is called microalbuminuria. Values above 300 mg/day (200 µg/min) are considered to represent overt proteinuria. Although the 24-hour urine collection was previously the gold standard for the detection of microalbuminuria [10,11], it has been suggested that screening can be more simply achieved by a timed urine collection or an early morning specimen to minimize changes in urine volume that occur during the day [10,12]. The effect of volume can be avoided entirely by calculation of the albumin-to-creatinine ratio in an untimed urine specimen. A value above 30 mg/g (or 0.03 mg/mg) suggests that albumin excretion is above 30 mg/day and therefore that microalbuminuria is probably present [13]. With standard units, the comparable value is 3.4 mg of albumin per mmol of creatinine. Microalbuminuria develops in 2 to 5 percent of patients of type 2 diabetes per year [14,15]. In type 2 diabetes, unlike type 1 diabetes, microalbuminuria is seldom reversible [16,17] but, instead, progresses to overt proteinuria in 20 to 40 percent of patients [18,19]. In 10 to 50 percent of patients with proteinuria, chronic kidney disease develops that ultimately requires dialysis or transplantation [20]. Forty to 50 percent of patients with type 2 diabetes who have microalbuminuria eventually die 8

9 of cardiovascular disease [21]; this is three times as high a rate of death from cardiac causes as among patients who have diabetes but have no evidence of renal disease [15]. Thus, microalbuminuria is a major risk factor for renal and cardiovascular events, and the early identification and treatment of patients at increased risk for microalbuminuria may be instrumental to limit the excess renal and cardiovascular disease associated with type 2 diabetes. Attempts at prevention of nephropathy in type 2 DM have focused on the prevention of microalbuminuria, the earliest clinical hallmark of nephropathy, or its progression to macroalbuminuria [22]. Microalbuminuria can be effectively reduced by drugs as suggested by BENEDICT trial [23]. Lewis et al. [24] reported on the use of irbesartan in patients with type 2 DM and nephropathy. A 33% decrease in proteinuria was seen over 2.6 yr, whereas the placebo group decreased 10%. A similar study using losartan in type 2 DM and nephropathy found a reduction of 35%, whereas the placebo group had a rise in proteinuria [25]. Parving et al. [26] reported on the use of irbesartan in a population of hypertensive patients with type 2 DM and microalbuminuria and found that microalbuminuria was decreased by 38% over 2 yr with only a change of 2% in the controls. Evidence suggests that insulin resistance precedes and probably contributes to the development of microalbuminuria in type 1 diabetic patients [27] and in nondiabetic subjects [28] and most probably, in type 2 diabetics also. Insulin resistance is a clinical state which is a component of several disorders. The term insulin resistance actually means, impaired insulin stimulated glucose disposal [29] as measured with the hyperinsulinaemic euglycemic clamp technique [30]. Insulin resistance is considered as a fundamental component of the pathogenesis of type 2 diabetes. The research has been focused on tissues responsible for 9

10 insulin-mediated glucose uptake, namely muscle and, to a minor degree, adipose tissue [31]. However, it is well known that not only muscle glucose uptake but also adipose tissue lipolysis and suppression of glucose production by liver are regulated by insulin. Several factors have been linked to the development of this insulin resistance like leptin, adiponectin, free fatty acids, inflammation and some genetic factors. Insulin resistant state characterized by hyperinsulinaemia is responsible for various disorders like impaired glucose tolerance and diabetes mellitus, hypertriglyceridaemia and low HDL cholesterol, sodium retention, hyperuricaemia, hypertension, fatty liver, polycystic ovary syndrome, endothelial dysfunction and microalbuminuria. WHO clinical criteria of metabolic syndrome include both insulin resistance and microalbuminuria as the important components of this syndrome [32]. Different mechanisms have been proposed to link insulin resistance to abnormal albuminuria, like glomerular hyperfiltration [33], endothelial dysfunction [34], and increased vascular permeability [35]. Association of microalbuminuria with insulin resistance and other cardiovascular risk factors such as increased waist circumference, high BMI, dyslipidaemia and presence of metabolic syndrome can also explain association between increased urinary albumin excretion and cardiovascular morbidity and mortality [36] in type 2 diabetes. The relationship between insulin resistance and urinary albumin excretion had been a matter of debate, because association between insulin resistance and microalbuminuria was suggested by few studies [37-44] and was not confirmed by others [45-48]. There has been a convincing evidence that measures which target insulin resistance or components of insulin resistance syndrome reduce urinary albumin excretion in patients of type 2 DM, thereby slowing the progression of diabetic nephropathy and reducing cardiovascular risk. Losing weight in 10

11 obese diabetics, treatment of hyperlipidaemias and treatment with metformin have all shown to reduce urinary albumin excretion, but data on insulin sensitizers, thiazolidinediones in reducing urinary albumin excretion in type 2 diabetics is very encouraging. PPAR-gamma agonists, the thiazolidinediones (eg, pioglitazone and rosiglitazone) appear to have a variety of beneficial effects in animal models of diabetic nephropathy, such as reductions in fibrosis, mesangial cell proliferation, and inflammation [49-51]. While the human data on outcomes are limited, PPAR-gamma agonists reduce urinary albumin excretion at various stages of nephropathy [52-54]. Pistrosch et al, 2005 [55] used a crossover study design that permitted a comparison of rosiglitazone with other oral antidiabetic agents providing similar glycemic control but not insulin sensitization. Rosiglitazone ameliorated renal glomerular endothelial dysfunction suggested by the reduction in filtration fraction and improved bioavailability of nitric oxide (NO). All the renal hemodynamic changes were correlated with a reduction in microalbuminuria. Considering the possibility that treating and modifying insulin resistance in patients of type 2 diabetes would improve urine albumin excretion, we planned to study this association in our population. The objective of our study was to investigate the relationship of insulin resistance with microalbuminuria in patients of type 2 DM and observe any difference between men and women in this association. We used homeostatic model assessment of insulin resistance (HOMA-IR), as a surrogate of hyperinsulinaemic euglycemic clamp, to estimate insulin resistance, using fasting plasma glucose and fasting serum insulin levels [56]. 11

12 REVIEW OF LITERATURE 1. Introduction to Insulin Resistance Insulin resistance is a state in which a given concentration of insulin is associated with a subnormal glucose response [57]. The term first came into use several years after the introduction of insulin therapy in 1922 to describe occasional diabetic patients who required increasingly large doses of insulin to control hyperglycaemia. Most of these patients developed insulin resistance secondary to antibodies directed against the therapeutic insulin, which was impure and derived from non-human species at that time [58]. Anti-insulin antibodies are rare in patients treated with recombinant human insulin. Therefore, the insulin resistance that we are going to discuss, rather than being a rare complication of treatment of diabetes, is a clinical condition, which is a component of several disorders. It is characterized by high serum insulin concentrations in association with blood glucose concentrations that are normal or high. Type 2 diabetes results from a failure on the part of the pancreatic beta cells to compensate adequately for the defect in insulin action in insulinresistant persons [2]. Even before the development of significant beta cell failure to cause diabetes mellitus in insulin resistant persons, the ability to maintain the compensatory hyperinsulinaemia necessary to prevent glucose tolerance in insulin resistant persons, is associated with a number of adverse outcomes, including cardiovascular disease (CVD), polycystic ovary syndrome (PCOS), non-alcoholic fatty liver disease (NAFLD), and possibly several forms of cancer. Moreover the fact that many newly diagnosed type 2 diabetic subjects already suffer from chronic complications of diabetes at the time of diagnosis [59], indicates that there may be a delay in diagnosis, in addition that the pre-diabetic condition is harmful to human health also. Thus, type 2 diabetes mellitus 12

13 represents only the tip of the iceberg of long existing metabolic disturbances with deleterious effects on the vascular system, tissues and organs [60]. 2. Multiple Sites of Insulin Resistance The term insulin resistance in humans is frequently used synonymously with impaired, insulin stimulated glucose disposal [29,57] as measured with the hyperinsulinaemic euglycemic clamp technique [30]. Insulin resistance is considered as a fundamental component of the pathogenesis of type 2 diabetes. The research has been focused on tissues responsible for insulin-mediated glucose uptake, namely muscle and, to a minor degree, adipose tissue [31]. However, it is well known that not only muscle glucose uptake but also adipose tissue lipolysis and suppression of glucose production by liver are regulated by insulin. Muscle Muscle is the major site for insulin mediated glucose disposal in the body. This abnormality in insulin action on muscle does not depend on whether or not the patient is obese [61]. Insulin resistance in muscle is present in majority of persons with glucose intolerance but this defect, by itself, accounts neither for the development of significant hyperglycaemia in patients with type 2 DM, nor for the severity of fasting hyperglycaemia in these persons[62]. Adipose Tissue Dysregulation of fat metabolism occurs very early in the development of insulin resistance and well before the onset of hyperglycemia in type 2 diabetes. It has been shown that ambient plasma 13

14 free fatty acid (FFA) concentrations are higher than normal in patients with type 2 DM, that is, there adipose tissue is also insulin resistant [63,64]. The increase in plasma FFA concentration that occurs when insulin resistant person is unable to maintain a state of compensatory hyperinsulinaemia, is primarily responsible for the development of significant hyperglycaemia in type 2 diabetic patients [65]. The increased availability and utilization of FFA contribute to the development of skeletal muscle insulin resistance [65]. Moreover, FFA have been shown to increase endogenous glucose production by stimulating key enzymes and providing energy for gluconeogenesis [66], while the glycerol released during triglyceride hydrolysis serves as a gluconeogenic substrate [67], thereby decreasing the ability of hyperglycaemia to suppress hepatic glucose production. Consequently, resistance to the antilipolytic action of insulin in adipose tissue resulting in excessive release of FFA and glycerol would have deleterious effects on glucose homeostasis. The rise in plasma FFA and glucose concentrations impair beta cell function. Glucotoxicity of hyperglycaemia is a well recognized phenomenon [68], but data also suggests that chronic increases in FFA concentration, also inhibit beta cell response to glucose [69]. Liver Role of the liver in the pathogenesis of hyperglycaemia in type 2 diabetics had been a bit controversial issue. It is generally thought that fasting hyperglycaemia in the patients with type 2 diabetes is directly related to an absolute increase in hepatic glucose production. A few studies have suggested that hepatic glucose production was increased by only approximately 30% with significant fasting hyperglycaemia [70]. Based on the analysis of an enormous data, Radziuk and Pye [71] concluded that values of glucose production after an overnight fast in 14

15 patients with type 2 DM will vary from the normal range to rates that are from 20% to 50% higher than normal, depending on the study population and the experimental conditions. 3. Molecular Mechanisms of Insulin Resistance Development of Type 2 diabetes is a multi-step process with strong genetic and environmental influences. Although the precise pathogenesis and the pathophysiological sequence resulting in insulin resistance is still largely unknown, recent studies have contributed to a deeper understanding of the underlying molecular mechanisms. In addition to classical biochemical in vitro studies, the use of gene targeting approaches in mice and the analysis of naturally occurring mutations in animal models and insulin resistant patients has shed some light on the molecular dysregulations that can contribute to insulin resistance and type 2 diabetes [72]. The detailed understanding of these basic pathophysiological mechanisms is critical for the development of novel therapeutic strategies to treat diabetes. Insulin signalling The insulin receptor consists of extracellular ligand binding and intracellular tyrosine kinase domains. Binding of insulin to the extracellular portion of the receptor activates its kinase activity resulting in autophosphorylation of specific intracellular tyrosine residues. This autophosphorylation step enables a variety of scaffolding proteins including insulin receptor substrate (IRS) proteins to bind to intracellular receptor sites and to become phosphorylated [73]. 15

16 Insulin Receptor Substrate (IRS) IRS-1 and other recently cloned IRS proteins (IRS-2, -3, -4) are phosphorylated upon insulin stimulation and have adaptor function between the insulin receptor and other cellular substrates such as the phosphatidylinositol 3-kinase (PI 3-kinase). Based on results from specific knockout models, the most important IRS proteins in the regulation of carbohydrate metabolism appear to be IRS-1 and -2 [74]. In humans, rare mutations of the IRS-1 protein are associated with insulin resistance [75] and disruption of the IRS-1 gene in mice results in insulin resistance mainly of muscle and fat [76]. Interestingly, IRS-2 knockout mice not only show insulin resistance of muscle, fat and liver, but also develop diabetes mellitus because of β-cell failure [77]. Therefore, it is tempting to speculate that dysfunction of IRS-2 and its downstream targets might represent a common feature of both peripheral insulin resistance and β-cell failure. PI 3-kinase/PKB-signalling Downstream of IRS-proteins, the phospho-inositide-3-kinase (PI 3- kinase) is a central mediator of the effects of insulin. Binding of PI 3- kinase to phosphorylated sites in IRS proteins leads to activation of PI 3- kinase. The activated PI 3-kinase generates 3 -phosphoinositides [phosphatidyl-inositol-3,4-bisphosphate (PIP2) and phosphatidyl-inositol- 3,4,5-trisphosphate (PIP3)] [78]. PIP2 and PIP3 bind to the phosphoinositide dependent kinase 1 (PDK1). Therefore, these two phospholipids may attract PDK1 and the putative PDK2 to the plasma membrane. Known substrates of the PDKs are the PKB. PKB mediates the effects of insulin on glucose transport, glycogen synthesis, protein synthesis, lipogenesis and suppression of hepatic gluconeogenesis. PKB regulates both, glucose uptake via facilitated glucose transporters 16

17 (GLUTs) and intracellular glucose metabolism in insulin sensitive tissues such as skeletal muscle [79]. Under non-stimulated conditions, PKB is located in the cytoplasm and stimulation with insulin results in translocation of PKB to the plasma membrane, where PKB may bind to PIP-2 and PIP-3 [103]. At the plasma membrane, PKB co-localizes with PDK and becomes activated by phosphorylation of its two principal regulatory sites, Thr308 and Ser473. Following activation, PKB detaches from the plasma membrane to affect metabolic processes such as glycogen synthesis and glucose transport. Parts of the activated PKB also translocate through the cytoplasm into the nucleus by an unknown mechanism to affect gene expression [79]. Schematic representation of insulin signalling is presented in figure-1. Protein and lipid phosphatases Termination of the insulin signal is critical for the maintenance of metabolic control. Signalling of the insulin receptor cascade is terminated by specific phosphatases. One of the key phosphatases in this context is the protein-tyrosine-phosphatase 1B (PTP1B). For example, mice lacking the PTP1B gene exhibit increased insulin sensitivity and fail to develop insulin resistance under a high-fat diet [80]. In addition, the inhibition of PTP1B-activity by systemic application of antisense oligonucleotides specific for PTP1B improved insulin sensitivity and glycaemic control in diabetic mice [81]. Other phosphatases relevant for the termination of insulin signalling include PTEN (phosphatase and tensin homologue) which inactivates the lipid products of the PI 3-kinase and also SHIP2, an inositol 5 -phosphatase. 17

18 Glucose Transporters Glucose transport is the rate limiting step for glucose utilization in muscle at most physiologic glucose and insulin levels [82]. Glucose transport into muscle and adipose cells occurs by facilitated diffusion that is mediated primarily by two members of the glucose transporter (GLUT/SLC2A) family of proteins. There are currently 12 identified members of this family that are further subcategorized into three classes [83]. The class I molecules GLUT-1 to GLUT-4 have been most extensively characterized. In tissues with insulin-sensitive glucose transport (muscle and adipose cells), GLUT-4 is the predominant glucose transporter. The large stimulatory effect of insulin in these tissues results from the unique targeting of GLUT-4. In the absence of insulin, GLUT-4 is sequestered in intracellular vesicles; in response to insulin and other stimuli, these vesicles translocate to the plasma membrane. Gene knockout studies in mice have demonstrated that reduction in GLUT-4 levels can cause insulin resistance and frank diabetes and transgenic over expression studies have shown that increased expression of GLUT-4 in muscle or fat can increase whole-body glucose disposal and ameliorate insulin resistance in transgenic mice. Glucose metabolism Effectors involved in insulin-dependent glucose disposal into fat and muscle About 75% of insulin-dependent postprandial glucose disposal occurs into the skeletal muscle [84]. Activation of PI 3-kinase results in translocation of GLUT4 into the plasma membrane with consecutive increase of glucose transport into muscle and fat. The potential role of PKB in the pathogenesis of insulin resistance has been of central interest during the recent years. Recent data from PKB knockout animal models 18

19 offer a clearer answer to the question whether PKB is required for normal glucose homeostasis. While disruption of PKBα (Akt1) in mice did not cause any significant perturbations in metabolism, mice with a knock out of the PKBβ (Akt2) isoform show insulin resistance ending up with a phenotype closely resembling Type 2 diabetes in humans [85]. Initially, these animals have impaired insulin-mediated glucose disposal and a very prominent lack of suppression of hepatic glucose production in response to insulin. Finally, they progress to develop a relative β-cell dysfunction and consecutively manifest diabetes [85]. Hepatic glucose production The fasting hyperglycaemia in patients with Type 2 diabetes is the clinical correlate of the increased glucose production by the liver because of insulin resistance. This is because of the lack of inhibition of the two key gluconeogenic enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and the glucose-6-phosphatase catalytic subunit (G6Pase). Insulin inhibits the expression of both of these enzymes at the transcriptional level [86] and it is widely accepted that this process is mediated via activation of PKB. Currently, probably the best characterized substrate of PKB is the GSK-3 (glycogen synthase kinase-3), a critical enzyme regulating glycogen synthesis. There is abundant evidence that PKB-mediated inhibition of GSK-3 is the key mechanism through which insulin promotes glycogen synthesis. The major part of glucose taken up from the blood after insulin stimulation is stored as glycogen in skeletal muscle. Dysregulated glycogen synthesis is a critical feature in diabetes mellitus as glycogen synthesis rates in diabetic patients are approximately 50% lower than in healthy subjects [87]. 19

20 The role of the adipose tissue in insulin resistance Currently, there is strong evidence that dysfunction of adipose tissue plays a crucial role in the development of insulin resistance and Type 2 diabetes. Both obesity and lipodystrophy lead to insulin resistance in muscle. Adipose tissue can modulate whole body glucose metabolism by regulating levels of circulating free fatty acids (FFA) and also by secreting adipokines, thereby acting as an endocrine organ. There is also clear evidence from clinical and biochemical studies that circulating FFA can impair insulin sensitivity in muscle. The levels of circulating FFA are inversely correlated with insulin sensitivity in humans [88]. 4. Adipokines, Insulin Sensitivity and Type 2 Diabetes Mellitus Adipose tissue, in addition to being a fat store, secretes a number of hormones and proteins collectively termed adipokines [89,90]. Several adipokines, including adiponectin, leptin, and interleukin (IL)-6, have been linked to the development of diabetes [91,92]. Several adiposederived cytokines (adipokines) have been suggested to act as a link between accumulated fat mass and altered insulin sensitivity[93]. Skeletal muscle is the largest tissue by mass regulated by insulin, and is responsible for >80% of insulin-stimulated glucose disposal. In both humans and rodents, there is a strong correlation between abnormal fatty acid (FA) metabolism and the development of insulin resistance in skeletal muscle. Identifying the link between excess stores of body fat and altered substrate metabolism in muscle has been difficult. Several adipose-derived cytokines (adipokines), including resistin and tumour necrosis factor alpha (TNF-a) have been implicated in impairing insulin 20

21 sensitivity in rodents. Conversely, two other adipokines, leptin and adiponectin, are known to increase insulin sensitivity in lean and obese rodents [94]. Leptin Leptin, the product of the ob gene, is a 16 kda cytokine-like peptide that is produced primarily by adipose tissue and has been shown to regulate food intake and energy expenditure in rodents [95]. Various rodent models which either fail to secrete functional leptin (ob/ob mice) or express the leptin receptor (db/db mice and fa/fa rats), are characterized by obesity and insulin resistance. Rodent models of lipoatrophy as well as human lipodystrophy [96], characterized by an absence or low level of circulating leptin, also demonstrate insulin resistance. Leptin appears to be an important factor in maintaining insulin sensitivity in skeletal muscle. This may be due to direct effects on GLUT4 protein content and various proteins of the insulin signalling cascade. In addition, the stimulatory effects of leptin on insulin sensitivity may also be due to the ability of leptin to prevent excess lipid accumulation as IMTG. The latter effect appears to be due to several factors, including reduced FA uptake and esterification, and increased lipolysis and FA oxidation [97,98]. High levels of circulating leptin characterize most cases of obesity suggesting the development of central and/or peripheral resistance. The existence of leptin resistance in human skeletal muscle was shown by the fact that leptin stimulated FA oxidation in isolated muscle strips from lean, but not obese humans [98]. However, of greater practical significance is that aerobic training can at least partially prevent the high fat diet-induced development of leptin resistance in rat muscle [99]. 21

22 Adiponectin Adiponectin (Ad) is a 35 kda adipokine with antidiabetic properties, mediated predominantly by its effects on muscle and liver FA and glucose metabolism. Adiponectin is expressed exclusively in adipose tissue and is lower in human and rodent models of obesity and type 2 diabetes [100]. In particular, the decrease in circulating Ad is most closely correlated to the increase in visceral fat [101]. Adiponectin levels are upregulated by the antidiabetic thiazolidinediones (TZDs) which stimulate PPAR gamma in adipocytes [102]. The ability of Ad to improve the diabetic condition appears to be due to its effects on both muscle (improved insulin stimulated glucose disposal) and liver (decreased glucose production). In addition to the observed relationship between reduced circulating Ad and impaired insulin sensitivity, various lines of evidence indicate the probable role of Ad as an insulin-sensitizing agent. In diabetic rodent models such as the db/db mouse, the administration of Ad ameliorates insulin resistance [100]. Resistance to Leptin and Adiponectin A Precursor to Insulin Resistance In summary, leptin and adiponectin have similar acute and chronic effects on muscle metabolism. Each promote FA oxidation, lower lipid stores (decreased FA transport and increased lipolysis), resulting in improved insulin sensitivity. These effects are mediated at least in part by the activation of the AMPK axis. It appears as though some degree of resistance to each of these adipokines in skeletal muscle occurs during the development of obesity. Hypothetically, this could lead to increased FA uptake and decreased oxidation, resulting in the accumulation of various lipid species leading to impaired insulin signalling. 22

23 Resistin Resistin is a recently discovered protein that is expressed and secreted from adipocytes and is present in the circulation. Resistin has been proposed to be a potential link between obesity and insulin resistance [103]. Initial reports found that circulating levels of resistin were elevated in two different genetic models of obesity (ob/ob and db/db) as well as in a diet-induced model of diabetes and obesity. It was also reported that administration of resistin impaired glucose homeostasis and insulin sensitivity in wild-type mice, while neutralization of resistin in diet induced obese mice reduced blood glucose levels and improved insulin sensitivity. Furthermore, the anti-diabetic TZDs [103] markedly reduced resistin gene expression and protein secretion. The potential relevance of resistin in the pathogenesis of insulin resistance however, has also been questioned. A recent study [104] demonstrated only a weak relationship in humans between serum resistin concentrations and wholebody insulin sensitivity in a wide variety of subjects (non-obese, obese and obese diabetics). TNF alpha TNF-a is a proinflammatory cytokine that is produced and secreted from adipocytes and plays a major role in mediating immune responses. TNF-a has been implicated as a critical mediator of insulin resistance, particularly in relation to obesity [105]. Long-term exposure to TNF-a induces insulin resistance in rodents, while neutralization of TNF-a improves insulin action in obese insulin-resistant rats. It has also been shown that TNF-a expression is increased in adipose tissue [106] and skeletal muscle of insulin resistant humans [107]. Despite strong evidence for an association between TNF-a and insulin resistance in rodent models of obesity and diabetes, it is controversial whether circulating levels of 23

24 TNF-a are elevated in obese and type 2 diabetic humans and whether increased TNF-a concentrations correlate with insulin action [108]. Plasminogen activator inhibitor Plasminogen activator inhibitor 1, an inhibitor of fibrinolysis, is another protein related to adipocytes. It is also secreted from endothelial cells, mononuclear cells, hepatocytes, and fibroblasts and has been associated with an increased risk for cardiovascular disease. A five-year prospective study of 2356 adults aged 70 to 79 years identified 143 new cases of diabetes [109]. Elevated levels of plasminogen activator inhibitor 1 were an independent predictor of onset of diabetes (odds ratio [OR] 1.35; 95% CI ) after controlling for components of the metabolic syndrome (body mass index, visceral fat, lipids, hypertension, and fasting glucose). Retinol-binding protein 4 Retinol-binding protein 4 (RBP4), another protein released from adipocytes, correlates with the degree of insulin resistance in patients with obesity, impaired glucose tolerance, or type 2 diabetes, as well as in non-obese subjects with [110] or without [111] a strong family history of type 2 diabetes. Exercise training reduced RBP4 levels in patients whose insulin resistance improved with exercise. An inverse relationship between GLUT4 in adipocytes and serum RBP4 has been demonstrated in the humans [110]. 5. Inflammation and Insulin Resistance Hotamisligil and colleagues [112] first showed that the proinflammatory cytokine TNF-α was able to induce insulin resistance. This was a 24

25 revolutionary idea, that a substance produced by fat and overproduced by expanded fat had local and potentially systemic effects on metabolism. The concept of fat as a site for the production of cytokines and other bioactive substances quickly extended beyond TNF-α to include leptin, IL-6, resistin, monocyte chemoattractant protein-1 (MCP-1), PAI-1, angiotensinogen, visfatin, retinol-binding protein-4, serum amyloid A (SAA), adiponectin and others. While leptin and adiponectin are true adipokines that appear to be produced exclusively by adipocytes, TNF-α, IL-6, MCP-1, visfatin, and PAI-1 are expressed as well at high levels in activated macrophages and/or other cells. These cytokines and chemokines activate intracellular pathways that promote the development of insulin resistance and T2DM. Inflammation, TNF-α and Peroxisome Proliferator Activator Receptor-γ (PPAR-γ) An interesting possible mechanistic connection is the ability of TNF-α and PPAR-γ to antagonize each other. Insulin sensitizers, thiazolidinediones that act by activating the nuclear hormone receptor PPAR-γ can suppress inflammatory response in general and inhibit the transcriptional activity of TNF-α promoter in particular [113]. These PPAR-γ agonists can also antagonize the effects of exogenously administered TNF-a on insulin action in cultured cells, whole animals and humans [114]. Importantly this activity is independent of the adipogenic activity of PPAR-y [115]. Therefore, it is a possibility that the elevated inflammatory response in general and increased TNF-a expression in particular might be the consequence of reduced PPAR-y activity in obesity. The ability of these antagonists to increase insulin sensitivity might be directly related, at least in part, to their anti-inflammatory capacity via 25

26 the TNF-a pathway. The mechanism by which PPAR-y activators increase insulin sensitivity is not well understood. It has been observed that use of these compounds in rodent models of obesity and insulin resistance result in reduction of elevated levels of TNF-a as well as TNF receptor expression [116]. It is possible that reduction of TNF-a expression is secondary to increased insulin sensitivity or other indirect effects of these compounds. Whether suppression of TNF-a is one of the mechanism by which these drugs act as insulin sensitizers, is not yet confirmed. 6. Fatty Acids and Insulin Resistance Excessive accumulation of body fat (obesity) and dietary fat intake are both associated with insulin resistance [117]. A likely mechanism involves release of one or more messengers originating from the adipose tissue (or from ingested fat) that inhibit insulin action on skeletal muscle and/or the liver. Several candidates for such a role have been proposed including leptin, TNF-α, resistin and free fatty acids (FFAs). Free Fatty Acids and Peripheral (Muscle) Insulin Resistance Plasma FFA levels are chronically elevated in obesity. The elevated plasma FFAs chronically inhibit insulin sensitivity, which improves on lowering of the FFA levels. This defect of fat mediated inhibition of insulin-stimulated glucose uptake may be due to transport or phosphorylation (hexokinase) defect or decreased muscle glycogen synthase activity [118]. 26

27 The long delay (3-4 hours) between the increase in plasma FFAs and appearance of insulin resistance gave rise to the notion that FFAs must be esterified inside muscle cells to cause insulin resistance. It was shown with 1H nuclear magnetic resonance spectroscopy (1H-MRS) that increases in plasma FFA levels resulted (within 4 hours) in an increase in intramyocellular triglyceride content that occurred concurrently with the onset of insulin resistance [119]. The mechanisms by which increased IMCL bring about insulin resistance are not precisely understood. Muscle triglycerides themselves need not necessarily affect insulin action directly in the muscle cell. Rather, they represent a reservoir of FFAs, which intramyocellularly may give rise to metabolites interfering with insulin signalling, mechanisms also not precisely understood. An increase in delivery of fatty acids can lead to an increase in diacylglycerol, malonyl CoA, long-chain acyl CoA and ceramides. These metabolites activate a serine/threonine kinase cascade (possibly initiated by protein kinase Cy) leading to phosphorylation of serine/threonine sites on insulin receptor substrates (IRS-1 and IRS-2), which in turn reduces the ability of the IRSs to activate PI 3-kinase (figure-2). As a consequence, glucose transport activity and other events downstream of insulin receptor signalling are decreased [120]. Free Fatty Acid and Hepatic Insulin Resistance Several studies have shown that increasing plasma FFAs will partially inhibit insulin-induced suppression of endogenous glucose production (EGP). Studies using euglycemic-hyperinsulinaemic clamping indicated that FFA-induced attenuation of insulin suppression of EGP (i.e. hepatic insulin resistance) was caused primarily by inhibition of insulin suppression of glycogenolysis [121]. They also suggested that 27

28 elevated plasma FFAs in patients with type 2 DM may be responsible at least in part for the relative (in relation to insulin) or absolute elevations in EGP in these patients. 7. Insulin Resistance Syndrome The fact that most of the insulin resistant persons are able to compensate for this abnormality by secreting enough insulin to overcome the insulin resistance does not mean that this state of chronic hyperinsulinaemia is benign. In 1988 [122] it was emphasized that although most insulin resistant/hyperinsulinaemic individuals were able to prevent the development of frank hyperglycaemia, they were at greatly increased risk to be somewhat glucose intolerant, dyslipidemic, with a high fasting triglyceride (TG) and low high density lipoprotein (HDL), hypertensive and at increased risk for cardiovascular disease. This cluster of abnormalities related to insulin resistance and compensatory hyperinsulinaemia was initially referred to as syndrome X, but in the subsequent years, has been given almost as many names as are the number of adverse consequences of insulin resistant states. More recently Adult Treatment Panel III has acknowledged the importance of insulin resistant state as cardiovascular disease risk factor, and has proposed specific diagnostic criteria for what they referred to as the metabolic syndrome [123]. Manifestations of the Insulin Resistance Syndrome Compensatory Hyperinsulinaemia The combination of insulin resistance and compensatory hyperinsulinaemia differentiates insulin resistance syndrome from type 2 28

29 DM, which develops in genetically predisposed individuals when they develop insulin secretory defect. There is no absolute criterion that defines an individual as being either insulin sensitive or insulin resistant. The ability of physiologic hyperinsulinaemia to enhance muscle glucose uptake varies continuously in healthy, non obese population and there is an approximate six fold difference between the most insulin sensitive and insulin resistant individual [124]. Insulin resistance is influenced by both lifestyle and genes. Two important lifestyle factors, obesity and level of physical activity modulate insulin action, they are not its cause. Insulin Resistance versus Compensatory Hyperinsulinaemia Many of the manifestations of insulin resistance syndromes results from actions of the compensatory hyperinsulinaemia on normally insulin sensitive tissues. For example hyperinsulinaemia acts on a normally insulin sensitive kidney to increase sodium reabsorption and decrease uric acid clearance, contributing to the hypertension and hyperuricaemia seen in patients with the insulin resistance syndrome [125]. Other examples are PCOS (polycystic ovary syndrome) and dyslipidaemia. Glucose Metabolism Most insulin resistant persons maintain normal glucose tolerance due to compensatory hyperinsulinaemia. However, the greater the magnitude of insulin resistance, higher will be the prevalence of impaired glucose tolerance (IGT) and impaired fasting glucose (IFG) [122,126]. Although insulin resistance is common to both type 2 DM and the insulin resistance syndrome, the two clinical entities are different in many ways. For example, CVD risk is increased in both disorders but diabetic microangiopathy occurs only with type 2 DM. 29

30 Lipoprotein Metabolism Various abnormalities in lipoprotein metabolism associated with insulin resistance are all closely related to changes in VLDL metabolism. Plasma Triglyceride Concentration Insulin resistance at muscle and adipose tissue leads to higher levels of insulin and FFAs, and these two changes stimulate hepatic VLDL-TG secretion, leading to an increase in plasma TG concentration in insulin resistant individuals [127]. High Density Lipoprotein Cholesterol (HDL-C) Increases in plasma TG concentration are usually associated with low HDL-C concentrations. In part, this is likely due to transfer, catalyzed by cholesteryl ester transfer protein, of cholesterol from HDL to VLDL; the higher the VLDL pool size, the greater the transfer rate from HDL to VLDL, and the lower the ensuing HDL-C concentration [128]. Low Density Lipoprotein (LDL) Particle Size LDL has a predominance of two particle sizes, larger LDL (pattern A) or smaller LDL (pattern B). Individuals with pattern B have higher plasma TG and lower HDL-C concentrations, and are at increased risk for CVD. There is evidence that people with pattern B type LDL are insulin resistant, glucose intolerant, hyperinsulinaemic, hypertensive, hypertriglyceridaemic and have a lower HDL-C concentration [129]. Non-alcoholic Fatty Liver Disease (NAFLD) Hepatic TG synthesis is increased in insulin resistant individuals, leading to an increase in the mass of TG per VLDL particle. If the rate of appearance of the newly synthesized TG cannot be entirely incorporated into VLDL particles and secreted, there will be an inevitable increase in 30

31 the amount of TG that will accumulate in the liver. NAFLD has become the most recently recognized consequence of insulin resistance syndrome both in diabetic and non-diabetic individuals [130]. Hemodynamic Insulin resistant and compensatory hyperinsulinaemia can predispose individuals to develop increased resting heart rate and hypertension via stimulation of the sympathetic nervous system [131]. Hyperinsulinaemia increases renal sodium in patients with insulin resistance, suggesting normal renal insulin sensitivity, though muscles of these individuals are resistant to insulin mediated glucose disposal, similarly causing hyperuricaemia as insulin decreases urinary uric acid clearance [125]. There is considerable evidence that patients with essential hypertension, as a group are insulin resistant and hyperinsulinaemic when compared with individuals with normal blood pressure [131,132]. Several prospective studies have shown that hyperinsulinaemia at baseline predicts the subsequent development of essential hypertension. That is why essential hypertension is considered an integral component of insulin resistance or metabolic syndrome [133]. Prothrombotic Elevated levels of plasminogen activator inhibitor-1 (PAI-1) are seen in association with myocardial infarction, as well as in patients with hypertriglyceridaemia and hypertension. It has been shown that increased PAI-1 concentrations are associated with insulin resistance and compensatory hyperinsulinaemia, with subsequent development of type 2 DM [109]. 31

32 Polycystic Ovary Syndrome (PCOS) PCOS is another example, where hyperinsulinaemia secondary to muscle and liver insulin resistance, adversely affects the normally insulin sensitive ovaries, resulting in excessive secretion of testosterone by the ovaries [134]. Endothelial Dysfunction Endothelium-dependent flow-mediated vasodilatation has been shown to be decreased in a variety of clinical syndromes characterized by insulin resistance [135]. Plasma concentration of cellular adhesion molecules (CAMs) that modulate the adherence of mononuclear cells to the endothelium, have been shown to be elevated in insulin resistant individuals [136]. There is also an evidence that plasma concentration of asymmetric dimethylarginine (ADMA), an inhibitor of nitric oxide synthase, was increased in insulin resistant hypertensive patients as compared to normal population [137]. 8. Metabolic Syndrome The metabolic syndrome is a multiplex risk factor for cardiovascular disease (CVD) and type 2 diabetes that reflects the clustering of individual risk factors resulting from obesity and insulin resistance [138]. Currently, this multiplex is thought to be composed of the following broadly stated metabolic risk conditions: atherogenic dyslipidemia, hypertension, glucose intolerance, proinflammatory state, and a prothrombotic state. Atherogenic dyslipidemia is itself an aggregate term encompassing elevated triglycerides and apolipoprotein B, increased small LDL particles, and reduced HDL. Insulin resistance, the associated 32

33 hyperinsulinemia and hyperglycemia, and adipocyte cytokines (adipokines) may also lead to vascular endothelial dysfunction, an abnormal lipid profile, hypertension, and vascular inflammation, all of which promote the development of atherosclerotic cardiovascular disease (CVD) [139]. A similar profile can be seen in individuals with abdominal obesity that do not have an excess of total body weight [140]. Definition Because metabolic syndrome traits co-occur, patients identified with one or just a few traits are likely to have other traits as well as insulin resistance. Whether it is valuable to assess insulin resistance in addition to more readily measured traits of the syndrome is uncertain. There are several definitions for the metabolic syndrome, leading to some difficulty in comparing data from studies using different criteria [32,141,142]. The National Cholesterol Education Program (NCEP/ATP III) and International Diabetes Federation (IDF) definitions are the most widely used. New International Diabetes Federation (IDF) Definition According to the new IDF definition, for a person to be defined as having the metabolic syndrome they must have [142,143]: Central obesity (defined as waist circumference 94cm for Europid men and 80cm for Europid women, with ethnicity specific values for other groups) plus any two of the following four factors: Raised TG level: 150 mg/dl (1.7 mmol/l), or specific treatment for this lipid abnormality 33