Roollee Foof Lcciirccuulaattiinngg Lleevveelss Ooff

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Roollee Foof Lcciirccuulaattiinngg Lleevveelss Ooff

Transcript Of Roollee Foof Lcciirccuulaattiinngg Lleevveelss Ooff

ROLE OF CIRCULATING LEVELS OF PALMITATE AND PALMITOLEATE IN DEVELOPMENT OF BETA CELL DYSFUNCTION AND INSULIN RESISTANCE IN PEDIATRIC OBESITY Johan Staaf
Supervisor: Peter Bergsten Department of Medical Cell Biology, Uppsala University
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Table of content
Abstract ......................................................................................................................... 4 Abbreviations ...............................................................................................................5 1. Introduction..............................................................................................................6 Obesity and T2DM – a “joint pandemic” of the 21st century.......................................6 Defining childhood obesity and the relevance of puberty .............................................6 Cellular mechanisms of beta cell dysfunction ...............................................................7 Glucose and lipid metabolism........................................................................................8 Consequences of insulin hypersecretion........................................................................9 Role of non esterified fatty acids on beta cell function................................................10 Role of non esterified fatty acids on insulin resistance ...............................................10 Hypotheses ..................................................................................................................11 2. Material and methods............................................................................................12 Study population ..........................................................................................................12 Sampling ......................................................................................................................12 Measurements of body mass index (BMI) and body composition................................12 Oral glucose tolerance test (OGTT) ............................................................................12 Measurements of NEFAs...........................................................................................13 Chemicals ..................................................................................................................... 13 Extraction of lipids.......................................................................................................13 Derivatization of NEFA ...............................................................................................14 Identification of NEFA by GC-MS...............................................................................14 Measurements of insulin sensitivity .........................................................................15 Measurements of beta cell function..........................................................................15 In vitro experiments on human islets .......................................................................15 Cell culture...................................................................................................................15 Palmitate medium and islet treatment .........................................................................15
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Glucose-stimulated insulin secretion (GSIS) ...............................................................15 Measurements of insulin secretion by ELISA ..............................................................16 Measurements of apoptosis..........................................................................................16 Statistical analysis and data presentation................................................................16 3. Results .....................................................................................................................17 Subjects characteristics ...............................................................................................17 Fasting insulin and glucose levels in study subjects....................................................18 Insulin and glucose levels in study subjects after glucose challenge ..........................19 Circulating palmitate and insulin levels ......................................................................22 Palmitate-induced beta cell dysfunction......................................................................24 Fasting palmitoleate and insulin levels in study subjects............................................25 beta cell function and insulin resistance......................................................................26 Palmitoleate and insulin sensitivity .............................................................................27 4. Discussion................................................................................................................29 Acknowledgement ......................................................................................................33 References ...................................................................................................................33
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Abstract Obesity and T2DM show a significant relationship and are affecting increasing number of individuals. Obesity (BMI>30 kg/m2) has been portrayed as a pandemic of the 21st century with a prevalence of more than 1 billion people. Juvenile obesity has increased considerably in the last decades and is linked to alternation in insulin levels. In these individuals early manifestations of beta cell dysfunction can be studied. We hypothesized that hypersecretion of insulin is predictive of future beta cell failure and precedes insulin resistance. Furthermore elevated levels of palmitate have adverse effects on beta cells, whereas high levels of palmitoleate have cytoprotective characteristics and promote insulin sensitivity. Using GC-MS, circulating levels of NEFAs were measured in 55 obese non-diabetic children. Fasting insulin, glucose and additional metabolic parameters were obtained. In addition, OGTT was conducted and allowed determinations of beta cell function and insulin sensitivity. To further examine palmitate‟s effects on beta cells, GSIS on human islet was measured after exposure to 0.5 mM palmitate for 0 (control), 1, 24 and 48 hours. Islet apoptosis was also measured after exposure to the fatty acid. In individuals with high palmitate levels, hypersecretion of basal and stimulated insulin levels was observed in the youngest subjects. Stimulated hypersecretion showed a significant decline with age, indicating a loss of beta cell mass and/or secretory function caused by ER-stress and oxidative stress. In vitro increased apoptosis was observed in islets after 48 hours culture with palmitate levels greater or equal to 0.5 mM. Islets displayed hypersecretion of basal and glucose stimulated insulin secretion after 1 hour, which was further accentuated after 24 hours. After 48 hours decreasing levels of insulin secretion were observed, however. Circulating palmitoleate levels correlated with high insulin sensitivity and low basal insulin levels, promoting metabolic deceleration and beta cell recovery. In conclusion, hypersecretion of insulin promotes anabolism, trapping young obese individuals in a vicious cycle, surging towards T2DM.
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Abbreviations ACC = Acetyl CoA Carboxylase ATP = Adenosine Tri Phosphate BMI = Body Mass Index BSA = Bovine Albumin Serum CPT 1 = Carnitine Palmitoyl Transferase 1 CRP = C - reactive protein EDTA = EthyleneDiamineTetraacetic acid ER = Endoplasmatic Reticulum FABP = Fatty Acid Binding Protein FAS = Fatty acid synthesis FFA = Free Fatty Acids FSH = Follicle Stimulating Hormone GC-MS = Gas Chromatography-Mass Spectrometry GLUT = Glucose Transporter GnRH = Gonadotropin Releasing Hormone GPR = G-Protein Receptor GSIS = Glucose Stimulated Insulin Secretion HDL = High Density Lipoprotein IFG = Impaired Fasting Glucose IGF-1 = Insulin Growth Factor-1 IGT = Impaired Glucose Tolerance LDL = Low Density Lipoprotein LH = Lutein Hormone mM = mille Molar mU/L = micro Units / Liter MUFA = Mono Unsaturated Fatty Acids NAD = Nicotinamide Adenine Dinucleotide NEFA = Non Esterified Fatty Acids NF-κB = Nuclear Factor – κ B OGTT = Oral Glucose Tolerance Test PDH = Pyruvate Dehydrogenase PERK = Pancreatic Endoplasmatic Reticulum Kinase PFK = Phosphofructokinase PPAR = Peroxisome Proliferator-Activated Receptor SCD 1 = Stearoyl CoA Desaturase 1 SNP = Single Nucleotide Polymorphism T2DM = Type 2 Diabetes Mellitus TAG = Triglyceride TNF-α = Tumor Necrosis Factor-α UPR = Unfolded Protein Response VLDL = Very Low Density Lipoprotein
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1. Introduction
Obesity and T2DM – a “joint pandemic” of the 21st century Obesity and type 2 diabetes mellitus (T2DM) is afflicting an increasing number of individuals on a global scale [1-3]. Obesity has been referred to as a pandemic of 21st century [4], not only affecting citizens of western countries, but also many people in countries in Africa and Asia [5]. It is estimated that more than 1 billion people suffer from obesity [4] and the prevalence is increasing rapidly [4-5]. During the first decade of the third millennium, the prevalence of obesity in Europe increased by 10% and is consuming a large proportion of healthcare resources [5]. Obesity has a complex etiology and affects individuals of all ages [4]. In recent decades a dramatic rise in childhood obesity has been observed [5]. In 2003, 22 million children worldwide, 5 years old or younger, were reported obese or overweight [4]. Numerous reports demonstrate a rising prevalence of paediatric obesity in Sweden the past decades [67]. However, recent data indicate that the trend in Sweden is levelling off [8]. Obese children have an augmented risk of future morbidity [4, 6] and obesity in itself can predict premature mortality and potentially counteract the current rise in life expectancy [9]. A strong relationship between obesity and prevalence of T2DM, not long ago considered a disease of the older generation, has been established [2]. WHO estimated that around 170 million people worldwide suffered from diabetes mellitus at the turn of the century and this number is expected to more than double by 2030 [10]. Up to 85% of children, who are diagnosed with T2DM, are obese or overweight [11]. Type 1 diabetes mellitus has been the most common type of diabetes observed in paediatric patients. With the growing obesity pandemic, T2DM is expected to surpass T1DM in children [2]. T2DM is the result of beta cell dysfunction and insulin resistance [12-15]. The exact mechanisms linking obesity with T2DM are not fully understood, however. In this study we propose that hyperinsulinemia, in essence hypersecretion of insulin by the beta cell, is particularity relevant for understanding early manifestation of T2DM in obese individuals. Also, we propose that early events of beta cell failure can be investigated by studying obese children.
Defining childhood obesity and the relevance of puberty The general method of defining obesity is by using body mass index (BMI) [16]. In adults, the BMI is simply calculated by dividing the weight (kilograms) by the height (meters) squared, thus obtaining the unit kg/m2. According to international standards, overweight is defined as BMI > 25kg/m2 and obesity as BMI > 30kg/m2, respectively. Cut-off points are related to increased health risks. When applying the BMI formula on children and adolescents, adjustments have to be made with regard to age and sex (ISO-BMI) [16], since children undergo rapid growth and development, especially during puberty. During puberty a decrease in insulin sensitivity of approximately 30% has been observed in both lean and obese adolescents [17]. The mechanisms behind this are not clear, though an appealing theory is that insulin sensitivity is reduced as a consequence of a transitory rise of growth hormone (GH) during puberty. GH has been shown to affect lipolysis and the concentration of free non esterified fatty acids (NEFA) in the bloodstream [17], which could potentially fuel insulin resistance. Interestingly, the compensatory increase in insulin secretion by the beta cells was lower than expected, signifying an attempt of protecting and preserving beta cell function [17]. In isolated beta cells, GH has been shown to causes a rise in insulin secretion [18], enhancing anabolic pathways and promoting insulin resistance.
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Cellular mechanisms of beta cell dysfunction T2DM is a metabolic disease with four main characteristics: beta cell dysfunction accompanied or not with inadequate insulin action leading to hyperglycemia and obesity [19]. Hyperinsulinemia, caused by beta cell dysfunction, has been proposed as a potential primary cause of early progression towards T2DM [15]. T2DM is a disease that slowly develops over an extended time period. This is due to the fact that beta cells possess a great reserve capacity, which enables insulin output to be increased dramatically in times of nutrient excess [20]. It has been demonstrated both in vitro and in vivo that glucose potentiates beta cell proliferation [20]. This is achieved by increase in beta cell mass, mitoses of existing beta cells and de novo generation of beta cells from progenitor cell within the pancreas [20]. At the time of diagnosis of T2DM, a 25-50% reduction in beta cell-mass can be observed, suggesting initial compensation for the decline in number of beta cells by hypersecretion of insulin from remaining beta cells [20].
Beta cell dysfunction arises from environmental and genetic factors [21]. Environmental factors are primarily chronically elevated levels of glucose and lipids, which lead to harmful effects including endoplasmatic reticulum (ER) and oxidative stress. This stress is caused by an increased metabolic demand, in terms of insulin secretion and biosynthesis, on beta cells (Fig 1.1) [22].
Figure 1.1 Hypersecretion of insulin is dependent on exogenous factor (hyperlipidaemia and hyperglycaemia) and endogenous factors (oxidative stress and ER-stress). Adapted from Rustenbeck et al [22]
Hypersecreting beta cells are showing different manifestations of intracellular stress (Fig 1.1), which elicit defense mechanisms including the unfolded protein response (UPR) [23]. Over time, the growing secretory demand on beta cells in obese individuals will promote apoptosis, with a subsequent loss of total beta cell mass, however [22]. Loss in beta cell mass is a sign of incipient T2DM. The exact pathway between hypersecretion of insulin and T2DM is still to be defined. Possible explanations could be cellular fatigue or inability to handle oxidative and ER-stress [15, 24]. ER-stress occurs when the ER is burdened by excess protein synthesis [15]. Secretory cells, beta cells included, have a very capable ER and are therefore particularly sensitive to ER-stress [15]. Some actually refers to diabetes as “misfolded protein disease” [25], demonstrating the important role of the ER in beta cell function
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[15]. Beta cells have a lot of ER transducer proteins, which main function is to intervene in case of ER-stress [15]. Pancreatic-ER kinase (PERK), which is a part of the UPR, senses protein folding status of the ER and can trigger “rescue cascades” involving phosphorylation of eIF2α [25]. This will temporarily halt protein synthesis, in order to deal with misfolded proteins within the ER, thus reducing insulin secretion. In vitro studies have demonstrated the role of ER-stress in beta cell dysfunction, where a knockout of PERK lead to beta cell apoptosis and elevated glucose levels [26].
High glucose levels cause increased glycolytic flux, which results in generation of reactive oxygen species (ROS) within mitochondria, generating oxidative stress [15]. Oxidative stress causes intracellular damage, affecting multiple part of the cell including the DNA. Ca2+ levels are disturbed, which compromise the entire secretory machinery, and eventually the beta cell will undergo apoptosis [15]. In vitro studies show increased oxidative stress in beta cells cultured in a glucolipotoxic milieu [15]. Patients with T2DM show increased levels of oxidative stress markers as well as markers for ER-stress [15], indicating its significance in pathogenesis and pathology of T2DM.
Glucose and lipid metabolism Elevated levels of glucose and fatty acids will fuel oxidative pathways. An intricate relationship between glucose oxidation and fat oxidation exists within cells, however [27]. In 1963, Philip Randle proposed a theory linking metabolism of glucose and fatty acids in the „Glucose-fatty acid cycle‟ [28]. He acknowledged that relationship of glucose- and fatty acid metabolism is reciprocal, hence elevated influx of fatty acids would lead to increased ratios of acetyl CoA/CoA and NADH/NAD+ in the mitochondria. This would result in decreased activity of pyruvate dehydrogenase (PDH) and elevated levels of citrate in the Krebs cycle [29]. High levels of citrate inhibit phosphofructokinase (PFK), a key regulatory enzyme in the glycolytic pathway. By inhibition of PFK, fructose-6-phosphate will accumulate within the cell, which will inhibit hexokinase and lead to reduced levels of glucose flux [29].
The main function of beta cells is the biosynthesis and secretion of insulin, which is tightly regulated by ambient glucose levels. High levels of glucose will cause increased glucose oxidation and lead to elevated levels of malonyl-CoA. MalonylCoA is a key building block in fatty acid biosynthesis (Fig 1.2). In addition, malonylCoA inhibits β-oxidation by suppressing the rate limiting enzyme carnitine palmitoyltransferase 1 (CPT1), thus preventing NEFAs from being transported into the mitochondria and undergo β-oxidation (Fig 1.2) [27]. Regulation and activity of CPT 1 play essential roles in maintenance of viability and secretory function in beta cells. Elevated levels of NEFAs, in particular palmitic acid (palmitate; C16:0), in combination with impaired CPT1 activity lead to intracellular accumulation and synthesis of lipids, which eventually precipitate beta cell dysfunction and apoptosis [27]. In contrast, it has been shown that via activation of GPR40 and subsequent peroxisome proliferator-activated receptor (PPAR)-signaling, NEFA actually stimulate CPT1 gene expression [3]. This would result in an increased β-oxidation, thus reduced intracellular levels of lipids.
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Figure 1.2 Increase in glucose oxidation causes depolarization of beta cells and insulin release but also lipid accumulation. High glucolytic flux will stimulate acetyl-CoA carboxylase (ACC), which will biosynthesize malonyl-CoA from acetyl-CoA. MalonylCoA promotes lipid biosynthesis and TAG formation and inhibits CPT 1, the rate limiting enzyme in β-oxidation. This will have adverse effects on the beta cells by accumulation of intracellular lipids, a concept known as lipotoxicity. Adapted from Sugden, Holness [30].
Consequences of insulin hypersecretion A prominent feature in obese children is elevated level of fasting insulin [31], which promotes accumulation of glucose and lipids and consequently further development of obesity [32]. Chronically elevated basal insulin levels have negative effects on glucose stimulated insulin secretion (GSIS), where reduction in the number of insulin vesicles present for instant release has been proposed as one mechanism [33]. Furthermore, chronically elevated basal insulin levels will lead to desensitization and internalization of peripheral insulin receptors [33], which will be compensated for by increased insulin release and further rise in basal insulin levels. An interesting theory is that high basal insulin levels also desensitize insulin receptors on beta cells, resulting in attenuated glucose sensitivity and impaired feedback regulation, which will result in insufficient first phase insulin secretion [33].
Factors that determine if an individual will hyper- or hyposecrete insulin at a given insulin sensitivity are not fully understood [33]. Many potential mechanisms have been suggested, including serum NEFA composition and concentration [34], insulin clearance rate [35], secretory signaling pathways [36], insulin receptor expression on beta cells [37] and alternations parasympathetic activity directed towards the pancreas [38]. Genetics has been shown to play a crucial role in
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predisposing dysinsulinemia; the gene coding for the pancreatic beta cell glucose carrier, GLUT2, has been identified as a strong candidate. GLUT2 is involved in regulating stimulated insulin secretion and a mutation in this gene trigger onset of diabetes mellitus [33]. The majority of known single nucleotide polymorphisms (SNP) linked to obesity and T2DM are connected with beta cell function [21].
Role of non esterified fatty acids on beta cell function Long-term exposure of beta cell to NEFAs, such as palmitate, causes beta cell apoptosis [39-40]. We wanted to examine the effects of palmitate in obese children and on islets in vitro. The term lipotoxicity is commonly used when addressing lipid‟s toxic effect on a cellular level. The loss of secretory function and viability of beta cells, when exposed to NEFAs for an extensive period of time, plays an important role in the development of T2DM [40]. However, the effects NEFAs have on beta cells depend on length and degree of saturation of the NEFA. Differences in beta cell response to long chain saturated or mono unsaturated fatty acids have been documented, indicating that they work through different intracellular mechanisms [40]. Cells treated with high levels of palmitate display morphological alterations in membrane structures within the cell, most likely the ER, manifestations of ER-stress, loss of viability and apoptosis [24, 41]. Moreover, experiments on isolated islets show that prolonged (24 hours) levels of elevated palmitate induce rise in basal insulin secretion [42], which is detrimental to the metabolic homeostasis. On the other hand, when exposing beta cells in vitro to mono-unsaturated fatty acids like palmitoleic acid (palmitoleate; C16:1), much less destructive effects on the cells are observed [40]. Thus, palmitoleate appears to mediate positive effects, or at least counteract the negative apoptotic effects of palmitate, when the two NEFAs are administered together [40]. Moreover, palmitoleate averts steatosis of the liver in mice, possibly by lowering the levels of lipogenic enzymes in the liver [43-44]. This endocrine communication between adipose tissue, myocytes and hepatocytes reflects the general action of a hormone. Indeed, palmitoleate has been referred to as a lipid hormone or „lipokine‟ [43].
Role of non esterified fatty acids on insulin resistance In the present study we set out to examine the effects of palmitate and palmitoleate on insulin resistance in obese individuals. Elevated levels of circulating NEFAs have been associated with development and progression of insulin resistance and impaired glucose tolerance although the exact mechanisms are poorly apprehended [29]. Possible explanations are effects exerted on the cellular plasma membrane and, perhaps more importantly, NEFAs‟ capability to operate as signalling molecules within cells [45]. It has been shown that palmitate aggravate glucose homeostasis by causing insulin resistance [29]. In contrast, palmitoleate has been linked to improved insulin sensitivity in vitro [29] and in adult humans, independent of age, sex and adiposity [43-44]. The function of glucose transporter (GLUT) 4 is dependent on insulin and is found in the plasma membrane of adipocytes, hepatocytes and skeletal muscle cells. Both palmitate and palmitoleate appears to have direct effects on insulin signalling and insulin dependent transport of glucose by GLUT4 [29]. It has been shown that palmitoleate‟s effect is partly mediated by recruitment of GLUT4 to the plasma membrane and retention of GLUT4, thus preventing down regulation of the important glucose carrier by internalization [29]. Fatty acid binding protein (FABP) 4 and 5, lipid chaperons in control of lipid traffic within cells [43], have both been found to repress palmitoleate synthesis [44]. The actions of FABP incorporate
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Beta CellsLevelsPalmitateGlucoseAcids