University of Ghana http://ugspace.ug.edu.gh BLOOD PERILIPIN A LEVELS IN DIABETIC PATIENTS AT KORLE-BU TEACHING HOSPITAL, GHANA. MICHAEL QUANSAH (10550529) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF MPHIL CHEMICAL PATHOLOGY DEGREE. JULY, 2017 University of Ghana http://ugspace.ug.edu.gh DECLARATION I, Michael Quansah, do hereby declare that this thesis is to be submitted in fulfilment of the requirements for an M.Phil. Degree in Chemical Pathology is the result of my own research conducted at the National Diabetic Research and Management Centre (Korle-Bu) , School of Biomedical and Allied Health Sciences Laboratory and Central Laboratory unit of Korle-Bu Teaching Hospital under the supervision of Dr. Seth Amanquah and Dr. Sylvester Yaw Oppong. However people’s works have been duly acknowledged. This thesis presents results of original research undertaken by me and neither all nor part of this thesis has been presented for another degree in this institution or elsewhere. ………………………………………… Date:……………… MICHAEL QUANSAH (STUDENT) ……………………………………… Date:……………. DR. SETH AMANQUAH (SUPERVISOR) ……………………………………………. Date:……………. DR. SYLVESTER YAW OPPONG (SUPERVISOR) i University of Ghana http://ugspace.ug.edu.gh ABSTRACT Perilipins are phosphoproteins that are found in fat cells where they are restricted at the uppermost layer of lipid droplets. Over-expression of perilipin proteins in fat cells cause the storage of triacylglyceride (TAG) to increase in connection with a reduction in fats and lipd breakdown that causes increases in weight and type 2 diabetes and cardiovascular diseases. There are two types of perilipin which have a common N-teminal amino acids sequence which are perilipin A (PLIN 1) and perilipin B (PLIN 2). PLIN1 is the prevailing form found in fat cells. PLIN1 protein is highly seen in human fat tissue and individual with increased in weight (obese subjects) had higher levels of PLIN1 than the lean subjects .The research aimed at measuring blood perilipin A levels in non-obese diabetic patients and obese diabetic patients. A total of 86 subjects including controls were recruited (42 obese diabetic case subjects and 42 non-obese diabetics as controls).Anthropometric measurement such as visceral and body fats was measured using digital body sensor .Human perilipin 1 ELISA kit was used to determine the levels of perilipin in both subjects. Lipid profile was determined using Mindray BS-120 fully automated chemistry analyzer as well as glycated hemoglobin was also measured. The mean age for the non-obese diabetic study group and obese diabetic group were 46.8 ± 8.2 and 47.5 ± 6.7 years respectively. The mean age difference between non-obese diabetics subjects and obese diabetics subjects was found insignificant (p=0.607). High mean differences in body mass index (BMI) was observed in the obese diabetics than in the non-obese diabetics (p<0.0001).The results in visceral and body fat was found to be significantly higher in case subjects than in the control subjects. (p<0.0001). The result in perilipin A levels between case subjects and control subjects was insignificant (p>0.6353) but perilipin A level was found to be increased in Non-obese diabetic patients(117.2 +47.3) than Obese diabetic patients (109.1+ 36.9). The difference observed ii University of Ghana http://ugspace.ug.edu.gh in the levels of perilipin A between male and female subjects was not significant (p>0.7353).The levels of perilipin was found to be higher in male than in female subjects. In conclusion, the levels of blood perilipin A observed in non-obese diabetic subjects were not significantly different from the obese diabetic subjects. iii University of Ghana http://ugspace.ug.edu.gh DEDICATION This research is dedicated to the late Miss Verma Marie Annan. iv University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENT This work owes its existence to the assistance received from many people. I therefore wish to register my heartfelt gratitude to all those, who in diverse ways contributed to the success of this project work. I would like to thank my supervisors, Dr. Seth Amanquah Department of Chemical Pathology, School of Biomedical and Allied Health sciences (SBAHS), University of Ghana. Dr. Sylvester Yaw Oppong Department of Medical Biochemistry (SBAHS), University of Ghana, whose earnest supervision, suggestions and constructive criticism have brought me this far. I am so grateful to them. I am thankful to the authors and publishers whose works were extremely useful in carrying out this project. My special thanks go to the staff of the National Diabetic Management and Research Center in Korle-Bu who were extremely helpful during collection of samples for the work. I am also grateful to the staff of the Department of Chemical Pathology, Central Laboratory, Korle-Bu (SBAHS) especially Auntie Yvoone Mr. George Kpentey, Mr. Isaac Boamah and Mr. Bernard Squire for helping me to use the ELISA KIT. I say “Merci” and may God bless you all. v University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENT DECLARATION………………………………………………………………………i ABSTRACT………………………………………………………………………….ii DEDICATION……………………………………………………………………….iv ACKNOWLEDGEMENT……………………………………………………………...v TABLE OF CONTENT…………………………………………………………………………..vi LIST OF FIGURES……………………………………………………………………………....ix LIST OF TABLES………………………………………………………………………………..xi LIST OF ABBREVIATION……………………………………………………………………..xii CHAPTER ONE………………………………………………………………………………….1 INTRODUCTION………………………………………………………………………………...1 1.1 Background………………………………………………………………………………….1 1.2 Problem statement…………………………………………………………………………...4 1.3 Justification………………………………………………………………………………….5 1.4 Hypothesis…………………………………………………………………………………...6 1.5 Aim…………………………………………………………………………………………..6 1.6 Specific objetives……………………………………………………………………………6 CHAPTER TWO………………………………………………………………………………….7 LITERATURE REVIEW…………………………………………………………………………7 2.1 Overview of Perilipin A (PLIN1)……………………………………………………………..7 2.2 The role of perilipin…………………………………………………………………………...8 vi University of Ghana http://ugspace.ug.edu.gh 2.3 The mechanism of Lipolysis…………………………………………………………………9 2.4 Obesity……………………………………………………………………………………….11 2.4.1 Prevalence of Obesity…………………………………………………………………..12 2.5 Overview of Diabetes Mellitus……………………………………………………………...13 2.5.1 Aetiology of diabetes mellitus………………………………………………………….13 2.5.2 Classification of Diabetes……………………………………………………………….15 2.5.3 Prevalence of diabetes mellitus…………………………………………………………15 CHAPTER THREE…………………………………………………...........................................17 MATERIALS AND METHOD………………………………………………………………….17 3.1. Study site…………………………………………………………………………………….17 3.2. Target Population……………………………………………………………………………17 3.3 Study design………………………………………………………………………………….18 3.4 Sample size determination…………………………………………………………………...18 3.5 Inclusion criteria……………………………………………………………………………..19 3.6 Exclusion criteria…………………………………………………………………………….19 3.7 Sample collection.....................................................................................................................19 3.7.1 Specimen collection, Transport and Storage...................................................................19 3.7.2 Anthropometric measurement…………………………………………………………..20 3.8 Biochemical Analysis of Sample…………………………………………………………….20 3.8.1 Measurement of Perilipin A/Perlipin 1(PLIN1)………………………………………..20 3.8.2 Measurement of Glucose………………………………………………………………..22 3.8.3. Measurement of Total Cholesterol (T. CHOL)…………………………………………23 3.8.4. Measurement of Triglyceride (TG)……………………………………………………..24 3.8.5. Measurement of High Density Lipoprotein (HDL) Cholesterol………………………..25 vii University of Ghana http://ugspace.ug.edu.gh 3.8.6 Measurement of low density lipoprotein (LDL-C) Cholesterol………………………...’25 3.8.7. Measurement of Glycated Hemoglobin (HbA1c)………………………………………25 3.8.8. Determination of HbA1c ……………………………………………………………….26 3.9 Data Handling………………………………………………………………………………..26 3.10 Statistical Analysis………………………………………………………………………….27 CHAPTER FOUR………………………………………………………………...28 RESULTS…………………………………………………………………………28 4.1: General Characteristics and background of the Study Population………………………….28 4.2: Biochemical indices of the subjects in the Study Population……………………………….30 4.3: Sociodemographic and clinical characteristics of males compared to females in the population………………………………………………………………………………………..31 4.4:Biochemical Parameters of males compare to females in the study population……………..32 4.5: Association of Perilipin A Levels with General Characteristics and Clinical Parameters of population Subjects..................................................................................................................................35 4.6:Association of Perilipin A levels with Biochemical Parameters of the Study Population…..38 4.7: Correlation plot of perilipin A levels against age,BMI, visceral fat, body fat and TG in male and female subjects…………………………………………………………………………………………………………………………….38 CHAPETR FIVE………………………………………………………………………………...43 DISCUSSION……………………………………………………………………………………43 5.1 CONCLUSION………………………………………………………………………………45 5.2 RECOMMENDATIONS.........................................................................................................45 5.3 LIMITATIONS........................................................................................................................46 REFERENCE……………………………………………………………………..47 APPENDICES…………………………………………………………………….62 viii University of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES PAGE Fig 1: The pathway of mechanism of lipolysis 9 Fig 2 : A correlation between age and perilipin A levels of obese male subjects 38 Fig 3:A correlation between age and peril ipin A levels of non-obese male subject38 Fig 4: A correlation between age and perilipin A levels of obese female subjects 38 Fig 5 : A correlation between age and perilipin A levels of non- obese female subjects 38 Fig 6 : A correlation between visceral fats and perilipin A levels of obese male subjects 39 Fig 7: A correlation between visceral fats and perilipin A levels of non-obese male subjects 39 Fig 8: A correlation between visceral fats and perilipin A levels of obese female subjects 39 Fig 9 : A correlation between visceral fats and perilipin A levels of non- obese female subjects 39 Fig 10 : A correlation between BMI and perilipin A levels of obese male subjects 40 Fig 11: A correlation between BMI and perilipin A levels of non-obese male subjects 40 Fig 12: A correlation between BMI and perilipin A levels of obese female subjects 40 Fig 13 : A correlation between BMI and perilipin A levels of non- obese female subjects 40 Fig 14 : A correlation between body fats and perilipin A levels of obese male subjects 41 ix University of Ghana http://ugspace.ug.edu.gh Fig 15: A correlation between body fats and perilipin A levels of non-obese male subjects 41 Fig 16: A correlation between body fats and perilipin A levels of obese female subjects. 41 Fig 17 : A correlation between body fats and perilipin A levels of non- obese female subjects 41 Fig 18 : A correlation between TG and perilipin A levels of obese male subjects 42 Fig 19: A correlation between TG and perilipin A levels of non-obese male subjects 42 Fig 20: A correlation between TG and perilipin A levels of obese female subjects 42 Fig 21: A correlation between TG and perilipin A levels of non- obese female subjects 42 x University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES Page TABLE 1: Demographic characteristics and clinical indices of the study population 29 TABLE 2: Biochemical indices of the study population 30 TABLE 3: Demographic information and clinical parameters of male and female in the study 32 TABLE 4: Biochemical indices of the study population between male and female 34 TABLE 5: Correlation of perilipin A levels with general characteristics and clinical parameters of the study population 35 TABLE 6: Correlation of perilipin A levels with biochemical parameters of the study population 37 xi University of Ghana http://ugspace.ug.edu.gh LIST OF ABBREVATIONS ADA……………………………………………………...American Diabetes Association β-cell……………………………………………………………………….…….beta cell BMI…………………………………………………………….………..Body Mass Index CDC……………………………………………………………Centre for Disease Control c AMP ……………………………………………………….Cyclic adenosintriphosphate DKA………………………………………………………………...Diabetes ketoacidosis DM………………………………………………………………………Diabetes Mellitus EDTA………………………………………………….Ethylene diamine tetra-acetic acid ELISA………………………………………………Enzyme linked immunesolbent assay FBG……………………………………………………………..…Fasting Blood Glucose FFA…………………………………………………………………………Free fatty acid Fig…………………………………………………………………………………..Figure GDM………………………………………………………..Gestational Diabetes Mellitus g/L……………………………………………………………………… Gram per liter GLU…………………………………………………..………………………….Glucose HbA1c……………………………………………………………...Glycated haemoglobin HDL……………………………………………………………..High Density Lipoprotein HRP………………………………………………………………..Horseradish Peroxidase HSL………………………………………………………………Hormone sensitive lipase IDA…………………………………………………….International Diabetes Association Kg/m2………………………………………………………….Kilogram per meter square xii University of Ghana http://ugspace.ug.edu.gh LDL…………………………………………………………...…Low Density Lipoprotein mL......…………………………………………………………………………...Milliliters mmol/L…………………………………………………………………Millimole per liter NDMRC……………………………National Diabetes Management and Research Center ng……………………………………………………………………………….Nanogram nm………………………………………………………………………………Nanometer oC………………………………………………………………………..…Degree Celsius %...........................................................................................................................Percentage OD………………………………………………………………………….Optical Density PKA………………………………………………………………………Protein kinase A PLIN 1…………………………………………………………………………Perilipin A PLIN 2………………………………………………………………………….Perilipin B SD…………………………………………………………………….Standard Deviation T1DM………………………………………………………….Type one diabetes mellitus T2DM………………………………………………………….Type two diabetes mellitus T.Chol……………………………………………………….…...………Total Cholesterol TG………………………………………………………......………….…….Triglyceride TMB…………………………………………………..3, 3’, 5, 5’, - tetramethyl benzidine T.Prot……………………………………………………….……………….Total Protein UK…………………………………………………………………….….United Kingdom VLDL-C…………………………………………………..Very Low Density Lipoprotein WHO……………………………………………………..……World Health Organization μL……………………………………………………………………………....Microliters xiii University of Ghana http://ugspace.ug.edu.gh >……………………………………………………………………….……..Greater Than <............................................................................................................................ Less Than xiv University of Ghana http://ugspace.ug.edu.gh CHAPTER ONE INTRODUCTION 1.1 Background Perilipins are phosphoproteins that are found in fat cells where they are restricted at the uppermost layer of lipid droplets. Over-expression of perilipin in fat cells causes the accumulation of triacylglyceride (TAG) to increase in connection with a reduction in fats and lipd breakdown that cause type 2 diabetes, weight gain and cardiovascular diseases. (Greenberg et al.,1998, Souza et al., 1998). There are two types of perilipin which have a common N-teminal amino acids sequence which are perilipin A (PLIN 1) and perilipin B (PLIN 2). Perilipin A is the prevailing form found in fat cells (Greenberg et al., 1993). Perilipin A protein is highly seen in human fat tissue and individual with increased in weight (obese subjects) had higher levels of perilipin A than the lean subjects (Kern et al., 2004). Studies relating expression of PLIN1 with obesity showed that the mRNA and protein of perilipin have a connection with obesity (Kern et al., 2004). Research by Tansey (2001) suggest that mice with perilipin eliminated, the unstimulated state of lipolysis was high, causing the mouse to decrease in weight that was unaffected by high fat diet and reduced the weight in db/db mouse (Tansey,2001). However, these mice also had their blood glucose increased and the body cells become resistance to the effect of insulin more frequently, due to the increas levels of free fatty acids (Tansey,2001). Plasma fatty acid levels was lower in the perilipin knock out mice than in mice with perilipin and the well being of the perilipin knockout mice attributed to the fact that there was an increase in plasma leptin and adiponectin levels (Sztaryd et al., 2012).People with increase body size are more prone to develop type 2 diabetes than lean people due to an increase in non-esterified free fatty acid as a result of lower levels of perilipin A in the fat cells . (Tansey, 2001). 1 University of Ghana http://ugspace.ug.edu.gh Blood perilipin A levels need to be known in diabetic subjects since most of the research work done was in adipocytes of both animal models and in humans. These insights will eventually help the pharmaceutical companies to design a drug to target the lipid droplet from breaking down by the hormone sensitive lipase (HSL) to prevent it from producing free fatty acids ,this will help in the management of obesity, diabetes and insulin resistance. (Tansey, 2001). Diabetes is one of the metabolic diseases resulting from inadequate secretion of insulin , action of insulin, or both. Diabetes is said to be the frontline of diseases and death in a population and worldwide, with an average rate of 346 million adults being affected. (WHO, 2012).World Health Organization (WHO, 2012) speculated that deaths resulting from diabetes will rise by two thirds between 2008 and 2030 (WHO, 2012). In 2010 adults with diabetes had a prevalence rate of 285 million (6.4%) in the world . The prevalence will rise from 285 to 439 million (7.7%) by 2030 (Shaw et al., 2012). The rate at which diabetes mellitus is increasing in the southern part of Ghana (Accra) has been found to be 6.3% (Amoah et al., 2002). In developed areas of Ghana, 6% of adults have type 2 diabetes and correlated with age and obesity. In urban areas of Ghana, type 2 diabetes mellitus affects large number of obese patients who have lower socioeconomic status due to hypertension and hyperlipidaemia (Danquah et al., 2012). About 23% of adults have been found to be obese due to ageing, gender, living in a developed environment and high income. (Amoah et al., 2002 ). Obesity has become a great contributor to the development of diabetes and hypertension; lack of control of diet could be due to the incomplete understanding of the pathogenesis of these diseases. Obesity is a condition of increase fat accumulation affecting the life and well-being of an ndividual (Gatineau and Mathrani, 2011). World Health Organization has a reference range for body mass index (BMI) . Individual with BMI of more than 25 kg/m2 is said to be overweight, and if the BMI is greater than the value of 30 kg/m2, the indivual is said to be obese. Obesity and overweight are 2 University of Ghana http://ugspace.ug.edu.gh found to be the leading causes of global deaths (Lokuruka, 2013). Obesity has been found to be one of the risk factor which causes coronary heart diseases, stroke, metabolic syndromes, osteoarthritis and cancer. The average rate of obesity conditions differ from population sub-group (Gatimen and Mathral, 2011). In 2015, the world health organization (WHO) predicted about 300 – 400 million adults were obese and about 1 billion were overweight and a new word has emerged to called “glo- besity” due to the global nature of the epidemic (Lokuruka, 2013). The incidence of obesity is at its peak in some parts of the world and figures that show the relationship of obesity in females and males in some countries are as follows, in the USA (35 females and 33 males, Russia Federation ;, 25 females and 10 males , Argentina ;.25 females and 28 male, UK; 24 females and 21 males) (Lokuruka, 2013). The percentage crude prevalence of adults in Accra and its rural environs who are 25 years with overweight and obesity was found to be 23.4 and 14.1% respectively (Amoah, 2003). while the national (Ghana) prevalence rate was reported to be 5.5% with females showing higher (7.4%) rates compared to 2.8% of males. Obesity is more common in the Southern part of Ghana compared to the Northern sector (Biritwum et al., 2003). The two most common types of fat depositions are the subcutaneous (abdominal and femoral) and intra abdominal (visceral and retoperitoneal) that is connected with increased plasma levels of low density lipoprotein cholesterol. Visceral fat is acquired from fat deposition in the middle of the body (Van Harmelen, 1997). Although several metabolic disorders are connected with adiposity, the adipocytes are important to human health. Fat cells undergo cycles of metabolic formation of fats and lipids breakdown to maintain energy balance depending on the bodys nutritional state. Increased rates of lipolysis result in higher rate of free fatty acid release into circulation. Accumulation of adipose tissue vary among individuals and are generally classified as fat body storage around buttocks, abdominal subcutaneous and visceral fat (intra abdominal fat deposition 3 University of Ghana http://ugspace.ug.edu.gh among nearby organs (Van Harmelen, 1997). The quality of the different forms of obesity is the increase in fat-cell size that correlate with the basal lipolysis which is increased in obese subjects (Arner, 2002). Non-esterified fatty acids derived from adipocyte breakdown causes insulin resistance in skeletal muscle and liver (Bergman et al., 2000). Fatty acids provide increase glucose production in the liver by providing a constant source of energy and substrate , also free fatty acids (FFA) promotes liver gluconeogenesis and insulin resistance (Williams et al.,1995). Although increased free fatty acids is present in obese individual, the mechanisms behind this phenomenon could not be properlly explained by changes in the action of the major lipolysis-regulating hormones, such as insulin, and catecholamines (Mottagui-Tabar et al., 2003). 1.2 Problem statement Diabetes is a major concern affecting the lives of people in a population. The number of diabetes cases have been constantly increasing over the past years. Diabetes is no longer the disease of the rich nations but it is steadily increasing everywhere especially in middle income countries (WH0, 2016). In addition diabetes mellitus and its complications have been a high health burden to individuals, families and nations in terms of finance and healthcare management. People with obesity are more susceptible to developing diabetes than non-obese people due to an increase in non-esterified free fatty acid as a result of low levels of perilipin in the adipocyte (Tansey, 2011) . Lower levels of perilipin has a direct link to an increased rate of adipocyte lipolysis in a study on Caucasian women with different BMI status and fat-cell volumes. The concentration of FFA obtained from fat cells lipolysis are present in overweight and obese individuals which is high in serum. One of the major problems for an individual to develop type 2 diabetes mellitus is an increase 4 University of Ghana http://ugspace.ug.edu.gh in plasma fatty acids (Boden, 1996) derived from adipose tissue lipolysis. Downregulaion of lipolysis has been one of the major problems causing resistance to insulin, hyperlipidermia, obesity and diabetes mellitus (Bergman et al., 1998). Changes in perilipin A levels may be shown to be involed in the susceptibility to obesity and diabetic complications. However, blood levels of perilipin A in diabetic patients are not fully understood. 1.3 Justification Changes in blood perilipin A levels may be implicated in diabetic complications and this further reduces productivity and may even increase mortality rates (Rodriguez-Segade et al., 1991). Studies show that in db/db mice, an absence in adipocyte lipolysis was attributed to absences of perilipin that was able to reverse obesity in db/db mice (Martinez, 2000). Although diabetes was not reversed in mice, the situation in humans is not known. Blood levels of perilipin A which may serve as early signs in the aetiogenesis of diabetic complications have however not been studied in Ghana. Considering the important role perilipin A play in the body’s lipolysis, there is the need to research into blood perilipin A levels in people with diabetes and their association with diabetic complications such as nephropathy and hypertension so as to improve the management of the disease. There is lack of data regarding changes in blood periliin A levels in patients with diabetes in Ghana. This work therefore sought to determine the blood perilipin A levels in obese diabetic and compare with non- obese diabetic individuals. The results may demonstrate the importance of perilipin in obese management and promote the search for agents that can inactivate perilipin as anti-obesity medications. 5 University of Ghana http://ugspace.ug.edu.gh 1.4 Hypothesis Diabetes does not influence the levels of blood perilipin A . 1.5 Aim The research is aimed at determining blood perilipin A levels in diabetic patient. 1.6 Specific objectives - To investigate blood perilipin A levels between obese and non-obese diabetic patients - To determine the relationship between blood perilipin A levels and the general(demography and clinical) characteristic of the study population. - To determine the relationship between biochemical indices (fasting blood glucose, lipid profile glycated haemoglobin) and blood perilipin A levels in the study population. 6 University of Ghana http://ugspace.ug.edu.gh CHAPTER TWO LITERATURE REVIEW 2.1 Overview of Perilipin A (PLIN1) Perilipin is a smaller lipid protein expressed by the humans perilipin gene. Perilipins are about 1-5 in size in microns in the pre-adipocytes or differentiating fibroblast (Brasaemle et al., 1997). A matured fat cells which contains the perilipin causes the cell to grow in fat droplets comprising of 95% of the primary fat cells . It is found in cell-line and models of animal that perilipin is important in the regulation of triglycerides (TAGs’) deposition and mobilization (Tansey et al.,2003). In rodents, there are four different types of perilipin and these perilipins are, perilipin A(PLIN 1) ,perilipin B (PLIN 2) , perilipin C (PLIN3) and perilipin D (PLIN 4) . (Souza et al.,1998) These perilipins are found in rodents due to splicing with a lower molecular weight but a common amino acid N-terminal. Perilipin A is mostly located in human adipose tissue, perilipin B is primarily seen in fat cells and steroidogenic cell whiles perilipin C and D are found in the steroidogenic cell. Perilipin A which is abundant is believed to perform the central physiological role but the less expressed Perilipin B may also play a lesser role in lipolysis.(Souza et al.,1998) Two types of perilipin are identified to have a common N-terminal amino acid (Greenberg et al., 1993) but differ in their sequences. In adipocytes, catcholamines binds to the adrenergic receptors which activate the protein kinase A (PKA) by cyclic AMP (cAMP) which phosphorilate perilipin, resulting in the removal of the perilipin from the uppermost surface of the lipid droplet causing hormone-sensitive lipase (HSL) to break down the triglyceride core (Londos et al.,1995). Mice adipocyte with no or decrease perilipin exhibit high rates of basal fats and lipid breakdown and restrict lipolytic response to adrenergic agonists (Tansey et al., 2001). Perilipin A protein is highly 7 University of Ghana http://ugspace.ug.edu.gh found in human fat tissue and individual who are obese shows higher levels of perilipin A as compare to lean individual (Kern et al., 2004). Animals who have their perilipin removed ,loss their protective effects around TAG under conditions of inactive (PKA) causes an increase in unstimulated lipolysis .(Tansey et al., 2001) Perilipin null mice are healthy, muscular and lean compared to mice with perilipin mice (Matinez-Botas et al., 2000). Further studies by Matinez-Botas and co workers in 2000 also found that mice without perilipin had an increase in metabolic rate and were resistant to high fat diet . (Matinez-Botas et al., 2000). The absence of perilipin was able to reduce the weight in db/db mice and was able to prevent increase in wieght as a result of high fat diet. (Tansy et al., 2004). Perilipin has been described as “the gate keeper of the adipocyte lipid storehouse” as it acts as a barrier to lipolysis in non stimulated cells. During fasting or by the action of catecholamines, perilipin gain phosphate group, causing the removal of protein from the surface of the lipid droplet, allowing hormone-sensitive lipase to breskdown the triglyceride (Souza et al ., 1998, Kern et al ., 2004). 2.2 The role of perilipin Perilipin is a protein that has the ability to protects the surface layer of the lipid core. In embryonic stem cell, perilipin gene is removed from mice by the technique of homologous recombination (Martinez et al., 2000) .After the process the mice grew lean indicating a decrease in fat tissue mass by 60-70%. The decrease in fat tissue was because of an increase in basal adipocyte lipolysis due to the absence of perilipin at the surface layer of the lipid core (Martinez et al., 2000). The absence of perilipin in the mice also had a different effect on lipid breakdwon, which shows that perilipin gives higher activated response to lipolytic stimulants. The levels of plasma fatty acid was low in the 8 University of Ghana http://ugspace.ug.edu.gh mice with perilipin than in the mice without perilipin and the well being of the mice without perilipn was due to the fact that there was high plasma leptin and adiponectin levels (Sztaryd et al.,2012). Research shows that the addition of perilipin A and PKA site of the three amino terminal (81,223,277) are required for maximum lipolytic response.When these sites and the phosphate group are removed , lipolysis is stop even in the activated cells (Souza et al.2003). Perilipin A functions to block the work or activities of HSL during lipolysis (Sztalryd et al., 2002). Perilipin A which is phosphorylated is required to function with HSL at the surface layer of lipid and to help in the removal of HSL in cultured embryonic fibroblasts however, mice without perilipin do not function to remove HSL under stimulated conditions. (Shen et al.,1999) Perilipin B which share similarities with perilipin A ,lacks the ability to protect TAG from lypolisis . (Shen et al.,1999). The function of Perilipin in humans is unclear and not well understood which give rise to less reports on Perilipin expression and function (Nishiu et al.,1998). It is known that women with subcutenous tissue, perilipin expression increases with a decrease in basal lipolysis (Mottagui-Tabar et al., 2003).Lipolysis differs between different adipose regions, thus in omental fat, unstimulated lipolysis is lower than in subcutaneous adipose tissue, but stimulation of catcholamines is high (Mottagui- Tabar et al., 2003). 2.3 The mechanism of Lipolysis Lipolysis involves the breaking down of fats and other lipids to release free fatty acids and glycerol. TAG are the major energy storage found in adipose tissue. Catecholamine hormones signal can give rise to a lipolytic response in fat cells. (Chaudhry et al., 2002).The catecholamine binds to beta- 3 adrenergic receptors on the upper layer of adipocyte. These receptors releases signals through a G coupled stimulatory protein to activate adenylyl cyclase.The activation of adenylyl cyclase causes 9 University of Ghana http://ugspace.ug.edu.gh the cyclic AMP to be activated .This leads to the addition of phosphate group to the perilipin at the surface layer of the lipid droplet. The reaction is blunted by insulin, which reduces the concentration of cAMP by activating phosphodiesterase 3B (Holm et al., 1997). Hormone sensitive lipase and perilipins are the two key determinant in lipolysis (Chaudhry et al., 2002). HSL is reomoved from the cytoplasmic compartment to surface layer of the lipid droplet in fat cell during lipolytic stimulation which breaks down the lipid droplet (Egan et al., 1992) and subsequent immunofluorescence studies shows that using anti-HSL antibodies in 3T3-L1 fat cells have been found that HSL is present throughout the cytoplasm of unstimulated cells, but it moves to the surface layer of lipid droplets upon lipolytic stimulation (Londos et al., 1999).It has been proposed by London and co-wokers that hormonal stimulation of lipids could be affected by substrate activation and other factors such as the mechanism of lipolytic activation by catecholamines (London et al,1999).Research work by Barber et al, predicted that catecholamines was expected to assist the removal of HSL, by some change in the lipid droplets observed in 3T3-L1 adipocytes and Leydig cells (Barber et al., 1995). Studies have showen that certain aspect of lipid droplets protein expression are different and are responsible for the differences in lipolytic response to catecholamines observed in adipocytes from different fat depositions (Morimoto et al., 1997). It has been proposed that in unstimulated cells, perilipin may affect the changes and activities of HSL by forming a protective covering around the TAG against HSL, whiles cAMP dependent protein kinase-phosphorylated perilipin may allow access to the HSL to the droplet (Londos et al., 1995). 10 University of Ghana http://ugspace.ug.edu.gh Fig 1 : Pathway of mechanism of lipolysis Source:The U.S.A National Library Medicine (2012). 2.4 Obesity Obesity and type 2 diabetes mellitus play a major role in non-adipose tissue such as skeletal muscle by causing resistance to insulin due to increase in fat storage in the body. (Samuel et al.,2012). Obesity is a condition of excessive fat storage in adipose tissue with an increase in BMI of more than 30kg/m2 which has an effect on health (WHO,2010). The distribution of fat storage in the body such as the waist and the peripheral pose a treat to the health of the individual. In general, risk factors such as obesity and diabetes causes early death and cardiovascular disease. Obesity also affacts the 11 University of Ghana http://ugspace.ug.edu.gh health of an individual through a psychosocial health due to societal stigma against fatness (WHO,2015). Obesity is one of the preventable cause of death in the world, with increasing incidence rate in adults and children, it has been found to be of the serious public health conditions in the world (Metcaf et al.,2011). 2.4.1 Prevalence of Obesity The rate at which obesity is rising globally has been a major issues in the world In 2008, adults who were 20 years and above were said to be obese with a figure of about 1.5 billion and out of this figure over 200 million men and 300 million women were said to be obese (Lieb et al.,2009). Research has shown that majority of the world’s population are obese and children who are below five years with a figure of 4 million were obese in 2010. (Martinelli et al.,2011). Research conducted in some African countries including Ghana showed that three million Ghanaians are overweight and obese. Ghana showed the highest number of obesity among the countries indicating that the the prevalenc of obesity in Ghana needs to be addressed. In Ghanaian population settings the rate of obesity is seen among the elderly, females and the people who lived in the developed areas. Research conducted in the southern part of Ghana (Accra) indicated an increased number of obesity (BMI > 30 kg/m2 ) of which 20.2% were females and 4.6% were males. (Abbasi et al., 2010). Obesity is associated with an increased with age between 55 to 64-year. People in residential area are considered to have higher BMI compared to people from the lower class areas.Individual in the Urban areas had higher BMI compared to rural areas. (Abbasi et al., 2010). From a larger body weight , the global prevalence of obesity has been mainly from societal factors that promote lifestyles of spending much time and the consumption of high-fat diets (WHO, 2010). 12 University of Ghana http://ugspace.ug.edu.gh 2.5 Overview of Diabetes Mellitus Diabetes is a metabolic condition which occur as a result of pancrease failing to produce insulin or the the production of the insulin is not enough to be used by the body. (WHO,2016). Diabetes is an important public health problem, which is characterized by hyperglycemia which is often accompanied by excessive thirst, polyuria and polyphagia (Celik & Zimmet, 2001). An increase in blood glucose, may destroy the heart, blood vessels, eyes, kidneys and nerves which leads to high level of plasma glucose (hyperglycemia). In diabetes, the body tissues have to metabolize protein and fat reserve for energy (Rizza, 2010). Diabetes is also associated with a defect in protein and lipid metabolism. In diabetes, amino acids catabolism increases, with the liver converting amino acids into glucose under stimulation of glucagon (Knop et al.,2010). There are three different types of diabetes namelly, type 1 (T1DM), type 2 (T2DM) gestational diabetes mellitus (GDM) (WHO, 2011). 2.5.1 Aetiology of diabetes mellitus Diabetes mellitus is said to result from an increase in glusoce level which is a metabolic conditions and different group of disorders with different underlying causes (Færch et al., 2013). Several factors have been identified as aetiology of type one diabetes; which are the genetic constitution, immune function, and the environment. Both the genetic and environmental factors also causes type two diabetes (Paramythiotis et al., 2003). It is believed that an individual inherits a susceptibility to develop either insulin dependent diabetes mellitus (IDDM) or non-insulin diabetes mellitus (NIDDM) and that environmental factors can eventually precipitate overt clinical disease (Ragoobirsingh and McGrowder, 2012). With autoimmune mechanism as a risk factor, the T 13 University of Ghana http://ugspace.ug.edu.gh lymphocytes (CD4+ and CD8+) infiltrate the pancreas and destroy the insulin (Akerblom et al., 2002).Type 2 diabetes mellitus (T2DM) is also known as non-insulin dependent diabetes mellitus, or (WHO and IDF, 2006). 2.5.2 Classification of Diabetes The three major types of diabetes are type 1 diabetes, type 2 diabetes, gestational diabetes and (WHO, 2011). Type 1 diabetes is also known as insulin-dependent which constituent for 5-10% of diabetes mellitus world-wide and is caused by autoimmune destruction of the beta cell ( β-cell) in the pancreas (Rother, 2007). Viral infection like Rubella and toxins are thought to trigger the immunological mediated destruction of the pancreas mainly caused by T-cell mediated autoimmune attack (ADA, 2006).The distruction of the Beta cell (β-cell) is relative, it is high in some individuals and low in others (ADA, 2006). Type 1 patients are prone to diabetes ketoacidosis (DKA), and can only be managed with insulin therapy (Gale, 2002). One of the features of type 2 DM is impaired insulin secretion or defective action at responsive tissue or both (WHO, 1999). Insulin resistance cause impaired glucose utilization. Type 2 DM is considered a lifestyle-dependent disease that is associated with a strong genetic factor (Diabetes Atlas, 2009).The prevalence of type-2 DM in the world and the variability among ethnic groups is due to many reasons including, the silent nature of type 2 DM, genetic factors, environmental factors, population ageing, unhealthy diet (amount and origin of dietary protein), overweight or obesity, history of gestational diabetes, impaired glucose metabolism, lack of physical , and lifestyle of having a lot of time, are all risk factors of type 2 DM (WHO, 1999). The lifestyles of people all around the world is changing rapidly because of rapid socio-economic development (Husseini et al., 2000). Excess weight, abdominal fat, in particular the visceral rather than subcutaneous depots, increases insulin 14 University of Ghana http://ugspace.ug.edu.gh requirements and compounds the problem of insulin resistance (Hodge et al., 2001).The risk factors in type 2 diabetes are; genetic predisposition, age, obesity, alcohol intake, history of gestational diabetes and life style. The life style which may pre-dispose people to type 2 diabetes include; intake of high carbohydrate and fatty foods, alcohol intake, and lack of physical exercise. Researchers have showed that having a large waist circumference correlate strongly with an increased risk of developing Type 2 diabetes which is not dependent of a person’s body mass index (The InterAct Consortium, 2012). Gestational diabetes mellitus (GDM) is said to be an increase in glucose levels with onset or first recognition during pregnancy (Ross, 2006). The negative outcome for developing GDM are pre diagnosis of gestational, impaired fasting glycaemia, a family history of having first Type 2 diabetes, motherly age, ethnicity, overweight and a history of previous pregnancy which resulted in a child with a high birth weight >4kg (Ross, 2006). Maternal obesity increases the outcome of a number of other pregnancy complications may set in , including preeclampsia and Caesarean delivery (Leddy et al., 2008). 2.5.3 Prevalence of diabetes mellitus According to Diabetes Atlas (2009), “Diabetes now affects seven percent of the world’s adult population. The regions with the highest comparative prevalence rates are North America, where 10.2 % of the adult population have diabetes, followed by the Middle East and North Africa Region with 9.3%. (Diabetes Atlas, 2009).The regions with the highest number of people living with diabetes are Western Pacific, where some 77 million people have diabetes and South East Asia with 59 million. India is the country with most people having diabetes, with a current figure of 50.8 15 University of Ghana http://ugspace.ug.edu.gh million, followed by China with 43.2 million. The rest are the United States (26.8 million), the Russian Federation (9.6 million), Brazil (7.6 million), Germany (7.5 million), Pakistan (7.1 million), Japan (7.1 million), Indonesia (7 million) and Mexico (6.8 million) (Diabetes Atlas, 2009). When it comes to the percentage of adult population living with diabetes, current data reveals the devastating impact of diabetes across the Gulf Region, where five of the Gulf States are among the top ten countries affected (Diabetes Atlas, 2009). The Pacific island nation of Nauru has the world’s highest rate of diabetes, with almost a third of its adult population (30.9%) living with the disease (Diabetes Atlas, 2009). It is followed by the United Arab Emirates (18.7%), Saudi Arabia (16.8%), Mauritius (16.2%), Bahrain (15.4%), Reunion (15.3%), Kuwait (14.6%), Oman (13.4%), Tonga (13.4%) and Malaysia (11.6%)” (Diabetes Atlas, 2009). 16 University of Ghana http://ugspace.ug.edu.gh CHAPTER THREE MATERIALS AND METHOD 3.1. Study site The study site was conducted at the National Diabetes Management and Research Centre (NDMRC), Korle-Bu Teaching Hospital. The Korle-Bu Teaching Hospital is the leading tertiary referral hospital in Accra with 1,600 bed capacity and 3,000 members of staff. The hospital serves the city of Accra (with a population of about three million) and the whole of the southern sector of Ghana. At the National Diabetes Management and Management Center (NDMRC), the number of daily out-patient attendance was 100 to 200 admissions daily (Personal Communication and Hospital Records). The Diabetic Clinic of the NDMRC, however, holds over 85,000 patient records, and each day, over 200 patients attend the clinic from in and around the area (NDMRC Records). The clinic manages both type 1 and type 2 diabetic cases, nonetheless, majority of the patients seen are type 2 diabetics reporting for routine check-up and management. 3.2. Target Population The study was carried out on patients diagnosed with diabetes according to WHO (1999) criteria undergoing management of their conditions in the out-patient’s clinic at NDMRC between January to March 2017. A total of 86 subjects including controls were recruited (43 obese type 2 diabetic subjects as case subjects and 43 non-obese type 2 diabetics as controls). Subjects who served as controls were screened to make sure that they were non-obese. The selection criteria for the subjects was based on response to the questionnaire, which were use to obtain information on patients socio- demographc characteristics, duration of the diabetes , drug usage and lifestyle. 17 University of Ghana http://ugspace.ug.edu.gh 3.3 Study design The design was a hospital based cross sectional study involving 46 obese type 2 diabetic patients and 46 non-obese type 2 diabetic patients from the National Diabetes Research and Management Centre . 3.4 Sample size determination Minimum sample size was determined using a standard case-control formular below 2        u  1 (1   1 )   2 (1   2 )  v     1 2 1 2 1     2    N   2 1    2 Where π1 = Proportion of cases exposed = 0.33 π2 = Proportion of controls exposed = 0.3 u = Sample proportion for cases = 1 v = Sample proportion for controls = 1 N = Sample size per group Outcome of interest is perilipin A in patients with diabetes mellitus. Assuming odds ratio of 2 among the cases, at a 5% significant level and a power of 80%, minimum sample size per group = 41. For this study, a total of 86 including 43 cases and 43 controls were recruited. 18 University of Ghana http://ugspace.ug.edu.gh 3.5 Inclusion criteria The diabetic subjects enlisted for the study were between age 30 to 60 years. The cases were type 2 diabetic patients who were obese and on management at the NDMRC. The controls consisted of non-obese type 2 diabetic subjects within the same age brackets. 3.6 Exclusion criteria Subjects above 60 and below 30 years as well as smokers and heavy drinkers were excluded from the study. Patients who are on lipid lowering therapeutic drugs and those who refuses consent were exempted from the study. 3.7 Sample collection 3.7.1 Specimen collection, Transport and Storage Blood samples (10 mL) was withdrawn from the diabetic subjects between 7:00 and 9:00am each day, according to Helsinki protocol declaration (2008). Two milliliters (2 mL) of whole blood was put into tube with sodium flouride. The plasma was separated from the whole blood for the estimation of glucose. Three milliliters (3 mL) of whole blood was also put into tubes containing ethylene diamine tetra-acetic acid (EDTA) for the determination of glycated haemoglobin (HbA1c). The rest of the five milliliters (5 mL) of whole blood was then transferred into tubes with serum separator for processing. Serum samples were aliquoted into 3 different sterile eppendorf tubes and stored at -20oC . 19 University of Ghana http://ugspace.ug.edu.gh 3.7.2 Anthropometric measurement The anthropometric characteristics measured in the population include age, sex, weight, height, body mass index (BMI), body fat and visceral fat . The Omron HBF-514 full body sensor and scale was used to measured the height,weight, visceral fat and body fat. 3.8 Biochemical Analysis of Sample The collected blood samples from each recruited subjects according to standardized venipuncture procedure was transferred into serum separator, EDTA and fluoride tubes respectively. The blood in the tubes containing the serum separator were allowed to clot and centrifuged. Sandwitch Enzyme-Linked Immunosorbent assay (ELISA) technique (Kono Biotech Co. LTD China) for the perilipin A was used to determine their serum perilipin A levels in the subjects. Fasting blood glucose, lipid profile of the subjects were analysed using VITROS system auto analyser (version 950). HbA1c was measured using Randox Daytona auto analyzer. HbA1c was determined using a latex agglutination inhibition assay. 3.8.1 Measurement of Perilipin A/Perlipin 1(PLIN1) Test principle The kit uses the sandwich ELISA technique to assay Human Perilipin A, PLIN A level in serum. It uses purified PLIN A antibody which is specific to Perilipin A to coat microtiter plate wells to make a solid-phase antibody. Standards or samples are added to the appropriate microtiter strip plate wells and combined to the specific antibody. An antibody specific for PLIN A, labelled with horseradish peroxidase (HRP) enzyme is added to wells to form an antibody-antigen enzyme-antibody complex after washing thoroughly. A 3, 3’, 5, 5’, - tetramethylbenzidine (TMB) substrate solution is added 20 University of Ghana http://ugspace.ug.edu.gh to be catalyzed by the enzyme HRP if an antibody-antigen enzyme-antibody complex is formed. TMB substrate becomes blue coloured when it is catalyzed by HRP enzyme and the reaction is terminated by the addition of a sulphuric acid solution with a yellow colour change measured spectrophotometrically at a wavelength of 450nm. The concentration of Human Perilipin A in the sample is determined by comparing the optical density of samples to the standard curve. Assay Procedure Aliquoted serum samples were brought to room temperature to thaw and vortexed for 10 seconds. The Human Perilipin A kit (Kono Biotech Co., Ltd, China) which was previously stored in the refrigerator at (2-8)oC was brought to room temperature. Different concentrations of the standard were prepared as per the protocol.The standards were prepared according to the table below 200ng/ml 5 standard 150 μL original density standard + 150 μL standard diluent 100ng/ml 4 standard 150 μL 5 standard + 150 μL standard diluent 30ng/ml 3 standard 150 μL 4 standard + 150 μL standard diluent 25ng/ml 2 standard 150 μL 3 standard +150 μL standard diluents 12.5ng/ml 1 standard 150 μL 2 standard + 150 μL standard diluent 21 University of Ghana http://ugspace.ug.edu.gh The first well was reserved as blank. 50µl of standard samples were added to the next five wells with the remaining wells filled with 50µl of test samples (prepared by adding 10µl of test samples to wells filled with 40µl of sample diluent, dilution factor of 5) and gently mixed. The plate was then closed with a closure plate membrane and incubated at 370C ( in an oven) for 30minutes. A 30-fold wash solution was prepared with distilled water and was used to wash the plates. After 30minutes of incubation, the closure plate membrane was uncovered, samples discarded and plate dried by swinging. Washing buffer was then added to each well for 30seconds and drained. This was repeated four additional times and drying done by patting after each procedure. 50µl of HRP-conjugate reagent was then added to each well, except the blank well and incubated for 30minutes at 370C after closing plate with closure plate membrane. This was followed by washing and patting plate five times as described earlier. 50µl of chromogen A and 50µl of chromogen B was added to each plates followed by incubation at 370C for 15 minutes avoiding light. 50µl of stop solution was then added to each well to stop the reaction with a colour change from blue to yellow. With the blank well taken as zero, the absorbance was read at 450nm using Microtiter Plate Reader within 15minutes of addition of stop solution. Four control samples were interspersed in test samples to validate the process. 3.8.2 Measurement of Glucose Principles of the Procedure The VITROS Glucose (GLU) slide method was performed using the VITROS GLU slides and Calibrator kit on VITROS Chemistry System (Johnson & Johnson, New Jersey, USA). Six microliters (6μ1) of sample was deposited on the slide and was eventually distributed by the spreading layer to the underlying layers. The oxidation of glucose is catalyzed by glucose oxidase 22 University of Ghana http://ugspace.ug.edu.gh to form hydrogen peroxide and gluconate.The reaction is followed by an oxidative coupling catalyzed peroxidase in the presence of dye precursors to produce a dye. The intensity of the dye is measured by reflected light at a wavelength of 540 nm. The Chemistry of glucose slides used in this study is as described by Curme et al., (1978). The incubation time and reaction condition for the entire reaction were 5 minutes and 37oC respectively. Reaction Sequence β-D-glucose + O2 + H2O glucose oxidase D – gluconic acid + H2O2 2H2O2 + 4 – aminoantipyrine + 1, 7 – dihydroxynaphthalene peroxidase red dye 3.8.3. Measurement of Total Cholesterol (T. CHOL) The total cholesterol was analyzed using Mindray BS-120 fully automated chemistry analyzer and human assay kit Germany. Principles of Reaction Total cholesterol was determined using cholesterol oxidase/peroxidase aminophenazone (CHOD-PAP) method as described by Lumb and Slavia (1993). Cholesterol is determined after enzymatic hydrolysis and oxidation. The indicator quinoneimine is formed from hydrogen peroxide and 4-aminophenizone in the presence of phenol and peroxidase. 23 University of Ghana http://ugspace.ug.edu.gh Reaction Sequence Cholesterol ester + H2O cholesterol ester hydrolase cholesterol + fatty acids cholesterol + O2 cholesterol oxidase cholestene-3-one + H2O2 2H2O2 + 4-amine-antipyrine+ phenol peroxidase qinoneeimine + 4H2O 3.8.4. Measurement of Triglyceride (TG) The total cholesterol was analyzed using Mindray BS-120 fully automated chemistry analyzer and human assay kit Germany. Principles of the Reaction Triglyceride was analyzed using glycerol phosphate oxidase/peroxidase aminophenazone (GPO-PAP) method. Trigylceride are determined after enzymatichydrolysis with lipases.Indicator quinoneimine formed from hydrogen peroxidase 4 –amino-antipyrine and 4-chlorolphenol under the catalytic influence of peroxidase. Reaction Sequence Triglyceride + H2O lipase glycerol + fatty acids Glycerol + ATP glycerol kinase glycerol-3-phosphate + ADP Glycerol-3-phosphate + O2 oxidase dihydroxyacetone phosphate + H2O2 24 University of Ghana http://ugspace.ug.edu.gh H2O2 + 4-aminoantipyrine peroxidase quinoneimine+HCL+ H2O 3.8.5. Measurement of High Density Lipoprotein (HDL) Cholesterol The measurement of HDL-C is based on the same method for measurement of total cholesterol as described by Trinder (1969). 3.8.6 Measurement of low density lipoprotein (LDL-C) Cholesterol The Friedwald Equation In the most widely used indirect method, total cholesterol, triglycerides and HDL- cholesterol are measured and LDL-cholesterol is calculated from the primary measurements using the empirical equation of Friedwald et al., (1972). In this study, LDL- cholesterol concentration of the samples were estimated using the equation given below, provided triglyceride concentration was not greater than 4.4 mmol/l. Results were expressed in mmol/l. LDL-C(mmol/L) = [TC (mmol/L) – [TG](mmol/L)/2.2- HDL(mmol/L) 3.8.7. Measurement of Glycated Hemoglobin (HbA1c) Principles of the Procedure The measuremnt of glycated haemoglobin (HbA1c) involved the following steps. Sample Pre-treatment The first step of the procedure involved the pre-treatment of the whole blood sample. This was done by adding 10 μL of whole blood to 400 μL haemoglobin denaturant reagent (1:41 dilution). This lysed red 25 University of Ghana http://ugspace.ug.edu.gh blood cells and caused hydrolysis of the haemoglobin by the action of a protease enzymes in the haemoglobin denaturant reagent. 3.8.8. Determination of HbA1c Latex agglutination inhibition assay was used to determine the HbA1c . The agglutinator, which consists of a synthetic polymer containing multiple copies of the immunoreactive portion of HbA1c, causes agglutination of latex coated with HbA1c specific mouse monoclonal antibodies. The agglutinator in the HbA1c R2 Reagent and the antibody coated micro particles in the HbA1c R1 Reagent will agglutinate in the absence of HbA1c in the sample, causing absorbance to increase. The rate of agglutination as it competes with the HbA1c agglutinator for antibody binding sites on the latex will slow down due to the presence of HbA1c . Hence, the increase in absorbance is inversely proportional to the concentration of HbA1c in the sample. An increase in absorbance due to agglutination was measured at a wavelenght of 700nm and the extent of agglutination was used to calculate the concentration of HbA1c from a calibration curve. The percentage (%) of HbA1c was then calculated using the g/dl HbA1c and total haemoglobin values. 3.9 Data Handling All data were entered into Microsoft excel spreadsheet for storage and subsequent analysis. Data were handled confidentially. Unique identifiers were used for computer based data entry. The supervisors and Investigator was ensured that all study forms together with all identification code lists were kept safe and confidential. All protocol was observed to ensure participants of complete anonymity and confidentiality of participants’ information before, during and after the research period. Questionnaires were kept under lock and key . 26 University of Ghana http://ugspace.ug.edu.gh 3.10 Statistical Analysis Data obtained from the research was analysed based on the research objectives. The Statistical Package for the Social Sciences (SPSS) version 20.0 was used. The students t-test was used to determine the means of blood perilipin A levels between obese diabetic and non-obese diabetic subjects.. For comparison of demographic, clinical and biochemical parameters, quantitative data was presented as mean and SD. Spearman’s correlation coefficient (r) was used to measure the strength of the association between the parameters. Differences was considered statistically significant at p<0.05. 27 University of Ghana http://ugspace.ug.edu.gh CHAPTER FOUR RESULTS 4.1: General Characteristics and background of the Study Population The subjects who were enlisted in the study, demographic and clinical indices have been presented in Table 1. The subjects comprised fourty-three (43) non-obese type 2 diabetics who were matched for age with fourty-three (43) obese type 2 diabetic subjects. Non-obese diabetics group comprised of 22(51.2%) males and 21(48.8%) females and for the obese diabetics group consisted of 21(48.8%) males and 22(51.2%) females. The mean age for the non-obese diabetic study group and obese diabetic group were 46.8 ± 8.2 and 47.5 ± 6.7 years respectively. The mean differnece between the ages of control (non-obese diabetics) and cases (obese diabetics) was insignificant (p=0.607) (Table 1). The difference between the mean body mass index was significantly higher in the obese diabetic subjects than in the lean diabetic subjects (p<0.0001). Also the visceral fat and the body fat were significantly higher in obese diabetic subject than in lean diabetic subjects (p<0.0001). (Table 1). The mean difference of duration of diabetes was significantly higher in obese diabetic subjects than in non-obese diabetic subjects.(p<0.0006) (Table 1). 28 University of Ghana http://ugspace.ug.edu.gh Table 1: Demographic characterstics and clinical indices of the study population Parameters Non-obese diabetics Obese diabetics p-values ( N=43 ) (N=43) Age (years) 46.8 + 8.2 47.5 + 6.7 0.607 Gender (male) 22(51.2%) 21(48.8%) (female) 21(48.8%) 22(51.2%) BMI (kg/m2) 23.0+ 1.4 31.2+ 1.9 <0.0001* Visceral fat (%) 5.6+ 3.4 16.7+8.1 <0.0001* Body fat (%) 13.8.1 + 6.9 26.8 +5.6 <0.0001* Duration of diabetes(years) 8.7+7.9 13.4+ 5.9 <0.0006* Data for the demographic and clinical parameters (Age, Gender, BMI, and duration of diabetes). Values hfor Age, BMI,Visceral fat , Body fat and duration of diabetes are represented as mean ± standard deviation while values for gender, was given as frequency (percentage). *difference between the means are significant (p<0.05). 29 University of Ghana http://ugspace.ug.edu.gh 4.2:Biochemical indices of the subjects in the Study Population The means of the measured biochemical indices of the subjects are presented in Table 2. Fasting blood glucose (FBG) was shown to be significantly higher in the obese diabetic than the non-obese diabetic subjects (p<0.0001). The mean glycated haemoglobin (HbA1c) was found to be significantly higher in the obese diabetic than the non-obese diabetic subjects (p<0.0001). The mean triglyceride levels, was significantly higher in the obese diabetic than in the non-obese diabetic subjects (p<0.0001). There was insignificant difference in perilipin A levels between obese diabetics and non-obese diabetics (p>0.6353).(Table 2) but perilipin A level was found to be increased in non-obese diabetic patients (117.2 +47.3) than Obese diabetic patients (109.1+ 36.9). LDL. TG. T. chol and VLDL which was higher in both subjects but T.chol/HDL, HDL and concertration of perilipin A were statistically insignificant(Table 2). Table 2: Biochemical indices of the study population Parameters Non-obese diabetics Obese diabetics p-values (N= 43) (N= 43) FBG(mmol/L) 7.2+ 1.6 10.6+ 1.1 <0.0001* HbA1C(%) 7.5+ 1.3 9.4+ 1.4 0.0001* HDL(mmol/L) 1.6 + 0.2 1.4+ 0.2 <0.005 TG(mmol/L) 1.9+ 0.9 2.5 +0.2 0.0001* LDL(mmol/L) 2.1 + 0.6 3.3 + 0.6 0.0001* 30 University of Ghana http://ugspace.ug.edu.gh VLDL(mmol/L) 1.8 + 0.9 1.3+ 0.7 0.0001* T,chol(mmol/L) 4.7 + 1.2 6.4+ 0.9 0.0001* T,chol/HDL(mmol/L) 4.3+ 1.1 3.8+ 1.2 0.0394 Conc.of Perilipin(ng/ml) 117.2 + 47.4 109.1 + 36.9 0.6353 The data represents the biochemical indices of the whole population. Results are in mean ± standard deviation. * The difference in mean is considered significant (p<0.05). FGB is fasting blood glucose, HbA1c is glycated hemoglobin, T.Chol is total cholesterol, TG is triglyceride, HDL is high density lipoprotein, VLDL is very low density lipoprotein and Conc is concentration 4.3: Sociodemographic and clinical characteristics of males compared to females in the population Sociodemographic and clinical characteristics of male and female subjects are represented in (Table 3). The result in table 3 shows a significant difference in the means between the males and females in terms of BMI,visceral fat, body fat and duration of diabetes (p = 0.0001 , p = 0.0001 ,p=0.0400 and p=0.0006 respectively). There was however no significant difference in means of age for both non-obese and obese diabetics in terms of gender (Table 3). 31 University of Ghana http://ugspace.ug.edu.gh Table 3: Demographic information and clinical parameters of males and females Parameters Non-obese diabetics Obese diabetics p-values Male(22) Female(21) Male(21) Female(22) Age (years) 46.5+ 8.8 47.1+ 7.7 51.5+6.1 43.72+4.9 0.6367 BMI (kg/m2) 23.1 +1.6 24.0+1.1 30.4+1.4 32.0+2.2 <0.0001* Visceral fat (%) 5.7+3.6 5.3+3.3 19.1+9.3 14.2+5.9 <0.0001* Body fat (%) 11.7+7.6 15.9+5.6 25.9+3.4 27.5+7.0 <0.0400* Duration of diabetes(years) 11.8+8.8 6.2+5.9 14.9+6.4 11.9+5.0 <0.0006* Data for the demographic and clinical parameters (Age, Gender, BMI, and duration of diabetes). Values for Age, BMI,Visceral fat , Body fat and duration of diabetes are given as mean ± standard deviation while values for gender, was given as frequency (percentage). *differences in means are significant (p<0.05). 4.4:Biochemical Parameters of males compare to females in the study population The means of the measured biochemical indices of the subjects were presented in Table 4. Fasting blood glucose (FBG) was observed to be significantly higher in both male and female obese diabetic than the male and female non-obese diabetic subjects (p<0.0001). The mean glycated haemoglobin (HbA1c) was found to be significantly higher in the obese diabetic than the non-obese diabetic subjects (p<0.0001) but HbA1C was high in female subjects than the male subjects. The means of triglyceride, Tchol/HDL and Tchol. however, was significantly higher in male subjects than the females subjects in both obese diabetic and non-obese diabetic subjects (p<0.0001). 32 University of Ghana http://ugspace.ug.edu.gh There was no significant difference in the levels of perilipin of male and female subjects between obese diabetics and non-obese diabetics (p>0.7353) (Table 4) though the levels of perilipin was found to be higher in male than in female subjects. Table 4: Biochemical indices of the study population between male and female Parameters Non-obese diabetics Obese diabetics p-values Male(22) Female(21) Male(21) Female(22) FBG(mmol/L) 7.3+ 1.5 7.2+1.4 10.9+1.4 10.3+ 0.7 <0.0001* HbA1C(%) 7.1+1.0 7.9+1.5 9.1+1.4 9.7+1.5 < 0.0001* HDL(mmol/L) 1.5+0.2 1.7+0.3 1.3+0.2 1.4+0.1 <0.0188 TG(mmol/L) 2.5+0.8 2.4+0.9 2.1+0.1 1.6+0.9 <0.0001* LDL(mmol/L) 2.3+0.6 1.9+0.5 3.2+0.6 3.4+0.5 < 0.00231 VLDL(mmol/L) 1.9+0.6 1.6+1.0 1.2+0.7 1.3+0.8 < 0.00211 T,chol(mmol/L) 5.3+1.1 4.2+0.9 6.7+0.9 6.0+0.8 < 0.0001 T,chol/HDL 4.5+1.0 4.2+1.1 4.2+1.2 3.4+1.1 < 0.0394 (mmol/L) 33 University of Ghana http://ugspace.ug.edu.gh Conc.of Perilipin123.4+46.2 111.4+48.9 109.7+30.1 108.5+43.1 0.7353 (ng/ml) The data represents the biochemical indices of the whole population. Results are in mean ± standard deviation. * The difference in mean is considered significant (p<0.05). FGB is fasting blood glucose, HbA1c is glycated hemoglobin, T.Chol is total cholesterol, TG is triglyceride, HDL is high density lipoprotein, VLDL is very low density lipoprotein and Conc is concentration 4.5:Association of Perilipin A Levels with General Characteristics and Clinical Parameters of population Subjects . The results obtained showed that there was significant association between age and perilipin A in non-obese diabetic subjects. Age was also found to negatively correlate with perilipin A levels in non-diabetic subjects. However there was no association between age and perilipin A in both subjects (Table 5). There was no significant relationship between body mass index of non-obese diabetic and obese diabetic subjects and their perilipin A levels (Table 5). The results as presented in Table 4 did not show any significant association between visceral fat and body fat of the subjects and their perilipin A levels. However, duration of diabetes was found not to correlate with perilipin A levels in both subjects (Table 5). 34 University of Ghana http://ugspace.ug.edu.gh Table 5:Correlation of Perilipin A levels with general characteristic and clinical parameters of the Population subjects Perilipin A levels Non-obese diabetic Obese diabetic (N=43) (N=43) Age(years) R -0.054 -0.048 P 0.672 0.758 BMI(kg/m2) R -0.113 -0.142 P 0.467 0.361 Visceral fat(%) R -0.054 -0.214 P 0.727 0.167 Body fat (%) R 0.148 -0.189 P 0.341 0.222 Duration of R -0.063 0.051 Diabetes(years) P 0.683 0.741 Data presented as Spearman’s correlation coefficient, r. *Correlation is significant at p<0.05; BMI: body mass index. R is Spearmans correlation coefficient and P is the p value at less than 0.05 35 University of Ghana http://ugspace.ug.edu.gh 4.6:Association of Perilipin A levels with Biochemical Parameters of the Study Population The results of the various associations of perilipin A levels with biochemical indices of the study population are presented in (Table 6). Low Density Lipoprotein (LDL), TG, the ratio: LDL/High Density Lipoprotein (HDL) as well as the ratio: Total Cholesterol (T.Chol)/HDL did not show any significant association with the perilipin of the study population. VLDL correlates significantly and inversely with perilipin in non-obese diabetic subjects but was not significant in obese diabetic subjects (p<0.05). Similarly, HDL correlates positively with perilipin in non-obese diabetic but did not correlate with obese diabetic subjects (p<0.05). (Table 6). As the fasting blood glucose level increased, perilipin levels also increased in the obese diabetic subjects.HbA1c correlated significantly, positively with perilipin in the non obese diabetic subjects (p<0.05) but inversely with obese diabetic subjects (p<0.05) (Table 6). Table 6:Correlation of Perilipin A levels with Biochemical parameters of the studypopulation Perilipin A levels Non-obese diabetics Obese diabetics (N= 43) (N= 43) FBG(mmol/L) R 0.078 0.116 P 0.066 0.007 * HbA1C (%) R 0.078 -0.008 P 0.006* 0.002* HDL(mmol/L) R 0.129 0.057 P 0.009 0.078 36 University of Ghana http://ugspace.ug.edu.gh TG(mmol/L) R -0.208 -0.214 P 0.179 0.166 LDL(mmol/L) R 0.136 0.065 P 0.383 0.678 VLDL(mmol/L) R -0.337 -0.262 P 0.026 * 0.090 T,chol(mmol/L) R 0.027 0.117 P 0.863 0.457 T,chol/HDL(mmol/L) R -0.269 0.192 P 0.081 0.216 Data presented as Spearman’s correlation coefficient, r. *Correlation is significant at p<0.05; BMI: body mass index. R is Spearmans correlation coefficient and P is the p value at less than 0.05 *mean difference is significant (p<0.05). FGB is fasting blood glucose, HbA1c is glycated 37 University of Ghana http://ugspace.ug.edu.gh 4.7: Correlation plot of perilipin A levels against age, BMI, visceral fat, body fat and TG in male and female subjects. 2 5 0 2 0 0 2 0 0 1 5 0 1 5 0 1 0 0 1 0 0 5 0 5 0 0 0 3 0 4 0 5 0 6 0 7 0 0 2 0 4 0 6 0 8 0 A G E (y r s ) A G E (y r s ) F ig 2 : A c o r r la t io n b e tw e e n a g e a n d p e r i l ip in le v e ls o f F ig 3 : A c o r re la t io n b e tw e e n a g e a n d p e r i l ip in le v e ls o f n o n -o b e s e d m a le o b e s e d m a le s u b je c ts w ith r -v a lu e = -0 .2 4 5 9 a n d p - v a lu e = 0 .2 8 2 7 s u b je c ts w ith r -v a lu e = -0 .1 3 8 9 a n d p - v a lu e = 0 .5 3 7 5 2 5 0 3 0 0 2 0 0 2 0 0 1 5 0 1 0 0 1 0 0 5 0 0 0 3 0 4 0 5 0 6 0 0 2 0 4 0 6 0 8 0 A G E (y r s ) A G E (y r s ) F ig 4 : A c o rre la tio n b e tw e e n a g e a n d p e r ilip in le v e ls in fe m a le F ig 5 : A c o r re la t io n b e tw e e n a g e a n d p e r i l ip in in n o n -o b e s e d f e m a le o b e s e d s u b je c ts w ith r -v a lu e = 0 .0 6 7 4 a n d p -v a lu e = 0 .7 6 5 7 s u b je c ts w ith r -v a lu e = 0 .0 1 8 4 2 a n d p -v a lu e = 0 .9 3 6 8 38 P E R IL IP IN C O N C P E R IL IP IN C O N C P E R IL IP IN C O N C P E R IL IP IN C O N C University of Ghana http://ugspace.ug.edu.gh 2 0 0 2 5 0 2 0 0 1 5 0 1 5 0 1 0 0 1 0 0 5 0 5 0 0 0 0 1 0 2 0 3 0 4 0 0 5 1 0 1 5 V IS C E R A L F A T V IS C E R A L F A T F ig 6 : A c o r re la t io n b e tw e e n v is c e ra l f a t a n d p e r i l ip in le v e ls in o b e s e d m a le F ig 7 : A c o r re la t io n b e tw e e n v is c e ra l f a t a n d p e r i l ip in le v e ls in n o n -o b e s e d m a le s u b je c ts w ith r -v a lu e = -0 .2 5 4 6 a n d p - v a lu e = 0 .2 6 5 4 s u b je c ts w ith r = - 0 .1 8 6 2 a n d p - v a lu e = 0 .4 0 6 7 2 5 0 3 0 0 2 0 0 2 0 0 1 5 0 1 0 0 1 0 0 5 0 0 0 0 5 1 0 1 5 2 0 2 5 0 5 1 0 1 5 V IS C E R A L F A T V IS C E R A L F A T F ig 8 : A c o rre la t io n b e tw e e n v is c e ra l fa t a n d p e rilip in le v e ls in F ig 9 : A c o r re la t io n b e tw e e n v is c e ra l f a t a n d p e r i l ip in le v e ls in o b e s e d fe m a le s u b je c ts w ith r -v a lu e = -0 .2 4 2 8 a n d p -v a lu e = 0 .2 7 6 3 n o n - o b e s e d f e m a le s u b je c ts w ith r - v a lu e = 0 .1 0 0 4 a n d p - v a lu e = 0 .6 6 4 9 39 P E R IL IP IN C O N C P E R IL IP IN C O N C P E R IL IP IN C O N C P E R IL IP IN C O N C University of Ghana http://ugspace.ug.edu.gh 2 0 0 2 5 0 2 0 0 1 5 0 1 5 0 1 0 0 1 0 0 5 0 5 0 0 0 2 6 2 8 3 0 3 2 3 4 3 6 1 8 2 0 2 2 2 4 2 6 2 8 B M I V A L U E B M I F ig 1 0 : A c o r re la t io n b e tw e e n B M I a n d p e r i l ip in in o b e s e d m a le F ig 1 1 : A c o r re la t io n b e tw e e n B M I a n d p e r i lip in le v e ls in n o n -o b e s e d m a le s u b je c ts w ith r= - 0 .3 2 1 5 a n d p = 0 .1 5 5 3 s u b je c ts w ith r= - 0 .0 8 9 3 a n d p = 0 .6 9 2 5 2 5 0 3 0 0 2 0 0 2 0 0 1 5 0 1 0 0 1 0 0 5 0 0 0 2 6 2 8 3 0 3 2 3 4 3 6 1 8 2 0 2 2 2 4 2 6 2 8 B M I V A L U E B M I F ig 1 2 : A c o r re la t io n b e tw e e n B M I a n d p e r il ip in le v e ls in o b e s e d F ig 1 3 : A c o r re la t io n b e tw e e n B M I a n d p e r i l ip in le v e ls in n o n - o b e s e d f e m a le f e m a le s u b je c ts w i th r = -0 .0 7 2 1 a n d p = 0 .7 4 9 9 s u b je c ts w ith r= - 0 .1 5 2 7 a n d p = 0 .5 0 8 7 40 P E R IL IP IN C O N C P E R IL IP IN C O N C P E R IL IP IN C O N C P E R IL IP IN C O N C University of Ghana http://ugspace.ug.edu.gh 2 5 0 2 0 0 2 0 0 1 5 0 1 5 0 1 0 0 1 0 0 5 0 5 0 0 0 0 1 0 2 0 3 0 4 0 2 0 2 5 3 0 3 5 4 0 B O D Y F A T B O D Y F A T F ig 1 4 : A c o r re la t io n b e tw e e n b o d y f a t a n d p e r i l ip in le v e ls in o b e s e d m a le F ig 1 5 : A c o r re la t io n b e tw e e n b o d y f a t a n d p e r i l ip in le v e ls in n o n -o b e s e d m a le s u b je c t w i th r = -0 .1 3 4 5 a n d p = 0 .5 6 1 2 s u b je c ts w ith r= - 0 .1 7 0 7 a n d p = 0 .4 4 7 5 2 5 0 3 0 0 2 0 0 2 0 0 1 5 0 1 0 0 1 0 0 5 0 0 0 0 1 0 2 0 3 0 4 0 0 5 1 0 1 5 2 0 2 5 B O D Y F A T B O D Y F A T F ig 1 6 : A c o r re la t io n b e tw e e n b o d y f a t a n d p e r il ip in le v e ls in o b e s e d f e m a le F ig 1 7 : A c o r re la t io n b e tw e e n b o d y f a t a n d p e r i l ip in le v e ls in n o n -o b e s e d f e m a le s u b je c ts r = - 0 .2 1 0 7 a n d p = 0 .3 4 6 6 s u b je c ts w i th r = 0 .5 0 4 5 a n d p = 0 .0 1 9 7 41 P E R IL IP IN C O N C P E R IL IP IN C O N C P E R IL IP IN C O N C P E R IL IP IN C O N C University of Ghana http://ugspace.ug.edu.gh 2 0 0 2 5 0 2 0 0 1 5 0 1 5 0 1 0 0 1 0 0 5 0 5 0 0 0 0 1 2 3 4 0 1 2 3 4 T G T G F ig 1 8 : A c o r r e la t io n b e tw e e n tr ig ly c e r id e s a n d p e r i l ip in le v e ls in o b e s e d F ig 1 9 : A c o r re la t io n b e tw e e n tr ig ly c e r id e s a n d p e r i l ip in le v e ls in m a le s u b je c ts w i th r= - 0 .2 5 1 6 a n d p = 0 .2 7 1 3 n o n -o b e s e d m a le s u b je c ts w i th r = -0 .0 2 4 5 a n d p = 0 .9 1 4 2 5 0 3 0 0 2 0 0 2 0 0 1 5 0 1 0 0 1 0 0 5 0 0 0 0 1 2 3 4 0 1 2 3 4 T G T G F ig 2 0 : A c o r r e la t io n b e tw e e n tr ig ly c e r id e s a n d p e r i l ip in le v e ls in o b e s e d f e m a le F ig 2 1 : A c o r re la t io n b e tw e e n t r ig ly c e r id e s a n d p e r i l ip in le v e ls in n o n -o b e s e d s u b je c ts w ith r= - 0 .2 1 1 5 a n d p = 0 .3 4 4 7 fe m a le s u b je c ts w i th r = -0 .3 7 7 4 a n d p = 0 .0 9 1 7 42 P E R IL IP IN C O N C P E R IL IP IN C O N C P E R IL IP IN C O N C P E R IL IP IN C O N C University of Ghana http://ugspace.ug.edu.gh CHAPETR FIVE DISCUSSION Perilipin in fat cells are found at the surface of the lipid droplet. Despite the position of the perilipin, their role in lipid hydrolysis and in blood is not understood. The experiments described in this study demonstrated that Perilipin A can be found in blood. Results from the study with 43 non-obese diabetic and 43 obese diabetic subjects showed a significantly higher BMI, visceral fat and body fats in obese diabetic than non-obese diabetic subjects (Table 1) due to an increased in total cholesterol and glucose level in their blood. (Table 2). This result was in support of findings by researchers (Wang et al., 2005; Knowler et al., 2002 ). BMI is identified as a pre-disposing factor in diabetic development (The InterAct Consortium, 2012). An increase in BMI is high in individuals with large amount of visceral fat (Kannel et al., 1991), which contributes to insulin resistance (Xu et al., 2003). These findings was in support that a higher BMI causes a high risk of type 2 diabetes and high blood pressure. Obesity is one of the negative outcome that has been established for hypertension, type 2 diabetes and dyslipidemia. Several changes of serum lipids and lipoproteins are frequently noted in overweight/obese individuals. The most common modifications are hypertriglyceridemia and decreased HDL-C levels (Elsawi et al.,2014). However, elevated glycated haemoglobin (≥ 6.5%) has been used to diagnose type-2 diabetes mellitus especially in obese individuals. This study was also carried out to determine the fasting lipid profiles and glycated hemoglobin in non-obese and obese subjects, in other to establish the effect of obesity on these biochemical parameters. The findings of the study showed that FBG, HbA1c, TC, LDL, and TG,T.chol/HDL were significantly higher in obese diabetic subjects than in the non-obese diabetic subjects (Table 2). This was in agreement with the findings of (McGill et al.,2002).TG, LDL and TC are risk factor for the 43 University of Ghana http://ugspace.ug.edu.gh development of cocronary heart disease and atherosclerosis. Higher increase of TG, LDL and TC in obese diabetic sujects stand higher risk of developing coronary heart disease and artherosclerosis than non-obese diabetic (Gaziano et al.,1997).The levels of prilipin A was found to be higer in obese diabetic than non- obese diabetic subjects (Table 2).This may be attributed to the fact that,perilipin acts like a barrier that protects the TG from been hydrolysed by hormone sensitive lipase which cause an increase in basal lipolysis and TG. Since obese diabetic subjects have a lower level of perilipin ,it may causes the obese diabetic subjects to exhibit a lower rates of basal lipolysis. This finding was consistent with Hellmer et al., 2012 who in their study showed that the adipocytes of obese subjects exhibit lower rates of basal lipolysis and numerous differences in metabolism and gene expression. In this study, female subjects in both obese and non-obese diabetics had significantly higher BMI as compared to the males (Table 3). This agrees with a study in Nigeria by Erasmus et al., (1992). The epidemiological survey strongly indicated that most of the female subjects lead sedentary lifestyle whereas the males were involved in a lot of manual jobs that involved physical activities. The mean HbA1C was higher in females in both obese and non-obese diabetic subjects than the males (Table 4). This type of result was also obtained by David et al (1984) because of chronic blood loss and sex difference in red blood cell life span (109 days), female hemoglobin can undergo greater degree of glycosylation compared to that of the male (David et al.,1984). In the comparism of levels of perilipin in gender , the males in both obese and non-obese diabetic subjects had a higher levels of perilipin than the females (Table 4) this may be due to the fact that the males had an increase in visceral fats and body fats as compare to the females which accommodate the perilipin from been expressed in the blood . Study done by Wang et al (2003) showed that perilipin mass in adipocytes of obese men was higher compared to women . 44 University of Ghana http://ugspace.ug.edu.gh In this study, both visceral fat and body fats had an effect on the level of perilipin in both non-obese and obese diabetic subjects but the effects was not significant. It was observed that the non-obese subjects who have lower visceral fat and body fats had an increase in perilipin A levels than the obese subjects.This may be due to the fact that obese subjects who had a higher visceral fats and body fat might be shielding the perilipin in the tissue from been expressed ,indicating a lower perilipin A level in blood. Since the body depot (visceral fats and body fats ) was high in obese diabetic subjects,it causes an increase in basal lipolysis which leads to a lower level of perilipin in blood than the non-obese diabetic subjects.(Table 5) . A study done by Arvisddon (2004) shows that the expression of perilipin was lower in fats cells compare with subcutaneouns due to high stimulated lipolysis. 5.1 CONCLUSION The findings of the study showed there was no significant difference in blood perilipin A between the non-obese and obese diabetic subjects. Blood perilipin A levels revealed an increased levels in males than the females between the two groups.The work revealed association between age .FBG and HbA1C with blood perilipin A. The rest of the parameters did not show any significant association with blood perilipin A levels. Thus the hypothesis that diabetes does not influence levels of blood perilipin A is accepted because the levels of blood perilipin A observed in non-obese diabetic subjects was not significantly different from the obese diabetic subjects. 5.2 RECOMMENDATIONS Future research should be performed in connection with the levels of perilipin A to complications of diabetes. Further research should be done to investigate the effects of blood perilipin levels to 45 University of Ghana http://ugspace.ug.edu.gh anti-diabetic drugs in diabetic patients. Larger sample size and different age categories with a smaller range of duration of the diabetes would be recommended as well as the measurement of free fatty acids and insulin levels. Further investigations with well structured prospective studies with incident type 2 diabetes as the outcome would be needed to make appropriate recommendations on the usefulness of fat depot as a first line diagnostic tool when monitoring an individual’s outcome of diabetes. 5.3 LIMITATIONS One of the challenges in the research was inadequate reference range for perilipin A levels of obese and lean diabetic Ghanaians. Restricted time and logistic of the selection of subjects in type 1 diabetes was a challenge. Another challenge was determining the correct duration of diabetes of the subjects. 46 University of Ghana http://ugspace.ug.edu.gh REFERENCE Abbasi, A., Corpeleijn, E., Postmus, D., Gansevoort, R.T., De Jong, P.E., Gans R.O.,(2010). 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Kern ,P.A, Di, Gregorio G, Lu T, Rassouli N, Ranganathan G.(2004). Perilipin expression in human adipose tissue is elevated with obesity. J Clin Endocrinol Metab ; 89: 1352–1358 53 University of Ghana http://ugspace.ug.edu.gh Knop, F., Vilsbol, T., Madsbad, S., Holst, J., and Krarup, T., (2010). Inappropriate suppression of glucagon during OGTT but not during isoglycaemic IV glucose infusion contributes to the reduced incretin effect in type 2 diabetes mellitus. Diabetologia, 50(4), 797-805. Knowler, W. C., Barrett Connor, E., Fowler, S. E., Hamman, R. F., Lachin J. M., Walker, E. A., Nahan, D. M., (2002). Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin. Diabetes Prevention Program Research Group. New England Journal of Medicine: 346(6):393-403. Lieb,W., Sullivan , l.m., Harris ,T.B., (2009) .Plasma leptin levels and incidence of heart failure cardiovascular diseases and total motility in elderly individuals . 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J., Forenstein, D., Chen, K. H, and Chan, L. (2000) Absence of perilipin results in leanness and reverses obesity in Leprdb/db mice. Nature Genetics 26, 474 – 479. Metcaf, B.S., Hosking, J., Fremeaux, A.E., Jeffery, A.N., Voss, L.D., Wilkin, T. J., (2011) . BMI was right all along:taller children really fatter(implications of making childhood BMI independednt of height ) International Journal of obesity : 35()541-7 Mottagui-Tabar ,S., Ryden, M., Lofgren, P., (2003) . Evidence for an important role of perilipin in the regulation of human adipocyte lipolysis. Diabetologia 2003; 46: 789–97. 55 University of Ghana http://ugspace.ug.edu.gh Morimoto, C., Tsuji, T., and Okuda, H.(1997) J. Lipid Res. 38,132-138. Nishiu, J., Tanaka, T., Nakamura, Y..(1998) Isolation and chromosomal mapping of the human homolog of perilipin (PLIN), a ratadipose tissue-specific gene, by differential display method. Genomics 1998; 48: 254–7. Ragoobirsingh, D., & McGrowder, D. A. (2012). A Prospective Study of Children Who were Admitted for Diabetes Mellitus at a Jamaican Hospital. Journal of Clinical and Diagnostic Research, 6(5): 832-834. Rizza, R.A., (2010). Pathogenesis of fasting and postprandial hyperglycemia in type 2 diabetes: implications for therapy. Diabetes, 59(11), 2697-2707. Rodriguez-Segade, S., Camina, M. F., Paz, J. M. and Del Rio, R., (1991). Abnormal serum immunoglobulin concentrations in patients with diabetes mellitus clin chim Acta. 203: 135-142 Ross, G., (2006). Gestational diabetes. Aust Fam Physician 35(6):392-6 Rother K. I (2007): "Diabetes Treatment ó Bridging the Divide". New England journal of medicine; 356 (15): 1499-1501. 56 University of Ghana http://ugspace.ug.edu.gh Samuel, P., Venkateswarlu, M., & SIVA, P. V. (2012). Lipid Profile Levels in Type 2 Diabetes Melli-tus from the Tribal Population of Adilabad in Andhra Pradesh, India. Journal of Clinical and Diagnostic Research, 2. Shaw J.E., Sicree R.A., and Zimmet P.Z (2012). Global estimates of the prevalence of diabetes for 2010 and 2030. Diabetes Res. Clin. Pract, 87:4-14. Shen, W. J., Sridhar, K., Bernlohr, D. A., and Kraemer, F. B. (1999) Interaction of rat hormone- sensitive lipase with adipocyte lipidbinding protein. Proc. Natl. Acad. Sci. U.S.A. 96, 5528 – 5532 Souza, S.C., De Vargas, L.M,, Yamamoto, M.T,, Lien, P,, Franciosa, M.D,, Moss, L.G, Large, V, Arner P (1998) Regulation of lipolysis in humans. Pathophysiological modulation in obesity, diabetes, and hyperlipidaemia. Diabetes Metab 24:409–418. Souza, S. C., Moitoso de Vargas, L. Yamamoto, M. T., Lien, P., Franciosa , M. D., Moss, L. G., and Greenberg, A. S., (2003) J. Biol. Chem. 273, 24665-24669 57 University of Ghana http://ugspace.ug.edu.gh Sztalryd, C., Xu, G., Doward, H., Tansey J, T., Contreras, J. A., Kimmel, A. R., and Londos, C. (2012) Perilipin A is essential for the translocation of hormone-sensitive lipase during lipolytic activation of adipocytes. J. 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Wang, Y., Rimm, E.B., Stampfer, M.J., Willett, W.C. and Hu, F.B., (2005). Comparison of abdominal adiposity and overall obesity in predicting risk of type 2 diabetes among men. Am J Clin Nutr. 81:555-563. 59 University of Ghana http://ugspace.ug.edu.gh Williams, D. R., Wareham, N. J., Brown, D.C., Byrne, C.D., Clark, P. M., Cox, L.J., Day, N.E., Hales, C.N., Palmer, C.R., (1995). Undiagnosed glucose intolerance in the community; the isle of Ely Diabetes Project. Diabetic Medicine; 12(1): 30-35. World Health Organization, (1999). Definition, Diagnosis and classification of Diabetes Mellitus and Its complications: Report of WHO Consultation. WHO/NCD/NCS/99.2. Geneva: WHO World Health Organization, (2006) Diabetes. Geneva: WHO. http: // www.who.int/ncd/dia/databases.4htm. World Health Organization, (2011). classification of Diabetes Mellitus and Its complications: Report of WHO Consultation. WHO/NCD/NCS/99.2. Geneva: WHO. World Health Organization, (2012). Waist circumference and waist-hip ratio: report of a WHO expert consultation, Geneva, 8-11 December 2008. 1-31. World Health Organization (2015). The World Obesity Reports, World Health Organization, (2016) Diabetes. Geneva: WHO. http: // www.who.int/ncd/dia/databases.4htm. 60 University of Ghana http://ugspace.ug.edu.gh Xu, H., Barnes, G.T., Yang, Q., Tan, G., Yang, D., Chou, C.J., Sole, J., Nicholas, A., Ross, J.s., Tartaglia, L.A. and Chen, H., (2003). Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance. J Clin Invest. 112:1821-1830. 61 University of Ghana http://ugspace.ug.edu.gh APPENDIX A INFORMED CONSENT FORM I, Michael Quansah of the Department of Chemical Pathology, University of Ghana School of Biological and Allied Health Sciences (SBAHS) wish to embark on a study entitled, Blood perilipin A levels in Diabetic patients at Korle –Bu Teaching Hospital. The purpose of this study is to investigate the concentration levels of perilipin A and Hormone sensitive lipase in obese diabetic and non-obese diabetic subjects. You will be asked few routine questions about your personal details and diabetic history. The benefits of the study are to educate you on the effect of Diabetes and its implications. It will be appreciated if you volunteer, though participation is entirely voluntary and strictly confidential. You may choose to withdraw from the study whenever you wish. Participants of the study will undergo an overnight fast after which 3 mls of blood will be withdrawn. Both procedures may involve slight discomfort. The amount of blood to be taken by phlebotomists and used for this research study will not exceed 3 mls. You will be assured of the strictest confidentiality of your personal information. This study will adhere to all applicable protocols and will maintain quality assurance in accordance with good laboratory practice. The blood samples collected will bear an identification code to ensure anonymity, confidentiality and ease of identification. There is the possibility that you might not benefit directly from participation. However, the information obtained and conclusions drawn will be applied in the adoption of relevant health policies as well as the appropriate care and management. You will incur no costs and you will also not be paid for participating in this study. However, you will be entitled to know the outcome of the laboratory results and this will be well explained to you. All data will be entered onto a lock/word-protected Microsoft Excel spreadsheet. Study questionnaires will be kept in a locked cabinet in a locked office. 62 University of Ghana http://ugspace.ug.edu.gh My contact numbers is 0243944269. You may call me for any further clarification. Thank you for the cooperation and anticipated compliance to the study requirements. Signature of Participant: ……………………… Date……………………………….. Signature of Researcher…………………… Date………………………………… 63 University of Ghana http://ugspace.ug.edu.gh APPENDIX B PARTICIPANT CONSENT RESPONSE Research title: Blood perilipin A levels in Diabetic patients at Korle –Bu Teaching Hospital. I, ………………………………………………… have been invited to take part in this research. I have been told of the purpose and procedure of this study which is to answer the questions raised about the differences in blood perilipin A levels. I have agreed to give information about personal information such as my educational background, diabetes history etc. I understand I will not be reimbursed monetarily for participating in this study. I am aware of the risk, dangers and discomforts that might be associated with the pain of blood collection. The study team will try to reduce the chances of those risks happening by employing trained phlebotomist. The arm will be sanitized before blood collection, and new sterile needles and gloves will be used for each participant. I promise to comply with the requirement of study and I consent accordingly. Signature……..……………. Thumbprint…………………………… 64 University of Ghana http://ugspace.ug.edu.gh APPENDIX C QUESTIONNAIRE Participant’s ID ……………………….. Date…………………………………… A. SOCIO –DEMOGRAPHIC STATUS 1. Age ………. (years) 30-39 [ ] 40-49[ ] 50 -59 [ ] exactly 60[ ] Gender : M ( ) F( ) 2. Marital Status i. single [ ] ii. Married [ ] iii. Divorced [ ]iv. Widowed [ ] v. Separated [ ] Other…………………….. 3. Religion i. Christian [ ] ii. Muslim [ ] iii.Others, please specify……………... 4. Educational Background i. No formal education [ ] ii. Basic education (middle/JHS) [ ] iii. SHS/O –Level [ ] iv. HND/Diploma Certificate [ ] v. Bachelor Degree [ ] vi. Post Degree[ ] 5. Employment Status: i Employed [ ] ii Unemployed [ ] iii Retired [ ] iv. Student [ ] 6. VISCERAL FAT…………………. BODY FATS…………………. BMI………………… B. MEDICAL HISTORY /ASSESSMENT OF DIABETIC COMPLICATIONS 7. Are you diabetic? [ ] Yes [ ] No , 7b. If yes which type, type 1 diabetes [ ] type 2 diabetes [ ] 7c. How long have you been diagnose of diabetes? …………………………………. 8. Do you have any medical condition? YES [ ] NO [ ] 65 University of Ghana http://ugspace.ug.edu.gh 9. If yes please state the condition ……………………………………………………… 10. Are you on medication? YES [ ] NO [ ] 11b.If yes, mention………………………………………………………………………… 12. Do you take other medication or herbal treatments? YES [ ] NO [ ] 12b.If yes, please state…………………………………………………… C. LIFE STYLE BEHAVIOURS 13.Do you smoke? YES [ ] NO [ ] 13b. if yes how often? Daily [ ] Weekly [ ] monthly [ ] occasionally [ ] 14. Do you drink alcohol? YES [ ] NO [ ] 14b. If yes how often Daily [ ] Weekly [ ] Monthly [ ] occasionally [ ] One servings of alcohol: 120ml of wine (½ medium glass of dry wine) 285ml of beer (1/2 large beer bottle, one full mini Guinness) 30ml (1 tot of spirit, whisky gin, akpeteshi and alcoholic bitters) 60ml of (brandy, vermouth, aperitif) 14c.On average, how many servings of alcohol do you take daily? ............................... 66 University of Ghana http://ugspace.ug.edu.gh 67 University of Ghana http://ugspace.ug.edu.gh 68 University of Ghana http://ugspace.ug.edu.gh 69