The impact of oleanolic acid on lipid metabolism
and mitochondrial biogenesis in fructose-induced
neonatal metabolic derangement model
M. M ABOTSENG
orcid.org/
0000-0003-3226-9314
Thesis submitted for the degree of the
Doctor of Philosophy in Science
with Biochemistry
at the North-West University
Supervisor:
Prof Emmanuel Mukwevho
Co-supervisor:
Dr Ademola Ayeleso
Graduation: July 2019
Student number: 16625978
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DECLARATION OF ORIGINALITY
I, Molefhi Moirapula Abotseng, do hereby state that this thesis, which has been submitted in fulfilment of the requirement for the award of PhD, is entirely my own work. I have exercised reasonable care to ensure that the work is original and has not been taken from the work of others. Therefore, I declare that any contribution, material and works of other people in this thesis have been cited and referenced accordingly.
This thesis is presented in fulfilment of the requirement for the degree of Doctor of Philosophy in Science with Biochemistry, North West University.
Student signature: ___________________ Signed on of day 2019
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ACKNOWLEDGEMENTS
All Glory to the Almighty God for His Strength and guidance for the successful completion of this research work.
Many thanks to my supervisor, Professor Emmanuel Mukwevho, for giving me an opportunity to work in his laboratory and under his esteemed guidance, as well as financial and moral supports. His warmth, availability and keen interest in scientific work created a conducive working environment for me. I also extend my sincere gratitude to my co-supervisor, Dr Ademola Ayeleso for his immense contributions and guidance towards this work. His patience and kindness played a crucial role in understanding the experimental protocols and eventually completing this work.
My appreciations to the Human Metabolomics Platform in the North West University, Potchefstroom campus for allowing me to use their facility to carry out part of the project. In particular, Professor Chris Vorster, Mr Graant Massdorp and Dr Zander Lindeque for training me on the use of the equipment. I want also to recognise Professor Kennedy Erlwanger for accepting me to collaborate with his PhD student. Dr Trevor T. Nyakudya, it was a great journey working with you and I have learned a lot from you. Many thanks to Professor Lesetja Motadi, Dr Sandile Fuku and Mr Nditsheni Rabambi for your time and assistance towards the completion of my study.
To my wife, Katlego Abotseng, I am indebted to you for your support and encouragement during this time of the study. I could not have accomplished this enormous task without your love and understanding as my study mate. My parents, my father Kooagile Abotseng (late) and mother Mmopiemang Abotseng, thank you very much for loving and raising me up, your unwavering support for education has been amazing. My father in-law, Lucas Sehlabane and mother in-law, Grace Sehlabane, I thank you for your encouragement. My siblings, thank you for your support and care at the time when I needed you the most. Last but not the least, I would like to convey my gratitude and thanks to all my colleagues in the Diabetes and Therapeutics Laboratory particularly Brian Munansangu, Mashudu G. Matumba, Simon Isaiah, Mmaheiine Molepo, Dr Taiwo B. Ayeleso and Dr Shesan J. Owonubi.
I am grateful to both North West University and University of Botswana for affording me a chance to undertake this study.
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ABSTRACT
Metabolic syndrome is a health condition emanating from a combination of metabolic disorders, which include diabetes, hypertension, dyslipidemia, insulin resistance and obesity. These metabolic disorders are commonly referred to as life-style or non-communicable diseases in some quarters. These conditions have become the leading cause of death in the recent years. The prevalence of metabolic syndrome is at an alarming rate in both the developing and developed countries across the globe. There is an overwhelming strain in the national health budget because of non-communicable diseases, big chunk of moneys are allocated in trying to address them. Metabolic diseases are attributed to a combination of contributing factors such as the type of food consumed, life style activities and genetic inheritance. These contributing factors are responsible for aggravating metabolic diseases. Diabetes and cardiovascular diseases are increasingly becoming silent killers among both young and older population. Obesity is another condition which is having devastating consequences in human health. Individuals gain an excessive amount of fat around the adnominal area, which end up tempering with the functions of vital organs such as the liver, heart and kidneys.
The metabolic diseases are caused by a number factors. One of the possible causes of metabolic diseases is genetic inheritance, this involves genetic materials in the form of DNA being passed form parents to their offspring. Individuals coming from families with history of metabolic disorders are susceptible to developing such diseases. There is undisputed evidence suggesting an increase of fructose consumption in food intake. This increase intake of fructose is marred with a lot of controversies and it is correlated to the rise in metabolic disorders and other related complications. Fructose and its metabolism form the cornerstone of this study. Fructose has been viewed as having metabolic programming effects. Metabolic programming is the window period of early health status, which is very delicate and can affect the state of health in adulthood life. In order to address the rapid increase of metabolic diseases, plants are commonly used as alternative or complementary remedy for chronic diseases, especially in African and Asian countries. Oleanolic acid (OA) is a plant phytochemical with many therapeutic potentials in several foods and medicinal plants. Therefore, OA has been used in this scientific research to determine its impact in lipid metabolism and mitochondrial biogenesis.
In this study, one week old neonatal experimental animals (Sprague Dawley rats) were subjected to different treatments for seven consecutive days with 10 mℓ/kg body mass of: DMSO with 0.5% (v/v) as Control, Oleanolic Acid (OA) with 60 mg/kg, High Fructose (HF) with 20% w/v,
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Oleanolic Acid + High Fructose (OA+HF) and Metformin + High Fructose (MET+HF) with 60 mg/kg. The pups were raised until they reached their adulthood life, fasted on 111th day and were sacrificed on 112th day. The Snowrex Electronic and Clover scales were used to measure the weekly weights of rats and the Contour Plus glucometer was used to determine the blood glucose levels. The skeletal muscle and liver samples were harvested from the sacrificed rats. The Gas Chromatography-Mass Spectrometry (GC-MS) was used to determine the levels of different fatty acids and Real Time quantitative Polymerase Chain Reaction (RT-qPCR) was used to evaluate the expression of genes related to lipid metabolism, mitochondrial biogenesis and inflammatory responses. Western blot was used for protein analysis while ChIP assay was used to assess protein-DNA interactions of MEF-2A to NRF-1 genes.
The results showed increased percentage weight gain and fasting blood glucose in high fructose-fed rats, while oleanolic acid and metformin treatments showed no significant differences when compared with the untreated controls. The concentration levels of omega 3 polyunsaturated fatty acids (docosahexaenoic, eicosapentaenoic, alpha linoleic acid) and omega 6 polyunsaturated fatty acid (eicosadienoic, calendic and gamma linoleic acid) in high fructose-fed rats were not significantly increased when compared with the normal control. However, high fructose-fed rats treated with oleanolic acid or metformin showed significantly increased concentration of these fatty acids. A similar result was also observed in the concentration levels of monounsaturtaed fatty acids (MUFA) in the high fructose-fed rats. The treatment of high fructose-fed rats with oleanolic acid showed increased significant levels of MUFA. Lipid oxidation associated genes assessed in this study were CPT-1, NRF-1, GLUT-4 and MEF-2A and were highly expressed when high fructose-fed rats were treated with oleanolic acid and metformin when compared with the control. Similarly, mitochondrial associated genes such as TIM, TOM, TFAM, PGC-1 and COX-2, also were highly expressed when the high fructose-fed rats were treated with oleanolic acid or metformin. On the contrary, lipid synthesis associated genes such as FAS, ACC-1 and SREBP-1c were highly expressed on the rats fed with high fructose alone. The insulin related genes such as IRS-1and PPARγ were highly expressed on the rats treated with oleanolic acid. These results indicated that, oleanolic acid possesses ability to alleviate metabolic dysfunctions and their related complications.
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LIST OF ABBREVIATIONS
ACC: Acetyl coenzyme -A carboxylase ADP: Adenosine diphosphate
AESC: Animal ethics screening committee AGE: Advanced glycation end products AHA: American heart association ALA: Alpha linoleic acid
AOAC: Association of official analytical chemists ATP: Adenotriphosphate
BMI: Body mass index
BSTFA: Bis (trimethylsilyl) triofuoroacetamide CAD: Coronary artery disease
cAMP: Cyclic adenosine monophosphate ChIP: Chromatin immunoprecipitation cDNA: Complimentary DNA
CHO: Carbohydrates
CKD: Chronic kidney disease CL: Cardiolipin
CoA: Coenzyme A CO2: Carbon dioxide COX: Cyclooxygenase
vi CPT: Carnitine palmitoyltransferase
CVD: Cardio vascular disease DEPC: Diethyl pyrocarbonate DHA: Docosahexaenoic acid DMSO: Dimethyl sulfoxide DNA: Deoxy ribonucleic acid DNL: De novo lipogenesis
DoHAD: Development origins of health and disease ECL: Enhanced chemiluminescence
EDTA: Ethylenediaminetetraacetic acid
EGIR: European group for the study of insulin resistance EPA: Eicosapentaenoic acid
ER: Endoplasmic reticulum FAME: Fatty acid methyl ester FAS: Fatty acid synthase F: Forward
FFA: Free fatty acids
GCFID: Gas chromatography flame ionization detector GC-MS: Gas chromatography mass spectrometry GLUT-4: Glucose transporter 4
GWAS: Genome wide association studies HDL: High density lipoprotein
vii HF: High fructose
HFCS: High fructose corn syrup HFD: High-fat diets
HPA: Hypothalamic-pituitary adrenal axis HRP: Horseradish peroxidase
IDF: International diabetes federation IgG: Immunoglobulins G
IN: Input sample IL: Interleukin
IP: Immunoprecipitation IR: Insulin resistance IS: Internal standard LA: Linoleic acid
LCFA: Long chain fatty acids LDL: Low density lipoproteins MCFA: Medium chain fatty acids MEF 2A: Myocyte enhancer factor-2 MET: Metformin
MetS: Metabolic syndrome MHC: Myosin heavy chain mRNA: Messenger RNA MPO: Myeloperoxidase
viii mtDNA: Mitochondrial DNA
mtTFAM: Mitochondrial TFAM MUFA: Monounsaturated fatty acids NAFLD: Non-alcoholic fatty liver disease NASH: Necroinflammation or steatohepatitis NCD: Non-communicable diseases
NCEP: National cholesterol educational panel NGT: Normal glucose tolerance
NRF 1: Nuclear respiratory factor 1 OA: Oleanolic acid
OGTT: Oral glucose tolerance test PBS: Phosphate buffered saline PC:Phosphatidylcholines
PCTP: Phosphatidylcholine transfer protein
PCR: Polymerase chain reaction PE: Phosphatidylethanolamine PG: Phosphatidylglycerol
PGC-1: Peroxisome proliferator-activated receptor γ-coactivator 1 PPAR: Peroxisome proliferator-activated receptors
PLT: Phospholipid transfer protein PUFA: Polyunsaturated fatty acids PLTP: Phospholipid transfer protein
ix RIPA: Radioimmunoprecipitation assay
RNS: Reactive nitrogen species ROS: Reactive oxygen species Rev: Reverse
RT: Room temperature
RT-qPCR: Real time quantitative polymerase chain reaction SD: Standard deviation
SDS PAGE: Sodium dodecyl sulfate polyacrymide gel electrophoresis SFA: Saturated fatty acids
STATS SA: Statistics South Africa
SREBP: Sterol regulatory element-binding protein TAG: Triacylglycerol
T2DM: Type 2 diabetes mellitus
TFAM: Mitochondrial transcription factor A TG: Triglyceride
TIM: Translocase of the inner membrane TFA: Trans-unsaturated fatty acids TMCS: Trimethylchlorosilane TNFα: Tumor necrosis factor α
TOM: Translocase of the outer membrane VLDL: Very low-density lipoprotein WAT: White adipose tissue
x WHO: World Health Organization
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS ... ii
ABSTRACT ... iii
LIST OF ABBREVIATIONS ... v
LIST OF TABLES ... xiii
LIST OF FIGURES ... xiv
CHAPTER 1 ... 1
INTRODUCTION AND BACKGROUND OF THE STUDY ... 1
1.1 Introduction ... 1
1.2. Background ... 2
1.3 Problem statement ... 4
1.4 Aim of the research... 5
1.5 Specific Objectives ... 5
1.6 Significance of the study ... 6
CHAPTER 2 ... 7
LITERATURE REVIEW ... 7
2.1. Metabolic syndrome: a global health problem ... 7
2.3 Metabolic programming ... 12
2.4 Metabolism related diseases ... 16
2.5 Metabolism of lipids and fatty acids ... 24
Phosphatidylcholine (PC) forms a large part of cellular membranes. ). ... 25
2.6 Livers and Muscles and their roles in metabolic diseases ... 30
2.7 Mitochondrial biogenesis ... 33
2.8 Fructose and its metabolism ... 39
2.9 Inflammation in metabolic disorders ... 41
2.10 Oleanolic acid ... 42
CHAPTER 3 ... 44
MATERIALS AND METHODS ... 44
3.1 Animal care ... 44
3.3 Weekly body mass for Sprague Dawley rats ... 46
3.4 Normal glucose level in Sprague Dawley rats ... 47
3.5 Gas Chromatography Mass Spectrometry (GS-MS) ... 47
3.6. Ribonucleic acid (RNA) analysis ... 48
3.7 cDNA synthesis ... 48
3.8 Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) ... 49
3.9 Chromatin immunoprecipitation (ChIP) assay ... 51
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3.11 Statistics ... 56
CHAPTER 4 ... 57
RESULTS ... 57
4.1 Chromatography analysis of lipids ... 57
4.2 Assessment of level of fatty acids ... 60
4.3 Assessment of level of Phospholipids ... 78
4.4 Assessment of genes involved in lipid metabolism ... 81
4.5 Assessment of Chromatin Immunoprecipitation ... 96
4.6 Assessing protein level of NRF-1 gene using western blot ... 98
CHAPTER 5: ... 99
DISCUSSIONS ... 99
CHAPTER 6 ... 106
6.1 STUDYCONCLUSION ... 106
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LIST OF TABLES
Table 3. 1: List of primers for RT-qPCR used in the study ... 49
Table 3.2: The reaction mixture components for RT-qPCR ... 50
Table 3.3: Thermal cycling conditions for the RT-qPCR ... 50
Table 3.4: ChIP assay primers sequence ... 53
Table 4.1: The effect of oral administration of oleanolic acid, high fructose diet and metformin on the average weights of the rats………...58
Table 4.2: The effects of neonatal oral administration of oleanolic acid, high fructose and metformin on the concentrations of blood glucose levels of the rats from 7th day to 112th day………..58
Table 4.3: The blood concentration of total cholesterol, low density lipoproteins and high density lipoproteins levels in neonatal rats……….59
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LIST OF FIGURES
Figure 2. 1: A flow diagram showing the stage of early in utero fetal programming to adulthood metabolic disorder...14 Figure 2. 2: A flow diagram showing the stages of early fetal programming to adulthood metabolic disorder...15 Figure 2. 3: Fructosemetabolised in the liver...40 Figure 3. 1: The diagram indicates arrangement of the five experimental groups of Sprague Dawley rats. .. ... 44 Figure 3. 2: Flow diagram showing the experimental groups, stages, and timing of dietary interventions………..……… 46 Figure 4. 1: The concentration of eicosapentaenoic acid (EPA) in 4.1a (skeletal muscle), 4.1b (liver), docosahexaenoic acid (DHA) 4.1c (skeletal muscle), 4.1d (liver) of Sprague Dawley rats .. 62 Figure 4. 2: The concentration of alpha linoleic acid (ALA) in 4.2a (skeletal muscle), 4.2b (liver) and stearodonoic acid 4.2c (skeletal muscle) and 4.2d (liver) of Sprague Dawley rats ... 64 Figure 4. 3: The concentration of eicosnadienoic acid, calendic acid and gamma linoleic acid in 4.3a (muscle), 4.3b (liver), 4.3c (muscle), 4.3d (liver) and 4.3e (muscle), 4.3f (liver) of Sprague Dawley rats. ... 67 Figure 4. 4: The concentration of arachidonic acid in 4.4 a (skeletal muscle) and 4.4b (liver) of Sprague Dawley rats. ... 68 Figure 4. 5: The concentration of elaidic acid, eicosenoic acid and oleic acid in 4.5a (skeletal muscle), 4.5b (liver), 4.5c (skeletal muscle), 4.5d (liver), 4.5e (skeletal muscle) and 4.5f (liver) of Sprague Dawley rats. ... 71 Figure 4. 6: The concentration of lauric acid, palmitic acid and caprylic acid in 4.6a (skeletal muscle) and 4.6b (liver), 4.6c (skeletal muscle) and 4.6d (liver), 4.6e (skeletal muscle) and 4.6f (liver) of Sprague Dawley rats. ... 74 Figure 4. 7: The concentration of stearic acid and myristic acid in 4.7a (skeletal muscle), 4.7b, 4.7c (skeletal muscle) and 4.7d (liver) of Sprague Dawley rats. ... 76 Figure 4. 8: The expression of phosphatidylethanolamine, phosphatidylcholine and phosphatidylglycerol in 4.8a (skeletal muscle), 4.8b (liver), 4.8c (skeletal muscle), 4.8d (liver), 4.9e (skeletal muscle) and 4.9f (liver) of Sprague Dawley rats. ... 79
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Figure 4. 9: The expression of CPT-1, MEF-2A, GLUT-4 and NRF-1 in 4.9a (skeletal muscle) and 4.9b (liver), 4.9c (skeletal muscle), 4.9d (liver), 4.9e (skeletal muscle), 4.9f (liver), 4.9g (skeletal muscle) and 4.9h (liver) of Sprague Dawley rats. ... 83 Figure 4. 10: The expression of FAS, SREBP-1 and PPARγ in 4.10a (skeletal muscle), 4.10b (liver), 4.10c (skeletal muscle), 4.10d (liver), 4.10e (skeletal muscle) and 4.10f (liver) of Sprague Dawley rats. ... 85 Figure 4.11: The expression of TIM, TOM, PGC-1, TFAM and COX-2 in 4.11a (skeletal muscle) and 4.11b (liver), 4.11a (skeletal muscle), 4.11b (liver), 4.11C (skeletal muscle), 4.11d (liver), 4.11e (skeletal muscle) and 4.11f (liver), of Sprague Dawley rats. ... 88 Figure 4.12: The expression of PPARγ and IRS-1 in 4.12a (skeletal muscle) and 4.12b (liver) of Sprague Dawley rats. ... 92 Figure 4. 13: The expression of TNF-α in 4.13a (skeletal muscle), 4.13b (liver), 4.13c (skeletal muscle) and 4.13d (liver) of Sprague Dawley rats. ... 94 Figure 4. 14: ChIP assay images 4.14a skeletal uscle and 4.14b liver. ... 95 Figure 4. 15: Western blot images. Figure 4.15a representing the skeletal muscle while figure 4.15b represent the liver………...98
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CHAPTER 1
INTRODUCTION AND BACKGROUND OF THE STUDY
1.1 Introduction
Metabolic syndrome (MetS) is defined as a group of interconnected metabolic and cardiovascular disorders characterized by obesity, insulin resistance (IR), type 2 diabetes mellitus (T2DM), dyslipidaemia, hypertension, atherosclerosis and myocardial infarction (O'Neill & O'Driscoll, 2015). In order to describe these disorders of metabolic origin, other terms such as the deadly quartet syndrome, insulin resistance and cardiovascular syndrome have been used (Hertel et al., 2011). There has been contention on the definition of metabolic syndrome by researchers, in order to provide a standard tool for defining MetS for researchers and clinicians. World Health Organization (1998) coined the term “metabolic syndrome” and provided the first complex and prescriptive definition of the metabolic disorder (Alberti & Zimmet, 1998). Presently, these metabolic disorders are detrimental to human health since they cause metabolic diseases. The past two decades have experienced the emergence of a modern epidemic, which is metabolic syndrome (MetS) and it has appalling consequences to the health of humans worldwide (Jaacks et al., 2016). This health epidemic has been indicated by the alarming increase in non-communicable diseases (NCD) in the world populations. During 2008, total of 57 million deaths occurred around the world (WHO, 2010). About 36 million (63%) of these deaths were due to NCDs, principally cardiovascular and metabolic diseases, cancer and chronic respiratory diseases (WHO, 2010). Nearly 80% of these NCD deaths (29 million) occurred in developing countries, which are characterised by low and middle income (WHO, 2004). The prevalence of NCDs is rapidly rising in the African continent and is projected to cause almost three-quarters as many deaths as communicable diseases by 2020, and even to be viewed as the most common causes of death by 2030 (WHO, 2015). South Africa recorded a total of 460 236 deaths in 2014 and the three leading causes of deaths were tuberculosis, diabetes mellitus and cerebrovascular diseases (STATS SA, 2015).
The consumption of fructose has increased significantly in the recent years and has been associated with development of obesity. Obesity is accumulation of fat in obesity causes systemic oxidative stress via the production of reactive oxygen species (ROS) from the accumulating adipose tissue (Frei & Higdon, 2003; Rani et al., 2016). The overproduction of ROS by adipocytes contributes to the development of metabolic disorders by decreasing the expression of anti-oxidant enzymes (Sankhla, 2017). Studies performed in adult animal models have shown that the activation of
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cytoprotective anti-oxidant genes can suppress the development of oxidative stress associated with MetS (Chen & Kunsch, 2004b; Furukawa et al., 2017a). Therapeutic approaches that reduce oxidative stress will, therefore, contribute to the improvement of glycaemic control and prevention of metabolic complications of MetS and T2DM (Pall & Levine, 2015).
1.2. Background
Modern lifestyles and obesity epidemic are the reasons for rising of metabolic diseases in both developed and developing countries (van Vliet-Ostaptchouk et al., 2014). In addition, the plentiful availability of low cost, highly sweetened nutrients and an increasingly sedentary lifestyle, are key factors responsible for the marked increase in the prevalence of metabolic disorders (Grundy, 1998; Hill & Peters, 1998; Wickelgren, 1998). High blood glucose, low high density lipoprotein level (HDL), abdominal obesity, elevated blood pressure, high serum triglycerides and insulin resistance increase chances of developing metabolic syndrome (Mukwevho & Joseph, 2014). These conditions are interconnected and share joint mechanisms and pathways and, are mostly common among people who are not physically active (Silverman & Deuster, 2014). Therefore, physical activities and diets play a crucial role in an individual’s health status. All of these risk factors occurring together, increase the risk of occurrence of metabolic disorders (Alberti et al., 2009). Throughout the world diabetes, obesity and overweight raised a great fear to the prevention of chronic non-communicable diseases. In some developing countries, non-communicable diseases present a double burden to the already existing problems of starvation and malnutrition (WHO, 2004).
Diet is an important integral part of human health and livelihood, and it is crucial to have elementary information about food types and their nutritional value in the human body. For instance, fructose intake is linked to the prevalence of metabolic dysfunctions (Ludwig et al., 2001; Schulze et al., 2004). The recent increase in the consumption of fructose, is primarily due to consumption of diets containing sucrose (50% fructose and 50% glucose) and high fructose corn syrup, which has a higher fructose content (typically 55% fructose) (Mazibuko et al., 2013). High-fructose corn syrup (HFCS) represents approximately 40% of all added sweeteners used by the manufacturers of soft drinks and juices, probably because it is relatively cost effective (Basciano et
al., 2005). South Africa as a country, has also experienced high fructose intake, with squash
concentrates, jams, cookies, carbonated soft drinks, sweets and breakfast cereals ranking as the most common sucrose-containing foods (Steyn & Temple, 2012). The breakdown of carbohydrates and lipids result in the production of energy which occurs in the mitochondria, carried out by
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different genes (Joseph, 2014). Mitochondria are the intracellular organelles that produce energy for cellular processes, through oxidative phosphorylation (Khitan & Kim, 2013). Exercise, cold exposure, caloric restriction and oxidative stress are commonly known environmental stresses and responsible for triggering mitochondrial biogenesis. The commonly known environmental stresses are responsible for cell division and differentiation (Jornayvaz & Shulman, 2010b). From a physiological perspective, the extent of fat deposition in animals depends on the relative intensities of triacylglycerol synthesis and storage of lipid and fatty acid oxidation (Wang et al., 2010).
The medicinal plants were used in the past to curb ailments before the introduction of modern medicines. However, in the recent past two decades, more focus has been projected towards the medicinal plants role in curbing life-style ailments. Therefore, the rich potential of plant metabolites to combat obesity and its complications has been under the spotlight and needs to be fully explored (de Melo et al., 2010). In developing countries over 80% of the population rely on traditional medicine for their primary daily health care needs, which are predominantly from plants (Gurib-Fakim, 2006). Plants that are rich in triterpenoid, are notably considered because they have shown antioxidant, antiinflammatory, analgesic, antipyretic, hepatoprotective, cardiotonic, sedative and tonic effectsand anti-glycosylation effects and anti-glycosylation effects (Wang et al., 2011). Oleanolic acid (OA) is the compound of interest in this study. Oleanolic acid exits widely in plants in the form of free acid for triterpenoid saponins (Liu, 1995). The intake of OA helps with improving metabolism, thus it has proven to be a powerful antioxidant by activating antioxidant defences (Xi et al., 2008). Anti-inflammatory, anti-hyperlipidaemia and hypoglycaemic effects are some of the pharmacological and biochemical properties shown by oleanolic acid (Liu, 1995), and an antioxidant effect works with the mechanisms of its hypoglycemic and hypolipidemic effects (Wang et al., 2011). Oleanolic acid has been reported to inhibit alpha-glucosidase activity, which controls the absorption of glucose in the intestines, and the protein tyrosine phosphatase 1B activity (Lu et al., 2010). This process is vital feature in the negative regulation of insulin pathway and a hopeful way of reducing diabetes and obesity.
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1.3 Problem statement
Metabolic syndrome (MetS) is a worldwide health epidemic that is rapidly increasing in recent times. It is a precursor of many metabolic diseases that are of a great concern. About 80% of 200 million people, are likely to die from metabolic and heart related sickness. People with metabolic disorders have five-fold more chances of developing type 2 diabetes, cardiovascular and metabolic diseases (Roberts et al., 2013). These diseases are classified as non-communicable diseases because they cannot be transmitted from one person to the other but they are perpetuated by factors such as life style, diet and genetic inheritance. These metabolic conditions are increasing at an alarming rate, in South Africa. In 2010, NCDs accounted for 39% of total deaths in the country and more than a third (36%) of these deaths occurred before the age of 60 years (SAMJ, 2014). These diseases are in part due to high consumption of high fat, fructose or carbohydrates containing food, which alters metabolic pathways in the cells. The contribution of mitochondrial dysfunction in insulin metabolic signalling is shown by gene studies, as causing a reduction in expression of genes regulating mitochondrial adenosine triphosphate (ATP) production (Patti et al., 2003). This dysfunction is associated with insulin resistance and type 2 diabetes mellitus (Mootha et al., 2003). Currently a number of anti-diabetic drugs such as metformin and thiazolidinediones (TZD) are used to manage diabetes. Metformin works by reducing amount of sugar released by the liver and improving how the body responds ti insulin (Feig & Moses, 2011). Metformin has low adherence rate and has less side effects when compared with other anti-diabetic drugs. Thiazolidinediones (TZDs) are used by people with tyepe 2 diabetes who need increased glucose-lowering beyond what metformin and other drugs can provide (Quinn et al., 2008). The usage of TZDs has dropped significantly in the past few years due to safety. The side effects of TZD include adverse cardiovascular events such as heart attack, congestive heart failure and stroke (Quinn et al., 2008). In view of rapid increase of metabolic diseases, there is need for researchers to come up with drugs, which are more effective, affordable and with less or no side effects. Alternative modalities are urgently needed to reduce the impact of the adverse health effects of metabolic syndrome. The use of phytochemical has attracted keen interest in the management of life-style associated diseases. Metabolic genes associated with lipid oxidation seem to be downregulated when there is a consumption of high fructose but the opposite happens when they are treated with oleanolic acid (Castellano et al., 2013). Therefore, this study focused on the use of oleanolic acid to prevent the development of high fructose-induced metabolic syndrome in neonatal rats.
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1.4 Aim of the research
The aim of the study was to investigate the influence of oleanolic acid on lipid metabolism and mitochondrial biogenesis in fructose-induced neonatal metabolic derangements in Sprague Dawley rats. This research study was focused on closing the existing gap of anti-diabetic drugs, which are not readily available due to being expensive and having side effects. Countries from the developing economies will be able afford to use oleanolic acid to treat metabolic syndrome. Therefore, the use of natural products as alternative treatment for human ailments is cheaper and readily available with less side effects. Phytochemicals are advocated for prevention of metabolic dysfunctions.
1.5 Specific Objectives
The specific objectives were to:
1. Measure the levels of saturated, monounsaturated and polyunsaturated fatty acids in the skeletal muscle and liver using Gas Chromatography Mass Spectrometry.
2. Evaluate the expression of genes in the skeletal muscle and liver responsible for lipid oxidation such as CPT-1, MEF-2A, NRF-1 and GLUT 4 using real time quantitative Polymerase Chain Reaction.
3. Study the expression of genes in the skeletal muscle and liver responsible for lipid synthesis such as FAS, ACC-1 and SREBP-1 using real time quantitative Polymerase Chain Reaction.
4. Assess the expression of genes in the skeletal muscle and liver responsible for mitochondrial biogenesis such as TIM, TOM, PGC-1, TFAM and COX-2 using real time quantitative Polymerase Chain Reaction.
5. Investigate the expression of genes in the skeletal muscle and liver responsible for inflammation such as TNFα and IL-6 using real time quantitative Polymerase Chain Reaction.
6. Evaluate the expression of genes related to insulin resistance such as PPARγ and IRS-1 in the skeletal muscle and liver using real time quantitative Polymerase Chain Reaction. 7. Assess the level of binding NRF-1 to MEF-2A cis- element in the skeletal muscle and
liver using Chromatin Immunoprecipitation assay (ChIP assay).
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1.6 Significance of the study
The recent years have witnessed the occurrence of modern health scourges, particularly the non-communicable diseases, with life threatening consequences to the health of humans worldwide. The metabolic disorders are as a consequential effect of consumption of high fructose containing foods, which alters metabolic pathways. For instance, high carbohydrates or lipids alter mitochondrial dysfunctions which, have deadly effects in the cellular functions (Jafri & Kumar, 2014). Also metabolic genes responsible for lipid oxidation seems to be downregulated when there is a consumption of high fructose but when there is treatment with oleanolic acid, it reverses the effects of fructose diet. Medicinal plants have been used since ancient times to treat various ailments, this was done with little scientific analysis or phytochemical understanding. Some plants used in the folk medicine have been found to contain some compounds able to stimulate the insulin-signalling pathways, which is important in metabolic related illnesses. The phytochemicals are biologically active compounds from plants that are known to be pharmacologically effective with fewer side effects unlike the convectional drugs. One of these plant phytochemicals is oleanolic acid. This study was initiated to provide evidence that oleanolic acid, indeed, can prevent the development of metabolic disorders. Previous studies on the beneficial pharmacological effects of OA on MetS have been done in adult rats and none have been done in neonatal rats, especially in the early post-natal period, which is considered as the critical phase of development during which epigenetic changes are likely to cause metabolic changes that exert lifelong effects into adulthood. Moreover, despite the widespread beneficial properties of OA, there seems to be limited knowledge on whether its administration in the neonatal phase could protect against development of metabolic syndrome. The findings can lead to further investigative studies in the clinical discipline that will, in turn lead to the development of health care products that prevent metabolic disorders and its related conditions at early stages of life.
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CHAPTER 2 LITERATURE REVIEW
2.1. Metabolic syndrome: a global health problem
Metabolic syndrome is viewed as one of the ever-increasing health problems worldwide. This increase is associated with the global epidemic of overweight, obesity, diabetes and insuli rsistance. With the elevated risks not only centred on metabolic diseases but also cardiovascular diseases, there is an urgent need to implement strategies that prevent this emerging global epidemic (Schwalm et al., 2016). The next sections will focus at the definition of metabolic syndrome, (MetS) as a global health problem and its prevalence.
2.1.1. Definition of metabolic syndrome
The concept of the metabolic syndrome has now been existing for many years. However, recently some similar definitions of the syndrome have been agreed upon, that has made it possible to compare its incidence among populations worldwide (Roberts et al., 2013). Consequently, when bringing the definition of MetS, there was need to consider key aspects such as genetic context, diet, intensity of physical activity, population age and sex structure, levels of over- and under-nutrition, and body shape (Cameron et al., 2004). The measurement technique and definition of metabolic syndrome used is particularly argumentative, and therefore alternative criteria for different populations need to be established (Zimmet et al., 2005).
Researchers and clinicians have proposed an inclusive definition for MetS and, in 1998, the World Health Organization coined the term “metabolic syndrome”, thus providing the first precise definition of the metabolic disorder which was revised in 1999 (Alberti & Zimmet, 1998). This definition provided by WHO defined MetS as “the metabolic condition accompanied by dyslipidaemia, high blood pressure, obesity or microalbuminuria” (Alberti & Zimmet, 1998). Different health professional bodies have made submissions in an attempt to give MetS definition suitable for their fields. The most common definitions of MetS after WHO include proposals made by: the European Group for the Study of Insulin Resistance (EGIR) (Balkau & Charles, 1999; Einhorn et al., 2003), the International Diabetes Federation (IDF) (Alberti et al., 2005) and the American Heart Association (AHA) (Grundy et al., 2004). There was a joint statement regarding the clinical definition of MetS released by the World Health Federation, International Atherosclerosis Society, the IDF task force on Epidemiology and Prevention, the National Heart,
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Lung, and Blood Institute and the International Association for the Study of Obesity (O'Neill & O'Driscoll, 2015). The following aspects were agreed upon as areas of commonality; the occurrence of three out of the following five risk factors: increased waist circumference, high blood pressure, high triglyceride levels, low high-density lipoprotein-cholesterol (HDLC) levels, high low-density lipoprotein (LDL) levels and persistent hyperglycaemia (O'Neill & O'Driscoll, 2015). The definition and classification criteria of MetS differ according to the research area from each of the proposed definitions, central obesity, dyslipidaemia, elevated blood pressure, glucose intolerance, IR and other biochemical markers were featuring. As a result of the vigorous consultations and engagements, metabolic syndrome (MetS) can be defined as a combination of metabolic related diseases which are characterized by increase in the risk to develop central obesity, insulin resistance (IR), type 2 diabetes mellitus (T2DM), dyslipidaemia, cardiovascular diseases (Spalding et al., 2013; Asrih & Jornayvaz, 2015; O'Neill & O'Driscoll, 2015). The following section addresses metabolic syndrome as worldwide health problem.
2.1.2 A global health problem
Metabolic syndrome is one of the fastest growing public health problems in both developed and developing countries (Kaur, 2014; Lao et al., 2014). This increase is associated with the global epidemic of obesity and diabetes. According to the World Health Organisation (WHO, 2015), developing countries will likely suffer from non-communicable epidemics in the 21st century. In developing countries, around 50% of people with metabolic ailments related are undiagnosed (WHO & Mathers, 2017). As a result, many do not receive adequate treatment and care to manage the disease, putting them at greater risk of serious complications and eventually death. The World Health Organization (WHO) has indicated that the world’s population living with obesity has doubled to 13% since the 1980s, affecting more than 600 million adults and 42 million children under the age of 5 years in 2014 (WHO, 2015). This indicates that obesity has become a global epidemic that is growing at an alarming rate. People with the syndrome are twice as likely to die from a macrovascular event and three times as likely to have ischemic heart disease and stroke compared with people without the syndrome (Oladejo, 2011). As a risk factor to metabolic syndrome, obesity results in high mortalities both in children and adults, and continues to reduce life expectancies of individuals across the globe (WHO, 2015). Another risk factor for metabolic syndrome is type 2 diabetes. According to International Diabetes Federation (IDF, 2017), the number of people with metabolic dysfunctions such as type 2 diabetes, is increasing in every country. In 2013, diabetes caused 5.1 million deaths and it affected at least 382 million people worldwide (Federation, 2017b). The number of people with diabetes is expected to reach 592
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million by the year 2035, with 80% of all diabetes cases occurring in low- to middle-income countries (Guariguata et al., 2014). It is estimated that one adult in every ten will have diabetes by 2035, equating to approximately three new cases every ten seconds or almost 10 million cases per year. It is reported that every six seconds, a person dies from diabetes.
The metabolic syndrome is intertwined with high chances of developing non-communicable diseases, and it is widely agreed that it is a growing and a pressing problem for society (Tonkin, 2004). The United Nations (2011), made a declaration for the first time in the history of humanity, that non-communicable diseases had overtaken infectious diseases as the main threat to human health globally (Rebollo et al., 2012). This meant that more resources such as money, skilled workers and research equipment, are directed towards combating non-communicable diseases. With these developments, National Cholesterol Education Panel (NCEP) has formally described and identified a number of risk factors associated with cardiovascular and metabolic disorders (Grundy et al., 2004). These factors included abdominal obesity, elevated triglycerides (TG) levels, low high density lipoprotein (HDL)-cholesterol levels, increased blood pressure, and higher fasting glucose (Edwards et al., 2001). Metabolic dysfunctions occur as a result of assimilation of these risk factors. It has been agreed upon that insulin resistance and obesity are actually part of one common pathologic mechanism of metabolic syndrome (Avramoglu et al., 2003; Rosmond, 2005). Evidence based from previous researches showed that metabolic syndrome process begins early in life programming and persist from childhood to adolescent/adult life (Bao et al., 1996; Freedman et
al., 2001). This results in life-style diseases such as type 2 diabetes, cardiovascular and metabolic
diseases. Better management of the metabolic syndrome should reflect in the awareness and reduction on the burden of the disease in global population which will be discussed in details in the next section.
2.1.3 The prevalence of metabolic syndrome
The underlying factors such as genetic and environmental contributed immensely to higher prevalence of metabolic conditions like heart related diseases, obesity, hypertension, hyperglycaemia, and dyslipidemia besides (Roberts et al., 2013). Metabolic syndrome affects the general population in great numbers, and brings along other metabolic related ailments. The main goal of clinical management in individuals who have difficulties associated with metabolic disorders is to reduce the risk for cardiovascular related symptoms since it would exacerbate the condition (Kaur, 2014).
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The International Diabetes Federation (IDF) postulated that about 25% of the world's population has been plague-ridden by MetS (O'Neill & O'Driscoll, 2015). These rough estimations widely depends on age, ethnicity, and gender of the population studied (Kaur, 2014). This sudden increase in the global prevalence of metabolic disorders and has added burden to the ailing public health sector in developing countries (Spalding et al., 2013). About 415 million people are currently suffering from T2DM worldwide, and IDF reported that this number is projected to grow over 642 millions by 2040 (Rahelic et al., 2017). More than a quarter of the world’s adult population is estimated to have MetS, the prevalence keeps on rising concurrently with obesity (Kaur, 2014; Asrih & Jornayvaz, 2015). The prevalence of metabolic syndrome has become frequent in Africans too, contrary to a popular belief that its trend is scarce (Fezeu et al., 2007). The prevalence of metabolic syndrome in African populations has initially been documented to be ranging from as low as 0% to as high as about 50% and it can even reach higher figures depending on the population setting (Okafor, 2012). In a survey study conducted among South African undergraduate students, it was observed that metabolic risk factors were evident in much younger population (60%) affecting older population (Smith et al., 2009). Since metabolic syndrome is spreading at an alrming rate across the globe, it is imperative to look at underlying mechanisms that are responsible for its development.
The development of MetS has been attributed to the complex interaction of several factors that include genetic factors, physical activities and diet (James et al., 2004; Vickers, 2011). Epidemiological studies have established that there is a recent increase in the prevalence of MetS across all ages including adolescents and young adults (Cameron et al., 2004; Dehghan et al., 2005). The genetic studies postulate that development of metabolic disorders in families or related individuals emanate from the fact that the metabolic risk factors may be genetically transmitted from parents to offspring (Groop, 2000; Bavaresco, 2003). This notion is supported by epigenetic studies, which show that changes occuring in the gene end up affecting its activity and expression (Yan et al., 2004; Youngson & Morris, 2013). Thrifty gene hypothesis, states that genetic selection favours energy-saving genotypes in an environment where there is adverse shortage of food supply (McCance et al., 1994). If such genotypes are exposed to an abundant food supply later on in adulthood, individuals will be susceptible to MetS (Neel, 1999). This proposes that poor or excessive exposure to nutrition early or post-natal can result in weight increase during adulthood leading to the development of MetS (Groop, 2000). Overregulation of certain genes such as FAS, ACC-1 and SREBP-1 have been known to be associated with development of MetS.
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2.1.3.1 Influence of gender and age on development metabolic syndrome
Age and gender are some of the variables, which have influence in the develoment of metabolic syndrome. Age has a crucial role in the development of MetS as the prevalence of MetS appears to be increasing with age and it is more frequent in males than in females (Ervin, 2009). A large epidemiological study in the United States of America found that the prevalence of MetS and its associated complications was lower in Afro-American men than their white counterparts and lower in white women than in Afro-American women (Ford et al., 2002). These studies show that there are many variables to be considered for fuller understanding of prevalence of metabolic syndrome.
2.1.3.2 Influence of physical activities on development metabolic syndrome
Physical activity is defined as a deliberate, organized and structured increase of energy usage by doing some activities which causes the body to move repetitively in order to be physically fit (Caspersen et al., 1985). Physical activity and diet have positive effect on weight control and are viewed as the cornerstones in the prevention and treatment of MetS (Lakka & Laaksonen, 2007). These activities include; sporting activities like walking, cycling, dancing, swimming; running house chores, and many others. Epidemiological studies have shown that an increase in physical activity and exercise promotes good health parameters such as improved BMI, lower blood pressure, triglyceride levels and HDLC (Babyak et al., 2000; Balducci et al., 2010). Intervention mechanisms like exercise increases insulin-stimulated glucose disposal and GLUT-4 protein content in obese patients with type 2 diabetes (Mukwevho & Joseph, 2014). In a study conducted by Shepherd et al. (1999), it showed that 45-60 min of cycling at 60-70% VO2max caused plasma membrane GLUT-4 protein increases in the vastus lateralis muscles of diabetic patients and non-diabetic subjects. It is also reported that exercise increases plasma membrane GLUT-4 content and glucose transport in insulin resistant obese zucker rats (Hansen et al., 1998). All these studies show that exercise effectively translocates GLUT-4 to the cell surface and increases glucose transport in insulin resistant and diabetic animals (Henry, 2003; Leguisamo et al., 2012). Research has confirmed that sedentary lifestyle choices, which entails decrease or no physical activities have been implicated as the major contributing factor to the rise in the prevalence MetS (Hu et al., 2004; Armitage et al., 2005).
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2.1.3.4 Influence of diet on development metabolic syndrome
Western lifestyles have infiltrated the developing countries, resulting in people gradually adopting sedentary lifestyles (Adediran et al., 2012). These sedentary lifestyles are characterised by physical inactivity. For instance, at work environment where one gets into an office and there is restricted movement for most part of the day. According to Xu (2010), an increase in physical activity and fitness were shown to protect against the development of MetS through their positive effects on lipid metabolism. The American College of Sports Medicine recommends 120-150 minutes of moderate exercise or physical activity per week in order to prevent excessive weight gain (Medicine, 2009). Exercise coupled with dietary interventions, can yield good results in reducing weight compared to dietary interventions only (Jakicic et al., 2008). Therefore, the combination of the use of exercise and the right choice of food has been advocated for in order to minimise the risk of developing of MetS. The high prevalence of MetS and obesity has been attributed to the excessive consumption of foods high in fats and fructose (Xu et al., 2010). A number of studies have been conducted that deal with the link between dietary choices and the risk of developing MetS (Khitan & Kim, 2013; Fontana & Partridge, 2015). They have shown that consumption of high fructose diets indeed is implicated in the development of MetS and obesity (Hu et al., 2004). It has been observed that dietary intake of high calorific processed foods has been associated with the Western life-style, which perpetuate MetS (Drewnowski & Specter, 2004). Western type of diet is characterised by fast foods with excessive refined sugars and grains, processed foodstuffs, fried foods and red meats.
2.3 Metabolic programming
Metabolic programming is a critical period of development with lifelong health implications, which may occur in utero or postnatal (Srinivasan et al., 2013). The developmental origins of health and disease (DoHAD) hypothesized that the environmental factors and conditions in critical stages of development in early life (pre-conceptual, prenatal, gestation and the early post-natal period) can lead to “programmed” permanent alterations in the state of health or disease later in adult life (Ramirez-Espinosa et al., 2011). During this period of development, the tissues and organs are being are created. It has been revealed that susceptibility to develop MetS and its related conditions is strongly affected by exposure to early life developmental environment during pregnancy or afterbirth life (Saben et al., 2016). In most mammalian species, organ development is not complete at birth, hence it continues in the immediate postnatal period (suckling period) (Kaung, 1994). For
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example, studies in rats show that the maturation of pancreatic islets and development of neuronal systems in the hypothalamus continue in the suckling period of the rats (Srinivasan et al., 2008).
Overnutrition or insufficient nutrition during suckling period results in permanent changes to certain structural and physiological metabolic functions of the fetus (Fernandez-Twinn & Ozanne, 2010). Studies in both humans and animals have also shown that the events during gestation and early post-natal stages may have long-term consequences for health (Patel & Srinivasan, 2011; Manridue et al., 2013). Although the precise mechanisms that support metabolic programming effects due to altered nutritional experiences in the immediate postnatal period may be not well understood, but altered hormonal levels have side effects (Chowdhury et al., 2016). Hormones such as insulin and leptin stimulate each other during early periods of life and altered levels of these hormones play role a in the development of MetS (Patel & Srinivasan, 2011).
2.3.1 In utero metabolic programing
The utero phase is when the offspring is still developing in the uterus of the mother or before the actual birth. Barker (2001), in his study, stated that changes in the uterine environment that the individual is exposed to, may result in development of metabolic disorders in adulthood. All these disorders may be originating from foetal development stage (Neitzke et al., 2011). The homeostatic ability of the foetus enables it to adapt its adverse environmental conditions in utero with permanent readjustments inorder to maximise its survival chances (Heindel et al., 2015). These conditions can either be translated through over-nutrition or under-nutrition, which will ultimately alter the genetic make-up of the offsprings (Barker et al., 1990). These adaptations could bring negative consenquential effects since they may be disadvantageous to postnatal life and may pose risks of having chronic non-communicable disease in adulthood (Gluckman et al., 2007). The fetal adaptations can be beneficial if the nutritional conditions are maintained simillar to those which it has been exposed to in utero. However, if the conditions during the early post-natal period are ideal and providing plenty nutrition, the mismatched offspring phenotype may not be able to deal with the altered environment resulting in the development of a wide range of metabolic diseases as shown in figure 2.1 (Pittenger et al., 2005).
The developmental origins of health and disease (DOHaD) hypothesis argues that the concept of poor nutrition status during pregnancy, is linked to the disease process in human population (Pearce
et al., 2013). A study carried out on rodents showed that nursing mothers fed with high fructose
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prone to developing obesity in adult life (Sedaghat et al., 2015). The changes in fetal physiology may be beneficial for short-term survival in utero, but they may be maladaptive in post natal life, contributing to metabolic disorder when offsprings are exposed to catch-up growth, diet-induced obesity and other metabolic factors as indicated in figure 2.1 (Hanson & Gluckman, 2015). In a study conducted in rats and mice treated with a non protein diet during pregnancy, the rodents produced offsprings that had low-birth weight and developed metabolic derangements with age (de Oliveira et al., 2016).
Figure 2. 3: A flow diagram showing the stage of early in utero fetal programming to adulthood
metabolic disorder. Adapted and modified from (Jones & Ozanne, 2009).
2.3.2 Neonatal metabolic programming
Neonatal phase is a stage when an offspring has been newly born or the first few weeks after birth. Providing offspring with a high carbohydrate or fructose formula milk diet immediately after birth can result in the development of metabolic diseases in adult life, which can be transmitted to the next generation even after females received normal diet after weaning (Srinivasan et al., 2013). This evidence demonstrates the significance of the postnatal life in pre-exposing offspring to MetS and its related metabolic disorders. The rats immediately after birth depend exclusively on the mother’s milk during early postnatal (neonatal) development, which is similar to last trimester of pregnancy in humans (Schneider et al., 2011). The neonatal rat model is thus well suited for investigating the effects of early life dietary manipulations on the programming of adverse
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metabolic outcomes in early and adulthood life. A study using neonatal rat models has shown that dietary manipulations during suckling can cause temporary or permanent alterations to physiological functions and increased susceptibility to diseases (Huynh et al., 2008). The neonatal rats are suitable target for investigating pharmacological and medicinal substances intervention against the diet-induced metabolic programming (Yamada & Chong, 2017). Therefore, a nutritional alterations occurring during the suckling period alone can be used as a sign for live-long health consequences. The ability of offspring during early periods to respond to monitored nutritional environment in order to enable them short-term survival is known as developmental plasticity (Patel & Srinivasan, 2011). In the end, such responses are detrimental because they condition the organism for adult-onset metabolic disorders as shown in figure 2.2. For example, hyperinsulinemia in the immediate postnatal period in rats has been associated with adult-onset obesity (Dorner & Plagemann, 1994). The direct (neonatal) exposure to high-fructose diets in early developmental phases modified the expression of genes and receptors that are involved in glucose and lipid metabolism (Manridue et al., 2013; Lembede et al., 2018).
Figure 2. 4: A flow diagram showing the stages of early fetal programming to adulthood metabolic
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2.4 Metabolism related diseases
Metabolic disease refers to any ailment or disorder that disrupt normal metabolism. The process of metabolism is responsible for oxidising food to energy for cellular functions. Metabolic diseases temper with the ability of the cell to partake in important biochemical reactions that involves the processing or transportation of food molecules such as proteins, fructose, starches or lipids (Gropper & Smith, 2012). The next session focused on: type 2 diabetes, cardiovascular diseases, non alcoholic liver diseases and obesity.
2.4.1 Type 2 Mellitus Diabetes (T2DM)
Type 2 diabetes mellitus is a chronic disease that is characterized by chronic hyperglycaemia with disturbances of carbohydrate, lipids, and protein metabolism resulting from defects in insulin secretion, insulin action, or both (WHO, 2000). Both type 2 diabetes and metabolic syndrome are non-communicable diseases that are on the rise in our recent times (WHO & Mathers, 2017). Early in the century, the prevalence rate of diabetes mellitus in Africa was 0-1% but today available data showed that this is no longer the position (Jung & Choi, 2014). Diabetes is usually diagnosed by symptoms such as levels of plasma glucose ≥200 mg/dl (11.1 mmol/l) and a fasting plasma glucose ≥126 mg/dl (7.0 mmol/l) (Gerstein et al., 2001). This means that individuals diagnosed with these symptoms are declared diabetic (Mellitus et al., 2010; Bailey et al., 2016). The hallmark of all forms of diabetes mellitus is abnormal insulin physiology, persistent hyperglycaemia, and relative lack of insulin (Farris et al., 2003; Ahmad, 2013). There are two main types of diabetes, being type 1 and type 2 diabetes mellitus. The type 1 diabetes mellitus is also referred to as insulin-dependent diabetes mellitus and is due to the inability of the pancreas to secrete insulin (Skyler et al., 2007). On the other hand, type 2 diabetes (T2DM), also called non-insulin dependent diabetes or adult-onset diabetes, is the most common form of diabetes accounting for approximately 90% of cases worldwide (Prentki & Nolan, 2006).
Type 2 diabetes mellitus (T2DM) is metabolic disease, which is regarded as a major cause of deaths. WHO (2016), estimatd that 422 million people are afflicted with T2DM and these figures are expected to reach 592 million by the year 2035. In the past, T2DM was regarded as a disease for developed countries, but current research showed that there is alarming spread of T2DM up to 90%–95% diabetes cases in developing countries (Association, 2013a). This new trend has been attributed to urbanization of the society (people moving from rural areas to towns looking for better life) and also changing lifestyles in general (Gwatkin et al., 1999). There is tremendous number of
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people afflicted by T2DM in sub-Saharan Africa, the recorded number was at 19.8 million in 2013 and the figure was projected to rise to 41.5 million by 2016 (International Diabetes Federation, 2006; Erasmus et al., 2012; Agyemang et al., 2016). The statistics showed that in South Africa, about 2.5 million people are diabetic and half of the number goes undiagnosed and the international diabetes foundation estimates that 5 million South Africans have pre-diabetes conditions (Federation, 2017a). In South Africa, the highest prevalence of diabetes is among the Indian population (11-13%) and followed by the Coloured community with 8-10%, while among the Black population it is estimated at 5-8% and amongst Whites 4% (Erasmus et al., 2012).
2.4.1.1 Role of Glucose transporter 4 (GLUT-4) in development of diabetes
Glucose transporter-4 (GLUT-4) is responsible for transporting extracellular glucose into insulin-sensitive cells (Chang et al., 2004). It is the predominant insulin-responsive glucose transporter isoform. GLUT-4 is predominatly found in the adipose tissue and the skeletal muscles (Ploug & Ralston, 2002). At cellular level the regulation of glucose depends on GLUT4-mediated glucose uptake, facilitative diffusion glucose transporter and is the major insulin-regulated glucose transporter in skeletal muscle, heart, and adipocytes (Castoldi et al., 2016; Moraes-Vieira et al., 2016). Maier & Gould (2000), reported that the expression of GLUT-4 in the skeletal muscles of type 2 diabetes patients is significantly reduced, indicating that such patients have less capability to process glucose. Type 2 diabetes in skeletal muscle results in insulin resistance, which impairs insulin-stimulated glucose disposal rate (Huang & Czech, 2007). The core underlying mechanism for type 2 diabetes is glucose transport system (Maier & Gould, 2000). Metabolic syndrome down regulates GLUT-4 in adipocytes of both humans and rodents, and this down regulation is one of the earliest events in the pathogenesis of insulin resistance, obesity, type 2 diabetes and other metabolic disorders (Garvey et al., 1998). GLUT-4 increases glucose transport after insulin activation and contraction in the skeletal muscle (Lauritzen et al., 2013). Facilitated transport using glucose transporter proteins feed cells with glucose through the plasma membrane.
Intervention mechanisms like exercise increases insulin-stimulated glucose disposal and GLUT4 protein content in obese patients with type 2 diabetes (Mukwevho & Joseph, 2014). In a study conducted by Shepherd et al. (1999), it showed that 45-60 min of cycling at 60-70% VO2max caused plasma membrane GLUT-4 protein increases in the vastus lateralis muscles of diabetic patients and non-diabetic subjects. It is also reported that exercise increases plasma membrane GLUT-4 content and glucose transport in insulin resistant obese zucker rats (Hansen et al., 1998).
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All these studies show that exercise effectively translocates GLUT-4 to the cell surface and increases glucose transport in insulin resistant and diabetic animals (Henry, 2003; Leguisamo et al., 2012)
2.4.1.2 Role of myocyte enhancer factor 2 (MEF-2A) in development of diabetes
Myocyte enhancer factor 2 (MEF-2) are proteins that function as the main transcriptional regulators of gene expression of genes related to skeletal muscle development, as well as fibre type control, and glucose uptake metabolism (Anderson et al., 2015). Myocyte-specific enhancer factor 2A is protein found in humans and it is encoded by the MEF-2A gene. The MEF-2A is a transcription factor in the MEF-2 family and in humans it is located on chromosome 15q26 (Chen, 2014). The MEF-2 transcription factors are associated with a number of diseases and they have a diversity of functions in a wide range of tissues. Changes made on the MEF-2 excerbate the development of metabolic and neurological disorders but more recently have been associated with cancer development and metabolic syndrome (Pon & Marra, 2016b). In addition, MEF-2A has been implicated in the genetic basis of metabolic disease. However, the genes that increase chances of developing of these diseases are not fully established. Prolonged activation of MEF-2A-dependent genes in myocytes may become maladaptive, contributing to pathological remodeling and accumulation of focal fibrosis in diabetes-induced cardiomyopathy (Chen et al., 2016). MEF-2A found in blood vessels during early mouse maturity promote skeletal muscle differentiation (Sturm, 2004).
Protein is the major functional and structural constituent found in all the cells of the body. All proteins are comprised of amino acid chains, which contain an amino nitrogen group. Amino acids function as precursors of many coenzymes, hormones, and nucleic acids. A diet with more protein (defined as 30% of calories) may or may not improve blood glucose level but appears to improve one or more cardiovascular risk factors (Beasley & Wylie-Rosett, 2013). While high protein intake does not necessarily lead to weight loss, in some cases, higher protein intake may influence diabetes risk through weight loss. For individuals with type 2 diabetes (T2DM), studies have demonstrated that moderate weight loss (5% of body weight) is associated with decreased insulin resistance, improved measures of glycaemia and lipemia, and reduced blood pressure (Association, 2013b). Higher protein intake may reduce risk of developing diabetes and improve metabolic control only when weight loss is achieved. When people with type 1 diabetes consume protein, there is an increase in postprandial pre-prandial blood glucose level (BGLs) and insulin requirements (Paterson
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et al., 2015). Two mechanisms have been proposed by which dietary protein may cause delayed
and sustained postprandial glycaemia excursions in people with type 1 diabetes: (1) the alteration of hormones, which affects glucose homeostasis, and (2) the conversion of amino acids to glucose by gluconeogenic pathways (Association, 2013b; Paterson et al., 2015). Meal protein content also influences a number of other hormones, such as cortisol, growth hormone, IGF-1 and ghrelin, but how these hormonal changes subsequently influence postprandial glucose levels is poorly understood (Nordt et al., 1991). High-protein meals produce increased cortisol concentrations, which in turn may increase insulin resistance and insulin requirements (Paterson et al., 2015).
MEF-2 transcription factor plays important functions in both physiological and pathological processes. Certain mutations in MEF-2A are responsible for causing some cardiovascular related diseases such as coronary artery disease, diabetes and myocardial infarction (Snyder et al., 2013). MEF-2A expression is upregulated in the diabetic myocardium. In animal study, MEF-2A inhibition improves cardiac function and remodeling in diabetic mice (Chen et al., 2016). Thus, prolonged activation of MEF-2A-dependent genes in myocytes may become maladaptive, contributing to pathological remodeling and accumulation of focal fibrosis in diabetes-induced cardiomyopathy (Chen et al., 2016). Cardiac MEF2A silencing may protect against cardiac fibrosis and improve myocardial function in diabetic mice (Chen et al., 2016). The proteins that bind to the MEF-2 target DNA sequence present in the regulatory regions of many genes. Researchers are busy with developing new medications that target MEF-2 family in order to treat MEF-2 associated human diseases, for instance, oleanolic acid is a new discovered phytochemical used for treating lung cancers by the help of inhibiting MEF-2A expression (Liu et al., 2014; Zhao et al., 2015).
2.4.2 Insulin resistance
Insulin resistance (IR) is a pathological condition in which cells fail to respond to insulin. Insulin resistance can be defined as the inability of insulin to stimulate glucose disposal, and when an insulin-resistant individual is unable to secrete sufficient insulin to overcome this defect, T2DM develops. Large amount of insulin circulating in the blood to maintain the normal blood glucose, result the liver and skeletal muscle receiving insufficient insulin (Gustafson et al., 2015). Several studies have shown that insulin resistance is a precusor of obesity, diabetes, cardiovascular and metabolic syndrome (Brüning et al., 2000; Furukawa et al., 2017b). The main function of insulin is stabilising energy balance in the body by acting on the brain using specific central receptors (Brüning et al., 2000). For instance, the brain responds more to foods that contain glucose, which is
