Chuturgoon, Anil Amichund.Nagiah, Savania.Naicker, Nikita.2020-03-312020-03-3120172017https://researchspace.ukzn.ac.za/handle/10413/17304Doctoral Degree. University of KwaZulu-Natal, Durban.Type two diabetes mellitus (T2D) is a significant cause of premature death and disability, accompanied with negative socio-economic impacts. This metabolic disorder is characterized by hyperglycaemia and defective insulin signalling. Long-term exposure to hyperglycaemia gives rise to altered fat metabolism and reactive oxygen species (ROS) generation. These precursors are central to the progression of dyslipidaemia and attenuated antioxidant (AO) response and detoxification system, respectively. Diabetic dyslipidaemia and oxidative stress (OS) are risk factors for the onset and progression of cardiovascular disease (CVD) and other diabetic complications. The treatment regimen for T2D comprises self-care and anti-diabetic drugs such as metformin. However, due to the lack of compliance to self-care recommendations and some undesirable side effects of metformin, there is the necessity for alternate therapy. Natural products have been used for the treatment of many disorders, including T2D. Trigonella foenum-graecum commonly known as fenugreek is a plant that possesses anti-diabetic effects. These effects are attributed to its bioactive compound – 4-hydroxyisoleucine (4-OH-lle), which constitutes approximately 80% of the bio-composition of the fenugreek seed. Despite these effects, biochemical and molecular effects of 4-OH-lle on insulin signalling, lipid metabolism, and ROS production is not well-documented. This study investigated the effects of 4-OH-lle in comparison to metformin and fenugreek seed extract (FSE) on hyperglycaemic human hepatoma (HepG2) cells and C57BL/6 male mice. Treatments were conducted under normoglycaemic and hyperglycaemic conditions as follows; control, 4-OH-lle (in vitro: 100ng/ml; in vivo: 100mg/kg Body weight) metformin (in vitro: 20mM; in vivo: 20mg/kg Body weight) and FSE (in vitro: 100ng/ml; in vivo: 100mg/kg Body weight) treatment groups. The experiments included; blood glucose measurements, lipid profile analysis, spectrophotometric assays (in vitro), western blotting for protein expression and qPCR for mRNA expression. First, to validate the effects on insulin signalling and glucose sensing, glucose levels were measured with completion of an oral glucose tolerance test. 4-OH-lle treatment attenuated glucose levels, and elevated the mRNA levels of glycogen synthase (GS) and glucokinase (Gck). This was followed by the investigation of the protein and gene expression of insulin signalling regulators: insulin receptor β (IRβ), insulin receptor substrate 1 (IRS1), phosphorylated protein kinase B (pAkt), phosphorylated glycogen synthase kinase 3α/β (pGSK3α/β) and glucose transport 2 (GLUT2). In in vivo hyperglycaemia, 4-OH-lle increased the expression of the investigated proteins and genes. The results showed that 4-OH-lle was just as potent as MF, and FSE in stimulating the insulin signalling cascade. Second, the effect of 4-OH-lle on dyslipidaemia was investigated by measuring mRNA levels of sterol regulatory binding element 1c (SREBP1c) and fatty acid synthase (FAS) – key factors in fatty acid metabolism. Both genes were up-regulated and correlated with the changes in triglyceride and cholesterol levels. Next the protein expression of proprotein convertase subtilisin-like/kexin type (PCSK9) - a regulator of low density lipoprotein cholesterol (LDLc) and peroxisome proliferator-activated receptor gamma (PPARG) – a regulator of high density lipoprotein (HDLc) was evaluated. The data showed that 4-OH-lle down-regulated protein and mRNA expression of PCSK9 and up-regulated protein expression of PPARG. The reduction in PCSK9 levels correlated with the changes observed in low density lipoprotein receptor (LDLr) and LDLc, whereas the increase in PPARG correlated with the elevated mRNA expression of apolipoprotein A1 (Apo A1) and HDLc. Together these results provide substantial evidence for the regulatory effect of 4-OH-lle, in comparison to metformin, and FSE on PCSK9, PPARG and related lipid factors. Finally, the effect of 4-OH-lle on redox status and AO response was assessed by measuring nuclear factor E2-related factor 2 (Nrf2). In both models, there was an increase in the protein expression of phosphorylated Nrf2 accompanied by an increase in mRNA levels of superoxide dismutase 2 (SOD2) and glutathione peroxidase (GPx), and GSH levels. Mitochondria play a central role in contributing to elevated ROS levels. While nuclear responses like Nrf2 regulate ROS, mitochondria possess their own maintenance proteins. These include mitochondrial Lon protease 1 (LonP1), Sirtuin 3 (SIRT3) and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) which play an integral role in combatting OS and mitochondrial dysfunction. The results showed that 4-OH-lle displayed a potent effect in inducing the AO response and increasing mitochondrial regulatory proteins. In conclusion, 4-OH-lle improved the compromised insulin signalling and the altered lipid profile as well as induced the AO response and mitochondrial maintenance proteins, in the presence of elevated glucose. Furthermore, the effect of 4-OH-lle was greater than the first-line drug; metformin and FSE, albeit in cultured human liver cells and a mouse model. Also, the crude seed extract displayed promising effects on all investigated parameters. Considering the active role of chronic hyperglycaemia in the onset and progression of CVD and diabetic complications, 4-OH-lle poses as a highly favourable alternate therapy in the treatment of T2D. Moreover, this has great importance in socio-economically challenged communities where T2D is a common disorder, access to healthcare facilities is limited, and plants serve as sources of easily accessible treatments.enTrigonella foenum-graecum seed extract.Type two diabetes mellitus.Glucose sensing.Fenugreek seed extract.4-Hydroxyisoleucine.Insulin signalling.Metformin.Thesis