The incidence of Type 2 Diabetes Mellittus (T2DM) is increasing world wide. In Africa the limited access to health care and the insidious course of the disease lead to more severe illness and diabetic complications. There is a need to find alternative approaches to treatment and prevention that address the problems and needs of Africa. Sutherlandia frutescens (S.frutescens) is a traditional herbal plant with known anti-diabetic properties, the precise mechanism of action of S.frutescens is not known. In order to develop new approaches for treatment and prevention of T2DM the pathophysiology of T2DM must be understood. T2DM is the final outcome of a multi-organ disease characterized by early defects in muscle, adipocytes, hepatocytes and pancreatic β-cells. In this study the role of the liver was investigated because of its central role in glucose and lipid metabolism. It is hard to differentiate between all the influences in an in vivo model, so the aim of this study was to develop an in vitro model of T2DM in Chang liver cells and to determine if S.frutescens can reverse the state of insulin resistance in this model. Different culture media conditions were screened to identify a method that can be used as the T2DM model in Chang liver cells. Serum free medium (MCBD-201) supplemented with human diabetic serum, (2.5%-10%), high insulin concentrations (0.1μM-1μM), high fructose concentrations (1-10mM). and a combination of high insulin and high fructose was used for this screening. Chang liver cells cultured in MCBD-201 medium supplemented with 1mM fructose and 0.1μM insulin showed reduced glucose uptake and increased lipid accumulation. The effect of two S.frutescens extracts, two anti-diabetic drugs, metformin and ciglitazone, and a hypolipidemic drug ciprofibrate were determined and shown to increase glucose uptake and reduce lipid accumulation. It was postulated that exposing the cells to excess nutrients in the form of high fructose would stimulate the cells to become adipogenic and accumulate lipids, which would interfere with the glucose uptake and induce insulin resistance. Gene expression of PPARγ, PPARα, and SREBP-1 transcription factors regulating lipid metabolism was determined in Chang liver cells cultured in insulin resistance inducing medium over a 48 hour time course. The expression of PPARγ, known to stimulate adipogenesis was increased after 6, 24 and 48 hours of exposure (P(H1)<0.0001). The expression of PPARα, known to stimulate β-oxidation expression, was significantly decreased after 24 hours of exposure (P(H1)<0.0001). The presence of the plant extracts in the insulin resistance inducing media protect against this increase in adipogenesis and decrease in β-oxidation after 48 hours of exposure by increasing PPARα expression and decreasing PPARγ expression. A PCR Array was performed which identified 32 more potential molecular targets of S.frutescens. Five of the 32 targets identified with the PCR Array were validated using qRT-PCR. These genes play a role in lipid and glucose metabolism and protection against oxidative stress and inflammation. In summary a cellular model of insulin resistace in hepatocytes has been established and the capacity of S.frutescens to reverse this process has been demonstrated by acting as a dual PPARγ/α agonist. New genes have been identified in the development of insulin resistance and as targets of S.frutescens.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:nmmu/vital:10339 |
| Date | January 2009 |
| Creators | Williams, Saralene Iona |
| Publisher | Nelson Mandela Metropolitan University, Faculty of Science |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis, Doctoral, PhD |
| Format | xx, 270 leaves, pdf |
| Rights | Nelson Mandela Metropolitan University |
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