Liver steatosis and insulin-resistance : reversal by Sutherlandia frutescens
- Authors: Clarke, Stephen
- Date: 2014
- Subjects: Insulin resistance , Diabetes -- Treatment
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10348 , http://hdl.handle.net/10948/d1020788
- Description: Type 2 diabetes mellitus (T2DM) is rapidly emerging as one of the greatest global health issues of the 21st century. Insulin-resistance is a condition associated with T2DM and in the cell it is defined as the inadequate strength of insulin signalling from the insulin receptor downstream to the final substrates of insulin action involved in multiple metabolic, gene expression, and mitogenic aspects of cellular function. To investigate the potential mechanisms involved in the development of insulin-resistance, two in vitro liver cell models were established using palmitate or a combination of insulin and fructose as inducers. The development of insulin-resistance was determined via the capacity of the hepatocytes to maintain normal glucose metabolism functionality by measuring hepatic gluconeogenesis and glycogenolysis. It was established that the treatments induced the development of insulinresistance after 24 hours chronic exposure. Previous studies have investigated the potential of Sutherlandia frutescens extracts as therapeutic agents for insulin-resistance. The aim of this study was thus to investigate the ability of a hot aqueous extract of S. frutescens to reverse the insulin-resistant state, via measuring gluconeogenesis and glycogenolysis, the associated changes in cellular physiology (lipid accumulation, oxidative stress, and acetyl- CoA levels), and changes in mRNA expression. The results showed that S. frutescens had a significant effect on reversing the insulin-resistant state in both models of insulin-resistance. Furthermore, S. frutescens was capable of reducing lipid accumulation in the form of triacylglycerol in the high insulin/fructose model, while this was unaffected in the palmitate model. However, S. frutescens did reduce the accumulation of diacylglycerol in the palmitate model. Oxidative stress, seen to be associated with the insulin-resistant state, was successfully treated using the extract, as indicated by a reduction in reactive oxygen species. However no change was seen in the nitric oxide levels, in either model. Interestingly, although S. frutescens had no effect on the level of acetyl-CoA in the insulin/fructose model, it was found to increase this in the palmitate model. It is suggested that this may be due to increased β-oxidation and metabolic activity induced by the extract. The analysis of mRNA expression gave some insight into possible mechanisms by which insulin-resistance develops, although the results were inconclusive due to high variability in samples and the possibility of the RNA being compromised. Future studies will address this issue. The results of this study reflect different proposed clinical causes of insulin-resistance through the responses seen in the two cell models. These indicate that liver steatosis and insulin-resistance are induced by high palmitate as well as high insulin and fructose levels, and reversed by S. frutescens. Therefore the potential of S. frutescens to be used as a therapeutic agent in the treatment of insulin-resistance is indicated by this study.
- Full Text:
- Date Issued: 2014
- Authors: Clarke, Stephen
- Date: 2014
- Subjects: Insulin resistance , Diabetes -- Treatment
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10348 , http://hdl.handle.net/10948/d1020788
- Description: Type 2 diabetes mellitus (T2DM) is rapidly emerging as one of the greatest global health issues of the 21st century. Insulin-resistance is a condition associated with T2DM and in the cell it is defined as the inadequate strength of insulin signalling from the insulin receptor downstream to the final substrates of insulin action involved in multiple metabolic, gene expression, and mitogenic aspects of cellular function. To investigate the potential mechanisms involved in the development of insulin-resistance, two in vitro liver cell models were established using palmitate or a combination of insulin and fructose as inducers. The development of insulin-resistance was determined via the capacity of the hepatocytes to maintain normal glucose metabolism functionality by measuring hepatic gluconeogenesis and glycogenolysis. It was established that the treatments induced the development of insulinresistance after 24 hours chronic exposure. Previous studies have investigated the potential of Sutherlandia frutescens extracts as therapeutic agents for insulin-resistance. The aim of this study was thus to investigate the ability of a hot aqueous extract of S. frutescens to reverse the insulin-resistant state, via measuring gluconeogenesis and glycogenolysis, the associated changes in cellular physiology (lipid accumulation, oxidative stress, and acetyl- CoA levels), and changes in mRNA expression. The results showed that S. frutescens had a significant effect on reversing the insulin-resistant state in both models of insulin-resistance. Furthermore, S. frutescens was capable of reducing lipid accumulation in the form of triacylglycerol in the high insulin/fructose model, while this was unaffected in the palmitate model. However, S. frutescens did reduce the accumulation of diacylglycerol in the palmitate model. Oxidative stress, seen to be associated with the insulin-resistant state, was successfully treated using the extract, as indicated by a reduction in reactive oxygen species. However no change was seen in the nitric oxide levels, in either model. Interestingly, although S. frutescens had no effect on the level of acetyl-CoA in the insulin/fructose model, it was found to increase this in the palmitate model. It is suggested that this may be due to increased β-oxidation and metabolic activity induced by the extract. The analysis of mRNA expression gave some insight into possible mechanisms by which insulin-resistance develops, although the results were inconclusive due to high variability in samples and the possibility of the RNA being compromised. Future studies will address this issue. The results of this study reflect different proposed clinical causes of insulin-resistance through the responses seen in the two cell models. These indicate that liver steatosis and insulin-resistance are induced by high palmitate as well as high insulin and fructose levels, and reversed by S. frutescens. Therefore the potential of S. frutescens to be used as a therapeutic agent in the treatment of insulin-resistance is indicated by this study.
- Full Text:
- Date Issued: 2014
The medicinal plant Sutherlandia Frutescens regulates gene expression to reverse insulin resistace in rats
- Authors: Fortuin, Melissa
- Date: 2013
- Subjects: Insulin resistance , Medicinal plants , Genetic regulation , Insulin resistance -- Animal models
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10349 , http://hdl.handle.net/10948/d1020823
- Description: Obesity can lead to Type 2 Diabetes, both conditions increase in association with physical inactivity and high-energy diets, resulting in elevated blood glucose, decreased insulin sensitivity and increased insulin resistance. Sutherlandia frutescens (S.frutescens), an anti-diabetic plant, reverses and prevents insulin resistance in a rat model and human cell culture model. Gene expression analysis in hepatocyte cultures, identified genes down regulated in insulin resistance and up regulated by S.frutescens. These included genes encoding vesicle transporter proteins, hypothesised to be linked to hepatic lipid accumulation and lipid droplet formation during insulin resistance. The aim of this study was to investigate critical genes involved in lipid droplet formation, vesicle assembly and transport in high fat diet (HFD)-induced insulin resistant rat liver tissue during the development of insulin resistance and the reversal of these changes by S.frutescens. Rats were fed a low fat diet (LFD) or HFD supplemented with S.frutescens for 2, 4 and 8 weeks. Rats fed a HFD for 12 weeks developed insulin resistance, confirmed by plasma glucose and insulin levels (compared to normal controls). Groups of these rats were gavaged with S. frutescens (50mg/kg BW), Metformin (13mg/kg BW) or water for a further 4 weeks and starved for 12 hours, anaesthetized and blood removed by heart puncture. Liver was stored in RNA-Later™ for qRT-PCR and snap-frozen in liquid nitrogen for western blotting and confocal microscopy analysis. Changes in expression of vesicle transporter genes VAMP3 and NSF were analysed by qRT-PCR and changes in the protein expression by western blotting analysis. Proteins were localised within the liver by confocal immunohistochemistry using ZEN lite™ software. Statistical analysis was performed using One-Way ANOVA and unpaired t-test. mRNA gene expression of vesicle transport components VAMP3, NSF and SNAP25 showed relatively moderate changes with considerable individual variation within control or experimental groups. Uncorrelated changes in mRNA and protein products were found and may be due to differential regulation by siRNA. Proteins also showed altered staining patterns in high fat diet rats that reverted towards normal on S. frutescens treatment, potentially reflecting functional changes associated with transport of lipid-filled vesicles.
- Full Text:
- Date Issued: 2013
- Authors: Fortuin, Melissa
- Date: 2013
- Subjects: Insulin resistance , Medicinal plants , Genetic regulation , Insulin resistance -- Animal models
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10349 , http://hdl.handle.net/10948/d1020823
- Description: Obesity can lead to Type 2 Diabetes, both conditions increase in association with physical inactivity and high-energy diets, resulting in elevated blood glucose, decreased insulin sensitivity and increased insulin resistance. Sutherlandia frutescens (S.frutescens), an anti-diabetic plant, reverses and prevents insulin resistance in a rat model and human cell culture model. Gene expression analysis in hepatocyte cultures, identified genes down regulated in insulin resistance and up regulated by S.frutescens. These included genes encoding vesicle transporter proteins, hypothesised to be linked to hepatic lipid accumulation and lipid droplet formation during insulin resistance. The aim of this study was to investigate critical genes involved in lipid droplet formation, vesicle assembly and transport in high fat diet (HFD)-induced insulin resistant rat liver tissue during the development of insulin resistance and the reversal of these changes by S.frutescens. Rats were fed a low fat diet (LFD) or HFD supplemented with S.frutescens for 2, 4 and 8 weeks. Rats fed a HFD for 12 weeks developed insulin resistance, confirmed by plasma glucose and insulin levels (compared to normal controls). Groups of these rats were gavaged with S. frutescens (50mg/kg BW), Metformin (13mg/kg BW) or water for a further 4 weeks and starved for 12 hours, anaesthetized and blood removed by heart puncture. Liver was stored in RNA-Later™ for qRT-PCR and snap-frozen in liquid nitrogen for western blotting and confocal microscopy analysis. Changes in expression of vesicle transporter genes VAMP3 and NSF were analysed by qRT-PCR and changes in the protein expression by western blotting analysis. Proteins were localised within the liver by confocal immunohistochemistry using ZEN lite™ software. Statistical analysis was performed using One-Way ANOVA and unpaired t-test. mRNA gene expression of vesicle transport components VAMP3, NSF and SNAP25 showed relatively moderate changes with considerable individual variation within control or experimental groups. Uncorrelated changes in mRNA and protein products were found and may be due to differential regulation by siRNA. Proteins also showed altered staining patterns in high fat diet rats that reverted towards normal on S. frutescens treatment, potentially reflecting functional changes associated with transport of lipid-filled vesicles.
- Full Text:
- Date Issued: 2013
In vitro effects of endogenous and exogenous cannabinoids on insulin resistance and secretion
- Authors: Gallant, Megan
- Date: 2009
- Subjects: Cannabinoids , Cannabis , Insulin resistance
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10324 , http://hdl.handle.net/10948/1076 , Cannabinoids , Cannabis , Insulin resistance
- Description: Type 2 diabetes mellitus results from a combination of insulin resistance and impaired insulin secretion. The aim of this study is to investigate the effect of endogenous and exogenous cannabinoids on insulin resistant cell lines, viz skeletal muscle (C2C12) and fat (3T3-L1), and to investigate the effects of these cannabinoids on insulin secretion in pancreatic β-cells (INS 1). Insulin resistance was induced in the cells using 20 ng/mL TNF-α (3T3-L1) and 100 nM insulin (C2C12). Insulin resistant cells were exposed to cannabinoids for 48 hours after which glucose uptake, RT-PCR and Western blot analysis was performed. Additionally, adipokine assays were performed on the 3T3-L1 cells. The insulin resistant 3T3-L1 and C2C12 cells had reduced glucose uptake, decreased IRS-1 and Glut-4 expression indicative of an insulin resistant state. The extract and THC significantly enhanced glucose uptake, IRS-1 and Glut-4 in 3T3-L1 and C2C12 cells. The extract and THC thus have the potential to be an insulin sensitizing agent. Interleukin-6 was significantly decreased by THC. INS 1 cells, cultured under normoglycemic conditions, were exposed to cannabinoids for 48 hours after which glucose-stimulated insulin secretion, radioimmunoassay, oxygen consumption, RT-PCR and Western blot analysis was performed. Insulin stimulatory index was not significantly affected after cannabinoid exposure, except by THC. The cannabinoids decreased insulin content, in a concentration dependent manner, but the inhibition mechanism remains elusive. The cannabinoid Treated cells showed insulin gene expression levels similar to the control, while only THC proved effective in significantly stimulating Glut-2 gene expression. Oxygen consumption studies showed levels lower than the control cells. Most of the cannabinoids inhibited insulin secretion under normoglycemia except THC, while the cannabinoids exhibited the potential to improve insulin resistant adipocyte and myocytes response to glucose and gene regulation.
- Full Text:
- Date Issued: 2009
- Authors: Gallant, Megan
- Date: 2009
- Subjects: Cannabinoids , Cannabis , Insulin resistance
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10324 , http://hdl.handle.net/10948/1076 , Cannabinoids , Cannabis , Insulin resistance
- Description: Type 2 diabetes mellitus results from a combination of insulin resistance and impaired insulin secretion. The aim of this study is to investigate the effect of endogenous and exogenous cannabinoids on insulin resistant cell lines, viz skeletal muscle (C2C12) and fat (3T3-L1), and to investigate the effects of these cannabinoids on insulin secretion in pancreatic β-cells (INS 1). Insulin resistance was induced in the cells using 20 ng/mL TNF-α (3T3-L1) and 100 nM insulin (C2C12). Insulin resistant cells were exposed to cannabinoids for 48 hours after which glucose uptake, RT-PCR and Western blot analysis was performed. Additionally, adipokine assays were performed on the 3T3-L1 cells. The insulin resistant 3T3-L1 and C2C12 cells had reduced glucose uptake, decreased IRS-1 and Glut-4 expression indicative of an insulin resistant state. The extract and THC significantly enhanced glucose uptake, IRS-1 and Glut-4 in 3T3-L1 and C2C12 cells. The extract and THC thus have the potential to be an insulin sensitizing agent. Interleukin-6 was significantly decreased by THC. INS 1 cells, cultured under normoglycemic conditions, were exposed to cannabinoids for 48 hours after which glucose-stimulated insulin secretion, radioimmunoassay, oxygen consumption, RT-PCR and Western blot analysis was performed. Insulin stimulatory index was not significantly affected after cannabinoid exposure, except by THC. The cannabinoids decreased insulin content, in a concentration dependent manner, but the inhibition mechanism remains elusive. The cannabinoid Treated cells showed insulin gene expression levels similar to the control, while only THC proved effective in significantly stimulating Glut-2 gene expression. Oxygen consumption studies showed levels lower than the control cells. Most of the cannabinoids inhibited insulin secretion under normoglycemia except THC, while the cannabinoids exhibited the potential to improve insulin resistant adipocyte and myocytes response to glucose and gene regulation.
- Full Text:
- Date Issued: 2009
The effects of a Kenyan antidiabetic plant on insulin homeostasis
- Authors: Suleiman, Khairunisa Yahya
- Date: 2009
- Subjects: Medicinal plants -- Kenya , Insulin resistance
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10326 , http://hdl.handle.net/10948/1055 , Medicinal plants -- Kenya , Insulin resistance
- Description: The metabolic disorder diabetes; is a global epidemic affecting people in developed countries and increasingly in developing countries. In two decades time, 350 million people will be diabetic at the current rate of prevalence. In a preliminary study, insulin resistant rats were treated with Prunus Africana (plant A) for 28 days. Plasma samples obtained from P. africana treated rats had increased insulin levels compared to normal and untreated insulin resistant rats (Karachi, 2009). The treatment of insulin resistant rats with P. africana also showed increased glucose uptake in rat adipose tissue (Karachi, 2009), suggesting that P. africana had anti-diabetic properties. The aim of the study was to investigate the mechanism of the anti-diabetic properties of P africana extract. Increased insulin secretion was confirmed by the increased Cpeptide concentration in plasma samples of rats treated with P. africana. In order to explain the high insulin levels, several hypothesis’ were investigated: (1) P. africana may increase insulin secretion in β cells, hence the effect of P. africana on insulin secretion by INS-1 cells was investigated; (2) P. africana may increase insulin secretion by prolonging the half-life of glucagon like peptide-1 (GLP-1) by decreasing dipeptidyl peptidase IV (DPP IV) activity; the effect of P. africana on DPP IV activity was determined spectrophotometrically, (3) P. africana may increase the half-life of insulin in the plasma by decreasing the activity of insulin degrading enzyme (IDE); the effect of P. africana on IDE in rat muscle and spleen samples was investigated. To explain the increased glucose uptake in adipose tissue observed in the previous study two parameters were investigated: (1) increased GLUT4 expression in P. africana treated rats; the effect of P. africana treatment on the expression of glucose transporter 4 (GLUT4) was determined using real-time polymerase chain reaction (RT-PCR), (2) P. africana may increase glucose utilization; the effect of P. africana on glucose utilization was determined in 3T3-L1 cells. The plant extract did not significantly increase insulin secretion by INS-1 cells in the absence of glucose. P. africana decreased DPP IV activity in rat plasma when compared to the untreated insulin resistant rats and this could be a mechanism by which insulin secretion is increased during plant treatment. P. africana decreased IDE activity (however not significantly) when compared to the untreated insulin resistant The effects of a Kenyan antidiabetic plant on insulin homeostasis KY Suleiman VII rats. P. africana appeared to have no effect on GLUT4 expression. The plant appeared to increase glucose utilization in 3T3-L1 cells in the absence of insulin suggesting that P. africana may have insulin like activity. In summary, this study indicates that P. africana is indirectly involved in inhibiting DDPIV. This in turn can increase the half life of GLP-1, which in turn can enhance the secretion of insulin. P. africana increases glucose utilization although there was no evidence that the GLUT 4 transporter has a higher expression in the plant treated rats. Further studies should be conducted to investigate the expression of GLUT1 under the same conditons.
- Full Text:
- Date Issued: 2009
- Authors: Suleiman, Khairunisa Yahya
- Date: 2009
- Subjects: Medicinal plants -- Kenya , Insulin resistance
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10326 , http://hdl.handle.net/10948/1055 , Medicinal plants -- Kenya , Insulin resistance
- Description: The metabolic disorder diabetes; is a global epidemic affecting people in developed countries and increasingly in developing countries. In two decades time, 350 million people will be diabetic at the current rate of prevalence. In a preliminary study, insulin resistant rats were treated with Prunus Africana (plant A) for 28 days. Plasma samples obtained from P. africana treated rats had increased insulin levels compared to normal and untreated insulin resistant rats (Karachi, 2009). The treatment of insulin resistant rats with P. africana also showed increased glucose uptake in rat adipose tissue (Karachi, 2009), suggesting that P. africana had anti-diabetic properties. The aim of the study was to investigate the mechanism of the anti-diabetic properties of P africana extract. Increased insulin secretion was confirmed by the increased Cpeptide concentration in plasma samples of rats treated with P. africana. In order to explain the high insulin levels, several hypothesis’ were investigated: (1) P. africana may increase insulin secretion in β cells, hence the effect of P. africana on insulin secretion by INS-1 cells was investigated; (2) P. africana may increase insulin secretion by prolonging the half-life of glucagon like peptide-1 (GLP-1) by decreasing dipeptidyl peptidase IV (DPP IV) activity; the effect of P. africana on DPP IV activity was determined spectrophotometrically, (3) P. africana may increase the half-life of insulin in the plasma by decreasing the activity of insulin degrading enzyme (IDE); the effect of P. africana on IDE in rat muscle and spleen samples was investigated. To explain the increased glucose uptake in adipose tissue observed in the previous study two parameters were investigated: (1) increased GLUT4 expression in P. africana treated rats; the effect of P. africana treatment on the expression of glucose transporter 4 (GLUT4) was determined using real-time polymerase chain reaction (RT-PCR), (2) P. africana may increase glucose utilization; the effect of P. africana on glucose utilization was determined in 3T3-L1 cells. The plant extract did not significantly increase insulin secretion by INS-1 cells in the absence of glucose. P. africana decreased DPP IV activity in rat plasma when compared to the untreated insulin resistant rats and this could be a mechanism by which insulin secretion is increased during plant treatment. P. africana decreased IDE activity (however not significantly) when compared to the untreated insulin resistant The effects of a Kenyan antidiabetic plant on insulin homeostasis KY Suleiman VII rats. P. africana appeared to have no effect on GLUT4 expression. The plant appeared to increase glucose utilization in 3T3-L1 cells in the absence of insulin suggesting that P. africana may have insulin like activity. In summary, this study indicates that P. africana is indirectly involved in inhibiting DDPIV. This in turn can increase the half life of GLP-1, which in turn can enhance the secretion of insulin. P. africana increases glucose utilization although there was no evidence that the GLUT 4 transporter has a higher expression in the plant treated rats. Further studies should be conducted to investigate the expression of GLUT1 under the same conditons.
- Full Text:
- Date Issued: 2009
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