Implementation of novel flow cytometric methods to assess the in vitro antidiabetic mechanism of a Sutherlandia Frutescens extract
- Authors: Elliot, Gayle Pamela
- Date: 2010
- Subjects: Insulin resistance -- South Africa , Insulin -- Therapeutic use -- South Africa , Medicinal plants -- South Africa , Non-insulin-dependent diabetes -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10304 , http://hdl.handle.net/10948/1439 , Insulin resistance -- South Africa , Insulin -- Therapeutic use -- South Africa , Medicinal plants -- South Africa , Non-insulin-dependent diabetes -- South Africa
- Description: The ability of insulin to stimulate glucose uptake into muscle and adipose tissue is central to the maintenance of whole-body glucose homeostasis. Deregulation of insulin action manifests itself as insulin resistance, a key component of type 2 diabetes. Insulin resistance is also observed in HIV patients receiving protease inhibitors. An agent that can reversibly induce an insulin-resistant state would be a very useful tool in developing model systems that mimic the pathogenesis of type 2 diabetes. Insulin resistance can arise from defects in insulin signal transduction, changes in the expression of proteins or genes that are targets of insulin action, cross talk from other hormonal systems or metabolic abnormalities. Deterioration of the insulin-receptor-signalling pathway at different levels leading to decreased levels of signalling pathway intermediates and/or decreased activation through phosphorylation accounts for the evolution from an insulin-resistant state to type 2 diabetes. In addition, defects in GLUT4 glucose transporter translocation are observed, further fuelling impairments in skeletal muscle glucose uptake. Levels of insulin-induced GLUT4 translocation in the skeletal muscle of type 2 diabetic patients are typically reduced by 90%. Many cellular pathways & their intermediates are in some way or another linked to insulin signalling. This study focused on three of these namely the PI3-kinase/Akt pathway, the Mitogen Activated Protein Kinase (MAPK) cascade and the AMP Kinase pathway, with successful monitoring of the PI3-K pathway. Investigations involved observing and evaluating the effects of various compounds as well as an indigenous medicinal plant, Sutherlandia frutescens on the activities of key insulin signalling pathway intermediates within the three fore mentioned pathways including Akt, AMPK and MEK1/2 as well as membrane surface GLUT4 levels. Scientific research has in the past leant heavily on Western blotting as the method of choice for gaining vital information relating to signal transduction pathways, however for research into cellular mechanisms the negatives of this method outweigh the positives. The drawbacks include a need for large amount of cells, multiple washing steps which may be disadvantageous to any weak and transient interactions as well as lysing of cells which may interfere with the maintenance of the subcellular localisation of a specific signalling event. Based on these, the need for a better method in terms of speed & reliability to monitor phosphorylation states of signal transduction pathway intermediates & GLUT4 translocation was evident and was one VII of the main aims & successes of this study. The method created used the mouse muscle cell line C2C12 in conjunction with the quick, sensitive method of flow cytometry which allowed us to monitor these processes in these cells through immune-labelling. Adherent cell cultures such as the C2C12 cell line pose the problem of possible damage to plasma membrane receptors (including insulin receptors) during harvesting to obtain a cell suspension for flow cytometry. We however used C2C12 mouse myocytes to optimize a method yielding insulin responsive cells in suspension that were successfully used for flow cytometry after immunelabelling of insulin signalling intermediates. Insulin (0.1μM) significantly raised the levels of both P-Akt and GLUT4 above basal levels. This effect was shown to be dose dependent. At a concentration of 50μg/ml, Sutherlandia frutescens was able to act as an insulin-mimetic in terms of its ability to increase P-Akt levels, GLUT4 translocation and glucose utilisation in an acute manner. These increases could be reduced with the addition of wortmannin, a PI3-K inhibitor. Therefore, these results suggest the mechanism of the plant extract’s insulin-like activity may be in part due to the activation of the insulin signalling pathway leading to GLUT4 translocation, which involves the phosphorylation of insulin receptor- and subsequent PI3-K activity, leading to P-Akt activity. These results provide further evidence of this plant extract’s anti-diabetic potential. The effect of Sutherlandia frutescens on insulin secretion, calcium signalling and proliferation in INS-1 rat pancreatic cells was also investigated and it was found to increase the activities of all of these processes. However no change in the levels of GLUT2 glucose transporter was seen. Ritonavir is prescribed by the South African Department of Health in co-formulation with other protease inhibitors within its second regime in the treatment of HIV and AIDS. Using C2C12 cells, ritonavir decreased glucose uptake acutely and had no effect on GLUT4 translocation however surprisingly increased P-Akt levels. In conclusion, it was found that Sutherlandia frutescens has antidiabetic benefits, diverse in nature depending on tissue type as well as length of time administered. The establishment of novel flow cytometry techniques to assess antidiabetic properties using in vitro cell culture was achieved. These methods will be useful in the future for the assessment of insulin sensitivity and in the identification of novel compounds that stimulate the insulin signalling pathways.
- Full Text:
- Date Issued: 2010
- Authors: Elliot, Gayle Pamela
- Date: 2010
- Subjects: Insulin resistance -- South Africa , Insulin -- Therapeutic use -- South Africa , Medicinal plants -- South Africa , Non-insulin-dependent diabetes -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10304 , http://hdl.handle.net/10948/1439 , Insulin resistance -- South Africa , Insulin -- Therapeutic use -- South Africa , Medicinal plants -- South Africa , Non-insulin-dependent diabetes -- South Africa
- Description: The ability of insulin to stimulate glucose uptake into muscle and adipose tissue is central to the maintenance of whole-body glucose homeostasis. Deregulation of insulin action manifests itself as insulin resistance, a key component of type 2 diabetes. Insulin resistance is also observed in HIV patients receiving protease inhibitors. An agent that can reversibly induce an insulin-resistant state would be a very useful tool in developing model systems that mimic the pathogenesis of type 2 diabetes. Insulin resistance can arise from defects in insulin signal transduction, changes in the expression of proteins or genes that are targets of insulin action, cross talk from other hormonal systems or metabolic abnormalities. Deterioration of the insulin-receptor-signalling pathway at different levels leading to decreased levels of signalling pathway intermediates and/or decreased activation through phosphorylation accounts for the evolution from an insulin-resistant state to type 2 diabetes. In addition, defects in GLUT4 glucose transporter translocation are observed, further fuelling impairments in skeletal muscle glucose uptake. Levels of insulin-induced GLUT4 translocation in the skeletal muscle of type 2 diabetic patients are typically reduced by 90%. Many cellular pathways & their intermediates are in some way or another linked to insulin signalling. This study focused on three of these namely the PI3-kinase/Akt pathway, the Mitogen Activated Protein Kinase (MAPK) cascade and the AMP Kinase pathway, with successful monitoring of the PI3-K pathway. Investigations involved observing and evaluating the effects of various compounds as well as an indigenous medicinal plant, Sutherlandia frutescens on the activities of key insulin signalling pathway intermediates within the three fore mentioned pathways including Akt, AMPK and MEK1/2 as well as membrane surface GLUT4 levels. Scientific research has in the past leant heavily on Western blotting as the method of choice for gaining vital information relating to signal transduction pathways, however for research into cellular mechanisms the negatives of this method outweigh the positives. The drawbacks include a need for large amount of cells, multiple washing steps which may be disadvantageous to any weak and transient interactions as well as lysing of cells which may interfere with the maintenance of the subcellular localisation of a specific signalling event. Based on these, the need for a better method in terms of speed & reliability to monitor phosphorylation states of signal transduction pathway intermediates & GLUT4 translocation was evident and was one VII of the main aims & successes of this study. The method created used the mouse muscle cell line C2C12 in conjunction with the quick, sensitive method of flow cytometry which allowed us to monitor these processes in these cells through immune-labelling. Adherent cell cultures such as the C2C12 cell line pose the problem of possible damage to plasma membrane receptors (including insulin receptors) during harvesting to obtain a cell suspension for flow cytometry. We however used C2C12 mouse myocytes to optimize a method yielding insulin responsive cells in suspension that were successfully used for flow cytometry after immunelabelling of insulin signalling intermediates. Insulin (0.1μM) significantly raised the levels of both P-Akt and GLUT4 above basal levels. This effect was shown to be dose dependent. At a concentration of 50μg/ml, Sutherlandia frutescens was able to act as an insulin-mimetic in terms of its ability to increase P-Akt levels, GLUT4 translocation and glucose utilisation in an acute manner. These increases could be reduced with the addition of wortmannin, a PI3-K inhibitor. Therefore, these results suggest the mechanism of the plant extract’s insulin-like activity may be in part due to the activation of the insulin signalling pathway leading to GLUT4 translocation, which involves the phosphorylation of insulin receptor- and subsequent PI3-K activity, leading to P-Akt activity. These results provide further evidence of this plant extract’s anti-diabetic potential. The effect of Sutherlandia frutescens on insulin secretion, calcium signalling and proliferation in INS-1 rat pancreatic cells was also investigated and it was found to increase the activities of all of these processes. However no change in the levels of GLUT2 glucose transporter was seen. Ritonavir is prescribed by the South African Department of Health in co-formulation with other protease inhibitors within its second regime in the treatment of HIV and AIDS. Using C2C12 cells, ritonavir decreased glucose uptake acutely and had no effect on GLUT4 translocation however surprisingly increased P-Akt levels. In conclusion, it was found that Sutherlandia frutescens has antidiabetic benefits, diverse in nature depending on tissue type as well as length of time administered. The establishment of novel flow cytometry techniques to assess antidiabetic properties using in vitro cell culture was achieved. These methods will be useful in the future for the assessment of insulin sensitivity and in the identification of novel compounds that stimulate the insulin signalling pathways.
- Full Text:
- Date Issued: 2010
Leonotis leonurus: the anticoagulant and antidiabetic activity of Leonotis leonurus
- Authors: Mnonopi, Nandipha
- Date: 2010
- Subjects: Medicinal plants -- South Africa , Materia medica, Vegetable -- South Africa , Diabetes -- Alternative treatment -- South Africa , Plant bioactive compounds , Leonotis leonurus -- Physiological aspects
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10323 , http://hdl.handle.net/10948/1194 , Medicinal plants -- South Africa , Materia medica, Vegetable -- South Africa , Diabetes -- Alternative treatment -- South Africa , Plant bioactive compounds , Leonotis leonurus -- Physiological aspects
- Description: Commercial marrubiin, aqueous and organic extracts of Leonotis leonurus were tested in vitro for their anticoagulant and antiplatelet activities. The aqueous extract inhibited platelet aggregation by 69.5 percent (100 μg/mL), while the organic extract (100 μg/mL) and marrubiin (5 μg/mL) showed 92.5 percent and 91.6 percent inhibition, respectively, by inhibiting the binding of fibrinogen to glycoprotein IIb/IIIa receptor in a concentration dependent manner. The extracts significantly prolonged activated partial thromboplastin time compared to untreated plasma controls. Fibrin and D-Dimer formation were drastically decreased. The extracts and marrubiin concentration-dependently inhibited calcium mobilization induced by collagen and thrombin. The formation of thromboxane A2 was also significantly reduced by both the extracts and marrubiin. Protein secretion and platelet adhesion were significantly reduced by both the extracts and marrubiin. The organic extract and marrubiin showed a more pronounced effect than the aqueous extracts in all the in vitro assays. The ex-vivo animal model confirmed the results obtained in vitro. Similar to the in vitro studies, activated partial thromboplastin time clotting time was prolonged by marrubiin and the number of aggregated platelets were significantly reduced relative to aspirin. The findings reflect that marrubiin largely contributes to the organic extract's anticoagulant and antiplatelet effect in vitro. INS-1 cells were cultured under normo- and hyperglycaemic conditions. Marrubiin and the two Leonotis leonurus extracts were screened for anti-diabetic activity in vitro. The stimulatory index of INS-1 cells cultured under hyperglycaemic conditions was significantly increased by 60 percent and 61 percent (p<0.01; n=5) in cells exposed to the organic extract (10 μg/mL) and marrubiin (500 ng/mL), respectively, relative to the normoglycaemic conditions. The gene expression of insulin was significantly increased by 76.5 and 71 percent, and of glucose transporter-2 by 93 and 92.5 percent for marrubiin and the organic extract, respectively, under the same conditions stipulated above (p<0.01; n=4). The extract and marrubiin similarly showed an increase in respiratory rate under hyperglycaemic conditions. Marrubiin increased insulin secretion, HDL-cholesterol, while it decreased total cholesterol, LDL-cholesterol and the atherogenic index in the in vivo rat model.
- Full Text:
- Date Issued: 2010
- Authors: Mnonopi, Nandipha
- Date: 2010
- Subjects: Medicinal plants -- South Africa , Materia medica, Vegetable -- South Africa , Diabetes -- Alternative treatment -- South Africa , Plant bioactive compounds , Leonotis leonurus -- Physiological aspects
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10323 , http://hdl.handle.net/10948/1194 , Medicinal plants -- South Africa , Materia medica, Vegetable -- South Africa , Diabetes -- Alternative treatment -- South Africa , Plant bioactive compounds , Leonotis leonurus -- Physiological aspects
- Description: Commercial marrubiin, aqueous and organic extracts of Leonotis leonurus were tested in vitro for their anticoagulant and antiplatelet activities. The aqueous extract inhibited platelet aggregation by 69.5 percent (100 μg/mL), while the organic extract (100 μg/mL) and marrubiin (5 μg/mL) showed 92.5 percent and 91.6 percent inhibition, respectively, by inhibiting the binding of fibrinogen to glycoprotein IIb/IIIa receptor in a concentration dependent manner. The extracts significantly prolonged activated partial thromboplastin time compared to untreated plasma controls. Fibrin and D-Dimer formation were drastically decreased. The extracts and marrubiin concentration-dependently inhibited calcium mobilization induced by collagen and thrombin. The formation of thromboxane A2 was also significantly reduced by both the extracts and marrubiin. Protein secretion and platelet adhesion were significantly reduced by both the extracts and marrubiin. The organic extract and marrubiin showed a more pronounced effect than the aqueous extracts in all the in vitro assays. The ex-vivo animal model confirmed the results obtained in vitro. Similar to the in vitro studies, activated partial thromboplastin time clotting time was prolonged by marrubiin and the number of aggregated platelets were significantly reduced relative to aspirin. The findings reflect that marrubiin largely contributes to the organic extract's anticoagulant and antiplatelet effect in vitro. INS-1 cells were cultured under normo- and hyperglycaemic conditions. Marrubiin and the two Leonotis leonurus extracts were screened for anti-diabetic activity in vitro. The stimulatory index of INS-1 cells cultured under hyperglycaemic conditions was significantly increased by 60 percent and 61 percent (p<0.01; n=5) in cells exposed to the organic extract (10 μg/mL) and marrubiin (500 ng/mL), respectively, relative to the normoglycaemic conditions. The gene expression of insulin was significantly increased by 76.5 and 71 percent, and of glucose transporter-2 by 93 and 92.5 percent for marrubiin and the organic extract, respectively, under the same conditions stipulated above (p<0.01; n=4). The extract and marrubiin similarly showed an increase in respiratory rate under hyperglycaemic conditions. Marrubiin increased insulin secretion, HDL-cholesterol, while it decreased total cholesterol, LDL-cholesterol and the atherogenic index in the in vivo rat model.
- Full Text:
- Date Issued: 2010
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