Effects of selected natural flavonoids on an optimized insulin resistance induction model on myotubes and hepatocytes in vitro
- Authors: Van de Venter, Ruben
- Date: 2023-12
- Subjects: Flavonoid , Insulin resistance , Myotubes and hepatocytes
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10948/63049 , vital:73085
- Description: Introduction: As the third largest life-threatening non-communicable disease, further research to improve T2DM treatment remains pivotal. The approved insulin sensitizing agents, metformin and thiazolidinediones, although effective, have an array of adverse effects. This leads to the growing need for safer insulin sensitizing agents. Aims: To induce insulin resistance, an insulin resistance induction (IRI) model consisting of palmitic acid (PA), fructose, and dexamethasone (DEX), was optimized. The model was utilized to compare the insulin sensitizing efficacy of chrysin, apigenin, luteolin, and quercetin. The most effective flavonoid was extrapolated based on the hydroxylation hypothesis and relevant SAR. The flavonoid was combined with metformin to evaluate potential insulin sensitizing synergism. Methods: C3A hepatocytes and L6 myotubes were exposed to the IRI models: IR1, IR2, and OIR, for 24 and 48h. Morphological features indicative of insulin resistance were monitored through high-content analysis (HCA). Thereafter, cells were exposed to OIR and concurrently treated with the selected flavonoids. Cytotoxicity, oxidative stress (OS), mitochondrial content, mitochondrial membrane potential (MMP), and lipid accumulation, were once again evaluated through HCA. Subsequently, quercetin was combined with metformin (QM); insulin resistant conditions established through OIR exposure, and QM administered as prophylaxis. The efficacy of QM was determined through changes in phosphorylated-Akt, glucose uptake, and gluconeogenesis. Results: IR1 and IR2 demonstrated excessive potency, whereas OIR consistently generated insulin resistant C3A/L6 cells by increasing OS (↑14%/20%), lipid accumulation (0.7-fold/1.5-fold), and decreasing MMP (↓4%/6%). Of the selected flavonoids, quercetin was most effective at ameliorating each of these parameters. Furthermore, QM demonstrated potential insulin sensitizing synergism, as it improved Akt phosphorylation (Thr308: ↑31%/↑17%; Ser473: ↑29%), glucose uptake (↑10%/ 5%), and suppressed hepatic gluconeogenesis (15↓%) more effectively compared to monotherapy. Conclusions: OIR is an effective model for the induction of complete early-stage insulin resistance in myotubes and hepatocytes. Quercetin demonstrates exceptional antidiabetic activity due its unique expression of hydroxyl groups. Lastly, QM, comparative to monotherapy, improves insulin sensitivity with enhanced efficacy. , Thesis (Ma) -- Faculty of Health Science, 2023
- Full Text:
- Date Issued: 2023-12
- Authors: Van de Venter, Ruben
- Date: 2023-12
- Subjects: Flavonoid , Insulin resistance , Myotubes and hepatocytes
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10948/63049 , vital:73085
- Description: Introduction: As the third largest life-threatening non-communicable disease, further research to improve T2DM treatment remains pivotal. The approved insulin sensitizing agents, metformin and thiazolidinediones, although effective, have an array of adverse effects. This leads to the growing need for safer insulin sensitizing agents. Aims: To induce insulin resistance, an insulin resistance induction (IRI) model consisting of palmitic acid (PA), fructose, and dexamethasone (DEX), was optimized. The model was utilized to compare the insulin sensitizing efficacy of chrysin, apigenin, luteolin, and quercetin. The most effective flavonoid was extrapolated based on the hydroxylation hypothesis and relevant SAR. The flavonoid was combined with metformin to evaluate potential insulin sensitizing synergism. Methods: C3A hepatocytes and L6 myotubes were exposed to the IRI models: IR1, IR2, and OIR, for 24 and 48h. Morphological features indicative of insulin resistance were monitored through high-content analysis (HCA). Thereafter, cells were exposed to OIR and concurrently treated with the selected flavonoids. Cytotoxicity, oxidative stress (OS), mitochondrial content, mitochondrial membrane potential (MMP), and lipid accumulation, were once again evaluated through HCA. Subsequently, quercetin was combined with metformin (QM); insulin resistant conditions established through OIR exposure, and QM administered as prophylaxis. The efficacy of QM was determined through changes in phosphorylated-Akt, glucose uptake, and gluconeogenesis. Results: IR1 and IR2 demonstrated excessive potency, whereas OIR consistently generated insulin resistant C3A/L6 cells by increasing OS (↑14%/20%), lipid accumulation (0.7-fold/1.5-fold), and decreasing MMP (↓4%/6%). Of the selected flavonoids, quercetin was most effective at ameliorating each of these parameters. Furthermore, QM demonstrated potential insulin sensitizing synergism, as it improved Akt phosphorylation (Thr308: ↑31%/↑17%; Ser473: ↑29%), glucose uptake (↑10%/ 5%), and suppressed hepatic gluconeogenesis (15↓%) more effectively compared to monotherapy. Conclusions: OIR is an effective model for the induction of complete early-stage insulin resistance in myotubes and hepatocytes. Quercetin demonstrates exceptional antidiabetic activity due its unique expression of hydroxyl groups. Lastly, QM, comparative to monotherapy, improves insulin sensitivity with enhanced efficacy. , Thesis (Ma) -- Faculty of Health Science, 2023
- Full Text:
- Date Issued: 2023-12
Evaluating antidiabetic properties of selected African medicinal plants in a cell-based model
- Sirkhotte, Saeedah, Reddy, Shanika
- Authors: Sirkhotte, Saeedah , Reddy, Shanika
- Date: 2023-12
- Subjects: Medicinal plants -- South Africa , Insulin resistance , Gluconeogenesis
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10948/62600 , vital:72827
- Description: The World Health Organisation lists diabetes as one of the major non-communicable diseases affecting the world, and its prevalence is expected to increase rapidly. Type II diabetes mellitus (T2DM) is characterised by insulin resistance and impaired blood glucose control. T2DM is of growing concern within South Africa, with 10.8% of the population currently diagnosed. The popularity, as well as availability, of traditional plant-based medicine in South Africa, might provide a solution. Antidiabetic potential is commonly tested by in vitro assays, however the methods to test this potential via gluconeogenesis are limited. This project aimed to optimise an in vitro hepatic gluconeogenesis model. In addition, this project aimed to determine the antidiabetic properties of Prunus africana, Hypoxis stellipilis, and Eriocephalus africanus by in vitro analysis. These properties were examined in human hepatoma cells (C3A cell line) and rat pancreatic cells (INS1 cell line) and methods included: cytotoxicity analysis, Amplex® red glucose oxidase assay, antibody staining, gene expression analysis by RT-qPCR, oxidative stress analysis, and calcium signaling for insulin release. Insulin resistance was successfully induced by exposing C3A hepatocarcinoma cells to a combination treatment of 50 μM dexamethasone, 1.25 mM fructose and 0.125 mM palmitic acid for a period of three days. Thereafter, gluconeogenesis was assessed using the Amplex® red glucose oxidase assay. The established model was effective in inducing insulin resistance and upregulating gluconeogenesis. Of the tested plant extracts, H. stellipilis showed the most potential as an antidiabetic treatment. It had low toxicity, significantly decreased hepatic glucose production and reduced the amount of phosphoenolpyruvate carboxykinase (PCK) and well as PCK gene expression, and reduced lipid content and reactive oxygen species (ROS) in the C3A cell line. H. stellipilis increased calcium signalling in INS1 rat insulinoma cells, however there was a decrease in expression of genes for insulin and glucose transporter 2 after 6 hr exposure. H. stellipilis appears be beneficial as an antidiabetic treatment. Although antidiabetic studies have been done on other Hypoxis species, this is the first study on the effects of H. stellipilis on gluconeogenesis and diabetes. , Thesis (MSc) -- Faculty of Science, School of Biomolecular & Chemical Sciences, 2023
- Full Text:
- Date Issued: 2023-12
- Authors: Sirkhotte, Saeedah , Reddy, Shanika
- Date: 2023-12
- Subjects: Medicinal plants -- South Africa , Insulin resistance , Gluconeogenesis
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10948/62600 , vital:72827
- Description: The World Health Organisation lists diabetes as one of the major non-communicable diseases affecting the world, and its prevalence is expected to increase rapidly. Type II diabetes mellitus (T2DM) is characterised by insulin resistance and impaired blood glucose control. T2DM is of growing concern within South Africa, with 10.8% of the population currently diagnosed. The popularity, as well as availability, of traditional plant-based medicine in South Africa, might provide a solution. Antidiabetic potential is commonly tested by in vitro assays, however the methods to test this potential via gluconeogenesis are limited. This project aimed to optimise an in vitro hepatic gluconeogenesis model. In addition, this project aimed to determine the antidiabetic properties of Prunus africana, Hypoxis stellipilis, and Eriocephalus africanus by in vitro analysis. These properties were examined in human hepatoma cells (C3A cell line) and rat pancreatic cells (INS1 cell line) and methods included: cytotoxicity analysis, Amplex® red glucose oxidase assay, antibody staining, gene expression analysis by RT-qPCR, oxidative stress analysis, and calcium signaling for insulin release. Insulin resistance was successfully induced by exposing C3A hepatocarcinoma cells to a combination treatment of 50 μM dexamethasone, 1.25 mM fructose and 0.125 mM palmitic acid for a period of three days. Thereafter, gluconeogenesis was assessed using the Amplex® red glucose oxidase assay. The established model was effective in inducing insulin resistance and upregulating gluconeogenesis. Of the tested plant extracts, H. stellipilis showed the most potential as an antidiabetic treatment. It had low toxicity, significantly decreased hepatic glucose production and reduced the amount of phosphoenolpyruvate carboxykinase (PCK) and well as PCK gene expression, and reduced lipid content and reactive oxygen species (ROS) in the C3A cell line. H. stellipilis increased calcium signalling in INS1 rat insulinoma cells, however there was a decrease in expression of genes for insulin and glucose transporter 2 after 6 hr exposure. H. stellipilis appears be beneficial as an antidiabetic treatment. Although antidiabetic studies have been done on other Hypoxis species, this is the first study on the effects of H. stellipilis on gluconeogenesis and diabetes. , Thesis (MSc) -- Faculty of Science, School of Biomolecular & Chemical Sciences, 2023
- Full Text:
- Date Issued: 2023-12
Evaluation of the antidiabetic potential, safety and efficacy of selected natural products using zebrafish larvae and in vitro bioassays
- Reddy, Shanika, Dambuza, Ntokozo
- Authors: Reddy, Shanika , Dambuza, Ntokozo
- Date: 2023-12
- Subjects: Traditional medicine -- Research -- South Africa , Medicinal plants -- South Africa -- Research , Insulin resistance
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10948/62575 , vital:72825
- Description: Abstract. , Thesis (MSc) -- Faculty of Science, School of Biomolecular & Chemical Sciences, 2023
- Full Text:
- Date Issued: 2023-12
- Authors: Reddy, Shanika , Dambuza, Ntokozo
- Date: 2023-12
- Subjects: Traditional medicine -- Research -- South Africa , Medicinal plants -- South Africa -- Research , Insulin resistance
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10948/62575 , vital:72825
- Description: Abstract. , Thesis (MSc) -- Faculty of Science, School of Biomolecular & Chemical Sciences, 2023
- Full Text:
- Date Issued: 2023-12
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 effect of 14 weeks of strength training on insulin resistance
- Authors: Cairncross, Joy Claudia
- Date: 2013
- Subjects: Insulin resistance , Diabetes -- Exercise therapy
- Language: English
- Type: Thesis , Masters , MA
- Identifier: vital:10104 , http://hdl.handle.net/10948/d1018276
- Description: Insulin resistance is a precursor to type II diabetes mellitus and in conjunction with dyslipidaemia, hypertension, and obesity, these abnormalities constitute the metabolic syndrome. Insulin resistance usually develops before these other diseases and therefore identifying and successfully treating insulin resistant patients may have potentially great preventive value. Insulin resistance, obesity, and subsequently type II diabetes mellitus have increased dramatically and have reached epidemic proportions. The incidence of diabetes, and in particular type II diabetes mellitus, is increasing in developing countries and throughout the world and this is mainly as a result of increasingly sedentary lifestyle and obesity in an aging population. The specific aim of this study was to explore and describe the effect of a 14-week strength-based resistance training programme on insulin resistance amongst individuals aged 25 to 68 years, who are pre-diabetic, have T2DM, and/or are overweight. The research approach used in this investigation was explorative, experimental, and quantitative in nature. The quasi-experimental design consisted of a pre-test and post-test for an experimental and comparison group who were chosen through convenience and snowball sampling. A total of 30 participants were involved in this study, 15 participants in each group. The following dependent variables were selected, namely: body weight; BMI; body composition; waist-to-hip ratio; total cholesterol levels; triglyceride levels; HOMA-IR; and muscle strength for upper and lower body. Pre-and post-test analysis was performed at the Biokinetics and Sport Science Unit, located at the Nelson Mandela Metropolitan University (NMMU). Blood samples of the participants were drawn by nurses at the Health Clinic at the Nelson Mandela Metropolitan University and these blood plasma samples were stored at the Department of Microbiology and Biochemistry at NMMU for later analysis of glucose and insulin. The experimental group trained three times per week for a period of fourteen weeks, performing strength training exercises with progressive increments in the intensity of the exercise. The control group remained sedentary throughout the intervention period. Analysis of the data was conducted utilizing descriptive and inferential statistics. Analysis of variance (ANOVA) was used as a hypothesis-testing procedure to evaluate the mean differences. The following dependent variables showed a decrease in mean values: body weight, body mass index, body fat percentage, waist minimum, cholesterol and insulin. However these differences in results were not practically and statistically significant. The following dependent variables showed an increase in mean values: hip maximum, arm relaxed, arm flexed, thigh circumference, glucose and HOMA-IR. However these results were not practically and statistically significant. The mean differences in the plasma insulin level, pre- to post-test, between both groups indicated that a significant difference (t = -1.77, p = 0.044) existed between them. Cohen‟s d revealed a value of 0.64, which indicates moderate practical significance. The only dependent variable which showed both statistical and practical significance was sum of skinfolds. The findings for sum of skinfolds revealed that the mean differences, from pre- to post-test, between both groups indicated that a significant difference (t = -2.30, p = 0.015) existed between them. Cohen‟s d revealed a value of 0.84, which indicated a large practical significance. Although the sample size was too small to indicate generalisations to the diabetic population as a whole, strength training should be furthermore explored as an alternative and successful modality in the existing range of options available to the health and exercise professional to address the needs of the person with T2DM. The researcher proposed that a bigger sample size be used for the experimental and control group, the intervention period increased as well as various differences related to frequency, intensity and duration of strength training could possibly result in significant changes.
- Full Text:
- Date Issued: 2013
- Authors: Cairncross, Joy Claudia
- Date: 2013
- Subjects: Insulin resistance , Diabetes -- Exercise therapy
- Language: English
- Type: Thesis , Masters , MA
- Identifier: vital:10104 , http://hdl.handle.net/10948/d1018276
- Description: Insulin resistance is a precursor to type II diabetes mellitus and in conjunction with dyslipidaemia, hypertension, and obesity, these abnormalities constitute the metabolic syndrome. Insulin resistance usually develops before these other diseases and therefore identifying and successfully treating insulin resistant patients may have potentially great preventive value. Insulin resistance, obesity, and subsequently type II diabetes mellitus have increased dramatically and have reached epidemic proportions. The incidence of diabetes, and in particular type II diabetes mellitus, is increasing in developing countries and throughout the world and this is mainly as a result of increasingly sedentary lifestyle and obesity in an aging population. The specific aim of this study was to explore and describe the effect of a 14-week strength-based resistance training programme on insulin resistance amongst individuals aged 25 to 68 years, who are pre-diabetic, have T2DM, and/or are overweight. The research approach used in this investigation was explorative, experimental, and quantitative in nature. The quasi-experimental design consisted of a pre-test and post-test for an experimental and comparison group who were chosen through convenience and snowball sampling. A total of 30 participants were involved in this study, 15 participants in each group. The following dependent variables were selected, namely: body weight; BMI; body composition; waist-to-hip ratio; total cholesterol levels; triglyceride levels; HOMA-IR; and muscle strength for upper and lower body. Pre-and post-test analysis was performed at the Biokinetics and Sport Science Unit, located at the Nelson Mandela Metropolitan University (NMMU). Blood samples of the participants were drawn by nurses at the Health Clinic at the Nelson Mandela Metropolitan University and these blood plasma samples were stored at the Department of Microbiology and Biochemistry at NMMU for later analysis of glucose and insulin. The experimental group trained three times per week for a period of fourteen weeks, performing strength training exercises with progressive increments in the intensity of the exercise. The control group remained sedentary throughout the intervention period. Analysis of the data was conducted utilizing descriptive and inferential statistics. Analysis of variance (ANOVA) was used as a hypothesis-testing procedure to evaluate the mean differences. The following dependent variables showed a decrease in mean values: body weight, body mass index, body fat percentage, waist minimum, cholesterol and insulin. However these differences in results were not practically and statistically significant. The following dependent variables showed an increase in mean values: hip maximum, arm relaxed, arm flexed, thigh circumference, glucose and HOMA-IR. However these results were not practically and statistically significant. The mean differences in the plasma insulin level, pre- to post-test, between both groups indicated that a significant difference (t = -1.77, p = 0.044) existed between them. Cohen‟s d revealed a value of 0.64, which indicates moderate practical significance. The only dependent variable which showed both statistical and practical significance was sum of skinfolds. The findings for sum of skinfolds revealed that the mean differences, from pre- to post-test, between both groups indicated that a significant difference (t = -2.30, p = 0.015) existed between them. Cohen‟s d revealed a value of 0.84, which indicated a large practical significance. Although the sample size was too small to indicate generalisations to the diabetic population as a whole, strength training should be furthermore explored as an alternative and successful modality in the existing range of options available to the health and exercise professional to address the needs of the person with T2DM. The researcher proposed that a bigger sample size be used for the experimental and control group, the intervention period increased as well as various differences related to frequency, intensity and duration of strength training could possibly result in significant changes.
- Full Text:
- Date Issued: 2013
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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