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.
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- Date Issued: 2013
Mutational analysis of the PacC binding sites within the aflR promoter in Aspergillus flavus
- Authors: Suleman, Essa
- Date: 2011
- Subjects: Mutation (Biology) , Genetic regulation , Proteins -- Synthesis , Microbiological synthesis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10336 , http://hdl.handle.net/10948/d1012683 , Mutation (Biology) , Genetic regulation , Proteins -- Synthesis , Microbiological synthesis
- Description: It is generally known that media containing simple sugars (sucrose, glucose) and organic nitrogen sources (ammonium) when buffered to acidic pH stimulates aflatoxin production in Aspergillus flavus & A. parasiticus while lactose, nitrate and an alkaline pH inhibit aflatoxin biosynthesis. It has been shown that pH of the growth medium is the most important regulatory factor for aflatoxin biosynthesis since media containing stimulatory carbon and/or nitrogen sources (sucrose and ammonia) do not enhance aflatoxin (or sterigmatocystin) production at alkaline pH. RNA interference (in A. flavus) of the pH regulatory transcription factor, PacC, resulted in aflatoxin production under acidic and alkaline pH conditions whilst wildtype Aspergillus flavus produced aflatoxins only under acidic conditions. This conclusively proved that PacC negatively regulates aflatoxin production at alkaline pH in A. flavus. However the exact mechanism involved in PacC repression of aflatoxin biosynthesis at alkaline pH still remains unknown. The AflR protein is essential for expression of several genes in the aflatoxin biosynthetic cluster. In the current study, sequence analysis of the aflR promoter indicated the presence of two putative PacC binding sites within the aflR promoter of A. flavus 3357WT located at positions -162 and -487 bp from the start codon. The presence of the PacC binding sites in the aflR promoter indicated a possible link between aflR expression and PacC regulation under alkaline conditions. Thus, in this study, it was hypothesized that at alkaline pH, PacC inhibits aflR expression by binding to one or both of the PacC binding sites within the aflR promoter. This in turn, would result in inhibition of aflatoxin biosynthesis since expression of several aflatoxin biosynthetic pathway genes is dependent on activation by AflR. The aim and objective of this study was to test the validity of this hypothesis i.e. that at alkaline pH PacC binds to one or both of its recognition sites within the aflR promoter thereby inhibiting aflR expression which subsequently would result in inhibition of aflatoxin biosynthesis. This was done by first mutating each individual and then both PacC binding sites in the A. flavus 3357 aflR promoter via Single-Joint PCR (SJ-PCR) and fusing the wildtype and each mutated aflR promoter to the Green Fluorescent Protein (gfp) gene and the trpC terminator to yield a functional expression vector. These constructs were then transformed into A. flavus 3357.5. Positive transformants were confirmed to express GFP by fluorescence microscopy and spectrofluorometry. Quantification of GFP protein levels of the various transformants in this study indicated that PacC negatively regulated aflR promoter activity at alkaline pH. RT-qPCR was performed on positive transformants after growth on SLS medium at acidic and alkaline pH to determine if PacC negatively regulated aflR promoter activity at alkaline pH and to determine whether PacC binds preferentially to one or both recognition sites within the aflR promoter. RT-qPCR analysis suggest that PacC binds non-preferentially to both recognition sites within the aflR promoter on sucrose and lactose media at alkaline pH, although mutation of PacC binding site 2 results in a slightly higher expression compared to mutation of PacC binding site 1. Increasing the concentration of an aflatoxin conducive nitrogen source stimulated aflR promoter activity but this was not sufficient to overcome negative regulation by PacC. It is generally known that repression of aflR expression results in repression of aflatoxin biosynthesis irrespective of pH. The results of this study strongly suggest that PacC negatively regulates aflR promoter activity at alkaline pH by binding to one or both PacC recognition sites within the aflR promoter. Since aflR promoter activity is repressed by PacC at alkaline pH, this substantiates the hypothesis that PacC represses aflatoxin biosynthesis by inhibiting expression of aflR. Furthermore, the results of this study indicated that there may be some PacC protein present in the active form at acidic pH irrespective of the carbon source and nitrogen source used in the growth medium. RT-qPCR analysis indicated that any active PacC present at acidic pH may cause repression of the aflR promoter based on the position of the PacC binding site relative to the aflR start codon, although it appears that PacC may have a higher affinity for PacC binding site 2 (which is closer to the aflR start codon).
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- Date Issued: 2011