What are some of the syndromes that can occur as a result of mutations in insulin receptors?
Leprechaunism, Rabson-Mendenhall syndrome, Type A insulin resistance
Where is insulin produced?
Beta cells of Islets of Langerhans
What is the immediate effect of insulin secretion into the blood stream?
Glucose uptake into muscle cells and adipocytes
What is the long term effect of insulin?
Increased expression of liver enzymes that synthesise glycogen and of adipocyte enzymes that synthesise triacylglycerides
How is the insulin receptor substrate activated?
Insulin binding to the alpha subunit of the insulin receptor results in autophosphorylation of the IRS
IRS acts as a docking site for many other proteins such as GRB2
GRB2 causes Ras proteins to phosphorylate kinases
Outline the steps on Ras-independent signalling
- PI3 binds to IRS and phosphorylates PI4,5BP to PI3,4,5TP and PI4P to PI3,4BP. This creates a docking site PKB
- PKB is recruited to the membrane and is phosphorylated by membrane associated kinases (e.g. PKD1)
- PKB goes through conformational change to become active and is released where it effects various proteins
How can you identify which genes are activated by insulin signalling?
Track changes in gene expression with microarrays before and after adding insulin to cells in culture
What happens when insulin levels are low?
PKB inhibits FOXO
FOXO binds to IRS near to PEPCK to activate its transcription
What happens when insulin levels are high?
PKB phosphorylates FOXO and inactivates it
Outline the steps of PCR
- Heat to separate strands
- Hybridisation of primers
- Add DNA polymerase, dATP, dGTP, dCTP and dTTP
- DNA synthesis from primers
How can PCR be used to isolate mRNA?
- Take mRNA sequence to be cloned
- Add the first primer, reverse transcriptase and deoxyribonucleoside triphosphates
- Separate strands and add a second primer
- PCR amplification
How do you carry out qPCR?
- Make cDNA from tissue
- Perform PCR in presence of fluorescent DNA dye
- Measure the fluorescence after each PCR cycle
What is promoter bashing?
A technique used to identify how promoters affect transcription of downstream genes
How is promoter bashing carried out?
- Make a transgene that uses a quantifiable reporter
- Luciferase uses a fluorescent substrate that is quantifiable
- Make a series of deletions and test responsiveness to insulin
- Use this in conjunction with EMSA and DNAse I protection
