19 - Insulin Signalling Flashcards
Mutations in the insulin receptor lead to which 3 clinical syndromes?
Leprechaunism
Rabson-Mendenhall syndrome
Type A insulin resistance
What is Leprechaunism?
Fatal within the first 2 years of life
Elfin-like facial appearance - protuberant ears and relatively large hands and feet
A decreased amount of subcutaneous fat and muscle mass
Skin is abnormal with increased hair growth
What is Rabson-Mendenhall syndrome?
Survival into second decade
Skin and teeth abnormalities
Hair overgrowth
Pineal hyperplasia
What is type A insulin resistance?
Survival into middle age and beyond
Where is insulin made and secreted?
Insulin is made in Beta cells of Islets and is secreted into the blood stream
What holds the insulin molecule together?
Disulphide bridges form between cysteines in an oxidative extracellular environment
What is the immediate effect of insulin production?
Glucose uptake from the blood into muscle cells and adipocytes
What is the long term effects of insulin production?
Increased expression of liver enzymes that synthesize glycogen
Increased expression of adipocyte enzymes that synthesize triacylglycerols
How does insulin result in activation of IRS method?
- Alpha and beta subunits are synthesized as a single polypeptide
- This single polypeptide is cleaved into two fragments
- Binding to insulin results in autophosphorylation
- Insulin receptor substrate (IRS) contains a phosphotyrosine binding domain
a. It is highly phosphorylated by the insulin receptor - IRS then acts as a docking site for other proteins
What other proteins does IRS act as a docking site for?
GRB2 to activate the RAS pathway
- Ras proteins phosphorylate kinases
- MAP kinases
- Transcription factors
What are PI-3 kinase subunits?
P85 - SH2 domain
P110 - kinase
Ras-independent signalling method
- After binding to IRS PI-3 kinase phosphorylates
a. PI 4,5-bisphosphate to PI 3,4,5- trisphosphate
b. PI 4-phosphate to PI 3,4-biphosphate - Creates a docking site for protein kinase B
- Once recruited to the membrane PKB is phosphorylated by membrane associated kinases
a. e.g. PKD1 - 3-phosphoinositide-dependent kinase - PKB goes through a confirmational change to become active
- PKB is released and effects many proteins
After binding to IRS PI-3 kinase phosphorylates what?
PI 4,5-bisphosphate to PI 3,4,5- trisphosphate
PI 4-phosphate to PI 3,4-biphosphate
What 4 proteins does PKB effect?
Glycogen synthase kinase (-)
Glucose transporter 4 (+)
FOXO (-)
Phosphoenolpyruvate carboxykinase (glucose synthesis enzyme)
How does insulin effect glycogen synthase kinase?
Insulin inactivates this channel so glycogen synthase can store glycogen
How does insulin effect glucose transporter 4?
Insulin causes translocation of these transporters to pull glucose out of blood
How does insulin effect FOXO?
Insulin inactivates the transcription factor - no longer stimulates PEPCK production
- Glucose synthesis enzyme
How do we identify which genes are activated by insulin signalling?
Track changes in gene expression using microarrays, before and after adding insulin to cells in culture
E.g. PEPCK is inhibited by insulin – happens through PI 3-kinase
For some transcriptional targets of insulin signalling we know the mechanism of regulation - low blood insulin levels
Foxo1 binds to an IRS (insulin response sequences) near to PEPCK
Activates its transcription
For some transcriptional targets of insulin signalling we know the mechanism of regulation - high blood insulin levels
Phosphorylation of Foxo1 by PKB
Inactivates it
What is PCR used for?
Clone DNA from minute samples
Quantify levels of RNA
PCR method
- Double stranded template
- 95oC - separates strands
- 55oC - hybridisation of primers
- 72oC - addition of dNTPS and DNA synthesis from primers
- Repeated through 25+ cycles
How are PCR primers designed?
Designed based upon known sequence
They must face each other and be on opposite strands
Define the target sequence
Where do heat stable DNA polymerases come from?
Most common one comes from Thermus aquaticus bacteria (TAQ)
Which PCR product increases exponentially?
End products after many cycles end up with primers on both end
- They are the exact length of the target sequence
- The amount of these defined size products increases exponentially
What are the two different ways to carry out PCR?
Isolate DNA
Isolate mRNA - reverse transcription polymerase chain reaction – RT-PCR
Quantitative PCR method
- Make cDNA from tissue
- Perform PCR in the presence of a fluorescent DNA dye
- Measure the fluorescence after each PCR cycle
- Machine measuring fluorescence has a threshold it can measure
- Fluorescence increases after each cycle
- Plateaus when dNTPs begin to run out
a. Gives you an idea of how much of your target is in the cDNA
Promoter bashing method
- Make a transgene that uses a quantifiable reporter
a. Example reporter – Luciferase
b. Inserted into cell line or model organism - Luciferase produces a fluorescent substrate that is quantifiable
- Make a series of deletions and test the responsiveness to insulin
Promoter bashing results
If you lose half the region of interest you do not get the same amount of Luciferase expressed
Use this in conjunction with EMSA and DNAse protection
