L19 - Insulin Signalling Flashcards
Leprechaunism is fatal after
Within the first two years of life
Describe symptoms seen in leprechaunism
Elfin like facial appearance with protuberant ears and relatively large hands and feet
Decreased ammount of subcuatneous fat and muscle mass
Skin abnormal with increased hair growth
Rabson-Mendenhall syndrome - life expect.
Survival into the second stage
Describe symptoms of Rabson-mendenhall syndrome
Skin and teeth abdnormalities, hair over growth and pineal hyperplasia
How long do people with type A insulin resistance survive
Into the middle age and beyond
Where is inuslin produced
In the beta cells of the islets
Then secreted into the bloodstream
What forms between _______ in insulin production
What environment does this require
Disulphide bridges form between cysteines
This requires an oxidative environment
What is the immediate effect of insulin release
glucose uptake from the blood into muscle cells and adipocytes
What is the effect of long term exposure to insulin
Increased expression of liver enzymes that synthesise glycogen and of adipocyte enzymes that synthesise triacyglycerols
Insulin results in activation of
IRS
Describe the synthesis of the two subunits of insulin
Aloha and beta subunits are synthesised as a single polypeptide which is then cleaved into two fragments
Binding of insulin to the receptor results in
Autophosphorylation
IRS stands for
What does it contain
What happens to this domain
Insulin receptor substrate
Phosphotyrosine binding domain (PTB)
This domain is highly phosphorylated by the insulin receptor
IRS acts as a …
Docking sites for many other protein such as GRB2 to activate the RAS pathway
What is PI-3 kinase made up of two subunits
P85
P110
P85 contains a
SH2 domain
P110 is a
Kinase
Describe what occurs when Pi3 kinase binds to IRS
Pi3 kinase phosophrylates Pi-4,5-biphosphate and Pi-4-phosphate
What happens when:
Pi-4-5-biphosphate is phroylated
Pi-4-phosphate is phosphorylated
Pi-3,4,5-triphosphate
Pi-3,4-biphosphate
Formation of Pi-4,5-BP and Pi-4-P creates a binding site for
Protein kinase B
Describe what haappens once protein kinase B is recruited to the membrane
What does this cause to happen
Phosphorylated by membrane associated kinases
Causes a confirmation change to become active and is then released and effects numerous proteins
What is the effect of active PKB on
GLycogen synthase kinase
Glucose transporter
FOXO
Decrease
Increase
Decrease
Phosphoenolpyruvate carboxykinase is what sort of enzyme
Glucose synthesis enzyme
Describe on of the way in which we can identify genes that are activated by insulin signalling
Can track changes in gene expression using microarrays comparing to before and after insluin is added to the cells in culture
Describe the action of FOXO when blood insulin levels are low
Foxo binds to IRES near to PEPCK to activate its transcription
Describe what occurs with FOXO1 at high blood insulin levels
Phosphorylation of FOXO1 by protein kinase B which inactivates it
What is PCR
Used to amplify the DNA
PCR is used to
Clone DNA from minute samples and to quantify the levels of RNA
For the first sequence describe how primers are designed
Based upon a known sequence - they must face each other and be on opposite strands thus defining the target sequence
What type of DNA polymerase must be used with PCR - where is this obtained from
Heat stable DNA polymerase
From the thermus aquaticus bacteria (TAQ)
How many ds strands produced after 1 orund of PCR
What about 2 cycles
What about 3 cycles
2
4
8
Describe the porcess of PCR
Heat DNA - causes the separation of the strands
Primers anneal
DNA synthesis by polymerase from the primer template junction
After 3 rounds of synthesis how many strands are there of the target sequence
8 (2 ds strands and 4 ss strands)
Describe how PCr may be used to make genomic clones
Identify DNA to be cloned
Sep strands and add primers
PCr amplification
Describe how PCR may be used to make cDNA clones
Isloate the mRNA
Add first primer and reverse transcriptase
DNTPs also required to be added
Separate strands - RNA/cDNA hybrid and add the second primer
Amplify with PCR
Descirbe quantitative PCR
make cDNA from tissue
Perform PCR in the presence of a fluoresecent DNA dye
Measure the fluoresecnce after each PCR cycle
what technique can be used an ana;yse the mechanism of trnascription regulation
Promoter bashing
Describe how promoter bashing is performed
Make a transgene that uses a quantifiable reporter then make a series of deletions - test the activity of reporter to identify the critical regions of regulation
Why is luciferase a good reporter for promoter bashing
An insulin receptor - produces a flourescent substrate which is quantifiable
What can promoter bashing be used in conjunction with
EMSA and DNAseI protection
