Insulin Signalling Flashcards
Mutations in the insulin receptor can cause
Leprechaunism - usually fatal within the first two years of life. Elf like facial appearance and large ears hands and feet. They have decreased fat and muscle mass and increased hair growth.
Rabson mendenhall syndrome- only survive into the second decade. Skin and teeth abnormalities and hair overgrowth. Pineal hyperplasia.
Type A insulin resistance causing diabetes and survival into middle age.
Where is insulin made
How is it made
What about disulphide bridges
In beta cells of islets in the pancreas and is secreted into the blood stream.
It is made as a short protein and is then cleaved by a series of proteases (PC3 endopeptidase, PC2, carboxylpeptidase). This makes pro insulin into insulin.
They can form between cysteines on proteins. Only in an oxidative environment so this won’t happen inside a cell. This helps to hold the structure of insulin together.
What are the immediate effects and long term effects of insulin.
The immeadiate effect is stimulation of glucose uptake into muscle cells and adipocytes.
Long term will affect transcription. Increased expression of liver enzymes that synthesise glycogen and of adipocyte enzymes that synthesise triacylglycerols. This is for storing energy.
What family is the insulin receptor in.
What is the structure like
Tyrosine kinase family.
It is a dimer.
Each half of the receptor is synthesised as one whole polypeptide and these are cleaved into two fragments which remain attached by disulphide bridges.
What does binding of insulin to the receptor cause
What is IRS and what does it do
Auto phosphorylation of the two intra cellular kinase domains causing activation of kinase activity.
The two dimers of the receptor are brought together.
Insulin receptor substrate. It works in a similar way to SH2. Contains a phosphotyrosine binding domain and this is phosphorylated by the insulin receptor.
It then acts as a docking site for other proteins.
Structure of PI3 kinase
Intracellular protein that has an SH2 domain on its p85 meaning it binds to phosphotyrosine docking site.
PI3 also has a P85 domain and a PIIO domain which has the kinase activity the PIIO is bigger.
How does insulin binding cause activation of PKB.
How does phosphoinositol get involved
Causes auto phosphorylation of the receptor and created docking sites for IRS.
IRS is phosphorylated and contains docking sites for PI3 kinase.
PI3 kinase will bind and it will be held up near the membrane and is now close enough to phosphorylate phosphoinositol.
This is held in the membrane and when it is phosphorylated it becomes a docking site for PKB.
PKB binds and is activates by phosphorylation by membrane bound kinases such as PKD1.
What happens to PKB when it is activated and what does it go on to do
And how does it cause glucose removal from the blood
PKB is released from the membrane and it goes on to phosphorylate other proteins.
GLUT4 is able to be released from its vesicles and into the cell after being phosphorylated.
This is how glucose is removed from the blood and it is very quick.
What happens when GSK3 is phosphorylated by PKB
What happens if there is too much glucose in the blood
This causes inactivation of GSK3 so it cannot go on to inactivate glycogen synthase by phosphorylating it.
But if we have too much glucose in the blood and we want to activate glycogen synthase to store away the glucose.
Glucose in the blood activated PKB which will inactivate GSK3. This means glycogen synthase will not be repressed and it can store away the extra glucose.
What does PKB do to FOXO
What is FOXOs normal job
If there is high glucose what needs to happen to FOXO
Is this a quick response
FOXO is a TF which is inactivated by PKB
It would go on to activate the gene PEPCK a glucose synthesis enzyme.
We don’t want FOXO to be active and make even more glucose so PKB inhibits FOXO.
This is slow and involves transcription
Can the insulin pathway activate or inactivate transcription
Both
What are IRS sequences and what do they do
How does this work for FOXO
They regulate the genes expression and it is where the TF binds. They are called insulin response sequences.
They can be upstream or downstream of the gene.
FOXO binds to an IRS near the PEPCK gene to activate its transcription where there are low blood insulin levels.
High insulin levels cause phosphorylation of FOXO by PKB which inactivates it.
What can PCR be used for
To amplify DNA and make more copies if it so it can be studied.
To quantify RNA to let us see which of our samples has more of a certain RNA.
What happens in the first cycle of PCR
What is the distance between the two primers called
The template DNA is heated at 95 to separate the strands.
A primer is designed and added at 55.
The DNA is synthesised at 72 and two double strands are made.
Target sequence
What DNA polymerase is used in PCR and where is it from.
How fast is DNA synthesis
How many copies does the second and third cycle make
How many cycles are done overall
It comes from thermus aquatics bacteria.
One or two kilobases per minute because that is how fast the polymerase can move.
Second makes four.
Third makes eight.
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