Insulin signalling (L5) Flashcards
What diseases (and symptoms) do mutations in insulin signalling lead to?
Leprechaunism - usually fatal within the first 2 years of life. - Elfin like facial features with large hands and feet. Decreased subcutaneous fat and muscle and increased hair growth.
Rabson-Mendenhall syndrome (survival into 2nd decade) skin and teeth abnormalities, hair overgrowth and pineal hyperplasia.
Type A insulin resistance - survival into middle age and beyond with the right treatment
How is insulin produced?
Insulin is made in beta cells in the islets of Langerhans in the pancreas and is secreted into the bloodstream. PC2 and 3 endoproteases cleave the proinsulin, and 2 arginines are then removed to make insulin. DIsulphide bridges form between 2 cysteines in an oxidative (extracellular) environment (intracellular is reductive) so it enables it to be secreted.
What is the effect of insulin?
Immediate effect - glucose moves from the blood into muscle cells and adipocytes to reduce the levels of sugar in the blood.
Insulin results in the activation of IRS. The insulin receptor is made of alpha and beta subunits. They’re first synthesised as a single polypeptide which is cleaved into 2 fragments, however, they stay held together through disulphide bonds. Binding of insulin to its receptor results in autophosphorylation. Insulin receptor substrate (IRS) contains a phosphotyrosine binding domain (PTB) and is highly phosphorylated by the insulin receptor. IRS then acts as a docking site for many other proteins, such as GRB2 to activate the RAS pathway.
Explain the Ras-independent signalling pathway that insulin works through?
PI-3 kinase is made up of 2 subunits: p85 which has an SH2 domain and P110 (a kinase) - the PI-3 pathway is independent to the RAS pathway but is also activated by insulins signalling. Upon binding to IRS, PI-3 kinase phosphorylates Pi-4, 5-biphosphate to Pi3,4,5 triphosphate and Pi4 to Pi4,5 biphosphate. This creates a docking site for PKB (protein kinase B). Once recruited to the membrane PKB is phosphorylated by a membrane-associated kinase (PKD1) which causes fusion with the membrane. PKB then goes through a conformational change to become active, and is then released and affects numerous proteins. Activation of insulin causes translocation and has an immediate effect (no new products need to be made so is very quick). FOXO is a TF that activates PEPCK (a glucose synthesis enzyme). So this is inhibited when glucose is high. GSK3 inhibits glycogen synthase so when glucose is high, insulin signalling inhibits GSK3 so you can increase glucose to glycogen conversion.
How can you identify genes involved in insulin signalling?
The insulin pathway is highly studied. You can identify the genes activated by insulin signalling by tracking changes in gene expression using microarrays. For some transcriptional targets of insulin signalling, we also know the exact mechanism of regulation E.g. when FOXO binds to an IRS near the PEPCK to activate its transcription. High levels of insulin lead to phosphorylation of FOXO1 by PKB (which inactivates it)
PCR is used to amplify DNA for research. You can clone DNA from small samples or quantify veles of RNA (qPCR)
To do this, you design primers based on known sequences. They must face each other and be on opposite strands- this defines the target sequence. You heat with DNA polymerase. You can also combine this with reverse transcriptase to make cDNA.
Explain how qPCR works.
- make cDNA from tissue
- perform PCR in the presence of a fluorescent DNA Dye
- Measure the fluorescence after each PCR cycle.
Enables you to quantify how much DNA you have.
Explain promoter bashing and what it’s used for.
When you take out chunks to find critical regions. Complementation methods. You make a transgene with a quantifiable receptor.
Luciferase produces a fluorescent substrate which is quantifiable. Then you make a series of deletions and test the responsiveness to insulin. USe with EMSA and DNAse. to identify how certain regions of a DNA strand, commonly promoters, affect the transcription of downstream genes. Under normal circumstances, proteins bind to the promoter and activate or repress transcription. In a promoter bashing assay, specific point mutations or deletions are made in specific regions of the promoter and the transcription of the gene is then measured. The contribution of a region of the promoter can be observed by the level of transcription. If a mutation or deletion changes the level of transcription, then it is known that that region of the promoter may be a binding site or another regulatory element
