LECTURE 19: Insulin signalling Flashcards
DISEASE: Leprechaunism
associated with mutations in the insulin receptor
- fatal within first 2 years of life
- large ears, hands and feet
- decreased amount of subcutaneous fat + muscle mass
- abnormal skin with increased hair growth
List some clinical syndromes associated with mutations in the insulin receptor
- leprechaunism
- rabson-mendenhall syndrome
- type A insulin resistance
Rabson-Mendenhall syndrome
associated with mutations in the insulin receptor
- survival into 2nd decade
- teeth + skin abnormalities
- pineal hyperplasia
- hair overgrowth
Ligands:
(A) are highly processed
(B) are always proteins
(C) always undergo dimerisation
(A) are highly processed
Which proteases and peptidases cleave proinsulin into insulin?
- PC3 endopeptidase
- PC2 endopeptidase
- carboxypeptidase
DEFINE: proteolysis
breakdown of peptides into smaller polypeptides or amino acids
Why are cysteine bridges not found inside the cell?
Intracellular environment is a reducing environment
What is the immediate effect of insulin?
- High blood glucose is recognised
- Beta cells in the Islets of Langerhans make and secrete insulin into the blood
- insulin binds to receptors on muscle cells and adipocytes - stimulates glucose uptake from the blood
What occurs after long term exposure to insulin?
- increased expression of liver enzymes synthesising glycogen from glucose
- increased expression of adipocyte enzymes synthesising triacylglycerols = stores energy from the glucose consumed
Insulin receptor structure
- RTK
- alpha and beta subunits attached by disulphide bridges
- alpha subunit = extracellular
- beta subunit = intracellular
Insulin signalling pathway (Ras-dependent signalling)
- insulin binds
- autophosphorylation
- kinase activity activated
- IRS binds to docking site on insulin receptor via PI domain. insulin receptor phosphorylates IRS.
- IRS = docking site for GRB2 and other proteins
- GRB2 binds to IRS –> activates Ras pathway –> activates MAP kinases –> transcription
What subunits are PI-3 kinase made up of?
- P85 = contains sh2 domain
- P110 = larger + kinase
What is the structure of phosphoinositol (PI)?
- lipid motif keeping PI in the membrane
- inositol serving as docking site once phosphorylated
FOXO
TF that binds to insulin response sequence to activate PEPCK transcription = glucose synthesis enzyme
PI3 kinase/inositol lipid pathway
- insulin binds to insulin receptor – dimerisation
- autophosphorylation of insulin receptor
- IRS binds to docking sites on insulin receptor
- IRS is phosphorylated by kinase activity of insulin receptor producing docking sites
- PI3 kinase recruited to membrane via docking site on IRS
- PI3 kinase phosphorylates PI 4,5 bisphosphate to PI 3,4,5 trisphosphate and phosphorylates PI 4-phosphate to PI 3,4 biphosphate - creates docking sites for PKB
- PKB recruited to membrane and is phosphorylated by PDK1, activating PKB
- PKB phosphorylates other components involved in insulin signalling
How would you identify transcriptional targets of the insulin pathway?
- take cells in tissue culture
- control group and group of cells with insulin added
- purify rna
- track changes in gene expression using microarrays before and after adding insulin to cells in culture
How would you identify the sequences that transcription factors bind?
- EMSA = electromobility shift assay
- DNAse I footprinting
- promoter bashing
What technique is used to quantify RNA levels?
qPCR
How quickly does DNA synthesis from primers take place?
1-2kb per minute
Quantitative PCR
- isolate mRNA from tissue
- convert to cDNA using reverse transcriptase
- use cDNA as a template for PCR. perform PCR in the presence of a fluorescent DNA dye
- DNA dye is integrated into PCR product as it is made
- measure quantity of target by measuring fluorescence
Promoter bashing
- start with a clone of a genomic fragment containing promoter of interest, gene of interest and sequences upstream, downstream and far away
- replace gene with luciferase in place of start codon of the gene
- insert gene into cell line or model organism - transgene made
- delete away parts of the promoter to find the critical region required to have 100% responsiveness
