Lecture 14 - Insulin Signalling Flashcards
How was insulin discovered?
Removed pancreas from dogs, dogs become diabetic, at this point they failed to identify and purify insulin
Banting and Best were able to purify an extract form the pancreas that reversed diabetes
Isletin was the original name for insulin, on injection this caused a drop-in blood glucose
Before insulin the only treatment was a VLCD, which lead to patients becoming severely malnourished
What are the effects of Insulin?
Regulation of ion flux
Stimulation receptor tyrosine kinase
Stimulation of receptor-substrate
Stimulation of glucose transport
Stimulation of enzyme activity (Glycogen synthase and pyruvate)
Stimulation of amino acid transport
Stimulation of protein synthesis
Regulation of gene expression
Stimulation of cell division
Insulin signalling is both acute and chronic
Insulin signalling can cause cell wide effects
How is insulin produced and what is its structure?
Insulin is produced as a single chain of 81-86 amino acids, it is not released as soon as it is made it is stored and processed
The final molecule consists of 51 amino acids in two chains joined by disulphide bonds
Insulin is stored in a hexatrimeric form complex with zinc
Insulin is first made as preproinsulin, then in the RER a 24 amino acid signal peptide is cleaved, the protein then folds and 3 sulphide bonds are made, creating proinsulin
Then in the Golgi, C-peptide is cleaved by Carboxypeptide-E and pro hormone convertase 1 and 2, which creates insulin which is then packaged into secretory granules
What is hyperglycaemia?
Increased glucose, stimulates insulin release, inhibits glucagon release
How do blood glucose levels change after a carbohydrate meal?
After around 30 minutes our blood glucose and insulin levels will have risen
Insulin declines in parallel with decline in blood glucose, eventually get to a point where we want to release glucose, which is when glucagon is released
Glucagon and insulin work as a balanced pair to oppose each other
How does glucose stimulate insulin secretion?
Extracellular glucose concentrations regulate insulin secretions
Glucokinase functions functions as the glucose sensor for insulin secretion
Glucokinase is a protein in the glycolysis pathway that phosphorylates glucose to glucose 6p
Glucose stimulate glucose-dependent insulin exocytosis
Glucose enters cells via Glut2
Once in the cell glucose is phosphorylated to glucose-6-phosphate, and then it is further metabolised by glycolysis and mitochondrial oxidation to generate ATP/ADP
The increase in ATP causes the Potassium-ATP channel to close
The closer of the Potassium-ATP channel causes the plasma membrane to become depolarised
Depolarisation of the membrane causes the calcium channel to open (Voltage Dependent Calcium Channel)
Once the calcium channel is open calcium ions rush into the cell
The increase of calcium in the cell causes endocytosis of the insulin granule, so insulin is released
What is the structure of the Insulin Receptor?
Consists of 2 alpha subunits joined by disulphide bonds and 2 beta subunits which are joined to the alpha subunits by disulphide bonds
The beta subunits pass through the membrane, resulting in an alpha helical portion of the membrane
Similar to enzyme linked receptors
What happens when different tyrosine residues are phosphorylated in Beta Subunit of Insulin Receptor?
Different tyrosine residues can be phosphorylated, each having a different function
What happens when tyrosine 960 is phosphorylated?
Required for substrate binding
What happens when tyrosine 1146, 1150 1151 are phosphorylated?
Phosphorylation leads to kinase activity
What happens when tyrosine 1293, 1294, 1336 are phosphorylated?
Weakens (attenuates) kinase activity
What happens when tyrosine 1316 and 1322 are phosphorylated?
May be associated with growth signal
What is IRS?
Insulin Receptor Substrate - It is what the insulin receptor works on
What band does IRS run as on a gel?
180-185kDa
What does the activated receptor to do IRS?
Phosphorylates on tyrosine
Phosphorylation on series may inhibit action
What does the IRS containing?
Phosphotyrosine binding (PTB) and PH domain
What is the function of the PH domain?
Can interact with lipids, which allows it to connect to the plasma membrane
What does the IRS become a docking protein for?
Proteins that contain SH2 domains
What do SH2 domains do?
Src Homology 2 Domain - Bind phosphotyrosine residues surrounded by unique protein sequences
What do SH3 domains do?
Src Homology 3 Domain - Bind specific to proline rich regions
Explain the Ras-dependent pathway:
Grb2 is a SH2/SH3 domain containing protein which interacts with SOS (Son of Sevenless)
SOS acts as a GDP/GTP exchange factor, then Ras is activated once bound GTP
This drives differentiation, survival and growth
Grb2 binds IRS because IRS has a Sh2 domain, so it can bind the phosphotyrosine on the insulin receptor substrate
Grb2 also has a Sh3 domain so it can also interact with the proline rich region on SOS which acts as a GDP/GTP exchange factor for Ras
GDP release activated by Ras-GEF
What is GDP release from Ras activated by?
Ras-GEF
Explain the Ras-independent pathway:
Leads to the production of Akt (Protein Kinase B), which leads to GLUT-4 transporters being translocated to the plasma membrane
Rad-Independent pathway cascade:
IRS interacts with PIK3 which then activates AKt which then activates the cascade
GLUT4 are glucose transporters that are activated to translocate to the cell membrane from Akt being produced in the Ras-independent pathway
Act when activated has many effects
Act acts on GSK3 (glucose synthase kinase), when this protein is phosphorylated it is in active
When GSK3 is active it inhibits glycogen synthase
When Akt phosphorylates GSK3 it becomes inactive, so it can no longer inhibit glycogen synthase, so we get increased production of glycogen
Akt also acts of mTor which regulates protein synthesis
Akt also activates PDE3B and HSL which controls the levels of fatty acids and glycerol
Explain the glucose cycle:
Removal of lactate from muscle to liver reduces “metabolic burden” on muscle
If glucose is in blood, it is taken up into cells and converted into pyruvate in glycolysis
But in muscle cells the glycose will be used to power them and be converted to lactate
So, this lactate needs to be recycled, so it is taken up by the kidney and liver cells, converted back to pyruvate and it then re-enters the glucose cycle, and it can then re-enter the blood
When is glucagon used?
When blood glucose levels are low, and we want glucose to be used
What else does glucagon act on?
Fat cells
What are adipocytes?
They are cells that store fats, but they also have to be able to release it
Glucagon binds to its GPCR which activates adenylyl cyclase
cAMP is made from ATP which is then able to activate protein kinase, which then activates the enzyme triacylglycerol cyclase which is able to break down triacylglycerol
What happens in diabetes?
We get: Increased free fatty acid levels Increased beta oxidation Pyruvate dehydrogenase is inhibited by acetyl-CoA Increased fatty acids to muscle Increase citrate cycle activity
What is diabetes?
Failure of insulin to lower blood glucose
Insulin fails to stop the liver producing glucose, and it fails to stimulate the muscle and adipocytes to take up glucose
What is the safe range of blood glucose levels?
2mM to 7mM
