Lecture 14 - Insulin Signalling

Question 1

How was insulin discovered?

Answer 1

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

Question 2

What are the effects of Insulin?

Answer 2

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

Question 3

How is insulin produced and what is its structure?

Answer 3

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

Question 4

What is hyperglycaemia?

Answer 4

Increased glucose, stimulates insulin release, inhibits glucagon release

Question 5

How do blood glucose levels change after a carbohydrate meal?

Answer 5

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

Question 6

How does glucose stimulate insulin secretion?

Answer 6

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

Question 7

What is the structure of the Insulin Receptor?

Answer 7

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

Question 8

What happens when different tyrosine residues are phosphorylated in Beta Subunit of Insulin Receptor?

Answer 8

Different tyrosine residues can be phosphorylated, each having a different function

Question 9

What happens when tyrosine 960 is phosphorylated?

Answer 9

Required for substrate binding

Question 10

What happens when tyrosine 1146, 1150 1151 are phosphorylated?

Answer 10

Phosphorylation leads to kinase activity

Question 11

What happens when tyrosine 1293, 1294, 1336 are phosphorylated?

Answer 11

Weakens (attenuates) kinase activity

Question 12

What happens when tyrosine 1316 and 1322 are phosphorylated?

Answer 12

May be associated with growth signal

Question 13

What is IRS?

Answer 13

Insulin Receptor Substrate - It is what the insulin receptor works on

Question 14

What band does IRS run as on a gel?

Answer 14

180-185kDa

Question 15

What does the activated receptor to do IRS?

Answer 15

Phosphorylates on tyrosine

Phosphorylation on series may inhibit action

Question 16

What does the IRS containing?

Answer 16

Phosphotyrosine binding (PTB) and PH domain

Question 17

What is the function of the PH domain?

Answer 17

Can interact with lipids, which allows it to connect to the plasma membrane

Question 18

What does the IRS become a docking protein for?

Answer 18

Proteins that contain SH2 domains

Question 19

What do SH2 domains do?

Answer 19

Src Homology 2 Domain - Bind phosphotyrosine residues surrounded by unique protein sequences

Question 20

What do SH3 domains do?

Answer 20

Src Homology 3 Domain - Bind specific to proline rich regions

Question 21

Explain the Ras-dependent pathway:

Answer 21

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

Question 22

What is GDP release from Ras activated by?

Answer 22

Ras-GEF

Question 23

Explain the Ras-independent pathway:

Answer 23

Leads to the production of Akt (Protein Kinase B), which leads to GLUT-4 transporters being translocated to the plasma membrane

Question 24

Rad-Independent pathway cascade:

Answer 24

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

Question 25

Explain the glucose cycle:

Answer 25

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

Question 26

When is glucagon used?

Answer 26

When blood glucose levels are low, and we want glucose to be used

Question 27

What else does glucagon act on?

Answer 27

Fat cells

Question 28

What are adipocytes?

Answer 28

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

Question 29

What happens in diabetes?

Answer 29

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

Question 30

What is diabetes?

Answer 30

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

Question 31

What is the safe range of blood glucose levels?

Answer 31

2mM to 7mM