19 - Insulin Signalling

Question 1

Mutations in the insulin receptor lead to which 3 clinical syndromes?

Answer 1

Leprechaunism
Rabson-Mendenhall syndrome
Type A insulin resistance

Question 2

What is Leprechaunism?

Answer 2

Fatal within the first 2 years of life
Elfin-like facial appearance - protuberant ears and relatively large hands and feet
A decreased amount of subcutaneous fat and muscle mass
Skin is abnormal with increased hair growth

Question 3

What is Rabson-Mendenhall syndrome?

Answer 3

Survival into second decade
Skin and teeth abnormalities
Hair overgrowth
Pineal hyperplasia

Question 4

What is type A insulin resistance?

Answer 4

Survival into middle age and beyond

Question 5

Where is insulin made and secreted?

Answer 5

Insulin is made in Beta cells of Islets and is secreted into the blood stream

Question 6

What holds the insulin molecule together?

Answer 6

Disulphide bridges form between cysteines in an oxidative extracellular environment

Question 7

What is the immediate effect of insulin production?

Answer 7

Glucose uptake from the blood into muscle cells and adipocytes

Question 8

What is the long term effects of insulin production?

Answer 8

Increased expression of liver enzymes that synthesize glycogen
Increased expression of adipocyte enzymes that synthesize triacylglycerols

Question 9

How does insulin result in activation of IRS method?

Answer 9

1. Alpha and beta subunits are synthesized as a single polypeptide
2. This single polypeptide is cleaved into two fragments
3. Binding to insulin results in autophosphorylation
4. Insulin receptor substrate (IRS) contains a phosphotyrosine binding domain
   a. It is highly phosphorylated by the insulin receptor
5. IRS then acts as a docking site for other proteins

Question 10

What other proteins does IRS act as a docking site for?

Answer 10

GRB2 to activate the RAS pathway

• Ras proteins phosphorylate kinases
• MAP kinases
• Transcription factors

Question 11

What are PI-3 kinase subunits?

Answer 11

P85 - SH2 domain

P110 - kinase

Question 12

Ras-independent signalling method

Answer 12

1. After binding to IRS PI-3 kinase phosphorylates
   a. PI 4,5-bisphosphate to PI 3,4,5- trisphosphate
   b. PI 4-phosphate to PI 3,4-biphosphate
2. Creates a docking site for protein kinase B
3. Once recruited to the membrane PKB is phosphorylated by membrane associated kinases
   a. e.g. PKD1 - 3-phosphoinositide-dependent kinase
4. PKB goes through a confirmational change to become active
5. PKB is released and effects many proteins

Question 13

After binding to IRS PI-3 kinase phosphorylates what?

Answer 13

PI 4,5-bisphosphate to PI 3,4,5- trisphosphate

PI 4-phosphate to PI 3,4-biphosphate

Question 14

What 4 proteins does PKB effect?

Answer 14

Glycogen synthase kinase (-)
Glucose transporter 4 (+)
FOXO (-)
Phosphoenolpyruvate carboxykinase (glucose synthesis enzyme)

Question 15

How does insulin effect glycogen synthase kinase?

Answer 15

Insulin inactivates this channel so glycogen synthase can store glycogen

Question 16

How does insulin effect glucose transporter 4?

Answer 16

Insulin causes translocation of these transporters to pull glucose out of blood

Question 17

How does insulin effect FOXO?

Answer 17

Insulin inactivates the transcription factor - no longer stimulates PEPCK production
- Glucose synthesis enzyme

Question 18

How do we identify which genes are activated by insulin signalling?

Answer 18

Track changes in gene expression using microarrays, before and after adding insulin to cells in culture
E.g. PEPCK is inhibited by insulin – happens through PI 3-kinase

Question 19

For some transcriptional targets of insulin signalling we know the mechanism of regulation - low blood insulin levels

Answer 19

Foxo1 binds to an IRS (insulin response sequences) near to PEPCK
Activates its transcription

Question 20

For some transcriptional targets of insulin signalling we know the mechanism of regulation - high blood insulin levels

Answer 20

Phosphorylation of Foxo1 by PKB

Inactivates it

Question 21

What is PCR used for?

Answer 21

Clone DNA from minute samples

Quantify levels of RNA

Question 22

PCR method

Answer 22

1. Double stranded template
2. 95oC - separates strands
3. 55oC - hybridisation of primers
4. 72oC - addition of dNTPS and DNA synthesis from primers
5. Repeated through 25+ cycles

Question 23

How are PCR primers designed?

Answer 23

Designed based upon known sequence
They must face each other and be on opposite strands
Define the target sequence

Question 24

Where do heat stable DNA polymerases come from?

Answer 24

Most common one comes from Thermus aquaticus bacteria (TAQ)

Question 25

Which PCR product increases exponentially?

Answer 25

End products after many cycles end up with primers on both end

• They are the exact length of the target sequence
• The amount of these defined size products increases exponentially

Question 26

What are the two different ways to carry out PCR?

Answer 26

Isolate DNA

Isolate mRNA - reverse transcription polymerase chain reaction – RT-PCR

Question 27

Quantitative PCR method

Answer 27

1. Make cDNA from tissue
2. Perform PCR in the presence of a fluorescent DNA dye
3. Measure the fluorescence after each PCR cycle
4. Machine measuring fluorescence has a threshold it can measure
5. Fluorescence increases after each cycle
6. Plateaus when dNTPs begin to run out
   a. Gives you an idea of how much of your target is in the cDNA

Question 28

Promoter bashing method

Answer 28

1. Make a transgene that uses a quantifiable reporter
   a. Example reporter – Luciferase
   b. Inserted into cell line or model organism
2. Luciferase produces a fluorescent substrate that is quantifiable
3. Make a series of deletions and test the responsiveness to insulin

Question 29

Promoter bashing results

Answer 29

If you lose half the region of interest you do not get the same amount of Luciferase expressed
Use this in conjunction with EMSA and DNAse protection