Insulin Signalling

Question 1

What are some of the syndromes that can occur as a result of mutations in insulin receptors?

Answer 1

Leprechaunism, Rabson-Mendenhall syndrome, Type A insulin resistance

Question 2

Where is insulin produced?

Answer 2

Beta cells of Islets of Langerhans

Question 3

What is the immediate effect of insulin secretion into the blood stream?

Answer 3

Glucose uptake into muscle cells and adipocytes

Question 4

What is the long term effect of insulin?

Answer 4

Increased expression of liver enzymes that synthesise glycogen and of adipocyte enzymes that synthesise triacylglycerides

Question 5

How is the insulin receptor substrate activated?

Answer 5

Insulin binding to the alpha subunit of the insulin receptor results in autophosphorylation of the IRS
IRS acts as a docking site for many other proteins such as GRB2
GRB2 causes Ras proteins to phosphorylate kinases

Question 6

Outline the steps on Ras-independent signalling

Answer 6

1. PI3 binds to IRS and phosphorylates PI4,5BP to PI3,4,5TP and PI4P to PI3,4BP. This creates a docking site PKB
2. PKB is recruited to the membrane and is phosphorylated by membrane associated kinases (e.g. PKD1)
3. PKB goes through conformational change to become active and is released where it effects various proteins

Question 7

How can you identify which genes are activated by insulin signalling?

Answer 7

Track changes in gene expression with microarrays before and after adding insulin to cells in culture

Question 8

What happens when insulin levels are low?

Answer 8

PKB inhibits FOXO

FOXO binds to IRS near to PEPCK to activate its transcription

Question 9

What happens when insulin levels are high?

Answer 9

PKB phosphorylates FOXO and inactivates it

Question 10

Outline the steps of PCR

Answer 10

1. Heat to separate strands
2. Hybridisation of primers
3. Add DNA polymerase, dATP, dGTP, dCTP and dTTP
4. DNA synthesis from primers

Question 11

How can PCR be used to isolate mRNA?

Answer 11

1. Take mRNA sequence to be cloned
2. Add the first primer, reverse transcriptase and deoxyribonucleoside triphosphates
3. Separate strands and add a second primer
4. PCR amplification

Question 12

How do you carry out qPCR?

Answer 12

1. Make cDNA from tissue
2. Perform PCR in presence of fluorescent DNA dye
3. Measure the fluorescence after each PCR cycle

Question 13

What is promoter bashing?

Answer 13

A technique used to identify how promoters affect transcription of downstream genes

Question 14

How is promoter bashing carried out?

Answer 14

1. Make a transgene that uses a quantifiable reporter
2. Luciferase uses a fluorescent substrate that is quantifiable
3. Make a series of deletions and test responsiveness to insulin
4. Use this in conjunction with EMSA and DNAse I protection