Glucose Sensing and Insulin Signalling

Question 1

What is the normal blood glucose concentration?

Answer 1

4-5 mM

Question 2

At what blood glucose concentration does brain activity become compromised?

Answer 2

Below 1-2 mM, the brain cannot sustain its activity (glucose-dependent so glucose homeostasis very important for brain)

Question 3

What happens if glucose concentration rises to 7-8 mM for an extended period of time?

Answer 3

. Proteins become glycated

. CNS and PNS nerve damage occurs

Question 4

How can you monitor glycaemic control in Type 1 Diabetics?

Answer 4

Concentration of HbA1c (glycated haemoglobin) can be used as a measure of long-term glycaemic control

Question 5

How many times does each channel protein (GLUT) span a membrane? What implications does this have?

Answer 5

12 times, which means that the cytoplasmic C and N termini are on the same side of the membrane (all GLUTs have same topology)

Question 6

Where is GLUT1 found?

Answer 6

In RBCs and blood brain barrier

Question 7

Where is GLUT2 found?

Answer 7

In the liver, pancreas, and intestines

Question 8

Where is GLUT3 found?

Answer 8

In the brain, neurones, and sperm

Question 9

Where is GLUT4 found?

Answer 9

In skeletal muscle, heart, and adipose

Question 10

Which GLUT transporters are insulin-dependent? Which are insulin-independent?

Answer 10

GLUT1,2,3 are insulin-independent, GLUT4 is insulin-dependent

Question 11

What is the role of the SGLT1 transporter? When do they take effect?

Answer 11

. Co-transporter in the small intestine involved in the co-transport of glucose with sodium, bringing glucose into cells of the s.intestine from the lumen against their concentration gradient
. Takes effect during fasting state when glucose concentration in lumen of intestine lower than that in the intestinal cells and blood

Question 12

What is the role of the GLUT2 transporter in the small intestine?

Answer 12

. In the fed state, calcium ions enter cells of the small intestine, triggering GLUT2 to insert into the cell membranes
. GLUT2 has a high Km (low affinity for glucose), so only transports glucose when the concentration of glucose in the lumen is high (i.e. in the fed state)
. Glucose is transported into the intestinal cells via GLUT2 (before SGLT1 transporter needed when concentration gradient reversed in fasting state)

Question 13

Which transporter do pancreatic β-cells have in their membrane?

Answer 13

GLUT2

Question 14

Describe the role and functionality of glucokinase in pancreatic β-cells

Answer 14

. Not inhibited by its product (G6P) in β-cells, unlike in the liver
. High Km (low affinity) and not inhibited by G6P means that it can keep producing G6P

Question 15

Describe how the entry of glucose into a pancreatic β-cell leads to insulin release.

Answer 15

. Glucose enters β-cell via GLUT2 transporter
. Glucokinase converts glucose to G6P (which goes on to yield ATP in glycolysis)
. High ATP:ADP ratio causes potassium ion channels in cell membrane to open and potassium ions diffuse out of cell down their concentration gradient
. This depolarises the cell membrane, triggering the opening of calcium ion channels
. Calcium ions diffuse into the β-cell, which stimulates the release of insulin-containing vesicles

Question 16

What is the function of SUR1? Why is it clinically important?

Answer 16

. SUR1 is a chaperone protein that promotes the insertion of potassium ion channels into β-cell membranes
. SUR1 reduces the Km (increases affinity) of potassium ion channels for ATP, so that the potassium ion channels open at lower ATP concentrations
. This means that more potassium ions leave the cell, more depolarisataion occurs, more calcium ion channels open, more calcium ions enter the cell, and more insulin is released
. This is clinically important because many drugs target SUR1 (e.g. activate it more to increase insulin production and lower blood glucose)

Question 17

Describe the structure of insulin.

Answer 17

. Polypeptide consisting of two chains (A and B), bound together by disulphide bridges (A chain also has disulphide bridge within its chain)
. Signal sequence present in chain

Question 18

What is the purpose of the signal sequence in insulin?

Answer 18

. Allows insulin to bind to endoplasmic reticulum to be processed
. It is cut off in the ER as it has no other function beyond this point

Question 19

Describe the structure of an insulin receptor.

Answer 19

. Consists of 4 subunits, 2 alpha (extracellular, ligand-binding) and 2 beta (embedded in membrane with tyrosine kinase activity)
. Two identical insulin binding sites (essentially a dimer) which can be activated by one insulin monomer
. Subunits linked by disulphide bridges

Question 20

Where are insulin receptors present?

Answer 20

Present everywhere because used for effects beside lowering blood glucose e.g. pro-growth
Different numbers of receptors in different areas depending on necessity e.g. RBCs don’t have many but adipocytes and hepatocytes have lots

Question 21

Why are diabetics advised to change the site of insulin injection from time to time?

Answer 21

. Insulin can promote cell division so if you inject it in the same place over a long period of time, there will be increased cell growth and a mass may arise

Question 22

How are insulin receptors (IRs) activated? What happens to the beta-subunits where this activation occurs?

Answer 22

. Insulin binds to the alpha subunit (outside membrane) of IR, causing a conformational change on the beta subunit (embedded in membrane)
. Beta subunit tyrosine kinase domains transphosphorylate each other

Question 23

What happens after the tyrosine kinase domains phosphorylate eachother?

Answer 23

. The tyrosine kinases act as docking sites for other proteins to bind to (e.g. IRS proteins and other proteins which all contain phosphotyrosine, which allows them to bind to TK)
. The proteins are phosphorylated

Question 24

What is the point of IRS proteins binding to TK domains?

Answer 24

. Act as docking sites to bind to other proteins and enzymes (i.e. PI3 kinase or Grb2)
. Signal from the original binding of insulin to the IR is amplified

Question 25

What do IRS proteins do when bound to insulin receptors?

Answer 25

. Two routes: one involving PI3 kinase, one involving Grb2

. IRS proteins recruit PI3 kinase, which contains SH2 domains which bind phosphotyrosine (IRS proteins also bind phosphotyrosine)
. PI3K phosphorylates PIP2to PIP3, and PIP3 activates AKT. AKT phosphorylates RGC, inactivating it so GLUT4 can insert into the membrane

. IRS proteins recruit Grb2, which activates SOS, which activates Ras GTPase
. Ras stimulates Raf, which activates ERK to promotes pro-growth

Question 26

Describe the structure and function of PI3 kinase.

Answer 26

. Two domains: P85 (binding domain) and P110 (catalystic domain)
. PI3 kinase phosphorylates PIP2 (a lipid in the plasma membrane) to convert it to PIP3

Question 27

What is the function of PIP3?

Answer 27

. Docking site for proteins containing PH domains
. Phosphorylates AKT
- AKT phosphorylates RGC
- Phosphorylation of RGC inactivates it to allow the insertion of GLUT4 into the plasma membrane of muscle and adipocyte cells (in its dephosphorylated state, RGC inhibits insertion of GLUT4 into the plasma membrane)

Question 28

Which enzyme antagonises PI3 kinase? What does this enzyme do?

Answer 28

. PTEN

. Phosphatase enzyme that dephosphorylates PIP3, converting it back to PIP2

Question 29

Name three cancer hotspot enzymes involved in insulin signalling

Answer 29

PI3 kinase, PTEN, and Ras GTPase (due to its involvement in pro-growth)

Question 30

What is Ras GTPase activated by and which kinase does it stimulate?

Answer 30

. Ras is activated by SOS (activated by Grb2, which is recruited by IRS proteins)
. Ras stimulates Raf, which activates ERK (kinase that promotes cell division)

Question 31

Which insulin signalling pathway is involved in glucose uptake and which pathway is involved in pro-growth?

Answer 31

. Glucose uptake- When IRS protein binds PI3 kinase, resulting in the conversion of PIP2 to PIP3. PIP3 recruits AKT, which phosphorylates RGC, inactivating it to allow the insertion of GLUT4 into the plasma membrane (muscle, heart, adipose)

. Pro-growth- IRS protein binds Grb2, which recruits SOS, which activates RasGTPase. RasGTPase recruits Raf, which activates ERK to promote cell division and pro-growth activities

Question 32

What is the function of RasGTPase?

Answer 32

. Stimulates Raf (a kinase), which activates ERK (a pro-growth kinase)

Question 33

What are the TORC and GSK3 kinases involved in?

Answer 33

. TORC involved in protein synthesis

. GSK3 involved in glycogen synthesis

Question 34

What effect do phosphatases have?

Answer 34

Phosphatases ‘switch off’ kinases, dephosphorylating proteins/enzymes (usually inactivates them)

Question 35

What do kinases do?

Answer 35

Phosphorylate things (usually activates them)

Question 36

Which kind of kinases result in activation in insulin signalling? Which result in deactivation?

Answer 36

. Kinases bound to tyrosine (tyrosine kinases) result in activation (i.e. Raf, ERK, TORC, GSK3 all bind to tyrosine domains and perpetuate insulin signalling from IRS)
. Kinases bound to other regions (serine/threonine kinases) result in deactivation in a similar way that phosphatases cause deactivation

Question 37

Describe the factors that activate serine/threonine kinases? What happens when these kinases are activated by these factors?

Answer 37

. Factors often associated with obesity and diabetes: Lipotoxicity, inflammation, hyperglycaemia, mitochondria dysfunction, ER stress activate serine/threonine kinases
. Serine/threonine kinases switch off the insulin signalling pathways so that GLUT4 doesn’t insert (=hyperglycaemia) and less cell division

Question 38

Why is diabetes often referred to as ‘chronic inflammation’?

Answer 38

Because inflammation inhibits the insulin pathway by activating serine/threonine kinases, causing resistance to insulin because GLUT4 not inserted into membrane in response to insulin. This is essentially Type II Diabetes.

Question 39

What are the holistic results of insulin signalling?

Answer 39

. Decrease blood glucose (Increased glycolysis, glycogenesis, cholesterol synthesis, FA and TAG synthesis, VLDL synthesis, decrease gluconeogenesis, FA oxidation, lipolysis)
. Increase protein synthesis and pro-survival/cell growth signals