WEEK 9 Insulin Signalling & Resistance

Question 1

2. Explain how hormone specificity is achieved in insulin signalling.

Answer 1

Model Answer:
Hormone specificity arises from the cell-specific expression of hormone receptors. The insulin receptor is uniquely expressed in insulin-responsive tissues, ensuring only those cells respond to insulin. Ligand-receptor binding is highly specific due to the structural compatibility of the ligand with its receptor binding site. For example, insulin binds only to the insulin receptor and not to PDGF receptors, and vice versa.

Question 2

1. What is insulin resistance and how does it contribute to metabolic disease?

Answer 2

Model Answer:
Insulin resistance is a pathological state where cells in target tissues such as muscle, liver, and adipose tissue no longer respond adequately to insulin. Despite insulin being present, glucose uptake in muscle is reduced, lipolysis in fat remains active, and hepatic glucose production is not suppressed. These dysfunctions lead to hyperglycemia, elevated free fatty acids, and contribute to conditions like type 2 diabetes and metabolic syndrome.

Question 3

3. Describe the role of protein phosphorylation in insulin signal transduction.

Answer 3

Model Answer:
Protein phosphorylation is central to insulin signalling. Upon insulin binding, the insulin receptor undergoes autophosphorylation on tyrosine residues, initiating a cascade. This creates binding sites for adapter proteins such as IRS1. IRS1 gets phosphorylated, providing docking sites for downstream effectors like PI3K. This leads to further phosphorylation events on serine/threonine residues, ultimately activating kinases like AKT that drive cellular responses like glucose uptake, glycogen synthesis, and inhibition of lipolysis.

Question 4

4. How does phosphorylation change protein-protein interactions in insulin signalling?

Answer 4

Model Answer:
Phosphorylation of tyrosine residues on IRS1 creates binding sites for SH2 and PTB domain-containing proteins (e.g., PI3K, Grb2, Nck). These domains specifically recognize phosphorylated tyrosines, allowing the recruitment and assembly of signalling complexes at the plasma membrane. These interactions are essential for propagating the signal downstream and are dynamically regulated by kinases and phosphatases.

Question 5

5. Explain how insulin signalling affects protein localisation.

Answer 5

Model Answer:
Insulin signalling causes significant changes in protein localisation. For example:
* PI3K is recruited to the plasma membrane via its SH2 domain binding phosphorylated IRS1.
* AKT translocates to the membrane through its PH domain binding PIP3.
* FOXO, a transcription factor, is phosphorylated by AKT, causing it to exit the nucleus and suppress transcription of gluconeogenic genes.
* GLUT4 vesicles are translocated to the plasma membrane, allowing glucose uptake.

Question 6

6. Outline the insulin signalling cascade and the role of AKT.

Answer 6

Model Answer:
1. Insulin binds to the insulin receptor → autophosphorylation.
2. IRS1 is phosphorylated → recruits PI3K.
3. PI3K converts PIP2 to PIP3.
4. PIP3 recruits AKT and PDK1 to the membrane.
5. AKT is phosphorylated and activated → phosphorylates multiple targets:
o AS160 → GLUT4 translocation
o PDE3B → inhibits lipolysis
o FOXO → nuclear exclusion, inhibits gluconeogenesis
o GSK3 → activation of glycogen synthase
AKT acts as a central hub for insulin-mediated effects.

Question 7

7. What are key defects in insulin signalling during insulin resistance?

Answer 7

Model Answer:
In insulin resistance:
* IRS1 phosphorylation is impaired or misdirected.
* GLUT4 translocation to the plasma membrane is defective.
* AKT activation is diminished.
* Lipolysis is not suppressed, leading to elevated free fatty acids.
* FOXO remains active in the nucleus, promoting gluconeogenesis.
Overall, this is due to a re-wiring of the insulin signalling network, not a single defective node, as revealed by phosphoproteomics studies.

Question 8

8. How has phosphoproteomics changed our understanding of insulin resistance?

Answer 8

Model Answer:
Phosphoproteomics, using mass spectrometry to measure phosphorylation across thousands of proteins, has revealed that insulin signalling involves a vast network of dynamically regulated phosphorylation events. In insulin resistance, it’s not a single defect but a global rewiring of signalling networks, with altered phosphorylation patterns on multiple proteins, reflecting systemic dysregulation across metabolic tissues.

Question 9

9. Explain how live-cell fluorescence microscopy can be used to study insulin signalling.

Answer 9

Model Answer:
Live-cell fluorescence microscopy uses GFP-tagged domains to observe real-time changes in protein localisation. Examples include:
* GFP-tagged PH domains show AKT recruitment to the membrane upon insulin stimulation (indicating PIP3 production).
* FOXO-mNeonGreen shows insulin-induced nuclear export of FOXO.
* pH-sensitive GLUT4-pHluorin shows vesicle fusion with the plasma membrane after insulin treatment.
These tools visualize the dynamics of protein localisation changes driven by insulin.

Question 10

10. Describe the experimental setup used to study insulin resistance in mice.

Answer 10

Model Answer:
* Mice from five genetic strains were fed either a chow or high-fat Western diet.
* Mice received insulin or saline injections.
* Soleus muscles were collected, and glucose uptake was assessed using radiolabeled 2-deoxyglucose.
* Phosphoproteomics was performed to quantify phosphorylation events.
Findings showed strain- and diet-dependent insulin resistance, with widespread phosphorylation changes, confirming signalling pathway rewiring.