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BMS 242 - Advanced Cell and Molecular > Insulin Signalling > Flashcards

Insulin Signalling Flashcards

1
Q

What receptors does insulin signalling involve?

A

Receptor tyrosine kinases

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2
Q

What type of conditions do mutations in insulin signalling result in?

What function are these mutations involved with?

A

Inherited conditions

Not always involved with sugar metabolism - other functions too (especially growth)

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3
Q

What are 3 conditions arising from mutations in insulin signalling?

A

1) Leprechaunism
2) Rabson-Mendenhall syndrome
3) Type A insulin resistance

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4
Q

What is the phenotype of Leprechaunsim and what is the life expectancy?

A
  • ‘elfin-like’ facial appearance
  • Large hands and feet
  • Decreased subcutaneous fat and muscle mass
  • Skin abnormal
  • Increased hair growth

Fatal within the first 2 years of life

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5
Q

What is the phenotype of Rabson-Mendenhall syndrome and what is the life expectancy?

A
  • Skin and teeth abnormalities
  • Hair outgrowth
  • Pineal hyperplasia

Survival into 2nd decade

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6
Q

Where is insulin made?

A

In Beta cells of the Islets of Langerhans

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7
Q

How is insulin made?

A

1) Synthesised as a short protein

2) Cleaved to a mature protein using proteases to remove the middle portion of protein

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8
Q

What are the proteases which cleave the insulin protein?

A

PC2, PC3, endoproteases, carboxypeptidase

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9
Q

Where are cysteine bridges formed and why can they form?

A

Present extracellularly

Cysteine have sulphur atom attached - oxidative environment of extracellular region allows disulphide bridges to form

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10
Q

Why can cysteine bridges not form intracellularly?

A

The intracellular environment is reductive

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11
Q

What ensures that the correct cysteine bridges form?

A

Enzymes in the ER

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12
Q

What is the function of cysteine bridges in insulin?

A

Hold the 2 ends of insulin together, after the middle portion has been cleaved out

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13
Q

What is the immediate effect of insulin?

A

Glucose uptake from the blood into muscle cells and adipocytes

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14
Q

What happens when there is long-term exposure to insulin?

A

Effects on transcription:
- Increased expression of liver enzymes that synthesise glycogen

  • Increased expression of adipocyte enzymes that synthesise triacylgylcerols
  • Stores the energy of glucose
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15
Q

What type of receptors is the insulin receptor?

A

RTK

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16
Q

How are the alpha and beta subunits of the insulin receptor synthesised?

How does it create the different subunits?

A

As a single polypeptide

Cleaved into 2 fragments

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17
Q

How is the insulin receptor different to canonical TK signalling?

A

Normal signaling involves phosphorylating many regions of the receptor to make docking sites for many different proteins

Insulin signalling - TK goes on to phosphorylate IRS which acts as the docking site, bound to the insulin receptor through a phosphorylated tyrosine

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18
Q

How is IRS held to the insulin receptor?

A
  • By its docking site through the PTB domain (phosphotyrosine binding domain)
  • Recognises the phosphoylated tyrosine on the insulin receptor and neighbouring amino acids
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19
Q

How is IRS held to the insulin receptor?

A
  • By its docking site through the PTB domain (phosphotyrosine binding domain)
  • Recognises the phosphoylated tyrosine on the insulin receptor and neighbouring amino acids
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20
Q

What is the structure of PI-3 kinase?

A

2 subunits:

  • P85 - containing the SH2 domain
  • P110 (larger) - kinase
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21
Q

What is phosphoinositol (PI)?

A

An intracellular molecule with a lipid anchor inserted into the membrane

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22
Q

When PI is phosphorylated, what can it act as?

A

A docking site

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23
Q

What happens to PI-3 kinase when insulin is bound to its receptor and why?

A

It is recruited to the membrane - binds to IRS which is phosphorylated by the insulin receptor

24
Q

What happens when PI-3 kinase binds to IRS?

A

Creates docking sites for protein kinase B, recruiting it to the membrane by:

1) Phosphorylating PI 4,5-biphosphoglycerate –> PI 3,4,5-trisphosphate
2) Phosphorylating PI 4-phosphate –> PI 3,4-biphosphate

25
What domains does protein kinase B have?
pH domain | Kinase domain
26
What are the 4 proteins that PKB phosphorylates? Which contribute to the immediate responses and which contribute to longer-term responses
1) Glycogen Synthase Kinase (GSK) - Immedaite 2) GLUcose Transporter 4 (GLUT4) - Immediate 3) FOXO - Longer-term 4)PhosphoEnolPyruvate carboxyKinase (glucose synthesis enzyme)
27
What happens to GLUT 4 when phosphorylated by PKB? (at high levels of glucose)
- Vesicles that contain GLUT4 (in an inactive state) fuse with the plasma membrane - GLUT4 fuse with the plasma membrane, more transport proteins in the membrane, pull glucose out of the blood
28
What happens to GSK when phosphorylated by PKB? (at high levels of glucose) What does this cause?
- GSK is inactivated by phosphorylation by PKB - GSK normally phosphorylates glycogen synthesis to inactivate it - So, at high glucose levels, PKB is activated, GSK is inactivated - This releases the inactivation of glucose synthase - Glucose is stored as glycogen
29
What is FOXO? What does it activate?
A transcription factor Activates PEPCK (Phosphoenolpyruvate carboxykinase)
30
What does PEPCK do?
Promotes glucose synthesis
31
What happens to FOXO when phosphorylated by PKB? (at high levels of glucose) What does this cause?
- Inhibition of FOXO by phosphorylation by PKB | - Inactivation of PEPCK, less glucose synthesis
32
How can we identify genes that are activated by insulin signalling?
- Track changes in gene expression before and after adding insulin to cells in culture (using microarrays) - Identify genes that are upregulated and downregulated - Identify which branch of signalling that transduces the insulin signalling
33
What determines if the insulin pathway activates or inactivates transcription?
Depends upon the enhancers located near the genes
34
What happens to FOXO when sugar in the blood is low?
FOXO1 binds to IRS near PEPCK Activate PEPCK Stimulating glucose synthesis
35
What are IRS and where are they present?
Insulin response sequences - present on DNA
36
What 3 methods can be used to identify where transcription factors bind on DNA?
1) ESMA 2) DNAse footprinting I 3) PCR
37
What 2 things can PCR do?
1) Amplify very small amounts of DNA - to be able to study | 2) Quantify levels of RNA
38
Describe the process of PCR
1) Start with a DNA template with part of the sequence known 2) Design 2 primers that bind to the sequence (one in each direction) 3) Heat the DNA to 95 degrees to denature the H bonds 4) Cool to 55 degrees to allow DNA to bind with primers 5) Heat to 72 degrees with a special DNA polymerase - allow DNA synthesis
39
How are the primers direct DNA synthesis?
Towards each other
40
What is the region between the 2 DNA primers?
The target sequence
41
Where does the DNA polymerase used in PCR come from?
Thermus aquaticus bacteria (TAQ)
42
What sized DNA does PCR not work for?
DNA above 10,000bp
43
What is difference between cycles 1 and 2 and cycle 3 in PCR?
In cycles 1 and 2: - Produce DNA that is longer than the target sequence In cycle 3: - Produce DNA that is the same length as the target sequence
44
What occurs to the target sequence after cycle 3?
Amount of that sequence increases exponentially and is more common than the longer DNA
45
How are the fragments of PCR separated?
Electrophoresis in a gel
46
What is the process of quantitive PCR?
1) Extract all the RNA from a given tissue 2) Perform rtPCR - Round 1: RNA --> DNA by reverse transcriptase - Followed by amplification of DNA by PCR 3) Once amplified, perform PCR in the presence of a fluorescent dye, which only binds to dsDNA 4) Produce a graph (number of cycles vs fluorescence) 5) Can compare different tissues (different graphs)
47
What is the idea behind qPCR?
- Direct relationship between the rate at which the PCR product is generated and the original concentration of the mRNA of interest - Higher concentration of starting mRNA = quicker for the fluorescence to appear
48
What is promoter bashing?
A way of looking at the genes regulatory sequence | To identify the transcription factor binding sites
49
What is promoter bashing used in conjunction with?
ESMA | DNAse footprining I
50
What is the process of promoter bashing?
1) Start with a GENOMIC CLONE of the gene of interest 2) Replace the gene with a reporter (Luciferase) 3) Insert this construct into a cell line or model organism (create a transgene) 4) Clone 5) Make a series of deletions and test the responsiveness to insulin
51
In promoter bashing, why do you start with a GENOMIC clone of the gene of interest?
Contains the promoter of interest
52
What does Luciferase do?
Encodes an enzyme which produces fluorescence when the substrate is added
53
Why in promoter bashing is the gene replaced with Luciferase?
- So this reporter is now under the control of the all the regulatory sequences of interest (including promoter of interest) - Certain levels of Luciferase at a given time
54
In the case of an insulin responsive gene, what happens to Luciferase expression after insulin has been added?
It is maximally activated
55
What does promoter bashing of an insulin responsive gene show?
- A critical region in the promoter which is needed to have insulin responsiveness at a 100% level (remove this portion - responsiveness to insulin is reduced dramatically) - Highlights the DNA important in the regulation of the gene

BMS 242 - Advanced Cell and Molecular (24 decks)

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