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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
Q

What domains does protein kinase B have?

A

pH domain

Kinase domain

26
Q

What are the 4 proteins that PKB phosphorylates?

Which contribute to the immediate responses and which contribute to longer-term responses

A

1) Glycogen Synthase Kinase (GSK)
- Immedaite

2) GLUcose Transporter 4 (GLUT4)
- Immediate

3) FOXO
- Longer-term

4)PhosphoEnolPyruvate carboxyKinase (glucose synthesis enzyme)

27
Q

What happens to GLUT 4 when phosphorylated by PKB? (at high levels of glucose)

A
  • 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
Q

What happens to GSK when phosphorylated by PKB? (at high levels of glucose)

What does this cause?

A
  • 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
Q

What is FOXO?

What does it activate?

A

A transcription factor

Activates PEPCK (Phosphoenolpyruvate carboxykinase)

30
Q

What does PEPCK do?

A

Promotes glucose synthesis

31
Q

What happens to FOXO when phosphorylated by PKB? (at high levels of glucose)

What does this cause?

A
  • Inhibition of FOXO by phosphorylation by PKB

- Inactivation of PEPCK, less glucose synthesis

32
Q

How can we identify genes that are activated by insulin signalling?

A
  • 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
Q

What determines if the insulin pathway activates or inactivates transcription?

A

Depends upon the enhancers located near the genes

34
Q

What happens to FOXO when sugar in the blood is low?

A

FOXO1 binds to IRS near PEPCK
Activate PEPCK
Stimulating glucose synthesis

35
Q

What are IRS and where are they present?

A

Insulin response sequences - present on DNA

36
Q

What 3 methods can be used to identify where transcription factors bind on DNA?

A

1) ESMA
2) DNAse footprinting I
3) PCR

37
Q

What 2 things can PCR do?

A

1) Amplify very small amounts of DNA - to be able to study

2) Quantify levels of RNA

38
Q

Describe the process of PCR

A

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
Q

How are the primers direct DNA synthesis?

A

Towards each other

40
Q

What is the region between the 2 DNA primers?

A

The target sequence

41
Q

Where does the DNA polymerase used in PCR come from?

A

Thermus aquaticus bacteria (TAQ)

42
Q

What sized DNA does PCR not work for?

A

DNA above 10,000bp

43
Q

What is difference between cycles 1 and 2 and cycle 3 in PCR?

A

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
Q

What occurs to the target sequence after cycle 3?

A

Amount of that sequence increases exponentially and is more common than the longer DNA

45
Q

How are the fragments of PCR separated?

A

Electrophoresis in a gel

46
Q

What is the process of quantitive PCR?

A

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
Q

What is the idea behind qPCR?

A
  • 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
Q

What is promoter bashing?

A

A way of looking at the genes regulatory sequence

To identify the transcription factor binding sites

49
Q

What is promoter bashing used in conjunction with?

A

ESMA

DNAse footprining I

50
Q

What is the process of promoter bashing?

A

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
Q

In promoter bashing, why do you start with a GENOMIC clone of the gene of interest?

A

Contains the promoter of interest

52
Q

What does Luciferase do?

A

Encodes an enzyme which produces fluorescence when the substrate is added

53
Q

Why in promoter bashing is the gene replaced with Luciferase?

A
  • 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
Q

In the case of an insulin responsive gene, what happens to Luciferase expression after insulin has been added?

A

It is maximally activated

55
Q

What does promoter bashing of an insulin responsive gene show?

A
  • 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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