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Cell and Molecular Biology > LECTURE 19: Insulin signalling > Flashcards

LECTURE 19: Insulin signalling Flashcards

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1
Q

DISEASE: Leprechaunism

A

associated with mutations in the insulin receptor

  • fatal within first 2 years of life
  • large ears, hands and feet
  • decreased amount of subcutaneous fat + muscle mass
  • abnormal skin with increased hair growth
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2
Q

List some clinical syndromes associated with mutations in the insulin receptor

A
  • leprechaunism
  • rabson-mendenhall syndrome
  • type A insulin resistance
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3
Q

Rabson-Mendenhall syndrome

A

associated with mutations in the insulin receptor

  • survival into 2nd decade
  • teeth + skin abnormalities
  • pineal hyperplasia
  • hair overgrowth
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4
Q

Ligands:
(A) are highly processed
(B) are always proteins
(C) always undergo dimerisation

A

(A) are highly processed

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5
Q

Which proteases and peptidases cleave proinsulin into insulin?

A
  • PC3 endopeptidase
  • PC2 endopeptidase
  • carboxypeptidase
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6
Q

DEFINE: proteolysis

A

breakdown of peptides into smaller polypeptides or amino acids

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7
Q

Why are cysteine bridges not found inside the cell?

A

Intracellular environment is a reducing environment

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8
Q

What is the immediate effect of insulin?

A
  1. High blood glucose is recognised
  2. Beta cells in the Islets of Langerhans make and secrete insulin into the blood
  3. insulin binds to receptors on muscle cells and adipocytes - stimulates glucose uptake from the blood
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9
Q

What occurs after long term exposure to insulin?

A
  • increased expression of liver enzymes synthesising glycogen from glucose
  • increased expression of adipocyte enzymes synthesising triacylglycerols = stores energy from the glucose consumed
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10
Q

Insulin receptor structure

A
  • RTK
  • alpha and beta subunits attached by disulphide bridges
  • alpha subunit = extracellular
  • beta subunit = intracellular
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11
Q

Insulin signalling pathway (Ras-dependent signalling)

A
  1. insulin binds
  2. autophosphorylation
  3. kinase activity activated
  4. IRS binds to docking site on insulin receptor via PI domain. insulin receptor phosphorylates IRS.
  5. IRS = docking site for GRB2 and other proteins
  6. GRB2 binds to IRS –> activates Ras pathway –> activates MAP kinases –> transcription
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12
Q

What subunits are PI-3 kinase made up of?

A
  • P85 = contains sh2 domain

- P110 = larger + kinase

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13
Q

What is the structure of phosphoinositol (PI)?

A
  • lipid motif keeping PI in the membrane

- inositol serving as docking site once phosphorylated

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14
Q

FOXO

A

TF that binds to insulin response sequence to activate PEPCK transcription = glucose synthesis enzyme

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15
Q

PI3 kinase/inositol lipid pathway

A
  1. insulin binds to insulin receptor – dimerisation
  2. autophosphorylation of insulin receptor
  3. IRS binds to docking sites on insulin receptor
  4. IRS is phosphorylated by kinase activity of insulin receptor producing docking sites
  5. PI3 kinase recruited to membrane via docking site on IRS
  6. PI3 kinase phosphorylates PI 4,5 bisphosphate to PI 3,4,5 trisphosphate and phosphorylates PI 4-phosphate to PI 3,4 biphosphate - creates docking sites for PKB
  7. PKB recruited to membrane and is phosphorylated by PDK1, activating PKB
  8. PKB phosphorylates other components involved in insulin signalling
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16
Q

How would you identify transcriptional targets of the insulin pathway?

A
  1. take cells in tissue culture
  2. control group and group of cells with insulin added
  3. purify rna
  4. track changes in gene expression using microarrays before and after adding insulin to cells in culture
17
Q

How would you identify the sequences that transcription factors bind?

A
  • EMSA = electromobility shift assay
  • DNAse I footprinting
  • promoter bashing
18
Q

What technique is used to quantify RNA levels?

A

qPCR

19
Q

How quickly does DNA synthesis from primers take place?

A

1-2kb per minute

20
Q

Quantitative PCR

A
  1. isolate mRNA from tissue
  2. convert to cDNA using reverse transcriptase
  3. use cDNA as a template for PCR. perform PCR in the presence of a fluorescent DNA dye
  4. DNA dye is integrated into PCR product as it is made
  5. measure quantity of target by measuring fluorescence
21
Q

Promoter bashing

A
  1. start with a clone of a genomic fragment containing promoter of interest, gene of interest and sequences upstream, downstream and far away
  2. replace gene with luciferase in place of start codon of the gene
  3. insert gene into cell line or model organism - transgene made
  4. delete away parts of the promoter to find the critical region required to have 100% responsiveness

Cell and Molecular Biology (20 decks)

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