2. Introduction to Lipids and Lipoprotein Metabolism (Part III)

Question 1

How can we know where a protein is found within a cell?

Answer 1

Attach a green fluorescent protein (GFP) to create a hybrid

Question 2

Where is the SWEET transporter found?

Answer 2

In the plasma membrane, according to the GFP

Question 3

How did scientists construct a synthetic transgene to direct the overexpression of human LDLR in the liver of mice? What was particular about this sequence?

Answer 3

• Portion of the LDLR as genomic sequence from the 5’ end
• Middle: RNA
• Portion of the LDLR as genomic sequence from the 3’ end
• The sequence is MUCH smaller (1/10)

Question 4

What do LDLR transgenic mice (overexpression of LDLR) fed a low cholesterol diet display?

Answer 4

Low blood LDL cholesterol

Question 5

What do LDLR transgenic mice (overexpression of LDLR) exhibit in terms of hypercholesterolemia?

Answer 5

Resistance to diet-induced hypercholesterolemia

Question 6

What does size-exclusion chromatography allow for? How?

Answer 6

• Separation of particles based on their size
• Large particles cannot enter the matrix and are excluded
• Small particles enter the matrix

Question 7

What kind of particles elute first in size-exclusion chromatography? Why elute later?

Answer 7

• Large first

- Small later

Question 8

Which lipoproteins would come out first in size-exclusion chromatography?

Answer 8

• Chylomicrons
• VLDL
• IDL/LDL
• HDL

Question 9

How are the VLDL and LDL levels of mice? Why?

Answer 9

• Low VLDL
• NO LDL
• Because they are very efficient at getting rid of lipoproteins in blood

Question 10

What does targeted gene disruption permit?

Answer 10

• Very specific mutations (gene deficiencies) to be created

- Allows direct assessment of gene function

Question 11

What is the manipulated ES injected in?

Answer 11

Into the blastocysts

Question 12

Differentiate a heterozygote and a chimera.

Answer 12

• Heterozygote: contains complete genomes of the same background
• Chimera: contains two genetic backgrounds

Question 13

What does the production of a chimera from a wild-type genome mouse and a modified genome mouse tell us?

Answer 13

• That the participation of the ES cell donor genome was limited, because the progeny mouse is mostly white
• Only portions of the mouse were formed by the ES cell that was manipulated

Question 14

What do we do to the mice that display a modified genome?

Answer 14

They are bred so we can uncover their particularities as a colony

Question 15

What was the proof of concept of what we find in human FH?

Answer 15

Targeted disruption of the LDLR gene in mice

Question 16

What do we need to do to go past the association between LDLR and FH?

Answer 16

• Start with normal
• Take out the gene encoding for the LDLR in mice
• Reproduce the disorder to have a proof of concept

Question 17

How are mice naturally resistant to diet-induced hypercholesterolemia?

Answer 17

Cyp7a1 gene

Question 18

What is a difference between human and mouse hypercholesterolemia?

Answer 18

Atherosclerosis is premature in humans, but mice are only more susceptible

Question 19

What happens when there is targeted gene disruption of the LDLR gene in mice when they are fed a low-fat diet? What about when they are fed a high-fat diet?

Answer 19

• They exhibit an increase in LDL concentration in the blood

- The response is elevated when they are fed a high-cholesterol and high-fat diet

Question 20

What are LXRs activated by? When would they be stimulated?

Answer 20

• Activated by oxysterols

- Stimulated when cellular oxysterol concentrations increase

Question 21

What is the effect of targeted disruption of the LXR-alpha gene in mice in terms of hepatic lipids?

Answer 21

• Liver mass increases (fatty liver)
• Cholesterol concentration increases substantially
• NO effect on TG metabolism

Question 22

How does the abundance of Cyp7a1 change if they are fed a diet high in cholesterol?

Answer 22

Increases

Question 23

How does the abundance of Cyp7a1 change if the mice undergo targeted disruption of the LXR-alpha gene?

Answer 23

• No difference

- Without LXR-alpha, there is no change

Question 24

Which SREBP is embryonic lethal? What does that tell you?

Answer 24

• SREBP-2

- Means that it is very important in the functioning of cells

Question 25

Where is NPC1L1 expressed at high levels in humans and mice?

Answer 25

• Liver and small intestine (humans)

- Mainly the small intestine (mice)

Question 26

What is the function of ezetimibe?

Answer 26

• Competitive inhibitor of NPC1L1

- Competes with cholesterol for uptake by enterocytes

Question 27

What is the function of ABCG5/G8?

Answer 27

Getting rid of cholesterol from enterocytes

Question 28

What is the function of NPC1L1?

Answer 28

Bringing in cholesterol from enterocytes

Question 29

How much cholesterol do mice absorb due to NPC1L1?

Answer 29

80% (20% does not go through NPC1L1)

Question 30

Is the remaining 20% of cholesterol that is not absorbed by NPC1L1 affected by ezetimibe?

Answer 30

No