2. Introduction to Lipids and Lipoprotein Metabolism (Part III) Flashcards
How can we know where a protein is found within a cell?
Attach a green fluorescent protein (GFP) to create a hybrid
Where is the SWEET transporter found?
In the plasma membrane, according to the GFP
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?
- 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)
What do LDLR transgenic mice (overexpression of LDLR) fed a low cholesterol diet display?
Low blood LDL cholesterol
What do LDLR transgenic mice (overexpression of LDLR) exhibit in terms of hypercholesterolemia?
Resistance to diet-induced hypercholesterolemia
What does size-exclusion chromatography allow for? How?
- Separation of particles based on their size
- Large particles cannot enter the matrix and are excluded
- Small particles enter the matrix
What kind of particles elute first in size-exclusion chromatography? Why elute later?
- Large first
- Small later
Which lipoproteins would come out first in size-exclusion chromatography?
- Chylomicrons
- VLDL
- IDL/LDL
- HDL
How are the VLDL and LDL levels of mice? Why?
- Low VLDL
- NO LDL
- Because they are very efficient at getting rid of lipoproteins in blood
What kind of lipoproteins does a non-transgenic mice consuming a low-fat diet contain?
ONLY HDL
What kind of lipoproteins does a non-transgenic mice consuming a high-fat diet contain?
- Accumulation of VLDL, IDL/LDL, and HDL
- There is NO spike in LDL, as they are extremely efficient
Transgenic mice display a low level of HDL. Would they develop atherosclerosis?
No, since there is no circulating LDL
What kind of lipoproteins does a transgenic mice consuming a low-fat diet contain?
- Much less LDL
- Because they express more LDLR, which increases the LDL pickup by the liver cells
What kind of lipoproteins does a transgenic mice consuming a high-fat diet contain?
- Little change in the amount of VLDL, and HDL
- They are resistant to hypercholesterolemia
What does targeted gene disruption permit?
- Very specific mutations (gene deficiencies) to be created
- Allows direct assessment of gene function
Differentiate global mutations and local mutations.
- Global: whole animal
- Local: specific tissues, or even specific cells
Describe the mechanism for targeted gene disruption.
- Embryonic stem cells are collected from the blastocysts
- They are grown in a dish
- Targeting vector (DNA) is directed into a specific site
- Cell is screened, isolated, and injected into a blastocyst
What does a targeting vector induce?
Induces a disruption of the local gene
What is the manipulated ES injected in?
Into the blastocysts
Differentiate a heterozygote and a chimera.
- Heterozygote: contains complete genomes of the same background
- Chimera: contains two genetic backgrounds
What does the production of a chimera from a wild-type genome mouse and a modified genome mouse tell us?
- 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
What do we do to the mice that display a modified genome?
They are bred so we can uncover their particularities as a colony
What was the proof of concept of what we find in human FH?
Targeted disruption of the LDLR gene in mice
What do we need to do to go past the association between LDLR and FH?
- Start with normal
- Take out the gene encoding for the LDLR in mice
- Reproduce the disorder to have a proof of concept
How are mice naturally resistant to diet-induced hypercholesterolemia?
Cyp7a1 gene
What is a difference between human and mouse hypercholesterolemia?
Atherosclerosis is premature in humans, but mice are only more susceptible
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?
- 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
What are LXRs activated by? When would they be stimulated?
- Activated by oxysterols
- Stimulated when cellular oxysterol concentrations increase
What genes involved in sterol and fat metabolism are controlled by LXR-alpha?
- Cyp7a1 (stimulates)
- SREBP-1c (stimulates)
- ABCA1 (stimulates)
- ABCG5/G8 (stimulates)
- ACAT (inhibits)
- NPC1L1 (inhibits)
What is the effect of targeted disruption of the LXR-alpha gene in mice in terms of hepatic lipids?
- Liver mass increases (fatty liver)
- Cholesterol concentration increases substantially
- NO effect on TG metabolism
How does the abundance of Cyp7a1 change if they are fed a diet high in cholesterol?
Increases
How does the abundance of Cyp7a1 change if the mice undergo targeted disruption of the LXR-alpha gene?
- No difference
- Without LXR-alpha, there is no change
Which SREBP is embryonic lethal? What does that tell you?
- SREBP-2
- Means that it is very important in the functioning of cells
Where is NPC1L1 expressed at high levels in humans and mice?
- Liver and small intestine (humans)
- Mainly the small intestine (mice)
What is the function of ezetimibe?
- Competitive inhibitor of NPC1L1
- Competes with cholesterol for uptake by enterocytes
What is the function of ABCG5/G8?
Getting rid of cholesterol from enterocytes
What is the function of NPC1L1?
Bringing in cholesterol from enterocytes
What does free cholesterol become within the enterocyte to induce LXR activity? What does that increase?
- Oxysterol
- ABCG5/G8
What transporter does free cholesterol induce directly to get rid of cholesterol?
ABCA1
Do bile acids have an impact on cholesterol absorption in mice?
Probably not too much
How much cholesterol do mice absorb due to NPC1L1?
80% (20% does not go through NPC1L1)
Is the remaining 20% of cholesterol that is not absorbed by NPC1L1 affected by ezetimibe?
No
