Topic 2 Flashcards
(86 cards)
1
Q
What are the two sources of cholesterol?
A
endogenously and exogenously (diet)
2
Q
Describe the mechanism of endogenous cholesterol synthesis.
A
acetyl coA:
1. HMG coA
2. ketone body -> HMG coA synthetase -> HMG coA
use HMG coA reductase to make melavonic acid
-statins competively inhibit HMG coA since it is the rate limiting enxyme/step to cholesterol synthesis
3
Q
Which cells can make cholesterol?
A
All cells in the body can which means all cells have HMG coA reductase.
4
Q
What are some exogenous sources of cholesterol?
A
eggs, cheese, meat
5
Q
What is the lipids pathway throughout the digestive tract?
A
oral cavity: lingual lipases
liver: produces bile acids (stored in the gal bladder)
(released in the small intestine)
pancreas: produces proteases and releases hydrolytic enzymes
small intestine: acquire nutrients from food
large TGs are carried by chylomicrons, VLDL, LDL, HDL and distributed in the body for use
stored in adipose tissue
6
Q
What is the process of esterifying cholesterol?
A
free cholesterol to esterified cholesterol (an example is cholesterol oleate):
add fatty acid and eliminates water.
7
Q
What is the function of bile acids?
A
emulsifies fat
soap to solubilize lipids
found in the small and large intestines
active bile salt activated lipase
8
Q
what is the function of lipases?
A
digest lipids
found in small intestine
9
Q
What is the process of conversion of bile acids to bile salts? (HESC)What is the end result?
A
hydroxylation of steroid nucleus
epimerization of 3b hydroxyl group
saturation of steroid nucleus
side chain cleavage
result: from chair confirmation to flat configuration
all the charged surfaces are on one side: hydrophobic react with oil and other phase reacts with water=detergent!
10
Q
What are macromolecules hydrolyzed to?
A
protein: amino acids
DNA/RNA: deoxy/ribonucleic acids
Carbohydrate: glucose/simple sugars
lipids: constituent subunits
11
Q
Breakdown the fate of diet derived lipids.
A
Hydrolysis (in lumen) -> absorpotion -> resynthesis (for enterocytes)
TG -> FA, MG -> TG
CE -> FA, cholesterol -> CE
PC -> lysoPC, FA -> PC (phosphotidylcholine?)
12
Q
How phospholipids broken down?
A
by phospholipase!
they become lysophospholipids (a phospholipid missing a phosphate group)
13
Q
How are diet-derived lipids processed?
A
the huge TG droplet is broken up/emulsified by bile salts so they form smaller lipid droplets.
they are then digested by lipase (hydrolysis) into lipid subunits and then arrange their selves into micelles where they enter into the cell of the enterocyte and are resynthesized/reconstituted.
14
Q
What are the specific receptors for each type of dietary lipids to be absorbed in the enterocyte?
A
cholesterol: NPC1L1
fatty acids: FATP4, CD36
phospholipids: MFSDA2A?
15
Q
How are chylomicrons assembled? Where does it occur?
A
cholesterol must be esterified to cholester esters (CE)
the congregation of TG and CE and FAs
assembly of the CM by MTP
this process must occur in a specialized area away from water (ER)
16
Q
Distinguish lipoprotein and apolipoprotein.
A
lipoprotein: non-cavlently bound complexes of lipids and proteins
apolipoprotein: the protein part of the lipoprotein
17
Q
How can lipoproteins be classified?
A
by the protein by the mobility (DNA electrophoresis) by the types of lipid densities by size by the source
18
Q
What is the lipoprotein profile of CM?
A
largest Apo B48: structural in chylomicrons assembled in the ER of enterocytes (intestinal lining) smallest density mostly TG
19
Q
What is the liprotein profile of VLDL?
A
second largest ApoB100: structural and is the ligand for VLDL and LDLR repackaged in the liver from CM remnants b mobility second least dense mostly TG
20
Q
What is the lipoprotein profile of LDL?
A
third largest ApoB100: structural and VLDL & LDLR ligand b mobility mostly CE 3rd least dense metabolllic byproduct of VLDL
21
Q
What is the lipoprotein profile of HDL?
A
smallest
most dense
Apo1A: structural & activates LCAT (which esterifies cholesterol)
alpha mobility
source: Apo A1 from CM that is shrunken and then attached to PL
22
Q
What are Apo C and E for?
A
They are found on all apolipoproteins = can jump to any species
ligands for LDLR and VLDLR
23
Q
List in ordinal form, the mobility of lipoproteins.
A
alpha: most mobile
pre-beta: intermediate
beta: least mobile
24
Q
What is the relationship between lipid, protein and density?
A
inversely related:
more lipid, less protein
more lipid, less dense
directly related:
more dense, more protein
25
Demyth the Good vs. Bad cholesterol.
Good cholesterol is often refered to as HDL while the bad chosterol is often referred to as LDL. However, cholesterol is cholesterol (CH in HDL is identical to CH in LDL) and the atherogenic aspect is the liproprotein that carries the cholesterol esters.
HDL:
- has a higher protein portion and phospholipid
- picks up CE from peripheral cells
- returns them to the liver directly/indirectly (CETP)
LDL:
-a high proportion is made of CE
26
Describe the exogenous pathway.
From dietary cholesterol.
goes to intestines, packaged into CM and then travels around the circulation to give off TG to muscles and adipose tissues. CM remnants return to the liver and then follows the endogenous pathway.
when liver transforms the cholesterol into bile acids, this is a terminal transformation. It is excreted (biliary cholesterol) with bile acids = makes the bile and released out to the intestines where it emulsified TG droplets. for absorption of it.
A portion of the bile salts are excreted with the cholesterol out via feces.
27
What is the endogenous pathway?
CM remnants go to the liver to be repackaged as VLDL. VLDL goes into the blood stream to give TG to the adipose and muscles tissues and then becomes IDL returning to the liver or become LDL which then builds in muscle and adipose tissues risk causing atherosclerosis.
HDL3 pick up excess unesterified cholesterol in tissues at the peripheries and esterifies the UC to CE using LCAT which means it is moved deep within the core of the HDL. It then can return it to the liver for disposal. It can be done either directly or by CETP which exchanges the CE for TGs with LDL or VLDL and then the LDL or VLDL return to the liver.
28
What is GPIHBP1 for?
It hydrolyzes CM using LPL so that FAs enter cell.
It is a dimer and tethered to the epithelial surface. One anchor binds the CM and the other binds the LPL and then the GPIHBP1 pulls them close so they can interact.
29
How can excess cholesterol be removed?
the reverse cholesterol transport pathway
- HDL can go to the peripheral cells and pick up UC which is then esterfied by LCAT to CE. This moves deep within the hydrophobic core of HDL. This way, HDL can continuously pick up more cholesterol around the cell since the surface is cleared of it. It then has a direct pathway to the liver or can use CETP to exchange CE for TGs with LDL. The LDL then returns to the liver via receptors and becomes UC again adding to the liver pool.
30
What is the difference between LCAT and ACAT?
Both esterifies cholesterol )making them active again)
LCAT: gets FAs sources from PC, only in PERIPHERAL cells
ACAT: gets acyl sources from acetyl coA, found in ALL cells
31
What is the fate of cholesterol in the liver?
unique: to have an unesterified cholesterol pool
- store as CE in lipid droplets
- lipoprotein assembly (VLDL)
- bile acid synthesis to exogenous pathway
- direct secretion into bile as biliary cholesterol (so then treated and mixed with dietary cholesterol and both identically absorbed by NPC1L1
32
What is the fate of endogenously synthesize and diet derived cholesterol?
diet+ biliary CH from bile go to intestine and enter blood stream via CM and then have cholesterol deposits. It then taken up by HDL which brings it back to the liver where it can be stored or secreted as bile acids where that can be excreted in feces but biliary CH is mixed with diet CH.
33
What is dyslipidemia? How can be get it?
abnormal lipid concentration
- hyper/hypo lipidemia
- hyper triglyceridemia
- hyper cholesterolemia
combined: hyper lipidemia = high triglycerides and cholersterol
acquire (malnutrition: abnormally high intake or deficiency) or genetic (gene variants/mutations)
34
Give an example of abetalipoproteinia?
an example of hypocholetserolemia.
undetectable CM circulating therefore there is malabsorption of fats and fat solbuble vitamins
dietary management: restrict long chained TG use medium chained TG so they can directly diffuse to cell and do not need CM to be absorbed by cells.
35
How can we recognize abetalipoproteinemia?
start shaped RBC, diarrhea,
36
What is MTP and apo B48 for?
both are lipid carriers.
MTP: carries TG, PL and CE so they the CM can be assembled
Apo B48: carry lipids
37
Describe the genetically acquire Abetalipoproteinemia.
-a mutation in the gene for MTP which is essential for CM assembly.
38
FH: familial hypercholesterolemia - how do you get it?
in born errors are:
- inherited genetic disorder
- due to one gene that affects one enzyme --> not true because the majority of the genome is composed of regulatory elements and for eg. MCM6 regulates the gene expression of LCT but also many others upstream from the start site it affects many.??
39
What is the consequence of epigenetics?
modifies the controls for genetic information use during this life and the next generation as well.
40
How can FH be identified
reliably diagnosed, use FH skin cells (fibroblasts) that can be grown and studied, lipoprotein isolates from blood of FH patients so that
the metabolism of cholesterol can be observed.
41
What are clinical features of FH?
xanthomas (tendon) and xanthelasmas (eye) deposits of cholesterol there
high cholesterol in the blood (becomes opaque)
42
What are epigenetics modifications?
DNA methylation
histone acetylation
RNA-based mechanism (siRNA?)
43
What is the difference between normal and FH cells?
normal:
1. low cellular CE
2. low rate of synthesis of UC
3. low HMG coA reductase activity
FH cells:
1. high cellular CE
2. high rate of UC synthesis
3. high activity of HMG coA reducatse
44
What is the response to lipid depletion?
normal:
1. mobilize CE stores
2. increase rate of UC synthesis
3. increased HMG coA reductase activity
FH cells:
1. no effect on CE stores (remains high)
2. no effect on UC synthesis (remains high)
3. no effect on HMG coA reductase activity (remains high)
45
What is the response to lipid addition?
normal:
1. slight increase CE stores
2. decrease rate of UC synthesis
3. decrease HMG coA reductase activity
FH cells:
1,2,3 no effect (always remains high)
46
What is the problem with FH cells when compared to normal cells?
They do not have the sensitivity to cholesterol OUTSIDE membrane which results in their inability to respond.
47
How can we track lipoproteins?
using radioactive tracers allows us to follow nutrients and observe their metabolism
1. tag the protein with I 125:
- can follow the protein
- can follow lipoprotein
2. tag the lipid component with H3:
- can follow the metabolism and lipid deposit paths
- can follow the lipoprotein itself
48
Wahat are the effects of LDL on cholesterol metabolism?
increase of LDL:
no effect on binding, internalization of LDL, hydrolysis of apo B and cholesteryl esters (metabolism of LDL via lysosome), no effect on cholesterol synthesis nor cholesterol esterification (for storage)
HOWEVER:
should affect cells normally
increased binding to receptors, internalization of at clathrin pit, hydrolysis of apo B and cholesteryl esters hydrolysis due to metabolism of all the LDL and then cholesterol esterification so that it can be stored.
49
How is HMGR activity and LDL in normal and FH cells?
homozygote: FH cells
normal: normal
no response in homozygote to inability to sense extracellular cholesterol (receptor problem)
while normal cells will increase HMGR activity if LDL is decreased
while decreasing HMGR activity proportionally by the amount of LDL that is added.
50
What is the genetic prbblem with FH?
4 different mutations
1. absence of LDLR (no synthesis so no binding)
2. inability to transport the receptor from ER to Golgi and then exocytose to cell surface (synthesis but no binding as well)
3. cannot bind LDL (defective receptor although it is synthesized and transported, it cannot bind.)
4. no clustering in the coated pit (synthesize, binds but not internalization!)
51
Identify the parts of the LDL receptor and the gene variation causing defect.
EGF repeats are recognized by the membrane protein so that it will bind: mutation 3 where it is synthesized and transported but unable to bind LDL
52
How does the cell respond to increased cellular cholesterol?
```
LDL receptors aggregated at the cloathrin pit
LDL binds (contains cholesteryl linoleate or cholesteryl oleate packagedwithin Apo100) and binds to LDLR.
Internalized as it is packaged into a vesicle and fused with lysozomes to hydrolysis contents. THe apoB100 and CE are hydrolyzed where the amino acids are recovered from the apoliporpotein and transferred to the ER where it contributes to an increase in cholesterol concentration. This increase causes the SREBP to be bound to INSIG and SCAP which stays within in the ER. There is a decrease in HMGR and LDLR. All the while, LCAT is activated to turn the cholesterol to cholesterol esters which can be stored in the cell.
```
53
Identify genetic disorders and the association to the lipoprotein.
Familial hypercholesterolemia: mutations 1-4 (no synthesis of LDLR, no transport to Golgi from ER, no binding and no internalization) Apo B or E receptors
Metabolic syndrome:
- defect CD36 (fatty acid synthesis)
- defect ApoA2
Hypertriglyceridemia:(metabolic syndrome) associated to LPL (lipoprotein lipase defect)
Hyperlipidemia (LDL/VLDL levels) : defect in Apo E
Familial defective ApoB100 Hypobetalipoproteinemia: defect with ApoB100
Low HDL: associated to
- defect ApoA1
- defect in LCAT
54
Describe cellular cholesterol homeostasis.
If cellular cholesterol increases:
- decrease of LDLR so there is less binding and internalization of LDL
- incease ABC A1 so that the cholesterol can be brought to the surface so that an HDL can pick up cholesterol from this peripheral tissue
- decrease in HMGR activity so less synthesis of cholesterol
- increased ACAT so that the cholesterol is stored as esterified cholesterol
IT IS TIGHTLY CONTROLLED WITHIN THE CELL
55
What is the difference between ACAT and LCAT?
ACAT: esterifies cholesterol in all cells (takes FAs from acyl chains?+ UC)
LCAT: esterifies cholesterol only in peripheral cells (take FAs (from PL) +UC)
56
What is SRE?
sterol response element
specific sequence in the DNA promoter region:
increased expression when cholesterol is depleted
decreased expression when cholesterol is in excess
eg of SREs: HMGR and LDLR
If this gene sequence is added to promoter regions of other genes (not related to lipid metabolism), the gene will still exhibit the same sensitivity to cholestertol status as it does normally
57
What is SREBP? Where is it found? How many isoforms?
sterol response element binding protein
binding specifically to SRE determined by the UC WITHIN the cell
activated when cholesterol decreases
it is a membrane bound precursor protein (SCAP-SREBP--- INSIG/COPII) that must be proteolytically cleaved (S1p & S2p)
THREE isoforms:
SREBP 1a, SREBP 1c & SREBP 2
58
Describe each SREBP isoform.
SREBP 1a: potent activator for all SREBP responsive genes
SREBP 1c: only activated genes involved in fatty acid synthesis (weaker)
SREBP 2: PREFERENTIALLYregulates genes of cholesterol synthesis
59
Connect LDL path within the cell and SREBP sterol sensing.
LDL comes into the cell. The vesicle contents are fused with lysosome so that the protein amino acids are taken up by ApoB and the cholesterol is unesterified. It flows to the ER as free cholesterol and HERE the SREBP senses the cellular sterol concentration. If it is high, it will stay in the ER bound to INSIG, if it is low, (means there is no LDL being internalized), SREBP-SCAP form a COPII vesicle to Golgi so that it can be proteolytically cleaved by S1P and S2P so the N terminal can go into the nucleus and influence gene expression. It binds to SRE as a transcription factor and regulates genes. Hence, it makes more HMGR gene so that it can make more cholesterol.
If there is not SREBP binding to SRE, HMGR is less expressed (no transcription) so decreased synthesis. There is increased synthesis of ACAT however to esterify the cholesterol so it can be stored within the cell.
60
What does high cellular cholesterol concentration also stimulate?
gene expression of ABC A1: to efflux cholesterol from cell, APO E: codes for apolipoprotein that serves as a ligand for LDL receptor (more uptake of LDL) for eg. in reverse cholesterol transport, there is increase in internalization of lipoprotein so there is more drainage of it which means it can be converted to bile acid for disposal as a terminal transformation), and Cyp7a1 (only expressed in mice this way), catalyzes first and rate limiting step of bile acid biosynthesis pathway
61
What are the shapes of nuclear receptors?
heterodimer (two different parts)
monomer (one unit)
homodimer (same)
62
How do nuclear receptors work?
1. binds to DNA with or without ligand
2. they bind to DNA at the HRE (hormone response element) half site
3. the space between the subunits of heterodimer or homodimer can range from 0-12 nucleotides
4. the half sites on the DNA can be arranged as
- direct repeat: same directions
- everted repeat: pointing away from each other
- inverted repeat: towards each other
*ESTROGEN: only estrogen can exist outside the nucleus, all other steroid/nuclear receptors are inside the nucleus
63
What are the 4 types of nuclear receptors?
steroid receptors: homodimers
Dimeric Orphan receptors: they have unknown physiologic ligand
RXR heterodimers: for most vitaminds and hormones, bila cids
monomeric/tethered orphand receptors: only one part is attached to the DNA, the other part of the receptor is attached to that
64
How do nuclear receptors regulate gene expression?
1. ligand binding or release
causes
2. recruitment/release of co activators or co repressors
causing
3. stimulation or repression of gene transcription
location: promoter upstream from start site
65
What are the combinations of nuclear receptor responses?
1. ligand binding - recruitment of co-activator - stimulation of gene transcription
2. ligand binding - recruitment of co repressor - repression of gene transcription
3. ligand release - release of co repressor - stimulate
4. ligand release - release co activator - repression
5. ligand binding - release co activator - repression
6. ligand binding - release co repressor - stimulation
7. ligand release - recruitment of coactivator - stimulation
8. ligand release - recruitment of co repressor - repression
66
What is LXR?
- has two isofroms (alpha and beta)
- activated by oxysterols which are produced during cholesterol metabolism/synthesis
- LXR alpha: predominantly expressed in tissues and cells - maintain lipid homeostasis
- LXR beta: distributed everywhere
alpha and beta LXRs are not identical but overlap with genes and ligand preferences
67
What genes does LXR regulate?
ABC A1: efflux of cholesterol from cells
Cyp7A1: bile acid synthesis
SREBP 1c: responds by stimulating genes involved with fatty acid metabolism and synthesis (fat metabolism)
68
Name some ways to conduct molecular analyses.
- gene cloning
- gene sequencing
- use SNPs (single nucleotide polymorphism - modify and cut the gene so there are variants)
69
What are SNPs and its effects?
this is a substitution of a single base in the gene causing an amino acid substitution which mean a different protein
results:
synonymous - one SNP at the same positions leads to the same polypeptide sequence
(a change in one base still synthesizes the same protein)
why? due to the wobble position which is at the third position in the amino acid codon. This position can change without changing the amino acid that corresponds to that sequence
non synonymous- one SNP at the same position leads to a different polypeptide sequence
(a change in one base leads to a different protein synthesized altogether)
why?
because the codon is changed which alters the amino acid that reads the codon
70
What are examples of Apo E polymorphism?
E7: hypertrigluceridemia
E4: Alzheimer's disease
SNP-219G: optic neuropathy
ApoE gene variants vary from individual to individual so each person have different variants and we do not know how that affects the genome. Different gene sequences give rise to different phenotypes and interactions with nutrients.
71
What are ESTs?
Expressed sequence tags
these are short cDNA sequences that are generated by cutting one end or both ends of a gene and tags things
Take a short cDNA and hybridize it, (more binding of DNA with RNA) then the more the gene is transcribe in the sample =can be seen using ESTs (as they tag the the DNA/RNA)
72
What is the difference between Q-PCR and microarray analysis?
Both study changes in gene expression between a gene variant and the normal.
or even mRNA species alterations due to changes in state of nutrition or metabolic status.
```
Q-PCR: quantitative
DNAse tx (denature DNA), reverse transcription+primers+probes= fluorescent cDNA product and quantification
```
microarray analysis: qualitative
reverseve transcription, take target cDNA and transcribe fluorescently labeled cRNA. Fragment into micro array analysis and hybridize = scan
73
What are results of microarray analysis? How to read them?
The test is done for control and experimental genes. The same process is applied and the results are compared.
For example: control genes at tagged red and experimental is tagged blue,
once it is hybridized,
if results for experimental show:
1. blue: the experimental gene is upregulated
2. red: the experimental gene (decreased so) is down regulated
3. purpe: there is no change in gene expression of experimental RNA since the control RNA will also have the results of purple. (there was an equal part mix of experimental and control genes so hybridization should show an equal mix (purple) if there is no additional up regulation or down regulation of the experimental gene in comparison to the control gene.
74
What is RNA-seq?
it provides the information of RNA sequences-genomic sequences still unidentified, shows relative abundance of transcripts
very powerful in determining: amount and which chromosomes/RNA the genes give rise to
75
What is proteomic analysis?
the study of proteins and enzyme alterations by the change of the state in nutrition or metabolic status
How? compare the protein the mass spectroscopy of control and experimental where you can see some proteins existing in one sample that does not exist in the control or darkened spots (greater expression of a protein in the control than in the experimental)
76
How does proteomic analysis procedures?
protein separation using 2D gel or HPLC
cleanup
mass spectroscopy
77
Why mice useful in research?
short developmental cycle so you can see effects of something throughout "life"
pure bread strains available
transgenics: can add new genes (human genes) or knock out
-provide info on metabolic pathways and regulation
-small, easy to house
-fully sequences genome and corresponding human genes are mapped
78
How are mice different from human (disadvantages)?
metabolic differences
different nutrient requirements
genetic differences
79
What is trangensis?
modifying genome of a species by adding from another or taking out
80
What is the process of trangenesis?
```
construct transgene
clone
grow/evaluate in vitro
microinject into embryo
allow the embryo transfer (pass the gene on to next generation)
analyse the genome of new mouse
```
81
What are two type of genes that can be introduced into a genome?
natural: from another species
synthetic: components from other genes or species
DNA solution is injected into the nucleus of fertilized eggs so it can be incorporated in to the development of the mouse
82
Give an example of transgenesis in mice.
overexpression of LDLR
synthetic gene: to overexpress LDLR in LIVER
compare mice with: low CH diet vs. standard diet
83
What were the results of LDLR overexpression transgenic mice?
Mice have natural HDL spike.
transgenic mice have lower HDL spike than normal on LF diet.
since reverse CH path is very efficient in depositing of cholesterol back to liver due to so many LDLR receptors, there is no need for that many HDL. HDL is also synthesized from CM ApoA1 being pinched of it but if the diet is low fat, not many CM in blood so less nascient HDL formed and less PL to bind to nascient HDL for the making of HDL.
transgene mice with high fat diet, have higher VLDL but not higher LDL because fat being packaged into CM and then feeding tissues, remnants are repackaged by liver to VLDL and then to LDL which is efficiently disposed of due to the overexpression of LDLR. HDL spike is a little bit higher.
LDLR transgene mice = resistance to diet induced hypercholesterolemia and have low concentration of LDL when on low fat diet
84
What are targeted gene disruptions?
Ko=knock outs
allows specific mutation to be created
mutation can be local or global (whole animal)
introduced at certain developmental stage
direct access to gene function
85
What is the process of targeted gene disruption?
get stem stells from blastocyst
multiply and save
take targeted vector and add to the ES cells, grow and inject into the blastocyst via microinjection, implant the blastocyst back into the mother.
86
What is a chimeria? Which type is better?
when the mouse has two genetic background (albino x agouti)
darker: the modified genome ES cell donor is more expressed, next generation will carry the modified genome too
middle: equivalent balance
do not want: light because that means the genetic makeup is mostly of albino
