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What are some sources of body cholesterol?
- Endogenous: de novo synthesis froma acetyl-CoA, All cells in the body are capable of making cholesterol
- Exogenous source: diet
What is targeted by cholesterol drugs?
What is the storage form of cholesterol?
How are lipids stored?
stored as triacylglycerols
What is the purpose of bile acids in lipid absorption?
- ERmulsification of fats and fat soluble-nutrients; activator of bile-salt activated lipase
- In large and small intestines
What is the purpose of lipase in lipid absorption?
- Digestion of lipids (cholesteryl esters, acylglycerols, phospholipids)
- In small intestine
How do bile acids change cholesterol?
saturation of sterol gets the molecule more planar
How are dietary lipids absorbed?
Requires specific membrane-bound transportes on the surfaces of enterocytes
What is the specific membrane-bound transporter for fatty acids?
What are lipoproteins?
non-covalent complexes of lipids and proteins
What is the difference between Apo B-48 and Apo B100?
Apo B-48 contains the first 48% of amino acids found in Apo B100
What is required in order to make HDL?
Which apolipoproteins are not found in HDL?
How are lipoproteins seperated in the lab?
Lipoproteins can be separated by agarose gel electrophoresis
Why do chylomicrons not fit in agarose gel electrophoresis?
they are too big
How can you tell fasting plasma from non-fasting plasma based on lipoproteins?
Fasting plasma shouldnt contain any chylomicrons
How are lipoproteins seperated?
According to density?
What determines a proteins density?
- Less lipid more protein- higher density
- More lipid, less protein - lower density
Which lipoproteins have the most protein?
What are lipoproteins mainly composed of?
Phospholipids, Triacylglycerols, cholesterol
Which is the 'bad' cholesterol?
How do good cholesterol and bad cholesterol interact?
'Good' cholesterol (HDL) is stable and carries 'bad' cholesterol (LDL) away from the arteries. The 'bad' cholesterol (LDL) sticks to artery walls and contributes to plaque build-up
How is HDL formed?
Chylomicrons lose fat, thus become shriveled up. Some phospholipid pinches off to get a smooth surface and Apo A goes with it
How does HDL transport cholesterol?
- HDL goes and gets cholesterol in peripheral tissue
- Liver either erabsorbs all the HDL particle or only the inside so the HDL can go again
What is the purpose of CETP?
CETP moves cholesterol from HDL to chylomicron reminants in exchange of a TG (that goes from remnant to HDL)
What is the reverse cholesterol transport pathway?
- UC (unesterified cholesterol) is taken from membrane of peripheral cells
- LCAT esterifies UC, becomes even more hydrophobic- stays insie HDL
- Receptor-mediated uptake by liver parenchymal cells
What happens in the indirect reverse cholesterol transport pathway?
After the cholesterol is esterified, it goes into LDL and then into liver parenchymal cells
How does cholesterol inhibit its own uptake?
High cholesterol intake inhibits bile acid synthesis, which decreases lipid intake, which decreases cholesterol intake
What is dyslipidemias?
Abnormal lipid concentration in the blood?
What is hyperlipidemia?
High concentration of lipids in blood
What is hypolipidemia?
Low concentration of lipids
What is combined hyperlipidemia?
High triglycerides and cholesterol
What is abetalipoproteinia?
- An example of hypocholesterolemia
- Low to undetectable circulating chylomicrons
- Malabsorption of fats and fat-soluble vitamins
- Dietary management: restriction of lonf-chain dietary TG, use medium chain TG as alternate source of fat
What are some clinical features of abetalipoproteinemia?
- chronic diarrhea (steatorrhea- cannot absorb fat)
- retinis pigmentosa (tunnel vision)
- Ataxia (neurological balance disorder)
- Star-shaped RBCs (acanthocytosis)
What can lead to abetalipoproteinemia?
mutation of ACAT (lipid metabolizing enzymes)
What is required for absorption of dietary lipids?
Requires specific membrane-bound transporters on the surfaces of enterocytes
What does exetimibe do?
- Blocks the absorption of cholesterol without affecting the absorption of other lipids
- does so by acting on NPC1L1 (cholesterol transport)
What is familial hypercholesterlemia (FH)?
- An example of hyercholesterolemia
- Some clinical features
- Lessons learned from studies using FH fibroblasts
- Molecular mechanism underlying the regulation of cholesterol biosynthesis
- Can be reliably diagnosed
What are some clinical features of familial hypercholesterolemia?
- High concentration of cholesterol in the blood
- Presence of xanthelasmas and tendon xanthomas
What is the difference between normal and FH fibroblasts
- Low cellular cholesterol concentration (as CE)
- Low rate of cholesterol (UC) synthesis
- Low HMG-CoA reductase activity
- FH cells:
- High cellular cholesterol (as CE) concentration
- High rate of cholesterol (UC) synthesis
- High HMG-CoA reductase activity
How do normal and FH fibroblasts respond to Lp depletion?
- Moilization of CE stores
- Increase rate of UC sythesis
- Increase in HMG-COA reductase activity
- Can sens how much cholesterol is outside
- FH cells:
- No effect on CE stores (high)
- No effet on rate of UC synthesis (high)
- No effect on HMG-CoA reductase activity (high)
- Cant sense cholesterol
How do normal and FH fibroblasts respod to Lp depletion?
- Normal cells:
- Slight increase inc ellular CE concentration
- Decrease in rate cholesterol synthesis
- Decrease in HMG-CoA reductase activity
- FH cells:
- No effect on CE stores (high)
- No effect on rate of UC synthesis (high)
- No effect on HMG-CoA reductast activity (high)
How are lipoproteins modified?
- Apolipoprotein can be labeled with radioactive tracer (such as I125)
- Lipids can also be labeled with radioactive tracers (such as H3; may be placed on the fatty acyl chains, or the cholesterol moiety etc)
What is abnormal about FH cells?
FH cells are unable to bind, internalize or metabolize LDL
What is FH caused by?
FH is a disease caused byt he absence of LDL-receptor function
How do cells respond to increased cellular cholesterol concentration?
- decrease in HMG-CoA reductase
- Increase in ACAT- more cholesterol to cholesteryl oleate
- Decrease in LDL receptors
What is the Sterol Response Element (SRE)?
- A short and specific DNA sequence located in the promoter region of genes whose expression is increased by cholesterol depletion and decreased by cholesterol excess (eg. HMG-CoA reductase and low density liporprotein receptor genes)
- Addition of this sequence to the promoter region of other genes confers sensitivity to cellular cholesterol status in the same way manner as HMGR and LDLR genes
What is SRE Binding Protein (SREBP)?
- A protein that binds specifically to the SRE sequence and stimulates transcription
- The binding of SREBP to SRE is dictated by the concentration of unesterified cholesterol within the cell
- SREBPs are activated in response to DECREASED cellular cholesterol concentration
- SREBP is synthesized as a membrane-bound precursor protein that must be proteolytically processed to release the DNA-binding domain
What are the three distinct isoforms of SREBP in mammalian cells?
- SREBP-1a, SREBP-1c - encoded by one cene
- SREBP-2- encoded by another gene
What is SREBP-1a?
A potent activator of ALL SREBP responsive genes
What is SREBP-1c?
Regulates genes involved in fatty acid synthesis rather than cholesterol synthesis; weaker activator compared to SREBP-1a
What is SREBP-2?
Preferentially regulates genes involved in cholesterol synthesis
What is ABCA1?
Codes for a transporter involved in the efflux of cholesterol from cells
What is APOE?
Codes for an apolipoprotein that can also serve as a ligand for the LDL receptor
What is Cyp7a1?
Only in mice, not in humans- codes for a microsomal enzyme that catalyzes the first and rate-imiting step in the classical bile acid biosynthetic pathway
What is LXR?
- A member of the NR superfamily of transcription factors
- two isoforms: alpha, beta
- Activated by oxysterols (generated during the synthesis of cholesterol, and during the metabolism of cholesterol)
- LXRalpha, predominantly expressed in tissues and cells that are important in maintaining lipid homeostasis
- LXRbeta- ubiquitous distribution
- LXRalpha and LXRbeta are not identical (and therefore different biological importance) but likely have overlapping targget genes and ligand preference
What genes are regulated by LXR?
- ABCA1- facilitates the efflux of cholesterol
- CYP7A1- responsible for catalyzing the first and rate-limiting step in the synthesis of bile acids from cholesterol in mice (not in humans)
- SREMP-1c, a transcription factor that regulates genes involved in fat metabolism
What are Q-PCR and microarray analysis used for?
Can be used to study which and how specirfic mRNA species are altered by changes in state of nutrition or metabolic status
What is Q-PCR limited by?
How does Q-PCR work?
DNase treatment --> reverse transcription + Primers + specific probes --> fluorescent cDNA product relative quantitation
How does microarray work?
reverse transcription --> target cDNA -(transcription)-> fluorescently labeled cDNA -(fragmentation)-> microrray -(hybridization)-> scan and quantitation
What are expressed sequence tags (ESTs)?
- Short sequences that are generated by sequencing either one or both ends of an expressed gene
- >40 million ESTs are document in public databases
- Some ESTs are identified (correspond to fully characterized genes); many correspond to unidentified genes
- Many genes involved in the acquisition and metabolism of nutrients are not yet knowm
What are single nucleotide polymorphosms (SNPs)?
Single base substitution in genes also often cause amino acid substitution in their encoded proteins, but the replacements do not necessrily result in the inactivation of the respective proteins
What are synonymous SNPs?
- Distince SNPs at the same position lead to the same polypeptide sequence
- DNA sequence is different, but same amino acid
What is nonsynonymous SNPs?
Distinct SNPs at the same position lead to different polypeptide sequences
What is proteomic anaysis used for?
Can be used to study which and how proteins/enzymes are altered by changes in state of nutrition or metabolic status
Why do we do proteomic analysis?
A diffeence in gene transcription is not necessarily associated with a change in mRNA levels and a change in mRNA levels is not always related to a change in protein levels
Why are engineered mouse models used in nutrition studies?
- Many purebred strains available; easy to standardize studies done in different lbs
- Short developmental cycle
- Can be genetically modified
How is transenesis done?
- Transfene construction
- Evaluation in vitro
- Embryo transfer
- Genomic analysis
What is transgenesis?
- Natural or synthetic genes introduced into another genome for permanent propagation
- The DNA solution is injected into the nucleus of fertilized eggs of the host species
What is targeted gene disruption?
- Creating gene 'knock-outs' involves a more complicated procedure compared to transgenesis
- Permits very specific mutation to be created
- Mutation can be global (whole animal) or local (specific tissue, or even specific cells)
- Mutations can also be introduced at certain stages of development
- Allows direct assessement of gene function
What happened with overexpression of LDL receptors in transgenic mice?
- Synthetic transgene to direct overexpression of LDL receptors in the liver
- LDLR transgenic mice fed a low cholesterol diet display low blood LDL concentration
- LDLR transfenic mice exhibit resistance to diet-induced hypercholesterolemia
What is the importance of LXRalpha on lipid metabolism?
- LXRs are activated by oxysterols
- LXR target genes are stimulated when cellular oxysterol concentrations increas
- Controls the expression of many genes involved in sterol and fat metabolism
What does NCP1L1 stand for?
Neimann-Pick C1 Like 1
What is NCP1L1?
- Gene expressed at high levels in the liver and small intestine (human); mainly in small intestine (mice)
- Transmembrane protein found in the apical surface of enterocytes (in the small intestine)
- A drug called exetimibe has been found to bind to NCP1L1 in vitro and interfere with the absorption of dietary cholesterol in vivo