Nutrition - topic 2

• Endogenous: de novo synthesis froma acetyl-CoA, All cells in the body are capable of making cholesterol
• Exogenous source: diet

HMG-CoA

Cholesteryl esters

stored as triacylglycerols

• ERmulsification of fats and fat soluble-nutrients; activator of bile-salt activated lipase
• In large and small intestines

• Digestion of lipids (cholesteryl esters, acylglycerols, phospholipids)
• In small intestine

saturation of sterol gets the molecule more planar

Requires specific membrane-bound transportes on the surfaces of enterocytes

FATP4, CD36

non-covalent complexes of lipids and proteins

Apo B-48 contains the first 48% of amino acids found in Apo B100

Apo A-1

Apo C's

Lipoproteins can be separated by agarose gel electrophoresis

they are too big

Fasting plasma shouldnt contain any chylomicrons

According to density?

• Less lipid more protein- higher density
• More lipid, less protein - lower density

HDL

Phospholipids, Triacylglycerols, cholesterol

LDL

'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

Chylomicrons lose fat, thus become shriveled up.  Some phospholipid pinches off to get a smooth surface and Apo A goes with it

Liver

• 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

CETP moves cholesterol from HDL to chylomicron reminants in exchange of a TG (that goes from remnant to HDL)

• 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

After the cholesterol is esterified, it goes into LDL and then into liver parenchymal cells

High cholesterol intake inhibits bile acid synthesis, which decreases lipid intake, which decreases cholesterol intake

Abnormal lipid concentration in the blood?

High concentration of lipids in blood

Low concentration of lipids

High triglycerides and cholesterol

• 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

• chronic diarrhea (steatorrhea- cannot absorb fat)
• retinis pigmentosa (tunnel vision)
• Ataxia (neurological balance disorder)
• Star-shaped RBCs (acanthocytosis)

mutation of ACAT (lipid metabolizing enzymes)

Requires specific membrane-bound transporters on the surfaces of enterocytes

• Blocks the absorption of cholesterol without affecting the absorption of other lipids
• does so by acting on NPC1L1 (cholesterol transport)

• 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

• High concentration of cholesterol in the blood
• Presence of xanthelasmas and tendon xanthomas

• Normal:
• 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

• Normal:
• 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

• 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)

• 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)

FH cells are unable to bind, internalize or metabolize LDL

FH is a disease caused byt he absence of LDL-receptor function

• decrease in HMG-CoA reductase
• Increase in ACAT- more cholesterol to cholesteryl oleate
• Decrease in LDL receptors

• 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

• 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

• SREBP-1a, SREBP-1c - encoded by one cene
• SREBP-2- encoded by another gene

A potent activator of ALL SREBP responsive genes

Regulates genes involved in fatty acid synthesis rather than cholesterol synthesis; weaker activator compared to SREBP-1a

Preferentially regulates genes involved in cholesterol synthesis

Codes for a transporter involved in the efflux of cholesterol from cells

Codes for an apolipoprotein that can also serve as a ligand for the LDL receptor

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

• 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

• 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

Can be used to study which and how specirfic mRNA species are altered by changes in state of nutrition or metabolic status

Primers

DNase treatment --> reverse transcription + Primers + specific probes --> fluorescent cDNA product relative quantitation

reverse transcription --> target cDNA -(transcription)-> fluorescently labeled cDNA -(fragmentation)-> microrray -(hybridization)-> scan and quantitation

• 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

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

• Distince SNPs at the same position lead to the same polypeptide sequence
• DNA sequence is different, but same amino acid

Distinct SNPs at the same position lead to different polypeptide sequences

Can be used to study which and how proteins/enzymes are altered by changes in state of nutrition or metabolic status

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

• Many purebred strains available; easy to standardize studies done in different lbs
• Short developmental cycle
• Can be genetically modified

• Transfene construction
• Cloning
• Evaluation in vitro
• Microinjection
• Embryo transfer
• Genomic analysis

• 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

• 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

• 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

• 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

Neimann-Pick C1 Like 1

• 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