Transcript 1) Lecture notes: effects of bile salts on cholesterol metabolism

Nutrition and Gene Expression
Lecture, Feb 27, 2014
Lipid Metabolism: Changes
in Gene Expression
and Application to Studies
in Guinea Pigs and Humans
MEASUREMENT OF SPECIFIC
mRNA LEVELS:
The most important method
in study of gene expression.
We will discuss RT-PCR in detail,
because of its widespread use.
MAKE THE INITIAL RNA
PROCESS TO mRNA:
this is measured
TRANSLATE TO PROTEIN
MODIFY THE PROTEIN
R
NOTE: other steps can also be
measured, if you want
The mechanism of how a gene is switched on is
very complicated and important. It usually requires
special proteins that bind to the REGULATORY ELEMENT
of the gene. These proteins are usually called
TRANSCRIPTION FACTORS. We will study these proteins
in detail, for the March and April lectures continue discuss
of these proteins throughout the semester.
If there in an increase in the mRNA for a protein, you
know the synthesis of that protein has been increased.
Lots of research just looks at that: increase in mRNA.
The newer methods are very interesting and powerful.
Reverse transcriptase-PCR (RT-PCR) is now the
method of choice for measuring levels of specific mRNA.
Northern blots, and the ribonuclease protection assay,
are two other methods for measuring specific mRNA.
The guinea pig paper used the ribonuclease protection assay
(RNase protection assay) because they wanted to measure
two kinds of mRNA at the same time:
-mRNA for LDL receptor
-mRNA for Cytochrome P7
Since RT-PCR is now extremely common, we will review
that method in detail.
Polymerase Chain Reaction (PCR):
TO MEASURE AMOUNT
OF ONE SPECIFIC mRNA
IN A CELL SAMPLE
CELL#1:
Few copies of
mRNA for a
certain protein
CELL#2:
Many copies of
mRNA for
that protein
LYSE CELLS
WITH INHIBITORS:
mRNA ISOLATED
INTACT
mRNA:
Sample #1
NOTE: Many other
kinds of mRNA
are also present!
mRNA:
Sample #2
The famous enzyme REVERSE TRANSCRIPTASE
is used to make a DNA copy of an RNA sequence.
Why is that called reverse transcriptase?
UUUCGAACCGGUCGUAUUCCGGG
mRNA
strand
Reverse
transcriptase
AAAGCTTGGCCAGCATAAGGCCC
Complentary
DNA strand
In this procedure, mRNA is REVERSE TRANSCRIBED
back into a matching DNA strand (called: cDNA).
With reverse transcriptase, non-specific primers
are often used to get cDNA for all the different kinds
of mRNA in the cell.
Specificity is achieved when we amplify only
one of the cDNA molecules.
You now have the COMPLEMENTARY DNA
STRAND TO YOUR INITIAL mRNA
AAAGCTTGGCCAGCATAAGGCCC
With short primers SPECIFIC for that cDNA, you can get
this replicated by a DNA polymerase.
TTTCGAA
AAAGCTTGGCCAGCATAAGGCCC
TTTCGAACCGGTCGTATTCCGGG
AAAGCTTGGCCAGCATAAGGCCC
Initial product
of PCR reaction
SPECIFIC PRIMER!
Need abundant dATP, dCTP, dTTP, dGTP
to provide bases for the new strand
TTTCGAA
AAAGCTTGGCCAGCATAAGGCCC
Activity of DNA polymerase
TTTCGAACCGGTCGTATTCCGGG
AAAGCTTGGCCAGCATAAGGCCC
The DNA polymerase has made a new DNA sequence,
that is complementary to your original sequence
Once you have a 2d DNA sequence, you will need a primer
to copy that sequence also.
No OH on
at this spot
on DNA
This OH only
present on the
ribose in RNA
RNA AND DNA, key differences:
-uracil on RNA, thymine on DNA
-ribose on DNA lacks OH group on ring
Strands will separate
at high temperature
Double-stranded
DNA
Heat to
90C
DNA polymerase makes a
new complementary
sequence to each strand
Cool to
65C
REPEAT
Heat
Cool
Each cycle: -heat to separate the strands
-cool and allow strand replication
Doubles the amount of DNA
After about 40 cycles, there is enough DNA to visualize
with simple ethidium bromide fluorescence on a gel.
Newer methods (Sybr Green, etc) measure the formation
of the product right within the PCR machine. We do that
now in Weed Hall and other labs (called real-time PCR).
The amount of cDNA that is made after many
cycles (typically, 40) is roughly proportional
to the amount of mRNA isolated from the
cells at the start of the experiment.
The cDNA sample is run on a gel, stained with
ethidium bromide, and quantified with
a fluorescence gel reader.
Sample1
Gel
migration
Sample2
WHICH BAND SHOWS
MORE STAINING?
Corn Fiber Oil Lowers Plasma Cholesterol by Altering
Hepatic Cholesterol Metabolism and Up-Regulating
LDL Receptors in Guinea Pigs
Tripurasundari Ramjiganesh, Suheeta Roy, Hedley C.
Freake, Jonathan C. McIntyre* and Maria Luz Fernandez
This study in guinea pigs uses a food rich in stanols to lower
plasma cholesterol.
The mechanism is complex, and involves recirculation of bile acids.
This interesting study, which used both plasma and liver samples,
showed some important effects on expression of genes in the liver
involved in the biochemistry of lipids.
BILE SALTS ARE NEEDED FOR DIGESTION OF FATS.
WE MAKE THEM IN THE LIVER FROM CHOLESTEROL.
SOME BILE SALT IS USUALLY REABSORBED AND USED AGAIN.
What would happen if the bile salts were NOT reasorbed?
The liver uses cholesterol (largely imported from the plasma
through the LDL receptor) to make bile acids needed
for lipid digestion in the small intestine.
MUCH OF THIS CHOLESTEROL IS OBTAINED BY GETTING
CHOLESTEROL-RICH LDL FROM THE PLASMA.
THE LDL ARE IMPORTED WITH THE LDL RECEPTOR ON
THE SURFACE OF THE HEPATOCYTE.
These compounds in corn fiber oil (called STANOLS)
can BLOCK the re-asorption of bile acids.
This has striking effects on metabolic events in the liver!
The liver has to make MORE BILE ACIDS..using
cholesterol from some source. LDL is a very good source.
LIPOPROTEINS CONSIST
LARGELY OF
CHOLESTEROL
The upper panels show intensity of mRNA for the
LDL receptor in liver, which increases with either
corn fiber oil, or with low cholesterol diet.
DISCUSSION: How has corn fiber oil changed the
biochemistryof the guinea pig?
-Which individual metabolic pathways are now altered?
-How could we determine if this were happening
in a person who added this to their diet?
WHAT KIND OF CHANGES IN GENE EXPRESSION
CAN BE TYPICALLY STUDIED IN HUMANS?
What questions were asked in the study of humans
placed on a weight-reduction program?
NOTE: Changes in biochemistry and gene expression
in the liver are a MAJOR topic in nutrition..but we
usually can’t measure that directly by getting
human liver biopsies!
What CAN we do instead? Important question.
STUDY USING CIRCULATING WBC IN HUMANS:
The Lowering of Plasma Lipids following a Weight
Reduction Program Is Related to Increased
Expression of the LDL Receptor and Lipoprotein
Lipase
Madhu Patalay, Ingrid E. Lofgren, Hedley C. Freake,
Sung I. Koo, and Maria Luz Fernandez
Journal of Nutrition, 135: 735-739, 2004
LDL-receptor gene, chromosome 19,
at P13.3
Lipoprotein lipase gene
Chromosome 8, at P22
Lipoprotein lipase (LPL) acts to convert
trigylcerides to glycerol and free fatty absorbs, which
are then transported into the cell.
Mononuclear cell isolation. Mononuclear
cells were isolated from whole blood
through centrifugation on a Ficoll gradient
according to the method of Boyum (21).
NOTE: Mononuclear cells are a class of white
cells. They are usually 3-5% of the total
WBC, and upon stimulation can become active
macrophages. Many diet studies look
at this class of WBC, because they are thought to
play a role in heart disease.
RT-PCR USED
FOR THIS
MEASUREMENT
DATA FROM TWO SUBJECTS IN THE STUDY: Shows
changes in specific mRNA levels for several genes.
DISCUSSION:
-Which CLASS of proteins changed in monocytes of subjects
on a weight-loss program?
-What can we conclude about metabolites available to monocytes,
which are active cells that absorb many components from
the bloodstream?