Nutrition and Gene Expression Lecture, Feb 12, 2015 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.

R MAKE THE INITIAL RNA PROCESS TO mRNA: this is measured TRANSLATE TO PROTEIN MODIFY THE PROTEIN 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 are an older, well-established method for measurement of the amount of specific mRNA. -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 mRNA: Sample #2 NOTE: Many other kinds of mRNA are also present!

The famous enzyme REVERSE TRANSCRIPTASE is used to make a DNA copy of an RNA sequence. Why is that called reverse transcriptase?

mRNA strand UUUCGAACCGGUCGUAUUCCGGG AAAGCTTGGCCAGCATAAGGCCC Reverse transcriptase 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. AAAGCTTGGCCAGCATAAGGCCC TTTCGAA AAAGCTTGGCCAGCATAAGGCCC TTTCGAACCGGTCGTATTCCGGG Initial product of PCR reaction

AAAGCTTGGCCAGCATAAGGCCC TTTCGAA AAAGCTTGGCCAGCATAAGGCCC TTTCGAACCGGTCGTATTCCGGG Need abundant dATP, dCTP, dTTP, dGTP to provide bases for the new strand Activity of DNA polymerase The DNA polymerase has made a new DNA sequence, that is complementary to your original sequence SPECIFIC PRIMER! Once you have a 2d DNA sequence, you will need a primer to copy that sequence also.

RNA AND DNA, key differences: -uracil on RNA, thymine on DNA -ribose on DNA lacks OH group on ring This OH only present on the ribose in RNA No OH on at this spot on DNA

Double-stranded DNA Heat to 90C Strands will separate at high temperature Cool to 65C DNA polymerase makes a new complementary sequence to each strand REPEAT HeatCool

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. WHICH BAND SHOWS MORE STAINING? Sample1 Sample2 Gel migration

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. 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

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: , 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.

DATA FROM TWO SUBJECTS IN THE STUDY: Shows changes in specific mRNA levels for several genes. RT-PCR USED FOR THIS MEASUREMENT

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?