1. PPARα Expression in Postprandial Python Liver
Introduction
Eric Cobb, MCDB: 4202-800
Background:
PPARα (Peroxisome Proliferator-Activated Receptor ahlpa) is a
type II nuclear receptor has been identified as the master
regulator of hepatic lipid metabolism. Additionally, PPARα has
been shown to regulate: fatty acid β-oxidation, glucose
metabolism, amino acid metabolism, bile acid synthesis and
hepatocyte proliferation. Similar to other members of the
nuclear receptor superfamily PPARα heterodimerizes with
retinoid X receptor (RXR) and binds to a DNA response element,
in this case the peroxisome proliferator response element
(PPRE). Corepressors are recruited to the PPARα/RXR complex,
preventing transcription of target genes until the a ligand is
present to bind PPARα. Natural ligands of PPARα include a
variety of dietary fatty acids (FA) and other molecules
resembling FA structures including Acetyl-CoAs, Enoyl-CoAs,
and endocannabinoids. Synthetic ligands such as fibrates is
used for the treatment of dyslipidemia associated with type II
diabetes mellitus. Upon ligand binding causes a conformational
change in the PPARα/RXR complex resulting in the dissociation
of corepressor. Thus allowing for essential coactivators such as
PBP/MED1, to promote transcription of genes such as CD36,
Scd1, and Cyp7a1 to name a few. PPARα has also shown to
influence other lipogenic genes such by regulating transcription
factors SREBP-1c and LXRα. Deficiency or absence of PPARα
results in hepatic steatosis and hypolipedemia, indicating that
PPARα is an essential gene for the regulation of lipid levels in
the liver.
Hypothesis:
Considering the high levels of TAGs and FAs in python serum
after 1 day post fed, genes involved in lipid uptake and break
down should increase in order to decrease serum lipid levels
back to normal by 6-10 days post fed. Because PPARα activate
plays such a large role in the regulation of lipid metabolism I
expect to see an increase in PPARα expression between 1 and 6
days post fed. As this is the range in time points showing a
decrease in serum lipid levels.
Department of Molecular, Cellular, and Developmental Biology
University of Colorado-Boulder
Quantitative PCR Results
The Burmese Python:
Burmese Python undergo drastic physiological changes after
feeding. Firstly, the python can digest a meal 1.6 times it’s own
body weight which would be equivalent to a 145lb man eating a
232lb meal. Despite the size of their meal, digestion is
completed only 6-10 days later. After only 1 day post fed, the
python shows an substantial increase in glucose metabolism, a
rapid decrease in pH, and increase an increase in triacylglyceride
(TAGs) levels that would be fatal to humans. Furthermore,
organs such as the liver and the heart nearly double in size
through hyperplasia and hypertrophy respectively. The python
proves to be an extreme example of regulation of physiology
that is worth exploring to uncover ways to improve lipid
regulation and healthy organ hyperplasia and hypertrophy in
humans. Which could be used for target therapies for diseases
like diabetes and heart disease.
Postprandial Python Serum Lipid LevelsIntroduction
PPARα Null Mice Results in Hepatic Steatosis
PPARα -/- fasted mice results in hepatic steatosis: (A) Image (Left) shows WT liver,
image (Right) shows 66hr fasted PPARα -/- liver. Figures (B,D,F) are Fed mice and
(C,E,G) are fasted 48 hr mice, all of which are stained with Oil Red O-stains (stains
neutral triglycerides and lipids. Figures (B,C) are PPAR +/+, Figures (D,E) are PPAR -
/-, and Figures (F,G) is a double knockout PPAR -/- and AOX -/-.
Methods
Primer Design:
• Obtained mRNA sequence of PPARα from NCBI
and mapped exons
• Ran a Nucleotide Blast of mRNA sequence and
assembled python PPARα transcript from
overlapping sequences
• Validated gene product using Expasy Translate and
Protein Blast
• Used Primer3 to design optimal primers and
validated them with Primer Blast
Forward Primer: 5’-CTTGTGGGGAAAGCCAGTAA-3’
Reverse Primer: 5’-CACTGGCAGCAGTGGAAAAT-3’
Experimental Design:
• Isolated RNA from liver tissue homogenates from different
time points (Fasted-15 days post fed) using a Quiagen
RNeasy Mini Kit protocol
• Determined the concentration and purity of our isolated
RNA using spectrophotometry to obtain 260/280
absorbance ratio
• Conducted an analysis of the integrity of our isolated RNA
using gel electrophoresis
• Synthesized cDNA from isolated RNA using Superscript III
reverse transcriptase
• Conducted PCR using our designed Primers and synthesized
cDNA and ran the products on a gel to determine if our
primers are amplifying products of the same size as the
target gene
• Assessed relative gene expression of target genes compared
to reference genes using quantitative PCR (qPCR)
Conclusion
Quantitative PCR Relative Expression of
PPARα: A) Expression of PPARα in python liver
(Fasted-15dpf) relative to GAPDH reference
gene expression. At 3dpf there is a 52 fold
increase in PPARα expression. Fasted expression
showed roughly one fold increase in expression.
All other time points all show higher relative
expression compared to GAPDH indicating that
PPARα is high in abundance in python liver. B)
Standard Curve for qPCR plate with the y-axis
representing cycle number and the x-axis Log
Starting Quantity. Target gene of unknown
samples amplified between cycles 16-21. All
samples were done in triplicate. C) Melt Peak
shows amplification of a single gene product
represented by the single peak between 75-80
degrees Celsius.
A)
B) C)
The dramatic increase in PPARα expression in python liver suggests
that PPARα may be essential for the transcription of genes involved
in FA metabolism and lipogenenesis. Thereby, preventing
lipotoxicity and hepatic steatosis. Considering CD36, a gene
regulated by PPARα, showed a 36 fold increase at the same time
point PPARα had significant expression suggests that PPARα
activates expression of CD36 to import circulating FAs.
Furthermore, this may suggest a positive feedback mechanism may
be promoting the efflux of PPARα ligands into hepatocytes
maximize lipid intake into the hepatocytes for conversion into bile
acids or break down into energy through β, ω-oxidation. This would
explain the substantial decrease in lipid levels in python serum
between 3 dpf and 6 dpf. However, the fact that PPARα shows high
expression at 3 dpf rather than at 1 dpf indicates that other genes
are promoting the break down of lipids in the liver. Possibly
contributing to the PPARα ligand pool, such as Acetyl-CoA’s, that
are then used to promote the expression seen at 3 dpf.
Alternatively, other nuclear receptors such as LXR and FXR may be
expressed earlier resulting in decreased levels of RXR available for
PPARα to heterodimerize with which could explain the decreased
expression levels at theses other time points. Further studies of
genes regulated by PPARα and genes that compete for binding to
RXR would help further solidify PPARα as a key regulator of lipid
metabolism in the python liver.
Sources and Acknowledgements: Background information: Peroxisome Proliferator-Activated Receptor Alpha Target Genes,
Rakhshandehroo et al., 2010; PPARα: energy combustion, hypolipidemia, inflammation and cancer, Pyper et al., 2010 Serum lipid leve
figure: Fatty Acids Identified in the Burmese Python Promote Beneficial Cardiac Growth, Leinwand et al., 2011:PPAR -/- figure:
http://www.jbc.org/content/275/37/28918.long. Acknowledgments: I would like to thank Professor Pamela Harvey for being such an
active leader in our research efforts in the Python Project, and I would like to thank Leslie Leinwand’s lab for their support.