Python Poster (1)
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Hepatocyte Nuclear Factor 4 and Bile Acid Transport
Anne Cathryn Cox | | Python Project Fall 2016
Standard Curve Melt Peak
WGS sequence mapped to Gallus gallus annotated HNF4A gene
Primers for HNF4A created from Whole Genome Shotgun (WGS) Python sequence
Primers validated in-silico using NCBI BLAST
Primer Design RNA Isolation
RNA isolated from fasted, 1, 3, and 10 day-post-fed python liver tissue using RNeasy column
Two python specimens used per sample to minimize error due to individual python abnormalities
cDNA Synthesis cDNA made from RNA using
(1) random hexamers, (2) oligo d(T), and (3) gene specific primers to find best synthesis route
Gene specific primers found as best synthesis protocol
cDNA synthesized for fasted, 1, 3, and 10 day-post-fed RNA (100% synthesis assumed)
Quantitative PCR qPCR performed using SYBR
Select (binds to dsDNA) on diluted fasted, 1, 3, and 10 days-post-fed cDNA
Standard curve created with known concentrations of pooled cDNA to interpolate starting quantity of unknowns
Number of PCR cycles required to amplify was correlated to starting quantity
Products from qPCR reaction were run on a 1% agarose gel with Ethidium Bromide
Product size verified as intended product from in-silico validation to verify that primers amplified correct gene
Bile Acid Transport and HNF4AHepatocyte nuclear factor 4 alpha (HNF4A) is one of several transcription factors that heavily regulate genes involving the transport of bile acids around the digestive system, largely from the liver where they are synthesized through the bile duct to the intestines to digest fats, and then back to the liver from the intestines. By focusing on a transcription factor for many transport proteins instead of one specific transport protein, expression levels can potentially be implied for many genes and not just one.
In knockout studies with mice, adult male mice had decreased levels of mRNA for NCTP (Sodium Taurocholrate Cotransporting Polypeptide) among others when HNF4A was rendered nonfunctional in the liver (Lu, Gonzalez, & Klaassen, 2010). NCTP, expressed less in the absence of HNF4A, is a transport pump and is the primary way in which bile acids recycle from the ileum and colon back into the hepatocyte for reuse. HNF4A has been shown to increase NCTP in other studies as well (Dietrich et al., 2007; Hayhurst, Lee, Lambert, Ward, & Gonzalez, 2001).
Python Bivittatus and Organ HypertrophyThe Burmese python has several key physiological processes that make it such a pertinent animal for research on human disease involving the metabolism of cholesterol and other fats, including metabolic syndrome, pathological cardiac hypertrophy, and hepatic steatosis (fatty liver disease).
The pythons digestive organs, including the heart, all grow in mass rapidly after consuming a meal (Secor, 2008). This organ growth is physiological and not pathological, and is attributed to a few select fatty acids present in the pythons serum that are not originally from the meal (Riquelme et al., 2011; Secor, 2008). Key insights from research on the Burmese python are already yielding new human implications for treating cardiac regression in cancer patients and cardiac hypertrophy (Riquelme et al., 2011). However, it is not well understood where these key fatty acids come from in the python serum, and how the python processes the large amount of fat in its postprandial system and serum. In order to address this, researchers from the Python Project under the lab of Dr. Leslie Leinwand are looking into specific genes that are known in humans to play a role in either the synthesis or transport of bile acids, which emulsify fats in the digestive system and allow them to be processed. By unlocking the key to how pythons process such large amounts of fat and remain healthy, human conditions such as metabolic syndrome can be looked at from another, perhaps more enlightening angle than previous research.
Figure from (Secor 2008)
Figure from (Riquelme et al., 2011)
In another knockout study, the serum chemistry of wild-type vs HNF4A-null mice was analyzed (Hayhurst et al., 2001). This study found that in addition to having decreased NCTP levels by western blot, HNF4A null mice had increased levels of ALT, triglycerides, total cholesterol, HDL cholesterol, and bile acids in their serum (Hayhurst et al., 2001). This leads to the conclusion that HNF4A must be important to getting bile acids out of an organisms serum and into its hepatocytes for use in the liver.
Another important function of HNF4A is to help hepatocyte nuclear factor 1 alpha (HNF1A), another important hepatic transcription factor, to bind its promoters on several other hepatic genes in liver cell nuclei (Eeckhoute, Formstecher, & Laine, 2004). When it does this, the genes which are activated by HNF1A, such as Cholesterol 7-Alpha-Hydroxylase (CYP7A1) are also upregulated. CYP7A1 plays a vital role and is the rate determining step in the conversion of cholesterol into bile acids.
Because of HNF4As varying functions in bile acid synthesis and export, it may hold key insights into fat digestion in the Burmese python.
Figure from (Dawson et al., 2009)
hepatocyte nuclear factor 4 alpha (HNF4A) expression in python liver tissue was tested using real-time PCR. HNF4A has two separate functions important to bile acid homeostasis in hepatocytes in humans: (1) to upregulate the transcription of transport proteins (largely NCTP) moving recycled bile acid into hepatic cells while downregulating transport proteins which move bile acids from hepatic cells into the canaliculus, and (2) to increase binding of HNF1A to its promoter thereby increasing the transcription of CYP7A1 and the downstream synthesis of bile acids. This research found that expression of HNF4A mRNA doubles from the fasted tissue at 1 day-post-fed (DPF), and then returns to normal by 3 DPF, suggesting that the python uses similar pathways of bile-acid sequestration in hepatocytes as humans.
Figure from (Riquelme et al., 2011)
Figure from (Halilbasic et al., 2013)
Over 1 in 5 adults in the US has metabolic syndrome (MetS), which is a collection of symptoms including some or all of the following: obesity, high fasting plasma glucose, high blood pressure, high triglyceride levels, and low HDL cholesterol levels (Beltrn-Snchez, Harhay, Harhay, & McElligott, 2013). Although this syndrome is well studied, there is still much to learn about how humans process fat in the liver. The Burmese python has several key physiological processes that make it such a pertinent animal for research on human disease involving the metabolism of cholesterol and other fats, including MetS, pathological cardiac hypertrophy, and hepatic steatosis (fatty liver disease). One of these processes is the rapid metabolism of serum fatty acids post-meal. To explore this process,
Primer DesignIn order to find successful primers that would amplify only the intended gene product, the
Real Time PCR
Gallus gallus annotated genome sequence of HNF4A was blasted against the python whole genome shotgun from NCBI, which produced the results shown here. The majority of the gene was able to be assembled using the corresponding contigs from the python WGS, mapping to the order of the Gallus gallus transcript.
The primers shown were chosen because they had acceptable length, melt temperature for real time PCR (~60C), low G-C content, and low self-complementarity. Additionally, they were in 2 different exons, which helps to distinguish mRNA from genomic contamination in the real time PCR experiment.
As an additional precaution, the three possible reading frames of the assembled putative python transcript were translated online with ExPASy to verify that an open reading frame existed.
Standards (Known starting concentrations of
gene-specific cDNA to generate standard curve)
10 ng/L1 ng/L
0.1 ng/L0.01 ng/L
Fasted1 day post fed3 days post fed10 days post fed
Unknowns(cDNA synthesized from python liver tissue at different time point after eating)
To measure changes in gene expression levels of HNF4A, gene specific cDNA from fasted, 1 dpf, 3 dpf, and 10 dpf was synthesized from pooled RNA from two different pythons using the designed primers. This cDNA was then used to set up a real time PCR plate as shown below, using known concentrations of pooled cDNA in order to create a standard curve to interpolate the starting cDNA concentrations of the unknown samples.
The data show that HNF4A is upregulated by approximately 2 fold one day after the python has had a meal. Additionally,
HNF4A remains slightly upregulated at 3 and 10 days post fed.
In subsequent experiments, the expression of NCTP could be measured using the same methodology used in this study. This would provide insights into how important NCTP is to bile acid homeostasis. Additionally, other bile acid transporters such as ASBT, OSTa/OSTb, and BSEP could be explored. Knowing the changes in expression for these genes would allow a more complete picture of bile acid transport in the postprandial Burmese python. Finally, HNF1A (hepatocyte nuclear factor 1 alpha) could be measured, to solidify the relationship between HNF4A and HNF1A.
100 bp200 bp
Product size from primer
design: 121 bp
The products of