The Factors of FATP and ACS in Lipid Uptake in
the Intestine and Liver
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Sean Crawford |
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Lipid uptake in the intestinal epithelium and liver has always been considered an act of diffusion. However, the recent discovery of several proteins that play a role in lipid uptake has challenged this notion. The major reason for this suspicion is that once a triglyceride has been processed in an enterocyte, it is secreted as a chylomicron particle, which is far too large to pass through the cell by diffusion. Therefore, a more complex process appears, involving the breakdown of triglycerides and cholesterol into fatty acids along the intestine. Once the fatty acid has been transported into the cell, it is then resynthesized into a triglyceride and an ApoB protein is added, creating the chylomicron.
Fatty Acid Transport Protein (FATP) and Acyl-CoA Synthetase (ACS) are believed to be the two principle proteins involved in the uptake of lipids along the plasma membrane of enterocytes in the gut and hepatocytes in the liver. FATP transports the fatty acid across the plasma membrane and ACS adds an Acyl-CoA group to the fatty acid to prevent it from exiting the cell. Both FATP and ACS have five isoforms in mice, while six isoforms of FATP exist in humans. Some isoforms are nerve-specifically expressed. FATP4 is believed to be strongly expressed in the intestine, and FATP5 is exclusively expressed in the liver. The main question to be asked is, which proteins are expressed in which cells? Once this answer is found, knockouts can be made for the genes encoding them or anti-peptides can be created which will prevent lipid uptake at the protein level in the cells. This can lead to effective treatment of obesity, artherosclerosis, diabetes, and other diseases.
The cells used as models for the intestine and liver were CaCo2 cells, and HepG2 cells, respectively. CaCo2 cells are fetal colonocytes, colon cancer cells. They display very similar characteristics to intestinal enterocytes, and can be grown to differentiation relatively quickly. This allows for the comparison of changes in expression due to differentiation. The cells become polarized upon differentiation and are expected to uptake lipids accordingly. HepG2 cells are human hepatocyte cells, having the exact same characteristics as living hepatocytes.
The process of determining which RNA is expressed in each cell is indirect. RNA from the cells is extracted and reverse transcribed creating cDNA. The cDNA is then amplified, using primers specific to the protein of interest, in a polymerase chain reaction (PCR). Primers are created based on the sequence of each protein in the human genome database, and a gel is run to determine if the amplified segments are the proper lengths. Only then can it be determined if the gene is expressed in the cell.
Once each primer set had been created, the RT-PCR reactions had to be done, and the segments measured by gel electrophoresis. Each primer set was run for both types of cells and the presence or absence of the RNA was recorded. In undifferentiated CaCo2 cells, FATP1 was faintly expressed, FATP2, 3, and 5 were expressed at higher levels, and FATP4 was strongly expressed. ACS4 was the only ACS not expressed. In 21 Day differentiated CaCo2 cells, all FATP isoforms were expressed, FATP1 being weakly expressed. All ACS isoforms were also expressed in the 21 Day cells. HepG2 cells produced positive results for FATP2, 3, and 5. All ACS isoforms were expressed at the same level, except FACL3 was seen more faintly than the others.
The only difficulty with a RT-PCR approach to analyzing protein expression is that it is not quantitative. There is either a positive or a negative result, which can only be compared to relative strengths of each other protein expressed in the same gel. In the future, steps will be taken for a quantitative approach for the proteins that are known in the cells. This can be done using radioisotopes when performing the PCR reactions. Also, there is a two-pronged attack at prohibiting lipid uptake. The first technique is at the DNA level, by splicing a strand of DNA so that the protein is never produced. The second method is to create anti-peptides that prohibit lipid uptake once the protein has already been synthesized.
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Natural Sciences Learning Center
Washington University - Biology
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