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Quantitative analysis of proteins facilitating fatty acid uptake: CD36, Caveolin-1, FATP4 and ACSL1

Schneider, Hannah Katarina

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Fatty acids are essential for the cellular metabolism but their uptake has to be tightly regulated to prevent fatty acid associated diseases. Proteins that facilitate fatty acid uptake include CD36, FATP4, ACSL1 and Caveolin-1. CD36, FATP4 and ACSL1 increase oleate uptake upon overexpression. CD36 is an integral transmembrane glycoprotein that directly facilitates fatty acid uptake across the plasma membrane. The structural protein Caveolin-1 binds fatty acids and is discussed as potential interaction partner for CD36. The acyl-CoA synthetases FATP4 and ACSL1 are localized intracellularly and activate fatty acids by esterification with coenzyme A, thus driving fatty acid uptake indirectly by metabolic trapping.

This study was designed to 1) quantify absolute protein amounts of CD36, FATP4 and ACSL1 proteins in overexpressing cell lines and calculate protein specific oleate uptake 2) test CD36, FATP4, ACSL1 and Caveolin-1 for cooperativity in facilitating fatty acid uptake.

MDCK cells were chosen as an unspecialized and unbiased model system and overexpressing cells were generated by retroviral and adenoviral infection. Infection of MDCK cells with increasing amounts of CD36 or FATP4 adenovirus resulted in enhanced fatty acid uptake as measured by incubation with radiolabeled oleic acid for three hours. Protein amounts of CD36 and FATP4 were analyzed by western blotting and absolute protein quantities were calculated by correlation to a recombinant protein standard. Increases in fatty acid uptake correlated to rising protein amounts of CD36 and FATP4 in accordance with rising adenovirus quantities used for infection.

Comparison of CD36 and FATP4 expression showed a significant difference in protein quantities whereas the difference in oleate uptake was much smaller: 13 ng CD36 protein increased oleate uptake by 20 % whereas 701 ng FATP4 protein resulted in 37 % enhancement. When CD36 and FATP4 were expressed at equal quantities, oleate uptake was still enhanced more by 10 ng CD36 protein as by 10 ng FATP4 protein (27 % versus 4 %, respectively), indicating that CD36 is more efficient in increasing fatty acid uptake than FATP4. The function of CD36 in fatty acid transport of MDCK cells was independent from Caveolin-1, as an 80 % knock down of Caveolin-1 did not alter CD36 dependent oleate uptake.

Immunofluorescence analysis confirmed the localization of CD36 to the plasma membrane whereas FATP4 localized to the endoplasmic reticulum and ACSL1 was expressed at mitochondria.Co-expression of CD36 and FATP4 enhanced oleate uptake significantly more than calculated from single protein overexpression, resulting in an increase by 49 pmol oleate/μg total protein for co-expressing cells as compared to 10 pmol oleate/μg total protein for CD36 and 21 pmol oleate/μg total protein for FATP4 overexpressing cells. Oleate uptake was similarly increased in CD36 and ACSL1 co-expressing cells whereas FATP4 and ACSL1 together enhanced fatty acid uptake less than calculated from single protein overexpression: oleate uptake was increased by 30 pmol oleate/μg total protein in co-expressing cells as compared to 26 pmol oleate/μg total protein in FATP4 and 15 pmol oleate/μg total protein in ACSL1 cells.

In conclusion, CD36, FATP1 and ACSL1 enhance fatty acid uptake by two different mechanisms: CD36 directly facilitates fatty acid transport across the plasma membrane whereas FATP4 and ACSL1 indirectly mediate fatty acid uptake by metabolic trapping of intracellular fatty acids. CD36 is expressed at significantly lower protein amounts than FATP4 but increases fatty acid uptake more than FATP4 when expressed at similar quantities. The two acyl-CoA synthetases FATP4 and ACSL1 target similar metabolic processes and therefore show no cooperativity in fatty acid uptake. As both enzyme act intracellularly, their impact on fatty acid uptake is limited by the transport rate of fatty acids across the plasma membrane. CD36 cooperates with FATP4 and ACSL1 in enhancing oleate uptake, probably due to an efficient combination of facilitated fatty acid transport across the plasma membrane by CD36 followed by immediate intracellular fatty acid activation by FATP4 and ACSL1.

Item Type: Dissertation
Supervisor: Fricker, Prof. Dr. Gert
Date of thesis defense: 17 December 2012
Date Deposited: 18 Jan 2013 07:32
Date: 14 January 2013
Faculties / Institutes: The Faculty of Bio Sciences > Institute of Pharmacy and Molecular Biotechnology
Subjects: 570 Life sciences
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