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Overexpression of CD36 and acyl-CoA synthetases FATP2, FATP4 and ACSL1 increases fatty acid uptake in human hepatoma cells.

Krammer J, Digel M, Ehehalt F, Stremmel W, F├╝llekrug J, Ehehalt R - Int J Med Sci (2011)

Bottom Line: FATP2 had the highest effect on B12 uptake of all proteins tested.Whereas [3H]-oleic acid uptake was increased after overexpression, CD36 had no effect on B12 uptake.Therefore these molecules are potential targets for treatment of nonalcoholic fatty liver disease (NAFLD) or steatohepatitis (NASH).

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, Hepatology and Infectious Diseases, University Hospital Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany.

ABSTRACT

Background: Understanding the mechanisms of long chain fatty acid (LCFA) uptake in hepatic cells is of high medical importance to treat and to prevent fatty liver disease (FLD). ACSs (Acyl-CoA synthetases) are a family of enzymes that catalyze the esterification of fatty acids (FA) with CoA. Recent studies suggest that ACS enzymes drive the uptake of LCFA indirectly by their enzymatic activity and could promote special metabolic pathways dependent on their localization.The only protein located at the plasma membrane which has consistently been shown to enhance FA uptake is CD36.

Aims: The current study investigated whether ACSs and CD36 could regulate hepatic LCFA uptake.

Methods and results: FATP2 and FATP4 were both localized to the ER of HuH7 and HepG2 cells as shown by double immunofluorescence in comparison to marker proteins. ACSL1 was located at mitochondria in both cell lines. Overexpression of FATP2, FATP4 and ACSL1 highly increased ACS activity as well as the uptake of [3H]-oleic acid and fluorescent Bodipy-C12 (B12) fatty acid. Quantitative FACS analysis showed a correlation between ACS expression levels and B12 uptake. FATP2 had the highest effect on B12 uptake of all proteins tested. CD36 was mainly localized at the plasma membrane. Whereas [3H]-oleic acid uptake was increased after overexpression, CD36 had no effect on B12 uptake.

Conclusion: Uptake of LCFA into hepatoma cells can be regulated by the expression levels of intracellular enzymes. We propose that ACS enzymes drive FA uptake indirectly by esterification. Therefore these molecules are potential targets for treatment of nonalcoholic fatty liver disease (NAFLD) or steatohepatitis (NASH).

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HuH7 cells with short time incubation of B12 fatty acid. Overexpression of RFP-FATP4, FATP2-HA and ACSL1-FLAG increased B12 uptake into the cell. B12 was accumulated and stored in lipid droplets. CD36-FLAG did not show any enhanced uptake of B12 compared to control cells.
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Figure 7: HuH7 cells with short time incubation of B12 fatty acid. Overexpression of RFP-FATP4, FATP2-HA and ACSL1-FLAG increased B12 uptake into the cell. B12 was accumulated and stored in lipid droplets. CD36-FLAG did not show any enhanced uptake of B12 compared to control cells.

Mentions: To compare FA uptake to the expression level of the individual protein at a single cell level, we performed FACS analysis as described 29. FATP2, CD36 and ACSL1 were transiently overexpressed and incubated for a short time with the fluorescent long chain fatty acid B12. FATP4 was used as RFP-construct, FATP2, CD36 and ACSL1 were marked by immunofluorescence. In initial experiments our cells were analysed by a confocal microscope and classified by visual characteristics. We could register an increased fluorescence level and thus increased B12 uptake upon overexpression of FATP2, FATP4 and ACSL1 compared to their non-expressing counterparts. Surprisingly, there was no significant uptake detectable upon overexpression with CD36 (figure 7).


Overexpression of CD36 and acyl-CoA synthetases FATP2, FATP4 and ACSL1 increases fatty acid uptake in human hepatoma cells.

Krammer J, Digel M, Ehehalt F, Stremmel W, F├╝llekrug J, Ehehalt R - Int J Med Sci (2011)

HuH7 cells with short time incubation of B12 fatty acid. Overexpression of RFP-FATP4, FATP2-HA and ACSL1-FLAG increased B12 uptake into the cell. B12 was accumulated and stored in lipid droplets. CD36-FLAG did not show any enhanced uptake of B12 compared to control cells.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198256&req=5

Figure 7: HuH7 cells with short time incubation of B12 fatty acid. Overexpression of RFP-FATP4, FATP2-HA and ACSL1-FLAG increased B12 uptake into the cell. B12 was accumulated and stored in lipid droplets. CD36-FLAG did not show any enhanced uptake of B12 compared to control cells.
Mentions: To compare FA uptake to the expression level of the individual protein at a single cell level, we performed FACS analysis as described 29. FATP2, CD36 and ACSL1 were transiently overexpressed and incubated for a short time with the fluorescent long chain fatty acid B12. FATP4 was used as RFP-construct, FATP2, CD36 and ACSL1 were marked by immunofluorescence. In initial experiments our cells were analysed by a confocal microscope and classified by visual characteristics. We could register an increased fluorescence level and thus increased B12 uptake upon overexpression of FATP2, FATP4 and ACSL1 compared to their non-expressing counterparts. Surprisingly, there was no significant uptake detectable upon overexpression with CD36 (figure 7).

Bottom Line: FATP2 had the highest effect on B12 uptake of all proteins tested.Whereas [3H]-oleic acid uptake was increased after overexpression, CD36 had no effect on B12 uptake.Therefore these molecules are potential targets for treatment of nonalcoholic fatty liver disease (NAFLD) or steatohepatitis (NASH).

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, Hepatology and Infectious Diseases, University Hospital Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany.

ABSTRACT

Background: Understanding the mechanisms of long chain fatty acid (LCFA) uptake in hepatic cells is of high medical importance to treat and to prevent fatty liver disease (FLD). ACSs (Acyl-CoA synthetases) are a family of enzymes that catalyze the esterification of fatty acids (FA) with CoA. Recent studies suggest that ACS enzymes drive the uptake of LCFA indirectly by their enzymatic activity and could promote special metabolic pathways dependent on their localization.The only protein located at the plasma membrane which has consistently been shown to enhance FA uptake is CD36.

Aims: The current study investigated whether ACSs and CD36 could regulate hepatic LCFA uptake.

Methods and results: FATP2 and FATP4 were both localized to the ER of HuH7 and HepG2 cells as shown by double immunofluorescence in comparison to marker proteins. ACSL1 was located at mitochondria in both cell lines. Overexpression of FATP2, FATP4 and ACSL1 highly increased ACS activity as well as the uptake of [3H]-oleic acid and fluorescent Bodipy-C12 (B12) fatty acid. Quantitative FACS analysis showed a correlation between ACS expression levels and B12 uptake. FATP2 had the highest effect on B12 uptake of all proteins tested. CD36 was mainly localized at the plasma membrane. Whereas [3H]-oleic acid uptake was increased after overexpression, CD36 had no effect on B12 uptake.

Conclusion: Uptake of LCFA into hepatoma cells can be regulated by the expression levels of intracellular enzymes. We propose that ACS enzymes drive FA uptake indirectly by esterification. Therefore these molecules are potential targets for treatment of nonalcoholic fatty liver disease (NAFLD) or steatohepatitis (NASH).

Show MeSH
Related in: MedlinePlus