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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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Related in: MedlinePlus

Bodipy fatty acid uptake of RFP-FATP4, CD36-FLAG, FATP2-HA and ACSL1-FLAG transfected HuH7 cells in arbitrary units based on FACS analyses. Bodipy fatty acid uptake was correlated to the protein expression level of transfected protein on a snigle cell basis. The gated overexpressing cells (in % of all cells gated) were multiplied with the corresponding overall mean of the fluorescence signal derived from the Bodipy fatty acid (SBodipy). HuH7 Cells transfeced with pcDNA3 were used as control and as background range and were set as base level of the x-axis. Uptake was performed with B12 fatty acid in experiments with FATP4, FATP2, ACSL1 and CD36 (CD36 B12) or with B16 (CD36 B16). The pictured diagram is representative for three independent experiments.
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Figure 9: Bodipy fatty acid uptake of RFP-FATP4, CD36-FLAG, FATP2-HA and ACSL1-FLAG transfected HuH7 cells in arbitrary units based on FACS analyses. Bodipy fatty acid uptake was correlated to the protein expression level of transfected protein on a snigle cell basis. The gated overexpressing cells (in % of all cells gated) were multiplied with the corresponding overall mean of the fluorescence signal derived from the Bodipy fatty acid (SBodipy). HuH7 Cells transfeced with pcDNA3 were used as control and as background range and were set as base level of the x-axis. Uptake was performed with B12 fatty acid in experiments with FATP4, FATP2, ACSL1 and CD36 (CD36 B12) or with B16 (CD36 B16). The pictured diagram is representative for three independent experiments.

Mentions: To find out, which one of the investigated proteins causes the quantitative highest FA uptake, we compared Bodipy fatty acid uptake to the fluorescent level of the overexpressed candidate protein on a single cell basis. The percentage of gated cells was multiplied with the mean of the fluorescence signal derived from B12. Presenting this data in arbitrary units showes that FATP2 overexpressing cells seem to have the quantitative highest uptake of fluorescent fatty acid compared to their expression levels (figure 9). This means less FATP2 within one cell is necessary to incease FA uptake to a similar level compared to other candidate proteins.


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)

Bodipy fatty acid uptake of RFP-FATP4, CD36-FLAG, FATP2-HA and ACSL1-FLAG transfected HuH7 cells in arbitrary units based on FACS analyses. Bodipy fatty acid uptake was correlated to the protein expression level of transfected protein on a snigle cell basis. The gated overexpressing cells (in % of all cells gated) were multiplied with the corresponding overall mean of the fluorescence signal derived from the Bodipy fatty acid (SBodipy). HuH7 Cells transfeced with pcDNA3 were used as control and as background range and were set as base level of the x-axis. Uptake was performed with B12 fatty acid in experiments with FATP4, FATP2, ACSL1 and CD36 (CD36 B12) or with B16 (CD36 B16). The pictured diagram is representative for three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198256&req=5

Figure 9: Bodipy fatty acid uptake of RFP-FATP4, CD36-FLAG, FATP2-HA and ACSL1-FLAG transfected HuH7 cells in arbitrary units based on FACS analyses. Bodipy fatty acid uptake was correlated to the protein expression level of transfected protein on a snigle cell basis. The gated overexpressing cells (in % of all cells gated) were multiplied with the corresponding overall mean of the fluorescence signal derived from the Bodipy fatty acid (SBodipy). HuH7 Cells transfeced with pcDNA3 were used as control and as background range and were set as base level of the x-axis. Uptake was performed with B12 fatty acid in experiments with FATP4, FATP2, ACSL1 and CD36 (CD36 B12) or with B16 (CD36 B16). The pictured diagram is representative for three independent experiments.
Mentions: To find out, which one of the investigated proteins causes the quantitative highest FA uptake, we compared Bodipy fatty acid uptake to the fluorescent level of the overexpressed candidate protein on a single cell basis. The percentage of gated cells was multiplied with the mean of the fluorescence signal derived from B12. Presenting this data in arbitrary units showes that FATP2 overexpressing cells seem to have the quantitative highest uptake of fluorescent fatty acid compared to their expression levels (figure 9). This means less FATP2 within one cell is necessary to incease FA uptake to a similar level compared to other candidate proteins.

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