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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, Füllekrug 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

Localisation of ACSL1 in HepG2 cells. For ACSL1-FLAG we used mouse anti-FLAG antibody (Sigma) and Cy2 or Cy3 fluorescently-labelled secondary antibodies. HepG2 cells were cotransfected with mito-RFP, a marker for mitochondria and a plasmamembrane marker protein (ssGFPglyc). There was an overlap with the mitochondria staining (mito-RFP) (A) and no concordance with plasmamembrane staining (ssGFPglyc) (B). In addition ACSL1 had no overlap with lipid droplets (adipophilin-mRFP1) (C) or peroxisome (peroxisomal-RFP) marker proteins (D). Results are representative for HuH7 cells as well. Bar: 10 µm.
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Figure 3: Localisation of ACSL1 in HepG2 cells. For ACSL1-FLAG we used mouse anti-FLAG antibody (Sigma) and Cy2 or Cy3 fluorescently-labelled secondary antibodies. HepG2 cells were cotransfected with mito-RFP, a marker for mitochondria and a plasmamembrane marker protein (ssGFPglyc). There was an overlap with the mitochondria staining (mito-RFP) (A) and no concordance with plasmamembrane staining (ssGFPglyc) (B). In addition ACSL1 had no overlap with lipid droplets (adipophilin-mRFP1) (C) or peroxisome (peroxisomal-RFP) marker proteins (D). Results are representative for HuH7 cells as well. Bar: 10 µm.

Mentions: ACSL1 is one of the most extensively characterized proteins of the ACSL-family. Nevertheless its localization is still debatable 27, 29, 48, 49. To specify the localization of FLAG-tagged ACSL1 we did a co-staining with the mitochondria-marker (mito-RFP) and found a co-localization in both HuH7 and HepG2 cell lines (figure 3). No overlap was found with plasma membrane (ssGFPglycTMDpcx), peroxisomes (peroxisomal-mRFP1), ER (mRFP-Sec61ß) or lipid droplets (adipophilin-mRFP1), where it has been previously described to be located as well 27.


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, Füllekrug J, Ehehalt R - Int J Med Sci (2011)

Localisation of ACSL1 in HepG2 cells. For ACSL1-FLAG we used mouse anti-FLAG antibody (Sigma) and Cy2 or Cy3 fluorescently-labelled secondary antibodies. HepG2 cells were cotransfected with mito-RFP, a marker for mitochondria and a plasmamembrane marker protein (ssGFPglyc). There was an overlap with the mitochondria staining (mito-RFP) (A) and no concordance with plasmamembrane staining (ssGFPglyc) (B). In addition ACSL1 had no overlap with lipid droplets (adipophilin-mRFP1) (C) or peroxisome (peroxisomal-RFP) marker proteins (D). Results are representative for HuH7 cells as well. Bar: 10 µm.
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Related In: Results  -  Collection

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

Figure 3: Localisation of ACSL1 in HepG2 cells. For ACSL1-FLAG we used mouse anti-FLAG antibody (Sigma) and Cy2 or Cy3 fluorescently-labelled secondary antibodies. HepG2 cells were cotransfected with mito-RFP, a marker for mitochondria and a plasmamembrane marker protein (ssGFPglyc). There was an overlap with the mitochondria staining (mito-RFP) (A) and no concordance with plasmamembrane staining (ssGFPglyc) (B). In addition ACSL1 had no overlap with lipid droplets (adipophilin-mRFP1) (C) or peroxisome (peroxisomal-RFP) marker proteins (D). Results are representative for HuH7 cells as well. Bar: 10 µm.
Mentions: ACSL1 is one of the most extensively characterized proteins of the ACSL-family. Nevertheless its localization is still debatable 27, 29, 48, 49. To specify the localization of FLAG-tagged ACSL1 we did a co-staining with the mitochondria-marker (mito-RFP) and found a co-localization in both HuH7 and HepG2 cell lines (figure 3). No overlap was found with plasma membrane (ssGFPglycTMDpcx), peroxisomes (peroxisomal-mRFP1), ER (mRFP-Sec61ß) or lipid droplets (adipophilin-mRFP1), where it has been previously described to be located as well 27.

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