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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

FACS analysis of fluorescent fatty acid uptake of transiently transfected HuH7 cells. Thespreadsheets correlated the level of expression with the amount of fluorescent fatty acid uptake (B12-FA). (A) HuH7 cells transfected with RFP-FATP4.Control cells were transfected with red fluorescent mRFP protein. RFP-FATP4 (r=0.73) enhanced fatty acid uptake depending on its relative level of expression in comparison to control cells only expressing mRFP (r=0.4). The low increase of the fatty acid uptake of control cells (r=0.4) could be explained by endogenous FATP4 expression in HuH7 cells. FATP2-HA (B) (r=0.92) and ACSL1-FLAG (C) (r=0.64) overexpressing cells enhanced the uptake of fatty acids as well. The overexpression of CD36-FLAG (D) (r=0.2) did not significantly correlate with the uptake of fatty acids. Control cells were transfected with pcDNA3 (E) (r=0.18). The pictured diagrams are representative for three independent experiments.
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Figure 8: FACS analysis of fluorescent fatty acid uptake of transiently transfected HuH7 cells. Thespreadsheets correlated the level of expression with the amount of fluorescent fatty acid uptake (B12-FA). (A) HuH7 cells transfected with RFP-FATP4.Control cells were transfected with red fluorescent mRFP protein. RFP-FATP4 (r=0.73) enhanced fatty acid uptake depending on its relative level of expression in comparison to control cells only expressing mRFP (r=0.4). The low increase of the fatty acid uptake of control cells (r=0.4) could be explained by endogenous FATP4 expression in HuH7 cells. FATP2-HA (B) (r=0.92) and ACSL1-FLAG (C) (r=0.64) overexpressing cells enhanced the uptake of fatty acids as well. The overexpression of CD36-FLAG (D) (r=0.2) did not significantly correlate with the uptake of fatty acids. Control cells were transfected with pcDNA3 (E) (r=0.18). The pictured diagrams are representative for three independent experiments.

Mentions: To correlate individual transfected cells with their special fluorescent intensity of B12-FA that has been taken up, data were transformed by a spreadsheet method (figure 8) 42. Each data point in figure 8 contains all cells with the same fluorescence value of the transfected protein (equals the level of expression) and the average B12-FA fluorescence of these cells (equals the amount of FA taken up). Control cells were transfected with mRFP and pcDNA and show the endogenous B12 uptake of HuH7 cells. The calculated slopes (m) and the correlation coefficients (r) confirm a strong linear correlation between the level of protein expression of RFP-FATP4, FATP2-HA and ACSL1-FLAG and B12 uptake.


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)

FACS analysis of fluorescent fatty acid uptake of transiently transfected HuH7 cells. Thespreadsheets correlated the level of expression with the amount of fluorescent fatty acid uptake (B12-FA). (A) HuH7 cells transfected with RFP-FATP4.Control cells were transfected with red fluorescent mRFP protein. RFP-FATP4 (r=0.73) enhanced fatty acid uptake depending on its relative level of expression in comparison to control cells only expressing mRFP (r=0.4). The low increase of the fatty acid uptake of control cells (r=0.4) could be explained by endogenous FATP4 expression in HuH7 cells. FATP2-HA (B) (r=0.92) and ACSL1-FLAG (C) (r=0.64) overexpressing cells enhanced the uptake of fatty acids as well. The overexpression of CD36-FLAG (D) (r=0.2) did not significantly correlate with the uptake of fatty acids. Control cells were transfected with pcDNA3 (E) (r=0.18). The pictured diagrams are representative for three independent experiments.
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Related In: Results  -  Collection

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Figure 8: FACS analysis of fluorescent fatty acid uptake of transiently transfected HuH7 cells. Thespreadsheets correlated the level of expression with the amount of fluorescent fatty acid uptake (B12-FA). (A) HuH7 cells transfected with RFP-FATP4.Control cells were transfected with red fluorescent mRFP protein. RFP-FATP4 (r=0.73) enhanced fatty acid uptake depending on its relative level of expression in comparison to control cells only expressing mRFP (r=0.4). The low increase of the fatty acid uptake of control cells (r=0.4) could be explained by endogenous FATP4 expression in HuH7 cells. FATP2-HA (B) (r=0.92) and ACSL1-FLAG (C) (r=0.64) overexpressing cells enhanced the uptake of fatty acids as well. The overexpression of CD36-FLAG (D) (r=0.2) did not significantly correlate with the uptake of fatty acids. Control cells were transfected with pcDNA3 (E) (r=0.18). The pictured diagrams are representative for three independent experiments.
Mentions: To correlate individual transfected cells with their special fluorescent intensity of B12-FA that has been taken up, data were transformed by a spreadsheet method (figure 8) 42. Each data point in figure 8 contains all cells with the same fluorescence value of the transfected protein (equals the level of expression) and the average B12-FA fluorescence of these cells (equals the amount of FA taken up). Control cells were transfected with mRFP and pcDNA and show the endogenous B12 uptake of HuH7 cells. The calculated slopes (m) and the correlation coefficients (r) confirm a strong linear correlation between the level of protein expression of RFP-FATP4, FATP2-HA and ACSL1-FLAG and B12 uptake.

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