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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

Oleate uptake of transient transfected HuH7 cells after 60 sec of incubation with oleate-BSA mix (in pmol oleate/µg protein). Controls for endogenous oleate uptake were transfected with GFP plasmid. The proteinoverexpression resulted in a significant increase in the rate of [3H]oleate uptake within 60 sec (p<0.05). Values are means ± SD of three independent experiments.
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Figure 5: Oleate uptake of transient transfected HuH7 cells after 60 sec of incubation with oleate-BSA mix (in pmol oleate/µg protein). Controls for endogenous oleate uptake were transfected with GFP plasmid. The proteinoverexpression resulted in a significant increase in the rate of [3H]oleate uptake within 60 sec (p<0.05). Values are means ± SD of three independent experiments.

Mentions: To investigate the LCFA uptake in HuH7 and HepG2 cells, we transiently expressed FATP2, FATP4, ACSL1 and CD36 and incubated the cells with different FA substrates. Transcription of FATP4, FATP2, ACSL1, and CD36 was measured by efficiency corrected real-time PCR relative to ß-actin to estimate the basal und overexpression rates. Transfection resulted in a several fold increase of mRNA expression: overexpression was 271-fold (ACSL1), 6777-fold (FATP2), 3165-fold (FATP4) and 181-fold (CD36) in HuH7 cells, respectively. To work with physiological fatty acids, we first used radio-labeled oleate in a 1:1 relation to BSA and incubated the cells for 60 sec with the mix. All proteins significantly increased the oleate uptake in HuH7 (figure 5) as well as in HepG2 cells (data not shown) in comparison to GFP overexpression. GFP served as control and did not show any influence on LCFA uptake compared to non-transfected cells.


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)

Oleate uptake of transient transfected HuH7 cells after 60 sec of incubation with oleate-BSA mix (in pmol oleate/µg protein). Controls for endogenous oleate uptake were transfected with GFP plasmid. The proteinoverexpression resulted in a significant increase in the rate of [3H]oleate uptake within 60 sec (p<0.05). Values are means ± SD of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Oleate uptake of transient transfected HuH7 cells after 60 sec of incubation with oleate-BSA mix (in pmol oleate/µg protein). Controls for endogenous oleate uptake were transfected with GFP plasmid. The proteinoverexpression resulted in a significant increase in the rate of [3H]oleate uptake within 60 sec (p<0.05). Values are means ± SD of three independent experiments.
Mentions: To investigate the LCFA uptake in HuH7 and HepG2 cells, we transiently expressed FATP2, FATP4, ACSL1 and CD36 and incubated the cells with different FA substrates. Transcription of FATP4, FATP2, ACSL1, and CD36 was measured by efficiency corrected real-time PCR relative to ß-actin to estimate the basal und overexpression rates. Transfection resulted in a several fold increase of mRNA expression: overexpression was 271-fold (ACSL1), 6777-fold (FATP2), 3165-fold (FATP4) and 181-fold (CD36) in HuH7 cells, respectively. To work with physiological fatty acids, we first used radio-labeled oleate in a 1:1 relation to BSA and incubated the cells for 60 sec with the mix. All proteins significantly increased the oleate uptake in HuH7 (figure 5) as well as in HepG2 cells (data not shown) in comparison to GFP overexpression. GFP served as control and did not show any influence on LCFA uptake compared to non-transfected cells.

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