Limits...
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).

Show MeSH

Related in: MedlinePlus

Localisation of FATP2 in HepG2 (A+C) and HuH7 (B) cells. The ER marker protein mRFP (red) was cotransfected. For FATP2-HA we used mouse anti-HA antibody from Santa Cruz and Cy2 fluorescently-labelled secondary antibody. FATP2 shows a reticular cellular structure representing ER membranes. There was no match with plasmamembrane marker (ssGFPglyc) (B) or peroxisomal marker proteins (peroxisomal-RFP) (C) in both cell lines. Bar: 10 µm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3198256&req=5

Figure 1: Localisation of FATP2 in HepG2 (A+C) and HuH7 (B) cells. The ER marker protein mRFP (red) was cotransfected. For FATP2-HA we used mouse anti-HA antibody from Santa Cruz and Cy2 fluorescently-labelled secondary antibody. FATP2 shows a reticular cellular structure representing ER membranes. There was no match with plasmamembrane marker (ssGFPglyc) (B) or peroxisomal marker proteins (peroxisomal-RFP) (C) in both cell lines. Bar: 10 µm.

Mentions: To study the role of FATP2, FATP4, ACSL1 and CD36 in LCFA transport of hepatoma cells we first investigated the localization of these proteins. So far the localization of FATP2 was described in peroxisomes as well as at the plasma membrane of rat, mouse and monkey cells 27, 34, 45 and additionally in the endoplasmatic reticulum (ER) of COS cells 46. FATP4 was originally reported to be located at the plasma membrane and act there like a transporter for LCFA 28. However, recent studies could show a localization in the ER of mouse small intestine, HeLa, Caco-2, Ptk2, MDCK, 3T3-L1 and COS cells 29, 47. For localization studies HA-tagged FATP2 as well as GFP-tagged FATP4 was transiently transfected in HuH7 and HepG2 cells. In these cells they showed a similar reticular cellular pattern typifying the ER membranes (figure 1 and 2). ER localization was assured by co-expression with the ER-marker mRFP-Sec61ß. FATP2 and FATP4 did neither co-localize with plasma membrane (ssGFPglycTMDpcx) nor peroxisomal (peroxisomal-mRFP1) marker 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, Füllekrug J, Ehehalt R - Int J Med Sci (2011)

Localisation of FATP2 in HepG2 (A+C) and HuH7 (B) cells. The ER marker protein mRFP (red) was cotransfected. For FATP2-HA we used mouse anti-HA antibody from Santa Cruz and Cy2 fluorescently-labelled secondary antibody. FATP2 shows a reticular cellular structure representing ER membranes. There was no match with plasmamembrane marker (ssGFPglyc) (B) or peroxisomal marker proteins (peroxisomal-RFP) (C) in both cell lines. Bar: 10 µm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Localisation of FATP2 in HepG2 (A+C) and HuH7 (B) cells. The ER marker protein mRFP (red) was cotransfected. For FATP2-HA we used mouse anti-HA antibody from Santa Cruz and Cy2 fluorescently-labelled secondary antibody. FATP2 shows a reticular cellular structure representing ER membranes. There was no match with plasmamembrane marker (ssGFPglyc) (B) or peroxisomal marker proteins (peroxisomal-RFP) (C) in both cell lines. Bar: 10 µm.
Mentions: To study the role of FATP2, FATP4, ACSL1 and CD36 in LCFA transport of hepatoma cells we first investigated the localization of these proteins. So far the localization of FATP2 was described in peroxisomes as well as at the plasma membrane of rat, mouse and monkey cells 27, 34, 45 and additionally in the endoplasmatic reticulum (ER) of COS cells 46. FATP4 was originally reported to be located at the plasma membrane and act there like a transporter for LCFA 28. However, recent studies could show a localization in the ER of mouse small intestine, HeLa, Caco-2, Ptk2, MDCK, 3T3-L1 and COS cells 29, 47. For localization studies HA-tagged FATP2 as well as GFP-tagged FATP4 was transiently transfected in HuH7 and HepG2 cells. In these cells they showed a similar reticular cellular pattern typifying the ER membranes (figure 1 and 2). ER localization was assured by co-expression with the ER-marker mRFP-Sec61ß. FATP2 and FATP4 did neither co-localize with plasma membrane (ssGFPglycTMDpcx) nor peroxisomal (peroxisomal-mRFP1) marker 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