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Uptake of long chain fatty acids is regulated by dynamic interaction of FAT/CD36 with cholesterol/sphingolipid enriched microdomains (lipid rafts).

Ehehalt R, Sparla R, Kulaksiz H, Herrmann T, Füllekrug J, Stremmel W - BMC Cell Biol. (2008)

Bottom Line: Floating experiments showed that there are two pools of FAT/CD36, one found in DRMs and another outside of these domains.Another candidate transporter, FATP4, was neither present in DRMs nor co-localized with FAT/CD36 at the plasma membrane.There is no direct interaction of FATP4 with lipid rafts or raft associated FAT/CD36.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Gastroenterology, University Hospital Heidelberg, INF 410, 69120 Heidelberg, Germany. robert_ehehalt@med.uni-heidelberg.de

ABSTRACT

Background: Mechanisms of long chain fatty acid uptake across the plasma membrane are important targets in treatment of many human diseases like obesity or hepatic steatosis. Long chain fatty acid translocation is achieved by a concert of co-existing mechanisms. These lipids can passively diffuse, but certain membrane proteins can also accelerate the transport. However, we now can provide further evidence that not only proteins but also lipid microdomains play an important part in the regulation of the facilitated uptake process.

Methods: Dynamic association of FAT/CD36 a candidate fatty acid transporter with lipid rafts was analysed by isolation of detergent resistant membranes (DRMs) and by clustering of lipid rafts with antibodies on living cells. Lipid raft integrity was modulated by cholesterol depletion using methyl-beta-cyclodextrin and sphingolipid depletion using myriocin and sphingomyelinase. Functional analyses were performed using an [3H]-oleate uptake assay.

Results: Overexpression of FAT/CD36 and FATP4 increased long chain fatty acid uptake. The uptake of long chain fatty acids was cholesterol and sphingolipid dependent. Floating experiments showed that there are two pools of FAT/CD36, one found in DRMs and another outside of these domains. FAT/CD36 co-localized with the lipid raft marker PLAP in antibody-clustered domains at the plasma membrane and segregated away from the non-raft marker GFP-TMD. Antibody cross-linking increased DRM association of FAT/CD36 and accelerated the overall fatty acid uptake in a cholesterol dependent manner. Another candidate transporter, FATP4, was neither present in DRMs nor co-localized with FAT/CD36 at the plasma membrane.

Conclusion: Our observations suggest the existence of two pools of FAT/CD36 within cellular membranes. As increased raft association of FAT/CD36 leads to an increased fatty acid uptake, dynamic association of FAT/CD36 with lipid rafts might regulate the process. There is no direct interaction of FATP4 with lipid rafts or raft associated FAT/CD36. Thus, lipid rafts have to be considered as targets for the treatment of lipid disorders.

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Cholesterol depletion inhibits [3H]-oleate uptake. COS cells were transiently transfected with FAT/CD36 and treated with 10 mM MβCD leading to a ~50% decrease in total cellular cholesterol (not shown). Afterwards [3H]-oleate uptake within 5 min was analysed. FAT/CD36 increased [3H]-oleate uptake significantly (p < 0.05). Cholesterol depletion decreased overall fatty acid uptake to less then 50% of control cells (p < 0.05). Data are expressed as mean and SEM of at least n = 6 experiments. The ratio has been arbitrarily set to 100% in cells that were not cholesterol depleted.
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Figure 1: Cholesterol depletion inhibits [3H]-oleate uptake. COS cells were transiently transfected with FAT/CD36 and treated with 10 mM MβCD leading to a ~50% decrease in total cellular cholesterol (not shown). Afterwards [3H]-oleate uptake within 5 min was analysed. FAT/CD36 increased [3H]-oleate uptake significantly (p < 0.05). Cholesterol depletion decreased overall fatty acid uptake to less then 50% of control cells (p < 0.05). Data are expressed as mean and SEM of at least n = 6 experiments. The ratio has been arbitrarily set to 100% in cells that were not cholesterol depleted.

Mentions: To study the role of lipid rafts in long chain fatty acid uptake we first analyzed again the effect of cholesterol depletion also in COS cells. Cholesterol depletion was done by treatment with the complexing agent methyl-β-cyclodextrin (MβCD) that extracts plasma membrane cholesterol. Immediately prior to testing uptake of [3H]-oleic acid, COS cells were treated with 10 mM MβCD for 30 min. By this the cholesterol levels could be reduced to 50% of those of control cells. To easily monitor the role of FAT/CD36 in fatty acid uptake cells were transiently tranfected with cDNA of FAT/CD36. Using lipofectamine as a mediator up to 50% of cells could be transfected. Overexpression of FAT/CD36 resulted in an increased overall uptake of [3H]-oleic acid by >20% within 5 min (Figure 1). Depletion of cholesterol with MβCD decreased fatty acid uptake by >50% in both control cells and FAT/CD36 transfected cells (Figure 1). These results show again that cholesterol is critically involved in [3H]-oleate uptake.


Uptake of long chain fatty acids is regulated by dynamic interaction of FAT/CD36 with cholesterol/sphingolipid enriched microdomains (lipid rafts).

Ehehalt R, Sparla R, Kulaksiz H, Herrmann T, Füllekrug J, Stremmel W - BMC Cell Biol. (2008)

Cholesterol depletion inhibits [3H]-oleate uptake. COS cells were transiently transfected with FAT/CD36 and treated with 10 mM MβCD leading to a ~50% decrease in total cellular cholesterol (not shown). Afterwards [3H]-oleate uptake within 5 min was analysed. FAT/CD36 increased [3H]-oleate uptake significantly (p < 0.05). Cholesterol depletion decreased overall fatty acid uptake to less then 50% of control cells (p < 0.05). Data are expressed as mean and SEM of at least n = 6 experiments. The ratio has been arbitrarily set to 100% in cells that were not cholesterol depleted.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2533316&req=5

Figure 1: Cholesterol depletion inhibits [3H]-oleate uptake. COS cells were transiently transfected with FAT/CD36 and treated with 10 mM MβCD leading to a ~50% decrease in total cellular cholesterol (not shown). Afterwards [3H]-oleate uptake within 5 min was analysed. FAT/CD36 increased [3H]-oleate uptake significantly (p < 0.05). Cholesterol depletion decreased overall fatty acid uptake to less then 50% of control cells (p < 0.05). Data are expressed as mean and SEM of at least n = 6 experiments. The ratio has been arbitrarily set to 100% in cells that were not cholesterol depleted.
Mentions: To study the role of lipid rafts in long chain fatty acid uptake we first analyzed again the effect of cholesterol depletion also in COS cells. Cholesterol depletion was done by treatment with the complexing agent methyl-β-cyclodextrin (MβCD) that extracts plasma membrane cholesterol. Immediately prior to testing uptake of [3H]-oleic acid, COS cells were treated with 10 mM MβCD for 30 min. By this the cholesterol levels could be reduced to 50% of those of control cells. To easily monitor the role of FAT/CD36 in fatty acid uptake cells were transiently tranfected with cDNA of FAT/CD36. Using lipofectamine as a mediator up to 50% of cells could be transfected. Overexpression of FAT/CD36 resulted in an increased overall uptake of [3H]-oleic acid by >20% within 5 min (Figure 1). Depletion of cholesterol with MβCD decreased fatty acid uptake by >50% in both control cells and FAT/CD36 transfected cells (Figure 1). These results show again that cholesterol is critically involved in [3H]-oleate uptake.

Bottom Line: Floating experiments showed that there are two pools of FAT/CD36, one found in DRMs and another outside of these domains.Another candidate transporter, FATP4, was neither present in DRMs nor co-localized with FAT/CD36 at the plasma membrane.There is no direct interaction of FATP4 with lipid rafts or raft associated FAT/CD36.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Gastroenterology, University Hospital Heidelberg, INF 410, 69120 Heidelberg, Germany. robert_ehehalt@med.uni-heidelberg.de

ABSTRACT

Background: Mechanisms of long chain fatty acid uptake across the plasma membrane are important targets in treatment of many human diseases like obesity or hepatic steatosis. Long chain fatty acid translocation is achieved by a concert of co-existing mechanisms. These lipids can passively diffuse, but certain membrane proteins can also accelerate the transport. However, we now can provide further evidence that not only proteins but also lipid microdomains play an important part in the regulation of the facilitated uptake process.

Methods: Dynamic association of FAT/CD36 a candidate fatty acid transporter with lipid rafts was analysed by isolation of detergent resistant membranes (DRMs) and by clustering of lipid rafts with antibodies on living cells. Lipid raft integrity was modulated by cholesterol depletion using methyl-beta-cyclodextrin and sphingolipid depletion using myriocin and sphingomyelinase. Functional analyses were performed using an [3H]-oleate uptake assay.

Results: Overexpression of FAT/CD36 and FATP4 increased long chain fatty acid uptake. The uptake of long chain fatty acids was cholesterol and sphingolipid dependent. Floating experiments showed that there are two pools of FAT/CD36, one found in DRMs and another outside of these domains. FAT/CD36 co-localized with the lipid raft marker PLAP in antibody-clustered domains at the plasma membrane and segregated away from the non-raft marker GFP-TMD. Antibody cross-linking increased DRM association of FAT/CD36 and accelerated the overall fatty acid uptake in a cholesterol dependent manner. Another candidate transporter, FATP4, was neither present in DRMs nor co-localized with FAT/CD36 at the plasma membrane.

Conclusion: Our observations suggest the existence of two pools of FAT/CD36 within cellular membranes. As increased raft association of FAT/CD36 leads to an increased fatty acid uptake, dynamic association of FAT/CD36 with lipid rafts might regulate the process. There is no direct interaction of FATP4 with lipid rafts or raft associated FAT/CD36. Thus, lipid rafts have to be considered as targets for the treatment of lipid disorders.

Show MeSH
Related in: MedlinePlus