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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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Effects of antibody cross-linking (x-link) and cholesterol depletion on [3H]-oleate uptake. Cells were transiently transfected with FLAG-FAT/CD36 and FATP4 and then treated or not for 30 min with 10 mM methyl-β-cyclodextrin (MβCD). Afterwards overall [3H]-oleate uptake within the first 5 min in presence or absence of anti-FAT/CD36 from Biosource was analysed. Quantification of three independent experiments is shown. The ratio has been arbitrarily set to 100% in cells neither cross-linked nor cholesterol depleted. Cross-linking increased overall fatty acid uptake significantly (p < 0.05). However under conditions of cholesterol depletion, antibody enhanced fatty acid uptake was not apparent.
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Figure 6: Effects of antibody cross-linking (x-link) and cholesterol depletion on [3H]-oleate uptake. Cells were transiently transfected with FLAG-FAT/CD36 and FATP4 and then treated or not for 30 min with 10 mM methyl-β-cyclodextrin (MβCD). Afterwards overall [3H]-oleate uptake within the first 5 min in presence or absence of anti-FAT/CD36 from Biosource was analysed. Quantification of three independent experiments is shown. The ratio has been arbitrarily set to 100% in cells neither cross-linked nor cholesterol depleted. Cross-linking increased overall fatty acid uptake significantly (p < 0.05). However under conditions of cholesterol depletion, antibody enhanced fatty acid uptake was not apparent.

Mentions: If long chain fatty acid uptake were to take place in lipid rafts, then antibody cross-linking should not only induce co-patching with raft markers at the surface of living cells and increase association between FAT/CD36 and DRMs. It should also increase LCFA uptake, provided that the antibody does not neutralize LCFA binding or transport To find this out, we analyzed the effect of antibody cross-linking on overall [3H]-oleate uptake. Cells were co-transfected with FATP4 and FAT/CD36 and [3H]-oleate uptake analyzed within 5 min in the presence of anti-FAT/CD36 from Biosource (Figure 6). Antibody enhanced cross-linking indeed increased [3H]-oleate uptake significantly. We next examined the effect of cholesterol depletion on antibody-induced [3H]-oleate uptake. Immediately prior to the uptake assay transfected cells were treated for 30 min with 10 mM methyl-β-cyclodextrin and again uptake within 5 min was analyzed. After cholesterol depletion with methyl-β-cyclodextrin, [3H]-oleate uptake was no longer enhanced by antibody cross-linking (Figure 6). Thus, under conditions in which rafts are supposed to be disrupted (cholesterol depletion) increased uptake of [3H]-oleate due to cross-linking with antibodies is not detectable. Thus, lipid raft integrity is important for FAT/CD36 function in fatty acid 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)

Effects of antibody cross-linking (x-link) and cholesterol depletion on [3H]-oleate uptake. Cells were transiently transfected with FLAG-FAT/CD36 and FATP4 and then treated or not for 30 min with 10 mM methyl-β-cyclodextrin (MβCD). Afterwards overall [3H]-oleate uptake within the first 5 min in presence or absence of anti-FAT/CD36 from Biosource was analysed. Quantification of three independent experiments is shown. The ratio has been arbitrarily set to 100% in cells neither cross-linked nor cholesterol depleted. Cross-linking increased overall fatty acid uptake significantly (p < 0.05). However under conditions of cholesterol depletion, antibody enhanced fatty acid uptake was not apparent.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Effects of antibody cross-linking (x-link) and cholesterol depletion on [3H]-oleate uptake. Cells were transiently transfected with FLAG-FAT/CD36 and FATP4 and then treated or not for 30 min with 10 mM methyl-β-cyclodextrin (MβCD). Afterwards overall [3H]-oleate uptake within the first 5 min in presence or absence of anti-FAT/CD36 from Biosource was analysed. Quantification of three independent experiments is shown. The ratio has been arbitrarily set to 100% in cells neither cross-linked nor cholesterol depleted. Cross-linking increased overall fatty acid uptake significantly (p < 0.05). However under conditions of cholesterol depletion, antibody enhanced fatty acid uptake was not apparent.
Mentions: If long chain fatty acid uptake were to take place in lipid rafts, then antibody cross-linking should not only induce co-patching with raft markers at the surface of living cells and increase association between FAT/CD36 and DRMs. It should also increase LCFA uptake, provided that the antibody does not neutralize LCFA binding or transport To find this out, we analyzed the effect of antibody cross-linking on overall [3H]-oleate uptake. Cells were co-transfected with FATP4 and FAT/CD36 and [3H]-oleate uptake analyzed within 5 min in the presence of anti-FAT/CD36 from Biosource (Figure 6). Antibody enhanced cross-linking indeed increased [3H]-oleate uptake significantly. We next examined the effect of cholesterol depletion on antibody-induced [3H]-oleate uptake. Immediately prior to the uptake assay transfected cells were treated for 30 min with 10 mM methyl-β-cyclodextrin and again uptake within 5 min was analyzed. After cholesterol depletion with methyl-β-cyclodextrin, [3H]-oleate uptake was no longer enhanced by antibody cross-linking (Figure 6). Thus, under conditions in which rafts are supposed to be disrupted (cholesterol depletion) increased uptake of [3H]-oleate due to cross-linking with antibodies is not detectable. Thus, lipid raft integrity is important for FAT/CD36 function in fatty acid 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