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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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Myriocin and sphingomyelinase treatment decreases [3H]-oleate uptake. COS cells were either grown before cotransfection of FAT/CD36 and FATP4 for 3 d in the presence or absence (control) of 5 μM myriocin or were treated after co-transfection for 15 min with 1 U sphingomyelinase. (A) Overexpressing FAT and FATP4 increased [3H]-oleate uptake significantly (p < 0.05). Myriocin and sphingomyelinase treatment inhibited [3H]-oleate uptake significantly (p < 0.05). Data are expressed as mean and SEM from n = 3 independent experiments. (B) Staining of COS cells with Rh-CTB after 3 d treatment with 5 μM myriocin. The amount of the ganglioside GM1 on the surface is reduced after myriocin treatment. Bar: 200 μm. Both images in panel B were recorded with the same exposure time.
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Figure 2: Myriocin and sphingomyelinase treatment decreases [3H]-oleate uptake. COS cells were either grown before cotransfection of FAT/CD36 and FATP4 for 3 d in the presence or absence (control) of 5 μM myriocin or were treated after co-transfection for 15 min with 1 U sphingomyelinase. (A) Overexpressing FAT and FATP4 increased [3H]-oleate uptake significantly (p < 0.05). Myriocin and sphingomyelinase treatment inhibited [3H]-oleate uptake significantly (p < 0.05). Data are expressed as mean and SEM from n = 3 independent experiments. (B) Staining of COS cells with Rh-CTB after 3 d treatment with 5 μM myriocin. The amount of the ganglioside GM1 on the surface is reduced after myriocin treatment. Bar: 200 μm. Both images in panel B were recorded with the same exposure time.

Mentions: COS cells were cultured in the presence of 5 μM myriocin, which inhibits the first step of de novo ceramide synthesis by interacting with the serine-palmitoyl transferase or treated with 1 U of sphingomyelinase that is supposed to deplete plasma membrane sphingolipids. Cells were either treated for 3 d with myriocin before cotransfection of FAT/CD36 and FATP4 or treated 1 d after cotransfection for 15 min with 1 U of sphingomyelinase. [3H]-oleate uptake within 3 min was measured. Both treatments resulted in a reduction of [3H]-oleate uptake by 17 ± 3% (myriocin) and 15 ± 2% (sphingomyelinase), respectively (Figure 2A). The ganglioside GM1 localizes to lipid rafts and its plasma membrane level is altered by inhibition of ceramide synthesis [28]. We monitored the effect of myriocin and sphingomyelinase by staining of the ganglioside GM1 with rhodamine-conjugated cholera toxin subunit B (Rh-CTB). Myriocin treatment led to a decrease in staining, suggesting a decreased amount of total sphingolipids (Figure 2B). Sphingomyelinase resulted in similar results (data not shown). Thus, [3H]-oleate uptake can also be altered by depletion of sphingolipids.


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)

Myriocin and sphingomyelinase treatment decreases [3H]-oleate uptake. COS cells were either grown before cotransfection of FAT/CD36 and FATP4 for 3 d in the presence or absence (control) of 5 μM myriocin or were treated after co-transfection for 15 min with 1 U sphingomyelinase. (A) Overexpressing FAT and FATP4 increased [3H]-oleate uptake significantly (p < 0.05). Myriocin and sphingomyelinase treatment inhibited [3H]-oleate uptake significantly (p < 0.05). Data are expressed as mean and SEM from n = 3 independent experiments. (B) Staining of COS cells with Rh-CTB after 3 d treatment with 5 μM myriocin. The amount of the ganglioside GM1 on the surface is reduced after myriocin treatment. Bar: 200 μm. Both images in panel B were recorded with the same exposure time.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Myriocin and sphingomyelinase treatment decreases [3H]-oleate uptake. COS cells were either grown before cotransfection of FAT/CD36 and FATP4 for 3 d in the presence or absence (control) of 5 μM myriocin or were treated after co-transfection for 15 min with 1 U sphingomyelinase. (A) Overexpressing FAT and FATP4 increased [3H]-oleate uptake significantly (p < 0.05). Myriocin and sphingomyelinase treatment inhibited [3H]-oleate uptake significantly (p < 0.05). Data are expressed as mean and SEM from n = 3 independent experiments. (B) Staining of COS cells with Rh-CTB after 3 d treatment with 5 μM myriocin. The amount of the ganglioside GM1 on the surface is reduced after myriocin treatment. Bar: 200 μm. Both images in panel B were recorded with the same exposure time.
Mentions: COS cells were cultured in the presence of 5 μM myriocin, which inhibits the first step of de novo ceramide synthesis by interacting with the serine-palmitoyl transferase or treated with 1 U of sphingomyelinase that is supposed to deplete plasma membrane sphingolipids. Cells were either treated for 3 d with myriocin before cotransfection of FAT/CD36 and FATP4 or treated 1 d after cotransfection for 15 min with 1 U of sphingomyelinase. [3H]-oleate uptake within 3 min was measured. Both treatments resulted in a reduction of [3H]-oleate uptake by 17 ± 3% (myriocin) and 15 ± 2% (sphingomyelinase), respectively (Figure 2A). The ganglioside GM1 localizes to lipid rafts and its plasma membrane level is altered by inhibition of ceramide synthesis [28]. We monitored the effect of myriocin and sphingomyelinase by staining of the ganglioside GM1 with rhodamine-conjugated cholera toxin subunit B (Rh-CTB). Myriocin treatment led to a decrease in staining, suggesting a decreased amount of total sphingolipids (Figure 2B). Sphingomyelinase resulted in similar results (data not shown). Thus, [3H]-oleate uptake can also be altered by depletion of sphingolipids.

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