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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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Effect of antibody cross-linking on association of FAT/CD36 to DRMs. 20 h after transient transfection of FAT/CD36, FATP4 or both COS cells were were lysed in 1% Triton X-100/TNE at 4°C. (A) After floatation in an OptiPrep step-gradient FAT/CD36 was found in two pools, in DRMs and in soluble membranes (lane 1–3). Co-expression of FATP4 resulted in an increased relative amount of FAT/CD36 found in DRMs (lane 6). Antibody cross-linking of FAT/CD36 using an mouse anti human FAT/CD36 antibody from Biosource shifted FAT/CD36 towards the DRM fractions (lane 6). No significant amount of FATP4 was found in DRMs, indicating that FAT/CD36 and FATP4 might be in distinct compartments within the cell. Flotillin-2, a typical raft protein was used as a control to estimate the quality of DRM isolation. The results are representative of three others experiments carried out independently. (B) Quantification; FATP4 expression and antibody-induced patching significantly increased the amount of FAT/CD36 in the top two fractions (DRM associated). The amount in the top two fractions was correlated to the total amount of protein in all fractions. Data are expressed as mean and SEM of n = 3 experiments. Asterisk indicates significant differences to cells transfected with FAT/CD36 only (p < 0.05).
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Figure 5: Effect of antibody cross-linking on association of FAT/CD36 to DRMs. 20 h after transient transfection of FAT/CD36, FATP4 or both COS cells were were lysed in 1% Triton X-100/TNE at 4°C. (A) After floatation in an OptiPrep step-gradient FAT/CD36 was found in two pools, in DRMs and in soluble membranes (lane 1–3). Co-expression of FATP4 resulted in an increased relative amount of FAT/CD36 found in DRMs (lane 6). Antibody cross-linking of FAT/CD36 using an mouse anti human FAT/CD36 antibody from Biosource shifted FAT/CD36 towards the DRM fractions (lane 6). No significant amount of FATP4 was found in DRMs, indicating that FAT/CD36 and FATP4 might be in distinct compartments within the cell. Flotillin-2, a typical raft protein was used as a control to estimate the quality of DRM isolation. The results are representative of three others experiments carried out independently. (B) Quantification; FATP4 expression and antibody-induced patching significantly increased the amount of FAT/CD36 in the top two fractions (DRM associated). The amount in the top two fractions was correlated to the total amount of protein in all fractions. Data are expressed as mean and SEM of n = 3 experiments. Asterisk indicates significant differences to cells transfected with FAT/CD36 only (p < 0.05).

Mentions: In initial experiments COS cells were transfected with FAT/CD36 and then extracted with Triton X-100 and subjected to OptiPrep™ step gradient centrifugation. Under those conditions in three independent experiments no significant effect of antibody cross-linking could be detected. FAT/CD36 was found in both Triton X-100 resistant and soluble fractions showing that there are two pools of FAT/CD36 in cellular membranes. However, when FATP4 was co-expressed with FAT/CD36 raft association of the FAT/CD36 was increased (Figure 5). Co-expression of the cytosolic green fluorescent protein (GFP) or the ER marker sec61-GFP [33] did not change DRM association of FAT/CD36 (data not shown). Performing cross-linking under co-expressing conditions revealed a clearly increased fraction of FAT/CD36 in the DRM fraction (Figure 5). Therefore, DRM association of FAT/CD36 can be increased by cross-linking with antibodies, which probably reflects increased raft affinity caused by oligomerization. Similar results have been demonstrated for other proteins, which by forming oligomers increase their raft association [34-39]. The fact that increased association of FAT/CD36 to DRMs occurs especially in FATP4 overexpressing cells indicates a promoting effect of FATP4 for FAT/CD36 raft association.


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)

Effect of antibody cross-linking on association of FAT/CD36 to DRMs. 20 h after transient transfection of FAT/CD36, FATP4 or both COS cells were were lysed in 1% Triton X-100/TNE at 4°C. (A) After floatation in an OptiPrep step-gradient FAT/CD36 was found in two pools, in DRMs and in soluble membranes (lane 1–3). Co-expression of FATP4 resulted in an increased relative amount of FAT/CD36 found in DRMs (lane 6). Antibody cross-linking of FAT/CD36 using an mouse anti human FAT/CD36 antibody from Biosource shifted FAT/CD36 towards the DRM fractions (lane 6). No significant amount of FATP4 was found in DRMs, indicating that FAT/CD36 and FATP4 might be in distinct compartments within the cell. Flotillin-2, a typical raft protein was used as a control to estimate the quality of DRM isolation. The results are representative of three others experiments carried out independently. (B) Quantification; FATP4 expression and antibody-induced patching significantly increased the amount of FAT/CD36 in the top two fractions (DRM associated). The amount in the top two fractions was correlated to the total amount of protein in all fractions. Data are expressed as mean and SEM of n = 3 experiments. Asterisk indicates significant differences to cells transfected with FAT/CD36 only (p < 0.05).
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Figure 5: Effect of antibody cross-linking on association of FAT/CD36 to DRMs. 20 h after transient transfection of FAT/CD36, FATP4 or both COS cells were were lysed in 1% Triton X-100/TNE at 4°C. (A) After floatation in an OptiPrep step-gradient FAT/CD36 was found in two pools, in DRMs and in soluble membranes (lane 1–3). Co-expression of FATP4 resulted in an increased relative amount of FAT/CD36 found in DRMs (lane 6). Antibody cross-linking of FAT/CD36 using an mouse anti human FAT/CD36 antibody from Biosource shifted FAT/CD36 towards the DRM fractions (lane 6). No significant amount of FATP4 was found in DRMs, indicating that FAT/CD36 and FATP4 might be in distinct compartments within the cell. Flotillin-2, a typical raft protein was used as a control to estimate the quality of DRM isolation. The results are representative of three others experiments carried out independently. (B) Quantification; FATP4 expression and antibody-induced patching significantly increased the amount of FAT/CD36 in the top two fractions (DRM associated). The amount in the top two fractions was correlated to the total amount of protein in all fractions. Data are expressed as mean and SEM of n = 3 experiments. Asterisk indicates significant differences to cells transfected with FAT/CD36 only (p < 0.05).
Mentions: In initial experiments COS cells were transfected with FAT/CD36 and then extracted with Triton X-100 and subjected to OptiPrep™ step gradient centrifugation. Under those conditions in three independent experiments no significant effect of antibody cross-linking could be detected. FAT/CD36 was found in both Triton X-100 resistant and soluble fractions showing that there are two pools of FAT/CD36 in cellular membranes. However, when FATP4 was co-expressed with FAT/CD36 raft association of the FAT/CD36 was increased (Figure 5). Co-expression of the cytosolic green fluorescent protein (GFP) or the ER marker sec61-GFP [33] did not change DRM association of FAT/CD36 (data not shown). Performing cross-linking under co-expressing conditions revealed a clearly increased fraction of FAT/CD36 in the DRM fraction (Figure 5). Therefore, DRM association of FAT/CD36 can be increased by cross-linking with antibodies, which probably reflects increased raft affinity caused by oligomerization. Similar results have been demonstrated for other proteins, which by forming oligomers increase their raft association [34-39]. The fact that increased association of FAT/CD36 to DRMs occurs especially in FATP4 overexpressing cells indicates a promoting effect of FATP4 for FAT/CD36 raft association.

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