Insa Buers

Adipophilin-enriched domains in the ER membrane are sites of lipid droplet biogenesis

The prevailing hypothesis of lipid droplet biogenesis proposes that neutral lipids accumulate within the lipid bilayer of the ER membrane from where they are budded off, enclosed by a protein-bearing phospholipid monolayer originating from the cytoplasmic leaflet of the ER membrane. We have used a variety of methods to investigate the nature of the sites of ER–lipid-droplet association in order to gain new insights into the mechanism of lipid droplet formation and growth. The three-dimensional perspectives provided by freeze-fracture electron microscopy demonstrate unequivocally that at sites of close association, the lipid droplet is not situated within the ER membrane; rather, both ER membranes lie external to and follow the contour of the lipid droplet, enclosing it in a manner akin to an egg cup (the ER) holding an egg (the lipid droplet). Freeze-fracture cytochemistry demonstrates that the PAT family protein adipophilin is concentrated in prominent clusters in the cytoplasmic leaflet of the ER membrane closely apposed to the lipid droplet envelope. We identify these structures as sites at which lipids and adipophilin are transferred from ER membranes to lipid droplets. These findings call for a re-evaluation of the prevailing hypothesis of lipid droplet biogenesis.

Butyrophilin controls milk fat globule secretion

The molecular mechanism underlying milk fat globule secretion in mammary epithelial cells ostensibly involves the formation of complexes between plasma membrane butyrophilin and cytosolic xanthine oxidoreductase. These complexes bind adipophilin in the phospholipid monolayer of milk secretory granules, the precursors of milk fat globules, enveloping the nascent fat globules in a layer of plasma membrane and pinching them off the cell. However, using freeze-fracture immunocytochemistry, we find these proteins in locations other than those previously inferred. Significantly, butyrophilin in the residual plasma membrane of the fat globule envelope is concentrated in a network of ridges that are tightly apposed to the monolayer derived from the secretory granule, and the ridges coincide with butyrophilin labeling in the globule monolayer. Therefore, we propose that milk fat globule secretion is controlled by interactions between plasma membrane butyrophilin and butyrophilin in the secretory granule phospholipid monolayer rather than binding of butyrophilin–xanthine oxidoreductase complexes to secretory granule adipophilin.

Identification of a putative lysosomal cobalamin exporter altered in the cblF defect of vitamin B12 metabolism

Vitamin B12 (cobalamin) is essential in animals for metabolism of branched chain amino acids and odd chain fatty acids, and for remethylation of homocysteine to methionine. In the cblF inborn error of vitamin B12 metabolism, free vitamin accumulates in lysosomes, thus hindering its conversion to cofactors. Using homozygosity mapping in 12 unrelated cblF individuals and microcell-mediated chromosome transfer, we identified a candidate gene on chromosome 6q13, LMBRD1, encoding LMBD1, a lysosomal membrane protein with homology to lipocalin membrane receptor LIMR. We identified five different frameshift mutations in LMBRD1 resulting in loss of LMBD1 function, with 18 of the 24 disease chromosomes carrying the same mutation embedded in a common 1.34-Mb haplotype. Transfection of fibroblasts of individuals with cblF with wild-type LMBD1 rescued cobalamin coenzyme synthesis and function. This work identifies LMBRD1 as the gene underlying the cblF defect of cobalamin metabolism and suggests that LMBD1 is a lysosomal membrane exporter for cobalamin.

Mutations in ABCD4 cause a new inborn error of vitamin B12 metabolism

Inherited disorders of vitamin B12 (cobalamin) have provided important clues to how this vitamin, which is essential for hematological and neurological function, is transported and metabolized. We describe a new disease that results in failure to release vitamin B12 from lysosomes, which mimics the cblF defect caused by LMBRD1 mutations. Using microcell-mediated chromosome transfer and exome sequencing, we identified causal mutations in ABCD4, a gene that codes for an ABC transporter, which was previously thought to have peroxisomal localization and function. Our results show that ABCD4 colocalizes with the lysosomal proteins LAMP1 and LMBD1, the latter of which is deficient in the cblF defect. Furthermore, we show that mutations altering the putative ATPase domain of ABCD4 affect its function, suggesting that the ATPase activity of ABCD4 may be involved in intracellular processing of vitamin B12.

Compartmentalization of proteins in lipid droplet biogenesis

Our existing understanding of the structure, protein organization and biogenesis of the lipid droplet has relied heavily on microscopical techniques that lack resolution and the ability to preserve native cellular and protein composition. The electron microscopic technique of freeze-fracture replica immunogold labeling (FRIL) overcomes these problems, and is currently providing new perspectives in the field. Because of the property of frozen lipids to deflect the fracture plane, en face views of the lipid droplet and its component layers are revealed for high resolution visualization. By means of immunogold labeling, proteins involved in the accretion and mobilization of lipids, notably the PAT family proteins, can be localized at and in the droplet. Application of this approach demonstrates that, contrary to prevailing wisdom, the PAT family proteins are not invariably restricted to the surface of the lipid droplet but can occur throughout the core. The notion that lipid droplet biogenesis involves neutral lipid accumulation within the ER membrane bilayer followed by budding off, enclosed by a protein-containing phospholipid monolayer, is not substantiated. Instead, lipid droplets appear to develop externally to both ER membranes at specialized sites in which the ER enwraps the droplet, and the facing leaflets of the ER membrane and droplet surface are enriched in adipophilin. PAT family proteins are not, as often stated, specific to the lipid droplet, but are widely present in the plasma membrane where, under conditions of lipid loading, they adopt a similar configuration to that of specialized sites in the ER. FRIL has further provided new insights into the mechanism of secretion of a special type of lipid droplet, the milk fat globule. These examples highlight the contribution of the FRIL technique to critical appraisal and development of concepts in the lipid droplet field.

Efficient non-viral transfection of THP-1 cells

Macrophages are an important part of the cellular immune system and play a key role during immune responses. Thus, macrophages are interesting targets in basic and clinical research. Primary monocytes or monocyte-derived macrophages do not proliferate on a suitable scale so that their use for functional studies in vitro is limited. Immortal proliferating cell lines, such as the human THP-1 monocytic leukemia cell line, are therefore often used instead of primary cells. Transfection is a useful tool to study the function of gene products, but transfection of THP-1 monocytes and pre-differentiated THP-1 macrophages with subsequent differentiation into mature THP-1 macrophages using phorbol esters is usually accompanied by a progressive loss of cell viability. In this study, we describe a simple and rapid approach for efficient transfection of THP-1 monocytes and pre-differentiated THP-1 macrophages using a modified Nucleofection-based approach. The protocol maintains cell viability and functionality, thus allowing efficient transfection of THP-1 cells combined with subsequent differentiation of transfected THP-1 cells into mature macrophages.

TIP47, a Lipid Cargo Protein Involved in Macrophage Triglyceride Metabolism

Objective—Uptake of lipids by macrophages (M&PHgr;) leads to lipid droplet accumulation and foam cell formation. The PAT family proteins are implicated in lipid droplet formation, but the precise function of the 47-kDa tail interacting protein (TIP47), a member of this family, is poorly defined. The present study was performed to determine the function of TIP47 in M&PHgr; lipid metabolism. Methods and Results—Freeze-fracture cytochemistry demonstrates that TIP47 is present in the plasma membrane of M&PHgr; and is aggregated into clusters when the cells are incubated with oleate. Suppression of adipophilin levels using siRNA knockdown leads to migration of TIP47 from a cytoplasmic pool to the lipid droplet. Further, reduction of TIP47 decreases triglyceride levels, whereas raising TIP47 levels by expression of EGFP-TIP47 shows the opposite effect. Conclusion—Our results show that the TIP47 protein levels directly correlate with triglyceride levels. We propose that TIP47 may act as a carrier protein for free fatty acids and in this way participates in conversion of M&PHgr; into foam cells.

SR-PSOX at sites predisposed to atherosclerotic lesion formation mediates monocyte-endothelial cell adhesion

OBJECTIVE The scavenger receptor SR-PSOX/CXCL16, which is identical to the chemokine CXCL16, is thought to be involved in atherogenesis. However, the presence and function of SR-PSOX/CXCL16 in the endothelium of atherosclerotic arteries has not been substantiated. METHODS AND RESULTS In rabbit aorta immunocytochemistry revealed SR-PSOX/CXCL16 primarily in the endothelium at sites predisposed to lesion formation, in the endothelium of early atherosclerotic lesions, and mainly in intimal macrophages of more developed lesions, indicating that SR-PSOX/CXCL16-expression shifts during atherogenesis. In addition to its function as scavenger receptor and chemokine, SR-PSOX mediated the adhesion of THP-1 monocytes to endothelial cells in vitro. Both THP-1 monocytes and endothelial cells express SR-PSOX/CXCL16, and THP-1 monocytes express CXCR6, the specific receptor for SR-PSOX/CXCL16. Anti-SR-PSOX/CXCL16 and anti-CXCR6 antibody block monocyte adhesion, showing that SR-PSOX/CXCL16-CXCR6 interaction mediates monocyte-endothelial cell adhesion. SR-PSOX/CXCL16 expression of endothelial cells is upregulated by pro-inflammatory cytokines, and is reversed by incubation with ciglitazone and lovastatin. CONCLUSIONS We suggest that SR-PSOX/CXCL16 may promote the adhesion of monocytes to the endothelium during early atherogenesis and that accumulating cytokines enhance SR-PSOX/CXCL16-mediated adhesion by upregulating SR-PSOX/CXCL16 expression. Manipulation of SR-PSOX/CXCL16 expression with anti-inflammatory agents may be of therapeutic value.

Topography of Lipid Droplet-Associated Proteins: Insights from Freeze-Fracture Replica Immunogold Labeling

Lipid droplets are not merely storage depots for superfluous intracellular lipids in times of hyperlipidemic stress, but metabolically active organelles involved in cellular homeostasis. Our concepts on the metabolic functions of lipid droplets have come from studies on lipid droplet-associated proteins. This realization has made the study of proteins, such as PAT family proteins, caveolins, and several others that are targeted to lipid droplets, an intriguing and rapidly developing area of intensive inquiry. Our existing understanding of the structure, protein organization, and biogenesis of the lipid droplet has relied heavily on microscopical techniques that lack resolution and the ability to preserve native cellular and protein composition. Freeze-fracture replica immunogold labeling overcomes these disadvantages and can be used to define at high resolution the precise location of lipid droplet-associated proteins. In this paper illustrative examples of how freeze-fracture immunocytochemistry has contributed to our understanding of the spatial organization in the membrane plane and function of PAT family proteins and caveolin-1 are presented. By revisiting the lipid droplet with freeze-fracture immunocytochemistry, new perspectives have emerged which challenge prevailing concepts of lipid droplet biology and may hopefully provide a timely impulse for many ongoing studies.

Postprandial triglyceride-rich lipoproteins regulate perilipin-2 and perilipin-3 lipid-droplet-associated proteins in macrophages ☆

Lipid accumulation in macrophages contributes to atherosclerosis. Within macrophages, lipids are stored in lipid droplets (LDs); perilipin-2 and perilipin-3 are the main LD-associated proteins. Postprandial triglyceride (TG)-rich lipoproteins induce LD accumulation in macrophages. The role of postprandial lipoproteins in perilipin-2 and perilipin-3 regulation was studied. TG-rich lipoproteins (TRLs) induced the levels of intracellular TGs, LDs and perilipin-2 protein expression in THP-1 macrophages and in Apoe(-/-) mice bone-marrow-derived macrophages with low and high basal levels of TGs. Perilipin-3 was only synthesized in mice macrophages with low basal levels of TGs. The regulation was dependent on the fatty acid composition of the lipoproteins; monounsaturated and polyunsaturated fatty acids (PUFAs) more strongly attenuated these effects compared with saturated fatty acids. In THP-1 macrophages, immunofluorescence microscopy and freeze-fracture immunogold labeling indicated that the lipoproteins translocated perilipin-3 from the cytoplasm to the LD surface; only the lipoproteins that were rich in PUFAs suppressed this effect. Chemical inhibition showed that lipoproteins induced perilipin-2 protein expression through the peroxisome proliferator-activated nuclear receptor (PPAR) PPARα and PPARγ pathways. Overall, our data indicate that postprandial TRLs may be involved in atherosclerotic plaque formation through the regulation of perilipin-2 and perilipin-3 proteins in macrophages. Because the fatty acid composition of the lipoproteins is dependent on the type of fat consumed, the ingestion of olive oil, which is rich in monounsaturated fatty acids, and fish oil, which is rich in omega-3 fatty acids, can be considered a good nutritional strategy to reduce the risk of atherosclerosis by LD-associated proteins decrease.