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Trends Endocrinol Metab. Author manuscript; available in PMC Jun 1, 2012.
Published in final edited form as:
PMCID: PMC3118920

Determinants of Adipophilin’s Function in Milk Lipid Formation and Secretion


In many species, the lactating mammary gland is one of the most lipogenic organs of the body. The majority of the lipid produced during lactation is secreted into milk by a novel process of membrane envelopment of cytoplasmic lipid droplets (CLDs). Adipophilin (ADRP/ADPH/PLIN2), a member of the perilipin (PAT) family of lipid droplet proteins, is hypothesized to play a pivotal role in both formation and secretion of milk lipids. Production of milk lipids is the only known example of CLD secretion, and the only process in which PAT family members undergo secretion. This review discusses emerging data about the structural and functional properties of adipophilin that determine its physiological actions and mediate its effects on milk lipid formation and secretion.

Adipophilin-enriched Cytoplasmic Lipid Droplets are Precursors of Milk Lipids

Lipids in milk supply the majority of the calories required for neonatal growth in many species [1], and are a primary source of the essential fatty acids needed for neonatal membrane synthesis and synthesis of eicosanoids and other bioactive lipid signalling molecules [2]. Milk lipids, which are primarily composed of triglycerides (98%) [3], exist in unique membrane-bound structures referred to as milk lipid globules (MLGs). Ultrastructural and biochemical studies have shown that MLGs are composed of a phospholipid bound triglyceride core, separated from a classical membrane bilayer by protein rich electron dense material that is assumed to be comprised of cytoplasmic domains of integral membrane proteins and associated peripheral proteins [4]. Analyses of isolated membranes from MLGs from multiple species have demonstrated that adipophilin is one of the most abundant proteins detected in MLGs [5-8].

Milk lipids originate from CLDs, which are secreted from specialized milk-producing epithelial cells by a unique membrane envelopment process that is distinct from the classical secretory pathway used for secretion of milk proteins, sugars and water [9] (Figure 1). The concept that CLDs are the source of milk lipids originated over 40 years ago by ultrastructural studies [10], which showed that CLDs are abundant structures in milk secreting cells of lactating rats; furthermore, seminal radiotracer experiments [11] showed that labeled fatty acids that initially incorporated into triglycerides near the endoplasmic reticulum rapidly appeared in CLDs scattered throughout these cells, and subsequently appeared in MLGs within the luminal space of alveoli. Electron micrographs of lactating mammary glands demonstrated that CLDs are completely enclosed by the plasma membrane before being released into the alveolar lumen by a membrane scission event [4].

Figure 1
Milk Component Secretion. Milk is a complex mixture of sugars, proteins, lipids, ions and other small molecules. These components enter milk through five types of transport processes: Pathway I - Major milk proteins such as casein, oligosaccharides, and ...

Proteomic analyses [6] documented that the overall protein composition of CLDs isolated from milk secreting cells of mice was similar to that of MLGs obtained from their milk, as expected if CLDs are precursors of MLGs. A correlative of this observation is that the process of secreting CLDs into milk appears to occur without significant compositional alteration, which again is consistent with envelopment of CLD in toto during secretion. These proteomic studies, which were the first to describe the CLD protein composition, showed that CLDs were enriched in specific types of proteins, and that adipophilin was a major protein component of CLD from the mammary gland and the liver. Subsequent proteomic studies of CLDs isolated from a variety of other mammalian cell types, including adipocyte-, Chinese hamster ovary K2-, lymphoblast- and hepatoma-cell lines, demonstrated variability in CLD protein compositions and suggesting that the protein content of these structures are influenced by the actions of cell-specific functions and/or the cell’s metabolic state [12-15]. Nevertheless, and consistent with the notion that adipophilin plays a common role in cellular lipid storage [16, 17], adipophilin has been shown to be a prominent protein component of CLD in most cells examined thus far [12-14], including milk secreting cells [18, 19]. Interestingly, in situ hybridization analysis documented that adipophilin transcript levels in these cells are markedly greater than in surrounding cell types, such as adipocytes and ductal epithelial cells [20], suggesting that in the mouse at least, high-level expression of adipophilin is a specialized function of milk secreting cells.

Adipophilin transcript and protein levels in milk secreting cells increase over 30-fold as the mammary gland undergoes differentiation into a secretory organ over the course of pregnancy [20]. Time course analyses showing that the pattern of adipophilin transcript expression in the differentiating mammary gland closely correlates with the appearance, growth and accumulation of CLDs in milk secreting cells, implicate adipophilin as a physiological regulator of milk lipid production [20, 21]. Indeed, microarray analysis indicates that adipophilin transcripts are among the most abundant present in the lactating mouse mammary gland, equivalent to that of other secreted milk proteins such as the caseins [22]. Interestingly adipophilin was originally identified as a highly induced gene in differentiating adipocytes [23]; furthermore, in 3T3-L1 cells, adipophilin induction correlated with the initial accumulation of CLDs [24]. Thus milk-secreting cells appear to be a second cell type in which adipophilin expression is functionally linked to the cell’s differentiation status and CLD accumulation. As yet, the functional significance of sustained elevated adipophilin expression in differentiated adipocytes is unclear since CLDs in these cells become coated by perilipin rather than adipophilin, and the expressed adipophilin undergoes rapid proteasomal degradation [24, 25]. In contrast, high-level adipophilin expression in differentiated milk secreting cells is presumably related to the constant secretion of adipophilin-coated CLDs into milk during lactation.

Other perilipin family members, including perilipin (PLIN1) and TIP47 (PLIN3), are also expressed in the mammary gland [19]. As expected from its tissue distribution pattern (Gene Atlas, EMBL-EPI), perilipin expression is confined to CLDs in mammary adipose [19]. TIP47 on the other hand is diffusely localized within the cytoplasm of mammary epithelial cells as well as in adipose, but not on CLDs [19]. Furthermore, in contrast to the surge in adipophilin expression, TIP47 transcripts decrease during mammary gland differentiation, which argue against a physiological role for TIP47 in CLD accumulation and secretion under normal conditions [20]. Although TIP47 is found in MLG in human milk [26], it is unclear whether it is present in these structures as a CLD-associated protein or if it arises from an adventitious inclusion of cytoplasm during CLD engulfment by the plasma membrane [4]. Therefore, it seems that adipophilin is the primary, if not exclusive, PAT family member on CLDs in milk secreting cells.

Functional Domains of Adipophilin

The identification of adipophilin as a major protein of CLDs and MLGs suggests it functions as an adaptor, linking CLDs to elements of the apical plasma to facilitate their secretion [4, 27]. Structure/function studies [28, 29] and structure prediction algorithms [17, 30] indicate that adipophilin possesses a distinct domain organization (Figure 2) expected of a protein with adaptor functions. Sequence alignment of adipophilin to other PAT family members has shown that it most closely relates to TIP47, with the two proteins sharing approximately 43% amino acid similarity [31]. Based on sequence comparisons to other PAT family members [17] and mutation analyses [28, 29], adipophilin is proposed to be composed of three principal domains: an unstructured N-terminal domain defined by a region of amino acid sequence similarity in the N-terminal tails of PAT family members [17] referred to as the PAT1 domain; an 11-mer helical repeat region in the N-terminal half of adipophilin that is hypothesized to mediate CLD binding [17, 28-30]; and a C-terminal four-helix bundle domain identified by alignment of the adipophilin C-terminal sequence to the C-terminal sequence of TIP47, whose crystal structure has been solved [30]. The corresponding three-dimensional structural features predicted by functional studies and sequence alignments (Figure 2) indicate that adipophilin is a bipartite protein composed of structurally independent N- and C-terminal regions, and the functions encoded by these regions are beginning to be to be defined.

Figure 2
Adipophilin Domains. Structure-function and structural modeling data show that adipophilin is composed of multiple domains with distinct structures and functions. (a) Computational-based model of adipophilin generated using Discovery Studio, version 2.5 ...

The PAT1 Domain

Consistent with the idea that adipophilin expression promotes CLD accumulation, adipophilin knockout mice displayed reduced hepatic TAG storage, deficits in CLD formation and resistance to diet-induced fatty liver disease [32, 33]. Unexpectedly, these knockout mice were found to express an N-terminally truncated form of adipophilin in their mammary glands during lactation, and have come to be known as adipophilin deficient or Δ2,3-ADPH mice [19]. Δ2,3- mice lack exons 2 and 3 that encode the initiating methionine and the first 75 residues of PAT1 domain, which as indicated in Figure 2, is predicted to form an unstructured region. The mechanism by which ADPH lacking the PAT1 domain (here referred to as ΔPAT-adipophilin) is produced in mammary glands of Δ2,3-ADPH mice is not well understood. However the adipophilin gene has been shown to encode a competent internal translation initiation site located in exon 5 that is capable of producing a truncated form of adipophilin that is similar in size to the ΔPAT-adipophilin found in mammary glands of Δ2,3-ADPH mice [19]. Intriguingly, a ΔPAT-adipophilin isoform of similar size was also found in mammary glands of wild type mice [19], as well as in extracts of mouse quadriceps muscle [14]. It is uncertain whether the ΔPAT-adipophilin isoforms found in tissues of wild type mice are generated from an alternative translation initiation site, or by the action of proteases. However, their presence demonstrates that truncated forms of adipophilin lacking the PAT1 domain exist naturally in some cells. As present it is unclear if ΔPAT forms of adipophilin are functionally distinct entities or accidents of biology. However, as discussed below, the PAT1 domain appears to have distinct functions. Thus ΔPAT-forms of adipophilin may serve distinct physiological functions in some tissues.

Δ2,3-ADPH mice have provided insights into the potential physiological roles of the adipophilin PAT1 domain and the regulation of CLD production and secretion by milk secreting cells. Immunolocalization studies, with C- and N-terminal specific anti-adipophilin antibodies, documented that adipophilin lacking the PAT1 domain localizes to CLDs in mammary glands of lactating Δ2,3-ADPH mice and is found on MLGs in their milk [19]. Thus the PAT1 domain is not absolutely required for adipophilin to bind to CLD, nor is it essential for CLD secretion. Cell culture experiments have also demonstrated that N-terminal truncations of adipophilin [34], perilipin [35] and TIP47 [36] lacking their respective PAT1 domains correctly localize to CLDs. In addition, S3-12, a PAT family member that lacks the evolutionarily conserved PAT-1 domain, binds to CLDs [37]. Thus there appears to be wide agreement that the PAT1 domain is not essential for PAT family members to bind CLD. Nevertheless, the adipophilin PAT1 domain possesses distinct functions that may influence the ability of adipophilin to promote CLD accumulation.

The finding that ΔPAT-adipophilin levels in mammary glands of Δ2,3-ADPH mice were significantly greater than that expected from ΔPAT-adipophilin transcript levels led to speculation that translational or post-translational mechanisms contribute to its regulation [19]. Studies demonstrating that native adipophilin undergoes rapid proteasome-dependent degradation unless bound to CLD [25] suggest that regulated proteolysis is a primary mechanism by which cells control adipophilin levels. Proteasome degradation of adipophilin is mediated by sequences within the PAT1 domain, and adipophilin variants lacking the PAT1 domain are significantly more stable than full-length adipophilin when not bound to CLD [34]. Importantly, replacing the N-terminal region of adipophilin with the homologous N-terminal region of TIP47, which is relatively resistant to proteasome degradation [34], produced a stable chimera (TIP47-ADPH). In contrast, replacing the N-terminal region of TIP47 with the homologous N-terminal region of adipophilin produced an unstable chimera (ADPH-TIP47) [34]. These findings suggest that the PAT1 domain of adipophilin contains a sequence, or sequences not found in the PAT1 domain of TIP47, that function in targeting adipophilin for rapid proteasome degradation. The absence of these sequences in ΔPAT-adipophilin may contribute to the greater than expected levels of this protein in mammary glands of Δ2,3-ADPH mice due to resistance to proteasome degradation [19].

Evidence from fibroblasts [38] and differentiating milk secreting cells [19] in Δ2,3-ADPH mice indicate that TIP47 binds to CLDs in cells deficient in adipophilin, compensating for loss of adipophilin in CLD production. By analogy to perilipin-null adipocytes, in which adipophilin replaces perilipin on the surface of CLD [39], the observation that TIP47 replaces adipophilin on the surface of CLDs in at least some cells of Δ2,3-ADPH mice, has led to the hypothesis that PAT proteins exhibit hierarchical affinities for CLD (perilipin > adipophilin > TIP47) [38]. This concept is supported by studies showing that exogenous expression of adipophilin in HEK293 cells displaces TIP47 from CLD [34, 40]. Evidence that the PAT1 domain of adipophilin is responsible for excluding TIP47 from CLDs was obtained in studies showing that full-length adipophilin but not ΔPAT-adipophilin prevents TIP47 from binding to CLD in HEK293 cells [34]. Further, studies showing that the adipophilin-TIP47 chimera, but not the TIP47-adipophilin chimera, excludes endogenous TIP47 from CLDs [34] provide evidence that differences in the PAT1 domains of adipophilin and TIP47 may account, in part, for presumptive differences in their CLD affinities. CLD accumulation in differentiating milk secreting cells is temporally regulated and characterized by histologically distinct phases [20, 21]. Observations that adipophilin induction, and the first appearance of CLDs in milk secreting cells, occurs 24 to 48 hours before induction of lipogenic processes and the major accumulation and growth of CLDs [20], suggest a model of milk lipid biogenesis in which the initial stage of CLD accumulation is mediated primarily by adipophilin stabilization of CLD [21]. This concept is supported by studies showing that simply increasing cellular adipophilin levels by preventing its proteasomal degradation is sufficient to induce CLD accumulation under conditions of basal triglyceride synthesis[25] [34]. The observation that over-expressed ΔPAT-adipophilin was unable to induce CLD accumulation under these same conditions [34] implicates the adipophilin PAT1 domain in the CLD stabilizing actions of adipophilin.

The CLD Targeting Domain

Several lines of evidence indicate that the CLD targeting function of PLIN2 localizes to a region of the N-terminal half of PLIN2 that does not include the PAT1 domain. The PAT1 domain itself is not capable of binding CLD [28, 29], and as indicated herein, Δ2,3-ADPH mice confirm that it is not required for the CLD binding functions of adipophilin [19]. Additionally, the C-terminal half of adipophilin is not required for targeting adipophilin to CLD [28, 29]. Systematic amino- and carboxy- terminal truncations of adipophilin [28, 29] and TIP47 [36] indicate that the essential CLD targeting region of these proteins is located within a stretch of hydrophobic residues arranged as tandem, 11-mer helical repeats [17] (Figure 2). These 11-mer repeats are predicted to form an atypical, slightly unwound helix with three complete turns/11 residues (α11/3)[41] and are believed to be responsible for targeting these proteins to specific lipid membrane structures. Due to lack of structural characterization, it is unclear whether all PAT proteins have the same number of 11-mer repeats or if the number of repeats influence affinity for CLDs.

The Four-Helix Bundle Domain

The preponderant localization of the known physiological functions of adipophilin within its N-terminal half raises questions about the functions of its C-terminus. The adipophilin C-terminus is predicted to encode a four-helix bundle domain with an adjacent hydrophobic motif comprised of a unique α/β fold based on sequence homology modeling (Figure 2) to the solved crystal structure of the TIP47 C-terminus [30]. Although we know little about the physiological functions of the four-helix bundle domains of adipophilin and TIP47, this motif is conserved structurally among exchangeable apolipoproteins [41] and is characterized as a reversible lipid membrane-binding domain [42]. Direct biochemical evidence that the adipophilin C-terminus also encodes an independently folding membrane binding domain has been obtained recently[43]. In studies using recombinantly expressed portions of the mouse adipophilin C-terminus and liposome binding assays, residues 172-425, which encompass the four-helix bundle domain, were shown to form a stable α-helical protein that directly binds to membrane bilayers [43]. Modeling studies suggested that the membrane binding properties of this region are mediated by residues located within helices 3 and 4 of the four-helix bundle domain [43].

Freeze fracture studies demonstrated the presence of adipophilin clusters in the cytoplasmic leaflets of endoplasmic reticulum and plasma membranes of macrophages [44], raising the possibility that the C-terminus of adipophilin mediates membrane interactions. In addition, observations that CLDs are often found in close proximity to ER, Golgi and mitochondria membranes [4, 6, 45] suggest that the adipophilin C-terminal four-helix bundle domain may function independently of the N-terminal CLD binding region as a mechanism for targeting CLDs to organelles involved in lipid metabolism. These concepts are supported by the observation that the C-terminal region of mouse adipophilin, encompassing the four-helix bundle, localized to the plasma membrane and to vesicle like structures within the cytoplasm when it was stably expressed in HEK293 cells [43]. Collectively, these data provide a steady stream of evidence that the adipophilin four-helix bundle is an independently functioning membrane binding domain.

The adipophilin C-terminus may also mediate CLD secretion by milk-secreting cells [27]. As indicated, adipophilin is a major protein component of MLGs found in the milk of numerous species. Adipophilin is localized on the inner leaflet of the membrane surrounding the lipid core of these structures as well as on the phospholipid monolayer that coats the lipid core [46]. Such dual localization is consistent with the possibility that adipophilin may direct interactions between the CLD and elements of the plasma membrane involved in their secretion. In vivo expression experiments support this hypothesis. Using adenoviral transduction to express GFP-fused forms of full-length and truncated adipophilin in milk secreting cells of lactating mice, it was shown that the N-terminal region of adipophilin directed binding to CLD. However the secretion of CLD coated by the N-terminal region of adipophilin appeared to be impaired relative to that of CLD coated by full-length adipophilin [43]. Along with evidence that the adipophilin C-terminus possesses membrane-binding functions, these observations support the concept that the C-terminal half of adipophilin mediates the membrane interactions that are important for CLD secretion.

Models of CLD secretion

The Tripartite Model

Adipophilin is hypothesized to participate in CLD secretion through formation of a tripartite complex with the transmembrane protein butyrophilin (BTN) and the ubiquitously expressed cytoplasmic enzyme xanthine oxidoreductase (XOR) [4, 27]. BTN is a type 1 transmembrane protein and member of the immunoglobulin superfamily [47, 48]. Mammary gland expression of BTN is induced prior to lactation at the end of secretory differentiation [49], and BTN specifically localizes to the apical plasma membrane of milk secreting cells [50]. XOR is a homodimeric purine oxidase that is highly expressed in the cytoplasm of mammary epithelial cells [51], and is an abundant structural component of the membrane envelope of MLG [4, 6]. Ablation of BTN or XOR in mice results in impaired CLD envelopment and secretion, leading to abnormal accumulation of large CLDs in milk secreting cells and reduced lipid content in milk [52, 53]. These independent observations suggest that BTN and XOR function in a similar pathway as adipophilin to achieve CLD secretion. Indeed, in lactating mice, adipophilin co-localizes with BTN and XOR on the apical plasma membrane at sites of CLD secretion [54], and biochemical studies show that adipophilin, BTN and XOR co-purify as a detergent-stable complex from the membrane envelope surrounding bovine MLGs [54].

As yet, the molecular nature of the adipophilin, BTN and XOR interactions are not completely understood. BTN is organized into multiple structural domains with two exoplasmic immunoglobulin folds, a single transmembrane region and a cytoplasmic domain [47]. The cytoplasmic domain of BTN specifically associates with endogenous XOR from cultured mouse mammary epithelial cells [55, 56]. Jeong et al [56] further defined the BTN/XOR interaction by demonstrating that the B30.2/PRY/SPRY region of the BTN cytoplasmic domain directly interacts with XOR with high affinity in vitro. However, interpreting the physiological significance of direct interactions between BTN and XOR in milk lipid secretion is complicated by detection of XOR in membrane fractions of mammary glands from lactating BTN−/− mice [56], arguing against the essential interaction between XOR and BTN. Furthermore, a clear molecular interaction between specific adipophilin domains and BTN or XOR is lacking, which provides opportunity for alternative mechanisms to explain CLD secretion.

The Butyrophilin Model

BTN accounts for more than 20% of the protein in the membrane envelop of MLGs [4], suggesting its importance for CLD secretion and MFG stability. Freeze fracture immunolabeling (FRIL) of isolated MFGs demonstrates the presence of BTN on both the exoplasmic face and inner leaflet of the membrane envelope surrounding MLG, as well as on the cytoplasmic face of the CLD phospholipid monolayer [46]. Based on differences in the distributions of immunogold labeled-BTN, -XOR and -adipophilin in the fracture planes of the membrane envelope and the CLD monolayer surface, BTN could be the primary determinant of CLD secretion, with its self-aggregation perhaps acting as the driving force of CLD envelopment [46]. Acceptance of this hypothesis is hindered by the findings that BTN and XOR are direct binding partners [55, 56] and by the finding that heterozygous XOR mice display severely impaired CLD secretion [53]. Furthermore, it remains to be determined if BTN alone is sufficient for achieving CLD secretion.

The Adipophilin Model

Neither the tripartite nor the butyrophilin models explain the initial interactions between CLD and elements of the apical plasma membrane involved in their secretion. As noted, adipophilin has been localized to ER membranes and the inner leaflets of the plasma membrane of macrophages and MLGs as well as to the nuclear membrane of macrophages [44]. Collectively these observations indicate that adipophilin-membrane interactions do not involve binding to specific membrane proteins. The observation that the adipophilin C-terminal region can bind to artificial membranes[43], raises the possibility that it may directly interact with the phospholipid bilayer of membranes. Such direct interactions between adipophilin and phospholipid bilayers suggest an alternative model of milk lipid secretion in which adipophilin acts as an adaptor between the CLD surface and the inner leaflet of the apical plasma membrane (Figure 3). In this model, the four-helix bundle motif in the adipophilin C-terminus and helical repeat elements in the N-terminus bind to the plasma membrane and CLD surface, respectively. Helical bundle motifs are known to be present in proteins that bend and deform the plasma membrane [57, 58]. Induction of membrane curvature is known to alter the structure and fluidity of membranes, which commonly results in the recruitment of other proteins to the site of deformation [57]. Therefore, in the adipophilin model of CLD secretion (Figure 3), direct association of adipophilin four-helix bundle with the apical plasma membrane is predicted to induce bending of the membrane and recruitment of other factors to the site of CLD secretion. Diffusion of BTN within the membrane then recruits XOR to complete tripartite complex formation and CLD secretion (Figure 3). It is important to emphasize that in this model, the adipophilin four-helix bundle motif is only proposed to initiate contact between the CLD and the apical plasma membrane that eventually lead to CLD secretion; BTN and/or XOR are hypothesized to be ultimately required for completion of the secretion process, and these proteins may or may not interact directly with adipophilin to accomplish this function. The prediction that the four-helix bundle motif mediates contact with the apical plasma membrane also leaves open the possibility that other four-helix bundle proteins, such as TIP47 or ΔPAT Adipophilin, may be competent to initiate CLD secretion, which would explain the relatively mild reduction in lipid content of milk from Δ2,3-ADPH mice [19]. Although transgenic loss of function studies are necessary to formally determine if CLD secretion specifically requires the adipophilin four-helix bundle, the observation that exogenous expression of adipophilin lacking its C-terminal region appears to impair milk lipid secretion provides support for this concept.

Figure 3
Adipophilin Adaptor Model of Milk Lipid Secretion. Current evidence suggests the following mechanism of milk lipid secretion: (a) Adipophilin functions as an adaptor to couple CLD to the cytoplasmic leaflet of the apical plasma membrane through independently ...

Concluding Remarks

There is considerable evidence that adipophilin functions as a physiological regulator of CLD accumulation in differentiating milk secreting cells and as an adaptor protein in the secretion of CLD to form milk lipids during lactation. Although adipophilin expression has been shown to be required for diet induced increases in hepatic triglycerides and CLD accumulation [32, 33], defining its physiological role in milk secreting cells has been complicated by the discovery of a truncated form of adipophilin lacking the PAT1 domain in mammary glands of adipophilin-null animals [19]. Nevertheless, studies indicating that the C-terminal four-helix bundle domain of adipophilin binds membranes, and that it is important for CLD secretion by milk secreting cells in the mouse mammary gland, support the concept that adipophilin functions as an adaptor in milk lipid secretion. However, further studies using bone fide adipophilin-null and/or transgenic mice expressing mutated forms of adipophilin will be required to define the specific physiological role(s) of adipophilin in milk lipid formation and secretion. Future efforts to define the structural features of adipophilin that mediate its interactions with CLD and membrane elements will provide the molecular framework needed to facilitate understanding of its physiological functions in the mammary gland and other organs.


Supported by grants from the National Institutes of Health, 2RO1-HD045962 and PO1-HD38129 to JLM. The authors thank E. Bales for assistance with the manuscript.


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