Cytoplasmic lipid droplet accumulation in developing mammary epithelial cells: roles of adipophilin and lipid metabolism

Abstract

PAT proteins (perilipin, adipophilin, and TIP47) are hypothesized to be critical regulators of lipid accumulation in eukaryotic cells. We investigated the developmental relationships between the expression of these proteins and cytoplasmic lipid droplet (CLD) accumulation in differentiating secretory epithelial cells in mouse mammary glands. Adipophilin (ADPH) specifically localized to CLD in differentiating and lactating mammary glands and was found exclusively in the secreted lipid droplet fraction of mouse milk. ADPH transcripts were selectively detected in secretory epithelial cells, and steady-state levels of both ADPH mRNA and protein increased during secretory differentiation in patterns consistent with functional linkage to CLD accumulation. TIP47 also was detected in secretory epithelial cells; however, it had a diffuse punctate appearance, and its mRNA and protein expression patterns did not correlate with CLD accumulation. Perilipin-positive adipose cells and steady-state levels of perilipin mRNA and protein decreased during mammary gland differentiation, suggesting a progressive loss of adipose lipid storage during this process.Collectively, these data demonstrate that increased ADPH expression is a specialized property of differentiated secretory epithelial cells and provide developmental evidence specifically linking increased ADPH expression to increased CLD accumulation. In addition, evidence is presented that the epithelial and adipose compartments of the mammary gland undergo concerted, developmentally regulated shifts in lipid metabolism that increase the availability of fatty acids necessary for lipid synthesis by milk-secreting cells.-

The Adipophilin C Terminus Is a Self-folding Membrane-binding Domain That Is Important for Milk Lipid Secretion

“…Parturition occurs on approximately day 19 of gestation and is also designated day 1 of lactation. Mammary tissue was removed from animals euthanized by carbon dioxide and cervical dislocation and frozen in liquid nitrogen or processed for immunofluorescence microscopy (8,19). Animal procedures were approved by the University of Colorado Anschutz Medical Campus Institutional Animal Care and Use Committee.…”

“…Mammary epithelial cells were transduced on pregnancy day 17 with the indicated adenovirus particles (24). Transduced mammary glands were excised on lactation day 2 and processed for immunofluorescence microscopy (8). Mammary gland sections were incubated with Alexa 594-or Alexa 488-labeled wheat germ agglutinin to detect luminal borders and with DAPI to detect nuclei (8).…”

“…PAT proteins have been shown to promote the formation and accumulation of CLD in a variety of tissues (9, 10) and are also implicated in the trafficking and secretion of CLD (2, 9). The lactating mammary gland is an ideal system to study the recurrent cycles of CLD formation, accumulation, and secretion, and previous work from our laboratory has implicated ADPH in each of these processes (5,8,11).…”

“…PAT proteins have been shown to promote the formation and accumulation of CLD in a variety of tissues (9, 10) and are also implicated in the trafficking and secretion of CLD (2, 9). The lactating mammary gland is an ideal system to study the recurrent cycles of CLD formation, accumulation, and secretion, and previous work from our laboratory has implicated ADPH in each of these processes (5,8,11).CLD are secreted during lactation by an apocrine process where the apical plasma membrane engulfs associated CLD, which are then released by a budding process to form MFG (2). Two key regulators of CLD secretion are the transmembrane protein butyrophilin (BTN) and the cytoplasmic homodimer, xanthine oxidoreductase (XOR).…”

“…Milk lipids are delivered to the newborn as milk fat globules (MFG) 4 derived from triglyceride-rich cytoplasmic lipid droplets (CLD) (2). Adipophilin (ADPH/ADRP/Adfp/Plin2), a member of the perilipin/PAT (perilipin/adipophilin/TIP47) family of proteins (3), is an abundant MFG protein (4 -6) and a prominent CLDassociated protein of mammary epithelial cells (7,8). PAT proteins have been shown to promote the formation and accumulation of CLD in a variety of tissues (9, 10) and are also implicated in the trafficking and secretion of CLD (2, 9).…”