. Lipid synthesis is induced during differentiation of the mammary gland into a secretory organ;and during lactation in humans and rodents the mammary gland is estimated to be among the most active lipogenic organs in the body. Milk lipid secretion is a tightly regulated process requiring synthesis, assembly and transport of triglyceride and cholesterol ester containing droplets (cytoplasmic lipid droplets -CLD) to the apical membrane of mammary epithelial cells where they are secreted by unique membrane budding mechanism. The long-term objectives of this proposal are to elucidate the molecular and cellular mechanisms underlying formation, transport, and secretion of CLD. We hypothesize that adipophilin (ADPH), a member of the perilipin (PAT) family of lipid droplet binding proteins, is required for CLD formation and secretion, thus integrating lipid synthesis and secretion during lactation. We propose to test this hypothesis by determining the functional consequences of selectively disrupting expression of ADPH and/or the closely related PAT protein, TIP47, in the mouse mammary gland using transgenic and adenoviral approaches. Transgenic mice expressing N- and C-terminally truncated forms of ADPH will be used to define effects of specific ADPH domains on milk lipid formation and secretion. Adenoviral vectors will be used to express variants of ADPH and/or TIP47, or mutations of specific functional domains of these proteins, in mammary glands of ADPH-null mice to identify molecular determinants that will rescue defects induced by absence of this protein. ADPH and TIP47 are both hypothesized to function in lipid accumulation in many mammalian cell types. Because lipid synthesis in the mammary gland is robust and developmentally regulated by well defined promoter systems, and mammary epithelial cells can be manipulated by transgenic and adenoviral techniques, the proposed studies offer an excellent opportunity to understand molecular interactions and structure-function relations of lipid storage in many cells and tissues. NICHD Health Relatedness: CLD are the source of milk lipids, which are required neonatal growth, and essential fatty acids and cholesterol needed for membrane synthesis, particularly in the CNS. However, there is increasing recognition of the general importance of CLDs in storage and intracellular trafficking of lipids in eukaryotic cells. These cellular structures are the primary storage depots for triglycerides in liver, adipose and muscle cells;and the source of triglycerides and cholesterol esters in serum lipoprotein particles secreted by the liver, and milk fat globules secreted by mammary epithelial cells. CLDs are important homeostatic elements in cellular and tissue lipid metabolism and may serve to limit the availability of potentially toxic free fatty acids. Furthermore, elevated CLD accumulation in non-adipose tissue is a prominent pathological feature of many human metabolic diseases, such as obesity, type-II diabetes and non-alcoholic hepatic steatosis, which are increasing health concerns for children. Characterization of the fundamental molecular and cellular mechanisms regulating normal formation and metabolism of CLD as proposed in this application is essential to understanding the molecular abnormalities contributing the pathophysiology of lipid metabolism disorders.

Public Health Relevance

The long-term objectives of this proposal are to elucidate the molecular and cellular mechanisms underlying formation, transport, and secretion of CLD. We hypothesize that adipophilin (ADPH), a member of the perilipin (PAT) family of lipid droplet binding proteins, is required for CLD formation and secretion, thus integrating lipid synthesis and secretion during lactation. We propose to test this hypothesis by determining the functional consequences of selectively disrupting expression of ADPH and/or the closely related PAT protein, TIP47, in the mouse mammary gland using transgenic and adenoviral approaches. Transgenic mice expressing mutated forms of ADPH will be used to define effects of specific ADPH domains on milk lipid formation and secretion. Adenoviral vectors will be used to express variants of ADPH and/or TIP47, or mutations of specific functional domains of these proteins, in mammary glands of ADPH-null mice to identify molecular determinants that will rescue defects induced by their absence.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
2R01HD045965-05A1
Application #
7683687
Study Section
Special Emphasis Panel (ZRG1-EMNR-A (02))
Program Officer
Grave, Gilman D
Project Start
2003-12-01
Project End
2014-07-31
Budget Start
2009-08-05
Budget End
2010-07-31
Support Year
5
Fiscal Year
2009
Total Cost
$318,426
Indirect Cost
Name
University of Colorado Denver
Department
Obstetrics & Gynecology
Type
Schools of Medicine
DUNS #
041096314
City
Aurora
State
CO
Country
United States
Zip Code
80045
Libby, Andrew E; Bales, Elise S; Monks, Jenifer et al. (2018) Perilipin-2 deletion promotes carbohydrate-mediated browning of white adipose tissue at ambient temperature. J Lipid Res 59:1482-1500
Monks, Jenifer; Dzieciatkowska, Monika; Bales, Elise S et al. (2016) Xanthine oxidoreductase mediates membrane docking of milk-fat droplets but is not essential for apocrine lipid secretion. J Physiol 594:5899-5921
Libby, Andrew E; Bales, Elise; Orlicky, David J et al. (2016) Perilipin-2 Deletion Impairs Hepatic Lipid Accumulation by Interfering with Sterol Regulatory Element-binding Protein (SREBP) Activation and Altering the Hepatic Lipidome. J Biol Chem 291:24231-24246
McManaman, James L (2014) Lipid transport in the lactating mammary gland. J Mammary Gland Biol Neoplasia 19:35-42
Kharbanda, Kusum K; Todero, Sandra L; Thomes, Paul G et al. (2014) Increased methylation demand exacerbates ethanol-induced liver injury. Exp Mol Pathol 97:49-56
Orlicky, David J; Monks, Jenifer; Stefanski, Adrianne L et al. (2013) Dynamics and molecular determinants of cytoplasmic lipid droplet clustering and dispersion. PLoS One 8:e66837
Crunk, Amanda E; Monks, Jenifer; Murakami, Aya et al. (2013) Dynamic regulation of hepatic lipid droplet properties by diet. PLoS One 8:e67631
McManaman, James L; Bales, Elise S; Orlicky, David J et al. (2013) Perilipin-2-null mice are protected against diet-induced obesity, adipose inflammation, and fatty liver disease. J Lipid Res 54:1346-59
McManaman, James L (2012) Milk lipid secretion: recent biomolecular aspects. Biomol Concepts 3:581-591
Wahlig, Jessica L; Bales, Elise S; Jackman, Matthew R et al. (2012) Impact of high-fat diet and obesity on energy balance and fuel utilization during the metabolic challenge of lactation. Obesity (Silver Spring) 20:65-75

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