2004 — 2013 |
Mcmanaman, James Lewis |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mechanisms of Milk Lipid Secretion @ University of Colorado Denver
DESCRIPTION (provided by applicant):. 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.
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0.958 |
2005 — 2009 |
Mcmanaman, James Lewis |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Developmental Regulation of Cytoplasmic Lipid Droplet Synthesis @ University of Colorado Denver
In mammary epithelial cells cytoplasmic lipid droplets that are formed and secreted into milk provide essential fatty acids and cholesterol for membrane synthesis and a large percentage of neonatal calories. Since the mammary gland is one of the most active lipogenic organs of the body it provides an ideal model system to understand the cellular and molecular processes involved in lipid droplet formation. The overall goal of this project is to define how lipids, triglycerides and cholesterol esters, synthesized in the endoplasmic reticulum, are packaged into cytoplasmic lipid droplets. The PAT family (Perilipin, Adipophilin, TIP47) of lipid droplet associated proteins and caveolins have been shown to play an essential role in the formation, stabilization and metabolism of lipid droplets in other tissues. In the mammary gland this process is developmentally coordinated and regulated by the hormones of pregnancy. We hypothesize that the endoplasmic reticulum of mammary alveolar epithelial cells undergoes structural alterations during pregnancy resulting in the formation of specific lipid droplet assembly domains that contain the enzymes of lipid synthesis as well as the proteins necessary for lipid droplet assembly. In Specific Aim 1 we use morphological techniques to define the temporal alterations in the structure of the endoplasmic reticulum during lipid droplet formation. These changes will be correlated with expression of lipid synthetic enzymes and lipid synthesis in Specific Aim 2. The expression levels of candidate genes will be altered in Specific Aim 3 to develop a precise molecular model for the interactions of proteins involved in formation of lipid droplets. These experiments are designed to identify the structural and biochemical processes involved in formation of lipid droplets and to provide a framework for understanding the role of hormones in this process. The unique aspect of this proposal is the hypothesis that CLD form within specialized domains in the ER containing all the synthetic machinery. If this hypothesis is correct it will substantially alter our current understanding of the cell biological mechanisms involved in the initiation of CLD formation.
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0.958 |
2007 — 2008 |
Mcmanaman, James Lewis |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Lactational Effectors of Triacylglycerol Mobilization @ University of Colorado Denver
[unreadable] DESCRIPTION (provided by applicant): The lactating mouse presents a remarkable model for the study of the regulation of the lipid content of adipose depots. During typical 20-day lactation the murine mammary gland secretes approximately one body weight equivalent of triacylglycerol into the milk. During this process the mouse becomes almost entirely lean, returning to its pre-lactation body composition after weaning. Although lactation related delipidation of adipose depots has long been recognized in both rodents and dairy species, the mechanisms involved are largely unknown and the differences between adipose depots in their response to lactation have received little attention. In the intact mouse, delipidation of the mammary fat pads occurs faster and more completely than does delipidation of the peripheral, non-mammary fat pads, a phenomenon we ascribe to cross-talk between the mammary epithelium and the mammary fat pad. When the mammary fat pad is cleared of epithelial elements, it becomes almost unresponsive to the systemic influences that lead to delipidation of the uterine and perirenal fat pads. Because the degree of delipidation in these fat pads appears to be roughly proportional to prolactin receptor expression as well as to expression of a prolactin downstream signaling molecule, SOCS-2, we hypothesize that the high level of circulating prolactin during lactation acts as the systemic lipolytic factor. To test this hypothesis, we will assess prolactin receptor signaling and direct effects of prolactin on various fat pads of virgin and mid-lactating mice; the prolactin receptor will also be expressed in cultured adipocytes. Specific cell based and directed gene array assays will be used to define prolactin effects on adipose metabolism. To test the hypothesis that an epithelial- derived paracrine factor is responsible for the delipidation of the intact mammary fat pad, we will use high- resolution proteomic approaches in conjunction with sensitive cell culture assays. We aim to identify substances secreted from cultured mammary epithelial cells that increase adipocyte lipolytic activity. Through these studies, we hope to gain valuable insight into how female reproductive hormones influence adipocyte biology as well as how the mammary epithelial compartment communicates with the mammary adipose compartment to regulate energy stores. In addition, the long term ramifications of this study should lead to better understanding the role of various adipose tissue depots in the genesis of such complex disorders as obesity, metabolic syndrome, and gestational diabetes. [unreadable] [unreadable] [unreadable]
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0.958 |
2013 — 2017 |
Maclean, Paul S. Mcmanaman, James Lewis |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Postnatal Actions of Maternal Obesity On Neonatal Metabolic Health @ University of Colorado Denver
DESCRIPTION (provided by applicant): Maternal obesity increases the risk for offspring to become obese, and there is substantial evidence to suggest that programming during both fetal and neonatal development contributes to this predisposition. Breastfeeding, the recognized gold standard for human neonatal nutrition, is associated with reduced childhood obesity risk. However, emerging evidence from human and animal studies suggests that maternal obesity may override the benefits of breastfeeding on the metabolic health and obesity risk of nursing offspring. Our study is directed at understanding the mechanisms by which maternal obesity influences the metabolic predisposition of their off-spring to obesity. We established a mouse model that allows us to define postnatal contributions of maternal obesity to neonatal metabolic health and obesity predisposition, distinguishing the specific effects of maternal obesity from those imparted by maternal consumption of a high fat (HF) obesigenic diet. Our data document that milk from obese dams selectively programs obesigenic changes in neonatal metabolism. In recent work we linked these changes to impaired de novo milk lipid synthesis due to inhibition of acetyl- CoA carboxylase-1 (ACC1), and the production of lipid-poor milk by obese dams. The overall goals of this proposal are to use obese mouse models in conjunction with innovative genetic manipulation and quantitative metabolic and imaging approaches to: (1) define the effects of maternal obesity on off-spring obesity predisposition; (2) detail the effects of milk frm obese dams on neonatal metabolism; (3) define the roles ACC1 and de novo lipogenesis in the obesity-associated alterations in milk that promote neonatal obesity. The detailed systematic investigation of the physiological and molecular mechanisms underlying postnatal effects of maternal obesity on neonatal metabolic health, as outlined in this proposal, form the foundation for development of new intervention strategies to prevent obesity.
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0.958 |
2018 — 2021 |
Mcmanaman, James Lewis Monks, Jenifer Ann |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Determits of Lactation Success @ University of Colorado Denver
Milk is a complex fluid capable of sustaining the total nutrition of the human infant for 6 months or longer, as well as providing protection against infection and common childhood illnesses during the critical early postnatal period. Milk lipids provide the majority of the calories required for neonatal growth, in addition to being the primary mechanism for transferring fat-soluble vitamins to infants and the source of essential fatty acids needed for neonatal membrane synthesis and the synthesis of eicosanoids and other bioactive lipid signalling molecules. To meet caloric and nutritional demands of newborns, a unique apocrine mechanism evolved in mammals to efficiently secrete large quantities of lipid into milk. Evidence from humans and animal models suggest that apocrine lipid secretion is also important for initiating and sustaining lactation, and that interference with this process increases the risk of lactation failure. Work from our laboratories indicates that apocrine lipid secretion involves three mechanistically distinct steps: (1) formation of specialized ?docking? contacts between CLD and the apical plasma membrane (APM); (2) membrane envelopment of docked CLD, driven in part by Golgi derived secretory vesicles; and (3) release of membrane enveloped CLD into the luminal space as trilaminar membrane coated structures, referred to as milk fat globules (MFG), by an apocrine scission process. We previously identified interactions between butyrophilin (Btn), a mammary gland specific transmembrane protein, the cytoplasmic redox enzyme, xanthine oxidoreductase (XOR), and the CLD coat protein, perilipin-2 (Plin2) as important for apocrine lipid secretion in mice. Importantly, we have demonstrated that genetic deletion of either of these proteins interferes with CLD secretion, and impairs or prevents lactation. The goals of this proposal are: (1) to investigate the mechanism of apocrine lipid secretion and test the hypothesis that prolactin and oxytocin independently regulate discrete steps of this process; (2) define how apocrine lipid secretion affects apical membrane properties, secretory vesicle trafficking, lactogenesis and lactation success; and (3) to define critical molecular determinants of interactions between XOR, Btn and Plin2 that mediate apocrine lipid secretion. The proposed studies take advantage of novel XOR, Btn and Plin2 knockout mouse models developed in our laboratories, quantitative high resolution imaging, and innovative physiological and biochemical analyses and cell culture models of apocrine lipid secretion to accomplish these goals.
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0.958 |