Source: CORNELL UNIVERSITY submitted to
LIPID STORAGE AND UTILIZATION IN MAMMALIAN CELLS
 
PROJECT DIRECTOR: Brown, W. J. Bensadoun, A.
 
PERFORMING ORGANIZATION
MOLECULAR BIOLOGY AND GENETICS
CORNELL UNIVERSITY
ITHACA,NY 14853
 
NON TECHNICAL SUMMARY: When dietary fats are in excess, cells convert fats into molecules called triglycerides that are stored within specialized intracellular compartments called Lipid Bodies. Excessive fat storage in Lipid Bodies results in obesity. Little is known about the cellular and molecular mechanisms responsible for Lipid Body formation. The purpose of these studies is to identify the cellular and molecular mechanisms responsible for the storage of fats in Lipid Bodies. Identifying molecules responsible for fat storage in Lipid bodies could lead to the discovery of pharmaceutical agents useful for treatment of chronic obesity.
 
OBJECTIVES: The overall objective of this research is to understand at the molecular level how stored triglycerides are metabolized within the LBs of mammalian cells. More specifically, we propose to examine the biological and biochemical functions of a newly identified family of neutral lipases, the PNPLA neutral lipases, which appear to be involved in the liberation of fatty acids from triglycerides that are stored in LBs. Our specific goals are encompassed in three specific aims that focus on the biological and biochemical functions of PNPLA1, 2 and 5. We have chosen to focus on these three proteins because we have extensive preliminary studies on PNPLA2 and because PNPLA1 and 5 are completely uncharacterized members of the family. Other PNPLA enzymes are being studied in other laboratories. Specific Aim 1: The goal of this aim is to elucidate the biological roles of PNPLA1, 2 and PNPLA5 in LB homeostasis by determining when and how these proteins associate with LBs, and whether or not they are required for LB formation, maintenance, or degradation in adipocytes and non-adipocytes. Specific Aim 2: The goal of this aim is to conduct a molecular dissection of PNPLA1, 2 and PNPLA5 to identify domains of the proteins that are required for their association with LBs and those that are required for catalytic activity (see Aim 3). Specific Aim 3: The goal of this aim is to determine the biochemical functions of PNPLA1 and PNPLA5 by examining their substrate preferences and catalytic properties. Both enzymes are predicted to be triglyceride lipases, but they may have activities against other substrates as well, which we will examine in detail. The basic cell and molecular biology research proposed here should have an important impact on our understanding of how cells store and utilize lipids. Moreover, if PNPLA1, 2 and PNPLA5 do play important roles in lipid storage/utilization, then one could easily imagine that they might be attractive pharmaceutical targets for treatment of chronic obesity, e.g., stimulation of PNPLA1, 2 and PNPLA5 should reduce lipid load in adipocytes.
 
APPROACH: Our general approach is to utilize cell and molecular biological methods to study the role of PNPLA2 and PNPLA5 on LB formation and degradation using two cultured mammalian cell model systems. The first is adipogenic mouse NIH 3T3-L1 cells, which can be induced in cell culture to differentiate into fat-storing adipocytes. We are fortunate that Dr. Andre Bensadoun, of the Division of Nutritional Sciences, has agreed to collaborate with us on this project. Dr. Bensadoun works on lipid metabolic enzymes, and he will provide important intellectual input and hands-on technical support for the project, including supplying us with timed, differentiated adipocytes. Because all mammalian cells can store triglycerides in LBs, we will also examine the roles of PNPLAs in commonly used cells lines such as HeLa cells. In fact, our preliminary studies show that both PNPLA2 and 5 are associated with LBs in HeLa cells. Similar to adipocytes, HeLa and other cells can be induced to store triglycerides and form large LBs by simply growing cells in excess fatty acids. Aim 1: To elucidate the biological roles of PNPLA2 and PNPLA5 in LB formation, the association of PNPLAs with LBs will be determined by fluorescence microscopy at various times after the induction of LB formation while exposed to fatty acids or degradation stimulated by catecholamines. Endogenous proteins will be detected by immunofluorescence with specific antibodies we have prepared. We will also express GFP-tagged fusion proteins. Translocation of PNPLAs from soluble cytoplasmic fractions to LBs will also be investigated during stimulated lipolysis by centrifugation in sucrose gradients. To determine if PNPLAs are involved in LB degradation, we will over-express tagged versions of the proteins and then perform biochemical assays for triglyceride levels and fluorescence microscopy to detect changes in LB size and number. Similar experiments to examine the effects of loss of PNPLAs will be done by siRNA knockdowns. As a control, we will also determine the effects of over-expressing catalytically inactive forms of the proteins (see below). Aim 2: To identify important domains of PNPLA2 and PNPLA5, various mutations, deletions, and truncations will be introduced by molecular biology. Some of the most important domains, e.g., residues involved in catalysis, targeting to LBs, etc., can be predicted by sequence comparisons, and these will be the first ones investigated. Mutant versions will be expressed in cultured cells to determine effects on LBs association and degradation, or purified from bacterial or insect cells to determine effects on catalytic activity (in Aim 3). Aim 3: To determine the enzymatic properties of PNPLA2 and PNPLA5, GST- or 6His-tagged versions of the proteins will be over-expressed in either E. coli or insect cells and purified using standard methods. Purified wild-type and mutant enzymes (as described above) will then be assayed to determine substrate specificity against triglycerides, diacylglycerides (the precursor to triglycerides), and various phospholipids.
 
CRIS NUMBER: 0205385 SUBFILE: CRIS
PROJECT NUMBER: NYC-165415 SPONSOR AGENCY: CSREES
PROJECT TYPE: HATCH PROJECT STATUS: TERMINATED MULTI-STATE PROJECT NUMBER: (N/A)
START DATE: Oct 1, 2005 TERMINATION DATE: Sep 30, 2008

GRANT PROGRAM: (N/A)
GRANT PROGRAM AREA: (N/A)

CLASSIFICATION
Knowledge Area (KA)Subject (S)Science (F)Objective (G)Percent
702701010305.1100%

CLASSIFICATION HEADINGS
KA702 - Requirements and Function of Nutrients and Other Food Components
S7010 - Biological Cell Systems
F1030 - Cellular biology
G5.1 - Ensure Access to Nutritious Food


RESEARCH EFFORT CATEGORIES
BASIC 100%
APPLIED (N/A)%
DEVELOPMENTAL (N/A)%

KEYWORDS: obesity; fat and lipid storage; adipocytes; lipid body; lipid droplet; lipases; patatin-like phospholipase domain containing protein a; transfection; microscopy; enzymology

PROGRESS: Oct 1, 2006 TO Sep 30, 2007
OUTPUTS: For the past year we have been conducting research on the storage of lipids (fats) in mammalian cells. Most of this work involves laboratory research using cultured mammalian cells as a model system for understanding lipid storage. In animals such as ourselves, excess fats are converted to and stored as molecules called triglycerides (TGs) within specialized cellular structures called lipid bodies (droplets). TG storage is especially prominent in professional fat storing cells called adipocytes, which are abundant in fat tissues. Our goals have been to characterize the functions of a newly discovered family of enzymes that mediate the breakdown of stored TGs. These efforts have resulted in new information on the fundamental process of lipid storage, which has direct implications for understanding obesity. Our results have been disseminated in various ways including published manuscripts and invited presentations at national and international meeting. These meetings include the 2007 FASEB summer conference on Lipid Droplets and the annual meeting of the American Society for Biochemistry and Molecular Biology. PARTICIPANTS: Deborah Brown of SUNY Stony Brook was a collaborator with us on focused aspects of this project.

IMPACT: 2006-10-01 TO 2007-09-30 Our research activities have primarily lead to a significant change in our knowledge about how lipids are stored as triglycerides (TGs). Our studies have clear implications for determining how humans and other mammals metabolize dietary fats, thus contributing fundamental knowledge that might lead to a better understanding of obesity. Moreover, our studies suggest that PNPLA2/ATGL and PNPLA5 could be attractive targets for gene knock-out studies in mice, whose purpose would be to see if the lack of PNPLAs has any effect on overall body size and lipid storage. Interestingly, another group has just published results on a PNPLA2/ATGL knock-out mouse and found although these mice had significantly increased fat deposits in cardiac tissue, with attending heart defects, they were not obese. In other words, other PNPLA family members must also be functioning to keep triglyceride amounts at normal levels. Thus, we await the results of knock out studies with other PNPLA members. Depending on the outcome of our studies, one could easily imagine long-term studies to develop specific drugs that target PNPLAs, to either inhibit or stimulate their activity as a therapeutic intervention for obesity. Such studies would have a clear impact on the general population and fit the CSREES goals for a healthy and well-nourished population.

PUBLICATION INFORMATION: 2006-10-01 TO 2007-09-30
Listenberger, L.L., A. G. Ostermeyer-Fay, E. B. Goldberg, W. J. Brown, and D. A. Brown. 2007. Adipocyte differentiation-related protein reduces the lipid droplet association of adipose triglyceride lipase and slows triacylglycerol turnover. J. Lipid Res. 48: 2751 - 2761.

PROJECT CONTACT INFORMATION
NAME: Hoffmann, M. P.
PHONE: 607-255-2224
FAX: 607-255-9499