Progress 10/01/11 to 09/30/16
Outputs
Target Audience:Scientists within the international biomedical research community Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Over the five years of the project, eight undergraduate students have received training in research including Rachael Aubin, Abdul Balaparya, Adam Ahmad Chaustre, Daniel Kurz, Rahyan Mahmud, Daniel Mascarenhas, Lauren Simon, and Brittney Suchan. Additionally, four graduate students have received training including Derek McMahon (PhD completed 2012), Dana Lee Daley (PhD student, dropped program in 2013), Mayda Hernandez (M.S. completed 2013), and Sarah Hassanien (M.S. completed 2014). How have the results been disseminated to communities of interest?Over the five years of the project, six peer-reviewed manuscripts, one book chapter, and two abstracts have been published. Additionally, nine invited talks have been presented at international conferences related to obesity, diabetes, and lipid metabolism, including the Keystone Symposium on Genetic and Molecular Basis of Obesity and Body Weight Regulation, the Annual Meeting of the American Society of Biochemistry and Molecular Biology, the Annual Meeting of the American Gastroenterological Association, and the FASEB Summer Research Conference on Lipid Droplets, and eight invited lectures have been presented at domestic and international universities or research institutes, including Albert Einstein College of Medicine Diabetes Research Center, the University of Pennsylvania Diabetes Research Center, and the Department of Chemistry and Cell Biology at the University of Utrecht in the Netherlands. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Impact: The project has increased knowledge of the biomedical research community by adding to a growing mechanistic model for how adipose (fat) tissue controls the storage of fat (triacylglycerols) and release of fatty acids when the body requires energy. Fat is stored within the cells of adipose tissue in structures called lipid droplets. The most abundant protein associated with these lipid droplets is perilipin 1. Perilipin 1 serves as a gatekeeper protein, controlling how much triacylglycerol is stored in the lipid droplets of adipocytes (fat cells) and how much is released in the form of fatty acids during exercise and fasting to supply a source of energy to muscle and other tissues. Perilipin 1 controls triacylglycerol storage and breakdown (lipolysis) by regulating the activity and access of various proteins, including enzymes that break down fat called lipases, to triacylglycerols stored in lipid droplets. These control mechanisms include the binding of lipases and accessory proteins directly to perilipin under specific conditions. Various hormones exert control over these conditions by altering the structure of perilipin through chemical modification (phosphorylation). Over the years, we have defined how phosphorylation of perilipin 1 is needed to recruit some proteins to the lipid droplet, while dispersing other proteins away from lipid droplets Thus, these studies have helped to unravel the complex mechanisms by which perilipin 1 accomplishes control of fat metabolism in adipose tissue, an important component of the maintenance of whole body energy balance. The maintenance of energy balance is critical to avoid pathological complications of obesity and insulin resistance that leads to type II diabetes. Although perilipin 1 itself is not a likely target for the development of new drug therapies, some of the enzymes that bind to perilipin may be viable future targets for drugs to treat obesity or type II diabetes. Specific Aim 1 was to investigate the role of perilipin 1 amino acid 517 (serine 517) in the lipid droplet targeting and association of perilipin and in perilipin's function to control lipolysis of stored triacylglycerols. Site directed mutagenesis was used to make 12 different mutations (amino acid substitutions) in serine 517 of perilipin, and to incorporate the cDNA for these mutated variants into adenoviral expression vectors. The adenoviral vectors were transduced into cultured fibroblasts and the titers of the vectors were adjusted to normalize the expression of all variants to the same level by measuring mRNA levels of the ectopic perilipin using reverse transcriptase and quantitative PCR. The mutated perilipin variants were expressed in mouse fibroblasts and the ability of the mutated perilipin to target to lipid droplets was tested using immunofluorescence microscopy. We found that perilipin with glutamate or lysine substitutions of amino acid 517 targeted to lipid droplets as well as unmodified perilipin (serine 517), whereas perilipin with an alanine substitution failed to target to lipid droplets. However, the addition of a FLAG epitope tag of DYKDDDDK immediately after an alanine residue at position 517 rescued lipid droplet targeting. Additionally, early experiments with methionine and cysteine substitutions at position 517 suggested low or absent levels of targeting of the mutated perilipins to lipid droplets. These data suggest that polar residues at the carboxyl terminus of perilipin enable the most efficient targeting of the nascent protein to lipid droplets. The next planned steps were to complete characterization of all mutated perilipin variants using microscopy and then to use subcellular fractionation to obtain more quantitative results. Finally, we planned to test the effects of these mutations on perilipin's function to control lipolysis, since serine 517 is a substrate for protein kinase A (PKA); however, the project was terminated in May of 2013 when the graduate student working on the project left the lab and terminated her graduate studies; there were insufficient funds remaining in the NIH grant funding the project to hire a new person to continue the project. Specific Aim 2 was to investigate the role of PKA-mediated phosphorylation of serine 492 in perilipin's function to control lipolysis. We made adenoviral vectors to drive expression of mutated variants of perilipin to test the function of serine 492, including an alanine substitution at position 492, a variant that has mutations to 5 other PKA site serine residues leaving serine 492 intact and a variant that has mutations in all 6 PKA site serine residues to block all PKA-mediated phosphorylation of perilipin. Additionally, we made adenoviral expression vectors to drive the expression of the two major lipases present in adipocytes, adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL). These vectors were then used to express either ATGL or HSL in cells that also expressed either wild-type (unmodified) perilipin or one of the three mutated variants, and lipolysis was measured in intact cells. We discovered that the phosphorylation of perilipin serine 492 is required for maximal hormonally stimulated lipolysis in cells expressing ectopic ATGL with or without co-expression of ectopic CGI-58, the co-activator of ATGL. Phosphorylation of perilipin serine 492 is also needed for maximal lipolysis catalyzed by the as yet unidentified triacylglycerol lipase(s) of fibroblasts; hence the mechanism by which this phosphorylation aids lipolysis is a general mechanism, and not specific to the release of CGI-58 from its binding site on perilipin 1 to enable its interaction with ATGL and co-activation of lipolysis (which is an additional known consequence of the phosphorylation of perilipin serine 492). In three additional projects that are closely related to Specific Aim 2, we studied the function of CGI-58, a perilipin binding protein and co-activator of ATGL. CGI-58 binds to the carboxyl terminus of perilipin 1 under basal conditions and, when hormones stimulate lipolysis, CGI-58 is released from the perilipin scaffold, in part by the PKA-mediated phosphorylation of serine 492. Upon release, CGI-58 binds and co-activates ATGL to enable triacylglycerol hydrolysis. In the first project, we established that CGI-58 lacks lysophosphatidic acid acyltransferase activity that had been reported previously, and that it binds phosphorylated phosphatidyl inositols (PIPs), including PI(3)P and PI(5)P, but not PI(4)P or PI(4,5)P. We found that CGI-58 binding of PIPs does not alter the co-activation function for ATGL, so it likely serves a different function such as enabling subcellular targeting. In the second project, we studied a novel human mutation of CGI-58 (His82Arg in human CGI-58, His84Arg in mouse CGI-58) that is responsible for causing a neutral lipid storage disorder (NLSD). Using purified recombinant CGI-58, or CGI-58 ectopically expressed in cells, we showed that this mutation does not reduce protein-protein interactions between ATGL and CGI-58, although it does eliminate the co-activation of ATGL by CGI-58. The mutation does not reduce the interaction of CGI-58 with perilipin 1 under basal conditions, and does not alter the dispersion of CGI-58 into the cytoplasm upon hormonal stimulation of cells to activate PKA. It also does not alter the recruitment of CGI-58 with or without bound ATGL to lipid droplets. Thus, we do not yet understand the mechanism by which this mutation causes NLSD. In the third project, we have identified a serine residue (serine 239) in CGI-58 that is phosphorylated by PKA when lipolysis is hormonally stimulated. We have shown that the function of this phosphorylation is to help disperse CGI-58 from its binding site on perilipin to enable its interaction with and co-activation of ATGL. The phosphorylation of both serine 492 and serine 517 of perilipin 1 is also critical for this control mechanism.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2016
Citation:
Brasaemle, D. and Wolins N. 2016. Isolation of cells by density gradient centrifugation. Current Protocols in Cell Biology 72:3.15.1-3.15.13. Editors Juan S. Bonifacino, Joe B. Harford, Jennifer Lippincott-Schwartz, and Kenneth M. Yamada, Publisher John Wiley & Sons, Inc. doi:10.1002/cpcb.10.
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:Scientists within the international biomedical research community Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training for 2015 included training of 1 MS. Degree student, who completed her degree in October 2014, and 5 undergraduate students through either part-time hourly work or an independent research project for credit. They assisted the Laboratory Researchers in my group, Dr. Anna Dinh and Dr. JaeYeon Chun with a variety of procedures. How have the results been disseminated to communities of interest?Events/Dissemination for 2015 included a presentation at the annual meeting of the American Gastroenterological Association during Digestive Disease Week 2015 in Washington, DC. The oral presentation used Powerpoint slides to disseminate information to researchers in the biomedical research community to advance understanding of the function of proteins within the perilipin family in the control of lipid metabolism in adipocytes and other tissues. What do you plan to do during the next reporting period to accomplish the goals?Plans: We will continue to characterize the transgenic mouse model of altered perilipin 1 function and perform pilot experiments to support grant proposal writing, as well as prepare a manuscript to describe our studies on the function of phosphorylation of serine 492 in perilipin 1 control of lipolysis using a cell culture model (described in previous annual reports).
Impacts
What was accomplished under these goals?
Activities for 2015 include study of a perilipin binding protein called CGI-58. CGI-58 is a co-activator of adipose triglyceride lipase (ATGL), a major adipose lipase. Under fed conditions, adipocytes synthesize triglycerides from excess calories that are metabolized to fatty acids. The triglycerides are stored in lipid droplets coated with perilipin 1. Under these conditions, CGI-58 binds to amino acids located in the carboxyl terminus of perilipin 1 and when bound, cannot interact with ATGL. This prevents the activation of ATGL, hence preventing lipolysis, or the breakdown of triglycerides, and ensures that triglycerides are stored. During fasting or extended exercise, lipolysis is increased when catecholamines bind to beta-adrenergic receptors, leading to increased intracellular levels of cAMP and the activation of protein kinase A (PKA). PKA phosphorylates perilipin 1 on multiple serine residues, particularly serine 492 and serine 517, resulting in the release of CGI-58 into the cytoplasm. This facilitates CGI-58 binding to ATGL; the lipase is activated and triglycerides are hydrolyzed, releasing fatty acids into circulation for use as energy substrates. We have discovered that CGI-58 is also phosphorylated on at least one serine residue (serine 239 in mouse CGI-58) in lipolytically stimulated cells, and this phosphorylation is important for the release of CGI-58 from perilipin 1 so that it can bind to and co-activate ATGL. In addition, the phosphorylation of serine residues in perilipin 1 is necessary for this process; previous studies have shown that the critical perilipin residues include serine 492 and serine 517. This work has now been published in the Journal of Lipid Research. Additionally, we have begun to characterize a mouse model of altered perilipin 1 function. This mouse expresses a mutated perilipin 1 transgene that has an alanine substitution for serine 492 in a PKA consensus sequence; the expression of this transgene is limited to adipose tissue by use of the adiponectin promoter sequence. We have shown in cell culture models that the PKA-mediated phosphorylation of perilipin 1 on serine 492 is required for maximal catecholamine-stimulated lipolysis. We are now testing the hypothesis that phosphorylation of serine 492 is essential for maximal lipolysis in vivo, using this mouse model. To ensure that the mutated variant of perilipin 1 is the only perilipin 1 that is expressed, we have bred the transgenic mice to perilipin null mice, and now have a small number of the desired animals. We are measuring the level of transgene expression to ascertain whether protein levels of the transgene can fully substitute for the loss of endogenous perilipin 1. We are also measuring body weight and body composition over time to determine whether the transgene can support the maintenance of normal fat content in the mice. Impacts for 2015 include the gain of knowledge that will help the biomedical research community to better understand how fat storage and release is controlled in adipose tissue and other tissues. We have gained a better understanding of the molecular mechanisms by which specific proteins control how fat is stored during feeding and how fat is metabolized during fasting and exercise. We have increased understanding of how a protein called perilipin 1 works by coordinating the activity of proteins that metabolize fat. These findings provide insight into normal physiological processes as well as how these processes change during obesity, leading to associated pathologies such as insulin resistance and type II diabetes mellitus.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Sahu-Osen, A., Montero-Moran, G., Schittmayer, M., Fritz, K., Dinh, A., Chang, Y.-F., Boeszoermenyi, A., Cornaciu, I., Russell, D., Oberer, M., Carman, G. M., Birner-Gruenberger, R., and Brasaemle, D. L. 2015 CGI-58/ABHD5 is phosphorylated on Ser-239 by protein kinase A: Control of subcellular localization. J. Lipid Res. 56, 109-121. DOI:10.1194/jlr.M055004.
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Scientists within the international biomedical research community Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Services for 2014 included training of 2 undergraduate students and 1 graduate student. The M.S. student, Sarah Hassanien, completed her M.S. degree for October, 2014. 2 undergraduate students received training through either part-time hourly work or as an independent research project for credit. They assisted the Laboratory Researcher, Anna Dinh, with a variety of procedures. How have the results been disseminated to communities of interest? Events/Dissemination for 2014 included presentations of seminars in the Department of Biochemistry at West Virginia University, at the Lipid MAPS consortium annual meeting in San Diego, CA, and at the FASEB summer research conference, Lipid Droplets: Metabolic Consequences of the Storage of Neutral Lipids, in Saxton’s River, VT. These oral presentations used Powerpoint slides to disseminate information to students and researchers in the scientific community to advance understanding of the function of perilipins in the control of lipid metabolism in adipocytes and other tissues. Products for 2014 included 1 manuscript, with an additional manuscript accepted for final publication in 2015. What do you plan to do during the next reporting period to accomplish the goals? We will work to complete projects on phosphorylation of serine 492 of perilipin and the histidine mutation of CGI-58 with the goal of writing and submitting manuscripts. We are also performing pilot experiments to support grant proposal writing.
Impacts
What was accomplished under these goals?
Impacts for 2014 include the gain of knowledge that will help the research community to better understand how adipose tissue controls fat storage and release. We have gained better understanding of how perilipin works to control fat metabolism in adipose tissue by controlling the interaction of various perilipin-binding proteins with lipid droplets, the major fat storing structures in adipocytes (fat cells). Activities for 2014 include study of a perilipin binding protein, CGI-58. CGI-58 is a co-activator of adipose triglyceride lipase (ATGL), a major adipose lipase. Under basal conditions when adipocytes primarily synthesize and store triglycerides, CGI-58 binds to an amino acid sequence in the carboxyl terminus of perilipin; perilipin, in turn, is bound to lipid droplets. From this location, CGI-58 cannot interact with ATGL, and hence cannot co-activate lipolysis; this serves to keep the rate of lipolysis low. When lipolysis is triggered by catecholamine binding to beta-adrenergic receptors, intracellular levels of cAMP rise, activating protein kinase A (PKA). PKA then phosphorylates perilipin on multiple serine residues; one consequence of perilipin phosphorylation is the release of CGI-58 into the cytoplasm. The released CGI-58 can then interact with and co-activate ATGL, and lipolysis of triglyceride stores ensues. For the past several years, we have studied the PKA-mediated phosphorylation of CGI-58 on serine 239. Working with collaborating scientists in Austria, we have completed this project. We have demonstrated that phosphorylation of serine 239 of (mouse) CGI-58 is important for release of CGI-58 from the perilipin scaffold during the stimulation of lipolysis. This phosphorylation neither increases nor impairs the interaction of CGI-58 with ATGL, or co-activation of lipolysis. A manuscript describing this work was submitted to the Journal of Lipid Research, and accepted for publication; it is now available online in pre-print form, and will be published in 2015. Additionally, we have continued to characterize a mutation of CGI-58 (His82Arg) that is responsible for a neutral lipid storage disorder in humans. We have found that the mutation blocks the co-activation of ATGL in vitro, but does not impair the physical interaction of mutated CGI-58 with ATGL, or the recruitment of these proteins to lipid droplets. Changes in knowledge for 2014 include new discoveries that the protein kinase A-mediated phosphorylation of serine 239 of mouse CGI-58 is important for efficient release from a binding site on perilipin 1. This adds to prior knowledge that phosphorylation of serine residues at positions 492 and 517 of mouse perilipin 1 facilitate the release of CGI-58 from lipid droplets into the cytoplasm. This release of CGI-58 enables interaction of CGI-58 with ATGL and the activation of lipolysis. The ultimate outcome of increasing adipose lipolysis is to release fatty acids into circulation for uptake by peripheral tissues for metabolism to drive ATP synthesis. These findings increase the knowledge of basic scientists regarding the biology and function of adipocytes, and the control of whole body energy homeostasis in animals. Maintaining balance in energy metabolism is critical to avoid pathological complications of obesity and insulin resistance.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
McMahon, D., Dinh, A., Kurz, D., Shah, D., Han, G.-S., Carman, G. M., and Brasaemle, D. L. 2014. Comparative Gene Identification 58 (CGI-58)/Alpha Beta Hydrolase Domain 5 (ABHD5) lacks acyltransferase activity. J. Lipid Res. 55, 1750-1760. DOI:10.1194/jlrM051151.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Brasaemle, D. Control of Lipolysis by Perilipin and its Binding Partners. Annual Meeting of the Lipid MAPS Consortium. May 13-14, 2014. San Diego, CA, 2014.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Brasaemle, D. Control of Lipolysis by Perilipin and its Binding Partners. FASEB Summer Research Conference , Lipid Droplets: Metabolic Consequences of the Storage of Neutral Lipids. July 27-Augst 1, 2014. Saxtons River, VT. 2014
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience: Scientists within the international biomedical research community Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Services for 2013 included training of 2 undergraduate students and 3 graduate students. How have the results been disseminated to communities of interest? Events/Dissemination for 2013 included presentation of seminars at 1 conference and 2 universities, including the Southeast Regional Lipid Conference in Callaway Gardens, GA, the Department of Biology at the University of Rochester, Rochester, NY and the Department of Chemistry and Cell Biology at the University of Utrecht in the Netherlands. These presentations disseminated knowledge through lectures accompanied by Powerpoint slides to students and researchers within the science community to advance understanding of the function of perilipin in the control of lipid metabolism in adipocytes. What do you plan to do during the next reporting period to accomplish the goals? We are working to complete additional experiments to respond to the review of a submitted manuscript on CGI-58 function; we anticipate that this manuscript will be re-reviewed and published in the first half of 2013. We are writing an additional manuscript on the phosphorylation of CGI-58 that will be submitted in the first half of 2013. We will then write a manuscript on the phosphorylation of serine 492 of perilipin for submission in the second half of 2013. We will continue experiments to study the H84R mutation of CGI-58 and the role of phosphorylation of serine 517 of perilipin in lipid droplet targeting and control of lipolysis.
Impacts
What was accomplished under these goals?
Activities for 2013 include continuation of a project investigating the role of serine 517 in perilipin targeting to lipid droplets and regulation of lipolysis. Adenoviral vectors for expression of 12 mutations of serine 517 of perilipin have been transduced into cells. Data to date show that unmodified perilipin, and perilipin with glutamate or lysine substitutions for serine 517 targets to lipid droplets. In contrast, perilipin with an alanine substitution for serine 517 fails to target to lipid droplets, but the addition of a FLAG epitope tag (DYKDDDDK) immediately following alanine at 517 rescues lipid droplet targeting, suggesting that polar or charged residues are required at the carboxyl terminus of perilipin A. Additionally, preliminary data suggest that perilipin with methionine or cysteine substitution for serine 517 target to lipid droplets, at least in some cells; these experiments will be repeated to determine consistency and establish quantitative data. Phosphorylation of serine 517 is proposed to be a critical regulator of lipolysis, although the mechanism for this effect is unknown; future studies will test this hypothesis using these targeting mutations of perilipin. Another project investigates the role of serine 492 in perilipin function, particularly lipolysis. Significant progress was made during 2013 to show that the phosphorylation of serine 492 is required for maximal hormonally stimulated lipolysis in cells expressing ectopic adipose triglyceride lipase (ATGL) with or without the co-expression of ectopic CGI-58, a co-activator of ATGL. Additionally, since the same observation was made in cells expressing only as yet uncharacterized endogenous neutral lipid lipases, the mechanism by which phosphorylation of serine 492 facilitates lipolysis is a general mechanism, and not specific to the release of CGI-58 binding from the perilipin scaffold to activate ATGL. A final project has been investigating a novel mutation in CGI-58 (His84Arg) that causes neutral lipid storage disorder in humans. We recently showed that this mutation does not reduce protein-protein interactions between ATGL and CGI-58, although it does impede activation of ATGL. The mutation does not reduce CGI-58 binding to perilipin.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Brasaemle, D. L. 2013. Perilipin 5: Putting the brakes on lipolysis. J. Lipid Res., 54:876-877. DOI:10.1194/jlrE036962
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: Activities for 2011/2012 include continuation of a project investigating the role of serine 517 in perilipin targeting to lipid droplets and regulation of lipolysis. Adenoviral vectors have been made for expression of 12 mutations of serine 517 of perilipin and are now being transduced into cells. The titer of adenovirus required for expression of equivalent levels of perilipin has been determined for most mutated variants by extracting RNA and using reverse transcriptase with quantitative PCR to determine the levels of mRNA. A preliminary study investigated the association of 5 of these variants with lipid droplets in cultured fibroblasts; unmodified perilipin, and perilipin with glutamate or lysine substitutions for serine 517 targeted to lipid droplets. In contrast, perilipin with an alanine substitution for serine 517 failed to target to lipid droplets, but the addition of a FLAG epitope tag (DYKDDDDK) immediately following alanine at 517 rescued lipid droplet targeting, suggesting that polar or charged residues are required at the carboxyl terminus of perilipin A. Phosphorylation of serine 517 is proposed to be a critical regulator of lipolysis, although the mechanism for this effect is unknown; future studies will test this hypothesis further. Another project investigates the role of serine 492 in perilipin function, particularly lipolysis. We have made and purified an adenoviral expression vector for adipose triglyceride lipase (ATGL), in preparation for studying the interaction of ATGL with perilipin and the effect of mutation of serine 492 on this interaction. Additionally, adenovirus for hormone-sensitive lipase (HSL) has been expanded for purification of virus to use in experiments. A final project has been investigating the activity of CGI-58 in co-activating adipose triglyceride lipase using an in vitro approach with purified recombinant protein. We have now shown that CGI-58 is not a lysophosphatidic acid acyltransferase as previously reported and that CGI-58 binds phosphorylated phosphoinositides. Events/Dissemination for 2011 included presentation of seminars at 3 international conferences and 3 universities, including the Keystone Symposium on "Genetic and Molecular Basis of Obesity and Body Weight Regulation" in Santa Fe, NM; the American Society of Biochemistry and Molecular Biology session on "Lipid Droplets: A Dynamic Subcellular Compartment" at Experimental Biology 2012, in San Diego, CA; the 4th LipidomicNet Annual Steering Committee Meeting in Regensburg, Germany; the Diabetes Research Center, University of Pennsylvania, Philadelphia, PA; the Department of Biomedical Engineering, Rutgers University, New Brunswick, NJ; and the Department of Biochemistry & Molecular Biology, Mayo Clinic College of Medicine, Scottsdale, AZ. These presentations disseminated knowledge through lectures accompanied by Powerpoint slides to students and researchers within the science community to advance understanding of the function of perilipin in the control of lipid metabolism in adipocytes. Services for 2011/2012 included training of 2 undergraduate students and 3 graduate students. Products for 2011/2012 included 3 manuscripts. PARTICIPANTS: Participants: PI: Dawn L. Brasaemle, Ph.D. Graduate Student: Derek McMahon Graduate Student: Dana Lee Daley Graduate Student: Mayda Hernandez Laboratory Researcher: Anna Dinh Undergraduate Student: Daniel Kurz Undergraduate Student: Daniel Mascarenhas Collaborators: Judith Storch, Ph.D., Rutgers George Carman, Ph.D., Rutgers Martin Yarmush, Ph.D., Rutgers J. Mark Brown, Ph.D., Wake Forest University, Winston-Salem, NC Nathan E. Wolins, Ph.D., Washington University School of Medicine, St. Louis, MO Ruth Birner-Gruenberger, Ph.D., Medical University of Graz, Graz, Austria TARGET AUDIENCES: Scientists within the international biomedical research community PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Changes in knowledge for 2011/2012 included new discoveries that CGI-58 is not a lysophosphatidic acid acyltransferase, as previously reported, and binds phosphorylated phosphatidyl inositols (PIPs) including PI(3)P and PI(5)P, but not PI(4)P or PI(4,5)P; the latter observations have been made using blots containing various species of lipids. PIPs are essential for a number of cellular functions including transduction of signals between proteins and targeting of proteins to specific membrane compartments. Thus, PIP binding may explain the role of CGI-58 in co-activating adipose triglyceride lipase (ATGL), perhaps by promoting either the interaction between the 2 proteins, or the targeting of the 2-protein complex to lipid droplets, where ATGL encounters its lipid substrates. Further testing established that when CGI-58 binds PIPs, it does not alter the co-activation of ATGL, so it is more likely that PIP binding influences subcellular localization of CGI-58. Additional new findings identify lysine as a permissible amino acid in position 517 (the carboxyl terminal-most residue) of perilipin A. Thus, either negatively (glutamate) or positively (lysine) charged amino acids allow targeting of perilipin to lipid droplets, while uncharged, non-polar (alanine) residues disrupt this targeting. Targeting of perilipin to lipid droplets is essential to its function in control of lipolysis. These findings increase the knowledge of basic scientists regarding the basic biology and functioning of adipocytes (fat cells) and the control of energy metabolism in animals. Balance of energy metabolism is critical to avoid pathological complications of obesity and insulin resistance, leading to diabetes. Changes in action for 2011: there were no significant changes in action for 2011/2012.
Publications
- Nativ, N. I., Maguire, T. J., Yarmush, G., Brasaemle, D. L., Henry, S. D., Guarrera, J. V., Berthiaume, F., and Yarmush, M. L. 2012. Liver defatting: an alternative approach to enable steatotic liver transplantation, Amer. J. Transplant., 12, 3176-3183. DOI:10.1111/j. 1600-6143.2012.04288.x
- Lord, C., Betters, J. L., Ivanova, P. T., Milne, S. B., Myers, D. S., Madenspacher, J., Chung S., Liu, M., Davis, M. A., Lee, R. G., Crooke, R. M., Graham, M. J., Parks, J. S., Brasaemle, D. L., Fessler, M. B., Brown, H. A., and Brown, J.M. 2012. CGI-58/ABHD5-derived signaling lipids regulate systemic inflammation and insulin action. Diabetes, 61, 355-363. DOI:10.2337/db11-0994.
- Brasaemle, D. L. and Wolins, N.E. 2012. Packaging of fat: an evolving model of lipid droplet assembly and expansion. J. Biol. Chem., 287, 2273-2279. DOI:10.1074/jbc.R111. 309088.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Activities for 2011 included a project investigating the role of serine 517 in perilipin targeting to lipid droplets and regulation of lipolysis. Adenoviral constructs have been made for expression of 12 mutations of serine 517 of perilipin and are now being transduced into cells. The titer of adenovirus required for expression of equivalent levels of perilipin for all constructs is being determined by extracting RNA and using reverse transcriptase with quantitative PCR to determine the level of mRNA for each construct. Once the appropriate amount of each virus has been determined, studies using the constructs will investigate the function of this residue. Phosphorylation of serine 517 is proposed to be a critical regulator of lipolysis, although the mechanism for this effect is unknown. Another project investigates the role of serine 492 in perilipin function, particularly lipolysis. We have constructed an adenoviral expression vector for the lipase, adipose triglyceride lipase (ATGL), in preparation for studying the interaction of ATGL with perilipin and the effect of mutation of serine 492 on this interaction. An additional component of this project is to study the interaction of the ATGL co-activator CGI-58 with perilipin containing mutations of serine 492. Serine 492 is very near to the binding site for CGI-58 on perilipin and may thus modulate this binding activity, and in doing so, alter the availability of CGI-58 to interact with ATGL. A collaborative project with Carole Sztalryd of the University of Maryland has studied the role of perilipin 5 in the control of lipolysis; a manuscript describing these studies was published in 2011. A final project has been investigating the activity of CGI-58 in co-activating adipose triglyceride lipase using an in vitro approach with purified recombinant protein. In 2011, this project expanded into an investigation of lipid binding activity of CGI-58 focusing on studying how lipid binding activity affects CGI-58's activity as a co-activator of ATGL. Events/Dissemination for 2011 included presentation (dissemination) of seminars at 5 international conferences and 2 universities, including the Diabetes Research Center, Albert Einstein College of Medicine, Bronx, NY; Department of Nutritional Sciences, Rutgers University, New Brunswick, NJ; 1st Annual Retreat for Review of the International PhD Program Metabolic and Cardiovascular Disease, Graz, Austria; 71st Scientific Sessions of the American Diabetes Association, San Diego, CA; Steenbock Symposium on Lipid Metabolism: Implications in Human Disease, University of Wisconsin-Madison, Madison, WI; Keystone Symposium on Lipid Biology and Lipotoxicity, Killarney, County Kerry, Ireland; and the New York Lipid and Vascular Biology Club, Rockefeller University, New York, NY. These presentations disseminated knowledge through lectures accompanied by Powerpoint slides to students and researchers within the science community to advance understanding of the function of perilipin in the control of lipid metabolism in adipocytes. Services for 2011 included training of 2 undergraduate students and 3 graduate students. Products for 2011 included 4 manuscripts. PARTICIPANTS: Participants: PI: Dawn L. Brasaemle, Ph.D. Graduate Student: Derek McMahon Graduate Student: Dana Lee Daley Graduate Student: Mayda Hernandez Laboratory Researcher: Anna Dinh Undergraduate Student: Daniel Kurz Undergraduate Student: Daniel Mascarenhas Collaborators: Judith Storch, Ph.D., Rutgers George Carman, Ph.D., Rutgers Carole Sztalryd, Ph.D., University of Maryland School of Medicine, Baltimore, MD TARGET AUDIENCES: Target Audience: Scientists within the international biomedical research community PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Changes in knowledge for 2011 included new discoveries that CGI-58 avidly binds phosphorylated phosphatidyl inositols (PIPs) including PI(3)P and PI(5)P, but not PI(4)P or PI(4,5)P; these observations have been made using blots containing various species of lipids. Currently, tryptophan quenching experiments are being used to confirm these observations and to determine the affinity of binding. CGI-58 has a very strong signal for tryptophan fluorescence due to the presence of 9 tryptophan residues in the protein; the signal is quenched when PI(3)P binds to it. Lipid binding occurs in a 1:1 molar ratio. PIPs are essential for a number of cellular functions including transduction of signals between proteins and targeting of proteins to specific membrane compartments. Thus, PIP binding may explain the role of CGI-58 in co-activating adipose triglyceride lipase (ATGL), perhaps by promoting either the interaction between the 2 proteins, or the targeting of the 2-protein complex to lipid droplets, where ATGL encounters its lipid substrates. These findings increase the knowledge of basic scientists regarding the basic biology and functioning of adipocytes (fat cells) and the control of energy metabolism in animals. Balance of energy metabolism is critical to avoid pathological complications of obesity and insulin resistance, leading to diabetes. Changes in action for 2011: there were no significant changes in action for 2011.
Publications
- Caviglia, J. M., Betters, J.L., Dapito, D. H., Lord, C.C., Sullivan, S. P., Chua, S., Sekowski, A., Yin, T., Mu, H., Shapiro, L., Brown, J.M., and Brasaemle, D. L. 2011. Adipose-selective overexpression of CGI-58 does not increase lipolysis or protect against diet-induced obesity. J. Lipid Res. 52, 2032-2042. DOI:10.1194/jlr.M019117.
- Brasaemle, D. L. 2011. DisseCCTing phospholipid function in lipid droplet dynamics. Cell Metab., 14, 437-438. DOI:10.1016/j.cmet.2011.09.002.
- Brasaemle, D.L. and Osborne, T.F. 2011. Chewing the fat: An expanding role for lipids in complex biological processes. ASBMB Today, October 2011 issue, 28-29.
- Wang, H., Bell, M., Sreenevasan, U., Hu, H., Liu, J., Dalen, K., Londos, C., Yamaguchi, T., Rizzo, M.A., Coleman, R. A., Gong, D., Brasaemle, D., and Sztalryd, C. 2011. Unique regulation of adipose triglyceride lipase (ATGL) by perilipin 5, a lipid droplet-associated protein. J. Biol. Chem. 286, 15707-15715. DOI:10.1074/jbc.M110.207779.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Activities for 2010 included a project on the role of serine 517 in perilipin targeting to lipid droplets and regulation of lipolysis. The project is at the stage of tool development. Adenoviral constructs have been made for expression of more than 12 mutations of serine 517, for testing in cells. Most of these adenoviral preparations have been amplified to the stage where they are ready to be transduced into cells; the remaining preparations are still undergoing amplification steps. In 2011, testing of the constructs in cells will begin. Phosphorylation of serine 517 has been proposed to be a critical regulator of lipolysis, although the mechanism for this effect is as yet unknown, and will be studied. Another project investigated the role of serine 492 in perilipin-mediated fragmentation and dispersion of lipid droplets following lipolytic stimulation of adipocytes. A high throughput assay was developed to investigate other genes involved in the process using siRNA technology. As a pilot project, 138 genes were investigated for participation in lipid droplet remodeling following phosphorylation of perilipin serine 492, which is required for maximal lipolysis. In 2011, the assay will be refined and functions of some of the 138 genes explored further. A collaborative project with Carole Sztalryd of the University of Maryland studied the role of perilipin 5 (OXPAT/MLDP5) in the control of lipolysis via interactions with adipose triglyceride lipase and CGI-58; a manuscript is currently under revision for re-submission in 2011. A final project has investigated acyl transferase activity of CGI-58 and the activation of adipose triglyceride lipase using an in vitro approach with purified recombinant proteins. Events/Dissemination for 2010 included presentation (dissemination) of the data obtained through our Hatch funded experiments in seminars presented at 5 different international conferences and invited lectureships including lectures at the Lipoprotein Metabolism Gordon Research Conference in Waterville Valley, NH in June, the 11th International Congress on Obesity in Stockholm, Sweden in July, the FASEB summer research conference on Lipid Droplets in Steamboat Springs, CO in July, the 2010 Scientific Sessions of the American Heart Association in Chicago, IL in November, and the Department of Medicine at the Wake Forest University School of Medicine in Winston-Salem, NC in December. These presentations disseminated knowledge through lectures accompanied by Powerpoint slides to students and researchers within the science community to advance understanding of the function of perilipin A in control of lipid metabolism in adipocytes. Services for 2010 included the training of 1 undergraduate student, and 2 graduate students, who were mentored and taught throughout the experiments that they performed. Products for 2010 included 3 manuscripts published as listed in Publications. PARTICIPANTS: Participants: PI: Dawn L. Brasaemle, Ph.D. Graduate Student: Derek McMahon Graduate Student: Danalee Daley Laboratory Researcher: Deanna Russell Laboratory Researcher: Anna Dinh Collaborators: Joseph Dixon, Ph.D., Rutgers Anita Brinker, Ph.D., Rutgers Judith Storch, Ph.D., Rutgers George Carman, Ph.D., Rutgers Carole Sztalryd, Ph.D., University of Maryland School of Medicine, Baltimore, MD TARGET AUDIENCES: Scientists within the international biomedical research community PROJECT MODIFICATIONS: Project Modifications: none
Impacts
Outcomes/Impact of the Project Changes in knowledge for 2010 included new discoveries that perilipin 5 recruits adipose triglyceride lipase to lipid droplets in non-adipose cells through a direct protein-protein interaction that reduces lipolysis. In contrast, interaction of adipose triglyceride lipase with CGI-58 increases lipolysis of stored triglyceride. Perilipin 5 was found to be a substrate for phosphorylation by protein kinase A, and the activation of protein kinase A increased lipolysis through an as yet uncharacterized mechanism. Finally, acyltransferase activity is not required for CGI-58 activation of adipose triglyceride lipase. These findings increase the knowledge of basic scientists regarding the control of energy metabolism in animals. Balance of energy metabolism is critical to avoid pathological complications of obesity and insulin resistance, leading to diabetes. Changes in action for 2010: there were no significant changes in action for 2010.
Publications
- Montero-Moran, G., Caviglia, J. M., McMahon, D., Subramanian, V., Rothenberg, A., Xu, Z., Lara-Gonzalez, S., Storch, J., Carman, G. M., and Brasaemle, D. L. 2010. CGI-58/ABHD5 is a Coenzyme A-dependent lysophosphatidic acid acyltransferase. J. Lipid Res. 51, 709-719. DOI:10.1194/jlrM001917.
- Kimmel, A. R., Brasaemle, D. L., McAndrews-Hill, M., Sztalryd, C., and Londos, C. 2009. Adoption of PERILIPIN as a unifying nomenclature for the mammalian PAT-family of intracellular lipid storage droplet proteins. J. Lipid Res., 51, 468-471. DOI:10.1194/jlr.R000034.
- Brasaemle, D. L. 2010. Lipolysis control: The plot thickens. Cell Metab. 11, 173-174. DOI:10.1016/j.cmet.2010.02.008.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Activities for 2009 included experiments to investigate the role of phosphorylation of perilipin A in lipolysis of triacylglycerols by hormone-sensitive lipase. We used truncated variants of perilipin and point mutations of serine residues in consensus sequences for protein kinase A (PKA) to identify the binding site for hormone-sensitive lipase (HSL) on PKA-phosphorylated perilipin A, and to study the effects of phosphorylated serine residues on lipase binding. We found that the 3 amino-terminal-most PKA sites must be phosphorylated on perilipin A to facilitate HSL binding to amino terminal sequences of perilipin between residues 1 and 121. This sequence of the protein is highly conserved in members of the perilipin protein family, including adipophilin, TIP47, and OXPAT; we demonstrated that this sequence in these other 3 proteins also binds hormone-sensitive lipase. Finally, we showed that binding of HSL to proteins in the perilipin family is not sufficient to increase lipolysis of triacylglycerols; PKA-mediated phosphorylation of HSL is required to activate the lipase. A second set of experiments identified and characterized the enzyme activity of a perilipin binding protein, CGI-58/ABHD5, as a Coenzyme A-dependent lysophosphatidic acid acyltransferase. Additional ongoing experiments investigate the role of PKA phosphorylation of CGI-58/ABHD5 in subcellular localization to lipid droplets, the role of amino acid 517 in perilipin subcellular localization and control of lipolysis, and the relationship between acyltransferase activity of CGI-58/ABHD5 and triacylglycerol homeostasis. Events/Dissemination for 2009 included presentation (dissemination) of the data obtained through our Hatch funded experiments in seminars presented at 7 conferences and universities including lectures at the Department of Physiology and Biophysics, Boston University School of Medicine in Boston, MS, the Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX, and the Department of Medical Sciences, University of Alberta, Edmonton, Alberta, and invited plenary lectures at the American Society of Biochemistry and Molecular Biology session on "Mechanisms for Lipid Storage and Transport" at Experimental Biology 2009, New Orleans, LA, the session on "Metabolism and Inflammation", at the 10th Annual Conference of Arteriosclerosis, Thrombosis and Vascular Biology Council of the American Heart Association, Washington, D.C., the conference on "Frontier Lipidology: Lipidomics in Health and Disease", Gothenburg, Sweden, and the 1st LipidomicNet Annual Steering Committee Meeting, Regensburg, Germany. These presentations disseminated knowledge through lectures accompanied by Powerpoint slides to researchers within the science community to advance understanding of the function of perilipin A in control of lipid metabolism in adipocytes. Services for 2009 included the training of 3 undergraduate students, 1 graduate student and a postdoctoral associate, who were mentored and taught throughout the experiments that they performed. Products for 2009 included 5 manuscripts published or accepted for publication and available online for 2009. PARTICIPANTS: Participants: PI: Dawn L. Brasaemle, Ph.D. Postdoctoral Associate: Gabriela Montero-Moran, Ph.D. Graduate Student: Derek McMahon Laboratory Researcher: Deanna Russell Collaborators: Joseph Dixon, Ph.D., Rutgers Anita Brinker, Ph.D., Rutgers Judith Storch, Ph.D., Rutgers George Carman, Ph.D., Rutgers Carole Sztalryd, Ph.D., University of Maryland School of Medicine, Baltimore, MD TARGET AUDIENCES: Scientists within the international biomedical research community PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Changes in knowledge for 2009 included new discoveries that the phosphorylation of serines 81, 222, and 276 of perilipin A is required for hormone-sensitive lipase binding to the amino terminus of perilipin and maximal stimulated lipolysis, and that increased lipolysis requires phosphorylation of hormone-sensitive lipase when the lipase binds to OXPAT on lipid droplets. Other findings included the identification of CGI-58/ABHD5 as a Coenzyme A-dependent lysophosphatidic acid acyltransferase, and the observation that CGI-58/ABHD5 is an important factor in the secretion of very low density lipoproteins from liver. Changes in action for 2009: there were no significant changes in action for 2009.
Publications
- Wang, H., Hu, L., Dalen, K., Dorward, H., Marcinkiewicz, A., Russell, D., Gong, D., Londos, C., Holm, C., Yamaguchi, T., Rizzo, M., Brasaemle, D. L., and Sztalryd, C. 2009. Activation of hormone-sensitive lipase requires two steps: protein phosphorylation and binding to the PAT-1 domain of lipid droplet coat proteins. J. Biol. Chem. 284, 32116-32125.
- Montero-Moran, G., Caviglia, J. M., McMahon, D., Subramanian, V., Rothenberg, A., Xu, Z., Lara-Gonzalez, S., Storch, J., Carman, G. M., and Brasaemle, D. L. 2009. CGI-58/ABHD5 is a Coenzyme A-dependent lysophosphatidic acid acyltransferase. In press, J. Lipid Res. Available online on October 2, 2009. DOI:10.1194/jlrM001917.
- Kimmel, A. R., Brasaemle, D. L., McAndrews-Hill, M., Sztalryd, C., and Londos, C. 2009. Adoption of PERILIPIN as a unifying nomenclature for the mammalian PAT-family of intracellular lipid storage droplet proteins. In press, available online J. Lipid Res., July 28, 2009 DOI:10.1194/jlr.R000034.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Activities for 2008 included experiments to investigate the role of phosphorylation of perilipin A in facilitation of lipolysis subsequent to remodeling of lipid droplets. Specifically, we used adenoviral expression vectors for four mutated forms of perilipin A to study the role of phosphorylation of serine 492 in facilitating lipolysis, including 1) a form of perilipin A with 5 out of 6 phosphorylation sites mutated so that only serine 492 can be phosphorylated, 2) a form of perilipin A that can be mutated on all 5 of the other sites but not serine 492, and 3) a form of perilipin A that cannot be phosphorylated. All mutated forms of perilipin were expressed in NIH 3T3 mouse fibroblasts and release of radiolabeled fatty acids was used to measure rates of lipolysis; cells expressing mutated forms of perilipin were compared to cells with unmodified ectopic perilipin (positive control) or Beta-galactosidase (negative control where lipid droplets are coated with adipophilin). A second line of experimentation has investigated the effects of perilipin phosphorylation on binding of a lipolytic factor called CGI-58, and mutated forms of CGI-58 to perilipin A. A third line of experimentation involved preparing a series of mutations in the final amino acid in perilipin A (aa 517) to study targeting and effects on lipolysis of this residue; we have now prepared expression vectors for 8 mutated variants of perilipin A for these experiments. Events/Dissemination for 2008 included presentation (dissemination) of the data obtained through our Hatch funded experiments in seminars presented at 10 different international conferences and invited lectureships including the Keystone Symposium on Molecular Control of Adipogenesis and Obesity, Banff, Alberta, Canada; the 2nd International Symposium on Lipid and Membrane Biology, Graz, Austria; Medical Diagnostic Laboratories, Hamilton, NJ; the Lipoprotein Metabolism Gordon Research Conference, Waterville Valley NH; the 2008 Kern Aspen Lipid Conference, Given Institute, Aspen CO; the 49th International Conference on the Biosciences of Lipids, Maastricht, The Netherlands; the South East Lipid Research Conference, Pine Mountain, GA; the Fall Research Symposium for recipients of Established Investigator Award, American Heart Association, New Orleans, LA; the 10th Annual Frontiers in Diabetes Research: Adipocyte Biology in Obesity and Diabetes, Naomi Berrie Diabetes Center, Columbia University; and the Gladstone Institute for Cardiovascular Disease, University of California, San Francisco, CA. These presentations disseminated knowledge through lectures accompanied by Powerpoint slides to students and researchers within the science community to advance understanding of the function of perilipin A in control of lipid metabolism in adipocytes. Services for 2008 included the training of 2 graduate students and a postdoctoral associate, who were mentored and taught through experiments that they performed. Products for 2008 included 2 full-length publications and an abstract. PARTICIPANTS: Participants: PI: Dawn L. Brasaemle, Ph.D. Postdoctoral Associate: Gabriela Montero-Moran, Ph.D. Graduate Student: Devon Golem Graduate Student: Xiaofang Liang Laboratory Researcher: Deanna Russell Laboratory Researcher: Sean Sullivan Collaborators: Joseph Dixon, Rutgers Anita Brinker, Rutgers TARGET AUDIENCES: Scientists within the international biomedical research community PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Outcomes/Impact of the Project Changes in knowledge for 2008 included new discoveries that the phosphorylation of serine 492 of perilipin A is required for maximal stimulated lipolysis. Additionally, phosphorylation of one or more of the other 5 sites is also essential for maximal lipolysis. In a second line of experimentation, we learned that the release of CGI-58 from perilipin A at the surfaces of lipid droplets depends upon the phosphorylation of CGI-58 by protein kinase A. Even when perilipin A is phosphorylated, if CGI-58 cannot also be phosphorylated, CGI-58 is not released into the cytoplasm. We are now testing the hypothesis that the subcellular location of CGI-58 is important for its function in facilitating lipolysis. Changes in action for 2008: there were no significant changes in action for 2008.
Publications
- Caviglia, J. M., Sparks, J., Toraskar, N., Brinker, A., Yin, T. C., Dixon, J., and Brasaemle, D. L. 2009. ABHD5/CGI58 facilitates the assembly and secretion of apolipoprotein B-lipoproteins by McA RH7777 rat hepatoma cells. In press, Biochim. Biophys. Acta-Molec. and Cell Biol. Lipids. Available online through: DOI:10.1016/j.bbalip.208.12.018
- Brasaemle, D. L., Subramanian, V., Garcia, A., Marcinkiewicz, A., and Rothenberg, A. 2009. Perilipin A and the Control of Lipolysis. In press, Molecular and Cellular Biochemistry. Available online through: DOI:10.1007/s1 1010-008-9998-8
- Brasaemle, D. L., Montero-Moran, G., Caviglia, J. M., McMahon, D., Liang, X., Subramanian, V., Dapito, D. H., and Sullivan, S. 2008. Lipid droplet associated proteins and control of triacylglycerol metabolism. Chem. Phys. Lipids 154, supplement: S4
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Activities for 2007 included experiments to investigate the role of phosphorylation of perilipin A in facilitation of lipolysis subsequent to remodeling of lipid droplets. Specifically, we completed the construction of viral expression vectors to study the role of phosphorylation of serine 492 including 1) a form of perilipin A with 5 out of 6 phosphorylation sites mutated so that only serine 492 can be phosphorylated, 2) a form of perilipin A that can be mutated on all 5 of the other sites but not serine 492, and 3) a form of perilipin A that cannot be phosphorylated. Preliminary tests of these expression vectors were conducted and the basic experimental design to study the effect of these forms of perilipin A on hormonally stimulated lipolysis were executed. A second line of experimental activities included use of a series of 6 synthetic peptides of 10 amino acids which contain the consensus sequences for phosphorylation to study the kinetics of phosphorylation. Larger
recombinant peptides of perilipin A with combinations of mutations of the phosphorylation sites were prepared for further study of the specificity and kinetics of the phosphorylation reaction. A third line of experimentation has investigated the effects of perilipin phosphorylation on binding of a lipolytic factor called CGI-58, and mutated forms of CGI-58 to perilipin A. A fourth line of experimentation involved preparing a series of mutations in the final amino acid in perilipin A (aa 517) to study targeting and effects on lipolysis of this residue. Events for 2007 included presentation (dissemination) of the data obtained through our Hatch funded experiments in seminars presented at 7 different international conferences and invited lectureships including the 6th International Conference on Lipid Binding Proteins at Simon Fraser University in Burnaby, British Columbia; the Molecular Membrane Biology Gordon Research Conference in Andover, NH; the Johonson & Johnson Skin Research
Center in Skillman, NJ; the Department of Biochemistry at the University of Medicine and Dentistry of New Jersey in Piscataway, NJ; the Children's Hospital Oakland Research Institute in Oakland, CA; the 2007 Annual Meeting of the Obesity Society in New Orleans, LA; and the December meeting of the New York Lipid and Vascular Biology Club at Rockefeller University, New York, NY. These lectures (with Powerpoint presentations) disseminated knowledge to the science community to advance understanding of the function of perilipin A in control of lipid metabolism in adipocytes. Services for 2007 included the training of 2 graduate students, a postdoctoral associate, and an undergraduate student who were mentored and taught throughout the experiments that they performed. Products for 2007 included the publication of a review article (listed in Publications) summarizing our own work in the context of the growing field of lipid droplet biology as part of the thematic review series on adipocyte
biology in the Journal of Lipid Research.
PARTICIPANTS: PI: Dawn L. Brasaemle, Ph.D. Postdoctoral Associate: Gabriela Montero-Moran, Ph.D. Graduate Student: Devon Golem Graduate Student: Xiaofang Liang Undergraduate student: Amanda Smith Laboratory Researcher: Deanna Russell Laboratory Researcher: Sean Sullivan
TARGET AUDIENCES: Scientists within the international biomedical research community
Impacts
Changes in knowledge for 2007 included new discoveries that the 6 phosphorylation sites of perilipin A are not equivalent substrates for cAMP-dependent protein kinase (PKA). For example, serine 517, the final amino acid in perilipin A is a relatively poor substrate for phosphorylation despite a current hypothesis that phosphorylation of this site is required for the regulation of lipolysis. By contrast serine 433 is a very good substrate. The remaining 4 sites are intermediate substrates. In a second line of experimentation, we learned that the binding of CGI-58 to perilipin A depends upon the status of CGI-58 phosphorylation by PKA. Even when perilipin A is phosphorylated, if CGI-58 cannot also be phosphorylated, CGI-58 remains bound to perilipin A on lipid droplets. We are now testing the hypothesis that the subcellular location of CGI-58 is important for its function in facilitating lipolysis. Changes in action for 2007 included a redesigning of a key experiment to
study lipolysis in cultured fibroblasts expressing ectopic perilipin A. Previously, we had assessed glycerol secretion as a measure of lipolysis and observed very small changes (10-20% increase) in release of glycerol when perilipin A is fully phosphorylated. A new series of experiments revealed greater changes in release of fatty acids, and assessment of fatty acids was begun as a measure of lipolytic activity. Phosphorylation of perilipin A promotes a 3- to 4-fold increase in the release of fatty acids into the culture medium; hence, much more significant changes can be measured. We have incorporated these changes in procedure into our new experimental design.
Publications
- Brasaemle, D. L. 2007. The perilipin family of structural lipid droplet proteins: Stabilization of lipid droplets and control of lipolysis. J. Lipid Res. 48:2547-2559.
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Progress 01/01/06 to 12/31/06
Outputs
Progress for 2006: In 2006, we have published a peer-reviewed paper related to the project. We also co-authored a review article and 2 short reviews with commentaries related to the project. Research findings published in 2006 include the observation that the phosphorylation of perilipin A on serine 492 drives the fragmentation and dispersion of lipid droplets in response to the stimulation of lipolysis in adipocytes. The activation of protein kinase A promotes the phosphorylation of perilipin A on as many as 6 serines scattered throughout the protein. Serine to alanine and serine to glutamic acid mutations of site #5 blocks this remodeling of lipid droplets; in contrast, mutations of sites 1, 2, 3, 4, and 6 either singly, or in combinations, has no effect on lipid droplet remodeling. New unpublished findings show that mutation of serine 492 at site #5 to alanine to block phosphorylation does not significantly impede lipolysis, suggesting that phosphorylation of the
other 5 sites may play a more important role in facilitating lipolysis. We are now making mutated forms of perilipin that can be phosphorylated on only site#5, to determine if this site plays any role in assisting lipolysis. Additionally, we have begun phosphopeptide mapping of phosphorylated perilipin A to determine which of the 6 sites is phosphorylated by protein kinase A.
Impacts
Our current research has added to our model of how the phosphorylation of perilipin A by protein kinase A works to control the lipolysis of triglycerides stored in adipocytes. There are 6 serines that may be phosphorylated by protein kinase A, and evidence from this project, and from other groups, suggest that the phosphorylation of different sites plays different roles in facilitating lipolysis. This project has begun to unravel the effects of phosphorylation of one of these serines, serine 492. We hypothesize that the massive remodeling of lipid droplets that accompanies the phosphorylation of serine 492 plays an important role in increasing the surface area of lipid droplets that is available to cytosolic lipases, leading to more extensive lipolysis. This research will contribute to a greater understanding of mechanisms that contribute to obesity.
Publications
- Marcinkiewicz, A., Gauthier, D., Garcia, A., and Brasaemle, D. L. 2006. Phosphorylation of serine 492 of perilipin A directs lipid droplet fragmentation and dispersion. J. Biol. Chem., 281, 11901-11909.
- Wolins, N. E., Brasaemle, D. L., Bickel, P. E. 2006. A proposed model of fat packaging by exchangeable lipid droplet proteins. FEBS Lett. 580, 5484-5491.
- Brasaemle, D. L. 2006. A Metabolic Push to Proliferate. Science, 313, 1581-1582.
- Brasaemle, D. L., and Hansen, J. C. 2006. Developmental Biology: Holding Pattern for Histones. Current Biology,16:R918-R920.
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Progress 01/01/05 to 12/31/05
Outputs
Research findings for 2005 that were compiled into a recently submitted manuscript included the findings that the phosphorylation of perilipin A on serine 492 drives the fragmentation and dispersion of lipid droplets in response to the stimulation of lipolysis in adipocytes. The activation of protein kinase A promotes the phosphorylation of perilipin A on as many as 6 serines scattered throughout the protein. Serine to alanine and serine to glutamic acid mutations of site #5 blocks this remodeling of lipid droplets; in contrast, mutations of sites 1, 2, 3, 4, and 6 either singly, or in combinations, has no effect on lipid droplet remodeling. Furthermore, during this remodeling, perilipin A remains associated with buoyant micro-lipid droplets, and does not disperse into the cytosol, as others have reported. We are now working to correlate this remodeling process to changes in triglyceride hydrolysis.
Impacts
Our current research has added to our model of how the phosphorylation of perilipin A by protein kinase A works to control the lipolysis of triglycerides stored in adipocytes. There are 6 serines that may be phosphorylated by protein kinase A, and evidence from this project, and from other groups, suggest that the phosphorylation of different sites plays different roles in facilitating lipolysis. This project has begun to unravel the effects of phosphorylation of one of these serines, serine 492. We hypothesize that the massive remodeling of lipid droplets that accompanies the phosphorylation of serine 492 plays an important role in increasing the surface area of lipid droplets that is available to cytosolic lipases, leading to more extensive lipolysis. This research will contribute to a greater understanding of mechanisms that contribute to obesity.
Publications
- Cohen, A. W., Schubert, W., Brasaemle, D. L., Scherer, P. E., and Lisanti, M. P. 2005. Caveolin-1 expression is essential for proper non-shivering thermogenesis in brown adipose tissue. Diabetes, 54, 679-686.
- Capozza, F., Combs, T., Cohen, A., Cho, Y. R., Park, S. Y., Schubert, W., Williams, T., Brasaemle, D. L., Jelicks, L., Scherer, P., Kim, J., and Lisanti, M. 2005. Caveolin-3 knockout mice show increased adiposity and whole-body insulin resistance, with ligand-induced insulin receptor instability in skeletal muscle. Am. J. Physiol. Cell Physiol. 288, C1317-1331.
- Brasaemle, D. L., and Wolins, N. E. 2005. Isolation of Lipid Droplets. Methods Chapter for Current Protocols in Cell Biology. Editors Juan S. Bonifacino, Mary Dasso, Joe B. Harford, Jennifer Lippincott-Schwartz, and Kenneth M. Yamada, Publisher John Wiley & Sons, Inc. Supplement 29, Unit 3.15; pages 3.15.1-3.15.12.
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Progress 01/01/04 to 12/31/04
Outputs
The newly published work establishes that 1) the amino and carboxyl termini of perilipin A serve a critical role in the function of perilipin A to protect stored triacylglycerols from lipolysis by cytosolic lipases, 2) that perilipin A serves to recruit caveolin-1 to lipid droplets in adipocytes stimulated with beta-adrenergic agonists, and that this complex is required to bring the catalytic sub-unit of protein kinase A to perilipin so that perilipin can be phosphorylated and promote lipolysis, 3) that hydrophobic sequences in the central region of perilipin A serve to target and anchor perilipin to lipid droplets, 4) that a 48-amino acid residue on perilipin A recruits a protein named CGI-58 to the lipid droplet, where it serves an as yet uncharacterized role in triacylglycerol catabolism, and 5) that adipocyte lipid droplets contain numerous proteins other than perilipin A and that the protein content changes during beta-adrenergic stimulation of adipocytes. During
2004, we have brought one additional project to a close and are now writing a manuscript showing the function of the the phosphorylation of serine 492 on perilipin in the fragmentation and dispersion of lipid droplets that follows the beta-adrenergic stimulation of adipocytes.
Impacts
Our current research has added to our model of how perilipin A works in adipocytes to control fat storage and lipolysis. Specifically, we have homed in on specific sequences of amino acids that 1) specify lipid droplet targeting and anchoring into the hydrophobic environment of the lipid droplet, and 2) control how much lipid an adipocyte stores in its lipid droplets. We have begun to unravel the complex process by which perilipins at the surface control how much energy is released from the adipocyte at times of need.
Publications
- Garcia, A., Subramanian, V., Sekowski, A., Love, M., and Brasaemle, D. L. 2004. The amino and carboxyl termini of perilipin A facilitate the storage of triacylglycerol. J. Biol. Chem. 279, 8409-8416.
- Cohen, A. W., Razani, B., Schubert, W., Williams, T. M., Wang, X. B., Iyengar, P., Brasaemle, D. L., Scherer, P. E., and Lisanti, M. P. 2004. Role of Caveolin-1 in the Regulation of Lipolysis and Lipid Droplet Formation. Diabetes 53, 1261-1270.
- Subramanian, V., Rothenberg, A., Gomez, C., Cohen, A. W., Garcia, A., Bhattacharyya, S., Shapiro, L., Dolios, G., Wang, R., Lisanti, M. P., and Brasaemle, D. L. 2004. Perilipin A mediates the reversible binding of CGI-58 to lipid droplets in 3T3-L1 adipocytes. J. Biol. Chem. 279, 42062-42071.
- Subramanian, V., Garcia, A., Sekowski, A., and Brasaemle, D. L. 2004. Hydrophobic sequences target and anchor perilipin A to lipid droplets. J. Lipid Res. 45, 1983-1991.
- Brasaemle, D. L., Dolios, G., Shapiro, L., and Wang, R. 2004. Proteomic analysis of proteins associated with lipid droplets in basal and lipolytically-stimulated 3T3-L1 adipocytes. J. Biol. Chem., 279, 46835-46842.
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Progress 01/01/03 to 12/31/03
Outputs
During 2003, we have published 4 papers resulting from the project; two of these papers were in press and available in an online format at the end of the previous reporting period, both of these papers are now available in journal format; one of these papers is now in press and available online. We have also published a collaborative paper on a related topic. The newly published work establishes that 1) the central domain of perilipin A contains multiple partially redundant sequence motifs that target and anchor perilipin A to lipid droplets, 2) the amino and carboxyl termini of perilipin A are essential to the function of perilipin A in protecting stored triacylglycerols from lipolysis by cytosolic lipases, 3) the phosphorylation of perilipin A facilitates stimulated lipolysis, allowing soluble lipases to gain contact with stored triacylglycerols, and 4) some classic fixatives used in morphological studies of cells damage lipid droplets leading to artefactual imaging
of surface proteins and stored lipids; paraformaldehyde fixatives are preferable to preserve lipid droplet structure. During the past year, we have brought several additional projects to a close, and are now writing additional manuscripts including: 1) a manuscript identifying the roles of the specific sequence elements in the central domain of perilipin A to lipid droplet targeting, and 2) a manuscript describing the role of the phosphorylation of serine 492 of perilipin A in lipid droplet remodeling that accompanies lipolysis.
Impacts
Our current research has added to our model of how perilipin A works in adipocytes to control fat storage and lipolysis. Specifically, we have identified sequences that 1) specify lipid droplet targeting, and embed the protein into the hydrophobic environment of the lipid droplet, and 2) serve critical roles in controlling how much lipid an adipocyte stores in its lipid droplets. Also, we have begun to unravel a very complex process by which perilipins at the surface of lipid droplets control how much energy is released from the adipocyte at times of need.
Publications
- Garcia, A., Sekowski, A., Subramanian, V., and Brasaemle, D. L. 2003. The central domain is required to target and anchor perilipin A to lipid droplets. J. Biol. Chem., 278:625-635.
- Tansey, J. T., Huml, A. M., Vogt, R., Davis, K. E., Jones, J. M., Fraser, K. A., Brasaemle, D. L., Kimmel, A. R., and Londos, C. 2003. Functional studies on native and mutated forms of perilipins: A role in protein kinase A-activated lipolysis of triacylglcyerols in CHO cells. J. Biol. Chem., 278:8401-8406.
- DiDonato, D., and Brasaemle, D. L. 2003. Fixation methods for the visualization of lipid droplets by immunofluorescence microscopy. J. Histochem. Cytochem., 51:773-780.
- Moreno, D. A., Ilic, N., Poulev, A. Brasaemle, D. L., Fried, S. K., and Raskin, I. 2003. Inhibitory effects of grape seed extract on lipases. Nutrition, The International Journal of Applied and Basic Nutritional Sciences 19:876-879.
- Garcia, A., Subramanian, V., Sekowski, A., Love, M., and Brasaemle, D. L. 2003. The amino and carboxyl termini of perilipin A facilitate the storage of triacylglycerol. In press, and available online: J. Biol. Chem. M311198200, available Nov. 10, 2003.
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Progress 01/01/02 to 12/31/02
Outputs
We have now published our first paper describing the role of the central domain of perilipin A in directing the targeting and anchoring of perilipins to lipid droplets. We are proceeding in three directions: 1) refining our study of elements of the central domain by doing more specific mutagenesis in this region, 2) writing a paper describing the role of the amino and carboxyl termini in serving the functions of perilipin of protecting stored triacylglycerols from soluble lipases, and 3) conducting studies of the role of the phosphorylation of perilipins in altering the protective functions of perilipin A.
Impacts
Our current research has advanced our understanding of how perilipins work to control fat storage in adipose tissue. We have now identified the portions of the molecule that define the protein as a protein that binds to lipid storage droplets; these findings will help to identify and understand other proteins that associate with lipid droplets and control the storage and release of triacylglycerols. Our continuing work aims to understand the mechanisms by which the protein prevents the lipolysis of the stored lipids at a molecular level. These studies will help us to understand how adipose cells are so efficient at storing fat.
Publications
- Cole, N. B., Murphy, D. D., Grider, T., Reuter, S., Brasaemle, D. L., and Nussbaum, R. L. 2002. Lipid droplet binding and oligomerization properties of the Parkinson's disease protein alpha-synuclein. J. Biol. Chem., 277:6344-6352.
- Garcia, A., Sekowski, A., Subramanian, V., and Brasaemle, D. L. 2002. The central domain is required to target and anchor perilipin A to lipid droplets. In press, J. Biol. Chem. Available online: 10.1074/jbc.M206602200, October 28, 2002.
- Tansey, J. T., Huml, A. M., Vogt, R., Davis, K. E., Jones, J. M., Fraser, K. A., Brasaemle, D. L., Kimmel, A. R., and Londos, C. 2002. Functional studies on native and mutated forms of perilipins: A role in protein kinase A-activated lipolysis of triacylglcyerols in CHO cells. In press, J. Biol. Chem. Available online: 10.1074/jbc.M211005200, December 10, 2002.
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Progress 01/01/01 to 12/31/01
Outputs
We continue to study the domains of perilipin A that function to target perilipin A to lipid droplets, mediate its association via hydrophobic interactions, and facilitate triacylglycerol storage by blocking the access of soluble neutral lipid lipases to stored lipid. We are currently preparing a manuscript describing the central domain that is required to target perilipins to droplets. We are now conducting more detailed analysis of the hydrophobic sequences within this central domain. We have refined our studies of the role of the flanking amino and carboxyl termini in lipid protection to home in on two shorter motifs within these domains. We are now studying these regions in more detail.
Impacts
Perilipins have been demonstrated to have critical importance in controlling how much fat is stored in adipose tissue. The current studies are aimed at understanding the molecular basis of how this protein works; the identification of portions of the protein which control its function may facilitate the design or discovery of small molecules that may alter perilipin function, and hence fat storage. These small molecules may provide promising leads for the development of new anti-obesity drugs.
Publications
- Wolins, N.E., Rubin, B., and Brasaemle, D. L. 2001. TIP47 associates with lipid storage droplets. J. Biol. Chem. 276:5101-5108.
- Cole, N.B., Murphy, D.D., Grider, T., Rueter, S., Brasaemle, D., Nussbaum, R. L. 2001. Lipid droplet binding and oligomerization properties of the Parkinson's disease protein alpha-synuclein. J. Biol. Chem. online, December 14, 2001.
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Progress 01/01/00 to 12/31/00
Outputs
The goal of these studies is to map the domains of perilipin A that serve the functions of targeting perilipin A to lipid droplets, mediating its tight association with lipid droplets, and fulfilling perilipin function by protecting stored neutral lipids from hydrolysis by soluble lipases. We have found that perilipin A reduces the rate of triacylglycerol hydrolysis by as much as 500%, and leads to the storage of 6-30-fold more triacylglycerol in cells when compared to the same cells lacking all perilipins. We have used deletion mutagenesis to map the domains of perilipin A responsible for these protective functions, and found that both the amino and carboxyl termini are required for full protection of stored lipids. By contrast, the central 25% of the amino acid sequence contains all of the signals required to target perilipin A to lipid droplets and mediate a tight association; this internal sequence can be fused to the soluble green fluorescent protein, and when
the fusion protein is expressed in cells, the fluorescent marker is found to target to lipid droplets. We have found that 3 20 amino acid hydrophobic motifs mediate targeting and lipid droplet association while the more highly charged termini serve the protective role.
Impacts
(N/A)
Publications
- Brasaemle, D. L., Levin, D. M., , Adler-Wailes, D. C., and Londos, C. 2000. The lipolytic stimulation of 3T3-L1 adipocytes promotes the translocation of cytosolic hormone-sensitive lipase to the surfaces of lipid storage droplets. Biochim. Biophys. Acta, 1483:251-262.
- Brasaemle, D. L., Rubin, B., Harten, I. A., Gruia-Gray, J., Kimmel, A. R., and Londos, C. 2000. Perilipin A increases triacylglycerol storage by decreasing the rate of triacylglycerol hydrolysis. J. Biol. Chem., 275:38486-38493.
- Wolins, N. E., Rubin, B., and Brasaemle, D. L. 2000. TIP47 associates with lipid storage droplets. J. Biol. Chem., 276:5101-5108.
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