Role of CIDE proteins in lipid droplet formation and adipocyte metabolism

Lead Research Organisation: Imperial College London
Department Name: Dept of Surgery and Cancer

Abstract

Population statistics indicate an ever-increasing number of obese people in western societies. The situation is alarming due to the side effects of excessive fat storage which are health risks for cardiovascular disease, diabetes, and even cancer. Excessive fat storage results from an imbalance between energy intake and energy expenditure. Research over the past 10 years has changed our view of adipose tissue so that today we consider it to be an active hormone-producing organ that communicates with the muscle, liver, pancreas, heart, and the brain. Furthermore, in people with excess body fat, adipocytes (fat cells) produce factors that cause metabolic disorders, such as diabetes. Mammals store excess energy in the form of fat, predominantly in adipocytes. Fat tissue comes in two types, white fat for storing energy and brown fat for burning fat. Until very recently human brown fat was considered only to be present in newborn babies. However, imaging methods have identified distinct brown fat depots above the collarbones and in the upper chest. The activity of this tissue was higher in thin compared to obese patients and was rapidly switched on by exposure to cold temperatures. These findings have ignited interest in this elusive tissue due to its potential to burn fat and therefore aid weight loss. There are a number of fundamental differences between brown and white fat cells including the size and shape of fat droplets. Although on first inspection they appear to be simple fat-containing structures, a more thorough analysis reveals the droplet to be surrounded by a membrane structure and coated by a complex mix of proteins. These proteins are the key to how the cell manages the lipids stored within the droplet. They determine whether the droplets formed are large or small and control the release of stored energy. A white adipocyte has a single large droplet that almost completely fills the space inside the cell. In contrast, brown adipocytes contain many small droplets. We will investigate how the CIDE family of proteins leads to the appearance of lipid droplets. The first member of the family, CIDEA, is present at high levels in brown adipocytes and initiates the appearance of lipid droplets. We aim to understand how CIDEA and the other family members associate with and control the size and shape of lipid droplets. In addition, we will define their roles in essential metabolic processes and study the importance of these genes in switching on and off of brown fat activity in the body's different fat depots. Our understanding of how genes control the appearance and activity of lipid droplets is crucial in the identification of new targets for the treatment of metabolic diseases such as obesity, fatty liver, and type 2 diabetes.

Technical Summary

Excessive lipid storage is a major contributory factor to the development of metabolic disease. Cellular lipid is stored in discrete lipid droplet organelles with abundant surface proteins that confer stability, prevent or promote coalescence, and facilitate storage or utilisation by regulation of metabolic processes. The major site of fat storage is white adipose tissue (WAT). In contrast, brown adipose tissue (BAT) is the site of energy dissipation by the process of thermogenesis. One of the fundamental differences between these two tissues is the presence a single large fat droplet in white adipocytes whereas brown adipocytes contain multiple small lipid droplets. We discovered that the BAT protein CIDEA surrounds lipid droplets in adipocytes and induces the formation of lipid droplets in non-adipogenic cell lines. Our objectives are to determine the biological roles of the CIDE gene family (CIDE-A, -B and -C) in the control of lipid droplet formation and energy metabolism. The proteins associated with lipid droplets induced by CIDE expression or present in white and brown adipocytes will be identified by mass spectrometry. The key structural features of the CIDE protein family required for lipid droplet appearance, size and shape will be assessed by expression of wild type and mutant versions. To determine the metabolic consequences of lipid droplet appearance we will study the effects of CIDE on beta-oxidation, lipolysis, and triglyceride synthesis and turnover. To explore the mechanism of CIDE action, we will screen for CIDEA-interacting proteins using yeast-2-hybrid and co-immunoprecipitation. To identify the pathways responsible of CIDE regulation, we will characterise CIDE promoter regulation by nuclear receptors, cytokine signalling, and cellular stress pathways. Furthermore, as a cold stimulus actively converts WAT depots to a more BAT-like phenotype we will determine if CIDE gene regulation is part of the cold-response programme.

Planned Impact

Who will benefit from this research? The identified beneficiaries of this proposal outside academia include those in industry and the commercial sector. New basic science findings in the control of cellular energy storage are of great interest to those in this sector. Because elevated fat storage in diseases such as obesity and fatty liver disease is linked with increased risk of diabetes, high blood pressure, heart disease and certain cancers, this work will be of importance to the health sector and to the general public, either from a general science education benefit, or for those who are directly impacted by these diseases. This work will also have educational benefits, including the training and skills that will be acquired by the appointed research associate on the project and in educating and inspiring interest in scientific careers in secondary school students. How will they benefit from this research? The area of research is the understanding of the actions of the CIDE gene family which promote the formation of lipid droplets in cells. Energy storage in cells has profound implications for a wide spectrum of diseases and therefore the CIDEs represent an important target for therapeutic intervention. The research in this proposal could identify new molecular targets and regulatory pathways that control lipid storage and energy metabolism which would be used by industry or academic labs. As CIDEA is selectively expressed in brown adipocytes we could validate a screen for transdifferentation of white to brown fat cells. This has important implications in obesity treatments due the energy dissipating nature of brown fat. The screen could be used to test for actions of ligands, hormones, small molecules and nutritional compounds on promoting brown adipocyte appearance by industrial partners or in a future application. The training of the appointed research associate would potentially benefit areas outside of academia by the development of transferable skills that they could then apply in any other employment sector. These include oral and written communication skills that will be acquired by presentation to both those in science and to the public. In addition the research associate will gain skills in project and time management, problem solving, information technology and mentoring. What will be done to ensure that they have the opportunity to benefit from this research? We will develop impact of this work by communicating our findings to industry and health sectors. This will be achieved by data presentation and networking at conferences such as the Keystone Symposium which is attended by industry members. In addition, we will take advantage of the Imperial Business Development department to initiate and develop contacts with interested industrial partners. 'Imperial Innovations' would enable us to achieve commercial impact of the research. They assist with patent development and application to the development of spin-out companies to help Imperial researchers discover and develop the commercial potential for their research. We will take advantage of opportunities to impact on the public awareness of the health benefits of this research and in education by continued participation in the Institute's Open Days to the public and secondary school students. The appointed research associate on this proposal will be involved in these activities and receive training from the Imperial Postdoc Development Centre to acquire transferable skills.

Publications

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Related Projects

Project Reference Relationship Related To Start End Award Value
BB/H020233/1 01/02/2011 28/02/2013 £394,944
BB/H020233/2 Transfer BB/H020233/1 01/03/2013 31/07/2014 £169,584
 
Description We have determined the key molecular actions of CIDEA required for lipid droplet (LD) enlargement. Fundamental differences between brown and white adipose tissues (BAT & WAT) include the high levels of CIDEA in BAT, and the multilocular LD morphology in BAT compared to the unilocular LD in WAT. This project facilitated the establishment of a multidisciplinary international research network including experts in biophysics, yeast biology, computer modelling, and adipose tissue morphology.

With a comprehensive structure-function analysis we have defined the CIDEA protein regions required for the discrete steps in its function to promote LD enlargement: (i) LD targeting, (ii) LD-LD docking, and (iii) LD fusion by fat transference.
A key finding is that the ability of CIDEA to enlarge LDs is dependent on it directly binding phosphatidic acid (PA), which is a cone-shaped anionic phospholipid that participates in membrane fusion by favouring its curvature.
We have computer modelled a C terminal amphipathic helix and determined experimentally that it is present and essential for CIDEA function in LD enlargement.
We determined experimentally that PA binds to the conserved amphipathic helix and computer simulations revealed that the helix was able to reorient itself to penetrate the LD phospholipid monolayer prior to becoming fully embedded in it. The same mechanism of PA binding was essential for function of the related WAT-expressed protein CIDEC/FSP27. Further, we revealed that a mutation in this gene (hCIDEC-E186X), which is associated with a human lipodystrophy, produces a protein that lacks PA binding and is defective in LD enlargement.
A surprising observation was that CIDEA mimics its adipocyte selective LD-enlargement activity when expressed in yeast cells (lacking an orthologous gene), has offered novel experimental approaches to unravel essential features of this process. This finding has significant biotechnological potential for increasing lipid yield for food and biodiesel production in microorganisms.
Focusing on adipose tissues in response to cold we undertook a targeted approach to assess gene expression changes that occur in BAT and WAT depots. We found that most LD proteins are expressed at higher levels in BAT, with the greatest differences observed for CIDEA. Prolonged cold exposure, which induces the appearance of brown-like adipocytes in WAT depots, was accompanied with the potentiation of the lipolytic machinery. However the major change detected in WAT was the enhancement of CIDEA mRNA levels. Together with the increase in CIDEC, it indicates that LD enlargement through LD-LD transference of fat is an important process during the WAT browning.
We revealed the existence of a LD remodelling futile cycle, with progressive reduction in LD size by lipolysis, followed by formation of new LDs, which were subjected to an enlargement process, likely to be CIDE-triggered. Importantly, this implies the release of substantial amounts of heat that will constitute a thermogenic system to facilitate the molecular mechanism for the unilocular to multilocular transformation during WAT browning.
Future directions include regenerative therapy as we found CIDEA highly expressed in stem cells coincident with the presence of large LDs (5?m). We aim to study the role of CIDEA and LDs in the metabolism of ES cells and their importance in maintaining pluripotency and self-renewal.
Exploitation Route The findings from this project can be taken further by academic researchers, industry, and the commercial sector. Our study has revealed a key molecular mechanism of lipid droplet enlargement. As lipid droplets are ubiquitous organelles present in all types of eukaryotic cells such as plants, mammals, algae and yeast our findings impact on many biological systems.
Energy storage in cells has profound implications for a wide spectrum of diseases and therefore the targeting of lipid droplets to affect cellular metabolism. Thus, further studies would target disorders of energy storage such as obesity and fatty liver disease as well as lipodystrophy, in which we revealed a new mechanistic link with a mutant form of CIDEC. Profound changes in cellular metabolism occur in both cancerous cells and stem cells, both linked with lipid droplet changes. Thus, lipid droplet manipulation could affect cancer cell viability and stem cell development. Lipids are toxic to cells, but packaging them into lipid droplets facilitated by CIDEA will be protective for the cell. Thus, in the biotechnology industry, CIDEA expression represents a valuable tool to increase the accumulation of yield of valuable lipids in microorganisms used for food production, biodiesel, bioplastics and lipid-based drugs.
Sectors Agriculture, Food and Drink,Chemicals,Energy,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description UK economic competitiveness is facilitated by training and skills acquisition. The project has succeeded in providing an excellent training of the appointed Post Doc David Barneda. He developed the molecular tools used in the study and through an international visit to Cornell University acquired additional skills in yeast manipulations. The wide range of skills acquired from the project (e.g. time management, problem solving, information technology, presentation skills, record keeping, budgeting, innovative thinking and mentoring) are applicable to career progression both within and outside academia. Thus, David has benefitted as an individual from the training, and the UK institutions where he applies his skills and training will benefit. David is continuing his scientific career at Queen Mary University of London. With this project, we have contributed to increasing public awareness and understanding of science. This was undertaken at fundraising events to raise the profile of our research with the general public. We have contributed to changing public perceptions of brown adipose tissue, which is currently limited. For this, I have given advice given to a scientific writer on a recently published article describing brown fat activation as a potential anti-obesity treatment. The discussion and advice given was to ensure the scientific accuracy of article at the same time as contributing to increasing public awareness and understanding of the importance of brown fat in health. The international multidisciplinary collaboration network (UK-USA-Italy-China) established through this project has served to enhance the research capacity and skills base. The management skills involved in coordinating a multicentre collaboration are important on a personal development perspective and are applicable to economic advancement. The network has served to raise the profile of UK research with a group of influential scientists and policy makers within their different fields.
First Year Of Impact 2013
Impact Types Societal,Economic

 
Description BBSRC Responsive Mode
Amount £293,592 (GBP)
Funding ID BB/P005209/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2017 
End 03/2020
 
Description International Workshop / ISIS Award
Amount £4,040 (GBP)
Funding ID BB/J010316/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2012 
End 05/2012
 
Title Conditionally Immortal Adipocyte Cell Lines 
Description We generated a new model of depot-specific adipocyte cultures. We used an adenovirus to introduce a temperature-sensitive SV40 large T-antigen (tsA58) into preadipocyte cultures prepared from different fat depots. In addition to making depot-specific adipocyte cell lines, we have generated cell lines from adipose tissues of genetically modified mice. 
Type Of Material Cell line 
Year Produced 2014 
Provided To Others? Yes  
Impact These adipocyte models allowed us to study adipocyte biology in a physiologically relevant cell without the continuous need to prepare primary cultures. Thus, this represents a contribution to the reduction of animal use. Data on these models were published in our papers in AJP-Endo and Metabolism, and Molecular Endocrinology. 
 
Title Yeast model of CIDEA expression 
Description Expression system for CIDEA in yeast cells 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact In conjuction with a range of yeast strains, this tool enabled us to determine the mechanism of action of CIDEA in promoting lipid droplet enlargement. The data obtained is included in a manuscript prepared for publication. 
 
Title Cold-dependent transcriptomes in adipose tissues 
Description Microarray analysis of gene expression in brown and white adipose tissues after exposure to warm (28 C) or cold (6 C). The dataset is available from NCBI's Gene Expression Omnibus. 
Type Of Material Database/Collection of data 
Year Produced 2013 
Provided To Others? Yes  
Impact The analysis of this dataset allowed us to identify key genes and pathways associated with the conversion of white to brite (brown-in-white) adipose tissue. The data was published in a paper in AJP-Endo and Metabolism. Researchers have already contacted me directly that they are utilizing the database to investigate gene expression changes due to cold in brown fat. 
 
Description Analysis of CIDEA by Circular Dichroism 
Organisation University of Warwick
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution I designed the peptides and the overall experimental approaches for the Circular Dichroism study with the assistance of Dr Ann Dixon.
Collaborator Contribution Dr Ann Dixon assisted with the completion of experiments using Circular Dichroism of wild type and mutant forms of CIDEA in the absence or presence of liposomes generated from different phospholipids. This allowed us to complete our study in determining the interaction of CIDEA with phosphatidic acid enabling us to address reviewer comments for our publication in Elife.
Impact This was a multidisciplinary collaboration with the output a publication in Elife (PMID: 26609809)
Start Year 2015
 
Description Brown Adipose Tissue Lipid Droplet Proteome 
Organisation Chinese Academy of Sciences
Country China 
Sector Public 
PI Contribution Intellectual input, experimental design and writing manuscript
Collaborator Contribution Prof Pingsheng Liu investigated the brown adipose tissue lipid droplet proteome from mice exposed to control or cold conditions.
Impact This collaboration identified that CIDEA protein levels were acutely regulated by cold with minor effects on mRNA. This collaboration is the first to determine a lipid droplet proteome in an adipose tissue. The data in a manuscript currently under consideration.
Start Year 2013
 
Description CIDEA protein biophysics 
Organisation University of Warwick
Country United Kingdom 
Sector Academic/University 
PI Contribution We performed cell-based protein structure-function assays. This identified an important functional domain. We provided the protein for analysis by Judith Klein-Seetharaman.
Collaborator Contribution Prof Judith Klein-Seetharaman performed circular dichroism spectroscopy and showed that CIDEA peptide folds to form an alpha helix.
Impact The biophysical analysis confirmed computer modelling and cell-based studies. These data are included in a manuscript prepared for submission.
Start Year 2013
 
Description Computer Modelling of CIDEA 
Organisation University of Pittsburgh
Country United States 
Sector Academic/University 
PI Contribution We undertook in vitro analysis of and an initial in silico analysis CIDEA protein structure-function relationship. We provided protein extract for experiments performed in the Kagan lab.
Collaborator Contribution In the lab of Prof Valerian Kagan, computer based modeling of interaction of CIDEA domains with lipid droplets was undertaken. In addition the group investigated in-gel interaction between CIDEA protein and phospholipids.
Impact The computer-based modelling represents a multi-disciplinary approach to the investigation of CIDEA. An amphiphathic helix was predicted, and identified as sufficient for lipid droplet interaction. The work from this collaboration is included in a manuscript prepared for publication.
Start Year 2013
 
Description Direct interaction between CIDE proteins and phosphatidic acid 
Organisation Babraham Institute
Country United Kingdom 
Sector Private 
PI Contribution My research team performed the experiments to investigate interactions between CIDE proteins and phosphatidic acid (PA) in vitro.
Collaborator Contribution Dr Nicholas Ktistakis provided intellectual input into the study of PA interaction with CIDE proteins. He provided reagents for the study (PA-conjugated beads) to permit study of interactions in vitro.
Impact This collaboration allowed us to validate a direct interaction between CIDEA and PA. These data have been presented at scientific meetings and included in a manuscript being prepared for publication.
Start Year 2011
 
Description Investigation of CIDE genes in yeast 
Organisation Cornell University
Country United States 
Sector Academic/University 
PI Contribution With the assistance of a BBSRC ISIS grant, a collaboration was established with Cornell to study the action of CIDEA using yeast as a model system. David Barneda travelled to the lab of Prof Susan Henry where he performed experiments to express CIDEA in yeast cells.
Collaborator Contribution Prof Susan Henry and her lab members provided input into experimental design for CIDEA expression systems in yeast. In addition, the provided yeast cell lines and expression constructs.
Impact Findings were that CIDEA was fully functional in promoting large lipid droplets in yeast. This is important as yeast do not have genes related to CIDEA in their genome. The data from this collaboration are included in a manuscript being prepared for submission.
Start Year 2012
 
Description Lipid droplet proteins in adipose tissues 
Organisation University of Ancona
Country Italy 
Sector Academic/University 
PI Contribution We analysed gene expression in adipose tissues provided by partners. We wrote manuscript for publication
Collaborator Contribution Prof Saverio Cinti and Dr Andrea Frontini provided adipose tissues from mice exposed to different temperatures. They also performed histological analysis of the tissues
Impact We determined the differential expression of genes encoding lipid droplet-associated proteins and their regulation by cold. The data are published in the journal Biochimica et Biophysica Acta.
Start Year 2010
 
Description Transcriptional Regulation in Brown and White Adipocytes 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Experimental analysis of the role transcriptional regulators in the expression of genes in adipocytes.
Collaborator Contribution Prof. M G Parker provided cell lines, intellectual input and antibody used in study of gene regulation and function in brown and a model of brown-in-white (brite/beige) adipocytes. The group of Prof Cinti performed the immunocytochemistry for CIDEA and UCP1 in adipose tissues.
Impact Our findings on the transcriptional targets of RIP140 including CIDEA were published in the journal Molecular Endocrinology.
Start Year 2007
 
Description Transcriptional Regulation in Brown and White Adipocytes 
Organisation University of Ancona
Country Italy 
Sector Academic/University 
PI Contribution Experimental analysis of the role transcriptional regulators in the expression of genes in adipocytes.
Collaborator Contribution Prof. M G Parker provided cell lines, intellectual input and antibody used in study of gene regulation and function in brown and a model of brown-in-white (brite/beige) adipocytes. The group of Prof Cinti performed the immunocytochemistry for CIDEA and UCP1 in adipose tissues.
Impact Our findings on the transcriptional targets of RIP140 including CIDEA were published in the journal Molecular Endocrinology.
Start Year 2007
 
Description Engagement at Institutional Fundraising Event 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Participants asked questions regarding the potential for brown adipose tissue as a target for obesity.

Participants reported that they increased their understanding of importance of brown adipose tissue.
Year(s) Of Engagement Activity 2012
 
Description Media interest in brown adipose tissue 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact An article written in the magazine "Red" describes the targetting of brown fat for weight loss. I provided advice on accuracy of parts of the article and commented on the potential for activating brown fat as an obesity therapy. The article is in press, so it currently unclear of impacts, but it is likely to increase knowledge of this area.

As the article is only now going to press, it is unclear as the the impacts. However, it is likely the readers will have greater understanding of the role of brown fat in adult humans.
Year(s) Of Engagement Activity 2014