Principal Investigator/Program Director (Last, first, middle):
424 R&R and PHS-398 Specific
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SF 424 R&R Face Page------------------------------------------------------------------------------------------
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Performance Sites---------------------------------------------------------------------------------------------
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Research & Related Other Project Information------------------------------------------------------------------
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Project Summary/Abstract (Description)----------------------------------------
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Public Health Relevance Statement (Narrative attachment)----------------------------------------
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Bibliography & References Cited----------------------------------------
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Facilities & Other Resources----------------------------------------
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Other Attachments----------------------------------------
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List of referees----------------------------------------
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Research & Related Senior/Key Person--------------------------------------------------------------------------
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Biographical Sketches for each listed Senior/Key Person----------------------------------------
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Research & Related Budget - Year 1----------------------------------------------------------------------------
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Research & Related Budget - Year 2----------------------------------------------------------------------------
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Research & Related Budget - Year 3----------------------------------------------------------------------------
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Research & Related Budget - Year 4----------------------------------------------------------------------------
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Budget Justification----------------------------------------
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Research & Related Budget - Cumulative Budget-----------------------------------------------------------------
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PHS 398 Specific Cover Page Supplement------------------------------------------------------------------------
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PHS 398 Checklist---------------------------------------------------------------------------------------------
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PHS 398 Career Development Award Supplemental Form------------------------------------------------------------
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Candidates Background----------------------------------------
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Career Goals and Objectives----------------------------------------
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Development Activities During Award Period----------------------------------------
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Training in the Responsible Conduct of Research----------------------------------------
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Statements by Mentor, Co-Mentors, Consultants, Contributors----------------------------------------
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Institutional Environment----------------------------------------
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Institutional Commitment to Career Development----------------------------------------
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Specific Aims----------------------------------------
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Research Strategy----------------------------------------
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Protection of Human Subjects----------------------------------------
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Women &Minorities----------------------------------------
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Planned Enrollment Table----------------------------------------
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Children----------------------------------------
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Vertebrate Animals----------------------------------------
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Select Agent Research----------------------------------------
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Principal Investigator/Program Director (Last, first, middle):
Consortium/Contractual----------------------------------------
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Resource Sharing Plan----------------------------------------
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Table of Contents
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Principal Investigator/Program Director (Last, first, middle):
Project Summary:
Non-alcoholic fatty liver disease (NAFLD) is an increasingly prevalent medical problem that
affects approximately a third of the US population. Patatin-like phospholipase domain
containing 3 (PNPLA3) was first implicated in the metabolism of hepatic triacylglycerides
(TAGs) when our lab found that a missense mutation that substitutes isoleucine at position 148
with methionine (I148M) was associated with non-alcoholic fatty liver disease in humans.
Homozygotes with the variant allele (148M) have a 2-fold higher risk of hepatic TAG
accumulation than homozygotes with the wild type allele (148I). The function of PNPLA3 and
the cause of I148M induced hepatic TAG accumulation remains unclear. Since fatty liver
represents an accumulation of fatty acids in the form of triglycerides, I will adapt and/or develop
targeted liquid chromatography-tandem mass spectrometry (LC-MS-MS) methods to measure
the concentrations of different classes of fatty acid-containing lipids in liver, adipose tissue, and
plasma from mouse models of PNPLA3-associated steatosis. Mice will be studied on both a
normal chow diet and on diets designed to exacerbate or alleviate PNPLA3-I148M associated
accumulation of hepatic TAG. I will then extend these LC-MS/MS-based methods to determine
the flux of each lipid class by measuring the rate of incorporation of deuterium from deuterated
water (D2O) in cultured cells that overexpress PNPLA3, and in genetically manipulated mouse
models. Alternative stable isotope labeling strategies, such as 13C-glycerol or 13C-fatty acid
incorporation, will also be employed to compliment the D2O incorporation data. Execution of
this research proposal will provide training in the application of metabolomics technology to
study human disease and shed insight into the function of PNPLA3 and its role in the
progression of fatty liver disease.
Project Description
Page 7
Principal Investigator/Program Director (Last, first, middle):
Project Narrative:
Fatty liver disease is a growing epidemic that afflicts a third of the US population and leads to
steatosis, cirrhosis, and liver failure. We have identified a gene named PNPLA3 that is strongly
associated with fatty liver disease in human, but the function of this gene remains unclear. I am
proposing experiments designed to determine the function of PNPLA3 and ultimately aid in the
development of effective medical therapies to prevent fatty liver disease.
Public Health Relevance Statement
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Principal Investigator/Program Director (Last, first, middle):
RESOURCES
FACILITIES: Specify the facilities to be used for the conduct of the proposed research. Indicate the performance sites and d escribe capacities,
pertinent capabilities, relative proximity, and extent of availability to the project. Under “Other,” identify support services such as machine shop,
electronics shop, and specify the extent to which they will be available to the project. Use continuation pages if neces sary.
- The mentor and co-mentor Drs. Hobbs and Dr. Jonathan Cohen, have 1,050 sq. ft. of laboratory
space in the McDermott Center for Human Growth and Development. Dr. Xie has 900 sq. ft. of laboratory
space adjacent to the laboratory of Drs. Hobbs and Cohen. The laboratories are equipped with an Amino
DW2C Dual wavelength spectrophotometer; a Beckman DU-70 scanning spectrophotometer; a Fluostar
Optima Multi-functional Fluorimeter; a Bio-Rad Biologic FPLC system; a Beckman HPLC system; Pharmacia
column chromatography and electrophoresis apparatus; Bio-Rad electrophoresis and blotting systems; two
Forma Scientific CO2 incubators, a sterile hood, an inverted microscope, liquid N2 storage chambers, a
Beckman scintillation counter, PCR machines, orbital shaking-incubators, bacterial incubators, -80 C freezers,
-20 C freezers, a chromatography refrigerator, refrigerators, low speed, high speed and ultraspeed centrifuges,
rotors, including 70.1Ti, 70Ti, 50.2Ti, 45Ti, type65, SW60, SW41Ti, SW40Ti, NVT65 rotors, and JA 20,17,14,
and 10 rotors. The investigators and their personnel have access to core facilities, including high speed and
ultra-centrifuges, cold/warm rooms, tissue culture rooms and a cleaning facility.
Laboratory:
Dr. Hobbs also has a laboratory (~600 sq. ft.) in the Department of Molecular Genetics. The Department of
Molecular Genetics has a real-time PCR machine and as Agilent gas chromatography/mass spectrometer. Dr.
Cohen also has ~600 sq. ft. of laboratory space in the Center for Human Nutrition.
Clinical:
Not applicable
The mouse colony will be housed in a secure vivarium requiring a combination card-key access to
approved users. A staff of veterinarians specializing in laboratory animal care provides expert assistance with
care and maintenance of the mouse colony. The facility has a total of 16,500 sq ft floor space, is self contained with respect to physical plant, and occupies the entire second floor of the investigators’ building,
providing convenient access to animals. Excellent surgical and barrier facilities are available as needed.
Animal:
The investigators’ laboratories are equipped with personal computers connected by local area
networks. The McDermott Center and the Medical School have computer support personnel and equipment to
support our additional computer needs.
Computer:
Office:
The PI and co-PIs have personal offices of 110 sq. ft. each adjoining their laboratories.
The McDermott Center provides administrative support and access to office equipment (fax,
photocopier, etc.). The McDermott Center has core facilities for DNA sequencing available on a cost-recovery
basis to Center faculty.
Other:
MAJOR EQUIPMENT: List the most important equipment items already available for this project, noting the location and pertinent capabilities of
each.
Core Facilities available at UT Southwestern (which are subsidized)
1. Protein Chemistry Technology Core peptide mapping - A fee-for-service facility for protein sequencing, and
protein modification analysis
2. Transgenic/Knockout- Technology Center Core Facility - A fee for service facility run by Dr. Robert
Hammer for the development of genetically modified mice.
3. Structural Biology Core Facility – A fee for service facility directed by Dr. Diana Tomchick, provides a
unique platform for biochemists and molecular biologists to engage in structural biology projects by making
macromolecular crystallography available as a general tool.
Facilities
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Principal Investigator/Program Director (Last, first, middle):
List of Referees:
Jeffrey McDonald, Ph.D.
Associate Professor of Molecular Genetics
UT Southwestern Medical Center
5323 Harry Hines Blvd. Dallas, TX 75390-9046
E-mail: Jeffrey.mcdonald@utsouthwestern.edu
Telephone: 214-638-8663
Elizabeth Parks, Ph.D.
Associate Professor of Nutrition
UT Southwestern Medical Center
5323 Harry Hines Blvd, Dallas, TX, 75390-9046
E-mail: Elizabeth.parks@utsouthwestern.edu
Telephone: 214 648-2054
Ralph Deberardinis, M.D., Ph.D.
Assistant Professor of Pediatrics and Genetics
UT Southwestern Medical Center
5323 Harry Hines Blvd., Dallas, TX, 75390-8502
E-mail: ralph.deberardinish@utsouthwestern.edu
Telephone: 214-633-1803
Donald Small, M.D.
Professor of Physiology and Biophysics
Boston University Medical Center
700 Albany Street, Boston, MA, 02118-2526
E-mail: dmsmall@bu.edu
Telephone: 617-638-4002
List of referees
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Principal Investigator/Program Director (Last, first, middle):
BIOGRAPHICAL SKETCH
Provide the following information for the Senior/key personnel and other significant contributors in the order listed on Form Page 2.
Follow this format for each person. DO NOT EXCEED FOUR PAGES.
NAME
POSITION TITLE
Postdoctoral Researcher II
eRA COMMONS USER NAME (credential, e.g., agency login)
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and
residency training if applicable.)
DEGREE
INSTITUTION AND LOCATION
MM/YY
FIELD OF STUDY
(if applicable)
University of Delaware, Newark DE
B.S.
06/06
Chemical Engineering
Boston University, Boston MA
Ph.D
01/12
Biophysics
University of Texas Southwestern Medical
Center, Dallas TX
Postdoctoral
Lipid Physiology
A. Personal Statement
I will serve as the principle investigator on this research training grant. My primary research interested is using
metabolomics and stable isotope labeling to study lipid metabolism and metabolic disease. I obtained a
bachelor’s degree in Chemical Engineering, where I specialized in Biochemical Engineering, and a doctoral
degree in Physiology and Biophysics under the mentorship of Dr. Donald Small. This education provided multidisciplinary training in quantitative biochemistry and physiology. In 2011, I began a post-doctoral fellowship at
UT Southwestern under the mentorship of Dr. Helen Hobbs and Dr. Jonathan Cohen in 2011. During my postdoctoral training I have specializing in using LC-MS/MS based metabolomics technology to study lipid
physiology, sterol metabolism and the progression of fatty liver disease. I hope to continue this training under
the Mentored Research Scientist Development Award in Metabolomics.
B. Positions and Honors
Positions and Employment:
2004-2006 Undergraduate Researcher, Department of Chemical Engineering, University of Delaware,
………………..Newark DE
2006-2011 Graduate Research Fellow, Department of Physiology and Biophysics, Boston University, Boston
………………..MA
2011Post-Doctoral Fellow, Department of Molecular Genetics, UT Southwestern Medical Center,
………………..Dallas, TX
Honors:
2006
2009,2012
2010
2010
2010
2011
Graduate Research Assistant Scholarship
Kern Aspen Lipid Conference Travel Award
Ruth Kirschstein Scholar
Henry J. Russek Student Achievement Award in Physiology and Biophysics
NIH-HGSRF Award
Deuel Lipid Conference Travel Award
Biosketches
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Principal Investigator/Program Director (Last, first, middle):
C. Publications
Interfacial Properties of a Complex Multi-Domain 490 Amino Acid Peptide Derived from Apolipoprotein B
(Residues 292-782); Mitsche, M.A., Wang, L, Jiang, G.Z., McKnight, C.J., Small, D.M; Langmuir, 25(4) pp
2322-2330, 2009
The adsorption of biological peptides and proteins at the oil/water interface. A potentially important but largely
unexplored field; Small, D.M., Wang, L., Mitsche, M.A.; Journal of Lipid Research, Vol. 50, S329-S334, 2009
Deposition of egg-PC to an Air/Water and Triolein/Water Interface; Mitsche, M.A, Small, D.M.; Biophysical
Journal, 96(3) pp.461a, 2009
Adsorption of Egg-PC to an Air/Water and Triolein/Water Interface: Use of the 2-Dimensional Phase Rule to
Estimate the Surface Composition of a Phospholipid/Triolein/Water Surface as a Function of Surface Pressure;
Mitsche, M.A., Wang, L., Small, D.M.; Journal of Physical Chemistry B, 114(9) pp 3276-3284
C-terminus of Apolipoprotein A-I Removes Phospholipids (PL) from a Triolein/PL/Water Interface, but
the N-Terminus does not: A possible mechanism for nascent HDL Assembly; Mitsche, M.A., Small,
D.M.; Biophysical Journal, 101(2) pp. 353-361, 2011 Feature Article
Interfacial Properties of the N-terminal Lipid –Binding Domains of ApolipoproteinB and Their Role in
Triacylglyceride-Rich Lipoprotein Assembly; Mitsche, M.A.; Ph.D. Thesis, Boston University 2012
Surface Pressure Dependent Remodeling of Amphipathic α-Helices at a Triolein/Water Interface; Mitsche,
M.A., Small, D.M.; Accepted to the Journal of Lipid Research on 1/23/13
D. Research Support
Ongoing Research Support
None
Completed Research Support
None
Biosketches
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Principal Investigator/Program Director (Last, first, middle):
Background Statement:
I began my technical education by studying chemical engineering at the University of
Delaware where I specialized in biochemical engineering. Studying engineering gave me a
strong fundamental education in physical and bio-chemistry, physics, mathematics, computer
science, and analytical reasoning, which have served me well since transitioning into biomedical
research during graduate school. During my undergraduate years, I came to realize that a
traditional scientific graduate education would be required if I wished to work in research and
development. Therefore, I decided to pursue a doctoral degree in biophysics at Boston
University. My graduate work, under the direction of Dr. Donald Small, focused on elucidating
the mechanism of lipoprotein remodeling by studying the surface chemistry of apolipoprotein
fragments at a triacylglyceride/water interface. Working with Dr. Small provided me with
extensive exposure to the field of lipid physiology, where I became particularly interested in
biology of intracellular lipid droplets and lipid metabolism in general. I came to the conclusion
that studying lipid droplet biology would require additional training in molecular genetics and
lipidomics. Lipidomics, and metabolomics in general, is of particular interest to me because it
will reveal fundamental aspects of lipid physiology that have been unapproachable to date and
my engineering training makes me uniquely suited to assist in the development of this
technology. Therefore, the opportunity to learning mass spectroscopy based lipidomics was my
primary objective in choosing a lab for my post-doctoral training.
In 2011 I joined the Hobbs-Cohen lab at the University of Texas Southwestern Medical
Center as a post-doctoral fellow. My post-doc began with training on a liquid chromatographytandem mass spectrometer (LC-MS/MS) and a method for measuring sterols developed by Dr.
Jeff McDonald. To date, my post-doctoral research has focused on using LC-MS/MS to study
sterol metabolism. My first project is aimed at defining the post-squalene biosynthetic pathway
of cholesterol. In the 1960’s two alternative post-squalene cholesterol biosynthetic pathways
were proposed, however the relative utilization of these two pathways remains unknown. With
the LC-MS/MS based sterol methods, most of the post-squalene sterol biosynthetic
intermediates can be measured, and the turnover of these intermediates can be measured by
labeling newly synthesized sterols with deuterated water (D2O). I have developed a method to
monitor sterol biosynthetic intermediates turnover in vivo using D2O and have applied it to
culture cells and mice to measure the kinetics of this pathway. Stable isotope labeling with
D2O will be directly translatable to other lipid classes, as proposed in this grant. My second
project is focused on measuring sterol metabolism and clearance in ABCG5/G8 mice. Our lab
has recently developed liver and intestine specific ABCG5/G8 knock-out mice. To measure the
tissue specific contribution of ABCG5/G8 to sterol clearance, I have been developing and
implementing stable isotope labeling experiments to measure the absorption and clearance of
cholesterol and plant sterols in ABCG5/G8 genetically manipulated mice. In addition to
continuing these projects, I began to develop methods for measuring lipid class relevant to other
research projects in the lab. Specifically I was interested in using LC-MS/MS to measure
triacylglycerides (TAG) to measure lipid species in mice over expressing PNPLA3 in the
liver. After successfully developing a method to measure individual TAG species, I observed a
large reduction in the percentage of poly-unsaturated fatty acid TAGs when PNPLA3 was over
expressed. I also used this method to measure turnover of TAG species using D2O. In this
proposal, I have developed a research plan to pursue these findings and develop methods to
measure other lipid classes to understand the function of PNPLA3 and, in the future, to use
these methods to study other proteins involved in intracellular lipid droplet biology.
Candidates Background
Page 69
Principal Investigator/Program Director (Last, first, middle):
Career Goals and Objectives:
The long-term goal of my career is to run a laboratory that focuses on using mass spectroscopy
for biomedical research and medical diagnosis. The primary objective of the lab will be two-fold. Firstly,
the lab will focus its independent research on using metabolomics and stable isotope labeling to
elucidate the molecular mechanism of the progression of metabolic disease. In particular, we will aim to
advance our understanding of the genetic and dietary contributions to lipid metabolism. Secondly, the
lab will use metabolomics and stable isotope labeling to both assist in the diagnosis of patients
presenting complex metabolic phenotypes and provide a service to other biomedical researchers.
Reaching this goal will require significant training in the next four years. I will need to become an expert
in development of LC-MS/MS based metabolomics methods and understand how to apply these
techniques to biomedical research. In addition, I will need training in molecular biology, genetics, and
animal husbandry to establish an independent lab studying lipid metabolism. This research proposal
outlines a series of experiments to elucidate the function of PNPLA3 that will provide excellent training
in the application of metabolomics and stable isotope labeling to biomedical research. The HobbsCohen lab and UT Southwestern is an ideal environment to develop these skills because many of the
required techniques are of molecular biology, genetics, and animal handling are already used in the lab
In addition, there is a growing group of metabolomics researchers in the department and throughout
the university.
Career Goals and Objectives
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Principal Investigator/Program Director (Last, first, middle):
Career Development/Training Activities During Award Period:
The majority of the training associated with
Sub-Aims Year 1
Year 2
Year 3
Year 4
this grant will be accomplished through the
Develop
Lipidomics
Methods
1A
successful execution of the research
Lipidomics of PNPLA3 Mice
1B
proposal. A timeline for the execution of this
Lipidomics on Custom Diets
1C
proposal is shown in Figure 1. This research
Human Plasma
1D
will give me training in LC-MS/MS based
D2O Labeling in Cells
2A
metabolomics, cell culture, and mouse
Alternative Labeling in Cells
2B
handling in addition to other skills required
D2O Labeling in PNPLA3 Mice
2C
to manage an independent laboratory. To
D2O Labeling on Custom Diets
assist in the execution of this proposal, an
2D
advisory committee of six members has been
Figure 1: Timeline of research proposal
assembled. This team includes Drs. David
Russell and Jeff McDonald who are experts in LC-MS/MS based lipidomics, Drs. Ralph DeBerardinis and
Elizabeth Park who are experts in measuring metabolite flux using stable isotopes, and Drs. Helen Hobbs
and Jonathan Cohen who are experts in lipid metabolism and PNPLA3. Regular attendance of seminars
at UTSW in metabolism, nutrition, and obesity and University Lecture will be essential for my training as
well. In addition, I will attend two conferences per year to be exposed to progress in lipid metabolism
and metabolomics.
Development Activities During Award Period
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Principal Investigator/Program Director (Last, first, middle):
Responsible Conduct of Research Training for Postdoctoral Scholars at UT Southwestern
I am currently enrolled in post-doctoral responsible conduct in research training at UT Southwestern. This
training will be completed in May of 2013. The format of the training is outlined below:
1. Format
a. Introductory Presentation on the Postdoctoral Certificate Training Program - attended by
all postdoctoral scholars within two months of postdoctoral appointment
b. Eight-week course of faculty led discussions - The eight-week course meets a requirement
for the Postdoctoral Certificate in Research, which must be completed within the first two years
of training at UT Southwestern. The course is offered in the Spring term. Participation is
monitored and pass/fail grade is assigned by faculty of the graduate school’s Postdoctoral
Affairs Office in conjunction with registration and academic certificate completion. Format: 90
minutes every week for eight weeks - 45-minute lecture followed by 45-minute faculty-led
discussion.
2. Subject Matter
a. Introductory Presentation
i. UT Southwestern Graduate School of Biomedical Science Policies on Academic Integrity
ii. Mentor/mentee responsibilities and relationships
iii. Importance of learning and following lab notebook policy
b. Eight-week course
i. Human Research Ethics and Genetics
ii. Use of Animals in Research
iii. Research misconduct, plagiarism, and peer review
iv. Record Keeping and Research Notebooks;
v. Authorship
vi. Data and Image Manipulation
vii. Conflict of Interest
viii. Collaboration, Technology Transfer, and Intellectual Property
3. Faculty Participation
a. Introductory Presentation – Deirdre Brekken, Ph.D.
b. Eight-week course
i. Humans: Fred Grinnell, Ph.D.
ii. Animals: Bart Carter, D.V.M.
iii. Misconduct: Jerry Neiderkorn, Ph.D.
iv. Record keeping: David Russell, Ph.D.
v. Authorship: Melanie Cobb, Ph.D.
vi. Images: Sandra Schmid, Ph.D.
vii. Conflicts: Stuart Ravnik, Ph.D.
viii. Technology: Philip Thomas, Ph.D.
4. Duration of Instruction
a. Introductory Presentation – 60 minutes
b. Eight-week course – 90 minutes: 45-minute lecture followed by 45-minute faculty-led
discussion
5. Frequency of Instruction
a. Introductory Presentation – one 60-minute session
b. Eight-week course – once per week for eight weeks
12/04/2012
Training in the Responsible Conduct of Research
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Principal Investigator/Program Director (Last, first, middle):
Statements by Mentor, Co-Mentors, Consultants, Contributors
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Principal Investigator/Program Director (Last, first, middle):
Statements by Mentor, Co-Mentors, Consultants, Contributors
Page 74
Principal Investigator/Program Director (Last, first, middle):
Statements by Mentor, Co-Mentors, Consultants, Contributors
Page 75
Principal Investigator/Program Director (Last, first, middle):
Statements by Mentor, Co-Mentors, Consultants, Contributors
Page 76
Principal Investigator/Program Director (Last, first, middle):
Merck Research Laboratories
Merck & Co., Inc.
2015 Galloping Hill Road
Kenilworth, NJ 07033-1310
T 908-298-4000
merck.com
February 27, 2013
To Whom It May Concern,
This letter affirms my willingness to serve as a consultant to
mass spectrometry methods he is developing as part of a KO1 application.
on the
I am currently the Director of Analytical Biochemistry, which is a group of 18 mass
spectrometry scientists focused on cardiometabolic research. I have considerable
experience with the analysis of lipid concentration and turnover by mass spectrometry
and also have several Ph.D. experts in the field in my group as regular consultants in
this area of research.
As part of an ongoing collaboration with the Hobbs-Cohen laboratory, my group has
already provided data and technical advice on lipids measurement to
, and
we look forward to continuing this relationship.
Sincerely,
Thomas P. Roddy, Ph.D.
Director of Analytical Biochemistry in Molecular Biomarkers
TPR/bf
Statements by Mentor, Co-Mentors, Consultants, Contributors
Page 77
Principal Investigator/Program Director (Last, first, middle):
Description of Institutional Environment
UT Southwestern Medical Center has a vibrant academic environment sustained by over 1,200 full time
faculty, including five Nobel laureates, nineteen members of the National Academy of Sciences,
nineteen members of the Institute of Medicine, thirteen members of the American Academy of Arts and
Sciences and twelve investigators of Howard Hughes Medical Institute. UTSW supports more than 3,500
research projects with annual funding over $400 million. These laboratories occupy approximately
582,000 square feet of space with more construction under way. As part of the total amount of funding
in 2001, the Texas legislature appropriated $9 million annually to the Institute for Innovations in Medical
Technologies. The Institute will include centers for genetics, molecular and cellular biotechnology, new
drug and vaccine development, computational biology, and advanced medical devices and imaging
technology. UTSW also has several core facilities including those for microarray, transgenic, DNA
sequencing, live cell imaging, flow cytometry, structural biology, and rapid biochemical kinetics
technology, in addition to a comprehensive library.
Nobel Laureates Drs. Joseph Goldstein and Michael Brown oversee the Department of Molecular
Genetics, which primarily focuses on the human genetics and molecular mechanisms underlying human
body energy homeostasis, including cholesterol metabolism, triglyceride metabolism and glucose
metabolism. This Department has been funded with a P01 grant >4 million/year for more than 30 years.
The department, where the applicant will perform research, has well-established programs related to
the applicant’s area of research, including faculty members and resources. Dr. Helen Hobbs, a HHMI
investigator, and Dr. Jonathan Cohen (co-mentors on this project) are active in NAFLD research. In
addition, the Department of Molecular Genetics is establishing a small molecule mass spectroscopy
based metabolomics facility. This includes three faculty members, Dr. Jeff McDonald, Dr. David Russell,
and Dr. Ruth Gordillo, who are currently working on expanding metabolomics research and four modern
tandem mass spectrometers. The applicant will have full-time access to a triple quadrupole mass
spectrometer. Furthermore, there are other investigators whose NAFLD research is funded by NIH, for
example Dr. Jay Horton, Dr. Jeff Browning, and Dr. Elizabeth Parks. These investigators all work closely
with Dr. Helen Hobbs and Dr. Jonathan Cohen, the applicant’s mentors. These resources provide a
unique environment for Dr.
training in NAFLD and metabolomics research.
The facilities and resources outlined in the resources section will be freely available to the applicant and
will allow the applicant to perform all of the experiments outlined in the grant proposal. The applicant
will also have many opportunities for his intellectual development. He will have access not only to
faculty members in the Department of Molecular Genetics, but also to accomplished investigators in
many other departments. He will attend multiple basic science seminars including the weekly University
Lecture Series, in which Nobel-caliber scientists (from within and outside the institution) present their
research. He will also attend weekly seminars on Nutrition, Metabolism and Obesity hosted by Center
for Human Nutrition, which attract world-renowned scientists. The applicant will present his work
periodically to other post-doctoral fellows and graduate students in weekly laboratory meetings.
Institutional Environment
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Principal Investigator/Program Director (Last, first, middle):
Institutional Commitment to Career Development
Page 79
Principal Investigator/Program Director (Last, first, middle):
Specific Aims:
Genetic studies provide compelling evidence that the patatin-like phospholipase domain
containing protein 3 (PNPLA3) confers susceptibility to fatty liver disease, a burgeoning health
problem throughout the world (1-3). A sequence variation in PNPLA3 (I148M) is associated with
the full spectrum of nonalcoholic fatty liver disease, including steatosis, nonalcoholic
steatohepatitis, cirrhosis and hepatocellular carcinoma, but the function of the protein remains
enigmatic (4). The overall objective of this proposal is to define the physiological role of
PNPLA3 using liquid chromatography-tandem mass spectroscopy (LC-MS/MS)-based
lipidomics and stable isotope labeling. Since fatty liver represents an accumulation of fatty
acids in the form of triglycerides (TAG), targeted LC-MS-MS methods will be adapted, optimized
and/or developed to measure concentrations of fatty acid-containing metabolites in liver,
adipose tissue, and plasma from mouse models of PNPLA3-associated steatosis. LC-MS/MSbased methods will also be used to determine the flux of each lipid class by measuring the rate
of incorporation of deuterium from deuterated water (D2O) into lipids in cultured cells and
genetically-manipulated mouse models that have been developed in our laboratory. Execution
of this research proposal will provide training in the application of metabolomics technology to
study a prevalent human disease and shed insight into the function of PNPLA3 and its role in
the progression of fatty liver disease.
Specific Aim 1: Determine the effect of PNPLA3-wt and the I148M on the lipidome of mice
Aim 1A: Develop LC-MS/MS methods to quantitate acyl-lipids
Aim 1B: Compare lipidomes of plasma, liver and adipose tissue in PNPLA3 mouse
models
Aim 1C: Determine effects of changes in dietary composition on hepatic lipid profile
Aim 1D: Compare lipidomes of plasma from humans homozygous for the non-risk (148I)
and the risk (148M) PNPLA3 variant.
Specific Aim 2: Define the metabolic basis of PNPLA3-associated fatty liver
Aim 2A: Measure lipid turnover using D2O in cultured hepatocytes expressing wt or
mutant PNPLA3
Aim 2B: Compare D2O with isotope labeled fatty acids and glycerol in cultured cells
Aim 2C: Determine lipid flux in PNPLA3 models using D2O
Aim 2D: Examine effects of changes in diet composition on lipid flux in mice
IMPACT:
Our previous work provided compelling evidence that genetic variation in PNPLA3 is a major
cause of fatty liver disease in the US population. The studies outlined in this proposal will
elucidate the metabolic mechanism by which the risk allele causes liver fat accumulation and
may provide the basis for new therapeutic options to prevent this potentially life-threatening
chronic disease.
Specific Aims
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Principal Investigator/Program Director (Last, first, middle):
RESEARCH STRATEGY
(a) Significance:
Non-alcoholic fatty liver disease (NAFLD) is an increasingly prevalent medical problem that affects
approximately a third of the US population (1). Hepatic steatosis alone has no known medical consequences.
However, the disease progresses to steatohepatitis and cirrhosis in a subset of individuals with steatosis.
Soon cirrhosis due to NAFLD will overtake hepatitis C as the major indication for liver biopsy (2).
Homozygosity for the PNPLA3-148M is associated with a 1.5-2-fold higher hepatic TAG content (3). The 148M
allele is associated with the full progression of NALFD, as well as alcoholic liver disease. In the Hispanic
population, where the prevalence of cirrhosis due to fatty liver disease is strikingly high, the frequency of the
PNPLA3 risk allele is 49% (4). Combatting this disease will require elucidation of the physiological role of
PNPLA3 and the mechanism by which the 148M allele results in liver disease. A series of studies using LCMS/MS based metabolomics and stable isotope labeling to elucidate the in vivo function of PNPLA3 and the
mechanism of 148M induced hepatic TAG accumulation is proposed. Understanding the functional role of
PNPLA3 in the progression of fatty liver disease is essential to designing effective treatment strategies of
NAFLD and to understand the progression of this disease.
(b) Innovation:
This proposal deploys innovative methods in novel animal models to determine the metabolic basis of fatty
liver disease, a major clinical disorder. A central aspect of the proposal is the development of LC-MS/MS
based methods to simultaneously determine both the concentrations and the flux of many lipid classes,
including triacylglycerides, phospholipids, cholesteryl esters, free fatty acids, diacylglycerides,
monoacylglycerides, and lysophospholipids. Currently, the overwhelming majority of biomedical studies assay
the concentrations of only a small number of individual metabolites. Such measurements provide limited
mechanistic insight into the underlying biological processes. Measurement of multiple metabolites in metabolic
pathways, referred to as metabolomics, is largely confined to a few specialized laboratories. Similarly,
conclusions regarding the flux of metabolites are frequently inferred from their concentrations, or from indirect
indices such as enzyme activities or mRNA levels assessed by real-time PCR. Here we will focus on lipid
metabolism, although the conceptual approach and methodology can be readily applied to other analytes.
LC-MS/MS metabolomics methods for the major lipid classes have been developed (for example by the
LIPID MAPS consortium (5)). These methods allow the rapid quantitation of hundreds of individual lipid
species, but extension of these techniques to determine rates of lipid flux has been very limited. We will
combine LC-MS/MS based lipidomics with stable isotope labeling, using deuterium or 13C to monitor the
incorporation of hydrogen or carbon into newly synthesized lipids. Hydrogen from water is incorporated into
most metabolites during biosynthesis. Therefore labeling with deuterated water (D2O) allows in vivo flux to be
determined on multiple metabolites simultaneously (6). This method can be used to determine the rate of
synthesis and turnover of essentially any molecule that can be measure by LC-MS/MS.
The second major innovation in this project is the and utilization of an animal model that recapitulates a
common form of human fatty liver disease. In 2008, the Hobbs-Cohen laboratory identified the first DNA
sequence variation associated with fatty liver disease (3). The variant substitutes methionine for isoleucine at
codon 148 in the gene encoding patatin-like domain-containing protein 3 (PNPLA3), and is associated with the
full spectrum of disease from steatosis to cirrhosis. Despite several mechanistic studies in genetically
manipulated mice that either overexpress or lack PNPLA3, the mechanism by which sequence variation in
PNPLA3 impacts liver fat content remains equivocal. Here we will pursue the metabolic underpinnings of
PNPLA3-associated fatty liver-disease using a novel mouse model in which the isoleucine codon at position
148 of the endogenous gene has been replaced with methionine. mRNA measurements using real-time PCR
indicate that expression of the modified allele is indistinguishable from that of the wild-type transcript. On high
carbohydrate diets, mice expressing the I148M allele have a two-fold increase in liver fat content, quantitatively
recapitulating the human phenotype.
The application of powerful metabolic flux measurements to a mouse model engineered to precisely
mimic the human disease-causing genetic variant should provide significant mechanistic insights into this
important disorder.
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(c) Approach:
Background and Rationale:
Patatin-like phospholipase domain containing protein 3 (PNPLA3) was first implicated in the
metabolism of hepatic triacylglycerides (TAGs) when our laboratory discovered that a missense variant in
PNPLA3 is strongly associated with non-alcoholic fatty liver disease (NAFLD). The culprit variant substitutes
methionine for isoleucine at position 148 (I148M) (3). PNPLA3 is a member of the patatin-like phospholipase
domain containing protein family and is primarily expressed in liver and adipose tissue in humans and mice (7).
The protein is tightly associated with membranes and partitions between the endoplasmic reticulum and lipid
droplets (8). PNPLA3 consists of a highly conserved N-terminal domain that shares sequence similarity with
patatin, a plentiful protein expressed in plants that has nonspecific acyl hydrolase activity. This domain
contains the catalytic dyad with a consensus serine lipase motif (Gly-X-Ser-X-Gly)(9). Substituting alanine for
the catalytic serine in the consensus motif (S47A) is predicted to yield a catalytically dead enzyme. PNPLA3 is
dramatically regulated by nutritional intake both at the transcriptional and the post-transcriptional level. During
fasting, the transcription of PNPLA3 is suppressed and the protein is actively degraded. Upon re-feeding,
PNPLA3 transcription is increased and degradation of the protein is retarded (10).
The phenotypic effect of the PNPLA3-I148M substitution is consistent with PNPLA3 playing a role in
fatty acid metabolism. Two hypotheses have been proposed for the molecular function of PNPLA3, which are
based primarily on in vitro assays. The first, suggested by Huang et al, proposes that PNPLA3 functions as an
acylglycerol hydrolase(11). Biochemical characterization of the purified protein reveals that the I148M
mutation causes a loss of hydrolytic activity. Arguing against a loss of function in PNPLA3 causing hepatic
steatosis is the finding that PNPLA3 knockout mice do not have increased hepatic TAG levels, even on a high
sucrose diet (12). The second hypothesis, suggested by Kumari et al, proposes that PNPLA3 has
lysophosphatidic acid acyl-transferase (LPAAT) activity (13). The I148M substitution increases LPAAT activity
when the protein is expressed in bacteria as a fusion protein with trigger factor. LPAAT catalyzes the
conversion of lysophosphatidic acid to phosphatidic acid, which is a key step in TAG biosynthesis. Evidence
that this activity is not responsible for the increase in hepatic TAG associated with the PNPLA3-I148M variant
is the finding that overexpression of the mutant (or wt) form of PNPLA3 in the livers of mice fails to promote
LPAAT activity (14). Thus, additional studies will be required to test the function of PNPLA3.
Our lab has established several model systems to investigate the function of PNPLA3. We have
adenoviral constructs to express wild type (wt), I148M, or S47A isoforms of PNPLA3 in cultured human
hepatocytes (HuH-7 cells) (11). We also have developed transgenic mice expressing similar amounts of wt
(PNPLA3wtTg) or mutant (PNPLA3148MTg) human PNPLA3 in either the liver or adipose tissue (14). The liverspecific PNPLA3148MTg mice have increased hepatic TAG content on a chow diet. The increase in hepatic TAG
is further exacerbated when the mice consume a high sucrose diet, which drives lipogenesis. In contrast, overexpression of similar amounts of wt PNPLA3 in the liver does not alter hepatic TAG levels (14). Since
overexpression of a heterologous protein may introduce artifacts, we have recently developed mice in which
either the I148M or the catalytically inactive S47A variant are knocked in to the mouse Pnpla3 gene. Neither of
these new mouse models develop hepatic steatosis on a chow diet, but ingestion of a diet high in sucrose is
associated with a ~2-fold increase in hepatic TAG content in both strains of genetically-modified mice (data not
shown). These mouse models, including the knockout (12), transgenic, and knock-in models, will be referred
to collectively as PNPLA3 genetically manipulated mice.
In this research proposal, high pressure liquid chromatography (HPLC) coupled to tandem mass
spectroscopy (LC-MS/MS) will be used to identify and measure metabolites that are influenced by expression
of PNPLA3 or the 148M variant. Then, a comprehensive analysis of the in vivo flux of fatty acid-containing
metabolites by stable isotope labeling will be performed. These experiments will reveal the molecular
mechanism of action of PNPLA3 and how the I148M mutation leads to hepatic lipid accumulation.
Preliminary Results:
Established methods to analyze TAG composition of tissues in PNPLA3 genetically manipulated mice:
The most robust phenotype associated with the I148M variant is accumulation of hepatic TAGs(3). Therefore,
we developed a LC MS/MS method to measure individual TAG species in cultured cells, plasma and tissues.
First, TAGs were separated from other lipids using a C-18 column with a linear solvent gradient (98% methanol
(MeOH)/2% dichloromethane to 86% MeOH/14% dichloromethane over 20 min). Under these conditions,
TAGs differing by a single double bond elute ~two minutes (min) apart with a peak width of ~20 seconds (sec),
shown in Figure 1. Using electrospray ionization, TAGs ionize without fragmentation so the number of
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Principal Investigator/Program Director (Last, first, middle):
carbons and double bonds in each TAG can be
determined based on the mass to charge ratio
in the first quadrapole (Q1) of the MS/MS.
Upon collision induced fragmentation in Q2, a
single fatty acid is lost from the TAG ion. The
fatty acid composition is determined by
scanning the mass of all fatty acids that could
potentially be lost from the ion in Q3. A library
of all measureable TAGs was created by pairing
the intact ion mass in Q1 with the mass of the
TAG missing one fatty acid in Q3. The library
generated from mouse liver included ~270
different TAG species. The fatty acid
Figure 1: Example chromatograph of selected 54
composition of each TAG was determined. A
carbon TAGs separated by HPLC. The TAGs are
similar approach was used to measure 24
cholesteryl esters species.
identified based on the number of carbons (1st
number), number of double bonds (2nd number), and
Altered TAG profile associated with hepatic
expression of PNPLA3:
the identity of the fatty acids (shown in parentheses).
The concentrations of 271 TAG species were
measured in the livers of wt, PNPLA3wtTg and PNPLA3148MTg mice (six 8-10 week old male mice/group). The
peak intensity of each species was normalized to an internal standard of 45:0 (15:0,15:0,15:0) and the ratio of
the mean peak intensity for the TAG in the PNPLA3wtTg to the wt mice was determined. No net change in TAG
level was observed between the two strains, but there were significant differences in the relative amounts of 47
of the TAG species that were measured (defined as a t-test p-value <0.05 with 6 mice per group) relative to the
wt mice. In general, the TAG that had an odd number of carbons or contained exclusively shorter chained fatty
acids (≤16 carbons) were increased while the levels of the TAG containing more than 4 double bonds were
decreased in the livers of the PNPLA3wtTg mice.
The livers of PNPLA3148MTg mice had an increase in the total TAG content and more than 60% of the TAG
species were increased relative to both the wt and wt-transgenic mice. The only species that were not
increased in PNPLA3wtTg mouse livers relative to wt were TAGs that contained more than 4 double bonds.
This trend of decreased polyunsaturated hepatic TAGs observed
in the livers of the PNPLA3wtTg mice
was accentuated in the PNPLA3148MTg
mice (Figure 2).
In vivo turnover of TAGs in
cultured hepatocytes (HuH7 cells)
expressing recombinant PNPLA3:
The rate of TAG synthesis in HuH-7
cells infected with recombinant
adenoviruses expressing wt or
mutant PNPLA3 was measured using
D2O labeling. Cells were grown in
DMEM plus 10% FCS and then 5% of
the medium was substituted with D2O
and grown for additional 24 hrs. Cells
were collected at the indicated time
points (Figure 2) and the TAG was
Figure 2: A) Example of a PUFA containing TAG that is
isolated, and 32 TAG species were
decreased in PNPLA3-ITg liver. B) Example of TAG increased in
assayed using LC-MS/MS as
PNPLA3-ITg liver. C) Fold change of hepatic TAGs in PNPLA3described above. The rate of
IMTg with the specified number of double bonds. Each point
increase in labeled triolein was
represents a different TAG species. D) Average fold change of
greater in the cells expressing
hepatic TAGs with a 0-10 double bonds in livers of PNPLA3PNPLA3-WT and slower in cells
WTTg (blue) and PNPLA3-IMTg (red) relative to wt mice.
expressing PNPLA3- I148M than in
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Principal Investigator/Program Director (Last, first, middle):
those infected with an empty virus. A higher rate of deuterium incorporation implies a faster rate of TAG
synthesis if the cells have equivalent concentrations of
TAGs. The cells over expressing I148M-PNPLA3 had a
A)
TAG content that was 80% higher than the cells
expressing PNPLA3-wt or the empty vector. The relative
TAG synthesis associated with expression of either
PNPLA3-WT or PNPLA3-I148M was equivalent and
about 25-30% higher than cells infected with an empty
vector (Figure 2B). Therefore, increased TAGs caused
by PNPLA3-148M cannot be solely attributed to
increased synthesis. Additional studies, such as
measuring turnover in the tissues of genetically-modified
mice and using alternative stable isotope labels (for
example fatty acids or glycerol), will be required to
B)
elucidate the function of PNPLA3.
Specific Aim 1: Determine the effect of PNPLA3-wt
and PNPLA3-I148M on the lipidome of mice
Rational: PNPLA3 plays a role in the metabolism of a
diverse set of metabolites comprising thousands of
chemically distinct molecules. The most abundant acyllipids in human tissues are phospholipids (PL), TAGs,
sphingolipids, and cholesteryl esters (CE). The
complexity of these lipid classes is due primarily to the
diversity of fatty acids. At least 50 different fatty acids
can be detected in human plasma (15). These fatty acids
Figure 3: Turnover of TAGs in cells
vary in length from 12 to 24 carbons and can contain up
overexpressing PNPLAS measured with
to 6 double bonds at any location in the fatty acid.
D2O. The turnover was measured in Huh-7
Molecules containing multiple fatty acid chains can
cells over expressing PNPLA3-wt or I148M.
incorporate a multitude of combination of fatty acids,
The fraction of TAGs newly synthesized (dresulting in thousands of fatty acid combinations for a
TAG/Total TAG) was determined using mass
single class of lipids. Traditional methods for isolating
isotopomer distribution analysis (MIDA).
and quantifying lipid species using thin layer
chromatography (TLC), gas chromatography (GC), and/or biochemical assays have traditionally been
insensitive, non-specific, and time consuming. LC-MS/MS provides an excellent alternative to these
approaches that will be more accurate, more sensitive and less laborious.
Modern triple quadrapole mass spectrometers permit single mass unit selectivity and sub-nanogram
sensitivity. Lipids that differ by only a single double bond or carbon can be distinguished based on their mass
to charge ratio (m/z), and each component fatty acid can be identified based on its neutral loss by collision
induced fragmentation. However, MS/MS alone is not sufficient to accurately quantify individual lipid species.
Due to natural isotopic natural abundance there is overlap between a lipid of interest and the M+2 isotopic
peak of a similar lipid with one additional double bond. This results in convolution of multiple lipid species at a
single mass. These lipids can be deconvoluted by incorporating a chromatographic separation (HPLC) prior to
the MS/MS. The HPLC eluent can be introduced directly into the MS via an atmospheric-pressure ionization or
electrospray source. Therefore, by combining HPLC and MS, individual lipid species can be quantified
accurately. This approach has been used by multiple investigators to develop LC-MS/MS techniques that can
quantify the concentration of many lipids in a biological sample.
Aim 1A: Develop LC-MS/MS Methods to Quantitate Acyl-Lipids
Here I will implement methods that have been developed by the LIPID MAPS consortium and others to
measure the concentration of fatty acid-containing lipids in plasma, liver and adipose tissue, including TAGs,
CEs, PLs, diacylglycerides (DAG), monoacylglycerides (MAG), lysophospholipids, acyl-CoAs, and free fatty
acids. An LC-MS/MS system (ABSciex Qtrap 4000 triple quadrapole with a Shimadzu LC20A HPLC) will be
used for these experiments. Since the LC-MS/MS methods for each class of lipids have been developed and
reported, it is anticipate that establishing these assays in our laboratory will take between 4 and 8 weeks per
lipid class. If problems arise during the implementation of methods, Dr. Jeff McDonald, a member of the LIPID
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Principal Investigator/Program Director (Last, first, middle):
MAPs consortium and a faculty member in my department, will be consulted (see letter). If we hit roadblocks, I
will consult Drs. Jose Castro and Tom Roddy, who are in the small molecule mass spectroscopy division at
Merck; these investigators are collaborating with our laboratory on another project and have agreed to help
troubleshoot any problems I encounter in setting up these assays or interpreting the data generated. Each
method will be designed to fully analyze a sample in less than 30 min, which will assist in rapid implementation
of methods to measure each lipid class in many samples.
Potential Problems and Alternative Strategies:
The methods that will be implemented have several potential drawbacks. First, it is impractical to obtain
authentic internal standards for each lipid species, thus it is not possible to calculate the absolute concentration
of each lipid. Where practical, standards will be used to quantify the absolute concentration of a lipid species.
Lipids for which no internal standard is available will be normalized to a single internal standard (specific to the
lipid class). This analysis will provide relative concentrations of each lipid species. Second, the fatty acid
double bond location (i.e. ω3, 6, or 9 fatty acids) cannot be differentiated using the proposed LC-MS/MS
method. If it is necessary to identify the location of the double bond, an orthogonal method, such as GC-flame
ionization detection (GC-FID), can be used. Our laboratory has an established method using an HP6890
instrument to measure saponified TAG composition using TLC and GC-FID (14). Lastly, using different LC
MS/MS methods for each lipid class can potentially be cumbersome if different columns and mobile phase
combination are used. To minimize this challenge, methods that utilize identical HPLC solvents and columns
will be developed.
Aim 1B: Compare lipidomes of plasma, liver and adipose tissue in the PNPLA3 mouse models
As described previously, our laboratory has established several lines of transgenic and knock-in mice
that express various forms of PNPLA3. This collection includes transgenic mice overexpressing PNPLA3-wt or
-148M in liver (under the control of the ApoE promoter) or in adipose tissue (under the control of the AP2
promoter) (14), and PNPLA3 148M and 47A knock-in mice (unpublished data). We also have Pnpla3-/- mice
that were obtained from Erin Kershaw and have been characterized extensively in our laboratory (12). To
glean insights into the effects of PNPLA3 on lipid composition, we will characterize and compare the lipidomes
of these different mouse strains.
To determine the effect of PNPLA3 expression on the lipidome of tissue, we will extract the lipids from
the liver, adipose tissue and plasma using the LC-MS/MS methods developed in Aim 1A. For each
experiment, we will use six 12-week-old male, chow-fed genetically-modified mice and an equal number of
wild-type littermate controls. Mice will be sacrificed and liver, WAT, and plasma will be collected and store at 80C. Tissues will be homogenized and lipids extracted using a Bligh-Dyer extraction. The concentration of
each lipid species will be measured by LC-MS/MS. The lipid level in each mouse model will be compared to
the wt littermate controls and significant differences between means will be determined by a t-test. In addition,
trends in the lipids most affected by PNPLA3 will be identified, such as a reduction in PUFA containing TAGs
shown in preliminary data.
Aim 1C: Determine effects of changes in dietary composition on hepatic lipid profile
The accumulation of hepatic TAGs in the PNPLA3148MtTg mice is increased when the mice are fed a highsucrose diet (14). Although these mice also develop steatosis on some chow diets, they do not develop
steatosis on all chow diets (unpublished data). When the mice are fed a chow diet that contains animal fat
source, the PNPLA3148MtTg have 50% higher hepatic TAGs than PNPLA3wtTg mice. However, when vegetable
fat replaces the animal fat, the PNPLA3148MtTg and PNPLA3wtTg mice have similar levels of hepatic TAG.
These differences are not caused by any differences in PNPLA3 mRNA or protein expression (unpublished
data). This suggests that PNPLA3 I148M induced NAFLD can potentially be alleviated by dietary modification.
To investigate the role of diet in PNPLA3 function, the lipidome of the PNPLA3 mouse models on the two
different chow diets will be characterized. For these experiments, I will use 8-10 week-old male mice (6/group)
that have been fed either of the two chow diets for four weeks. The tissues will be processed and the results
analyzed exactly as described above.
I will also examine the effect of a high-sucrose,+/- polyunsaturated fatty acids (PUFA) on tissue lipid
composition. Since over-expression of PNPLA3 results in a reduction in TAG: PUFAs, supplementing PUFAs
into a high-sucrose diet may result in less accumulation of hepatic TAGs in the PNPLA3148MtTg mice. Other
dietary modifications may also be tested, depending on results obtained from the studies described in Aim 1B.
The choice of mouse models will also depend on the results of Aim 1B. Likely, I will use the I148M knock-in
mice for these studies.
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Principal Investigator/Program Director (Last, first, middle):
Aim 1D: Lipidome of human plasma with PNPLA3 II or MM
We have collected plasma samples from age-, sex- and race-matched subjects who are homozygotes for the
PNPLA3-148I or the PNPLA3-148M variant (100/group). We will analyze the lipidome of these samples using
the methods developed in Aim 1A. The lipids from 100 μL of plasma will be extracted with a Bligh-Dyer
extraction and concentrated. After extraction, the samples will be analyzed by LC-MS/MS and the level of
each lipid species will be quantified. Metabolites that differ significantly between the two genotypes will be
identified using a t-test. A separate set of samples taken from the Dallas Heart Study will be analyzed to
confirm any observed associations. These results will then be compared to the plasma lipidomes of the
PNPLA3 mouse models, which will allow us to address two questions:
1) Are there any changes in the profile of acyl-containing lipids that relate to the susceptibility allele in
PNPLA3?
2) How well do the lipid distributions in the plasma of the mouse models expressing the PNPLA3-I148M variant
reflect those of humans with the same variant?
Specific Aim 2: Role of PNPLA3 in Lipid Flux
Rationale: Regulated synthesis and catabolism of metabolites, known as metabolic flux, is the central function
of metabolism. Measuring metabolic flux has traditionally been a technically challenging aspect of metabolic
research. A standard method to measure metabolic flux is with radioactive isotope labeling. For example, the
flux of TAGs in vivo can be measured by injecting tritium-labeled water into the circulation of an animal. The
tritium is incorporated into TAGs. After an hour of labeling the animal is sacrificed, the organs of interest are
collected, the lipids are extracted, TAGs are isolated from a TLC plate, and the radioactivity of the TAGs is
measured. Using this methodology, the rate of TAG synthesis can be measured and compared between
groups of animals. Usually, flux is not measured directly, but rather is inferred from the measurement of the
concentration of a metabolite. It is assumed that an increased in the amount of a metabolite is the result of
either an increase in synthesis or a decrease in catabolism. This assumption is derived from the law of first
order kinetics, which states that for a molecule to accumulate in a steady state system there must either be an
increased rate of appearance (i.e. synthesis) or a decreased rate of disappearance (i.e. catabolism or
clearance). In some biological systems this is a valid assumption. However, this assumption is not valid for
PNPLA3148MTg mice.
When PNPLA3 is overexpressed in mouse liver (the PNPLA2wtwtTg mice) there is no excess
accumulation of hepatic TAGs relative to wt mice (14). The PNPLA3148MTg mice have ~60% more hepatic TAG
than PNPLA3wtTg mice. Assuming first order kinetics, one would predict that the PNPLA148MTg mice have a
higher rate of TAG synthesis than the PNPLA3wtTg or wt mice, and the PNPLA3wtTg and wt mice have similar
TAG synthesis rates. When tritiated water was used to measure TAG synthesis, the two strains of transgenic
mice had similar rates of hepatic TAG synthesis, and both transgenic models had ~30% higher synthesis rate
than wt mice (14). Therefore, a more refined model is required to describe the mechanism of PNPLA3-I148M
induced NAFLD.
Using radioisotope labeling has numerous technical limitations that hinder the ability to develop and
validate models of the mechanism of PNPLA3. To limit exposure to radioactivity, labeling experiments are
generally performed on a very short time scale, for example, after only one hour for TAG synthesis. The halflife of mouse hepatic TAGs is ~3 days. Therefore, the labeling protocol only samples the most rapidly turningover pool of TAGs. Tritiated water labeling also cannot be directly translated into human due to the toxicity of
radioactivity. Finally, the radio-labeling methods for measuring flux are inherently non-specific and cannot
utilize modern metabolomics technologies. Stable isotope labeling is an alternative approach to measuring
metabolic flux in vivo and does not have the same technical drawbacks associated with radio-labeling.
The principle of stable isotope labeling is similar to radio-labeling. A stable isotope labeled substrate,
such as deuterated water (D2O), is administered to an animal. The isotope label, which has a higher molecular
weight than a non-labeled substrate, will incorporate into metabolites via biosynthesis causing an increase in
the molecular mass of the newly synthesized metabolites (5). The mass shift is measured using a mass
spectrometer and the fraction of newly synthesized metabolites is calculated using mass isotopomer
distribution analysis (MIDA) (16). Combining LC-MS/MS based metabolomic methods with stable isotope
labeling will allow the measurement of metabolic flux of thousands of molecules simultaneously and will be a
powerful tool for understanding the mechanism of PNPLA3. In this aim, in vivo stable isotope labeling will be
used to measure the role of PNPLA3 on lipid flux in cultured hepatoma cells overexpressing PNPLA3 and in
PNPLA3 genetically manipulated mouse models.
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Principal Investigator/Program Director (Last, first, middle):
Aim 2A: Measure Lipid Flux using D2O in Cultured Cells Over-Expressing PNPLA3
In this set of experiments,
cultured hepatocytes
overexpressing recombinant
PNPLA3-wt, PNPLA3-I148M and
PNPLA3-S47A will be used to
assess the effect of the I148M
variants on lipid flux. D2O will be
used to monitor the flux of fatty
acid-containing metabolites in
these cells.
HuH-7 cells will be
infected with recombinant
Figure 4: Experimental plan for in vivo lipid labeling using D2O in HuH-7
adenoviruses expressing the
cells infected with PNPLA3-wt, PNPLA3-148M, PNPLA3-47A, or an
various forms of human
empty virus, as described in Aim 2A.
PNPLA3. Cells will be grown for
3 days in DMEM plus 10% FCS
medium. On day 4 the medium will be supplemented with 5% D2O. The cells will be harvested at several time
points over the next 48 hours. Twelve time points in duplicate will be harvested for each condition. The time
points will be exponentially spaced with the majority of the cells harvested in the first 8 hours. After harvesting
the cells, the lipids will be isolated using a Bligh-Dyer extraction and then the lipids will be analyzed by LCMS/MS using the methods developed in Specific Aim 1. This experimental plan is illustrated in Figure 4.
Five isotopomers (M=0 to M=4) will be monitored for each lipid species to determine the change in the isotopic
distribution. If any problems arise during the development of these experiments, I will consult with Drs.
Elizabeth Parks and Ralph Deberadinis (see letters), who are experts in using stable isotopes to measure
metabolic flux.
The relationship between the fraction of molecules newly synthesized and time is based on the
change in the isotopic distribution. Based on this relationship, flux parameters (e.g., metabolite turnover, halflife and rate of synthesis) will be quantified. The effect of PNPLA3 on the flux of lipid will be determined by
comparing the quantitated rate of synthesis and half-life in each group of cells infected with different viruses
containing PNPLA3. To assist in data analysis and quantification, Matlab algorithms have been programed for
MIDA and kinetic parameter regression.
This approach will be applied to most metabolites measured by the LC-MS/MS methods developed
in Aim 1A. With these tools, it is feasible to monitor the turnover of hundreds, if not thousands, of individual
metabolite species in vivo.
Potential Limitations and Alternative Strategies:
Quantifying the rate of synthesis of each lipid species requires using MIDA or other similar techniques. To
apply MIDA, the number of label incorporation sites in a molecule (N; i.e. the number of deuterium atoms that
can incorporate into a single lipid) must be assigned (17). Deuterium can be metabolically incorporated at
numerous steps during lipid assembly. For example, deuterium can be incorporated in TAGs during fatty acid
synthesis, fatty acid elongation, or during esterification. Therefore, there is uncertainty about the value of N for
most lipids. To overcome this limitation the fraction of the water pool that is labeled with D2O (p) must be
assigned. The value of p can be calculated based on a molecule with a known N value, for example
cholesterol, or determined by GC-MS. After assigning a value of p, N of each lipid species can be calculated
using a non-linear regression. The uncertainty associated with this regression complicates direct comparison
between different lipids species. To overcome this uncertainty, alternative stable isotope labels with a known
N value, such as 13C labeled fatty acids or glycerol, will be used (see Aim 2B). These alternative labeling
strategies will also provide an orthogonal method to confirm that any results are not due to artifacts associates
with using D2O labeling.
Aim 2B: Alternative stable isotope labeling strategies in cultured cells over-expressing PNPLA3
While D2O is an excellent isotope label for monitoring the flux of most metabolites, there are some limitations
to this approach (see above). Glycerolipids can also be labeled by providing stable isotope labeled
metabolites, such as 13C-fatty acids or 13C-glycerol, which will be incorporated into a lipid species in a known
stoichiometric ratio (18). These labels also monitor a subtly different process than water labeling. Lipid
biogenesis involves two broad steps: 1) de novo fatty acid synthesis and 2) assembly and reassembly of lipids.
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Principal Investigator/Program Director (Last, first, middle):
The rate-limiting step in de novo lipid biogenesis is fatty acid synthesis. Since deuterium from D2O incorporates
primarily during fatty acid synthesis, D2O only monitors this process. Lipid assembly and reassembly,
commonly known as “futile cycling”, occurs at a faster rate. By labeling with the preformed lipid components,
such as fatty acids and glycerol, the rate of lipid assembly and fatty acid exchange can be measured in vivo.
To address the functional mechanism of PNPLA3, 13C-3-glycerol, 13C-U-palmitate, 13C-U- oleate, and 13C-Uarachidonate will be used to monitor lipid flux in cultured hepatoma cells overexpressing wt and mutant
PNPLA3. HuH-7 cells will be infected with adenoviruses expressing PNPLA3-wt, PNPLA3-148M, PNPLA347A, or an empty virus and cultured for 3 days in DMEM with 10% FCS. On day 4, stably isotope label
substrate will be added to each dish of cultured cells. After adding the labeled substrate, cells will be
harvested over a time course. Appropriate timing of sampling and concentration of stable isotope labeled
substrate will be determined empirically. After harvesting, lipids will be extracted and analyzed by LC-MS/MS.
Rate of incorporation will be determined by analyzing the fraction of each lipid species with incorporated label.
Each group of infected cells will be compared to each other and to the incorporation when D2O was used for
labeling. If necessary, these experiments will be extended to primary hepatocytes or to the PNPLA3
genetically-manipulated mouse models.
Aim 2C: Lipid flux in PNPLA3 genetically manipulated mouse using D2O
Although many facets of PNPLA3 biology can be addressed in cultured cells, an animal model must be
utilized to define the bona fide physiological function of the protein. D2O will be used to measure the
metabolic flux in genetically manipulated mouse models. The experiments will be conducted in a similar
manner to the cell culture experiments proposed in Aim 2A. The body water pool of the mice will be enriched
by injecting 500 μL of D2O (in physiological saline) into the circulation of 6 mice of each genotype. After
injection, the water source of the mice will be supplemented with 6% D2O. This causes a rapid enrichment of
D2O in body water to around 5%(6). After injection, tissue samples from the animals will be collected in a time
course with exponentially timed sampling. For example collections may be made at 0, 1, 2,4, 7, 10, 14, 24, 32
and 48 hours after injection of D2O. In the first series of experiments, 10 μL of plasma per mouse will be
collected at each time point. Lipids will be extracted from plasma and analyzed by LC-MS/MS. This
experimental plan is illustrated in
Figure 5. The rate of synthesis
and half-life of each measureable
lipid will be determined based on
the rate of incorporation of
deuterium. Each mouse model
will be compared based on the
quantified rate of synthesis and
on the qualitative characteristics
of the incorporation. Plasma will
be analyzed before individual
tissues because fewer mice are
Figure 5: Experimental plan for in vivo lipid labeling using D2O in
needed for each experiment,
PNPLA3 genetically manipulated mice, as described in Aims 2C and 2D
allowing more efficient
optimization of the dosing and
collection procedures.
Once the optimal time course of sampling is determined, the experiment will be repeated using 10 to
20 mice per group. Each mouse will be injected with 500 μL D2O and tissues collected at the pre-specified
time points. Lipids from the liver and WAT will be analyzed by LC-MS/MS and the rates of synthesis of
selected lipids will be determined based on rate of deuterium incorporation. The exact number of mice per
group and timing of the sampling will be dependent on the rate of incorporation in plasma and the availability of
genetically manipulated mice. The results obtained from the different groups of mice will be compared based
on the quantitative rate of synthesis and the qualitative characteristics of deuterium incorporation. This
experiment will reveal the influence PNPLA3 and the I148M variant on lipid flux. The metabolic role of the
protein should be apparent from these results.
Aim 2D: Dietary Influence on Lipid Flux and the Contribution of PNPLA3
PNPLA3 function is influenced by diet; therefore dietary modification may provide a potential
therapeutic strategy to alleviate NAFLD in homozygote MM patients. In Aim 1C, we will examine the effect of
differences in diets on the lipidome in the PNPLA3 genetically-modified mouse models. Ideally, a dietary
Research Strategy
Page 88
Principal Investigator/Program Director (Last, first, middle):
modification will be identified that lowers hepatic TAGs in either the PNPLA3148MTg or the PNPLA3-I148M
knock-in mice. When such a diet is identified, in vivo lipid flux in mice consuming that diet will be measured.
This experiment will be conducted as described in Aim 2C. Six animals per group per diet will be injected with
500 μL D2O and the water will be supplemented with D2O. At 10 time points, 10 μL of plasma per mouse will
be collected. The plasma lipids will be extracted and analyzed by LC-MS/MS to determine the rate of
deuterium incorporation. The rate of synthesis will be determined based on the rate of deuterium
incorporation. Mouse models on different diet will be quantitatively compared based on the rate of lipid
synthesis. If necessary, a time course of tissue collection after D2O administration. This will depend on the
correlation between liver and plasma lipid turnover, established in Aim1C. By measuring the lipid flux in these
animals the metabolic mechanism by which dietary change modifies PNPLA3 148M pathology will be
determined.
Research Strategy
Page 89
Principal Investigator/Program Director (Last, first, middle):
Protection of Human Subjects:
In sub-aim 1D of this research proposal I will analyze 200 human plasma samples. These
samples include 100 homozygote patients with PNPLA3 II and MM. I will use approximately
100 μL of plasma from each subject to determine the effect of PNPLA3 I148M on the human
lipidome. The samples were collected last year, so analysis of the plasma samples will cause
no additional risk to the subjects. The cohort of subjects includes approximately 50% female
and 33% African Americans, 33% Hispanics, and 33% whites. No children were used in this
study because they are not afflicted with atherosclerosis.
Protection of Human Subjects
Page 90
Principal Investigator/Program Director (Last, first, middle):
Inclusion of Women and Minorities:
The subjects for this proposal comprise an equal portion of women and men. In addition, the
study is racially diverse including equal portions of African America, Hispanic, and White
subjects. No gender or racial background was excluded from this study.
Women &Minorities
Page 91
Principal Investigator/Program Director (Last, first, middle):
Targeted/Planned Enrollment:
This proposal uses a databank of plasma samples that have already been collected from a
gender and ethnically diverse population of subjects. No additional enrollment of patients is
expected for this research plan.
Planned Enrollment Table
Page 92
Principal Investigator/Program Director (Last, first, middle):
Inclusion of Children:
Children were excluded from this research study. Children were excluded because they do not
generally exhibit risk of atherosclerosis, so the profile of their lipidome will be irrelevant to this
study.
Children
Page 93
Principal Investigator/Program Director (Last, first, middle):
Vertebrate Animals
Description of Animals:
In my research plan, I propose using wild type C57BL/6 mice or genetically modified mice that
have been bred into a C57BL/6 background. The genetically modified mice will include
PNPLA3 knock-out mice, PNPLA3 liver and adipose tissue specific transgenic mice, and
PNPLA3 I148M and S47A knock-in mice. The mice will be at least 8 weeks old for all
experiments. I will use both female and male mice. I will use approximately 500 mice for this
research proposal.
Why Animals:
Lipid metabolism is a complex process that is intrinsically linked to systematic biological factors
such as intracellular signaling, diet, immune system, etc. Although cell culture models can
provide some insight into the function of PNPLA3, animal models will be required to determine
the true function. Lower organisms, such as flies or c. elegans, do not recapulate human lipid
physiology therefore a rodent model will be required.
Veterinary care of animals:
The animals will be housed in UT Southwestern animal housing facility located in the Y building
2nd floor and exposed to 12 hour light and 12 hour dark cycle. Normal chow or specialized diets
and water will be freely available to the animals. Cages will be routinely changed to ensure a
clean environment for the animals.
Procedures to minimize discomfort, stress or pain of anmials:
Mice will be sacrificed anaesthetized by inhaling isoflurane in a big chamber. All animal
procedures will be approved by UT Southwestern’s institutional animal care and use committee
(IACUC) before executing the experiment.
Animal Euthasnasia:
Surplus mice that do not qualify for proposed experiments will be euthanized via either carbon
dioxide or isoflurane inhalation. These two methods are consistent with the AVMA Guidelines of
Euthanasia, 2012.
Vertebrate Animals
Page 94
Principal Investigator/Program Director (Last, first, middle):
Select Special Agents:
No select agents will be used in this research proposal
Select Agent Research
Page 95
Principal Investigator/Program Director (Last, first, middle):
Consortium/Contractual Arrangement:
This research proposal does not involve a consortium of researchers. All research will be
performed at UT Southwestern.
Consortium/Contractual
Page 96
Principal Investigator/Program Director (Last, first, middle):
Resource Sharing Plan:
A resource sharing plan is not required for this proposal because it does not involve developing
model organisms, genome wide association studies, and it does not request more than
$500,000 per year.
Resource Sharing Plan
Page 97
SUMMARY STATEMENT
( Privileged Communication )
PROGRAM CONTACT:
Richard Okita
301-594-3827
okitar@nigms.nih.gov
Release Date:
Application Number:
Principal Investigator
05/29/2013
1 K01 GM109317-01
PHD
Applicant Organization: UT SOUTHWESTERN MEDICAL CENTER
Review Group: ZRG1 IMST-K (50)
Center for Scientific Review Special Emphasis Panel
Mentored Research Scientist Development Award in Metabolomics
Meeting Date: 05/22/2013
Council: AUG 2013
Requested Start: 09/01/2013
Project Title:
SRG Action:
Next Steps:
Human Subjects:
Animal Subjects:
Gender:
Minority:
Children:
Project
Year
1
2
3
4
___________
TOTAL
RFA/PA: RM12-025
PCC: P140RO
Dual PCC: 1ME201
Dual IC(s): RM
Determining the Function of PNPLA3 Utilizing Metabolomics and Stable Isotope
Labe
Impact Score: 38
Visit http://grants.nih.gov/grants/next_steps.htm
30-Human subjects involved - Certified, no SRG concerns
44-Vertebrate animals involved - SRG concerns
1A-Both genders, scientifically acceptable
1A-Minorities and non-minorities, scientifically acceptable
3A-No children included, scientifically acceptable
Clinical Research - not NIH-defined Phase III Trial
Direct Costs
Requested
95,559
96,926
98,334
99,783
_______________
390,602
Estimated
Total Cost
103,204
104,680
106,201
107,766
_______________
421,850
ADMINISTRATIVE BUDGET NOTE: The budget shown is the requested budget and has not been
adjusted to reflect any recommendations made by reviewers. If an award is planned, the costs will be
calculated by Institute grants management staff based on the recommendations outlined below in the
COMMITTEE BUDGET RECOMMENDATIONS section.
1 K01 GM109317-01
2
ZRG1 IMST-K (50)
1K01GM109317-01
SCIENTIFIC REVIEW OFFICER'S NOTES
VERTEBRATE ANIMAL UNACCEPTABLE
RESUME AND SUMMARY OF DISCUSSION: This application for a K01 mentored research scientist
development award in metabolomics proposes to extend the principal investigator’s training in
biochemical engineering, biophysics, molecular genetics and lipidomics by utilizing targeted liquid
chromatography-tandem mass spectrometry (LC-MS-MS) methods to determine the flux of different
classes of fatty acid-containing lipids in liver, adipose tissue, and plasma in mouse models of PNPLA3associated steatosis. This project will be carried out under the direction of Professors Helen Hobbs and
Jonathan Cohen at the University of Texas Southwestern Medical Center. Reviewers noted that the
applicant is highly regarded by his advisors and mentors and is a productive and enthusiastic research
scientist. While early in his career, he was considered to have demonstrated innovation by developing
methods for the analysis of multiple triglyceride species and also has demonstrated an appreciation for
the need to combine metabolomics concentration measurements with flux measurements. The
mentoring team was viewed as having significant experience in training PhD and postdoctoral students
as well as excellent publication and funding records. While the career development plan addresses
several areas of importance to establishing an independent research career, it was felt that it did not
address the PI’s lack of background in biological sciences and did not define a plan to establish an
independent career. The research plan was considered to be outstanding and was noted to do an
excellent job of combining metabolomics with flux analysis. The complementarity of the in vitro and in
vivo uses of labeling and metabolomics to evaluate the pathways of lipid metabolism with changes in
PNPLA3 function was specifically cited as a strength of the application. While the environment at
UTSW was considered to be outstanding, the institutional commitment to
was noted to be
weak. At the conclusion of the discussion, enthusiasm for the strengths of the sponsors, the applicant,
the research plan and the institutional environment was dampened somewhat by weakness described
for the training plan and institutional support and as a result, the panel noted the potential overall
impact/merit of the proposal to be excellent.
DESCRIPTION (provided by applicant): Non-alcoholic fatty liver disease (NAFLD) is an increasingly
prevalent medical problem that affects approximately a third of the US population. Patatin-like
phospholipase domain containing 3 (PNPLA3) was first implicated in the metabolism of hepatic
triacylglycerides (TAGs) when our lab found that a missense mutation that substitutes isoleucine at
position 148 with methionine (I148M) was associated with non-alcoholic fatty liver disease in humans.
Homozygotes with the variant allele (148M) have a 2-fold higher risk of hepatic TAG accumulation than
homozygotes with the wild type allele (148I). The function of PNPLA3 and the cause of I148M induced
hepatic TAG accumulation remains unclear. Since fatty liver represents an accumulation of fatty acids
in the form of triglycerides, I will adapt and/or develop targeted liquid chromatography-tandem mass
spectrometry (LC-MS-MS) methods to measure the concentrations of different classes of fatty acidcontaining lipids in liver, adipose tissue, and plasma from mouse models of PNPLA3-associated
steatosis. Mice will be studied on both a normal chow diet and on diets designed to exacerbate or
alleviate PNPLA3-I148M associated accumulation of hepatic TAG. I will then extend these LC-MS/MSbased methods to determine the flux of each lipid class by measuring the rate of incorporation of
deuterium from deuterated water (D2O) in cultured cells that overexpress PNPLA3, and in genetically
manipulated mouse models. Alternative stable isotope labeling strategies, such as 13C-glycerol or 13Cfatty acid incorporation, will also be employed to compliment the D2O incorporation data. Execution of
this research proposal will provide training in the application of metabolomics technology to study
human disease and shed insight into the function of PNPLA3 and its role in the progression of fatty liver
disease.
1 K01 GM109317-01
3
ZRG1 IMST-K (50)
PUBLIC HEALTH RELEVANCE: Fatty liver disease is a growing epidemic that afflicts a third of the
US population and leads to steatosis, cirrhosis, and liver failure. We have identified a gene named
PNPLA3 that is strongly associated with fatty liver disease in human, but the function of this gene
remains unclear. I am proposing experiments designed to determine the function of PNPLA3 and
ultimately aid in the development of effective medical therapies to prevent fatty liver disease.
CRITIQUE 1:
Candidate: 2
Career Development Plan/Career Goals & Objectives/Plan to Provide Mentoring: 2
Research Plan: 1
Mentor(s), Co-Mentor(s), Consultant(s), Collaborator(s): 1
Environment and Institutional Commitment to the Candidate: 2
Overall Impact: This is an application from a postdoctoral scholar who wants to receive training to be
able to run a service metabolomics lab with expertise in stable isotope technology. This will allow
metabolomic data to be complemented by flux measurements. This variation of metabolomics has great
potential for the discovery of new regulatory processes. This training will take place in a Genetics lab
which generated very interesting data on the mechanism of liver steatosis. The involvement of experts
in lipidomics, metabolism and stable isotope technology, as well as the rich environment of UTSW,
bodes well for the success of this project.
1. Candidate:
Strengths
The applicant,
, has received excellent training in Biophysics of lipids with Dr Donald
Small. He should have no problems mastering the metabolomics techniques of his project.
Weaknesses
It is not clear how much
has learned about metabolism and isotopic techniques
before starting his postdoctoral scholarship. He will not get such training from the experts in
Genetics who will co-mentor him (Dr Hobbs and Cohen who initiated this excellent project on
the consequences of mutation in PNPLA3). It will thus be important that
get training
in metabolism from Drs Parks and DeBerardinis, and from other metabolic experts at UTSW. On
page 70,
acknowledges that he needs training in a number of disciplines (molecular
biology, genetics and animal husbandry) but does not mention metabolism.
2. Career Development Plan/Career Goals & Objectives/Plan to Provide Mentoring:
Strengths
career plan (briefly outlined page 70) is to run a service lab which will assist other
investigators. “Independent research” is just mentioned passing by. There is nothing wrong in
running a service lab. Actually, many metabolomics units are set up to provide services to
biological investigators. In fairness to the applicant, it is a bit early in his career to have a clear
idea of a possible future independent research.
Weaknesses
None noted
3. Research Plan:
1 K01 GM109317-01
4
ZRG1 IMST-K (50)
Strengths
The applicant correctly points out that classical metabolomic studies yield only vague
conclusions on the impact of a perturbation on metabolomics fluxes. It is erroneous to interpret
variations in relative concentrations as reflecting corresponding variations in fluxes. Assessment
of variations in metabolic fluxes requires isotopic tracer studies conducted under rigorous
conditions to avoid artifacts and errors in data interpretation. The association of metabolomics
and mass isotopomer analysis provides a powerful tool to identify new regulatory mechanisms
and new pathways. The applicant understands this very well, and benefits from the mentorship
of an expert in lipidomics, Dr Jeffrey McDonald, and experts in mass isotopomer analysis, Drs
Elizabeth Parks and Ralph DeBerardinis.
The planning of the components and phases of the project is very logical with the right mixture
of in vivo and in vitro experiments, using rodent and human materials.
The project centers on lipidomics. Although there a many types of lipids, the number of their
building blocks is reasonable, and is made up of fairly few types of compounds. Thus the
analytical techniques to be used can crank out data rapidly without having to identify totally
unknown compounds.
Weaknesses
One would have liked to see more detail on the measurements of isotopic enrichments of lipids
containing many carbons, where the naturally-enriched M1, M2 mass isotopomers have similar
abundances as the unlabeled M peak.
The section on the use of other tracers than D2O is only sketchily outlined.
4. Mentor(s), Co-Mentor(s), Consultant(s), Collaborator(s):
Strengths
Dr McDonald has much expertise in lipidomics
Drs Parks and DeBerardinis have great expertise in mass isotopomer analysis applied to
lipogenesis and to pathway regulation
Should the need arise, there is much expertise in metabolic NMR at UTSW (Drs Craig Malloy
and Shawn Burgess)
Weaknesses
Because the synthesis of complex lipids involves low concentrations of the CoA esters of the
fatty acids, metabolomics and labeling of acyl-CoAs might provide useful information on the
synthesis of complex lipids.
5. Environment and Institutional Commitment to the Candidate:
Strengths
The environment at UTSW is outstanding
Weaknesses
None noted
Protections for Human Subjects:
Acceptable Risks and Adequate Protections
Deidentified human plasma samples will be used.
1 K01 GM109317-01
5
ZRG1 IMST-K (50)
Data and Safety Monitoring Plan (Applicable for Clinical Trials Only):
Not Applicable (No Clinical Trials)
Inclusion of Women, Minorities and Children:
G1A - Both Genders, Acceptable
M1A - Minority and Non-minority, Acceptable
C3A - No Children Included, Acceptable
Vertebrate Animals:
Acceptable
Biohazards:
Not Applicable (No Biohazards)
Training in the Responsible Conduct of Research:
Acceptable
Comments on Format (Required):
Appropriate
Comments on Subject Matter (Required):
Appropriate and extensive
Comments on Faculty Participation (Required; not applicable for mid- and senior-career awards):
Excellent distribution of faculty expertise
Comments on Duration (Required):
adequate
Comments on Frequency (Required):
adequate
Resource Sharing Plans:
Not Applicable (No Relevant Resources)
Budget and Period of Support:
Recommend as Requested
CRITIQUE 2:
Candidate: 2
Career Development Plan/Career Goals & Objectives/Plan to Provide Mentoring: 4
Research Plan: 2
Mentor(s), Co-Mentor(s), Consultant(s), Collaborator(s): 2
1 K01 GM109317-01
6
ZRG1 IMST-K (50)
Environment and Institutional Commitment to the Candidate: 4
Overall Impact: Although relatively new to the use of LC/MS/MS for metabolomics research,
e is off to a good start, has demonstrated his skill and innovation in developing methods for the
analysis of multiple triglyceride species. He has demonstrated an appreciation for the need to combine
metabolomics concentration measurements with flux measurements. The project describes the use of
deuterium labeling as a means for making these flux measurements. This methodology is generalizable
to other metabolites and is translatable to human investigation. The project encompasses in vitro cell
studies, in vivo transgenic mouse studies, and evaluation of human blood samples of patients known to
have genetic variation relevant to this study. Thus, this project strongly supports the career
development in the use of metabolomics. The mentors are outstanding with experience in mentorship
to successful transition of previous mentees to independent careers. However, the closely knit goals of
this project with those of mentors may make it difficult down the road for
to assert his
independence of thought in the execution of this study. The proposal suffers from a lack of any formal
training in LC/MS/MS, metabolomics, statistical analyses of metabolomics data, or career development
courses. As well, institutional support is ambiguous in its commitment to dedicated time or
independence after the K01 is completed.
1. Candidate:
Strengths
is well regarded by his advisors and mentors as a highly motivated and productive
researcher with a strong potential to become an independent investigator.
Track record in the hands-on use of LC/MS/MS with the ability to develop new analytical
approaches is shown in the preliminary data on the TG assays.
Good grasp of the use of the metabolite labeling from deuterium oxide as a means to measure
metabolic fluxes.
Weaknesses
None noted
2. Career Development Plan/Career Goals & Objectives/Plan to Provide Mentoring:
Strengths
Advisory team covering all aspects of the research plan will provide guidance in meeting the
goals of the research and in transitioning to an independent career.
Drs Hobbs and Cohen experience with mentorship is well established and provides training in
lab etiquette, integrity in data collection and recording, data presentation, manuscript and grant
preparation, and scheduled evaluation of progress towards short and long-term goals.
Weaknesses
The research is intertwined closely with the work of Hobbs and Cohen, and does not establish a
unique niche for
that is distinct from his mentors.
There are no plans for formal training in the field of metabolomics and the statistical tools
currently used.
3. Research Plan:
Strengths
1 K01 GM109317-01
7
ZRG1 IMST-K (50)
The use of metabolomics analysis to evaluate changes in lipid metabolism in transgenic mice
targeting the relevant pathways is a powerful approach to understand the nuances of metabolic
regulation.
Metabolomics, when combined with the use of deuterium labeling to assess metabolic fluxes
significantly strengthens the project. A much better understanding of metabolic regulation is
obtained when flux measurements are combined with concentration measurements.
Includes provisions for translation to humans, with aim 1D to compare lipid profiles in humans
with the PNPLA3 mutant mice.
In vitro and in vivo complement each other in the use of the metabolomics and labeling
approaches to evaluate the pathways of lipid metabolism with changes in PNPLA3 function.
The experimental plan is relevant to
goal of developing his expertise in the use of
metabolomics and isotopic tracers for the study of human metabolic diseases.
Weaknesses
None noted
4. Mentor(s), Co-Mentor(s), Consultant(s), Collaborator(s):
Strengths
The mentorship by Drs. Hobbs and Cohen is a strength in that the lab has a good track record
in mentoring of post-docs on their way to establishing independent research.
Mentorship in mass spectroscopy and metabolomics will be provided by Jeff McDonald, Ph.D.
This will be a continuation of the training already provided by Dr. McDonald for the use of
LC/MS/MS to measure triglycerides, and deuterium incorporation into sterols. This has been a
productive training period and it can be expected to continue in that vein.
Weaknesses
None noted
5. Environment and Institutional Commitment to the Candidate:
Strengths
UTSW is an outstanding research institution with renowned experts in the field of research that
will pursue during this K01 award period.
Weaknesses
The amount of protected time for research is unspecified, and is stated only as “will provide
protected time for
to pursue his research.
No indication that the institution has a commitment to independence of research following this
K01.
Protections for Human Subjects:
Acceptable Risks and Adequate Protections
Data and Safety Monitoring Plan (Applicable for Clinical Trials Only):
Not Applicable (No Clinical Trials)
1 K01 GM109317-01
8
ZRG1 IMST-K (50)
Inclusion of Women, Minorities and Children:
G1A - Both Genders, Acceptable
M1A - Minority and Non-minority, Acceptable
C3A - No Children Included, Acceptable
Samples are from previously approved study of PNPLA3 patients.
Vertebrate Animals:
Unacceptable
Justification for the numbers of mice is not given.
Biohazards:
Not Applicable (No Biohazards)
Training in the Responsible Conduct of Research:
Acceptable
Comments on Format (Required):
Format is 45-min lecture followed by 45-min faculty led discussion.
Comments on Subject Matter (Required):
Covers ethical and regulatory considerations for use of animals and humans in research.
Authorship, data, conflict of interests, and collaboration issues are discussed.
Comments on Faculty Participation (Required; not applicable for mid- and senior-career awards):
8 faculty members lead the discussions
Comments on Duration (Required):
8-week course
Comments on Frequency (Required):
Once per week.
Resource Sharing Plans:
Not Applicable (No Relevant Resources)
Budget and Period of Support:
Recommend as Requested
CRITIQUE 3:
Candidate: 3
Career Development Plan/Career Goals & Objectives/Plan to Provide Mentoring: 3
Research Plan: 3
Mentor(s), Co-Mentor(s), Consultant(s), Collaborator(s): 3
1 K01 GM109317-01
9
ZRG1 IMST-K (50)
Environment and Institutional Commitment to the Candidate: 3
Overall Impact: It is impressive to see a chemical engineer geared up to pursue a career in clinical
biology. The proposal is organized well with two specific aims to reach the goal. Both strategies are laid
out well to secure the productive outcome of the proposed plan. Metabolomics flux analysis further
strengthens this proposal.
1. Candidate:
Strengths
Candidate possesses needed expertise to reach the proposed goal.
Already actively involved in the same project and showed the credibility with one recent paper in
J. Lipid Research.
Weaknesses
Given that the stable isotope labeling experiments require critical assessment of reaction
conditions; it would be helpful to know the experience candidate possesses on this aspect.
2. Career Development Plan/Career Goals & Objectives/Plan to Provide Mentoring:
Strengths
Applicant understands the areas of expertise that he/she need to gain.
The time line is proposed well in the figure 1.
Weaknesses
In order to conduct meaningful metabolism studies on any particular platform, it is necessary to
have either the knowledge or a plan for pathway analysis. This proposal will be benefited much
more if the lipid biosynthetic pathway analysis was included. Candidate is planning on
conducting flux analysis may be that will provide the hints needed.
3. Research Plan:
Strengths
Aims are defined well in the proposal
Weaknesses
If possible include mass spec imaging in parallel with flux analysis. This would strengthen the
outcome to seek pathway analysis.
4. Mentor(s), Co-Mentor(s), Consultant(s), Collaborator(s):
Strengths
Productive team with all necessary expertise.
Weaknesses
None noted
5. Environment and Institutional Commitment to the Candidate:
Strengths
1 K01 GM109317-01
10
ZRG1 IMST-K (50)
Contain all the support needed.
Weaknesses
None noted
Protections for Human Subjects:
Acceptable Risks and Adequate Protections
Data and Safety Monitoring Plan (Applicable for Clinical Trials Only):
Not Applicable (No Clinical Trials)
Inclusion of Women, Minorities and Children:
G1A - Both Genders, Acceptable
M1A - Minority and Non-minority, Acceptable
C3A - No Children Included, Acceptable
Vertebrate Animals:
Unacceptable
Number of animals are not justified by a power analysis
Biohazards:
Acceptable
Training in the Responsible Conduct of Research:
Acceptable
Comments on Format (Required):
Appropriate
Comments on Subject Matter (Required):
Appropriate
Comments on Faculty Participation (Required; not applicable for mid- and senior-career awards):
Appropriate
Comments on Duration (Required):
Appropriate
Comments on Frequency (Required):
Appropriate
Resource Sharing Plans:
Not Applicable (No Relevant Resources)
1 K01 GM109317-01
11
ZRG1 IMST-K (50)
THE FOLLOWING RESUME SECTIONS WERE PREPARED BY THE SCIENTIFIC REVIEW
OFFICER TO SUMMARIZE THE OUTCOME OF DISCUSSIONS OF THE REVIEW COMMITTEE ON
THE FOLLOWING ISSUES:
PROTECTION OF HUMAN SUBJECTS (Resume): ACCEPTABLE
The application proposes the use of existing samples which previously were collected for use in other
unrelated studies. All samples and data cannot be linked to identifiable living beings and no code can
allow re-identification by investigators involved in the studies described in this application. The E4
exemption applies.
INCLUSION OF WOMEN PLAN (Resume): ACCEPTABLE
Both genders are adequately recruited and this was noted to be acceptable.
INCLUSION OF MINORITIES PLAN (Resume): ACCEPTABLE
Both minorities and non-minorities are adequately recruited and this was noted to be acceptable.
INCLUSION OF CHILDREN PLAN (Resume): ACCEPTABLE
Children were not recruited for this study and this was noted to be acceptable
VERTEBRATE ANIMAL (Resume): UNACCEPTABLE
The five issues concerning protections of vertebrate animals were not adequately addressed. While
the number of animals to be used in each Aim was stated, the application did not justify this number
and this was noted to be Unacceptable.
SCIENTIFIC REVIEW OFFICER'S NOTES:
The Training in the Responsible Conduct of Research Section was complete and was noted to be
Acceptable.
The application stated that a Resource Sharing Plan was not required and this was noted to be
Acceptable.
COMMITTEE BUDGET RECOMMENDATIONS: The budget was recommended as requested.
NIH has modified its policy regarding the receipt of resubmissions (amended applications).
See Guide Notice NOT-OD-10-080 at http://grants.nih.gov/grants/guide/notice-files/NOT-OD10-080.html.
The impact/priority score is calculated after discussion of an application by averaging the
overall scores (1-9) given by all voting reviewers on the committee and multiplying by 10. The
criterion scores are submitted prior to the meeting by the individual reviewers assigned to an
application, and are not discussed specifically at the review meeting or calculated into the
overall impact score. Some applications also receive a percentile ranking. For details on the
review process, see http://grants.nih.gov/grants/peer_review_process.htm#scoring.