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Determining the consequences of sirtuin activity on liver metabolism in a mouse model of obesity

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Title:
Determining the consequences of sirtuin activity on liver metabolism in a mouse model of obesity
Creator:
Kendrick, Agnieszka A
Place of Publication:
Denver, CO
Publisher:
University of Colorado Denver
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xi, 57 leaves : ; 28 cm

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Sirtuins ( lcsh )
Liver -- Metabolism ( lcsh )
Obesity ( lcsh )
Liver -- Metabolism ( fast )
Obesity ( fast )
Sirtuins ( fast )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Bibliography:
Includes bibliographical references (leaves 51-57).
Thesis:
Department of Chemistry
Statement of Responsibility:
by Angieszka A. Kendrick.

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Full Text
DETERMINING THE CONSEQUENCES OF SIRTUIN ACTIVITY ON LIVER
METABOLISM IN A MOUSE MODEL OF OBESITY
by
Agnieszka A. Kendrick
B.S., Wroclaw University, Poland 2005
A thesis submitted to the
University of Colorado Denver
in partial fulfillment of the
requirements for the degree of
Master of Science
Chemistry
2010


This thesis for the Master of Science
Degree by
Agnieszka A. Kendrick
has been approved
by
Karen R. J
Duglas F.
Mark Anderson
a.
V -

Date


Kendrick, Agnieszka A (M.S., Chemistry, CLAS)
Determining the consequences of Sirtuin activity on liver metabolism in a mouse
model of obesity.
Thesis directed by Assistant Professor Karen R. Jonscher and Professor Douglas F.
Dyckes.
ABSTRACT
A proteomics approach was employed to investigate the role of global protein
acetylation in dysregulation of hepatic fuel sensing upon exposure to high fat. Using
immunoaffinity enrichment and mass spectrometry, 35 hyper-acetylated proteins in
the livers of obese mice were observed, about 30% of which were novel. Fatty liver
led to significant hyper-acetylation of mitochondrial proteins involved in
gluconeogenesis, mitochondrial oxidative phosphorylation, and liver injury. Although
protein expression of SIRT1 and SIRT3 was unchanged, SIRT3 activity was
significantly down regulated in obese livers, with a concomitant 30% decrease in
NAD+ levels. No change in histone acetyltransferase activity was observed.
Additionally, acetylation levels were increased in SIRTS^' mice fed a high fat diet.
Results presented here suggest that reduced SIRT3 activity in obesity may play an
important role in fatty liver disease through hyper-acetylation of important
mitochondrial proteins involved in energy metabolism.


This abstract accurately represents the content of the candidates thesis. We
recommend its publication.


DEDICATION
I dedicate this thesis to my husband, James, who provides me with never-
ending support, love and inspiration. I also dedicate it to my son, Ian, who brightens
my every day and makes my life lull of fun and excitement.


ACKNOWLEDGEMNT
I wish to thank my advisor, Karen R. Jonscher, for her support and
contribution to my research. I would especially like to thank her for giving me the
opportunity to be a part of this exciting study. I would also like to thank Douglas F.
Dyckes, Marc A. Donsky, and Jed E. Friedman for all of their valuable advice and
support. Lastly, I would like to express my gratitude to Mizanoor Rahman and
Mahua Choudhury for showing me how exciting and interesting biochemical research
can be!


LIST OF CONTENTS
LIST OF FIGURES.......................................................6
LIST OF TABLES........................................................7
ABBREVIATIONS.........................................................9
INTRODUCTION.........................................................12
BACKGROUND...........................................................14
2.1 Acetylation.....................................................14
2.2 Sirtuins........................................................17
2.3 Obesity and mitochondrial dysfunction...........................19
2.4 MS based proteomics...........................................20
2.5 Research aims...................................................23
RESULTS..............................................................25
3.1 The identification of acetylation changes in liver mitochondria.25
3.2 Validation of MS results via Western blotting...................30
3.3 Determination of sirtuin expression and activity................31
3.4 FLAT and other HDAC activity....................................32
3.5 Correlation of NAD+ levels with body weight changes.............33
3.6 The identification of acetylation levels in SIRT3'/' mice fed a high fat diet ....34
DISCUSSION...........................................................36
CONCLUSIONS AND FUTURE INVESTIGATIONS................................42
MATERIALS AND METHODS................................................44
6.1 Reagents........................................................44
6.2 Animals and sample extraction...................................44
6.3 Immunoprecipitation, 1D electrophoresis and Western blot........45
vii


6.4 In-gel digestion and mass spectrometry..................................46
6.5 NAD+ quantification.....................................................48
6.6 HAT and HDAC activity assays............................................48
6.7 SIRT1 and 3 activity assays.............................................49
6.8 Statistical analysis....................................................50
BIBLIOGRAPHY..................................................................51
Vlll


LIST OF FIGURES
1. Reversible acetylation of a lysine residue.............................14
2. Reaction catalyzed by sirtuin enzymes...................................17
3. An alternative pathway of sirtuin activity.............................18
4. The schematic overview of an ion trap mass spectrometer................22
5. Proteins in liver homogenates from mice on a high fat diet are more highly
acetylated than those from mice fed a control diet.....................26
6. Representative fragmentation mass spectra manually validated for protein
identification..........................................................27
7. The overall expression and acetylation levels of selected proteins.....31
8. Changes in SIRT1 and 3 expressions and activities.......................32
9. HAT and HDAC class I and II activities in control and obese livers......33
10. NAD* levels in lean mice as compared to obese counterparts...34
11. The acetylation levels of proteins of interest in SIRT3A mice fed a high fat
diet....................................................................35
12. Functional classification of proteins identified via mass spectrometry.......37
13. Cellular localization and functional description of CPS1, Aldolase B, PC
and SIRT3
39


LIST OF TABLES
Table
1. The localization and size of human sirtuins [26]....................18
2. Hyperacetylated proteins in livers from high fat fed mice compared to chow-
fed mice...........................................................29
x


ABBREVIATIONS
SIRT sirtuin, silent information regulator
HDAC histone deacetylase
HAT histone acetyl transferase
NAD+- nicotinamide adenine dinucleotide
NADH nicotinamide adenine dinucleotide reduced form
Ac acetyl moiety
CH chow diet (control diet)
HF high fat diet
TSA trichostatin
DAC deacetylase
ART adenosyl ribose transferase
BMI body mass index
ROS reactive oxygen species
IP immunoprecipitation
PC pyruvate carboxylase
Aldolase B fructose biphosphate aldolase B
CPS1 carbamoyl-phosphate synthase
ACoS2 acetyl -Coenzyme synthase 2
GDH glutamate dehydrogenase
DTT dithiothreitol
SREBS sterol regulatory element binding proteins
MS mass spectrometry
xi


INTRODUCTION
Nearly 40 million American adults are obese and over 50 % of the population
is overweight. The risks associated with high body mass index include increased
incidence of Type 2 Diabetes, cardiovascular disease, cancer, fatty liver and other
diseases. Recent studies suggest that there is a direct connection between obesity and
decreased activity of sirtuins [1]. Sirtuins are NAD+ dependent histone deacetylases
that are directly involved in gene silencing via covalent interactions with chromatin -
the main component of the DNA packaging pocket [2]. There are seven sirtuin
isoforms: SIRTs 1, 2, 6 and 7 are located in the nucleus while SIRTs 3, 4 and 5 are
mitochondrial, although SIRT3 was reported to shuttle from the nucleus to
mitochondria with cellular stress [3]. Under conditions of caloric restriction, sirtuins
have been shown to increase life span [4, 5] and influence important metabolic
pathways such as fat mobilization in human cells [6], insulin secretion [7],
gluconeogenesis [7-9], and fatty acid oxidation in muscle [1,7, 10].
In contrast to the well-studied role of sirtuins, particularly SIRT1, in models
of caloric restriction, relatively little is known about sirtuin activity under conditions
of caloric excess, where different organs, including the liver, play a critical role in
diseases such as type 2 diabetes, non-alcoholic fatty liver disease and inflammation.
To investigate this paradigm, a proteomics approach was employed in which proteins
extracted from the livers of mice fed either a high fat or control diet were
immunoprecipitated with lysine acetylated antibody, separated using ID
electrophoresis, digested with trypsin and subjected to mass spectrometry.
Acetylation levels of selected proteins were confirmed with Western blotting and
12


sirtuin activity was associated with NAD+ levels and measurements of HAT or
HDAC activity.
The data suggest that acetylation levels of a number of important metabolic
proteins are increased in liver samples from obese mice (high fat diet) and decreased
in livers of control-fed mice, supporting the hypothesis that sirtuin activity is
decreased in obesity. Data presented here additionally point to a reduction in SIRT3
activity as a potential regulator of a network of proteins implicated in both
mitochondria] and cytosolic metabolism. These proteins may play an important role
in the progression of diabetes, inflammation, and fatty liver disease under conditions
of chronic nutrient excess.
13


BACKGROUND
2.1 Acetylation
Acetylation is a type of protein post-translational modification in which an
acetyl group is attached to the side chain of one of the amino acids. Although acetyl
groups can bind to lysine, arginine, histidine and even tyrosine residues, the most
commonly acetylated amino acid is lysine. Historically, acetylation was only assigned
to histones and was shown to have a major influence on regulation of structure and
function of chromatin, with subsequent effects on DNA packaging and gene
expression [11]. The reactions leading to addition or removal of an acetyl moiety are
catalyzed by histone acetyltransferases, or histone deacetylases, respectively (Figure
Figure 1: Reversible acetylation of a lysine residue.
Acetylation (attachment of an acetyl group) is a reversible covalent modification
catalyzed by histone acetyltransferases (HATs). HAT activity can be inhibited by
interaction of sodium butyrate (NaBu) with the acetylation reaction. The removal of
an acetyl group (deacetylation) is catalyzed by histone deacetylases (HDACs) and can
be inhibited or activated depending on the type of HDAC enzyme used.
1).
0
NH
3
9
NH
; i!' y i u fI:
HAT
HDAC
-r
Lys
Ac -Lys
14


Current studies identify several non-histone targets of HATs and HDACs and
therefore suggest a more global influence of lysine acetylation on cellular function
[12]. It is also important to mention that modification of lysine via acetylation or
deacetylation has a regulatory influence on other possible lysine or adjacent amino
acid modifications, like phosphorylation, ubiquitination, sumoylation, methylation,
and butyrylation [13-15]. The attachment of an acetyl moiety at the side chain amino
acid prevents the lysine residue from undergoing different types of modifications.
Acetylation removes positive charges, in the case of histones this reduces their
attraction to DNA. This makes it easier for RNA polymerase and transcription
factors to approach the promoter region. As a result, histone acetylation is well-
known to improve transcription while histone deacetylation, generally, suppresses
transcription [16]. Some studies suggest that phosphorylation (which appears on
serine residue) and acetylation of histone H3 are coupled and could influence
transcriptional response of H3 [17]. Additionally, some members of HD AC family
like HDAC6 have been shown to play a regulatory role in ubiquitin-dependent
pathways [15, 18].
HATs are proteins that are responsible for the acetylation of both histone and
non-histone proteins, they are classified into two major groups: HAT-A localized in
the nucleus and responsible for gene regulation, and HAT-B which appear in the
cytoplasm and are responsible for modification of newly synthesized core histones
[19]. Histone acetyltransferases are a superfamily of about 30 enzymes classified into
subfamilies based on similarities in sequences, size of active site, and the presence or
absence of other protein domains [19]. The three families of HATs are: GNATs,
300/CBPs and MYSTs enzymes [20]. HAT enzymes employ a mechanism based on
transferring an acetyl group from acetyl-CoA to a specific lysine residue within a core
of histones [21]. The function of HATs is associated with DNA repair [20-22],
transcription [11, 20], tumor suppression [23] and folding of chromatin fiber [22].
15


Histone deacetylases comprise a much larger family of enzymes than HATs.
HDACs mediate reversible post-translational modification of histones and non-
histone proteins in which acetyl groups are detached from lysine amino acid residues.
Eighteen proteins belonging to the HDAC family are grouped into three classes [20].
Class I and II share analogous catalytic activity and function through a Zn2+
dependent mechanism, whereas class III activity is facilitated by an NAD+ dependent
mechanism. Members of class I (HDAC 1, 2, 3, and 8) are relatively small proteins
localized almost entirely in the nucleus. Proteins belonging to class II (HDAC 4, 5, 6,
7, 9, and 10) are much larger in size and have the ability to shuttle in and out of the
nucleus [18]. Class I and II are homologous to yeast deacetylases RPD3 and HDA1,
respectively; and unlike class III, are inhibited by trichostatin.
Acetylation of proteins on lysine residues has been shown to play an
important role in regulating nuclear processes such as transcription, DNA repair and
gene expression [12, 13, 21-25], however the emerging role of this modification in
mediating functions of other cellular processes has been the subject of recent
investigations. A global proteomics screen revealed 1750 proteins conserved in
mitochondria, nucleus, cytoplasm and other cellular fractions to contain 3600
acetylation sites overall [12]. Additionally, acetylation has been shown to be more
abundant in mitochondria than other cellular organs [14] and, interestingly, this
posttranslational modification is more likely to occur in larger macromolecular
complexes [12]. Considering the number of different proteins modified by
acetylation, those studies suggest that acetylation could play an important role in
many functional processes within the cell.
16


2.2 Sirtuins
Sirtuins (silent information regulators SIRTs) comprise a large histone
deacetylase family, with homologs as varied as yeast, mouse and human. Sirtuins are
class III HDACs and are NAD+ dependent deacetylases. The mechanism of sirtuin
action (Figure 2) is based on the transfer of an acetyl group from an acetylated protein
to NAD+ with simultaneous cleavage of nicotinamide. The products of this reaction
are nicotinamide, deacetylated protein and O-acetylated ADP-ribose.
Figure 2: Reaction catalyzed by sirtuin enzymes.
In the presence of NAD+, sirtuins catalyze the deacetylation of lysine-acetylated
proteins, with concomitant cleavage of nicotinamide from the NAD+ moiety. The
reaction products are 2-O-acetyl A DP ribose, nicotinamide and deacetylated protein.
The binding of the acetyl group to the 2-OH position of ribose is highly selective and
is only accomplished when catalyzed with yeast Sir2 or SIRTs 1,2, 3, and 5.
P
Nh )

/0
OH Oh'
NAD '
h3c
^Protein r.il
NH S- Si
Sirt2 3.S
Lysine-acetylated
protein
.-O OH
H?0 J Y O
r2/Sirtl \
4
OH ^
2-O-acetylated
ADPR
h2n
Nicotinamide
^Protein tail
Deacetylated protein
Additionally, some members of sirtuin family can act as ADP-ribose
transferases (Figure 3). In this alternative mechanism, sirtuins catalyze addition of the
ADP-ribose portion of NAD+ to a protein with subsequent elimination of
nicotinamide from the nucleotide. Currently, only SIRTs 4 and 6 have been reported
to employ the ADP-ribose transferase mechanism, while SIRTs 2 and 3 utilize both
DAC and ART mechanisms [26].
17


Figure 3: An alternative pathway of sirtuin activity.
SIRTs 2, 3, 4 and 6 catalyze a reaction in which the ADP-ribose portion of NAD+ is
attached into a protein and nicotinamide is cleaved.

ka::
AL)P noose arotiM".

Mammalian sirtuins are separated into groups according to location in the cell,
mechanism, size and function (Table 1). SIRTs 3, 4 and 5 are strictly mitochondrial,
and are involved in variety of mitochondrial processes [27]. SIRT2 appears in the
cytoplasm and but is also known to interact with some of the nuclear proteins via a
shuttling mechanism [27]. SIRT7 is located in the nucleolus and interacts with rRNA.
Table 1: The localization and size of human sirtuins [26].
The two mechanisms of sirtuin action are DAC (deacetylase) and ART (mono-ADP-
ribosyl transferase).
Name Location Function Size (kDa)
SIRT1 Nucleus DAC 62.0
SIRT2 Cytoplasm ART and DAC 41.5
SIRT3 Mitochondria DAC and ART 43.6
SIRT4 Mitochondria ART 35.2
SIRT5 Mitochondria DAC 33.9
SIRT6 Heterochromatin ART 39.1
SIRT7 Nucleolus ??? 44.8
SIRT1, the homolog of yeast Sir2, is the most widely investigated member of
the sirtuin family. Yeast Sir2 was initially described to be an essential part of
transcriptional silencing at the silent mating loci and telomeres [26]. Studies show
18


that Sir2 has an important role in extending life span under caloric restriction in yeast
[5]. Both Sir2 and human SIRT1 are NAD+ dependent deacetylases. SIRT1 is
localized to the nucleus, but is also capable of shuttling to the cytoplasm, and it is
involved in several different nuclear and cytoplasmic processes [26, 27]. SIRT1 can
promote cell survival via an interaction with proapoptotic factors like p53 and FOXO
[4, 8,9], and can also regulate glucose regulated insulin secretion in pancreatic-p cells
[28]. The majority of studies involving SIRT1 and its function have been performed
in caloric restriction models [2, 4, 6, 7, 25, 29, 30], and showed that resveratrol, the
main component of red wine, is the most powerful SIRT1 activator [26]. SIRT3, a
mitochondrial homolog of yeast Sir2, activates acetyl-CoA synthetase 2 via
deacetylation of a key lysine residue on the enzyme [31]. Over-expression of SIRT3
results in increased cellular respiration and decreased mitochondrial membrane
potential, with a concomitant decrease in the production of reactive oxygen species
[3, 9, 32-34]. Mice lacking SIRT3 have impaired ATP production and mitochondrial
respiration [32-34], suggesting a key role for SIRT3 as a metabolic regulator of
mitochondrial function. SIRT3 appears to be the primary mediator of mitochondrial
acetylation since no significant changes in acetylation status were detectable in mice
lacking SIRT4 and SIRT5 [32]. A decrease in SIRT3 has been described in brown
adipose tissue of genetically obese animals [33], however its role in the liver has not
been studied under conditions of caloric excess.
2.3 Obesity and mitochondrial dysfunction
Individuals with a body mass index of 30 kg/m2 or more are defined to be
obese, and obesity is one of the dominant public health problems in our century. Diets
rich in fat and lack of exercise are considered to be the major causes of increasing
body weight that may lead to serious co-morbid medical conditions such as
cardiovascular disease [1, 35], hypertension [36], respiratory disease [37], depression
19


[38], non alcoholic steatohepatitis [39], and insulin resistance, leading to type 2
diabetes mellitus [40]. Each of these medical conditions may be linked to
mitochondrial dysfunction, potentially caused by increased P-oxidation of fatty acids,
induction of reactive oxygen species, and generation of ketone bodies.
Major metabolic pathways dysregulated by high fat diet take place either
inside or within the boundaries of mitochondria, resulting in mitochondrial
dysfunction [10, 41]. The mitochondrion is a double-layered cellular organelle found
in almost all eukaryotes. It is a unique organelle, with its own genome and a wide
range of mitochondria-specific proteins. A variety of processes take place in this
organelle, including those related to energy conversion and production of ATP,
apoptosis, calcium signaling and storing, steroid synthesis and cellular proliferation,
among others. The role of mitochondria in diseases associated with obesity is
primarily related to the energy conversion processes that take place inside
mitochondria, particularly the citric acid cycle and oxidative phosphorylation, and the
connection of these pathways to glycolysis and gluconeogenesis.
Mitochondria can be found nearly in every organ, however they are most
densely localized in liver, which contains about 1,000-2,000 mitochondria per cell.
The key role of the liver in regulation of metabolic pathways and production of ATP,
acetyl-CoA and pyruvate is likely enabled by its relatively high mitochondrial
content. In the case of a diet rich in fat, the production of acetyl-CoA and pyruvate
increases significantly [42].
2.4 MS based proteomics
Determination of gene function and expression on a protein level can be
achieved using a combined biochemical and molecular biology approach called
proteomics. Proteomics analysis very often utilizes mass spectrometry as a sensitive
20


and reproducible method in identification and quantification of proteins, their
modifications, and protein-protein interactions. MS-based proteomics was initially
applied to small subsets of purified proteins But currently, due to the development of
more robust instrumentation and bioinformatic techniques, large scale proteomics
approaches are possible [43]. Mass spectrometric data allows determination of the
molecular masses of proteins and peptides, their post translational modifications, and
even concentrations. Nanoelectrospray ion trap mass spectrometry is a common
method of choice in protein identification because of the small amount of sample
required, low flow rates, good sensitivity, the ability to obtain multiple fragmentation
spectra (MS, MS/MS, MS"), and increased signal-to-noise ratio due to the high
accumulation of ions. There are some limitations in using an ion trap spectrometer,
such as 1/3 rule. Low mass cutoff, commonly termed the 1/3 rule, does not allow
trapping of fragment masses that are 28-30% below the mass of parent ion. However,
considering the large size of proteins and relatively large size of peptides, this
restriction does not have a significant influence on the sensitivity of ion trap
instrument.
The main components of an ion trap mass spectrometer are shown in Figure 4
and may be categorized into three groups: an ion source, a mass analyzer, and a
detector. The role of an ion source is to either convert gas phase sample molecules
into ions or move the existing ions from solution into the gas phase. Some of the
common ionization techniques used are chemical ionization (Cl), matrix-assisted
laser desorption ionization (MALDI), atmospheric ionization (AI), and electrospray
ionization (ESI). ESI is especially suitable for large biological molecules that are hard
to vaporize or ionize. In the ESI source, a sample is sprayed into the source chamber
to form droplets which are further bombarded with high temperature nebulizing gas to
remove the solvent. Molecules are subsequently ionization. The charged droplets are
passed through the capillary to the skimmer, octopoles, and lenses which help
21


concentrate and focus ion droplets. Finally, the ions reach the mass analyzer which
sorts them according to their mass by applying electromagnetic fields. In this case it
would be an ion trap, which collects the ions and then releases them according to their
mass-to-charge ratio. An ion trap has the ability to fragment precursor ions and obtain
fragmentation spectra via isolation and induction of a particular ion (mainly the most
intense signal ion) and generation of product ions. The final part of the analysis is the
detector, which is responsible for the production of mass spectra based on the
measurement of the value of an indicator quantity.
It is important to separate the analytical sample into smaller fractions to
simplify the analyzed fractions before the introduction to the mass spectrometer. The
sample can be separated into smaller portions using high performance liquid
chromatography and then directly delivered to the ion source (online analysis). In the
liquid chromatographic method, peptides with different masses and charges will elute
at different retention times and can be further detected and fragmented in the ion trap.
Samples can also be initially separated into smaller fractions, using offline ID- or 2D-
LC separation as well as ID or 2D electrophoresis. It is also crucial to digest the
sample before mass spectrometry with an enzyme that cleaves the protein at specific
amino-acid residues and therefore reduces the size of the analyzed protein fractions.
Figure 4: The schematic overview of an ion trap mass spectrometer.
ON SOURCE ION TRANSPORT AND -OCIiSiNG MASS ANALYZE* Cf-f-:'!
JU

22


After generation of the fragmentation data, each spectrum is assigned a charge
and a mass value based on the known information of the mass and charge of the
parent ion. Further, the amino acid sequence corresponding to the specific spectrum
is identified by comparison of mass and charge list from each spectrum to the peptide
fragmentation spectrum enclosed in a database. The protein identity is determined
based on the number of unique peptides (peptides specific for only this particular
protein), spectral intensity, and number of peptides that were observed with removal
of redundant observations.
2.5 Research aims
Sirtuins, NAEH-dependent deacetylases, regulate lipid and glucose metabolism
in liver and increased SIRT1 activity in caloric-restricted models has been linked to
extended life span in several species [5, 26, 44]. Some of the members of the SIRT
family can deacetylate mitochondrial proteins, suggesting that posttranslational
modification by sirtuins may have global effects on energy metabolism. SIRT3 has
recently emerged as a mediator of global lysine acetylation in the mitochondria [32]
and has been also shown to regulate basal ATP levels in the mitochondria [34]. In
recent years, several studies have been published showing the influence of sirtuins on
longevity [5, 44], inflammation [26], and mitochondrial regulation in normal or
caloric restricted models [2, 4, 6, 7, 9, 25, 29-34, 45] however, the potential role of
sirtuins in high fat models has not been extensively explored. Taking into
consideration the above results, the following hypothesis was pursued: sirtuin activity
is decreased in obesity, leading to increased protein acetylation The goal of the
study presented here was to use a high fat feeding model to assess changes in
acetylation levels of mitochondrial or cytoplasmic proteins, and elucidate a sirtuin
family member potentially responsible for hyper-acetylation of some of those
proteins.
23


The following research aims were pursued:
Research Aim 1: Identify the hyper-acetylated proteins in lean and obese animals
using mass spectrometry.
Research Aim 2: Use immunoblotting to validate MS results.
Research Aim 3: Quantify NAD+ levels in the liver tissue.
Research Aim 4: Correlate the activity of sirtuins, other HDACs and HATs with
protein acetylation levels.
Research Aim 5: Determine which particular sirtuin family member may be the
primary mediator of protein acetylation levels in a model of obesity.
24


RESULTS
3.1 The identification of acetylation changes in liver mitochondria
A targeted proteomic approach was employed to evaluate the acetylation
changes of liver mitochondrial proteins between control and high fat fed animals.
Livers from mice fed either control or a high fat diet (45 kcal % fat, 12 weeks) were
harvested and homogenized. In the case of animals on a high fat diet, only tissues
from animals that showed significant mass gain were selected for the analysis.
Proteins modified by lysine acetylation were immunoprecipitated with anti-
acetyllysine antibody, followed by separation by one dimensional gel electrophoresis
(Figure 5). Bands showing differential staining between the control and HF fed mice
were excised, proteins digested with trypsin, and analyzed by tandem mass
spectrometry using an Agilent Ultra quadrupole ion trap. Product ion spectra (Figure
6) were searched with SpectrumMill against the SwissProt database. 35 proteins
showing differential acetylation levels were identified with high confidence (Table 1).
Some of the identified proteins, such as carbamoyl-phosphate synthase, uricase,
pyruvate carboxylase and ATP synthase, are known to contain acetylated lysines [14].
Some of the proteins observed have not been previously reported to be acetylated.
25


Figure 5: Proteins in liver homogenates from mice on a high fat diet are more
highly acetylated than those from mice fed a control diet.
Proteins were first immunoprecipitated with acetyllysine antibody and then separated
using ID gel electrophoresis using a 10% SDS-PAGE gel. Bands showing differential
staining are identified. These bands are excised, in gel-digested, and analyzed using
tandem mass spectrometry.
HF CH
250k Da
5.ND 1
150k Da
BAND 2
100k Da *'
75kDa
37kDa
ND i
26


Figure 6: Representative fragmentation mass spectra manually validated for
protein identification.
A Pyruvate Carboxylase (PC), B Carbamoyl-phosphate synthase (CPS 1), and C -
Fructose biphosphate aldolase B (Aldolase B). The precursor ions for peptides
presented in the spectras were fragmented, and the resulting spectra are shown. The
masses of the theoretical fragment ions are listed with the observed ions in bold. All
fragment ions were required to have a signal-to-noise ratio of at least 5. The y-type
fragment ions (charge is retained on C terminal end of peptide) and b-type fragment
ions (charge is retained on N terminal end of peptide) are shown.
A

1275
'359 CC2 966 923 687
'455
565
1*55
563 536
359
-*lL ~ ^ it Vi ~i ini-1
27


489 546 611 639 713
b 261 218 417 488 617 745 858 995 1 1 10 1239 1296 1442
Phe Lue Gly Val Ala Glu Gin Leu His Asn Glu Gly Phe Lys
y 1442 1329 1272 1173 1102 972 844 731 594 480 351 294 147
y2< 721 664 636 587 551 487 423 366 298 241 176 147 74
481 444 425 391 368 325 282 244
yll
y9
l kibhk (Lai. u -lla
bl 2
yoo 4 Ofl Ana BOO 1 ooo 1 zoii 1400 m ly
b 185 300 428 53) 628 715 802 915 9B6 1099 1227 1356 1471 1542 1656 1727 1140 1910
lie Ala Asp Gii Pro Ser Set Lai Ala lie Gn Ghi Asn Ala Asn Ala Leu Ab Arg
y 197! 1901 1786 1657 1555 1458 1371 12(4 1171 1099 966 858 729 615 544 430 359 246 175
2+ 986 950 893 829 778 729 685 642 586 550 493 429 365 308 272 2'5 180 123 88
- 2
D^5
018
jbU. jlL-
28


Table 2: Hyperacetylated proteins in livers from high fat fed mice compared to
chow-fed mice.
Thirty five high scoring proteins identified from differentially stained gel bands. The
total number of peptide spectra that were observed that may be derived from a protein
are summarized as Spectra #, while Distinct Peptides # reflects the number of
peptides that were observed, with removal of redundant observations. Summed
MS/MS Score results from addition of peptide scores for the highest scoring distinct
peptides for each protein. All proteins reported had at least two distinct peptides
whose sequence assignments were manually validated. The accession number is
linked to the SwissProt database, where more information regarding the structure and
function of the protein may be investigated. Proteins marked with an asterisk have not
been previously reported as acetylated based on UniProtKB/Swiss-Prot database
information.
' j VS Ms \ \ P-.-lcn \i u .r- \ .i : .

is 1 n- i .. iMM' " *.>M 1 -M- | i .;] h.m.n pii.'.l, s uili.i', ,i-t: u. .!: ..i i. ill iln r.mu:i ..: 1 pun i.i'.-i
r s >,. i1 s > : Mr,s< ; 1111 < ^ ri, \ ,i; ^ irS, \ \ i.-s ;ji i?,.v i-.Mivir.i: nrv-v rs. r
1 .. i *i': 1 \ II \ ii: li.i'C 'ii-*'." i: ri .1", cnni: ,.! rv-.iM'i
i; 4 s ; s ' 1 'in\ 1 i i ' kctn.u \ ( > \ lh-.-l.lsw ir t,n !' .-II Jr.:
44 a'r( s i *;4-in j [{,.,; in... j ni ('c \ ci s s u L h \ : m i 'v i . :
- *'';: i \ is Ow!,-. .
' i ' 4 [ i n '-i j k l).i i -r. -:i! .i '.'s i"s. i-. r
i. ,,| 4 : 1 i*' 4 si--i j 11 s J , v . :,:cln [j u; r v 1 \ s Ini', r: P>. '.Jr..
! r.l 1' 4 _li S V ' :-m :4- A Jcilo s v ill.'Ill ; S 'ts-lll.1'4
> 1 '< <' !> ;< -in. IVr->\ rv.i.'M' -
44: 4 ns.*/l ] \u.', 1 1 \ ..,1.1'. .1: Mils k 1 .is ,
j j;,,.,- ris\ (. ||n : -k pii'.u v i- -' \ tircl.i'w !i. r. i.> \ .1
| Sr- II; ;*n ;m : v M' kl).i Knl s:-,.. .. r-i-Iw'i ii' ,i! rr.\ i.-s,.'
i 1 '! 14 1 1 ],n/.it mti :.u r 1 cpln
- - ; ; s[,4 S;n-s..'n j-r.'icii. iv.ji-..- -in.; r i.n :
i.r,.: ( 1 S .
; L' W> S -1 - i* j hJl.h \ i 4 nin.'siK i:m c si m:\is,
i" <> 4<- 4 -MS > }h> \> j S ,k! c lies . i'iis'i :i mK \ n 1 !u..!.. is :r : \ pc '
i__ ... ;' :a :*: s i-' Sc i.il.iin iiv n!:: w: .i ^ s.
- s; ,, j: t <
t. : ;P;MS | (|
S 4 ? . hT:. .r ;
j . ; iwi v -
";iS 4 ;*><'!! 1 U .It 'll", k ml.' i k 1).: n.'U-n!
' n i*s 1 ; S,-1 11,- 11; v .tl,- ,H" ' "! nr s ', i .1-,'. I'l H", .ii i.1 p ii: S"
4 j ; s s :* s r- 4 M i \ 11 s i n; li.i',- u;'uimi i .i in ii". "ini ri.i: piv, .un "
j - ; ! 4 4" 'ii s I iss'lsr. ' ,un p k11*h 1.1 \ ml, ,:i/ \ in \ n1
; ; S S ' 4 is | linysil ; | .4,.^ _.s;v.r N.. -v... .N1(-
: : > >s '4 I :u v" 1 mi -.r.. 1.- .in: j.,. m t-'cr .Jin r.^ i;r' -'
: | 's 4 .jv,-ui: ( Hi.> j 1 H.is. 1 \ \ n:s.'
i ;4 >i' 4 :> i 111.il.it per" n:.i'4
4 'is "H" - nsRii'i < ; H -n \ !;,; j.ii1 \ ,!,np. Ir, ,1;" m,>,'
^ i. ' k ir ,'l", 1', ': , -.i'' p;"i .-in I >s \ ; li.- 'i,. 4
29


3.2 Validation of MS results via Western blotting
Several of the proteins identified by mass spectrometry that are especially
relevant to metabolic function were further validated for both protein expression and
acetylation changes by Western blotting. Proteins from HF and CH liver
homogenates were immunoprecipitated using acetyllysine antibody, then probed for
specific protein identity by Western blotting (Figure 7). Additionally the overall
expression levels of those proteins were determined using the same Western blotting
approach but without prior enrichment by immunoprecipitation with acetyllysine
antibody. As shown in Figure 7A, expression of CPS1 significantly decreased (2-fold
decrease, p < 0.01) with obesity while acetylation levels increased, suggesting that
CPS1 is hyper-acetylated in the livers of obese mice. Although protein expression of
PC and Aldolase B appeared to be unchanged between samples, acetylation of both
proteins were increased in HF (Figure 7B and C), suggesting these proteins are also
hyper-acetylated in obesity.
30


Figure 7: The overall expression and acetylation levels of selected proteins.
Lysine acetylated proteins were immunoprecipitated from liver extracts of high fat
(HF) and chow diet (CH) mice. Cell lysates were also Western blotted and probed
with (A) CPS1, (B) Aldolase B, and (C) PC. For each set of three Westerns the upper
band indicates acetylated-protein, the middle represents the overall expression of that
protein, and the bottom band shows the loading control for the protein expression
experiments. Blots shown in here are representative of three separate experiments that
showed similar results. The bar graph represents total expression changes of CPS1
(n=6, p < 0.01).
CH
HF
CH
- Ac-Aldolase B
- Aldolase B
-GAPDH
HF
3.3 Determination of sirtuin expression and activity
In order to determine whether sirtuin activity might account for these changes
in protein acetylation status, commercially available activity assays were used to
measure the activities of SIRT1 and 3 in liver homogenates. The expression of those
sirtuins was evaluated using Western blotting with SIRT1 and 3 antibodies in the
same set of samples. The activities of other sirtuins could not be determined due to
the unavailability of activity assays. The measurements revealed that the expression
of SIRT1 and SIRT3 were unchanged in CH and HF livers. Although SIRT1 activity
also remained unchanged, SIRT3 activity was significantly (p<0.01) decreased in HF
samples (Figure 8).
31


Figure 8: Changes in SIRT1 and 3 expressions and activities.
Liver homogenates were assayed for the influence of specific sirtuins on acetylation
levels of the identified proteins. High fat and chow samples were Western blotted and
probed with (A) anti-SIRTl and (B) anti-SIRT3 to assess total cellular levels of those
proteins. Additionally 40 pg of cell lysates were incubated with fluorescent sirtuin
substrates and fold changes in enzymes activities were determined (n = 6; # p<0.01).
CH HF
Sirtl
-GAPDH
3.4 HAT and other HDAC activity
The acetylation or deacetylation of proteins can be influenced by HAT or
HDAC enzymes, respectively. In order to ascertain whether the observed protein
hyperacetylation might be due to HAT or HDAC class 1 and 2 activity instead of
sirtuin activity, the same sets of samples as the ones used previously were analyzed
using commercially available HAT and HDAC activity assays.
Figure 9 reveals that HAT and HDAC I and II activities are unchanged in
obesity, suggesting that reduction in deacetylase activity, most likely that of SIRT3, is
primarily responsible for the observed protein hyper-acetylation.
32


Figure 9: HAT and HD AC class I and U activities in control and obese livers.
40 |ig of cell lysates were assessed for changes in (A) HAT or (B) HDAC activities.
The results are presented in a bar graph with error bars indicating SEM from six
biological replicates (3 chow and 3 high fat). For both experiments, HeLa cell
extracts provided an experimental control.
B
3.5 Correlation of NAD+ levels with body weight changes
NAD+ concentration is a major modulator of class III HDACs [46],
suggesting that changes in sirtuin activity might be linked to changes in NAD+ levels.
The levels of NAD+ in liver samples were measured as a function of weight gain in
mice. Overall, NAD+ levels were significantly lower in HF mice by 30% (Figure
IOA) and appeared to decrease linearly with increasing body weight change (Figure
IOB) .
33


Figure 10: NAD* levels in lean mice as compared to obese counterparts.
About 20 mg of HF and CH liver lysates were used to evaluate the concentration of
NAD* in obese and control animals. (A) Quantification of NAD* levels in CH and HF
samples shows significantly higher concentrations in control diet samples as
compared to fat. The data presented in a bar graph contain error bars indicating
SEM from six biological replicates (3 chow and 3 high fat; # < 0.05). (B) The
correlation between body weight and NAD* concentration for six samples (n = 6; R2
= 0.8765). Three separate experiments were performed with similar results.
Representative data are presented.
A B
Change in body weight [g]
3.6 The identification of acetylation levels in SIRTS^' mice fed a high fat diet
To test whether the hyper-acetylation of proteins observed under high fat-fed
conditions is related to reduced SIRT3 activity, liver extracts from wild-type and
SIRT3-deficient mice were prepared, proteins were immunoprecipitated using anti-
acetyllysine antibodies and probed for PC, Aldolase B and CPS1 by Western blot.
Under chow fed conditions there was very little change in the acetylation status of
selected proteins in SirtS^' mice as compared to WT mice (Figure 11). However,
following high fat feeding, Western blots for PC, Aldolase B and CPS1 revealed that
these proteins had exaggerated hyper-acetylation in the absence of SIRT3 as
compared to WT high fat-fed mice. There was no concomitant change in protein
34


concentration. These results support the hypothesis that the combination of
diminished SIRT3 activity and low NAD+ levels leads to increased lysine acetylation
in obesity.
Figure 11: The acetylation levels of proteins of interest in SIRT3+ mice fed a
high fat diet.
Wild type (WT) and SIRT37' (KO) animals were fed either high fat diet (60% fat) or
regular chow for 8-9 weeks. Liver homogenates were immunoprecipitated with
acetyllysine and immunoblotted with anti-PC, anti-Aldolase B and anti-CPS 1.
Additionally cell lysates were Western blotted and probed with (A) CPS1, (B)
Aldolase B, and (C) PC. For each set of three Westerns the upper band indicates
acetylated-protein, the middle represents the overall expression of that protein, and
the bottom band shows the loading control for the protein expression experiments.
A HF CH
- Ac-CPS 1
- CPS1
-GAPDH
WT KO WT KO
- Ac-PC
- PC
-GAPDH
C HF CH
WT KO WT KO
... -Ac-Aldolase B
1 j - Aldolase B
-GAPDH
35


DISCUSSION
In the current study, targeted proteomics revealed increased levels of lysine-
acetylation modification of proteins in high fat fed mice as compared with chow fed
controls. In summary, increased protein acetylation and decreased NAD+ levels in
obese mice correlated with decreased SIRT3 activity and, based on those results, one
could postulate that acetylation-based protein modification impairs protein function,
potentially leading to obesity-associated diseases such as diabetes, inflammation,
cardiovascular and fatty liver disease. The targeted proteomics approach identified 35
proteins hyper-acetylated in livers of animals fed a high fat diet. A subset of the
proteins identified using this approach, such as CPS1, uricase, PC and ATP synthase,
have previously been shown to contain acetylated lysines [14]. Functional
classificationFigure 12) of high scoring protein identifications from the excised bands
suggests that amino acid metabolism, gluconeogenesis, fatty acid metabolism, TCA
cycle enzymes, redox regulation and stress response pathways may be mediated by
acetylation in response to obesity.
36


Figure 12. Functional classification of proteins identified via mass spectrometry.
The proteins were separated into functional groups based on information contained in
the SwissProt database.
Fatty Acid
Metabolism
4%
Urea Cycle A .
4 ---------- Synthesis
4%
Based on the data from HAT and HDAC activity assays, acetylation changes
are likely not due to changes in HAT or HDAC class I and II activities. Additionally,
the decreased NAD+ concentration in obese animals suggests that one or more
members of the sirtuin family may play an important role in the control of acetylation
levels in a high fat diet model of obesity. In order to elucidate which member of the
sirtuin family could be responsible for the acetylation changes, the expression and
activities of SIRT1 and 3 were measured. The data presented in here support recent
reports that mitochondrial SIRT3 may be one of the key regulators of acetylation
levels in mitochondria [2, 9, 31-34].
SIRT3 is a NAD+ dependent deacetylase crucial for maintaining basal ATP
levels in mitochondria [34]. Recent studies suggest that SIRT3 might be responsible
for global changes in acetylation of several mitochondrial proteins [32]. The
depletion of the SIRT3 gene in mice causes increased acetylation of proteins present
in mitochondrial-rich tissues and, analogous to SIRT1, the SIRT3 gene has been
shown to be involved in regulating longevity [34]. SIRT3-deficient mice have been
37


generated and, although there is striking mitochondrial protein hyper-acetylation,
SIRT3 deficient mice generally appear to be metabolically unremarkable under basal
conditions [9, 32]. In contrast, during stress conditions SIRT3 appeared to be
necessary in preventing heart dysfunction through modulation of cellular levels of
ROS [9], suggesting that SIRT3 might play a role in stress response pathways.
Some of the ROS-scavenging antioxidase enzymes, such as catalase (a known
SIRT3 target [9]), uricase, peroxiredoxin and glutathione peroxidase, have been
reported to be hyper-acetylated in fed but not fasted mice. It is likely that down-
regulation of these antioxidant systems by hyper-acetylated mediators would tend to
further increase ROS levels and oxidative stress. Given that ROS and oxidative stress
are common in obesity [47], the results suggest that excessive acetylation and reduced
SIRT3 activity may impair the function or activity of oxidative stress defense
proteins, thereby contributing to liver injury and accumulation of damaged proteins.
High fat diet also led to hyper-acetylation of proteins involved in
gluconeogenesis and ureagenesis, including PC, Aldolase-B and CPS-1 (Figure 13).
Aldolase B is a bifunctional enzyme that participates in both glycolysis and
gluconeogenesis.
38


Figure 13: Cellular localization and functional description of CPS1, Aldolase B,
PC and SIRT3.
ASPA!- 'AT;
nac*. ahginiovx c i\/r; / ;
SHU Tit . '
H < iXIDATK :\
LSI- A
Pyruvate carboxylase is an enzyme involved in the transformation of pyruvate
to oxaloacetate in the first step of gluconeogenesis [8] and is an important regulator of
the TCA cycle. Carbamoyl-phosphate synthase is a liver mitochondrial matrix protein
and a regulatory enzyme in the urea cycle. CPS1 is crucial in the process of
detoxification of excess ammonia from an organism via regulation of the production
of carbamoyl phosphate. Carbamoyl phosphate is the first reactant in a series of
reactions leading to the production of urea, a relatively non-toxic compound from the
highly toxic precursor compound, ammonia. The active sites of this enzyme are
connected by a molecular tunnel containing several lysines [48]. It is possible that
acetylation of one or more of these lysines influences the metabolism of ammonia
through the enzyme, resulting in toxic buildup in the liver. Recently, CPS1 was
39


shown to be a target of SIRT5 [30], however data presented here suggest that it may
be additionally modified by SIRT3.
Gluconeogenesis is increased in obesity due to insulin resistance [49], and
lysine acetylation of PC and Aldolase B may disrupt glycolysis or gluconeogenesis
pathways, leading to increased production of glucose and development of Type 2
diabetes. The consequences of up regulation of gluconeogenesis are increased levels
of acetyl-CoA from excess glucose, as well as an increased risk of obesity.
Therefore, post translational modifications of pyruvate carboxylase and Aldolase B
might lead to important changes in the gluconeogenesis pathway.
Several heat shock proteins were identified: HSP60, HSP70 and HSP71. Heat
shock proteins are ubiquitously expressed in the cytoplasm and nucleus and are
named based on their molecular weight and their capability of being induced by heat.
Heat shock proteins are present in the cell under normal conditions and are
responsible for stabilization of partially folded proteins and binding to partially
synthesized proteins, preventing them from aggregating and becoming nonfunctional
[50]. In stress-related conditions like extreme temperature variance, viral infection,
energy depletion, ROS overproduction, and acidosis, heat shock binding factors
(present in the cytosol) separate from the heat shock proteins and translocate to the
nucleus where they cause increased production of heat shock proteins [51]. The
overproduction of heat shock proteins plays an important role in cell protection from
stress via inhibition of apoptosis, repair and refolding of stress-damaged proteins [50,
51].
In addition to the heat shock proteins, GRP78, a hyper-acetylated target in
fatty liver [52], was also identified. GRP78, glucose related protein, is the ER
homologue of HSP70 with a conserved ATPase domain and a peptide-binding
domain [53], As a chaperone, GRP78 directly interacts with all three ER stress
40


sensors (inositol-requiring protein 1 IRE1, PKR-like endoplasmic reticulum kinase
- PERK, and activating transcription factor ATF-6) and maintains them in inactive
forms in non-stressed cells [54]. Stable over expression of GRP78 inhibits the
activation of sterol regulatory element binding proteins and the genes under their
regulation [55]. SREBPs are transcription factors localized to the nuclear envelope
and endoplasmic reticulum that are responsible for binding to sterol regulatory DNA
sequences, regulating the expression of enzymes involved in lipid homeostasis [56].
Hyper-acetylation of GRP78 may be a potential factor affecting lipid metabolism as
well as the ER stress response in fatty livers [53].
There is emerging evidence that mitochondrial dysfunction plays a key role in
the physiopathology of non-alcoholic steatohepatitis [39]. Notably, impaired
mitochondrial respiration has been shown to induce the ER stress response and
upregulate GRP78[57]. The acetylation of the mitochondrial electron transport chain
protein ATP synthase and the TCA cycle enzyme fumarate hydratase were showed
here to be increased. Modification of these proteins by hyper-acetylation might lead
to dysfunctional mitochondrial respiration and subsequent impairment in lipid
oxidation and TCA cycle activity [1].
41


CONCLUSIONS AND FUTURE INVESTIGATIONS
Targeted proteomics revealed increased levels of lysine-acetylated proteins in
the livers of obese mice as compared with normal controls. Results from proteomics
studies and enzymatic activity assays show a correlation between increased protein
acetylation, decreased NAD+ levels and decreased SIRT3 activity in obese mice. It
could be postulated that this protein modification may impair or regulate protein
function, potentially leading to obesity-associated diseases such as diabetes, cancer,
cardiovascular and fatty liver disease. One could suggest that posttranslational
modification by sirtuins may have global effects on energy metabolism, especially
gluconeogenesis and lipogenesis. Given previous observations demonstrating SIRT3
as a modulator of ACoS2, Complex I and GDH [8, 32], and results presented here
could suggest that the list of SIRT3 targets might extend to other mitochondrial
proteins including some acetylated proteins identified in this study.
Further studies are necessary to identify other potential SIRT3 targets and to
evaluate the influence of acetylation/deacetylation on their function. In order to
perform such studies, SIRT3 deficient and/or SIRT3 over expressed models, fed a
regular and high fat diet, would be required. It is important to evaluate the ATP,
NAD+ and NAD7NADH levels in those animals in addition to identifying and
quantifying changes in specific proteins hyper-acetylated in SIRT3 deficient and over
expressed mice. One could also consider evaluating expression changes of varies
mitochondrial metabolites like oxaloacetate, pyruvate, acetyl-CoA and others.
Additionally it would be worthwhile to assess the changes in oxidative or ER stress in
high fat vs. control animals.
42


Taken together, these findings show that high fat diet has a striking effect on
the acetylation status of numerous cytosolic and mitochondrial proteins in association
with reduced hepatic NAD+ levels and attenuated SIRT3 activity. SIRT3 depletion
enhances these posttranslational modifications, supporting its key role in chronic fat
overload-mediated metabolic protein acetylation. Given the complexity and cross-talk
of biochemical pathways involved in cellular metabolism, care must be taken in
future work to identify important metabolites and quantitatively assess changes in
their concentrations when proteins mediating those pathways are acetylated. The
experimental challenges are significant; however, this hyper-acetylation of hepatic
metabolic proteins in obesity may underlie susceptibility to fatty liver, inflammation,
and diabetes. Understanding the molecular pathogenesis of these diseases will
ultimately lead to the development of effective therapies for these conditions.
43


MATERIALS AND METHODS
6.1 Reagents
Polyclonal anti-acetylated lysine antibody was purchased from Cell Signaling
(Danvers, MA). All other antibodies were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA). HRP-conjugated goat anti-rabbit and anti-mouse immunoglobulin
G were purchased from Bio-Rad Laboratories (Hercules, CA). Protease inhibitors
were obtained from Sigma (St. Louis, MO). HPLC-grade water, acetonitrile and
formic acid were purchased from VWR (Denver, CO). Sequencing grade trypsin was
bought from Promega (Madison, WI), iodoacetamide and dithiothreitol were from
Sigma. SIRT1 and SIRT3 fluorometric assays were obtained from Biomol (Plymouth
Meeting, PA), NAD+ quantification was performed usign a kit from BioVision
(Mountain View, CA), and a HAT activity assay kit was purchased from Upstate
Biotechnology (Lake Placid, NY). Normal and high fat chow diets for the animals
were purchased from Research Diet, Inc. (New Brunswick, NJ).
6.2 Animals and sample extraction
Animal protocols were approved by the University of Colorado Institutional
Animal Care and Use Committee in accordance with National Institute of Health
guidelines. Animals were a generous gift from Dr. Mizanoor Rahman and Dr. Carrie
McCurdy. 5-6 week old male C57BL/6 SVJ mice were fed either a control chow or
high fat diet (45 kcal% fat, Research Diets) for 16 weeks. The wild type and SIRT3 _/
mice [3] were fed a diet containing 60 kcal% fat for 8-9 weeks. Livers were
harvested immediately from anesthetized mice and snap frozen at -70 C in liquid
44


nitrogen before analysis. Mice were sacrificed by pentobarbital over-dose
following treatment. Livers (around 200 mg each) were mixed with 500 pi of lysis
buffer A (10 Mm HEPES (pH 7.9), 0.5 mM KC1, 10 mM MgCl2, 0.5 mM DTT, 0.1%
IGEPAL CA-630, 0.5 mM phenylmethylsulfonyl fluoride) and homogenized. The
homogenates were collected and centrifuged at 7,000 rpm in 4C for 10 min. The
supernatant was then collected and transferred into fresh tubes, and the remaining
pellet was combined with 200 pi of buffer B (20 mM HEPES (pH 7.9), 25% glycerol,
1.5 mM MgCl2, 420 mM NaCl, 0.5 mM DTT, 0.2 mM EDTA, 0.5 mM
phenylmethylsulfonyl fluoride, 4 pM leupeptin), and was subsequently centrifuged
(14,000 rpm at 4C for 30 min). Protein concentration was measured using a standard
colorimetric Bradford reagent assay (BioRad, CA). The intensity of samples and
bovine serum albumin (BSA) standards was measured in a 96-well plate at 595 nm
using a microplate reader. Data were analyzed using Gen5 data analysis software and
the concentration was calculated based on the BSA standard curve.
6.3 Immunoprecipitation, ID electrophoresis and Western blot
The concentration of previously extracted samples was adjusted to 1 pg/pl
and 20 pi of sample was used for Western blotting and 500 pi for
immunoprecipitation (IP). Samples for IPs were incubated overnight (in a shaker at 4
C) with 2 pi of antibody. The next day, 40 pi of agarose beads (Upstate, MA) were
added into each sample and the mixtures were incubated under the same conditions
for additional 2 hrs. Following incubation, the agarose-Ab-protein complex was
centrifuged (4,000 rpm, 10 min) and the pellet was collected. Each sample was
washed four times with ice cold RIPA buffer and the extraction of
immunoprecipitated proteins using Laemmli sample buffer (Bio-Rad, CA) and heat
denaturation was performed directly before 1-dimensional gel electrophoresis.
Samples were loaded onto 10% polyacrylamide SDS gels (Criterion ReadyMade gel,
45


BioRad, CA) and constant voltage of 180V was applied for 1 hr to separate the
proteins. The gel was subsequently washed with water and fixed overnight in 100 ml
of 40% ethanol/ 10% acetic acid solution. Following staining with Flamingo
fluorescence stain (Bio-Rad, CA) proteins were visualized using a LabScan imager
and bands exhibiting changes in image density between HF and CH samples were
further processed for analysis by mass spectrometry.
To perform Western blotting, the concentration of previously extracted
samples was adjusted to 1 pg/pl and 20 pi of Laemmli buffer was added to 20 pi of
each sample. Samples were incubated for about 5 min in a 100 C water bath then
loaded into a 10% gel. Gels were run for approximately 1 hour (170 V, constant
voltage) and 100V was applied for 30 min to transfer proteins onto a PVDF
membrane.
Membranes were washed with TBS-T wash buffer (Tris buffered saline with
0.1% Tween20) for 5 min and blocked for 2 hours (5 % nonfat milk in wash buffer) at
room temperature. After blocking, membranes were washed again in TBS-T buffer
and incubated overnight (4C) with primary antibody (dilution: from 1:300 to 1:800).
The next day, membranes were washed again and incubated with the secondary
antibody (dilution: 1:10,000) for 1 hour at room temperature. Finally, the membranes
were washed and protein-antibody interactions were detected by chemiluminescence
and X-ray film processing. The densitometry of each band was quantified using
Image Quant 4.0 (GE Healthcare, PA), GAPDH levels provided a loading control.
6.4 In-gel digestion and mass spectrometry
Each band excised from the 10 % SDS gel was sliced into 1mm3 cubes,
destained (1:1 acetonitrile in 50 mM ammonium bicarbonate), and dried in a vacuum
centrifuge for approximately one hour. The dried slices were transferred into fresh
46


microfuge tubes and disulfide bonds were reduced using 1.5 mg/ml solution of DTT
in 25 mM ammonium bicarbonate (one hour at 37 C). Following reduction and
removal of excess reducing reagent, gel samples were cooled to room temperature
and incubated in the dark for an additional hour with alkylating reagent (10 mg/ml
iodoacetamide in 25 mM ammonium bicarbonate). The alkylating solution was
subsequendy removed and gel samples were additionally destained and freeze dried.
Samples were then rehydrated with 0.2 mg/ml of trypsin in 50 mM ammonium
bicarbonate, excess trypsin was removed, and samples were incubated overnight (37
C) with a slight excess of 50 mM ammonium bicarbonate/10% acetonitrile. The next
day, peptides were extracted using 1% trifluoroacetic acid and 60% acetonitrile,
concentrated under vacuum, and stored at -20C prior to analysis using an Agilent
LC/MS Utra Ion Trap.
The in gel-digested samples were analyzed by reverse phase nanospray LC-
MS/MS (Agilent 1100 HPLC, 75 um ID x 15 cm separating column, Zorbax Cig). 5-8
ul of each sample was loaded onto an enrichment column (Agilent, Zorbax 300SB-
C18, 300 pm x 5 mm, 5 pm) using 3% acetonitrile in 0.1% formic acid and washed
for 2 min to remove salts, then a switching valve was toggled to allow peptides to
bind to the separating column. Nano-spray was induced using a capillary voltage of
1550 V applied to a fused silica emitter (PicoTip, New Objective, Inc., Woburn, MA)
with an 8 um aperture. Peptides were eluted into the mass spectrometer using a
gradient of increasing buffer B (90% ACN, 0.1% formic acid) at a flow rate of 300
nLVmin. The gradient was ramped from 3% to 8% B in three min, then from 8% to
40% B over 40 min. Finally, Buffer B was increased to 90% in 5 min, held for 5 min
and returned to initial conditions. Spectra were collected over an range of 350-
18000 Da (Agilent LC/MSD Ultra Trap). Three MS/MS spectra were collected for
the three most abundant values, then those masses were excluded from analysis
for 1 min and the next three most abundant values were selected for
47


fragmentation in order to maximize the dynamic range of peptides sequenced in the
mixture. Over 10,000 MS/MS spectra were typically obtained during a run.
Compound lists of the resulting spectra were generated using an intensity threshold of
10,000 and a minimum of 0.2% relative abundance. Similar spectra observed within a
5 scan window were grouped. The compound lists were searched against a SwissProt
database (UniProtKB/Swiss-Prot Release 57.6; 495,880 entries) using the
SpectrumMill (Agilent Technologies, Inc., Santa Clara, CA) search engine.
Parameters used in the database search were as follows: mouse taxonomy,
carbarn idomethylation as a fixed modification, monoisotopic mass, peptide mass
tolerance of 1.2 Da, fragment ion mass tolerance of 0.6 Da, peptide charge +2 and +3,
and allowance of up to 2 missed tryptic cleavages. Each spectral assignment was
manually confirmed and only accepted if the signal-to-noise ratio of fragment ions
was at least 5.
6.5 NAD+ quantification
Quantification of NAD+ levels in livers was performed using a colorimetric
assay according to the manufacturers protocol. Standard curves generated with a
known amount of purified NADH were used to compute the total tissue concentration
of NAD+ and NADH. The amount of NAD+ was calculated by subtraction of the
NADH concentration from the total NAD (NAD+ + NADH) measurement.
6.6 HAT and HDAC activity assays
HDAC activity was measured using a commercially available assay (Biomol
International LP) according to manufacturers instructions. Approximately 40 pg of
protein from cellular extracts were incubated (15 min., 37 C) with 500 pM Color de
Lys substrate containing acetylated lysine chain. Subsequently, 10 ul of developer
48


was added to each sample and the reaction mixture was again incubated at 37 C for
15 min. The absorbance was read at 405 nm using a plate reader. In a subsequent
experiment, 40 pg of protein from the same samples were first incubated with 5 pi of
1 mM TSA (HDAC I and II inhibitor) and the same procedure as above was followed
to measure the contribution of HDAC III activity. HeLa nuclear extracts with or
without TSA were used as positive and negative controls.
The activity of histone acetyltransferases was established using a colorimetric
assay (BioVision Research Products, CA) according to manufacturers instructions.
Samples were extracted without DTT, as this compound strongly interferes with
colorimetric plate reading. 40 pg of protein from control and high fat samples were
used in the analysis, 10 pi of 10 mg/ml of HeLa cell nuclear extract, provided by the
manufacturer, served as a positive control. The samples were mixed in a U-shaped
96-well plate with 65 pi of assay solution and the plate was read (440 nm OD) after 2
hours of incubation at room temperature.
6.7 SIRT1 and 3 activity assays
The enzymatic activity of SIRT1 and SIRT3 were assayed using commercially
available SIRT1 and SIRT3 activity assays with a few modifications from the
manufacturers protocol. In brief, 40 pg of protein from liver homogenates were
mixed with Fluor de Lys-SIRTl or Fluor de Lys-SIRT2 substrates in 37 C for 45 min
in a 96-well plate. Subsequently, each sample was mixed with 25 ul of developer and
incubated for an additional 45 min. The activity of each sirtuin was measured using a
fluorometric microplate reader at 350nm/450nm.
49


6.8 Statistical analysis
Data from Western blots and activity assays were combined from 3 separate
experiments. Statistical analysis was performed using an unpaired t test and/or one-
way ANOVA. Differences with p < 0.05, p < 0.01, and p < 0.001 were determined to
be statistically significant.
50


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Full Text

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DETERMINING THE CONSEQUENCES OF SIRTUIN ACTIVITY ON LIVER METABOLISM IN A MOUSE MODEL OF OBESITY by Agnieszka A. Kendrick B.S., Wroc/aw University, Poland 2005 A thesis submitted to the University of Colorado Denver in partial fulfillment of the requirements for the degree of Master of Science Chemistry 2010

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This thesis for the Master of Sdence Degree by Agnieszka A. Kendrick has been approved by Date

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Kendrick, Agnieszka A (M.S., Chemistry, CLAS) Determining the consequences of Sirtuin activity on liver metabolism in a mouse model of obesity. Thesis directed by Assistant Professor Karen R. Jonscher and Professor Douglas F. Dyckes. ABSTRACT A proteomics approach was employed to investigate the role of global protein acetylation in dysregulation of hepatic fuel sensing upon exposure to high fat. Using immunoaffinity enrichment and mass spectrometry, 35 hyper-acetylated proteins in the livers of obese mice were observed, about 30% of which were novel. Fatty liver led to significant hyper-acetylation of mitochondrial proteins involved in gluconeogenesis, mitochondrial oxidative phosphorylation, and liver injury. Although protein expression of SIRTI and SIRT3 was unchanged, SIRT3 activity was significantly down regulated in obese livers, with a concomitant 30% decrease in NAD+ levels. No change in histone acetyltransferase activity was observed. Additionally, acetylation levels were increased in SIRT3-1mice fed a high fat diet. Results presented here suggest that reduced SIRT3 activity in obesity may play an important role in fatty liver disease through hyper-acetylation of important mitochondrial proteins involved in energy metabolism.

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This abstract accurately represents the content of the candidates' thesis. We recommend its publication.

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DEDICATION I dedicate this thesis to my husband, James, who provides me with never ending support, love and inspiration. I also dedicate it to my son, Ian, who brightens my every day and makes my life full of fun and excitement.

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ACKNOWLEDGEMNT I wish to thank my advisor, Karen R. Jonscher, for her support and contribution 'to my research. I would especially like to thank her for giving me the opportunity to be a part of this exciting study. I would also like to thank Douglas F. Dyckes, Marc A. Donsky, and Jed E. Friedman for all of their valuable advice and support. Lastly, I would like to express my gratitude to Mizanoor Rahman and Mahua Choudhury for showing me how exciting and interesting biochemical research can be!

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LIST OF CONTENTS LIST OF FIGURES ....................................................................................................... 6 LIST OF TABLES ......................................................................................................... 7 ABBREVIATIONS ....................................................................................................... 9 INTRODUCTION ....................................................................................................... 12 BACKGROUND ......................................................................................................... 14 2.1 Acetylation ........................................................................................................ 14 2.2 Sirtuins .............................................................................................................. 17 2.3 Obesity and mitochondrial dysfunction ........................................................... 19 2.4 MS-based proteomics ..................................................................................... 20 2.5 Research aims ................................................................................................... 23 RESULTS .................................................................................................................... 25 3.1 The identification of acetylation changes in liver mitochondria ...................... 25 3.2 Validation ofMS results via Western blotting ................................................ .30 3.3 Determination of sirtuin expression and activity ............................................. .31 3.4 HAT and other HDAC activity ......................................................................... 32 3.5 Correlation ofNAD+ levels with body weight changes .................................... 33 3.6 The identification of acetylation levels in SIRT3""'mice fed a high fat diet ... .34 DISCUSSION .............................................................................................................. 36 CONCLUSIONS AND FUTURE INVESTIGATIONS ............................................ .42 MATERIALS AND METHODS ................................................................................. 44 6.1 Reagents ............................................................................................................. 44 6.2 Animals and sample extraction .......................................................................... 44 6.3 Immunoprecipitation, I D electrophoresis and Western blot ............................ .45 vii

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6.4 In-gel digestion and mass spectrometry ........................................................... .46 6.5 NAD+ quantification ............................................ ....... ......................... ....... ..... 48 6.6 HAT and HDAC activity assays ....................................................................... 48 6.7 SIRTI and 3 activity assays .................................. ... ......................................... 49 6.8 Statistical analysis ............................................................................................. 50 BIBLIOORAPHY ........................................................................................................ 51 viii

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LIST OF FIGURES Figure 1. Reversible acetylation of a lysine residue .................................................... 14 2. Reaction catalyzed by sirtuin enzymes ........................................................ 17 3. An alternative pathway ofsirtuin activity .................................................... 18 4. The schematic overview of an ion trap mass spectrometer .......................... 22 5. Proteins in liver homogenates from mice on a high fat diet are more highly acetylated than those from mice fed a control diet. .................................. 26 6. Representative fragmentation mass spectra manually validated for protein identification ............................................................................................. 27 7. The overall expression and acetylation levels of selected proteins .............. 31 8. Changes in SIRT1 and 3 expressions and activities ..................................... 32 9. HAT and HDAC class I and II activities in control and obese livers .......... 33 10. NAD+ levels in lean mice as compared to obese counterparts ................... 34 II. The acetylation levels of proteins of interest in SIRT3_,_ mice fed a high fat diet. ......... ... .. .................................... ................... 35 12. Functional classification of proteins identified via mass spectrometry ..... 37 13. Cellular localization and functional description of CPS 1, Aldolase B, PC and SIRT3 ................................................................................................. 39 ix

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LIST OF TABLES Table I. The localization and size ofhuman sirtuins [26] ......................................... 18 2. Hyperacetylated proteins in livers from high fat fed mice compared to chowfed mice ..................................................................................................... 29 X

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ABBREVIATIONS SIRT-sirtuin, silent information regulator HDAC histone deacetylase HAT histone acetyl transferase NAD+-nicotinamide adenine dinucleotide NADH nicotinamide adenine dinucleotide reduced form Ac acetyl moiety CH-chow diet (control diet) HF high fat diet TSA trichostatin DAC-deacetylase ART-adenosyl ribose transferase BMI body mass index ROS reactive oxygen species IP immunoprecipitation PC pyruvate carboxylase Aldolase B fructose biphosphate aldolase B CPS I carbamoyl-phosphate synthase ACoS2 acetyl-Coenzyme synthase 2 GDH glutamate dehydrogenase DTT -dithiothreitol SREBS sterol regulatory element binding proteins MS -mass spectrometry xi

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INTRODUCTION Nearly 40 million American adults are obese and over 50% of the population is overweight. The risks associated with high body mass index include increased incidence of Type 2 Diabetes, cardiovascular disease, cancer, fatty liver and other diseases. Recent studies suggest that there is a direct connection between obesity and decreased activity of sirtuins [1]. Sirtuins are NAD+ dependent histone deacetylases that are directly involved in gene silencing via covalent interactions with chromatin the main component of the DNA packaging pocket [2]. There are seven sirtuin isofonns: SIRTs 1, 2, 6 and 7 are located in the nucleus while SIRTs 3, 4 and 5 are mitochondrial, although SIRT3 was reported to shuttle from the nucleus to mitochondria with cellular stress [3]. Under conditions of caloric restriction, sirtuins have been shown to increase life span [4, 5] and influence important metabolic pathways such as fat mobilization in human cells [ 6], insulin secretion [7], gluconeogenesis [7-9], and fatty acid oxidation in muscle [1, 7, 10]. In contrast to the well-studied role of sirtuins, particularly SIRTI, in models of caloric restriction, relatively little is known about sirtuin activity under conditions of caloric excess, where different organs, including the liver, play a critical role in diseases such as type 2 diabetes, non-alcoholic fatty liver disease and inflammation. To investigate this paradigm, a proteomics approach was employed in which proteins extracted from the livers of mice fed either a high fat or control diet were immunoprecipitated with lysine acetylated antibody, separated using 1 D electrophoresis, digested with trypsin and subjected to mass spectrometry. Acetylation levels of selected proteins were confinned with Western blotting and 12

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sirtuin activity was associated with NAD+ levels and measurements of HAT or HDAC activity. The data suggest that acetylation levels of a number of important metabolic proteins are increased in liver samples from obese mice (high fat diet) and decreased in livers of control-fed mice, supporting the hypothesis that sirtuin activity is decreased in obesity. Data presented here additionally point to a reduction in SIRT3 activity as a potential regulator of a network of proteins implicated in both mitochondrial and cytosolic metabolism. These proteins may play an important role in the progression of diabetes, inflammation, and fatty liver disease under conditions of chronic nutrient excess. 13

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BACKGROUND 2.1 Acetylation Acetylation is a type of protein post-translational modification in which an acetyl group is attached to the side chain of one of the amino acids. Although acetyl groups can bind to lysine, arginine, histidine and even tyrosine residues, the most commonly acetylated amino acid is lysine. Historically, acetylation was only assigned to histones and was shown to have a major influence on regulation of structure and function of chromatin, with subsequent effects on DNA packaging and gene expression [11]. The reactions leading to addition or removal of an acetyl moiety are catalyzed by histone acetyltransferases, or histone deacetylases, respectively (Figure I). Figure 1: Reversible acetylation of a lysine residue. Acetylation (attachment of an acetyl group) is a reversible covalent modification catalyzed by histone acetyltransferases (HATs). HAT activity can be inhibited by interaction of sodium butyrate (NaBu) with the acetylation reaction. The removal of an acetyl group (deacetylation) is catalyzed by histone deacetylases (HDACs) and can be inhibited or activated depending on the type of HDAC enzyme used. HAT HDAC 1\ Lys S r t .. :::!<,..:.-.:":' '"l AcLys 14

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Current studies identify several non-histone targets of HATs and HDACs and therefore suggest a more global influence of lysine acetylation on cellular function [12]. It is also important to mention that modification of lysine via acetylation or deacetylation has a regulatory influence on other possible lysine or adjacent amino acid modifications, like phosphorylation, ubiquitination, sumoylation, methylation, and butyrylation [ 13-15]. The attachment of an acetyl moiety at the side chain amino acid prevents the lysine residue from undergoing different types of modifications. Acetylation removes positive charges, in the case of histones this reduces their attraction to DNA. This makes it easier for RNA polymerase and transcription factors to approach the promoter region. As a result, histone acetylation is well known to improve transcription while histone deacetylation, generally, suppresses transcription [ 16]. Some studies suggest that phosphorylation (which appears on serine residue) and acetylation of histone H3 are coupled and could influence transcriptional response of H3 [17]. Additionally, some members of HDAC family like HDAC6 have been shown to play a regulatory role in ubiquitin-dependent pathways [15, 18]. HATs are proteins that are responsible for the acetylation of both histone and non-histone proteins, they are classified into two major groups: HAT-A localized in the nucleus and responsible for gene regulation, and HAT -B which appear in the cytoplasm and are responsible for modification of newly synthesized core histones [ 19]. Histone acetyltransferases are a superfamily of about 30 enzymes classified into subfamilies based on similarities in sequences, size of active site, and the presence or absence of other protein domains [19]. The three families of HATs are: GNATs, 300/CBPs and MYSTs enzymes [20]. HAT enzymes employ a mechanism based on transferring an acetyl group from acetyl-CoA to a specific lysine residue within a core of histones [21 ]. The function of HATs is associated with DNA repair [20-22], transcription [ 11, 20], tumor suppression [23] and folding of chromatin fiber [22]. 15

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Histone deacetylases comprise a much larger family of enzymes than HATs. HDACs mediate reversible post-translational modification of histones and non histone proteins in which acetyl groups are detached from lysine amino acid residues. Eighteen proteins belonging to the HDAC family are grouped into three classes [20]. Class I and II share analogous catalytic activity and function through a Zn2+ dependent mechanism, whereas class III activity is facilitated by an NAD+ dependent mechanism. Members of class I (HDAC 1, 2, 3, and 8) are relatively small proteins localized almost entirely in the nucleus. Proteins belonging to class II (HDAC 4, 5, 6, 7, 9, and I 0) are much larger in size and have the ability to shuttle in and out of the nucleus [18]. Class I and II are homologous to yeast deacetylases RPD3 and HDA1, respectively; and unlike class III, are inhibited by trichostatin. Acetylation of proteins on lysine residues has been shown to play an important role in regulating nuclear processes such as transcription, DNA repair and gene expression [ 12, 13, 21-25], however the emerging role of this modification in mediating functions of other cellular processes has been the subject of recent investigations. A global proteomics screen revealed 1750 proteins conserved in mitochondria, nucleus, cytoplasm and other cellular fractions to contain 3600 acetylation sites overall [12]. Additionally, acetylation has been shown to be more abundant in mitochondria than other cellular organs [14] and, interestingly, this posttranslational modification is more likely to occur in larger macromolecular complexes [12]. Considering the number of different proteins modified by acetylation, those studies suggest that acetylation could play an important role in many functional processes within the cell. 16

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2.2 Sirtuins Sirtuins (silent information regulators SIRTs) comprise a large histone deacetylase family, with homologs as varied as yeast, mouse and human. Sirtuins are class III HDACs and are NAo+dependent deacetylases. The mechanism of sirtuin action (Figure 2) is based on the transfer of an acetyl group from an acetylated protein to NAD+ with simultaneous cleavage of nicotinamide. The products of this reaction are nicotinamide, deacetylated protein and 0-acetylated ADP-ribose. Figure 2: Reaction catalyzed by sirtuin enzymes. In the presence of NAD+, sirtuins catalyze the deacetylation of lysine-acetylated proteins, with concomitant cleavage of nicotinamide from the NAD+ moiety. The reaction products are 2-0-acetyl ADP ribose, nicotinamide and deacetylated protein. The binding of the acetyl group to the 2-0H position of ribose is highly selective and is only accomplished when catalyzed with yeast Sir2 or SIRTs I, 2, 3, and 5. OH Of NAD-c Lysineacetylated protein 0 OH _H,o . q Sir2/Sirtl Sirt2 lS OH 2-0-acetylated ADPR u rJNH2 + N Nicotinamide tail H 2 N Deacetylated protein Additionally, some members of sirtuin family can act as ADP-ribose transferases (Figure 3). In this alternative mechanism, sirtuins catalyze addition ofthe ADP-ribose portion of NAD+ to a protein with subsequent elimination of nicotinamide from the nucleotide. Currently, only SIRTs 4 and 6 have been reported to employ the ADP-ribose transferase mechanism. while SIRTs 2 and 3 utilize both DAC and ART mechanisms [26]. 17

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Figure 3: An alternative pathway ofsirtuin activity. SIRTs 2, 3, 4 and 6 catalyze a reaction in which the ADP-ribose portion of NAD+ is attached into a protein and nicotinamide is cleaved. Mammalian sirtuins are separated into groups according to location in the cell, mechanism, size and function (Table I). SIRTs 3, 4 and 5 are strictly mitochondrial, and are involved in variety of mitochondrial processes [27]. SIRT2 appears in the cytoplasm and but is also known to interact with some of the nuclear proteins via a shuttling mechanism [27]. SIRT7 is located in the nucleolus and interacts with rRNA. Table 1: The localization and size of human sirtuins [26]. The two mechanisms of sirtuin action are DAC (deacetylase) and ART (mono-ADP ribosyl transferase). Name Location Function Size (kDa) SIRTl Nucleus DAC 62.0 SIRT2 Cytoplasm ARTandDAC 41.5 SIRT3 Mitochondria DACandART 43.6 SIRT4 Mitochondria ART 35.2 SIRTS Mitochondria DAC 33.9 SIRT6 Heterochromatin ART 39.1 SIRT7 Nucleolus ??? 44.8 SIRTI, the homolog of yeast Sir2, is the most widely investigated member of the sirtuin family. Yeast Sir2 was initially described to be an essential part of transcriptional silencing at the silent mating loci and telomeres [26]. Studies show 18

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that Sir2 has an important role in extending life span under caloric restriction in yeast [5]. Both Sir2 and human SIRTI are NAD+ dependent deacetylases. SIRTI is localized to the nucleus, but is also capable of shuttling to the cytoplasm, and it is involved in several different nuclear and cytoplasmic processes [26, 27]. SIRTI can promote cell survival via an interaction with proapoptotic factors like p53 and FOXO [ 4, 8, 9], and can also regulate glucose regulated insulin secretion in pancreatic-P cells [28]. The majority of studies involving SIRTI and its function have been performed in caloric restriction models [2, 4, 6, 7, 25, 29, 30], and showed that resveratrol, the main component of red wine, is the most powerful SIRT1 activator [26]. SIRT3, a mitochondrial homolog of yeast Sir2, activates acetyl-CoA synthetase 2 via deacetylation of a key lysine residue on the enzyme [31 ]. Over-expression of SIRT3 results in increased cellular respiration and decreased mitochondrial membrane potential, with a concomitant decrease in the production of reactive oxygen species [3, 9, 32-34]. Mice lacking SIRT3 have impaired A TP production and mitochondrial respiration [32-34], suggesting a key role for SIRT3 as a metabolic regulator of mitochondrial function. SIRT3 appears to be the primary mediator of mitochondrial acetylation since no significant changes in acetylation status were detectable in mice lacking SIRT4 and SIRT5 [32]. A decrease in SIRT3 has been described in brown adipose tissue of genetically obese animals [33], however its role in the liver has not been studied under conditions of caloric excess. 2.3 Obesity and mitochondrial dysfunction Individuals with a body mass index of 30 kg/m2 or more are defined to be obese, and obesity is one of the dominant public health problems in our century. Diets rich in fat and lack of exercise are considered to be the major causes of increasing body weight that may lead to serious co-morbid medical conditions such as cardiovascular disease [1, 35], hypertension [36], respiratory disease [37], depression 19

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[38], non alcoholic steatohepatitis [39], and insulin resistance, leading to type 2 diabetes mellitus [40]. Each of these medical conditions may be linked to mitochondrial dysfunction, potentially caused by increased P-oxidation of fatty acids, induction of reactive oxygen species, and generation of ketone bodies. Major metabolic pathways dysregulated by high fat diet take place either inside or within the boundaries of mitochondria, resulting in mitochondrial dysfunction [I 0, 41]. The mitochondrion is a double-layered cellular organelle found in almost all eukaryotes. It is a unique organelle, with its own genome and a wide range of mitochondria-specific proteins. A variety of processes take place in this organelle, including those related to energy conversion and production of ATP, apoptosis, calcium signaling and storing, steroid synthesis and cellular proliferation, among others. The role of mitochondria in diseases associated with obesity is primarily related to the energy conversion processes that take place inside mitochondria, particularly the citric acid cycle and oxidative phosphorylation, and the connection of these pathways to glycolysis and gluconeogenesis. Mitochondria can be found nearly in every organ, however they are most densely localized in liver, which contains about 1,000-2,000 mitochondria per cell. The key role ofthe liver in regulation of metabolic pathways and production of ATP, acetyl-CoA and pyruvate is likely enabled by its relatively high mitochondrial content. In the case of a diet rich in fat, the production of acetyi-CoA and pyruvate increases significantly [42]. 2.4 MS-based proteomics Determination of gene function and expression on a protein level can be achieved using a combined biochemical and molecular biology approach called proteomics. Proteomics analysis very often utilizes mass spectrometry as a sensitive 20

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and reproducible method in identification and quantification of proteins, their modifications, and protein-protein interactions. MS-based proteomics was initially applied to small subsets of purified proteins But currently, due to the development of more robust instrumentation and bioinformatic techniques, large scale proteomics approaches are possible [43]. Mass spectrometric data allows determination of the molecular masses of proteins and peptides, their post translational modifications, and even concentrations. Nanoelectrospray ion trap mass spectrometry is a common method of choice in protein identification because of the small amount of sample required, low flow rates, good sensitivity, the ability to obtain multiple fragmentation spectra (MS, MS/MS, MSn), and increased signal-to-noise ratio due to the high accumulation of ions. There are some limitations in using an ion trap spectrometer, such as "1/3 rule". Low mass cutoff, commonly termed the "1/3 rule", does not allow trapping of fragment masses that are 28-30% below the mass of parent ion. However, considering the large size of proteins and relatively large size of peptides, this restriction does not have a significant influence on the sensitivity of ion trap instrument. The main components of an ion trap mass spectrometer are shown in Figure 4 and may be categorized into three groups: an ion source, a mass analyzer, and a detector. The role of an ion source is to either convert gas phase sample molecules into ions or move the existing ions from solution into the gas phase. Some of the common ionization techniques used are chemical ionization (CI), matrix-assisted laser desorption ionization (MALDI), atmospheric ionization (AI), and electrospray ionization (ESI). ESI is especially suitable for large biological molecules that are hard to vaporize or ionize. In the ESI source, a sample is sprayed into the source chamber to form droplets which are further bombarded with high temperature nebulizing gas to remove the solvent. Molecules are subsequently ionization. The charged droplets are passed through the capillary to the skimmer, octopoles, and lenses which help 21

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concentrate and focus ion droplets. Finally, the ions reach the mass analyzer which sorts them according to their mass by applying electromagnetic fields. In this case it would be an ion trap, which collects the ions and then releases them according to their mass-to-charge ratio. An ion trap has the ability to fragment precursor ions and obtain fragmentation spectra via isolation and induction of a particular ion (mainly the most intense signal ion) and generation of product ions. The final part ofthe analysis is the detector, which is responsible for the production of mass spectra based on the measurement of the value of an indicator quantity. It is important to separate the analytical sample into smaller fractions to simplify the analyzed fractions before the introduction to the mass spectrometer. The sample can be separated into smaller portions using high performance liquid chromatography and then directly delivered to the ion source (online analysis). In the liquid chromatographic method, peptides with different masses and charges will elute at different retention times and can be further detected and fragmented in the ion trap. Samples can also be initially separated into smaller fractions, using offline 10or 20LC separation as well as I 0 or 20 electrophoresis. It is also crucial to digest the sample before mass spectrometry with an enzyme that cleaves the protein at specific amino-acid residues and therefore reduces the size ofthe analyzed protein fractions. Figure 4: The schematic overview of an ion trap mass spectrometer. 0 N S 0 ll R C;:: I !J N T R A. N S P () R T AN f) ;:: 0 C 1i S! N C, ASS 4 L Y Z E R [) l I ' .... .. ) ) II F. : n : .:.:-: 22

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After generation of the fragmentation data, each spectrum is assigned a charge and a mass value based on the known information of the mass and charge of the parent ion. Further, the amino acid sequence corresponding to the specific spectrum is identified by comparison of mass and charge list from each spectrum to the peptide fragmentation spectrum enclosed in a database. The protein identity is determined based on the number of unique peptides (peptides specific for only this particular protein), spectral intensity, and number of peptides that were observed with removal of redundant observations. 2.5 Research aims Sirtuins, NAD+-dependent deacetylases, regulate lipid and glucose metabolism in liver and increased SIRTl activity in caloric-restricted models has been linked to extended life span in several species [5, 26, 44]. Some of the members of the SIRT family can deacetylate mitochondrial proteins, suggesting that posttranslational modification by sirtuins may have global effects on energy metabolism. SIRT3 has recently emerged as a mediator of global lysine acetylation in the mitochondria [32] and has been also shown to regulate basal ATP levels in the mitochondria [34]. In recent years, several studies have been published showing the influence of sirtuins on longevity [5, 44], inflammation [26], and mitochondrial regulation in normal or caloric restricted models [2, 4, 6, 7, 9, 25, 29-34, 45] however, the potential role of sirtuins in high fat models has not been extensively explored. Taking into consideration the above results, the following hypothesis was pursued: sirtuin activity is decreased in obesity, leading to increased protein acetylation The goal of the study presented here was to use a high fat feeding model to assess changes in acetylation levels of mitochondrial or cytoplasmic proteins, and elucidate a sirtuin family member potentially responsible for hyper-acetylation of some of those proteins. 23

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The following research aims were pursued: Research Aim 1: Identify the hyper-acetylated proteins in lean and obese animals using mass spectrometry. Research Aim 2: Use immunoblotting to validate MS results. Research Aim 3: Quantify NAD+ levels in the liver tissue. Research Aim 4: Correlate the activity of sirtuins, other HDACs and HATs with protein acetylation levels. Research Aim 5: Determine which particular sirtuin family member may be the primary mediator of protein acetylation levels in a model of obesity. 24

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RESULTS 3.1 The identification of acetylation changes in liver mitochondria A targeted proteomic approach was employed to evaluate the acetylation changes of liver mitochondrial proteins between control and high fat fed animals. Livers from mice fed either control or a high fat diet (45 kcal% fat, 12 weeks) were harvested and homogenized. In the case of animals on a high fat diet, only tissues from animals that showed significant mass gain were selected for the analysis. Proteins modified by lysine acetylation were immunoprecipitated with anti acetyllysine antibody, followed by separation by one dimensional gel electrophoresis (Figure 5). Bands showing differential staining between the control and HF fed mice were excised, proteins digested with trypsin, and analyzed by tandem mass spectrometry using an Agilent Ultra quadrupole ion trap. Product ion spectra (Figure 6) were searched with SpectrumMill against the SwissProt database. 35 proteins showing differential acetylation levels were identified with high confidence (Table I). Some of the identified proteins, such as carbamoyl-phosphate synthase, uricase, pyruvate carboxylase and ATP synthase, are known to contain acetylated lysines [14]. Some of the proteins observed have not been previously reported to be acetylated. 25

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Figure 5: Proteins in liver homogenates from mice on a high fat diet are more highly acetylated than those from mice fed a control diet. Proteins were first immunoprecipitated with acetyllysine antibody and then separated using ID gel electrophoresis using a 10% SDS-PAGE gel. Bands showing differential staining are identified. These bands are excised. in gel-digested, and analyzed using tandem mass spectrometry. HF CH 250k D a .. 1 50kDa B.i::dJD / 1 00kDa 75k0a BAiJD .1 S.<.\rJD ') r; .....:.. 37kDa 8/i.ND / 26

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Figure 6: Representative fragmentation mass spectra manually validated for protein identification. A Pyruvate Carboxylase (PC), B Carbamoyl-phosphate synthase (CPS 1 ), and C Fructose biphosphate aldolase B (Aldolase B). The precursor ions for peptides presented in the spectras were fragmented, and the resulting spectra are shown. The masses of the theoretical fragment ions are listed with the observed ions in bold. All fragment ions were required to have a signal-to-noise ratio of at least 5. The y-type fragment ions (charge is retained on C terminal end of peptide) and b-type fragment ions (charge is retained on N terminal end of peptide) are shown. A !ll 12i5 :m 1 :ss 359 ')59 1CCl 966 923 687 750 565 5SJ 535 !85 I .nl ! -'----'----------27

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bl-489 546 611 639 713 b 261 :J18 417 488 617 745 858 995 1110 12:J9 1296 1442 B Phe Lue Gly Val Ala Glu Gin L<'U His. Asn Glu Gly Ph<' Ly y 1442 U29 1272 1173 1102 972 1144 7:J1 594 480 351 294 147 y2< 721 664 636 S87 551 487 42l 366 298 241 176 147 74 y3+ 481 444 425 191 368 325 282 244 2 0 yll y6 1 1 !) "' yS 0 y7 -;; ;;; c b9 1 0 y9 y3 ylj; .. bll y12 0 5 b2 b3 y4 n1 IL ISS 300 428 531 628 715 802 915 986 1099 1227 1356 1471 1542 1656 1727 1140 1910 c lie Ala Asp Gr. Cys Pro Ser Ser le\1 Ala lie Gin Gu AY: AI.! .\sri Ala le\1 Ala Arg y 1971 1901 1786 1657 1555 1458 1l71 1114 1171 1099 986 151 729 615 544 430 )59 246 175 yl ')g(i 95() 89l 829 778 729 685 642 586 550 493 429 365 3C@ m 2'" ) 180 123 88 3-y13 biO ylO c 2" b11 Oi) -!: y14 .. I )'9 s y16
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Table 2: Hyperacetylated proteins in livers from high fat fed mice compared to chow-fed mice. Thirty five high scoring proteins identified from differentially stained gel bands. The total number of peptide spectra that were observed that may be derived from a protein are summarized as "Spectra #", while "Distinct Peptides #" reflects the number of peptides that were observed, with removal of redundant observations. "Summed MS/MS Score" results from addition of peptide scores for the highest scoring distinct peptides for each protein. All proteins reported had at least two distinct peptides whose sequence assignments were manually validated. The accession number is linked to the SwissProt database, where more information regarding the structure and function of the protein may be investigated. Proteins marked with an asterisk have not been previously reported as acetylated based on UniProtKB/Swiss-Prot database information. r.._ -,, I .,, i .. I :_,.,, '" I ;,, ,. I 'I u: I I : I' it I ->+ :,. i' ,, \\ l':n \I\\ .: ),[I ---",,I_' : __ lr''-1--,,;I 1'1. i "''' ( .: : .l _i_J_ __ !) I t--Jhl i lllll."ih.._ i :.1[;,; "-\ Lj(!' .. L,.._' ------------'i' ,,-jllu.._l, : -:h" ,,iJ,l.l, )I :I, hll.: 'lh .----+-----+--: '(I -+--_____ II;'J:; -'--__ __ ni,:;r' 11 !'' 11'\'hr i ,::J/\11:, \ :1:..:.:-, : ... :: 1; ... i t. :; :..: \', , l :,l; ;; r, t.,-1 .. __ .. .. 1 >-.;,..; I I:! -I i -----------------29

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3.2 Validation ofMS results via Western blotting Several of the proteins identified by mass spectrometry that are especially relevant to metabolic function were further validated for both protein expression and acetylation changes by Western blotting. Proteins from HF and CH liver homogenates were immunoprecipitated using acetyllysine antibody, then probed for specific protein identity by Western blotting (Figure 7). Additionally the overall expression levels of those proteins were determined using the same Western blotting approach but without prior enrichment by immunoprecipitation with acetyllysine antibody. As shown in Figure 7A, expression of CPS I significantly decreased (2-fold decrease, p < 0.01) with obesity while acetylation levels increased, suggesting that CPS 1 is hyper-acetylated in the livers of obese mice. Although protein expression of PC and Aldolase B appeared to be unchanged between samples, acetylation of both proteins were increased in HF (Figure 78 and C), suggesting these proteins are also hyper-acetylated in obesity. 30

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Figure 7: The overall expression and acetylation levels of selected proteins. Lysine acetylated proteins were immunoprecipitated from liver extracts of high fat (HF) and chow diet (CH) mice. Cell lysates were also Western blotted and probed with (A) CPSI, (B) Aldolase B, and (C) PC. For each set ofthree Westerns the upper band indicates acetylated-protein, the middle represents the overall expression of that protein, and the bottom band shows the loading control for the protein expression experiments. Blots shown in here are representative of three separate experiments that showed similar results. The bar graph represents total expression changes of CPS I (n=6, p < 0.01). A CH HF B I-Ac-CPS1 CPS1 r---= -----------'GAPDH c CH HF CH HF Ac-Aidolase B 1-JAldolase B i .... niL_ 1GAPDH CH HF Ac-PC 1---i-PC I 1-GAPDH ._____ ____ _, 3.3 Determination of sirtuin expression and activity In order to determine whether sirtuin activity might account for these changes in protein acetylation status, commercially available activity assays were used to measure the activities of SIRTI and 3 in liver homogenates. The expression of those sirtuins was evaluated using Western blotting with SIRTI and 3 antibodies in the same set of samples. The activities of other sirtuins could not be determined due to the unavailability of activity assays. The measurements revealed that the expression ofSIRTl and SIRT3 were unchanged inCH and HF livers. Although SIRTl activity also remained unchanged, SIRT3 activity was significantly (p
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Figure 8: Changes in SIRTl and 3 expressions and activities. Liver homogenates were assayed for the influence of specific sirtuins on acetylation levels of the identified proteins. High fat and chow samples were Western blotted and probed with (A) anti-SIRTl and (B) anti-SIRT3 to assess total cellular levels of those proteins. Additionally 40 J.lg of cell lysates were incubated with fluorescent sirtuin substrates and fold changes in enzymes activities were determined (n = 6; # p.-.. Q) :;: 01 0 .75 -0)0.75 -c: > c .... cu :;:; IU "5 0 .50 U..c IU U 0 .50 t:"C C")'C ,__ .. iii g 0 .25 .: 0 0.25 -r -0 .00 CH HF 0 .00 CH HF 3.4 HAT and other HDAC activity The acetylation or deacetylation of proteins can be influenced by HAT or HDAC enzymes, respectively. In order to ascertain whether the observed protein hyperacetylation might be due to HAT or HDAC class 1 and 2 activity instead of sirtuin activity, the same sets of samples as the ones used previously were analyzed using commercially available HAT and HDAC activity assays. Figure 9 reveals that HAT and HDAC I and II activities are unchanged in obesity, suggesting that reduction in deacetylase activity, most likely that of SIRT3, is primarily responsible for the observed protein hyper-acetylation. 32

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Figure 9: HAT and HDAC class I and U activities in control and obese livers. 40 J.lg of cell lysates were assessed for changes in (A) HAT or (B) HDAC activities. The results are presented in a bar graph with error bars indicating SEM from six biological replicates (3 chow and 3 high fat). For both experiments, HeLa cell extracts provided an experimental control. A 0.125 >-(I) 0.100 -C) ::; c -IU 0 075 t).c" IU U 1-"C 0 050 ->t -c cao .so (J(J ct ::2o 25 co 0 00 -,---CH 3.5 Correlation ofNAD+ levels with body weight changes -r HF NAD+ concentration is a major modulator of class III HDACs [46], suggesting that changes in sirtuin activity might be linked to changes in NAD+ levels. The levels of NAD+ in liver samples were measured as a function of weight gain in mice. Overall, NAD+ levels were significantly lower in HF mice by 30% (Figure 1 OA) and appeared to decrease linearly with increasing body weight change (Figure lOB). 33

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Figure 10: NAD+ levels in lean mice as compared to obese counterparts. About 20 mg of HF and CH liver lysates were used to evaluate the concentration of NAD+ in obese and control animals. (A) Quantification ofNAD+ levels in CH and HF samples shows significantly higher concentrations in control diet samples as compared to fat. The data presented in a bar graph contain error bars indicating SEM from six biological replicates (3 chow and 3 high fat; # < 0.05). (B) The correlation between body weight and NAD+ concentration for six samples (n = 6; R2 = 0.8765). Three separate experiments were performed with similar results. Representative data are presented. A 1 25 (.) 1 00 c ...... ocn u E 75 +tn 0C50 <('-' z 25 0 T # -,-CH HF 8 ...... C'l E -C'l c -100 (.) c 0 (.) + c 50 ................ ..... ........ .... ..... ........... ........... ... ... <( z 0 10 20 30 Change in body weight [g) 3.6 The identification of acetylation levels in SIRT3-tmice fed a high fat diet To test whether the hyper-acetylation of proteins observed under high fat-fed conditions is related to reduced SIRT3 activity, liver extracts from wild-type and SIRT3-deficient mice were prepared, proteins were immunoprecipitated using anti acetyllysine antibodies and probed for PC, Aldolase 8 and CPSI by Western blot. Under chow fed conditions there was very little change in the acetylation status of selected proteins in Sirt3-1mice as compared to WT mice (Figure II). However, following high fat feeding, Western blots for PC, Aldolase B and CPS I revealed that these proteins had exaggerated hyper-acetylation in the absence of SIRT3 as compared to WT high fat-fed mice. There was no concomitant change in protein 34

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concentration. These results support the hypothesis that the combination of diminished SIRT3 activity and low NAD+ levels leads to increased lysine acetylation in obesity. Figure 11: The acetylation levels of proteins of interest in SIRT3_,_ mice fed a high fat diet. Wild type (WT) and SIRT3_,_ (KO) animals were fed either high fat diet (60% fat) or regular chow for 8-9 weeks. Liver homogenates were immunoprecipitated with acetyllysine and immunoblotted with anti-PC, anti-Aldolase B and anti-CPS I. Additionally cell lysates were Western blotted and probed with (A) CPSI, (B) Aldolase B, and (C) PC. For each set of three Westerns the upper band indicates acetylated-protein, the middle represents the overall expression of that protein, and the bottom band shows the loading control for the protein expression experiments. A 8 HF WT HF CH WT CH KO j -----Ac-PC '--------' Ac-Aidolase 8 ;.__.......,: _.-Aldolase 8 .___ ___ __, 35

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DISCUSSION In the current study, targeted proteomics revealed increased levels of lysine acetylation modification of proteins in high fat fed mice as compared with chow fed controls. In summary, increased protein acetylation and decreased NAD+ levels in obese mice correlated with decreased SIRT3 activity and, based on those results, one could postulate that acetylation-based protein modification impairs protein function, potentially leading to obesity-associated diseases such as diabetes, inflammation, cardiovascular and fatty liver disease. The targeted proteomics approach identified 35 proteins hyper-acetylated in livers of animals fed a high fat diet. A subset of the proteins identified using this approach, such as CPS1, uricase, PC and ATP synthase, have previously been shown to contain acetylated lysines [ 14]. Functional classificationFigure 12) of high scoring protein identifications from the excised bands suggests that amino acid metabolism, gluconeogenesis, fatty acid metabolism, TCA cycle enzymes, redox regulation and stress response pathways may be mediated by acetylation in response to obesity. 36

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Figure 12. Functional classification of proteins identified via mass spectrometry. The proteins were separated into functional groups based on infonnation contained in the SwissProt database. R edox J Regulation 21% Based on the data from HAT and HDAC activity assays, acetylation changes are likely not due to changes in HAT or HDAC class I and II activities. Additionally, the decreased NAD+ concentration in obese animals suggests that one or more members of the sirtuin family may play an important role in the control of acetylation levels in a high fat diet model of obesity. In order to elucidate which member of the sirtuin family could be responsible for the acetylation changes, the expression and activities of SIRTI and 3 were measured. The data presented in here support recent reports that mitochondrial SIRT3 may be one of the key regulators of acetylation levels in mitochondria [2, 9, 31-34]. SIRT3 is a NAD+ dependent deacetylase crucial for maintaining basal A TP levels in mitochondria [34]. Recent studies suggest that SIRT3 might be responsible for global changes in acetylation of several mitochondrial proteins [32]. The depletion of the SIRT3 gene in mice causes increased acetylation of proteins present in mitochondrial-rich tissues and, analogous to SIRTI, the SIRT3 gene has been shown to be involved in regulating longevity [34]. SIRT3-deficient mice have been 37

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generated and, although there is striking mitochondrial protein hyper-acetylation, SIRT3 deficient mice generally appear to be metabolically unremarkable under basal conditions [9, 32]. In contrast, during stress conditions SIRT3 appeared to be necessary in preventing heart dysfunction through modulation of cellular levels of ROS [9], suggesting that SIRT3 might play a role in stress response pathways. Some of the ROS-scavenging antioxidase enzymes, such as catalase (a known SIRT3 target [9]), uricase, peroxiredoxin and glutathione peroxidase, have been reported to be hyper-acetylated in fed but not fasted mice. It is likely that down regulation of these antioxidant systems by hyper-acetylated mediators would tend to further increase ROS levels and oxidative stress. Given that ROS and oxidative stress are common in obesity [ 4 7], the results suggest that excessive acetylation and reduced SIRT3 activity may impair the function or activity of oxidative stress defense proteins, thereby contributing to liver injury and accumulation of damaged proteins. High fat diet also led to hyper-acetylation of proteins involved in gluconeogenesis and ureagenesis, including PC, Aldolase-B and CPS-1 (Figure 13). Aldolase B is a bifunctional enzyme that participates in both glycolysis and gluconeogenesis. 38

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Figure 13: Cellular localization and functional description of CPSl, Aldolase B, PC and SIRT3 . ,t.,[. ,;. J ,_-. ... ..... .. li'il \ ,n E ;.' 1 '- ,l'' - J.:l-J' f'.' J :. .... :;:.-c.:.j' ;-. .]: .. :(_ : .. 1 _)c<: 11.]!f v I I II IV Ill c,,e o .. K' H" "-" Pyruvate carboxylase is an enzyme involved in the transfonnation of pyruvate to oxaloacetate in the first step of gluconeogenesis [8] and is an important regulator of the TCA cycle. Carbamoyl-phosphate synthase is a liver mitochondrial matrix protein and a regulatory enzyme in the urea cycle. CPS 1 is crucial in the process of detoxification of excess ammonia from an organism via regulation of the production of carbamoyl phosphate. Carbamoyl phosphate is the first reactant in a series of reactions leading to the production of urea, a relatively non-toxic compound from the highly toxic precursor compound, ammonia. The active sites of this enzyme are connected by a molecular tunnel containing several lysines [48]. It is possible that acetylation of one or more of these lysines influences the metabolism of ammonia through the enzyme, resulting in toxic buildup in the liver. Recently, CPS I was 39

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shown to be a target of SIRT5 [30], however data presented here suggest that it may be additionally modified by SIRT3. Gluconeogenesis is increased in obesity due to insulin resistance [49). and lysine acetylation of PC and Aldolase B may disrupt glycolysis or gluconeogenesis pathways, leading to increased production of glucose and development of Type 2 diabetes. The consequences of up regulation of gluconeogenesis are increased levels of acetyl-CoA from excess glucose, as well as an increased risk of obesity. Therefore, post translational modifications of pyruvate carboxylase and Aldolase B might lead to important changes in the gluconeogenesis pathway. Several heat shock proteins were identified: HSP60, HSP70 and HSP71. Heat shock proteins are ubiquitously expressed in the cytoplasm and nucleus and are named based on their molecular weight and their capability of being induced by heat. Heat shock proteins are present in the cell under normal conditions and are responsible for stabilization of partially folded proteins and binding to partially synthesized proteins, preventing them from aggregating and becoming nonfunctional [50]. In stress-related conditions like extreme temperature variance, viral infection, energy depletion, ROS overproduction, and acidosis, heat shock binding factors (present in the cytosol) separate from the heat shock proteins and translocate to the nucleus where they cause increased production of heat shock proteins [51]. The overproduction of heat shock proteins plays an important role in cell protection from stress via inhibition of apoptosis, repair and refolding of stress-damaged proteins [50, 51]. In addition to the heat shock proteins, GRP78, a hyper-acetylated target in fatty liver [52]. was also identified. GRP78, glucose related protein, is the ER homologue of HSP70 with a conserved A TPase domain and a peptide-binding domain [53]. As a chaperone, GRP78 directly interacts with all three ER stress 40

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sensors (inositol-requiring protein 1 IRE1, PKR-like endoplasmic reticulum kinase PERK, and activating transcription factor ATF -6) and maintains them in inactive forms in non-stressed cells [54]. Stable over expression of GRP78 inhibits the activation of sterol regulatory element binding proteins and the genes under their regulation [55]. SREBPs are transcription factors localized to the nuclear envelope and endoplasmic reticulum that are responsible for binding to sterol regulatory DNA sequences, regulating the expression of enzymes involved in lipid homeostasis [56]. Hyper-acetylation of GRP78 may be a potential factor affecting lipid metabolism as well as the ER stress response in fatty livers [53]. There is emerging evidence that mitochondrial dysfunction plays a key role in the physiopathology of non-alcoholic steatohepatitis [39]. Notably, impaired mitochondrial respiration has been shown to induce the ER stress response and upregulate G RP78 [57]. The acetylation of the mitochondrial electron transport chain protein A TP synthase and the TCA cycle enzyme fumarate hydratase were showed here to be increased. Modification of these proteins by hyper-acetylation might lead to dysfunctional mitochondrial respiration and subsequent impairment in lipid oxidation and TCA cycle activity [1]. 41

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CONCLUSIONS AND FUTURE INVESTIGATIONS Targeted proteomics revealed increased levels of lysine-acetylated proteins in the livers of obese mice as compared with normal controls. Results from proteomics studies and enzymatic activity assays show a correlation between increased protein acetylation, decreased NAD+ levels and decreased SIRT3 activity in obese mice. It could be postulated that this protein modification may impair or regulate protein function, potentially leading to obesity-associated diseases such as diabetes, cancer, cardiovascular and fatty liver disease. One could suggest that posttranslational modification by sirtuins may have global effects on energy metabolism, especially gluconeogenesis and lipogenesis. Given previous observations demonstrating SIRT3 as a modulator of ACoS2, Complex I and GDH [8, 32], and results presented here could suggest that the list of SIRT3 targets might extend to other mitochondrial proteins including some acetylated proteins identified in this study. Further studies are necessary to identify other potential SIRT3 targets and to evaluate the influence of acetylationldeacetylation on their function. In order to perform such studies, SIRT3 deficie11t and/or SIRT3 over expressed models, fed a regular and high fat diet, would be required. It is important to evaluate the ATP, NAD+ and NAD+/NADH levels in those animals in addition to identifying and quantifying changes in specific proteins hyper-acetylated in SIRT3 deficient and over expressed mice. One could also consider evaluating expression changes of varies mitochondrial metabolites like oxaloacetate, pyruvate, acetyl-CoA and others. Additionally it would be worthwhile to assess the changes in oxidative or ER stress in high fat vs. control animals. 42

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Taken together, these fmdings show that high fat diet has a striking effect on the acetylation status of numerous cytosolic and mitochondrial proteins in association with reduced hepatic NAD+ levels and attenuated SIRT3 activity. SIRT3 depletion enhances these posttranslational modifications, supporting its key role in chronic fat overload-mediated metabolic protein acetylation. Given the complexity and cross-talk of biochemical pathways involved in cellular metabolism, care must be taken in future work to identify important metabolites and quantitatively assess changes in their concentrations when proteins mediating those pathways are acetylated. The experimental challenges are significant; however, this hyper-acetylation of hepatic metabolic proteins in obesity may underlie susceptibility to fatty liver, inflammation, and diabetes. Understanding the molecular pathogenesis of these diseases will ultimately lead to the development of effective therapies for these conditions. 43

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MATERIALS AND METHODS 6.1 Reagents Polyclonal anti-acetylated lysine antibody was purchased from Cell Signaling (Danvers, MA). All other antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). HRP-conjugated goat anti-rabbit and anti-mouse immunoglobulin G were purchased from Bio-Rad Laboratories (Hercules, CA). Protease inhibitors were obtained from Sigma (St. Louis, MO). HPLC-grade water, acetonitrile and fonnic acid were purchased from VWR (Denver, CO). Sequencing grade trypsin was bought from Promega (Madison, WI), iodoacetamide and dithiothreitol were from Sigma. SIRT1 and SIRT3 fluorometric assays were obtained from Biomol (Plymouth Meeting, PA), NAD+ quantification was performed usign a kit from Bio Vision (Mountain View, CA), and a HAT activity assay kit was purchased from Upstate Biotechnology (Lake Placid, NY). Normal and high fat chow diets for the animals were purchased from Research Diet, Inc. (New Brunswick, NJ). 6.2 Animals and sample extraction Animal protocols were approved by the University of Colorado Institutional Animal Care and Use Committee in accordance with National Institute of Health guidelines. Animals were a generous gift from Dr. Mizanoor Rahman and Dr. Carrie McCurdy. S-6 week old male C57BU6 SVJ mice were fed either a control chow or high fat diet (45 kcal% fat, Research Diets) for 16 weeks. The wild type and SIRT3 -/mice [3] were fed a diet containing 60 kcal% fat for 8-9 weeks. Livers were harvested immediately from anesthetized mice and snap frozen at -70 C in liquid 44

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nitrogen before analysis. Mice were sacrificed by pentobarbital over-dose following treatment. Livers (around 200 mg each) were mixed with 500 Jll of lysis buffer A (10 Mm HEPES (pH 7.9), 0.5 mM KCI, 10 mM MgC}z, 0.5 mM OTT, 0.1% IGEPAL CA-630, 0.5 mM phenylmethylsulfonyl fluoride) and homogenized. The homogenates were collected and centrifuged at 7,000 rpm in 4oC for 10 min. The supernatant was then collected and transferred into fresh tubes, and the remaining pellet was combined with 200 )11 of buffer B (20 mM HEPES (pH 7.9), 25% glycerol, 1.5 mM MgCl2. 420 mM NaCI, 0.5 mM DTT. 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 4 J.IM leupeptin), and was subsequently centrifuged (14,000 rpm at 4oC for 30 min). Protein concentration was measured using a standard colorimetric Bradford reagent assay (BioRad, CA). The intensity of samples and bovine serum albumin (BSA) standards was measured in a 96-well plate at 595 nm using a microplate reader. Data were analyzed using GenS data analysis software and the concentration was calculated based on the BSA standard curve. 6.3 Immunoprecipitation, lD electrophoresis and Western blot The concentration of previously extracted samples was adjusted to 1 Jlg/Jll and 20 Jll of sample was used for Western blotting and 500 Jll for immunoprecipitation (IP). Samples for IPs were incubated overnight (in a shaker at 4 C) with 2 Jll of antibody. The next day, 40 Jll of agarose beads (Upstate, MA) were added into each sample and the mixtures were incubated under the same conditions for additional 2 hrs. Following incubation, the agarose-Ab-protein complex was centrifuged (4,000 rpm, 10 min) and the pellet was collected. Each sample was washed four times with ice cold RIP A buffer and the extraction of immunoprecipitated proteins using Laemmli sample buffer (Bio-Rad, CA) and heat denaturation was performed directly before !-dimensional gel electrophoresis. Samples were loaded onto 10% polyacrylamide SDS gels (Criterion ReadyMade gel, 45

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BioRad, CA) and constant voltage of 180V was applied for 1 hr to separate the proteins. The gel was subsequently washed with water and fixed overnight in 100 ml of 40% ethanol/ 10% acetic acid solution. Following staining with Flamingo fluorescence stain (Bio-Rad, CA) proteins were visualized using a LabScan imager and bands exhibiting changes in image density between HF and CH samples were further processed for analysis by mass spectrometry. To perform Western blotting, the concentration of previously extracted samples was adjusted to 1 J.Ig/J.Il and 20 J.Il of Laemmli buffer was added to 20 J.Il of each sample. Samples were incubated for about 5 min in a 100 C water bath then loaded into a 10% gel. Gels were run for approximately 1 hour (170 V, constant voltage) and 1 OOV was applied for 30 min to transfer proteins onto a PVDF membrane. Membranes were washed with TBS-T wash buffer (Tris buffered saline with 0.1% Tween20) for 5 min and blocked for 2 hours (5 % nonfat milk in wash buffer) at room temperature. After blocking, membranes were washed again in TBST buffer and incubated overnight (4C) with primary antibody (dilution: from 1:300 to 1:800). The next day, membranes were washed again and incubated with the secondary antibody (dilution: 1:10,000) for 1 hour at room temperature. Finally, the membranes were washed and protein-antibody interactions were detected by chemiluminescence and X-ray film processing. The densitometry of each band was quantified using Image Quant 4.0 (GE Healthcare, PA), GAPDH levels provided a loading control. 6.4 In-gel digestion and mass spectrometry Each band excised from the 10 % SDS gel was sliced into 1mm3 cubes, destained (1: 1 acetonitrile in 50 mM ammonium bicarbonate), and dried in a vacuum centrifuge for approximately one hour. The dried slices were transferred into fresh 46

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microfuge tubes and disulfide bonds were reduced using 1.5 mg/ml solution of DTI in 25 mM ammonium bicarbonate (one hour at 37 C). Following reduction and removal of excess reducing reagent, gel samples were cooled to room temperature and incubated in the dark for an additional hour with alkylating reagent (1 0 mglml iodoacetamide in 25 mM ammonium bicarbonate). The alkylating solution was subsequently removed and gel samples were additionally destained and freeze dried. Samples were then rehydrated with 0.2 mg/ml of trypsin in 50 mM ammonium bicarbonate, excess trypsin was removed, and samples were incubated overnight (37 C) with a slight excess of 50 mM ammonium bicarbonate/10% acetonitrile. The next day, peptides were extracted using 1% trifluoroacetic acid and 60% acetonitrile, concentrated under vacuum, and stored at -20C prior to analysis using an Agilent LC/MS Utra Ion Trap. The in gel-digested samples were analyzed by reverse phase nanospray LC MS/MS (Agilent 1100 HPLC, 75 urn ID x 15 em separating column, Zorbax C18). 5-8 ul of each sample was loaded onto an enrichment column (Agilent, Zorbax 300SB C18, 300 J.Im x 5 nun, 5 J.Im) using 3% acetonitrile in 0.1% fonnic acid and washed for 2 min to remove salts, then a switching valve was toggled to allow peptides to bind to the separating column. Nano-spray was induced using a capillary voltage of 1550 V applied to a fused silica emitter (PicoTip, New Objective, Inc., Woburn, MA) with an 8 urn aperture. Peptides were eluted into the mass spectrometer using a gradient of increasing buffer B (90% ACN, 0.1% fonnic acid) at a flow rate of 300 nUmin. The gradient was ramped from 3% to 8% B in three min, then from 8% to 40% B over 40 min. Finally. Buffer B was increased to 90% in 5 min, held for 5 min and returned to initial conditions. Spectra were collected over an range of 35018000 Da (Agilent LC/MSD Ultra Trap). Three MS/MS spectra were collected for the three most abundant values, then those masses were excluded from analysis for 1 min and the next three most abundant values were selected for 47

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fragmentation in order to maximize the dynamic range of peptides sequenced in the mixture. Over I 0,000 MS/MS spectra were typically obtained during a run. Compound lists of the resulting spectra were generated using an intensity threshold of 10,000 and a minimum of0.2% relative abundance. Similar spectra observed within a 5 scan window were grouped. The compound lists were searched against a SwissProt database (UniProtKB/Swiss-Prot Release 57.6; 495,880 entries) using the SpectrumMill (Agilent Technologies, Inc., Santa Clara, CA) search engine. Parameters used in the database search were as follows: mouse taxonomy, carbamidomethylation as a fixed modification, monoisotopic mass, peptide mass tolerance of 1.2 Da, fragment ion mass tolerance of 0.6 Da, peptide charge + 2 and + 3, and allowance of up to 2 missed tryptic cleavages. Each spectral assignment was manually confirmed and only accepted if the signal-to-noise ratio of fragment ions was at least 5. 6.5 NAD+ quantification Quantification of NAD+ levels in livers was performed using a colorimetric assay according to the manufacturer's protocol. Standard curves generated with a known amount of purified NADH were used to compute the total tissue concentration of NAD+ and NADH. The amount of NAD+ was calculated by subtraction of the NADH concentration from the total NAD (NAD+ + NADH) measurement. 6.6 HAT and HDAC activity assays HDAC activity was measured using a commercially available assay (Biomol International LP) according to manufacturer's instructions. Approximately 40 J.lg of protein from cellular extracts were incubated ( 15 min., 3 7 C) with 500 J.1M Color de Lys substrate containing acetylated lysine chain. Subsequently, 10 ul of developer 48

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was added to each sample and the reaction mixture was again incubated at 37 C for 15 min. The absorbance was read at 405 nm using a plate reader. In a subsequent experiment, 40 jlg of protein from the same samples were first incubated with 5 Ill of 1 mM TSA (HDAC I and II inhibitor) and the same procedure as above was followed to measure the contribution of HDAC III activity. HeLa nuclear extracts with or without TSA were used as positive and negative controls. The activity of histone acetyltransferases was established using a colorimetric assay (BioVision Research Products, CA) according to manufacturer's instructions. Samples were extracted without DTI, as this compound strongly interferes with colorimetric plate reading. 40 jlg of protein from control and high fat samples were used in the analysis, 10 jll of 10 mglml of He La cell nuclear extract, provided by the manufacturer, served as a positive control. The samples were mixed in a U-shaped 96-well plate with 65 jll of assay solution and the plate was read (440 nm OD) after 2 hours of incubation at room temperature. 6.7 SIRTI and 3 activity assays The enzymatic activity ofSIRT1 and SIRT3 were assayed using commercially available SIRTI and SIRT3 activity assays with a few modifications from the manufacturer's protocol. In brief, 40 jlg of protein from liver homogenates were mixed with Fluor de Lys-SIRTJ or Fluor de Lys-SIRT2 substrates in 37 C for 45 min in a 96-well plate. Subsequently, each sample was mixed with 25 ul of developer and incubated for an additional 45 min. The activity of each sirtuin was measured using a fluorometric microplate reader at 350nm/450nm. 49

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6.8 Statistical analysis Data from Western blots and activity assays were combined from 3 separate experiments. Statistical analysis was performed using an unpaired t test and/or one way ANOVA. Differences with p < 0.05, p < 0.01, and p < 0.001 were determined to be statistically significant. 50

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