O-LINKED BETA–N-ACETYLGLUCOSAMINE (O-GLCNAC) AND THE
MITOCHONDRION
By
CHRISTOPHER E. CALDERON
JOHN C. CHATHAM, COMMITTEE CHAIR
SCOTT W. BALLINGER
VICTOR M. DARLEY-USMAR
LORI L. McMAHON
MARTIN E. YOUNG
A THESIS
Submitted to the graduate faculty of The University of Alabama at Birmingham,
in partial fulfillment of the requirements for the degree of
Master of Science
BIRMINGHAM, ALABAMA
2013
Copyright by
Christopher E. Calderon
2013
O-LINKED BETA–N-ACETYLGLUCOSAMINE (O-GLCNAC) AND THE
MITOCHONDRION
CHRISTOPHER E. CALDERON
CELL, DEVELOPMENTAL, AND INTEGRATIVE BIOLOGY
ABSTRACT
O-linked beta–N-acetylglucosamine (O-GlcNAc) is a dynamic and ubiquitous
posttranslational modification of serine and threonine residues on nuclear and
cytoplasmic proteins. O-GlcNAc has emerged as an important regulator of cellular
processes such as cell signaling, transcription, translation, apoptosis, and cell cycle
regulation, among others. O-GlcNAc is thought to be a contributor to pathologies such as
hyperglycemia and insulin resistance. O-GlcNAc has been viewed as an indicator of
cellular energy levels and is associated with diabetic complications under nutrient excess.
Other studies have shown that a variety of stress stimuli increase the levels of protein OGlcNAc in mammalian cells, and this increase is associated with cytoprotection.
Inhibiting O-GlcNAcylation decreased cell survival in response to stress, while increased
O-GlcNAcylation augmented cell survival. Many of these studies demonstrated that this
cytoprotection was associated with mitochondrial proteins.
Compared to the nucleo-cytoplasm, much less work has been done in elucidating
the potential role of O-GlcNAc in the mitochondria. In cardiac myocytes chronically
exposed to high levels of glucose, complexes of the mitochondrial respiratory chain were
shown to be O-GlcNAc modified. However these modifications were associated with
impaired mitochondrial respiratory function. Hyperglycemia also induces mitochondrial
iii
superoxide production, which was shown to increase hexosamine biosynthesis and OGlcNAcylation. Reactive oxygen species production can also lead to mitochondrial
respiratory dysfunction and loss of mitochondrial membrane potential. It is possible that
O-GlcNAcylation of respiratory chain complexes is involved in cardioprotection. While
these results may seem contradictory, O-GlcNAcylation may simply be a constitutive
element in non-pathological cellular functions, including oxidative phosphorylation, and
perturbations in homeostatic O-GlcNAc signaling may lead to cell and mitochondrial
dysfunction.
Despite the current progress, the effect of O-GlcNAc on the many functions that
mitochondria perform is still relatively unknown. It is increasingly apparent that
mitochondrial proteins are O-GlcNAc modified and that this modification appears to
modulate the function of these proteins.
Keywords: O-GlcNAc, Hexosamine Biosynthesis, Cytoprotection, Mitochondria,
Reactive Oxygen Species
iv
DEDICATION
To my mother and aunt, who have had the biggest impact in shaping the man that
I am today.
To Heather Minkel, who has provided me with much support through the easy and
difficult times.
Finally, to Dr. John Chatham, who has exhibited great patience with me and truly
believed in me. Thank you for giving me a shot.
v
ACKNOWLEDGEMENTS
What is the fundamental skill that a scientist acquires in graduate school? In my
opinion, it is the ability to learn how to learn. Very few other vocations combine the skills
necessary to identify a problem and then devise a method to test solutions to that problem
in the context of physical and empirical reality. This skill is translatable to virtually any
setting one can imagine. Armed with the scientific method, I am equipped to properly
deal with the everyday occurrences of life. To this, I am thankful for the opportunity to
stand on the shoulders of the giants, such as Karl Popper, for their enormous influence on
the formation of the modern scientific inquiry and empirical falsification.
It is important that I thank Randy Seay, since I wouldn’t be here if it weren’t for
his recruitment of me to move to Alabama for graduate school. Not only has it been the
most important decision toward my career and personal growth, I also enjoy the state
which I now call home.
Dale Benos, Mark Bevensee, Kevin Kirk, and Lori McMahon. Thank you for
your support through some interesting and difficult times.
Asaf Stein and Colin Reily. You guys have helped me shape my worldview by
using the rigorous scientific process from our graduate training to understand objective
reality. Each of us brought something different to the table and esprit de corps means
something to me now. You created a monster. Because of you two, I probably could have
gotten a PhD in Economics, Constitutional Law, and Classical History by now.
vi
Heather Minkel. Thanks for your patience. You believed in me and encouraged
me to believe in myself. I ran three marathons with your help, and have become quite the
outdoorsman. It’s fitting how I have learned to be a man, from a woman.
I have had the pleasure to work with quite a few great scientists. David
Westbrook, Michelle Johnson, Gloria Benavides, Shawn Galin, and Brad Hill have all
given me great technical and philosophical guidance. Thank you.
I’d like to thank my past and present lab mates. The Chatham lab objectives were
a collaborative effort and having the various expertise available helped in my own
studies. Most notable is Luyun Zou. She has taught me the majority of the methods used
in this work. But equally important, she is a friend. I have a tendency to zone out into my
little world, and she was always willing to help with anything and it was really nice to
take breaks and talk about random things. I’m going to miss her most.
I’d like to thank the members of my committee. Victor Darley-Usmar, Scott
Ballinger, Lori McMahon, Martin Young, and Peter Smith. You were all very
challenging but fair. I really enjoyed my meetings with you. I could sense that you all
believed in my skills and abilities and cared about me as an individual. Thank you.
Finally, I’d like to thank Charlye and John Chatham. You took a shot with me. I
know it hasn’t always paid dividends. I just want to thank you both for believing in me
and not giving up on me. Dr. Chatham has been a great mentor that has provided the
framework thinking critically on ideas and questions in general, a lesson I will use
wherever I end up. My only regret is that I couldn’t do more.
vii
TABLE OF CONTENTS
Page
ABSTRACT .....................................................................................................................iii
DEDICATION .................................................................................................................v
ACKNOWLEDGEMENTS .............................................................................................vi
LIST OF FIGURES .........................................................................................................xi
LIST OF ABBREVIATIONS ..........................................................................................xii
CHAPTER
1 INTRODUCTION ........................................................................................................1
The Hexosamine Biosynthetic Pathway and O-GlcNAc Modification..............3
Discovery of the O-Linked GlcNAc modification ....................................3
The Hexosamine Biosynthetic Pathway ...................................................4
O-GlcNAc versus other forms of Glycosylation.......................................7
O-GlcNAc Duality: Pathology versus Protection ..............................................10
O-GlcNAc and Mitochondrion ...........................................................................12
Summary ............................................................................................................16
2 METHODS ...................................................................................................................18
Cell Culture .........................................................................................................18
Neonatal Rat Ventricular Myocytes....................................................................19
Western Blot Analysis ........................................................................................20
Inhibition of O-GlcNAcase .................................................................................22
DMNQ ................................................................................................................22
viii
Seahorse Extracellular Flux Assay ......................................................................23
Mitochondrial Isolation ........................................................................................28
Mitoplast Isolation ...............................................................................................31
MTT Cell Viability Assay ...................................................................................32
Statistical Analysis ...............................................................................................34
3 RESULTS .....................................................................................................................35
Introduction ..........................................................................................................35
Mitochondrial O-GlcNAc-ome and Mitochondrial Protein
Targets of Modification............................................................................36
Mitochondrial O-GlcNAcylation: Respiratory Function
and Cytoprotection...................................................................................36
Results ..................................................................................................................37
O-GlcNAcase pharmacological inhibition ..............................................37
Dynamic O-GlcNAcylation of NRVM mitochondria................................39
Detection of O-GlcNAc modifications in C2C12 mitoplasts ...................39
Optimization of the XF-24 mitochondrial function assay
in C2C12 myoblasts ................................................................................41
Thiamet-G and PUGNAc on oxygen consumption
in intact C2C12 myoblasts .......................................................................45
The effect of DMNQ on oxygen consumption in
C2C12 myoblasts .....................................................................................45
Cytotoxicity of DMNQ and ThG ..............................................................49
4 DISCUSSION ...............................................................................................................52
ix
Summary ..............................................................................................................52
O-GlcNAcylation of Mitochondrial Proteins ...........................................52
O-GlcNAcylation and Mitochondrial Function .......................................53
Future Directions .................................................................................................54
LIST OF REFERENCES .................................................................................................56
x
LIST OF FIGURES
Figure
Page
1-1
O-GlcNAc catalyzing enzymes.......................................................................... 2
1-2
The Hexosamine Biosynthetic Pathway ........................................................... 5
1-3
O-GlcNAc versus other Glycosylations ........................................................... 7
1-4
Prospective O-GlcNAc/ROS signaling based on current literature ................... 15
2-1
XF-24 Analyzer measures oxygen and pH changes in live, intact cells ............ 25-26
2-2
Mitochondrial function assay ............................................................................. 29
2-3
Simplified schematic of mitoplast isolation method .......................................... 33
3-1
Inhibition of OGA with ThG and PUGNAc increases
cellular O-GlcNAc ...............................................................................................38
3-2
Dynamic O-GlcNAcylation of NRVM mitochondria..........................................40
3-3
O-GlcNAcylation of C2C12 mitoplasts ...............................................................42
3-4
Optimization of oligomycin and FCCP in C2C12 ...............................................43
3-5
OCR of C2C12 at different cell densities normalized by protein ........................44
3-6
Thiamet-G and PUGNAc on oxygen consumption in intact cells .......................46
3-7
XF-24 parallel plate O-GlcNAc Western blot .....................................................47
3-8
DMNQ dose response on C2C12 OCR ...............................................................48
3-9
Cytotoxicity of DMNQ ........................................................................................50
3-10
MTT cytotoxicity of ThG ...................................................................................................... 51
xi
LIST OF ABBREVIATIONS
A/A
antimycin/antibiotic
ANOVA
analysis of variance
ATP
adenosine triphosphate
BSA
bovine serum albumin
°C
degrees Celsius
C2C12
mouse myoblast cell line
CO2
carbon dioxide
DMEM
Dulbecco's Modified Eagle Medium
DMNQ
2,3-dimethoxy-1,4-napthoquinone
DMSO
dimethyl sulfoxide
ECAR
extracellular acidification rate
EGTA
ethylene glycol tetra acetic acid
eNOS
endothelial nitric oxide synthase
ER
endoplasmic reticulum
ETC
electron transport chain
FBS
fetal bovine serum
FCCP
carbonyl cyanide-p-trifluoromethoxyphenylhydrazone
Frc-6-P
fructose-6-phosphate
g
gram
xii
GAPDH
glyceraldehyde 3-phosphate dehydrogenase
GFAT
glutamine fructose-6-phosphate amidotransferase
Glc-6-P
glucose- GlcN-6-6-phosphate
GlcN-6-P
glucosamine-6-phosphate
GLUT
glucose transporter
H2O2
hydrogen peroxide
HAT
histone acetyltransferase
HBP
hexosamine biosynthetic pathway
HRP
horseradish peroxidase
I/R
ischemia/reperfusion
IgG
immunoglobulin G
IgM
immunoglobulin M
L
liter
M
molar
min
minute(s)
mL
milliliter
mM
millimolar
mOGT
mitochondrial O-GlcNAc transferase isoform
mPTP
mitochondrial permeability transition pore
MTT
Thiazoyl blue Tetrazolium bromide
NADH
nicotinamide adenine dinucleotide
ncOGT
nucleo-cytoplasmic O-GlcNAc transferase isoform
nm
nanometer
xiii
NO
nitric oxide
NRVM
neonatal rat ventricular myocytes
OCR
oxygen consumption rate
OGA
β-N-acetylglucosaminidase
OGA-L
long O-GlcNAcase isoform
OGA-S
short O-GlcNAcase isoform
O-GlcNAc
O-Linked Beta–N-Acetylglucosamine
OGT
O-GlcNAc transferase
OSCP
oligomycin sensitive-conferring protein
PBS
phosphate buffered saline
PUGNAc
O- (2-Acetamido-2-deoxy-D-glucopyranosylidene) amino Nphenyl carbamate
PVDF
polyvinylidene difluoride
RNA
ribonucleic acid
ROS
reactive oxygen species
SDS
sodium dodecyl sulfate
SERCA
sarco/endoplasmic reticulum Ca2+ ATPase
ser/thr
serine/threonine
sOGT
short O-GlcNAc transferase isoform
Thiamet-G
ThG
TPR
tetratricopeptide repeat
UDP-GlcNAc
uridine diphosphate N-acetylglucosamine
VDAC
voltage dependent anion channel
xiv
XF-24
extracellular Flux Analyzers
μL
microliter
μM
micromolar
xv
CHAPTER 1
INTRODUCTION
O-linked beta–N-acetylglucosamine (O-GlcNAc) is a dynamic and ubiquitous
posttranslational modification of serine and threonine residues. O-GlcNAc-transferase
(OGT) catalyzes the addition of uridine diphosphate N-acetylglucosamine (UDPGlcNAc) [5], the end product of the hexosamine biosynthetic pathway (HBP), onto
proteins. The removal of O-GlcNAc is regulated via a unique hexosaminidase, β-Nacetylglucosaminidase (OGA), a process similar to kinases and phosphatases in
phosphorylation (Figure 1-1). O-GlcNAc is emerging as an important regulator of
cellular processes such as cell signaling, transcription, translation, apoptosis, cell
proliferation and differentiation, among others [6]. Abnormal amounts of OGlcNAcylation are associated with insulin resistance and glucose toxicity in diabetes [7,
8]. O-GlcNAc also seems to play a role in neurodegenerative diseases [9], and many
oncogenic proteins and tumor suppressor proteins are also regulated by O-GlcNAcylation
[10-12]. Paradoxically, reports have indicated that O-GlcNAc may be cytoprotective as
well [2, 13]. An increasing amount of nucleo-cytoplasmic enzymes have been identified
as substrates for O-GlcNAcylation [14, 15], augmenting our understanding in both cell
signaling and O-GlcNAc biology.
1
OGT
Unmodified
Protein
O-GlcNAc
Protein
OGA
Figure 1-1: O-GlcNAc catalyzing enzymes. O‐GlcNAc‐transferase (OGT) catalyzes the addition of UDP‐GlcNAc, onto ser/thr residues of naked proteins. The removal of O‐GlcNAc is regulated via a unique hexosaminidase, β‐N‐
acetylglucosaminidase (OGA), in a process similar to kinases and phosphatases in phosphorylation. 2
The Hexosamine Biosynthetic Pathway and O-GlcNAc Modification
Discovery of the O-Linked GlcNAc modification
The O-GlcNAc modification was discovered to exist as a dynamic and ubiquitous
posttranslational modification of serine and threonine residues on nuclear and
cytoplasmic proteins in an attempt to characterize potential differentiation and
histocompatibility effects of the GlcNAc moiety on the cell surface of thymic
lymphocytes. Torres and Hart discovered that the majority of terminal GlcNAc residues
resided primarily in the nucleo-cytoplasm [16]. This discovery prompted further inquiry
into the nature of O-linked GlcNAcylation distinct from other forms of glycosylation that
form sugar complexes on proteins exposed to the cell surface or in the endoplasmic
reticulum (ER) and Golgi apparatus.
Subsequent studies have been and still are limited by the detection methods of OGlcNAc. Unlike O-phosphorylation, which is identified via immunodetection using
antibodies specific to phospho-proteins [17], O-GlcNAc modifications were identified by
labeling O-GlcNAc with UDP[3H]Gal using galactosyltransferase [16]. This method
positively identifies O-GlcNAc modifications, but is very time consuming. The
development of the RL2 antibody identified a group of nuclear pore complex
glycoproteins that contain the O-GlcNAc modification [18]. The CTD 110.6
immunoglobulin M (IgM) antibody was raised against an O-GlcNAc–modified peptide
from the large subunit ribonucleic acid (RNA) polymerase II CTD [19]. CTD 110.6
displays a broader immunoreactivity to O-GlcNAc than RL2, however it has limited
immunoprecipitation, a method useful in identifying specific O-GlcNAc modified
proteins, utility because of the IgM isotype. Recently, Anti-O-GlcNAc, clone 9D1.E4(10)
3
immunoglobulin G (IgG) antibody was developed with the broad immunoreactivity to OGlcNAc with the IgG isotype [19]. Another technical limitation inhibiting the study of OGlcNAc modification is the fact that there is currently no antibody available that
recognizes a specific protein O-GlcNAc modification. This limitation makes it relatively
difficult to identify specific sites of modifications as compared to O-phosphorylation.
The Hexosamine Biosynthetic Pathway
O-GlcNAcylation is dependent on the availability of UDP-GlcNAc, the precursor
for all amino sugars [20], synthesis by the HBP [21]. Glucose enters the cell by way of
the GLUT family of glucose transporters. The GLUT transporters are a group of
membrane proteins that facilitate the transport of glucose and related hexoses across the
polar molecule impermeable plasma membrane [22]. Glucose is then phosphorylated by
hexokinase into glucose-6-phosphate (Glc-6-P). The majority of Glc-6-P is metabolized
by glycolysis, but it can also enter a myriad of different pathways for glycogen synthesis,
ribonucleotide synthesis, and hexosamine synthesis [23]. A portion of Glc-6-P enters the
HBP after it is converted to fructose-6-phosphate (Frc-6-P) by phosphofructokinase [5,
24, 25]. Frc-6-p is converted to glucosamine-6-phosphate (GlcN-6-P) by glutamine:
fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme of the HBP.
GlcN-6-P is then acetylated by glucosamine-6-phosphate N-acetyltransferase to yield Nacetylglucosamine 6-phosphate, which is then isomerized by phosphoacetylglucosamine
mutase to create N-acetylglucosamine 1-phosphate. This product is then uridinylated by
the uridine-5’-diphosphate (UDP)-GlcNAc pyrophosphorylase to form the final product
of the HBP, N-acetylglucosamine-6-
4
Glucose
ATP
ADP
Hexokinase
Glucose 6-phosphate
Glc-6-P Isomerase
6-phosphofructokinase
GLYCOLYSIS
Fructose 6-phosphate
Fructose 1,6-diphosphate
ADP
ATP
Glutamine
GFAT
Glutamate
Glucosamine
Hexokinase
ATP
ADP
Glucosamine 6-phosphate
Acetyl CoA
CoA
GlcN-6-P
N-acetyltransferase
N-acetylglucosamine 6-phosphate
Phosphoacetylglucosamine
mutase
N-acetylglucosamine 1-phosphate
UTP
PPi
UDP-N-acetylglucosamine
pyrophosphorylase
UDP-N-acetylglucosamine
(UDP-GlcNAc)
N-linked
Glycosylation
O-GlcNAc
O-linked
Glycosylation
Figure 1­2: The Hexosamine Biosynthetic Pathway 5
phosphate, or UDP-GlcNAc. UDP-GlcNAc is the precursor for all amino-glucose
molecules and is used for making proteoglycan, glycolipids, and glycosaminoglycans
(Figure 1-2) [23].
GFAT exists in two known isoforms, GFAT 1 and GFAT 2, both having a mass
of approximately 77 kDa [26] and the two isoforms are transcribed from different genes
(Chromosomal location GFAT1: 2p13i; GFAT2: 5q) [20]. Reports suggest that the
transcription factor Sp1 plays an important role in the regulation of GFAT gene
expression [27], which, interestingly, is known to be O-GlcNAc modified [28-31]. Tissue
distribution analyses revealed GFAT1 to be more highly expressed in the placenta,
pancreas, and testis than GFAT2. GFAT2 was expressed throughout the central nervous
system, especially in the spinal cord, and heart compared to GFAT 1 [26].
The literature widely cites that the HBP consumes 2%-5% of the total glucose
population in the cell [20, 25, 32-34]. The investigators that determined those data
studied glucose-induced desensitization of GLUT in cultured adipocytes [35, 36].
Glucose flux into the HBP in other cell types and tissues in vivo, however, is currently
unknown. GFAT activity regulates glucose entry into the HBP, which is balanced by a
number of factors. GFAT activity is moderated by the concentration of Frc-6-P [20],
feedback inhibition from the HBP product UDP-GlcNAc [37], the concentration of
GFAT protein in the cell [38], and cAMP dependent phosphorylation [39].
Exogenous glucosamine can be taken up by the cell through the glucose
transporter family (specifically GLUT 1, 2, and 4). Interestingly, the liver and pancreatic
beta cell expressing GLUT 2 was shown to have a higher affinity for glucosamine over
glucose [40]. Glucosamine is then phosphorylated by hexokinase yielding GlcN-6-P,
6
bypassing the rate-limiting step operated by GFAT. Glucosamine provides a method to
study the affect of glucose flux through the HBP [20, 41]. Inhibition of GFAT by the
substrate analogue azaserine and inhibition of GFAT protein synthesis by antisense RNA
have been useful tools in studying the role of the HBP in the context of high glucose [20,
42].
O-GlcNAc versus other forms Glycosylation
Once UDP-GlcNAc is formed, a portion is transported into the Golgi apparatus
via the UDP-GlcNAc transporter to act as a substrate for both N-glycosylation and Oglycosylation of membrane bound proteins [23]. UDP-GlcNAc is also used as a substrate
for the O-GlcNAc modifications of intracellular proteins. Unlike both N- and Oglycosylation, O-GlcNAc modification occurs outside of the Golgi apparatus [23]. It is a
single amino sugar moiety, as opposed to a branched network of sugars found in N- and
O-glycosylation. It is also bound to serine/threonine (ser/thr) residues as opposed to
asparagine residues, as found in N-glycosylation. Finally, O-GlcNAc is added to and
removed from proteins in a dynamic fashion, which is contrasted in permanent N- and Oglycosylation (Figure 1-3) [23].
The O-GlcNAc cycling enzymes, OGT and OGA, are specific for OGlcNAcylation and the enzymes involved in the formation of the N- and O- glycosylation
are different [43]. OGT has three known splice variants that are encoded on the X
chromosome [44, 45]. Each of the splice variants share a common catalytic domain but
have distinctive N-terminal regions which are essential for proper targeting to the
nucleus, cytoplasm or mitochondria. The largest of the splice variants is the nucleo-
7
Figure 1-3: O-GlcNAc versus other Glycosylations
8
cytoplasmic OGT (ncOGT) at 110 kDa followed by the mitochondria OGT (mOGT) at
103 kDa [46] and the 78 kDa short OGT (sOGT), which is identical to ncOGT sans the
histone acetyltransferase domain [5]. Deletion of OGT in mammals is embryonically
lethal [47]. Over-expression of OGT in muscle and fat of transgenic mice induces insulin
resistance [48]. There is no known consensus sequence for OGT specificity, but it is
known that it can act on many different protein substrates in a regulated fashion with
tetratricopeptide (TPR) domains, transient protein: protein interactions of the catalytic
subunit to form enzyme complexes, competes with tyrosine and ser/thr phosphorylation,
as well as acting in parallel with phosphorylation [49, 50]. A screen of anti-apoptotic
genes in leukemia cells identified sOGT as an anti-apoptotic marker and a dominant
negative inhibitor of OGT [51]. Global over-expression of mOGT triggers apoptosis,
while over-expression of ncOGT or a catalytically inactive form of OGT does not induce
apoptosis. These overexpression systems were associated with increased cellular OGlcNAc, but mitochondrial O-GlcNAc was not determined [50].
O-GlcNAcase is a highly conserved hexosaminidase encoded by a single gene on
chromosome 10 and has two splice variants, a long (OGA-L) and a short version (OGAS) [52]. The long form consists of a catalytic domain, an OGT binding domain, and a
histone acetyltransferase (HAT) domain [53]. In addition, the short form is identical to
the long form sans HAT domain. The HAT domain seems to assist in OGA interaction
with transcription machinery [54, 55]. OGA can form complexes with other enzymes to
confer substrate specificity [6]. OGA-L is a nucleo-cytoplasmic protein. There is
evidence that OGA-S is localized to the nucleus, but also near lipid droplets where OGA-
9
S interacts with the ubiquitin proteasome system [52]. There is currently no known OGA
isoform in the mitochondria.
O-GlcNAc Duality: Pathology versus Protection
O-GlcNAc has emerged as an important regulator of cellular processes such as
cell signaling, transcription, translation, apoptosis, and cell cycle regulation, among
others. Historically, the O-GlcNAc modification has been associated with pathologies
such as diabetes and hyperglycemia because it was studied in the context of nutrient
excess. O-GlcNAc has been viewed as an indicator of cellular energy levels and is
associated with diabetic complications. As a consequence of elevated extracellular
glucose due to hyperglycemia, increased flux through the HBP will occur, augmenting
subsequent availability of UDP-GlcNAc. O-GlcNAc modification of cellular proteins
increases, and these modifications may alter a protein’s activity, half-life, localization,
and/or phosphorylation state. Because of this, sustained augmented O-GlcNAc has been
implicated in conditions such as cardiomyocyte dysfunction and hypertrophy, vascular
dysfunction, Alzheimer’s, and inflammation [4, 24, 48].
In 1991, the HBP was found to be required for insulin insensitivity in adipocytes
[56]. The authors discovered that glucose, insulin, and glutamine were all required for
insulin resistance and it was confirmed by using inhibitors of GFAT. The effect was
reproduced when adipocytes were treated with glucosamine, a molecule that bypasses
GFAT in the hexosamine biosynthesis pathway.
10
After OGT was identified, it was also found to be associated with insulin
resistance. Moderate overexpression of OGT caused insulin resistance in mice [48].
Overexpression of OGT in cardiomyocytes induced diabetic related cardiac dysfunction,
increased O-GlcNAcylation and impaired calcium signaling [57]. In 2008, researchers
found the molecular mechanism that regulates insulin signaling through OGT and OGlcNAc. OGT contains a phosphoinositide-binding domain. After insulin exposure,
phosphatidylinositol 3,4,5-trisphosphate recruits OGT from the nucleus to the plasma
membrane, where OGT O-GlcNAc modifies key enzymes of the insulin pathway [8].
While the initial studies relating O-GlcNAc modifications to cellular processes
were largely supportive of a negative role of the modification in pathology, many studies
have since shown that a variety of stress stimuli increased the levels of protein O-GlcNAc
in mammalian cells, and these increases were associated with cytoprotection. In 2003, a
study showed that inhibiting O-GlcNAc modification consistently decreased cell survival
in response to a variety of cellular stressors, while increased O- GlcNAc modifications
augmented cell survival [2]. This is consistent with other reports showing that preischemic treatment with glucosamine or O-GlcNAcase inhibitor (PUGNAc) protects
against ischemia- reperfusion (I/R) injury in both isolated rat hearts and rat ventricular
myocytes (NRVM) [58], and this protection is associated with elevated levels of OGlcNAc modification and elevated expression of the anti-apoptotic protein Bcl-2 [59].
Others have shown that PUGNAc treatment attenuates the hydrogen peroxide (H2O2)
induced decrease in mitochondrial membrane potential [60]. This protection is also
associated with an increase in O-GlcNAc modification of voltage dependent anion
11
channel (VDAC), a major component of the mitochondrial permeability transition pore
[3].
O-GlcNAc and Mitochondrion
Many measures of cellular health relate to the energy state of the cell. When
assessing O-GlcNAc roles in cellular biology the mitochondrion has been an understudied area. A variety of stress stimuli such as heat shock, UV irradiation, ethanol, and
NaCl increase the levels of protein O-GlcNAc in mammalian cells. Inhibiting O-GlcNAc
modification decreases cell survival in response to stress, while increased O-GlcNAc
modifications augmented cell survival [2, 61]. As previously mentioned, elevated OGlcNAc also protects against stress in both isolated rat hearts and NRVM [58], and this
protection was associated with elevated levels of total O-GlcNAc. Both glucosamine and
OGT over-expression increased basal and I/R-induced O-GlcNAc levels, significantly
decreased cellular injury, and attenuated loss of cytochrome c [59]. Augmenting OGlcNAc also attenuated the loss of mitochondrial membrane potential induced by H2O2
and was associated with an increase in mitochondrial Bcl-2 levels. Decreasing cellular OGlcNAc had the reverse affect [59]. Increasing cellular O-GlcNAc by inhibiting OGlcNAcase with PUGNAc or knocking down OGA with RNAi attenuated the posthypoxic loss of mitochondrial membrane potential [60]. Many of these measures of
cellular health and viability rely on measures of mitochondrial health. With the exception
of VDAC [3], however, little had previously been shown to be directly linking changes in
the mitochondria to O-GlcNAc addition to associated proteins.
12
More has been elucidated in this relationship in recent years. Augmenting OGlcNAc reduces calcium-induced mitochondrial permeability transition pore (mPTP)
activation in adult cardiac mitochondria. Reducing O-GlcNAc levels diminished the
recovery of mitochondrial membrane potential following re-oxygenation following
ischemia. Protein levels of the putative components of mPTP remain unchanged, and the
exact mechanism of O-GlcNAc and mPTP remain unknown. VDAC, an integral member
of mPTP, was revealed by immunoprecipitation to be O-GlcNAc modified [4, 13, 29,
59]. mOGT was discovered [49] to be an active transferase, although no mitochondrial
O-GlcNAc accumulation was initially found [46]. This claim de-incentivized further
study until the mitochondrion was suggested to contribute to the cardioprotective
properties of O-GlcNAc [3, 59].
In cardiac myocytes chronically exposed to high levels of glucose, mitochondrial
respiratory chain members such as NDUFA9 of complex I, subunits core 1 and core 2 of
complex III, and the mitochondrial DNA-encoded subunit I of complex IV were shown to
be O-GlcNAc modified. Mitochondrial O-GlcNAc modification is associated with
impaired activity of complex I, III, and IV in addition to lower mitochondrial calcium and
cellular ATP content. Mitochondrial function improved with expression of OGlcNAcase. These mitochondrial respiratory function data are somewhat limited,
however, because the experiments were performed on isolated mitochondria removed
from the affects of an intact cell [62].
There is evidence of crosstalk between O-GlcNAc and redox signaling. The
cytochrome c oxidase complex reduces oxygen to water, however small amounts of
superoxide anion and H2O2 are produced by the electron transport chain (ETC) [63].
13
Reactive oxygen species (ROS) are also produced by activation of oxidative enzymes
such as NADPH oxidase and xanthine oxidase [4, 64, 65]. Production of ROS by protonpumping complexes is greatest at high membrane potentials; it has been proposed that
mitochondria regulate their activity to maintain the membrane potential within a narrow
range that balances ATP production against oxidant generation [66]. It has been
demonstrated that ROS act as important signaling molecules and that the general
oxidative environment of the cell helps govern various cellular processes [67]. ROS can
also be increased with stress [4], and as previously stated, O-GlcNAc attenuates the H2O2
induced loss of mitochondrial membrane potential, and many of the stressors O-GlcNAc
protects against have a strong ROS component [13, 29, 59]. Hyperglycemia-induced
mitochondrial superoxide overproduction inhibits glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) and activates the hexosamine pathway, increasing cellular OGlcNAcylation and the O-GlcNAcylation of Sp1 [30]. The activation of the HBP is
presumably due the inhibition of GAPDH redirecting Frc-6-P from glycolysis to UDPGlcNAc formation. In another report, the same investigators found that hyperglycemia
inhibited endothelial nitric oxide synthase (eNOS), which catalyzes the production of the
free radical and signaling molecule nitric oxide (NO), due to increased O-GlcNAcylation
of eNOS [1]. Taken together, these facts indicate that the O-GlcNAc and reactive species
signaling systems have a concordant role in regulating mitochondrial function and stress
protection (Figure 1-4).
14
Hyperglycemia
Heat
ROS
Inflammation
Resistance to ROS
and Mito dysfunction
Hypoxia
ROS
O-GlcNAcylation
Mito and Cyto Proteins
O-GlcNAc
Figure 1-4: Prospective O-GlcNAc/ROS signaling based on current literature. Stressors
such as hyperglycemia [1], ROS, heat, inflammation and hypoxia [2] increase production of
mitochondrial ROS. This leads to an increase in O-GlcNAcylation of mitochondrial and
cytosolic proteins. O-GlcNAcylation of these proteins (e.g. VDAC [3]) leads to downstream
mitochondrial protection. Adapted from Laczy et al. [4]
15
Summary
In the relatively short period of time that the O-GlcNAc posttranslational
modification has been a subject of scientific inquiry, it has been revealed to be a
fundamental signaling pathway involved in a diverse set of cellular processes in all
metazoans. Despite the current progress, the effect of O-GlcNAc on the many functions
that mitochondria perform is still relatively unknown. It is increasingly apparent that
mitochondrial proteins are O-GlcNAc modified and that this modification appears to
modulate the function of these proteins and that these modifications are associated with
cytoprotection.
A sizeable majority of studies focused on O-GlcNAcylation has examined OGlcNAc in the context of hyperglycemia, insulin resistance, and diabetes. Since OGlcNAc is associated with those conditions, the predominant view of O-GlcNAc has
been as a nutrient sensor and a negative factor in hyperglycemia related pathologies. This
is similar to studies of reactive species demonstrating their role in aging, tissue damage,
and disease. However, reactive species are now known to be key signaling agents. The
concept of “redox tone” is now used to describe redox-signaling pathways that balance
between cytoprotection and cell death through apoptosis by changing the status of thiols
on signaling proteins. Redox tone establishes a baseline for other signals under normal
biological conditions.
Newer reports seem to imply that O-GlcNAcylation is not simply a negative
regulator of nutrient excess, but also a key signaling mechanism in normal biological
conditions. The recent findings of O-GlcNAc acting as a cytoprotective factor support
that implication. The O-GlcNAc signaling system, similar to reactive species, is involved
16
in both cytoprotection and cell death. This evidence supports the idea of “O-GlcNAc
tone”, where the balance of the O-GlcNAc state plays a role in cell survival. The reports
demonstrating O-GlcNAcylation of mitochondrial proteins and other proteins involved in
mitochondrial processes lend credence to a relationship between the cellular redox and OGlcNAc statuses. As previously stated, O-GlcNAc and the HBP are activated in response
to superoxide overproduction. O-GlcNAc cycling enzymes and their complexes may be
redox sensitive, and inversely oxidant-producing enzymes may be O-GlcNAc modified.
Despite the these promising revelations regarding the involvement of O-GlcNAc
in balancing cell protection and cell death, there are some important fundamental
questions that have yet to be answered. Namely, what is the role of cellular and
mitochondrial O-GlcNAc on non-pathological mitochondrial respiration? Are the inner
mitochondrial membrane proteins involved in electron transport O-GlcNAc modified?
Do the cytoprotective properties of O-GlcNAc involve modification of respiratory
enzymes?
Therefore, we proposed that increases in mitochondrial O-GlcNAc modifications
are protective against ROS induced mitochondrial respiratory dysfunction. We began
testing this by demonstrating O-GlcNAcylation of proteins located in the inner
mitochondrial subfraction of C2C12 mouse myoblasts. Then we sought to determine the
effect of altering O-GlcNAc levels in C2C12 on mitochondrial respiratory function under
basal conditions and demonstrate that that acute increases in mitochondrial protein OGlcNAc modifications are protective against ROS induced mitochondrial respiratory
dysfunction.
17
CHAPTER 2
METHODS
Cell Culture
C2C12 mouse myoblast cells are an immortal cell line of mouse skeletal
myoblasts derived from mouse satellite cells [68]. Satellite cells are progenitor cells
located in the space between the sarcolemma and the basal lamina that differentiate into
myoblasts which then fuse to existing multinucleated skeletal muscle fibers [69]. C2C12s
are robust and grow rapidly on untreated cell culture dishes and have a relatively large
amount of mitochondria. For these practical reasons, C2C12s were chosen for our
studies.
C2C12s were cultured in Dulbecco’s modified Eagles medium (DMEM)
containing 5.5mM glucose, 20% fetal bovine serum (FBS), and 1% Antimycin/Antibiotic
(A/A) in untreated cell culture dishes. These cells differentiate well into myocytes by
replacing FBS with 2% horse serum. Cells were grown to about 70% - 80% confluence
(C2C12 cells will begin to differentiate when reaching confluency) in a 37°C, 5% CO2
incubator before being split. Prior to experimentation, FBS was lowered to 1% to
synchronize cell growth. Dr. Andrew Paterson at UAB donated the C2C12 line to our lab.
18
Neonatal Rat Ventricular Myocytes
Primary cultures of NRVMs were obtained from 2- to 3-day-old neonatal
Sprague-Dawley rats by isolating myocytes from cardiac tissue using collagenase
dissociation. Pups were rinsed in ethanol and decapitated. Hearts were removed and atria,
fat, and connective tissue were discarded. Remaining ventricles were diced in perfusion
buffer on ice with a razor blade.

Place minced hearts in 25 mL flask with 5 mL perfusion buffer and 5 ml enzyme
buffer - Worthington Collagenase II (Worthington # CLS-2) and perfusion buffer.

Incubate for 15 minutes (min) in 37° C water bath with shaking.

Remove flasks from incubator at room temperature and allow tissue clumps to
settle, and then aspirate the supernatant and discard.

Add 10 mLs of warm enzyme buffer then incubate for 18 min in water bath.

Aspirate and discard supernatant as in step 3.

Save Subsequent Incubations

Add 10 mLs of enzyme buffer and incubate for 22 min. After the first 14 min,
resuspend tissue fragments with a transfer pipette until only small pieces of tissue
are left.

Save the supernatant with transfer pipette and funnel into a 50 mL conical tube
with a filter. Rinse filter with perfusion buffer and spin down for 5 min at
1500rpm.

Begin next incubation by repeating step 6

Aspirate supernatant from spun cells and gently resuspend in 5 mLs of FBS and
place in the cell culture incubator with a loosened cap.
19

Repeat steps 6-8 twice more for a total of three incubations.

Combine the 3x 50 ml conical tubes containing cells suspended in FBS and spin.
Resuspend cells in 20 mLs of overnight media (DMEM + 18% final concentration
M199 (M 4530) 15% FBS, 1%A/A) per rat.

Place cell suspension in a dish and place in the incubator for 10 min to remove
fibroblasts.

Filter suspension into 50 ml tube. Count and plate cells. Place in incubator
overnight.

Next day: Wash 1x with warm PBS, replace with growth media.
Western Blot Analysis
To measure total cellular and mitochondria O-GlcNAc levels as well as specific
enzymes of interest, total protein were separated on SDS-PAGE gels and transferred to
PVDF membranes. The following protocol was used:
Cell Lysate Preparation

Aspirate cell culture media and wash 2x with 1xPBS.

Place culture dishes on ice and add RIPA buffer supplemented with Thiamet-G
(ThG) and PUGNAc (OGA inhibitors), and protease inhibitor cocktail (Sigma
P8340). Total volume added was dependent on size of the culture well.

Scrape cells using a cells scraper and incubate on ice for 30 min.

Centrifuge at 14,000 g for 15 min at 4°C.

Transfer supernatant (cell lysate) to fresh tube.
Protein Measurement
20

Bio-Rad DC Protein Assay kit (Bio-Rad # 500-0114)

Using a 96 well plate, pipette 5 μL BSA standards into wells in triplicate.

Pipette 2.5 μL samples into wells in triplicate.

Add 25 μL of Reagent A from the kit followed by 200 μL of Reagent B.

Incubate for 15 min on rotator with medium rpm setting.

Read absorbance using spectrophotometry set on 750 nm

Calculate protein concentration
Samples prepared for SDS-Page were separated in 7.5% polyacrylamide gels at
100mV using the Bio-Rad Mini Protean 3 system and transferred to PVDF membranes
for immunodetection.

PVDF incubated overnight with primary antibody in a dilution optimized for
C2C12s in 1%casein-PBS in cold room on shaking platform. Membrane must be
dry before going into the primary incubation solution and all bubbles must be
removed from membrane before being placed on the shaker.

Following overnight incubation, the membrane is washed 3 times for 10 min with
1X PBS at room temperature

Membranes are incubated with appropriate dilution of HRP-conjugated secondary
antibody in 1% casein-PBS for 1 hour at room temperature.

Following 1 hr incubation, the membrane should be washed 3 times for 15 min in
1X PBS at room temperature.

The membranes are then placed into ECL (SuperSignal West Pico-Pierce
Biotech.) for 5 min and signal detected with HyBlot CL autoradiography films
(Denville Scientific).
21

Membranes are washed 3-5 min in 1X PBS following detection and equilibrated
in methanol
The following primary antibodies were used for this study:

anti-O-GlcNAc CTD110.6 (Pierce #24565

anti-O-GlcNAc clone 9D1.E4(10) (Millipore # 05-1245)

anti-Complex IV (Invitrogen # 459600)

anti-SERCA (Abcam # ab3625)

anti-GAPDH (Abcam # ab9485)

anti-Porin (MitoSciences # MSA03)
Inhibition of O-GlcNAcase
OGA inhibition with PUGNAc (Sigma # A7229) and Thiamet-G (ThG) (SD
Chem Molecules # DC-001329) are known to increase cellular O-GlcNAcylation.
PUGNAc is has been the gold standard in the O-GlcNAc field to induce OGlcNAcylation [70]. ThG is relatively newer and has been reported to be 37,000-fold
more selective for OGA over other hexosaminidases [71]. Inhibition of OGA will
increase total cellular O-GlcNAc levels by disrupting the removal of O-GlcNAc from
proteins.
DMNQ
DMNQ was used (2,3-dimethoxy-1,4-napthoquinone) (Enzo Life Sciences ALX420-027) as a redox-cycling agent that induces intracellular superoxide anion formation
[72]. DMNQ does not react with free thiol groups, is non-alkylating and adduct-forming
22
in contrast to other quinones. Thus, DMNQ is a valuable tool for the generation of ROS
in order to study the role of ROS in various cell functions.
Seahorse Extracellular Flux Assay
The Extracellular Flux Analyzer (XF-24) manufactured by Seahorse Biosciences
(Billerica, MA) is a device that provides real time oxygen and pH measurements in intact
cells. The XF-24 is advantageous compared to traditional methods of measuring
mitochondrial function because it avoids the harsh treatment of mitochondria during
isolation procedures. Mitochondria are also subject to influence from the rest of the cell,
which makes measuring mitochondrial function in intact cells valuable [73]. The XF-24
uses a specialized microplate that has 24-wells with a surface area similar to a 96-well
plate. The instrument uses a plunger containing both an oxygen and a pH sensor, which is
lowered into the well, to create a transient 7 μL chamber. The rate of oxygen
consumption and the change in pH can be measured simultaneously in this chamber. The
plunger remains lowered while taking a series of measurements for a defined period of
time, and upon completion of the measurements, the plungers are raised and the media reequilibrates within the well restoring oxygen and pH levels to baseline. The rate of
oxygen consumption and extracellular acidification can be calculated to obtain the
oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) (Figure 2-1
A and B).
XF-24 analyzer was used to measure respiratory function in C2C12 cells. A C2C12
optimized mitochondrial respiratory function assay was adapted from the Victor DarleyUsmar lab in UAB [74]. In preliminary studies, we have found that 40,000 cells/well is
23
the optimum seeding density for obtaining reproducible OCR. C2C12 cells were plated
with 100 μL of DMEM media supplemented with 20% FBS for 3 hours, then 150 μL of
media was added to reach a final volume of 250 μL. This stepwise addition of media
allows the cells to adhere to the floor of the well as opposed to the walls of the wells. In
order to correct for fluctuations in background and temperature across the plate, 4 wells
containing media alone were used as controls (temperature controls). Overnight
incubation of the Seahorse extracellular flux assay cartridge with XF calibrant (provided
by Seahorse Biosciences, premade solution with a known pH to calibrate sensors in the
XF) was also performed.
After 24 hours incubation at 37°C/5% CO2, 230 μL of media was removed from
each well leaving approximately 10 μL of media to avoid shocking the cells. 1 mL of
Seahorse media (8.28g/L DMEM, 1g/L Glucose, 0.11g/L Sodium Pyruvate, pH 7.4 at
37°C) was used to gently wash the cells. After removal of the wash media, 640 μL of
Seahorse media was added to reach a final volume of 660 μL. Cells were allowed to
equilibrate to the buffer free Seahorse media for one hour prior to experimentation.
Mitochondrial respiratory function was assayed using basal measurements were
recorded, followed by the administration of oligomycin (1μM), FCCP (1μM), and
antimycin A (10μM) which allows for the observation of defects in activity that is
coupled to ATP production, maximal OCR, respiratory reserve capacity, the proportion
of maximal activity that is being used at basal conditions, and level of non-mitochondrial
oxygen consumption.
Oligomycin inhibits ATP synthase by binding to the Oligomycin Sensitiveconferring Protein (OSCP). This inhibition has the effect of decreasing oxygen
24
A
Plungers
Plunger rises to
equilibrate
media
pH sensor
Temporary 7μL
microchamber is
formed.
O2 Sensor
Cells
Well 1
Well 2
25
B
Figure 2-1: XF-24 Analyzer measures oxygen and pH changes in live, intact cells. (A) The XF-24 uses plungers
containing both oxygen and a pH sensor, which is lowered into the well, to create a transient 7 μL chamber. The plungers
remain lowered while taking a series of measurements for a defined period of time, and upon completion of the
measurements, the plungers are raised and the media re-equilibrates within the well restoring oxygen and pH levels to
baseline. (B) The measurements made when the plungers are lowered can be used to determine the rate of change in oxygen
and pH to calculate OCR and ECAR. Figure (B) is adapted from Seahorse Biosciences©.
26
consumption since it prevents proton translocation into the matrix through ATP synthase
[75]. The result of this provides the rate of oxygen consumption used for ATP production
in the mitochondria.
Carbonyl cyanide-p- trifluoromethoxyphenylhydrazone (FCCP) is a proton
ionophore and serves to uncouple mitochondria. FCCP binds a proton from the
intermembrane space and translocate to the matrix where it dissociates to make a weak
base and releases the proton. This allows mitochondrial oxygen consumption to be
limited solely by the availability of oxidizable substrate and the capacity of the ETC by
removing ATP synthase as the limiting factor of respiration. This provides the
mitochondria’s maximal oxygen consumption capacity. Reserve capacity is calculated by
subtracting the maximal OCR by basal OCR.
Antimycin A is the final compound administered for the mitochondrial respiratory
function assay. It inhibits cytochrome C reductase (complex III), preventing the oxidation
of ubiquinol and subsequent disruption of cytochrome C oxidase (complex IV) from
receiving electrons. This final step essentially shuts down ETC giving the nonmitochondrial OCR.
Stock solutions of the following compounds in ethanol were diluted in Seahorse media to
yield the following concentrations for injection: oligomycin (9.8 μg/ml), FCCP (21.6
μM), and antimycin A (128 μM). For each solution 75 μL was loaded into the appropriate
port on the Seahorse flux pack cartridge (ports B, C, and D were loaded with oligomycin,
FCCP, and antimycin A, respectively). Port D was loaded with the compound being
tested. This resulted in a final concentration in the well of 1 μg/ml oligomycin, 1 μM
FCCP, and 10μM antimycin A, all of which elicit their maximal effect within a minute of
27
exposure. Oligomycin and FCCP concentrations were determined by two optimization
experiments to find the lowest exposure that would elicit the maximal response in C2C12
cells. Figure 2-2 demonstrates the mitochondrial function assay in C2C12 mouse
myoblasts. Basal OCR readings were recorded for 100 min followed by the
administration of the mitochondrial function assay to elucidate the mitochondrial
functional parameters of the C2C12 mouse myoblast cell line.
The flux cartridge was then inserted into the Seahorse first for calibration,
followed by the XF-24 microplate containing C2C12 cells. The Seahorse was
programmed to take 4 baseline readings with and without OGA inhibition before making
sequential injections from ports A, B, C and D. A brief mixing and one or two readings
followed each injection. Compounds of interest were injected from port A first, followed
by a 2 hour incubation and measurement period before making sequential additions of
oligomycin, FCCP, and antimycin A.
Mitochondrial Isolation
Mitochondria were isolated using these basic key steps: (1) rupturing of cells by
mechanical and/or chemical means, (2) differential centrifugation at low speed to remove
debris and extremely large cellular organelles, and (3) centrifugation at a higher speed to
isolate and collect mitochondria. The following is the expanded protocol:

Isolation Buffer:

220 mM Mannitol

70 mM Sucrose

5 mM MOPS
28
Antimycin A
FCCP
Untreated C2C12 mitochondrial function assay
RESERVE
CAPACITY
Oligomycin
ATP
BASAL
LEAK
MAX RESP.
CAPACITY
NON‐MITOCHONDRIAL OXYGEN CONSUMPTION
Figure 2-2: Mitochondrial function assay. Basal OCR measurements were recorded from C2C12 mouse myoblasts followed by
administration of oligomycin (1 µg/ml), FCCP (1 µM) and Antimycin-A (10 µM). Data from a mitochondrial function assay
includes the basal respiration, ATP-linked respiration, proton leak respiration, maximal respiration, reserve capacity, and nonmitochondrial oxygen consumption.
29

1 mM EGTA

Adjust pH to 7.4 with KOH
Homogenates

150 mg heart tissue powder per 1.5 mL ice-cold Buffer A supplemented with 25% PIC and 0.2 % BSA

Homogenize (Polytron homogenizer) at 1* setting for 10 sec

Incubate on ice for 5 min, at the end of 5 min vortex (medium-5 sec) the tubes

Centrifuge the tubes at 750xg for 10 min at 4C

Save and collect the supernatant into an Eppendorf tube (repeat step 4 if the
lysates are not homogenous

Centrifuge the supernatant at 6,000xg for 10 min at 4C

Keep supernatant (=crude cytosolic fraction/post-mitochondrial fraction) store at 80C

Resuspend the mitochondrial pellet (pipette and vortex for 10 sec) in 250 µL
Buffer A supplemented with 2% PIC (no BSA)

Centrifuge the samples at 6,000xg for 10 min at 4C

Repeat steps 8-9 once more

Resuspend the pellet in the lysis buffer (100-150 µL) used for further assays. E.g.
T-PER

Incubate the samples on ice for 30min (vortex /10 min)

Centrifuge the mitochondrial fraction to remove debris (2,500xg for 5 min)

Store samples at -80°C
30
Mitoplast Isolation
Mitoplasts were isolated from C2C12 myoblasts using the protocol established in
the laboratory of Dr. Victor Darley-Usmar. Mitoplasts were isolated using digitonin to
solubilize outer mitochondrial membranes followed by the administration of the bacterial
protease nagarse to catabolize any loosely associated outer mitochondrial membrane and
cytosolic proteins. Differential centrifugation in a gradient buffer then purified
mitoplasts (Figure 2-3). Mitoplast material was processed through Western blot to
observe O- GlcNAcylation. To confirm enrichment and purity of the mitoplast fraction,
GAPDH (cytosolic marker), VDAC (outer mitochondrial membrane marker), and
Complex IV subunit 1 (inner mitochondrial protein marker) antibodies were used for
Western blot analysis.
Confluent cells grown in a monolayer were switched to low serum for 24 hours
prior to isolation. A 6-well plate (7 x 106 cells) was washed with 2ml ice-cold PBS
solution/well. Cells were scraped in 1 ml (167 µL/well) of isolation media prior to the
addition of Digitonin (100 g/ 106 cells). Tubes were mixed by inversion and centrifuged
on a bench centrifuge for approximately 30 sec. The supernatant was discarded and fresh
buffer (0.5 mL) containing nagarse (10 g/ 106 cells) was added and mixed end over end
for 30 sec prior to the addition of 4 mg BSA to quench the Nagarse. Samples were then
centrifuged for 5 min at 10,000-x g at 4ºC. The supernatant was discarded and fresh
isolation media containing 4 mg BSA + 1x PIC + 1mM PMSF was used to wash the
pellets. The final wash did not contain BSA. Centrifuge for 1 min at 500-x g; transfer the
flow through material to a clean tube and spin for 10 min at 10,000 x g. Discard
supernatant and reconstitute the pellet for assay.
31
MTT Cell Viability Assay
The thiazoyl blue tetrazolium bromide (MTT) assay is a colorimetric assay for
measuring the activity of cellular enzymes that reduce the tetrazolium dye to its Make a
stock of MTT at 2 mg/ml in 1 X PBS and sterile filter. Cover with foil and store this
stock at 4 ºC.

Add 5 ml of MTT stock to 20 ml of media to get a 0.4 mg/ml MTT solution when
ready to use.

Treat cells with the desired reagent for the desired amount of time.

Remove media by aspirating off with a sterile pipette.

Wash once with fresh sterile media (200 µL/well).

Add 200 µL of 0.4 mg/mL MTT solution per well with multichannel pipette.

Incubate plate for 3 hours at 37ºC in cell culture incubator.

Remove media carefully to avoid removing cells from the wells.

Add 100 µL of DMSO per well with multichannel pipette and shake plate gently
to dissolve Formazan crystals.

Reincubate the plate for 15-30 min.

Read at 550 nm

To calculate % viability, assume A550nm of untreated cells are 100% viable and
normalize all conditions to those values.
32
Mitochondria
Mitoplast
Digitonin
solubilization of
mitochondrial
outer membrane
Differential
centrifugation to
purify mitoplast
population
Nagarse proteolysis
of loosely associated
non-mitoplast
proteins
Figure 2-3: Simplified schematic of mitoplast isolation method. Mitoplasts
were isolated using digitonin to solubilize outer mitochondrial membranes
followed by the administration of the bacterial protease nagarse to catabolize
any loosely associated outer mitochondrial membrane and cytosolic proteins.
Differential centrifugation in a gradient buffer then purified mitoplasts.
33
Statistical Analysis
Seahorse experiments and MTT assays will be performed using a sample size of
5. Mitoplast fractionation, immunoprecipitation, enzyme activity will all be performed
using a sample size of 3 and repeated to confirm results. Analysis of Variance (ANOVA)
will be used to compare more than two sample means. Significant differences will be
determined at p < 0.05.
34
CHAPTER 3
RESULTS
Introduction
Despite the current progress [3, 4, 13, 49, 59, 62], the effect of O-GlcNAc on the
many functions that mitochondria perform is still relatively unknown. The mitochondrialocalized OGT (mOGT) was discovered [49] to be an active transferase, although no
mitochondrial O-GlcNAc accumulation was found [46]. This was most likely due to the
investigators using the RL2 anti-O-GlcNAc antibody, which is selective for O-GlcNAc
modified proteins in the nuclear compartment. There is also currently no known
mitochondrial isoform. These facts de-incentivized the study of the mitochondrial OGlcNAc-ome until the mitochondrion was suggested to contribute to the cardioprotective
properties of O-GlcNAc [3]. Subsequent studies have identified specific mitochondrial
proteins such as VDAC [13], mitochondrial aconitase 2 [4], NADH dehydrogenase
[ubiquinone] 1 alpha subcomplex subunit 9 [62], and ATP synthase,
ubiquinolcytochrome c reductase [76]. All of these proteins, however, are either located
on the outer mitochondrial membrane or encoded in the nuclear genome, allowing for the
possibility of the cytosolic O-GlcNAc cycling enzymes to modify these proteins. The
characteristics of the mitochondrial O-GlcNAc cycling pathway remain unclear.
35
Mitochondrial O-GlcNAc-ome and Mitochondrial protein targets of modification
Based on the literature showing that alterations in O-GlcNAc are associated with
mitochondrial function and modification of specific proteins, as well as preliminary data
showing that O-GlcNAc affects respiration, it is reasonable to hypothesize that specific
proteins involved in respiration are modified. Identifying mitochondrial proteins that are
O-GlcNAc modified under basal and stress conditions is fundamental to understanding
the O-GlcNAc system in the mitochondria. To confirm alterations of mitochondrial OGlcNAc in response to each method of altering cellular and mitochondrial O-GlcNAc,
mitoplast fractionation followed by Western blotting was performed. In order to induce
an increase in cellular O-GlcNAcylation, OGA inhibitors PUGNAc and ThG were used.
Mitochondria were isolated and O-GlcNAc Western blots were performed to determine
potential changes in mitochondrial O-GlcNAc.
Isolating mitoplasts are necessary in order to test the hypothesis that inner
mitochondrial membrane and/or matrix proteins are indeed O-GlcNAc modifiable.
Mitoplasts are isolated mitochondria where the outer mitochondrial membrane is
digested, leaving the inner mitochondrial membrane and its contents. This procedure is
helpful because it ensures that loosely associated cytosolic and other organelle proteins
do not contaminate the mitochondrial fraction, especially if antibodies are sensitive
enough to detect small traces of contaminants.
Mitochondrial O-GlcNAcylation: Respiratory Function and Cytoprotection
It is currently unknown whether O-GlcNAc modification of cellular and/or
mitochondrial proteins plays a role in homeostatic mitochondrial respiration. It is
36
possible that there is a homeostatic level of O-GlcNAc modification for normal
mitochondrial function, while extreme deviation of this range is detrimental. O-GlcNAc
has been implicated in mitochondrial redox signaling [4]. Oxidative phosphorylation and
ATP production are vital functions of the mitochondria. Along with producing ATP,
oxidative phosphorylation maintains membrane potential and also produces ROS. It is
possible that the protective effect seen with O-GlcNAc treatment in response to stress is
due to the modification of key complexes in oxidative phosphorylation. Altering the
whole cell and mitochondrial O-GlcNAc state may also affect basal mitochondrial
respiratory function. The method and duration of changing O-GlcNAc is important in
predicting the effect on mitochondrial function. The Seahorse Bioscience XF24 Analyzer
was used to investigate the impact of altering global and mitochondrial O-GlcNAc on
basal mitochondrial respiratory function in intact C2C12 myoblasts, and to determine
mitochondrial O-GlcNAcylation protection of respiratory function in response to ROS,
using DMNQ. O-GlcNAc levels were elevated by pharmacologically inhibiting OGA.
Results
O-GlcNAcase pharmacological inhibition
OGA inhibition with PUGNAc and ThG are known to increase cellular OGlcNAc modifications. PUGNAc is has been the gold standard in the O-GlcNAc field to
induce O-GlcNAc modifications. ThG is relatively newer and has been reported to be
37,000-fold more selective for OGA over other hexosaminidases[71]. In Figure 3-1,
C2C12 myoblasts were to ThG (1-50 µM) or PUGNAc (100 µM) to determine a
37
Figure 3-1: Inhibition of OGA with ThG and PUGNAc increases cellular OGlcNAc. OGA was inhibited in C2C12 cells for 1hr with ThG (1-50µM) or PUGNAc
(100µM). All methods of OGA inhibition increased cellular O-GlcNAc. Maximal
cellular O-GlcNAcylation was achieved with 10µM Thiamet-G.
concentration of ThG to use for the following experiments. 10µM ThG achieved an equal
amount of O-GlcNAc modification as 50µM ThG and PUGNAc.
38
Dynamic O-GlcNAcylation of NRVM mitochondria
To examine the mitochondrial O-GlcNAc and if it could be modulated by OGA
inhibition, NRVM were treated with 1 µM ThG for one hour before mitochondria were
isolated. Whole cell and mitochondrial fractions were immunostained for total O-GlcNAc
to observe the dynamic nature of mitochondrial protein O-GlcNAcylation. GAPDH and
CoxIV were used to determine purity of mitochondrial fractions. Figure 3-2 demonstrates
that O-GlcNAc can be increased in NRVM mitochondria by inhibiting OGA. It is
interesting to note that a mitochondrial OGA has yet to be identified. It is possible that
there is an unknown protein that is located in the mitochondria with hexosaminidase
activity that can be inhibited by ThG. Another possible explanation is that the positive OGlcNAc staining observed is due to outer mitochondrial membrane proteins that are
susceptible to the cytosolic O-GlcNAc cycling enzymes (OGT and OGA) or OGlcNAcylated cytosolic proteins loosely associated with the mitochondria. To answer
this question, the fractionation of mitoplasts was employed.
Detection of O-GlcNAc modifications in C2C12 mitoplasts
In Figure 3-3, mitoplasts were isolated from C2C12 myoblasts. Extracts were
separated, and then analyzed by Western blotting. Whole cell controls were used to
control for purity and enrichment in the mitoplast fraction. Total O-GlcNAc was
measured using anti-O-GlcNAc antibody CTD110.6. Parallel membranes were stained
for Complex IV subunit 1 (inner mitochondrial membrane marker), GAPDH (cytosolic
39
Whole Cell
Mitochondria
Thiamet‐G (1µM) ‐ + ‐ +
Anti‐O‐GlcNAc
CTD 110.6
GAPDH
~37kDa
CoxIV
~17kDa
Figure 3-2: Dynamic O-GlcNAcylation of NRVM Mitochondria. NRVM
mitochondria were isolated, whole cell and mitochondrial fractions were
immunostained for total O-GlcNAc to observe the dynamic nature of mitochondrial
protein O-GlcNAcylation. 1µM Thiamet-G increased O-GlcNAc modification in
NRVM mitochondrial fractions.
40
protein marker), and VDAC (outer mitochondrial membrane marker). As expected,
endogenous O-GlcNAc was detected in mitoplasts. These data indicate that there are OGlcNAcylated proteins on and inside of the inner mitochondrial membrane.
Optimization of the XF-24 mitochondrial function assay in C2C12 myoblasts
We optimized the conditions for measuring oxygen consumption and testing
mitochondrial respiratory function using the Seahorse XF 24 for C2C12 myoblasts.
Figure 3-4 demonstrates the dose response curves of oligomycin and FCCP in C2C12
myoblasts for the mitochondrial function assay using the Seahorse XF-24. These data
determined the concentration of both reagents to be used in mitochondrial function
studies. Next, oxygen consumption rates of C2C12 at different cell densities were
determined. Basal measurements were recorded, followed by injection of oligomycin (1
µg/ml), FCCP (1 µM) and Antimycin-A (10 µM), which initiate the mitochondria
respiratory function assay. This assay determines basal activity that is coupled to ATP
production, maximal oxygen consumption (OCR), respiratory reserve capacity, the
proportion of maximal activity that is being used at basal conditions, and the level of nonmitochondrial oxygen consumption. Figure 3-5 demonstrates a typical mitochondrial
respiratory function assay on 10k, 20k, 40k, and 60k cells when normalized to the
amount of protein. As expected, there is a linear relationship between cell density and
OCR, as well as cell density and total protein. OCR normalized to protein showed that
40k cell density was provided sufficient OCR measurements while maintaining a
confluent monolayer.
41
Figure 3-3: O-GlcNAcylation of C2C12 mitoplasts. Mitoplasts were
isolated from C2C12s. A western blot analysis revealed endogenous
mitoplast O-GlcNAc. Complex IV, GAPDH, and VDAC were used to
measure enrichment and purity of mitoplast preparation.
42
Figure 3-4: Optimization of oligomycin and FCCP in C2C12. A. % OCR of
C2C12 seeded at 40K cells/well. Four basal measurements then oligomycin (0.1, 0.5,
1, 1.5 µg/ml) were injected. B. % OCR of C2C12 seeded at 40K cells/well. Four basal
measurements then oligomycin (1 µg/ml), FCCP (0.1, 0.5, 1, 2 µM) were injected.
Data is the mean ±sem, n=5 per group.
43
B
A
O
F
A
C
Figure 3-5: OCR of C2C12 at different cell densities
normalized by protein. Panel A: OCR of C2C12 seeded
at 10, 20, 40 and 60K cells/well normilized by protein.
Panel B shows the OCR at rate 4 normalized vs cell
density. Panel C: Protein vs cell density by well. Data is
the mean ±sem, n=5 per group.
44
Thiamet-G and PUGNAc on oxygen consumption in intact C2C12 myoblasts
We began looking at the role of cellular O-GlcNAc on the respiration of C2C12
myoblasts under basal conditions. We used the Seahorse XF24 analyzer to measure
mitochondrial respiratory function in the presence of OGA inhibitors ThG (1M and
10M) and PUGNAc (100M). Basal oxygen consumption was measured prior to
exposure to OGA inhibitors for 1hr. Then the mitochondrial respiratory function assay
was performed. Inhibition of OGA with 100M PUGNAc and 1M ThG do not affect
any of the mitochondrial respiratory function parameters. 10M ThG, however, does
decrease reserve capacity (maximal OCR-basal OCR) significantly (Figure 3-5). These
data are consistent with the idea that the degree and duration of O-GlcNAc alteration is a
relevant factor when studying its affect on mitochondrial function and dysfunction. More
concentrations of ThG remain to be performed. A parallel plate was processed via
Western blot to ensure that OGA inhibition was attained (Figure 3-7).
The effect of DMNQ on oxygen consumption in C2C12 myoblasts
To test the hypothesis that acute increases in mitochondrial O-GlcNAc protects
against ROS induced mitochondrial respiratory dysfunction, the mitochondria respiration
assay was used to measure respiration in response to the redox cycling agent DMNQ,
which generates superoxide and hydrogen peroxide. In bovine epithelial cells, DMNQ
stimulated the basal OCR and proton leak-dependent OCR as well as inhibited maximal
OCR [77]. This effect was mirrored in C2C12 cells (Figure 3-8). Basal, ATP-linked, and
proton leak OCR levels were increased in a similar fashion with all DMNQ
concentrations (5, 10, and 20 M).
45
46
Figure 3-7: XF-24 parallel plate O-GlcNAc Western blot. OGA inhibitor
parallel plate 3.5hr ThG or PUGNAc followed by OCR readings. The purpose of
this procedure is to ensure that O-GlcNAc is increased by the OGA inhibitors
during XF 24 experiments. OGA inhibition (Thiamet-G and PUGNAc) increased
protein O-GlcNAc levels.
47
Figure
3-8: DMNQ dose response on C2C12 OCR. OCR of C2C12 cells seeded at 40k
cells/well treated with DMNQ for 1h normalized to protein. Data is the mean ±sem, n=3-5
per group. *p value <0.005 vs control.
48
Maximal OCR and reserve capacity were decreased in a dose dependent manner.
These data demonstrates that DMNQ is a viable ROS stressor for future experiments
studying O-GlcNAc cytoprotection.
Cytotoxicity of DMNQ and ThG
To determine if ThG is cytoprotective against DMNQ induced ROS we used the
MTT assay. Optimization of DMNQ and ThG in the MTT assay was necessary. First,
varying doses of DMNQ (Figure 3-9) were administered to C2C12 myoblasts over 6 or
12 hrs followed by the MTT assay. While 1 hr was sufficient for changes in
mitochondrial respiration as seen in figure 3-8, the time necessary for DMNQ to be
cytotoxic must be longer. As shown, DMNQ was cytotoxic in a dose and time dependent
manner. We then examined any potential cytotoxic effects of varying doses of ThG
across the same time points. ThG was only able accomplish significant cytotoxicity at
10mM, an order of magnitude higher than normally used to increase cellular O-GlcNAc
levels.
49
Absorbance (570nM) % change from Ctrl
DMNQ
Figure 3­9: Cytotxicity of DMNQ. C2C12 myoblasts were plated in 96 wells then treated with varying doses of DMNQ for either 6 or 12 hrs. Data expressed as a % of 0 DMNQ. n=3, where n designates a different experiment run on separate occasions and values were averaged. 50
Absorbance (570nM) % change from Ctrl
ThG
Figure 3­10: MTT cytotoxicity of ThG. C2C12 myoblasts were plated in 96 wells then treated with varying doses of ThG for either 6 or 12 hrs. Data expressed as a % of 0 ThG. n=8, where n designates an individual well. 51
CHAPTER 4
DISCUSSION
Summary
O-GlcNAcylation of Mitochondrial Proteins
OGA inhibition with ThG and PUGNAc was shown to significantly increase
cellular O-GlcNAc levels in C2C12 myoblasts. Mitochondrial protein O-GlcNAcylation
was increased with OGA inhibition. We also were able to discover O-GlcNAc positive
staining in mitoplast fractions, however, whether O-GlcNAc in this fraction can be
increased with OGA inhibition remains to be determined. The latter is a key factor in
elucidating the role of O-GlcNAc in the mitochondria since it proves that inner
mitochondria contain O-GlcNAcylated proteins. This implies that there is indeed OGlcNAc signaling, but also raises questions about the nature of dynamic O-GlcNAc
signaling in mitochondria.
O-GlcNAcylation and Mitochondrial Function
It is possible that the protective effect seen with O-GlcNAc treatment in response
to stress is due to the modification of key complexes in oxidative phosphorylation.
Altering the whole cell and mitochondrial O-GlcNAc state may also affect basal
mitochondrial respiratory function. The method and duration of changing O-GlcNAc is
important in predicting the effect on mitochondrial function. We show that treatment with
10 μM Thiamet-G for one-hour decreases reserve capacity in intact C2C12 myoblasts.
Lower doses did not alter any of the respiratory parameters, although O-GlcNAc levels
52
increased. It is currently unknown whether O-GlcNAc modification of cellular and/or
mitochondrial proteins plays a role in homeostatic mitochondrial respiration. The
Seahorse Bioscience XF24 Analyzer was used to begin the investigation of the impact of
altering global and mitochondrial O-GlcNAc on basal mitochondrial respiratory function
in intact C2C12 myoblasts, and to determine if mitochondrial O-GlcNAcylation protects
respiratory function in response to ROS. In this report, we demonstrate the optimization
of the XF-24 mitochondrial function assay in C2C12 myoblasts. The O-GlcNAc levels
were elevated by pharmacologically inhibiting OGA using ThG, while basal
measurements were recorded, followed by the mitochondria respiratory function assay.
Respiratory function was correlated with changes in cellular protein O-GlcNAcylation.
DMNQ induced mitochondrial dysfunction was tested using the XF-24. Basal,
ATP-linked, and proton leak OCR levels were increased in a similar fashion with all
DMNQ concentrations (5, 10, and 20 M). Maximal OCR and reserve capacity were
decreased in a dose dependent manner. These data demonstrates that DMNQ is a viable
ROS stressor for studying O-GlcNAc cytoprotection.
Following those studies, cytotoxicity of ThG and DMNQ were tested using the
MTT assay in C2C12 myoblasts. As expected, ThG did not exhibit cytotoxic effects in
experimental concentrations across 6 and 12hrs of exposure. DMNQ, however, exhibited
cytotoxicity in a dose dependent manner.
Conclusion
As discussed in the introduction, the existence of O-GlcNAc signaling in the
mitochondria appears to be a relevant modification potentially affecting the function of a
53
myriad of mitochondrial functions. The goal of this study was to begin characterizing the
basic characteristics of O-GlcNAc cycling in the mitochondria, the consequences of basal
and pathological increases of O-GlcNAc on mitochondrial function, and to determine if
O-GlcNAc protects mitochondrial function in the presence of DMNQ induced oxidative
stress. Understanding the basic characteristics of O-GlcNAc signaling in the
mitochondria and its relationship with the preservation of mitochondrial homeostasis can
prove fruitful in the development of therapeutic interventions related to mitochondrial
dysfunction. Taken together, these data lay the groundwork for further study of OGlcNAcylation in the mitochondria and its potential cytoprotection and maintenance of
mitochondrial function.
Future Directions
Identifying mitochondrial proteins that are O-GlcNAc modified under basal and
stress conditions is fundamental to understanding the O-GlcNAc system in the
mitochondria. To achieve this goal, a targeted candidate strategy would be useful to
identify those proteins that become O-GlcNAcylated in the mitochondria in response to
alterations of mitochondrial O-GlcNAc and in response to ROS. This strategy includes
immunoprecipitation of targets using the protocol established in our lab, followed by total
O-GlcNAc immunoblotting. Targets for immunoprecipitation are members of complexes
of the respiratory chain previously found to be modified with hyperglycemia [62]. To
determine the affect of O-GlcNAc on function, an enzyme activity assay may be
performed to determine if the modification alters activity.
54
The current study focuses on OGA inhibition to increase cellular O-GlcNAc
levels. A transgenic approach to modulated O-GlcNAc levels would be beneficial. Global
and mitochondrial targeted over-expression of OGT, OGA, dominant negative OGT and
dominant negative OGA (to test the loss of function of these two enzymes [78, 79]) on
C2C12 myoblasts would provide a much more comprehensive picture of mitochondrial
O-GlcNAc signaling. Each of these interventions can then be studied using the XF-24 to
determine their effect on basal mitochondrial respiration as well as ROS induced
mitochondrial dysfunction. To confirm alterations of mitochondrial O-GlcNAc in
response to each method of altering cellular and mitochondrial O-GlcNAc, mitoplast
fractionation followed by Western blotting may be performed.
55
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
Du, X.L., et al., Hyperglycemia inhibits endothelial nitric oxide synthase activity
by posttranslational modification at the Akt site. J Clin Invest, 2001. 108(9): p.
1341-8.
Zachara, N.E., et al., Dynamic O-GlcNAc modification of nucleocytoplasmic
proteins in response to stress. A survival response of mammalian cells. J Biol
Chem, 2004. 279(29): p. 30133-42.
Ngoh, G.A., et al., Non-canonical glycosyltransferase modulates post-hypoxic
cardiac myocyte death and mitochondrial permeability transition. J Mol Cell
Cardiol, 2008. 45(2): p. 313-25.
Laczy, B., et al., Protein O-GlcNAcylation: a new signaling paradigm for the
cardiovascular system. Am J Physiol Heart Circ Physiol, 2009. 296(1): p. H1328.
Hanover, J.A., M.W. Krause, and D.C. Love, The hexosamine signaling pathway:
O-GlcNAc cycling in feast or famine. Biochim Biophys Acta. 1800(2): p. 80-95.
Butkinaree, C., K. Park, and G.W. Hart, O-linked beta-N-acetylglucosamine (OGlcNAc): Extensive crosstalk with phosphorylation to regulate signaling and
transcription in response to nutrients and stress. Biochim Biophys Acta. 1800(2):
p. 96-106.
Copeland, R.J., J.W. Bullen, and G.W. Hart, Cross-talk between GlcNAcylation
and phosphorylation: roles in insulin resistance and glucose toxicity. Am J
Physiol Endocrinol Metab, 2008. 295(1): p. E17-28.
Yang, X., et al., Phosphoinositide signalling links O-GlcNAc transferase to
insulin resistance. Nature, 2008. 451(7181): p. 964-9.
Gong, C.X., F. Liu, and K. Iqbal, O-GlcNAc cycling modulates
neurodegeneration. Proc Natl Acad Sci U S A, 2012. 109(43): p. 17319-20.
Caldwell, S.A., et al., Nutrient sensor O-GlcNAc transferase regulates breast
cancer tumorigenesis through targeting of the oncogenic transcription factor
FoxM1. Oncogene, 2010. 29(19): p. 2831-42.
Slawson, C., R.J. Copeland, and G.W. Hart, O-GlcNAc signaling: a metabolic
link between diabetes and cancer? Trends Biochem Sci, 2010. 35(10): p. 547-55.
Wang, K., S.-R. Ho, and A. Paterson, O-GlcNAc Causes Increased levels of
Tumor Suppressor Protein p53 in Human Breast Cancer MCF-7 Cells. FASEB J,
2010. 24(566.8).
Jones, S.P., et al., Cardioprotection by N-acetylglucosamine linkage to cellular
proteins. Circulation, 2008. 117(9): p. 1172-82.
Hahne, H., A. Moghaddas Gholami, and B. Kuster, Discovery of O-GlcNAcmodified proteins in published large-scale proteome data. Mol Cell Proteomics,
2012. 11(10): p. 843-50.
Nandi, A., et al., Global identification of O-GlcNAc-modified proteins. Anal
Chem, 2006. 78(2): p. 452-8.
56
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
Torres, C.R. and G.W. Hart, Topography and polypeptide distribution of terminal
N-acetylglucosamine residues on the surfaces of intact lymphocytes. Evidence for
O-linked GlcNAc. J Biol Chem, 1984. 259(5): p. 3308-17.
Hart, G.W., et al., Cross talk between O-GlcNAcylation and phosphorylation:
roles in signaling, transcription, and chronic disease. Annu Rev Biochem, 2011.
80: p. 825-58.
Snow, C.M., A. Senior, and L. Gerace, Monoclonal antibodies identify a group of
nuclear pore complex glycoproteins. J Cell Biol, 1987. 104(5): p. 1143-56.
Comer, F.I., et al., Characterization of a mouse monoclonal antibody specific for
O-linked N-acetylglucosamine. Anal Biochem, 2001. 293(2): p. 169-77.
Schleicher, E.D. and C. Weigert, Role of the hexosamine biosynthetic pathway in
diabetic nephropathy. Kidney Int Suppl, 2000. 77: p. S13-8.
Marsh, S.A., L.J. Dell'Italia, and J.C. Chatham, Activation of the hexosamine
biosynthesis pathway and protein O-GlcNAcylation modulate hypertrophic and
cell signaling pathways in cardiomyocytes from diabetic mice. Amino Acids,
2011. 40(3): p. 819-28.
Bell, G.I., et al., Molecular biology of mammalian glucose transporters. Diabetes
Care, 1990. 13(3): p. 198-208.
in Essentials of Glycobiology, A. Varki, et al., Editors. 2009: Cold Spring Harbor
(NY).
Wells, L., K. Vosseller, and G.W. Hart, A role for N-acetylglucosamine as a
nutrient sensor and mediator of insulin resistance. Cell Mol Life Sci, 2003. 60(2):
p. 222-8.
Chatham, J.C., et al., Hexosamine biosynthesis and protein O-glycosylation: the
first line of defense against stress, ischemia, and trauma. Shock, 2008. 29(4): p.
431-40.
Oki, T., et al., cDNA cloning and mapping of a novel subtype of
glutamine:fructose-6-phosphate amidotransferase (GFAT2) in human and mouse.
Genomics, 1999. 57(2): p. 227-34.
Sayeski, P.P., et al., Cloning and partial characterization of the mouse
glutamine:fructose-6-phosphate amidotransferase (GFAT) gene promoter.
Nucleic Acids Res, 1997. 25(7): p. 1458-66.
Lim, K. and H.I. Chang, O-GlcNAc modification of Sp1 inhibits the functional
interaction between Sp1 and Oct1. FEBS Lett, 2009. 583(3): p. 512-20.
Ngoh, G.A., et al., O-GlcNAc signaling in the cardiovascular system. Circ Res.
107(2): p. 171-85.
Du, X.L., et al., Hyperglycemia-induced mitochondrial superoxide
overproduction activates the hexosamine pathway and induces plasminogen
activator inhibitor-1 expression by increasing Sp1 glycosylation. Proc Natl Acad
Sci U S A, 2000. 97(22): p. 12222-6.
Lim, K. and H.I. Chang, O-GlcNAc inhibits interaction between Sp1 and Elf-1
transcription factors. Biochem Biophys Res Commun, 2009. 380(3): p. 569-74.
Darley-Usmar, V.M., L.E. Ball, and J.C. Chatham, Protein O-linked beta-Nacetylglucosamine: a novel effector of cardiomyocyte metabolism and function. J
Mol Cell Cardiol, 2012. 52(3): p. 538-49.
57
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
Kang, J.G., et al., O-GlcNAc protein modification in cancer cells increases in
response to glucose deprivation through glycogen degradation. J Biol Chem,
2009. 284(50): p. 34777-84.
Anderson, J.W., R.J. Nicolosi, and J.F. Borzelleca, Glucosamine effects in
humans: a review of effects on glucose metabolism, side effects, safety
considerations and efficacy. Food Chem Toxicol, 2005. 43(2): p. 187-201.
Marshall, S., V. Bacote, and R.R. Traxinger, Discovery of a metabolic pathway
mediating glucose-induced desensitization of the glucose transport system. Role
of hexosamine biosynthesis in the induction of insulin resistance. J Biol Chem,
1991. 266(8): p. 4706-12.
Marshall, S., V. Bacote, and R.R. Traxinger, Complete inhibition of glucoseinduced desensitization of the glucose transport system by inhibitors of mRNA
synthesis. Evidence for rapid turnover of glutamine:fructose-6-phosphate
amidotransferase. J Biol Chem, 1991. 266(16): p. 10155-61.
McKnight, G.L., et al., Molecular cloning, cDNA sequence, and bacterial
expression of human glutamine:fructose-6-phosphate amidotransferase. J Biol
Chem, 1992. 267(35): p. 25208-12.
Sayeski, P.P. and J.E. Kudlow, Glucose metabolism to glucosamine is necessary
for glucose stimulation of transforming growth factor-alpha gene transcription. J
Biol Chem, 1996. 271(25): p. 15237-43.
Zhou, J., et al., Regulation of glutamine:fructose-6-phosphate amidotransferase
by cAMP-dependent protein kinase. Diabetes, 1998. 47(12): p. 1836-40.
Uldry, M., et al., GLUT2 is a high affinity glucosamine transporter. FEBS Lett,
2002. 524(1-3): p. 199-203.
Zou, L., et al., Glucosamine improves cardiac function following traumahemorrhage by increased protein O-GlcNAcylation and attenuation of NF{kappa}B signaling. Am J Physiol Heart Circ Physiol, 2009. 296(2): p. H515-23.
Kolm-Litty, V., et al., High glucose-induced transforming growth factor beta1
production is mediated by the hexosamine pathway in porcine glomerular
mesangial cells. J Clin Invest, 1998. 101(1): p. 160-9.
Spiro, R.G., Protein glycosylation: nature, distribution, enzymatic formation, and
disease implications of glycopeptide bonds. Glycobiology, 2002. 12(4): p. 43R56R.
Nolte, D. and U. Muller, Human O-GlcNAc transferase (OGT): genomic
structure, analysis of splice variants, fine mapping in Xq13.1. Mamm Genome,
2002. 13(1): p. 62-4.
Lazarus, B.D., D.C. Love, and J.A. Hanover, Recombinant O-GlcNAc transferase
isoforms: identification of O-GlcNAcase, yes tyrosine kinase, and tau as isoformspecific substrates. Glycobiology, 2006. 16(5): p. 415-21.
Love, D.C., et al., Mitochondrial and nucleocytoplasmic targeting of O-linked
GlcNAc transferase. J Cell Sci, 2003. 116(Pt 4): p. 647-54.
Shafi, R., et al., The O-GlcNAc transferase gene resides on the X chromosome
and is essential for embryonic stem cell viability and mouse ontogeny. Proc Natl
Acad Sci U S A, 2000. 97(11): p. 5735-9.
58
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
McClain, D.A., et al., Altered glycan-dependent signaling induces insulin
resistance and hyperleptinemia. Proc Natl Acad Sci U S A, 2002. 99(16): p.
10695-9.
Hanover, J.A., et al., Mitochondrial and nucleocytoplasmic isoforms of O-linked
GlcNAc transferase encoded by a single mammalian gene. Arch Biochem
Biophys, 2003. 409(2): p. 287-97.
Shin, S.H., D.C. Love, and J.A. Hanover, Elevated O-GlcNAc-dependent
signaling through inducible mOGT expression selectively triggers apoptosis.
Amino Acids. 40(3): p. 885-93.
Fletcher, B.S., et al., Functional cloning of SPIN-2, a nuclear anti-apoptotic
protein with roles in cell cycle progression. Leukemia, 2002. 16(8): p. 1507-18.
Keembiyehetty, C.N., et al., A lipid-droplet-targeted O-GlcNAcase isoform is a
key regulator of the proteasome. J Cell Sci, 2011. 124(Pt 16): p. 2851-60.
Hart, G.W., M.P. Housley, and C. Slawson, Cycling of O-linked beta-Nacetylglucosamine on nucleocytoplasmic proteins. Nature, 2007. 446(7139): p.
1017-22.
Gao, Y., et al., Dynamic O-glycosylation of nuclear and cytosolic proteins:
cloning and characterization of a neutral, cytosolic beta-N-acetylglucosaminidase
from human brain. J Biol Chem, 2001. 276(13): p. 9838-45.
Hanover, J.A., Glycan-dependent signaling: O-linked N-acetylglucosamine.
FASEB J, 2001. 15(11): p. 1865-76.
Garvey, W.T., et al., Pretranslational suppression of a glucose transporter
protein causes insulin resistance in adipocytes from patients with non-insulindependent diabetes mellitus and obesity. J Clin Invest, 1991. 87(3): p. 1072-81.
Clark, R.J., et al., Diabetes and the accompanying hyperglycemia impairs
cardiomyocyte calcium cycling through increased nuclear O-GlcNAcylation. J
Biol Chem, 2003. 278(45): p. 44230-7.
Liu, J., R.B. Marchase, and J.C. Chatham, Increased O-GlcNAc levels during
reperfusion lead to improved functional recovery and reduced calpain
proteolysis. Am J Physiol Heart Circ Physiol, 2007. 293(3): p. H1391-9.
Champattanachai, V., R.B. Marchase, and J.C. Chatham, Glucosamine protects
neonatal cardiomyocytes from ischemia-reperfusion injury via increased protein
O-GlcNAc and increased mitochondrial Bcl-2. Am J Physiol Cell Physiol, 2008.
294(6): p. C1509-20.
Ngoh, G.A., et al., Unique hexosaminidase reduces metabolic survival signal and
sensitizes cardiac myocytes to hypoxia/reoxygenation injury. Circ Res, 2009.
104(1): p. 41-9.
Zachara, N.E. and G.W. Hart, O-GlcNAc a sensor of cellular state: the role of
nucleocytoplasmic glycosylation in modulating cellular function in response to
nutrition and stress. Biochim Biophys Acta, 2004. 1673(1-2): p. 13-28.
Hu, Y., et al., Increased enzymatic O-GlcNAcylation of mitochondrial proteins
impairs mitochondrial function in cardiac myocytes exposed to high glucose. J
Biol Chem, 2009. 284(1): p. 547-55.
Raha, S. and B.H. Robinson, Mitochondria, oxygen free radicals, disease and
ageing. Trends Biochem Sci, 2000. 25(10): p. 502-8.
59
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
Kelley, E.E., et al., Hydrogen peroxide is the major oxidant product of xanthine
oxidase. Free Radic Biol Med, 2010. 48(4): p. 493-8.
Zhang, H., et al., Angiotensin II-induced superoxide anion generation in human
vascular endothelial cells: role of membrane-bound NADH-/NADPH-oxidases.
Cardiovasc Res, 1999. 44(1): p. 215-22.
Kadenbach, B., et al., New extension of the Mitchell Theory for oxidative
phosphorylation in mitochondria of living organisms. Biochim Biophys Acta.
1800(3): p. 205-12.
Gutierrez, J., et al., Free radicals, mitochondria, and oxidized lipids: the
emerging role in signal transduction in vascular cells. Circ Res, 2006. 99(9): p.
924-32.
Yaffe, D. and O. Saxel, Serial passaging and differentiation of myogenic cells
isolated from dystrophic mouse muscle. Nature, 1977. 270(5639): p. 725-7.
Morgan, J.E. and T.A. Partridge, Muscle satellite cells. Int J Biochem Cell Biol,
2003. 35(8): p. 1151-6.
Zou, L., et al., The protective effects of PUGNAc on cardiac function after
trauma-hemorrhage are mediated via increased protein O-GlcNAc levels. Shock,
2007. 27(4): p. 402-8.
Yuzwa, S.A., et al., A potent mechanism-inspired O-GlcNAcase inhibitor that
blocks phosphorylation of tau in vivo. Nat Chem Biol, 2008. 4(8): p. 483-90.
Dranka, B.P., B.G. Hill, and V.M. Darley-Usmar, Mitochondrial reserve capacity
in endothelial cells: The impact of nitric oxide and reactive oxygen species. Free
Radic Biol Med, 2010. 48(7): p. 905-14.
Zhang, J., et al., Measuring energy metabolism in cultured cells, including human
pluripotent stem cells and differentiated cells. Nat Protoc, 2012. 7(6): p. 1068-85.
Higdon, A.N., et al., Hemin causes mitochondrial dysfunction in endothelial cells
through promoting lipid peroxidation: the protective role of autophagy. Am J
Physiol Heart Circ Physiol, 2012. 302(7): p. H1394-409.
Hundal, T., B. Norling, and L. Ernster, The oligomycin sensitivity conferring
protein (OSCP) of beef heart mitochondria: studies of its binding to F1 and its
function. J Bioenerg Biomembr, 1984. 16(5-6): p. 535-50.
Park, J., et al., Proteomic analysis of O-GlcNAc modifications derived from
streptozotocin and glucosamine induced beta-cell apoptosis. J Biochem Mol Biol,
2007. 40(6): p. 1058-68.
Dranka, B.P., B.G. Hill, and V.M. Darley-Usmar, Mitochondrial reserve capacity
in endothelial cells: The impact of nitric oxide and reactive oxygen species. Free
Radic Biol Med. 48(7): p. 905-14.
Wang, K., et al., Increased O-GlcNAc causes disrupted lens fiber cell
differentiation and cataracts. Biochem Biophys Res Commun, 2009. 387(1): p.
70-6.
Goldberg, H.J., et al., Posttranslational, reversible O-glycosylation is stimulated
by high glucose and mediates plasminogen activator inhibitor-1 gene expression
and Sp1 transcriptional activity in glomerular mesangial cells. Endocrinology,
2006. 147(1): p. 222-31.
60