THE EFFECT OF GLUTATHIONE ON HIGH MOLECULAR WEIGHT ... · the effect of glutathione on high...
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THE EFFECT OF GLUTATHIONE ON HIGH MOLECULAR WEIGHT ADIPONECTIN SECRETION FROM 3T3-L1 ADIPOCYTES DURING ENDOPLASMIC RETICULUM
STRESS
By
BRANDON EUDY
A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
UNIVERSITY OF FLORIDA
2016
© 2016 Brandon Eudy
To persistence
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ACKNOWLEDGMENTS
I want to first thank Dr. Percival for accepting me into her lab and making my
endeavors possible. Your willingness to let me pursue this project and discover its ins
and outs of pursuing this work on my own have allowed me to learn so much. I also
appreciate Dr. Henken and Dr. Anton for serving on my committee and offering
encouragement and providing me with additional insight to my project. I thank all of my
lab and class mates who have made this experience even more enjoyable. Cheryl,
thank you for trying to keep me in line. Mom and Dad, I appreciate your support
throughout the past two years and for helping me make this possible.
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TABLE OF CONTENTS page
ACKNOWLEDGMENTS ............................................................................................ 4
LIST OF TABLES ..................................................................................................... 6
LIST OF FIGURES ................................................................................................... 7
LIST OF ABBREVIATIONS ....................................................................................... 9
ABSTRACT ............................................................................................................ 12
CHAPTER
1 INTRODUCTION ................................................................................................. 14
Introduction, Obesity, and the Metabolic Syndrome ............................................. 14
Endoplasmic Reticulum Stress in the Adipocyte .................................................. 15
Adiponectin ...................................................................................................... 19
Glutathione....................................................................................................... 20
Glutathione and ER Stress ................................................................................ 22
Specific Aims.................................................................................................... 25
2 MATERIALS AND METHODS .............................................................................. 28
Cell Culture and Treatments .............................................................................. 28
Measurement of Total Glutathione ..................................................................... 29
Measurement of Total and HMW Adiponectin ..................................................... 29
Western Blot Analysis ....................................................................................... 30
RNA Isolation and RT-qPCR ............................................................................. 31
Oil Red O Staining ............................................................................................ 31
3 RESULTS ........................................................................................................... 34
Glutathione Uptake Into 3T3-L1 Adipocytes and Effect of ER Stress on Glutathione Concentrations ............................................................................ 34
Effect of Glutathione Pre-treatment on UPR Markers in 3T3-L1 Adipocytes During ER Stress ........................................................................................... 35
Glutathione Does not Attenuate the Decrease in HMW Adiponectin Secretion Caused by Treatment With Palmitate .............................................................. 36
Glutathione Pre-treatment Lowers Ero-1α Gene Expression ................................ 36
4 DISCUSSION ...................................................................................................... 45
LIST OF REFERENCES ......................................................................................... 54
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BIOGRAPHICAL SKETCH ...................................................................................... 61
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LIST OF TABLES Table page 2-1 Primer sequences used for RT-qPCR. ........................................................... 33
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LIST OF FIGURES
Figure page 1-1 Thiol-mediated protein retention. Proteins such as adiponectin may form
disulfide bonds with the ER chaperone ERp44................................................ 27
3-1 Oil Red O staining of 3T3-L1 adipocytes......................................................... 38
3-2 Glutathione uptake into 3T3-L1 adipocytes ..................................................... 39
3-3 Intracellular glutathione concentrations in 3T3-L1 adipocytes during ER stress ........................................................................................................... 40
3-4 GSH pre-treatment prevents induction of CHOP during palmitate-induced ER stress ........................................................................................................... 41
3-5 GSH pre-treatment does not decrease XBP1s mRNA expression during palmitate induced ER stress .......................................................................... 42
3-6 Adiponectin secretion, Total and HMW ........................................................... 43
3-7 GSH pre-treatment decreases ERo-1α mRNA expression ............................... 44
4-1 Hypothetical mechanism of action explaining how increased intracellular glutathione alters UPR signaling .................................................................... 53
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LIST OF ABBREVIATIONS
µg microgram
µL microliter
µM micromolar
ARE Antioxidant response element
ATF4 Activating transcription factor 4
ATF6 Activating transcription factor 6
AMPK adenosine monophosphate kinase
ANOVA Analysis of variance
BCL2 B-cell lymphoma 2
Bid BH3 interacting-domain death protein
BMI Body mass index
BSA Bovine serum albumin
C Celsius
CHOP CAAT/enhancer binding homologous protein
ddH2O Double distilled water
DM1 Differentiation medium 1
DM2 Differentiation medium 2
DMEM Dulbecco’s Modified Eagle Media
DR5 Death receptor 5
DsbA-L Disulfide bond A oxidoreductase-like protein
DXM Dexamethasone
eIF2α Eukaryotic translation initiation factor 2 alpha
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ELISA Enzyme-linked immunosorbent assay
ER Endoplasmic reticulum
ERp44 Endoplasmic reticulum resident protein 44
Ero-1α Endoplasmic reticulum oxidoreductase alpha
GCL Glutamate cysteine ligase
GRP78 78 kDa glucose-regulated protein
GR Glutathione reductase
GSH Reduced glutathione
GSSG Oxidized glutathione
GSTP Glutathione-S-transferase Pi
HDL High-density lipoprotein
IBMX Isobutyl-1-methylxanthine
IRE1 Inositol requiring kinase 1
IRS-1 insulin receptor substrate-1
JNK c-Jun N-terminal kinase
kDa Kilodalton
KEAP1 Kelch like-ECH-associated protein 1
HMW High-molecular weight
MetS Metabolic syndrome
MPA Metaphosphoric acid
mRNA Messenger RNA
Nmoles nanomoles
NAC n-acetyl-cysteine
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NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells
NRF2 Nuclear factor (erythroid-derived 2)-like 2
PBS Phosphate-buffered saline
PDI protein disulfide isomerase
PERK Double stranded RNA-activated protein kinase-like ER kinase
PPARα peroxisome proliferator-activated receptor alpha
RIPA Radio immunoprecipitation assay
RNA Ribonucleic acid
RNS Reactive nitrogen species
ROS Reactive oxygen species
RT-qPCR Real time quantitative polymerase chain reaction
RPL13A Ribosomal Protein L13a
SDS Sodium dodecyl sulfate
siRNA Small-interfering RNA
TNF-α Tumor necrosis factor alpha
TZD Thiazolidinedione
UPR Unfolded protein response
WAT White adipose tissue
WT Wild-type
XBP1 X-box binding protein 1
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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science
THE EFFECT OF GLUTATHIONE ON HIGH MOLECULAR WEIGHT ADIPONECTIN SECRETION FROM 3T3-L1 ADIPOCYTES DURING ENDOPLASMIC RETICULUM
STRESS
By
Brandon Eudy
August 2016
Chair: Susan S. Percival Major: Food Science and Human Nutrition
Obesity and metabolic syndrome are serious conditions afflicting many in the US
and worldwide. These conditions are often associated with chronic low-grade
inflammation, oxidative stress, and endoplasmic reticulum (ER) stress. Glutathione
(GSH) is an important intracellular antioxidant and also assists protein folding in the ER.
Little attention has been given to GSH concerning possible roles in metabolic health.
GSH has been shown to be associated with higher levels of the insulin-sensitizing
adipokine, adiponectin, in human plasma. Adiponectin exists in several isoforms. The
high molecular weight (HMW) form of adiponectin is thought to have the most potent
insulin sensitizing effect. Whether GSH plays a role in adiponectin secretion and
oligomerization has not been previously investigated. GSH may protect against
metabolic dysregulation by ameliorating ER stress and increasing HMW adiponectin
secretion in stressed cells. The aims of this study were to determine the effects of
intracellular GSH concentration on ER Stress and HMW adiponectin secretion in 3T3-L1
adipocytes. ER stress was induced by treatment with palmitate (1 mM) and the effect of
GSH pre-treatment (1 mM) was also investigated. To show that palmitate induced ER
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stress, XBP1s mRNA expression and CHOP protein expression were measured. mRNA
expression of the ER chaperone ERO-1α was measured to evaluate the effects of
palmitate and GSH treatment on protein folding. Adiponectin was measured by ELISA.
GSH pretreatment did not spare secretion of HMW adiponectin into the cell culture
medium. GSH pre-treatment prevented induction of CHOP and ERO-1α, but had no
effect on splicing of XBP1. Given that CHOP is implicated in ER stress associated cell
death, the intracellular GSH concentration may direct cell fate in response to ER stress.
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CHAPTER 1 INTRODUCTION
Introduction, Obesity, and the Metabolic Syndrome
Our current food environment is characterized by nutritional abundance. The typical
Western diet is comprised of meals high in fat and carbohydrates, and includes a great
deal of snacking. Taken together, these factors have played a part in the increased
prevalence of chronic disease observed over the past several decades including obesity
and diabetes. These disease states are part of the metabolic syndrome (MetS), a term
used to characterize the presence of a combination of the following conditions: high
blood glucose, high blood pressure, low high-density lipoprotein (HDL), high body mass
index (BMI), and high serum triglycerides (1). These pathologies can wreak havoc in
overall health and are a serious problem in US. Data from the 2003-2006 National
Health and Nutrition Examination Survey show prevalence of MetS in the US to be
approximately 34% (2). Numerous lifestyle interventions, drugs, and surgeries are
available as treatments, but vary in effectiveness. Obesity represents a particularly
difficult condition to treat. This may stem from the multi-factorial nature behind what
causes this pathology. Although excess energy consumption is certainly implicated,
genetics and other factors also play a role (3-5). For example, obesity is accompanied
by oxidative stress and chronic low-grade inflammation. Furthermore, obese individuals
have been shown to have altered gut microbiota and altered ratios of important
hormones including leptin and adiponectin (6-8).
Another perturbation that occurs at the cellular level in response to chronic over nutrition
is endoplasmic reticulum (ER) stress. Indeed, adipose tissue isolated from obese mice
and humans show heightened markers of ER stress (9). Furthermore, ER stress
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causes induction of the unfolded protein response (UPR), which may potentially activate
several inflammatory pathways. Therefore, ER stress may provide a crucial link
between obesity, adipose tissue inflammation, and insulin resistance.
Insulin resistance is a hallmark of metabolic disease. Of interest is the adipokine,
adiponectin. Adiponectin is secreted by white adipose tissue (WAT) and plays roles in
glucose metabolism and insulin sensitivity. However, higher BMI is known to be
associated with lower concentrations of circulating adiponectin. In particular, the high-
molecular weight (HMW) isoform of adiponectin seems to be closely correlated with
metabolic health. Treatment of diabetic subjects with thiazolidinediones (TZDs)
improves insulin sensitivity by increasing circulating HMW adiponectin concentrations.
Therefore, novel treatment for obesity and related pathologies may focus on increasing
HMW adiponectin secretion from adipocytes. Glutathione is an important intracellular
antioxidant and may be protective against ER stress. For example, GSH may regulate
ER chaperones which are involved in the synthesis and secretion of HMW adiponectin
from the ER. The aim of this study is to determine whether increased glutathione
concentrations in adipocytes may favorably modulate the UPR induced by palmitate
treatment and blunt the decrease in HMW adiponectin seen in this condition.
Endoplasmic Reticulum Stress in the Adipocyte
The ER is an organelle found in all eukaryotic cells and serves as the location for
synthesis, assembly, and secretion of new proteins. Homeostasis of this system must
be carefully maintained in order to meet cellular protein demand, while avoiding
overwhelming the secretory pathway. In times of increased stress, UPR signaling is
induced primarily by activation of three ER transmembrane proteins in response to
accumulation of misfolded proteins in the ER lumen: double stranded RNA-activated
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protein kinase-like ER kinase (PERK), inositol requiring kinase 1 (IRE1), and activating
transcription factor 6 (ATF6.) Although each of these proteins activates its own unique
signal transduction pathway, they share the same means of activation and converge
upon many of the same transcriptional targets. Therefore, these pathways together
encompass the entire UPR signaling pathway. These proteins normally bind to 78 kDa
glucose-regulated protein (GRP78), the master regulator of the UPR. When
concentrations of misfolded proteins are high inside of the ER lumen, GRP78
dissociates from these proteins and binds misfolded proteins instead, allowing for
activation of PERK, IRE1, and ATF6. Increased phosphorylation of PERK and IRE1 are
strong indicators of ongoing ER stress, as is proteolytic cleavage of ATF6 (10).
UPR activation is a dynamic process that determines cell fate during ER stress. The
UPR begins as an adaptive pathway with emphasis on cell survival. However, as
greater amounts of cellular stress accrue, the UPR experiences a paradigm shift
towards promoting cell death (11). The dynamics of the UPR involved in determining
cell fate are complex and this introduction will only briefly highlight key targets. In short,
splicing of the X-box binding protein 1 (XBP1) transcript by IRE-1 promotes cell survival
by increasing lipid synthesis and upregulating ER chaperones (12, 13) The PERK
pathway is also implicated in cell survival. PERK phosphorylates the transcription factor
Nuclear factor (erythroid-derived 2)-like 2 (NRF2), which triggers its dissociation from
Kelch like-ECH-associated protein 1 (KEAP1.) Free NRF2 then interacts with
antioxidant response elements (ARE) in the promoter regions of many antioxidant
genes causing their upregulation. PERK is also thought to be protective by transiently
phosphorylating eIF2α, temporarily halting synthesis of new proteins.
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A central protein related to ER stress associated cell death is CAAT/enhancer binding
homologous protein (CHOP)(13). CHOP is located downstream of PERK and
upregulated during prolonged ER stress. CHOP can reduce expression of BCL-2 anti-
apoptotic proteins and has also been shown to upregulate death receptor-5 (DR5), both
of which factors promote a pro-apoptotic phenotype (14, 15). Furthermore, CHOP may
be involved in mediating other forms of cell death in response to ER stress (16).
Pyroptosis is a type of programmed cell death that shares features of apoptosis and
necrosis and is a typical response of cells to unresolvable inflammation. Cleavage of
caspase-1 is a key feature defining pyroptosis (17). CHOP is thought to be involved in
caspase-1 cleavage, resulting in cell death by pyroptotis. Treatment of primary mouse
hepatocytes with tunicaymcin resulted in caspase-1 cleavage. However, knockdown of
Chop using siRNA ameliorated this effect (18). Finally, ER stress may also promote cell
death by increasing intracellular ROS and Ca2+ release into the cytosol and
mitochondria (11).
The UPR is highly responsive to nutritional status. Therefore it is logical that
dysregulated energy metabolism would disrupt this pathway, particularly in the
adipocyte. ER stress was first linked with obesity by Ozcan et al. in 2004 (10). Ob/ob
mice and mice fed high fat diets both showed increased PERK phosphorylation, JNK
activation, and GRP78 protein expression in adipose tissue. Several factors may
contribute to obesity-induced ER stress. In obesity, adipocytes face hypoxia and
increased concentrations of free fatty acids, both of which factors further contribute to
ER stress. Indeed, in-vitro studies have shown that the saturated fatty acid (SFA),
palmitate activates the UPR (19, 20). The mechanism by which palmitate induces ER
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stress is not well understood. However, one hypothesis points to accumulation of SFA
in the ER membrane, which may interfere with fluidity by replacing phosphatidylcholine
residues (21). Increased UPR activation causes changes in inflammatory signaling to
immune cells. All three UPR pathways have been shown to upregulate nuclear factor
kappa-light-chain-enhancer of activated B cells (NF-κB) and c-Jun N-terminal kinase
(JNK) (22). Both of these pathways are pro-inflammatory and may inhibit tyrosine
phosphorylation of insulin receptor substrate-1 (IRS-1), leading to insulin resistance.
Inflammation and insulin resistance are two downstream consequences of ER stress
important to the adipocyte that will be discussed below.
Chronic UPR activation may directly cause insulin resistance. In 2001, Harding et al.
showed that ER stress in Perk -/- mice, was marked with a compensatory increase in
phosphorylation of IRE1. Perk -/- mice experienced heightened type-2 diabetes disease
severity and increased apoptosis of pancreatic β-cells (23). Furthermore, UPR
activation in adipose tissue of mice interferes with IRS-1 signaling by IRE1 dependent
JNK activation (10). This observation suggests that obesity associated ER stress may
have direct consequences on insulin sensitivity and link obesity with type-2 diabetes.
Prolonged ER stress may also contribute to adipocyte cell death, which is implicated in
inflammation and metabolic disease. When a cell is not able to cope with ER stress, cell
death is favored over adaptation. Cinti et al. showed macrophage infiltration in adipose
tissue was 90% localized to adipocytes showing morphological features of necrosis
(24). On the contrary, adipocyte apoptosis has also been shown to cause increased
macrophage infiltration. (25). Inactivation of the pro-apoptotic protein BH3 interacting-
domain death agonist (Bid) reduced macrophage infiltration and TNF-α concentrations
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in adipose tissue of obese mice. Furthermore, insulin resistance was higher in wild-type
mice compared to their Bid-/- littermates. These data suggest multiple cell death
pathways may be activated in adipocytes during obesity-induced ER stress. However, it
was later verified by ultrasound imaging that adipocytes undergo pyroptosis in obesity-
induced ER stress, a cell death pathway which shares features of apoptosis and
necrosis (16). Therefore, future research should critically consider the mechanism
behind cell death in adipocytes. In summary, adipocyte cell death promotes
inflammation in WAT and further increases insulin resistance in humans and animal
models. Therefore, targeting cell death in the adipocyte may have implications in
resolving metabolic dysregulation.
Adiponectin
Adiponectin is recognized as an anti-inflammatory adipokine, or adipo-cytokine secreted
by adipocytes. It was discovered in 1995 by four independent labs (26-29). Since its
discovery, adiponectin has been studied due to its inverse correlation with obesity, type-
2 diabetes, and MetS. Obese and diabetic subjects have been shown to have lower
circulating concentrations of adiponectin (30). This association has garnered interest in
adiponectin as a target for prevention or treatment of metabolic diseases including
obesity and type-2 diabetes. Interestingly, adiponectin concentrations are shown to be
higher in women than men and are also higher in centenarians and their offspring (31).
The presence of higher levels of adiponectin in centenarians has harbored questions as
to whether adiponectin may also contribute to longevity.
Disulfide bonding is crucial in the assembly of higher order complexes of adiponectin
(32). The HMW form, which is an octadecamer consisting of eighteen adiponectin
monomers, has been shown to behave differently than the other forms and may be
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most relevant to metabolic health. The liver and skeletal muscle are key tissues where
adiponectin exerts its insulin sensitizing effects. Adiponectin exerts insulin sensitizing
properties through the adenosine monophosphate kinase (AMPK) and peroxisome
proliferator-activated receptor alpha (PPARα) pathways by binding to its
transmembrane protein receptors AdipoR1 and AdipoR2 (33, 34). AdipoR1 shows high
affinity for globular adiponectin while AdipoR2 shows high affinity for the HMW isoform
(35).
There is some evidence that ER stress alters assembly and secretion of adiponectin in
adipocytes. 3T3-L1 adipocytes subjected to hypoxic conditions, similar to what is seen
in obese adipose tissue, have been shown to have increased GRP78 mRNA
expression, indicating ER stress (36). Importantly, adiponectin mRNA expression was
reduced in this cell culture model. Other in-vitro studies have also observed
relationships between ER stress and adiponectin concentrations. Liu et al. observed
increased ER stress in 3T3-L1 cells treated with thapsigargin along with decreased
adiponectin concentrations (37). Another study using human adipose stem cells showed
that ER stress induced by palmitate decreased secretion of the HMW form of
adiponectin specifically into the cell culture medium (19). Total adiponectin was also
decreased in cell lysates. These experiments show that ER stress results in decreased
adiponectin concentrations and secretion into the extracellular environment.
Glutathione
Glutathione is a tripeptide consisting of glutamate, cysteine, and glycine,
covalently bonded in that respective order. Glutathione has spurred much interest over
the past 40 years because it is a powerful intracellular antioxidant and plays an
important role in maintaining cellular redox status (38). GSH exerts antioxidant activity
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by reacting with intracellular reactive oxygen species (ROS) and reactive nitrogen
species (RNS), subsequently converting them to more benign forms at the cost of its
own oxidation to glutathione disulfide (GSSG). Oxidized glutathione may be recycled by
enzymatic activity of glutathione reductase (GR) and NADPH as a corresponding
source of reducing equivalents. GSH is also involved in reduction of hydrogen peroxide
and other various lipid peroxides by acting as a substrate for the enzyme glutathione
peroxidase. Besides its antioxidant activity, glutathione is an important storage form of
sulfur and cysteine. Glutathione also assists in the removal of xenobiotics. In the
intracellular environment, glutathione exists primarily in the reduced form, but in the
extracellular environment, oxidized glutathione is the predominant form. The ratio of
GSH : GSSG also varies by cellular compartment (39, 40). For example in the cytosol
and mitochondria, GSH may represent over 98% of the total glutathione pool. This may
reflect the role of GSH in ameliorating oxidative stress in these compartments,
especially the mitochondria. In the endoplasmic reticulum (ER), as much as 50% of total
glutathione has been reported to be as GSSG (41). This is essential as the ER requires
a slightly oxidizing environment to ensue correct protein folding. In many cases of
chronic disease, the intracellular GSH : GSSG ratio is lower than normal and is an
indicator of oxidative stress. However, the total amount of intracellular glutathione may
be more telling of a cell’s potential to deal with oxidative stress due to the high efficiency
of GR. Therefore, boosting intracellular glutathione concentrations may be an effective
means of reducing the damage caused by ROS in many cell and tissue types.
Glutathione is present in dietary sources, but poorly available. It is found in animal
sources such as beef and poultry, but quickly degraded by proteases in the stomach
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and poorly absorbed by cells. Consuming foods rich in the amino acids cysteine and
glycine, or foods known to increase GSH biosynthesis, such as alliums and cruciferous
vegetables may be the optimal way to increase GSH through dietary means. In-vivo
studies often show GSH to be poorly absorbed. However, not all studies show this.
Kovaks-Nolan et al. found GSH to undergo transport across the epithelium in humans
(42). However, glutathione was increased only in liver and red blood cells, not in
plasma. Another ex-vivo study showed exogenous GSH was absorbed into isolated
kidney cells treated with the glutathione synthetase inhibitor, buthionine sulfoxomine.
These cells did not show signs of GSH depletion, implying that exogenous GSH was
indeed absorbed.(43). These data suggests that GSH is able to be transported across
membranes in these cell types. However, Future studies are needed to understand the
conditions which allows optimal cellular GSH uptake and determine the differences in
GSH transport between cell types.
Recently, supplemental forms of GSH with increased bioavailability have recently
been developed as dietary supplements for human use. GSH in this form has been
shown to be highly bioavailable. A clinical trial showed that 1000mg GSH / day
significantly increased glutathione concentrations in PBMC after 1, 3, and 6 months of
supplementation compared to a placebo (44). The maximum increase in lymphocyte
GSH was approximately 30%. However, whether GSH was increased in other tissues,
such as the adipose tissue was not measured and should be a subject of future studies.
Glutathione and ER Stress
Reducing cell death in adipose tissue is a potential target in alleviating adipose
tissue inflammation and insulin resistance. ER stress has been shown to be implicated
in adipocyte cell death and CHOP is an important transcription factor in this process
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(45). Furthermore, ER stress is also known to deplete intracellular glutathione
concentrations due to increased protein folding demand, ROS production, and initiation
of cell death during severe ER stress (46, 47).
The PERK pathway of the UPR is highly sensitive to misfolded proteins in the ER
lumen as well as oxidative stress. Therefore, bolstering intracellular GSH concentrations
may protect against PERK-eIF2α -ATF4-CHOP activation by preventing the
accumulation of misfolded proteins and ameliorating oxidative stress.
Although glutathione contributes to the maintenance of the redox state inside of
the ER lumen, it is not well understood how modulating intracellular glutathione
concentrations affects the UPR. Giordano et al. recently showed that treating HepG2
cells with glutathione ethyl ester showed no decrease in GRP78 or CHOP mRNA
expression (48). However, a study performed in mice showed that treatment with the
GSH precursor, n-acetyl-cysteine (NAC) reduced oxidative stress and ER stress after
liver ischemia reperfusion (49). ER stress severity was determined by GRP78, AFT4,
and CHOP protein expression. Interestingly, GSH concentrations were lower in the mice
who did not receive NAC treatment. The above studies are insightful, but do not clearly
show whether increased GSH concentrations may independently reduce ER stress or
modulate the UPR. More research is needed to elucidate how GSH may alter UPR
activation and contribute to determining cell fate during ER stress.
GSH may also have protective effects indirectly related to the UPR. For example, GSH
may directly reduce non-native disulfide bonds (aka misfolded proteins) which
accumulate during ER stress (41). GSH may also reduce oxidized protein disulfide
isomerases (PDI), which catalyze the formation of disulfide bonds in newly assembled
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proteins. Together, these processes show that GSH supports rearrangement of disulfide
bonds, which is crucial for correct protein assembly (50, 51). Therefore, bolstering
intracellular GSH concentrations may ameliorate ER stress by assisting in protein
folding. ERp44 and ERo-1α are key proteins located in the ER that regulate protein
folding. Erp44 is a member of the PDI family and binds to proteins, retaining them in the
ER until they become correctly folded. Ero-1α works oppositely of ERp44 by binding to it
and releasing any previously bound proteins for secretion from the cell. ERo-1α can
also facilitate disulfide bonding in proteins and is thought to be rate limiting factor in this
process. ERp44 and ERo-1α together form a system known as thiol-mediated protein
retention, which is crucial for proper assembly of proteins requiring significant post-
translational modifications (52). ERo-1α mRNA expression is upregulated in ER stress
by the PERK and ATF6 pathways (53). Increased ERO-1α activity may interfere with
thiol-mediated protein retention, which could cause premature release of proteins from
the ER (54). Indeed, previous work in human adipose stem cells seems to support this.
In response to palmitate-induced ER stress, these cells showed lower secretion of
HMW adiponectin in the cell culture medium (19). Additionally, ERo-1α mRNA
expression was upregulated.
In summary, GSH is able to reduce PDI proteins including ERp44, which could
influence disulfide rearrangement. GSH also regulates protein folding by
counterbalancing ERo-1α activity, possibly preventing secretion of immature proteins.
Therefore, it is tempting to speculate that bolstering intracellular concentrations of GSH
may antagonize ERo-1α activity or prevent its upregulation. This could allow for the
complete assembly of HMW adiponectin to occur before it is secreted from the cell.
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Specific Aims
Specific Aim I. Investigate the effect of augmenting total glutathione
concentrations on glutathione depletion during palmitate-induced ER stress in 3T3-L1
adipocytes. It is not known whether extracellular GSH is readily taken up by 3T3-L1
adipocytes. Therefore, I propose to incubate cells with extracellular GSH and measure
glutathione concentrations in the cell after 20 hours. Palmitate, in the free fatty acid form
will be used to induce ER stress in 3T3-L1 adipocytes for 24 hours. A time course
experiment will be conducted and intracellular glutathione concentrations will be
measured in cells pre-treated with GSH or vehicle.
Specific Aim II. Determine effect of GSH on UPR activation during palmitate-
induced ER stress. The role of intracellular GSH status in protecting against ER stress
is not fully understood. Furthermore, it is not known whether GSH may modulate the
UPR in response to ER Stress. Therefore, I propose to investigate the effect of
augmented intracellular glutathione concentrations on UPR activation in 3T3-L1 cells
during palmitate-induced ER stress. CHOP protein expression and splicing of XBP1 will
be measured to assess activation of the PERK and IRE1 arms of the UPR respectively.
Specific Aim III. Determine the effect of GSH on adiponectin secretion and Ero-
1α mRNA expression during palmitate-induced ER stress. Pre-treating 3T3-L1
adipocytes undergoing ER stress with GSH may improve secretion of HMW adiponectin
by reducing the negative consequences of ER stress. It is hypothesized that GSH
treatment will accomplish this by ameliorating oxidative stress and assisting in protein
folding. In addition to its antioxidant potential, GSH may catalyze disulfide
rearrangement in misfolded proteins. GSH also works antagonistically of Ero-1α, a
protein that has been shown to be involved in regulating adiponectin secretion. Several
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antioxidant compounds including tocopherols, (-)-catechin from green tea polyphenols,
and hydroxycinnamic acid derivatives have previously been shown to increase
adiponectin concentrations in 3T3-L1 cells (55-57). However, the increases shown in
these studies may have resulted from increased adiponectin mRNA expression, rather
than post-translational events. Furthermore, HMW adiponectin was not measured, nor
was secretion of adiponectin into conditioned medium.
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Figure 1-1. Thiol-mediated protein retention. Proteins such as adiponectin may form disulfide bonds with the ER chaperone ERp44. This allows retention of the target protein in the ER lumen, allowing for the required post-translational modifications to take place. Ero-1α acts antagonistically of ERp44 in facilitating thiol-mediated retention by binding to it and releasing the previously bound protein.
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CHAPTER 2 MATERIALS AND METHODS
Cell Culture and Treatments
3T3-L1 cells were obtained from the American Type Culture Collection (ATCC®
CL173™) and subcultured in Dulbecco’s Modified Eagle Media (DMEM) containing 4.5
g/L glucose and 584 mg/L L-glutamine (Cellgro), supplemented with 1% Penicillin (100
U/ml) / Streptomycin (100 µg/ml) (Sigma), 10% calf serum, and incubated at 37°C in 5%
CO2. Once fibroblasts were 2 days post-confluent, differentiation into adipocytes was
initiated by switching to differentiation medium 1 (DM1.) This was Day 0. DM1 consists
of DMEM supplemented with 10% fetal bovine serum (FBS), 0.5 mM 3-Isobutyl-1-
methylxanthine (IBMX) (Sigma), 1 µM dexamethasone (DXM) (Sigma), and 1.5 µg/ml
insulin (Sigma). Cells were incubated in DM1 for 48 hours before being switched to a
post-differentiation medium consisting of DMEM supplemented with 10% FBS, and 1.5
µg/ml insulin. A stock solution of glutathione (50 mM) was prepared by dissolving GSH
(Sigma) in deionized water. The solution was then sterilized using a 0.2µ syringe filter
and stored at -80°C in 0.5 ml aliquots. For glutathione pre-treatments, mature
adipocytes (day 9) were treated with 1 mM GSH or equivalent volume of saline solution
for 20 hours. ER stress was induced by treating cells with 1 mM palmitate (Sigma.)
For ER stress experiments, cells were cultured in DMEM supplemented with 10% FBS,
1% Penicillin/Streptomycin, and 0.75% fatty acid-free bovine serum albumin (BSA) (58).
Palmitic acid was dissolved in 0.1M aqueous NaOH and incubated at 37°C for 45
minutes before being quickly dissolved in medium supplemented with 0.75% BSA at
37°C. The resulting medium was incubated overnight before being used the following
29
day. To induce ER stress, palmitate was added to a final concentration of 1 mM.
Medium containing 0.75% BSA served as a control for these experiments.
Fatty acid-free BSA (Sigma) was purchased or prepared by the method adapted from
Chen (59). Briefly, 5g BSA was dissolved in 50ml deionized H2O and pH was lowered to
3.0 using 0.1 M HCl. 2.5g activated charcoal (Sigma) was then added. The resulting
slurry was magnetically stirred at 4°C overnight before being transferred to multiple
microfuge tubes and centrifuged at 13,000 rpm for 20 minutes. The supernatant was
collected and pH was adjusted to 7.0 by addition of 0.1 M NaOH. The resulting solution
was then sterile filtered using a 0.2µ filter and degassed before being stored at 4°C.
Thapsigargin (Santa Cruz Biotechnology) was used as a positive control for ER stress
marker experiments. A 1 mM stock solution was created by dissolving thapsigargin in
DMSO and the final concentration of thapsigargin used in experiments was 1 µM.
Measurement of Total Glutathione
Total intracellular glutathione was measured using a commercial assay kit (Cayman
Chemical). Cells were washed with cold phosphate-buffered saline (PBS) before
addition of cold MES buffer. Cells were then scraped and transferred to a 1.5 ml
Eppendorf tube before being sonicated twice in ten second bursts. To cell lysates, an
equal volume of metaphosphoric acid (MPA) was added. After 5 minutes of incubation
with MPA, lysates were centrifuged for 4 minutes at 12,000 rpm to precipitate proteins.
Deproteinated cell lysates were stored at -80°C for future analysis.
Measurement of total and HMW adiponectin
Total and HMW adiponectin was measured in conditioned medium using a commercial
ELISA kit (Alpco Adiponectin (Mouse) Total, HMW ELISA.) This protocol was modified
from the method designed Harris et al (60) which adapted the original protocol to be
30
more suitable for measuring adiponectin in conditioned medium. To measure HMW
adiponectin, 50 µL of conditioned medium was incubated with 100 µL protease solution
before being neutralized by 100 µL sample pre-treatment buffer. This protease
specifically digests lower-order isoforms of adiponectin, isolating the HMW isoform for
analysis. The sample was then diluted 25 times before being assayed according to the
standard protocol. To measure total adiponectin, 50 µL of conditioned medium was
added to 200 µL sample pre-treatment buffer. The sample was then diluted 75 times
before being assayed according to the standard protocol.
Western Blot Analysis
Cells were washed with cold PBS before being scraped in 1xRIPA buffer supplemented
with protease inhibitors (Pierce) and phosphatase inhibitors (PhosSTOP, Sigma). The
cell lysate was then sonicated twice in ten second bursts. Cellular debris were pelleted
by centrifugation at 12,000 rpm for 20 minutes at 4°C. The Bradford method (Biorad)
was used to determine total protein concentrations. Cell lysates containing 40 µg protein
were dissolved in SDS running buffer (Thermo Fisher Scientific) and boiled at 70°C for
10 minutes before being loaded onto a 12% Bis-Tris gel for electrophoresis. Samples
were ran in duplicate against Molecular weight standards (Precision Plus, dual color,
Bio-Rad) and MagixMark XP Western Protein Standards (Invitrogen.) The gel was then
transferred onto a nitrocellulose membrane (Biorad) before being blocked for 1 hour in
blocking buffer consisting of TBST (tris-buffered saline, pH 7.4 and 0.01% Tween-20)
supplemented with 5% BSA or non-fat dry milk. The blocked membranes were washed
and incubated with primary antibodies overnight at 4°C. Membranes were probed as
follows: CHOP (1:1000) (Cell Signaling Technologies). Following incubation with
primary antibodies, membranes were subsequently probed with the correct secondary
31
antibody: anti-rabbit secondary antibody conjugated to horseradish peroxidase (1:2000)
(Cell Signaling Technologies). α-tubulin was used as a protein loading standard. The
primary antibody was directed against stripped membranes as follows: (1:5000) (Sigma)
before probing with the secondary antibody, goat anti-mouse secondary antibody
conjugated to horseradish peroxidase (1:2000) (Cell Signaling Technologies).
Membrane-bound antibodies were detected using an electrochemiluminescent detection
reagent (Thermo Fisher Scientific).
RNA isolation and RT-qPCR
Cells were lysed in cold RNAzol RT lysis buffer (Sigma) and stored at -80°C for future
analysis. Frozen cell lysates were thawed by placing on a dry heat block set at 37°C for
5 minutes. Lysates were then centrifuged at 12,000 rpm for 5 minutes and residual
lipids were removed. RNA isolation was then carried out by the manufacturer’s protocol
and checked for purity. cDNA was synthesized using a commercial high-capacity
reverse-transcription kit (Applied Biosystems.) RT-qPCR was performed using Power
SYBR Green PCR Master Mix (Applied Biosystems) and the CFX96 Real-Time PCR
system (Bio-Rad) according to the manufacturer’s instructions. The standard curve
method was used for quantitation of results. Standard curves featured the original
sample and three 10-fold serial dilutions of the original sample. mRNA concentrations
were standardized to the housekeeping gene RPL13a. Primer sequences are shown in
Table 2-1.
Oil Red O Staining
Mature 3T3-L1 adipocytes (Day 9) were stained with Oil Red O by the following method
(Derived from biolonza.) Cells were gently washed with cold PBS before a 2%
paraformaldehyde solution in PBS was added. Cells were incubated at room
32
temperature for 90 minutes. Cells were then washed with ddH2O before 1 ml of a
working solution of Oil Red O was added for 5 minutes. Cells were then washed again
with ddH2O before observed under a phase-contrast microscope at 100x magnification.
Photos were taken with a digital camera (Canon).
Statistical Analysis
Statistics were performed using SigmaPlot 11. Data are expressed as mean ± SD or
mean ± SE. Student’s T-test was used to determine whether glutathione treatment
significantly increased intracellular glutathione concentrations. A one-way ANOVA was
used to determine differences in gene expression, protein expression, and adiponectin
secretion between treatment groups. A two-way analysis of variance (ANOVA) was
used to determine differences in intracellular glutathione concentrations during ER
stress. Time and treatment group were factors and glutathione concentrations were the
quantity being compared. For post-hoc analysis, the Holm-Sidak method was used. A p-
value of <0.05 was considered significant for these experiments.
33
Table 2-1. Primer sequences used for RT-qPCR.
Gene Forward Primer (5’ 3’) Reverse Primer (5’ 3’)
XBP1s
XBP1u
ERo-1α
RPL13A
TGAGTCCGCAGCAGGT
GCTTGGGAATGGACACGCTG
CGATATACAGTCCCCCGATG
GCAAGTTCACAGAGGTCCTCAA
TGTCAGAGTCCATGGGAAGA
GCACATAGTCTGAGTGCTGCG
ACTTTTTCCTCGCCCAGAAG
GGCATGAGGCAAACAGTCTTTA
34
CHAPTER 3 RESULTS
The aim of this study was to investigate any protective effects of bolstering
glutathione concentrations in 3T3-L1 adipocytes during ER stress. 3T3-L1 adipocytes
were grown to maturity. The presence of lipid droplet accumulation was shown using Oil
Red O staining (Figure 3-1). An aqueous solution of glutathione (1 mM) was used as
pre-treatment before treatment with palmitate (1 mM) to induce ER stress. Following
these treatments, glutathione concentrations in the cell were measured. To investigate
the effect of increasing glutathione concentrations on UPR signaling, CHOP and XBP1s
were measured by western blotting and RT-qPCR respectively. Adiponectin secretion
was measured by ELISA and gene expression of the ER chaperone, ERO-1α was
measured using RT-qPCR.
Glutathione uptake into 3T3-L1 adipocytes and effect of ER stress on glutathione concentrations
Uptake of GSH into mature 3T3-L1 adipocytes was determined by incubating
cells with GSH (1 mM) or vehicle (saline) for 20 hours. Cells were washed with PBS
before being lysed in cold MES buffer. Intracellular glutathione was measured using a
commercial colorimetric assay. Glutathione concentrations were reported as nmoles
glutathione / mg cellular protein. Total protein was determined using the Bradford
method. To determine how palmitate-induced ER stress affects intracellular glutathione
concentrations, a time course experiment was performed. Cells were pre-treated with or
without GSH as described above. Fresh media was then added containing palmitate (1
mM) or vehicle (0.75% BSA) and cells were incubated for 6, 12, or 24 hours. Cells were
lysed and assayed for glutathione concentrations as described above. 20 hours
following incubation with GSH, cells showed a significant increase in glutathione
35
concentrations (approximately 60%), implying uptake of reduced glutathione into the cell
(Figure 3-2). During ER stress, glutathione concentrations decreased after 12, and 24
hours. GSH pre-treatment did not ameliorate this effect (Figure 3-3).
Effect of glutathione pre-treatment on UPR markers in 3T3-L1 adipocytes during ER stress
CHOP protein expression was measured to assess activation of the PERK
pathway of the UPR. 3T3-L1 adipocytes were treated with 1 mM palmitate or vehicle
(0.75% BSA) for 12 hours. Additionally, the effect of 20 hours pre-treatment with 1 mM
GSH was investigated. Cells were lysed in cold 1XRIPA buffer supplemented with
protease and phosphatase inhibitors. Total protein content was determined using the
Bradford method. 3T3-L1 adipocytes treated with thapsigargin for 12 hours were used
as a positive control. Densitometric analysis was performed on protein bands. 3T3-L1
cells treated with 1 mM palmitate showed an approximate 50% increase CHOP
induction at 12 hours (Figure 3-4). Interestingly, GSH pre-treatment completely
ameliorated this effect.
Activation of the IRE-1 pathway of the UPR results in cleavage of a 28 base pair
intron from XBP1 (XBP1u), resulting in the spliced variant of the gene, XBP1s. XBP1s
and XBP1u mRNA expression was measured to assess activation of the IRE1 pathway
of the UPR. 3T3-L1 adipocytes were treated with 1 mM palmitate or vehicle (0.75%
BSA) for 12 hours. Additionally, the effect of 20 hours pre-treatment with 1 mM GSH or
vehicle was observed. Total RNA was isolated using RNAzol RT lysis buffer. 1 µg RNA
was reverse transcribed using a commercial kit. RT-qPCR was performed and results
were quantified using the standard curve method. Data were normalized to the
housekeeping gene, RPL13a. Expression of XBP1s was approximately 6 fold higher in
36
cells treated with palmitate compared to controls (Figure 3-5, A). GSH pre-treatment
had no significant effect on XBP1s mRNA expression. No significant change in XBP1u
was observed in cells treated with palmitate or pre-treated with GSH (Figure 3-5, B).
Glutathione does not attenuate the decrease in HMW adiponectin secretion caused by treatment with palmitate
A study by Mondal et al. showed human adipose stem cells secrete less HMW
adiponectin under ER stress (19). To determine if ER stress has a similar effect on
HMW adiponectin secretion in 3T3-L1 adipocytes, adiponectin was measured in
conditioned medium after 24 hours of treatment with palmitate (1 mM.) The effect of 20
hours of GSH pre-treatment (1 mM) was also investigated. Adiponectin concentrations
were quantified using ELISA and standardized to total cellular protein to obtain the units
of ng adiponectin / mg cellular protein. Total protein was determined using the Bradford
method. Total and HMW adiponectin were both quantified.
Treatment with palmitate did not significantly decrease total adiponectin secretion
from 3T3-L1 adipocytes after 24 hours Figure 3-6, A. However, a significant decrease in
HMW adiponectin was observed (~60%) (Figure 3-6, B). GSH pre-treatment did not
ameliorate this effect. Surprisingly, GSH pretreatment also resulted in a small, but
significant decrease in total adiponectin secretion relative to non-palmitate treated
controls.
Glutathione pre-treatment lowers ERo-1α gene expression
ERo-1α is one of the primary oxidoreductases found in the ER and thought to be
the rate limiting enzyme in disulfide bond formation. Here, ERo-1α mRNA expression
was measured to determine if ER stress induced by palmitate upregulates ERo-1α gene
expression in 3T3-L1 cells and whether GSH pre-treatment has influences ERo-1α gene
37
expression. 3T3-L1 adipocytes were treated with 1 mM palmitate or vehicle (0.75%
BSA) for 12 hours. Additionally, the effect of 20 hours pre-treatment with 1 mM GSH or
vehicle was observed. Total RNA was isolated using RNAzol RT lysis buffer. 1 µg RNA
was reverse transcribed using a commercial kit (Applied Biosystems.) RT-qPCR was
performed and results were quantified using the standard curve method. Data were
normalized to the housekeeping gene, RPL13a. ERo-1α mRNA expression was not
significantly affected by palmitate treatment, indicating that this gene was not
upregulated in ER stress (Figure 3-7). However, GSH pre-treatment suppressed ERo-
1α gene expression by approximately 80%.
38
Figure 3-1. Oil Red O staining of 3T3-L1 adipocytes. Photos were taken Day 9 after differentiation using a phase contrast microscope (100x magnification) and a digital camera (Canon). Oil Red O dye selectively stains lipids a red color.
39
Treatment Group
Vehicle GSH
nm
ole
s G
luta
thio
ne /
mg p
rote
in
0
2
4
6
8
10
12
14
Figure 3-2. Glutathione uptake into 3T3-L1 adipocytes. Mature 3T3-L1 adipocytes were incubated with 1 mM GSH or vehicle (saline) for 20 hours. Cells were lysed in MES buffer and deproteinated using 5% MPA. The supernatant was analyzed for total glutathione concentrations. Data represent three independent experiments and are reported as mean ± SD analyzed by the Student’s T-test (P=0.026).
*
40
Time (hours)
6 12 24
nm
ole
s G
luta
thio
ne /
mg p
rote
in
0
1
2
3
4
5
6
Vehicle
Palmitate
Palmitate-GSH
Figure 3-3. Intracellular glutathione concentrations in 3T3-L1 adipocytes during ER
stress. Mature 3T3-L1 adipocytes were incubated with 1 mM GSH or vehicle (saline) for 20 hours. The media was then removed and cells were incubated with fresh media supplemented with palmitate (1 mM) or vehicle (0.75% BSA) for 6, 12, or 24 hours. Cells were lysed in MES buffer and deproteinated using 5% MPA. The supernatant was analyzed for total glutathione concentrations. Data represent three independent experiments and are reported as mean ± SD analyzed by two-way ANOVA. Different lowercase letters denote significant differences between treatment groups (P<0.05 vs. treatment).
a
b b b
b
a a
a
a
41
Figure 3-4. GSH pre-treatment prevents induction of CHOP during palmitate-induced ER stress. 3T3-L1 adipocytes were pre-treated with 1 mM GSH or vehicle for 20 hours prior to treatment with 1 mM palmitate or vehicle for 12 hours. CHOP protein expression in cell lysates was measured by western blotting and normalized to α-tubulin. Cells treated with thapsigargin were used as a positive control. Data represent densitometric analysis of relative densities of each protein band. Results are reported as mean ± SD of three independent experiments analyzed by ANOVA (P<0.001 for all significant differences).
Different lowercase letters denote significant differences between treatment groups.
Treatment Group
Vehicle Palmitate Palmitate-GSH
CH
OP
exp
ressio
n (
arb
itra
ry u
nits)
0.0
0.5
1.0
1.5
2.0
2.5
b
a
c
CHOP
α-tubulin
Veh Palm Palm-GSH (+) Thap
~27
kDa
~50
42
Treatment Group
Vehicle Palmitate Palmitate-GSH Thapsigargin
XB
P1
u m
RN
A e
xpre
ssio
n (fo
ld in
cre
ase
)
0.0
0.5
1.0
1.5
2.0
2.5
Treatment Group
Vehicle Palmitate Palmitate-GSH Thapsigargin
XB
P1s m
RN
A e
xpre
sssio
n (
fold
incre
ase)
0
2
4
6
8
10
12
Figure 3-5. GSH pre-treatment does not decrease XBP1s mRNA expression during
palmitate induced ER stress. 3T3-L1 adipocytes were treated with 1 mM palmitate for 12 hours. The effect of 20 hours pre-treatment with GSH was also investigated. RT-qPCR was performed to assess splicing of the XBP1 transcript. Data were normalized to RPL13A and reported as a fold increase from vehicle treated cells. Results were reported as mean ± SD of three independent experiments (P <0.01) (A). XBP1u mRNA was also measured.
No significant changes were shown in cells treated with palmitate or palmitate + GSH (B). Different lowercase letters denote significant differences between treatment groups.
b
a
a,b
A B
a
a
a
43
Treatment Group
Vehicle Palmitate Palmitate-GSH
ng A
dip
onectin /
mg p
rote
in
0
200
400
600
800
1000
1200
1400
Treatment Group
Vehicle Palmitate Palmitate-GSH
ng A
dip
onectin /m
g p
rote
in
0
100
200
300
400
500
600
Figure 3-6. Adiponectin secretion, Total and HMW. 3T3-L1 adipocytes were pre-treated
with 1 mM GSH or vehicle (saline) for 20 hours prior to treatment with 1 mM palmitate or vehicle (0.75% BSA) for 24 hours. Conditioned media was collected and centrifuged for 4 minutes at 12,000 rpm to pellet cell debris. Total (A) and HMW (B) adiponectin was quantitated using ELISA and standardized to total cellular protein. Results are reported as mean ± SD of three independent experiments analyzed using ANOVA. P-values are (P=0.029) and (P<0.001) for total and HMW adiponectin respectively.
Different lowercase letters denote significant differences between treatment groups.
a
a,b
b a
b
a
b
A B
44
Treatment Group
Vehicle Palmitate Palmitate - GSH Thapsigargin
Ero
-1m
RN
A e
xpre
ssio
n (
fold
change)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Figure 3-7. GSH pre-treatment decreases ERo-1α mRNA expression. ERo-1α mRNA expression was measured using RT-qPCR. 3T3-L1 adipocytes were treated with 1 mM glutathione or vehicle (saline) before being treated with1 mM palmitate or vehicle (0.75% BSA) for 12 hours. Data were normalized to RPL13A and reported as a fold change from vehicle treated cells. Results were reported as mean ± SD of three independent experiments. (P = 0.015).
Different lowercase letters denote significant differences between treatment groups.
a
a
b
45
CHAPTER 4 DISCUSSION
Glutathione is one of the major endogenous antioxidants in the human body.
Many chronic diseases, including obesity and type-2 diabetes are associated with
oxidative stress, ER stress, glutathione depletion, and lower antioxidant capacity.
Recently, GSH supplements with higher bioavailability have been proven to increase
intracellular GSH in lymphocytes, erythrocytes, and buccal cells (44). Whether the same
effect would be seen in cells of peripheral tissues remains to be determined. The ability
of various cell types to uptake GSH from the extracellular environment would be one
important factor. Therefore, one of the aims of this study was to show that extracellular
GSH is able to influence intracellular glutathione concentrations in 3T3-L1 adipocytes. It
was also investigated whether higher concentrations of glutathione may confer
resistance to ER stress in this same cell line. Mature adipocytes were pre-treated with
or without GSH before being treated with the free fatty acid, palmitate. Palmitate is
known to induce both ER stress and oxidative stress and was used in this study to
mimic high concentrations of fatty acids that adipocytes may be exposed to during
obesity (61). In addition, ER stress has been shown to decrease HMW adiponectin
secretion; therefore, the effect of GSH on adiponectin secretion during ER stress was
also evaluated.
Dietary sources of glutathione are known to have poor bioavailability.
Furthermore, concentrations of glutathione in the extracellular environment are between
one and three orders of magnitude lower than that inside of the cell (62). This, in
combination with the hydrophilic nature of glutathione and lack of specific importers has
popularized the idea that glutathione uptake into cells is poor. Conversely, a small
46
number of studies have reported GSH uptake into cells (42, 43). These data support the
idea that 3T3-L1 adipocytes uptake GSH from the extracellular environment. Incubating
3T3-L1 adipocytes with 1 mM GSH for 20 hours resulted in an approximate 60%
increase in total glutathione concentrations. Numerous possible glutathione transporters
have been identified, many of which are non-specific (63). It is possible than non-
specific transporters such as the organic ion transporter family (OAT) may mediate GSH
uptake across the plasma membrane. The exact roles and mechanisms of these
transporters with respect to GSH are still being investigated. Alternatively, GSH may not
cross the plasma membrane intact. 3T3-L1 cells and many other cell types express
plasma membrane ƴ-glutamyl transpeptidase, which may cleave GSH, resulting in free
cysteine. Cysteine may then be taken up by the cell via amino acid transporters. An
increase in intracellular cysteine could also explain the increased glutathione
concentrations reported here.
ER stress is involved in the pathology of obesity and is associated with oxidative
stress and glutathione depletion. Glutathione is able to neutralize ROS and is thought to
contribute to proper folding of proteins by maintaining the pool of reduced PDI in the
ER. Although glutathione may contribute to proper protein folding, other studies have
questioned its importance. Tsunoda et al. showed that GSH was dispensable to many
protein folding features of the ER, but may still be important in folding large proteins with
several disulfide bonds (40). It was hypothesized that treatment with palmitate would
deplete glutathione concentrations in 3T3-L1 adipocytes and pre-treatment with GSH
would ameliorate this effect. Indeed, after 12 or 24 hours of palmitate treatment,
intracellular glutathione concentrations decreased by ~50%. However, GSH pre-
47
treatment did not ameliorate this effect. Interestingly, there was a trend towards lower
glutathione concentrations 6 hours following palmitate treatment in cells pre-treated with
GSH. The non-allosteric feedback inhibition of glutamate cysteine ligase (GCL) by
glutathione may explain this. These data are in line with previous studies showing that
saturated free fatty acids induce oxidative stress to cells by UPR signaling or other
pathways (64). These data also suggests that any protective effects of augmenting
glutathione concentrations during ER stress is likely to occur early during the onset of
stress or by a mechanism that does not rely only on free GSH acting as an antioxidant
or assisting in protein folding.
Although, pre-treating 3T3-L1 adipocytes with GSH did not prevent glutathione
depletion during palmitate-induced ER stress, GSH pre-treatment did show other
biological effects, such as significantly decreasing induction of CHOP, a downstream
target of the PERK-eIF2α-ATF4 signaling pathway associated with ER stress induced
cell death. The spliced variant of XBP1 (XBP1s) was also measured as a marker for
activation of the IRE1 pathway of the UPR and was upregulated in response to
palmitate. GSH pre-treatment did not significantly affect XBP1s mRNA expression.
XBP1s binds to ER stress response elements in its target genes and is involved in lipid
synthesis, endoplasmic reticulum associated protein degradation (ERAD) and initiation
of adipogenesis (65). Therefore, XBP1s is thought to promote cellular adaptation to ER
stress. XBP1s overexpression in murine embryonic fibroblasts conferred resistance to
ER stress and insulin resistance. Furthermore, obese XBP1+/- mice showed insulin
resistance and increased PERK phosphorylation compared to WT mice (10). Seeing
that GSH pre-treatment completely ameliorated CHOP expression, but did not affect
48
XBP1s, it is tempting to speculate that increased intracellular GSH concentrations may
modulate UPR signaling in response to ER stress, possibly conferring stress resistance
and promoting adaptation over cell death.
Adipocyte cell death has been shown to be associated with adipose tissue
inflammation and insulin resistance during obesity. It has been shown that adipocytes
undergoing cell death in obese adipose tissue show morphological features of
pyroptosis, a programmed form of cell death resulting in inflammation (16). Mice lacking
CHOP seem to be protected against pyroptotic cell death (66). Therefore, CHOP
induction may be crucial in adipocyte pyroptosis. The fact that GSH strongly prevented
CHOP induction by palmitate treatment points to a possible role of GSH in reducing
adipocyte cell death during obesity. The mechanism by which GSH prevents CHOP
induction is not known. However, CHOP is more highly induced in mice lacking
glutathione S-transferase Pi (GSTP), a protein catalyzing S-glutathionylation of proteins,
compared to WT mice during ER stress. This implies that protein S-glutathionylation of
proteins upstream of CHOP may regulate its expression. (67). Protein S-
glutathionylation has been shown to occur when cells are facing oxidative stress and
may influence protein function (67, 68). The binding of GSH to cysteine residues on
proteins occurs more readily when intracellular glutathione concentrations are high and
the redox environment is mildly oxidizing in order to facilitate disulfide bonding.
Conversely, protein de-glutathionylation is favored under reducing conditions and can
be mediated by redox proteins including thioredoxins and glutaredoxins.
It was recently reported that S-glutathionylation of key ER stress proteins can
alter UPR signaling. Bone marrow-derived dendritic cells and murine embryonic
49
fibroblasts cells lacking GSTP showed increased mRNA and protein expression of
CHOP and IRE1 in response to treatment with the pharmacological ER stress inducers
thapsigargin or tunicamycin (67). Therefore, higher glutathione concentrations may be
ER protective by increasing S-glutathionylation of UPR proteins. However, further
studies are needed to confirm that excess glutathione concentrations directly increases
S-glutathionylation of proteins. Interestingly, GSH pre-treatment did not significantly
affect XBP1s gene expression even though IRE1 expression has been shown to be
regulated by S-glutathionylation. Therefore, the PERK pathway of UPR signaling may
be more susceptible to glutathione concentrations than IRE1.
Besides acting as a reservoir for energy storage, adipocytes partake a signaling
role by secreting adipokines such as leptin and adiponectin. Adiponectin is an anti-
inflammatory adipokine with insulin sensitizing properties and is inversely correlated
with BMI. ER stress is associated with decreased adiponectin secretion. Mondal et al.
showed that murine adipose stem cells secreted less adiponectin (total and HMW)
during ER stress (19). Furthermore, this decrease was associated with a small, but
significant increase in mRNA expression of the redox active protein ERo-1α. ERo-1α
may prevent folding of adiponectin into its HMW form by interfering with thiol mediated
protein retention and causing premature secretion of adiponectin from the cell (52).
Therefore, I aimed to determine whether GSH pre-treatment may increase HMW and
total adiponectin secretion during ER stress, perhaps by antagonizing ERo-1α activity.
GSH pre-treatment did not have any effect on HMW adiponectin secretion after 24
hours of incubation with palmitate in 3T3-L1 adipocytes. Interestingly, GSH pre-
treatment slightly decreased total adiponectin secretion relative to vehicle and cells
50
treated with palmitate alone. There was no significant difference between adiponectin
concentrations in conditioned media between palmitate and vehicle treated cells at 24
hours. Also, GSH pretreatment resulted in decreased ERo-1α mRNA expression
relative to both vehicle and palmitate treated cells. Therefore, it is possible that GSH
may regulate ERo-1α at the transcriptional level. I hypothesized that GSH may act
antagonistically of ERo-1α by maintaining a pool of reduced PDI in the ER. This could
allow for proper assembly of HMW adiponectin before the protein is secreted. In this
case, I expected that GSH would increase HMW adiponectin secretion from 3T3-L1
adipocytes during ER stress. It was also hypothesized that ERo-1α would be
upregulated during ER stress, as CHOP is known to upregulate ERo-1α mRNA
expression (69). However, I did not see this occurrence, perhaps because palmitate is a
mild ER stress inducer compared to pharmacological agents such as thapsigargin. The
observed suppression of ERo-1α mRNA expression by GSH may also explain why total
adiponectin secretion was lower in GSH pre-treated cells. ERo-1α is the rate limiting
enzyme in disulfide bond formation in the ER. Therefore, decreased ERo-1α expression
could actually prevent disulfide bonding and secretion of many proteins, including
adiponectin.
This study pointed out potential roles of GSH as a signaling molecule outside of
its normal roles as an antioxidant and in assisting with protein folding. Few studies have
investigated how increasing glutathione status in the cell affects ER stress related
outcomes. Furthermore, it was shown that exogenous GSH was able to increase
intracellular GSH, perhaps by directly crossing the plasma membrane. Although, these
data show that higher initial glutathione concentrations may not prevent its depletion by
51
palmitate, protein bound glutathione was not measured and may represent an additional
pool of intracellular glutathione. GSH pretreatment ameliorated CHOP induction in 3T3-
L1 adipocytes during ER stress. Increased S-glutathionylation of proteins is a plausible
mechanism. However, future studies should determine whether S-glutathionylation is
depended on glutathione concentrations, in addition to GSTP activity. Furthermore,
exactly which UPR proteins are glutathionylated also remains to be determined. GSH
has been shown bind to transcription factors c-jun and NF-kB, inhibiting their function
(70). It is also plausible that GSH could bind to the transcription factor ATF4, preventing
it from upregulating CHOP. This study showed that GSH pre-treatment did not increase
HMW adiponectin secretion from 3T3-L1 adipocytes during ER stress. The lack of effect
of GSH on HMW adiponectin secretion suggests that redox chaperones other than Ero-
1α may be more important in maintaining secretion of HMW adiponectin during ER
stress. Indeed, disulfide bond A oxidoreductase-like protein (DsbA-L) has been shown
to localize in the ER and prevent the decrease in HMW adiponectin secretion by
thapsigargin-induced ER stress in 3T3-L1 adipocytes (71, 72). Therefore, future studies
should investigate regulation of this chaperone during ER stress. A limitation of this
study is that intracellular adiponectin concentrations were not measured. Therefore, it
cannot be known whether the decrease in HMW adiponectin secretion was due to
impaired synthesis or secretion. Finally, GSH suppressed ERo-1α mRNA expression,
which is in line with previous studies showing that ERo-1α is upregulated by CHOP
during ER stress. Therefore, ERo-1α may also be regulated by S-glutathionylation of its
upstream proteins.
52
In summary, GSH may play a role in determining cell fate during ER stress by
tilting UPR signaling towards survival, rather than cell death. This could be favorable
during obesity and MetS, as adipocyte cell death is linked to inflammation and insulin
resistance. Therefore, bolstering intracellular glutathione concentrations in the adipocyte
may help prevent these comorbidities. Future animal studies could point out whether
highly bioavailable oral glutathione supplements may be effective in increasing
adipocyte glutathione concentrations. Glutathione may also be increased by hormetic
compounds in food that target the NRF2 pathway, such as curcumin and sulphoraphane
(73).
53
Figure 4-1. Hypothetical mechanism of action explaining how increased intracellular
glutathione alters UPR signaling. Increased intracellular glutathione in the presence of oxidative stress favors S-glutathionylation of proteins containing cysteine residues. ATF4 (mouse and human) contains multiple cysteine residues which may serve as targets for S-glutathionylation, perhaps inhibiting the DNA binding activity of the protein. Downstream effects include decreased CHOP and Ero-1α expression. Decreased availability of Ero-1α may prevent release of thiol-retained proteins from the ER, including adiponectin.
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BIOGRAPHICAL SKETCH
Brandon Eudy was born in 1991 in New Bern, North Carolina. He attended North
Carolina State University from 2009-2013, where he graduated cum laude with a BS in
chemistry. During his undergraduate career, he developed an avid interest in the field of
nutrition, which motivated him to pursue his MS in the Department of Food Science and
Human Nutrition at the University of Florida. After graduating, Brandon will continue his
education by pursuing a PhD in nutritional sciences to prepare him for a career in
nutrition science research.