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Metabolic shift in lung alveolar cell mitochondria after exposure to environmental toxicants
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Metabolic shift in lung alveolar cell mitochondria after exposure to environmental toxicants
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Content
METABOLIC SHIFT IN LUNG ALVEOLAR CELL MITOCHONDRIA AFTER
EXPOSURE TO ENVIRONMENTAL TOXICANTS
by
Amit Rajendra Agarwal
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)
DECEMBER 2013
Copyright 2013 Amit Rajendra Agarwal
i
न क म र् णामन ार म् भान्न ैष् क म् य � प
ु रु षो ऽश्न
ु त े ।
न च संन् य सन ा द े व � स� द्ध ं सम� ध ग च् छ� त
“One does not attain freedom from the bondage of Karma by merely abstaining from work. No
one attains perfection by merely giving up work”- Bhagavad Gita (3:04)
ii
DEDICATION
This thesis is dedicated to my parents Rajendra and Rita Agarwal
and my mentor Madhu Mhasawde.
iii
ACKNOWLEDGMENTS
I would like to acknowledge my heartfelt appreciation to the committee
reviewing my thesis, Dr. Enrique Cadenas, Dr. Wei-Chiang Shen, and Dr.
Curtis Okamoto. Thank you for providing me with great support and help
during various stages of my doctoral research. Dr. Cadenas, I could not
have had a better mentor than you.
I would also like to thank my current and former lab members Dr. Jerome
Garcia, Dr. Li Peng Yap, Dr. Ryan Hamilton, Dr. Juliana Hwang, Dr. Fei
Yin, Dr. Chen Li and Tianyi Jiang for all their help over the years. Special
thanks to summer students, Ryan Martinez and Dolores Almeida for their
help.
I am also grateful to my parents Rajendra and Rita Agarwal; little sister
Neha; and my wife Payal for the love, encouragement and continuous
support to keep me going.
iv
TABLE OF CONTENTS
Epigraph i
Dedication ii
Acknowledgements iii
List of Tables vi
List of Figures vii
Abstract ix
Preface xi
Chapter One: General Introduction
Chronic Obstructive Pulmonary Disease (COPD)
Free radicals and electrophiles in cigarette smoke
Role of mitochondrial dysfunction in COPD
Mitochondrion driven apoptosis in COPD
The cellular redox status: maintaining cellular health
Glycolysis: Providing fuel to mitochondrion
Pulmonary surfactant: Holding the alveoli together
1
1
1
4
10
11
12
14
Chapter Two: Cigarette smoke induced alterations in cellular redox status
independent of inflammatory response
Introduction
Experimental procedures
Results
Discussion
24
24
25
33
46
Chapter Three: Cigarette smoke induced metabolic shift in lung alveolar cell
mitochondrion
Introduction
Experimental procedures
Results
Discussion
51
51
53
58
71
v
Chapter Four: Acrolein mediated shift in substrate utilization in lung alveolar
cell mitochondrion
Introduction
Experimental procedures
Results
Discussion
77
77
78
85
95
Chapter Five: Conclusions and Future directions
Conclusions
Future directions
102
102
108
References 111
vi
LIST OF TABLES
Table 1. Redox genes significantly (p<0.05) up-regulated after 8 weeks
exposure to cigarette smoke
35
Table 2. Effect of cigarette smoke exposure on pyridine nucleotide
levels in lung homogenate and mitochondrial fraction
41
Table 3. Effect of cigarette smoke exposure on GSH and GSSG levels
in lung homogenate and mitochondrial fraction
42
Table 4.Genes significantly (p<0.05) up-regulated after 8 weeks
exposure to cigarette smoke
57
Table 5. Effect of cigarette smoke exposure on mitochondrial
respiratory chain complexes activity and expression
61
Table 6. Effect of CS exposure on ATP production in alveolar type II
cells
65
Table 7. Effect of CS exposure on Spare Respiratory capacity in
alveolar type II cells in Air/4, 8 wk CS exposure
66
vii
LIST OF FIGURES
Figure 1. COPD expenditure (billions) 2
Figure 2. COPD pathology 3
Figure 3. Redox modulation of thiols 8
Figure 4. Glucose metabolism in type II alveolar cells 13
Figure 5. Pulmonary surfactant and alveolar epithelium 15
Figure 6. Composition of pulmonary surfactant 18
Figure 7. Cellular processing of pulmonary surfactant 20
Figure 8. CS exposure leads to airspace enlargement in A/J mice 32
Figure 9. Changes in gene expression after CS exposure
34
Figure 10. Changes in metabolic flux following exposure to CS 37
Figure 11. Changes in GAPDH and G6PDH activity following exposure to CS 38
Figure 12. Oxidative modification of GAPDH following CS exposure 39
Figure 13. Changes in NADPH generating enzymes in the mitochondrion 44
Figure 14. Changes in levels of lung proinflammatory mediators 45
Figure 15. Changes in metabolism, OxPhos and transport related genes expression after CS
exposure
56
Figure 16. Changes in the expression and activity of mitochondrial complexes 60
Figure 17. CS exposure alters mitochondrial oxygen consumption rates in type II alveolar
cells
63
Figure 18. Effect of CE on OCR while metabolizing different substrates 64
Figure 19. CS exposure alters mitochondrial extracellular acidification rates in type II
alveolar cells
67
Figure 20. CS exposure leads to an increase in the expression of CD36 in type II alveolar
cells
69
viii
Figure 21. CS exposure leads to an increase in the expression of CPT1 in type II alveolar
cells
70
Figure 22. CS exposure leads to decreased levels of phosphatidylcholine and an increase in
PLA
2
activity in type II alveolar cells
72
Figure 23. Acrolein-induced cytotoxicity in RLE-6TN, pAT2, and H441 cells 83
Figure 24. Acrolein-induced apoptosis/necrosis in RLE-6TN, pAT2, and H441 cells 84
Figure 25. Acrolein induced mitochondrial toxicity in RLE-6TN cells 87
Figure 26. Acrolein induced mitochondrial toxicity in pAT2 cells 88
Figure 27. Acrolein induced mitochondrial toxicity in H441 cells 89
Figure 28. Acrolein induced inhibition of glycolytic metabolism in RLE-6TN, pAT2, and
H441 cells
91
Figure 29. Acrolein induced GAPDH inhibition in RLE-6TN, pAT2 and H441 cells 92
Figure 30. Acrolein induced increase in β-oxidation in Type II alveolar cells 94
Figure 31. Effect of acrolein on phosphatidylcholine levels in type II alveolar cells 96
Figure 32. Effect of acrolein on phospholipase A
2
activity in type II alveolar cells 97
Figure 33. Effect of cigarette smoke and acrolein on enzymes of glycolysis, pentose
phosphate pathway, and surfactant (phosphatidylcholine) levels
107
ix
ABSTRACT
Cigarette smoking (CS) leads to alteration in cellular redox status, a hallmark in the
pathogenesis of chronic obstructive pulmonary disease (COPD). The current study was
undertaken to determine the role of CS in the development of mitochondrial dysfunction
due to oxidative stress as a consequence of altered redox status. Male A/J mice were
exposed to CS generated by a smoking machine for 4 or 8 weeks. A recovery group was
exposed to CS for 8 weeks and allowed to recover for 2 week. Our data indicate that
short-term cigarette smoke exposure leads to altered metabolism of glucose due to
oxidative modification of GAPDH, a central glycolytic enzyme and a concurrent increase
in the pentose phosphate pathway of glucose metabolism. On the other hand, the activity
and expression of mitochondrial respiratory chain complexes II, IV, and V were found to
increase after 8 weeks of CS exposure. Microarray analysis of gene expression in mouse
lungs after exposure to CS for 8 weeks revealed upregulation of a group of genes
involved in metabolism, electron transfer chain, oxidative phosphorylation, mitochondrial
transport and dynamics, and redox regulation. To follow up on the source of substrates
for mitochondrial respiratory chain mediated oxidative phosphorylation, we studied the
effect of CS on primary alveolar Type II (pAT2) cells isolated from mice exposed to CS.
The Type II alveolar cells showed a decrease in mitochondrial respiration while
metabolizing glucose and increased respiration on fatty acids (palmitate). This metabolic
shift was also observed in RLE-6TN and primary alveolar type II cells in response to
acrolein exposure. pAT2 cells also showed an increase in expression of FAT/CD36 and
CPT1 after CS exposure. Phosphatidylcholine levels were found to decrease after CS
x
exposure alongwith an increase in the cytosolic PLA
2
activity. These changes in
surfactant phospholipid biosynthesis and metabolism were also observed in type II
alveolar cells after acrolein exposure. Thus, palmitate present in alveolar cells for
surfactant synthesis could serve as an energy substrate in the event of altered glucose
metabolism in alveolar cells. This ability to utilize alternate substrates for energy
production was found to be specific to alveolar cells as clara (club) cells did not shown a
similar increase in palmitate metabolism after acrolein exposure.
xi
PREFACE
The overarching goal of the research project detailed in this dissertation is to gain
mechanistic insights into the development of COPD in order to identify targets which
may help in delaying the onset of the disease. I believe that the metabolic shift identified
in lung alveolar cell mitochondria in response to environmental toxicants exposure may
be the main reason for the surfactant deficiency observed in COPD patients.
In chapter 1, I introduce three components of lung physiology - mitochondrial
metabolism, surfactant biosynthesis and cells redox status. A multitude of pathways
converge on these three components for maintaining lung physiology and any alteration
in the normal functioning of these components may lead to dire consequences as
observed in COPD.
In chapter 2, I aimed at characterizing the alterations in cellular redox
microenvironment to develop a timeline for COPD development. The duration of tobacco
smoke exposure used in our study showed an adaptive response in mice and its ability to
recover most of the effects that developed during from the exposure. The Translational
Lab at the USC School of Pharmacy under the direction of Dr. Liqin Zhao helped us with
the micro-array analysis of genes altered by cigarette smoke exposure. This alteration in
redox status of the cell was also found to be independent of any inflammatory response.
The cytokine analysis was performed by colleagues at the University of Rochester, Dr.
Isaac Sundar in the laboratory of Dr. Irfan Rahman. A portion of the work presented in
this chapter was published in American Journal of Physiology – Lung Cellular and
Molecular Biology titled “Short-term cigarette smoke exposure induces reversible
xii
changes in energy metabolism and cellular redox status independent of inflammatory
responses in mouse lungs”.
In chapter 3, I studied the effects of cigarette smoke exposure on mitochondrial
metabolism and oxidative phosphorylation. The utilization of different substrates in
alveolar cells was studied using the 96 well XF Extracellular Flux Analyzer in the
laboratory of Dr. Roberta Brinton. The surfactant biosynthesis pathway is suggested to be
the source for mitochondrial substrates in alveolar type II cells isolated from mice
exposed to cigarette smoke. This metabolic shift was also found to be reversed in the
recovery group.
In chapter 4, I explored the effect of acrolein on lung alveolar cell mitochondria. A
similar metabolic shift to the one observed with cigarette smoke exposure was found in
the alveolar cells RLE-6TN and pAT2. Interestingly, H441 cells which are club cells and
are not involved in surfactant biosynthesis did not show increase in fatty acid metabolism
after acrolein exposure. The mitochondrial respiration on different substrates was studied
using the 24 well XF Extracellular Flux Analyzer in the Translational Lab at USC School
of Pharmacy. Dr. Fei Yin helped with the FACS analysis to study cytotoxicity in cells
after acrolein exposure. A portion of the work presented in this chapter was published in
American Journal of Physiology – Lung Cellular and Molecular Biology titled
“Metabolic shift in lung alveolar cell mitochondria following acrolein exposure”.
In chapter 5, I derive the final conclusions from the project and suggest some studies
which could be carried out in the future to build on this work.
1
CHAPTER ONE: GENERAL INTRODUCTION
Chronic Obstructive Pulmonary Disease (COPD) – COPD, the third leading cause of
mortality in United States (Hoyert and Xu, 2012), is characterized by an irreversible and
progressive airflow limitation along with an abnormal inflammatory response to toxicants
(GOLD definition) (Pauwels et al., 2001) (Fig. 2). 15 million US adults are estimated to
suffer from COPD (CDC, 2012) and another 24 million have evidence of impaired lung
function indicating an underdiagnosis of COPD. Oxidant/antioxidant imbalance is
believed to be one of the major causes in the development of COPD as it can be caused
from inflammation, which potentiates proteolytic damage, induces cell death, and inhibits
cell repair (Rangasamy et al., 2009; Yoshida and Tuder, 2007). COPD is also considered
to be one of the most important risk factors for lung cancer among smokers (Young et al.,
2009) and approximately, 85-90% of COPD deaths are caused by smoking.
Free radicals and electrophiles in cigarette smoke - Cigarette smoke is composed of
more than 5000 different chemicals and is a rich source of oxidants and free radicals,
including acrolein, nicotine, nitrogen-dioxide, nitric oxide, nitrosamines, and polycyclic
aromatic hydrocarbons, along with additives such as glycerin and sugars (Haussmann,
2012). CS contains about 500 ppm NO that can be oxidized to form the more reactive
NO
2
.
. ONOO
–
can also be formed under these conditions (Repine et al., 1997; Stevenson
et al., 2006). Superoxide anion (O
2
.-
) could also be generated from the water
2
Fig. 1. COPD expenditure (billions). Direct medical costs may include
preventive, diagnostic, and treatment services related to COPD.
Indirect costs relate to morbidity and mortality costs. Morbidity costs
are defined as the value of income lost from decreased productivity,
restricted activity, absenteeism, and bed days. Mortality costs are the
value of future income lost by premature death (CDC, 2012).
3
Fig. 2 .COPD pathology. COPD is the co-existence of two diseases at
the same time - chronic bronchitis and emphysema. Adapted from
Siddiqui et al; 2012. (Siddiqui and Usmani, 2012)
4
soluble components of the tar phases which could also dismutate to form hydrogen
peroxide (H
2
O
2
). The amount of reactive oxygen species (ROS) released from CS is
estimated to be ~384 nmol in research grade cigarettes to 414 nmol in commercially-
available cigarettes without filters and 288 nmol in cigarette with filters (Ou and Huang,
2006). End-product specific chemical post-translational modifications of proteins, such as
protein carbonylation, glutathionylation and tyrosine nitration, are fingerprints of specific
ROS and RNS damage caused by cigarette smoke. COPD patients have been shown to
have increased protein bond nitrotyrosine in sputum (Sugiura et al., 2004) and bronchial
mucosa (Ricciardolo et al., 2005). Nitrotyrosine formation was also shown to be reversed
by corticosteroids leading to improved airway function (Ricciardolo et al., 2005).
Role of mitochondrial dysfunction in COPD – Mitochondrial dysfunction and
oxidative stress are known to be responsible for a number of diseases. Mitochondria play
a central role in development of pathology because of its role as powerhouses of the cell
generating ATP by oxidative phosphorylation and reactive oxygen and nitrogen species.
Mitochondria are the major cellular source of oxidants – O
2
.–
was found to be the
stoichiometric precursor of mitochondrial H
2
O
2
released through ubisemiquinone
autoxidation (Boveris and Cadenas, 1975; Boveris et al., 1976; Cadenas et al.,
1977) and also by reverse electrol transfer at NADH-dehydrogenase (Turrens and
Boveris, 1980) after the initial suggestion by Chance and Boveris of the
production of H
2
O
2
by intact mitochondria (Boveris et al., 1972). O
2
.–
is released
5
physiologically from two sites in the electron transport chain; complex I and III
(Muller et al., 2004; Treberg et al., 2011).
Cu,Zn-superoxide dismutase present in
the intermembrane space can also convert the O
2
.–
released into this compartment
(Han et al., 2001) to H
2
O
2
and this production is regulated by the mitochondrial
metabolic state and intra-mitochondrial steady-state concentration of
.
NO.
Although O
2
.–
is not a strong oxidant, it could lead to formation of the more
reactive oxygen and nitrogen species and also propagation of chain reactions
(Turrens, 2003).
The oxidants released by the mitochondria have also been shown to be
beneficial for cellular adaptation and stress response. Under physiological
hypoxic conditions, H
2
O
2
is known to signal the transcription of hypoxia
inducible factors 1 (Chandel et al., 1998) which promotes the expression of
erythropoietin, vascular endothelial growth factor and glycolytic enzymes to
maintain ATP levels. Mitochondrial H
2
O
2
released as a result of starvation has
also been shown to regulate autophagy by oxidizing and inactivating cysteine
protease HsAtg4 (Scherz-Shouval et al., 2007). Reactive oxygen species
production has also been helpful in eliminating infection by macrophage
activation through the MAPK pathway (Emre et al., 2007). Oxidant-induced JNK
activation inhibits insulin signaling and up-regulates the production of pro-
inflammatory cytokines through activation of the transcriptional factors NF-κB
and AP-1 (Konner and Bruning, 2011). Mitochondrial reactive oxygen species
have also been suggested to be involved in the early stages of T-cell activation
(Chaudhri et al., 1986) but this has not been shown in vivo. Thus low levels of
6
reactive oxygen species could play a role in the cellular adaptation of stress and
under physiological conditions could be important regulators of homeostasis
(Sena and Chandel, 2012).
Another feature common in patients with COPD is the skeletal muscle
dysfunction and the decrease in endurance and muscle strength associated with it
(Kim et al., 2008; Mador and Bozkanat, 2001; Meyer et al., 2013; Rabinovich and
Vilaro, 2010). This effect has also been attributed to the defects in the electron
transport chain in skeletal muscle mitochondria (Puente-Maestu et al., 2009;
Puente-Maestu et al., 2012).
Regulation of mitochondrial functions by nitric oxide – Cytochrome oxidase
(COX, complex IV) catalyzes the oxidation of cytochrome c and the reduction of
O
2
to water while pumping protons out of the matrix. Nitric oxide competes with
O
2
for the binuclear center CuB/a3 of COX leading to its competitive inhibition
(Antunes et al., 2004; Brown and Cooper, 1994). Energy demands are known to
drive respiration but the kinetic control of ATP production is dependent on the
availability of ADP to F
1
-ATPase and O
2
and
.
NO to COX (Boveris et al., 1999).
Thus, NO
.
is able to create a metabolic hypoxic state where O
2
cannot be
adequately used for ATP production (Moncada and Erusalimsky, 2002). NO
.
formed during inflammation and neurodegenerative diseases would be able to
inhibit mitochondrial respiration and also increase the formation of O
2
.–
and
ONOO
–
(Poderoso et al., 1996). Nitrative stress and the increase in NO
.
in
exhaled air have also been shown to affect the small airway function in patients
with COPD (Brindicci et al., 2005; Hirano et al., 2013; Schafroth Torok and
7
Leuppi, 2007). NO
.
in cigarette smoke is also known to contribute to nicotine
addiction (Comhair et al., 2005) by increasing the nicotine absorption. NO
.
is also
released endogenously in the brain from nicotine (Krukoff, 1999), which helps
reduce symptoms of stress and increase synaptic dopamine levels. This is
supported by the attenuation of symptoms of nicotine abstinence by NOS
inhibitors (Malin et al., 1998).
Chemical modification of redox sensitive mitochondrial proteins: A
number of mitochondrial proteins such as Mn-SOD (Comhair et al., 2005),
NADH dehydrogenase complex (Brown and Borutaite, 2004)
and aconitase (Han
et al., 2005)
have been shown to be susceptible to chemical modification due to
oxidative and nitrosative stress. The three mechanisms by which such a
modification may occur are shown in Fig. 3. Complex I activity can be inhibited
due to post-translation modifications such as nitration by ONOO
-
and S-
glutathionylation (Murray et al., 2003; Taylor et al., 2003). Aconitase activity has
been shown to be inhibited due to S-glutathionylation under oxidative and
nitrosative stress (Han et al., 2005). Although no specific modification of
mitochondrial proteins have been observed in COPD, the components of cigarette
smoke have been shown to affect the activities of various complexes of the
mitochondrial respiratory chain. The aldehyde adducts and nitrotyrosine formed
from cigarette smoke is known to post-translationally modify the activity of
histone deacetylases, leading to the ineffectiveness of corticosteroids and increase
in the release of pro-inflammatory cytokines in macrophages (Yang et al., 2006).
8
Fig. 3. Redox modulation of thiols. The GSH/GSSG redox couple can
regulate protein function through the reversible formation of mixed
disulfides that can occur mainly through three mechanisms. (i) thiol
disulfide exchange between protein sulfhydryl groups and GSSG, (ii)
GSH reduction of protein sulfenic acids, and (iii) the nucleophilic
attack of thiolate anion on the S-NO bond of GSNO.
9
Cigarette smoke is also known to cause mitochondrial fragmentation
affecting the ability of mitochondria to respond to oxidative stress through
fission/fusion (Hara et al., 2013; Hoffmann et al., 2013). The high reactivity of
cigarette smoke components and the presence of a high number of proteins that
are susceptible to chemical protein post-translational modifications in
mitochondria suggest that mitochondria may play a central role in the
development of COPD pathology.
The impairment of mitochondrial protein function lead to an increase in
the production of reactive oxygen and nitrogen species (over physiological
levels), along with a decrease in energy production and a collapse of the
mitochondrial membrane potential, further leading to an initiation of the
commitment phase of mitochondrion-driven apoptosis.
Cell death or survival is determined largely by mitochondria, where a number of
signaling and transcriptional pathways converge. This highlights the central role of
mitochondria in the development of disease pathology like COPD by executing cell death
and maintenance of cellular homeostasis by propagating oxidants and also activating
cellular antioxidant defenses to neutralize ROS. Damage to the mitochondrial proteins
that remains unrepaired may lead to decline in cellular energy, increased oxidant
production and irreversible cell dysfunction and death, even after smoking cessation.
10
Mitochondrion-driven apoptosis in COPD – The redox sensitive sulphydryl groups in
adenine nucleotide translocase (ANT) may play a role as sensors of oxidative stress by
modulating into a non-specific structural pore and becoming a part of the mitochondrial
permeability transition pore, PTP (Halestrap, 2000; Halestrap et al., 2000). This leads to
the opening of the PTP and uncoupling of mitochondrial respiration. The mitochondrial
swelling and rupture of the outer mitochondrial membrane releases cytochrome c and
activates the apoptotic pathways (Cadenas, 2004). A number of studies have suggested
the role of apoptosis in COPD pathogenesis due to the presentation of endothelial and
epithelial apoptotic cells in the lung of patients with COPD (Demedts et al., 2006;
Henson et al., 2006). In-utero exposure to tobacco smoke leads to activation of
mitochondrial caspase-9 and down regulation of the antiapoptotic proteins Bcl-2 and Bcl-
x
L
, further leading to regulation of cytochrome c release from non-descript pores in
mitochondria, as shown in the lungs of infant monkeys (Zhong et al., 2006).
The pretreatment of lung epithelial cells with staurosporin (an apoptosis inducer
without any effect on mitochondrial respiratory chain) leads to a shift from apoptosis to
necrosis, in response to cigarette smoke exposure (van der Toorn et al., 2007). This shift
has also been observed in bronchial epithelial cells (Slebos et al., 2006). It may be
surmised that apoptosis in COPD is mitochondrion-driven and lung cells have a “switch”
point, which can be defined by the Nernst potential, characterized a complete collapse of
mitochondrial membrane potential, and cellular ATP levels and cell death by necrosis.
11
The cellular redox status: maintaining cellular health - Glutathione (GSH) is a small
hydrophilic molecule that is synthesized in the cytosol from glycine, glutamate, and
cysteine in a two-step process by the enzymes γ-glutamylcysteine synthetase and GSH
synthase (Griffith, 1999). Lung alveolar epithelium is one of the last defenses of the body
to protect against inhaled environmental toxicants from reaching systemic circulation.
Thus the epithelial lining fluid requires higher levels of glutathione to protect against
oxidative damage from inhaled pollutants (Cantin et al., 1987b; Rahman, 1999).
Indeed, as the most abundant non-protein thiol (Han et al., 2003), GSH plays a
central role in maintaining cellular redox health. This GSH however, cannot be
transported into the cells as a tripeptide but requires the activity of γ-
glutamyltranspeptidase to release constitutive amino acids (Griffith et al., 1979b). These
amino acids can then be transported intracellularly and reassembled to form the
functional tripeptide (Seelig et al., 1984a). The oxidation product of GSH, GSSG, is
reduced back to GSH by glutathione reductase, a NADPH-dependent enzyme
ubiquitously distributed throughout the cell and tissues. Under non-oxidative or
nitrosative stress conditions, the concentration of GSSG is negligible at 1/100
th
of the
total GSH pool (Schafer and Buettner, 2001a). The other major small protein containing a
redox active disulfide in the active site is thioredoxin. The thioredoxin system consists of
NADPH, thioredoxin (Trx) and thioredoxin reductase (TrxR), with TrxR involved in
reducing the oxidized (Trx S-S) disulfide to a reduced Trx (SH)2 dithiol (Holmgren,
1985). The system is of major importance in cancer because of its involvement in the
synthesis of deoxyribonucleotides for DNA synthesis and antioxidant defense through
repair of oxidatively damaged proteins. The thioredoxin system has 10-fold higher K
m
12
than glutaredoxins, in functioning as electron donor in the deoxribonucleotide synthesis
mostly up-regulated in tumors (Rahman et al., 2006).
Oxidative stress has also been implicated to be a key component in a range of
disorders such as cancer, atherosclerosis, arthritis, Alzheimer’s disease and diabetes
(Klatt and Lamas, 2000b). The redox sensitive sulfhydryl groups in the vast majority of
protein cysteine residues can be regulated by the GSH/GSSG redox couple through the
reversible formation of mixed disulfides (Schafer and Buettner, 2001a). This reversible
formation of protein mixed disulfides has been suggested to protect the critical sulfhydryl
residues from irreversible damage due to oxidative stress (Klatt and Lamas, 2000b). A
number of glycolytic proteins such as GAPDH, triosephosphate isomerase and aldolase
also have been suggested to be down regulated by glutathionylation in hepatocytes in
response to oxidative and nitrosative stress (Fratelli et al., 2003; Ito et al., 2003).
Glycolysis: Providing fuel to mitochondrion – Lungs are mainly dependent on glucose
as the principal substrate for energy generation (Fischer, 1984). GAPDH, a redox
sensitive central glycolytic enzyme with cysteine in its active site has a number of diverse
physiological functions in the cell. It is involved in DNA base-excision repair (Meyer-
Siegler et al., 1991), as a membrane fusion facilitator (Glaser and Gross, 1995), defective
RNA export to nucleus (Singh and Green, 1993), and in initiating cell death (Hara et al.,
2005; Ishitani and Chuang, 1996; Sawa et al., 1997). S-thiolation of GAPDH has also
been shown to reroute the metabolic flux to the cytosolic pentose phosphate pathway
13
(PPP) (Ralser et al., 2007; Shenton and Grant, 2003), responsible for 10-20% of the
glucose metabolism.
Fig. 4. Glucose metabolism in type II alveolar cells. Glucose is the
principal substrate utilized by the lungs for ATP production. The rate
limiting step in the pentose phosphate pathway helps in the production
for NADPH and maintaining the reducing environment in the cell.
The surfactant biosynthetic pathway also originates from the
glycolysis pathway.
14
The regulatory step catalyzed by glucose-6-phosphate dehydrogenase (G6PDH) functions
to produce NADPH (Fig. 4), which is utilized to maintain the cellular redox status in the
reduced form by regenerating GSH and thioredoxin and in the process protecting the cell
from oxidative damage. This reducing power may also be utilized in detoxification
reactions and biosynthesis of fatty acids to reduce the double bonds of the intermediates
(Fischer, 1984). NADPH could also be released by the activity of NADP
+
-dependent
isocitrate dehydrogenase, malic enzyme, and alcohol dehydrogenase. NADP
+
-dependent
isocitrate dehydrogenase – 2 (IDH2) and nicotinamide nucleotide transhydrogenase
(NNT) are the only mitochondrial sources for NADPH. Oxidative metabolism in lungs
maintains ATP content at levels comparable to other metabolically active organs and any
alteration may influence the lungs response to oxidative stress (Fischer, 1984).
Pulmonary surfactant: Holding the alveoli together - The major energy-dependent
processes in lungs are phagocytosis by alveolar macrophages, synthesis of dipalmitoyl
lecithin, bronchoconstriction and secretion (Fischer, 1984). 60% of the lung alveolus is
composed of Type II cells and are responsible for the surfactant synthesis and secretion
(Crapo et al., 1983; Fischer, 1976) (Fig. 5). Type I cells are involved in gaseous exchange
and form the alveolar wall. The pulmonary surfactant is composed of 90% lipids, 10%
protein, and trace amounts of carbohydrates. A complete breakdown of surfactant
components is shown in Fig. 6. Dipalmitoyl phosphatidyl choline constitutes half of the
lipid content and is an important constituent in reducing surface tension at the air-liquid
interface (Schurch S, 1992; Yu and Possmayer, 2003). A number of studies have
15
Fig. 5. Pulmonary surfactant and alveolar epithelium. The synthesis of
pulmonary surfactant takes place in the type II alveolar cells and is
then secreted on the surface to form a thin layer. The synthesized
surfactant is stored in lamellar bodies and the surfactant layer is also
known to contain macrophages to protect against inhaled pathogens.
Adapted from Piknova et al (Piknova et al., 2002).
16
suggested that smoking leads to an alteration in the levels of surfactant components
leading to a number of diseases like cystic fibrosis (Meyer et al., 2000), asthma (Cheng et
al., 2001; Enhorning et al., 2000; Hohlfeld et al., 1999), allergic alveolitis (Jouanel et al.,
1981), and COPD (Kaup et al., 1990). Pulmonary surfactant contains four major types of
surfactant proteins SP-A, SP-B, SP-C and SP-D (Scott, 2004). SP-A and SP-D are known
to have carbohydrate binding domains to bind with pathogens such as viruses, bacteria,
fungi, thus promoting phagocytosis by macrophages (Mason et al., 1998). The SP-B and
SP-C are hydrophobic in nature and help in maintaining a surface active film over the
alveolus and thus assisting in the normal biophysical function of the lung (Timothy E,
1998). Thus the surfactant layer not only prevents the collapse of the alveolar sacs during
exhalation but also forms a protective barrier against inhaled pathogens.
Surfactant biosynthesis – The biosynthetic pathway for surfactant phospholipid
synthesis originates from the glycolytic pathway (Fig. 4) and thus, glucose plays a
crucial role in providing the glycerol backbone for the biosynthesis of
phosphatidylcholine (Rooney, 1979; Rooney, 1984). This process takes place
exclusively in the type II alveolar cells and is regulated by enzymatic (Batenburg
and Haagsman, 1998) and hormonal control (Rooney, 1984; Rooney et al., 1994).
A number of hormones such as thyroid hormone, estrogen, glucocorticoids and
prolactin have been shown to increase the biosynthesis of surfactant lipid (Akella
and Deshpande, 2013). The synthesis of surfactant is also highly dependent on
the availability of fatty acids which are taken up into the alveolar cells from
17
circulation through fatty acid binding proteins like CD36 (Guthmann et al., 1999;
Ibrahimi and Abumrad, 2002).
Phosphatidic acid (PA) is one of the key intermediates and important
precursor for the synthesis of surfactant phospholipid (Wykle et al., 1977). The de
novo synthesis of phosphatidic acid can take place through two different
pathways; the glycerol-3 phosphate (G3P) pathway and the dihydroxyacetone
phosphate (DHAP) pathway. G3P, formed from DHAP by utilizing NADH by the
enzyme G3P dehydrogenase is converted to lysophosphatidic acid (lyso-PA)
through acylation by G3P acyltransferase. 60 % of the lyso-PA, however, is
formed by the DHAP pathway by generating acyl-DHAP using DHAP-
acyltransferase which is further reduced by acyl-DHAP reductase in an NADPH-
dependent reaction (Mason, 1978). Lyso-PA formed by these two pathways can
be converted to Phosphatidic acid by the endoplasmic reticulum enzyme 1-
acylglycerol-3-phosphate acyltransferase. The PA biosynthesis is known to take
place in the mitochondria, ER, or the peroxisomes depending on the destiny of the
synthesized phospholipids or the need for the cell. It has also been suggested that
the synthesis of membrane phospholipids takes place in the ER and the secretory
lipids are synthesized in the mitochondria or peroxisomes (Athenstaedt and
Daum, 1999).
Phosphatidylcholine (PC) which forms 80% of the surfactant phospholipid
is formed from Di-acyl glycerol (DAG) and CDP Choline (Agassandian and
Mallampalli, 2013). CDP Choline is synthesized in a two-step process after the
phosphorylation of choline; obtained primarily from diet, and converting it to
18
Fig. 6. Composition of pulmonary surfactant. 90% of the pulmonary
surfactant is composed of phospholipids and 10% proteins. Adapted
from Agassandian et al (Agassandian and Mallampalli, 2013) .
19
Choline Phosphate by the activity of choline kinase. Choline phosphate is then
converted to CDP-choline by the rate limiting step in the biosynthesis of PC,
catalyzed by CTP: phosphocholine cytidylyltransferase utilizing CTP (Kent,
1997). PA converted to DAG by phosphatidate phosphatase serves as an
important intermediate in the biosynthesis of PC because of its role in signaling
pathways such as the PI3K/Akt pathway.
The formation of PC from DAG and CDP choline takes place by the
activity of choline phosphotransferase which is located primarily in the ER (Li
and Vance, 2008). The di-palmitoyl phosphatidylcholine (DPPC) is the major
surface active component in the pulmonary surfactant. The DPPC synthesis from
the above explained pathway will depend on the fatty acyl species added during
the synthesis of PA. The presence of unsaturated fatty acyl species in newly
synthesized PC would require a remodeling step catalyzed by phospholipase A
2
(PLA
2
) and is also considered to be the predominant source of DPPC (den
Breejen et al., 1989).
Surfactant proteins form only 5% of the pulmonary surfactant composition
but are involved in important functions including providing protection against
inhaled pathogens (Mason et al., 1998) and maintaining a surface active film at
the air liquid interface (Timothy E, 1998). SP-A is involved in the maintenance of
surfactant integrity by inhibition the activity of sPLA
2
during lung injury (Chabot
et al., 2003). SP-A is highly conserved and is also involved in homeostatic control
of surfactant secretion by providing negative feedback (Wright and Dobbs, 1991).
SP-B deficiency is lethal in humans and along with SP-C is responsible for the
20
Fig. 7. Cellular processing of pulmonary surfactant. The surfactant
synthesized in the ER is stored in the lamellar bodies (LB) and
released at the cell surface to form a surface active film by exocytosis.
The components of the surfactant can be reutilized after uptake.
Adapted from Miyoshi, 2001 (Miyoshi, 2001) .
21
maintenance of surfactant film (Beers et al., 2000). SP-D, a member of collectin
family along with SP-A, is composed of 4 homotrimeric units and is also involved
in the providing innate immunity to the alveolar epithelium by promoting
phagocytosis and stimulating cytokine production by immune cells (Wright,
1997).
DPPC combines with minute quantities of phosphatidylglycerol,
phosphatidylserine and phosphatidylethanolamine along with surfactant proteins
A, B, C and D to form the secretable pulmonary surfactant. The surfactant once
synthesized can be stored in alveolar cell specific organelles called lamellar
bodies which are lysosome like organelles and released at the cell surface by
exocytosis (Weaver et al., 2002).
Regulation of surfactant secretion – The trafficking of the pulmonary surfactant
from the lamellar bodies to the apical surface and its release through exocytosis is
still not completely understood (Fig. 7). The evidence for golgi apparatus
dependent (Chevalier and Collet, 1972) and independent trafficking of pulmonary
surfactant (Osanai et al., 2001) has further complicated the matters. The
exocytosis of the lamellar bodies’ contents on the cell surface to form a thin film
is known to be facilitated by ABC transporters (Kaltenborn et al., 2012). A
number of physiological stimuli are known to increase the surfactant secretion
such as labor (Enhorning et al., 1985; Hallman et al., 1985), hyperventilation
(Decramer et al., 2005; Gross and Smith, 1981), mechanical stretch (Leberl et al.,
2013), as well as paracrine stimulation by ATP release from alveolar type I cells
22
(Rice et al., 1987). Three distinct signaling mechanisms are said to be involved in
the regulation of surfactant secretion; activation of cAMP-dependent protein
kinase through the activation of adenylate cyclase and cAMP, protein kinase C
activation and the activation of Ca
2
+
-calmodulin-dependent protein (Rooney,
2001). The reorganization of cytoskeleton in type II alveolar cells by annexin
proteins binding to lamellar bodies may also be involved in regulation of
surfactant secretion. Annexin 2 (Singh et al., 2004), annexin 5 (Sohma et al.,
2001), and annexin 7 (Singh et al., 2004) are the annexin proteins involved in this
reorganization and this notion is also supported by the colocalization of annexin 7
with ABCA3 (Gerelsaikhan et al., 2011; Gerelsaikhan et al., 2012) and fusion
protein attachment receptor, SNAP23 (Sohma et al., 2001).
Surfactant phospholipid metabolism – The pulmonary surfactant released
at the cell surface needs to be undergo a regulated metabolic cycle to clear the
invading pathogens and replenish the air-liquid interface with fresh surfactant
(Wright, 1997). This released surfactant can then either be recycled by type II
alveolar cells or degraded by the alveolar macrophages (Hallman et al., 1981).
The recycling of pulmonary surfactant was found to be the major pathway for
surfactant clearance in newborn rabbits (Jacobs et al., 1982) which shifted to
increased degradation in adult rabbits (Jacobs et al., 1985; Jacobs et al., 1988).
This increase in the amount of recycled surfactant PC is also observed in pigs
(Martini et al., 1999). The reuptake of phospholipids and SP-A is thought to be
via clathrin-coated endocytosis (Wissel et al., 2001), but inhibition of clathrin
23
mediated endocytosis have also found to have no effect on ABCA3 mediated
uptake of SP-A and transferrin (Bates et al., 2000).
24
CHAPTER TWO: CIGARETTE SMOKE INDUCED ALTERATIONS IN CELLULAR
REDOX STATUS INDEPENDENT OF INFLAMMATORY RESPONSE
INTRODUCTION
Glutathione (GSH) is the most abundant, low molecular weight, non-protein thiol
that is synthesized in the cytosol from glycine, glutamate, and cysteine in a two-step
process by the enzymes γ-glutamylcysteine synthetase and GSH synthase (Griffith and
Mulcahy, 1999) and plays a central role in the maintenance of the cellular redox status
(Meister, 1994). Over 98% of the glutathione is considered to be in the reduced form
under non-oxidative and nitrosative stress conditions (Schafer and Buettner, 2001b). The
normal alveolar epithelial lining fluid in the lungs is known to contain even higher levels
of GSH than in plasma, in order to protect from oxidative damage from inhaled pollutants
and inflammatory cells (Cantin et al., 1987a; Rahman, 1999). However, GSH cannot be
taken up into the cells but requires degradation to its constitutive amino acids by γ-
glutamyl transpeptidase (Griffith et al., 1979a) and their further intracellular reassembly
by γ-glutamylcysteine synthetase (γ-GCS/GCL) and glutathione synthetase (Seelig et al.,
1984b). GSH thus synthesized can be freely transported to the extracellular compartment.
It may be expected that alterations in intracellular GSH levels control extracellular
GSH/GSSG ratios.
CS-induced lung inflammatory processes are likely to be preceded by changes in
energy metabolism and the redox status of the cell. A number of studies have focused on
the effect of CS on glutathione homeostasis in alveolar cells (Rahman et al., 1995a;
Rahman et al., 1995b; Rahman and MacNee, 1999) or development of inflammation
25
(Edirisinghe et al., 2008; Moodie et al., 2004; Sarir et al., 2009), but none has highlighted
the importance of cytosolic metabolic events preceding redox and inflammatory changes.
Thus, this study was aimed at characterizing the redox changes and identifying metabolic
modulators in initial stages of exposure to cigarette smoke; the experimental model
consisted of mice exposed to cigarette smoke (CS) under controlled conditions for 4- and
8 weeks followed by a 2-week recovery period.
EXPERIMENTAL PROCEDURES
Exposure of mice to CS – Male A/J mice (Jackson Laboratories) 8 weeks of age (n
= 108) were exposed to CS for 4 weeks (n = 20), 8 weeks (n = 20) or 8 weeks + 2 weeks
recovery (n = 20). The control group (n = 48) was kept in a filtered air environment,
whereas the remaining mice were exposed whole body to
cigarette smoke generated by
Kentucky 3R4F reference cigarettes (Tobacco
Research Institute, University of Kentucky,
Lexington, KY),
which has the content of 11 mg of total particulate matter
and 0.73 mg of
nicotine each. Before use, the cigarettes were
kept for 48 h in a standardized atmosphere
humidified with a
mixture of 70% glycerol and 30% water. The smoke concentration and
the duration of exposure were gradually increased in the first week in order to acclimatize
the mice to tobacco smoke. A mixture of sidestream smoke (89%) and mainstream smoke
(11%),
mimicking an exposure to ETS, was produced by using a smoking
machine
(model TE-10; Teague Enterprises, Davis, CA), where
each smoldering cigarette was
puffed for 2 s, once every min
for a total of 9 puffs. The machine was adjusted
to burn 6
cigarettes at one time, 6 h a day and 5 days a week (Monday to Friday) for a period of
26
either 4 weeks or 8 weeks. Recovery group was exposed to CS for 8 weeks and then
allowed to recover for 2 weeks without smoke exposure. Sidestream
and mainstream
smokes were aspirated in a mixing chamber before
distribution to two exposure
chambers. Under those conditions,
the total particulate matter in the exposure chambers
was 80-90 mg/m
3
(Sundar et al., 2010; Yao et al., 2010). The position
of the cages in the
exposure chambers was rotated daily. All animal protocols were approved by the
Department of Animal Resources at the University of Southern California.
Exposure to CS led to a 30-35% decrease in body weight that returned to normal
levels during 2 weeks recovery period after CS exposure. Food intake was observed to
decrease with smoking, but water intake was normal.
Lung morphometry – The lungs of mice sacrificed after pentobarbital overdose
were inflated using 1% low-melting agarose and then fixed in 10% buffered formalin
overnight. The lungs were then paraffin-embedded to cut 5 µm sections and the slides
were stained with hematoxylin and eosin (H&E). The images were acquired using a Zeiss
Axioskop microscope 5x lens. The mean linear intercept was determined using computer-
assisted morphometry with ImageJ software.
Isolation of lung mitochondria –Lung mitochondria were isolated by differential
centrifugation. The mice were sacrificed and the lung excised and rinsed in 2 ml of
mitochondrial isolation buffer (MIB) (sucrose 250 mM, HEPES 20 mM, EDTA 1 mM,
EGTA 1 mM, Protease inhibitor 0.25%, BSA 0.5%, pH 7.4) and then homogenized in 10
ml of MIB. The homogenate was centrifuged at 3110 rpm for 5 min at 4°C to remove
27
tissue debris and nuclei and yield a supernatant enriched in mitochondria. The pellet was
re suspended in 10 ml and the above process repeated until no visible pellet was seen.
The supernatant collected was then centrifuged at 12,500 rpm for 10 min at 4°C to isolate
mitochondria. To remove any cytosolic contaminants, the mitochondrial pellet was re-
suspended in MIB and again pelleted by centrifugation at 9000 rpm for 8 min at 4°C
(Velsor et al., 2006).
TLDA gene expression profiling – Taqman low-density array (TLDA) cards were
custom manufactured at Applied Biosystems (Foster City, CA), and loaded with Taqman
expression assays for target genes divided into four functional groups and 4 assays for
candidate control genes. Total RNA was isolated from mouse lung tissues using the
PureLink RNA Mini Kit (Invitrogen, Carlsbad, CA). RNA quantity and quality were
analyzed using the Experion RNA StdSens Analysis Kit on an Experion Automated
Electrophoresis System (Bio-Rad, Hercules, CA). The integrity of RNA samples was
assessed by the Relative Quality Indicator. RNA to cDNA synthesis was prepared using
the High Capacity RNA-to-cDNA Master Mix (Applied Biosystems) on a MyCycler
Thermal Cycler (Bio-Rad). Taqman real-time qRT-PCR reactions were performed on 100
ng cDNA samples mixed with the TaqMan Universal PCR Master Mix 2X (Applied
Biosystems), under the thermal cycling conditions: stage 1: AmpErase UNG activation at
50 °C / 2 min / 100% ramp; stage 2: AmpliTaq gold DNA polymerase activation at 94.5
°C / 10 min / 100% ramp; stage 3: melt at 97 °C / 30 s / 50% ramp, followed by
anneal/extend at 59.7 °C / 1 min / 100% ramp, for 40 cycles. Fluorescence was detected
28
on an ABI 7900HT Fast Real-Time PCR System equipped with the Sequence Detection
System Software Version 2.4 (Applied Biosystems).
Data were analyzed using the RQ Manager Version 1.2 and DataAssist Version 3.0
(Applied Biosystems). Relative gene expression levels or fold changes relative to the
comparison group (control) were calculated by the 2
– ∆∆Ct
method, with Ct denoting
threshold cycle. Selection of the endogenous control gene for normalization was based on
the control stability measure (M), which indicates the expression stability of control
genes on the basis of non-normalized expression levels. M was calculated using the
geNorm algorithm; genes with the lowest M values have the most stable expression. Four
samples (collected from 4 animals) per group were included in the analysis. For each
sample, ∆Ct was calculated as the difference in Ct of the target gene and the endogenous
control gene. For each treatment group, mean 2
– ∆Ct
was calculated as the mean of 2
– ∆Ct
of
the 4 samples in the group. Fold change (2
– ∆ ∆Ct
) was then calculated as mean 2
– ∆Ct (treatment
group)
/ mean 2
– ∆Ct (comparison group)
. Fold change values greater than one indicate a positive
expression or up-regulation relative to the comparison group. Fold change values less
than one indicate a negative expression or down-regulation relative to the comparison
group. Fold regulation was used to represent the fold change results in a biologically
meaningful way. For fold change values greater than one (up-regulation), the fold
regulation is equal to the fold change; for fold change values less than one (down-
regulation), the fold regulation is the negative inverse of the fold change Hierarchical
clustering diagrams graphically displayed clusters of treatment groups as well as target
29
genes. Distances between treatment groups / target genes, shown in maximum linkage,
were calculated by clustering algorithm Pearson’s correlations based on the ∆Ct values.
Quantification of pyridine nucleotides in lung homogenate and mitochondria –
HPLC analyses of NAD
+
, NADH, NADP
+
, NADPH were carried out as previously
described (Klaidman et al., 1995) with modifications. An HPLC ZORBAX C
18
analytical
column (5 µM, 4.5 × 250 mm) and a guard column (Agilent Technologies, Santa Clara,
CA) were used for analysis in an Agilent 1100 series HPLC system and a Hitachi
Fluorescence Spectrophotometer. All other chemicals were obtained from Sigma. An
HPLC gradient time program was used with 100% ammonium acetate (0.2 M, pH 5.5)
and 0% HPLC grade methanol as initial condition followed by increasing concentration
of methanol to 6.8% over time. Pure NADH, NAD
+
,
NADPH, and NADP
+
at a
concentration of 1 mg/ml were used for standardization of the method. The pyridine
nucleotides were quantified using the fluorescence spectrophotometer at excitation 330
nm and emission 460 nm. This was repeated several times to generate a standard curve.
Cell homogenates and mitochondrial fractions were stored in 200 µl of 0.2 M KCN/well
prior to chloroform extraction that was carried out at 14000 rpm in a microcentrifuge at
4°C. The mitochondrial fraction was energized with glutamate/malate (5 mM) before
adding the KCN solution. The aqueous supernatant was collected, containing the soluble
pyridine nucleotides and extracted thrice to remove the lipids and proteins. The aqueous
solution was then filtered through a 0.45 µm positively charged filter (Pall Life Sciences,
Port Washington, NY) to remove the DNA and RNA. On diluting the filtrate with 0.2 M
ammonium acetate it was injected on a HPLC C
18
column.
30
Quantification of thiols in lung homogenate and mitochondria – GSH and GSSG
were detected using HPLC with a coulometric electrochemical detector from ESA
(Chelmsford, MA) as described previously (Yap et al., 2010). ESA offers CoulArray
systems that utilize between 4 and 16 channels. A 4-channel electrochemical array was
employed for the simultaneous detection of GSH and GSSG. The retention times for
GSH and GSSG were at ~6.5 and ~28.5 min, respectively. GSH was monitored and
detected at 500 mV and 700 mV; whereas GSSG was detected at 900 mV. Cell
homogenates and mitochondrial fractions were stored in 200 µl of 5% meta-phosphoric
acid containing 25 mM ammonium sulfamate for precipitation of proteins. Before
measurement of thiols, mitochondria were energized with glutamate/malate (5 mM). The
samples were then stored at –70°C until analysis.
Cytokine analysis – The level of proinflammatory mediators, such as neutrophilic
chemokine keratinocyte chemoattractant (KC) and macrophage-monocyte chemotactic
protein (MCP-1), in whole lung homogenates were measured by enzyme-linked
immunoabsorbent assay (ELISA) using respective dual-antibody kits (R&D Systems,
Minneapolis, MN) according to the manufacturer’s instructions. The results were
expressed in samples as pg/mg protein (Rajendrasozhan et al., 2010; Sundar et al., 2010).
Western blot analyses – Lung mitochondrion were lysed in RIPA buffer
containing Tris-HCl (50 mM), NP-40 (1%), sodium deoxycholate (0.25%), NaCl (150
mM), EDTA (1 mM), 5% protease inhibitor and 5% phosphatase inhibitor cocktail, pH
31
7.4. Samples were denatured in reducing sample buffer or non-reducing sample buffer
(Pierce, Rockford, IL) and separated on 12 -15% SDS-PAGE gels. Proteins were
transferred onto PVDF membrane using the criterion system (Bio-Rad, Hercules,
CA) and then probed in 1:1000 concentration against antibodies for G6PDH, GAPDH
(Abcam Inc, Cambridge, MA), NNT (Mitosciences, Eugene, OR) or IDH-2 (Proteintech
Group, Inc., Chicago, IL). Chemidoc XRS+ imaging system (Bio-Rad, Hercules, CA)
was used to obtain western blot images after developing using a chemiluminesence kit
(Pierce, Rockford, IL).
Immunoprecipitation – The glutathionylated proteins were pulled down using a
coated plate immunoprecipitation kit (Thermo Scientific, Rockford, IL) and then
separated in a 10% SDS-PAGE gel. After transfer on a PVDF membrane the immunoblot
was probed using anti-GAPDH (Abcam, Cambridge, MA) and anti-GSH (Virogen,
Watertown, MA) antibodies. DTT (25 mM) was added to the samples as a negative
control.
Enzyme activity assays – GAPDH activity in the cytosolic fraction was measured
according to established methods (Ghezzi et al., 2002) with some modifications: cytosol
was lysed in RIPA buffer with the addition of protease inhibitors and freeze-thawed 3
times. The protein concentration was measured using the Bradford assay. GAPDH
activity was detected by monitoring the increase in NADH formation
spectrophotometrically at 340 nm in the presence of 250 μM NAD
+
and 50 mg/ml
glyceraldehyde-3-phosphate and 100 µg of cytosolic protein. Glucose-6-phosphate
32
Control 4
8 Recovery
0
30
60
Control 4 8 Recovery
Mean Linear Intercept
(µM)
CS Exposure
(weeks)
** **
A
B
Fig. 8. CS exposure leads to airspace enlargement in A/J mice. (A) H&E stained images
of lung sections from Air/CS exposed mice obtained using Zeiss Axioskop microscope
at 5X magnification. The mean linear intercept (MLI) determined using ImageJ (B) as
described in the Experimental Procedures section. **p < 0.01 compared to control was
found, as evaluated using t-test. ANOVA statistical analysis was also performed and p <
0.001 was found.
33
dehydrogenase (G6PDH) activity in the cytosolic fraction was measured as previously
described (Garcia-Nogales et al., 1999) with some modifications: cytosol was lysed in
RIPA buffer with the addition of protease inhibitors and freeze-thawed three times. 100
µg of protein was mixed with the reaction mixture containing 100 µM NADP, 3.4 mM
glucose 6-phosphate, and reaction buffer to a final volume of 1 ml. The absorbance was
monitored at 340 nm for 300 s. Enzyme activity was calculated from the slopes and
expressed in nmol/min/mg protein with an extinction coefficient of 6.22.
Statistical analyses – Two sample t-test assuming unequal variances was
performed to determine statistical significance along with ANOVA statistical analysis.
Statistics are indicated in the legends of appropriate figures and tables.
RESULTS
CS exposure leads to air space enlargement in A/J mice – H&E staining of lungs sections
from Air/CS exposed mice revealed an increase in the mean linear intercept (MLI) by 37
(from 38.3 to 52.6 µM) and 43% (55.07 µM) after 4 and 8 wk of CS exposure,
respectively. This increase in air space enlargement was found to be reducing by 15%
(46.8 µM) in the recovery group indicating the ability of lungs to repair the alveolar
damage (Fig. 8 A,B).
34
Fig. 9. Changes in gene expression after CS exposure. TLDA gene
expression profiling and data analyses were carried out as described in
the Materials and Methods section. Shown in this figure are genes
involved in redox homeostasis. Green indicates genes with minimum
expression and red with maximum expression. The mean 2
–ΔCt
values
for each target gene were statistically compared between the treatment
and comparison group using Student’s t-test. Data shown represents
three control mice (Cnt1, 2 and 4, four mice exposed to CS for 8
weeks (Smo1, 2, 3 and 4) and four recovered mice (Rec 1, 2 and 3).
Gene symbols are indicated.
35
Table 1. Redox genes significantly (p<0.05) upregulated after 8 weeks
exposure to cigarette smoke. *Genes that did not recover after
cigarette smoke exposure
36
Effect of CS on gene expression profile – Changes in the expression of a focused group of
genes involved in redox regulation in mouse lung tissues after 8 weeks smoke exposure
was comparatively analyzed using qRT-PCR-based Taqman low-density arrays. 4 genes
involved in cytosolic redox regulation (Sod1, Gpx1, Gpx4, Gsr) were found to be
significantly (p < 0.05) up regulated after 8 wk of CS exposure. Prdx 5 was the only gene
involved in redox regulation which did not recover back in the recovery group (Fig. 9,
Table 1).
Effect of CS on enzymes of glycolysis and pentose phosphate pathway – GAPDH
expression (Fig. 10A,B) and activity (Fig. 11) were decreased after CS exposure; the
down-regulation of GAPDH implies decrease in the rate of glycolysis and is expected to
redirect glucose metabolism to the pentose phosphate pathway (Ralser et al., 2007;
Tristan et al., 2011); accordingly, G6PDH expression (Fig. 10A,B) and activity (Fig. 11)
were steadily increased due to exposure to CS. Two weeks recovery after CS exposure
resulted in an increased of GAPDH expression and activity (higher than in control levels)
and a progressive decrease of G6PDH towards control levels (Fig. 10 and 11).
Oxidative modification of GAPDH on CS exposure – The down-regulation of GAPDH
activity was further probed for reversible oxidative modification following CS exposure.
The presence of two redox-sensitive cysteines’ (Cys
149
and Cys
153
) in the catalytic site of
GAPDH (Mohr et al., 1999) along with the high levels of electrophiles in CS renders the
enzyme susceptible to S-glutathionylation following CS exposure. This was confirmed by
37
Fig. 10. Changes in metabolic flux following exposure to CS. (A)
Expression of G6PDH and GAPDH was observed using western blot
analyses in cytosolic fraction of mice lungs exposed to CS after 4
weeks, 8 weeks and 8 weeks smoke exposure with 2 week recovery.
(B) The blots were quantified using UN-SCAN-IT gel 6.1 as
described in the Materials and Methods section **p < 0.01, #p < 0.05,
was found as evaluated using t-test.
38
Fig. 11. Changes in GAPDH and G6PDH activity following
exposure to CS. G6PDH enzyme activity was measured by
monitoring the formation of NADPH at 340 nm as described in
Materials and Methods section.; GAPDH enzyme activity was
measured by monitoring the consumption of NAD at 340 nm as
described in the Materials and Methods section. *p < 0.05, **p <
0.01, ***p < 0.001 compared to control; ##p < 0.01, ###p <
0.001 compared to 8 weeks smoke exposed; and †††p < 0.001
compared to 4 weeks smoke exposed was found as evaluated
using t-test.
39
Fig. 12. Oxidative modification of GAPDH following CS
exposure. Immunoprecipitation of cytosolic glutathionylated
proteins was performed as described in the Materials and
Methods section and then probed with anti-GAPDH antibody.
40
pulling-down the glutathionylated proteins from lung cytosol and immunoblotting with
anti-GAPDH antibody (Fig. 12).
Effect of CS on NADPH/NADP
+
, NADH/ NAD
+
, and GSH/GSSG ratios in cell
homogenates – Changes in GAPDH (anaerobic glycolysis) and G6PDH (pentose
phosphate pathway) activities resulted in changes in pyridine nucleotides levels, for
NAD
+
and NADP
+
are cofactors for the GAPDH- and G6PDH-catalyzed reactions,
respectively. Hence, in agreement with increases in G6PDH activity, NADPH levels
increased with exposure to CS and start to decrease after 8 weeks of CS exposure (Table
2). NADPH/NADP
+
values, however, decreased after 4 weeks of CS exposure due to an
increase in the NADP
+
levels.
The increased NADPH/NADP
+
values in the recovery
group, suggest a more reducing environment. NADH/NAD
+
values were also found to
decrease with CS exposure due to increasing levels of NAD
+
, probably due to a
decreased utilization by GAPDH. NAD
+
levels also decreased in the recovery group with
increased GAPDH activity (Table 2). NADPH is the ultimate electron donor for the thiol-
based systems that control the cellular redox state: consequently, GSH/GSSG values were
found to follow the same trend where the ratio increases after 4 weeks of CS exposure
and starts decreasing after 8 weeks of CS exposure (Table 3).
Effect of CS exposure on mitochondrial pyridine nucleotides and thiols – In
mitochondria, a steady increase in NADPH/NADP
+
values was observed upon CS
exposure, but these values dropped to normal levels immediately after removal of CS-
induced stress (Table 2). The increase in the levels of NADPH was mainly responsible
41
_______________________________________________________________________________________________________________________
Control
____
Weeks of Exposure
____
Recovery ANOVA
4 8
Homogenate
NADPH 0.12 ± 0.02 0.18 ± 0.04 0.12± 0.03 0.09 ± 0.02 p < 0.01
NADP
+
0.12 ± 0.06 0.21 ± 0.02 0.10 ± 0.03
††
0.06 ± 0.01 p < 0.01
NADPH/NADP
+
1.11 ± 0.03 0.87 ± 0.14 1.19 ± 0.03 1.68 ± 0.06 NS
NADH 0.05 ± 0.02 0.05 ± 0.01 0.08 ± 0.02 0.04 ± 0.01
#
p < 0.05
NAD
+
0.14 ± 0.01 0.34 ± 0.17 0.21 ± 0.05
†
0.14 ± 0.03 p < 0.05
NADH/NAD
+
0.37 ± 0.15 0.17 ± 0.06 0.37 ± 0.09 0.31 ± 0.12 NS
__________________________________________________________________________________________________________________
Mitochondrial fraction
NADPH 1.08 ± 0.37 2.10 ± 0.41
**
2.73 ± 0.77
**
0.92 ± 0.23
##
p < 0.001
NADP
+
1.04 ± 0.29 1.07 ± 0.42 0.92 ± 0.29 1.50 ± 0.50 NS
NADPH/NADP
+
1.11 ± 0.47 2.07 ± 0.37
**
3.04 ± 0.59
***,†
0.68 ± 0.34
###
p < 0.001
NADH 0.81 ± 0.30 1.84 ± 0.37
**
1.83 ± 0.37
***
0.82 ± 0.18
##
p < 0.001
NAD
+
1.16 ± 0.48 1.01 ± 0.31 1.31 ± 0.45 1.29 ± 0.31 NS
NADH/NAD
+
0.76 ± 0.33 1.86 ± 0.23
***
1.48 ± 0.39
*
0.65 ± 0.13
#
p < 0.001
_______________________________________________________________________________________________________________________
Table 2. Effect of cigarette smoke exposure on pyridine nucleotide
levels in lung homogenate and mitochondrial fraction. NS Not
significant. *p < 0.05, **p < 0.01, ***p< 0.001 compared to control;
#p < 0.05, ##p < 0.01, ###p < 0.001 com-pared to 8 weeks smoke
exposed; and †p < 0.05, ††p < 0.01 compared to 4 weeks smoke
exposed as evaluated using t-test. ANOVA statistical analysis was also
performed and shown in the right-most column. The values are
expressed in nmol/mg of protein.
42
_______________________________________________________________________________________________________________________
____
Weeks of Exposure
____
Control 4 8 Recovery ANOVA
Homogenate
GSH 7.66 ± 0.86 3.77 ± 0.60
**
5.52 ± 1.74 5.58 ± 0.83
*
p < 0.05
GSSG 0.85 ± 0.10 0.31 ± 0.07
**
0.60 ± 0.34 1.10 ± 0.23 p < 0.05
GSH/GSSG 9.03 ± 0.46 12.20 ± 0.69
**
10.26 ± 3.78 5.16 ± 1.07
*
p < 0.05
__________________________________________________________________________________________________________________
Mitochondrial fraction
GSH 0.37 ± 0.08 0.63 ± 0.05
*
0.38 ± 0.05
††
0.47 ± 0.22 NS
GSSG 0.18 ± 0.04 0.18 ± 0.07 0.06 ± 0.03
*
0.11 ± 0.02 NS
GSH/GSSG 2.10 ± 0.27 3.70 ± 1.24 6.53 ± 2.39 4.11 ± 1.98 p < 0.05
_______________________________________________________________________________________________________________________
Table 3. Effect of cigarette smoke exposure on GSH and GSSG levels
in lung homogenate and mitochondrial fraction. NS Not significant.
*p < 0.05, **p < 0.01, compared to control and ††p < 0.01 compared
to 4 weeks smoke exposed as evaluated using t-test. ANOVA
statistical analysis was also performed and shown in the right-most
column. The values are expressed in nmol/mg protein.
43
for the increase in this ratio, thus suggesting an expanded mitochondrial capacity to
handle a redox stress; this may play an important role under more prolonged exposure to
CS. The NADH/NAD
+
ratio also showed a similar increase with exposure to CS and
decreased in the recovery period (Table 2). Mitochondrial sources of NADPH entail the
activities of IDH2 and NNT, the expression of which was found to increase upon CS
exposure (Fig. 13A,B). Thus, these two enzymes contribute to the highly reduced
environment in the mitochondrion due to CS exposure. Mitochondrial GSH/GSSG values
also showed a steady increase after 4 and 8 weeks of CS exposure, but unlike the
NADPH/NADP
+
values, they do not decrease in the recovery group (Table 3). This may
be an adaptive response by the mitochondrion to protect the redox-sensitive protein thiols
of the mitochondrial respiratory chain from the oxidative stress for a longer period of
time.
Effect of CS on the levels of proinflammatory mediators in mouse lung – The CS-induced
redox changes and associated alteration in mitochondrial energetics may precede the
inflammatory responses in the lung. This notion was addressed by assessing the lung
proinflammatory response in 4 and 8 weeks CS exposed group and the recovery groups.
The levels of proinflammatory mediators, such as KC (neutrophil) and MCP-1
(monocyte-macrophage) were measured using the whole lung homogenates. KC levels
were not significantly altered in control and CS exposed 4 weeks, 8 weeks and recovery
mice groups (Fig. 14A). The levels of MCP-1 remained unaltered in 4 weeks CS exposed
mice compared to control group. After 8 weeks CS exposure and in the recovery mice
groups, the levels of MCP-1 was significantly reduced in the lung (p < 0.01) (Fig. 14B).
44
Fig. 13. Changes in NADPH generating enzymes in the
mitochondrion. (A) Western blot analyses of NADPH-
generating enzymes were performed in mitochondria as
described in the Material and Methods section. (B) Blot
quantification; *p < 0.05, **p < 0.01, ***p < 0.001 compared to
control was found as evaluated using t-test.
45
Fig. 14. Changes in levels of lung proinflammatory mediators
The levels of proinflammatory mediators KC (A) and MCP-1
(B) were measured by ELISA in whole lung homogenates of
control and CS-exposed mice after 4 weeks, 8 weeks, and 8
weeks followed by 2 weeks recovery. Data are mean ± SEM.
**p < 0.01, significant compared to control group.
46
The overall data on the levels of proinflammatory mediators reflect on lack of
macrophage and neutrophil recruitment into the lung as confirmed by KC and MCP-1
data after short-term CS exposure (4 weeks, 8 weeks, and recovery) in male A/J mice.
Thus the alterations in redox and mitochondrial energetics seen above are independent of
proinflammatory responses after sub-chronic CS exposure in mouse lungs.
DISCUSSION
The increase in MLI after CS exposure indicates destruction of alveoli and an
enlargement of air spaces which are indications for an emphysematous lung. This
increase was however, reversible after smoking cessation and the alveolar repair was
almost complete after 2 weeks of smoking withdrawal (Fig. 8 A,B). CS exposure is
known to contribute to pulmonary cell senescence which may impair the ability of lungs
to repair the alveolar damage and this may require chronic exposure (Miro et al., 1999;
Scholz and Rhoades, 1971). A number of groups have established a 6 month period of
chronic exposure in mice using this model of smoke exposure system to induce disease
pathology comparable to human disease (Anbarasi et al., 2005).
CS exposure for short periods (4 and 8 weeks) lead to changes in glucose
metabolism, entailing an inhibition of the key glycolytic enzyme, GADPH, due to
glutathionylation (Fig. 12) and re-routing glucose metabolism to the pentose phosphate
pathway, with increased expression and activity of G6PDH (Fig 10 and 11). This
metabolic change is accompanied by a redox response, encompassed by changes in the
cellular and mitochondrial levels of pyridine nucleotides and GSH. Of note, mitochondria
47
appear to maintain a more reducing environment in response to CS exposure, as revealed
by increasing NADPH levels and NADPH/NADP
+
values (Table 2) and the expression of
NADPH-generating enzymes (IDH2 and NNT) (Fig. 13). NNT has been recently shown
to be a critical determinant of the cellular redox status (Freeman et al., 2006; Pedersen et
al., 2008) and maintenance of the mitochondrial energy–redox axis (Yin et al., 2012).
Accordingly, the energy- and redox changes are integrated in a co-dependent manner. For
example, inactivation of GAPDH due to oxidative stress has been shown to reroute the
metabolic flux to the pentose phosphate pathway in C. elegans (Ralser et al., 2007).
Likewise, activation of G6PDH promoted survival by balancing the energy metabolism
between the pentose phosphate pathway and glycolytic pathways in neurons (Bolanos
and Almeida, 2010; Bolanos et al., 2008).
The redox-sensitive cysteinyl groups in the vast majority of proteins can be
regulated by the GSH/GSSG redox couple through the formation of mixed disulfides
(Schafer and Buettner, 2001b), which has been suggested to protect critical sulfhydryl
moieties from irreversible damage due to oxidative stress (Klatt and Lamas, 2000a). The
decrease in activity of GAPDH without much change in the expression, suggested a post-
translational modification that contributed to altered metabolism of glucose. This notion
was confirmed by the reversible inactivation of GAPDH by S-glutathionylation (Fig. 12),
as shown previously by a number of studies (Ralser et al., 2007; Tristan et al., 2011)
under oxidative stress conditions. The presence of a large number of electrophiles in
cigarette smoke along with two redox-sensitive cysteines (Cys
149
and Cys
153
) in the
catalytic site of GAPDH (Mohr et al., 1999) renders it more susceptible to oxidative
48
modifications. It is also one of the several instances in which the gene expression profile
does not correlate with the protein expression (Ralser et al., 2009).
A number of redox related genes were found to be significantly upregulated after
8 weeks of CS exposure, which include Gpx1, Gpx3, Gpx4, Sod1, Sod2, Prdx5, and Gsr
(Fig. 8 and Table 1). The increased expression of Sod 1 and Sod 2 indicate the existent
oxidative stress after 8 weeks of CS exposure. The glutathione peroxidases (Gpx1, Gpx3,
and Gpx4), peroxiredoxin 5, and glutathione reductase are all dependent on NADPH as
the ultimate electron donor. Gpx 3 is the extracellular isoenzyme form of glutathione
peroxidases and along with Prdx 5 did not recover back after 2 weeks of CS exposure
termination. Gpx4 encodes for the protein specifically involved in the catalytic reduction
of lipid peroxides and is present in cytosol and in the mitochondrial inter-membrane
space. Peroxiredoxin 6 and glutaredoxin 1, which are specifically expressed in lungs and
are actively involved in protecting lung from CS exposure (Chung et al., 2010; Sundar et
al., 2010), were unfortunately not included in the low-density gene array. Most of the
gene changes induced by CS exposure were reversed in the recovery group indicating
that the point of no return had not yet been reached and longer duration (6 months) of CS
exposure may be required to cause irreversible damage.
Earlier reports have shown CS-mediated increase in release of pro-inflammatory
mediators due to recruitment of inflammatory cells, such as the macrophages and
neutrophils into the lung after acute CS exposure (Rajendrasozhan et al., 2010;
Rangasamy et al., 2004b; Sundar et al., 2010; Yao et al., 2008). In the above mentioned
studies, mice were exposed to ~300 mg/m
3
TPM mainstream CS for 3 days (acute CS
exposure) and they showed significant increase in neutrophil influx into the lung which
49
was further supported by increased pro-inflammatory mediators KC and MCP-1 levels in
CS exposed WT mice compared to air group control (Fig. 14). Hwang et al also showed
no significant difference in lung histopathology by hemotoxylin and eosin staining
between air- or CS-exposed WT mouse lungs after 8 weeks of CS exposure, which was
assessed by determining the Lm. These mice did not show either any goblet cell
metaplasia as determined by Periodic acid-Schiff (PAS) staining (Hwang et al., 2011). In
the present study, no significant changes were seen in the levels of KC in the lungs of
mice exposed to CS for 4 weeks and 8 weeks compared to controls (Fig. 14). The reasons
for this disparity is not unknown, but it may be due to different concentration of CS
exposures, side-stream versus main-stream CS exposures, and time of sacrifice of
animals (e.g., previous studies sacrificed animals 24 h post-last exposures (see below),
whereas the current study sacrificed animals immediately after CS exposure. Our data
shows that the MCP-1 levels remain similar to control until 4 weeks CS exposure and
later the levels of MCP-1 were significant decrease after 8 weeks CS and in the recovery
group. This suggests that macrophage response in the lung was not recovered implicating
the presence of macrophages in bronchoalveolar lavage fluid.
It is likely that the A/J mice exposed to CS from Teague cigarette smoke machine
(model TE-10; Teague Enterprises, Davis, CA) causes either very less or no neutrophil
influx into the lung. Furthermore, A/J mice were exposed to a lower dose of CS (~80-90
TPM; mixture of side-stream and main-stream CS) that might have caused considerably
lower cellular infiltration into the lung, which is reflected in the lower levels of
cytokine/chemokine production. Earlier report by Yao et al. also suggests that CS-
mediated inflammatory and oxidative response is strain-dependent in mice, C57BL/6J
50
mice being highly susceptible as compared to A/J, AKR/J, and CD-1 mice that were
moderately susceptible strains; and the 129Svj strain were resistant to acute CS exposure
(Yao et al., 2008). The highly resistant C57BL/6J mice also required 6 months of CS
exposure to observe a significant increase in airspace enlargement (Yao et al., 2010; Yao
et al., 2012).
The experimental model in this study entails an acute response of A/J mice to CS
exposure (4 and 8 weeks) and cannot be equated to that for COPD, which requires 6
months of CS exposure to observe ~23% increase in alveolar diameter (Ou and Huang,
2006). However, the energy- and redox changes described in the short-term exposure in
this study set the platform for the development of pathology, essentially inflammatory
responses found under long-term exposures. This also indicates that most of the redox
changes observed in short-term exposure can be reversed upon cessation of smoking.
It may be surmised that CS-mediated alterations in redox regulation in mouse
lung are early events that are independent of pro-inflammatory effects of CS. This study
provided the evidence that the initial redox changes and lung responses observed due to
the acute CS exposure are independent of lung inflammatory response. The highly
reducing environment in the homogenate after 4 weeks of smoke exposure indicated a
cellular response to counteract oxidative stress by up-regulating the pentose phosphate
shunt and, thereby, the availability of NADPH to support removal of oxidizing species;
the increase in the mitochondrial energy-transducing capacity suggested a compensatory
response to decreased glycolysis and supply of substrate to mitochondria. These findings
have implications for the understanding the pathogenesis of COPD and early onset of CS-
induced alterations in energy- and redox metabolism.
51
CHAPTER THREE: CIGARETTE SMOKE INDUCED METABOLIC SHIFT IN
LUNG ALVEOLAR CELL MITOCHODNRIA
INTRODUCTION
Glucose is an important substrate utilized by lung alveolar type II cells, not only
for generation of energy but also for biosynthesis of pulmonary surfactant (Fischer,
1984). The biosynthesis and secretion of pulmonary surfactant is an important function of
type II alveolar cells, helping in reducing the surface tension and airway resistance at the
air-liquid interface and promoting efficient gas exchange (Rooney, 1984). Alterations in
the processing or secretion of pulmonary surfactant have been shown to cause adult
respiratory distress syndromes (Sakata et al., 2003; Yoval-Sanchez and Rodriguez-
Zavala, 2012), chronic obstructive pulmonary disease (COPD) (Sarkar and Hayes, 2009),
pulmonary edema (Fukuda et al., 1999), and infectious diseases (cystic fibrosis (Pison et
al., 1990), pneumonia (Pison et al., 1989)). It also forms a protective barrier over the
alveolar epithelium to prevent infection caused by inhaled particles and micro-organisms
(Lusuardi et al., 1992). The alveolar type II cells which are considered stem cells in an
adult lung (Nakos et al., 1997) are also actively involved in alveolar repair in response to
injury (Wright, 1997); they cover 60% of the alveolar epithelium (Crapo et al., 1983) and
differentiate in to type I cells following acute injury (Evans et al., 1975).
Cigarette smoke contains more than 5000 chemicals (Haussmann, 2012) some of
which include acrolein, nicotine, nitric oxide, nitrogen-dioxide, polycyclic aromatic
hydrocarbons, nitrosamines, etc. along with additives such glycerin and sugars (Stevens
and Maier, 2008). Exposure to CS leads to an oxidant/antioxidant imbalance in the lungs
52
which forms the basis for the development of COPD (Rahman and Adcock, 2006).
However, as shown in chapter 2, cytosolic metabolic events precede any redox or
inflammatory changes in the lungs flowing CS exposure. A number of glycolytic
enzymes are sensitive to CS-induced oxidative stress and any alterations in their
functional activity may affect the ATP production. CS and its constituents have also been
shown to inhibit mitochondrial respiratory complexes affecting mitochondrial energy
production and contributing to cell death (Barkauskas et al., 2013). Acrolein, one of the
major constituents of CS has been shown to inhibit Complexes I and II in hepatocytes
(Sun et al., 2006) and brain mitochondria (Picklo and Montine, 2001). Nicotine is a
competitive inhibitor for Complex I (Castranova et al., 1988) and nitric oxide is known to
react with superoxide anion in mitochondria to yield peroxynitrile which could
irreversibly modify mitochondrial proteins (Ghafourifar and Cadenas, 2005). The
susceptibility of mitochondrial proteins to oxidative damage and subsequent dysfunction
after exposure to free radicals from CS is well-documented (Haussmann, 2012;
Hoffmann et al., 2001; van der Toorn et al., 2007). The ability of lung mitochondria to
utilize palmitate under conditions of starvation indicates its adaptability to utilize
alternate substrates for energy production (Cormier et al., 2001; Rhoades, 1975). Thus it
could be expected that CS exposure could induce a similar shift in substrate utilization to
meet energy demands. We tested this notion by exposing mice to CS under controlled
conditions for 4 and 8 wk and isolating type II alveolar cells to measure mitochondrial
respiration on different substrates.
53
EXPERIMENTAL PROCEDURES
Isolation of primary alveolar type II cells – Primary alveolar type II cells were
isolated from mice exposed to CS/filtered air using dispase (BD Biosciences, Bedford,
MA) digestion – agar instillation method: mice were sacrificed after pentobarbital
overdose and the abdominal cavity was opened. The renal artery was severed to allow
blood to flow through. The tissue around the trachea was cleared and a suture was placed
below the trachea. A small incision was made at the top of the trachea to allow a needle
to pass through. The needle was held in place by tying a knot with the suture thread. 20
ml of PBS was injected through the left ventricle to perfuse the lungs and then 0.5 ml of 1
% low-melting agarose (Sigma, St. Louis, MO) was injected into the lungs through the
trachea. 5 ml of dispase was then injected into the lungs for digestion and the lungs were
excised and incubated in another 3 ml of dispase for 45 min. The lungs were then
chopped with fine-tipped forceps in wash medium containing a 1:1 mixture of Dulbecco's
modified Eagle's medium and Ham's F- 12 (DME/F-12; Sigma) containing 0.01 %
DNAase, 1 mM L-glutamine, 100 U/ml sodium penicillin G, and 100 μg/ml
streptomycin. The resulting cell mixture was then passed through cell strainers of pore
size 100, 40, 20, 15 and 10 µM. The cells were then incubated with biotinylated anti-
macrophage antibodies (anti-CD45, anti-CD45.1, anti-CD45.2, anti-Ter 119, and anti-
CD16/32; BD Biosciences) for 30 min to separate the other cell types. The purified cells
were then seeded on plates coated with Laminin-I (Trevigen, Gaithersburg, MD) in
medium containing DMEM/F-12, 1 mM L-glutamine, 0.25 % bovine serum albumin (BD
Biosciences), 10 mM HEPES, 0.1 mM nonessential amino acids, 0.05% insulin-
transferrin-sodium selenite (Roche, Basel, Switzerland), and 100 μg/ml Primocin
54
(Invitrogen, Carlsbad, CA) supplemented with 10% newborn bovine serum (Omega
Scientific, Tarzana, CA). The contaminating fibroblasts were removed by changing to
serumless medium after 3 days of seeding.
XF extracellular metabolic flux analysis – Mitochondrial respiration was measured
using the XF Extracelllular Fux Analyzer from Seahorse Biosciences (North Billerica,
MA) according to manufacturer’s protocol. The type II cells (20,000/well) were seeded
directly into XF96 plates after isolation from the CS/Air exposed mice and the
mitochondrial respiration was measured in KHB buffered medium, pH 7.4 supplemented
with 2.5 mM glucose and 0.5 mM carnitine to facilitate the uptake of palmitate-BSA. The
substrates (glucose (25mM), pyruvate (2 mM), palmitate-BSA (200µM), BSA) were
added through the first port to obtain basal respiration. Oligomycin (4µM), FCCP (1µM)
and rotenone (1µM) were added one by one to measure ATP production, maximal
respiration rate and non-mitochondrial respiration, respectively. All values were
normalized to control well by measuring the protein concentration using Bradford assay
after the metabolic flux analysis.
Enzymatic activities - The activities for Complex I, II, and IV were determined using
microplate assay kits (Abcam Inc, Cambridge, MA) in which the respective complexes
were immunocaptured in the microplate wells and the enzyme activity determined by
following the change in absorbance at 450 nm, 600 nm, and 550 nm, respectively. ATP
synthetase (Complex V) activity was assayed as previously described (Pullman et al.,
1960) with minor modifications, in a mixture containing 50 mM Hepes, pH 8.0, 5 mM
55
MgSO
4
, 250 mM sucrose, 0.5 M ATP, 0.5 M sodium phosphoenolpyruvate, 0.4 µg/µl
antimycin A dissolved in ethanol, and 0.7 mM NADH. The reaction was initiated by the
addition of 10 µg of lung mitochondria in each well. All the chemicals were purchased
from Sigma Aldrich (St. Louis, MO) and the activities are expressed in µmol/min/mg
protein.
Western blot analyses – Western blot analysis was performed as described earlier
using total oxphos rodent NNT (Mitosciences, Eugene, OR)
Immunofluorescence – Immunofluorescence experiments’ were performed on cells
fixed with 3.7% formaldehyde at RT for 10 min followed by permeabilization with 90%
methanol for 5 min. The cells were then blocked using 3% FBS for 30 min followed by
incubation with anti-CD36 (1:50; Novus Biologicals, Littleton, CO) or anti CPT1a (1:50;
Abcam, Cambridge,MA) antibody for 1 h. The FITC-conjugated secondary antibodies
(1:500) were then incubated along with DAPI (1:1000) for 1 h at RT in dark. The cells
were the washed with PBS three times and the images were procured using a BD
Pathway 435 High-Content Bioimager (BD Biosciences, Bedford, MA).
Total phosphatidylcholine assay – The levels of phosphatidylcholine were measured
in total cell lysates after centrifuging the lysate at 14,000 g to remove cell membrane
contamination using kits from Abnova (Taipei, Taiwan). The lysis buffer used contained
50 mM Tris·HCl, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA,
56
Fig. 15. Changes in metabolism, OxPhos and transport related genes
expression after CS exposure. TLDA gene expression profiling and
data analyses were carried out as described in the Materials and
Methods section. Green indicates genes with minimum expression and
red with maximum expression. The mean 2
–ΔCt
values for each target
gene were statistically compared between the treatment and
comparison group using Student’s t-test. Data shown represents three
control mice (Cnt1, 2 and 4, four mice exposed to CS for 8 weeks
(Smo1, 2, 3 and 4) and four recovered mice (Rec 1, 2 and 3). Gene
symbols are indicated.
57
Table 4. Genes significantly (p<0.05) upregulated after 8 weeks
exposure to cigarette smoke. *Genes that did not recover after
cigarette smoke exposure
58
10% protease inhibitor, and 10% phosphatase inhibitor cocktail, pH 7.4. The OxiRed
probe generated after phosphatidylcholine hydrolysis and its subsequent oxidation was
measured calorimetrically at 570 nm.
Phospholipase A
2
(PLA
2
) activity assay - PLA
2
activity was measured in cell lysates after
CS exposure using kits available from Cayman Chemicals (Ann Arbor, MI) according to
manufacturer’s protocol. Cell lysates were centrifuged at 14,000 g to prevent
contamination from cell membrane and the supernatant was used for analysis. The lysis
buffer used contained 50 mM Tris.HCl, 1% NP-40, 0.25% sodium deoxycholate, 150
mM NaCl, 1 mM EDTA, 10% protease inhibitor, and 10% phosphatase inhibitor cocktail,
pH 7.4. The free thiol released after hydrolysis of arachidonoyl thio-PC at the sn-2
position by PLA
2
was detected at 414 nm using DTNB.
Statistical analyses – Students t-test assuming unequal variances was performed as
indicated in the figure legends. ANOVA statistical analysis was also performed as
indicated in the figure legends. Results are mean ± SD from a minimum of 3
experiments.
RESULTS
Effect of CS on gene expression profile – The effect of CS exposure on a group of
genes involved in metabolism, electron transfer chain, and oxidative phosphorylation,
mitochondrial transport and dynamics were comparatively analyzed using qRT-PCR-
59
based Taqman low-density arrays as described before. 27 genes were significantly up-
regulated (Table 4) by smoke exposure (p < 0.05), including 3 genes involved in
mitochondrial redox regulation (Sod2, Prdx5, Glrx2) as well as 11 genes involved in
metabolism (Hk1, Gapdh, Idh1, Sdhc, Pdk3, Pla2g4a, Lrp8, Hsd17b10, Acad1, Acadm,
Decr1), and the gene encoding the assembly factor for the F
1
component of mitochondrial
ATP synthase (Atpaf2). The upregulation of mitochondrial transport-related genes
(Vdac3, Tomm40, Tomm20, Slc25a20, Ucp2, Cpt1a, Abca1) along with mitochondrial
fusion genes (Opa1 and Mfn1) suggests increased mitochondrial dynamics after CS
exposure (Fig. 15). However, the ratio of nuclear protein (globin) DNA to mitochondrial
protein (Cox II) –an index of mitochondrial biogenesis– showed no significant difference
after CS exposure (data not shown). Four other genes including Tomm40, Pla2g4a, Gpx3,
and Sdhc were found to not recover in the recovery group.
Effect of CS on mitochondrial electron-transport complexes – Exposure to CS elicited
an increase in the expression of Complexes II, III, IV, and V (ATPase) (Fig. 16; Table 5).
The activity of Complexes II, III, IV, and V (ATPase) increased at 8 weeks of CS and
returned to slightly below control values in the recovery group (Table 5). The expression
and activity of Complex I was found to decrease after 8 weeks of CS exposure and did
not return to control levels in the recovery group (Table 5).
CS exposure alters mitochondrial oxygen consumption rates in type II alveolar cells –
The effect of cigarette smoke on mitochondrial respiration was examined on confluent
60
Fig.16. Changes in the expression and activity of mitochondrial
complexes. Western blot analysis of mitochondrial complexes was
performed as described in the Materials and Methods section.
Control 4 8 Recovery
CS exposure
(weeks)
61
Table 5. Effect of cigarette smoke exposure on mitochondrial
respiratory chain complexes activity and expression. Activity is
expressed in µmol/min/mg protein and expression as percentage of
control. *p < 0.05, **p < 0.01, ***p < 0.001 compared to control; #p
< 0.05, ###p < 0.001 compared to 8 weeks smoke exposed; and †p <
0.05, †††p < 0.001 compared to 4 weeks smoke exposed as evaluated
using t-test. ANOVA statistical analysis was also performed and
shown in the right-most column.
62
monolayers of cells isolated from mice exposed to Air/CS; the cells were provided with
glucose and pyruvate/glucose/pyruvate/palmitate-BSA in KHB buffered medium and the
increase or decrease in mitochondrial respiration after addition of substrates were noted.
The decrease in basal respiration after addition of 4 µM oligomycin served as an
indicator for ATP production and was not found to change significantly while utilizing
glucose and pyruvate (Table 6). The basal respiration on glucose and pyruvate was found
to increase significantly by 10% after 8 wk of CS exposure (Fig. 18B), but the ATP
production did not change significantly (Table 6) indicating an increase in proton leak
during the electron transfer through the respiratory complexes. 1 µM FCCP was added to
uncouple the mitochondrial respiration from ATP production and the maximal respiration
rate was recorded. The difference between maximal respiration rate and basal respiration
is indicated as spare respiratory capacity in Table 7. The spare respiratory capacity was
found to increase on pyruvate and palmitate-BSA, although not significantly, after 8 wk
of CS exposure (Table 7). The change in respiration on glucose or pyruvate when
supplied individually was more dramatic. The basal mitochondrial respiration was found
to decrease significantly by 13 and 10% on glucose after 4 and 8 wk of CS exposure,
respectively indicating a decrease in pyruvate formation from glycolysis (Fig. 18 A,B).
This decrease was reversed when supplied with pyruvate directly and a 5 and 10%
increase in mitochondrial respiration was found after 4 and 8 wk of CS exposure,
respectively (Fig. 18 A,B). Accordingly, the ATP production was found to decrease while
metabolizing glucose and increase while utilizing pyruvate (Table 6). The ability of
alveolar type II cells mitochondria to utilize pyruvate after CS exposure at a higher rate
when supplied directly signifies an increasing need of substrates for ATP production and
63
Fig. 17. CS exposure alters mitochondrial oxygen consumption rates
in type II alveolar cells. Original trace showing changes in oxygen
consumption rate (OCR) on glucose/palmitate-BSA in Air/8 wk CS
exposed alveolar type II cells.
64
Fig. 18. Effect of CS on OCR while metabolizing different substrates.
Changes in OCR on glucose (25 mM) and pyruvate (2 mM), glucose
(25 mM), pyruvate (2 mM), or palmitate-BSA (200 µM) and BSA
measured using the XF Extracellular Flux Analyzer as described in the
Experimental Procedures section after (A) 4 wk, (B) 8 wk and (C)
8wk smoke exposure + 2 wk recovery. *p < 0.05, **p < 0.01 and ***p
< 0.001 compared to control was found as evaluated using t-test.
A
B
C
65
Table 6. Effect of CS exposure on ATP production in alveolar type
II cells. % Change in basal respiration after addition of oligomycin
is indicated as ATP production in the above table. *p < 0.05 and
**p < 0.01 compared to control was found as evaluated using t-
66
Table 7. Effect of CS exposure on Spare Respiratory capacity in
alveolar type II cells in Air/4, 8 wk CS exposure. % Change in basal
respiration after addition of FCCP is indicated as spare respiratory
capacity in the above table.
67
Fig. 19. CS exposure alters mitochondrial extracellular acidification
rates in type II alveolar cells. Changes in Extracellular acidification
rate (ECAR) on glucose (25 mM) and pyruvate (2 mM), or glucose
measured using the XF Extracellular Flux Analyzer as described in the
Experimental Procedures section after (A) 4 wk, (B) 8 wk and (C)
8wk smoke exposure + 2 wk recovery. *p < 0.05 and **p < 0.01
compared to control was found as evaluated using t-test.
68
an alteration in glycolysis. This notion is also supported by the increase in basal
respiration and ATP production on palmitate-BSA by 8 and 13 % after 4 wk of CS
exposure and 13 and 9 % after 8 wk of CS exposure, respectively (Fig. 18A,B, Table 6).
The mitochondrial respiration on pyruvate was still higher in the recovery group but was
reversed when supplied with palmitate-BSA.
CS exposure alters extracellular acidification rates in type II alveolar cells – The
extracellular acidification rates which serve as an indicator for glycolysis were not found
to change significantly after 4 wk of CS exposure on glucose or glucose and pyruvate
(Fig. 19A), but decreased significantly on glucose after 8 wk of CS exposure (Fig. 19B).
This alteration in glycolysis was reversed in the recovery group and an increase was
observed while metabolizing glucose and pyruvate or glucose alone (Fig. 19C).
CS exposure leads to an increased transport of palmitate into type II alveolar cells
mitochondria – The uptake of palmitate in type II alveolar cells is mediated by CD36
receptor on the cell surface and the expression of the receptor was measured by staining
the cells with anti-CD36 antibody using Immunofluorescence technique. The expression
of CD36 receptor was found to increase by 38 and 83% after 4 and 8 wk of CS exposure
indicating an increase in the uptake of fatty acids in the type II alveolar cells (Fig. 20
A,B). This increase was also reversed in the recovery group. The translocation of fatty
acids into the mitochondria takes place by a two-step process mediated by the carnitine-
palmitoyl transferase system. The expression of carnitine-palmitoyl transferase-1 was
69
CS exposure
(weeks)
0
110
220
Control 4 8 Recovery
*
*
% Fluorescence intensity
(AU)
CS exposure
(weeks)
Fig. 20. CS exposure leads to an increase in the expression of
CD36 in type II alveolar cells. Immunofluorescence analysis
for changes in the expression of (A) CD36 in alveolar type II
cells performed by staining for DAPI (blue) and FITC labeled
CD36 (green). The changes in expression were quantified using
ImageJ software as shown in (B) CD36. *p < 0.05 compared to
control was found as evaluated using t-test. ANOVA statistical
analysis was also performed and p < 0.001 was found.
B
A
70
CS exposure
(weeks)
Fig. 21. CS exposure leads to an increase in the expression of
CPT1 in type II alveolar cells. Immunofluorescence analysis for
changes in the expression of (A) CPT1 in alveolar type II cells
performed by staining for DAPI (blue) and FITC labeled CPT1
(green). The changes in expression were quantified using
ImageJ software as shown in (B) CPT1. *p < 0.05 compared to
control was found as evaluated using t-test. ANOVA statistical
analysis was also performed and p < 0.01 was found.
B
A
0
110
220
Control 4 8 Recovery
*
*
% Fluorescence intensity
(AU)
CS exposure
(weeks)
71
also found to increase significantly by 69 % after 8 wk of CS exposure and this increase
was also found to be reversible in the recovery group (Fig. 21 A,B).
CS exposure leads to an increased phosphatidylcholine breakdown in type II alveolar
cells – The levels of phosphatidylcholine which is the major surfactant phospholipid was
found to decrease significantly by 61 % after 8 wk of CS exposure indicating a decrease
in the surfactant biosynthesis (Fig. 22A). This decrease was not found to be reversed in
the recovery group. The release of palmitate from the sn-2 position of
phosphatidylcholine could explain the decrease in the levels of phosphatidylcholine as
well as the increased mitochondrial respiration on palmitate-BSA. Accordingly, a
significant increase in PLA
2
activity after 8 wk of CS exposure was found (Fig. 22B).
DISCUSSION
The metabolic changes observed in whole lung homogenates are expected to decrease
substrate supply (in the form of pyruvate-derived glycolysis) to mitochondria; however,
an enhanced mitochondrial energy-transducing capacity –expressed as an increased
expression of Complexes II, III, IV and ATPase (complex V) and increased activity of
Complexes II, IV and V– were observed (Fig. 16 and Table 5); this may be viewed as a
compensatory response to the limited substrate (pyruvate) supply to mitochondria. The
significant upregulation of Atpaf2 gene, encoding the ATP synthase mitochondrial F
1
complex assembly factor 2 (Table 4), supports the increased expression and activity of
ATPase (Fig. 16 and Table 5). The activity and expression of Complex I was found to
72
Fig. 22. CS exposure leads to decreased levels of phosphatidylcholine
and an increase in PLA
2
activity in type II alveolar cells. Changes in
(A) phosphatidylcholine levels and (B) PLA
2
activity after Air/CS
exposure in type II alveolar cells as described in the Experimental
Procedures section. *p < 0.05 and **p < 0.01 compared to control was
found as evaluated using t-test. ANOVA statistical analysis was also
performed and p < 0.001 for phosphatidylcholine and p < 0.05 for
PLA
2
activity was found.
73
decrease after 8 weeks of CS exposure, indicating a Complex II-driven oxidative
phosphorylation after CS exposure. The gene expression profile also showed an
upregulation of a number of metabolic genes, involved in glycolysis and tricarboxylic
acid cycle, such as hexokinase-1, GAPDH, succinate de-hydrogenase, and isocitrate
dehydrogenase-1, as well as enzymes of the mitochondrial fatty acid oxidation pathway
including acyl-CoA dehydrogenase for medium and long chain fatty acids and 2,4-
dienoyl CoA reductase-1. The exposure to CS also leads to a significant increase in the
gene expression of pyruvate dehydrogenase kinase-3, indicating an inactivation of
pyruvate dehydrogenase (i.e., phosphorylation of pyruvate dehydrogenase in the E1α
subunit leads to its inactivation) that may be due to reduced pyruvate levels from
glycolysis as surmised by the decrease in glycolysis and increase in pentose phosphate
pathway (Fig. 15).
The percent changes in basal respiration after addition of substrates though small,
indicate an ability of alveolar cell mitochondria to respond to the external stress
conditions. The difference between basal respiration on glucose and palmitate-BSA is
exacerbated not only due to altered glycolysis but also due to an increase in β-oxidation
after CS exposure. This indicates a mechanism in alveolar type II cells to shift from
pyruvate (glycolysis) to palmitate (most likely from dipalmitoyl-phosphatidylcholine) for
energy production. The decrease in basal respiration after 4 and 8 wk of CS exposure
while metabolizing glucose (Fig. 17 and 18 A,B) can be attributed to the inactivation of
glycerdehye-3 phosphate dehydrogenase (GAPDH); a central glycolytic enzyme, due to
glutathionylation as shown before (Fig. 12). This is also supported by the decrease in
ECAR after 8 wk of CS exposure in type II alveolar cells (Fig. 19B) while metabolizing
74
glucose. The ATP production was found to decrease accordingly after 4 and 8 wk of CS
exposure (Table 6). As glutathionylation is a reversible post-translational modification,
the decrease in basal respiration and ATP production were found to be reversed in the
recovery group. The values for spare respiratory capacity were found to be higher during
the presence of pyruvate as the substrates were readily available for complexes I and II
while the mitochondria respired at its maximal capacity, whereas, glucose and palmitate
needed to be metabolized to acyl-co-A by glycolysis and β-oxidation, respectively before
being able to enter the Krebs cycle (Table 7).
The biosynthesis and secretion of pulmonary surfactant takes place exclusively in the
type II alveolar cells and the glycerol backbone for phosphatidylcholine synthesis also
originates from glycolysis pathway (Rooney, 1984). The phosphatidylcholine synthesized
in the cells is then stored in lamellar bodies and also serves as structural markers for the
identification of type II alveolar cells. Although glucose is the preferred substrate for
energy production in type II alveolar cells under normal conditions (Bassett et al., 1981;
Fischer, 1984), palmitate has been shown to be preferred under altered physiologic states
such as starvation (Cormier et al., 2001; Rhoades, 1975). Thus an increase in palmitate
metabolism may be able to support the decrease in substrate availability due to altered
glycolysis. A number of β-oxidation related genes such as Acadl and Acadm were also
found to be significantly up regulated after 8 wk of CS exposure (Fig 15). Type II
alveolar cells mostly depend on circulation for fatty acid uptake and are known to express
a specific receptor for the uptake of palmitate known as CD36 or fatty acid translocase
(FAT) (Leberl et al., 2013; Teague et al., 1994). We found a significant, dose-dependent
and reversible increase in the expression of CD36 on the alveolar type II cells after 4 and
75
8 wk of CS exposure (Fig. 20 A,B). The uptake of palmitate into the mitochondria is a
two-step process catalyzed by CPT1 on the outer mitochondrial membrane and was also
found to be up regulated as indicated by its increased expression (Fig 21 A,B). Cpt1a and
Slc25a20 were also found to be up-regulated after 8 wk CS exposure (Fig. 15, Table 4).
Slc25a20 is a gene which encodes for the carnitine/acylcarnitine translocase which is
located on the inner mitochondrial membrane and helps in the translocation of acyl
carnitine into the mitochondrial matrix; it was also found to increase significantly after 8
wk of CS exposure (Table 4). Acylcarnitine is then converted to carnitine by the activity
of CPT2 to release Acyl Co A which could then enter the β-oxidation cycle.
Phosphatidylcholine represents ~80% of the surfactant phospholipid (Rooney, 1984;
Yu and Possmayer, 2003) and the levels were found to decrease significantly after 8 wk
of CS exposure (Fig. 22A). Surfactant deficiency as a result of alterations in the levels of
phospholipids or inactivation of surfactant proteins is a common observation in smokers
and patients with COPD (More et al., 2010; Ohlmeier et al., 2008). This decrease in
phosphatidylcholine was found to be due to increase in PLA
2
activity after 4 and 8 wk of
CS exposure (Fig. 22B). PLA
2
is known to release the fatty acid at the sn-2 position of the
phospholipid which is mostly palmitate for surfactant phosphatidylcholine (Bartling,
2013; Chilosi et al., 2013). The released palmitate can be converted to palmitoyl-Co A by
the activity of palmitoyl-Co A synthase and then taken up into the mitochondrion by the
CPT system.
These alterations in mitochondrial substrate utilization may have implications in
affecting the efficient gas exchange at the air liquid-interface and may provide a target to
delay the onset of CS induced alveolar damage. The prevention of GAPDH inactivation
76
may promote glycolysis induced mitochondrial respiration and prevent the increase in β-
oxidation of fatty acids. This would also help to utilize phosphatidylcholine for surfactant
biosynthesis and thus delay the onset of COPD.
77
CHAPTER FOUR: ACROLEIN MEDIATED SHIFT IN SUBSTRATE
UTILIZATION IN LUNG ALVEOLAR CELL MITOCHONDRIA
INTRODUCTION
Acrolein, an important major constituent of CS and responsible for more than 80% of
the non-cancerous effects of CS (Haussmann, 2012), is also produced in ambient air from
fuel combustion and cooking oils (Stevens and Maier, 2008). Acrolein has the ability to
form adducts with cellular nucleophiles such as glutathione leading to its subsequent
depletion (Horton et al., 1997; Lam et al., 1985; Rudra and Krokan, 1999; Slater et al.,
1996) and could also be released endogenously after lipid peroxidation and polyamines
metabolism (Stevens and Maier, 2008). It also has the ability to cause protein oxidation
as indicated by the increase in protein carbonylation in retinal pigment epithelium cells
(Jia et al., 2007). Low-dose acrolein exposure is known to induce apoptosis via both the
intrinsic (Tanel and Averill-Bates, 2005; Tanel and Averill-Bates, 2007) and the extrinsic
pathway (Roy et al., 2010). However, acrolein inhibits apoptosis in neutrophils
(Finkelstein et al., 2001) and causes necrosis at higher doses only, in pro B lymphocytes,
indicating its cell-type specificity (Kern and Kehrer, 2002). It also forms adducts with
glyceraldehye-3-phosphate dehydrogenase (GAPDH), (Martyniuk et al., 2011) adenine
nucleotide translocase (ANT) and ATP synthase δ chain (Wu et al., 2011) leading to their
inhibition. Acrolein is also known to functionally inhibit the mitochondrial pyruvate
dehydrogenase (PDH) complex and Krebs cycle enzyme like α-ketoglutarate
dehydrogenase (α-KGDH) in rat liver mitochondria (Sun et al., 2006) and purified
enzyme preparations (Pocernich and Butterfield, 2003). In addition to these
78
mitochondrial complexes I and II are also inhibited by acrolein contributing to
mitochondrial dysfunction and decrease in energy production in rat hepatocytes (Sun et
al., 2006) and brain mitochondria (Picklo and Montine, 2001). It can also modulate stress
responses by inhibiting NF-κB-mediated gene expression (Li et al., 1999; Moon, 2011)
and conjugating with nucleic acid bases like deoxyguanosine (Chung et al., 1984).
Acrolein also triggered endoplasmic reticulum stress through the activation of eIF2α,
ATF-3 and -4, and Gadd153/CHOP without the up regulation of the protective ER
chaperones GRP78 and GRP94 eventually leading to cell death by mitochondrion driven
apoptosis (Mohammad et al., 2012a).
This study is aimed at establishing changes in energy metabolism when cells are
exposed to acrolein under controlled conditions; experiments were performed on isolated
mouse primary type II alveolar cells (pAT2), rat lung epithelial cells (RLE-6TN), and
human lung adenocarcinoma cells (H441).
EXPERIMENTAL PROCEDURES
Cell lines – Experiments in this study were carried out in RLE-6TN (CRL-2300) and
H441 (HTB-174) cell lines obtained from American Type Culture Collection. Rat lung
epithelial (RLE-6TN) cell line, derived from Type II alveolar cells, was cultured in
Ham’s F-12 media supplemented with 2 mM L-glutamine, 0.01 mg/ml bovine pituitary
extract, 5 µg/ml insulin, 0.002 5 µg /ml insulin-like growth factor, 1.25 µg/ml transferrin,
2.5 ng/ml EGF, 5% penicillin-streptomycin, and 10% fetal bovine serum. The Clara cell-
like human bronchial epithelial (H441) cell line, derived from pericardial fluid of a
79
patient with papillary adenocarcinoma, was cultured in RPMI-1640 media supplemented
with 5% penicillin-streptomycin and 10% fetal bovine serum. All the supplements were
obtained from Sigma Aldrich (St. Louis, MO).
Isolation of primary alveolar Type II (pAT2) cells – pAT2 cells were isolated from
male A/J mice (Jackson Laboratories) 8-12 weeks of age using dispase (BD Biosciences,
Bedford, MA) digestion - agar instillation method (Demaio et al., 2009): mice were
sacrificed after pentobarbital sodium overdose and the abdominal cavity was opened to
sever the renal artery and drain blood. PBS was then perfused through the ventricles,
followed by 5 ml dispase and 0.5 ml of 1% low-melting point agarose (Sigma, St. Louis,
MO) into the lungs through the trachea. Lungs were then excised and incubated in 3 ml
of dispase for 45 min following which were chopped in wash medium containing a 1:1
mixture of Dulbecco's modified Eagle's medium and Ham's F-12 (DME/F-12; Sigma)
supplemented with 0.01 % DNAase, 1 mM L-glutamine, 100 U/ml sodium penicillin G,
and 100 μg/ml streptomycin. The cell mixture was then passed through cell strainers of
pore size 100, 40, 20, 15, and 10 µm and the resulting cell suspension was then
centrifuged at 300 g for 10 min at 10°C. The pellet was re-suspended in the wash medium
supplemented with 10% fetal bovine serum (Gemini Bio-products, West Sacramento,
CA) and incubated with biotinylated anti-macrophage antibodies (anti-CD45, anti-
CD45.1, anti-CD45.2, anti-Ter 119, and anti-CD16/32; BD Biosciences) for 30 min. The
antibody-bound macrophages were separated using streptavidin conjugated magnetic
beads (Promega, Madison, WI) and the cells were incubated on petri dishes previously
coated with mouse IgG (Sigma St. Louis, MO) for 2 h at 37°C. The unattached cells were
80
then seeded on plates coated with Laminin-1 (Trevigen, Gaithersburg, MD) in Complete
Mouse Medium (CMM) containing DMEM/F-12, 1 mM L-glutamine, 0.25 % bovine
serum albumin (BD Biosciences), 10 mM HEPES, 0.1 mM nonessential amino acids,
0.05% insulin-transferrin-sodium selenite (Roche, Basel, Switzerland), and 100 μg/ml
Primocin (Invitrogen, Carlsbad, CA) supplemented with 10% newborn bovine serum
(Omega Scientific, Tarzana, CA). After 3 days of seeding, the medium was changed to
serumless CMM to remove any contaminating fibroblasts.
Acrolein treatments – All acrolein exposures were done in the respective cell-culture
medium for 4 h in a humidified 5% CO
2
-95% air incubator at 37°C. The exposure period
of 4 h was based on time course experiments and the cell-doubling time where longer
duration of exposure showed higher cytotoxicity and cell proliferation.
Cell viability assays –
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) - Cell viability
was assessed by measuring the ability of the cells to reduce MTT after acrolein exposure.
After exposure period the medium was removed and the cells were washed twice with
PBS and then replaced with HEPES buffered toxicity medium containing 5 mM HEPES,
154 mM NaCl, 4.6 mM KCl, 2.3 mM CaCl
2
, 1.1 mM MgCl
2
, 33 mM glucose, 5 mM
NaHCO
3
, 1.2 mM Na
2
HPO
4,
pH 7.4, and 0.5 mg/ml of MTT (Sigma Aldrich, St. Louis,
MO). The cells were incubated for 90 min in the above media and after incubation the
resulting formation of formazan crystals was measured by dissolving in DMSO and
reading the absorbance at 490 nm in a microplate spectrophotometer.
81
FACS – Cells were incubated with FITC conjugated Annexin V (apoptosis marker) for
15 min at RT and propidium iodide (necrosis marker) post acrolein treatment using
Anexin-V FITC apoptosis detection kit from Calbiochem (San Diego, CA) according to
manufacturer’s instructions. Flow cytometric analysis was performed using FACSDiva
from BD Biosciences (San Jose, CA).
XF extracellular metabolic flux analysis – Mitochondrial respiration was measured
using XF Extracellular Flux Analyzer from Seahorse Biosciences (North Billerica, MA)
according to manufacturer’s protocol. Cells were seeded in XF-24 or XF-96 plates 1 day
before the experiment and treated with acrolein the next morning. For basal
mitochondrial respiration on glucose and pyruvate, the cells were washed with DMEM
buffer, pH 7.4, containing 25 mM glucose and 2 mM pyruvate and the decrease in O
2
levels was measured in the medium immediately surrounding the cells. 4 µM oligomycin
and 1 µM FCCP were added through one of the ports to measure the H
+
leak and
maximal respiration capacity, respectively. For mitochondrial respiration on
glucose/pyruvate the initial respiration was measured in DMEM medium without
glucose/pyruvate to get baseline respiration, and the increase/decrease in mitochondrial
respiration after addition of substrates was measured to obtain basal respiration values.
The respiration on palmitate-BSA/BSA was measured similarly in KHB buffered
medium, pH 7.4, containing 2.5 mM glucose and 0.5 mM carnitine to facilitate palmitate-
BSA uptake; palmitate-BSA was added to a final concentration of 200 µM. ECAR data
was validated by using 2-deoxyglucose. All values were normalized to control by
82
measuring the protein concentrations using Bradford assay post XF Extracellular Flux
analysis.
GAPDH and G6PDH activities – The activities for GAPDH and G6PDH were
measured in buffer containing 100mM Tris/HCl and 5mM sodium arsenate, pH 8.6. 100
µg of cell lysate was added along with 100 µM NADP and 3.4 mM glucose-6-phosphate
in a final reaction volume of 1 ml for G6PD activity. The formation of NADPH was
monitored spectrophotometrically at 340 nm. For GAPDH activity, 100 µg of cell lysate
was added along with 250 µM NAD
+
and 15 µl of 50 mg/ml of glyceraldehyde-3-
phosphate to a final reaction volume of 1 ml. The formation of NADH was monitored
spectrophotometrically at 340 nm and the enzyme activity was calculated from the slopes
obtained with an extinction coefficient of 6.22.
Total phosphatidylcholine assay – The levels of phosphatidylcholine were measured
using kits available from Abnova (Taipei, Taiwan) using manufacturer’s protocol. Cell
lysates were centrifuged at 14,000 g to prevent contamination from cell membrane and
the supernatant was used for analysis. The OxiRed probe generated after
phosphatidylcholine hydrolysis and its subsequent oxidation was measured
calorimetrically at 570 nm.
Phospholipase A
2
(PLA
2
) activity assay - PLA
2
activity was measured in cell lysates
after acrolein treatment using kits available from Cayman Chemicals (Ann Arbor, MI)
83
Fig. 23. Acrolein-induced cytotoxicity in RLE-6TN, pAT2, and H441
cells. The cytotoxicity of acrolein was studied by measuring the
ability of the cells to reduce MTT as described in the Experimental
Procedures section. *p < 0.05, **p < 0.01, ***p < 0.001 compared to
control of the respective cells as evaluated using t-test. (●) RLE-6TN,
(▲) pAT2, (■) H441 cells.
84
Fig. 24. Acrolein-induced apoptosis/necrosis in RLE-6TN, pAT2, and
H441 cells. FACS analysis traces for pAT2 cells showing control (A),
20 µM acrolein (B). The number of cells that did not label with FITC
conjugated Annexin V and propidium iodide were quantified and
plotted as shown in (C).
85
according to manufacturer’s protocol. Cell lysates were centrifuged at 14,000 g to prevent
contamination from cell membrane and the supernatant was used for analysis. The free
thiol released after hydrolysis of arachidonoyl thio-PC at the sn-2 position by PLA
2
was
detected at 414 nm using DTNB.
Statistical analyses – Students t-test assuming unequal variances was performed
along with ANOVA to determine statistical significance as indicated in the figure
legends. Results are mean ± SD from a minimum of 3 experiments.
RESULTS
Acrolein induces cytotoxicity in RLE-6TN, H441, and primary AT2 cells – The effect
of acrolein on cell viability was measured by studying the ability of cells to reduce MTT
after 4 h acrolein exposure (Fig. 23). RLE-6TN and pAT2 cells were more susceptible to
acrolein-induced cell death than H441 cells. The IC
50
for RLE-6TN and pAT2 cells was
~40 µM and ~50 µM acrolein, respectively, whereas that for H441 cells was ~200 µM.
This indicates that H441 cells are more resistant to acrolein than the alveolar Type II cells
(RLE-6TN and pAT2). The higher resistance of H441 cells to acrolein may be partly
accounted for their having Clara cell-like morphology, mostly found in the bronchial
epithelium, therefore more resistant to environmental toxicants as they would be exposed
to higher concentrations compared to the distal alveolar epithelial cells. These
cytotoxicity results were also confirmed by labeling the cells for Annexin V and
propidium iodide to identity cells undergoing apoptosis or necrosis. There was no
86
significant increase in the number of apoptotic/necrotic cells in any of the cell lines (Fig
24).
Effect of acrolein on mitochondrial metabolism in RLE-6TN, H441, and primary AT2
cells – The effect of acrolein on mitochondrial respiration was examined on confluent
monolayers of cells (RLE-6TN, H441, and primary AT2) exposed to acrolein for 4 h;
oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were
measured by XF-extracellular flux analysis as indicators of mitochondrial
respiration/oxidative phosphorylation and glycolysis, respectively. Assays were
performed in the presence of 25 mM glucose and 2 mM pyruvate. The basal
mitochondrial respiration decreased in a dose-dependent manner in RLE-6TN (Fig.
25A,B), pAT2 (Fig. 26A), and H441 (Fig. 27A) cells after 4 h acrolein exposure. The
concentration of acrolein needed to reduce mitochondrial respiration by ~25% in H441
cells was 10-fold higher than that in Type II cells (RLE-6TN and pAT2). Addition of 4
µM oligomycin did not completely inhibit the OCR as a result of H
+
leak across the inner
mitochondrial membrane. The oxygen consumption contributing to ATP production
(basal respiration – proton leak) also decreased in a dose-dependent manner in RLE-6TN,
pAT2, and H441 cells following 4 h acrolein exposure. 1 µM FCCP uncoupled the
mitochondrial respiration and the oxygen consumption reached its maximal capacity as
shown in Fig. 25A,B, 26A, and 27A. The spare respiratory capacity (maximal respiration
– basal respiration) of the cells did not change significantly in RLE-6TN, pAT2, and
H441 cells after 4 h acrolein exposure. Of note, basal OCR values differed with cell type:
H441 cells are the largest in size and thus showed the highest basal OCR values
87
Fig. 25. Acrolein induced mitochondrial toxicity in RLE-6TN cells.
Change in Oxygen Consumption Rate (OCR) (A, B) and Extracellular
acidification rate (ECAR) (C) on glucose and pyruvate after 4 h
acrolein exposure in RLE-6TN cells meas-ured using the XF
Extracellular Flux Analyzer as described in the Experimental
Procedures section. *p < 0.05, **p < 0.01 compared to control was
found as evaluated using t-test. The original trace (A) was quantified
for basal respiration, proton leak, ATP production, maximal
respiration, and spare respiratory capacity for control and acrolein
treated cells as shown in (B). (●) control, (○) plus 15 µM acrolein.
88
Fig. 26. Acrolein induced mitochondrial toxicity in pAT2 cells.
Change in Oxygen consumption rate (OCR) (A) and Extracellular
acidification rate (ECAR) (B) on glucose and pyruvate after 4 h
acrolein exposure in pAT2 cells measured using the XF Extracellular
Flux Analyzer as described in the Experimental Procedures section. *p
< 0.05 compared to control was found as evaluated using t-test.
89
Fig. 27. Acrolein induced mitochondrial toxicity in H441 cells.
Change in Oxygen consumption rate (OCR) (A) and Extracellular
acidification rate (ECAR) (B) on glucose and pyruvate after 4 h
acrolein exposure in H441 cells measured using the XF Extracellular
Flux Analyzer as described in the Experimental Procedures section. *p
< 0.05 compared to control was found as evaluated using t-test.
90
compared to RLE-6TN and pAT2 cells, whereas pAT2 cells showed the lowest basal
OCR values possibly due to their primary nature.
The increase in basal ECAR values in the two cancerous cell lines (RLE-6TN (Fig.
25C) and H441 Fig. 27B)) upon acrolein exposure was not statistically significant,
whereas the decrease in basal ECAR in pAT2 cells (Fig. 26B) upon exposure to 20 µM
acrolein was statistically significant indicating altered glycolysis. These data were
validated by supplementing RLE-6TN cells with 2-deoxyglucose, an inhibitor of
glycolysis, that decreased basal ECAR values by ∼80% (data not shown).
Acrolein-mediated inhibition of glycolysis in RLE-6TN, H441, and primary AT2 cells
– 4 h acrolein exposure resulted in a decrease of basal respiration on glucose by 25, 12,
and 20 % in RLE-6TN, pAT2, and H441 cells (compared to their respective controls),
respectively (Fig. 28A-C). Basal respiration when cells were metabolizing pyruvate
increased by 22 and 20% in RLE-6TN and pAT2 cells, respectively after 15 µM acrolein
exposure for 4 h (Fig. 28A,B). The increase in basal respiration on pyruvate was not
observed in H441 cells after 100 µM acrolein exposure for 4 h (Fig. 28C). This indicated
an alteration of glucose metabolism in all the three cell types leading to decrease
substrate (glucose) availability for mitochondrial respiration; bypassing glycolysis by
providing mitochondrial substrate (pyruvate) resulted in an OCR increase.
Acrolein inhibits GAPDH activity and up-regulates G6PDH activity – The decrease
in glucose metabolism observed in Fig. 5 was partly due to a dose-dependent inhibition
91
Fig. 28. Acrolein induced inhibition of glycolytic metabolism in RLE-
6TN, pAT2, and H441 cells. Changes in OCR on glucose or pyruvate,
measured in (A) RLE-6TN, (B) pAT2, and (C) H441 cells after 15 µM
(RLE-6TN and pAT2) and 100 µM (H441 cells) acrolein exposure
using the XF Extracellular Flux Analyzer as described in the
Experimental Procedures section. *p < 0.05, **p < 0.01 compared to
control of the respective cells was found as evaluated using t-test.
92
Fig. 29. Acrolein induced GAPDH inhibition in RLE-6TN, pAT2 and
H441 cells. Changes in GAPDH (□) and G6PD (■) activity, measured
by monitoring the consumption and formation of NADH and
NADPH, respectively at 340nm using UV spectrophotometer as
described in the Experimental Procedures section in (A) RLE-6TN,
(B) pAT2 and (C) H441 cells. *p < 0.05, **p < 0.01 compared to
control of the respective cells was found as evaluated using t-test.
93
of glyceraldehyde-3P-dehydrogenase (GAPDH) activity in RLE-6TN, pAT2, and H441
cells after 4 h acrolein exposure (Fig. 29A-C). Type II alveolar cells were more
susceptible to acrolein toxicity entailing a decrease by ~72 and 50% in RLE-6TN and
pAT2 cells, respectively (following 4 h exposure to 15 µM acrolein). The activity of
glucose-6P-dehydrogenase (G6PDH) –the only regulatory control of the pentose
phosphate pathway– increased in the primary AT2 cells (Fig. 29B) with decreasing
GAPDH activity, but not in the two immortalized cell lines (RLE-6TN and H441) (Fig.
29A,C). This may be due to the pentose phosphate pathway providing ribose-P for
nucleic acid synthesis and continuous proliferation, already high in the two immortalized
cell lines. As mentioned above, the decrease in GAPDH activity may account for the
decrease in basal respiration on glucose in the three cell types.
Acrolein exposure leads to an increase in mitochondrial metabolism of palmitate in
Type II alveolar cells – Mitochondrial respiration on glucose was compared with that on
200 µM palmitate-BSA complex following acrolein exposure. RLE-6TN and pAT2 cells
showed an increase in oxygen consumption on palmitate-BSA (Fig. 30A,B) by 65 and
29%, respectively, compared to control after 10 µM acrolein exposure, whereas H441
cells respiring on palmitate-BSA showed a dose-dependent decrease in OCR (Fig. 30C)
upon acrolein exposure. The OCR on glucose was found to decrease in RLE-6TN, pAT2,
and H441 cells (Fig. 30A-C). This indicated that the rate of β-oxidation increased in Type
II alveolar cells after acrolein exposure but not in bronchoalveolar (H441) cells.
Etomoxir, an inhibitor of carnitine-palmitoyl transferase I, inhibited OCR (basal
respiration, ATP turnover, and maximal respiratory capacity) (data not shown). This
94
Fig. 30. Acrolein induced increase in β-oxidation in Type II alveolar
cells. Changes in OCR on glucose (■) or palmitate-BSA (■),
measured in (A) RLE-6TN, (B) pAT2, and (C) H441 cells after 4 h
acrolein exposure, measured using the XF Extracellular Flux Analyzer
as described in the Experimental Procedures section. *p < 0.05, **p <
0.01, ***p < 0.001 compared to control of the respective cells was
found as evaluated using t-test. (■) control, BSA alone. ANOVA
statistical analysis was also performed and p < 0.01 for RLE-6TN
cells (glucose), p < 0.01 for pAT2 cells (glucose), p < 0.001 for pAT2
cells (palmitate-BSA), p < 0.001 for H441 cells (glucose), and p <
0.001 for H441 cells (palmitate-BSA) was found.
95
supports the β-oxidation of palmitate in these cells; the inhibition of the oligomycin
effect indicated that palmitate metabolism was ATP linked.
Effect of acrolein on levels of surfactant lipids – The biosynthesis of surfactants takes
place exclusively in Type II alveolar cells and thus the levels of phosphatidylcholine
(major surfactant phospholipid (Agassandian and Mallampalli, 2013)) were measured
following acrolein exposure. The levels of phosphatidylcholine were found to decrease
by 43 and 58% in RLE-6TN (Fig. 31A) and pAT2 (Fig. 31B) cells, respectively, after
exposure to 15 µM acrolein.
Effect of acrolein on phospholipase A
2
activity – Phospholipase A
2
catalyzes the
release of fatty acids from the sn-2 position of phosphatidylcholine; acrolein exposure (15
µM) resulted in a 2.5- and 1.5-fold increase in phospholipase A
2
activity in RLE-6TN
and pAT2 cells, respectively (Fig. 32).
DISCUSSION
The doses of acrolein used in the study were based on the MTT cell viability assay
and the OCR values for the three cell types. Physiologically, it has been shown that
acrolein may reach the concentrations of 80 µM in the respiratory tract lining fluid in
smokers (Eiserich et al., 1995) and up to 180 µM in plasma of patients with renal failure
(Sakata et al., 2003). Low-dose of acrolein (maximum 20 µM) did not decrease the cell
viability by more than 10% in Type II cells (Fig. 23) but decreased the mitochondrial
96
Fig. 31. Effect of acrolein on phosphatidylcholine levels in type II
alveolar cells. Changes in the levels of phosphatidylcholine levels
in RLE-6TN (A) and pAT2 (B) cells following 4 h acrolein
exposure as described in the Experimental Procedures section. *p
< 0.05, and ***p < 0.001 compared to control was found as
evaluated using t-test. ANOVA statistical analysis was also
performed and p < 0.05 for RLE-6TN cells and p < 0.001 for
97
Fig. 32. Effect of acrolein on phospholipase A2 activity in type II
alveolar cells. Changes in PLA2 activity in RLE-6TN cells (A) and
pAT2 cells (B) following 4 h acrolein exposure as described in the
Experimental Procedures section. *p < 0.05 and **p < 0.01
compared to control was found as evaluated using t-test. ANOVA
statistical analysis was also performed and p < 0.01 for RLE-6TN
cells was found.
98
respiration by ~50% (Fig. 25 A,B and Fig 26 A). In H441 cells the maximum dose used
(120 µM) decreased the cell viability by ~20% and mitochondrial respiration by ~27%
(Fig. 27A). The reduction of MTT is catalyzed by mitochondrial aldehyde dehydrogenase
that is also susceptible to inactivation by acrolein (Yoval-Sanchez and Rodriguez-Zavala,
2012). Thus, to prevent a false positive for cell death, acrolein-induced apoptosis/necrosis
was measured using FACS analysis. The highest doses of 20 µM (RLE-6TN and pAT2)
and 120 µM (H441) cells did not elicit any increase in apoptotic / necrotic cells (Fig 24A-
C). Higher doses of acrolein were highly cytotoxic to H441 cells and would have thus
changed the cell numbers beyond comparison with the Type II alveolar cells after
acrolein exposure. This study shows that low-dose acrolein exposure for 4 h leads to
alterations in glucose metabolism partly due to GAPDH inactivation (Fig. 28A-C and
29A-C) and up-regulation of the activity of the rate-limiting enzyme of the pentose
phosphate pathway, G6PDH, in pAT2 (Fig. 29B). This inactivation of GAPDH is most
likely due to acrolein electrophilic attack on thiol cysteine residues in the enzyme
(Martyniuk et al., 2011; Nakamura et al., 2012), rather than glutathionylation as shown
before (Fig. 12) due to the ability of acrolein to deplete GSH without its oxidation to
GSSG (Lam et al., 1985). The metabolism of palmitate present in the form of
phosphatidylcholine, a major surfactant– may meet the energy demands in alveolar Type
II cells (Fig. 30 A,B).
The basal and maximal respiration values were found to decrease in a dose-dependent
manner following acrolein exposure for 4 h in RLE-6TN, pAT2, and H441 cells. ATP
production (oligomycin-sensitive respiration) was also found to decrease as a result of a
decrease in substrate availability after acrolein exposure in RLE-6TN (Fig. 25A,B), pAT2
99
(Fig. 26A), and H441 cells (Fig. 27A). The decrease in OCR in RLE-6TN and pAT2 cells
respiring on glucose and the increase in cells respiring on pyruvate indicated impairment
in glucose metabolism (upstream of pyruvate formation), i.e. GAPDH inactivation. These
deficiencies were overcome by RLE-6TN and pAT2 cells respiring on pyruvate (Fig.
28A,B). OCR did not increase in H441 cells respiring on pyruvate, thus indicating altered
glucose metabolism (downstream of pyruvate formation, i.e., changes in the pyruvate
dehydrogenase complex) leading to decreased substrate supply to the tricarboxylic acid
cycle and reducing equivalents to the electron-transfer chain (Fig. 28C). This notion is
supported by the lack of effect of acrolein on OCR
BASAL RESPIRATION
values in the presence
of pyruvate (Fig. 28C).
Acrolein exposure in RLE-6TN and pAT2 cells results in a decrease in OCR while
respiring on glucose and an increase while metabolizing palmitate-BSA (Fig. 30A,B).
This indicates the ability of alveolar cell mitochondrion to utilize palmitate for energy
production after decreased glycolysis. The H441 cells however, showed a dose-dependent
decrease in OCR after acrolein exposure while metabolizing palmitate-BSA (Fig. 30C),
which may be due to the absence of palmitate in the form of surfactant
phosphatidylcholine in these types of cells. The limited number of glycogen stores and
absence of gluconeogenesis (Fischer, 1984), along with the presence of palmitate in
surfactant phosphatidylcholine in Type II alveolar cells renders them an effective
alternate fuel source for mitochondrion.
The increase in G6PD activity in pAT2 cells after acrolein exposure indicated a shift
of glucose metabolism towards the pentose phosphate pathway (Fig. 29B). This
upregulation of pentose phosphate pathway would also support an increase in the levels
100
of NADPH to counteract acrolein-induced oxidative stress (Table 2) (Ralser et al., 2009).
The immortalized cell lines (RLE-6TN and H441) would require a continuous supply of
ribose-5-P for the synthesis of nucleic acid bases and nucleotides, provided by the
pentose phosphate pathway. Thus, the G6PD activity in RLE-6TN and H441 cells did not
change significantly following acrolein exposure (Fig. 29 A,C).
The down-regulation of GAPDH activity would be expected to promote surfactant
biosynthesis through glycerol-3-phosphate pathway. However, the levels of
phosphatidylcholine decreased with increasing acrolein exposure, thus indicating a
decrease in the levels of surfactant as phosphatidylcholine represents ~80% of the
surfactant phospholipid (Rooney, 1984; Yu and Possmayer, 2003) (Fig. 31). This
decrease in the levels of phosphatidylcholine may be attributed either to the down-
regulation of choline-phosphate cytidylyltransferase, the rate-limiting enzyme in the
biosynthesis of phosphatidylcholine (Agassandian and Mallampalli, 2013; Rooney et al.,
1994) or its degradation by cytosolic phospholipase A
2
to furnish fatty acids for
mitochondrial β-oxidation and energy production. Acrolein has been shown to down-
regulate ANX1 gene which would lead to an upregulation of phospholipase A
2
activity in
rat lung epithelial cells (Sarkar and Hayes, 2009). This is also supported by the increase
in phospholipase A
2
activity observed in sheep after lung injury (Fukuda et al., 1999). In
accordance with these results, we found a substantial increase in PLA
2
activity after
acrolein exposure in RLE-6TN and pAT2 cells (Fig. 32). The released palmitate
converted to palmitoyl-Co A by the activity of palmitoyl-Co A synthase, can then be
translocated to the mitochondrion by carnitine-palmitoyl transferases I and II.
Alternatively, inhibition of glycolysis alone may be sufficient to increase fatty acid
101
oxidation and utilization of palmitate (Rhoades, 1975), as it can be surmised by the
substantial increase in fatty acid oxidation in RLE-6TN cells after inhibiting glycolysis
with 2-deoxy glucose for 1h (data not shown).
COPD is characterized by surfactant impairment that may be due to alterations in the
levels of phospholipids or inactivation of surfactant proteins (More et al., 2010; Ohlmeier
et al., 2008). Cigarette smoke exposure has also been shown to affect the levels of
surfactant protein (SP-D) in mice and in A549 cells (Hirama et al., 2007). A number of
studies have focused on mitochondria due to the toxicity mediated by acrolein-induced
oxidative stress in brain (Dong et al., 2013; Luo and Shi, 2005; Picklo and Montine,
2001), heart (Anderson et al., 2012; Wu et al., 2011), liver (Mohammad et al., 2012b;
Sun et al., 2006; Watanabe et al., 1992), eyes (Feng et al., 2010; Jia et al., 2007; Liu et
al., 2007), and lungs (Jia et al., 2009; Roy et al., 2010); however, the effect of acrolein on
energy metabolism, specifically in lung alveolar cells, has not been highlighted before.
The findings from this study summarized in Fig. 33 have implications for understanding
the acrolein-mediated bioenergetic alterations in alveolar cells that may affect the
surfactant biosynthesis pathway and energy production, possibly providing a mechanism
for reduced surfactant secretion in respiratory diseases.
102
CHAPTER FIVE: CONCLUSIONS AND FUTURE DIRECTIONS
CONCLUSIONS AND PERSPECTIVES
COPD is a preventable disease, but the increasing mortality suggests a lack of
general understanding of the pathophysiology of the disease. Great strides have been
made to try and treat the disease, but most of the therapies available in the clinic only
help in the management of the disease symptoms. The underlying cause is still unknown
and untreated. With the studies detailed in this dissertation we are beginning to seek
answers for some of the symptoms commonly observed in COPD such as surfactant
deficiency; served to address the hypothesis that early stage cigarette smoke and
environmental pollutants exposure reroute the utilization of fatty acids from surfactant
synthesis to energy production in order to counteract oxidative stress, thus leading to
inefficient gaseous exchange in alveoli and reduced lung function.
Short-term CS exposure in A/J mice showed an upregulation of a number of
antioxidant defense related redox genes to prevent the oxidative stress-induced damage to
redox sensitive proteins. The decrease in the GSH/GSSG ratio in homogenate after 8 wk
CS exposure indicates a possibility of increased oxidative stress with longer duration of
CS exposure. The role of oxidative stress in the pathogenesis of COPD is well established
(Ou and Huang, 2006; Rahman and Adcock, 2006; Rahman et al., 2006), but antioxidant
therapies have not succeeded in treating the disease. This may be partly due to lack of
recognition of the metabolic component within an energy-redox axis. A large clinical trial
with more than 500 COPD patients showed no efficacy of antioxidant treatment in
improving lung function and reducing exacerbations (Decramer et al., 2005). The reason
103
for this may be that antioxidant therapy is given to patients after having developed an
emphysematous lung. The oxidative modification of proteins may be irreversible and thus
increasing the antioxidant defense or cells redox status may not be able to repair the
damage. Thus antioxidant therapy should be tested in patients on the path to develop
COPD. This task is made difficult by the lack of any biomarkers or diagnostic tests which
would better predict lung health. The situation is further complicated by the diversity of
patient population in the number of cigarettes they smoke, the brand of cigarettes and
smoking history.
2 weeks of smoking cessation in mice was found to reverse most of the changes in
gene expression and enzyme activity after 8 weeks of CS exposure. This recovery may
either be due to the low duration of CS exposure or the young age of mice. COPD
development in humans suggests more of the latter. Young smokers are able to repair the
damage that may have been caused in the lungs, but old smokers with an age-related
reduction in pulmonary function may have an impaired repair process contributing to the
development of disease. Oxidative stress and the generation of ROS may play a critical
role in damaging the DNA and promoting chronic inflammation. Some of the
characteristics evident in senescent cells are frequently observed in alveolar cells which
could be described as premature/accelerated aging of the lungs in response to
environmental toxicants exposure (Bartling, 2013; Chilosi et al., 2013). Some of the
interventions known for lifespan extension such as reduced insulin sensitivity, decreased
glucose metabolism/calorie restriction, exercise, and increased antioxidant defense should
thus improve lung health delay the progression of disease.
104
Mitochondrial dynamics were also found to be affected with CS exposure. The
increase in transcription of mitochondrial fusion genes Mfn-1 and Opa-1 after CS
exposure with increased expression of mitochondrial complexes indicate an adaptive
response to meet the increasing energy demands as a result of the impending stress. The
induction of mitochondrial metabolism in response to reduced glucose metabolism is
suggested to help in increasing life span in lower species such as Caenorabditis elegans
and Drosophila melanogaster. Thus shot-term (8wk) CS exposure may induce increased
mitochondrial fusion to remove the damaged components and prevent the initiation of the
apoptotic pathway, but longer duration may promote mitochondrial fission leading to
decreased functional capability contributing to reduced exercise capacity and chronic
inactivity.
Surfactant impairment has been associated with the pathophysiology of COPD for
a long time, but its biosynthesis has never been looked as a potential target to prevent this
deficiency. This is also because of the physiologic regulation by feedback inhibition of
surfactant secretion in response to increased levels of surfactant components at the air
liquid interface (Rice et al., 1987). Artificial surfactant delivery has also been successful
in treatment of diseases such as the infant respiratory distress syndrome (Enhorning et al.,
1985; Hallman et al., 1985). Small molecule modulators of the surfactant biosynthetic
pathway and the enzymes involved in these pathways have not been explored. Our study
provides a mechanism for the altered levels of surfactant phospholipid as a result of
oxidative modification of the glycolytic enzyme, GAPDH. The early stages of CS
exposure would have sufficient glutathione to reversibly modify GAPDH by
glutathionylation, but longer exposure duration would lead to oxidation of glutathione
105
and thus irreversible modification by adduct formation or nitrosylation. Thus prevention
of GAPDH inactivation may support the utilization of pyruvate in place of fatty acids for
ATP production and the surfactant deficiency would be delayed (Fig. 33).
COPD has never been looked as a metabolic disease. The increase in fatty acid
metabolism in response to environmental toxicants exposure signifies a stress response to
maintain the production of ATP. The increased effort required in the work of breathing
and impairment of gas exchange leads to reduced exercise capacity and chronic
inactivity. The increase in β-oxidation is also supported at the transcriptional level as
shown by the increase in genes encoding proteins involved in medium and long chain
fatty acid metabolism and those involved in the transport of fatty acids into the
mitochondria. The increase in β-oxidation in cells other than alveolar in response to
altered glycolytic metabolism, may explain the decrease in body weight frequently
observed in smokers. The ability of CS constituents to cross the placental barrier and the
blood brain barrier is well established. Thus, alterations in systemic glycolytic
metabolism or increased insulin resistance should not be surprising. The decrease in body
weight observed in mice after CS exposure may either be the cause or effect of low food
intake. The metabolic shift towards the non-energy producing pathway; the pentose
phosphate pathway also suggests the CS-induced calorie restriction may play an
important role in the development of disease. The link between acute effects of CS and
cardiovascular diseases is also well established. The exact mechanism with which CS and
its components increase the predisposition to cardiovascular disease is largely unknown
but the major effects observed include thrombosis, inflammation, oxidation of LDL and
atherosclerosis.
106
The repeated injuries to the alveolar epithelium due to chronic exposure to
environmental toxicants may be expected to promote an accumulation of oxidatively
damaged and misfolded proteins in the endoplasmic reticulum. Although, the unfolded
protein response is mediated towards maintenance of protein homeostasis, the inability of
the ubiquitin proteasome degradation pathway to clear the damaged proteins may lead to
altered cellular functions and eventually, cell death. Redox homeostasis also plays an
important role in preventing the development of ER stress where reduced glutathione acts
as a net reductant in the ER by reducing disulfide bonds and maintaining the
oxidoreductases in the reduced state. The identification of ER stress as a key component
in the pathogenesis of COPD is recent and would require further exploration to identify
targets to impede the progression of disease.
The presence of a number of animal models also increase the complications
associated in developing an accurate timeline for development of COPD. As reviewed by
Leberl et al, a number of tobacco smoke exposure studies have utilized different smoke
exposure systems, brands of cigarettes, animal models, and strains of animals (Leberl et
al., 2013). With the advent of Teague smoke exposure system in 1994; most of the
studies are performed utilizing this system which helped maintain consistent levels of
total particulate matter (TPM) and mimic human exposure. But we are still far away from
an animal model which could accurately mimic the progression of disease as observed in
humans.
One of the major issues in the management of COPD is the current focus in
research laboratories and clinics which disregards the need to develop therapies treating
the root-cause of the disease. A cursory look at the current ongoing clinical trials and the
107
Fig. 33. Effect of cigarette smoke and acrolein on enzymes of
glycolysis, pentose phosphate pathway and surfactant
(phosphatidylcholine) levels. Acrolein exposure leads to a metabolic
shift from the glycolytic pathway to the pentose phosphate pathway.
The decreased pyruvate from glycolytic metabolism forces the
mitochondrion to utilize alternate substrates for energy production,
i.e., palmitate from phosphatidylcholine; this may lead to a decrease in
the levels of surfactants following exposure to environmental
toxicants.
108
research articles emanating from various laboratories suggest a huge emphasis on the
management of symptoms such as exacerbations, bronchoconstriction and inflammation.
The therapies used involve β
2
adrenergic receptor agonists or anticholinergics for
bronchodilation, steroids for anti-inflammatory effect, or antioxidants. Forced expiratory
volume (FEV
1
) is the most commonly used measure for diagnosis and treatment of
COPD. The increasing mortality suggests a significant need to develop targeted therapies
to not only treat symptoms of COPD but also the underlying mechanism contributing to
development of the disease.
FUTURE DIRECTIONS
Some of the future studies which may help build on this research project are as
follows:-
Chronic cigarette smoke exposure studies – Cigarette smoke exposure for 16 to 24 wk
would help develop a timeline for cellular redox changes and COPD development in
lungs of A/J mice. A number of studies have shown that, CS exposure in the Teague
smoke exposure system requires 24 wk to develop COPD pathophysiology. This would
help develop therapeutics which could be beneficial to treat COPD. The current exposure
system helps establish the molecular basis of the disease and provides an insight into the
physiological changes associated with short-term CS exposure. A number of compounds
could be tested to prevent GAPDH inactivation as it could help delay the onset of the
pathophysiological conditions in the lungs. Some of these therapeutics include:
109
N-acetyl cysteine
N-acetyl cysteine is a glutathione precursor that effectively maintains cellular glutathione
levels and prevents the depletion observed with acrolein toxicity and chronic CS
exposure. It is also used as a mucolytic agent in COPD management. Along with its
indirect antioxidant effect, NAC can also directly interact with electrophiles in CS and
form NAC disulfide, thus neutralizing the deleterious effects of CS (Dekhuijzen and van
Beurden, 2006). NAC has also been shown to rescue the inflammatory effects of CS in
some studies.
Deprenyl
Deprenyl is a selective and irreversible Monoamine Oxidase B inhibitor, which at 10
times lower doses is known to prevent the S-nitrosylation of GAPDH. Nitric oxide being
110
one of the major constituents of CS may be expected to cause S-nitrosylation of the
active site cysteines in GAPDH. The prevention of GAPDH S-nitrosylation may help
prevent the translocation of GAPDH to the nucleus and initiation of the death cascade
(Hara et al., 2005).
Lipoic acid
Lipoic acid has the ability to oxidize the cysteine residues on Keap 1 protein leading to its
dissociation from Nrf2. Lipoic acid increases Nrf-2 driven transcription of phase II-
enzymes related to detoxification and antioxidant response. This action of lipoic acid is
known to protect retinal pigment epithelium cells and human lung fibroblasts from
acrolein toxicity. Genetic ablation of Nrf2 is also know to enhance susceptibility to
cigarette smoke induced emphysema in mice (Rangasamy et al., 2004a).
In addition to these the MAPK pathway leads to transcriptional activation of a
number of pro-inflammatory cytokines in response to cigarette smoke exposure. The
changes in the levels of pro-inflammatory cytokines were not significant after 8 wk of CS
exposure, but longer duration of exposure may be expected to induce significant
inflammatory response. A number of MEK and Raf kinase inhibitors could be utilized to
prevent the activation of this pro-inflammatory pathway.
111
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Abstract (if available)
Abstract
Cigarette smoking (CS) leads to alteration in cellular redox status, a hallmark in the pathogenesis of chronic obstructive pulmonary disease (COPD). The current study was undertaken to determine the role of CS in the development of mitochondrial dysfunction due to oxidative stress as a consequence of altered redox status. Male A/J mice were exposed to CS generated by a smoking machine for 4 or 8 weeks. A recovery group was exposed to CS for 8 weeks and allowed to recover for 2 week. Our data indicate that short-term cigarette smoke exposure leads to altered metabolism of glucose due to oxidative modification of GAPDH, a central glycolytic enzyme and a concurrent increase in the pentose phosphate pathway of glucose metabolism. On the other hand, the activity and expression of mitochondrial respiratory chain complexes II, IV, and V were found to increase after 8 weeks of CS exposure. Microarray analysis of gene expression in mouse lungs after exposure to CS for 8 weeks revealed upregulation of a group of genes involved in metabolism, electron transfer chain, oxidative phosphorylation, mitochondrial transport and dynamics, and redox regulation. To follow up on the source of substrates for mitochondrial respiratory chain mediated oxidative phosphorylation, we studied the effect of CS on primary alveolar Type II (pAT2) cells isolated from mice exposed to CS. The Type II alveolar cells showed a decrease in mitochondrial respiration while metabolizing glucose and increased respiration on fatty acids (palmitate). This metabolic shift was also observed in RLE-6TN and primary alveolar type II cells in response to acrolein exposure. pAT2 cells also showed an increase in expression of FAT/CD36 and CPT1 after CS exposure. Phosphatidylcholine levels were found to decrease after CS exposure along with an increase in the cytosolic PLA₂ activity. These changes in surfactant phospholipid biosynthesis and metabolism were also observed in type II alveolar cells after acrolein exposure. Thus, palmitate present in alveolar cells for surfactant synthesis could serve as an energy substrate in the event of altered glucose metabolism in alveolar cells. This ability to utilize alternate substrates for energy production was found to be specific to alveolar cells as clara (club) cells did not shown a similar increase in palmitate metabolism after acrolein exposure.
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Agarwal, Amit Rajendra
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Metabolic shift in lung alveolar cell mitochondria after exposure to environmental toxicants
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Molecular Pharmacology and Toxicology
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11/19/2014
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acrolein
alveolar cells
GAPDH
mitochondria
oxidative stress
pulmonary surfactant
respiration