Leptin is key to peroxynitrite-mediated stress and Kupffer cell activation in experimental non-alcoholic steatohepatitis

Environmental Health and Disease Laboratory, Department of Environmental Health Sciences, University of South Carolina, Columbia 29208 USA
Journal of Hepatology (Impact Factor: 11.34). 11/2012; 58(4). DOI: 10.1016/j.jhep.2012.11.035
Source: PubMed
ABSTRACT
Background & aims:
Progression from steatosis to steatohepatitic lesions is hypothesized to require a second hit. These lesions have been associated with increased oxidative stress, often ascribed to high levels of leptin and other proinflammatory mediators. Here we have examined the role of leptin in inducing oxidative stress and Kupffer cell activation in CCl4-mediated steatohepatitic lesions of obese mice.

Methods:
Male C57BL/6 mice fed with a high-fat diet (60%kcal) at 16 weeks were administered CCl₄ to induce steatohepatitic lesions. Approaches included use of immuno-spin trapping for measuring free radical stress, gene-deficient mice for leptin, p47 phox, iNOS and adoptive transfer of leptin primed macrophages in vivo.

Results:
Diet-induced obese (DIO) mice, treated with CCl4 increased serum leptin levels. Oxidative stress was significantly elevated in the DIO mouse liver, but not in ob/ob mice, or in DIO mice treated with leptin antibody. In ob/ob mice, leptin supplementation restored markers of free radical generation. Markers of free radical formation were significantly decreased by the peroxynitrite decomposition catalyst FeTPPS, the iNOS inhibitor 1400W, the NADPH oxidase inhibitor apocynin, or in iNOS or p47 phox-deficient mice. These results correlated with the decreased expression of TNF-alpha and MCP-1. Kupffer cell depletion eliminated oxidative stress and inflammation, whereas in macrophage-depleted mice, the adoptive transfer of leptin-primed macrophages significantly restored inflammation.

Conclusions:
These results, for the first time, suggest that leptin action in macrophages of the steatotic liver, through induction of iNOS and NADPH oxidase, causes peroxynitrite-mediated oxidative stress thus activating Kupffer cells.

Full-text

Available from: Ronald P Mason, Jun 11, 2015
Leptin is key to peroxynitrite-mediated oxidative stress and
Kupffer cell activation in experimental non-alcoholic steatohepatitis
Saurabh Chatterjee
1,2,
, Douglas Ganini
2
, Erik J. Tokar
3
, Ashutosh Kumar
2
, Suvarthi Das
1
,
Jean Corbett
2
, Maria B. Kadiiska
2
, Michael P. Waalkes
3
, Anna Mae Diehl
4
, Ronald P. Mason
2
1
Environmental Health and Disease Laboratory, Department of Environmental Health Sciences, University of South Carolina, Columbia,
SC 29208, USA;
2
Free Radical Metabolism Group, Laboratories of Toxicology and Pharmacology, National Institute of Environmental Health
Sciences, Research Triangle Park, NC 27709, USA;
3
Inorganic Toxicology Group, Division of National Toxicology Program, National Institute
of Environmental Health Sciences, Research Triangle Park, NC 27709, USA;
4
Division of Gastroenterology, Duke University,
Durham, NC 27707, USA
Background & Aims: Progression from steatosis to steatohepatit-
ic lesions is hypothesized to require a second hit. These lesions
have been associated with increased oxidative stress, often
ascribed to high levels of leptin and other proinflammatory medi-
ators. Here we have examined the role of leptin in inducing oxi-
dative stress and Kupffer cell activation in CCl
4
-mediated
steatohepatitic lesions of obese mice.
Methods: Male C57BL/6 mice fed with a high-fat diet (60% kcal)
at 16 weeks were administered CCl
4
to induce steatohepatitic
lesions. Approaches included use of immuno-spin trapping for
measuring free radical stress, gene-deficient mice for leptin,
p47 phox, iNOS and adoptive transfer of leptin primed macro-
phages in vivo.
Results: Diet-induced obese (DIO) mice, treated with CCl
4
increased serum leptin levels. Oxidative stress was significantly
elevated in the DIO mouse liver, but not in ob/ob mice, or in
DIO mice treated with leptin antibody. In ob/ob mice, leptin sup-
plementation restored markers of free radical generation. Mark-
ers of free radical formation were significantly decreased by the
peroxynitrite decomposition catalyst FeTPPS, the iNOS inhibitor
1400W, the NADPH oxidase inhibitor apocynin, or in iNOS or
p47 phox-deficient mice. These results correlated with the
decreased expression of TNF-alpha and MCP-1. Kupffer cell
depletion eliminated oxidative stress and inflammation, whereas
in macrophage-depleted mice, the adoptive transfer of leptin-
primed macrophages significantly restored inflammation.
Conclusions: These results, for the first time, suggest that leptin
action in macrophages of the steatotic liver, through induction of
iNOS and NADPH oxidase, causes peroxynitrite-mediated oxida-
tive stress thus activating Kupffer cells.
Ó 2012 European Association for the Study of the Liver. Published
by Elsevier B.V. All rights reserved.
Introduction
Leptin’s role as a proinflammatory adipocytokine has gained
attention in non-alcoholic steatohepatitis. Circulating leptin
levels are elevated in obesity and NASH. Leptin-induced cyto-
kine release, especially of IL-1 and TNF-
a
, has been shown in
microglia and monocytes [1,2]. Leptin acts on Kupffer cells,
the resident macrophages, by binding to its functional receptor
in the liver and inducing the release of TNF-
a
, TGF-beta and IL-
15 [3–5].
Despite wide-ranging reports of leptin’s role in inflamma-
tion and release of inflammatory mediators, its role in inducing
oxidative stress in the liver remains unclear. There are reports
regarding leptin-induced reactive oxygen species formation by
different cell types, including endothelial cells, cardiomyocytes
and hepatic stellate cells [6–8]. These studies focused on reac-
tive oxygen species formation but the mechanisms of free rad-
ical species generation and their link to exacerbated
inflammation through Kupffer cell activation are not com-
pletely understood. Since leptin is known to induce both
NADPH oxidase and iNOS, the resultant superoxide and nitric
oxide can react at a diffusion-controlled rate to produce perox-
ynitrite, a strong physiological oxidant. Peroxynitrite can form
several free radical species including
OH,
CO
3
and
NO
2
radi-
cals, depending on the pathophysiological microenvironment
[9–11].
Based on the available studies on the role of leptin in oxi-
dative stress induction and inflammation in steatohepatitis,
we hypothesized that leptin-induced peroxynitrite and its
ensuing free radical formation play a major role in early liver
injury in obesity. Here we show that CCl
4
administration in
diet-induced obese mice increases circulating levels of leptin;
we also demonstrate that heightened levels of leptin contribute
significantly to the pathogenesis of the resultant liver damage
by activating NADPH oxidase, inducing iNOS, and activating
release of TNF-
a
and MCP-1 from Kupffer cells by peroxyni-
trite-dependent mechanisms. Furthermore, we prove that leptin
exerts its free radical formation and proinflammatory effects
mainly by acting on macrophages and Kupffer cells of obese
mice.
Journal of Hepatology 2013 vol. 58
j
778–784
Keywords: Adipocytokines, Kupffer cell; Oxidative stress; Tyrosine nitration;
NADPH oxidase; ob/ob mice.
Received 7 March 2012; received in revised form 16 November 2012; accepted 20
November 2012; available online 1 December 2012
Corresponding author. Address: Environmental Health and Disease Laboratory,
Department of Environmental Health Sciences, University of South Carolina,
Columbia, SC 29208, USA. Tel.: +1 803 777 8120; fax: +1 803 777 3199.
E-mail address: schatt@mailbox.sc.edu (S. Chatterjee).
Research Article
Page 1
Materials and methods
Obese mice
Custom DIO adult male, pathogen-free, with a C57BL6/J background (Jackson
Laboratories, Bar Harbor, Maine) were used as models of diet-induced obesity.
The mice were fed a high-fat diet (60% kcal) from 6 weeks until 16 weeks. All
experiments were conducted in the 16-week age group. Age-matched lean
controls were fed with a diet having 10% kcal fat. The animals were housed,
one animal per cage before any experimental use. Mice bearing the disrupted
OB gene (leptin) (B6.V-Lep<ob>/J) (Jackson Laboratories), disrupted p47 phox
(B6.129S2-Ncf1
tm1shl
N14) (Taconic, Cranbury, NJ) genes, or disrupted NOS2
(B6.129P2-Nos2
tm1Lau
/J, Jackson Laboratories; C57BL6 background) were fed with
a high-fat diet and treated identically to DIO obese mice. Mice had ad libitum
access to food and water and were housed in a temperature-controlled room at
23–24 °C with a 12-h light/dark cycle. All animals were treated in strict
accordance with the NIH Guide for the Humane Care and Use of Laboratory
Animals, and the experiments were approved by the institutional review board.
Induction of liver injury in obese mice
DIO mice or high-fat-fed gene-specific knockout mice at 16 weeks were adminis-
tered carbon tetrachloride (0.8 mmoles/kg, diluted in olive oil) through the intra-
peritoneal route. This model is a free radical-based mechanistic model for non-
alcoholic steatohepatitis [12].
Administration of allopurinol, FeTPPS and 1400W
Allopurinol, a specific inhibitor of xanthine oxidase, was administered in a single
bolus dose of 35 mg/kg through the i.p. route, 30 min prior to carbon tetrachlo-
ride treatment [13]. In other studies, the iNOS inhibitor 1400W was administered
through the intraperitoneal route at a dose of 10 mg/kg, 1 h before carbon tetra-
chloride treatment, using an intraperitoneal route [14]. FeTPPS was administered
at 30 mg/kg, 1 h prior to CCl
4
treatment [10,15].
Administration of mouse recombinant leptin and leptin neutralization
ob/ob mice received recombinant leptin (100
l
g/mice) twice daily for 5 days prior
to CCl
4
administration through the intraperitoneal route. Leptin antibody was
used to neutralize the circulating leptin in DIO mice. DIO mice were treated for
2 days prior to CCl
4
administration either with 100
l
g of control mouse IgM or
with mouse leptin Abs intraperitoneally in a total volume of 100
l
l of PBS [16].
Isolation of Kupffer cells
Kupffer cells were isolated as per the protocol by Froh et al. [17]. Qualitative
screening for Kupffer cells was carried out with immunoreactivity against a
CD68 antibody. Cultures with >80% CD68-positive cells were used for the
experiments.
Enzyme-linked immunosorbent assay
Immuno-spin trapping, a method for detection of free radical formation, was
used, and immunoreactivity for DMPO nitrone adducts and nitrotyrosine was
detected in liver homogenates and Kupffer cell lysates using standard ELISA
[10].
Western blot analysis
Liver homogenates were resolved in 4–10% Bis-Tris gels using SDS–PAGE, and
subjected to Western blot analysis.
Histopathology
For each animal, sections of the liver were collected and fixed in 10% neutral
buffered formalin. For histological examinations, formalin-fixed liver sections
were stained with hematoxylin/eosin (H&E) and observed under a light
microscope.
Real-time reverse transcription–polymerase chain reaction analysis
Gene expression levels in tissue samples were measured by real-time reverse
transcription–polymerase chain reaction analysis as described in Supplementary
material.
Confocal laser scanning microscopy (Zeiss LSM 510 UV meta)
Frozen tissue sections after formalin fixation were analyzed by confocal micros-
copy, Zeiss LSM710-UV meta (Carl Zeiss, Inc., Oberkochen, Germany), using a
Plan-NeoFluor 40/1.3/40 Oil DIC objective with different zoom levels.
Macrophage depletion by GdCl
3
and liposomal clodronate
Mice were injected with gadolinium chloride (10 mg/kg) through the i.v. route
24 h prior to CCl
4
treatment, as described by Rai et al. [18]. Liposomal clodronate
was injected through intravenous injections at a dose of 4
l
l/g of mice (Clopho-
some™; Formumax, Pao Alto, CA), 24 h prior to CCl
4
treatment.
Adoptive transfer of leptin primed cells
Mouse non-parenchymal cells (mostly Kupffer cells) were isolated as per Froh
et al. [17]. Cells were washed and plated in 35 mm
2
dishes using 10% FBS contain-
ing DMEM with mouse recombinant leptin (500 ng/ml). The dose was selected on
the basis of the concentration used by Wang et al. (10–100 nmoles/L) [4]. The cells
were harvested at 18 h and 1 10
6
cells/mouse were injected through the tail
vein into macrophage depleted mice. The recipient mice were macrophage-
depleted by the administration of the macrophage toxin gadolinium chloride.
Statistical analyses
All in vivo experiments were repeated three times with 3 mice per group (N = 3;
data from each group of three mice was pooled). The statistical analysis was car-
ried out by analysis of variance (ANOVA) followed by a post hoc test. Quantitative
data from Western blots, as depicted by the relative intensity of the bands, were
analyzed by performing a Student’s t test. p <0.05 was considered statistically
significant.
Results
Increased leptin levels cause oxidative and nitrosative stress in DIO-
steatohepatitic mice
Our results indicated that diet-induced obese mice had signifi-
cantly higher leptin levels as compared to lean control mice
(Fig. 1A), which is in line with human studies [19]. Our previous
study established a free radical-based mechanistic model of non-
alcoholic steatohepatitis where low-dose CCl
4
administration
induced non-alcoholic steatoheaptitis in obese mice [12]. Fur-
thermore, DIO mice treated with CCl
4
had significantly higher
serum leptin levels when compared to both untreated DIO and
CCl
4
-treated lean control mice (Fig. 1A). The study showed that
leptin deficiency significantly decreased protein radical forma-
tion. We found that when CCl
4
-treated leptin deficient mice
and CCl
4
-treated DIO mice were administered a neutralizing anti-
body against leptin, they had significantly decreased protein
DMPO nitrone adduct formation, a measurement of free radical
formation on proteins (Fig. 1B), compared to DIO mice treated
with CCl
4
only [20]. Treating leptin deficient mice with a seven-
day course of recombinant leptin increased their protein radical
formation in response to CCl
4
administration to levels that were
comparable to those in wild type DIO mice (Fig. 1B).
Since protein 3-nitrotyrosine formation originates from tyro-
syl radicals reacting with reactive nitrogen species [21],we
JOURNAL OF HEPATOLOGY
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Page 2
probed nitrotyrosine formation as a stable post-translational
oxidative modification. Results from laser scanning confocal
microscopy showed both sinusoidal and centrolobular 3-
nitrotyrosine immunoreactivity in DIO mouse livers treated
with CCl
4
and in leptin deficient mice treated with CCl
4
and
recombinant leptin (Fig. 1C). However, leptin deficient mice
and DIO mice administered leptin monoclonal antibody, simi-
larly treated with CCl
4
, had 3-nitrotyrosine immunoreactivity
primarily in the sinusoids.
Finally, quantification of fluorescent intensities of 3-nitrotyro-
sine immunoreactivity from these groups showed that when
CCl
4
-treated leptin deficient mice and CCl
4
-treated DIO mice
were administered a neutralizing antibody against leptin, they
had significantly decreased 3-nitrotyrosine formation (Fig. 1D)
compared to DIO mice treated with CCl
4
. However, nitrotyrosine
reactivity increased significantly following a seven-day course of
recombinant leptin treatment.
Serum levels of leptin might have originated from both the
adipose tissue and liver since Western blot analysis of leptin
showed significant increases in both liver and adipose tissue fol-
lowing CCl
4
treatment (Supplementary Fig. 2A).
Leptin augments proinflammatory cytokines that are markers of
Kupffer cell activation
Oxidative stress can play a significant role in Kupffer cell activa-
tion [12,22,23]. Kupffer cell activation can be marked by
increased release of TNF-
a
and monocyte chemoattractant pro-
tein-1 (MCP-1). Thus we analyzed leptin-induced effects on
TNF-
a
and MCP-1 in DIO mice treated with CCl
4
. We found that
CCl
4
-treated leptin deficient mice and CCl
4
-treated DIO mice,
administered neutralizing antibody against leptin, had signifi-
cantly decreased TNF-
a
and MCP-1 levels (Supplementary
Fig. 1A) compared to DIO mice treated with CCl
4
alone. However,
pre-treating leptin deficient mice with a seven-day course of
recombinant leptin restored their post-CCl
4
TNF-
a
and MCP-1
levels to those of wild type DIO mice that were treated with
CCl
4
(Supplementary Fig. 1B). Liver activation of NF
j
B was also
increased in DIO mice treated with CCl
4
as seen from increased
translocation of p65 unit to the nucleus (Supplementary Fig. 2C).
Peroxynitrite from NADPH oxidase and iNOS activity is a key
regulator of Kupffer cell activation in steatohepatitic injury
When leptin deficient mice and DIO mice, administered a mono-
clonal antibody against leptin, were treated with CCl
4
, their levels
of p47 phox and iNOS mRNA expression were significantly less
(Fig. 2A and B) compared to DIO mice treated with CCl
4
. When
leptin deficient mice were pretreated with recombinant leptin
before CCl
4
exposure, expression of p47 phox mRNA increased
significantly from the leptin deficient level to a point near to,
but less than that of (hyperleptinemic) DIO mice treated with
CCl
4
(Fig. 2A), while iNOS mRNA expression also increased signif-
icantly when compared to leptin deficient mice identically trea-
ted, and was comparable to that in DIO mice treated with CCl
4
(Fig. 2B). The significant increase in mRNA expression of both
p47 phox and iNOS in DIO mice was observed 24 h following
treatment with CCl
4,
thus coinciding with the increase in protein
radical formation and nitrotyrosine formation. Since we observed
a significant leptin-dependent increase in both p47 phox and iNOS
mRNA expression, we explored the role of peroxynitrite in the
formation of protein radicals and tyrosine nitration. Peroxynitrite
is a key oxidant species, and nitric oxide formed from iNOS reacts
with superoxide, in a diffusion controlled rate, to form peroxyni-
trite. We used the peroxynitrite decomposition catalyst FeTPPS
administered in vivo to assess the role of peroxynitrite as
reported by Chatterjee et al. [15] and others [24,25]. In DIO mice
treated with CCl
4
and administered FeTPPS, protein radical for-
mation and tyrosine nitration were significantly decreased
(Fig. 2C and D). They also decreased significantly when mice were
administered the NADPH oxidase inhibitor apocynin or the iNOS
inhibitor 1400W. When fed with a high-fat diet and treated with
CCl
4
, mice that lacked the NADPH oxidase subunit p47 phox or
iNOS had significantly decreased protein radical formation and
tyrosine nitration compared to DIO mice treated with CCl
4
(Fig. 2C and D).
Confocal laser scanning microscopy showed decreased protein
radical adducts and nitrotyrosine immunoreactivity in the cen-
trolobular regions of CCl
4
-treated mouse livers, after administra-
tion of FeTPPS, compared to CCl
4
-treated DIO mice after vehicle
treatment only (without FeTPPS) (Fig. 2E). FeTPPS also had a sig-
nificant effect in reducing both mRNA and protein levels of pro-
inflammatory cytokines that exacerbate sterile inflammation in
0
5000
10,000
15,000
20,000
25,000
30,000
Arbitrary fluorescent units
123
0
45
90
135
180
Serum leptin levels
(ng/ml)
A B
DC
1
0
5000
10,000
15,000
20,000
25,000
30,000
DMPO nitrone adducts
(relative light units)
Lean control
DIO mice
DIO mice
DIO mice
ob/ob mice
DIO mice
ob/ob mice
ob/ob mice
CCl
4
-
treated
CCl
4
-treated
*
*
*
*
*
*
*
+ rLeptin
+ Leptin mAb
CCl
4
-treated
CCl
4
-treated
+ rLeptin
+ Leptin mAb
+ rLeptin+ Leptin mAb
Fig. 1. Increased leptin levels cause free radical and nitrosative stress in DIO-
steatohepatitic mice. Lean control or high-fat fed diet induced obese (DIO) mice
were treated with either olive oil (vehicle) or CCl
4
. (A) Serum levels at 24 h post
CCl
4
. (B) Liver homogenates from DIO, ob/ob, leptin supplemented and DIO mice
injected with leptin antibody were subjected to immuno-spin trapping and anti-
DMPO immunoreactivity was measured using ELISA. (C) Frozen liver slices were
analyzed for 3-nitrotyrosine immunoreactivity. (D) Relative fluorescence inten-
sities of 3-nitrotyrosine immunoreactivity from mouse livers (n = 4).
p <0.05.
(This figure appears in color on the web.)
Research Article
780
Journal of Hepatology 2013 vol. 58
j
778–784
Page 3
steatohepatitis. In CCl
4
-treated DIO mice administered FeTPPS,
both TNF-
a
and MCP-1 mRNA expression and protein levels were
significantly decreased, compared to DIO mice treated with CCl
4
alone (Figs. 2F and 3A–D). Similarly, pharmacologically inhibiting
either iNOS or NADPH oxidase or using p47 phox and iNOS gene-
deleted mice, resulted in significantly decreased TNF-
a
and MCP-
1 mRNA and protein levels post-CCl
4
treatment, compared to DIO
mice treated with CCl
4
(2F and 3A–D).
Leptin-mediated protein radical and 3-nitrotyrosine forma-
tion and release of the proinflammatory cytokines TNF-
a
and
MCP-1 are Kupffer cell and macrophage dependent.
Resident liver macrophages (primarily Kupffer cells) and infil-
trating neutrophils and other immune cells that exhibit macro-
phage-like function are found to express both the vascular form
of NADPH oxidase and iNOS. Consistent with that concept, we
found that protein radical formation and tyrosine nitration were
significantly reduced upon administration of the macrophage
toxin GdCl
3
or liposomal clodronate to DIO mice before treatment
with CCl
4
(Fig. 4A and B; Supplementary Fig. 6), and they were
not restored upon further supplementation of recombinant lep-
tin. However, they were significantly increased after adoptive
transfer of leptin-primed Kupffer cells into DIO mice treated with
CCl
4
or ob/ob mice (Fig. 4A and B; Supplementary Fig. 6). Since
TNF-
a
and MCP-1 release from macrophages contribute to ste-
atohepatitic lesions in obesity following treatment with CCl
4
,it
was important to explore whether the leptin effect on release
of the cytokines was also macrophage dependent. Results indi-
cated that administration of macrophage toxin GdCl
3
to DIO mice
before treatment with CCl
4
, significantly decreased TNF-
a
and
MCP-1 release (Fig. 4C and D; Supplementary Fig. 6). These
results thus clearly suggested that leptin was acting through
macrophages in the CCl
4
-treated DIO mouse liver in exacerbating
the steatohepatitic lesions in these mice.
Leptin contributes to steatohepatitic lesions in CCl
4
-treated DIO mice
To examine the effect of leptin in inducing steatohepatitic lesions
following CCl
4
administration in DIO mice, we compared the
histopathology of steatohepatitis in leptin deficient mice and
mice fed with a high-fat diet. DIO mice with higher leptin levels,
1
0
2
4
6
mRNA expression
(x fold DIO)
mRNA expression
(x fold DIO)
1
0.0
1.0
2.0
0
10
20
30
40
DIO
p47 phox
KO
DIO
iNOS
KO
DIO + APO
DIO
+ 1400W
DIO + FeTPPS
DIO
DIO
mRNA expression
(x fold DIO)
A B
E F
DIO mice
ob/ob mice
DIO mice
ob/ob mice
+ rLeptin
+ Leptin mAb
CCl
4
-treated
+ rLeptin
+ Leptin mAb
CCl
4
-treated
*
*
*
*
*
*
*
*
p47 phox iNOS
+ APO
+ 1400W
+ FeTPPS
CCl
4
-treated
0
5000
10,000
15,000
20,000
25,000
30,000
35,000
0
2000
6000
10,000
14,000
18,000
22,000
+ APO
+ 1400W
+ FeTPPS
No DMPO
DMPO nitrone adducts
(relative light units)
Nitrotyrosine adducts
(relative light units)
C D
DIO mice
DIO iNOS KO
DIO p47 phox KO
DIO mice
DIO iNOS KO
DIO p47 phox KO
CCl
4
-treated
CCl
4
-treated
*
*
Protein radical adducts
3-nitrotyrosine adducts
CCl
4
-treated
DIO DIO DIO + FeTTPS
TNF-α
MCP-1
*
Fig. 2. Peroxynitrite from NADPH oxidase and iNOS activity is a key regulator in Kupffer cell activation in steatohepatitic injury. (A) Quantitative real-time PCR
analysis of p47 phox and (B) iNOS mRNA expression in DIO, DIO + CCl
4
, ob/ob mice, with or without leptin supplementation, and DIO + CCl
4
mice treated with leptin
monoclonal antibody. (C) Liver homogenates from CCl
4
-treated DIO mice, either challenged with the peroxynitrite decomposition catalyst FeTPPS or iNOS inhibitor 1400W
or NADPH oxidase inhibitor apocynin; or using iNOS and p47 phox knockout mice, were assayed for anti-DMPO immunoreactivity, and (D) for 3-nitrotyrosine
immunoreactivity using ELISA. (E) Localization of DMPO nitrone adducts (red, upper panel) or nitrotyrosine adducts (green, lower panel) was analyzed by confocal laser
scanning microscopy of liver slices from CCl
4
-treated mice, with or without the peroxynitrite decomposition catalyst FeTPPS, and is a representative of liver slices collected
from 4 mice (n = 4). (F) Inhibitors of peroxynitrite decrease macrophage activation. Liver homogenates were assayed for TNF-
a
and MCP-1 mRNA expression. The figure
represents data from 3 mice/group and
p <0.05. (This figure appears in color on the web.)
JOURNAL OF HEPATOLOGY
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Page 4
treated with CCl
4
had centrolobular necrosis and showed markers
of early steatohepatitic injury (Fig. 4Ei). When DIO mice that
received FeTPPS (Fig. 4Eii), or leptin deficient mice (ob/ob)
(Fig. 4Eiii) were administered CCl
4
, they showed steatosis, peri-
portal necrosis and occasional necrotic areas around the central
vein (Zone III), with little or no leukocyte accumulation compared
to wild type DIO mice treated with CCl
4
(Fig. 4Ei), which had cen-
trilobular necrosis, infiltration of leukocytes, and ballooned hepa-
tocytes. This result suggests that leptin contributes significantly
in inducing early inflammatory lesions in DIO mice treated with
CCl
4
. Consistent with this result, serum ALT levels in DIO mice
significantly increased compared to leptin deficient mice admin-
istered CCl
4
(Supplementary Fig. 2B). Kupffer cell aggregation
near the perivenular regions, a hallmark of early steatohepatitic
lesions, was also evident by confocal microscopy of CD68 positive
cells (Supplementary Fig. 7). Histological Activity Index score was
found to be significantly higher for DIO mice treated with CCl
4
when compared to ob/ob mice (Supplementary Table 1).
Discussion
This is the first report that shows that leptin-mediated protein
radical formation, tyrosine nitration and activation of Kupffer
cells are caused by peroxynitrite formation, and demonstrates
that this process exacerbates CCl
4
-induced steatohepatitic lesions
in diet-induced obesity. We found that exposing mice with DIO to
a potential ‘‘2nd hit’’ from CCl
4
caused a twofold increase in cir-
culating leptin levels (Fig. 1). This result was central to our sub-
sequent investigations, which established that leptin-induced
formation of peroxynitrite was key to drive the ensuing inflam-
matory processes. Higher leptin levels, as a result of CCl
4
admin-
istration in DIO mice, might be due to the release of IL-beta, as
shown previously by others [26]. Since diet-induced obese mice
that were treated with CCl
4
had higher leptin levels (2-fold higher
than vehicle-treated obese mice), and leptin is known to cause
0
5
10
15
20
25
30
35
DMPO nitrone adducts
(relative light units x10
3
)
0
200
400
600
800
1000
0
2000
4000
6000
4x image 4x image 4x image
A
B
C D
E
DIO mice
DIO + GdCl
3
ob/ob + GdCl
3
DIO mice
DIO + GdCl
3
ob/ob + GdCl
3
DIO mice
DIO + GdCl
3
ob/ob + GdCl
3
CCl
4
-treated
CCl
4
-treated CCl
4
-treated
CCl
4
+++
GdCl
3
++
Leptin
+
DIO
DIO + CCl
4
+ GdCl
3
+ leptin primed MØ
ob/ob + CCl
4
+ GdCl
3
+ leptin primed MØ
*
*
*
rLeptin
rLeptin
primed MØ
(through i.v. route)
*
rLeptin
rLeptin
primed MØ
(through i.v. route)
*
rLeptin
rLeptin
primed MØ
(through i.v. route)
*
Serum TNF-α (pg/ml)
Serum MCP-1 (pg/ml)
(i) (ii) (iii)
Fig. 4. Leptin action through liver macrophages and Kupffer cells is respon-
sible for peroxynitrite chemistry and subsequent free radical formation in
steatohepatitc injury. Macrophage toxin gadolinium chloride was used to
deplete liver macrophages and was followed by CCl
4
treatment in DIO mice. In
certain experimental groups, which had their macrophages depleted, either only
leptin or leptin primed macrophages were infused through tail vein. (A) Whole
liver homogenates from these mice were then analyzed for anti-DMPO immu-
noreactivity by ELISA. (B) Liver slices of mice from the above experimental set-up
and adoptive transfer of leptin or leptin primed macrophages were subjected to
confocal laser scanning microscopy to analyze 3-nitrotyrosine immunoreactivity
and localization. Analysis of serum TNF-
a
(C), and MCP-1 (D) was assayed by
ELISA. The figure represents data from 3 mice/group and
p <0.05 was considered
statistically different. (E) H&E of the liver of obese, CCl
4
-treated mice adminis-
tered either saline (i) or FeTPPS (ii) or CCl
4
-treated ob/ob mice (iii). The images
represent both a 4 magnification and a 20 magnification (inset). MØ,
macrophage. (This figure appears in color on the web.)
1
0
1000
2000
3000
34
Serum MCP-1 (pg/ml)
0
200
400
600
800
1000
1200
Serum TNF-α (pg/ml)
TNF-α/β-actin
MCP-1/β-actin
A
B
C
D
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.0
0.2
0.4
0.6
0.8
1.0
+ APO
+ 1400W
+ FeTPPS
DIO mice
DIO iNOS KO
DIO p47 phox KO
DIO mice
WT mice
DIO iNOS KO
DIO p47 phox KO
DIO mice
WT mice
DIO iNOS KO
DIO p47 phox KO
CCl
4
-treated
+ APO
+ 1400W
+ FeTPPS
CCl
4
-treated
+ APO
+ 1400W
+ FeTPPS
CCl
4
-treated
+ APO
+ 1400W
+ FeTPPS
CCl
4
-treated
* *
* *
Liver TNF-α Liver MCP-1
TNF-α
β-actin
MCP-1
β-actin
Fig. 3. Leptin-induced oxidative stress and peroxynitrite formation activate
Kupffer cells. Serum and liver tissue (A and C) TNF-
a
and (B and D) MCP-1
analyses following blockade of peroxynitrite formation. The figure represents
data from 3 mice/group and
p <0.05.
Research Article
782
Journal of Hepatology 2013 vol. 58
j
778–784
Page 5
oxidative stress [27], we investigated the contribution of leptin to
the protein radical formation and tyrosine nitration in early ste-
atohepatitis. In ob/ob mice and DIO mice, treatment with a neu-
tralizing antibody against leptin significantly reduced protein
radical formation, whereas leptin supplementation for 7 days
restored higher protein radical formation and tyrosine nitration,
suggesting that leptin is responsible for the redox imbalance in
the liver in early steatohepatitic injury (Fig. 1B–D). This result
assumes significance since leptin is known to cause oxidative
stress in different tissues and cells in response to inflammatory
stress [6–8]. Furthermore, leptin-mediated reactive oxygen spe-
cies generation was found to activate stellate cell proliferation,
a known phenomenon in steatohepatitic injury [8]. However, lit-
tle is known with regard to the redox mechanisms by which
Kupffer cells promote liver inflammation and fibrosis in obesity.
Our results indicated that in addition to leptin’s involvement in
protein radical formation and tyrosine nitration, it is also
involved in activating Kupffer cell release of TNF-
a
and MCP-1
(Supplementary Fig. 1), but how the cytokine release and Kupffer
cell activation are related to the redox processes initiated by lep-
tin remained unknown at this point.
Therefore, to establish the mechanism of free radical forma-
tion by leptin action, we analyzed the mRNA expression of
enzymes that are significant contributors to production of free
radical species in inflammatory microenvironments. We found
that levels of expression of inducible NOS and p47 phox mRNA
were significantly elevated in early steatohepatitic injury and
leptin-supplemented ob/ob mice (Fig. 2A–B). In addition, ob/ob
mice had less induction of mRNA for these enzymes. This data
suggested that nitric oxide and superoxide radicals play a signif-
icant role in the protein radical formation process by leptin. Fur-
thermore, since protein radical formation and tyrosine nitration
were localized in Kupffer cells [12], there was a possibility that
Kupffer cells, which are known to express both NADPH oxidase
and inducible NOS, might form peroxynitrite from superoxide
and nitric oxide, respectively, at a diffusion-controlled rate. Such
a reaction is feasible since both species are found in the same cell
at the same time. Our results showed that the peroxynitrite
decomposition catalyst FeTPPs, the NADPH oxidase inhibitor
apocynin and the iNOS inhibitor 1400W, all significantly inhib-
ited protein radical formation and tyrosine nitration (Fig. 2C
and D). High-fat fed mice, depleted of iNOS and the NADPH oxi-
dase subunit p47 phox, and treated with CCl
4,
showed significant
decreases in protein radical formation and tyrosine nitration,
thus confirming the role of these enzymes in the formation of
peroxynitrite.
The role of peroxynitrite formation in the activation of Kupffer
cells was established by examining subsequent TNF-
a
and MCP-1
mRNA expression in the presence of FeTPPS and inhibitors of
iNOS and NADPH oxidase, as well as the use of gene-deficient
mice for both iNOS and p47 phox (Fig. 2F). The radicals that are
generated from peroxynitrite form stable post-translational mod-
ifications of proteins that can affect their function and contribute
to the activation of inflammatory pathways [15]. In the present
model of early steatohepatitic injury, Kupffer cell activation
(there was an increase in both CD68 and F4/80 immunoreactivity
in liver tissues of CCl
4
-administered DIO mice as compared to DIO
and ob/ob mice; Supplementary Fig. 3.) and production of the
inflammatory cytokines TNF-
a
and MCP-1 might have resulted
from loss or gain of function of the proteins that modulated the
induction response of these genes; however, this point remains
speculative at present. Though there may be multiple sources
of these cytokines, including stellate cells, Kupffer cells were
found to be a principal source of MCP-1 as shown in Supplemen-
tary Fig. 4B.
Although much of our attention in this study was focused on
the ability of either resident macrophages or infiltrating leuko-
cytes to promote leptin-induced formation of peroxynitrite, the
data so far is incomplete without further confirmatory evidence
of their involvement in leptin-induced redox changes, inflamma-
tion, and progression of steatohepatitic injury. To establish the
site of leptin action, we first depleted DIO mice of macrophages
by pre-treating them with GdCl
3
and then adoptively transferred
leptin-primed Kupffer cells into both DIO mice and leptin defi-
cient ob/ob mice. Results showed that protein radical formation,
tyrosine nitration, and release of proinflammatory cytokines
were restored in macrophage-depleted mice only when leptin
primed Kupffer cells were adoptively transferred (Fig. 4). Further-
more, supplementation with leptin only (without macrophages)
in these mice had no effect. Similarly, macrophage-depleted mice
that were leptin deficient exhibited increased protein radical
formation, tyrosine nitration and release of proinflammatory
cytokines, following adoptive transfer of leptin-primed macro-
phages, suggesting the essential roles of both leptin and macro-
phages in potentiating redox-mediated steatohepatitic injury
(Fig. 4). Our results also establish that leptin and macrophages
are symbiotic in their actions in promoting inflammation in the
fatty liver, and it might be speculated that the process involves
multiple signaling mechanisms and intermediary molecules [28].
Finally, we report a leptin-mediated oxidative stress mecha-
nism for inflammatory events promoted by the formation of per-
oxynitrite in macrophages of the steatohepatitic liver.
Financial support
This work has been supported by a K99-R00, NIH pathway to
Independence Award (4R00ES019875-02 to Saurabh Chatterjee)
and the Intramural Research Program of the National Institutes
of Health and the National Institute of Environmental Health Sci-
ences (Z01 ES050139-13 to Ronald P. Mason).
Conflict of interest
The authors who have taken part in this study declared that they
do not have anything to disclose regarding funding or conflict of
interest with respect to this manuscript.
The underlying research reported in the study was funded by
the NIH Institutes of Health. This article may be the work product
of an employee or group of employees of the National Institute of
Environmental Health Sciences (NIEHS), National Institutes of
Health (NIH), however, the statements, opinions or conclusions
contained therein do not necessarily represent the statements,
opinions or conclusions of NIEHS, NIH or the United States
government.
Acknowledgements
The authors gratefully acknowledge James Clark, Tiwanda Marsh,
Jeoffrey Hurlburt, Jeff Tucker, and Ralph Wilson for excellent
JOURNAL OF HEPATOLOGY
Journal of Hepatology 2013 vol. 58
j
778–784
783
Page 6
technical assistance. We also sincerely thank Dr. Ann Motten and
Mary Mason for help in the careful editing of this manuscript.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.jhep.2012.
11.035.
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    • "The higher levels of TNF-α in obese patients stimulate adipocytes to secrete monocyte chemoattractant protein-1 (MCP-1) leading to macrophage recruitment [110]. Then, the macrophages cytokine signaling promotes lipolysis and the release of pro-inflammatory adipokines like leptine, which complete a vicious circle by recruiting and promoting new macrophage activation [111]. The alterations on gut microbiota composition and barrier function resulting in an increase permeability for bacterial endotoxin, gut dysbiosis, has been implicated in chronic metabolic disorders such as obesity, MS, diabetes, and cardiovascular diseases [112]. "
    [Show abstract] [Hide abstract] ABSTRACT: The term nonalcoholic fatty liver disease (NAFLD) encompasses diseases of lipid accumulation in the liver not due to excessive alcohol consumption. NALFD encompasses a series of diseases, from simple liver steatosis to steatohepatitis to cirrhosis. NAFLD is a growing public health burden due to its increasing prevalence and its liver and cardiovascular morbimortality. NAFLD is the liver manifestation of the metabolic syndrome, a cluster of conditions that multiply cardiovascular risk with a common patophysiological pathway centered on insulin resistance. The relationship between obesity, hypertension, endothelial dysfunction and dyslipidemia with NAFLD are analyzed, as well as other diseases associated with the metabolic syndrome that act as development or progression factors NAFLD. Pharmacological and non-pharmacological options for the management of this disease are discussed with an emphasis in their impact in insulin resistance, lowering of liver enzymes and improvement of liver histology.
    No preview · Chapter · Jan 2016
  • Source
    • "The study is significant since it identifies a new mediator for stellate cell activation and proliferation crucial for NASH progression. We and others have shown previously that metabolic oxidative stress increases systemic and hepatic leptin levels over and above the levels found in obesity [23,24]. Increased leptin can perform an array of functions or dysfunctions that affect a myriad of pathways causing dysregulation in immune, endothelial and metabolic functions in NASH [38] . "
    [Show abstract] [Hide abstract] ABSTRACT: Metabolic oxidative stress via CYP2E1 can act as a second hit in NASH progression. Our previous studies have shown that oxidative stress in NASH causes higher leptin levels and induces purinergic receptor X7 (P2X7r). We tested the hypothesis that higher circulating leptin due to CYP2E1-mediated oxidative stress induces P2X7r. P2X7r in turn activates stellate cells and causes increased proliferation via modulating Glut4, the glucose transporter, and increased intracellular glucose. Using a high fat diet-fed NAFLD model where bromodichloromethane (BDCM) was administered to induce CYP2E1-mediated oxidative stress, we show that P2X7r expression and protein levels were leptin and CYP2E1 dependent. P2X7r KO mice had significantly decreased stellate cell proliferation. Human NASH livers showed marked increase in P2X7r, and Glut4 in α-SMA positive cells. NASH livers had significant increase in Glut4 protein and phosphorylated AKT, needed for Glut4 translocation while leptin KO and P2X7r KO mice showed marked decrease in Glut4 levels primarily in stellate cells. Mechanistically stellate cells showed increase in phosphorylated AKT, Glut4 protein and localization in the membrane following administration of P2X7r agonist or leptin+P2X7r agonist, while use of P2X7r antagonist or AKT inhibitor attenuated the response suggesting that leptin-P2X7r axis in concert but not leptin alone is responsible for the Glut4 induction and translocation. Finally P2X7r-agonist and leptin caused increase in intracellular glucose and consumption by increasing the activity of hexokinase. In conclusion, the study shows a novel role of leptin-induced P2X7r in modulating Glut4 induction and translocation in hepatic stellate cells, that are key to NASH progression.
    Full-text · Article · Oct 2015 · Biochimica et Biophysica Acta
  • Source
    • "While EPR methods have long been the gold standard for detection of free radicals in vitro, very few EPR experiments have demonstrated free radical formation intracellularly. Immuno-spin trapping, however, has been used successfully to detect and visualize protein and DNA radical adducts in cells, tissues, and animals131415161718. The major disadvantage of immuno-spin trapping compared to direct EPR, however, is that no information on the chemical structure of the radical is obtained. "
    [Show abstract] [Hide abstract] ABSTRACT: Xenobiotic metabolism can induce the generation of protein radicals, which are believed to play an important role in the toxicity of chemicals and drugs. It is therefore important to identify chemical structures capable of inducing macromolecular free radical formation in living cells. In this study, we evaluated the ability of four structurally related environmental chemicals, aniline, nitrosobenzene, N,N-dimethylaniline, and N,N-dimethyl-4-nitrosoaniline (DMNA), to induce free radicals and cellular damage in the hepatoma cell line HepG2. Cytotoxicity was assessed using lactate dehydrogenase assays, and morphological changes were observed using phase contrast microscopy. Protein free radicals were detected by immuno-spin trapping using in-cell western experiments and confocal microscopy to determine the subcellular locale of free radical generation. DMNA induced free radical generation, lactate dehydrogenase release, and morphological changes in HepG2 cells, whereas aniline, nitrosobenzene, N,N-dimethylaniline did not. Confocal microscopy showed that DMNA induced free radical generation mainly in the cytosol. Preincubation of HepG2 cells with N-acetylcysteine and 2,2′-dipyridyl significantly prevented free radical generation on subsequent incubation with DMNA, whereas preincubation with apocynin and dimethyl sulfoxide had no effect. These results suggest that DMNA is metabolized to reactive free radicals capable of generating protein radicals which may play a critical role in DMNA toxicity. We propose that the captodative effect, the combined action of the electron-releasing dimethylamine substituent, and the electron-withdrawing nitroso substituent, leads to a thermodynamically stabilized radical, facilitating enhanced protein radical formation by DMNA.
    Full-text · Article · Nov 2014 · Free Radical Biology and Medicine
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