Obesity in a model of gpx4 haploinsufficiency
uncovers a causal role for lipid-derived
aldehydes in human metabolic disease and
cardiomyopathy
ot
az
Jitka A. Virag 2, Ethan J. Anderson 1,4,*
The prevalence of obesity is rapidly spreading throughout the world
with over 28% of adults having a body mass index (BMI) of 25 kg/
type 2 diabetes, a
prevalence of car-
diomyopathy in obese patients (w40% by some estimates) [3], with
‘preclinical’ myocardial damage [4] and diastolic dysfunction already
iversity, Greenville, NC,
East Carolina University,
., Greenville, NC 27834,
se; WT, wild type; CNTL,
in; TG, triglycerides; EF,
ejection fraction; FS, fractional shortening; Nrf2, nuclear factor (erythroid-derived 2)-like 2; IL-1b, interleukin-1 beta; IL-6, interleukin-6; TNF-a, tumor necrosis factor-a;
iNOS, inducible nitric oxide synthase; RAGE, receptor for advanced glycation end products; Coll1a1, collagen, type I, alpha; Coll4a1, collagen, type IV, alpha 1; b-MHC, b
myosin heavy chain; TGF-b1, transforming growth factor beta 1; TGF-b2, transforming growth factor beta 2
Original article1Department of Pharmacology & Toxicology, East Carolina University, Greenville, NC, United States 2Department of Physiology, East Carolina Un
United States 3Department of Public Health, East Carolina University, Greenville, NC, United States 4East Carolina Diabetes and Obesity Institute,
Greenville, NC, United States 5East Carolina Heart Institute, East Carolina University, Greenville, NC, United States
*Corresponding author. Department of Pharmacology & Toxicology, Brody School of Medicine, East Carolina University, BSOM 6S-11, 600 Moye Blvd
United States. E-mail: andersonet@ecu.edu (E.J. Anderson).
Abbreviations: PUFA, polyunsaturated fatty acids; LPPs, lipid peroxidation end products; GPx4, glutathione peroxidase 4; HFHS, high fat, high sucro
control; BMI, body mass index; 4-HNE, 4-hydroxynonenal; ROS, reactive oxygen species; RNS, reactive nitrogen species; HDL, high-density lipoprote1. INTRODUCTION m2 [1,2]. In addition to the increased risk for
growing number of studies have reported a highABSTRACT
Objective: Lipid peroxides and their reactive aldehyde derivatives (LPPs) have been linked to obesity-related pathologies, but whether they have
a causal role has remained unclear. Glutathione peroxidase 4 (GPx4) is a selenoenzyme that selectively neutralizes lipid hydroperoxides, and
human gpx4 gene variants have been associated with obesity and cardiovascular disease in epidemiological studies. This study tested the
hypothesis that LPPs underlie cardio-metabolic derangements in obesity using a high fat, high sucrose (HFHS) diet in gpx4 haploinsufficient mice
(GPx4þ/) and in samples of human myocardium.
Methods: Wild-type (WT) and GPx4þ/ mice were fed either a standard chow (CNTL) or HFHS diet for 24 weeks, with metabolic and car-
diovascular parameters measured throughout. Biochemical and immuno-histological analysis was performed in heart and liver at termination of
study, and mitochondrial function was analyzed in heart. Biochemical analysis was also performed on samples of human atrial myocardium from
a cohort of 103 patients undergoing elective heart surgery.
Results: Following HFHS diet, WT mice displayed moderate increases in 4-hydroxynonenal (HNE)-adducts and carbonyl stress, and a 1.5-fold
increase in GPx4 enzyme in both liver and heart, while gpx4 haploinsufficient (GPx4þ/) mice had marked carbonyl stress in these organs
accompanied by exacerbated glucose intolerance, dyslipidemia, and liver steatosis. Although normotensive, cardiac hypertrophy was evident with
obesity, and cardiac fibrosis more pronounced in obese GPx4þ/mice. Mitochondrial dysfunction manifesting as decreased fat oxidation capacity
and increased reactive oxygen species was also present in obese GPx4þ/ but not WT hearts, along with up-regulation of pro-inflammatory and
pro-fibrotic genes. Patients with diabetes and hyperglycemia exhibited significantly less GPx4 enzyme and greater HNE-adducts in their hearts,
compared with age-matched non-diabetic patients.
Conclusion: These findings suggest LPPs are key factors underlying cardio-metabolic derangements that occur with obesity and that GPx4
serves a critical role as an adaptive countermeasure.

 2015 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords Glutathione peroxidase 4; Lipid peroxidation; Obesity; Mitochondria; Inflammation; Human heartLalage A. Katunga 1,3, Preeti Gudimella 1, Jimmy T. Efird 3,5, Sc
Cherese Beatty 1, Timothy M. Darden 1, Kathleen A. Thayne 1, HReceived March 24, 2015  Revision received April 8, 2015  Accepted April 14, 201
http://dx.doi.org/10.1016/j.molmet.2015.04.001
MOLECULAR METABOLISM 4 (2015) 493e506 
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www.molecularmetabolism.comt Abernathy 1, Taylor A. Mattox 1,
aim Alwair 5, Alan P. Kypson 5,5  Available online 22 April 2015
pen access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 493
evident in young adult (35 yrs) obese/diabetic patients [5] and even
children [6]. Importantly, these cardiac derangements appear in the
absence of detectable coronary disease or hypertension. Together,
these findings strongly indicate a need for more careful examination
of potential mechanisms that underlie cardiomyopathy in obese
individuals.
Polyunsaturated fatty acids (PUFAs) are continuously oxidized in vivo
through both enzymatic and non-enzymatic reactions [7,8], forming
lipid peroxidation end products (LPPs) such as isoprostanes, isofurans,
thromboxanes, and a,b-unsaturated aldehydes [9], all of which have
2. RESULTS
2.1. GPx4 deficiency in obesity leads to enhanced lipid
peroxidation and carbonyl stress in liver, exacerbating insulin
resistance and steatosis
To test our hypothesis that LPPs underlie obesity-related pathologies
we used a GPx4-deficient (GPx4þ/) mouse model. GPx4þ/ mice are
phenotypically indistinguishable from WT in the absence of an exog-
enous stressor but more susceptible to damage from radiation and
environmental toxicants [43,44]. The rationale for using this mouse
Original articlebiological effects [10e13]. These aldehydes cause post-translational
modifications of proteins through Michael addition with lysines and
histidines, and through covalent modification of sulfhydryl groups (e.g.,
cysteines) to form carbonyl adducts [14,15]. Due to this high degree of
reactivity, many of these aldehydes have been proposed to be etiologic
agents in disease [7,14,16e21]. 4-hydroxynonenal (HNE) is a a,b-
unsaturated aldehyde derived from n-6 PUFA peroxidation, and HNE
levels increase proportionally with ROS/RNS levels [22]. One recent
report showed that HNE-modified albumin is significantly increased in
the serum of type 2 diabetic patients [23].
Mitochondrial membranes are abundant with unsaturated fatty
acids, which are prone to peroxidation [24e26]. The mitochondrion
is also the primary source of cellular ROS, making it a major
source of LPPs [27]. As a countermeasure, mitochondria contain an
elaborate system of antioxidant and LPP-detoxifying enzymes. One
of these is glutathione peroxidase 4 (GPx4), which resides in the
mitochondrial inner membrane (in addition to nucleus and cytosol)
to specifically scavenge lipid hydroperoxides [28e30]. GPx4 is one
of a few antioxidant enzymes known to neutralize both simple and
complex lipid hydroperoxides (e.g., Cholesterol hydroperoxide)
[31,32]. It is the only member of the GPx super-family that is
indispensable during development, with embryonic lethality of GPx4
null mice occurring at stage E7.5 [33e35]. In recent genetic and
epidemiological studies, variants of gpx4 that result in diminished
content and/or catalytic activity have been associated with obesity
[36], cardiovascular disease [37,38] and inflammation [39,40].
However, no experimental studies to date have explored GPx4 in
the context of obesity or its related pathologies, and this may be a
significant oversight given the well-known association between
mitochondrial-derived oxidative stress and metabolic disease
[41,42]. In this study, we tested the hypothesis that LPPs are a
causal factor underlying cardio-metabolic derangements in
obesity by investigating the effect of a long-term high fat, high
sucrose (HFHS) diet in a mouse model of gpx4 haploinsufficiency
(GPx4þ/) and in samples of human atrial myocardium obtained
from non-diabetic and diabetic patients undergoing elective heart
surgery.
Table 1 e Body composition and metabolic parameters.
Variables WT
CNTL
WT
HFHS
Terminal body weight (g) 34.5  1.3 48.0  2.2*
Fat mass (g) 8.4  1.0 17.7  0.9*
Lean mass (g) 21.6  0.4 26.0  1.2*
Body fat (%) 24.0  2.1 36.9  0.5*
Fasting glucose (mg/dL) 141.3  4.1 168.6  14.2*
Cholesterol (mg/dL) 51.0  8.6 72.8  6.5
Triglycerides (mg/dL) 46.8  6.3 44.0  10.6
HDL cholesterol (mg/dL) 74.9  8.0 96.7  7.2
All values expressed as mean  S.E.M., n ¼ 4e6 per group. * P<0.05 vs. WT-CNTL vs.
494 MOLECULAR METABOLISM 4 (2015) 493e506 
 2015 The Authors. Published by Elsevier GmbH.model was that it mimics the effect of previously identified gpx4 gene
variants on GPx4 enzyme levels and activity in human cells [36,38,45].
Following HFHS diet no significant differences in adiposity or weight
gain were observed between WT and GPx4þ/ mice, although obese
GPx4þ/ mice had substantial dyslipidemia and fasting hyperglycemia
with HFHS diet (Table 1). Whole body energy expenditure, determined
by VO2 and VCO2 using indirect calorimetry, was not different between
WT and GPx4þ/ mice, either with CNTL or HFHS diet (data not
shown). Glucose intolerance was exacerbated in the GPx4þ/ mice
compared with WT (Figure 1A), although serum insulin levels were
increased with HFHS diet to equal extent regardless of genotype
(Figure 1B). GPx4 enzyme levels in liver of WT mice increased
significantly as a result of HFHS diet, although GPx4þ/ mice contain
significantly less enzyme than WT (Figure 1C and Supplemental
Figure 1A). In parallel with the increase in GPx4, levels of HNE-
adducts in liver increased in WT with HFHS diet but were signifi-
cantly more pronounced in GPx4þ/ mice compared with WT. To
determine whether the increased lipid peroxidation in the GPx4þ/
mice corresponded to a more severe liver pathology following HFHS
diet, liver triglycerides and collagen were measured by oil red O and
picrosirius red staining, respectively. GPx4þ/ mice displayed greater
liver steatosis (Figure 1D) and fibrosis (Figure 1E) than WT mice
following HFHS diet.
2.2. Cardiac structural remodeling and fibrosis are exacerbated by
GPx4 deficiency in obesity
Previous studies have shown that HFHS diet-induced obesity causes
significant cardiac hypertrophy and left ventricular (LV) diastolic
dysfunction in rodent models [46e48]. Here, HFHS diet caused an
increase in cardiac mass to a similar extent between WT and GPx4þ/
mice (Figure 2A,E). However, compared with WT, GPx4þ/ mice
display significantly greater cardiomyocyte diameter (Figure 2E,F) and
fibrosis (Figure 2B,C) following HFHS diet. Obese mice also have
increased levels of serum brain natriuretic peptide (BNP, Figure 2G),
indicative of ventricular wall stress. This evidence of cardiomyopathy
exists in the absence of any changes in LV systolic function (Figure 2H),
blood pressure (Figure 2I) or heart rate (Figure 2J) in the mice following
GPx4þ/-CNTL GPx4þ/
HFHS
p-value
35.8  4.2 48.5  1.2*x <0.0001
10.0  2.9¤ 19.4  0.8*x 0.0001
22.3  1.4¤ 24.9  0.4* 0.004
31.3  3.0* 40.0  0.8*x <0.0001
148.0  3.7 204.8  11.3*¤x 0.007
34.0  8.7 103.5  17.8*x 0.01
¤x55.5  1.5 99.5  11.3* 0.003
53.1  9.4 118.4  14.4*x 0.009
¤ P<0.05 vs. WT-HFHS. x P<0.05 vs. GPX4þ/- CNTL.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
www.molecularmetabolism.com
lu
in
ine
upHFHS diet, indicating that any cardiomyopathy which might be present
Figure 1: Glycemic control and liver biochemistry/pathology. Shown in A are oral g
diet. In C are representative immunoblots of GPx4 and HNE-adducts in liver from 4
Representative images of liver triglycerides stained with oil red O D, and collagen sta
representative of 16 image fields captured per mouse, n ¼ 2e3 mice per treatment gro
genotype, yP < 0.05 vs. WT-HFHS.in these mice is not due to increased afterload.
Recently, in two independent experimental models of cardiac hyper-
trophy it was observed that GPx4 and Thioredoxin Reductase (TxnRd)
were the only antioxidant seleno-enzymes to increase in the heart,
suggesting that they are critical for cardiac compensatory response to
hypertrophic stimuli [49,50]. A significant increase in GPx4 mRNA
(Figure 3A) and enzyme content (Figure 3B and Supplemental
Figure 1B) in myocardium of WT mice with HFHS diet was observed.
Conversely, GPx4þ/ mice had lower levels of mRNA that did not
correspond to any significant change in GPx4 enzyme levels following
HFHS diet. Levels of HNE-adducts (Figure 3C) and protein carbonyls
(Figure 3C) do increase moderately in WT hearts with HFHS diet,
although GPx4 deficiency leads to a striking increase in this protein
carbonylation with obesity. To determine if GPx4 deficiency and
augmented carbonyl stress in obesity triggered a compensatory redox
adaptive response in the heart, expression of a number of antioxidant
genes were examined. GPx4þ/ mice were found to have increased
expression of several antioxidant genes following HFHS diet
(Figure 3D).
2.3. GPx4 deficiency causes mitochondrial abnormalities and up-
regulation of cardiac inflammation and fibrosis signaling pathways
with obesity
Mitochondrial dysfunction, as characterized by decreased ATP and/or
respiration combined with increased ROS, has been linked to several
obesity-related pathologies, including insulin resistance and cardio-
myopathy, and recent studies have implicated a role for mitochondrial
protein carbonylation in mediating this dysfunction [51e53]. Param-
eters of mitochondrial function in permeabilized LV myofibers were
examined to assess the relationship between GPx4 deficiency,
carbonyl stress, and mitochondria in the obese myocardium. GPx4
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www.molecularmetabolism.comenzyme content was measured in the mitochondrial fraction isolated
cose tolerance tests, along with fed-state serum insulin levels B after 24 weeks on the
dividual mice in each group, along with the corresponding b-actin loading control.
d with picrosirius red under polarized light E are shown for each group. Images are
. Data shown as mean  S.E.M., n ¼ 7e8 mice per group. *P < 0.05 vs. CNTL withinfrom whole hearts. GPx4 increased in cardiac mitochondria of WT mice
with HFHS diet, while total levels of GPx4 were lower and remained
that way following HFHS diet in the GPx4þ/ mice (Figure 4A and
Supplemental Figure 2A).
It was expected that mitochondrial GPx4 deficiency would potentially
lead to a greater degree of mitochondrial membrane lipid peroxidation
and reactive aldehyde formation. Here, the amount of mitochondrial
HNE-adducts were greater in GPx4þ/ hearts than WT, and the HFHS
diet increased the relative amount of these adducts in both WT and
GPx4þ/ mice (Figure 4B and Supplemental Figure 2B). These
increased HNE-adducts may have functional consequences since
recent studies have documented that mitochondrial HNE-adducts
correspond to decreased oxidative phosphorylation in diabetic heart
mitochondria [74,75]. Moreover, another report highlights the impor-
tance of mitochondrial fatty acid b-oxidation as a metabolic ‘sink’ for
HNE [72]. In WT hearts, both basal and maximal ADP-stimulated
palmitoyl-L-carnitine supported respiration was markedly increased
following HFHS diet, while these rates were unchanged with HFHS diet
in the GPx4þ/ mice (Figure 4B).
In a previous study we reported that the selenoenzyme thioredoxin
reductase-2 (TxnRd2) increased in rat heart with HFHS diet [54],
corresponding to a decreased rate of mitochondrial H2O2 emission in
the obese rat heart. Other groups have also reported decreased
mitochondrial ROS with obesity/diabetes in mouse [55] and rat models
[56]. Given our previous findings, combined with the fact that TxnRd2
expression was increased in GPx4þ/ hearts with HFHS diet
(Figure 3E), we sought to ascertain whether a similar adaptation in
redox buffering was occurring in cardiac mitochondria of these mice.
Here, using paired H2O2 emission experiments in the absence and
presence of the TxnRd2 inhibitor auranofin, a dramatic (w4-fold) in-
crease in total H2O2 emission was observed in permeabilized
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Original articlemyofibers prepared from obese GPx4þ/ mice fed HFHS diet, while a
decrease in this ROS emission was observed in the obese WT mice
following HFHS diet (Figure 4C). Auranofin exposure significantly
increased the rate of mitochondrial ROS in the obese WT mice but not
obese GPx4þ/ mice.
Chronic, low-grade inflammation in the obese heart is known to cause
profibrotic and hypertrophic signaling. Although it has been difficult to
fully ascertain the exact nature of the relationship between inflam-
mation and cardiac remodeling, some investigators have proposed that
activation of the receptor for advanced glycation end products (RAGE)
may play a role. RAGE belongs to a family of pattern recognition re-
ceptors for AGE and has been studied extensively in the context of
diabetes-related pathologies [57,58]. Increased expression of RAGE is
present in the hearts of obese GPx4þ/ mice (Figure 5A), and this is
consistent with the increased carbonyl stress present in these hearts
since reactive aldehydes such as HNE are known to potentiate AGE
formation [59]. Activation of RAGE has been shown to induce chronic
activation of nuclear factor kappa-light-chain-enhancer of activated B
cells (NFkB), which, in turn, activates further inflammatory pathways.
Furthermore, there is extensive crosstalk between the RAGE and
Transforming Growth Factor-Beta (TGF-b) family of proteins [60e62].
Notably, the hearts of obese GPx4þ/ mice also displayed greater pro-
inflammatory cytokine expression and genes involved in fibrosis and
hypertrophy compared with WT following HFHS diet, while only a
modest increase in a few select genes was observed with HFHS diet in
WT hearts (Figure 5A & B).
Figure 2: Cardiac structural and functional parameters. Panels shown are represen
polarized light B,C, and Masson’s Trichrome stained cardiac tissue D, from mice within eac
and serum BNP levels G. Cardiac contractility H, mean arterial pressure I, and heart rate J
mouse, n ¼ 2e3 mice per treatment group. Data in E-J are shown as mean  S.E.M., n ¼
GPx4þ/--CNTL.
496 MOLECULAR METABOLISM 4 (2015) 493e506 
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adducts and relative risk (RR) for diabetes in humans
Next, we sought to determine whether there is an association between
GPx4 and lipid-derived carbonyl stress in human heart and, also, to
examine whether there is a connection between these parameters and
human metabolic disease. In a small pilot study ofw20 patients, we
previously reported greater levels of HNE-protein adducts in the hearts
of hyperglycemic type 2 diabetic patients, and these increased HNE-
adducts were accompanied by depletion of cardiac glutathione and
increased mitochondrial ROS production [63]. Here, using samples of
atrial myocardium obtained from patients (n ¼ 103) undergoing
elective cardiac surgery (Table 2), we observed that diabetic patients
had significantly less cardiac GPx4 than non-diabetic patients
(Figure 6A), and this corresponded with greater levels of HNE-adducts
in the diabetic hearts (Figure 6B). A multivariable regression analysis
was performed on quartiles of cardiac GPx4 and tertiles of HNE-
adducts, and cardiac GPx4 was found to be negatively associated
while HNE-adducts positively associated with RR for diabetes in these
patients (Table 3). To determine if hyperglycemia was associated with
cardiac GPx4 or HNE-adducts in these patients, a multiple linear
regression model was applied, adjusting for sex. No significant as-
sociation between cardiac GPx4 or HNE-adduct levels and HbA1c was
observed in these patients (Table 3 and Supplemental Figure 1).
Similar to what was observed with the WT-HFHS mice, cardiac GPx4
content was positively correlated with cardiac HNE-adducts in these
patients (Figure 6C), suggesting that GPx4 is an adaptive response of
tative images of whole hearts A, cardiac collagen stained with picosirius red under
h study group. Shown in E are heart weight/tibia length ratio, cardiomyocyte diameter F,
are shown for each group Images are representative of 16 image fields captured per
6e8 mice per group. *P < 0.05 vs. WT-CNTL, yP < 0.05 vs. WT-HFHS, xP < 0.05 vs.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Figure 3: Cardiac GPx4, protein carbonylation and redox signaling. Shown here is GPx4 mRNA expression A and protein content B in whole hearts from mice used in this
study. Representative immunoblot of HNE-adducts and hydrazide-labeled protein carbonyls C are shown from 3 individual mice in each group, Expression of redox and phase II
detoxifying genes are shown in D. Data shown in A & D are means  S.E.M, n ¼ 5e7 per group. *P < 0.05 vs. WT-CNTL, ¤ P < 0.05 vs. WT-HFHS, xP < 0.05 vs. GPx4þ/--CNTL.
CAT-Catalase; TrxII- Thioredoxin reductase II; GR- Glutathione Reductase; GPX1- Glutathione Peroxidase1; NQO1- NAD(P)H:quinine oxidoreductase; HO-1- Heme oxygenase-1;
GSTA1-glutathione s-transferase-1.
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497
Figure 4: Cardiac mitochondrial GPx4 and functional parameters. A representative immunoblot of GPx4 protein A and HNE-adducts B along with corresponding COX IV loading
control are shown of isolated cardiac mitochondria obtained from mice used in this study. In C are maximal ADP-stimulated rates of mitochondrial respiration (JO2) supported by
palmitoyl-L-carnitine in permeabilized cardiac myofibers from these mice. Shown in D are rates of mitochondrial H2O2 emission (mito-JH2O2) in phosphorylating state supported by
palmitoyl-L-carnitine þ 100 mM ADP, in the absence () and presence (þ) of the TxnRd2 inhibitor Auranofin. Data shown in C & D are means  S.E.M, N ¼ 4e6 per group.
*P < 0.05 vs. all other groups for each respiratory state, yP < 0.05 vs. WT-HFHS, xP < 0.05 vs. GPx4þ/--CNTL, #P < 0.05 for Auranofin effect within group.
Original article
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www.molecularmetabolism.com
the tissue to persistent oxidative stress. Interestingly, the ratio of HNE-
adducts/GPx4 was much higher in diabetic than non-diabetic hearts
(Figure 6D), indicating that the GPx4 adaptive response has been over-
whelmed or compromised in these patients.
3. DISCUSSION
Only an increased understanding of the mechanisms underlying
obesity-related pathologies will enable physicians to better manage
diseases associated with this emerging epidemic. Lipid peroxides and
their reactive aldehyde derivatives have been postulated to play a
causal role in obesity-related cardio-metabolic diseases, and emerging
epidemiologic data has reported a link between polymorphisms and
mutations in gpx4 and human diseases, including obesity. Together,
these previous findings suggest that GPx4 may be an adaptive
component that serves a protective role against the persistent oxidative
stress in obesity. The present study tested this hypothesis in a
translational model of HFHS diet-induced obese GPx4þ/ (i.e. GPx4
deficient) mice, combined with cardiac tissue samples obtained from
patients undergoing elective cardiac surgery. The findings from this
study demonstrate that GPx4 deficiency with diet-induced obesity
leads to significant increases in lipid peroxide-derived aldehydes,
corresponding to more severe cardio-metabolic derangements
including glucose intolerance, dyslipidemia, liver steatosis, and cardiac
hypertrophy and fibrosis. Abnormalities in mitochondrial function in
hearts from the obese GPx4þ/ mice implicate a role for the enzyme in
protecting that critical organelle from oxidative damage in obesity.
Figure 5: Cardiac inflammatory and pro-fibrotic/hypertrophic gene expression.
Expression of pro-inflammatory A, pro-fibrotic and hypertrophy genes B are shown in
hearts from all four groups of mice in this study. B-MHC- b-Myosin Heavy Chain;
Coll1a1-Collagen 1a1; Coll4a1-Collagen 4a1; TGFb1 and 2 -Transforming Growth
Factorb 1 and 2; IL-b-Interleukin-1 Beta; IL-6 -Interleukin 6; TNF-a-Tumor Necrosis
Factor-alpha; iNOS-Inducible nitric oxide synthase; RAGE- Receptor for Advanced
Glycation End Products. All target genes normalized to 18S ribosome RNA. Data shown
are means  S.E.M, N ¼ 4e6 per group. *P < 0.05 vs. WT-CNTL, ¤ P < 0.05 vs. WT-
HFHS, xP < 0.05 vs. GPx4þ/--CNTL.
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www.molecularmetabolism.comFurthermore, the adverse cardiac remodeling observed in the obese
GPx4þ/ mice was accompanied by up-regulation of genes involved in
cardiac inflammation and remodeling, illustrating the importance of
carbonyl stress as a causal factor in these pathways. Lastly, the
relationship between decreased cardiac GPx4 and increased HNE-
adducts and human metabolic disease was confirmed by studying
cardiac tissue samples obtained from non-diabetic and diabetic pa-
tients. Collectively, these observations allow for a proposed model of
GPx4 as a protective adaptation in obesity (as shown in the Graphical
Abstract). Clearly, when lipid peroxidation increases as a result of
persistent oxidative stress in obesity/nutrient overload, GPx4 is upre-
gulated to compensate (Figures 1 and 3). However, over time and
possibly due to underlying genetic causes, GPx4 becomes over-
whelmed in an obese individual and HNE-adducts increase (along with
numerous other LPPs that were not measured in this study). The
consequence of this is increased inflammation, mitochondrial
dysfunction (and likely potentiation of mitochondrial ROS), and the
broad cardio-metabolic pathologies that are known to follow (e.g.,
diabetes, cardiomyopathy, liver disease).
These findings stand in contrast to preceding studies of diet-induced
obesity in antioxidant-deficient models. One recent study of high fat-
fed GPx1-deficient mice (whole body GPx1/) found that systemic
oxidative stress increased with obesity as expected, but these mice
were actually protected from insulin resistance and liver steatosis [98].
In a study using high fat diet in SOD2þ/ mice (also exhibiting greater
oxidative stress), insulin sensitivity was again unchanged with obesity,
although a modest effect on pancreatic b-cell release of insulin was
observed [99]. Another intriguing study used high fat diet in a
glutathione-deficient (Gclm/) mouse model. The authors found that
despite a huge increase in systemic oxidative stress with the diet,
these mice were also protected from increased adiposity, insulin
resistance and steatosis, possibly due to an increase in basal energy
expenditure [100]. Given these recent findings, the fact that GPx4
haploinsufficiency alone can induce such a damaging effect on cardio-
metabolic parameters with HFHS diet implies that lipid peroxides and
their LPP derivatives are a distinctive form of oxidative stress that must
play a very prominent etiological role in obesity-related cardio-meta-
bolic diseases.
Deleterious effects of lipid-derived aldehydes in biological systems
have been well known for many years. For example, nucleophilic
attack of cysteine residues by aldehydes has negative ramifications on
metabolic pathways in the cell [14,64]. A recent mass spectrometric
analysis of cysteine oxidation status in hearts from mice fed a dia-
betogenic diet for 8 months showed that w40% of total oxidized
cysteines are mitochondrial in origin [65]. Proteomic analysis of
mitochondria from diabetic rat hearts reveals an increased formation of
carbonyl adducts (e.g. HNE, MDA) with complexes of the electron
transport chain and respiratory complexes, and these adducts corre-
spond to a reduction in enzyme activity [18]. Our data suggest that
GPx4 protects against cardiac mitochondrial dysfunction with obesity
as the obese GPx4þ/ mice had very high levels of carbonyl stress in
their hearts (Figure 3), particularly HNE-adducts in the cardiac mito-
chondria (Figure 4B), accompanied by mitochondrial dysfunction, as
compared with obese WT mice. Several studies have shown that GPx4
preserves ATP production and attenuates cytochrome c release in the
mitochondria under oxidative stress [66e68]. Cardiac-specific over-
expression of mitochondrial GPx4 is protective in models of cardiac
ischemia/reperfusion injury and in a streptozotocin (STZ)-induced
diabetes model [69,70]. GPx4 exists as both short (i.e., mitochondrial-
localized) and long (i.e., cytosolic-localized) isoforms, yet only deletion
of the former is lethal in mice [67]. Thioredoxin Reductase is the only
pen access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 499
other essential selenoenzyme identified to date, and this enzyme also
has both mitochondrial (TxnRd2) and cytosolic (TxnRd1) isoforms [71].
In the present study, increased mitochondrial H2O2 emission was
observed in hearts of lean GPx4þ/ mice following the inhibition of
TxnRd2 with auranofin (Figure 4C), although auranofin did not signif-
icantly affect the rate in the obese GPx4þ/ mice (which was already
much greater than all other groups). This suggests that mitochondrial
lipid peroxide-scavenging with GPx4 and the thioredoxin redox couple
are both important adaptive responses of cardiac mitochondria in
obesity-related cardiac hypertrophy and that TxnRd2 cannot appro-
priately compensate for GPx4 deficiency within cardiac mitochondria if
animals are obese.
A recent study showed that fatty acid b-oxidation is a novel HNE
clearance pathway in heart [72]. Considering the diminished rates of
maximal palmitoyl carnitine-supported respiration observed in the
obese GPx4þ/ hearts, it is plausible that the elevated carbonyl stress
observed in the hearts of these mice is a result of both increased
mitochondrial ROS production (which initiates more lipid peroxidation)
as well as a disruption of aldehyde detoxification pathways. The
decreased GPx4 and correspondingly increased HNE-adducts seen
here in myocardium of diabetic patients support our previous obser-
vations of decreased palmitoyl-L-carnitine supported respiration and
increased mitochondrial ROS in diabetic human hearts [42,73]. A link
between mitochondrial HNE-adduct formation and decreased oxidative
phosphorylation in diabetic heart mitochondria has recently been made
[74,75], and our findings suggest that GPx4 deficiency may underlie
these oxidative modifications. High variability in dietary fat may also be
Table 2 e Patient clinical and demographic characteristics.
Variables Diabetes
n (%)
No diabetes
n (%)
p-value
Age
Mean  SD 64  9.28 61  11 0.49
Median (IQR) 65 (16) 60 (17)
Sex
Female 7 (14) 1 (2)
Male 42 (85) 53 (97) 0.026
Race
White 41 (83) 47 (86) 0.78
Black 8 (16) 7 (13)
BMI
Mean  SD 31  6.6 30  5.3 0.46
Median (IQR) 30 (7.4) 29 (4.5)
Preoperative medications
b-blockers 34 (69) 41 (75) 0.87
ACEi/ARBs 15 (31) 22 (41) 0.79
Diuretics 21 (43) 20 (37) 0.69
CCBs 10 (20) 13 (24) 0.89
Diabetic medications 43 (87) 2 (4) <0.0001
Insulin 32 (65) 2 (4)
Metformin 15 (31) 0
Sulfonylureas 4 (8) 0
Glitazones 1 (2) 0
GLP-1 agonist 2 (4) 0
DDP-4 inhibitor 3 (6) 0
Statins 41 (83) 46 (84) 0.94
Nitrates 19 (39] 32 (59) 0.65
Heart failure 8 (16) 5 (9) 0.31
LVEF (%) 51.8  3.9 53.6  6.4 0.87
Sample size N ¼ 103 for GPx4, N ¼ 61 for HNE-adduct.
Figure 6: GPx4 content and HNE-adducts in human myocardium. Total GPx4 enzym
diabetic and diabetic patients undergoing elective heart surgery. Shown in C is associati
HNE-adduct to GPx4 enzyme are depicted in D. Each symbol corresponds to one individ
computed using Friedman’s nonparametric test for central tendency, adjusting for sex.
Original article
500 MOLECULAR METABOLISM 4 (2015) 493e506 
 2015 The Authors. Published by Elsevier GmbH.a contributor to the greater levels of cardiac HNE-adducts in the dia-
betic patients, as it has been reported that diets consisting of high fat
mixed with carbohydrate, or high fat alone, can lead to drasticallye content A and HNE-adducts B are shown in atrial myocardium obtained from non-
on between HNE-adducts and GPx4 enzyme in these heart samples, and the ratio of
ual patient (N ¼ 103, see Table 2 for demographics and clinical variables). P-value
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
www.molecularmetabolism.com
different outcomes in HNE accumulation in oxidative tissues [101].
Other studies directed at examining the role that alternative aldehyde-
producing (e.g., monoamine oxidase) and detoxifying enzymes (e.g.,
aldehyde dehydrogenases) may have in pathologies of obese/diabetic
hearts may also be useful [64]. In addition, an improved understanding
of the regulation of selenocysteine status may serve as a major clue to
uncovering the mechanisms that govern the progression to mal-
adaptive remodeling in hearts of patients with metabolic disease.
Epidemiological studies of Keshan’s disease, a cardiomyopathy
endemic to regions of China, confirm that the heart is highly sus-
ceptible to injury resulting from disturbances to selenocysteine en-
zymes. Cardiomyopathy in Keshan’s disease manifests as acute
Table 3 e Multivariable analysis of GPx4 and HNE-adducts in human heart.
GPx4 content (ug/ml)
Diabetes
n (%)
No diabetes
n (%)
RR (95% CI)a
Mean  SD 6.2  4.2 8.7  5.9 P ¼ 0.034b
Median (IQR) 6.0 (5.2) 6.7 (6.6)
Q1 (3.9) 17 (35) 9 (17) 1.0 Referent
Q2 (>3.9e6.3) 10 (20) 15 (28) 0.65 (0.37e1.1)
Q3 (>6.3e9.4) 13 (27) 13 (24) 0.74 (0.47e1.2)
Q4 (>9.4) 9 (18) 17 (31) 0.58 (0.31e1.1)
Ptrend ¼ 0.063c
HNE content (mM)
Mean  SD 18  8.3 14  9.8 P ¼ 0.028b
Median (IQR) 16 (11) 11 (12)
T1 (9.0) 3 (10) 21 (47) 1.0 Referent
T2 (>9.0e17) 14 (47) 11 (24) 4.2 (1.4e13)
T3 (>17) 13 (43) 13 (29) 3.9 (1.2e12)
Ptrend ¼ 0.050c
a Relative Risk (RR) and 95% Confidence Interval (CI), adjusted for sex.
b P-value computed using Friedman’s Nonparametric test for central tendency,
adjusting for sex.
c P-value computed using the likelihood ratio trend test, adjusting for sex.cardiac fibrosis and mitochondrial dysfunction [76], a phenotype that is
strikingly similar to the GPx4þ/ mice in the present study.
Aside from measurements of liver steatosis and serum lipids, our study
did not explore the potential contribution of altered lipid metabolism
that might exist in these mice. Lipotoxicity is a consistent feature of
cardio-metabolic disease in both humans and animal models of obesity
[77e82] and therefore may contribute to the severe liver and cardiac
pathology observed in the obese GPx4þ/ mice. The obese GPx4þ/
mice do not possess significantly greater adiposity than obese WT, as
determined by % body fat. However, we cannot exclude that there may
have been differences in adipose distribution between groups. It is also
known that GPx4 directly modulates the lipoxygenase and cyclo-
oxygenase II enzyme systems, and, when viewed in this context, the
increased cardiac inflammation present in obese GPx4þ/ mice sup-
ports a potential role for prostaglandins and other oxidized lipids in
mediating the severe metabolic pathologies in this model. Thus, we
cannot exclude the contribution of these mediators in the obese
GPx4þ/ mice. Other studies have documented that proteolysis and
autophagy become compromised with carbonyl stress, and the
diminished activity of these cellular processes may also explain the
more severe phenotype observed in the obese GPx4þ/ hearts [83,84].
None of these alternative pathways were explored in the present study.
It is anticipated that these findings will serve to lay a foundation for
future mechanistic studies directed at understanding how LPPs
contribute to obesity-related pathologies and the adaptive role of GPx4.
Considering that the most severe effects observed in the obese GPx4þ/
MOLECULAR METABOLISM 4 (2015) 493e506 
 2015 The Authors. Published by Elsevier GmbH. This is an o
www.molecularmetabolism.com mice were evident in oxidative tissues (i.e., liver, heart, muscle), it
will be very important in future studies to identify the cell- and tissue-
specific role of GPx4 in mediating lipid peroxidation and carbonyl stress
with obesity. Furthermore, our findings have clinical implications as
they suggest that patients exhibiting decreased GPx4 expression and
activity might be predisposed to obesity-related cardio-metabolic
diseases. Indeed, clinical studies and human cell models have shown
that GPx4 expression and activity vary widely in response to envi-
ronmental and nutritional cues [38,45,85e88]. Finally, although our
understanding of the role of lipid-derived aldehydes and carbonyl
stress in the etiology of obesity-related diseases is in its infancy, it is
important to recognize that compounds that scavenge HNE and other
lipid-derived aldehydes specifically, such as histidine-containing di-
peptides, may be very useful in this regard [89,90,102] and have
experienced some success in pre-clinical testing and even clinical
trials [91e93]. Thus, further research into novel therapeutics targeting
carbonyl stress will be important for mitigating the numerous cardio-
metabolic diseases that accompany this rising epidemic of obesity.
4. METHODS
4.1. Mouse model and study design
Animal care and experimental procedures were performed with
approval from the Institutional Animal Care and Use Committee of East
Carolina University and were in compliance with the National Institutes
of Health’s Guide for Care and Use of Laboratory Animals. C57BL6/J
female mice (Jackson Laboratory) were crossed to male GPx4þ/mice
and pups were genotyped by polymerase chain reaction (PCR) using
primers described in [43]. At 8e12 weeks, WT and GPx4þ/ male and
female age-matched littermates (n ¼ 6) were randomly assigned to
groups and individually housed. Mice were fed either control (CNTL-
TD110367) or high fat high sucrose (HFHS-TD110365) diet from
Harlan-Teklad Laboratories (Madison, WI) ad libidum for 25 weeks.
The composition of this diet was a special formulation consisting of
mixed saturated and n-6 PUFA (44.6% kcal/g fat), with high sucrose
(34% kcal/g) content [54]. Mice were housed at controlled temperature
and a 12hr light/dark cycle was maintained. At the end of the diet
period, all live animal metabolic and echocardiographic measurements
were made 1e2 weeks prior to tissue collection to allow for a ‘wash-
out period’ to minimize artifact attributable to additional stress that
may have been introduced by these procedures.
4.2. Patient enrollment and myocardial tissue collection
The Institutional Review Board of the Brody School of Medicine at East
Carolina University approved all aspects of this study. Adult patients
undergoing primary, non-emergent elective coronary artery bypass graft
(CABG) or CABG/valve surgery between January 2011 and December
2014 were enrolled for this study. Demographic and clinical variables
for these patients, including preoperative cardiovascular and metabolic
drugs, are listed in Table 2 according to diabetes status. Those having
prior cardiac surgery, history of arrhythmia, or severely enlarged atria
(>4.0 cm) were excluded from the study. Atrial appendage biopsies
were obtained from each patient as previously described [94], directly
prior to institution of cardiopulmonary bypass. Myocardium was
dissected from the endocardial side of the biopsy, rinsed briefly and
frozen in liquid N2. All samples were rapidly processed this way to
minimize protein and mRNA degradation across patients.4.3. Metabolic parameters
Body weight of the mice was recorded on a weekly basis throughout
HFHS diet. Body composition was analyzed by nuclear-MRI (EchoMRI
pen access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 501
700 Echo Medical Systems, Houston, TX). An oral glucose tolerance
test (dose-0.5 g/kg) was performed following aw4 h fast in week 22
of the HFHS diet period. Glucose levels were measured using a
standard glucometer (OneTouch UltraMini, LifeScan, Milpitas, CA) in
blood collected from a tail nick. Serum cholesterol, high-density li-
poprotein (HDL), and triglycerides (TG) were analyzed using UniCel DxC
600 (Beckman Coulter, Indianapolis, IN).
4.4. Assessment of cardiovascular function
At end of the diet intervention, blood pressure was determined by non-
invasive tail-cuff plethysmography (SC1000, Hattaras Instruments,
Cary, NC). Mice were acclimated to the procedure for 3 days, and the
average of blood pressure readings made over 3 consecutive days was
recorded. A single recording was an average of 10 measurements after
5 acclimatization cycles, with duration no longer than 10 min, at
approximately the same time every day. Also at this time, high-
resolution echocardiography was performed on fully conscious mice
using a 30 MHz transducer (Vevo 2100, VisualSonics, Toronto, ON) to
assess cardiac function and structural parameters in vivo. Parasternal
long- and short-axis views were captured in M-mode and recordings
analyzed using system software.
4.5. Liver and cardiac histology
Mice were anesthetized with isoflurane. Hearts (n ¼ 2/group) were
stopped with KCl, fixed in Zinc Fixative then embedded in paraffin.
Next, they were cut cross-sectionally, stained in Hematoxylin and eosin
or picosirus red (collagen), then viewed under polarized light. Livers
from these animals were fixed, embedded and stained in a similar
fashion, with the exception that oil red O was used for liver tri-
glycerides. Images of each heart were captured at 20X. The car-
diomyocyte diameter of 8e10 myocytes from 5 captured images per
animal was analyzed using Image J (http://imagej.nih.gov/ij/) by an
individual blinded to the treatment group.
4.6. Preparation of permeabilized cardiac myofibers and
mitochondrial function measurements
All procedures for these measurements have been described in detail
by our group previously [42,54,73,95]. The mice were anesthetized
with isoflurane and rapidly euthanized by pneumothorax. Blood was
collected for serum analysis via cardiac puncture. Whole hearts were
dissected, weighed, and a small portion of the left ventricle (LV) was
cut for preparation of permeabilized myofibers (w30 mg) and placed
in Buffer X containing (in mM): 7.23 K2EGTA, 2.77 CaK2EGTA, 20
Imidazole, 20 Taurine, 5.7 ATP, 14.3 Phosphocreatine, 6.56 MgCl2-
6H2O and 50 MES (pH 7.1, 295 mOsm). Of the remaining LV/septum,
one portion was used for standard mitochondrial isolation, and another
was snap frozen in liquid N2 and stored at 80 C for further
biochemical analysis. Following permeabilization in 50 mg/ml saponin
for 30 min, fibers were transferred to Buffer Z containing (in mM):
110 K-MES, 35 KCl, 1 EGTA, 5 K2HPO2, 3 MgCl2-6H2O, and 5 mg/ml
BSA (pH 7.4, 295 mOsm) and remained in Buffer Z with 20uM
Blebbstatin on a rotator at 4 C until analysis (<1.5 h). Fibers were
then transferred to an Oroboros O2K (Oroboros Instruments, Innsbruck,
Austria) where respiration was recorded in Buffer Z þ 20uM Bleb-
bistatin, 20 mM Creatine at 30 C. Respiration (JO2) measurements
were performed following addition of 125uM palmitoyl-
carnitine þ 1 mM malate followed by 4 mM ADP. H2O2 emission was
measured using a spectrofluorometer (Photon Technology In-
Original articlestruments, Birmingham, NJ) in Buffer Z þ 20uM Blebbistatin, 10 mM
Amplex Red, 1 U/ml horseradish peroxidase, 25 U/ml superoxide dis-
mutase. For H2O2 measurements, respiratory substrates used were
502 MOLECULAR METABOLISM 4 (2015) 493e506 
 2015 The Authors. Published by Elsevier GmbH.125 palmitoyl-carnitine, 2 mM Malate þ100 mM ADP, 5 mM glucose
and 1 U/ml hexokinase (i.e., phosphorylating state) followed by
sequential addition of 5 mM glutamate then 5 mM succinate. Once
mitochondrial experiments completed, fibers were collected, rinsed in
ddH2O to remove excess salts, and lyophilized (Labconco, Kansas City,
MO). Fibers were then weighted on a microbalance (Mettler Toledo,
Denver, CO) and mitochondrial JO2 (oxygen consumption rate) and
JH2O2 (peroxide emission rate) were normalized to dry weight.
4.7. Real-time qPCR of gene expression
Cardiac samples (w10 mg) were homogenized in glass grinders
(Kimble Chase, Vineland, NJ) then subjected to Proteinase K digestion.
All materials used in RNA extraction were provided in RNeasy Fibrous
Tissue Mini Kit (Qiagen Inc, Valencia, CA cat# 74704). Reverse Tran-
scription was performed using the iScript  cDNA Kit (Biorad Labo-
ratories, Hercules, CA cat# 170e8891). The SsoAdvanced SYBER 
Green reaction cocktail was used in the amplification and detection of
DNA in RT-PCR. All protocols were performed according to product
specifications unless otherwise stated. Cycle threshold (C(t)) values
were converted to relative gene expression levels using the 2-DDC(t)
method and normalized to the level of 18S ribosomal RNA. Data are
reported as fold-change in gene expression (arbitrary units)  S.E.M.
relative to WT-CNTL mice (n ¼ 4e5 per group). The sequences and
references for the primers used in qRT-PCR are listed in Supplemental
Table 1.
4.8. Mitochondria isolation
The technique applied has been modified from methods previously
described [96,97]. Briefly, w100 mg of cardiac tissue was excised
and added to a petri dish with ice. The tissue was minced for 4e5 min,
until it could pass through a pipette, and subjected to a brief (2 min)
trypsin incubation. The mixture was transferred to a 50 mL conical
tube and allowed to settle. The supernatant was removed and dis-
carded. The remaining mixture was resuspended in 3 mL of Mito-
chondria Isolation Medium (MIM) (300 mM Sucrose, 10 mM Na-
HEPES, 0.2 mM EDTA) with BSA, then transferred to a pre-chilled
Dounce homogenizer and slowly homogenized for a total of w10e
12 counts. The homogenate was then subjected to a series of differ-
ential centrifugation steps. The fractions of isolated mitochondria ob-
tained (mixed subsarcolemmal and intermyofibrillar) were
resuspended in a small volume of MIM þ BSA and frozen at 80 for
biochemical analysis.
4.9. Immunoblot and enzyme-linked immunosorbent assay (ELISA)
Cardiac tissue was homogenized in TEET Buffer (10 mM Tris, 1 mM
EDTA, 1 mM EGTAþ 0.5% Triton X-100), then loaded on 4e20% pre-
cast polyacrylamide SDS gel (Biorad, Hercules, CA) under reducing
conditions. Protein was transferred to PVDF membranes (Millipore,
Bellerica, CA) and incubated with primary antibodies for GPx4 and a-
tubulin (Abcam, Cambridge, UK), COX IV and b-actin (Cell Signaling
Technology, Danvers MA), and HNE-adduct (Percipio Biosciences).
Membranes were incubated using infrared fluorophore-conjugated
secondary antibodies (LiCor Biosciences, Lincoln, NE), scanned using
Odyssey Clx Infrared Imaging System (Li-Cor) and analyzed by
densitometry using Image J (NIH). The absolute quantities of GPx4 and
HNE-modified protein adducts in human cardiac tissue was deter-
mined by a quantitative ELISA approach developed in our lab, as
described previously [54]. Fixed concentrations of HNE-adducts were
made by incubating 4-hydroxynonenal (Cayman Chemical, Ann Arbor,
MI) with BSA. A standard curve of both recombinant GPx4 (AbFrontier,
Seoul, Korea) and HNE-modified BSA were then incubated on
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
www.molecularmetabolism.com
immunolon-coated 96-well assay plate (Fisher Scientific) along with
diluted cardiac protein. Samples were incubated overnight at 4 C, and
subsequently washed with PBSþ0.05% Tween-20 and blocked for 2 h
with BSA or NB4025 (NOF America, White Plains, NY) at 37 C.
Samples were then incubated with primary antibodies for GPx4 and
HNE adduct for 2 h at 37 C. Samples were washed with PBSþ0.05%
Tween-20 and incubated with HRP-conjugated secondary antibody for
2 h at 27 C. Following this incubation, samples were washed as
before and incubated with Amplex Red (10 mM). Concentrations of
GPx4 and HNE-adducts in the samples were determined by fitting to
the standard curve within each plate and normalized to total protein
concentration.
4.10. Hydrazide labeling of carbonyl-modified proteins
Hydrazide Cy5.5 dye (GE Healthcare Bio-Sciences, Pittsburgh, PA) was
diluted in dimethyl sulfoxide (DMSO) at a stock concentration 1 mg/ml.
Tissue was homogenized under an anaerobic chamber (Coy Laboratory
Products, Grass Lake, MI) in 10 mM sodium phosphate buffer, con-
taining 0.1% Triton X-100, and de-gassed using dry vacuum pump
(Welch No. 25228e01, Monroe, LA). Dye was added to half of the
homogenate (1:10). The samples were left under anaerobic conditions
for 2 h and incubated overnight on orbital shaker at 4 C. The other
aliquot of tissue homogenate was used to quantify protein content.
Portions of the labeled cardiac tissue homogenate were subjected to
SDS-PAGE on 10% SDS polyacrylamide gel. The gels were scanned
using Odyssey Clx Infrared Imaging System (Li-Cor) and Cy5.5-labeled
proteins were analyzed by densitometry using Image J (NIH).
4.11. Statistical analysis
All animal data are presented as mean  SEM. Statistical analysis on
mouse model variables were performed with GraphPad Prism
(GraphPad Prism, La Jolla, Ca.). One way ANOVA was performed on
continuous variables followed by NewmaneKeuls post test, with
a < 0.05 considered statistically significant. In the human data
analysis, categorical variables are presented as frequency and per-
centage and continuous variables were presented as mean standard
deviation, mediation, median and interquartile range. Fisher exact and
Chi (c2) procedures were used to compute statistical significance of
group comparisons for categorical variables. Deuchler-Wilcoxon was
used for continuous variables. Biochemical variables were divided into
quartiles (GPx4) or tertiles (HNE) and analyzed using a robust Poisson
regression model (with relative risk as the measure of association).
Missing values for all clinical and biochemical variable were imputed
using the iterative expectation-maximization (EM) algorithm as
described in a recent study [94]. Variables that were statistically sig-
nificant in the univariable analysis were included into the multivariable
analysis. P-value was computed using Friedman’s Nonparametric test
for central tendency while adjusting for age. Ptrend was computed using
likelihood ratio trend test, adjusting for age and sex. Statistical sig-
nificance was defined as P < 0.05. SAS Version 9.3 was used for all
analyses of human biochemical and clinical variables.
FUNDING
This research was supported in part by National Institutes of Health
grant 1R01HL122863 to E.J.A
CONFLICT OF INTERESTThe authors have no conflict of interest to declare.
MOLECULAR METABOLISM 4 (2015) 493e506 
 2015 The Authors. Published by Elsevier GmbH. This is an o
www.molecularmetabolism.comACKNOWLEDGMENTS
This authors would like to thank Amy Bullock for assistance with animal care and
breeding and Joany Oswald Zary for assistance with histology.
APPENDIX A. SUPPLEMENTARY DATA
Supplementary data related to this article can be found at doi:10.1016/j.molmet.
2015.04.001.
REFERENCES
[1] Ng, M., Fleming, T., Robinson, M., Thomson, B., Graetz, N., Margono, C.,
et al., 2014. Global, regional, and national prevalence of overweight and
obesity in children and adults during 1980e2013: a systematic analysis for
the global burden of disease study 2013. Lancet 384(9945):766e781. http://
dx.doi.org/10.1016/S0140-6736(14)60460-8.
[2] Danaei, G., Finucane, M.M., Lu, Y., Singh, G.M., Cowan, M.J., Paciorek, C.J.,
et al., 2011. National, regional, and global trends in fasting plasma glucose
and diabetes prevalence since 1980: systematic analysis of health exami-
nation surveys and epidemiological studies with 370 country-years and 2.7
million participants. Lancet 378(9785):31e40. http://dx.doi.org/10.1016/
S0140-6736(11)60679-X.
[3] Kiencke, S., Handschin, R., von Dahlen, R., Muser, J., Brunner-Larocca, H.P.,
Schumann, J., et al., 2010. Pre-clinical diabetic cardiomyopathy: prevalence,
screening, and outcome. European Journal of Heart Failure 12(9):951e957.
http://dx.doi.org/10.1093/eurjhf/hfq110.
[4] Selvin, E., Lazo, M., Chen, Y., Shen, L., Rubin, J., McEvoy, J.W., et al., 2014.
Diabetes mellitus, prediabetes, and incidence of subclinical myocardial
damage. Circulation 130(16):1374e1382. http://dx.doi.org/10.1161/
CIRCULATIONAHA.114.010815.
[5] Khan, J.N., Wilmot, E.G., Leggate, M., Singh, A., Yates, T., Nimmo, M., et al.,
2014. Subclinical diastolic dysfunction in young adults with type 2 diabetes
mellitus: a multiparametric contrast-enhanced cardiovascular magnetic
resonance pilot study assessing potential mechanisms. European Heart
Journal-Cardiovascular Imaging jeu121.
[6] Porcar-Almela, M., Codoner-Franch, P., Tuzon, M., Navarro-Solera, M.,
Carrasco-Luna, J., Ferrando, J., 2015. Left ventricular diastolic function and
cardiometabolic factors in obese normotensive children. Nutrition, Meta-
bolism & Cardiovascular Diseases 25(1):108e115. http://dx.doi.org/10.1016/
j.numecd.2014.08.013.
[7] Catala, A., 2009. Lipid peroxidation of membrane phospholipids generates
hydroxy-alkenals and oxidized phospholipids active in physiological and/or
pathological conditions. Chemistry and Physics of Lipids 157(1):1e11. http://
dx.doi.org/10.1016/j.chemphyslip.2008.09.004.
[8] Guichardant, M., Chantegrel, B., Deshayes, C., Doutheau, A., Moliere, P.,
Lagarde, M., 2004. Specific markers of lipid peroxidation issued from n-3 and n-
6 fatty acids. Biochemical Society Transactions 32(Pt 1):139e140 doi: 10.1042/.
[9] Guichardant, M., Bacot, S., Moliere, P., Lagarde, M., 2006. Hydroxy-alkenals
from the peroxidation of n-3 and n-6 fatty acids and urinary metabolites.
Prostaglandins Leukotrienes and Essential Fatty Acids 75(3):179e182. http://
dx.doi.org/10.1016/j.plefa.2006.05.006.
[10] Refsgaard, H.H., Tsai, L., Stadtman, E.R., 2000. Modifications of proteins by
polyunsaturated fatty acid peroxidation products. Proceedings of National
Academy of Sciences USA 97(2):611e616.
[11] West, J.D., Marnett, L.J., 2005. Alterations in gene expression induced by the
lipid peroxidation product, 4-hydroxy-2-nonenal. Chemical Research inToxicology 18(11):1642e1653.
[12] Uchida, K., 2003. 4-hydroxy-2-nonenal: a product and mediator of oxidative
stress. Progress in Lipid Research 42(4):318e343.
pen access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 503
[13] Uchida, K., 2000. Role of reactive aldehyde in cardiovascular diseases. Free
Radical Biology and Medicine 28(12):1685e1696.
[14] LoPachin, R.M., Gavin, T., Petersen, D.R., Barber, D.S., 2009. Molecular
mechanisms of 4-hydroxy-2-nonenal and acrolein toxicity: nucleophilic tar-
gets and adduct formation. Chemical Research in Toxicology 22(9):1499e
1508 doi: 10.1021/tx900147g.
[15] Januszewski, A.S., Alderson, N.L., Metz, T.O., Thorpe, S.R., Baynes, J.W.,
2003. Role of lipids in chemical modification of proteins and development of
complications in diabetes. Biochemical Society Transactions 31(Pt 6):1413e
1416 doi: 10.1042/.
[16] Blair, I.A., 2008. DNA adducts with lipid peroxidation products. Journal of
Biological Chemistry 283(23):15545e15549 doi: 10.1074/jbc.R700051200.
[17] Siems, W., Grune, T., 2003. Intracellular metabolism of 4-hydroxynonenal.
Molecular Aspects of Medicine 24(4e5):167e175.
[18] Chavez, J.D., Wu, J., Bisson, W., Maier, C.S., 2011. Site-specific proteomic
analysis of lipoxidation adducts in cardiac mitochondria reveals chemical
diversity of 2-alkenal adduction. Journal of Proteomics. http://dx.doi.org/
10.1016/j.jprot.2011.03.031.
[19] Minko, I.G., Kozekov, I.D., Harris, T.M., Rizzo, C.J., Lloyd, R.S., Stone, M.P.,
2009. Chemistry and biology of DNA containing 1, N(2)-deoxyguanosine
adducts of the alpha,beta-unsaturated aldehydes acrolein, crotonaldehyde,
and 4-hydroxynonenal. Chemical Research in Toxicology 22(5):759e778 doi:
10.1021/tx9000489.
[20] Maier, C.S., Chavez, J., Wang, J., Wu, J., 2010. Protein adducts of aldehydic
lipid peroxidation products identification and characterization of protein ad-
ducts using an aldehyde/keto-reactive probe in combination with mass
spectrometry. Methods in Enzymology 473:305e330. http://dx.doi.org/
10.1016/S0076-6879(10)73016-0.
[21] Nair, J., Barbin, A., Velic, I., Bartsch, H., 1999. Etheno DNA-base adducts
from endogenous reactive species. Mutation Research 424(1e2):59e69.
[22] Esterbauer, H., Schaur, R.J., Zollner, H., 1991. Chemistry and biochemistry of
4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radical
Biology and Medicine 11(1):81e128.
[23] Shimozu, Y., Hirano, K., Shibata, T., Shibata, N., Uchida, K., 2011. 4-
hydroperoxy-2-nonenal is not just an intermediate but a reactive molecule
that covalently modifies proteins to generate unique intramolecular oxidation
products. Journal of Biological Chemistry 286(33):29313e29324. http://
dx.doi.org/10.1074/jbc.M111.255737.
[24] Paradies, G., Petrosillo, G., Pistolese, M., Di Venosa, N., Serena, D.,
Ruggiero, F.M., 1999. Lipid peroxidation and alterations to oxidative meta-
bolism in mitochondria isolated from rat heart subjected to ischemia and
reperfusion. Free Radical Biology and Medicine 27(1e2):42e50.
[25] Fry, M., Green, D.E., 1981. Cardiolipin requirement for electron transfer in
complex I and III of the mitochondrial respiratory chain. Journal of Biological
Chemistry 256(4):1874e1880.
[26] Chicco, A.J., Sparagna, G.C., 2007. Role of cardiolipin alterations in mito-
chondrial dysfunction and disease. American Journal of Physiology e Cell
Physiology 292(1):C33eC44. http://dx.doi.org/10.1152/ajpcell.00243.2006.
[27] Anderson, E.J., Katunga, L.A., Willis, M.S., 2012. Mitochondria as a source
and target of lipid peroxidation products in healthy and diseased heart.
Clinical and Experimental Pharmacology and Physiology 39(2):179e193.
http://dx.doi.org/10.1111/j.1440-1681.2011.05641.x, 10.1111/j.1440-
1681.2011.05641.x.
[28] Imai, H., Hirao, F., Sakamoto, T., Sekine, K., Mizukura, Y., Saito, M., et al.,
2003. Early embryonic lethality caused by targeted disruption of the mouse
PHGPx gene. Biochemical and Biophysical Research Communications 305(2):
278e286.
[29] Brigelius-Flohe, R., Maiorino, M., 2012. Glutathione peroxidases. Biochimica
Original articleet Biophysica Acta. http://dx.doi.org/10.1016/j.bbagen.2012.11.020,
10.1016/j.bbagen.2012.11.020.
504 MOLECULAR METABOLISM 4 (2015) 493e506 
 2015 The Authors. Published by Elsevier GmbH.[30] Brigelius-Flohe, R., 1999. Tissue-specific functions of individual glutathione
peroxidases. Free Radical Biology and Medicine 27(9e10):951e965.
[31] Maiorino, M., Thomas, J.P., Girotti, A.W., Ursini, F., 1991. Reactivity of
phospholipid hydroperoxide glutathione peroxidase with membrane and li-
poprotein lipid hydroperoxides. Free radical research communications 1(12e
13 Pt):131e135.
[32] Maiorino, M., Gregolin, C., Ursini, F., 1990. Phospholipid hydroperoxide
glutathione peroxidase. Methods in Enzymology 186:448e457.
[33] Scimeca, M.S., Lisk, D.J., Prolla, T., Lei, X.G., 2005. Effects of gpx4 haploid
insufficiency on GPx4 activity, selenium concentration, and paraquat-induced
protein oxidation in murine tissues. Experimental Biology and Medi-
cine(Maywood) 230(10):709e714.
[34] Morgan, E.E., Faulx, M.D., McElfresh, T.A., Kung, T.A., Zawaneh, M.S.,
Stanley, W.C., et al., 2004. Validation of echocardiographic methods for
assessing left ventricular dysfunction in rats with myocardial infarction.
American Journal of Physiology - Heart and Circulatory Physiology 287(5):
H2049eH2053 doi: 287/5/H2049 [pii] 10.1152/ajpheart.00393.2004.
[35] Ran, Q., Van Remmen, H., Gu, M., Qi, W., Roberts 2nd, L.J., Prolla, T., et al.,
2003. Embryonic fibroblasts from Gpx4þ/- mice: a novel model for studying
the role of membrane peroxidation in biological processes. Free Radical
Biology and Medicine 35(9):1101e1109.
[36] Ruperez, A.I., Olza, J., Gil-Campos, M., Leis, R., Mesa, M.D., Tojo, R., et al.,
2014. Association of genetic polymorphisms for glutathione peroxidase genes
with obesity in spanish children. Journal of Nutrigenetics and Nutrigenomics
7(3):130e142 doi: 000368833.
[37] Polonikov, A.V., Vialykh, E.K., Churnosov, M.I., Illig, T., Freidin, M.B.,
Vasil’eva, O.V., et al., 2012. The C718T polymorphism in the 3’-untranslated
region of glutathione peroxidase-4 gene is a predictor of cerebral stroke in
patients with essential hypertension. Hypertension Research 35(5):507e512
doi: 10.1038/hr.2011.213.
[38] Crosley, L.K., Bashir, S., Nicol, F., Arthur, J.R., Hesketh, J.E., Sneddon, A.A.,
2013. The single-nucleotide polymorphism (GPX4c718t) in the glutathione
peroxidase 4 gene influences endothelial cell function: interaction with se-
lenium and fatty acids. Molecular Nutrition & Food Research 57(12):2185e
2194 doi: 10.1002/mnfr.201300216.
[39] Du, X.H., Dai, X.X., Xia Song, R., Zou, X.Z., Yan Sun, W., Mo, X.Y., et al.,
2012. SNP and mRNA expression for glutathione peroxidase 4 in kashin-beck
disease. British Journal of Nutrition 107(2):164e169. http://dx.doi.org/
10.1017/S0007114511002704.
[40] Villette, S., Kyle, J.A., Brown, K.M., Pickard, K., Milne, J.S., Nicol, F., et al.,
2002. A novel single nucleotide polymorphism in the 3’ untranslated region of
human glutathione peroxidase 4 influences lipoxygenase metabolism. Blood
Cells, Molecules & Diseases 29(2):174e178 doi: S1079979602905565.
[41] Houstis, N., Rosen, E.D., Lander, E.S., 2006. Reactive oxygen species have a
causal role in multiple forms of insulin resistance. Nature 440(7086):944e
948 doi: nature04634.
[42] Anderson, E.J., Lustig, M.E., Boyle, K.E., Woodlief, T.L., Kane, D.A., Lin, C.T.,
et al., 2009. Mitochondrial H2O2 emission and cellular redox state link
excess fat intake to insulin resistance in both rodents and humans. Journal of
Clinical Investigation 119(3):573e581 doi: 10.1172/JCI37048.
[43] Yant, L.J., Ran, Q., Rao, L., Van Remmen, H., Shibatani, T., Belter, J.G., et al.,
2003. The selenoprotein GPX4 is essential for mouse development and
protects from radiation and oxidative damage insults. Free Radical Biology
and Medicine 34(4):496e502.
[44] Ran, Q., Liang, H., Ikeno, Y., Qi, W., Prolla, T.A., Roberts 2nd, L.J., et al.,
2007. Reduction in glutathione peroxidase 4 increases life span through
increased sensitivity to apoptosis. Journals of Gerontology Series A: Biological
Sciences and Medical Sciences 62(9):932e942 doi: 62/9/932.[45] Meplan, C., Crosley, L.K., Nicol, F., Horgan, G.W., Mathers, J.C., Arthur, J.R.,
et al., 2008. Functional effects of a common single-nucleotide polymorphism
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
www.molecularmetabolism.com
(GPX4c718t) in the glutathione peroxidase 4 gene: interaction with sex.
American Journal of Clinical Nutrition 87(4):1019e1027.
[46] Qin, F., Siwik, D.A., Luptak, I., Hou, X., Wang, L., Higuchi, A., et al., 2012.
The polyphenols resveratrol and S17834 prevent the structural and functional
sequelae of diet-induced metabolic heart disease in mice. Circulation
125(14):1757e1764. http://dx.doi.org/10.1161/CIRCU-
LATIONAHA.111.067801. S1-6.
[47] Burgmaier, M., Sen, S., Philip, F., Wilson, C.R., Miller 3rd, C.C., Young, M.E.,
et al., 2010. Metabolic adaptation follows contractile dysfunction in the heart
of obese zucker rats fed a high-fat “western” diet. Obesity (Silver Spring)
18(10):1895e1901 doi: 10.1038/oby.2009.500.
[48] Wilson, C.R., Tran, M.K., Salazar, K.L., Young, M.E., Taegtmeyer, H., 2007.
Western diet, but not high fat diet, causes derangements of fatty acid
metabolism and contractile dysfunction in the heart of wistar rats.
Biochemical Journal 406(3):457e467 doi: BJ20070392.
[49] Hoffmann, F.W., Hashimoto, A.S., Lee, B.C., Rose, A.H., Shohet, R.V.,
Hoffmann, P.R., 2011. Specific antioxidant selenoproteins are induced in
the heart during hypertrophy. Archives of Biochemistry and Biophysics
512(1):38e44. http://dx.doi.org/10.1016/j.abb.2011.05.007, 10.1016/
j.abb.2011.05.007.
[50] Berndt, C., Lillig, C.H., Holmgren, A., 2007. Thiol-based mechanisms of the
thioredoxin and glutaredoxin systems: implications for diseases in the car-
diovascular system. American Journal of Physiology e Heart and Circulatory
Physiology 292(3):H1227eH1236 doi: 01162.2006. [pii] 10.1152/
ajpheart.01162.2006.
[51] Curtis, J.M., Hahn, W.S., Stone, M.D., Inda, J.J., Droullard, D.J.,
Kuzmicic, J.P., et al., 2012. Protein carbonylation and adipocyte mitochon-
drial function. Journal of Biological Chemistry 287(39):32967e32980 doi:
10.1074/jbc.M112.400663.
[52] Frohnert, B.I., Bernlohr, D.A., 2013. Protein carbonylation, mitochondrial
dysfunction, and insulin resistance. Advances in Nutrition 4(2):157e163 doi:
10.3945/an.112.003319.
[53] Hill, B.G., Dranka, B.P., Zou, L., Chatham, J.C., Darley-Usmar, V.M., 2009.
Importance of the bioenergetic reserve capacity in response to cardiomyocyte
stress induced by 4-hydroxynonenal. Biochemical Journal 424(1):99e107.
http://dx.doi.org/10.1042/BJ20090934.
[54] Fisher-Wellman, K.H., Mattox, T.A., Thayne, K., Katunga, L.A., La
Favor, J.D., Neufer, P.D., et al., 2013. Novel role for thioredoxin
reductase-2 in mitochondrial redox adaptations to obesogenic diet and
exercise in heart and skeletal muscle. Journal of Physiology 591(Pt 14):
3471e3486. http://dx.doi.org/10.1113/jphysiol.2013.254193, 10.1113/
jphysiol.2013.254193.
[55] Rindler, P.M., Plafker, S.M., Szweda, L.I., Kinter, M., 2013. High dietary fat
selectively increases catalase expression within cardiac mitochondria.
Journal of Biological Chemistry 288(3):1979e1990 doi: 10.1074/
jbc.M112.412890.
[56] Bhatt, N.M., Aon, M.A., Tocchetti, C.G., Shen, X., Dey, S., Ramirez-
Correa, G., et al., 2015. Restoring redox balance enhances contractility in
heart trabeculae from type 2 diabetic rats exposed to high glucose.
American Journal of Physiology e Heart and Circulatory Physiology 308(4):
H291eH302 doi: 10.1152/ajpheart.00378.2014.
[57] Xie, J., Mendez, J.D., Mendez-Valenzuela, V., Aguilar-Hernandez, M.M.,
2013. Cellular signalling of the receptor for advanced glycation end products
(RAGE). Cell Signaling 25(11):2185e2197. http://dx.doi.org/10.1016/
j.cellsig.2013.06.013.
[58] Fritz, G., 2011. RAGE: a single receptor fits multiple ligands. Trends in
Biochemical Sciences 36(12):625e632. http://dx.doi.org/10.1016/
j.tibs.2011.08.008, 10.1016/j.tibs.2011.08.008.[59] Thorpe, S.R., Baynes, J.W., 2003. Maillard reaction products in tissue pro-
teins: new products and new perspectives. Amino Acids 25(3e4):275e281.
http://dx.doi.org/10.1007/s00726-003-0017-9.
MOLECULAR METABOLISM 4 (2015) 493e506 
 2015 The Authors. Published by Elsevier GmbH. This is an o
www.molecularmetabolism.com[60] Biernacka, A., Dobaczewski, M., Frangogiannis, N.G., 2011. TGF-beta
signaling in fibrosis. Growth Factors 29(5):196e202. http://dx.doi.org/
10.3109/08977194.2011.595714.
[61] Dobaczewski, M., Gonzalez-Quesada, C., Frangogiannis, N.G., 2010. The
extracellular matrix as a modulator of the inflammatory and reparative
response following myocardial infarction. Journal of Molecular and Cellular
Cardiology 48(3):504e511. http://dx.doi.org/10.1016/j.yjmcc.2009.07.015,
10.1016/j.yjmcc.2009.07.015.
[62] Frangogiannis, N.G., 2012. Matricellular proteins in cardiac adaptation and
disease. Physiological Reviews 92(2):635e688. http://dx.doi.org/10.1152/
physrev.00008.2011, 10.1152/physrev.00008.2011.
[63] Anderson, E.J., Kypson, A.P., Rodriguez, E., Anderson, C.A., Lehr, E.J.,
Neufer, P.D., 2009. Substrate-specific derangements in mitochondrial
metabolism and redox balance in the atrium of the type 2 diabetic human
heart. Journal of the American College of Cardiology 54(20):1891e1898 doi:
S0735-1097(09)02758-2 [pii] 10.1016/j.jacc.2009.07.031.
[64] LoPachin, R.M., Gavin, T., 2014. Molecular mechanisms of aldehyde toxicity:
a chemical perspective. Chemical Research in Toxicology 27(7):1081e1091
doi: 10.1021/tx5001046.
[65] Behring, J.B., Kumar, V., Whelan, S.A., Chauhan, P., Siwik, D.A.,
Costello, C.E., et al., 2014. Does reversible cysteine oxidation link the
western diet to cardiac dysfunction? FASEB Journal 28(5):1975e1987 doi:
10.1096/fj.13-233445.
[66] Liang, H., Ran, Q., Jang, Y.C., Holstein, D., Lechleiter, J., McDonald-
Marsh, T., et al., 2009. Glutathione peroxidase 4 differentially regulates the
release of apoptogenic proteins from mitochondria. Free Radical Biology and
Medicine 47(3):312e320. http://dx.doi.org/10.1016/
j.freeradbiomed.2009.05.012.
[67] Liang, H., Yoo, S.E., Na, R., Walter, C.A., Richardson, A., Ran, Q., 2009. Short
form glutathione peroxidase 4 is the essential isoform required for survival
and somatic mitochondrial functions. Journal of Biological Chemistry 284(45):
30836e30844. http://dx.doi.org/10.1074/jbc.M109.032839.
[68] Cole-Ezea, P., Swan, D., Shanley, D., Hesketh, J., 2012. Glutathione
peroxidase 4 has a major role in protecting mitochondria from oxidative
damage and maintaining oxidative phosphorylation complexes in gut
epithelial cells. Free Radical Biology and Medicine 53(3):488e497.
http://dx.doi.org/10.1016/j.freeradbiomed.2012.05.029, 10.1016/
j.freeradbiomed.2012.05.029.
[69] Baseler, W.A., Dabkowski, E.R., Jagannathan, R., Thapa, D., Nichols, C.E.,
Shepherd, D.L., et al., 2013. Reversal of mitochondrial proteomic loss in type
1 diabetic heart with overexpression of phospholipid hydroperoxide gluta-
thione peroxidase. American Journal of Physiology. Regulatory, Integrative
and Comparative Physiology 304(7):R553eR565. http://dx.doi.org/10.1152/
ajpregu.00249.2012.
[70] Dabkowski, E.R., Williamson, C.L., Hollander, J.M., 2008. Mitochondria-
specific transgenic overexpression of phospholipid hydroperoxide glutathione
peroxidase (GPx4) attenuates ischemia/reperfusion-associated cardiac
dysfunction. Free Radical Biology and Medicine 45(6):855e865. http://
dx.doi.org/10.1016/j.freeradbiomed.2008.06.021, 10.1016/
j.freeradbiomed.2008.06.021.
[71] Conrad, M., 2009. Transgenic mouse models for the vital selenoenzymes
cytosolic thioredoxin reductase, mitochondrial thioredoxin reductase and
glutathione peroxidase 4. Biochimica et Biophysica Acta 1790(11):1575e
1585. http://dx.doi.org/10.1016/j.bbagen.2009.05.001.
[72] Li, Q., Sadhukhan, S., Berthiaume, J.M., Ibarra, R.A., Tang, H., Deng, S., et al.,
2013. 4-hydroxy-2(E)-nonenal (HNE) catabolism and formation of HNE adducts
are modulated by beta oxidation of fatty acids in the isolated rat heart. Free
Radical Biology and Medicine 58:35e44. http://dx.doi.org/10.1016/j.freeradbiomed.2013.01.005.
[73] Anderson, E.J., Rodriguez, E., Anderson, C.A., Thayne, K., Chitwood, W.R.,
Kypson, A.P., 2011. Increased propensity for cell death in diabetic human
pen access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 505
Original articleheart is mediated by mitochondrial-dependent pathways. American Journal
of Physiology e Heart and Circulatory Physiology 300(1):H118eH124. http://
dx.doi.org/10.1152/ajpheart.00932.2010.
[74] Lashin, O.M., Szweda, P.A., Szweda, L.I., Romani, A.M., 2006. Decreased
complex II respiration and HNE-modified SDH subunit in diabetic heart. Free
Radical Biology and Medicine 40(5):886e896. http://dx.doi.org/10.1016/
j.freeradbiomed.2005.10.040.
[75] Sverdlov, A.L., Elezaby, A., Behring, J.B., Bachschmid, M.M., Luptak, I.,
Tu, V.H., et al., 2015. High fat, high sucrose diet causes cardiac mito-
chondrial dysfunction due in part to oxidative post-translational modification
of mitochondrial complex II. Journal of Molecular and Cellular Cardiology 78:
165e173. http://dx.doi.org/10.1016/j.yjmcc.2014.07.018.
[76] Loscalzo, J., 2014. Keshan disease, selenium deficiency, and the seleno-
proteome. New England Journal of Medicine 370(18):1756e1760. http://
dx.doi.org/10.1056/NEJMcibr1402199.
[77] Stanley, W.C., Dabkowski, E.R., Ribeiro Jr., R.F., O’Connell, K.A., 2012. Di-
etary fat and heart failure: moving from lipotoxicity to lipoprotection. Circu-
lation Research 110(5):764e776. http://dx.doi.org/10.1161/
CIRCRESAHA.111.253104, 10.1161/CIRCRESAHA.111.253104.
[78] Ghosh, S., Rodrigues, B., 2006. Cardiac cell death in early diabetes and its
modulation by dietary fatty acids. Biochimica et Biophysica Acta 1761(10):
1148e1162. http://dx.doi.org/10.1016/j.bbalip.2006.08.010.
[79] Borisov, A.B., Ushakov, A.V., Zagorulko, A.K., Novikov, N.Y., Selivanova, K.F.,
Edwards, C.A., et al., 2008. Intracardiac lipid accumulation, lipoatrophy of
muscle cells and expansion of myocardial infarction in type 2 diabetic pa-
tients. Micron 39(7):944e951. http://dx.doi.org/10.1016/
j.micron.2007.11.002.
[80] Sharma, S., Adrogue, J.V., Golfman, L., Uray, I., Lemm, J., Youker, K., et al.,
2004. Intramyocardial lipid accumulation in the failing human heart re-
sembles the lipotoxic rat heart. FASEB Journal 18(14):1692e1700 doi: 18/
14/1692 [pii] 10.1096/fj.04-2263com.
[81] Stanley, W.C., Recchia, F.A., 2010. Lipotoxicity and the development of heart
failure: moving from mouse to man. Cell Metabolism 12(6):555e556.
S1550-4131(10)00408-0 [pii] 10.1016/j.cmet.2010.11.016.
[82] Opie, L.H., Knuuti, J., 2009. The adrenergic-fatty acid load in heart failure.
Journal of the American College of Cardiology 54(18):1637e1646, 10.1016/
j.jacc.2009.07.024; 10.1016/j.jacc.2009.07.024.
[83] Hill, B.G., Haberzettl, P., Ahmed, Y., Srivastava, S., Bhatnagar, A., 2008.
Unsaturated lipid peroxidation-derived aldehydes activate autophagy in
vascular smooth-muscle cells. Biochemical Journal 410(3):525e534. http://
dx.doi.org/10.1042/BJ20071063.
[84] Okada, K., Wangpoengtrakul, C., Osawa, T., Toyokuni, S., Tanaka, K.,
Uchida, K., 1999. 4-hydroxy-2-nonenal-mediated impairment of intracellular
proteolysis during oxidative stress. identification of proteasomes as target
molecules. Journal of Biological Chemistry 274(34):23787e23793.
[85] Imai, H., 2004. Biological significance of lipid hydroperoxide and its reducing
enzyme, phospholipid hydroperoxide glutathione peroxidase, in mammalian
cells. Yakugaku Zasshi 124(12):937e957.
[86] Nakamura, T., Imai, H., Tsunashima, N., Nakagawa, Y., 2003. Molecular
cloning and functional expression of nucleolar phospholipid hydroperoxide
glutathione peroxidase in mammalian cells. Biochemical and Biophysical
Research Communications 311(1):139e148.
[87] Sneddon, A.A., Wu, H.C., Farquharson, A., Grant, I., Arthur, J.R., Rotondo, D.,
et al., 2003. Regulation of selenoprotein GPx4 expression and activity in
human endothelial cells by fatty acids, cytokines and antioxidants. Athero-
sclerosis 171(1):57e65.
[88] Frustaci, A., Francone, M., Petrosillo, N., Chimenti, C., 2013. High prevalence
of myocarditis in patients with hypertensive heart disease and cardiac506 MOLECULAR METABOLISM 4 (2015) 493e506 
 2015 The Authors. Published by Elsevier GmbH.deterioration. European Journal of Heart Failure 15(3):284e291. http://
dx.doi.org/10.1093/eurjhf/hfs169.
[89] Aldini, G., Orioli, M., Rossoni, G., Savi, F., Braidotti, P., Vistoli, G., et al., 2011.
The carbonyl scavenger carnosine ameliorates dyslipidaemia and renal
function in zucker obese rats. Journal of Cellular and Molecular Medicine
15(6):1339e1354. http://dx.doi.org/10.1111/j.1582-4934.2010.01101.x,
10.1111/j.1582-4934.2010.01101.x.
[90] Menini, S., Iacobini, C., Ricci, C., Scipioni, A., Blasetti Fantauzzi, C.,
Giaccari, A., et al., 2012. D-carnosine octylester attenuates atherosclerosis
and renal disease in ApoE null mice fed a western diet through reduction of
carbonyl stress and inflammation. British Journal of Pharmacology 166(4):
1344e1356. http://dx.doi.org/10.1111/j.1476-5381.2012.01834.x,
10.1111/j.1476-5381.2012.01834.
[91] Kass, D.A., Shapiro, E.P., Kawaguchi, M., Capriotti, A.R., Scuteri, A.,
deGroof, R.C., et al., 2001. Improved arterial compliance by a novel advanced
glycation end-product crosslink breaker. Circulation 104(13):1464e1470.
[92] Little, W.C., Zile, M.R., Kitzman, D.W., Hundley, W.G., O’Brien, T.X.,
Degroof, R.C., 2005. The effect of alagebrium chloride (ALT-711), a novel
glucose cross-link breaker, in the treatment of elderly patients with diastolic
heart failure. Journal of Cardiac Failure 11(3):191e195 doi:
S1071916404009017.
[93] Tsujita, K., Shimomura, H., Kaikita, K., Kawano, H., Hokamaki, J.,
Nagayoshi, Y., et al., 2006. Long-term efficacy of edaravone in patients with
acute myocardial infarction. Circulation Journal 70(7):832e837 doi:
JST.JSTAGE/circj/70.832.
[94] Anderson, E.J., Efird, J.T., Davies, S.W., O’Neal, W.T., Darden, T.M., BS
Thayne, K.A., Katunga, L.A., et al., 2014. Monoamine oxidase is a major
determinant of redox balance in human atrial myocardium and is associated
with postoperative atrial fibrillation. Journal of the American Heart Association
(JAHA) 3(1):e000713. http://dx.doi.org/10.1161/JAHA.113.000713.
[95] Anderson, E.J., Neufer, P.D., 2006. Type II skeletal myofibers possess unique
properties that potentiate mitochondrial H(2)O(2) generation. American
Journal of Physiology. Cell physiology 290(3):C844eC851.
[96] Frezza, C., Cipolat, S., Scorrano, L., 2007. Organelle isolation: functional
mitochondria from mouse liver, muscle and cultured fibroblasts. Nature
Protocols 2(2):287e295 doi: nprot.2006.478.
[97] Gostimskaya, I., Galkin, A., 2010. Preparation of highly coupled rat heart
mitochondria. Journal of visualized experiments(43) pii: 2202. doi(43):
10.3791/2202.
[98] Merry, T.L., Tran, M., Stathopoulos, M., Wiede, F., Fam, B.C., Dodd, G.T.,
et al., 2014 May 10. High-fat-fed obese glutathione peroxidase 1-deficient
mice exhibit defective insulin secretion but protection from hepatic stea-
tosis and liver damage. Antioxid Redox Signal 20(14):2114e2129.
[99] Kang, L., Dai, C., Lustig, M.E., Bonner, J.S., Mayes, W.H.,
Mokshagundam, S., et al., 2014 Nov. Heterozygous SOD2 deletion impairs
glucose-stimulated insulin secretion, but not insulin action, in high-fat-fed
mice. Diabetes 63(11):3699e3710.
[100] Kendig, E.L., Chen, Y., Krishan, M., Johansson, E., Schneider, S.N.,
Genter, M.B., et al., 2011 Dec 15. Lipid metabolism and body composition in
Gclm(-/-) mice. Toxicology and Applied Pharmacology 257(3):338e348.
[101] Li, Q., Tomcik, K., Zhang, S., Puchowicz, M.A., Zhang, G.F., 2012 Mar 15.
Dietary regulation of catabolic disposal of 4-hydroxynonenal analogs in rat
liver. Free Radical Biology and Medicine 52(6):1043e1053.
[102] Aldini, G., Carini, M., Yeum, K.J., Vistoli, G., 2014 Apr. Novel molecular
approaches for improving enzymatic and nonenzymatic detoxification of 4-
hydroxynonenal: toward the discovery of a novel class of bioactive com-
pounds. Free Radical Biology and Medicine 69:145e156.This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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