heme oxygenase-1 drives metaflammation and insulin...
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Heme Oxygenase-1 DrivesMetaflammation and Insulin Resistance inMouse and ManAlexander Jais,1,12 Elisa Einwallner,1,12 Omar Sharif,1,2 Klaus Gossens,3 Tess Tsai-Hsiu Lu,3 Selma M. Soyal,4
David Medgyesi,3,5 Daniel Neureiter,4 Jamile Paier-Pourani,6 Kevin Dalgaard,3 J. Catharina Duvigneau,7
Josefine Lindroos-Christensen,1 Thea-Christin Zapf,1,11 Sabine Amann,1 Simona Saluzzo,1,2 Florian Jantscher,1
Patricia Stiedl,8 Jelena Todoric,1 Rui Martins,1,2 Hannes Oberkofler,4 Simone Muller,7 Cornelia Hauser-Kronberger,4
Lukas Kenner,1,7,8 Emilio Casanova,1,8 Hedwig Sutterluty-Fall,1 Martin Bilban,1 Karl Miller,9 Andrey V. Kozlov,6
Franz Krempler,9 Sylvia Knapp,1,2 Carey N. Lumeng,10 Wolfgang Patsch,4 Oswald Wagner,1 J. Andrew Pospisilik,3,*and Harald Esterbauer1,*1Medical University of Vienna, 1090 Vienna, Austria2CeMM, Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria3Max Planck Institute of Immunobiology and Epigenetics, 79108 Freiburg, Germany4Paracelsus Medical University, 5020 Salzburg, Austria5BIOSS Centre of Biological Signalling Studies, University of Freiburg, 79104 Freiburg, Germany6Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, 1200 Vienna, Austria7University of Veterinary Medicine Vienna, 1210 Vienna, Austria8Ludwig Boltzmann Institute for Cancer Research, 1090 Vienna, Austria9General Hospital Hallein, 5400 Hallein, Austria10University of Michigan, Ann Arbor, MI 48109, USA11Present address: Phillips University Marburg, 35043 Marburg, Germany12Co-first authors*Correspondence: [email protected] (J.A.P.), [email protected] (H.E.)
http://dx.doi.org/10.1016/j.cell.2014.04.043
SUMMARY
Obesity and diabetes affect more than half abillion individuals worldwide. Interestingly, thetwo conditions do not always coincide and themolecular determinants of ‘‘healthy’’ versus ‘‘un-healthy’’ obesity remain ill-defined. Chronic meta-bolic inflammation (metaflammation) is believed tobe pivotal. Here, we tested a hypothesized anti-inflammatory role for heme oxygenase-1 (HO-1)in the development of metabolic disease. Surpris-ingly, in matched biopsies from ‘‘healthy’’ versusinsulin-resistant obese subjects we find HO-1 tobe among the strongest positive predictors ofmetabolic disease in humans. We find that hepa-tocyte and macrophage conditional HO-1 deletionin mice evokes resistance to diet-induced insulinresistance and inflammation, dramatically reducingsecondary disease such as steatosis and livertoxicity. Intriguingly, cellular assays show thatHO-1 defines prestimulation thresholds for in-flammatory skewing and NF-kB amplification inmacrophages and for insulin signaling in hepa-tocytes. These findings identify HO-1 inhibition asa potential therapeutic strategy for metabolicdisease.
INTRODUCTION
The American Medical Association recently voted to recognize
obesity as a disease (http://www.ama-assn.org). Interestingly,
up to one in four individuals currently labeled as obese are actu-
ally metabolically healthy (Stefan et al., 2013), scoring normal on
indices of insulin resistance and systemic inflammation. Impor-
tantly, the factors determining ‘‘healthy’’ versus ‘‘unhealthy’’
obesity remain ill-defined.
Adipose tissuemass, and in particular ratios of ‘‘good’’ subcu-
taneous versus ‘‘bad’’ visceral depots are strongly correlated
with disease; whereas visceral fat expansion is associated with
metabolic disease (Tchkonia et al., 2013), expansion associated
with insulin sensitizers (Virtue and Vidal-Puig, 2008), transplanta-
tion of subcutaneous adipose (Gavrilova et al., 2000), and even
massive subcutaneous expansion (Kusminski et al., 2012) have
all been shown to be disease sparing. Indeed, pronounced
loss of white fat is—with rare exceptions (Pospisilik et al.,
2010)—linked to insulin resistance (Garg, 2011). Using insulin
sensitivity as the hallmark of healthy obesity several controlling
factors have emerged (Johnson and Olefsky, 2013). Visceral
adipocyte hypertrophy and associated hypoxia and fibrosis are
detrimental (Sun et al., 2013). Saturated free fatty acids and
ectopic lipid storage in liver and muscle impair insulin action
(Samuel and Shulman, 2012). Further, reactive oxygen species
(Houstis et al., 2006), mitochondrial function (Szendroedi et al.,
2012), endoplasmic reticulum stress (Hotamisligil, 2010), and
gut microbiota (Burcelin et al., 2012) all provide influence.
Cell 158, 25–40, July 3, 2014 ª2014 Elsevier Inc. 25
Importantly, many—if not all—insulin resistance drivers identi-
fied to date converge uniformly upon inflammation and proin-
flammatory signaling (Lumeng and Saltiel, 2011; Odegaard and
Chawla, 2013; Solinas and Karin, 2010). Such chronic inflamma-
tion, so-called ‘‘metaflammation,’’ currently provides a unifying
theory for unhealthy obesity (Gregor and Hotamisligil, 2011).
The concept of metaflammation was born out of the discovery
that tumor necrosis factor-alpha (TNF-a) potently inhibits adi-
pose insulin signaling (Hotamisligil et al., 1993). Subsequent
work implicated additional hallmark activities including inhibitor
of kB kinase (Yuan et al., 2001), c-Jun amino-terminal kinases
(Hirosumi et al., 2002), and protein kinase R (Nakamura et al.,
2010). Two landmark papers described macrophage infiltrates
in adipose as the source of insulin-resistance inducing TNF-a
(Weisberg et al., 2003; Xu et al., 2003). Such infiltrates have since
been characterized as classically activated, proinflammatory
M1-like macrophages (Lumeng et al., 2007; Solinas et al.,
2007), which have proven pivotal to the emergence of metabolic
disease (Patsouris et al., 2008). Importantly, studies now impli-
cate metaflammation as a central feature of human insulin resis-
tance (Capel et al., 2009; Qatanani et al., 2013).
Comparisons of ‘‘cold’’ metaflammation with classical ‘‘hot’’
inflammation reveal few differences (Calay and Hotamisligil,
2013). Here, we tested heme oxygenase-1 (HO-1), a historically
anti-inflammatory molecule that catalyzes heme breakdown to
CO, biliverdin, and free iron (Abraham and Kappas, 2008). In
addition to heme, HO-1 is induced by numerous stressors
including endotoxin, cytokines, and oxidants (Abraham and
Kappas, 2008). In humans, HO-1 loss-of-function leads to early
death (Yachie et al., 1999). Deletion in mice results in high peri-
natal mortality and increased susceptibility to inflammation
(Kapturczak et al., 2004). Interestingly, several studies have
shown that chemical HO-1 induction ameliorates obesity and
diabetes in rodents (Li et al., 2008; Ndisang et al., 2009). How-
ever, these results have failed validation in more specific genetic
models (Huang et al., 2013; Huang et al., 2012), and human
studies show similarly conflicting results (Bao et al., 2010; Shak-
eri-Manesch et al., 2009).
Here, using conditional mouse genetics we show that HO-1
is in fact proinflammatory, potently and independently driving
insulin resistance in the hepatic and macrophage compart-
ments. Against expectations, we find that HO-1 is among the
top predictors of metabolic disease in humans and mice and,
intriguingly, that hepatocyte and macrophage loss-of-function
leads to resistance to diet-induced metaflammation, insulin
resistance, and steatosis. The results indicate that HO-1 is in
fact necessary for the development of metaflammation and
metabolic disease, and call for a re-evaluation of numerous find-
ings in the field. They identify HO-1 as a candidate biomarker for
stratification of metabolically healthy and unhealthy obesity and
provide a framework for selective, personalized therapy.
RESULTS
HO-1 Expression Predicts Insulin Resistance in Mouseand ManOver the last years we collected a unique resource of matched
serum, liver, and adipose biopsies from well-characterized indi-
26 Cell 158, 25–40, July 3, 2014 ª2014 Elsevier Inc.
viduals for studying ‘‘healthy’’ versus ‘‘diseased’’ obesity. Re-
cruited subjects were required to be clinically obese (BMI >
30), to exhibit normal fasting glucose levels, to have no overt
inflammation, and to be free of any medications. Critically,
whereas obese insulin-resistant (obIR) individuals exhibited
clearly elevated insulin resistance (HOMA-IR R 5), obese insu-
lin-sensitive (obIS) individuals were required to show no signs
of systemic insulin resistance, i.e., HOMA-IR % 2. We thus bio-
psied 17 obIS and 27 obIR individuals well-matched for age and
BMI (Table 1) and complemented the samples with biopsies from
6 nonobese individuals. Consistent with their insulin resistance,
the obIR group displayed minimally elevated waist circumfer-
ence, visceral adiposity, triglyceride to HDL ratio, as well as
GLUT4 and HSD11B1 mRNA expression by qPCR (Table 1).
Thus, we assembled a clinical resource to probe early determi-
nants of insulin resistance in obesity.
Interestingly, and in contrast to our initial expectations, we
found elevated HO-1 expression in obIR individuals in both
the liver and visceral fat compartments (Figures 1A and 1B).
HO-1 levels correlated directly with metabolic dysregulation,
increasing progressively from lean to obIS and through to obIR
states (Figures 1A and 1B). Importantly, regression analyses
including seven ‘‘intermediate’’ insulin resistance subjects
(HOMA-IR > 2 and < 5) revealed that liver and adipose HO-1
expression explain 21.7% and 43.3% of HOMA-IR, relationships
maintained in both males and females, and independent of waist
circumference or visceral fat (Tables S1 and S2 available online).
These findings were confirmed on the protein level, and impor-
tantly, placed HO-1 within both the hepatocyte and Kupffer cell
compartments in liver and in macrophages in adipose (Figures
1C and 1D). Of note, HO-1 protein was elevated in both hepatic
compartments in obIR individuals (Figures 1C and 1E). In
adipose, HO-1 was particularly evident in inflammatory
macrophage crown-like structures (Figure 1D). Thus, liver and
adipose HO-1 are potent independent predictors of insulin resis-
tance in man.
Since a proinflammatory role for HO-1 contradicted bulk liter-
ature on the enzyme, we performed an unbiased search for data
sets to validate our findings. We identified five data sets, two in
liver and three in adipose, comparing obese and/or insulin-resis-
tant adult humans with controls, and mined them for any pattern
of HO-1 expression. Impressively, HO-1 was increased in the
metabolically compromised setting in five of five studies. HO-1
was increased in livers of obese versus lean individuals (Fig-
ure 1F) (Pihlajamaki et al., 2009) and of diabetic versus nondia-
betic subjects (Figure 1G) (Misu et al., 2010). In adipose, HO-1
was elevated in insulin-resistant obese subjects (Figure 1H)
(NCBI, GEO data set GDS3665). In a rare opportunity to control
for genetic background in the human setting, we mined adipose
expression profiles from monozygotic twins discordant for BMI
and observed HO-1 again to be consistently higher in the obese
sibling (Figure 1I) (Pietilainen et al., 2008). Finally, in a landmark
survey of adipose from 387 morbidly obese individuals, HO-1
scored 3rd of all insulin-resistance risk genes (Figure 1J) (Qata-
nani et al., 2013). Thus, liver and adipose tissue HO-1 expression
consistently predict metabolic dysfunction in humans.
To test conservation, we set out to generate a parallel data set
in mice. Similar to humans, mice exhibit substantial HO-1
Table 1. Characteristics of the Study Population
Trait Nonobese obIS obIR pa pb pc
Male/female (n) 3/3 2/15 10/17 0.088 0.089 0.090
Age (years) 40.5 (3.3) 39.2 (2.3) 38.8 (2.3) n.s. n.s. n.s.
BMI (kg/m2) 25.2 (0.9) 40.9 (1.2) 45.0 (0.9) n.d. n.d. n.s.
HOMA-IR 1.0 (0.2) 1.4 (0.1) 7.0 (0.3) n.d. 0.023 n.d.
Waist circumference (cm) 87.8 (2.8) 119.1 (3.4) 132.9 (2.9) n.d. n.d. 0.035
Hip circumference (cm) 101.2 (2.8) 130.5 (2.0) 135.1 (2.2) n.d. n.d. n.s.
Waist/hip ratio 0.87 (0.03) 0.91 (0.02) 0.99 (0.02) <0.001 0.042 0.078
SAT (cm) 2.3 (0.6) 3.1 (0.2) 3.5 (0.2) n.d. n.d. n.s.
VAT (cm) 1.8 (0.4) 5.3 (0.7) 6.9 (0.5) n.d. n.d. 0.025
Insulin (pmol/l) 31.8 (5.9) 44.9 (2.2) 201.1 (10.6) n.d. 0.027 n.d.
Glucose (mmol/l) 4.8 (0.2) 4.8 (0.1) 5.5 (0.1) n.d. n.s. n.d.
Cholesterol (mmol/l) 5.6 (0.4) 4.8 (0.2) 4.9 (0.2) n.s. n.s. n.s.
Triglycerides (mmol/l) 1.3 (0.4) 1.2 (0.1) 2.2 (0.3) 0.002 n.s. 0.006
HDL cholesterol (mmol/l) 1.8 (0.2) 1.5 (0.1) 1.2 (0.1) <0.001 n.s. 0.007
LDL cholesterol (mmol/l) 3.2 (0.2) 2.8 (0.2) 2.8 (0.2) n.s. n.s. n.s.
Triglyceride/HDL ratio 1.6 (0.4) 2.1 (0.3) 4.7 (0.7) <0.001 n.s. 0.001
C-reactive protein (mg/l) 4.5 (3.1) 5.6 (1.1) 7.2 (0.1) n.s. n.s. n.s.
GLUT4 mRNA (AU) 1.36 (0.27) 1.00 (0.10) 0.53 (0.05) <0.001 n.s. 0.001
HSD11B1 mRNA (AU) 0.63 (0.18) 1.00 (0.15) 2.28 (0.35) <0.001 n.s. <0.001
Data are numbers of observations or unadjusted and untransformed means (SEM). Two-sided p values obtained from Chi-square test or ANOVA
(Scheffe-Test for subgroup comparisons adjusted for age and sex) for categorical or continuous data, respectively.acomparison among all groups.bnonobese controls versus obese insulin-sensitive (obIS).cobIS versus obese insulin-resistant (obIR); participants (total) n = 50 (6/17/27; nonobese/obIS/obIR; n = 48 (6/16/26) for SAT and VAT; obIS, HOMA-IR
% 2.0; obIR, HOMA-IR > = 5.0; n.s., not significant; n.d., not determined; SAT, subcutaneous adipose tissue; VAT, visceral adipose tissue; AU, arbitrary
unit. See also Tables S1 and S2.
expression in the lung, kidney, and spleen and, interestingly, in
metabolic organs including the liver and white adipose tissue
(Figure S1A). To explore mouse HO-1 variation with metabolic
disease, we generated a large cohort of high-fat-diet (HFD)-
treated male C57BL/6J mice, stratified them according to
glucose (Figure S1B) and insulin tolerance (Figure S1C) and
selected subgroups exhibiting either insulin sensitivity (IS) or in-
sulin resistance (IR) despite comparable weight gain on the HFD
regimen (Figure S1D). Of note, and in complete agreement with
our human findings, HO-1 expression in liver (Figure 1K) and
adipose (Figure 1L) was directly proportional to severity of meta-
bolic dysregulation. Again, measures were confirmed on the pro-
tein level, in both liver (Figure 1M) and epididymal adipose tissue
(Figure 1N). Importantly, the obese-IR and -IS groups did not
differ in body weight (Figure S1D), and were matched for age
and developmental environment. Thus, hepatic and adipose tis-
sue HO-1 predict insulin resistance in mouse and man.
Hepatocyte HO-1 Knockout Mice Are InsulinHypersensitiveTo directly assess the role of HO-1 in the etiology of metabolic
disease, we generated mice bearing a conditional allele for
HO-1 (HO-1fl/fl) (Figures S2A and S2B). First, we crossed them
to Albumin-Cre transgenic mice (Alb-Cre) to obtain liver (hepato-
cyte)-specific HO-1 knockouts (Lhoko). Efficient deletion was
confirmed on the DNA, protein and activity levels (Figures
2A, 2B, and S2C). Functional deletion of HO-1 was confirmed
in vivo by examining HO-1 expression in whole livers upon hemin
injection, a heme analog known to induce HO-1 expression and
activity (Figure 2C). Thus, we generated hepatocyte-specific
HO-1 mutant mice.
Lhoko mice were born at Mendelian ratios, developed
normally, and exhibited normal liver morphology as well as
serum parameters (Figures S2D and S2E). Importantly, Lhoko
animals exhibited completely normal glucose and insulin toler-
ance relative to their littermate controls (Figures S2F and S2G),
showing no evidence of HO-1 imposed anti-inflammatory tone
in the naive state. Remembering that our observations of
disease predicting HO-1 were made in metabolically stressed
individuals, we challenged Lhoko mice with HFD. With respect
to adiposity, Lhoko mice showed no difference in HFD-induced
body weight gain relative to littermates (Figure S2H). Similarly,
they maintained unremarkable oral glucose tolerance (Fig-
ure 2D). Intriguingly though, insulin tolerance, fasting insulin,
and HOMA-IR all indicated significantly increased insulin sensi-
tivity in the HFD-treated Lhoko animals (Figures 2D and 2E), a
clear indication of improved metabolic health. In agreement
with these findings, serum measures of hepatotoxicity including
alanine transaminase (ALAT), aspartate transaminase (ASAT),
and ASAT/ALAT ratios were clearly improved (Figures 2F). To
understand the insulin sensitivity on a cellular level, an insulin
bolus was administered directly into the portal vein of
Cell 158, 25–40, July 3, 2014 ª2014 Elsevier Inc. 27
Figure 1. HO-1 Levels Predict Insulin Resistance in Mouse and Man
(A andB) qPCR analysis ofHO-1mRNA in (A) liver and (B) visceral adipose biopsies obtained from obese insulin-sensitive (obIS) and obese insulin-resistant (obIR)
humans.
(C and D) Immunohistochemical staining for HO-1 in (C) liver and (D) obIR visceral fat biopsies. Scale bar, 50 mm.
(E) Percentage of HO-1 positive hepatocytes in obIS and obIR liver biopsies.
(F–I) HO-1 mRNA levels obtained from public microarray data sets; (F and G) liver (H and I) adipose tissue. Numbers in bars represent study participants.
(legend continued on next page)
28 Cell 158, 25–40, July 3, 2014 ª2014 Elsevier Inc.
HFD-treated Lhoko and control animals, and liver biopsies
taken over 10 min. Intriguingly, loss of HO-1 induced stark
augmentation and acceleration of key upstream insulin
signaling activation events including insulin receptor (INSR)
and AKT phosphorylation (Figure 2G). The effects were highly
reproducible, and evident within 2 min of injection. Indicating
more widespread positive effects of hepatic HO-1 deletion,
examinations of muscle and adipose responses in the same
animals revealed moderately elevated pAKT (Figure 2G). In
keeping with these improvements in metabolic function, oil
red O staining and biochemical measures revealed attenuated
liver steatosis in HFD-treated Lhoko animals (Figures 2H and
2I). Gluconeogenic and lipogenic programs were largely unal-
tered (Figure S2I). Together, these findings comprise direct ge-
netic evidence that loss of hepatocyte HO-1 in vivo induces
proximal insulin signaling hypersensitivity and resistance to
HFD-induced metabolic sequelae.
To corroborate these data, we also performed the opposite
genetic experiment, acutely overexpressing HO-1 via tail vein
injection of adenoviral HO-1 (Figures S2J and S2K). Critically,
and in direct support of the human and Lhoko data, 1 week of
�5-fold HO-1 overexpression in unchallenged chow-fed
C57BL/6Jmice triggered glucose intolerance, insulin resistance,
and elevated HOMA-IR relative to Adeno-LacZ injected controls
(Figures 2J, 2K, and S2L). Thus, genetic gain- and loss-of-func-
tion studies in vivo indicate that hepatocyte HO-1 exacerbates
insulin resistance, steatosis, liver damage and metabolic
disease.
Macrophage HO-1 Knockout Mice Resist MetabolicDiseaseConsidering the observed increases in macrophage HO-1 in in-
sulin-resistant liver and adipose in both mouse and man, and
the central role of macrophages in metaflammation, we next
tested the contribution of macrophage HO-1 toward insulin
resistance. We crossed our conditional HO-1 mice to macro-
phage deleting LysM-Cre transgenic animals, creating macro-
phage HO-1 knockout (Macho) mice. Equal and efficient deletion
was confirmed in naive, as well as lipopolysaccharide (LPS)/
interferon (IFN)g-driven proinflammatory (M1) and interleukin
(IL)-4/IL-13-driven anti-inflammatory (M2) bone-marrow-derived
macrophages (BMDMs; Figures 3A–3C). Similar to Lhoko ani-
mals, Macho mice were born at Mendelian ratios, developed
normally, exhibited normal white blood cell counts, and ap-
peared robust and healthy (Figures S3A and S3B and data not
shown). Importantly, metabolic profiling was equally unremark-
able in the unchallenged state with no detectable alterations in
body weight, food intake, glucose tolerance, insulin sensitivity,
or plasma-free fatty acid levels (Figures S3A–S3F). Thus, macro-
phageHO-1 is dispensable for normal growth, development, and
metabolic homeostasis.
(J) Rank order of 968 visceral adipose tissue genes correlating with HOMA-IR and
genes) obesity. p values for Spearman correlation coefficients (HOMA-IR versus
significance cut-off at p < 0.01, corrected for multiple testing.
(K and L) qPCR analysis of HO-1 expression in (K) liver, and (L) epididymal adipos
(M and N) Immunoblot analysis for HO-1 in (M) liver, and (N) epididymal adipose
Results are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S1
As mentioned above, bulk literature would have predicted, if
anything, enhanced inflammation upon HO-1 deletion. In an
attempt to unmask such a phenotype, we again turned to HFD.
Intriguingly, and again in contrast to expectations, we observed
marked increases in glucose clearance and insulin sensitivity in
Macho animals after a 16 week HFD regimen (Figures 3D and
3E). Indeed, even body weight gain was slightly reduced
(Figure 3D)—despite no measurable change in food intake (Fig-
ure S3G). Importantly, all aspects of improved glucose homeo-
stasis were evident before body weight differences emerged
(data not shown) and, independently, when comparing body-
weight-matched animals at the experiment’s end (Figure 3F).
Interestingly, the metabolic improvements elicited by macro-
phage HO-1 loss were systemic. Both liver morphology and his-
tology revealed clear reductions in hepatic steatosis in Macho
animals (Figure 3G), improvements confirmed by reduced mea-
sures of lipid content and liver toxicity, as well as lipogenic and
gluconeogenic profiles (Figures 3H, 3I, and S3H). Specifically,
expression of the lipogenic factors Pparg, Srebf1, Cd36, and
Fasn were reduced in HFD-Macho livers as well as G6pc, which
mediates hepatic glucose output (Figure 3H). Expression of in-
flammatory Tnf, Il6, and Il1b were equally reduced (Figure 3H),
a contrast to elevated anti-inflammatory Il10 (Figure 3H). Impor-
tantly, HO-1 expression, whichwe show here to be proinflamma-
tory, was not only reduced in the myeloid compartment, but
importantly also in hepatocytes of the Macho mice (Figure 3G),
indicating that macrophage HO-1 is necessary for effective
transmission of inflammatory status to target tissues. Consistent
with our previous findings, liver, muscle, and fat responses to in-
traportal insulin were significantly enhanced in the HFD-treated
Macho mice (Figure S3I). These findings are in direct agreement
with our observations that HO-1 expression in both hepatocytes
and macrophages of human obese liver predicts disease
severity. An aside on the observed body weight difference,
Macho animals exhibited a minor increase in energy expenditure
(Figure 3J) independent of any altered activity (Figure 3K) or res-
piratory quotient (Figure S3J). These data are consistent with the
observed system-wide improvements in metabolic homeosta-
sis. In all, the data of both mouse models and the human cohort
implicate myeloid HO-1 as an effector of metabolic disease.
Thus, macrophage HO-1 is necessary for the manifestation of
HFD-induced metaflammation.
Reduced Metaflammation and Improved Adipose TissueArchitecture in Macho MiceSteatosis results in part from fatty acid spill-over resulting from
inflammation, insulin resistance, and improper lipolysis. In line
with the reduced steatosis, HFD-Macho mice exhibited reduced
fasted and fed serum-free fatty acids (Figure 4A). Further, exam-
ination of HFD-Macho epididymal fat revealed dramatic
improvements in metaflammation. Included were near-absence
predicting metabolically healthy (251 protective genes) and unhealthy (717 risk
expression values) obtained from public data set. Dotted line represents the
e obtained from obese IS and obese IR C57BL/6J mice after 16 weeks on HFD.
obtained from obese IR and obese IS C57BL/6J mice after 16 weeks of HFD.
and Tables S1 and S2.
Cell 158, 25–40, July 3, 2014 ª2014 Elsevier Inc. 29
of crown-like structures (Figure 4B), reduced adipocyte size
(Figures 4B and 4C), and a substantial reduction in macrophage
infiltration (Figure 4D). Importantly, these healthy metabolic attri-
butes were manifest in the face of clear obesity. Consistent with
this ‘‘healthy’’ obesity profile, HFD-Macho animals exhibited
clear reductions in the systemic indicators of metaflammation,
namely TNF-a, IL-1b, MCP-1, and RANTES (Figure 4E). Adipo-
nectin (Adipoq) and retinol binding protein-4 (Rbp4) mRNA levels
were increased in HFD-Macho mouse adipose, whereas leptin
(Lep) levels were not affected (Figure 4F). Notably, the Rbp4
mRNA changes were not manifest on the protein level (Fig-
ure 4G). Thus, Macho mice exhibit reduced systemic and adi-
pose tissue metaflammation.
The human andmouse data to this point suggested a predom-
inantly myeloid and hepatic role for HO-1 in metaflammation and
insulin resistance. We also largely ruled out the contribution of
HO-1 in muscle, adipocyte, and pancreatic b-cell compart-
ments, as targeted deletion in these depots did not affect
HFD-challenged body weight accumulation, glucose, or insulin
tolerance (Figures S4A–S4C).
Next, we asked how HO-1 elicits metaflammation. We purified
and FACS analyzed the nonadipocyte stromal vascular fraction
(SVF) from epididymal adipose of HFD-treated Macho and con-
trol mice. In line with the reduced evidence for metaflammation,
HFD-Macho adipose tissue contained a 2-fold reduction in
proinflammatory CD11c+ and CCR2+ macrophage infiltrates,
while protective M2-like MGL+ macrophage numbers were
increased (Figures 4H–4J and Figure S4D). These observations
were equally reflected in qPCR measures of Cd68, Itgax/
Cd11c and the chemokine receptors Ccr2 and Ccr5 in whole-
adipose RNA preparations (Figures S4E and S4F), as well as
confocal evidence of improved adipose tissue architecture
(Figure 4K). In support of a conserved system in humans, re-
examination of our human adipose biopsies revealed an increase
in total macrophage number and M1-like marker expression
(Table S3). Importantly, HO-1 exhibited a tight positive correla-
tion with M1-like marker expression (TNF, ITGAX/CD11c),
whereas M2-like markers showed no correlation (MRC1/
CD206) or an inverse relationship (CLEC10A/CD301, CCL18)
(Table S4). Importantly, these findingswere independent of waist
circumference and again maintained after correcting for visceral
fat. Thus, macrophage HO-1 is necessary to induce proinflam-
matory macrophage skewing and disruption of adipose tissue
architecture.
Figure 2. Hepatocyte HO-1 Knockout Mice are Insulin Hypersensitive
(A) HO-1 immunoblot of liver obtained from control and Lhoko mice.
(B) HO activity in liver homogenates, n R 6 per group.
(C) Immunohistochemical staining for HO-1 in livers of vehicle and hemin injecte
(D) Oral glucose and insulin tolerance tests on HFD.
(E) HOMA-IR of control and Lhoko mice after 16 weeks on HFD.
(F) Serum levels of liver enzymes after 16 weeks on 60% HFD, n R 4 per group.
(G) In vivo activation of liver insulin signaling. HFD-fed mice were injected with in
blotting. A representative result from three independent experiments is shown.
(H) Liver triglyceride content, n = 3 per group.
(I) H&E, oil red O and HO-1 staining of representative liver sections obtained from
(J) Insulin tolerance tests in AdLacZ and AdHO-1 injected C57BL/6J animals, n =
(K) HOMA-IR of AdLacZ and AdHO-1 injected C57BL/6J animals, n = 7 per grou
Results are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S2.
HO-1 Loss Induces an Anti-Inflammatory MetabolicSignature and Blunts NF-kB SignalingNext, to gain insight into the attenuated metaflammatory pheno-
type, we harvested and in vitro differentiated bone marrow
progenitors from unchallenged, chow-fed Macho animals and
controls intomacrophages (BMDMs). Using TNF-a, we activated
BMDMs and examined NF-kB signaling. Using phospho-IkBa as
a surrogate for activation, we found a clearly diminished NF-kB
response in Macho BMDMs upon induction (Figure 5A). Impor-
tantly, this finding was confirmed using more direct assays as
well: RelA/p65 nuclear translocation was reduced (Figure 5B),
its target-sequence binding by EMSA was reduced (Figure 5C),
and NF-kB target gene expression including Nfkbia/IkBa, Tnf,
and Nos2/iNos was reduced (Figure 5D). Further, chromatin
immunoprecipitation measures of endogenous NF-kB promoter
binding at known target genes Nos2 and Tnf also showed clear
reductions (Figure 5E). Thus HO-1-deficient BMDMs have
blunted NF-kB signaling.
We and others have previously shown that oxidative meta-
bolism is a signature of activated M2-like macrophages, that
activatedM1-like proinflammatory cells display amore glycolytic
metabolic profile, and that direct alterations in metabolism can
regulate NF-kB signaling potential (Haschemi et al., 2012; Rodrı-
guez-Prados et al., 2010; Vats et al., 2006). Intriguingly, when
assessing these parameters in naive BMDMs, we found clearly
elevated basal and spare respiratory capacity, as well as ATP-
turnover in the HO-1-deficient cells (Figures 5F and 5G). Finding
all three hallmarks of an M2 ‘‘aerobic signature’’ already in non-
stimulated Macho BMDMs indicated that HO-1 is required for
proper metabolic programming in the most basal cellular state,
and that such ‘‘pre-’’programming may potently influence
macrophage skewing and thus whole-body inflammation.
Indeed, we observed consistent reductions in TNF-a, IL-1b,
and IL-6 secretion in Macho BMDMs relative to controls (Fig-
ure 5H). Thus, HO-1 is required for metabolic programming of
naive macrophages, and thus, for proper TNF-a- and LPS-
induced skewing and activation.
HO-1 Suppresses OxPhos and ROS SignalingWe next asked whether mitochondrial changes might constitute
a common molecular link between the liver and myeloid pheno-
types (Cheng et al., 2009). Indeed, when repeating our measures
on both systems, parallels were evident for essentially all
measures. Like Macho BMDMs, primary Lhoko hepatocytes
d control and Lhoko mice. Scale bar, 100 mm.
sulin, tissue biopsies taken at the indicated times, and analyzed by immuno-
HFD-fed control and Lhoko mice. Scale bar, 200 mm.
7 per group, p value 1 sided.
p.
Cell 158, 25–40, July 3, 2014 ª2014 Elsevier Inc. 31
exhibited increased basal, spare as well as maximal respiratory
capacity (Figures 6A and S5A). Notably, these effects were inde-
pendent of any obvious underlying changes in coupling effi-
ciency (Figure S5B), mitochondrial DNA content (Figure S5C),
mitochondrial mass (Figure S5D), and respiratory chain stoichi-
ometry (Figure S5E). Interestingly, qPCR analysis revealed clear
andwidespread upregulation of the entire mitochondrial reactive
oxygen species (ROS) detoxification system, from glutathione
processing to the mitochondrial-specific superoxide dismutase
2 (Sod2), again in both systems (Figure 6B), indicating that the
increased mitochondrial flux was coupled to the induction of
ROS quenching machinery. Notably, these changes were
confirmed directly, with the findings of increased SOD activity
(Figure 6C) and indirectly, with an �50% increase in intracellular
H2O2, the product of SOD, and an increase of cytosolic ROS,
again in both knockout cell types (Figures 6D and S5F). Recent
studies have suggested that mild ROS elevations activate mito-
chondrial respiration (Mailloux et al., 2013). If ROS balance was
altering oxidative capacity (Figure 6A) then mitochondrial func-
tion in the knockout cells should be particularly sensitive to anti-
oxidants. In line with this idea, 1 hr preadministration of the
potent ROS quencher N-acetyl-cysteine (NAC) reverted HO-1-
deficient mitochondrial functional parameters to control levels
(Figure 6E). Thus, HO-1 regulates acute ROS thresholding of
mitochondrial respiration.
ROS have previously been linked to INSR activation via impair-
ment of protein tyrosine phosphatase nonreceptor type 1
(PTPN1/PTP1B) (Tiganis, 2011) and to nuclear factor-erythroid-
derived 2-like (NFE2L2/NRF2) function via impairment of the
inhibitory protein kelch-like ECH-associated protein 1 (KEAP1)
(Kobayashi et al., 2006). Because activation of INSR and NRF2
alone would explain the majority of phenotypes observed, we
tested their activation state in our most naive HO-1 knockout
models. Importantly, we found increased nuclear translocation
(Figure 6F) and target-binding activity (Figure 6G) of NRF2 in
Macho BMDMs and decreased PTP1B activity (Figure 6H) and
acute INSR activation (Figure 6I) in naive, chow-fed Lhoko livers.
Notably, in vitro reconstitution of HO-1 expression completely
reverted mitochondrial respiratory changes (Figure S5H), down-
stream pINSR increases (Figure S5G), and TNF-a and IL-1b
blunting (Figure S5I) on a timescale paralleling the 1–2 days ex-
pected for adenoviral HO-1 re-expression. This data set sup-
ports a mechanism where HO-1 continuously limits activation
of key signaling systems through an acute and ROS-dependent
Figure 3. Macrophage HO-1 Knockout Mice Resist Metabolic Disease
(A) qPCR analysis of HO-1mRNA levels in BMDMs generated from control and M
for 24 hr.
(B) Immunoblot for HO-1 in naive BMDMs derived from indicated genotypes.
(C) HO activity in naive BMDMs generated from LFD-fed control and Macho mic
(D) Body weight gain of control and Macho mice on 60% HFD.
(E) Oral glucose and insulin tolerance tests, corresponding blood glucose and in
(F) Oral glucose and insulin tolerance tests, corresponding blood glucose and in
(G) Liver aspects and stainings of representative liver sections obtained fromHFD
Scale bar, 50 mm.
(H) Relative mRNA levels of inflammatory, lipogenic, and gluconeogenic genes in
(I) Liver triglyceride and cholesterol content, n R 5 per group.
(J and K) Energy expenditure and activity of body-weight-matched control and M
Results are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S3.
mechanism. Again all key phenotypes were observed in naive
unchallenged cells, lending further support to the notion that
HO-1 contributes to the ‘‘pre’’-programming of cellular function.
DISCUSSION
Here, we show that HO-1 drives insulin resistance in mouse and
man. In the absence of macrophage HO-1 animals fail to develop
metaflammation and key hallmarks of metabolic disease. The
findings redefine the current view of HO-1, have substantial
implications for the stratification of healthy and unhealthy
obesity, and open the door for HO-1 inhibitors as a new thera-
peutic strategy for obesity and diabetes.
Intersecting strongpatient datawith genetic evidence fromfive
tissue-specific HO-1 deletion models, as well as overexpression
and genetic rescue scenarios, we find a clear proinflammatory
role for HO-1 in metabolic control and thus break dogma.
Induction of HO-1 by systemic cobalt-protoporphyrin (CoPP)
administration has previously been reported to ameliorate
obesity and diabetes in mice (Li et al., 2008). Indeed, much of
our knowledge of HO-1 biology has relied on CoPP. That said,
key studies raise concerns about CoPP specificity as an ‘‘HO-1
agonist.’’ Specifically, CoPP acts indirectly, by decreasing
BACH1 and increasing NRF2 protein levels (Shan et al., 2006).
CoPP inhibits ALAS1 function (Zheng et al., 2008), and recent
data also suggest effects on FOXO1 (Liu et al., 2013). Together,
NRF2, ALAS1 and FOXO1 impact tens if not hundreds of down-
stream targets including heme synthesis, mitochondrial respira-
tion, cytochrome function, and much more. Also, CoPP blocks
HO-1’s interaction with the key proinflammatory mediator IFN
regulatory factor 3 (IRF3) (Koliaraki and Kollias, 2011).
In the liver, we found that HO-1 deficiency promotes insulin
sensitivity and reduces diet-induced fatty liver disease. This
overall finding is in line with a study showing that in the context
of IRS1/IRS2 double-knockout, HO-1 links insulin signaling dys-
regulation to impaired mitochondrial function (Cheng et al.,
2009). Critically, we observed a brake-like, negative regulator
effect on insulin activation even in healthy, chow-fed animals.
Also in macrophages, HO-1 loss blunted NF-kB signaling poten-
tial in the unstimulated state, even in naive, not-yet polarized
cells. These findings indicate a novel role for HO-1 in the meta-
bolic regulation of cellular signaling thresholds. It will be inter-
esting to see whether additional signaling pathways exhibit
similar threshold control via HO-1.
acho mice, naive and polarized with LPS/IFNg (M1-like) or IL-4/IL-13 (M2-like)
e, n = 3 per group.
sulin levels, n = 10–15 per genotype.
sulin levels in body-weight-matched animals, n = 7 per genotype.
-fed control andMachomice for H&E, oil red O, HO-1 (red), andMAC-2 (brown).
liver samples, n = 4 per group.
acho mice, n = 7 per genotype.
Cell 158, 25–40, July 3, 2014 ª2014 Elsevier Inc. 33
Mechanistically, the signal thresholding effects of HO-1
appear to have common ground. HO-1 loss increases mitochon-
drial respiration, antioxidant capacity, and consequently, H2O2
production. Our findings are consistent with detailed examina-
tions of redox regulation of NRF2 and PTP1B in the literature
and their downstream effects on INSR and NF-kB responses
(Kobayashi et al., 2006; Thimmulappa et al., 2006; Tiganis,
2011). Here, we add HO-1 as a new upstream effector of such
redox-dependent signal ‘‘preconditioning.’’
Corroborating these notions, macrophage HO-1 knockout
mice have been independently reported elsewhere to exhibit
an anti-inflammatory phenotype (Tzima et al., 2009). Using
polyI:C and LPS as stimuli, the authors found that HO-1 was
necessary for full activation of thioglycollate elicited peritoneal
macrophages. Our study and theirs, together, provide definitive
genetic evidence that HO-1 is necessary for both ‘‘cold’’ and
‘‘hot’’ inflammation. Intriguingly, Tzima et al. reported no change
of NF-kB activation, a contrast perhaps attributable to differ-
ences in M1-like activation between the BMDMs used here,
and thioglycollate elicited peritoneal macrophages (Ghosn
et al., 2010). Our data are also in line with improvements in
glucose handling observed in HFD-fed recipient mice trans-
planted with HO-1 haploinsufficient bone marrow (Huang et al.,
2012). Notably, our observation of an M2-like aerobic signature
in unstimulated Macho BMDMs indicates that HO-1 regulates
cell fate skewing even prior to stimulation, and therefore that
HO-1 may fine tune pro- versus anti-inflammatory cell ratios. It
will be interesting to see if HO-1 and associatedmetabolic skew-
ing in precursor cell populations will predict outcome in classical
inflammatory disorders.
Along similar lines, our data suggest a trigger-like role for HO-1
in metaflammation and insulin resistance and thus indicate high
prognostic value for detecting disease onset. The observations
are certainly generalizable, corroborated by multiple indepen-
dent patient cohorts including human diabetes (Misu et al.,
2010), generalized obesity (Qatanani et al., 2013), and obese
monozygotic twins (Pietilainen et al., 2008). These data and our
own, show that liver and adipose HO-1 predict disease severity
independently of age, sex, and degree of adiposity, and that they
correlate specifically with pro- but not anti-inflammatory
markers. Careful examination reveals that even our obIS group
showed earliest signs of insulin resistance relative to nonobese
Figure 4. Macrophage HO-1 Knockout Mice Resist Metaflammation
(A) Serum-free fatty acid levels measured in fed and fasted states on HFD, n R
(B) H&E staining of epididymal adipose of HFD fed control and Macho mice. Sca
(C) Adipocyte size distribution in epididymal fat of HFD fed control and Macho mi
n = 6 per group. n = number of adipocytes.
(D) Immunofluorescence staining for MAC-2 and confocal imaging of whole-m
experiments is shown. Scale bar, 200 mm.
(E) Serum levels of proinflammatory cytokines, n R 3 per group.
(F) qPCR analysis of adipokines in epididymal fat isolated from control and Mach
(G) Serum adipokine levels in control and Macho mice, n = 4 per group.
(H–J) Flow cytometric analysis of the SVF isolated from epididymal white adipose t
cells and examined for CD11b coexpression to obtain total macrophage numbe
subpopulations is presented; (J) distribution of CD11c+ MGL� and CD11c� MGL
(K) Immunofluorescence staining for perilipin (red) andMAC-2 (green) of whole-mo
is shown. Scale bar, 100 mm.
Results are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S4,
controls with elevations in HOMA-IR, insulin and nonsignifcant
increases in GLUT4 and HSD11B1 mRNA (Table 1). HO-1 had
significant predictive power in distinguishing these groups.
Thus, it will be most interesting to evaluate the predictive power
of blood HO-1 levels in detecting pre- and early disease states.
Ending actually with a theorectical and technical unknown, our
study leaves unanswered the potential role of Kupffer cells, and
Kupffer cell HO-1, in the etiology of steatosis, particularly in the
Macho animals. While reported to delete strictly in the circulating
macrophage compartment of the liver (Maeda et al., 2005), we
findwhat appears to be LysM-Cremediated deletion in the entire
liver myeloid compartment, including Kupffer cells, specifically
after long-term HFD. Increased promiscuity of Cre-drivers
upon HFD would certainly not be a new concept. The findings
indicate that the stark improvements observed in HFD-Macho
livers could well be supported by HO-1 deletion in Kupffer cells.
EXPERIMENTAL PROCEDURES
Human Study Population
Study subjects included 51 obese patients and 6 nonobese controls that
underwent weight-reducing surgery or elective surgical procedures such as
cholecystectomy. Participants were included if they had fasting plasma
glucose levels <7.0 mmol/l, no history of diabetes, no medications, no weight
changes >3% during the previous 2 months, C-reactive protein (CRP) levels
<20 mg/l, and normal blood leukocyte counts. For further information, see
the Extended Experimental Procedures.
Metabolically Healthy and Unhealthy Obese Mice
Age- and body-weight-matched C57BL/6J mice were selected based on their
response in glucose and insulin tolerance tests after 16 weeks on HFD.
Glucose and Insulin Tolerance Tests
Glucose and insulin tolerance tests were performed as described in the
Extended Experimental Procedures.
Generation of Tissue-Specific HO-1 Knockout Mice
All mouse models are described in the Extended Experimental Procedures.
Animals were kept on a 12 hr light/dark cycle with free access to food and
water and housed in accordance with international guidelines. Dietary inter-
ventions started at 6 weeks of age using diets which contained 10% and
60% calories of fat (Research Diets, D12450B and D12492).
Heme Oxygenase Activity
HO activity was determined as outlined in the Extended Experimental
Procedures.
7 per group.
le bar, 200 mm.
ce as determined by quantitative morphometry of H&E stained tissue sections,
ount epididymal fat pads. A representative result from three independent
o mice, n = 4 per group.
issue (WAT) of HFD fed control andMachomice; (H) Cells were gated for F4/80+
rs; (I) The number of CD11c+, CCR2+ and MGL+ adipose tissue macrophage+ adipose macrophages, n = 3 mice per group.
unt epididymal fat. A representative result from three independent experiments
Tables S4 and S5.
Cell 158, 25–40, July 3, 2014 ª2014 Elsevier Inc. 35
In Vivo Liver Insulin Signaling
For examination of in vivo insulin signaling, mice were fasted for 2 hr, anesthe-
tized and injected with insulin. Liver, muscle and adipose were removed at the
indicated times.
Indirect Calorimetry
Indirect calorimetry and activity measurements were performed as reported
elsewhere (Pospisilik et al., 2010) and as described in the Extended Experi-
mental Procedures.
Flow Cytometry
SVF cells were obtained by collagenase digestion from epididymal adipose
tissue as described (Todoric et al., 2011). FACS analysis was performed as
described (Lumeng et al., 2007) and outlined in the Extended Experimental
Procedures.
Nuclear Translocation Assay
Naive BMDMswere stimulated with 10 ng/ml TNF-a or medium only for 30 and
60 min, fixed in ice-cold methanol, washed, and stained with anti-RelA/p65
antibody (Santa Cruz Biotechnology). Nuclei were counterstained with DAPI.
Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared from BMDMs as described in the Extended
Experimental Procedures. EMSA was performed using the NF-kB EMSA
kit (LI-COR Biosciences) according to the manufacturer’s instructions. For
supershift assays anti-RelA/p65 (Santa Cruz Biotechnology), anti-p50
(Millipore), and anti-rabbit IgG (Vector Laboratories) was used. Unlabeled
double-stranded NF-kB probe was used to test specific binding. Samples
were separated on a nondenaturing polyacrylamide gel. NF-kB DNA binding
was visualized using the LI-COR Odyssey Imaging System.
Chromatin Immunoprecipitation Assay
ChIP assayswere performed using theMAGnify ChIP Kit (Life Technologies) as
detailed by the manufacturer’s instruction and as described in the Extended
Experimental Procedures.
Oxygen Consumption Assay and Bioenergetic Profile
OCR was measured using the XF24 Flux Analyzer (Seahorse Bioscience) as
described (Teperino et al., 2012). For details see Extended Experimental
Procedures.
PTP1B Activity Assay
PTP1B activity was determined as outlined in the Extended Experimental
Procedures.
Hydrogen Peroxide Measurements
Hydrogen peroxide (H2O2) production was measured using the Amplex Red
Hydrogen Peroxide/Peroxidase Assay Kit (Invitrogen).
Figure 5. Attenuated NF-kB Signaling and Anti-Inflammatory Metaboli
(A) pIkBa and IkBaweremeasured by immunoblotting in BMDMs from control and
the indicated times. b-Actin was used as internal loading control.
(B) RelA/p65 nuclear translocation assay. Naive BMDMs were stimulated with TN
immunofluorescent staining. Nuclei were stained with DAPI, and the percentage o
Scale bar, 50 mm.
(C) EMSA. Nuclear extracts were prepared from control and Macho BMDMs stimu
binding was verified by adding a 100-fold excess of unlabeled double-stranded
assays using anti-RelA/p65 and anti-p50 antibodies.
(D) RelativemRNA expression of NF-kB target genes in BMDMs generated from LF
for 60 min, n = 3 per group.
(E) qPCR analysis of NF-kB targets Tnf andNos2 in chromatin immunoprecipitatio
(F and G) Oxygen consumption rates (OCR) and mitochondrial function of naive
(H) Cytokine secretion of LPS stimulated control and Macho BMDMs, n = 3 per
Results are mean ± SEM n = 2–3 independent experiments. *p < 0.05, **p < 0.01
Superoxide Dismutase Assay
Superoxide dismutase (SOD) activity was measured using a commercially
available test kit (Cayman) according to manufacturer’s instructions.
Adenovirus Experiments
BMDM rescue experiments were performed using RGD-fiber-modified adeno-
virus particles (Vector Biolabs). In vivo grade viruses were produced to target
livers and isolated primary Lhoko hepatocytes. For details, refer to the
Extended Experimental Procedures.
NRF2 DNA-Binding Activity
The NRF2 TransAM ELISA-kit (Active Motif) was used to evaluate NRF2 DNA-
binding activity according to the manufacturer’s protocol.
Statistical Analysis
Data are expressed as mean ± standard error of the mean (SEM) unless other-
wise specified. Statistical analyses were performed as described in the
Extended Experimental Procedures. All reported p values are two-tailed
unless stated otherwise. p < 0.05 was considered to indicate statistical
significance.
Other Methods
See Extended Experimental Procedures.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Extended Experimental Procedures, six
tables, and five figures and can be found with this article online at http://dx.
doi.org/10.1016/j.cell.2014.04.043.
AUTHOR CONTRIBUTIONS
H.E. conceptualized the study. W.P., O.W., and J.A.P. contributed to the study
design. H.E. and J.A.P. supervised the study and wrote the manuscript. A.J.
analyzed the macrophage model. E.E. analyzed liver and muscle phenotypes.
O.S. and S.K. performed and analyzed NF-kB immunoblots and bandshift as-
says. K.G. performed in vivo adenovirus rescue experiments. S.M.S., D.N.,
J.T., H.O., C.H.-K, L.K., and W.P. contributed human data. D.M. performed
PTP1B activity assays. K.M., F.K., and W.P. contributed human samples.
T.T.-H.L., K.D., S.S., P.S., R.M., S.M., E.C., and M.B. contributed to some of
the experiments. J.P-P. and A.V.K. measured mitochondrial ROS production.
J.C.D. measured HO activity. J.L.-C. and T.-C.Z. analyzed adipose HO-1
knockout mice. S.A. contributed b-cell data. F.J. and H.S.-F. generated
adenoviral particles. C.N.L. helped with FACS andwhole-mount immunohisto-
chemistry. A.J., E.E., O.S., W.P., and O.W. were involved in manuscript
preparation.
c Signature in Macrophage HO-1 KO Mice
Machomice fedwith LFD. Naive cells were stimulatedwith TNF-a (10 ng/ml) for
F-a (10 ng/ml) for the indicated times and analyzed for RelA/p65 localization by
f nuclear RelA/p65 positive cells was counted. Arrows show nuclear RelA/p65.
lated with TNF-a (10 ng/ml) for the indicated times. Specific RelA/p65 and p50
probe (cold oligo) harboring the NF-kB consensus site as well as supershift
D fed control andMachomice after stimulation with vehicle or TNF-a (10 ng/ml)
ns prepared from control andMacho BMDMs stimulatedwith TNF-a (10 ng/ml).
BMDMs derived from control and Macho mice, n = 9 per group.
group.
, ***p < 0.001.
Cell 158, 25–40, July 3, 2014 ª2014 Elsevier Inc. 37
ACKNOWLEDGMENTS
This research was supported by the Vienna Science and Technology Fund
(J.A.P. and H.E.), the Max-Planck Society (J.A.P.), the Austrian Diabetes
Association (H.E.), the Medical Scientific Fund of the Mayor of the City of
Vienna (E.E.), the Osterreichische Gesellschaft fur Laboratoriumsmedizin
und Klinische Chemie (E.E.), and a grant from the National Institutes of Health
(DK090262, C.N.L.). The authors are grateful to J. Husa, J. Kacerovsky, I.
Miller, G. Mitterer, M. Ozsvar Kozma, andM. Ruf for critical technical and theo-
retical help.
Received: October 7, 2013
Revised: March 3, 2014
Accepted: April 18, 2014
Published: July 3, 2014
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5F is shown, n = 5 for hepatocytes, n = 9 for macrophages.
ckout hepatocytes and macrophages as well as their corresponding controls,
ive BMDMs, n = 4 per group.
group.
tocytes and naive BMDMs isolated from Lhoko and Macho animals as well as
asurements. Note that for untreated hepatocytes the same experiment as in (A)
l BMDMs and analyzed for NRF2, Histone H3 (nuclear marker) and GAPDH
rol BMDMs, n = 4 per genotype.
cytes (Hep) as well as liver biopsies of LFD and HFD fed Lhoko and control
, liver biopsies taken at the indicated times, and analyzed by immunoblotting. A
Cell 158, 25–40, July 3, 2014 ª2014 Elsevier Inc. 39
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Supplemental Information
EXTENDED EXPERIMENTAL PROCEDURES
ReagentsUnless otherwise stated all chemicals and reagents were obtained from SIGMA.
Human Study PopulationStudy subjects included 51 obese patients and 6 nonobese controls that underwent weight reducing surgery or elective surgical
procedures such as cholecystectomy. Participants were included if they had fasting plasma glucose levels < 7.0 mmol/l, no history
of diabetes, no medications, no weight changes > 3% during the previous 2 months, C-reactive protein (CRP) levels < 20 mg/l and
normal blood leukocyte counts. All study subjects provided informed consent and study protocols were approved by the local Ethics
Committee. Tissue biopsies from visceral adipose tissue, obtained during surgery, were collected in RNA-later (Life Technologies)
and stored at�80�C until further processing. In a subset of 43 obese study participants liver biopsies were obtained at the beginning
of the surgical intervention. Glucose, insulin, lipid parameters, high-sensitive CRP andHOMA-IR indexwere determined as described
(Oberkofler et al., 2004; Todoric et al., 2011). Abdominal subcutaneous and visceral fat thickness were determined by ultrasonogra-
phy using the HDI 3000 CV System (ATL) (Oberkofler et al., 2010; Pontiroli et al., 2002).
Quantitative PCRqPCRwas performed as described (Todoric et al., 2011). In brief, total RNA andDNAwere extracted from respective tissues and cells
using RNA and DNA isolation kits (RNeasy, QIAGEN; TRIzol, Invitrogen). Isolated total RNAwas reverse-transcribed into cDNA using
commercially available kits (Applied Biosystems). qPCR reactions were performed using the iQ SYBR Green Supermix (Bio-Rad
Laboratories). Postamplification melting curve analysis was performed to check for unspecific products and primer-only controls
were included to ensure the absence of primer dimers. For normalization threshold cycles (Ct-values) were normalized to acidic
ribosomal phosphoprotein P0 (Rplp0) within each sample to obtain sample-specific DCt values ( = Ct gene of interest - Ct Rplp0).
2�DDCt values were calculated to obtain fold expression levels, where DDCt = (DCt treatment - DCt control). Human GLUT4 and
CD68 transcripts were quantified using TaqMan gene expression assays Hs00168966_1 and Hs00154355_m1 (Applied Biosystems,
Foster City, CA), respectively. Other human and mouse primers used are listed in Tables S5 and S6.
ImmunohistochemistryHuman and mouse tissues were fixed in 4% phosphate-buffered formalin and embedded into paraffin. Tissue sections were depar-
affinized and rehydrated prior to antigen unmasking using Target Retrieval Solution, pH 6.0 (Dako). Endogenous peroxidase activity
was quenched with incubation of 3% hydrogen peroxide for 10 min. Sections were blocked with Biotin-Blocking System (Dako) and
normal serum obtained from the secondary antibody host animal. Blocked sections were incubated with anti-HO-1 (1:500; ab13243,
Abcam), anti-CD68 (1:500; M0718, Dako) and anti-MAC-2 (1:500; CL8942AP, Cedarlane). Secondary antibody staining was per-
formed using the EnVision Detection System (Dako) and 3,30-diaminobenzidine as chromogenic substrate (Roche Molecular
Biochemicals) according to the manufacturer’s instructions. HO-1 positive cells were evaluated using the particle analysis module
incorporated in ImageAccess 9 (IMAGIC). HO-1 positive cells per all hepatocytes were counted on three different high-power fields
(4003 magnification).
Mouse and Human Multiple Tissue RNA LibrariesTotal RNA was isolated from tissues of three 6 week old male C57BL/6J mice to analyze HO-1 tissue distribution. A human multiple
tissue RNA library (Life Technologies) was used to analyze the tissue distribution of human HO-1 expression in tissues corresponding
to the mouse library.
Metabolically Healthy and Unhealthy Obese MiceObese age- and body-weight-matched C57BL/6J mice were selected based on their response in oral glucose and insulin tolerance
tests after 16 weeks on HFD. Epididymal fat pads and livers were removed and RNA and protein isolated to measure HO-1
expression.
Glucose and Insulin Tolerance TestsFollowing an overnight fast, mice were administered glucose (1 g/kg) by oral gavage, and blood samples for glucose and insulin
determination were collected from the tail vein at the indicated times. Insulin tolerance was assessed after a 2 hr fast by intraperito-
neal administration of human regular insulin (0.75 U/kg) and blood glucose monitoring. Low-dose insulin tolerance tests (0.1 U/kg)
were performed in adenovirus injectedmice to assess hepatic insulin sensitivity. Glycemia was assessed using a Accu-Chek (Roche)
glucometer. Plasma insulin levels were determined using the Ultrasensitive Mouse Insulin ELISA kit (Mercodia).
Western Blot AnalysisProteins were extracted from tissues by homogenizing in RIPA buffer (0.5% NP-40, 0.1% sodium deoxycholate, 150 mM NaCl,
50 mM Tris-HCl, pH 7.5) containing protease inhibitors (Complete Mini, Roche). The homogenate was cleared by centrifugation at
Cell 158, 25–40, July 3, 2014 ª2014 Elsevier Inc. S1
4�C for 30 min at 15,000 g and the supernatant containing the protein fraction recovered. Protein concentration in the supernatant
was determined using the BCA Protein Assay Kit (Pierce). 20 mg of proteins were resolved by SDS-PAGE and transferred to PVDF
membranes (GE Healthcare). Membranes were blocked with 5% BSA in Tris-buffered saline containing 0.2% Tween-20 (TBS-T),
and incubated with primary antibodies at 4�C over night. The following antibodies were used (all from Cell Signaling unless indicated
otherwise): anti-HO-1 (1:5,000; ab13243, Abcam), anti-phospho-Insulin receptor (pTyr1162/1163) (1:1,500; sc-25103, Santa Cruz
Biotechnology), anti-insulin receptor (1:1,500; Cat.-No. 3025), anti-phospho-AKT (pSer473) (1:1,500; Cat.-No. 9271), anti-phos-
pho-AKT (pThr308) (1:1,500; Cat.-No. 9275), anti-AKT (1:1,500; Cat.-No. 9272), anti-phospho-IkBa (pSer32/36) (1:1,000; Cat.-No.
9246), anti-ikBa (1:1,000; sc-371, Santa Cruz Biotechnology), anti-NRF2 (1:1,000; Cat.-No. 12721), anti-Histone H3 (1:1,000; Cat-
No. 4499), anti-b-Actin (1:500; A5441, Sigma), anti-GAPDH (1:2,500; ab9485, Abcam), anti-HSC-70 (1:5,000; sc-7298, Santa Cruz
Biotechnology). Expression levels of 5 different OXPHOScomplexes (CI subunit NDUFB8, CII-30kDa, CIII-Core protein 2, CIV subunit
I and CV alpha subunit) were analyzed using the MitoProfile Total OXPHOS Rodent WB Antibody Cocktail (Abcam). Incubated
membranes were washed and probed with the appropriate anti-igG-horseradish peroxidase-linked (HRP) secondary antibody
(NA 934, anti-rabbit IgG, 1:20,000; NA 931, anti-mouse IgG, 1:20,000; GE Healthcare). Antigen-specific binding of antibodies was
detected with SuperSignal West Femto and Pico Kits (Pierce) using a ChemiDoc XRS Imager (Bio-Rad). Image analysis was per-
formed using Image Lab Software Version 3.0.1 (Bio-Rad).
Generation of Tissue-Specific HO-1 Knockout MiceMicewith aconditionalHO-1alleleweregeneratedbyhomologous recombination. The targeting vectorwasdesigned to introduce two
LoxPsitesflankingexon2of theHO-1gene locus.Eliminationof exon2 leads toaconsecutive frameshift in exon3, anearly stopcodon,
thus resulting in a truncatedpeptideconsistingof 9aminoacids. The targetingconstructwaselectroporated intoC57BL/6Nembryonic
stemcells. TheprimaryEScellswerescreenedandpotentially targetedcloneswereexpanded.Southernblot confirmationwasused to
select correctly targeted cells that were subjected to removal of the Frt-flanked neomycin cassette by Flp-recombinase, and sub-
sequently injected into blastocysts. Offsprings were tested for germline transmission and two correctly targeted mouse lines were
established. C57BL/6N founder mice were backcrossed onto the more insulin resistance prone C57BL/6J background using speed
congenics (purity > 99.9%). Hepatocyte-specific HO-1 knockout (Lhoko) mice were generated by crossing the conditional HO-1
line with Alb-Cre transgenic mice expressing Cre recombinase under the control of the albumin promoter (Postic et al., 1999). Mice
with a targeted deletion in macrophages (Macho) were generated by crossing the conditional HO-1 line with LysM-Cre knockin
mice having the Cre recombinase inserted into the first coding ATG of the lysozyme 2 gene (Clausen et al., 1999). Muscle-, adipose-
and beta-cell targeted HO-1 knockout mice were generated using Mck-cre (Bruning et al., 1998), Fabp4/aP2-cre (He et al., 2003)
and Rip-cre (Postic et al., 1999) knockin mice, respectively. Heterozygous breeding schemes (male HO-1fl/+;Cre+/� X female HO-
1fl/+;Cre�/�) were used to generate Lhoko andMacho animals, muscle (Muhoko), adipose (Fahoko) and b-cell (Bhoko) HO-1 knockout
mice as well as their respective wild-type (HO-1+/+;Cre�/�), conditional (HO-1fl/fl;Cre�/�) and Cre control littermates (HO-1+/+;Cre+/�).Offspringswere found tobebornat expectedMendelian ratiosandnodifference in survival rateswasobserved.Animalswerekeptona
12 hr light/dark cycle with free access to food andwater and housed in accordancewith international guidelines. Dietary interventions
started at 6 weeks of age using standardized low- and high-fat diets which contained 10% and 60% calories of fat, respectively
(Research Diets Inc., D12450B and D12492). Animal studies were approved by Austrian and German governments.
Heme Oxygenase ActivityHO activity was determined as reported elsewhere (Kozlov et al., 2010). In brief, aliquots of frozen liver (Lhoko) or cell pellets (Macho)
werehomogenizedusingaElvehjempotterwithPTFEpestle in abuffer 1:10 (wt/vol) containing300mMsucrose, 20mMTris, and2mM
EDTAat pH7.4. Protein concentrationswere determinedusingBradford reagent andBSAas standard. For the activity assay, 100 ml of
homogenate (containing about 1 mg of protein) was added to a reaction mixture containing 500 nmol NADPH in a 100mMpotassium
phosphate buffer (pH 7.4) supplementedwith 1mMEDTAand 20 nmol of hemin. 30 ml rat kidney cytosolic (RKC) fractionwas added to
the reaction to provide sufficient biliverdin reductase activity. RKCwas prepared as described elsewhere (McCoubrey, 2001). The re-
action mixture was incubated under constant agitation for 30 min at 37�C in darkness. After adding 0.2 assay volumes of saturated
potassiumchloride, the formedbilirubinwas extracted into chloroform (4 assay volumes). Sampleswere then centrifuged, the organic
phase harvested for subsequent spectrophotometric determination of bilirubin concentrations. Sampleswere scanned three times to
calculate the absorption difference between 450 and 520 nm. All samples were run in duplicates and corrected for the absorption
measured in corresponding samples incubatedon ice.Bilirubin standardcurvesweregeneratedbyspiking knownamountsof bilirubin
into sample homogenates. Heme oxygenase activity was calculated as nanomol bilirubin formed per milligram protein per 30 min.
Hemin InjectionHemin (2.5 mg/ml) was dissolved in 10% ammonium hydrochloride in 0.15M sodium chloride and injected intraperitoneally into mice
(10 ml/g body weight). Livers were harvested 12 hr postinjection to test HO-1 induction.
Mouse Laboratory Parameters and CytokinesAlanine transaminase (ALT), Aspartate transaminase (AST), cholinesterase (ChE), alkaline phosphatase (ALKP), triglycerides and
cholesterol were quantified with tests certified for in vitro diagnostics at the Department of Laboratory Medicine of the Medical
S2 Cell 158, 25–40, July 3, 2014 ª2014 Elsevier Inc.
University of Vienna. Free fatty acids were measured using the nonesterified fatty acid (NEFA) kit (Wako Chemicals) according to the
provided protocol. Blood samples were analyzed on an automated hematology analyzer (CELL-DYN 3500, Abbott Laboratories) and
validated manually with Giemsa stained blood smears. IL-6, TNF-a, IL-1b and IL-10 ELISA kits (all from Biolegend) were used to
determine the respective cytokine levels in mouse serum and supernatants derived from cultured macrophages according to the
protocol provided by the manufacturer. For hepatic triglyceride and cholesterol measurements, total lipids were extracted from
50 mg liver as previously described (Folch et al., 1957). Serum leptin, adiponectin and RBP4 levels were quantified using commer-
cially available ELISA kits (Millipore, R&D Systems).
Oil Red O StainingFor staining of neutral lipids, liver cryosections were stained with oil red O (1% w/v isopropanol, diluted 3:2 in PBS) for 1 hr at room
temperature according to standard procedures.
In Vivo Insulin SignalingFor examination of in vivo insulin signaling, mice were fasted for 2 hr, anesthetized and injected with human regular insulin (1 U/kg,
Novo Nordisk). Liver, muscle and adipose biopsies were removed at the indicated times, flash-frozen in liquid nitrogen and stored at
�80�C until further processing.
Indirect CalorimetryIndirect calorimetry and activity measurements were performed as reported elsewhere (Pospisilik et al., 2010). In brief, body-weight-
matched mice were placed in metabolic cages connected to an open-circuit, indirect calorimetry system combined with the deter-
mination of spontaneous activity by beam breaking (Oxylet, Panlab-Bioseb). The animals were accustomed to the apparatus during
the first 24 hr, followed by measurements. Oxygen consumption and carbon dioxide production were recorded using a computer-
assisted data acquisition program (Metabolism 2.2.01, Panlab Harvard Apparatus).
Generation of Bone-Marrow-Derived MacrophagesBone marrow cells were obtained from femurs and tibias of 6-10 week old control and Macho mice. Bone-marrow-derived macro-
phages (BMDMs) were generated in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 10% supernatant derived
fromM-CSF transduced L929 confluent cells as described (Weischenfeldt and Porse, 2008; Zanoni et al., 2009). At day 7 postinduc-
tion macrophages were collected and cultured in the presence or absence of stimuli in RPMI-1640 medium supplemented with 10%
FBS. BMDMs were polarized for 24 hr with 10 ng/ml LPS and 20 ng/ml Interferon-g (R&D Systems) to generate classically activated
M1 macrophages. Alternatively activated M2 macrophages were generated using IL-4/IL-13 (20 ng/ml each; R&D Systems). For
NF-kB signaling experiments macrophages were treated with 10 ng/ml TNF-a (Peprotech) for the indicated times.
Histology, Adipocyte Size, and NumberFor tissue sections, hematoxylin and eosin (H&E) staining was performed on 5 mm paraffin sections of tissues fixed for 16 hr in 4%
phosphate-buffered formalin at 4�C. Adipocyte size distribution was determined by semi-automated morphometry. In brief, three
fields of view of 3 different sections (200 mm intervals) per animal were quantified. Epididymal fat pads from 4 animals per group
were analyzed. Semi-automated morphometry (Axiovision morphometry software, Zeiss) was used to define and quantify fat cells
based on shape, size and presence of a lipid droplet.
Whole-Mount ImmunofluorescenceWhole-mount epididymal fat samples were processed and stained as described (Lumeng et al., 2007; Todoric et al., 2011). In brief,
mice (three per group) were perfused with 1% paraformaldehyde for fixation before dissecting fat pads. Samples were incubated for
1 hr at room temperature with 5% normal goat serum (Dako) in 0.3% PBS-T to block unspecific binding sites. After blocking, tissues
were incubated overnight at 4�C with primary and secondary antibodies diluted in PBS containing 5% BSA. Anti-perilipin (Cat.-Nr.
9349, Cell Signaling Technology) and anti-MAC-2 antibodies (Cat.-Nr. CL8942AP, Cedarlane Laboratories) were used to stain adipo-
cyte lipid droplets and macrophages, respectively. The following secondary antibodies were used for immunofluorescence staining:
fluorescein anti-rat IgG (Cat.-Nr. FI-4001, VECTOR Laboratories), Alexa Fluor-594 goat anti-rabbit IgG (Cat.-Nr. A11037, Life Tech-
nologies). For control experiments, the primary antibody was substituted with preimmune serum. Stained tissues were mounted in
chamber slides and subjected to confocal imaging analysis on a LSM 700 Laser Scanning Microscope (Zeiss).
Flow CytometryStromal vascular cells (SVCs) were obtained by collagenase digestion from epididymal adipose tissue as described previously
(Lumeng et al., 2007; Pospisilik et al., 2010). In brief, minced fat depots were digested at 37�C for 30 min in a shaking water bath
with a cocktail consisting of 1 mg/ml collagenase II (Worthington) in DMEM containing 3% fatty-acid-free BSA and DNase I
(100 Units/ml). After digestion, the slurry was passed through a 100 mm cell strainer (Becton Dickinson) and centrifuged at 200 g
for 5 min to separate SVCs and adipocyte fractions. Adipocyte fractions were examined by microscopy to detect adherent cells.
Digestion was continued for another 15 min until adipocyte fractions were free of adherent cells to guarantee isolation of most
Cell 158, 25–40, July 3, 2014 ª2014 Elsevier Inc. S3
adipose macrophages. Cells were then pelleted by centrifugation at 200 g for 5 min, resuspended in 0.5 ml red blood cell lysis buffer
and incubated for 5 min prior to resuspension in sorting buffer (PBS with 2% FBS) at a concentration of 106 cells/ml. Isolated cells
were incubated with Fc Block (BD Biosciences) prior to staining with conjugated antibodies for 30 min at 4�C followed by 2 washes in
sorting buffer. Cells were resuspended in sorting buffer supplemented with Fixable Viability Dye eFluor (eBioscience) and subjected
to FACS analysis (LSR Fortessa, BDBiosciences). The following antibodies were used for staining adipose tissuemacrophages: anti-
F4/80 PE/Cy7 (Cat.-Nr. 123113, BioLegend), anti-CD11b Percp/Cy5.5 (Cat.-Nr. 101228, BioLegend), anti-CD11c Alexa Fluor700
(Cat.-Nr. 56-0114-82, eBioscience), anti-CCR2 APC (Cat.-Nr. FAB5538A, R&D Systems) and anti-MGL Alexa Fluor 647 (Cat.-Nr.
MCA2392A647T, AbD Serotec). Viable cells were gated and analyzed using FlowJo (Tree Star).
Nuclear Translocation AssayTo assess nuclear RelA/p65 translocation naive BMDMs were seeded in 8-well chamber slides (BD Biosciences). Cells were either
stimulated with 10 ng/ml TNF-a or medium only as control for 30 and 60 min, fixed in ice-cold methanol for 10 min, washed and
stained overnight at 4�Cwith anti-RelA/p65 antibody (sc-372, Santa Cruz Biotechnology). After several washes cells were incubated
with a fluorescent secondary antibody (Alexa Fluor 488, Life Technologies) for 1 hr at room temperature. Nuclei were counterstained
with DAPI. Images were obtained using a Zeiss Axio Imager A1 microscope and evaluated in a double-blinded manner by two
different researchers.
Electrophoretic Mobility Shift AssayNuclear extracts were prepared from 1x107 BMDMs. Adherent cells were washed with ice-cold PBS, transferred and washed three
times in 1 ml of ice-cold hypotonic buffer (10 mMHEPES pH 7.9, 1.5 mMMgCl2, 10mMKCl) to induce swelling. Thereafter, the pellet
was resuspended in hypotonic buffer supplemented with 0.1% Nonidet P-40 and incubated on ice for 5 min to release nuclei. Sub-
sequently, samples were centrifuged at 4�C to pellet nuclei and the cytoplasmic fraction was discarded. Nuclei were resuspended in
50 ml of ice-cold, high-salt buffer (20mMHEPES pH7.9, 1.5mMMgCl2, 420mMNaCl, 25%glycerol) and incubated for 15min at 4�C.Disrupted nuclei were centrifuged at full speed in a table top centrifuge for 15 min at 4�C to isolate the nuclear protein fraction. EMSA
was performed using the NF-kB EMSA kit and an IRDye 700 labeled double-stranded probe harboring the NF-kB consensus site (LI-
COR Biosciences) according to the manufacturer’s instructions. For the binding assay, 5 mg of nuclear extracts were added to bind-
ing buffer (10 mM Tris, 50 mM KCl, 1 mM DTT, pH 7.5) together with 50 ng poly (dI:dC), 2.5 mM DTT/0.25% Tween-20 and 0.5 pmol
IRDye labeled probe in a total volume of 20 ml. For supershift assays 0.2 mg of the following antibodies were used: anti-RelA/p65 (sc-
372, Santa Cruz Biotechnology), anti-p50 (06-886, Millipore) and anti-rabbit IgG (S-5000, Vector Laboratories). Unlabeled double-
stranded NF-kB probe was used at a 100-fold molar excess to test specific binding. Samples were incubated at room temperature
for 15min and separated on a prerun nondenaturing polyacrylamide gel in TGE buffer (25mMTris-HCl pH 8.0, 190mMglycine, 1mM
EDTA) for 3 hr at 100 V. NF-kB DNA binding was visualized using the Odyssey Imaging System (LI-COR Biosciences).
Chromatin Immunoprecipitation AssayChromatin immunoprecipitation (ChIP) assayswere performed using theMAGnifyChIPKit (Life Technologies) as detailed by theman-
ufacturer’s instruction. In brief, 2 3 106 cells were crosslinked for 10 min at 37�C by adding formaldehyde (methanol-free, Thermo
Scientific) into the culturing media to a final concentration of 1%. Reactions were stopped by adding glycine to a final concentration
of 0.125 M. Cells were harvested, washed twice with ice-cold PBS and lysed in 100 ml lysis buffer containing protease inhibitors. Cell
lysates were transferred to microTUBEs (Covaris) and subjected to shearing on the Covaris S2 sonicator (4 cycles; cycle time: 60 s,
peak power: 140, duty factor: 5, cycles/burst: 200). Dynabeads (Life Technologies) were precoupled with anti-RelA/p65 (sc-372,
Santa Cruz Biotechnology) or rabbit IgG according to the protocol provided. 3 mg of antibody was used for each ChIP experiment.
Sheared chromatin DNA was diluted to 200,000 cells per ChIP reaction and incubated with antibody-coupled beads at 4�Cwith con-
stant rotation for 4 hr. Input controls were set aside for each IP sample. Bound chromatin waswashedwith a series of washing buffers
included in the kit and formaldehyde crosslinkingwas reversed using Proteinase K. DNAwas purified usingDNApurificationmagnetic
beads provided in the kit and eluted in 150 ml of DNA Elution Buffer. Precipitated DNA was subjected to quantitative PCR using
primers 50-CCCCAGATTGCCACAGAATC-30 and 50-CCAGTGAGTGAAAGGGACAG-30 for the Tnf promoter (NF-kB site 1) (Kuwata
et al., 2006) and 50-CCTAGTGAGTCCCAGTTTTGAAGT-30 and 50-CATCAGGTATTTATACCCCTCCAG-30 for the proximal NF-kB
site in the Nos2/iNos promoter (Guo et al., 2008). Data were analyzed according to the MAGnify ChIP Kit (Life Technologies) manual.
Oxygen Consumption Assay and Bioenergetic ProfileAnalysis of oxygen consumption rates (OCR) was performed using the XF24 Flux Analyzer (Seahorse Bioscience) as reported
previously (Haschemi et al., 2012; Teperino et al., 2012). In brief, naive bone-marrow-derivedmacrophages and primary hepatocytes
were seeded into XF 24-well cell culture microplates and allowed to recover for 24 hr. A final volume of 600 ml of buffer-free Assay
Medium (Seahorse Bioscience) was added to each well prior to the experimental protocol. Cells were then transferred to a CO2-free
incubator and maintained at 37�C for 1 hr before starting the assay. Following instrument calibration, cells were transferred to the
XF24 Flux Analyzer to record cellular oxygen consumption rates. The measurement protocol consisted of 2 min mixture, 2 min
wait and 4 min OCR measurement times (macrophages), and 4 min mixture, 0 min wait and 3 min OCR measurement times (hepa-
tocytes). For the mitochondrial stress test ATP synthase was inhibited by injection of 1 mM oligomycin, followed by 3 mM
S4 Cell 158, 25–40, July 3, 2014 ª2014 Elsevier Inc.
FCCP-induced mitochondrial uncoupling to determine the spare/maximal respiratory capacity. Nonmitochondrial respiration was
determined after rotenone/antimycin A injection (1 mMeach). At the end of the assay, the mediumwas carefully aspirated and cellular
DNA measured using CyQuant (Life Technologies) to adjust for potential differences in cell densities. For acute ROS-quenching
experiments, 10 mMbuffered N-acetyl-cysteine (NAC) was added to naive BMDMs or primary hepatocytes 1 hr prior to mitochondrial
stress tests and OCR measurements. For adenoviral rescue experiments, primary hepatocytes isolated from Lhoko animals were
transduced with Adeno-LacZ (AdLacZ) or Adeno-HO-1 (AdHO-1) virus particles (10 pfu/cell). Mitochondrial stress tests and OCR
measurements were initiated 48 hr after transduction.
Primary Hepatocyte IsolationHepatocytes were isolated and cultured according to the protocols provided by the manufacturer of Liver Perfusion Medium and
Liver Digest Medium, respectively (Invitrogen). In brief, mice were anaesthetized by intraperitoneal injection of ketamine-xylazine
(10% ketamine, 5% xylazine). The liver was perfused in situ with Liver Perfusion Medium (Invitrogen), and digested using Liver Digest
Medium (Invitrogen). Digested livers were removed, minced, and filtered through a 70 mm cell strainer (BD Biosciences). Filtered
hepatocytes were washed in William’s E medium supplemented with 5% FBS, purified from the nonparenchymal cells by centrifu-
gation and plated in the respective experimental dishes. After 4 hr of incubation adherent hepatocytes were switched to serum-free
RPMI-1640 and incubated over night. Experiments were performed on the following day.
PTP1B Activity AssaySamples were homogenized in immunoprecipitation buffer (20 mM Tris pH 7.6, 150mMNaCl, 0.5 mMEDTA, 10% glycerol, protease
inhibitors, and 1% Triton X-100. Lysates were cleared by centrifugation for 15 min at 20.000 g. Liver (2 mg) and hepatocytes (1 mg)
samples were precleared for 30 min at 4�C by incubation with 20 ml protein G coupled Dynabeads (Life Technologies). Precleared
samples were incubated for 1 hr at 4�C with goat anti-PTP1B (Santa Cruz Biotechnology). Next, 20 ml protein G coupled Dynabeads
were added to capture PTP1B and incubated for 2 hr at 4�C. Samples were washed two times with immunoprecipitation buffer and
two times with phosphatase assay buffer (25 mM HEPES pH 7.2, 50 mM NaCl, 2 mM EDTA). Total PTP1B activity was measured
using 50 mM of para-Nitrophenylphosphate (New England BioLabs) as a substrate according to the manufacturer’s instructions.
Subsequently, immunoprecipitated PTP1B samples were boiled and subjected to immunoblotting using rabbit anti-PTP1B (Abcam).
PTP1B signals were quantified by densitometry. Total PTP1B activity results were normalized to the amount of PTP1B in the precip-
itates to obtain specific PTP1B activity values (1 Unit = hydrolysis of 1 nmol of para-Nitrophenylphosphate in 1 min).
Hydrogen Peroxide MeasurementsHydrogen peroxide (H2O2) production was measured using the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Invitrogen)
according to the protocol provided by the manufacturer.
Superoxide Dismutase AssaySamples were sonicated and centrifuged at 1,500 g for 5 min at 4�C in ice-cold HEPES buffer (20 mM) containing EGTA (1 mM),
mannitol (210 mM) and sucrose (70 mM). Superoxide dismutase (SOD) activity was measured using a commercially available test
kit (Cayman) according to manufacturer’s instructions.
Cytosolic ROS and Mitochondrial ContentBMDMs and primary hepatocytes were cultured in Lab-Tek II Chambered Coverglass polystyrene media chambers mounted to ex-
trathin borosilicate cover glass for optimum high-power inverted microscopy (Nalge Nunc). Cytosolic ROS were quantified as
communicated recently (Kuznetsov et al., 2011). In brief, cells were loaded for 20 min with 5 mM 20,70-Dichlorofluorescin diacetate
(DCF-DA; Invitrogen). Stained cells were imaged with an inverted confocal microscope (LSM 510, Zeiss), the AxioVision software
package (Zeiss) was used to analyze recorded images as described (Kuznetsov et al., 2011). Results were expressed as arbitrary
fluorescence intensity units. MitoTracker Deep Red 633 (500 nM, staining time 40min at 37�C) was used to analyze themitochondrial
content in isolated primary hepatocytes. In BMDMs mitochondrial content was determined using MitoTracker Green FM (Life Tech-
nologies) according to the manufacturer’s instructions. Stained cells were washed once with serum-free RPMI-1640 and incubated
at 37�C for 30min. Thereafter, cells were washed extensively with PBS, resuspended in FACS buffer (PBS, 2mMEDTA, 2%FBS) and
analyzed with the LSR II Flow Cytometer (Becton Dickinson). Result were analyzed using the FlowJo software package (Tree Star).
Adenovirus ExperimentsThe macrophage tropic RGD-fiber-modified (Dmitriev et al., 1998) Ad(RGD)-HO-1 and Ad(RGD)-GFP obtained commercially from
Vector Biolabs were used for Macho-BMDM rescue experiments. BMDMs were infected with 100 pfu/cell on day 4 of differentiation.
Experiments were performed on day 7 of differentiation, i.e., 3 days after adenoviral transduction. For adenovirus experiments target-
ing livers and isolatedprimary Lhokohepatocytes, in vivogradeHO-1 (AdHO-1) andLacZ (AdLacZ) expressing viruseswereproduced
according to published protocols (Hardy et al., 1997). In brief, Sfi I-digested Adlox plasmid DNAwas cotransfected with psi5 DNA into
Cre8 cells using FuGENE 6 Transfection Reagent (Roche). Three days after transfection cells were collected by centrifugation and
recombined viruses extracted from cell pellets by four freeze and thaw cycles. Cell debris was removed by centrifugation. HEK293
Cell 158, 25–40, July 3, 2014 ª2014 Elsevier Inc. S5
cells (ATCC) were used to amplify adenoviral particles. Amplified AdHO-1 and AdLacZ were purified by cesium chloride density-
gradient ultracentrifugation, collected from the gradient, diluted 1:1 in 2x storage buffer (10 mM Tris pH 8.0, 100 mM NaCl, 0.1%
BSA, 50% glycerol) and stored in small aliquots at �20�C. Primary Lhoko hepatocytes were infected with 10 plaque forming units
(pfu)/cell. Transduced cells were incubated for 48 hr before experiments were started. In vivo tail vein injections were performed
as described (Pospisilik et al., 2007). In brief, a total volumeof 200ml (0.23 109 pfu/g bodyweight) of PBS-dilutedAdHO-1 andAdLacZ
particles was injected into the tail vein of recipient mice. OGTT and ITT were performed on day 5 and 7 postinjection.
NRF2 DNA-Binding ActivityThe NRF2 TransAM ELISA-kit (Active Motif) was used to evaluate NRF2 DNA-binding activity according to the manufacturer’s pro-
tocol. In brief, 8 mg of nuclear extracts isolated from naive BMDMswere incubated for 1 hr in 96 well plates on which oligonucleotides
containing the antioxidant response element (ARE) consensus-binding site were immobilized. After binding and washing, NRF2 was
detected using an antibody specific for active, i.e., DNA-bound NRF2. Addition of a horseradish peroxidase (HRP) coupled second-
ary antibody was used to quantitate-binding activity by spectrophotometry.
Statistical AnalysisData are expressed as mean ± standard error of the mean (SEM) unless otherwise specified. Statistical significance was tested by
Student’s t test or ANOVAwith Scheffe’s post hoc testing where appropriate. Correlations were tested by linear regression. Categor-
ical datawere summarizedby frequencies andanalyzedby theChi-square test. TheWilcoxon ranksum testwasused toanalyzepublic
human data sets and energy expenditure data. Heteroskedasticity of human data was tested by the Breusch-Pagan/Cook-Weisberg
test, normal distribution of quantitative traits were tested by the Shapiro-Francia normality test. Logarithmic transformations were
made if the equal variance and normality assumptions were rejected. Adjustments for confounding variables were performed as indi-
cated. All figures andmouse statistical analyses were generated using Prism 6 (GraphPad). Human data were analyzed with Stata 12
(StataCorp). All reported p values are two-tailed unless stated otherwise. p < 0.05 was considered to indicate statistical significance.
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Figure S1. Conserved HO-1 Tissue Expression Profile and Metabolically Healthy and Unhealthy Obese C57BL/6J mice, Related to Figure 1
(A) Quantitative PCR analysis of HO-1 mRNA in human and mouse tissue libraries.
(B and C) Stratification of obese insulin-sensitive (IS) and obese insulin-resistant (IR) C57BL/6J mice fed a 60% fat diet for 16 weeks; (B) oral glucose tolerance
tests, and (C) intraperitoneal insulin tolerance tests, n = 6 per group.
(D) Body weight of obese IS and obese IR mice after 16 weeks on high fat diet. Results presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.
Cell 158, 25–40, July 3, 2014 ª2014 Elsevier Inc. S7
Figure S2. Generation of Conditional HO-1 Mice, Lhoko Low-Fat Diet Studies, Lipogenic and Gluconeogenic Program, and Adenoviral
Injections, Related to Figure 2
(A) Shown (top to bottom) are wild-type, targeted, conditional (floxed) and knockout HO-1 gene loci.
(B) Southern blot of correctly targeted ES cell clone.
(C) PCR for tissue-specific HO-1 deletion in Lhoko mice.
(D) Hematoxylin-eosin (H&E) staining of liver sections obtained from control and Lhoko mice fed a LFD for 16 weeks.
(E) Serum levels of liver enzymes, n = 4 per genotype.
(F) Oral glucose tolerance tests on LFD.
(G) Insulin tolerance tests on LFD.
(H) Body weight gain of control and Lhoko mice on HFD.
(I) Gluconeogenic and lipogenic program in HFD fed Lhoko livers, n = 4 per genotype.
(J) Relative HO-1 mRNA levels in AdLacZ and AdHO-1 transduced liver biopsies, n = 4 per group.
(K) HO-1 immunoblot of AdLacZ and AdHO-1 transduced liver biopsies, n = 3 per group.
(L) Oral glucose tolerance tests and corresponding blood glucose and insulin levels in AdLacZ and AdHO-1 transduced C57BL/6J animals, n = 7 per group. Two-
sided p values obtained from unpaired t test. Results are mean ± SEM. *p < 0.05.
S8 Cell 158, 25–40, July 3, 2014 ª2014 Elsevier Inc.
Figure S3. Characterization of Macrophage HO-1 Knockout Mice, Related to Figure 3
(A) Body weight gain of control and Macho mice on LFD.
(B) White blood cell counts (WBC) in LFD fed control and Macho mice, n = 4 per group.
(C) Daily food intake (% of body weight, BW) of lean control and Macho mice, n = 8 per group.
(D) Oral glucose tolerance tests and corresponding blood glucose and insulin levels on LFD.
(E) Insulin tolerance tests on LFD.
(F) Serum-free fatty acid levels measured in fed and overnight fasted states of control and Macho mice on LFD, n = 6 per group. (G) Daily food intake (% BW) of
control andMachomice on HFD, n = 8 per group. (H) Serum levels of liver enzymes determined in control andMachomice after 16weeks onHFD, n = 3 per group.
(I) In vivo activation of liver insulin signaling. HFD fed mice were injected with insulin, tissue biopsies taken at the indicated times, and analyzed for pINSR, INSR,
pAKT and AKT by immunoblotting. (J) Respiratory quotient (RQ) of body-weight-matched control and Macho animals, n = 7 per genotype. Results are mean ±
SEM. *p < 0.05.
Cell 158, 25–40, July 3, 2014 ª2014 Elsevier Inc. S9
Figure S4. Muscle-, Adipose-, and Beta-Cell HO-1 Knockout Animals and Macrophage Markers in Macrophage HO-1 Knockout Mice,
Related to Figure 4
(A) Muscle-HO1 knockout mice (Muhoko) on HFD: body weight gain, glucose and insulin tolerance tests.
(B) Adipose-HO1 knockout mice (Fahoko) on HFD: body weight gain, glucose and insulin tolerance tests.
(C) Beta-cell-HO1 knockout mice (Bhoko) on HFD: body weight gain, glucose and insulin tolerance tests.
(D) Gating strategy used in flow cytometry quantification of total macrophage numbers and their subpopulations. Stromal vascular cells obtained from epididymal
fat pads were gated for F4/80+/CD11b+ cells to obtain total macrophage numbers. Double-positive cells were examined for CD11c, MGL and CCR2 expression.
The percentage of CD11c+ and MGL+ cells within the F4/80+/CD11b+ macrophage population is indicated. A representative result from three independent
experiments is shown.
(E) Relative mRNA expression of HO-1/Hmox1 and macrophage markers Cd68, Cd11c/Itgax and Mgl2 in epididymal adipose tissue from HFD fed control and
Macho mice, n = 4 per group. (F) Relative mRNA expression of cytokine receptors in epididymal adipose tissue from HFD fed control and Macho mice, n = 4 per
group. Results are mean ± SEM. *p < 0.05, **p < 0.01.
S10 Cell 158, 25–40, July 3, 2014 ª2014 Elsevier Inc.
Figure S5. Mitochondrial Profiling of HO-1 Knockout Animals and HO-1 Rescue Studies, Related to Figure 6
(A and B) Basal respiration, maximal respiration, spare respiratory capacity (spare) and coupling efficiency of primary hepatocytes as well as naive BMDMs
derived from Lhoko and Macho animals and their littermate controls, n = 5 for hepatocytes, n = 9 for macrophages.
(C) Relative mitochondrial DNA (mtDNA) levels of HO-1 knockout hepatocytes and macrophages as well as their corresponding controls, n = 3 per group.
(D)MitoTracker green andMitoTracker deep red fluorescencewas recorded tomeasuremitochondrial mass of HO-1 knockoutmacrophages and hepatocytes as
well as their corresponding controls. AU; arbitrary units.
(E) Protein extracts from hepatocytes andmacrophages were analyzed using an antibody cocktail for protein components of the electron transport chain. b-Actin
was used as internal loading control, n = 4 per genotype.
(F) Cytosolic ROS measurements in HO-1 knockout macrophages and hepatocytes as well as their corresponding controls; FI, fluorescence intensity.
(G) In vitro activation of hepatocyte insulin signaling in AdLacZ and AdHO-1 transduced primary hepatocytes isolated from hepatocyte HO-1 knockout animals.
Primary hepatocytes were stimulated with insulin and analyzed for pINSR, INSR, pAKT and AKT by immunoblotting. HO-1 was used to control for adenoviral
HO-1 expression, n = 3 per group.
(H) Oxygen consumption rates (OCR) and mitochondrial function of AdLacZ and AdHO-1 transduced primary hepatocytes. Cells were derived from hepatocyte
HO-1 knockout animals (Lhoko) and transduced in vitro, n = 6 per group.
(I) Cytokine secretion of LPS (10 ng/ml) stimulated Macho BMDMs transduced with fiber type AdGFP and AdHO-1 virus particles, n = 3 per group. Results are
mean ± SEM. *p < 0.05, **p < 0.001.
Cell 158, 25–40, July 3, 2014 ª2014 Elsevier Inc. S11
1
Table S1. Correlations Between Adipose HO-1 mRNA and Anthropometric and Metabolic
Characteristics, Related to Table 1 and Figure 1
All
(n = 57)
Female, all
(n = 39)
Female, premeno
(n = 35)
Male
(n = 18)
Beta/R p Beta/R p Beta/R p Beta/R p
HOMA-IR 0.658 <0.001 0.596 <0.001 0.554 <0.001 0.751 <0.001
HOMA-IR (+ waist adjusted) 0.509 <0.001 0.535 <0.001 0.504 <0.001 0.648 0.021
HOMA-IR (+ VAT adjusted) 0.596 <0.001 0.504 0.004 0.483 0.007 0.887 0.006
BMI (kg/m2) 0.448 0.004 0.227 0.212 0.236 0.217 0.720 0.007
Waist circumference (cm) 0.454 0.003 0.139 0.444 0.130 0.503 0.715 0.004
Hip circumference (cm) 0.370 0.019 0.134 0.458 0.123 0.536 0.667 0.010
Waist/hip ratio 0.423 0.015 0.081 0.687 0.091 0.695 0.662 0.005
SAT (cm) 0.010 0.949 0.054 0.754 0.086 0.636 0.100 0.735
VAT (cm) 0.563 0.002 0.440 0.003 0.434 0.005 0.873 <0.001
Insulin (pmol/L) 0.663 <0.001 0.597 <0.001 0.555 <0.001 0.741 <0.001
Glucose (mmol/L) 0.319 0.024 0.339 0.052 0.333 0.073 0.379 0.098
Cholesterol (mmol/L) -0.177 0.148 -0.141 0.357 -0.062 0.674 -0.389 0.084
Triglycerides (mmol/L) 0.558 <0.001 0.504 <0.001 0.511 0.001 0.622 <0.001
HDL cholesterol (mmol/L) -0.443 <0.001 -0.307 0.038 -0.242 0.136 -0.750 <0.001
LDL cholesterol (mmol/L) -0.256 0.059 -0.295 0.072 -0.233 0.172 -0.333 0.209
Triglyceride/HDL ratio 0.605 <0.001 0.531 <0.001 0.542 0.001 0.740 <0.001
C-reactive protein (mg/L) 0.339 0.010 0.294 0.064 0.284 0.103 0.494 0.005
Standardized beta-coefficients (Beta/R) and two-sided p-values obtained from regression analysis
adjusted for age and sex. Normal distribution of traits tested by the Shapiro-Francia normality test.
Non-normal distributed parameters log10-transformed. Participants (total) n = 57 (6/17/27/7; non-
obese/obIS/obIR/intermediate); obIS, HOMA-IR <=2.0; obIR, HOMA-IR >=5.0; intermediate,
HOMA-IR >2.0 and <5.0; premeno, premenopausal; SAT, subcutaneous adipose tissue; VAT,
visceral adipose tissue.
2
Table S2. Correlations Between Liver HO-1 mRNA and Anthropometric and Metabolic
Characteristics in Obese Study Participants, Related to Table 1 and Figure 1
All
(n = 43)
Female, all
(n = 29)
Female, premeno
(n = 26)
Male
(n = 14)
Beta/R p Beta/R p Beta/R p Beta/R p
HOMA-IR 0.466 <0.001 0.451 0.003 0.517 0.009 0.523 0.011
HOMA-IR (+ waist adjusted) 0.374 0.005 0.373 0.042 0.412 0.016 0.430 0.012
HOMA-IR (+ VAT adjusted) 0.453 0.001 0.436 0.007 0.456 0.007 0.616 0.092
BMI (kg/m2) 0.515 0.007 0.551 0.003 0.472 0.004 0.285 0.377
Waist circumference (cm) 0.435 0.079 0.303 0.219 0.250 0.347 0.292 0.412
Hip circumference (cm) 0.226 0.209 0.272 0.242 0.297 0.252 0.125 0.681
Waist/hip ratio 0.328 0.185 0.188 0.485 0.119 0.700 0.317 0.242
SAT (cm) -0.015 0.940 0.174 0.246 0.185 0.235 -0.066 0.873
VAT (cm) 0.256 0.130 0.090 0.553 0.067 0.694 0.622 0.090
Insulin (pmol/L) 0.450 <0.001 0.408 0.007 0.426 0.008 0.540 0.011
Glucose (mmol/L) 0.371 0.032 0.580 <0.001 0.624 <0.001 -0.007 0.985
Cholesterol (mmol/L) -0.006 0.969 0.031 0.873 0.064 0.759 0.103 0.717
Triglycerides (mmol/L) 0.183 0.196 0.054 0.744 -0.013 0.943 0.696 0.006
HDL cholesterol (mmol/L) -0.473 <0.001 -0.407 0.004 -0.320 0.020 -0.678 <0.001
LDL cholesterol (mmol/L) 0.114 0.424 0.204 0.263 0.226 0.259 0.093 0.737
Triglyceride/HDL ratio 0.331 0.014 0.205 0.205 0.112 0.517 0.756 0.003
C-reactive protein (mg/L) 0.250 0.112 0.362 0.069 0.271 0.223 -0.028 0.932
Standardized beta-coefficients (Beta/R) and two-sided p-values obtained from regression analysis
adjusted for age and sex. Normal distribution of traits tested by the Shapiro-Francia normality test.
Non-normal distributed parameters log10-transformed. Participants (total) n = 43 (15/21/7;
obIS/obIR/intermediate); obIS, HOMA-IR <=2.0; obIR, HOMA-IR >=5.0; intermediate, HOMA-IR
>2.0 and <5.0; premeno, premenopausal; SAT, subcutaneous adipose tissue; VAT, visceral
adipose tissue.
3
Table S3. Comparison of Relative Adipose mRNA Expression Levels in Obese Study
Participants, Related to Table 1 and Figure 4
Category Symbol/Alias obIS obIR p* p† p‡
Heme Oxygenase-1 HMOX1/HO-1 1.00 (0.08) 1.47 (0.09) <0.001 <0.001 0.001
Insulin Resistance SLC2A4/GLUT4 1.00 (0.10) 0.53 (0.05) <0.001 0.002 0.001
HSD11B1 1.00 (0.15) 2.28 (0.35) <0.001 <0.001 <0.001
Macrophage CD68 1.00 (0.11) 1.83 (0.18) <0.001 <0.001 <0.001
ITGAX/CD11c 1.00 (0.32) 1.52 (0.26) 0.012 0.028 0.067
TNF 1.00 (0.18) 1.23 (0.16) 0.248 0.291 0.629
MRC1/CD206 1.00 (0.11) 1.16 (0.09) 0.172 0.333 0.172
CLEC10a/MGL/CD301 1.00 (0.20) 1.37 (0.57) 0.295 0.345 0.418
CCL18 1.00 (0.21) 0.72 (0.11) 0.021 0.067 0.033
Data are fold changes and presented as unadjusted and untransformed mean (SEM). Two-sided
p-values obtained from ANOVA. Heteroskedasticity tested by the Breusch-Pagan/Cook-Weisberg
test. Normal distribution of RNA data was tested by the Shapiro-Francia normality test. Non-normal
distributed parameters log10-transformed. *adjusted for age and sex; †adjusted for age, sex and
waist circumference; ‡adjusted for age, sex and visceral adipose tissue; participants (total) n = 44
(17/27; obIS/obIR); n = 42 (16/26) for visceral adipose adjustments; obIS, HOMA-IR <=2.0; obIR,
HOMA-IR >=5.0.
4
Table S4. Correlations Between Visceral Adipose HO-1 mRNA and Select Visceral Adipose
Transcript Levels in Obese Study Participants, Related to Table 1 and Figure 4
Gene Symbol/Alias Beta/R* p* Beta/R† p† Beta/R‡ p‡
SLC2A4/GLUT4 -0.506 <0.001 -0.479 <0.001 -0.431 0.002
HSD11B1 0.414 0.006 0.385 0.005 0.348 0.025
CD68 0.630 <0.001 0.609 <0.001 0.590 <0.001
ITGAX/CD11c 0.588 <0.001 0.577 <0.001 0.461 <0.001
TNF 0.471 0.003 0.464 0.003 0.379 0.018
MRC1/CD206 0.102 0.490 0.077 0.631 -0.014 0.936
CLEC10a/MGL/CD301 -0.350 0.028 -0.354 0.029 -0.379 0.018
CCL18 -0.562 <0.001 -0.552 <0.001 -0.583 0.001
Standardized beta-coefficients (Beta/R) and two-sided p-values obtained from multiple regression
analysis. Normal distribution of RNA data was tested by the Shapiro-Francia normality test. Non-
normal distributed parameters were log10-transformed. *adjusted for age and sex; †adjusted for
age, sex and waist circumference; ‡adjusted for age, sex and visceral adipose tissue; obese
participants (total) n = 51 (17/27/7; obIS/obIR/intermediate); n = 49 (16/26/7) for visceral adipose
adjustments; obIS, HOMA-IR <=2.0; obIR, HOMA-IR >=5.0; intermediate, HOMA-IR >2.0 and <5.0.
5
Table S5. Human PCR Primer, Alphabetical Order, Related to Quantitative PCR in Extended
Experimental Procedures
Gene/Alias Forward Sequence (5’-3’) Reverse Sequence (5’-3’)
CD68 GCTACATGGCGGTGGAGTACAA ATGATGAGAGGCAGCAAGATGG
CCL18 TGCTCCTGTGCACAAGTTGGTAC TTGTGGAATCTGCCAGGAGGTATAG
CLEC10A/MGL AAGGCACACCTAGGCCACTG CTTCAGGTCTTGCACCAGCTG
HMOX1/HO-1 ACTGCGTTCCTGCTCAACATC GCTCTGGTCCTTGGTGTCATG
HSD11B1 TCATTCTCAACCACATCACCAACACT CCAGCCAGAGAGGAGACGACAAC
ITGAX/CD11c CCTTGGTCCAGCTCTTCCTG GCTAAGGCTGTGAACAGGAGG
MRC1/CD206 CACACCATCGAGGAATTGGAC TAAGCCGATCCACAATTCGTC
RPLP0/ARP GTCATCCAGCAGGTGTTCGAC CTCCAGGAAGCGAGAATGCAG
SLC2A4/GLUT-4 GTCTCGGTGTTGTTGGTGGAG GAGCCACAGTCATCAGGATGG
TNF GAGGAGGACGAACATCCAACC TTGAGCCAGAAGAGGTTGAGG
6
Table S6. Mouse PCR Primer, Alphabetical Order, Related to Quantitative PCR in Extended
Experimental Procedures
Gene/Alias Forward Sequence (5’-3’) Reverse Sequence (5’-3’)
Acaca TTGAGAAGGTTCTTATCGCCAAC GACCACCGACGGATAGATCG
Acacb TCCGTAATGAACGTGCCATC CATCTTGATGTACTCTGCGTTGG
Acly CTGTGCCACCATGTTCTCCTC AGGCCTGGTTCTTGGCTACTG
Acox1 GCCCAACTGTGACTTCCATT GGCATGTAACCCGTAGCACT
Adipoq CGTGATGGCAGAGATGGCACTC CCTTAGGACCAAGAAGACCTGCATC
Ccr1 GCAGGTGACTGAGGTGATTGC AGCTGCCGAAGGTACTTCCAG
Ccr2 GGCATTGGATTCACCACATG TATGCCGTGGATGAACTGAGG
Ccr3 CTGGCCTTGTACAGCGAGATC AACACAGCATGGACGATAGCC
Ccr5 CAACATTGTCCTCCTCCTGACC GAGTCTCTGTTGCCTGCATGG
Cd36 GGAGCAACTGGTGGATGGTT TTGAGACTCTGAAAGGATCAGCA
Cd68 GCTACATGGCGGTGGAGTACAA ATGATGAGAGGCAGCAAGATGG
Cpt1a TTCAATACTTCCCGCATCCC TGTGGTACACGACAATGTGCCT
Dgat2 TGGCATAAGGCCCTATTTGG ATGGTGTCTCGGTTGACAGG
Fasn GGAGGTGGTGATAGCCGGTAT TGGGTAATCCATAGAGCCCAG
Foxo1 GCGTGCCCTACTTCAAGGATAA GTGGCGAATTGAATTCTTCCA
G6pc CCGGATCTACCTTGCTGCTCACTTT TAGCAGGTAGAATCCAAGCGCGAAAC
Gclc CCGCTGTCCAAGGTTGACGA GTTGCCGCCTTTGCAGATGTC
Glcm CGTGAAGAGCAGGGGAATCA AGCTGGAGTTAAGAGCCCCT
Gpam CAGTATCCCATCTCTGGGTTTG GTCTCGCCAGCCATCCTCT
Gpx1 GACACCAGGAGAATGGCAAGA TCACCATTCACTTCGCACTTC
Gpx4 GTTCCTGGGCTTGTGTGCATC GTCCTTGGCTGAGAATTCGTG
Gsr CCACGGCTATGCAACATTCG AATCAGGATGTGTGGAGCGG
Hmox1/HO-1 GCCGAGAATGCTGAGTTCATG TGGTACAAGGAAGCCATCACC
Il1b TGTGCAAGTGTCTGAAGCAGC TGGAAGCAGCCCTTCATCTT
Il6 CCAGAGATACAAAGAAATGATGG ACTCCAGAAGACCAGAGGAAAT
Il10 GCCAGAGCCACATGCTCCTA GTCCAGCTGGTCCTTTGTTTG
Itgax/Cd11c CCATGCTGGCTGTAGATGACC GTCATCCTGGCAGATGTGGTC
Lep CAATGACATTTCACACACGCAGTCG GGTGAAGCCCAGGAATGAAGTC
7
Me1 CTCATAGGAGTTGCTGCAATTGG CGTTGAAGGCAGCCATATCC
Mgl2 GGTCATCTCCGTGATTGGAT TCCCTCTTCTCCAGTGTGCT
mt-Nd2 AGGGATCCCACTGCACATAG CTCCTCATGCCCCTATGAAA
Ndufv1 CTTCCCCACTGGCCTCAAG CCAAAACCCAGTGATCCAGC
Nfkbia GGAGACTCGTTCCTGCACTTGG AACAAGAGCGAAACCAGGTCAGG
Nos2/iNos AATCTTGGAGCGAGTTGTGG CAGGAAGTAGGTGAGGGCTTG
Nqo1 CGCCTGAGCCCAGATATTGT GCACTCTCTCAAACCAGCCT
Pck1 CACCATCACCTCCTGGAAGA GGGTGCAGAATCTCGAGTTG
Prdx3 CCGTTTGGTAAAGGCGTTCC GGCTTGATCGTAGGGGACTC
Prdx5 TCGTCGGCTGAAAAGGTTCT ATCTGGCTCCACGTTCAGTG
Ppara AAGGCCTCAGGGTACCACTAC CCAGCTTCAGCCGAATAGTT
Pparg CACCAGTGTGAATTACAGCAAATC AGCTGATTCCGAAGTTGGTG
Rbp4 ACTGCAGGGTGAGCAGCTT TATGACCCTTCTCGTCCACA
Scd1 CCGGGAGAATATCCTGGTTT CACTGGCAGAGTAGTCGAAGG
Sod2 AAGGTCGCTTACAGATTGCTGC AGCGGAATAAGGCCTGTTGTTC
Srebf1 GCATGCCATGGGCAAGTAC TGTTGCCATGGAGATAGCATCT
Tnf TCGAGTGACAAGCCTGTAGCC TTGAGATCCATGCCGTTGG
Txn2 GACCGCGGCTAGAGAAGATG AGGCACAGCTGACACCTCATA