Control of adipose tissue inflammation through TRB1.

Abstract

Based on its role as an energy storage compartment and endocrine organ, white adipose tissue (WAT) fulfills a critical function in the maintenance of whole-body energy homeostasis. Indeed, WAT dysfunction is connected to obesity-related type 2 diabetes triggered at least partly by an inflammatory response in adipocytes. The pseudokinase tribbles (TRB) 3 has been identified by us and others as a critical regulator of hepatic glucose homeostasis in type 2 diabetes and WAT lipid homeostasis. Therefore, this study aimed to test the hypothesis that the TRB gene family fulfills broader functions in the integration of metabolic and inflammatory pathways in various tissues.
To determine the role of TRB family members for WAT function, we profiled the expression patterns of TRB13 under healthy and metabolic stress conditions. The differentially expressed TRB1 was functionally characterized in loss-of-function animal and primary adipocyte models.
Here, we show that the expression of TRB1 was specifically upregulated during acute and chronic inflammation in WAT of mice. Deficiency of TRB1 was found to impair cytokine gene expression in white adipocytes and to protect against high-fat diet-induced obesity. In adipocytes, TRB1 served as a nuclear transcriptional coactivator for the nuclear factor kappaB subunit RelA, thereby promoting the induction of proinflammatory cytokines in these cells.
As inflammation is typically seen in sepsis, insulin resistance, and obesity-related type 2 diabetes, the dual role of TRB1 as both a target and a (co) activator of inflammatory signaling might provide a molecular rationale for the amplification of proinflammatory responses in WAT in these subjects.

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Control of Adipose Tissue Inflammation Through TRB1
Anke Ostertag,1Allan Jones,1Adam J. Rose,1Maria Liebert,1Stefan Kleinsorg,1Anja Reimann,1
Alexandros Vegiopoulos,1Mauricio Berriel Diaz,1Daniela Strzoda,1Masahiro Yamamoto,2
Takashi Satoh,2Shizuo Akira,2and Stephan Herzig1
OBJECTIVE—Based on its role as an energy storage compart-
ment and endocrine organ, white adipose tissue (WAT) fulfills
a critical function in the maintenance of whole-body energy
homeostasis. Indeed, WAT dysfunction is connected to obesity-
related type 2 diabetes triggered at least partly by an inflamma-
tory response in adipocytes. The pseudokinase tribbles (TRB) 3
has been identified by us and others as a critical regulator of
hepatic glucose homeostasis in type 2 diabetes and WAT lipid
homeostasis. Therefore, this study aimed to test the hypothesis
that the TRB gene family fulfills broader functions in the integra-
tion of metabolic and inflammatory pathways in various tissues.
RESEARCH DESIGN AND METHODS—To determine the
role of TRB family members for WAT function, we profiled the
expression patterns of TRB13 under healthy and metabolic stress
conditions. The differentially expressed TRB1 was functionally
characterized in loss-of-function animal and primary adipocyte
models.
RESULTS—Here, we show that the expression of TRB1 was
specifically upregulated during acute and chronic inflammation
in WAT of mice. Deficiency of TRB1 was found to impair
cytokine gene expression in white adipocytes and to protect
against high-fat diet–induced obesity. In adipocytes, TRB1 served
as a nuclear transcriptional coactivator for the nuclear factor ?B
subunit RelA, thereby promoting the induction of proinflamma-
tory cytokines in these cells.
CONCLUSIONS—As inflammation is typically seen in sepsis,
insulin resistance, and obesity-related type 2 diabetes, the dual
role of TRB1 as both a target and a (co) activator of inflammatory
signaling might provide a molecular rationale for the amplifica-
tion of proinflammatory responses in WAT in these subjects.
Diabetes 59:1991–2000, 2010
A
ized in the storage of chemical energy such as triglycerides
(1), thereby providing a reserve storage compartment for
excess nutrients as a protective measure against starva-
tion (2). In addition, white adipocytes also serve as critical
dipose tissue can be subdivided into two dis-
tinct categories, white and brown. Whereas
brown adipose tissue (BAT) dissipates energy
as heat, white adipose tissue (WAT) is special-
endocrine cells, secreting a variety of adipokines to con-
trol energy intake and expenditure at the systemic level
(3). Importantly, under both acute and chronic metabolic
stress, as exemplified by sepsis and obesity-related type 2
diabetes, respectively, impaired WAT function is charac-
terized by WAT tissue inflammation and an increased
release of proinflammatory cytokines from this tissue as
well as dysregulation of the energy balance (4). Indeed,
sepsis induces the metabolic rate by 30–60% in humans
(5), and obesity has been linked to enhanced macrophage
recruitment and T-cell receptor rearrangements in WAT
(6,7). Additionally, proinflammatory cytokine expression
is activated in adipocytes, per se, in response to inflam-
matory cues, including bacterial lipopolysaccharide (LPS)
(8). Interestingly, recent evidence suggests that chroni-
cally elevated levels of LPS resulting from alterations in
the gut microbiota during obesity contribute to obesity-
related insulin resistance (9), promoting the idea that
common molecular pathways may underlie the proinflam-
matory responses in adipocytes and WAT under both
acute and chronic metabolic disturbances (10).
The tribbles (TRB) protein family consists of three
related serine/threonine kinase-like proteins, TRB1, -2, and
-3, which have been highly conserved throughout evolu-
tion and serve as critical regulators of cell-cycle progres-
sion during development in Drosophila and Xenopus (11).
In mammals, TRB3 was originally identified as a critical
checkpoint in hepatic glucose homeostasis during fasting
and type 2 diabetes, mediated through its regulatory
function on Akt/protein kinase B in the insulin signaling
pathway (12). Consistently, subsequent studies have pro-
posed TRB3 as a determinant of insulin sensitivity in liver,
skeletal muscle, and WAT (13–15) and of lipolysis in WAT
by triggering the degradation of acetyl-CoA carboxylase
via association with an E3 ubiquitin ligase (16). Interest-
ingly, both TRB2 and TRB3 were found to suppress white
adipocyte differentiation in an insulin-/peroxisome prolif-
erator–activated receptor (PPAR) ?–dependent manner
(17,18). Apart from hormonal pathway control, all three
tribbles family members have been implicated in the
activation of macrophages and, particularly, TRB3 in the
cellular endoplasmatic reticulum stress response (19–22),
overall supporting the hypothesis that the TRB family may
fulfill broader but largely unexplored functions in the
integration of metabolic and inflammatory pathways in
various tissues.
RESEARCH DESIGN AND METHODS
Recombinant adenoviruses. Adenoviruses expressing a TRB1-specific or
-nonspecific short-hairpin RNA (shRNA) under the control of the U6 promoter
were cloned as described previously (23,24). Viruses were purified by the
caesium chloride method and dialyzed against PBS containing 10% glycerol
prior to use. TRB1 shRNA forward 5?-CACCGGGCTATGTTGACTCAGAAATC
GAAATTTCTGAGTCAACATAGCCC-3?; TRB1 shRNA reverse 5?-AAAAGGGC
TATGTTGACTCAGAAATTTCGATTTCTGAGTCAACATAGCCC-3?.
From the
Metabolic Control, DKFZ-ZMBH Alliance, German Cancer Research Center
Heidelberg, Heidelberg, Germany; and the2Department of Host Defense,
Graduate School of Medicine, Osaka University, Suita, Osaka, Japan.
Corresponding author: Stephan Herzig, s.herzig@dkfz.de.
Received 15 October 2009 and accepted 4 May 2010. Published ahead of
print at http://diabetes.diabetesjournals.org on 3 June 2010. DOI:
10.2337/db09-1537.
A.J. and A.J.R. contributed equally to this article.
© 2010 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit,
and the work is not altered. See http://creativecommons.org/licenses/by
-nc-nd/3.0/ for details.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1Emmy Noether and Marie Curie Research Group, Molecular
ORIGINAL ARTICLE
diabetes.diabetesjournals.orgDIABETES, VOL. 59, AUGUST 2010 1991

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Animal experiments. Male 8- to 12-week-old C57BL6, db/db, ob/ob, toll-like
receptor (TLR) 4, tumor necrosis factor (TNF) receptor (TNFR)1/2, jun
NH2-terminal kinase (JNK), p50, and TNFR1/interleukin (IL)-1? receptor
knockout mice were obtained from Charles River Laboratories (Brussels,
Belgium). Mice with TRB1 haploinsufficiency have been described previously
(19). All animals were maintained on a 12-h light-dark cycle with regular
unrestricted diet. For LPS experiments, animals were fasted for 18 h with free
access to water and injected with 20 mg/kg body weight LPS. In each
experiment, three to seven animals received identical treatments and were
analyzed 2.5 h after LPS administration. For tumor induction in the cachexia
model, 1.5 ? 106colon 26 (C26) cells (25) in PBS were injected subcutane-
ously into 10-week-old CD2F1 mice (Charles River Laboratories). Control
mice were injected with heat-inactivated C26 cells. Subcutaneous implanta-
tion of C26 cells promoted severe reduction of body weight, as well as skeletal
muscle and adipose tissue mass. In addition, C26 mice displayed reduced
levels of serum triglycerides and substantial hepatic steatosis as described
(26). In high-fat diet experiments, TRB1?/?mice and wild-type littermates
were either fed a standard diet (D12450B, 10% energy from fat; Research Diets,
New Brunswick, NJ) or a high-fat diet (D12492, 60% energy from fat; Research
Diets) for a period of 13 weeks. Insulin and glucose tolerance tests were
performed as described previously (27). Organs including liver, epididymal fat
pads, and gastrocnemius muscles were collected after the corresponding time
period, snap frozen, and used for further analysis. Total body fat content was
determined by an Echo magnetic resonance imaging body composition
analyzer (Echo Medical Systems, Houston, TX). Animal handling and experi-
mentation was done in accordance with National Institutes of Health guide-
lines and approved by local authorities.
Blood metabolites. Serum levels of glucose, insulin, triglycerides, choles-
terol, and cytokines were determined using an automatic glucose monitor
(One Touch; Lifescan, Neckargemu ¨nd, Germany) or commercial kits (MP
Biomedicals, Orangeburg, NY; Mercodia, Uppsala, Sweden; Sigma, Munich,
Germany; Randox, Wako, Neuss, Germany; Millipore, Schwalbach, Germany).
Cell culture and transient transfection assays. 3T3-L1 preadipocytes and
HEK293 cells were transfected using lipofectamine (Invitrogen, Karlsruhe,
Germany) reagent according to the manufacturer’s instructions. Cell extracts
were prepared 48 h after transfection, and luciferase assays were performed
as described (23), normalizing to the activity from cotransfected ?-galactosi-
dase expression plasmid. Primary stromal-vascular fractions (SVFs) were
isolated from mouse epididymal fat depots, cultured, and differentiated into
mature primary adipocytes (essentially) as described (28). Cells were infected
with recombinant adenoviruses at a multiplicity of infection of 1,000 and
stimulated with 1.5 ng/ml TNF-? (Biomol, Hamburg, Germany). Cytokine-
conditioned medium was obtained by stimulating RAW264.7 mouse macro-
phages with 100 ng/ml LPS for 3.5 h and collecting the medium. Medium
harvested from RAW264.7 cells not exposed to LPS was used as the control ?
in all experiments.
Analysis of the macrophage-derived conditioned medium and control was
done by the Milliplex MAP Kit against mouse cytokines and chemokines
(Millipore, Schwalbach, Germany). 3T3-L1 and primary adipocytes were
treated with pharmacological inhibitors (50 ?mol/l parthenolide, 50 ?mol/l
SP600125, 50 ?mol/l PD98059, 10 ?mol/l SB202190; Calbiochem, Darmstadt,
Germany) 30 min prior to stimulation with conditioned medium or control.
Cells were lysed in QIAzol (QIAgen, Hilden, Germany), and RNA was isolated
according to standard procedures.
Cell separation studies of adipose tissue macrophages (ATMs) from
epididymal fat pads of LPS- or PBS-injected mice were done by magnetic
immunoaffinity isolation using anti-CD11b antibodies, conjugated to magnetic
beads (MACS Cell Separation System; Miltenyi Biotec). Following isolation of
ATMs from the SVFs using positive selection columns (MS columns; Miltenyi
Biotec), the remaining cells were eluted as the SVFs. For the analysis of
mRNA expression levels, eluted cells (CD11b-positive [?] and SVFs) and the
floating adipocyte fraction were resuspended in QIAzol reagent.
Quantitative Taqman RT-PCR. Total RNA was extracted from homoge-
nized mouse WAT or cell lysates using QIAzol and the RNeasy (Qiagen,
Hilden, Germany) kit. cDNA was prepared by reverse transcription using
Superscript II (Invitrogen) and Oligo dT primer (Fermentas, St. Leon-Rot,
Germany). cDNAs were amplified using assay-on-demand kits and an ABI
Prism 7300 sequence detector (Applied Biosystems, Darmstadt, Germany).
RNA expression data were quantified according to the ?Ctmethod as
described (29) and normalized to levels of TATA-box binding protein RNA.
Protein analysis. Protein was extracted from frozen organ samples or
cultured adipocytes in 2 ? SDS-8 mol/l urea cell lysis buffer, and 20–30 ?g of
protein were loaded onto 10% SDS–polyacrylamide gels and blotted onto
nitrocellulose membranes. Western blot assays were performed as described
(23) using antibodies specific for TRB1 (30), RelA, P300, ?-actin (Santa Cruz,
Heidelberg, Germany), or valosin-containing protein (VCP) (Abcam, Cam-
bridge, U.K.).
Chromatin immunoprecipitation assay. 3T3-L1 preadipocytes were trans-
fected with a plasmid encoding Flag-tagged TRB1, stimulated with condi-
tioned medium or control for 6 h and fixed with formaldehyde 48 h after
transfection, and chromatin immunoprecipitation assays were performed as
described (31) using Flag-specific antibodies (Upstate, Lake Placid, NY) or
nonspecific anti-HA antibody (Santa Cruz). Precipitated DNA fragments were
analyzed by PCR amplification, as described above, using primers directed
against the IL-6, IL-1?, and TNF-? promoters. Primers against cytokine
promoter regions lacking a RelA recognition site were used as negative
controls as described (32). Primer sequences were mIL-6_forward TGTGT
GTCGTCTGTCATGCG; mIL-6_reverse AGCTACAGACATCCCCAGTCTC;
mIL-6_A forward (without nuclear factor [NF]-?B) CCTACTTTCAAGCCTG
GAATC; mIL-6_A reverse (without NF-?B) TCAAGTCTTCTAGGCTGGGTC;
mIL-1?_forward TGCCCATTTCCACCACG; mIL-1b_reverse TGCTACCCTGAA
ATAATTTCTAATCCC; mIL-1b_forward (without NF-?B) CCCAAGGGAAAATT
TCACAGC; mIL-1? _reverse (without NF-?B) ACCACTGCAGGGTTTGTTGTC;
mTNF-?_forwardCCCCCGCGATGGAGAAGAAACCGAGA;
GCTAGTCCCTTGCTGTCCTCGCTGA.
Plasmids. Wild-type or mutated NF-?B reporter plasmids have been de-
scribed previously (32). Flag-TRB1 expression vector was generated by
PCR-based standard procedures and cloned into pcDNA3.1 (Invitrogen) using
standard protocols.
Glutathione-S-transferasepulldown
(GST) fusion proteins (pGEX5.1, pGEX5.1_GST_p65, pGEX5.1_GST_p65
RHD_1-305, and pGEX5.1_GST_p65_TA_441-551) (32) were produced in BL21
cells and affinity purified using glutathione Sepharose (Amersham Bio-
sciences, Darmstadt, Germany). In vitro transcription/translation was per-
formed using the TNT T7/T3 quick-coupled transcription/translation system
(Promega, Mannheim, Germany), according to the manufacturer’s instruc-
tions, and GST and in vitro translated proteins were incubated at 4°C
overnight. After extensive washing, GST-precipitated proteins were separated
by SDS-PAGE and detected by autoradiography.
Immunoprecipitation. HEK293 cells were cotransfected with a RelA expres-
sion vector plus Flag-TRB1 or an empty Flag vector. Subsequently, cells were
lysed, centrifuged, and the supernatant was incubated with anti-FLAG M2
agarose (Sigma) for 2 h. The immunoprecipitates were subsequently analyzed
by Western blot as described.
Statistical analysis. Statistical analyses were performed using a two-way
ANOVA with Bonferroni-adjusted posttests or Student t test in one-factorial
designs, respectively. The significance level was at P ? 0.05.
mTNF-?_reverse
assay.
Glutathione-S-transferase
RESULTS
TRB1 expression in WAT is elevated in acute and
chronic inflammation. To initially explore the expres-
sion profiles of TRB family members under conditions of
metabolic and, particularly, WAT dysfunction, we ex-
tracted total RNA from various tissues of wild-type or
db/db mice, the latter representing a standard model for
obesity and the metabolic syndrome (33). Quantitative
PCR analysis confirmed the previously reported upregula-
tion of TRB3 expression in livers of obese mice (12),
thereby verifying the experimental system (data not
shown). In contrast, hepatic mRNA levels of TRB1 and
TRB2 remained unchanged under these conditions (data
not shown).
Intriguingly, TRB1 mRNA expression was found to be
significantly elevated in WAT of db/db animals compared
with wild-type controls (Fig. 1A). In contrast, TRB2 and
TRB3 showed no difference in their WAT expression levels
(Fig. 1A), suggesting a specific impact of obesity-related
conditions on TRB1 adipose tissue expression. Indeed,
correlating with increasing, age-dependent overweight
condition a higher expression of WAT TRB1 levels was
also observed in a second, early-onset obesity mouse
model (supplemental Fig. 1A, available at http://diabetes.
diabetesjournals.org/cgi/content/full/db09-1537/DC1). Ex-
pression was, however, not affected by late-onset obesity
as associated with high-fat diet feeding of adult wild-type
mice (supplemental Fig. 1C). Furthermore, TRB1 mRNA
levels were found to be elevated in WAT of tumor-bearing
cachectic mice, while TRB2 and TRB3 again remained
TRB1 COACTIVATES PROINFLAMMATORY GENES
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Page 3

unchanged (Fig. 1B). Despite contrary states of energy
availability, obesity actually shares many phenotypic fea-
tures with severe wasting conditions such as cancer
cachexia, including insulin resistance, hepatic steatosis,
and particularly chronic inflammation (34). To test the
hypothesis that proinflammatory conditions represent a
common trigger for TRB1 expression in WAT, we pro-
voked extensive proinflammatory cytokine production and
signaling in mice by injecting sublethal doses of LPS. As
shown in Fig. 1C and D, LPS treatment stimulated TRB1
mRNA and protein expression in WAT compared with
controls (Fig. 1C and D), demonstrating that the expres-
sion of TRB1 in WAT is specifically induced by proinflam-
matory conditions, thereby representing a distinguishing
feature from other TRB family members.
TRB1 expression is under the control of cytokine
signaling in an adipocyte-autonomous manner. WAT
is composed of a variety of different cell types, including
mature adipocytes and the so-called SVF, comprising
macrophages, endothelial cells, and corresponding pro-
genitors (1). Cell separation studies using WAT explants
from LPS-treated or nontreated wild-type mice demon-
strated that TRB1 mRNA expression was specifically in-
duced by LPS in whole WAT depots, mature adipocytes,
and in the SVF, but not in CD11b?macrophage-enriched
cellular fractions (Fig. 2A), indicating that adipocytes
indeed represent the major site of TRB1 regulation in
response to proinflammatory signaling in WAT. To specif-
ically explore potential signaling pathways involved in
TRB1 induction under proinflammatory conditions in adi-
pocytes, we isolated the SVFs from WAT depots of
wild-type and TLR4 knockout mice, the latter deficient
in the cellular LPS receptor (35). Isolated SVFs were
differentiated into mature and primary adipocytes (data
not shown) and treated either with cytokine-enriched
conditioned medium from LPS-treated macrophages or
LPS (supplementary Fig. 2A). Whereas conditioned me-
dium efficiently stimulated TRB1 expression in both
primary wild-type and TLR4 knockout adipocytes, heat-
inactivated conditioned medium (supplementary Fig.
2B) and LPS treatment had no effect in either cell type
(Fig. 2B), supporting the hypothesis that LPS/TLR4
signaling, per se, is not responsible for the induction of
TRB1 under proinflammatory conditions but most likely
LPS-triggered cytokines such as TNF-? and ILs. Inter-
estingly, ablation of either TNFR1 and -2 or TNFR1 and
IL-1? receptor in primary adipocytes had no effect on
TRB1 mRNA induction in response to conditioned me-
dium treatment (supplementary Fig. 2C), arguing that
the induction of TRB1 expression in WAT is driven by
multiple proinflammatory mediators in a combinatorial
manner.
In this respect, the activator protein (AP) 1 and NF-?B
transcriptional complexes represent common integration
sites for divergent upstream proinflammatory signaling
pathways in various cell types (10). Whereas the condi-
tioned medium-dependent TRB1 induction in primary adi-
pocytes was not affected by genetic deficiency for the AP1
upstream kinase JNK1 (supplemental Fig. 1D), knockout
of the NF-?B subunit p50 significantly but incompletely
0.0
0.5
1.0
1.5
2.0
2.5
db/db
wt
TRB1TRB2 TRB3
**
***
***
relative mRNA levelsA
0
1
2
3
4
5
PBS
LPS
TRB1TRB2
relative mRNA levels
C
TRB3
D
B
PBSLPS
VCP
TRB1
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
TRB1TRB2TRB3
relative mRNA levels
C26
Control
0.0
0.5
1.0
1.5
PBS LPS
TRB1 (AU)
FIG. 1. TRB1 expression in WAT is elevated in acute and chronic inflammation. A–C: Quantitative PCR analysis of TRB1, TRB2, or TRB3 mRNA
levels in abdominal WAT of wild-type (wt) and obese db/db mice (A), healthy control and tumor-bearing cachectic (C26) CD2F1 mice (B), and
control (PBS)- and LPS-injected C57BL6 mice (means ? SE, n ? 5). *P < 0.05; **P < 0.01; ***P < 0.001. D: Western blot of WAT extracts from
two representative control (PBS)- or LPS-injected animals as in C using antibodies against TRB1 or valosin-containing protein (VCP). Relative
amounts of TRB1 protein levels normalized to VCP protein levels shown. AU, arbitrary units.
A. OSTERTAG AND ASSOCIATES
diabetes.diabetesjournals.org DIABETES, VOL. 59, AUGUST 20101993

Page 4

impaired TRB1 gene expression upon cytokine stimulation
in primary adipocytes (Fig. 2C), suggesting that the NF-?B
transcriptional complex may represent a critical check-
point for TRB1 gene regulation in these cells and that
other NF-?B components apart from p50 are essential in
this context. To test this hypothesis directly, we treated
mature adipocytes derived from differentiated 3T3-L1
preadipocytes with various inhibitors for specific intracel-
lular signaling pathways upon conditioned medium expo-
sure. Inhibitors of p38 (SB202190), extracellular signal–
regulated kinase (ERK) (PD98059), and protein kinase A
pathways elicited no effect on conditioned medium–trig-
gered TRB1 mRNA levels (Fig. 2D and data not shown).
However, specific pharmacological inhibition of the NF-?B
signaling axis by parthenolide as well as the non–isoform-
specific JNK inhibitor SP600125 (36) completely elimi-
nated the effect of conditioned medium on TRB1 mRNA
levels in differentiated 3T3-L1 adipocytes as well as in
SVF-derived primary adipocytes from wild-type mice (Fig.
2D and E; supplementary Fig. 2E), thereby demonstrating
that the cell autonomous induction of TRB1 in white
adipocytes under proinflammatory conditions is deter-
mined in a combinatorial fashion by distinct proinflamma-
tory axes, including NF-?B and JNK signaling.
TRB1 controls cytokine gene expression in WAT. The
data thus far established TRB1 as a novel output gene of
the proinflammatory pathway in WAT, prompting us to
investigate the functional relevance of these findings in an
in vivo setting. Due to the high perinatal mortality of
homozygous TRB1 knockout mice on the C57BL6 back-
ground strain (unpublished data, obtained from T. Satoh,
Japan), we studied mice with haploinsufficiency for TRB1
(19), displaying an ?50% reduction in whole-body TRB1
mRNA levels (supplementary Fig. 3A). Notably, both TRB2
and TRB3 mRNA levels in WAT of these mice were
substantially lower compared with TRB1 and not affected
by TRB1 deficiency (supplmentary Fig. 3A). Under basal
conditions, TRB1 haploinsufficiency slightly increased food
consumption (supplementary Fig. 3B) but had no effect on
body weight; total body fat content, as determined by
magnetic resonance technology (Fig. 4A and B); blood
glucose levels (supplementary Fig. 3C); serum insulin
levels (supplementary Fig. 3D); or serum cholesterol
(supplementary Fig. 3E), as well as nonesterified fatty
acid (supplementary Fig. 3F) and triglyceride (supple-
mentary Fig. 3G) levels as compared with wild-type
littermates. In WAT, key genes in glucose and lipid
regulatory pathways, including GLUT4, acetyl-carbox-
ylase-2, fatty acid synthase, and fatty acid–binding
protein-4, were not different when comparing wild-type and
TRB1 haploinsufficient animals (supplementary Fig. 3H).
In contrast, WAT mRNA expression of IL-1?, TNF-?, and
relative TRB1 mRNA levels
A
C
D
0
1
2
3
4
5
6
CM
+++
n.s.
n.s.
PD98059
+
SB202190
+
Parthenolide
+
3T3-L1 adipocytesPrimary adipocytes
-
B
0.0
LPS
M
CM
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
-
-
-----
----
+
--
+
--
-
+
+
+
-
+
-
n.s.
wtTLR4 ko
TRB1 relative mRNA levels
0.0
M
CM
0.5
1.0
1.5
2.0
2.5
3.0
3.5
+
-
+
+
-
+
wtp50 ko
--
TRB1 relative mRNA levels
0.0
CM
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Parthenolide
+
no inhibitor
**
*
-
-+
TRB1 relative mRNA levels
TRB1 relative mRNA levels
0
1
2
3
4
5
6
7
8
9
10
11
12
13
PBS
LPS
+
-++++
+
-
+
-
+
-
----
WAT
CD11b+
SVFAdipocytes
*
***
**
*
Primary adipocytesPrimary adipocytes
E
FIG. 2. TRB1 expression is under the control of cytokine signaling in an adipocyte-autonomous manner. A: Quantitative PCR analysis of TRB1
mRNA levels in WAT depots, mature adipocytes after separation from SVFs, SVF- or CD11b?-enriched cellular fractions from WAT depots. B and
C: Quantitative PCR analysis of TRB1 mRNA levels in primary adipocytes derived from the stromal-vascular WAT fractions of wild-type (wt) and
TLR4 (B) or p50 (C) knockout (ko) mice. Cells were treated with LPS and LPS-conditioned (conditioned medium [CM]) or non–LPS-conditioned
(control [M]) macrophage supernatant as indicated. D and E: Quantitative PCR analysis of TRB1 mRNA levels in mature adipocytes derived from
3T3–L1 preadipocytes (D) or the SVF of wild-type mice (E). Cells were pretreated with inhibitors against p38 (SB202190), the ERK (PD98059),
and NF-?B (parthenolide) pathways and subsequently stimulated with LPS-conditioned (conditioned medium [CM]) or non–LPS-conditioned
(control [M]) macrophage supernatant as indicated (means ? SE, n ? 5). *P < 0.05; **P < 0.01; ***P < 0.001. n.s., non significant.
TRB1 COACTIVATES PROINFLAMMATORY GENES
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Page 5

plasminogen activator inhibitor (PAI)-1 was significantly
reduced in TRB1 haploinsufficient mice under basal and/or
LPS-stimulated conditions (Fig. 3A), supporting the hy-
pothesis that TRB1 is specifically required for the execu-
tion of the proinflammatory program in WAT.
Given the variable impact of TRB1 haploinsufficiency on
WAT cytokine gene expression (Fig. 3A), which can most
likely be explained by differential effects of TRB1 on gene
expression in different WAT cell types including macro-
phages (19), we next sought to determine the cell auton-
omous role of TRB1 in proinflammatory responses
specifically in adipocytes. To this end, we infected 3T3-
B
C
0
1
2
3
4
5
6
7
relative mRNA levels
0
10
20
30
40
50
60
70
80
relative mRNA levelsrelative mRNA levels
relative mRNA levels
0.0
0.5
1.0
1.5
2.0
relative mRNA levels
-+-+-+-+-+-+-+-+
-
+
-
+
TNFα
Primary adipocytes
0
5000
10000
15000
20000
25000
30000
-+-+
(pg/ml)
D
VCP
TRB1
shNCshTRB1
IL-6
*
PAI-1
*
IL-6
******
IL-1βIFN-β
***
TNFα
****
PAI-1
Primary adipocytes
TNFα TNFα
0
10000
20000
30000
40000
50000
(pg/ml)
-+-+
*
shNC
shTRB1
*
*
A
wt
TRB1
+/-
IL-1β
TNFα
PAI-1
p < 0.07
0
10
20
30
40
50
60
70
LPS
-+-+
relative mRNA levels
0
25
50
75
100
125
150
175
LPS
-+-+
***
*
relative mRNA levels
0
10
20
30
40
LPS
-+-+
relative mRNA levels
0
5
10
15
0
5
10
15
20
25
FIG. 3. TRB1 controls cytokine gene expression in WAT. A: Quantitative
PCR analysis of IL-1?, TNF-?, and PAI-1 mRNA levels in WAT of
wild-type (wt) or TRB1 heterozygous knockout mice (TRB1?/?) under
basal or LPS-injected (20 mg/kg) conditions (means ? SE, n ? 6–7). B:
Western blot of extracts from primary mature adipocytes derived from
the stromal-vascular WAT fraction of wild-type mice using antibodies
against TRB1 or valosin-containing protein (VCP). Cells were infected
with a nonspecific control (NC) or TRB1-specific shRNA adenovirus at
a multiplicity of infection of 1,000. C: Quantitative PCR analysis of IL-6,
IL-1?, interferon (IFN)-?, TNF-?, and PAI-1 mRNA levels in primary
mature adipocytes derived from the stromal-vascular WAT fraction of
wild-type mice. Cells were infected with a nonspecific control (NC) or TRB1-specific shRNA adenovirus at a multiplicity of infection of 1,000 and
exposed to TNF-? for 6 h prior to harvesting. D: Release of IL-6 and PAI-1 from the same cells as in C was measured by enzyme-linked
immunosorbent assay (means ? SEM, n ? 3). *P < 0.05; **P < 0.01; ***P < 0.001.
A. OSTERTAG AND ASSOCIATES
diabetes.diabetesjournals.org DIABETES, VOL. 59, AUGUST 20101995