Retinol-binding protein 4 inhibits insulin signaling in adipocytes by inducing proinflammatory cytokines in macrophages through a c-Jun N-terminal kinase- and toll-like receptor 4-dependent and retinol-independent mechanism.
ABSTRACT Retinol-binding protein 4 (RBP4), the sole retinol transporter in blood, is secreted from adipocytes and liver. Serum RBP4 levels correlate highly with insulin resistance, other metabolic syndrome factors, and cardiovascular disease. Elevated serum RBP4 causes insulin resistance, but the molecular mechanisms are unknown. Here we show that RBP4 induces expression of proinflammatory cytokines in mouse and human macrophages and thereby indirectly inhibits insulin signaling in cocultured adipocytes. This occurs through activation of c-Jun N-terminal protein kinase (JNK) and Toll-like receptor 4 (TLR4) pathways independent of the RBP4 receptor, STRA6. RBP4 effects are markedly attenuated in JNK1-/- JNK2-/- macrophages and TLR4-/- macrophages. Because RBP4 is a retinol-binding protein, we investigated whether these effects are retinol dependent. Unexpectedly, retinol-free RBP4 (apo-RBP4) is as potent as retinol-bound RBP4 (holo-RBP4) in inducing proinflammatory cytokines in macrophages. Apo-RBP4 is likely to be physiologically significant since RBP4/retinol ratios are increased in serum of lean and obese insulin-resistant humans compared to ratios in insulin-sensitive humans, indicating that higher apo-RBP4 is associated with insulin resistance independent of obesity. Thus, RBP4 may cause insulin resistance by contributing to the development of an inflammatory state in adipose tissue through activation of proinflammatory cytokines in macrophages. This process reveals a novel JNK- and TLR4-dependent and retinol- and STRA6-independent mechanism of action for RBP4.
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ABSTRACT: The discovery of adipokines has revealed adipose tissue as a central node in the inter-organ crosstalk network, which mediates the regulation of multiple organs and tissues. Adipose tissue is a true endocrine organ that produces and secretes a wide range of mediators regulating adipose tissue function in an auto-/paracrine manner and important distant targets like the liver, skeletal muscle, the pancreas and the cardiovascular system. In metabolic disorders such as obesity, enlargement of adipocytes leads to adipose tissue dysfunction and a shift in the secretory profile with an increased release of pro-inflammatory adipokines. Adipose tissue dysfunction has a central role in the development of insulin resistance, type 2 diabetes and cardiovascular diseases. Besides the well-acknowledged role of adipokines in metabolic diseases, and the increasing number of adipokines being discovered in the last years, the mechanisms underlying the release of many adipokines from adipose tissue remain largely unknown. In order to combat metabolic diseases, it is crucial to better understand how adipokines can modulate adipose tissue growth and function. Therefore, we will focus on adipokines with a prominent role in auto-/paracrine crosstalk within the adipose tissue such as RBP4, HO-1, WISP2, SFRPs and chemerin. In order to depict the endocrine crosstalk between adipose tissue with skeletal muscle, the cardiovascular system and the pancreas, we will report the main findings regarding the direct effects of adiponectin, leptin, DPP4 and visfatin on skeletal muscle insulin resistance, cardiovascular function and β-cell growth and function. This article is protected by copyright. All rights reserved.Acta Physiologica 02/2014; · 4.38 Impact Factor
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ABSTRACT: There has been an upsurge of interest in the adipocyte coincident with the onset of the obesity epidemic and the realization that adipose tissue plays a major role in the regulation of metabolic function. The past few years, in particular, have seen significant changes in the way that we classify adipocytes and how we view adipose development and differentiation. We have new perspective on the roles played by adipocytes in a variety of homeostatic processes and on the mechanisms used by adipocytes to communicate with other tissues. Finally, there has been significant progress in understanding how these relationships are altered during metabolic disease and how they might be manipulated to restore metabolic health.Cell 01/2014; 156(1-2):20-44. · 31.96 Impact Factor
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ABSTRACT: Retinol-binding protein 4 (RBP4), a novel adipokine secreted by adipocytes and the liver, has elevated levels in type 2 diabetes mellitus (T2DM). However, its association with human metabolic diseases remains controversial. The present study was designed to investigate the associations of plasma RBP4 levels with oxidative stress, inflammatory markers, and metabolic syndrome (MetS) in a Chinese population. We evaluated plasma RBP4 levels in a cross-sectional sample of 1748 Chinese men and women aged 50 to 70 years in Guangzhou using an in-house developed and validated sandwich ELISA. Plasma glucose, insulin, lipid profile, serum adiponectin, adipocyte fatty acid-binding protein (A-FABP), 8-iso-prostaglandin F2alpha (8-iso PGF2alpha), 13-(S)-hydroxyoctadecadienoic acid (13-HODE), high-sensitivity C-reactive protein (hsCRP), interleukin 6 (IL6), monocyte chemotactic protein 1 (MCP1) and tumor necrosis factor alpha (TNFalpha) were all measured. MetS was defined according to the updated National Cholesterol Education Program Adult Treatment Panel III criteria for Asian Americans. Circulating RBP4 levels were positively correlated with A-FABP (r = 0.104, P < 0.001), 8-iso PGF2alpha (0.236, P < 0.001), and 13-HODE (0.204, P < 0.001) and were inversely correlated with HDL cholesterol (r = -0.072, P = 0.004). After multivariable adjustment, the RBP4 levels were strongly associated with MetS and its components. The ORs (95% CIs) for the comparisons of the extreme quartiles of RBP4 were 3.46 (2.87, 4.42) for MetS, 5.92 (4.47, 8.02) for hypertriglyceridemia, 1.42 (1.11, 1.68) for reduced HDL cholesterol, 1.87 (1.48, 2.36) for central obesity and 2.74 (2.15, 3.36) for hyperglycemia (all P < 0.001). When we further controlled for adipokines, markers of oxidative stress and proinflammatory response, the association of RBP4 with central obesity was abolished but not the association with other MetS components. Plasma RBP4 levels are associated with an adverse profile of oxidative stress and inflammatory markers and an increased risk of MetS in this Chinese population. These associations are independent of conventional risk factors.Diabetology and Metabolic Syndrome 02/2014; 6(1):25. · 1.92 Impact Factor
Retinol-Binding Protein 4 Inhibits Insulin Signaling in Adipocytes by
Inducing Proinflammatory Cytokines in Macrophages through a c-Jun
N-Terminal Kinase- and Toll-Like Receptor 4-Dependent
and Retinol-Independent Mechanism
Julie Norseen,aTetsuya Hosooka,a* Ann Hammarstedt,bMark M. Yore,aShashi Kant,cPratik Aryal,aUrban A. Kiernan,d
David A. Phillips,dHiroshi Maruyama,a* Bettina J. Kraus,a* Anny Usheva,aRoger J. Davis,cUlf Smith,band Barbara B. Kahna
Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts,
USAa; Lundberg Laboratory for Diabetes Research, Center of Excellence for Metabolic and Cardiovascular Research, Department of Molecular and Clinical Medicine, the
Sahlgrenska Academy, University of Gothenburg, Gothenburg, Swedenb; Howard Hughes Medical Institute and Program in Molecular Medicine, University of
Massachusetts Medical School, Worcester, Massachusetts, USAc; and ThermoFisher Scientific, Inc., Tempe, Arizona, USAd
type 2 diabetes could lead to development of new prevention and
treatment approaches. Multiple mechanisms may contribute, in-
pokines) (1, 15, 29), infiltration of white adipose tissue (WAT)
with proinflammatory macrophages (42), and aberrant lipid de-
position in tissues such as muscle and liver (51). These mecha-
nisms are not mutually exclusive. For example, adipokines can
affect inflammation and lipid deposition in tissues (15).
lin-resistant humans and mouse models, and genetic or pharma-
cologic elevation of serum RBP4 causes insulin resistance in nor-
mal mice (19, 31, 65). Although many studies show strong
correlations of serum RBP4 levels with obesity and the severity of
insulin resistance (9, 16, 27, 35), others do not (8, 17, 32, 46), as
populations of human subjects or from methodological issues
with RBP4 assays (18, 32, 64). Many studies also show that serum
RBP4 levels correlate with other components of the metabolic
disease (26), and intra-abdominal fat mass (9, 31, 36, 56). Strong
associations have been demonstrated in large-scale population
studies support a potential role for RBP4 in causing insulin resis-
besity is a major risk factor for insulin resistance, which is a
tance in humans. A study in approximately 6,500 aging adults
showed that a gain-of-function single nucleotide polymorphism
risk of type 2 diabetes (58). This SNP increases RBP4 promoter
activity and is positively associated with RBP4 expression in adi-
pose tissue and with body mass index (BMI) (40).
resistance in mice (45, 65, 68), the molecular mechanism is not un-
Received 30 August 2011 Returned for modification 27 September 2011
Accepted 6 March 2012
Published ahead of print 19 March 2012
Address correspondence to Barbara B. Kahn, firstname.lastname@example.org.
*Present address: T. Hosooka, Division of Diabetes and Endocrinology,
Department of Internal Medicine, Kobe University Graduate School of Medicine,
Kobe, Japan; B. J. Kraus, University Hospital Würzburg, Department of Internal
Medicine I, Würzburg, Germany; H. Maruyama, Mitsubishi Tanabe Pharma
Corporation, Toda, Saitama, Japan.
J.N. and T.H. contributed equally to this article.
Supplemental material for this article may be found at http://mcb.asm.org/.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
mcb.asm.org0270-7306/12/$12.00 Molecular and Cellular Biologyp. 2010–2019
of RBP4-injected mice (65). Incubation of isolated adipocytes with
RBP4 reduces the sensitivity to insulin-stimulated extracellular sig-
nal-regulated kinase (ERK) phosphorylation (43). These data pro-
vide some mechanistic insights into RBP4-mediated insulin resis-
Obesity is a state of chronic, low-grade inflammation, and
macrophages are thought to play an important role in maintain-
ing this state in adipose tissue (42, 61, 63). Many molecules se-
matory pathways in RBP4-induced insulin resistance. RBP4 ex-
correlate with subclinical inflammation, including serum levels
(3) and adipose tissue expression (66) of proinflammatory cyto-
kines. Lifestyle intervention can reduce serum RBP4 levels in par-
allel with improvement in markers for subclinical inflammation
(3, 22). At the cellular level, a recent study showed that RBP4
other study showed that the RBP4/retinol complex stimulates
JAK2/STAT5 signaling and expression of suppressor of cytokine
signaling 3 (SOCS3) (4), which has been implicated in insulin
It is not known whether RBP4’s effects on insulin action are
retinol (vitamin A) dependent. RBP4 delivers retinol to target tis-
fundamental effects on cellular function, including gene tran-
scription (37), differentiation, proliferation (20), and immune
function (44). Retinol deficiency, which is accompanied by low
RBP4 levels, results in impaired immunity (60). The majority of
RBP4 in serum is retinol bound (holo-RBP4), although a small
amount is not bound to retinol (apo-RBP4). While the contribu-
of serum RBP4 that is apo-RBP4 is increased in obese people (39)
and the ratio of RBP4 to retinol is increased in people with type 2
Determination of the role of retinol in the effects of RBP4 on
insulin resistance is critical for discerning the mechanisms of
RBP4 action. Here we show that RBP4 can act independently of
retinol to impair insulin signaling in adipocytes indirectly, by in-
ducing proinflammatory cytokine production from macro-
phages. This is mediated, in part, by the Toll-like receptor 4
(TLR4) cell surface receptor and not by the RBP4 receptor,
STRA6, and involves the c-Jun N-terminal protein kinase (JNK)
signaling pathway. These studies enhance our understanding of
establish that RBP4, independent of retinol, has important bio-
MATERIALS AND METHODS
inol-bound RBP4 (holo-RBP4) was expressed in Escherichia coli and pu-
rified as described previously (65). The endotoxin level of this recombi-
as the ambient endotoxin levels in reverse-osmosis double-deionized wa-
ter as quantitatively measured by the Limulus amoebocyte lysate test
(Lonza Limulus amoebocyte lysate QCL-1000; catalog no. 50-647 U). To
generate retinol-free (apo-RBP4) or retinol-reconstituted (add-back)
This step was repeated twice more with 1-h incubations of 40% butanol–
60% diisopropyl ether. The resulting retinol-stripped RBP4 (apo-RBP4)
was then incubated at 30°C overnight with either 100 mM retinol in eth-
anol to generate add-back RBP4 or with an equal volume of ethanol to
generate apo-RBP4. The holo, apo, and add-back forms of RBP4 were
purified by high-performance liquid chromatography (HPLC), and both
Western blotting after separation by nondenaturing gel electrophoresis
and by fluorescent spectrometry. After HPLC purification, RBP4 was di-
alyzed against 1? phosphate-buffered saline (PBS) for 4 h using a Slide A
Lyzer dialysis cassette (Thermo Scientific, catalog no. 66370). The dialy-
sate buffer was used as a vehicle control in experiments where indicated.
The protein was stored at ?80°C and protected from exposure to light.
Culture and coculture of 3T3L1 adipocytes and RAW264.7 macro-
phages. 3T3L1 preadipocyte culture and differentiation were performed
Dulbecco’s modified Eagle’s medium (DMEM), which was free of retinol
and other retinoids and contained 10% fetal bovine serum (FBS) and
antibiotics. Incubations with RBP4 were done under serum-free condi-
tions, except for specific experiments to compare RBP4 effects with and
without serum. Coculture of 3T3L1 adipocytes and RAW264.7 macro-
phages was performed as described previously with slight modifications
(55). In the direct contact system, 3T3L1 adipocytes were cultured in a
3T3L1 adipocytes. Cells were cultured overnight prior to treatment with
RBP4, inhibitors, or antibodies. In the noncontact Transwell system,
were plated in the upper Transwell inserts containing a 0.4-?m porous
membrane (Corning), and the cells were cultured overnight and then
used for the experiments.
Generation of primary mouse macrophages. To generate peritoneal
macrophages, PBS was injected into the peritoneal cavity. After massage
of the abdomen, the PBS was collected and then centrifuged at 1,000 rpm
for 5 min, and the cells were resuspended in RPMI supplemented with
macrophages were used for further studies.
Bone marrow-derived macrophages (BMDMs) were harvested from
the bone marrow was flushed from the bone with DMEM supplemented
with 10% FBS. The bone marrow was resuspended into a single-cell sus-
pension, centrifuged for 5 min at 1,000 rpm, and then washed twice in
with 20% FBS and 30% L929 supernatant, plated at 107cells per 10-cm
plate, and cultured for 3 days. After 3 days, fresh DMEM–20% FBS–30%
L929 was added to the cells. After 5 days, the plate was washed once with
PBS and the macrophages were used for further study.
Primary human macrophage differentiation. Primary human
monocytes were isolated by Ficoll gradient centrifugation and adhered to
6-well plates in RPMI 1640 medium supplemented with glutamine and
sodium pyruvate. After 1 h of incubation at 37°C, nonadherent cells were
removed by repeated washings. Adherent cells were incubated with mac-
rophage-specific medium (Invitrogen) containing glutamine, PEST and
granulocyte-macrophage colony-stimulating factor (GM-CSF; R&D Sys-
tems) for 3 days, after which the medium was replaced with fresh macro-
phage-specific serum-free medium (SFM) without GM-CSF. The cells
were then incubated for additional 3 days to generate well-differentiated
no. 420119 and 401489). Cocultured 3T3L1 adipocytes and RAW264.7
stimulation. For quantitative PCR analysis of cytokine expression in
Mechanism by Which RBP4 Causes Insulin Resistance
May 2012 Volume 32 Number 10 mcb.asm.org 2011
RAW264.7 macrophages, these cells were pretreated with 1 ?M or 5 ?M
Neutralizing antibodies. Neutralizing antibodies for tumor necrosis
factor alpha (TNF-?), interleukin-6 (IL-6), and monocyte chemoattrac-
tant protein 1 (MCP-1) were obtained from R&D Systems (catalog no.
and RAW264.7 macrophages were serum starved for 2 h, pretreated with
1 ?g/ml or 5 ?g/ml of each neutralizing antibody or with combination of
5 ?g/ml of neutralizing antibodies for TNF-?, IL-6, and MCP-1 for 30
min, and then incubated with RBP4 for 24 h before insulin stimulation.
Western blotting. Cells were lysed with 20 mM Tris-HCl (pH 7.5),
137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 1% NP-40, 10% glycerol, 1
mM sodium orthovanadate, 5 mM NaF, 5 mM ?-glycerophosphate, and
protease inhibitor, including 1 mM phenylmethylsulfonyl fluoride
(PMSF) and 10 ?g/ml aprotinin. Lysates were subjected to immunoblot-
ting with the indicated antibody. The following antibodies from Cell Sig-
no. 2370), P-IKK?/? (catalog no. 2078), JNK (catalog no. 9252), P-JNK
(catalog no. 9251), P-p38 (catalog no. 9211), p38 (catalog no. 9212), P-
ERK (catalog no. 9101), and ERK (catalog no. 9102).
Measurement of cytokine secretion. Cytokines (IL-2, IL-8, Il-12p70,
IL-1?, GM-CSF, gamma interferon [IFN-?], IL-6, IL-10, and TNF-?)
secreted into conditioned medium from human macrophages were mea-
sured by a human proinflammatory 9-plex assay (Meso Scale Discovery).
In addition, MCP-1 secreted into conditioned medium was measured by
an enzyme-linked immunosorbent assay (ELISA) according to the man-
ufacturers’ instructions (Biosource).
rophages or mouse peritoneal macrophages were measured by ELISAs
from Biosource (TNF-?, catalog no. KMC3011; IL-6, catalog no.
KMC0061; and MCP-1, catalog no. KMC1011).
Luciferase assay. The pNF?B-Luc vector was purchased from Clon-
control for transfection efficiency, using Fugene6 transfection reagent
from Roche (catalog no. 1815091). Luciferase activity in medium was
Mx1-Cre or C57 Mx1-Cre JNK1fl/flJNK2?/?mice (10) 4 weeks of age
were treated with 5 intraperitoneal injections of 20 ?g/g body weight
poly(I-C) over 5 to 7 days. Four weeks after treatment, bone marrow-
derived macrophages (BMDMs) were harvested and cultured. JNK1 de-
letion was confirmed by Western blotting. BMDMs were serum starved
for 2 h prior to treatment with 50 ?g/ml RBP4 for 24 h. The culture
medium was then collected, and the levels of secreted cytokines were
measured by ELISA.
Generation of TLR4?/?macrophages. TLR4?/?peritoneal macro-
phages were harvested as described above from male C57/Bl TLR4?/?
at the University of Massachusetts Medical School).
diabetic offspring of one parent with type 2 diabetes and one nondiabetic
parent, as determined by oral glucose tolerance test (OGTT) (6). Height
and weight were measured to the nearest centimeter and 0.1 kg, respec-
tively, and body mass index (BMI) was calculated as kg body weight di-
vided by height (m) squared. Fasting blood samples were drawn after an
overnight fast before an OGTT (75 g glucose) to evaluate glucose toler-
by standard laboratory methods. At 60 min after a glucose bolus, a eugly-
cemic-hyperinsulinemic clamp was initiated and carried out for the next
120 min (insulin infusion of 40 mU m?2min?2) to evaluate insulin sen-
glucose at various rates according to the blood glucose measurements
performed at 5-min intervals. The mean amount of glucose infused dur-
ing the last hour was used to calculate the rate of whole-body glucose
101; Akern SRI, Florence, Italy).
as previously described (48) with minor modifications. Briefly, 50 ?l of
serum was mixed with 450 ?l of PBS, and 0.5 ml ethanol was spiked with
approximately 50 pmol of internal standard (IS) (2.5 ?l of 20 ?M retinyl
acetate in acetonitrile). Retinol was extracted twice in 2.5 ml hexane. The
organic solvent was evaporated under a stream of nitrogen, and retinol
extracts were resuspended in 50 ?l of acetonitrile.
Serum retinol and the internal standard were separated as previously
described (28). Briefly, we used reverse-phase chromatography (Zorbax
SB-C18, 2.1 by 50 mm, 1.8 ?m) under isocratic conditions (89% acetoni-
trile [ACN]–H2O–0.01% formic acid, 0.5 ml/min) on an Agilent 1100/
were measured by UV absorbance at 325 nm and quantified using a stan-
dard curve. The injection volume was 10 ?l, and all samples were mea-
sured in duplicate. Retinol eluted at 1.14 min, and retinyl acetate (IS)
eluted at 1.94 min.
(MSIA) from 20 ?l of serum, as previously described (30, 64). Serum
retinol and RBP4 measurements were done in duplicate.
Quantitative PCR. An RNeasy kit (Qiagen) was used for RNA isola-
tion from human macrophages. Gene expression was analyzed with the
ABI Prism sequence detection system (TaqMan; Applied Biosystems).
software (Applied Biosystems) (see Table S1 in the supplemental mate-
rial). Each sample was run in duplicate, and the quantity of a particular
gene in each sample was normalized to ribosomal 18S RNA.
For studies of mouse macrophages, RNA was extracted using Tri-
Reagent (MRC, catalog no. TR 118) and cDNA was generated with ran-
dom hexamers (Clontech, catalog no. 639506). Quantitative real-time
PCR was performed with the ABI Prism sequence detection system. All
primers and probes used were obtained from Applied Biosystems, and
identification (ID) numbers for each gene are listed as follows: TNF-?,
Mm00443258-m1; IL-6, Mm99999064-m1; MCP-1, Mm00441242-m1;
and GAPDH (glyceraldehyde-3phosphate dehydrogenase), 4352339E-
0902020. Expression levels of mRNA were normalized to those of
rophages. We first examined whether RBP4 induces proinflam-
matory cytokines in macrophages. RAW264.7 macrophages (Fig.
1A) or primary mouse peritoneal macrophages (Fig. 1B) were
incubated with recombinant mouse holo-RBP4 (50 ?g/ml for 24
h) at a concentration present in serum of some insulin-resistant
humans (19, 31), and the levels of cytokines secreted into culture
medium were measured. RBP4 treatment markedly increased
TNF-?, IL-6, and MCP-1 secretion in both types of macrophages
(Fig. 1A and B). Maximal effects were achieved with 50 ?g/ml
the incubation medium using this concentration of RBP4 was
tested as a separate control and showed no effects on cytokine
expression or secretion (data not shown).
Since retinol has many roles in cellular function, determina-
tion of whether the effect of RBP4 to induce cytokines is retinol
dependent is mechanistically important. To investigate this, we
1C). As a control to ensure that the retinol-stripping process did
not damage the RBP4 protein, we also added back retinol to an
aliquot of apo-RBP4 (add-back RBP). The presence or absence of
Norseen et al.
mcb.asm.org Molecular and Cellular Biology
retinol in all forms of RBP4 was verified by fluorimetry (see Fig.
S1A in the supplemental material). We confirmed that apo-RBP4
remained free of retinol at the end of the incubation of the cells
with RBP4 by nondenaturing gel electrophoresis and Western
blotting (see Fig. S1B). Mouse holo-RBP4 increased the produc-
tion of TNF-? and IL-6 by 3- to 5-fold and MCP-1 by nearly
2-fold. Surprisingly, mouse apo-RBP4 increased the secretion of
TNF-? and MCP-1 by greater than 10-fold while increasing IL-6
secretion by greater than 100-fold (Fig. 1C). Apo-RBP4 with ret-
inol added back (add-back RBP4) had similar effects to the origi-
to generate apo-RBP4 did not alter the function of the protein.
in human macrophages and if this effect is retinol dependent.
secretion of TNF-?, IL-6, and MCP-1 as well as the cytokines
was the only cytokine we tested which was not induced by RBP4
treatment. To test whether increased cytokine secretion in re-
sponse to RBP4 reflects increased production, we measured ex-
pression of a subset of cytokines. In agreement with increased
cytokine secretion, both holo- and apo-RBP4 induced expression
of TNF-?, IL-6, MCP-1, and IL-1? (Fig. 1E). Since peroxisome
proliferator-activated receptor ? (PPAR?) is a negative regulator
of proinflammatory pathways in macrophages (23), we also ex-
MCP-1 from RAW264.7 macrophages. Retinol was rebound to apo-RBP4 as an additional control (add-back). (D) Induction of cytokine secretion in primary
human macrophages after stimulation with human holo- or apo-RBP4 or add-back RBP4 relative to vehicle control. (E) Induction of TNF-?, IL-6, MCP-1,
errors (SE). For panels A and B, n ? 3 per group; *, P ? 0.05 versus vehicle by t test. For panel C, n ? 3 per group; *, P ? 0.05 versus vehicle; #, P ? 0.05 versus
apo-RBP4 by ANOVA with post hoc analysis. For panels D and E, n ? 7 per group. *, P ? 0.05 versus vehicle by ANOVA with Wilcoxon analysis.
Mechanism by Which RBP4 Causes Insulin Resistance
May 2012 Volume 32 Number 10 mcb.asm.org 2013
amined if PPAR? expression is altered with RBP4 stimulation.
PPAR? expression was decreased by 50 to 70% after treatment
with holo- or apo-RBP4 or add-back RBP4 (Fig. 1E). One inter-
pretation of these data is that reduced PPAR? expression may be
involved in the induction of a proinflammatory state by RBP4.
These experiments were done under serum-free cell culture
conditions. Since serum is present in vivo, we repeated the exper-
iments in the presence of serum and obtained similar results (see
ence of serum in the cell culture medium does not affect RBP4
stimulation of cytokine secretion from macrophages.
RBP4 inhibits insulin signaling in 3T3L1 adipocytes cocul-
tured with RAW264.7 macrophages. Macrophages can play a
critical role in the development of insulin resistance through in-
teracting with adipocytes (61, 63). We next tested whether RBP4
affects communication between adipocytes and macrophages. In
trast, when 3T3L1 adipocytes were cocultured in a contact system
intermingled with macrophages, RBP4 treatment suppressed Akt
phosphorylation (S473 and T308) by 30 to 40% at both submaxi-
mal and maximal insulin concentrations (Fig. 2C and D). These
inhibition of insulin-stimulated Akt phosphorylation in adi-
The signaling results in Fig. 2C and D are from the mixture of
adipocytes and macrophages, although the macrophages contrib-
ute relatively little to the total cellular protein. However, to defin-
itively show that the effect on Akt phosphorylation occurs in adi-
tact, Transwell system. RBP4 treatment suppressed insulin-de-
pendent Akt phosphorylation by 30% in adipocytes cocultured
in the direct contact system, but not in adipocytes alone (Fig. 2E
and F). These results imply that macrophage-derived proinflam-
matory cytokines induced by RBP4 may mediate the impairment
in insulin signaling. To test this, we next investigated the effect of
neutralizing antibodies against proinflammatory cytokines on
RBP4-mediated inhibition of insulin signaling in adipocytes
tibodies for TNF-?, IL-6, or MCP-1 (1 or 5 ?g/ml) for 30 min
insulin-stimulated Akt phosphorylation compared to the level
with control IgG (Fig. 2G and H). Each of these antibodies had a
significant effect, and treatment with all three neutralizing anti-
FIG 2 RBP4 indirectly inhibits insulin signaling in adipocytes by stimulating
proinflammatory cytokine secretion from macrophages. (A) Representative
Western blot of total Akt and phosphorylated Akt (S473 and T308) in 3T3L1
adipocytes after direct treatment with holo-RBP4 (50 ?g/ml for 24 h) and
insulin stimulation (100 nM for 10 min). (B) Quantification of experiments
PBS and stimulated with insulin. (C) Representative Western blot of total Akt
and phosphorylated Akt (S473 and T308) from contact coculture of 3T3L1
adipocytes and RAW264.7 macrophages after treatment with holo-RBP4 (50
?g/ml for 24 h) and insulin stimulation (10 nM or 100 nM for 10 min). (D)
Quantification of experiments shown in panel C (n ? 3). All conditions were
insulin conditions for no RBP4 by two-way analysis of variance (ANOVA)
with post hoc analysis. (E) Representative Western blot of total Akt and phos-
phorylated Akt (S473) from noncontact Transwell coculture of 3T3L1 adi-
pocytes and RAW264.7 macrophages after treatment with holo-RBP4 (50
iments shown in panel E (n ? 4). All conditions were normalized to cells
0.05 versus no RBP4 in the absence or presence of macrophages; #, P ? 0.05
versus absence of macrophages with same RBP4 treatment by two-way
ANOVA with post hoc analysis. (G) Representative Western blot of total Akt
and phosphorylated Akt (S473) from contact coculture of 3T3L1 adipocytes
and RAW264.7 macrophages after treatment with neutralizing antibodies
24 h) treatment and then insulin (100 nM for 10 min). (H) Quantification of
expressed as means ? SE except for panel H, in which n ? 2.
Norseen et al.
mcb.asm.orgMolecular and Cellular Biology
signaling (Fig. 2G and H). These findings indicate that RBP4 can
inhibit insulin signaling in adipocytes by inducing proinflamma-
tory cytokines in macrophages.
sought to determine which pathways RBP4 activates in macro-
phages. We first tested the I?B kinase (IKK)/NF-?B pathway,
which plays a central role in cytokine production in macrophages
(57). RBP4 increased IKK?/? phosphorylation in cultured mac-
rophages (Fig. 3A) and NF-?B transcriptional activity, as deter-
mined by a 2-fold increase in NF-?B luciferase activity (Fig. 3B).
Lipopolysaccharide (LPS) served as a positive control.
We next examined whether RBP4 activates other proinflam-
matory pathways. Both apo-RBP4 and holo-RBP4 treatment of
macrophages activated the JNK, p38, and ERK pathways, with a
release in RAW264.7 macrophages (Fig. 1C), mouse apo-RBP4
had a much greater effect than holo-RBP4. In contrast, neither
apo-RBP4 nor holo-RBP4 activated the Akt pathway in macro-
phages (Fig. 3C). These results suggest that both holo- and apo-
RBP4 activate proinflammatory pathways at several steps.
RBP4 induces proinflammatory cytokines through the JNK
pathway in macrophages. We next sought to determine which
pathway is responsible for RBP4-mediated induction of proin-
flammatory cytokines in macrophages and for RBP4-mediated
inhibition of insulin signaling in adipocytes that are cocultured
with macrophages. IKK/NF-?B and JNK play central roles in in-
flammatory pathways leading to insulin resistance (2, 7, 24). We
therefore tested the effect of specific inhibitors for JNK or IKK on
RBP4-mediated inhibition of insulin signaling in 3T3L1 adi-
pocytes cocultured with macrophages. Insulin-dependent Akt
phosphorylation was again inhibited by RBP4 treatment (Fig. 4A
and B). A JNK-specific inhibitor reversed this RBP4-dependent
ner, while an IKK-specific inhibitor did not (Fig. 4A and B). The
JNK inhibitor also blocked RBP4-mediated induction of proin-
flammatory cytokines TNF-?, IL-6, and MCP-1 in a concentra-
tion-dependent manner (Fig. 4C).
To further determine the role of JNK in RBP4 induction of
cytokines, we studied the effects in primary mouse macrophages
lacking JNK1 and JNK2. In control macrophages with functional
JNK1/2, holo-RBP4 stimulated IL-6, MCP-1, and TNF-? secre-
tion and apo-RBP4 had a greater effect on IL-6 and MCP-1 (Fig.
the JNK1?/?JNK2?/?macrophages, the induction of IL-6 and
MCP-1 secretion by both holo- and apo-RBP4 was reduced by 35
to 75% relative to control macrophages, demonstrating a JNK-
dependent effect for both holo- and apo-RBP4. TNF-? induction
cells. Taken together, these results suggest that RBP4 inhibits in-
sulin signaling in adipocytes (Fig. 4A and B) by inducing proin-
flammatory cytokines in macrophages, in part through the JNK
pathway (Fig. 4C and D) and that this does not require that RBP4
be bound to retinol.
To understand whether this effect was mediated by STRA6
(stimulated by retinoic acid 6), the only known specific receptor
for RBP4 (5), we measured STRA6 expression by quantitative
not find expression of STRA6 in cultured or primary mouse mac-
rophages from either the peritoneum or the bone marrow from
several mouse strains (data not shown). Furthermore, we did not
detect STRA6 in cultured or primary human macrophages (data
To determine whether another cell surface receptor could be
involved, we tested the role of the TLR4 receptor since phosphor-
ylation of JNK, ERK, and p38 and subsequent cytokine secretion
can be initiated by stimulation of TLR4 (21). A previous publica-
tion indicated that TLR4 was involved in cytokine secretion from
macrophages stimulated with holo-RBP4 (11). We sought to test
whether TLR4 is necessary for the effects of both holo- and apo-
RBP4 in primary macrophages (Fig. 4E). In wild-type primary
mouse macrophages, holo- and apo-RBP4 and add-back RBP4
stimulate IL-6 and TNF-? secretion. However, in TLR4?/?mac-
rophages, IL-6 and TNF-? secretion is attenuated by 60 to 80%
(Fig. 4E). The partial suppression of IL-6 secretion in TLR4?/?
rophages. In contrast, TNF-? secretion is also attenuated in the
TLR4?/?macrophages but not in the JNK1?/?JNK2?/?macro-
phages, suggesting that another pathway downstream of TLR4
may also be involved.
The RBP4/retinol ratio is elevated in insulin-resistant hu-
mans independent of obesity. Since the cytokine-induced im-
pairment of insulin signaling by RBP4 is retinol independent, we
wanted to know the potential physiological significance of apo-
RBP4. Apo-RBP4 has been shown to be elevated in humans with
FIG 3 RBP4 activates proinflammatory signaling pathways in macrophages.
(A) Western blot of total and phosphorylated IKK?/IKK? from RAW264.7
macrophages treated with holo-RBP4 (50 ?g/ml) for the indicated times. (B)
Quantification of NF-?B promoter activity in RAW264.7 macrophages trans-
?g/ml for 18 h). LPS (10 ng/ml) was used as a positive control. AU, arbitrary
(?-Gal) expression. *, P ? 0.05 versus PBS. (C) Western blot of total and
phosphorylated Akt, ERK, p38, and JNK from RAW264.7 cells that were
treated with either apo- or holo-RBP4 (50 ?g/ml) for the indicated times.
Mechanism by Which RBP4 Causes Insulin Resistance
May 2012 Volume 32 Number 10mcb.asm.org 2015
obesity, type 2 diabetes, and/or renal failure (12, 13, 39). To de-
termine whether apo-RBP4 is elevated in association with obesity
the ratio of apo- to holo-RBP4 in insulin-sensitive and insulin-
resistant lean humans and in insulin-resistant obese subjects. The
lean insulin-resistant and obese insulin-resistant subjects were
matched for equivalent levels of insulin resistance, as determined
by the glucose disposal rate during a euglycemic-hyperinsuline-
mic clamp (Table 1).
Our data show that total RBP4 levels are increased by 2.5- to
3-fold in both lean and obese insulin-resistant subjects (Fig. 5A).
The magnitudes of elevation in lean and obese insulin-resistant
subjects are similar. While total serum retinol levels do not vary
significantly between insulin-sensitive and insulin-resistant lean
level in the lean groups combined (Fig. 5B). We calculated the
RBP4 is increased in the setting of insulin resistance even in lean
subjects and that it does not require obesity for this elevation to
alter RBP4, retinol, or RBP4/retinol ratio in any group.
Mounting evidence indicates that stimulation of proinflamma-
inflammation in adipose tissue in the setting of obesity-induced
insulin resistance (42, 55, 62, 63). However, there is debate over
what factors establish and maintain this inflammation. Our study
provides evidence that the elevated RBP4 levels associated with
insulin resistance and obesity (19, 31, 47, 65) may contribute to
either initiating or sustaining this proinflammatory state by acti-
vating macrophages. Through this mechanism, RBP4 indirectly
Akt in 3T3L1 adipocytes from direct contact coculture with RAW264.7 macrophages. Cells were pretreated with JNK or IKK inhibitor (1 or 10 ?M for 30 min),
and then human holo-RBP4 was added (50 ?g/ml for 24 h) prior to insulin (100 nM for 10 min). (B) Quantification of experiments shown in panel A. All
conditions were normalized to cells treated with PBS and then stimulated with insulin. Data are means ? SE. *, P ? 0.05 versus insulin-stimulated (?) with no
expression (n ? 3 to 4). *, P ? 0.05 versus no RBP4; #, P ? 0.05 versus no JNK inhibitor; ?, P ? 0.05 versus RBP4 plus 1 ?M JNK inhibitor, one-way ANOVA.
wild-type mice (open circles) or two mice with TLR4 deleted (TLR4 KO) (closed circles), and the experiments were done in duplicate. The bars show the mean
with individual values indicated by circles (n ? 2). *, P ? 0.05 versus vehicle (dialysate) treatment within the same genotype; #, P ? 0.05 for TLR4 KO versus
control with same treatment; ?, P ? 0.05 versus holo- and add-back RBP4 treatment within same genotype by two-way ANOVA with post hoc analysis.
TABLE 1 Anthropometric and metabolic characteristics of human
subjects included in Fig. 5
Result for groupa:
Gender (no. male/female)
GIR (mg/kg LBM ? min)b
42.2 ? 2.4
22.3 ? 0.5
19.3 ? 0.7
4.14 ? 0.07
36.6 ? 2.3
22.3 ? 0.5
8.19 ? 0.5
4.18 ? 0.05
39.0 ? 2.6
31.1 ? 0.3
8.09 ? 0.8
4.09 ? 0.12
aAll subjects were nondiabetic. Except for gender, data are expressed as means ? SE.
n ? 8 per group.
bThe glucose infusion rate (GIR) was determined by euglycemic-hyperinsulinemic
clamp as described previously (6). Lean body mass (LBM) was determined by
Norseen et al.
mcb.asm.orgMolecular and Cellular Biology
impairs insulin action in adipocytes. Furthermore, this effect of
RBP4 is mediated, in part, through the JNK and TLR4 pathways
and is independent of retinol binding to RBP4.
We show that RBP4 induces expression and secretion of pro-
inflammatory cytokines, including TNF-?, IL-6, MCP-1, IFN-?,
PPAR?, a negative regulator of inflammation, indicates that re-
duction in PPAR? could be part of the mechanism for the proin-
flammatory effects of RBP4. Similar to the effects in obesity,
whereby localized adipose tissue inflammation promotes insulin
resistance (42), we find that RBP4-induced cytokine production
from macrophages causes insulin resistance in adipocytes, and
sidered an anti-inflammatory cytokine. This may be a compensa-
ous induction of pro- and anti-inflammatory cytokines also
occurs in the inflammation induced by a high-fat diet (14).
Multiple redundant and overlapping signaling pathways me-
activates the p38, ERK, NF-?B, and JNK signaling pathways in
greatest decrease in RBP4-stimulated cytokine secretion and in
RBP4 on stimulation of IL-6 and MCP-1 in JNK1?/?JNK2?/?
macrophages strongly supports a critical role for JNK in RBP4-
induced inflammation. The persistent effect on TNF-? produc-
tion in these macrophages is not surprising because TNF-? pro-
duction is stimulated by multiple signaling pathways, but IL-6
production is more indicative of JNK activation (59).
A recent report indicated that holo-RBP4 induces TNF-? and
IL-6 through a TLR4-dependent pathway (11). We show that
TLR4 is necessary for complete stimulation of cytokine release
from macrophages treated with either holo- or apo-RBP4. The
fact that RBP4-induced TNF-? secretion is attenuated in
TLR4?/?macrophages but not in JNK1?/?JNK2?/?macro-
phages indicates another pathway downstream of TLR4 may be
completely blocked in TLR4?/?macrophages suggests that addi-
tional parallel pathways may be involved.
tissues and retinol has many important effects on the immune
adipose tissue inflammation was retinol dependent. However, we
find that apo-RBP4 elicits a cytokine response as robust as that of
holo-RBP4 in macrophages. While we observe a 10- to 100-fold-
enhanced effect of mouse apo-RBP4 in the mouse macrophage
rophages (Fig. 1D). The effect of human apo-RBP4 in primary
mouse macrophages is only about 50 to 80% greater than that of
holo-RBP4 (Fig. 4D and E). The reason for the differential effects
phages in a retinol-independent manner.
Our data show an increase in serum apo-RBP4 (Fig. 5C) in
both lean and obese insulin-resistant subjects. This implies that
elevation of RBP4, and not a concordant increase in retinol, is
associated with insulin resistance. It is not obesity per se that is
associated with increased apo-RBP4 but rather insulin resistance.
Interestingly, glucose-insulin infusion did not alter the levels of
retinol, RBP4, or the RBP4/retinol ratio in lean or obese subjects,
underscoring the relative stability of these levels.
The data in this paper demonstrate a novel alternative mecha-
nism for RBP4 action and demonstrate that the RBP4 protein
directly induces proinflammatory signaling pathways indepen-
dent of retinol. Furthermore, we find that the effects of RBP4 to
impair insulin signaling in adipocytes are indirect since they re-
quire the presence of macrophages (Fig. 2A to F). In contrast, a
SOCS3 expression (4). This mechanism is retinol-dependent and
did not detect STRA6 expression in any of the macrophages used
overall, the role of STRA6 in RBP4 actions and retinoid biology is
FIG 5 The ratio of apo-RBP4 to holo-RBP4 is elevated in human subjects with insulin resistance but not altered by insulin-glucose infusion during a
euglycemic-hyperinsulinemic clamp. Values in serum from lean insulin-sensitive (I.S.), lean insulin-resistant (I.R.), and obese insulin-resistant nondiabetic
immunoassay (A), and total serum retinol (?M) was measured by HPLC (B). (C) The molar concentration of serum RBP4 and retinol was used to calculate the
molar ratio of RBP4 to retinol in serum for each subject. Data are means ? SE, with individual values shown (n ? 8 per group). *, P ? 0.05 versus lean
insulin-sensitive subjects both before and after euglycemic-hyperinsulinemic clamp, as determined by two-way ANOVA with post hoc analysis. #, P ? 0.05 for
retinol values in obese subjects versus the two lean groups combined. The lean groups were combined because there was no difference in retinol values between
the lean groups. The retinol values for each subject before and after clamp were averaged for comparison of obese versus lean serum retinol values by t test.
Mechanism by Which RBP4 Causes Insulin Resistance
May 2012 Volume 32 Number 10mcb.asm.org 2017
findings of Berry et al. (4) since the effects of the JAK/STAT path-
way in adipocytes may be retinol and STRA6 dependent, whereas
the effects of RBP4 on cytokine production in macrophages are
are also indicated by our finding that RBP4 increases ERK activa-
RBP4 impairs insulin-stimulated ERK signaling in adipocytes
(43). Importantly, both direct and indirect effects of RBP4 on
adipocyte insulin signaling may contribute to insulin resistance.
RBP4 induces a proinflammatory response in macrophages
through JNK- and TLR4-dependent pathways, and the resulting
findings provide insights into the cellular mechanisms by which
RBP4 causes insulin resistance and could lead to new therapeutic
approaches to reduce obesity-related inflammation and insulin
We thank Madhumita Das for providing Mx1-Cre JNK1fl/flJNK2?/?
Jiang for assistance with TLR4?/?macrophage isolation. We also thank
Simon Dillon at the Beth Israel Deaconess Medical Center Genomics and
ment of serum retinol and Odile D. Peroni for critical assistance with
This work was supported by National Institutes of Health grants
NIDDK F32 DK091041 (to J.N.) and NIDDK R37 DK43051 (to B.B.K).
T.H. was supported by a Research Fellowship from the Manpei Suzuki
Diabetes Foundation, Japan. B.J.K. was supported by a Research Fellow-
ship from the German Cardiac Society. R.J.D. is an investigator of the
Howard Hughes Medical Institute.
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