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Diabetes Volume 63, October 2014
Tanit L. Gabriel,1 Marc J. Tol,1 Roelof Ottenhof,1 Cindy van Roomen,1 Jan Aten,2 Nike Claessen,2
Berend Hooibrink,3 Barbara de Weijer,4 Mireille J. Serlie,4 Carmen Argmann,5 Leonie van Elsenburg,1
Johannes M.F.G. Aerts,1 and Marco van Eijk1
Lysosomal Stress in Obese
Adipose Tissue Macrophages
Contributes to MITF-Dependent
Gpnmb Induction
OBESITY STUDIES
Diabetes 2014;63:3310–3323 | DOI: 10.2337/db13-1720
and point toward a crucial role for MITF in driving part of
the ATM phenotype.
In obesity, adipose tissue (AT) contains crown-like
structures where macrophages surround nonviable adipocytes. To understand how AT macrophages (ATMs)
contribute to development of insulin resistance, we
examined their character in more detail. In silico analysis of F2 mouse populations revealed significant correlation between adipose glycoprotein nonmetastatic
melanoma protein B (Gpnmb) expression and body
weight. In obese mice and obese individuals, Gpnmb
expression was induced in ATMs. Cultured RAW264.7
cells were used to obtain insight into the mechanism
of Gpnmb regulation. Gpnmb was potently induced
by lysosomal stress inducers, including palmitate and
chloroquine, or Torin1, an inhibitor of mammalian target
of rapamycin complex 1 (mTORC1). These stimuli also
provoked microphthalmia transcription factor (MITF)
translocation to the nucleus, and knockdown of MITF
by short hairpin RNA indicated its absolute requirement
for Gpnmb induction. In agreement with our in vitro data,
reduced mTORC1 activity was observed in isolated
ATMs from obese mice, which coincided with increased
nuclear MITF localization and Gpnmb transcription.
Aberrant nutrient sensing provokes lysosomal stress,
resulting in attenuated mTORC1 activity and enhanced
MITF-dependent Gpnmb induction. Our data identify
Gpnmb as a novel marker for obesity-induced ATM
infiltration and potentiator of interleukin-4 responses
Obesity is characterized by a state of low-grade inflammation, which is an essential contributing factor to
insulin resistance (IR) (1). Both adaptive and innate immunity contribute to this inflammatory response. Approximately a decade ago, increased macrophage numbers
were first described in obese adipose tissue (AT) (2). In
recent years, virtually all cells of the immune system
have been described in AT. In lean AT, a crucial role is
played by eosinophils, innate lymphoid type 2 cells, invariant natural killer T cells, and regulatory T cells, which promote an anti-inflammatory environment that warrants
insulin sensitivity (3–6). During obesity, AT becomes
populated by proinflammatory Th1 T cells (both CD4+
and CD8+), neutrophils, mast cells, and B cells, promoting IR (7–11). During diet-induced obesity (DIO), a phenotypical change occurs in AT macrophages (ATMs)
characterized by a shift toward a more proinflammatory
state (12,13). One typical characteristic of inflamed
obese AT is the presence of crown-like structures
(CLSs), which consist, in part, of actively recruited
proinflammatory ATMs, surrounding dead adipocytes
(2,14). Another characteristic of inflamed obese ATMs
1Department
Corresponding author: Marco van Eijk, [email protected].
of Medical Biochemistry, Academic Medical Center, University of
Amsterdam, Amsterdam, the Netherlands
2Department of Pathology, Academic Medical Center, University of Amsterdam,
Amsterdam, the Netherlands
3Department of Cell Biology, Academic Medical Center, University of Amsterdam,
Amsterdam, the Netherlands
4Department of Endocrinology and Metabolism, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands
5Department of Genetics and Genomic Sciences, Icahn Institute for Genomics and
Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY
Received 9 November 2013 and accepted 21 April 2014.
This article contains Supplementary Data online at http://diabetes
.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1720/-/DC1.
© 2014 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.
diabetes.diabetesjournals.org
is lipid accumulation, as these cells must buffer the fat
spillover from dying adipocytes (15,16).
Glycoprotein nonmetastatic melanoma protein B
(Gpnmb) is a transmembrane protein expressed by several
cell types, including macrophages (17). It is also referred
to as osteoactivin, hematopoietic growth factor–inducible
neurokinin 1, and the dendritic cell–associated heparin
sulfate proteoglycan–dependent integrin ligand (18–21).
Gpnmb has been implicated in regulation of both adaptive and innate immunity (22–24). Interestingly, Gpnmb
induction has also been observed in a mouse model of
Gaucher disease, a macrophage lipid–storage disease characterized by deficient lysosomal acid b-glucosidase and IR
in humans (25,26). Presumably, in this case, Gpnmb induction is triggered by the lysosomal accumulation of
nondegradable glycosphingolipids in macrophages. Recently, the lysosome regained scientific interest as it was
discovered that the transcription factor EB (TFEB) plays
a crucial role in driving lysosomal biogenesis (27). TFEB
belongs to the microphthalmia transcription factor
(MITF) E subfamily of basic helix-loop-helix leucine zipper
transcription factors to which MITF, transcription factor
E3, and transcription factor EC also belong (28).
Given the insight that Gpnmb seemingly tracts with
two key characteristics of obese settings, immune function and lipid exposure, we first investigated a possible
link between Gpnmb expression and metabolic-related
traits using an in silico approach. Next, we analyzed
Gpnmb expression in AT of lean versus obese mice
(genetically induced and high-fat diet [HFD]–induced
models for murine obesity) as well as in human AT compartments. Finally, we addressed the mechanism of transcriptional regulation for Gpnmb expression in ATMs. We
report Gpnmb as a novel marker for obesity-induced ATM
infiltration and point toward a crucial role for lysosomal
stress and MITF in driving part of the ATM phenotype. In
addition, we provide evidence that Gpnmb potentiates
interleukin (IL)-4–mediated arginase-1 induction.
RESEARCH DESIGN AND METHODS
In Silico Analysis
We investigated two independently generated mouse F2
populations: an F2 intercross between CAST/Ei 3 C57BL/6J
(CTB6F2; n = 408; males and females combined) and an
F2 intercross between C57BL/6J 3 C3H/HeJ both on an
apolipoprotein E–null background (BHF2; n = 135;
females only) (29,30). We queried GeneNetwork (http://
www.genenetwork.org/webqtl/main.py), which is a webbased program archiving these data sets (31), using the
following microarray and phenotype data sets: 1) UCLA
CTB6/B6CTF2 adipose (2005) mlratio, 2) UCLA BHF2
adipose female mlratio, and 3) CTB6F2 published phenotypes. Published phenotypes from the BHF2 were imported into the database ([30] and http://www.sagebase
.org/index.php). The sole Gpnmb probe on each of the
adipose databases (record 10024406500) was selected,
and either the top ;500 Gpnmb covariates from each
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3311
experiment or the correlation to all scored phenotypes
were determined. To reduce trait redundancy wherever
possible only trait:gene correlations using the normalized
or transformed trait expression values are shown.
Heatmap transformations of trait:gene correlations were
performed using the gplots package available from http://
www.r-project.org/.
Animals
C57BL/6J control mice and leptin-deficient obese (Ob/Ob)
mice (C57BL/6J background) were obtained from Harlan.
Animals (n = 6 per group unless stated otherwise) were fed
a commercial chow diet (AM-II). Studies were performed
using 12-week-old male mice.
For DIO studies, 6-week-old male C57Bl/6J mice were
fed either an HFD (D12492, Research Diets Inc.; consisting of 20 kcal % protein, 20% carbohydrates, and 60% fat)
or low-fat diet (LFD; D12450B, Research Diets Inc.;
consisting of 20 kcal % protein, 70% carbohydrates, and
10% fat) for 16 weeks. Principles of laboratory animal care
were followed, and approval for the study was obtained
from the local ethical committee for animal experiments.
Preparation of Stromal Vascular Fractions
Epididymal white AT (EWAT) of lean, Ob/Ob, and LFDand HFD-fed mice was dissected, minced, and exposed to
collagenase II (Sigma)/DNase I (Roche) treatment for 25
min. The cell suspension was passed through a 70 mm
(lean) or 100 mm (obese) cell strainer (BD Falcon) and
washed in Hanks’ balanced salt solution/2% FCS. The
obtained pellet is referred to as the stromal vascular fraction (SVF). Erythrocytes were removed from the SVF using red cell erythrocyte lysis buffer (Sigma).
FACS Sorting and CD11b+ Selection
SVF were incubated with rat anti-mouse CD16/CD32
mouse Fc–blocking reagent (BD Biosciences) for 10 min
and subsequently double labeled with rat anti-mouse
macrophage galactose–specific lectin/CD301 Alexa 647
(Bio-Connect) and rat anti-mouse F4/80 fluorescein isothiocyanate (eBioscience). From lean and LFD SVFs,
CD301+/F4/80+ cells were sorted, and from Ob/Ob and
HFD-fed mice SVFs, CD301+/F4/80+ and CD301lo/F4/80+
cells were sorted using a FACSAria instrument (BD Biosciences). Positive selection of ATMs from SVF was performed with anti-CD11b conjugated magnetic microbeads
(Miltenyi Biotec) using MACS according to the manufacturer’s protocol.
Human AT
Twenty obese (BMI ranging from 34.8 to 61.3 kg/m2)
Caucasian women with a mean age of 40.5 (26–50) years
who were scheduled for Roux-en-Y gastric bypass surgery
were included. Patients were eligible if they had no Diagnostic and Statistical Manual of Mental Disorders IV
diagnosis, were older than 18 years, understood the objective of the study, and gave informed consent. Biopsies
of the subcutaneous (SC), omental (OM), and mesenteric
(MES) fat compartment were taken at the beginning of
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Lysosomal Stress, Obese ATM Phenotype, and MITF
the surgical procedure after an overnight fast. The control
group consisted of age- and sex-matched female subjects
scheduled for elective cholecystectomy. They were lean
(BMI 20–24.4 kg/m2) and had a normal glucose tolerance
test, HOMA-IR, and HbA1c. The samples were snap-frozen
in liquid nitrogen and thereafter stored at 280°C for subsequent analysis. The women participated in a study on
the short-term metabolic effects of bariatric surgery. The
study was approved by the Medical Ethical Committee of
the Academic Medical Center.
RNA Extraction and Real-time PCR
Total RNA was extracted from total fat-, SVF-, and FACSsorted ATMs using TRIzol (Invitrogen) reagent and the
NucleoSpin II extraction kit, which included an RNasefree DNase step (Macherey Nagel). RNA concentrations
were measured using the NanoDrop spectrophotometer
(NanoDrop Technologies). Equal amounts of RNA were
used to synthesize cDNA according to the manufacturer’s
method (Invitrogen). Gene-specific analysis was done by
real-time RT-PCR using an iCycler MyiQ system (Bio-Rad).
Expression levels in mouse and human were normalized
to those of acidic ribosomal phosphoprotein 36B4, also
referred to as P0.
Cell Culture Experiments
RAW264.7 from American Type Culture Collection were
cultured in DMEM/10% FCS supplemented with penicillinstreptomycin. Palmitate and oleate (Sigma) were coupled
to BSA Fraction V fatty acid–free (Roche) as described
previously (32). IL-4 (R&D Systems), IL-10 (PeproTech),
and interferon-g (IFN-g; PeproTech) were used at
50 ng/mL, and lipopolysaccharide (LPS) from Salmonella minnesota R 595 (Alexa) was used at 100 ng/mL.
BSA-coupled lipids were used at 500 mmol/L; chloroquine
(CQ) at 40 mmol/L; bafilomycin at 50 nmol/L; concanamycin
A at 2 nmol/L; tunicamycin at 1, 5, and 10 mg/mL;
thapsigargin at 1, 10, and 100 nmol/L; and Torin at
250 nmol/L. b-Hexosaminidase activity was determined
in cell-free supernatant using 4MU-b- D-6-sulpho-2acetamido-2-deoxy-glucopyranoside as substrate (Sigma).
MITF knockdown in RAW264.7 cells was achieved using
a lentiviral approach. The short hairpin RNA (shRNA)
containing pKLO.1 puro lentiviral vectors were derived
from the Department of Human Genetics of the Academic
Medical Center. High-titer lentiviral stocks were generated by calcium phosphate–mediated transfection of the
purified plasmids and packaging vectors pMDL/pRRE,
pRSV-Rev, and pMD2.VSVG into HEK-293T cells. Supernatants were collected 48 and 72 h posttransfection and
subjected to ultracentrifugation to obtain concentrated
viral stocks. Titers were determined with p24 ELISA and
by means of real-time PCR using primers directed at proviral DNA in genomic host-cell DNA. The following
shRNA-encoding sequences targeting murine Mitf were
used in this study: Mitf shRNA#1 TRCN0000095285
(59-GCAGTACCTTTCTACCACTTT-39); Mitf shRNA#2
TRCN0000095288 (59-GCAAATACGTTACCCGTCTCT-39);
Diabetes Volume 63, October 2014
and Gpnmb shRNA TRCN0000011929 (59-CCGAATAAAC
AGATATGGCTA-39). The noncoding MISSION NonTarget Control Vector (SHC002, 59-CAACAAGATGAA
GAGCACCAA-39) served as an internal control in our
experiments. Raw264.7 cells were seeded at 40% confluency and the following day exposed to optimized
lentiviral titers for 4 h. After 24 h, transduced cells
were selected with 5 mg/mL puromycin for 2 days,
and experiments were performed immediately thereafter. Mouse plasma Gpnmb levels were determined by
ELISA (R&D Systems).
Immunohistochemistry
EWAT was fixed in 4% formaldehyde, phosphate-buffered
at pH 7.0, and embedded in paraffin. Deparaffinized
tissue sections (4 mm) were blocked for endogenous peroxidase activity by immersion in 0.3% H2O2 in methanol
for 20 min. For immunostaining, sections were incubated
with primary goat IgG anti-mouse Gpnmb antigen affinitypurified polyclonal antibody (AF2330; R&D Systems) diluted in phosphate-buffered primary antibody diluent
(ScyTek Laboratories) followed by rabbit anti-goat IgG
(Dako) and horseradish peroxidase–conjugated swine
anti-rabbit IgG (Dako). Bound horseradish peroxidase activity was visualized using diaminobenzidine as substrate.
Sections were counterstained with either hematoxylin or
methyl green and were mounted in Pertex. RAW264.7
were fixed in 4% formaldehyde and permeabilized in
0.2% Tween/PBS followed by endogenous peroxidase inactivation and staining with anti-MITF antibody (Exalpha
Biologicals Inc.).
Western Blot Analysis
Cell lysates were prepared in radioimmunoprecipitation
assay buffer (150 mmol/L NaCl, 50 mmol/L Tris-HCl pH
7.4, 2 mmol/L EDTA, 0.5% deoxycholaat, 1 mmol/L
Na3VO4, 20 mmol/L NaF, and 0.5% Triton X-100) supplemented with protease inhibitor cocktail (Roche) and phenylmethylsulfonyl fluoride (PMSF). Primary antibodies
used were anti-Gpnmb (R&D Systems), anti-tubulin-a
(Cedarlane Laboratories Limited), anti-MITF (Exalpha
Biologicals Inc.), anti-lamin A/B (Santa Cruz Biotechnology Inc.), anti-4E-BP1 (Cell Signaling Technology Inc.),
anti-phospho-4E-BP-1 (thr37/46) (Cell Signaling Technology Inc.), anti-gapdh (Cell Signaling Technology Inc.),
and matching secondary IRDye-conjugated antibodies
(Westburg BV) were used for detection in an Odyssey
version 3.0 (LI-COR Inc.).
Cytosolic and nuclear fractions were prepared as
follows. Cells pellets were washed in PBS and taken up
in 500 mL buffer (25 mmol/L HEPES, 5 mmol/L KCl,
0.5 mmol/L MgCl2, 1 mmol/L dithiothreitol, and 1 mmol/L
PMSF) and allowed to swell on ice for 15 min. Next,
200 mL buffer containing 0.35% Triton X-100 was added
and after 15 min rotation centrifuged for 30 s at maximum speed. Supernatant contains cytosolic fraction. Pellets
were resuspended in 25 mmol/L HEPES, 350 mmol/L NaCl,
10% sucrose, 0.1% Triton X-100, 1 mmol/L dithiothreitol,
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and 1 mmol/L PMSF and rotated for 1 h; centrifuge and
supernatant contains nuclear proteins.
Statistical Analysis
Values presented in figures represent means 6 SEM. Statistical analysis of the two groups was assessed by Student
t test (two tailed). Level of significance is depicted in the
figures with the actual P values. P values ,0.05 were
considered significant unless otherwise stated.
RESULTS
Adipose Gpnmb mRNA Expression Correlates With
Metabolic Traits in Mouse F2 Populations
Inbred mouse strains display significant inherent genetic
diversity affecting their susceptibility to various diseases,
including aspects of metabolic syndrome. F2 intercrosses of
such inbred mice consequently produce genotypically distinct offspring randomly segregating for these genetic and
varying disease susceptibilities. As a result, these populations of mice provide an experimental model for correlating
phenotypic trait expression to generate biological insight. In
our in silico analysis, we used data from two independent F2
intercrosses, namely, the CTB6F2 and the BHF2 (29,30,33).
We determined the relationship between the adipose
Gpnmb gene expression levels and that of various metabolic
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traits scored in the F2 individuals. A Heatmap transformation of the correlation coefficients is shown in Fig. 1A. The
most striking observation was the positive correlation of
Gpnmb to body weight (r = 0.66 and r = 0.43; P value
,5.0 3 10210) in both the BHF2 and CTB6F2 crosses, respectively. In contrast, Gpnmb expression was most significantly inversely correlated to the ratio of glucose to insulin
(r = 20.47 and r = 20.25; P value ,5.0 3 1025) in both the
BHF2 and CTB6F2 crosses, respectively. An example of the
correlation plot for the BHF2 cross is shown in Fig. 1B.
The results of this correlation analysis strongly suggest
a link between the expression of Gpnmb in AT and markers
of insulin sensitivity.
Gpnmb Is Highly Expressed in Macrophages in AT of
Obese Mice and Humans
To extend and validate the observations of Gpnmb in the
mouse F2 populations, we investigated Gpnmb expression
in two independent models for murine obesity, namely,
the high-fat DIO model and the genetically obese mouse
model (Ob/Ob). By using quantitative PCR (qPCR),
a dramatic increase of Gpnmb mRNA was observed in
EWAT of 12-week-old Ob/Ob mice and 16-week-old HFDfed DIO mice when compared with lean or LFD control
groups (all n = 6 per group) (Fig. 2A). Active recruitment
Figure 1—In silico analysis of Gpnmb expression reveals positive correlation with body weight and negative correlation with insulin
sensitivity measures. A: A Heatmap transformation of the correlation coefficients (P, Pearson; Rho; Spearman) of adipose Gpnmb expression to various metabolic traits scored in individuals from two independent mouse F2 intercrosses (BHF2 and CTB6F2). B: Example of
correlation plot between adipose Gpnmb expression and body weight scored in BHF2 individuals. BMD, bone mineral density; FFA,
free fatty acid; Glu, glucose; Ins, insulin; TC, total cholesterol; TG, triglyceride; UC, unesterified cholesterol.
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Lysosomal Stress, Obese ATM Phenotype, and MITF
Diabetes Volume 63, October 2014
Figure 2—Gpnmb is induced in obese AT. A: Gpnmb expression in obese EWAT of leptin-deficient Ob/Ob mice and mice fed an HFD for 16
weeks (n = 6 per group; depicted are mean 6 SEM). B: F4/80 induction in obese EWAT. C: F4/80 correlates significantly with Gpnmb in
EWAT. D: Gpnmb is enriched in SVF of obese mice. E: F4/80 induction in obese SVF. White bars refer to lean and LFD control groups; the
gray bars refer to Ob/Ob and HFD groups. F: Immunohistochemical analysis reveals Gpnmb-positive CLSs in EWAT of Ob/Ob mice but not
in lean control mice; bar size = 200 mm. (G) F4/80 and (H) Gpnmb are highly induced in EWAT. The white bars refer to the LFD group, and
the gray bars refer to the HFD group. I: Gpnmb is not induced in matured 3T3-L1 adipocytes, whereas peroxisome proliferator–activated
receptor g2 and adiponectin are. (J) CD68 and (K) Gpnmb expression is induced in human AT compartments (lean AT, n = 6; obese AT, n =
20; depicted are mean 6 SEM). *P < 0.05; **P < 0.01. White bars refer to the SC, light gray bars refer to the MES, and dark gray bars refer
to the OM adipose compartments. BAT, brown AT; Pparg2, peroxisome proliferator–activated receptor g2.
of inflammatory macrophages into AT results in the formation of CLSs, a hallmark of AT inflammation (2,14). As
expected, the macrophage content, which was analyzed by
qPCR as F4/80 expression, increased in DIO and Ob/Ob
AT when compared with lean and LFD-fed AT (Fig. 2B).
Interestingly, Gpnmb expression correlated well with F4/
80 expression in total AT (r2 = 0.94; P , 0.0001) (Fig. 2C).
Next, we prepared SVF of EWAT to narrow down the
cells possibly expressing Gpnmb. A clear enrichment of
Gpnmb and F4/80 expression in the SVF of both obese
mouse models was observed (n = 6 per group) (Fig. 2D and
E). By using anti-Gpnmb antibodies, we demonstrated
highly Gpnmb-positive CLSs (Fig. 2F). In male mice, highest expression of both F4/80 and Gpnmb was observed in
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the EWAT compartment (Fig. 2G and H). We also analyzed other macrophage-rich tissues and only found
a modest 10-fold induction in liver on HFD feeding, but
not in lung and spleen (Supplementary Fig. 1). In cultured
3T3-L1 adipocytes, Gpnmb was virtually absent and not
induced during maturation, whereas both peroxisome
proliferator–activated receptor g2 and adiponectin were
induced as anticipated (Fig. 2I). In plasma, Gpnmb levels
were not different between the LFD (13 6 4.6 ng/mL)
and HFD (12 6 3.7 ng/mL), despite the striking increase
in EWAT.
Analysis of human lean and obese AT samples, including SC, MES, and OM AT compartments, revealed
a significant increase of both CD68 (as a marker of
macrophages) and Gpnmb expression in the SC and MES
fat of the obese subjects compared with the lean controls
(Fig. 2J and K). When combining all AT compartments, we
observed a strong correlation between CD68 and Gpnmb
(r = 0.68; P , 0.0001). Interestingly, in both SC AT and
MES AT, Gpnmb significantly correlated with BMI (r =
0.46 [P = 0.021] and r = 0.44 [P = 0.044], respectively).
During obesity, a shift toward a more proinflammatory
phenotype is observed in ATMs (13). Murine ATM populations can for instance be defined by differences in
macrophage galactose N-acetyl-galactosamine–specific lectin 1 (MGL1) expression (12). By FACS sorting, F4/
80+MGL1+ fractions were isolated from lean and LFD
SVF, representing alternatively activated resident M2
macrophages (Fig. 3A and B). From Ob/Ob and HFD-fed
SVF, F4/80+MGL1+ (M2) macrophages were sorted as
well as inflammatory-recruited (M1) macrophages F4/
80+MGL1lo (Fig. 3C and D). Using qPCR, we reconfirmed
that the sorted cells were indeed F4/80+, either positive or
negative for MGL1 expression (Fig. 3E and F) and that the
inflammatory-recruited (M1) macrophages F4/80+MGL1lo
expressed high levels of CD11c (Fig. 3G).
A striking induction of Gpnmb was observed in obese
ATMs, both the F4/80+MGL1+ and F4/80+MGL1lo sorted
population (Fig. 3H). Of interest, no correlation was observed with FIZZ1, an alternatively activated macrophage
marker, which was highly present in lean ATMs but reduced to a large extent in both ATM populations isolated
from Ob/Ob and HFD mice (Fig. 3I). The latter is in line
with the lean Th2 adipose environment (eosinophilderived IL-4), which is lost in obese AT (3). The Gpnmb
expression pattern in obese ATMs showed a remarkable
similarity with osteopontin expression, a marker previously reported to be induced in ATMs in obese AT
(Fig. 3J) (34).
Gpnmb Is Induced in Macrophages by Palmitate and CQ
It is known that the development of obesity is associated with
both immunological and metabolic changes such as hypoxia
and lipotoxicity. To get more insight in the regulation of
Gpnmb expression in obese ATMs, several experiments were
performed using macrophage-like RAW264.7 cells. During
obesity, the inflammatory status of AT switches from a Th2-
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prone environment (eosinophil-derived IL-4 and regulatory
T cell–derived IL-10) toward a Th1 environment (T cell–
derived IFN-g). It was observed that Gpnmb was neither
induced by the Th2 cytokines IL-4 and IL-10 nor the
Th1-driving factors IFN-g, LPS, or a combination of IFN-g
with LPS. Analysis of macrophage polarization markers
such as arginase-1 (Th2) and nitric oxide synthase (Th1)
confirmed that the stimuli worked (Fig. 4A–C). The role of
hypoxia as a possible inducer of Gpnmb expression was
studied by using the hypoxia mimetic cobalt chloride
(CoCl2) at 100 mmol/L. As a reflection of the hypoxic status
of the cells, an increase of GLUT-1 (fourfold) was observed.
In contrast, we observed that Gpnmb expression was reduced, suggesting hypoxia does not control Gpnmb expression (Fig. 4D). Acute endoplasmic reticulum (ER) stress as
a possible Gpnmb inducer was explored by using thapsigargin (1–100 nmol/L) and tunicamycin (1–10 mg/mL), but
neither induced Gpnmb, whereas the ER stress marker
CHOP was clearly dose-dependently induced (Fig. 4E).
During the development of obesity, adipocyte dysfunction occurs and macrophages, among others, increasingly
scavenge the adipocyte-derived lipids. This lipotoxic
environment was mimicked by loading RAW cells with
BSA-coupled palmitate. Palmitate condensation to serine
via the action of serine palmitoyltransferase initiates
sphingolipid synthesis. Indeed after 7 h of lipid challenge,
increased formation of dihydroceramide (10-fold) was
detected when compared with BSA alone or oleate
(Fig. 5A). Interestingly, Gpnmb was strongly induced by
the palmitate treatment, whereas oleate did not affect
Gpnmb expression. At the level of protein, the effect of
palmitate was confirmed by immunoblot analysis (Fig. 5B
and C). To address if the effect was caused by palmitate
itself or a newly synthesized sphingolipid, we used
myriocin, which inhibits serine palmitoyltransferase. We
observed that myriocin reduced de novo synthesis of
sphingolipids, whereas it did not lower Gpnmb protein,
suggesting that Gpnmb induction was not caused by
a newly derived sphingolipid (Fig. 5D and E). Given the
overexpression of Gpnmb in mouse models of Gaucher
disease, a lysosomal storage disorder, we studied the potential impact of lysosomal stress on Gpnmb expression
(25). To address if lysosomal dysfunction could account
for adipose Gpnmb induction, RAW cells were treated for
24 h with the lysosomotropic agent CQ. Targeting of
lysosomes was confirmed by analysis of b-hexosaminidase
activity in the medium of cells, which increased fourfold.
Interestingly, we observed a striking induction of Gpnmb
at both the mRNA and the protein level (Fig. 5F–H).
When using the lysosomal vacuolar-type H+-ATPase inhibitors bafilomycin and concanamycin A, we also observed
a potent induction of Gpnmb (Supplementary Fig. 2).
MITF Is Required for the Induction of Gpnmb
What regulates Gpnmb expression in ATMs is unknown.
The association with lysosomal stress—which we define
as a dysfunction of lysosomes, which can be reached by
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impairment of lysosomal pH regulation or through subtle
overloading with lipids—and suggested connections in
literature prompted us to address if MITF controlled
Gpnmb induction in obese ATMs. Interestingly, MITF
gene expression was increased in total EWAT of obese
mice when compared with lean EWAT (Fig. 6A). Its
macrophage-derived origin in total EWAT was strengthened, as MITF expression correlated significantly with the
macrophage marker F4/80 (r2 = 0.90; P , 0.0001) and
with Gpnmb (r2 = 0.88; P , 0.0001), hence reflecting
ATM load. In obese SVF, MITF was increased at the
mRNA and protein level (Fig. 6B and C). In sorted
ATMs, MITF mRNA expression levels were not different
between studied populations (Fig. 6D). In other words,
per macrophage, MITF is not different, but it reflects increased macrophage load in total EWAT during obese conditions. Increased Gpnmb protein in obese ATMs seems
not to be accompanied by increased MITF, suggesting that
posttranscriptional regulation of MITF can occur during
obesity. Recent evidence suggests active regulation of
MITF localization from cytosol to the nucleus (35,36).
This prompted us to study if palmitate and CQ-mediated
induction of Gpnmb also required translocation of MITF
to the nucleus. Therefore, RAW264.7 cells were incubated
with CQ (40 mmol/L), BSA, or BSA-coupled palmitate.
Next, MITF protein was analyzed by immunoblot in
whole-cell extracts, cytosol, or nuclear-enriched fractions.
A clear shift from the cytosol to nuclei of MITF protein
was observed in CQ-pulsed and palmitate-loaded cells
(Fig. 6E). This was further confirmed by immunocytochemistry (Fig. 6F).
To ensure that Gpnmb indeed was solely induced in
a MITF-dependent manner, we used a lentiviral knockdown
strategy targeting MITF. We confirmed efficient MITF
knockdown on protein level compared with cells exposed
to control virus. Importantly, MITF depletion completely
abrogated the induction of Gpnmb following palmitate
loading and CQ stimulation (Fig. 6G and H). Interestingly,
MITF knockdown also prevented CQ-mediated induction of
Ccl3 and osteopontin gene expression (Fig. 6I). Osteopontin
was already found to behave as Gpnmb in sorted ATMs (Fig.
3J). The important ATM defining marker CD11c, but also
tumor necrosis factor, was not induced by CQ in a MITFdependent manner (data not shown).
Mammalian Target of Rapamycin Complex 1–Derived
Signals Control Gpnmb Expression
Very recently, it became clear that active mammalian
target of rapamycin complex 1 (mTORC1) at the site of
Figure 3—Sorting of ATM populations from lean and obese adipose EWAT. SVF labeled with anti-F4/80 and anti-MGL1/CD301
antibodies were FACS sorted. Representative FACS profiles are
depicted. A: Lean ATMs (F4/80-Mgl1+ [M2]). B: LFD control ATMs
(F4/80-Mgl1+ [M2]). C: Ob/Ob ATMs (both F4/80-Mgl1+ [M2] and
F4/80-Mgllo [M1]). D: HFD ATMs (both F4/80-Mgl1+ [M2] and F4/80Mgllo [M1]). By qPCR, the sorted ATM populations were reanalyzed.
E: F4/80 expression. F: MGL1 expression. G: CD11c expression.
H: Gpnmb expression. I: FIZZ1 expression. J: Osteopontin expression. Data are depicted as mean 6 SEM. n = 6 per group.
White bars refer to lean and LFD M2 ATMs, gray bars refer to Ob/Ob
and HFD M2 ATMs, and black bars refer to Ob/Ob and HFD M1
ATMs. OPN, osteopontin.
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Figure 4—Gpnmb expression is not induced in RAW264.7 cells by Th1, Th2 polarizing factors, or hypoxia induced by CoCl2. A: Th2- and
Th1-skewing cytokines do not induce Gpnmb expression. B: Arginase-1 induction by IL-4. C: NOS induction by Th1-skewing factors.
D: Hypoxic conditions induce GLUT-1 but not Gpnmb. E: Acute ER stress stimuli induce CHOP but fail to induce Gpnmb. Data are depicted
as mean 6 SEM. n = 3 per group. **P < 0.01; ***P < 0.001.
the lysosomal membrane sequesters MITF in the cytosol
by means of phosphorylation and that inhibition of the
kinase activity of mTORC1 resulted in the nuclear
localization of MITF (35). mTORC1 is widely considered
as the master regulator of metabolism (37). To address if
mTORC1 activity regulated Gpnmb induction in RAW
cells, we made use of the inhibitor Torin1. Inhibition of
the kinase activity of mTORC1 was confirmed by analyzing the phosphorylation of its substrate 4E binding
protein-1 (Fig. 7A). At the level of gene expression, a clear
induction of Gpnmb was detected, which also occurred at
the level of protein (Fig. 7B and C). In agreement, MITF
rapidly translocated from the cytosol to the nucleus upon
incubation with Torin1 (Fig. 7D and E). Importantly,
MITF knockdown also prevented induction of Gpnmb
by Torin1 (Fig. 7F). Of note, rapamycin failed to induce
Gpnmb, suggesting autophagy is not the driving factor of
Gpnmb induction.
Lastly, we analyzed if CD11b+-selected obese ATMs
also showed alterations in the mTORC1 pathway. We observed that 4E binding protein-1 phosphorylation was
attenuated in obese versus lean ATMs (Fig. 7G and H).
Importantly, Gpnmb was strongly induced in obese ATMs,
whereas MITF protein levels were unchanged, reflecting
a similar amount of both lean and obese macrophages in
our analysis.
Gpnmb Potentiates Alternative Macrophage Activation
We earlier (Fig. 4) demonstrated that Gpnmb was not induced by cytokines or LPS. Here we addressed the question
if Gpnmb itself could play a functional role in polarizing
RAW264.7 cells. To this end, Gpnmb-directed shRNA was
used, which efficiently knocked down Gpnmb in RAW cells,
both at the level of RNA and protein (Fig. 8A). When basal
levels of Gpnmb were knocked down, no difference was
observed in the capacity of LPS (6-h stimulation) to induce
iNOS when compared with scrambled control cells (Fig.
8A). Interestingly, the response to IL-4, analyzed as
arginase-1 expression, was reduced by ;80% when
Gpnmb was knocked down. To address if Gpnmb protein
induction could potentiate the IL-4 response, RAW cells
were first cultured with CQ or Torin to induce Gpnmb
(Fig. 8B) and subsequently pulsed with IL-4 for 6 h. A
striking sevenfold to eightfold induction of arginase-1
was observed, which again was suppressed when Gpnmb
was targeted by shRNA.
DISCUSSION
It is well established that the low-grade inflammation that
develops during obesity can be attributed to both innate
and adaptive immune cells. In lean AT, a crucial role is
played by eosinophils, innate lymphoid type 2 cells,
invariant natural killer T cells, and regulatory T cells all
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Diabetes Volume 63, October 2014
Figure 5—Gpnmb is induced by palmitate and CQ. A: Dihydroceramide induction following loading of cells with palmitate. B: Palmitate, but
not oleate, induces Gpnmb gene expression. C: Induction of Gpnmb protein by palmitate. D: The serine palmitoyltransferase inhibitor
myriocin inhibits de novo sphingolipid synthesis as ceramide levels are reduced. E: Myriocin does not prevent induction of Gpnmb. F: The
lysosomotropic agent CQ causes increased levels of b-hexosaminidase. CQ induces (G) Gpnmb mRNA and (H) protein. Data are depicted
as mean 6 SEM. n = 3 per group. ***P < 0.001. CER, ceramide; DihydroCer, dihydroceramide; hex, hexosaminidase; Myr, myriocin.
involved in promoting an anti-inflammatory environment
and insulin sensitivity (3–6). Obese AT becomes invaded
by inflammation-promoting Th1 T cells (both CD4+ and
CD8+), neutrophils, mast cells, and B cells, which leads to
IR (7–11). A hallmark of inflamed obese AT is the increased recruitment of macrophages, which are organized
in so-called CLSs, consisting of inflammatory macrophages surrounding dysfunctional adipocytes (2,14).
Given their pathophysiological role in metabolic disease,
characterization of these macrophages and identification
of markers is important. Here we report the exclusive and
massive expression of Gpnmb in obese ATMs. Gpnmb
expression was shown to reflect MITF activation as a response to lipid and lysosomal stress.
The evidence for this was obtained by experiments
with cultured RAW264.7 cells. Increased lipid pressure on
macrophages, caused by spillover from adipocytes occurring during the development of obesity, could be mimicked by loading RAW cells with palmitate. Importantly,
we found that Gpnmb was induced by palmitate. Increased lipid load in lysosomes has already been connected to Gpnmb induction in a mouse model of Gaucher
disease (25). In these studies, it has been postulated that
Gpnmb may serve as a biomarker for Gaucher disease. In
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Figure 6—Nuclear translocation of MITF drives adipose Gpnmb expression. A: MITF gene expression is induced in obese AT. White bars
refer to LFD control mice, and gray bars refer to HFD mice. B: MITF gene expression increases in the SVF of HFD-fed obese mice. Data are
depicted as mean 6 SEM. n = 6 per group. C: MITF protein is higher in obese SVF as assessed by Western blot analysis. D: MITF
expression is not different in lean and obese FACS-sorted ATM populations. E: Palmitate loading and CQ induce nuclear localization of
MITF in RAW cells, as demonstrated by Western blot analysis of cytosolic versus nuclei-enriched fractions. F: By immunohistochemistry,
this was confirmed at the cellular level. Culture of RAW in BSA and medium showed cytosolic MITF staining (see arrows), whereas culture
with palmitate and CQ revealed MITF presence in nuclei, as indicated by the arrows. G: MITF knockdown blunts palmitate-driven Gpnmb
induction compared with a scrambled control. H: MITF knockdown prevents CQ-mediated induction of Gpnmb. I: MITF knockdown also
prevents CQ-mediated induction of osteopontin and Ccl3 gene expression. White bars refer to scrambled control cells, light gray bars refer
to MITF knockdown cells, dark gray bars refer to scrambled control cells stimulated with CQ, and black bars refer to MITF knockdown cells
stimulated with CQ. Data are depicted as mean 6 SEM. n = 3 per group. ctrl, control; CYTO, cytosolic; Gapdh, glyceraldehyde-3phosphate dehydrogenase; NF, nuclei-enriched fractions; OPN, osteopontin; Palm, palmitate; scr, scrambled; sh-Ctrl, scrambled control;
sh-MITF, MITF knockdown. ***P < 0.001.
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Figure 7—In vitro inhibition of mTORC1 kinase activity with Torin1 induces Gpnmb expression in a MITF-dependent manner. A: Torin1
inhibits TORC1 kinase activity, as evidenced by reduced phosphorylation of 4E binding protein-1. (B) Gpnmb mRNA and (C) protein are
induced following mTORC1 inhibition. D: Torin1 induces nuclear localization of MITF, as demonstrated by Western blot analysis of cytosolic
versus nuclei-enriched fractions. E: By immunohistochemistry, this was confirmed at the cellular level. In DMSO-treated cells, MITF is
cytosolic (see arrows) and Torin1 stimulation results in nuclear presence (see arrows). F: Knockdown of MITF prevents Torin1-mediated
induction of Gpnmb. mTORC1 activity is reduced in vivo in obese ATMs. G. Western blot analysis revealed that CD11b+ selected ATMs
from obese mice show increased levels of Gpnmb compared with lean ATMs and reduced mTORC1 activity as phosphorylation of 4E
binding protein-1 is reduced. H: Quantification of phosphorylation status of 4E binding protein-1. Data are depicted as mean 6 SEM. n = 4
per group. ***P < 0.0001. CYTO, cytosolic; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NF, nuclei-enriched fractions; phospho,
phosphorylation; sh-Ctrl, scrambled control; sh-MITF, MITF knockdown.
obese mice, we were not able to demonstrate increased
levels of Gpnmb in the circulation, despite the several
hundred–fold induction in EWAT. Additional studies in
human obese subjects are still worthwhile, as in our human cohort, only a modest induction of Gpnmb was
detected and CLSs were only found in limited numbers.
A possible role for lysosomal dysfunction was further
demonstrated, as it was found that CQ very potently
triggered Gpnmb induction. Our lysosome-centered view
was further strengthened, as neither mimicking of a hypoxic environment nor culture with cytokines nor acute
induction of ER stress induced Gpnmb. We realize that
chronic lysosomal stress also impacts on other cellular
compartments. It therefore cannot be excluded that
such later events sustain Gpnmb expression. We postulate
a role for the lysosome in driving part of the ATM phenotype. In this context, it is important to note that obesity activates a program of lysosomal-dependent lipid
metabolism in ATMs independently of classic activation
(38). Interestingly, in our in silico KEGG pathway enrichment analysis, it was also found that Gpnmb expression
was linked to the lysosome in both the BHF2 and the
CTB6F2 crosses (data not shown).
Recently, it was demonstrated that lysosomal genes
display coordinated transcriptional regulation with an
important role for TFEB in lysosomal biogeneses
(27,39,40). MITF belongs to the same family of transcription factors as TFEB, and we found MITF to be essential
for Gpnmb induction in the macrophage cell line
RAW264.7, as MITF knockdown blunted induction by
the tested stimuli. Importantly, osteopontin and Ccl3,
but not tumor necrosis factor and CD11c, were induced
by CQ in an MITF-dependent manner, pointing toward a
crucial role of MITF in driving the ATM phenotype under
obese conditions. In FACS-sorted ATMs, Gpnmb followed
osteopontin expression, which has been described to be
involved in potentiating Mcp1/Ccl2-mediated monocyte
chemoattraction (34).
Finally, we found that mTORC1 inhibition also induced
Gpnmb expression in an MITF-dependent manner.
mTORC1 is a central regulator of cellular metabolism,
and it has been shown recently that its activation occurs
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Figure 8—Gpnmb potentiates IL-4–mediated arginase-1 induction. A: shRNA directed against Gpnmb lowers basal Gpnmb expression
and hampers IL-4–mediated arginase-1 expression but not LPS-mediated iNOS induction. B: Induction of Gpnmb potentiates IL-4–
mediated arginase-1 increment. Data are depicted as mean 6 SEM. n = 3 per group. Arg1, arginase-1; KD, knockdown; scr, scrambled.
at the site of the lysosomal membrane (37,41). Our data
propose a unifying model in which lysosomal stress
(palmitate and CQ treatment) and perturbed signaling
cascades (mTORC1 inhibition) could lead to MITF translocation to the nucleus with subsequent induction of
Gpnmb. The observations that in RAW264.7 cells, endogenous MITF shifts from the cytosol to nucleus upon
mTORC1 inhibition and CQ treatment is in agreement
with recent MITF overexpression studies (35,36). Importantly, our in vitro studies on MITF and Gpnmb induction
were confirmed in vivo. It was found that during obesity,
phosphorylation of the downstream target of mTORC1,
4E-BP-1, was reduced. This causes MITF to shift from the
cytosol to nuclei in ATMs, and consequently, Gpnmb induction occurred. Similar MITF-dependent regulation of
Gpnmb was recently reported in other cell types like melanoblasts and osteoclasts (42–45).
Thus far, it is unknown what the function of Gpnmb is
in obesity. It has been suggested that Gpnmb is involved
in controlling immune responses. In the context of
adaptive immunity, Gpnmb has been described to repress
T-cell activation (22). Additional evidence comes from
studies in mice homozygous for a spontaneous mutation
within the Gpnmb locus causing a truncated form of
Gpnmb. These mice exhibit autoimmune pigmentary
glaucoma and compromising ocular immunosuppression,
which is manifested by deficient anterior chamber–
associated immune deviation (18,46). In the context of innate immunity, Gpnmb was demonstrated to be involved in
recognition of dermatophytic fungi (24). Overexpression of
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Lysosomal Stress, Obese ATM Phenotype, and MITF
Gpnmb dampens LPS responses, suggesting that Gpnmb
negatively regulates inflammatory macrophage responses
(17). We now provide evidence that Gpnmb potentiates
IL-4–mediated induction of the alternative macrophage
marker arginase-1. Ablation of Gpnmb drastically reduces
IL-4–driven arginase-1 induction. Possibly, adipose Gpnmb
is involved in preventing inflammation from derailing completely, thus contributing to the counterinflammation
phase, which was recently proposed (47). Alternatively,
Gpnmb is involved in enhancing the phagocytic/remodeling
capacity of local ATMs. Recently, a connection was made
with repair after kidney tissue damage and regulation of
required phagocytosis (48). It is important to note in this
context that arginase-1 is an important mediator of tissue
repair and wound healing (49). In follow-up experiments,
we plan to study in mice with macrophage-specific inactivation of Gpnmb the physiological impact in these macrophages. At present, these appropriate animals are not
available. These are required to elucidate whether Gpnmb
has a causal role in lipid handling in macrophages and
whether this protein controls the inflammatory status of
macrophages upon lysosomal overload.
Gpnmb has been implicated in several disease processes,
including cancer, glaucoma, and renal damage and repair
(18,44,46,48,50). Here we have provided evidence that
Gpnmb is linked to obesity-driven AT inflammation as well.
In conclusion, we propose that adipose lipid spillover
provokes continuous pressure on the lysosomal compartment of resident macrophages, with consequential aberrations in signaling cascades closely linked to lysosome
function, such as mTORC1. Active mTORC1 normally
sequesters MITF in the cytosol, but as a result of mTORC1
inhibition, MITF translocates to the nucleus where it
initiates a transcriptional program including Gpnmb and
hence impacts on the ATM phenotype.
Acknowledgments. The authors thank Dr. N. Zelcer from the Department of
Medical Biochemistry, University of Amsterdam, for critical reading of the manuscript.
Funding. This work was supported by the Dutch Diabetes Foundation (grant
number 2009.80.016).
Duality of Interest. No potential conflicts of interest relevant to this article
were reported.
Author Contributions. T.L.G. and M.J.T. researched data and reviewed/
edited the manuscript. R.O., C.v.R., J.A., N.C., B.H., B.d.W., M.J.S., C.A., and
L.v.E. researched data. J.M.F.G.A. contributed to discussion and reviewed/edited the
manuscript. M.v.E. wrote the manuscript and researched data. M.v.E. is the
guarantor of this work and, as such, had full access to all the data in the study and
takes responsibility for the integrity of the data and the accuracy of the data analysis.
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