Heat Shock Protein 60 as a Mediator of Adipose Tissue Inflammation and Insulin Resistance

Abstract

The stress protein heat shock protein 60 (Hsp60) induces secretion of proinflammatory mediators from murine adipocytes. This study aimed to study Hsp60 as a mediator of adipose tissue inflammation and skeletal muscle cell (SkMC) insulin sensitivity and to quantify plasma Hsp60 concentrations in lean and obese individuals. Regulation of Hsp60 release and Hsp60-induced cytokine secretion and signaling was measured in human adipocytes and SkMCs. Adipocytes exhibited higher Hsp60 release than preadipocytes and SkMCs, which was further stimulated by cytokines and Toll-like receptor (TLR)-4 activation. Hsp60 activated extracellular signal-related kinase (ERK)-1/2, Jun NH(2)-terminal kinase (JNK), p38, nuclear factor (NF)-κB, and impaired insulin-stimulated Akt phosphorylation in adipocytes. Furthermore, Hsp60 stimulated adipocytes to secrete tumor necrosis factor-α, interleukin (IL)-6, and IL-8. In SkMCs, Hsp60 activated ERK1/2, JNK, and NF-κB and inhibits insulin signaling and insulin-stimulated glucose uptake. SkMCs released IL-6, IL-8, and monocyte chemoattractant protein-1 on Hsp60 stimulation. Plasma Hsp60 was higher in obese males than in lean males and correlated positively with BMI, blood pressure, leptin, and homeostasis model assessment-insulin resistance. In summary, Hsp60 is released by human adipocytes, increased in plasma of obese humans, and induces insulin resistance. This is accompanied by activation of proinflammatory signaling in human adipocytes and SkMCs. Thus, Hsp60 might be a factor underlying adipose tissue inflammation and obesity-associated metabolic disorders.

Full text

Heat Shock Protein 60 as a Mediator of Adipose Tissue
Inflammation and Insulin Resistance
Tina Märker,
1
Henrike Sell,
2
Pia Zilleßen,
1
Anja Glöde,
1
Jennifer Kriebel,
1
D. Margriet Ouwens,
2
Piet Pattyn,
3
Johannes Ruige,
4
Susanne Famulla,
2
Michael Roden,
1,5
Jürgen Eckel,
2
and
Christiane Habich
1
The stress protein heat shock protein 60 (Hsp60) induces secre-
tion of proinflammatory mediators from murine adipocytes. This
study aimed to study Hsp60 as a mediator of adipose tissue in-
flammation and skeletal muscle cell (SkMC) insulin sensitivity
and to quantify plasma Hsp60 concentrations in lean and obese
individuals. Regulation of Hsp60 release and Hsp60-induced
cytokine secretion and signaling was measured in human adi-
pocytes and SkMCs. Adipocytes exhibited higher Hsp60 release
than preadipocytes and SkMCs, which was further stimulated by
cytokines and Toll-like receptor (TLR)-4 activation. Hsp60 activated
extracellular signal–related kinase (ERK)-1/2, Jun NH
2
-terminal
kinase (JNK), p38, nuclear factor (NF)-kB, and impaired insulin-
stimulated Akt phosphorylation in adipocytes. Furthermore,
Hsp60 stimulated adipocytes to secrete tumor necrosis factor-a,
interleukin (IL)-6, and IL-8. In SkMCs, Hsp60 activated ERK1/2,
JNK, and NF-kB and inhibits insulin signaling and insulin-stimulated
glucose uptake. SkMCs released IL-6, IL-8, and monocyte chemo-
attractant protein-1 on Hsp60 stimulation. Plasma Hsp60 was higher
in obese males than in lean males and correlated positively with
BMI, blood pressure, leptin, and homeostasis model assessment–
insulin resistance. In summary, Hsp60 is released by human adi-
pocytes, increased in plasma of obese humans, and induces insulin
resistance. This is accompanied by activation of proinflamma-
tory signaling in human adipocytes and SkMCs. Thus, Hsp60
might be a factor underlying adipose tissue inflammation and
obesity-associated metabolic disorders. Diabetes 61:615–625,
2012
Obesity is frequently accompanied by metabolic
disturbances such as insulin resistance and
other components of the metabolic syndrome
(1). Enlarged adipose tissue mass, especially in
the visceral compartment, is one of the major risk factors
for the development of type 2 diabetes (2). Adipocytes
from obese subjects are characterized by altered meta-
bolic and endocrine function with increased secretion of
proinflammatory adipokines such as tumor necrosis factor
(TNF)-a, interleukin (IL)-6, and resistin (3,4). However,
until now, the physiological signals triggering the secretion
of proinflammatory mediators from adipocytes remain
largely unknown. The stress protein heat shock protein
60 (Hsp60) has been described as a potent inductor of
proinflammatory mediators in innate immune cells such as
macrophages and in adipocytes (5–8). Furthermore, ele-
vated Hsp60 concentrations have been measured in the
circulation of individuals with type 2 diabetes (9). Thus,
Hsp60 could be a potential trigger of human adipocyte in-
flammation. Because insulin resistance is typical for obesity
emerging early in the development of the metabolic syn-
drome and is highly associated with increased visceral ad-
ipose tissue mass, this study also aims at characterizing
Hsp60 in the context of skeletal muscle insulin resistance.
Here, we describe for the first time that Hsp60 is released
from adipocytes and can therefore be identified as a novel
adipokine, mediating paracrine proinflammatory effects on
adipocytes as well as endocrine effects on other cell types
such as skeletal muscle cell (SkMC). These findings are
supported by our results that circulating Hsp60 levels
are higher in obese individuals with and without type 2
diabetes than in lean individuals. The current study pro-
vides evidence that Hsp60 contributes to a negative
crosstalk between adipose tissue and skeletal muscle.
RESEARCH DESIGN AND METHODS
Cell cultures. Primary human preadipocytes were obtained from sub-
cutaneous adipose tissue from lean or overweight females undergoing elective
plastic surgery (BMI 28.1 61.1 kg/m
2
, age 42.4 62.8 years) and from PromoCell
(Heidelberg, Germany) and were differentiated in vitro to adipocytes as de-
scribed before (10). For isolation of mature adipocytes and the stromavascular
fraction, the protocol was modified by decreasing the collagenase digestion
period to 45 min. Mature adipocytes were collected by careful aspiration of the
upper phase, while the lower phase was centrifuged at 1,100gto obtain the
stromavascular fraction. All protocols were approved by the local ethics
committee, and all participants gave written informed consent. Primary hu-
man SkMCs derived from healthy individuals (male: 16 and 21 years of age;
female: 33 and 37 years of age) were obtained from PromoCell, cultivated, and
differentiated as described before (10).
Antibodies and reagents. Antibodies against phospho–extracellular signal–
related kinase (ERK)-1/2 (Thr202/Tyr204), phospho-p38 (Thr180/Tyr182),
phospho-SAPK/JNK (p46,Thr183/Tyr185), phospho–nuclear factor (NF)-kB
(p65, Ser536), phospho-Akt (Ser473), phospho-GSK3a/b(Ser21/9), and b-actin
(clone 13E5) were obtained from Cell Signaling Technology (Danvers, MA).
Antitubulin antibodies were obtained from Calbiochem (Merck Biosciences,
Schwalbach, Germany). Hsp60 antibodies (clone 24/HSP60) were purchased
from BD Biosciences (San Diego, CA), and horseradish peroxidase–conjugated
goat anti-rabbit and goat anti-mouse secondary antibodies were from Pierce
Thermo Scientific (Bonn, Germany). Recombinant human Hsp60 was ob-
tained from Loke Aps Diagnostics (Risskov, Denmark) or from StressGen
Biotechnologies (Victoria, BC, Canada). Lipopolysaccharide (LPS) (Escher-
ichia coli, 026:B6), lipoteichoic acid, ovalbumin (OVA), and porcine insulin
were purchased from Sigma-Aldrich (Steinheim, Germany). Recombinant
TNF-a,IL-1b, and interferon (IFN)-gwere obtained from Miltenyi Biotech
(Bergisch Gladbach, Germany). The enzyme-linked immunosorbent assay
From the
1
Institute for Clinical Diabetology, German Diabetes Center, Leibniz
Center for Diabetes Research at the Heinrich-Heine-University Düsseldorf,
Düsseldorf, Germany; the
2
Institute of Clinical Biochemistry and Pathobio-
chemistry, German Diabetes Center, Leibniz Center for Diabetes Research
at the Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany; the
3
De-
partment of Gastrointestinal Surgery, Ghent University Hospital, Ghent,
Belgium; the
4
Department of Endocrinology, Ghent University Hospital,
Ghent, Belgium; and the
5
Department of Metabolic Diseases, Heinrich-
Heine-University Düsseldorf, Düsseldorf, Germany.
Corresponding author: Christiane Habich, christiane.habich@online.de.
Received 12 November 2010 and accepted 8 December 2011.
DOI: 10.2337/db10-1574
This article contains Supplementary Data online at http://diabetes
.diabetesjournals.org/lookup/suppl/doi:10.2337/db10-1574/-/DC1.
T.M. and H.S. contributed equally to this article.
Ó2012 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.
diabetes.diabetesjournals.org DIABETES, VOL. 61, MARCH 2012 615
ORIGINAL ARTICLE

(ELISA) kit for phospho–insulin receptor substrate (IRS)-1 (Ser307) was pur-
chased from Cell Signaling Technology.
Hsp60 expression. Primary human subcutaneous adipocytes and SkMCs
obtained at different differentiation time points (day 0–14 and day 0–8, re-
spectively) were lysed in a buffer containing 50 mmol/L HEPES, pH 7.4, 1%
Triton, and protease inhibitors (Roche Diagnostics, Mannheim, Germany). Cell
lysates were analyzed for Hsp60 expression by Western blot using the Lumi-
Imager system (Roche Diagnostics).
Adipocyte release of Hsp60. To analyze the release of Hsp60 from human
preadipocytes, mature adipocytes and SkMC 3.5 310
6
cells were seeded in
75-cm
2
cell culture flasks for differentiation, and adipocyte conditioned me-
dium (CM) was generated as described earlier (11). Adipocyte CM was col-
lected and concentrated 200-fold by Amicon Ultra centrifugal filter units
(Millipore, Schwalbach, Germany) before Western blot analysis. To induce
Hsp60 release upon inflammatory stress or Toll-like receptor (TLR) activation,
cells were treated with LPS (1 mg/mL); lipoteichoic acid (5 mg/mL); a
cytokine mixture composed of TNF-a,IL-1b,andIFN-g(1,000 units/mL for
each cytokine); or each cytokine individually for 48 h. Concentrated cell
culture supernatants were analyzed for Hsp60 concentrations by ELISA
(Cusabio Biotech, Newark, DE).
Hsp60 binding and inhibition. For Hsp60 binding studies, 0.5 310
6
human
adipocytes or SkMCs were either directly incubated with DyLight649-labeled
(Pierce, Rockford, IL) Hsp60 (Hsp60*, 45 min, 4°C) or preincubated with un-
labeled Hsp60 or OVA as described before (12).
Assessment of insulin sensitivity of human SkMCs and adipocytes.
SkMCs were used for glucose uptake experiments at 4 days after start of
differentiation. Uptake of 2-deoxyglucose was measured for 2 h after 30 min
exposure to insulin (100 nmol/L) as described before (13). To analyze the ef-
fect of Hsp60 on Akt and GSK3a/bphosphorylation, human subcutaneous adi-
pocytes and SkMCs were preincubated with medium or Hsp60 (0.5–20 mg/mL)
for 24 h. Afterward, cells were stimulated with insulin (100 nmol/L) for 10 min.
To investigate the Hsp60-induced activation of signal proteins, cells were
stimulated for 0–60 min with medium, 2.5 nmol/L TNF-a(positive control), or
Hsp60 (1–20 mg/mL). Subsequently, cells were washed with cold PBS, lysed
(1–2 h, 4°C), sonified, and centrifuged (15 min, 10,000g, 4°C). For detection of
activated signal proteins, appropriate antibodies were applied. Signals were
visualized by the Lumi-Imager system.
Quantification of inflammatory mediators. Human subcutaneous adipo-
cytes or SkMCs (1 310
5
cells each) were seeded and differentiated in 48-well
cell culture plates and exposed to control medium, 0.001–20 mg/mL recom-
binant human Hsp60 (StressGen Biotechnologies) or 1 mg/mL LPS. After 24 h,
concentrations of TNF-a, monocyte chemoattractant protein (MCP)-1, regulated
on activation normal T cell expressed and secreted (RANTES), macrophage
inflammatory protein-1a(MIP-1a), IL-6, and IL-8 were measured in cell super-
natants by multiplex beads assay (Luminex, Austin, TX).
Studies on Hsp60 in humans. Hsp60 concentrations were determined by
ELISA(CusabioBiotech)inplasmaobtainedfrom18lean(BMI23.465.6
kg/m
2
, age 56 614 years) and 23 obese (15 without and 8 with type 2 diabetes)
men (BMI 44.5 65.6 kg/m
2
, age 51 614 years) in the fasted state before
undergoing abdominal surgery at Ghent University Hospital. Anthropometric
measurements were performed during preoperative examination. Subjects
gave written informed consent to participate in this study, which was ap-
proved by the Ethical Review Board of the Ghent University Hospital and
conducted according to most recent version of the Declaration of Helsinki.
Adipose tissue lysates from paired subcutaneous and visceral fat of lean and
obese individuals with or without type 2 diabetes were prepared as described
before (14).
Statistical analysis. Data were expressed as means 6SEM. Statistical
analysis was performed using the Student ttest or ANOVA. Correlations were
performed by Pearson product-moment correlation. Statistical analyses were
done with JMP (SAS Institute, Cary, NC) or Prism (GraphPad Software, San
Diego, CA). Differences were considered statistically significant at P,0.05.
RESULTS
Inflammatory stress induces release of Hsp60 by
primary human subcutaneous adipocytes. Human
subcutaneous adipocytes express Hsp60 in comparable
amounts in all differentiation stages (day 0–14) (Fig. 1A).
Mature adipocytes freshly isolated from adipose tissue ex-
press significantly higher amounts of Hsp60 compared with
the stromavascular fraction (Fig. 1B). To examine whether
human subcutaneous adipocytes release Hsp60, a consider-
able amount of cell supernatant, termed “conditioned me-
dium,”was harvested, concentrated 200-fold, and analyzed
by Western blot, with adiponectin as a positive control (Fig.
1C). Hsp60 was already detected in 1 mL concentrated CM
with increasing signal in 3 and 5 mL concentrated CM (Fig.
1C). Of note, Hsp60 concentration in CM was lower than
adiponectin, since Hsp60 (in contrast to adiponectin) was
only detectable by ELISA in concentrated CM. Adipocytes
at day 14 of differentiation released about three times more
Hsp60 than preadipocytes at day 0 (Fig. 1D). Moreover,
human adipocytes were treated with proinflammatory cy-
tokines (TNF-a,IL-1b, and IFN-g, individually or as a mix-
ture) to induce the release of Hsp60 under inflammatory
conditions. The application of single cytokines evoked
asignificant Hsp60 secretion by IL-1b(3.9 60.8-fold) and
TNF-a(3.7 60.3-fold), whereas the effect of IFN-gwas
negligible (1.1 60.3-fold) compared with medium control
(Fig. 1E). Exposure of adipocytes to the cytokine mixture
led to a 3.1 60.9-fold secretion of Hsp60. The application of
single cytokines to human preadipocytes revealed similar
tendencies as for adipocytes (data not shown). Treatment
of adipocytes with LPS as a TLR4 agonist stimulated Hsp60
release, whereas lipoteichoic acid as a TLR2 agonist had no
effect on Hsp60 secretion (Fig. 1F).
Hsp60 binds to primary human subcutaneous adipocytes.
Applying fluorescent-labeled Hsp60 (Hsp60*) to human
preadipocytes and mature adipocytes revealed specific
binding of Hsp60* to both cell populations (Fig. 2). Specificity
was proven through inhibition of Hsp60*-binding by pre-
incubation with the unlabeled ligand (Hsp60) up to 85.0% for
preadipocytes (Fig. 2A) and up to 81.4% for adipocytes (Fig.
2B), whereas incubation with OVA was without any effect.
Hsp60 affects insulin signaling in primary human
subcutaneous adipocytes. Human adipocytes revealed
a dose-dependent significant decrease of insulin-stimulated
Akt phosphorylation up to 55.0 614.1% by 10 mg/mL Hsp60
(Fig. 3A). To elucidate the effect of Hsp60 on Akt phos-
phorylation, different mitogen-activated protein (MAP)
kinases and the NF-kB pathway were investigated (Fig. 3B–
F). Medium and TNF-a(2.5 nmol/L) were used as controls.
A4.660.7-fold activation of the MAP kinase ERK1/2 oc-
curred after a 10-min exposure to Hsp60 (1 mg/mL) (Fig.
3C). JNK activation reached its maximum after 10 min of
incubation with 10 mg/mL Hsp60, since the phosphorylation
increased 2.1 61.2-fold over medium control (Fig. 3D).
Activation of the MAP kinase p38 increased after 30 min (10
mg/mL Hsp60) up to 3.8 60.7-fold (Fig. 3E), whereas the
NF-kB pathway reached its activation peak (4.8 62.8-fold)
already after 10 min at the highest Hsp60 concentration
(Fig. 3F).
Hsp60 induces the release of inflammatory mediators
by primary human subcutaneous adipocytes. Human
adipocytes were exposed to different Hsp60 concentra-
tions (0.001–20 mg/mL) to identify stimulatory Hsp60 con-
centrations and to investigate dose-dependent Hsp60
effects. Hsp60 concentrations ,0.5 mg/mL did not result in
a measurable secretion of proinflammatory mediators
(data not shown). Treatment with Hsp60 (.0.5 mg/mL) led
to a dose-dependent significant secretion of TNF-a(up to
14.0 66.3-fold), RANTES (up to 7.1 62.0-fold), MIP-1a
(up to 15.6 63.5-fold), and IL-8 (up to 15.2 62.4-fold) by
preadipocytes compared with untreated cells (Fig. 4A–F).
In mature adipocytes, Hsp60-stimulated secretion of TNF-a
(up to 20.0 69.1-fold), MCP-1 (up to 22.7 67.3-fold),
RANTES (up to 1,900.0 6302.0-fold), MIP-1a(up to 140.0 6
59.1-fold), IL-6 (up to 32.0 63.5-fold), and IL-8 (up to 2.4 6
0.5-fold) and occurred in a concentration-dependent manner
compared with unstimulated adipocytes (Fig. 4A–F).
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FIG. 1. Hsp60 expression and release by primary human subcutaneous adipocytes. A: The Hsp60 expression in primary human subcutaneous
adipocytes was analyzed at different differentiation time points (day 0–14) with anti-Hsp60 and anti–b-actin antibodies (loading control) by
Western blot analysis. Data represent means 6SEM (n=3–4) and were normalized to b-actin and compared with day 0. B: Mature adipocytes and
stromavascular fraction (SVF) were analyzed for Hsp60 expression. Data represent means 6SEM (n= 3) and were normalized to b-actin. *P<
0.05 vs. stromavascular fraction. C: Cell supernatants of primary human subcutaneous adipocytes were collected and 200-fold concentrated, and
Hsp60 release was investigated by Western blot analysis. Adiponectin was used as a positive control. D: Concentrated supernatants from pre-
adipocytes and adipocytes were analyzed for their Hsp60 content by ELISA. Data represent means 6SEM (n‡4), *P<0.05 vs. day 0. Eand F: Cell
supernatants of cytokine-treated (IFN-g,IL-1b, TNF-a; individually and as a mixture; each 1,000 units/mL) and TLR agonist–treated (LPS 1 mg/mL,
lipoteichoic acid [LTA] 5 mg/mL) adipocytes were analyzed by ELISA. Data represent means 6SEM (n= 3); *P<0.05 vs. medium control.
T. MÄRKER AND ASSOCIATES
diabetes.diabetesjournals.org DIABETES, VOL. 61, MARCH 2012 617

Hsp60 impairs insulin signaling and glucose uptake in
primary human SkMCs. Hsp60-treated human SkMCs
revealed a significant dose-dependent decrease in insulin-
stimulated Akt phosphorylation from 77.3 67.9% (for 1 mg/mL
Hsp60) to 50.3 64.6% (for 20 mg/mL Hsp60) compared
with insulin control (Fig. 5A). Moreover, Hsp60 impaired
insulin-stimulated phosphorylation of GSK3aand GSK3b
(Fig. 5Band C). Our results demonstrate a significantly in-
creased IRS-1 phosphorylation after application of insulin
and Hsp60 (154.1 612.4-fold) compared with insulin alone
(126.8 67.7-fold; Fig. 5D). Furthermore, Hsp60 exposure to
human SkMCs significantly decreased insulin-stimulated
glucose uptake (2,477.0 629.3 cpm/well) compared with
insulin-stimulated control (2,731.0 6134.2 cpm/well; Fig.
5E). Hsp60 treatment increases basal glucose uptake com-
pared with control (1,491 cpm compared with 1,292 cpm).
In previous studies, we also observed that SkMCs treated
with adipocyte-CM display increased basal glucose uptake.
Because both Hsp60 and adipocyte CM activate inflam-
matory and stress signaling pathways, one might suggest
that activation of these pathways is responsible for this
effect. To elucidate the role of Hsp60 in the activation of
MAP kinases, Hsp60 and TNF-awere applied to SkMCs
(Fig. 6A). Maximal activation of the MAP kinases ERK1/2
and JNK was reached after 30 min of incubation with 20
mg/mL Hsp60 (2.4 60.3-fold increase of ERK1/2 and 2.4 6
0.2-fold increase of JNK activation above medium con-
trol;Fig.6A–C). The NF-kB pathway was activated in
a more prolonged manner, with a maximal activation
(2.3 60.5-fold over medium control) after 60 min of ex-
posure to 20 mg/mL Hsp60 (Fig. 6D). Activation of the
MAP kinase p38 by Hsp60 could not be observed (data
not shown).
Hsp60 is expressed, but not released, from human
SkMCs. Immunoblot analysis of unstimulated SkMC lysates
revealed a consistent expression of Hsp60 in all analyzed
maturation states (day 0–8; Fig. 7A). Hsp60 expression in
SkMCs is donor dependent and slightly, but not significantly,
lower than in adipocytes (data not shown). To investigate if
human SkMCs themselves serve as a source for extracellu-
lar Hsp60, cell supernatants of unstimulated SkMCs were
generated, concentrated to the same extent as the CM from
adipocytes, and analyzed by ELISA. Hsp60 release by hu-
man SkMCs was not detectable.
Hsp60 binds to human SkMCs and induces the release
of cytokines. Hsp60* was applied to human myoblasts
and found to bind specifically to these cells (Fig. 7B). The
Hsp60* binding signal was drastically reduced (81.6% in-
hibition) only in the presence of unlabeled Hsp60, but not
with OVA as the control (Fig. 7B). Human SkMCs were
exposed to different Hsp60 concentrations (0.001–20 mg/mL),
and measurable cytokine levels resulted from Hsp60 con-
centrations $0.5 mg/mL. Hsp60 (20 mg/mL) induced a sig-
nificant release of MCP-1 (1.2 60.2 ng/mL) compared with
medium control (0.1 60.0 ng/mL), IL-8 (1.7 60.4 ng/mL)
compared with medium control (0.1 60.0 ng/mL), and IL-6
(0.4 60.1 ng/mL) compared with medium control (0.1 6
0.0 ng/mL) from human SkMCs (Fig. 7C–E).
Hsp60 levels are elevated in plasma of obese
individuals. Plasma Hsp60 levels were higher in obese
(20.3 611.0 ng/mL Hsp60) than in lean (12.6 611.0 ng/mL
Hsp60) men (Fig. 8A; for clinical characterization of patients,
see Supplementary Table 1).Within the obese group, Hsp60
levels did not differ between males with (n= 13) or without
(n= 8) type 2 diabetes (data not shown). Nevertheless,
plasma Hsp60 related weakly but positively with BMI (P=
0.04; r= 0.34), diastolic (P= 0.04; r= 0.35) and systolic
(P=0.03;r= 0.35) blood pressure, plasma leptin (P=0.04;
r= 0.34), and homeostasis model assessment–insulin re-
sistance (HOMA-IR) (P=0.05;r= 0.35) and inversely with
quantitative insulin sensitivity check index (QUICKI) (P=
0.04; r= 0.38). Hsp60 expression in visceral relative to
subcutaneous adipose tissue was greater in obese patients
with type 2 diabetes (n=8)thaninobese(n= 7) or lean
(n= 9) patients without type 2 diabetes (Fig. 8B).
DISCUSSION
The current study demonstrates that Hsp60 is a novel
adipokine that could contribute to inflammatory processes
in an autocrine manner within adipose tissue and to the
development of peripheral insulin resistance in an endo-
crine fashion. The observation that Hsp60 is not only
expressed but also released clearly indicates that Hsp60 is
an intracellular chaperone and also a secretion product.
Other heat shock proteins can also be secreted from viable
cells such as cardiomyocytes, glial cells, and peripheral
blood mononuclear cells (15,16). To date, the origin of
FIG. 2. Hsp60 binding to primary human subcutaneous adipocytes. Aand B: Preadipocytes and mature adipocytes were incubated with 100 nmol/L
Hsp60-DyLight649 (Hsp60*) in the absence or presence of 1 mmol/L unlabeled Hsp60 or OVA. Fluorescence intensities of the cells were plotted
against cell counts and determined by fluorescence-activated cell sorter analysis.
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FIG. 3. Effect of Hsp60 on insulin signaling in primary human subcutaneous adipocytes. A: Human adipocytes were treated with medium or dif-
ferent Hsp60 concentrations (0.5–10 mg/mL) for 24 h. After stimulation with insulin (100 nmol/L, 10 min), total cell lysates were analyzed for Akt
activation. The relative Akt phosphorylation after insulin stimulation was set at 100%. Lanes were excised from a single Western blot and dis-
played in the presented order. B: Representative Western blots of p-ERK1/2, p-JNK, p-p38, p-NFkB after stimulation with medium, Hsp60, or TNF-a.
b-Actin was used for normalization. C–F: Human adipocytes were treated with medium, Hsp60 (1 and 10 mg/mL), or TNF-a(2.5 nmol/L) for 0–60
min. Total cell lysates were analyzed for activation of the MAP kinases ERK1/2, JNK, p38, and NF-kB. Data represent the means 6SEM of three
independent experiments, were normalized to b-actin, and were compared with medium; *P<0.05; **P<0.01 vs. the corresponding insulin-
stimulated control (A) and medium control (C–F), respectively. ■, Medium control; ▲,1mg/mL Hsp60; D,10mg/mL Hsp60.
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circulating Hsp60 remains elusive, but adipose tissue might
be considered as one of the sources. It has been reported
that circulating levels of Hsp60 were elevated in subjects
with inflammatory diseases such as arthritis, atherosclerosis,
or type 2 diabetes (9,17,18). Here, we demonstrate that
obese patients with or without diabetes also have higher
plasma Hsp60 concentrations, suggesting that adipose
tissue is a possible source of circulating Hsp60. Analysis of
Hsp60 levels in concentrated cell culture supernatants con-
firmed adipocytes as a putative origin of circulating Hsp60,
since Hsp60 was mainly released from untreated human
adipocytes, in a lesser extent from preadipocytes, but not
from human SkMCs. The latter could be explained by
Hsp60 concentrations below the detection limit, even in
highly concentrated CM from SkMCs. However, further
investigations are needed to clarify Hsp60 sources in vivo.
Furthermore, we simulated inflammation by applying a
cytokine mixture to human adipocytes, to induce the re-
lease of Hsp60. To elucidate the individual contribution of
these cytokines to these processes, cells were also exposed
to each cytokine individually. Hsp60 secretion was ob-
served after the application of TNF-aand IL-1bin com-
parable amounts to that obtained after incubation with
the cytokine mixture, but not after IFN-gtreatment. These
results indicate that macrophages/monocytes and adipo-
cytes themselves might be an inducer of Hsp60 release by
adipocytes, since they have been described as sources of
TNF-aand IL-1b(7,19). Besides IL-1band TNF-a, other
stimuli for the regulation of Hsp60 expression might be
considered. Increased Hsp60 expression was reported for
primary human astrocytes in response to cytokines as
diverse as IL-1band TNF-abut also IL-4, IL-6, and IL-10
(20).
In general, the amounts of Hsp60 released by adipocytes
are relatively low and not measurable in unconcentrated
CM by ELISA, a problem that is often encountered in
unconcentrated cell culture supernatants. In fact, various
known adipokines that are found in high concentrations in
the circulation are relatively low in adipocyte CM (21).
Nevertheless, it can be hypothesized that extracellular
levels of Hsp60 mediating auto- and paracrine effects
might be significantly higher. Furthermore, the fact that
circulating Hsp60 levels are higher in obese subjects than
in lean control subjects and that Hsp60 concentrations are
correlated with circulating leptin further supports the as-
sumption that adipocytes are a source for circulating
Hsp60 in vivo.
Hsp60-binding studies revealed that Hsp60 binds spe-
cifically and in a dose-dependent manner to adipocytes,
representing the typical characteristics of a ligand-receptor
interaction. These findings are consistent with results
obtained from the murine adipocyte cell line 3T3-L1 (12).
Attempts to characterize the Hsp60 receptor structure(s)
on innate immune cells identified TLR2, TLR4, and CD14
as components responsible for the proinflammatory ef-
fects of Hsp60 (22–25). Activation of TLR4 but not TLR2,
FIG. 4. Hsp60-induced release of inflammatory mediators from primary human subcutaneous adipocytes. Human preadipocytes and mature adi-
pocytes remained untreated (medium control) or were exposed to increasing concentrations of Hsp60 (0.5–20 mg/mL) or LPS (1 mg/mL). After 24 h,
TNF-a(A), MCP-1 (B), RANTES (C), MIP-1a(D), IL-6 (E), and IL-8 (F) concentrations were determined in cell culture supernatants by
multiplex-beads assay. The data show means 6SEM from three independent experiments; *P<0.05; **P<0.01 vs. the corresponding medium
control.
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FIG. 5. Effect of Hsp60 on insulin signaling in human SkMCs. A–C: SkMCs were treated with medium or Hsp60 (1–20 mg/mL) for 24 h. After
stimulation with insulin (100 nmol/L, 10 min), total cell lysates were analyzed for Akt (A) and GSK3a/b(Band C) activation. The relative Akt and
GSK3a/bphosphorylation, respectively, after insulin stimulation was set at 100%. Data represent means 6SEM (n=3–6), were normalized to
tubulin, and were compared with the insulin-stimulated control; *P<0.05; **P<0.01; ***P<0.001 vs. the corresponding insulin-stimulated
control. D–E: Skeletal muscle cells were cultured for 24 h in the absence or presence of Hsp60 (20 mg/mL). IRS-1 phosphorylation (D) and glucose
uptake (E) were assessed after acute stimulation with insulin, as outlined in RESEARCH DESIGN AND METHODS. Means 6SEM of three to four in-
dependent experiments are shown; *P<0.05 vs. insulin-stimulated control.
T. MÄRKER AND ASSOCIATES
diabetes.diabetesjournals.org DIABETES, VOL. 61, MARCH 2012 621

on the other hand, induces the release of Hsp60 from
adipocytes. The identity of the Hsp60 receptor complex on
human adipocytes, however, remains elusive.
Because Hsp60 can be released from human sub-
cutaneous adipocytes and moreover binds specifically to
human adipocytes, autocrine effects of Hsp60 can be as-
sumed. We obtained Hsp60-mediated activation of the MAP
kinases ERK1/2, JNK, and p38 and the transcription factor
NF-kB in human adipocytes. The activation of similar MAP
kinase patterns has been described for other adipokines,
such as chemerin and MCP-1 (26), suggesting that different
adipokines are capable of inducing similar proinflammatory
responses. Moreover, Hsp60 induced secretion of proin-
flammatory mediators. All these Hsp60-mediated effects
may indicate that Hsp60 could be involved in adipose tissue
inflammation by both inducing acute proinflammatory sig-
naling and an enhanced release of proinflammatory media-
tors. The resulting elevated adipokine levels, e.g., for TNF-a,
IL-6, or MCP-1, are well described to be obesity-related
and may indirectly induce the development of insulin
FIG. 6. Impact of Hsp60 on the activation of signaling pathways in human SkMCs. A–D: Human SkMCs were treated with medium, Hsp60 (10 and 20
mg/mL), or TNF-a(2.5 nmol/L) for 0–60 min. Representative Western blots of total cell lysates for activation of the MAP kinases ERK1/2 (B), JNK
(C), and NF-kB(D) are depicted in A. Data represent means 6SEM of three independent experiments, were normalized to tubulin, and were
compared with the unstimulated control; *P<0.05; **P<0.01 vs. the corresponding unstimulated medium control. ■,Mediumcontrol;D,10mg/mL
Hsp60; ▲,20mg/mL Hsp60; ●, 2.5 nmol/L TNF-a.
Hsp60 AND INSULIN RESISTANCE
622 DIABETES, VOL. 61, MARCH 2012 diabetes.diabetesjournals.org

resistance in human adipocytes and other insulin-sensitive
tissues (1,27). The first evidence that Hsp60 might con-
tribute to these processes is given, since Hsp60 provoked
a significant decrease of the insulin-stimulated Akt and
GSK3a/bphosphorylation in human adipocytes (Akt) and
SkMCs (Akt, GSK3a/b), thereby triggering the develop-
ment of an insulin resistance. These findings were con-
firmed by our results demonstrating significant effects of
Hsp60 on IRS-1 phosphorylation as well as Hsp60-mediated
impairment of glucose uptake in human SkMCs, indicating
Hsp60 as a putative mediator in the development of insulin
resistance. Therefore, Hsp60 might have direct effects on
insulin signaling and also may indirectly induce insulin re-
sistance by increasing proinflammatory adipokines, known
to interfere with insulin signaling.
There are several putative mechanisms by which Hsp60
and cytokine concentrations could reach pathophysiolog-
ical levels in obese and/or type 2 diabetic patients. Heat
shock proteins are involved in the activation of innate
immune cells and in the resulting macrophage-infiltration
of adipose tissue by the release of chemokines such as
MCP-1 (5,6,28). Macrophage infiltration positively corre-
lated with increased adipocyte size and body mass in hu-
man subcutaneous tissue, leading to elevated cytokine
levels, which finally may contribute to the development of
insulin resistance in adipocytes (29,30). Therefore, besides
adipose tissue macrophages, adipocytes themselves must
be considered as important players in the development of
obesity-related insulin resistance (31). However, other
mechanisms such as obesity-related hypoxia might con-
tribute to inflammatory processes in adipose tissue. Pre-
viously, it was depicted that hypoxia is associated with an
increased Hsp60 expression in human vessels (32) and that
Hsp60 was translocated to the plasma membrane in the
heart (33). Because hypoxia has been reported to occur in
obese individuals (34), one might speculate that hypoxic
conditions could contribute to elevated Hsp60 expression
levels, possibly leading to an increased release of Hsp60
observed in obese individuals.
Several studies indicate that circulating Hsp60 levels are
increased not only in patients with type 2 diabetes but also
in patients with coronary heart disease (35). In vitro
studies further underline that Hsp60 has endocrine effects
on cardiomyocytes. Hsp60 was found to be increased early
in heart failure accompanied by increased release from
cardiomyocytes, where it induces apoptosis via TLR4
(36,37). Furthermore, Hsp60 induces proliferation of vas-
cular smooth muscle cells (38), which might also contrib-
ute to cardiovascular disease (39). In the current study, we
revealed a positive association between elevated Hsp60
concentrations and blood pressure, which might contrib-
ute to the development of cardiovascular diseases. Our
study is the first demonstrating that Hsp60 might also be
a relevant mediator for skeletal muscle and adipose tissue
insulin resistance, thereby contributing to the develop-
ment of type 2 diabetes. In an acute way, Hsp60 activates
proinflammatory signaling cascades in primary human
FIG. 7. Hsp60 binding capacity and Hsp60 reactivity to human SkMCs.
A: Hsp60 expression in SkMCs was investigated at different differen-
tiation time points (day 0–8) with antibodies directed against Hsp60
and tubulin (loading control) by Western blot analysis. B: Human
SkMCs were incubated with 100 nmol/L Hsp60-DyLight649 (Hsp60*) in
the absence or presence of 1 mmol/L unlabeled Hsp60 or OVA. Fluo-
rescence intensities of the cells were plotted against cell counts and
determined by fluorescence-activated cell sorter analysis. C–E: SkMCs
were treated with medium, increasing Hsp60 concentrations (0.5–20
mg/mL) or LPS (1 mg/mL). After 24 h, MCP-1 (C), IL-8 (D), and IL-6 (E)
concentrations were measured in cell culture supernatants by multiplex-
beads assay. The data show means 6SEM from three independent
determinations; *P<0.05; ***P<0.001 vs. the corresponding me-
dium control.
T. MÄRKER AND ASSOCIATES
diabetes.diabetesjournals.org DIABETES, VOL. 61, MARCH 2012 623

SkMCs similarly to MCP-1 and chemerin (10,26). Pro-
longed incubation with Hsp60 induces insulin resistance at
the level of Akt and GSK3a/bin these cells at both phys-
iological and pathophysiological concentrations. These
findings could be verified through Hsp60-mediated im-
paired glucose uptake in SkMCs and a positive association
between Hsp60 concentration and HOMA-IR, as well as
a negative correlation with QUICKI. Most interestingly,
Hsp60 treatment of SkMCs results in a marked secretion of
myokines such as MCP-1, which is associated with skeletal
muscle inflammation (40). Increased release of MCP-1 in
Hsp60-treated SkMCs is in line with a reported increase in
MCP-1 secretion in these cells after stimulation with adi-
pocyte CM (41). Because Hsp60 is released by adipocytes,
Hsp60 content in adipocyte CM might at least partly ex-
plain its effect on MCP-1 release.
In summary, inflammatory stress induces the release of
Hsp60 by human adipocytes, and Hsp60 exerts autocrine/
paracrine effects on adipocytes characterized by an in-
creased release of proinflammatory adipokines, increased
inflammatory signaling, and insulin resistance. Further-
more, the current study reveals that Hsp60 has endocrine
effects on SkMCs, inducing insulin resistance. Our clinical
data reveal positive associations of circulating Hsp60 con-
centrations with BMI, leptin, HOMA-IR, and blood pres-
sure. Therefore, there is rising evidence that circulating
Hsp60 levels are increased in obesity, leading to the con-
ception that Hsp60 might represent a novel adipokine in-
volved in adipose tissue inflammation, thereby contributing
to the development of insulin resistance in human adipo-
cytes and SkMCs.
ACKNOWLEDGMENTS
This work was supported by the Bundesministerium für
Gesundheit and by the Ministerium für Innovation, Wissen-
schaft, Forschung und Technologie des Landes Nordrhein-
Westfalen, in part by a grant from the Bundesministerium
für Bildung und Forschung (BMBF) to the German Center
for Diabetes Research (DZD e.V.), European Union COST
(European Cooperation in Science and Technology), the
Commission of the European Communities (Collaborative
Project ADAPT [Adipokines as Drug Targets to Combat
Adverse Effects of Excess Adipose Tissue]), and the Deut-
sche Forschungsgemeinschaft (DFG).
No potential conflicts of interest relevant to this article
were reported.
T.M. and H.S. researched data and wrote the manu-
script. P.Z., A.G., J.K., and S.F. researched data. D.M.O.,
P.P., and J.R. performed clinical research. T.M., H.S., and
C.H. designed and initiated the experimental procedures.
M.R., J.E., and C.H. contributed to discussion and reviewed
the manuscript. C.H. is the guarantor of this work and, as
such, had full access to all of the data in the study and takes
responsibility for the integrity of the data and the accuracy
of the data analysis.
The authors thank Professor Jutta Liebau, Department
of Plastic Surgery, Florence-Nightingale-Hospital Düsseldorf,
and Dr. Christoph Andree, Department of Plastic Surgery,
Sana-Hospital Düsseldorf-Gerresheim, for support in ob-
taining adipose tissue samples. The technical assistance of
Jutta Brüggemann, Angelika Horrighs, Andrea Cramer, and
Manuela Elsen is gratefully acknowledged.
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