Osteopontin deficiency protects against obesity-induced hepatic steatosis and attenuates glucose production in mice.

Abstract

Obesity is strongly associated with the development of non-alcoholic fatty liver disease (NAFLD). The cytokine osteopontin (OPN) was recently shown to be involved in obesity-induced adipose tissue inflammation and reduced insulin response. Accumulating evidence links OPN to the pathogenesis of NAFLD. Here we aimed to identify the role of OPN in obesity-associated hepatic steatosis and impaired hepatic glucose metabolism.
Wild-type (WT) and Opn (also known as Spp1) knockout (Opn (-/-)) mice were fed a high-fat or low-fat diet to study OPN effects in obesity-driven hepatic alterations.
We show that genetic OPN deficiency protected from obesity-induced hepatic steatosis, at least in part, by downregulating hepatic triacylglycerol synthesis. Conversely, absence of OPN promoted fat storage in adipose tissue thereby preventing the obesity-induced shift to ectopic fat accumulation in the liver. Euglycaemic-hyperinsulinaemic clamp studies revealed that insulin resistance and excess hepatic glucose production in obesity were significantly attenuated in Opn (-/-) mice. OPN deficiency markedly improved hepatic insulin signalling as shown by enhanced insulin receptor substrate-2 phosphorylation and prevented upregulation of the major hepatic transcription factor Forkhead box O1 and its gluconeogenic target genes. In addition, obesity-driven hepatic inflammation and macrophage accumulation was blocked by OPN deficiency.
Our data strongly emphasise OPN as mediator of obesity-associated hepatic alterations including steatosis, inflammation, insulin resistance and excess gluconeogenesis. Targeting OPN action could therefore provide a novel therapeutic strategy to prevent obesity-related complications such as NAFLD and type 2 diabetes.

Full text

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ARTICLE
Osteopontin deficiency protects against obesity-induced
hepatic steatosis and attenuates glucose production in mice
F. W. Kiefer & S. Neschen & B. Pfau & B. Legerer & A. Neuhofer & M. Kahle &
M. Hrabé de Angelis & M. Schlederer & M. Mair & L. Kenner & J. Plutzky & M. Zeyda &
T. M. Stulnig
Received: 19 December 2010 /Accepted: 4 April 2011 /Published online: 12 May 2011
# The Author(s) 2011. This article is published with open access at Springerlink.com
Abstract
Aims/hypothesis Obesity is strongly associated with the
development of non-alcoholic fatty liver disease (NAFLD).
The cytokine osteopontin (OPN) was recently shown to be
involved in obesity-induced adipose tissue inflammation
and reduced insulin response. Accumulating evidence links
OPN to the pathogenesis of NAFLD. Here we aimed to
identify the role of OPN in obesity-associated hepatic
steatosis and impaired hepatic glucose metabolism.
Methods Wild-type (WT) and Opn (also known as Spp1)
knockout (Opn−/−) mice were fed a high-fat or low-fat diet
to study OPN effects in obesity-driven hepatic alterations.
Results We show that genetic OPN deficiency protected
from obesity-induced hepatic steatosis, at least in part, by
downregulating hepatic triacylglycerol synthesis. Converse-
ly, absence of OPN promoted fat storage in adipose tissue
thereby preventing the obesity-induced shift to ectopic fat
accumulation in the liver. Euglycaemic–hyperinsulinaemic
clamp studies revealed that insulin resistance and excess
hepatic glucose production in obesity were significantly
attenuated in Opn−/−mice. OPN deficiency markedly
improved hepatic insulin signalling as shown by enhanced
insulin receptor substrate-2 phosphorylation and prevented
upregulation of the major hepatic transcription factor
Forkhead box O1 and its gluconeogenic target genes. In
addition, obesity-driven hepatic inflammation and macro-
phage accumulation was blocked by OPN deficiency.
Conclusions/interpretation Our data strongly emphasise
OPN as mediator of obesity-associated hepatic alterations
including steatosis, inflammation, insulin resistance and
excess gluconeogenesis. Targeting OPN action could
therefore provide a novel therapeutic strategy to prevent
obesity-related complications such as NAFLD and type 2
diabetes.
Keywords Gluconeogenesis.High-fat diet.Inflammation.
Insulin resistance.Non-alcoholic fatty liver disease
Abbreviations
2-[14C]DG
ALT
FOXO1
GINF
GWAT
HF
ITT
LF
2-Deoxy-D-[1-14C]glucose
Alanine aminotransferase
Forkhead box O1
Glucose infusion rate
Gonadal white adipose tissue
High-fat diet
Insulin tolerance test
Low-fat diet
Electronic supplementary material The online version of this article
(doi:10.1007/s00125-011-2170-0) contains supplementary material,
which is available to authorised users.
F. W. Kiefer:B. Pfau:B. Legerer:A. Neuhofer:M. Zeyda:
T. M. Stulnig (*)
Department of Medicine III,
Clinical Division of Endocrinology and Metabolism,
Medical University of Vienna,
Waehringer Guertel 18-20,
1090 Vienna, Austria
e-mail: thomas.stulnig@meduniwien.ac.at
S. Neschen:M. Kahle:M. Hrabé de Angelis
Institute of Experimental Genetics,
Helmholtz Zentrum Muenchen,
German Research Center for Environmental Health,
Neuherberg, Germany
M. Schlederer:M. Mair:L. Kenner
Ludwig Boltzmann Institute for Cancer Research,
Vienna, Austria
J. Plutzky
Department of Medicine, Brigham and Women’s Hospital Boston,
Cardiovascular Division, Harvard Medical School,
Boston, MA, USA
Diabetologia (2011) 54:2132–2142
DOI 10.1007/s00125-011-2170-0

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MCP-1
NAFLD
NASH
NF-κB
OPN
PPAR
PGC
Monocyte chemoattractant protein-1
Non-alcoholic fatty liver disease
Non-alcoholic steatohepatitis
Nuclear factor κB
Osteopontin
Peroxisome proliferator-activated receptor
Peroxisome proliferator-activated receptor
coactivator
Serum amyloid P
Sterol regulatory element-binding protein 1
Subcutaneous white adipose tissue
Wild-type
SAP
SREBP-1c
SWAT
WT
Introduction
The obesity epidemicistightlylinkedtoa spectrumofhepatic
disorders collectively known as non-alcoholic fatty liver
disease (NAFLD). NAFLD has become an important public
health issue because of its high prevalence and association
with serious cardiometabolic abnormalities, including meta-
bolicsyndrome, type2 diabetesand coronaryheart disease [1,
2]. NAFLD spans a spectrum from simple hepatic steatosis
through non-alcoholic steatohepatitis (NASH) ultimately
leading to liver fibrosis and cirrhosis [3].
Increased energy intake exceeding energy dissipation
promotes ectopic triacylglycerol storage in non-fat organs
such as skeletal muscle and liver [4]. Triacylglycerol
homeostasis in the liver is regulated by a complex interplay
between hepatic plasma NEFA uptake and de novo fatty
acid and triacylglycerol synthesis (lipogenesis) on the one
hand, as well as fatty acid oxidation and triacylglycerol
export by VLDLs on the other hand. Hepatic steatosis
develops when the rate of hepatic fatty acid input (uptake
and synthesis) exceeds the rate of fatty acid output
(oxidation and secretion) [5].
Steatosis is associated with hepatic insulin resistance, i.e.
the reduced sensitivity of the liver to the suppressive effects
of insulin on glucose and VLDL triacylglycerol production.
Decreased ability of insulin to suppress the hepatic output
of glucose and VLDL contributes to hyperglycaemia and
hyperlipidaemia, intrinsic features of the metabolic syn-
drome. As such, hepatic steatosis is regarded as the hepatic
component of the metabolic syndrome [4]. Insulin resis-
tance in liver, adipose tissue, and skeletal muscle is directly
related to intrahepatic triacylglycerol content. However, it
still remains a matter of debate whether insulin resistance is
a cause or consequence of NAFLD [6, 7].
In addition to lipid accumulation, another hallmark of
NAFLD is hepatic inflammation resulting in NASH.
Similar to its effects on adipose tissue, obesity induces
inflammatory alterations in the liver as reflected by a higher
abundance of immune cells such as macrophages [8],
increased cytokine production [3] and by activation of the
nuclear factor κB (NF-κB) pathway [9]. Adipose tissue
inflammation and unfavourable adipokine secretion are
enhanced in individuals with NAFLD compared with
individuals with normal intrahepatic triacylglycerol content
[10], indicating that inflammatory factors released by the
adipose tissue could be involved in the pathogenesis of
NAFLD and hepatic insulin resistance [11]. Only recently
liver macrophages were reported to be increased in number
and to promote hepatic steatosis and insulin resistance in
obesity [12]. Chemokines such as monocyte chemoattrac-
tant protein-1 (MCP-1) contribute to obesity-induced
recruitment of macrophages to the liver [12, 13].
Osteopontin (OPN) is a multifunctional protein produced
in numerous cells including activated macrophages and T
cells, osteoclasts, smooth muscle, endothelial cells, and also
hepatocytes [14, 15]. OPN induces the production of a
variety of proinflammatory cytokines and chemokines in
peripheral blood mononuclear cells [16] and supports
migration of monocytes/macrophages [15]. Systemic con-
centrations as well as adipose content of OPN are
significantly elevated in obese patients and mice [17, 18].
IthasrecentlybeensuggestedthatgeneticOPNdeficiencyand
antibody-mediated neutralisation improve obesity-associated
adipose tissue inflammation and glucose tolerance [19, 20].
Notably, OPN content is also markedly upregulated in the
liver in obesity, and hepatic OPN levels correlate with liver
triacylglycerol content [18, 21–23]. Within the liver, OPN is
predominantly produced in inflammatory cells but also
hepatocytes [24]. A role for OPN in the pathogenesis of
NAFLD was suggested in mice fed a methionine- and
choline-deficient diet [22, 23]. Recently, antibody-mediated
OPN neutralisation was shown to protect against high-fat
diet-induced hepatic macrophage infiltration [19] and D-
galactosamine-induced inflammatory liver injury [24]. How-
ever, a functional role of OPN in obesity-associated hepatic
steatosis and liver insulin sensitivity still remains unclear.
Here we show that genetic OPN deficiency prevents high-
fat diet-induced hepatic lipid accumulation and enhances
hepatic insulin action to suppress gluconeogenesis. Obesity-
driven liver inflammation, as well as deleterious signal
transduction pathways related to hepatic insulin resistance, is
attenuated in obese Opn (also known as Spp1)−/−mice.
Hence, targeting OPN action in vivo could provide a novel
therapeutic strategy to combat obesity-associated hepatic
steatosis and related metabolic complications.
Methods
Animals and diet C57BL/6J wild-type (WT) and B6.Cg-
Spp1tm1Blh/J (here referred to as Opn knockout [Opn−/−])
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mice were purchased from Charles River Laboratories
(Sulzfeld, Germany). At 7 weeks of age, male littermates
were placed for 24 weeks on a high-fat (HF, 60% of energy
from fat, D12492, Research Diets, New Brunswick, NJ,
USA) and a low-fat diet (LF, 10% of energy from fat,
D12450B, Research Diets) to induce obesity or to serve as
lean controls, respectively. All mice were housed in a
specific pathogen-free facility that maintained a 12 h light/
dark cycle. Mice had free access to food and water and food
intake was monitored. The protocol was approved by the
local ethics committee for animal studies and the Federal
Ministry for Science and Research and followed the
guidelines on accommodation and care of animals formu-
lated by the European Convention for the Protection of
Vertebrate Animals Used for Experimental and Other
Scientific Purposes.
Insulin treatment in mice WT and Opn−/−mice were kept
on HF for 24 weeks. After 6 h fasting, mice were
intraperitoneally injected with 1.5 U/kg insulin (Actrapid,
Novo Nordisk, Bagsværd, Denmark) and saline, respec-
tively, 15 min prior to being killed (n=5 per group). The
liver was removed and immediately snap frozen for later
immunoblot analyses.
Metabolic measurements Plasma glucose, triacylglycerol,
and NEFA concentrations were measured in EDTA plasma
using an automated analyser (Falcor 350, A. Menarini
Diagnostics, Florence, Italy). We used commercially avail-
able ELISA kits to determine plasma insulin (Mercodia AB,
Uppsala, Sweden) and serum amyloid P (SAP) (Alpco
Diagnostics, Windham, NH, USA). Plasma concentrations
of alanine aminotransferase (ALT) were measured using the
Reflotron analysis system (Roche, Mannheim, Germany).
Insulin tolerance tests (ITTs) were performed after a 3 h
fasting period. Blood glucose concentrations were mea-
sured before and 30, 60, 90 and 120 min after an
intraperitoneal injection of recombinant human insulin
(Actrapid, Novo Nordisk, 0.75 U/kg body weight for the
HF group and 0.25 U/kg for the LF group, respectively).
Euglycaemic–hyperinsulinaemic clamp studies WT and
Opn−/−mice were fed a HF diet for 24 weeks in order to
induce obesity. One week prior to clamp experiments an
intravenous silicone catheter was inserted into the right
jugular vein of weight-matched mice (n=10 per group).
Mice were overnight fasted prior to the clamp procedure. In
vivo experiments lasted for 240 min and consisted of a
120 min basal period followed by a 120 min euglycaemic–
hyperinsulinaemic clamp as previously described [25, 26].
Briefly, the clamp period was initiated by a 600 pmol/kg,
3 min prime, followed by a 36 pmol kg−1min−1continuous
infusion of insulin (Huminsulin Normal, Lilly Deutschland,
Bad Homburg, Germany) raising plasma insulin concen-
trations within a physiological range. By means of a
variable 20% glucose infusion ‘steady state’ (minute 90–
120) conditions for plasma glucose concentration and
specific activity were achieved. At minute 70, a single
intravenous 2-deoxy-D-[1-14C]glucose (2-[14C]DG,
370 kBq) injection was administered. Plasma samples for
determination of insulin-stimulated plasma [3H3]glucose,
3H2O and 2-[14C]DG concentrations were collected at t=
77.5, 80, 85, 90, 100, 110, and 120 min of the glucose
clamp, and basal [3H3]glucose concentrations in the final
10 min of the basal period. At the end of each experiment,
organs (liver, gonadal white adipose tissue [GWAT], and M.
gastrocnemius) were immediately dissected, and snap
frozen.
Tissue 2-[14C]DG uptake 2-[14C]DG injected during the
steady state conditions of the euglycaemic–hyperinsulinae-
mic clamp experiments resulted in intracellular accumula-
tion of 2-[14C]DG-6-phosphate, which was separated from
2-[14C]DG using ion-exchange columns (Bio-Rad, Hercu-
les, CA, USA). Tissue 2-[14C]DG uptake was calculated
from the plasma 2-[14C]DG AUC and tissue 2-[14C]DG-6-
phosphate content as previously described [26].
Determination of liver triacylglycerol content Liver triacyl-
glycerol was determined following lipid extraction as de-
scribed previously [27], modified by using a commercially
available enzymatic reagent (A. Menarini Diagnostics).
Histology and immunohistochemistry Liver samples were
fixed with neutral buffered 4% paraformaldehyde and
subsequently paraffin embedded. Haematoxylin–eosin
staining was performed in liver as described [22]. NAFLD
activity scores were determined by a certified pathologist
blinded to mouse genotype and dietary intervention. After
dewaxing and rehydration, immunohistochemical staining
for MAC-2 (Serotec, Oxford, UK) was performed using the
ABC kit (Vector Laboratories, Burlingame, CA, USA)
according to the manufacturer’s recommendations. As a
negative control, staining was performed on selected
sections with isotype control. Samples were analysed with
standard light microscopy.
Immunoprecipitation and immunoblotting Standard immu-
noblotting techniques were applied as described previously
[28] using specific antibodies against forkhead box O1
(FOXO1; Santa Cruz Biotechnology, Santa Cruz, CA,
USA), thymoma viral proto-oncogene 1 (AKT) and pAKT
(ser473) (Cell Signaling, Danvers, MA, USA). IRS-2 was
immunoprecipitated from liver tissue extracts (anti-IRS-2
polyclonal antibody, Cell Signaling). IRS-2 tyrosine phos-
phorylation was determined using anti-phosphotyrosine
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mAb (4G10) horse radish peroxidase (HRP) conjugate
(Upstate, Lake Placid, NY, USA).
Reverse transcription and gene expression Adipose tissue
and liver was homogenised in TRIzol reagent (Invitrogen,
Carlsbad, CA, USA) and RNA was isolated and transcribed
to cDNA according to the manufacturer’s protocol [19].
Gene expression normalised to Ubiquitin C was analysed
by quantitative real-time RT-PCR on an ABI Prism 7000
cycler using commercial Assays-on-Demand kits (all
Applied Biosystems, Foster City, CA, USA). Gene expres-
sion was compared with WT HF, which was considered to
be 100%.
Statistics All data are given as means±SE. All data were
normally distributed by visual inspection. Comparisons
between genotypes on the same diet were assessed by
unpaired two-tail Student’s t tests. Multiple regression
analysis was performed to evaluate association between
liver and GWAT weight in WT and Opn−/−mice. A p value
of 0.05 or less was considered to be statistically significant.
Results
OPN deficiency protects from obesity-induced hepatic
steatosis To assess the role of OPN in obesity-associated
NAFLD and hepatic insulin resistance, WT and Opn−/−
mice were fed either the HF or LF diet for 24 weeks to
induce obesity or to serve as lean controls, respectively.
Whereas OPN deficiency did not affect body weight gain or
feed efficiency, liver weight was significantly lower in
obese Opn−/−mice compared with obese WT mice
(Table 1). Systemic concentrations of glucose, triacylgly-
cerols and NEFA did not differ between the two genotypes,
while the obesity-induced rise in circulating plasma insulin
was drastically blunted in Opn−/−mice to one third of the
levels measured in WT mice. ALT, a marker of hepatocyte
damage, was similarly reduced in obese Opn−/−mice
(Table 1). HF feeding induced severe lipid accumulation
in livers of WT mice as determined by liver histology and
quantification of hepatic triacylglycerol content. Strikingly,
Opn−/−mice on HF were markedly protected from diet-
induced hepatic steatosis (Fig. 1)
OPN deficiency counteracts hepatic insulin resistance and
excessive glucose production We examined whether lack of
OPN affects obesity-associated hepatic insulin resistance
and gluconeogenesis. First we performed ITTs in WT and
Opn−/−mice showing that the insulin effect on plasma
glucose was comparable in lean mice irrespective of the
genotype. Notably, glucose clearance following insulin
injection in obese HF-fed animals was significantly acceler-
ated in Opn−/−mice compared with WT controls, indicat-
ing improved whole-body insulin sensitivity (Fig. 2a, b).
In order to directly evaluate insulin sensitivity and the
relative contribution of the liver to this metabolic
phenotype we performed euglycaemic–hyperinsulinaemic
clamp tests in obese WT and Opn−/−mice. An increased
glucose infusion rate (GINF) was required in obese OPN
deficient mice to maintain euglycaemia during the clamp,
confirming improved whole-body insulin sensitivity com-
pared with WT controls (Fig. 2c). Basal glucose produc-
tion was unchanged between both genotypes but,
importantly, insulin-mediated suppression of hepatic glu-
cose production was significantly enhanced in obese
Opn−/−mice compared with WT animals (Fig. 2d, e).
Whole-body glycolytic activity under insulin-stimulated
conditions was unaffected by OPN deficiency (Fig. 2f). To
determine if organs other than liver contributed to the
Table 1 Body characteristics and plasma variables
VariableWT LF Opn−/−LF WT HF Opn−/−HF
Body mass (g)
Liver mass (g)
Feed efficiency
Glucose (mmol/l)
Insulin (pmol/l)
Triacylglycerol (mmol/l)
NEFA (μmol/l)
ALT (U/l)
Serum amyloid P (ng/ml)
32.5±1.0
1.34±0.07
2.08±0.41
7.50±0.06
55.8±8.0
0.41±0.03
224.5±13.2
15.1±1.6
32.7±5.8
30.7±0.7
1.41±0.06
1.42±0.45
6.92±0.69
40.5±4.3
0.38±0.01
271.3±27.0
11.0±2.1
34.6±6.1
50.8±0.6
2.08±0.11
8.35±1.24
15.15±0.88
448.2±65.9
0.64±0.06
328.7±26.2
53.9±6.9
72.9±10.9
50.6±0.9
1.76±0.10*
8.22±1.92
14.77±0.76
134.7±24.9***
0.67±0.09
286.3±31.9
31.6±4.3**
40.6±8.7*
Data are expressed as mean±SEM
WT and Opn−/−mice were fed LF or HF for 24 weeks (n=10 per group). Body and liver mass were measured. Feed efficiency was determined as
weight gain per food consumption. Blood samples were obtained after 3 h fasting period and analysed for depicted plasma variables
Significant differences between genotypes on the same diet are indicated as *p≤0.05, **p≤0.01, ***p≤0.001
Diabetologia (2011) 54:2132–21422135

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metabolic improvement we measured insulin-stimulated
2-[14C]DG uptake both in GWAT and skeletal muscle,
without detecting a difference between WT and Opn−/−mice
(Fig. 2g, h). Hence, attenuated hepatic steatosis in obese
OPN deficient mice is paralleled by improved hepatic insulin
sensitivity and restored insulin-mediated suppression of
hepatic glucose production.
Reduced hepatic triacylglycerol synthesis may contribute to
the absence of hepatic steatosis in OPN deficiency In order
to investigate possible mechanisms underlying the preven-
tion of hepatic steatosis in OPN deficient mice, we analysed
the expression of genes relevant for fatty acid and
triacylglycerol homeostasis in the liver. Markers of fatty
acid synthesis such as Fasn and Acaca did not differ
between obese WT and Opn−/−mice (Fig. 3a). Neither was
enhanced fatty acid oxidation likely to contribute to
reduced hepatic steatosis since important transcription
factors driving β-oxidation were unchanged or even down-
regulated as shown for Ppara and Ppargc1a, respectively
(Fig. 3a). However, gene expression of the major transcrip-
tional regulator of hepatic lipogenesis Srebf1 was markedly
decreased in Opn−/−compared with WT animals (Fig. 3b).
This was similarly found to be the case for Pparg (Fig. 3c),
a hepatic sterol regulatory element-binding protein 1
(SREBP-1c) target gene [29]. Given the downregulation
of hepatic Pparg mRNA expression, we next examined its
transcriptional targets Dgat1 and -2, which catalyse the
formation of triacylglycerols from diacylglycerol and acyl-
coenzyme A [30]. Notably, Dgat1 expression was signifi-
cantly downregulated in obese Opn−/−livers while Dgat2
followed the same trend, although the change was not
significant (Fig. 3d, e). These results strongly emphasise
that reduced hepatic triacylglycerol synthesis by down-
regulation of Dgat expression could contribute to the
absence of hepatic steatosis in obese OPN-deficient mice.
Lack of OPN favours lipid accumulation in visceral fat
instead of liver Decreased hepatic triacylglycerol content in
obese Opn−/−mice (Fig. 1) indicates the prevention of
ectopic fat accumulation, suggesting orthotopic fat storage
in adipose tissue. Subcutaneous white adipose tissue
(SWAT) weight was similar in both genotypes on the same
diet, whereas GWAT weight was strikingly increased in
Opn−/−compared with WT animals, both on HF diets
(Fig. 4a, b). Since liver weight and steatosis were
significantly reduced in obese Opn−/−vs WT mice (Table 1
and Fig. 1), we next investigated a possible relationship
between liver and GWAT mass. Multiple regression
analyses revealed no association between liver and GWAT
mass in lean animals (r2=0.05, p=0.28, Fig. 4c). Notably,
liver mass was strongly negatively associated with GWAT
weight in obese WT and Opn−/−mice (r2=0.42, p=0.008,
Fig. 4d), indicating that storage of excess lipids in gonadal
adipose tissue prevents hepatic steatosis in the absence of
OPN. Taken together, OPN deficiency inhibits ectopic fat
deposition in liver thereby protecting from deleterious
effects on hepatic metabolism.
Genetic OPN deletion improves hepatic insulin signalling
in obesity and regulates gluconeogenic enzyme expression
via FOXO1 HF-induced obesity impairs insulin-stimulated
tyrosine phosphorylation of IRSs, thereby causing insulin
resistance [31]. Since IRS-2 is the predominant IRS in liver,
we studied insulin-stimulated hepatic IRS-2 tyrosine phos-
phorylation in obese WT and Opn−/−mice, 15 min
following intraperitoneal injection of 1.5 U/kg insulin or
saline. Probably as a consequence of markedly elevated
basal plasma insulin levels in WT mice, hepatic IRS-2
phosphorylation was pronounced under unstimulated con-
ditions, but exogenous insulin failed to further stimulate
0
2
4
6
8
10
12
WT Opn−/−
WT Opn−/−
*
LFHF
14
Triacylglycerol/(AU) mg tissue
WT
Opn−/−
LF
HF
a
b
Fig. 1 OPN deficiency protects against obesity-induced hepatic
steatosis. WT and Opn−/−mice were kept either on LF or HF diet
for 24 weeks (n=10 per group). a Haematoxylin–eosin staining on
liver paraffin sections. Representative pictures are given in 20-fold
magnification. b Hepatic triacylglycerol content was determined in
lipid extraction by an enzymatic test. *p≤0.05 between genotypes.
AU, arbitrary unit
2136 Diabetologia (2011) 54:2132–2142

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IRS-2 phosphorylation (Fig. 5a). Conversely, tyrosine-
phosphorylated IRS-2 was barely detectable at baseline in
HF-fed OPN deficient mice but significantly increased
following insulin stimulation (Fig. 5a). In addition, insulin-
stimulated serine phosphorylation of AKT, which mediates
signal transduction downstream of IRS, was markedly more
enhanced in Opn−/−vs WT mice (Fig. 5b). These data
strongly indicate that impaired hepatic insulin signal
transduction in obesity is improved in the absence of
OPN. Intact insulin signalling is required for the inactiva-
tion of the transcription factor Foxo1 [32], a potent
regulator of hepatic gluconeogenesis. Improved insulin
signalling in obese Opn−/−livers, together with a down-
regulation of peroxisome proliferator-activated receptor
gamma coactivator (PGC)-1α (Fig. 3a), a transcriptional
coactivator of FOXO1 [33], prompted us to examine
hepatic abundance of FOXO1 in WT and OPN-deficient
mice. Foxo1 mRNA and protein levels were significantly
enhanced in obese WT livers, whereas absence of OPN
protected from HF-induced hepatic FOXO1 upregulation
(Fig. 5c, d). In addition, expression of the FOXO1 targets
G6pc and Mttp was upregulated in obese WT livers but
remained close to lean levels in OPN-deficient mice
(Fig. 5e, f). Thus, downregulation of hepatic FOXO1 and
gluconeogenic enzyme production probably contributes to
the beneficial effects of OPN deficiency on glucose and
lipid homeostasis in obesity.
OPNdeficiencypreventsobesity-mediatedhepaticinflammation
and macrophage accumulation In order to assess NAFLD
disease stage, liver histology was performed and the
NAFLD activity score was determined by a certified
pathologist. Mice on a normal chow diet did not show
any alterations in liver histology irrespective of the
genotype (electronic supplementary material [ESM]
Fig. 1). However, 24 weeks of HF feeding significantly
enhanced hepatic steatosis (Fig. 1), lobular inflammation
and hepatocyte ballooning in WT but not in Opn−/−mice
(Fig. 6a, b). In addition, circulating concentrations of the
liver-derived inflammatory marker serum amyloid P (SAP)
were highly elevated in the plasma of obese WT, compared
with OPN deficient, animals (Table 1). Increasing evidence
links macrophage accumulation in the liver to hepatic
steatosis and insulin resistance [12, 34]. OPN was recently
shown to mediate obesity-induced adipose tissue macro-
phage accumulation (19; 20). HF feeding raised gene
50
110
130
WT
Opn−/−
GINF (% of WT HF)
HF
70
90
*
WT
Opn−/−
Suppression of HGP
(% of WT HF)
HF
*
0
30
150
180
60
90
120
WT
Opn−/−
Basal HGP (% of WT HF)
HF
0
20
100
120
40
60
80
WT
Opn−/−
Glycolysis (% of WT HF)
HF
0
20
100
120
40
60
80
WT
Opn−/−
2-DG uptake (nmol g− 1min− 1)
HF
0
5
25
10
15
20
WT
Opn−/−
2-DG uptake (nmol g− 1min− 1)
HF
0
5
25
10
15
20
acd
efg
b
h
**
0
5.5
11.1
0
30 60
90
120
Time (min)
Blood glucose (mmol/l)
**
*
2.8
8.3
0
200
400
600
1,000
WT Opn−/−
WT
AUC (mmol/l×min)
Opn−/−
*
LF
HF
800
Fig. 2 OPN deficiency reduces obesity-induced hepatic insulin
resistance and glucose production. WT and Opn−/−mice were kept
either on LF or HF for 24 weeks. a, b An ITT was performed in WT
(solid lines) and Opn−/−mice (dashed lines) after feeding LF
(triangles) or HF (squares) (n=10 per group). Glucose concentrations
during ITT (a) and area under the curve are given (b). c–
h Euglycaemic–hyperinsulinaemic clamp studies were conducted in
mice kept on HF (n=10 per group). GINF (c) was measured during
steady state conditions as well as basal hepatic glucose production
(HGP; d), insulin-mediated suppression of HGP (e) and whole-body
glycolysis (f). g, h Tissue-specific glucose uptake was analysed by
enrichment of phosphorylated 2-[14C]DG in M. gastrocnemius (g) and
GWAT (h). *p≤0.05; **p≤0.01
Diabetologia (2011) 54:2132–2142 2137

Page 7

expression of the macrophage marker Emr1 significantly
less in OPN-deficient than in WT livers (Fig. 6c). Hepatic
gene expression of Ccl2 was markedly increased in obesity
in WT but not in Opn−/−animals (Fig. 6d). Immunohisto-
chemical analysis of liver sections revealed a sparse number
of macrophages in lean mice without any genotypic
difference (not shown). In contrast, hepatic macrophages
were highly abundant in obese WT mice and were clustered
in close vicinity to lipid droplets (Fig. 6e) resembling the
so-called crown-like structures described in obese adipose
tissue [35]. However, only a few such structures were found
in livers of obese Opn−/−mice, where macrophages were
less abundant and located rather individually throughout the
tissue (Fig. 6e). Macrophages are the major source of
obesity-driven cytokine production not only in adipose
tissue but also in liver [36]. Hepatic mRNA expression of
inflammatory Tnf and Tgfb1, known to be crucially
involved in pathogenesis and progression of NAFLD [37,
38] was increased with HF in WT mice but significantly
less in Opn−/−mice (Fig. 6f, g). Taken together, these
results reveal that OPN is not only a mediator of hepatic
steatosis but also of obesity-driven inflammatory alterations
including macrophage accumulation in the liver.
Discussion
The growing prevalence of obesity and related inflamma-
tory and metabolic disorders such as NAFLD and type 2
diabetes demands novel therapeutic approaches. Osteopon-
tin is an inflammatory cytokine, hepatic content of which is
upregulated in the course of human and murine hepatic
steatosis and various models of liver injury [18, 21, 39].
Indirect evidence suggests that OPN is involved in hepatic
steatosis, but a functional role has not been demonstrated.
Here we show that genetic OPN deficiency prevents high-
Liver weight (g)
1.0
1.2
1.4
1.6
GWAT weight (g)
0.2 0.4 0.6 0.8 1.0 1.2
Liver weight (g)
1.5
2.0
GWAT weight (g)
1.0 1.5 2.0 2.5 3.0 3.5
2.5
0
0.5
1.0
1.5
2.0
2.5
3.0
WT Opn−/−
WT
SWAT weight (g)
Opn−/−
LF
HF
0
0.5
1.0
1.5
2.0
2.5
3.0
WT Opn−/−
WT
GWAT weight (g)
Opn−/−
LF
HF
***
a
cd
b
Fig. 4 Lack of OPN prevents hepatic lipid accumulation by
orthotopic storage in GWAT. WT and Opn−/−mice were kept either
on LF or HF for 24 weeks (n=10 per group). SWAT (a) and GWAT
(b) mass was measured immediately after dissection. Multiple
regression analyses were performed between liver and GWAT mass
of lean (r2=0.05, p=0.28; c) and obese (r2=0.42, p=0.008; d). WT,
white circles; Opn−/−, black circles. ***p≤0.001
a
b
0
20
40
60
80
100
120
WT Opn−/−
WT
Dgat1 mRNA expression
Opn−/−
*
LF HF
LF
HF
0
20
40
60
80
100
120
WT Opn−/−
WT
Dgat2 mRNA expression
Opn−/−
0
20
40
60
80
100
120
WT Opn−/−
WT
Pparg mRNA expression
Opn−/−
*
LF HF
0
20
40
60
80
100
120
WT Opn−/−
WT
Srebf1 mRNA expression
Opn−/−
***
LF
HF
**
0
20
40
60
80
100
120
Fasn
Relative mRNA expression
Acaca
Ppara
Ppargc1a
*
FA synthesisFA oxidation
c
de
Fig. 3 OPN deficiency interferes with expression of lipogenic
markers. WT and Opn−/−mice were kept either on LF or HF for
24 weeks (n=10 per group). Hepatic mRNA expression of Fasn and
Acaca and the FA oxidation markers Ppara and Ppargc1a (a); white
bars: WT HF, black bars: Opn−/−HF; as well as of transcription factors
regulating lipid metabolism (Srebf1 and Pparg; b, c) and enzymes
controlling triacylglycerol synthesis (Dgat1 and -2; d, e) was
analysed. *p≤0.05, **p≤0.01, ***p≤0.001. Gene expression is
shown as percentages relative to WT HF
2138 Diabetologia (2011) 54:2132–2142

Page 8

fat diet-induced hepatic steatosis and inflammation, and
markedly improves insulin signalling and insulin sensitivity
in the liver, thereby improving hepatic glucose and lipid
homeostasis. Thus, our data indicate that OPN is causally
linked to obesity-induced detrimental hepatic alterations.
We and others have recently reported that hepatic OPN
production is significantly upregulated in murine HF-
induced and genetic obesity [18, 23] and correlates with
the severity of hepatic steatosis in humans [21]. Here we
demonstrate that Opn−/−mice are protected from obesity-
induced hepatic steatosis (Fig. 1). Hepatic steatosis is
characterised by an imbalance between fatty acid input
and output [5]. Expression of marker genes indicated
unchanged fatty acid oxidation and synthesis between WT
and Opn−/−livers in obesity despite decreased SREPB-1c
levels in obese OPN-deficient mice (Fig. 3a, b). Interest-
ingly, HF feeding upregulated hepatic production of the
lipogenic factor Pparg in WT but not in Opn−/−, which
could be the result of decreased SREBP-1c production and/
or the lack of OPN (Fig. 3c). Consequently, the expression
of Dgat1, which is regulated by peroxisome proliferator-
activated receptor (PPAR)-γ [29] and catalyses triacylgly-
cerol formation [30], was significantly decreased (Fig. 3d).
Hence, our data point toward unaltered transcriptional
regulation of fatty acid metabolism, but reduced hepatic
triacylglycerol synthesis, underlying the lack of steatosis in
OPN deficiency. Given that NEFA concentrations as well as
feed efficiency (Table 1), respiratory quotient and energy
expenditure (data not shown and [20]) were unchanged
between both genotypes, increased fatty acid dissipation
was unlikely to occur in Opn−/−mice. Thus, we hypoth-
esised that, in the absence of OPN, plasma NEFA were
preferentially distributed toward non-hepatic tissue. Indeed,
GWAT weight was strikingly increased in obese Opn−/−
mice (Fig. 4b) indicating that OPN deficiency facilitates
orthotopic fat storage in adipose tissue thereby preventing
its ectopic accumulation in the liver. This conclusion was
corroborated by multiple regression analyses revealing a
strong negative association between liver and GWAT
weight in obese mice from both genotypes (Fig. 4d).
Visceral fat is metabolically highly active, in contrast to
SWAT. The enlargement of GWAT, which is regarded as the
visceral fat depot in mice, is frequently associated with
insulin resistance [40]. We assume that the reduction of
hepatic lipid content occurring in OPN deficient animals
(Fig. 1) is more relevant for insulin sensitivity than the
accompanying increase in GWAT mass (Fig 4b). Observa-
tions of decreased inflammatory alterations in GWAT of
obese Opn−/−mice [20] could contribute to the mitigation
of metabolic perturbation occurring with GWAT enlarge-
ment in the absence of OPN.
Notably, decreased steatosis in obese Opn−/−mice was
paralleled by improved whole-body insulin sensitivity,
which was mainly due to reduction in hepatic insulin
resistance and glucose production as revealed by euglycae-
mic–hyperinsulinaemic clamp studies (Fig. 2). Enhanced
Insulin:
++
ab
0
20
40
60
80
100
120
WT Opn−/−
WT
Foxo1 mRNA expression
Opn−/−
**
LF HF
c
d
FOXO1
ß-Actin
80 kDa
40 kDa
WT Opn−/−
WT Opn−/−
WT
Opn−/−
HF
LF
**
0
20
40
60
80
100
120
WT Opn−/−
WT
G6pc mRNA expression
Opn−/−
LF HFLFHF
0
20
40
60
80
100
120
WT Opn−/−
WT
Mttp mRNA expression
Opn−/−
***
e
+
++
WT HF
Opn−/−HF
IP: IRS-2
IB: pY
IRS-2
__
_
__
_
+
pAKT
AKT
Insulin:
__
++
_
+
__
++
_
+
WT HF
Opn−/−HF
f
Fig. 5 OPN deficiency ameliorates hepatic insulin signalling and
determines FOXO1 gluconeogenic enzyme levels in obesity. WT
and Opn−/−mice were kept on HF for 24 weeks. 15 min prior to
sacrifice mice were intraperitoneally injected either with insulin
(1.5 U/kg) or saline (n=5 per group). IRS-2 tyrosine phosphorylation
(pY) was analysed in liver tissue extracts following immunoprecipi-
tation (IP) of IRS-2. Representative immunoblots (IB) are given (a).
Phosphorylation (ser473) of AKT was determined in livers of obese
WT and Opn−/−mice after intraperitoneal insulin injection. Represen-
tative immunoblots are given (b). Hepatic mRNA (c) and protein (d)
levels of FOXO1 were determined in lean and obese WT and Opn−/−
mice (n=10 per group). A representative blot is given. e, f mRNA
expression of FOXO1 transcriptional targets G6pc (e) and Mttp (f)
were analysed in livers of lean and obese WT and Opn−/−mice. **p≤
0.01, ***p≤0.001. Gene expression is shown as percentages relative
to WT HF
Diabetologia (2011) 54:2132–21422139

Page 9

insulin-stimulated phosphorylation of IRS-2 and AKT in
OPN deficient, compared with WT, livers provided addi-
tional evidence for ameliorated hepatic insulin signalling
(Fig. 5). A hallmark of hepatic insulin action is reduction of
gluconeogenesis, which is downstream regulated by the
transcription factor FOXO1 and its coactivator PGC-1α.
Both FOXO1 and PGC-1α were downregulated in livers
from obese Opn−/−compared with WT mice, as were the
FOXO1 target genes G6pc and Mttp (Fig. 3a and Fig. 5)
indicating abated gluconeogenesis. These results are in line
with our previous observation that antibody-mediated OPN
neutralisation reduces hepatic gluconeogenic enzyme pro-
duction in HF-fed WT mice [19].
An obvious parallel between obesity-related pathologies
of adipose tissue and liver pertains to the emerging role of
inflammation. Evidence is growing that hepatic macro-
phages such as Kupffer cells are critically implicated in the
pathogenesis and progression of NAFLD [34]. Only
recently, chemical depletion of Kupffer cells was shown
to prevent HF-induced hepatic steatosis and insulin resis-
tance [12, 41, 42]. Here we demonstrate that OPN
deficiency markedly counteracts obesity-induced hepatic
inflammation and macrophage accumulation as assessed by
NAFLD activity score, SAP concentrations and expression
of macrophage markers, respectively (Fig. 6, Table 1).
These data concur with recent reports showing that genetic
OPN deletion and antibody-mediated OPN neutralisation
antagonise macrophage recruitment in obese adipose tissue
[19, 20]. Macrophages are the major source of obesity-
associated inflammatory cytokine production in liver as
shown for adipose tissue [36, 40]. OPN deficiency inhibited
HF-induced upregulation of hepatic Tnf and Tgfb1 gene
expression (Fig. 6f, g), both of which are predominantly
derived from hepatic macrophages and were repeatedly
reported to underlie NAFLD progression [37, 38]. Lack of
OPN also downregulated hepatic gene expression of MCP-1
(Fig. 6d), a chemokine that promotes hepatic macrophage
infiltration and steatosis [13, 43]. Since OPN and MCP-1
cooperate in monocyte chemotaxis [15, 44], abrogation of
inflammatory cell migration is likely to account for reduced
abundance of macrophages in livers from HF-fed Opn−/−
mice. Moreover, deficiency and neutralisation of OPN
improved liver injury and fibrosis elicited by methionine-
and choline-deficient diet, concanavalin A, and D-galactos-
amine [22, 24, 45, 46] indicating a general implication of
OPN in inflammatory liver damage.
In conclusion, at least two mechanisms are likely to
contribute to the improvement of obesity-related hepatic
steatosis and metabolic perturbation during OPN deficiency.
On the one hand lack of OPN reduces hepatic triacyl-
glycerol accumulation and fosters lipid storage in adipose
tissue. On the other hand, hepatic inflammation is
attenuated in the absence of OPN. Hence, OPN is shown
here as a novel mediator of obesity-associated hepatic
inflammation, steatosis and insulin resistance. Blocking
OPN effects in vivo could normalise altered hepatic lipid
a
b
LF HF
0
20
40
60
80
100
120
WT Opn−/−
WT
Emr1 mRNA expression
Opn−/−
-
*
0
20
40
60
80
100
120
WT Opn−/−
WT Opn−/−
*
LFHF
140
Ccl2 mRNA expression
0
0
1
1
2
2
3
3
4
4
5
5
7
6
6
******
**
****
Steatosis
Lobular
inflammation
Hepatocyte
ballooning
7
NAS
WT HF
Opn−/−HF
c
20×
40×
d
Fig. 6 Obesity-induced hepatic inflammation and macrophage accu-
mulation is antagonised by OPN deficiency. WT and Opn−/−mice
were kept either on LF or HF for 24 weeks (n=10 per group).
a Representativehaematoxylin–eosinstainingofliverparaffinsectionsis
given. Black arrows indicate hepatocyte ballooning, red arrows point to
portal leucocyte infiltrate. b NAFLD activity score (NAS) was
determined by a certified pathologist. White bars, WT HF; black bars
Opn−/−HF. c, d Hepatic mRNA expression of the macrophage marker
Emr1 (c) andthemonocyterecruitingchemokineCcl2 (d) was measured.
e Macrophages were stained brown by immunohistochemistry using
MAC-2 monoclonal antibody on paraffin sections of livers isolated from
HF-fed mice. Representative pictures are given. f, g mRNA expression
of the inflammatory markers Tnf (f) and Tgfb1 (g) was analysed in livers
of WT and Opn−/−mice. *p≤0.05, **p≤0.01, ***p≤0.001. Gene
expression is shown as percentages relative to WT HF
2140Diabetologia (2011) 54:2132–2142

Page 10

and glucose homeostasis in obesity and therefore prevent
progression of NAFLD and development of type 2 diabetes.
Acknowledgements
thology, Medical University of Vienna), L-I Ionasz (Clinical Division
of Endocrinology and Metabolism, Department of Medicine III,
Medical University of Vienna, Austria) N. Boche (Institute of
Experimental Genetics, Helmholtz Zentrum Muenchen, German
Research Center for Environmental Health) and the German Mouse
Clinic, for excellent technical expertise. This work was supported by
the Austrian Science Fund (project no. P18776-B11 and as part of the
CCHD doctoral programme W1205-B09), the European Community"s
7th Framework Programme (FP7/2007–2013) under grant agreement
no. 201608 (all to T.M. Stulnig), and the National Genome Research
Network of the German Federal Ministry of Education and Research
(BMBF, NGFNplus grant, 01GS0850). This study was in part
supported by a grant from the German Federal Ministry of Education
and Research (BMBF) to the German Center for Diabetes Research
(DZD e.V.).
We thank H. Schachner (Department of Pa-
Duality of interest
interest associated with this manuscript.
The authors declare that there is no duality of
Open Access
Creative Commons Attribution Noncommercial License which per-
mits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
This article is distributed under the terms of the
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