From the Institut National de la Santé et de la Recherche Médicale U145, IFR 50, Avenue de Valombrose, 06107 Nice Cédex 2, France
Received for publication, November 26, 2000, and in revised form, April 5, 2001
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ABSTRACT |
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Alterations in the expression level of genes may
contribute to the development and pathophysiology of obesity. To
find genes differentially expressed in adipose tissue during obesity,
we performed suppression subtractive hybridization on epididymal fat
mRNA from goldthioglucose (GTG) obese mice and from their lean
littermates. We identified the secreted protein
acidic and rich in cysteine
(SPARC), a protein that mediates cell-matrix interactions and plays a
role in modulation of cell adhesion, differentiation, and angiogenesis.
SPARC mRNA expression in adipose tissue was markedly increased
(between 3- and 6-fold) in three different models of obesity,
i.e. GTG mice, ob/ob mice, and AKR mice, after
6 weeks of a high fat diet. Immunoblotting of adipocyte extracts
revealed a similar increase in protein level. Using a SPARC-specific
ELISA, we demonstrated that SPARC is secreted by isolated adipocytes.
We found that insulin administration to mice increased SPARC mRNA
in the adipose tissue. Food deprivation had no effect on SPARC
expression, but after high fat refeeding SPARC mRNA levels were
significantly increased. Our results reveal both hormonal and
nutritional regulation of SPARC expression in the adipocyte, and
importantly, its alteration in obesity. Finally, we show that purified
SPARC increased mRNA levels of plasminogen activator inhibitor 1 (PAI-1) in cultured rat adipose tissue suggesting that elevated
adipocyte expression of SPARC might contribute to the abnormal
expression of PAI-1 observed in obesity. We propose that SPARC is a
newly identified autocrine/paracrine factor that could affect key
functions in adipose tissue physiology and pathology.
Obesity results from a chronic imbalance between energy intake and
energy expenditure. This syndrome is commonly associated with increased
risks of cardiovascular problems and metabolic abnormalities, including
hypertension, hyperinsulinemia, insulin resistance, and type 2 diabetes
(1, 2). Obesity is defined as a pathological excess of fat mass.
Adipose tissue grows throughout life by increasing the size and volume
of preexisting adipocytes and de novo recruitment of
preadipocytes. Adipose tissue is the major site for energy storage and
disposal and is also implicated in glucose homeostasis. Today, it is
well recognized that adipocytes play a central role in metabolism
through the secretion of signaling factors that regulate food intake
and metabolic efficiency (3, 4).
Recent research on obesity has revealed that body weight regulation is
a complex phenomenon that is partly genetically defined. Body weight is
determined by multiple interactions between genes and environmental
factors such as dietary composition, total caloric intake, and exercise
(1, 5). Multiple genes appear to contribute to the pathogenesis of
obesity (6). Several studies have identified the existence of monogenic
obesity in mice and, more recently, cases of monogenic obesity in
humans have been reported. However, the mutations identified so far
explain only a minute proportion of human obesity syndromes (6).
Despite intensive efforts, the nature of the molecular defects leading
to the occurrence and progression of the common obesity is still poorly understood.
It is generally accepted that alterations in gene expression may
contribute to the pathogenesis and physiopathology of this syndrome.
Numerous studies have focused on the expression of specific candidate
genes at the RNA level (7-9). For example, it has been shown that
obesity is associated with elevated expression of plasminogen activator
inhibitor 1 (PAI-1)1 or tumor
necrosis factor One attractive strategy for finding obesity-related genes involves
examining differential gene expression in adipose tissue of obese
animal models. Here we employed the power of the SSH method (19) to
identify mRNA that are differentially expressed in the adipose
tissue of non-obese mice compared with obese ones. We used the GTG
obese mice, which is a well studied model of obesity (20). In these
mice, obesity is obtained after the injection of GTG, which destroys
part of the hypothalamus and thus induces hyperphagia with ensuing
obesity, hyperinsulinemia, and insulin resistance. With this approach,
we found that the expression of SPARC (also known as osteonectin or
BM-40) is strongly elevated in the adipocyte of obese mice. We studied
also the modulation of SPARC expression in physiologic and
pathophysiologic states of the adipose tissue in rodents. Finally, we
demonstrated that SPARC might be implicated in the regulation of PAI-1
mRNA expression in the adipose tissue.
Animals and Experimental Design--
Male OF1 mice were from
Iffa-Credo (L'Arbresle, France). Male AKR/OlaHsd mice, male
C57BL/6OlaHsd ob/ob mice and their lean controls
(C57BL6OlaHsd +/?) were obtained at 4-10 weeks of age from Harlan
France (Gannat, France). Wistar rats (150-200 g in body weight) were
obtained from Iffa-Credo. Four animals were kept per cage in a
temperature-controlled room (22 °C) with a 12-hour light-dark cycle.
Water and food (standard laboratory chow diet from UAR, Epinay-S/Orge,
France) were available ad libitum, except when indicated.
GTG obese mice were generated by double injection of goldthioglucose
(Sigma, St Louis, MO) administered to 3-week-old OF1 mice as previously
described (20). Matched lean littermate controls were studied in
parallel. The development of high fat diet-induced obesity was studied
in the AKR/OlaHsd mice (21). At 5 weeks of age, mice were randomly
assigned to receive either a chow diet containing 12% of calories from
fat or a Western high fat diet (Teklad Adjusted Calories TD 97363, Harlan Teklad, Madison WI) containing 42% of calories from fat. For 10 weeks the mice were weighed twice a week. After 3, 6, and 10 weeks on these diets animals were euthanatized by cervical dislocation, and the epididymal fat depots were removed, weighed, and
processed for preparation of total RNA. In some experiments, normal OF1
mice (20-22 weeks old) were deprived of food for 24 h. Fasted
mice were injected intraperitoneal with either insulin (0.04 IU
Actrapid Hmge, Novo-Nordisc, Denmark) or saline. In other experiments,
fasted mice were refed with chow diet or high fat diet for 6 h.
Tissues and RNA Extraction--
The tissues were surgically
dissected, rapidly removed, and processed for RNA extraction. Total RNA
was isolated using the TRIzol reagent following the manufacturer's
instructions (Life Technologies, Cergy Pontoise, France).
Poly(A)+ mRNA purification was carried out from total
RNA using the Oligotex mRNA kit (Qiagen, Valencia, CA) with two
rounds of elution.
For preparation of isolated adipocytes, freshly obtained epididymal fat
was minced and digested with the Liberase Blendzyme 3 (Roche Molecular
Biochemicals, Indianapolis, IN) as previously described (22).
Adipocytes were separated from stromal vascular cells by
centrifugation. The stromal vascular fraction was washed three times
with Krebs-Ringer bicarbonate (KRB) buffer. Total RNA was extracted
from the two fractions as described above.
Suppression Subtractive Hybridization--
SSH between obese and
lean white adipose tissue RNA was performed with the PCR-Select
cDNA subtraction kit (CLONTECH, Palo Alto, CA)
according to the manufacturer's protocol. We used 2 µg of epididymal
white adipose tissue mRNA from 4 obese GTG mice (body weight
60 ± 3 g) as tester and 2 µg of mRNA from 8 OF1 mice (body weight 35 ± 2 g) as driver. After hybridization,
differential transcripts were amplified by suppression PCR (19). PCR
products were cloned into the pCR 2.1 vector using the TOPO TA cloning kit (Invitrogen, Groningen, The Netherlands) and screened by Southern blot analysis for differentially regulated genes. Plasmid DNA from two
hundred random clones were arrayed on nitrocellulose filters using a
dot-blot apparatus (Biometra Inc, Tampa, FL) and hybridized with
tester, driver, and subtracted cDNA probes. Approximatively 20 of
the clones that differentially hybridized were sequenced and subjected
to Northern blotting. Partial cDNA sequences were determined and
compared with entries in the GenBankTM and mouse expressed
sequence tag databases using the advanced BLAST homology search program.
cDNA--
cDNA clones for GAPDH and aP2 were gifts from
C. Dani (CNRS UMR 6543 Nice, France). The cDNA clone for murine
leptin was a gift from J. Auwerx (IGBMC, Illkirch, France). cDNA
probes for mouse SPARC and PAI-1 were obtained by RT-PCR from adipose
tissue using the SuperScript one-step RT-PCR system kit (Life
Technologies). Appropriate fragments of these cDNAs were labeled
with [ Northern Blot Analysis and RT-PCR--
Northern blots were
performed according to standard protocols (23). Membranes were
hybridized overnight at 42 °C with the indicated
32P-labeled cDNA probes and exposed to Kodak bioMax MR
film for 3-24 h at Protein Analysis--
Western blotting was performed on whole
cell lysates from freshly isolated adipocytes. Protein extracts were
obtained by mixing 200 µl of fat cell suspension with 200 µl of
Laemmli buffer (3% SDS, 70 mM Tris, pH 7, 11% glycerol).
The samples were incubated for 1 h at 90 °C, and the protein
concentration was assayed by bicinchoninic acid technique (Pierce).
Proteins were separated by SDS-polyacrylamide gel electrophoresis and
analyzed by immunoblot using a monoclonal antibody to SPARC
(Hematologic Technologies, Essex Junction, VT). Immunoreactive proteins
were detected by enhanced chemiluminescence. SPARC levels in the
conditioned medium of rat adipocytes were measured with the ELISA kit
from Hematologic Technologies according to the manufacturer's
instructions. After cell fractionation, 200 µl of adipocytes were
resuspended in 2 ml of Dulbecco's modified Eagle's medium
supplemented with 4% (w/v) bovine serum albumin (fraction V, Intergen
Compagny, Purchase, NY) and 25 mM Hepes, pH 7.4. Cells were
subsequently cultured in suspension in polypropylene tubes in a
humidified incubator at 37 °C under 5% CO2. Trypan blue
exclusion was tested to measure cell viability. After 16 h, the
cell-conditioned medium was collected, concentrated 10-fold using the
ultrafree-15 centrifugal filters device (Millipore), and analyzed by
ELISA. The assay was repeated three times.
Adipose Tissue Culture--
Fat pads from Wistar rats were
dissected under sterile conditions, washed in KRB buffer, minced
finely, and incubated (1 ml of media per gram of tissue) in 6-well
tissue culture plates containing Dulbecco's modified Eagle's medium
supplemented with 0.5% (w/v) bovine serum albumin and 25 mM Hepes, pH 7.4. The fat tissue samples were left
unstimulated or stimulated with 20 µg/ml of human purified osteonectin/SPARC (Calbiochem-Novabiochem Corp.) or 10 ng/ml of recombinant human transforming growth factor- SSH Reveals SPARC as a Newly Identified Gene Differentially
Expressed in GTG and ob/ob Mice--
The purpose of our work was to
reveal genes differentially expressed during obesity. We used SSH to
detect changes in mRNA expression in white adipose tissue of obese
GTG mice and their lean controls. cDNAs were prepared by reverse
transcription and subjected to subtractive hybridization as previously
described (Ref. 19; and "Materials and Methods"). A minilibrary was
generated by randomly subcloning the subtracted PCR products. Over two
hundred cDNA plasmids from the library were then subjected to
differential screening using lean and obese subtracted cDNAs as
probes. Subsequent analysis on Northern blots identified and confirmed
six gene products differentially expressed in adipose tissue of GTG
obese mice (data not shown). Primary DNA analysis of one of those
clones revealed a sequence corresponding to that present in the
3'-untranslated region of the mouse SPARC gene (25). SPARC is a
non-structural component of the extracellular matrix that modulates
cell-matrix interaction (26, 27). During embryogenesis its expression is time- and tissue-specific. In the adult, SPARC expression has been
associated with wound repair, tumorigenesis, and cataractogenesis (28,
29). Because of its reported functional properties and pattern of
expression, SPARC was chosen for further characterization. Northern
blot hybridization with a cDNA probe encoding the full-length SPARC
protein confirmed increased steady-state levels of the major SPARC
transcript (2.2 kilobases) in adipose tissue of obese GTG mice. Adipose
tissue of obese mice showed approximately a 5- to 6-fold increase in
the expression of the main SPARC message (Fig. 1A, lanes 1 and
2). An increased level of SPARC mRNA was also observed
in the subcutaneous adipose tissue of obese mice (data not shown).
SPARC expression was also examined in liver and skeletal muscle, two
other insulin-responsive tissues implicated in glucose homeostasis. No
expression was observed in the liver (Fig. 1A, lanes
3 and 4). In contrast, SPARC messages were detected in
skeletal muscle but levels were lower than in white fat. However, the
amount of SPARC mRNA expression in this tissue was no different
between lean and obese mice (Fig. 1A, lanes 5 and
6).
To test the hypothesis that obesity might be associated to abnormal
expression of SPARC in adipose tissue, we looked at SPARC mRNA
levels in the ob/ob mice and their lean littermates (+/?). In the genetic ob/ob model, obesity results from the
disruption of the leptin signaling system (30). The ob/ob
mouse lacks functional leptin and as a consequence is associated with
hyperphagia, massive obesity, hyperinsulinemia, and severe insulin
resistance (31). As shown in Fig. 1B, in ob/ob
mice the two SPARC transcripts were induced ~3-fold compared with
lean counterparts (lanes 1 and 2). This
demonstrates that up-regulation of SPARC mRNA expression in obese
white fat does not depend on a functional leptin signaling system.
Besides adipocytes, fat tissue contains several other cell types,
including preadipocytes, endothelial cells, smooth muscle cells,
fibroblasts, mast cells, and macrophages. To determine the source of
SPARC expression in adipose tissue, fat pads from lean (+/?) and
ob/ob mice were digested with collagenase and then subjected
to differential centrifugation to separate mature fat cells from
non-adipose cells. The amount of SPARC mRNA associated with each
cell fraction was analyzed by Northern blot. Expression of the
adipocyte fatty acid binding protein aP2 was shown as a positive marker
of the adipocyte fraction. Fig. 1C shows that the majority
of SPARC mRNA was fractionated with the adipocytes (lanes
1-4) and as expected, SPARC expression was increased in adipocytes of obese mice (lanes 3 and 4). SPARC
mRNA was also detected, but to a lesser degree in the stromal
vascular fraction (lanes 5 and 6). Because of the
relatively strong signal in the adipocyte fraction as compared with the
stromal vascular fraction, we conclude that SPARC mRNA expression
and its increase in the adipose tissue of obese mice are mainly found
in adipocytes. In situ hybridization is needed to localize
more precisely SPARC mRNA in the different adipose tissue components.
Expression of SPARC mRNA during Development of High Fat
Diet-induced Obesity--
To gain further insight into the regulation
of SPARC in obesity, we examined SPARC expression in a model of
diet-induced obesity. We studied the development of obesity in the
obesity-prone AKR strain in response to high fat diet (21). AKR mice
were divided into a normal diet group and a high fat diet group, and
assigned to receive the two diets for up to 10 weeks. Fig.
2A shows the body weight gain
within the two groups of mice. As previously described, on a high fat
diet AKR mice developed a marked obesity compared with mice fed on a
standard chow (21). At the end of the study, fat pads from fat fed
animals weighed ~4-fold more than those of control animals (1.9 ± 0.2 versus 0.4 ± 0.05; Fig. 2B).
Northern blot experiments were performed to compare the level of SPARC
expression in adipose tissue from mice after 10 weeks of feeding the
two diets (Fig. 2C). Feeding mice the fat diet resulted in
an increase in the level of the major SPARC transcript (lanes
1 and 2). In parallel, we studied SPARC expression in
the adipose tissue of AKR mice after 3, 6, and 10 weeks of high fat feeding. Fig. 2D illustrates the fat pad weights throughout
the study. SPARC mRNA was detected by Northern analysis of total
RNA isolated from the fat pads (Fig. 2E). SPARC levels were
markedly increased at early time points in this diet (lane
3). After 10 weeks, SPARC levels tended to decrease but remained
higher than those observed after 3 weeks on this diet (lanes
2 and 4).
Our results show that SPARC mRNA is up-regulated in a model of
dietary obesity, and demonstrate that changes in SPARC expression appears relatively early in the development of obesity. Taken together,
our results suggest that elevation of SPARC gene expression in the
adipose tissue of mice characterizes both genetic and acquired obesity
either induced by hyperphagia or by high fat diet.
Expression and Secretion of SPARC by the Adipocyte--
Next we
confirmed by immunoblotting that the protein SPARC was also increased
in the adipocytes of obese mice (Fig.
3A). SPARC expression was
between 3- and 4-fold higher in protein extracts of ob/ob
mouse adipocytes as compared with that of lean adipocytes. Because
SPARC is a secreted molecule, we examined the ability of adipocytes to
secrete SPARC ex vivo. The concentration of SPARC antigen in
16 h-conditioned medium of freshly isolated rat adipocytes was measured
by a SPARC-specific ELISA (Fig. 3B). A ~7-fold higher amount of SPARC was observed in adipocyte-conditioned medium compared with control medium. This suggests that in our experimental system mature fat cells produce and secrete SPARC. We also found that the
secretion of SPARC by adipocytes isolated from ob/ob mice was higher than that of adipocytes from lean mice (data not shown).
Expression of the SPARC-related Gene SC1 in Adipose Tissue of Obese
Mice--
As elevated expression of SPARC in adipocytes is likely to
be associated with the increase in adipose mass that occurs in obesity,
we investigated whether the expression of SC1 (also known as hevin), a
SPARC-related protein, is also modulated in the adipose tissue of obese
mice. SPARC and SC1 are members of a small family of proteins that are
defined by a variable NH2-terminal domain, followed by two
conserved domains, a follistatin-like and an extracellular calcium-binding domain (32). SC1 shows the highest similarity with
SPARC (70% identity at the amino acid level) and has been proposed to
functionally compensate for the loss of SPARC in SPARC-null mice (33).
RT-PCR studies of SC1 message level in white adipose tissue from
20-week-old obese GTG and ob/ob mice compared with that of
lean counterparts are presented in Fig.
4. SC1 mRNA was expressed in the
white adipose tissue of the two lean strains (lanes 1 and
3). However, contrary to SPARC, SC1 mRNA expression was
not affected by obesity at least at the time point studied (lanes
1 and 3 versus lanes 2 and 4, respectively).
This suggests that altered adipogenic expression of SPARC could have
specific functional consequences in obesity.
Effects of Insulin Administration and Food Intake on SPARC mRNA
Expression--
Because obesity in both humans and rodents is often
associated with hyperinsulinemia (1, 2), we hypothesized that insulin could have an effect on the elevation of SPARC induced by obesity. We
examined SPARC expression in the adipose tissue of fasted control mice
after insulin administration. RNA was extracted from the fat pads at 2 and 4 h after injection of insulin. SPARC expression was examined
by Northern blot (Fig. 5A). As
a positive control for adipocyte genes induced by insulin, we analyzed
the expression of ob gene coding for leptin (34). Insulin
induced a time-dependent increase in both SPARC and
ob messages. More precisely, mice injected with insulin for
4 h showed a 3.3-fold increase in the expression of SPARC mRNA
in the adipose tissue (lanes 1 and 3). This
suggests that insulin could have a key role as a specific stimulus of
SPARC gene expression in adipose cells. However, the insulin effect on
SPARC expression could be because of changes in glycemia rather than to
a direct effect of insulin. Indeed, insulin induces a hypoglycemic
response that can be associated with counter-regulatory hormonal
mechanisms. Further studies are needed to more precisely define the
regulatory role of insulin on SPARC expression. Because food intake
increases plasma insulin concentration, we also examined the effect of
fasting and refeeding on SPARC gene expression. Mice were first divided
into a fed control group and a fasted one. After an overnight fast, the
second group of animals was then divided into three groups; one group
that served as a fasting control and two other groups that were refed
with standard diet or with high fat diet for 6 h. SPARC and leptin
expression in fat pads were analyzed by Northern blot (Fig.
5B). Food deprivation for 24 h did not markedly affect
SPARC mRNA levels (lanes 1 and 2). Whereas
refeeding mice with the standard diet induced a modest increase in the
level of SPARC messages (lane 4), high fat refeeding doubled
SPARC levels compared with fasted controls (lanes 3 and 5). As previously shown (34), under fasting conditions,
ob mRNA amount was decreased to barely detectable levels
(lanes 1 and 2) but was restored to normal within
6 h after refeeding with both diets (lanes 4 and
5). Because it is widely accepted that a high fat diet tends
to cause obesity, our findings suggest that consumption of a diet rich
in fat could participate in the elevation of SPARC expression in the
adipose tissue of mice.
SPARC Modulates PAI-1 mRNA Expression in Adipose
Tissue--
Finally, we approached the issue of SPARC action on the
adipose tissue. SPARC has been shown to modulate the expression of a
subset of extracellular matrix proteins implicated in angiogenesis (35). SPARC also stimulates PAI-1 biosynthesis by bovine endothelial cells (36). Because increased expression of PAI-1 by the adipose tissue
is associated with obesity (11, 37, 38), we looked at the effect of
SPARC on PAI-1 gene expression in cultured rat adipose tissue. At the
same time we studied the effect of TGF- We used the SSH strategy to isolate candidate genes that are
dysregulated in the adipose tissue of obese mice. One of the genes
revealed codes for SPARC, a matricellular protein that it thought to
modulate cell-matrix interaction (27). We have demonstrated that SPARC
is a newly identified factor secreted by the adipocyte and that its
expression is strongly elevated in several models of experimental
obesity. These observations suggest that elevated SPARC gene expression
may be a general feature associated with these disorders. Further, we
have shown that SPARC expression may be regulated in response to
signals that modulate adipose tissue size such as insulin or high fat
food. It would be now interesting to determine whether obesity in
humans is also associated with altered expression of SPARC in the
adipose tissue.
In an attempt to understand the physiological role of SPARC in the
adipose tissue we studied the effect of SPARC on PAI-1 expression. Such
a SPARC action has been previously described in bovine endothelial
cells (35, 36). Similarly, we found that addition of SPARC to cultured
whole adipose tissue induced an increase in PAI-1 mRNA levels.
PAI-1 is the primary inhibitor of fibrinolysis, and its elevation has
been associated with an increased risk for thrombotic disease (40).
Up-regulation of PAI-1 expression by the adipose tissue is a major
contributor to the elevation in plasma PAI-1 observed in obesity (11,
37, 38). The regulation of PAI-1 expression in the adipose tissue has
been shown to be partly controlled by TNF- Because of the multitude of functional properties attributed to SPARC,
it is possible that SPARC impacts on several cellular processes in the
adipose tissue. It has been shown that SPARC can influence the
expression and/or activity of specific proteins that regulate cellular
interactions with the extracellular matrix (26, 28). For example, SPARC
has been reported to bind many components of the extracellular matrix
such as thrombospondin and certain types of collagen (26), and also to
stimulate the production of matrix metalloproteinases (42, 43). In
addition, SPARC possesses a strong anti-adhesive activity. Indeed,
SPARC affects cell shape by disrupting focal contacts and lowering
adhesion to the matrix and to neighboring cells (26, 28). These
observations suggest that SPARC could increase matrix plasticity and
facilitate adipose tissue remodeling. Another characteristic of SPARC
is the binding of cytokines and growth factors, and modulation of cellular responses induced by these molecules (26, 28). In this regard,
SPARC could have a modulatory action on the growth factors that mediate
adipocyte hyperplasia and adipose tissue growth. Finally, a role of
SPARC in the neovascularization of the adipose tissue should be
considered. Angiogenesis is prerequisite for fat mass development (44).
Although factors with angiogenic activities such as vascular
endothelial growth factor (VEGF) and leptin are known to be secreted by
adipose cells (45-47), the mechanisms that promote angiogenesis in
obesity are largely unknown. Interestingly, SPARC has been implicated
in the regulation of angiogenesis during wound healing and tumor growth
(29). These observations are in favor of a role of SPARC in
angiogenesis in the adipose tissue of obese mice.
In conclusion, SPARC is a newly identified autocrine and/or paracrine
factor of the adipose tissue that may affect key functions of this
tissue. We are currently addressing the physiological role of SPARC in
the adipose tissue and its implication in pathological conditions of
the adipose tissue such as adipocyte hypertrophism or hyperplasia,
vascular remodeling, and development of obesity-associated complications.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(TNF-
) in the adipose tissue (10, 11).
Interestingly, these defects have been associated with the
insulin-resistant state and cardiovascular risk, two obesity-related complications (12, 13). At present, a limited number of studies have
screened for new genes of which the expression is modulated by obesity
and thus might contribute to the obese phenotype (14-18).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP using the Rediprime II kit (Amersham
Pharmacia Biotech, Buckinghamshire, UK) and were used as probes in
Northern blotting.
70 °C. Blots were stripped and reprobed with
GAPDH cDNA probe or 18 S rRNA probe as control for RNA integrity
and loading. All Northern blots were repeated at least three times. Semiquantitative RT-PCR assays were carried out as described (24). Data
regarding gene sequences were obtained from GenBankTM.
Total RNA was treated with Rnase-free DNase, and first-strand cDNA
was generated from 1 µg of RNA by using the reverse transcriptase system from Promega (Madison, WI). The optimal number of cycles for
each primer set was determined to keep signal amplification in the
linear range. For internal control we amplified GAPDH mRNA along
with the target mRNA. PCR was performed with a GeneAmp PCR system
2400 (Cetus-Perkin Elmer, Foster City, CA). Products were separated
with 1.2% agarose gel electrophoresis, stained with ethidium bromide,
and photographed using ultraviolet light.
1 (TGF-
1). The plates were incubated at 37 °C under 5% CO2 for 6 h. The samples were collected, total RNA was isolated, and were
analyzed by Northern blot.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Expression of SPARC mRNA in lean and
obese mice. A, Northern blot analysis was performed
using 15 µg of total RNA isolated from white adipose tissue
(WAT), liver, and skeletal muscle of lean or GTG animals
(n = 4 for each group). Levels of SPARC mRNA
(indicated by arrows) were analyzed using a
32P-labeled SPARC cDNA probe. GAPDH mRNA is shown
as a control for the loading and integrity of RNA. B,
Northern blot analysis of SPARC mRNA levels (15 µg of RNA/lane)
in white adipose tissue of 4-month-old ob/ob mice
(n = 4) and of their lean counterparts (+/?)
(n = 8). GAPDH mRNA is shown as a control for the
loading and integrity of RNA. C, adipocytes and stromal
vascular fraction from lean (+/?) or ob/ob mice were
separated by collagenase digestion followed by centrifugation. 15 µg
of total RNA isolated from the whole fat tissue or from each cellular
fraction was analyzed for expression of SPARC by Northern blot. The
blot was subsequently probed with aP2 and GAPDH as controls.
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Fig. 2.
Expression of SPARC mRNA during
development of high fat diet-induced obesity in AKR mice.
Initially, the obesity-prone AKR strain was fed a standard chow diet.
At 5 weeks of age, half of the mice were switched to a high fat diet.
After 3, 6, and 10 weeks on these diets, animals (n = 6 per group) were euthanatized and the epididymal fat depots were removed
and weighed. Total RNA was prepared and examined for the expression of
SPARC by Northern blot hybridization. The blots were subsequently
probed with GAPDH as a control. A, growth curves of high fat
and chow fed animals. B and D, epididymal fat pad
weights of AKR mice maintained on chow or on high fat diet. Values are
mean ± S.E. (n = 6 per group). C and
E, representative Northern blots.
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Fig. 3.
Expression and secretion of SPARC by the
adipocytes. A, isolated mouse adipocytes from lean or
ob/ob mice were prepared by digestion of epididymal fat with
collagenase. 50 µg of adipocyte protein extract was separated by
SDS-polyacrylamide gel electrophoresis. Immunoblot analysis was
performed using a monoclonal antibody to SPARC and revealed using
enhanced chemiluminescence. Molecular mass markers are indicated as
well as the band corresponding to SPARC. B, analysis of
conditioned medium derived from isolated rat adipocytes by an ELISA
specific for SPARC. The level of SPARC in the growth medium is also
shown. Results are averaged from three independent experiments.
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Fig. 4.
Expression of SC1 mRNA in the adipose
tissue of lean and obese mice. 1 µg of total white adipose
tissue RNA from 20-week-old obese mice (GTG and ob/ob mice)
(n = 4 each) and from their lean counterparts
(n = 8 each) was reverse transcribed and amplified by
PCR (40 cycles) in the presence of specific primers for SC1 and for
GAPDH as an internal control. The products were subjected to agarose
gel electrophoresis in the presence of ethidium bromide and were
subsequently photographed using ultraviolet light.
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Fig. 5.
Effect of insulin and refeeding on SPARC
mRNA expression. A, after an overnight fast, OF1
mice were divided into three groups (n = 3 each). One
group served as a fasting control and was injected intraperitoneal with
saline, whereas the other groups were injected intraperitoneal with
insulin. 2 or 4 h later, mice were euthanatized, epididymal fat
pads were removed, and total RNA was isolated and analyzed for SPARC
expression by Northern blot (15 µg of RNA per lane). The blot was
subsequently probed with GAPDH and the product of ob gene as
controls. B, OF1 mice were divided into four groups
(n = 3 each). A fed group was allowed free access to
food. The other groups were fasted for a 24-h period. At the end
of the cycle two groups of fasted mice were refed with chow diet or
with high fat diet for 6 h whereas the other group served as a
fasting control. 15 µg of adipose tissue RNA from each group was used
for analysis of SPARC and leptin (ob gene) mRNA
expression by Northern blot. A GAPDH cDNA probe was used as a
control.
1, which has been shown to
induce PAI-1 expression in this tissue (38, 39). Samples of whole
adipose tissue were exposed to purified SPARC or TGF-
1 for 6 h,
and PAI-1 mRNA expression was analyzed by Northern blot (Fig.
6). Addition of SPARC led to a 1.8-fold
increase in PAI-1 mRNA accumulation after correction for
variability in the 18 S rRNA signal (lane 2), and this
increment was comparable with that obtained with TGF-
1 (lane
3). Our observation demonstrates that the adipose tissue responds
to exogenous SPARC and that SPARC may function as a novel signaling
molecule for this tissue.
View larger version (44K):
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Fig. 6.
Effect of SPARC on PAI-1 mRNA expression
in cultured adipose tissue. Rat adipose tissue was cultured in
medium alone (control) or medium containing 20 µg/ml SPARC or 10 ng/ml TGF- 1 for 6 h. 15 µg of total RNA isolated from each
sample was analyzed for PAI-1 mRNA expression by Northern blot. RNA
was hybridized with an 18 S rRNA probe as a control. Fold increase was
determined after densitometric scanning of the representative
autoradiogram. Values (lane 2, × 1.8; lane 3, × 1.9) were obtained after normalization to the signal obtained with the
18 S rRNA probe.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and TGF
-1 (38, 39,
41). Here we show that SPARC could be a regulator of PAI-1 expression
also in this tissue. More importantly, our findings suggest that
overproduction of SPARC by the adipocyte could be an event that
contributes to increased PAI-1 levels in conditions associated with
obesity. Whereas the relationship between SPARC and PAI-1 production by
adipose tissue has to be further analyzed, it is likely that aberrant
expression of SPARC may contribute to the impairment of the
fibrinolytic system observed in obesity. The mechanism(s) by which
SPARC affects the expression of PAI-1 is at present unknown. One
hypothesis is that SPARC could exert its effects through a
TGF
1-dependent pathway. Indeed, it was shown that SPARC
regulates TGF
1 expression in mesangial cells (24). However, we were
unable to detect a change in TGF
1 mRNA levels in our
experimental system and under the conditions tested (data not shown).
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ACKNOWLEDGEMENTS |
---|
We thank Georges Manfroni for his expertise in animal care, and Dr. Marcel Deckert and Dr. Pascal Peraldi for discussion and critical review of the manuscript. We also thank Dr. Johan Auwerx and Dr. Christian Dani for providing cDNAs.
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FOOTNOTES |
---|
* This work was supported in part by the Institut National de la Santé et de la Recherche Médicale, Université de Nice-Sophia-Antipolis, a grant from Groupe Merck-Lipha (Lyon, France), and Research Technical Development Program Grant QLGI-CT-1999-00674 from the European Community.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
33-4-93-81-54-47; Fax: 33-4-93-81-54-32; E-mail: tartare@unice.fr.
§ Recipient of a doctoral fellowship from the Ministère de l'Enseignement Supérieur et de la Recherche (France).
¶ Supported by Merck-Lipha (Lyon, France).
Published, JBC Papers in Press, April 9, 2001, DOI 10.1074/jbc.M010634200
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ABBREVIATIONS |
---|
The abbreviations used are:
PAI-1, plasminogen
activator inhibitor-1;
SPARC, secreted protein acidic and rich in
cysteine;
TGF-1, transforming growth factor-
1;
GTG, goldthioglucose;
SSH, suppression subtractive hybridization;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
PCR, polymerase chain
reaction;
RT-PCR, reverse transcription-PCR;
ELISA, enzyme-linked
immunosorbent assay.
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