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Introduction
Traditionally, adipose tissue is only considered to be
energy storage. When energy is required, free fatty acids
(FFA) are released from adipose triglyceride stores into the
circulation by lipolysis and
oxidization[1]. Recently, adipose tissue has also being recognized as an important endocrine
organ with the discoveries of several adipocyte-secreted
molecules. Using these molecules, adipocytes are able to
communicate with other tissues and organs to regulate lipid
and glucose metabolism, energy balance and insulin
action[2,3].
Resistin (Retn, NM_022984) is a newly discovered
adipo-cyte hormone (adipocytokine) involved in the development
of insulin resistance[4]. Several studies have shown resistin
as a potential link between obesity and type 2 diabetes
mellitus (T2DM)[5,6]. Recently, direct functional effects of resistin
on some of insulin target organs were described, such as L6
skeletal muscle cells, 3T3-L1 adipocytes and the
liver[7_9].
Resistin-binding peptide (RBP) was coded by 3 cDNA
fragments identified by screening a cDNA phage display
library of rat multiple tissues. Three cDNA fragments have
the same 11 bp 5' sequence. Studies have shown that RBP
can bind to resistin specifically. Overexpression of RBP in
3T3-L1 pre-adipocytes can inhibit the stimulatory role of
resistin on adipocytes
differentiation[10]. All these data suggest that RBP may be the therapeutic blockers of insulin
resistance in diabetic patients.
However, no data are currently available on potential
direct paracrine or autocrine effects of resistin on adipose tissue,
and there are no known reports on the relationship of RBP
and resistin. In this study, we examined the effect of rat
resistin protein (rResistin) on rat white adipose tissue (WAT)
in primary cultures to demonstrate direct actions of resistin
on lipid metabolism and adipocytokine expression. In
addition, we provide the evidence that RBP
administration can antagonize these roles of resistin.
Materials and methods
Adipose tissue culture conditions Epididymal adipose
tissue fragments from male Sprague-Dawley (180_220 g) rats
were placed in serum-free M199 (Gibco, Grand Island, NY,
USA). Minced tissue of known mass (150 mg) was plated
with 5 mL medium containing 30 ng/mL rResistin (Alexis,
San Diego, CA, USA) or combined with RBP (N-AWIL-C,
Sangon, Shanghai, China) with varying concentrations
(1×10-12 mol/L, 1×10-10
mol/L, 1×10-8 mol/L) in each 60 mm
Petri dish. The dishes were maintained at 37 °C and 5%
CO2: 95% O2. Culture medium supernatant and adipose tissue were
collected respectively from each dish after 24 h.
Measurement of lipolysis in WAT fat pads Adipose
tissue lipolysis was determined using FFA levels released into
the culture medium supernatant[11]. FFA in conditioned
medium were measured using an available colorimetric kit
(Randox laboratories Co, Antrim, UK). Each experiment was
repeated 6 times.
Measurement of adipocytokine levels secreted in WAT
The levels of TNF-α and adiponectin were measured by ELISA
kit. In brief, we dispensed 100 µL of standards and
specimens into appropriate wells and dispensed 50 µL of enzyme
conjugate reagent into each well. We incubated the wells at
18_25 °C for 90 min, and rinsed and emptied the microtiter
wells with washing buffer. After washing 5 times, we removed
residual water droplets and dispensed 50 µL of color A and
color B reagent into each well. We gently mixed for 5 s and
incubated it at 18_25 °C for 15 min. The reaction was stopped
by adding 50 µL of stop solution to each well and then
gently mixed for 30 s. The optical density was recorded at 450 nm
within 20 min. Each experiment was repeated at least 3 times.
RT-PCR assay for TNF-α and adiponectin RNA was
prepared from WAT with Trizol reagent (Invitrogen, Carlsbad,
CA, USA) according to the manufacturer's instructions. The
RNA content was quantified by UV spectrophotometer at
260 nm, and samples were separated by electrophoresis on
1% agarose gels, stained with 0.1 mg/L ethidium bromide
and visualized under UV light for confirmation of RNA
integrity. Total RNA of 1 µg was reverse transcripted into
first-strand complementary DNA (fscDNA) by using an
Oligo(dT)15 primer (Promega, Madison, WI, USA). The fscDNA
was amplified by PCR. The PCR was carried out in a total
volume of 20 µL, containing 20 mmol/ L Tris-HCl, 50 mmol/L
KCl, 1.5 mmol/ L MgCl2, 0.2 mmol/ L dNTP (Promega, Madison,
WI, USA), 0.6 mmol/L of each primer, and 2.5 units of
Ex-Taq DNA polymerase (TaKaRa, Dalian, Liaoning, China). The
mRNA expression of the housekeeping gene β-actin was
used as an internal standard. Primer sequences and PCR
reaction conditions are shown in Table 1. Denaturing,
annealing, and extension reactions were performed at 94 ºC
for 30 s, at Ta ºC for 30 s, and at 72 ºC for 1 min. The PCR
pro-ducts were analyzed on 1.5% agarose gel and stained with
ethidium bromide. The levels of gene mRNA were expressed
as the ratio of the gene to β-actin.
Statistical analysis All data are expressed as mean±SEM.
Data were analyzed using one-way ANOVA of the SPSS 10.0
statistic software package (Spss, Chicago, IL, USA). The
threshold of significance was defined as
P<0.05.
Results
rResistin and RBP regulates lipolysis in WAT fat pads
ex vivo Lipolysis of adipose tissue was determined using
FFA released in the culture medium supernatant. As shown
in Figure 1A, rResistin increase FFA release in fat pads from
Sprague-Dawley rats after 24 h, where resistin was a strong
stimulation of lipolysis. To ascertain whether this effect was
antagonized or enhanced by RBP, a similar experiment was
performed combined with varying concentrations of RBP.
As shown in Figure 1B, FFA released in the fat pads
decreased after RBP was added as expected, whereas there
was no difference between the 3 concentrations of RBP
(P>0.05).
Effects of rResistin and RBP treatment on
TNF-α expression in WAT It has been demonstrated that
TNF-α is an important pro-inflammatory adipocytokine and can lead
to insulin resistance[12]. Studies have found that it activates
resistin mRNA expression in
vitro[13]. To determine whether resistin is capable of direct activating the expression of
TNF-α in WAT, we examined its protein secretion and gene expression after exposure 24 h to rResistin. As shown in
Figure 2A and 2B, the incubation of adipose tissue with
rResistin resulted in the induction of TNF-α. As expected,
TNF-α secretion decreased after RBP was added into the
medium, indicating RBP can antagonize the role of resistin
on TNF-α, but this inhibitory effect was limited to RBP with
the higher concentration of 1×10-8
mol/L. Moreover, the
expression change of genes was consistent with the protein
level (Figure 3).
Effects of rResistin and RBP treatment on adiponectin
expression in WAT Given a direct negative correlation
between adiponectin and insulin
resistance[14], we investigated whether resistin itself directly affects the production
of adiponectin in adipose tissue. Analysis of the level of
adiponectin in the culture supernatant by ELISA after
treatment for 24 h with rResistin revealed decreased secretion in
primary cultures of in vitro adipose tissue (Figure 4A).
Consistent with the protein level, adiponectin mRNA expression
was also significantly downregulated (Figure 5). In contrast,
the combined treatment with RBP resulted in an increase of
protein secretion (Figure 4B) and gene expression (Figure 5).
Although there was no difference between the 3
concentrations of RBP, there was a concentration-dependent increase.
Discussion
Globally, the prevalence of obesity is escalating, and
insulin resistance resulting from increased adipose tissue mass
has been identified as a key factor that could drive parallel
rises in T2DM prevalence[15]. Although it is not yet entirely
clear how obesity produces insulin resistance/T2DM, it has
become clear that a failure of lipid homeostasis plays an
important role[16].
We suggest here that resistin, a newly found
adipocyto-kine in adipose tissue, influences adipose tissue lipid
metabolism. It is well known that resistin is expressed mainly
in WAT in rodents. The expression of resistin mRNA in
adipose tissue and protein levels in circulation were
significantly elevated in diet-induced obesity mice and several
obese animal models, including db/db and
ob/ob mice[4]. These initial observations strongly suggest that resistin would play
important roles in adipocytes and adipose tissues, and its
abnormal expression would be linked to lipid metabolism
disorders frequently found in obese animals. Therefore, our
studies imply that overproduced and secreted resistin from
obesity has direct autocrine effects on adipose tissue.
Recently, many studies have suggested that overproduced
FFA from adipocytes cause peripheral (muscle, liver) insulin
resistance, which are linked with metabolic diseases such as
T2DM and cardiovascular disease[17,18]. Thus, our
experiments strongly suggest that an abnormal increase of resistin
in obese subjects would promote circulation plasma FFA
release, which might eventually accelerate lipolysis and
contribute to the onset of insulin resistance.
In this study, we also demonstrate that administration of
RBP decreases high levels of FFA, although there was no
obvious correlation between FFA levels and RBP
concen-tration. Recently, evidence has suggested that drugs that
cause a sustained reduction in elevated plasma FFA
concentration may represent an effective modality for the
prevention of T2DM[19]. A similar idea has been demonstrated that
the regulation of fatty acid metabolism may be a target for
obesity treatment[20]. From the above points, our results
indicate that RBP can antagonize the role of resistin in fatty
acid metabolism and may have a potential role in improving
insulin resistance by decreasing FFA release.
In spite of these findings, it has been poorly
demonstrated that resistin is able to affect the functions of adipose
tissues, and RBP can antagonize the roles of resistin on the
development of insulin resistance. As a major tissue for
whole-body energy homeostasis, many increasing studies
have found that adipose tissue is also an important
endocrine organ for expressing various adipocytokines,
including leptin, resistin, TNF-α, adiponectin and
interleukin-6[2,3]. It is well known that certain adipocytokines are important in
provoking the insulin resistance found in many obese and
diabetic subjects. For example, a positive relationship between obesity, insulin resistance and adipose tissue mRNA
levels of TNF-α has clearly been established in rodent
models[12]. An increased level of TNF-α in obese states leads to
insulin resistance via a number of mechanisms, including
decreased insulin-stimulated glucose transport or insulin
signaling[21,22]. Unlike TNF-α, adiponectin expression levels
are inversely correlated with obesity, T2DM and
atherosclerosis[23,24]. Furthermore, increasing evidence has
demonstrated that adiponectin is a key player in the
insulin-sensitizing mechanism[14].
In this paper, we demonstrate that the expressions of
TNF-α and adiponectin are changed in resistin treated
adipose tissues. Treatment with resistin in WAT elevated the
protein secretion and mRNA expression of TNF-α. In contrast,
both protein secretion and mRNA expression of adiponectin
decreased. Therefore, the observations show that resistin
has a direct regulating effect on the expressions of
adipocytokines in adipose tissue, but the molecular mechanisms
underlying these results remain elucidated. In addition, the
previous idea that resistin would induce insulin resistance
in obesity was confirmed by our observation that resistin
treatment changed the levels of adipocytokines in adipose
tissue. Taken together, these results strongly suggest that
aberrantly high expression of resistin in adipose tissues of
obesity would result in insulin resistance not only by
increasing FFA levels, but also by modulating adipocytokine
expressions.
In accordance with the former results, our studies of RBP
on the endocrine function of WAT show that RBP has an
antagonizing role on resistin in adipose tissue endocrine.
However, our studies failed to find a positive correlation
between RBP concentration and its antagonizing role. The
results of adipose tissue gene expression changes is
consistent with its protein secretion, which suggests that
RBP may perform its roles though the regulation of gene transcription
in cells. Maybe, there were 2 mechanisms; first, as a specific
bind peptide of resistin, RBP could direct combine to resistin
in culture medium supernatant and inhibit the regulation
effect of resistin on adipose tissue endocrine function,
thereby modulates indirectly the expressions of
TNF-α and adiponectin. Second, RBP compounded in this experiment is
a short peptide and our previous studies have indicated that
RBP is able to change the subcellular localization of resistin
and inhibit resistin-induced differentiation of 3T3-L1
pre-adipocytes[10]. Thus, it is likely that RBP exerts its
antagonizing on resistin through going into adipocytes directly.
However, these 2 hypotheses require further investigation.
In summary, we have shown that resistin directly affects
the fatty acid metabolism and adipocytokine expressions of
WAT through the autocrine or paracrine pathway. Our
findings that resistin treatment elevates FFA release and changes
adipocytokine expression levels in adipose tissue provides
strong evidence that overproduced resistin in obesity
induces lipid metabolism disorders and insulin resistance.
Nevertheless, this study provides the first evidence that RBP
is able to antagonize these effects of resistin on adipose
tissue. Further work is needed to investigate the mechanisms
underlying these effects.
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