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Introduction
Resistin, a novel, cysteine-rich hormone mainly secreted
by adipose tissues, has been implicated in obesity and type
2 diabetes[1]. Recent research demonstrated that
recombinant resistin protein impaired insulin action in normal mice
and cultured adipocytes and immunoneutralization of resistin
improved insulin action in mice with diet-induced
obesity[1]. Plasma resistin levels were increased in
db/db, ob/ob and diet-induced obese
mice[1], while resistin mRNA levels in obese rodents were often found to be
decreased[2_ 4]. Resistin is also believed to be a thiazolidinedione (TZD)-regulated
protein, a new class of insulin sensitizing drugs, as TZD
treatment suppressed resistin expression in 3T3-L1
adipo-cytes and in white adipose tissues of mice fed with a high-fat diet[1].
The pathophysiological role of resistin in human has not
been fully elucidated. The putative human homologue of
resistin is only 59% identical to mouse resistin at the amino
acid level. The source of resistin appears to differ between
human and mice[1,5], with research suggesting macrophages,
while adipocytes in mice, are the principal source of resistin
in human. This reinforces the notion that adipose tissue,
rather than simply adipocytes, functions as a dynamic
endocrine organ. Resistin (also called "found in inflammatory
zone 3", FIZZ3) has been implicated in a low-grade
inflammatory condition associated with
obesity[6]. Increased plasma resistin concentration has been observed in
obese[7] and diabetic
people[8]. Moreover, TZD treatment resulted in
decreased plasma resistin concentration in patients with type
2 diabetes[9], suggesting resistin plays an important role in
the etiology of insulin resistance and
diabetes[10].
In our study, we utilized the single commercial resistin
protein to examine its effects on glucose uptake in skeletal
muscle cells. Additionally, considering the higher
expression of the resistin gene in human macrophages, and the fact
that some rodent adipocytes may make a more complex
condition (eg the alteration of cytokines secretion, the different
polymeric forms of resistin[11]) besides the increased resistin
secretion, we applied the cultured supernatant from 293-T
cells transfected with resistin-expressing vectors to further
assess the effect of the complex condition on glucose
uptake in skeletal muscle cells.
Materials and methods
Antibodies Monoclonal antibody (6×His) was purchased
from Clontech (Mountain View, CA, USA). Monoclonal
Antibody (anti-flag) was from Sigma (St Louis, MO,
USA). Primary polyclonal GLUT1, GLUT4, and SNAP23 antibodies
and horseradish peroxidase-conjugated secondary
antibodies were purchased from Santa Cruz (Santa Cruz, CA,
USA). IRS-1 polyclonal antibody was from Cell Signaling (Danvers,
MA, USA). Phospho-specific polyclonal antibody against
IRS-1 (Tyr612) was from Biosource (Camarillo, CA,
USA). These primary antibodies were respectively diluted for
Western blotting [resistin (anti-His), 1:8000, (anti-Flag), 1:1000;
GLUT4, 1:700; GLUT1, 1:600; IRS-1, 1:800; Phospho-IRS-1
Tyr612, 1:500; SNAP23, 1:800], as well as all secondary
antibodies (1:2500).
Plasmid construction Full-length rat Resistin were
amplified by reverse transcriptase PCR (RT-PCR, 58 ºC, 33
cycles). Primer sequence were forward, 5'-GCA GGA TCC
ACC ACC ATG AAG AAC CTT TCA T-3', and reverse, 5'-TAT CTC GAG CGG GAA CCA ACC CGC-3', and contained
BamH I and the Xho I sites, respectively. The PCR products
were then subcloned into the eukaryotic expression vector
pcDNA3.1Myc/His(B) (Invitrogen, Carlsbad, CA, USA).
Cell culture and transient transfection 293-T cells were
cultured in Dulbecco's modified Eagle's medium (DMEM)
containing 10% fetal bovine serum (FBS), 100
µg/mL penicillin and streptomycin until reaching 70%_80%
confluence. The cells were then cultured with serum free media (SFM,
MP Biomedicals, Seven Hills, NSW, Australia) and
transfected with expression vectors, utilizing
FugeneTM 6 transfection reagent (Roche, Basel, BS, Switzerland). To confirm
transfection, the 293-T cell supernatant was collected 24 h
post-transfection, centrifuged (700 g for 5 min) then probed
for resistin expression by Western blotting, together with
the single commercial resistin. Recombinant resistin
concentration in the cultured supernatant was quantified by
ELISA (YuanXiang Medical Instruments, Shanghai, China).
Differentiation of myoblasts L6 rat myoblasts
(ATCC, Manassas, VA, USA) were maintained in DMEM
supplemented with 10% FBS and differentiated into myotubes by
exposure to DMEM supplemented with 2% FBS.
Myogenic differentiation to myotubes was confirmed morphologically
and biochemically as previously
described[12]. Morphological differentiation parameters (alignment, elongation, and
fusion) were assessed by light microscopy after staining
with May-Grünwald Giemsa (JianCheng Biochemical Institute,
Nanjing, China). Myogenic differentiation was determined
biochemically by measuring creatine kinase (CK) activity
using a spectrophotometric-based kit (JianCheng
Biochemical Institute, Nanjing, China).
2-Deoxyglucose uptake assay Myotubes were cultured
in 24-well plates and treated with single commercial resistin
(130 ng/mL , 0_24 h, Alexis, San Diego, CA, USA) or cultured
supernatant (0_24 h) contained in conditioned medium (50%
cultured supernatant from 293-T cells transfected with
resistin-expressing vectors and 50% SFM), or without them
for periods of 0_24 h, then incubated for 15 min with or
without insulin (10 nmol/L). Uptake of
2-deoxy-D-[3H]glucose (CIC, Beijing, China) was assayed for 10 min as previously
described[13] with minor
modifications. Briefly, the cells were washed with ice-cold phosphate-buffered saline, and then
200 µL NaOH (1 mol/L) was added to each
well. Aliquots of the cell lysate were transferred to the scintillation vials for
radioactivity counting and the remainder was used for the
protein assay. Non-specific uptake was determined in the
presence of cytochalasin B (10 µmol/L) and was subtracted
from all values.
Western blotting L6 myotube cells were grown in 6-well
plates, treated with single resistin, cultured supernatant
containing recombinant resistin, or without them for 2 h
followed with or without insulin (100 nmol/L, 15 min).
The total or phosphorylated protein was extracted as previously
described[14]. The plasma membrane (PM) protein was extracted
using the Eukaryotic Membrane Protein Extraction Reagent
(Pierce, Rockford, IL, USA). After SDS-PAGE, the proteins
(20 µg/lane) were electrophoretically transferred to a
nitrocellulose membrane (Whatman, London, UK). Blocked with
TBST (Tris-Buffered Saline Tween-20; 0.14 mol/L NaCl, 0.02
mol/L Tris base, pH 7.6, and 0.1% Tween) containing 3%
BSA (Bovine serum albumin) for 1 h at room temperature, the
membrane was hybridized with primary antibodies at a
appropriate dilution at 4 ºC overnight.
The membrane was then washed with TBST for 5 min and repeated 5
times. Then, the membranes were incubated with horseradish
peroxidase-conjugated secondary antibodies for 1 h at room temperature,
washed with TBST and developed with ECL (Enhanced chemiluminescence; Amersham, Picataway,
UK).
Protein assay The total protein content of the cell
extracts or cultured supernatant was determined with
BCATM Protein Assay Reagent (Pierce, Rcokford, IL, USA).
Statistical analysis All data are expressed as mean±SD.
Data were analyzed by one-way ANOVA or Student's
t-test utilizing the SPSS 10.0 statistic software package (SPSS Inc,
Chicago, IL, USA) with P<0.05 considered significant.
Results
Identification of single commercial resistin and resistin
in culture supernatant of resistin-transfected 293-T cells
Single commercial resistin and resistin in culture
supernatant of resistin transfected 293-T cells were identified by
Western blot analysis. They both existed mainly as trimers
(Figure 1). The resistin concentration of the culture
supernatant was 260 ng/mL quantified by ELISA.
L6 myoblast differentiation Morphological changes to
myotube formation in L6 myoblasts were noted 7 d following
the induction of myogenic differentiation.
May-Grünwald Giemsa staining data suggested that over 90% of the cells
contained more than 1 nucleus (Figure 2A). CK activity
increased gradually after the induction of differentiation
(Figure 2B).
Effects of resistin on basal and insulin-stimulated
glucose uptake in L6 myotubes Resistin decreased basal
and insulin-stimulated glucose uptake, with a significant
decrease elicited by resistin at as little as 2 h (Figure 3A, 3B).
An approximate 25% decrease in basal glucose uptake was
observed in L6 myotubes after 24 h resistin treatment.
Although insulin increased basal glucose uptake, the
magnitude of the response was significantly attenuated
(approxi-mately 1.81 and 1.1-fold in the control and the 24 h
resistin-stimulated cells, respectively). Glucose uptake following
insulin exposure was reversed to lower the basal values when
treated with resistin for 2, 6, 12, and 18 h. A trend towards
partial recovery of the insulin response at later time-points
was observed. Additionally, the exposure of the cells to the
cultured supernatant containing recombinant resistin, at a
concentration of 130 ng/mL in our research, had a greater
effect on inhibition of basal and insulin-stimulated glucose
uptake (Figure 3C). Resistin treatment did not affect cell
viability as assessed by
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium
bromide (MTT) assay (control: 1.0±
0.07; single resistin: 0.96±0.08; cultured supernatant:
0.96±0.07).
Effects of resistin on basal and insulin-stimulated GLUT4
translocation to the PM We consequently examined the
effects of resistin treatment on GLUT4 translocation to the
PM in response to insulin in L6 myotubes. The results
demonstrated that resistin decreased insulin-stimulated GLUT4
translocation to the plasma, but did not alter basal GLUT4
translocation (Figure 4). Our data suggested that 2 h resistin
treatment did not alter the total content of either GLUT4
(control: 1.0±0.18; single resistin: 1.0±0.17; cultured
supernatant: 1.03±0.19) or GLUT1 (control: 1.0±0.2; single
resistin: 1.02±0.2; cultured supernatant: 1.09±0.21) in L6
myotubes (Figure 4A, 4C).
Effects of resistin on insulin-stimulated IRS-1 tyrosine
phosphorylation We examined the IRS-1 tyrosine
phosphorylation status to further elucidate the effects of resistin (2 h)
on IRS-1 in L6 myotubes. As seen in Figure 5, single resistin
greatly inhibited insulin-stimulated IRS-1 tyrosine
phosphorylation; meanwhile, the cultured supernatant
containing recombinant resistin resulted in a much greater
inhibition at a concentration of 130 ng/mL in our research, both
without affecting the total IRS-1 protein content.
Effects of resistin on SNAP23 expression
SNAP23 is a key SNAREs (SNAP receptors) component and is required
for docking and/or fusion of intracellular vesicles to the
PM. As shown in Figure 6, resistin treatment (2 h) decreased the
SNAP23 total content in L6 myotubes, in the presence or
absence of insulin. There was an approximate 0.23-fold
decrease in protein content, relative to the control, resulting
from exposure to resistin, while a 0.35-fold decrease was noted
following exposure to the cultured supernatant
treatment. Our results also suggested that insulin did not alter SNAP23
protein content (Figure 6).
Discussion
In the current study we examined the effects of resistin
and cultured supernatant containing recombinant resistin
on glucose uptake in rat skeletal muscle cells.
Considerable debate exists regarding the role of resistin in the
pathophysiology of insulin resistance in human and animals, and
whether resistin acts primarily in muscles, liver or
fat[15_18]. Recent
research [19], however, implicates resistin function in
skeletal muscles, for example; adenoviral overexpression of
murine resistin, at supraphysiological concentrations for
7 d, resulted in glucose intolerance, hyperinsulinemia, and
an impaired ability of insulin to lower blood glucose in male
Wistar rats. Furthermore, the inhibition of resistin activity in
transgenic mice expressing a dominant inhibitory version of
the protein improved insulin sensitivity and glucose
tolerance in mice[17], while the injection of resistin into mice
resulted in impaired glucose tolerance and insulin
action[1].
Here we investigated the effect of resistin on basal and
insulin-stimulated glucose-uptake in skeletal muscle cells,
with resistin treated for 0_24 h. We observed an inhibition
of basal or insulin-stimulated glucose uptake in L6 rat
skeletal muscle cells when incubated with resistin. These
results were similar to those noted following acute resistin
treatment in L6 rat skeletal muscle
cells[20]. Furthermore, we measured the level of GLUT4 on the plasma membrane in L6
cells. It is well known that insulin-stimulated glucose uptake
is mediated by the translocation of insulin-sensitive glucose
transporters (GLUT4) from intracellular vesicles to the PM.
Our results indicated that the effect of resistin on
insulin-stimulated glucose uptake involved a decrease in the extent
of translocation of GLUT4 to PM. Trying to explain the
decreased basal glucose uptake, we found no overall change
in the GLUT1 or GLUT4 protein content.
To further investigate the mechanism of how resistin
decreased insulin-stimulated GLUT4 translocation and
glucose uptake, we examined the protein content and tyrosine
phosphorylation level of IRS-1, which plays a central role in
metabolic effects of insulin[21]. Intriguingly, resistin
decreased insulin-stimulated tyrosine phosphorylation of
IRS-1, without changes on IRS-1 total protein content. It
has been previously reported that the insulin-activated PI3K
(Phosphoinositide Kinase-3)-Akt/PKB (Protein Kinase B)
pathway resulted in a translocation of GLUT4 from the
cytosol to the PM[22]. Research has also suggested that
Tyr612 phosphorylation of IRS-1 generates the major docking site
for PI3K mediated insulin signaling[23]. Based on previous
work documenting the effect of Tyr612 phosphorylation
status on insulin signaling[24], we speculated that
the down-regulated level of phosphorylated Tyr612 in L6 cells
incubated with resistin might impair the IRS-1/PI3K-Akt
signaling pathway activation in response to insulin, suggesting a
potential mechanism by which resistin decreases
insulin-stimulated GLUT4 translocation and glucose uptake.
A decrease of basal glucose uptake was observed,
although no changes were noted in the content of GLUT4
and GLUT1 on the plasma membrane. To further elucidate
this effect, we investigated SNAP23 protein content and
found that its expression was downregulated when treated
with resistin. It has been suggested that SNAP23 promotes
the docking and fusion of secretory vesicles to the PM and
controls vesicle transport by retention mechanisms and
dynamic sorting[25,26]. We speculated that downregulated
SNAP23 might decrease the rate of the GLUT4 vesicle
recycling through the cytosol to the PM, Then, the downregulated
recycling rate noted in the GLUT4 vesicles lead to a decreased
cyclic utilization rate of GLUT4 for glucose transport, per
unit time; no variability was noted in the PM protein content
of GLUT4.
As mentioned above, 2 kinds of research systems, single
resistin and supernatant containing recombinant resistin from
transfected 293-T cells, were performed and both acquired
similar results. However, interestingly, the incubation of L6
cells with the cultured supernatant containing recombinant
resistin from transfected 293-T cells, at a concentration of
130 ng/mL in our research, resulted in a greater inhibition of
glucose uptake, SNAP23 expression, and Tyr612
phosphorylation of IRS-1, compared to single resistin treatment (130
ng/mL). As SNAP23, IRS-1 and glucose uptake, several main
steps in this bio-pathway, from up to down, were different.
We inferred it might just be due to the difference between
the single resistin and the cultured supernatant. We
proposed 2 possible explanations: first, the different polymer
styles of resistin (different polymer styles of resistin
obviously had different
bio-activity[11]); the other is the cultured
supernatant which included some cofactors of resistin.
However, in our research, the main polymer styles of resistin
protein in the 2 research system were both trimers, and we
believed the co-factors worked. Compared with the single
commercial resistin that might just act as a cytokine working
outside of skeletal muscle cells, resistin-transfected 293-T
cells might have both secreted resistin in cultured
supernatant and prior-secreted resistin in cells. Moreover, previous
research[11] in our laboratory suggested that the resistin
protein contained a putative leucine zipper, which may also act
as a transcription factor to regulate the expression of genes
associated with metabolism. In this way, the prior-secreted
resistin could lead to consequent changes in the cultured
supernatant. The consequent changes, together with the
secreted resistin, would play roles on skeletal muscle cells
when the cultured supernatant was added. These data lead
us to believe that the cultured supernatant from transfected
293-T cells contained additional effectors that facilitated the
physiological effects of resistin. Further research, however,
is needed to verify this hypothesis.
In summary, our results demonstrated that resistin
inhibited basal and insulin-stimulated glucose uptake. Our
results also suggested these effects were mediated by tyrosine
phosphorylation of IRS-1 and increased SNAP23 protein
content or as some yet-unidentified mediators. Our results
also showed that the inhibitory effects of glucose uptake
following exposure to cultured supernatant containing
recombinant resistin from transfected 293-T, at concentration
of 130 ng/mL in our research, were greater than those noted
in resistin-treated L6 cells. Our data demonstrated that resistin
is a potential direct regulator of glucose homeostasis.
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