Introduction
White adipose tissue is innervated by sensory nerves
(1–5), however, the roles they exert in this
tissue remain to be fully elucidated (1,2,5). It
may be possible that these sensory innervations inform the central
nervous system of the correct size of fat stored in the peripheral
white adipose tissue. It was previously demonstrated that substance
P (SP) and calcitonin gene-related peptide are expressed in sensory
neurons (4,5).
SP is a conserved 11-amino-acid peptide (6) and is a member of the tachykinin
family of neurotransmitters (7).
Previous studies have defined the role of SP as a pain transmitter,
and it was demonstrated that SP and its specific receptor,
neurokinin 1 receptor (NK-1R), are expressed in nervous tissues
(8,9). However, a burgeoning body of evidence
has revealed that NK-1R is also expressed in a variety of
non-neuronal cell types including endothelial cells (10), monocytes (11), macrophages (11) and adipocytes (12). Therefore, novel roles identified
for SP in non-neuronal cells have been reported, including immune
modulation (13), mobilization of
bone-marrow-derived stem cells (14), wound healing (15,16),
and the regulation of insulin signaling (17).
Adipocyte dysfunction following the onset of insulin
resistance is associated with type 2 diabetes (18). These dysfunctions may contribute to
insulin resistance in the peripheral tissues, including adipose
tissue, through mechanisms including the release of non-esterified
fatty acids, glycerol, proinflammatory cytokines and proteins,
which induce the development of insulin resistance (19–21).
Notably, insulin resistance leads to a decrease in the uptake of
glucose and in the expression level of glucose transporter 4
(GLUT4) in adipose tissue (22,23).
Signaling pathways, which regulate energy
homeostasis, are associated with the development of insulin
resistance. The AMP-activated protein kinase (AMPK) is a key
protein associated with these signaling pathways (24). Indeed, AMPK is dysregulated in
animals and humans with type 2 diabetes, and its pharmacological
activation is one of the therapeutic targets which has been
identified for the treatment of this condition (25). The activation of AMPK following its
phosphorylation on residue Thr-172 occurs when intracellular ATP
levels decrease (26,27). Notably, AMPK promotes the
trans-localization of GLUT4 to the plasma membrane (28) and also increases the expression of
GLUT4 (29,30).
The level of SP varies under pathological
conditions, including type 2 diabetes. Previous studies
demonstrated that the level of SP in the serum from patients with
type 2 diabetes (31), in skin
biopsies from patients with types 1 and 2 diabetes (32), and in heart tissue from patients
with type 1 diabetes (33), is
markedly lower compared with the controls, although a previously
published study contradicted this evidence (34). Therefore, it is possible that the
decreased expression of SP in various different tissues from
patients with type 2 diabetes may be associated with pathological
features, including insulin resistance, through the regulation of
cellular functions in the peripheral tissues, including adipose
tissue. Although a previous study suggested that SP decreases the
insulin-mediated uptake of fatty acids in 3T3-L1 cells (12), the aim of the present study was to
investigate the role of SP in the process of lipid accumulation in
3T3-L1 cells during their differentiation into adipocytes in
response to a high concentration of glucose, under different medium
conditions and using different concentrations of SP.
Materials and methods
Cells and cell culture
The 3T3-L1 preadipocytes were purchased from
American Type Culture Collection (Manassas, VA, USA; cat. no.
CL-173) and the cells were maintained in Dulbecco's modified
Eagle's medium (DMEM; GE Healthcare Life Sciences, Little Chalfont,
UK) with 25 mM glucose, supplemented with 10% fetal bovine calf
serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA,
USA), 100 U/ml penicillin/100 μg/ml streptomycin (Gibco;
Thermo Fisher Scientific, Inc.). The cells were incubated at 37°C
in a humidified atmosphere, containing 5% CO2, and their
subcultures were performed at <70% confluence. The cells in
passage numbers P10 to P20 were used for subsequent
experiments.
Differentiation of 3T3-L1 preadipocytes
into adipocytes
The 3T3-L1 preadipocytes were cultured until they
reached 100% confluence under normal culture conditions. At 48 h
following the attainment of confluence, the cells were cultured
with DMEM containing 25 mM glucose and supplemented with 10%
heat-inactivated FBS, 5 μg/ml insulin, 5 μM
dexamethasone and 5 μM rosiglitazone (all from
Sigma-Aldrich, St. Louis, MO, USA) for 48 h. Subsequently, the
cells were incubated for 48 h with DMEM (25 mM glucose),
supplemented with 10% FBS and 5 μg/ml insulin. The medium
was subsequently exchanged with DMEM (25 mM glucose), supplemented
with 10% FBS on every other day for 4 days. If necessary, DMEM
containing a different concentration of glucose (5.5 mM) was used
for the differentiation of 3T3-L1 preadipocytes into
adipocytes.
Use of SP in experiments
SP was purchased from EMD Millipore (San Diego, CA,
USA; cat. no. 05-23-0600), and was prepared with 5% acetic acid
(Sigma-Aldrich). When required, SP was added to the 3T3-L1 cells at
various concentrations whenever the medium was exchanged.
5-Bromo-2′-deoxyuridine (BrdU)
incorporation assay
The 3T3-L1 cells were seeded onto fibronectin-coated
cover-slips (1 μg/ml) in 24-well plates at a density of
4×103 cells/well. The cells were initially incubated for
24 h with normal culture medium, prior to an incubation of 18–24 h
duration under conditions of serum starvation. The cells were
subsequently treated with SP for 48 h. For the final 6 h of the
incubation period, 20 μM BrdU (Sigma-Aldrich) was added to
the cells. The cells were subsequently prepared for the
immunocytochemical analysis using BrdU by fixation in 4%
paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA)
in phosphate-buffered saline (PBS) for 10 min on ice. The fixed
cells were incubated with 2 N HCl for 15 min at room temperature
and washed with PBS vigorously. Following permeabilization with
0.2% Triton X-100 (Affymetrix, Inc., Santa Clara, CA, USA), the
cells were treated with blocking solution (5% non-fat milk in PBS
with 0.1% Triton X-100) for 30 min at room temperature.
Subsequently, the cells were incubated with primary mouse
monoclonal anti-BrdU antibody (1:20, cat. no. #11-170-376-001;
Roche Diagnostics GmbH, Mannheim, Germany) for 1.5 h at room
temperature. Following three washes with 1% non-fat milk in PBS
with 0.1% Triton X-100, the secondary antibody, Invitrogen
Alexa-488 anti-mouse immunoglobulin G1 (Thermo Fisher Scientific,
Inc.), was added, and the cells were incubated for a further 45 min
at room temperature. Finally, the samples were mounted using
Invitrogen ProLong® Gold Antifade mounting solution with
4′,6-diamidino-2-phenylindole (Thermo Fisher Scientific, Inc.), and
left to dry overnight prior to observation. Images were captured
using a fluorescence microscope (DMI4000; Leica, Solms, Germany),
and the total number of cells and BrdU-positive cells were
counted.
RNA isolation and reverse
transcription-quantitative polymerase chain reaction (RT-qPCR)
SP (0, 10, 100 or 300 nM) was added to the 3T3-L1
cells on the initial day of differentiation, and the total RNA was
extracted from the cells on day 2 following the induction of
adipogenesis using the Invitrogen TRIzol™ reagent (Thermo Fisher
Scientific, Inc.), according to the manufacturer's protocol.
Aliquots of 5 μg total RNA were used for single-strand cDNA
synthesis using the Invitrogen Superscript First-Strand cDNA
Synthesis system (Thermo Fisher Scientific, Inc.), according to the
manufacturer's protocol. RT-qPCR was performed using the Invitrogen
Power SYBR Green PCR Master mix (Thermo Fisher Scientific, Inc.).
The ribosomal protein 36B4 gene from the mouse was used as an
endogenous control. The following primers were used to detect the
expression of peroxisome proliferator-activated receptor-γ
(PPAR-γ), adipocyte protein 2 (aP2) and 36B4 protein: PPAR-γ,
sense: 5′-CGC TGA TGC ACT GCC TAT GA-3′ and antisense: 5′-AGA CCT
CCA CAG AGC TGA TTCC-3′; aP2, sense: 5′-CAT GGC CAA GCC CAA CAT-3′
and antisense: 5′-CGC CAA GTT TGA AGG AAA TC-3′; 36B4, sense:
5′-GAA CAT CTC CCC CTT CTC CTT-3′ and antisense: 5′-GCA GGG CCT GCT
CTG TGAT-3′.
Oil Red O staining
To assess adipogenesis in the 3T3-L1 cells, Oil Red
O staining was performed on differentiated cells. Oil Red O
solution (0.3%; Sigma-Aldrich) was prepared by dissolving Oil Red O
in 60% isopropanol (Daejung Chemicals & Metals, Co., Ltd.,
Shiheung, Korea). The differentiated cells were fixed in 4%
paraformaldehyde (Electron Microscopy Sciences) in PBS for 10 min
at room temperature, and subsequently washed with PBS. The fixed
cells were incubated with Oil Red O solution for 30 min at room
temperature. Images of the stained cells were captured using a
light microscope (DMI4000; Leica, Solms, Germany). To further
quantify the Oil-Red O-stained lipid drops, the stained cells were
rinsed twice with 60% isopropanol and subsequently dried. The
stains were eluted with 1 ml isopropanol for 10 min at room
temperature and the optical density was measured at 490 nm using an
absorbance plate reader (Spectramax190; Molecular Devices; Thermo
Fisher Scientific, Inc.).
Western blotting
The cells were rinsed twice with ice-cold PBS and
lysed with 2X SDS loading buffer [100 mM Tris-HCl (pH 6.8), 4%
(w/v) SDS, 0.2% (w/v) bromophenol blue, 20% glycerol and 200 mM
β-mercaptoethanol]. Subsequently, the cell lysates were denatured
at 92°C for 10 min. The denatured protein samples were separated
using 10% SDS polyacrylamide gel electrophoresis and transferred
onto nitrocellulose membranes (Whatman, Dassel, Germany. Following
blocking with 5% non-fat milk in 20 mM Tris buffer, containing 0.1%
Tween-20 (TBS-T), the membranes were incubated with primary
antibody diluted with TBS-T buffer, including 5% non-fat milk,
overnight at 4°C. The following primary antibodies were used:
Rabbit monoclonal anti-phosphorylated AMPK (1:1,000, cat. no.
#2535; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit
monoclonal anti-phosphorylated Akt (1:4,000, cat. no. #4060; Cell
Signaling Technology, Inc.,), rabbit polyclonal anti-GLUT4 (1:500,
cat. no. ab65976; Abcam, Cambridge, UK) and mouse monoclonal
anti-α-tubulin antibody (1:5,000, cat. no. T5618; Sigma-Aldrich).
Subsequently, the membranes were incubated with goat anti-rabbit
(1,5,000; cat. no. 7074; Cell Signaling Technology, Inc.) or goat
anti-mouse IgG (1:10,000; cat. no. 170-6516; Bio-Rad Laboratories,
Inc., Hercules, CA, USA) horseradish peroxidase-conjugated
secondary antibodies at room temperature for 30 min. The target
proteins were visualized using an enhanced chemiluminescence
detection kit (EMD Millipore). The band densities were measured
using ImageJ software (NIH, Bethesda, MD, USA). If required, the
stripping of the membranes was performed using Restore™ Western
Blot Stripping buffer (Thermo Fisher Scientific, Inc.) for 15 min
at room temperature. The membranes were reblotted using an antibody
raised against α-tubulin.
Statistical analysis
The data are expressed as the mean ± standard
deviation or the mean ± standard error of the mean. An unpaired
Student's t-test was used to evaluate differences between the two
groups. All statistical analyses were performed using GraphPad
Prism version 5.01 software (GraphPad Software, Inc., San Diego,
CA, USA; http://www.graphpad.com). P<0.05 was
considered to indicate a statistically significant difference.
Results
SP causes no effect on the proliferation
of the 3T3-L1 preadipocytes
A previous study demonstrated that SP increases the
cellular proliferation of mesenteric preadipocytes (35). Therefore, whether or not SP
affected the proliferation of the 3T3-L1 preadipocytes was examined
using a BrdU incorporation assay. The 3T3-L1 preadipocytes were
revealed to express NK-1R, which is a receptor of SP (data not
shown) (12). As shown in Fig. 1, SP caused no effect on the
cellular proliferation of 3T3-L1 cells, irrespective of the
concentration of SP.
 | Figure 1Cellular proliferation of 3T3-L1
preadipocytes treated with SP. (A) BrdU incorporation assays were
performed to examine whether SP affected the proliferation of
3T3-L1 preadipocytes. The 3T3-L1 preadipocytes were treated with
various concentrations of SP (0, 0.1, 1, 10, 100, 300 or 600 nM;
denoted in Fig. 1A as SP 0, SP
0.1, SP 1, SP 10, SP 100, SP 300 and SP 600, respectively). The
BrdU staining is in green and nuclei are illustrated by the
staining in blue. (B) Graph illustrating the quantitative analysis
of the data (expressed as the mean ± standard deviation). SP,
substance P; BrdU, bromodeoxyuridine; DAPI,
4′,6-diamidino-2-phenylindole. |
SP causes no af fect on the dif
ferentiation of the 3T3-L1 preadipocytes
Whether SP regulated the differentiation of 3T3-L1
preadipocytes into adipocytes was subsequently examined. The 3T3-L1
cells were incubated with the differentiation medium, which
included 10% FBS, 5 μg/ml insulin, 5 μM dexamethasone
and 5 μM rosiglitazone (36), for 2 days. SP failed to affect the
expression level of PPAR-γ or aP2 (Fig. 2), which were previously used as
markers for differentiated adipocytes (37). Therefore, it is possible that SP
does not promote the differentiation of 3T3-L1 cells into
adipocytes.
 | Figure 2Effect of SP on the expression levels
of PPAR-γ and aP2 in the 3T3-L1 cells. Reverse
transcription-quantitative polymerase chain reaction was performed
to observe the expression levels of (A) PPAR-γ and (B) aP2 in
3T3-L1 cells, which were treated with different concentrations of
SP (0, 10, 100 or 300 nM; illustrated by the labels SP 0, SP 10, SP
100 and SP 300, respectively) under the differentiation medium
conditions for 2 days. Ribosomal protein 36B4 gene was used as an
internal control. Two independent experiments were performed and
the quantitative results are shown as the mean ± standard error of
the mean. aP2, adipocyte protein 2; CON, control; PPAR-γ,
peroxisome proliferator-activated receptor-γ; SP, substance P. |
SP upregulates the accumulation of lipids
in the 3T3-L1 preadipocytes
Although SP failed to promote the differentiation of
the 3T3-L1 cells, it was hypothesized that SP may affect the
accumulation of lipids in these cells. Therefore, the effects of SP
on the accumulation of lipids in the 3T3-L1 adipocytes were
analyzed using Oil Red O staining. SP was added to the 3T3-L1 cells
every other day during the differentiation of the cells into
adipocytes. Compared with the undifferentiated control cells, the
accumulation of lipids increased markedly in the differentiated
cells without SP treatment (Figs. 3A
and B). Notably, the treatment with SP increased the
accumulation of lipids in the 3T3-L1 adipocytes by ~0.3-fold
compared with the untreated differentiated 3T3-L1 cells (Figs. 3A and B), even though SP failed to
promote the differentiation of 3T3-L1 cells into adipocytes
(Fig. 2). In addition, an
SP-mediated increase in the accumulation of lipids was observed in
the differentiated 3T3-L1 adipocytes, which were cultured with
medium, containing a high concentration of glucose (25 mM),
however, not in the cells which were cultured in medium containing
a normal concentration of glucose (5 mM; Fig. 3C). These results suggested that SP
may increase the accumulation of lipids in adipocytes in a manner
which is dependent on the concentration of glucose.
 | Figure 3Effect of SP on lipid accumulation in
the 3T3-L1 adipocytes. (A and B) The 3T3-L1 cells were cultured in
the absence or presence of 600 nM SP (SP 0 or SP 600) under the
differentiation medium conditions for 8 days. The accumulation of
lipids was analyzed using Oil Red O staining of the 3T3-L1
adipocytes maintained in the presence of a high concentration of
glucose (25 mM). (A) Representative images are shown (scale bar,
100 μm), and (B) illustrates the quantification of the
results (the mean values are indicated by the red horizontal bars;
*P<0.0001, compared with CON). (C) The accumulation
of lipids was analyzed by Oil Red O staining of the 3T3-L1
adipocytes, which had differentiated in the absence or presence of
600 nM SP (SP 0 or SP 600) under the differentiation medium
conditions, including the presence of either 5 or 25 mM Glu, for 8
days. Three independent experiments were performed, and the
quantitative results are shown as the mean ± standard error of mean
[*P<0.003, compared with SP 0; N.S, not significant
(P>0.6)]. Undifferentiated 3T3-L1 cells were used as the control
in all the experiments. CON, control; SP, substance P; OD, optical
density; Glu, glucose. |
SP regulates the activity of AMPK and
Akt, and the expression levels of GLUT4
It is well known that AMPK is a key regulator in the
metabolism of glucose and fatty acids in various cell types,
including adipocytes (27,38). Therefore, whether SP regulated the
activity of AMPK in the 3T3-L1 cells was examined. Indeed, SP was
revealed to induce the activation of AMPK in a dose-dependent
manner (Fig. 4A). Notably,
however, the activity of Akt was downregulated by SP (Fig. 4B). These results are in very good
agreement with a previous report, which demonstrated that the
regulation of AMPK activity is associated with the regulation of
Akt activity (39). It is also
known that AMPK promotes the translocalization of GLUT4 to the
plasma membrane (28), and
furthermore, that AMPK increases the expression level of GLUT4
(29,30). It is noteworthy that SP increased
the expression level of GLUT4 in 3T3-L1 cells (Fig. 4C). Therefore, these results
suggested that SP may modulate glucose uptake in 3T3-L1 adipocytes,
and that this is associated with the activity of AMPK.
 | Figure 4Effect of SP on the protein
expression levels of p-AMPK, p-Akt and GLUT4 in the 3T3-L1
adipocytes. (A–C) The 3T3-L1 adipocytes were treated with various
concentrations of SP (0, 100, 300 or 600 nM; denoted by the labels
SP 0, SP 100, SP 300 and SP 600, respectively). Western blotting
was performed to observe the phosphorylation levels of (A) p-AMPK
and (B) p-Akt, and (C) the protein expression level of GLUT4.
α-Tubulin was used as an internal control. The numbers underlying
the gel in (C) indicate the protein expression values relative to
the control (SP 10). p-, phosphorylated; AMPK, AMP-activated
protein kinase; GLUT4, glucose transporter 4; SP, substance P. |
Discussion
The present study has demonstrated the ability of
the neurotransmitter SP to increase the accumulation of lipids in
3T3-L1 preadipocytes in the presence of a high concentration of
glucose, although not under normal glucose conditions. This
increase in the accumulation of lipids was associated with an
SP-mediated increase in the expression level of GLUT4 following the
activation of AMPK.
AMPK exerts an essential role in the regulation of
glucose uptake by adipocytes and muscle cells (30,40,41).
A previous report demonstrated that the activation of AMPK
increases glucose uptake, upregulates the expression of GLUT4 in
3T3-L1 adipocytes, and that the activation of AMPK is independent
of the insulin receptor-mediated signaling pathway (41). The present study also suggested
that the SP-mediated activation of AMPK increased glucose uptake by
means of an increased expression of GLUT4 in the 3T3-L1 adipocytes.
Although it remains to be fully elucidated whether insulin
signaling functions in association with SP in order to increase the
accumulation of lipids in 3T3-L1 adipocytes (the differentiation
medium used in this study contained insulin), SP was able to induce
this effect only under high glucose conditions. Notably, SP
decreased the activation of Akt in the adipocytes. Akt activity is
required for the differentiation of mouse embryonic fibroblasts and
3T3-L1 cells into adipocytes (42,43),
and the presence of the constitutively active mutation of Akt is
sufficient to induce the differentiation of the 3T3-L1 cells
(44). However, it is also known
that the insulin-mediated Akt signaling pathway is not a major
pathway for the induction of glucose uptake by adipocytes,
according to a previous report (41). Therefore, Akt was not be expected
to be involved in the SP-mediated accumulation of lipids following
glucose uptake. In the present study, the SP-mediated increases
observed in the accumulation of lipids and in the expression level
of GLUT4 may occur via AMPK activation, as the exercise-mediated
activation of AMPK was revealed to increase glucose uptake,
following the intracellular translocation of GLUT4 to the plasma
membrane (45,46).
Defects in the innervation of adipose tissues impair
the formation of blood vessels, damage the architecture of adipose
tissues and reduce the insulin-sensitivity, and the metabolism of
adipocytes (47). The defects in
innervation also include defects in the sensory neurons that
express SP, which may be involved in the regulation of cellular
functions in the adipose tissues. The present study suggested that
SP may rescue insulin-sensitivity and glucose tolerance in the
adipose tissue of patients with diabetes through the AMPK signaling
pathway. In addition, SP may be useful for the therapeutic
treatment of diabetes in order to control insulin resistance.
Acknowledgments
The present study was supported by the Korean Health
Technology R&D Project (Ministry of Health and Welfare,
Republic of Korea; no. HI13C1479), the Basic Science Research
Program through the National Research Foundation of Korea funded by
the Ministry of Education (no. NRF-2012R1A1A2042265) and the Bio
& Medical Technology Development Program of the NRF funded by
the Ministry of Science, ICT & Future Planning (no.
NRF-2012M3A9C6050485).
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