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Research Article
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Effects of Fenugreek Seeds on Adipogenesis and Lipolysis in Normal and Diabetic Rats |
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Ahmad Ghorbani,
Mousa-Al-Reza Hadjzadeh,
Ziba Rajaei
and
Seyed Bamdad Zendehbad
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ABSTRACT
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Several studies support hypolipidemic effect of fenugreek
in normal and diabetic subjects. However, very little is known about the possible
direct action of fenugreek on adipose tissue. The present study was designed
to investigate the effects of fenugreek seeds on adipogenesis and lipolysis.
Preadipocytes were isolated from adipose tissue of normal rats and differentiated
to adipocyte in the presence of ethanolic extract of fenugreek seeds. The effect
of this extract on lipolysis was also evaluated in fat tissue isolated from
diabetic rats. Fenugreek led to a significant reduction in lipid droplet accumulation
as evaluated with Oil Red O staining. Incubation of preadipocytes with the extract
for 24 h resulted in significant decrease in cell viability. The extract, even
at high concentrations (up to 1000 μg mL-1), had virtually no
significant effect on lipolysis. The present data demonstrated that fenugreek
seed inhibits formation of new differentiated adipocytes from precursor cells
through an anti-proliferative effect on preadipocytes.
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Received: March 16, 2013;
Accepted: April 04, 2013;
Published: November 26, 2013
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INTRODUCTION
Adipose tissue is a highly dynamic organ that has an important role in regulation
of energy metabolism. The adipose mass depends on the number and the size of
adipocytes and is determined by the rate of preadipocytes proliferation; differentiation
of the preadipocytes into mature adipocytes; the balance between lipogenesis
and lipolysis within individual mature adipocyte; and the rate of adipocyte
apoptosis (Smith and Ravussin, 2006). Expansion of fat
mass can be occurring by an increase in both fat cell size and number of adipocytes.
Obesity is a result of body fat mass expansion and is still one of the main
health problems all over the world. Based on World Health Organization
reports, in 2008, about 1.4 billion people in the world suffer from overweight.
Also, it is estimated that 2.8 million men and women die annually from obesity
related diseases (WHO, 2013). Numerous studies have shown
that obesity increases risk of cardiovascular diseases, stroke, metabolic syndrome
and type-2 diabetes (Chiang et al., 2011; Lavie
et al., 2009). The mass of adipose tissue can be reduced by increasing
lipolysis, inhibiting adipogenesis and inducing apoptosis of fat cells (Rayalam
et al., 2008).
Several plants have been shown to have direct effects on lipolysis, preadipocytes
differentiation and adipocyte life cycle (Rayalam et
al., 2008; Andersen et al., 2010). Trigonella
foenum-graecum (fenugreek), an annual herb in the family Fabaceae, has a
long history for amelioration of abnormalities in glucose and lipid homeostasis
(Fatima et al., 2004; Ghorbani
and Rakhshandeh, 2012). Many studies support hypolipidemic effect of fenugreek
seed in diabetic subjects (Kassaian et al., 2009).
It has also reported that fenugreek reduces the body weight gain and hyperlipidaemia
induced by high-fat diet (Handa et al., 2005;
Al-Matubsi et al., 2011). However, very little
is known about the possible direct action of fenugreek on adipose tissue. Up
to now, decrease of fat accumulation in differentiating 3T3-L1 cell line is
the only report on action of fenugreek on adipocytes (Vijayakumar
et al., 2010). Therefore, the present study was designed to investigate
the effects of fenugreek seeds on adipogenesis and lipolysis in normal and diabetic
animals, respectively.
MATERIALS AND METHODS
Chemicals and reagents: Dulbeccos Modified Eagles Medium (DMEM)
and fetal calf serum was purchased from Gibco (Carlsbad, CA). Streptozotocin
(STZ) was obtained from Enzo Life (USA). Penicillin-streptomycin, type-II collagenase,
3-(4,5-Dimethyl-2-thiazolyl)-2,5- Diphenyl-2H-tetrazolium bromide (MTT), fatty
acid-free bovine serum albumin fraction V, 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic
acid sodium salt (HEPES), glycerol assay reagent, and isoproterenol were provided
from Sigma (USA). Dimethyl sulfoxide (DMSO) and 3-isobutyl-1-methylxanthine
(IBMX) were purchased from Fluka Chemical Co. Indomethacin and human insulin
were kindly provided by EXIR Company (Iran).
Preparation of extracts: The fenugreek seeds were purchased from a local
market. The seeds were cleaned and grounded to fine powder with a blender. The
macerated extract was prepared by suspension of 100 g of the powder in 70% ethanol
and incubation for 72 h at 37°C. The extract was then dried on a water bath
and the yield dissolved in DMSO.
Animals: Male albino Wistar rats (280-330 g) were used for each experiment.
They were housed in a room with controlled lighting (12 h light/12 h darkness)
and temperature (22 ±2°C). The animals were given standard pellets
diet and water ad libitum. All animal experiments were done according
to the ethical guidelines of the animal care of the Mashhad University of Medical
Sciences, Iran. Effect of fenugreek extract on adipogenesis and lipolysis was
studied on adipose tissue isolated from normal and diabetic rats, respectively.
For induction of diabetes, the animals were given a single dose of STZ (55 mg
kg-1, i.p.). Two days after STZ injection, induction of diabetes
was confirmed by measuring fasting blood glucose. Rats were considered to be
diabetic if they had blood glucose concentration of 250 mg dL-1 or
higher (Shafiee-Nick et al., 2012).
Preadipocyte preparation and culture: Subcutaneous adipose tissue sample
was excised from normal rats under ether anesthesia. The tissue was sliced into
small pieces and washed with phosphate-buffered saline. The tissue pieces were
then digested in phosphate-buffered saline containing collagenase (2 mg mL-1)
under shaking (60 cycles min-1) at 37°C. After centrifugation,
the floated adipocytes were discarded and the stromal cells were suspended in
DMEM medium supplemented with 10% fetal calf serum, 100 units mL-1
penicillin and 100 μg mL-1 streptomycin and seeded (104
cells/well) in 12-well plates. After 24 h of incubation, the medium was changed
into differentiation medium consisting of DMEM supplemented with 3% fetal calf
serum, 250 μM IBMX, 66 μM biotin, 34 μM d-pantothenate, 1 μM
dexamethasone, 0.2 μM insulin and 5 μM indomethacin. The cells were
maintained in the differentiation medium for 3 days and then exposed to the
adipocyte maintenance medium consisting of DMEM supplemented with 3% fetal calf
serum, 66 μM biotin, 34 μM d-pantothenate, 1 μM dexamethasone
and 0.2 μM insulin. The cells were cultured in adipocyte maintenance medium
for 9 days and the medium was changed every 3 days (Yu et
al., 2011). To study effect of fenugreek extract on adipogenesis, the
differentiation and adipocyte maintenance medium were supplemented with varying
concentrations of the extract or vehicle (1% DMSO).
Oil red O staining: Oil Red O was used to stain intracellular triglyceride
droplets in differentiated adipocytes. After 12 days of differentiation, the
cells were fixed using 10% formalin and then stained by Oil Red O solution.
After several washing with distilled water, the stain was eluted from cells
using isopropanol and its optical density was read at 540 nm (Yu
et al., 2011).
Cell viability assay: The effect of fenugreek extract on viability of
the stromal cells was determined using MTT colorimetric assay as previously
described (Mortazavian and Ghorbani, 2012; Mortazavian
et al., 2012). The cells were seeded (5000/well) in 96-well culture
plates containing DMEM medium supplemented with 10% fetal calf serum, 100 units/mL
penicillin and 100 μg mL-1 streptomycin. After 24 h, the medium
was changed by fresh one containing various concentrations of fenugreek extract
and the cells were further incubated for 24 h. At the end of treatment, the
MTT was added to the cell media at final concentration of 0.5 mg mL-1
and the cells incubated for 2 h. Then, the absorbance of formazan dye was measured
at 545 nm. The assay was done in triplicate and repeated twice times.
Lipolysis studies: The effect of fenugreek extract on lipolysis was
evaluated using an ex-vivo organ culture method (Ghorbani
et al., 2011, 2013). After seven days of
STZ injection, the retroperitoneal adipose tissues were excised from diabetic
rats. The tissues were minced into uniform small pieces of about 5 mg. The tissue
pieces were washed, dried on the gauze, and weighted precisely. The tissues
were then distributed into 24-well culture plate (100 mg/well) and bathed with
1 mL Krebs-Ringer bicarbonate buffer containing 5.5 mM glucose, 25 mM HEPES
and 2% (w/v) bovine serum albumin. The wells were left untreated (basal lipolysis)
or treated with isoproterenol (stimulated lipolysis) and incubated in the absence
or presence of fenugreek extract at 37°C in a humidified chamber under constant
shaking for 90 min. At the end of the incubation, concentration of glycerol
in the media was measured by an enzymatic method.
Statistical analysis: The results are presented as the mean±standard
error. The values were compared using the one-way analysis of variance followed
by Tukeys post hoc test. Results were considered to be statistically significant,
if the p-values were under 0.05.
RESULTS
Effect of fenugreek on adipogenesis: Exposure of differentiating cells
to the fenugreek extract led to a significant reduction in lipid droplet accumulation
as evaluated with Oil Red O staining (Fig. 1). The presence
of 50, 200 and 400 μg mL-1 of the extract in the culture medium
decreased the lipid droplet content from 100±11% (untreated cells) to
57±12% (p < 0.05), 64±6.6% (p<0.05) and 60±5% (p<0.05),
respectively (Fig. 2).
Effect of fenugreek on viability of preadipocytes: Incubation of preadipocytes
in the presence of fenugreek extract resulted in significant decrease in cell
viability (Fig. 3).
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Fig. 1(a-b): |
Oil Red O staining of differentiated adipocytes after treatment
with fenugreek extract. Preadipocytes were isolated from normal rats and
cultured for 12 days in the presence (a) or absence (b) of fenugreek extract
(50 μg mL-1) in adipogenic media. Lipid-containing Oil Red
O-positive cells appear in non-treated cells (original magnification,x200) |
The extract at 10, 50, 100, 200 and 400 μg mL-1 led to 12%,
39% (p <0.001), 54% (p<0.001), 51% (p<0.001) and 50% (p<0.001) decrease
in cell viability, respectively.
Effect of fenugreek on lipolysis: Figure 4a demonstrates
the effect of fenugreek extract on basal lipolysis.
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Fig. 2: |
Effect of fenugreek extract on lipid droplet accumulation
in differentiating preadipocyte isolated from normal rats. The lipid accumulation
was estimated by measuring the optical density of Oil Red O stain eluted
from cells. Data are mean±SEM of two independent experiments performed
in triplicate. *p<0.05 vs vehicle |
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Fig. 3: |
Effect of fenugreek extract on viability of preadipocytes
isolated from normal rats. The cells cultured for 24 h in the presence of
the extract. The bars show percent of cell viability as compared with untreated
cells (vehicle). Data are mean±SEM of two independent experiments
performed in triplicate. *p<0.001 vs vehicle |
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Fig. 4(a-b): |
Effects of fenugreek extract on basal and stimulated lipolysis
in diabetic rats, (a): Lipolysis in retroperitoneal adipose tissue was assessed
in the presence of 100 μg mL-1 fenugreek extract or 1 μM
isoproterenol, (b) and (c), Isoproterenol-induced lipolysis in the adipose
tissue was assessed in the presence or absence of fenugreek extract. Results
are expressed relative to basal and isoproterenol-induced lipolysis. The
data are presented as means±SEM of 6 independent experiments. *p<0.001
vs vehicle |
The presence of this extract (100 μg mL-1) in the tissue medium
did not change basal glycerol release (95±13% and 100±3.4% for
fenugreek and vehicle, respectively). To examine the effect of fenugreek on
stimulated lipolysis, the lipolytic activity was also evaluated in the presence
of isoproterenol, a nonselective beta adrenergic receptor agonist which induces
lipolysis. As expected, isoproterenol led to a significant elevation (2.87 fold,
p<0.001) in lipolysis.
As shown in Fig. 4b, the extract at concentrations of 10,
100 and even 1000 μg mL-1 had virtually no significant effect
on the stimulated lipolysis. Therefore the level of glycerol release was still
higher than that of basal level.
DISCUSSION
The mass of adipose tissue is determined with the balance between accumulation
of lipid droplet (through lipogenesis and formation of new adipocytes from precursor
cells) and reduction of fat content (through lipolysis and adipocyte apoptosis).
The present study showed that macerated extract of fenugreek inhibits formation
of new differentiated fat cells from preadipocytes. This finding is in agreement
with earlier reports that fenugreek inhibits lipid accumulation in differentiating
3T3-L1 cells (Vijayakumar et al., 2010) and
reduces the body weight gain induced by high-fat diet (Handa
et al., 2005). The results from MTT assay demonstrated that anti-proliferative
action of the extract is most probably responsible for inhibition of new adipocytes
formation. In line with this finding, Al-Daghri and coworkers reported that
fenugreek extract induces cellular death at concentrations of ≥30 μg
mL-1 (Al-Daghri et al., 2012).
Breakdown of triglycerides (lipolysis) in adipose tissue is a highly regulated
process. The regulation of this process is essential for maintaining body homeostasis
and prevention of various diseases development. Although, a variety of factors
affect on lipolysis, insulin and catecholamines are the main anti-lipolytic
and pro-lipolytic hormones, respectively (Large et al.,
2004). Previously it has been shown that fenugreek stimulates insulin signaling
pathways in some tissue (Mohammad et al., 2006;
Vijayakumar et al., 2005). To test whether this
plant can induce anti-lipolytic effect in fat tissue, hydrolysis of triglyceride
was investigated in the tissue incubated with the seed extract. This investigation
was done using an ex vivo organ culture model which has certain advantages
over isolated adipocyte because it retains autocrine, paracrine, cell-cell and
cell-matrix interactions (Ghorbani et al., 2013).
Yet, because the limited amount of adipose tissue obtained from diabetic rats,
it was impossible to test varying concentration of the extract on basal lipolysis.
Beside, the results showed that fenugreek (with the tested concentrations) has
no effect on basal or catecholamine-stimulated lipolysis in adipose tissue isolated
from diabetic rats. Therefore, its beneficial effect on diabetic dyslipidemia
is mediated by other mechanism suggested above.
In conclusion, the present study demonstrated that macerated extract of fenugreek
seed inhibits formation of new differentiated adipocytes from precursor cells.
This effect of fenugreek is mediated through its anti-proliferative action on
preadipocytes.
ACKNOWLEDGMENTS
This work was supported by a grant from Research Council of Mashhad University
of Medical Sciences, Mashhad, IRAN. The authors declare that they have no conflict
of interest.
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