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Department of Nutrition, University of Tennessee, Knoxville, TN 37996
3To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • agouti • fatty acid synthase • adipose tissue • adipocyte differentiation • obesity • humans
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Jackson 1991
). However,
ectopic expression of agouti in animal models such as
Ay and
Avy or in transgenic mice
overexpressing agouti under the control of ß-actin
promoter results in a yellow coat color, as well as obesity,
hyperinsulinemia, insulin resistance, increased body length and
susceptibility to neoplasia, which is collectively called "yellow
obese" syndrome (Klebig et al. 1995
, Yen et al. 1994
).
Much attention has been directed toward central mechanisms of agouti’s
action. Recent evidence indicates that chronic antagonism of central
melanocortin 4-receptor may be responsible for the hyperphagia and
obesity in yellow agouti mutants (Fan et al. 1997
,
Huszar et al. 1997
). However, the human homologue of
agouti is expressed in both adipose tissue (Kwon et al. 1994
) and pancreas (Xue et al.1999
) in which
it exerts important peripheral effects.
We showed previously that recombinant agouti protein exhibits potent
lipogenic and antilipolytic effects in human adipocytes via a
calcium-dependent mechanism (Jones et al. 1996
,
Kim et al. 1996
, Xue et al. 1998
),
thereby promoting lipid storage in adipose tissue. Previous data from
our laboratory also demonstrated that agouti protein stimulates calcium
signaling and serves as a potent insulin secretagogue in both the
pancreatic ß-cell line and human pancreatic islets (Xue et al. 1999
). Accordingly, we propose that this effect of agouti on
insulin release, combined with its effect on adipocyte lipid
metabolism, may represent a peripheral mechanism for agouti in the
development of obesity. Consistent with this, although transgenic mice
expressing agouti only in adipose tissue did not become
obese, hyperinsulinemia in these mice produced by either daily insulin
injection (Mynatt et al. 1997
) or sucrose feeding
(Zemel et al. 1999
) did produce significant obesity in
the transgenic mice, but not in their nontransgenic littermates.
However, this hypothesis is based on in vitro studies of human
adipocytes and human pancreatic islets. Little is known regarding its
significance in vivo.
In this study, we investigated the relationship between agouti expression and adipocyte fatty acid synthase (FAS) expression and activity. We report here that human adipocyte agouti is up-regulated during human preadipocyte differentiation. In addition, there is a strong correlation between human adipose tissue agouti protein content and FAS activity as well as between human adipose tissue agouti mRNA and FAS mRNA level.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Human subcutaneous preadipocytes were supplied by Zen-Bio (Research Triangle Park, NC). To differentiate into mature adipocytes, preadipocytes were plated at a density of 30,000 cells/cm2 with preadipocyte medium [Dulbecco’s modified Eagle medium (DMEM)/Ham’s F-10 nutrient broth (Ham’s F-10), 1:1 (v/v) containing 10% fetal calf serum, 15 mmol/L HEPES and antibiotics] and allowed to grow to confluence in 1–2 d. Cells were then induced to differentiate with differentiation medium [DMEM/Ham’s F-10, 1:1 (v/v) containing 10% fetal bovine serum, 15 mmol/L HEPES, 33 µmol/L biotin, 17 µmol/L pantothenate, 100 nmol/L insulin, 1 µmol/L dexamethasone, 0.25 mmol/L isobutyl methylxanthine, 1 µmol/L BRL49653 and antibiotics]. After 3 d of differentiation, cells were maintained in adipocyte medium [DMEM/Ham’s F-10, 1:1 (v/v) containing 3% fetal bovine serum, 15 mmol/L HEPES, 33 µmol/L biotin, 17 µmol/L pantothenate, 100 nmol/L insulin, 1 µmol/L dexamethasone and antibiotics] until fully differentiated.
Human adipose tissue.
Human subcutaneous adipose tissue was obtained during liposuction from nonobese and mildly obese patients, with body mass indices ranging from 21 to 31 kg/m2. This protocol was approved by the Institutional Review Board for Human Subjects and the Committee for Research Participation of the University of Tennessee.
Agouti protein measurement.
Human preadipocytes and mature adipocytes were rinsed once with Hank’s
balanced salt solution (Life Technologies, Grand Island, NY) and
homogenized in sucrose buffer (250 mmol/L sucrose, 1 mmol/L
dithiothreitol, 1 mmol/L EDTA and 100 µmol/L
phenylmethylsulfonyl fluoride). Human adipose tissue was homogenized in
sucrose buffer. Fat-free infranatant was obtained by centrifugation
at 18,500 x g for 60 min using a Sorval RMC 14
microcentrifuge (Sorval, Newtown, CT). Agouti protein was measured
using whole-cell homogenates from pre- and mature adipocyte
preparations and the fat-free infranatant from human adipose tissue
via an agouti RIA kit (Phoenix, Mountain View, CA), similar to the
approach used recently for the assessment of tissue leptin content
(Claycombe et al. 2000
).
Fatty acid synthase (FAS) activity determination.
FAS activity was determined spectrophotometrically using fat-free infranatant from human adipose tissue by measuring the oxidation rate of NADPH as described previously (Moustaid et al. 1988).
Protein measurement.
Total protein was determined by a modified Bradford method
using Coomassie blue dye (Pierce, Rockford, IL) (Bradford 1976
).
Northern blot analysis.
Total RNA from human adipose tissue was isolated using CsCl2 density centrifugation. RNA from different fat samples were electrophoresed on the same gel (1%) and transferred to nylon membranes (New England Nuclear, Boston, MA). Membranes were hybridized with 32P-labeled human FAS, agouti and ß-actin (Clontech, Palo Alto, CA) cDNA probe at 42°C overnight using Ultra Hyb solution (Ambion, Austin, TX). Membranes were washed with 2X saline-sodium citrate (SSC) at 42°C for 15–30 min, 0.1X SSC at 42°C for 15–30 min and then 0.1X SSC at 60°C for 30–60 min. Finally, membranes were exposed to X-ray film (New England Nuclear) at -80°C. Autoradiograms were analyzed by densitometric scanning.
Statistics.
All data are expressed as means ± SEM Data were analyzed using the procedures of the SPSS (Chicago, IL). Different agouti protein content in pre- and mature adipocyte was compared by independent sample t test. The relationships between adipose tissue agouti expression and FAS expression and agouti protein and FAS activity were analyzed by linear regression. Pearson correlation coefficients were calculated to determine the relationship between variables. A P-value < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, agouti protein level was four times more abundant in human mature
adipocytes than in preadipocyte (19.18 ± 2.46 vs. 4.07 ± 0.51 pg/µg protein, P < 0.005),
suggesting that agouti is up-regulated during human adipocyte
differentiation.
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) comparable to those found in adipocytes obtained by differentiating
cultured human stromal vascular preadipocytes (Fig. 1)
. Human adipose
tissue agouti protein content varied >10-fold among different
individuals (Fig. 2A
). Over this range, adipose tissue
agouti content was correlated with FAS activity (Fig. 2A
,
r = 0.782, P < 0.005).
|
, r = 0.846, P
< 0.002). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), indicating a possible physiologic role for agouti in
human adipose tissue. We showed previously that the FAS mRNA level is
significantly higher in adipose tissue from obese
Avy mice than in lean (a/a)
controls (Jones et al. 1996
). In addition, recombinant
agouti protein stimulated both the expression and activity of adipocyte
FAS in vitro via a Ca2+-dependent mechanism
(Jones et al. 1996
). Further, the FAS promoter contains
an agouti response element that also responds to KCl, a membrane
depolarization agent (Claycombe et al. 1997
). We also
demonstrated that agouti protein inhibited both basal- and
agonist-stimulated lipolysis in human adipose tissue via a
Ca2+-dependent mechanism (Xue et al. 1998
). These in vitro data suggest a coordinated regulation of
adipocyte lipid metabolism by agouti, which may in turn promote lipid
storage in adipose tissue.
Numerous reports have demonstrated that adipose tissue synthesizes
various proteins and hormones, participating in energy regulation
through a network of endocrine, paracrine and autocrine signals
[reviewed in Fried and Russel (1998)
and
Mohamed-Ali et al. (1998)
]. Leptin, for example, is a
satiety factor produced by adipocyte and regulates body weight through
its effects on food intake in hypothalamus. In addition, adipose tissue
has been also shown to produce factors that act in an endocrine and
paracrine/autocrine manner to modulate its own metabolism, preadipocyte
proliferation and differentiation, and multiple systemic effects. These
factors include the cytokines, interleukin-6 and tumor necrosis
factor-
, acylation-stimulating protein, angiotensin II,
prostaglandins, sex steroids and plasminogen activator inhibitor-1
[reviewed in Fried et al. (1998)
, Hwang et al. (1997)
and Mohamed-Ali et al. 1998)
].
Fatty acid synthase is the key enzyme in de novo lipogenesis. Both
human liver and adipose tissue exhibit substantially lower FAS activity
than that found in rats (Weiss et al. 1986
).
Nonetheless, significant de novo lipogenesis is well documented in
human adipocytes. Human adipocytes contain substantial levels of FAS
activity which is sensitive to both nutritional and hormonal modulation
(Moustaid et al. 1996
). Studies in humans fed a
high carbohydrate diet demonstrated that total body fat synthesis
significantly exceeded hepatic de novo lipogenesis, suggesting that
adipose tissue may be the major site for fat synthesis (Aarsland et al. 1997
). Further, another study showed that adipose tissue
accounts for up to 40% of whole-body lipogenesis under this
condition (Chascione et al. 1987
). The effect of
carbohydrate intake on adipose tissue lipogenesis is qualitatively
similar in humans and in rats. Quantitatively, rats have higher rates
of lipogenesis, which may be explained in part by the higher metabolic
rate in rats than in humans (Chascione et al. 1987
).
Moreover, FAS levels are elevated in obese animals (Guichard et al. 1992
). Thus, induction of adipocyte lipogenesis may
contribute to obesity.
In this study, we demonstrated that human adipocyte agouti is
up-regulated during differentiation. In addition, we showed a
strong correlation between agouti expression and FAS expression and
activity in human adipose tissue. It is possible that agouti and FAS
are regulated in a similar manner. FAS is regulated primarily by
hormonal and nutritional factors at the transcription level. It is
increased by feeding, thyroid hormone and insulin, and decreased by
starvation, cAMP and polyunsaturated fatty acids [reviewed in
Sul and Wang (1998)
]. However, it is not known at
present which factor(s) may modulate adipocyte agouti expression and
how it is regulated. Further, because agouti is a paracrine/autocrine
factor that does not enter the general circulation (Wolff 1963
) and because it has been shown to regulate FAS expression
and activity in vitro (Jones et al. 1996
), it is
possible that agouti may be another adipocyte-synthesized factor,
which may contribute in part to the regulation of adipocyte lipid
metabolism.
In summary, we have found that human adipocyte agouti is
up-regulated during adipocyte differentiation. In addition, there
is a strong correlation between human adipose tissue agouti protein
content and FAS activity as well as between human adipose tissue agouti
mRNA and FAS mRNA level. Consistent with this, we have shown that
agouti regulates adipocyte lipid metabolism in vitro (Jones et al. 1996
, Xue et al. 1998
). These data suggest
that agouti may be an additional adipocyte factor, which modulates
adipocyte lipid metabolism via a paracrine/autocrine mechanism.
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FOOTNOTES |
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2 Supported in part by Knoll Pharmaceutical Company, Weight Risk Investigators Study Council and the Tennessee Agricultural Experiment Station.
Manuscript received January 20, 2000. Initial review completed March 24, 2000. Revision accepted June 1, 2000.
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