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J. Biol. Chem., Vol. 277, Issue 19, 16347-16350, May 10, 2002
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From the Division of Biochemistry and Molecular Biology, Louisiana State University, Baton Rouge, Louisiana 70803
Received for publication, February 26, 2002, and in revised form, March 11, 2002
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Acetyl-CoA carboxylase catalyzes the first
committed step in the synthesis of long chain fatty acids. In this
study, we observed that treatment of 3T3-L1 cells with biotin
chloroacetylated at the 1' nitrogen reduced the enzymatic activity of
cytosolic acetyl-CoA carboxylase and concomitantly inhibited the
differentiation of 3T3-L1 cells in a dose-dependent manner.
Treatment with chloroacetylated biotin blocked the induction of
PPAR Obesity is characterized by an increase in the number and size of
adipocytes (1). During the course of adipogenesis the activities of
several lipogenic enzymes such as acetyl-CoA carboxylase, fatty acid
synthase, and ATP citrate lyase are increased (2). The up-regulation of
these enzymes suggests they could be targets for anti-obesity agents.
For example, it has been shown recently that mice treated with
inhibitors of fatty acid synthase resulted in decreased food intake and
weight loss (3). The hypothesis that acetyl-CoA carboxylase could be a
target for anti-obesity agents was strengthened by a recent study
demonstrating that mice lacking the gene coding for the mitochondrial
isoform of acetyl-CoA carboxylase lost weight despite eating more food
(4). In this study, we have demonstrated a pharmalogical regulation of
acetyl-CoA carboxylase activity and inhibition of adipocyte
differentiation in 3T3-L1 cells.
Acetyl-CoA carboxylase is a biotin-dependent enzyme
that catalyzes the following two-step mechanism.
, STAT1, and STAT5A expression that normally occurs with
adipogenesis. Moreover, addition of chloroacetylated biotin inhibited
lipid accumulation, as judged by Oil Red O staining. Our results
support recent studies that indicate that acetyl-CoA
carboxylase may be a suitable target for an anti-obesity therapeutic.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
The first half-reaction is carried out by the biotin carboxylase
component of acetyl-CoA carboxylase and involves the
ATP-dependent carboxylation of biotin with bicarbonate
serving as the source of CO2. The carboxyl group is
transferred from biotin to acetyl-CoA to form malonyl-CoA in the second
half-reaction, which is catalyzed by carboxyltransferase. For both
half-reactions, biotin remains covalently linked to the enzyme through
an amide bond to a specific lysine residue of the biotin carboxyl
carrier protein and is designated as enzyme-biotin in the reaction
scheme. Mammalian acetyl-CoA carboxylase contains all three components
on a single polypeptide (5).
We have recently synthesized a bisubstrate analog inhibitor of
the carboxyltransferase component of bacterial acetyl-CoA carboxylase (Fig. 1) (6). Since human acetyl-CoA
carboxylase is now a target for anti-obesity drugs, the question arose
as to whether the bisubstrate analog we synthesized would inhibit
mammalian acetyl-CoA carboxylase and in turn act to reduce lipid
accumulation. Unfortunately, the bisubstrate analog contains the
nucleotide ADP and therefore is not permeable to the cell membrane.
However, the precursor to the analog, a
chloroacetylated biotin derivative
(CABI)1 (Fig. 1), is
sufficiently hydrophobic to diffuse across the cell membrane. The
results of this study clearly demonstrate that treatment of 3T3-L1
cells with CABI inhibits the activity of acetyl-CoA carboxylase.
Moreover, we have shown that CABI treatment inhibits the adipocyte
differentiation of 3T3-L1 cells by blocking the induction of PPAR
and other adipocyte transcription factors.
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EXPERIMENTAL PROCEDURES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Materials--
Dulbecco's modified Eagle's medium (DMEM) was
purchased from Invitrogen. Bovine and fetal bovine serum (FBS)
were obtained from Sigma and Invitrogen, respectively. PPAR was a
mouse monoclonal antibody from Santa Cruz Biotechnology. STAT
antibodies were monoclonal IgGs purchased from Transduction
Laboratories or polyclonal IgGs from Santa Cruz Biotechnology.
Streptavidin linked to horseradish peroxidase (HRP) was from Pierce.
HPLC was performed using a Waters HPLC system equipped with a Waters
996 photodiode array detector. Analytical HPLC used a Discovery C-18
column (15 cm × 4 mm, 5 µm) purchased from Supelco. All other
reagents were from Sigma or Aldrich.
Cell Culture-- Murine 3T3-L1 preadipocytes were plated and grown to 2 days postconfluence in DMEM with 10% bovine serum. Medium was changed every 48 h. Cells were induced to differentiate by changing the medium to DMEM containing 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine (MIX), 1 µM dexamethasone (DEX), and 1.7 µM insulin. After 48 h this medium was replaced with DMEM supplemented with 10% FBS, and cells were maintained in this medium until utilized for experimentation. CABI was dissolved in Me2SO and added to the cell culture medium at a 1 to 1000 dilution. Vehicle additions were performed in every experiment.
Synthesis of Chloroacetylated Biotin-- Synthesis of chloroacetylated biotin was as described previously (7).
Preparation of Whole Cell Extracts-- Monolayers of 3T3-L1 adipocytes were rinsed with phosphate-buffered saline and then harvested in a nondenaturing buffer containing 150 mM NaCl, 10 mM Tris, pH 7.4, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.5% Nonidet P-40, 1 µM phenylmethylsulfonyl fluoride, 1 µM pepstatin, 50 trypsin inhibitory milliunits of aprotinin, 10 µM leupeptin, and 2 mM sodium vanadate for Western blot analysis. Samples were extracted for 30 min on ice and centrifuged at 10,000 × g at 4 °C for 15 min. Supernatants containing whole cell extracts were analyzed for protein content using a BCA kit (Pierce) according to the manufacturer's instructions. This procedure was modified to prepare extracts for enzymatic analysis. The nondenaturing buffer contained 150 mM KCl, instead of NaCl, and there was no Triton X-100, Nonidet P-40, or any protease or phosphatase inhibitors included in the buffer. The cell monolayers were scraped in this buffer and were homogenized with 16 strokes in a Dounce homogenizer. The homogenates were centrifuged at 10,000 × g for 5 min, and the supernatants were saved as cytosolic extract and used to assay acetyl-CoA carboxylase activity.
Gel Electrophoresis and Immunoblotting-- Proteins were separated in 5, 7.5, or 12% polyacrylamide (acrylamide from National Diagnostics) gels containing sodium dodecyl sulfate (SDS) according to Laemmli (8) and transferred to nitrocellulose (Bio-Rad) in 25 mM Tris, 192 mM glycine, and 20% methanol. Following transfer, the membrane was blocked in 4% milk for 1 h at room temperature. Results were visualized with HRP-conjugated secondary antibodies (Sigma) or streptavidin linked to HRP and enhanced chemiluminescence (Pierce).
Enzyme Assays--
The activity of acetyl-CoA carboxylase from
3T3-L1 cell lysates was determined using a fixed time assay. Assays
were performed by measuring the loss in acetyl-CoA and/or the
production of malonyl-CoA at 5 min intervals for 20 min, using reverse
phase HPLC. The rate of conversion of acetyl-CoA to malonyl-CoA was
found to be linear for 20 min, and velocities were calculated by linear
regression analysis of the malonyl-CoA concentration with respect to
time. The reaction mixture contained 50 mM Tris, pH 7.5, 6 µM acetyl-CoA, 2 mM ATP, 7 mM
KHCO3, 8 mM MgCl2, 1 mM
dithiothreitol, and 1 mg/ml bovine serum albumin. Cell lysates
were preincubated (30 min, 25 °C) with bovine serum albumin (2 mg/ml) and potassium citrate (10 mM). Reactions were
initiated by transferring 50 µl of preincubated cell lysate to the
reaction mixture (final volume 200 µl) and incubated for 5-20 min at
25 °C. Reactions were terminated by addition of 50 µl 10%
perchloric acid. Following termination of the reaction the samples were
centrifuged (3 min, 10,000 × g) and analyzed by HPLC.
A mobile phase of 10 mM KH2PO4, pH
6.7 (solvent A), and MeOH (solvent B) was used. The flow rate
was 1.0 ml/min, and the gradient was as follows: hold at 100% solvent
A for 1 min followed by a linear gradient to 30% solvent B
over the next 5 min, then hold at 30% solvent B for 5 min.
Using this method the retention times were 7.5 and 9.0 min for
malonyl-CoA and acetyl-CoA, respectively.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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To test the ability of CABI to reduce acetyl-CoA carboxylase
activity, confluent 3T3-L1 preadipocytes were treated for 4 h with
10 µM CABI, and whole cell extracts were prepared and
immediately used to measure acetyl-CoA carboxylase activity by
analytical reverse phase HPLC. As shown in Fig.
2A, the activity of acetyl-CoA carboxylase from cells treated with CABI was 0.30 nmol of
malonyl-CoA/min·mg, and cells treated with Me2SO had an
activity of 1.40 nmol/min·mg. Thus, treatment of preadipocytes with
CABI resulted in a 79% reduction in acetyl-CoA carboxylase activity.
These samples were also analyzed for the expression of acetyl-CoA
carboxylase using streptavidin HRP (Fig. 2B). These results
clearly demonstrate that the reduced activity of acetyl-CoA carboxylase
is not due to altered expression levels of the enzyme.
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Since treatment of 3T3-L1 preadipocytes with CABI reduced acetyl-CoA
carboxylase activity, we examined the effects of CABI on the
adipogenesis of these cells. At 2 days postconfluence, the 3T3-L1 cells
were exposed to the normal differentiation mixture (FBS, MIX, DEX, and
insulin) for 48 h in the presence or absence of various doses of
CABI. After 48 h, the cells were maintained in 10% FBS in DMEM. A
vehicle addition of Me2SO was also performed. CABI or
Me2SO was added to the cells in a 1 to 1000 dilution into the cell culture medium every 24 h. The isolation of whole cell extracts and Oil Red O staining was performed 1 week following treatment with the induction mixture. Adipogenesis was assessed by
examining the expression of several transcription factors, PPAR and
STATs 1 and 5A, which are highly induced during adipogenesis (9), and
by examining lipid accumulation, as judged by Oil Red O staining.
Optimal differentiation, as judged by PPAR expression and Oil red O
staining, was achieved when 3T3-L1 cells were exposed to the normal
differentiation mixture containing FBS, MIX, DEX, and insulin, which
resulted in 100% adipocyte conversion as reported previously (10).
However, we observed a dose-dependent inhibition of
differentiation in the presence of CABI. Exposure of differentiating adipocytes to 17 or 8 µM CABI blocked the induction of
PPAR expression (Fig. 3) and lipid
accumulation as judged by Oil Red O staining (Fig.
4). Parallel with the 100% conversion,
STATs 1 and 5A were highly expressed, and the induction in expression
of these two transcription factors was also inhibited in a
dose-dependent manner by CABI treatment. The specificity of
this treatment is demonstrated by examining the expression of STAT3, a
protein whose expression is not substantially regulated during
differentiation. As shown in Fig. 3, the expression of STAT3 was
unaffected by CABI treatment and is shown as a loading control.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The novel observations in this study include the ability of CABI
to diffuse into 3T3-L1 cells and reduce the activity of cellular acetyl-CoA carboxylase and to inhibit the adipogenesis of 3T3-L1 cells
in a dose-dependent manner by blocking the induction of PPAR expression. To account for these observations we suggest that
upon entering the cell, CABI reacts with endogenous coenzyme A to form
the bisubstrate analog CABI-CoA. Once formed the bisubstrate analog
inhibits the cytosolic and/or mitochondrial isoforms of acetyl-CoA
carboxylase. This hypothesis is supported by the observations that CABI
had no effect on acetyl-CoA carboxylase activity in isolated cellular
extracts (data not shown). However, the bisubstrate analog CABI-CoA was
indeed able to inhibit acetyl-CoA carboxylase activity when added to
isolated cellular extracts in vitro (data not shown). Moreover, there
is precedent for enzyme inhibitors forming intracellularly by reaction
of a precursor with a metabolite. Examples include finasteride, which
inhibits 5
reductase (11), isoniazid, which inhibits the
mycobacterial enzyme InhA (12), and a bisubstrate analog inhibitor of
serotonin N-acetyltransferase (13). The first two examples
are used clinically to treat benign prostatic hypertrophy and
tuberculosis, respectively. Our attempts to detect CABI-CoA in extracts
from CABI-treated 3T3-L1 cells have been unsuccessful using HPLC with
absorption optics. However, studies are under way to detect the
bisubstrate analog CABI-CoA in cellular extracts with more sensitive methods.
The observation that the expression of STAT3 was unaffected by CABI is very important, because it suggests that CABI is not acting as a nonspecific alkylating agent. Moreover, the fact that the level of acetyl-CoA carboxylase did not decrease with CABI treatment further indicates that CABI is not exerting a general toxic effect. It should be noted that the inhibition of differentiation by CABI was reversible. If the addition of CABI was not repeated every 24 h, the cells started to differentiate (data not shown).
We have recently determined the inhibition constant of the bisubstrate analog, CABI-CoA, for bacterial acetyl-CoA carboxylase is 23 µM (6). If the inhibition constant for the murine acetyl-CoA carboxylase in 3T3-L1 cells is similar to the bacterial enzyme, then it is tempting to speculate how an inhibitor with such a modest Ki can result in such a significant biological effect. The answer begins by noting the importance of acetyl-CoA carboxylase for cell growth and the recent demonstration that a gene knock-out of cytosolic acetyl-CoA carboxylase is embryonic lethal (4). Our goal was not to abolish the activity of all cellular acetyl-CoA carboxylase and induce cell death. Instead, the objective was to attenuate the activity of acetyl-CoA carboxylase and prevent lipid accumulation. To this end, a molecule with a 23 µM Ki value would serve this purpose, while a molecule with a nM or lower Ki value would have greater cytotoxicity.
In summary, the results presented in this paper are the first
demonstration of a link between a pharmalogical modulation of cytosolic
acetyl-CoA carboxylase and inhibition of adipogenesis. These studies
support the gene knock-out experiments in mice, which indicate that
acetyl-CoA carboxylase is a very promising target for anti-obesity
agents (4). It will be interesting to examine the effects of CABI on
other types of cells in which acetyl-CoA carboxylase is up-regulated
such as breast cancer cells (14). We are currently searching for
alterative solvents for CABI and trying to synthesize a more soluble
precursor so that these compounds can be tested in rodents.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants R01DK5268-02 (to J. M. S.) and GM51261 (to G. L. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biological
Sciences, Rm. 202 Life Sciences Bldg., Louisiana State University, Baton Rouge, LA 70803. Tel.: 225-578-1749; Fax: 225-578-2597; E-mail:
jsteph1@lsu.edu.
Published, JBC Papers in Press, March 20, 2002, DOI 10.1074/jbc.C200113200
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ABBREVIATIONS |
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The abbreviations used are: CABI, a chloroacetylated biotin derivative; PPAR, peroxisome proliferator-activated receptor; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; STAT, signal transducers and activators of transcription; HRP, horseradish peroxidase; HPLC, high performance liquid chromatography; MIX, 3-isobutyl-1-methylxanthine; DEX, dexamethasone.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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