From the Centre de Recherches Biomédicales des Cordeliers, Université Pierre et Marie Curie, UPRES-A 7079 CNRS, 15 rue de l'Ecole de Médecine, 75270 Paris, Cedex 06, France
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ABSTRACT |
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In an effort to identify novel mRNAs
modulated during the course of adipose conversion, we have used a
simplified differential display technique and have isolated a cDNA
encoding an amine oxidase tremendously expressed in the adipocyte, the
semicarbazide-sensitive amine oxidase (SSAO). The predicted amino acid
sequence (765 amino acids) is likely to be the homologue of the human
placental amine oxidase and of the partially known sequence of the rat
adipocyte membrane amine oxidase. SSAO mRNAs are present in several
tissues, but strikingly, the highest levels of gene expression are
found in adipose tissue and aorta. Enzyme transcript levels are barely detectable in preadipocytes but are induced several hundred-fold during
the adipocyte differentiation of 3T3-L1 or 3T3-F442A cells and of rat
precursor primary cultures. These changes in transcript levels parallel
a sharp increase in SSAO enzyme activity. The biochemical properties of
the SSAO present in 3T3-L1 or 3T3-F442A adipocytes closely resemble the
features of the SSAO activity previously described in white and brown
adipose tissues. Interestingly, SSAO mRNA levels and enzyme
activity drop in response to effectors of the cAMP pathway and to the
cytokine tumor necrosis factor- Adipose tissue is the primary site for triacylglycerol storage in
mammals and exerts a key role in the control of energy balance. To
perform these essential functions, adipocytes express a large pattern
of enzymes and accessory proteins that concur in lipogenesis or
lipolysis. Moreover, these metabolic pathways are under the tight
control of a variety of hormones. Several major discoveries have
supported the idea that, besides its classical energetic functions, the
adipocyte is also able to exert regulatory functions through the
secretion of paracrine or endocrine factors.
In vivo a process of recruitment and conversion of
mesenchymal cells into adipocytes can occur throughout life. Studies on adipogenesis have been greatly facilitated by the availability of
established preadipose cell lines (1-3). When cultured under appropriate conditions, committed preadipocytes differentiate into
adipocytes mimicking the white phenotype. The highly specialized functions of the mature adipocyte are acquired during the course of
adipose differentiation. The emergence of the adipocyte phenotype is
linked to the coordinated expression of a large number of genes and
proteins. So far, a great number of genes have been identified and
related to the molecular mechanisms of fat cell differentiation. They
include genes coding for enzymes and structural proteins, transcription
factors, hormone receptors, and secreted proteins (4, 5).
The scope of this study was to identify novel genes that are
differentially expressed during the adipose conversion process. For
this purpose, we used an mRNA differential display technique (6)
adapted according to the simple protocol of Sokolov and Prockop (7).
The key element of this technique is to generate cDNA fragments by
reverse transcription with random hexanucleotides and then to perform a
polymerase chain reaction in the presence of primers arbitrary in
sequence. This approach allowed us to clone the cDNA of the murine
semicarbazide-sensitive amine oxidase (SSAO),1 an amine oxidase
related to the copper-containing amine oxidase family. This cDNA is
the murine homolog of the recently described human placental amine
oxidase (8) and of the partially identified cDNA of the rat
adipocyte plasma membrane amine oxidase (9). These enzymes are known to
oxidize primary amines by molecular oxygen to generate the
corresponding aldehyde, ammonia, and hydrogen peroxide. During
differentiation of 3T3-F442A or 3T3-L1 preadipose cells, there was a
dramatic increase in SSAO mRNA levels correlated with a striking
induction in enzyme activity. The same pattern of expression was
observed during adipose conversion of mesenchymal adipocyte precursors.
This profile of SSAO expression was quite different from those of two
well known copper-containing amine oxidases, diamine oxidase and
polyamine oxidase. Interestingly, SSAO expression appeared to be highly
modulated by environmental conditions. Tissue distribution of SSAO
mRNA indicated a preferential expression in white adipose tissue,
brown adipose tissue, and aorta. Given the peculiar tissue expression
profile, its functional properties (10), and the characteristics of its
modulation, this amine oxidase could represent an important
intermediate in the physiology of adipose and vascular tissues.
Cell Lines and Cell Culture--
Stocks of murine 3T3-C2
fibroblasts (11), and of 3T3-L1 (1) or 3T3-F442A (2) preadipocytes were
maintained in DMEM containing 25 mM D-glucose
and supplemented with 10% donor calf serum. For experiments, cells
were grown in DMEM containing 10% fetal calf serum. Differentiation of
3T3-F442A cells was initiated by the addition of 1 µg/ml insulin at
confluence. For 3T3-L1 cells, induction of differentiation was achieved
by a treatment with dexamethasone (0.25 µM), insulin
(1 µg/ml), and 1-methyl-3-isobutylxanthine (0.1 mM) for
the 48-h period following confluence (12). After removal of induction
mixture, cells were refed in DMEM supplemented with 10% fetal calf
serum and 1 µg/ml insulin. At day 7 after confluence, more than 90%
of 3T3-L1 or 3T3-F442A cells had the morphology of mature adipocytes.
3T3-C2 cells kept a fibroblastic shape at all of the culture stages.
Primary cultures of rat preadipose cells were seeded from the stroma
vascular fraction derived from the isolation of rat adipocytes
according to the procedure of Rodbell (13). Cultures were maintained
and allowed to differentiate as described previously (14).
Cloning of SSAO cDNA--
A partial cDNA was first
isolated using the general procedure of mRNA differential display
described by Liang and Pardee (6) and modified according to Sokolov and
Prockop (7). Total cellular RNA was prepared (15) from 3T3-C2 cells and
3T3-F442A cells harboring different phenotypes. One µg of total RNA
was reverse-transcribed with 400 units of Moloney murine leukemia
virus-reverse transcriptase (Life Technologies, Inc.) in a 20-µl
volume, consisting of 400 µM of each dNTP, 10 µM of random hexanucleotides, 3 mM
MgCl2, 50 mM Tris-HCl, pH 8.3, and 1 unit of
RNase inhibitor/µg of RNA. After a 1-h incubation at 42 °C,
Moloney murine leukemia virus-reverse transcriptase was
heat-inactivated. One µl of the cDNA sample was amplified by PCR
in a DNA thermal cycler 9600 (Perkin-Elmer). PCR was performed in a
25-µl volume containing 20 mM Tris-HCl, pH 8.55, 16 mM (NH4)2SO4, 2.5 mM MgCl2, a 125 µM concentration of each dNTP, 12.5 pmol of each of the two oligonucleotide primers, and
1 unit of Taq polymerase (Life Technologies). A set of
10-23-mer primers with an arbitrary but defined sequence was used. The
sequences of the 20-mer oligonucleotides that allowed us to identify an 840-bp cDNA (see "Results") were 5'-CACTGGCCTCGATCTACTCC-3' and 5'-GGGAGAAGGTCGTCCTCGTC-3'. cDNAs were denatured for 5 min at 94 °C and submitted to 40 cycles of amplification (one cycle: 94 °C for 30 s, 45 °C for 1 min, and 72 °C for 1 min)
followed by a final extension of 10 min at 72 °C. PCR products were
separated on a 2% agarose gel and stained by ethidium bromide.
cDNA fragments were then isolated from the gel using the Geneclean
II kit (Bio 101, Inc.). The purified DNA was reamplified by using the
same primer pair, subsequently cloned into the T/A cloning pGEMT vector (Promega), and submitted to sequencing (see below).
To isolate the full-length SSAO cDNA, 3'- and 5'-RACE-PCR
strategies were employed (16, 17). The 3'-end of the SSAO cDNA was
obtained using the 3'-RACE-PCR. Two µg of total cellular RNA were
reverse transcribed in the presence of an oligo(dT)15
adaptor primer (17). The first PCR assay was performed with a first adaptor primer and a primer A specific for SSAO. A second round of
amplification was carried out using a second adaptor primer and the
same specific primer A. The 5'-end of the cDNA was identified by
5'-RACE-PCR. The first strand of cDNA was synthesized from 1 µg
of poly(A)+ RNA in the presence of a primer B specific for
the 5'-end of the 840-bp fragment. A homopolymeric A tail was then
appended to the 5'-end of the first strand cDNA using recombinant
terminal deoxynucleotidyl transferase (Life Technologies). The second
strand of the tailed cDNA was obtained using the
oligo(dT)15 adaptor primer. The PCR amplification was then
performed with the first adaptor (as in 3'-RACE above) and the specific
primer B. A second round of amplification was carried out with the
second adaptor primer and the primer B. The sequences of the primers
used were as follows:
5'-CCAGTGAGCAGAGTGACGAGGACTCGAGCTCAAGCT15-3' for the oligo(dT)15 adaptor primer; 5'-CCAGTGAGCAGAGTGACG-3' and
5'-GAGGACTCGAGCTCAAGC-3' for the first and second adaptor primers,
respectively (17); 5'-TCCCCAACACGGTGACTGTG-3' for the specific primer A
defined on the 3'-end of the 840-bp cDNA derived from the
differential display; 5'-TCAAACTGGTCCTCCAGCTG-3' for the specific
primer B defined on the 5'-end of the 840-bp fragment. Products from
3'- and 5'-RACE-PCR were then analyzed on a 1% agarose gel, and the
DNA bands of interest were identified by Southern blot. These bands
were purified by Geneclean II kit and subloned in a pGEMT easy vector.
Sequences of the cDNAs derived from differential display or from
5'-RACE- and 3'-RACE-PCR were determined by dideoxysequencing with
Sequenase version 2.0 (U.S. Biochemical Corp.). Final sequencing was
achieved by Genome Express (Montreuil, France). GenBankTM
data base searches were performed using the Blastn procedure provided
by the Infobiogen Service Center (Villejuif, France).
Northern Blots--
RNA was extracted from cultured cells as
described by Cathala et al. (15) and from tissues by the
acid phenol-chloroform procedure (18). Ten µg of total RNA from
cultured cells and 20 µg of total RNA from tissues were
electrophoresed through a 1.2% agarose gel containing 2.2 M formaldehyde and transferred to nylon membranes
(Nylon-plus; Schleicher & Schuell). Methylene blue staining of blots
was carried out to ensure the similarity in RNA loading. Hybridization
to probes labeled by random priming (19) was carried out as described
by Church and Gilbert (20). Final washing was performed in 0.2 × SSC, 0.1% SDS for 15 min at 60 °C. The 840-bp SSAO DNA fragment,
initially PCR-amplified by differential display, was used as a probe.
Glycerol-3-phosphate dehydrogenase and adipocyte lipid-binding protein
cDNAs were a generous gift of Dr. H. Green (21).
Enzyme Assays--
3T3-L1, 3T3-F442A, and 3T3-C2 cells were
washed twice in phosphate-buffered saline and then harvested and
homogenized in 40 mM sodium phosphate buffer, pH 7.4 (20 strokes in a Dounce homogenizer, pestle B). White adipocytes from
1-month-old lean (Fa/fa) or obese (fa/fa) male
Zücker rats were isolated from inguinal fat pads by collagenase
digestion (13) and washed in phosphate-buffered saline. After
homogenization in 40 mM sodium phosphate buffer, pH 7.4, the fat cake was removed by a brief centrifugation, and homogenates
were kept at
SSAO activity was tested by H2O2 production on
cell homogenates by the fluorimetric method of Matsumoto et
al. (23). The assay was performed in a final volume of 100 µl
consisting of 40 mM sodium phosphate buffer, pH 7.4, 1 mM homovanillic acid, 1 mM sodium azide, 5 µg/ml peroxidase (Sigma), and 10-50 µg of cell homogenate. Except
otherwise mentioned, SSAO activity was measured under conditions of
monoamine oxidase (MAO)-A and -B inhibition by pargyline (1 mM). When indicated, a SSAO inhibitor was preincubated with
cell extract for 20 min at 37 °C prior to the addition of the
substrate. Incubation was initiated by the addition of the substrate
and carried out in triplicate for 1 h at 37 °C. The reaction
was stopped by the addition of 1 mM semicarbazide. After
cooling, 1.2 ml of 0.1 N NaOH was added, and the
fluorescence intensity was measured with excitation at 323 nm and
emission at 426 nm. As blank tests, assay mixtures without substrate
were incubated. Preliminary experiments were performed to ensure that SSAO activity was tested at the initial rates of the reaction. Proportionality between enzyme activity and amounts of homogenate was
also established.
Apparent kinetic constants for SSAO substrates were determined within
the following concentration ranges: 10-500 µM for
benzylamine, 100 µM to 5 mM for methylamine,
1-30 mM for N1-acetylputrescine,
300 µM to 10 mM for histamine, 100 µM to 10 mM for tyramine, and 30 µM to 3 mM for Cloning and Sequencing of SSAO in Murine 3T3 Preadipose Cell
Lines: A Differentiation-dependent Amine
Oxidase--
mRNA differential display has a potential power for
identifying genes that are differentially expressed in a variety of
in vivo and in vitro systems (6). In this
context, the simplified mRNA differential display procedure of
Sokolov and Prockop (7) seemed particularly convenient for the
isolation of novel genes expressed during the course of adipose
conversion of 3T3 preadipose cell lines. For this purpose, total RNA
was prepared from undifferentiated or fully differentiated 3T3-F442A
cells and from growing or resting 3T3-C2 cells that do not
differentiate and keep a fibroblastic shape. cDNAs derived from
these RNAs were then used in the differential display PCR assay in the
presence of different sets of primers arbitrary in sequence and of
various lengths. Among several PCR products detected on agarose gels, a
fragment of 840 bp (Fig. 1A),
particularly abundant in adipocytes but absent in nonadipose cells, was
therefore explored further. Indeed, using this 840-bp DNA fragment as a
probe in Northern analysis, we confirmed that the related mRNA
species, with an apparent size of 4.2 kilobases, was highly expressed
in mature 3T3-F442A adipocytes but remained virtually undetectable in
the 3T3-F442A preadipocytes and 3T3-C2 fibroblastic cell line, even in
the resting state (Fig. 1B). This profile indicated that
expression of this mRNA species was
differentiation-dependent but was not related to withdrawal
from the cell cycle.
The nucleotide sequence of the cDNA fragment was determined and
analysis from the Blastn GenBankTM sequence data bank
revealed high identities between the sequence of the 840-bp DNA
fragment and those of several members of the copper-containing amine
oxidase family such as the human placental amine oxidase (8), the
bovine serum amine oxidase (25), and the human kidney diamine oxidase
(26). Using 5'- and 3'-RACE-PCR strategies (see "Experimental
Procedures"), we identified a full-length cDNA clone of 4210 bp.
Both strands were entirely sequenced. Sequence analysis and
overlapping, open reading frame identification and translation, and
sequence alignments were performed using the BISANCE program of
Infobiogen (Villejuif, France). The first in frame ATG codon is found
at position 166 from the 5'-end of the cDNA. The ATG initiate a
long open reading frame extending up to position 2460.
The sequence of the resulting 765-amino acid protein was compared
with those of several other related amine oxidases (Fig. 2). This protein displays the typical
features of the copper-containing amine oxidase family. Thus, the
pre-TPQ-Tyr is located at position 471 in the protein sequence. This
important tyrosine residue is found within the NYDY consensus sequence
present in all amine oxidases shown in Fig. 2. The TPQ-amine oxidases
have been shown to contain three histidine residues involved in copper
binding. On the alignment presented, we identified these residues at
positions 520, 522, and 684. The predicted amino acid sequence shows a
high identity with the partially cloned rat (vp97) (95% identity) (9) and with the human placental amine oxidase (83% identity) (8). These
findings, together with the extensive enzyme characterization, which
was further documented in 3T3 adipocytes (see below), indicated that we
have cloned the murine SSAO. Murine SSAO shares with rat membrane-bound
amine oxidase vp97 and human placental amine oxidase a short 5-amino
acid intracellular segment (positions 1-5) and a hydrophobic
transmembrane domain (positions 6-26) that probably ensures anchoring
in the plasma membrane. The level of identity is lower between murine
SSAO and bovine serum amine oxidase (79% identity) and human kidney
diamine oxidase (35% identity), especially in the N-terminal portion.
This difference is the consequence of the fact that bovine serum amine
oxidase and diamine oxidase are secreted enzymes, while SSAO is
membrane-associated. During the preparation of this manuscript, a new
search of the GenBankTM sequence data base revealed the
identity of murine SSAO with vascular adhesion protein-1 (VAP-1), whose
mRNA and gene have been cloned from nonadipose tissues from mouse
and human (27-29). VAP-1 has been implicated in lymphocyte binding to
endothelial cells in the immune system (30).
Pattern of SSAO mRNA Expression in Various Rat Tissues--
So
far, tissue distribution of SSAO has been documented only on the basis
of its enzyme activity. Thus, it was important to verify whether the
distribution of the SSAO mRNA was identical to that of the enzyme
activity referred as SSAO. The level of SSAO expression was examined in
a variety of rat tissues by Northern analysis. Results are shown in
Fig. 3. As assessed by methylene blue
staining of ribosomal mRNAs, equivalent amounts of total RNA were
present in each lane. SSAO mRNA was mainly found in white adipose
tissue, brown adipose tissue, and aorta and found at lower levels in
lung and skeletal muscle. A long exposure of the same blot also
revealed limited amounts of SSAO mRNA in brain, heart, intestine,
and kidney. The preferential expression of SSAO transcripts in adipose
tissue and aorta, and at a lesser extent in lung and skeletal muscle is
in good agreement with the previously described tissue distribution of
SSAO activity in different species (10, 31-39).
Regulation of SSAO mRNA Expression and Enzyme Activity during
Adipocyte Differentiation--
Due to the very high SSAO mRNA
levels in white adipocyte tissue, we investigated the temporal pattern
of SSAO gene expression and enzyme activity in several types of
preadipose cells. We first examined SSAO gene expression during adipose
conversion of 3T3-F442A and 3T3-L1 cell lines. Total RNA was harvested
from cells at various intervals before and after cell confluence (day
0). As shown in Fig. 4, A and
B, SSAO mRNA was virtually absent in preadipocytes, and
its level dramatically increased during adipocyte differentiation of
3T3-F442A or 3T3-L1 cells. In the two cell lines, SSAO transcripts were
clearly induced between days 3 and 5 and rose almost to a plateau level
8 days after confluence. Various times of autoradiographic exposure led
us to estimate an approximately 100-500-fold induction during the
adipose conversion process. When compared with the emergence of
adipocyte lipid-binding protein and glycerol-3-phosphate dehydrogenase
mRNAs, two well characterized markers of the adipose conversion
process (21, 40), SSAO mRNA induction occurred after adipocyte
lipid-binding protein transcript emergence and was concomitant with
that of the glycerol-3-phosphate dehydrogenase transcript. Thus, SSAO
transcript expression appeared to represent a late event of
adipogenesis. The high abundance of SSAO mRNA in adipose tissue
described above prompted us to determine whether emergence of SSAO
mRNA was also differentiation-specific in primary culture of rat
adipocytes. Fig. 4C shows that SSAO mRNA levels were
also strongly induced during differentiation of these rat precursor
cells.
We then examined whether changes in mRNA levels of SSAO were
reflected by changes in the corresponding enzyme activity. Cell homogenates were prepared from 3T3-L1 cells at various times before and
after cell confluence. Extracts were also obtained from
undifferentiated or differentiated 3T3-F442A cells and from growing and
resting 3T3-C2 cells (Fig. 5). Enzyme
assays were performed in the presence of an excess of pargyline to
prevent an interference of MAO activity. Since over 97% of benzylamine
oxidase activity was inhibited by prior incubation with 100 µM semicarbazide, this activity will be referred as SSAO
activity. Until day 2 of confluence, SSAO activity was very low, but it
was strongly induced by day 4 of confluence and reached a maximal level
between days 6 and 8. There was a 50-100-fold increase in SSAO
activity during differentiation of 3T3-L1 preadipocytes. In agreement
with previous studies (9, 10, 35-37), subcellular fractionation of
3T3-L1 adipocytes indicated that SSAO activity was preferentially
present in the crude membranes but existed at very low levels in the
cytosol (data not shown). During the differentiation of 3T3-F442A
cells, an even more dramatic rise in SSAO specific activity was
observed (Fig. 5). By contrast, no increase in enzyme activity could be
detected in 3T3-C2 cells, a cell line unable to undergo adipose
conversion. Thus, results shown in Figs. 4 and 5 emphasize the close
parallel between the pattern of SSAO gene expression and that of enzyme
activity.
To explore the specificity of SSAO induction during the course of
adipose conversion, we also examined in 3T3 cell lines the pattern of
activities of two other members of the amine oxidase family, diamine
oxidase and polyamine oxidase (Table I).
Diamine oxidase activity, estimated with putrescine as a substrate,
clearly decreased during adipose conversion of 3T3-L1 cells but
remained stable in 3T3-C2 cells. Polyamine oxidase activity, measured
with N1-acetylspermidine as a substrate,
moderately increased by about 2-fold during differentiation of 3T3-L1
or 3T3-F442A cells. Since the same magnitude of enzyme activity
induction was observed in quiescent 3T3-C2 cells, as compared with the
growing 3T3-C2 fibroblasts, the moderate increase of polyamine oxidase
activity in 3T3-L1 or 3T3-F442A adipocyte is not the consequence of the
differentiation process. Taken together, our results demonstrate that,
among the mRNAs and enzyme activities of amine oxidases tested,
SSAO is specifically and dramatically induced during adipocyte
differentiation.
Biochemical Characterization of SSAO Activity in 3T3-L1 and
3T3-F442A Adipocytes--
To further characterize SSAO activity of
3T3-L1 and 3T3-F442A mature adipocytes, we analyzed a variety of
substrates and inhibitors. We first investigated the deaminating
properties of homogenates prepared from 3T3-L1 and 3T3-F442A adipocytes
in the presence of benzylamine, methylamine,
N1-acetylputrescine, histamine, tyramine, and
A range of potential inhibitors of SSAO was also tested against a
submaximal concentration (50 µM) of benzylamine in cell homogenates of 3T3-L1 adipocytes. Inhibitors were present in the assay
20 min prior to the benzylamine addition. Except when the MAO component
of benzylamine oxidase activity was tested, an excess of the MAO
inhibitor pargyline (1 mM) was included during the preincubation and incubation periods. As shown in Table
III, SSAO activity was sensitive toward
carbonyl reagents, such as semicarbazide or aminoguanidine. Otherwise,
Thus, our biochemical characterization of SSAO activity derived from
substrate and inhibitor studies demonstrates the close relationship
between the enzyme activity present in 3T3-L1 or 3T3-F442A adipocytes
and that previously described in white and brown adipose tissue
(35-37).
Regulation of SSAO mRNA Content and Enzyme Activity by
Effectors of the cAMP Signaling Pathway and TNF- Alteration in SSAO Gene Expression and Enzyme Activity in an Animal
Model of Genetic Obesity--
To analyze whether SSAO gene expression
could be modified in obesity, we examined SSAO mRNA content and
enzyme activity in inguinal white adipose tissue samples from
1-month-old lean (Fa/fa) and obese (fa/fa) male
Zücker rats. A ~3-fold reduction in SSAO transcripts was
observed in white adipose tissue of obese rats as compared with their
lean littermates (Fig. 7A). A
parallel decrease in enzyme activity was also detected (Fig.
7B). Since less than 5% of benzylamine oxidase activity was
resistant to semicarbazide in lean or obese animals, this activity
essentially represented that of SSAO. These results suggest that SSAO
expression is clearly dysregulated in obesity from the fa/fa
rat.
The differential display technique has the potential power for
identifying genes that are differentially expressed in a variety of
situations including cellular differentiation. By studying differential
gene expression during the adipose conversion process, we have
identified an adipose mRNA species present at high levels in
adipocyte and virtually absent from preadipocyte. The present study
reports the cloning and the sequencing of the corresponding full-length
cDNA. This cDNA encodes the murine homolog of the human
placental amine oxidase whose properties as protein have not been
examined by the authors (8). It is also the homolog of a partial rat
adipocyte cDNA sequence referred as vp97 and corresponds to a major
integral glycoprotein of adipocyte plasma membrane (9). In addition, we
here report the extensive biochemical characterization of the related
amine oxidase enzyme activity. The biochemical properties of the
enzyme, its modulation by important signaling molecules, and its
dysregulation in an animal model of obesity suggest that it could play
an important role in adipocyte homeostasis.
The amine oxidase described here is known as SSAO. It is highly
susceptible to inhibition by semicarbazide and is usually assayed using
benzylamine as a substrate. Amine oxidases, which are distributed
across all phyla, can be classified on the basis of their biochemical
properties, now complemented with gene studies. In this regard, the
complete SSAO cDNA sequence reported in this study contains all
hallmarks of a gene encoding a class of enzymes with a new prosthetic
group belonging to the topaquinone-amine oxidase family (49, 50). The
presence of the 6-hydroxydopa quinone as the true cofactor at the
active site of this family was first reported by Klinman and
colleagues, who studied bovine plasma amine oxidase (51). In this
study, we identify the amino acid sequence adjacent to SSAO cofactor
site by comparison of the translated sequence of SSAO cDNA with
sequences of known cofactor sites for other
copper(II)-topaquinone-containing amine oxidases (49, 50). It is now
recognized that this quinone (oxidized form of the
trihydroxyphenylamine) is formed by co- or post-translational modification of a specific tyrosyl residue within the polypeptide chain
of the immature enzyme (49, 50).
The chemical nature of a set of compounds inhibiting SSAO activity as
well as the order of potency of these substances (Table II) are quite
consistent with the current knowledge of quinoenzymes. Reagents that
are able to react with the oxidized form of trihydroxyphenylamine and
to derivatize it as an hydrazone are strong inhibitors of SSAO
activity. The enzyme has the lowest apparent Ki values toward phenelzine and hydrazine, whereas phenylhydrazine is 2 orders of magnitude less potent than these compounds. However in our
endeavor to provide a useful guide to relative potencies, we should
mention that apparent Ki values may depend both on
the nature of the inhibitory process and the form of the enzyme
(soluble or particulate as well as crude or purified preparations). As
far as the irreversible inhibitor semicarbazide is concerned, the molar
ratio of inhibitor over enzyme concentration can be critical.
In this study, we have distinguished SSAO activity from that of other
amine oxidases. Indeed, clorgyline and pargyline, which are classical
effectors of MAOs (amine:oxygen oxidoreductase (deaminating) (flavin-containing)) do not inhibit the SSAO quinoenzyme activity (Table II). Under MAO blockade, the residual amine oxidase activity (i.e. resistant to semicarbazide inhibition) has never
exceeded 5% for the total amine oxidase activity, whatever kind of
substrate was used. This strongly suggests that SSAO activity
represents a major amine oxidase in 3T3 adipocytes. The substrate
specificities of MAOs overlap to some extent and complicate enzyme
activity analysis. SSAO can oxidize both aromatic and aliphatic primary amines. The nonphysiological amine benzylamine is the best known substrate. Methylamine and N1-acetylputrescine
are both physiological amines and are also significantly oxidized by
SSAO. Finally, adipocyte SSAO does not seem to exhibit a narrow
substrate specificity, since histamine as well as tyramine can also be
degraded by the enzyme. Although methylamine and aminoacetone, which
are both amine products of intermediary metabolism, have been proposed
as putative candidates (37, 52-55), the nature of the endogenous
substrate(s) of SSAO remains so far speculative. Since adipose tissue
is a privileged site of SSAO gene expression and represents a major
component of body weight composition, we can question the potential
involvement of this enzyme in the removal and/or the detoxification
toward specific amines. Molecular cloning of the SSAO cDNA will
probably help in the identification of new physiological or
nonphysiological substrates.
Beside its role of degradation and/or scavenger of bioactive amines,
the precise physiological functions of SSAO remain open to discussion.
Several lines of evidence support the idea that SSAO could play an
important role in the control of energy balance in adipose tissue. (i)
SSAO is highly and preferentially expressed in white and brown adipose
tissue. This observation is consistent with the dramatic induction of
the enzyme during the adipose conversion process. (ii) Two recent
reports have identified SSAO as an integral protein mainly expressed in
adipocyte plasma membrane but also colocalized with GLUT4
transporter-containing vesicles (9, 10). It is noteworthy that SSAO,
through hydrogen peroxide production, can activate glucose transport in
isolated rat adipocytes (56). This further documents that in
adipocytes, hydrogen peroxide can mimic some insulin effects. It is
well established that hydrogen peroxide not only activates glucose
transport but also stimulates glucose C-1 oxidation (57), glucose
incorporation into glycogen (58), and lipogenesis (59) and inhibits
lipolysis (60-62). Further investigations are required to demonstrate
whether SSAO, which is able to produce hydrogen peroxide on the outer
of the plasma membrane, is involved in these metabolic pathways. (iii) SSAO gene expression and the corresponding enzyme activity are markedly
altered by cAMP and TNF- During the preparation of this manuscript, a murine cDNA with an
identical sequence to our adipose SSAO mRNA species was reported and appeared in the data base. Starting from a purified protein with
lymphocyte adhesion properties, VAP-1 (30), Jalkanen's group has
isolated the related human and murine cDNAs (27, 28). VAP-1 is
expressed in high endothelial venules from peripheral lymphatic tissues
and appears to play an important role in lymphocyte trafficking (30).
From comparative analysis of various sequences, the product of VAP-1
cDNA appears to display a dual function of lymphocyte adhesion and
amine oxidase activity. While substantial differences exist concerning
the substrate specificity between VAP-1 overexpressed in transfected
cells and the enzyme in the native adipocyte context, all of these
findings underline the multiple physiological roles of the VAP-1/SSAO
according to the nature of the cell type.
So far, amine oxidases have been considered as widely distributed in
animal tissues and organs on the basis of enzyme activity measurements.
Due to the use of nonselective substrates and inhibitors, the probing
of these oxidases with modern molecular biological methodologies seems
much more appropriate. In our study, after prolonged autoradiography of
tissue blots, low amounts of SSAO mRNA were detected in brain,
heart, intestine, and kidney. An intermediate level of expression was
seen in lung and skeletal muscle. Overall, the preferential SSAO gene
expression was observed in adipose tissue and aorta. The high abundance
of SSAO mRNA in aorta agrees with previous reports on enzyme
activity (31-33, 76). Several works converge to indicate that SSAO is
essentially present in smooth muscle cells (32, 33, 76, 77). The
presence of SSAO in the blood vessel wall and its location in the
plasma membrane (33, 78) support a possible scavenging role with respect to circulating amines. Alternatively, the generation of the
reactive molecule, hydrogen peroxide, at the cell surface could be of
importance in modulating signals controlling the activity of this
contractile tissue. Otherwise, the presence of SSAO in the smooth
muscle cells from the media of aorta might be relevant in the
pathophysiology of vascular diseases, including atherosclerosis. The
cloning of adipocyte murine SSAO should permit us to further explore
the involvement of this protein under physiological or diseased states.
, indicating that two major signaling
molecules of adipose tissue development and metabolism can control SSAO
function. Moreover, the expression of SSAO transcripts and activity are
clearly down-regulated in white adipose tissue from obese Zücker
rats. Because of its known stimulatory effect on glucose transport, its
biochemical properties and its pattern of expression and regulation,
SSAO could play an important role in the regulation of adipocyte homeostasis.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C until use. Protein content was assayed
according to Lowry et al. (22) using bovine serum albumin as
a standard.
-phenylethylamine. To determine IC50 and Ki values for amine
oxidase inhibitors, SSAO activity was determined in the presence of 50 µM benzylamine as a substrate. The concentration ranges
of inhibitors used were as follows: 300 nM to 1 mM for semicarbazide and aminoguanidine, 0.1 nM
to 1 µM for phenelzine, 0.3 nM to 3 µM for hydrazine, 1 nM to 100 µM for hydralazine, 3 nM to 300 µM for phenylhydrazine, 100 nM to 1 mM for iproniazide, 300 nM to 1 mM
for
-aminopropionitrile, 10 nM to 100 µM for benserazide, and 0.1 nM to 1 mM for pargyline and clorgyline. Kinetic parameters were
determined using the nonlinear regression analysis curve-fitting
procedure of the ENZFITTER program (Biosoft-Elsevier, Cambridge, United
Kingdom). Polyamine oxidase was measured under conditions identical to
those for SSAO, except that N1-acetylspermidine
was used as a substrate. Diamine oxidase was tested radiochemically by
the conversion of [1,4-14C]putrescine dihydrochloride in
14C-labeled
1-pyrroline (24).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Differential display of mRNAs from
undifferentiating or differentiating 3T3 cells. A,
total RNA was prepared from undifferentiated (P, 1 day
before confluence) or differentiated (A, day 8 after
confluence) 3T3-F442A cells and from growing (G, 1 day
before confluence) or resting (R, day 8 after confluence)
3T3-C2 fibroblasts. mRNA differential display reactions were
carried out as mentioned under "Experimental Procedures." PCR
products were visualized by ethidium bromide staining. Molecular size
markers (in bp) are indicated on the right. The
arrow shows the position of the 840-bp PCR fragment
differentially expressed in 3T3-F442A adipocytes. B,
Northern analysis of total RNA derived from preconfluent (1 day before
confluence) and day 8 postconfluent 3T3-F442A or 3T3-C2 cells. Total
RNA was electrophoresed (10 µg/lane), blotted to nylon membranes, and
hybridized to the radiolabeled 840-bp DNA fragment. Methylene blue
staining of the 18 S ribosomal RNA was performed to assess the
equivalence in RNA loading. The apparent size of the transcript is
indicated on the right.
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Fig. 2.
Deduced amino acid sequence of murine SSAO
and alignment with other cloned members of the copper-containing amine
oxidase family. The numbering on the right
of each row corresponds to the last amino acid. The
consensus sequence including the pre-TPQ-Tyr is boxed, and
the Pre-TPQ-Tyr itself is underlined. The
asterisks denote the position of the histidine residues
involved in copper binding. The residues corresponding to potential
N-glycosylation sites are in boldface
type (8). The putative transmembrane domain of murine SSAO
is underlined. mSSAO, murine
semicarbazide-sensitive amine oxidase; vp97, partial
sequence of the rat adipocyte membrane-bound amine oxidase (9);
hPAO, human placental amine oxidase (8); bSAO,
bovine serum amine oxidase (25); hDAO, human kidney diamine
oxidase (26).
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Fig. 3.
Tissue distribution of SSAO mRNA in
rat. Total RNA from various rat tissues was tested in Northern
analysis (20 µg/lane) was as described in the legend of Fig. 1.
Tissues are designated as follows. A, aorta; B,
brain; BF, brown adipose tissue; WF, white
adipose tissue; H, heart; I, intestine;
K, kidney; Li, liver; Lu, lung;
M, skeletal muscle. A and B correspond
to a short (24 h) or long (5 days) autoradiographic exposure,
respectively. C represents the methylene blue staining of 18 S ribosomal RNAs derived from the blot.
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Fig. 4.
SSAO mRNA levels during adipose
conversion of 3T3 cell lines and of rat precursor primary
cultures. Northern blot analysis was performed on total RNA (10 µg/lane) extracted from 3T3-F442A (A) or 3T3-L1
(B) cells at the indicated times relative to confluence (day
0). The same blots were hybridized to the 840-bp SSAO DNA probe or to
adipocyte lipid-binding protein (aP2) or
glycerol-3-phosphate dehydrogenase (G3PDH)
32P-labeled cDNA probes. Equivalence in RNA loading in
each lane was assessed by methylene blue staining. SSAO mRNA levels
were also tested in undifferentiated or differentiated primary cultures
of rat precursor cells (C).
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Fig. 5.
Development of SSAO activity during the
course of adipose conversion. Cell homogenates were prepared from
3T3-L1, 3T3-F442A, and 3T3-C2 cells at the indicated times relative to
confluence (day 0). SSAO activity was measured by
H2O2 production as described under
"Experimental Procedures." Results are expressed in nmol of
H2O2/h/mg of protein and represent the
mean ± S.E. of four independent experiments.
Amine oxidase activities in 3T3 subclones
-phenylethylamine (Table II). When the
amine oxidase assay was performed with a prior incubation with 100 µM semicarbazide, only 0.5-5% of the total amine
oxidase activity was resistant to inhibition by this compound (data not
shown). This observation indicated that in 3T3-L1 and 3T3-F442A
adipocytes, the amine oxidase activity responsible for the deamination
of these substrates corresponded mainly to SSAO activity. The rank
order of Vmax values for the different substrates was as follows: benzylamine > methylamine > N1-acetylputrescine >
-phenylethylamine > tyramine > histamine. The lowest
Km value was measured with benzylamine, while the
highest Km values were observed with
N1-acetylputrescine and histamine. SSAO was not
able to oxidize diamines and polyamines such as putrescine, spermine,
spermidine, and cadaverine (data not shown).
Kinetic parameters of SSAO activity in 3T3-L1 or 3T3-F442A adipocytes
-phenylethylamine were performed as described under "Experimental
Procedures," with a prior incubation with 1 mM pargyline.
Since less than 5% of amine oxidase activity remained when cell
extracts were preincubated in the presence of 100 µM
semicarbazide, it almost certainly corresponded to the SSAO activity.
Km and Vmax values were
calculated by the nonlinear regression analysis curve fitting procedure
of the ENZFITTER program. Intrinsic activities (IA) were calculated by
standardizing the Vmax value of each substrate to
that of benzylamine. Results represent the mean ± S.E. of 4-6
separate experiments performed in duplicate or triplicate.
-aminopropionitrile, an inhibitor of lysyl oxidase (42), was a poor
inhibitor of SSAO activity. By contrast, hydrazine derivatives
displayed the lowest Ki values for SSAO inhibition.
Phenelzine and hydrazine appeared to be the most potent compounds, with
Ki values ranging from 1 to 3 nM.
Finally, benzylamine oxidase assays performed with prior incubations
with various concentrations of pargyline or clorgyline indicated that
these two MAO inhibitors were virtually inefficient to block SSAO
activity. Under our experimental conditions, more than 90% of
benzylamine oxidase activity was resistant to 1 mM
clorgyline or pargyline. This result indicated that in 3T3 adipocytes,
benzylamine oxidase activity was essentially related to SSAO
activity, while the MAO activity component was negligible.
Inhibition of SSAO activity by various compounds
in 3T3-L1
Adipocytes--
cAMP has been demonstrated to exert a key role in the
control of gene expression in adipocytes (43-46). Likewise, TNF-
has a pleiotropic effect on adipocyte development and metabolism (47, 48). Thus, we examined whether effectors of the cAMP signaling pathway
and TNF-
could modulate SSAO gene expression and activity in
adipocytes. 3T3-L1 adipocytes (day 8 postconfluence) were shifted in a
defined medium and then exposed or not exposed to (
)-isoproterenol, forskolin, or TNF-
. The regulation of SSAO gene expression was analyzed by Northern blot after a 24-h treatment (Fig.
6A). For measurement of SSAO
activity, cell homogenates were prepared after a 48-h effector exposure
(Fig. 6B). Isoproterenol and forskolin provoked a clear
decrease in SSAO mRNA levels, while TNF-
dramatically reduced
SSAO mRNA abundance. These variations in transcript levels were
reflected by parallel changes in SSAO activity. Interestingly, insulin
was not able to modulate SSAO gene expression and activity under
similar experimental conditions (data not shown).
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Fig. 6.
Regulation of SSAO gene expression and enzyme
activity by effectors of the cAMP signaling pathway and
TNF- in 3T3-L1 adipocytes. At day 8 following confluence, 3T3-L1 adipocytes were shifted for 24 h in a
defined medium consisting of DMEM/Ham's F-12 medium (2:1, v/v) and
0.1% bovine serum albumin. Adipocytes were then exposed or not exposed
(control (C)) to 10 µM (
)-isoproterenol
(Iso), 10 µM forskolin (FSK), or 1 nM TNF-
. A, total RNA was prepared after a
24-h treatment and tested for SSAO mRNA content. Methylene blue
staining of the 18 S ribosomal RNA was performed to assess the
equivalence in RNA loading. B, cell homogenates were
obtained after a 48-h treatment and used to measure SSAO activity. More
than 95% of the enzyme activity was inhibited by prior incubation with
100 µM semicarbazide. Results are expressed as the
percentage of SSAO activity measured in control cells and represent the
mean ± S.E. of 4-7 independent experiments. SSAO activity was
12.62 ± 0.74 nmol of H2O2/h/mg of protein
in control adipocytes. *, p < 0.01, isoproterenol- or
forskolin-treated versus control adipocytes. **,
p < 0.001, TNF-
-treated versus control
adipocytes (Student's t test).
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Fig. 7.
SSAO gene expression and enzyme activity in
white adipose tissue from lean and obese Zücker rats.
A, 10 µg/lane of total RNA from lean and obese rat
inguinal fat pads were examined for SSAO mRNA content. Methylene
blue staining of the 18 S ribosomal RNA was performed to control the
similarity in RNA loading. B, SSAO enzyme activity of white
adipose tissue homogenates from lean and obese Zücker rats was
measured as described under "Experimental Procedures." Enzyme
assays were performed in the absence ( ) or in the presence (+) of 100 µM semicarbazide (SCZ). Results are expressed
in nmol of H2O2/h/mg of protein and represent
the mean ± S.E. of eight individual determinations in each group.
*, p < 0.001, obese versus lean animals
(Student's t test).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, two signaling molecules that exert marked
effects on adipose tissue development and metabolism. Cyclic AMP, the
messenger produced by activation of the
-adrenergic system, can
acutely activate lipolysis, but it also negatively regulates several
enzymes of the lipogenic pathway (43, 44, 63, 64) and interferes with
adipocyte differentiation (12, 46, 65). TNF-
has also profound
effects on lipid metabolism and adipose conversion. Beside its
antilipogenic (66-68) and lipolytic (69) action on mature adipocytes,
TNF-
potently inhibits adipocyte differentiation (70). This cytokine
is responsible, at least in part, for the insulin resistance observed
in obesity (71, 72). (iv) Finally, SSAO transcript levels and enzyme
activity are clearly dysregulated in white adipose tissue from obese
Zücker rats. Given the overproduction of TNF-
in white adipose
tissue from obese humans and animals (including the fa/fa
model) (71, 73-75) and the inhibitory effect of TNF-
on SSAO
expression in 3T3-L1 adipocytes, we speculate that the reduction in
SSAO levels detected in obese rats might be related, at least in part,
to an excess of local or systemic TNF-
. Taken together, these
observations support the view that SSAO could be of importance in
adipocyte physiology. The exact involvement of this enzyme in the main
metabolic pathways of fat cells remains to be clarified.
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ACKNOWLEDGEMENTS |
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We thank Dr. J. B. Michel and Dr. M. P. Jacob for the gift of aorta total RNA and Dr. R. Pecquery for providing of rat preadipocyte RNA. Dr. P. Djian is gratefully acknowledged for critical review of the manuscript.
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FOOTNOTES |
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* This work was supported by the Centre National de la Recherche Scientifique, Université Paris VI, and the Fondation pour la Recherche Médicale.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF115411.
Recipient of grants from the Ministère de l'Enseignement
Supérieur et de la Recherche, and the Association pour la
Recherche sur le Cancer.
§ To whom correspondence should be addressed. Tel.: 33-1-42-34-68-74; Fax: 33-1-46-34-59-73; E-mail: pairault{at}infobiogen.fr.
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ABBREVIATIONS |
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The abbreviations used are: SSAO, semicarbazide-sensitive amine oxidase; 3' and 5'-RACE, 3'- and 5'-rapid amplification of cDNA ends; DMEM, Dulbecco's modified Eagle's medium; MAO, monoamine oxidase; PCR, polymerase chain reaction; TPQ, topaquinone (6-hydroxydopa quinone); bp, base pair(s); TNF, tumor necrosis factor; VAP-1, vascular adhesion protein-1.
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