Molecular Cloning of a Major mRNA Species in Murine 3T3 Adipocyte Lineage
DIFFERENTIATION-DEPENDENT EXPRESSION, REGULATION, AND IDENTIFICATION AS SEMICARBAZIDE-SENSITIVE AMINE OXIDASE*

Marthe MoldesDagger , Bruno Fève, and Jacques Pairault§

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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-alpha , 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 -80 °C until use. Protein content was assayed according to Lowry et al. (22) using bovine serum albumin as a standard.

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 beta -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 beta -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 Delta 1-pyrroline (24).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (57K):
[in this window]
[in a new window]
 
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.

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).


View larger version (68K):
[in this window]
[in a new window]
 
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).

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).


View larger version (60K):
[in this window]
[in a new window]
 
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.

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.


View larger version (42K):
[in this window]
[in a new window]
 
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).

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.


View larger version (21K):
[in this window]
[in a new window]
 
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.

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.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Amine oxidase activities in 3T3 subclones
Cell homogenates were prepared from undifferentiated (day 1 before confluence) or differentiated (day 8 after confluence) 3T3-L1 or 3T3-F442A cells and from growing (day 1 before confluence) or resting (day 8 after confluence) 3T3-C2 fibroblasts. SSAO, diamine oxidase (DAO), and polyamine oxidase (PAO) activities were measured as described under "Experimental Procedures." Results are expressed in nmol of H2O2/h/mg of protein and represent the mean ± S.E. of 4-7 independent experiments.

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 beta -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 > beta -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).

                              
View this table:
[in this window]
[in a new window]
 
Table II
Kinetic parameters of SSAO activity in 3T3-L1 or 3T3-F442A adipocytes
Cell extracts were obtained from mature 3T3-L1 and 3T3-F442A adipocytes. Enzyme activity towards benzylamine, methylamine, N1-acetyl putrescine, histamine, tyramine, and beta -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.

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, beta -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.

                              
View this table:
[in this window]
[in a new window]
 
Table III
Inhibition of SSAO activity by various compounds
Cell extracts were prepared from mature 3T3-L1 adipocytes. 3T3-L1 cell homogenates were preincubated for 20 min at 37 °C in the presence of various concentrations of each inhibitor. Pargyline at 1 mM was also present to inhibit MAO activity, except when the enzyme activity was tested with various concentrations of clorgyline or pargyline. Benzylamine (50 µM) was then added as substrate for 1 h. IC50 values were obtained by computer analysis with the ENZFITTER program. Apparent Ki values of SSAO towards the different compounds were calculated by the equation of Cheng and Prusoff (41). Results represent the mean ± S.E. of 3-5 independent experiments.

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-alpha 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-alpha has a pleiotropic effect on adipocyte development and metabolism (47, 48). Thus, we examined whether effectors of the cAMP signaling pathway and TNF-alpha 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-alpha . 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-alpha 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).


View larger version (45K):
[in this window]
[in a new window]
 
Fig. 6.   Regulation of SSAO gene expression and enzyme activity by effectors of the cAMP signaling pathway and TNF-alpha 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-alpha . 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-alpha -treated versus control adipocytes (Student's t test).

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.


View larger version (30K):
[in this window]
[in a new window]
 
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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-alpha , two signaling molecules that exert marked effects on adipose tissue development and metabolism. Cyclic AMP, the messenger produced by activation of the beta -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-alpha has also profound effects on lipid metabolism and adipose conversion. Beside its antilipogenic (66-68) and lipolytic (69) action on mature adipocytes, TNF-alpha 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-alpha in white adipose tissue from obese humans and animals (including the fa/fa model) (71, 73-75) and the inhibitory effect of TNF-alpha 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-alpha . 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.

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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

Dagger 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.

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
  1. Green, H., and Kehinde, O. (1974) Cell 1, 113-116
  2. Green, H., and Kehinde, O. (1976) Cell 7, 105-113[Medline] [Order article via Infotrieve]
  3. Négrel, R., Grimaldi, P., and Ailhaud, G. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 6054-6058[Abstract]
  4. Ailhaud, G., Grimaldi, P., and Négrel, R. (1992) Annu. Rev. Nutr. 12, 207-233[CrossRef][Medline] [Order article via Infotrieve]
  5. Cornelius, P., MacDougald, O. A., and Lane, M. D. (1994) Annu. Rev. Nutr. 14, 99-129[CrossRef][Medline] [Order article via Infotrieve]
  6. Liang, P., and Pardee, A. B. (1992) Science 257, 967-971[Medline] [Order article via Infotrieve]
  7. Sokolov, B. P., and Prockop, D. J. (1994) Nucleic Acids Res. 22, 4009-4015[Abstract]
  8. Zhang, X., and MacIntire, W. S. (1996) Gene (Amst.) 179, 279-286[CrossRef][Medline] [Order article via Infotrieve]
  9. Morris, N. J., Ducret, A., Aebersold, R., Ross, S. A., Keller, S. R., and Lienhard, G. E. (1997) J. Biol. Chem. 272, 9388-9392[Abstract/Free Full Text]
  10. Enrique-Tarancon, G., Marti, L., Morin, N., Lizcano, J. M., Unzeta, M., Sevilla, L., Camps, M., Palacin, M., Testar, X., Carpene, C., and Zorzano, A. (1998) J. Biol. Chem. 273, 8025-8032[Abstract/Free Full Text]
  11. Green, H., and Kehinde, O. (1975) Cell 5, 19-27[Medline] [Order article via Infotrieve]
  12. Rubin, C. S., Hirsch, A., Fung, C., and Rosen, O. M. (1978) J. Biol. Chem. 253, 7570-7578[Medline] [Order article via Infotrieve]
  13. Rodbell, M. (1964) J. Biol. Chem. 239, 375-380[Free Full Text]
  14. Deslex, S., Négrel, R., and Ailhaud, G. (1987) Exp. Cell. Res. 168, 15-30[Medline] [Order article via Infotrieve]
  15. Cathala, C., Savouret, J. F., Mendez, B., Karin, M., Martial, J. A., and Baxter, J. D. (1983) DNA 2, 329-335[Medline] [Order article via Infotrieve]
  16. Frohman, M. A., Dush, M. K., and Martin, G. R. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8998-9002[Abstract]
  17. Frohman, M. A. (1995) in PCR primer: A Laboratory Manual (Dieffenbach, C. W., and Dveksler, G. S., eds), pp. 381-409, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  18. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159[CrossRef][Medline] [Order article via Infotrieve]
  19. Feinberg, A. P., and Vogelstein, B. (1983) Anal. Biochem. 132, 6-13[Medline] [Order article via Infotrieve]
  20. Church, G. M., and Gilbert, W. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1991-1995[Abstract]
  21. Spiegelman, B. M., Frank, M., and Green, H. (1983) J. Biol. Chem. 258, 10083-10089[Abstract/Free Full Text]
  22. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275[Free Full Text]
  23. Matsumoto, T., Furuta, T., and Nimura, Y. (1982) Biochem. Pharmacol. 31, 2207-2209[Medline] [Order article via Infotrieve]
  24. Okuyama, T., and Kobayashi, Y. (1961) Arch. Biochem. Biophys. 96, 242-250
  25. Mu, D., Medzihradszky, K. F., Adams, G. W., Mayer, P., Hines, W. M., Burlingame, A. M., Smith, A. J., Cai, D., and Klinman, J. P. (1994) J. Biol. Chem. 269, 9926-9932[Abstract/Free Full Text]
  26. Barbry, P., Champe, M., Chassande, O., Munemitsu, S., Champigny, G., Lingueglia, E., Maes, P., Frelin, C., Tartar, A., Ullrich, A., and Lazdunski, M. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7347-7351[Abstract]
  27. Bono, P., Salmi, M., Smith, D. J., and Jalkanen, S. (1998) J. Immunol. 160, 5563-5571[Abstract/Free Full Text]
  28. Smith, D. J., Salmi, M., Bono, P., Hellman, J., Leu, T., and Jalkanen, S. (1998) J. Exp. Med. 188, 17-27[Abstract/Free Full Text]
  29. Bono, P., Salmi, M., Smith, D. J., Leppänen, I., Horelli-Kuitunen, N., Palotie, A., and Jalkanen, S. (1998) J. Immunol. 161, 2953-2960[Abstract/Free Full Text]
  30. Salmi, M., and Jalkanen, S. (1992) Science 257, 1407-1409[Medline] [Order article via Infotrieve]
  31. Lewinsohn, R., Bohm, K. H., Glover, V., and Sandler, M. (1978) Biochem. Pharmacol. 27, 1857-1863[CrossRef][Medline] [Order article via Infotrieve]
  32. Lewinsohn, R. (1981) J. Pharm. Pharmacol. 33, 569-575[Medline] [Order article via Infotrieve]
  33. Lyles, G. A., and Singh, I. (1985) J. Pharm. Pharmacol. 37, 637-643[Medline] [Order article via Infotrieve]
  34. Elliott, J., Callingham, B. A., and Sharman, D. F. (1989) Biochem. Pharmacol. 38, 1507-1515[CrossRef][Medline] [Order article via Infotrieve]
  35. Barrand, M. A., and Callingham, B. A. (1982) Biochem. Pharmacol. 31, 2177-2184[Medline] [Order article via Infotrieve]
  36. Raimondi, L., Pirisino, R., Ignesti, G., Capecchi, S., Banchelli, G., and Buffoni, F. (1991) Biochem. Pharmacol. 41, 467-470[CrossRef][Medline] [Order article via Infotrieve]
  37. Raimondi, L., Pirisino, R., Banchelli, G., Ignesti, G., Conforti, L., Romanelli, E., and Buffoni, F. (1992) Comp. Biochem. Physiol. 102B, 953-960
  38. Lizcano, J. M., Fernandez de Arriba, A., Tipton, K. F., and Unzeta, M. (1996) Biochem. Pharmacol. 52, 187-195[CrossRef][Medline] [Order article via Infotrieve]
  39. Lizcano, J. M., Tipton, K. F., and Unzeta, M. (1998) Biochem. J. 331, 69-78[Medline] [Order article via Infotrieve]
  40. Bernlohr, D. A., Angus, C. W., Lane, M. D., Bolanowski, M. A., and Kelly, T. J. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 5468-5472[Abstract]
  41. Cheng, Y., and Prusoff, W. H. (1973) Biochem. Pharmacol. 22, 3099-3108[CrossRef][Medline] [Order article via Infotrieve]
  42. Pinnell, S. R., and Martin, G. R. (1968) Proc. Natl. Acad. Sci. U. S. A. 61, 708-712[Medline] [Order article via Infotrieve]
  43. Spiegelman, B. M., and Green, H. (1981) Cell 24, 503-510[CrossRef][Medline] [Order article via Infotrieve]
  44. Antras, J., Lasnier, F., and Pairault, J. (1991) Mol. Cell. Endocrinol. 82, 183-190[CrossRef][Medline] [Order article via Infotrieve]
  45. Cornelius, P., Marlowe, M., Call, K., and Pekala, P. H. (1991) J. Cell. Physiol. 146, 298-308[Medline] [Order article via Infotrieve]
  46. Wang, H. Y., Watkins, D. C., and Malbon, C. C. (1992) Nature 258, 334-337
  47. Beutler, B., and Cerami, A. (1989) Annu. Rev. Immunol. 7, 625-655[CrossRef][Medline] [Order article via Infotrieve]
  48. Grunfeld, C., and Feingold, K. R. (1991) Trends Endocrinol. Metab. 2, 213-219
  49. Klinman, J. P., and Mu, D. (1994) Annu. Rev. Biochem. 63, 299-344[CrossRef][Medline] [Order article via Infotrieve]
  50. Klinman, J. P. (1996) J. Biol. Chem. 271, 27189-27192[Free Full Text]
  51. Janes, S. M., Mu, D., Wenner, D., Smith, A. J., Kaur, S., Malby, D., Burlingame, A. L., and Klinman, J. P. (1990) Science 248, 981-987[Medline] [Order article via Infotrieve]
  52. Precious, E., Gunn, C. E., and Lyles, G. A. (1988) Biochem. Pharmacol. 37, 707-713[CrossRef][Medline] [Order article via Infotrieve]
  53. Lyles, G. A., and Chalmers, J. (1992) Biochem. Pharmacol. 43, 1409-1414[CrossRef][Medline] [Order article via Infotrieve]
  54. Conforti, L., Raimondi, L., and Lyles, G. A. (1993) Biochem. Pharmacol. 46, 603-607[Medline] [Order article via Infotrieve]
  55. Lyles, G. A., and Chalmers, J. (1995) Biochem. Pharmacol. 49, 416-419[CrossRef][Medline] [Order article via Infotrieve]
  56. Czech, M. P., Lawrence, J. C., Jr., and Lynn, W. S. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 4173-4177[Abstract]
  57. Czech, M. P., Lawrence, J. C., Jr., and Lynn, W. S. (1974) J. Biol. Chem. 249, 1001-1006[Abstract/Free Full Text]
  58. Lawrence, J. C., and Larner, J. (1978) J. Biol. Chem. 253, 2104-2113[Medline] [Order article via Infotrieve]
  59. May, J. M., and de Haën, C. (1979) J. Biol. Chem. 254, 9017-9021[Abstract]
  60. Livingston, J. N., Gurny, P. A., and Lockwood, D. H. (1977) J. Biol. Chem. 252, 560-562[Abstract]
  61. Little, S. A., and de Haën, C. (1980) J. Biol. Chem. 255, 10888-10895[Abstract/Free Full Text]
  62. Muchmore, D. B., Little, S. A., and de Haën, C. (1982) Biochemistry 21, 3886-3892[Medline] [Order article via Infotrieve]
  63. Dobson, D. E., Groves, D. L., and Spiegelman, B. M. (1987) J. Biol. Chem. 262, 1804-1809[Abstract/Free Full Text]
  64. Paulauskis, J. D., and Sul, H. S. (1988) J. Biol. Chem. 263, 7049-7054[Abstract/Free Full Text]
  65. Russell, T. R., and Ho, R. J. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 4516-4520[Abstract]
  66. Kawakami, M., Pekala, P. H., Vine, W., Lane, M. D., and Cerami, A. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 912-916[Abstract]
  67. Pekala, P. H., Kawakami, M., Vine, W., Lane, M. D., and Cerami, A. (1983) J. Exp. Med. 157, 1360-1365[Abstract]
  68. Pape, M. E., and Kim, K. H. (1988) Mol. Endocrinol. 2, 395-403[Abstract]
  69. Patton, J. S., Shepard, H. M., Wilking, H., Lewis, G., Aggarwal, B. B., Eessalu, T. E., Gavin, L. A., and Grunfeld, C. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8313-8317[Abstract]
  70. Torti, F. M., Dieckmann, B., Beutler, B., Cerami, A., and Ringold, G. M. (1985) Science 229, 867-869[Medline] [Order article via Infotrieve]
  71. Hotamisligil, G. S., Shargill, N. S., and Spiegelman, B. M. (1993) Science 259, 87-91[Medline] [Order article via Infotrieve]
  72. Uysal, K. T., Wiesbrock, S. M., Marino, M. W., and Hotamisligil, G. S. (1997) Nature 389, 610-614[CrossRef][Medline] [Order article via Infotrieve]
  73. Hamann, A., Benecke, H., Le Marchand-Brustel, Y., Susulic, V. S., Lowell, B. B., and Flier, J. S. (1995) Diabetes 44, 1266-1273[Abstract]
  74. Hotamisligil, G. S., Arner, P., Caro, J. F., Atkinson, R. L., and Spiegelman, B. M. (1995) J. Clin. Invest. 95, 2409-2415[Medline] [Order article via Infotrieve]
  75. Kern, P. A., Saghizadeh, M., Ong, J. M., Bosch, R. J., Deem, R., and Simsolo, R. B. (1995) J. Clin. Invest. 95, 2111-2119[Medline] [Order article via Infotrieve]
  76. Ryder, T. A., MacKenzie, M. L., Pryse-Davies, J., Glover, V., Lewinsohn, R., and Sandler, M. (1979) Histochemistry 62, 93-100[Medline] [Order article via Infotrieve]
  77. Hysmith, R. M., and Boor, P. J. (1985) J. Cardiovasc. Pharmacol. 9, 668-674
  78. Wibo, M., Duong, A. T., and Godfraind, T. (1980) Eur. J. Biochem. 112, 87-94[Abstract]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.