1 Gifford Laboratories, Touchstone Center for Diabetes Research, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75390-8854; and 2 Veterans Affairs Medical Center, Dallas, Texas 75216
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ABSTRACT |
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Morbid obesity is the result of massive expansion of white adipose tissue (WAT) and requires recruitment of adipocyte precursor cells and their supporting infrastructure. To characterize the change in the expression profile of the preexisting WAT at the start of obesity, when adipocyte hypertrophy is present but hyperplasia is still minimal, we employed a cDNA subtraction screen for genes differentially expressed in epididymal fat pads harvested 1 wk after the start of a 60% fat diet. Ninety-six genes were upregulated by at least 50% above the WAT of control rats receiving a 4% fat diet. Of these genes, 30 had not previously been identified. Sixteen of the 96 genes, including leptin, adipocyte complement-related protein 30 kDa, and resistin, were predicted to encode a signal peptide. Ten of the 16 had been previously identified in other tissues and implicated in cell growth, proliferation, differentiation, cell cycle control, and angiogenesis. One was a novel gene. Twenty-nine novel fragments were identified. Thus, at the onset of high-fat-diet-induced obesity in rats, adipose tissue increases its expression of factors previously implicated in the expansion of nonadipocyte tissues and of several uncharacterized novel factors. The only one of these thus far characterized functionally was found to promote lipogenesis.
obesity; adipocyte hormones; adipocyte hyperplasia; adipocyte hypertrophy
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INTRODUCTION |
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DIET-INDUCED OBESITY (DIO), the single most common health abnormality in the United States, is a progressive disorder that begins with hypertrophy of preexisting adipocytes and then enters a phase of cellular expansion during which new fat cells are recruited from the preadipocyte population (6). The capacity to expand the adipocyte population is truly remarkable. For example, some humans are capable of increasing their adipose tissue weight by more than two times their lean body weight; this must surely rank as the most massive expansion of any tissue in all of nature. The adipocyte expansion process, we reasoned, must require a radical modification in gene expression within the preexisting adipocytes so as to recruit adipocyte precursor cells to initiate the differentiation process and to generate the infrastructure required to sustain the new tissue.
The gene expression profile of adipose tissue has been studied previously in various rodent models of established obesity (34, 23). However, in established obesity, the adipose gene expression profile reflects both the preexisting hypertrophic fat cells and the newly recruited adipocytes in various stages of maturity. There are to our knowledge no studies at the very onset of DIO when white adipose tissue (WAT) consists largely of preexisting adipocytes in a hypertrophic state with only a minimal admixture of newly recruited fat cells. To obtain such tissue, we examined fat tissue harvested from overnourished normal lean rats 1 wk after starting a high-fat diet, a time that hypertrophy of adipose tissue was near-maximal and hyperplasia was minimal. Using a cDNA subtraction screen, we identified several classes of genes that are upregulated during this initial phase of rat DIO.
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MATERIALS AND METHODS |
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Animals.
Male Sprague-Dawley rats (Charles River, MA) were employed. From 6 wk
of age, rats were given either their usual diet of 50 g/day standard
chow (Tekland FG rodent diet) containing 24.5% protein, 51%
carbohydrate, and 4% fat or 50 g/day of a high-fat diet containing
24.5% protein, 7.5% carbohydrate, and 60% fat. Both groups weighed
116 g at the start of the diets. The rats were killed at 7, 14, and 21 days later, at which time the control group weighed 120, 125, and 132 g and the high-fat-fed group weighed 126, 138, and
157 g, respectively. Epididymal fat pads were removed, weighed,
and immediately stored at 72°C in liquid nitrogen.
cDNA subtraction.
Total RNA was isolated from a pool of epididymal fat pads from each
group using TRIzol according to the manufacturer's protocol (Life
Technologies, Gaithersburg, MD). Poly(A) mRNA was further purified
using the mRNA isolation kit from Ambion (Austin, TX). PCR-based cDNA
subtraction was carried out with the cDNA Subtraction Kit (Clontech,
Palo Alto, CA), according to the manufacturer's protocol.
Normalization of cDNA pools between two groups was achieved in parallel
experiments by adding [-32P]dCTP and then counting the
radioactivity and monitoring band patterns. Subtracted cDNAs were
subcloned into TA cloning vector pCR2.1 (Invitrogen) and sequenced
using an automatic DNA sequencer. The DNA sequences obtained were
analyzed using National Institutes of Health GenBank BLAST. We obtained
220 clones from the subtracted library, and 104 of these clones were
successfully sequenced. Eight clones were found to be repeated clones.
Reverse Northern.
Cloned cDNAs in Escherichia coli bacteria were isolated by a
Maniprep kit (Qiagen). DNA (0.1 µg) from each purified clone was
spotted in duplicate on a HyBond N+ nylon membrane
(Amersham) and cross-linked by ultraviolet light. [32P]cDNA probes were synthesized using a template of 2 µg of poly(A) mRNA from adipose tissue of rats fed the 60% fat diet
or 4% fat control diet. A mixture of 1.5 µl random hexamer primer, 1 µl dNTP (10 mM each), 1 µl Moloney murine leukemia virus reverse transcriptase (200 U/µl), and 4 µl [-32P]dCTP (10 mCi/ml) was incubated at 42°C for 1 h. Labeled probe (50,000 counts/min) from each reaction was applied separately to the prepared
nylon membrane and hybridized overnight at 65°C. Washed filters were
exposed in a Phosphoimager, and each clone was individually quantified
by software from Molecular Dynamics. Calculations were based on at
least two independent experiments.
Northern blot. Northern blotting was carried out by a conventional method (27). cDNA fragments for Northern blotting were generated by conventional PCR cloning and were confirmed by DNA sequencing. The probes were excised from the TA cloning vector and gel purified. The intensity of the blot signals was calculated using a Phosphoimager GS-363 (Bio-Rad).
RT-PCR.
RT-PCR was used to semiquantify the expression of genes previously
reported by others to be increased in obesity but that did not appear
in our differential screen. We employed the Advantage RT-for-PCR kit as
instructed by the manufacturer (Clontech). Briefly, 2 µg of
reverse-transcribed cDNA was used for each PCR reaction, and, in most
cases, -actin was used for normalization as described previously
(14). Primer sequences used for the RT-PCR were as follows: tumor necrosis factor (TNF)-
gene
5'-TAAGTACTTGGGCAGGTTGA and 5'-AGATCATCTTCTCAAAACTC (GenBank
X66539); insulin-like growth factor (IGF)-I gene
5'-GCCACAGCCGGACCAGAGAC and 5'-AAAGACAAT GTCGGAATGTT (GenBank
M17714); macrophage colony-stimulating factor (MCSF) gene
5'-CTCTGTCGGGGCCTTCAACC and 5'-GCTGGAGGATCCCTCGGACT (GenBank
M64592); AMP-activated kinase (AMPK)
1-subunit
gene 5'-GGTCCTGGTGGTTTCTGTTG and 5'-ATGATGTCAGATGGTGAATT
(GenBank NM019142); AMPK
2-subunit gene
5'-CACTGTATAAACTGTTCATC and 5'-ATGATGTCAGAT GGTGAATT
(GenBank U12149); sterol regulatory element-binding protein (SREBP)-1c
gene 5'-GGAGCCATGGATTGCACATT and 5'-AGGAAGGCTTC CAGAGAGGA (GenBank
L16995).
Histology. Cryosections of epididymal fat pad were stained with hematoxylin and eosin. Photomicrographs were captured with Sony CCD-IRIS at ×40 amplification. Cell size was measured under a Nikon Optiphot Microscope. The diameter of 40 cells in two different microscopic fields was determined in fat pads from two rats from each group. Statistical analysis was done by STATVIEW Software.
Triglyceride and DNA content of fat tissue. Epidydimal fat pads were resected, weighted, and immediately frozen in liquid nitrogen. About 100 mg of tissue were homogenized with a Polytron in 4 ml of buffer containing 18 mM Tris · HCl (pH 7.5), 300 mM D-mannitol, and 5 mM EGTA. DNA was measured by the method of Hopcroft et al. (11). Triglyceride (TG) was measured by sigma diagnostic kit.
Cloning and transfection. The encoding region of AK1 was obtained by means of RT-PCR. Briefly, the cDNA was reversely transcribed from isolated rat adipose total RNA. With the use of the synthesized rat adipose cDNA as template and the primers described above, the PCR reaction was carried out, and its product was subcloned into a pEF6/V5-His TOPO mammalian expression vector (Invitrogen). By using a cDNA fragment from the PCR-subtracted clones, we screened the Uni-ZAP XR cDNA library of rat adipocytes (Stratagene) and isolated a 1.4-kb gene called "lipogenin." Sequence analysis showed no homology to any known gene in the GenBank. It is predicted to encode a peptide with 130 amino acids. The open reading frame was cloned using PCR and ligated into pEF6/V5-his TOPO vector. The constructs were confirmed by DNA sequencing.
To establish a stable cell line expressing a gene of interest, 3T3-L1 cells were transfected with a V5-tagged cDNA or empty vector using the Lipofectin kit (Qiagen) and subjected to 5 µg/ml blasticidin selection. Clonal cells were collected after 1 wk of drug selection, and protein expression was confirmed by Western blot using anti-V5 antibody as previously described (18). For the TNF- ![]() |
RESULTS |
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Chronology of the hypertrophy and hyperplasia of white adipose
tissue during high-fat feeding.
After 2 wk of high-fat feeding, the wet weight of the fat pad had
increased threefold (Fig. 1A),
but cell size had reached a plateau (Fig. 1B). This implied
that any subsequent increase in wet weight was largely the result of
hyperplasia. The histological appearance and size of epididymal
adipocytes was consistent with this interpretation (Fig.
1C). As an additional index of TG content per cell, we
measured the TG-to-DNA ratio, which appeared to be leveling off after
the 1st wk (Fig. 1D), also suggesting that hyperplasia was
becoming increasingly important.
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Differential expression of genes encoding products involved in
mitochrondrial respiration or nutrient metabolism.
We then grouped the previously known upregulated genes into functional
categories. The largest group of upregulated genes was involved in
mitochondrial functions (Table 1). These
included stearyl-CoA desaturase, which was increased sevenfold,
cytochrome c oxidase subunits, cytochrome b,
ATPase synthases, and ATPases. Cytosolic malate dehydrogenase, carboxyl
esterase and glycerol 3-phosphate dehydrogenase, and UDP-glucose
pyrophosphorylase and aldose reductase were also upregulated. Two
proteins potentially involved in nucleotide biosynthesis,
phosphoribosyl pyrophosphate synthase-associated protein and
adenine nucleotide translocator gene, were also increased.
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Differential expression of matricellular/cell skeletal proteins. Expression of a class of genes encoding proteins associated with cytoskeletal structure of cells was increased. These included radixin (35), actin-like protein 3, genes similar to mouse evectin 2 (15), and nischarin protein (Ref. 1 and Table 1).
Some of the upregulated matricellular/cell skeletal genes were predicted to encode secreted proteins (Table 1). These included the secreted proteases, cathepsins K, L, and S (21, 5), a gene similar to human protease inhibitor 12, and the secreted metalloproteinase, A disintegrin and metalloproteinase domain with thrombospondin type-1 modules (Adamts)5, expressed during embryogenesis (13). Adamts5 was increased eightfold, more than any other gene. It may be noteworthy that the knockout of Adamts1, which has functional domains identical to Adamts5, causes adipose tissue malformation and abnormal leanness (30). Type XV and pre-Differential expression of genes related to protein metabolism and electrolyte exchange. Several genes related to protein metabolism were augmented in the early stage of DIO, including the two ribosomal proteins elongation factor 1 and proteasome activator PA28 and a gene similar to human regulator of nonsense transcripts 2. The protein degradation-associated protein, mouse double minute 2, was increased 2.4-fold.
The mRNA of one protein involved in ion exchange, Na-K-Cl cotransporter, was increased (Table 1).Differential expression of genes encoding signal transducing
proteins and transcription factors.
Genes encoding signal transduction components, RhoGAP5, STAT2, and
B-cell receptor-associated protein, were increased in the DIO rats
(Table 1). One gene similar to mouse muscle-specific transcriptional
activator- and another similar to human nuclear factor NF45 were
also upregulated (Table 1), as was insulin-induced gene 1, which had
been reported previously to be the most abundant insulin-inducible gene
in regenerating rat liver and which is believed to regulate cell/tissue
growth (Table 1 and Ref. 25).
Characterization of a novel member of a TNF--induced gene
family.
Among the upregulated gene fragments was one that bore a 96% homology
to an uncharacterized mouse cDNA called AK1 (Table 1). Because it had
44% homology with a TNF-
-induced adipocyte-related protein
(20), we studied its TNF-
responsiveness by expressing it in 3T3-L1 cells. As shown in Fig.
3A, AK1 protein was easily detected by Western blot of cell lysates, and its mRNA was dramatically elevated when the cells were cultured with TNF-
for 8 h (Fig. 3B).
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Expression of genes of lipogenesis. Despite the sevenfold increase in the expression of stearyl-CoA desaturase in adipocytes of the high-fat-fed rats, no increase in expression of genes encoding lipogenic transcription factors or lipogenic enzymes was detected in the subtractive screen.
To further explore this issue, we semiquantified by RT-PCR the mRNA of SREBP-1c, an insulin-responsive transcription factor that increases the expression of lipogenic enzymes in liver (29). It was lower in the adipocytes of the rats on a high-fat diet. We also semiquantified the expression of subunits of AMPK, which might be reduced during increased lipogenesis, since it can block fatty acid synthesis by phosphorylating acetyl-CoA carboxylase (39). However, the mRNA of theDifferential expression of mRNA encoding known adipocyte hormones.
Sixteen differentially expressed mRNAs were predicted to encode
proteins with signal peptides. Three of them were previously known
adipocyte-specific hormones {leptin (40), resistin, and Adipocyte complement-related protein 30 kDa (Acrp30) [Ref.
9; AdipoQ (12) and adiponectin
(2)]; Table 2}.
Surprisingly, neither IGF-I nor TNF-, previously shown to be
upregulated in adipose tissue of high-fat-diet-fed rats (19,
22), was among these. However, semiquantitative RT-PCR revealed
a slight induction of these two factors after 1 wk of high-fat feeding.
MCSF mRNA, reported to be elevated in the adipose tissue of humans
during rapid weight gain (17), was undetectable by RT-PCR
in rat adipose tissue at this phase of obesity. This may reflect a
species difference and/or a difference in the time of sample
collection. None of 11 other known secretory products of adipose tissue
reviewed by Frühbeck et al. (8) turned up in our
screen at this early stage of obesity.
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Differential expression of genes encoding proteins secreted by nonadipocytes. Ten genes upregulated in the differential screen had previously been identified in nonadipose tissues and were also upregulated in adipose tissue at the onset of DIO.
We noted a 3.2-fold increase in the expression of the calvasculin gene (S-100, metastasin PEL-98, 18A2, 42A, or p9Ka), which encodes a protein secreted by bovine aortic smooth muscle (38), had not been shown previously to be increased in early obesity. It may be involved in cell growth and differentiation (37, 38) and has been implicated in the metastases of certain neoplasms such as breast cancer and malignant gliomas (26, 10). Genes of three other secreted proteins were increased more than fourfold. One of these, osteonectin, also known as secreted protein acidic and rich in cysteine (SPARC), had previously been identified by Tartare-Deckert et al. (34) in ob/ob and AKR mice and in mice with obesity induced by gold thioglucose. Finally, a threefold increase in a previously undiscovered gene was noted. It caused an increase in oil red O staining when transfected into 3T3-L1 cells and was therefore called lipogenin. ![]() |
DISCUSSION |
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The goal of this study was to identify genes of WAT that are upregulated at the very start of obesity. We used Sprague-Dawley rats and harvested their adipocytes after the 1st wk of high-fat feeding rather than after obesity was fully established. This timing was based on the belief that the gene expression profile of adipose tissue would largely reflect hypertrophy of the preexisting adipocytes, undistorted by mRNA from newly recruited maturing adipocytes. In fact, enlarged adipocytes seemed to be close to a peak in size at that time (Fig. 1).
A total of 96 genes was increased by 1.5-fold or more in WAT harvested at that time (Tables 1 and 2). Twenty-five of these genes are involved in mitochondrial respiration, carbohydrate/protein metabolism, thermogenesis, or ion transport in the adipocytes and/or the stromal vascular cells.
Based on prediction of a signal peptide sequence, 16 of the 96 upregulated mRNAs were classified as encoding secretory peptides. Three of these were previously identified hormones of WAT {leptin (7), resistin (33), and Acrp30 [Ref. 9; adiponectin (2) and adipoQ (12)]}. Inasmuch as Acrp30 has previously been reported to be downregulated in obesity of much longer duration (2, 9, 12), it is possible that its expression changes in the course of obesity. Because the proteolytic cleavage product of Acrp30 increases fatty acid oxidation in muscle and other potential target tissues (9), actions that should protect nonadipose tissues from lipotoxicity, the late decline in its expression could be a factor in the development of the lipotoxic complications that appear late in obesity (33).
Two adipocyte products previously implicated in preadipocyte maturation [IGF-I (19, 16, 24, 28, 32) and MCSF (17)] were not detected in our subtraction screen. IGF-I was increased 1.5-fold when semiquantified by RT-PCR, but MCSF could not be detected at this stage of the disorder.
Osteonectin or SPARC (4), a secreted protein previously shown by Tartare-Deckert et al. (34) to be increased in DIO (34), was also upregulated in our screen. The secreted proteases, cathepsins K, L, and S, were expressed at high levels, and, like osteonectin, they have been implicated in cell growth, proliferation, differentiation, cell cycle control, and angiogenesis. Leptin has also been reported to have angiogenic activity (3, 31) and could be of importance in the development of the stromal vascular elements required to support expansion of the adipocyte population in DIO. However, in ob/ob mice, the absence of leptin does not appear to impede their obesity.
An additional 40 genes were upregulated during the early phase of obesity, but a complete sequence has thus far been obtained in only 11. One of these, predicted to be a secreted protein, is similar to a mouse embryonic stem cell cDNA without a known function. Another of these is predicted to encode a novel secretory protein with a possible signal peptide; it was called lipogenin based on the fact that it increases oil red O staining when transfected in 3T3-L1 cells.
In summary, our findings are most consistent with release from hypertrophic adipocytes of an array of factors that are also secreted by nonadipose tissues. These gene products may be relatively ubiquitous promoters of cell growth and provide stromal vascular support during expansion of a tissue. Other gene products, such as lipogenin, not known to be expressed in other tissues, are more likely to be involved in lipogenesis and the differentiation of fat cells. This catalogue of genes upregulated at the start of obesity should be helpful in the further exploration of the biology of DIO.
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ACKNOWLEDGEMENTS |
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We thank Susan Kennedy for secretarial services and Shirley Waggoner and Kay McCorkle for technical support. We thank Drs. Cai Li and Christopher Newgard for critical review of this manuscript.
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FOOTNOTES |
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This work was supported by Department of Veterans Affairs Institutional Support Grant SMI 821-109, National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-02700-37, and the National Institutes of Health Grant 5POI-DK-58398.
Address for reprint requests and other correspondence: R. H. Unger, Center for Diabetes Research, Univ. of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-8854 (E-mail: Roger.Unger{at}UTSouthwestern.edu).
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.
First published February 11, 2002;10.1152/ajpendo.00516.2001
Received 16 November 2001; accepted in final form 4 February 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Alahari, SK,
Lee JW,
and
Juliano RL.
Nischarin, a novel protein that interacts with the integrin alpha5 subunit and inhibits cell migration.
J Cell Biol
151:
1141-1154,
2000
2.
Arita, Y,
Kihara S,
Ouchi N,
Takahashi M,
Maeda K,
Miyagawa J,
Hotta K,
Shimomura I,
Nakamura T,
Miyaoka K,
Kuriyama H,
Nishida M,
Yamashita S,
Okubo K,
Matsubara K,
Muraguchi M,
Ohmoto Y,
Funahashi T,
and
Matsuzawa Y.
Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity.
Biochem Biophys Res Commun
257:
76-83,
1999.
3.
Bouloumie, A,
Drexler HC,
Lafontan M,
and
Busse R.
Leptin, the product of OB gene, promotes angiogenesis.
Circ Res
83:
1059-1066,
1998
4.
Brekken, RA,
and
Sage EH.
SPARC, a matricellular protein: at the crossroads of cell-matrix.
Matrix Biol
19:
569-580,
2000[ISI][Medline].
5.
Denhardt, DT,
Hamilton RT,
Parfett CL,
Edwards DR,
St. Pierre R,
Waterhouse P,
and
Nilsen-Hamilton M.
Close relationship of the major excreted protein of transformed murine fibroblasts to thiol-dependent cathepsins.
Cancer Res
46:
4590-4593,
1986[Abstract].
6.
Faust, IM,
Johnson PR,
Stern JS,
and
Hirsch J.
Diet-induced adipocyte number increase in adult rats: a new model of obesity.
Am J Physiol Endocrinol Metab Gastrointest Physiol
235:
E279-E286,
1978
7.
Frederich, RC,
Hamann A,
Anderson B,
Lollman B,
Lowell BB,
and
Flier JS.
Leptin levels reflect body lipid content in mice: evidence for diet-induced resistance to leptin action.
Nat Med
1:
1311-1314,
1995[ISI][Medline].
8.
Frühbeck, G,
Gomez-Ambrosi J,
Muruzabal FJ,
and
Burrell MA.
The adipocyte: a model for integration of endocrine and metabolic signaling in energy metabolism regulation.
Am J Physiol Endocrinol Metab
280:
E827-E847,
2001
9.
Fruebis, J,
Tsao TS,
Javorschi S,
Ebbeta-Reed D,
Erickson MRS,
Yen FT,
Bihain BE,
and
Lodish HF.
Proteolytic cleavage product of 30-kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causes weight loss in mice.
Proc Natl Acad Sci USA
98:
2005-2010,
2001
10.
Gunnersen, JM,
Spirkoska V,
Smith PE,
Danks RA,
and
Tan SS.
Growth and migration markers of rat C6 glioma cells identified by serial analysis of gene expression.
Glia
32:
146-154,
2000[ISI][Medline].
11.
Hopcroft, DW,
Mason DR,
and
Scott RS.
Standardization of insulin secretion for pancreatic islets: validation of a DNA assay.
Horm Metab Res
17:
559-561,
1985[ISI][Medline].
12.
Hu, E,
Liang P,
and
Spiegelman BM.
AdipoQ is a novel adipose-specific gene dysregulated in obesity.
J Biol Chem
271:
10697-10703,
1996
13.
Hurskainen, TL,
Hirohata S,
Seldin MF,
and
Apte SS.
ADAM-TS5, ADAM-TS6 and ADAM-TS7, novel members of a new family of zinc metalloproteases.
J Biol Chem
274:
25555-25563,
1999
14.
Kakuma, T,
Wang ZW,
Pan W,
Unger RH,
and
Zhou YT.
Role of leptin in peroxisome proliferators-activated receptor gamma coactivator-1 expression.
Endocrinology
141:
4576-4582,
2000
15.
Krappa, R,
Nguyen A,
Burrois P,
Deretic D,
and
Lemke G.
Evectins: vesicular proteins that carry a pleckstrin homology domain and localize to post-Golgi membranes.
Proc Natl Acad Sci USA
96:
4633-4638,
1999
16.
Kras, KM,
Hausman DB,
Hausman GJ,
and
Martin RJ.
Adipocyte development is dependent upon stem cell recruitment and proliferation of preadipocytes.
Obesity Res
7:
491-497,
1999[Abstract].
17.
Levine, JA,
Jensen MD,
Eberhardt NL,
and
O'Brien T.
Adipocyte macrophage colony-stimulating factor is a mediator of adipose tissue growth.
J Clin Invest
101:
1557-1564,
1998
18.
Li, J,
DeFea K,
and
Roth RA.
Modulation of insulin receptor substrate-1 tyrosine phosphorylation by an Akt/phosphatidylinositol 3-kinase pathway.
J Biol Chem
274:
9351-9356,
1999
19.
Marques, BG,
Hausman DB,
Latimer AM,
Kras KM,
Grossman BM,
and
Martin RJ.
Insulin-like growth factor I mediates high-fat diet-induced adipogenesis in Osborne-Mendel rats.
Am J Physiol Regulatory Integrative Comp Physiol
278:
R654-R662,
2000
20.
Moldes, M,
Lasnier F,
Gauthereau X,
Klein C,
Parault J,
Feve B,
and
Chambaut-Guerin A-M.
Tumor necrosis factor-alpha-induced adipose-related protein (TIARP), a cell-surface protein that is highly induced by tumor necrosis factor-alpha and adipose conversion.
J Biol Chem
276:
33938-33946,
2001
21.
Moran, MT,
Schofield JP,
Hayman AR,
Shi GP,
Young E,
and
Cox TM.
Pathologic gene expression in Gaucher disease: up-regulation of cysteine proteinases including osteoclastic cathepsin K.
Blood
98:
1969-1978,
2000.
22.
Morin, CL,
Eckel RH,
Marcel T,
and
Pagliassotti MJ.
High fat diets elevate adipose tissue-derived tumor necrosis factor-alpha activity.
Endocrinology
138:
4665-4671,
1997
23.
Nadler, ST,
Stoehr JP,
Schueler KL,
Tanimoto G,
Yandell BS,
and
Atti AD.
The expression of adipogenic genes is decreased in obesity and diabetes mellitus.
Proc Natl Acad Sci USA
97:
11371-11376,
2000
24.
Nougues, J,
Rayne Y,
Barenton B,
Chery T,
Garandel V,
and
Soriano J.
Differentiation of adipocyte precursors in a serum-free medium is influenced by glucocorticoids and endogenously produced insulin-like growth factor-I.
Int J Obes Relat Metab Disord 1
7:
159-167,
1993.
25.
Peng, Y,
Schwarz EJ,
Lazar MA,
Genin A,
Spinner NB,
and
Taub R.
Cloning, human chromosomal assignment and adipose and hepatic expression of the CL-6/INSIG1 gene.
Genomics
43:
278-284,
1997[ISI][Medline].
26.
Rudland, PS,
Platt-Higgins A,
Renshaw C,
West CR,
Winstanley JH,
Robertson L,
and
Barraclough R.
Prognostic significance of the metastasis-inducing rotein S100A4 (p9Ka) in human breast cancer.
Cancer Res
60:
1595-1603,
2000
27.
Sambrook, J,
Fritsch EF,
and
Manniatis T.
Molecular Cloning. A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989.
28.
Schmidt, W,
Poll-Jordan G,
and
Loffler G.
Adipose conversion of 3T3-L1 cells in a serum-free culture system depends on epidermal growth factor, insulin-like growth factor I, corticosterone and cyclic AMP.
J Biol Chem
265:
15489-15495,
1990
29.
Shimomura, I,
Bashmakov Y,
Ikemoto S,
Horton JD,
Brown MS,
and
Goldstein JL.
Insulin selectively increases SREBP-1c mRNA in the livers of rats with streptozotocin-induced diabetes.
Proc Natl Acad Sci USA
96:
13656-13661,
1999
30.
Shindo, T,
Kurihara H,
Kuno K,
Yokoyama H,
Wada T,
Kurihara Y,
Imai T,
Wang Y,
Ogata M,
Nishimatsu H,
Moriyama N,
Oh-hashi Y,
Morita H,
Ishikawa T,
Nagai R,
Yazaki Y,
and
Matsushima K.
ADAMTS-1: a metalloproteinase-disintegrin essential for normal growth, fertility and organ morphology and function.
J Clin Invest
105:
1345-1352,
2000
31.
Sierra-Honigmann, MR,
Nath AK,
Murakami C,
Garcia-Cardena G,
Papapetropoulos A,
Sessa WC,
Madge LA,
Schechner JS,
Schwabb MB,
Polverini PJ,
and
Flores-Riveros JR.
Biological action of leptin as an angiogenic factor.
Science
281:
1683-1686,
1998
32.
Smith, PJ,
Wise LS,
Berkowitz R,
Wan C,
and
Rubin CS.
Insulin-like growth factor-I is an essential regulator of the differentiation of 3T3-L1 adipocytes.
J Biol Chem
263:
9402-9408,
1988
33.
Steppan, CM,
Bailey ST,
Bhat S,
Brown EJ,
Banerjee RR,
Wright CM,
Patel HR,
Ahima RS,
and
Lazar MA.
The hormone resistin links obesity to diabetes.
Nature
409:
307-312,
2001[ISI][Medline].
34.
Tartare-Deckert, S,
Chavey C,
Monthousel MN,
Gautier N,
and
Van Obberghen E.
The matricellular protein SPARC/osteonectin, as a newly identified factor upregulated in obesity.
J Biol Chem
276:
22231-22237,
2001
35.
Tsukita, S,
Hieda Y,
and
Tsukiita S.
A new 82-kD barbed end capping protein (radixin) localized in the cell-to-cell adherens junction: purification and characterization.
J Cell Biol
108:
2369-2382,
1989[Abstract].
36.
Unger, RH,
and
Orci L.
Disease of liporegulation: new perspective on obesity and related disorders.
FASEB J
15:
312-321,
2001
37.
Van Eldik, LJ,
Christie-Pope B,
Bolin LM,
Shooter EM,
and
Whetsell WO, Jr.
Neurotrophic activity of S-100 beta in cultures of dorsal root ganglia from embryonic chick and fetal rat.
Brain Res
542:
280-285,
1991[ISI][Medline].
38.
Watanabe, Y,
Usuda N,
Tsugane S,
Kobayashi R,
and
Hidaka H.
Calvasculin, an encoded protein from mRNA termed pEL-98, 18A2, 42A or p9Ka, is secreted by smooth muscle cells in culture and exhibits Ca2+-dependent binding to 36-kDa microfibril-associated glycoprotein.
J Biol Chem
267:
17136-17140,
1992
39.
Winder, WW,
and
Hardie DG.
AMP-activated protein kinase, a metabolic master switch: possible roles in Type 2 diabetes.
Am J Physiol Endocrinol Metab
277:
E1-E10,
1999
40.
Zhang, Y,
Proenca R,
Maffei M,
Barone M,
Leopold L,
and
Friedman JM.
Positional cloning of the mouse obese gene and its human homologue.
Nature
372:
425-432,
1994[ISI][Medline].