Gene expression profile of rat adipose tissue at the onset of high-fat-diet obesity

Jinping Li1, Xinxin Yu1, Wentong Pan1, and Roger H. Unger1,2

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 [alpha -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.

Differentially expressed genes that might encode secreted proteins were identified by prediction of signal peptide sequences using the Prediction Servers Center for Biological Sequence Analysis (www.cbs.dtu.dk/services1).

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 [alpha -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, beta -actin was used for normalization as described previously (14). Primer sequences used for the RT-PCR were as follows: tumor necrosis factor (TNF)-alpha 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) alpha 1-subunit gene 5'-GGTCCTGGTGGTTTCTGTTG and 5'-ATGATGTCAGATGGTGAATT (GenBank NM019142); AMPK alpha 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-alpha stimulation assay, 3T3-L1 adipocytes were serum-starved overnight and then treated with 1 ng/ml mouse TNF-alpha (Sigma) for 8 h. The total RNA was isolated, and Northern blot was carried out with a [32P]cDNA probe of the AK1 gene as described above.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Assessment of the chronology of hypertrophy and hyperplasia of adipocytes in rats fed a high-fat (60%) diet (HFD). A: change in the wet weight (mean ± SE) of rat epididymal fat pad at 1, 2, and 3 wk after a 4 or 60% fat diet (n = 3 experiments). B: comparison of fat cell diameter at the 1-, 2-, and 3-wk time points (mean ± SE; n = 40). C: fat pad histology after 1, 2, and 3 wk of the 60% fat diet. D: ratio of triglyceride (TG) to DNA, reflecting the fat per cell in the epididymal fat pad from rats on a 4 or 60% fat diet.

Based on these findings, we decided to harvest the fat tissue at the end of the 1st wk of the high-fat-diet feeding. We considered an increase of 50% or more in expression of a gene to constitute significant upregulation. By this criterion, 96 genes were differentially upregulated after 1 wk of high-fat feeding; this was confirmed by reverse Northern blot (Fig. 2A). Representative positive clones were reconfirmed by conventional Northern blotting (Fig. 2B) in adipose tissue derived from different DIO and control rats.


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Fig. 2.   A: representative reverse Northern blot. Equal amounts of the subcloned gene fragments from the subtractive library were spotted on two different pieces of nylon membranes and hybridized with the 32P-labeled, reversely transcribed cDNA probes for >18 h as described. Each image represents one cloned gene. The autoradiographs were obtained using the Phosphoimager. B: conventional Northern blotting of epididymal fat obtained from different rats on 4 or 60% fat diets 1 wk after feeding. An equal amount of RNA was loaded as judged by the band intensities of 18S and 28S RNA. Differences observed are expressed as the mean ± SE of the fold differences.

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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Table 1.   Effect of high-fat diet on mRNA in adipose tissue of genes encoding nonsecretory proteins

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-alpha 2 (I) collagen, which could provide new stroma for the expanding adipocyte population, were also increased.

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-alpha 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-alpha -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-alpha -induced adipocyte-related protein (20), we studied its TNF-alpha 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-alpha for 8 h (Fig. 3B).


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Fig. 3.   A: expression of AK1 in 3T3-L1 cells. V5-tagged Ak1 and empty vector were transfected in 3T3-L1 cells. Cells were lysed after drug selection. AK1 protein was detected by Western blot using a V-5 antibody; the result is representative of 2 independent experiments. B: 3T3-L1 cells overexpressing AK1 were differentiated and cultured with or without tumor necrosis factor (TNF)-alpha for 8 h. AK1 mRNA was detected by Northern blot.

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 the alpha 1- or alpha 2-isoforms of AMPK did not differ from controls. These results imply that the increase within adipocyte TG was not derived from increased fatty acid synthesis in adipocytes but rather from fatty acid synthesized in the liver and then transported to adipocytes in the form of very low-density lipoproteins. We speculate that, had the rats been fed a high-carbohydrate diet rather than a high-fat diet, genes controlling de novo fatty acid synthesis might have been upregulated in adipocytes.

Differential 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-alpha , 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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Table 2.   Effect of high-fat diet on expression in adipose tissues of genes encoding secreted proteins

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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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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DISCUSSION
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Am J Physiol Endocrinol Metab 282(6):E1334-E1341