Adipose Gene Expression Patterns of Weight ... - Wiley Online Library

Adipose Gene Expression Patterns of Weight Gain Suggest Counteracting Steroid Hormone Synthesis Evert M. Van Schothorst,* Nicole Franssen-van Hal,† Mirjam M. Schaap,* Jeroen Pennings,* Barbara Hoebee,* and Jaap Keijer†

Abstract VAN SCHOTHORST, EVERT M., NICOLE FRANSSENVAN HAL, MIRJAM M. SCHAAP, JEROEN PENNINGS, BARBARA HOEBEE, AND JAAP KEIJER. Adipose gene expression patterns of weight gain suggest counteracting steroid hormone synthesis. Obes Res. 2005;13:1031–1041. Objective: To identify early molecular changes in weight gain, using analysis of gene expression changes in adipose tissue of mice fed well-defined humanized (Western) highfat and low-fat (control) diets during a short (3- to 5-week) time interval. Research Methods and Procedures: An adipose-enriched cDNA microarray was constructed and used for the expression analyses of visceral adipose tissues of wildtype young adult C57BL/6J male mice on different diets. Results: Mice on a high-fat diet had significantly higher body weight (at most, 9.6% greater) and adipose tissue weights compared with mice on a control diet. Gene expression analyses revealed 31 transcripts significantly differentially expressed in visceral adipose tissue between the diet groups. Most of these genes were expressed more on the high-fat diet. They mainly encode proteins involved in cellular structure (e.g., myosin, procollagen, vimentin) and lipid metabolism (e.g., leptin, lipoprotein lipase, carbonic anhydrase 3). This increase in gene expression was accompanied by a decrease in oxidative phosphorylation and car-

Received for review October 29, 2004. Accepted in final form April 8, 2005. The costs of publication of this article were defrayed, in part, by the payment of page charges. This article must, therefore, be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. *Laboratory of Toxicology, Pathology, and Genetics, National Institute of Public Health and the Environment (RIVM), Bilthoven, The Netherlands and †Food Bioactives Group, RIKILT Institute of Food Safety, Wageningen, The Netherlands. Address correspondence to Evert M. van Schothorst, Laboratory of Toxicology, Pathology, and Genetics, National Institute of Public Health and the Environment (RIVM), PO Box 1, 3720 BA Bilthoven, The Netherlands. E-mail: [email protected] Copyright © 2005 NAASO

bohydrate metabolism (ATP citrate lyase). Importantly, genes belonging to steroid hormone biosynthesis (3␤-hydroxysteroid dehydrogenase-1, cholesterol side-chain cleavage cytochrome P450, and steroid-11␤-hydroxylase) were all expressed less in mice on a high-fat diet. Discussion: A short time period of 3 to 5 weeks of high-fat feeding altered gene expression patterns in visceral adipose tissue in male mice. Gene expression changes indicate initiation of adipose tissue enlargement and the down-regulation of adipose steroid hormone biosynthesis. The latter suggests a mechanism by which initial progression toward weight gain is counteracted. Key words: overweight, diet, mouse, genomics, corticosterone

Introduction Obesity is a recent epidemic that has important implications because it increases the risk of morbidity by type II diabetes, hypertension, and cardiovascular disease (1). Although a few monogenic causes for obesity are known, representing no more than 5% of all obese subjects (2), susceptibility to obesity and weight gain is largely determined by complex interactions among genes, lifestyle, and diet. Weight gain resulting in obesity is characterized by a long-term positive energy balance that leads to increased lipid storage and enlargement of adipose tissue. To study weight gain, representing the initial stages of obesity, we used the C57BL/6J diet-induced obesity mouse model. This model has been used to study obesity, leptin resistance, insulin resistance, and body fat accumulation using different diets (3). However, a number of these diets contain an extremely high fat content (4,5), which does not mimic the Western human diet, one of the important environmental factors in human obesity. We used a well-defined humanOBESITY RESEARCH Vol. 13 No. 6 June 2005

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Diet-Induced Adipose Gene Expression, Van Schothorst et al.

ized (Western) high-fat diet (HFD)1 and control low-fat diet (CD), with the main difference being the quantity of dietary fat and not the composition. Several in vivo studies on obesity have addressed changes after a relative long time period, e.g., 5 to 6 months (6,7), when the obese physiological phenotype is established. We were interested in more subtle and early changes and focused on mice with mild weight gain obtained after 3 to 5 weeks of HFD. This is particularly relevant because it was recently shown that normal to overweight humans refraining from weight gain showed markedly improved cardiovascular parameters (8). Other rodent adipose gene expression studies have focused either on long-term HFD or extreme HFDs, or have used subcutaneous adipose tissue (4,5,9). The first aim of this study was to characterize phenotypic differences of mice in the initial phase of diet-induced weight gain. Second, we determined differential gene expression in visceral adipose tissues from these mice, because this depot is considered to be the most potent adipose tissue contributing to obesity risk factors (1). For this purpose, an adipose-enriched cDNA microarray was constructed and used to study the expression of thousands of adipose genes simultaneously. This resulted in genes specifically and significantly up- and down-regulated by an HFD in murine visceral adipose tissue. Importantly, three genes within the corticosterone biosynthesis pathway were all down-regulated, suggesting an opposite effect on the initial progression toward weight gain.

Research Methods and Procedures Animals Wildtype C57BL/6J male mice were purchased from Harlan (Zeist, The Netherlands) at 9 weeks of age. Housing facilities were maintained at 22 °C to 23 °C and 50% to 60% relative humidity, with a 12-hour light/dark cycle according to guidelines for use and care as given by the Dutch Animal Experimentation Act (1996). All mice were housed singly. Access to water and food (Hope Farms, Woerden, The Netherlands) was ad libitum, and body weights and food consumption were measured three times a week. During the experiment, mice were fed with either a humanized HFD (Table 1) or a humanized CD (Table 1). The HFD is based on the average human intake in The Netherlands (Dutch Food Consumption Survey, 1998). Lard and corn oil were used as sources for fatty acids. The CD had the same composition as the HFD except that a percentage of the fat in the HFD was exchanged for dextrose in the CD. Both diets were equally supplemented with vitamins and minerals.

1 Nonstandard abbreviations: HFD, high-fat diet; CD, control low-fat diet; FR, fold ratio; BW: body weight; WAT, white adipose tissue; EST, expressed sequence tag.

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Table 1. Diet composition Total Composition

CD

HFD

Fat Lard Corn oil Dextrose Caseine Starch Crude protein (g/kg) Sugar ⫹ starch (g/kg) Crude fat (g/kg) Total C8–C22 (g/kg) Gross energy (kcal/kg)

5.0% 3.5% 1.5% 54.3% 20.0% 10.0% 176.0 579.3 51.0 51.0 3740.2

21.0% 14.7% 6.3% 38.3% 20.0% 10.0% 176.0 435.3 207.0 214.5 4600.4

All percentages are given as w/w%.

After a 1-week quarantine and acclimation period in which all mice were fed the CD, three mice were killed (t ⫽ 0, Group C0). The remaining mice were randomly divided into two groups. One group received an HFD (n ⫽ 6) and the other the CD (n ⫽ 6). One-half of each group was killed at Day 21 (Groups C1 and HF1) and the other one-half at either Day 30 (Group C2) or Day 40 (Group HF2). All animals were (non-fasted) dissected between 9:00 and 10:00 AM. Blood was collected, and glucose measurements were done using a Dex glucose meter (Bayer, Germany). Plasma samples were isolated or removed, adipose tissues were weighted, and several organs were snap frozen in liquid nitrogen and stored at – 80 °C. A portion of each visceral adipose tissue was formalin-fixed and paraffinembedded, sectioned (10 ␮m), and stained with hematoxylin and eosin for histological analyses. Plasma Measurements Insulin, leptin, and corticosterone measurements were performed using ELISA (DSL, Veghel, The Netherlands). Total triglycerides and total free fatty acids were measured using a Hitachi 912 automatic analyzer (Roche, Almere, The Netherlands). Statistical Analyses The relationships between time and diet (CD or HFD) as independent variables and body weights, organ weights, plasma values, and fat pad weights as dependent variables were analyzed using mixed model ANOVA, using PROC MIXED in SAS (version 8.2; SAS Institute, Cary, NC). Days of follow-up were log-transformed to obtain approximate linear relationships.


RNA Isolation Total RNA from adipose tissues was isolated using Trizol according to the manufacturer (Gibco/BRL, Breda, The Netherlands), with the following modifications: tissue was homogenized using pestle and mortar (Fisher Scientific, Breda, The Netherlands) on liquid nitrogen. After chloroform extraction in the presence of glycogen (8 ␮g/mL), an additional phenol/chloroform/isoamylalcohol (pH 7.9) extraction step was introduced. Quality and quantity were checked using a spectrophotometer and agarose-gel analysis. Construction of a cDNA Library using a Suppression Subtraction Hybridization Procedure Wildtype C57BL/6J males and females were fed ad libitum a standard SRM-A chow diet (Hope Farms). Mice were dissected between 10 weeks and 24 months of age. Adipose tissues and lungs were removed and snap frozen in liquid nitrogen and used to isolate total RNA. After DNase treatment and purification of total RNA using RNeasy columns (Qiagen, Leusden, Netherlands), samples were pooled (tester RNA). A suppression-subtracted hybridization was performed using the SMART PCR cDNA Synthesis kit and PCR-Select cDNA Subtraction kit (Clontech, Palo Alto, CA). Briefly, a pool of total RNA isolated from lung tissue from four animals was used as driver, and both tester and driver were digested with RsaI before the adaptor ligation and subsequent subtraction procedure. After two rounds of polymerase chain reaction, purified fragments were ligated into a TA cloning vector (pGEM T-easy; Promega, Madison, WI) and were used for transformation into Escherichia coli DH5-␣ cells (Stratagene, San Diego, CA). Cells were plated on LB-agar containing 100 ␮g/mL ampicillin, 80 ␮g/mL X-gal, and 0.5 mM isopropyl-beta-D-thiogalactopyranoside and grown overnight at 37 °C. White colonies were randomly selected by a colony picker (Flexys, Genomic Solutions, Cambridgeshire, United Kingdom) and inoculated in 96-well plates as published before (10). Polymerase chain reaction was performed on 1-␮L input as described (10). Microarray Manufacturing Amplified adipose-enriched cDNA fragments were precipitated and diluted in 15 ␮L 5⫻ SSC spotting buffer and spotted as described before (11). In addition, Luciferase and Salmonella cDNA were arrayed as positive and negative clones, respectively (12). Furthermore, a set of 27 cDNA clones with established functional relevance in fat metabolism and differentiation was included, sequence-verified, and arrayed in duplicate. Fourteen genes were represented by oligos (60-mers) and were spotted in duplicate as well, whereas the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase was represented by three different oligos, each spotted in quadruplicate. In total, 3136 spots (56 ⫻ 56) were arrayed.

Sequencing of cDNA Clones All spotted cDNA clones were sequenced using the Big Dye Terminator kit according to the manufacturer (Applied Biosystems, Foster City, CA). Sequences were verified against public and/or commercial databases using BLAST-n and BLAST-x (13). Sample Preparation and Labeling We performed a duplicate labeling and subsequent microarray hybridization for each individual mouse adipose tissue sample. Total RNA samples were purified using RNeasy columns (Qiagen) and labeled as described (14,15), with some minor modifications. Briefly, 20 ␮g of each sample was used in duplicate for cDNA synthesis using 2.0 ␮g oligo-dT primer (21-mer) in the presence of aminoallyl dUTP. Luciferase mRNA (1.0 ng) was spiked into all RNA samples to check the quality of the labeling reaction. Samples were labeled using fluorochrome Cy5, whereas a reference pool was labeled with Cy3. This reference pool consisted of a mixture of total RNA of all four adipose tissues from mice of 12 months of age, mixed 1:1 with total RNA derived from cultured 3T3-L1 cells at different differentiation stages. Purification, precipitation, and denaturation of labeled cDNA were performed as described (10). Before hybridization, the Cy5- and Cy3-labeled samples were mixed 1:1 (vol/vol). Microarray prehybridization and hybridization were performed at 42 °C and subsequently washed and scanned as described (11). The software package ArrayVision (Imaging Research, St. Catharines, Ontario, Canada) was used for image analysis. Average spot intensities were collected for each individual spot and stored for further data processing in Microsoft Excel (Redmond, WA). Microarray Data Analyses For each slide, the overall quality was checked by several criteria. Apart from visual inspection of the images and Bland-Altman plots, quality inspection included comparing average signal and background values and the number of clones expressed. Clones were considered expressed if the signal value was more than twice the background value. For most slides, ⬎95% of the clones showed expression in both Cy3 and Cy5. Slides with ⬍80% expressed spots in either channel or irregular Bland-Altman plots were rejected and not used for further analysis. Data were log-transformed and normalized by global Loess correction of the ln(Cy5/Cy3) ratio using S-plus. For subsequent statistical analysis, we considered only those groups in which at least five replicate slides passed the quality control to ensure that the calculations were sufficiently reliable. Spot data were compared between groups and spots that showed a fold ratio (FR) of ⬎2.0 with a p value ⬍0.05 (unpaired two-tailed Student’s t tests) were OBESITY RESEARCH Vol. 13 No. 6 June 2005

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considered significant. Because some genes had several different clones spotted on the array, the average FR was calculated per gene, and genes with a FR of ⬎1.5 and at least one significant spot were selected for further study.

Results Phenotype Body weights (BWs) of young adult male mice after acclimatization were identical at the start of the experiment (Figure 1A, t ⫽ 0 days). Already after 2 days, a significant difference was observed in BWs between the HFD and CD groups (data not shown). During the whole experiment, BWs of HF-fed mice remained higher than mice fed the control diet and reached, at most, a 9.6% increase (after 19 days; HFD, 28.9 ⫾ 1.6 g vs. control, 26.1 ⫾ 1.6 g). However, the amount of food consumed was not significantly different between the groups (3.19 ⫾ 0.14 vs. 3.17 ⫾ 0.25 g/d) during the time interval of a maximum of 40 days. This reflects energy intake (a mean difference of 2.46 kcal/d) as the most likely cause for changes in BW. The surplus weight gain in the HFD groups resulted mainly from differences in adipose tissue weight, and significant differences in absolute and relative adipose tissue weights were observed for all investigated white adipose tissues (WATs) and for interscapular brown adipose tissue (p ⬍ 0.0005; Table 2; Figure 1, B–G). Organ weights of liver (Table 2), heart, lungs, muscle, and kidneys were not significant higher in the HFD groups (data not shown). Animals fed an HFD had significantly higher blood glucose levels (p ⫽ 0.0015; Table 2), even within the short time interval and the non-fasted state at time of dissection. Plasma levels of insulin, total free fatty acids, and total triglycerides were not significantly increased in the HFD group (Table 2). However, insulin, total triglycerides, and blood glucose levels were increased in the HFD2 group, suggesting an early transition from a normal to a diabetic physiology. Corticosterone plasma levels were not significantly different, whereas leptin plasma levels showed a significant increase in the HFD groups (Table 2; Figure 1H). Morphological examination showed an enlargement and fat-filling of visceral adipocytes in both HFD groups compared with the CD groups (Figure 2). cDNA Library To evaluate many genes simultaneously for their expression in adipose tissue, we constructed a murine adipose tissue-enriched cDNA library by a subtraction suppression hybridization procedure. The library was amplified and spotted on glass slides, and all clones were subsequently sequenced. This microarray contains 3136 spots, of which 1034


2938 are derived from our cDNA library. Of these 2938 cDNA clones, 2650 sequences (90%) were verified against databases, which resulted in 1742 sequences representing 568 named genes. Furthermore, 567 expressed sequence tags (ESTs) with unknown function and 80 unknown sequences are present. Of these 568 named genes, 384 are present uniquely, whereas only 223 ESTs are present uniquely. A full list of all named genes can be found in Appendix A, available online at the Obesity Research website (www.obesityresearch.org). Gene Expression Patterns Labeled samples (in duplicate) of all individual mouse visceral fat depots were hybridized against a common reference pool on cDNA microarrays. Quality checks excluded the diet groups C0 and C1 from further analyses. Data analyses were performed on the diet groups C2, HF1, and HF2 to obtain a set of significantly differentially expressed genes. As part of the selection criteria, we used an FR of 2.0 and Student’s t test analyses. This resulted in a total of 102 cDNA clones that were differentially expressed. They represent, in total, 31 transcripts: 24 named genes and 7 ESTs with unknown function. Only one gene was significantly differentially expressed between the two HFD groups (leptin). Therefore, all transcripts were grouped according to their differential expression between HFD and CD groups, i.e., either significantly higher (Table 3, A–C) or lower expression (Table 3, D–F). Of the 23 upregulated transcripts, 12 were more highly expressed in both HFD groups (Table 3A). These included procollagen type III, vimentin, haptoglobin, and lipoprotein lipase, among others. Genes showing a significantly higher expression after only 3 weeks on HFD (Group HF1) and a smaller increase in expression after a longer time period (Group HF2) were myosin, carbonic anhydrase 3, and secreted acidic cysteine rich glycoprotein (Table 3B). In contrast, some genes showed significantly higher expression only in the HF2 group, although their expression was higher in the HF1 group compared with the C2 group (Table 3C). These included XPA binding protein 1, a metalloprotease, and leptin. In total, seven transcripts were less expressed in one or both HFD groups. Importantly, three genes of the corticosterone biosynthesis pathway, comprising all genes of this pathway on the array, were less expressed in both HFD groups (Table 3D): 3␤-hydroxysteroid dehydrogenase-1, cytochrome P450, family 11, subfamily a, polypeptide 1, and cytochrome P450, family 11, subfamily b, polypeptide 1. Plasma corticosterone levels were not significantly altered (Table 2). This suggests a reduction of intracellular corticosterone levels thwarting the initial increase in adipose tissue mass.


Figure 1: Phenotypic characteristics. All data are represented per diet group (x-axes, n ⫽ 3 for all groups) as mean ⫾ SD. iBAT, interscapular brown adipose tissue.


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‡ ND ‡ NS * NS * NS NS NS ‡ ‡ † ‡ †

NS

0.85 ⫾ 0.23 1.04 ⫾ 0.03 § 0.26 ⫾ 0.14 2.47 ⫾ 1.81 480.47 ⫾ 42.03 0.66 ⫾ 0.09 0.51 ⫾ 0.01 3.4 ⫾ 0.8 1.4 ⫾ 0.4 1.5 ⫾ 0.2 0.5 ⫾ 0.2 1.5 ⫾ 0.4

24.8 ⫾ 1.3 0

C0 24.9 ⫾ 1.2 21 25.9 ⫾ 1.8 1.0 ⫾ 0.7 1.05 ⫾ 0.21 1.10 ⫾ 0.04 7.1 ⫾ 1.2 0.23 ⫾ 0.02 5.13 ⫾ 2.61 370.23 ⫾ 28.01 0.68 ⫾ 0.13 0.34 ⫾ 0.10 3.9 ⫾ 0.6 1.7 ⫾ 0.3 1.6 ⫾ 0.3 0.6 ⫾ 0.2 1.5 ⫾ 0.3

C1 24.9 ⫾ 1.2 21 29.9 ⫾ 1.9 5.0 ⫾ 0.8 2.34 ⫾ 0.53 1.19 ⫾ 0.12 10.9 ⫾ 2.6 0.34 ⫾ 0.13 19.47 ⫾ 10.25 420.00 ⫾ 52.19 0.53 ⫾ 0.06 0.38 ⫾ 0.03 7.8 ⫾ 1.3 3.0 ⫾ 0.6 3.2 ⫾ 0.6 1.6 ⫾ 0.2 2.1 ⫾ 0.2

HF1

Diet group

All data is represented by mean ⫾ SD. Statistical analyses were performed between the CD and HFD groups using PROC MIXED. NS, not significant; ND, not determined because of model; iBAT, interscapular brown adipose tissue. *p ⱕ 0.005, †p ⱕ 0.0005, ‡p ⱕ 0.0001. §Not determined. ¶Plasma values.

BW at day t ⫽ 0 (g) Killed at day BW (g) at death Weight gain (g) Total WAT (g) Liver (g) Blood glucose (mM) Insulin (ng/mL)¶ Leptin (ng/mL)¶ Corticosterone (ng/mL)¶ Triglycerides (mM)¶ Free fatty acids (mM)¶ Total WAT/BW (%) Subcutaneous WAT/BW (%) Epididymal WAT/BW (%) Visceral WAT/BW (%) iBAT/BW (%)

HFD vs. CD

Table 2. Phenotypic characteristics

25.0 ⫾ 1.2 30 27.7 ⫾ 1.3 2.7 ⫾ 0.8 1.26 ⫾ 0.18 1.14 ⫾ 0.07 7.3 ⫾ 1.6 0.31 ⫾ 0.14 12.73 ⫾ 2.57 423.18 ⫾ 65.32 0.72 ⫾ 0.15 0.34 ⫾ 0.04 4.5 ⫾ 4.0 1.7 ⫾ 0.1 2.0 ⫾ 0.4 0.9 ⫾ 0.1 1.0 ⫾ 0.1

C2

25.2 ⫾ 1.4 40 31.6 ⫾ 2.2 6.4 ⫾ 1.9 2.76 ⫾ 0.75 1.22 ⫾ 0.03 10.4 ⫾ 2.4 0.85 ⫾ 0.34 30.10 ⫾ 12.65 358.50 ⫾ 28.80 1.89 ⫾ 0.60 0.45 ⫾ 0.02 8.7 ⫾ 0.9 3.2 ⫾ 0.5 3.7 ⫾ 1.1 1.7 ⫾ 0.4 2.1 ⫾ 0.4

HF2



Figure 2: Morphology of visceral adipose tissue. Ten-micrometer sections of formalin-fixed, paraffin-embedded hematoxylin and eosin–stained visceral adipose tissues. All photographs were made using a magnification of ⫻40. C, control low fat; HF, high fat.

Four transcripts showed significantly decreased expression only in the HF1 group (and non-significantly decreased expression in Group HF2), and they encoded ATP citrate lyase and CUG triplet repeat, RNA binding protein 2, in addition to two ESTs (Table 3E). Finally, NADH dehydrogenase subunit 6 showed decreased expression only in Group HF2 and non-significantly decreased expression in Group HF1 (Table 3F). In general, about one-half of the selected genes were represented by several different cDNA clones (Table 3, as denoted by an asterisk). In all cases, all clones of a given gene showed a similar expression pattern, confirming the observed differentially regulated genes. The significant up-regulation of leptin mRNA in a time(HF2 ⬎ HF1) and diet- (HF2 ⬎ C2) dependent way (Table 3C) accompanied its significant rise in plasma protein levels (Table 2; Figure 1H). Although, in the HF1 group, its mRNA level was only marginally up-regulated compared with the C2 group (FR ⫽ 1.16), its mean plasma protein level increased 1.53 times (19.47 ⫾ 10.25 vs. 12.73 ⫾ 2.57). Likewise, between the HF2 and the C2 diet groups, mRNA levels were 2.52 times higher, and corresponding protein levels had a comparable factor of 2.36. This confirms the observed mRNA changes in relation to its functional property, the protein level. Moreover, leptin is the only transcript that was significantly increased between the

HF1 and HF2 groups (FR HF2/HF1 ⫽ 2.17), relating to the increase in WAT mass in time (Figure 1, C–F) and its rise in protein level (Figure 1H).

Discussion Mice on an HFD for a time interval of 3 to 5 weeks showed significant differences in BW and adipose tissue weights and differential gene expression patterns in visceral adipose tissue compared with control mice. The weight difference between both groups was, at most, 9.6% and corresponded to mild weight gain/overweight in humans. The gene expression data indicated that there were hardly any changes between the HF1 and HF2 time-points. Probably, adipose gene expression patterns after 3 to 5 weeks of HFD already indicate a number of changes that may last until the obese state. This makes sense physiologically because (increasing degrees of) overweight and not solely obesity is an important predictor of metabolic complications (1). In general, the functional classes/metabolic pathways of cellular structure [e.g., amyloid ␤ (A4) precursor protein, Sparc/osteonectin, myosin, and procollagen type III; Table 3, A–C] and lipid biosynthesis (Lpl and Car3; Table 3, A–C) are up-regulated on an HFD. At the same time, ATP citrate lyase mRNA levels were decreased (Table 3E), which is in agreement with a reduction in the metabolism of OBESITY RESEARCH Vol. 13 No. 6 June 2005

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C

Myosin, light polypeptide 1, alkali; atrial, embryonic

Myl1*

2.02

2.03

Significantly increased expression in group HF2 and non-significant increase in group HF1 PET112l PET112-like (yeast) 1.52 Ob* Leptin 1.16 1.48 Adamts5* A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 5 Mup3* Major urinary protein 3 1.71 Xab1 XPA binding protein 1 1.90

Mrc1

Carbonic anhydrase 3 Secreted acidic cysteine rich glycoprotein Mannose receptor 1

Car3* Sparc

2.29 2.09

Significantly increased expression in group HF1 and non-significant increase in group HF2 Aldh1a1* Aldehyde dehydrogenase family 1, 2.31 subfamily A1

2.92 2.83 2.68 2.59 2.50 2.38 2.23 2.18 2.16 2.08 2.07 2.01 2.01

HF1/C2

B

Dynamin 1-like

Lipoprotein lipase Sarcospan

in groups HF1 and HF2 Vimentin Procollagen, type III, alpha 1 Major urinary protein 1 Amyloid beta (A4) precursor protein Haptoglobin

Gene name

Significantly increased expression Vim* Col3a1 Mup1* App Hp* Clone A130050C04 Lpl* Sspn Clone 5930433F13 Clone BM932941 Clone D130006O04 Clone A730058K20 Dnm1l

Symbol

FR

A

Group

Table 3. Microarray data

Mm.52275 Mm.277072 Mm.112933

Mm.250267 Mm.270472

2.12 2.08

Mm.1000

Mm.2019

Mm.300 Mm.291442

Mm.4514

Mm.268000 Mm.249555 Mm.347469 Mm.277585 Mm.26730 Mm.261423 Mm.1514 Mm.49689 Mm.324242 Mm.259830 Mm.31274 Mm.213293 Mm.218820

Unigene

2.93 2.52 2.24

1.88

1.59

1.97 1.97

1.97

3.39 2.10 4.32 3.45 2.03 3.00 2.21 2.94 2.02 2.28 2.09 2.18 2.12

HF2/C2

Transporter Gtpase with role in NER

Protein biosynthesis Lipid metabolism Extracellular matrix

Mediator of cell cycle check-point Cytoskeleton

Lipid metabolism/glycolysis/ gluconeogenesis/pyruvate metabolism Energy metabolism Extracellular matrix

Cytoskeleton Cell adhesion Transporter Cell adhesion Acute phase response Unknown Lipid metabolism Cytoskeleton Unknown Unknown Unknown Unknown Cytoskeleton

Function


All selected genes are represented by their gene expression pattern on an HFD. Genes per group are sorted based on descending FRs in the relevant diet group. * Gene is represented by at least two cDNA clones on the microarray.

Energy metabolism

Citric acid cycle Unknown Unknown Mm.282039 Mm.101586

Significantly decreased expression in group HF2 and non-significant decrease in group HF1 mt-Nd6* NADH dehydrogenase subunit 6 ⫺1.96 ⫺2.13

Pre-MRA splicing factor Mm.147091

F

Cyp11b1

Significantly decreased expression in group HF1 and non-significant decrease in group HF2 Cugbp2 CUG triplet repeat, RNA binding ⫺3.13 ⫺1.96 protein 2 Acly* ATP citrate lyase ⫺2.50 ⫺1.47 Riken 061001N22 ⫺2.00 ⫺1.35 Clone RP23–166P10 ⫺2.00 ⫺1.67

2.56 ⫺3.03

Mm.302865 2.04 ⫺3.13 Cyp11a*

Cholesterol side-chain cleavage, CYP450, 11a Cytochrome P450, 11b1

2.78

E

D

Significantly decreased expression in groups HF1 and HF2 Hsd3b1* 3␤-Hydroxysteroid dehydrogenase-1

⫺3.13

Mm.140811

Steroid hormone biosynthesis Steroid hormone biosynthesis Steroid hormone biosynthesis


glucose into fatty acids. Furthermore, oxidative phosphorylation was reduced (NADH dehydrogenase subunit 6; Table 3F). All these processes lead in the same direction, namely hypertrophy of adipocytes. Indeed, morphology showed larger adipocytes (Figure 2) when mice were fed an HFD. Comparison of our results with other published expression data (4,5,16 –20) is difficult because of the fact that there are (large) differences among the studies. For example, there are differences in diet composition, duration of intervention, use of fasted/non-fasted animals, or age of animals, or different adipose tissue depots or cell fractions [e.g., adipose tissue vs. (pre)adipocytes] were analyzed. Furthermore, it should be kept in mind that we performed all gene expression analyses on total adipose tissue, i.e., a mixture of adipocytes, preadipocytes, and stromal cells. These fractions show their own particular gene expression pattern in mice and humans (16,18). However, several significantly higher expression genes found by us (e.g., leptin, haptoglobin, Sparc, and Lpl) were also found to be up-regulated in other studies in (obese) rodents (4,5,17,19). This exemplifies that some of the observed gene expression changes obtained during a short time period using human physiological levels of dietary fats involve, in fact, pivotal differentially regulated genes, which seem to maintain their expression well into the obese state. Some of the genes known to be more highly expressed in obese mice are aP2, resistin, adiponectin, sterol regulatory element-binding protein-1, and peroxisome proliferator activated receptor ␥ (16). We observed higher expression on an HFD for all these genes, although they did not meet an FR of 2.0 and a p value ⬍0.05 to be significantly differentially expressed (data not shown). A significant down-regulation on an HFD of three enzymes involved in corticosterone biosynthesis was observed. In rodents, this pathway contains four enzymes that mediate the conversion of cholesterol into corticosterone (Figure 3), of which the Cyp11a gene product performs the first and rate-limiting step. The gene steroid 21 hydroxylase (Cyp21a1) is lacking on the microarray. The expression of genes involved in steroid hormone metabolism in adipose tissue has been reported in humans (21) and rats (22), but differential expression was not studied or observed. A possible explanation for this discrepancy is the site of adipose tissue. We observed visceral WAT expression of these genes in mice fed the humanized CD and HFD. In an additional experiment, expression was observed in adipose samples exclusively of visceral origin from mice fed a standard chow diet. No or very low expression levels were observed in subcutaneous, gonadal, and brown adipose tissue (data not shown). Indeed, no differential expression in epididymal OBESITY RESEARCH Vol. 13 No. 6 June 2005

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Acknowledgments We thank Dr. N.J.D. Nagelkerke for statistical analyses on phenotypic measurements, C.W. Schot and C. Moolenbeek for animal handling, Dr. R.B. Beems for histology, and J.W.J.M. Cremers, P. van Impelen, H.W. Verharen, and H.M. Hodemaekers for expert help (Bilthoven). We also thank the microarray unit at RIKILT, Wageningen, for help with all array experiments.

Figure 3: Corticosterone biosynthesis pathway. Simplified diagram of the major rodent corticosterone biosynthesis pathway (bold). Genes present on the cDNA microarray are underlined.

WAT of obese vs. normal weight mice (23) or no expression at all in (obese) rodent adipose tissue has been observed (16,19,20,23,24). Transgenic mice overexpressing hydroxysteroid 11-␤ dehydrogenase (Hsd11b1; Figure 3) in adipocytes have been shown to have local elevated levels of active corticosterone and visceral obesity (25), although human data show conflicting results (26). Furthermore, two studies have reported decreased murine adipose expression levels of Hsd11b1 on an HFD, resulting in lower corticosterone levels in WAT (24,27). Our results strengthen the proposed adaptive response to an HFD (27) by decreased gene expression of the enzymes mediating the biosynthesis of corticosterone. Interestingly, it has been found that leptin acts as an inhibitory signal in gene regulation of Cyp11a within the adrenal gland and testis (28). We speculate here that leptin down-regulates the Cyp11a gene within visceral adipose tissue as well. As the adaptive response to HFD diminishes in time, it might play a role only in the initial progress from a normal physiological state toward weight gain/overweight. The role of corticosterone conversion in adipose tissue is intensively being studied. Our findings indicate that de novo corticosterone synthesis can occur. This warrants further research. In conclusion, using a constructed adipose tissue-enriched cDNA microarray, we obtained insight into the early progression of obesity and associated metabolic disturbances. Some of these disturbances seem to be counteracted initially by decreased adipose tissue gene expression of corticosterone biosynthesis. 1040


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