Obesity-related Differential Gene Expression in the

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Research Articles

Obesity-related Differential Gene Expression in the Visceral Adipose Tissue Ancha Baranova1; Rochelle Collantes1,2; Shobha J. Gowder1; Hazem Elariny1,2; Karen Schlauch1,2; Abraham Younoszai1,2; Steve King1; Manpreet Randhawa1; Sitapati Pusulury1; Tariq Alsheddi1; Janus P. Ong1,2; Lisa M. Martin1,2; Vikas Chandhoke1; Zobair M. Younossi1,2 1Center

for the Study of Genomics in Liver Diseases, Molecular and Microbiology Department, George Mason University; 2Center for Liver Diseases, Inova Fairfax Hospital, Falls Church, VA, USA

Background:This study investigates the expression patterns in human adipose tissue, and identifies genes that may be involved in the abnormal energy homeostasis. Methods: Subjects were prospectively recruited from morbidly obese patients undergoing bariatric surgery and from non-obese organ donors. Extensive clinical data and visceral fat specimens were obtained from each subject at the time of surgery. A group of 50 obese patients and 9 non-obese controls were selected for further study. Two custom two-color cDNA microarrays were produced with 40,173 human individual cDNA clones. Microarray experiments were performed for each sample, and a selected group of gene expression values were confirmed with real-time RT-PCR. Results: A comparison of gene expression profiles from obese and non-obese patients identified 1,208 genes with statistically significant differential expression between the 2 groups. Most prominent among these genes are multiple glycolysis enzyme encoding genes; others are involved in oxysterol biosynthesis and signaling, or are ATP-binding transporters and solute carriers. Conclusion: Differential gene expression in the adipose tissue of morbidly obese patients includes genes related to lipid and glucose metabolism, membrane transport, and genes promoting the cell cycle. These findings are a first step toward clarifying the molecular pathogenesis of obesity and identifying potential targets for therapeutic intervention. Key words: Morbid obesity, adipose tissue, microarrays, gene expression Reprint requests to: Zobair M. Younossi, MD, MPH, Center for Liver Diseases, Inova Fairfax Hospital, 3300 Gallows Road, Falls Church, VA 22042, USA. Fax: (703) 698-3482 or (703) 208-6655; e-mail: [email protected]

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Introduction The global epidemic of obesity emphasizes the necessity of understanding the molecular mechanism of energy regulation. Adipocyte differentiation is regulated by a complex interplay of transcriptional factors. The exact triggers for changes that lead to the adipogenic cascade of events are not known. Fat metabolism is affected by many factors, including hormone sensitive lipase, catecholamines, and insulin. Additionally, visceral adipose tissue also produces many peptides, bioactive products, and cytokines, which can have an important impact on other organs.1 Because of its complexity, the regulatory network for human adipose cells is well-suited for an investigation with gene expression profiling, a method used for studying the molecular nature of multifaceted diseases. Most microarray-based expression studies published to date have used cultured human adipocytes, which have important differences from visceral fat tissue obtained from obese patients,2,3 and have been performed on a small group of genes4 or pooled samples.5 Also, subcutaneous and visceral deposits of white adipose tissue are metabolically different,19 resulting in profound differences in their expression profiles. We have examined the molecular differences between samples of visceral adipose tissue obtained from obese subjects during bariatric surgery and lean subjects during organ donation surgery, producing expression profiles for nearly 40,000 human mRNAs in human adipose tissue. © FD-Communications Inc.

Obesity-related Differential Gene Expression in Visceral Adipose Tissue

Methods and Procedures Patient Selection The study enrolled patients undergoing bariatric surgery at Inova Fairfax Hospital from April 2003 to May 2004. Extensive clinical and laboratory data were collected on each patient after obtaining informed consent. Diabetes mellitus was defined as a clinically established diagnosis treated with either dietary modification or anti-diabetic medications or both. Omental fat specimens were obtained at the time of bariatric surgery and immediately snapfrozen with liquid nitrogen to be used later for RNA extraction. Control samples of visceral adipose were obtained from non-obese (BMI 0.05 0.05 >0.05 NA* NA*

Obesity-related Differential Gene Expression in Visceral Adipose Tissue

A separate analysis of the obese diabetics was performed to minimize the confounding effect that diabetes may have on gene expression among the obese patients.10 Comparisons of the gene expression values in the previously diagnosed diabetics and normoglycemic obese individuals did not reveal significantly differential gene expression, validating our strict inclusion criteria (see Methods). On the other hand, comparisons between the diabetic obese group and the controls, and between the normoglycemic group and the controls revealed 1,454 and 1,114 differentially expressed genes, respectively. A Venn diagram showing results from these comparisons is presented in Figure 1.

Classification of Differentially Expressed Genes All genes with significant differential expression were subdivided into two categories: known human genes and ESTs/predicted genes. cDNAs representing known human genes were further subdivided according to the biological processes in which they

DMO

MO

118

758

137

528 425

31

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NGO Figure 1. Venn diagram depicting pairwise intersections of significantly differentially expressed genes obtained in the three comparisons: morbidly obese (MO) vs controls (dark circle), obese diabetics (DMO) vs controls (grey circle) and normoglycemic obese (NGO) vs controls (pale circle).

are involved (http://gunston.gmu.edu/liverdisease/ research_obesity.html, Supplementary Table 1). cDNAs representing ESTs/predicted genes were localized to the human genome by BLAST searches and were subdivided accordingly (http://gunston. gmu.edu/liverdisease/research_obesity.html, Supplementary Table 2). Known human genes that were differentially expressed in all three comparisons (N = 528) are represented by the central section in the Venn diagram (Figure 1) and listed in http://gunston.gmu.edu/liverdisease/research _obesity.html, Supplementary Table 3. A subset of genes differentially expressed in all three comparisons was used for hierarchical clustering of obese and control patients. Here, Eisen’s Cluster and TreeView programs were used, with the correlation coefficient and average agglomerative method. This set of genes, which includes 15 obesity up-regulated and 57 obesity down-regulated genes, clearly groups together all but one control within the tightest cluster (Figure 2 and Supplementary Table 4).*

Validation of Gene Expression Data by Qualitative Real-time PCR To validate our microarray technology, we obtained Quantitative Real-time PCR measurements of the mRNA level of 15 genes: GUK1, ADH1B, CH25H, PFKP, AWP1, PGK1, LDHA, FABP4, CCNB2, GSN, FIGF, NTS, VMD2 ABCA8, Adiponectin (APMI) (Table 2). In all but one case (ABCA8), the real-time PCR fold differences were in complete correspondence with the microarray data. In the case of ABCA8, contradictory results were obtained in cDNA hybridizations to two different ABCA8related probes (AA634308 located at the very 3’ of the gene and W74070 overlapping with ABCA8 coding exons), with significantly different gene expression values for each. Those clones may represent alternative isoforms of mRNA ABCA8 that are regulated independently of major isoform(s) representing coding exons of this gene. The results of four representative Real-time PCR experiments quantifying messages for genes PGK1, PFKP, GSN and FIGF are depicted in Figure 3. *This paper is associated with four large Supplementary Tables that contain lists of differentially expressed genes. These Supplementary Tables are available on: http://gunston.doit.gmu.edu/liverdisease/research_obesity.html

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Baranova et al Table 2. Sequences of PCR primers used in Real Time PCR experiments. (F: Forward pimer; R: Reverse primer) Guanylate kinase 1 (GUK1) Alcohol dehydrogenase IB (class I), beta polypeptide (ADH1B) Cholesterol 25-hydroxylase (CH25H) Phosphofructokinase, platelet (PFKP) Protein associated with PRK1 (AWP1) Phosphoglycerate kinase 1 (PGK1) Lactate dehydrogenase A (LDHA) Fatty acid binding protein 4, adipocyte (FABP4) Cyclin B2( CCNB2) Gelsolin (GSN) c-fos induced growth factor/vascular endothelial growth factor D (FIGF) Neurotensin (NTS) Vitelliform macular dystrophy (Best disease, bestrophin) (VMD2) ATP-binding cassette, sub-family A (ABC1), member 8 (ABCA8) Adiponectin (APM1) 18S

Discussion The results obtained in this study suggest differential expression in genes encoding key glycolysis enzymes phosphoglycerate kinase 1 (PGK1), platelet type of phosphofructokinase (PFKP), fructose-bisphosphate aldolase A (ALDOA), and 6-Phosphofructo-2kinase/fructose 2,6-bisphosphatase (PFKFB4). Upregulation of these enzymes leads to an increase in intracellular glycerol-3-phosphate concentrations, which is necessary for storable triacylglycerol formation.11 Cholesterol uptake in obese adipocytes is also stimulated by LDLR and APOC3 up-regulation. The latter gene encodes apolipoprotein CIII, adversely affecting lipoprotein lipase activity.12 The adipocytes from obese individuals also demonstrated profound changes in the expression of various ATP-binding transporters and solute carri762

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F: TTGCTCGTGCCATACAGG R:CAGCGTGTCCCATACCAC F:GGTGGGCAGAGAAGACAGAAAC R:CACATCCTCAATGGAAAAGGG F: CCAGACGCTCATATACTGC R:TCTTCGACATGGAGTTCTTC F:CACCAACCTGTGTGTGATC R:TTGAGGTAGGCGTACTTCTG F:AGAATAGGGAAGGCAGAAAG R:CCAGTGGAACAAAGCATAG F:GAAGAAGGGAAGGGAAAAG R:GTGCCAAAAGCATCATTGAC F:GGAGTGGAATGAATGTTGC R:CAGGATGTGTAGCCTTTGAG F:GGTACCTGGAAACTTGTCTC R:CCCATTCACACTGATGATC F: AGAGCAGAGCAGTAATCCC R:GTCCACTCCAAGTTTAGGC F: GCCCATGATCATCTACAAG R: CATCGTTGGAGTTCAGTG F:GAGTGGGTAGTGGTGAATG R:CAATGTGGACTGAGATGATC F:GAGGGAACATGTGCTTTAC R:GCTACTCCTGGCTTTCAG F:AACTGAGCCTACCACACAAC R:GAGTACGCAAGGTGTTCATC F: AGCTTGAAGAGCCACAAAACC R:AAGGAGCACAAGGACCATACAG F:CCATTCGCTTTACCAAGATC R: AAGAGGCTGACCTTCACATC F: AGGAATTCCCAGTAAGTGCG R: GCCTCACTAAACCATCCAA

ers, including up-regulation of insulin-responsive facilitated glucose transporter GLUT4/SLC2A4. This up-regulation may reflect an overall increase in insulin levels in obese individuals necessary to maintain normal blood glucose despite their insulin resistance. An increase in insulin resistance may lead to GLUT4 up-regulation and a subsequent increase of adiposity with further advancement to a pro-diabetic state, promoting a metabolic vicious circle. We also observed an increase in EHD2 expression, encoding GLUT4-interacting protein. In addition, we detected a decrease in adipocyte-specific ABCC9 expression, which encodes the regulatory SUR2A subunit of the K(ATP) channel, stimulating insulin release when closed, and an increase in ENSA expression, which encodes endosulfine alpha serving as an endogenous equivalent of sulfonylurea that closes this K(ATP) channel. Earlier

Obesity-related Differential Gene Expression in Visceral Adipose Tissue

Figure 2. Hierarchical clustering of obese and control patients across a subset of 72 genes differentially expressed in all three comparisons. Genes are listed from top to bottom; patients from left to right. Control subjects are identified with a rectangle. Clusters were generated using Cluster and TreeView with the correlation coefficient and the average agglomerative method. Green indicates a down-regulation of a gene between obese patients and controls; red an up-regulation, and black a non-response. Obesity Surgery, 15, 2005

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Baranova et al PGK1

GSN

CCNB2

FIGF

Figure 3. Bar graphs representing ratios of average expression levels in MO and CONTROL patients obtained by microarray technology (black bars) and by RT-PCR (white bars). The dashed line represents a ratio equal to 1 (no difference in gene expression between the two groups). The upper two graphs present up-regulation in obese patients; the lower two graphs present down-regulation in obese patients.

work demonstrated components of the sulfonylurea system in human adipose tissue, but the physiological importance of this system remains unclear.13 Oxysterol biosynthesis, which depends on cholesterol 25-hydroxylase CH25H,14 is another pathway with apparently decreased expression in the adipocytes of obese individuals. Defects in oxysterol-dependent SREBPs suppression can lead to SREBPs-dependent activation of cholesterol synthesis and ectopic up-regulation of melanin produc764

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tion.15 Melanin production has not been connected to adipocytes before, but obese adipocytes display significant increases in messages encoded by genes related to tyrosinase-related protein 1(TYRP1) and dopachrome tautomerase (DCT). Recent studies indicate that adipose tissue is a highly active endocrine organ.1 In accordance with previous studies,16 we observed adiponectin mRNA down-regulation in the adipose samples from obese patients. Basal adiponectin mRNA levels in obese

Obesity-related Differential Gene Expression in Visceral Adipose Tissue

patients (51.2 ± 33.4) was lower than that of nonobese controls (98.8 ± 21.0) when measured by realtime PCR. Our microarray analysis also revealed a significant increase in expression of genes involved in sex hormone conversion (AKR1C2) and cortisol biosynthesis (H6PD). This study also points to three functional gene classes that are differentially regulated in adipocytes of obese individuals. We found significant overexpression in six protein biosynthesis genes and in 23 of 24 cell cycle related genes. Furthermore, our comparison of obese and non-obese individuals showed a significant decrease in the expression of all five angiogenesis-promoting proteins. The potential importance of angiogenesis in obesity is interesting, because expanding adipose tissue represents one of the few sites with active angiogenesis in adults. We observed a paradoxical decrease in the expression of pro-angiogenic genes PDGFD, ANGPTL1, FIGF, CYR61 and PDGFRA. On the other hand, we observed up-regulated mRNA expression for type 2 pro-angiogenic methionine aminopeptidase (MetAP2)17 and adipose specific vascular marker prohibitin18 in obese adipocytes. Most of the genes discussed above were differentially expressed in all three comparisons performed in this study (morbidly obese vs controls; diabetic obese vs controls; normoglycemic obese vs controls), and are represented by the central section in the Venn diagram of Figure 1. In conclusion, our microarray expression profiling reveals specific genes that are differentially expressed in the adipose tissue of morbidly obese individuals compared to non-obese controls. Molecular pathways involved in glucose utilization, cholesterol biosynthesis, steroid hormone metabolism, and possibly angiogenesis, are deregulated in human obesity. If confirmed in future studies, these patterns of gene expression can contribute to our understanding of the pathogenesis of obesity and provide potential targets for future therapy.

References 1. Yang YS, Song HD, Li RY et al. The gene expression profiling of human visceral adipose tissue and its secretory functions. Biochem Biophys Res Commun 2003; 300: 839-46. 2. Soukas A, Socci ND, Saatkamp BD et al. Distinct transcrip-

tional profiles of adipogenesis in vivo and in vitro. J Biol Chem 2001; 276(36): 34167-74. 3. Permana PA, Del Parigi A, Tataranni PA. Microarray gene expression profiling in obesity and insulin resistance. Nutrition 2004; 20: 134-8. 4. Linder K, Arner P, Flores-Morales A et al. Differentially expressed genes in visceral or subcutaneous adipose tissue of obese men and women. J Lipid Res 2004; 45: 148-54. 5. Gomez-Ambrosi J, Catalon V, Diez-Caballero A et al. Gene expression profile of omental adipose tissue in human obesity. FASEB J 2004; 18: 215-7. 6. Baranova A, Hammursund M, Ivanov D et al. Distinct organization of the candidate tumor suppressor gene RFP2 in human and mouse: multiple mRNA isoforms in both species- and human-specific antisense transcript RFP2OS. Gene 2003; 321: 103-12. 7. Epstein CB, Hale IV W, Butow R. Numerical methods for handling uncertainty in microarray data: an example analyzing perturbed mitochondrial function in yeast. Methods in Cell Biology 2001; 65: 439-52. 8. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. Journal of the Royal Statistical Society Series B 1995; 57: 289-300. 9. Eisen MB, Spellman PT, Brown PO et al. Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci USA 1998; 95: 14863-8. 10. Golay A, Felber JP. Evolution from obesity to diabetes. Diabetes Metab 1994; 20: 3-14. 11. Forest C, Tordjman J, Glorian M et al.Fatty acid recycling in adipocytes: a role for glyceroneogenesis and phosphoenolpyruvate carboxykinase. Biochem Soc Trans 2003; 31 (Pt 6): 1125-9. 12. Jong MC, Rensen PC, Dahlmans VE et al. Apolipoprotein C-III deficiency accelerates triglyceride hydrolysis by lipoprotein lipase in wild-type and apoE knockout mice. J Lipid Res 2001; 42: 1578-85. 13. Gabrielsson BG, Karlsson AC, Lonn M et al. Molecular characterization of a local sulfonylurea system in human adipose tissue. Mol Cell Biochem 2004;258(1-2):65-71. 14. Lund EG, Kerr TA, Sakai J et al. cDNA cloning of mouse and human cholesterol 25-hydroxylases, polytopic membrane proteins that synthesize a potent oxysterol regulator of lipid metabolism. J Biol Chem 1998; 273: 34316-27. 15. Hall AM, Krishnamoorthy L, Orlow SJ. 25-hydroxycholesterol acts in the Golgi compartment to induce degradation of tyrosinase. Pigment Cell Res 2004; 17: 396-406. 16. Kern PA, Di Gregorio GB, Lu T et al. Adiponectin expression from human adipose tissue: relation to obesity, insulin resistance, and tumor necrosis factor-alpha expression. Diabetes 2003; 52: 1779-85. 17. Klein CD, Folkers G. Understanding the selectivity of fumagillin for the methionine aminopeptidase type II. Oncol Res 2003; 13: 513-20. 18. Kolonin MG, Saha PK, Chan L et al. Reversal of obesity by targeted ablation of adipose tissue. Nat Med 2004; 10: 625-32. 19. Vohl MC, Sladek R, Robitaille J et al. A survey of genes differentially expressed in subcutaneous and visceral adipose tissue in men. Obes Res 2004; 12: 1217-22.

(Received March 25, 2005; accepted May 11, 2005) Obesity Surgery, 15, 2005

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