PDF icon Download PDF
Horm Metab Res 2007; 39(7): 495-500
DOI: 10.1055/s-2007-982502
Original Basic

© Georg Thieme Verlag KG Stuttgart · New York

Validation of Endogenous Control Genes in Human Adipose Tissue: Relevance to Obesity and Obesity-associated Type 2 Diabetes Mellitus

V. Catalán 1 , J. Gómez-Ambrosi 1 , F. Rotellar 2 , C. Silva 3 , A. Rodríguez 1 , J. Salvador 3 , M. J. Gil 4 , J. A. Cienfuegos 2 , G. Frühbeck 1 , 3
  • 1Metabolic Research Laboratory, Clínica Universitaria de Navarra, University of Navarra, Pamplona, Spain
  • 2Department of Surgery, Clínica Universitaria de Navarra, University of Navarra, Pamplona, Spain
  • 3Department of Endocrinology, Clínica Universitaria de Navarra, University of Navarra, Pamplona, Spain
  • 4Department of Biochemistry, Clínica Universitaria de Navarra, University of Navarra, Pamplona, Spain
Further Information

Publication History

received 17. 10. 2006

accepted 29. 1. 2007

Publication Date:
05 July 2007 (online)

Also available ateRef
   Permissions and Reprints
Preview

Abstract

The aim of the present study was to test the influence of obesity and the presence of type 2 diabetes mellitus (T2DM) on the expression of ten housekeeping genes and of the 18S rRNA in a group of human adipose tissue samples from the omental and subcutaneous depot. Adipose tissue biopsies were obtained by laparoscopic surgery from lean and obese patients. After the extraction, mRNA levels in adipose tissue samples were quantified by real-time PCR using the commercial Human Endogenous Control Plates. From the genes analyzed, 18S rRNA exhibited the most stable expression levels in both depots regardless of the pathophysiological conditions of obesity and obesity-associated T2DM. Contrarily, GAPD was the gene with the highest variation in its expression levels, being upregulated (8.0-fold) in the obese group and downregulated (3.5-fold) in obesity-associated T2DM. Our results show that 18S rRNA may be the most suitable gene for normalization in expression studies performed in human adipose tissue samples obtained from patients suffering from obesity and/or obesity-associated T2DM, whereas GAPD is less appropriate for comparison purposes under these circumstances.

Key words

Obesity - type 2 diabetes mellitus - real-time PCR - adipose tissue - housekeeping gene expression

References

  • 1 Frühbeck G, Gómez-Ambrosi J, Muruzábal FJ, Burrell MA. The adipocyte: a model for integration of endocrine and metabolic signaling in energy metabolism regulation.  Am J Physiol Endocrinol Metab. 2001;  280 E827-E847
    PubMedSearch in Google Scholar
  • 2 Soukas A, Socci ND, Saatkamp BD, Novelli S, Friedman JM. Distinct transcriptional profiles of adipogenesis in vivo and in vitro.  J Biol Chem. 2001;  276 34167-34174
    CrossrefPubMedSearch in Google Scholar
  • 3 Rajala MW, Scherer PE. Minireview: the adipocyte-at the crossroads of energy homeostasis, inflammation, and atherosclerosis.  Endocrinology. 2003;  144 3765-3773
    CrossrefPubMedSearch in Google Scholar
  • 4 Whitehead JP, Richards AA, Hickman IJ, Macdonald GA, Prins JB. Adiponectin-a key adipokine in the metabolic syndrome.  Diabetes Obes Metab. 2006;  8 264-280
    CrossrefPubMedSearch in Google Scholar
  • 5 Frühbeck G, Gómez-Ambrosi J. Control of body weight: a physiologic and transgenic perspective.  Diabetologia. 2003;  46 143-172
    PubMedSearch in Google Scholar
  • 6 Gabrielsson BL, Carlsson B, Carlsson LM. Partial genome scale analysis of gene expression in human adipose tissue using DNA array.  Obes Res. 2000;  8 374-384
    PubMedSearch in Google Scholar
  • 7 Gómez-Ambrosi J, Catalán V, Diez-Caballero A. et al . Gene expression profile of omental adipose tissue in human obesity.  FASEB J. 2004;  18 215-217
    PubMedSearch in Google Scholar
  • 8 Linder K, Arner P, Flores-Morales A, Tollet-Egnell P, Norstedt G. Differentially expressed genes in visceral or subcutaneous adipose tissue of obese men and women.  J Lipid Res. 2004;  45 148-154
    PubMedSearch in Google Scholar
  • 9 Urs S, Smith C, Campbell B, Saxton AM, Taylor J, Zhang B, Snoddy J, Jones Voy B, Moustaid-Moussa N. Gene expression profiling in human preadipocytes and adipocytes by microarray analysis.  J Nutr. 2004;  134 762-770
    PubMedSearch in Google Scholar
  • 10 Vandesompele J, Preter K De, Pattyn F, Poppe B, Roy N Von, Paepe A De, Speleman F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.  Genome Biol. 2002;  3 R34
    PubMedSearch in Google Scholar
  • 11 Szabo A, Perou CM, Karaca M, Perreard L, Quackenbush JF, Bernard PS. Statistical modeling for selecting housekeeper genes.  Genome Biol. 2004;  5 R59
    CrossrefPubMedSearch in Google Scholar
  • 12 Bustin SA. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays.  J Mol Endocrinol. 2000;  25 169-193
    CrossrefPubMedSearch in Google Scholar
  • 13 Suzuki T, Higgins PJ, Crawford DR. Control selection for RNA quantitation.  Biotechniques. 2000;  29 332-337
    PubMedSearch in Google Scholar
  • 14 Boucher J, Quilliot D, Praderes JP, Simon MF, Gres S, Guigne C, Prevot D, Ferry G, Boutin JA, Carpene C, Valet P, Saulnier-Blache JS. Potential involvement of adipocyte insulin resistance in obesity-associated up-regulation of adipocyte lysophospholipase D/autotaxin expression.  Diabetologia. 2005;  48 569-577
    CrossrefPubMedSearch in Google Scholar
  • 15 Poitou C, Viguerie N, Cancello R, Matteis R De, Cinti S, Stich V, Coussieu C, Gauthier E, Courtine M, Zucker JD, Barsh GS, Saris W, Bruneval P, Basdevant A, Langin D, Clement K. Serum amyloid A: production by human white adipocyte and regulation by obesity and nutrition.  Diabetologia. 2005;  48 519-528
    CrossrefPubMedSearch in Google Scholar
  • 16 Viguerie N, Vidal H, Arner P, Viguerie N, Vidal H, Arner P, Holst C, Verdich C, Avizou S, Astrup A, Saris WH, Macdonald IA, Klimcakova E, Clement K, Martinez A, Hoffstedt J, Sorensen TI, Langin D. Nutrient-Gene Interactions in Human Obesity-Implications for Dietary Guideline (NUGENOB) project. Adipose tissue gene expression in obese subjects during low-fat and high-fat hypocaloric diets.  Diabetologia. 2005;  48 123-131
    CrossrefPubMedSearch in Google Scholar
  • 17 Plomgaard P, Keller P, Keller C, Pedersen BK. TNF-alpha, but not IL-6, stimulates plasminogen activator inhibitor-1 expression in human subcutaneous adipose tissue.  J Appl Physiol. 2005;  98 2019-2023
    CrossrefPubMedSearch in Google Scholar
  • 18 Liu YM, Lacorte JM, Viguerie N, Poitou C, Pelloux V, Guy-Grand B, Coussieu C, Langin D, Basdevant A, Clement K. Adiponectin gene expression in subcutaneous adipose tissue of obese women in response to short-term very low calorie diet and refeeding.  J Clin Endocrinol Metab. 2003;  88 5881-5886
    CrossrefPubMedSearch in Google Scholar
  • 19 Schoof E, Stuppy A, Harig F, Carbon R, Horbach T, Stohr W, Rascher W, Dotsch J. Comparison of leptin gene expression in different adipose tissues in children and adults.  Eur J Endocrinol. 2004;  150 579-584
    CrossrefPubMedSearch in Google Scholar
  • 20 Gabrielsson BG, Johansson JM, Lonn M, Jernas M, Olbers T, Peltonen M, Larsson I, Lonn L, Sjostrom L, Carlsson B, Carlsson LM. High expression of complement components in omental adipose tissue in obese men.  Obes Res. 2003;  11 699-708
    CrossrefPubMedSearch in Google Scholar
  • 21 Huan JN, Li J, Han Y, Chen K, Wu N, Zhao AZ. Adipocyte-selective reduction of the leptin receptors induced by antisense RNA leads to increased adiposity, dyslipidemia, and insulin resistance.  J Biol Chem. 2003;  278 45638-45650
    CrossrefPubMedSearch in Google Scholar
  • 22 Engeli S, Bohnke J, Feldpausch M, Gorzelniak K, Heintze U, Janke J, Luft FC, Sharma AM. Regulation of 11beta-HSD genes in human adipose tissue: influence of central obesity and weight loss.  Obes Res. 2004;  12 9-17
    PubMedSearch in Google Scholar
  • 23 Blouin K, Richard C, Belanger C, Dupont P, Daris M, Laberge P, Luu-The V, Tchernof A. Local androgen inactivation in abdominal visceral adipose tissue.  J Clin Endocrinol Metab. 2003;  88 5944-5950
    CrossrefPubMedSearch in Google Scholar
  • 24 Vohl MC, Sladek R, Robitaille J, Gurd S, Marceau P, Richard D, Hudson TJ, Tchernof A. A survey of genes differentially expressed in subcutaneous and visceral adipose tissue in men.  Obes Res. 2004;  12 1217-1222
    PubMedSearch in Google Scholar
  • 25 Sjoholm K, Palming J, Olofsson LE, Gummesson A, Svensson PA, Lystig TC, Jennische E, Brandberg J, Torgerson JS, Carlsson B, Carlsson LM. A microarray search for genes predominantly expressed in human omental adipocytes: adipose tissue as a major production site of serum amyloid A.  J Clin Endocrinol Metab. 2005;  90 2233-2239
    CrossrefPubMedSearch in Google Scholar
  • 26 Minehira K, Vega N, Vidal H, Acheson K, Tappy L. Effect of carbohydrate overfeeding on whole body macronutrient metabolism and expression of lipogenic enzymes in adipose tissue of lean and overweight humans.  Int J Obes Relat Metab Disord. 2004;  28 1291-1298
    CrossrefPubMedSearch in Google Scholar
  • 27 Carlsson E, Fredriksson J, Groop L, Ridderstrale M. Variation in the calpain-10 gene is associated with elevated triglyceride levels and reduced adipose tissue messenger ribonucleic acid expression in obese Swedish subjects.  J Clin Endocrinol Metab. 2004;  89 3601-3605
    CrossrefPubMedSearch in Google Scholar
  • 28 Linscheid P, Seboek D, Zulewski H, Keller U, Muller B. Autocrine/paracrine role of inflammation-mediated calcitonin gene-related peptide and adrenomedullin expression in human adipose tissue.  Endocrinology. 2005;  146 2699-2708
    CrossrefPubMedSearch in Google Scholar
  • 29 Hammarstedt A, Jansson PA, Wesslau C, Yang X, Smith U. Reduced expression of PGC-1 and insulin-signaling molecules in adipose tissue is associated with insulin resistance.  Biochem Biophys Res Commun. 2003;  301 578-582
    CrossrefPubMedSearch in Google Scholar
  • 30 Gabrielsson BG, Olofsson LE, Sjogren A, Jernas M, Elander A, Lonn M, Rudemo M, Carlsson LM. Evaluation of reference genes for studies of gene expression in human adipose tissue.  Obes Res. 2005;  13 649-652
    CrossrefPubMedSearch in Google Scholar
  • 31 Giusti V, Verdumo C, Suter M, Gaillard RC, Burckhardt P, Pralong F. Expression of peroxisome proliferator-activated receptor-gamma1 and peroxisome proliferator-activated receptor-gamma2 in visceral and subcutaneous adipose tissue of obese women.  Diabetes. 2003;  52 1673-1676
    CrossrefPubMedSearch in Google Scholar
  • 32 Selvey S, Thompson EW, Matthaei K, Lea RA, Irving MG, Griffiths LR. Beta-actin-an unsuitable internal control for RT-PCR.  Mol Cell Probes. 2001;  15 307-311
    CrossrefPubMedSearch in Google Scholar
  • 33 Lupberger J, Kreuzer KA, Baskaynak G, Peters UR, Coutre P, Schmidt CA. Quantitative analysis of beta-actin, beta-2-microglobulin and porphobilinogen deaminase mRNA and their comparison as control transcripts for RT-PCR.  Mol Cell Probes. 2002;  16 25-30
    CrossrefPubMedSearch in Google Scholar
  • 34 Warrington JA, Nair A, Mahadevappa M, Tsyganskaya M. Comparison of human adult and fetal expression and identification of 535 housekeeping/maintenance genes.  Physiol Genomics. 2000;  2 143-147
    PubMedSearch in Google Scholar
  • 35 Thellin O, Zorzi W, Lakaye B, Borman B De, Coumans B, Hennen G, Grisar T, Igout A, Heinen E. Housekeeping genes as internal standards: use and limits.  J Biotechnol. 1999;  75 291-295
    CrossrefPubMedSearch in Google Scholar
  • 36 Bustin SA. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems.  J Mol Endocrinol. 2002;  29 23-39
    CrossrefPubMedSearch in Google Scholar
  • 37 Bustin SA, Benes V, Nolan T, Pfaffl MW. Quantitative real-time RT-PCR - a perspective.  J Mol Endocrinol. 2005;  34 597-601
    CrossrefPubMedSearch in Google Scholar
  • 38 Tricarico C, Pinzani P, Bianchi S, Paglierani M, Distante V, Pazzagli M, Bustin SA, Orlando C. Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies.  Anal Biochem. 2002;  309 293-300
    CrossrefPubMedSearch in Google Scholar
  • 39 Einstein FH, Atzmon G, Yang XM, Ma XH, Rincon M, Rudin E, Muzumdar R, Barzilai N. Differential responses of visceral and subcutaneous fat depots to nutrients.  Diabetes. 2005;  54 672-678
    CrossrefPubMedSearch in Google Scholar
  • 40 Giusti V, Suter M, Verdumo C, Gaillard RC, Burckhardt P, Pralong FP. Molecular determinants of human adipose tissue: differences between visceral and subcutaneous compartments in obese women.  J Clin Endocrinol Metab. 2004;  89 1379-1384
    CrossrefPubMedSearch in Google Scholar
  • 41 Lefebvre AM, Laville M, Vega N, Riou JP, Gaal L, Auwerx J, Vidal H. Depot-specific differences in adipose tissue gene expression in lean and obese subjects.  Diabetes. 1998;  47 98-103
    CrossrefPubMedSearch in Google Scholar
  • 42 Wells JC, Fuller NJ. Precision of measurement and body size in whole-body air-displacement plethysmography.  Int J Obes Relat Metab Disord. 2001;  25 1161-1167
    CrossrefPubMedSearch in Google Scholar
  • 43 Barber RD, Harmer DW, Coleman RA, Clark BJ. GAPDH as a housekeeping gene: analysis of GAPDH mRNA expression in a panel of 72 human tissues.  Physiol Genomics. 2005;  21 389-395
    CrossrefPubMedSearch in Google Scholar
  • 44 Harmelen V, Elizalde M, Ariapart P, Bergstedt-Lindqvist S, Reynisdottir S, Hoffstedt J, Lundkvist I, Bringman S, Arner P. The association of human adipose angiotensinogen gene expression with abdominal fat distribution in obesity.  Int J Obes Relat Metab Disord. 2000;  24 673-678
    CrossrefPubMedSearch in Google Scholar
  • 45 Simon MF, Daviaud D, Pradere JP, Gres S, Guigne C, Wabitsch M, Chun J, Valet P, Saulnier-Blache JS. Lysophosphatidic acid inhibits adipocyte differentiation via lysophosphatidic acid 1 receptor-dependent down-regulation of peroxisome proliferator-activated receptor gamma2.  J Biol Chem. 2005;  280 14656-14662
    CrossrefPubMedSearch in Google Scholar
  • 46 Radonic A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A. Guideline to reference gene selection for quantitative real-time PCR.  Biochem Biophys Res Commun. 2004;  313 856-862
    CrossrefPubMedSearch in Google Scholar
  • 47 Solanas M, Moral R, Escrich E. Unsuitability of using ribosomal RNA as loading control for Northern blot analyses related to the imbalance between messenger and ribosomal RNA content in rat mammary tumors.  Anal Biochem. 2001;  288 99-102
    CrossrefPubMedSearch in Google Scholar
  • 48 Rolland V, Liepvre X Le, Houbiguian ML, Lavau M, Dugail I. C/EBP alpha expression in adipose tissue of genetically obese Zucker rats.  Biochem Biophys Res Commun. 1995;  207 761-767
    CrossrefPubMedSearch in Google Scholar
  • 49 Rolland V, Dugail I, Liepvre X Le, Lavau M. Evidence of increased glyceraldehyde-3-phosphate dehydrogenase and fatty acid synthetase promoter activities in transiently transfected adipocytes from genetically obese rats.  J Biol Chem. 1995;  270 1102-1106
    CrossrefPubMedSearch in Google Scholar
  • 50 Barroso I, Benito B, Garci-Jimenez C, Hernandez A, Obregon MJ, Santisteban P. Norepinephrine, tri-iodothyronine and insulin upregulate glyceraldehyde-3-phosphate dehydrogenase mRNA during Brown adipocyte differentiation.  Eur J Endocrinol. 1999;  141 169-179
    CrossrefPubMedSearch in Google Scholar
  • 51 Zhu G, Chang Y, Zuo J, Dong X, Zhang M, Hu G, Fang F. Fudenine, a C-terminal truncated rat homologue of mouse prominin, is blood glucose-regulated and can up-regulate the expression of GAPDH.  Biochem Biophys Res Commun. 2001;  281 951-956
    CrossrefPubMedSearch in Google Scholar
  • 52 Zhong H, Simons JW. Direct comparison of GAPDH, beta-actin, cyclophilin, and 28S rRNA as internal standards for quantifying RNA levels under hypoxia.  Biochem Biophys Res Commun. 1999;  259 523-526
    CrossrefPubMedSearch in Google Scholar
  • 53 Ke LD, Chen Z, Yung WK. A reliability test of standard-based quantitative PCR: exogenous vs endogenous standards.  Mol Cell Probes. 2000;  14 127-135
    CrossrefPubMedSearch in Google Scholar
  • 54 Roche E, Assimacopoulos-Jeannet F, Witters LA, Perruchoud B, Yaney G, Corkey B, Asfari M, Prentki M. Induction by glucose of genes coding for glycolytic enzymes in a pancreatic beta-cell line (INS-1).  J Biol Chem. 1997;  272 3091-3098
    CrossrefPubMedSearch in Google Scholar
  • 55 Nasrin N, Ercolani L, Denaro M, Kong XF, Kang I, Alexander M. An insulin response element in the glyceraldehyde-3-phosphate dehydrogenase gene binds a nuclear protein induced by insulin in cultured cells and by nutritional manipulations in vivo.  Proc Natl Acad Sci USA. 1990;  87 5273-5277
    CrossrefPubMedSearch in Google Scholar
  • 56 Alexander MC, Lomanto M, Nasrin N, Ramaika C. Insulin stimulates glyceraldehyde-3-phosphate dehydrogenase gene expression through cis-acting DNA sequences.  Proc Natl Acad Sci USA. 1988;  85 5092-5096
    CrossrefPubMedSearch in Google Scholar
  • 57 Dugail I, Quignard-Boulange A, Bazin R, Liepvre X Le, Lavau M. Adipose-tissue-specific increase in glyceraldehyde-3-phosphate dehydrogenase activity and mRNA amounts in suckling pre-obese Zucker rats.  Effect of weaning. Biochem J. 1988;  254 483-487
    PubMedSearch in Google Scholar
  • 58 Gorzelniak K, Janke J, Engeli S, Sharma AM. Validation of endogenous controls for gene expression studies in human adipocytes and preadipocytes.  Horm Metab Res. 2001;  33 625-627
    Thieme ConnectPubMedSearch in Google Scholar

Correspondence

G. FrühbeckR Nutr, MD, PhD 

Department of Endocrinology

Clínica Universitaria de Navarra

University of Navarra

Avda. Pío XII, 36

31008 Pamplona

Spain

Phone: +34/948/25 54 00 (ext. 44 84)

Fax: +34/948/29 65 00

Email: gfruhbeck@unav.es