Gene Microarray Study Corroborates Proteomic Findings in Rodent Islet Cells Alessandra K. Cardozo,† Laurence Berthou,† Mogens Kruhøffer,‡ Torben Ørntoft,‡ Mark R. Nicolls,§ and Decio L. Eizirik*,† Laboratory of Experimental Medicine, Universite´ Libre de Bruxelles, 808 Route de Lennik, B-1070, Brussels, Belgium, Molecular Diagnostic Laboratory, Department of Clinical Biochemistry, Aarhus University Hospital, Skejby, Aarhus N, Denmark, and Division of Pulmonary Sciences and Critical Care Medicine and Biochemical Mass Spectrometric Facility, University of Colorado Health Sciences Center, Denver, CO, USA Received April 3, 2003
As reported in an issue of Journal of Proteome Research,1 mass spectrometry has been used to identify numerous proteins in pancreatic islets. Our group studies beta cell gene expression, and we were interested in whether proteins described in this study could be found at the level of the transcriptome. Microarray analysis is a powerful technique for quantitative measurements of the expression of thousand genes in parallel.2 However, in crude tumor biopsies, only a subset of transcripts correlate with protein levels,3 and it is still unknown how frequently mRNA expression correlates with amount of protein in well-differentiated cells. To address this issue, we presently compared data from mouse primary islet proteins obtained by proteomic analysis1 with RNA data from FACS purified primary rat beta cells obtained by microarray analysis4,5 (Rasschaert J, Liu D, Cardozo AK, Kutlu B, Eizirik DL, manuscript in preparation). Keywords: pancreatic islets • pancreatic beta cells • proteomic analysis • microarray analysis • diabetes mellitus • Alzheimer’s disease
Pancreatic islets were isolated from 10-week-old male Wistar rats by collagenase digestion, and islet beta cells were purified by autofluorescence-activated cell sorting (FACStar, BectonDickinson and Co., Sunnyvale, CA).6 FACS-purified beta cells were cultured in HAM’s F10 medium for 2 days prior to total RNA extraction. We used a pool of purified beta cells corresponding to 4-6 experiments (around 3 × 105 cells per group) for each independent microarray analysis. Array analysis was performed in duplicate using the U34-A rat array from Affymetrix (www.affymetrix.com). The present results on mRNA expression are means ( SEM of 5 independent microarray analysis of control beta cells4,5 (Rasschaert J, Liu D, Cardozo AK, Kutlu B, Eizirik DL, manuscript in preparation). A mRNA was considered as “expressed” based on hybridization to perfect match and mismatch probes, as described in detail in reference.7 The protein database at NCBI (pBlast) was used to find the rat orthologues of the described mouse proteins.1 On the basis of the rat protein accession numbers, a link to nucleotide database allowed us to find the nucleotide sequences related to these proteins. We then used the interactive
query from the Affymetrix site (www.affymerix.com), to verify whether these genes were included on the U34-A array. After this, we evaluated whether these mRNAs were considered as “expressed” based on 5 independent experiments (see above).
* To whom correspondence should be addressed. E-mail: deizirik@ ulb.ac.be. Fax: 32-2-555 62 39. † Laboratory of Experimental Medicine, Universite Libre de Bruxelles. ‡ Molecular Diagnostic Laboratory, Department of Clinical Biochemistry, Aarhus University Hospital. § Division of Pulmonary Sciences and Critical Care Medicine and Biochemical Mass Spectrometric Facility, University of Colorado Health Sciences Center.
An interesting finding from both the islet proteomic1 and microarray analysis4,5 (Rasschaert J, Liu D, Cardozo AK, Kutlu B, Eizirik DL, manuscript in preparation) is that beta cells express several neuronal related genes, including genes potentially involved in the pathogenesis of Alzheimer’s disease (Table 1). Moreover, besides the genes described in Table 1,
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2003 American Chemical Society
The rat orthologues from the mouse proteins were obtained using the protein database at NCBI and link to pBlast. Departing from the rat protein accession number, a link to nucleotide database provided us with the corresponding gene accession numbers. The accession numbers of 9 proteins were not found at NCBI because they were removed from these database due to standard genome annotation processing. Seventy nine rat orthologue genes were found from a total of 88 mouse proteins. From these 79 rat genes, 63 were present in the U34-A array and 60 were considered as expressed in FACS purified rat beta cells (Table 1), i.e., a correlation of 95% between mRNA data from rat beta cells and protein data from mouse islet cells. It is possible that missing mRNA data may reflect that beta cells are a subset of cells present in pancreatic islets and that some proteins identified in the proteomic study may be unique to nonbeta cell constituents of islets (e.g., alpha cells, delta cells).
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Table 1: Correlation between Proteins Expressed in Mouse Islets (previously described in ref 1) and mRNAs Expressed in FACS-Purified Rat Beta Cells Identified by Microarray Analysis4,5 (Rasschaert J, Liu D, Cardozo AK, Kutlu B, Eizirik DL, manuscript in preparation) acc N° protein
3219998 2506545 6671666 10946870 6671539 20861909 13637776 6753052 7304889 13277612 21263374 6680748 15214215 12831229 6680836 6753406 3287958 1169475 13626388 18202285 6679078 20831568 6752954 6680027 20825772 6679937 6680045 12848861 13937391 6679987 13624307 9789985 6678916 6678918 6680343 6679439 547923 20178035 20853786 20882127 7305371 129729 119531 6679237 6755074 7709980 20889049 20861909 20845230 20908717 20897385 6678359 6678469 7106439 6678619 9256646 13384794 6678559 6755963
acc N° mRNA
X54793 M14050 L11930 D10854 M12919 U73174 X02610 V01225 AI171167 D42137 J05210 X56133 D89514 U41853 X53363 X16054 L07281 M81088 AA860024 AA892801 M91597 J04473 X52815 AI179613 M91652 M17701 U88324 U03390 AA817892 X74401 M11942 J05031 AI010480 AF093773 AA891785 M19533 AI010083 S63233 X16554 M22631 M83745 X02918 M86870 U32314 AA818951 M15185 AI639479 U73174 AA892532 AA800250 AA859980 U09256 V01227 X03369 D30739 S55305 AA926137 U11760 AF048828
description
precursora
60 kDa heat shock protein, mitchondrial 78 kDa glucose-regulated protein precursor (GRP 78)a adenylyl cyclase-associated CAP protein Aldehyde reductase aldolase 1, A isoformb similar to aldose reductaseb alpha enolaseb amylase 2, pancreatic annexin A4; annexin IV annexin A5 ATP citrate (pro-S) lyaseb ATP synthase, H+ transporting mitochondrial F1, alpha subunit bifunctional purine biosynthesis protein PURH calcium binding protein, 140 kDa; 170 kDa GRP precursor; 150 kDa oxygen reg. prot. calreticulina carboxyl ester lipase carboxypeptidase H precursor elongation factor 1-alpha 1 elongation factor 1-gamma elongation factor 2b expressed in nonmetastatic cells 2, protein (NM23B) fumarate hydratase 1 actin, gamma, cytoplasmic glutamate dehydrogenaseb similar to glutamine synthetasea,b glyceraldehyde-3-phosphate dehydrogenase guanine nucleotide binding protein beta 1 subunitb guanine nucleotide binding protein beta 2 related sequence guanine nucleotide binding protein, beta 2 subunit guanosine diphosphate (GDP) dissociation inhibitor 3 GDI beta heat shock protein 70 kDaa isovaleryl coenzyme A dehydrogenaseb malate dehydrogenase, mitochondrialb malate dehydrogenase, soluble isocitrate dehydrogenase 2 (NADP+), mitochondrialb peptidylprolyl isomerase A; cyclophilin A peroxiredoxin 1a phosphoglycerate mutase 1b phosphoribosyl pyrophosphate synthetase 1a similar to propionyl CoA-carboxylase alpha-subunit proprotein convertase subtilisin/kexin type 1 protein disulfide isomerase precursora,b protein disulfide isomerase precursor A4 (Erp72)a pyruvate decarboxylaseb pyruvate kinase 3 S-adenosylhomocysteine hydrolase aldo-keto reductase family 1, member C12 similar to glutathione reductase 1b similar to protein disulfideisomerase-related protein [RIKEN cDNA 1700015E05] similar to succinate dehydrogenase complex, subunit A, flavoprotein T complex protein 1a Transketolase tubulin alpha 6 tubulin, beta 5b tyrosine 3-monooxygenase (tyrosine hydroxylase) [epsilon polypeptide] tyrosine 3-monooxygenase/tryptophan 5-monooxygenase [gamma polypeptide] ubiquinol - cytochrome c reductase core proteina valosin-containing proteina voltage dependent anion channel 1b
expression (mean)
SEM
1316 3190 188 1638 3496 90 2580 2011 116 371 3481 1179 80 357 632 216 3428 2517 1689 1889 2137 657 3057 925 941 3292 394 1954 831 635 2637 208 796 2714 498 1249 810 2828 123 70 835 2772 625 256 3420 524 76 90 2487 438 132 480 863 150 973 1349 147 1064 697
128 71 21 127 481 7 183 245 14 40 410 41 6 34 84 50 529 270 153 330 108 42 288 54 42 715 26 123 29 60 119 14 59 127 27 122 89 207 13 7 78 225 59 29 746 61 7 7 144 25 12 24 89 16 66 100 12 85 85
Data are mean ( SEM of 5 different independent experiments, each analyzed by microarray in duplicate. a Genes potentially implicated in the pathogenesis of Alzheimer’s disease. b mRNA identified by more then one probe from the microarray.
several other neuronal-related genes were identified as expressed in beta cells. These include, among others, glutamate receptor, Pax-6, synapsin I, neurofilament proteins, neurodegeneration associated proteins, nicotinic acetylcoline receptor β4 subunit, N-methyl-D-aspartate receptor 1, synaptic glycoprotein, survival motor neuron, brain acyl-CoA synthethase II, neuron-specific protein PEP-19, and neuronatin alpha. The 554
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expression of similar genes in beta cells and neurons is probably due to the presence of concordant transcriptional activators8 (Isl-1, Pax-6, beta2) and/or absence of comparable transcriptional repressors9 (NRSF/REST) in both cell types. Of note, presenilin-1 mRNA expression was also identified in our array analysis of rat beta cells. Presenilin-1 and 2, AbetaPP and beta-site cleaving enzyme have been previously identified in
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Proteomic Findings in Rodent Islet Cells
pancreatic beta cells and have central importance in the development of amyloid deposits in Alzheimer’s disease10. In conclusion, the present data suggest a close correlation between basal mRNA and protein expression in pancreatic islets cells, as evaluated by proteomic and microarray analysis. Pancreatic beta cells express a large number of neuronal genes and proteins. It will be of interest to understand the function of these neuronal proteins in beta cells in both normal and pathogenic states.
References (1) Nicolls, M. R.; D’Antonio, J. M.; Hutton, J. C.; Gill, R. G.; Czwornog, J. L.; Duncan, M. W. J. Proteome Res. 2003, 2, 199-205. (2) Lipshutz, R. J.; Fodor, S. P.; Gingeras, T. R.; Lockhart, D. J. Nat. Genet. 1999, 21, 20-24.
(3) Orntoft, T. F.; Thykjaer, T.; Waldman, F. M.; Wolf, H.; Celis, J. E. Mol. Cell. Proteomics 2002, 1, 37-45. (4) Cardozo, A. K.; Kruhoffer, M.; Leeman, R.; Orntoft, T.; Eizirik, D. L. Diabetes 2001, 50, 909-920. (5) Cardozo, A. K.; Heimberg, H.; Heremans, Y.; Leeman, R.; Kutlu, B.; Kruhoffer, M.; Orntoft, T.; Eizirik, D. L. J. Biol. Chem. 2001, 276, 48 879-48 886. (6) Pipeleers, D. G.; in't Veld, P. A.; Van de Winkel, M.; Maes, E.; Schuit, F. C.; Gepts, W. Endocrinology 1985, 117, 806-816. (7) Affymetrix Inc. GeneChip Analysis Suite verion 3.3 User Guide 1999, 478-479. (8) Yamaoka, T.; Itakura, M. Int. J. Mol. Med. 1999, 3, 247-261. (9) Atouf, F.; Czernichow, P.; Scharfmann, R. J. Biol. Chem. 1997, 272, 1929-1934. (10) Figueroa, D. J.; Shi, X. P.; Gardell, S. J.; Austin, C. P. J. Alzheimers Dis. 2001 3, 393-396.
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