Accumulation of triglyceride in islets may contribute to the loss of glucose-stimulated insulin secretion (GSIS) in some forms of type 2 diabetes (Diraison et al., Biochem J 373:769–778, 2004). Here, we use adenoviral vectors and oligonucleotide microarrays to determine the effects of the forced expression of SREBP1c on the gene expression profile of rat islets. Sterol regulatory element binding protein-1c (SREBP1c) overexpression led to highly significant (P < 0.1 with respect to null adenovirus) changes in the expression of 1,238 genes or expressed sequence tags, of which 1,180 (95.3%) were upregulated. By contrast, overexpression of constitutively active AMP-activated protein kinase (AMPK), expected to promote lipolysis, altered the expression of 752 genes, of which 702 (93%) were upregulated. To identify specific targets for SREBP1c or AMPK, we eliminated messages that were 1) affected in the same direction by the expression of either protein, 2) changed by less than twofold, or 3) failed a positive false discovery test; 206 SREBP1c-regulated genes (195; 95% upregulated) and 48 AMPK-regulated genes (33; 69% upregulated) remained. As expected, SREBP1c-induced genes included those involved in cholesterol (6), fatty acid (3), and eicosanoid synthesis. Interestingly, somatostatin receptor (sstr1) expression was increased by SREBP1c, whereas AMPK induced the expression of peptide YY, the early endocrine pancreas marker.

Sterol regulatory element binding proteins (SREBPs) including the splice variants SREBP1a and SREBP1c, as well as SREBP2 (encoded by a distinct gene), are involved in the regulation of fatty acid and cholesterol synthesis in a variety of mammalian tissues (1). Members of the basic helix-loop-helix leucine zipper (bHLH-Zip) family of transcription factors, SREBPs, are synthesized as an endoplasmic reticulum-bound precursor that is proteolytically processed in the presence of an SREBP-cleavage activating protein (SCAP) to release the active, nuclearly targeted NH2-terminal domain (1,2).

Pancreatic islets and β-cells express the SREBP1c isoform exclusively, under the control of ambient glucose concentrations (35). Forced overexpression of SREBP1c leads to the accumulation of triglyceride and inhibition of glucose-stimulated insulin secretion (GSIS) from both pancreatic islets (6) and β-cell lines (3,4,7). This is associated with the accumulation of mRNAs encoding fatty acid synthase and acetyl-CoA carboxylase I as well as peroxisome proliferator-activated receptor-γ and uncoupling protein-2 (UCP2) (1.4-fold) (6). The latter change may contribute to decreases in glucose oxidation and glucose-induced increases in intracellular ATP content in SREBP1c overexpressing β-cells, consistent with a role for SREBP1c-mediated increases in fatty acid and triglyceride synthesis under conditions of “lipotoxicity” (8) or “glucolipotoxicity” (9). On the other hand, no changes in the expression of the key β-cell transcription factor pancreatic duodenum homeobox-1 (PDX-1) or in the glucose transporter, Glut2 or glucokinase, were apparent after overexpression of SREBP1c in islets (6), although these genes were reported to be downregulated by SREBP1c in a study of candidate genes in the INS-1 cell line (4).

While many SREBP target genes have recently been defined in a combinatorial analysis of gene profiles of livers from SREBP2 and SREBP1a-expressing mice (10), no information on the targets for SREBP1c in the islet or β-cell are presently available at the transcriptome level. Nevertheless, some of the genes identified as SREBP targets in the liver study, including fatty acid synthase and acetyl-CoA carboxylase, were also upregulated by SREBP1c in β-cells (3,4) and in islets (6), suggesting that this may represent a useful approach to understanding the mechanisms by which SREBP1c overexpression inhibits insulin secretion from islets.

AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase, activated by phosphorylation on threonine-172 (11) when the cellular energy charge is low and the AMP/ATP ratio is high (12). In direct contrast to the effects of SREBP1, AMPK activation is associated with the suppression of lipid synthesis and the activation of fatty acid oxidation. Targets for phosphorylation and inhibition by AMPK include enzymes involved in cholesterol (hydoxymethylglutaryl-CoA reductase) and fatty acid (acetyl-CoA carboxylase) synthesis (12). Moreover, activation of AMPK suppresses the expression of lipogenic genes in the liver (13). Increases in the concentrations of glucose (1417) or amino acids (18) inhibit AMPK activity in islet β-cells, while metformin (17) activates the enzyme in islets. Forced increases in AMPK activity inhibit the expression of the preproinsulin and liver-type pyruvate kinase gene (15) in clonal β-cells, and inhibit insulin secretion acutely (7,1618).

Here, we compare the effects on the gene expression profile of transducing pancreatic islets with adenoviral forms of activated SREBP1c (6) or AMPK (16).

Materials.

Collagenase (type V) was obtained from Sigma (Poole, Dorset, U.K.). Culture medium, fetal bovine serum (FBS), and glutamine were obtained from Gibco (Glasgow, U.K.). Histopaque solutions and antibiotics were from Sigma.

Islet preparation and culture.

Male Wistar rats (200–225 g) were sacrificed by cervical dislocation. Islets were isolated by pancreatic distension as described previously (6) and purified using Histopaque gradient solutions (10 ml of 1.119 g/l, 6 ml of 1.083 g/l, 6 ml of 1.077 g/l). After centrifugation for 20 min at 1,000g, islets were removed from the top layer and washed once with Dulbecco’s modified Eagle’s medium (DMEM). Isolated islets were cultured in suspension for 16 h in DMEM containing 30% (vol/vol) FBS, 11 mmol/l glucose, 2 mmol/l glutamine, 100 IU/ml penicillin, and 100 mg/ml streptomycin and were incubated at 37°C with 95% air and 5% CO2.

Amplification of recombinant adenoviruses and islet infection.

Adenoviruses encoding constitutively active SREBP1c (amino acids 1–403, wild-type sequence) (3), constitutively active AMPK (16), or only enhanced green fluorescent protein (eGFP; “Null” virus) were amplified as described (6). SREBP1c and AMPK adenoviruses also express eGFP, under a distinct CMV promoter. Virus particles were purified on cesium chloride gradients before infection at a multiplicity of infection (MOI) of 30 (SREBP1c) or 100 (AMPK) viral particles per cell (6). Islets were infected with adenoviruses the day after isolation and cultured for 24 h in DMEM containing 10% FBS and 11 mmol/l glucose and were then cultured for a further 32 h in DMEM containing 3 mmol/l glucose before RNA extraction. Triplicate experiments were performed for each condition.

Microarray analysis.

Total RNA (at least 5 μg/sample) was extracted from rat islets (1,000 islets/condition) using TRIzol reagent (Invitrogen Life Technologies, Paisley, U.K.) according to Affymetrix recommendations (Affymetrix, Santa Clara, CA). Processing of total RNA (5 μg) was performed at the Bioinformatics Facility of the University of Wales College of Medicine at Cardiff (http://www.cf.ac.uk/biosi/research/molbiol/Arrayer/index.html), as described (AffymetrixGenechip Expression Analysis Manual; Affymetrix).

Quality of total RNA was first controlled using an AgilentChip (Agilent Technologies, West Lothian, U.K.). Double-stranded cDNA was synthesized using a T7-oligo(dt) primer. The resultant cDNA was purified using Phase Lock Gel phenol/chloroform extraction and precipitation with ethanol. Biotin-labeled cRNA was synthesized using an RNA Transcript labeling kit (Enzo BioarrayHigh Yield; Enzo Life Sciences, Farmingdale, NY). In vitro transcription reactions were carried out at 37°C for 5 h, and the labeled cRNA obtained purified using RNeasy columns (Qiagen). The cRNA was fragmented in fragmentation buffer for 35 min at 94°C. Fragmented cRNA (10–11 μg/probe array) was used to hybridize to rat 230A GeneChip arrays (Affymetrix) at 45°C for 24 h in a hybridization oven with constant rotation (60 rpm). The chips were then washed and stained using Affymetrix fluidics stations. Staining was performed using streptavidin phycoerythrin conjugate (SAPE; Molecular Probes, Eugenes, OR), followed by the addition of biotinylated antibody to streptavidin (Vector Laboratories, Burlingame, CA). Probe arrays were scanned using fluorometric scanners. The scanned images were inspected and analyzed using established quality control measures.

MAS 5.0 software (Affymetrix) was used to obtain an expression signal and a present/absent/medium call status for every probe set on each of the hybridized chips. Probe sets with an absent/medium call in two of the three replicates in at least one of the two states (Treatment and Control) were removed from the subsequent analysis.

Log2-transformation of the data were performed (19) to allow the normalization of intensities and before statistical Student’s t test (two tailed; assuming unequal variances). Positive false discovery rates (pFDR) were calculated as described (20).

Semiquantitative RT-PCR.

One microgram of total RNA, prepared as described above, was reverse-transcribed. First-strand cDNA synthesis was performed as described (21) using oligo (15)-dT primers and reverse transcriptase (500 units Moloney-murine leukemia virus; Promega) in a total volume of 50 μl. PCR reactions were performed in a total volume of 50 μl, comprising 5 μl cDNA product, 0.2 mmol/l of each nucleotide triphosphate, 20 pmol of each primer, and 0.875 units Taq polymerase (Expand High Fidelity; Roche). Oligonucleotide primer sequences and PCR conditions used in this study are available upon request. PCR was performed at 95°C for 2 min, followed by determined cycles for each target gene at 95°C for 1 min, 1 min at the melting temperature (determined for each target gene; not shown), and 72°C for 1 min. The last cycle was followed by a final extension step at 72°C for 10 min.

PCR products were separated on 1% (wt/vol) agarose gels containing ethidium bromide (0.5 μg/ml) and quantitated by digital imaging (ImageQuant; Molecular Dynamics).

To identify SREBP1c-regulated genes in isolated pancreatic islets, we used adenoviruses to express a recombinant, truncated, and constitutively active form of SREBP1c, comprising the nuclear NH2-terminal domain (amino acids 1–403) (1). As described in detail elsewhere (6), infection with this virus leads to the expression of SREBP1c in 20–30% of cells, the majority (>70%) being insulin-positive β-cells at the islet periphery.

After transduction with a SREBP1c-encoding or null (eGFP-encoding) adenoviruses and culture for 32 h at 3 mmol/l glucose, 8,748 of a total of 15,923 probe sets were retained for analysis. In comparison to null adenovirus-infected islets, SREBP1c affected the expression of 2,992 genes significantly (P < 0.05); of these, 1,238 genes were altered highly significantly (P < 0.01). Of the latter group, the vast majority (1,180, or 95.3%) were upregulated. By contrast, overexpression of a constitutively active form of AMPK catalytic subunit (amino acids 1–312, T172D, corresponding to the identical NH2-terminal domains of AMPKα1 and α2) (22) altered the expression of 2,531 genes significantly (P < 0.05) and 752 genes highly significantly (P < 0.01). Of these, 702 (93.4%) were upregulated.

To identify genes that may be direct targets for SREBP1c in the islet, as opposed to genes whose expression results from the indirect effects of lipid accumulation in response to transduction with SREBP1 (3,6), we next applied three further criteria to increase the stringency of the search. Specifically, genes were included only if their expression 1) changed highly significantly (P < 0.01) in response to SREBP1c overexpression but did not change significantly and in the same direction in response to both SREBP1c and AMPK overexpression (since AMPK overexpression also inhibits insulin secretion and glucose-stimulated increases in intracellular ATP [16,22] this allowed correction for the effects of SREBP1c on these parameters); 2) was altered by at least 2.0-fold (increased or decreased) by either SREBP1c or AMPK; and 3) passed a positive false discovery rate test (P < 0.05). This led to the identification of 206 genes that were regulated by SREBP1c (Table 1); of these, 195 (94.6%) were increased. Within this group, six genes were upregulated by SREBP1c and downregulated by AMPK. Of the SREBP1c-regulated genes identified, 82 were identified functionally (Table 1) using Pathway Assist (Stratagene) software.

Genes affected highly significantly by either activated SREBP1c or AMPK are illustrated in Fig. 1. Of those affected by SREBP1c, the majority were involved in either metabolism or metabolic signaling, with smaller numbers involved in growth or apoptosis. Interestingly, 7% of SREBP1c-stimulated genes were involved in the immune response, an observation that may reflect either the presence of immune cells with the isolated islets or the activation of these genes in endocrine cells, or both. Genes involved in the neural regulation of insulin secretion were among those strongly affected by SREBP1c, including receptors for the inhibitory neuropeptides somatostatin (sstr1) and galanin (Table 1). These and other changes identified in the microarrays were also verified by semiquantitative RT-PCR analysis (underlined in Tables 1 and 2).

A high-stringency analysis, as described above, revealed that of 48 genes affected uniquely by AMPK overexpression, 33 (70%) were highly significantly upregulated. As shown in Table 2 and Fig. 1B, a greater proportion of AMPK-affected genes were involved in cellular functions, including adhesion and growth. It should be stressed, however, that under the conditions used here (3 mmol/l glucose) AMPK is significantly activated in β-cells (1418,22) such that further increases in AMPK activity as a result of adenoviral transduction may be relatively small (22). Future studies will be required to explore the effects of changes in AMPK activity over the physiological range of glucose concentrations (17).

As observed in analyses of livers from transgenic mice overexpressing SREBP1a or SREBP2 (10), expression of SREBP1c in isolated rat islets led to changes in the expression of a large number of genes (>1,200, representing 14% of all genes called present in the islet preparations used). It seems likely that many of these are changed as a result of the nonspecific effects of lipid accumulation and the consequent metabolic changes (10). Thus, SREBP1c expression in either clonal β-cells (3) or primary rat islets (6) leads to a decrease in ATP levels and to an inhibition of insulin secretion. To control for these changes, we used as a control condition islets overexpressing AMPK. While the effects of AMPK expression on lipid synthesis are likely to be directly opposed to those of SREBP1c (12), the expression of AMPK also leads to a reduction in glucose oxidation and GSIS (1618,22). Thus, by this combinatorial approach, we sought to identify genes likely to be under the direct control of SREBP1c.

In contrast to the changes observed after SREBP1c expression (see below), the effects of AMPK overexpression were relatively modest with the levels of only three genes (prostaglandin E synthase, lipoprotein lipase, and peptide YY precursor) changing by >3.0-fold. Further studies will be required to determine whether the induction of PPY, a neuropeptide present at very early stages in islet development (23), may reflect dedifferentiation of the islet cell population.

The findings reported here in respect to the effects of SREBP1c overexpression are in broad agreement with those reported for the livers of transgenic SREBP-expressing mice, including genes involved in cholesterol and fatty acid biosynthesis (Table 1). However, other, unsuspected SREBP1c-regulated genes were also identified in islets. These include the glycine transporter; solute transporter family 6, member 6 (NM_017206; Table 1); and receptors for the inhibitory neuropeptides somatostatin (receptor 1, sstr1, NM_012719) and galanin (receptor 2, NM_019172), which were not detectably expressed in the liver (10). Because sstr1 (24) is localized largely to β-cells within the islet, upregulation of this receptor may render islets more susceptible to inhibition by endogenous somatostatin. By contrast, type 2 galanin receptors (NM_019172) are usually linked to phospholipase C activation and the activation of insulin release (25).

We did not, however, observe any significant effect of SREBP1c overexpression on the levels of mRNAs encoding a number of key β-cell transcription factors such as pdx-1 or Nkx6.1, or on the key “glucose sensing” genes glut2/slc2a2 or glucokinase, in line with previous studies in islets (6) but in contrast to INS-1 cells (4). Moreover, we detected no significant changes in the expression of acetyl-CoA carboxylase I, consistent with only very small changes observed previously observed by RT-PCR (6). Finally, no large changes in the expression of mitochondrial enzymes were detected, although UCP2 mRNA levels were increased 2.0-fold, albeit at the limit of significance (P = 0.025) (6).

We also detected changes in the expression of a number of proapoptotic genes (6). These included GADD45β, the p75-like apoptosis-inducing death domain protein, PLAIDD, and prostaglandin D2 synthase. On the other hand, expression of T-cell death-associated gene and BAX inhibitor-1 (Table 1) were decreased in SREBP1c-infected islets. Further exploration of the impact of SREBP1c on islet cell apoptosis may thus be warranted given reports of decreases in β-cell mass in type 2 diabetes (26).

FIG. 1.

The functional distribution of gene clusters affected by overexpression of SREBP1c (A) or AMPK (B) in pancreatic islets. See text for further details.

FIG. 1.

The functional distribution of gene clusters affected by overexpression of SREBP1c (A) or AMPK (B) in pancreatic islets. See text for further details.

TABLE 1

Genes up- or downregulated in active SREBPI overexpressing rat islets compared with Null virus-infected islets

Affymetrix number/gene functionGenBank accession no.Gene/protein nameFold change
Metabolism    
    Steroid biosynthesis    
        1382492 a at AA866404 Estradiol 17 beta-dehydrogenase 8† 2.64 
    Proteolysis    
        1367651_at NM_134334 Cathepsin D 2.01 
        1370220_at BI283159 Retinoid-inducible serine carboxypeptidase 2.15 
        1387213_at NM_133559 Proprotein convertase subtilisin/kexin type 4 3.34 
    Phospholipid synthesis    
        1387900_at D82928 CDP-diacylglycerol-inositol 3-phosphatidyltransferase 2.12 
    Carbohydrate metabolism    
        1370870_at M30596 Malic enzyme 1 3.10 
    Cholesterol biosynthesis and uptake    
        1386990_at NM_057137 Phenylalkylamine Ca2+ antagonist (emopamil) binding protein 2.81 
        1367662_at NM_031682 Hydroxysteroid (17-beta) dehydrogenase 10 2.26 
        1368878_at NM_053539 Isopentenyl-diphosphate delta isomerase 2.57 
        1367667_at NM_031840 Testis-specific farnesyl pyrophosphate synthetase 2.36 
        1387017_at NM_017136 Squalene epoxidase 2.51 
        1376089_at B1294974 Low density lipoprotein receptor 3.00 
    Fatty acid synthesis    
        1367707_at NM_017332 Fatty acid synthase 7.68 
        1388108_at BE116152 Fatty acid elongase 2 4.25 
        1387630_at NM_134382 Fatty acid elongase 1 2.05 
    Eicosanoid synthesis    
        1367851 at J04488 Prostaglandin D2 synthase 12.78 
    Glycerolipid metabolism    
        1371793_at AI008697 Acetylcholinesterase 2.54 
    Glycosaminoglycan metabolism    
        1371894_at AI409037 N-acetylglucosamine-6-sulfatase (EC 3.1.6.14)* 2.17 
    Lipid metabolism    
        1371012_at AJ245707 2-hydroxyphytanoyl-CoA lyase 2.14 
        1371748 BI284306 Iysophosphatidic acid acyltransferase alpha* 2.10 
    Polyphosphate metabolism    
        1388126_at BG380493 Multiple inositol polyphosphate histidine phosphatase, 1 2.14 
    Protein amino acid dephosphorylation    
        1387444_at NM_133592 Brain-enriched membrane-associated protein tyrosine BEM-2 2.66 
Transcription regulation    
        1368122_at NM_053438 Zinc finger protein 103 2.08 
        1368247_at NM_031971 Heat shock 70kD protein 1A 0.17 
        1368489_at NM_012953 Fos-like antigen 1 0.44 
        1373439_at AI178491 Lamin B receptor 2.29 
        1386910_a_at AF311054 Apurinic/apyrimidinic endonuclease 1 2.46 
        1370912_at BI278231 Hsp70.2 mRNA for heat shock protein 70 0.15 
        1371907_at BE101624 Nuclear protein SR-25* 2.10 
        1374119_at BI279615 E74-like factor 3* 2.77 
        1391454_at BF286105 HCF-binding transcription factor Zhangfei* 2.09 
    Ribosomal proteins    
        1372094_at BG380556 Ribosomal protein L18 2.03 
    Signal transduction    
        1368495_at NM_013413 Relaxin 1 3.94 
        1368185_at NM_013189 Guanine nucleotide binding protein, alpha z subunit 2.34 
Growth    
        1367894_at NM_022392 Growth response protein (CL-6) 4.79 
        1368056_at U24150 Tuberous sclerosis 2 2.09 
        1370747_at D14839 Fibroblast growth factor 9 2.01 
        1368109_at NM_031337 Sialyltransferase 9 2.86 
        1370989_at AI639318 Ret proto-oncogene 4.31 
        1370163 NM_012615 Ornithine decarboxylase 1 2.10 
    Epidermal differentiation    
        1388506_at AW144509 Desmoplakin (DP)* 2.30 
Immune response    
        1387396_at NM_053469 Hepcidin antimicrobial peptide 3.53 
        1371213_at AJ005023 Histocompatibility 2, Q region locus 10 2.40 
        1372070_at BM389261 Gamma-interferon inducible lysosomal thiol reductase precursor* 2.85 
        1388255 NM_012645 RT1 class Ib gene (Aw2) 2.50 
    Metalloproteinase    
        1398275_at NM_031055 Matrix metalloproteinase 9 0.17 
        1388204_at M60616 Matrix metalloproteinase 13 0.21 
Cell adhesion    
        1368269_at NM_012975 Lectin, galactose binding, soluble 4 3.92 
        1390366_at BI291884 Protocadherin LKC precursor* 3.54 
        1386947_at NM_031334 Cadherin 1 2.28 
    Platelet adhesion    
        1386955 NM_053930 Glycoprotein Ib (platelet), beta polypeptide 2.80 
Apoptosis    
        1368860_at NM_017180 T-cell death-associated gene 0.32 
        1377080_at AI598730 p75-like apoptosis-inducing death domain protein PLAIDD 2.52 
        1371309_at BI276999 Testis enhanced gene transcript (BAX inhibitor 1) 2.17 
        1370694_at AB020967 SKIP3 0.33 
        1367478_at AI600136 Serine protease HTRA2* 2.55 
    Activation of map kinase/apoptosis    
        1372016_at BI287978 GADD45 beta* 13.40 
Cytoarchitecture    
    Cytoskeleton organization and biogenesis    
        1371530_at BF281337 Keratin 8 2.61 
    Metalloproteinase    
        1398275_at NM_031055 Matrix metalloproteinase 9 0.17 
        1388204_at M60616 Matrix metalloproteinase 13 0.21 
Intracellular signaling    
    Amino acid transporter activity    
        1368778_at NM_017206 Solute carrier family 6, member 6 2.24 
    Electron transport    
        1367705_at AF319950 Glutaredoxin 1 (thioltransferase) 2.28 
        1387414_at NM_024141 Dual oxidase 2 2.74 
        1389339_at BF284062 Thioredoxin-like 2* 2.24 
    Fatty acid transport    
        1367789_at NM_053580 Fatty acid transport protein 2.09 
    GPCRs Class A Rhodopsin-like    
        1368758_a_at NM_019172 Galanin receptor 2 4.31 
    G-protein signaling    
        1369770_at NM_012719 Somatostatin receptor 1 4.85 
        1368574_at NM_016991 Adrenergic receptor, alpha 1b 2.21 
        1368869_at BG663107 AKAP12/SSeCKS 6.18 
    Ion transport    
        1387104_at NM_031548 Sodium channel, nonvoltage-gated, type 1, alpha polypeptide 2.16 
        1368606_at NM_030838 Organic anion transporting polypeptide 3 3.10 
        1386911_at NM_012505 ATPase, Na+K+ transporting, alpha 2 4.38 
    Intracellular signaling cascade    
        1370041_at NM_053440 Stathmin-like 2 2.84 
        1369097_s_at NM_012769 Guanylate cyclase 1, soluble, beta 3 2.91 
        1374812_at AA818197 Protein tyrosine phosphatase non-receptor type 13 (Ptpn 13) 2.55 
    Microtubule-based processes    
        1388670_at BI286860 Kinesin-like protein KIF1A (Axonal transporter of synaptic vesicles)* 2.17 
    Protein folding    
        1370319_at U68544 Peptidylprolyl isomerase F2.52  
        1372462_at AI412322 t-complex protein 1 5.10 
    Protein binding    
        1372658_at AB091769 Desmuslin 3.40 
    Lipid transport    
        1372604_at BI289459 Apolipoprotein L, 3* 2.44 
        1367519_at BG373087 Oxysterol-binding protein-like protein 2* 2.19 
    MAPK cascade    
        1368964_at NM_030856 Leucine rich repeat protein 3, neuronal 2.22 
    Ubiquitin cycle    
        1374808_at BG375471 Hypothetical protein FLJ21156* 2.45 
Others    
        1367793_at NM_024131 D-dopachrome tautomerase 3.13 
        1367850_at NM_053843 Fc receptor, IgG, low affinity III 5.83 
        1370448_at L20468 Cerebroglycan 3.36 
        1387917_at U19614 Lamina-associated polypeptide IC 3.03 
        1370956_at BM390253 Decorin 0.11 
        1367793_at NM_024131 D-dopachrome tautomerase 3.13 
        1374107_at AI229668 ElaC homolog 2 2.35 
        1387356_at NM_031823 Wolfram syndrome 1 2.43 
        1371354_at AI710682 TPHUCC troponin C, cardiac and slow skeletal muscle* 2.10 
        1372607_at BM384157 Nucleotide binding protein 2* 2.69 
        1398962_at BM388453 CGI-67 protein* 2.67 
        1399068_at AA957069 Hypothetical protein FLJ22709* 2.12 
        1388327_at AI232357 Uncharacterized hematopoietic stem/progenitor cells protein MDS029* 2.05 
        1386160_at AI639401 Trichohyalin* 2.88 
        1377016_at BE106888 Hypothetical protein MGC11256* 3.20 
        1374292_at BF414124 Hypothetical protein FLJ22222* 2.16 
        1374109_at AI230466 Hypothetical protein MGC4618* 2.71 
        1374005_at AI233232 Hypothetical protein FLJ12436* 2.09 
Affymetrix number/gene functionGenBank accession no.Gene/protein nameFold change
Metabolism    
    Steroid biosynthesis    
        1382492 a at AA866404 Estradiol 17 beta-dehydrogenase 8† 2.64 
    Proteolysis    
        1367651_at NM_134334 Cathepsin D 2.01 
        1370220_at BI283159 Retinoid-inducible serine carboxypeptidase 2.15 
        1387213_at NM_133559 Proprotein convertase subtilisin/kexin type 4 3.34 
    Phospholipid synthesis    
        1387900_at D82928 CDP-diacylglycerol-inositol 3-phosphatidyltransferase 2.12 
    Carbohydrate metabolism    
        1370870_at M30596 Malic enzyme 1 3.10 
    Cholesterol biosynthesis and uptake    
        1386990_at NM_057137 Phenylalkylamine Ca2+ antagonist (emopamil) binding protein 2.81 
        1367662_at NM_031682 Hydroxysteroid (17-beta) dehydrogenase 10 2.26 
        1368878_at NM_053539 Isopentenyl-diphosphate delta isomerase 2.57 
        1367667_at NM_031840 Testis-specific farnesyl pyrophosphate synthetase 2.36 
        1387017_at NM_017136 Squalene epoxidase 2.51 
        1376089_at B1294974 Low density lipoprotein receptor 3.00 
    Fatty acid synthesis    
        1367707_at NM_017332 Fatty acid synthase 7.68 
        1388108_at BE116152 Fatty acid elongase 2 4.25 
        1387630_at NM_134382 Fatty acid elongase 1 2.05 
    Eicosanoid synthesis    
        1367851 at J04488 Prostaglandin D2 synthase 12.78 
    Glycerolipid metabolism    
        1371793_at AI008697 Acetylcholinesterase 2.54 
    Glycosaminoglycan metabolism    
        1371894_at AI409037 N-acetylglucosamine-6-sulfatase (EC 3.1.6.14)* 2.17 
    Lipid metabolism    
        1371012_at AJ245707 2-hydroxyphytanoyl-CoA lyase 2.14 
        1371748 BI284306 Iysophosphatidic acid acyltransferase alpha* 2.10 
    Polyphosphate metabolism    
        1388126_at BG380493 Multiple inositol polyphosphate histidine phosphatase, 1 2.14 
    Protein amino acid dephosphorylation    
        1387444_at NM_133592 Brain-enriched membrane-associated protein tyrosine BEM-2 2.66 
Transcription regulation    
        1368122_at NM_053438 Zinc finger protein 103 2.08 
        1368247_at NM_031971 Heat shock 70kD protein 1A 0.17 
        1368489_at NM_012953 Fos-like antigen 1 0.44 
        1373439_at AI178491 Lamin B receptor 2.29 
        1386910_a_at AF311054 Apurinic/apyrimidinic endonuclease 1 2.46 
        1370912_at BI278231 Hsp70.2 mRNA for heat shock protein 70 0.15 
        1371907_at BE101624 Nuclear protein SR-25* 2.10 
        1374119_at BI279615 E74-like factor 3* 2.77 
        1391454_at BF286105 HCF-binding transcription factor Zhangfei* 2.09 
    Ribosomal proteins    
        1372094_at BG380556 Ribosomal protein L18 2.03 
    Signal transduction    
        1368495_at NM_013413 Relaxin 1 3.94 
        1368185_at NM_013189 Guanine nucleotide binding protein, alpha z subunit 2.34 
Growth    
        1367894_at NM_022392 Growth response protein (CL-6) 4.79 
        1368056_at U24150 Tuberous sclerosis 2 2.09 
        1370747_at D14839 Fibroblast growth factor 9 2.01 
        1368109_at NM_031337 Sialyltransferase 9 2.86 
        1370989_at AI639318 Ret proto-oncogene 4.31 
        1370163 NM_012615 Ornithine decarboxylase 1 2.10 
    Epidermal differentiation    
        1388506_at AW144509 Desmoplakin (DP)* 2.30 
Immune response    
        1387396_at NM_053469 Hepcidin antimicrobial peptide 3.53 
        1371213_at AJ005023 Histocompatibility 2, Q region locus 10 2.40 
        1372070_at BM389261 Gamma-interferon inducible lysosomal thiol reductase precursor* 2.85 
        1388255 NM_012645 RT1 class Ib gene (Aw2) 2.50 
    Metalloproteinase    
        1398275_at NM_031055 Matrix metalloproteinase 9 0.17 
        1388204_at M60616 Matrix metalloproteinase 13 0.21 
Cell adhesion    
        1368269_at NM_012975 Lectin, galactose binding, soluble 4 3.92 
        1390366_at BI291884 Protocadherin LKC precursor* 3.54 
        1386947_at NM_031334 Cadherin 1 2.28 
    Platelet adhesion    
        1386955 NM_053930 Glycoprotein Ib (platelet), beta polypeptide 2.80 
Apoptosis    
        1368860_at NM_017180 T-cell death-associated gene 0.32 
        1377080_at AI598730 p75-like apoptosis-inducing death domain protein PLAIDD 2.52 
        1371309_at BI276999 Testis enhanced gene transcript (BAX inhibitor 1) 2.17 
        1370694_at AB020967 SKIP3 0.33 
        1367478_at AI600136 Serine protease HTRA2* 2.55 
    Activation of map kinase/apoptosis    
        1372016_at BI287978 GADD45 beta* 13.40 
Cytoarchitecture    
    Cytoskeleton organization and biogenesis    
        1371530_at BF281337 Keratin 8 2.61 
    Metalloproteinase    
        1398275_at NM_031055 Matrix metalloproteinase 9 0.17 
        1388204_at M60616 Matrix metalloproteinase 13 0.21 
Intracellular signaling    
    Amino acid transporter activity    
        1368778_at NM_017206 Solute carrier family 6, member 6 2.24 
    Electron transport    
        1367705_at AF319950 Glutaredoxin 1 (thioltransferase) 2.28 
        1387414_at NM_024141 Dual oxidase 2 2.74 
        1389339_at BF284062 Thioredoxin-like 2* 2.24 
    Fatty acid transport    
        1367789_at NM_053580 Fatty acid transport protein 2.09 
    GPCRs Class A Rhodopsin-like    
        1368758_a_at NM_019172 Galanin receptor 2 4.31 
    G-protein signaling    
        1369770_at NM_012719 Somatostatin receptor 1 4.85 
        1368574_at NM_016991 Adrenergic receptor, alpha 1b 2.21 
        1368869_at BG663107 AKAP12/SSeCKS 6.18 
    Ion transport    
        1387104_at NM_031548 Sodium channel, nonvoltage-gated, type 1, alpha polypeptide 2.16 
        1368606_at NM_030838 Organic anion transporting polypeptide 3 3.10 
        1386911_at NM_012505 ATPase, Na+K+ transporting, alpha 2 4.38 
    Intracellular signaling cascade    
        1370041_at NM_053440 Stathmin-like 2 2.84 
        1369097_s_at NM_012769 Guanylate cyclase 1, soluble, beta 3 2.91 
        1374812_at AA818197 Protein tyrosine phosphatase non-receptor type 13 (Ptpn 13) 2.55 
    Microtubule-based processes    
        1388670_at BI286860 Kinesin-like protein KIF1A (Axonal transporter of synaptic vesicles)* 2.17 
    Protein folding    
        1370319_at U68544 Peptidylprolyl isomerase F2.52  
        1372462_at AI412322 t-complex protein 1 5.10 
    Protein binding    
        1372658_at AB091769 Desmuslin 3.40 
    Lipid transport    
        1372604_at BI289459 Apolipoprotein L, 3* 2.44 
        1367519_at BG373087 Oxysterol-binding protein-like protein 2* 2.19 
    MAPK cascade    
        1368964_at NM_030856 Leucine rich repeat protein 3, neuronal 2.22 
    Ubiquitin cycle    
        1374808_at BG375471 Hypothetical protein FLJ21156* 2.45 
Others    
        1367793_at NM_024131 D-dopachrome tautomerase 3.13 
        1367850_at NM_053843 Fc receptor, IgG, low affinity III 5.83 
        1370448_at L20468 Cerebroglycan 3.36 
        1387917_at U19614 Lamina-associated polypeptide IC 3.03 
        1370956_at BM390253 Decorin 0.11 
        1367793_at NM_024131 D-dopachrome tautomerase 3.13 
        1374107_at AI229668 ElaC homolog 2 2.35 
        1387356_at NM_031823 Wolfram syndrome 1 2.43 
        1371354_at AI710682 TPHUCC troponin C, cardiac and slow skeletal muscle* 2.10 
        1372607_at BM384157 Nucleotide binding protein 2* 2.69 
        1398962_at BM388453 CGI-67 protein* 2.67 
        1399068_at AA957069 Hypothetical protein FLJ22709* 2.12 
        1388327_at AI232357 Uncharacterized hematopoietic stem/progenitor cells protein MDS029* 2.05 
        1386160_at AI639401 Trichohyalin* 2.88 
        1377016_at BE106888 Hypothetical protein MGC11256* 3.20 
        1374292_at BF414124 Hypothetical protein FLJ22222* 2.16 
        1374109_at AI230466 Hypothetical protein MGC4618* 2.71 
        1374005_at AI233232 Hypothetical protein FLJ12436* 2.09 

Genes whose expression was also measured by RT-PCR are underlined. Genes with strong similarity to homosapiens (*) or mus protein (#) are specified.

TABLE 2

Genes up- or downregulated in AMPK overexpressing rat islets compared with Null virus-infected islets

Affymetrix number gene functionGenBank accession numberGene/protein nameFold change
Metabolism    
    Proteolysis and peptidolysis    
        1387503_at NM_053526 Carboxypeptidase N, polypeptide 1, 50kD 2.38 
        1372440_at BI275818 Serine (or cysteine) proteinase inhibitor, clade E, member 2 2.04 
        1399085_at NM_005857 Zinc metalloproteinase Ste24 homolog* 2.15 
    Protein amino acid glycosylation    
        1370714_a_at NM_147205 Sialyltransferase 1 0.50 
    Prostaglandin metabolism    
        1368014_at NM_021583 Prostaglandin E synthase 0.29 
    Nitrogen metabolism    
        1388324_at NM_182668 Nitrilase 1 2.12 
Intracellular signaling    
    Calcium ion binding    
        1386865_at NM_012946 SPARC-like 1 2.60 
        1389533_at AA944398 Fibulin 22.27  
    Protein transport    
        1370539_at NM_153317 GTPase Rab8b 0.46 
    Copper ion transport    
        1368046_at NM_133600 Solute carrier family 31 (copper transporters), member 1 0.35 
    Lipid transport    
        1370862_at NM_138828 Apolipoprotein E 2.15 
        1386965_at NM_012598 Lipoprotein lipase 3.18 
Cytoarchitecture    
    Protein complex assembly    
        1369597_at NM_021847 Vesicle-associated membrane protein, associated protein B and C 0.49 
Cell adhesion    
    1387854_at NM_053356 Procollagen, type I, alpha 2 2.38 
    1373345_at NM_182816 Transmembrane protein AMIGO2 2.60 
    1373232_at BC035608 NID2_HUMAN Nidogen-2 precursor (NID-2) (Osteonidogen)* 0.33 
    1389020_at NP_005536 Immunoglobulin superfamily containing leucine-rich repeat* 2.33 
Transcription regulation    
        1389778_a_at NM_017103 Transcription elongation factor B (SIII), polypeptide 3 0.48 
        1374335_at NM_019185 GATA binding protein 6 2.15 
        1389555_at NM_007109 Transcription factor 19 (SCI)* 2.14 
        1373303_at XM_231361 SC35-interacting protein I* 0.48 
    Chaperone activity    
        1389487_at BC064920 DnaJ homolog subfamily B member 12* 0.44 
Apoptosis    
        1387122_at NM_012760 Pleiomorphic adenoma gene-like 1 (PLAGLI) 2.39 
Growth    
    Cell growth/immune response    
        1387316_at NM_030845 GroI 0.17 
        1388469_at AA945615 Rat insulin-like growth factor I 2.02 
Cell proliferation/regulation of cell cycle    
        1369968_at NM_017066 Pleiotrophin 2.23 
        1393907_at AA998735 Peptide YY precursor (PYY)* 4.17 
Others    
        1367749_at NM_031050 Lumican 2.42 
        1375813_at BE102687 Rattus norvegicus similar to NAKAP95 (LOC299569), mRNA 0.34 
        1373755_at NM_019083 Hypothetical protein FLJ10287* 2.50 
        1376486_at XM_231127 Similar to SH3-domain GRB2-like endophilin B2 (LOC311848)* 2.11 
Affymetrix number gene functionGenBank accession numberGene/protein nameFold change
Metabolism    
    Proteolysis and peptidolysis    
        1387503_at NM_053526 Carboxypeptidase N, polypeptide 1, 50kD 2.38 
        1372440_at BI275818 Serine (or cysteine) proteinase inhibitor, clade E, member 2 2.04 
        1399085_at NM_005857 Zinc metalloproteinase Ste24 homolog* 2.15 
    Protein amino acid glycosylation    
        1370714_a_at NM_147205 Sialyltransferase 1 0.50 
    Prostaglandin metabolism    
        1368014_at NM_021583 Prostaglandin E synthase 0.29 
    Nitrogen metabolism    
        1388324_at NM_182668 Nitrilase 1 2.12 
Intracellular signaling    
    Calcium ion binding    
        1386865_at NM_012946 SPARC-like 1 2.60 
        1389533_at AA944398 Fibulin 22.27  
    Protein transport    
        1370539_at NM_153317 GTPase Rab8b 0.46 
    Copper ion transport    
        1368046_at NM_133600 Solute carrier family 31 (copper transporters), member 1 0.35 
    Lipid transport    
        1370862_at NM_138828 Apolipoprotein E 2.15 
        1386965_at NM_012598 Lipoprotein lipase 3.18 
Cytoarchitecture    
    Protein complex assembly    
        1369597_at NM_021847 Vesicle-associated membrane protein, associated protein B and C 0.49 
Cell adhesion    
    1387854_at NM_053356 Procollagen, type I, alpha 2 2.38 
    1373345_at NM_182816 Transmembrane protein AMIGO2 2.60 
    1373232_at BC035608 NID2_HUMAN Nidogen-2 precursor (NID-2) (Osteonidogen)* 0.33 
    1389020_at NP_005536 Immunoglobulin superfamily containing leucine-rich repeat* 2.33 
Transcription regulation    
        1389778_a_at NM_017103 Transcription elongation factor B (SIII), polypeptide 3 0.48 
        1374335_at NM_019185 GATA binding protein 6 2.15 
        1389555_at NM_007109 Transcription factor 19 (SCI)* 2.14 
        1373303_at XM_231361 SC35-interacting protein I* 0.48 
    Chaperone activity    
        1389487_at BC064920 DnaJ homolog subfamily B member 12* 0.44 
Apoptosis    
        1387122_at NM_012760 Pleiomorphic adenoma gene-like 1 (PLAGLI) 2.39 
Growth    
    Cell growth/immune response    
        1387316_at NM_030845 GroI 0.17 
        1388469_at AA945615 Rat insulin-like growth factor I 2.02 
Cell proliferation/regulation of cell cycle    
        1369968_at NM_017066 Pleiotrophin 2.23 
        1393907_at AA998735 Peptide YY precursor (PYY)* 4.17 
Others    
        1367749_at NM_031050 Lumican 2.42 
        1375813_at BE102687 Rattus norvegicus similar to NAKAP95 (LOC299569), mRNA 0.34 
        1373755_at NM_019083 Hypothetical protein FLJ10287* 2.50 
        1376486_at XM_231127 Similar to SH3-domain GRB2-like endophilin B2 (LOC311848)* 2.11 

Genes whose expression was also measured by RT-PCR are underlined. Genes with strong similarity to Homo sapiens (*) are specified.

This article is based on a presentation at a symposium. The symposium and the publication of this article were made possible by an unrestricted educational grant from Servier.

This study was supported by grants from the Wellcome Trust (Project 062321; Programme 067081/Z/02/Z, Prize Studentship to E.M.), the Biotechnological and Biological Research Council, the Juvenile Diabetes Research Foundation International, and the Medical Research Council (U.K.). G.A.R. and I.L. are grateful to the Wellcome Trust for Research Leave and Advanced Fellowships, respectively, and F.D. is a recipient of the European Union for a Marie Curie Fellowship.

We thank Rebecca Rowe for excellent technical assistance.

1.
Osborne TF: Sterol regulatory element-binding proteins (SREBPs): key regulators of nutritional homeostasis and insulin action.
J Biol Chem
275
:
32379
–32382,
2000
2.
Goldstein JL, Rawson RB, Brown MS: Mutant mammalian cells as tools to delineate the sterol regulatory element-binding protein pathway for feedback regulation of lipid synthesis.
Arch Biochem Biophys
397
:
139
–148,
2002
3.
Andreolas C, daSilvaXavier G, Diraison F, Zhao C, Varadi A, Lopez-Casillas F, Ferré P, Foufelle F, Rutter GA: Stimulation of acetyl-CoA carboxylase gene expression by glucose requires insulin release and sterol regulatory element binding protein 1c in MIN6 β-cells.
Diabetes
51
:
2536
–2545,
2002
4.
Wang H, Maechler P, Antinozzi PA, Herrero L, Hagenfeldt-Johansson KA, Bjorklund A, Wollheim CB: The transcription factor SREBP-1c is instrumental in the development of beta-cell dysfunction.
J Biol Chem
278
:
16622
–16629,
2003
5.
Schuit F, Flamez D, De Vos A, Pipeleers D: Glucose-regulated gene expression maintaining the glucose-responsive state of beta-cells.
Diabetes
51 (Suppl. 3)
:
S326
–S332,
2002
6.
Diraison F, Parton L, Ferre P, Foufelle F, Briscoe CP, Leclerc I, Rutter GA: Over-expression of sterol regulatory element binding protein-1c or culture with 5-aminoimidazole-4-carboxamide ribonucleoside induces lipogenesis and decreases glucose-stimulated insulin secretion from rat pancreatic islets.
Biochem J
373
:
769
–778,
2004
7.
Eto K, Yamashita T, Matsui J, Terauchi Y, Noda M, Kadowaki T: Genetic manipulations of fatty acid metabolism in beta-cells are associated with dysregulated insulin secretion.
Diabetes
51 (Suppl. 3)
:
S414
–S420,
2002
8.
Robertson RP, Harmon J, Tran PO, Poitout V: Beta-cell glucose toxicity, lipotoxicity, and chronic oxidative stress in type 2 diabetes.
Diabetes
53
:
S119
–S124,
2004
9.
Prentki M, Corkey BE: Are the beta-cell signaling molecules malonyl-CoA and cystolic long-chain acyl-CoA implicated in multiple tissue defects of obesity and NIDDM?
Diabetes
45
:
273
–283,
1996
10.
Horton JD, Shah NA, Warrington JA, Anderson NN, Park SW, Brown MS, Goldstein JL: Combined analysis of oligonucleotide microarray data from transgenic and knockout mice identifies direct SREBP target genes.
Proc Natl Acad Sci U S A
100
:
12027
–12032,
2003
11.
Hawley SA, Boudeau J, Reid JL, Mustard KJ, Udd L, Makela TP, Alessi DR, Hardie DG: Complexes between the LKB1 tumor suppressor, STRADalpha/beta and MO25alpha/beta are upstream kinases in the AMP-activated protein kinase cascade.
J Biol
2
:
28
,
2003
12.
Rutter GA, Da Silva Xavier G, Leclerc I: Roles of 5′-AMP-activated protein kinase (AMPK) in mammalian glucose homeostasis.
Biochem J
375
:
1
–16,
2003
13.
Leclerc I, Kahn A, Doiron B: The 5′-AMP-activated protein kinase inhibits the transcriptional stimulation by glucose in liver cells, acting through the glucose response complex.
FEBS Lett
431
:
180
–184,
1998
14.
Salt IP, Johnson G, Ashcroft SJ, Hardie DG: AMP-activated protein kinase is activated by low glucose in cell lines derived from pancreatic beta cells, and may regulate insulin release.
Biochem J
335
:
533
–539,
1998
15.
daSilvaXavier G, Leclerc I, Salt IP, Doiron B, Hardie DG, Kahn A, Rutter GA: Role of AMP-activated protein kinase in the regulation by glucose of islet beta-cell gene expression.
Proc Natl Acad Sci U S A
97
:
4023
–4028,
2000
16.
daSilvaXavier G, Leclerc I, Varadi A, Tsuboi T, Moule SK, Rutter GA: Role for AMP-activated protein kinase in glucose-stimulated insulin secretion and preproinsulin gene expression.
Biochem J
371
:
761
–774,
2003
17.
Leclerc I, Woltersdorf WW, Da Silva Xavier G, Rowe RL, Cross SE, Korbutt GS, Rajotte RV, Smith R, Rutter GA: Metformin, but not leptin, regulates AMP-activated protein kinase in pancreatic islets: impact on glucose-stimulated insulin secretion.
Am J Physiol Endocrinol Metab
286
:
E1023
–E1031,
2004
18.
Leclerc I, Rutter GA: AMP-activated protein kinase: a new β-cell glucose sensor? Regulation by amino acids and calcium ions.
Diabetes
53 (Suppl. 3)
:
S67
–S74,
2004
19.
Hoyle DC, Rattray M, Jupp R, Brass A: Making sense of microarray data distributions.
Bioinformatics
18
:
576
–584,
2002
20.
Storey JD: A direct approach to false discovery rates.
J R Stat Soc, Series B
64
:
479
–498,
2002
21.
Akli S, Chelly J, Lacorte JM, Poenaru L, Kahn A: Seven novel Tay-Sachs mutations detected by chemical mismatch cleavage of PCR-amplified cDNA fragments.
Genomics
11
:
124
–134,
1991
22.
Tsuboi T, DaSilva XG, Leclerc I, Rutter GA: 5′ AMP-activated protein kinase controls insulin-containing secretory vesicle dynamics.
J Biol Chem
278
:
52042
–52051,
2003
23.
Upchurch BH, Aponte GW, Leiter AB: Expression of peptide YY in all four islet cell types in the developing mouse pancreas suggests a common peptide YY-producing progenitor.
Development
120
:
245
–252,
1994
24.
Kumar U, Sasi R, Suresh S, Patel A, Thangaraju M, Metrakos P, Patel SC, Patel YC: Subtype-selective expression of the five somatostatin receptors (hSSTR1-5) in human pancreatic islet cells: a quantitative double-label immunohistochemical analysis.
Diabetes
48
:
77
–85,
1999
25.
Gundlach AL: Galanin/GALP and galanin receptors: role in central control of feeding, body weight/obesity and reproduction?
Eur J Pharmacol
440
:
255
–268,
2002
26.
Butler AE, Janson J, Bonner-Weir S, Ritzel R, Rizza RA, Butler PC: Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes.
Diabetes
52
:
102
–110,
2003