Posters: Gene Chips and Microarrays
1080-P
Identification of Regulatory Networks Specifying Cell-Specific Control of Glucagon and GLP-1 Synthesis and Secretion
GRACE FLOCK, MARLENA MAZIARZ, XIEMIN CAO, DANIEL J. DRUCKER. Toronto, ON, Canada
The proglucagon-derived peptides are expressed in a highly tissue- specific manner in islet alpha and gut enteroendocrine cells. Although glucagon, GLP-1, and GLP-2 are derived from the same proglucagon gene, energy ingestion and nutrient assimilation represses proglucagon biosynthesis in the [alpha] cell, but stimulates the synthesis and secretion of GLP-1 from the gut L cell. To delineate regulatory networks utilized by specialized proglucagon- producing endocrine cells for regulated PGDP biosynthesis and secretion, we analyzed gene expression profiles and transcriptional networks in murine islet [alpha]TC, [beta]TC and gut GLUTag cell lines. 1841 genes were differentially expressed in islet [alpha]TC versus GLUTag cells (932 genes up and 909 down-regulated in [alpha]TC vs. GLUTag cells, respectively). From a total of 1139 genes corresponding to transcription factor networks represented on the Affymetrix GeneChip array MG-U74Av2, we detected subsets of transcription factors and transcription related genes expressed either exclusively in [alpha]TC alone (6), GLUTag alone (10), both [alpha]TC and GLUTag (1) or expressed in all 3 cell lines (164). These analyses identified a large number of transcription factor classes not previously associated with alpha or enteroendocrine cell biology. For example, the basic domain TF Nfib was expressed exclusively in alphaTC cells, whereas 17 basic domain TFs were expressed in all 3 cell lines. Similarly, TFs such as PPARgc1 and GATA2 were restricted to [alpha]TC and not expressed in [beta]TC or GLUTag cells. In contrast, although the caudal factor Cdx-2 was restricted to GLUTag cells, 29 HLH TFs were detected across all 3 cell lines. Our analyses have delineated new TFs and genes representing new candidates involved in control proglucagon biosynthesis and cell signaling. These studies provide a new conceptual framework for identifying molecular targets differentially regulating the nutrient-regulated synthesis and secretion of glucagon and GLP-1 in distinct islet and enteroendocrine cell types.
1081-P
Expression Profiling of Spleen Leukocytes Reveal Strain-Specific Patterns in NOD, NON and C57B1/6 Mice
IVAN C. GERLING, JIAN WU, DANA R. MARSHALL, NATALIYA LENCHIK. Memphis, TN
The focus of this study was characterizing molecular events in spleen leukocytes of NOD female mice at the earliest stages of development of autoimmunity. We used the Affymetrix MOE430A microarray to evaluate the expression of 22,000 mouse genes and ESTs. Transcriptomes of pooled spleen leukocytes of 2-and 4-week- old NOD, NON, and C57B1/6 female mice were compared. Each group consisted of five independent replicates (30 samples total) and was analyzed using GeneSpring software.
Two-way-ANOVA (age x strain) with Bonferroni correction identified 164 genes showing a strain effect and 59 genes showing an age effect (p<0.05). Nine genes appeared in both lists and one showed a statistically significant interaction. Hierarchical clustering of the 164 strain specific genes showed that much of the transcriptome followed a pattern of one strain showing relatively low expression compared to the other two strains. Another group of genes showed relatively high expression in C57B1/6 mice compared to NOD and NON. Strain-specific differences were found in, interferon induced transcripts, H-2 genes, Toll like receptors, killer cell receptor like, and many genes of unknown function.
NON mice express H2-IE, whereas NOD and C57B1/6 are H2-IE null types. We examined the transcriptome of 4-wk-old NOD mice vs a single group consisting of 2-wk-old NOD and 2- and 4-wk-old C57B1/6 mice. We hypothesized that unique expression patterns should be detectable in NOD mice at 4-wks, the time immediately preceding detectable pancreatic pathology. We identified 53 differentially expressed genes including IL12a, H2-Ab1, H2-K, Mmp12, chemokine ligands, PKC zeta ,unique variable T-cell receptor and immunoglobulin regions, nucleolar autoantigen, and the NK receptor Klra17.
This analysis documents previously known as well as novel differences in gene expression between the three mouse strains. The data clearly demonstrate immune activation at 4 weeks in NOD spleen leukocytes, and suggest activation as early as 2 weeks. These patterns point toward numerous NOD-specific defects in immune regulation and activation of specific immune responses.
1082-P
Microarray Gene Expression Profiling in Human Islets Treated with Glucagon-Like Peptide-1
HONGXIANG HUI, DAVID M. HARLAN, BOAZ HIRSHBERG, JOHN D. ZHENG, NASIF KHOURY, EUGENIO D’AMICO, XIAONING ZHAO, CHARLES WANG, RICCARDO PERFETTI. Los Angeles, CA; Bethesda, MD
Objective: To identify the gene expression profile associated with the action of GLP-1 on human islets. Methods: Freshly isolated human islets from 3 cadaveric donors were treated with 10 nmol GLP- 1, or control, for 22 h. Biotinylated RNA was synthesized using the BioArray high yield RNA transcript labeling kit and the RNA was then hybridized with 12 Human U133A chip containing 23000 genes (Affymetrix array). Results: Microarray analysis identified the expression of about 7,000 genes transcribed in human islets. 83 genes were found to be differentially expressed by 2 fold or more (up or down regulation) after treatment with GLP-1. Real-time PCR for 5 genes (BARX1, CSPG2, GAS-2, ZFP, and NKX6-1) showed the results that were consistent with microarray analysis. The pool of 83 genes that were found regulated by GLP-1 included: binding proteins (25 genes), enzymes (17 genes), transporter proteins (8 genes), signal transduction molecules (5 genes), proliferation related factors (5 genes), apoptosis related proteins (5 genes), and differentiation factors (7 genes). In addition, up- or down- regulated genes involved clusters of factors that are part of signal transduction pathways (G-proteins, etc.), mitochondrial metabolism (fatty acid beta-oxidation, oxidoreductase activity, etc), ions channel activity (Ca^sup 2+^ channel and K+ channel) as well as exocytosis. Conclusion: GLP-1 has been shown enhanced islets function and expand [beta]-cell mass. The identification of molecules mediating its biological effects may lead to identifying new targets to improve function and viability of insulin producing cells.
1083-P
Alterations in Hepatic Gene Expression Profiles in db/db Mice are Not Normalized by Blood Glucose Lowering Drugs
MICHAEL LOFFLER, MARTIN BILBAN, REIMERS MARK, WERNER WALDHAUSL, THOMAS M. STULNIG. Vienna, Austria; Huddinge, Sweden
Human type 2 diabetes and appropriate mouse models such as db/db mice express a variety of genes differently to non-diabetic controls. Agonists of the nuclear receptors peroxisome proliferator- activated receptor (PPAR) [gamma], PPAR[alpha], and liver X receptors (LXR), considerably reduce blood glucose concentrations in patients and/or db/db mice by regulating gene transcription. However, it is not known to which extent these glucose-lowering drugs normalize gene expression. Therefore, we analyzed hepatic gene expression profiles of obese diabetic db/db mice treated with agonists for PPAR[gamma](rosiglitazone [Rosi]), PPAR[alpha] (Wy14643 [Wy]), and LXR (T0901317 [T09]), and lean non-diabetic littermates (ab/+; 3 animals per group) using individual high-density 12k oligonucleotide arrays. All three drugs significantly lowered blood glucose levels (untreated db/db, 53416 mg/dL [mean S.D.]; Rosi, 2046 mg/dL; Wy, 22330 mg/dL; T09, 20014 mg/dL; ab/+, 1383 mg/dL). To compare profiles, genes with a fold change of > or =1.5/< or =-1.5 were selected at p < 0.01 according to robust multiarray analysis thereby obtaining estimated false discoveiy rates of approximately 10% (6.9-12.6%). Expression of 262 genes was significantly altered in db/db mice compared to lean controls. Drug treatment of db/db mice regulated 282 (Rosi), 553 (Wy), and 628 (T09) genes, respectively. However, from the genes changed in db/db mice compared to lean controls, only 13%, 16%, and 26% were regulated by treatment of db/db mice with Rosi, Wy, and T09, respectively (32% by any drug). From the genes differently expressed in db/db mice only 21 were changed by all drugs including genes involved in gluconeogenesis and the glucocorticoid-activating enzyme 11[beta]- hydroxysteroid dehydrogenase type 1 (Hsd11b1). In conclusion, these data provide a comprehensive view on alterations in hepatic gene expression profiles in db/db mice and reveal that blood glucose lowering drugs fail to normalize diabetes associated deviations in gene expression.
1084-P
Effect of Native LDL and Modified LDL on mRNA Expression of Genes Involved in Lipid Metabolism in Human Retinal Pericytes
KANGMO LU, WEIWEI SONG, YAN HUANG, JEREMY BARTH, SCOTT ARGRAVES, TIMOTHY J. LYONS. Oklahoma City, OK; Charleston, SC
Dyslipidemia, including the presence of small dense LDL, is associated with diabetic retinopathy. Capillary leakage, an early feature of diabetic retinopathy, results in the exposure of retinal pericytes to modified (glycated and/or oxidized) LDL, and in vitro- modified LDL causes apoptosis in cultured retinal pericytes. Using gene microarray (quadruplicate experiments) and confirmation by quant\itative real-time RT-PCR analysis, we compared the effects of normal and modified LDL on the mRNA levels of genes involved in lipid metabolism in human retinal pericytes. Cells were exposed to serum-free medium (SFM), or SFM plus native LDL(N-LDL), glycated LDL (G-LDL), or heavily-oxidized-glycated LDL(HOG-LDL) (all at 100ug protein/ml) for 24hr. There were no significant differences in expression of lipid-related genes between N-LDL and G-LDL. Compared to SFM, N-LDL down-regulated three sterol-related genes (LDL receptor (LDLR) by 70%; sterol-C4-methyl oxidase-like(SC4MOL) by 80%; and isopentenyl-diphosphate delta isomerase (IDI 1) by 70%, and up-regulated three genes related to fatty acid metabolism (fatty acid desaturase 1 (FADS-1) by 40%; fatty acid binding protein 4 (FABP 4) by 140%; and short-chain dehydrogenase/reductase 1 (SDR 1) by 70%). By comparison, HOG-LDL had a greater inhibiting effect than N-LDL on SC4MOL and IDI 1, but no effect on LDLR. HOG-LDL strongly up-regulated FADS-1, FADS-2, FABP 4 and strongly down-regulated SDR 1 and ATP-binding cassette, sub-family A, member 6 (ABCA6). Compared with N-LDL, HOG-LDL up-regulated FADS-1 (230%), FADS-2 (190%), FABP 4 (170%), SC4MOL (100%), and IDI-1 (70%), and down-regulated SDR-1 (60%) and ABCA6 (50%). Thus, HOG-LDL has effects on the expression of multiple genes in pericytes, many of which are independent of serum deprivation. Exposure to extravasated, modified LDL may contribute to pericyte loss in diabetic retinopathy. ADA Funded Research
1085-P
Molecular Determinants of GLP-1-Secretion Revealed through Proteomic and Genomic Characterization of Murine Enteroendocrine Cells
MARLENA MAZIARZ, GRACE FLOCK, THOMAS KISLINGER, ANDREW EMILI, DANIEL J. DRUCKER. Toronto, ON, Canada
The pleiotropic properties of GLP-1 that converge on lowering of blood glucose have fostered interest in strategies directed at enhancing GLP-1 action however the molecular determinants of enteroendocrine L cells important for nutrient sensing and the control of GLP-1 synthesis and secretion remain poorly understood. We employed both mass spectrometry and gene expression arrays to characterize murine enteroendocrine GLUTag and islet [alpha]TC-1 cells. Proteomic analysis of pooled cellular fractions detected 1888 proteins expressed in [alpha]TC-1 vs 1499 in GLUTag cells; 1199 proteins were detected in both cell lines. Gene expression analyses were performed using the Affymetrix MG-U74Av2 array containing 12,488 murine probe sets per slide. 5118 genes were identified as present in [alpha]TC-1 cells and 5358 were detected in GLUTag cells; 4690 genes were common to both cell lines. We assessed the sensitivity and specificity of proteomic vs genomic approaches; 506 GLUTag proteins were identified by MS alone but not included on the expression arrays. 359 proteins were identified as present by MS but as absent by gene chips, 887 genes/proteins were identified as present by both methods, 353 transcripts were identified as present by gene chips but as absent by MS, and lastly, 4118 expressed genes were identified as present by gene chips, but were not found in the MS list of proteins. Proteins involved in nucleic acid and protein binding, transcription, signal transduction, cell growth and maintenance were more frequently identified by MS whereas membrane and mitochondrial proteins and proteins involved in regulation of cell signaling were more likely identified by gene chip profiling. These findings have identified multiple new molecular targets for regulation of GLP-1 secretion. Furthermore, the inherent limitations of either MS or array chip technology alone are considerably minimized by combining both technologies for characterization of the murine enteroendocrine cell
1086-P
Phytanic Acid Improves Metabolic Parameters and Modulates Hepatic Gene Expression In Vivo (DIO C57BL/6J Mice)
MAREIKE PRELLER, YING WANG, MARIE-FRANCE CHAMPY, JOHAN AUWERX, VOLKER ELSTE, BEAT FLUEHMANN, PETER WEBER, SANDRA R. TEIXEIRA. Kaiseraugst, Switzerland; Illkirch, France
Phytanic acid (PYA) is a diet-derived agonist for peroxisome proliferator-activated receptors (PPAR) alpha and gamma. The purpose of this study was to examine the in vivo effects of PYA and a TZD (BRL49653), which is a known specific PPARgamma agonist, on body weight, metabolic parameters and hepatic gene expression in DIO C57BL/6J mice.
Forty-eight male C57BL/6J mice were assigned to 4 treatment groups (n=12/group). One group received a chow diet (designated as lean control, LC), while the other groups received a high fat (HF) diet. One of the 3 HF groups served as fat control, whereas the other 2 groups additionally received 150 rnpk PYA or 10 mpk TZD. Liver was collected from 5 mice/group at wk 23 for Affymetrix GeneChip analysis (mouse U74Av2 arrays). A selection of relevant genes was validated by quantitative real-time RT-PCR (TaqMan).
Mice receiving HF gained more weight than the LC mice. HF feeding resulted in significant regulation of 407 hepatic genes (fold change<0.5 or >1.5, p-value<0.05) with 32% down- and 68% upregulated. Treatment with PYA or TZD improved metabolic parameters (lower fasting blood glucose and plasma total cholesterol) and reverted the expression of many genes close to LC levels. While most genes were regulated similarly by PYA and TZD, some were regulated differentially by the two compounds. Specifically, SCD1, SREBP1 (transcription factor for lipogenic genes) and its downstream target SPOT14 were decreased only by PYA. Adipsin (an enzyme necessary for the synthesis of acylation-stimulating protein) was upregulated by TZD, but decreased by PYA. This differential regulation of lipid- metabolism genes suggests the possibility for differential lipid accumulation in the liver.
The hepatic gene profile and the improvement in metabolic parameters brought about by PYA suggest a promising role for this natural compound in the prevention of metabolic syndrome X.
1087-P
Distinct Patterns of Transcriptional Regulation in Skeletal Muscle and Adipose Tissue from Humans with Type 2 Diabetes: Evidence for a Role of Inflammation
JIN SHANG, FANG ZHANG, BRIAN E. LAVAN, ANTHONY C. SCHWEITZER, JOSEPH YU, JERRY M. OLEFSKY, THOMAS A. GUSTAFSON, SHONNAA. MOODIE. Hayward, CA; San Diego, CA
Type 2 Diabetes is characterized by a loss of insulin sensitivity in skeletal muscle and adipose tissue. While defects in several biological processes have been identified, the molecular pathogenesis of type 2 diabetes in these two tissues remains unclear. We used Affymetrix oligonucleotide microarrays to analyze the global gene expression changes characteristic of type 2 diabetes in skeletal muscle and subcutaneous adipose tissue. Compared with control subjects, the diabetic subjects had profound gene expression changes in both tissues. We performed Pfam HMM search, gene ontology (GO) term association and pathway analysis on the transcripts that were significantly dysregulatcd in order to identify potential drugable targets. Expression of known diabetic genes was examined. PDK4 (2.9 FC), RAD (1.8 FC) and PGC-1a (0.62 FC) were found to be dysregulated in skeletal muscle, whereas LEPTIN (1.34 FC), PPARg (0.66 FC), IRS1 (0.54 FC) and 11b-HSD1 (1.7 FC) were found to be dysregulated in adipose tissue. Further analysis with GO term association using MAPPFinder identified multiple pathways and biological processes dysregulated in these two tissues. GO terms associated with energy metabolism appeared to be significantly down- regulated in skeletal muscle, whereas GO terms associated to immune and inflammatory response were up-regulated concomitant with a downregulation of genes involved in lipid and cholesterol biogenesis in adipose. Obesity-induced inflammatory changes in rodent adipose tissue have recently been reported. Our study clearly demonstrates that a similar inflammatory state exists in human diabetic subcutaneous adipose tissue. Identifying global patterns of gene expression changes in diabetic skeletal muscle and adipose tissue will lead us to identify pathways important in pathogenesis of insulin resistance and diabetes. Furthermore, modulation of these pathways may represent potential novel therapeutic targets for the treatment of type 2 diabetes.
1088-P
Changes in Transcriptomes and Proteomes upon Activation of CD4 and CDS T Lymphocytes in Normal and Type 2 Diabetic Subjects
FRANKIE B. STENTZ, ABBAS E. KITABCHI, CHARIS N. ZAMBER. Memphis, TN
T lymphocytes (T cells) are normally devoid of insulin receptors and are insulin insensitive; however, upon activation develop de novo insulin receptors and become insulin responsive and metabolize glucose in response to insulin in a dose-dependent manner. The degree by which these events differ in diabetic and nondiabetic, as well as the mechanism of this activation are not known.
We analyzed the genes expressed (transcriptome) and proteins translated (proteome) during T cell activation in 5 male African- American newly diagnosed Type 2 diabetes and compared the results to 5 matched normal subjects, none of whom were on any medications. RNA and protein were extracted from both the nonactivated and activated CD4 and CDS T cells. The genes expressed were determined using Affymetrix human U133 A microarray chips and data analyzed using GCOS and GeneSifter software. Proteins translated were analyzed using the Cyphergen mass spectrometry system.
Greater than 2000 genes were increased or decreased by 2 fold or greater in CD4 and CD8 T cells with activation. Many of the genes ontologies of insulin signaling and glucose metabolism were not expressed until the T cells were activated. The insulin receptor, insulin degrading enzyme, Akt, IRS-1 and 1RS-2 genes expressed were decreased at least 2 fold in the diabetic subjects in activated T cells compared to normal subjects. Transcriptomes of enzymes involved in the glycolytic pathway such as hexokinase and glyceraldehyde-3- ph\osphate were also decreased 2 fold or greater in the diabetic T cells. The proteome profile of the normal vs diabetic showed differences in protein concentrations. Proteins corresponding to the molecular weight of the above translated transcriptomes were decreased in concentration in the diabetic T cells by 2 fold or greater compared to normal subjects.
We conclude changes in transcriptomes and proteomes (in particular, insulin signaling and glucose metabolizing pathways) occur upon activation of CD4 and CD8 T cells which are decreased 2 fold or greater in diabetic compared to normal subjects.
1089-P
Leptin Induced or Suppressed Genes in Myogenic Cells Using High- Density Oligonucleotide Arrays
A. LULU STRAT, B. DAN RODGERS, MICHAEL V. DODSON, ARIEH GERTLER, RODNEY A. HILL. Moscow, ID; Pullman, WA; Rehovot, Israel
Leptin modulates energy substrate utilization favoring fatty acid oxidation over glucose oxidation, an important role in energy partitioning in muscle. In the present study we investigated leptin effects on gene expression in primary bovine myogenic cells (BMC) by cross-species hybridization to Human Genome U133A Array (HG-U133A). BMC were grown in DMEM, 0.3mM BSA, 0.3mM Free Fatty Acids (0.1mM each palmitic, oleic, linoleic) and treated with 60nM leptin 6h. The analysis showed the following pattern of detection: from 22283 probe sets 14.9% were detected as present (P), 83.3% as absent (A) and 1.7% marginal (M). The change in the functional status of these genes was: 46 (0.2%) were induced (increased, I) de novo [A/ M[implies]P, MI/I] and include structural, functional and metabolic muscle tissue genes; expression of 90 (0.4%) genes was decreased (D) [P[implies]A/M, MD/D] and include cell cycle progression, extracellular matrix, adhesion molecules and functional genes; 215 (1%) increased [P[implies]P, MI/I] and include mainly structural, functional and metabolic genes; 129 (0.6%) decreased [P[implies]P, MD/D] including functional genes. The profiling pattern revealed no change of genes for the molecules involved in the leptin signaling pathway but only in pathway modulators (decreased serine-threonine kinase and tyrosine phosphatase), induction of the metabolic genes involved in known leptin effects and major induction of myogenic differentiation genes.
1090-P
Gene Expression Analysis of Subcutaneous Adipose Tissue in Human Obesity Suggests a Role for Inflammation
FANG ZHANG, FRANCINE M. GREGOIRE, JIN SHANG, GRACE V. PEREZ, BRIAN E. LAVAN, ANTHONY C. SCHWEITZER, THOMAS A. GUSTAFSON, SHONNA A. MOODIE. Hayward, CA
In an effort to identify genes involved in the pathogenesis of obesity and obesity-induced insulin resistance, we analyzed global gene expression profiles in human subcutaneous adipose tissues from obese (BMI>30) and lean (BMI<25) subjects. Subcutaneous adipose tissues were profiled using Affymetrix commercial HG-U133 A and B genechips. 1,047 probesets were found to be differentially expressed with statistical significance in obese adipose tissue compared with control, with 879 probesets up-regulated and 168 probesets down- regulated. Expression levels of known obesity-linked genes were examined. In agreement with published reports, FAS (0.58FC) was found to be down-regulated whereas leptin (2.9FC) was found to be up- regulated in obese samples. Analysis using association with Gene Ontology terms and Pfam HMM revealed potential "drugable" targets within the dysregulated gene pool. Global gene-expression profile analysis using MAPPFinder identified multiple biological processes significantly up-regulated in adipose tissue from obese subjects, including defense response, immune response, complement activation and inflammatory response. Further analysis revealed that macrophage- specific or -enriched genes, such as MAC-1 (1.6FC), CD68 (1.8FC) and MCP-1 (1.9FC), were up-regulated in human obese adipose tissues. This suggests that macrophage-related inflammatory activity is activated in human adipose tissue in obesity and this activation may be an early event that contributes to the pathogenesis of obesityinduced insulin resistance. Upregulation of inflammatory genes and macrophage-specific genes in human adipose tissue is in agreement with recent reports from rodent models of obesity and diabetes. In summary, this gene expression profiling study reveals that obesity results in profound changes in gene expression in human adipose tissue. Functional pathway analysis will lead to better understanding of the pathogenesis of obesity and obesity-induced insulin resistance, and lead to identification of novel therapeutic targets.
Copyright American Diabetes Association Jun 2004