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Gene expression profiles of Beta-cell enriched tissue obtained by laser capture microdissection from subjects with type 2 diabetes.

Marselli L, Thorne J, Dahiya S, Sgroi DC, Sharma A, Bonner-Weir S, Marchetti P, Weir GC - PLoS ONE (2010)

Bottom Line: There were few changes in the major genes associated with cell cycle, apoptosis or endoplasmic reticulum stress.There was differential expression of genes associated with pancreatic regeneration, most notably upregulation of members of the regenerating islet gene (REG) family and metalloproteinase 7 (MMP7).Some of the genes found in GWAS studies to be related to T2D were also found to be differentially expressed.

View Article: PubMed Central - PubMed

Affiliation: Section on Islet Transplantation and Cell Biology, Research Division, Joslin Diabetes Center and the Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT

Background: Changes in gene expression in pancreatic beta-cells from type 2 diabetes (T2D) should provide insights into their abnormal insulin secretion and turnover.

Methodology/principal findings: Frozen sections were obtained from cadaver pancreases of 10 control and 10 T2D human subjects. Beta-cell enriched samples were obtained by laser capture microdissection (LCM). RNA was extracted, amplified and subjected to microarray analysis. Further analysis was performed with DNA-Chip Analyzer (dChip) and Gene Set Enrichment Analysis (GSEA) software. There were changes in expression of genes linked to glucotoxicity. Evidence of oxidative stress was provided by upregulation of several metallothionein genes. There were few changes in the major genes associated with cell cycle, apoptosis or endoplasmic reticulum stress. There was differential expression of genes associated with pancreatic regeneration, most notably upregulation of members of the regenerating islet gene (REG) family and metalloproteinase 7 (MMP7). Some of the genes found in GWAS studies to be related to T2D were also found to be differentially expressed. IGF2BP2, TSPAN8, and HNF1B (TCF2) were upregulated while JAZF1 and SLC30A8 were downregulated.

Conclusions/significance: This study made possible by LCM has identified many novel changes in gene expression that enhance understanding of the pathogenesis of T2D.

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Related in: MedlinePlus

Real-time PCR analysis of the regenerating islet genes Reg 1 alpha, Reg 1 beta, Reg 3 alpha, and Reg 3 gamma.The assay was performed on samples from four T2D subjects (D) and four controls (C). Panels A–D show the relative gene expression of each single sample. In panels a–d data are reported as mean ± SE ratios of relative expression values from T2D samples (D) over values from control samples (C). The statistical significance was evaluated by the two-tailed Student's t-test using the dCt values (not performed with Reg 3 alpha and Reg 3 gamma since the expression was not detectable in two control samples each).
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pone-0011499-g003: Real-time PCR analysis of the regenerating islet genes Reg 1 alpha, Reg 1 beta, Reg 3 alpha, and Reg 3 gamma.The assay was performed on samples from four T2D subjects (D) and four controls (C). Panels A–D show the relative gene expression of each single sample. In panels a–d data are reported as mean ± SE ratios of relative expression values from T2D samples (D) over values from control samples (C). The statistical significance was evaluated by the two-tailed Student's t-test using the dCt values (not performed with Reg 3 alpha and Reg 3 gamma since the expression was not detectable in two control samples each).

Mentions: A variety of genes potentially involved in pancreatic islet development and regeneration were evaluated (Table S3). Genes belonging to the regenerating islet gene (REG) family were upregulated in samples of T2D subjects (Table S3); among them, REG1A and REG3G were those with the highest differential expression, and REG3G was the gene with the highest expression variability among diabetic subjects (Table S3). The expression of REG1A, REG1B, REG3A, and REG3G was quantified by real-time PCR, performed on samples from four T2D subjects and four controls, which, confirmed the differential expression between the two groups (Fig. 3 A–D). The roles of Reg family of proteins continue to be intriguing but poorly understood. Reg was first found in the islets after partial pancreatectomy in rats [59]. Reg1 has been implicated in beta-cell growth by association and by administration of the peptide [60], and a knock-out resulted in reduced islet mass [61]. Reg has also been found in beta-cells in mice with new onset type 1 diabetes [62]. Reg3A (HIP/PAP) is expressed in multiple tissues and has been associated with liver regeneration; it has been found to have mitogenic and anti-apoptotic functions [63]. The peptides human proIslet peptide (HIP) and islet neogenesis associated protein (INGAP) are related to sequences in REG3A, and are candidates for inducing neogenesis [64]. We also found that the transcription factor SOX9 was upregulated in samples from T2D donors (Table S3), and this differential expression of SOX9 was confirmed by real-time PCR (Fig. 4 A). This finding is of interest because SOX9 is known to be present in pancreatic duct cells and has been implicated in islet development by maintaining pancreatic progenitors [65]. The metalloproteinase MMP7 was upregulated in samples of T2D subjects (Table S3). The real-time PCR performed in samples obtained from four T2D subjects and four controls confirmed the differential expression of MMP7 between the two groups (Fig. 4 B). MMP-7 is involved in development, cancer invasion and sending epithelial signals to stromal cells [66], [67].


Gene expression profiles of Beta-cell enriched tissue obtained by laser capture microdissection from subjects with type 2 diabetes.

Marselli L, Thorne J, Dahiya S, Sgroi DC, Sharma A, Bonner-Weir S, Marchetti P, Weir GC - PLoS ONE (2010)

Real-time PCR analysis of the regenerating islet genes Reg 1 alpha, Reg 1 beta, Reg 3 alpha, and Reg 3 gamma.The assay was performed on samples from four T2D subjects (D) and four controls (C). Panels A–D show the relative gene expression of each single sample. In panels a–d data are reported as mean ± SE ratios of relative expression values from T2D samples (D) over values from control samples (C). The statistical significance was evaluated by the two-tailed Student's t-test using the dCt values (not performed with Reg 3 alpha and Reg 3 gamma since the expression was not detectable in two control samples each).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2903480&req=5

pone-0011499-g003: Real-time PCR analysis of the regenerating islet genes Reg 1 alpha, Reg 1 beta, Reg 3 alpha, and Reg 3 gamma.The assay was performed on samples from four T2D subjects (D) and four controls (C). Panels A–D show the relative gene expression of each single sample. In panels a–d data are reported as mean ± SE ratios of relative expression values from T2D samples (D) over values from control samples (C). The statistical significance was evaluated by the two-tailed Student's t-test using the dCt values (not performed with Reg 3 alpha and Reg 3 gamma since the expression was not detectable in two control samples each).
Mentions: A variety of genes potentially involved in pancreatic islet development and regeneration were evaluated (Table S3). Genes belonging to the regenerating islet gene (REG) family were upregulated in samples of T2D subjects (Table S3); among them, REG1A and REG3G were those with the highest differential expression, and REG3G was the gene with the highest expression variability among diabetic subjects (Table S3). The expression of REG1A, REG1B, REG3A, and REG3G was quantified by real-time PCR, performed on samples from four T2D subjects and four controls, which, confirmed the differential expression between the two groups (Fig. 3 A–D). The roles of Reg family of proteins continue to be intriguing but poorly understood. Reg was first found in the islets after partial pancreatectomy in rats [59]. Reg1 has been implicated in beta-cell growth by association and by administration of the peptide [60], and a knock-out resulted in reduced islet mass [61]. Reg has also been found in beta-cells in mice with new onset type 1 diabetes [62]. Reg3A (HIP/PAP) is expressed in multiple tissues and has been associated with liver regeneration; it has been found to have mitogenic and anti-apoptotic functions [63]. The peptides human proIslet peptide (HIP) and islet neogenesis associated protein (INGAP) are related to sequences in REG3A, and are candidates for inducing neogenesis [64]. We also found that the transcription factor SOX9 was upregulated in samples from T2D donors (Table S3), and this differential expression of SOX9 was confirmed by real-time PCR (Fig. 4 A). This finding is of interest because SOX9 is known to be present in pancreatic duct cells and has been implicated in islet development by maintaining pancreatic progenitors [65]. The metalloproteinase MMP7 was upregulated in samples of T2D subjects (Table S3). The real-time PCR performed in samples obtained from four T2D subjects and four controls confirmed the differential expression of MMP7 between the two groups (Fig. 4 B). MMP-7 is involved in development, cancer invasion and sending epithelial signals to stromal cells [66], [67].

Bottom Line: There were few changes in the major genes associated with cell cycle, apoptosis or endoplasmic reticulum stress.There was differential expression of genes associated with pancreatic regeneration, most notably upregulation of members of the regenerating islet gene (REG) family and metalloproteinase 7 (MMP7).Some of the genes found in GWAS studies to be related to T2D were also found to be differentially expressed.

View Article: PubMed Central - PubMed

Affiliation: Section on Islet Transplantation and Cell Biology, Research Division, Joslin Diabetes Center and the Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT

Background: Changes in gene expression in pancreatic beta-cells from type 2 diabetes (T2D) should provide insights into their abnormal insulin secretion and turnover.

Methodology/principal findings: Frozen sections were obtained from cadaver pancreases of 10 control and 10 T2D human subjects. Beta-cell enriched samples were obtained by laser capture microdissection (LCM). RNA was extracted, amplified and subjected to microarray analysis. Further analysis was performed with DNA-Chip Analyzer (dChip) and Gene Set Enrichment Analysis (GSEA) software. There were changes in expression of genes linked to glucotoxicity. Evidence of oxidative stress was provided by upregulation of several metallothionein genes. There were few changes in the major genes associated with cell cycle, apoptosis or endoplasmic reticulum stress. There was differential expression of genes associated with pancreatic regeneration, most notably upregulation of members of the regenerating islet gene (REG) family and metalloproteinase 7 (MMP7). Some of the genes found in GWAS studies to be related to T2D were also found to be differentially expressed. IGF2BP2, TSPAN8, and HNF1B (TCF2) were upregulated while JAZF1 and SLC30A8 were downregulated.

Conclusions/significance: This study made possible by LCM has identified many novel changes in gene expression that enhance understanding of the pathogenesis of T2D.

Show MeSH
Related in: MedlinePlus