Gene Expression Profiles of Beta-Cell Enriched Tissue Obtained by Laser Capture Microdissection from Subjects with Type 2 Diabetes

Abstract

BackgroundChanges in gene expression in pancreatic beta-cells from type 2 diabetes (T2D) should provide insights into their abnormal insulin secretion and turnover.Methodology/Principal FindingsFrozen 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/SignificanceThis study made possible by LCM has identified many novel changes in gene expression that enhance understanding of the pathogenesis of T2D.

Are we overestimating the loss of beta cells in type 2 diabetes?

“…In accordance with previous studies [13,14], light microscopy revealed that the islets varied in terms of size, insulin-and glucagon-positive areas, and they either retained a normal architecture or instead displayed sparse architecture (ESM Fig. 2).…”

“…In addition, in carefully performed examinations, morphological variations in type 2 diabetic islets have also been reported [13,14]. The present study confirms the presence of such alterations by the use of insulin immunocytochemistry and the measurement of fractional pancreatic beta cell area (a surrogate of beta cell mass) [10].…”

“…Islet isolation and functional studies Isolated islets were prepared as described previously [12,13], and insulin secretion determined by batch incubation (45 min) in response to 3.3 and 16.7 mmol/l glucose [12]. In addition, morphological, functional and ultra-structural studies were performed with islets from three independent non-diabetic donors after 24 h incubation with 22.2 mmol/l glucose.…”

Islet inflammation in type 2 diabetes

“…In addition, the length of the culture period from isolation to molecular assessments may play a role [35], and, finally, the endocrine cell heterogeneity of the islets precludes the clear determination of the properties of the beta cells [36]. A possible solution to some of these problems is the use of beta cells from frozen pancreas samples, as obtained by laser capture microdissection [37,38]. Marselli et al [38] published a study comparing the transcriptomics of laser microdissected beta cells prepared from the pancreas of ten type 2 diabetic and ten non-diabetic organ donors.…”

“…A possible solution to some of these problems is the use of beta cells from frozen pancreas samples, as obtained by laser capture microdissection [37,38]. Marselli et al [38] published a study comparing the transcriptomics of laser microdissected beta cells prepared from the pancreas of ten type 2 diabetic and ten non-diabetic organ donors. The expression of genes encoding inflammatory and/or immunomodulatory mediators, and that of markers of inflammatory cells were evaluated.…”