Differential gene expression between Zucker Fatty rats and Zucker Diabetic Fatty rats: a potential role for the immediate-early gene Egr-1 in regulation of beta cell proliferation

Abstract

The -cell failure that characterises type 2 diabetes is likely to involve altered expression of many genes. We aimed to identify global changes in gene expression underlying -cell dysfunction in pre-diabetic Zucker Diabetic Fatty rat islets, followed by functional studies to verify our findings. Gene expression profiles in islets from 6-week-old Zucker Diabetic Fatty rats and Zucker Fatty rat controls were analysed using Affymetrix microarrays. Totally 977 genes were found to be differentially regulated, comprising large groups of membrane and structural proteins, kinases, channels, receptors, transporters, growth factors and transcription factors. We are particularly interested in transcription factors, which can have profound effects on cellular function. Thus a subset of those with no role yet defined in the -cell was selected for further study namely the immediate-early gene Egr-1, PAG608, rCGR19 and mSin3b. Tissue specificity of these factors varied but interestingly Egr-1 expression was highly enriched in the pancreatic islet. To determine a possible role of Egr-1 in the -cell, Egr-1 expression in INS-1 cells was silenced using RNA interference (RNAi). Glucose-stimulated insulin secretion in these cells was then measured using ELISA and cell proliferation was measured by [ 3 H]thymidine incorporation. Small interfering RNA (siRNA)-mediated silencing of the Egr-1 gene inhibited its induction by glucose but had no observable effect on glucose-stimulated insulin secretion. However, Egr-1 gene silencing did inhibit proliferation of INS-1 cells in a glucose-independent manner. Our studies have revealed a role for Egr-1 and suggest that reduced Egr-1 gene expression may contribute to decreased -cell proliferation and the consequent -cell failure observed in the later stages of type 2 diabetes.

Deriving at candidate genes of metabolic stress from pancreas of WNIN/GR-Ob mutant rats

“…These transcription factors have been reported to participate in apoptosis, proliferation, differentiation and stress responses, [25][26][27][28][29] which is in line with studies reported by Garnett et al 24 where they showed an upregulation of 41 transcription factors, in pre diabetic ZDF islets, in comparison with Zucker-Fatty islets. 24 Accelerated aging has been an yet another well documented phenomenon in these mutants with their average life span shortened to 1.5 y as compared with 2.5-3 y seen in Controls and Leans. 5,6 This is accompanied by increase in episodes of cataract, 6 cancers, 5,6 infertility, 5,6 and infection 6 marking an early onset of degenerative diseases in these mutants.…”

“…24 The mutants demonstrated an upregulation of 18 transcription factors (Table 1D) as compared with controls. These transcription factors have been reported to participate in apoptosis, proliferation, differentiation and stress responses, [25][26][27][28][29] which is in line with studies reported by Garnett et al 24 where they showed an upregulation of 41 transcription factors, in pre diabetic ZDF islets, in comparison with Zucker-Fatty islets. 24 Accelerated aging has been an yet another well documented phenomenon in these mutants with their average life span shortened to 1.5 y as compared with 2.5-3 y seen in Controls and Leans.…”

“…Transcription factors have profound and multiple effects on phenotypic traits 24 and dysregulation of transcription factors affects multiple metabolic pathways. 24 The mutants demonstrated an upregulation of 18 transcription factors (Table 1D) as compared with controls.…”

Egr-1 binds the GnRH promoter to mediate the increase in gene expression by insulin

“…Consistent with its role as an immediate early gene, Egr-1 mRNA levels rise and peak early after stimulation with insulin then decline quickly, with a corresponding pattern of protein expression (Figure 1). This time course of insulin induced increase in Egr-1 mRNA levels has been observed in many tissues (Barroso and Santisteban, 1999;Tan et al, 2003;Silverman and Collins, 1999;Leung-Theung-Long et al, 2005;Garnett et al, 2005) as has its dependence on the MAP kinase pathway (Leung-Theung-Long et al, 2005;Garnett et al, 2005;Keeton et al, 2003). The GnRH mRNA level peak after one hour of insulin treatment corresponds with the time of maximal Egr-1 protein levels, suggesting that higher protein levels, rather than protein activation, promote binding of Egr-1 to the GnRH promoter.…”

“…A variety of factors can stimulate Egr-1 gene expression, including cellular injury, radiation, and growth factors (Gashler and Sukhatme, 1995). Insulin regulates Egr-1 gene expression in a variety of tissues including hepatic, bone, pancreatic beta cells and vascular endothelial tissue (Barroso and Santisteban, 1999;Tan et al, 2003;Silverman and Collins, 1999;Leung-Theung-Long et al, 2005;Garnett et al, 2005). The induction of Egr-1 gene expression by insulin is primarily mediated by the MAP kinase pathway.…”