Transcriptional regulation of healthy and diabetic pancreatic beta-cell gene expression

Abstract

Hyperglycemia, deficient insulin secretion, and insulin resistance are the characteristic pathogenetic features of type 2 diabetes (T2D), hallmarked by functional and survival defects of the insulin-producing islet beta-cell. The primary pathogenesis of T2D probably involves both genetic and environmental forces, and hyperglycemia and very commonly hyperlipidemia might further aggravate beta-cell dysfunction. To gain a deeper understanding of the precise nature of the cellular and molecular defects responsible for T2D, it is necessary to pin down the molecular mechanisms by which glucotoxicity and lipotoxicity adversely impact rodent and human beta-cells. This was the over-arching aim of the current work. Additionally, the influence of antidiabetic drugs, with alleged ability to protect and preserve beta-cell function and viability, on the above pathways was addressed.

Paper I, II Long term exposure to free fatty acids (FFAs) may alter beta-cell signaling and gene expression, precipitating beta-cell failure through impaired insulin synthesis and secretion in combination with apoptosis, a phenomenon commonly referred to as lipotoxicity. It is still unclear whether beta-cell failure and apoptosis are induced by increased FFAs alone, in synergy with glucose, or if predisposing genetic factors are required. Also, the protective effects of the PPARγ agonist pioglitazone (a thiazolidinedione) and the GLP-1 receptor agonist exenatide (an incretin mimetic) against FFA-induced beta-cell dysfunction and apoptosis are still debated. We sought to investigate the long term effect of pioglitazone and exenatide on global alterations in gene expression, with/out palmitate to evoke beta-cell lipotoxicity, in human and rat diabetic and non-diabetic islets. Microarray interrogation and qRT-PCR analyses revealed alterations in several functional categories, most importantly epigenetic regulation of gene expression that seems to regulate beta-cell function and survival. Furthermore, FFAs seem to contribute and play an important role in the development of beta-cell failure in synergy with hyperglycemia, in obese individuals, or in subjects who are genetically more predisposed to T2D. Considering that the nutritional state directly induces epigenetic modifications, pioglitazone and exenatide appear to normalize these epigenetic misregulations and may protect the beta-cell from lipotoxic insult. The epigenetic modifications of the genome provide new promising targets for clinical diagnostics and also therapeutic purposes in T2D.

Paper III Chronic hyperglycemia is believed to be an important determinant of beta-cell dysfunction that may become irreversible over time (known as glucotoxicity). The objective of this study was to investigate the expression of glucose-regulated key genes in islets from spontaneously diabetic Goto-Kakizaki (GK) rats compared to normoglycemic Wistar rats, by using the microarray and qRT-PCR technology. We identified significant changes in islet mRNAs involved in glucose sensing, phosphorylation, incretin action, glucocorticoid handling, ion transport, mitogenesis and apoptosis that clearly distinguish diabetic animals from controls. Our results identify key elements in glucose-regulated gene expression in beta-cells, revealing substantial qualitative and quantitative differences in gene expression between healthy and diabetic beta-cells, which may have implications for our understanding of the etiology and treatment of human T2D. Such markers may be of predictive and therapeutical value in clinical settings in efforts aiming at conferring beta-cell protection against apoptosis, impaired regenerative capacity and functional suppression occurring in diabetes.

Paper IV Pancreatic and duodenal homeobox-1 (PDX-1) transcription factor is critical for embryonic development of the pancreas and normal islet function. In beta-cells, PDX-1 regulates a number of genes involved in maintaining beta-cell identity and function, including insulin. Glucose homeostasis requires tight regulation of insulin synthesis and secretion. The homeodomain of the PDX-1 protein plays a critical role in DNA binding and glucose-dependent regulation of insulin gene transcription. The molecular basis of PDX-1 nuclear translocation, however, remains poorly understood. In this study, we aimed to identify amino acid sequences responsible for the nuclear translocation of mouse PDX-1 by using site-directed mutagenesis of putative phosphorylation sites and positively charged amino acid residues in putative nuclear localization signal (NLS) motifs of GFP-tagged PDX-1. We demonstrate that the NLS motif RRMKWKK is necessary, and in conjunction with the integrity of the ´helix 3` domain of the PDX-1 homeodomain is sufficient, for the nuclear import of PDX-1 in the beta-cell.