Type 2 diabetes (T2D) is a metabolic disorder characterized by beta cell dysfunction and insulin resistance in fat, muscle and liver cells. It is thought that hyperglycemia and hyperlipidemia are two major factors contributing to the development of beta cell dysfunction in T2D. However, clinical studies in diabetic patients and diabetic rat/mouse models have shown that beta cell dysfunction takes place before the development of hyperglycemia or an increase in circulating levels of plasma free fatty acids (FFAs). It therefore seems that neither ‘glucotoxicity’ nor ‘lipotoxicity’ can fully explain the onset of beta cell dysfunction in T2D.
Recent studies have shown that the development of insulin resistance in pancreatic beta cells may be a key link in fully understanding the development of beta cell dysfunction in T2D. Tissue-specific knockout of the insulin receptor (InsR) gene in mouse pancreatic beta cells produces defects in insulin secretion similar to that in T2D. This evidence strongly supports the notion that insulin resistance in these cells may lead to pancreatic beta cell dysfunction. However, detailed investigations regarding the precise mechanisms by which the insulin deficiency may occur have not yet been undertaken. Therefore, the major aims of this project were to determine whether the development of insulin resistance in pancreatic beta cells contributes to beta cell dysfunction, and to investigate the molecular mechanisms underlying the functional changes that occur in response to impaired insulin signalling.
To address these aims, an INS-1 InsR knockdown pancreatic beta cell line (InsRβKD cells) was established by knocking down InsR expression in INS-1 cells with anti InsRβ lentiviral small hairpin RNA (InsRβshRNA). The resultant InsRβKD cells demonstrated a significantly reduced expression of InsR as determined by quantitative real-time polymerase chain reaction (qPCR) and Western blot. Upon fasting for glucose, these cells also exhibited significantly decreased insulin secretion in response to glucose stimulation. In accordance with reduced insulin production, glucose uptake efficiency also decreased, as indicated by a 3[H] - 2-deoxyglucose assay. Furthermore, InsRβKD cells had a dramatic decrease in glucose transporter 2 (SLC2A2, herein referred to as GLUT2) and pancreatic duodenal homeobox (Pdx1) messenger RNA (mRNA) expression compared to control cells.
In summary, this project established a novel insulin resistant pancreatic beta cell line and provided a more reliable model for investigations into pancreatic beta cell failure in type 2 diabetes. Our data suggests that reduced insulin expression and decreased glucose stimulated insulin secretion (GSIS) are the results of reduced glucose uptake in InsRβKD cells, which is mainly mediated by decreased GLUT2 expression. Furthermore, preliminary experiment indicated that decreased Pdx1 expression may contribute to the reduction of GLUT2 expression in InsRβKD cells. Ultimately this study showed that pancreatic beta cell insulin resistance contributes to the development of beta cell dysfunction by impairing pancreatic beta cell glucose sensitivity and Pdx1- GLUT2 pathway. These impairments may contribute to the reduced GSIS observed in diabetes.