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Abstract
Type 2 diabetes (T2D) is a metabolic disorder characterized by beta cell dysfunction and insulin resistance in fat, muscle and liver cells. Recent studies have shown that the development of insulin resistance in pancreatic beta cell lines may contribute to beta cell dysfunction in T2D. However, there still is a lack of detailed investigations regarding the mechanisms by which insulin deficiency may contribute in diabetes. In this study, we firstly established a stable insulin receptor knockdown cell line in pancreatic beta cells INS-1 (InsRβKD cells) using anti InsRβ small hairpin RNA (InsRβ-shRNA) encoded by lentiviral vectors. The resultant InsRβKD cells demonstrated a significantly reduced expression of InsRβ as determined by real-time PCR and Western blotting analyses. Upon removing glucose from the medium, these cells exhibited a significant decrease in insulin gene expression and protein secretion in response to 20 mM glucose stimulation. In accordance with this insulin reduction, the glucose uptake efficiency as indicated by a 3[H]-2-deoxy-d-glucose assay also decreased. Furthermore, InsRβKD cells showed a dramatic decrease in glucose transporter 2 (GLUT2, encoded by SLC2A2) and pancreatic duodenal homeobox (Pdx1) mRNA expression compared to the controls. These data collectively suggest that pancreatic beta cell insulin resistance contributes to the development of beta cell dysfunction by impairing pancreatic beta cell glucose sensation through the Pdx1- GLUT2 pathway. InsRβKD cells provide a good model to further investigate the mechanism of β-cell dysfunction in T2D.
Highlights
Diabetes is one of the fastest growing chronic diseases worldwide [1]
Other studies using a small interfering RNA to knockdown insulin receptor (IRKD) in MIN6 cells described a significant decrease in glucose-stimulated insulin secretion (GSIS) and a reduced cell proliferation [8,9]
The DNA fragment cut from the pLL3.7 vector with no insert gave a band of 450 bp, while the digest containing the InsR β (InsRβ) Small Hairpin RNA (shRNA) gave a slight bigger band of 500 bp (Figure 1D)
Summary
Diabetes is one of the fastest growing chronic diseases worldwide [1]. Type 2 diabetes (T2D) accounts for over 90% with more of the pre-diabetes population exhibiting an elevation of fasting glucose and/or impaired glucose tolerance [2]. Impaired response to insulin action in fat, muscle and liver cells induces a breakdown of fat, a failure of glycogenesis, and excess insulin secretion at early stages of diabetes [4,5]. Long-term compensating insulin secretion to insulin resistance may eventually lead to pancreatic beta cell failure [6]. Previous studies have suggested that neither peripheral tissue insulin resistance nor glucolipotoxicity fully explain the onset of beta cell dysfunction showing a delayed and insufficient insulin section to hyperglycemia in T2D. Deletion (knock out) of InsR expression results in a complete loss of insulin action through life, which is different from progressive impaired insulin action on beta cells. It is necessary to set up a long-term InsR knockdown model in vitro to investigate the molecular mechanism underlying pancreatic beta cell dysfunction
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