Knocking down Insulin Receptor in Pancreatic Beta Cell lines with Lentiviral-Small Hairpin RNA Reduces Glucose-Stimulated Insulin Secretion via Decreasing the Gene Expression of Insulin, GLUT2 and Pdx1

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.

Keywords: RNA interference; Type 2 diabetes; glucose uptake; insulin receptor; insulin resistance; insulin secretion; pancreatic beta cell dysfunction; pancreatic beta cells; shRNA.

Figure 1

Lentiviral vector and predicted InsRβ shRNAs. (A) The linear structure of pLL3.7; the shRNA expression cassette is under the control of U6 promoter and eGFP gene is under the CMV promoter. (B) The annealed shRNA expression cassette. (C) The finished stem-loop structure of a representative shRNA. (D) Agarose gel electrophoresis shows a 500 bp band cut from a positive InsRβ-pLL3.7 while band cut from pLL3.7 was 450 bp. (E) Sequencing results confirms the inserted shRNA-3 sequence is correct.

Figure 2

The morphology of transduced INS-1 cells. The transduced INS-1 cells showed strong green fluorescence (A) under a fluorescent microscope. The same view of the cells under white light field (B) show the cell morphology of transduced cells, indicating most of the cells were GFP-positive. From the view of morphology under the light microscope, the control un-transduced INS-1 cells (C) and InsRβKD INS-1 cells (D) were no much differences. The scale bar is 50 μm and all images share the same scale.

Figure 3

The stability of transduced INS-1 cells. After initial transductions of INS-1 cells with lentiviruses, GFP positive cells were sorted by FACS (left panels). After one-month culture of the positive cells, the InsRβKD and LV-7-14-INS-1 cells show high percentages of GFP-positive (right panels), suggesting the transduction is stable.

Figure 4

InsR expression in transduced cells. (A) qPCR result of measuring InsR mRNA levels in the 3 cell lines. The quantity level was firstly normalized to that of endogenous control glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and then expressed relative to that in INS-1 cells. (B) A representative Western blot analysis of InsR protein levels in INS-1, 7-14 INS-1, and InsRβKD cells. GAPDH protein was similarly analysed as the loading control. (C) The densitometry analysis of band intensities of InsR relative to these of GAPDH. * p < 0.05, n = 3.

Figure 5

InsRα and insulin mRNA expression, and insulin content in transduced cells. InsRα (A) and insulin (B) mRNA expressions were measured using qPCR. The mRNA expressions were normalized to that of GAPDH and then to that of INS-1 cells. (C) ELISA result of insulin levels in INS-1, 7-14 INS-1, and InsRβKD cells. InsRβKD cells showed a reduction of insulin levels compared to the controls. ** p < 0.01, n = 3.

Figure 6

GSIS and GLUT2 expression in transduced cells. (A) ELISA results of insulin secretion induced by 2 and 20 mM glucose and 25 mM KCl in INS-1, 7-14 INS-1, and InsRβKD cells. Compared to controls, InsRβKD cells showed significantly reduced insulin secretion at 20 mM glucose and 25 mM KCl stimulations. (B) GLUT2 mRNA expression by qPCR analysis, which was normalized to GAPDH expression and then to that of INS-1 cells. (C) A representative result of Western blot analysis for GLUT2 protein expression. (D) The densitometry analysis of band intensity of GLUT2 relative to GAPDH. * p < 0.05, ** p < 0.01, n = 3.

Figure 7

Radioactive 2-deoxyglucose uptake and Pdx1 expression in transduced cells. (A) Radioactivity of radioactive 2-deoxyglucose uptake expressed as CPM by a microplate scintillation counter. These results were then expressed relative to those in INS-1 cells. (B) The Pdx1 mRNA levels by qPCR analysis that was normalized to those of GAPDH and then to INS-1 cells. ** p < 0.01, n = 3.