Hyperinsulinemic hypoglycemia subtype glucokinase V91L mutant induces necrosis in β-cells via ATP depletion

Abstract

Hyperinsulinemic hypoglycemia subtype glucokinase (GCK-HH) is caused by an activating mutation in glucokinase (GCK) and has been shown to increase β-cell death. However, the mechanism of β-cell death in GCK-HH remains poorly understood. Here, we expressed the GCK-HH V91L GCK mutant in INS-1 832/13 cells to determine the effect of the mutation on β-cell viability and the mechanisms of β-cell death. We showed that expression of the V91L GCK mutant in INS-1 832/13 cells resulted in a rapid glucose concentration-dependent loss of cell viability. At 11 mM D-glucose, INS-1 832/13 cells expressing V91L GCK showed increased cell permeability without significant increases in Annexin V staining or caspase 3/7 activation, indicating that these cells are primarily undergoing cell death via necrosis. Over-expression of SV40 large T antigen, which inhibits the p53 pathway, did not affect the V91L GCK-induced cell death. We also found that non-phosphorylatable L-glucose did not induce rapid cell death. Of note, glucose phosphorylation coincided with a 90% loss of intracellular ATP content. Thus, our data suggest that the GCK V91L mutant induces rapid necrosis in INS-1 cells through accelerated glucose phosphorylation, ATP depletion, and increased cell permeability.

Keywords: Beta-cell death; Glucokinase; Hyperinsulinemic hypoglycemia; Hyperinsulinemic hypoglycemia subtype glucokinase; Necrosis.

Fig. 1

V91L GCK expression in INS-1 832/13 cells induces D-glucose-dependent cell death. (A) Immunoblot of HEK293T cells transduced with SIN-SFFV-GCK or SIN-SFFV-GCK-V91L and selected by puromycin. (B) Cell viability of INS-1 832/13 cells transduced with increasing SIN-SFFV-GCK-V91L vector titers and cultured with 11 mM D-glucose for 48 h after transduction (n = 8 per vector titer). (C) Immunoblot of non-transduced INS-1 832/13 cells and INS-1 832/13 cells transduced with SIN-SFFV-GCK or SIN-SFFV-GCK-V91L following puromycin selection. (D) Cell viability of non-transduced control INS-1 832/13 cells, INS-1 832/13 GCK cells, and INS-1 832/13 GCK V91L cells after culturing in increasing D-glucose concentrations for 24 h (n = 4 per group). (E) Real-time cell viability of INS-1 832/13, INS-1 832/13 GCK, and INS-1 832/13 GCK V91L cultured with 0 mM and 11 mM D-glucose (n = 6–7 per group). (F) Real-time Annexin V luminescence of INS-1 832/13, INS-1 832/13 GCK, and INS-1 832/13 GCK V91L cultured with 0 mM and 11 mM D-glucose (n = 6–7 per group). (G) Real-time cell permeability of INS-1 832/13, INS-1 832/13 GCK, and INS-1 832/13 GCK V91L cultured with 0 mM and 11 mM D-glucose (n = 6–7 per group). Real-time cell viability, Annexin V luminescence, and cell permeability measurements were taken 30 min, 1 h, and every 3 h after glucose addition for 48 h. Puromycin was supplemented at 25 µg/ml media as a positive cell death control. Results shown as line graphs with mean ± SEM. *p < 0.05 against control, # p < 0.05 against respective 0 mM D-glucose group, @ p < 0.05 against all other groups.

Fig. 2

D-glucose-induces cell death in INS-1 832/13 GCK V91L cells with minimal caspase 3/7 activation. Representative images from fluorescent live-cell imaging taken at 0 h and 48 h after media change. Non-transduced INS-1 832/13 control cells and INS-1 832/13 GCK V91L cells were imaged with 0 mM or 11 mM D-glucose. Caspase 3/7 activation was visualized using CellEvent Caspase-3/7 Green Detection Reagent (Invitrogen). Propidium iodide staining was visualized using the pSIVA Real-Time Apoptosis Fluorescent Microscopy Kit (Bio-Rad). Scale bar represents 100 µm.

Fig. 3

Glucose phosphorylation is required for D-glucose-induced cell death in INS-1 832/13 GCK V91L cells. (A) Immunoblot of INS-1 832/13 cells transduced with lentiviral vectors expressing the SV40 small and large T antigens. (B) Cell viability of non-transduced INS-1 832/13 cells, INS-1 832/13 GCK cells, and INS-1 832/13 GCK V91L cells 48 h after transduction with lentiviral vectors expressing the SV40 small and large T antigens in 0 mM or 11 mM D-glucose (n = 7 per group). (C) Cell viability of non-transduced INS-1 832/13 cells, INS-1 832/13 GCK cells, and INS-1 832/13 GCK V91L cells cultured for 24 h with 0 mM D-glucose, 11 mM D-glucose, 11 mM 2-DG, or 11 mM L-glucose (n = 7 per group). (D) Intracellular ATP content of non-transduced INS-1 832/13 cells, INS-1 832/13 GCK cells, and INS-1 832/13 GCK V91L cells after 1 h culture with 0 mM D-glucose, 11 mM D-glucose, 11 mM 2-DG, or 11 mM L-glucose (n = 7 per group). Results shown as bar graph with mean ± SEM. *p < 0.05.