Insulin-like growth factor binding protein-3 is a novel mediator of apoptosis in insulin-secreting cells

Abstract

Insulin-like growth factor binding protein-3 (IGFBP-3) is emerging as a critical regulator of cell survival. There has been no study which directly examined the potential role for this major growth factor in the programmed cell death (apoptosis) of insulin-secreting cells. To determine whether IGFBP-3 mediates apoptosis in insulin-secreting cells, we performed a rigorous series of experiments with the rat insulinoma (RIN) cell line m5F and the hamster insulin-secreting tumor (HIT) T-15. Within 24 h exogenous IGFBP-3 induced significant DNA fragmentation in RIN and HIT cells, at doses ranging from 4.4 to 2000 ng/ml (P<0.05) without a classic dose-response relationship. DNA fragmentation induced by rhIGFBP-3 occurred in the presence of immunoglobulin to block the type 1 IGF receptor. As detected by flow cytometry for Annexin V exposure to the cell surface, rhIGFBP-3 treatment doubled the proportion of apoptotic HIT cells from 1.7 +/- 0.4% (serum-free control) to 3.4 +/- 0.2% (P<0.02), an effect completely reversed by co-treatment with 1000 ng/ml rhIGF-I. Immunofluorescent microscopy disclosed that pro-inflammatory Th1 cytokines increased intranuclear aggregation of endogenous IGFBP-3. Cytokine-induced DNA fragmentation was completely blocked by relatively brief pre-treatment with antisense IGFBP-3 phosphorothioate oligodeoxynucleotides. In conclusion, we have presented the first evidence that IGFBP-3 contributes to cytokine-mediated apoptosis in insulin-secreting cells.

Fig. 1

DNA fragmentation ELISA of HIT cells exposed to serum-free basal (B) conditions or cytokines.

Fig. 2

Western ligand blots for IGFBPs in conditioned media of RIN cultures after 72 h with serum-free baseline (B) conditions or IL-1β (I). Cytokine treatment induced release of IGFBP-3 into conditioned media. Densitometry was performed by quantification of all pixels in the IGFBP-3 bands using the black channel, and all values below a level of 190 were summated. Vertical bars represent the total black pixel count in the IGFBP-3 band for each experiment.

Fig. 3

DNA fragmentation ELISA of HIT and RIN cultures in the presence of rhIGFBP-3 (BP3), rhIGF-I (IGF), neutralizing antibody to the type 1 IGF receptor (aIR), or genistein (G).

Fig. 4

DNA fragmentation ELISA of RIN cells exposed to rhIGFBP-3 in the presence of calphostin (CAL).

Fig. 5

Representative FACS for HIT cultures. Histogram compiles mean (±SE) data of Annexin V-positive cell populations (lower right panels) from triplicate FACS experiments, each counting 10,000 gated events per condition.

Fig. 6

Immunofluorescent microscopy of HIT cell cultures under serum-free conditions (A–C) and after treatment with TNF-α (D–F). Green stain depicts endogenous murine IGFBP-3, and DAPI stains heterochromatin blue. Histograms (G–H) depict means ± SD.

Fig. 7

DNA fragmentation ELISA of RIN (A) and HIT (B) cultures.