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. 2002 Jun 11;99(12):8236–8241. doi: 10.1073/pnas.122686299

Figure 3.

Figure 3

FLIP protects human β cells from glucose-induced apoptosis and restores β cell proliferation. (A) Human islets were cultured on extracellular matrix-coated dishes for 4 days in 5.5 and 33.3 mM glucose (control) after transfection with an expression vector coding for FLIP (FLIP) with or without addition of the antagonistic Fas antibody ZB4 (FLIP + ZB4). Results are means ± SE of the relative number of Ki-67-positive (1) and TUNEL-positive β cells (2) per islet normalized to control incubations at 5.5 mM glucose alone (100%; in absolute values: 1.63 Ki-67-positive β cells per islet and 0.30 TUNEL-positive β cells per islet). The mean number of islets scored from each donor was 32 for each treatment condition. Islets were isolated from six organ donors. *, P < 0.001 relative to islets at 5.5 mM glucose; **, P < 0.01 relative to islets at 33.3 mM glucose; §, P < 0.01 relative to FLIP transfected islets at 33.3 mM glucose. (B) Islets were exposed for 4 days to media containing 5.5 mM glucose (1, 2, 7, 10) or 33.3 mM glucose (3, 4, 8, 11) or 33.3 mM glucose and after transfection with FLAG-tagged FLIP (5, 6, 9, 12). Detection of β cell proliferation with anti-Ki-67 (orange; 1, 3, 5) and with anti-insulin antibody (green; 2, 4, 6). Double-immunostaining (7–9) for insulin (orange) and DNA fragmentation by the TUNEL assay (black). Double-immunostaining (10–12) for cleaved caspase-3 (red) and insulin (green). The red arrows mark cells stained positive for Ki-67 and for insulin; the blue arrows mark β cells nuclei stained positive for the TUNEL reaction; the white arrows mark β cells stained positive for cleaved caspase-3 (×250, with higher magnifications of Ki-67+- (1–6) or TUNEL+- (8) β cells in the inserts. (C) Double-immunostaining for cytokeratin 19 (1) and insulin (2) in human islets cultured for 4 days in 5.5 mM glucose. The red arrows mark ductal cells (×250). (D) Islets were transfected with FLAG-tagged FLIP and exposed for 4 days to media containing 33.3 mM glucose. Double-immunostaining for FLAG (1) and insulin (2) or FLIP (3) and FLAG (4). In 3 and 4, images are of a selected field in which essentially all cells were seen to have been transfected (FLAG positive). (×200) (1, 2); confocal microscopy (3, 4).