High Glucose Causes Apoptosis in Cultured Human Pancreatic Islets of Langerhans
A Potential Role for Regulation of Specific Bcl Family Genes Toward an Apoptotic Cell Death Program
Article Figures & Tables
Figures
- FIG. 1.
A-E: Validation of RT-PCR for amplification of Bcl and housekeeping genes in human islets cultured in high glucose versus normal glucose. Cycle titration curves of RT-PCR amplification for housekeeping genes, cyclophyllin (A) and α-tubulin (B), and Bcl family genes, Bcl-xl (C), Bad (D), and Bid (E), in human islets treated under normal glucose (NG5, •) or high glucose (HG5, ○) conditions. Means ± SD of two separate experiments of relative expression of the genes are shown in graph.
- FIG. 2.
A-H: Assessment of apoptosis in human islets cultured in high glucose versus normal glucose. A: Human islets (400 per experimental point) were cultured under HG5, NG5 (7 preparations), and NG5+MAN5 (3 preparations) conditions. At the end of treatment, the islets were dissociated and the cells were fixed and analyzed for apoptosis after propidium iodide staining, by flow cytometry. The extent of apoptosis was increased in HG5 islets compared with NG5 islets and NG5+MAN5 (**P < 0.01 and *P < 0.05 by ANOVA). The data represent the means ± SD from four independent preparations (NG5 and HG5) and three independent preparations (NG5, HG5, and NG5+MAN5), respectively, analyzed in duplicate; each experimental point was evaluated twice by flow cytometry counting 10,000 events. B: Fluorescent DNA fragmentation assays and immunostaining for insulin were performed in dissociated human islets cultured under HG5 and NG5 conditions to analyze apoptosis (TUNEL) and relative abundance of β-cells and non–β-cells. Fluorescence microscopic fields were photographed individually and merged to detect β-cell death under NG5 and HG5 conditions. The extent of apoptosis was increased in HG5 islets compared with NG5 islets (*P < 0.05, by ANOVA). The data represent the means ± SD from four independent preparations. The number of apoptotic cells was evaluated by counting the number of TUNEL-positive cells in each field (100 cells for field) by two independent researchers in a blinded manner on a total of 103 dissociated cells from each preparation. In C-H, dissociated β-cells from human islets (insulin staining, red), treated as described above, are shown. Fluorescent DNA fragmentation was detected by TUNEL (green). Representative fields for NG5 and HG5 are C-E and F-H, respectively.
- FIG. 3.
A-D: Immunoelectron microscopy of human islets cultured in high glucose versus normal glucose. Immunoelectron microscopic analysis of islets was performed as described in research design and methods. A: Human pancreatic β-cells cultured under NG5 conditions show densely granulated insulin-containing cells. B: Intense gold-immunolabeling of insulin-containing granules. C: β-cells appeared completely degranulated after exposure to HG5 conditions. D: β-cells cultured under HG5 conditions showed features of a proapoptotic stage.
- FIG. 4.
A-E: Bcl family gene regulation in human islets cultured in high versus normal glucose. Expression of Bcl-2, Bcl-xl, Bad, Bid, and Bik mRNA was detected by RT-PCR and quantified by FluorImager analysis of ethidium bromide signal. In each experiment, band densities were normalized against cyclophyllin, and the results are expressed as mRNA level to NG1 control islets (NG1 = 100%). A: Bcl-2. B: Bcl-xl (HG5 vs. NG5, **P < 0.01). C: Bad (HG5 vs. NG5, **P < 0.01). D: Bid (HG5 vs. NG5, ***P < 0.001). E: Bik (HG5 vs. NG5, **P < 0.01). One representative gel is also shown. Islets from six donors were analyzed. Means ± SD of relative expression of the genes are shown in bar graph. Statistical analysis was performed by ANOVA.
- FIG. 5.
A-H: Immunofluorescence analysis of Bcl family genes in normal human pancreas. Insulin-positive signal was used as a marker of islets of Langerhans. Sections were stained with the following antibodies: anti-Bid (A); anti-Bad (C); anti-Bik (E); and anti–Bcl-xl (G). The same sections were double stained with anti-insulin antibodies to identify islets of Langerhans (B, D, F, and H). Note the robust expression of Bid and Bad in islets, and expression of Bik and Bcl-xl in both endocrine and exocrine cells (original magnification 150×).
- FIG. 6.
A-N: Immunofluorescence analysis of Bcl family genes in human islets of Langerhans. Human pancreatic sections were stained with the following antibodies: anti-Bid (A); anti-Bad (E); anti-Bik (G); anti–Bcl-xl (L); anti-insulin (B and D); antiglucagon (H); and antichromogranin (M). Note the expression of proteins in the cytoplasmic compartment of cells. Merged confocal sections showed almost complete colocalization of Bid and Bad with insulin (C and F, respectively), a partial colocalization of Bik with glucagon (I), and a partial colocalization of Bcl-xl with chromogranin (N) (original magnification 330×).
Tables
- TABLE 1
Insulin and proinsulin secretion and content of human islets of Langerhans cultured in NG5, HG5, and NG5+MAN5
NG5 HG5 NG5+MAN5 Chronic insulin release (nmol/l/mg/day) 925.50 ± 129.10* 2,843.00 ± 337.02 1,060.40 ± 186.50* Basal insulin secretion (nmol/l/mg/min) 0.0574 ± 0.007 0.043 ± 0.007 0.064 ± 0.004 Stimulated insulin release (nmol/l/mg/min) 0.49 ± 0.035† 0.129 ± 0.035 0.68 ± 0.07† Insulin content (nmol/l/mg/min) 101 ± 25‡ 23.9 ± 5.7‡ 284.1 ± 16.7‡ Chronic proinsulin release (nmol/l/mg/day) 0.26 ± 0.14§ 1.83 ± 0.131 0.39 ± 0.12§ Proinsulin content (nmol/l/mg/min) 15.10 ± 0.02∥ 6.60 ± 0.01∥ 39.05 ± 0.03∥ Data are means ± SD of three separate experiments in three different human islet preparations and analyzed by ANOVA.
* NG5 vs. HG5 and NG5+MAN5 vs. HG5 (P < 0.01);
† NG5 vs. HG5 and NG5+MAN5 vs. HG5 (P < 0.01);
‡ NG5 vs. HG5, NG5+MAN5 vs. HG5, NG5 vs. NG5+MAN5 (P < 0.01);
§ NG5 vs. HG5 and NG5+MAN5 vs. HG5 (P < 0.001);
∥ NG5 vs. HG5, NG5+MAN5 vs. HG5, NG5 vs. NG5+MAN5 (P < 0.001).
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