Increased transcriptional preproinsulin II beta-cell activity in neonatal nonobese diabetic mice: in situ hybridization analysis

Abstract

In the prediabetic nonobese diabetic (NOD) mouse, a spontaneous model of type 1 diabetes, we previously reported transient postweaning hyperinsulinemia followed by progressive islet hyperplasia. A modified in situ hybridization technique was used to determine whether these effects were accompanied by changes in insulin transcriptional activity as a function of age. We found that NOD neonates express higher levels of preproinsulin II primary transcripts than age-matched C57BL/6 mice, but this difference disappeared within the first wk of age. To manipulate insulin transcriptional activity in NOD neonates, NOD mothers were treated with insulin during the last two wk of gestation. A down-regulation of beta-cell hyperactivity was observed in female NOD neonates but not in male neonates. By contrast, the same insulin treatment applied to NODscid (severe combined immunodeficiency) mothers, devoid of functional lymphocytes but showing like NOD mice postweaning hyperinsulinemia, increased transcriptional beta-cell activity in both sexes of neonates. In conclusion, NOD mice exhibit successive and transient signs of beta-cell hyperactivity, reflected as early as birth by high transcriptional preproinsulin II activity and later, from weaning to around 10 wk of age, by hyperinsulinemia. Of note, when thinking in terms of in utero disease programming, the NOD neonatal transcriptional beta-cell hyperactivity could be modulated by environmental (maternal and/or fetal) factors.

Figure 1

Preproinsulin II primary transcript (ppIns II) expression is restricted to the nucleus of a subpopulation of β-cells in the islet. In situ hybridization for the ppIns II was carried out on paraffin-embedded sections as described in the materials and methods. Serial pancreatic sections for (A) in situ hybridization and (B) immunostaining for proinsulin and glucagon: the specific signal of in situ hybridization is localized in β-cell nuclei (A), when compared to proinsulin (brown) and glucagon (black) staining (B), using an anti-sense probe, specific signal for ppIns II were restricted to the nucleus of a subset of β-cells (C), when in situ hybridization was carried out with a sense probe (D), there was a complete absence of staining. Magnification factors: (A) and (B) x400, (C) and (D) x1600.

Figure 2

Transcriptional activity is increased in neonatal NOD mice. In situ hybridization for the ppIns II was carried out on paraffin-embedded sections as described in the materials and methods. A VIDAS image analysis system was used to semi-quantify the number and intensity of stained nuclei. The number of positive nuclei per islet area was measured at 4 different thresholds (25, 50, 75, 100) of staining intensity. These threshold values were arbitrarily assigned to encompass the range of staining intensities. The graph shows the number of nuclei stained per islet area at different arbitrarily assigned thresholds of intensity in NOD (●-●) and C57Bl/6 (○-○) mice during the early developmental period. Means ± SEM of 10 mice per age group in each strain, p-values from 0.02 to 0.00003.

Figure 3

Maternal insulin treatment during the two last weeks of gestation decreases the degree of insulitis. In both vehicle- or insulin-treated groups, islets were examined and the respective percentages of intact, peri-infiltrated and severely damaged islets were determined. Values are means ± SEM (n = 951 and n = 984 islets in vehicle- and insulin-treated NOD mothers, respectively, p = 0.025 and p = 0.028 for unaffected islets and islets showing insulitis, respectively).

Figure 4

Insulin treatment of NOD mothers diminishes ppIns II primary transcript expression in neonatal NOD females but not in males. Results are expressed as in Figure 2. The graph shows the number of nuclei stained per islet area at one threshold of intensity (50), in 1-day-old NOD neonates from mothers treated with vehicle (□) or insulin (■, 1U/100g bw). Means ± SEM of 4 mice per treatment group in each sex; p < 0.01.

Figure 5

Insulin treatment of NODscid mothers increases ppIns II primary transcript expression in both sexes of NODscid neonates. Results are expressed as in Figure 4 at one threshold of intensity (50). Means ± SEM of 4 mice per treatment group in each sex; p < 0.05.