Impairment of rat fetal beta-cell development by maternal exposure to dexamethasone during different time-windows

Abstract

Aim: Glucocorticoids (GCs) take part in the direct control of cell lineage during the late phase of pancreas development when endocrine and exocrine cell differentiation occurs. However, other tissues such as the vasculature exert a critical role before that phase. This study aims to investigate the consequences of overexposure to exogenous glucocorticoids during different time-windows of gestation for the development of the fetal endocrine pancreas.

Methods: Pregnant Wistar rats received dexamethasone acetate in their drinking water (1 µg/ml) during the last week or throughout gestation. Fetuses and their pancreases were analyzed at day 15 and 21 of gestation. Morphometrical analysis was performed on pancreatic sections after immunohistochemistry techniques and insulin secretion was evaluated on fetal islets collected in vitro.

Results: Dexamethasone given the last week or throughout gestation reduced the beta-cell mass in 21-day-old fetuses by respectively 18% or 62%. This was accompanied by a defect in insulin secretion. The alpha-cell mass was reduced similarly. Neither islet vascularization nor beta-cell proliferation was affected when dexamethasone was administered during the last week, which was however the case when given throughout gestation. When given from the beginning of gestation, dexamethasone reduced the number of cells expressing the early marker of endocrine lineage neurogenin-3 when analyzed at 15 days of fetal age.

Conclusions: GCs reduce the beta- and alpha-cell mass by different mechanisms according to the stage of development during which the treatment was applied. In fetuses exposed to glucocorticoids the last week of gestation only, beta-cell mass is reduced due to impairment of beta-cell commitment, whereas in fetuses exposed throughout gestation, islet vascularization and lower beta-cell proliferation are involved as well, amplifying the reduction of the endocrine mass.

Figure 1. Maternal body weight during pregnancy.

Dexamethasone was administered to the mother during the last week of gestation (DEXL) or throughout gestation (DEX). Values are means ± SEM, n = 4, **P<0.01, *P<0.05 vs Controls (C).

Figure 2. Islet size distribution in the 21-day-old fetuses.

Effect of dexamethasone on size-frequency distribution of the 21-day-old rat pancreatic islets. Values are means ± SEM, n = 9, * P<0.05, ** P<0.01, vs C.

Figure 3. Effect of dexamethasone on BrdU incorporation in beta-cells of 21-day-old fetuses.

(A) Beta-cells in S phase (blue) were counted on pancreatic section in colocalisation with insulin (brown). Beta-cells that are proliferating are indicated with arrowheads. (B) Percentage of proliferating beta-cells over total beta-cells. A total of 4500 to 6000 nuclei were counted in each group. Values are means ± SEM, n = 9 **P<0.01 vs Controls. Scale bar = 50 µm.

Figure 4. Effect of dexamethasone on NEUROG3 Expression in pancreas of 15-day-old fetuses.

(A) Detection of NEUROG3 positive cells in the duct network at day 15 (brown; arrowheads in insets), n = 6, Scale bar = 25 µm. (B) The number of positive nuclei was evaluated per pancreas surface area (100 µm2). Values are means ± SEM, **P<0.01 vs Controls. Scale bar = 20 µm.

Figure 5. Effect of dexamethasone on islet vascularization of 21 day-old fetuses.

(A and B) Semi-thin section (1 µm) of a pancreatic islet. Scale bar = 20 µm. (A) Inset: magnification of a blood vessel containing an erythrocyte (*). The arrow shows an endothelial cell. (B) The islet is surrounded by exocrine tissue and 6 blood vessels are highlighted in red in this islet. (C) Volumic and (D) numerical density of islet blood vessels as measured on semi-thin sections. Values are means ± SEM, n = 9,* P<0.05, vs Controls.

Figure 6. Effect of dexamethasone given to the mother throughout gestation on fetal neoformed islets.

(A) Dexamethasone reduced the islet insulin content. Values are the means of 9 observations pooled from three independent cultures (n = 3). Bars represent SEM,* P<0.05 vs C. (B) Dexamethasone also reduced the size of islets after the culture ,* P<0.05 vs C. (C) The ratio islets insulin content per islet size was increased by dexamethasone treatment. Values are means of 100 observations pooled from 3 independent cultures. Bars represent SEM,* P<0.05. (D) Insulin secretion capacity of fetal islets. Islets were incubated in Krebs-Ringer medium containing glucose at 2.5 or 16.7 mmol/L. Values are the means of 9 observations pooled from three independent cultures (n = 3). SEM, ** P<0.01.