Insulin regulation of messenger ribonucleic acid for the surfactant-associated proteins in human fetal lung in vitro.

Abstract

Respiratory distress syndrome (RDS) is primarily caused by an immaturity in the synthesis and secretion of surfactant by the fetal lung type II cell. Fetal hyperinsulinemia associated with maternal diabetes places the newborn at an increased risk of developing RDS, and therefore, it has been hypothesized that insulin inhibits type II cell differentiation. We have previously shown that insulin inhibits the accumulation of surfactant-associated protein A (SP-A), the major surfactant-associated protein, in human fetal lung explants maintained in vitro. In the present study, we used Northern blot analysis to evaluate the effects of insulin on the content of SP-A messenger RNA (mRNA) as well as on the content of mRNA for the hydrophobic surfactant-associated proteins SP-B and SP-C in human fetal lung explants maintained in vitro. Lung explants were maintained in serum-free medium with or without added insulin (0.25-2500 ng/ml) for up to 6 days. We observed that insulin, at concentrations of 25-2500 ng/ml, significantly inhibited the accumulation of SP-A mRNA when compared to controls (P less than 0.01). The inhibitory effect of insulin on SP-A mRNA accumulation was dose dependent with an approximately 75% inhibition observed at 2500 ng/ml. Insulin, at the concentration of 2500 ng/ml, significantly inhibited the accumulation of SP-B mRNA by approximately 30% when compared to control levels (P less than 0.01) but had no effect at lower concentrations. Insulin had no significant effect on SP-C mRNA levels at any concentration tested. Our findings provide evidence that insulin may delay fetal lung development by inhibiting SP-A and SP-B gene expression. A deficiency of these proteins in pulmonary surfactant may account for the increased incidence of RDS in infants of diabetic mothers.

Intrauterine growth restriction delays surfactant protein maturation in the sheep fetus

“…As fetal hyperinsulinemia associated with maternal diabetes is associated with a reduction in SP-A protein in amniotic fluid (42) and an increased risk of developing RDS in the newborn, it has been hypothesized that insulin inhibits type II cell differentiation. Subsequently, it was shown that insulin administration to human fetal lung tissue explants inhibits both SP-A and SP-B gene expression (8). As the IUGR fetus is characterized by hypoglycemia and concomitant hypoinsulinemia (40), this pathway is unlikely to be the mechanism for the observed inhibition of SP mRNA or protein expression in the present study.…”

SP-A2 Gene Expression in Human Fetal Lung Airways

“…The frozen tissues (50-100 mg) were homogenized in 1 ml of RNA extraction mixture, and total RNA was isolated as described previously (22,23). The precipitated total RNA pellet was washed with 1 ml 75% ethanol, air-dried, and resuspended in 25 ml sterile diethylpropylcarbonate-treated water (24).…”

“…The precipitated total RNA pellet was washed with 1 ml 75% ethanol, air-dried, and resuspended in 25 ml sterile diethylpropylcarbonate-treated water (24). Following quantitation of total RNA by determining the ultraviolet (UV) absorbence at 260 nm, equal amounts of total RNA (10 g) from each sample were separated by gel electrophoresis on a 1.0% agarose/5% formaldehyde gel, and was capillary transferred to Nytran Plus membranes (Schleicher & Schuell, Keene, NH) using a phosphate buffer as described previously (23). The ethidium bromide-stained ribosomal RNA bands in the agarose gel were photographed on a UV lightbox.…”

In Situ Hybridization of SP-A mRNA in Adult Human Conducting Airways

“…Fetal lung explants (prepared from distal lung tissue) that had been maintained in vitro for 6 days and an adult lung adenocarcinoma cell line (NCI-H441 cells) were used as positive controls for some experiments [15,16]. Both the cultured fetal lung explants and the H44l cells produce both human SP-A proteins (SP-Al and SP-A2) and SP-B [11,17]. The fetal lung explants also produce SP-C [17].…”

“…Both the cultured fetal lung explants and the H44l cells produce both human SP-A proteins (SP-Al and SP-A2) and SP-B [11,17]. The fetal lung explants also produce SP-C [17].…”