Growth deficits in cystic fibrosis mice begin in utero prior to IGF-1 reduction

Abstract

Growth deficits are common in cystic fibrosis (CF), but their cause is complex, with contributions from exocrine pancreatic insufficiency, pulmonary complications, gastrointestinal obstructions, and endocrine abnormalities. The CF mouse model displays similar growth impairment despite exocrine pancreatic function and in the absence of chronic pulmonary infection. The high incidence of intestinal obstruction in the CF mouse has been suggested to significantly contribute to the observed growth deficits. Previous studies by our group have shown that restoration of the cystic fibrosis transmembrane conductance regulator (CFTR) in the intestinal epithelium prevents intestinal obstruction but does not improve growth. In this study, we further investigate growth deficits in CF and gut-corrected CF mice by assessing insulin-like growth factor 1 (IGF-1). IGF-1 levels were significantly decreased in CF and gut-corrected CF adult mice compared to wildtype littermates and were highly correlated with weight. Interestingly, perinatal IGF-1 levels were not significantly different between CF and wildtype littermates, even though growth deficits in CF mice could be detected late in gestation. Since CFTR has been suggested to play a role in water and nutrient exchange in the placenta through its interaction with aquaporins, we analyzed placental aquaporin expression in late-gestation CF and control littermates. While significant differences were observed in Aquaporin 9 expression in CF placentas in late gestation, there was no evidence of placental fluid exchange differences between CF and control littermates. The results from this study indicate that decreased IGF-1 levels are highly correlated with growth in CF mice, independent of CF intestinal obstruction. However, the perinatal growth deficits that are observed in CF mice are not due to decreased IGF-1 levels or differences in placenta-mediated fluid exchange. Further investigation is necessary to understand the etiology of early growth deficits in CF, as growth has been shown to be a significant factor in disease outcomes.

Fig 1. Intestinal correction of CFTR does not improve growth.

(A) Weight of CF mice, gut-corrected CF mice and control littermates was assessed up to 40 days of age (n≥10 of each sex). Weight was significantly decreased in CF and gut-corrected CF mice compared to control animals at every age measured (P<0.001) with equal numbers of each sex used. (B) Length and (C) inguinal fat weight of CF mice, gut-corrected CF mice and control littermates was assessed between 6–7 weeks of age (n≥10 of each sex). Both length and inguinal fat weight was significantly decreased in CF and gut-corrected CF mice compared to control animals independent of sex (* = P<0.05; data represent mean±SE). (D) Survival of CF and gut-corrected CF between 10–42 days of age. All deaths were the result of intestinal obstruction as verified by postmortem examination.

Fig 2. IGF-1 levels are reduced in CF and gut-corrected CF mice and correlate with growth.

(A) Igf1 expression and (B) IGF-1 serum levels are significantly reduced at 6 weeks of age in both CF and gut-corrected CF mice compared to control littermates (* = P<0.05; n≥6; data represent mean±SE). IGF-1 levels were significantly correlated with weight in (C) CF and (D) gut-corrected CF mice (P<0.05; n≥10).

Fig 3. Reduced growth in CF mice occurs prenatally in the absence of reduced IGF-1.

(A) A Mendelian distribution was observed in fetuses originating from Cftr heterozygote matings. (n>100) (B) Weight was significantly reduced in newborn CF mice and e18 fetuses compared to control littermates but the groups did not differ at e15. (C) IGF-1 levels were not significantly different between CF and control littermates perinatally (e18 and newborn) but IGF-1 levels were significantly reduced between CF and control littermates at 3 weeks of age. (* = P<0.05; n≥15; data represent mean±SE)

Fig 4. Placental aquaporin expression in CF mice.

Aquaporin(Aqp) 1,3,8 and 9 expression was evaluated in placentas from e15 and e18 CF and control littermates. Only Aqp9 at e18 was significantly different between placentas from CF and control littermates (* = P<0.005) Each measurement is an average of six mice completed in replicates (data represent mean±SEM).

Fig 5. Placental-mediated fluid exchange is not different between CF and control fetuses.

(A) Fetal body and placental water weight at e18 was not significantly different between CF and control fetuses. (B) Equilibration of ²H₂O between dam and CF and control fetuses at e18 were not significantly different. (n≥10; data represent mean±SEM).