Consequences of a Maternal High-Fat Diet and Late Gestation Diabetes on the Developing Rat Lung

Abstract

Rationale: Infants born to diabetic or obese mothers are at risk of respiratory distress and persistent pulmonary hypertension of the newborn (PPHN), conceivably through fuel-mediated pathogenic mechanisms. Prior research and preventative measures focus on controlling maternal hyperglycemia, but growing evidence suggests a role for additional circulating fuels including lipids. Little is known about the individual or additive effects of a maternal high-fat diet on fetal lung development.

Objective: The objective of this study was to determine the effects of a maternal high-fat diet, alone and alongside late-gestation diabetes, on lung alveologenesis and vasculogenesis, as well as to ascertain if consequences persist beyond the perinatal period.

Methods: A rat model was used to study lung development in offspring from control, diabetes-exposed, high-fat diet-exposed and combination-exposed pregnancies via morphometric, histologic (alveolarization and vasculogenesis) and physiologic (echocardiography, pulmonary function) analyses at birth and 3 weeks of age. Outcomes were interrogated for diet, diabetes and interaction effect using ANOVA with significance set at p≤0.05. Findings prompted additional mechanistic inquiry of key molecular pathways.

Results: Offspring exposed to maternal diabetes or high-fat diet, alone and in combination, had smaller lungs and larger hearts at birth. High-fat diet-exposed, but not diabetes-exposed offspring, had a higher perinatal death rate and echocardiographic evidence of PPHN at birth. Alveolar mean linear intercept, septal thickness, and airspace area (D2) were not significantly different between the groups; however, markers of lung maturity were. Both diabetes-exposed and diet-exposed offspring expressed more T1α protein, a marker of type I cells. Diet-exposed newborn pups expressed less surfactant protein B and had fewer pulmonary vessels enumerated. Mechanistic inquiry revealed alterations in AKT activation, higher endothelin-1 expression, and an impaired Txnip/VEGF pathway that are important for vessel growth and migration. After 3 weeks, mortality remained highest and static lung compliance and hysteresis were lowest in combination-exposed offspring.

Conclusion: This study emphasizes the effects of a maternal high-fat diet, especially alongside late-gestation diabetes, on pulmonary vasculogenesis, demonstrates adverse consequences beyond the perinatal period and directs attention to mechanistic pathways of interest. Findings provide a foundation for additional investigation of preventative and therapeutic strategies aimed at decreasing pulmonary morbidity in at-risk infants.

Fig 1. Experimental paradigm.

Female Sprague Dawley rats were fed either control (CD) or high-fat (HF) diet at least 4 weeks prior to mating and bred with normal males. A positive vaginal swab for spermatozoa was deemed gestational day zero of pregnancy (GD0). On GD14, dams were injected with either citrate buffer (CB) vehicle or streptozotocin (STZ) to induce diabetes, which was controlled with sliding scale insulin twice daily. Offspring from four groups were analyzed on day one of life (NB1) or cross fostered to normal dams and analyzed at 3 weeks of age.

Fig 2. Perinatal mortality rate.

Perinatal mortality is expressed by group as a scatterplot. Individual marks represent the percent of each litter that is dead by newborn day one. The average mortality rate is represented by the median (horizontal bar). * indicates diet associated differences by 2-way ANOVA (p<0.05). CD-CB, controls; CD-STZ, diabetes exposed; HF-CB, high-fat diet exposed; HF-STZ, combination (high-fat and diabetes) exposed. N = 11–13 litters per group.

Fig 3. Alveolar and vessel histology in newborn lungs.

(A) Representative hematoxylin and eosin staining in fixed newborn rat lung (20X). Comparison of (B) mean linear intercept, (C) septal thickness, and (D) airspace area (D₂) as quantified using ImageJ to analyze five images per lung with four animals per litter (n = 2–4 litters/group). (E) Representative immunohistochemistry of fixed newborn rat lung processed with von Willebrand factor (vWF) stain to identify endothelial cells (indicated by arrows; see inset for staining details). (F) vWF-positive vessels were quantified from five representative images per lung with four animals per litter (n = 2–4 litters/group). Data are expressed as mean±SEM across treatment groups. * indicates significant diet associated changes by 2-way ANOVA. Significance set at p<0.05. CD-CB, controls; CD-STZ, diabetes exposed; HF-CB, high-fat diet exposed; HF-STZ, combination (high-fat and diabetes) exposed.

Fig 4. Maturity assessment in newborn lungs.

(A) Representative glycogen deposition in fixed newborn rat lung stained using PAS stain (60X). (B) SDS-PAGE and immunoblot of newborn rat lung lysates using anti-T1α, anti-SP-B and anti-SP-C with β-actin as a loading control. Each lane represents equal protein contributions of multiple offspring (n = 2–4) from a single litter. Densitometric analysis of (C) T1α (D) SP-B and (E) SP-C protein expression. Data are expressed as mean±SEM. * indicates significant diet and ^± diabetes associated changes by 2-way ANOVA. Significance set at p<0.05. CD-CB, controls; CD-STZ, diabetes exposed; HF-CB, high-fat diet exposed; HF-STZ, combination (high-fat and diabetes) exposed.

Fig 5. Alveolar and vessel histology in 3-week-old lungs.

Quantification of (A) mean linear intercept and (B) airspace area (D₂) using ImageJ to analyze five images per lung across four animals per litter (n = 4 litters). Representative images of 3 week-old rat lungs stained to identify (C) collagen deposition with trichrome staining (10X) and (D) endothelial cells using immunohistochemistry for von Willebrand factor (vWF) (endothelial cells are indicated by arrows; see inset for details). (E) vWF-positive vessels were quantified from five representative images per lung with four animals per litter (n = 4 litters). Data are expressed as mean±SEM across treatment groups. CD-CB, controls; CD-STZ, diabetes exposed; HF-CB, high-fat diet exposed; HF-STZ, combination (high-fat and diabetes) exposed.

Fig 6. Pulmonary function testing in 3-week-old offspring.

Respiratory physiology was evaluated in 3-week-old offspring via mechanical ventilation using the FlexiVent FX system. Computer-controlled perturbations measured (A) pressure-volume curves, (B) PV loop area (C) static compliance and (D) airway resistance. Quantifiable data are expressed as mean±SEM (n = 3–7 per group). A significant interaction effect was found in all measures by two-way ANOVA and differences that remained significant by one-way ANOVA with Dunnett’s post-test comparison to controls are indicated by ^†. Significance set at p<0.05. CD-CB, controls; CD-STZ, diabetes exposed; HF-CB, high-fat diet exposed; HF-STZ, combination (high-fat and diabetes) exposed.