Interaction between maternal and offspring diet to impair vascular function and oxidative balance in high fat fed male mice

Abstract

Aims: To determine the impact of maternal and post-weaning consumption of a high fat diet on endothelium-dependent vasorelaxation and redox regulation in adult male mouse offspring.

Methods: Female C57BL6J mice were fed an obesogenic high fat diet (HF, 45% kcal fat) or standard chow (C, 21% kcal fat) pre-conception and throughout pregnancy and lactation. Post-weaning, male offspring were continued on the same diet as their mothers or placed on the alternative diet to give 4 dietary groups (C/C, HF/C, C/HF and HF/HF) which were studied at 15 or 30 weeks of age.

Results: There were significant effects of maternal diet on offspring body weight (p<0.004), systolic blood pressure (p = 0.026) and endothelium-dependent relaxation to ACh (p = 0.004) and NO production (p = 0.005) measured in the femoral artery. With control for maternal diet there was also an effect of offspring post-weaning dietary fat to increase systolic blood pressure (p<0.0001) and reduce endothelium-dependent relaxation (p = 0.022) and ACh-mediated NO production (p = 0.007). There was also a significant impact of age (p<0.005). Redox balance was perturbed, with altered regulation of vascular enzymes involved in ROS/NO signalling.

Conclusions: Maternal consumption of a HF diet is associated with changes in vascular function and oxidative balance in the offspring of similar magnitude to those seen with consumption of a high fat diet post-weaning. Further, this disadvantageous vascular phenotype is exacerbated by age to influence the risk of developing obesity, raised blood pressure and endothelial dysfunction in adult life.

Figure 1. Phenotype of male mouse offspring at 15 and 30 weeks of age.

Dams were assigned to either a high fat (HF) diet or standard chow (C) for 4 weeks before conception and during gestation and lactation. At weaning, offspring were assigned to C or HF to give four dietary groups C/C, HF/C, C/HF and HF/HF. Bar graphs represent mean ± SEM for (A) Body weight from 15 week C/C n = 9, HF/C n = 8, C/HF n = 7, HF/HF n = 9 and 30 week C/C n = 10, HF/C n = 10, C/HF n = 10, HF/HF n = 10 offspring, and (B) Systolic blood pressure measured by tail-cuff plethysmography from 15 week and 30 week offspring (C/C n = 7, HF/C n = 7, C/HF n = 5, HF/HF n = 9). Statistical comparisons were by ANOVA for the effects of maternal and offspring diet and age (see Table 2) followed by analysis using post hoc Dunnett’s multiple comparison tests for HF/C, C/HF and HF/HF vs. C/C. Values significantly different between high fat fed offspring groups and control offspring (C/C) at 15 or 30 weeks of age are indicated by ** p<0.001.

Figure 2. Impact of maternal HF-feeding on offspring vasoconstrictor responses in the femoral artery.

Cumulative dose response curves and maximum response (%) to the vasoconstrictor noradrenaline (NA) measured in the femoral artery of male mice offspring from four dietary groups at (A) 15 weeks of age and (B) 30 weeks of age C/C (○) H/FC (•) C/HF (□) or HF/HF (▪). Data are mean ± SEM (n = 6 per group).

Figure 3. Impact of maternal HF-feeding on offspring vasorelaxation in the femoral artery.

Cumulative dose response curves and % maximum response to the vasodilator (ACh) measured in the femoral artery of male mice offspring from four dietary groups at (A) 15 weeks (○ C/C n = 6, • HF/C n = 7, □ C/HF n = 6, ▪ HF/HF n = 7) and (B) 30 weeks of age. (○ C/C n = 8, • HF/C n = 6, □ C/HF n = 8, ▪ HF/HF n = 7). Data are mean ± SEM. Values significantly different between high fat fed offspring groups and control offspring (C/C) are indicated by * p<0.05, **p<0.001.

Figure 4. Impact of maternal HF-feeding on offspring NO-component of the ACh-induced vasorelaxation.

Femoral dilation to ACh alone (open columns) or in the presence of L-NAME (100 µM) (shaded columns) in the four male offspring groups at 15 weeks of age. Within offspring group statistical comparisons were made using a paired t test. Data are mean ± SEM, (n = 4–6 per group). * p<0.01.

Figure 5. Impact of maternal HF-feeding on offspring NO production in femoral artery.

(A) Basal NO production and (B) ACh-stimulated NO production in femoral arteries as detected by using 4,5-diaminofluorescein diacetate (DAF-FM) normalised to NO production by C/C offspring group (n = 3 per group). Data are expressed as mean ± SEM. Statistical comparisons were by ANOVA for the effects of maternal and offspring diet and age followed by comparisons using Dunnett’s Multiple Comparison Test for HF/C, C/HF and HF/HF vs. C/C. Values significantly different between high fat fed offspring groups and control offspring (C/C) at 15 or 30 weeks of age are indicated by * p<0.05, **p<0.01. (Please see Figure S4 for confocal images of DAF staining in the four offspring groups.).

Figure 6. Impact of maternal HF-feeding on offspring redox regulation.

Superoxide generation in (A) fresh femoral artery segments from 15 weeks (n = 4 per group) and (B) vastus muscle from 30 week old offspring (n = 5 per group) assessed using the redox-sensitive dye dihydroethidium (DHE) staining (5 µM in PBS). Representative confocal images obtained from femoral artery and vastus segments from the four offspring groups are shown above each bar graph. Scale bar = 250 µm. Bar graphs represent mean ± SEM. Statistical comparisons were by ANOVA for the effects of maternal and offspring diet followed by comparisons using Dunnett’s Multiple Comparison Test for HF/C, C/HF and HF/HF vs. C/C. * p<0.05).

Figure 7. Impact of maternal HF-feeding on offspring redox regulation. in the liver.

NADPH activity in liver homogenates estimated using a lucigenin chemiluminescence assay. Values are expressed as mean light units (AU) per mg protein. Each determination was performed in triplicate and results obtained were normalized for total protein. Bar graphs represent mean ± SEM from n = 6 per group.

Figure 8. Impact of maternal HF-feeding on offspring Nox2 and HO-1 protein expression.

A, Representative immunoblots of Nox2, HO-1 and α-tubulin. B, Densitometric analyses of Nox2 and HO-1 relative to α-tubulin and normalised to C/C in aortic tissue lysates from 30 week old male offspring from the four dietary groups. Data are mean ± SEM from C/C n = 5, HF/C n = 5 C/HF n = 5, HF/HF n = 4 offspring per group.

Figure 9. Schematic of processes by which the pre-natal environment ‘primes’ for vascular dysfunction in the offspring.