Effects of maternal diet-induced obesity on metabolic disorders and age-associated miRNA expression in the liver of male mouse offspring

Abstract

Objective: This study investigated the effect of maternal obesity on aged-male offspring liver phenotype and hepatic expression of a programmed miRNA.

Methods: A mouse model (C57BL/6 J) of maternal diet-induced obesity was used to investigate fasting-serum metabolites, hepatic lipid content, steatosis, and relative mRNA levels (RT-PCR) and protein expression (Western blotting) of key components involved in hepatic and mitochondrial metabolism in 12-month-old offspring. We also measured hepatic lipid peroxidation, mitochondrial content, fibrosis stage, and apoptosis in the offspring. To investigate potential mechanisms leading to the observed phenotype, we also measured the expression of miR-582 (a miRNA previously implicated in liver cirrhosis) in 8-week-old and 12-month-old offspring.

Results: Body weight and composition was similar between 8-week-old offspring, however, 12-month-old offspring from obese mothers had increased body weight and fat mass (19.5 ± 0.8 g versus 10.4 ± 0.9 g, p < 0.001), as well as elevated serum levels of LDL and leptin and hepatic lipid content (21.4 ± 2.1 g versus 12.9 ± 1.8 g, p < 0.01). This was accompanied by steatosis, increased Bax/Bcl-2 ratio, and overexpression of p-SAPK/JNK, Tgfβ1, Map3k14, and Col1a1 in the liver. Decreased levels of Bcl-2, p-AMPKα, total AMPKα and mitochondrial complexes were also observed. Maternal obesity was associated with increased hepatic miR-582-3p (p < 0.001) and miR-582-5p (p < 0.05). Age was also associated with an increase in both miR-582-3p and miR-582-5p, however, this was more pronounced in the offspring of obese dams, such that differences were greater in 12-month-old animals (-3p: 7.34 ± 1.35 versus 1.39 ± 0.50, p < 0.0001 and -5p: 4.66 ± 1.16 versus 1.63 ± 0.65, p < 0.05).

Conclusion: Our findings demonstrate that maternal diet-induced obesity has detrimental effects on offspring body composition as well as hepatic phenotype that may be indicative of accelerated-ageing phenotype. These whole-body and cellular phenotypes were associated with age-dependent changes in expression of miRNA-582 that might contribute mechanistically to the development of metabolic disorders in the older progeny.

Fig. 1. Body weight, body composition, hepatic lipid content and steatosis grades in 12-month-old offspring.

Average body weight (a), average absolute lean mass (b), average absolute fat mass (c), fat mass of intra-abdominal, retroperitoneal and epididymal fat depots (d), total hepatic lipid content (e), hepatic lipid content as percentage of liver weight (f), representative liver sections stained with H&E and percentage of steatosis grades in the CC group (g) and OC group (h) at 12 months of age. CC 12 m: mother received control diet and offspring-fed control diet after weaning up to 52 weeks of age; OC 12 m: mother received obesogenic diet and offspring-fed control diet after weaning up to 52 weeks of age. Values are presented as means ± SEM; CC 12 m: n = 7–13 and OC 12 m: n = 8–9 animals from independent litters for each group. Data were analyzed by unpaired student’s t test or Mann–Whitney U test. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Fig. 2. AMPKα protein expression and mitochondrial phenotype in the liver of 12-month-old offspring.

Protein levels of p-AMPKα (Thr172) and total AMPKα (a), representative images of protein expression (b), and relative protein expression of citrate synthase and mitochondrial OXPHOS complexes (c) in the liver of the offspring at 12 months of age. CC 12 m: mother received control diet and offspring-fed control diet after weaning up to 52 weeks of age; OC 12 m: mother received obesogenic diet and offspring-fed control diet after weaning up to 52 weeks of age. Values are presented as means ± SEM; CC 12 m: n = 9 and OC 12 m: n = 9 animals from independent litters for each group. Data were analyzed by unpaired student’s t test. *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig. 3. Hepatic fibrosis and apoptosis in 12-month-old offspring.

Quantification of Picrosirius red staining (a), relative mRNA levels of Col1a1, Col3a1 and Col4a1 (b), representative images of protein expression (c), relative protein expression of p-SAPK/JNK, Bax and Bcl-2 (d), and percentage of TUNEL-positive cells (e) in the liver of the offspring at 12 months of age. CC 12 m: mother received control diet and offspring-fed control diet after weaning up to 52 weeks of age; OC 12 m: mother received obesogenic diet and offspring fed control diet after weaning up to 52 weeks of age. Values are presented as means ± SEM; CC 12 m: n = 7–9 and OC 12 m: n = 8–9 animals from independent litters for each group. Data were analyzed by unpaired student’s t test or Mann–Whitney U test or Kolmogorov–Smirnov test. *p < 0.05 and **p < 0.01.

Fig. 4. Age-related changes in the miR-582 hepatic expression in the 8-week-old and 12-month-old offspring.

Relative expressions of miR-582-3p and miR-582-5p in the liver of the offspring at eight weeks and 12 months of age (a) and relative mRNA expression of phosphodiesterase 4D in the liver of the offspring at 12 months of age (b). CC 8w: mother received control diet and offspring-fed control diet after weaning up to eight weeks of age; OC 8w: mother received obesogenic diet and offspring-fed control diet after weaning up to eight weeks of age. CC 12 m: mother received control diet and offspring-fed control diet after weaning up to 52 weeks of age; OC 12 m: mother received obesogenic diet and offspring-fed control diet after weaning up to 52 weeks of age. Values are presented as means ± SEM; CC 8w: n = 8 and OC 8w: n = 8; CC 12 m: n = 8–9 and OC 12 m: n = 9 animals from independent litters for each group. Data were analyzed by Mann–Whitney U test or two-way ANOVA. *p < 0.05 and ****p < 0.0001.