Enhanced Steatosis and Fibrosis in Liver of Adult Offspring Exposed to Maternal High-Fat Diet

Abstract

Early life exposures can increase the risk of developing chronic diseases including nonalcoholic fatty liver disease. Maternal high-fat diet increases susceptibility to development of steatosis in the offspring. We determined the effect of maternal high-fat diet exposure in utero and during lactation on offspring liver histopathology, particularly fibrosis. Female C57Bl/6J mice were fed a control or high-fat diet (HFD) for 8 weeks and bred with lean males. Nursing dams were continued on the same diet with offspring sacrificed during the perinatal period or maintained on either control or high-fat diet for 12 weeks. Increased hepatocyte proliferation and stellate cell activation were observed in the liver of HFD-exposed pups. Offspring exposed to perinatal high-fat diet and high-fat diet postweaning showed extensive hepatosteatosis compared to offspring on high-fat diet after perinatal control diet. Offspring exposed to perinatal high-fat diet and then placed on control diet for 12 weeks developed steatosis and pericellular fibrosis. Importantly, we found that exposure to perinatal high-fat diet unexpectedly promotes more rapid disease progression of nonalcoholic fatty liver disease, with a sustained fibrotic phenotype, only in adult offspring fed a postweaning control diet.

Figure 1

Maternal diet exposure model. Scheme for breeding and experimental diet exposure for each group of offspring. Male and female mice were included in each group.

Figure 2

Maternal HFD exposure leads to prolonged stellate cell activation and increased cellular proliferation in perinatal offspring liver. (A) Hematoxylin and eosin (H&E) staining of representative liver from female mice fed HFD for 8 weeks prior to mating. (B) Body weight, liver weight, and body weight/liver weight ratio of maternal CD- or HFD-exposed offspring at p7. (C) Histology and IHC of offspring liver at p0, p7, p14, and p21 exposed to maternal CD or HFD. Representative photomicrographs shown with type of staining labeled on left. Scale bars: 100 μm (Sirius red), 50 μm (H&E, α-SMA, and PCNA). (D) Liver triglyceride levels at p0, p7, p14, and p21 from offspring exposed to maternal CD or HFD (n = 3–4 males per group). (E) TUNEL staining of offspring liver at p21 exposed to maternal CD or HFD. Scale bars: 100 μm. (F) CK19 and PCNA staining of offspring liver at p21 exposed to maternal CD or HFD, highlighting PCNA-positive cholangiocytes in HFD-exposed offspring. Scale bars: 20 μm. (G) Quantitation of PCNA-positive hepatocytes in p21 offspring liver exposed to maternal CD or HFD. *p < 0.05 HFD versus CD.

Figure 3

Progressive pathology in adult offspring previously exposed to maternal HFD. (A) Body weight of male and female mice over the time course of postweaning diet exposure (n = 4–9 per group). (B) Final liver weights of male and female mice after perinatal CD or HFD exposure followed by 12 weeks of postweaning CD or HFD. (C) Fasting glucose levels of mice at three time points during the course of postweaning diet exposure. (D) Average food intake (grams/week) over the course of the study. (E) Histology and IHC of adult offspring liver following perinatal CD or HFD exposure and 12 weeks of postweaning CD or HFD. Top row: H&E staining. Scale bars: 50 μm. Middle row: Sirius red staining. Scale bars: 100 μm. Bottom row: IHC for α-SMA. Scale bars: 50 μm. (F) Morphometric analysis of Sirius red staining (n = 5 livers per group with three fields/liver analyzed). (G) Photomicrograph of proteome array performed on pooled protein lysates (n = 3) from liver of offspring from each group. White asterisks denote loading control dots. Colored boxes identify select proteins on the array. (H) Representative photomicrograph of Western blot for osteopontin in adult offspring liver from each group. GAPDH is shown as a loading control. *p < 0.05 HFD/CD versus CD/CD, #p < 0.05 HFD/CD versus CD/HFD, &p < 0.05 HFD/CD versus HFD/HFD, %p < 0.05 HFD/HFD versus CD/CD.

Figure 4

Gene expression of markers of fibrosis, inflammation, and oxidative stress in adult offspring. (A) RT-PCR data for various factors involved in fibrosis. (B) RT-PCR data for proinflammatory cytokines. (C) RT-PCR data for NF-κB. (D) RT-PCR data for lipocalin-2. All data are presented as relative expression normalized to actin expression with secondary normalization to the average for CD/CD male. *p < 0.05, **p < 0.01, ***p < 0.001. Col1a1, collagen type 1 alpha 1; Col3a1, collagen type 3 alpha 1; TGF-β, transforming growth factor-β, MMP, matrix metalloprotease; Spp1, osteopontin; CTGF, connective tissue growth factor; IL, interleukin; NFκb1, nuclear factor kappa B; Lcn2, lipocalin-2.

Figure 5

Increased hepatocyte turnover and ductular reaction in liver of HFD/CD offspring. (A) Staining and IHC of adult offspring liver following perinatal CD or HFD exposure and 12 weeks of postweaning CD or HFD. Representative photomicrographs shown with type of staining labeled on the left. Scale bars: 100 μm for all images. (B) Quantitation of PCNA-positive hepatocytes from each group (n = 4–5 livers per group with three fields/liver counted). (C) Quantitation of TUNEL-positive hepatocytes from each group (n = 3–4 livers per group with three fields/liver counted). (D) Morphometric analysis of 4-HNE staining (n = 3 livers per group with three fields/liver analyzed). (E) Morphometric analysis of CK19 staining (n = 3 livers per group with three fields/liver analyzed). *p < 0.05 versus CD/CD, #p < 0.05 versus CD/HFD, &p < 0.05 versus HFD/HFD.