Transgenerational impact of maternal obesogenic diet on offspring bile acid homeostasis and nonalcoholic fatty liver disease

Abstract

Studies show maternal obesity is a risk factor for metabolic syndrome and nonalcoholic fatty liver disease (NAFLD) in offspring. Here we evaluated potential mechanisms underlying these phenotypes. Female C57Bl6 mice were fed chow or an obesogenic high-fat/high-sucrose (HF/HS) diet with subsequent mating of F₁ and F₂ female offspring to lean males to develop F₂ and F₃ generations, respectively. Offspring were fed chow or fibrogenic (high transfat, cholesterol, fructose) diets, and histopathological, metabolic changes, and bile acid (BA) homeostasis was evaluated. Chow-fed F₁ offspring from maternal HF/HS lineages (HF/HS) developed periportal fibrosis and inflammation with aging, without differences in hepatic steatosis but increased BA pool size and shifts in BA composition. F₁, but not F₂ or F₃, offspring from HF/HS showed increased steatosis on a fibrogenic diet, yet inflammation and fibrosis were paradoxically decreased in F₁ offspring, a trend continued in F₂ and F₃ offspring. HF/HS feeding leads to increased periportal fibrosis and inflammation in chow-fed offspring without increased hepatic steatosis. By contrast, fibrogenic diet-fed F₁ offspring from HF/HS dams exhibited worse hepatic steatosis but decreased inflammation and fibrosis. These findings highlight complex adaptations in NAFLD phenotypes with maternal diet.

Keywords: bile acid metabolism; fatty liver; liver fibrosis; maternal high-fat/high-sucrose diet.

Fig. 1.

Breeding scheme and experimental design. Top, F₀ female mice were fed high-fat/high-sucrose (HF/HS) diet or chow and bred with F₀ male mice fed chow. All mice from the F₁ through F₃ generations were fed chow. For baseline analysis (bottom), offspring were evaluated at 8 and 40 wk old (data presented in Figs. 2–5). Bottom, nonalcoholic steatohepatitis (NASH) model of high-transfat, cholesterol, fructose (HTFC) diet feeding in male offspring (data presented in Figs. 6–9). Diets: Chow [kcal: 13% fat, 62% carbohydrates (3.2% sucrose), and 25% protein], HF/HS [kcal: 59% fat, 26% carbohydrates (17% sucrose), and 15% protein], and HTFC [kcal: 40% fat, 40% carbohydrate (20% fructose), 20% protein, with 2% cholesterol content]. TG, triglyceride; Chol, cholesterol.

Fig. 2.

Body and liver weights in F₁–F₃ offspring from maternal high-fat/high-sucrose (HF/HS) diet lineage. A: body weight of male and female offspring from maternal control (CON) and HF/HS lineage from F₁, F₂, and F₃ generations. B: liver weight of male and female offspring from maternal CON and HF/HS lineage from F₁, F₂, and F₃ generations. C: liver weight-to-body weight (LW/BW) ratio of male and female offspring from maternal CON and HF/HS lineage from F₁, F₂, and F₃ generations. Quantitative data are presented as means ± SD with n ≥ 10 in each group and ≥5 separate litters represented in each group. P values are as indicated.

Fig. 3.

Liver triglyceride and cholesterol levels in F₁–F₃ offspring after maternal high-fat/high-sucrose (HF/HS) diet exposure. A: liver triglyceride levels from male and female offspring from maternal control (CON) and HF/HS lineage from F₁, F₂, and F₃ generations. Triglyceride levels normalized to tissue weight. B: total liver cholesterol levels in male and female offspring (F₁–F₃) from maternal CON and maternal HF/HS lineages. Cholesterol levels normalized to tissue weight. Quantitative data are presented as means ± SD with n ≥ 4 in each group and ≥4 separate litters represented in each group.

Fig. 4.

Periportal fibrosis and inflammation in offspring exposed to maternal high-fat/high-sucrose (HF/HS) diet. A: representative photomicrographs of picrosirius red (PSR) staining. B: representative photomicrographs of immunohistochemistry (IHC) for cytokeratin 19 (CK19). Arrows point to infiltrate around CK19-positive bile duct. C: representative photomicrographs of IHC for CD45, CD11b, and F4/80 in consecutive sections from maternal control (CON) and HF/HS offspring liver. D: quantitative analysis of PSR staining. E: no. of CD11b-positive cells per portal area (5 portal areas counted in each of 3 offspring/group). Quantitative data are presented as means ± SD with n = 5 (from 5 separate litters) in each group for PSR quantification and n = 3 (from 3 separate litters) in each group for CD11b cell counts. P values are as indicated.

Fig. 5.

Altered bile acid (BA) homeostasis in offspring exposed to maternal high-fat/high-sucrose (HF/HS) diet. BA pool size in male and female offspring (F₁–F₃) exposed to maternal control (CON) or HF/HS diet. Quantitative data are presented as means ± SD with n ≥ 4 in each group. P values are as indicated.

Fig. 6.

Histopathological progression of nonalcoholic fatty liver disease (NAFLD) in F₁ offspring from maternal control (CON) and high-fat/high-sucrose (HF/HS) diet-fed lineage. A: weekly body weights during 16 wk of chow or high-transfat, cholesterol, fructose (HTFC) diet feeding. B: liver weight and liver weight-to-body weight ratio after 16 wk of chow or HTFC diet feeding C: representative photomicrographs of hematoxylin and eosin (H&E), picrosirius red (PSR), and CD45 staining of liver from maternal CON and maternal HF/HS lineage offspring fed HTFC diet. D: quantification of %area positive for PSR in maternal CON and maternal HF/HS offspring fed HTFC diet. E: quantitative PCR for Col1a1 in liver from maternal CON and HF/HS lineage offspring fed chow or HTFC diet. Levels are presented as fold change to maternal CON chow-fed group as control. F: NAFLD Activity Score (NAS) (steatosis, inflammation, ballooning, and total) and fibrosis scoring on liver from maternal CON and HF/HS offspring fed chow or HTFC diet. G: hepatic triglyceride and cholesterol levels from offspring fed chow or HTFC diet. H: quantification of %area with large or small lipid droplets in liver from offspring fed HTFC diet. Five separate litters represented in each group with triglyceride and cholesterol levels; ≥6 separate litters represented for all other quantitative data. P values are as noted on each graph. All data are from male offspring.

Fig. 7.

Bile acid (BA) homeostasis in F₁ offspring during Western diet feeding. A: BA pool size in offspring fed chow or high-transfat, cholesterol, fructose (HTFC) diet. B: intrahepatic BA composition (%total) with key to individual species on right. C: quantitative PCR for Cyp7a1, Cyp27a1, and Cyp3a11 from liver of offspring fed chow or HTFC diet. Levels are presented as fold change to maternal control (CON) chow-fed group as control. D: quantitative PCR (qPCR) for small heterodimer protein (Shp) from liver of offspring fed chow or HTFC diets. Levels are presented as fold change to maternal CON chow-fed group as control. E: qPCR for several BA transporters (BSEP, NTCP, Oatp2, and Oatp4) from liver of offspring fed chow or HTFC diet. Levels are presented as fold change to maternal CON chow-fed group as control. F: serum fibroblast growth factor 15 (FGF15) levels (left) and qPCR for liver fibroblast growth factor receptor 4 (FGFR4, right). G: serum levels of 7α-hydroxy-4-cholesten-3-one (C-4) from offspring of maternal CON or fed high-fat/high-sucrose (HF/HS) diet lineage-fed chow diet. H: circulating BAs in offspring fed chow or HTFC diet. Quantitative data are presented as means ± SD with n ≥ 4 in each group. For BA pool size, ≥4 separate litters are represented in each group. For all other data, ≥6 separate litters are represented in each group. P values are as noted on each graph. All data are from male offspring.

Fig. 8.

Histopathological progression of nonalcoholic fatty liver disease (NAFLD) and bile acid (BA) homeostasis in F₂ offspring from maternal control (CON) and fed high-fat/high-sucrose (HF/HS) diet lineage. A: weekly body weights during 16 wk of chow or high-transfat, cholesterol, fructose (HTFC) diet feeding. B: liver weight and liver weight-to-body weight ratio after 16 wk of chow or HTFC diet feeding C: representative photomicrographs of hematoxylin and eosin (H&E) and picrosirius red (PSR) staining of liver from maternal CON and maternal HF/HS lineage offspring fed HTFC diet. D: quantification of %area positive for PSR in maternal CON and maternal HF/HS offspring fed HTFC diet. E: fibrosis scoring on liver from offspring fed chow or HTFC diet. F: hepatic triglyceride and cholesterol levels from offspring fed chow or HTFC diet. G: measurement of BA pool size and serum BAs in offspring fed chow or HTFC diet. Quantitative data are presented as means ± SD with n ≥ 5 in each group with ≥5 separate litters represented in each group. P values are as noted on each graph. All data are from male offspring.

Fig. 9.

Histopathological progression of nonalcoholic fatty liver disease (NAFLD) and bile acid (BA) homeostasis in F₃ offspring from maternal control (CON) and high-fat/high-sucrose (HF/HS) diet lineage. A: weekly body weights during 16 wk of chow or high-transfat, cholesterol, fructose (HTFC) diet feeding. B: liver weight and liver weight-to-body weight ratio after 16 wk of chow or HTFC diet feeding. C: representative photomicrographs of hematoxylin and eosin (H&E) and picrosirius red (PSR) staining of liver from maternal CON and maternal HF/HS lineage offspring fed HTFC diet. D: quantification of %area positive for PSR in maternal CON and maternal HF/HS offspring fed HTFC diet. E: fibrosis scoring on liver from offspring fed chow or HTFC diet. F: hepatic triglyceride and cholesterol levels from offspring fed chow or HTFC diet. G: measurement of BA pool size and serum BAs in offspring fed chow or HTFC diet. Quantitative data are presented as means ± SD with n ≥ 5 in each group and ≥5 separate litters represented in each group. P values are as noted on each graph. All data are from male offspring.