High maternal adiposity during pregnancy programs an imbalance in the lipidome and predisposes to diet-induced hepatosteatosis in the offspring

Abstract

Background: Exposure to high maternal adiposity in utero is a significant risk factor for the later-life development of metabolic syndrome (MetS), including non-alcoholic fatty liver disease (NAFLD). We have previously shown that high pre-pregnancy adiposity programs adipose tissue dysfunction in the offspring, leading to spillover of fatty acids into the circulation, a key pathogenic event in obesity-associated MetS. Herein, we hypothesized that programming of adipose tissue dysfunction in offspring born to overweight dams increases the risk for developing NAFLD.

Results: Females heterozygous for leptin receptor deficiency (Hetdb) were used as a model of high pre-pregnancy adiposity. Female wild-type (Wt) offspring born to Hetdb pregnancies gained significantly more body fat following high-fat/fructose diet (HFFD) compared with Wt offspring born to Wt dams. HFFD increased circulating free fatty acids (FFA) in male offspring of control dams, while FFA levels were similar in HFFD-fed offspring from Wt dams and CD or HFFD-fed Wt offspring from Hetdb dams. Despite female-specific protection from diet-induced FFA spillover, both male and female offspring from Hetdb dams were more susceptible to diet-induced hepatosteatosis. Lipidomic analysis revealed that CD-offspring of overweight dams had decreased hepatic polyunsaturated FA (PUFA) levels compared with control offspring. Changes to saturated FA (SFA) and the de novo lipogenic (DNL) index were diet driven; however, there was a significant effect of the intrauterine environment on FA elongation and Δ9 desaturase activity.

Conclusion: High maternal adiposity during pregnancy programs a susceptibility to diet-induced hepatosteatosis.

Keywords: Developmental Programming; Lipidomics; Maternal Obesity; NAFLD.

Figure 1. High pre-pregnancy adiposity programs an obesogenic phenotype in the offspring

Body composition was measured by TD-NMR in Wt offspring born to Wt (Wt/Wt dam) or Het_db (Wt/Het_db) dams following control diet (CD) or high-fat/fructose diet (HFFD). Fat mass in (A) male and (B) female offspring. Lean body mass in (C) male and (D) female offspring. Fasting levels of plasma free fatty acids were measured in (E) male and (F) female offspring. Main effects of intrauterine environment and diet were determined by two-way ANOVA, with Sidak’s multiple comparison test to determine differences between groups.

Figure 2. Offspring born to overweight dams are more susceptible to diet-induced hepatic steatosis

Following control diet (CD) or high-fat/fructose diet (HFFD), H&E-stained liver sections from Wt offspring born to Wt (Wt/Wt dam) or Het_db (Wt/Het_db) dams were evaluated by an experienced pathologist. Representative H&E-stained sections of the liver from (A) male and (B) female offspring. The distribution of scores for steatosis (0-3) in (C) male and (D) female offspring. The distribution of scores for hepatocellular ballooning (0-2) in (E) male and (F) female offspring. A contingency table was created to perform a chi-square test to determine differences in score count between groups (n = 6–9/group). **P<0.01 Wt/Het_db offspring CD versus HFFD.

Figure 3. Expression of lipogenic, pro-fibrotic and pro-inflammatory genes in the liver

qPCR was used to measure the expression of (A,G) transforming growth factor β (Tgfβ); (B,H) pro-collagen I (PcI); (C,I) pro-collagen III (PcIII); (D,J) tumor necrosing factor α (Tnfα); (E,K) stearoyl-CoA 9 desaturase (Scd1); and (F,L) sterol regulatory element binding protein-1 (Srebp1c);in livers of control diet (CD) or high-fat/fructose diet (HFFD)-fed Wt male and female offspring born to Wt (Wt/Wt dam) or Het_db (Wt/Het_db) dams. Differences were assessed by two-way ANOVA with intrauterine environment and diet as main effects, while Sidak’s multiple comparison test determined differences between groups. P-values for Tgfβ and PcI in males and PcII in females represent results from log-transformed data.

Figure 4. Individual FFA and TG species in hepatic lipid fractions

The Folch method was used to extract lipids from liver samples of 12-week-old male offspring for the separation and identification of individual lipid classes by thin layer chromatography. (A) Major lipid species present in abundance in FFA, including saturated FA: palmitic acid (16:0) and stearic acid (18:0); monounsaturated FA: palmitoleic acid (16:1), oleic acid (18:1ϖ9) and vaccenic acid (18:1ϖ7); and polyunsaturated FA: linoleic acid (18:2) and arachidonic acid (20:4). (B) Heat map showing geometric means of major individual FFA lipid species. (C) Minor lipid species present in trace amounts in FFA, including polyunsaturated FAs: γ-linolenic acid (18:3ω6), α-linolenic acid (18:3ω3), dihomo-γ-linolenic acid (20:3ω6) and docosahexaenoic acid (22:6). (D) Heat map showing geometric means of minor individual FFA lipid species. (E) Major lipid species present in TAG. (F) Heat map showing geometric means of minor individual TAG species. (G) Minor lipid species present in the TAG fraction, including polyunsaturated FAs: γ-linolenic acid (18:3ω6), α-linolenic acid (18:3ω3), dihomo-γ-linolenic acid (20:3ω6) and docosahexaenoic acid (22:6). (H) Heat map showing geometric means of minor individual TAG species. Main effects of intrauterine environment and diet were determined by two-way ANOVA, with Sidak’s multiple comparison test to determine differences between groups (n=5–9/group). *P<0.05: **P<0.01; ***P<0.001; ****P<0.0001 HFFD versus CD (same intrauterine environment); ^#P<0.05; ^##P<0.01; ^###P<0.001 Wt/Wt dam versus Wt/Het_db dam (same diet).

Figure 5. Effects of intrauterine environment and diet on lipogenic enzyme activity

Lipidomic profiles of livers collected from 12-week-old male offspring fed a control diet (CD) or high-fat/fructose diet (HFFD) were used to indirectly measure the activity of lipogenic enzymes. The Δ9 desaturation index, reflecting stearoyl-CoA desaturase activity, was calculated as the ratios of (A) palmitoleic acid (C16:1) to and palmitic acid (16:0) and (B) oleic acid (18:1) to stearic acid (18:0). (C) The de novo lipogenic (DNL) index was calculated as the ratio of palmitic acid (16:0) to linoleic acid (18:2). (D) The elongation index was calculated as the ratio of stearic acid (18:0) to palmitic acid (16:0). Main effects of intrauterine environment and diet were determined with two-way ANOVA with Sidak’s multiple comparison test to determine differences between groups (n=5–9).

Figure 6. Effects of intrauterine environment and diet on MUFA, SFA and PUFA in hepatic lipid fractions

The sum of (A) monounsaturated fatty acids (MUFAs), (B) saturated fatty acids (SFAs) and (C) polyunsaturated fatty acids (PUFAs) were calculated and expressed as % of total lipid species. (D) The ratio of the sum of SFA relative to the sum of PUFA. Main effects of intrauterine environment and diet were determined by two-way ANOVA with Sidak's multiple comparison test to determine differences between groups (n=5–9).