Lipid overload during gestation and lactation can independently alter lipid homeostasis in offspring and promote metabolic impairment after new challenge to high-fat diet

Abstract

Background: Nutritional status in early life is critically involved in the metabolic phenotype of offspring. However the changes triggered by maternal consumption of high-fat diet (HFD) in pre- or postnatal period should be better understood. Here we evaluated whether maternal HFD consumption during gestation and lactation could differently affect liver miR-122 and miR-370 expression leading to metabolic damages observed in offspring. Moreover, we investigate whether early overnutrition program offspring to more harmful response to HFD in later life.

Methods: Female mice were fed either a standard chow (SC) diet or a HFD three weeks before and during mating, gestation and/or lactation. Offspring were evaluated on the delivery day (d0), in a cross-fostering model at day 28 (d28) and in adult life, after a re-challenge with a HFD (d82).

Results: In vitro analysis using liver cell line showed that palmitate could induced decrease in miR-122 and increase in miR-370 expression. Newborn pups (d0) from obese dams showed a decrease in lipid oxidation markers (Cpt1a and Acadvl), an increase in triacylglycerol synthesis markers (Agpat and Gpam), as well as lower miR-122 and higher miR-370 hepatic content that was inversely correlated to maternal serum NEFA and TAG. Pups fostered to SC dams presented an increase in body weight and Agpat/Gpam expression at d28 compared to pups fostered to HFD dams and an inverse correlation was observed between miR-122 hepatic expression and offspring serum TAG. In adult life (d82), the reintroduction of HFD resulted in higher body weight gain and hepatic lipid content. These effects were accompanied by impairment in lipid and glucose metabolism, demonstrated by reduced Cpt1a/Acadvl and increased Agpat/Gpam expression, lower glucose tolerance and insulin sensitivity.

Conclusion: Our data suggest that both gestational and lactation overnutrition results in metabolic changes that can permanently alter lipid homeostasis in offspring. The presence of fatty acids in maternal blood and milk seem to be responsible for modulating the expression of miR-122 and miR-370, which are involved in liver metabolism. These alterations significantly increase susceptibility to obesity and ectopic lipid accumulation and lead to a more harmful response to HFD in offspring.

Keywords: Maternal obesity; Metabolic programming; NAFLD; microRNAs.

Fig. 1

Experimental Protocol. Experimental protocol followed to investigate the effect of maternal overnutrition on offspring metabolism, during gestation or lactation. Offspring of control (C-D) and HFD (HF-D) dams were evaluated at d0 (C and H) and d28 (CC, CH, HH and HC) (a). Details of the protocol followed to obtain cross-fostered groups (b). Experimental protocol followed to evaluate the metabolic complication in offspring after re-challenge to adult life in nutritional overload at d82 (CC, CC-HF, HH, and HH-HF) (c)

Fig. 2

Fatty acid exposure induces miRNAs modulation in mouse and human liver cell lines. MicroRNA level (qRT-PCR) - miR-122 and miR-370 - from Hepa1c1c7 mouse hepatoma cell line (1x10⁸cells/mL) (a) and HepG2 human hepatoma cell line (1x10⁸cells/mL) (b) 6hs after exposure to palmitic acid (500uM). For relative gene expression analysis, U6snRNA was used as endogenous control. Values are means (n = 3–6) + - SEM. Student's t-test was used to compare Control and Palmitic Acid groups. Different letters indicate statistical significance between groups (p ≤ 0.05)

Fig. 3

Maternal HFD consumption at gestation period leads newborn offspring to altered miRNAs and lipid metabolism-related gene expression. Body weight (a), lee index of obesity (LIO) (b), serum lipids (CHOL and TAG - c), fasting glucose (d) and serum insulin (e), mRNA levels (qRT-PCR) of hepatic Cpt1a and Acadvl (f), and Agpat and Gpam (g), microRNA level (qRT-PCR) of hepatic miR-122 and miR-370 (h) from newborn offspring from C and H groups. Correlation analysis between hepatic miR-122 from d0 offspring vs. serum NEFA (i) and TAG (j) from dams. For relative gene expression analysis, β-Actin and U6snRNA were used as endogenous controls. Values are means (n = 5–12) + - SEM. Student's t-test was used in all analyses to compare C and H groups. Different letters indicate statistical significance between groups (p ≤ 0.05)

Fig. 4

Maternal HFD consumption at gestation or lactation independently alters miRNAs and lipid-related gene expression. Body weight (a), adiposity (b), caloric intake (c), fasting glucose (d) and serum lipids (CHOL and TAG - e), mRNA levels (qRT-PCR) of hepatic Cpt1a and Acadvl (f), and Agpat and Gpam (g), hepatic total lipid content (h), microRNA level (qRT-PCR) of hepatic miR-122 and miR-370 (i), correlation analysis between hepatic miR-122 and serum TAG (j) from recently weaned unfostered offspring (CC and HH) and crossfostered offspring (CH and HC) at d28. For relative gene expression analysis, β-Actin and U6snRNA were used as endogenous controls. One-way ANOVA was used in all analyses to compare CC, CH, HH and HC groups. Values are means (n = 5–8) + - SEM. Different letters indicate statistical significance between groups (p ≤ 0.05)

Fig. 5

Metabolic programming and hepatic miRNAs modulation by maternal HFD consumption persists into adult life of offspring. Body weight (a), adiposity (b), caloric intake (c), fasting glucose (d), mRNA levels (qRT-PCR) of hepatic Cpt1a and Acadvl (e) and Agpat and Gpam (f), serum lipids (CHOL and TAG - g), total hepatic lipid content (h), microRNA level (qRT-PCR) of hepatic miR-122 and miR-370 (i) from adult offspring from CC and HH groups at d82. For relative gene expression analysis, β-Actin and U6snRNA were used as endogenous controls. Values are means (n = 5–8) + - SEM. Student's t-test was used in all analyses to compare CC and HH groups. Different letters indicate statistical significance between groups (p ≤ 0.05)

Fig. 6

Maternal HFD consumption leads offspring to increased metabolic complications when exposed to HFD in adult life. Weight gain (a), body weight (b), adiposity (c), caloric intake (d), serum parameters (CHOL, TAG and leptin) (e), fasting glucose (f), area under curve (AUC) of GTT (g) and PTT (h), glucose clearance during ITT (i), mRNA levels (qRT-PCR) of hepatic Cpt1a and Acadvl (j) and Agpat and Gpam (k), total hepatic lipid content (l) from adult offspring after exposure to HFD for 40 days at d82 (CC-HF and HH-HF groups). For relative gene expression analysis, β-Actin was used as endogenous control. Values are means (n = 5–8) + - SEM. Student's t-test was used in all analyses to compare CC-HF and HH-HF groups. Different letters indicate statistical significance between groups (p ≤ 0.05)