Transcriptome analysis reveals liver metabolism programming in kids from nutritional restricted goats during mid-gestation

Abstract

Background: Maternal nutrient restriction during pregnancy causes a metabolic disorder that threatens the offspring's health in humans and animals. However, the molecular mechanism of how undernutrition affecting hepatic metabolism of fetal or postnatal offspring is still unclear. We aimed to investigate transcriptomic changes of fetal livers in response to maternal malnutrition in goats during mid-gestation and to explore whether these changes would disappear when the nutrition was recovered to normal level during mid-gestation using goats (Capra hircus) as the experimental animals.

Methods: Fifty-three pregnant goats were subjected to a control (100% of the maintenance requirements, CON) or a restricted (60% of the maintenance requirements on day 45 to day 100 of gestation and then realimentation, RES) diet. A total of 16 liver samples were collected from fetal goats on day 100 of gestation and goat kids of postnatal day 90 to obtain hepatic transcriptional profiles using RNA-Seq.

Results: Principal component analysis of the hepatic transcriptomes presented a clear separation by growth phase (fetus and kid) rather than treatment. Maternal undernutrition up-regulated 86 genes and down-regulated 76 genes in the fetal liver of the FR group as compared to the FC group. KEGG pathway analysis showed the DEGs mainly enriched in protein digestion and absorption, steroid biosynthesis, carbohydrate digestion and absorption and bile secretion. A total of 118 significant DEGs (fold change > 1.2 and FDR < 0.1) within KR vs. KC comparison was identified with 79 up-regulated genes and down-regulated 39 genes, and these DEGs mainly enriched in the biosynthesis of amino acids, citrate cycle, valine, leucine and isoleucine biosynthesis and carbon metabolism.

Conclusion: Hepatic transcriptome analysis showed that maternal undernutrition promoted protein digestion and absorption in the fetal livers, while which restrained carbohydrate metabolism and citric acid cycle in the livers of kid goats after realimentation. The results indicate that maternal undernutrition during mid-gestation causes hepatic metabolism programming in kid goats on a molecular level.

Keywords: Goat; Hepatic metabolism; Maternal undernutrition; RNA-sequencing.

Figure 1. Hepatic transcriptomic profiles of expressed genes (with average FPKM > 1 of the group and FPKM > 1 in at least one animal) in goat fetuses and kids.

(A) Venn diagram showing the overlap of expressed genes in livers of goats from FC and FR groups. (B) Venn diagram showing the overlap of expressed genes in livers of goats from KC and KR groups. (C) PCA plot of expressed genes for the liver samples of goats from FC, FR, KC and KR groups. The X and Y-axis represent the first two principle components. The percentage value in the bracket represents the percentage of variance explained by that principle component. (D) Number of differential expressed genes between FC and FR group and KC and KR group.

Figure 2. Gene ontology (GO) and KEGG pathway analyses of the DEGs in the liver of fetal goats.

(A) The significant GO terms (biological processes) associated with the identified DEGs in fetal goats between FC and FR groups. The vertical axis represents the GO category, and the horizontal axis represents the -Log2 (P-value) of the significant GO terms. The number represented amount of DEGs enriched in the corresponding GO terms. (B) The significant pathways associated with the identified DEGs in fetal goats between FC and FR groups. The vertical axis represents the pathway category, and the horizontal axis represents the -Log2 (P-value) of the significant pathways. The number represented amount of DEGs enriched in the corresponding pathways.

Figure 3. Gene ontology (GO) and KEGG pathway analyses of the DEGs in the liver of kid goats.

(A) The significant GO terms (biological processes) associated with the identified DEGs in kid goats between KC and KR groups. The vertical axis represents the GO category, and the horizontal axis represents the -Log2 (P-value) of the significant GO terms. The number represented amount of DEGs enriched in the corresponding GO terms. (B) The significant pathways associated with the identified DEGs in kid goats between KC and KR groups. The vertical axis represents the pathway category, and the horizontal axis represents the -Log2 (P-value) of the significant pathways. The number represented amount of DEGs enriched in the corresponding pathways.

Figure 4. Expression (log2 FPKM) of genes involved in hepatic metabolism of goats from each group.

(A) Heat map for the expression of genes involved in carbohydrate metabolism of goats from each group. (B) Heat map for the expression of genes involved in lipid metabolism of goats from each group. (C) Heat map for the expression of genes involved in cellular energy metabolism of goats from each group. Green color represents lower expression and red color represents higher expression. Data are expressed as means ±SD, *P < 0.05.

Figure 5. Validation of metabolic and immune DEGs in the liver of fetal and kid goats by quantitative real-time PCR (qRT-PCR).

(A) The relative gene expression of DEGs related to hepatic metabolism and immunity by qRT-PCR in the fetal goats. (B) The relative gene expression of DEGs related to hepatic metabolism and immunity by qRT-PCR in the kid goats. Values are presented as mean with their standard errors. *P < 0.05.