Hepatic Metabolic Profile Reveals the Adaptive Mechanisms of Ewes to Severe Undernutrition during Late Gestation

Abstract

The mechanisms underlying the adaption of liver metabolism to the undernutrition in ewes during late gestation remain unclear. This research aimed to explore the adaptive mechanisms of liver metabolism by hepatic metabolome analysis in pregnant ewes to the negative energy balance induced by severe feed restriction. Twenty ewes carrying multiple fetuses and gestating for 115 days were fed normally or restricted to a 30% feed level (10 ewes in each group) for 15 days. All ewes were sacrificed and hepatic samples were collected and analyzed by liquid chromatography-mass spectrometry. Both the principal components analysis and partial least squares of discriminant analysis of hepatic metabolites showed the clear separation between ewes in the control and severely feed-restricted groups. The metabolic profile demonstrated that the proportions of differential metabolites between the two groups in fatty acids and lipids, organic acids, and amino acids and derivatives were 61.11%, 16.67%, and 11.11%, respectively. Enriched pathways of differential metabolites were mainly involved in fatty acids and amino acids metabolism and biosynthesis. Correlation networks of differential metabolites revealed that general metabolic pattern was changed apparently and mainly based on fatty acids and lipids in the livers of feed-restricted ewes. The accumulation and oxidation of long-chain fatty acids were intensified in the livers of feed-restricted ewes, while those of medium-chain fatty acids were the opposite. In general, severe feed restriction significantly affected the levels of hepatic metabolites and altered the overall metabolic pattern. Furthermore, fatty acids oxidation as well as the utilization of amino acids and organic acids were intensified to adapt to the negative energy balance during late gestation.

Keywords: adaptive mechanisms; amino acid; fatty acid; lipid metabolism; undernutrition.

Figure 1

Blood biochemical indicators of ewes in the control group (CON, fed at the normal level, n = 10) and treated group (TR, restricted to a 30% level of feed intake, n = 10) before and after intervention. (a) Glucose level. (b) NEFAs level. (c) BHBA level. Values are means with their standard errors represented by vertical bars. * Mean values with asterisks were significantly different between the two groups (p < 0.05, independent-sample t-test). NEFAs, non-esterified fatty acids; BHBA, beta-hydroxybutyric acid.

Figure 2

Organ indexes of ewes in the control group (CON, fed at the normal level, n = 10) and treated group (TR, restricted to a 30% level of feed intake, n = 10). Values are means with their standard errors represented by vertical bars. * Mean values with asterisks were significantly different between the two groups (p < 0.05, independent-sample t-test).

Figure 3

Principal components analysis (PCA) and partial least squares of discriminant analysis (PLS-DA) of hepatic metabolites for ewes in the control group (CON, fed at the normal level, n = 8) and treated group (TR, restricted to a 30% level of feed intake, n = 8). (a) PCA score scatter plot; (b) PLS-DA score scatter plot (predictive ability parameter (Q²) (cum) = 0.945, goodness-of-fit parameter (R²) (Y) = 0.980); (c) PLS-DA loading scatter plot. Only the differential metabolites distributed near to the $M2.DA(CON) or $M2.DA(TR) were shown in the loading scatter plot.

Figure 4

Metabolome view map of the differential metabolites identified in the hepatic tissues between the control group (CON, fed at the normal level, n = 8) and treated group (TR, restricted to a 30% level of feed intake, n = 8). Power calculation had identified a required sample size of 8 ewes per group in order to enable detection of an effect size of 1.94 for most of the cognitive test scores with 95% power and a type I error of 5%. The x-axis represents the pathway impact, and the y-axis represents the pathway enrichment. The larger size indicates higher pathway enrichment, and the darker color indicates higher pathway impact values.

Figure 5

Correlation networks of hepatic metabolites for ewes in the control group (CON, fed at the normal level, n = 8) (a) and treated group (TR, restricted to a 30% level of feed intake, n = 8) (b) based on Spearman’s correlation coefficients (|r| > 0.8 and p < 0.05). Node size and color corresponds to the degree and classification, respectively. Red lines denote positive correlations, while blue lines denote negative correlations.

Figure 6

The gene expressions of acyl-CoA synthases and dehydrogenases in the hepatic tissues of ewes in the control group (CON, fed at the normal level, n = 10) and treated group (TR, restricted to a 30% level of feed intake, n = 10). Values are means with their standard errors represented by vertical bars. * Mean values with asterisks were significantly different between the two groups (p < 0.05, independent-sample t-test). ACSL, long-chain acyl-CoA synthase; ACADL, long-chain acyl-CoA dehydrogenase; ACSM, medium-chain acyl-CoA synthase; ACADM, medium-chain acyl-CoA dehydrogenase; ACSS, short-chain acyl-CoA synthase; ACADSB, short and branch-chain acyl-CoA dehydrogenase.

Figure 7

Overview of metabolic alteration related to energy conversion in the hepatic tissues of feed-restricted ewes during late gestation. The red font indicates the increased metabolites while the green font indicates the decreased metabolites in feed-restricted ewes compared with ewes in the control group. TCA, tricarboxylic acid cycle; LysoPC, lysophosphatidyl choline; LysoPE, lysophosphatidyl ethanolamine; BHBA, beta-hydroxybutyric acid; PEP, phosphoenolpyruvate.