Genetic Variant in Flavin-Containing Monooxygenase 3 Alters Lipid Metabolism in Laying Hens in a Diet-Specific Manner

Abstract

Genetic variant T329S in flavin-containing monooxygenase 3 (FMO3) impairs trimethylamine (TMA) metabolism in birds. The TMA metabolism that under complex genetic and dietary regulation, closely linked to cardiovascular disease risk. We determined whether the genetic defects in TMA metabolism may change other metabolic traits in birds, determined whether the genetic effects depend on diets, and to identify genes or gene pathways that underlie the metabolic alteration induced by genetic and diet factors. We used hens genotyped as FMO3 c.984 A>T as well as those with the homozygous normal genotype. For each genotype, hens were provided with either a corn-soybean meal basal diets (SM), which contains lower levels of TMA precursor, or the basal diets supplemented with 21% of rapeseed meal (RM), which contains higher levels of TMA precursor. An integrative analysis of metabolomic and transcriptomic was used to explore the metabolic patterns of FMO3 genetic variant in hens that were fed the two defined diets. In birds that consumed SM diets, the T329S mutation increased levels of plasma TMA and lipids, FMO3 mRNA levels, and the expression of genes involved in long chain polyunsaturated fatty acid biosynthesis. In birds that consumed RM diets, the T329S mutation induced fishy odor syndrome, a repression in LXR pathway and a reciprocal change in lipid metabolism. Variations in TMA and lipid metabolism were linked to the genetic variant in FMO3 in a diet-specific manner, which suggest FMO3 functions in TMA metabolism and lipid homeostasis. LXR pathway and polyunsaturated fatty acid metabolism are two possible mechanisms of FMO3 action in response to dietary TMA precursor.

Keywords: dietary TMA precursor; flavin-containing monooxygenase 3; genetic variant; laying hen; lipid metabolism; trimethylamine.

Figure 1

The genetic variant T329S in FMO3 alters trimethylamine metabolism in birds. (a) TMA levels in egg yolks, (b) TMA levels in cecal chime, (c) hepatic FMO3 mRNA levels, (d) ability of hepatic FMO, (e) plasma TMAO levels, (f) plasma TMA levels. Laying hens homozygous normal for FMO3 (AA genotype) and mutant hens genotyped as FMO3 c.984 A>T (i.e. T239S mutation; TT genotype) were fed either the corn-soybean meal basal diets (SM; < 0.15 mg/g sinapine) or the basal diets supplemented with 21% of rapeseed meal (RM; 1.33 mg/g sinapine) for 8 weeks. Data are presented as mean ± SEM. Data represent means from 6 replicates with 6 eggs each (a). Cecal chyme (b) and liver samples (c-d) from hens was analyzed, n = 6 birds/group. (e-f) Plasma samples from hens was analyzed, data are the mean of 6 replicates, and 3 birds from each replicate were analyzed. Significance was measured with Student's t-test. *P < 0.05. trimethylamine, TMA; trimethylamine-N-oxide, TMAO.

Figure 2

Metabolic profiling in chickens reveals distinct variations in lipid metabolism linked to the T329S genetic variation in FMO3. Hens were fed the soybean meal (SM) diets (a, b) or rapeseed meal (RM) diets (c, d) (n = 6 birds/groups). (a, c) OPLS-DA score plots based on ¹H NMR spectra of plasma samples from AA and TT genotype hens. tP is the first principal component, and tO is the second orthogonal component. ((b, d) Corresponding coefficient loading plots according to OPLS-DA analysis with the metabolites labeled from plasma samples of AA and TT hens. (e) Hierarchical clustering heat-map of Spearman's correlations among 13 identified differential metabolites. (f) Heatmap showing the degree of change (left) and fold change (FC, right) in the TT genotype hens compared with the AA genotype hens. An asterisk in the FC heatmap indicate a significant change (P < 0.05). Metabolites: 1, lipid (high-density lipoprotein); 2, lipid (low-density lipoprotein); 3, lipid (very low-density lipoprotein); 4, 3-hydroxybutyrate; 5, lipid (saturated fatty acids); 6, lactate; 7, lipid (unsaturated fatty acids); 8, acetone; 9, glutamate; 10, lipid (polyunsaturated fatty acids); 11, trimethylamine N-oxide; 12, β-glucose; 13, α-glucose.

Figure 3

Effect of the T329S genetic variation in FMO3 on plasma and liver lipids in birds. (a-d) Data are the mean of 6 replicates, and 3 birds from each replicate were analyzed, (e-f) Liver samples from hens was analysed, n = 6 birds/group. (a) Plasma total triglyceride (TG) levels. (b) Plasma cholesterol levels. (c) Plasma high-density lipoprotein cholesterol levels. (d) Plasma low-density lipoprotein cholesterol levels. (e) Hepatic total triglyceride levels. (f) Hepatic cholesterol levels. Data are presented as mean ± SEM. Significance was measured with student's t-test. *P < 0.05.

Figure 4

Genetic variation in FMO3 alters hepatic transcriptional profiling in a diet-specific manner. Hepatic genes involved in lipid metabolism are highlighted. (a) Number of differentially expressed genes (DEGs) between AA and TT hens under the corn-soybean meal basal (SM) diets or rapeseed meal (RM) diets (n = 6 birds/groups). The DEGs were identified by the combined cut-offs of P < 0.05 and fold change > 2. (b) Venn diagrams showing 11 shared DEGs induced by genotypes. (c) Fold change analysis showing expression patterns of the 11 shared DEGs. Fold changes were determined by comparing the change in gene expression (reads per kilobase per million mapped reads, RPKM) of TT hens relative to AA hens, and were log₂ transformed. (d, e) Heatmaps of expression data generated from the read counts of lipid metabolism-related genes in AA and TT genotype hens along with fold change (FC; to the right) analysis under the SM (d) or RM diets (e). Each column in the heatmap represents an individual animal. * P < 0.05; ** P < 0.01; *** P < 0.001.