How dietary arachidonic- and docosahexaenoic- acid rich oils differentially affect the murine hepatic transcriptome

Abstract

Introduction: Herein, we expand our previous work on the effects of long chain polyunsaturated fatty acids (LC-PUFA) on the murine hepatic transcriptome using novel statistical and bioinformatic approaches for evaluating microarray data. The analyses focuses on key differences in the transcriptomic response that will influence metabolism following consumption of FUNG (rich in 20:4n6), FISH (rich in 20:5n3, 22:5n3, and 22:6n3) and COMB, the combination of the two.

Results: Using a variance-stabilized F-statistic, 371 probe sets (out of 13 K probe sets in the Affymetrix Mu11K chip set) were changed by dietary treatment (P < 0.001). Relative to other groups, COMB had unique affects on murine hepatic transcripts involved in cytoskeletal and carbohydrate metabolism; whereas FUNG affected amino acid metabolism via CTNB1 signaling. All three diets affected transcripts linked to apoptosis and cell proliferation, with evidence FISH may have increased apoptosis and decreased cell proliferation via various transcription factors, kinases, and phosphatases. The three diets affected lipid transport, lipoprotein metabolism, and bile acid metabolism through diverse pathways. Relative to other groups, FISH activated cyps that form hydroxylated fatty acids known to affect vascular tone and ion channel activity. FA synthesis and delta 9 desaturation were down regulated by COMB relative to other groups, implying that a FA mixture of 20:4n6, 20:5n3, and 22:6n3 is most effective at down regulating synthesis, via INS1, SREBP, PPAR alpha, and TNF signaling. Heme synthesis and the utilization of heme for hemoglobin production were likely affected by FUNG and FISH. Finally, relative to other groups, FISH increased numerous transcripts linked to combating oxidative such as peroxidases, an aldehyde dehydrogenase, and heat shock proteins, consistent with the major LC-PUFA in FISH (20:5n3, 22:5n3, 22:6n3) being more oxidizable than the major fatty acids in FUNG (20:4n6).

Conclusion: Distinct transcriptomic, signaling cascades, and predicted affects on murine liver metabolism have been elucidated for 20:4n6-rich dietary oils, 22:6n3-rich oils, and a surprisingly distinct set of genes were affected by the combination of the two. Our results emphasize that the balance of dietary n6 and n3 LC-PUFA provided for infants and in nutritional and neutraceutical applications could have profoundly different affects on metabolism and cell signaling, beyond that previously recognized.

Figure 1

Principle component analysis scatter plot showing the five arrays used, represented by 371 most highly differentiated probe sets. Spatially closest arrays have the most similar genomic profiles. Super imposed are numbers of probes sets significantly different at P < 0.001 in six pair wise comparisons. Probes differing between arrays would not necessarily be the same in each comparison. PUFAConrol, Control diet; PUFAFungal, arachidonate-rich fungal oil; PUFAFish, fish oil; PUFACombo, combination diet.

Figure 2

Hierarchical clustering of significant probes. Left, probe clusters; top, diet clusters; center, heat map, where red = high; black = middle; green = low. As expected, the two CONT groups clustered together.

Figure 3

Pathway analysis. Figures 3–7 represent signaling pathways for 58 focus genes selected from Table 3 by Ingenuity Systems software (Redwood City, CA. The following signaling cascades are shown: JUN, TNF, and CDKN2A signaling cascade affecting: DNA replication; recombination and repair; immune response; and cell cycle (Fig. 3); TGFB1 signaling cascade affecting: cell morphology; cancer; and tumor morphology (Fig. 4); CTNB1 signaling cascade affecting: cell signaling; gene expression; and cell cycle (Fig. 5); INS1/hRAS signaling cascade affecting: carbohydrate metabolism; endocrine disorders; and metabolic disease (Fig. 6); and MYC signaling cascade affecting: viral function; gene expression; and cell Cycle (Fig. 7). Differentiating groups (per Table 3) are overlaid onto the signaling diagrams, and abbreviated: FU, fungal; FI, fish oil; CO, combination diet. When CO was the differentiating group, absolute differences between FU and FI are indicated. Intracellular location of focus genes (subscripts) are annotated: C, cytoplasm; E, extracellular; N, nucleus; P, plasma membrane; U, unknown. Major canonical functional/signaling categories associated with genes in the figures identified by the software, are shown in yellow boxes.

Figure 4

Pathway analysis. Figures 3-7 represent signaling pathways for 58 focus genes selected from Table 3 by Ingenuity Systems software (Redwood City, CA. The following signaling cascades are shown: JUN, TNF, and CDKN2A signaling cascade affecting: DNA replication; recombination and repair; immune response; and cell cycle (Fig. 3); TGFB1 signaling cascade affecting: cell morphology; cancer; and tumor morphology (Fig. 4); CTNB1 signaling cascade affecting: cell signaling; gene expression; and cell cycle (Fig. 5); INS1/hRAS signaling cascade affecting: carbohydrate metabolism; endocrine disorders; and metabolic disease (Fig. 6); and MYC signaling cascade affecting: viral function; gene expression; and cell Cycle (Fig. 7). Differentiating groups (per Table 3) are overlaid onto the signaling diagrams, and abbreviated: FU, fungal; FI, fish oil; CO, combination diet. When CO was the differentiating group, absolute differences between FU and FI are indicated. Intracellular location of focus genes (subscripts) are annotated: C, cytoplasm; E, extracellular; N, nucleus; P, plasma membrane; U, unknown. Major canonical functional/signaling categories associated with genes in the figures identified by the software, are shown in yellow boxes.

Figure 5

Pathway analysis. Figures 3-7 represent signaling pathways for 58 focus genes selected from Table 3 by Ingenuity Systems software (Redwood City, CA. The following signaling cascades are shown: JUN, TNF, and CDKN2A signaling cascade affecting: DNA replication; recombination and repair; immune response; and cell cycle (Fig. 3); TGFB1 signaling cascade affecting: cell morphology; cancer; and tumor morphology (Fig. 4); CTNB1 signaling cascade affecting: cell signaling; gene expression; and cell cycle (Fig. 5); INS1/hRAS signaling cascade affecting: carbohydrate metabolism; endocrine disorders; and metabolic disease (Fig. 6); and MYC signaling cascade affecting: viral function; gene expression; and cell Cycle (Fig. 7). Differentiating groups (per Table 3) are overlaid onto the signaling diagrams, and abbreviated: FU, fungal; FI, fish oil; CO, combination diet. When CO was the differentiating group, absolute differences between FU and FI are indicated. Intracellular location of focus genes (subscripts) are annotated: C, cytoplasm; E, extracellular; N, nucleus; P, plasma membrane; U, unknown. Major canonical functional/signaling categories associated with genes in the figures identified by the software, are shown in yellow boxes.

Figure 6

Pathway analysis. Figures 3-7 represent signaling pathways for 58 focus genes selected from Table 3 by Ingenuity Systems software (Redwood City, CA. The following signaling cascades are shown: JUN, TNF, and CDKN2A signaling cascade affecting: DNA replication; recombination and repair; immune response; and cell cycle (Fig. 3); TGFB1 signaling cascade affecting: cell morphology; cancer; and tumor morphology (Fig. 4); CTNB1 signaling cascade affecting: cell signaling; gene expression; and cell cycle (Fig. 5); INS1/hRAS signaling cascade affecting: carbohydrate metabolism; endocrine disorders; and metabolic disease (Fig. 6); and MYC signaling cascade affecting: viral function; gene expression; and cell Cycle (Fig. 7). Differentiating groups (per Table 3) are overlaid onto the signaling diagrams, and abbreviated: FU, fungal; FI, fish oil; CO, combination diet. When CO was the differentiating group, absolute differences between FU and FI are indicated. Intracellular location of focus genes (subscripts) are annotated: C, cytoplasm; E, extracellular; N, nucleus; P, plasma membrane; U, unknown. Major canonical functional/signaling categories associated with genes in the figures identified by the software, are shown in yellow boxes.

Figure 7

Pathway analysis. Figures 3-7 represent signaling pathways for 58 focus genes selected from Table 3 by Ingenuity Systems software (Redwood City, CA. The following signaling cascades are shown: JUN, TNF, and CDKN2A signaling cascade affecting: DNA replication; recombination and repair; immune response; and cell cycle (Fig. 3); TGFB1 signaling cascade affecting: cell morphology; cancer; and tumor morphology (Fig. 4); CTNB1 signaling cascade affecting: cell signaling; gene expression; and cell cycle (Fig. 5); INS1/hRAS signaling cascade affecting: carbohydrate metabolism; endocrine disorders; and metabolic disease (Fig. 6); and MYC signaling cascade affecting: viral function; gene expression; and cell Cycle (Fig. 7). Differentiating groups (per Table 3) are overlaid onto the signaling diagrams, and abbreviated: FU, fungal; FI, fish oil; CO, combination diet. When CO was the differentiating group, absolute differences between FU and FI are indicated. Intracellular location of focus genes (subscripts) are annotated: C, cytoplasm; E, extracellular; N, nucleus; P, plasma membrane; U, unknown. Major canonical functional/signaling categories associated with genes in the figures identified by the software, are shown in yellow boxes.