1476-511X-5-10 1476-511X Research <p>How dietary arachidonic- and docosahexaenoic- acid rich oils differentially affect the murine hepatic transcriptome</p> Berger Alvin aberger2@nc.rr.com Roberts A Matthew mrsci68@yahoo.com Hoff Bruce hoff@biodiscovery.com

Department of Nutrition, University of North Carolina, Chapel Hill, NC 27599, USA

Head of Biochemistry, Metabolon, Inc., 800 Capitola Drive, Suite 1, Durham, NC 27713, USA

Director, Nestle Corporate Venture Funds, Acquisitions & Business Development, Nestle S.A., 55 Avenue Nestle, 1800 Vevey, Switzerland

Director of Analytical Sciences, BioDiscovery, Inc., 100 North Sepulveda Blvd., Suite 1230, El Segundo, CA 90245, USA

 Lipids in Health and Disease 1476-511X 2006 5 1 10 http://www.lipidworld.com/content/5/1/10 16623957 10.1186/1476-511X-5-10 13 3 2006 20 4 2006 20 4 2006 2006 Berger et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

Background

Microarrays and related technologies such as RT-PCR have accelerated our ability to understand the effects of long chain polyunsaturated fatty acids (LC-PUFA) and their derivatives on the transcriptome, implied metabolome, and lipid signaling cascades in various species and tissues 12.

Transcription factors indicated thus far include peroxisome proliferator activated receptors (PPARs), hepatic nuclear-4α (HNF-4α), nuclear factor κβ (NF-κβ), retinoid X receptor α (RXRα), sterol regulatory element binding protein-1c (SREBP-1c), and liver X receptors (LXR) 13. Several studies have examined effects of LC-PUFA on the focused and global transcriptome 4, with some examining the effects of n6/n3 LC-PUFA ratios using precursors of 20:4n6 (such as 18:2n6), and precursors of 22:6n3 (such as 18:3n3) or 22:6n3 itself 56789. We are not aware of works comparing arachidonic acid (AA), eicosapentaenoic (EPA)/docosahexaenoic acids (DHA), and the combination of AA and EPA/DHA in liver and other tissues of mice nor other organisms.

In the present study and previous works, we fed mice diets enriched with fungal oil enriched in AA (FUNG), fish oil (FISH), or a combination of the two (COMB). In our first study, we examined the microarray transcriptional profile in liver and hippocampus, focusing on genes affecting lipid metabolism via known transcriptional signatures (PPARs, SREBPS, etc.), and provided supporting lipidomic data 1. We documented and refined our statistical approaches used to select differentially regulated genes 1011. Thereafter, we focused in hepatic and hippocampal genes implicated in: behavior 12; cancer etiology 1314; and obesity 15. Lastly, we examined changes to all differentially regulated genes in liver 16 and hippocampus 17. These works have been described in reviews 318192021. What is still lacking is a more comprehensive evaluation of how n6 and n3 LC-PUFA differentially affect the murine hepatic transcriptome and how such events might translate to affects on metabolism 4. To address this challenge, we re-evaluated our original microarray data 1 using new statistical approaches, pathway mapping, and updated literature.

Results and discussion

Comparison of diets on a genomic scale

In mouse liver, there were 371 probe sets varying between diets using an F-statistic (P < 0.001; GeneSight™ software (BioDiscovery, Inc.). Sets were evaluated by principal component analysis (PCA) analysis (Fig. 1) and retained 63% of the variance of the original data.

<p>Figure 1</p>

Principle component analysis scatter plot showing the five arrays used, represented by 371 most highly differentiated probe sets

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.

The number of significantly different genes between pairs of diets (F-statistic; P < 0.001) is superimposed on the PCA plot (Fig 1). The two replicate control (CONT) groups were very similar based on PCA, as expected. Numbers of pair wise probes differing between CONT and -FUNG, -FISH, and -COMB were 160, 204, and 208, respectively (P < 0.001; Fig. 1), indicating CONT and FUNG were most similar to one another. Numbers of probes differing between FUNG-FISH, FUNG-COMB, and FISH-COMB were 127, 127, and 153, respectively (P < 0.001). At P < 0.001, only 13 genes (0.001 × 13,000) are expected to appear by chance.

Using set intersection analysis on probes from pair wise comparisons, 20-, 27- and 44 probes differentiated FUNG, FISH and COMB from one another, respectively; most being down regulated (Table 1). 63349_s in FUNG-COMB, and C78039 in FUNG-FISH overlapped between sets.

<p>Table 1</p>

Number of up- and down-regulated probes that differentiated each treatment group from all other groups. Trends in regulation for probes differentiating treatment groups. The smallest set of probes (a subset of probes described in Fig. 1) differentiating each group (FUNG, FISH, COMB) from all others at P < 0.001 was determined with set intersection analysis. There is one row for each set of differentiating probes. Columns show numbers of genes in the list, up (+) and down (-) regulated. Almost all differentiating genes were down-regulated in pair wise comparisons.

Group vs.:

GROUP

# Differentiating Genes

CONT

FUNG

FISH

COMB

FUNG

20

+0/-20

+0/-20

+0/-20

FISH

27

+5/-22

+6/-21

+5/-22

COMB

44

+1/-43

+2/-42

+1/-43

Hierarchical cluster analysis on the 371 probes indicated the two CONT groups clustered together (Fig. 2). FISH was more similar to CONT than FUNG or COMB, which clustered together. However, cross bar height shows FISH is not profoundly closer to CONT, than FUNG or COMB.

<p>Figure 2</p>

Hierarchical clustering of significant probes

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.

 <p>Figure 3</p>

Pathway analysis

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.

Pathway analysis

Pathway analysis (Figs. 3, 4, 5, 6, 7) was performed on selected transcripts differentiating the groups (Table 3; described in next section). It is not always possible from transcript data alone to correctly predict whether a pathway will be up or down regulated since post translational and dimerization events, and promoter/enhancer sequences ultimately affect DNA binding activation/signal transduction. The various diets provided different ratios of n6/n3 LC-PUFA. Thus, the dietary groups affected similar global signaling pathways; but differentially affected down stream signaling cascades and the magnitude/direction of change to specific transcripts. Generalized descriptions of the signaling cascades are provided below; detailed descriptions of specific transcripts are included in the subsequent section, "Comparison of diets on an individual gene level." Unreferenced literature emanates from Ingenuity software directly. Uppercase symbols refer to proteins; lower case symbols refer to genes; gene abbreviations are found in Table 3.

<p>Figure 4</p>

Pathway analysis

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.

 <p>Figure 5</p>

Pathway analysis

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.

 <p>Figure 6</p>

Pathway analysis

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.

 <p>Figure 7</p>

Pathway analysis

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.

 <p>Table 3</p>

Genes differentiating FUNG, FISH, AND COMB. Genes differentiating FUNG, FISH, and COMB. Shown are genes differentiating each group from all other groups, for which annotations were available. Unigene numbers lack Mm. prefix. Fold changes, relative to the differentiating group, are as follows: red, up regulation; green, down regulation. For example, for tcfe3, COMB was the differentiating group. Transcript levels for tcfe3 were 6.2 fold less for COMB relative to CONT; 7.8 fold less for COMB relative to FUNG; and 11.1 fold less for COMB relative to FISH; and FISH increased transcript levels relative to FUNG. Biological categories (e.g., Structural Role, Phosphatases and kinases) were derived from gene ontology (GO) nomenclature. Within each biological category, rows were sorted by differentiating group (FUNG, FISH, COMB), up or down regulation, and thereafter, alphabetically. ***P≤0.001; **0.001<P≤0.005; *0.005<P≤0.010. A more extensive version of this table including GO biological processes and GO molecular functions is available upon request.

Fold Change: Differentiating group vs. other groups:

Gene Symbol

Name

Unigene

Differentiating Group

CONT

FUNG

FISH

COMB

Structural role (adhesion, microtubule, actin)

Sn

sialoadhesin

1374

COMB

-2.7***

-2.6**

-2.8***

Catns

catenin src

198356

FUNG

-1.5*

-1.7**

-1.6*

Diaph1

diaphanous homolog 1 (Drosophila)

3282

FUNG

-3.5***

-4.9***

-4.2***

acta2

actin, alpha 2, smooth muscle, aorta

16537

COMB

-3.9***

-5.0***

-4.9***

arhgef7

Rho guanine nucleotide exchange factor (GEF7)

3439

COMB

-3.4**

-3.7**

-3.8**

cryaa

crystallin, alpha A

1228

FISH

-2.8***

-3.1**

-4.7***

homer2

homer homolog 2 (Drosophila)

228

FISH

-60.5***

-50.5***

-68.9***

tubb2

tubulin, beta 2

246377

COMB

-3.6***

-2.2**

-3.8***

tubb3

tubulin, beta 3

40068

COMB

-2.7***

-2.0**

-2.9***

eln

elastin

111845

FUNG

-19.0**

-16.6*

-19.1*

dvl2

dishevelled 2, dsh homolog (Drosophila)

5114

FISH

-29.4***

-47.3**

-21.9*

numbl

numb-like

49224

FUNG

-27.3***

-58.3***

-22.9***

hps1

Hermansky-Pudlak syndrome 1 homolog (human)

218381

COMB

-3.3***

-3.2**

-3.7***

plod3

procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3

137885

FUNG

-4.7***

-5.2***

-4.5***

hip1r

huntingtin interacting protein 1 related

149954

COMB

-9.1***

-8.2**

-7.0**

Phosphatases and kinases

ppp1ca

protein phosphatase 1, catalytic subunit, alpha isoform

1970

FUNG

-18.5***

-12.3***

-23.4***

ppp2cb

protein phosphatase 2a, catalytic subunit, beta isoform

7418

FISH

1.9***

1.6*

1.9***

ppm1b

protein phosphatase 1B, magnesium dependent, beta isoform

849

FISH

-27.4**

-48.3***

-57.5***

ptpn1

protein tyrosine phosphatase, non-receptor type 1

2668

FUNG

-10.3***

-28.4***

-13.3***

dusp1

dual specificity phosphatase 1

2404

COMB

-2.2**

-3.2***

-2.6***

dusp9

dual specificity phosphatase 9

16479

COMB

-53.8***

-34.1***

-70.8***

camk2b

calcium/calmodulin-dependent protein kinase II, beta

4857

FISH

-8.8***

-8.6***

-4.5**

ptpra

protein tyrosine phosphatase, receptor type, A

587

COMB

-10.2***

-12.2**

-8.0*

fgfr3

fibroblast growth factor receptor 3

6904

FISH

-3.3**

-3.7**

-3.3*

plk1 (plk)

polo-like kinase homolog, (Drosophila)

16525

COMB

-1.9***

-2.1***

-2.0***

ccnb1

cyclin B1

22569

FUNG

-46.7***

-19.5***

-58.6***

cdk4

cyclin-dependent kinase 4

6839

FISH

-2.8***

-2.5**

-3.4***

ntrk1

neurotrophic tyrosine kinase, receptor, type 1

129850

COMB

-2.84***

-3.31***

-3.7***

G protein coupled signaling

rgs16

regulator of G-protein signaling 16

181709

COMB

2.2***

1.9*

2.9***

gnat1

guanine nucleotide binding protein, alpha transducing 1

69061

COMB

-20.4***

-18.8***

-17.9**

Transcription factors

notch2

Notch gene homolog 2, (Drosophila)

57005

FUNG

-5.0***

-6.6***

-3.4*

barx1

BarH-like homeobox 1

42241

FUNG

-23.5***

-62.5***

-15.1***

tcfeb (tfeb)

transcription factor EB

2305

FUNG

-4.3***

-6.5***

-5.1***

nfkbia

nuclear factor of kappa light chain gene enhancer in B-cells inhibitor, alpha

8884

FISH

-1.6**

-2.1***

-1.9***

jund1

Jun proto-oncogene related gene d1

1175

FISH

-7.2**

-18.6***

-14.0***

jun

Jun oncogene

482

FISH

-4.7***

-5.5***

-4.2***

yy1

YY1 transcription factor

3868

FISH

-3.2***

-5.7***

-4.4***

tef

thyrotroph embryonic factor

19258

FISH

-21.9***

-18.4**

-24.2**

usf2

upstream transcription factor 2

15781

FISH

-5.6***

-6.9***

-5.5***

hoxa13

homeo box A13

8056

FISH

-2.5***

-2.2*

-2.6**

tcea2

transcription elongation factor A (SII), 2

24245

FISH

-8.7**

-16.8***

-33.3***

foxa3; hnf-3g

forkhead box A3

42260

COMB

-2.2***

-2.8***

-2.4***

tcf7

transcription factor 7, T-cell specific

31630

COMB

2.5**

2.1*

2.6**

tcf2

transcription factor 2

7226

COMB

-55.0***

-46.5***

-89.1***

atf5

activating transcription factor 5

1566

COMB

-3.1***

-1.9***

-2.0***

gtf2i

general transcription factor II I

22593

COMB

-11.4***

-6.8**

-20.6***

asb6

ankyrin repeat and SOCS box-containing protein 6

27656

COMB

-20.8***

-13.3**

-16.0***

mllt1

myeloid/lymphoid or mixed lineage-leukemia translocation to 4 homolog (Drosophila)

148748

COMB

-9.8***

-15.0***

-12.2***

ighmbp2

immunoglobulin mu binding protein 2

3179

COMB

-5.3***

-4.3*

-6.3***

nr2c2

nuclear receptor subfamily 2, group C, member 2

3535

COMB

-37.9***

-81.7***

-19.72***

tcfe3 (tfe3)

transcription factor E3

25762

COMB

-6.2***

-7.8***

-11.1***

miz1 (pias2)

Msx-interacting-zinc finger

6370

COMB

-1.9*

-3.3***

-2.9***

jmj

jumonji

25059

COMB

-8.7***

-14.6***

-7.1**

mRNA and rRNA splicing and processing

sf3a2

splicing factor 3a, subunit 2, 66kD

34039

FUNG

1.6**

2.0***

1.7**

nono

non-POU-domain-containing, octamer binding protein

21559

FUNG

-4.6***

-6.2***

-10.6***

srp9

signal recognition particle 9 kDa

89927

COMB

-3.1**

-2.7*

-3.3**

bop1

block of proliferation 1

4283

FISH

-10.1*

-11.9*

-14.0*

zfp385 (znf385)

zinc finger protein 385

14099

COMB

-2.3***

-2.3***

-1.7**

Amino acid and protein metabolism

dct

dopachrome tautomerase

19987

FUNG

-4.8***

-4.5***

-2.1***

mrpl23

mitochondrial ribosomal protein L23

12144

COMB

-16.4***

-13.9***

-18.7***

rpl28

ribosomal protein L28

3111

COMB

-1.8***

-1.5**

-1.8***

eef2

eukaryotic translation elongation factor 2

27818

FISH

-4.9***

-3.0***

-4.0***

eef1a1 (2 probes)

eukaryotic translation elongation factor 1 alpha 1

196614

FUNG

-1.6*** to -1.7***

-1.6** to -2.1***

-1.5* to -1.9***

eprs

glutamyl-prolyl-tRNA synthetase

154511

COMB

-16.2***

-17.3***

-18.9***

sars1

seryl-aminoacyl-tRNA synthetase 1

28688

FISH

-4.5***

-4.5**

-4.2*

ppil2

peptidylprolyl isomerase (cyclophilin)-like 2

218627

COMB

-3.4***

-4.6***

-3.7***

ywhaq

tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, theta polypeptide

14722

FUNG

-4.0**

-4.4*

-7.3***

oat

ornithine aminotransferase

13694

FUNG

1.7***

1.5*

1.7***

lamp2

lysosomal membrane glycoprotein 2

486

FUNG

-12.7***

-15.6***

-45.0***

ang

angiogenin (2 probes)

202665

FISH

-1.5* to -2.7***

-1.8*** to -4.5***

-1.8*** to -2.1*

kai1 (cd82)

kangai 1 (suppression of tumorigenicity 6, prostate)

4261

FISH

-1.7***

-2.3***

-1.5*

psmb10

proteasome (prosome, macropain) subunit, beta type 10

787

FISH

-22.7***

-47.8***

-14.0**

folh1

folate hydrolase

7522

FUNG

-10.8**

-13.3*

-21.1**

mcpt7 (tpsab1)

mast cell protease 7

3301

COMB

-3.5***

-2.9***

-3.5***

Lipid metabolism and P450s

acas2

acetyl-Coenzyme A synthetase 2 (ADP forming)

22719

COMB

-4.8***

-2.0*

-2.9***

scd1 (2 probes)

stearoyl-Coenzyme A desaturase 1

140785

COMB

-1.5** to -1.8***

-1.4* to -1.7***

-1.6*** to -1.7***

elovl3; cig30

elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 3

21806

FISH

1.9***

2.2***

3.4***

fasn

fatty acid synthase

3760

FUNG

-1.8***

-1.4*

-3.2***

acly (2 probes)

ATP citrate lyase (ACL)

25316

COMB

-3.6 to -5.2***

-1.9 to -2.0***

-2.0 to -2.3***

lypla3

lysophospholipase 3

25492

FISH

-32.4***

-25.9***

-23.9***

thrsp

thyroid hormone responsive SPOT14 homolog (Rattus)

28585

COMB

-1.8***

-2.3***

-1.7***

cyp4a10

cytochrome P450, 4a10

10742

FISH

4.7***

1.7**

1.7**

cyp24

cytochrome P450, 24

6575

FUNG

-8.6**

-15.1***

-19.3***

Carbohydrate metabolism

lgals3

lectin, galactose binding, soluble 3

2970

FUNG

-4.0***

-5.5***

-4.8***

idua

iduronidase, alpha-L-

3054

COMB

-14.7***

-15.7***

-12.2**

gaa

glucosidase, alpha, acid

4793

COMB

-3.1***

-2.5**

-2.3**

g6pc

glucose-6-phosphatase, catalytic

18064

COMB

-1.9***

-2.3***

-3.0***

pkm2

pyruvate kinase, muscle

2635

FISH

-23.3***

-48.8***

-12.2**

bat2

HLA-B associated transcript 2

20304

COMB

-9.6***

-4.8**

-7.7***

Transport (lipid and other)

abce1

ATP-binding cassette, sub-family E (OABP), member 1

5831

FISH

2.7***

2.5**

2.5**

fabp5

fatty acid binding protein 5, epidermal

741

FISH

-12.1***

-2.0**

-1.8***

hspg2

perlecan (heparan sulfate proteoglycan 2)

7257

COMB

-6.7***

-5.5***

-3.5**

dbi

diazepam binding inhibitor

2785

COMB

-1.4**

-1.5**

-1.8***

cd36

CD36 antigen

18628

FISH

2.4***

2.5***

3.8***

rab5c

RAB5C, member RAS oncogene family

29829

FISH

-3.6***

-3.6***

-4.0***

ap4s1

adaptor-related protein complex AP-4, sigma 1

116858

FUNG

-24.3*

-54.2***

-22.6*

slc4a2

solute carrier family 4 (anion exchanger), member 2

4580

FUNG

3.0**

3.9**

5.3***

slc10a1

solute carrier family 10 (sodium/bile acid cotransporter family), member 1

104295

FISH

-1.6**

-1.7**

-1.7**

apoc1

apolipoprotein C-I

182440

FUNG

-1.6***

-1.7***

-1.5**

apoe

apolipoprotein E

156335

FISH

1.5***

1.4*

1.8***

slc27a1

solute carrier family 27 (fatty acid transporter), member 1

7206

FUNG

-2.0**

-2.2**

-2.3**

apoa4

apolipoprotein A-IV

4533

COMB

-17.2***

-7.0***

-5.4***

hba-a1

hemoglobin alpha, adult chain 1

196110

FISH

1.5***

1.4*

2.0***

hbb-b1

hemoglobin, beta adult major chain

233825

COMB

-1.7***

-1.7***

-2.3***

aqp8

aquaporin 8

9970

COMB

-3.6***

-1.7**

-1.7**

Immune response/oxidative stress

c5r1

complement component 5, receptor 1

137488

FISH

-79.3***

-165.2***

-87.7***

cd72

CD72 antigen

88200

FISH

-46.2***

-38.9**

-41.1**

aldh1a7

aldehyde dehydrogenase family 1, subfamily A7

14609

FISH

1.9***

1.6**

1.6**

mif

macrophage migration inhibitory factor

2326

FISH

-2.3***

-3.1***

-1.9***

prdx2

peroxiredoxin 2

42948

FISH

-1.6***

-1.7**

-1.6**

hspb1

heat shock protein 1

13849

FISH

-4.0***

-5.8***

-6.3***

hspcb

heat shock protein 1, beta

2180

FUNG

-1.5*

-1.6*

-1.7**

hpxn

hemopexin

3485

COMB

-1.5***

-1.9***

-1.5**

Heme synthesis

alas1

aminolevulinic acid synthase 1

19143

FUNG

3.9***

2.1***

1.6**

alas2

aminolevulinic acid synthase 2, erythroid

140509

FUNG

-2.7**

-2.9**

-2.8**

Calcium, sodium, zinc ion binding

tpt1

tumor protein, translationally-controlled 1

254

FUNG

-1.6**

-1.7**

-1.6*

fxyd2

FXYD domain-containing ion transport regulator 2

22742

COMB

-29.9***

-57.5***

-20.8*

zfp312

zinc finger protein 312

34644

FISH

-6.0*

-8.2*

-12.8**

slc30a3

solute carrier family 30 (zinc transporter), member 3

1396

FISH

-4.2**

-5.3**

-6.8***

s100a3

S100 calcium binding protein A3

703

COMB

-3.0***

-4.0***

-4.4***

rhbdl4

rhomboid like gene 4 (Drosophila)

219535

FISH

-3.3**

-4.3***

-3.4**

Miscellaneous

serpinc1

serine (or cysteine) proteinase inhibitor, clade C (antithrombin), member 1

30025

FISH

1.7***

1.8***

2.5***

ssr2

signal sequence receptor, beta

7091

FUNG

-5.0***

-8.0***

-5.5**

ssr1

signal sequence receptor, alpha

138725

FISH

-6.2***

-5.9***

-6.3***

surf1

surfeit gene 1

6874

FISH

-2.4***

-2.4**

-2.3**

wbp1

WW domain binding protein 1

1109

FISH

-3.9***

-2.6***

-3.5***

JUN/TGIF/TNF/CDKN2A signaling (Fig. 3)

JUN/TGIF/TNF/CDKN2A proteins affect DNA replication, recombination and repair, immune responses, and cell cycle. JUN is involved in signaling cascades including: B cell, chemokine, EGF, hypoxia, IGF-1, IL-6, neurotrophin/TRK, TGFβ, and toll-like signaling. Down stream of JUN, TGIF is involved in death receptor signaling; TNF has roles in apoptosis and cAMP signaling; CDKN2A is involved in G2/M DNA damage check point regulation.

FUNG and COMB increased jund relative to FISH (FISH deceased jund), similar to that reported in other models comparing effects of n6 and n3 LC-PUFA on jun 22. Jund was decreased by FISH which in turn led to decreased yy1 and cryaa (crystallin, alpha A); decreased homer2 (homer homolog 2 Drosophila) and hspb1 (heat shock protein 1) (via RHOA); and decreased psmb10 (proteasome subunit, beta type 10) and cd82 (Kai1, kangai 1, suppression of tumorigenicity 6, prostate) (via TNF).

FUNG decreased diaph1 (diaphanous homolog 1) (via JUN target RHOA); decreased fasn (fatty acid synthase) and slc27a1 (solute carrier family 27 fatty acid transporter member 10) (via JUN target TNF) COMB decreased pias2 (miz1, Msx-interacting-zinc finger) (via JUN); increased rgs16 (regulator of G-protein signaling 16) (via JUN target TNF); and decreased dusp1 (dual specificity phosphatase 1) and znf385 (zinc finger protein 385) (via JUN target CDKN2A). Rgs16 is known to be decreased in response to G1/S activation in mouse liver, hence an increase could have a role in preventing cell progression 23.

TGFB1 signaling (Fig. 4)

TGFB1 and JUN signaling are intertwined. TGFB1 is involved in IL-6 signaling and valine/leucine/isoleucine biosynthesis. FUNG decreased eln (elastin) (via TGFB1); and decreased ighmbp2 (immunoglobulin mu binding protein 2) relative to FISH (via TGFB1 target HGF). FISH decreased fabp5 (fatty acid binding protein 5, epidermal) (via TGFB1) and subsequently abce1 (ATP-binding cassette, sub-family E member 1) (via FABP-target SNCA); and decreased hoxa13 (homeo box A13) (via TGFB1 target BMP2). Human ABCE1 has recently been shown to be essential for in vitro and in vivo translation of mRNA, and to bind the initiation factors eIF2α and eIF5 24.

COMB decreased ighmbp2, eprs (glutamyl-prolyl-tRNA synthetase), tpsab1 (mast cell protease 7) (via TGFB1), gnat1 (guanine nucleotide binding protein, alpha transducing 1) (via TGFB1 target IKBKb); and g6pc (glucose-6-phosphatase, catalytic) and hpx (hemopexin) (via TGFB1 target LEP).

CTNB1 signaling (Fig. 5)

CTNB1 is involved in PI3K/AKT and WNT/β catenin signaling. FUNG decreased lgals3 (lectin, galactose binding, soluble 3), dct (dopachrome tautomerase), and bat2 (HLA-B associated transcript 2) (relative to FISH) and increased oat (ornithine aminotransferase) (all via CTNB1); it also increased sf3a2 (splicing factor 3a, subunit 2) (via CTNB1 target SF3A1). FISH decreased dvl2 (dishevelled 2, dsh homolog) (via CTNB1). COMB increased tcf7 (transcription factor 7, T-cell specific), and decreased plk1 (polo-like kinase homolog) and bat2 (all via CTNB1); and decreased tubb2 and increased tubb3 (via CTNB1 target TUBBA1).

INS1/HRAS signaling (Fig. 6)

INS1 is involved in numerous signaling cascades: G protein, insulin, JNK/STAT, and PPAR. hRAS is an INS1 target involved in the following signaling cascades: B and T cell, EAF, estrogen, FGF, IL-2, insulin, integrin, neurotrophin/TRK, PI3K/AKT, SAPK/JNK, sterol biosynthesis, TGFβ, and VEGF. In response to growth factors, hRAS signaling involves the following activation cascade: hRAS-RAF(MAPKKK)-MEK (MAPKK)-ERK (MAPK).

FUNG decreased apoc1 (apolipoprotein C-I) (via INS1); decreased numbl (numb-like) (via INS1 target APP); decreased alas2 (aminolevulinic acid synthase 2, erythroid) (via INS1 target STAT3); and decreased pp1ca (protein phosphatase 1, catalytic subunit, alpha isoform) (via INS1 target hRAS).

FISH increased cd36 (CD36 antigen) and decreased camk2b (calcium/calmodulin-dependent protein kinase II, beta) (via INS1); decreased fgfr3 (fibroblast growth factor receptor 3) (via INS1 target STAT3); and decreased slc30a3 (solute carrier family 30) (via INS1 target hRAS). With respect to this last observation, DHA and fish oil can decrease RAS plasma membrane localization, RAS-GTP binding, and p42/44 ERK signaling in colonocytes 25.

COMB decreased acly (ATP citrate lyase) and thrsp (via INS1); decreased foxa3 (forkhead box A3) (via INS1 target FOXA2); and decreased ntrk1 (neurotrophic tyrosine kinase, receptor, type 1) (via INS1 target HRAS).

MYC signaling (Fig. 7)

MYC is involved in various signaling cascades: cell cycle G1/S check point regulation, p38 MAPK, and PDGF. Down stream of MYC, CREBP is a PPAR target involved in TGFβ and NFKβ signaling.

FISH decreased bop1 (block of proliferation 1) (via MYC) and decreased tcea2 (via MYC target POL2RA). Consistent with our findings, fish oil can decrease cMYC protein 26. FUNG decreased lamp2 (via MYC); and decreased tcf2 relative to FISH (transcription elongation factor A SII) (via MYC target PCAF). COMB decreased dbi (diazepam binding inhibitor) (via MYC) and decreased tcf2 (transcription factor 2) (via MYC targets PCAF and CREBP).

Comparison of diets on an individual gene level

Herein, we focus on genes implicated in pathway analysis (Figs 3, 4, 5, 6, 7) and linkable to specific functions (Tables 2, 3). For brevity, signaling cascades cannot be described in their entirety. In Table 2, pathways are selected based on enrichment analysis. Table 3 includes genes that are individually most differentially regulated between diets. Up and down regulation are always relative to the other two groups even if not explicitly stated for brevity (e.g., down regulation by FUNG indicates down regulation by FUNG relative to FISH and COMB). Unreferenced literature emanates directly from Ingenuity- or Affymetrix Netaffx programs. For readability, gene and protein abbreviations are followed by full names in brackets.

<p>Table 2</p>

Genes differentiating FUNG, FISH, and COMB, using gene ontology classifications and enrichment analysis. Genes differentiating FUNG, FISH, and COMB, using gene ontology (GO) classifications and enrichment analysis. Differentially regulated genes (GEA model) were subjected to enrichment analysis to select GO terms "enriched with" these genes. Two broad GO categories included are "Biological Process" and "Molecular Function". Specific GO terms are included and specific genes within each gene term differentiating the diets are parenthesized, alphabetized, and separated by commas. Rows are sorted for consistency with Table 3 where possible. Differentiating genes were selected at P < 0.001, and GO terms computed at P < 0.01; P is the probability a random gene for each GO term will have as many genes with the same GO term as the actual list. The biological process category "Metabolism" was excluded as it was too general. Genes in Tables 2-3 are not identical because of the different statistical approaches utilized. Enrichment analysis placed acadm (acetyl-Coenzyme A dehydrogenase, medium chain (MCAD) in both the FA oxidation and Electron transport categories; it was removed from the latter.

Process/Differentiating group(s)

FUNG

FISH

COMB

Biological process classifications

Amino acid metabolism

agxt, alas2, bcat1, oat

Acetyl-CoA biosynthesis

acas2, acly

acas2, acly

Fatty acid and lipid synthesis

elovl2, elovl3, fasn, scd1

Fatty acid oxidation

acadm, cpt1a, cpt2

acox1, cpt2, ech1

cpt1a, cpt2, acsl1(facl2)

Carbohydrate metabolism

foxa3, gck

Electron transport (Cyt P450 metabolism)

por, cyp2a4, cyp2b9, cyp3a11, cyp3a16, cyp3a41, cyp4a10

Lipid transport

apoa4, apoc2, osbpl5, pltp

Heme biosynthesis

alad, alas1, alas2

Oxygen transport

hba-a1, hbb-b1

Peroxidase reaction

gpx1, mpo, prdx2

Response to heat, stress, inflammation

hspa8, hspcb, hspb1, hspb8

Molecular function classifications

Structural constituent of cytoskeleton

acta1, acta2, krt1-13, tuba1, tubb2, tubb3, tubb5

Transaminase

agxt, bcat1, oat

Lyase

acly, fasn, pck1, umps

Transferase, transferring groups other than amino-acyl groups

elovl2, lce-pending

Acyltransferase

alas1, alas2, cpt1a, cpt2, fasn, gpam

alas1, alas2, cpt1a, cpt2, fasn, gpam

Monooxygenase/P450

cyp2a4, cyp2b9, cyp3a11, cyp3a41, cyp3a16, cyp4a10

5-aminolevulinate synthase

alas1, alas2

alas1, alas2

alas1, alas2

Oxygen transporter

hba-a1, hbb-b1

Oxidoreductase

acadm, aldh1a1, cyp2a4, cyp2b9, cyp3a11, cyp3a16, cyp3a41, cyp4a10, dct, fasn, gpx1, por, sdh1

Heat shock protein

cryac, hspa8, hspb1, hspcb

Acetate-coa ligase

acas2, facl2

Aspartic-type endopeptidase

ctse, mela

RAS small monomeric gtpase

hras1, rras

Aldehyde dehydrogenase NAD+

aldh1a1, aldh1a7, aldh2

Antioxidant

prdx2, prdx4

Hormone

gh, igf2, prlpe, sct

Peptide hormone

igf2, npy, sct

Cathepsin S, L, K

ctsl, ctss

Globin

hba-a1, hbb-b1

Hydroxymethylglutaryl-CoA synthase

hmgcs1, hmgcs2

Structural role

DIAPH1 has a role in actin cytoskeleton organization and biogenesis; and inhibits apoptosis 2728. FUNG down regulated diaph1 (diaphanous homolog 1) which may induce apoptosis.

FUNG down regulated plod3 (pro-collagen-lysine, 2-oxoglutarate 5-dioxygenase 3), coding lysyl hydroxylase, involved in collagen synthesis 29. Fish oil consumption can increase collagen synthesis 30, and FISH could increase collagen synthesis via plod3. FUNG also down regulated eln (elastin) and catns (catenin src), having roles in cell adhesion.

FISH decreased homer2 involved in actin binding via glutamate receptor signaling. DVL2 (dishevelled 2, dsh homolog) acts on the cytoskeleton and induces apoptosis via MAPK9 and -10 and CTNB1/WNT/β/Catenin signaling (Fig. 5). FISH dramatically decreased dvl2 which may decrease apoptosis.

COMB differed from other groups with respect to effects on the cytoskeleton, using molecular function enrichment analysis (Table 2). Specifically, COMB down regulated: acta2 (actin, alpha 2) involved in smooth muscle contraction; arhgef7 (Rho guanine nucleotide exchange factor) and Sn (sialoadhesin) having roles in cell adhesion; and tubb2- and 3 (tubulins β 2 and -3), with roles in microtubule movement. Tubb 2 and 3 are known to be induced in response to G1/S activation in mouse liver 23.

Phosphatases and kinases

PTP-1β (protein tyrosine phosphatase 1β, non-receptor type 1) is a tyrosine phosphatase which: interacts with EGF and PDGF receptors; regulates insulin and leptin signaling; and has affects on LDL-cholesterol and obesity in humans 31. FUNG decreased ptpn1, which could affect cell proliferation.

CDK1 (cyclin dependent kinase 1) activation results in mitosis following CDK1-cyclin-B1 complex formation. FUNG decreased ccnb1 (cyclin B1) (proportionately increased by FISH), and this could impair CDK1 activation altering mitotic progression. CDK4 is a serine/threonine protein kinase required for G1-S cell cycle transition. Its activity is controlled by D-cyclins and CDK inhibitor p16 (INK4a). CDK4 also phosphorylates RB (retinoblastoma) protein which affects cell cycle progression. FISH down regulated cdk4 (cyclin-dependent kinase 4), which could inhibit cell cycle progression.

PPP1ca/PPP2C-β (protein phosphatases 1ca and -2b) are serine/threonine phosphatases with myosin phosphatase activity; and roles in glycogen metabolism, RNA processing, and cell cycle regulation. FISH increased ppp1ca and ppp2cb which could affect cell proliferation.

Camk2b (Calcium/calmodulin-dependent protein kinase IIβ, CaM-KII, CKII) is required for cell division via INS1 signaling (Fig. 6). Specifically, CKII phosphorylates SAG (sensitive to apoptosis gene), leading to degradation of IκBα and P27KIP1, and cell proliferation via G1/S phase transition 32. SAG also inhibits lipid oxidant-induced apoptosis by inhibiting metal ion-induced release of cytochrome c and activating caspase. Camk2b was down regulated with FISH (and possibly its oxidation products acting on SAG), which may induce apoptosis/inhibit cell proliferation. CKII can also phosphorylate numb/numbl proteins in vitro; and in vivo, Cam-KI phosphorylates numbl in rat liver. Numbl was down regulated by FUNG (increased by FISH) via INS1-APP signaling (Fig. 6). The Cam-KII-NUMBL kinase cascade may affect axonal growth. NUMBL also interacts with additional binding proteins in liver (e.g., 14-3-3 proteins), thus new functions for numbl are being discovered 33. CKII may also affect FA β-oxidation by altering CPT-1 (carnitine paltmitoyl transferase) phosphorylation 34.

DUSPs (dual specificity phosphatases or mitogen-activated protein kinase phosphatase, MKPs) negatively regulate MAPK/ERK-, SAPK/JNK-, and p38- mitogen activated protein kinase-induced cellular proliferation 35. Dusps 1 and -9 were down regulated by COMB, which could increase proliferation 36.

PTPRA (protein tyrosine phosphatase, receptor type, A) de-phosphorylates and activates Src family tyrosine kinases, and may regulate integrin signaling, cell adhesion and proliferation. Ptpra was down regulated by COMB (increased with FISH) which could affect cell proliferation.

PLK1 (polo-like kinase homolog) acts through CTNB1 signaling (Fig. 5) to influence G2/M DNA damage check point regulation and induce mitotic entry of cells. NTRK1 (neurotrophic tyrosine kinase, receptor, type 1) acts through INS1/hRAS signaling (Fig. 6) to affect the cell cycle via protein tyrosine kinase signaling. Plk1 and ntrk1 were decreased by COMB. Plk is known to be increased in response to G1/S activation in mouse liver (induced by partial partial hepatectomy), hence a decrease in plk1could decrease cell progression 23.

Transcription factors

Fish oil may decrease map kinase activity (p38, p44/42, JNK/SAPK) leading to decreased AP-1 (activator protein 1) binding activity, down regulated IL-6- and TNF signaling, and decreased mitosis 223738. jund1 was down regulated by FISH, which could inhibit cell cycle progression.

USF-2 (upstream stimulating factor 2) is an E-box binding factor which activates many of the same genes activated by the basic helix-loop helix (bHLH) transcription factor, SREBP-1. USF-2 is post-translationally phosphorylated 39 and may repress MYC-induced proliferation and transformation 4041. FISH down regulated usf2 (confirmed with real time-polymerase chain reaction, RT-PCR) 1, which could inhibit proliferation. USF-2 also binds fasn (fatty acid synthase) E-box 42. Using a different gene selection model, FISH down regulated nfy (Nuclear factor Y). NF-Y binds Y-box motifs in FA synthase 1 (and inverted CCAAT/ATTGG motifs in peroxisomal β-oxidation transcripts) 43. Taken together, FISH may down regulate fasn and decrease FA synthesis, via SREBP/SRE, USF-2/E-box, and NFY/Y-box interactions.

NFKBIA (nuclear factor of kappa light chain gene enhancer in B-cells inhibitor, alpha) inhibits apoptosis by inhibiting apoptosis activator NFKB1 (nuclear factor of kappa light chain gene enhancer in B-cells 1, p105). FISH down regulated nfkbia which could activate apoptosis.

HNF3γ is implicated in obesity, hyperlipidemia, and diabetes. It regulates glucagon transcription, insulin resistance, and pancreaticγ cell function 44. Oxidative stress activates the related transcription factor FoxO3a, which in turn activates sterol carrier protein 2 (SCP2), protecting FA from further oxidation 45; however, HNF3γ regulation is not known. COMB down regulated HNF3γ and the gluconeogenic transcript g6pc (glucose-6-phosphatase, catalytic (G6Pase), which could affect the balance between glycolysis and gluconeogenesis.

Activating transcription factors (ATF) are basic region-leucine zipper (bZIP) proteins 46. ATF5 can repress human cAMP-induced gene transcription 47 and may inhibit apoptosis and promote G1/S transitions 23. COMB decreased atf5 would could increase apoptosis and inhibit G1/S progression. Using a different gene selection model 1, COMB decreased the related factor, atf4 48.

Amino acid metabolism

Enrichment analysis indicated FUNG differed from other groups via changes to transcripts involved in amino acid metabolism and having transaminase function (Table 2).

Ornithine-delta-aminotransferase (OAT) has role in arginine/proline metabolism, and the urea cycle, acting through CTNB1 (Fig. 5). OAT converts L-ornithine to 2-ketoglutarate 49. FUNG increased oat which could reduce ornithine for polyamine (putrescine, spermidine, and spermine) synthesis via ornithine decarboxylase, having subsequent affects on cell proliferation. DCT (dopachrome tautomerase) and SF3A1/SF3A2 (splicing factors 3a) also act through CTNB1 (Fig. 5) and affect tyrosine metabolism. FUNG decreased dct and increased sf3a 2.

EPRS (glutamyl-prolyl-tRNA synthetase) has roles in glutamate metabolism, and also death receptor signaling, cell cycle G1/S check point regulation, and leukotriene LTC4 synthesis. Eprs was down regulated by COMB via TGFB1 signaling (Fig. 4), which would affect amino acid metabolism and cell cycle regulation. TPSAB1 (mast cell protease 7) has roles in proteolysis and WNT/β catenin signaling. Tbsab1 was also down regulated by COMB via TGFB1 (Fig. 4).

Fatty acid and phospholipid metabolism

As previously reported, LC-PUFA studied in our microarray experiments 1 likely increased FA β-oxidation via PPARα 50; decreased FA synthesis via down regulation of SREBP and its signaling cofactors 51; and increased glucose synthesis via PCK1 (phosphoenolpyruvate carboxykinase 1). In starvation experiments in mice, similar sets of transcripts were altered in the liver: apoa4, cyp4a14, and ech1 were increased; and scd1, fasn, and fabp5 were decreased 52.

In response to G1/S activation in mouse liver (induced by partial partial hepatectomy) 23, apoa4, cyp4a14, and ech1 were increased; and fasn, thrsp, and fabp5 were decreased 52. LC-PUFA feeding thus mimicked the fasted metabolic state, and a state in which normally quiescent hepatocytes are dividing 53. All groups up regulated mitochrondrial and peroxisomal FA β-oxidation transcripts (Tables 2, 3) 1, but no obvious trends concerning up or down regulation of β-oxidation emerged between groups in the present re-investigation.

Interestingly, COMB (providing a mixture of fatty acids in FUNG and FISH) differed most from the other groups with respect to transcripts involved in FA synthesis (Table 2). COMB down regulated: acetyl CoA biosynthesis via acas2 (acetyl-Coenzyme A synthetase 2); FA biosynthesis via thrsp (spot14); entry of AcCoA into the cytoplasm for FA synthesis via acly (ATP citrate lyase); and FA synthesis via fasn. Fasn was down regulated more with FUNG than FISH. COMB also down regulated scd1 (stearoyl-Coenzyme A desaturase 1) which could result in less monounsaturated FA being stored in triacylglycerol, cholesterol ester, and phospholipids (PL) pools, and potentially decreased obesity 54. Our PL lipidomic data were consistent with the changes in SCD 115. FISH increased FA elongation via up regulation of elovl3 (a long-chain fatty acyl elongase); this elongase may be SREBP1a-regulated 155.

LYPLA2 (lysophospholipase 3) hydrolyzes mitogenic lysoPL such as lysophosphatidylcholine 56. FISH down regulated lypla2, which could increase PLA2-induced mitogenicity and affect other cellular processes.

Cytochrome P450 metabolism

Using enrichment analysis (Table 2), FUNG affected numerous P450 transcripts, including por (P450 cytochrome oxidoreductase), cyp2a4, -2b9, -3a11, -3a16, -3a41, and -4a10 (up regulation of 4a10 confirmed with RTPCR; 1).

CYP4 is activated by PPARα-RXR. Murine CYP4A-10 can form ω/ω-1 hydroxylated 20:4n6 and hydroxylated-epoxylated 20:4n6 5758. Similar products from 20:5n3 and 22:6n3 have not been reported. These FA derivatives can affect: ion channel activity; regulate vascular tone and systemic blood pressures; and inhibit oxidative stress in mouse liver models 5859. FISH up regulated cyp4a10 (verified with RT-PCR) 1, and the related cyp 4a14 using a different gene selection model 1, which may result in the physiological changes noted above.

Calcitriol (1,25 (OH2) D3; vitamin D) binds vitamin D receptor (VDR). VDR-RXR heterodimers then bind VDRE (vitamin D-responsive elements) in genes in various organs including liver. Activated VDR regulates calcium and phosphate homeostasis and inhibits cell growth. In hepatocytes, via xenobiotic-responsive elements (ER6, DR3, and DR4), VDR also induces cyps 3a4, -2b6 and 2c9 60.

Cyp24 degrades 1,25(OH)2D3 to 1,24,25(OH)3D3, a product with decreased hormonal activity. Cyp24 expression is activated by 1,25(OH)2D3, phorbol esters, and JNK (c-Jun N-terminal kinase), acting on VDRE 61; and PXR (pregnane X receptor) agonists 62.

FUNG down regulated cyp24 which could increase 1,25(OH)2D, and decrease mitogenesis. FUNG did not up regulate cyps 2b6, -2c9, and -3a4, but up regulated family members, cyp2b9, -3a11 and -3a16, which are also major drug detoxifying enzymes 1.

Lipid transport

FATP-1 (fatty acid transport protein 1; slc27a1) transports FA across the cell membrane in liver. Thereafter efflux is prevented by CoA esterification, via ACS (acyl CoA synthase) 63. Slc27a1 and acs are PPARα regulated 63. Slc27a1 was down regulated by FUNG, likely through PPARα, and this could lead to less FA transported into the liver for various purposes including triacylglycerol formation.

Through PPARα, CD36 is implicated in long chain free FA uptake in adipocytes and liver. CD36 also affects cell proliferation, angiogenesis, and tumor metastasis 6465; and is a receptor for thrombospondin, collagen type I, and oxidized LDL on macrophages 66. Contrary to work in cancer cells where the fish oil component DHA reduced cd36 67, herein, FISH up regulated cd36 which could increase uptake of FA into liver, and increase cell proliferation.

APOC1 inhibits lipoprotein lipase dependent triacylglycerol hydrolysis in mice, independent of very-low-density lipoprotein receptor and apoCIII 68. FUNG decreased apoc1, which could lead to a lowering of circulating triacylglycerol.

E-FABP (fatty acid binding protein 5, epidermal) transports FA intracellularly in liver and other organs 69. Fabp5 may be PPARα regulated based on clustering analysis 1 and the fact that a PPARα ligand decreased fabp 70. Herein, FISH decreased fabp5 (verified with RT-PCR) 1, similarly to that reported in rats 70. FISH-induced decrease in fabp5 could alter intracellular LC-PUFA transport 71.

Hepatic uptake of bile acids is mediated by various proteins including SLC (solute carrier family) members. Slc10a1 was down regulated by FISH, which could decrease hepatic uptake of bile acids during bile acid recycling, increasing circulating cholesterol levels 72. Slc4a2 was up regulated by FUNG, which could affect biliary bicarbonate secretion, possibly via HNF signaling-HNF1α is regulated by Slc4a2 in human cells 73. The related factor, hnf-3g, was increased by FUNG relative to COMB. AQP8 (aquaporin 8) facilitates hepatic bile secretion 74. COMB down regulated aqp8 (aquaporin 8) possibly through SREBP1a 5575. Decreased AQP8 could decrease biliary cholesterol secretion and water secretion into bile 55 and conceivably increase circulating cholesterol.

COMB affected transcripts involved in apolipoprotein metabolism (apoa4, apoc2), PL transport (pltp, phospholipid transfer protein), and oxysterol binding (osbpl5, oxysterol binding protein-like 5) (Table 2, enrichment analysis). COMB down regulated apoa4 and apoa4 precursor (confirmed with RT-PCR) 1, likely through PPARα 7076. Changes to apoa4 mediated by COMB could affect reverse cholesterol transport from periphery to liver, since APOA4 is an HDL component.

APOE is involved in cholesterol transport and has anti-inflammatory properties 77. The FISH-induced increase in apoe could enhance delivery of LDL cholesterol to liver and decrease serum triacylglycerol.

Heme synthesis and oxygen transport

ALAS1 and -2 (aminolevulinic acid synthases 1 and -2) control the rate limiting steps in heme synthesis; ALAD (aminolevulinate, delta dehydratase) is the second enzyme in this pathway. Alas1 and alas2 were respectively increased- and decreased with FUNG, complicating the interpretation of how groups affected heme production.

There was evidence the diets affected heme utilization. Hbb-a1 (hemoglobin alpha, adult chain 1) and hbb-b1 (hemoglobin, beta adult major chain) are classes of hemoglobin. Heme is incorporated into not only hemoglobulins for oxygen transport; but also eicosanoid enzymes; and lipid peroxidases and heme-binding proteins which reduce damaging lipid peroxides 78. Hbb-a1 was increased with FISH and hbb-b1 was decreased with COMB (FISH>FUNG) indicating more heme may be incorporated into hemoglobin with FISH relative to FUNG.

Responses to oxidative stress and inflammation

FISH increased transcripts utilized to combat oxidative stress. This is expected since key FA in FISH having 5–6 double bonds vs. 2–4 double bonds for the key FA in FUNG, are more oxidizable 79. Peroxidase transcripts increased by FISH included gpx1 (glutathione peroxidase 1), prdx2, prdx4 (peroxiredoxins), and mpo (myeloperoxidase). Peroxiredoxins are a new family of antioxidant proteins 80. Prdx2 is a putative biomarker for progression of metastasis in melanoma cells, emphasizing its importance in controlling oxidative stress 81. MPO is a heme-containing protein with roles in atherosclerosis and Alzheimer's disease 82.

Additional proteins with roles in oxidative stress were aldh1a7 (aldehyde dehydrogenases, increased with FISH), hspcb (a heat shock protein, decreased with FUNG), hspb1 (HSP25/P27, decreased with FISH), mif (macrophage migration inhibitory factor, decreased with FISH), c5r1 (complement component 5, receptor 1, dramatically decreased with FISH), cd72 (CD72 antigen, dramatically decreased with FISH), cryaa (crystallin, alpha A, decreased with FISH), and hpxn (hemopexin, decreased with COMB, FUNG>FISH) 78. Using enrichment analysis (Table 2), additional transcription factors linked to oxidative stress distinguishing FISH, included: aldh1a1, aldh2 (aldehyde dehydrogenases), hspa8, and hspb8 (heat shock proteins). Globally, FISH had the most profound effects, increasing oxidative stress markers.

The increase in aldha7 was expected since more reactive aldehydes, such as 4-hydroxy-2-nonenal, are expected to be formed from reactions between oxygen radicals and the highly oxidizable LC-PUFA in fish oil, including DHA. Aldehyde dehydrogenases prevent lipid damage by destroying reactive aldehydes, and may be SREBP1a regulated 5583. Oxidant stress inducing drugs are known to increase aldh1a1 in rat liver, as well as transcripts for hsp (see below) 84. Transcripts for aldehyde dehydrogenases, hsp, and cd36 (all were increased with FISH) are also known to increase with high fat feeding in mouse liver, consistent with the fact that high fat induces an oxidant stress 85.

Changes to HSPs would enable cell survival and recovery in response to oxidative stress and have been previously found to be changed following fish oil consumption 86. HSPs also have roles in cytoskeletal actin dynamics in response to stress 87 and may account for changes to some cytoskeletal transcripts observed herein. Hspb1 encodes HSP25 and HSP27 88. LC-PUFA may affect transcription as well as post-translational phosphorylation of HSPs via ERK, PKCδ, TGFβ, p38, and MK2. MK2 (map kinase 2; also known as MAPKAPK2 or mitogen activated protein kinase-activated protein kinase 2) is activated by ERK. These signaling cascades are partly shown in Figs. 3–4. HSPs in turn activate transcription of antioxidant defense genes such as SOD2 (Mn superoxide dismutase). This in turn activates expression of inflammatory factors including TNFα(tumor necrosis factor α), IL1β (interleukin 1 β), and NFκβ (nuclear factor kappa beta) 89. Changes to these transcripts up- and down stream of HSPs were not found but the upstream activator of HSPs, junD, was decreased with FISH (Fig. 3).

LC-PUFA, particularly from fish oil, affect neutrophil and macrophage functioning. Down regulation of mif (macrophage migration inhibitory factor) by FISH could increase hypersensitivity 90, opposite that expected for fish oil which typically down regulates immune responses. C5R1 (complement factor 5a receptor) promotes local inflammation and is a potent chemo-attractant for neutrophils and macrophages 91. Its down regulation by FISH would decrease some inflammatory responses. CD72 prevents differentiation of naive B cells into plasma cells, blocking production of low-affinity antibodies. It also activates map kinase and BCR (B cell antigen receptor)-mediated calcium influx. Down regulation of cd72 with FISH may promote apoptosis/inhibit proliferation of B cells 92.

Summary and conclusion (Table 4)

 <p>Table 4</p>

Summary of genes differentiating FUNG, FISH, and COMB. Summary of genes differentiating FUNG, FISH, and COMB. Included are a subset of the most important genes selected from Table 3. The predicted global metabolic effect (up or down regulation) mediated by the differentiating group relative to other groups, is shown in the first column. The 2nd column shows the differentiating group. The differentially regulated genes, and their direction of change, are shown in column 3, with signaling cascades in column 4. For example, relative to other groups, FUNG up regulated apoptosis via down regulation of the apoptosis inhibitor diap1, via JUN-RHOA signaling. TF, transcription factor.

Metabolic effect of gene change

Differentiating group

Gene changes

Signaling pathway

Amino acid: tyrosine & ornithine metabolism

FUNG

dct↓, sf3a2↑, oat↑

CTNB1

Protein synthesis

FUNG

eef1a1↓

Apoptosis↑

FUNG

Diaph1↓ (apoptosis inhibitor)

JUN-RHOA

Apoptosis↓

FISH

dvl2↓

CTNB1/MAPK9 and -10 activation

Apoptosis↑

FISH

Camk2b↓

INS1/hRAS/SAG/IκBα, P27KIP1

Apoptosis↑

FISH

Nfkbia↓

NFκB signaling

Apoptosis↑/↓ G1/S progression

COMB

atf4↓, atf5↓

bZIP TF

Biliary bicarbonate secretion↓

FISH<FUNG

slc4a2↓

Bile acid uptake by hepatocytes↓

FISH

slc10a1↓

Biliary cholesterol secretion↓

COMB

aqp8↓

SREBP1a

Carbohydrate metabolism

COMB

foxa3↓, hnf3g↓, g6pc↓

foxa3: INS1-FOXA2

Carbohydrate metabolism

COMB; FUNG<FISH

bat2↓

CTNB1

Cell proliferation↑

FUNG<FISH

dusp9↓

JUN-CDKN2A. DUSP phosphatases inhibit MK

Cell proliferation↑

FUNG

ppp1ca↓

INS1-hRAS

Cell proliferation↑

FUNG>FISH

Ptpra↑

activates Src tyr kinases

Cell proliferation↑

FUNG<FISH

ptpn1↓, ppp2cb↓

Cell proliferation↓

FUNG<FISH; COMB

ighmbp2↓

TGFβ 1-HGF

Cell proliferation↓

FUNG

cyp24↓ (↑1,25(OH)2D3)

VDRE-PXR signaling

Cell proliferation↓

FUNG

ccnb1↓ (codes cyclin B1)

CDK1 activation

Cell proliferation↓

FUNG

tcfeb↓, barx1↓, notch2↓

Cell proliferation↓

FUNG<FISH; COMB

tcfe3↓

bHLH-zip TF

Cell proliferation↑

FISH

bop1↓

MYC

Cell proliferation↑/Lipid transport↑

FISH

cd36↑

PPARα signaling

Cell proliferation↑/Lipid transport↑

FISH

abce1↑

FABP-SNCA

Cell proliferation↓

FISH

tcea2↓

MYC-POL2RA

Cell proliferation↓

FISH

usf2↓

bHLH TF

Cell proliferation↓

FISH

jund↓, yy1↓

bHLHzip TF: IL6-TNF signaling

Cell proliferation↓

FISH

hoxa13↓

TGFβ 1-BMP2

Cell proliferation↓

FISH

fgfr3↓

INS1-STAT3

Cell proliferation↓

FISH

cdk4↓

D-cyclin-INK4a

Cell proliferation↓

FISH

tef↓

Cell proliferation

FISH

rab5c↓ (phosphatase; GTPase)

Cell proliferation

FISH

eef2↓ (activity depends on P state)

Cell proliferation↓

COMB

plk1↓ (Zn finger)

CTNB1

Cell proliferation↓

COMB

Rgs16↑

JUN-TNF-G protein coupled receptor

Cell proliferation

COMB

atf5↓

bzip TF

Cell proliferation

COMB

ntrk1↓ (receptor tyr kinase)

INS1/hRAS

Cell proliferation

COMB

eprs↓ (activity depends on P state)

TGFβ 1-IKBKb

Clotting↓

FISH

serpinc1↑

Collagen synthesis↓

FUNG

plod3↓

Cytoskeletal effects

COMB

acta2↓, arhgef7↓, sn↓, tubb2↓, tubb3↓

CTNB1

FA β-Oxidation↑

ALL

acadm↑, crat↑, cpt1a↑, cpt2↑, ech1↑

PPARα signaling

AcetylCoA biosynthesis↓

COMB

acas2↓

AcylCoa binding↓

COMB

dbi↓

MYC

FA synthesis/desaturation↓

COMB

acly↓ thrsp↓, fasn (FUNG<FISH)↓, scd↓

INS1, SREBP, PPARα, TNF

FA elongation↑

FISH

elovl2/3↑

Cyt P450 metabolism↑

FISH

4a10↑, 4a14↑ (affect ion channel activity, vascular tone)

PPARα signaling

FA transported into liver↓

FUNG

slc27a1↓

JUN-TNF-PPARα

Lipid transport↓

FISH

fabp5↓

TGFβ 1-PPARα

Lipoprotein metabolism

FUNG

apoc1↓

INS1

Lipoprotein metabolism: reverse cholesterol transport↑

FISH

apoe↑ (↓ cholesterol and TAG)

Lipoprotein metabolism: transport↓

COMB

apoa4↓

PPARα signaling

Galactose binding↓

FUNG

lgal3↓

CTNB1

Heme synthesis

FUNG

alas1↑, alas2↓

INS1-STAT3

Hemoglobin synthesis↑

FISH>FUNG

hba-a1↑, hbb-b1↑

Immune system

COMB

tcf2↓, tcf7↑

MYC-PCAF-CREBP; CTNB1

Oxidative stress

COMB

hpx↓

TGFβ 1-LEP

Oxidative stress: aldehyde dehydrogenases↑

FISH

aldh1a7↑, aldh2↑ aldh1a1↑

SREBP1a

Oxidative stress: peroxidases↑

FISH

gpx1↑, prdx2↑, prdx4↑, mpo↑

Oxidative stress: heat shock proteins

FISH vs FUNG

hspb1 (HSP25/27)↓, hspcb↑

ERK, PKCδ, TGFβ, p38, MK2/TNFα, IL1β, NFκB

Cytoskeletal-, amino acid-, heme- and carbohydrate metabolism

COMB decreased numerous transcripts implicated in the cytoskeleton, including acta2, arhgef7, sn, tubb2, and tubb3, via CTNB1 signaling. FUNG had the most profound affects on amino acid metabolism affecting tyrosine (dct, sf3a2) and ornithine (oat) metabolism, via CTNB1 signaling. Heme synthesis was likely altered via FUNG (alas1 increased, alas2 decreased; via INS1-STAT3 signaling). Hemoglobin synthesis may have been increased with FISH relative to FUNG (hba-a1, hbb-b1). Carbohydrate metabolism was most profoundly affected by COMB via decreases to foxa3 (INS1 signaling), bat2 (CTNB1 signaling), hnf3g, and g6pc.

Lipid metabolism and transport

All three diets increased numerous transcripts involved in FA β-oxidation (acadm, aldh1a1, aldh1a7, aldh2, crat, cpt1a, cpt2, and ech1) and activated by PPARα. FA synthesis (acly thrsp, fasn) and desaturation (scd) were down regulated by COMB relative to other groups, implying that a FA mixture of AA, EPA, and DHA is most effective at down regulating synthesis through INS1, SREBP, PPARα, and TNF signaling. FISH up regulated elongation of FA via elovl2/3. Lipid transport was also altered by the dietary groups. FUNG decreased slc27a1 which could decrease transport of FA into the liver; whereas FISH increased cd36 (via PPARα signaling), which could increase transport of FA into the liver. FISH decreased fabp5 which could affect intracellular fat transport (via TGFβ-PPARα signaling); and increased abce1 (via FABP-SNCA signaling). Understanding changes to apolipoprotein transcripts is complicated since mice and humans metabolize cholesterol and triacylglycerol differently (e.g., mice carry most of their cholesterol in HDL particles) and apolipoprotein polymorphisms have large impacts on cholesterol metabolism. FUNG decreased apoc1 (via INS1 signaling) which could lead to reduced circulating triacylglycerol. FISH increased apoe which could enhance delivery of cholesterol and triacylglycerol to liver, decreasing serum cholesterol and triacylglycerol. COMB decreased apoa4 (via PPARα signaling), which could affect transport of cholesterol to the liver. Bile acid metabolism was affected by all three diets. Relative to FUNG, FISH may have decreased biliary bicarbonate secretion (slc4a2) and bile acid uptake by hepatocytes (slc10a1); whereas, COMB may have decreased biliary cholesterol secretion (aqp8). Relative to other groups, FISH activated cyps 4a10 and -14 (via PPARα signaling), which hydroxylate AA and possibly DHA, forming products which affect vascular tone and ion channel activity.

Cell proliferation and apoptosis

Interpreting changes to transcripts implicated in cell proliferation is particularly complicated since transcribed proteins are ultimately regulated by pos-translational modifications and protein-protein interactions. FISH may have decreased cell proliferation via decreased tcea2 (MYC-POL2RA signaling), usf2 (bHLH transcription factor), junD, yy1 (bHLHzip transcription factor and IL6-TNF signaling), hoxa13 (TGFB1-BMP2 signaling), fgfr3 (INS1-STAT3 signaling), cdk4 (D-cyclin-INK4a signaling) and tef. FISH also decreased two phosphatase transcripts (rab5c, eef2) linked to cell proliferation; and altered three transcripts that might increase cell proliferation (decreased bop1, increased cd36 and abce1). FUNG may have increased cell proliferation via changes to phosphates including dusp9 (JUN-CDKN2A signaling), ppp1ca (INS1-hRAS), ptpn, and ppp2cb; and the kinase and ptpra (Src tyrosine kinase signaling). Contrastingly, FUNG could have also decreased cell proliferation via decreased ighmbp2 (TGFB1-HGF signaling), cyp24 (VDRE signaling), ccnb1 (less CDK1 activation), and tcfe3 (bHLH-zip TF). COMB also affected transcripts implicated in cell proliferation (see Table 4 for details). Relative to other groups, FISH likely increased apoptosis via camk2b (INS1/hRAS signaling), and nfkbia (NFκB signaling), but there was also evidence of changes to transcripts that would decrease apoptosis (e.g., via decreased dvl2). FUNG and COMB may also have increased apoptosis, but through different signaling cascades than noted for FISH.

Oxidative stress and inflammation

With respect to combating oxidative stress, relative to other groups, FISH increased various peroxidases (gpx1, prdx2, prdx4, mpo); increased an aldehyde dehydrogenase (aldh1a7, possibly via SREBP1a signaling); and affected heat shock proteins (hspb1 coding HSP25/27 was decreased, hspcb was increased). These observations are consistent with the major LC-PUFA in FISH (20:5n3, 22:5n3, 22:6n3) being more oxidizable than the major FA in FUNG (20:4n6; and small amounts of 22:4n6 and 22:5n6 formed endogenously following consumption).

Conclusion

Overall, diets rich in 20:4n6, 20:5n3/22:6n-3, and the combination of the two, had unique affects on the murine hepatic transcriptome, signaling cascades, and the predicted metabolome. The balance of dietary n6 and n3 LC-PUFA used in nutritional/neutraceutical applications could have profoundly different affects on metabolism and cell signaling, beyond that previously recognized. Future studies are needed to evaluate the effects of various ratios of n6 and n3 LC-PUFA on the transcriptome in diverse species, and in numerous tissues.

Methods

Experimental diets, feeding and dissection conditions for obtaining mouse liver, nucleic acid preparation, and gene expression analysis using the Murine 11k GeneChip have been previously published 11011. The above information is summarized; new procedures are described in detail.

Diets

Diets contained 90% fat-free AIN93G rodent diet, 0.4% milk fat, 1.2% palm olein, 1.9% sunflower oil, 1.5% soybean oil and 2.1-5.1% medium chain triacylglycerol oil 1. Medium chain triacylglycerol oil in CONT was partly replaced with: 1.1% fungal oil (providing 0.5 dietary wt% AA and 1.0 en% AA) in FUNG; 1.9% fish oil (providing 0.5 dietary wt% DHA and 1.0 en% DHA) in FISH; and 1.1% fungal oil and 1.9% fish oil in COMB. AA and DHA levels were provided at levels known to affect neurotransmitter levels and behavior in rats 93; and were not excessively high (2–3 fold higher than that recommended for infants, with slower Δ 6 desaturase activity than rodents).

Statistical and gene selection procedures

Principle component analysis and hierarchical clustering

PCA was performed with GeneSight™ software (BioDiscovery, Inc.; Fig. 1; P < 0.001). Probe set measurements from Mu11k A and B arrays were combined, creating 13 K probe sets. To reduce data dimensionality, 371 probe sets differentiating the four groups from one another was pre-selected from the 13 K following: log transformation; centering values on each array by subtraction of the array mean value; and selecting probe sets whose expression was most significantly affected by diet, using an F-statistic. To address the paucity of replicate arrays (only two replicates for CONT), residuals derived from multiple genes were binned with a global error assessment (GEA) model 11. Within-group variance was calculated per gene by combining 500 residual measurements from genes of similar intensity. Set intersection was used to find the smallest set of probes differentiating the four groups from one another (Table 1; P < 0.001). Tables 2, 3 described below include GEA-selected genes (P ≤ 0.001, 0.001 < P ≤ 0.005, 0.005 < P ≤ 0.010 as indicate by asterisks in the Table 3 legend). Hierarchical clustering included the 371 probes and used Euclidean distance metric and average linkage (Fig. 2).

Fold change calculations

Labeled RNA from 5 pooled mice per group was hybridized to Mu11K Affymetrix Chip arrays "A" and "B", each with 6.5 K probe sets. Expression values for each probe set were calculated with Affymetrix software. Data were log transformed discarding non-positive values, and log mean values subtracted to compare arrays. For CONT, two Mu11k chips were used to calculate a mean; a single Mu11k chip was used for other groups. Differential regulation for each dietary pair comparison was computed by subtracting normalized, log transformed values. Differential expression (DE) values were converted to fold change (FC) values: if DE > 0, FC = antilog(DE); if DE < 0, FC = -antilog(-DE).

Enrichment analysis

Enrichment analysis was performed on differentiating gene lists, ranking gene ontology (GO) terms by frequency of occurrence (Table 2) 94, using BioDiscovery, Inc. software. Gene lists and GO term assignments from Affymetrix IDs were mapped to gene symbols to minimize false positives resulting when a large number of probe sets correspond to a single gene. Differentiating genes were selected at P < 0.001, and the most significant GO terms were computed at P < 0.01. P is the false positive rate, the probability of a random gene for each GO term having as many genes with the same GO term as the actual list 94.

Consistency analysis

Chip sets A and B above had multiple oligonucleotide probe sets for some genes with Affymetrix's Unigene cluster mapping. Two differentially regulated genes (hba-a1, acly) had multiple probe sets but the direction of differential regulation was consistent in pair wise comparisons (Table 3).

Pathway analysis (Figs. 3, 4, 5, 6, 7)

Pathway analysis was performed (Ingenuity Systems software, Redwood City, CA) to derive biological and signaling connections amongst the differentially regulated genes in Table 3. Of the 127 genes in Table 3, the program selected 66 focus genes having direct- and 132 having indirect connections, totaling 198 genes for pathway analysis. Direct connections require any two nodes (genes or gene products) to make direct physical contact. Indirect connections permit intermediate factors between nodes. The 198 genes were divided into 13 networks. Networks 1–5 had 15-, 12-, 13-, 13- and 5 focus genes, respectively. The remaining 8 networks had 1 gene each and were not considered. Network diagrams including these 58 focus genes and some connection genes were redrawn for simplicity (Figs. 3, 4, 5, 6, 7). The 5 networks were independent from one another, except for an indirect connection via JUN linking Figs. 3, 4. Abbreviations were from GeneCards 95.

Functional, biological, and pathway information sources

Functional and biological information on genes was obtained from: Afffymetrix Netaffx Analysis Center 96; EMBL Bioinformatic Harvester 97; GeneCards 9598; MedMiner 99100; and NCBI Entrez Gene 101. Additional pathway information was obtained from: Biocarta 102; Cell Signaling Technology 103; Genmapp 104; Kegg 105; MetaCyc (106; Reactome 107; and RefViz 108.

Abbreviations

AA, arachidonic acid; AKT, akt murine thymoma viral oncogene; AP-1, activator protein 1; APP, amyloid beta (A4) precursor protein; bHLH, basic helix-loop helix; BMP2, bone morphogenetic protein 2; bZIP, basic region-leucine zipper; CDK, cyclin dependent kinase; COMB, combination of FUNG+FISH; CONT, control; CPT-1, carnitine paltmitoyl transferase; CREBP, cAMP response element binding protein; CTNB1, catenin (cadherin-associated protein), beta 1; cyt, cytochrome; DE, differential expression; DHA, docosahexaenoic acid; EAF, ELL associated factor; EGF, epidermal growth factor; ERK, extracellular signal-regulated protein kinases; EPA, eicosapentaenoic acid; FA, fatty acid; FC, fold change; FGF, fibroblast growth factor; FISH, fish oil; FOXA2, forkhead box A2; FUNG, fungal; GEA, global error assessment; GO, gene ontology; HGF, hepatocyte growth factor; hRAS, Harvey rat sarcoma; IκBα inhibitor of kappa light polypeptide gene enhancer in B-cells; IKBKb, inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta; IL, interleukin; INS1, insulin 1; JNK, c-Jun N-terminal kinase; JUN, jun proto-oncogene; LC-PUFA, long chain polyunsaturated fatty acids; LEP, leptin obesity homolog, mouse; LTC4, leukotriene C4; LXR, liver X receptor; LXRE, LXR responsive elements; MAPK, mitogen activated protein kinase; MK2, MAPKAPK2, MAPKAP kinase 2; MKP, mitogen-activated protein kinase phosphatase; MYC, myc myelocytomatosis viral oncogene; neurotrophin/TRK, neurotrophic tyrosine kinase, receptor; NF-κβ, nuclear factor κβ; P27, KIP1, cyclin-dependent kinase inhibitor 1B; p38, a MAPK; PCA, principal component analysis; PCAF, p300/CBP-associated factor; PDGF, platelet derived growth factor; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PL, phospholipids; POL2RA, RNA polymerase II subunit; PPAR, peroxisome proliferator activated receptor; PXR, pregnane X receptor; RT-PCR, real time-polymerase chain reaction; RXR, retinoid X receptor; SAG (CKBBP1//ROC2/RBX2), sensitive to apoptosis gene; SAPK, stress-activated protein kinase (same as JNK); SF3A1, splicing factor 3a, subunit 1; SNCA, synuclein, alpha (non A4 component of amyloid precursor); SOD2, super oxide dismutase 2; SRE(BP), sterol regulatory element (binding protein); STAT3, signal transducer and activator of transcription 3; TF, transcription factor; TGFβ, transforming growth factor, beta; TGIF, transforming growth factor, beta, induced factor; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor; WNT, wingless-type MMTV integration site family. Gene symbol abbreviations for differentially regulated genes are found in Table 3 and the text.

Competing interests

The author(s) declare that they have no competing interests.

Authors' contributions

AB designed the animal experiments, provided overall coordination and wrote the manuscript; MR organized and conducted all laboratory and microarray analysis; MR with an extended team, developed the GEA gene selection model used in the present work; and BH provided all statistical and computational tools and performed the gene- and pathway- statistical analyses.

Acknowledgements

The authors thank the Nestlé Research Center in Lausanne, Switzerland, for their support of the original microarray work.

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