Background
Methods
Animals and dietary feeding paradigm
Euthanasia procedures
Liver trichrome staining and TAG content
Liver RNA-seq methods
Liver NF-κB pathway phosphoprotein determination
Serum cytokine profile determination
Statistics and bioinformatics for liver RNA-seq data
Statistics for non-RNA-seq data
Results
Phenotypic changes following 6 weeks of WD versus CTL feeding
Variable | CTL | WD |
p-value |
---|---|---|---|
Body and tissue masses
| |||
Body mass at sacrifice (g) | 329 ± 7 | 350 ± 12 | 0.002 |
Omental fat mass (g) | 0.25 ± 0.03 | 0.38 ± 0.03 | <0.001 |
Perirenal fat mass (g) | 1.70 ± 0.14 | 2.95 ± 0.40 | <0.001 |
Dietary data
| |||
Total kcal consumed | 2,488 ± 32 | 2,849 ± 38 | <0.001 |
Total protein consumed (g) | 100 ± 1 | 110 ± 1 | <0.001 |
Total carbohydrate consumed (g) | 461 ± 6 | 307 ± 4 | <0.001 |
Total fat consumed (g) | 40 ± 1 | 134 ± 2 | <0.001 |
Total sucrose consumed (g) | 8.5 ± 0.1 | 215 ± 3 | <0.001 |
Total cholesterol consumed (g) | 0.099 ± 0.001 | 1.27 ± 0.02 | <0.001 |
Liver characteristics | |||
Fat deposition (Oil-Red O area; AUs) | 167.6 ± 51.3 | 701.3 ± 139.5 | 0.004 |
Liver TAGs (nmol/g tissue) | 3.34 ± 0.25 | 7.53 ± 0.58 | <0.001 |
WD feeding disrupts the hepatic expression of genes involved with cholesterol and fatty acid synthesis
Transcript | Protein function | WD/CTL fold-change | FDR value | Nominal p-value |
---|---|---|---|---|
Top transcripts up-regulated with WD feeding (FDR < 0.05, > 2.0 fold-change)
| ||||
Stearoyl-CoA desaturase (delta-9-desaturase) (Scd) | Involved in fatty acid biosynthesis, primarily the synthesis of oleic acid; linked to inhibition of CPT-1A and suppression of mitochondrial fatty acid oxidation | 28.392 | 0.01 | 0.00004 |
PDZ and LIM domain 1 (Pdlim1) | Cytoskeletal protein that may act as an adapter that brings other proteins (like kinases) to the cytoskeleton | 2.741 | 0.006 | 0.00002 |
Cytochrome P450, family 2, subfamily C, polypeptide 18 (Cyp2c18) | This gene encodes a member of the cytochrome P450 superfamily of enzymes which are monooxygenases that catalyze reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids | 2.504 | 0.01 | 0.00003 |
Platelet factor 4 (Pf4) | Chemotactic molecule for neutrophil and monocyte attraction | 2.031 | 0.04 | 0.0004 |
Top transcripts down-regulated with WD feeding (FDR < 0.05, < −2.0 fold-change)
| ||||
Cytochrome P450, family 51, subfamily A, polypeptide 1 (Cyp51a1) | This gene encodes a member of the cytochrome P450 superfamily of enzymes which are monooxygenases that catalyze reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids | −8.426 | 0.0001 | 0.00000006 |
Transmembrane 7 superfamily member 2 (Tm7sf2) | Involved in the conversion of lanosterol to cholesterol | −8.381 | 0.007 | 0.00002 |
3-hydroxy-3-methylglutaryl-CoA synthase 1 (Hmgcs1) | This enzyme condenses acetyl-CoA with acetoacetyl-CoA to form HMG-CoA, which is the substrate for HMG-CoA reductase | −6.824 | 0.005 | 0.00001 |
Methylsterol monooxygenase 1 (Msmo1) | The protein is localized to the endoplasmic reticulum membrane and is believed to function in cholesterol biosynthesis | −6.482 | 0.002 | 0.000002 |
Zinc finger protein 48 (Znf48) | May be involved in transcriptional regulation | −6.458 | 0.00006 | 0.00000001 |
Insulin induced gene 1 (Insig1) | Mediates feedback control of cholesterol synthesis | −4.711 | 0.03 | 0.0002 |
RUN and SH3 domain containing 1 (Rusc1) | Seems to be involved in signaling pathways that are regulated by the prolonged activation of MAPK; may be involved in regulation of the NF-kappa-B pathway | −4.705 | 0.0001 | 0.00000008 |
Farnesyl diphosphate synthase (Fdps) | Involved in cholesterol synthesis | −4.275 | 0.0003 | 0.0000003 |
Fatty acid desaturase 1 (Fads1) | Component of a lipid metabolic pathway that catalyzes biosynthesis of unsaturated fatty acids polyunsaturated fatty acids | −3.735 | 0.004 | 0.000007 |
Proprotein convertase subtilisin/kexin type 9 (Pcsk9) | Acts via a non-proteolytic mechanism to enhance the degradation of the hepatic low-density lipoprotein receptor | −3.558 | 0.0001 | 0.0003 |
Cysteine sulfinic acid decarboxylase (Csad) | Plays a role in multiple biological processes as the rate-limiting enzyme in taurine biosynthesis | −3.023 | 0.049 | 0.0005 |
ATP citrate lyase (Acly) | ATP citrate-lyase is the primary enzyme responsible for the synthesis of cytosolic acetyl-CoA in many tissues; has a central role in de novo lipid synthesis | −2.879 | 0.049 | 0.0006 |
Acyl-CoA synthetase short-chain family member 2 (Acss2) | Activates acetate so that it can be used for lipid synthesis or for energy generation | −2.426 | 0.0001 | 0.00000003 |
Lanosterol synthase (2,3-oxidosqualene-lanosterol cyclase) (Lss) | Catalyzes the cyclization of (S)-2,3 oxidosqualene to lanosterol, a reaction that forms the sterol nucleus | −2.388 | 0.02 | 0.00008 |
Deoxyribonuclease II beta (Dnase2b) | Gene is ubiquitously expressed but liver function unknown | −2.344 | 0.02 | 0.0002 |
Phosphomevalonate kinase (Pmvk) | Involved in the cholesterol biosynthetic pathway | −2.278 | 0.004 | 0.000006 |
UDP glucuronosyltransferase 2 family, polypeptide A3 (Ugt2a3) | UDP-glucuronosyltransferases are involved in the elimination of potentially toxic xenobiotics and endogenous compounds | −2.129 | 0.009 | 0.00003 |
WD feeding does not affect systemic or hepatic pro-inflammatory markers, but modestly affects markers of the acute phase protein response
Transcript | CTL RPKM mean | WD RPKM mean | WD/CTL Fold-change | Nominal p-value |
---|---|---|---|---|
Select genes involved in IL-6 signaling
| ||||
IL-6
| not on RNA-seq readout | |||
IL-6R
| 29.55 | 30.89 | 1.05 | 0.80 |
gp130
| 26.04 | 23.44 | −1.11 | 0.44 |
Jak1
| 34.9 | 34.1 | −1.02 | 0.66 |
Jak2
| 1.87 | 1.81 | −1.03 | 0.78 |
Jak3
|
2.02
|
2.78
|
1.37
|
0.034
|
Stat3
| 15.43 | 16.20 | 1.05 | 0.68 |
Socs3
| 2.49 | 3.44 | 1.38 | 0.47 |
Select genes involved in TNF-α signaling
| ||||
TNF-α
| 0.02 | 0.03 | 1.56 | 0.38 |
Tnfrsf1b
| 3.91 | 2.88 | −1.36 | 0.08 |
Traf2
| 6.96 | 7.06 | 1.01 | 0.81 |
Rela (p65)
| 10.56 | 10.64 | 1.01 | 0.89 |
Nfkbia (IkBa)
| 18.57 | 18.35 | −1.01 | 0.90 |
Select genes involved in IL-1β signaling
| ||||
IL-1β
|
1.99
|
2.93
|
1.47
|
0.013
|
IL-1R1
| 6.38 | 6.79 | 1.07 | 0.75 |
Traf6
| 1.45 | 1.30 | −1.12 | 0.23 |
Mapk8 (JNK1)
| 2.24 | 1.98 | −1.13 | 0.32 |
Mapk9 (JNK2)
| 18.27 | 19.25 | 1.05 | 0.52 |
Select genes encoding acute phase response proteins
| ||||
Apcs
|
1048
|
862
|
−1.22
|
0.047
|
Apoh
|
3874
|
3401
|
−1.07
|
0.006
|
C2
| 51.0 | 47.0 | −1.09 | 0.10 |
C9
| 517 | 614 | 1.19 | 0.10 |
Crp
| not on RNA-seq readout | |||
F2
| 1051 | 1060 | 1.01 | 0.86 |
Fth1
| 1817 | 1969 | 1.08 | 0.18 |
Hmox1
| 16.3 | 17.0 | 1.05 | 0.62 |
Hp
|
1299
|
1603
|
1.24
|
0.0002
|
Hpx
| 2667 | 3086 | 1.16 | 0.11 |
IL-1rn
| 13.7 | 11.5 | −1.19 | 0.35 |
Lbp
|
538
|
350
|
−1.54
|
0.002
|
Mur2
| 432 | 392 | −1.10 | 0.31 |
Pf4
|
2.48
|
5.04
|
2.03
|
0.0004
|
Plg
| 944 | 868 | −1.08 | 0.11 |
Sod2
|
41.8
|
47.2
|
1.13
|
0.013
|
Select genes representative of macrophage markers
| ||||
Cd68
| 12.69 | 12.94 | 1.02 | 0.86 |
Emr1 (F4/80)
| 4.33 | 4.89 | 1.13 | 0.32 |
Itgax (Cd11c)
|
0.27
|
0.42
|
1.53
|
0.044
|
Itgam (Cd11b)
| 0.37 | 0.37 | −1.01 | 0.92 |