Supplementary

Modulation of the Liver Carbonylome by the Combined Effect of Marine Omega-3 PUFAs and Grape Polyphenols Supplementation in Rats Fed an Obesogenic High Fat and High Sucrose Diet

Lucía Méndez 1,*, Silvia Muñoz 1, Bernat Miralles-Pérez 2, M. Rosa Nogués 2, Sara Ramos- Romero 3,4, Josep L. Torres 3 and Isabel Medina 1

Figure S1. Determination of the linear range for the quantification of albumin in rat plasma base on Coomassie intensity. Bands were analyzed by using LabImage 1D (Kapelan Bio-Imaging Solutions, Halle, Germany).

Supplementary Table S1. Pathways and processes enriched by FOM, GSE and FOM&GSE in rats fed either HFHS or STD diet. "Count" is the number of with membership in the given ontology term. "%" is the percentage of all of the user-provided genes that are found in the given ontology term (only input genes with at least one ontology term annotation are included in the calculation). "Log10(P)" is the p-value in log base 10. "Log10(q)" is the multi-test adjusted p-value in log base 10.1. HFHS: high-fat and high-sucrose diet; STD: standard diet; GSE: grape seed polyphenols extract; FOM: fish oil mixture.

Background Other significant members in the Supplement GO Category Description Count % Log10(P) Log10(q) diet Symbols cluster (in order of significance) HFHS DIETS

protein homooligomerization; organic acid catabolic process; carboxylic acid catabolic process; Acadl, protein complex oligomerization; GO Otc, Hpd, small molecule liver development; hepaticobiliary HFHS FOM GO:0044282 Biological 5 50.00 -6.32 -2.06 Aldh2, catabolic process system development; small Processes Upb1, molecule biosynthetic process; Cat, Pgd carboxylic acid biosynthetic process; organic acid biosynthetic process; gland development GO Otc, Hpd, cellular amino acid alpha-amino acid metabolic HFHS FOM GO:0006520 Biological 4 40.00 -4.97 -1.01 Upb1, metabolic process process Processes Nit2 alcohol metabolic process, organic Cat, Hpd, GO hydroxy compound metabolic Aldh2, HFHS FOM GO:0042737 Biological drug catabolic process 3 30.00 -4.18 -0.52 process, response to acid chemical, Acadl, Processes response to toxic substance, drug Otc, Actb metabolic process response to metal ion, response to GO Alb, Cat, nutrient levels, response to HFHS FOM GO:0007584 Biological response to nutrient 3 30.00 -3.05 0.00 Otc extracellular stimulus, response to Processes inorganic substance

1Gene names in green colour represent that decreased their carbonylation level by the supplement and gene names in red colour represent proteins that increased their carbonylation level by the supplement GO monocarboxylic acid Acadl, HFHS FOM GO:0032787 Biological 3 30.00 -2.52 0.00 metabolic process Nit2, Pgd Processes R-RNO- Reactome Innate Immune Cat, Actb, HFHS FOM 3 30.00 -2.21 0.00 168249 Gene Sets System Nit2 organic acid catabolic process, carboxylic acid catabolic process, cellular amino acid biosynthetic process, cellular amino acid metabolic process, alpha-amino Acadl, acid catabolic process, cellular GO Otc, Got2, small molecule amino acid catabolic process, HFHS GSE GO:0044282 Biological 6 75.00 -9.00 -4.75 Hpd, catabolic process carboxylic acid biosynthetic Processes Aldh2, process Upb1 organic acid biosynthetic process, of amino acids and derivatives, alpha-amino acid metabolic process, small molecule biosynthetic process Cat, drug catabolic process, antibiotic GO Acadl, metabolic process, organic hydroxy alcohol metabolic HFHS GSE GO:0006066 Biological 4 50.00 -5.21 -1.80 Aldh2, compound metabolic process, process Processes Akr1c9, steroid metabolic process, drug Hpd metabolic process GO Cat, protein HFHS GSE GO:0051260 Biological 4 50.00 -4.86 -1.65 Acadl, protein complex oligomerization homooligomerization Processes Otc, Upb1 GO cellular ketone Cat, Got2, HFHS GSE GO:0042180 Biological 3 37.50 -4.17 -1.12 cofactor metabolic process metabolic process Akr1c9 Processes GO Otc, Got2, liver development, hepaticobiliary HFHS GSE GO:0048732 Biological gland development 4 50.00 -4.01 -1.03 Aldh2, system development Processes Upb1 response to acid chemical; response GO Cat, Otc, to peptide hormone; response to HFHS GSE GO:0032868 Biological response to insulin 3 37.50 -3.53 -0.67 Got2, peptide; response to toxic Processes Aldh2 substance GO Otc, Hpd, cellular amino acid alpha-amino acid metabolic HFHS FOM&GSE GO:0006520 Biological 4 36.36 -4.78 -0.65 Upb1, metabolic process process Processes Nit2 protein homooligomerization, organic acid catabolic process, Acadl, carboxylic acid catabolic process, GO small molecule Otc, Hpd, protein complex oligomerization, HFHS FOM&GSE GO:0044282 Biological 4 36.36 -4.48 -0.65 catabolic process Upb1, small molecule biosynthetic Processes Cat, Pgd process, carboxylic acid biosynthetic process, organic acid biosynthetic process response to inorganic substance, response to metal ion, response to Alb, Cat, GO nutrient levels, response to Otc, HFHS FOM&GSE GO:0007584 Biological response to nutrient 4 36.36 -4.31 -0.65 extracellular stimulus, response to Hspd1, Processes hypoxia, response to decreased Tf, Actb oxygen levels, response to oxygen levels, response to toxic substance R-RNO- Reactome Vesicle-mediated Alb, Tf, HFHS FOM&GSE 3 27.27 -2.55 0.00 5653656 Gene Sets transport Actb GO monocarboxylic acid Acadl, HFHS FOM&GSE GO:0032787 Biological 3 27.27 -2.39 0.00 metabolic process Nit2, Pgd Processes R-RNO- Reactome Innate Immune Cat, Actb, HFHS FOM&GSE 3 27.27 -2.08 0.00 168249 Gene Sets System Nit2 STANDARD DIETS

STD FOM No significant pathways/process enriched Cat, Got2, KEGG STD GSE rno01200 Carbon metabolism 4 40.00 -6.38 -2.12 Tkt, Pgd, cofactor metabolic process Pathway Akr1c9 Adh1, GO cellular aldehyde Tkt, Pgd, monocarboxylic acid metabolic STD GSE GO:0006081 Biological 3 30.00 -5.18 -1.45 metabolic process Akr1c9, process Processes Nit2 glutamine family amino acid metabolic process; biosynthesis of Otc, Got2, GO glutamine family amino acids, alpha-amino acid Nit2, Tkt, STD GSE GO:0009064 Biological amino acid metabolic 3 30.00 -5.11 -1.45 metabolic process, cellular amino Adh1, Processes process acid metabolic process, small Pgd molecule catabolic process, small molecule biosynthetic process response to activity, antibiotic Adh1, metabolic process, response to GO Cat, Got2, antibiotic, response to ethanol, response to toxic STD GSE GO:0009636 Biological 5 50.00 -4.66 -1.17 Hspd1, alcohol metabolic process, response substance Processes Actb, to alcohol, organic hydroxy Akr1c14 compound metabolic process, drug metabolic process response to insulin, response to nutrient, response to acid chemical, Cat, Otc, GO response to peptide hormone, Got2, STD GSE GO:0032868 Biological response to insulin 3 30.00 -3.21 -0.13 response to peptide, response to Hspd1, Processes nutrient levels, response to Adh1 extracellular stimulus, response to inorganic substance R-RNO- Reactome Innate Immune Cat, Actb, STD GSE 3 30.00 -2.21 0.00 168249 Gene Sets System Nit2 glutamine family amino acid GO P4hb, alpha-amino acid metabolic process; cellular amino STD FOM&GSE GO:1901605 Biological 5 62.50 -7.98 -3.79 Otc, Ass1, metabolic process acid metabolic process; arginine Processes Got2, metabolic process; alpha-amino Fah, acid catabolic process; small Aldh2 molecule catabolic process; cellular amino acid catabolic process; organic acid catabolic process; carboxylic acid catabolic process alpha-amino acid biosynthetic process; cellular amino acid biosynthetic process; Biosynthesis of amino acids; Metabolism of amino acids and derivatives; response to acid chemical; response to peptide hormone; gland development; response to peptide; Otc, Ass1, KEGG liver development; hepatobiliary STD FOM&GSE rno00220 Arginine biosynthesis 3 37.50 -7.19 -3.63 Got2, Cat, Pathway system development; response to Aldh2 insulin; carboxylic acid biosynthetic process; organic acid biosynthe