Fig 1.
PCA score plot of urinary metabolome data.
Urine from mice fed diets containing intact (I) or hydrolyzed (H) casein at 16% energy or 32% energy was analyzed using LC-MS in negative mode.
Table 1.
Summary of the top 50 VIP features in urine identified from an OPLSDA model with intact versus hydrolyzed casein as discriminating variables.
Fig 2.
Response patterns of selected LC-MS features in urine.
Urine from mice fed diets containing intact (I) or hydrolyzed (H) casein, at 16% energy or 32% energy from protein: glucuronic acid and sulphate conjugate, m/z 435.141 (A); glucuronic acid conjugate, m/z 399.141 (B); sulphate conjugate, m/z 339.066 (C); glycine conjugate, m/z 214.108 (D); glycine and sulphate conjugate, m/z 323.074 (E); salicyluric acid (a glycine conjugate, F), m/z 194.045.
Fig 3.
Liver gene expression related to Phase II metabolism.
Gene expression in livers from mice fed diets containing intact (I) or hydrolyzed (H) casein at 16% energy or 32% energy from protein was analyzed (n = 6–8).). Gene abbreviations: Abcc3, ATP-binding casette, sub-family c, member 3; Glyat, Glycine-N-acyltransferase; Sult1a1, Sulfotransferase Family, Cytosolic, 1A, Phenol-Preferring, Member 1; Ugt1a5, UDP glucuronosyltransferase 1 family, polypeptide A5; Ugt1a6b, UDP glucuronosyltransferase 1 family, polypeptide A6B; Ugt2b34, UDP glucuronosyltransferase 2 family, polypeptide B34. * Significant effect of hydrolysis, p < 0.05.
Fig 4.
Liver lipogenic gene expression and lipid composition.
PCA score plot (A) and loading plot (B) of liver genes related to lipogenesis and lipid composition data from mice fed diets containing intact (I) or hydrolyzed (H) casein, at 16% energy or 32% energy from protein (n = 5–6). Furthermore, selected data of liver gene expression (C-F) and lipid composition (J-M) are shown. These data are presented as means ± SE and p values of the effects of protein level (L), protein form (F) and the interaction (L × F) are shown in the inserts. Abbreviations: PS, phosphatidylserine; PI, phosphatidylinositol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; CL, cardiolipin; SM, sphingomyelin; LPC, lyso-phosphatidylcholine; FFA, free fatty acids; Chol, cholesterol (free); SE, sterylester (sterol + fatty acid); TAG, triacylglycerol. Gene abbrevations: Srebf1, Sterol regulatory element binding transcription factor 1; Acaca, Acetyl-Coenzyme A carboxylase alpha; Fasn, Fatty acid synthase; Scd1, Stearoyl-Coenzyme A desaturase 1.
Fig 5.
Summary of Metabolic alterations induced by ingestion of extensively hydrolyzed casein in mice.
Metabolites given in red decreased after intake of hydrolyzed casein, whereas metabolites in blue increased. Mice fed diets with hydrolyzed casein have reduced plasma glucose levels [24] as well as reduced liver glucose and lactate concentrations [30]. Concomitantly, mice fed hydrolyzed casein diets exhibit a higher liver glycogen concentration [30], indicating a shift in glucose metabolism from glycolysis toward UDP-glucose synthesis, which is the intermediate substrate used both for glycogen- and D-glucuronic acid synthesis. The increased urinary abundance of D-glucuronic acid conjugated substances identified in the present study further supports such a switch in glucose metabolism by hydrolyzed casein feeding. The liver glutathione and taurine levels were shown to be increased in mice fed hydrolyzed casein [30], indicating higher hepatic cysteine availability in these mice. As cysteine is the precursor for sulphate synthesis, higher hepatic cysteine availability could explain the higher urinary content of sulphate-conjugated molecules in the hydrolyzed casein fed mice observed in the present study. In summary, these metabolic alterations induced by intake of hydrolyzed casein facilitate both the reduced plasma glucose levels and increased urinary levels of Phase II conjugated molecules.
Fig 6.
Liver TBARS and gene expression related to oxidative stress and glucose metabolism.
TBARS (A), expression of Nrf-2 (B) and p53 target genes (C) in livers from mice fed diets containing intact (I) or hydrolyzed (H) casein, at 16% energy or 32% energy from protein (n = 6–8). * Significant effect of hydrolysis, p < 0.05.