Urinary loss of tricarboxylic acid cycle intermediates as revealed by metabolomics studies:an underlying mechanism to reduce lipid accretion by whey protein ingestion?

[Image: see text] Whey protein intake is associated with the modulation of energy metabolism and altered body composition both in human subjects and in animals, but the underlying mechanisms are not yet elucidated. We fed obesity-prone C57BL/6J mice high-fat diets with either casein (HF casein) or whey (HF whey) for 6 weeks. At equal energy intake and apparent fat and nitrogen digestibility, mice fed HF whey stored less energy as lipids, evident both as lower white adipose tissue mass and as reduced liver lipids, compared with HF-casein-fed mice. Explorative analyses of 48 h urine, both by (1)H NMR and LC–MS metabolomic platforms, demonstrated higher urinary excretion of tricarboxylic acid (TCA) cycle intermediates citric acid and succinic acid (identified by both platforms), and cis-aconitic acid and isocitric acid (identified by LC–MS platform) in the HF whey, relative to in the HF-casein-fed mice. Targeted LC–MS analyses revealed higher citric acid and cis-aconitic acid concentrations in fed state plasma, but not in liver of HF-whey-fed mice. We propose that enhanced urinary loss of TCA cycle metabolites drain available substrates for anabolic processes, such as lipogenesis, thereby leading to reduced lipid accretion in HF-whey-fed compared to HF-casein-fed mice

Intake of Hydrolyzed Casein is Associated with Reduced Body Fat Accretion and Enhanced Phase II Metabolism in Obesity Prone C57BL/6J Mice

<div><p>The amount and form of dietary casein have been shown to affect energy metabolism and lipid accumulation in mice, but the underlying mechanisms are not fully understood. We investigated 48 hrs urinary metabolome, hepatic lipid composition and gene expression in male C57BL/6J mice fed Western diets with 16 or 32 energy% protein in the form of extensively hydrolyzed or intact casein. LC-MS based metabolomics revealed a very strong impact of casein form on the urinary metabolome. Evaluation of the discriminatory metabolites using tandem mass spectrometry indicated that intake of extensively hydrolyzed casein modulated Phase II metabolism associated with an elevated urinary excretion of glucuronic acid- and sulphate conjugated molecules, whereas glycine conjugated molecules were more abundant in urine from mice fed the intact casein diets. Despite the differences in the urinary metabolome, we observed no differences in hepatic expression of genes involved in Phase II metabolism, but it was observed that expression of <i>Abcc3</i> encoding ATP binding cassette c3 (transporter of glucuronic acid conjugates) was increased in livers of mice fed hydrolyzed casein. As glucuronic acid is derived from glucose and sulphate is derived from cysteine, our metabolomic data provided evidence for changes in carbohydrate and amino acid metabolism and we propose that this modulation of metabolism was associated with the reduced glucose and lipid levels observed in mice fed the extensively hydrolyzed casein diets.</p></div

Liver gene expression related to Phase II metabolism.

<p>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: <i>Abcc3</i>, ATP-binding casette, sub-family c, member 3; <i>Glyat</i>, Glycine-N-acyltransferase; <i>Sult1a1</i>, Sulfotransferase Family, Cytosolic, 1A, Phenol-Preferring, Member 1; <i>Ugt1a5</i>, UDP glucuronosyltransferase 1 family, polypeptide A5; <i>Ugt1a6b</i>, UDP glucuronosyltransferase 1 family, polypeptide A6B; <i>Ugt2b34</i>, UDP glucuronosyltransferase 2 family, polypeptide B34. * Significant effect of hydrolysis, p < 0.05.</p

PCA score plot of urinary metabolome data.

<p>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.</p

Liver lipogenic gene expression and lipid composition.

<p>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: <i>Srebf1</i>, Sterol regulatory element binding transcription factor 1; <i>Acaca</i>, Acetyl-Coenzyme A carboxylase alpha; <i>Fasn</i>, Fatty acid synthase; <i>Scd1</i>, Stearoyl-Coenzyme A desaturase 1.</p

Response patterns of selected LC-MS features in urine.

<p>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.</p

Summary of Metabolic alterations induced by ingestion of extensively hydrolyzed casein in mice.

<p>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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118895#pone.0118895.ref024" target="_blank">24</a>] as well as reduced liver glucose and lactate concentrations [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118895#pone.0118895.ref030" target="_blank">30</a>]. Concomitantly, mice fed hydrolyzed casein diets exhibit a higher liver glycogen concentration [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118895#pone.0118895.ref030" target="_blank">30</a>], 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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118895#pone.0118895.ref030" target="_blank">30</a>], 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.</p

Summary of the top 50 VIP features in urine identified from an OPLSDA model with intact versus hydrolyzed casein as discriminating variables.

<p>^a Hydrolyzed, most abundant in urine from mice fed hydrolyzed casein; Intact, most abundant in urine from mice fed intact casein; Diet, feature detected in the diet</p><p>Summary of the top 50 VIP features in urine identified from an OPLSDA model with intact versus hydrolyzed casein as discriminating variables.</p