Soy Protein Isolate Feeding Research Articles

Hepatic Proteomics Analysis of Nonalcoholic Fatty Liver Disease Obese Rat Model After Short- and Long-Term Soy Protein Isolate Feeding.

To identify possible mechanisms involved in the development and progression of nonalcoholic fatty liver disease (NAFLD), we conducted shotgun proteomics analysis on liver of obese Zucker rats fed either casein (CAS) or soy protein isolate (SPI) for 8 and 16 weeks. Rats (7 weeks old, n = 8-9/group) were randomly assigned to either a CAS-based or an SPI-based diet. Rats were killed after 8 or 16 weeks of feeding and livers were stored at -80°C. Ingenuity Pathway Analysis (IPA) software was used to facilitate interpretation of proteomics data. Predictions of activation or inhibition of molecules in the data were made based on activation z-score and P value of overlap (P < .05). Activation z-scores ≥2.0 indicate that a molecule is predicted to be activated, whereas activation z-scores of less than or equal to -2.0 indicate that a target molecule is predicted to be inhibited. Upstream regulator analysis with IPA revealed Neuregulin 1 (NRG1) to be the top activated protein in (z-score = 2.48, P < .05), and MKNK1 as the top inhibited protein (z-score = -2.83, P < .05) in SPI diet compared with CAS diet after both 8 and 16 weeks of SPI feeding. Regulator effects analysis also predicted that some proteins would be participating, directly or indirectly, in the inhibition of immune response functions (such as leukocyte migration) and lipid metabolism (such as synthesis of lipids) in SPI-fed rats relative to CAS-fed rats. Our results suggest that SPI diet modifies the expression of proteins that could be involved in the reduction of NAFLD.

Effects of Short and Long-Term Soy Protein Isolate Intake on Hepatic Cytochrome P450 Expression in Obese Zucker Rats

ObjectivesTo determine the effects feeding for 8 (short-term) and 16 weeks (long-term) soy protein isolate on hepatic CYP gene expression. Methods7-weeks old rats were randomly assigned to either a casein (CAS) or a soy protein isolate (SPI) diet. They were provided the diets ad libitum for 8 and 16 weeks. Rats were euthanized and livers were stored at – 80°C. RNA was extracted from liver samples, and sequenced to obtain transcriptomic data (RNAseq). Ingenuity Pathway Analysis software (IPA, Qiagen, CA) was used in the analysis of global gene expression data. This analysis includes predictions of activation or inhibition of molecules or upstream regulators and functions based on a generated z-score and p-value of overlap (P = 0.05). Z-scores were consider significant when > 2 (activation) and < –2 (inhibition). ResultsComparing short- vs long-term feeding revealed an increase in the number of down-regulated CYP genes from only 3 at 8 weeks of SPI diet to 10 at 16 weeks of same diet (P < 0.05). In contrast, upregulated CYP gene numbers showed a small increase in long-term SPI diet compared to short-term, from 14 genes at 8 weeks to 17 genes at 16 weeks (P < 0.05). In addition, we present a predicted activation of the transcription factor Aryl hydrocarbon receptor (AHR, activation z-score = 2.146, P = 4.20E-11), linked to the subsequent activation or up-regulation of various CYPs genes, indirectly leading to the activation and inhibition of two main metabolic functions under SPI feeding: conversion of lipid (lipid metabolism) –predicted to be activated (z-score = 2.089, P = 2.77E-08), and recruitment of phagocytes (inflammatory response) –predicted to be inhibited (z-score = –2.311, P = 2.10E-05). ConclusionsThrough global gene expression analysis we showed that gene expression of drug-metabolizing cytochrome P450 genes was modified in genetically obese Zucker rats after being fed a soy-based diet for short- and long-term, and that this change could have an important role in attenuation of liver steatosis. Further research is needed to corroborate these results. Funding SourcesThis study was supported in part by the College of Medicine’s University Medical Group (RH) and the Arkansas Biosciences Institute (WB, RH).

Liver Proteomics Analysis After Short- and Long-Term Soy Protein Isolate Feeding Using Obese Zucker Rat Model

ObjectivesTo identify possible mechanisms involved in the development and progression of NAFLD through protein expression (shotgun proteomics) analysis on liver samples of obese Zucker rats fed with either casein (CAS) or soy protein isolate (SPI) during 8 and 16 weeks. Methods7 weeks old rats (n = 8–9 per group) were randomly assigned to an either CAS-based or a SPI-based diet. Rats were sacrificed after 8 weeks or 16 weeks of SPI feeding. Livers were immediately obtained and stored at –80 C. Ingenuity Pathway Analysis (IPA) software was used to facilitate interpretation of proteomics data. Predictions of activation or inhibition of molecules in the data was made based on activation z-score and P value of overlap (P < 0.05). Activation z-scores > 2.0 indicate that a molecule is activated, whereas activation z-scores of < –2.0 indicate that a target molecule is inhibited. ResultsUpstream regulator analysis by IPA revealed 6 molecules predicted to be activated (z-scores between 2 and 2.8) and 9 inhibited (z-scores between –2 and –2.6) in SPI vs CAS-fed rats in proteomics data at 8 weeks of SPI feeding. In contrast, at 16 weeks of SPI feeding there were 12 molecules activated (z-scores between 2 and 2.5) and 18 inhibited (z-scores between –2 and –2.8) in SPI vs CAS-fed. All p values were <0.05. Regulator effects analysis also revealed that some of these molecules would be participating, directly or indirectly, in the inhibition of the immune response of cells (such as IL27 and CSF2) and synthesis of lipids (CEBPA, Ins1 and IRF8) in SPI-fed rats. ConclusionsThese molecules and their downstream target proteins may provide clues by which soy protein produces the observed attenuation of liver steatosis that can be tested in future experiments in this obese rat model. Funding SourcesThis study was supported in part by the College of Medicine’s University Medical Group (RH) and the Arkansas Biosciences Institute (WB, RH).

Effects of Diet Containing Soy Protein Isolate on Liver Metabolic Methylation Status Using Obese Zucker Rat Model (P08-033-19)

ObjectivesThe obesity epidemic is continuing to grow in the United States and world for past two decades. There is a link between obesity and chronic diseases development such as diabetes, cardiovascular disease, certain types of cancer and liver diseases. Previously, we reported that obesity caused a significant increase liver steatosis and feeding soy protein isolate (SPI) reduced liver steatosis. The mechanism of SPI protection against liver steatosis is less known. We hypothesize that soy protein diet will reduce development of liver steatosis caused by obesity in part by changing methylation status. The objective of the present study was to investigate the effects of SPI feeding on liver metabolic methylation status using obese zucker rat model. MethodsAfter one week of acclimation, five weeks old female lean and obese Zucker rats (n = 8/group) were randomly fed AIN-93-G diet with either casein (CAS as control) or SPI as source of protein for 22 weeks. Rats were weighted twice per week. Liver sample metabolites concentrations were measured using HPLC with Electrochemical Detection and LC-MS. ResultsOur results shows that; 1) obesity increased body weight significantly (P < 0.001) for both CAS and SPI diets; 2) Obese SPI-fed rats significantly (P < 0.001) reduced liver steatosis compared to obese CAS-fed rats. Also, our results show that liver metabolic profile in lean SPI-fed rats significantly (P < 0.025) increased SAM/SAH ratio (methylation ratio) compare to CAS-fed rats. Obese SPI-fed rats significantly (P < 0.001) decrease level of Homocysteine in liver and increase significantly (P < 0.001) Methionine/Homocysteine ratio. ConclusionsIn summary we showed that SPI diet can reduce liver steatosis by changing methylation status and improved metabolism of Homocysteine, toxic intracellular compound, through remethylation to Methionine. Funding SourcesArkansas Children’s Research Institute’s University Medical Group Fund grant program and Arkansas.

EB 2017 Article: Soy protein isolate feeding does not result in reproductive toxicity in the pre-pubertal rat testis.

The isoflavone phytoestrogens found in the soy protein isolate used in soy infant formulas have been shown to have estrogenic actions in the developing male reproductive tract resulting in reproductive toxicity. However, few studies have examined potential estrogenicity of soy protein isolate as opposed to that of pure isoflavones. In this study, we fed weanling male Sprague-Dawley rats a semi-purified diet with casein or soy protein isolate as the sole protein source from postnatal day 21 to 33. Additional groups were fed casein or soy protein isolate and treated s.c. with 10 µg/kg/d estradiol via osmotic minipump. Estradiol treatment reduced testis, prostate weights, and serum androgen concentrations ( P < 0.05). Soy protein isolate had no effect. Estradiol up-regulated 489 and down-regulated 1237 testicular genes >1.5-fold ( P < 0.05). In contrast, soy protein isolate only significantly up-regulated expression of 162 genes and down-regulated 16 genes. The top 30 soy protein isolate-up-regulated genes shared 93% concordance with estradiol up-regulated genes. There was little overlap between soy protein isolate down-regulated genes and those down-regulated by estradiol treatment. Functional annotation analysis revealed significant differences in testicular biological processes affected by estradiol or soy protein isolate. Estradiol had major actions on genes involved in reproductive processes including down-regulation of testicular steroid synthesis and expression of steroid receptor activated receptor (Star) and cytochrome P450 17α-hydroxylase/(Cyp17a1). In contrast, soy protein isolate primarily affected pathways associated with macromolecule modifications including ubiquitination and histone methylation. Our results indicate that rather than acting as a weak estrogen in the developing testis, soy protein isolate appears to act as a selective estrogen receptor modulator with little effect on reproductive processes. Impact statement Soy protein isolate (SPI) is the sole protein used to make soy-based infant formulas. SPI contains phytoestrogens, which are structurally similar to estradiol. These phytoestrogens, daidzein, genistein, and equol, fit the definition of endocrine-disrupting compounds, and at high concentrations, have estrogenic actions resulting in reproductive toxicity in the developing male, when provided as isolated chemicals. However, few animal studies have examined the potential estrogenicity of SPI as opposed to pure isoflavones. In this study, SPI feeding did not elicit an estrogenic response in the testis nor any adverse outcomes including reduced testicular growth, or androgen production during early development in rats when compared to those receiving estradiol. These findings are consistent with emerging data showing no differences in reproductive development in males and female children that received breast milk, cow's milk formula, or soy infant formula during the postnatal feeding period.

Uterine responses to feeding soy protein isolate and treatment with 17β-estradiol differ in ovariectomized female rats

There are concerns regarding reproductive toxicity from consumption of soy foods, including an increased risk of endometriosis and endometrial cancer, as a result of phytoestrogen consumption. In this study, female rats were fed AIN-93G diets made with casein (CAS) or soy protein isolate (SPI) from postnatal day (PND) 30, ovariectomized on PND 50 and infused with 5μg/kg/d 17β-estradiol (E2) or vehicle. E2 increased uterine wet weight (P<0.05). RNAseq analysis revealed that E2 significantly altered expression of 1991 uterine genes (P<0.05). SPI feeding had no effect on uterine weight and altered expression of far fewer genes than E2 at 152 genes (P<0.05). Overlap between E2 and SPI genes was limited to 67 genes. Functional annotation analysis indicated significant differences in uterine biological processes affected by E2 and SPI and little evidence for recruitment of estrogen receptor (ER)α to the promoters of ER-responsive genes after SPI feeding. The major E2 up-regulated uterine pathways were carcinogenesis and extracellular matrix organization, whereas SPI feeding up-regulated uterine peroxisome proliferator activated receptor (PPAR) signaling and fatty acid metabolism. The combination of E2 and SPI resulted in significant regulation of 504 fewer genes relative to E2 alone. The ability of E2 to induce uterine proliferation in response to the carcinogen dimethybenz(a)anthracene (DMBA) as measured by expression of PCNA and Ki67 mRNA was suppressed by feeding SPI (P<0.05). These data suggest that SPI is a selective estrogen receptor modulator (SERM) interacting with a small sub-set of E2-regulated genes and is anti-estrogenic in the presence of endogenous estrogens.

Mammary Gland Morphology and Gene Expression Differ in Female Rats Treated with 17β-Estradiol or Fed Soy Protein Isolate

Soy foods have been suggested to have both positive health benefits and potentially adverse effects as a result of their content of phytoestrogens. However, studies on the estrogenicity of soy foods are lacking. Here we directly compared the effects of soy protein isolate (SPI), the protein in soy infant formula, with those of 17β-estradiol (E2), on global gene expression profiles and morphology in the female rat mammary gland. Rats were fed AIN-93G diets containing casein or SPI beginning on postnatal d 30. Rats were ovariectomized on postnatal d 50 and treated with 5 μg/kg/d E2 or vehicle for 14 d. Microarray analysis revealed that E2 treatment altered expression of 780 genes more than or equal to 2-fold (P < 0.05), whereas SPI feeding altered expression of only 53 genes more than or equal to 2-fold. Moreover, the groups had only 10 genes in common to increase more than or equal to 2-fold. The combination of SPI feeding and E2 altered expression of 422 genes and reversed E2 effects on many mRNAs, including those involved in the c-myc signaling pathway, cyclin D1, and Ki67. ERα binding to its response element on the Tie-2/Tek and progesterone receptor promoters was increased by E2, but not SPI, and this promoter binding was suppressed by the combination of E2 + SPI for the Tie-2/Tek promoter but increased for the progesterone receptor promoter (P < 0.05). SPI reduced the ratio of epithelial to fat pad area and E2 + SPI reduced both epithelial and fat pad area (P < 0.05). These data suggest that SPI is only minimally estrogenic in the rat mammary gland even in the absence of endogenous estrogens.

Enhanced Expression and Glucocorticoid-Inducibility of Hepatic Cytochrome P450 3A Involve Recruitment of the Pregnane-X-Receptor to Promoter Elements in Rats Fed Soy Protein Isolate

Previous studies and Expt. 1 of the current study demonstrate that diets made with soy protein isolate (SPI) enhance the glucocorticoid-inducibility of hepatic cytochrome P450 (CYP)3A-dependent monooxygenase activities (P < 0.05) compared with diets made with casein (CAS). To determine the underlying molecular mechanism, in a second experiment, we analyzed the time course of dexamethasone (DEX)-induction of hepatic CYP3A mRNA expression on postnatal d (PND) 25 and PND60 in male and female rats fed SPI- or CAS-based diets. After 50 mg/kg DEX, CYP3A1 mRNA expression increased >200-fold in SPI-fed males and females at PND25 compared with a 100-fold increase in CAS-fed rats (P < 0.05). The DEX-induced increase in CYP3A1 mRNA in SPI-fed rats on PND60 was also greater than that in CAS-fed rats. The induction by DEX of CYP3A2 mRNA was 1- to 3-fold greater in rats fed SPI compared with those fed CAS on PND25 (P < 0.05). Quantitation of newly synthesized CYP3A1 RNA transcripts by nuclear run-on analysis demonstrated a greater rate of basal transcription in SPI-fed compared with CAS-fed rats on PND60 accompanied by greater binding of the pregnane X receptor (PXR) to a response element on the CYP3A1 promoter in SPI-fed compared with CAS-fed rats (P < 0.05). These data suggest that increased hepatic CYP3A expression and inducibility following SPI feeding involves recruitment of PXR to its response element and suggests that soy consumption has potential effects on metabolism and transport of a wide variety of drugs and on bile acid homeostasis via proteins regulated by this transcription factor.

Feeding soy protein isolate (SPI) does not result in an estrogenic gene expression profile in the mammary of ovariectomized (OVX) female rats

Concerns of increased breast cancer risk in women consuming soy exist because of the perceived estrogenicity of soy isoflavones. Female Sprague‐Dawley rats (N = 20/group) were fed AIN‐93G diets with casein or SPI as the protein from PND30. On PND50 rats were OVX and 10/group infused s.c. with 5 μg/kg/d 17β‐estradiol (E2) to restore physiological levels. On PND64, mammary glands were collected and total mRNA isolated for microarray analysis. Array data were normalized using GeneSpring. Statistical analysis of genes expressed greater than 1.5‐fold revealed 651 genes regulated by E2 (P&lt; 0.05) compared to 27 genes regulated by SPI with only 6 in common between the two treatments. A sub‐set of 164 E2‐regulated genes were further modulated by SPI feeding and 126 genes were only affected by the combination of SPI + E2. Data were confirmed by real time RT‐PCR and suggest that gene signatures of E2 and SPI have little overlap. SPI feeding did significantly modulate responses to E2. In most cases SPI antagonized E2 effects. This included ERβ‐specific genes such as Knsl2 and Top2a and the cell proliferation mediator cyclin D1. USDA CRIS 6251‐51000‐007‐03S.

Hepatic gene expression following consumption of soy protein isolate in female Sprague–Dawley rats differs from that produced by 17β-estradiol treatment

Although soy foods have been recognized as an excellent source of protein, there have been recent concerns regarding potential adverse effects of isoflavone phytochemicals found in soy products, which are known to bind and activate estrogen receptors. Here, we used global hepatic gene expression profiles in ovariectomized female Sprague-Dawley rats treated with 17beta-estradiol (E(2)) or fed with soy protein isolate (SPI) as a means of estimating potential estrogenicity of SPI. Female Sprague-Dawley rats were fed AIN-93G diets containing casein (CAS) or SPI starting at postnatal day (PND) 30. Rats were ovariectomized on PND 50 and infused with E(2) or vehicle in osmotic pumps for 14 d. Microarray analysis was performed on liver using Affymetrix GeneChip Rat 230 2.0. Serum E(2) levels were within normal ranges for the rat and SPI feeding did not increase uterine wet weight in the absence or presence of E(2). SPI feeding altered (P<0.05, >or=+/-1.5-fold) the expression of 82 genes, while E(2) treatment altered 892 genes. Moreover, only 4% of E(2)-affected genes were also modulated by SPI, including some whose expression was reversed by SPI feeding. The interaction between E(2) and SPI uniquely modulated the expression profile of 225 genes including the reduction of those involved in fatty acid biosynthesis or glucocorticoid signaling and an induction of those involved in cholesterol metabolism. The different hepatic gene signatures produced by SPI feeding compared with E(2) and the lack of increase in uterine wet weight in rats fed with SPI suggest that SPI is not estrogenic in these tissues.

Estrogenic status modulates the effect of soy on hepatic responses to 7,12-dimethylbenz(a)anthracene (DMBA)

We examined the influence of estradiol (E2) status and soy protein isolate (SPI) intake on the hepatic responses altered by 7,12-dimethylbenz(a)anthracene (DMBA, a polycyclic aromatic hydrocarbon [PAH]). Sprague–Dawley rats were ovariectomized (OVX) at PND50 and infused with E2 or vehicle for 14 d and gavaged with 50 mg/kg DMBA or vehicle 24 h before sacrifice at PND64. Rats were fed an AIN-93G diet made with SPI or casein as sole protein source throughout the study. Basal AhR protein levels were reduced ( P < 0.05) by SPI feeding irrespective of the E2 status. However, DMBA increased ( P < 0.05) AhR-induced CYP1A1 gene expression in OVX, SPI-fed rats, but reduced ( P < 0.05) CYP1A1 in OVX + E2, SPI-fed rats. Chromatin-immunoprecipitation demonstrated lower ( P < 0.05) DMBA-mediated recruitment of estrogen receptor alpha to the CYP1A1 promoter by SPI feeding in the presence of E2, suggesting an estrogen-like action of SPI on DMBA-mediated signaling in the absence of E2. Further, microarray analysis (Rat 230-2.0 Affymetrix-GeneChip™) revealed 231 genes common to SPI + DMBA and SPI + E2 + DMBA (normalized to E2) treatments. AhR-activated genes ( CYP1A1, CYP1A2, and NQO1) were down-regulated by SPI + E2 + DMBA compared to SPI + DMBA. Unique interactions among SPI, DMBA and E2 altered the expression profile of 316 genes, not observed by either treatment alone. Our data suggest that although E2 status does not effect soy-mediated AhR degradation, it modulates the effects of soy on many genes, including CYP1A1.

Temporary consumption of diet with unbalanced amino acid pattern affects long-lasting growth retardation correlated with oxidative stress response associated gene expression in juvenile pigs

The present study was conducted to study whether oxidative stress is the trigger of long-term physiological effects of temporary consumption of a soy protein isolate (SPI) based diet with an imbalanced amino acid pattern. Hepatic expression of 19 genes that are involved in and co-regulated with oxidative stress response and showing diet-associated expression after chronic SPI feeding using quantitative RT-PCR, growth and liver composition were investigated in a model of protein-underfeeding juvenile pigs, which were fed a casein (CAS) based diet for four weeks subsequent to a four week consumption of an SPI diet in comparison with chronically CAS fed animals. Temporary feeding of SPI diet resulted in prolonged up-regulation of genes involved in oxidative/cellular stress response (glutathione-S-transferase, peptide methionine sulfoxide reductase, calnexin, organic anion transport polypeptide 2). Cluster analysis of gene expression data indicated persistent SPI-related co-regulation of the genes involved in stress response with genes involved in the regulation of protein biosynthesis and in neuronal signalling for at least four weeks after replacement of SPI by CAS. Gene expression data are negatively correlated with body weight and liver protein content. Significant association of oxidative stress responsiveness with growth retardation and liver composition underline the possible impact of diet-affected oxidative stress for long-lasting deleteriously metabolic consequences.

Studies on persisting effects of soy-based compared with amino acid-supplemented casein-based diet on protein metabolism and oxidative stress in juvenile pigs

Juvenile growing pigs were studied to explore whether a soy-based diet can induce persistent physiological alterations, especially in protein and energy metabolism, nutrient oxidation and redox homeostasis. In former studies we have shown that in juvenile pigs chronically fed protein diets based on either casein (CAS) or soy protein isolate (SPI), the SPI diet significantly decreases growth rate and increases oxidative stress responsiveness as compared to CAS. In addition, here we show that chronic feeding of SPI vs. CAS diet decreases whole body protein synthesis (WBPS) (p = 0.007) and hepatic gene expression associated with protein synthesis. To study persistent SPI effects, a three-period feeding experiment was designed: In the test group 18 pigs received the CAS diet for 24 days (period 1), followed by 31 days on the SPI diet (period 2) and further 31 days on the CAS diet (period 3). In the control group 18 pigs were fed the CAS diet throughout the three periods (86 days). Temporary consumption of SPI diet results in persistent changes of protein metabolism and oxidative stress responsiveness. After switching back from SPI to CAS diet the decrease of WBPS of the test group vs. control group was of borderline significance (p = 0.061), transcript levels of hepatic gene expressions of leucine aminopeptidase, endopeptidase 24.16, glutathione-S-transferase and peptide methionine sulfoxide reductase were increased. In liver tissue, total glutathione was increased and thiobarbituric acid reactive substances were decreased in the test vs. control group. In conclusion, results suggest that SPI-induced changes in protein and amino acid metabolism as well as in redox homeostasis and antioxidative potential in growing pigs persist 4 weeks after the cessation of SPI feeding.

Soy Protein Suppresses Gene Expression of Acetyl-CoA Carboxylase Alpha from Promoter PI in Rat Liver

Dietary soy protein isolate (SPI) reduces hepatic lipogenesis by suppressing gene expression of lipogenic enzymes, including acetyl-CoA carboxylase (ACC). In order to elucidate the mechanism of this regulation, the effect of dietary SPI on promoter (PI and PII) specific gene expression of ACC alpha was investigated. Rats were fed experimental diets containing SPI or casein as a nitrogen source. SPI feeding decreased the hepatic contents of total ACC mRNA as well as triglyceride (TG) content, but dietary SPI affected the amount of sterol-regulatory element binding protein (SREBP)-1 mRNA and protein very little. The amount of ACC mRNA transcribed from PII promoter containing SRE was not significantly affected by dietary protein, while a significant decrease in PI-generated ACC mRNA content was observed in rats fed the SPI diet. These data suggest that SPI feeding decreased the hepatic contents of ACC alpha mRNA mainly by regulating PI promoter via a nuclear factor(s) other than SREBP-1.

Effect of a soy protein diet on protein and energy metabolism and organ development in protein-restricted growing pigs

Two feeding experiments were carried out with castrated male pigs weighing between 10 and 30 kg to study acute and persisting dietary effects on growth and on protein and energy metabolism in growing pigs. Pigs were fed semi-synthetic isoenergetic and isonitrogenous diets at 50% protein requirement with either soy protein isolate (SPI) or casein (CAS) as sole protein source. Intake of protein and ME amounted to 9% w/w and 1800 kJ · kg BW − 0.62 · d − 1 in Exp. 1, respectively, and 9% w/w and 1430 kJ · kg BW − 0.62 · d − 1 in Exp. 2. The CAS diet was supplemented by Lys, Met, Thr and Trp. In Exp. 1 (acute effects), 18 pigs received the CAS diet for 24 days (period 1); 9 pigs were then switched to a SPI diet whereas 9 pigs continued on the CAS diet for another 31 days (period 2). In Exp. 2, a third period of 31 days was added in which the SPI group was switched back to CAS diet. The control group was fed on the CAS diet throughout Exp. 2 (86 days). Altogether the majority of parameters were not affected neither comparing SPI with CAS in Exp. 1 nor inspecting possible persistence of effects in Exp. 2. In detail, in Exp. 1 SPI compared to CAS feeding resulted in a lower efficiency of protein utilisation and lower protein retention. Attendant upon the lower protein retention an increased energy retention as fat was only observed in tendency. SPI feeding caused a decreased body weight, thyroid weight and increased hepatic carbohydrate content that persisted after the diet was changed back to CAS (Exp. 2).