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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 21, 2002 as doi:10.1096/fj.01-0734fje. |
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* Research Units Molecular Biology,
Nutritional Physiology ‘Oskar Kellner’, and
# Genetics and Biometrics, Research Institute for the Biology of Farm Animals, D-18196 Dummerstorf; Germany
2Correspondence: Research Unit Molecular Biology, Research Institute for the Biology of Farm Animals, Wilhelm-Stahl-Allee 2, D-18196 Dummerstorf, Germany. E-mail: schwerin{at}fbn-dummerstorf.de
SPECIFIC AIMS
This study aims to investigate the molecular basis for physiological effects of different dietary amino acid patterns in relation to optimal health and performance by investigating diet-associated hepatic gene expression in growing pigs fed restricted protein diets based on either casein (CAS) or soy protein isolate (SPI). Hepatic diet-dependently displayed expressed sequence tags (EST) preselected by the mRNA differential display technique (DDRT-PCR) were used as molecular probes in real-time PCR with the LightCycler® to identify differences in mRNA expression profiles.
PRINCIPAL FINDINGS
1. Using DDRT-PCR, 86 hepatic diet-related expressed nucleotide sequences were identified
The mRNA differential display technique was applied to characterize the general postabsorptive expression patterns in liver of growing pigs fed SPI or CAS added to otherwise identical diets. Data of 86 differentially displayed nucleotide sequences were submitted to GenBank and have been assigned the accession numbers BG695746-BG695798. Subsequent FASTA search of GenBank/EMBL database showed that 38 of these EST (44.2%) were similar to previously described genes whereas 18 EST (20.9%) did not match any database entries. The other EST showed a similarity to EST, repetitive and mitochondrial DNA elements, or other sequences (PAC, BAC) already deposited in database. Physiological function of the EST was identified on the basis of their sequence similarity with known genes. The majority of these sequences (>90%) exhibited homology to genes involved in oxidative stress response, protein and amino acid metabolism, nervous and cellular signal transfer, regulation of transcription and translation, membrane transport, and fat and energy metabolism.
2. Soy protein diet enhances hepatic expression of gene associated with oxidative stress
To study the effect of both protein diets on postabsorptive hepatic gene expression, mRNA abundance of the differentially displayed sequences showing similarities to already known genes was analyzed by real-time PCR using LightCycler® (Roche, Mannheim, Germany). Among these EST, six diet-related displayed genes involved in the metabolism of stress response exhibit significant changes in transcription level and indicate increased oxidative stress and/or cellular stress response in pigs fed the SPI vs. CAS diet. These genes include the glutathione-S-transferase gene involved in detoxification, the peptide methionine sulfoxide reductase gene involved in repair of oxidized physiologically essential macromolecules, two genes (calnexin, heat shock transcription factor 1) that are involved in cellular stress response, and two genes (apolipoprotein A-I (apoAI), organic anion transport polypeptide 2) promoting cholesterol efflux from tissues to the liver and excretion. In pigs fed the SPI diet, the mean hepatic transcription levels of calnexin, organic anion transport polypeptide 2, glutathione-S-transferase, and peptide methionine sulfoxide reductase were two- to threefold higher (all P<0.03) than in pigs fed the CAS diet (Fig. 1
). By contrast, the mRNA abundance of the heat shock transcription factor 1 was significantly higher in the CAS group (P<0.01). In the SPI group, transcriptional up-regulation of apoAI was of borderline significance (P=0.066).
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3. Individual specificity of diet-related hepatic gene expression patterns
Responses on the SPI diet show extreme interindividual variability. As shown for glutathione-S-transferase, mRNA abundance of individual SPI animals range from values comparable to CAS animals to 17.4-fold increased concentration (Fig. 1)
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4. Expression of genes associated with oxidative stress shows coregulation with genes involved in regulation of gene expression and neuronal signaling
From results of a hierarchical cluster analysis based on first-principal components of clusters, a tree diagram was drawn to display the correlation among gene expression data of 33 EST analyzed across 14 pigs fed the two different protein diets.
Hierarchical cluster analysis resulted in clustering of genes related to oxidative stress response with genes related to regulation of gene expression and neuronal signaling. The genes related to regulating gene expression include three genes involved in regulation of transcription [DNA methyltransferase, nuclear pore-associated protein, TLS (translocated in liposarcoma)-associated protein]; a gene involved in termination of translation (eukaryotic release factor 1); and a gene involved in replication (replication factor C2). Genes related to neuronal signaling include two involved in neuron apoptosis (endopeptidase 24.16, retinal binding like protein), and four involved in neuronal development (transmembrane 4, NT2, CGI-139 protein, LC3 polypeptide).
CONCLUSIONS AND SIGNIFICANCE
It is well known that soy protein vs. milk protein diets affect growth, body composition, and health, but the molecular regulative mechanisms for these parameters are insufficiently known.
Our findings indicate complex diet-associated modifications of intermediary processes and a high interindividual variability in response to both diets. As shown in this study, soy protein vs. the CAS diet enhances hepatic gene expression associated with oxidative stress in growing pigs fed restricted protein diets (Fig. 2
). This could be related to differences in protein amino acid patterns or to soy-associated bioactive constituents such as isoflavones, saponins, phytosterols. However, we assume that the metabolic effects of both protein-restricted diets can be attributed mainly to the imbalanced amino acid pattern of SPI compared to CAS feeding. The main differences are increased content of methionine, proline, phenylalanine, and tyrosine in the CAS diet and of cystine, glycine, aspartic acid, and arginine in the SPI diet. Since we used highly purified SPI produced for infant formula, effects of soy-associated constituents like trypsin inhibitors and lectins could be largely excluded due to heat treatment under mild alkaline conditions. Thus, in SUPRO® 1610, levels of total isoflavones or trypsin inhibitors were > 200-fold lower than other soy protein diets reported. It has been shown recently that methionine and, to a smaller degree, cysteine deprivation result in overexpression of asparagine synthetase, the CCAAT/enhancer binding protein homologous protein, and of the c-Jun amino-terminal kinase 1 in human cells. Depletion of arginine and cysteine leads to induction of insulin-like growth factor binding protein 1 mRNA. The SPI used here is characterized by reduced concentrations of methionine and about twice as much cystine and arginine as CAS.
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The up-regulation of peptide methionine sulfoxide reductase and glutathione-S-transferase observed in the SPI diet corresponds to published findings on accumulation of peptide methionine sulfoxide on oxidative stress and on the role of glutathione-S-transferases in oxidative stress response due to dietary depletion of sulfur amino acids in humans and mice. Peptide-bound methionine is readily oxidized to methionine sulfoxide by reactive oxygen species. Oxidation of surface exposed methionine thus serves to protect other functionally essential residues from oxidative damage. The peptide methionine sulfoxide reductase catalyzes the reduction of methionine sulfoxide to methionine in proteins.
Glutathione in mitochondria is the only defense available against endogenously generated hydrogen peroxide. Depletion of glutathione renders the cell more susceptible to oxidative stress. Decrease in the activity of antioxidant enzymes and phase II metabolizing enzymes such as glutathione-S-transferase is strongly associated with an increased microsomal lipid peroxidation in rats. The up-regulation of peptide methionine sulfoxide reductase and glutathione-S-transferase by the SPI diet increases the scavenging efficiency of the system. Observed increased transcript levels of the apoAI and the organic anion transport polypeptide 2 are in line with the assumed increased oxidative stress effects in the SPI group. ApoAI is the major protein component of high density lipoprotein (HDL) and inversely correlated with the risk of premature atherosclerosis. The initial stage of oxidation of HDL is accompanied by the lipid hydroperoxide-dependent, selective oxidation of two of the three methionine residues of apoAI to methionine sulfoxides that probably initiates an increased activity of the peptide methionine sulfoxide reductase as observed in the present study. ApoAI promotes cholesterol efflux from tissues to the liver for excretion. Therefore, it is not surprising that the organic anion transport polypeptide 2 which is involved in the transport of taurocholic acid, a biological detergent synthesized in the liver from cholesterol, is up-regulated as well in pigs fed soy protein isolates. This suggests further that cholesterol metabolism is affected by SPI and agrees with the observed hypocholesterolemic effect of soy. The oxidative stress response enhancing effect of SPI diet as compared with CAS diet is supported by the SPI related up-regulation of the calnexin acting as a chaperone in assisting protein assembly and/or in the retention of unassembled protein subunits and the feed back down-regulated heat shock factor 1.
Clustering the gene expression data indicates a close coregulation of the genes related to oxidative stress response with genes related to the regulation of gene expression and in neuronal signaling. Many studies have emphasized the critical importance of oxidative stress as part of pathophysiological mechanism, such as in Alzheimer’s disease, and demonstrated significant association of oxidative stress with proliferation, neurodegeneration and regulation of gene expression. The presented results contribute to the search of the molecular basis of nutrient function and mechanisms that underlie nutritionally related diseases. The elucidation of diet-dependent changes in gene expression improves our understanding of the molecular basis for differences in amino acid requirements in relation to optimal health and performance in human and livestock. However, the high interindividual variability of the transcription levels observed emphasizes that although there are significant differences between the corresponding mean values, the overall transcriptional response is animal specific and the result of the interaction of co- and/or alternatively regulated genes. Thus, this can partly explain the known biological variation of requirement values. We conclude that under our dietary conditions, i.e., an imbalanced amino acid pattern at 50% protein requirement level, the relative deficiency (methionine, proline, phenylalanine, tyrosine) or surplus of amino acids (cystine, glycine, aspartic acid, arginine) led to changes in metabolism related to an enhanced endogenous production of reactive oxygen species. This is associated with induction of the scavenging systems at the molecular level.
It remains to be determined whether and how long the observed diet-dependent changes in expression profiles persist after termination of the dietary challenge, whether the transcriptional modulation is translated into protein expression and function and whether it results in a physiological response which maintains whole-body oxidative/antioxidative balance.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0734fje; to cite this article, use FASEB J. (June 21, 2002) 10.1096/fj.01-0734fje 
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