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Long-term-selected DUhTP mice represent a non-inbred model for inborn physical high-performance without previous training. Abundance of hepatic mRNA in 70-day male DUhTP and control mice was analyzed using the Affymetrix mouse array 430A 2.0. Differential expression analysis with PLIER corrected data was performed using AltAnalyze. Searching for over-representation in biochemical pathways revealed cholesterol metabolism being most prominently affected in DUhTP compared to unselected control mice. Furthermore, pathway analysis by AltAnalyze plus PathVisio indicated significant induction of glycolysis, fatty acid synthesis and cholesterol biosynthesis in the liver of DUhTP mice versus unselected control mice. In contrast, gluconeogenesis was partially inactivated as judged from the analysis of hepatic mRNA transcript abundance in DUhTP mice. Analysis of mRNA transcripts related to steroid hormone metabolism inferred elevated synthesis of progesterone and reduced levels of sex steroids. Abundance of steroid delta isomerase-5 mRNA (Hsd3b5, FC 4.97) was increased and steroid 17-alpha-monooxygenase mRNA (Cyp17a1, FC -11.6) was massively diminished in the liver of DUhTP mice. Assessment of steroid profiles by LC-MS revealed increased levels of progesterone and decreased levels of sex steroids in serum from DUhTP mice versus controls. Analysis of hepatic mRNA transcript abundance indicates that sterol regulatory element-binding protein-1 (SREBP-1) may play a major role in metabolic pathway activation in the marathon mouse model DUhTP. Thus, results from bioinformatics modeling of hepatic mRNA transcript abundance correlated with direct steroid analysis by mass spectrometry and further indicated functions of SREBP-1 and steroid hormones for endurance performance in DUhTP mice.

Purpose: Long-distance runners have increased needs of energy supply. To unravel genetically based mechanisms required for efficient energy supply, we have analyzed hepatic metabolism of mice characterized by the inborn capacity to perform as long-distance runners., Methods: The mouse model had been established by phenotypic selection for high treadmill performance for 90 generations and was characterized by approximately 3.8-fold higher running capacities (Dummerstorf high Treadmill Performance mouse line [DUhTP]) compared with unselected and also untrained controls (Dummerstorf Control mouse line [DUC]). From 7-wk-old male mice, serum and liver samples were collected and analyzed for messenger RNA, protein, and metabolite levels, respectively., Results: In livers from DUhTP mice, we identified significantly higher messenger RNA transcript levels of peroxisome proliferator-activated receptor delta and higher protein levels of sirtuin-1, acetyl-CoA-synthetase, acetyl-CoA-carboxylase, phosphoenolpyruvate carboxykinase, and glutamate-dehydrogenase, suggesting higher gluconeogenesis and lipogenesis in DUhTP mice. In fact, higher hepatic levels of glycogen and triglycerides as well as higher concentrations of carbohydrate, fatty acid, and cholesterol metabolites were found in DUhTP mice. In parallel, in DUhTP mice, which did not have access to running wheels, a marked hyperlipidemia (cholesterol = 160% ± 8%, triglycerides = 174% ± 14% of controls, respectively), and abdominal obesity (DUhTP = 0.396 ± 0.019 g, DUC = 0.291 ± 0.019 g) were found., Conclusions: From our data, we conclude that the physiological basis of genetically fixed higher endurance-running performance in DUhTP marathon mouse is related to increased hepatic gluconeogenesis and lipogenesis. Expression of sirtuin 1 as well as of gluconeogenic and lipogenic key enzymes may be related to peroxisome proliferator-activated receptor delta. Metabolic adaptations presented in our study represent inborn features of superior endurance-running performance.

Maternal nutrition during gestation has important effects on offspring gene expression mediated by DNA methylation. In order to evaluate the effect of restricted and excess protein intake during gestation, hepatic gene expression and DNA methylation of key metabolic genes NR3C1, PPARα, HMGCR, PGC1α, INSR and CYP2C34 were investigated. Liver samples of German Landrace offspring were collected at Gestational Day 95, at birth, at weaning and from finisher pigs. Gene expression in foetal liver revealed significant differences between the control group (CO) and the low-protein group (LP) in HMGCR (P<.0001), INSR (P=.0003), NR3C1 (P=.020) and PGC1α (P=.003). At birth INSR (P=.032), PPARα (P=.0006) and CYP2C34 (P<.0001) showed significant differences between LP and CO. CYP2C34 was significantly increased in the high-protein group (HP) compared to CO (P=.001). At weaning, INSR was significantly higher expressed in LP than in CO (P=.018). HMGCR showed a significant decrease of transcript amount in HP compared to CO (P=.0006). Furthermore, we studied the question whether gene expression differences between distinct diet groups are a result of differential DNA methylation status. CpG sites in the 5'-flanking region of CYP2C34 showed a significant positive correlation with transcript amount in LP (nt -137: R=0.67, P<.0001; nt -112: R=0.54, P=.003). In NR3C1 methylation, differences in the CpG island were negatively correlated with gene expression data in LP (R=-0.34, P=.032). The mean of methylation of PPARα over CpG sites from nt -220 to -11 was significantly increased in the LP group compared with CO (P=.043). These data suggest an influence of DNA methylation in nutrient-dependent transcriptional regulation of NR3C1, PPARα and CYP2C34., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Cortisol is a steroid hormone with important roles in regulating immune and metabolic functions and organismal responses to external stimuli are mediated by the glucocorticoid system. Dysregulation of the afferent and efferent axis of glucocorticoid signaling have adverse effects on growth, health status, and well-being. Glucocorticoid secretion and signaling show large interindividual variation that has a considerable genetic component; however, little is known about the underlying genetic variants. Here, we used trait-correlated expression analysis, screening for expression quantitative trait loci (eQTL), genome-wide association (GWA) studies, and causality modeling to identify candidate genes in porcine liver and muscle that affect or respond to plasma cortisol levels. Through trait-correlated expression, we characterized transcript activities in many biological functions in liver and muscle. Candidates from the list of trait-correlated expressed genes were narrowed using only those genes with an eQTL, and these were further prioritized by determining whether their expression was predicted to be related to variation in plasma cortisol levels. Using network edge orienting (NEO), a causality modeling algorithm, 26 of 990 candidates in liver were predicted to affect and 70 to respond to plasma cortisol levels. Of 593 candidates in muscle that were correlated with cortisol levels and were regulated by eQTL, 2 and 25 were predicted as effective and responsive, respectively, to plasma cortisol levels. Comprehensive data integration has helped to elucidate the complex molecular networks contributing to cortisol levels and thus its subsequent metabolic effects. The discrimination of up- and downstream effects of transcripts affecting or responding to plasma cortisol concentrations improves the understanding of the biology of complex traits related to growth, health, and well-being.

Maternal diet during gestation is known to affect offspring phenotype induction. In the present study the influence of maternal protein restriction and excess during gestation on offspring candidate gene expression was analysed. German Landrace gilts were fed control, low protein (LP) or high protein (HP) diet throughout gestation (n = 18 per diet group). After birth piglets were cross-fostered and lactated by control diet fed nursing sows. Samples of offspring liver tissue were taken at foetal, newborn, weaning and finishing phase (n = 16, respectively). Transcript amount of selected candidate genes related to cell cycle and cell proliferation was estimated by quantitative real-time PCR. Maternal protein restriction influenced gene expression of candidate genes CCND2, GADD45B, GALK1, GSTP1, MARCKS, MGMT, NEAT1, PSEN1, SNX1 and TRPM7 in liver from foetuses, newborn piglets, weaned and/or finisher pigs. In the offspring of mothers fed a HP diet expression of target genes was affected exclusively in finisher pigs showing increased transcript amount of CCND2, GALK1, MARCKS, SNX1 and TRPM7. The results of the present study clearly show a long-lasting impact of the maternal protein supply during gestation on offspring candidate genes. Remarkably, effects of gestational HP diet became evident in finisher pigs while LP supply already alters genes expression in foetal tissue. Thus it is suggested that LP and HP supply affect the offspring in utero by different physiological mechanisms with the consequence of late effects in case of prenatal protein excess in contrast to early effects in case of protein restriction.

Recent evidence indicates that maternal nutrition during pregnancy influences gene expression in offspring through epigenetic alterations. In the present study we evaluated the effect of protein excess and deficiency during porcine pregnancy on offspring hepatic and skeletal muscular expression patterns of key genes of methionine metabolism (DNMT1, DNMT3a, DNMT3b, BHMT, MAT2B and AHCYL1), condensin I subunit genes (NCAPD2, NCAPG and NCAPH), important for chromosome condensation and segregation, global DNA methylation and gene-specific DNA methylation. German Landrace sows were randomly assigned to control (CO), high protein (HP) and low protein (LP) diet groups. Tissue samples of offspring were collected from fetal (dpc95), newborn (dpn1), weanling (dpn28) and finisher pigs (dpn188). Gene expression of DNMT1, DNMT3a and DNMT3b was influenced by both HP and LP diets, indicating an involvement of DNA methylation in fetal programming by maternal protein supply. Moreover, hepatic global methylation was significantly affected by protein restriction at dpc95 (p = 0.004) and by protein excess at dpn188 (p = 0.034). Gene expression in fetal liver was significantly different between CO and LP for NCAPD2 (p = 0.0005), NCAPG (p = 0.0009) and NCAPH (p < 0.0001). In skeletal muscle, LP fetuses had significantly altered gene expression of NCAPD2 (p = 0.020) and NCAPH (p = 0.001), compared with CO. Furthermore, NCAPG was differentially methylated among LP, HP and CO; indeed, a significant positive correlation was detected with transcript amount in fetal pigs (r = 0.47, p = 0.002). These data demonstrate that both restriction and excess dietary protein during pregnancy alters the offspring's epigenetic marks and influences gene expression.

The liver plays a central role in the regulation of the metabolic status, partitioning of nutrients, and expenditure of energy. To gain insight into hepatic metabolic pathways and key transcripts affecting traits related to body composition, liver expression profiles were compared of pigs of two breeds, the obese German Landrace (DL) and the lean Pietrain (Pi). Porcine oligonucleotide microarray were hybridized with liver cRNAs obtained at peripubertal age (180 days of age) and prenatal stages (35, 63, and 91 days postconception) that represent three developmental stages of liver, i.e., period of differentiation, period of metabolic activity, and period of glycogen accumulation. In terms of the number of genes regulated between DL and Pi, the most striking distinctions were found at peripubertal age with upregulation of key genes of lipid metabolism pathways (FASN, ACSS2, ACACA) in obese DL pigs and upregulation of genes of cell growth and/or maintenance, and protein syntheses, as well as cell proliferation pathways (PPARD, POU1F1, IGF2R), in lean Pi pigs. Moreover, time course analysis of breed-dependent expression profiles revealed breed-typical temporal regulation from prenatal stages to peripubertal age of genes assigned to biological processes involving lipid pathways and cell activity, i.e., breed differences are already initiated during early prenatal development. Information about mRNA expression levels of the two breeds differing in body composition, partitioning and utilization of nutrients and energy reveals functional candidate genes for traits related to obesity and leanness.

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.

The present study aimed at identifying bovine hepatic and intestinal DNA sequences expressed breed specifically as potential functional candidate genes for nutrient transformation. Transcript levels of 29 expressed sequence tags (ESTs) were analysed comparatively in the liver and intestine of growing Charolais and German Holstein bulls by real-time RT-PCR. In previous studies, these ESTs were characterized as differentially displayed in mRNA differential display of cows varying in metabolic type and harbouring single nucleotide polymorphisms. Breed-specific gene expression levels indicate significantly increased hepatic metabolic activity in Charolais and increased intestinal metabolic activity in German Holstein bulls. Transcript levels of six functional genes measured in liver (NDUFB8, NACA, UAP1, SAH) and intestine (FUS/TLS, APOC3), respectively, support this assumption. The observed coincidence of metabolic type-specific expressed ESTs with variant ESTs showing breed-specific allele distribution points to functional genetic variants located in the vicinity of the analysed sequences. In addition, location of most of the breed specifically expressed ESTs within chromosome regions known to be affecting carcass and growth traits in cattle supports the putative candidate gene character of the ESTs identified.

This study aims to identify hepatic genes affecting traits related to muscularity and obesity by combining expression analyses, association studies, and gene mapping. Functional candidate genes with trait-associated expression were obtained by hybridising custom made application-specific cDNA microarrays with targets of discordant sib pairs of a porcine experimental population. Out of 238 genes addressed, nine genes were regulated by the factor>or=2 between the sib pairs. Differential gene expression was independently confirmed for selected genes by real time RT-PCR. Transcript levels of four genes (APOH, PEDF, SLCO1B3, TBG) were significantly different between the phenotype groups. Screening for trait associated markers within TBG and APOH by comparative sequencing of discordant sib pairs revealed a SNP at position nt 778 (A>C) (N229H) of TBG. No polymorphism in APOH was detected. Association analysis confirmed effects of TBG on carcass traits statistically. Allocating TBG to a QTL region on chromosome X revealed genetic evidence for the effect. Moreover, our results indicate that there are probably two polymorphisms segregating-one (N229H) altering binding capability of TBG and another still to be detected altering the transcription rate of TBG.

In a previous investigation we showed by expression profiling based on transcription analysis using differential display RT-PCR (DDRT-PCR) and real-time RT-PCR that a soy protein diet (SPI) significantly changes the hepatic transcription pattern compared with a casein diet (CAS). The present study was conducted to determine whether the transcriptional modulation is translated into protein expression. The hepatic mRNA abundance of four genes (EP24.16, LC3, NPAP60L, RFC2) that showed diet-related expression in previous DDRT-PCR experiments was analyzed by real-time RT-PCR. Two pigs that showed the most prominent SPI-related changes of transcription and two casein-fed pigs were selected and their hepatic protein pattern was studied comparatively by two-dimensional gel electrophoresis and peptide mass fingerprinting. The two-dimensional protein gel electrophoresis revealed a predominant SPI-associated upregulation of protein expression that corresponded to the results of the mRNA study. Of 380 diet-related protein spots displayed, 215 appeared exclusively or enlarged in the two SPI pigs; 10 of 39 diet-related expressed protein spots extracted could be identified by peptide mass fingerprinting and database search. Compared with the transcriptomics approach, the proteomics approach led in part to the identification of the same diet-associated expressed molecules (plasminogen, trypsin, phospholipase A2, glutathione-S-transferase alpha, retinal binding protein) or at least molecules belonging to the same metabolic pathways (protein and amino acid metabolism, oxidative stress response, lipid metabolism). The present results at the proteome level confirm SPI-related increased oxidative stress response and significant effects on protein biosynthesis already observed at the transcriptome level.

Understanding the basis for differences in nutrient requirements and for nutrient effects on health and performance requires an appreciation of the links between nutrition and gene expression. We developed and applied molecular probes to characterize diet-associated postabsorptive hepatic gene expression in growing pigs chronically fed protein-restricted diets based on either casein (CAS) or soy protein isolate (SPI). Eighty-eight expressed sequence tags (ESTs) were identified on the basis of diet-related changes in expression, by using an mRNA differential display method. Expression profiling based on transcription analysis by real-time reverse transcriptase-polymerase chain reaction showed that the SPI diet significantly changed the pattern of gene expression as compared with the CAS diet and allowed identification of coregulated genes. The expression of six genes involved in the metabolism of stress response (glutathione S-transferase, peptide methionine sulfoxide reductase, apolipoprotein A-I, organic anion transport polypeptide 2, calnexin, heat shock transcription factor 1) exhibited significant changes in the transcription level and indicated an increased oxidative stress response in pigs fed the SPI diet. Hierarchical clustering of gene expression data of all 33 ESTs analyzed across 14 pigs fed the two different diets resulted in clustering of genes related to the oxidative stress response with genes related to the regulation of gene expression and neuronal signaling.

mRNA differential display was applied to identify hepatic and intestinal expressed sequence tags (ESTs) in lactating cows of different metabolic types (milk type, meat/milk type, meat type) that are potentially associated with energy turnover and involved in the regulation of these processes. Altogether, 277 ESTs (liver: 161, intestine: 116) were identified. For 150 transcripts (liver: 99, intestine: 51), the sequences showed similarity to previously described genes and ESTs. Many of these homologous sequences are reported to be involved in hepatic metabolism. Ninety-four ESTs (liver: 43, intestine: 51) did not match with any database entries. Semi-quantitative RT-PCR revealed quantitative differences in transcript represented by randomly chosen ESTs in liver samples of animals of the Holstein and Charolais breeds. One hundred twenty-two ESTs were mapped physically by using a bovine-hamster somatic cell hybrid panel (SCP) and a 5000-rad bovine whole genome radiation hybrid panel (WGRH). These ESTs were assigned to the bovine syntenic groups and positioned in the recently established RH-based ordered comparative map of the cattle and human genomes. The mapped, differentially expressed sequence tags are a useful prerequisite for cloning of genetic variation underlying economic traits.