Skeletal muscle PGC-1α1 modulates kynurenine metabolism and mediates resilience to stress-induced depression.

Depression is a debilitating condition with a profound impact on quality of life for millions of people worldwide. Physical exercise is used as a treatment strategy for many patients, but the mechanisms that underlie its beneficial effects remain unknown. Here, we describe a mechanism by which skeletal muscle PGC-1α1 induced by exercise training changes kynurenine metabolism and protects from stress-induced depression. Activation of the PGC-1α1-PPARα/δ pathway increases skeletal muscle expression of kynurenine aminotransferases, thus enhancing the conversion of kynurenine into kynurenic acid, a metabolite unable to cross the blood-brain barrier. Reducing plasma kynurenine protects the brain from stress-induced changes associated with depression and renders skeletal muscle-specific PGC-1α1 transgenic mice resistant to depression induced by chronic mild stress or direct kynurenine administration. This study opens therapeutic avenues for the treatment of depression by targeting the PGC-1α1-PPAR axis in skeletal muscle, without the need to cross the blood-brain barrier.

Modeling the transport of nuclear proteins along single skeletal muscle cells.

Skeletal muscle cells contain hundreds of myonuclei within a shared cytoplasm, presenting unique challenges for regulating gene expression. Certain transcriptional programs (e.g., postsynaptic machinery) are segregated to specialized domains, while others (e.g., contractile proteins) do not show spatial confinement. Furthermore, local stimuli, such as denervation, can induce transcriptional responses that are propagated along the muscle cells. Regulated transport of nuclear proteins (e.g., transcription factors) between myonuclei represents a potential mechanism for coordinating gene expression. However, the principles underlying the transport of nuclear proteins within multinucleated cells remain poorly defined. Here we used a mosaic transfection model to create myotubes that contained exactly one myonucleus expressing a fluorescent nuclear reporter and monitored its distribution among all myonuclei. We found that the transport properties of these model nuclear proteins in myotubes depended on molecular weight and nuclear import rate, as well as on myotube width. Interestingly, muscle hypertrophy increased the transport of high molecular weight nuclear proteins, while atrophy restricted the transport of smaller nuclear proteins. We have developed a mathematical model of nuclear protein transport within a myotube that recapitulates the results of our in vitro experiments. To test the relevance to nuclear proteins expressed in skeletal muscle, we studied the transport of two transcription factors-aryl hydrocarbon receptor nuclear translocator and sine oculis homeobox 1-and found that their distributions were similar to the reporter proteins with corresponding molecular weights. Together, these results define a set of variables that can be used to predict the spatial distributions of nuclear proteins within a myotube.

Mitochondrial NDUFA4L2 is a novel regulator of skeletal muscle mass and force.

The hypoxia-inducible nuclear-encoded mitochondrial protein NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4-like 2 (NDUFA4L2) has been demonstrated to decrease oxidative phosphorylation and production of reactive oxygen species in neonatal cardiomyocytes, brain tissue and hypoxic domains of cancer cells. Prolonged local hypoxia can negatively affect skeletal muscle size and tissue oxidative capacity. Although skeletal muscle is a mitochondrial rich, oxygen sensitive tissue, the role of NDUFA4L2 in skeletal muscle has not previously been investigated. Here we ectopically expressed NDUFA4L2 in mouse skeletal muscles using adenovirus-mediated expression and in vivo electroporation. Moreover, femoral artery ligation (FAL) was used as a model of peripheral vascular disease to induce hind limb ischemia and muscle damage. Ectopic NDUFA4L2 expression resulted in reduced mitochondrial respiration and reactive oxygen species followed by lowered AMP, ADP, ATP, and NAD+ levels without affecting the overall protein content of the mitochondrial electron transport chain. Furthermore, ectopically expressed NDUFA4L2 caused a ~20% reduction in muscle mass that resulted in weaker muscles. The loss of muscle mass was associated with increased gene expression of atrogenes MurF1 and Mul1, and apoptotic genes caspase 3 and Bax. Finally, we showed that NDUFA4L2 was induced by FAL and that the Ndufa4l2 mRNA expression correlated with the reduced capacity of the muscle to generate force after the ischemic insult. These results show, for the first time, that mitochondrial NDUFA4L2 is a novel regulator of skeletal muscle mass and force. Specifically, induced NDUFA4L2 reduces mitochondrial activity leading to lower levels of important intramuscular metabolites, including adenine nucleotides and NAD+ , which are hallmarks of mitochondrial dysfunction and hence shows that dysfunctional mitochondrial activity may drive muscle wasting.

Neuronal cell-based high-throughput screen for enhancers of mitochondrial function reveals luteolin as a modulator of mitochondria-endoplasmic reticulum coupling.

BACKGROUND: Mitochondrial dysfunction is a common feature of aging, neurodegeneration, and metabolic diseases. Hence, mitotherapeutics may be valuable disease modifiers for a large number of conditions. In this study, we have set up a large-scale screening platform for mitochondrial-based modulators with promising therapeutic potential. RESULTS: Using differentiated human neuroblastoma cells, we screened 1200 FDA-approved compounds and identified 61 molecules that significantly increased cellular ATP without any cytotoxic effect. Following dose response curve-dependent selection, we identified the flavonoid luteolin as a primary hit. Further validation in neuronal models indicated that luteolin increased mitochondrial respiration in primary neurons, despite not affecting mitochondrial mass, structure, or mitochondria-derived reactive oxygen species. However, we found that luteolin increased contacts between mitochondria and endoplasmic reticulum (ER), contributing to increased mitochondrial calcium (Ca2+) and Ca2+-dependent pyruvate dehydrogenase activity. This signaling pathway likely contributed to the observed effect of luteolin on enhanced mitochondrial complexes I and II activities. Importantly, we observed that increased mitochondrial functions were dependent on the activity of ER Ca2+-releasing channels inositol 1,4,5-trisphosphate receptors (IP3Rs) both in neurons and in isolated synaptosomes. Additionally, luteolin treatment improved mitochondrial and locomotory activities in primary neurons and Caenorhabditis elegans expressing an expanded polyglutamine tract of the huntingtin protein. CONCLUSION: We provide a new screening platform for drug discovery validated in vitro and ex vivo. In addition, we describe a novel mechanism through which luteolin modulates mitochondrial activity in neuronal models with potential therapeutic validity for treatment of a variety of human diseases.

Genome-wide association studies pathway-based meta-analysis for residual feed intake in beef cattle.

Genome-wide association studies (GWASes) have been performed to search for genomic regions associated with residual feed intake (RFI); however inconsistent results have been obtained. A meta-analysis may improve these results by decreasing the false-positive rate. Additionally, pathway analysis is a powerful tool that complements GWASes, as it enables identification of gene sets involved in the same pathway that explain the studied phenotype. Because there are no reports on GWAS pathways-based meta-analyses for RFI in beef cattle, we used several GWAS results to search for significant pathways that may explain the genetic mechanism underlying this trait. We used an efficient permutation hypothesis test that takes into account the linkage disequilibrium patterns between SNPs and the functional feasibility of the identified genes over the whole genome. One significant pathway (valine, leucine and isoleucine degradation) related to RFI was found. The three genes in this pathway-methylcrotonoyl-CoA carboxylase 1 (MCCC1), aldehyde oxidase 1 (AOX1) and propionyl-CoA carboxylase alpha subunit (PCCA)-were found in three different studies. This same pathway was also reported in a transcriptome analysis from two cattle populations divergently selected for high and low RFI. We conclude that a GWAS pathway-based meta-analysis can be an appropriate method to uncover biological insights into RFI by combining useful information from different studies.

Foetal development of skeletal muscle in bovines as a function of maternal nutrition, foetal sex and gestational age.

To determine the effects of maternal nutrition on modifications of foetal development of the skeletal muscle and possible increase in the potential of skeletal muscle growth in cattle, gestating cows were either fed 190% NRC recommendations (overnourished; ON) or 100% NRC recommendation (control; CO). Interaction between maternal nutrition (MN) and the foetal sex (FS) was also investigated. Foetuses were necropsied at four different time points throughout gestation (139, 199, 241 and 268 days of gestation) to assess the mRNA expression of myogenic, adipogenic and fibrogenic markers in skeletal muscle. Phenotypic indicators of the development of skeletal muscle fibres, intramuscular lipogenesis and collagen development were also evaluated. Modifications in mRNA expression of skeletal muscle of foetuses were observed in function of MN and FS despite the lack of effect of MN and FS on foetal weight at necropsy. Maternal ON increased the mRNA expression of the myogenic marker Cadherin-associated protein, beta 1 (CTNNB1) and adipogenic markers Peroxissome proliferator-activated receptor gamma (PPARG) and Zinc finger protein 423 (ZNF423) at midgestation. However, no differences on foetal skeletal muscle development were observed between treatments at late gestation indicating that a compensatory development may have occurred on CO foetuses making the effect of MN on skeletal muscle development not significant at late gestation. Moreover, our data have shown an evidence of sexual dimorphism during foetal stage with a greater skeletal muscle development in male than in female foetuses. In conclusion, providing a higher nutritional level to pregnant cows changes the trajectory of the development of skeletal muscle during midgestation, but apparently does not change the potential of post-natal growth of muscle mass of the offspring, as no differences in skeletal muscle development were observed in late gestation.

Peroxisome Proliferator-activated Receptor γ Coactivator-1 α Isoforms Selectively Regulate Multiple Splicing Events on Target Genes.

Endurance and resistance exercise training induces specific and profound changes in the skeletal muscle transcriptome. Peroxisome proliferator-activated receptor γ coactivator-1 α (PGC-1α) coactivators are not only among the genes differentially induced by distinct training methods, but they also participate in the ensuing signaling cascades that allow skeletal muscle to adapt to each type of exercise. Although endurance training preferentially induces PGC-1α1 expression, resistance exercise activates the expression of PGC-1α2, -α3, and -α4. These three alternative PGC-1α isoforms lack the arginine/serine-rich (RS) and RNA recognition motifs characteristic of PGC-1α1. Discrete functions for PGC-1α1 and -α4 have been described, but the biological role of PGC-1α2 and -α3 remains elusive. Here we show that different PGC-1α variants can affect target gene splicing through diverse mechanisms, including alternative promoter usage. By analyzing the exon structure of the target transcripts for each PGC-1α isoform, we were able to identify a large number of previously unknown PGC-1α2 and -α3 target genes and pathways in skeletal muscle. In particular, PGC-1α2 seems to mediate a decrease in the levels of cholesterol synthesis genes. Our results suggest that the conservation of the N-terminal activation and repression domains (and not the RS/RNA recognition motif) is what determines the gene programs and splicing options modulated by each PGC-1α isoform. By using skeletal muscle-specific transgenic mice for PGC-1α1 and -α4, we could validate, in vivo, splicing events observed in in vitro studies. These results show that alternative PGC-1α variants can affect target gene expression both quantitatively and qualitatively and identify novel biological pathways under the control of this system of coactivators.

Performance strategies affect mammary gland development in prepubertal heifers.

In Brazil, the majority of dairy cattle are Holstein × Gyr (H×G). It is unknown whether excessive energy intake negatively affects their mammary development to the same extent as in purebred Holsteins. We hypothesized that mammary development of H×G heifers can be affected by dietary energy supply. We evaluated the effect of different average daily gains (ADG) achieved by feeding different amounts of a standard diet during the growing period on biometric measurements, development of mammary parenchyma (PAR) and mammary fat pad (MFP), and blood hormones. At the outset of this 84-d experiment, H×G heifers (n = 18) weighed 102.2 ± 3.4 kg and were 3 to 4 mo of age. Heifers were randomly assigned to 1 of 3 ADG programs using a completely randomized design. Treatments were high gain (HG; n = 6), where heifers were fed to gain 1 kg/d; low gain (LG; n = 6), where heifers were fed to gain 0.5 kg/d; and maintenance (MA; n = 6), where heifers were fed to gain a minimal amount of weight per day. Heifers were fed varying amounts of a single TMR to support desired BW gains. Over the 84 d, periodic biometric and blood hormone measurements were obtained. On d 84, all heifers were slaughtered and carcass and mammary samples were collected. At the end, HG heifers weighed the most (181 ± 7.5 kg), followed by LG (146 ± 7.5 kg) and MA (107 ± 7.5 kg) heifers. The ADG were near expected values and averaged 0.907, 0.500, and 0.105 ± 0.03 kg/d for HG, LG, and MA, respectively. In addition, body lengths, heart girths, and withers heights were affected by dietary treatment, with MA heifers generally being the smallest and HG heifers generally being the largest. Body condition scores differed by treatment and were highest in HG and lowest in MA heifers; in vivo subcutaneous fat thickness measurement and direct analysis of carcass composition supported this. The HG heifers had the heaviest MFP, followed by LG and then MA heifers. Amount of PAR was highest in LG heifers and was the same for HG and MA heifers. The percentage of udder mass occupied by PAR was lowest in HG heifers, differing from LG and MA heifers. Composition of MFP was not evaluated. Regarding PAR composition, no differences in ash or DM were found. On the other hand, CP concentration of PAR for HG heifers was lower than that for LG heifers, which was lower than that for MA heifers. Regarding the fat content, HG treatment was higher than LG and MA treatment, which did not differ from each other. In PAR, differences in relative abundance of genes related to both stimulation and inhibition of mammary growth were observed to depend on dietary treatment, sampling day, or both. The same can be said for most of the blood hormones that were measured in this experiment. In this experiment, high ADG achieved by feeding different amounts of a standard diet during the growing period negatively affected mammary development.

Intestinal development of bovine foetuses during gestation is affected by foetal sex and maternal nutrition.

We aimed to evaluate the effects of maternal nutrition (MN) and foetal sex on the intestinal development of bovine foetuses throughout different days of gestation (DG). Forty-four multiparous, dry Holstein × Gyr cows with average initial body weight of 480 ± 10 kg were fed the same diet of either restricted feeding at 1.15% of body weight (CO, n = 24) or fed ad libitum (overnourished, ON, n = 20). Six cows from CO group and five cows from ON group were slaughtered at 139, 199, 241 and 268 DG, and foetuses were necropsied to evaluate the intestinal development. The mass, length and density of foetal intestines were not affected by MN (p ≥ 0.260). An interaction between MN and DG was observed for the villi length of jejunum (p = 0.006) and ileum (p < 0.001). Villi length of jejunum and ileum was higher (p < 0.10) in foetuses from ON-fed cows than in foetuses from CO-fed cows at 139 DG. However, at 199 DG, the villi length of jejunum and ileum of foetuses from CO-fed cows was higher than in foetuses from ON-fed cows. Despite these differences, MN did not affect the villi length of jejunum and ileum at 268 DG (p > 0.10). Female foetuses had greater small intestine mass (p = 0.093), large intestine mass (p = 0.022), small intestine mass in proportion to body mass (p = 0.017) and large intestine mass in proportion to body mass (p < 0.001) than male foetuses. Female foetuses had also longer small intestine (p = 0.077) and greater small intestine density (p = 0.021) and villi length of jejunum (p = 0.001) and ileum (p = 0.010) than males. We conclude that MN affects the pathway for the development of foetal villi length throughout the gestation in bovine foetuses without changing the final villi length. Female foetuses had higher intestinal mass, density and villi length than males during the foetal phase in bovines.

Effects of nutrient intake level on mammary parenchyma growth and gene expression in crossbred (Holstein × Gyr) prepubertal heifers.

This study investigated the effects of increased nutrient intake levels on prepubertal mammary parenchyma development in crossbreed (Holstein × Gyr) dairy heifers. Eighteen heifers age 3 to 4 mo were fed 1 of 3 nutrient intake levels (n=6 per treatment) designed to sustain an average daily gain of 0.0kg/d (maintenance, MA), 0.5kg/d (low gain, LG), or 1.0kg/d (high gain, HG). Serum blood samples collected on d 42 and 84 after a 12-h fast were analyzed for triglycerides, leptin, insulin, and insulin-like growth factor 1 (IGF-1). Liver and mammary parenchyma were biopsied on d 42 and harvested on d 84 for gene expression analysis. Parenchyma samples were also used for biochemical and histological analysis. Mammary parenchyma weight was lower in HG than in MA or LG heifers, but mammary extraparenchymal fat was greater in HG heifers than in other groups. Heifers fed the HG diet had a greater fraction of ether extract in their parenchyma than the others and a smaller fraction of crude protein in their parenchyma than MA heifers. Moreover, the HG and LG heifers had greater body fat mass than MA heifers. Nutrient intake level had no effect on the number of intraparenchymal adipocytes. Heifers fed the HG diet had greater serum IGF-1 than the others, and serum insulin was lower in the MA than the HG or LG heifers. Liver GHR, IGF1, and IGFBP3 mRNA expression was higher, but IGFBP2 mRNA was lower in HG heifers than in others. The parenchyma mRNA expression of lipogenic markers, such as CD36, ACCA, FASN, and ADIPOR1, was upregulated by nutrient intake level. Significant nutrient intake × time interactions for lipogenic genes during the experimental period indicated variable gene expression depending on the time point of prepubertal mammary gland development. Overall, our data suggest that enhancing nutrient intake increased body fat accumulation and lipogenesis in the mammary gland to the detriment of parenchyma growth. Moreover, increased lipogenesis in the parenchyma of HG heifers may indicate that fat accumulation occurred because of adipocyte hypertrophy and not differences in adipogenesis. The implications of these results for milk yield needs to be elucidated.

miR-34a/SIRT1/p53 is suppressed by ursodeoxycholic acid in the rat liver and activated by disease severity in human non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) comprises a spectrum of stages from simple steatosis to non-alcoholic steatohepatitis (NASH). However, disease pathogenesis remains largely unknown. microRNA (miRNA or miR) expression has recently been reported to be altered in human NASH, and modulated by ursodeoxycholic acid (UDCA) in the rat liver. Here, we aimed at evaluating the miR-34a/Sirtuin 1(SIRT1)/p53 pro-apoptotic pathway in human NAFLD, and to elucidate its function and modulation by UDCA in the rat liver and primary rat hepatocytes. METHODS: Liver biopsies were obtained from NAFLD morbid obese patients undergoing bariatric surgery. Rat livers were collected from animals fed a 0.4% UDCA diets. Primary rat hepatocytes were incubated with bile acids or free fatty acids (FFAs) and transfected with a specific miRNA-34a precursor and/or with a p53 overexpression plasmid. p53 transcriptional activity was assessed by ELISA and target reporter constructs. RESULTS: miR-34a, apoptosis and acetylated p53 increased with disease severity, while SIRT1 diminished in the NAFLD liver. UDCA inhibited the miR-34a/SIRT1/p53 pathway in the rat liver in vivo and in primary rat hepatocytes. miR-34a overexpression confirmed its targeting by UDCA, which prevented miR-34a-dependent repression of SIRT1, p53 acetylation, and apoptosis. Augmented apoptosis by FFAs in miR-34a overexpressing cells was also inhibited by UDCA. Finally, p53 overexpression activated miR-34a/SIRT1/p53, which in turn was inhibited by UDCA, via decreased p53 transcriptional activity. CONCLUSIONS: Our results support a link between liver cell apoptosis and miR-34a/SIRT1/p53 signaling, specifically modulated by UDCA, and NAFLD severity. Potential endogenous modulators of NAFLD pathogenesis may ultimately provide new tools for therapeutic intervention.

Nutrient supplementation of beef female calves at pre-weaning enhances the commitment of fibro-adipogenic progenitor cells to preadipocytes.

We aimed to evaluate the impact of nutrient supplementation of beef female calves at pre-weaning on adipogenic determination. Thirty-four female calves were assigned to two experimental treatments: Control (CON, n = 17), where animals were supplemented only with mineral mixture; Supplemented (SUP, n = 17), where animals received energy-protein supplement containing minerals (5 g/kg of BW per day) of their body weight. Animals were supplemented from 100 to 250 days of age, and muscle samples were biopsied at the end of the supplementation period. Regarding the performance variables, there were no differences between treatments for initial body weight (P = 0.75). The final body weight (P = 0.07), average daily gain (P = 0.07), rib eye area (P = 0.03), and rib fat thickness (P = 0.08) were greater in SUP female calves compared with CON treatment. The number of fibro-adipogenic progenitor cells (P = 0.69) did not differ between treatments, while a greater number of intramuscular pre-adipocytes were observed in SUP than CON female calves (P = 0.01). The expression of miRNA-4429 (P = 0.20) did not differ between treatments, while the expression of miRNA-129-5p (P = 0.09) and miRNA-129-2-3p (P = 0.05) was greater in CON than SUP female calves. Our results suggest that nutrient supplementation at early postnatal stages of development enhances the commitment of fibro-adipogenic progenitor cells into the adipogenic lineages allowing to an increase in intramuscular fat deposition potential of the animals later in life.

Dietary nucleotide supplementation as an alternative to in-feed antibiotics in weaned piglets.

Nucleotides are important to cell growth and division and are crucial to the rapid proliferation of such cells as the intestinal mucosa and immune cells. Accordingly, the nucleotide requirements of animals are high during periods of rapid growth and periods of stress like post-weaning period. Thus, nucleotide supplementation may be a possible alternative to in-feed antibiotics as growth promoter in this phase. The study aimed to evaluate dietary nucleotide supplementation as an alternative to in-feed antibiotics on performance and gut health of weaned piglets. Ninety-six 21-day-old piglets, weighing 7.44 ±â€¯0.65 kg, were allocated into 1 of 3 treatments (8 pens per treatment; 4 pigs per pen) in a 14-day trial. Dietary treatments consisted of control: corn-soybean meal-based diet; nucleotides: control +2 g/kg of a nutritional additive with purified nucleotides; and antibiotic: control +0.8 g/kg of antibiotic growth promoter based on colistin and tylosin. Performance variables and fecal score were not affected (P > 0.05) by supplementing nucleotide or antibiotic. Nucleotides treatment had similar effect to antibiotic and superior to control (P < 0.05) on enhancing duodenum villus height, jejunum crypt depth, and reduction of Paneth cellular area. Duodenum and ileum of animals supplemented with nucleotides or antibiotics had higher (P < 0.05) number of proliferating cells than did those of control animals, whereas the jejunum of animals that received antibiotic diets presented more (P < 0.05) proliferating cells than either the nucleotides or control animals. Jejunum of nucleotide-treated piglets showed a greater number of apoptotic cells than those fed antibiotic or control diets (P < 0.05). Nucleotides and antibiotic treatments decreased the B lymphocyte counts in duodenum and ileum (P < 0.05) but increased in the jejunum (P < 0.05), when compared to the control treatment. Relative abundance of mitogen-activated protein kinases-6, haptoglobin, and tumor necrosis factor-α mRNA was not influenced (P > 0.05) by treatments. In the ileal, antibiotic supplementation reduced total bacteria quantification compared to nucleotide supplementation or the control (P < 0.05), whereas nucleotides supplementation increased enterobacteria proliferation compared to the antibiotic or control diets (P < 0.05). However, nucleotides and antibiotic reduced (P < 0.05) colon total bacteria quantification when compared to control. These results suggest that the nucleotides source used to weaned piglets improved gut health by modulating the local immune response and modulating intestinal mucosa development, and, therefore, nucleotides may be an alternative to antibiotics as growth promoters.

Endurance exercise training-responsive miR-19b-3p improves skeletal muscle glucose metabolism.

Skeletal muscle is a highly adaptable tissue and remodels in response to exercise training. Using short RNA sequencing, we determine the miRNA profile of skeletal muscle from healthy male volunteers before and after a 14-day aerobic exercise training regime. Among the exercise training-responsive miRNAs identified, miR-19b-3p was selected for further validation. Overexpression of miR-19b-3p in human skeletal muscle cells increases insulin signaling, glucose uptake, and maximal oxygen consumption, recapitulating the adaptive response to aerobic exercise training. Overexpression of miR-19b-3p in mouse flexor digitorum brevis muscle enhances contraction-induced glucose uptake, indicating that miR-19b-3p exerts control on exercise training-induced adaptations in skeletal muscle. Potential targets of miR-19b-3p that are reduced after aerobic exercise training include KIF13A, MAPK6, RNF11, and VPS37A. Amongst these, RNF11 silencing potentiates glucose uptake in human skeletal muscle cells. Collectively, we identify miR-19b-3p as an aerobic exercise training-induced miRNA that regulates skeletal muscle glucose metabolism.

Effects of Tryptophan Supplementation and Exercise on the Fate of Kynurenine Metabolites in Mice and Humans.

The kynurenine pathway of tryptophan (TRP) degradation (KP) generates metabolites with effects on metabolism, immunity, and mental health. Endurance exercise training can change KP metabolites by changing the levels of KP enzymes in skeletal muscle. This leads to a metabolite pattern that favors energy expenditure and an anti-inflammatory immune cell profile and reduces neurotoxic metabolites. Here, we aimed to understand if TRP supplementation in untrained vs. trained subjects affects KP metabolite levels and biological effects. Our data show that chronic TRP supplementation in mice increases all KP metabolites in circulation, and that exercise reduces the neurotoxic branch of the pathway. However, in addition to increasing wheel running, we did not observe other effects of TRP supplementation on training adaptations, energy metabolism or behavior in mice. A similar increase in KP metabolites was seen in trained vs. untrained human volunteers that took a TRP drink while performing a bout of aerobic exercise. With this acute TRP administration, TRP and KYN were higher in the trained vs. the untrained group. Considering the many biological effects of the KP, which can lead to beneficial or deleterious effects to health, our data encourage future studies of the crosstalk between TRP supplementation and physical exercise.

Identification of microRNAs during rat liver regeneration after partial hepatectomy and modulation by ursodeoxycholic acid.

New gene regulation study tools such as microRNA (miRNA or miR) analysis may provide unique insights into the remarkable ability of the liver to regenerate. In addition, we have previously shown that ursodeoxycholic acid (UDCA) modulates mRNA levels during liver regeneration. Bile acids are also homeotrophic sensors of functional hepatic capacity. The present study was designed to determine whether miRNAs are modulated in rats following 70% partial hepatectomy (PH) and elucidate the role of UDCA in regulating miRNA expression during liver regeneration (LR). Total RNA was isolated from livers harvested at 3-72 h following 70% PH or sham operations, from both 0.4% (wt/wt) UDCA and control diet-fed animals. By using a custom microarray platform we found that several miRNAs are significantly altered after PH by >1.5-fold, including some previously described as modulators of cell proliferation, differentiation, and death. In particular, expression of miR-21 was increased after PH. Functional modulation of miR-21 in primary rat hepatocytes increased cell proliferation and viability. Importantly, UDCA was a strong inducer of miR-21 both during LR and in cultured HepG2 cells. In fact, UDCA feeding appeared to induce a sustained increase of proliferative miRNAs observed at early time points after PH. In conclusion, miRNAs, in particular miR-21, may play a significant role in modulating proliferation and cell cycle progression genes after PH. miR-21 is additionally induced by UDCA in both regenerating rat liver and in vitro, which may represent a new mechanism behind UDCA biological functions.

Impacts of protein supplementation during late gestation of beef cows on maternal skeletal muscle and liver tissues metabolism.

Since nutritional requirements are increased at the end of gestation to meet the demands of the pregnant uterus, pregnant beef cows are susceptible to mobilization of body reserves (mainly fat and amino acids (AAs)) and to alter the metabolism of nutrients in the liver and muscle to support such demands. The objective of this study was to evaluate the effect of CP supplementation on maternal nutrient metabolism in the late gestation of beef cows grazing a low-quality pasture. Forty-three pregnant Nellore cows gestating male fetuses (average age = 6 years; average weight = 544 kg) at 193 ± 30 (mean ± SD) days (d) of gestation were divided into eight groups (experimental units, with four to five cows each). Treatments were (1) control (CON, n = 4): pasture-based (PB) diet without CP supplementation and (2) supplemented (SUP, n = 4): PB diet daily supplemented with 2 g/kg of BW of a 43.5% CP supplement. Liver and skeletal muscle biopsies were performed at 265 days of gestation and samples were collected for mRNA expression. On day 280 of gestation, blood samples were collected to assess plasma levels of AA. The CON-fed cows tended to have greater (P = 0.057) total circulating AA than SUP-fed cows. The circulating glycogenic AA was greater (P = 0.035) in CON than in SUP cows. CON cows was greater for histidine (P = 0.015), methionine (P = 0.007) and alanine (P = 0.036) than SUP cows. The CON- and SUP-fed showed no differences for gluconeogenesis, fatty acid transport and signaling axis markers in the liver. The mRNA expression of markers for skeletal muscle synthesis, p7056k (P = 0.060) and GSK3B (P = 0.096), tended to be greater in cows from CON than SUP group. No differences were found for mRNA expression of markers for skeletal muscle degradation. We conclude that CP supplementation to CP-restricted late-pregnant beef cows reduces the maternal tissue mobilization and changes the profile of plasma circulating AA and the mRNA expression of markers for the synthesis of skeletal muscle tissue.

Effect of ractopamine and conjugated linoleic acid on performance of late finishing pigs.

The dietary inclusion of feed additives to improve the carcass characteristics of the final product is of great importance for the pork production chain. The aim of our study was to evaluate the effects of the association of ractopamine (RAC) and conjugated linoleic acid (CLA) on the performance traits of finishing pigs during the last 26 days prior to slaughter. In total, 810 commercial hybrid barrows were used. Animals were distributed among treatments according to a randomised block design in a 3 × 3 factorial arrangement, with three RAC levels (0, 5 or 10 ppm) and three CLA levels (0, 0.3 or 0.6%). Pigs fed the diet with 5 ppm RAC had higher average daily feed intake (ADFI) (2.83 kg; P < 0.05) when compared with those fed 10 ppm RAC and the control diet (2.75 and 2.74 kg, respectively). Lower ADFI values (P < 0.01) were observed with the diets containing CLA compared with the control diet with no CLA (2.73 and 2.75 v. 2.85 kg/day, respectively). The average daily weight gain of pigs fed 5 and 10 ppm RAC was +148 and +173 g/dayhigher (P < 0.001), respectively, than those fed the control diet. Dietary RAC levels influenced (P < 0.001) feed conversion ratio (FCR), which was reduced as RAC levels increased, with the pigs fed 10, 5 and 0 ppm RAC presenting FCR values of 2.57, 2.71 and 3.05, respectively. FCR also improved (P < 0.05) with the inclusion of 0.6% CLA relative to the control diet (2.70 v. 2.84, respectively). There was a significant interaction between CLA × RAC levels (P < 0.01) for final BW, loin eye area (LEA) (P < 0.05) and backfat thickness (BT) (P < 0.05). The treatments containing 10 ppm RAC + 0.6% or 0.3% CLA increased LEA and reduced BT. In conclusion, the level of 10 ppm inclusion of RAC increased the overall performance parameters of pigs and therefore improved production efficiency. The combined use of RAC and CLA promoted a lower feed conversion ratio as well as better quantitative carcass traits, as demonstrated by the higher LEA and lower BT. The dietary inclusion of CLA at 0.3% improved feed efficiency, however, without affecting LEA or BT yields.

Dietary L-arginine supplementation increased mammary gland vascularity of lactating sows.

The present study aimed to evaluate the mechanisms modulated by dietary arginine supplementation to sows during lactation regarding antioxidant capacity and vascularization of mammary glands. At 109 days of gestation, animals were transferred to individual farrowing crates equipped with manual feeders and automatic drinker bowls. Environmental temperature and humidity inside the farrowing rooms were registered every 15 min. At farrowing, sows were assigned in a completely randomized design to a control diet (CON) or the CON diet supplemented with 1.0% L-arginine (ARG). A total of three gilts and two sows were fed the CON diet, whereas three gilts and three sows were fed ARG diets. Sows were fed a fixed amount of 6.0 kg/day, subdivided equally in four delivery times (0700, 1000, 1300 and 1600 h) for 21 days. At weaning, sows were slaughtered and mammary tissue samples and blood from the pudendal vein were collected. Data were analyzed considering each sow as an experimental unit. Differences were considered at P<0.05. L-arginine fed sows presented lower messenger RNA (mRNA) expression for prolactin receptor (P=0.002), angiopoietin1 (P=0.03) and receptor tyrosine kinase (P=0.01); higher mRNA expression for prostaglandin synthase 1 (P=0.01); a trend of decrease for glucocorticoid receptor (P=0.06) and IGF receptor 1 (P=0.07); and a trend (P=0.05) for an increased glutathione peroxidase mRNA expression. The angiopoietin2:angiopoietin1 mRNA ratio tended to increase (P=0.07) in ARG fed sows. L-arginine fed sows had greater (P=0.04) volumetric proportion of blood vessels and a trend of enhance (P=0.07) in the number of blood vessels per mm2. These findings show that 1.0% ARG supplementation to sows activates proliferative mechanisms, may improve mammary tissues' angiogenesis and tended to increase mRNA expression of genes that encode antioxidant enzymes in mammary gland of sows.

Skeletal muscle PGC-1α1 reroutes kynurenine metabolism to increase energy efficiency and fatigue-resistance.

The coactivator PGC-1α1 is activated by exercise training in skeletal muscle and promotes fatigue-resistance. In exercised muscle, PGC-1α1 enhances the expression of kynurenine aminotransferases (Kats), which convert kynurenine into kynurenic acid. This reduces kynurenine-associated neurotoxicity and generates glutamate as a byproduct. Here, we show that PGC-1α1 elevates aspartate and glutamate levels and increases the expression of glycolysis and malate-aspartate shuttle (MAS) genes. These interconnected processes improve energy utilization and transfer fuel-derived electrons to mitochondrial respiration. This PGC-1α1-dependent mechanism allows trained muscle to use kynurenine metabolism to increase the bioenergetic efficiency of glucose oxidation. Kat inhibition with carbidopa impairs aspartate biosynthesis, mitochondrial respiration, and reduces exercise performance and muscle force in mice. Our findings show that PGC-1α1 activates the MAS in skeletal muscle, supported by kynurenine catabolism, as part of the adaptations to endurance exercise. This crosstalk between kynurenine metabolism and the MAS may have important physiological and clinical implications.