Myeloperoxidase expression in diencephalon is potentially associated with fear-related behavior in chicks of laying hen.

Preventing feather pecking (FP) in adult laying hens is important for the welfare of intensively poultry farming. Fear-related behavior in growing female layer chicks may predict FP in adult hens. In this study, in two representative laying breeds (White Leghorn [WL] and Rhode Island Red [RIR]) that have different FP frequencies, we identified a candidate gene associated with fear-related behavior in chicks and FP in adult hens. In the tonic immobility test and open-field test, the behavioral activity was lower in WL chicks than in RIR chicks (P < 0.01), suggesting that WL chicks were more fearful than RIR chicks. Based on previous studies, 51 genes that have been found to be differentially expressed in the brain between high- and low-FP populations were chosen, and their expression levels were screened in the chick diencephalon. This analysis revealed that myeloperoxidase (MPO) gene expression level was higher in WL chicks than that in RIR chicks (P < 0.05). Furthermore, STRING analysis predicted the gene network including MPO and MPO-related genes and revealed the association of these genes with fear-related behavior. These results suggest that MPO is potentially associated with fear-related behavior in growing female layer chicks and FP in adult hens.

Effect of Dietary 4-Phenylbuthyric Acid Supplementation on Acute Heat-Stress-Induced Hyperthermia in Broiler Chickens.

Hot, humid weather causes heat stress (HS) in broiler chickens, which can lead to high mortality. A recent study found that HS causes endoplasmic reticulum (ER) stress. However, the possible involvement of ER stress in HS-induced physiological alterations in broiler chickens is unclear. This study aimed to evaluate the effect of the dietary supplementation of 4-phenylbutyric acid (4-PBA), an alleviator of ER stress, in acute HS-exposed young broiler chickens. Twenty-eight 14-day-old male broiler chickens (ROSS 308) were divided into two groups and fed either a control diet or a diet containing 4-PBA (5.25 g per kg of diet feed) for 10 days. At 24 days old, each group of chickens was kept in thermoneutral (24 ± 0.5 °C) or acute HS (36 ± 0.5 °C) conditions for 2 h. The results showed that thermoneutral birds supplemented with 4-PBA exhibited no negative effects in terms of broiler body weight gain and tissue weight compared to non-supplemental birds. HS increased body temperature in both the control and 4-PBA groups, but the elevation was significantly lower in the 4-PBA group than in the control group. The plasma non-esterified fatty acid concentration was significantly increased by HS treatment in non-supplemental groups, while the increase was partially attenuated in the 4-PBA group. Moreover, 4-PBA prevented HS-induced gene elevation of the ER stress markers GRP78 and GRP94 in the skeletal muscle. These findings suggest that the 4-PBA effect may be specific to the skeletal muscle in HS-exposed birds and that 4-PBA supplementation attenuated HS-induced muscle ER stress, which could be associated with a supplementation of the body temperature elevation and lipolysis.

XBP1u Is Involved in C2C12 Myoblast Differentiation via Accelerated Proteasomal Degradation of Id3.

Myoblast differentiation is an ordered multistep process that includes withdrawal from the cell cycle, elongation, and fusion to form multinucleated myotubes. Id3, a member of the Id family, plays a crucial role in cell cycle exit and differentiation. However, in muscle cells after differentiation induction, the detailed mechanisms that diminish Id3 function and cause the cells to withdraw from the cell cycle are unknown. Induction of myoblast differentiation resulted in decreased expression of Id3 and increased expression of XBP1u, and XBP1u accelerated proteasomal degradation of Id3 in C2C12 cells. The expression levels of the cyclin-dependent kinase inhibitors p21, p27, and p57 were not increased after differentiation induction of XBP1-knockdown C2C12 cells. Moreover, knockdown of Id3 rescued myogenic differentiation of XBP1-knockdown C2C12 cells. Taken together, these findings provide evidence that XBP1u regulates cell cycle exit after myogenic differentiation induction through interactions with Id3. To the best of our knowledge, this is the first report of the involvement of XBP1u in myoblast differentiation. These results indicate that XBP1u may act as a "regulator" of myoblast differentiation under various physiological conditions.

Effect of dipeptide on intestinal peptide transporter 1 gene expression: An evaluation using primary cultured chicken intestinal epithelial cells.

Peptide transporter 1 (PepT1) is a transporter responsible for absorbing dipeptide and tripeptide in enterocytes and is upregulated by dipeptide in mammals. It has not been certain whether intestinal PepT1 expression is responsive to dipeptides in chickens because of the lack of in vitro study using the cultured enterocytes. This study established a primary culture model of chicken intestinal epithelial cells (IECs) in two-dimensional monolayer culture using collagen gel by which the response of chicken PepT1 gene expression to dipeptide stimuli was evaluated. The cultured chicken IECs showed the epithelial-like morphology attached in a patch-manner and exhibited positive expression of cytokeratin and epithelial cadherin, specific marker proteins of epithelial cells. Moreover, the chicken IECs exhibited the gene expression of intestinal cell type-specific marker, villin1, mucin 2, and chromogranin A, suggesting that the cultured IECs were composed of enterocytes as well as goblet and enteroendocrine cells. PepT1 gene expression was significantly upregulated by synthetic dipeptide, glycyl-l-glutamine, in the cultured IECs. From the results, we herein suggested that dipeptide is a factor upregulating PepT1 gene expression in chicken IECs.

Oleuropein suppresses mitochondrial reactive oxygen species generation possibly via an activation of transient receptor potential V1 and sirtuin-1 in cultured chicken muscle cells.

This study investigated the intracellular mechanism governing the effects of oleuropein (OLE), a phenolic compound of Olea europaea, on mRNA expression of avian uncoupling protein (avUCP) and mitochondrial biogenesis-related factors, and reactive oxygen species (mitROS) generation in a primary cultured chicken muscle cells. The OLE-treated cells exhibited increases in Avucp and ATP5a1z expression and a decrease in mitROS generation (p < 0.05), while the effects was canceled by sirtuin-1 (SIRT1) or transient receptor potential vanilloid 1 (TRPV1) inhibitors, EX-527 or BCTC, respectively. Intracellular Ca2+ concentration was significantly increased by OLE, while the induction was canceled by BCTC. The study also found that TRPV1 was expressed in the cell membrane and endoplasmic reticulum (ER), and Ca2+ could be released from ER in the OLE-treated cells. The OLE-treated cells exhibited increases in the phosphorylation ratio of AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) protein content. EX-527 and BCTC inhibitors canceled the effects of OLE on p-AMPK ratio and PGC-1α content, while EX-527 SIRT did not change PGC-1α content. The results suggest that the OLE effects may be due to Ca2+ release, possibly from TRPV1 at ER, and increased p-AMPK ratio, followed by SIRT1 activation and PGC-1α protein expression.

Peg1/Mest, an imprinted gene, is involved in mammary gland maturation.

Imprinted genes, which are specific to mammals, play important roles in cell proliferation, differentiation, ontogeny, and other phenomena. Moreover, these genes are considered crucial in the research of mammalian evolution. In the current study, we investigated the association between the expression of paternally imprinted gene paternally expressed 1/mesoderm-specific transcript (Peg1/Mest) and the maturation of the mammary gland. Quantitative real-time polymerase chain reaction analysis of Peg1/Mest gene expression at different stages of mouse mammary gland maturation revealed that its expression increased during gestation but decreased during lactation. Immunohistochemical staining demonstrated that Peg1/Mest was expressed in mammary epithelial cells. We measured expression levels of Peg1/Mest and E-cadherin during mammary alveoli formation using immunofluorescence staining a cell model for mammary alveoli formation in a 3D culture system. We found that the onset of E-cadherin expression roughly coincided with the peak of Peg1/Mest expression. Moreover, we discovered that the formation and proliferation of alveoli were suppressed in Peg1/Mest knockdown mammary epithelial cells. These results suggest that Peg1/Mest plays a certain role in mammary alveoli formation. To clarify the role of Peg1/Mest in the lactogenic differentiation of mammary epithelial cells, we examined the lactogenic differentiation capability of Peg1/Mest-overexpressing HC11 cells. Application of a differentiation-inducing stimulus did not increase ß-casein expression in Peg1/Mest-overexpressing HC11 cells. The current study for the first time reports the involvement of an imprinted gene in mammary gland maturation.

IRE1-XBP1 Pathway of the Unfolded Protein Response Is Required during Early Differentiation of C2C12 Myoblasts.

In skeletal muscle, myoblast differentiation results in the formation of multinucleated myofibers. Although recent studies have shown that unfolded protein responses (UPRs) play an important role in intracellular remodeling and contribute to skeletal muscle differentiation, the involvement of IRE1-XBP1 signaling, a major UPR signaling pathway, remains unclear. This study aimed to investigate the effect of the IRE1-XBP1 pathway on skeletal muscle differentiation. In C2C12 cells, knockdown of IRE1 and XBP1 in cells remarkably suppressed differentiation. In addition, apoptosis and autophagy were dramatically enhanced in the XBP1-knockdown cells, highlighting the participation of IRE1-XBP1 in cell survival maintenance with differentiation stimuli during skeletal muscle differentiation. In myogenic cells, we demonstrated that the expression of CDK5 (cyclin-dependent kinase 5) is regulated by XBP1s, and we propose that XBP1 regulates the expression of MyoD family genes via the induction of CDK5. In conclusion, this study revealed that IRE1-XBP1 signaling plays critical roles in cell viability and the expression of differentiation-related genes in predifferentiated myoblasts and during the early differentiation phase.

The unfolded protein response is involved in both differentiation and apoptosis of bovine mammary epithelial cells.

The unfolded protein response (UPR) describes a process involved in the homeostasis of endoplasmic reticulum (ER) and the differentiation of secretory cells. At present, the roles of UPR in the mammary gland tissue of dairy cattle are unknown. In the current study, we investigated the expression of UPR-related genes in Holstein cows during the developmental and lactating stages of the mammary gland tissue. To investigate the roles of UPR during the differentiation of mammary epithelial cells (MEC), we used MAC-T cells, a line of MEC. We collected samples of mammary gland tissue in dairy cows by biopsy during the late gestation and lactation periods and examined the expression of UPR-related genes by quantitative real-time PCR. Expression levels of the spliced X-box binding protein 1 (XBP1) and activating transcription factor 4 (ATF4) were found to be significantly higher in the mammary gland tissue 10 d before delivery compared with 40 d before delivery. An investigation before and after differentiation in MAC-T cells showed that the expression of ATF4 increased after differentiation of MEC, whereas that of the spliced XBP1 did not significantly change. Western blot analysis revealed that the differentiation-inducing stimulus induced phosphorylation of eukaryotic initiation factor 2α (eIF2α) but reduced that of protein kinase RNA-like endoplasmic reticulum kinase (PERK). Additionally, in ATF4-knockdown bovine MEC, differentiation was significantly suppressed; ATF4 knockdown also significantly suppressed the expression of glucocorticoid and insulin receptors. These results revealed that ER stress-independent ATF4 is involved in the cell differentiation mechanism, either directly or indirectly, via the control of the expression of lactogenic hormone receptors in bovine MEC. Immediately after parturition, gene expression levels of the spliced XBP1, ATF4, and C/EBP homologous protein (CHOP) markedly increased in mammary gland tissue, with a strong negative correlation between expression of CHOP and initial milk yield; CHOP is an apoptosis-related protein induced by ER stress. The above findings indicate that UPR is intrinsically associated with apoptosis of MEC, thus affecting the differentiation of these cells, as well as milk yield.

The role of unfolded protein response in differentiation of mammary epithelial cells.

The accumulation of misfolded proteins in the ER provokes ER stress by increasing the demand for energy, chaperones, and other proteins that are needed to fold client proteins or to degrade unfoldable secretory cargo. This stress activates a signaling network called the unfolded protein response (UPR). However, recent accumulated data suggested that the UPR also provides important signals for regulating cell differentiation and maturation. However, the relationship between UPR and mammary gland development has not been fully elucidated. To define the involvement of the UPR in mammary gland development, mammary glands were collected from non-pregnant mice, at days 5, 10 and 15 of pregnancy, at days 1 and 7 of lactation, and the expression patterns of UPR-related genes were determined by real-time PCR. We found that the mRNA expression of ATF4 and XBP1 significant increased during pregnancy. Moreover, we found that both ATF4 and XBP1 proteins are expressed in mammary epithelial cells by immunohistological analysis. In order to know the role of ATF4 and XBP1 in the differentiation of mammary epithelial cell, we performed gene knockdown experiment using HC11 cells. We found that ATF4 or XBP1 knockdown suppressed the mRNA expression of beta-casein and lactogenic hormone receptor in differentiating HC11 cells. Our results demonstrate that XBP1 and ATF4, which are UPR-related transcription factors, directly or indirectly participate in cell differentiation mechanisms through the regulation of the expression of lactogenic hormone receptors in mouse mammary epithelial cells.

Subcellular Localization and Polymorphism of Bovine FABP4 in Bovine Intramuscular Adipocytes.

Fatty acid binding protein 4 (FABP4) I74 V, a gene polymorphism associated with unsaturated fatty acid contents, was discovered in Japanese Black cattle. Individuals with FABP4 I/I genotype contain a significantly high level of palmitoleic acid compared to those with FABP4 V/V genotype. It remains unknown how the FABP4 polymorphism leads to different palmitoleic acid contents. We overexpressed FABP4 of different genotypes in bovine intramuscular preadipocytes and examined whether the intracellular localization of FABP4 and the expression levels of lipid metabolism-related genes were different among cells expressing different genotypes. Nuclear localization was observed for the FABP4 V/V, while the FABP4 I/I almost did not. The cells expressing FABP4 of different genotypes were comparable in terms of the expression levels of genes involved in lipid metabolism. FABP4 I/I was localized in most of the lipid droplets 4 days after differentiation induction, whereas approximately 25% lipid droplet co-localized with FABP4 in cells expressing FABP4 V/V. The lipid droplet size increased when palmitoleic acid was added compared to the size observed when palmitic acid was added. These results suggest that lipid droplet enlargement caused by palmitoleic acid and genotype-dependent differences in the fatty acid transporting capacity underlie variations in palmitoleic acid content among FABP4 polymorphisms.

ALS-Linked P56S-VAPB Mutation Impairs the Formation of Multinuclear Myotube in C2C12 Cells.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disorder that affects upper and lower motor neurons. Since motor neurons target skeletal muscles, the maintenance system of muscles is disturbed in ALS; however, the mechanism by which this occurs is unknown. In the present study, we investigated the effects of ALS-associated P56S-vesicle-associated membrane protein-associated protein B (VAPB) (P56S-VAPB) on the IRE1-XBP1 pathway, which is involved in the unfolded protein response (UPR) of the mouse myoblast cell line (C2C12 cells). Experiments with C2C12 cells transfected with wild-type wt-VAPB and P56S-VAPB expression vectors showed reduced myotube formation and aberrant myonuclear position in cells expressing P56S-VAPB. Activity of the IRE1-XBP1 pathway in the cells visualized with the ERAI system revealed that the pathway was disrupted in cells expressing P56S-VAPB, whereas the IRE1-XBP1 pathway activity was enhanced in the differentiation process of normal C2C12 cells. These results suggest that disruption of the IRE1-XBP1 pathway is a cause for the reduced myotube formation in P56S-VAPB-expressing cells. The expression level of the VAPB protein has been reported to be reduced in the neurons of patients with ALS. Therefore, it is expected that the IRE1-XBP1 pathway is also impaired in muscle tissues of patients with ALS, which causes a disturbance in the muscle maintenance system.

ALS-associated P56S-VAPB mutation restrains 3T3-L1 preadipocyte differentiation.

Amyotrophic lateral sclerosis (ALS), which is the most common motor neuron disease in adults, is a neurodegenerative disease that involves the selective and systematic death of upper and lower motor neurons. In addition to the motor neuron death, altered metabolic functions, such as dyslipidemia, have also been reported for ALS patients; however, the underlying mechanism remains unknown. In the present study, we investigated the effects of ALS-associated P56S-vesicle-associated membrane proteinassociated protein B (VAPB), P56S-VAPB on 3T3-L1 preadipocyte differentiation and on the expression of differentiation-associated genes and unfolded protein response (UPR)-related genes. Experiments with 3T3-L1 cells transfected with wild-type (Wt)-VAPB and P56S-VAPB expression vectors showed that the size of lipid droplets was markedly smaller in P56S-VAPB-expressing cells, although fat accumulated intracellularly. In P56S-VAPB-expressing cells, increased the expression of PPARγ2, aP2, and C/EBPα, the genes deeply involved in adipocyte differentiation, was not observed. Furthermore, the expression levels of the UPR-related ATF4 and CHOP genes were found to be enhanced in the P56S-VAPB-expressing cells. From these results, P56S-VAPB was found to suppress adipocyte differentiation by promoting the activation of the ATF4-CHOP pathway. Given previous reports showing increased ATF4 and CHOP expression levels in neurons of ALS patients, results from the present study suggest that dyslipidemia is caused by enhanced ATF4-CHOP pathway in the adipose tissue of ALS patients.

Dexamethasone and acetate modulate cytoplasmic leptin in bovine preadipocytes.

Hormonal and nutrient signals regulate leptin synthesis and secretion. In rodents, leptin is stored in cytosolic pools of adipocytes. However, not much information is available regarding the regulation of intracellular leptin in ruminants. Recently, we demonstrated that leptin mRNA was expressed in bovine intramuscular preadipocyte cells (BIP cells) and that a cytoplasmic leptin pool may be present in preadipocytes. In the present study, we investigated the expression of cytoplasmic leptin protein in BIP cells during differentiation as well as the effects of various factors added to the differentiation medium on its expression in BIP cells. Leptin mRNA expression was observed only at 6 and 8 days after adipogenic induction, whereas the cytoplasmic leptin concentration was the highest on day 0 and decreased gradually thereafter. Cytoplasmic leptin was detected at 6 and 8 days after adipogenic induction, but not at 4 days after adipogenic induction. The cytoplasmic leptin concentration was reduced in BIP cells at 4 days after treatment with dexamethasone, whereas cytoplasmic leptin was not observed at 8 days after treatment. In contrast, acetate significantly enhanced the cytoplasmic leptin concentration in BIP cells at 8 days after treatment, although acetate alone did not induce adipocyte differentiation in BIP cells. These results suggest that dexamethasone and acetate modulate the cytoplasmic leptin concentration in bovine preadipocytes.