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Mild heat stimulation is defined as a stimulation that occurs several degrees above optimal cell culture or body temperatures. Muscle hypertrophy in C2C12 cells is reportedly facilitated by 39 °C mild heat stimulation. However, the underlying molecular mechanisms by which 39 °C mild heat stimulation promotes muscle hypertrophy remains elusive. In this study, we aimed at understanding the details of these mechanisms. First, we found that 39 °C mild heat stimulation has little or no effect on Myogenesis-Related Factor (MRF) expression both in C2C12 and mouse primary satellite cells. Therefore, we hypothesized that 39 °C mild heat stimulation promotes muscle hypertrophy through myokines, essential components in myogenesis. Interestingly, we observed muscle hypertrophy in the group cultured at 37 °C in conditioned medium from 39 °C compared to the group cultured at 37 °C in conditioned medium from 37 °C in the case of both C2C12 and mouse primary satellite cells. These results suggest that 39 °C mild heat stimulation promotes muscle hypertrophy through myokines, once released in the culture medium. Finally, we identified Decorin as a hypertrophy-inducing myokine candidate. Therefore, in this study, we demonstrated that 39 °C mild heat stimulation contributes to muscle hypertrophy through enhancing Decorin gene expression in C2C12 and mouse primary satellite cells.

Objective: Cells have increased susceptibility to activation of apoptosis when suffering heat stress (HS). An effective supplementation strategy to mimic heat-induced apoptosis of bovine mammary epithelial cells (MECs) is necessary to maintain optimal milk production. This study aimed to investigate possible protective effects of the anti-apoptotic activity of 5-aminolevulinic acid (5-ALA) against HS-induced damage of bovine MECs.Methods: Bovine MECs were pretreated with or without 5-ALA at concentrations of 10, 100, and 500 μM for 24 h followed by HS (42.5°C for 24 h and 48 h). Cell viability was measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Real-time quantitative polymerase chain reaction and Western blotting were used to explore the regulation of genes associated with apoptosis, oxidative stress, and endoplasmic reticulum (ER) stress genes.Results: We found that 5-ALA induces cytoprotection via inhibition of apoptosis markers after HS-induced damage. Pretreatment of bovine MECs with 5-ALA resulted in dramatic upregulation of mRNA for nuclear factor erythroid-derived 2-like factor 2, heme oxygenase-1, and NAD(P)H quinone oxidoreductase 1, all of which are antioxidant stress genes. Moreover, 5-ALA pretreatment significantly suppressed HS-induced ER stress-associated markers, glucose-regulated protein 78, and C/EBP homologous protein expression levels.Conclusion: 5-ALA can ameliorate the ER stress in heat stressed bovine MEC via enhancing the expression of antioxidant gene.

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.

The aim of this study was to determine the effect of thermal stimulation at 39°C on the fusion and elongation of skeletal muscle cells. During a 5 day differentiation process of C2C12 cells, nine groups subjected to varying lengths of thermal stimulation at 39°C were established. Afterward, all groups were immunostained using anti-muscle heavy-chain antibody to test for myotube formation. Quantification of the myotube area demonstrated a significant increase in the group subjected to thermal stimulation at 39°C during the latter half of the differentiation compared with the control group, but the fusion index was significantly higher in the group that received hyperthermic treatment during the first half of the differentiation period. Moreover, the longitudinal length of myotubes was significantly increased in the groups that were subjected to thermal stimulation at 39°C during the latter half of the differentiation period. The distance between the center of myotubes and the nucleus farthest away from the center was substantially extended in the group receiving thermal stimulation at 39°C only on the fourth day of the differentiation. Together, these results demonstrate that thermal stimulation at 39°C facilitates myoblast fusion and elongation.

[BACKGROUND/OBJECTIVES] Alzheimer's disease is a neurodegenerative disease that induces symptoms such as a decrease in motor function and cognitive impairment. Increases in the aggregation and deposition of amyloid beta protein (Aβ) in the brain may be closely correlated with the development of Alzheimer's disease. In this study, the effects of an adzuki bean extract on the aggregation of Aβ were examined; moreover, the anti-Alzheimer's activity of the adzuki extract was examined. [MATERIALS/METHODS] First, we undertook thioflavin T (ThT) fluorescence analysis and transmission electron microscopy (TEM) to evaluate the effect of an adzuki bean extract on Aβ42 aggregation. To evaluate the effects of the adzuki extract on the symptoms of Alzheimer's disease in vivo, Aβ42-overexpressing Drosophila were used. In these flies, overexpression of Aβ42 induced the formation of Aβ42 aggregates in the brain, decreased motor function, and resulted in cognitive impairment. [RESULTS] Based on the results obtained by ThT fluorescence assays and TEM, the adzuki bean extract inhibited the formation of Aβ42 aggregates in a concentration-dependent manner. When Aβ42-overexpressing flies were fed regular medium containing adzuki extract, the Aβ42 level in the brain was significantly lower than that in the group fed regular medium only. Furthermore, suppression of the decrease in motor function, suppression of cognitive impairment, and improvement in lifespan were observed in Aβ42-overexpressing flies fed regular medium with adzuki extract. [CONCLUSIONS] The results reveal the delaying effects of an adzuki bean extract on the progression of Alzheimer's disease and provide useful information for identifying novel prevention treatments for Alzheimer's disease.

Insulin-like growth factor-1 (IGF-1) plays a key role in proliferation and galactopoiesis in mammary epithelial cells (MEC), but its definitive functions on endoplasmic reticulum (ER) during protein synthesis remain unknown. The present study aimed to elucidate the effects of IGF-1 on ER biogenesis in MEC in vitro and examined the expression of ER biogenesis-associated genes in the mammary gland during early lactation. We treated mammary alveolar cells–large T antigen cells (immortalized bovine MEC line established via stable transfection with simian virus-40 large T-antigen) with IGF-1 and examined ER biogenesis using the fluorescence intensity of an ER tracker and quantitative real-time PCR. We found IGF-1 significantly increased ER tracker staining and upregulated mRNA levels of ER biogenesis-related genes, such as CHKA (choline kinase α), PCYT1A (choline-phosphate cytidylyltransferase A), and SURF4 (surfeit locus protein 4). We focused on unfolded protein response to explore molecular mechanisms by which IGF-1 induces ER biogenesis. We found IGF-1 significantly increased mRNA levels of the XBP1 splicing form (XBP1s). Based on western blot analysis, IGF-1 induced the expression of (inositol-requiring kinase 1 α) protein, upstream of XBP1s, and phosphorylated-IRE1α. The inhibition of IRE1 endoribonuclease activity with 4-methylumbelliferone 8-carbaldehyde (4μ8C) significantly suppressed the increase in ER tracker fluorescence and ER biogenesis-related gene expression induced by IGF-1. Also, IGF-1–induced XBP1s and ER biogenesis-associated gene expression was inhibited by rapamycin, an inhibitor of mTORC1 (mammalian target of rapamycin complex 1), indicating that IRE1-XBP1 activation by IGF-1 is mediated by mTORC1. Moreover, to clarify the expression of XBP1s and ER biogenesis-associated genes expression under normal physiological conditions, mammary gland tissue from biopsies of dairy cows during late gestation and lactation were analyzed. In vivo data highlighted the significant increases in the mRNA levels of XBP1s and ER biogenesis-related genes in mammary gland tissue immediately after calving through 6 wk of lactation. The mRNA levels of IGF1R (IGF-1 receptor) in mammary glands increased during 6 wk of lactation. Therefore, the present study indicated for the first time that IGF-1 induces ER biogenesis by activating the IRE1-XBP1 axis under the regulation of mTORC1 in bovine MEC line.

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 β-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.

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 present study aimed to investigate the mechanism underlying the effect of adzuki bean extract on the differentiation of 3T3-L1 adipocytes. Treatment of these adipocytes with the extract significantly suppressed the expressions of the Pparγ, C/ebpα, and Fabp4 genes that are involved in adipogenesis (adipocyte differentiation). Oil Red O staining revealed that fat accumulation within these adipocytes is reduced significantly when the cells are treated with the extract. To explore the impact of adzuki bean extract on the differentiation of 3T3-L1 adipocytes, we investigated the effect of adzuki bean extract on the expression of cycle-related factors. We observed reduced expression of cyclin D1 mRNA and phosphorylated Rb (pRb), a downstream factor of cyclin D1 at 24 h after the application of a differentiation-inducing stimulus. Meanwhile, at 72 h after the stimulation, the expression of pRb increased with an increase in the amount of adzuki bean extract. The proliferation of cells declined significantly on treatment with the extract at 16 h after the stimulation but accelerated significantly at 72 h after the stimulation. These results suggest that adzuki bean extract contains an active ingredient that suppresses the differentiation of adipocytes. As a possible mechanism, the extract may alter the mitotic clonal expansion (MCE) which is essential for adipogenesis.

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.

Fatty acids play important roles in the regulation of endoplasmic reticulum (ER) stress-induced apoptosis in different cells. Currently, the effects of fatty acids on bovine mammary epithelial cells (MEC) remain unknown. Our study examined bovine MEC viability and measured unfolded protein response (UPR)-related gene and protein expressions following fatty acid treatments. To evaluate the role of fatty acids, we treated MAC-T cells (a line of MEC) with 100 to 400 μM of saturated (palmitic and stearic acid) and unsaturated (palmitoleic, oleic, linoleic, and linolenic acid) fatty acids and 1 to 5 mM of short- and medium-chain fatty acids (acetic, propionic, butyric, and octanoic acid). Thereafter, we determined UPR-related gene expression using quantitative real-time PCR. Palmitic acid stimulated expression of XBP1s, ATF4, ATF6A, and C/EBP homologous protein (CHOP). Stearic acid increased expression of XBP1s and CHOP and decreased expression of ATF4 and ATF6A. Results of Western blot analysis and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that palmitic and stearic acid reduced MAC-T cell viability and induced extreme ER stress by increasing the protein expression of ER stress markers, such as phospho-PKR-like endoplasmic reticulum kinase, phospho-eIF2α, cleaved CASP-3, and CHOP. Among unsaturated long-chain fatty acids, palmitoleic acid increased expression of ATF4 and ATF6A. Oleic acid increased expression of XBP1s, ATF4, and ATF6A. Linoleic and linolenic acids increased expression of XBP1s, ATF4, and ATF6A but decreased expression of XBP1s and ATF6A at the highest dose. Although palmitoleic, oleic, and linoleic acid decreased CHOP expression, only palmitoleic acid increased MAC-T cell viability. Therefore, unsaturated long-chain fatty acids did not induce severe ER stress. Acetic, propionic, and butyric acids decreased expression of ATF4, ATF6A, and CHOP and increased XBP1s expression. Although only octanoic acid increased ATF4 and ATF6A expressions, it lowered expression of XBP1s and CHOP. Although fatty acid treatment did not increase the levels of ER stress proteins, butyric and octanoic acids reduced cell viability, possibly because of early differentiation. These results suggest that saturated fatty acids play important roles in MEC viability by inducing severe ER stress compared with unsaturated fatty acids. In addition, acetic and propionic acids (short- and medium-chain fatty acids) reduced ER stress. Therefore, the present study reflects the new insight that serum fatty acid concentration plays an important role in maintaining the lactation physiology of dairy cows.

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&ndash, XBP1 signaling, a major UPR signaling pathway, remains unclear. This study aimed to investigate the effect of the IRE1&ndash, 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&ndash, 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&ndash, 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 conservation of mammary gland physiology by maintaining the maximum number of mammary epithelial cells (MECs) is of the utmost importance for the optimum amount of milk production. In a state of negative energy balance, palmitic acid (PA) reduces the number of bovine MECs. However, there is no effective strategy against PA-induced apoptosis of MECs. In the present study, 5-aminolevulinic acid (5-ALA) was established as a remedial agent against PA-induced apoptosis of MAC-T cells (an established line of bovine MECs). In PA-treated cells, the apoptosis-related genes BCL2 and BAX were down- and upregulated, respectively. The elevated expression of major genes of the unfolded protein response (UPR), such as CHOP, a proapoptotic marker (C/EBP homologous protein), reduced the viability of PA-treated MAC-T cells. In contrast, 5-ALA pretreatment increased and decreased BCL2 and BAX expression, respectively. Moreover, cleaved caspase-3 protein expression was significantly reduced in the 5-ALA-pretreated group in comparison with the PA group. The downregulation of major UPR-related genes, including CHOP, extended the viability of MAC-T cells pretreated with 5-ALA and also reduced the enhanced intensity of the PA-induced expression of phospho-protein kinase R-like ER kinase. Moreover, the enhanced expression of HO-1 (antioxidant gene heme oxygenase) by 5-ALA reduced PA-induced oxidative stress (OxS). HO-1 is not only protective against OxS but also effective against ER stress. Collectively, these findings offer new insights into the protective effects of 5-ALA against PA-induced apoptosis of bovine MECs.

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.

Mild heat stress (39 &deg, C&ndash, 40 &deg, C) can positively regulate cell proliferation and differentiation. Indeed, mild heat treatment at 39 &deg, C enhances the less-permeable tight junctions (TJs) formation and milk production in mammary epithelial cells. However, the molecular mechanisms of this response have not yet been delineated. In this study, the involvement of temperature-sensitive transient receptor potential vanilloid 4 (TRPV4) in the increase of &beta, casein and TJ protein-encoding gene expression in response to mild heat treatment (39 &deg, C) has been explored using HCll mouse mammary epithelial cells. Severe heat treatment (41 &deg, C) induced the transcriptional level of Chop (C/EBP homologous protein, proapoptotic marker) and reduced the cell viability. It is speculated that the difference in unfolded protein response (UPR) gene expression upon stimulation at 39 &deg, C vs. 41 &deg, C controls cell survival vs. cell death. The accumulation of Trpv4 mRNA was significantly higher in 39 &deg, C heat treatment cells. The &beta, casein, Zo-1 (zona occludens-1), Ocln (occludin), and Cldn3 (claudin 3) transcript levels were significantly increased in response to the addition of a selective TRPV4 channel agonist (GSK1016790A) at 37 &deg, C. TRPV4 stimulation with GSK1016790A also increased the X-box-binding protein 1 splicing form (Xbp1s) at the transcript level. The increase in the mRNA levels of &beta, casein, Zo-1, Ocln, and Cldn3 in response to 39 &deg, C heat treatment was suppressed by XBP1 knockdown. Moreover, the transcript level of Trpv4 was significantly increased at Day 15 of gestation, and its expression declined after 1 day of lactation. TRPV4 is activated not only by temperature but also by mechanical forces, such as cell stretching and shear stress, which guide mammary epithelial development in a normal mammary gland. These findings provide new insights of the possible function of TRPV4 in mammary gland development.

It has been reported that the expression of β-casein, a representative milk protein, increases when mammary epithelial cells are exposed to mild heat stress at 39°C. However, the direct effects and detailed molecular mechanisms have not yet been elucidated. In this study, we investigated the relationship between an increase in β-casein expression and the unfolded protein response (UPR) under mild heat stress. After reaching confluence, HC11 cells were incubated at 37°C (control) or 39°C (mild heat stress) without differentiation medium, and the expression levels of β-casein and UPR-related genes were assessed. It was revealed that, even with this mild heat treatment (39°C), β-casein expression in HC11 cells increased at the transcriptional level without differentiation induction. The expression levels of X-box binding protein 1 (XBP1) and activating transcription factor 6 alpha (ATF6α) were significantly higher in cells cultured at 39°C compared to those cultured at 37°C. Moreover, the increase in β-casein mRNA expression levels by mild heat treatment was suppressed in XBP1 or ATF6α knockdown cells generated by siRNA for XBP1 or ATF6α respectively. Thus, these results demonstrate that ATF6α and XBP1 is involved in the increase of β-casein expression following mild heat treatment.

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.

Advance online publication 6 January 2015, Here, we report a simple and low-cost oral oligodeoxynucleotide (ODN) delivery system targeted to the gut Peyer's patches (PPs). This system requires only Dulbecco's modified eagle's medium, calcium chloride, ODNs, and basic laboratory equipment. ODN nanocapsules (ODNcaps) were directly delivered to the PPs through oral administration and were taken up by macrophages in the PPs, where they induced an immune response. Long-term continuous oral dosing with inhibitory/suppressive ODNcaps (iODNcaps, "iSG3caps" in this study) was evaluated using an atopic dermatitis mouse model to visually monitor disease course. Administration of iSG3caps improved skin lesions and decreased epidermal thickness. Underlying this effect is the ability of iSG3 to bind to and prevent phosphorylation of signal transducer and activator of transcription 6, thereby blocking the interleukin-4 signaling cascade mediated by binding of allergens to type 2 helper T cells. The results of our iSG3cap oral delivery experiments suggest that iSG3 may be useful for treating allergic diseases., Article, MOLECULAR THERAPY. 23(2):297-309 (2015)

Milk synthesis is known to be modulated by peptide hormones such as prolactin (PRL), growth hormone (GH), and insulin-like growth factor I (IGF-I). Previous studies suggested that PRL and IGF-I acted directly on mammary epithelial cells and were involved in lactation. Meanwhile, GH is thought to be indirectly involved in lactation by stimulating the secretion of IGF-I. It is controversial as growth hormone receptors (GHR) is expressed in the mammary epithelial cells. In order to clarify whether GH acted directly on mammary gland tissue, we investigated the prolactin receptors (PRLR), IGF-I receptors (IGF-IR), and GHR as well as the gene expression levels of the downstream signaling molecule for each receptor in the mammary gland tissue of Holstein cows during different stages of lactation. The results revealed that the mRNA expressions of PRLR and IGF-IR were highest during early lactation, and the mRNA expression of the GHR was highest during mid-lactation. We also found that the expression profiling of the signal transducer and activator of transcription 5 (STAT5) genes was similar to that of the GHR gene. On the other hand, the expression profiling of the PRLR gene was similar to that of the SHP2 gene. These results suggest that GH acts on the mammary glands directly, milk synthesis and secretion are chiefly stimulated in mid-lactation, and the timing of the action is different for PRL and IGF-I.

Previous studies demonstrated that single nucleotide polymorphisms (SNPs) in exon 5 of the bovine growth hormone (GH) gene affected live weight and carcass traits in Japanese Black cattle. The aims of the present study were to identify SNPs in the promoter region of the GH gene and to analyze their effects on carcass traits and intramuscular (i.m.) fatty acid compositions in Japanese Black cattle (511 steers and 252 heifers; 30.5±0.1 months old). SNPs in the promoter region of the GH gene were detected at g.253, g.303, and g.502 and classified into 4 haplotypes, i.e. haplotype P (T-C-C), Q (C-C-C), R (C-C-T), and S (T-T-C). Among genotypes at g.253, genotype TT had the highest carcass weight (CW), rib thickness (RT), and C14:0 and C16:1 contents, but the lowest C18:0 content. The rib eye area (REA) in genotype TT was higher compared with that in genotype CC. Genotype CC had the lowest C16:0 content and higher C18:1 and MUFA contents compared with those in genotype TT. Among genotypes at g.303, genotype CC had the highest C14:0, C16:0, and C16:1 contents, but the lowest C18:1 content. Genotype TT had the lowest CW and RT, but the highest C18:0 content. At both locations, the heterozygous animals showed intermediate values for most traits. Regarding GH promoter diplotypes, diplotype PP had higher CW, C16:0, and C16:1 than diplotype SS and tended to have the lowest C18:0 compared with diplotype QQ and SS. In conclusion, SNPs at g.253 and g.303 in the GH promoter region affects carcass traits in Japanese Black cattle.

Glucagon-like peptide-1 (GLP-1) released from intestinal L cells in response to nutrient ingestion inhibits both gastrointestinal emptying and gastric acid secretion and promotes satiety. The main biological effect of GLP-1 is the stimulation of insulin secretion (thereby fulfilling the criterion for an incretin hormone) in order to reduce blood glucose levels in mammalian species. Chicken GLP-1 receptor (cGLP-1R) has also been identified in various tissues by gene expression analysis. Although certain effects of GLP-1 in mammals and birds are consistent, e.g., inhibition of food intake, whether GLP-1 has the same insulinotropic activity in chickens as in mammals is debated. Moreover, the expression of cGLP-1R in chicken pancreatic B cells has not been reported. The localization of cGLP-1R and its mRNA in pancreatic islets is studied by triple-immunofluorescence microscopy and in situ hybridization. Triple-immunofluorescence microscopy with antisera against cGLP-1R, somatostatin and insulin or glucagon revealed that cGLP-1R protein was exclusively localized in D cells producing somatostatin in chicken pancreatic islets. The D cells were localized in peripheral areas of the pancreatic islets and cGLP-1R mRNA was detected in the same areas, indicating that cGLP-1R mRNA was also expressed in D cells. This is the first report to demonstrate that cGLP-1R is expressed by D cells, not B cells as in mammals. Our study suggests that chicken GLP-1 performs its insulinotropic activity by a different mode of action from that of the mammalian hormone.

Influences of a specific dietary nutrient on glucagon-like peptide (GLP)-1-containing cells in the chicken intestine are not yet clear. Significance of dietary protein level on GLP-1-containing cells in the chicken ileum was investigated. Chickens fed control or experimental diets of varying protein levels were examined using immunohistochemical and morphometrical techniques. We show that the protein ingestion had an impact on the activities of GLP-1-immunoreactive cells in the chicken ileum. Weight gains declined with decreasing dietary crude protein (CP) levels, but no significant differences were detected in the daily feed intake and villous height. GLP-1-immunoreactive cells with a round or oval shape were frequently observed in the lower CP level groups (4.5% and 0%). Frequencies of occurrence of GLP-1-immunoreactive cells were 41.1 ± 4.1, 38.5 ± 4, 34.8 ± 3.1 and 34.3 ± 3.7 (cells/mm(2) , mean ± SD) for dietary CP level of 18%, 9%, 4.5% and 0% groups, respectively and significant differences were recognized between the control and lower CP level groups (P

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.

Proteins from buckwheat (Fagopyrum esculentum) are strong allergens that can cause serious symptoms, including anaphylaxis, in patients with hypersensitivity. In this study, we successfully developed a modified lactic acid bacterial vector (pNSH) and a recombinant strain of Lactococcus lactis NZ9000 (NZ9000) that produced a major allergenic storage protein of buckwheat, Fagag1 (61.2 kDa, GenBank accession number AF152003), with or without a green fluorescent protein (GFP) tag. GFP fluorescence allows for rapid, simple, and accurate measurement of target protein expression by microscopy or fluorimetry. We describe a convenient method for production of rGFP-Fagag1 fusion and rFagag1 proteins with a good yield in an advantageous probiotic host. We found that in vitro treatment of splenocytes isolated from buckwheat crude protein-immunized mice with rFagag1 increased the expression of allergic inflammation cytokines such as IL-4, IL-13, and IL-17 F. Because it was less antigenic, rGFP-Fagag1 protein from NZ9000 might be of limited use; however, rFagag1 from NZ9000 evoked a robust response as measured by induction of IL-4 and IL-17 F expression levels. The observed allergic activity is indicative of a Th2 cell-mediated immune response and is similar to the effects induced by exposure to buckwheat crude protein. Our results suggest that expression of rFagag1 in NZ9000 may facilitate in vivo applications of this system aimed at improving the specificity of immunological responses to buckwheat allergens., Article, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY. 97(8):3603-3611 (2013)

In this study, we successfully developed a recombinant strain of Lactococcus lactis NZ9000 (NZ9000) that produced green fluorescent protein fused to α(S1)-casein (GFP-α(S1)Cas). A modified lactic acid bacterial vector (pNZ8148#2) was constructed by inserting genes for GFP and α(S1)-casein, a major cow's milk allergen, and the resulting vector, pNZ8148#2-GFP-α(S1)Cas, was applied to the expression of recombinant GFP-α(S1)Cas protein (rGFP-α(S1)Cas) in NZ9000. After inducing expression with nisin, the production of rGFP-α(S1)Cas was confirmed by confocal laser microscopic analysis, and the expression conditions were optimized based on fluorescent analysis and western blotting results. Moreover, the in vitro treatment of splenocytes isolated from α-casein (≥70 % α(S)-casein)-immunized mice with rGFP-α(S1)Cas resulted in increased IL-13 mRNA expression. The observed allergic activity is indicative of the Th2-cell mediated immune response and is similar to the effects induced by exposure to α-casein. Our results suggest that the expression of rGFP-α(S1)Cas in NZ9000 may facilitate in vivo applications of this system aimed at improving the specificity of immunological responses to specific milk allergen.

Drosophila N-cadherin (CadN) is an evolutionarily conserved, atypical classical cadherin, which has a large complex extracellular domain and a catenin-binding cytoplasmic domain. We have previously shown that CadN regulates target selection of R7 photoreceptor axons. To determine the functional domains of CadN, we conducted a structure–function analysis focusing on its in vitro adhesive activity and in vivo function in R7 growth cones. We found that the cytoplasmic domain of CadN is largely dispensable for the targeting of R7 growth cones, and it is not essential for mediating homophilic interaction in cultured cells. Instead, the cytoplasmic domain of CadN is required for maintaining proper growth cone morphology. Domain swapping with the extracellular domain of CadN2, a related but non-adhesive cadherin, revealed that the CadN extracellular domain is required for both adhesive activity and R7 targeting. Using a target-mosaic system, we generated CadN mutant clones in the optic lobe and examined the target-selection of genetically wild-type R7 growth cones to CadN mutant target neurons. We found that CadN, but neither LAR nor Liprin-α, is required in the medulla neurons for R7 growth cones to select the correct medulla layer. Together, these data suggest that CadN mediates homophilic adhesive interactions between R7 growth cones and medulla neurons to regulate layer-specific target selection.

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.

Leptin mRNA is expressed in not only adipocytes but also mammary epithelial cells and leptin protein is present in milk. Although milk leptin is thought to influence metabolism or the immune system in neonates, there is little information about the regulation of leptin expression in mammary epithelial cells. We examined the effect of growth hormone (GH) and/or lactogenic hormone complex (DIP; dexamethasone, insulin and prolactin) on leptin mRNA expression in mammary epithelial cells. We used a bovine mammary epithelial cell (BMEC) clonal line, which was established from a 26-day pregnant Holstein heifer. We confirmed that the mRNA was expressed in BMECs and the expression was significantly reduced by GH and/or DIP, when the cells were cultured on both plastic plates and cell culture inserts at days 2 and 7 after stimulation with lactogenic hormones. GH and/or DIP significantly increased level of alpha-casein mRNA in BMECs after 7 days on the cell culture inserts, but no mRNA expression was detected at day 2. GH and DIP significantly stimulated the secretion of alpha-casein from BMEC on cell culture inserts at 3.5 and 7 days. However, neither alpha-casein mRNA expression nor secretion was observed in the BMECs cultured on plastic dishes, even in the presence of GH or/and DIP. These results indicate that GH and DIP can directly reduce leptin mRNA expression in both undifferentiated and functionally differentiated bovine mammary epithelial cell.

We investigated the action of bisphenol A (BPA) on cellular GH release and content, cell number, GHmRNA expression, and concentrations of cellular cyclic AMP ([cAMP]c) and calcium ion ([Ca2+]c) in primary cultured ovine anterior pituitary cells. The following results were found: (1) BPA as well as nonylphenol (NP) at 10(-6) to 10(-3) M significantly and concentration-dependently suppressed basal and GHRH-stimulated GH release, and the cellular GH content, (2) BPA suppressed the cell number in a time- and concentration-dependent manner, (3) 10(-4)M BPA suppressed GHmRNA expression to 68% of control (BPA-free), and abolished GHRH (10(-8) M)-induced increases in [cAMP]c and [Ca2+]c. From these findings we conclude that BPA possesses a suppressing action on GH synthesis and release, and this suppressing action is probably related to impairment of cellular signal transduction systems in ovine anterior pituitary cells.

The effects of acetate and butyrate on leptin and leptin receptor (OB-R) expression in bovine and rat anterior pituitary were examined. In bovine tissues, leptin gene expression using RT-PCR was observed in fat and anterior pituitary but not in liver. Isolated anterior pituitary cells cultured in Dulbecco’s modified Eagle’s medium (DMEM) for 3 days were further cultured for 48 h in DMEM containing 10 mM acetate or butyrate or without any fatty acids as control. Western blot analysis revealed that the abundance of leptin protein was greater in the presence of acetate and butyrate than that for the control culture. Leptin abundance was increased in a dose- and time-dependent manner in bovine anterior pituitary cells. However, leptin expression in rat cells, of which the basal level was much greater than that in ovine cells, was significantly decreased by the culture with butyrate. In addition, we studied the effects of both fatty acids on OB-R mRNA expression using semi-quantitative RT-PCR. The results showed that butyrate significantly decreased the expression in both bovine and rat cells. These findings indicate that acetate and butyrate enhance leptin expression in bovine, but not in rat anterior pituitary cells while butyrate suppresses OB-Ra expression in both rat and bovine pituitaries.

The aim of the study was to establish in vitro a bovine mammary epithelial cell (MEC) clone, able to respond to mitogenic growth factors and to lactogenic hormones. Mammary tissue from a 200-d pregnant Holstein cow was used as a source of MEC, from which a clone was established through a process of limiting dilution. When plated on plastic, the cells assumed a monolayer, cobblestone, epithelial-like morphology, with close contact between cells. Inclusion of IGF-1 and EGF in the media significantly increased the number of cells 5 d after plating. All cells stained strongly for cytokeratin and moderately for vimentin at young and old passage stages, indicating the epithelial nature of this cell clone. When the cells were plated at a high density on a thin layer of a commercial extracellular matrix preparation (Matrigel), lobular, alveoli-like structures developed within approximately 5 d, with a clearly visible lumen. When cells were plated onto Matrigel in differentiation media (containing lactogenic hormones), detectable quantities of α-casein were present in the media and particularly on the lumen side of the structures. Omission of one of the lactogenic hormones (insulin, prolactin or hydrocortisone) reduced α-casein release to the limit of detection of the assay used. Lactoferrin was also produced when the cells were plated on Matrigel, again principally on the lumen side of the lobules, though this was independent of the lactogenic hormones. By passage 40, the cells had senesced, and it was not possible to induce α-casein or lactoferrin production. This study notes the establishment of a functional bovine mammary epithelial cell clone, which is responsive to mitogenic and lactogenic hormones and an extracellular matrix.

Thirty-two male Holstein calves were used to investigate the effects of nutritional conditions around weaning and aging on carbonic anhydrase (CA) activity in the parotid gland and epithelium from the rumen and abomasum. We fed calf starter and lucerne hay as well as milk replacer (group N) or fed milk replacer either with (group S) or without (group M) administration of short-chain fatty acids (SCFA) through polypropylene tubing into the forestomach until 13 weeks of age. The diets were fed at 1000 hours and 1600 hours, and SCFA were administrated after milk replacer feeding at 1600 hours. Slaughter and tissue sampling were carried out between 1300 hours and 1430 hours at 1, 3, 7, 13, and 18 weeks of age. Tissue samples from five adult (1.5-2.0 years-old) Holstein steers were obtained from a local abattoir. In group N, CA activity in the parotid gland gradually and significantly increased toward the adult value, whilst in the epithelium from the rumen and abomasum, adult values were reached at 3 and 7 weeks of age, respectively. At 13 weeks, the activity for group N was significantly higher than that for the other two groups in the parotid gland, but there was no significant difference in the epithelium from the rumen and abomasum. The concentration of the carbonic isozyme VI in the parotid gland also changed with age but, in contrast to CA activity, had not reached adult levels by 13 weeks of age. In groups M and S, parotid saliva did not show any change toward an alkaline pH or toward a reciprocal change in the concentrations between Cl(-) and HCO(3)(-), even at 13 weeks of age. From these results we conclude that a concentrate-hay based diet around weaning has a crucial role in CA development in the parotid gland, but not in the epithelium of the rumen and abomasum.

In order to know the effects of weaning and volatile fatty acid feeding on gastric leptin expression, we investigated the expression of leptin and CCK receptor mRNA in the bovine rumen, abomasum and duodenum using RT-PCR in 3-week-old pre-weaning, 13-week-old post-weaning and adult animals. Leptin mRNA was expressed in the rumen and abomasum of 3-week-old pre-weaning animals, but it was abolished in 13-week-old and adult animals. In the duodenum, leptin expression was observed in the 3-, 13-week-old and adult animals. In the rumen, CCK A receptor mRNA was expressed in 3-week-old animals, but not in 13-week-old and adult animals. In the abomasum, CCK B receptor expression gradually decreased from 3-week-old to adult animals. Expression of CCK B receptor and of CCK A receptor was slight in the rumen and abomasum, respectively. In the next study, we examined the effect of weaning of 6 weeks or non-weaning (fed on milk replacer alone (milk) or milk replacer with volatile fatty acids (milk+VFA) until 13 weeks old) on leptin mRNA expression in the rumen and abomasum. In 13-week-old calf rumen and abomasum, leptin mRNA expression was detected in non-weaning milk-fed animals at 13 weeks old, although it was not observed in weaning and non-weaned milk+VFA-fed animals. The change in CCK A receptor expression in the rumen was similar to those of leptin mRNA expression. CCK B receptor transcription in the abomasum of milk-fed animals was higher than that of the weaning and milk+VFA-fed animals. These results indicate that leptin expression is coincident with CCK receptor expression in calf stomachs, and that leptin and CCK receptor mRNA expression are affected by the change in the physiological status brought about by weaning and VFA feeding.

The objective of this study was to investigate the relationship between marbling and serum leptin concentrations. First, we compared serum leptin concentrations between Japanese Black cattle and Holstein calves, and found that the mean serum concentration was significantly higher (P

BACKGROUND/OBJECTIVES: This study was conducted to investigate the effects of fermented soybean (FS) extract on adipocyte differentiation and fat accumulation using cultured 3T3-L1 adipocytes. MATERIALS/METHODS: 3T3-L1 adipocytes were treated with FS and nonfermented soybean (NFS) extract during differentiation for 10 days in vitro. Oil red O staining was performed and glycerol-3-phosphate dehydrogenase (GPDH) activity was measured for analysis of fat accumulation. Expressions of adipogenic genes were measured. RESULTS: Soluble extract of soybean fermented with Aspergillus oryzae GB107 contained higher levels of low-molecular-weight protein than conventional soybean protein did. FS extract (50 μg/ml) inhibited adipocyte differentiation and fat accumulation during differentiation of 3T3-L1 preadipocytes for 10 days in vitro. Significantly lower GPDH activity was observed in differentiated adipocytes treated with the FS extract than those treated with NFS extract. Treatment with FS extract resulted in decreased expression levels of leptin, adiponectin, and adipogenin genes, which are associated with adipogenesis. CONCLUSIONS: This report is the first to demonstrate that the water-soluble extract from FS inhibits fat accumulation and lipid storage in 3T3-L1 adipocytes. Thus, the soybean extract fermented with A. oryzae GB107 could be used to control lipid accumulation in adipocytes.

The present experiment was carried out to investigate the effects of exogenous adenosine 5'-triphosphate (ATP) and growth hormone (GH) on cellular H(+) efflux rate (extracellular acidification rate) and Ca(2+) concentration ([Ca(2+)](c)) in cloned bovine mammary epithelial cells (bMEC) raised from the mammary gland of a 26-day-pregnant Holstein heifer. Perifusion of 2-day cultured cells with a medium containing ATP (10, 100 and 1000 micromol/l) for 30 min caused a significant and concentration-dependent increase in the cellular H(+) efflux rate. ATP application (100 micromol/l) caused a transient and large increase in [Ca(2+)](c) in all cells. In contrast, perifusion with a medium containing bovine GH at 10, 50 and 250 ng/ml for 30 min caused a significant decrease in the cellular H(+) efflux rate in a concentration-dependent manner. However, bovine GH application (50 ng/ml) caused a small decrease followed by an increase, in some cases, in [Ca(2+)](c). In bMEC treated with lactogenic hormones (1 microgram/l prolactin, 1 nmol/ml dexamethasone and 5 microgram/ml insulin) for 2 days, the increased H(+) efflux rate induced by ATP was significantly reduced, whereas the negative response induced by GH was inversely and significantly changed to the positive. Treatment of the cells with lactogenic hormones reduced the increase in [Ca(2+)](c) induced by ATP stimulation, while it enhanced the increase in [Ca(2+)](c) induced by GH stimulation. Application of ATP or GH did not cause any significant changes in [pH](c). Treatment with lactogenic hormones enhanced GH receptor (GHR) transcription that was determined by RT-PCR. From these results, we conclude that exogenous application of ATP and GH causes prompt and significant responses in H(+) transport and [Ca(2+)](c) that were significantly changed in the opposite direction by the treatment with lactogenic hormones. The lactogenic hormone treatment also enhanced GHR transcription, which may change post-receptor signal transduction systems for both agents in the bMEC.

Influences of a specific dietary nutrient on glucagon-like peptide (GLP)-1-containing cells in the chicken intestine are not yet clear. Significance of dietary protein level on GLP-1-containing cells in the chicken ileum was investigated. Chickens fed control or experimental diets of varying protein levels were examined using immunohistochemical and morphometrical techniques. We show that the protein ingestion had an impact on the activities of GLP-1-immunoreactive cells in the chicken ileum. Weight gains declined with decreasing dietary crude protein (CP) levels, but no significant differences were detected in the daily feed intake and villous height. GLP-1-immunoreactive cells with a round or oval shape were frequently observed in the lower CP level groups (4.5% and 0%). Frequencies of occurrence of GLP-1-immunoreactive cells were 41.1 ± 4.1, 38.5 ± 4, 34.8 ± 3.1 and 34.3 ± 3.7 (cells/mm(2) , mean ± SD) for dietary CP level of 18%, 9%, 4.5% and 0% groups, respectively and significant differences were recognized between the control and lower CP level groups (P0.05). Multiple regression analysis indicated a significant correlation between the daily protein intake and frequencies of occurrence of GLP-1-immunoreactive cells. The protein ingestion is one of the signals that influence GLP-1-containing cells in the chicken small intestine.

Leptin is thought to be not only a satiety factor but also a stimulator of angiogenesis. We examined leptin, PPARγ2, and vascular endothelial growth factor (VEGF) expression in bovine intramuscular preadipocyte (BIP) cells during proliferation. The cells were seeded at 0.85 × 10(4) cells/cm(2) and collected every day until the fifth day after passage. Leptin mRNA was present in the cells between days 2 and 4, as indicated by RT-PCR analysis. Western blot analysis showed a band for leptin at approximately 16 kDa on all of the days during growth, and the cytoplasmic concentration of leptin was highest on day 2 and decreased gradually thereafter. A PPARγ2 band at approximately 54 kDa was also observed on all days. The concentration was highest on day 2 and decreased thereafter, which is similar to the expression pattern of leptin. In constant, the expression level of VEGF protein did not change while in culture. We have demonstrated that BIP cells can synthesize both leptin and PPARγ2, with maximal synthesis occurring during maximal proliferation. Given the role of leptin in angiogenesis, we speculate that leptin is involved in the neovascularization of adipose tissue, because new organization of adipose tissue requires the growth of new blood vessels.

Metazoan development requires complex mechanisms to generate cells with diverse function. Alternative splicing of pre-mRNA not only expands proteomic diversity but also provides a means to regulate tissue-specific molecular expression. The N-Cadherin gene in Drosophila contains three pairs of mutually-exclusive alternatively-spliced exons (MEs). However, no significant differences among the resulting protein isoforms have been successfully demonstrated in vivo. Furthermore, while the N-Cadherin gene products exhibit a complex spatiotemporal expression pattern within embryos, its underlying mechanisms and significance remain unknown. Here, we present results that suggest a critical role for alternative splicing in producing a crucial and reproducible complexity in the expression pattern of arthropod N-Cadherin. We demonstrate that the arthropod N-Cadherin gene has maintained the three sets of MEs for over 400 million years using in silico and in vivo approaches. Expression of isoforms derived from these MEs receives precise spatiotemporal control critical during development. Both Drosophila and Tribolium use ME-13a and ME-13b in “neural” and “mesodermal” splice variants, respectively. As proteins, either ME-13a- or ME-13b-containing isoform can cell-autonomously rescue the embryonic lethality caused by genetic loss of N-Cadherin. Ectopic muscle expression of either isoform beyond the time it normally ceases leads to paralysis and lethality. Together, our results offer an example of well-conserved alternative splicing increasing cellular diversity in metazoans., Author Summary Animal development requires complex mechanisms to generate many different types of cells. Alternative splicing is a process by which a single gene could produce several protein variants under particular circumstances. It is a useful means to generate a diversified set of proteins in different cell types. In this report, we showed that the alternative splicing of the arthropod N-Cadherin gene has been maintained for over 400 million years. The switch of expression patterns of two distinct variants is also well conserved in arthropods. As proteins, these two N-Cadherin splice variants have similar ability to rescue the embryonic lethality caused by genetic loss of N-Cadherin. However, when the expression of either isoforms was prolonged in muscles where the endogenous expression ceased beyond certain stages, it leads to larval lethality, suggesting the importance of precise spatiotemporal regulation of N-Cadherin splice-variant expression. This finding is particularly important because it offers an example of well-conserved alternative splicing increasing cellular diversity in animals.