Effect of porcine glucagon‐like peptides‐2 on tight junction in GLP‐2R + IPEC‐J2 cell through the PI3k/Akt/mTOR/p70S6K signalling pathway

Because of rare glucagon‐like peptide‐2 (GLP‐2) receptor (+) cells within the gut mucosa, the molecular mechanisms transducing the diverse actions of GLP‐2 remain largely obscure. This research identified the naturally occurring intestinal cell lines that endogenously express GLP‐2R and determined the molecular mechanisms of the protective effects of GLP‐2‐mediated tight junctions (TJ) in GLP‐2R (+) cell line. (i) Immunohistochemistry results showed that GLP‐2R is localised to the epithelia, laminae propriae and muscle layers of the small and large bowels of newborn piglets. (ii) GLP‐2R expression was apparent in the cytoplasm of endocrine cells in IPEC‐J2 cell lines. (iii) The protein expressions of ZO‐1, claudin‐1, occludin, p‐PI₃K, p‐Akt, p‐mTOR and p‐p70^S6K significantly (p < 0.05) increased in GLP‐2‐treated IPEC‐J2 cells, and all of them significantly (p < 0.05) decreased when LY‐294002 or rapamycin was added. GLP‐2 improves intestinal TJ expression of GLP‐2R (+) cells through the PI₃k/Akt/mTOR/p70^S6K signalling pathway.     

Dysregulated expression of the key effectors of the mammalian target of rapamycin signalling pathway in cutaneous papillomas of dogs

Cutaneous papillomas (CP) are one of the most common skin neoplasms in dogs. Different murine models have shown that persistent activation of the phosphatidylinositol 3‐kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway has a central role in the development and progression of CP. The purpose of this study were to evaluate the immunohistochemical expression pattern of two key molecules involved in the PI3K/Akt/mTOR signalling pathway, pAkt^Ser473, and pS6^Ser235/236, on 36 canine specimens of CP using a tissue microarray. The results show that the PI3K/Akt/mTOR signalling pathway is persistently activated in CP of dogs, pointing to this pathway as a potential therapeutic target.     

mTOR对信号通路调控的研究进展

哺乳动物雷帕霉素靶蛋白(mTOR)信号通路是最近新出现的细胞内重要信号途径,该途径在进化上高度保守,主要通过PI3K/Akt/mTOR信号通路磷酸化激活来调控细胞分裂、促进转录、信号翻译等,从而控制蛋白合成来调节细胞生长。mTOR作为一种重要的调节基因通过调节细胞周期、蛋白质合成、细胞能量代谢等多种途径发挥重要的生理功能,在细胞增殖、生长、分化过程中起着中心调控点的作用。     

Transcriptomic gene profiling of porcine muscle tissue depending on histological properties

In pig, the histological profile of muscle tissue, especially the proportion of individual fiber types, is one of the main factors affecting meat quality properties. In the present research, RNA sequencing (RNA‐seq) by using next generation sequencing method was applied to estimate the whole gene expression profile of Longissimus lumborum muscle of pigs (Large White breed) differing in the percentage of two fiber types (slow‐twitch (type I) fibers and fast‐twitch glycolytic (type IIB) fibers). The RNA‐seq approach allowed us to identify 355 differentially expressed genes (DEGs) indicated as significant (false discovery rate‐adjusted P < 0.05) using three types of software: DESeq2, edgeR and baySeq. Detected genes and pathways deregulated in muscle depending on tissue microstructure were associated with: metabolic processes – 158 genes; cellular processes – 122; biological regulation – 62; localization – 51; and 35 genes with developmental processes. The DEGs were included in: PI3K‐Akt; FoxO and MAPK signaling pathways, regulation of actin cytoskeleton, lysine degradation and insulin signaling pathway as well as mTOR and Hippo signaling pathways. These results highlight the mainly metabolic pathways related to glucose metabolism and contraction processes of muscle cells. Detection of genes involved in variation of fiber‐type distribution will be useful in understanding of the genetic factors affecting muscle structure, metabolic process and indirectly, meat quality traits.     

葡萄糖对鹅原代肝细胞GPX1和SOD1基因表达及酶活力的影响

In order to investigate the effect of glucose on oxidative stress in cultured goose primary hepatocytes, partial gene sequences of GPX1 and SOD1 genes were cloned, and the goose primary hepatocytes were treated with different concentrations（0（control group）, 5, 25 and 35 mmol/L） of glucose for 48 h, and then enzyme activities of GPX and SOD and mRNA levels of GPX1 and SOD1 were examined. The results showed that goose GPX1 gene sequence had the highly similarity with Zebra Finch, and SOD1 had the highly similarity with Red Jungle fowl. Compared with the control group, 5 mmol/L glucose had no significant effect on GPX enzyme activity （P>0.05）, but 25 and 35 mmol/L glucose could significantly decrease GPX enzyme activity (P＜0.05).5 mmol/L glucose could significantly increase SOD enzyme activity （P＜0.05）, but 25 and 35 mmol/L glucose had no significant effects on SOD enzyme activity（P＞0.05）.35 mmol/L glucose could significantly increase mRNA expression of GPX1 and SOD1 genes （P＜0.05）, but 5 and 25 mmol/L glucose had no significant effects on the two genes（P＞0.05）.In conclusion, the high concentration glucose （35 mmol/L） could induce intracellular oxidative stress in goose primary hepatocytes.     

Ankyrin repeat and suppressor of cytokine signaling (SOCS) box-containing protein (ASB) 15 alters differentiation of mouse C2C12 myoblasts and phosphorylation of mitogen-activated protein kinase and Akt

Ankyrin repeat and suppressor of cytokine signaling box-containing protein (ASB) 15 is a novel ASB gene family member predominantly expressed in skeletal muscle. We have previously reported that overexpression of ASB15 delays differentiation and alters protein turnover in mouse C(2)C(12) myoblasts. However, the extent of ASB15 regulation of differentiation and molecular pathways underlying this activity are unknown. The extracellular signal-regulated kinase (Erk) 1/2 and phosphatidylinositol-3 kinase-Akt (PI3K/Akt; Akt is also known as protein kinase B) signaling pathways have a role in skeletal muscle growth. Activation (phosphorylation) of the Erk1/2 signaling pathway promotes proliferation, whereas activation of the PI3K/Akt signaling pathway promotes myoblast differentiation. Accordingly, we tested the hypothesis that ASB15 controls myoblast differentiation through its regulation of these kinases. Stably transfected myoblasts overexpressing ASB15 (ASB15+) demonstrated decreased differentiation, whereas attenuation of ASB15 expression (ASB15-) increased differentiation. However, ASB15+ cells had less abundance of the phosphorylated mitogen-activated protein kinase (active) form, despite decreased differentiation relative to control myoblasts (ASB15Con). The mitogen-activated protein kinase kinase inhibitor, U0126, effectively decreased mitogen-activated protein kinase phosphorylation and stimulated differentiation in ASB15- and ASB15Con cells. However, inhibition of the Erk1/2 pathway was unable to overcome the inhibitory effect of overexpressing ASB15 on differentiation (ASB15+), suggesting that the Erk1/2 pathway is likely not the predominant mediator of ASB15 activity on differentiation. Expression of ASB15 also altered phosphorylation of the PI3K/Akt pathway, as ASB15+ and ASB15- cells had decreased and increased Akt phosphorylation, respectively. These data were consistent with observed differences in differentiation. Administration of IGF-I, a PI3K/Akt activator, in ASB15+ was able to partially override the previously observed phenotype of delayed differentiation, whereas administration of the PI3K/ Akt inhibitor, LY294002, decreased phosphorylation of Akt and differentiation of all cell lines similar to the untreated ASB15+ myoblasts. These results provide initial evidence that ASB15 has a role in early myoblast differentiation and that its effects may be mediated in part by the PI3K/Akt signal transduction pathway.     

Evaluation of a point‐of‐care blood glucose monitor in healthy goats

Objective

To assess agreement between a point‐of‐care glucometer (POCG) and a laboratory chemistry analyzer for blood glucose measurements in goats.

Design

Prospective study.

Setting

University teaching hospital.

Animals

Eighteen healthy adult goats.

Investigations

Whole blood samples were obtained via jugular venipuncture prior to premedication with xylazine and butorphanol (T0), following premedication (T20), and after 1 hour of inhalant anesthesia (T60). Each sample was tested with a POCG and a laboratory analyzer (HITA). Agreement was assessed using concordance correlation coefficients and calculation of bias and 95% limits of agreement.

Measurements and Main Results

Mean blood glucose concentration at T0 was 3.9 ± 0.6 mmol/L (70 ± 10 mg/dL; POCG) and 2.9 ± 0.4 mmol/dL (53 ± 8 mg/dL; HITA). Glucose concentrations at T20 were 6.7 ± 2.4 mmol/L (121 ± 43 mg/dL) and 5.4 ± 2.1 mmol/L (97 ± 37 mg/dL) and at T60 were 5.7 ± 1.7 mmol/L (102 ± 31 mg/dL) and 4.7 ± 1.3 mmol/L (85 ± 24 mg/dL) when measured with the POCG and HITA, respectively. The POCG overestimated blood glucose compared to the HITA. The bias ± SD was 1.08 ± 0.53 mmol/L (19.4 ± 9.5 mg/dL) (95% LOA 0.04 to 2.11 mmol/L [0.7 to 38.0 mg/dL]) and the concordance correlation coefficient was 0.82. After correcting the results of the POCG using a mixed‐effects linear model, the bias was 0.0 ± 0.38 mmol/L (0.0 ± 6.8 mg/dL) (95% LOA ± 0.74 mmol/L [± 13.4 mg/dL]) and the concordance correlation coefficient was 0.98.

Conclusions

The POCG overestimated blood glucose concentrations in goats, compared to the HITA, but when the POCG concentrations were corrected, the agreement was excellent.     

猪瘟病毒非结构蛋白NS5A与Beclin1相互作用并促进病毒增殖

为探究宿主蛋白Beclin1在猪瘟病毒(classical swine fever virus, CSFV)非结构蛋白NS5A激活细胞自噬反应过程中的作用及具体分子机制,本研究在感染CSFV及表达NS5A蛋白的ST细胞中,利用qRT-PCR方法检测Beclin1、PI3K/Akt通路相关因子表达变化情况;利用激光共聚焦、Co-IP及GST-pulldown等方法研究Beclin1与NS5A相互作用关系;通过在ST细胞中过表达或敲低Beclin1,研究其对CSFV复制的影响。结果表明,ST细胞感染猪瘟病毒或外源表达NS5A蛋白,Beclin1转录和蛋白表达水平均显著升高,且PI3K/Akt通路相关因子表达水平与之呈正相关。此外,CSFV NS5A蛋白与Beclin1蛋白在细胞中存在共定位且具有相互作用。最后,作者发现在细胞中过表达Beclin1,对CSFV复制起到明显促进作用;反之,利用siRNA敲低Beclin1后,抑制PI3K/Akt通路活化,CSFV增殖表现出明显抑制效应。以上结果表明,Beclin1蛋白对CSFV复制具有促进作用,其机制是通过与NS5A的相互作用调控PI3K/Ak...     

Effects of methionine on muscle protein synthesis and degradation pathways in broilers

This study investigated the hypothesis that supplementation of methionine (Met) to broiler diets increases muscle growth due to regulation of molecular pathways related to protein synthesis and degradation depending on the Met source. Day‐old male Cobb‐500 broilers (n = 240) were phase‐fed three different wheat–soya bean meal‐based basal diets during days 1–10, 11–21 and 22–35. Basal diets (Met‐ group, Met + Cys concentration 15% below NRC recommendations) were supplemented with 0.10% or 0.40% Met either as DL‐Met (DLM) or DL‐2‐hydroxy‐4‐(methylthio) butanoic acid (DL‐HMTBA) (equimolar comparison). Breast muscle weights were lower in the Met‐ group compared to all Met‐supplemented groups and were lower in broilers supplemented with 0.10% of DL‐HMTBA compared to the other groups fed Met‐supplemented diets. However, the expression of genes or relative phosphorylation and thus activation state of proteins involved in the somatotropic axis, the mammalian target of rapamycin (mTOR) pathway of protein synthesis, the ubiquitin–proteasome pathway (UPP) and autophagy–lysosomal pathway of protein degradation, the GCN2/eIF2a pathway involved in the inhibition of protein synthesis and in the myostatin–Smad2/3 pathway involved in myogenesis were not affected by Met source. Feeding diets with suboptimum Met + Cys concentrations, however, decreased expression of GHR and IGF1 in liver and muscle and increased that of MURF1 involved in the UPP in the broiler's muscle at day 10 and 21, while that of FOXO and atrogin‐1 and FOXO phosphorylation remained unaffected. Additionally, suboptimum dietary Met concentrations increased expression of the autophagy‐related genes ATG5 and BECN1 at day 35. Met supplementation neither affected gene expression nor phosphorylation of proteins involved in the GNC2/eIF2a and mTOR pathways. These data indicate that protein synthesis was not affected on the molecular level, while protein degradation was marginally affected by dietary Met dosage.     

Effects of rumen‐protected folic acid and dietary protein level on growth performance,ruminal fermentation,nutrient digestibility and hepatic gene expression of dairy calves

This study evaluated the effects of rumen‐protected folic acid (RPFA) supplementation and dietary protein level on growth performance, ruminal fermentation, nutrient digestibility and hepatic gene expression in calves. Forty Holstein male calves (161 ± 5.7 days of age and 192 ± 5.4 kg of body weight) were assigned to one of four groups in a randomized experimental design with a 2 × 2 factorial arrangement. Moderate crude protein (130.1 g CP/kg [MCP] or high crude protein (150.2 g CP/kg [HCP]) diets were fed without (RPFA?) or with 3.6 mg FA (RPFA+) as RPFA per kg dietary dry matter (DM). Calves were fed a total mixed ration with a corn silage to concentrate ratio of 50:50 on a DM basis. The CP×RPFA interaction was not significant for any of the studied variables. The unchanged DM intake, higher average daily gain and lower feed conversion ratio were observed for HCP or RPFA+. Ruminal pH was lower, and total volatile fatty acids (VFA) concentration was higher for HCP or RPFA+. Acetate proportion was higher, and propionate proportion was lower for HCP or RPFA+. As a result, the higher acetate to propionate ratio was observed. Ruminal ammonia N was higher for HCP, but was lower with RPFA supplementation. The higher digestibility of DM, OM, CP and NDF was observed. Blood glucose and insulin were unchanged, but albumin, total protein, GH and IGF‐1 were higher. Similarly, the higher hepatic expression of GH, IGF‐1, GHR, IGF‐1R, PI3K, mTOR and P70S6K was observed for HCP or RPFA+. The results indicated that increasing dietary CP content or supplementation with RPFA promoted growth performance of calves by improving nutrient utilization and up‐regulating hepatic expression of gene related to protein synthesis.     

PS48 promotes in vitro maturation and developmental competence of porcine oocytes through activating PI3K/Akt signalling pathway

Oocyte maturation plays a vitally important role in porcine reproduction. Regrettably, the quality of oocytes matured in vitro is weaker than that of in vivo matured oocytes. We collected and cultivated porcine cumulus oocyte complexes (COCs) in vitro with phosphoinositide-dependent kinase 1 (PDK1) activator 5-(4-chloro-phenyl)-3-phenyl-pent-2-enoic acid (PS48), whose concentrations were 0, 2, 5, 10 and 20 µM to investigate whether the phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) signalling pathway would impact the oocyte quality. The results showed that 10 µM PS48 increased the oocyte proportion of metaphase II (MII) stage and improved the expansion of cumulus cells (CCs). What's more, the activation of PI3K/Akt signalling pathway could regulate the expression of maturation-related genes and proteins. The results of quantitative real-time PCR showed that 10 µM PS48 increased the mRNA and protein levels of Akt and regulated maturation-related genes, including cyclin B1, MOS, BMP15, GDF9, CDC2, mTOR, BAX, BCL2 and caspase-3. The results of Western blot indicated that 10µM PS48 increased the protein abundance of Akt, phosphorylation of Akt Thr308 (p-Akt^Thr308) and cyclin B1, but decreased the protein abundance of pro-apoptotic BAX. These results suggested that adding 10 µM PS48 to mature culture medium could promote the maturation of porcine oocytes, potentially through activating the PI3K/Akt signalling pathway.     

牦牛IGFBP4对肝细胞增殖的调控及功能分析

旨在探究牦牛胰岛素样生长因子结合蛋白4(Bos grunniens insulin-like growth factor binding protein 4, BgIGFBP4)在牦牛肝细胞增殖及小鼠生长中的作用。本研究构建了pET-28a-BgIGFBP4原核表达载体，对其进行表达、鉴定，用终浓度为0.002、0.02、0.2、2、20 μg·mL^-1的BgIGFBP4蛋白处理肝细胞，采用CCK8法、细胞集落形成试验检测BgIGFBP4蛋白对肝细胞增殖能力的影响。再根据试验结果选取浓度蛋白处理肝细胞，检测24、48、72 h时肝细胞活性、肝细胞上清生长类激素和肝细胞PI3K-Akt信号通路的变化。选取30日龄((18±1)g)健康雄性KM小鼠，试验组每2 d灌喂100 μL 50 μg·mL^-1 BgIGFBP4蛋白，对照组用等量0.9%的生理盐水进行灌喂，每组40只，共80只。试验前进行饥饿处理12 h，试验周期为28 d，在试验28 d时检测各项生长性能指标、血清生长类激素水平和肝PI3K-Akt信号通路的变化。结果显示，获得大小约28.86 ku的BgIGFBP4蛋白，并纯化鉴定得到该目的蛋白。2 μg·mL^-1 BgIGFBP4蛋白可促进牦牛肝细胞增殖及集落形成。与对照组相比，试验组(2 μg·mL^-1BgIGFBP4蛋白处理)肝细胞上清GH、VEGF含量显著升高(P<0.05)，肝细胞中PI3K-Akt信号通路细胞增殖相关基因ERBB2、IRS1、PIK3R1、AKT1、RAF1、MAPK3转录水平显著升高(P<0.05)。与对照组相比，试验组(100 μL 50 μg·mL^-1 BgIGFBP4蛋白处理)小鼠平均日增重显著增加、料重比显著降低，肝、脾、肺、肾、小肠器官指数显著增加(P<0.05)。试验组小鼠血清GH、ISN、VEGF含量显著升高(P<0.05)，小鼠肝中PI3K-Akt信号通路相关基因ERBB2、IRS1、PIK3R1、AKT1、RAF1、MAPK3的转录水平显著升高(P<0.05)。综上表明，BgIGFBP4可通过调控PI3K-Akt信号通路促进牦牛肝细胞增殖、并能促进小鼠生长。这为深入研究IGFBP4在牦牛肝生长发育过程中的作用提供了参考。     

Activation of Akt/protein kinase B mediates the protective effects of mechanical stretching against myocardial ischemia-reperfusion injury

Akt/protein kinase B is a well-known cell survival factor and activated by many stimuli including mechanical stretching. Therefore, we evaluated the cardioprotective effect of a brief mechanical stretching of rat hearts and determined whether activation of Akt through phosphatidylinositol 3-kinase (PI3K) is involved in stretch-induced cardioprotection (SIC). Stretch preconditioning reduced infarct size and improved post-ischemic cardiac function compared to the control group. Phosphorylation of Akt and its downstream substrate, GSK-3β, was increased by mechanical stretching and completely blocked by wortmannin, a PI3K inhibitor. Treatment with lithium or SB216763 (GSK-3β inhibitors) before ischemia induction mimicked the protective effects of SIC on rat heart. Gadolinium (Gd³⁺), a blocker of stretch-activated ion channels (SACs), inhibited the stretch-induced phosphorylation of Akt and GSK-3β. Furthermore, SIC was abrogated by wortmannin and Gd³⁺. In vivo stretching induced by an aorto-caval shunt increased Akt phosphorylation and reduced myocardial infarction; these effects were diminished by wortmannin and Gd³⁺ pretreatment. Our results showed that mechanical stretching can provide cardioprotection against ischemia-reperfusion injury. Additionally, the activation of Akt, which might be regulated by SACs and the PI3K pathway, plays an important role in SIC.     

Blood Metabolite Profiles in Cycling and Non‐cycling Friesian–Sanga Cross‐bred Cows Grazing Natural Pasture During the Post‐partum Period

An experiment was conducted to investigate the effect of plasma concentrations of the metabolic hormones [Growth hormone (GH), insulin and insulin‐like growth factor –I (IGF‐I)] and nutritional metabolites (Glucose, cholesterol, total protein, albumin, globulin, urea and creatinine) on the resumption of post‐partum ovarian activity in sixteen Friesian–Sanga cows grazing extensively on native grassland. Blood samples were taken from cows from week 1 to 16 post‐partum. Cows were classified as having resumed ovarian activity when a plasma progesterone concentration of ≥ 1.0 ng/ml was recorded for two consecutive weekly samples. Based on the resumption of ovarian activity, cows were classified as early‐cycling, late‐cycling or non‐cycling. The concentrations of the metabolic hormones were measured from week 1 to 10, while those of the nutritional metabolites were measured during week 1, 3, 5, 7 and 9 during the study period. The concentrations of the metabolic hormones, GH and insulin were similar (p > 0.05) in the three ovarian activity groups, likewise the concentrations of the nutritional metabolites, glucose, total protein, globulin, urea and creatinine. Plasma IGF‐I concentration was higher (p < 0.001) in early‐cycling (18.7 ± 0.74 ng/ml) than in late‐cycling (12.4 ± 0.75 ng/ml) and non‐cycling (10.4 ± 0.91 ng/ml) cows. Plasma cholesterol concentrations were significantly lower (p < 0.05) in early‐cycling (1.94 ± 0.15 mmol/l) compared with late‐cycling (2.48 ± 0.12 mmol/l) and non‐cycling (2.61 ± 0.11 mmol/l) cows. For plasma albumin concentrations, the levels recorded for early‐cycling cows were higher (40.7 ± 2.85 g/l) than in late‐cycling (34.4 ± 1.97 g/l) and non‐cycling (33.6 ± 2.66) cows. The results suggest that cows with lower plasma concentrations of IGF‐I and albumin, but higher plasma cholesterol concentrations were at risk of delayed resumption of post‐partum ovarian activity.     

Study on Effect of IGF-1 on Proliferation and Apoptosis of Bovine Mammary Epithelial Cells by Adding Inhibitor

The test was aimed to study the effect of insulin-like growth factor 1(IGF-1) on the proliferation of bovine mammary epithelial cells (BMECs),and provide a theoretical basis for the subsequent regulating mammary gland development using IGF-1. The optimal IGF-1 concentration for inhibiting BMECs apoptosis was obtained by measuring the apoptosis rate at 12, 24, 48 and 72 h after the addition of IGF-1 at four groups (0, 10, 50 and 100 μg/mL). Then divided into six groups:BMECs group, BMECs+IGF-1 group, BMECs+LY294002 group, BMECs+IGF-1+LY294002 group, BMECs+RAPA group and BMECs+IGF-1+RAPA group,and the apoptosis rates were detected by flow cytometry. The results showed that the heterologous IGF-1 had an inhibitory effect on the apoptosis of BMECs with an optimal concentration of 50 μg/mL. The apoptosis rates of BMECs+IGF-1+LY294002 and BMECs+IGF-1+RAPA groups were extremely significantly higher than BMECs+IGF-1 group (P<0.01). This study suggested that IGF-1 could activate the PI3K/Akt/mTOR signaling pathway and thereby inhibit the apoptosis of BMECs. Moreover, IGF-1 might also promote the "repair" mechanism for the inhibited PI3K/Akt/mTOR signaling pathway, and therefore make it reparticipate in BMECs life process.     

Leucine‐induced promotion of post‐absorptive EAA utilization and hepatic gluconeogenesis contributes to protein synthesis in skeletal muscle of dairy calves

The high rate of protein synthesis in skeletal muscle of dairy calves can benefit their first lactation even lifetime milk yield. Since the rate of protein synthesis is relatively low in the post‐absorptive state, the aim of this research was to determine whether leucine supplementation could increase the post‐absorptive essential amino acid (EAA) utilization and protein synthesis in the skeletal muscle. Ten male neonatal dairy calves (38 ± 3 kg) were randomly assigned to either the control (CON, no leucine supplementation, n = 5) or supplementation with 1.435 g leucine/L milk (LEU, n = 5). Results showed that leucine significantly increased the length and protein concentration in longissimus dorsi (LD) muscle, whereas it decreased creatinine concentration and glutamic‐oxalacetic transaminase (GOT) activity. Compared to the control group, leucine supplementation also reduced the glutamic‐pyruvic transaminase (GPT) activity. Supplementation of leucine improved the phosphorylation of mammalian target of rapamycin (mTOR), eukaryotic initiation factor 4E‐binding protein 1 (4EBP1) and substrates ribosomal protein S6 kinase 1 (p70S6K). Supplementation of leucine resulted in increased concentrations of glucose, methionine, threonine, histidine and EAAs and decreased concentration of arginine in serum. Liver glucose concentration was higher and pyranic acid was lower in LEU compared to CON. In conclusion, leucine supplementation can promote post‐absorptive EAA utilization and hepatic gluconeogenesis, which contributes to protein synthesis in skeletal muscle of dairy calves.     

Effect of nutrient intake on intramuscular glucose metabolism during the early growth stage in cross‐bred steers (Japanese Black male × Holstein female)

The objective was to investigate the impact of nutrient intake during the early growth period on the expression of glucose metabolism‐related genes in skeletal muscle of cross‐bred cattle. From 1.5 to 5 months of age, group H (n = 7) animals were intensively fed a high‐protein and low‐fat milk replacer [crude protein (CP) 28%; ether extracts (EE) 18%; max: 2.0 kg, 12 l/day], and group R (n = 7) animals were fed a restricted amount of normal milk replacer (CP 25%; EE 23%; max 0.5 kg, 4 l/day). From 6 to 10 months of age, group H cattle were fed a high‐nutrition total mixed ration mainly prepared from grain feed, and group R cattle were fed only roughage. Blood samples were taken from each animal at three biopsy times (1.5, 5 and 10 months of age), and the blood plasma concentration of glucose and insulin was analysed. In glucose concentration, there were no significant differences; however, the concentrations of insulin were higher in group H than in group R at 5 and 10 months of age. Muscle samples were taken by biopsy from longissimus thoracis muscle (LT) at 1.5, 5 and 10 months of age. We analysed mRNA expression levels using the quantitative real‐time polymerase chain reaction (PCR) assay for glucose transporters (GLUT1 and GLUT4), insulin receptor, phosphatidylinositol 3‐kinase (PI‐3K), protein kinase B (PKB, also known as Akt), hexokinase 1 (HK1) and tumour necrosis factor alpha (TNFα). Although no differences were detected at 1.5 and 5 months of age, at 10 months of age, GLUT1, HK1 and TNFα mRNA expression levels were significantly higher in group H than in group R. These results suggested Glut1 that affects insulin‐independently mediated glucose uptake was more responsive to improved nutrition during early growth stage than GLUT4 that insulin‐dependently mediated glucose uptake in LT of cattle.     

Pyridoxine regulates hair follicle development via the PI3K/Akt,Wnt and Notch signalling pathways in rex rabbits

This study was conducted to evaluate the effect of pyridoxine on the development of hair follicles in Rex rabbits and the underlying molecular mechanism. Two hundred 3-month-old Rex rabbits were randomly divided into 5 groups and fed diets supplemented with 0, 5, 10, 20, or 40 mg/kg pyridoxine. The hair follicle density on the dorsal skin and the gene and protein expression levels of components of the phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB or Akt), Wnt, Notch and bone morphogenetic protein (BMP) signalling pathways were measured. In addition, free hair follicles were isolated from Rex rabbits and cultured with pyridoxine in vitro to measure hair shaft growth. Furthermore, dermal papilla cells (DPC) were isolated from the skin of Rex rabbits and cultured with pyridoxine in vitro to measure the gene and protein expression levels of components of the PI3K/Akt, Wnt, Notch and BMP signalling pathways. The results showed that the addition of dietary pyridoxine significantly increased the total follicle density, secondary follicle density, and secondary-to-primary ratio (S/P, P < 0.05), that the growth ratio of hair stems was promoted by pyridoxine in basic culture medium, and that the growth length of tentacle hair follicles cultured in the pyridoxine group was longer than that in the control group (P < 0.05). In addition, pyridoxine changed the DPC cycle progression and promoted cell proliferation, and appropriate concentrations of pyridoxine (10 and 20 μmol/L) significantly inhibited cell apoptosis (P < 0.05). Pyridoxine significantly affected the gene expression of components of the PI3K/Akt, Wnt and Notch signalling pathways in the skin and DPC of Rex rabbits (P < 0.05), increased the levels of phosphorylated catenin beta 1 (CTNNB1) and Akt, and decreased the level of phosphorylated glycogen synthase kinase 3 beta (GSK-3β) (P < 0.05). Therefore, the molecular mechanism by which pyridoxine promotes hair follicle density in Rex rabbits probably occurs through activation of the PI3K/Akt, Wnt and Notch signalling pathways, prolonging hair follicle growth and delaying the onset of telogen.     

添加通路阻断剂对IGF-1影响奶牛乳腺上皮细胞增殖与凋亡的研究

试验旨在研究胰岛素样生长因子-1（insulin-like growth factor 1,IGF-1）对奶牛乳腺上皮细胞（bovine mammary epithelial cells,BMECs）增殖的影响,为后期利用IGF-1调控乳腺发育奠定理论基础。以奶牛乳腺上皮细胞为材料,首先分4组分别外源添加0（对照组）、10、50、100 μg/mL IGF-1且分别培养12、24、48、72 h,测定抑制BMECs凋亡率的最佳浓度;然后分6组：单纯BMECs组、BMECs+IGF-1组、BMECs+LY294002组、BMECs+IGF-1+LY294002组、BMECs+RAPA组和BMECs+IGF-1+RAPA组,采用流式细胞术测定各组细胞凋亡率。结果表明,外源性添加IGF-1对BMECs凋亡率具有抑制作用,最佳浓度为50 μg/mL;BMECs+IGF-1+LY294002与BMECs+IGF-1+RAPA组细胞凋亡率均极显著高于BMECs+IGF-1组（P<0.01）。推断IGF-1能够活化PI3K/Akt/mTOR信号通路,参与BMECs凋亡的调控作用,进而抑制BMECs细胞凋亡;IGF-1可能会对被抑制PI3K/Akt/mTOR信号通路产生"修复"机制,使其能够重新参与BMECs的生命进程。     

IGF-I retards proper development of acinar structures formed by bovine mammary epithelial cells via sustained activation of Akt kinase

Insulin-like growth factor-I is involved in mammary gland development, promoting proliferation and inhibiting apoptosis of mammary epithelial cells (MECs). Mitogenic actions of IGF-I are mainly mediated by the phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway. We have found that in the presence of IGF-I bovine BME-UV1 MECs cultured on reconstituted basement membrane form large spheroids with disrupted polarity and no cavity in the center. These cells showed enhanced phosphorylation of Akt, decreased level of cleaved caspase-3, and sustained proliferative activity throughout the 16-d period of 3-dimensional culture. Inhibition of the PI3K/Akt pathway by a specific inhibitor of PI3K, LY294002, resulted in the restoration of the normal acinar phenotype. However, this effect was noted only when LY294002 was added in the second week of 3-dimensional culture, which corresponded with the time of cell cycle arrest and polarity formation under control conditions. Normal development of acini was also obtained when BME-UV1 cells were treated simultaneously with IGF-I and 17β-estradiol. The addition of 17β-estradiol regulated Akt activation, enabling the subsequent initiation of polarization processes. 17β-Estradiol also increased the level of IGFBP-3 protein in MECs cultured on Matrigel in the presence of IGF-I. The presented results indicate important interactions between signaling pathways activated by estrogen and IGF-I, which regulate alveologenesis in bovine mammary gland.