The effect of polymorphism in gene of insulin-like growth factor-Ⅰ on the serum periparturient concentration in Holstein dairy cows

Objective:To investigate the relationship between polymorphism within the 5'-untranslated region(5'-UTR)of IGF-I gene and its periparturient concentration in Iranian Holstein dairy cows.Methods:Blood samples(5 mL,n=37)were collected by caudal venipuncture from each animal into sample lubes containing the EDTA and DNA was extracted from blood.In order to measure ICF-I concentration the collection of blood samples(n=111)was also done at 14 d before calving(prepartum),25 and 45 d postpartum.Results:We found evidence for a significant effect of C to T mutation in position 512 of IGF-I gene on its serum concentration in dairy cows in Iran.Cows with CC genotype had significantly higher concentration(Mean-SD)of IGF-I at 14 d prepartum(91.8±18.1)μg/L compared to those with TT genotype(73.3±14.4)μg/L(P=0.04).A significant trend(quadratic)was found for IGF-I concentration,as higher in CC cows compared to ones with TT genotype,during the 14 d before calving to 45 d postpartum(P=0.01).Conclusions:We concluded that C/T transition in the promoter region of IGF-I gene can influence the serum concentration of ICF-I in periparturient dairy cows.

Mitochondrial protection by low doses of insulin-like growth factor-Ⅰin experimental cirrhosis

AIM： To characterize the mitochondrial dysfunction in experimental cirrhosis and to study whether insulin-like growth factor-Ⅰ （IGF-Ⅰ ） therapy （4 wk） is able to induce beneficial effects on damaged mitochondria leading to cellular protection. METHODS： Wistar rats were divided into three groups： Control group, untreated cirrhotic rats and cirrhotic rats treated with IGF-Ⅰ treatment （2 μg/1O0 g bw/d）. Mitochondrial function was analyzed by flow cytometry in isolated hepatic mitochondria, caspase 3 activation was assessed by Western blot and apoptosis by TUNEL in the three expedmental groups. RESULTS： Untreated cirrhotic rats showed a mitochondrial dysfunction characterized by a significant reduction of mitochondrial membrane potential （in status 4 and 3）; an increase of intramitochondrial reactive oxigen species （ROS） generation and a significant reduction of ATPase activity. IGF-Ⅰ therapy normalized mitochondrial function by increasing the membrane potential and ATPase activity and reducing the intramitochondrial free radical production. Activity of the electron transport complexes Ⅰ and Ⅲ was increased in both cirrhotic groups. In addition, untreated cirrhotic rats showed an increase of caspase 3 activation and apoptosis. IGF- Ⅰ therapy reduced the expression of the active peptide of caspase 3 and resulted in reduced apoptosis. CONCLUSION： These results show that IGF- Ⅰ exerts a mitochondrial protection in experimental cirrhosis leading to reduced apoptosis and increased ATP production.

Effects of ethanol on insulin-like growth factor-Ⅰ system in primary cultured rat hepatocytes: Implications of JNK1/2 and alcoholdehydrogenase

AIM: To evaluate the effects of ethanol on the insulin- like growth factor-Ⅰ (IGF-Ⅰ) system involved in c-Jun N-terminal kinase (JNK1/2) and alcoholdehydrogenase (ADH) activity in primary cultured rat hepatocytes. METHODS: Hepatocytes isolated from male Sprague-Dawley rats were incubated with various concentrations of ethanol for different durations of time. The cells were pretreated with SP600125 (10 μmol/L) and 4-MP (200 μmol/L), and then treated with ethanol (200 mmol/L). We then measured IGF-Ⅰ secretion, IGF-Ⅰ mRNA expression, cell viability and JNK1/2 activity by radioimmunoassay, RT-PCR, MTT assay and Western blot, respectively (n = 6). RESULTS: Ethanol induced the activity of phospho (p)-JNK1/2, reaching a maximum at 60 min and then decreasing at 180 min. The effects of ethanol on the IGF-Ⅰ system were increased at 60 min (secretion: 7.11 ± 0.59 ng/mg protein vs 4.91 ± 0.51 ng/mg, mRNA expression: 150.2% ± 10.2% vs 101.5% ± 11.3%, P = 0.045) and then decreased at 180 min (secretion: 3.89 ± 0.25 ng/mg vs 5.4 ± 0.54 ng/mg protein; mRNA expression: 41.5% ± 10.4% vs 84.7% ± 12.1%, P = 0.04), however cell viability was decreased in a dose- and time-dependent manner. SP600125 blocked the ethanol-induced changes (at 60 min). Additionally, 4-methylpyrazole prevented the ethanol-induced decreases in the IGF-Ⅰ system, cell viability and p-JNK1/2 activity (at 180 min). CONCLUSION: This study suggests that ethanol- induced p-JNK1/2 activation is associated with the IGF-Ⅰ system and cell viability in hepatocytes. Furthermore, alcohol dehydrogenase is involved in the relationship between ethanol-induced inactivation of p-JNK1/2 and the changes of the IGF-Ⅰ system and cell viability.

Reactivation of the insulin-like growth factor-Ⅱsignaling pathway in human hepatocellular carcinoma

Constitutive activation of the insulin-like growth factor (IGF)-signaling axis is frequently observed in human hepatocellular carcinoma(HCC).Especially the over- expression of the fetal growth factor IGF-Ⅱ,IGF-Ⅰ receptor(IGF-IR),and cytoplasmic downstream effectors such as insulin-receptor substrates(IRS)contribute to proliferation,anti-apoptosis,and invasive behavior. This review focuses on the relevant alterations in this signaling pathway and independent in vivo models that support the central role IGF-Ⅱsignaling during HCC development and progression.Since this pathway has become the center of interest as a target for potential anti-cancer therapy in many types of malignancies,various experimental strategies have been developed,including neutralizing antibodies and selective receptor kinase inhibitors,with respect to the specific and efficient reduction of oncogenic IGF-Ⅱ/IGF-IR-signaling.

The expression of insulin-like growth factor-Ⅰ mRNA and polypeptide in rat osteoblasts with exposure to parathyroid hormone

Objective To investigate the insulin-like growth factor-Ⅰ (IGF-Ⅰ) gene and polypeptide expression in cultured rat osteoblast (ROB) and the role of IGF-Ⅰ in mediating the cell-to-cell communication by mimicking the pharmacokinetics of parathyroid hormone (PTH). Methods The ROB was cultured with three kinds of treatment: (1) Control (Ctr), the cells were cultured without PTH during the first 6 hours and the subsequent 42 hours in a 48-hour cycle; (2) Intermittent exposure to PTH (Itm), the cells were cultured with PTH during the first 6 hours, but without PTH in the subsequent 42 hours; and (3) Continuous exposure to PTH (Ctu), the cells were cultured with PTH during the first 6 hours and the subsequent 42 hours. Results The bone-forming activities of ROB were increased in Itm and inhibited in Ctu. The IGF-Ⅰ mRNA content in Itm cells was elevated only during the first 6 hours and that in Ctu cells was elevated at any time during an incubation cycle. The free IGF-Ⅰ concentration in the medium of Itm cells was generally higher and that of the Ctu cells was generally lower compared with those of the Ctr cells. The IGF-Ⅰ antibody significantly reduced the alkaline phosphatase activity within the cells of Ctr and Itm. Conclusions PTH rapidly and constantly stimulates the IGF-Ⅰ gene transcription of osteoblast. There was an obvious discrepancy between the IGF-Ⅰ mRNA content within the osteoblast and the free IGF-Ⅰ level around the osteoblast in either mode of PTH action. The IGF-Ⅰ might be important for osteoblast-osteoblast communication and bone-forming activity, not only in intermittent PTH administration, but also in the physiological functioning of osteoblasts.

翘嘴鲌胰岛素样生长因子-Ⅰ的克隆及序列分析

为探讨胰岛素样生长因子-Ⅰ(Insulin like growth factor-Ⅰ,IGF-Ⅰ)对于翘嘴鲌(Culter alburnus)生长性状的影响,对其DNA序列进行了克隆。翘嘴鲌IGF-Ⅰ全长14567 bp,由5个外显子和4个内含子组成。其5个外显子长度分别为298、160、182、36和1360 bp。推测的阅读框为486 bp,编码由161个氨基酸组成的IGF-Ⅰ前体蛋白。前体肽由信号肽、成熟肽、E肽三部分组成,其中信号肽44个氨基酸,成熟肽70个氨基酸,E肽47个氨基酸。成熟肽由B、C、A、D四个区域组成,其中B结构域和A结构域的保守性最高,在这2个区域包含由6个半胱氨酸残基形成的3个二硫键。翘嘴鲌B区域还包含保守的IGF-Ⅰ受体识别序列(Phe B23-Tyr B24-Phe B25)。E肽的长度表明翘嘴鲌IGF-Ⅰ属Ea-2型。同源性分析表明翘嘴鲌与鲤科鱼类的IGF-Ⅰ编码氨基酸同源性较高,为94%—100%,但在聚类分析中翘嘴鲌并不是首先和鲌亚科的鱼类聚集在一起。Real-time qPCR组织特异性表达结果显示IGF-ⅠmRNA在肝脏组织中的表达量最高,脾、心脏、精巢、脑次之,肾、鳃、胃和卵巢中表达量较低。翘嘴鲌IGF-Ⅰ基因4个内含子长度分别为1170、9364、251和1746 bp。相对外显子来说,种间内含子变异较大,其中第三内含子变异最大。翘嘴鲌IGF-Ⅰ基因中包含6个微卫星,(GATG)5AATAT(ATAG)11位于第一内含子中,(CT)8、(TTA)5、(AC)13、(TG)12和(ATT)5位于第二内含子中。其中4个微卫星位点具有多态性,将它们在120尾同塘养殖的翘嘴鲌中进行基因型与生长性状的关联性分析,均未达到显著水平(P>0.05)。结果为进一步研究该基因的表达、功能及其转录调控特征奠定了分子基础。

宽鳍鱲胰岛素样生长因子-Ⅰ的克隆及繁殖期前后表达分析

为探讨胰岛素样生长因子-Ⅰ(Insulin like growth factor-Ⅰ,IGF-Ⅰ)对宽鳍鱲(Zacco platypus)繁殖期前后生长性状的影响,开展了宽鳍鱲IGF-Ⅰ基因序列的克隆及表达定位分析。宽鳍鱲IGF-Ⅰ基因全长13707 bp,包含5个外显子、4个内含子,其中5个外显子长度分别为222、160、182、36和829 bp;内含子长度分别为1194、7771、254和1879 bp,推测开放阅读框为486 bp,编码161个氨基酸。实时荧光定量PCR(Real-time qPCR,RT-qPCR)结果显示,宽鳍鱲IGF-ⅠmRNA在肝脏组织中的表达水平最高,其次是性腺、脾脏、心脏和脑,在肾脏中表达水平最低。在7—8月,处于繁殖期的性成熟宽鳍鱲性腺中IGF-Ⅰ表达水平显著上升,繁殖期过后回落至最低值。在其他组织中IGF-Ⅰ表达水平在生长发育过程和繁殖期前后波动不大,且雄鱼大多数组织中IGF-Ⅰ基因平均表达水平高于雌鱼。荧光原位杂交技术(Fluorescence in situ hybridization,FISH)定位显示,宽鳍鱲IGF-Ⅰ基因基本为胞浆阳性,少数为核阳性,在肝组织中呈全胞质性分布,在性腺组织的精母细胞、卵泡膜及卵泡液中阳性表达。上述结果表明宽鳍鱲IGF-Ⅰ基因表达模式具有性别差异性,推测精巢中IGF-Ⅰ在繁殖期的高表达是宽鳍鱲雄性成体大于雌性成体的原因之一。研究结果为宽鳍鱲的性二态和人工繁育的研究提供参考资料。

Study on the Cloning, Expression, and Bioactivity of Recombinant Chicken IGF-Ⅰ

The DNA sequence encoding the chicken＇s insulin-like growth factor Ⅰ （IGF-Ⅰ） was amplified with the reverse transcription polymerase chain reaction （RT-PCR）, which was then cloned into vector pMD18-T and sequenced. The sequencing result showed that there was 100% homology among the documented sequences and the sequence reported in this article, which was successfully inserted into the expressing plasmid pRLC and was highly expressed in E.coli. The Tricine-SDS- PAGE result showed that the cloned recombinant protein was expressed in the form of inclusion bodies in the E.coli cell with molecular weight of 7.6 kD and was amount to 23% of the whole protein in the E.coli cell. Western blotting indicated that recombinant protein had the antigenicity of IGF- Ⅰ. The inclusion bodies were subsequently dissolved in 7 M guanidine chloride and renatured with dilution in refolding buffer containing 0.5 M arginine. To obtain pure protein, the renatured chicken IGF- Ⅰ was desalting by Hiprep 26/10 and purified by Hiprep Sephacryl S-200 chromatography. The biological activities of IGF- Ⅰ product were assayed in NIH 3T3 cells and osteoblastic cells of embryonic chicken by using MTT method. The results show that the expressed IGF- Ⅰ can obviously stimulate NIH3T3 cells and osteoblastic cells to proliferate at the concentration ranging from 100, 200, 400 to 800 ng mL^-1, suggesting that the protein has its biological activities.

Branched-chain amino acids partially recover the reduced growth of pigs fed with protein-restricted diets through both central and peripheral factors

The objective of this study was to assess the growth efficiency of pigs fed with protein-restricted diets supplemented with branched-chain amino acids(BCAA)and limiting amino acids(LAA)above the rec-ommended levels.Following 2 weeks of adaptation,48 young barrows were weight matched and randomly assigned to 6 treatments(8 pigs/treatment)for 4 weeks:positive control(PC)with standard protein,negative control(NC)with very low protein containing LAA(i.e.,Lys,Met,Thr and Trp)at rec-ommended levels,and NC containing LAA 25%(L25),LAA 50%(L50),LAA+BCAA(i.e.,Leu,Ile and Val)25%(LB25)and LAA+BCAA 50%(LB50)more than recommendations.Feed intake(FI)and body weight(BW)were measured daily and weekly,respectively.At week 6,blood samples were collected,all pigs euthanized and tissue samples collected.The data were analyzed by univariate GLM or mixed procedure(SPSS)and the means were separated using paired Student's t-test followed by Benjamini-Hochberg correction.Relative to PC,NC had decreased FI,BW,unsupplemented plasma essential amino acids,serum insulin-like growth factor-I(IGF-I)and hypothalamic neuropeptide Y(NPY)(P<0.01).Compared to NC,L25 or L50,LB50 had increased BW and serum IGF-I and decreased plasma serotonin and both LB25 and LB50 had higher FI,plasma BCAA,hypothalamic 5-hydroxytryptamine-receptor 2A and NPY and jejunal 5-hydroxytryptamine-receptor 7(P<0.01).Overall,supplementation of protein-restricted diets with increased levels of dietary BCAA partially recovered the negative effects of these diets on growth through improved IGF-I concentration and FI,which was associated with changed expression of serotonin receptors,blood AA and hypothalamic NPY.

Recombinant proteins secreted from tissue-engineered bioartificial muscle improve cardiac dysfunction and suppress cardiomyocyte apoptosis in rats with heart failure

Background Tissue-engineered bioartificial muscle-based gene therapy represents a promising approach for the treatment of heart diseases. Experimental and clinical studies suggest that systemic administration of insulin-like growth factor-1 （IGF-1） protein or overexpression of IGF-1 in the heart exerts a favorable effect on cardiovascular function. This study aimed to investigate a chronic stage after myocardial infarction （MI） and the potential therapeutic effects of delivering a human IGF-1 gene by tissue-engineered bioartificial muscles （BAMs） following coronary artery ligation in Sprague-Dawley rats.Methods Ligation of the left coronary artery or sham operation was performed. Primary skeletal myoblasts were retrovirally transduced to synthesize and secrete recombinant human insulin-like growth factor-1 （rhIGF-1）, and green fluorescent protein （GFP）, and tissue-engineered into implantable BAMs. The rats that underwent ligation were randomly assigned to 2 groups： MI-IGF group （n=6） and MI-GFP group （n=6）. The MI-IGF group received rhIGF-secreting BAM （IGF-BAMs） transplantation, and the MI-GFP group received GFP-secreting BAM （GFP-BAMs） transplantation. Another group of rats served as the sham operation group, which was also randomly assigned to 2 subgroups： S-IGF group （n=6）and S-GFP group （n=6）. The S-IGF group underwent IGF-1-BAM transplantation, and S-GFP group underwent GFP-BAM transplantation. IGF-1-BAMs and GFP-BAMs were implanted subcutaneously into syngeneic rats after two weeks of operation was performed. Four weeks after the treatment, hemodynamics was performed. IGF-1 was measured by radioimmunoassay, and then the rats were sacrificed and ventricular samples were subjected to immunohistochemistry. Reverse transcriptase-polymerase chain reaction （RT-PCR） was used to examine the mRNA expression of bax and Bcl-2. TNF-α and caspase 3 expression in myocardium was examined by Western blotting.Results Primary rat myoblasts were retrovirally transduced to secrete rhlGF-1 and tissue-engineered into implantable BAMs containing parallel arrays of postmitotic myofibers. In vitro, they secreted consistent levels of hIGF （0.4-1.2 μg·BAM-1·d-1）. When implanted into syngeneic rat, IGF-BAMs secreted and delivered rhIGF. Four weeks after therapy,the hemodynamics was improved significantly in MI rats treated with IGF-BAMs compared with those treated with GFP-BAMs. The levels of serum IGF-1 were increased significantly in both MI and sham rats treated with IGF-BAM. The mRNA expression of bax was lower and Bcl-2 expression was higher in MI-IGF group than MI-GFP group （P ＜0.05）.Western blotting assay showed TNF-α and caspase 3 expression was lower in MI-IGF group than MI-GFP group after therapy.Conclusions rhIGF-1 significantly improves left ventricular function and suppresses cardiomyocyte apoptosis in rats with chronic heart failure. Genetically modified tissue- engineered BAMs provide a method delivering recombinant protein for the treatment of heart failure.