Altered muscle development and expression of the insulin-like growth factor system in growth retarded fetal pigs

We have used a porcine model of spontaneous differential fetal growth to investigate the effects of fetal size on muscle development. We hypothesized that altered muscle development may occur in small fetuses as a consequence of modified expression of selected genes of the insulin-like growth factor system. We examined the development of the Longissimus muscle (m. Longissimus) in small fetuses and their average sized littermates. We collected small for gestational age fetuses and their average sized sibling on days 45, 65 and 100 of gestation (term is 113-116 days). Small fetuses had significantly lower body weight at all three stages of gestation (p<0.05) and significantly reduced secondary to primary muscle fibre ratio in m. Longissimus on day 100 (p<0.05) compared to their littermates. On day 65, the expression of insulin-like growth factor receptor 1 and insulin-like growth factor binding protein 3 were significantly higher (p<0.05) in m. Longissimus of the small fetuses compared with their average sized littermates. On day 100, the expression of insulin-like growth factor receptor 1 remained significantly higher (p=0.001), in addition to significantly higher levels of insulin-like growth factor receptor 2 and insulin-like growth factor binding protein 5 in the small fetuses (p<0.05). No difference in levels of myogenin was observed between the small and average sized littermates. In conclusion, we demonstrate that reduced fetal muscle development is associated with an increased expression of several genes of the insulin-like growth factor system in small fetuses in mid to late gestation.     

Investigation of the insulin-like growth factor system in the avian epiphyseal growth plate

Components of the insulin-like growth factor (IGF) system were investigated in chondrocytes isolated from the avian growth plate. The genes for IGF-I, IGF-II, type 1 IGF receptor (IGF-R), IGF binding protein-2 (IGFBP-2), IGFBP-3, IGFBP-5 and IGFBP-7 were found to be expressed in both proliferative and hypertrophic chondrocytes. The expression of IGF-II in proliferative chondrocytes was extremely high relative to IGF-I. Although IGF-I expression was significantly increased in hypertrophic chondrocytes, the level was still low relative to IGF-II. In cell culture, IGF-I stimulated proteoglycan synthesis and increased the expression of Indian hedgehog (Ihh) and type X collagen, markers of chondrocyte differentiation. IGF-II was found to be equally efficacious in stimulating proteoglycan biosynthesis. These observations suggest that IGF-II may play a significant role in avian growth plate physiology, which is consistent with several reports on mammalian endochondral bone growth.     

Endometrial expression of the insulin-like growth factor system during uterine involution in the postpartum dairy cow

Rapid uterine involution in the postpartum period of dairy cows is important to achieve a short interval to conception. Expression patterns for members of the insulin-like growth factor (IGF) family were determined by in situ hybridisation at day 14 ± 0.4 postpartum (n = 12 cows) to investigate a potential role for IGFs in modulating uterine involution. Expression in each uterine tissue region was measured as optical density units and data were analysed according to region and horn. IGF-I mRNA was localized to the sub-epithelial stroma (SES) of inter-caruncular and caruncular endometrium. Both IGF-II and IGF-1R expression was detected in the deep endometrial stroma (DES), the caruncular stroma and myometrium. IGFBP-2, IGFBP-4 and IGFBP-6 mRNAs were all localised to the SES of inter-caruncular and caruncular uterine tissue, and in the DES and caruncular stroma, with IGFBP-4 mRNA additionally expressed in myometrium. IGFBP-3 mRNA was only detectable in luminal epithelium. IGFBP-5 mRNA was found in myometrium, inter-caruncular and caruncular SES and caruncular stroma. These data support a role for IGF-I and IGF-II in the extensive tissue remodelling and repair which the postpartum uterus undergoes to return to its non-pregnant state. The differential expression of binding proteins between tissues (IGFBP-3 in epithelium, IGFBP-2, -4, -5 and -6 in stroma and IGFBP-4 and -5 in myometrium) suggest tight control of IGF activity within each compartment. Differential expression of many members of the IGF family between the significantly larger previously gravid horn and the previously non-gravid horn may relate to differences in their rate of tissue remodelling.     

Basic principles of muscle development and growth in meat-producing mammals as affected by the insulin-like growth factor (IGF) system

This presentation aims to describe how the basic events in prenatal muscle development and postnatal muscle growth are controlled by the insulin-like growth factor system (IGF). The prenatal events (myogenesis) cover the rate of proliferation, the rate and extent of fusion, and the differentiation of three myoblast populations, giving rise to primary fibers, secondary fibers, and a satellite cell population, respectively. The number of muscle fibers, a key determinant of the postnatal growth rate, is fixed late in gestation. The postnatal events contributing to myofiber hypertrophy comprise satellite cell proliferation and differentiation, and protein turnover. Muscle cell cultures produce IGFs and IGF binding proteins (IGFBPs) in various degrees depending on the origin (species, muscle type) and state of development of these cells, suggesting an autocrine/paracrine mode of action of IGF-related factors. In vivo studies and results based on cell lines or primary cell cultures show that IGF-I and IGF-II stimulate both proliferation and differentiation of myoblasts and satellite cells in a time and concentration-dependent way, via interaction with type I IGF receptors. However, IGF binding proteins (IGFBP) may either inhibit or potentiate the stimulating effects of IGFs on proliferation or differentiation. During postnatal growth in vivo or in fully differentiated muscle cells in culture, IGF-I stimulates the rate of protein synthesis and inhibits the rate of protein degradation, thereby enhancing myofiber hypertrophy. The possible roles and actions of the IGF system in regulating and determining muscle growth as affected by developmental stage and age, muscle type, feeding levels, treatment with growth hormone and selection for growth performance are discussed.     

胰岛素样生长因子系统在动物生产中的研究进展

GH的促生长作用要通过肝脏、软骨等组织产生生长因子，即胰岛紊样生长因子(insulin-like growth factor，IGF)来介导。IGF系统与动物的生长发育、生产关系密切。作者综述了近年来IGF系统在动物生产方面的研究进展。     

Nutritional regulation of the genes encoding the acid-labile subunit and other components of the circulating insulin-like growth factor system in the sheep

In sheep, perinatal maturation of the endocrine arm of the insulin-like growth factor (IGF) system is characterized by two developmental events. First, concentrations of circulating IGF-I increase rapidly after birth and become responsive to changes in nutrition and growth hormone (GH). Second, the liver initiates synthesis of a serum protein called the acidlabile subunit (ALS). The acid-labile subunit promotes the endocrine actions of IGF-I and -II by recruiting them to long-lived complexes of 150 kDa. In this study, we examined the effect of nutrition on hepatic expression of the ALS gene around the time of birth and later in life. Expression of genes encoding other components of the circulating IGF system was also measured. At d 130 of fetal life, fetuses suffering from chronic undernutrition caused by placental insufficiency had lower expression of the ALS and IGF-I genes than well-nourished fetuses, but they did not have any changes in the expression of the IGF-binding protein (IGFBP)-2 or IGFBP-3 genes. In early postnatal life, hepatic gene expression was analyzed between d 12 and 38 in lambs fed a milk replacer at levels sustaining weight gains of 150 or 337 g/d. The lower plane of nutrition decreased the expression of the ALS, IGF-I, and GH receptor genes and increased the expression of the IGFBP-2 gene; expression of the IGFBP-3 gene was not affected by nutrition at this stage of life. Finally, hepatic gene expression was measured in 3-mo-old lambs offered ad libitum levels of a balanced diet or of a diet limiting for both energy and protein. Although the rate of growth of the lambs fed the limiting diet was reduced by 38%, the only effect detected in hepatic gene expression was a ninefold increase in the abundance of IGFBP-2 mRNA. Overall, these results indicate that undernutrition during late fetal and early postnatal life delays hepatic expression of the ALS gene and final maturation of the endocrine IGF system.     

Exogenous pituitary and recombinant growth hormones induce insulin and insulin-like growth factor 1 resistance in pig adipose tissue

In the present study, pigs were treated daily for 7 days with exogenous porcine growth hormone (pGH; 70 micrograms/kg BW) in order to determine whether pGH induced insulin and insulin-like growth factor 1 (1GF-1) resistance in pig adipose tissue. In the first experiment, pituitary-derived pGH (ppGH) decreased basal and insulin-stimulated lipogenesis by 50%. Insulin sensitivity decreased more than 90% as the result of pGH treatment. Sensitivity and responsiveness to IGF-1 were decreased 50% by ppGH. In a second experiment, pigs were treated daily (70 micrograms/kg BW) with exogenous pituitary pGH (ppGH) or recombinant pGH (rpGH) for 7 days in order to determine if the effects of pGH were intrinsic properties of the hormone. Both rpGH and ppGH caused similar decreases in basal rates of lipogenesis, insulin- and IGF-1-stimulated lipogenesis, and insulin and IGF-1 responsiveness in pig adipose tissue. In summary, the decrease in adipose tissue growth of pigs treated chronically with pGH is due in large part to the suppression of fatty acid synthesis and a decrease in the ability of insulin to stimulate lipid synthesis in pig adipocytes. These responses are intrinsic properties of pGH since the effects of rpGH mimicked those of ppGH. The role and importance of a decrease in IGF-1 responsiveness remains to be resolved.     

Epitope-tagged insulin-like growth factor-I expression in muscle

Development of a recombinant insulin like growth factor I (IGF-I) that is distinguishable from its endogenous counterpart would provide a powerful tool for delineating the role of IGF in myogenesis. Therefore, the objective of this study was to create an epitope-tagged IGF-I that retains biological activity and determine whether expression of this construct is possible in muscle tissue following direct DNA injection. Expression vectors were created that encoded porcine IGF-I containing a T7 (11-amino acid) epitope-tag (TIGF). Immunoreactivity of the purified recombinant TIGF was confirmed using monoclonal antibodies. Biological activity was evaluated by examining differentiation of myoblasts cultured with TIGF or transfected with TIGF plasmid DNA. Addition of purified TIGF to myoblast cultures stimulated (P < 0.05) muscle creatine kinase levels similar to insulin (10(-5) M). Likewise, transfection of L6A1 with TIGF DNA hastened (P < 0.01) differentiation compared to control pcDNA-transfected myoblasts. The integrity of the recombinant protein was confirmed using a sandwich-configured enzyme linked immunosorbent assay. Finally, recombinant TIGF DNA was injected in porcine muscle and the ability to detect TIGF protein was evaluated. TIGF expression was detected in muscle fibers of injected porcine muscle. These data show that a T7 amino acid tag placed on the amino terminus of the IGF-I protein remains intact during processing and does not interfere with the biological activity of the molecule. Use of this DNA construct is an excellent tool for investigating the role of IGFs in control muscle development and provides a model to investigate other regulators of animal growth.     

猪肌肉组织表达序列标签(ESTs)的分析

研究构建了民猪肌肉组织cDNA文库,并在文库中随机挑选克隆进行测序,获得107个高质量的ESTs。经生物信息学分析,所研究的107个ESTs中,有98个单一克隆,其中71个为人类及其他物种的同源序列,23个为猪的已知ESTs,4个为未知ESTs。对这4个未知ESTs进行开放阅读框预测并进行BLASTn分析,没有找到高度同源的氨基酸序列。对已知功能基因表达谱的构建和分析结果表明:最多的是未分类,占47.88%;然后依次是基因/蛋白表达占26.76%、细胞代谢占9.86%、细胞结构/迁移占8.45%、细胞/机体防御占4.23%和细胞信号/传导占2.82%。     

Proteolysis of insulin-like growth factor binding proteins during preovulatory follicular development in cattle

The present study was conducted to evaluate changes in follicular fluid (FF) insulin-like growth factor binding protein (IGFBP) proteolytic activity and levels of steroids and IGFBP during follicular development in cattle. Estrous cycles of cows were synchronized with two injections of prostaglandin F2alpha (PGF) 11 d apart and follicular growth monitored via daily rectal ultrasonography in order to identify the dominant follicle. All cows were ovariectomized 48 hr after the second injection of PGF. Follicular fluid was collected individually for all follicles > 5 mm and pooled for small (1 to 5 mm) follicles. Follicular fluid estradiol and androstenedione levels were greater (P < 0.05) and progesterone and IGFBP-3 levels not different (P > 0.10) in large dominant than in small (1 to 5 mm) or large (>5 mm) subordinate follicles, whereas IGFBP-2, -4 and -5 levels were less (P < 0.05) in large dominant than in small or large subordinate follicles. To evaluate proteolysis of IGFBPs, FF was incubated with recombinant human (125) I-labeled IGFBP-2, -3, -4, and -5 and proteins separated by 12% SDS-PAGE. Follicular fluid caused little or no proteolysis of (125)I-lableled IGFBP-2 or -3. However, cleavage of (125)I-labeled IGFBP-4 and -5 by FF from large dominant follicles was greater (P < 0.05) than by FF from small or large subordinate follicles indicating that a protease to IGFBP-4 and -5 exists in estrogen dominant follicles. We conclude that lower levels of IGFBP-2 in estrogen dominant follicles of cattle are not due to increased proteolysis, whereas decreases in IGFBP-4 and -5 levels are likely due, in part, to increased protease activity. Changes in IGFBP may alter levels of bioavailable IGFs that stimulate steroidogenesis and mitogenesis in developing bovine follicles.     

IGF-1和IGFBP-3在5个地方猪种血液中含量及其与猪肉质性状相关性分析

为研究胰岛素样生长因子-1 (IGF-1)和胰岛素样生长因子结合蛋白-3 (IGFBP-3)在血液中的含量及其与胴体和肉质表型指标的相关性,采用ELISA技术检测了丫杈猪、乌金猪、成华猪、雅南猪和藏猪血液中IGF-1和IGFBP-3的含量,同时测定了肌纤维直径和眼肌面积.结果显示:藏猪肌纤维直径最小,且显著低于丫杈猪、...