Acute handling disturbance modulates plasma insulin-like growth factor binding proteins in rainbow trout (Oncorhynchus mykiss)

The effects of acute stressor exposure on proximal (growth hormone [GH]) and distal (insulin-like growth factor-I [IFG-I] and insulin-like growth factor-binding proteins [IFGBPs]) components of the somatotropic axis are poorly understood in finfish. Rainbow trout (Oncorhynchus mykiss) were exposed to a 5-min handling disturbance to mimic an acute stressor episode, and levels of plasma GH, IGF-I, and IGFBPs at 0, 1, 4, and 24 h post-stressor exposure were measured. An unstressed group was also sampled at the same clock times (09:00, 10:00, 13:00, and 08:00 [the following day]) as acute stress sampling to determine temporal changes in the above somatotropic axis components. The acute stressor transiently elevated plasma cortisol and glucose levels at 1 and 4 h post-stressor exposure, whereas no changes were seen in the unstressed group. Plasma GH levels were not affected by handling stress or sampling time in the unstressed animals. Plasma IGF-I levels were significantly depressed at 1 and 4 h post-stressor exposure, but no discernible temporal pattern was seen in the unstressed animals. Using a western ligand blotting technique, we detected plasma IGFBPs of 21, 32, 42, and 50 kDa in size. The plasma levels of the lower-molecular-weight IGFBPs (21 and 32 kDa) were unaffected by handling stressor, nor were there any discernible temporal patterns in the unstressed animals. By contrast, the higher-molecular-weight IGFBPs (42 and 50 kDa) were affected by stress or time of sampling. Levels of the 42-kDa IGFBP levels significantly decreased over the sampling period in unstressed control animals, but this temporal drop was eliminated in stressed animals. Levels of the 50-kDa IGFBPs also decreased significantly over the sampling time in unstressed trout, whereas handling disturbance transiently increased levels of this IGFBP at 1 h but not at 4 and 24 h post-stressor exposure compared with the control group. Overall, our results suggest that acute stress adaptation involves modulation of plasma IGF-1 and high-molecular-mass IGFBP levels (42 and 50 kDa) in rainbow trout.     

Effects of short- and long-term fasting on plasma and stomach ghrelin,and the growth hormone/insulin-like growth factor I axis in the tilapia, Oreochromis mossambicus

Ghrelin is a highly conserved peptide hormone secreted by the stomach, which is involved in the regulation of food intake and energy expenditure. Ghrelin stimulates growth hormone (GH) release, and increases appetite in a variety of mammalian and non-mammalian vertebrates, including several fish species. Studies were conducted to investigate the effect of feeding and fasting on plasma and stomach ghrelin, and the growth hormone/insulin-like growth factor I (IGF-I) axis in the Mozambique tilapia, a euryhaline teleost. No postprandial changes in plasma and stomach ghrelin levels or stomach ghrelin mRNA levels were observed. Plasma levels of GH, IGF-I and glucose all increased postprandially which agrees with the anabolic roles of these factors. Fasting for 4 and 8 d did not affect ghrelin levels in plasma or stomach. Plasma GH was elevated significantly after 4 and 8 d of fasting, while plasma IGF-I levels were reduced. Plasma ghrelin levels were elevated significantly after 2 and 4 wk of fasting, but no change was detected in stomach ghrelin mRNA levels. Four weeks of fasting did not affect plasma GH levels, although plasma IGF-I and glucose were reduced significantly, indicating that GH resistance exists during a prolonged nutrient deficit (catabolic state). These results indicate that ghrelin may not be acting as a meal-initiated signal in tilapia, although it may be acting as a long-term indicator of negative energy balance.     

The preliminary study on the effects of growth hormone and insulin‐like growth factor‐I on κ‐casein synthesis in bovine mammary epithelial cells in vitro

The effects of growth hormone (GH) and insulin‐like growth factor‐I (IGF‐I) on protein synthesis and gene expression of κ‐casein in bovine mammary epithelial cell in vitro were studied. The treatments were designed as follows: the growth medium without serum was set as the control group, while the treatments were medium supplemented with GH (100 ng/ml), IGF‐I (100 ng/ml), and GH (100 ng/ml) + IGF‐I (100 ng/ml). The quantity of κ‐casein protein was measured by ELISA, and the κ‐casein gene (CSN3) expression was examined by real‐time quantitative PCR (RT‐qPCR). Compared with the control group, all the experimental groups had greater (p < 0.05) expression of CSN3. The concentration of κ‐casein followed a similar response as CSN3, but the difference between the treatments and the control was not statistically significant (p > 0.05). Furthermore, no synergistic effect of GH and IGF‐I was observed for both the κ‐casein concentration and CSN3 expression. It is therefore concluded that GH or IGF‐I can independently promote the expression of CSN3 in bovine mammary epithelial cells in vitro.     

Interactions of amino acids and hormones regulate the balance between growth and milk protein synthesis in lactating rats fed diets differing in protein content

Insulin-like growth factor-I (IGF-I), growth hormone (GH), and prolactin (PRL) play important roles in milk protein synthesis, and their plasma concentrations were reported to be affected by dietary protein intake. To investigate the relationship between circulating amino acid (AA) and concentrations of these hormones, 18 Wistar rats aged 14 wk were assigned to a low (LP; 9% protein), standard (SP; 21% protein), or high-protein (HP; 35% protein) diet from parturition through day 15 of lactation. Plasma, liver, pituitary gland, skeletal muscle, and mammary gland samples were collected at the end of treatment. Circulating and hepatic IGF-I concentrations increased linearly with elevated dietary protein concentrations (P < 0.0001). Rats receiving the HP diet had higher circulating GH (P < 0.01) and pituitary PRL concentrations (P < 0.0001) but lower pituitary GH concentration (P < 0.0001) relative to those in rats receiving the LP and SP diets. Pearson correlation test performed on composed data across treatments showed that several circulating AAs were correlated with circulating and tissue concentrations of IGF-I, GH, and PRL. Multiple linear regression analyses identified Leu, Gln, Ala, Gly, and Arg as the main AAs associated with hormone responses (R² = 0.37 ~ 0.80; P < 0.05). Rats fed the LP and HP diets had greater Igf1 and Ghr gene expression in skeletal muscle than those fed the SP diets (P < 0.01). However, LP treatment decreased Prlr mRNA abundance in mammary glands as compared with the SP and HP treatments (P < 0.05). The HP diets increased AA transporter expression (P < 0.01) but decreased mammalian target of rapamycin (P < 0.05) and 70 kDa ribosomal protein S6 kinase 1 (P < 0.01) phosphorylation in mammary glands as compared with the LP and SP diets. The results of the present study suggested that several circulating AAs mediated the effects of dietary protein supply on concentrations of IGF-I, GH, and PRL, which in turn altered the metabolism status in peripheral tissues including the lactating mammary glands.     

草原红牛IGFBP3基因多态性及与部分屠宰性状的相关性分析

以草原红牛为研究对象,采用PCR-SSCP方法检测IGFBP3基因的多态性,并将不同基因型与部分屠宰性状进行相关分析.结果表明在第2内含子发现多态性位点,有AA、AB和BB 3种基因型;测序结果显示在8 069 bp处存在C→T的碱基突变;该位点的多态性对草原红牛的眼肌面积有显著影响(P<0.05),AA型显著高于BB型(P<0.05),但与AB型之间差异不显著(P>0.05);其他屠宰性状在不同基因型间差异不显著.由此可以初步推断IGFBP3基因可能是影响草原红牛肉质性状的一个主效基因或是一个与影响肉质性状的QTL连锁的基因.     

Real-time RT-PCR quantification of pregnancy-associated plasma protein-A mRNA abundance in bovine granulosa and theca cells: effects of hormones in vitro

Ovarian follicular growth and dominance are controlled by a series of hormonal and intraovarian events including a decrease in intrafollicular IGF-binding proteins −2, −4 and −5 levels. Proteolytic enzymes such as pregnancy-associated plasma protein-A (PAPP-A) degrade IGFBPs and increase bioavailability of IGF-I and -II during follicular development. The objective of this study was to determine the effect of IGF-I, IGF-II, insulin (INS), LH, FSH, estradiol (E2), leptin or cortisol on ovarian PAPP-A mRNA levels. Granulosa (GC) from small (SM) (1–5 mm) and large (LG) (8–22 mm) follicles as well as theca cells (TC) from LG follicles were collected from bovine ovaries and cultured for 48 h in medium containing 10% FCS and then treated with various hormones in serum-free medium for an additional 24 h. Cells were treated with various concentrations (3–500 ng/ml) and combinations of IGF-I, IGF-II, FSH, LH, E2, INS, leptin and (or) cortisol for 24 h (Experiments 1–10). PAPP-A mRNA levels were measured using quantitative real-time RT-PCR. In SM-GC and LG-GC, none of the treatments significantly affected (P > 0.10) PAPP-A mRNA abundance. In LG-TC, IGF-I, LH or cortisol did not affect (P > 0.10) PAPP-A mRNA levels, whereas INS with or without LH decreased (P < 0.05) PAPP-A mRNA. E2 alone decreased PAPP-A mRNA levels in LG-TC, and E2 amplified the insulin-induced inhibition of PAPP-A mRNA abundance in LG-TC. We conclude that control of PAPP-A mRNA abundance in granulosa and theca cells differs, and that E2 may be part of an intraovarian negative feedback system which may reduce the bioavailable IGFs in the theca layer during growth and selection of follicles.     

Effects of ghrelin and its analogues on chicken ovarian granulosa cells

The aim of these in vitro experiments was (1) to examine the effects of ghrelin on the basic functions of ovarian cells (proliferation, apoptosis, secretory activity); (2) to determine the possible involvement of the GHS-R1a receptor and PKA- and MAPK-dependent post-receptor intracellular signalling cascades; (3) to identify the active part of the 28-amino acid molecule responsible for the effects of ghrelin on ovarian cells. We compared the effect of full-length ghrelin 1-28, a synthetic activator of GHS-R1a, GHRP6, and ghrelin molecular fragments 1-18 and 1-5 on cultured chicken ovarian cells. Indices of cell apoptosis (expression of the apoptotic peptide bax and the anti-apoptotic peptide bcl-2), proliferation (expression of proliferation-associated peptide PCNA), and expression of protein kinases (PKA and MAPK) within ovarian granulosa cells were analysed by immunocytochemistry. The secretion of progesterone (P(4)), testosterone (T), estradiol (E(2)) and arginine-vasotocin (AVT) by isolated ovarian follicular fragments was evaluated by RIA/EIA. It was observed that accumulation of bax was increased by ghrelin 1-28, GHRP6 and ghrelin 1-18, but not by ghrelin 1-5. Expression of bcl-2 was suppressed by addition of ghrelin 1-28, GHRP6 and ghrelin 1-5, but promoted by ghrelin 1-18. The occurrence of PCNA was reduced by ghrelin 1-28, GHRP6, ghrelin 1-18 and ghrelin 1-5. An increase in the expression of MAPK/ERK1, 2 was observed after addition of ghrelin 1-28, GHRP6 and ghrelin 1-18, but not ghrelin 1-5. The accumulation of PKA decreased after treatment with ghrelin 1-28 and increased after treatment with GHRP6 and ghrelin 1-18 but not ghrelin 1-5. Secretion of P(4) by ovarian follicular fragments was decreased after addition of ghrelin 1-28 or ghrelin 1-5 but stimulated by GHRP6 and ghrelin 1-18. Testosterone secretion was inhibited by ghrelins 1-28 and 1-18, but not by GHRP6 or ghrelin 1-5. Estradiol secretion was reduced after treatment with ghrelin 1-28 but stimulated by ghrelins 1-18 and 1-5; GHRP6 had no effect. AVT secretion was stimulated by ghrelin 1-28, GHRP6 and ghrelin 1-18, but inhibited by ghrelin 1-5. The comparison of the effects of the four ghrelin analogues on nine parameters of ovarian cells suggest (1) a direct effect of ghrelin on basic ovarian functions-apoptosis, proliferation, steroid and peptide hormone secretion; (2) that the majority of these effects can be mediated through GHS-R1a receptors; (3) an effect of ghrelin on MAPK- and PKA-dependent intracellular mechanisms, which can potentially mediate the action of ghrelin at the post-receptor level; (4) that ghrelin residues 5-18 may be responsible for the major effects of ghrelin on the avian ovary.     

Regulation of protein metabolism by insulin: value of different approaches and animal models

Insulin induces protein accretion by stimulating protein synthesis and inhibiting proteolysis. However, the mechanisms of regulation of protein metabolism by insulin are complex and still not completely understood. The use of approaches combining hyperinsulinemic clamp and isotopic methods, or measurement of the activation of intracellular kinases involved in insulin signaling, in addition to the use of different animal models in a comparative physiology process, provide better understanding of the potential regulation of protein metabolism by insulin. Studies using the clamp technique in lactating goats have shown a clear inhibitory effect of insulin on proteolysis, with an interaction between the effects of insulin and amino acids. Such studies revealed that the insulin-inhibited proteolysis is improved in lactating goats, this adaptative process limiting the mobilization of body protein under the conditions of amino acid deficit which occurs during early lactation. Insulin signaling studies in growing chickens have also provided some interesting features of insulin regulation compared to mammals. Refeeding or insulin injection leads to the activation of the early steps of insulin receptor signaling in the liver but not in the muscle. Muscle p70 S6 kinase, a kinase involved in the insulin activation of protein synthesis, was found to be markedly activated in response to insulin and to refeeding, suggesting that other signaling pathways than those classically described in mammalian muscles may be involved in signal transduction. Finally, although the role of insulin has been doubtful and has long been considered to be minor in ruminants and in avian species, this hormone clearly regulates protein metabolism in both species.     

Expression of tumor necrosis factor-alpha and cyclooxygenase-2 mRNA in porcine split-thickness wounds treated with epidermal growth factor by quantitative real-time PCR

Epidermal growth factor (EGF) accelerates the re-epithelialization of damaged epidermal cell layers in a wound, so it especially shortens the duration of wound healing. The effect of EGF on pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and cyclooxygenase-2 (COX-2), levels during wound healing has not been reported. We investigated the relationship between exogenous EGF treatment and the expression of TNF-alpha and COX-2 mRNA in porcine split-thickness wounds by real-time PCR. Twenty split-thickness wounds were created on the back of five pigs. Fifteen wounds were treated twice daily with EGF ointments (1 microg/g, 10 microg/g, and 50 microg/g) for 10 days and five wounds were untreated. Healing time until full-epithelialization was evaluated. We performed a quantitative analysis of TNF-alpha and COX-2 mRNA expression in wound biopsies using real-time PCR. Topical application of 1 microg/g EGF accelerated re-epithelialization more than treatments of EGF at 10 microg/g and 50 microg/g, and no treatment. The levels of TNF-alpha and COX-2 mRNA were significantly greater in wounds treated with 1 microg/g than those with 10 microg/g and 50 microg/g EGF, and no treatment. Topical treatment of EGF influences the level of TNF-alpha and COX-2 mRNA within porcine split-thickness wounds. EGF-dependent slightly up-regulation of TNF-alpha and COX-2 mRNA expression during the inflammatory phase of healing may create an optimal molecular environment for wound healing.     

Effects of N-methyl-D, L-aspartate on secretion of growth hormone-releasing hormone from the boar hypothalamic-preoptic area and median eminence in vitro

N-methyl-D, L-aspartate (NMA) elicited secretion of growth hormone (GH)-releasing hormone from both the hypothalamic-preoptic area and the median eminence that were collected from boars. We suggest that the previously described increase in GH secretion that follows peripheral treatment of swine with NMA is attributable, at least in part, to NMA-stimulated secretion of GH-releasing hormone from the central nervous system.