IGF-I mRNA Research Articles

Nutritional status affects Igf1 regulation of skeletal muscle myogenesis, myostatin, and myofibrillar protein degradation pathways in gopher rockfish (Sebastes carnatus)

Insulin-like growth factor-1 (Igf1) regulates skeletal muscle growth in fishes by increasing protein synthesis and promoting muscle hypertrophy. When fish experience periods of insufficient food intake, they undergo slower muscle growth or even muscle wasting, and those changes emerge in part from nutritional modulation of Igf1 signaling. Here, we examined how food deprivation (fasting) affects Igf1 regulation of liver and skeletal muscle gene expression in gopher rockfish (Sebastes carnatus), a nearshore rockfish of importance for commercial and recreational fisheries in the northeastern Pacific Ocean, to understand how food limitation impacts Igf regulation of muscle growth pathways. Rockfish were either fed or fasted for 14 d, after which a subset of fish from each group was treated with recombinant Igf1 from sea bream (Sparus aurata). Fish that were fasted lost body mass and had lower body condition, reduced hepatosomatic index, and lower plasma Igf1 concentrations, as well as a decreased abundance of igf1 gene transcripts in the liver, increased hepatic mRNAs for Igf binding proteins igfbp1a, igfbp1b, and igfbp3a, and decreased mRNA abundances for igfbp2b and a putative Igf acid labile subunit (igfals) gene. In skeletal muscle, fasted fish showed a reduced abundance of intramuscular igf1 mRNAs but elevated gene transcripts encoding Igf1 receptors A (igf1ra) and B (igf1rb), which also showed downregulation by Igf1. Fasting increased skeletal muscle mRNAs for myogenin and myostatin1, as well as ubiquitin ligase F-box only protein 32 (fbxo32) and muscle RING-finger protein-1 (murf1) genes involved in muscle atrophy, while concurrently downregulating mRNAs for myoblast determination protein 2 (myod2), myostatin2, and myogenic factors 5 (myf5) and 6 (myf6 encoding Mrf4). Treatment with Igf1 downregulated muscle myostatin1 and fbxo32 under both feeding conditions, but showed feeding-dependent effects on murf1, myf5, and myf6/Mrf4 gene expression indicating that Igf1 effects on muscle growth and atrophy pathways is contingent on recent food consumption experience.

MP30-19 HUMAN SPERM IGF2 mRNA LEVELS INVERSELY CORRELATE WITH EMBRYO GROWTH VELOCITY DURING EARLY STAGE OF EMBRYO DEVELOPMENT

You have accessJournal of UrologyCME1 Apr 2023MP30-19 HUMAN SPERM IGF2 mRNA LEVELS INVERSELY CORRELATE WITH EMBRYO GROWTH VELOCITY DURING EARLY STAGE OF EMBRYO DEVELOPMENT Rossella Cannarella, Scott Lundy, Nina Desai, Sarah Vij, and Aldo E. Calogero Rossella CannarellaRossella Cannarella More articles by this author , Scott LundyScott Lundy More articles by this author , Nina DesaiNina Desai More articles by this author , Sarah VijSarah Vij More articles by this author , and Aldo E. CalogeroAldo E. Calogero More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003258.19AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: IGF2 is a potent growth factor capable of influencing cell division and modulating numerous cellular functions. IGF2 mRNA has been identified in human spermatozoa, but little is known about its function in in early embryo development. The aim of this study is to evaluate whether IGF2 levels in spermatozoa is associated with early growth kinetics of human embryos in couples undergoing ICSI. METHODS: This study was conducted on patients undergoing ICSI treatment with fresh oocytes and fresh ejaculated spermatozoa. Couples with infertility due to suspected female factor (e.g., impaired oocyte quality as in patients with severe endometriosis and premature ovarian failure) were excluded from the study. Following clinical use, a surplus aliquot of the semen sample used for ICSI was used to analyze the levels of IGF2 mRNA. The kinetic parameters of each embryo were correlated with the amount of IGF2 mRNA measured in the same swim-up sperm sample used for ICSI. RESULTS: 16 patients were enrolled and a total of 131 embryos were collected. Patients were divided into two groups based on the βhCG test results. We found no statistically significant difference in IGF2 mRNA levels and embryo morphokinetic parameters between the two groups (Table 1). At the correlation analysis, we found a significant negative correlation between sperm IGF2 mRNA levels and the time of pronuclei (tPN) fading (r= -0.2572, p= 0.0078, n=106 embryos), and between sperm IGF2 mRNA and t2 (r= -0.2368, p= 0.0145, n=106 embryos). In contrast, sperm IGF2 mRNA levels were not significantly correlated with tPN, t3, t4, t5, t6, t7, t8, t9, t morula (tM), t blastocyst (tB), and time of expanded blastocyst (tEB) (Table 1). CONCLUSIONS: These results suggest a role for sperm IGF2 mRNA in the very early embryo development (tPN fading -t2). This evidence may help in identify the etiology of apparently idiopathic couple infertility. Indeed, since prolonged t2 has been associated with a poor chance of embryo development into a viable blastocyst, a low level of sperm IGF2 mRNA level can delay t2 and lead to no blastocyst formation. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e398 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Rossella Cannarella More articles by this author Scott Lundy More articles by this author Nina Desai More articles by this author Sarah Vij More articles by this author Aldo E. Calogero More articles by this author Expand All Advertisement PDF downloadLoading ...

382 The Role of the IL-6-IGF-II Axis in Systemic Sclerosis-Associated Lung Fibrosis

OBJECTIVES/GOALS: Interleukin (IL)-6 is produced in excess in Systemic Sclerosis (SSc). Likewise, microarray analysis of Insulin-like Growth Factor (IGF)-II-treated NL fibroblasts revealed increased expression of the basic helix-loop-helix transcription factor, BHLHB2. Our goal is to delineate the role of BHLHB2 in the fibrotic response to IGF-II and IL-6. METHODS/STUDY POPULATION: Primary lung fibroblasts were cultured from human lung tissues at 37oC and 5% CO2. Cell cultures were stimulated with IL-6. Gene expression was measured using quantitative PCR (qPCR). IGF-II mRNA expression levels after IL-6 stimulation were compared with those of the housekeeping gene PPIB (Peptidylprolyl Isomerase B). Western blot was performed on nuclear and chromatin-bound subcellular fractions from treated lung fibroblasts. BHLHB2 protein levels were assayed in response to IGF-II in comparison to PBS as vehicle control. RESULTS/ANTICIPATED RESULTS: Results: Our results show that IL-6 increases IGF-II levels in fibroblasts. In turn, IGF-II increases BHLHB2 nuclear localization. We further show that IL-6 increases BHLHB2 levels and its nuclear localization in lung fibroblasts. Our findings are novel since the role of the transcription factor BHLHB2 in the IL-6 induced/ IGF-II-mediated fibrotic response in SSc lung disease remains unexplored. DISCUSSION/SIGNIFICANCE: Our findings may provide a rationale for combination therapy to block IL-6 and IGF-II function concomitantly and thus halt the progression of SSc pulmonary fibrosis (PF). Our findings may have wide implications for lung fibrosis associated with various diseases, since SSc-PF, is characterized by the activation of common fibrotic pathways.

Eccentric and concentric exercises induce different adaptions in adipose tissue biology.

Alterations in adipose tissue (AT) metabolism related to inflammation and adipokine's production lead to perturbations in its capacity to store lipids and release fatty acids (FA) during feeding/fasting transition or during exercise. Exercise has a beneficial effect on AT metabolism, but conventional trainings are not always suitable for patients with functional limitations. Dynamic eccentric (ECC) exercise prevents the accumulation of AT and may then overcome those limitations. Consequently, this study aimed at investigating AT's adaptations after ECC training. Nine-week-old male rats were randomly assigned to a control sedentary or three-trained groups for which treadmill slopes modulated exercise oxygen consumption (VO2) and mechanical work (n = 15 per group): (1) + 15% uphill-concentric group (CONC), (2) - 15% downhill group (ECC15, same mechanical work as CONC) and (3) - 30% downhill group (ECC30, same VO2, or oxygen cost as CONC). Body composition and energy expenditure (EE) were measured before and after 8weeks of training. Subcutaneous AT was collected to study total FA profile and gene expression. Higher total EE was driven by lean mass gain in trained animals. In AT, there was a decrease in arachidonic acid with CONC or ECC15 training. Increased adiponectin, leptin, lipases, Glut4 and Igf1 mRNA levels in ECC15 group suggested major metabolic adaption in AT. In conclusion, ECC could induce beneficial modifications in AT fatty acid profile and the expression of key genes related to metabolism and insulin sensitivity.

Liver insulin-like growth factor-1 mediates effects of low-intensity vibration on wound healing in diabetic mice.

Chronic wounds in diabetic patients are associated with significant morbidity and mortality; however, few therapies are available to improve healing of diabetic wounds. Our group previously reported that low-intensity vibration (LIV) could improve angiogenesis and wound healing in diabetic mice. The purpose of this study was to begin to elucidate the mechanisms underlying LIV-enhanced healing. We first demonstrate that LIV-enhanced wound healing in db/db mice is associated with increased IGF1 protein levels in liver, blood, and wounds. The increase in insulin-like growth factor (IGF) 1 protein in wounds is associated with increased Igf1 mRNA expression both in liver and wounds, but the increase in protein levels preceded the increase in mRNA expression in wounds. Since our previous study demonstrated that liver was a primary source of IGF1 in skin wounds, we used inducible ablation of IGF1 in the liver of high-fat diet (HFD)-fed mice to determine whether liver IGF1 mediated the effects of LIV on wound healing. We demonstrate that knockdown of IGF1 in liver blunts LIV-induced improvements in wound healing in HFD-fed mice, particularly increased angiogenesis and granulation tissue formation, and inhibits the resolution of inflammation. This and our previous studies indicate that LIV may promote skin wound healing at least in part via crosstalk between the liver and wound. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

U-shaped association between serum IGF2BP3 and T2DM: A cross-sectional study in Chinese population.

To clarify the expression of N6-methyladenosine (m6 A) modulators involved in the pathogenesis of type 2 diabetes mellitus (T2DM). We further explored the association of serum insulin-like growth factor 2 mRNA-binding proteins 3 (IGF2BP3) levels and odds of T2DM in a high-risk population. The gene expression data set GSE25724 was obtained from the Gene Expression Omnibus, and a cluster heatmap was generated by using the R package ComplexHeatmap. Differential expression analysis for 13 m6 A RNA methylation regulators between nondiabetic controls and T2DM subjects was performed using an unpaired t test. A cross-sectional design, including 393 subjects (131 patients with newly diagnosed T2DM, 131 age- and sex-matched subjects with prediabetes, and 131 healthy controls), was carried out. The associations between serum IGF2BP3 concentrations and T2DM were modeled by restricted cubic spline and logistic regression models. Two upregulated (IGF2BP2 and IGF2BP3) and 5 downregulated (methyltransferase-like 3 [METTL3], alkylation repair homolog protein 1 [ALKBH1], YTH domain family 2 [YTHDF2], YTHDF3, and heterogeneous nuclear ribonucleoprotein [HNRNPC]) m6 A-related genes were found in islet samples of T2DM patients. A U-shaped association existed between serum IGF2BP3 levels and odds of T2DM according to cubic natural spline analysis models, after adjustment for body mass index, waist circumference, diastolic blood pressure, total cholesterol, and triglyeride. Multivariate logistic regression showed that progressively higher odds of T2DM were observed when serum IGF2BP3 levels were below 0.62 ng/mL (odds ratio 3.03 [95% confidence interval 1.23-7.47]) in model 4. Seven significantly altered m6 A RNA methylation genes were identified in T2DM. There was a U-shaped association between serum IGF2BP3 levels and odds of T2DM in the general Chinese adult population. This study provides important evidence for further examination of the role of m6 A RNA methylation, especially serum IGF2BP3 in T2DM risk assessment.

Diet, sex, and genetic predisposition to obesity and type 2 diabetes modulate motor and anxiety-related behaviors in mice, and alter cerebellar gene expression

Obesity and type 2 diabetes (T2D) are serious health problems linked to neurobehavioral alterations. We compared motor function, anxiety-related behavior, and cerebellar gene expression in TALLYHO/Jng (TH), a polygenic model prone to insulin resistance, obesity, and T2D, and normal C57BL/6 J (B6) mice. Male and female mice were weaned onto chow or high fat (HF) diet at 4 weeks of age (wk), and experiments conducted at young (5 wk) and old (14 – 20 wk) ages. In the open field, distance traveled was significantly lower in TH (vs. B6). For old mice, anxiety-like behavior (time in edge zone) was significantly increased for TH (vs B6), females (vs males), and for both ages HF diet (vs chow). In Rota-Rod testing, latency to fall was significantly shorter in TH (vs B6). For young mice, longer latencies to fall were observed for females (vs males) and HF (vs chow). Grip strength in young mice was greater in TH (vs B6), and there was a diet-strain interaction, with TH on HF showing increased strength, whereas B6 on HF showed decreased strength. For older mice, there was a strain-sex interaction, with B6 males (but not TH males) showing increased strength compared to the same strain females. There were significant sex differences in cerebellar mRNA levels, with Tnfα higher, and Glut4 and Irs2 lower in females (vs males). There were significant strain effects for Gfap and Igf1 mRNA levels with lower in TH (vs B6). Altered cerebellar gene expression may contribute to strain differences in coordination and locomotion.

Berberine promotes IGF2BP3 ubiquitination by TRIM21 to induce G1/S phase arrest in colorectal cancer cells

The increasing incidence of colorectal cancer (CRC) has become a major global public health burden. The natural drug Berberine (BBR) has shown potential in preventing CRC, and IGF2 mRNA binding protein 3 (IGF2BP3) may be a target of BBR. This study aims to investigate the mechanisms of BBR acting on IGF2BP3 to improve CRC. The results showed that IGF2BP3 played an important role in the development of CRC. BBR down-regulated IGF2BP3 expression and inhibited CRC growth in mice. Cell thermodynamic stability analysis (CETSA) and drug affinity responsive target stability (DARTS) analysis showed BBR may bind to IGF2BP3. BBR may induce structural changes in IGF2BP3 and decrease its protein stability in cytoplasm. The results from Co-Immunoprecipitation (Co-IP) suggested that BBR promoted the ubiquitination of IGF2BP3 by tripartite motif-containing protein 21 (TRIM21). Through RNA binding protein Immunoprecipitation (RIP) assay, it was found BBR inhibited the stabilization of CDK4/CCND1 mRNA by IGF2BP3 and promoted G1/S phase arrest in CRC cells. Overexpression of IGF2BP3 in vitro and in vivo attenuated the inhibition of CRC growth by BBR. This work demonstrated the potential of BBR targeting to IGF2BP3 in improving CRC and provided a new strategy for clinical treatment on CRC as well as novel anticancer drug design based on IGF2BP3 and TRIM21.

Detection of multiple biomarkers associated with satellite cell fate in the contused skeletal muscle of rats for wound age estimation.

From the perspective of forensic wound age estimation, experiments related to skeletal muscle regeneration after injury have rarely been reported. Here, we examined the time-dependent expression patterns of multiple biomarkers associated with satellite cell fate, including the transcription factor paired box 7 (Pax7), myoblast determination protein (MyoD), myogenin, and insulin-like growth factor (IGF-1), using immunohistochemistry, western blotting, and quantitative real-time PCR in contused skeletal muscle. An animal model of skeletal muscle contusion was established in 30 Sprague-Dawley male rats, and another five rats were employed as non-contused controls. Morphometrically, the data obtained from the numbers of Pax7 + , MyoD + , and myogenin + cells were highly correlated with the wound age. Pax7, MyoD, myogenin, and IGF-1 expression patterns were upregulated after injury at both the mRNA and protein levels. Pax7, MyoD, and myogenin protein expression levels confirmed the results of the morphometrical analysis. Additionally, the relative quantity of IGF-1 protein > 0.92 suggested a wound age of 3 to 7days. The relative quantity of Pax7 mRNA > 2.44 also suggested a wound age of 3 to 7days. Relative quantities of Myod1, Myog, and Igf1 mRNA expression > 2.78, > 7.80, or > 3.13, respectively, indicated a wound age of approximately 3days. In conclusion, the expression levels of Pax7, MyoD, myogenin, and IGF-1 were upregulated in a time-dependent manner during skeletal muscle wound healing, suggesting the potential for using them as candidate biomarkers for wound age estimation in skeletal muscle.

Postnatal exposure to a glyphosate-based herbicide interferes with the development and growth of the mammary gland of pre-pubertal Ewe lambs

The aim of the present study was to evaluate whether early postnatal exposure to a glyphosate-based herbicide (GBH) alters pre-pubertal mammary development in Friesian lambs. To this end, from postnatal day 1–14, ewe lambs were exposed subcutaneously or orally to GBH (2 mg/kg bw/day) or vehicle (control) and mammary gland biopsies were obtained at 45 days of age. GBH-exposed lambs exhibited larger mammary ducts and less area occupied by terminal duct lobular units than controls, accompanied by an increase in the area of adipocytes in the mammary stroma. Lambs subcutaneously exposed to GBH showed increased protein expression of estrogen receptor alpha; however, both GBH-exposed groups had decreased mRNA expression of this receptor. Control lambs showed nuclear progesterone receptor (PR) protein expression, whereas GBH-exposed animals showed cytoplasmic PR expression; both GBH-exposed groups exhibited decreased mRNA expression of PR. GBH-exposed lambs also had decreased epithelial cell proliferation. Regarding insulin-like growth factors, both groups showed similar IGF-1 mRNA and protein expression but decreased expression of its receptor, and increased IGFBP5 expression. In addition, phosphorylated AKT was only observed in the mammary gland of control lambs. Our results show that early postnatal exposure to GBH, regardless of the exposure route, affects the IGF-1 system and the AKT/protein kinase B pathway, interfering with steroid hormone receptor expression and cell proliferation. This consequently modifies the growth and development of the pre-pubertal mammary gland of Frisian lambs.

RF07 | PSUN384 Hepatocyte Deletion of IGF2 Prevents DNA Damage and Tumor Formation in Hepatocellular Carcinoma

Abstract Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide with a 18% 5-year survival rate. Mice lacking the splicing factor SRSF3 in hepatocytes (SKO mice) overexpress IGF2 and are predisposed to developing HCC with age. Loss of Igf2 in the SKO background prevented hepatic fibrosis and inflammation, and completely prevented tumor formation. This was associated with decreased proliferation, apoptosis and DNA damage, and restored DNA repair enzyme expression. Mechanistically, IGF2 treatment of HepG2 cells in vitro caused DNA damage and decreased DNA repair enzyme expression, and tumors from the SKO mice show mutational signatures consistent with double strand break and defective mismatch repair. Looking at human data, HCC patients with high IGF2 mRNA expression had worse survival compared to patients with normal IGF2 expression. The patients also showed a switch in IGF2 promoter usage that correlated with increased expression. HCC patients having high SRSF3 mRNA expression also showed poor survival as did patients with alterations in known SRSF3-dependent splicing events. The level of SRSF3 protein was decreased 6-fold in human HCC tissues compared with normal liver tissues. The results indicate that IGF2 overexpression in conjunction with reduced SRSF3 splicing activity could be a major cause of DNA damage and driver of liver cancer. Presentation: Saturday, June 11, 2022 1:18 p.m. - 1:23 p.m., Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Effects of maternal HF diet and absence of TRPC1 gene on mouse placental growth and fetal intrauterine growth retardation (IUGR)

Placental tissue intracellular calcium (Ca2+) regulates placental development and growth. Maternal high-fat diet (HFD) results in placental lipid accumulation, increased inflammation, reduced nutrient transport expression, and intrauterine growth restriction (IUGR). Currently, whether maternal HFD differentially affects placental and fetal growth and development under reduced Ca2+ influx is not yet known. We hypothesized that maternal HFD feeding decreases placental growth and development resulting in IUGR and that reduction of Ca2+ influx in the placenta worsens maternal HFD-induced placental dysfunction and IUGR. Three-week-old female B6129SF2/J wild type (WT) and transient receptor potential canonical 1 (TRPC1) protein deficient (KO) mice were fed normal fat (NF, 16 kcal % fat) and high fat (HF, 45 kcal % fat) diets for 12 weeks prior to mating with NF diet fed male mice. Fetuses and placentae were examined at mid- (D12) and late- (D18) gestation. At D12, maternal HFD had no effects on placental or fetal weight changes in WT and TRPC1 KO mice while absence of TRPC1 resulted in decreased placental and fetal weights. At D18, maternal HFD increased placental weights in both TRPC1 KO and WT mice, in part, by moderately increasing placental tissue triacylglyceride (TAG, P=.0632). At D12, mRNA expression of key placental growth factors including IGF1, PLGF, and VEGF were increased in WT compared to TRPC1 KO mice while IGF2 and VEGF mRNA expression were increased at D18. Results presented in our study demonstrated that maternal HFD increased placental weight, in part, due to increased lipid concentration resulting in IUGR and via an additive adverse effect of genotype and maternal HFD. Future studies are needed to determine the signaling mechanism underlying Ca2+ influx reduction-induced placental dysfunction and IUGR.

Differential mRNA expression in the induction of DNA damage, G2/M arrest, and cell death by zerumbone in HepG2/C3A cells

Zerumbone (ZER) is a phytochemical with antioxidant and antiproliferative properties. This study evaluated the cytoxicity of ZER combined with chemotherapeutic agents and the expression of mRNA genes related to cell cycle, cell death, xenobiotic metabolism, DNA damage, and endoplasmic reticulum (ER) stress in HepG2/C3A cells. ZER was cytotoxic (IC50, 44.31 μM). ZER-induced apoptosis was related to BBC3 and ERN1 upregulation (ER stress), and its antiproliferative effects were attributable to MYC, IGF1, and NF-kB mRNA inhibition. ZER-induced G2/M arrest and DNA damage was associated with mRNA expression of cell cycle (CDKN1A) and DNA damage (GADD45A) genes. Increased CYP1A2 and CYP2C19 mRNA expression suggested ZER metabolization, and reduced CYP1A1 and CYP2D6 expression indicated a longer time of action of ZER in the cell, enhancing its pharmacological effect. ZER downregulated TP53, PARP1, BIRC5 (apoptosis), and MAP1LC3A (autophagy). In apoptosis assay, the data of the association treatments with ZER suggested antagonism. In cytotoxicity assay, the data of the association treatments with ZER suggested synergism action to cisplatin and antagonism action to doxorubicin and 5-fluorouracil. Thus, ZER has potential for application in chemotherapy as it modulates mRNA targets; however, it may not have the desired efficiency when combined with other chemotherapeutic agents.

Imp interacts with Lin28 to regulate adult stem cell proliferation in the Drosophila intestine.

Stem cells are essential for the development and long-term maintenance of tissues and organisms. Preserving tissue homeostasis requires exquisite control of all aspects of stem cell function: cell potency, proliferation, fate decision and differentiation. RNA binding proteins (RBPs) are essential components of the regulatory network that control gene expression in stem cells to maintain self-renewal and long-term homeostasis in adult tissues. While the function of many RBPs may have been characterized in various stem cell populations, how these interact and are organized in genetic networks remains largely elusive. In this report, we show that the conserved RNA binding protein IGF2 mRNA binding protein (Imp) is expressed in intestinal stem cells (ISCs) and progenitors in the adult Drosophila midgut. We demonstrate that Imp is required cell autonomously to maintain stem cell proliferative activity under normal epithelial turnover and in response to tissue damage. Mechanistically, we show that Imp cooperates and directly interacts with Lin28, another highly conserved RBP, to regulate ISC proliferation. We found that both proteins bind to and control the InR mRNA, a critical regulator of ISC self-renewal. Altogether, our data suggests that Imp and Lin28 are part of a larger gene regulatory network controlling gene expression in ISCs and required to maintain epithelial homeostasis.

Effects of Decreasing Fishmeal as Main Source of Protein on Growth, Digestive Physiology, and Gut Microbiota of Olive Flounder (Paralichthys olivaceus).

Simple SummaryThe demand for fishmeal is increasing due to aquaculture development, but the supply is unstable. This indicates the need to reduce the fishmeal content in the feed and develop an optimal fish-feed formulation through substitutes. However, most studies on reducing fishmeal content in feed were conducted at the laboratory level. In this study, the application of a low-fishmeal diet as feed to olive flounder was evaluated in terms of growth-related factors, digestive physiology, and microbiota raised for five months in a fish farm using four feed formulations- FM70 [control (CON), 70% fishmeal], FM45 (45% fishmeal), FM35A (35% fishmeal), and FM35B (35% fishmeal + insect meal). There was no difference in growth-related factors, digestive physiology, and gut microbiota diversity compared with the CON-fed fish. Therefore, reducing the fishmeal content of the feed by up to 35% does not adversely affect growth and physiological characteristics under farm conditions.In olive flounder (Paralichthys olivaceus), growth performance, expression of growth-related factors, digestive physiology, and gut microbiota were assessed under farm conditions in the fish fed diets with low levels of fishmeal. Four experimental diets were prepared, FM70 [control (CON), 70% fishmeal], FM45 (45% fishmeal), FM35A (35% fishmeal), and FM35B (35% fishmeal + insect meal), and fed to the fish for five months. The CON-fed fish had the highest plasma GH, but IGF-1 and hepatic IGF-1 mRNA expression of the olive flounder fed diets with low-fishmeal levels did not significantly differ among diets. The intestinal villus length, muscular thickness, and the number of goblet cells were statistically similar, and ocular examination of hepatopancreas showed no discernable difference in all experimental diets. The chymotrypsin content of FM35B-fed fish is significantly lower, but trypsin and lipase contents are similar. The diversity of gut microbiota did not differ among groups, although the FM35B group had a higher composition of Firmicutes. Thus, a diet with reduced fishmeal content and several alternative protein sources can be used as feed ingredients in feed formulation for olive flounder reared under typical aquaculture farm conditions.

The insulin-like growth factor 1 stimulates ovarian steroidogenesis and oocyte maturation in spotted steed Hemibarbus maculatus

The insulin-like growth factor 1 (IGF1) plays important roles in fish reproduction. To understand the role of IGF1 in steroidogenesis and oocyte maturation, we investigated expression pattern and functions of Igf1 during in vitro maturation of spotted steed (Hemibarbus maculatus) oocytes. Igf1 expression quantified by qPCR and western blotting revealed that both Igf1 mRNA and protein was significantly increased in mature follicles. The immunofluorescence analyses showed IGF1 protein signal was detected in the theca cells of ovarian follicles during vitellogenesis stage, and in both theca cells and granulosa cells of matured oocytes. Incubation of ovarian follicles with recombinant IGF1 (rIGF1) significantly enhanced the rate of spotted steed oocyte germinal vesicle breakdown (GVBD), a marker of oocyte maturation. qPCR analysis showed that the transcription of steroidogenesis-related genes (3β-hsd, 20β-hsd, Cyp17a and Cyp19a1a) were influenced after IGF1 treatment. In addition, the expression profiles of receptors of Igf1, Fsh and Lh indicated that a cross talk response to IGF1 induced oocyte maturation. Furthermore, IGF1 treatment did not affect ERK protein expression but significantly upregulated CyclinB and AKT protein expression. All together, these findings help us to understand the molecular mechanism of IGF1 in regulation ovarian steroidogenesis and oocyte maturation of spotted steed.

The expression of IGFBP-5 in the reproductive axis and effect on the onset of puberty in female rats

Insulin-like growth factor-binding protein-5 (IGFBP-5) has recently been shown to alter the reproductive capacity by regulating insulin-like growth factor (IGF) bioavailability or IGF-independent effects. The present study aimed to investigate the effect and mechanism of IGFBP-5 on the onset of puberty in female rats. Immunofluorescence and real-time quantitative PCR were used to determine the expression and location of IGFBP-5 mRNA and protein distribution in the infant's hypothalamus-pituitary-ovary (HPO) axis prepuberty, peripuberty, puberty and adult female rats. Prepubertal rats with IGFBP-5 intracerebroventricular (ICV) were injected to determine the puberty-related genes expression and the concentrations of reproductive hormones. Primary hypothalamic cells were treated with IGFBP-5 to determine the expression of puberty-related genes and the Akt and mTOR proteins. Results showed that Igfbp-5 mRNA and protein were present on the HPO axis. The addition of IGFBP-5 to primary hypothalamic cells inhibited the expression of Gnrh and Igf-1 mRNAs (P < 0.05) and increased the expression of AKT and mTOR protein (P < 0.01). IGFBP-5 ICV-injection delayed the onset of puberty, reduced Gnrh, Igf-1, and Fshβ mRNAs, and decreased the concentrations of E2, P4, FSH,serum LH levels and the ovaries weight (P < 0.05). More corpus luteum and fewer primary follicles were found after IGFBP-5 injection (P < 0.05).

Initiation of muscle protein synthesis was unrelated to simultaneously upregulated local production of IGF-1 by amino acids in non-proliferating L6 muscle cells.

BackgroundIGF-1 is considered an important regulator of muscle protein synthesis. However, its role in stimulation of muscle protein synthesis by amino acids (AA) is not clear, despite pronounced alterations in IGF-1 mRNA expression and signaling in muscle tissues by feeding. This study evaluates the role of locally produced IGF-1 and IGF-1 signaling when skeletal muscle protein synthesis is activated by increased amino acid availability in confluent, non-proliferating cells.MethodsL6 skeletal muscle cells were subjected to amino acid starvation (24 h, 0.14 mM) followed by 18 h amino acid refeeding in Low AA (0.28 mM) or High AA concentrations (9 mM). Protein synthesis rates were estimated by L-[U-14C]-phenylalanine incorporation into cellular proteins. IGF-1 and IGF-1 receptor mRNA expression were quantified by real time PCR. SiRNA knockdown, antibodies and chemical inhibitors were used to attenuate muscle IGF-1 production and signaling.ResultsHigh AA concentrations (9mM) increased IGF-1 mRNA expression (+ 30%, p<0.05) and increased L-[U-14C]-phenylalanine incorporation compared to Low AA in confluent, non-proliferating muscle cells. Blocking IGF-1 signaling by chemical inhibitors reduced IGF-1 mRNA upregulation (~50%, p< 0.01), without decrease of protein synthesis. SiRNA knockdown of IGF-1 reduced protein synthesis, mainly explained by reduced cell proliferation. High AA or IGF-1 inhibitors did not change IGF-1 receptor mRNA expressions.ConclusionAmino acids increased IGF-1 mRNA expression and stimulated muscle protein synthesis. However, simultaneous upregulation of IGF-1 mRNA did not relate to increased protein synthesis by amino acids. The results indicate that increased IGF-1 mRNA expression is rather a covariate to amino acid initiation of protein synthesis in non-proliferating muscle cells; effects that may be related to unrecognized metabolic activities, such as transport of amino acids.

The Effect of Commercial Genetic Selection on Somatotropic Gene Expression in Broilers: A Potential Role for Insulin-Like Growth Factor Binding Proteins in Regulating Broiler Growth and Body Composition.

The somatotropic axis influences growth and metabolism, and many of its effects are a result of insulin-like growth factor (IGF) signaling modulated by IGF-binding proteins (IGFBPs). Modern commercial meat-type (broiler) chickens exhibit rapid and efficient growth and muscle accretion resulting from decades of commercial genetic selection, and it is not known how alterations in the IGF system has contributed to these improvements. To determine the effect of commercial genetic selection on somatotropic axis activity, two experiments were conducted comparing legacy Athens Canadian Random Bred and modern Ross 308 male broiler lines, one between embryonic days 10 and 18 and the second between post-hatch days 10 and 40. Gene expression was evaluated in liver and breast muscle (pectoralis major) and circulating hormone concentrations were measured post-hatch. During embryogenesis, no differences in IGF expression were found that corresponded with difference in body weight between the lines beginning on embryonic day 14. While hepatic IGF expression and circulating IGF did not differ between the lines post-hatch, expression of both IGF1 and IGF2 mRNA was greater in breast muscle of modern broilers. Differential expression of select IGFBPs suggests their action is dependent on developmental stage and site of production. Hepatic IGFBP1 appears to promote embryonic growth but inhibit post-hatch growth at select ages. Results suggest that local IGFBP4 may prevent breast muscle growth during embryogenesis but promote it after hatch. Post-hatch, IGFBP2 produced in liver appears to inhibit body growth, but IGFBP2 produced locally in breast muscle facilitates development of this tissue. The opposite appears true for IGFBP3, which seems to promote overall body growth when produced in liver and restrict breast muscle growth when produced locally. Results presented here suggest that paracrine IGF signaling in breast muscle may contribute to overall growth and muscle accretion in chickens, and that this activity is regulated in developmentally distinct and tissue-specific contexts through combinatorial action of IGFBPs.

Brief Research Report: Effect of Triiodothyronine on Hepatic Growth Hormone Receptor Expression in Primary Bovine Hepatocytes.

In previous studies, triiodothyronine (T3) was found to be lower in cows with ketosis and an effect of T3 on Growth Hormone Receptor (GHR) expression is described, e. g., in a human hepatoma cell line. Therefore, this study aimed to test whether T3 affects GHR messenger RNA (mRNA) expression in a well-established bovine hepatocyte model. Hepatocytes were kept in a sandwich culture and stimulated for 6 days with constant (10 μg/ml) or decreasing (from 10 to 5 μg/ml) T3 concentrations, and GHR, as well as IGF-1 mRNA expression, was measured using real-time polymerase chain reaction (RT-PCR). We could confirm in vitro that T3 has a stimulatory effect on GHR1A mRNA expression.