Jiwen Wang

The role of insulin and glucose in goose primary hepatocyte triglyceride accumulation

SUMMARY In order to obtain some information on how fatty liver arises in geese, we investigated the role of insulin and glucose in triglyceride (TG) accumulation in goose primary hepatocytes. Goose primary hepatocytes were isolated and treated with insulin and glucose. Compared with the control group, 100 and 150 nmol l–1 insulin increased TG accumulation, acetyl-CoA carboxylase-α (ACCα) and fatty acid synthase (FAS) activity, and the mRNA levels of sterol regulatory element-binding protein-1 (SREBP-1), FAS and ACCα genes. Insulin at 200 nmol l–1 had an inhibiting effect on TG accumulation and the activity of ACC and FAS, but increased the gene expression of SREBP-1, FAS and ACCα. We also found that high glucose (30 mmol l–1) increased the TG level, ACC and FAS activity, and the mRNA levels of SREBP-1 and FAS. However, there was no effect of high glucose on ACCα mRNA level. In addition, the interaction between insulin and glucose was observed to induce TG accumulation, ACC and FAS activity, and gene expression of SREBP-1, FAS and ACCα, and increase SREBP-1 nuclear protein level and binding of nuclear SREBP-1 and the SRE response element of the ACC gene. The result also indicated that the glucose-induced TG accumulation decreased after 96 h when the hepatocytes were cultured with 30 mmol l–1 glucose. In conclusion, insulin and glucose may affect hepatic lipogenesis by regulating lipogenic gene expression and lipogenic enzyme activity in goose hepatocytes, and SREBP-1 might play an important role in the synergetic activation of lipogenic genes. We propose that the utilization of accumulated TG in hepatocytes is the reason for the reversible phenomenon in goose hepatocellular steatosis.

In ovo feeding of IGF-1 to ducks influences neonatal skeletal muscle hypertrophy and muscle mass growth upon satellite cell activation.

To investigate reasons for the muscle increase observed when eggs are treated by IGF‐1 and whether or not satellite cell activation is specific to different types of myofibers, duck eggs were administrated with IGF‐1. After injection, during the neonatal stages, the duck breast muscle and leg muscle were isolated for analysis. The muscle weight, muscle fiber diameter (MFD), cross‐sectional area (CSA), the number of myofibers per unit area (MFN) and frequency of satellite cell activation and mitosis at the embryo stage of 27 days (27E) and the postnatal stage of 2 days after hatching (P2D) were determined. In addition, expression of two important myogenic transcription factors MyoD and Myf5 were detected and compared in the two types of muscle tissues. Results indicated that IGF‐1 administration increased the duck body weight, MFD, CSA, MFN, and quantity of activated satellite cells and mitotic nuclei in the two types of muscle tissues. The MyoD and Myf5 expressed at a higher level in IGF‐1‐treated muscle. IGF‐1 stimulated muscle weight growth more in the leg muscle than in the breast muscle. These results indicate that in ovo feeding of IGF‐1 can stimulate duck growth and, especially, lead to increased muscle hypertrophy. These increases appear to be mainly dependent on the activation of satellite cells, some of which proliferate and fuse to the myofiber, enabling increased muscle mass. IGF‐1 can indirectly affect satellite cells by regulating the expression of two important myogenic transcription factors, MyoD and Myf5, which help activate satellite cells. J. Cell. Physiol. 227: 1465–1475, 2012.

MyoD expression profile and developmental differences of leg and breast muscle in Peking duck (Anas platyrhynchos Domestica) during embryonic to neonatal stages.

In order to investigate the developmental differences between the duck breast muscle and leg muscle tissues during the embryonic stage to neonatal stages, as well as the expression profile of MyoD between the two muscle tissues, the morphologic characteristics in the two muscle tissues during duck embryo stages at E14, E18, E22, E27 and D7 were compared through the muscle paraffin sections. The coding domain sequence of duck MyoD gene was cloned, and then the expression of MyoD in duck leg muscle and breast muscle during embryo stage on E10, E14, E18, E22, E27 and D7 was detected using qRT-PCR method. Results showed that the developmental status of the duck breast muscle in embryonic phrases lag behind that of leg muscle. The CDS of duck MyoD gene consists of 894 nucleotides, and showed relatively high similarity with the gene of other species. The MyoD mRNA expressed in both kinds of muscle tissues and the expression profile had a similar trend, although the expression level of MyoD in the breast muscle was significantly higher than that in the leg muscle at each developmental stages (p<0.05). Results suggested that MyoD might have potential functions in controlling muscle fiber phenotype during the secondary myogenesis of muscle development. These fundamental works may provide some valuable clues for knowing the roles of MyoD in the myogenesis and the muscle fiber type differentiation in birds.

Leptin exerts proliferative and anti-apoptotic effects on goose granulosa cells through the PI3K/Akt/mTOR signaling pathway.

Leptin was known as a pivotal regulator for the control of food intake and energy expenditure. However, leptin has also been found to be involved in the regulation of female reproductive system through interactions with pathways in the hypothalamic-hypophyseal axis and direct action at the ovarian level. In the present study, granulosa cells from goose ovarian preovulatory (F1-F3) follicles were cultured with leptin (0, 1, 10 or 100ng/ml). The proliferative and anti-apoptotic actions of leptin in granulosa cells were revealed by CCK-8, BrdU and TUNEL assays. Quantitative real-time PCR and Western blot analyses further indicated that leptin treatment led to increased expression of cyclin D1, cyclin D2, cyclin D3 and bcl-2, and decreased expression of p21 and caspase-3. The effects were involved in the activation of the PI3K/Akt/mTOR signaling pathway, as leptin treatment enhanced the expression of PI3K, Akt1, Akt2, Raptor, mTOR, S6K and p-S6K. Moreover, blockade of the PI3K/Akt/mTOR pathway attenuated the influences of leptin on proliferation and apoptosis of granulosa cells, considering that activated factors by leptin were inhibited in the presence of either 20μM LY294002 (a PI3K inhibitor) or 10μM rapamycin (an mTOR inhibitor). In addition, leptin had a modulatory effect on the expression of its receptor at the transcriptional and translational levels, and blockade of PI3K/Akt/mTOR inhibited both basal and leptin-induced Lepr gene and protein expression. These findings suggest that leptin exerts its proliferative and anti-apoptotic effects on goose granulosa cells through the PI3K/Akt/mTOR signaling pathway via interaction with its receptor.

In ovo administration of rhIGF-1 to duck eggs affects the expression of myogenic transcription factors and muscle mass during late embryo development

In ovo administration of IGF-1 to poultry eggs has effective roles on post hatching muscle development. However, the secondary muscle development stages at the late embryo development stage are important for muscle fiber formation and differentiation. To investigate the roles of in ovo administration of IGF-1 on duck secondary muscle development, we injected rhIGF-1 into duck eggs in hatching at day 12. After administration on days 18, 21, 24, and 27 in hatching (E18d, E21d, E24d, and E27d, respectively), muscle samples were isolated, and the muscle tissue weight, muscle fiber parameters, and myoblast proliferation rate in leg and breast muscle were analyzed. Additionally, the expression levels of the transcription factors MyoG and MRF4 were detected using qPCR. Results show that embryo body weight and muscle fiber parameters, including muscle fiber diameter (MFD) and the number of myofibers per unit area, are upregulated in IGF-1-treated groups. Moreover, the transcription factors MyoG and MRF4 are expressed at higher levels in the experimental groups compared with the control groups. These results suggest that in ovo administration of IGF-1 to poultry eggs can mediate the expression of MyoG and MRF4, induce myoblast proliferation, and finally influence muscle development during the secondary muscle development stages.

Developmental expression and alternative splicing of the duck myostatin gene

Myostatin (MSTN) plays a key role in the negative regulation of muscle growth and development during embryogenesis. The MSTN genes have different genetic characteristics in vertebrates: sole gene in mammals, gene duplication in fish, and alternative splicing in birds. To investigate the alternative splicing sites and developmental expression patterns of the duck MSTN genes, the mRNA and genome sequences were cloned, and the expression patterns were detected during breast muscle and leg muscle development by real-time PCR. In our study, four alternatively spliced forms of MSTN mRNA were found in the developing skeletal muscle of Peking duck, including two novel alternatively spliced transcripts, MSTN-c and MSTN-d. As a result of alternative splicing at the common GT-AG processing sites, MSTN-b and MSTN-c retained only the N-terminal TGFβ-propeptide superfamily domains. However, MSTN-d was not missing these domains, in contrast to MSTN-a. The real-time PCR results showed that there was no significant difference between breast muscle and leg muscle in MSTN-a mRNA expression, also in MSTN-b and MSTN-c. MSTN-a and MSTN-b have significant higher expressions than MSTN-c and MSTN-d, suggesting that they play the major role during embryo muscle development.

Role of mammalian sirtuin 1 (SIRT1) in lipids metabolism and cell proliferation of goose primary hepatocytes.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Authors. It has come to the attention of the corresponding author that there are two errors in Section 3.1 of the Results section titled “Effect of overfeeding on gene expression and enzyme activity of several genes in liver”. The first error is that the article contains the wrong number of overfeeding days. The second error is that there are incorrect correlations between liver weight, lipids content in live and plasma metabolic substrates because of the wrong overfeeding days. The authors take responsibility for them and apologize to the readership of Molecular and Cellular Endocrinology.

Molecular cloning, expression profile and transcriptional modulation of two splice variants of very low density lipoprotein receptor during ovarian follicle development in geese (Anser cygnoide)

Very low density lipoprotein receptor (VLDLR)-mediated endocytosis of plasma lipoproteins into the ovary is essential for ovarian follicle development. Two splice variants of VLDLR have been identified in several species, yet little is known about their distinctive roles in ovarian developing follicles. In the present study, the full-length cDNAs of two splice isoforms of VLDLR were obtained from geese (Anser cygnoide) ovaries using the RACE method. The longer isoform (TypeI VLDLR) is 3141bp and contains five conserved structural domains, while the other (TypeII VLDLR) lacks 90bp encoding for the O-linked sugar domain. TypeII VLDLR was predominantly expressed in the ovary, with greater amounts of mRNA in theca and granulosa cells from early stages of follicle development but decreased during vitellogenesis. However, there was minimal expression of the TypeI VLDLR gene in theca cells and expression was almost undetectable in granulosa cells throughout follicle development. Yolk VLDL concentrations decreased as stage of development advanced while yolk triglyceride and cholesterol concentrations increased in a follicular size-dependent manner. The significant correlations between transcripts of TypeII VLDLR and yolk lipids supported its important role on yolk lipid deposition. In addition, in vitro experiments suggested that exogenous cholesterol, 25-hydroxycholesterol and mevinolin (a highly potent competitive inhibitor of HMG-CoA) treatment could significantly alter TypeII VLDLR gene expression in granulosa cells from both pre-hierarchical and pre-ovulatory follicles. Collectively, data from the present study indicate that TypeII VLDLR is more important for the transport of plasma lipoproteins into developing follicles than TypeI VLDLR, and provide new evidence about the influence of steroids in modulating VLDLR gene expression in ovarian cells.

Molecular cloning and in silico analysis of the duck (Anas platyrhynchos) MEF2A gene cDNA and its expression profile in muscle tissues during fetal development

The role of myogenic enhancer transcription factor 2a (MEF2A) in avian muscle during fetal development is unknown. In this work, we cloned the duck MEF2A cDNA sequence (GenBank accession no. HM460752) and examined its developmental expression profiles in cardiac muscle, non-vascular smooth muscle and skeletal muscle. Duck MEF2A cDNA comprised 1479 bp encoding 492 amino acid residues. In silico analysis showed that MEF2A contained MADS (MCM1, AGAMOUS, DEFICIENS and SRF - serum response factor), MEF2 and mitogen-activated protein kinase (MAPK) transcription domains with high homology to related proteins in other species. Modified sites in these domains were conserved among species and several variants were found. Quantitative PCR showed that MEF2A was expressed in all three muscles at each developmental stage examined, with the expression in smooth muscle being higher than in the other muscles. These results indicate that the conserved domains of duck MEF2A, including the MADS and MEF2 domains, are important for MEF2A transcription factor function. The expression of MEF2A in duck smooth muscle and cardiac muscle suggests that MEF2A plays a role in these two tissues.

Role of leptin in the regulation of sterol/steroid biosynthesis in goose granulosa cells

Leptin is critical for reproductive endocrinology. The aim of this study is to assess the expression patterns of leptin receptor (Lepr) during ovarian follicle development and to reveal the mechanism by which leptin affects steroid hormone secretion in goose granulosa cells. Transcripts of Lepr were ubiquitous in all tested tissues, with pituitary and adrenal glands being the predominant sites. Goose ovarian follicles were divided into several groups by diameter including prehierarchical (4 to 6, 6 to 8, and 8 to 10 mm) and hierarchical (F5-F1) follicles. Lepr gene expression was significantly higher in granulosa cells than in theca cells from follicles of 4 to 8 mm in diameter. Expression of Lepr in granulosa cells decreased gradually as follicles developed, with fluctuating expression in F5 and F3 follicles. Lepr mRNA in theca cells underwent a slight decrease from the 6- to 8-mm cohorts to F5 follicle and then exhibited a transient increase and declined later. In vitro experiments in cultured goose granulosa cells showed that estradiol release was significantly stimulated, whereas progesterone increased slightly and testosterone decreased dramatically after leptin treatment. In accordance with the data for steroids, expression of Lepr, Srebp1, Cyp51, StAR, and Cyp19a1 were induced by the addition of leptin, and the concomitant changes in Hmgcs1, Dhcr24, Cyp11a1, 17β-hsd, Cyp17, and 3β-hsd gene expression were seen. These results suggested that leptin is involved in the development of goose ovarian follicles, and leptins effect on steroid hormone secretion could be due to altered sterol/steroidogenic gene expression via interaction with its receptor.