Chunchun Han

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.

The Regulation of Lipid Deposition by Insulin in Goose Liver Cells Is Mediated by the PI3K-AKT-mTOR Signaling Pathway.

Background We previously showed that the fatty liver formations observed in overfed geese are accompanied by the activation of the PI3K-Akt-mTOR pathway and an increase in plasma insulin concentrations. Recent studies have suggested a crucial role for the PI3K-Akt-mTOR pathway in regulating lipid metabolism; therefore, we hypothesized that insulin affects goose hepatocellular lipid metabolism through the PI3K-Akt-mTOR signaling pathway. Methods Goose primary hepatocytes were isolated and treated with serum-free media supplemented with PI3K-Akt-mTOR pathway inhibitors (LY294002, rapamycin, and NVP-BEZ235, respectively) and 50 or 150 nmol/L insulin. Results Insulin induced strong effects on lipid accumulation as well as the mRNA and protein levels of genes involved in lipogenesis, fatty acid oxidation, and VLDL-TG assembly and secretion in primary goose hepatocytes. The stimulatory effect of insulin on lipogenesis was significantly decreased by treatment with PI3K-Akt-mTOR inhibitors. These inhibitors also rescued the insulin-induced down-regulation of fatty acid oxidation and VLDL-TG assembly and secretion. Conclusion These findings suggest that the stimulatory effect of insulin on lipid deposition is mediated by PI3K-Akt-mTOR regulation of lipogenesis, fatty acid oxidation, and VLDL-TG assembly and secretion in goose hepatocytes.

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.

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 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.

Effects of palmitic acid on lipid metabolism homeostasis and apoptosis in goose primary hepatocytes

Studies have shown that not only does palmitic acid promote triglyceride (TG) accumulation, but it also affects cell viability in in vitro steatosis models. However, to what degree these effects are mediated by steatosis in goose primary hepatocytes is unknown. In this study, the effects of palmitic acid on the lipid metabolism homeostasis pathway and on apoptosis were determined. The authors measured the mRNA levels of genes involved in TG synthesis, lipid deposition, fatty acid oxidation and the assembly and secretion of VLDL–TG in goose primary hepatocytes. The results indicated that palmitic acid can significantly reduce the activity of goose hepatocytes, and that palmitic acid had a significant effect on TG accumulation; however, with increasing palmitic acid concentrations, the extracellular TG and extracellular VLDL concentration gradually decreased. With increasing palmitic acid concentrations, the gene expression levels of DGAT1, DGAT2, PPARα, CPT-1, FoxO1 and MTTP (which regulate hepatic TG synthesis, fatty acid oxidation and the assembly and secretion of VLDL–TGs) first increased and then decreased; the change in PLIN gene expression was palmitic acid dose-dependent, similar to the regulatory mode of intracellular TG accumulation. In conclusion, this study clearly shows that palmitic acid can promote TG accumulation and induce apoptosis in goose primary hepatocytes, and this effect may be related to the lipid metabolism pathway.

Cloning and expression of stearoyl-CoA desaturase 1 (SCD-1) in the liver of the Sichuan white goose and landes goose responding to overfeeding

The EST sequence of goose (Anser cygnoides) Stearoyl-CoA desaturase 1(SCD-1) was obtained from a subtractive cDNA library. To further investigate the role of SCD-1 in lipid metabolism in geese, 5′-RACE and 3′-RACE were carried out in this study to obtain the complete cDNA sequence of goose SCD-1, which contained a 29-bp 5′ UTR, a 1074-bp open reading frame (ORF) encoding 357 amino acids, and a 125-bp 3′ UTR. The expression of SCD-1 was measured in several tissues, and the effects of overfeeding on the expression of SCD-1 were studied. The results of real time RT-PCR demonstrated that, compared to the brain, goose SCD-1 mRNA was more abundant in the liver. Overfeeding markedly increased the mRNA expression of SCD-1 in the liver of Sichuan White and Landes geese, and gene expression was markedly higher in the Sichuan White goose than in the landes goose. The mRNA abundance of SCD-1 in the liver had significant positive correlations with triacylglycerol (TG) content in liver lipids and in the levels of plasma insulin and very low-density lipoproteins (VLDL) levels in Sichuan white geese. However, the mRNA abundance of SCD-1 in the livers of Landes geese had only significant positive correlations with the TG content in liver lipids. In conclusion, SCD-1 is not only critical for hepatic steatosis in geese but is also important for the difference in lipid deposition in the livers of the two breeds.