GBA1-dependent membrane glucosylceramide reprogramming promotes liver cancer metastasis via activation of the Wnt/ß-catenin signalling pathway.

The effect of glucosylceramide (GlcCer) reprogramming on liver cancer metastasis remains poorly understood. In this study, we demonstrated that the protein expression of GBA1, which catalyses the conversion of GlcCer to ceramide, was downregulated in liver cancer tissue. A clinical relevance analysis revealed that low expression of GBA1 was associated with the metastatic potential of liver cancer cells. Furthermore, loss- and gain-of-function studies confirmed that low expression of GBA1 promoted metastasis of liver cancer both in vitro and in vivo. Mechanistic studies indicated that low expression of GBA1 enhanced the metastatic ability of liver cancer by promoting the epithelial-mesenchymal transition (EMT), in which Wnt signalling pathway is involved. In the plasma membrane (PM), GBA1-dependent GlcCer reprogramming increased LRP6 location in the PM leading to an interaction between GlcCer and LRP6, subsequently promoting LRP6 phosphorylation at Ser1490, and finally activating the Wnt/ß-catenin signalling pathway. To our knowledge, this is the first time to be found that GlcCer interacted with a protein. In addition, the results of mass spectrometry indicated that GlcCer d18:1/18:0 was the most notably increased studied species in the PM when GBA1 was downregulated, suggesting that GlcCer d18:1/18:0 may be the major functional lipid that promotes GBA1-dependent liver cancer metastasis. Thus, GBA1-mediated GlcCer reprogramming in the PM promotes metastasis of liver cancer via activation of the Wnt/ß-catenin signalling pathway, upregulation of GBA1 may be a potential therapeutic strategy to combat liver cancer metastasis.

Low-grade elevation of palmitate and lipopolysaccharide synergistically induced ß-cell damage via inhibition of neutral ceramidase.

High concentrations of free fatty acids (FFAs) or lipopolysaccharide (LPS) could lead to ß-cell apoptosis and dysfunction, while low-grade elevation of FFAs or LPS, which are more common in people with type 2 diabetes mellitus (T2DM) or obesity, have no obvious toxic effect on ß-cells. Palmitate is a component closely related to metabolic disorders in FFAs. Recent studies have found that low-grade elevation of palmitate and LPS synergistically affects the sphingolipid signaling pathway by activating Toll-like receptor 4 (TLR4) and further enhances the expression of inflammatory cytokines in immune cells. Previous studies demonstrated that sphingolipids also played an important role in the occurrence and development of T2DM. This study aimed to investigate the synergistic effects of low-grade elevation of palmitate and LPS on viability, apoptosis and insulin secretion in the rat pancreatic ß-cell line INS-1 or islets and the role of sphingolipids in this process. We showed that low-grade elevation of palmitate or LPS alone did not affect the viability, apoptosis, glucose-stimulated insulin secretion (GSIS) or intracellular insulin content of INS-1 cells or islets, while the combination of the two synergistically inhibited cell viability, induced apoptosis and decreased basal insulin secretion in INS-1 cells or islets. Treatment with palmitate and LPS markedly upregulated TLR4 protein expression and downregulated neutral ceramidase (NCDase) activity and protein expression. Additionally, low-grade elevation of palmitate and LPS synergistically induced a significant increase in ceramide and a decrease in sphingosine-1-phosphate. Blocking TLR4 signaling or overexpressing NCDase remarkably attenuated INS-1 cell injury induced by the combination of palmitate and LPS. However, inhibition of ceramide synthase did not ameliorate injury induced by palmitate and LPS. Overall, we show for the first time that low-grade elevation of palmitate and LPS synergistically induced ß-cell damage by activating TLR4 signaling, inhibiting NCDase activity, and further modulating sphingolipid metabolism, which was different from a high concentration of palmitate-induced ß-cell injury by promoting ceramide synthesis.

Biomineralized Composite Liquid Crystal Fiber Scaffold Promotes Bone Regeneration by Enhancement of Osteogenesis and Angiogenesis.

Liquid crystals (LCs) are appealing biomaterials for applications in bone regenerative medicine due to their tunable physical properties and anisotropic viscoelastic behavior. This study reports a novel composite poly (L-lactide) (PLLA) scaffold that is manufactured by a simple electrospinning and biomineralization technique that precisely controls the fibrous structure in liquid LC phase. The enriched-LC composites have superior mineralization ability than neat PLLA; furthermore BMSC cells were inoculated onto the HAP-PLLA/LC with hydroxyapatite (HAP) composite scaffold to test the capability for osteogenesis in vitro. The results show that the PLLA/LC with HAP produced by mineralization leads to better cell compatibility, which is beneficial to cell proliferation, osteogenic differentiation, and expression of the angiogenic CD31 gene. Moreover, in vivo studies showed that the HAP-PLLA/LC scaffold with a bone-like environment significantly accelerates new and mature lamellar bone formation by development of a microenvironment for vascularized bone regeneration. Thus, this bionic composite scaffold in an LC state combining osteogenesis with vascularized activities is a promising biomaterial for bone regeneration in defective areas.

Endoplasmic Reticulum-Associated Degradation (ERAD) Has a Critical Role in Supporting Glucose-Stimulated Insulin Secretion in Pancreatic ß-Cells.

The molecular underpinnings of ß-cell dysfunction and death leading to diabetes are not fully elucidated. The objective of the current study was to investigate the role of endoplasmic reticulum-associated degradation (ERAD) in pancreatic ß-cells. Chemically induced ERAD deficiency in the rat insulinoma cell line INS-1 markedly reduced glucose-stimulated insulin secretion (GSIS). The mechanistic basis for this effect was studied in cells and mice lacking ERAD as a consequence of genetic ablation of the core ERAD protein SEL1L. Targeted disruption of SEL1L in INS-1 cells and in mouse pancreatic ß-cells impaired ERAD and led to blunted GSIS. Additionally, mice with SEL1L deletion in ß-cells were chronically hyperglycemic after birth and increasingly glucose intolerant over time. SEL1L absence caused an entrapment of proinsulin in the endoplasmic reticulum compartment in both INS-1 cells and mouse pancreatic ß-cells. Both folding-competent and folding-deficient proinsulin can physiologically interact with and be efficiently degraded by HRD1, the E3 ubiquitin ligase subunit of the ERAD complex. GSIS impairment in insulinoma cells was accompanied by a reduced intracellular Ca2+ ion level, overproduction of reactive oxygen species, and lowered mitochondrial membrane potential. Together, these findings suggest that ERAD plays a pivotal role in supporting pancreatic ß-cell function by targeting wild-type and folding-deficient proinsulin for proteosomal degradation. ERAD deficiency may contribute to the development of diabetes by affecting proinsulin processing in the ER, intracellular Ca2+ concentration, and mitochondrial function.

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.

Five novel variants of GPR103 and their expression in different tissues of goose (Anser cygnoides).

GPR103 plays an important role in various tissues, while little information is available about the alternative splicing (AS) of its mRNA. In the present study, we used genomic PCR to identify the partial genomic locus of goose (Anser cygnoides) GPR103 and rapid amplification of cDNA ends (RACE)-PCR to identify five GPR103 variants, including the full-length variant (aGPR103-n) and four alternatively spliced variants (aGPR103-va, -vb, -vc and -vd). Sequence analysis showed that aGPR103-va and -vd are less likely to undergo nonsense-mediated mRNA decay, suggesting that they may be translated into truncated proteins. Quantitative real-time PCR (qRT-PCR) analysis revealed that the five variants are widely distributed in the brain and peripheral tissues of geese and show specific expression patterns. Thus, we here provide the first account of the GPR103 genomic locus and illustrate its transcriptional diversity and widespread distribution in geese.

RETRACTED: 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.

The cloning, characterization, and expression profiling of the LRP8 gene in duck (Anas platyrhynchos).

Low-density lipoprotein receptor-related protein 8 (LRP8) is a member of the low-density lipoprotein receptor gene family that functions in body lipoprotein homeostasis. In this study, reverse transcription-polymerase chain reaction, rapid amplification of cDNA ends, and real-time PCR were performed to characterize the duck LRP8 gene. The cDNA of duck LRP8 contained a 14-bp 5' UTR, a 2754-bp open reading frame, and a 189-bp 3' UTR. The duck LRP8 encoded a protein of 917 amino acid residues composed of five functional domains and resembling other members of the LDLR family, and it displayed high nucleotide and amino acid homology to the LRP8 sequences in other avian species. The mRNA expression level of LRP8 was greater in duck extra-hepatic adipose tissue than in the liver. The peak expression values of LRP8 in both liver and adipose tissues occurred at week 1 and were significantly higher than the values observed during any other week (p < 0.05). Differences in the expression patterns of LRP8 mRNA from weeks 2 to 8 of growth were observed in different organs. A consistent low expression was observed in the liver, and fluctuating expression was observed in the subcutaneous adipose tissue (up- and then down-regulated) and abdominal adipose tissue (down-, then up-, then down-regulated). These findings suggest that LRP8 might play more important roles in regulating lipid metabolism in extra-hepatic adipose tissues than in the liver during early growth after hatching in the duck.

De novo lipogenesis in the liver and adipose tissues of ducks during early growth stages after hatching.

In vivo de novo lipogenesis (DNL) in the liver and adipose tissues of ducks during early developmental stages after hatching has not previously been investigated. In this study, female Peking ducks (Anas platyrhynchos) at weeks 1 to 8 post-hatching were selected for experimentation. We measured the mRNA levels of 6 DNL-related genes in the duck liver, subcutaneous adipose tissue and abdominal adipose tissue by real-time PCR during the 8 weeks. Correlations of the plasma triacylglycerol (TG) and very low density lipoprotein (VLDL) concentrations with fat deposition at these sites were also detected during growth. Our results showed that fat content was highest in the subcutaneous adipose tissue and lowest in the liver during the growth period we studied. Additionally, plasma VLDL and TG were significantly associated with lipid content in adipose tissue (P<0.05), but not in the liver. Lastly, in the growing birds, the expression levels of lipogenic genes (with the exceptions SREBP-1c and SCD1) were much higher in the liver than in the adipose tissues, and the maximal expression levels of these genes occurred at week 4 or 5 at these sites. These findings indicated that the main site of DNL is always the liver in post-hatching ducks, and adipose tissues are of little importance for DNL. Taken together, our results suggested that the plasma lipoproteins contribute greatly to fat deposition in adipose tissues originating from hepatic lipogenesis.

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 TGFß-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.

Effects of linoleate on cell viability and lipid metabolic homeostasis in goose primary hepatocytes.

Studies have shown linoleate could not only promote cell viability but also affect lipid metabolism in mammals. However, to what degree these effects are mediated by steatosis in goose primary hepatocytes is unknown. In this study, the effect of linoleate on the lipid metabolic homeostasis pathway was determined. We measured the mRNA levels of genes involved in triglyceride synthesis, lipid deposition, ß-oxidation, and assembly and secretion of VLDL-TGs in goose (Anser cygnoides) primary hepatocytes. Linoleate significantly increased goose hepatocyte viability, and linoleate at 0.125 mM, 0.25 mM, 0.5 mM and 1.0 mM all showed a significant effect on TG accumulation. However, with increasing linoleate concentrations, the extracellular TG concentration and extracellular VLDL gradually decreased. DGAT1, DGAT2, PPARα, PPARγ, FoxO1, MTP, PLIN and CPT-1 mRNA was detected by real-time PCR. With increasing linoleate concentrations, the changes in DGAT1, DGAT2, PPARα and CPT-1 gene expression, which regulates hepatic TG synthesis and fatty acid oxidation, first increased and then decreased. Additionally, FoxO1 and MTP gene expression was reduced with increasing linoleate concentrations, and the change in PLIN gene expression was increased at all concentrations, similar to the regulation of intracellular TG accumulation. In conclusion, linoleate regulated TG accumulation and increased hepatocyte viability. The data suggest that linoleate does promote goose hepatocyte viability and steatosis, which may up-regulate TG synthesis-relevant gene expression, suppress assembly and secretion of VLDL-TGs, and increase fatty acid oxidation properly to function of goose primary hepatocytes.

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.

Screening and identification of differentially expressed genes in goose hepatocytes exposed to free fatty acid.

The overaccumulation of triglycerides in hepatocytes induces hepatic steatosis; however, little is known about the mechanism of goose hepatic steatosis. The aim of this study was to define an experimental model of hepatocellular steatosis with TG overaccumulation and minimal cytotoxicity, using a mixture of various proportions of oleate and palmitate free fatty acids (FFAs) to induce fat-overloading, then using suppressive subtractive hybridization and a quantitative PCR approach to identify genes with higher or lower expression levels after the treatment of cells with FFA mixtures. Overall, 502 differentially expressed clones, representing 21 novel genes and 87 known genes, were detected by SSH. Based on functional clustering, up- and down-regulated genes were mostly related to carbohydrate and lipid metabolism, enzyme activity and signal transduction. The expression of 20 selected clones involved with carbohydrate and lipid metabolism pathways was further studied by quantitative PCR. The data indicated that six clones similar to the genes ChREBP, FoxO1, apoB, IHPK2, KIF1B, and FSP27, which participate in de novo synthesis of fatty acid and secretion of very low density lipoproteins, had significantly lower expression levels in the hepatocytes treated with FFA mixtures. Meanwhile, 13 clones similar to the genes DGAT-1, ACSL1, DHRS7, PPARα, L-FABP, DGAT-2, PCK, ACSL3, CPT-1, A-FABP, PPARß, MAT, and ALDOB had significantly higher expression levels in the hepatocytes treated with FFA mixtures. These results suggest that several metabolic pathways are altered in goose hepatocytes, which may be useful for further research into the molecular mechanism of goose hepatic steatosis.

Identification of differentially expressed genes between hepatocytes of Landes geese (Anser anser) and Sichuan White geese (Anser cygnoides).

In this study, we examined gene expression in order to identify genes that are differentially expressed between the hepatocytes of Sichuan White geese and Landes geese. We hypothesized that such genes may be involved in the different predispositions between these two species to develop hepatic steatosis. RNA was isolated from primary hepatocytes of the two species, and suppression subtractive hybridization was employed to screen for genes that showed differences in mRNA expression. We built and characterized two reciprocal cDNA libraries that were enriched in genes up-regulated in Landes geese or Sichuan White geese. Using dot blot analysis we identified 128 of 600 randomly selected sequences that demonstrated differential expression between the two species. Of these differentially expressed genes, 115 sequences shared high homology with 46 known genes and 13 sequences corresponded to eight novel expressed sequence tags (ESTs). Based on functional clustering, up and down-regulated genes were mostly related to lipid metabolism, nuclear mRNA splicing, enzyme activity and transcription control. The expression of 18 selected clones was further studied by quantitative PCR. The data showed that eight clones similar to the genes ACSL5, CTGF, CIDEA, PPARγ, PCK, GSTS1, RPS4X, and THBS1 had significantly higher expression levels in the hepatocytes of Landes geese. In contrast, seven clones similar to the genes ADH5, YBX1, ASAH1, UCB, AOPVLDL, SCD-1, and ELOVL-6 had significantly higher expression levels in the hepatocytes of Sichuan White geese.

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

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-alpha (ACCalpha) and fatty acid synthase (FAS) activity, and the mRNA levels of sterol regulatory element-binding protein-1 (SREBP-1), FAS and ACCalpha 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 ACCalpha. 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 ACCalpha 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 ACCalpha, 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.