Bovine Intramuscular Preadipocytes Research Articles

LncBNIP3 Inhibits Bovine Intramuscular Preadipocyte Differentiation via the PI3K-Akt and PPAR Signaling Pathways.

Intramuscular fat (IMF) content is an economic trait in beef cattle that improves the meat quality. Studies have highlighted the correlation between long noncoding RNAs (lncRNAs) and IMF development. In this study, lncBNIP3 knockdown promoted bovine intramuscular preadipocyte differentiation. RNA-seq analysis of intramuscular preadipocytes with lncBNIP3 knockdown identified 230 differentially expressed genes. The PI3K-Akt and PPAR signaling pathways were enriched. lncBNIP3 interference promoted mRNA and protein expression of key genes in PI3K-Akt signaling pathway. LncBNIP3 interference reversed the effects of an AKT-inhibitor MK-2206 on Akt protein expression and lipid droplet accumulation, promoted mRNA and protein expression of essential genes in the PPAR signaling pathway, and ameliorated the inhibitory effects of a PPARg antagonist GW9662 on lipid accumulation. Therefore, lncBNIP3 inhibition of bovine intramuscular preadipocyte differentiation is likely mediated via the PI3K-Akt and PPAR signaling pathways. This study identified a valuable lncRNA with functional roles in IMF accumulation and revealed new strategies to improve beef quality.

Bta-miR-330 promotes bovine intramuscular pre-adipocytes adipogenesis via targeting SESN3 to activate the Akt-mTOR signaling pathway

Consumers are more inclined to choose beef with a high intramuscular fat content (IMF), which regulated by lots of factors. It is very significant to find a miRNA that plays a key role in the accumulation of IMF. In our study, we found that bta-miR-330 was highly expressed in Japanese black cattle and differentially expressed at intramuscular pre-adipocytes differentiation processes. Furthermore, we transfected the bta-miR-330 mimic & inhibitor in intramuscular pre-adipocytes. The results showed that bta-miR-330 inhibits the proliferation but promotes the adipogenesis of intramuscular pre-adipocytes. Subsequently, our study showed that bta-miR-330 binds to SESN3, which inhibits the adipogenesis of intramuscular pre-adipocytes. Moreover, we established the mechanism that bta-miR-330 promotes the adipogenesis of intramuscular pre-adipocytes by targeting SESN3 to activate the Akt-mTOR signaling pathway. Overall, our results revealed that bta-miR-330-SESN3-Akt-mTOR axis plays an important role in adipogenesis of intramuscular pre-adipocytes, which provides a molecular basis for increasing IMF content in beef cattle.

CircSSBP2 acts as a MiR-2400 sponge to promote intramuscular preadipocyte proliferation by regulating NDRG1.

Intramuscular fat (IMF) is a critical factor in beef quality. IMF is mainly distributed between muscle fibres and its accumulation can affect the marbling and meat quality of beef. IMF formation and deposition is a complex process and in recent years a group of non-coding RNAs (ncRNAs), known as circRNAs, have been discovered to play an important role in regulating intramuscular fat deposition. CircRNAs form a covalent loop structure after reverse splicing of precursor mRNAs. They can act by adsorbing miRNAs, thereby reducing their repressive effects on downstream target genes. Based on high-throughput sequencing of circRNAs in intramuscular fat of Qinchuan and Japanese black cattle, we identified a novel circSSBP2 that is differentially expressed between the two species and associated with adipogenesis. We show that circSSBP2 knockdown promotes bovine intramuscular preadipocyte proliferation, whereas overexpression inhibits bovine intramuscular preadipocyte proliferation. We also show that circSSBP2 can act as a molecular sponge for miR-2400 and that miR-2400 overexpression promotes bovine intramuscular preadipocyte proliferation. Furthermore, N-myc downstream-regulated gene 1 (NDRG1) was identified as a direct target gene of miR-2400, and NDRG1 interference promoted the proliferation of bovine intramuscular preadipocytes. In conclusion, our results suggest that circSSBP2 inhibits the proliferation of bovine intramuscular preadipocytes by regulating the miR-2400/NDRG1 axis.

Long Non-Coding RNA BNIP3 Inhibited the Proliferation of Bovine Intramuscular Preadipocytes via Cell Cycle.

The intramuscular fat (or marbling fat) content is an essential economic trait of beef cattle and improves the flavor and palatability of meat. Several studies have highlighted the correlation between long non-coding RNAs (lncRNAs) and intramuscular fat development; however, the precise molecular mechanism remains unknown. Previously, through a high-throughput sequencing analysis, we found a lncRNA and named it a long non-coding RNA BNIP3 (lncBNIP3). The 5' RACE and 3' RACE explored 1945 bp total length of lncBNIP3, including 1621 bp of 5'RACE, and 464 bp of 3'RACE. The nucleoplasmic separation and FISH results explored the nuclear localization of lncBNIP3. Moreover, the tissue expression of lncBNIP3 was higher in the longissimus dorsi muscle, followed by intramuscular fat. Furthermore, down-regulation of lncBNIP3 increased the 5-Ethynyl-2'- deoxyuridine (EdU)-EdU-positive cells. The flow cytometry results showed that the number of cells in the S phase was significantly higher in preadipocytes transfected with si-lncBNIP3 than in the control group (si-NC). Similarly, CCK8 results showed that the number of cells after transfection of si-lncBNIP3 was significantly higher than in the control group. In addition, the mRNA expressions of proliferative marker genes CyclinB1 (CCNB1) and Proliferating Cell Nuclear Antigen (PCNA) in the si-lncBNIP3 group were significantly higher than in the control group. The Western Blot (WB) results also showed that the protein expression level of PCNA transfection of si-lncBNIP3 was significantly higher than in the control group. Similarly, the enrichment of lncBNIP3 significantly decreased the EdU-positive cells in the bovine preadipocytes. The results of flow cytometry and CCK8 assay also showed that overexpression of lncBNIP3 inhibited the proliferation of bovine preadipocytes. In addition, the overexpression of lncBNIP3 significantly inhibited the mRNA expressions of CCNB1 and PCNA. The WB results showed that the overexpression of lncBNIP3 significantly inhibited the expression of the CCNB1 protein level. To further explore the mechanism of lncBNIP3 on the proliferation of intramuscular preadipocytes, RNA-seq was performed after interference with si-lncBNIP3, and 660 differentially expressed genes (DEGs) were found, including 417 up-regulated DEGs and 243 down-regulated DEGs. The KEGG pathway analysis showed that the cell cycle was the most significant pathway for the functional enrichment of DEGs, followed by the DNA replication pathway. The RT-qPCR quantified the expression of twenty DEGs in the cell cycle. Therefore, we speculated that lncBNIP3 regulated intramuscular preadipocyte proliferation through the cell cycle and DNA replication pathways. To further confirm this hypothesis, the cell cycle inhibitor Ara-C was used to inhibit DNA replication of the S phase in intramuscular preadipocytes. Herein, Ara-C and si-lncBNIP3 were simultaneously added to the preadipocytes, and the CCK8, flow cytometry, and EdU assays were performed. The results showed that the si-lncBNIP3 could rescue the inhibitory effect of Ara-C in the bovine preadipocyte proliferation. In addition, lncBNIP3 could bind to the promoter of cell division control protein 6 (CDC6), and down-regulation of lncBNIP3 promoted the transcription activity and the expression of CDC6. Therefore, the inhibitory effect of lncBNIP3 on cell proliferation might be understood through the cell cycle pathway and CDC6 expression. This study provided a valuable lncRNA with functional roles in intramuscular fat accumulation and revealed new strategies for improving beef quality.

Effects of the CDC10 (Septin 7) Gene on the Proliferation and Differentiation of Bovine Intramuscular Preadipocyte and 3T3-L1 Cells.

Intramuscular fat content and marbling affecting meat quality are important economic traits in beef cattle. CDC10 (cell division cycle 10 or Septin 7), a member of the septin family involved in cellular proliferation, was considered as a functional and positional candidate gene for beef marbling. In a previous study, we revealed that the expression levels of CDC10 were also positively correlated with marbling scores in Japanese Black cattle. However, the regulatory mechanism of the CDC10 gene on IMF deposition in cattle remains unclear. In the present study, flow cytometry, EdU proliferation assays, and Oil Red O staining results showed that overexpression of CDC10 could promote the differentiation of bovine intramuscular preadipocyte (BIMP) and 3T3-L1 cells, whereas knockdown of CDC10 resulted in the opposite consequences. Furthermore, quantitative PCR and Western blotting results showed that overexpression of CDC10 could promote the expression levels of adipogenic marker genes PPARγ and C/EBPα at both mRNA and protein levels in BIMP and 3T3-L1 cells, whereas knockdown of CDC10 resulted in the opposite consequences. Our results provide new insights into the regulatory roles of CDC10 in adipocytes in animals.

ACOX1, regulated by C/EBPα and miR-25-3p, promotes bovine preadipocyte adipogenesis.

Acyl-coenzyme A oxidase 1 (ACOX1) is the first and rate-limiting enzyme in peroxisomal fatty acid β-oxidation of fatty acids. Previous studies have reported that ACOX1 was correlated with the meat quality of livestock, while the role of ACOX1 in intramuscular adipogenesis of beef cattle and its transcriptional and post-transcriptional regulatory mechanisms remain unclear. In the present study, gain-of-function and loss-of-function assays demonstrated that ACOX1 positively regulated the adipogenesis of bovine intramuscular preadipocytes. The C/EBPα-binding sites in the bovine ACOX1 promoter region at −1142 to −1129 bp, −831 to −826 bp, and −303 to −298 bp were identified by promoter deletion analysis and site-directed mutagenesis. Electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) further showed that these three regions are C/EBPα-binding sites, both in vitro and in vivo, indicating that C/EBPα directly interacts with the bovine ACOX1 promoter and inhibits its transcription. Furthermore, the results from bioinformatics analysis, dual luciferase assay, site-directed mutagenesis, qRT-PCR, and Western blotting demonstrated that miR-25-3p directly targeted the ACOX1 3’UTR (3’UTR). Taken together, our findings suggest that ACOX1, regulated by transcription factor C/EBPα and miR-25-3p, promotes adipogenesis of bovine intramuscular preadipocytes via regulating peroxisomal fatty acid β-oxidation.

MicroRNA-378 regulates adipogenic differentiation in bovine intramuscular preadipocytes by targeting CaMKK2

ABSTRACT Intramuscular fat, as one of the most important palatability attribute of beef carcase, is the primary determinant of beef quality. The research of adipogenesis mechanism would provide new insight into intramuscular fatty deposition. Here, the role of microRNA-378 was investigated during bovine adipogenic differentiation. It was revealed that miR-378 expression exists variably in bovine major tissue and organs by RT-qPCR. It was predicted that miR-378 targets CaMKK2, as an AMPKα kinase, by DIANA Tools. For better research, primary preadipocytes with stable transfection for up-/down-regulated expression of miR-378 were constructed by lentiviral vectors with GFP gene. The analyses of qPCR showed that PPARγ and adiponectin mRNA levels increased, but C/EBPβ, pref-1 and CaMKK2 mRNA levels decreased during adipogenic differentiation. When miR-378 was overexpressed, preadipocytes proliferation became slower, there are more cellular lipid droplets, and PPARγ and C/EBPβ mRNA levels were higher, but pref-1, adiponectin and CaMKK2 were lower than control groups. Luciferase assay and western blot analysis validated that miR-378 binds the nucleotide sites of the 3′- untranslated region of CaMKK2, which inhibits the mRNA and protein expression of CaMKK2. These findings suggest that miR-378 promotes adipogenic differentiation in bovine intramuscular preadipocytes by targeting CaMKK2 via AMPK signalling pathway.

Bioinformatics analysis and transcriptional regulation of TORC1 gene through transcription factors NRF1 and Smad3 in bovine preadipocytes

Intramuscular fat is the an important factor that defines meat quality; however, enhancing its deposition without increasing the other three adipose depots (subcutaneous, visceral, and intermuscular fat) is a challenge for animal science and the meat industry. The TORC1 is a key regulator of adipogenesis and its regulation in bovine intramuscular preadipocytes has not been studied. The TORC1 is a member of the gene family that codes for a binding proteins which regulate transcription of cAMP which, is a key regulator of adipogenesis. In the present study, expression and sub-cellular localization of the TORC1 gene was analyzed in bovine preadipocytes. Bioinformatics tools were applied to characterize TORC1. To investigate the molecular mechanism of bovine TORC1 gene regulation, we cloned a 1008 bp of the 5’UTR regulatory region into a luciferase reporter vector. Different fragments were amplified using 5’UTR unidirectional deletion of the TORC1 promoter. Site directed mutation, dual luciferase reporter assay, RNAi interference and DNA-protein interaction (EMSA) were used to validate the regulatory roles of Smad3 and NRF1 in the regulation of TORC1 gene in bovine preadipocytes. The core promoter region of the TORC1 gene was identified at a location −410 to −155 bp upstream of transcription start site. Different vectors were constructed through serial deletion of the 5’UTR flanking region and in combination with site directed mutations and transcription interference through siRNA or shRNA, two transcription factors of NRF1 and Smad3 were found to be repressors in the promoter of the TORC1 gene. These findings were further confirmed through Electrophoretic Mobility Shift Assay (EMSA) within nuclear extracts of bovine adipocytes. The core promoter region of the TORC1 gene, spanning from −410 to −155 bp upstream of the transcription start site was specified in this study and this information will provide opportunity for the improvement of intramuscular fat in cattle.

Melatonin promotes triacylglycerol accumulation via MT2 receptor during differentiation in bovine intramuscular preadipocytes

Melatonin (N-acetyl-5-methoxytryptamine) is a derivative of tryptophan which is produced and secreted mainly by the pineal gland and regulates a variety of important central and peripheral actions. To examine the potential effects of melatonin on the proliferation and differentiation of bovine intramuscular preadipocytes (BIPs), BIPs were incubated with different concentrations of melatonin. Melatonin supplementation at 1 mM significantly increased peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein (C/EBP) β, and C/EBPα expression and promoted the differentiation of BIPs into adipocytes with large lipid droplets and high cellular triacylglycerol (TAG) levels. Melatonin also significantly enhanced lipolysis and up-regulated the expression of lipolytic genes and proteins, including hormone sensitive lipase (HSL), adipocyte triglyceride lipase (ATGL), and perilipin 1 (PLIN1). Moreover, melatonin reduced intracellular reactive oxygen species (ROS) levels by increasing the expression levels and activities of superoxide dismutase 1 (SOD1) and glutathione peroxidase 4 (GPX4). Finally, the positive effects of melatonin on adipogenesis, lipolysis, and redox status were reversed by treatment with luzindole, anantagonist of nonspecific melatonin receptors 1 (MT1) and 2 (MT2), and 4-phenyl-2-propionamidotetraline (4P-PDOT), a selective MT2 antagonist. These results reveal that melatonin promotes TAG accumulation via MT2 receptor during differentiation in BIPs.

Subcellular Localization and Polymorphism of Bovine FABP4 in Bovine Intramuscular Adipocytes

ABSTRACTFatty acid binding protein 4 (FABP4) I74 V, a gene polymorphism associated with unsaturated fatty acid contents, was discovered in Japanese Black cattle. Individuals with FABP4 I/I genotype contain a significantly high level of palmitoleic acid compared to those with FABP4 V/V genotype. It remains unknown how the FABP4 polymorphism leads to different palmitoleic acid contents.We overexpressed FABP4 of different genotypes in bovine intramuscular preadipocytes and examined whether the intracellular localization of FABP4 and the expression levels of lipid metabolism-related genes were different among cells expressing different genotypes. Nuclear localization was observed for the FABP4 V/V, while the FABP4 I/I almost did not. The cells expressing FABP4 of different genotypes were comparable in terms of the expression levels of genes involved in lipid metabolism. FABP4 I/I was localized in most of the lipid droplets 4 days after differentiation induction, whereas approximately 25% lipid droplet co-localized with FABP4 in cells expressing FABP4 V/V. The lipid droplet size increased when palmitoleic acid was added compared to the size observed when palmitic acid was added. These results suggest that lipid droplet enlargement caused by palmitoleic acid and genotype-dependent differences in the fatty acid transporting capacity underlie variations in palmitoleic acid content among FABP4 polymorphisms.

Effect of mevalonic acid on cholesterol synthesis in bovine intramuscular and subcutaneous adipocytes.

Mevalonic acid (MVA) is a key material in the synthesis of cholesterol; indeed, intracellular cholesterol synthesis is also called the mevalonic acid pathway. 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) is an essential enzyme in cholesterol biosynthesis. This study suggests that MVA may play an important role in the differentiation of bovine adipose tissue in vivo. We investigated differential mRNA expression in bovine intramuscular preadipocytes (BIPs) and bovine subcutaneous preadipocytes (BSPs) by culturing cells from the longissimus dorsi muscle and subcutaneous fat tissues of Luxi yellow cattle. The morphology of lipid accumulation of bovine preadipocytes was detected by Oil Red O staining, and total cholesterol (TC), low-density lipoprotein cholesterol (LDLC), and high-density lipoprotein cholesterol (HDLC) levels were measured. Temporospatial expression of HMGR was investigated by real-time quantitative polymerase chain reaction (PCR). The TC, LDLC, and HDLC content did not significantly differ over time but increased slowly with increasing MVA concentration. HMGR expression increased over time and with increasing concentrations of MVA. MVA increased adipose cell proliferation in a dose-dependent and time-dependent manner. MVA stimulated HMGR expression in two cell types and its influence on adipocyte differentiation.

Retinoic acids change gene expression profiles of bovine intramuscular adipocyte differentiation, based on microarray analysis.

Beef marbling is caused by intramuscular deposition and it is an economically important trait in the beef industry. Vitamin A (VA) is an important feed supplement for cattle, but it can hinder marbling if provided in excess. In cattle, VA forms various derivatives such as all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9cRA). Therefore, we investigated the genes involved in bovine intramuscular adipogenesis after VA treatment with ATRA and 9cRA. Differential gene expression levels were validated by microarray analysis in a clonal bovine intramuscular preadipocyte (BIP) cell line derived from the intramuscular adipose tissue of Japanese Black cattle. BIP cells were harvested 6 days after adipogenic stimulation with either 1 μmol/L ATRA, 1 μmol/L 9cRA or non-retinoic acid control. The ATRA- and 9cRA-treated cells exhibited reduced transcription of genes involved in the circulatory system and muscle development compared with the no retinoic acid (RA) treatment. In addition, the ATRA- and 9cRA-treated cells exhibited increased transcription of genes involved in the immune system, protein kinase B signaling and responses to various stimuli. These results demonstrate the lower expression of muscle development in ATRA- and 9cRA-treated BIP cells during adipogenesis.

Stimulatory effect of plasma samples from fattening cattle on adipogenesis-related gene expression in preadipocyte cells.

It is desirable to produce beef with high levels of monounsaturated fatty acids (MUFA), as this is related to fat softness and palatability. However, the physiology of MUFA synthesis in bovine fat during the fattening process remains to be established. In this study, in order to elucidate the relationship between plasma components and the fatty acid composition of intramuscular fat, we investigated the effect of plasma obtained from fattening cattle on the messenger RNA (mRNA) expressions of the adipogenesis-related gene in a clonal bovine intramuscular preadipocyte line (BIP cells). The mRNA expressions of stearoyl-CoA desaturase, adipocyte Protein 2, peroxisome proliferator-activated receptor gamma and sterol regulatory element-binding protein 1 in BIP cells were significantly higher following treatment with those plasma samples collected from the cattle with the highest diaphragmatic unsaturated fatty acids to saturated fatty acids ratio (US/S). Furthermore, the concentration of nonesterified fatty acid (NEFA) in the plasma samples had an inverse correlation with carcass diaphragmatic US/S. These results indicate that cattle with a low ratio of US/S in fat may be discriminated from the population of fattening cattle before slaughter by measuring the effect of their plasma on gene expression in BIP cells as well as their plasma concentration and composition of NEFA.

Dexamethasone and acetate modulate cytoplasmic leptin in bovine preadipocytes.

Hormonal and nutrient signals regulate leptin synthesis and secretion. In rodents, leptin is stored in cytosolic pools of adipocytes. However, not much information is available regarding the regulation of intracellular leptin in ruminants. Recently, we demonstrated that leptin mRNA was expressed in bovine intramuscular preadipocyte cells (BIP cells) and that a cytoplasmic leptin pool may be present in preadipocytes. In the present study, we investigated the expression of cytoplasmic leptin protein in BIP cells during differentiation as well as the effects of various factors added to the differentiation medium on its expression in BIP cells. Leptin mRNA expression was observed only at 6 and 8 days after adipogenic induction, whereas the cytoplasmic leptin concentration was the highest on day 0 and decreased gradually thereafter. Cytoplasmic leptin was detected at 6 and 8 days after adipogenic induction, but not at 4 days after adipogenic induction. The cytoplasmic leptin concentration was reduced in BIP cells at 4 days after treatment with dexamethasone, whereas cytoplasmic leptin was not observed at 8 days after treatment. In contrast, acetate significantly enhanced the cytoplasmic leptin concentration in BIP cells at 8 days after treatment, although acetate alone did not induce adipocyte differentiation in BIP cells. These results suggest that dexamethasone and acetate modulate the cytoplasmic leptin concentration in bovine preadipocytes.

Effect of retinoic acid on gene expression profiles of bovine intramuscular preadipocytes during adipogenesis

To investigate genes involved in intramuscular adipogenesis in ruminants, 16 genes with dramatic variable expression were selected. These were selected from the differentiation- and proliferation-phase libraries of our previous serial analysis of gene expression (SAGE) studies of a clonal bovine intramuscular preadipocyte (BIP) cell line. We harvested the BIP cells over 12 days after adipogenic stimulation with all-trans retinoic acid (ATRA). Quantitative real-time PCR confirmed the earlier SAGE study results of the expression patterns of 15 of the genes. On day 6, TG accumulation increased significantly in the BIP cells but was completely inhibited in the 3T3-L1 cells (the monogastric reference). ATRA enhanced expression levels of six genes whereas it suppressed expression of eight genes on day 3 of adipogenesis in the BIP cells. Forty-eight hours after transfection, the messenger RNA expression level of the adipose differentiation-related protein (ADFP), encoded by one of the upregulated genes, in the ADFP small interference RNA (siRNA)-transfected cells was 3.5% of that in negative control-transfected cells. Also, 6 days after induction the TG level in the ADFP siRNA-transfected cells was 21.8% lower than that in negative control-transfected cells. This analysis of gene expression profiles after ATRA treatment will contribute to our understanding of the molecular mechanisms involved in bovine intramuscular adipogenesis.

Proliferating bovine intramuscular preadipocyte cells synthesize leptin

Leptin is thought to be not only a satiety factor but also a stimulator of angiogenesis. We examined leptin, PPARγ2, and vascular endothelial growth factor (VEGF) expression in bovine intramuscular preadipocyte (BIP) cells during proliferation. The cells were seeded at 0.85 × 104 cells/cm2 and collected every day until the fifth day after passage. Leptin mRNA was present in the cells between days 2 and 4, as indicated by RT-PCR analysis. Western blot analysis showed a band for leptin at approximately 16 kDa on all of the days during growth, and the cytoplasmic concentration of leptin was highest on day 2 and decreased gradually thereafter. A PPARγ2 band at approximately 54 kDa was also observed on all days. The concentration was highest on day 2 and decreased thereafter, which is similar to the expression pattern of leptin. In constant, the expression level of VEGF protein did not change while in culture. We have demonstrated that BIP cells can synthesize both leptin and PPARγ2, with maximal synthesis occurring during maximal proliferation. Given the role of leptin in angiogenesis, we speculate that leptin is involved in the neovascularization of adipose tissue, because new organization of adipose tissue requires the growth of new blood vessels.

MEAT SCIENCE AND MUSCLE BIOLOGY SYMPOSIUM: The influence of extracellular matrix on intramuscular and extramuscular adipogenesis1

The extracellular matrix (ECM) and specific ECM components can have a major influence on cell growth, development, and phenotype. The influence of the ECM and ECM components on adipogenesis in vivo and in vitro will be reviewed in this paper. Engelbreth-Holm-Swarm substratum and laminin per se markedly increased attachment, spreading, and hypertrophy of preadipocytes in serum-free primary cultures of porcine adipose tissue stromal-vascular cells. Furthermore, primary cultures of stromal-vascular cells showed that preadipocytes express ECM components after preadipocyte recruitment. Staining for plant lectins, type IV collagen, and laminin in fetal pig adipose tissue demonstrates that adipocyte reactivity for laminin was strong throughout fetal development and was similar for developing adipocytes and vasculature. However, lectin binding and type IV collagen reactivity of blood vessels preceded that for adipocytes. Therefore, these studies indicated that the ECM and in particular laminin may play a critical role in morphological aspects of preadipocyte development. Specific inhibitors and modulators of collagen synthesis have been used to evaluate the role of collagens in the differentiation of bovine intramuscular preadipocytes (BIP) and other preadipocyte cell lines. Triglyceride accretion of BIP cells was inhibited by a general inhibitor of collagen biosynthesis, whereas specific inhibitors or modulators of type IV collagen inhibited 3T3-L1 cell differentiation. Further study revealed that compared with collagens types I to IV, type V and VI collagens have an important and active role in BIP adipogenesis. The growth of intramuscular bovine adipose tissue may be dependent on collagen newly synthesized and organized by the adipocytes per se. The role of extracellular or ECM proteolysis in regulating adipogenesis also will be reviewed in this paper. Many members of the matrix metalloproteinase (MMP) family are expressed by adipocytes, and specific inhibition of MMP-9 greatly reduces adipogenesis in vitro. Possibly, MMP and other proteases regulate turnover of key adipocyte ECM proteins that are involved in the regulation of preadipocyte proliferation and differentiation.

Effects of MicroRNA-143 in the differentiation and proliferation of bovine intramuscular preadipocytes

MicroRNAs (miRNAs) are short non-coding RNA that post-transcriptionally regulates gene expression. miRNA-143 has been proposed to play a role in the differentiation of preadipocytes. However, effects and mechanism of miRNA-143 in the differentiation of mammals intramuscular adipocytes is unknown. In this study, the fibroblast-like preadipocytes were cultured from the marbling muscle tissue of holstein steers by the ceiling culture method. The in vitro studies showed that the fibroblast-like preadipocytes could differentiated into mature adipocytes with up-regulated expression of miRNA-143. Furthermore, the transfection of the fibroblast-like preadipocytes with miRNA-143 antisense inhibitor induced a significant suppression of differentiation, and indicated by decreased storage of lipid droplets and down-regulated expression of key adipocytes regulatory genes such as CCAAT/enhancer-binding protein-α (C/EBPα) and fatty acid binding proteins-4 (FABP-4). On the contrary, cells proliferation were increased with miRNA-143 inhibitor transfection. Taken together, our study provide the first evidence for stimulation of endogenous miRNA-143 in the differentiation of bovine intramuscular fat, which in part contribute to the regulated expression of adipocyte genes.

Differentially expressed genes during bovine intramuscular adipocyte differentiation profiled by serial analysis of gene expression

Beef marbling or intramuscular fat deposition is an economically important carcass trait in Japanese Black cattle. To investigate genes involved in intramuscular adipogenesis, differential gene expression during adipogenesis in a clonal bovine intramuscular preadipocyte (BIP) cell line was profiled with serial analysis of gene expression (SAGE). We sequenced 75 283 tags for the proliferation phase (day 0) and 81 878 tags for the differentiation phase (4 days after adipogenic stimulation: day 4). A comparison of the unique SAGE tag frequencies between the day 0- and day 4-libraries revealed that 878 (2.8%) of the 30 989 unique putative transcripts were expressed at significantly different levels (P < 0.05); 401 tags (1.4%) were up-regulated and 477 tags (1.2%) were down-regulated in the day 4-library relative to the day 0-library. We confirmed up-regulation of 10 tags of the genes that were up-regulated in the previous subtraction cloning studies in BIP cells [Animal Science Journal, 76 (2005) 479]. Of the 878 differentially expressed tags, 377 were identified in the bovine RefSeq library and 356 were assigned a bovine draft genomic sequence. Fifteen tags were mapped in previously detected beef marbling quantitative trait loci (QTL) regions [Mammalian Genome, 18 (2007) 125]. These genes may be involved in the adipogenic processes of beef marbling.

Effect of SIRT1 on Apoptosis of Bovine Preadipocytes*

Sirtuin type 1 (SIRTl), a novel regulatory factor of adipocyte and myocyte, interacts with target the forkhead box O family 1 (FoxOl) to modulate cell proliferation and differentiation, aging, apotosis and metabolism. The effect of nicotinamide (NAM, inhibitor of SIRT1) on the apoptosis of bovine preadipocytes was investigated with Acridine Orange (AO) staining, the flow cytometry detection and quantitative real-time PCR (qPCR). The results showed that NAM inhibited the differentiation of bovine subcutaneous preadipocytes (BSP) and bovine intramuscular preadipocytes (BIP), and their apoptosis rates were higher than control. SIRT1 influenced the apoptosis of bovine preadipocytes by regulating FoxO1 and adipocyte marker genes. The different mechanisms of SIRT1 and its related genes exist in BSP and BIP.