Differentiation Of Bovine Preadipocytes Research Articles

LncRNA BlncAD1 Modulates Bovine Adipogenesis by Binding to MYH10, PI3K/Akt Signaling Pathway, and miR-27a-5p/CDK6 Axis.

Research on adipogenesis will help to improve the meat quality of livestock. Long noncoding RNAs (lncRNAs) are involved in mammalian adipogenesis as epigenetic modulators. In this study, we analyzed lncRNA expression during bovine adipogenesis and detected 195 differentially expressed lncRNAs, including lncRNA BlncAD1, which was significantly upregulated in mature bovine adipocytes. Gain- and loss-of-function experiments confirmed that BlncAD1 promoted the proliferation, apoptosis, and differentiation of bovine preadipocytes. RNA pull-down revealed that the nonmuscle myosin 10 (MYH10) is a potential binding protein of BlncAD1. Then, we elucidated that loss of BlncAD1 caused increased ubiquitination of MYH10, which confirmed that BlncAD1 regulates adipogenesis by enhancing the stability of the MYH10 protein. Western blotting was used to demonstrate that BlncAD1 activated the PI3K/Akt signaling pathway. Bioinformatic analysis and dual-luciferase reporter assays indicated that BlncAD1 competitively absorbed miR-27a-5p. The overexpression and interference of miR-27a-5p in bovine preadipocytes displayed that miR-27a-5p inhibited proliferation, apoptosis, and differentiation. Further results suggested that miR-27a-5p targeted the CDK6 gene and that BlncAD1 controlled the proliferation of bovine preadipocytes by modulating the miR-27a-5p/CDK6 axis. This study revealed the complex mechanisms of BlncAD1 underlying bovine adipogenesis for the first time, which would provide useful information for genetics and breeding improvement of Chinese beef cattle.

Multiple omics analysis reveals the regulation of SIRT5 on mitochondrial function and lipid metabolism during the differentiation of bovine preadipocytes

Preadipocyte differentiation represents a critical stage in adipogenesis, with mitochondria playing an undeniable pivotal role. Given the intricate interplay between transcription and metabolic signaling during adipogenesis, the regulation of sirtuin 5 (SIRT5) on mitochondrial function and lipid metabolism was revealed via multiple omics analysis. The findings suggest that SIRT5 plays a crucial role in promoting mitochondrial biosynthesis and maintaining mitochondrial function during preadipocyte differentiation. Moreover, SIRT5 modulates the metabolic levels of numerous bioactive substances by extensively regulating genes expression associated with differentiation, energy metabolism, lipid synthesis, and mitochondrial function. Finally, SIRT5 was found to suppress triacylglycerols (TAG) accumulation while enhancing the proportion and diversity of unsaturated fatty acids, and providing conditions for the expansion and stability of membrane structure during mitochondrial biosynthesis through numerous gene regulations. Our findings provide a foundation for the identification of crucial functional genes, signaling pathways, and metabolic substances associated with adipose tissue differentiation and metabolism.

CircADAMTS16 Inhibits Differentiation and Promotes Proliferation of Bovine Adipocytes by Targeting miR-10167-3p.

Circular RNAs (CircRNAs) are covalently closed-loop non-coding RNA (ncRNA) molecules present in eukaryotes. Numerous studies have demonstrated that circRNAs are important regulators of bovine fat deposition, but their precise mechanisms remain unclear. Previous transcriptome sequencing studies have indicated that circADAMTS16, a circRNA derived from the a disintegrin-like metalloproteinases with the thrombospondin motif 16 (ADAMTS16) gene, is high expressed in bovine adipose tissue. This gives a hint that the circRNA may be involved in the process of bovine lipid metabolism. In this study, the targeting relationship between circADAMTS16 and miR-10167-3p was verified using a dual-luciferase reporter assay. Then, the functions of circADAMTS16 and miR-10167-3p in bovine adipocytes were explored through gain-of-function and lose-of-function. The mRNA expression levels of genes were detected by real-time quantitative PCR (qPCR), and lipid droplet formation was phenotypically evaluated by Oil Red O staining. Cell proliferation and apoptosis were detected using CCK-8, EdU, and flow cytometry. We demonstrated that circADAMTS16 targeted binding to miR-10167-3p. The up-regulation of circADAMTS16 inhibited the differentiation of bovine preadipocytes, and the overexpression of miR-10167-3p promoted the differentiation of bovine preadipocytes. Meanwhile, CCK-8 and EdU results indicated that circADAMTS16 promoted adipocyte proliferation. Subsequently, flow cytometry analysis showed that circADAMTS16 promoted cell transition from G0/G1 phase to S phase, and inhibited cell apoptosis. However, up-regulation of miR-10167-3p inhibited cell proliferation and promoted apoptosis. Briefly, circADAMTS16 inhibited the differentiation and promotes the proliferation of bovine adipocytes by targeting miR-10167-3p during bovine fat deposition, which provides new insights into the mechanism of circRNAs regulation of beef quality.

Overexpression of DGAT2 Regulates the Differentiation of Bovine Preadipocytes

Triacylglycerols (TAGs) are a major component of intramuscular fat. Diacylglycerol O-acyltransferase 2(DGAT2) expression determines the rate of TAG synthesis. The purpose of this study was to investigate the role of DGAT2 in the differentiation of Yanbian cattle preadipocytes and lipid metabolism-related signalling pathways. Bovine preadipocytes were infected with overexpression and interfering adenovirus vectors of DGAT2. The effects on the differentiation of Yanbian cattle preadipocytes were examined using molecular and transcriptomic techniques, including differentially expressed genes (DEGs) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis. DGAT2 overexpression significantly increased (p < 0.05) intracellular TAG, adiponectin, and lipid droplet (LD) contents. Moreover, it upregulated (p < 0.05) peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein α, and fatty acid binding protein 4 mRNA expression. In contrast, DGAT2 knockdown reduced intracellular TAG and LD content and downregulated (p < 0.05) C/EBPβ, mannosyl (alpha-1,3-)-glycoproteinbeta-1,2-N-acetylglucosaminyltransferase, lipin 1,1-acylglycerol-3-phosphate O-acyltransferase 4, and acetyl-CoA carboxylase alpha mRNA expression. Between DGAT2-overexpressing preadipocytes and normal cells, 208 DEGs were identified, including 106 upregulated and 102 downregulated genes. KEGG pathway analysis revealed DEGs mainly enriched in PPAR signalling and AMP-activated protein kinase pathways, cholesterol metabolism, and fatty acid biosynthesis. These results demonstrated that DGAT2 regulated preadipocyte differentiation and LD and TAG accumulation by mediating the expression of adipose differentiation-, lipid metabolism-, and fatty acid synthesis-related genes.

MiR-33a Inhibits the Differentiation of Bovine Preadipocytes through the IRS2-Akt Pathway.

Several microRNAs (miRNAs) are known to participate in adipogenesis. However, their role in this process, especially in the differentiation of bovine preadipocytes, remains to be elucidated. This study was intended to clarify the effect of microRNA-33a (miR-33a) on the differentiation of bovine preadipocytes by cell culture, real-time fluorescent quantitative PCR (qPCR), Oil Red staining, BODIPY staining, and Western blotting. The results indicate that overexpression of miR-33a significantly inhibited lipid droplet accumulation and decreased the mRNA and protein expression of adipocyte differentiation marker genes such as peroxisome proliferator-activated receptor gamma (PPARγ), sterol regulatory element-binding protein 1 (SREBP1), and fatty acid-binding protein 4 (FABP4). In contrast, the interference expression of miR-33a promoted lipid droplet accumulation and increased the expression of marker genes. Additionally, miR-33a directly targeted insulin receptor substrate 2 (IRS2) and regulated the phosphorylation level of serine/threonine kinase (Akt). Furthermore, miR-33a inhibition could rescue defects in the differentiation of bovine preadipocytes and the Akt phosphorylation level caused by small interfering IRS2 (si-IRS2). Collectively, these results indicate that miR-33a could inhibit the differentiation of bovine preadipocytes, possibly through the IRS2-Akt pathway. These findings might help develop practical means to improve the quality of beef.

TGFβ3 regulates adipogenesis of bovine subcutaneous preadipocytes via typical Smad and atypical MAPK signaling pathways

BackgroundTransforming growth factor β3 (TGFβ3) is a member of TGFβ superfamily. Studies have shown that TGFβ is involved in adipose tissue biology. Whether TGFβ3 is a crucial regulator in adipogenic differentiation of bovine preadipocytes remains unclear. The main objective of this study was to investigate the effect of TGFβ3 on the adipogenic differentiation of bovine preadipocytes as well as the underlying mechanisms. ResultsPrimary subcutaneous preadipocytes derived from bovine subcutaneous adipose tissue were isolated and used as a cellular model. The results showed that TGFβ3 significantly promoted the proliferation of bovine preadipocytes and markedly decreased the lipid accumulation and expression levels of key adipogenic marker genes (PPARγ, c/EBPα, and FABP4) during adipocyte differentiation. In addition, TGFβ3 significantly increased the phosphorylation levels of Smad2, Smad3, JNK, and p38 in bovine preadipocytes, which could be eliminated by SB525334 (a TGFβRI inhibitor). Further studies showed that Smad3 and p38 mediated the inhibitory effect of TGFβ3 on differentiation, whereas Smad2 attenuated the anti-adipogenic effect of TGFβ3. ConclusionsTo sum up, our findings indicate that TGFβ3 is an important regulator of adipogenesis in bovine. TGFβ3 promotes the proliferation and regulates the differentiation of bovine preadipocytes through Smad2/3 and p38 signaling pathways.How to cite: Yang L, Wang H, Hao W, et al. TGFβ3 regulates adipogenesis of bovine subcutaneous preadipocytes via typical Smad and atypical MAPK signaling pathways. Electron J Biotechnol 2023;61. https://doi.org/10.1016/j.ejbt.2022.11.001.

CircRNA Profiling Reveals an Abundant circBDP1 that Regulates Bovine Fat Development by Sponging miR-181b/miR-204 Targeting Sirt1/TRARG1.

The proliferation and differentiation of preadipocytes is an important factor determining bovine fat development, which is closely related to the feed conversion ratio, carcass traits, and beef quality. The purpose of this study was to identify the effects of candidate circRNA and miRNA on the proliferation and differentiation of bovine preadipocytes in order to provide basic materials for molecular breeding in cattle. circRNA sequencing was performed on bovine adipocyte samples at different differentiation time points, and a total of 1830 differentially expressed circRNAs were identified. Among them, circBDP1, derived from the bovine BDP1 gene, has potential binding sites for miR-204 (known as a regulator of bovine fat development) and miR-181b, which gives us a hint that circBDP1 may regulate bovine fat development by adsorbing miR-204 and miR-181b. Here, our results revealed that circBDP1 overexpression promoted the proliferation and differentiation of bovine preadipocytes. The miRNA profile of bovine adipocytes at different differentiation time points was also analyzed using the small RNA sequencing method, and a total of 89 differentially expressed miRNAs were identified, including miR-204 and miR-181b. As expected, dual-luciferase reporter results showed that circBDP1 competitively adsorbed miR-181b and miR-204. Overexpression and interference of miR-181b in bovine preadipocytes and 3T3-L1 showed that miR-181b promoted the proliferation and differentiation of preadipocytes. Further results displayed that miR-181b and miR-204 simultaneously targeted the SIRT1 gene, and miR-204 also targeted the 3' UTR region of the TRARG1 gene. In summary, this study found that miR-181b and miR-204 were involved in fat development by targeting SIRT1 and TRARG1. The results of this study will lay a foundation for the research of fat development and beef cattle industry.

Overexpression of cholinergic receptor nicotinic gamma subunit inhibits proliferation and differentiation of bovine preadipocytes.

Muscle acetylcholine receptors have five alpha subunits (α, β, δ, ε, or γ), and cholinergic receptor nicotinic gamma subunit (CHRNG) is the γ subunit. It may also play an essential role in biological processes, including cell differentiation, growth, and survival, while the role of CHRNG has not been studied in the literature. Therefore, the purpose of this study is to clarify the effect of CHRNG on the proliferation and differentiation of bovine preadipocytes. We constructed a CHRNG overexpression adenovirus vector and successfully overexpressed it on bovine preadipocytes. The effects of CHRNG on bovine preadipocyte proliferation were detected by Edu assay, cell counting Kit-8 (CCK-8), real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), Western blot and other techniques. We also performed oil red O, RT-qPCR, Western blot to explore its effect on the differentiation of preadipocytes. The results of Edu proliferation experiments showed that the number of EDU-positive cells in the overexpression group was significantly less. CCK-8 experiments found that the optical density values of the cells in the overexpression group were lower than those of the control group, the mRNA levels of proliferating cell nuclear antigen (PCNA), cyclin A2 (CCNA2), cyclin B1 (CCNB1), cyclin D2 (CCND2) decreased significantly after CHRNG gene overexpression, the mRNA levels of cyclin dependent kinase inhibitor 1A (CDKN1A) increased significantly, and the protein levels of PCNA, CCNB1, CCND2 decreased significantly. Overexpression of CHRNG inhibited the differentiation of bovine preadipocytes. The results of oil red O and triglyceride determination showed that the size and speed of lipid droplets accumulation in the overexpression group were significantly lower. The mRNA and protein levels of peroxisome proliferator activated receptor gamma (PPARγ), CCAAT enhancer binding protein alpha (CEBPα), fatty acid binding protein 4 (FABP4), fatty acid synthase (FASN) decreased significantly. Overexpression of CHRNG in bovine preadipocytes inhibits the proliferation and differentiation of bovine preadipocytes.

MiR-302b promotes bovine preadipocyte differentiation and inhibits proliferation by targeting CDK2

MicroRNAs have been recently reported to act as key regulators of adipogenesis, a multifactorial complex process. One miRNA, miR-302b, is an important regulator of cell proliferation and differentiation and controls cancer development, but we speculate that miR-302b may also regulate bovine adipogenesis. Herein we have evaluated the role of this miRNA in bovine adipocyte differentiation using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR), Oil Red O staining, a dual-luciferase reporter. CDK2 was identified as the target gene of miR-302b, and miR-302b agomir promoted mRNA and protein expression levels of adipocyte-specific genes. In addition, a CCK-8 kit was used to show that miR-302b agomir, but not the negative control, inhibits preadipocyte proliferation. In conclusion, miR-302b promotes bovine preadipocyte differentiation and inhibits proliferation by targeting CDK2.

Effect of Neudesin Neurotrophic Factor on Differentiation of Bovine Preadipocytes and Myoblasts in a Co-Culture System.

Simple SummaryMarbling beef refers to the red and white beef formed by depositing fat between muscle fibers, and “marbling” is an important factor affecting beef quality. Therefore, we established a co-culture system of adipocytes and myocytes to mimic the microenvironment of marbling beef in vivo. However, Neudesin neurotrophic factor (NENF) was not detected in the co-culture system, but was detected in both adipocytes and myoblasts cultured separately. Further studies revealed that NENF knockdown inhibits adipogenesis and promotes myogenesis in separately cultured preadipocytes and myoblasts. However, because the monoculture system does not include the interaction of bovine adipocytes and myoblasts in the formation of marbling beef, in this study we investigated the effect of recombinant protein NENF on the differentiation of adipocytes and myoblasts in the co-culture system. The addition of NENF inhibited the formation of lipid droplets in co-cultured adipocytes but had no significant effect on myotube formation. These results were different from, and even conflicted with, those in the monocultures, which suggested that regulation of NENF expression in the same cell type changed along with the cell microenvironment and the molecular mechanism of marbling beef formation cannot be fully revealed through studies on the monoculture system.In this study, we successfully established a co-culture system of bovine preadipocytes and myoblasts to explore the effect of exogenous addition of Neudesin neurotrophic factor (NENF) recombinant protein on the differentiation of adipocytes and myoblasts in co-culture. The optimal concentration of NENF recombinant protein was 100 pg/mL. NENF promoted the differentiation of bovine preadipocytes and inhibited the differentiation of bovine myoblasts when the cells were cultured separately. After adding NENF recombinant protein to the co-culture system, the accumulation of lipid droplets in bovine preadipocytes decreased, but the differentiation of bovine myoblasts did not change significantly. The results of real-time quantitative PCR (RT-qPCR) and Western blot showed that the expression levels of adipogenesis-related factors such as PPARγ, FABP4 and FASN were significantly down-regulated at the mRNA and protein levels in adipocytes, while myogenic marker genes such as MYOD1, MYOG and MYHC had no significant changes at the mRNA or protein levels in myoblasts. This differs from, and potentially conflicts with, the monoculture system, where NENF expression in each cell type changed with the cell microenvironment. Consequently, the molecular mechanism of marbling beef formation cannot be fully revealed using monocultures of adipocytes or myocytes.

MiR-381 Targets KCTD15 to Regulate Bovine Preadipocyte Differentiation In Vitro.

MicroRNAs (miRNAs) are small, single-stranded, noncoding RNAs ~21 to ~23 nucleotides in length and have become a popular research topic in recent years due to their regulation of gene expression and many physiological processes, including fat metabolism; however, the precise functional mechanisms underlying their regulation of fat metabolism are not fully understood. Here, we identified miR-381, which specifically targets the 3' untranslated region (3' UTR) of potassium channel tetramerization-domain-containing 15 (KCTD15) , and verified the mechanism regulating its expression and participation in adipogenesis. We used a dual luciferase-reporter assay and transfection-mediated miR-381 overexpression and inhibition in Yanbian yellow cattle preadipocytes to investigate the role of miR-381 in adipogenesis. The results showed that miR-381 directly targets the 3' UTR of KCTD15 and downregulates its expression. Additionally, miR-381 overexpression using an miRNA mimic promoted triglyceride accumulation and upregulated adipogenic peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT enhancer-binding protein α (C/EBPα) at both the protein and mRNA levels, whereas miR-381 inhibition produced the opposite effect. These results indicated that miR-381 regulates the differentiation of Yanbian yellow cattle preadipocytes by inhibiting KCTD15 expression, thereby highlighting the importance of miRNA-mediated regulation of adipogenesis. Furthermore, our findings suggested that miR-381 and its target gene(s) might represent new targets for investigating intramuscular fat deposits in cattle and treating human obesity.

MiR-145 reduces the activity of PI3K/Akt and MAPK signaling pathways and inhibits adipogenesis in bovine preadipocytes

Adipose tissue is the largest metabolic organ because of adipogenesis controlled by numerous miRNAs. MiR-145 is classified into the same cluster with famous miR-143. However, few studies have investigated the role of miR-145 in adipogenesis. In the current study, we observed that the expression of miR-145 was downregulated during bovine adipogenesis in vivo and in vitro. The results of RNA-Seq analysis showed that miR-145 mainly disturb the PI3K/Akt and MAPK signaling pathways in bovine preadipocytes. MiR-145 inhibited bovine preadipocyte differentiation and downregulated phosphorylation level of Akt and ERK1/2 proteins. Furthermore, insulin, as a powerful inducer initiating adipogenesis and an activator of the PI3K/Akt and MAPK signaling pathways, was able to rescue the downregulation of Akt and ERK1/2 phosphorylation levels caused by miR-145. Taken together, our findings suggest that miR-145 is a potent inhibitor of adipogenesis that may function by reducing the activity of PI3K/Akt and MAPK signaling pathways.

SIRT6 cooperates with SIRT5 to regulate bovine preadipocyte differentiation and lipid metabolism via the AMPKα signaling pathway

Preadipocyte differentiation and lipid synthesis are critical steps for intramuscular fat (IMF) deposition and lipid metabolism homeostasis. IMF content of beef not only determines the ratio of muscle to adipose, but also determines the beef quality, flavor, and sensory characteristics. Maintaining lipid metabolism homeostasis is the key means of preventing and treating diabetes, obesity, and other metabolic diseases. SIRT6, which is an ADP-ribosyltransferase and NAD+-dependent deacetylase of acetyl and long-chain fatty acyl groups, playing central roles in lipid and glucose metabolism, is closely related to the occurrence of diabetes and obesity caused by overnutrition and aging. This study was based on bovine preadipocyte differentiation and an obese mice model, and comprehensively used transcriptome sequencing (RNA-seq) and morphological identification methods to explore the effects of inhibition of SIRT6 on differentiation and lipid synthesis, and related molecular mechanisms. Additionally, the feedback synergistic regulation of SIRT5 and SIRT6 on differentiation and lipid deposition was analyzed. The results showed that in the differentiation process of bovine preadipocytes, inhibition of SIRT5 significantly promoted SIRT6 expression. In addition, SIRT6 inhibited bovine preadipocyte differentiation and lipid synthesis, cooperating with SIRT5 to decrease lipid deposition, and repressed cell cycle arrest of preadipocytes. Moreover, in vivo verification experiments also obtained consistent results. Furthermore, SIRT6 inhibited preadipocyte differentiation and lipid deposition by activating the adenosine monophosphate activated protein kinase alpha (AMPKα) pathway. The above results provided a novel approach for understanding the functions of SIRT6 in regulating bovine adipocyte differentiation and lipid metabolism, as well as a new target for the treatment of diabetes and obesity in a clinical setting.

SIRT5 inhibits bovine preadipocyte differentiation and lipid deposition by activating AMPK and repressing MAPK signal pathways

SIRT5 (sirtuin 5) is located in the mitochondria and plays an important role in biological processes such as maintaining the balance of lipid metabolism and promoting fatty acid oxidation mobilization. In this study, the bovine preadipocyte differentiation and obese mouse models were constructed; combined with transcriptome sequencing (RNA-seq) and morphological identification, the regulatory and molecular mechanisms underlying the effects of SIRT5 on bovine preadipocyte differentiation and lipid metabolism were studied. The results reveal that during the differentiation of preadipocytes, SIRT5 inhibited the expression of key genes that promote lipid formation and differentiation in fatty acid biosynthesis and PPAR pathways. SIRT5 significantly activated the adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) pathway and repressed the mitogen-activated protein kinase (MAPK) pathway. Eventually, SIRT5 significantly inhibited the differentiation of bovine preadipocytes and simultaneously inhibited lipid synthesis and lipid deposition in adipocytes. The verification experiments performed using obese mice also yielded consistent results in vivo.

IGFBP7 downregulation or overexpression effect on bovine preadipocyte differentiation

The insulin-like growth factor binding-protein 7 (IGFBP7) has binding affinities to IGFs and is able to either positively or negatively regulate the IGFs signaling pathway. It also plays a crucial role in cell growth, differentiation and development in an IGF-independent manner. Herein, we investigated the specific regulation of the gene encoding for IGFBP7during the differentiation process of the adipocyte cells of the Yan Yellow Cattle by interfering with or by overexpressing the IGFBP7 gene. As a result, we found that the mRNA expression levels of IGFBP7 were significantly increased during the formation of progenitor cells. In addition, the expression levels of the lipoprotein lipase (LPL) and transcription factors (PPARγ, C/EBPα) were also significantly increased. IGFBP7 gene overexpression and RNA interfering promoted and inhibited respectively the lipid accumulation and triglyceride production in mature adipocytes, and the expression of the LPL and transcription factors (PPARγ, C/EBPα). The changes in the protein expression levels of IGFBP7 and adipogenic factors were in accord with the changes observed in the mRNA levels. In conclusion, our results indicate that IGFBP7 plays an important regulatory role in the differentiation of preadipocyte cells.

Adipogenesis associated Mth938 domain containing (AAMDC) protein expression is regulated by alternative polyadenylation and microRNAs.

The post-transcriptional events regulating expression of the adipogenesis associated Mth938 domain containing (AAMDC) protein are poorly understood. Here, we find that AAMDC expresses three isoforms due to alternative polyadenylation (APA) and alternative splicing (AS). Luciferase assay revealed that expression of the two isoforms with short and long 3'UTRs is differentially controlled. Further analyses showed that the short isoform displays higher translation efficiency and that miR-2428/664a reduces expression of the long isoform, indicating that APA-mediated shortening of the AAMDC 3'UTR renders the short isoform insusceptible to miRNA-mediated suppression due to loss of the binding sites for miR-2428/664a. Finally, we demonstrate that the short AAMDC isoform promotes bovine preadipocyte differentiation. Collectively, our findings indicate that AAMDC is post-transcriptionally regulated through APA and microRNAs.

Effects of puerarin on the AKT signaling pathway in bovine preadipocyte differentiation.

ObjectivePuerarin has the potential of regulating the differentiation of preadipocytes, but its mechanism of action has not yet been elucidated. Adipocytes found in adipose tissue, the main endocrine organ, are the main sites of lipid deposition, and are widely used as a cell model in the study of in vitro fat deposition. This study aimed to investigate the effects of puerarin on adipogenesis in vitro.MethodsPuerarin was added to the culture medium during the process of adipogenesis. The proliferation and differentiation of bovine preadipocytes was measured through cell viability and staining with oil red O. The content of triacylglycerol was measured using a triglyceride assay kit. The mRNA and protein expression levels of adipogenic genes, peroxisome proliferator-activated receptor-γ (PPARγ) and CCAAT/enhancer-binding protein-α, were measured using quantitative real-time polymerase chain reaction and western blotting, respectively.ResultsThe addition of puerarin significantly increased adipogenesis of bovine preadipocytes and enhanced the mRNA and protein level expression of PPARγ (p<0.01). The expression of P-Akt increased after adipogenic hormonal induction, whereas puerarin significantly increased PPARγ expression by promoting the Akt signaling component, P-Akt. The mechanism of adipogenesis was found to be related to the phosphorylation level of Ser473, which may activate the downstream signaling of the Akt pathway.ConclusionPuerarin was able to promote the differentiation of preadipocytes and improve fat deposition in cattle. The mechanism of adipogenesis was found to be related to the phosphorylation level of Ser473.

MicroRNA-224 impairs adipogenic differentiation of bovine preadipocytes by targeting LPL

In this study, the potential functions of miR-224 in regulating adipogenic differentiation were explored in bovine preadipocytes. Comparative transcriptome analysis between castrated male cattle with increased intramuscular fat (IMF) and intact male cattle revealed that miR-224 and LPL were abnormally expressed, correlating negatively, and LPL was a predicted target of miR-224. A dual luciferase reporter assay confirmed the negative targeting regulatory relationship between miR-224 and LPL. When miR-224 was either overexpressed or silenced, qRT-PCR showed a negative regulatory effect on LPL. mRNA expression levels of the fat-formation-related biomarkers C/EBPα, C/EBPβ, PPARγ, FASN and PLIN1 decreased when miR-224 was overexpressed, while the opposite effect occurred and adipogenic differentiation followed when miR-224 was inhibited. Oil Red O staining. Triglyceride (TG) levels and immunostaining revealed that the accumulation of lipid droplets decreased or increased accordingly. Taken together, our data demonstrated that miR-224 regulated the adipogenic differentiation of bovine preadipocytes by targeting LPL. This provides insight into the molecular basis of IMF deposition in beef cattle.

Effects of PSMA1 on the differentiation and lipid deposition of bovine preadipocytes

ABSTRACT In this study, our goal was to clarify the role of proteasomal subunit α-1 (PSMA1) in both the differentiation of preadipocytes and the accumulation of lipids in adipocytes. Preadipocytes from healthy one-day-old calves were collected, isolated, and cultured in vitro. The expression pattern of the PSMA1 gene was explored during the differentiation of bovine preadipocytes firstly. Then, the expression of the PSMA1 gene was inhibited by transfection of a chemically synthesized small interfering RNA (siRNA) before differentiation. After induction [...]

Ectopical expression of FABP4 gene can induce bovine muscle-derived stem cells adipogenesis

Fatty acid binding protein 4 (FABP4) plays a key role in Fatty acid catabolism in mammals. Findings from our previous studies have indicated that FABP4 neither affect the differentiation of bovine preadipocytes nor does it change the expression of upstream genes. To investigate whether ectopically expressed FABP4 can induces Muscle-Derived Stem Cells (MDSCs) lipid synthesis and understand the regulatory mechanism behind it. In this study, adenoviruses infection is achieved to ectopically expressed FABP4 in bovine MDSCs, RNA-seq analyses at the very early stages of induction were performed to reveal gene expression level changes during MDSCs transdifferentiation. Results showed FABP4 can induce bovine Muscle-Derived Stem Cells transdifferentiation into adipocyte-like cells, 23 genes' expression levels changed after 24 h inducing although there is no significant change in cell phenotypes. Along with induction time, more differently expressed genes (256 genes changes after 48 h induction) were screened out. These genes should be at the downstream of signal pathways and be regulated by the 23 genes identified before. Our findings may provide a unique new model for studying the molecular control of cattle cross-talk between adipose and skeletal muscle.