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The list of standard abbreviations for JDS is available at adsa.org/jds-abbreviations-24. Nonstandard abbreviations are available in the Notes. Postpartum cows experience lipolysis in adipose tissue due to negative energy balance, and accumulation of free fatty acids leads to metabolic stress in adipose tissue. Ferroptosis is a type of cell death triggered by excessive buildup of iron-dependent lipid peroxides and is involved in the occurrence and development of various metabolic diseases in nonruminants. However, whether ferroptosis occurs in the adipose tissue of ketotic cows and the regulatory mechanisms behind ferroptosis are still unclear. Despite multiple studies demonstrating the significant involvement of hypoxia-inducible-factor-1α (HIF-1α) in regulating cellular dysfunction, its specific function in the adipose tissue of ketotic dairy cows remains uncertain, particularly its regulation of oxidative stress and ferroptosis. This study aimed to explore the effect of HIF-1α on oxidative stress and ferroptosis in bovine subcutaneous adipose tissue and isolated adipocytes. The adipose tissue of clinical ketosis cows (n = 15) with a serum BHB concentration of 3.13 m M (interquartile range = 0.14) and healthy cows (n = 15) with a serum BHB concentration of and 0.58 m M (interquartile range = 0.13) was collected. The results showed that the concentrations of lipid peroxidation malondialdehyde (MDA), reactive oxygen species (ROS), Fe2+, and total iron were increased in adipose tissue of cows with ketosis, but the contents of glutathione (GSH) were reduced. Furthermore, the protein levels of HIF-1α, heme oxygenase 1 (HMOX1), catalase (CAT), superoxide dismutase 1 (SOD1), acyl-CoA synthetase 4, and nuclear factor erythroid-derived 2-like 2 (NFE2L2) exhibited higher abundance in adipose tissue obtained from cows with ketosis, whereas the protein abundance of solute carrier family 7 member 11 (SLC7A11), glutamate-cysteine ligase catalytic subunit (GCLC), kelch-like ECH-associated protein 1, glutamate-cysteine ligase regulatory subunit (GCLM), and glutathione peroxidase 4 (GPX4) were lower. To simulate the ferroptosis state of adipose tissue in ketotic cows, primary bovine adipocytes were isolated from the adipose tissue of healthy cows and cultured with erastin to construct the ferroptosis model. Adipocytes were cultured with either an adenovirus overexpressing HIF-1α or small interfering RNA targeting HIF for 48 h, followed by exposure to erastin (1 μ M) for 24 h. Treatment with erastin led to higher protein abundance of CAT, SOD1, NFE2L2 and HMOX1, and it inhibited the protein expression levels of GCLC, SLC7A11, GCLM, GPX4, and kelch-like ECH-associated protein 1. Furthermore, erastin treatment elevated the levels of ROS, MDA, Fe2+, and total iron and reduced the content of GSH. The overexpression of HIF-1α reversed the erastin-induced decreases in the protein abundance of GPX4 and SLC7A11, as well as the levels of MDA, ROS, Fe2+, and total iron, while significantly increasing protein abundance and content of CAT, SOD1, NFE2L2, HMOX1, GCLC, GCLM, GPX4, SLC7A11, and GSH. Conversely, the silencing of HIF-1α further exacerbated the erastin-induced levels of MDA, ROS, Fe2+, and total iron, while inhibiting the upregulation of SOD1, CAT, NFE2L2 and HMOX1. Collectively, these findings suggest that activation of HIF-1α may function as an adaptive mechanism to mitigate ferroptosis and alleviate oxidative stress in adipose tissue. [ABSTRACT FROM AUTHOR]

Investigating the involvement of long non-coding RNAs (lncRNAs) and epigenetic processes in bovine adipocytes can provide valuable new insights into controlling adipogenesis in livestock. Long non-coding RNAs have been associated with forming chromatin loops that facilitate enhancer-promoter interactions during adipogenesis, as well as regulating important adipogenic transcription factors like C/EBPα and PPARγ. They significantly influence gene expression regulation at the post-transcriptional level and are extensively researched for their diverse roles in cellular functions. Epigenetic modifications such as chromatin reorganization, histone alterations, and DNA methylation subsequently affect the activation of genes related to adipogenesis and the progression of adipocyte differentiation. By investigating how fat deposition is epigenetically regulated in beef cattle, scientists aim to unravel molecular mechanisms, identify key regulatory genes and pathways, and develop targeted strategies for modifying fat deposition to enhance desirable traits such as marbling and meat tenderness. This review paper delves into lncRNAs and epigenetic factors and their role in regulating bovine adipocytes while focusing on their potential as targets for genetic improvement to increase production efficiency. Recent genomics advancements, including molecular markers and genetic variations, can boost animal productivity, meeting global demands for high-quality meat products. This review establishes a foundation for future research on understanding regulatory networks linked to lncRNAs and epigenetic changes, contributing to both scholarly knowledge advancement and practical applications within animal agriculture. [ABSTRACT FROM AUTHOR]

Investigating the involvement of long non-coding RNAs (lncRNAs) and epigenetic processes in bovine adipocytes can provide valuable new insights into controlling adipogenesis in livestock. Long non-coding RNAs have been associated with forming chromatin loops that facilitate enhancer-promoter interactions during adipogenesis, as well as regulating important adipogenic transcription factors like C/EBPa and PPARγ. They significantly influence gene expression regulation at the post-transcriptional level and are extensively researched for their diverse roles in cellular functions. Epigenetic modifications such as chromatin reorganization, histone alterations, and DNA methylation subsequently affect the activation of genes related to adipogenesis and the progression of adipocyte differentiation. By investigating how fat deposition is epigenetically regulated in beef cattle, scientists aim to unravel molecular mechanisms, identify key regulatory genes and pathways, and develop targeted strategies for modifying fat deposition to enhance desirable traits such as marbling and meat tenderness. This review paper delves into lncRNAs and epigenetic factors and their role in regulating bovine adipocytes while focusing on their potential as targets for genetic improvement to increase production efficiency. Recent genomics advancements, including molecular markers and genetic variations, can boost animal productivity, meeting global demands for high-quality meat products. This review establishes a foundation for future research on understanding regulatory networks linked to lncRNAs and epigenetic changes, contributing to both scholarly knowledge advancement and practical applications within animal agriculture. [ABSTRACT FROM AUTHOR]

ABSTRACT: High-yielding dairy cows in early lactation often encounter difficulties in meeting the energy requirements essential for maintaining milk production. This is primarily attributed to insufficient dry matter intake, which consequently leads to sustained lipolysis of adipose tissue. Fatty acids released by lipolysis can disrupt metabolic homeostasis. Autophagy, an adaptive response to intracellular environmental changes, is considered a crucial mechanism for regulating lipid metabolism and maintaining a proper cellular energy status. Despite its close relationship with aberrant lipid metabolism and cytolipotoxicity in animal models of metabolic disorders, the precise function of diacylglycerol o-acyltransferase 1 (DGAT1) in bovine adipose tissue during periods of negative energy balance is not fully understood, particularly regarding its involvement in lipolysis and autophagy. The objective of the present study was to assess the effect of DGAT1 on both lipolysis and autophagy in bovine adipose tissue and isolated adipocytes. Adipose tissue and blood samples were collected from cows diagnosed as clinically ketotic (n = 15) or healthy (n = 15) following a veterinary evaluation based on clinical symptoms and serum concentrations of BHB, which were 3.19 mM (interquartile range = 0.20) and 0.50 mM (interquartile range = 0.06), respectively. Protein abundance of DGAT1 and phosphorylation levels of unc-51-like kinase 1 (ULK1), were greater in adipose tissue from cows with ketosis, whereas phosphorylation levels of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) were lower. Furthermore, when adipocytes isolated from the harvested adipose tissue of 15 healthy cows were transfected with DGAT1 overexpression adenovirus or DGAT1 small interfering RNA followed by exposure to epinephrine (EPI), it led to greater ratios and protein abundance of phosphorylated hormone-sensitive triglyceride lipase (LIPE) to total LIPE and adipose triglyceride lipase (ATGL), while inhibiting the protein phosphorylation levels of ULK1, PI3K, AKT, and mTOR. Overexpression of DGAT1 in EPI-treated adipocytes reduced lipolysis and autophagy, whereas silencing DGAT1 further exacerbated EPI-induced lipolysis and autophagy. Taken together, these findings indicate that upregulation of DGAT1 may function as an adaptive response to suppress adipocytes lipolysis, highlighting the significance of maintaining metabolic homeostasis in dairy cows during periods of negative energy balance.

Objective The present study was executed to explore the molecular mechanism of fibroblast growth factor 10 (FGF10) gene in bovine adipogenesis. Methods The bovine FGF10 gene was overexpressed through Ad-FGF10 or inhibited through siFGF10 and their negative control (NC) in bovine adipocytes, and the multiplicity of infection, transfection efficiency, interference efficiency were evaluated through quantitative real-time polymerase chain reaction, western blotting and fluorescence microscopy. The lipid droplets, triglycerides (TG) content and the expression levels of adipogenic marker genes were measured during preadipocytes differentiation. The differentially expressed genes were explored through deep RNA sequencing. Results The highest mRNA level was found in omasum, subcutaneous fat, and intramuscular fat. Moreover, the highest mRNA level was found in adipocytes at day 4 of differentiation. The results of red-oil o staining showed that overexpression (Ad-FGF10) of the FGF10 gene significantly (p

Unsaturated fatty acids (UFAs) in beef play a vital role in promoting human health. Long-chain fatty acyl-CoA synthase 1 (ACSL1) is a crucial gene for UFA synthesis in bovine adipocytes. To investigate the protein expression profile during UFA synthesis, we performed a proteomic analysis of bovine adipocytes by RNA interference and non-interference with ACSL1 using label-free techniques. A total of 3558 proteins were identified in both the NC and si-treated groups, of which 1428 were differentially expressed proteins (DEPs; fold change ≥ 1.2 or ≤ 0.83 and p-value < 0.05). The enrichment analysis of the DEPs revealed signaling pathways related to UFA synthesis or metabolism, including cAMP, oxytocin, fatty acid degradation, glycerol metabolism, insulin, and the regulation of lipolysis in adipocytes (p-value < 0.05). Furthermore, based on the enrichment analysis of the DEPs, we screened 50 DEPs that potentially influence the synthesis of UFAs and constructed an interaction network. Moreover, by integrating our previously published transcriptome data, this study established a regulatory network involving differentially expressed long non-coding RNAs (DELs), highlighting 21 DEPs and 13 DELs as key genes involved in UFA synthesis. These findings present potential candidate genes for further investigation into the molecular mechanisms underlying UFA synthesis in bovines, thereby offering insights to enhance the quality of beef and contribute to consumer health in future studies. [ABSTRACT FROM AUTHOR]

Objective: The present study was executed to explore the molecular mechanism of fibroblast growth factor 10 (FGF10) gene in bovine adipogenesis. Methods: The bovine FGF10 gene was overexpressed through Ad-FGF10 or inhibited through siFGF10 and their negative control (NC) in bovine adipocytes, and the multiplicity of infection, transfection efficiency, interference efficiency were evaluated through quantitative real-time polymerase chain reaction, western blotting and fluorescence microscopy. The lipid droplets, triglycerides (TG) content and the expression levels of adipogenic marker genes were measured during preadipocytes differentiation. The differentially expressed genes were explored through deep RNA sequencing. Results: The highest mRNA level was found in omasum, subcutaneous fat, and intramuscular fat. Moreover, the highest mRNA level was found in adipocytes at day 4 of differentiation. The results of red-oil o staining showed that overexpression (Ad-FGF10) of the FGF10 gene significantly (p<0.05) reduced the lipid droplets and TG content, and their downregulation (siFGF10) increased the measurement of lipid droplets and TG in differentiated bovine adipocytes. Furthermore, the overexpression of the FGF10 gene down regulated the mRNA levels of adipogenic marker genes such as CCAAT enhancer binding protein alpha (C/EBPα), fatty acid binding protein (FABP4), peroxisome proliferator-activated receptor-γ (PPARγ), lipoprotein lipase (LPL), and Fas cell surface death receptor (FAS), similarly, down-regulation of the FGF10 gene enriched the mRNA levels of C/EBPα, PPARγ, FABP4, and LPL genes (p<0.01). Additionally, the protein levels of PPARγ and FABP4 were reduced (p<0.05) in adipocytes infected with Ad-FGF10 gene and enriched in adipocytes transfected with siFGF10. Moreover, a total of 1,774 differentially expressed genes (DEGs) including 157 up regulated and 1,617 down regulated genes were explored in adipocytes infected with Ad-FGF10 or Ad-NC through deep RNA-sequencing. The top Kyoto encyclopedia of genes and genomes pathways regulated through DEGs were the PPAR signaling pathway, cell cycle, base excision repair, DNA replication, apoptosis, and regulation of lipolysis in adipocytes. Conclusion: Therefore, we can conclude that the FGF10 gene is a negative regulator of bovine adipogenesis and could be used as a candidate gene in marker-assisted selection. [ABSTRACT FROM AUTHOR]

Unsaturated fatty acids (UFAs) in beef play a vital role in promoting human health. Long-chain fatty acyl-CoA synthase 1 (ACSL1) is a crucial gene for UFA synthesis in bovine adipocytes. To investigate the protein expression profile during UFA synthesis, we performed a proteomic analysis of bovine adipocytes by RNA interference and non-interference with ACSL1 using label-free techniques. A total of 3558 proteins were identified in both the NC and si-treated groups, of which 1428 were differentially expressed proteins (DEPs; fold change ≥ 1.2 or ≤ 0.83 and p-value < 0.05). The enrichment analysis of the DEPs revealed signaling pathways related to UFA synthesis or metabolism, including cAMP, oxytocin, fatty acid degradation, glycerol metabolism, insulin, and the regulation of lipolysis in adipocytes (p-value < 0.05). Furthermore, based on the enrichment analysis of the DEPs, we screened 50 DEPs that potentially influence the synthesis of UFAs and constructed an interaction network. Moreover, by integrating our previously published transcriptome data, this study established a regulatory network involving differentially expressed long non-coding RNAs (DELs), highlighting 21 DEPs and 13 DELs as key genes involved in UFA synthesis. These findings present potential candidate genes for further investigation into the molecular mechanisms underlying UFA synthesis in bovines, thereby offering insights to enhance the quality of beef and contribute to consumer health in future studies.

ABSTRACT: Adipose tissue of ketotic dairy cows exhibits greater lipolytic rate and signs of inflammation, which further aggravate the metabolic disorder. In nonruminants, the endoplasmic reticulum (ER) is a key organelle coordinating metabolic adaptations and cellular functions; thus, disturbances known as ER stress lead to inflammation and contribute to metabolic disorders. Enhanced activity of diacylglycerol O-acyltransferase 1 (DGAT1) in murine adipocytes undergoing lipolysis alleviated ER stress and inflammation. The aim of the present study was to investigate the potential role of DGAT1 on ER stress and inflammatory response of bovine adipose tissue in vivo and in vitro. Adipose tissue and blood samples were collected from cows diagnosed as clinically ketotic (n = 15) or healthy (n = 15) following a veterinary evaluation based on clinical symptoms and serum concentrations of β-hydroxybutyrate, which were 4.05 (interquartile range = 0.46) and 0.52 mM (interquartile range = 0.14), respectively. Protein abundance of DGAT1 was greater in adipose tissue of ketotic cows. Among ER stress proteins measured, ratios of phosphorylated PKR-like ER kinase (p-PERK) to PERK and phosphorylated inositol-requiring enzyme 1 (p-IRE1) to IRE1, and protein abundance of cleaved ATF6 protein were greater in adipose tissue of ketotic cows. Furthermore, ratios of phosphorylated RELA subunit of NF-κB (p-RELA) to RELA and phosphorylated c-jun N-terminal kinase (p-JNK) to JNK were greater, whereas protein abundance of NF-κB inhibitor α (NFKBIA) was lower in adipose tissue of ketotic cows. In addition, mRNA abundance of proinflammatory cytokines including TNF and IL-6 was greater in adipose tissue of ketotic cows. To better address mechanistic aspects of these responses, primary bovine adipocytes isolated from the harvested adipose tissue of healthy cows were subjected to lipolysis-stimulating conditions via incubation with 1 μM epinephrine (EPI) for 2 h. In another experiment, adipocytes were cultured with DGAT1 overexpression adenovirus and DGAT1 small interfering RNA for 48 h, respectively, followed by EPI (1 μM) exposure for 2 h. Treatment with EPI led to greater ratios of p-PERK to PERK, p-IRE1 to IRE1, p-RELA to RELA, p-JNK to JNK, and cleaved ATF6 protein, whereas EPI stimulation inhibited protein abundance of NFKBIA. Furthermore, treatment with EPI upregulated the secretion of proinflammatory cytokines into culture medium, including TNF-α and IL-6. Overexpression of DGAT1 in EPI-treated adipocytes attenuated ER stress, the activation of NF-κB and JNK signaling pathways, and the secretion of inflammatory cytokines. In contrast, silencing DGAT1 further aggravated EPI-induced ER stress and inflammatory responses. Overall, these data indicated that activation of DGAT1 may act as an adaptive mechanism to dampen metabolic dysregulation in adipose tissue. As such, it contributes to relief from ER stress and inflammatory responses.

Long-chain fatty acyl-CoA synthase 1 (ACSL1) plays a vital role in the synthesis and metabolism of fatty acids. The proportion of highly unsaturated fatty acids in beef not only affects the flavor and improves the meat's nutritional value. In this study, si-ACSL1 and NC-ACSL1 were transfected in bovine preadipocytes, respectively, collected cells were isolated on the fourth day of induction, and then RNA-Seq technology was used to screen miRNAs related to unsaturated fatty acid synthesis. A total of 1,075 miRNAs were characterized as differentially expressed miRNAs (DE-miRNAs), of which the expressions of 16 miRNAs were upregulated, and that of 12 were downregulated. Gene ontology analysis indicated that the target genes of DE-miRNAs were mainly involved in biological regulation and metabolic processes. Additionally, KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis identified that the target genes of DE-miRNAs were mainly enriched in metabolic pathways, fatty acid metabolism, PI3K-Akt signaling pathway, glycerophospholipid metabolism, fatty acid elongation, and glucagon signaling pathway. Combined with the previous mRNA sequencing results, several key miRNAmRNA targeting relationship pairs, i.e., novel-m0035-5p--ACSL1, novelm0035-5p--ELOVL4, miR-9-X--ACSL1, bta-miR-677--ACSL1, miR-129-X--ELOVL4, and bta-miR-485--FADS2 were screened via the miRNA-mRNA interaction network. Thus, the results of this study provide a theoretical basis for further research on miRNA regulation of unsaturated fatty acid synthesis in bovine adipocytes. [ABSTRACT FROM AUTHOR]

Background: To identify differentially expressed genes (DEGs) between muscle and adipose in cattle, we analyzed the data from the RNA sequencing of three Angus×Qinchuan crossbred cattle. Results: Searched the Gene Expression Omnibus (GEO) for a microarray dataset of Yan yellow cattle, GSE49992. After the DEGs were identified, we used STRING and Cytoscape to construct a protein–protein interaction (PPI) network, subsequently analyzing the major modules of key genes. In total, 340 DEGs were discovered, including 21 hub genes, which were mainly enriched in muscle contraction, skeletal muscle contraction, troponin complex, lipid particle, Z disc, tropomyosin binding, and actin filament binding. Conclusions: In summary, these genes can be regarded as candidate biomarkers for the regulation of muscle and adipose development.How to cite: Wang S, Raza SHA, Mei C, et al. Transcriptome profiling reveals differential expression of genes potentially involved in muscle and adipose tissue development of cattle. Electron J Biotechnol 2020;48. https://doi.org/10.1016/j.ejbt.2020.09.004.

Marbling influences the taste and tenderness of meat and is the main determinant of carcass quality in many countries. This study aims to investigate the influence of KLF6 (Kruppel Like Factor 6) and associated molecular mechanisms on lipid metabolism in bovine adipocytes. In the current study, KLF6 gene expression was down regulated via siRNA (small interfering RNA) in bovine adipocytes in vitro. Subsequently, adipogenic cells were collected from the culture media after 9 days, and subjected to fluorescent imaging and RNA sequencing. After confirming that KLF6 was down regulated in bovine adipocytes by siRNA, differential gene expression analysis was used to characterize the infuence of KLF6 on gene expression profiles in bovine adipocytes. A total of 10,812 genes were characterized as differentially expressed genes (DEGs) of which, 109 were up-regulated and 62 were down-regulated genes. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis identified that the DEGs were associated with lipid metabolism, carbohydrate metabolism, cell growth and death, cancer, and the signaling pathways for calcium, AMPK (Adenosine Monophosphate-Activated Protein Kinase), PI3K-Akt (Phosphatidylinositol 3-kinase), PPAR (Peroxisome proliferator-activated receptors), MAPK (mitogen-activated protein kinase), cAMP (Cyclic adenosine monophosphate), and Wnt (Wingless-related integration site). Similarly, gene ontology analysis indicated that down-regulation of KLF6 gene significantly up regulated the genes that regulate adipogenesis, differentiation and regulation of adipocytes and homeostasis of bovine adipocytes, specifically regulating the cell-type specific apoptotic action, negative regulation of apoptotic pathways, programmed cell death, and growth. Results indicate that KLF6 has a role in regulating lipid metabolism in bovine adipocytes. These findings provide evidence that may inform further investigations into molecular mechanisms that underlie the role of bovine KLF6 gene in regulating adipogenesis. • KLF6 knockdown in bovine adipocytes via siRNA led to differential expression of a large cohort of genes (~10,812 and 171 of DEGs), of which 109 were significantly up-regulated and another 62 were significantly down-regulated. • Pathway analysis identified DEGs to be linked with a variety of different pathways including neuroactive ligand-receptor interaction pathway, calcium signaling pathway, cAMP pathway, chemokine signaling pathway and Wnt signaling pathway. • Gene Ontology (GO) term analysis of the DEGs indicated that KLF6 knockdown significantly downregulates genes involved in adipogenesis-related processes in bovine adipocytes. • Overall, RNA-seq analysis of siKLF6 transfected bovine adipocytes provided several novel insights into KLF6 's involvement in lipid metabolism that warrant further investigation [ABSTRACT FROM AUTHOR]

Long-chain acyl-CoA synthetase 1 (ACSL1) is a member of the acyl-CoA synthetase family that plays a vital role in lipid metabolism. We have previously shown that the ACSL1 gene regulates the composition of unsaturated fatty acids (UFAs) in bovine skeletal muscle, which in turn regulates the fatty acid synthesis and the generation of lipid droplets. Here, we used RNA-Seq to screen circRNAs that regulated the expression of ACSL1 gene and other UFA synthesis-related genes by RNA interference and noninterference in bovine adipocytes. The results of KEGG pathway analysis showed that the parental genes of differentially expressed (DE)-circRNAs were primarily enriched in the adipocytokine signaling pathway. The prediction results showed that novel_circ_0004855, novel_circ_0001507, novel_circ_0001731, novel_circ_0005276, novel_circ_0002060, novel_circ_0005405 and novel_circ_0004254 regulated UFA synthesis-related genes by interacting with the related miRNAs. These results could help expand our knowledge of the molecular mechanisms of circRNAs in the regulation of UFA synthesis in bovine adipocytes. • RNA-Seq was used to screen circRNAs that regulated UFA synthesis-related genes in bovine adipocytes. • 39 differentially expressed (DE)-circRNAs were detected between the NC and si-treated samples. • Differentially expressed (DE)-circRNAs regulated UFA synthesis-related genes by interacting with the related miRNAs. • Bioinformatics was performed to conduct the circRNA-miRNA-mRNA co-expression network. [ABSTRACT FROM AUTHOR]

The enzyme long-chain acyl-CoA synthetase 1 (ACSL1) is essential for lipid metabolism. The ACSL1 gene controls unsaturated fatty acid (UFA) synthesis as well as the formation of lipid droplets in bovine adipocytes. Here, we used RNA-Seq to determine lncRNA and mRNA that regulate UFA synthesis in bovine adipocytes using RNA interference and non-interference with ACSL1. The corresponding target genes of differentially expressed (DE) lncRNAs and the DE mRNAs were found to be enriched in lipid and FA metabolism-related pathways, according to GO and KEGG analyses. The differentially expressed lncRNA- differentially expressed mRNA (DEL-DEM) interaction network indicated that some DELs, such as TCONS_00069661, TCONS_00040771, TCONS_ 00035606, TCONS_00048301, TCONS_001309018, and TCONS_00122946, were critical for UFA synthesis. These findings assist our understanding of the regulation of UFA synthesis by lncRNAs and mRNAs in bovine adipocytes.

Abstract Background Dairy cows with metabolic disorder in peripartal period display high inflammatory levels. Adipose tissue is a major endocrine organ, which is closely related to systemic inflammation. Retinoic acid (RA), an active metabolite of vitamin A, has shown potential therapeutic immunomodulatory properties. The objective of the study was to examine the effect of all-trans-RA (ATRA), the biologically most active metabolite of vitamin A, on lipopolysaccharide (LPS)-induced bovine adipocytes inflammatory responses and elucidate the underlying mechanisms. Primary cultured bovine adipocytes were treated with ATRA in the presence or absence of LPS. The treated cells were examined for the inflammatory responses and the activity of transforming growth factor beta 1 (TGFβ1) /Smad3 signaling pathway. Results LPS treatment significantly decreased the expression levels of TGFβ1/Smad3 components and increased the content of pro-inflammatory cytokines. Treatment with ATRA could over-activate TGFβ1/Smad3 signaling pathway in bovine adipocytes and reversed the over-production of pro-inflammatory cytokines and inhibition of anti-inflammatory cytokines induced by LPS. Importantly, inhibition of TGFβ1/Smad3 signaling diminished the effects of ATRA on suppressing the proinflammatory responses induced by LPS. Furthermore, activation of TGFβ1/Smad3 signaling further extended the effects of ATRA on suppressing the proinflammatory responses on LPS stimulation. Conclusion In conclusion, ATRA stimulates TGFβ1/Smad3 signaling pathway and further suppresses bovine adipocytes inflammatory responses induced by LPS.

This study investigated the effect of ELOVL6 (elongation of very long chain fatty acids protein 6) and its underlying mechanism on lipid metabolism in bovine adipocytes. The ELOVL6 gene was overexpressed in bovine adipocytes by adenoviruses, and RNA sequencing was performed. Overexpression of ELOVL6 showed reduced proportions of C14:0 (Myristic) and C16:0 (palmitate) fatty acids and increased proportions of C18.0 (stearate) and C20:4n6 (arachidonic) fatty acids in adipocytes. In addition, a total of 2170 differentially expressed genes (DEGs) were found, containing 1802 up-regulated and 368 down-regulated genes. KEGG pathway analysis revealed that the down-regulated genes were linked with the regulation of lipolysis and the Wnt signaling pathway. The up-regulated genes were mainly involved in the FoxO signaling pathway; the PI3K-Akt signaling pathway; and the cAMP signaling pathway. In conclusion, our results suggest that ELOVL6 could affect the fatty acid composition in bovine adipocytes. We identified numerous related DEGs and pathways, which may provide a basis for studying the function and molecular mechanism of the ELOVL6 gene in regulating lipid metabolism. • ELOVL6 regulate lipid metabolism in bovine adipocytes. • ELOVL6 is a positive regulator of C18.0 and C20:4n6 and negative regulator of C14:0 and C16:0 fatty acids. • ELOVL6 regulate lipogenesis and lipolysis through Wnt, cAMP, FoxO, and PI3K-Akt signaling pathways in bovine adipocytes. • ELOVL6 gene could be used as a candidate gene for the improvement of meat quality in cattle breed improvement program. [ABSTRACT FROM AUTHOR]

Intramuscular fat is a real challenge for the experts of animal science to improve meat quality traits. Research on the mechanism of adipogenesis provides invaluable information for the improvement of meat quality traits. This study investigated the effect of bta-miR-149-5p and its underlying mechanism on lipid metabolism in bovine adipocytes. Bovine adipocytes were differentiated and transfected with bta-miR-149-5p mimics or its negative control (NC). A total of 115 DEGs including 72 upregulated and 43 downregulated genes were identified in bovine adipocytes. The unigenes and GO term biological processes were the most annotated unigene contributor parts at 80.08%, followed by cellular component at 13.4% and molecular function at 6.7%. The KEGG pathways regulated by the DEGs were PI3K-Akt signaling pathway, calcium signaling pathway, pathways in cancer, MAPK signaling pathway, lipid metabolism/metabolic pathway, PPAR signaling pathway, AMPK signaling pathway, TGF-beta signaling pathway, cAMP signaling pathway, cholesterol metabolism, Wnt signaling pathway, and FoxO signaling pathway. In addition to this, the most important reactome enrichment pathways were R−BTA−373813 receptor CXCR2 binding ligands CXCL1 to 7, R−BTA−373791 receptor CXCR1 binding CXCL6 and CXCL8 ligands, R−BTA−210991 basigin interactions, R−BTA−380108 chemokine receptors binding chemokines, R−BTA−445704 calcium binding caldesmon, and R−BTA−5669034 TNFs binding their physiological receptors. Furthermore, the expression trend of the DEGs in these pathways were also exploited. Moreover, the bta-miR-149-5p significantly (p < 0.01) downregulated the mRNA levels of adipogenic marker genes such as CCND2, KLF6, ACSL1, Cdk2, SCD, SIK2, and ZEB1 in bovine adipocytes. In conclusion, our results suggest that bta-miR-149-5p regulates lipid metabolism in bovine adipocytes. The results of this study provide a basis for studying the function and molecular mechanism of the bta-miR-149-5p in regulating bovine adipogenesis.

Perilipin 1 (PLIN1) is a protein encoded by the PLIN1 gene in eukaryotes. PLIN1 is a member of the PAT protein family, a family of proteins related to lipid droplet (LD) surface proteins. PLIN1 phosphorylation plays a vital role during fat metabolism of adipose tissue lipolysis and fat storage in adipocytes. However, to further explore the regulation of the PLIN1 gene on the proliferation, differentiation and lipid metabolism of bovine adipocytes. In this study, the mRNA expression of PLIN1, at day six, was the highest during bovine adipocyte differentiation. Moreover, PLIN1 can promote the proliferation and differentiation of preadipocytes in cattle. On the sixth day, after transfection with, and overexpression of, the PLIN1 gene in bovine preadipocytes via adenovirus, cell samples were collected, and transcriptome sequencing was performed. A total of 1923 differentially expressed genes were detected. Through GO and KEGG pathway analysis, the differentially expressed genes were established to be mainly enriched in the AMPK, Wnt, and PPAR signaling pathways related to fat proliferation and differentiation. In conclusion, at the transcriptional level, PLIN1 plays an important role in regulating fat proliferation and metabolism. Additionally, the sequencing results screened new differentially expressed genes related to fat metabolism, providing theoretical support for molecular breeding of Qinchuan beef cattle.

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. [ABSTRACT FROM AUTHOR]

The transition period in dairy cows (3 weeks prepartum until 3 weeks postpartum) is associated with substantial mobilization of energy stores, which is often associated with metabolic diseases. Nicotinic acid (NA) is an antilipolytic and lipid-lowering compound used to treat dyslipidaemia in humans, and it also reduces non-esterified fatty acids in cattle. In mice the G-protein coupled receptor 109A (GPR109A) ligand NA positively affects the secretion of adiponectin, an important modulator of glucose and fat metabolism. In cattle, the corresponding data linking NA to adiponectin are missing. Our objective was to examine the effects of NA on adiponectin and AMPK protein abundance and the expression of mRNAs of related genes such as chemerin, an adipokine that enhances adiponectin secretion in vitro. Differentiated bovine adipocytes were incubated with pertussis toxin (PTX) to verify the involvement of GPR signaling, and treated with 10 or 15 µM NA for 12 or 24 h. NA increased adiponectin concentrations (p = 0.001) and the mRNA abundances of GPR109A (p = 0.05) and chemerin (p = 0.01). Pre-incubation with PTX reduced the adiponectin response to NA (p = 0.001). The NA-stimulated secretion of adiponectin and the mRNA expression of chemerin in the bovine adipocytes were suggestive of GPR signaling-dependent improved insulin sensitivity and/or adipocyte metabolism in dairy cows. [ABSTRACT FROM AUTHOR]

We hypothesized that lubabegron fumarate (LUB) (Experior, Elanco Animal Health, Greenfield, IN) would act as an antagonist to β-adrenergic receptor (β-AR) subtypes in primary bovine subcutaneous (s.c.) and intramuscular (i.m.) adipocytes differentiated in culture. This study employed LUB, dobutamine (DOB, a selective β1-agonist), salbutamol (SAL, a selective β2-agonist), and propranolol (PRO, a non-selective β-AR antagonist). Preadipocytes were isolated by standard techniques from bovine longissimus muscle and overlying s.c. adipose tissue and differentiated to adipocytes for 14 d. The adipocyte source x stage of differentiation interaction was significant for β-adrenergic receptors-1 (ADRB1) (P = 0.001) and ADRB2 (P = 0.01) in that expression of ADRB1 and ADRB2 was greater in s.c. adipocytes than in s.c. preadipocytes; expression of the ADRB1-3 did not change after differentiation of i.m. adipocytes. CCATT/enhancer-binding protein alpha (CEBPA) expression increased upon differentiation in both s.c. and i.m. adipocytes (P = 0.006). The source x stage of differentiation interaction was significant for peroxisome proliferator-activated receptor gamma (PPARG) (P ≤ 0.001) and fatty acid binding protein-4 (FABP4) (P = 0.004). Expression of PPARG increased after differentiation of s.c. preadipocytes to adipocytes, but PPARG expression did not change with differentiation of i.m. preadipocytes to adipocytes. FABP4 expression increased after differentiation of both s.c. and i.m. adipocytes, but FABP4 expression increased to a greater extent in s.c. adipocytes. In s.c. adipocytes, DOB elevated cAMP and glycerol production and protein kinase A (PKA) activity, and SAL increased PKA activity; these effects were abolished by LUB and PRO (P < 0.001). Incubation of i.m. adipocytes with SAL increased cAMP production and PKA activity, which was attenuated by LUB and PRO (P ≤ 0.006). In s.c. adipocytes, SAL, LUB + SAL, and LUB + DOB upregulated hormone sensitive lipase (HSL) (P < 0.001) and perilipin (P = 0.002) gene expression. In i.m. adipocytes, DOB and LUB + DOB increased HSL gene expression (P = 0.001) and LUB + SAL depressed adipose triglyceride lipase expression below control levels (P = 0.001). These results demonstrate that LUB is a β-AR antagonist at the β1-AR and β2-AR subtypes in s.c. adipocytes, and that s.c. and i.m. exhibit different responses to β-AA and LUB., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Simple Summary: This is a novel study that explored the function of bta-miR-149-5p in the lipid metabolism of bovine adipocytes. Previously, we explored the role of bta-miR-149-5p gene in proliferation and differentiation of bovine adipocytes; however, the underlying mechanism of bta-miR-149-5p function in the regulation of lipid metabolism and adipogenesis in bovine adipocytes is unexplored. A transcriptomic study with integration of bioinformatics study was performed to fully explore the function of bta-miR-149-5p microRNA in bovine adipogenesis. This study will help the scientific community deal with lipogenesis for the breed improvement program of cattle for the provision of healthy meat to consumers. Intramuscular fat is a real challenge for the experts of animal science to improve meat quality traits. Research on the mechanism of adipogenesis provides invaluable information for the improvement of meat quality traits. This study investigated the effect of bta-miR-149-5p and its underlying mechanism on lipid metabolism in bovine adipocytes. Bovine adipocytes were differentiated and transfected with bta-miR-149-5p mimics or its negative control (NC). A total of 115 DEGs including 72 upregulated and 43 downregulated genes were identified in bovine adipocytes. The unigenes and GO term biological processes were the most annotated unigene contributor parts at 80.08%, followed by cellular component at 13.4% and molecular function at 6.7%. The KEGG pathways regulated by the DEGs were PI3K-Akt signaling pathway, calcium signaling pathway, pathways in cancer, MAPK signaling pathway, lipid metabolism/metabolic pathway, PPAR signaling pathway, AMPK signaling pathway, TGF-beta signaling pathway, cAMP signaling pathway, cholesterol metabolism, Wnt signaling pathway, and FoxO signaling pathway. In addition to this, the most important reactome enrichment pathways were R−BTA−373813 receptor CXCR2 binding ligands CXCL1 to 7, R−BTA−373791 receptor CXCR1 binding CXCL6 and CXCL8 ligands, R−BTA−210991 basigin interactions, R−BTA−380108 chemokine receptors binding chemokines, R−BTA−445704 calcium binding caldesmon, and R−BTA−5669034 TNFs binding their physiological receptors. Furthermore, the expression trend of the DEGs in these pathways were also exploited. Moreover, the bta-miR-149-5p significantly (p < 0.01) downregulated the mRNA levels of adipogenic marker genes such as CCND2, KLF6, ACSL1, Cdk2, SCD, SIK2, and ZEB1 in bovine adipocytes. In conclusion, our results suggest that bta-miR-149-5p regulates lipid metabolism in bovine adipocytes. The results of this study provide a basis for studying the function and molecular mechanism of the bta-miR-149-5p in regulating bovine adipogenesis. [ABSTRACT FROM AUTHOR]

Simple Summary: In bovine preadipocytes, the overexpression of the PLIN1 gene promotes the expression of FASN, PPARγ, ACC, LPL, FABP4, DGAT2, and C/EBPβ at the mRNA level, inhibiting the expression of fat metabolism genes such as PLIN2 and ATGL. Furthermore, after overexpression of PLIN1, the oil red O-staining results showed that the number of lipid droplets (LDs) in fat cells, their volume and triglyceride content were increased. The elevation in triglyceride content indicates that PLIN1 can promote the accumulation of triglyceride in bovine preadipocytes and has an important regulatory role in fat metabolism. After transfection with the adenovirues Ad-PLIN1 and Ad-NC (empty virus), RNA-seq was used to analyze the differentially expressed genes in bovine preadipocytes. A total of 1923 differentially expressed genes were detected, and GO and KEGG signaling pathways were analyzed for differentially expressed genes. Perilipin 1 (PLIN1) is a protein encoded by the PLIN1 gene in eukaryotes. PLIN1 is a member of the PAT protein family, a family of proteins related to lipid droplet (LD) surface proteins. PLIN1 phosphorylation plays a vital role during fat metabolism of adipose tissue lipolysis and fat storage in adipocytes. However, to further explore the regulation of the PLIN1 gene on the proliferation, differentiation and lipid metabolism of bovine adipocytes. In this study, the mRNA expression of PLIN1, at day six, was the highest during bovine adipocyte differentiation. Moreover, PLIN1 can promote the proliferation and differentiation of preadipocytes in cattle. On the sixth day, after transfection with, and overexpression of, the PLIN1 gene in bovine preadipocytes via adenovirus, cell samples were collected, and transcriptome sequencing was performed. A total of 1923 differentially expressed genes were detected. Through GO and KEGG pathway analysis, the differentially expressed genes were established to be mainly enriched in the AMPK, Wnt, and PPAR signaling pathways related to fat proliferation and differentiation. In conclusion, at the transcriptional level, PLIN1 plays an important role in regulating fat proliferation and metabolism. Additionally, the sequencing results screened new differentially expressed genes related to fat metabolism, providing theoretical support for molecular breeding of Qinchuan beef cattle. [ABSTRACT FROM AUTHOR]

Low intramuscular adipose tissue (marbling) continues to be challenge for improving beef quality in Chinese cattle. Highly marbled meat is very desirable; hence, methods to increase IMF content have become a key aspect of improving meat quality. Therefore, research on the mechanism of adipogenesis provides invaluable information for the improvement of meat quality. This study investigated the effect of TORC2 and its underlying mechanism on lipid metabolism in bovine adipocytes. The TORC2 gene was downregulated in bovine adipocytes by siRNA, and RNA sequencing was performed. Downregulation of TORC2 negatively affected bovine adipocyte differentiation. In addition, a total of 577 DEGs were found, containing 146 up-regulated and 376 down-regulated genes. KEGG pathway analysis revealed that the DEGs were linked with neuroactive ligand-receptor interaction pathway, calcium signaling pathway, cAMP pathway, chemokine signaling pathway and Wnt signaling pathway. Gene Ontology (GO) term analysis of the DEGs showed that down-regulation of TORC2 gene significantly suppressed the genes regulating important GO terms of adipogenesis-related processes in bovine adipocytes, especially regulation of biological activity, regulation of primary metabolic process, regulation of multicellular organismal process, cell adhesion, lipid metabolic process, secretion, chemical homeostasis, regulation of transport, cell-cell signaling, cAMP metabolic process, cellular calcium ion homeostasis, fat cell differentiation, and cell maturation. In conclusion, our results suggest that TORC2 at least in part regulates lipid metabolism in bovine adipocytes. The results of this study provide a basis for studying the function and molecular mechanism of the TORC2 gene in regulating adipogenesis. • Total 577 DEGs were found including 146 up and 376 down-regulated genes in adipocytes transfected with siTORC2 and siNC. • The DEGs were linked with neuroactive ligand-receptor interaction, calcium signaling, cAMP, and Wnt signaling pathways. • The GO term analysis of the DEGs showed the role of TORC2 gene in adipogenesis-related processes in bovine adipocytes. • RNA-seq analysis explored the effect of TORC2 on lipid metabolism in bovine adipocytes. [ABSTRACT FROM AUTHOR]

Alpha/beta hydrolase domain 5 (ABHD5) plays a significant role in intracellular lipid metabolism, which is regulated by a complex network of transcription factors. The transcriptional regulation of the ABHD 5 gene in cattle and other livestock, however, has not been previously investigated. Investigations in humans and animal models indicate that the transcription factors zinc finger E-box binding homeobox 1 (ZEB1) and cAMP-response element binding protein (CREB) may play important roles in the transcriptional regulation of ABHD 5 in cattle. Our comparison of the sequence similarities in the transcription factor binding sites in Bos taurus , Bos indicus , Bos mutus, and Homo sapiens revealed high homology. Based on the data collected by the Cistrome Data Browser and its visualization window, we found that ZEB1 and CREB have significant ChIP-seq enrichments in the 5'-untranslated region (5' UTR) of the human ABHD 5 gene. In bovine adipocytes, we detected ZEB1 and CREB binding sites in the ABHD 5 gene. Mutations in the ZEB1 and CREB binding sites significantly reduced the promoter activity ( p  < 0.05 and p  < 0.01, respectively). Moreover, electrophoretic mobility shift assays and chromatin immunoprecipitation (ChIP) assays demonstrated the binding of the transcription factors in vivo and in vitro , respectively. And overexpression or silencing the expression of the ZEB1 and CREB, respectively, resulted in significant changes to the ABHD 5 promoter activity. Collectively, these results indicate that ZEB1 and CREB are important transcription factors that regulate ABHD 5 gene expression in bovine adipocytes. They further our understanding of the transcriptional regulation and biological functions of the bovine ABHD 5 gene.

The TORC2 gene is a member of the transducer of the regulated cyclic adenosine monophosphate (cAMP) response element binding protein gene family, which plays a key role in metabolism and adipogenesis. In the present study, we confirmed the role of TORC2 in bovine preadipocyte proliferation through cell cycle staining flow cytometry, cell counting assay, 5-ethynyl-2′-deoxyuridine staining (EdU), and mRNA and protein expression analysis of proliferation-related marker genes. In addition, Oil red O staining analysis, immunofluorescence of adiponectin, mRNA and protein level expression of lipid related marker genes confirmed the role of TORC2 in the regulation of bovine adipocyte differentiation. Furthermore, the transcription start site and sub-cellular localization of the TORC2 gene was identified in bovine adipocytes. To investigate the underlying regulatory mechanism of the bovine TORC2, we cloned a 1990 bp of the 5' untranslated region (5′UTR) promoter region into a luciferase reporter vector and seven vector fragments were constructed through serial deletion of the 5′UTR flanking region. The core promoter region of the TORC2 gene was identified at location −314 to −69 bp upstream of the transcription start site. Based on the results of the transcriptional activities of the promoter vector fragments, luciferase activities of mutated fragments and siRNAs interference, four transcription factors (CCAAT/enhancer-binding protein C/BEPγ, X-box binding protein 1 XBP1, Insulinoma-associated 1 INSM1, and Zinc finger protein 263 ZNF263) were identified as the transcriptional regulators of TORC2 gene. These findings were further confirmed through Electrophoretic Mobility Shift Assay (EMSA) within nuclear extracts of bovine adipocytes. Furthermore, we also identified that C/EBPγ, XBP1, INSM1 and ZNF263 regulate TORC2 gene as activators in the promoter region. We can conclude that TORC2 gene is potentially a positive regulator of adipogenesis. These findings will not only provide an insight for the improvement of intramuscular fat in cattle, but will enhance our understanding regarding therapeutic intervention of metabolic syndrome and obesity in public health as well. [ABSTRACT FROM AUTHOR]

Background: Dairy cows with metabolic disorder in peripartal period display high inflammatory levels. Adipose tissue is a major endocrine organ, which is closely related to systemic inflammation. Retinoic acid (RA), an active metabolite of vitamin A, has shown potential therapeutic immunomodulatory properties. The objective of the study was to examine the effect of all-trans-RA (ATRA), the biologically most active metabolite of vitamin A, on lipopolysaccharide (LPS)-induced bovine adipocytes inflammatory responses and elucidate the underlying mechanisms. Primary cultured bovine adipocytes were treated with ATRA in the presence or absence of LPS. The treated cells were examined for the inflammatory responses and the activity of transforming growth factor beta 1 (TGFβ1) /Smad3 signaling pathway. Results: LPS treatment significantly decreased the expression levels of TGFβ1/Smad3 components and increased the content of pro-inflammatory cytokines. Treatment with ATRA could over-activate TGFβ1/Smad3 signaling pathway in bovine adipocytes and reversed the over-production of pro-inflammatory cytokines and inhibition of anti-inflammatory cytokines induced by LPS. Importantly, inhibition of TGFβ1/Smad3 signaling diminished the effects of ATRA on suppressing the proinflammatory responses induced by LPS. Furthermore, activation of TGFβ1/Smad3 signaling further extended the effects of ATRA on suppressing the proinflammatory responses on LPS stimulation. Conclusion: In conclusion, ATRA stimulates TGFβ1/Smad3 signaling pathway and further suppresses bovine adipocytes inflammatory responses induced by LPS. [ABSTRACT FROM AUTHOR]

The elongation of very long chain fatty acids protein 6 (ELOVL6) gene encodes a key enzyme that plays a role in lipogenesis through the catalytic elongation of both saturated and monounsaturated fatty acids. Previous studies have described the high expression of bovine ELOVL6 in adipose tissues. However, transcriptional regulation and the functional role of ELOVL6 in lipid metabolism and adipocyte proliferation remain unexplored. Here, a 1.5 kb fragment of the 5'-untranslated region promoter region of ELOVL6 was amplified from the genomic DNA of Qinchuan cattle and sequenced. The core promoter region was identified through unidirectional 5'-end deletion of the promoter plasmid vector. In silico analysis predicted important transcription factors that were then validated through site-directed mutation and small interfering RNA interference with an electrophoretic mobility shift assay. We found that the binding of KLF6 and PU.1 transcription factors occurred in the region -168/+69. Both perform a vital regulatory function in the transcription of bovine ELOVL6. Overexpression of ELOVL6 significantly upregulated the expression of peroxisome proliferator activated receptor γ (PPARγ), but inhibited the expression of fatty acid-binding protein 4 (FABP4), while silencing of ELOVL6 negatively regulated the messenger RNA expression level of PPARγ, FABP4, ACSL, and FATP1. In addition, ELOVL6 promotes adipocyte proliferation by regulating the cell-cycle genes' expression. Taken together, these findings provide useful information about the transcriptional regulation and functional mechanisms of bovine ELOVL6 in lipid metabolism and adipocyte proliferation in Qinchuan cattle., (© 2019 Wiley Periodicals, Inc.)

PLIN2 (Perilipin-2) is a protein that can anchor on the membrane of lipid droplets (LDs), playing a vital role in the early formation of LDs and in the regulation of LD metabolism in many types of cells. However, little research has been conducted in cattle adipocytes. In the present study, we found that the expression of PLIN2 mRNA peaks at Day 2 during cattle adipocyte differentiation (p < 0.01), but PLIN2 protein levels maintain high abundance until Day 4 and then decrease sharply. We first built an interaction model using PyMOL. The results of a pull-down assay indicated that bovine PLIN2 and CGI-58 (ABHD5, α/β hydrolase domain-containing protein 5) had an interaction relationship. Furthermore, Bimolecular Fluorescence Complementation-Flow Cytometry (BiFC-FC) was used to explore the function of the PLIN2-CGI-58 interaction. Interestingly, we found that different combined models had different levels of fluorescence intensity; specifically, PLIN2-VN173+CGI-58-VC155 expressed in bovine adipocytes exhibited the highest level of fluorescence intensity. Our findings elucidate the PLIN2 expression pattern in cattle adipocytes, the protein structure and the function of protein–protein interactions (PPI) as well as highlight the characteristics of bovine PLIN2 during the early formation and accumulation of lipid droplets. [ABSTRACT FROM AUTHOR]