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Overexpression of DGAT2 Regulates the Differentiation of Bovine Preadipocytes.

ABSTRACT: 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.

SUBMITTER: Guo PP

PROVIDER: S-EPMC10093762 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Overexpression of DGAT2 Regulates the Differentiation of Bovine Preadipocytes.

Guo Pan-Pan PP Jin Xin X Zhang Jun-Fang JF Li Qiang Q Yan Chang-Guo CG Li Xiang-Zi XZ

Animals : an open access journal from MDPI 20230329 7

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 ...[more]

PMID: 37048451

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Project description:ObjectiveMuscle 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.MethodsWe 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.ResultsThe 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.ConclusionOverexpression of CHRNG in bovine preadipocytes inhibits the proliferation and differentiation of bovine preadipocytes.

Project description:Intramuscular fat (IMF) is closely related to the tenderness, juiciness, and flavor of beef, and is an important indicator for beef quality assessment internationally. The main components of skeletal intramuscular fat (IMF) are phospholipids and triacylglycerols (TAG), and the final step of TAG biosynthesis is catalyzed by diacylglycerol acyltransferase 2 (DGAT2). To explore the effect of DGAT2 on the differentiation of bovine muscle satellite cells (BSCs) and its role in the signaling pathway related to lipid metabolism, the adenovirus overexpression and interference vector of the DGAT2 gene was constructed in this study, and the overexpression adenovirus Ad-DGAT2 and interfering adenovirus sh-DGAT2 were used to infect BSCs. Overexpression of DGAT2 resulted in a significant increase in the contents of TAG and ADP, and the mRNA and protein expression levels of PPARγ, C/EBPα, and SREBF1 (p < 0.05). Interfering with the expression of DGAT2 reduced the intracellular TAG content and lipid droplet accumulation. Furthermore, the mRNA and protein expression levels of PPARγ, C/EBPα, and SREBF1 (p < 0.05) were significantly downregulated. Transcriptome sequencing showed that a total of 598 differentially expressed genes (DEGs) were screened in BSCs infected with Ad-DGAT2, and these DEGs included 292 upregulated genes and 306 downregulated genes. A total of 49 DEGs were screened in BSCs infected with sh-DGAT2, and these DEGs included 25 upregulated and 24 downregulated genes. KEGG enrichment analysis showed that the DEGs, after overexpression of DGAT2, were mainly enriched in the PPAR signaling pathway, and the fat digestion and absorption, glycerophospholipid metabolism, fatty acid biosynthesis, and AMPK signaling pathways. The DEGs obtained after interfering with DGAT2 were mainly enriched in the metabolic pathways, such as the PPAR signaling pathway and PI3K/AKT signaling pathway. In summary, our study demonstrated that the lipid droplet formation, TAG accumulation, and adipogenic gene expression in BSCs overexpressing DGAT2 were higher than those in the control cells. These results highlight the important role of DGAT2 in regulating BSCs during adipogenic transdifferentiation and underscore the complexity of intramuscular adipogenesis.

Project description:BackgroundAdipose tissue has been proven to play a crucial role in wound healing, while kindlin-2, an integrin-associated protein, has been shown to regulate cell adhesion, migration, and differentiation. This study aimed to explore its involvement in the cell differentiation of 3T3-L1 preadipocytes and its role in wound healing.MethodsCell adhesion, Cell Counting Kit-8 (CCK-8), Transwell, and in vitro wound healing assays, along with adipogenic and osteogenic differentiation induction were performed in 3T3-L1 preadipocytes in which kindlin-2 was knocked down or overexpressed. In vivo, kindlin-2 (+/-) transgenic mice were constructed, and wound healing was analyzed by immunohistochemistry (IHC) in a mouse dorsal wound model. Real-time polymerase chain reaction (RT-PCR) and western blotting were performed to analyze the expression of adipokines and adipogenic markers in mouse wound tissues. Adipogenic differentiation induction of adipose tissue stromal vascular fraction (SVF) were performed, and the expression of adipogenic markers in SVF was detected by western blotting. The target signaling pathway highly related to adipogenic differentiation was explored by computational biology and verified by western blotting.ResultsKnockdown of kindlin-2 was found to inhibit the adhesion, migration, and adipogenic differentiation of 3T3-L1 preadipocytes while promoting their osteogenic differentiation. In contrast, kindlin-2 overexpression resulted in increased adhesion, migration, and adipogenic differentiation of 3T3-L1 preadipocytes while reducing osteogenic differentiation. In vivo, downregulation of kindlin-2 inhibited adipogenesis in kindlin-2 transgenic mice, resulting in delayed wound healing by inhibiting inflammation, angiogenesis, collagen remodeling, and wound contraction. Mechanistically, we found that kindlin-2 could regulate adipogenic differentiation through PI3K/AKT/mTOR signaling pathway.ConclusionsOur study revealed the essential role that kindlin-2 has in the differentiation and wound healing of 3T3-L1 preadipocytes, which offers a theoretical basis for further research and a novel strategy for wound healing.

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Overexpression of DGAT2 Regulates the Differentiation of Bovine Preadipocytes.

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Overexpression of <i>DGAT2</i> Regulates the Differentiation of Bovine Preadipocytes.

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