Effect of the ACAA1 Gene on Preadipocyte Differentiation in Sheep

Acetyl-CoA acyltransferase 1 (ACAA1) functions as a key regulator of fatty acid β-oxidation in peroxisomes by catalyzing the cleavage of 3-ketoacyl-CoA to acetyl-CoA and acyl-CoA, which participate in the extension and degradation of fatty acids. Thus, ACAA1 is an important regulator of lipid metabolism and plays an essential role in fatty acid oxidation and lipid metabolism. Our previous study findings revealed that ACAA1 is closely associated with the peroxisome proliferator-activated receptor (PPAR) signaling and fatty acid metabolism pathways, which are involved in fat deposition in sheep, leading to our hypothesis that ACAA1 may be involved in fat deposition by regulating lipid metabolism. However, the associated molecular mechanism remains unclear. In the present study, to assess the potential function of ACAA1 in sheep preadipocyte differentiation, we knocked down and overexpressed ACAA1 in sheep preadipocytes and evaluated the pattern of ACAA1 gene expression during preadipocyte differentiation by qRT-PCR. ACAA1 was significantly expressed in the early stage of adipocyte differentiation, and then its expression decreased. ACAA1 deficiency increased lipid accumulation and the triglyceride content and promoted sheep preadipocyte differentiation, whereas ACAA1 overexpression inhibited adipogenesis and decreased lipid accumulation and the triglyceride content. Simultaneously, we demonstrated that ACAA1 deficiency upregulated the expressions of the adipogenic marker genes PPARγ and C/EBPα in sheep preadipocytes, but ACAA1 overexpression inhibited the expressions of these markers, indicating that ACAA1 affects lipid metabolism by regulating adipogenic marker genes. Our results may promote a better understanding of the regulation of adipogenesis by ACAA1.

Adipogenic differentiation potential of porcine bone marrow mesenchymal stem cells grown in different basal media formulations

Bone marrow-derived porcine mesenchymal stem cells (pMSC) are precursors of multiple lineage cells. However, the differentiation potential of pMSC might vary due to the culture media in which they are isolated and grown. In this study the effects of αMEM, aDMEM, M199, αMEM/M199, aDMEM/M199 and αMEM/aDMEM was determined on pMSC expression of adipogenic marker genes PPARγ, C/EBPα and ApN at passage 5 and 10; and differentiation potential of pMSC at passage 5. Relative expressions of the genes were determined by qPCR assay. Adipogenic differentiation progress was determined by microscopic evaluation and accumulation of lipid vesicles in cells after day 21 differentiation was confirmed by Oil Red-O staining. Results indicated varying expressions of adipogenic marker genes at passage 5 and 10 in pMSC grown in different culture media. Adipogenic differentiation characteristics also varied in different culture media, grown cells depending on the expression of adipogenic marker genes. Classification in order of differentiation potential revealed that the pMSC grown in M199 and αMEM/M199 was the best, followed by aDMEM/M199 and αMEM/aDMEM the intermediate and αMEM and aDMEM the least suitable media. Effect of media interaction analysis indicated that M199 media might have played a significant role in up-keeping of the desired marker gene expression and adipogenic differentiation potential of the pMSC. In conclusion, adipogenic differentiation potential of pMSC varied due to the differences in basal media composition and the M199 played a significant role in skewing pMSC towards adipogenic lineage.

Deoxynivalenol Exposure Suppresses Adipogenesis by Inhibiting the Expression of Peroxisome Proliferator-Activated Receptor Gamma 2 (PPARγ2) in 3T3-L1 Cells

Deoxynivalenol (DON)—a type B trichothecene mycotoxin, mainly produced by the secondary metabolism of Fusarium—has toxic effects on animals and humans. Although DON’s toxicity in many organs including the adrenal glands, thymus, stomach, spleen, and colon has been addressed, its effects on adipocytes have not been investigated. In this study, 3T3-L1 cells were chosen as the cell model and treated with less toxic doses of DON (100 ng/mL) for 7 days. An inhibition of adipogenesis and decrease in triglycerides (TGs) were observed. DON exposure significantly downregulated the expression of PPARγ2 and C/EBPα, along with that of other adipogenic marker genes in 3T3-L1 cells and BALB/c mice. The anti-adipogenesis effect of DON and the downregulation of the expression of adipogenic marker genes were effectively reversed by PPARγ2 overexpression. The repression of PPARγ2′s expression is the pivotal event during DON exposure regarding adipogenesis. DON exposure specifically decreased the di-/trimethylation levels of Histone 3 at lysine 4 in 3T3-L1 cells, therefore weakening the enrichment of H3K4me2 and H3K4me3 at the Pparγ2 promoter and suppressing its expression. Conclusively, DON exposure inhibited PPARγ2 expression via decreasing H3K4 methylation, downregulated the expression of PPARγ2-regulated adipogenic marker genes, and consequently suppressed the intermediate and late stages of adipogenesis. Our results broaden the current understanding of DON’s toxic effects and provide a reference for addressing the toxicological mechanism of DON’s interference with lipid homeostasis.

LncRNA HCG11 inhibits adipocyte differentiation in human adipose-derived mesenchymal stem cells by sponging miR-204-5p to upregulate SIRT1

Background: LncRNAs have been discovered to play a key role in adipogenesis, vital in regulating adipose developmen t. Numerous evidences show that adipogenesis is the leading cause of obesity, while the role of lncRNA HLA complex group 11 ( HCG11 ) in adipocyte differentiation has not been elucidated. Methods: hAdMSCs were used to establish a model of cell differentiation in vitro . Expression of lncRNA HCG11 was detected by RT-qPCR analysis. Transfection of the shRNA targeting HCG11 or pcDNA-HCG11 into hAdMSCs was also assessed. The adipogenic marker proteins C/EBPα, FABP4 and PPARγ2 and important inflammatory factors IL-6 and TNF-α were detected by Western blot. Bioinformatics analysis predicted the target genes of HCG11 and mir-204-5p, which was confirmed by luciferase reporter gene analysis and RNA pull-down analysis. Results: Here we show that lncRNA HCG11 was decreased as the degree of adipogenesis. The expression of C/EBPα, FABP4 and PPARγ2 were significantly downregulated transfected with pcDNA-HCG11 in hAdMSCs at different stages, while knockdown lncRNA HCG11 can promote adipocyte differentiation. In addition, miR-204-5p was a potential target gene of HCG11 and SIRT1 could directly target miR-204-5p. Overexpression of SIRT1 or transfected with agonists of SIRT1 (Res) significantly inhibited adipogenic marker protein levels and inflammatory responses, and the proliferation of hAdMSCs were also inhibited. pcDNA-HCG11 and miR-204-5p mimic were co-transfected into hAdMSCs, we found that miR-204-5p mimic reversed the suppressor effect of pcDNA-HCG11. Conclusion: Our findings showed that HCG11 negatively regulated cell proliferation, inflammatory responses and adipogenesis by miR-204-5p/SIRT1 axis. And our findings may provide a new target for the study of adipogenesis in hAdMSCs and obesity.

LncRNA HCG11 Inhibits Adipocyte Differentiation in Human Adipose-Derived Mesenchymal Stem Cells by Sponging miR-204-5p to Upregulate SIRT1

Long noncoding RNAs (lncRNAs) have been discovered to play a key role in adipogenesis, while the role of lncRNA human leukocyte antigen complex group 11 (HCG11) in adipocyte differentiation has not been studied clearly. We used human adipose-derived mesenchymal stem cells (hAdMSCs) to establish a model of cell differentiation in vitro and found that expression of lncRNA HCG11 was decreased during adipogenesis through real-time quantitative polymerase chain reaction analysis. Then, hAdMSCs were transfected with pcDNA-HCG11 or HCG11-shRNA (sh-HCG11); the adipogenic marker proteins were detected by Western blot, and the activity of lipogenesis enzymes was detected by spectrophotometry. The expression of CCAAT-enhancer-binding protein α, fatty acid-binding protein, peroxisome proliferator-activated receptor gamma 2 and the levels of acetyl coenzyme A carboxylase and fatty acid synthase FAS were significantly downregulated in hAdMSCs at different stages transfected with pcDNA-HCG11, while knockdown of lncRNA HCG11 promoted adipocyte differentiation. Bioinformatic analysis indicated that miR-204-5p was a potential target gene of HCG11, which was confirmed by luciferase reporter gene analysis and RNA pull-down analysis. In addition, miR-204-5p directly targeting the 3′-untranslated region of SIRT1 was also predicted by StarBase and verified by luciferase reporter gene analysis. Enforced expression of miR-204-5p negatively regulated the SIRT1 protein level. Furthermore, SIRT1 overexpression significantly inhibited adipogenic marker protein, levels of lipogenesis enzymes, and the proliferation of hAdMSCs. When pcDNA-HCG11 and miR-204-5p mimic were co-transfected into hAdMSCs, we found that the miR-204-5p mimic reversed the suppressor effect of pcDNA-HCG11. Taken together, we found that HCG11 negatively regulated cell proliferation and adipogenesis by the miR-204-5p/SIRT1 axis. Our findings might provide a new target for the study of adipogenesis in hAdMSCs and obesity.

miR-135a-5p inhibits 3T3-L1 adipogenesis through activation of canonical Wnt/β-catenin signaling

MicroRNAs are endogenous, conserved, and non-coding small RNAs that function as post-transcriptional regulators of fat development and adipogenesis. Adipogenic marker genes, such as CCAAT/enhancer binding protein α (Cebpa), peroxisome proliferator-activated receptor γ (Pparg), adipocyte fatty acid binding protein (Ap2), and fatty acid synthase (Fas), are regarded as the essential transcriptional regulators of preadipocyte differentiation and lipid storage in mature adipocytes. Canonical Wnt/β-catenin signaling is recognized as a negative molecular switch during adipogenesis. In the present work we found that miR-135a-5p is markedly downregulated during the process of 3T3-L1 preadipocyte differentiation. Overexpression of miR-135a-5p impairs the expressions of adipogenic marker genes as well as lipid droplet accumulation and triglyceride content, indicating the importance of miR-135a-5p for adipogenic differentiation and adipogenesis. Further studies show that miR-135a-5p directly targets adenomatous polyposis coli (Apc), contributes to the translocation of β-catenin from cytoplasm to nucleus, and then activates the expressions of cyclin D1 (Ccnd1) and Cmyc, indicating the induction of canonical Wnt/β-catenin signaling. In addition, inhibition of APC with siRNA exhibits the same effects as overexpression of miR-135a-5p. Our findings demonstrate that miR-135a-5p suppresses 3T3-L1 preadipocyte differentiation and adipogenesis through the activation of canonical Wnt/β-catenin signaling by directly targeting Apc. Taken together, these results offer profound insights into the adipogenesis mechanism and the development of adipose tissue.

Ceiling culture-derived proliferative adipocytes retain high adipogenic potential suitable for use as a vehicle for gene transduction therapy

Adipose tissue is expected to provide a source of proliferative cells for regenerative medicine and cell-transplantation therapies using gene transfer manipulation. We have recently identified ceiling culture-derived proliferative adipocytes (ccdPAs) from the mature adipocyte fraction as cells suitable as a therapeutic gene vehicle because of their stable proliferative capacity. In this study, we examined the capability of adipogenic differentiation of the ccdPAs compared with stromal vascular fraction (SVF)-derived progenitor cells (adipose-derived stem cells, ASCs) with regard to their multipotential ability to be converted to another lineage and therefore their potential to be used for regenerative medicine research. After in vitro passaging, the surface antigen profile and the basal levels of adipogenic marker genes of the ccdPAs were not obviously different from those of the ASCs. However, the ccdPAs showed increased lipid-droplet accumulation accompanied with higher adipogenic marker gene expression after stimulation of differentiation compared with the ASCs. The higher adipogenic potential of the ccdPAs than the ASCs from the SVF was maintained for 42 days in culture. Furthermore, the difference in the adipogenic response was enhanced after partial stimulation without indomethacin. These results indicate that the ccdPAs retain a high adipogenic potential even after in vitro passaging, thus suggesting the commitment of ccdPAs to stable mature adipocytes after autotransplantation, indicating that they may have potential for use in regenerative and gene-manipulated medicine.