OBJECTIVES: To investigate the effect of pachymic acid on brown/beige adipocyte differentiation and lipid metabolism in preadipocytes. METHODS: 3T3-L1 MBX cells were induced to differentiate into beige adipocytes using a brown cocktail method. The impact of pachymic acid on the viability of 3T3-L1 MBX cells was evaluated using the CCK-8 assay. The formation of lipid droplets following treatment with pachymic acid was observed by oil red O staining. The mRNA expression levels of key browning genes, including uncoupling protein (Ucp) 1, the peroxisome proliferator activated receptor-γ coactivator (Pgc)-1α, and the PR domain-containing protein 16 (Prdm16), as well as the mRNA expression of sterol regulatory element-binding protein (Srebp) 1c, acetyl-coA carboxylase (Acc), fatty acid synthase (Fas), and hormone-sensitive triglyceride lipase (Hsl), adipose triglyceride lipase (Atgl), and carnitine palmitoyltransferase (Cpt) 1 were detected by quantitative reverse transcription polymerase chain reaction. The protein expression of Ucp1, Pgc-1a, and Prdm16 was detected by Western blotting. RESULTS: The 3T3-L1 MBX cells were induced in vitro to form beige adipocytes with high expression of key browning genes(Ucp1, Pgc-1α, and Prdm16), and beige adipose-marker genes (Cd137, Tbx1, and Tmem26). Concentrations range of 0-80 μmol/L pachymic acid were non-cytotoxic to 3T3-L1 MBX cells. Pachymic acid treatment significantly inhibited the differentiation of 3T3-L1 MBX cells, resulting in a notable decrease in lipid accumulation. There was a marked increase in the expression of key browning genes and their proteins products, such as Ucp1, Pgc-1α, and Prdm16, while the expressions of fat synthesis-related genes Srebp1c, Acc and Fas were significantly decreased (all P<0.05). The expressions of lipolysis-related genes (Hsl, Atgl, and Cpt1) were significantly increased (all P<0.05). Treatment with 20 μmol/L pachymic acid showed the most pronounced effect. CONCLUSIONS Pachymic acid can inhibit fat synthesis and promote lipid decomposition by regulating the brown formation and lipid differentiation of preadipocytes.
Animals ; Lipid Metabolism/drug effects* ; Mice ; Cell Differentiation/drug effects* ; Adipocytes, Beige/drug effects* ; 3T3-L1 Cells ; Adipocytes, Brown/drug effects* ; Triterpenes/pharmacology* ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha ; Uncoupling Protein 1 ; Sterol Regulatory Element Binding Protein 1/metabolism*

Saponins associated with Panax notoginseng (P. notoginseng) demonstrate significant therapeutic efficacy across multiple diseases. However, certain high-yield saponins face limited clinical applications due to their reduced pharmacological efficacy. This study synthesized and evaluated 36 saponin-1,2,3-triazole derivatives of ginsenosides Rg1/Rb1 and notoginsenoside R1 for anti-adipogenesis activity in vitro. The research revealed that the ginsenosides Rg1-1,2,3-triazole derivative a17 demonstrates superior adipogenesis inhibitory effects. Structure-activity relationships (SARs) analysis indicates that incorporating an amidyl-substituted 1,2,3-triazole into the saponin side chain via Click reaction enhances anti-adipogenesis activity. Additionally, several other derivatives exhibit general adipogenesis inhibition. Compound a17 demonstrated enhanced potency compared to the parent ginsenoside Rg1. Mechanistic investigations revealed that a17 exhibits dose-dependent inhibition of adipogenesis in vitro, accompanied by decreased expression of preadipocytes. Peroxisome proliferator-activated receptor γ (PPARγ), fatty acid synthase (FAS), and fatty acid binding protein 4 (FABP4) adipogenesis regulators. These findings establish the ginsenoside Rg1-1,2,3-triazole derivative a17 as a promising adipocyte differentiation inhibitor and potential therapeutic agent for obesity and associated metabolic disorders. This research provides a foundation for developing effective therapeutic approaches for various metabolic syndromes.
Adipogenesis/drug effects* ; Triazoles/chemical synthesis* ; Ginsenosides/chemical synthesis* ; Saponins/chemical synthesis* ; Animals ; Mice ; Structure-Activity Relationship ; PPAR gamma/genetics* ; 3T3-L1 Cells ; Adipocytes/metabolism* ; Panax notoginseng/chemistry* ; Drug Design ; Molecular Structure ; Humans ; Cell Differentiation/drug effects* ; Fatty Acid-Binding Proteins/genetics*

Plant polysaccharides are effective components that widely present in traditional Chinese medicine(TCM), exhibiting rich biological activities. However, as most plant polysaccharides cannot be directly absorbed and utilized by the human digestive system, it is now believed that their mode of action mainly involves interaction with intestinal microbiota, leading to the production of functional small molecules. The efficacy of Astragalus polysaccharide(APS) is extensive, including weight loss, improvement of fatty liver, reduction of blood lipids, and enhancement of insulin sensitivity, which may also be related to the regulation of intestinal microbiota. Adipose tissue senescence is an important characteristic of the physiological aging process in the body, often occurring prior to the aging of other important organs. Its main features include the accumulation of senescent cells and exacerbation of inflammation within the tissue. Therefore, to explore the potential protective effects of APS on aging, the improvement of adipose tissue aging phenotype in naturally aging mice was observed using APS, and combined with metagenomic metabolomics, corresponding microbial metabolic functional molecules were identified. Furthermore, functional tests in cell aging models were conducted. The results showed that APS significantly improved the adipocyte aging characteristics of naturally aging mice: specifically reducing aging-induced adipocyte hypertrophy; decreasing the protein expression of aging markers cyclin-dependent kinase inhibitor p21(P21) and multiple tumor suppressor 1(P16); lowering the tissue inflammation reaction. Metagenomic metabolomic analysis of serum from mice in each group revealed that APS significantly increased the content of indole-3-lactic acid(ILA) in naturally aging mice. Further in vitro studies showed that ILA could improve the aging of 3T3-L1 mouse embryonic fibroblasts induced by bleomycin, reduce the protein expression of the aging marker P21, alleviate inflammation, and enhance the ability of preadipocytes to mature. Therefore, APS had the efficacy of protecting naturally aging mice, and its action may be related to the increase in the intestinal microbiota metabolite ILA. This study suggested that TCM may serve as an important entry point for explaining the mechanism of action of TCM by regulating intestinal microbiota and their functional metabolites.
Animals ; Mice ; Aging/drug effects* ; Adipose Tissue/metabolism* ; Polysaccharides/pharmacology* ; Indoles/pharmacology* ; Male ; Astragalus Plant/chemistry* ; 3T3-L1 Cells ; Humans ; Adipocytes/cytology* ; Mice, Inbred C57BL ; Cellular Senescence/drug effects* ; Drugs, Chinese Herbal/administration & dosage* ; Gastrointestinal Microbiome/drug effects*

OBJECTIVE: To investigate the effect of adipocytes in the bone marrow microenvironment of patients with multiple myeloma (MM) on the pathogenesis of MM. METHODS: Bone marrow adipocytes (BMA) in bone marrow smears of health donors (HD) and newly diagnosed MM (ND-MM) patients were evaluated with oil red O staining. The mesenchymal stem cells (MSC) from HD and ND-MM patients were isolated, and in vitro co-culture assay was used to explore the effects of MM cells on the adipogenic differentiation of MSC and the role of BMA in the survival and drug resistance of MM cells. The expression of adipogenic/osteogenic differentiation-related genes PPAR-γ, DLK1, DGAT1, FABP4, FASN and ALP both in MSC and MSC-derived adipocytes was determined with real-time quantitative PCR. The Western blot was employed to detect the expression levels of IL-6, IL-10, SDF-1α, TNF-α and IGF-1 in the supernatant with or without PPAR-γ inhibitor. RESULTS: The results of oil red O staining of bone marrow smears showed that BMA increased significantly in patients of ND-MM compared with the normal control group, and the BMA content was related to the disease status. The content of BMA decreased in the patients with effective chemotherapy. MM cells up-regulated the expression of MSC adipogenic differentiation-related genes PPAR-γ, DLK1, DGAT1, FABP4 and FASN, but the expression of osteogenic differentiation-related gene ALP was significantly down-regulated. This means that the direct consequence of the interaction between MM cells and MSC in the bone marrow microenvironment is to promote the differentiation of MSC into adipocytes at the expense of osteoblasts, and the cytokines detected in supernatant changed. PPAR-γ inhibitor G3335 could partially reverse the release of cytokines by BMA. Those results confirmed that BMA regulated the release of cytokines via PPAR-γ signal, and PPAR-γ inhibitor G3335 could distort PPAR-γ mediated BMA maturation and cytokines release. The increased BMA and related cytokines effectively promoted the proliferation, migration and drug resistance of MM cells. CONCLUSION The BMA and its associated cytokines are the promoting factors in the survival, proliferation and migration of MM cells. BMA can protect MM cells from drug-induced apoptosis and plays an important role in MM treatment failure and disease progression.
Humans ; Osteogenesis/genetics* ; Bone Marrow/metabolism* ; Multiple Myeloma/metabolism* ; Drug Resistance, Neoplasm ; Peroxisome Proliferator-Activated Receptors/pharmacology* ; Cell Differentiation ; Adipogenesis ; Cytokines/metabolism* ; Adipocytes/metabolism* ; Bone Marrow Cells/metabolism* ; Cells, Cultured ; PPAR gamma/pharmacology* ; Tumor Microenvironment

The growth, differentiation and proliferation of adipose cells run through the whole life process. Dysregulation of lipid metabolism in adipose cells affects adipose tissue immunity and systemic energy metabolism. Increasingly available data suggest that lipid metabolism is involved in regulating the occurrence and development of various diseases, such as hyperlipidemia, nonalcoholic fatty liver disease, diabetes and cancer, which pose a major threat to human and animal health. Hypoxia inducible factor (HIF) is a major transcription factor mediating oxygen receptors in tissues and organs. HIF can induce disease by regulating lipid synthesis, fatty acid metabolism and lipid droplet formation. However, due to the difference of hypoxia degree, time and mode of action, there is no conclusive conclusion whether it has harmful or beneficial effects on the development of adipocytes and lipid metabolism. This article summarizes the regulation of hypoxia stress mediated transcription regulators and regulation of adipocyte development and lipid metabolism, aiming to reveal the potential mechanism of hypoxia induced changes in adipocyte metabolism pathways.
Animals ; Humans ; Lipid Metabolism ; Adipocytes/metabolism* ; Adipose Tissue/metabolism* ; Hypoxia/metabolism* ; Transcription Factors/metabolism*

OBJECTIVE: To establish a simple, low-cost and time-saving method for primary culture of mature white adipocytes from mice. METHODS: Mature white adipocytes were isolated from the epididymis and perirenal area of mice for primary culture using a modified mature adipocyte culture method or the ceiling culture method. The morphology of the cultured mature adipocytes was observed using Oil Red O staining, and the cell viability was assessed with CCK8 method. The expression of PPARγ protein in the cells was detected with Western blotting, and the mRNA expressions of CD36, FAS, CPT1A and FABP4 were detected using RT-qPCR. RESULTS: Oil Red O staining showed a good and uniform morphology of the adipocytes in primary culture using the modified culture method, while the cells cultured using the ceiling culture method exhibited obvious morphological changes. CCK8 assay showed no significant difference in cell viability between freshly isolated mature white adipocytes and the cells obtained with the modified culture method. Western blotting showed that the freshly isolated adipocytes and the cells cultured for 72 h did not differ significantly in the expression levels of PPARγ protein (P=0.759), which was significantly lowered in response to treatment with GW9662 (P < 0.001). GW9662 treatment of the cells upregulated mRNA expressions of CD36 (P < 0.001) and CPT1A (P=0.003) and down-regulated those of FAS (P=0.001) and FABP4 (P < 0.001). CONCLUSION We established a convenient and time-saving method for primary culture mature white adipocytes from mice to facilitate further functional studies of mature adipocytes.
Male ; Mice ; Animals ; Adipocytes, White/metabolism* ; PPAR gamma/metabolism* ; RNA, Messenger ; Cell Differentiation ; 3T3-L1 Cells

Overexpression of Krüppel like factor 2 (Klf2) or Klf7 inhibits adipocyte formation. However, it remains unclear whether Klf2 regulates klf7 expression in adipose tissue. In this study, oil red O staining and Western blotting were employed to study the effect of Klf2 overexpression on the differentiation of chicken preadipocytes. The results showed that Klf2 overexpression inhibited the differentiation of chicken preadipocytes induced by oleate and the expression of pparγ, while promoted klf7 expression in chicken preadipocytes. Spearman correlation analysis was used to study the correlation between the expression data of klf2 and klf7 in the adipose tissue of both human and chicken. The results showed that there was a significantly positive correlation between the expression of klf2 and klf7 in adipose tissues (r > 0.1). Luciferase reporter assay showed that overexpression of Klf2 significantly promoted the activity of chicken klf7 promoter (-241/-91, -521/-91, -1 845/-91, -2 286/-91, -1 215/-91; P < 0.05). In addition, the activity of klf7 promoter (-241/-91) reporter in chicken preadipocytes was significantly positively correlated with the amount of klf2 overexpression plasmid transfected (T_au=0.917 66, P=1.074×10^-7). Moreover, Klf2 overexpression significantly promoted the mRNA expression of klf7 in chicken preadipocytes (P < 0.05). In conclusion, upregulation of klf7 expression might be one of the pathways that Klf2 inhibits chicken adipocyte differentiation, and the sequence from -241 bp to -91 bp upstream chicken klf7 translation start site might mediate the regulation of Klf2 on klf7 transcription.
Animals ; Humans ; Chickens/genetics* ; Kruppel-Like Transcription Factors/metabolism* ; Transcription Factors/metabolism* ; Adipocytes/metabolism* ; Adipose Tissue/metabolism*

The aim of this study was to investigate the effect of activating transcription factor 3 (ATF3) on the differentiation of intramuscular preadipocytes in goat, and to elucidate its possible action pathway at the molecular level. In this study, the recombinant plasmid of goat pEGFP-N1-ATF3 was constructed, and the intramuscular preadipocytes were transfected with liposomes. The relative expression levels of adipocyte differentiation marker genes were detected by quantitative real-time PCR (qRT-PCR). After transfection of goat intramuscular preadipocytes with the goat pEGFP-N1-ATF3 overexpression vector, it was found that the accumulation of lipid droplets was inhibited, and the adipocyte differentiation markers PPARγ, C/EBPα and SREBP1 were extremely significantly down-regulated (P < 0.01), while C/EBPβ and AP2 were significantly down-regulated (P < 0.05). The ATF3 binding sites were predicted to exist in the promoter regions of PPARγ, C/EBPα and AP2 by the ALGGEN PROMO program. The overexpression of goat ATF3 inhibits the accumulation of lipid droplets in intramuscular preadipocytes, and this effect may be achieved by down-regulating PPARγ, C/EBPα and AP2. These results may facilitate elucidation of the regulatory mechanism of ATF3 in regulating the differentiation of goat intramuscular preadipocytes.
3T3-L1 Cells ; Activating Transcription Factor 3/pharmacology* ; Adipocytes ; Adipogenesis/genetics* ; Animals ; CCAAT-Enhancer-Binding Protein-alpha/pharmacology* ; Cell Differentiation ; Goats ; Mice ; PPAR gamma/metabolism*

We used CRISPR/Cas9 to delete plin1 of 3T3-L1 preadipocyte, to observe its effect on lipolysis in adipocytes and to explore regulatory pathways. We cultured 3T3-L1 preadipocytes, and the plin1 knockout vectors were transfected by electroporation. Puromycin culture was used to screen successfully transfected adipocytes, and survival rates were observed after transfection. The optimized "cocktail" method was used to differentiate 3T3-L1 preadipocytes. The glycerol and triglyceride contents were determined by enzymatic methods. The changes in lipid droplet form and size were observed by Oil red O staining. The protein expression of PLIN1, PPARγ, Fsp27, and lipases was measured by Western blotting. RT-PCR was used to measure the expression of PLIN1 and lipases mRNA. After the adipocytes in the control group were induced to differentiate, the quantity of tiny lipid droplets was decreased, and the quantity of unilocular lipid droplets was increased and arranged in a circle around the nucleus. Compared with the control group, the volume of unilocular lipid droplets decreased, and the quantity of tiny lipid droplets increased after induction of adipocytes in the knockout group. The expression of PLIN1 mRNA and protein in the adipocytes was significantly inhibited (P<0.05); glycerol levels increased significantly (0.098 4±0.007 6), TG levels decreased significantly (0.031 0±0.005 3); mRNA and protein expression of HSL and ATGL increased (P<0.05); PPARγ and Fsp27 expression unchanged in adipocytes. The above results indicate that the knockout of plin1 enhances the lipolysis of 3T3-L1 adipocytes by exposing lipids in lipid droplets and up-regulating lipases effects.
3T3-L1 Cells ; Adipocytes ; metabolism ; Animals ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Lipase ; metabolism ; Lipolysis ; genetics ; Mice ; Perilipin-1 ; genetics ; metabolism

The present study was aimed to clarify the signaling molecular mechanism by which fibroblast growth factor 21 (FGF21) regulates leptin gene expression in adipocytes. Differentiated 3T3-F442A adipocytes were used as study object. The mRNA expression level of leptin was detected by fluorescence quantitative RT-PCR. The phosphorylation levels of proteins of signal transduction pathways were detected by Western blot. The results showed that FGF21 significantly down-regulated the mRNA expression level of leptin in adipocytes, and FGF21 receptor inhibitor BGJ-398 could completely block this effect. FGF21 up-regulated the phosphorylation levels of ERK1/2 and AMPK in adipocytes. Either ERK1/2 inhibitor SCH772984 or AMPK inhibitor Compound C could partially block the inhibitory effect of FGF21, and the combined application of these two inhibitors completely blocked the effect of FGF21. Neither PI3K inhibitor LY294002 nor Akt inhibitor AZD5363 affected the inhibitory effect of FGF21 on leptin gene expression. These results suggest that FGF21 may inhibit leptin gene expression by activating ERK1/2 and AMPK signaling pathways in adipocytes.
3T3 Cells ; Adenylate Kinase ; Adipocytes ; metabolism ; Animals ; Down-Regulation ; Fibroblast Growth Factors ; metabolism ; Leptin ; metabolism ; MAP Kinase Signaling System ; Mice ; Phosphorylation ; Signal Transduction