Adipogenesis In 3T3-L1 Preadipocytes Research Articles

Comparative Study on the Differentiation Effect of Adipogenesis in 3T3-L1 Preadipocyte by 65 Herbal Medicine Prescriptions

Comparative Study on the Differentiation Effect of Adipogenesis in 3T3-L1 Preadipocyte by 65 Herbal Medicine Prescriptions

Tyrosol May Prevent Obesity by Inhibiting Adipogenesis in 3T3-L1 Preadipocytes

Tyrosol (TR), a major polyphenol found in extra virgin olive oil (EVOO), exerts several antioxidant effects. However, only scarce evidences are present regarding its activity on adipocytes and obesity. This study evaluated the role of TR in adipogenesis. Murine 3T3-L1 preadipocytes were incubated with TR (300 and 500 μM), and TR administration inhibited adipogenesis by downregulation of several adipogenic factors (leptin and aP2) and transcription factors (C/EBPα, PPARγ, SREBP1c, and Glut4) and by modulation of the histone deacetylase sirtuin 1. After complete differentiation, adipocytes treated with 300 and 500 μM TR showed a reduction of 20% and 30% in lipid droplets, respectively. Intracellular triglycerides were significantly reduced after TR treatment (p < 0.05). Mature adipocytes treated with TR at 300 and 500 μM showed a marked decrease in the inflammatory state and oxidative stress as shown by the modulation of specific biomarkers (TNF, IL6, ROS, and SOD2). TR treatment also acted on the early stage of differentiation by reducing cell proliferation (~40%) and inducing cell cycle arrest during Mitotic Expansion Clonal (first 48 h of differentiation), as shown by the increase in both S1 phase and p21 protein expression. We also showed that TR induced lipolysis by activating the AMPK-ATGL-HSL pathway. In conclusion, we provided evidence that TR reduces 3T3-L1 differentiation through downregulation of adipogenic proteins, inflammation, and oxidative stress. Moreover, TR may trigger adipose tissue browning throughout the induction of the AMPK-ATGL-UCP1 pathway and, subsequently, may have promise as a potential therapeutic agent for the treatment and prevention of obesity.

Anti‐adipogenesis and metabolism‐regulating effects of heat‐inactivated Streptococcus thermophilus MN‐ZLW‐002

Metabolic syndrome and obesity have become serious threats to public health worldwide. This study was conducted to evaluate the anti-adipogenesis and metabolism-regulating effects of heat-inactivated Streptococcus thermophilus MN-ZLW-002 (MN-ZLW-002), which can be used as a yogurt starter. In vitro study suggested that MN-ZLW-002 stimulated the RAW264.7 macrophages to produce significant amounts of interleukin (IL)-6, IL-10 and tumour necrosis factor (TNF)-α and induced intense phosphorylation of P38, p44/42 MAPK and nuclear factor κB. MN-ZLW-002-stimulated RAW264.7-conditioned medium (CM) notably suppressed the differentiation and adipogenesis of 3T3-L1 pre-adipocytes. The 12-week in vivo study suggested that orally administered MN-ZLW-002 significantly reduced the weight gain of mice caused by the high-fat diet (HFD) at weeks 3-8; decreased fasting blood glucose levels at week 4 and week 8; decreased serum total triglyceride level at week 12. MN-ZLW-002 also reduced serum IL-1β and chemokine ligand 3 levels in the HFD-fed mice. These findings suggest that heat-inactivated MN-ZLW-002 can suppress adipocytes differentiation and lipid accumulation by regulating the immune response, possibly via the release of cytokines, particularly TNF-α; MN-ZLW-002 can improve metabolism-related indicators in the early stage of HFD intervention and regulate the related pro-inflammatory immune response.

An in vitro study reveals the anti-obesity effects of 7- methoxy-3-methyl-5-((E)-prop-1-enyl)-2-(3,4,5-trimethoxyphenyl)-2,3-dihydrobenzofuran from Myristica fragrans

Adipogenesis, the maturation process of preadipocytes, is closely associated with the development of obesity and other complex metabolic syndromes. Herein, we investigated the effect of 7- methoxy-3-methyl-5-((E)- prop-1-enyl)-2-(3,4,5-trimethoxyphenyl)-2,3-dihydrobenzofuran (TM), a benzofuran, isolated from the mace of Myristica fragrans Houtt on adipogenesis in 3T3-L1 preadipocytes to extrapolate whether this compound has any anti-obesity potential. For this, 3T3-L1 preadipocytes were induced to differentiate in the presence of various concentrations of TM (1, 5, 10 μM) and analyzed for triglyceride (TG) accumulation and the expression of proteins and genes involved in lipogenesis and lipolysis associated with adipogenesis. Results showed that TM significantly reduced TG accumulation and expression of marker proteins of adipocyte differentiation (peroxisome proliferator-activated receptor γ, CCAAT/enhancer-binding protein α, and fatty acid-binding protein 4) and increased the secretion of glycerol in a dose-dependent manner. There was a significant dose-dependent decrease in the expression of fatty acid synthase, stearoyl-CoA desaturase-1, sterol regulatory element-binding transcription factor 1c, and acetyl-CoA carboxylase 1 and an increase in carnitine palmitoyltransferase 1, acyl-CoA oxidase, and peroxisome proliferator-activated receptor α in TM treated cells. The phosphorylation of cAMP-activated protein kinase was also increased, which in turn activated the phosphorylation of acetyl-CoA carboxylase in mature adipocytes. Also, there was an increase in glucose uptake by TM, suggesting its insulin-sensitizing potential. This is the first report on the anti-obesity effects of TM from Myristica fragrans on adipogenesis and lipid metabolism in 3T3-L1 adipocytes and demands detailed in vivo study for developing TM as anti-obesity therapeutics.

Inula britannica Inhibits Adipogenesis of 3T3-L1 Preadipocytes via Modulation of Mitotic Clonal Expansion Involving ERK 1/2 and Akt Signaling Pathways

The flower of Inula britannica contains various phenolic compounds with prophylactic properties. This study aimed to determine the anti-adipogenic effect of an I. britannica flower aqueous extract (IAE) and its underlying mechanisms in the 3T3-L1 preadipocytes and to identify the phenolic compounds in the extract. Treatment with IAE inhibited the adipogenesis of 3T3-L1 preadipocytes by showing a dose-dependently suppressed intracellular lipid accumulation and significantly mitigated expression levels of lipogenesis- and adipogenesis-associated biomarkers including transcription factors. IAE exerted an anti-adipogenic effect through the modulation of the early phases of adipogenesis including mitotic clonal expansion (MCE). Treatment with IAE inhibited MCE by arresting the cell cycle at the G0/G1 phase and suppressing the activation of MCE-related transcription factors. Furthermore, IAE inhibited adipogenesis by regulating the extracellular signal-regulated kinase 1/2 and Akt signaling pathways. Protocatechuic acid, chlorogenic acid, kaempferol-3-O-glucoside, and 6-methoxyluteolin, which are reported to exhibit anti-adipogenic properties, were detected in IAE. Therefore, modulation of early phases of adipogenesis, especially MCE, is a key mechanism underlying the anti-adipogenic activity of IAE. In summary, the anti-obesity effects of IAE can be attributed to its phenolic compounds, and hence, IAE can be used for the development of anti-obesity products.

NOTCH Receptors and DLK Proteins Enhance Brown Adipogenesis in Mesenchymal C3H10T1/2 Cells

The NOTCH family of receptors and ligands is involved in numerous cell differentiation processes, including adipogenesis. We recently showed that overexpression of each of the four NOTCH receptors in 3T3-L1 preadipocytes enhances adipogenesis and modulates the acquisition of the mature adipocyte phenotype. We also revealed that DLK proteins modulate the adipogenesis of 3T3-L1 preadipocytes and mesenchymal C3H10T1/2 cells in an opposite way, despite their function as non-canonical inhibitory ligands of NOTCH receptors. In this work, we used multipotent C3H10T1/2 cells as an adipogenic model. We used standard adipogenic procedures and analyzed different parameters by using quantitative-polymerase chain reaction (qPCR), quantitative reverse transcription-polymerase chain reaction (qRT-PCR), luciferase, Western blot, and metabolic assays. We revealed that C3H10T1/2 multipotent cells show higher levels of NOTCH receptors expression and activity and lower Dlk gene expression levels than 3T3-L1 preadipocytes. We found that the overexpression of NOTCH receptors enhanced C3H10T1/2 adipogenesis levels, and the overexpression of NOTCH receptors and DLK (DELTA-like homolog) proteins modulated the conversion of cells towards a brown-like adipocyte phenotype. These and our prior results with 3T3-L1 preadipocytes strengthen the idea that, depending on the cellular context, a precise and highly regulated level of global NOTCH signaling is necessary to allow adipogenesis and determine the mature adipocyte phenotype.

Anthocyanin-Enriched Riceberry Rice Extract Inhibits Cell Proliferation and Adipogenesis in 3T3-L1 Preadipocytes by Downregulating Adipogenic Transcription Factors and Their Targeting Genes.

Riceberry rice (Oryza sativa L.) is a new pigmented variety of rice from Thailand. Despite its high anthocyanin content, its effect on adipogenesis and adipocyte function remains unexplored. We investigated whether Riceberry rice extract (RBE) impacted cell proliferation by examining viability and cell cycle, using preadipocyte 3T3-L1 cells. To test RBE’s effect on adipocyte formation, cells were cultured in adipogenic medium supplemented with extract and adipocyte number and triglyceride levels were quantified. Furthermore, Akt1 phosphorylation along with RT-qPCR and intracellular calcium imaging were performed to obtain an insight into its mechanism of action. The effect of RBE on adipocyte function was investigated using glucose uptake and lipolysis assays. Treatment of cells with RBE decreased preadipocyte number without cytotoxicity despite inducing cell cycle arrest (p < 0.05). During adipogenic differentiation, RBE supplementation reduced adipocyte number and triglyceride accumulation by downregulating transcription factors (e.g., PPARγ, C/EBPα, and C/EBPβ) and their target genes (p < 0.05). The Akt1 phosphorylation was decreased by RBE but insignificance, however, the extract failed to increase intracellular calcium signals. Finally, the treatment of adipocytes with RBE reduced glucose uptake by downregulating Glut4 mRNA expression and enhanced isoproterenol-induced lipolysis (p < 0.05). These findings suggest that RBE could potentially be used in the treatment of obesity by inhibiting adipocyte formation and proliferation.

Effect of treatment with conditioned media derived from C2C12 myotube on adipogenesis and lipolysis in 3T3-L1 adipocytes.

Regular exercise is an effective strategy that is used to prevent and treat obesity as well as type 2 diabetes. Exercise-induced myokine secretion is considered a mechanism that coordinates communication between muscles and other organs. In order to examine the possibility of novel communications from muscle to adipose tissue mediated by myokines, we treated 3T3-L1 adipocytes with C2C12 myotube electrical pulse stimulation-conditioned media (EPS-CM), using a C2C12 myotube contraction system stimulated by an electrical pulse. Continuous treatment with myotube EPS-CM promoted adipogenesis of 3T3-L1 pre-adipocytes via the upregulation of the peroxisome proliferator-activated receptor-gamma (PPARγ) 2 and PPARγ-regulated gene expression. Furthermore, our results revealed that myotube EPS-CM induces lipolysis and secretion of adiponectin in mature adipocytes. EPS-CM obtained from a C2C12 myoblast culture did not induce such changes in these genes, suggesting that contraction-induced myokine(s) secretion occurs particularly in differentiated myotubes. Thus, contraction-induced secretion of myokine(s) promotes adipogenesis and lipid metabolism in 3T3-L1 adipocytes. These findings suggest the possibility that skeletal muscle communicates to adipose tissues during exercise, probably by the intermediary of unidentified myokines.

NADP modulates RNA m6A methylation and adipogenesis via enhancing FTO activity.

Metabolism is often regulated by the transcription and translation of RNA. In turn, it is likely that some metabolites regulate enzymes controlling reversible RNA modification, such as N6-methyladenosine (m6A), to modulate RNA. This hypothesis is at least partially supported by the findings that multiple metabolic diseases are highly associated with fat mass and obesity-associated protein (FTO), an m6A demethylase. However, knowledge about whether and which metabolites directly regulate m6A remains elusive. Here, we show that NADP directly binds FTO, independently increases FTO activity, and promotes RNA m6A demethylation and adipogenesis. We screened a set of metabolites using a fluorescence quenching assay and NADP was identified to remarkably bind FTO. In vitro demethylation assays indicated that NADP enhances FTO activity. Furthermore, NADP regulated mRNA m6A via FTO in vivo, and deletion of FTO blocked NADP-enhanced adipogenesis in 3T3-L1 preadipocytes. These results build a direct link between metabolism and RNA m6A demethylation.

Mir-22-3p/KLF6/MMP14 axis in fibro-adipogenic progenitors regulates fatty infiltration in muscle degeneration.

Fibro/adipogenic progenitors (FAPs) are the main cellular source of fatty degeneration in muscle injury; however, the underlying mechanism of FAP adipogenesis in muscle degeneration needs to be further examined. Matrix metalloproteinase 14 (MMP-14) has been reported to induce the adipogenesis of 3T3-L1 preadipocytes, but whether MMP-14 also regulates the differentiation of FAPs remains unclear. To investigate whether and how MMP-14 regulates FAP adipogenesis and fatty infiltration in muscle degeneration, we examined MMP-14 expression in degenerative muscles and tested the effect of MMP-14 on FAP adipogenesis in vitro and in vivo. As expected, MMP-14 enhanced FAP adipogenesis and fatty infiltration in degenerative muscles; moreover, blocking endogenous MMP-14 in injured muscles facilitated muscle repair. Further investigations revealed that Kruppel-like factor 6 (KLF6) was a transcription factor associated with MMP-14 and acted as an "on-off" switch in the differentiation of FAPs into adipocytes or myofibroblasts. Moreover, KLF6 was the target gene of miR-22-3p, which was downregulated during FAP adipogenesis both in vitro and in vivo, and overexpression of miR-22-3p markedly prevented FAP adipogenesis and attenuated fatty degeneration in muscles. Our study revealed that miR-22-3p/KLF6/MMP-14 is a novel pathway in FAP adipogenesis and that inhibiting KLF6 is a potential strategy for the treatment of muscular degenerative diseases.

Kurozu melanoidin, a novel oligoglucan-melanoidin complex from Japanese black vinegar, suppresses adipogenesis in vitro

• This study identifies an anti-adipogenic factor in the black vinegar kurozu. • The factor, kurozu melanoidin (KZM), consists of an oligoglycan and a melanoidin. • KZM inhibits adipocyte differentiation of 3 T3-L1 cells in vitro . • KZM may, in part, account for anti-obesity effects of kurozu. The black vinegar “kurozu” has an anti-obesity potential, but its active principle is unknown. Here, we isolate and characterize a novel oligoglucan-melanoidin complex, designated kurozu melanoidin (KZM), that suppresses adipogenesis in vitro . KZM was isolated by organic solvent extraction, followed by size-exclusion chromatography. Its average molecular mass was estimated to be 1,090 ± 30 Da using polyethylene glycols as standards. The combination of spectroscopic and chemical analyses revealed the presence of an oligoglucan and a melanoidin structures in KZM. The glucan moiety consisted mainly of 1 → 4 linkage. The MALDI-TOF-MS analysis identified an array of ion peaks that differed in m / z by ~ 162.055 [C 6 H 12 O 6 − H 2 O], indicating a variation of glucan chain length. Prominent signals around m / z 500–600 that were unassignable to glucan units suggested ion peaks for the melanoidin moiety. KZM significantly suppressed adipogenesis of 3T3-L1 preadipocytes at 100 μM.

Effects of 23-epi-26-deoxyactein on adipogenesis in 3T3-L1 preadipocytes and diet-induced obesity in C57BL/6 mice

BackgroundThe ethanolic extract of Actaea racemosa L. (Cimicifuga racemosa (L.) Nutt.) has recently been reported to ameliorate obesity-related insulin resistance, hyperlipidemia, and fatty liver in rodents. However, it remains unclear which A. racemosa components are responsible for these beneficial effects. PurposeWe aimed to examine the anti-obesity potential of 23-epi-26-deoxyactein (DA), which is contained in the ethanolic extracts of A. racemosa. Study design and methodsTo evaluate the effects of DA on adipogenesis in 3T3-L1 preadipocytes and diet-induced obesity in C57BL/6 mice, in vitro and in vivo tests were performed. For in vitro assessment, we used Oil red O staining that showed lipid accumulation in differentiated 3T3-L1 cells. For in vivo tests, male 5-week-old C57BL/6 mice were fed with low-fat diet (LFD), high-fat diet (HFD), HFD with 10 mg/kg/d luteolin (LU; positive control drug), HFD with 1 mg/kg/d DA, and HFD with 5 mg/kg/d DA for 12 weeks, respectively. Glucose and insulin tolerance tests were performed at week 17. The lipid deposition of adipose tissue and liver was visualized by hematoxylin and eosin staining. Real-time PCR showed mRNA levels of genes involved in adipogenesis, lipogenesis, and lipolysis. AMPK signaling and SIRT1-FOXO1 pathway were assessed by real-time PCR and western blot. Results10 μM DA and 20 μM LU treatments inhibited 3T3-L1 adipogenesis through down-regulating the expression of C/ebpα, C/ebpβ, and Pparγ, which are the critical adipogenic transcription factors. The in vivo results showed that 5 mg/kg/d DA and 10 mg/kg/d LU significantly lowered body weight gain, fat mass, and liver weight in HFD-fed mice. Meanwhile, DA and LU also reduced insulin resistance and serum lipoprotein levels in HFD-fed mice. Mechanistic studies showed that DA and LU promoted adipocyte lipolysis in mice through activating the AMPK signaling and SIRT1-FOXO1 pathway. ConclusionThe in vitro results indicate that 10 μM DA suppresses adipogenesis in 3T3-L1 preadipocytes. The in vivo treatment with 5 mg/kg/d DA ameliorates diet-induced obesity in mice, suggesting that DA is a promising natural compound for the treatment of obesity and related metabolic diseases.

Anti-Obesity Effects of Tanshinone I from Salvia miltiorrhiza Bunge in Mice Fed a High-Fat Diet through Inhibition of Early Adipogenesis.

Tanshinone I (Tan I) is a diterpenoid isolated from Salvia miltiorrhiza Bunge and exhibits antitumor effects in several cancers. However, the anti-obesity properties of Tan I remain unexplored. Here, we evaluated the anti-obesity effects of Tan I in high-fat-diet (HFD)-induced obese mice and investigated the underlying molecular mechanisms in 3T3-L1 cells. HFD-induced obese mice were orally administrated Tan I for eight weeks, and body weight, weight gain, hematoxylin and eosin staining and serum biological parameters were examined. The adipogenesis of 3T3-L1 preadipocytes was assessed using Oil Red O staining and measurement of intracellular triglyceride (TG) levels, and mitotic clonal expansion (MCE) and its related signal molecules were analyzed during early adipogenesis of 3T3-L1 cells. The administration of Tan I significantly reduced body weight, weight gain, and white adipocyte size, and improved obesity-induced serum levels of glucose, free fatty acid, total TG, and total cholesterol in vivo in HFD-induced obese mice. Furthermore, Tan I-administered mice demonstrated improvement of glucose metabolism and insulin sensitivity. Treatment with Tan I inhibited the adipogenesis of 3T3-L1 preadipocytes in vitro, with this inhibition mainly occurring at an early phase of adipogenesis through the attenuation of MCE via cell cycle arrest at the G1/S phase transition. Tan I inhibited the phosphorylation of p38, extracellular signal-regulated kinase (ERK), and Akt during the process of MCE, while it stimulated the phosphorylation of AMP-activated protein kinase. Furthermore, Tan I repressed the expression of CCAAT-enhancer-binding protein β (C/EBPβ), histone H3K9 demethylase JMJD2B, and subsequently cell cycle genes. Moreover, Tan I regulated the expression of early adipogenic transcription factors including GATAs and Kruppel-like factor family factors. These results indicate that Tan I prevents HFD-induced obesity via the inhibition of early adipogenesis, and thus improves glucose metabolism and insulin sensitivity. This suggests that Tan I possesses therapeutic potential for the treatment of obesity and obesity-related diseases.

Weight Regain in Formerly Obese Mice Hastens Development of Hepatic Steatosis Due to Impaired Adipose Tissue Function.

Weight regain after weight loss is common, and there is evidence to suggest negative effects on health because of weight cycling. This study sought to investigate the impact of weight regain in formerly obese mice on adipose tissue architecture and stromal cell function. A diet-switch model was employed for obesity induction, weight loss, and weight regain in mice. Flow cytometry quantified adipose tissue leukocytes in adipose tissue. Liver and adipose tissue depots were compared to determine tissue-specific effects of weight cycling. Epididymal white adipose tissue of formerly obese mice failed to expand in response to repeat exposure to high-fat diet and retained elevated numbers of macrophages and T cells. Weight regain was associated with disproportionally elevated liver mass, hepatic triglyceride content, serum insulin concentration, and serum transaminase concentration. These effects occurred despite an extended 6-month weightloss cycle and theydemonstrate that formerly obese mice maintain durable alterations in their physiological response to weight regain. Conditioned media from epididymal adipose tissue of formerly obese mice inhibited adipogenesis of 3T3-L1 preadipocytes, suggesting a potential mechanism to explain failed epididymal adipose tissue expansion during weight regain. Metabolic abnormalities related to defects in adipose tissue expansion and ongoing dysfunction manifest in formerly obese mice during weight regain.

Anti-adipogenic Effect of β-Carboline Alkaloids from Garlic (Allium sativum).

Garlic (Allium sativum L.) is utilized worldwide for culinary and medicinal use and has diverse health benefits. As part of our ongoing research to identify bioactive components from natural resources, phytochemical analysis of the methanolic extract of garlic led to the isolation and characterization of six compounds: Three eugenol diglycosides (1–3) and three β-carboline alkaloids (4–6). In particular, the absolute configurations of β-carboline alkaloids (5 and 6) were established by gauge-including atomic orbital nuclear magnetic resonance chemical shift calculations, followed by DP4+ analysis. Here, we evaluated the effects of compounds 1–6 on 3T3-L1 preadipocyte adipogenesis and lipid metabolism. 3T3-L1 adipocyte differentiation was evaluated using Oil Red O staining; the expression of adipogenic genes was detected using RT-qPCR. Among compounds 1–6, (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (6) inhibited 3T3-L1 preadipocyte adipogenesis and reduced the expression of adipogenic genes (Fabp4, PPARγ, C/EBPβ, Adipsin, and Adipoq). Moreover, it markedly decreased the actylation of α-tubulin, which is crucial for cytoskeletal remodeling during adipogenesis. Anti-adipogenic effects were observed upon treatment with compound 6, not only during the entire process, but also on the first two days of adipogenesis. Additionally, treatment with compound 6 regulated the expression of genes involved in adipocyte lipid metabolism, decreasing the lipogenic gene (SREBP1) and increasing lipolytic genes (ATGL and HSL). We provide experimental evidence of the health benefits of using (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid obtained from garlic to prevent excessive adipogenesis in obesity.

Adipogenic activity of 2-ethylhexyl diphenyl phosphate via peroxisome proliferator-activated receptor γ pathway

Recent studies have shown that exposure to some organophosphates, such as triphenyl phosphate (TPHP) and diphenyl phosphate (DPHP), can affect adipogenesis in preadipocytes. 2-Ethylhexyl diphenyl phosphate (EHDPP), an organophosphate, is frequently detected in various environmental media. However, there is less information about the toxicity effects and the mechanism by which EHDPP affects preadipocytes. In the present study, we investigated whether EHDPP could induce differentiation in 3T3-L1 preadipocytes through the peroxisome proliferator-activated receptor γ (PPARγ) signaling pathway. The fluorescence competitive binding assay and the dual-luciferase reporter gene assay were used to assess the binding affinity and activation of PPARγ, and the results showed that EHDPP can bind to the ligand binding domain of PPARγ (PPARγ-LBD) and activate PPARγ in vitro. Exposure to EHDPP for 10 days extensively induced adipogenesis in 3T3-L1 preadipocytes as assessed by lipid accumulation and gene expression of adipogenic markers of fatty acid binding protein 4 (FABP4), lipoprotein lipase (Lpl), adiponectin (Adip), and fatty acid synthase (Fasn). Furthermore, the preadipocytes differentiation was blocked by the PPARγ-specific antagonist GW9662, indicating that the PPARγ signaling pathway plays an important part in 3T3-L1 cell differentiation induced by EHDPP. Taken together, EHDPP can bind to PPARγ-LBD, activate PPARγ receptor, and induce cell differentiation via the PPARγ signaling pathway in 3T3-L1 preadipocytes.

S-nitrosoglutathione inhibits adipogenesis in 3T3-L1 preadipocytes by S-nitrosation of CCAAT/enhancer-binding protein \u03b2

Murine 3T3-L1 adipocytes share many similarities with primary fat cells and represent a reliable in vitro model of adipogenesis. The aim of this study was to probe the effect of S-nitrosoglutathione (GSNO) on adipocyte differentiation. Adipogenesis was induced with a mixture of insulin, dexamethasone, and 3-isobutyl-1-methylxanthine in the absence and presence of increasing GSNO concentrations. Biochemical analysis after 7 days of differentiation showed a prominent anti-adipogenic effect of GSNO which was evident as reduced cellular triglycerides and total protein content as well as decreased mRNA and protein expression of late transcription factors (e.g. peroxisome proliferator activated receptor γ) and markers of terminal differentiation (e.g. leptin). By contrast, the nitrosothiol did not affect mRNA and protein expression of CCAAT/enhancer-binding protein β (C/EBPβ), which represents a pivotal early transcription factor of the adipogenic cascade. Differentiation was also inhibited by the NO donor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate. Biotin switch experiments showed significantly increased S-nitrosation of C/EBPβ variants indicating that posttranslational S-nitrosative modification of this transcription factor accounts for the observed anti-adipogenic effect of NO. Our results suggest that S-nitrosation might represent an important physiological regulatory mechanism of fat cell maturation.

Promoting differentiation and lipid metabolism are the primary effects for DINP exposure on 3T3-L1 preadipocytes

Diisononyl phthalate (DINP) is a high-molecular-weight phthalate, and has been recently introduced as di-(2-ethyl hexyl) phthalate (DEHP) substitute and commonly used in a large variety of plastic items. The fat tissue is an important target for DINP exposure, however, very little is understood about its toxicity and mechanism(s) in adipocyte cells. Therefore, the present work aimed to investigate the role of DINP in adipogenesis using 3T3-L1 preadipocytes. DINP exposure for 10 days extensively induced adipogenesis in 3T3-L1 preadipocytes to adipocytes as assessed by lipid accumulation and gene expression of adipogenic markers. The RT-qPCR results showed that DINP could upregulate the expression of peroxisome proliferator-activated receptor-gamma (PPARγ), CCAAT/enhancer-binding protein alpha (C/EBPα) and C/EBPβ, while the expression of sterol regulatory element binding transcription factor 1 (SREBF1) and C/EBPδ was not affected. The DINP-induced adipogenesis could be inhibited by using the selective PPARγ antagonist GW9662. The RNA-seq analysis was used to study the systemic toxicities of DINP on preadipocytes. A total of 1181 differently expressed genes (DEGs) (640 genes were up-regulated, 541 genes were down-regulated) were detected in 3T3-L1 preadipocytes under 50 μM DINP. The GO enrichment showed the GO term of “fat cell differentiation” was the most significantly affected metabolic functions, and the KEGG pathway enrichment showed the PPAR pathway was the top affected pathway. The interactive pathway (iPath) analysis showed that the changed metabolic pathways were focus on the lipid metabolism.

Ivermectin decreases triglyceride accumulation by inhibiting adipogenesis of 3T3-L1 preadipocytes

Ivermectin, a member of the avermectins, is one of the most used anti-parasitic agents, and acts by binding to glutamate-gated chloride channels in invertebrate nerve cells. There is limited information, however, on the effects of ivermectin in non-neural cell, such as adipocytes. The present work aimed to investigate the role of ivermectin in adipogenesis using 3T3-L1 preadipocytes. Ivermectin inhibited the differentiation of preadipocytes and triglyceride (TG) accumulation. In particular, the treatment of ivermectin at the middle to late adipogenic differentiation period (day 2–8) was correlated with the inhibition of fat accumulation. Ivermectin treatment also significantly modulated the mRNA expression of key markers in adipogenesis, fatty acid synthesis, uptake, and oxidation, and enhanced the gene expression of two subunits of the glycine receptor (GlyR). Specifically, the protein levels of peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein alpha (C/EBPα), and acetyl-CoA carboxylase (ACC) were reduced. Interestingly, the suppression of TG accumulation by ivermectin was partially abolished by rosiglitazone, a specific PPARγ agonist, but Z-guggulsterone, a selective FXR antagonist, failed to rescue the ivermectin-induced effect on adipogenesis. Lastly, ivermectin prevented adipogenesis induced by permethrin and fipronil. In conclusion, ivermectin inhibits adipogenesis of 3T3-L1 preadipocytes partially via PPARγ & GlyR-dependent, but not FXR-dependent, pathway.

Clitoria ternatea Flower Petal Extract Inhibits Adipogenesis and Lipid Accumulation in 3T3-L1 Preadipocytes by Downregulating Adipogenic Gene Expression.

Clitoria ternatea (commonly known as blue pea) flower petal extract (CTE) is used as a natural colorant in a variety of foods and beverages. The objective of study was to determine the inhibitory effect of CTE on adipogenesis in 3T3-L1 preadipocytes. The phytochemical profiles of CTE were analyzed by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Anti-adipogenesis effect of CTE was measured by using Oil Red O staining, intracellular triglyceride assay, quantitative real-time PCR and western blot analysis in 3T3-L1 adipocytes. Cell cycle studies were performed by flow cytometry. Lipolysis experiments were performed using a colorimetric assay kit. In early stages, CTE demonstrated anti-adipogenic effects through inhibition of proliferation and cell cycle retardation by suppressing expression of phospho-Akt and phospho-ERK1/2 signaling pathway. The results also showed that CTE inhibited the late stage of differentiation through diminishing expression of adipogenic transcription factors including PPARγ and C/EBPα. The inhibitory action was subsequently attenuated in downregulation of fatty acid synthase and acetyl-CoA carboxylase, causing the reduction of TG accumulation. In addition, CTE also enhanced catecholamine-induced lipolysis in adipocytes. These results suggest that CTE effectively attenuates adipogenesis by controlling cell cycle progression and downregulating adipogenic gene expression.