(+)-Episesamin inhibits adipogenesis and exerts anti-inflammatory effects in 3T3-L1 (pre)adipocytes by sustained Wnt signaling,down-regulation of PPARγ and induction of iNOS

Obesity and its associated health risks still demand for effective therapeutic strategies. Drugs and compositions derived from Oriental medicine such as green tea polyphenols attract growing attention. Previously, an extract from the Japanese spice bush Lindera obtusiloba (L. obtusiloba) traditionally used for treatment of inflammation and prevention of liver damage was shown to inhibit adipogenesis. Aiming for the active principle of this extract (+)-episesamin was identified, isolated and applied in adipogenic research using 3T3-L1 (pre)adipocytes, an established cell line for studying adipogenesis. With an IC₅₀ of 10 μM (+)-episesamin effectively reduced the growth of 3T3-L1 preadipocytes and decreased hormone-induced 3T3-L1 differentiation as shown by reduced accumulation of intracellular lipid droplets and diminished protein expression of GLUT-4 and vascular endothelial growth factor. Mechanistically, the presence of (+)-episesamin during hormone-induced differentiation provoked a reduced phosphorylation of ERK1/2 and β-catenin along with a reduced protein expression of peroxisome proliferator-activated receptor γ and a strongly increased protein expression of iNOS. Treatment of mature adipocytes with (+)-episesamin resulted in a reduction of intracellular stored lipid droplets and induced the proapoptotic enzymes caspases-3/-7. Besides interfering with adipogenesis, (+)-episesamin showed anti-inflammatory activity by counteracting the lipopolysaccharide- and tumor necrosis factor α-induced secretion of interleukin 6 by 3T3-L1 preadipocytes. In conclusion, (+)-episesamin seems to be the active drug in the L. obtusiloba extract being responsible for the inhibition of adipogenesis and, thus, should be evaluated as a novel potential complementary treatment for obesity.     

A water-soluble extract of Petalonia binghamiae inhibits the expression of adipogenic regulators in 3T3-L1 preadipocytes and reduces adiposity and weight gain in rats fed a high-fat diet

We previously showed that an ethanolic extract of the edible brown algae Petalonia binghamiae promotes the differentiation of 3T3-L1 preadipocytes and decreases hyperglycemia in streptozotocin-induced diabetic mice. Here, we report that a water-soluble extract of P. binghamiae thalli, prepared by enzymatic digestion, inhibits preadipocyte differentiation and adipogenesis in a dose-dependent manner. In differentiating 3T3-L1 preadipocytes, the extract (designated PBEE) decreased the expression of peroxisome proliferator-activated receptor γ, CCAAT/enhancer-binding proteins α and β, and fatty acid-binding protein aP2. It also inhibited the mitotic clonal expansion process of adipocyte differentiation, and it inhibited insulin-stimulated uptake of glucose into mature 3T3-L1 adipocytes by reducing phosphorylation of insulin receptor substrate-1. In rats with high-fat diet (HFD)-induced obesity, PBEE exhibited potent anti-obesity effects. In this animal model, increases in body weight and fat storage were suppressed by the addition of PBEE to the drinking water at 500 mg/L for 30 days. PBEE supplementation reduced serum levels of glutamic pyruvic and glutamic oxaloacetic transaminases and increased the serum level of high-density lipoprotein cholesterol. Moreover, it significantly decreased the accumulation of lipid droplets in liver tissue, suggesting a protective effect against HFD-induced hepatic steatosis. Taken together, these data demonstrate that PBEE inhibits preadipocyte differentiation and adipogenesis in cultured cells and in rodent models of obesity.     

Phoenixin-14 stimulates differentiation of 3T3-L1 preadipocytes via cAMP/Epac-dependent mechanism

Phoenixin-14 (PNX) is a newly discovered peptide produced by proteolytic cleavage of the small integral membrane protein 20 (Smim20). Previous studies showed that PNX is involved in controlling reproduction, pain, anxiety and memory. Furthermore, in humans, PNX positively correlates with BMI suggesting a potential role of PNX in controlling fat accumulation in obesity. Since the influence of PNX on adipose tissue formation has not been so far demonstrated, we investigated the effects of PNX on proliferation and differentiation of preadipocytes using 3T3-L1 and rat primary preadipocytes. We detected Smim20 and Gpr173 mRNA in 3T3-L1 preadipocytes as well as in rat primary preadipocytes. Furthermore, we found that PNX peptide is produced and secreted from 3T3-L1 and rat primary adipocytes. PNX increased 3T3-L1 preadipocytes proliferation and viability. PNX stimulated the expression of adipogenic genes (Pparγ, C/ebpβ and Fabp4) in 3T3-L1 adipocytes. 3T3-L1 preadipocytes differentiated in the presence of PNX had increased lipid content. Stimulation of cell proliferation and differentiation by PNX was also confirmed in rat preadipocytes. PNX failed to induce AKT phosphorylation, however, PNX increased cAMP levels in 3T3-L1 cells. Suppression of Epac signalling attenuated PNX-induced Pparγ expression without affecting cell proliferation. Our data show that PNX stimulates differentiation of 3T3-L1 and rat primary preadipocytes into mature adipocytes via cAMP/Epac-dependent pathway. In conclusion our data shows that phoenixin promotes white adipogenesis, thereby may be involved in controlling body mass regulation.     

Extracellular redox environments regulate adipocyte differentiation

Oxidized extracellular redox states have been associated with many diseases related to obesity, including heart disease and diabetes, but relatively little is known about the relationship between extracellular redox states and obesity. In 3T3-L1 preadipocytes, oxidizing extracellular redox potentials (E_h) increased intracellular and mitochondrial reactive oxygen species (ROS) production. 3T3-L1 adipocytes showed a greater response to extracellular E_h, producing more intracellular ROS, than preadipocytes. 3T3-L1 adipocytes also produced more extracellular ROS and re-regulated the extracellular E_h to a more oxidizing state than preadipocytes. During 3T3-L1 differentiation, cellular glutathione and mitochondrial thioredoxin-2 become oxidized, suggesting that adipogenesis may be enhanced under conditions promoting intracellular and mitochondrial compartment oxidation. Under various extracellular E_h, 3T3-L1 adipogenesis, as determined by lipid accumulation and the expression of early genetic markers of adipogenesis, was sensitive to the extracellular redox environment, where it was enhanced under oxidizing conditions and lower under reducing conditions. Using a diet-induced obesity mouse model, plasma was collected before and after the 8 week diet regimens. Plasma GSH E_h was unchanged as a consequence of weight gain but plasma cystiene (Cys) E_h was significantly oxidized in overweight animals. Data presented here show that adipocytes/excessive adipose preferentially alter extracellular E_h to a more oxidized state in vivo and in vitro and may promote further adipogenesis.     

Distinct hypoxic regulation of preadipocyte factor-1 (Pref-1) in preadipocytes and mature adipocytes

Preadipocyte factor-1 (Pref-1) is a secretory soluble protein, which exerts pleiotropic effects on maintenance of cancer stem cell characteristics and commitment of mesenchymal stem cell lineages by inhibiting adipogenesis. Observations that obesity renders the microenvironment of adipose tissues hypoxic and that hypoxia inhibits adipogenesis lead us to investigate whether hypoxia increases the expression of anti-adipogenic Pref-1 in preadipocytes, mature adipocytes, and adipose tissues from obese mouse. In 3T3-L1 preadipocytes, hypoxia induces Pref-1 by a hypoxia-inducible factor 1 (HIF-1)-dependent mechanism accompanied by increase in the levels of the active histone mark, acetylated H3K9/14 (H3K9/14Ac). Adipogenesis increased the levels of the heterochromatin histone mark, trimethylated H3K27 (H3K27me3), whereas it decreased the levels of H3K4me3 and H3K9/14Ac euchromatin marks of the mouse Pref-1 promoter. However, differently from preadipocytes, in mature adipocytes hypoxia failed to reverse the repressive epigenetic changes of Pref-1 promoter and to increase its expression. Short term (8 weeks) high fat diet (HFD) increased HIF-1α protein in subcutaneous and epididymal adipose tissues, but did not increase Pref-1 expression. Unlike in 3T3-L1 preadipocytes, HIF-1α did not increase Pref-1 expression in adipose tissues in which mature adipocytes constitute the main population. Interestingly, long term (35 weeks) HFD increased Pref-1 in serum but not in obese adipose tissues. This study suggests that Pref-1 is an endocrine factor which is synergistically increased by obesity and age.     

Coculture with BJ fibroblast cells inhibits the adipogenesis and lipogenesis in 3T3-L1 cells

Mouse or human fibroblasts are commonly used as feeder cells to prevent differentiation in stem or primary cell culture. In the present study, we addressed whether fibroblasts can affect the differentiation of adipocytes. We found that the differentiation of 3T3-L1 preadipocytes was strongly suppressed when the cells were cocultured with human fibroblast (BJ) cells. BrdU incorporation analysis indicated that mitotic clonal expansion, an early event required for 3T3-L1 cell adipogenesis, was not affected by BJ cells. The 3T3-L1 cell expression levels of peroxisome proliferator-activated receptor γ2, CCAAT/enhancer-binding protein alpha (C/EBPα), sterol regulatory element binding protein-1c, and Krüppel-like factor 15, but not those of C/EBPβ or C/EBPδ, were decreased by coculture with BJ cells. When mature 3T3-L1 adipocytes were cocultured with BJ cells, their lipid contents were significantly reduced, with decreased fatty acid synthase expression and increased phosphorylated form of acetyl-CoA carboxylase 1. Our data indicate that coculture with BJ fibroblast cells inhibits the adipogenesis of 3T3-L1 preadipocytes and decreases the lipogenesis of mature 3T3-L1 adipocytes.     

LMO4 modulates proliferation and differentiation of 3T3-L1 preadipocytes

Previous microarray analyses revealed that LMO4 is expressed in 3T3-L1 preadipocytes, however, its roles in adipogenesis are unknown. In the present study, using RT-PCR sequencing and quantitative real-time RT-PCR, we confirmed that LMO4 gene is expressed in 3T3-L1 preadipocytes and its expression peaks at the early stage of 3T3-L1 preadipocyte differentiation. Further analyses showed that LMO4 knockdown decreased the proliferation of 3T3-L1 preadipocytes, and attenuated the differentiation of 3T3-L1 preadipocytes, as evidenced by reduced lipid accumulation and down-regulation of PPARγ gene expression. Collectively, our findings indicate that LMO4 is a novel modulator of adipogenesis.     

Rehmannia inhibits adipocyte differentiation and adipogenesis

Rehmannia glutinosa, a Traditional Chinese Medicine (TCM), has been used to increase physical strength. Here, we report that Rehmannia glutinosa extract (RE) inhibits adipocyte differentiation and adipogenesis. RE impairs differentiation of 3T3-L1 preadipocytes in a dose-dependent manner. At the molecular level, treatment with RE inhibits expression of the key adipocyte differentiation regulator C/EBPβ, as well as C/EBPα and the terminal marker protein 422/aP2, during differentiation of preadipocytes into adipocytes. Additionally, RE inhibits the mitotic clonal expansion (MCE) process of adipocyte differentiation, and RE prevents localization of C/EBPβ to the centromeres. RE also prevents high fat diet (HFD) induced weight gain and adiposity in rats. Taken together, our results indicate that Rehmannia glutinosa extract inhibits preadipocyte differentiation and adipogenesis in cultured cells and in rodent models of obesity.     

Caudatin suppresses adipogenesis in 3T3-L1 adipocytes and reduces body weight gain in high-fat diet-fed mice through activation of hedgehog signaling

BackgroundThe regulative effects of caudatin, a C-21 steroid that is identified from Cynanchum bungee roots, on adipogenesis and obesity have not been studied. Many studies have demonstrated that the activation of hedgehog (Hh) signaling can help prevent obesity. Therefore, we hypothesized that caudatin can inhibit adipogenesis and obesity via activating the Hh signaling pathway.MethodsTo investigate the effects of caudatin on adipogenesis in 3T3-L1 preadipocytes and high-fat diet induced obesity in C57BL/6 mice, in vitro and in vivo experiments were performed. For in vitro evaluation, Oil red O staining were used to represent lipid accumulation in differentiated 3T3-L1 adipocytes. For in vivo assessment, male 5 week-old C57BL/6 mice were fed with standard chow diet, high-fat diet (HFD), HFD with 25 mg/kg caudatin, HFD with 1mg/kg purmorpharmine for 10 weeks, respectively. Hh signaling and key adipogenic marker involved in adipogenesis were evaluated by real-time PCR and western blot. The adipocyte size of white adopose tissue and lipid storage of liver were visualized by hematoxylin and eosin staining. In addition, the expression of Gli1 and peroxisome proliferator-activated receptor γ (PPARγ) in white adipose tissue were investigated by immunohistochemistry staining.ResultsCaudatin suppressed the accumulation of lipid droplets and downregulated the expression of key adipogenic factors, i.e., peroxisome proliferator-activated receptor γ PPARγ and CCAAT-enhancer binding protein α (C/EBPα), through activating Hh signaling in differentiated 3T3-L1 cells. Furthermore, caudatin and the Hh activator purmorpharmine significantly decreased body weight gain and white adipose tissue (WAT) weight in HFD-induced mice and affected adipogenic markers and Hh signaling mediators in WAT, which were in line with the in vitro experimental results.ConclusionTo our best knowledge, it is the first report to demonstrate that caudatin downregulated adipocyte differentiation and suppressed HFD-induced body weight gain through activating the Hh signaling pathway, suggesting that caudatin can potentially counteract obesity.     

Increasing cAMP levels of preadipocytes by cyanidin-3-glucoside treatment induces the formation of beige phenotypes in 3T3-L1 adipocytes

Obesity is a serious health problem and a major risk factor for the onset of several diseases such as heart disease, diabetes, stroke and cancer. The conversion of white adipocytes to brown-like adipocytes, also called beige or brite adipocytes, by pharmacological and dietary compounds has gained attention as an effective treatment for obesity. Cyanidin-3-glucoside (Cy3G), a polyphenolic compound contained in black soybean, blueberry and grape, has several antiobesity effects. However, there are no reports on the role of Cy3G in the induction of differentiation of preadipocytes to beige adipocytes and corresponding phenotypes. Here, the formation of beige adipocyte phenotypes following treatment with Cy3G was evaluated using 3T3-L1 adipocytes. Cy3G induced phenotypic changes to white adipocytes, such as increased multilocular lipid droplets and mitochondrial content. Additionally, the expression of mitochondrial genes (TFAM, SOD2, UCP-1 and UCP-2), UCP-1 protein and beige adipocyte markers (CITED1 and TBX1) in 3T3-L1 adipocytes was increased by Cy3G. Furthermore, Cy3G promoted preadipocyte differentiation by up-regulating of C/EBPβ through the elevation of the intracellular cAMP levels. These results indicated that Cy3G elevates the intracellular cAMP levels, which induces beige adipocyte phenotypes. This is the first report on the effect of Cy3G on induction of differentiation of preadipocytes into beige adipocyte phenotypes.     

Reversine increases the plasticity of lineage-committed preadipocytes to osteogenesis by inhibiting adipogenesis through induction of TGF-β pathway in vitro

Reversine has been reported to reverse differentiation of lineage-committed cells to mesenchymal stem cells (MSCs), which then enables them to be differentiated into other various lineages. Both adipocytes and osteoblasts are known to originate from common MSCs, and the balance between adipogenesis and osteogenesis in MSCs is reported to modulate the progression of various human diseases, such as obesity and osteoporosis. However, the role of reversine in modulating the adipogenic potential of lineage-committed preadipocytes and their plasticity to osteogenesis is unclear. Here we report that reversine has an anti-adipogenic function in 3T3-L1 preadipocytes in vitro and alters cell morphology and viability. The transforming growth factor-β (TGF-β) pathway appears to be required for the anti-adipogenic effect of reversine, due to reversine-induced expression of genes involved in TGF-β pathway and reversal of reversine-inhibited adipogenesis by inhibition of TGF-β pathway. We show that treatment with reversine transformed 3T3-L1 preadipocytes into MSC-like cells, as evidenced by the expression of MSCs marker genes. This, in turn, allowed differentiation of lineage-committed 3T3-L1 preadipocytes to osteoblasts under the osteogenic condition in vitro. Collectively, these findings reveal a new function of reversine in reversing lineage-committed preadipocytes to osteogenesis in vitro, and provide new insights into adipose tissue-based regeneration of osteoblasts.     

Green Tea Polyphenol Epigallocatechin Gallate Inhibits Adipogenesis and Induces Apoptosis in 3T3‐L1 Adipocytes

Objective: Green tea catechins have been shown to promote loss of body fat and to inhibit growth of many cancer cell types by inducing apoptosis. The objective of this study was to determine whether epigallocatechin gallate (EGCG), the primary green tea catechin, could act directly on adipocytes to inhibit adipogenesis and induce apoptosis. Research Methods and Procedures: Mouse 3T3‐L1 preadipocytes and mature adipocytes were used. To test the effect of EGCG on viability, cells were incubated for 3, 6, 12, or 24 hours with 0, 50, 100, or 200 μM EGCG. Viability was quantitated by MTS assay. To determine the effect of EGCG on apoptosis, adipocytes were incubated for 24 hours with 0 to 200 μM EGCG, then stained with annexin V and propidium iodide and analyzed by laser scanning cytometry. Both preadipocytes and adipocytes were also analyzed for apoptosis by terminal deoxynucleotidyl transferase dUTP nick‐end labeling assay. To determine the effect of EGCG on adipogenesis, maturing preadipocytes were incubated during the 6‐day induction period with 0 to 200 μM EGCG, then stained with Oil‐Red‐O and analyzed for lipid content. Results: EGCG had no effect on either viability or apoptosis of preconfluent preadipocytes. EGCG also did not affect viability of mature adipocytes; however, EGCG increased apoptosis in mature adipocytes, as demonstrated by both laser scanning cytometry and terminal deoxynucleotidyl transferase dUTP nick‐end labeling assays. Furthermore, EGCG dose‐dependently inhibited lipid accumulation in maturing preadipocytes. Discussion: These results demonstrate that EGCG can act directly to inhibit differentiation of preadipocytes and to induce apoptosis of mature adipocytes and, thus, could be an important adjunct in the treatment of obesity.     

Fat-specific protein 27, a novel lipid droplet protein that enhances triglyceride storage

Fat-specific protein (FSP)27/Cidec is most highly expressed in white and brown adipose tissues and increases in abundance by over 50-fold during adipogenesis. However, its function in adipocytes has remained elusive since its discovery over 15 years ago. Here we demonstrate that FSP27/Cidec localizes to lipid droplets in cultured adipocytes and functions to promote lipid accumulation. Ectopically expressed FSP27-GFP surrounds lipid droplets in 3T3-L1 adipocytes and colocalizes with the known lipid droplet protein perilipin. Immunostaining of endogenous FSP27 in 3T3-L1 adipocytes also confirmed its presence on lipid droplets. FSP27-GFP expression also markedly increases lipid droplet size and enhances accumulation of total neutral lipids in 3T3-L1 preadipocytes as well as other cell types such as COS cells. Conversely, RNA interference-based FSP27/Cidec depletion in mature adipocytes significantly stimulates lipolysis and reduces the size of lipid droplets. These data reveal FSP27/Cidec as a novel adipocyte lipid droplet protein that negatively regulates lipolysis and promotes triglyceride accumulation.     

The adipokine Chemerin induces lipolysis and adipogenesis in bovine intramuscular adipocytes

The adipokine Chemerin is reported to regulate adipogenesis and glucose homeostasis in vivo and in 3T3-L1 cells. Our team is focused on the role of Chemerin in metabolism and intramuscular adipocyte differentiation because intramuscular fat is the basic material for the formation of marbling in livestock and poultry meat. In this study, bovine intramuscular mature adipocytes were cultured in medium with Chemerin, and the process of lipolysis of mature adipocytes and the adipogenesis of de-differentiated preadipocytes were investigated. The results showed that Chemerin induced significant lipolytic metabolism in intramuscular mature adipocytes, indicated by increased levels of glycerol, FFA, and up-regulated expression of the lipolysis critical factors HSL, LPL, and leptin. Meanwhile, the expressions of adipogenic key factors PPARγ, C/EBPα, and A-FABP were decreased by Chemerin during lipolysis or dedifferentiation in mature adipocytes. The de-differentiated preadipocytes could re-differentiate into mature adipocytes. Intriguingly, the formation of cells’ lipid droplets was promoted by Chemerin during preadipocyte differentiation. In addition, mRNA and protein expressions of PPARγ, C/EBPα, and A-FABP were up-regulated by Chemerin during preadipocytes differentiation. These results suggest that Chemerin promotes lipolysis in mature adipocytes and induces adipogenesis during preadipocyte re-differentiation, further indicating a dual role for Chemerin in the deposition of intramuscular fat in ruminant animals.

     

Hydroxysafflor yellow A (HYSA) inhibited the proliferation and differentiation of 3T3-L1 preadipocytes

Hydroxysafflor yellow A (HSYA), a main component of safflor yellow, has been demonstrated to prevent steroid-induced avascular necrosis of femoral head by inhibiting primary bone marrow-derived mesenchymal stromal cells adipogenic differentiation induced by steroid. In this study, we investigate the effect of HSYA on the proliferation and adipogenesis of mouse 3T3-L1 preadipocytes. The effects of HSYA on proliferation and differentiation of 3T3-L1 cells and its possible mechanism were studied by 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide spectrophotometry, Oil Red O staining, intracellular triglyceride assays, real-time quantitative RT-PCR, transient transfection and dual luciferase reporter gene methods. HSYA inhibited the proliferation of 3T3-L1 preadipocytes and cell viability greatly decreased in a dose and time dependent manner. HSYA (1 mg/l) notably reduced the amount of intracellular lipid and triglyceride content in adipocytes by 21.3 % (2.13 ± 0.36 vs 2.71 ± 0.40, P < 0.01) and 22.6 % (1.33 ± 0.07 vs 1.72 ± 0.07, P < 0.01) on days 8 following the differentiation, respectively. HSYA (1 mg/l) significantly increased hormone-sensitive lipase (HSL) mRNA expression and promoter activities by 2.4- and 1.55-fold, respectively (P < 0.01), in differentiated 3T3-L1 adipocytes. HSYA inhibits the proliferation and adipogenesis of 3T3-L1 preadipocytes. The inhibitory action of HYSA on adipogenesis may be due to the promotion of lipolytic-specific enzyme HSL expression by increasing HSL promoter activity.