Ferulic Acid Stimulates Adipocyte-Specific Secretory Proteins to Regulate Adipose Homeostasis in 3T3-L1 Adipocytes

Obesity has recently emerged as a public health issue facing developing countries in the world. It is caused by the accumulation of fat in adipose, characterized by insulin resistance, excessive lipid accumulation, inflammation, and oxidative stress, leading to an increase in adipokine levels. Herein, we investigated the capacity of a bioactive polyphenolic compound (ferulic acid (FA)) to control adipocyte dysfunction in 3T3-L1 adipocytes (in vitro). Key adipocyte differentiation markers, glycerol content, lipolysis-associated mRNA, and proteins were measured in experimental adipocytes. FA-treated adipocytes exhibited downregulated key adipocyte differentiation factors peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAT enhancer binding-proteins—α (C/EBP-α) and its downstream targets in a time-dependent manner. The FA-treated 3T3-L1 adipocytes showed an increased release of glycerol content compared with non-treated adipocytes. Also, FA treatment significantly up-regulated the lipolysis-related factors, including p-HSL, and p-perilipin, and down-regulated ApoD, Sema3C, Cxcl12, Sfrp2, p-stearoyl-CoA desaturase 1 (SCD1), adiponectin, and Grk5. Also, the FA treatment showed significantly down-regulated adipokines leptin, chemerin, and irisin than the non-treated cells. The present findings indicated that FA showed significant anti-adipogenic and lipogenic activities by regulating key adipocyte factors and enzyme, enhanced lipolysis by HSL/perilipin cascade. FA is considered a potent molecule to prevent obesity and its associated metabolic changes in the future.     

Anti-Obesity Potential of Ponciri Fructus: Effects of Extracts,Fractions and Compounds on Adipogenesis in 3T3-L1 Preadipocytes

Background: Ponciri Fructus, a crude drug consisting of the dried immature fruits of Poncirus trifoliata (L.) Raf., is a popular folk medicine used for the treatment of allergy and gastrointestinal disorders in Korea and China. In this study, the anti-adipogenic activity of extracts and isolated compounds were evaluated using 3T3-L1 preadipocytes. Methods: Dried immature fruits were extracted and fractionated into n-hexane, ethyl acetate (EtOAc), n-butanol and water-soluble fractions. The ethanol extract and fractions were tested for anti-adipogenic activity in the 3T3-L1 cell line. The active fractions (n-hexane and EtOAc fractions) were further subjected to chromatographic techniques to isolate and identify active compounds. Furthermore, the isolated compounds were evaluated for their anti-adipogenic activity. Results: Altogether, seven compounds, including two flavonoids, one phytosteroid and four coumarin derivatives, were isolated. Ethanol extract, n-hexane fraction, EtOAc fraction and three isolated compounds (phellopterin, oxypeucedanin and poncirin) showed significant anti-adipogenic activity as observed by reduced lipid deposition in differentiated 3T3-L1 cells. Further, oxypeucedanin downregulated the key adipogenic markers, such as peroxisome proliferator-activated receptors proteins γ (PPAR-γ), sterol response element binding proteins-1 (SREBP-1), CCAAT/enhancer binding proteins-α (C/EBP-α), adipocyte-specific lipid binding proteins (FABP-4), adipocyte fatty acid binding proteins (aP2), lipoprotein lipase (LPL) and leptin. Conclusion: This study indicated that the ethanol extract, hexane fraction and ethyl acetate fraction of P. trifoliata fruits possess strong anti-adipogenic activity, containing the active compounds such as phellopterin, oxypeucedanin and poncirin. Further research is recommended to explore their efficacy and safety in animal and clinical models.     

Inhibitory Effect of Acer truncatum Bunge Seed Coat Extract on Fatty Acid Synthase,Differentiation and Lipid Accumulation in 3T3-L1 Adipocytes

Acer truncatum Bunge is now widely cultivated throughout the world. Fatty acid synthase (FAS) is a potential target in the treatment of both obesity and cancer. Only a few FAS inhibitors have been reported. In this study, the inhibitory effect of A. truncatum seed coat (ESA) on FAS and the inhibition mechanisms were investigated using a FAS activity assay and an enzyme kinetics study. The main chemicals of ESA were analyzed with UPLC-MS/MS. The effects of ESA on 3T3-L1 adipocyte differentiation and lipid accumulation were investigated using Oil red O staining. We first identified seven main compounds (quinic acid, malic acid, gentisic acid, procyanidin dimer, procyanidin trimer, catechin, and quercetin) from 50% ethanol extracts of seed coats of A. truncatum (ESAs), which were then found to inhibit 3T3-L1 adipocyte differentiation at the concentration of 50 μg/mL. ESA obviously reduced the visible triglyceride droplets accumulation, and dramatically decreased the number of the adipocytes at a comparatively high concentration. It is suggested that the effects are due to the inhibition of FAS by ESA; FAS activity is inhibited by ESA at a half inhibition concentration (IC₅₀) of 0.57 μg/mL, which is lower than that of classically known FAS inhibitors. Meanwhile, ESA displayed different inhibition kinetics and reacting sites for FAS. These results provide new clues for the development of novel products for obesity treatment and a scientific basis for the full use of byproducts for future industrial production of vegetable oil.     

Amber Extract Reduces Lipid Content in Mature 3T3-L1 Adipocytes by Activating the Lipolysis Pathway

Amber—the fossilized resin of trees—is rich in terpenoids and rosin acids. The physiological effects, such as antipyretic, sedative, and anti-inflammatory, were used in traditional medicine. This study aims to clarify the physiological effects of amber extract on lipid metabolism in mouse 3T3-L1 cells. Mature adipocytes are used to evaluate the effect of amber extract on lipolysis by measuring the triglyceride content, glucose uptake, glycerol release, and lipolysis-related gene expression. Our results show that the amount of triacylglycerol, which is stored in lipid droplets in mature adipocytes, decreases following 96 h of treatment with different concentrations of amber extract. Amber extract treatment also decreases glucose uptake and increases the release of glycerol from the cells. Moreover, amber extract increases the expression of lipolysis-related genes encoding perilipin and hormone-sensitive lipase (HSL) and promotes the activity of HSL (by increasing HSL phosphorylation). Amber extract treatment also regulates the expression of other adipocytokines in mature adipocytes, such as adiponectin and leptin. Overall, our results indicate that amber extract increases the expression of lipolysis-related genes to induce lipolysis in 3T3-L1 cells, highlighting its potential for treating various obesity-related diseases.     

Anti-Obesity Natural Products Tested in Juvenile Zebrafish Obesogenic Tests and Mouse 3T3-L1 Adipogenesis Assays

(1) Background: The obesity epidemic has been drastically progressing in both children and adults worldwide. Pharmacotherapy is considered necessary for its treatment. However, many anti-obesity drugs have been withdrawn from the market due to their adverse effects. Instead, natural products (NPs) have been studied as a source for drug discovery for obesity, with the goal of limiting the adverse effects. Zebrafish are ideal model animals for in vivo testing of anti-obesity NPs, and disease models of several types of obesity have been developed. However, the evidence for zebrafish as an anti-obesity drug screening model are still limited. (2) Methods: We performed anti-adipogenic testing using the juvenile zebrafish obesogenic test (ZOT) and mouse 3T3-L1 preadipocytes using the focused NP library containing 38 NPs and compared their results. (3) Results: Seven and eleven NPs reduced lipid accumulation in zebrafish visceral fat tissues and mouse adipocytes, respectively. Of these, five NPs suppressed lipid accumulation in both zebrafish and 3T3-L1 adipocytes. We confirmed that these five NPs (globin-digested peptides, green tea extract, red pepper extract, nobiletin, and Moringa leaf powder) exerted anti-obesity effects in diet-induced obese adult zebrafish. (4) Conclusions: ZOT using juvenile fish can be a high-throughput alternative to ZOT using adult zebrafish and can be applied for in vivo screening to discover novel therapeutics for visceral obesity and potentially also other disorders.     

Anti-Obesity Effects of a Mixture of Atractylodes macrocephala and Amomum villosum Extracts on 3T3-L1 Adipocytes and High-Fat Diet-Induced Obesity in Mice

Since the potential of (3:1) mixtures of Atractylodes macrocephala and Amomum villosum extracts has been proposed in the management of obesity, the purpose of present study was to investigate the effects of AME:AVE (3:1) mixture on weight loss, obesity-related biochemical parameters, adipogenesis and lipogenesis related proteins in 3T3-L1 cells and HFD-induced obesity in a mouse model. Treatment with AME:AVE (3:1) mixture inhibited lipid accumulation. Furthermore, the treatment with 75 and 150 mg/kg of AME:AVE (3:1) significantly decreased the body weight gain, white adipose tissue (WAT) weight, and plasma glucose level in HFD-induced obese mice. Moreover, treatment with 75 and 150 mg/kg AME:AVE (3:1) also significantly lowered the size of adipocytes in adipose tissue and reduced the lipid accumulation in liver. AME:AVE (3:1) treatment significantly decreased the expression of proteins related to adipogenesis and lipogenesis in 3T3-L1 adipocytes and WAT of HFD-induced obese mice. These results suggest that the AME:AVE herbal mixture (3:1) has anti-obesity effects, which may be elicited by regulating the expression of adipogenesis and lipogenesis-related proteins in adipocytes and WAT in HFD-induced obesity in mice.     

Inhibitory effects of constituents from Euphorbia lunulata on differentiation of 3T3-L1 cells and nitric oxide production in RAW264.7 cells

A new flavonol galactopyranoside, myricetin 3-O-(2',3'-digalloyl)-β-D-galactopyranoide (1), and 23 known constituents, including myricetin 3-O-(2'-galloyl)-β-D-galactopyranoide (2), myricitrin (3), myricetin (4), quercetin 3-O-(2', 3'-digalloyl)-β-D-galactopyranoide (5), quercetin 3-O-(2'-galloyl)-β-D-galactopyranoide (6), hyperin (7), isoquercetrin (8), quercetin (9), kaempferol (10), apigenin (11), luteolin (12), 3-O-methylquercetin (13), 5,7,2',5'-tetrahydroxyflavone (14), 1,3,4,6-tetra-O-galloyl-β-D-glucose (15), 1,2,6-tri-O-galloyl-β-D-glucose (16), 1,3,6-tri-O-galloyl-β-D-glucose (17), gallic acid (18), protocatechuic acid (19), 3,4,5-trimethoxybenzoic acid (20), 2,6-dihydroxyacetophenone (21), 3,3'-di-O-methylellagic acid (22), ellagic acid (23) and esculetin (24) were isolated from Euphorbia lunulata Bge. Their structures were determined by spectroscopic analysis. Isolated hydrolysable tannins, flavonoids, and flavonol galactopyranoside gallates showed significant inhibition of the differentiation of 3T3-L1 preadipocytes and triglyceride accumulation in maturing adipocytes, and nitric oxide production in RAW 264.7 cells.     

Elecampane (Inula helenium) Root Extract and Its Major Sesquiterpene Lactone,Alantolactone, Inhibit Adipogenesis of 3T3-L1 Preadipocytes

Recent studies have shown that Nur77 and AMPKα play an important role in regulating adipogenesis and isoalantolactone (ISO) dual-targeting AMPKα and Nur77 inhibits adipogenesis. In this study, we hypothesized that Inula helenium (elecampane) root extract (IHE), which contains two sesquiterpene lactones, alantolactone (ALA) and ISO, as major compounds, might inhibit adipogenesis. Here, we found that ALA and IHE simultaneously target AMPKα and Nur77 and inhibited adipogenic differentiation of 3T3-L1 cells, accompanied by the decreased expression of adipocyte markers. Further mechanistic studies demonstrated that IHE shares similar mechanisms of action with ISO that reduce mitotic clonal expansion during the early phase of adipogenic differentiation and decrease expression of cell cycle regulators. These results suggest that IHE inhibits adipogenesis, in part, through co-regulation of AMPKα and Nur77, and has potential as a therapeutic option for obesity and related metabolic dysfunction.     

Anthraquinones from Morinda officinalis roots enhance adipocyte differentiation in 3T3-L1 cells

To search for anti-diabetic and insulin-sensitising natural products, the effect on adipocyte differentiation was investigated by assessing fat accumulation in 3T3-L1 preadipocytes using Oil Red O staining. Fractionation and separation of n-hexane and CHCl? fractions of Morinda officinalis (Rubiaceae) using several chromatographic methods led to the isolation of three anthraquinones, 1,2-dimethoxyanthraquinone (1), alizarin-2-methyl ether (2) and rubiadin-1-methyl ether (3). Among them, alizarin-2-methyl ether (2) showed the strongest enhancing activity, followed by rubiadin-1-methyl ether (3) and 1,2-dimethoxyanthraquinone (1). At a concentration of 100?μM, alizarin-2-methyl ether (2) enhanced adipocyte differentiation by up to 131% (compared to insulin-treated cells). Thus, these compounds could be beneficial in the treatment of diabetes.     

Inhibitory effects of constituents from Morus alba var. multicaulis on differentiation of 3T3-L1 cells and nitric oxide production in RAW264.7 cells

A new arylbenzofuran, 3',5'-dihydroxy-6-methoxy-7-prenyl-2-arylbenzofuran (1), and 25 known compounds, including moracin R (2), moracin C (3), moracin O (4), moracin P (5), artoindonesianin O (6), moracin D (7), alabafuran A (8), mulberrofuran L (9), mulberrofuran Y (10), kuwanon A (11), kuwanon C (12), kuwanon T (13), morusin (14), kuwanon E (15), sanggenon F (16), betulinic acid (17), uvaol (18), ursolic acid (19), β-sitosterol (20), oxyresveratrol 2-O-β-D-glucopyranoside (21), mulberroside A (22), mulberroside B (23), 5,7-dihydroxycoumarin 7-O-β-D-glucopyranoside (24), 5,7-dihydroxycoumarin 7-O-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranoside (25) and adenosine (26), were isolated from Morus alba var. multicaulis Perro. (Moraceae). Their structures were determined by spectroscopic methods. The prenyl-flavonoids 11-14, 16, triterpenoids 17,18 and 20 showed significant inhibitory activity towards the differentiation of 3T3-L1 adipocytes. The arylbenzofurans 1-10 and prenyl-flavonoids 11-16 also showed significant nitric oxide (NO) production inhibitory effects in RAW264.7 cells.     

A new diarylheptanoid from Alpinia officinarum promotes the differentiation of 3T3-L1 preadipocytes

A new diarylheptanoid, namely trans-(4R,5S)-epoxy-1,7-diphenyl-3-heptanone (1), and a new natural product, 7-(4″-hydroxy-3″-methoxyphenyl)-1-phenyl-hepta-4E,6E-dien-3-one (2), were obtained from the aqueous extract of Alpinia officinarum Hance, together with three other diarylheptanoids, 5-hydroxy-1,7-diphenyl-3-heptanone (3), 1,7-diphenyl-4E-en-3-heptanone (4) and 5-methoxy-1,7-diphenyl-3-heptanone (5). The structures were characterised mainly by analysing their physical data including IR, NMR and HRMS. This study highlights that the 4,5-epoxy moiety in 1 is rarely seen in diarylheptanoids. In addition, the five isolates were tested for their differentiation activity of 3T3-L1 preadipocytes. The results showed that these compounds could dose-dependently promote adipocyte differentiation without cytotoxicity (IC₅₀ > 100 μM).     

Anti-adipogenic activity of Cordyceps militaris in 3T3-L1 cells

Inhibition of adipocytes differentiation is suggested to be an important strategy for prevention and/or treatment of obesity. In our present study, Cordyceps militaris showed significant inhibitory activity on adipocyte differentiation in 3T3-L1 preadipocytes as assessed by measuring fat accumulation using Oil Red O staining. Activity-guided fractionation led to the isolation of cordycepin (1), guanosine (2) and tryptophan (3) as active compounds. All the three compounds were more effective in the prevention of early stage of adipogenesis than in lipolysis. In addition, combinational treatment of three compounds significantly increased anti-adipogenic activity.     

Anti-Obesity Effect of Dyglomera® Is Associated with Activation of the AMPK Signaling Pathway in 3T3-L1 Adipocytes and Mice with High-Fat Diet-Induced Obesity

Dyglomera^® is an aqueous ethanol extract of the fruit pods of Dichrostachys glomerata, a Cameroonian spice. Several studies have shown its anti-diabetic and anti-obesity effects. However, the underlying mechanisms for such effects remain unclear. Thus, the objective of this study was to investigate the anti-obesity effect of Dyglomera^® and its underlying mechanisms in mice with high-fat diet-induced obesity and 3T3-L1 adipocytes. Our results revealed that Dyglomera^® inhibited adipogenesis and lipogenesis by regulating AMPK phosphorylation in white adipose tissues (WATs) and 3T3-L1 adipocytes and promoted lipolysis by increasing the expression of lipolysis-related proteins. These results suggest that Dyglomera^® can be used as an effective dietary supplement for treating obesity due to its modulating effect on adipogenesis/lipogenesis and lipolysis.     

Effect of Cortisol on Calpains in the C2C12 and 3T3-L1 Cells

The present study was carried out to understand the effect of cortisol on calpain system in the C2C12 and 3T3-L1 adipocyte cells under co-culture system. Cells were co-cultured by using transwell inserts with a 0.4 μm porous membrane to separate C2C12 and 3T3-L1 preadipocyte cells. Each cell type was grown independently on the transwell plates. Following cell differentiation, inserts containing 3T3-L1 cells were transferred to C2C12 plates. Ten microgram per milliliter of cortisol was added to the medium. Following treatment for 3 days, the cells in the lower well were harvested for analysis. Calpains such as μ-calpain, m-calpain, and calpastatin were selected for the analysis. RT-PCR results indicated the significant increase in the mRNA expression of μ-calpain, m-calpain, and calpastatin. In addition, the confocal microscopical investigation indicated the cortisol treatment increases calpain expression in the C2C12 and 3T3-L1 cells. Taking all these together, cortisol treatment with co-culture system shows most reliable status of calpains expression in the cells, which is quite distinct from one-dimensional monocultured cells.     

Flavonol acylglycosides from flower of Albizia julibrissin and their inhibitory effects on lipid accumulation in 3T3-L1 cells

Obesity is a serious health problem worldwide. We investigated the anti-obesity effect of the flower of Albizia julibrissin DURAZZ. (Leguminosae). A 90% EtOH extract of the flower inhibited adipogenesis in 3T3-L1 preadipocytes, as well as the activity of glycerol-3-phosphate dehydrogenase (GPDH) activity. New flavonol acylglycosides (1-4) and eighteen known compounds (5-22) were isolated by bioassay-directed fractionation. These new glycosides were elucidated to be 3″-(E)-p-coumaroylquercitrin (1), 3″-(E)-feruloylquercitrin (2), 3″-(E)-cinnamoylquercitrin (3), and 2″-(E)-cinnamoylquercitrin (4) on the basis of spectroscopic and chemical analysis. These compounds inhibited adipogenesis in 3T3-L1 preadipocytes. In particular, 2 exhibited potent inhibitory effects on triglyceride accumulation. Furthermore, GPDH activity was inhibited by 2. Additionally, 2 inhibited glucose uptake in 3T3-L1 adipocytes. These results indicate that the 90% EtOH extract and compounds isolated from the flower of A. julibrissin inhibit adipogenesis in 3T3-L1 preadipocytes and may have anti-obesity effect through the inhibition of preadipocyte differentiation.