5‐Hydroxyferulic acid methyl ester isolated from wasabi leaves inhibits 3T3‐L1 adipocyte differentiation

To investigate the compounds present in wasabi leaves (Wasabia japonica Matsumura) that inhibit the adipocyte differentiation, activity‐guided fractionation was performed on these leaves. 5‐Hydroxyferulic acid methyl ester ( 1 : 5‐HFA ester), one of the phenylpropanoids, was isolated from wasabi leaves as a compound that inhibits the adipocyte differentiation. Compound 1 suppressed the intracellular lipid accumulation of 3T3‐L1 cells without significant cytotoxicity. Gene expression analysis revealed that 1 suppressed the mRNA expression of 2 master regulators of adipocyte differentiation, PPARγ and C/EBPα. Furthermore, 1 downregulated the expression of adipogenesis‐related genes, GLUT4, LPL, SREBP‐1c, ACC, and FAS. Protein expression analysis revealed that 1 suppressed PPARγ protein expression. Moreover, to investigate the relationship between the structure and activity of inhibiting the adipocyte differentiation, we synthesized 12 kinds of phenylpropanoid analog. Comparison of the activity among 1 and its analogs suggested that the compound containing the substructure that possess a common functional group at the ortho position such as a catechol group exhibits the activity of inhibiting the adipocyte differentiation. Taken together, our findings suggest that 1 from wasabi leaves inhibits adipocyte differentiation via the downregulation of PPARγ.     

3,4-Oxo-isopropylidene-shikimic acid promotes adiopkine expression during murine 3T3-L1 fibroblast differentiation into adipocytes

Objective3,4-Oxo-isopropylidene-shikimic acid (ISA), a derivative of shikimic acid, has exhibited ameliorative effect on cognitive impairment in experimental animal models of dementia. This study investigated the effect of ISA on lipid accumulation and adipokine secretion during differentiation of 3T3-L1 fibroblasts to adipocytes.Methods3T3-L1 cells were cultured and treated with ISA (50–800 μM) from days 3–8. Lipid accumulation and triglyceride content were measured. Gene expression of adipokines (adiponectin, leptin, and resistin), CCAAT/enhancer binding protein (C/EBP) β, C/EBP α and peroxisome proliferator-activated receptor γ (PPAR γ) and PPAR target genes, including adipocyte fatty acid binding protein (aP2) and fatty acid synthase (FAS) were investigated.ResultsISA promoted 3T3-L1 fibroblast differentiation to adipocytes and increased triglyceride content by 26%. On mechanistic levels, ISA increased expressions of C/EBP β, PPAR γ, C/EBP α, aP2 and FAS. Moreover, ISA stimulated expressions of adipokines secreted by adipocytes, including adiponectin, leptin, and resistin.ConclusionsThese findings demonstrated that ISA promoted adipogenesis by up-regulating expressions of C/EBP β, PPAR γ, C/EBP α, aP2 and FAS, and also stimulated adipokines during adipocyte differentiation. Further study should clarify the relationship between stimulation of adipokines and cognitive enhancing effect of ISA.     

Inhibition of preadipocyte differentiation and lipid accumulation by Orengedokuto treatment of 3T3-L1 cultures

Obesity is a major cause of metabolic syndrome and is due to an increase in the number and hypertrophy of adipocytes. Accordingly, inhibition of the differentiation and proliferation of adipocytes may be used in the treatment and prevention of metabolic syndrome. This study investigated the effects of 50 commonly used Kampo medicines on the differentiation of 3T3-L1 preadipocytes to search for a drug with an antiobesity effect. Kampo medicines were screened, and the strongest differentiation-inhibitory effect was noted with Orengedokuto. To explore the active ingredients in Orengedokuto, the effects of four crude drug components of Orengedokuto were investigated. It was found that the differentiation-inhibitory effect of Orengedokuto was accounted for by Coptidis rhizome and Phellodendri cortex. Furthermore, berberine, a principal ingredient common to Coptidis rhizome and Phellodendri cortex, showed a differentiation-inhibitory effect. The effect of berberine involves an inhibition of the mRNA and protein expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα). Moreover, berberine inhibited lipid accumulation in adipocytes. These findings suggest that an antiobesity effect could be a new indication for Orengedokuto and that its active ingredient is berberine, with a mechanism involving the inhibition of PPARγ and C/EBPα expression.     

Antiobesity effect of baicalin involves the modulations of proadipogenic and antiadipogenic regulators of the adipogenesis pathway

In this study, the antiobesity effects of baicalin, 5,6‐dihydroxyflavone‐7‐glucuronic acid, were characterized using an in vitro system of adipogenesis, i.e. fat cell formation. Baicalin‐treatment of 3T3‐L1 preadipocytes was shown to inhibit triglyceride accumulation and lipid droplet formation during induced adipogenesis. Microarray analyses showed that baicalin modulated the expression of genes located in pathways such as adipogenesis, cholesterol biosynthesis, focal adhesion and others. In the adipogenesis pathway, treatment with baicalin significantly down‐regulated terminal differentiation markers of adipocytes including fatty acid binding protein 4. The effects of baicalin on the core part of the adipogenesis pathway, however, were paradoxical; the expression levels of CCAAT/enhancer binding protein (C/EBP)β and C/EBPδ were up‐regulated, while the expression levels of the peroxisome proliferator‐activated receptor (PPAR)γ and C/EBPα were down‐regulated. The antiadipogenic mechanisms of baicalin can be explained by its effects on the upstream part of adipogenesis pathway; baicalin not only up‐regulates the antiadipogenic regulators, C/EBPγ, C/EBP homologous protein and Kruppel‐like factor (KLF)2, but also down‐regulates the proadipogenic regulator, KLF15. The overall effects of baicalin on these upstream regulators of adipogenesis were antiadipogenic, resulting in the down‐regulation of downstream genes and the inhibition of cellular fat accumulation. Copyright © 2009 John Wiley & Sons, Ltd.     

Brassinin,a phytoalexin in cruciferous vegetables,suppresses obesity‐induced inflammatory responses through the Nrf2‐HO‐1 signaling pathway in an adipocyte‐macrophage co‐culture system

The aim of this study was to investigate the effect of brassinin (BR), a phytoalexin found in plants belonging to the Brassicaceae family, on the obesity‐induced inflammatory response and its molecular mechanism in co‐culture of 3T3‐L1 adipocytes and RAW264.7 macrophages. BR effectively suppressed lipid accumulation by down‐regulating the expression of adipogenic factors, which in turn, were regulated by early adipogenic factors such as CCAAT‐enhancer‐binding protein‐β and Kruppel‐like factor 2. Production of inflammatory cytokines and reactive oxygen species, induced by adipocyte‐conditioned medium, was significantly decreased in BR‐treated cells. This effect of BR was more prominent in contact co‐culture of adipocytes and macrophages with a 90% and 34% reduction in IL‐6 and MCP‐1 levels, respectively. BR also restored adiponectin expression, which was significantly reduced by culturing adipocytes in macrophage‐conditioned medium. In the transwell system, BR increased the protein levels of nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2) and its target molecule, hemoxygenase‐1 (HO‐1), by 55%–93% and 45%–48%, respectively, and also increased Nrf2 translocation into the nucleus. However, knockdown of Nrf2 or HO‐1 in RAW264.7 cells restored this BR‐mediated inhibition of IL‐6 and MCP‐1 production. These results indicated that BR inhibited obesity‐induced inflammation via the Nrf2‐HO‐1 pathway.     

Piperine inhibits adipocyte differentiation via dynamic regulation of histone modifications

Previously, we reported that piperine, one of the major pungent components in black pepper, attenuates adipogenesis by repressing PPARγ activity in 3T3‐L1 preadipocytes. However, the epigenetic mechanisms underlying this activity remain unexplored. Here, gene set enrichment analysis using microarray data indicated that there was significant downregulation of adipogenesis‐associated and PPARγ target genes and upregulation of genes bound with H3K27me3 in response to piperine. As shown by Gene Ontology analysis, the upregulated genes are related to lipid oxidation and polycomb repressive complex 2 (PRC2). Chromatin immunoprecipitation assays revealed that PPARγ (and its coactivators), H3K4me3, and H3K9ac were less enriched at the PPAR response element of three adipogenic genes, whereas increased accumulation of H3K9me2, H3K27me3, and Ezh2 was found, which likely led to the reduced gene expression. Further analysis using three lipolytic genes revealed the opposite enrichment pattern of H3K4me3 and H3K27me3 at the Ezh2 binding site. Treatment with GSK343, an Ezh2 inhibitor, elevated lipolytic gene expression by decreasing the enrichment of H3K27me3 during adipogenesis, which confirms that Ezh2 plays a repressive role in lipolysis. Overall, these results suggest that piperine regulates the expression of adipogenic and lipolytic genes by dynamic regulation of histone modifications, leading to the repression of adipocyte differentiation.     

Bromophenol (5‐bromo‐3,4‐dihydroxybenzaldehyde) isolated from red alga Polysiphonia morrowii inhibits adipogenesis by regulating expression of adipogenic transcription factors and AMP‐activated protein kinase activation in 3T3‐L1 adipocytes

The aim of the present study was to investigate the effect of 5‐bromo‐3,4‐dihydroxybenzaldehyde (BD) isolated from Polysiphonia morrowii on adipogenesis and differentiation of 3T3‐L1 preadipocytes into mature adipocytes and its possible mechanism of action. Levels of lipid accumulation and triglyceride were significantly lower in BD treated cells than those in untreated cells. In addition, BD treatment reduced protein expression levels of peroxisome proliferator‐activated receptor‐γ, CCAAT/enhancer‐binding proteins α, and sterol regulatory element‐binding protein 1 compared with control (no treatment). It also reduced expression levels of adiponectin, leptin, fatty acid synthase, and fatty acid binding protein 4. AMP‐activated protein kinase activation was found to be one specific mechanism involved in the effect of BD. These results demonstrate that BD possesses inhibitory effect on adipogenesis through activating AMP‐activated protein kinase signal pathway.     

Vitexin, orientin and other flavonoids from Spirodela polyrhiza inhibit adipogenesis in 3T3-L1 cells

To investigate the adipogenesis inhibitory effect on lipid accumulation, 3T3-L1 cells were treated with fractions and isolated flavonoids of Spirodela polyrhiza. An ethanol extract of S. polyrhiza was fractionated into three fractions. The butanol soluble fraction (SPB) exhibited potent antiadipogenesis activity and decreased C/EBPα and PPARγ protein expression level in 3T3-L1 cells without significant cytotoxicity. The flavonoids were isolated from SPB and their chemical structures were identified as chrysoeriol (1), apigenin (2), luteolin (3), vitexin (4), cosmosin (5), orientin (6) and luteolin-7-O-β-d-glucoside (7) by spectroscopic analysis. Studies on the adipogenesis and intracellular triglyceride accumulation inhibitory effect showed that compounds 4 and 6 had the highest activity and decreased C/EBPα and PPARγ protein expression level in 3T3-L1 cells. These results suggest that the flavonoids isolated from SPB, especially compounds 4 and 6, contribute to the inhibitory activity of S. polyrhiza in 3T3-L1 cells.     

Effect of mollugin on apoptosis and adipogenesis of 3T3-L1 preadipocytes

The effect of mollugin, isolated from the roots of Rubia cordifolia L., on cell viability, apoptosis and adipogenesis in 3T3‐L1 preadipocytes was investigated. The inhibitory effect of mollugin (40–60 µm ) on cell viability was more significant in differentiated adipocytes than in 3T3‐L1 preadipocytes. In 3T3‐L1 cells, the cytotoxicity of mollugin was accompanied by apoptotic events including mitochondrial membrane potential (Δψm) loss and activation of caspase‐9, ‐3 and ‐7, leading to PARP degradation. Although the presence of 20 µm mollugin during induced adipocytic differentiation of 3T3‐L1 cells for 6 days failed to affect the cell viability, it could almost completely abrogate the differentiation‐associated morphology change and intracellular lipid accumulation. A similar level of inhibition was observed, when 20 µm mollugin was present during the early stage (D0–D2) of the differentiation period. In addition, the expression of C/EBPα, PPARγ1 and PPARγ2 was significantly down‐regulated. The presence of 20 µm mollugin during either middle stage (D2–D4) or late stage (D4–D6) of the differentiation period, however, caused the inhibition to a lesser extent. These results indicated that mollugin at high concentrations (40–60 µm ) exerted cytotoxicity via inducing apoptosis, whereas mollugin at a low concentration (20 µm ) suppressed adipocytic differentiation without exerting cytotoxicity in 3T3‐L1 preadipocytes. Copyright © 2010 John Wiley & Sons, Ltd.     

Berberine inhibits SREBP‐1‐related clozapine and risperidone induced adipogenesis in 3T3‐L1 cells

Weight gain is a common and potentially serious complication associated with the treatment of second generation antipsychotics such as clozapine and risperidone. Increased peripheral adipogenesis via the SREBP‐1 pathway could be one critical mechanism responsible for antipsychotic drug‐induced weight gain. Berberine, a botanic alkaloid, has been shown in our previous studies to inhibit adipogenesis in cell and animal models. MTT was used to determine the cytotoxic effects of clozapine and risperidone in combination with berberine. Differentiation of 3T3‐L1 cells was monitored by Oil‐Red‐O staining and the expression of SREBP‐1 and related proteins was determined by real‐time RT‐PCR and western blotting. The results showed that neither clozapine nor risperidone, alone or in combination with berberine had significant effects on cell viability. Eight days treatment with 15 μm clozapine increased adipogenesis by 37.4% and 50 μm risperidone increased adipogenesis by 26.5% during 3T3‐L1 cell differentiation accompanied by increased SREBP‐1, PPARγ, C/EBPα, LDLR and Adiponectin gene expression. More importantly, the addition of 8 μm berberine diminished the induction of adipogenesis almost completely accompanied by down‐regulated mRNA and protein expression levels of SREBP‐1‐related proteins. These encouraging results may lead to the use of berberine as an adjuvant to prevent weight gain during second generation antipsychotic medication. Copyright © 2010 John Wiley & Sons, Ltd.     

Genistein suppresses adipogenesis of 3T3‐L1 cells via multiple signal pathways

Genistein, an isoflavone, was shown to have therapeutic effects for obesity, diabetes and cardiovascular diseases. This study investigated the effect and underlying mechanism of genistein on adipogenesis in 3T3‐L1 preadipocytes. Genistein inhibited lipid accumulation and decreased the nonesterified fatty acid (NEFA) content of 3T3‐L1 on day 6 after the induction of differentiation with methylisobutylxanthine, dexamethasone and insulin (MDI). Genistein recovered nitric oxide (NO) release suppressed by MDI and the results were consistent with the expression of endothelial NO synthase (eNOS) assayed by western blotting. Pretreatment with genistein inhibited the phosphorylation of p38 mitogen‐activated protein kinase (p38 MAPK) stimulated with 10 µg/mL of insulin. Furthermore, genistein inhibited the expression of fatty acid synthase (FAS) from 178% of the MDI group to 74%. SB203580, a p38 inhibitor, mimicked the FAS inhibition effect of genistein, suggesting that the inhibitory effect of genistein on FAS was partially via the p38 pathway. On the other hand, genistein abolished the phosphorylation of janus‐activated kinase 2 (JAK2) in response to MDI. AG490, a JAK2 inhibitor, suppressed the expression of CCAAT/enhancer binding protein alpha (C/EBPα), a marker of adipocyte differentiation. The findings suggest that genistein attenuates the differentiation of 3T3‐L1 involving multiple signal pathways. Copyright © 2008 John Wiley & Sons, Ltd.