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.     

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.     

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.     

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.     

Constituents from Terminalia species increase PPARα and PPARγ levels and stimulate glucose uptake without enhancing adipocyte differentiation

Ethnopharmacological relevance

The fruits of Terminalia bellerica Roxb. (Combretaceae) and T. chebula Retz. (Combretaceae) are important components of triphala, a popular Ayurvedic formulation, for treating diabetes in Indian traditional medicine.

Aim of the study

The aim of this study was to evaluate the effects of the constituents of T. bellerica and T. chebula fruit extracts on PPARα and PPARγ signaling/expression, cellular glucose uptake and adipogenesis.

Materials and methods

PPARα and PPARγ signaling and expression (luciferase assay and western blot) and the insulin-stimulated uptake of 2-NBDG were determined in HepG2 cells. The effects on adipogenesis were determined in 3T3-L1 cells by Oil red O staining and measurement of lipid content by AdipoRed reagent.

Results

Out of the 20 compounds, two ellagitannins, chebulagic acid (1) and corilagin (2), and three gallotannins, 2,3,6-tri-O-galloyl-β-d-glucose (3), 1,2,3,6-tetra-O-galloyl-β-d-glucose (4), and 1,2,3,4,6-penta-O-galloyl-β-d-glucose (5), showed the enhancement of PPARα and/or PPARγ signaling. Two of the gallotannins (4 and 5) also increased PPARα and PPARγ protein expression, while all three (3–5) enhanced insulin-stimulated glucose uptake into HepG2 cells. Compound 1,2,3,6-tetra-O-galloyl-β-d-glucose (4) was the most potent in increasing cellular glucose uptake (9.92-fold increase at 50 μM). In the test for adipogenesis, 3–5 did not enhance the differentiation of 3T3-L1 preadipocytes but inhibited the adipogenic effect of rosiglitazone.

Conclusion

Three gallotannins (3–5) from Terminalia fruits acting as enhancers of both PPARα and PPARγ signaling increased insulin-stimulated glucose uptake without inducing the adipogenesis, with 1,2,3,6-tetra-O-galloyl-β-d-glucose (4) being the most effective in stimulating glucose uptake and 1,2,3,4,6-penta-O-galloyl-β-d-glucose (5) being most effective in increasing PPAR protein expression.     

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.     

A fraction of Acorus calamus L. extract devoid of beta-asarone enhances adipocyte differentiation in 3T3-L1 cells

The effects of fractions partitioned from the ethanol extract of Acorus calamus L. (AC) on adipocyte differentiation were investigated using cultured mouse 3T3-L1 preadipocytes. The degree of differentiation was evaluated by measuring the cellular triglycerides and protein expression of the glucose transporter GLUT4 in 3T3-L1 cells. The ethyl acetate fraction of the AC extract (ACE) was found to enhance adipocyte differentiation as did rosiglitazone. The results of further fractionation of ACE indicated that the active fraction does not consist of beta-asarone, which is a toxic component of this plant. This finding suggests that ACE has potential insulin-sensitizing activity like rosiglitazone, and may improve type 2 diabetes.     

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.