Ryunhwa Kang

AICAR, an activator of AMPK, inhibits adipogenesis via the WNT/β-catenin pathway in 3T3-L1 adipocytes

AMP-activated protein kinase (AMPK) is known to sense the cellular energy state and regulates various cellular energy metabolism pathways through its activation by AMP, an indicator of a low-energy state. 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), an activator of AMPK, efficiently inhibited the adipogenesis of 3T3-L1 cells. To elucidate its possible mechanism of action, the expression levels of β-catenin and other members of the WNT/β-catenin pathway were analyzed during the adipogenesis of 3T3-L1 cells in the presence or absence of AICAR. It was found that AICAR significantly enhanced β-catenin expression and its nuclear accumulation. Transfection of β-catenin small interfering RNA (siRNA) significantly prevented the effects of AICAR on the expression of various genes. The expression of the major genes of adipogenesis including the peroxisome proliferator-activated receptor (PPAR)γ, the CCAAT/enhancer binding protein (C/EPB)α, the fatty acid binding protein (FABP)4 and lipoprotein lipase (LPL), which were all reduced by AICAR treatment, were significantly recovered in β-catenin siRNA-transfected cells. Among the members of the WNT/β-catenin pathway, the expression of low density lipoprotein receptor-related protein (LRP)6, dishevelled (DVL)2 and DVL3 were significantly up-regulated by AICAR treatment, whereas the expression of AXIN was down-regulated. The present study provides compelling evidence that AICAR inhibits adipogenesis through the modulation of the WNT/β-catenin pathway.

Platycodin D inhibits adipogenesis of 3T3‐L1 cells by modulating kruppel‐like factor 2 and peroxisome proliferator‐activated receptor γ

In this study, platycodin D was found to inhibit intracellular triglyceride accumulation in 3T3‐L1 cells with an IC50 of 7.1 μM. The expression levels of genes involved in lipid metabolism such as fatty‐acid‐binding protein 4 and lipoprotein lipase were significantly downregulated following treatment with platycodin D. Treatment with platycodin D also resulted in a reduction of Peroxisome proliferator‐activated receptor(PPAR)γ expression and its binding to target DNA sequence. Among the various upstream regulators of PPARγ, the expression of Kruppel‐like factor(KLF)2, an anti‐adipogenic factor, was significantly upregulated following platycodin D treatment. When the upregulation of KLF2 was inhibited by KLF2 siRNA, the expression and binding of PPARγ to its target sequence were significantly recovered under these conditions. The results of this study suggested that anti‐adipogenic effect of platycodin D involves the upregulation of KLF2 and subsequent downregulation of PPARγ. Copyright

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

Shikonin inhibits fat accumulation in 3T3-L1 adipocytes.

Shikonin, 5,6‐dihydroxyflavone‐7‐glucuronic acid, is the main ingredient of Lithospermum erythrorhizon Sieb. et Zucc, and was reported to have various biological activities including antiinflammatory, anticancer, antimicrobial and others. This study aimed to elucidate, for the first time, the antiobesity activity of shikonin and its mechanism of action. Shikonin was found to inhibit fat droplet formation and triglyceride accumulation in 3T3‐L1 adipocytes. The half inhibitory concentration, IC50, for the inhibition of triglyceride accumulation was found to be 1.1 μM. The expression of genes involved in lipid metabolism, such as FABP4 and LPL, were significantly inhibited following shikonin treatment. Shikonin also inhibited the ability of PPARγ and C/EBPα, the major transcription factors of adipogenesis, to bind to their target DNA sequences. The expressions of mRNA and protein of PPARγ and C/EBPa were significantly down‐regulated following shikonin treatment. Among the upstream regulators of adipogenesis, only SREBP1C was found to be down‐regulated by shikonin. The results of this study suggest that shikonin down‐regulates the expression of SREBP1C and subsequently the expression of PPARγ and C/EBPα. Together, these changes result in the down‐regulation of lipid metabolizing enzymes and reduced fat accumulation. Copyright

SH21B, an anti-obesity herbal composition, inhibits fat accumulation in 3T3-L1 adipocytes and high fat diet-induced obese mice through the modulation of the adipogenesis pathway

ETHNOPHARMACOLOGICAL RELEVANCE SH21B is an anti-obesity composition composed of seven herbs: Scutellaria baicalensis Georgi, Prunus armeniaca Maxim, Ephedra sinica Stapf, Acorus gramineus Soland, Typha orientalis Presl, Polygala tenuifolia Willd and Nelumbo nucifera Gaertner. It has been used for the treatment of obesity in traditional medical clinics in Korea, but its anti-obesity effects and mechanism of action have not been studied until now. AIM OF THE STUDY This study was conducted to confirm the anti-obesity effects of SH21B and to elucidate its mechanism of action. MATERIALS AND METHODS 3T3-L1 adipocytes and high fat diet-induced obese mice were treated with SH21B, and its effect on gene expression was analyzed using microarray technology, real-time PCR and western blotting experiments. RESULTS SH21B significantly inhibited fat accumulation in 3T3-L1 adipocytes and reduced adipose tissue and serum triglyceride levels in high fat diet-induced obese mice. Microarray analyses showed that SH21B affected more genes in the adipogenesis pathway than any other pathway studied. SH21B significantly decreased the expression of major transcription factors of the adipogenesis pathway and resulted in the down-regulation of lipid metabolizing enzymes involved in the transport, uptake and synthesis of lipids. CONCLUSIONS SH21B inhibits fat accumulation by down-regulating the expression of genes involved in the adipogenesis pathway.

A promoter polymorphism -2122C>T of CHI3L1 is associated with serum low density lipoprotein cholesterol level in Korean subjects.

OBJECTIVES This study assessed the effects of 10 tagging single nucleotide polymorphisms (SNPs) of the CHI3L1 gene on serum LDL cholesterol levels in 290 Korean subjects. DESIGN AND METHODS Genotyping analyses of SNPs were conducted by TaqMan® method. The effects of the promoter SNP on mRNA expression and nuclear factor binding were measured by real-time PCR and electrophoretic mobility shift assay, respectively. RESULTS Among 10 tagging SNPs, -2122C>T SNP (rs946261) in the promoter region was significantly associated with serum LDL cholesterol level (P=0.005). The T allele of -2122C>T was associated with significantly increased mRNA expressions in peripheral blood cells of the subjects, and also increased a nuclear factor binding measured by an electrophoretic mobility shift assay. CONCLUSIONS -2122C>T of CHI3L1, a promoter SNP which affects the mRNA expression and nuclear factor binding, is significantly associated with serum LDL cholesterol levels in Korean subjects.

A Study on the Gene Expression in Shikonin-Induced Inhibition of Adipogenesis

Shikonin, a component of Lithospermum erythrorhizon Sieb. et Zucc, exerts various characteristics such as anti-inflammatory, anti-cancer and anti-obesity functions. To elucidate the molecular mechanism of shikonin-induced inhibition of adipogenesis, we analyzed the mRNA expression level of various adipogenesis-related factors including C/EBPs (CCAAT/enhancerbinding proteins) and (peroxisome proliferator-activated receptor ). The data showed that mRNA expressions of C/ and C/ were only slightly changed by shikonin treatment, but mRNA expressions of and C/ were significantly down-regulated. Then, we tested whether upstream regulators of C/ and were involved in anti-adipogenesis of shikonin. C/ and CHOP (C/EBP homologous protein), which are upstream regulators of C/, were not affected by shikonin treatment. On the contrary, the mRNA level of KROX20 was markedly down-regulated by shikonin treatment. These results suggest that KROX20 might regulate downstream factors of adipogenesis through C/-independent pathway. The expression of KLF15 (Kruppel-like factor15), which is a member of KLF family and is a upstream regulator of , was dramatically decreased by shikonin treatment, but KLF2 was not changed. Shikonin had no impact on the expression of KLF5 in the early stage of adipogenesis, but shikonin increased expression of KLF5 in the late stage of adipogenesis. Even though mRNA expression of KLF5 was moderately changed by shikonin treatment, its effect may be small compared with the effect of KLF15, which was markedly inhibited. Taken together, these results suggest that shikonin inhibits adipogenesis through the down-regulation of and C/, which is mediated by the down-regulation of two pro-adipogenic factors, KROX20 and KLF15.

Effects of Platycodin D on Gene Expressions of Pro-adipogenic and Anti-adipogenic Regulators in 3T3-L1 Cells

Platycodin D, a major component of Platycodi radix, is known to have various activities including anti-inflammatory, anti-hyperlipidemic, anti-tumor activities and others. Recently, it was reported that platycodin D inhibits fat accumulation and adipogenesis. The aim of this study was to investigate whether various adipogenic regulators are modulated by platycodin D treatment during the adipogenesis of 3T3-L1 cells. mRNA levels of terminal markers of adipogenesis such as ADIPOQ (adiponectin) and GLUT (glucose transporter) 4, which were quantified by real time PCR, were decreased by platycodin D treatment. mRNA expression of PPAR (peroxisome proliferator-activated receptor) and C/EBP (CCAAT/enhaner binding protein) , which are central transcription factors of adipogenesis, were also decreased by platycodin D treatment. To elucidate the detailed molecular mechanism of platycodin D-induced inhibition of adipogenesis, we analyzed mRNA expression of upstream regulators of PPAR and C/EPB. mRNA levels of the pro-adipogenic regulators, KROX20 and KLF (Kruppel-like factor) 15 were markedly down-regulated by platycodin D treatment. On the other hand, mRNA expression of CHOP (C/EBP homologous protein), an anti-adipogenic regulator, was significantly up-regulated by platycodin D treatment. mRNA levels of other pro-adipogenic regulators, C/EBP and C/EPB, were not affected by platycodin D treatment, and another anti-adipogenic regulator, C/EBP was also not affected by platycodin D treatment. Taken together, these results suggest that platycodin D-induced inhibition of adipogenesis is mediated by gene interactions including the down-regulation of pro-adipogenic regulators, KROX20 and KLF15, and the up-regulation of an anti-adipogenic regulator, CHOP.

Transcriptome Analyses for the Anti-Adipogenic Mechanism of an Herbal Composition

SH21B is a natural composition composed of seven herbs: Scutellaria baicalensis Georgi, Prunus armeniaca Maxim, Ephedra sinica Stapf, Acorus gramineus Soland, Typha orientalis Presl, Polygala tenuifolia Willd and Nelumbo nucifera Gaertner (Ratio 3:3:3:3:3:2:2). In our previous study, we reported that SH21B inhibited adipogenesis and fat accumulation in 3T3-L1 cells through modulation of various regulators in the adipogenesis pathway. The aim of this study was to analyze the transcriptome profiles for the anti-adipogenic effects of SH21B in 3T3-L1 cells. Total RNAs from SH21B-treated 3T3-L1 cells were reverse-transcribed into cDNAs and hybridized to Affymetrix Mouse Gene 1.0 ST array. From microarray analyses, we identified 2,568 genes of which expressions were changed more than two-fold by SH21B, and the clustering analyses of these genes resulted in 9 clusters. Three clusters among the 9 showed down-regulation by SH21B (cluster 4, cluster 6 and cluster 9), and two clusters showed up-regulation by SH21B (cluster 7 and cluster 8) during the adipogenesis of 3T3-L1 cells. It was found that many genes related to cell proliferation and adipogenesis were included in these clusters. Clusters 4, 6 and 9 included genes which were related with adipogenesis induction and cell cycle arrest. Clusters 7 and 8 included genes related to cell proliferation as well as adipogenesis inhibition. These results suggest that the mechanisms of the anti-adipogenic effects of SH21B may be the modulation of genes involved in cell proliferation and adipogenesis.

A Study on the Gene Expression of Adipogenic Regulators by an Herbal Composition

In our previous study, it was reported that an herbal mixture, SH21B, inhibits fat accumulation and adipogenesis both in vitro and in vivo models of obesity. SH21B is a mixture composed of seven herbs: Scutellaria baicalensis Georgi, Prunus armeniaca Maxim, Ephedra sinica Stapf, Acorus gramineus Soland, Typha orientalis Presl, Polygala tenuifolia Willd, and Nelumbo nucifera Gaertner (Ratio 3:3:3:3:3:2:2). The aim of this study was to investigate the detailed molecular mechanisms of the effects of SH21B on various regulators of the adipogenesis pathway. During the adipogenesis of 3T3-L1 cells, SH21B significantly decreased the expression levels of central transcription factors of adipogenesis, such as peroxisome proliferator-activated receptor (PPAR)γ and CCAAT/enhancer binding protein (C/EBP)α. To elucidate the detailed molecular mechanism of the anti-adipogenic effects of SH21B, we examined the expression levels of the various pro-adipogenic or anti-adipogenic regulators of adipogenesis upstream of PPARγ and C/EBPα. The mRNA levels of Krox20 and Kruppel-like factor (KLF) 15, which are pro-adipogenic regulators, were significantly down-regulated by SH21B treatment, whereas the mRNA levels of C/EBPβ and KLF5 were not changed. KLF2 and C/EBP homologous protein (CHOP), which are anti-adipogenic regulators, were significantly up-regulated by SH21B treatment. These results suggest that the molecular mechanism of the anti-adipogenic effect of SH21B involves both the down-regulations of pro-adipogenic regulators, such as Krox20 and KLF15, and the up-regulations of anti-adipogenic regulators, such as KLF2 and CHOP, which results in the suppression of central transcription factors of adipogenesis including PPARγ and C/EBPα.