Sungmin Bae

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.

The anti-adipogenic effects of (-)epigallocatechin gallate are dependent on the WNT/β-catenin pathway

(-)Epigallocatechin gallate (EGCG) is the most abundant catechin in green tea and reportedly has anti-obesity and anti-adipogenic effects. In this study, we determined that the up-regulation of the WNT/β-catenin pathway is the anti-adipogenic mechanisms of EGCG in 3T3-L1 cells. EGCG treatment down-regulates the expression of major genes involved in the adipogenesis pathway including peroxisome proliferator-activated receptor (PPAR)γ, CCAAT/enhancer binding protein (C/EBP)α, fatty acid binding protein (FABP)4 and fatty acid synthase (FASN), while up-regulating the nuclear level of β-catenin. Knockdown of β-catenin using small interfering (si) RNA attenuated the inhibitory effects of EGCG on intracellular lipid accumulation. β-catenin siRNA transfection also recovered terminal adipocyte markers such as FABP4, FASN, lipoprotein lipase and adiponectin, which were down-regulated by EGCG. The DNA binding activities as well as the expression levels of PPARγ and C/EBPα, which were down-regulated by EGCG, were significantly restored by β-catenin siRNA transfection. In addition, we found that EGCG efficiently up-regulates the WNT/β-catenin pathway. Among the members of the WNT/β-catenin pathway, the expressions of low density lipoprotein receptor-related protein (LRP)5, LRP6, disheveled (DVL)2 and DVL3 were significantly up-regulated, while AXIN expression was down-regulated by EGCG, and the phosphorylation of glycogen synthase kinase 3β was increased. These results suggest that EGCG activates the WNT/β-catenin pathway, resulting in the up-regulation of β-catenin, which down-regulates the major genes of the adipogenesis pathway. Taken together, our findings clearly show that the anti-adipogenic effects of EGCG are, at least partially, dependent on the WNT/β-catenin pathway.

WNT/β-catenin pathway is modulated in asthma patients and LPS-stimulated RAW264.7 macrophage cell line

In the present study, we investigated the possibility that the WNT/β-catenin pathway plays a role in inflammatory responses both in an human inflammatory condition and in an in vitro inflammation model. First, we analyzed gene expression patterns of the peripheral blood cells from asthma patients compared with those from normal subjects using microarray analyses. We found that intracellular signaling molecules of the WNT/β-catenin pathway were significantly changed in asthma patients compared with the levels in the controls. Next, we determined whether major components of the WNT/β-catenin pathway were involved in the lipopolysaccharide (LPS)-induced inflammatory response of the RAW264.7 macrophage cell line. Among the members of WNT/β-catenin pathway, the protein levels of low-density lipoprotein receptor-related protein (LRP) 6, dishevelled (DVL) 2, and AXIN1, which were measured using western blotting, did not significantly change in the presence of LPS. In contrast, the LPS induced a rapid phosphorylation of glycogen synthase kinase (GSK) 3β and accumulation of β-catenin protein. It was found that β-catenin plays a significant role in the LPS-induced inflammatory response through the performance of small interfering RNA (siRNA) transfection experiments. The mRNA level of IL-6 was significantly elevated in β-catenin siRNA-transfected cells compared with that in control siRNA-transfected cells after LPS treatment. Furthermore, nuclear factor-κB (NF-κB) activity was also significantly increased in β-catenin siRNA-transfected cells compared with the level seen in control siRNA-transfected cells. Taken together, these results suggest that β-catenin plays a role as a negative regulator, preventing the overproduction of inflammatory cytokines such as IL-6 in LPS-induced inflammatory responses.

Anti-adipogenic effects of 1,25-dihydroxyvitamin D3 are mediated by the maintenance of the wingless-type MMTV integration site/β-catenin pathway

1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the active metabolite of vitamin D, was found to have anti-adipogenic activity, however, its mechanism of action has not been fully elucidated. In this study, 3T3-L1 preadipocytes were differentiated in the presence and absence of 1,25(OH)2D3, and the expression of the genes and proteins of the wingless-type MMTV integration site (WNT)/β-catenin pathway were analyzed. While the expression of the members of the WNT/β-catenin pathway were significantly downregulated during the adipogenesis of untreated 3T3-L1 cells, 1,25(OH)2D3 treatment was found to maintain the WNT/β-catenin pathway. Among the members of the WNT/β-catenin pathway, the levels of WNT10B and disheveled (DVL)2 as well as the phosphorylation of glycogen synthase kinase (GSK)3β were maintained by 1,25(OH)2D3 treatment. The levels of nuclear β-catenin, which were downregulated during adipogenesis, were also maintained by 1,25(OH)2D3 treatment. The results of this study suggested that the anti-adipogenic effect of 1,25(OH)2D3 was mediated by the maintenance of the WNT/β-catenin pathway, which was normally downregulated during adipogenesis.

WNT/β-catenin pathway mediates the anti-adipogenic effect of platycodin D, a natural compound found in Platycodon grandiflorum

AIMS This study was conducted to suggest the role of WNT/β-catenin pathway in the anti-adipogenic effect of platycodin D, a natural compound found in Platycodon grandiflorum. MAIN METHODS Gene knockdown experiments using small interfering RNA (siRNA) transfection were conducted to elucidate crucial role of β-catenin in the anti-adipogenic effects of platycodin D. Real-Time PCR and Western blot were used to analyze the expression levels of mRNAs and proteins in the WNT/β-catenin pathway. KEY FINDINGS During the adipocyte differentiation of 3 T3-L1 cells, members of the WNT/β-catenin pathway were normally down-regulated, whereas platycodin D significantly reinstated the WNT/β-catenin pathway. The mRNA and protein expressions of disheveled (DVL) 2, which stabilize β-catenin, were increased by platycodin D treatment, but the protein level of AXIN, which induces the degradation of β-catenin, was decreased in platycodin D-treated cells. The nuclear level of β-catenin was normally down-regulated during adipogenesis, but platycodin D treatment led to the accumulation of β-catenin in the nucleus which resulted in the up-regulation of its target genes, cyclin D (CCND) 1 and peroxisome proliferator-activated receptor gamma (PPAR)γ. The anti-adipogenic effects of platycodin D were significantly attenuated in β-catenin siRNA-transfected cells compared with those of control siRNA-transfected cells. β-catenin siRNA transfection significantly recovered the levels of PPARγ, CCAAT/enhancer binding protein (C/EBP)α and fatty acid binding protein (FABP)4 as well as intracellular lipid droplet formation, all of which were reduced by platycodin D treatment. SIGNIFICANCE WNT/β-catenin pathway can be used as a therapeutic target of natural compounds for the regulation of adipogenesis.

Shikonin inhibits adipogenesis by modulation of the WNT/β-catenin pathway

AIM Our previous study showed for the first time that shikonin, a natural compound isolated from Lithospermun erythrorhizon Sieb. Et Zucc, inhibits adipogenesis and fat accumulation. This study was conducted to investigate the molecular mechanism of the anti-adipogenic effects of shikonin. MAIN METHODS Gene knockdown experiments using small interfering RNA (siRNA) transfection were conducted to elucidate the crucial role of β-catenin in the anti-adipogenic effects of shikonin. KEY FINDINGS Shikonin prevented the down-regulation of β-catenin and increased the level of its transcriptional product, cyclin D1, during adipogenesis of 3T3-L1 cells, preadipocytes originally derived from mouse embryo. β-catenin was a crucial mediator of the anti-adipogenic effects of shikonin, as determined by siRNA-mediated knockdown. Shikonin-induced reductions of the major transcription factors of adipogenesis including peroxisome proliferator-activated receptor γ and CCAAT/enhancer binding protein α, and lipid metabolizing enzymes including fatty acid binding protein 4 and lipoprotein lipase, as well as intracellular fat accumulation, were all significantly recovered by siRNA-mediated knockdown of β-catenin. Among the genes located in the WNT/β-catenin pathway, the levels of WNT10B and DVL2 were significantly up-regulated, whereas the level of AXIN was down-regulated by shikonin treatment. SIGNIFICANCE This study clearly shows that shikonin inhibits adipogenesis by the modulation of WNT/β-catenin pathway in vitro, and also suggests that WNT/β-catenin pathway can be used as a therapeutic target for obesity and related diseases using a natural compound like shikonin, even though the in vivo effects of shikonin and its clinical significance remain to be elucidated.

β-Catenin mediates the anti-adipogenic effect of baicalin

beta-Catenin reportedly inhibits adipogenesis through the down-regulations of peroxisome proliferator-activated receptor (PPAR)gamma and CCAAT/enhancer binding protein (C/EBP)alpha. We report that baicalin, a natural flavonoid compound, inhibits adipogenesis by modulating beta-Catenin. During 3T3-L1 cell adipogenesis, beta-Catenin was down-regulated, but baicalin treatment maintained beta-Catenin expression. Anti-adipogenic effects of baicalin were significantly attenuated by beta-Catenin siRNA transfection. beta-Catenin siRNA rescued the reduced expressions of PPARgamma, C/EBPalpha, fatty acid binding protein 4 and lipoprotein lipase by baicalin. Furthermore, baicalin modulated members of the WNT/beta-Catenin pathway by maintaining the expressions of low-density lipoprotein receptor-related protein 6, disheveled (DVL)2 and DVL3. These findings suggest that beta-Catenin mediates the anti-adipogenic effects of baicalin.

Copy number variation of CCL3L1 influences asthma risk by modulating IL-10 expression.

BACKGROUND Copy number of Chemokine ligand 3-like 1 (CCL3L1) is associated with various immune disorders. This study was conducted to assess the role of CCL3L1 in asthma by both association analyses of human subjects and in vitro functional analyses. METHODS We analyzed the copy number of the CCL3L1 gene in 533 Korean subjects (372 controls and 161 asthma patients) by real-time PCR, and investigated the effect of recombinant CCL3L1 protein on THP-1 human monocytic cells that were stimulated with house dust mite extract. RESULTS The mean copy number of CCL3L1 in asthma subjects was significantly lower than that of control subjects (3.18 vs. 3.75, p=0.001). A low copy number of ≤1 was significantly associated with increased asthma risk with an odds ratio of 2.47, and a high copy number of ≥5 was associated with decreased asthma risk with an odds ratio of 0.40. Subjects with ≤1 copy of CCL3L1 had significantly lower mRNA levels of CCL3L1 in peripheral blood cells, and significantly higher serum IgE levels (p<0.05). In the house dust mite extract-simulated THP-1 monocytic cells, CCL3L1 protein dose-dependently up-regulated the expression of IL-10, an anti-inflammatory cytokine. CONCLUSION Copy number of CCL3L1 may influence asthma risk by modulating IL-10 expression.

CD53, a suppressor of inflammatory cytokine production, is associated with population asthma risk via the functional promoter polymorphism -1560 C>T.

BACKGROUND In this study, the association of asthma with CD53, a member of the tetraspanin family, was assessed for the first time in a mechanism-based study. METHODS Genetic polymorphisms of CD53 were analyzed in 591 subjects and confirmed in a replication study of 1001 subjects. CD53 mRNA and protein levels were measured in peripheral blood leukocytes, and the effects of the promoter polymorphisms on nuclear factor binding were examined by electrophoretic mobility shift assay. Cellular functional studies were conducted by siRNA transfections. RESULTS Among tagging SNPs of CD53, the -1560 C>T in the promoter region was significantly associated with asthma risk. Compared with the CC genotype, the CT and TT genotypes were associated with a higher asthma risk, with odd ratios of 1.74 (P=0.009) and 2.03 (P=0.004), respectively. These findings were confirmed in the replication study with odd ratios of 1.355 (P=0.047) and 1.495 (P=0.039), respectively. The -1560 C>T promoter SNP had functional effects on nuclear protein binding as well as mRNA and protein expression levels in peripheral blood leukocytes. When CD53 was knocked down by siRNA in THP-1 human monocytic cells stimulated with house dust mite, the production of inflammatory cytokines as well as NFκB activity was significantly over-activated, suggesting that CD53 suppresses over-activation of inflammatory responses. CONCLUSIONS The -1560 C>T SNP is a functional promoter polymorphism that is significantly associated with population asthma risk, and is thought to act by directly modulating nuclear protein binding, thereby altering the expression of CD53, a suppressor of inflammatory cytokine production.

Beta-catenin promoter polymorphism is associated with asthma risk in Korean subjects.

OBJECTIVES The effects of β-catenin promoter haplotypes on its mRNA expression levels and asthma risks were investigated in Korean subjects. DESIGN AND METHODS The genotype analyses were conducted by a Taqman method for 684 Korean subjects, 400 controls and 284 with asthma. Measurement of mRNA expression levels in peripheral blood nucleated cells were conducted on subjects whose buffy coat fractions were available (n=185). Logistic regression analyses were conducted to test the associations of the β-catenin promoter haplotypes with asthma risks. RESULTS Four SNPs, -10,288C>T (rs7630377), -6,426C>G (rs9859392), -4,361G>C (rs9870255), and -765G>A (rs3864004), were identified in the promoter region of the β-catenin gene, and three common haplotypes were constructed from them. Haplotype ht1[CCGG] was associated with decreased β-catenin mRNA expression levels and a lower asthma risk with an odds ratio of 0.53, while ht2[TGCA] was associated with increased mRNA expression levels and a higher asthma risk with an odds ratio of 2.34. Ht3[TCGG] had no significant effects on both. CONCLUSIONS Our findings show that β-catenin promoter polymorphism affects its mRNA expression levels, and also is significantly associated with the asthma risk of Korean subjects.