Anti-Melanogenic and Anti-Inflammatory Effects of 2'-Hydroxy-4',6'-dimethoxychalcone in B16F10 and RAW264.7 Cells.

Chalcone is a type of flavonoid compound that is widely biosynthesized in plants. Studies have shown that consuming flavonoids from fruits and vegetables or applying individual ingredients reduces the risk of skin disease. However, the effects of chalcone on melanogenesis and inflammation have not been fully investigated. The aim of this study was to evaluate the anti-melanogenic and anti-inflammatory effects of 2'-hydroxy-3,4'-dimethoxychalcone (3,4'-DMC), 2'-hydroxy-4,4'-dimethoxychalcone (4,4'-DMC), 2'-hydroxy-3',4'-dimethoxychalcone (3',4'-DMC), and 2'-hydroxy-4',6'-dimethoxychalcone (4',6'-DMC). Among the derivatives of 2'-hydroxy-4'-methoxychalcone, 4',6'-DMC demonstrated the most potent melanogenesis-inhibitory and anti-inflammatory effects. As evidenced by various biological assays, 4',6'-DMC showed no cytotoxicity and notably decreased the expression of tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2 enzymes. Furthermore, it reduced cellular melanin content and intracellular tyrosinase activity in B16F10 melanoma cells by downregulating microphthalmia-associated transcription factor (MITF), cAMP-dependent protein kinase (PKA), cAMP response element-binding protein (CREB), p38, c-Jun N-terminal kinase (JNK), ß-catenin, glycogen synthase kinase-3ß (GSK3ß), and protein kinase B (AKT) proteins, while upregulating extracellular signal-regulated kinase (ERK) and p-ß-catenin. Additionally, treatment with 4',6'-DMC significantly mitigated the lipopolysaccharide (LPS)-induced expression of NO, PGE2, inflammatory cytokines, COX-2, and iNOS proteins. Overall, 4',6'-DMC treatment notably alleviated LPS-induced damage by reducing nuclear factor kappa B (NF-κB), p38, JNK protein levels, and NF-kB/p65 nuclear translocation. Finally, the topical applicability of 4',6'-DMC was evaluated in a preliminary human skin irritation test and no adverse effects were found. These findings suggest that 4',6'-DMC may offer new possibilities for use as functional ingredients in cosmeceuticals and ointments.

The Effects of 2'-Hydroxy-3,6'-Dimethoxychalcone on Melanogenesis and Inflammation.

In this study, we demonstrated that 2'-hydroxy-3,6'-dimethoxychalcone (3,6'-DMC) alleviated α-MSH-induced melanogenesis and lipopolysaccharides (LPS)-induced inflammation in mouse B16F10 and RAW 264.7 cells. In vitro analysis results showed that the melanin content and intracellular tyrosinase activity were significantly decreased by 3,6'-DMC, without cytotoxicity, via decreases in tyrosinase and the tyrosinase-related protein 1 (TRP-1) and TRP-2 melanogenic proteins, as well as the downregulation of microphthalmia-associated transcription factor (MITF) expression through the upregulation of the phosphorylation of extracellular-signal-regulated kinase (ERK), phosphoinositide 3-kinase (PI3K)/Akt, and glycogen synthase kinase-3ß (GSK-3ß)/catenin, and downregulation of the phosphorylation of p38, c-Jun N-terminal kinase (JNK), and protein kinase A (PKA). Furthermore, we investigated the effect of 3,6'-DMC on macrophage RAW264.7 cells with LPS stimulation. 3,6'-DMC significantly inhibited LPS-stimulated nitric oxide production. 3,6'-DMC also suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 on the protein level. In addition, 3,6'-DMC decreased the production of the tumor necrosis factor-α and interleukin-6. Successively, our mechanistic studies revealed that 3,6'-DMC also suppressed the LPS-induced phosphorylation of the inhibitor of IκBα, p38MAPK, ERK, and JNK. The Western blot assay results showed that 3,6'-DMC suppresses LPS-induced p65 translocation from cytosol to the nucleus. Finally, the topical applicability of 3,6'-DMC was tested through primary skin irritation, and it was found that 3,6'-DMC, at 5 and 10 µM concentrations, did not cause any adverse effects. Therefore, 3,6'-DMC may provide a potential candidate for preventing and treating melanogenic and inflammatory skin diseases.

HM15912, a Novel Long-Acting Glucagon-Like Peptide-2 Analog, Improves Intestinal Growth and Absorption Capacity in a Male Rat Model of Short Bowel Syndrome.

Extensive bowel resection caused by various diseases that affect the intestines, such as Crohn's disease, volvulus, and cancer, leads to short bowel syndrome (SBS). Teduglutide is the only approved glucagon-like peptide-2 (GLP-2) drug for SBS; however, it requires daily administration. A novel GLP-2 analog with a prolonged duration of action to reduce dosing frequency and promote a greater efficacy may provide patients with a better quality of life. In the present study, the sustained exposure of HM15912 was characterized in normal male rats. The efficacy of HM15912 on intestinal growth and absorption capacity was also evaluated in normal male mice, rats, and SBS rats. HM15912 exhibited a remarkably extended half-life (42.3 hours) compared with teduglutide (0.6 hours) in rats. Despite somewhat lower in vitro potency on GLP-2 receptor than human GLP-2 or teduglutide, this longer-lasting mode of action promotes HM15912 to be more effective in terms of small intestinal growth than existing GLP-2 analogs even with a less frequent dosing interval of as little as once a week in rodents, including SBS rats. Furthermore, the small intestinal weight was approximately doubled, and the D-xylose absorption was significantly increased after pre-treatment of existing GLP-2 analogs on the market or under clinical development followed by HM15912 in rodents. These results indicate that HM15912 possesses a significant small bowel trophic effect driven by continuously increased exposure, supporting that HM15912 may be a novel treatment option with greater efficacy and the longest dosing interval among existing GLP-2 analogs for SBS with intestinal failure. SIGNIFICANCE STATEMENT: HM15912, a novel long-acting glucagon-like peptide-2 (GLP-2) analog, has a significant small bowel hypertrophic effect in rodents with a reduced frequency of administration compared to the existing GLP-2 analogs on the market or currently under clinical development. This study supports the possibility that HM15912 could be administered much less frequently than other long-acting GLP-2 analogs for patients with short bowel syndrome.

Porcine Circovirus (PCV) Genotype 2d-Based Virus-like Particles (VLPs) Induced Broad Cross-Neutralizing Antibodies against Diverse Genotypes and Provided Protection in Dual-Challenge Infection of a PCV2d Virus and a Type 1 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV).

As PCV2d infection has been continuously reported in swine farms in which pigs were vaccinated with PCV2a- or 2d-based vaccines, we attempted to develop a novel vaccine using a PCV2d-based capsid to enhance its protective efficacy. In this study, recombinant virus-like particles (VLPs) of rPCV2a, rPCV2b and rPCV2d were synthesized from the capsid proteins of PCV2a, PCV2b and PCV2d field isolates, respectively. A cross-neutralization assay between the VLPs induced antisera and the field isolates demonstrated the broad cross-neutralizing activities of the rPCV2d-induced antisera. Then, the protective efficacy of rPCV2d as a vaccine candidate was investigated in commercial pigs by rPCV2d vaccination and a single- or dual-challenge infection using a PCV2d strain and a type 1 PRRSV strain. High levels of anti-PCV2d IgG and neutralizing antibodies were induced 3 weeks after vaccination. After the challenge infection, the average ADWG values of the vaccinated group were higher than those of the unvaccinated group. None or a significantly low amount of (p < 0.05) reduced PCV2 genomic DNA was found in the blood, saliva and tissues of the vaccinated pigs, when compared to the unvaccinated group. Moreover, macroscopic and microscopic lesions in the tissues were significantly (p < 0.05) reduced in the vaccinated groups. This study therefore suggests that rPCV2d may be highly useful for the control of diverse field genotypes.

Preparation, characterization, and pharmacological study of a novel long-acting FGF21 with a potential therapeutic effect in obesity.

FGF21 (Fibroblast Growth Factor 21), which is expressed in the liver, adipose tissue, and pancreas, has been widely known as a therapeutic candidate for metabolic diseases. Though FGF21 is crucial to glucose, lipid, and energy homeostasis, it is not straightforward to develop a new drug with FGF21 due to its short half-life in serum. Here, we derived a novel long-acting FGF21 (LAPS-FGF21), which is chemically conjugated to the human IgG4 Fc fragment for longer half-life in serum. The recombinant human IgG4 Fc fragment and FGF21 were prepared by the refolding of inclusion body and periplasmic expression in Escherichia coli overexpression systems, respectively. The efficacy study of LAPS-FGF21 in a Diet-Induced Obesity (DIO) mouse model revealed that LAPS-FGF21 reduced body weight effectively accompanied by improved glucose tolerance in a dose-dependent manner. The administration of LAPS-FGF21 also improved the blood profiles with a significant reduction in cholesterol and triglyceride levels. Additionally, the pharmacokinetic (PK) studies of LAPS-FGF21 using normal ICR mice demonstrated that the half-life of LAPS-FGF21 was approximately 64-fold longer than FGF21. Taken together, the LAPS-FGF21 could be a feasible drug candidate with excellent bodyweight loss efficacy and longer dosing interval by half-life increase in serum.

HM10660A, a long-acting hIFN-α-2b, is a potent candidate for the treatment of hepatitis C through an enhanced biological half-life.

Interferon-α (IFN-α) has been widely used for the treatment of infections due to the hepatitis C virus (HCV). Because of the short half-life of IFN-α in serum, it must be administered three times per week. To increase the half-life of IFN-α, the immunoglobulin G4 (IgG4) Fc fragment (HMC001) was conjugated with human IFN-α-2b to develop a long-acting IFN-α-2b, HM10660A. An analysis of the antiviral efficacy of HM10660A in a human hepatocyte-engrafted mouse model found that HM10660A reduced serum HCV titers more effectively than a commercially available peginterferon α-2a (PEGASYS®) and IFN-α-2b. Pharmacokinetic (PK) and pharmacodynamic (PD) studies of HM10660A using monkeys demonstrated that the half-life of HM10660A was approximately 2-fold longer than commercially available peginterferon α-2a, which is approved for a once-weekly regimen. Moreover, the IFN-mediated induction profiles of neopterin and 2', 5'-oligoadenylate synthase (OAS) in normal cynomolgus monkeys indicated that HM10660A had enhanced antiviral activity and a prolonged duration of action compared with peginterferon α-2a. Considering the improved PK and PD properties, HM10660A can most likely be dosed every two or four weeks, providing superior antiviral efficacy and convenience for patients with HCV.

Anti-adipogenic activity of berberine is not mediated by the WNT/ß-catenin pathway.

Adipogenesis is a differentiation process from preadipocytes to adipocytes, accompanied by the inductions of adipogenic transcription factors and lipid metabolizing enzymes. Among cellular pathways regulating adipogenesis, the WNT/ß-catenin pathway is well-known as a suppressor of adipogenesis. Berberine (BBR) is an isoquinoline alkaloid component of the medicinal plants including Coptis chinensis and Coptis japonica with diverse biological activities. This study was conducted to elucidate whether the anti-adipogenic effect of BBR is mediated by the WNT/ß-catenin pathway. The results of the present study confirmed that BBR efficiently inhibited adipogenesis of 3T3-L1 cells. However, the anti-adipogenic effects of BBR were not accompanied by the modulations of the WNT/ß-catenin pathway members including WNT10B, LRP6, DVL2, GSK3ß and ß-catenin. When ß-catenin was knocked down by its siRNA transfection, the anti-adipogenic effects of BBR including the expression of adipogenic transcription factors and lipid metabolizing enzymes as well as the intracellular fat accumulation were not affected at all. The results of this study showed that the anti-adipogenic effect of BBR is not mediated by the WNT/ß-catenin pathway.

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.

The anti-adipogenic effects of (-)epigallocatechin gallate are dependent on the WNT/ß-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/ß-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 ß-catenin. Knockdown of ß-catenin using small interfering (si) RNA attenuated the inhibitory effects of EGCG on intracellular lipid accumulation. ß-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 ß-catenin siRNA transfection. In addition, we found that EGCG efficiently up-regulates the WNT/ß-catenin pathway. Among the members of the WNT/ß-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 3ß was increased. These results suggest that EGCG activates the WNT/ß-catenin pathway, resulting in the up-regulation of ß-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/ß-catenin pathway.

Anti-adipogenic effects of 1,25-dihydroxyvitamin D3 are mediated by the maintenance of the wingless-type MMTV integration site/ß-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)/ß-catenin pathway were analyzed. While the expression of the members of the WNT/ß-catenin pathway were significantly downregulated during the adipogenesis of untreated 3T3-L1 cells, 1,25(OH)2D3 treatment was found to maintain the WNT/ß-catenin pathway. Among the members of the WNT/ß-catenin pathway, the levels of WNT10B and disheveled (DVL)2 as well as the phosphorylation of glycogen synthase kinase (GSK)3ß were maintained by 1,25(OH)2D3 treatment. The levels of nuclear ß-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/ß-catenin pathway, which was normally downregulated during adipogenesis.

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

OBJECTIVES: The effects of ß-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 ß-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 ß-catenin gene, and three common haplotypes were constructed from them. Haplotype ht1[CCGG] was associated with decreased ß-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 ß-catenin promoter polymorphism affects its mRNA expression levels, and also is significantly associated with the asthma risk of Korean subjects.

WNT/ß-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/ß-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/ß-catenin pathway were significantly changed in asthma patients compared with the levels in the controls. Next, we determined whether major components of the WNT/ß-catenin pathway were involved in the lipopolysaccharide (LPS)-induced inflammatory response of the RAW264.7 macrophage cell line. Among the members of WNT/ß-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) 3ß and accumulation of ß-catenin protein. It was found that ß-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 ß-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 ß-catenin siRNA-transfected cells compared with the level seen in control siRNA-transfected cells. Taken together, these results suggest that ß-catenin plays a role as a negative regulator, preventing the overproduction of inflammatory cytokines such as IL-6 in LPS-induced inflammatory responses.

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.

WNT/ß-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/ß-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 ß-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/ß-catenin pathway. KEY FINDINGS: During the adipocyte differentiation of 3 T3-L1 cells, members of the WNT/ß-catenin pathway were normally down-regulated, whereas platycodin D significantly reinstated the WNT/ß-catenin pathway. The mRNA and protein expressions of disheveled (DVL) 2, which stabilize ß-catenin, were increased by platycodin D treatment, but the protein level of AXIN, which induces the degradation of ß-catenin, was decreased in platycodin D-treated cells. The nuclear level of ß-catenin was normally down-regulated during adipogenesis, but platycodin D treatment led to the accumulation of ß-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 ß-catenin siRNA-transfected cells compared with those of control siRNA-transfected cells. ß-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/ß-catenin pathway can be used as a therapeutic target of natural compounds for the regulation of adipogenesis.

AICAR, an activator of AMPK, inhibits adipogenesis via the WNT/ß-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-ß-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 ß-catenin and other members of the WNT/ß-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 ß-catenin expression and its nuclear accumulation. Transfection of ß-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 ß-catenin siRNA-transfected cells. Among the members of the WNT/ß-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/ß-catenin pathway.

Shikonin inhibits adipogenesis by modulation of the WNT/ß-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 ß-catenin in the anti-adipogenic effects of shikonin. KEY FINDINGS: Shikonin prevented the down-regulation of ß-catenin and increased the level of its transcriptional product, cyclin D1, during adipogenesis of 3T3-L1 cells, preadipocytes originally derived from mouse embryo. ß-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 ß-catenin. Among the genes located in the WNT/ß-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/ß-catenin pathway in vitro, and also suggests that WNT/ß-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.

The WNT/ß-catenin pathway mediates the anti-adipogenic mechanism of SH21B, a traditional herbal medicine for the treatment of obesity.

AIM OF THE STUDY: This study was conducted to elucidate the molecular mechanisms of SH21B, a traditional Korean herbal medicine commonly used for the treatment of obesity. MATERIALS AND METHODS: 3T3-L1 preadipocytes were differentiated into adipocytes in the presence or absence of SH21B. Changes in mRNA or protein levels were analyzed using microarray, real-time polymerase chain reaction and western blotting analyses. Small interference (si)RNA transfection experiments were conducted to elucidate the essential role of ß-catenin. RESULTS: Microarray analyses showed that components of the WNT/ß-catenin pathway including ß-catenin, cyclin D1 and dishevelled 2 were up-regulated more than two-fold as a result of SH21B treatment during adipogenesis, which were confirmed by real-time PCR and western blotting. Modulation of the WNT/ß-catenin pathway by SH21B resulted in the nuclear accumulation of ß-catenin. Both intracellular lipid droplet formation and expressions of adipogenic genes including PPARγ, C/EBPα, FABP4 and LPL, which were inhibited by SH21B, were significantly recovered by ß-catenin siRNA transfection. CONCLUSIONS: SH21B modulates components of the WNT/ß-catenin pathway during adipogenesis, and ß-catenin plays a crucial role in the anti-adipogenic mechanism of SH21B.

Beta-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.