Leptin promotes methionine adenosyltransferase 2A expression in hepatic stellate cells by the downregulation of E2F-4 via the ß-catenin pathway.

Most obese patients develop hyperleptinaemia. Leptin, mainly produced by adipocytes, demonstrates a promotional role in liver fibrosis. Hepatic stellate cell (HSC) activation, a key step in liver fibrogenesis, requires global reprogramming of gene expression. The remodeling of DNA methylation is a mechanism of the epigenetic regulation of gene expression. The biosynthesis of S-adenosylmethionine, a principle biological methyl donor, is catalyzed by methionine adenosyltransferase (MAT) such as MATⅡ which has been shown to promote HSC activation in vitro. This study was mainly aimed to determine the effect of leptin on MAT2A expression (the catalytic subunit of MATⅡ) in HSCs. Results showed that MAT2A knockdown reduced leptin-induced HSC activation and liver fibrosis in the leptin-deficient mouse model. Leptin promoted MAT2A expression in HSCs and increased MAT2A promoter activity. The axis of the ß-catenin pathway/E2F-4 mediated the effect of leptin on MAT2A expression. Leptin-induced ß-catenin signaling reduced E2F-4 expression and thus abated E2F-4 binding to MAT2A promoter at a site around -2779 bp, leading to an increase in the MAT2A promoter activity. These data might shed more light on the mechanisms responsible for liver fibrogenesis in obese patients with hyperleptinaemia.

Leptin suppresses microRNA-122 promoter activity by phosphorylation of foxO1 in hepatic stellate cell contributing to leptin promotion of mouse liver fibrosis.

Adipocytokine leptin promotes hepatic stellate cell (HSC) activation (a key step in liver fibrogenesis) and liver fibrosis. microRNA-122 (miR-122) is the most abundant liver-specific miRNA and was demonstrated to inhibit liver fibrosis and reduced HSC proliferation. Our previous study revealed that leptin reduced miR-122 level in HSCs. This study was aimed to investigate whether leptin affected miR-122 promoter and the underlying mechanisms in HSCs. Results showed that leptin inhibited miR-122 promoter activity. Forkhead box protein O1(FoxO1) bound to miR-122 promoter at a site around - 56 and thus promoted miR-122 promoter activity, which could be suppressed by leptin-induced phosphorylation of FoxO1 at serine 256. The PI3K/Akt signaling pathway was involved in leptin-induced phosphorylation of FoxO1 and the effect of leptin on miR-122 expression. Furthermore, FoxO1 increased miR-122 and pri-miR-122 (primary miR-122) levels in HSCs in vivo, and reduced leptin-induced HSC activation and liver fibrosis in ob/ob mouse (leptin deficient) model. In conclusion, leptin suppressed microRNA-122 expression by PI3K/Akt/foxO1 axis in HSCs. These results have potential implications for clarifying the mechanisms for liver fibrogenesis in obese patients with hyperleptinaemia.

(R)-panaxadiol by whole cells of filamentous fungus Absidia coerulea AS 3.3382.

The microbial transformation of 20(R)-panaxadiol (PD) by the fungus Absidia coerulea AS 3.3382 afforded three new and three known metabolites. The structures of the metabolites were characterized as 3-oxo-20(R)-panaxadiol (1), 3-oxo-7ß- hydroxyl-20(R)-panaxadiol (2), 3-oxo-22ß-hydroxyl-20(R)-panaxadiol (3), 3-oxo- 7ß,22ß-dihydroxyl-20(R)-panaxadiol (4), 3-oxo-7ß,24ß-dihydroxyl-20(R)-panaxadiol (5), and 3-oxo-7ß,24α-dihydroxyl-20(R)-panaxadiol (6). Among them, 2-4 were new compounds. In addition, compounds 3 and 4 exhibited significant anti-hepatic fibrosis activity.

GATA binding protein 2 mediates leptin inhibition of PPARγ1 expression in hepatic stellate cells and contributes to hepatic stellate cell activation.

Hepatic stellate cell (HSC) activation is a crucial step in the development of liver fibrosis. Peroxisome-proliferator activated receptor γ (PPARγ) exerts a key role in the inhibition of HSC activation. Leptin reduces PPARγ expression in HSCs and plays a unique role in promoting liver fibrosis. The present studies aimed to investigate the mechanisms underlying leptin regulation of PPARγ1 (a major subtype of PPARγ) in HSCs in vivo and in vitro. Results revealed a leptin response region in mouse PPARγ1 promoter and indicated that the region included a GATA binding protein binding site around position -2323. GATA binding protein-2 (GATA-2) could bind to the site and inhibit PPARγ1 promoter activity in HSCs. Leptin induced GATA-2 expression in HSCs in vitro and in vivo. GATA-2 mediated leptin inhibition of PPARγ1 expression by its binding site in PPARγ1 promoter in HSCs and GATA-2 promoted HSC activation. Leptin upregulated GATA-2 expression through ß-catenin and sonic hedgehog pathways in HSCs. Leptin-induced increase in GATA-2 was accompanied by the decrease in PPARγ expression in HSCs and by the increase in the activated HSC number and liver fibrosis in vivo. Our data might suggest a possible new explanation for the promotion effect of leptin on liver fibrogenesis.

Biotransformation of 20(S)-protopanaxatriol by Mucor racemosus and the anti-cancer activities of some products.

OBJECTIVE: To produce new derivatives of 20(S)-protopanaxatriol by fungal biotransformation. RESULT: Biotransformation of 20(S)-protopanaxatriol (1) by Mucor racemosus AS 3.205 afforded six products. Their structures were elucidated on the basis of extensive spectroscopic analyses. M. racemosus could selectively catalyze dehydrogenation at C-12 and further hydroxylation at C-7, C-11, and C-15, as well as rearrangement of double bond at C-26. Two of these new compounds exhibited potent inhibitory activity against SH-SY5Y and HepG2 cell lines. CONCLUSION: Biotransformation by M. racemosus AS 3.205 was an effective approach to produce new derivatives of 20(S)-protopanaxatriol.

Microbial transformation of 20(S)-protopanaxatriol by Absidia corymbifera and their cytotoxic activities against two human prostate cancer cell lines.

Seven hydroxylates of 20(S)-protopanaxatriol (1) transformed by Absidia corymbifera AS 3.3387 were isolated and identified by spectral methods including 2D-NMR. Among them, 7ß-hydroxyl-20(S)-protopanaxatriol (2), 7α-hydroxyl-20(S)-protopanaxatriol (3), and 7ß, 15α-dihydroxyl-20(S)-protopanaxatriol (7) are new compounds. The metabolites 2, 6, 7, and 8 showed the more potent inhibitory effects against DU-145 and PC-3 cell lines than the substrate.

p38 mitogen-activated protein kinase and liver X receptor-α mediate the leptin effect on sterol regulatory element binding protein-1c expression in hepatic stellate cells.

Leptin, a key hormone in regulating energy homeostasis, is mainly produced by adipocytes. Cogent evidence indicates a unique role of leptin in the promotion of liver fibrosis. Hepatic stellate cell (HSC) activation is a pivotal step in the process of liver fibrosis. Sterol regulatory element binding protein (SREBP)-1c, a critical transcription factor for lipid synthesis and adipocyte differentiation, functions as a key transcription factor in inhibition of HSC activation. SREBP-1c is highly expressed in quiescent HSCs and downregulated upon HSC activation. The aim of this study is to examine the effect of leptin on SREBP-1c gene expression in HSCs in vitro and in vivo and elucidate the underlying mechanisms. The results of the present study demonstrated that leptin strongly inhibited SREBP-1c expression in HSCs in vivo and in vitro. p38 MAPK was involved in leptin regulation of SREBP-1c expression in cultured HSCs. Leptin-induced activation of p38 MAPK led to the decreases in liver X receptor (LXR)-α protein level, activity and its binding to the SREBP-1c promoter, which caused the downregulation of SREBP-1c expression. Moreover, leptin inhibition of SREBP-1c expression via p38 MAPK increased the expression of alpha1(I) collagen in HSCs. Our results might provide new insights into the mechanisms of the unique role of leptin in the development of liver fibrosis and might have potential implications for clarifying the molecular mechanisms underlying liver fibrosis in diseases in which circulating leptin levels are elevated such as nonalcoholic steatohepatitis, type 2 diabetes mellitus and alcoholic cirrhosis.

ß-Catenin pathway is required for TGF-ß1 inhibition of PPARγ expression in cultured hepatic stellate cells.

Hepatic stellate cell (HSC) activation is a key step in process of liver fibrosis. Transforming growth factor-ß1 (TGF-ß1) is the most powerful mediator of HSC activation and plays a central role in liver fibrosis. Peroxisome proliferator-activated receptor-γ (PPARγ) is an important regulator of adipocyte differentiation and has been proposed as a crucial factor for inhibition of HSC activation. The effect of TGF-ß1 on PPARγ in HSCs is largely unknown. This study is aimed to examine whether TGF-ß1 can influence PPARγ expression, focusing on the role of ß-catenin pathway, a key pathway linked to adipogenesis, in TGF-ß1 regulation of PPARγ in cultured HSCs. Our results demonstrated that TGF-ß1 evidently inhibited PPARγ expression and activity in cultured HSCs, which were mediated through ß-catenin pathway. TGF-ß1 promoted ß-catenin expression and also increased the stability of ß-catenin protein through ERK1/2/glycogen synthase kinase-3ß (GSK-3ß) axis in cultured HSCs. Moreover, TGF-ß1 inhibition of PPARγ expression by ß-catenin pathway caused the increase in alpha1(1) collagen and tissue inhibitor of matrix metalloproteinase expression. These results indicated for the first time that TGF-ß1 could down-regulate PPARγ expression through ß-catenin pathway and subsequently contributed to the increase in alpha1(1) collagen level in cultured HSCs.

[Curcumin inhibits iron overload-induced hepatocytic apoptosis and nuclear factor-κB activity].

OBJECTIVE: Iron is an essential micronutrient for human beings but its overload induces various diseases of liver, the main body storage site for iron, such as liver fibrosis. Curcumin is a natural polyphenol derived from turmeric and has been used widely. Its pharmacological action has attracted great attention in recent years. The apoptosis of rat cultured hepatocytes was induced by FeNTA (ferric nitrilotriacetate)-induced Iron overload. The present study was to examine the effect of curcumin at low concentrations on FeNTA-induced apoptosis of hepatocytes and elucidate the underlying mechanisms. METHODS: After the incubation of hepatocytes with 100 µmol/L FeNTA in the presence or absence of 1 - 10 µmmol/L of curcumin, a series of analyses were performed, including the analyses of hepatocytic apoptosis, the expressions of proteins relating with the regulations of cell apoptosis, caspase-3 activity, the production of reactive oxygen species (ROS) and nuclear factor NF-κB activity. RESULTS: Curcumin reduced the FeNTA-induced hepatocytic apoptosis by 46.65% and significantly down-regulated the protein levels of Bcl-2 and Bcl-XL. In contrast, it had no effect on the protein levels of Bax and Bad. The curcumin treatment reduced FeNTA-caused production of ROS and caspase-3 activity by 45.01% and 59.71% respectively. And the NF-κB activity was also inhibited. CONCLUSION: Curcumin at low concentrations reduces iron overload-caused hepatocytic apoptosis and NF-κB activity, the key regulatory transcription factor for the inflammation-related gene expression in cultured hepatocyte.

Type I interferons and TGF-ß cooperate to induce liver fibrosis during HIV-1 infection under antiretroviral therapy.

Liver diseases have become a major comorbidity health concern for people living with HIV-1 (PLWH) treated with combination antiretroviral therapy (cART). To investigate if HIV-1 infection and cART interact to lead to liver diseases, humanized mice reconstituted with progenitor cells from human fetal livers were infected with HIV-1 and treated with cART. We report here that chronic HIV-1 infection with cART induced hepatitis and liver fibrosis in humanized mice, associated with accumulation of M2-like macrophages (M2LMs), elevated TGF-ß, and IFN signaling in the liver. Interestingly, IFN-I and TGF-ß cooperatively activated human hepatic stellate cells (HepSCs) in vitro. Mechanistically, IFN-I enhanced TGF-ß-induced SMAD2/3 activation in HepSCs. Finally, blockade of IFN-I signaling reversed HIV/cART-induced liver diseases in humanized mice. Consistent with the findings in humanized mice with HIV-1 and cART, we detected elevated markers of liver injury, M2LMs, and of IFN signaling in blood specimens from PLWH compared with those of healthy individuals. These findings identify the IFN-I/M2LM/HepSC axis in HIV/cART-induced liver diseases and suggest that inhibiting IFN-I signaling or M2LM may provide a novel therapeutic strategy for treating HIV/cART-associated liver diseases in PLWH treated with antiretroviral therapy.

Microbial carbonylation and hydroxylation of 20(R)-panaxadiol by Aspergillus niger.

20(R)-panaxadiol (PD) was metabolised by the fungus Aspergillus niger AS 3.3926 to its C-3 carbonylated metabolite and five other hydroxylated metabolites (1-6). Their structures were elucidated as 3-oxo-20(R)-panaxadiol (1), 3-oxo-7ß-hydroxyl- 20(R)-panaxadiol (2), 3-oxo-7ß,23α-dihydroxyl-20(R)-panaxadiol (3), 3,12-dioxo- 7ß,23ß-dihydroxyl-20(R)-panaxadiol (4), 3-oxo-1α,7ß-dihydroxyl-20(R)-panaxadiol (5) and 3-oxo-7ß,15ß-dihydroxyl-20(R)-panaxadiol (6) by spectroscopic analysis. Among them, compounds 2-6 were new compounds. Pharmacological studies revealed that compound 6 exhibited significant anti-hepatic fibrosis activity.

[SGP polymorphism in cultivated naked barley from Qinghai-Tibet plateau in China and the relationship between SGPs and starch content].

Starch granule proteins (SGPs) are minor components bound with starch granule, which mutation may be related to starch properties. This study investigated the variation of SGPs in cultivated naked barley from Qinghai-Tibet Plateau in China for the first time, and the relationship between SGPs and starch content was preliminarily done. Ten major SGPs and 16 types of patterns were present in 66 cultivated naked varieties, indicating SGPs in cultivated naked barley from Qinghai-Tibet Plateau in China are polymorphic. SGPs in Tibet and Sichuan naked barley were greatly different and SGPs were specific to origin of site. Significance test analysis demonstrates SGPs described in this study except for SGP1 may be related with the variation of starch content in different naked barley.

Biotransformation of 20(R)-panaxatriol by Mucor racemosus and the anti-hepatic fibrosis activity of some products.

Biocatalysis of 20(R)-panaxatriol (PT) was performed by the fungus Mucor racemosus. Six metabolites (1-6) including five new compounds were obtained, and their structures were elucidated as 20(R),25-epoxy-12ß,24ß-dihydroxydammaran-3,6-dione (2), 20(R),25-epoxy-12ß,22ß-dihydroxydammaran-3,6-dione (3), 20(R),25-epoxy-23ß-hydroxydammaran-3,6,12-trione (4), 20(R),25-epoxy-12ß,23α- dihydroxydammaran-3,6-dione (5), and 20(R),25-epoxy-12ß-hydroxydammaran-3,6,23-trione (6) by spectroscopic analysis. Pharmacological studies revealed that compounds 2, 3 and 5 exhibited significant antihepatic fibrosis activity, while 4 and 6 showed cytotoxicity against HSC-T6 cells.

Leptin reduces microRNA-122 level in hepatic stellate cells in vitro and in vivo.

Obese patients, often accompanied by hyperleptinemia, are more likely to develop liver fibrosis. Leptin, an adipocyte-derived hormone, augments inflammatory in liver and promotes hepatic stellate cell (HSC) activation (a key step for liver fibrogenesis) and liver fibrosis. microRNA-122 (miR-122) is the most abundant liver-specific miRNA and can attenuate liver fibrosis. This study examined the effect of leptin on miR-122 level in HSCs in vivo and in vitro. Results demonstrated that leptin reduced the levels of both miR-122 (mature miR-122) and primary miR-122 (pri-miR-122). The effects of leptin on the levels of miR-122 and pri-miR-122 were through at least hedgehog pathway. Leptin-induced decrease in sterol regulatory element-binding protein-1c (SREBP-1c) has been shown to contribute to leptin-induced HSC activation. We revealed a mutual promotional effect between SREBP-1c and miR-122. Further experiments indicated that miR-122 inhibited leptin-induced liver fibrosis in leptin-deficient mouse model. These data have potential implications for clarifying the mechanisms of hepatic fibrogenesis associated with elevated leptin level in human such as obese patients.

Curcumin regulates peroxisome proliferator-activated receptor-γ coactivator-1α expression by AMPK pathway in hepatic stellate cells in vitro.

Curcumin exerts an inhibitory effect on hepatic stellate cell (HSC) activation, a key step for liver fibrogenesis, and on liver fibrosis by up-regulation of peroxisome proliferator-activated receptor-γ (PPARγ) expression. PPARγ plays a crucial role in suppression of HSC activation. Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) functions as a co-activator for PPARγ. Therefore, researches on the effect of curcumin on PGC-1α might contribute to understanding of the mechanisms underlying curcumin inhibition of HSC activation and liver fibrosis through PPARγ. The present study aimed to investigate the effect of curcumin on PGC-1α expression in HSCs in vitro and examine the underlying molecular mechanisms by western blot, reat-time PCR, and transfection. Our results showed that curcumin stimulation increased PGC-1α expression and the effects of curcumin on PGC-1α expression were correlated with the activation of adenosine monophosphate-activated protein kinase (AMPK). Curcumin increased superoxide dimutase-2 (SOD2) transcription and activity by AMPK/PGC-1α axis. Moreover, PGC-1α was demonstrated to inhibit α1(I) collagen (a marker for liver fibrosis) transcription in cultured HSCs. These results demonstrated the promotion effect of curcumin on PGC-1α expression through AMPK pathway, which led to the increases in PPARγ activity and in SOD-2 transcription and activity. These data might suggest a possible new explanation for the inhibitory effect of curcumin on HSC activation and on liver fibrogenesis.

Curcumin regulates delta-like homolog 1 expression in activated hepatic stellate cell.

Hepatic stellate cell activation is a key cellular event in the development of liver fibrosis. Recently, Delta-like homolog 1 (DLK1) protein level has been shown to increase in HSC activation and serve as a new contributor to HSC activation and liver fibrosis. Curcumin, a natural yellow polyphenol, possesses therapeutic roles in many diseases including liver fibrosis and has long been used in traditional medicine. The present study was aimed to elucidate the effect of curcumin on DLK1 expression in HSCs in vitro and in vivo, which is still unknown. Our results demonstrated that curcumin reduced DLK1 expression in culture-activated HSCs and in rat model of liver fibrosis. The inhibitory effect of curcumin on DLK1 expression may be mediated in part by interruption of Shh signaling pathway, which contributes to the promotion effect of curcumin on the expression of PPAR-gamma, a key factor in inhibiting HSC activation. Our results in this study may reveal a new mechanisms through which curcumin exerts its inhibitory effect on HSC activation and liver fibrosis.

Curcumin affects ß-catenin pathway in hepatic stellate cell in vitro and in vivo.

OBJECTIVES: Emerging evidence indicates that Wnt/ß-catenin pathway is linked to the fibrosis of different organs including liver fibrosis. ß-Catenin promotes hepatic stellate cells (HSCs) activation, a key event in the development of liver fibrosis, and has emerged as a novel mediator of fibrosis. Curcumin, a natural active ingredient derived from turmeric, possesses an inhibitory effect on liver fibrosis. This study is aimed to examine whether curcumin affects ß-catenin expression/activity in HSCs and explores the underlying mechanisms. METHODS: The researchers used Western blot, real-time PCR, transfection assay and electrophoretic mobility shift assay and employed cultured HSCs and rat model of liver injury. KEY FINDINGS: Results showed that curcumin could reduce ß-catenin protein level in HSCs in vitro and in vivo. Both ß-catenin transactivation activity and DNA-binding activity were suppressed by curcumin. Moreover, nuclear ß-catenin protein level was decreased by curcumin treatment. Further experiments suggested that delta-like homologue 1 contributed to curcumin inhibition of ß-catenin transactivation activity in cultured HSCs. CONCLUSIONS: Curcumin affects ß-catenin pathway in HSCs and might suggest a possible new explanation for the effects of curcumin on HSC activation and liver fibrosis.

Stat3 pathway correlates with the roles of leptin in mouse liver fibrosis and sterol regulatory element binding protein-1c expression of rat hepatic stellate cells.

Leptin, the adipocyte-derived hormone, plays an unique role in promoting liver fibrosis. Hepatic stellate cell (HSC) activation is the key step in liver fibrogenesis and sterol regulatory element binding protein-1c (SREBP-1c, a pivotal transcription factor for adipocyte differentiation) exerts a critical function in inhibition of HSC activation. Stat3 pathway is the main pathway induced by leptin and its role in liver fibrogenesis is controversial. Our previous results demonstrated the inhibitory effect of leptin on SREBP-1c expression in HSCs. The present study aimed to explore the role of Stat3 pathway in leptin-induced liver fibrogenesis in mouse model, focusing on examining the effect of leptin-induced Stat3 pathway on SREBP-1c expression in HSCs in vitro and in vivo. Results suggested that Stat3 pathway mediated the promotional role of leptin in liver fibrosis in mouse and was involved in leptin inhibition of SREBP-1c expression in HSCs. Leptin-induced Stat3 activation was, at least partially, ERK pathway-dependent in cultured HSCs and was correlated positively with ß-catenin activity and negatively with liver X receptor α expression and activity which influenced SREBP-1c expression in HSCs. The decrease in SREBP-1c expression by leptin-induced Stat3 pathway led to the increase in the marker for HSC activation and in α1(I) collagen expression in HSCs. In summary, the effect of leptin-induced Stat3 pathway on SREBP-1c expression in HSCs might contribute to the role of leptin in liver fibrosis in mouse, thus advancing understanding of the mechanisms of liver fibrogenesis associated with leptin.

The ß-catenin pathway contributes to the effects of leptin on SREBP-1c expression in rat hepatic stellate cells and liver fibrosis.

BACKGROUND AND PURPOSE: Liver fibrosis is commonly associated with obesity and most obese patients develop hyperleptinaemia. The adipocytokine leptin has a unique role in the development of liver fibrosis. Activation of hepatic stellate cells (HSCs) is a key step in hepatic fibrogenesis and sterol regulatory element-binding protein-1c (SREBP-1c) can inhibit HSC activation. We have shown that leptin strongly inhibits SREBP-1c expression in rat HSCs. Hence, we aimed to clarify whether the ß-catenin pathway, the crucial negative regulator of adipocyte differentiation, mediates the effects of leptin on SREBP-1c expression in HSCs and in mouse liver fibrosis. EXPERIMENTAL APPROACH: HSCs were prepared from rats and mice. Gene expressions were analysed by real-time PCR, Western blot analysis, immunostaining and transient transfection assays. KEY RESULTS: Leptin increased ß-catenin protein but not mRNA levels in cultured HSCs. Leptin induced phosphorylation of glycogen synthase kinase-3ß at Ser(9) and subsequent stabilization of ß-catenin protein was mediated, at least in part, by ERK and p38 MAPK pathways. The leptin-induced ß-catenin pathway reduced SREBP-1c expression and activity but did not affect protein levels of key regulators controlling SREBP-1c activity, and was not involved in leptin inhibition of liver X receptor α. In a mouse model of liver injury, the ß-catenin pathway was shown to be involved in leptin-induced liver fibrosis. CONCLUSIONS AND IMPLICATIONS: The ß-catenin pathway contributes to leptin regulation of SREBP-1c expression in HSCs and leptin-induced liver fibrosis in mice. These results have potential implications for clarifying the mechanisms of liver fibrogenesis associated with elevated leptin levels.