Yoon Mee Yang

Decrease of microRNA‐122 causes hepatic insulin resistance by inducing protein tyrosine phosphatase 1B, which is reversed by licorice flavonoid

Protein tyrosine phosphatase 1B (PTP1B) inhibits hepatic insulin signaling by dephosphorylating tyrosine residues in insulin receptor (IR) and insulin receptor substrate (IRS). MicroRNAs may modulate metabolic functions. In view of the lack of understanding of the regulatory mechanism of PTP1B and its chemical inhibitors, this study investigated whether dysregulation of specific microRNA causes PTP1B‐mediated hepatic insulin resistance, and if so, what the underlying basis is. In high‐fat‐diet‐fed mice or hepatocyte models with insulin resistance, the expression of microRNA‐122 (miR‐122), the most abundant microRNA in the liver, was substantially down‐regulated among those predicted to interact with the 3′‐untranslated region of PTP1B messenger RNA (mRNA). Experiments using miR‐122 mimic and its inhibitor indicated that miR‐122 repression caused PTP1B induction. Overexpression of c‐Jun N‐terminal kinase 1 (JNK1) resulted in miR‐122 down‐regulation with the induction of PTP1B. A dominant‐negative mutant of JNK1 had the opposite effect. JNK1 facilitated inactivating phosphorylation of hepatocyte nuclear factor 4α (HNF4α) responsible for miR‐122 expression, as verified by the lack of HNF4α binding to the gene promoter. The regulatory role of JNK1 in PTP1B induction by a decrease in miR‐122 level was strengthened by cell‐based assays using isoliquiritigenin and liquiritigenin (components in Glycyrrhizae radix) as functional JNK inhibitors; JNK inhibition enabled cells to restore IR and IRS1/2 tyrosine phosphorylation and insulin signaling against tumor necrosis factor alpha, and prevented PTP1B induction. Moreover, treatment with each of the agents increased miR‐122 levels and abrogated hepatic insulin resistance in mice fed a high‐fat diet, causing a glucose‐lowering effect. Conclusion: Decreased levels of miR‐122 as a consequence of HNF4α phosphorylation by JNK1 lead to hepatic insulin resistance through PTP1B induction, which may be overcome by chemical inhibition of JNK. (HEPATOLOGY 2012;56:2209–2220)

Inhibition of SREBP-1c-mediated hepatic steatosis and oxidative stress by sauchinone, an AMPK-activating lignan in Saururus chinensis

Sauchinone, as an AMP-activated kinase (AMPK)-activating lignan in Saururus chinensis, has been shown to prevent iron-induced oxidative stress and liver injury. Sterol regulatory element binding protein-1c (SREBP-1c) plays a key role in hepatic steatosis, which promotes oxidative stress in obese subjects. Previously, we identified the role of AMPK in liver X receptor-alpha (LXRalpha)-mediated SREBP-1c-dependent lipogenesis. Because sauchinone as an antioxidant has the ability to activate AMPK, this study investigated its effects on SREBP-1c-dependent lipogenesis in hepatocytes and in high-fat diet (HFD)-induced hepatic steatosis and oxidative injury. Sauchinone prevented the ability of an LXRalpha agonist (T0901317) to activate SREBP-1c, repressing transcription of the fatty acid synthase, acetyl-CoA carboxylase, stearoyl-CoA desaturase-1, ATP-binding cassette transporter A1, and LXRalpha genes. Consistent with this, an HFD in mice caused fat accumulation in the liver with SREBP-1c induction, which was attenuated by sauchinone treatment. Also, sauchinone had the ability to inhibit oxidative stress as shown by decreases in thiobarbituric acid-reactive substance formation, nitrotyrosinylation, and 4-hydroxynonenal production. Moreover, it prevented not only the liver injury, but also the AMPK inhibition elicited by HFD feeding. These results demonstrate that sauchinone has the capability to inhibit LXRalpha-mediated SREBP-1c induction and SREBP-1c-dependent hepatic steatosis, thereby protecting hepatocytes from oxidative stress induced by fat accumulation.

Identification of a novel class of dithiolethiones that prevent hepatic insulin resistance via the adenosine monophosphate-activated protein kinase-p70 ribosomal S6 kinase-1 pathway.

Several established liver diseases of various causes are highly associated with hepatic insulin resistance, which is characterized by the desensitization of target cells to insulin. Peripheral insulin resistance is observed in most patients who have cirrhosis. Conversely, insulin‐resistant diabetic patients are at increased risk for developing liver disease. Current therapeutic interventions in insulin resistance are limited and therefore likely to be advanced by new tailor‐made drugs. Oltipraz, a prototype dithiolthione, inhibits transforming growth factor β1 and has the ability to regenerate cirrhotic liver. We investigated the effects of oltipraz and synthetic dithiolthiones on hepatic insulin resistance and the molecular basis of action. Oltipraz and other dithiolethione compounds were tested on tumor necrosis factor α (TNF‐α)–induced insulin resistance and glucose homeostasis in vitro and in vivo via immunoblotting, plasmid transfection, kinase analysis, and functional assays. Oltipraz treatment inhibited the ability of TNF‐α to activate p70 ribosomal S6 kinase‐1 (S6K1) downstream of mammalian target of rapamycin, thus preventing insulin receptor substrate‐1 serine phosphorylation and protecting insulin signals. Moreover, oltipraz activated AMP‐activated protein kinase (AMPK), whose inhibition by a dominant negative mutant abolished S6K1 inhibition and protected insulin signaling, indicating that AMPK activation leads to S6K1 inhibition. In hepatocyte‐derived cell lines, oltipraz inhibited glucose production. Oltipraz prevented hepatic insulin resistance in C57BL/6 mice challenged with endotoxin (or TNF‐α), leptin‐deficient mice, and mice fed a high‐fat diet. Synthetic dithiolethiones comparably inhibited insulin resistance. Conclusion: Our findings led to the identification of dithiolethione compounds that prevent insulin resistance through a mechanism involving AMPK‐mediated S6K1 inhibition and thereby sensitize hepatic insulin response. (HEPATOLOGY 2007.)

Rimonabant, a Cannabinoid Receptor Type 1 Inverse Agonist, Inhibits Hepatocyte Lipogenesis by Activating Liver Kinase B1 and AMP-Activated Protein Kinase Axis Downstream of Gαi/o Inhibition

Liver X receptor-α (LXRα) and its target sterol regulatory element-binding protein-1c (SREBP-1c) play key roles in hepatic lipogenesis. Rimonabant, an inverse agonist of cannabinoid receptor type 1 (CB1), has been studied as an antiobesity drug. In view of the link between CB1 and energy metabolism, this study investigated the effect of rimonabant on LXRα-mediated lipogenesis in hepatocytes and the underlying basis. Rimonabant treatment inhibited CYP7A1-LXRα response element gene transactivation and an increase in LXRα mRNA level by the LXRα agonist N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl]-benzenesulfonamide (T0901317) in HepG2 cells. Rimonabant consistently attenuated the activation of SREBP-1c and its target gene induction. The reversal by CB1 agonists on rimonabants repression of SREBP-1c supported the role of CB1 in this effect. Rimonabant inhibited the activation of SREBP-1c presumably via Gαi/o inhibition, as did pertussis toxin. Adenylyl cyclase activator forskolin or 8-bromo-cAMP treatment mimicked the action of rimonabant, suggesting that Gαi/o inhibition causes repression of SREBP-1c by increasing the cAMP level. Knockdown or chemical inhibition of protein kinase A (PKA) prevented the inhibition of LXRα by rimonabant, supporting the fact that an increase in cAMP content and PKA activation, which catalyzes LXRα inhibitory phosphorylation, might be responsible for the antilipogenic effect. In addition, rimonabant activated liver kinase B1 (LKB1), resulting in the activation of AMP-activated protein kinase responsible for LXRα repression. Moreover, PKA inhibition prevented the activation of LKB1, supporting the fact that PKA regulates LKB1. In conclusion, rimonabant has an antilipogenic effect in hepatocytes by inhibiting LXRα-dependent SREBP-1c induction, as mediated by an increase in PKA activity and PKA-mediated LKB1 activation downstream of CB1-coupled Gαi/o inhibition.

Inhibition of liver X receptor-α-dependent hepatic steatosis by isoliquiritigenin, a licorice antioxidant flavonoid, as mediated by JNK1 inhibition.

Isoliquiritigenin (ILQ), a flavonoid obtained from Glycyrrhizae species, has an antioxidant effect. This study investigated the potential of ILQ for inhibiting liver X receptor-α (LXRα)-mediated lipogenesis and steatosis in hepatocytes and its underlying molecular basis. Treatment with ILQ antagonized the ability of an LXRα agonist (T0901317) to activate sterol regulatory element binding protein-1c (SREBP-1c), thereby repressing transcription of fatty acid synthase, acetyl-CoA carboxylase, ATP-binding cassette transporter-A1, and stearoyl-CoA desaturase-1. ILQ treatment inhibited activating phosphorylation of JNK1 elicited by palmitate or TNFα. JNK1, but not JNK2, increased LXRα phosphorylation at serine residues, promoting LXRα activation. The ability of ILQ to inhibit JNK1 downstream of ASK1-MKK7 led to the repression of T0901317-inducible LXRα and SREBP-1c activation. In mice fed a high-fat diet, ILQ treatment inhibited hepatic steatosis, as shown by a decrease in fat accumulation and repression of lipogenic genes. The results of blood biochemistry and histopathology confirmed attenuation of high-fat diet-induced liver injury by ILQ. Moreover, ILQ inhibited oxidative stress, as indicated by decreases in thiobarbituric acid-reactive substance formation, iNOS and COX2 induction, and nitrotyrosinylation. Our results demonstrate that ILQ has the ability to repress LXRα-dependent hepatic steatosis through JNK1 inhibition and protect hepatocytes from oxidative injury inflicted by fat accumulation.

Transactivation of genes encoding for phase II enzymes and phase III transporters by phytochemical antioxidants.

The induction of phase II enzymes and phase III transporters contributes to the metabolism, detoxification of xenobiotics, antioxidant capacity, redox homeostasis and cell viability. Transactivation of the genes that encode for phase II enzymes and phase III transporters is coordinatively regulated by activating transcription factors in response to external stimuli. Comprehensive studies indicate that antioxidant phytochemicals promote the induction of phase II enzymes and/or phase III transporters through various signaling pathways, including phosphoinositide 3-kinase, protein kinase C, and mitogen-activated protein kinases. This paper focuses on the molecular mechanisms and signaling pathways responsible for the transactivation of genes encoding for these proteins, as orchestrated by a series of transcription factors and related signaling components.

Inhibition of LXRα-dependent steatosis and oxidative injury by liquiritigenin, a licorice flavonoid, as mediated with Nrf2 activation.

Liver X receptor-α (LXRα) functions as a major regulator of lipid homeostasis through activation of sterol regulatory element binding protein-1c (SREBP-1c), which promotes hepatic steatosis and steatohepatitis. NF-E2-related factor 2 (Nrf2) is the crucial transcription factor that is necessary for the induction of antioxidant enzymes. This study investigated the potential of liquiritigenin (LQ), a hepatoprotective flavonoid in licorice, to inhibit LXRα-induced hepatic steatosis, and the underlying mechanism of the action. LQ treatment attenuated fat accumulation and lipogenic gene induction in the liver of mice fed a high fat diet. Also, LQ had the ability to inhibit oxidative liver injury, as shown by decreases in thiobarbituric acid reactive substances formation and nitrotyrosinylation. Moreover, LQ treatment antagonized LXRα agonist (T0901317)-mediated SREBP-1c activation, and transactivation of the lipogenic target genes. LQ was found to activate Nrf2, and the ability of LQ to inhibit LXRα-mediated SREBP-1c activation was reversed by Nrf2 deficiency, which supports the inhibitory role of Nrf2 in LXRα-dependent lipogenesis. Consistently, treatment with other Nrf2 activators or forced expression of Nrf2 also inhibited LXRα-mediated SREBP-1c activation. Our results demonstrate that LQ has an efficacy to activate Nrf2, which contributes to inhibiting the activity of LXRα that leads to SREBP-1c induction and hepatic steatosis.

Abrogation of Hyperosmotic Impairment of Insulin Signaling by a Novel Class of 1,2-Dithiole-3-thiones through the Inhibition of S6K1 Activation

A previous study from this laboratory showed that oltipraz and synthetic dithiolethiones prevent tumor necrosis factor-α-induced hepatic insulin resistance via AMP-activated protein kinase-dependent p70S6 kinase (S6K) 1 inhibitory pathway. This study investigated whether oltipraz and a novel class of 1,2-dithiole-3-thiones were capable of preventing insulin resistance induced by hyperosmotic stress, thereby enhancing insulin-dependent signals, and, if so, whether the restoration of insulin signal was mediated with the inhibition of S6K1 activity stimulated by hyperosmotic stress. In HepG2 cells, oltipraz treatment inhibited insulin receptor substrate (IRS) 1 serine phosphorylation, a marker of insulin resistance, induced by sorbitol-, mannitol-, or sodium chloride-induced hyperosmotic stress. Consequently, this allowed cells to restore insulin signals, which was evidenced by decrease in the ratio of serine to tyrosine phosphorylations of IRS1 and increase in the phosphorylations of Akt and glycogen synthase kinase (GSK) 3β. Hyperosmotic stress markedly activated S6K1; S6K1 activation was completely abolished by oltipraz pretreatment. An experiment using dominant-negative S6K1 supports the essential role of S6K1 in the hyperosmolarity-stimulated phosphorylation of IRS1. Transfection of constitutive active mutant S6K1 eliminated the protective effect of oltipraz on GSK3β phosphorylation, indicating that oltipraz restores insulin signaling by inhibiting S6K1 activation. A variety of synthetic 1,2-dithiole-3-thione derivatives also inhibited S6K1 activity and insulin resistance induced by hyperosmotic stress in HepG2 cells. The results of this study demonstrate that a novel class of 1,2-dithiole-3-thiones improve insulin sensitivity under the condition of hyperosmotic stress, which results from the inhibition of S6K1 activation.

Gα12 gep oncogene deregulation of p53-responsive microRNAs promotes epithelial-mesenchymal transition of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has a poor prognosis owing to aggressive phenotype. Gα12 gep oncogene product couples to G-protein-coupled receptors, whose ligand levels are frequently increased in tumor microenvironments. Here, we report Gα12 overexpression in human HCC and the resultant induction of zinc-finger E-box-binding homeobox 1 (ZEB1) as mediated by microRNA deregulation. Gα12 expression was higher in HCC than surrounding non-tumorous tissue. Transfection of Huh7 cell with an activated mutant of Gα12 (Gα12QL) deregulated microRNA (miRNA or miR)-200b/a/429, -194-2/192 and -194-1/215 clusters in the miRNome. cDNA microarray analyses disclosed the targets affected by Gα12 gene knockout. An integrative network of miRNAs and mRNA changes enabled us to predict ZEB1 as a key molecule governed by Gα12. Decreases of miR-200a/b, -192 and -215 by Gα12 caused ZEB1 induction. The ability of Gα12 to decrease p53 levels, as a result of activating protein-1 (AP-1)/c-Jun-mediated mouse double minute 2 homolog induction, contributed to transcriptional deregulation of the miRNAs. Gα12QL induced ZEB1 and other epithelial–mesenchymal transition markers with fibroblastoid phenotype change. Consistently, transfection with miR-200b, -192 or -215 mimic prevented the ability of Gα12QL to increase tumor cell migration/invasion. In xenograft studies, sustained knockdown of Gα12 decreased the overall growth rate and average volume of tumors derived from SK-Hep1 cell (mesenchymal-typed). In HCC patients, miR-192, -215 and/or -200a were deregulated with microvascular invasion or growth advantage. In the HCC samples with higher Gα12 level, a correlation existed in the comparison of relative changes of Gα12 and ZEB1. In conclusion, Gα12 overexpressed in HCC causes ZEB1 induction by deregulating p53-responsive miRNAs, which may facilitate epithelial–mesenchymal transition and growth of liver tumor. These findings highlight the significance of Gα12 upregulation in liver tumor progression, implicating Gα12 as an attractive therapeutic target.

Gα12gep oncogene inhibits FOXO1 in hepatocellular carcinoma as a consequence of miR-135b and miR-194 dysregulation.

The high mortality rate of hepatocellular carcinoma (HCC) is associated with its fast-growing malignancy. In tumor microenvironments, certain GPCRs are coupled to Gα12 for signal transduction. Given the role of forkhead box O1 (FOXO1) in the inhibition of various tumors, this study investigated whether increase of Gα12 in HCC causes FOXO1 repression, and if so, whether this event occurs through microRNA dysregulation. Overexpression of an active mutant of Gα12 (Gα12QL) decreased FOXO1 levels, whereas knockdown of Gα12 had the opposite effect. Of the microRNAs targeting FOXO1, miR-135b levels were markedly increased by Gα12 signaling, which led to FOXO1 repression as shown by the experiments using mimic, antisense oligonucleotide or siRNA. Gα12QL increased the primary form of miR-135b by activating JunB (or c-Jun)/AP-1. Consistently, knockdown of JunB (or c-Jun) decreased miR-135b levels, thereby increasing FOXO1. Moreover, Gα12QL induced MDM2, the deficiency of which facilitated FOXO1 accumulation. In addition, Gα12QL repressed miR-194 cluster gene products (194/192/215), which contributed to MDM2-mediated FOXO1 repression. In functional assays, Gα12QL facilitated tumor cell growth with alterations in cell cycle-associated protein levels, which was antagonized by enforced expression of FOXO1. In human HCCs, FOXO1 levels were decreased as compared with the surrounding liver tissue. Moreover, decrease of FOXO1 or miR-194 was statistically significant between stages T1 and T2, whereas increase of miR-135b discriminated tumor stage T3a versus T1/T2. In conclusion, Gα12gep oncogene inhibits FOXO1, which may result from the inhibition of FOXO1 de novo synthesis by miR-135b in conjunction with MDM2-mediated destabilization of FOXO1.