James J. Potter

Inositol Pyrophosphates Inhibit Akt Signaling, Thereby Regulating Insulin Sensitivity and Weight Gain

The inositol pyrophosphate IP7 (5-diphosphoinositolpentakisphosphate), formed by a family of three inositol hexakisphosphate kinases (IP6Ks), modulates diverse cellular activities. We now report that IP7 is a physiologic inhibitor of Akt, a serine/threonine kinase that regulates glucose homeostasis and protein translation, respectively, via the GSK3β and mTOR pathways. Thus, Akt and mTOR signaling are dramatically augmented and GSK3β signaling reduced in skeletal muscle, white adipose tissue, and liver of mice with targeted deletion of IP6K1. IP7 affects this pathway by potently inhibiting the PDK1 phosphorylation of Akt, preventing its activation and thereby affecting insulin signaling. IP6K1 knockout mice manifest insulin sensitivity and are resistant to obesity elicited by high-fat diet or aging. Inhibition of IP6K1 may afford a therapeutic approach to obesity and diabetes.

A randomized placebo controlled trial of vitamin E for alcoholic hepatitis

BACKGROUND/AIMS The effect of vitamin E administration on clinical and laboratory parameters of liver function and on markers of fibrogenesis was assessed in patients with mild to moderate alcoholic hepatitis in a double blind placebo controlled randomized trial. METHODS Twenty-five patients received 1000 I.U. of vitamin E per day, while 26 patients received placebo for 3 months. The patients were followed for 1 year after entry into the trial. RESULTS Vitamin E did not result in significant greater decreases in serum aminotransferases and serum bilirubin or in greater increases in serum albumin as compared with placebo. Prothrombin time did not change, while serum creatinine remained in the normal range. Monocyte nuclear nuclear factor-kappa B binding activity decreased in patients who remained abstinent, regardless of whether they received vitamin E. As regards markers of hepatic fibrogenesis, vitamin E treatment decreased serum hyaluronic acid (P<0.05) while serum aminoterminal peptide of type III procollagen did not change in either group. Four patients in the treatment group and five in the placebo group died during the 1-year study. CONCLUSIONS Vitamin E treatment improves serum hyaluronic acid but has no beneficial effects on tests of liver function in patients with mild to moderate alcoholic hepatitis.

Yes-Associated Protein Regulates the Hepatic Response After Bile Duct Ligation

Human chronic cholestatic liver diseases are characterized by cholangiocyte proliferation, hepatocyte injury, and fibrosis. Yes‐associated protein (YAP), the effector of the Hippo tumor‐suppressor pathway, has been shown to play a critical role in promoting cholangiocyte and hepatocyte proliferation and survival during embryonic liver development and hepatocellular carcinogenesis. Therefore, the aim of this study was to examine whether YAP participates in the regenerative response after cholestatic injury. First, we examined human liver tissue from patients with chronic cholestasis. We found more‐active nuclear YAP in the bile ductular reactions of primary sclerosing cholangitis and primary biliary cirrhosis patient liver samples. Next, we used the murine bile duct ligation (BDL) model to induce cholestatic liver injury. We found significant changes in YAP activity after BDL in wild‐type mice. The function of YAP in the hepatic response after BDL was further evaluated with liver‐specific Yap conditional deletion in mice. Ablating Yap in the mouse liver not only compromised bile duct proliferation, but also enhanced hepatocyte necrosis and suppressed hepatocyte proliferation after BDL. Furthermore, primary hepatocytes and cholangiocytes isolated from Yap‐deficient livers showed reduced proliferation in response to epidermal growth factor in vitro. Finally, we demonstrated that YAP likely mediates its biological effects through the modulation of Survivin expression. Conclusion: Our data suggest that YAP promotes cholangiocyte and hepatocyte proliferation and prevents parenchymal damage after cholestatic injury in mice and thus may mediate the response to cholestasis‐induced human liver disease. (HEPATOLOGY 2012;56:1097–1107)

Leptin enhances the effect of transforming growth factor β in increasing type I collagen formation

Hepatic fibrosis produced by carbon tetrachloride and by Schistosoma masoni is markedly decreased in leptin deficient ob/ob mice as compared to control mice. Leptin is present in activated rat stellate cells, which are the principal collagen producing cells in the liver. The purpose of this study was to identify the leptin receptor and to determine the effects of leptin on type I collagen expression in the human stellate cell line, LX-1. Leptin protein was detected in the LX-1 cells. The leptin receptor (OB-R) was demonstrated by immunofluorescent staining and confocal microscopy. However, only the short forms (Ob-R(s)), but not the long forms (Ob-R(l)), of leptin receptor mRNA expression were detected. Leptin increased alpha(1)(I) collagen mRNA and type I collagen production. Leptin did not increase TGFbeta1 mRNA or protein in the cultured LX-1 cells. Leptin, however, increased TGFbeta type II receptor mRNA and protein and augmented the effect of TGFbeta1 on collagen production. In conclusion, this study shows that the effect of leptin in increasing type I collagen production in stellate cells is mediated by actions of leptin in increasing the effectiveness of TGFbeta on fibrogenesis by means of an enhancement of the TGFbeta type II receptor.

Histone deacetylase inhibition suppresses the transforming growth factor β1–induced epithelial‐to‐mesenchymal transition in hepatocytes

Transforming growth factor β1 (TGFβ1) plays a crucial role in the induction of the epithelial‐to‐mesenchymal transition (EMT) in hepatocytes, which contributes to the pathogenesis of liver fibrosis. The inhibition of the TGFβ1 cascade suppresses EMT and the resultant fibrosis. In this study, we focus on EMT‐induced fibrosis in hepatocytes and the epigenetic regulation of the type I collagen gene. Histone acetylation is an important, major epigenetic mechanism that modulates gene transcription. We evaluated the epigenetic regulation of type I collagen in alpha mouse liver 12 hepatocytes (an untransformed mouse cell line) that had undergone EMT after treatment with TGFβ1. The histone deacetylase inhibitor trichostatin A (TSA) inhibited EMT; this was reflected by the preservation of epithelial markers and function (E‐cadherin and albumin). Fibrosis, the ultimate outcome of EMT, was abolished by TSA; this was indicated by the inhibition of type I collagen deposition. TSA exerted its anti‐EMT effects by deactivating the mothers against decapentaplegic homolog 3 (Smad3)/Smad4 transcription complex and by interfering with p300, a coactivator of the type I collagen promoter, and preventing its binding to Smad3. TSA also restored Friend leukemia virus integration 1, an inhibitor of the type I collagen gene. TGFβ1‐induced EMT and its inhibition by TSA were replicated in human primary hepatocytes. Conclusion: Histone deacetylase inhibition abrogates TGFβ1‐induced EMT in hepatocytes and reverses EMT‐induced fibrosis by epigenetic modulation of type I collagen. (HEPATOLOGY 2010)

Influence of leptin in the development of hepatic fibrosis produced in mice by Schistosoma mansoni infection and by chronic carbon tetrachloride administration

BACKGROUND/AIMS Leptin, a product of the obese (ob) gene is present in activated stellate cells. This study investigated whether leptin is essential for the development of hepatic fibrosis caused by various agents. METHODS Control and ob/ob mice were infected with Schistosoma mansoni or were administered chronic carbon tetrachloride to cause hepatic fibrosis. RESULTS Fibrosis developed in both ob/ob and control mice. However, the amount of histologically detectable fibrosis and the increase in liver hydroxyproline content was significantly greater in both models of fibrosis for treated controls than for treated ob/ob mice. Fibrosis was associated with higher secretion of TGFbeta1 from spleen cells of treated control than treated ob/ob mice. Chronic leptin administration in ob/ob mice infected with Schistosoma mansoni resulted in an increase in the amount of fibrosis caused by Schistosoma mansoni, eliminating any significant differences in the amount of fibrosis between infected ob/ob mice and control mice. It also eliminated any significant difference in TGFbeta1 secretion between the infected ob/ob and infected control mice. CONCLUSIONS This study shows that leptin deficiency decreases but does not eliminate hepatic fibrosis produced by Schistosoma mansoni and carbon tetrachloride administration. The effect of leptin in potentiating fibrogenesis is most likely mediated by TGFbeta1.

Lack of inducible nitric oxide synthase leads to increased hepatic apoptosis and decreased fibrosis in mice after chronic carbon tetrachloride administration

The role of nitric oxide (NO) in liver injury and fibrosis is unclear. The purpose of this study was to determine whether inducible NO synthase deficiency (iNOS−/−) affects liver injury and fibrosis produced in mice by chronic carbon tetrachloride (CCl4) administration. Wild‐type (WT) or iNOS−/− mice were subjected to biweekly CCl4 injections over 8 weeks, whereas controls were given isovolumetric injections of olive oil. Serum aminotransferases were lower after CCl4 in the iNOS−/− than in the WT mice, which correlated with decreased necrosis on liver histology. There was increased apoptosis, a lower number of stellate cells, and a lesser degree of fibrosis after CCl4 in the iNOS−/− as compared with the WT mice. α1(I) collagen messenger RNA (mRNA) was markedly increased after CCl4 in the WT and to a significantly lesser extent in the iNOS−/− mice. Liver matrix metalloproteinase‐9 (MMP‐9) mRNA and MMP‐2 mRNA were increased more in the WT than in the iNOS−/− mice after CCl4. Also tissue inhibitor metalloproteinase 1 (TIMP‐1) mRNA was increased to a much greater extent in the WT than in the iNOS−/− mice after CCl4 (P < 0.05). However, MMP‐9 and TIMP‐1 protein, determined by western blot, were similarly increased after CCl4 in both groups of mice. Conclusion: NO protects against CCl4‐induced apoptosis. In the absence of iNOS, there is decreased necrosis, increased apoptosis, and reduced liver fibrosis. (HEPATOLOGY 2008;47:2051–2058.)

Characteristics of alcohol dehydrogenase in fat-storing (Ito) cells of rat liver

Fat storing (Ito) cells, located in the perisinusoidal spaces of the liver and the main storage site of vitamin A, have been associated with fibrogenesis. The mechanisms of alcoholic liver disease, although mostly unknown, do involve ethanol metabolism. This study examined the ability of fat-storing cells to metabolize ethanol. Alcohol dehydrogenase activity was detected in fat-storing cells of the rat liver. The enzyme was demonstrated also by enzyme-linked immunosorbent assay and immunohistochemical staining. The enzyme in fat-storing cells is similar to the hepatocyte enzyme in its Michaelis-Menten constants for substrates and coenzymes and in the competitive inhibition by ethanol of retinol oxidation. It differs from the hepatocyte enzyme by its greater susceptibility to inhibition by 4-methylpyrazole and by having a single isoelectric point of 9.5 as compared with multiple isoelectric points in the hepatocyte ranging from 6.9 to 8.8. The ability of the fat-storing cell to oxidize ethanol and the inhibitory effect of ethanol on retinol oxidation may be important in the pathogenesis of alcoholic liver disease.

1,25-dihydroxyvitamin D3 and its nuclear receptor repress human α1(I) collagen expression and type I collagen formation

Vitamin D deficiency is common in chronic liver disease particularly in those with severe liver fibrosis.

Depression of alcohol dehydrogenase activity in rat hepatocyte culture by dihydrotestosterone

Hepatocytes harvested from castrated rats retained a higher alcohol dehydrogenase (EC 1.1.1.1) activity than hepatocytes harvested from normal rats during 7 days of culture. Dihydrotestosterone (1 microM) decreased the enzyme activity, after 2 and 5 days of culture, in hepatocytes from castrated and control animals respectively. Dihydrotestosterone decreased the enzyme activity to similar values in both groups of hepatocytes by the end of 7 days of culture. Testosterone (1 microM) had no effect on the enzyme activity in normal hepatocytes and only a transitory effect in decreasing the enzyme activity in hepatocytes from castrated animals. The increases in alcohol dehydrogenase activity after castration and their suppression by dihydrotestosterone were associated with parallel changes in the rate of ethanol elimination. Additions of substrates of the malate-aspartate shuttle or dinitrophenol did not modify ethanol elimination. These observations indicate that dihydrotestosterone has a direct suppressant effect on hepatocyte alcohol dehydrogenase and that the enzyme activity is a major determinant of the rate of ethanol elimination.