Jiawen Huang

Inhibiting triglyceride synthesis improves hepatic steatosis but exacerbates liver damage and fibrosis in obese mice with nonalcoholic steatohepatitis

In the early stages of nonalcoholic fatty liver disease (NAFLD), triglycerides accumulate in hepatocytes. Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step in hepatocyte triglyceride biosynthesis. DGAT2 antisense oligonucleotide (ASO) treatment improved hepatic steatosis dramatically in a previous study of obese mice. According to the 2‐hit hypothesis for progression of NAFLD, hepatic steatosis is a risk factor for nonalcoholic steatohepatitis (NASH) and fibrosis. To evaluate this hypothesis, we inhibited DGAT2 in a mouse model of NASH induced by a diet deficient in methionine and choline (MCD). Six‐week‐old genetically obese and diabetic male db/db mice were fed either the control or the MCD diet for 4 or 8 weeks. The MCD diet group was treated with either 25 mg/kg DGAT2 ASO or saline intraperitoneally twice weekly. Hepatic steatosis, injury, fibrosis, markers of lipid peroxidation/oxidant stress, and systemic insulin sensitivity were evaluated. Hepatic steatosis, necroinflammation, and fibrosis were increased in saline‐treated MCD diet–fed mice compared to controls. Treating MCD diet–fed mice with DGAT2 ASO for 4 and 8 weeks decreased hepatic steatosis, but increased hepatic free fatty acids, cytochrome P4502E1, markers of lipid peroxidation/oxidant stress, lobular necroinflammation, and fibrosis. Progression of liver damage occurred despite reduced hepatic expression of tumor necrosis factor alpha, increased serum adiponectin, and striking improvement in systemic insulin sensitivity. Conclusion: Results from this mouse model would suggest accumulation of triglycerides may be a protective mechanism to prevent progressive liver damage in NAFLD. (HEPATOLOGY 2007.)

Fate-Mapping Evidence that Hepatic Stellate Cells are Epithelial Progenitors in Adult Mouse Livers

Liver injury activates quiescent hepatic stellate cells (Q‐HSC) to proliferative myofibroblasts. Accumulation of myofibroblastic hepatic stellate cells (MF‐HSC) sometimes causes cirrhosis and liver failure. However, MF‐HSC also promote liver regeneration by producing growth factors for oval cells, bipotent progenitors of hepatocytes and cholangiocytes. Genes that are expressed by primary hepatic stellate cell (HSC) isolates overlap those expressed by oval cells, and hepatocytic and ductular cells emerge when HSC are cultured under certain conditions. We evaluated the hypothesis that HSC are a type of oval cell and, thus, capable of generating hepatocytes to regenerate injured livers. Because Q‐HSC express glial fibrillary acidic protein (GFAP), we crossed mice in which GFAP promoter elements regulated Cre‐recombinase with ROSA‐loxP‐stop‐loxP‐green fluorescent protein (GFP) mice to generate GFAP‐Cre/GFP double‐transgenic mice. These mice were fed methionine choline‐deficient, ethionine‐supplemented diets to activate and expand HSC and oval cell populations. GFP(+) progeny of GFAP‐expressing precursors were characterized by immunohistochemistry. Basal expression of mesenchymal markers was negligible in GFAP(+)Q‐HSC. When activated by liver injury or culture, HSC downregulated expression of GFAP but remained GFP(+); they became highly proliferative and began to coexpress markers of mesenchyme and oval cells. These transitional cells disappeared as GFP‐expressing hepatocytes emerged, began to express albumin, and eventually repopulated large areas of the hepatic parenchyma. Ductular cells also expressed GFAP and GFP, but their proliferative activity did not increase in this model. These findings suggest that HSC are a type of oval cell that transitions through a mesenchymal phase before differentiating into hepatocytes during liver regeneration.

Role for Hedgehog signaling in hepatic stellate cell activation and viability

Hepatic stellate cells (HSC) have a complex phenotype that includes both neural and myofibroblastic features. The Hedgehog (Hh) pathway has been shown to direct the fate of neural and myofibroblastic cells during embryogenesis and during tissue remodeling in adults. Therefore, we hypothesized that Hh signaling may regulate the fate of HSC in adults. In this study, we find that freshly isolated stellate cells from adult Patched-lacZ transgenic mice exhibit β-galactosidase activity, indicating Hh pathway activity. Transcripts of Hh ligands, the Hh pathway receptor, and Hh-regulated transcription factors are expressed by stellate cells from mice, rats, and humans. Transfection experiments in a cell line using a Hh-inducible luciferase reporter demonstrate constitutive Hh pathway activity. Moreover, neutralizing antibodies to Hh increase apoptosis, while viability is restored by treatment with Hh ligand. In vitro treatment of primary stellate cells with cyclopamine (Cyc), a pharmacologic inhibitor of the Hh pathway, inhibits activation and slightly decreases cell survival, while a single injection of Cyc into healthy adult mice reduces activation of HSC by more than 50% without producing obvious liver damage. Our findings reveal a novel mechanism, namely the Hh pathway, that regulates the activation and viability of HSC.

Oval cells compensate for damage and replicative senescence of mature hepatocytes in mice with fatty liver disease

Hepatic steatosis may have a generally benign prognosis, either because most hepatocytes are not significantly injured or mechanisms to replace damaged hepatocytes are induced. To determine the relative importance of these mechanisms, we compared hepatocyte damage and replication in ethanol‐fed and ob/ob mice with very indolent fatty liver disease to that of healthy control mice and PARP‐1‐/‐ mice with targeted disruption of the DNA repair enzyme, poly(ADP‐ribose) polymerase. Compared to the healthy controls, both groups with fatty livers had significantly higher serum alanine aminotransferase values, hepatic mitochondrial H2O2 production, and hepatocyte oxidative DNA damage. A significantly smaller proportion of the hepatocytes from fatty livers entered S phase when cultured with mitogens. Moreover, this replicative senescence was not reversed by treating cultured hepatocytes with agents (i.e., betaine or leptin) that improve liver disease in intact ethanol‐fed or leptin‐deficient mice. Hepatocytes from PARP1‐/‐ mice also had more DNA damage and reduced DNA synthesis in response to mitogens. However, neither mice with fatty livers nor PARP‐1‐deficient mice had atrophic livers. All of the mice with senescent mature hepatocytes exhibited hepatic accumulation of liver progenitor (oval) cells and oval cell numbers increased with the demand for hepatocyte replacement. Therefore, although hepatic oxidant production and damage are generally increased in fatty livers, expansion of hepatic progenitor cell populations helps to compensate for the increased turnover of damaged mature hepatocytes. In conclusion, these results demonstrate that induction of mechanisms to replace damaged hepatocytes is important for limiting the progression of fatty liver disease. (HEPATOLOGY 2004;39:403–411.)

Hedgehog-mediated mesenchymal-epithelial interactions modulate hepatic response to bile duct ligation

In bile duct-ligated (BDL) rodents, as in humans with chronic cholangiopathies, biliary obstruction triggers proliferation of bile ductular cells that are surrounded by fibrosis produced by adjacent myofibroblastic cells in the hepatic mesenchyme. The proximity of the myofibroblasts and cholangiocytes suggests that mesenchymal–epithelial crosstalk promotes the fibroproliferative response to cholestatic liver injury. Studying BDL mice, we found that bile duct obstruction induces activity of the Hedgehog (Hh) pathway, a system that regulates the viability and differentiation of various progenitors during embryogenesis. After BDL, many bile ductular cells and fibroblastic-appearing cells in the portal stroma express Hh ligands, receptor and/or target genes. Transwell cocultures of an immature cholangiocyte line that expresses the Hh receptor, Patched (Ptc), with liver myofibroblastic cells demonstrated that both cell types produced Hh ligands that enhanced each others viability and proliferation. Further support for the concept that Hh signaling modulates the response to BDL was generated by studying PtcLacZ mice, which have an impaired ability to constrain Hh signaling due to a heterozygous deficiency of Ptc. After BDL, PtcLacZ mice upregulated fibrosis gene expression earlier than wild-type controls and manifested an unusually intense ductular reaction, more expanded fibrotic portal areas, and a greater number of lobular necrotic foci. Our findings reveal that adult livers resurrect developmental signaling systems, such as the Hh pathway, to guide remodeling of the biliary epithelia and stroma after cholestatic injury.

Hepatic fibrogenesis requires sympathetic neurotransmitters

Background and aims: Hepatic stellate cells (HSC) are activated by liver injury to become proliferative fibrogenic myofibroblasts. This process may be regulated by the sympathetic nervous system (SNS) but the mechanisms involved are unclear. Methods: We studied cultured HSC and intact mice with liver injury to test the hypothesis that HSC respond to and produce SNS neurotransmitters to promote fibrogenesis. Results: HSC expressed adrenoceptors, catecholamine biosynthetic enzymes, released norepinephrine (NE), and were growth inhibited by α- and β-adrenoceptor antagonists. HSC from dopamine β-hydroxylase deficient (Dbh−/−) mice, which cannot make NE, grew poorly in culture and were rescued by NE. Inhibitor studies demonstrated that this effect was mediated via G protein coupled adrenoceptors, mitogen activated kinases, and phosphatidylinositol 3-kinase. Injury related fibrogenic responses were inhibited in Dbh−/− mice, as evidenced by reduced hepatic accumulation of α-smooth muscle actin+ve HSC and decreased induction of transforming growth factor β1 (TGF-β1) and collagen. Treatment with isoprenaline rescued HSC activation. HSC were also reduced in leptin deficient ob/ob mice which have reduced NE levels and are resistant to hepatic fibrosis. Treating ob/ob mice with NE induced HSC proliferation, upregulated hepatic TGF-β1 and collagen, and increased liver fibrosis. Conclusions: HSC are hepatic neuroglia that produce and respond to SNS neurotransmitters to promote hepatic fibrosis.

Endoplasmic reticulum stress, hepatocyte CD1d and NKT cell abnormalities in murine fatty livers

The liver regulates lipid homeostasis and is enriched with natural killer T (NKT) cells that respond to lipid antigens. Optimal maturation and activation of NKT cells requires their interaction with lipid antigens that are presented by cluster of differentiation-1 (CD-1) molecules on antigen-presenting cells. Hepatocytes express CD1d and present lipid antigens to NKT cells. Depletion and dysregulation of hepatic NKT cells occurs in mice with fatty livers. Herein, we assess whether reduced CD1d content on steatotic hepatocytes contributes to fatty liver-associated NKT cell abnormalities. We show that despite expressing normal levels of CD1d mRNA, fatty hepatocytes from ob/ob mice have significantly less CD1d on their plasma membranes than normal hepatocytes. This has functional significance because ob/ob hepatocytes are less able to activate CD1d-restricted T-cell responses in vitro, and CD1d-reactive NKT cells are reduced in ob/ob livers. Events in the endoplasmic reticulum (ER) normally regulate CD1d trafficking to plasma membranes. Hepatic steatosis has been associated with ER stress. To determine if ER stress reduces CD-1 accumulation on hepatocytes, we evaluated hepatic ER stress in ob/ob mice and treated cultured hepatocytes and lean mice with tunicamycin to induce ER stress. Lipid accumulation and ER stress occurred in the livers of both ob/ob and tunicamycin-treated mice. Tunicamycin caused dose-dependent decreases in hepatocyte CD1d, inhibited hepatocyte activation of CD1d-restricted T-cell responses, depleted liver populations of CD1d-reactive NKT cells and promoted Th-1 polarization of hepatic cytokine production. In conclusion, ER stress-related decreases in hepatocyte CD1d contribute to NKT cell dysregulation in fatty livers.

STAT-3 Overexpression and p21 Up-Regulation Accompany Impaired Regeneration of Fatty Livers

Fatty liver is an important cause of morbidity in humans and is linked to impaired liver regeneration after liver injury, but the mechanisms for impaired liver regeneration remain unknown. In the normal liver, the interleukin (IL)-6/STAT-3 pathway is thought to play a central role in regeneration because this pathway is disrupted in IL-6-deficient mice that exhibit impaired liver regeneration after 70% partial hepatectomy (PH). To determine whether inhibition of STAT-3 is involved in fatty liver-related mitoinhibition, regenerative induction of STAT-3 was compared in normal mice and leptin-deficient ob/ob mice that have fatty livers and markedly impaired liver regeneration after PH. In both groups, two waves of STAT-3 activation were observed, the first in endothelia and the second in hepatocytes. Before PH, a significantly higher percentage of ob/ob endothelial and hepatocyte nuclei expressed phosphorylated (activated) STAT-3. After PH, phospho-STAT-3 accumulated in liver nuclei of lean mice and this response was markedly exaggerated in ob/ob mice. Moreover, a striking inverse correlation was noted between hepatocyte nuclear accumulation of phospho-STAT-3 and DNA synthesis (as assessed by bromodeoxyuridine labeling), as well as cyclin D1 mRNA induction and protein expression. In contrast, STAT-3 activation was positively correlated with p21 protein expression in both groups of mice. Because these results link exaggerated STAT-3 activation with impaired hepatocyte proliferation, STAT-3 inhibition cannot be a growth-arrest mechanism in ob/ob fatty livers. Rather, hyperinduction of this factor may promote mitoinhibition by up-regulating mechanisms that impede cell cycle progression.

Diacylglycerol acyltranferase 1 anti‐sense oligonucleotides reduce hepatic fibrosis in mice with nonalcoholic steatohepatitis

Retinyl ester (RE) stores decrease during hepatic stellate cell (HSC) activation and liver fibrosis. Although retinol esterification is mostly catalyzed by lecithin:retinol acyltransferase (LRAT), diacylglycerol acyltransferase (DGAT)1 also does this. In previous reports, LRAT−/− mice had reduced hepatic RE but neither excessive HSC activation nor liver fibrosis, and DGAT1−/− mice had increased liver levels of RE and retinol. We sought to clarify the role of DGAT1 in liver fibrosis. Expression of DGAT1/2 was compared by real time PCR in freshly isolated, primary mouse HSCs and hepatocytes. To induce nonalcoholic steatohepatitis (NASH) and liver fibrosis, adult male db/db mice were fed methionine choline–deficient (MCD) diets. Half were treated with DGAT1 antisense oligonucleotide (ASO); the rest were injected with saline. Results were compared with chow‐fed controls. Inhibition of DGAT1 in liver had no effect on hepatic triglyceride content or liver necroinflammation but reduced HSC activation and liver fibrosis in mice with NASH. To evaluate the role of DGAT1 in HSC activation, HSC were isolated from healthy rats treated with DGAT1 ASO or saline. DGAT1 was expressed at relatively high levels in HSCs. HSC isolated from DGAT1 ASO‐treated rats had reduced DGAT1 expression and increased messenger RNA (mRNA) levels of LRAT and cellular retinol binding protein‐1. During culture, they retained more vitamin A, had repressed collagen a2 (I) transcriptional activity, and expressed less collagen a1 (I) and a2 (I) mRNA. Conclusion: DGAT1 may be a therapeutic target in NASH because inhibiting DGAT1 favorably altered. HSC retinoid homeostasis and inhibited hepatic fibrosis in mice with NASH. (HEPATOLOGY 2007.)

Sustained activation of Rac1 in hepatic stellate cells promotes liver injury and fibrosis in mice

Rac, a small, GTP‐binding protein in the Rho family, regulates several cellular functions, including the activation of NADPH oxidase, a major intracellular producer of reactive oxygen species (ROS). Hepatic stellate cells (HSCs) isolated from mice that are genetically deficient in NADPH oxidase produce less ROS, and their activation during chronic liver injury is abrogated, resulting in decreased liver fibrosis. Therefore, we hypothesized that HSC ROS production and activation would be enhanced, and fibrosis worsened, by increasing Rac expression in HSCs. To achieve this, we used transgenic mice that express constitutively active human Rac1 under the control of the α‐smooth muscle actin (α‐sma) promoter, because α‐sma expression is induced spontaneously during HSC activation. Transgene expression was upregulated progressively during culture of primary Rac‐transgenic HSCs, and this increased HSC ROS production as well as expression of activation markers and collagen. Similarly, Rac mice treated with carbon tetrachloride (CCl4) accumulated greater numbers of activated HSCs and had more liver damage, hepatocyte apoptosis, and liver fibrosis—as well as higher mortality—than CCl4‐treated wild‐type mice. In conclusion, sustained activation of Rac in HSCs perpetuates their activation and exacerbates toxin‐induced liver injury and fibrosis, prompting speculation that Rac may be a therapeutic target in patients with cirrhosis. (HEPATOLOGY 2006;44:1267–1277.)