Huizhi Lin

Cytokines and the pathogenesis of non-alcoholic steatohepatitis

Histopathological characteristics distinguish steatohepatitis from other causes of chronic liver injury. For years, the main cause of steatohepatitis was thought to be excessive consumption of alcohol. Increasingly, steatohepatitis is being diagnosed in individuals who deny alcohol abuse. Arbitrarily, “non-alcoholic” steatohepatitis (NASH) is subcategorised into “primary” and “secondary” NASH.1 Primary NASH refers to steatohepatitis that is associated with the dysmetabolic syndrome (that is, obesity, type 2 diabetes, dyslipidaemia).2 Primary NASH is thought to be the predominant form of NASH, afflicting at least as many individuals in the USA as chronic hepatitis C. Secondary NASH refers to steatohepatitis that accompanies other syndromes (for example, lipodystrophy) or that is caused by certain drugs (for example, amiodarone).3 Accumulating evidence suggests that common mechanisms may mediate the pathogenesis of alcohol induced steatohepatitis and primary NASH.4 Although unproven, it is likely that primary and secondary NASH also share common pathogenic mechanisms. ### Obesity Obesity, especially visceral adiposity, is a major risk factor for NASH in humans.5 Even if they do not have an elevated body mass index, patients with NASH are very likely to be insulin resistant.2 Basic research on fat cell (adipocyte) biology provides some clues about why obesity, insulin resistance, and NASH may be interrelated. Obesity increases adipose tissue mass. It has long been known that adipose tissue is a source of free fatty acids that are delivered to the liver and a depot for triglycerides that are synthesised by hepatocytes and released into the blood. Only recently however has the neuroendocrine role of adipose tissue been appreciated. Fat produces hormones, such as leptin, resistin, and adiponectin, that regulate metabolism in other tissues, as well as fat itself. Fat is also a source of neurotransmitters, such as noradrenaline and angiotensin II. In addition, because adipocytes produce immunomodulatory cytokines, such as …

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.)

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.

Norepinephrine regulates hepatic innate immune system in leptin‐deficient mice with nonalcoholic steatohepatitis

It is not known why natural killer T (NKT) cells, which modulate liver injury by regulating local cytokine production, are reduced in leptin‐deficient ob/ob mice. NKT cells express adrenoceptors. Thus, we hypothesize that the low norepinephrine (NE) activity of ob/ob mice promotes depletion of liver NKT cells, thereby sensitizing ob/ob livers to lipopolysaccharide (LPS) toxicity. To evaluate this hypothesis, hepatic NKT cells were quantified in wild‐type mice before and after treatment with NE inhibitors, and in dopamine β‐hydroxylase knockout mice (which cannot synthesize NE) and ob/ob mice before and after 4 weeks of NE supplementation. Decreasing NE activity consistently reduces liver NKT cells, while increasing NE has the opposite effect. Analysis of hepatic and thymic NKT cells in mice of different ages demonstrate an age‐related accumulation of hepatic NKT cells in normal mice, while liver NKT cells become depleted after birth in ob/ob mice, which have increased apoptosis of hepatic NKT cells. NE treatment inhibits apoptosis and restores hepatic NKT cells. In ob/ob mice with reduced hepatic NKT cells, hepatic T and NKT cells produce excessive T helper (Th)‐1 proinflammatory cytokines and the liver is sensitized to LPS toxicity. NE treatment decreases Th‐1 cytokines, increases production of Th‐2 cytokines, and reduces hepatotoxicity. Studies of CD1d‐deficient mice, which lack the receptor required for NKT cell development, demonstrate that they are also unusually sensitive to LPS hepatotoxicity. In conclusion, low NE activity increases hepatic NKT cell apoptosis and depletes liver NKT cells, promoting proinflammatory polarization of hepatic cytokine production that sensitizes the liver to LPS toxicity. (HEPATOLOGY 2004;40:434–441.)

Chronic Ethanol Exposure Potentiates Lipopolysaccharide Liver Injury Despite Inhibiting Jun N-terminal Kinase and Caspase 3 Activation

Although ethanol is known to sensitize hepatocytes to tumor necrosis factor (TNF) lethality, the mechanisms involved remain controversial. Recently, others have shown that adding TNFα to cultures of ethanol-pretreated hepatocytes provokes the mitochondrial permeability transition, cytochrome crelease, procaspase 3 activation, and apoptosis. Although this demonstrates that ethanol can sensitize hepatocytes to TNF-mediated apoptosis, the hepatic inflammation and ballooning hepatocyte degeneration that typify alcohol-induced liver injury suggest that other mechanisms might predominate in vivo. To evaluate this possibility, acute responses to lipopolysaccharide (LPS), a potent inducer of TNFα, were compared in mice that had been fed either an ethanol-containing or control diet for 5 weeks. Despite enhanced induction of cytokines such as interleukin (IL)-10, IL-15, and IL-6 that protect hepatocytes from apoptosis, ethanol-fed mice exhibited a 4–5-fold increase in serum alanine aminotransferase after LPS, confirming increased liver injury. Six h post-LPS histology also differed notably in the two groups, with control livers demonstrating only scattered apoptotic hepatocytes, whereas ethanol-exposed livers had large foci of ballooned hepatocytes, inflammation, and scattered hemorrhage. No caspase 3 activity was noted during the initial 6 h after LPS in ethanol-fed mice, but this tripled by 1.5 h after LPS in controls. Procaspase 8 cleavage and activity of the apoptosis-associated kinase, Jun N-terminal kinase, were also greater in controls. In contrast, ethanol exposure did not inhibit activation of cytoprotective mitogen-activated protein kinases and AKT or attenuate induction of the anti-apoptotic factors NF-κB and inducible nitric oxide synthase. Consistent with these responses, neither cytochrome c release, an early apoptotic response, nor hepatic oligonucleosomal DNA fragmentation, the ultimate consequence of apoptosis, was increased by ethanol. Thus, ethanol exacerbates TNF-related hepatotoxicity in vivo without enhancing caspase 3-dependent apoptosis.

Norepinephrine and neuropeptide Y promote proliferation and collagen gene expression of hepatic myofibroblastic stellate cells.

The mechanisms initiating and perpetuating the fibrogenic response in the injured liver are not well understood. Hepatic stellate cells are activated by liver injury to become proliferative and fibrogenic myofibroblasts. Emerging evidence suggests that the sympathetic nervous system may play a role in the development of cirrhosis. It is not known, however, whether this requires a direct interaction between sympathetic neurotransmitters and stellate cell receptors, or results indirectly, from sympathetic effects on the vasculature. Using cultured hepatic stellate cells, we show that the sympathetic neurotransmitters, norepinephrine and neuropeptide Y, markedly stimulate the proliferation of activated, myofibroblastic, hepatic stellate cells. Norepinephrine, but not neuropeptide Y, also induces collagen gene expression. In conclusion, physiologically relevant concentrations of sympathetic neurotransmitters directly modulate the phenotype of hepatic stellate cells. This suggests that targeted interruption of sympathetic nervous system signaling in hepatic stellate cells may be useful in constraining the fibrogenic response to liver injury.

Norepinephrine induces hepatic fibrogenesis in leptin deficient ob/ob mice

Leptins actions on certain cells require a leptin-inducible neurotransmitter, norepinephrine (NE). NE modulates hepatic fibrosis. Therefore, decreased NE may explain why leptin deficiency inhibits hepatic fibrosis. We manipulated adrenergic activity in leptin-deficient ob/ob mice, leptin-sufficient, dopamine beta-hydroxylase deficient (Dbh(-/-)) mice, and HSC cultures to determine if leptin requires NE to activate HSC and induce hepatic fibrosis. ob/ob mice have chronic liver injury, but reduced numbers of HSC. Supplemental leptin increases HSC, suggesting that leptin-dependent, injury-related factors permit expansion of HSC populations. NE also increases HSC numbers and activation, normalizing fibrogenesis. When fed hepatotoxic diets, NE-deficient Dbh(-/-) mice fail to accumulate activated HSC and have impaired fibrogenesis unless treated with adrenergic agonists. NE acts directly on HSC to modulate leptins actions because leptin increases HSC proliferation and prazosin, an alpha-adrenoceptor antagonist, inhibits this. Thus, leptin permits injury-related increases in adrenergic activity and requires NE to activate HSC and induce hepatic fibrogenesis.

Acetylcholine promotes the proliferation and collagen gene expression of myofibroblastic hepatic stellate cells

The mechanisms that initiate and perpetuate the fibrogenic response, during liver injury, are unclear. Animal studies, however, strongly support a role for the autonomic nervous system (ANS) in wound healing. Therefore, the ANS may also mediate the development of cirrhosis. Hepatic stellate cells (HSC), the livers major matrix-producing cells, are activated by injury to become proliferative, fibrogenic myofibroblasts. HSC respond to sympathetic neurotransmitters by changing phenotype, suggesting that HSC may be the cellular effectors of ANS signals that modulate hepatic fibrogenesis during recovery from liver damage. We show here that the parasympathetic neurotransmitter acetylcholine markedly stimulates the proliferation of myofibroblastic HSC and induces HSC collagen gene expression in these cells. By extending evidence that HSC are direct targets of the ANS, these results support the proposed neuroglial role of HSC in the liver and suggest that interrupting ANS signalling may be useful in constraining the fibrogenic response to liver injury.