An endoplasmic reticulum protein, Nogo‐B, facilitates alcoholic liver disease through regulation of kupffer cell polarization

Nogo‐B (Reticulon 4B) is an endoplasmic reticulum (ER) resident protein that regulates ER structure and function. Because ER stress is known to induce M2 macrophage polarization, we examined whether Nogo‐B regulates M1/M2 polarization of Kupffer cells and alters the pathogenesis of alcoholic liver disease (ALD). M1 and M2 phenotypes were assessed in relation to Nogo‐B expression and disease severity in liver specimens from ALD patients (NCT01875211). Liver specimens from wild‐type (WT) and Nogo‐B knockout (KO) mice fed a control or Lieber‐DeCarli ethanol liquid diet (5% ethanol) for 6 weeks were analyzed for liver injury and steatosis. Kupffer cells isolated from WT and Nogo‐B KO mice were assessed for M1 and M2 activation. A significant positive correlation was observed between Nogo‐B positive Kupffer cells and disease severity in ALD patients (n = 30, r = 0.66, P = 0.048). Furthermore, Nogo‐B–positive Kupffer cells were correlated with M1 activation (inducible nitric oxide synthase) (r = 0.50, P = 0.05) and negatively with markers of M2 status (CD163) (r = −0.48, P = 0.07) in these patients. WT mice exhibited significantly increased liver injury (P < 0.05) and higher hepatic triglyceride levels (P < 0.01) compared with Nogo‐B KO mice in response to chronic ethanol feeding. Nogo‐B in Kupffer cells promoted M1 polarization, whereas absence of Nogo‐B increased ER stress and M2 polarization in Kupffer cells.  Nogo‐B is permissive of M1 polarization of Kupffer cells, thereby accentuating liver injury in ALD in humans and mice. Nogo‐B in Kupffer cells may represent a new therapeutic target for ALD. (Hepatology 2017;65:1720‐1734).

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abstract: We compared the cirrhosis-prediction accuracy of an ultrasonographic scoring system (USSS) combining six representative sonographic indices with that of liver stiffness measurement (LSM) by transient elastography, and prospectively investigated the correlation between the USSS score and LSM in predicting cirrhosis.  Two hundred and thirty patients with chronic liver diseases (187 men, 43 women; age, 50.4±9.5 y, mean±SD) were enrolled in this prospective study. The USSS produces a combined score for nodularity of the liver surface and edge, parenchyma echogenicity, presence of right-lobe atrophy, spleen size, splenic vein diameter, and abnormality of the hepatic vein waveform. The correlations of the USSS score and LSM with that of a pathological liver biopsy (METAVIR scoring system: F0-F4) were evaluated.  The mean USSS score and LSM were 7.2 and 38.0 kPa, respectively, in patients with histologically overt cirrhosis (F4, P=0.017) and 4.3 and 22.1 kPa in patients with fibrotic change without overt cirrhosis (F0-F3) (P=0.025). The areas under the receiver operating characteristic (ROC) curves of the USSS score and LSM for F4 patients were 0.849 and 0.729, respectively. On the basis of ROC curves, criteria of USSS ≥6: LSM ≥17.4 had a sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of 89.2%:77.6%, 69.4%:61.4%, 86.5%:83.7%, 74.6%:51.9% and 0.83:0.73, respectively, in predicting F4.  The results indicate that this USSS has comparable efficacy to LSM in the diagnosis of cirrhosis.

title: Design and validation of a histological scoring system for nonalcoholic fatty liver disease
abstract: Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis in the absence of a history of significant alcohol use or other known liver disease. Nonalcoholic steatohepatitis (NASH) is the progressive form of NAFLD. The Pathology Committee of the NASH Clinical Research Network designed and validated a histological feature scoring system that addresses the full spectrum of lesions of NAFLD and proposed a NAFLD activity score (NAS) for use in clinical trials. The scoring system comprised 14 histological features, 4 of which were evaluated semi‐quantitatively: steatosis (0‐3), lobular inflammation (0‐2), hepatocellular ballooning (0‐2), and fibrosis (0‐4). Another nine features were recorded as present or absent. An anonymized study set of 50 cases (32 from adult hepatology services, 18 from pediatric hepatology services) was assembled, coded, and circulated. For the validation study, agreement on scoring and a diagnostic categorization (“NASH,” “borderline,” or “not NASH”) were evaluated by using weighted kappa statistics. Inter‐rater agreement on adult cases was: 0.84 for fibrosis, 0.79 for steatosis, 0.56 for injury, and 0.45 for lobular inflammation. Agreement on diagnostic category was 0.61. Using multiple logistic regression, five features were independently associated with the diagnosis of NASH in adult biopsies: steatosis (P = .009), hepatocellular ballooning (P = .0001), lobular inflammation (P = .0001), fibrosis (P = .0001), and the absence of lipogranulomas (P = .001). The proposed NAS is the unweighted sum of steatosis, lobular inflammation, and hepatocellular ballooning scores. In conclusion, we present a strong scoring system and NAS for NAFLD and NASH with reasonable inter‐rater reproducibility that should be useful for studies of both adults and children with any degree of NAFLD. NAS of ≥5 correlated with a diagnosis of NASH, and biopsies with scores of less than 3 were diagnosed as “not NASH.” (HEPATOLOGY 2005;41:1313–1321.

title: Reticulon 4B (Nogo‐B) facilitates hepatocyte proliferation and liver regeneration in mice
abstract: Nogo‐B, also known as reticulon 4B, promotes liver fibrosis and cirrhosis by facilitating the transforming growth factor β (TGF‐β) signaling pathway in activated hepatic stellate cells. The aim of this study was to determine the role of Nogo‐B in hepatocyte proliferation and liver regeneration. Partial hepatectomy (PHx, 70% resection) was performed in male wild‐type (WT) and Nogo‐A/B knockout mice (referred to as Nogo‐B KO mice). Remnant livers were isolated 2 hours, 5 hours, and 1, 2, 3, 7, and 14 days after PHx. Hepatocyte proliferation was assessed by Ki67 labeling index. Quantitative real‐time polymerase chain reaction was performed for genes known to be involved in liver regeneration. Hepatocytes isolated from WT and Nogo‐B KO mice were used to examine the role of Nogo‐B in interleukin‐6 (IL‐6), hepatocyte growth factor (HGF), epidermal growth factor (EGF), and TGF‐β signaling. Nogo‐B protein levels increased in the regenerating livers in a time‐dependent manner after PHx. Specifically, Nogo‐B expression in hepatocytes gradually spread from the periportal toward the central areas by 7 days after PHx, but receded notably by 14 days. Nogo‐B facilitated IL‐6/signal transducer and activator of transcription 3 signaling, increased HGF‐induced but not EGF‐induced hepatocyte proliferation, and tended to reduce TGF‐β1–induced suppression of hepatocyte proliferation in cultured hepatocytes. Lack of Nogo‐B significantly induced TGF‐β1 and inhibitor of DNA binding expression 1 day after PHx and IL‐6 and EGF expression 2 days after PHx. Lack of Nogo‐B delayed hepatocyte proliferation but did not affect the liver‐to‐body ratio in the regenerative process.  Nogo‐B expression in hepatocytes facilitates hepatocyte proliferation and liver regeneration. (HEPATOLOGY 2013)

title: Mouse model of chronic and binge ethanol feeding (the NIAAA model)
abstract: Chronic alcohol consumption is a leading cause of chronic liver disease worldwide, leading to cirrhosis and hepatocellular carcinoma. Currently, the most widely used model for alcoholic liver injury is ad libitum feeding with the Lieber-DeCarli liquid diet containing ethanol for 4–6 weeks; however, this model, without the addition of a secondary insult, only induces mild steatosis, slight elevation of serum alanine transaminase (ALT) and little or no inflammation. Here we describe a simple mouse model of alcoholic liver injury by chronic ethanol feeding (10-d ad libitum oral feeding with the Lieber-DeCarli ethanol liquid diet) plus a single binge ethanol feeding. This protocol for chronic-plus-single-binge ethanol feeding synergistically induces liver injury, inflammation and fatty liver, which mimics acute-on-chronic alcoholic liver injury in patients. This feeding protocol can also be extended to chronic feeding for longer periods of time up to 8 weeks plus single or multiple binges. Chronic-binge ethanol feeding leads to high blood alcohol levels; thus, this simple model will be very useful for the study of alcoholic liver disease (ALD) and of other organs damaged by alcohol consumption.

title: Plasma endotoxin concentrations in patients with alcoholic and non-alcoholic liver disease: reevaluation with an improved chromogenic assay.
abstract: Plasma endotoxin concentration was measured in 85 patients with alcoholic liver disease (alcoholic cirrhosis (n = 64), alcoholic hepatitis without cirrhosis (n = 11), fatty liver (n = 10), and in patients with non-alcoholic cirrhosis (n = 15]. Endotoxin concentration was determined with an improved chromogenic substrate assay, using individual standard curves for each plasma sample. In patients with alcoholic cirrhosis the mean endotoxin concentration was significantly higher than in patients with non-alcoholic cirrhosis (p less than 0.05). In addition, distinctly higher endotoxin concentrations (greater than 20 pg/ml) were more frequently observed in patients with alcoholic cirrhosis than in non-alcoholic cirrhosis (34.4 vs. 14.3%, p less than 0.05). Mean endotoxin concentration was not significantly higher in cirrhotics with ascites or esophageal varices as compared with the subgroup without ascites or esophageal varices. The endotoxin concentration did not correlate with serum bilirubin, prothrombin concentration or serum enzyme activities. In patients with alcoholic liver disease, however, endotoxin concentration revealed a negative correlation (p less than 0.05) with the concentration of high density lipoprotein cholesterol. On admission endotoxin concentrations in alcoholics with fatty liver were similarly elevated as observed in alcoholic cirrhosis. In six out of 12 patients with fatty liver or alcoholic hepatitis, in whom a second sample of plasma was investigated after 6 to 8 days, endotoxemia was no longer detectable; in the remaining patients, the endotoxin concentration decreased markedly. The results indicate that, irrespective of the stage of liver disease, alcohol abuse favours the development of endotoxemia. They support the hypothesis that gut-derived endotoxins might play a role in the initiation and aggravation of alcohol-induced liver disease.

title: Increased intestinal permeability to macromolecules and endotoxemia in patients with chronic alcohol abuse in different stages of alcohol-induced liver disease.
abstract: No information is yet available about the influence of alcohol abuse on the translocation of larger molecules (Mr>1200) through the intestinal mucosa in man. The present study aimed to determine the intestinal permeability to macromolecules in patients with chronic alcohol abuse and mild to more advanced stages of liver disease, and to measure the concentration of endotoxins in the plasma, as these compounds derive from the intestinal flora and are suspected to contribute to the development of alcoholic liver disease (ALD).



The permeability to polyethylene glycol Mr 400, Mr 1500, Mr 4000, and Mr 10,000 and endotoxin plasma concentrations were measured in 54 patients with alcoholic liver disease, 19 of them with cirrhosis, and in 30 non-alcoholic healthy controls.



Permeability to polyethylene glycol Mr 400 was found to be unchanged in patients with ALD in comparison to healthy controls, whereas polyethylene glycol Mr 1500 and Mr 4000 were recovered in about twice as high concentrations in the urine of ALD patients (p<0.01). Polyethylene glycol Mr 10,000 was detected significantly less frequently in urine from healthy controls (0/30) than in urine of patients with alcoholic liver disease (20/54, p<0.01). Endotoxin concentrations in the plasma of alcoholics were increased more than 5-fold compared to healthy controls (p<0.01).



The results of this study indicate that alcohol abuse impairs the function of the intestinal barrier, which might enhance the translocation of bacterial toxins, thereby contributing to inflammatory processes in alcoholic liver disease.

title: Exacerbation of alcoholic liver injury by enteral endotoxin in rats
abstract: Increased gut permeability (leaky gut) and endotoxin‐mediated Kupffer cell activation are proposed as the mechanisms of alcoholic liver injury. Although ethanol feeding is shown to sensitize the liver for injury induced by parental administration of lipopolysaccharide (LPS), how enteral LPS loading affects alcoholic liver injury is yet to be tested. The present study provides direct evidence for enhanced entrance to portal circulation of LPS enterally administered to the intragastric ethanol infusion model. Portal and systemic blood endotoxin levels increased to 43.0 ± 4.1 and 6.2 ± 4.3 pg/mL at 2 hours following enteral LPS administration (5 mg/kg) in alcohol‐fed animals, while no such increases were observed in pair‐fed controls. However, endotoxin levels in systemic blood of alcohol‐fed rats were reduced to 0 to 1.5 pg/mL 16 hours after LPS administration. Weekly enteral administration of LPS to the model for 9 weeks exacerbated an increase in plasma alanine transaminase (ALT) levels (227 ± 75 vs. 140 ± 70; P < .01), mononuclear infiltration (25 ± 22 vs. 6.4 ± 4.4/10 mm2; P = .02), sinusoidal congestion, and spotty necrosis, and induced diffuse coagulative necrosis and centrilobular fibrosis in some animals. Reverse‐transcription polymerase chain reaction (RT‐PCR) analysis confirmed the LPS effect at the tissue level by demonstrating accentuated induction of tumor necrosis factor α (TNF‐α) and Cox‐2 mRNA. In conclusion, enteral LPS administration potentiates alcoholic liver necrosis, inflammation, and fibrosis despite efficient endotoxin clearance by the liver and mild systemic endotoxemia that occurs episodically following enteral LPS challenge.

title: Macrophages in inflammation and its resolution
abstract: Macrophages are highly plastic leukocytes that differentiate from monocytes following their entry into extravascular tissues. Macrophages can enter various tissues under inflammatory or non-inflammatory conditions and assume different functions and phenotypes according to the cues they receive from the environment. The notion that inflammation in general and macrophage responses in particular affect physiological phenomena that were previously considered to be not immune-related has enhanced and broadened our understanding of macrophage function during inflammation and its resolution. 
 
This volume brings together 14 manuscripts that cover various aspects of macrophage function during inflammation and its resolution, as well as in several pathologic states for which a significant, long-lasting, macrophage-mediated immune response plays a significant role. Two of the manuscripts present original research on macrophage phagocytosis and its implications. Five provide an overview of macrophage function during inflammation and its resolution, with an emphasis on the modulatory role of particular elements in this response, such as apoptotic leukocytes, specific pathogens, hypoxia, and hormone receptors. The remaining seven manuscripts outline the role of macrophages during inflammation and its resolution in different tissues, including the lung, cardiovascular and adipose tissues, injured skeletal muscle and neuronal tissues, and synovial and oral cavities. 
 
The two original research articles are devoted to the consequences of particle engulfment by macrophages. Labrousse et al. (2011) describe a novel experimental strategy in which they use micro-patterned immune complexes to trigger frustrated phagocytosis and thereby determine spatial parameters in lysosomal movement and fusion. Janko et al. (2011) report on the cumulative binding of CRP and anti-CRP antibodies to the surface of secondary necrotic cells. This binding leads to a pro-inflammatory cytokine response following engulfment by macrophages, implying a potential role for these elements in the etiology of systemic lupus erythematosus. 
 
Of the review articles that discuss the regulation of macrophage differentiation and function by discrete events, two cover the interaction between macrophages and apoptotic leukocytes during the resolution of inflammation. Korns et al. (2011) outline the regulation of apoptotic cell clearance by macrophages (efferocytosis) and the environmental cues that promote the efferocytic capabilities of macrophages. The second manuscript by Ariel and Serhan (2012) reviews the impact of apoptotic cell sensing and disposal by macrophages on the switches in functional phenotypes displayed by these cells. The effect of another environmental factor, hypoxia, on monocyte/macrophage activation, and differentiation through transcriptional and translational modulation is covered by Rahat et al. (2011). Lugo-Villarino et al. (2011) discuss the pathogenesis and co-mortality displayed by two macrophage-inhabiting microbes (HIV and Mycobacterium Tuberculosis) and their influence on macrophage polarization. Patel et al. (2011) review the role of melanocortin receptor expression by macrophages in anti-inflammation and the resolution of inflammation, with attention given to melanocortin receptor agonists as therapeutic agents. 
 
Several review articles discuss the function of macrophages during inflammation and/or its resolution within distinct anatomical sites, taking into account the unique features of these tissue-specific macrophages, in particular the distinct environments in which they reside and their interactions with neighboring cells. Claria et al. (2011) review current knowledge on the contribution of macrophages to the inflammatory state characterizing adipose tissue and the phenotypic changes observed in macrophages during obesity. Kennedy et al. (2011) discuss macrophage polarization occurring within the synovial space of arthritic joints and its modulation by cytokines, transcription factors, and pro-resolving lipid mediators. The article from Bosurgi et al. (2011) describes the multiple actions of macrophages in injured skeletal muscle, where the effects of these cells are a double-edged sword and can either promote healing and repair or lead to fibrosis and fat replacement. Herold et al. (2011) survey the indispensable role of macrophages in the resolution and termination of inflammation in lung infection and injury as well as the molecular pathways involved in these processes. Proper termination of inflammatory events and clearance of apoptotic cells are also critical to the cardiovascular system, as reviewed by Thorp (2012), and defects in macrophage efferocytosis can lead to atherosclerosis and myocardial infarction. While monocyte-derived macrophages and resident microglia were previously considered to be detrimental in brain inflammation and injury, recent advances reviewed by Jung and Schwartz (2012) suggest an opposite role for these macrophage-like cells, with a positive impact on brain maintenance and repair. Finally, Hasturk et al. (2012) outline the reciprocal interaction between periodontal disease and chronic inflammatory illnesses and the role that macrophages play in mediating these chronic inflammatory diseases. 
 
Altogether, the articles in this volume portray the complexity of the multiple roles played by macrophages and members of their lineage during inflammation and its resolution, and their manipulation by the injured milieu. These topics are currently heavily studied, and advances in the field, facilitated by state-of-the-art genetics and optical technologies, will undoubtedly continue to contribute to our understanding of the immune system's response to foreign insults, trauma, and inflammatory disorders.

title: Molecular Mechanism for Adiponectin-dependent M2 Macrophage Polarization
abstract: The anti-inflammatory effects of globular adiponectin (gAcrp) are mediated by IL-10/heme oxygenase 1 (HO-1)-dependent pathways. Although full-length (flAcrp) adiponectin also suppresses LPS-induced pro-inflammatory signaling, its signaling mechanisms are not yet understood. The aim of this study was to examine the differential mechanisms by which gAcrp and flAcrp suppress pro-inflammatory signaling in macrophages. Chronic ethanol feeding increased LPS-stimulated TNF-α expression by Kupffer cells, associated with a shift to an M1 macrophage polarization. Both gAcrp and flAcrp suppressed TNF-α expression in Kupffer cells; however, only the effect of gAcrp was dependent on IL-10. Similarly, inhibition of HO-1 activity or siRNA knockdown of HO-1 in RAW264.7 macrophages only partially attenuated the suppressive effects of flAcrp on MyD88-dependent and -independent cytokine signatures. Instead, flAcrp, acting via the adiponectin R2 receptor, potently shifted the polarization of Kupffer cells and RAW264.7 macrophages to an M2 phenotype. gAcrp, acting via the adiponectin R1 receptor, was much less effective at eliciting an M2 pattern of gene expression. M2 polarization was also partially dependent on AMP-activated kinase. flAcrp polarized RAW264.7 macrophages to an M2 phenotype in an IL-4/STAT6-dependent mechanism. flAcrp also increased the expression of genes involved in oxidative phosphorylation in RAW264.7 macrophages, similar to the effect of flAcrp on hepatocytes. In summary, these data demonstrate that gAcrp and flAcrp utilize differential signaling strategies to decrease the sensitivity of macrophages to activation by TLR4 ligands, with flAcrp utilizing an IL-4/STAT6-dependent mechanism to shift macrophage polarization to the M2/anti-inflammatory phenotype.

title: Cannabinoid CB2 receptors protect against alcoholic liver disease by regulating Kupffer cell polarization in mice
abstract: Activation of Kupffer cells plays a central role in the pathogenesis of alcoholic liver disease. Because cannabinoid CB2 receptors (CB2) display potent anti‐inflammatory properties, we investigated their role in the pathogenesis of alcoholic liver disease, focusing on the impact of CB2 on Kupffer cell polarization and the consequences on liver steatosis.  In keeping with this, genetic ablation of CB2 enhanced hepatic induction of M1 gene signature and blunted the induction of M2 markers. CB2 also modulated alcohol‐induced fatty liver, as shown by the reduction of hepatocyte steatosis in JWH‐133‐treated mice and its enhancement in CB2−/− animals. Studies in isolated Kupffer cells and cultured macrophages further demonstrated that CB2 inhibits M1 polarization and favors the transition to an M2 phenotype. In addition, conditioned‐medium experiments showed that preventing M1 polarization in CB2‐activated macrophages protects from lipid accumulation in hepatocytes. Heme oxygenase‐1 (HO‐1) mediated the anti‐inflammatory effects of CB2 receptors. Indeed, alcohol‐fed mice treated with JWH‐133 showed increased hepatic expression of macrophage HO‐1, as compared to vehicle‐treated counterparts. In keeping with this, JWH‐133 induced HO‐1 expression in cultured macrophages, and the HO‐1 inhibitor, zinc protoporphyrin, blunted the inhibitory effect of JWH‐133 on lipopolysaccharide‐induced nuclear factor‐kappa B activation and M1 polarization. Altogether, these findings demonstrate that CB2 receptors display beneficial effects on alcohol‐induced inflammation by regulating M1/M2 balance in Kupffer cells, thereby reducing hepatocyte steatosis via paracrine interactions between Kupffer cells and hepatocytes. These data identify CB2 agonists as potential therapeutic agents for the management of alcoholic liver disease. (HEPATOLOGY 2011;)

title: GRP78 expression inhibits insulin and ER stress-induced SREBP-1c activation and reduces hepatic steatosis in mice.
abstract: Hepatic steatosis is present in insulin-resistant obese rodents and is concomitant with active lipogenesis. Hepatic lipogenesis depends on the insulin-induced activation of the transcription factor SREBP-1c. Despite prevailing insulin resistance, SREBP-1c is activated in the livers of genetically and diet-induced obese rodents. Recent studies have reported the presence of an ER stress response in the livers of obese ob/ob mice. To assess whether ER stress promotes SREBP-1c activation and thus contributes to lipogenesis, we overexpressed the chaperone glucose-regulated protein 78 (GRP78) in the livers of ob/ob mice using an adenoviral vector. GRP78 overexpression reduced ER stress markers and inhibited SREBP-1c cleavage and the expression of SREBP-1c and SREBP-2 target genes. Furthermore, hepatic triglyceride and cholesterol contents were reduced, and insulin sensitivity improved, in GRP78-injected mice. These metabolic improvements were likely mediated by restoration of IRS-2 expression and tyrosine phosphorylation. Interestingly, GRP78 overexpression also inhibited insulin-induced SREBP-1c cleavage in cultured primary hepatocytes. These findings demonstrate that GRP78 inhibits both insulin-dependent and ER stress-dependent SREBP-1c proteolytic cleavage and explain the role of ER stress in hepatic steatosis in obese rodents.

title: Nogo‐B: A potential indicator for hepatic cirrhosis and regulator in hepatic stellate cell activation
abstract: To evaluate plasma Nogo‐B levels in liver cirrhotic patients and declare a novel molecular basis by which Nogo‐B modulates hepatic stellate cell (HSC) activation.

title: Recent advances in alcoholic liver disease II. Minireview: molecular mechanisms of alcoholic fatty liver.
abstract: Alcohol has long been thought to cause fatty liver by way of altered NADH/NAD(+) redox potential in the liver, which, in turn, inhibits fatty acid oxidation and the activity of tricarboxylic acid cycle reactions. More recent studies indicate that additional effects of ethanol both impair fat oxidation and stimulate lipogenesis. Ethanol interferes with DNA binding and transcription-activating properties of peroxisome proliferator-activated receptor-alpha (PPARalpha), as demonstrated with cultured cells and in ethanol-fed mice. Treatment of ethanol-fed mice with a PPARalpha agonist can reverse fatty liver even in the face of continued ethanol consumption. Ethanol also activated sterol regulatory element binding protein 1, inducing a battery of lipogenic enzymes. These effects may be due in part to inhibition of AMP-dependent protein kinase, reduction in plasma adiponectin, or increased levels of TNF-alpha in the liver. The understanding of these ethanol effects provides new therapeutic targets to reverse alcoholic fatty liver.

title: Molecular mechanisms of alcoholic fatty liver: role of peroxisome proliferator-activated receptor alpha.
abstract: Normal function of the peroxisome proliferator-activated receptor alpha (PPARalpha) is crucial for the regulation of hepatic fatty acid metabolism. Fatty acids serve as ligands for PPARalpha, and when fatty acid levels increase, activation of PPARalpha induces a battery of fatty acid-metabolizing enzymes to restore fatty acid levels to normal. Hepatic fatty acid levels are increased during ethanol consumption. However, results of in vitro work showed that ethanol metabolism inhibited the ability of PPARalpha to bind DNA and activate reporter genes. This observation has been further studied in mice. Four weeks of ethanol feeding of C57BL/6J mice also impairs fatty acid catabolism in liver by blocking PPARalpha-mediated responses. Ethanol feeding decreased the level of retinoid X receptor alpha (RXRalpha) as well as the ability of PPARalpha/RXR in liver nuclear extracts to bind its consensus sequence, and the levels of mRNAs for several PPARalpha-regulated genes were reduced [long-chain acyl coenzyme A (acyl-CoA) dehydrogenase and medium-chain acyl-CoA dehydrogenase] or failed to be induced (acyl-CoA dehydrogenase, liver carnitine palmitoyl-CoA transferase I, very long-chain acyl-CoA synthetase, very long-chain acyl-CoA dehydrogenase) in livers of the ethanol-fed animals. Consistent with this finding, ethanol feeding did not induce the rate of fatty acid beta-oxidation, as assayed in liver homogenates. Inclusion of WY14,643, a PPARalpha agonist, in the diet restored the DNA-binding activity of PPARalpha/RXR, induced mRNA levels of several PPARalpha target genes, stimulated the rate of fatty acid beta-oxidation in liver homogenates, and prevented fatty liver in ethanol-fed animals. Blockade of PPARalpha function during ethanol consumption contributes to the development of alcoholic fatty liver, which can be overcome by WY14,643.

title: Inducible nitric oxide synthase is required in alcohol-induced liver injury: studies with knockout mice.
abstract: Oxidative stress contributes to early alcohol-induced liver injury, and superoxide (O(2)*-) production from NADPH oxidase plays a key role. However, the production of the free radical nitric oxide (NO*) by inducible nitric oxide synthase (iNOS) could also be involved.



To test this hypothesis, iNOS knockout (B6.129P2-Nos2 (tm1 Lau)) and wild-type mice were fed high-fat control or ethanol-containing diets for 4 weeks.



Mean body weight gains were not significantly different between treatment groups, and average urine ethanol concentrations were similar in wild-type and iNOS knockout mice. After 4 weeks, serum alanine aminotransferase (ALT) levels were increased significantly about 4-fold over control values (29 +/- IU/L) by enteral ethanol (113 +/- 20) in wild-type mice; this effect of ethanol was significantly blunted in iNOS knockout mice (50 +/- 9). Similar protective effects against liver damage were observed if wild-type mice were treated with the iNOS inhibitor N -(3-aminomethyl)benzyl-acetamindine (1400W). Enteral ethanol also caused severe fatty accumulation, mild inflammation, and necrosis in the liver in wild-type mice but had no effect in iNOS knockout mice. The accumulation of 4-hydroxynonenal (lipid peroxidation) and 3-nitrotyrosine (reactive nitrogen species formation) protein adducts caused by alcohol was completely blocked in iNOS knockout mice.



These data strongly support the hypothesis that iNOS is required for the pathogenesis of early alcohol-induced hepatitis by production of nitric oxide-derived pro-oxidants (e.g., peroxynitrite).