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Leptin is an adipokine with roles in food intake and energy metabolism through its actions on neurons in the hypothalamus. The role of leptin in obesity and cardiovascular disorders is well documented. However, its influence on liver conditions such as cholestasis is poorly understood. The effects of exogenous leptin and leptin-neutralizing antibody on biliary hyperplasia, hepatic fibrosis, and inflammation in the multidrug resistance protein 2 knockout (Mdr2KO) mouse model of cholestasis were assessed by quantifying markers specific for cholangiocytes, activated hepatic stellate cells (HSCs), and cytokines. Serum and hepatic leptin were increased in Mdr2KO mice compared with FVB/NJ (FVBN) controls, and exogenous leptin enhanced biliary hyperplasia and liver fibrosis in Mdr2KO and FVBN mice. Leptin administration increased hepatic expression of C-C motif chemokine ligand 2 and IL-6 in Mdr2KO mice. In contrast, leptin-neutralizing antibody reduced intrahepatic bile duct mass and decreased HSC activation in Mdr2KO mice compared with FVBN controls. Sex-related differences were noted, with female Mdr2KO mice having more leptin than males. In cholangiocytes and LX2 cells in vitro, leptin increased phosphorylated Akt and stimulated cell proliferation. Leptin receptor siRNA and inhibitors of Akt phosphorylation impaired leptin-induced cell proliferation and proinflammatory cytokines. The current data suggest that leptin is abnormally increased in cholestatic mice, and excess leptin increases ductular reaction, hepatic fibrosis, and inflammation via leptin receptor–mediated phosphorylation of Akt in cholangiocytes and HSCs.

Galanin (Gal) is a peptide with a role in neuroendocrine regulation of the liver. In this study, we assessed the role of Gal and its receptors, Gal receptor 1 (GalR1) and Gal receptor 2 (GalR2), in cholangiocyte proliferation and liver fibrosis in multidrug resistance protein 2 knockout (Mdr2KO) mice as a model of chronic hepatic cholestasis. The distribution of Gal, GalR1, and GalR2 in specific liver cell types was assessed by laser-capture microdissection and confocal microscopy. Galanin immunoreactivity was detected in cholangiocytes, hepatic stellate cells (HSCs), and hepatocytes. Cholangiocytes expressed GalR1, whereas HSCs and hepatocytes expressed GalR2. Strategies were used to either stimulate or block GalR1 and GalR2 in FVB/N (wild-type) and Mdr2KO mice and measure biliary hyperplasia and hepatic fibrosis by quantitative PCR and immunostaining of specific markers. Galanin treatment increased cholangiocyte proliferation and fibrogenesis in both FVB/N and Mdr2KO mice. Suppression of GalR1, GalR2, or both receptors in Mdr2KO mice resulted in reduced bile duct mass and hepatic fibrosis. In vitro knockdown of GalR1 in cholangiocytes reduced α-smooth muscle actin expression in LX-2 cells treated with cholangiocyte-conditioned media. A GalR2 antagonist inhibited HSC activation when Gal was administered directly to LX-2 cells, but not via cholangiocyte-conditioned media. These data demonstrate that Gal contributes not only to cholangiocyte proliferation but also to liver fibrogenesis via the coordinate activation of GalR1 in cholangiocytes and GalR2 in HSCs.

Hepatic encephalopathy describes an array of neurological complications that arise due to liver insufficiency. The pathogenesis of hepatic encephalopathy shares a longstanding association with hyperammonemia and inflammation, and recently, aberrant bile acid signaling has been implicated in the development of key features of hepatic encephalopathy. These key features include neuronal dysfunction, neuroinflammation and blood-brain barrier permeability. This review summarizes the findings of recent studies demonstrating a role for bile acids in the pathogenesis of hepatic encephalopathy via one of three main bile acid receptors and speculates on the possible downstream consequences of aberrant bile acid signaling.

Acute liver failure is a devastating consequence of hepatotoxic liver injury that can lead to the development of hepatic encephalopathy. There is no consensus on the best model to represent these syndromes in mice, and therefore the aim of this study was to classify hepatic and neurological consequences of azoxymethane- and thioacetamide-induced liver injury. Azoxymethane-treated mice were euthanized at time points representing absence of minor and significant stages of neurological decline. Thioacetamide-treated mice had tissue collected at up to 3 days following daily injections. Liver histology, serum chemistry, bile acids, and cytokine levels were measured. Reflexes, grip strength measurement, and ataxia were calculated for all groups. Brain ammonia, bile acid levels, cerebral edema, and neuroinflammation were measured. Finally, in vitro and in vivo assessments of blood‐brain barrier function were performed. Serum transaminases and liver histology demonstrate that both models generated hepatotoxic liver injury. Serum proinflammatory cytokine levels were significantly elevated in both models. Azoxymethane-treated mice had progressive neurological deficits, while thioacetamide-treated mice had inconsistent neurological deficits. Bile acids and cerebral edema were increased to a higher degree in azoxymethane-treated mice, while cerebral ammonia and neuroinflammation were greater in thioacetamide-treated mice. Blood‐brain barrier permeability exists in both models but was likely not due to direct toxicity of azoxymethane or thioacetamide on brain endothelial cells. In conclusion, both models generate acute liver injury and hepatic encephalopathy, but the requirement of a single injection and the more consistent neurological decline make azoxymethane treatment a better model for acute liver failure with hepatic encephalopathy.

Background & Aims Hepatic encephalopathy is a serious neurologic complication of acute and chronic liver diseases. We previously showed that aberrant bile acid signaling contributes to the development of hepatic encephalopathy via farnesoid X receptor (FXR)-mediated mechanisms in neurons. In the brain, a novel alternative bile acid synthesis pathway, catalyzed by cytochrome p450 46A1 (Cyp46A1), is the primary mechanism by which the brain regulates cholesterol homeostasis. The aim of this study was to determine if FXR activation in the brain altered cholesterol homeostasis during hepatic encephalopathy. Methods Cyp7A1-/- mice or C57Bl/6 mice pretreated with central infusion of FXR vivo morpholino, 2-hydroxypropyl-β-cyclodextrin, or fed a cholestyramine-supplemented diet were injected with azoxymethane (AOM). Cognitive and neuromuscular impairment as well as liver damage and expression of Cyp46A1 were assessed using standard techniques. The subsequent cholesterol content in the frontal cortex was measured using commercially available kits and by Filipin III and Nile Red staining. Results There was an increase in membrane-bound and intracellular cholesterol in the cortex of mice treated with AOM that was associated with decreased Cyp46A1 expression. Strategies to inhibit FXR signaling prevented the down-regulation of Cyp46A1 and the accumulation of cholesterol. Treatment of mice with 2-hydroxypropyl-β-cyclodextrin attenuated the AOM-induced cholesterol accumulation in the brain and the cognitive and neuromuscular deficits without altering the underlying liver pathology. Conclusions During hepatic encephalopathy, FXR signaling increases brain cholesterol and contributes to neurologic decline. Targeting cholesterol accumulation in the brain may be a possible therapeutic target for the management of hepatic encephalopathy.

Acute liver failure is a serious consequence of acetaminophen (APAP)-induced hepatotoxic liver injury with high rates of morbidity and mortality. Transforming growth factor beta 1 (TGFβ1) is elevated during liver injury and influences hepatocyte senescence during APAP-induced hepatotoxicity. This study investigated TGFβ1 signaling in the context of inflammation, necrotic cell death, and oxidative stress during APAP-induced liver injury. Male C57Bl/6 mice were injected with 600 mg/kg APAP to generate liver injury in the presence or absence of the TGFβ receptor 1 inhibitor, GW788388, 1 h prior to APAP administration. Acetaminophen-induced liver injury was characterized using histological and biochemical measures. Transforming growth factor beta 1 expression and signal transduction were assessed using immunohistochemistry, Western blotting and ELISA assays. Hepatic necrosis, liver injury, cell proliferation, hepatic inflammation, and oxidative stress were assessed in all mice. Acetaminophen administration significantly induced necrosis and elevated serum transaminases compared with control mice. Transforming growth factor beta 1 staining was observed in and around areas of necrosis with phosphorylation of SMAD3 observed in hepatocytes neighboring necrotic areas in APAP-treated mice. Pretreatment with GW788388 prior to APAP administration in mice reduced hepatocyte cell death and stimulated regeneration. Phosphorylation of SMAD3 was reduced in APAP mice pretreated with GW788388 and this correlated with reduced hepatic cytokine production and oxidative stress. These results support that TGFβ1 signaling plays a significant role in APAP-induced liver injury by influencing necrotic cell death, inflammation, oxidative stress, and hepatocyte regeneration. In conclusion, targeting TGFβ1 or downstream signaling may be a possible therapeutic target for the management of APAP-induced liver injury.

Background Acute liver failure resulting from drug-induced liver injury can lead to the development of neurological complications called hepatic encephalopathy (HE). Hepatic transforming growth factor beta 1 (TGFβ1) is upregulated due to liver failure in mice and inhibiting circulating TGFβ reduced HE progression. However, the specific contributions of TGFβ1 on brain cell populations and neuroinflammation during HE are not known. Therefore, the aim of this study was to characterize hepatic and brain TGFβ1 signaling during acute liver failure and its contribution to HE progression using a combination of pharmacological and genetic approaches. Methods C57Bl/6 or neuron-specific transforming growth factor beta receptor 2 (TGFβR2) null mice (TGFβR2ΔNeu) were treated with azoxymethane (AOM) to induce acute liver failure and HE. The activity of circulating TGFβ1 was inhibited in C57Bl/6 mice via injection of a neutralizing antibody against TGFβ1 (anti-TGFβ1) prior to AOM injection. In all mouse treatment groups, liver damage, neuroinflammation, and neurological deficits were assessed. Inflammatory signaling between neurons and microglia were investigated in in vitro studies through the use of pharmacological inhibitors of TGFβ1 signaling in HT-22 and EOC-20 cells. Results TGFβ1 was expressed and upregulated in the liver following AOM injection. Pharmacological inhibition of TGFβ1 after AOM injection attenuated neurological decline, microglia activation, and neuroinflammation with no significant changes in liver damage. TGFβR2ΔNeu mice administered AOM showed no effect on liver pathology but significantly reduced neurological decline compared to control mice. Microglia activation and neuroinflammation were attenuated in mice with pharmacological inhibition of TGFβ1 or in TGFβR2ΔNeu mice. TGFβ1 increased chemokine ligand 2 (CCL2) and decreased C-X3-C motif ligand 1 (CX3CL1) expression in HT-22 cells and reduced interleukin-1 beta (IL-1ß) expression, tumor necrosis factor alpha (TNFα) expression, and phagocytosis activity in EOC-20 cells. Conclusion Increased circulating TGFβ1 following acute liver failure results in activation of neuronal TGFβR2 signaling, driving neuroinflammation and neurological decline during AOM-induced HE. Electronic supplementary material The online version of this article (10.1186/s12974-019-1455-y) contains supplementary material, which is available to authorized users.

Hepatic cholestasis is associated with a significant suppression of the hypothalamus-pituitary-adrenal axis (HPA). In the present study, we tested the hypothesis that activation of the HPA axis by corticosterone treatment can reverse liver inflammation and fibrosis in a multidrug resistance protein 2 knockout (MDR2KO) transgenic mouse model of hepatic cholestasis. Friend Virus B NIH-Jackson (FVBN) control and MDR2KO male and female mice were treated with vehicle or corticosterone for two weeks, then serum and liver analyses of hepatic cholestasis markers were performed. Indicators of inflammation, such as increased numbers of macrophages, were determined. MDR2KO mice had lower corticotropin releasing hormone and corticosterone levels than FVBN controls in the serum. There was a large accumulation of CD68 and F4/80 macrophages in MDR2KO mice livers, which indicated greater inflammation compared to FVBNs, an effect reversed by corticosterone treatment. Intrahepatic biliary duct mass, collagen deposition and alpha smooth muscle actin (αSMA) were found to be much higher in livers of MDR2KO mice than in controls; corticosterone treatment significantly decreased these fibrosis markers. When looking at the gender-specific response to corticosterone treatment, male MDR2KO mice tended to have a more pronounced reversal of liver fibrosis than females treated with corticosterone.