Behnam Saberi

Role of JNK Translocation to Mitochondria Leading to Inhibition of Mitochondria Bioenergetics in Acetaminophen-induced Liver Injury

Previously, we demonstrated JNK plays a central role in acetaminophen (APAP)-induced liver injury (Gunawan, B. K., Liu, Z. X., Han, D., Hanawa, N., Gaarde, W. A., and Kaplowitz, N. (2006) Gastroenterology 131, 165–178). In this study, we examine the mechanism involved in activating JNK and explore the downstream targets of JNK important in promoting APAP-induced liver injury in vivo. JNK inhibitor (SP600125) was observed to significantly protect against APAP-induced liver injury. Increased mitochondria-derived reactive oxygen species were implicated in APAP-induced JNK activation based on the following: 1) mitochondrial GSH depletion (maximal at 2 h) caused increased H2O2 release from mitochondria, which preceded JNK activation (maximal at 4 h); 2) treatment of isolated hepatocytes with H2O2 or inhibitors (e.g. antimycin) that cause increased H2O2 release from mitochondria-activated JNK. An important downstream target of JNK following activation was mitochondria based on the following: 1) JNK translocated to mitochondria following activation; 2) JNK inhibitor treatment partially protected against a decline in mitochondria respiration caused by APAP treatment; and 3) addition of purified active JNK to mitochondria isolated from mice treated with APAP plus JNK inhibitor (mitochondria with severe GSH depletion, covalent binding) directly inhibited respiration. Cyclosporin A blocked the inhibitory effect of JNK on mitochondria respiration, suggesting JNK was directly inducing mitochondrial permeability transition in isolated mitochondria from mice treated with APAP plus JNK inhibitor. Addition of JNK to mitochondria isolated from control mice did not affect respiration. Our results suggests that APAP-induced liver injury involves JNK activation, due to increased reactive oxygen species generated by GSH-depleted mitochondria, and translocation of activated JNK to mitochondria where JNK induces mitochondrial permeability transition and inhibits mitochondria bioenergetics.

Signal Transduction Pathways Involved in Drug-Induced Liver Injury

Hepatocyte death following drug intake is the critical event in the clinical manifestation of drug-induced liver injury (DILI). Traditionally, hepatocyte death caused by drugs had been attributed to overwhelming oxidative stress and mitochondria dysfunction caused by reactive metabolites formed during drug metabolism. However, recent studies have also shown that signal transduction pathways activated/inhibited during oxidative stress play a key role in DILI. In acetaminophen (APAP)-induced liver injury, hepatocyte death requires the sustained activation of c-Jun kinase (JNK), a kinase important in mediating apoptotic and necrotic death. Inhibition of JNK using chemical inhibitors or knocking down JNK can prevent hepatocyte death even in the presence of extensive glutathione (GSH) depletion, covalent binding, and oxidative stress. Once activated, JNK translocates to mitochondria, to induce mitochondria permeability transition and trigger hepatocyte death. Mitochondria are central targets where prodeath kinases such as JNK, prosurvival death proteins such as bcl-xl, and oxidative damage converge to determine hepatocyte survival. The importance of mitochondria in DILI is also observed in the Mn-SOD heterozygous (+/-) model, where mice with less mitochondrial Mn-SOD are sensitized to liver injury caused by certain drugs. An extensive body of research is accumulating suggesting a central role of mitochondria in DILI. Drugs can also cause redox changes that inhibit important prosurvival pathways such as NF-kappaB. The inhibition of NF-kappaB by subtoxic doses of APAP sensitizes hepatocyte to the cytotoxic actions of tumor necrosis factor (TNF). Many drugs will induce liver injury if simultaneously treated with LPS, which promotes inflammation and cytokine release. Drugs may be sensitizing hepatocytes to the cytotoxic effects of cytokines such as TNF, or vice versa. Overall many signaling pathways are important in regulating DILI, and represent potential therapeutic targets to reduce liver injury caused by drugs.

Protein kinase C (PKC) participates in acetaminophen hepatotoxicity through c-jun-N-terminal kinase (JNK)-dependent and -independent signaling pathways

This study examines the role of protein kinase C (PKC) and AMP‐activated kinase (AMPK) in acetaminophen (APAP) hepatotoxicity. Treatment of primary mouse hepatocytes with broad‐spectrum PKC inhibitors (Ro‐31‐8245, Go6983), protected against APAP cytotoxicity despite sustained c‐jun‐N‐terminal kinase (JNK) activation. Broad‐spectrum PKC inhibitor treatment enhanced p‐AMPK levels and AMPK regulated survival‐energy pathways including autophagy. AMPK inhibition by compound C or activation using an AMPK activator oppositely modulated APAP cytotoxicity, suggesting that p‐AMPK and AMPK regulated energy survival pathways, particularly autophagy, play a critical role in APAP cytotoxicity. Ro‐31‐8245 treatment in mice up‐regulated p‐AMPK levels, increased autophagy (i.e., increased LC3‐II formation, p62 degradation), and protected against APAP‐induced liver injury, even in the presence of sustained JNK activation and translocation to mitochondria. In contrast, treatment of hepatocytes with a classical PKC inhibitor (Go6976) protected against APAP by inhibiting JNK activation. Knockdown of PKC‐α using antisense (ASO) in mice also protected against APAP‐induced liver injury by inhibiting JNK activation. APAP treatment resulted in PKC‐α translocation to mitochondria and phosphorylation of mitochondrial PKC substrates. JNK 1 and 2 silencing in vivo decreased APAP‐induced PKC‐α translocation to mitochondria, suggesting PKC‐α and JNK interplay in a feed‐forward mechanism to mediate APAP‐induced liver injury. Conclusion: PKC‐α and other PKC(s) regulate death (JNK) and survival (AMPK) proteins, to modulate APAP‐induced liver injury. (Hepatology 2014;59:1543‐1554)

Current Management of Alcoholic Hepatitis and Future Therapies.

Abstract Alcohol is one of the most common etiologies of liver disease, and alcoholic liver disease overall is the second most common indication for liver transplantation in the United States. It encompasses a spectrum of disease, including fatty liver disease, alcoholic hepatitis (AH), and alcoholic cirrhosis. AH can range from mild to severe disease, with severe disease being defined as: Discriminant Function (DF) ≥ 32, or Model for End-stage Liver Disease (MELD) ≥ 21, or presence of hepatic encephalopathy. Management of the mild disease consists mainly of abstinence and supportive care. Severe AH is associated with significant mortality. Currently, there is no ideal medical treatment for this condition. Besides alcohol cessation, corticosteroids have been used with conflicting results and are associated with an inherent risk of infection. Overall steroids have shown short term benefit when compared to placebo, but they have no obvious long term benefits. Pentoxifylline does not improve survival in patients with severe AH and is no longer recommended based on the results of the STOPAH (Steroid Or Pentoxifylline for Alcoholic Hepatitis) trial. Anti-tumor necrosis factor (TNF) agents are associated with increased risk of life threatening infections and death. Currently, early stage trials are underway, mainly targeting novel pathways based on disease pathogenesis, including modulation of innate immune system, inhibition of gut-liver axis and cell death pathways, and activation of transcription factor farnesyl X receptor (FXR). Future treatment may lie in human induced pluripotent stem cell (iPSC) technology, which is currently under investigation for the study of pathogenesis, drug discovery, and stem cell transplantation. Liver transplantation has been reported with good results in highly selected patients but is controversial due to limited organ supply.

Influence of vitamin D on liver fibrosis in chronic hepatitis C: A systematic review and meta-analysis of the pooled clinical trials data

AIM To investigate the relationship between vitamin D and liver fibrosis in hepatitis C-monoinfected or hepatitis C virus (HCV)-human immunodeficiency virus (HIV) co-infected patients. METHODS Pertinent studies were located by a library literature search in PubMed/Embase/Cochrane/Scopus/LILACS by two individual reviewers. Inclusion criteria: (1) studies with patients with HCV or co-infected HCV/HIV; (2) studies with patients ≥ 18 years old; (3) studies that evaluated liver fibrosis stage, only based on liver biopsy; and (4) studies that reported serum or plasma 25(OH)D levels. Studies that included pediatric patients, other etiologies of liver disease, or did not use liver biopsy for fibrosis evaluation, or studies with inadequate data were excluded. Estimated measures of association reported in the literature, as well as corresponding measures of uncertainty, were recorded and corresponding odds ratios with 95%CI were included in a meta-analysis. RESULTS The pooled data of this systematic review showed that 9 of the 12 studies correlated advanced liver disease defined as a Metavir value of F3/4 with 25(OH) D level insufficiency. The meta-analysis indicated a significant association across studies. CONCLUSION Low vitamin D status is common in chronic Hepatitis C patients and is associated with advanced liver fibrosis.

Isolated arterioportal fistula presenting with variceal hemorrhage

We report a case of life-threatening hematemesis due to portal hypertension caused by an isolated arterioportal fistula (APF). Intrahepatic APFs are extremely rare and are a cause of presinusoidal portal hypertension. Etiologies for APFs are comprised of precipitating trauma, malignancy, and hereditary hemorrhagic telangiectasia, but these were not the case in our patient. Idiopathic APFs are usually due to congenital vascular abnormalities and thus usually present in the pediatric setting. This is one of the first cases of adult-onset isolated APF who presented with portal hypertension and was successfully managed through endoscopic hemostasis and subsequent interventional radiological embolization.

Prediction of histologic alcoholic hepatitis based on clinical presentation limits the need for liver biopsy

The clinical presentation of alcoholic hepatitis (AH) can be mimicked by other alcoholic liver diseases. The aim of this study was to identify clinical features that predict AH on liver biopsy. Biopsies from patients hospitalized for presumed severe AH were used to identify a derivation cohort (101 patients) and validation cohort (71 patients). Using histologic scores for hepatocyte ballooning, Mallory‐Denk bodies, and lobular inflammation, 95 patient biopsies (55%) were classified as definite AH, 55 (32%) as possible AH, and 22 (13%) as no AH. Survival was similar among the groups, but mortality was significantly increased for patients with fatty change ≤50% on initial liver biopsy. An analysis limited to uninfected patients with definite AH or no AH in the derivation cohort identified a greater leukocyte count at admission and radiographic evidence of liver surface nodularity as independent predictors of definite AH on biopsy (P < 0.05). In the derivation cohort, the leukocyte count thresholds for ensuring 100% specificity for diagnosing definite AH were 10 × 109/L if the liver surface was nodular and 14 × 109/L if the liver surface was smooth, with a sensitivity of 76% and an area under the receiver operator characteristic curve of 0.88. In the validation cohort, these thresholds had a specificity of 86%, a sensitivity of 59%, and an area under the receiver operator characteristic curve of 0.72. Conclusion: The combination of an elevated leukocyte count and a nodular liver surface in the absence of active infection retrospectively identified patients with a high likelihood of histologic AH for whom liver biopsy may not be necessary. For patients with suspected severe AH who do not fulfill these criteria, liver biopsy is important to exclude other variants of alcoholic liver disease. (Hepatology Communications 2017;1:1070–1084)

Protein kinase C (PKC) participates in acetaminophen hepatotoxicity through JNK dependent and independent signaling pathways

This study examines the role of protein kinase C (PKC) and AMP‐activated kinase (AMPK) in acetaminophen (APAP) hepatotoxicity. Treatment of primary mouse hepatocytes with broad‐spectrum PKC inhibitors (Ro‐31‐8245, Go6983), protected against APAP cytotoxicity despite sustained c‐jun‐N‐terminal kinase (JNK) activation. Broad‐spectrum PKC inhibitor treatment enhanced p‐AMPK levels and AMPK regulated survival‐energy pathways including autophagy. AMPK inhibition by compound C or activation using an AMPK activator oppositely modulated APAP cytotoxicity, suggesting that p‐AMPK and AMPK regulated energy survival pathways, particularly autophagy, play a critical role in APAP cytotoxicity. Ro‐31‐8245 treatment in mice up‐regulated p‐AMPK levels, increased autophagy (i.e., increased LC3‐II formation, p62 degradation), and protected against APAP‐induced liver injury, even in the presence of sustained JNK activation and translocation to mitochondria. In contrast, treatment of hepatocytes with a classical PKC inhibitor (Go6976) protected against APAP by inhibiting JNK activation. Knockdown of PKC‐α using antisense (ASO) in mice also protected against APAP‐induced liver injury by inhibiting JNK activation. APAP treatment resulted in PKC‐α translocation to mitochondria and phosphorylation of mitochondrial PKC substrates. JNK 1 and 2 silencing in vivo decreased APAP‐induced PKC‐α translocation to mitochondria, suggesting PKC‐α and JNK interplay in a feed‐forward mechanism to mediate APAP‐induced liver injury. Conclusion: PKC‐α and other PKC(s) regulate death (JNK) and survival (AMPK) proteins, to modulate APAP‐induced liver injury. (Hepatology 2014;59:1543‐1554)

A 45-Year-Old Man With Recurrent Nausea, Vomiting, and Abdominal Distension

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Bilal Hameed, Uma Mahadevan, and Kay Washington, Section Editors 61 62 63 64 A 45-Year-Old Man With Recurrent Nausea, Vomiting, and Abdominal Distension 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 Nuha Alammar, Sonali Palchaudhuri, and Behnam Saberi

Protein kinase C (PKC) participates in acetaminophen hepatotoxicity through c-jun-N-terminal kinase (JNK)-dependent and -independent signaling pathways: Saberi, Ybanez, et al.

This study examines the role of protein kinase C (PKC) and AMP‐activated kinase (AMPK) in acetaminophen (APAP) hepatotoxicity. Treatment of primary mouse hepatocytes with broad‐spectrum PKC inhibitors (Ro‐31‐8245, Go6983), protected against APAP cytotoxicity despite sustained c‐jun‐N‐terminal kinase (JNK) activation. Broad‐spectrum PKC inhibitor treatment enhanced p‐AMPK levels and AMPK regulated survival‐energy pathways including autophagy. AMPK inhibition by compound C or activation using an AMPK activator oppositely modulated APAP cytotoxicity, suggesting that p‐AMPK and AMPK regulated energy survival pathways, particularly autophagy, play a critical role in APAP cytotoxicity. Ro‐31‐8245 treatment in mice up‐regulated p‐AMPK levels, increased autophagy (i.e., increased LC3‐II formation, p62 degradation), and protected against APAP‐induced liver injury, even in the presence of sustained JNK activation and translocation to mitochondria. In contrast, treatment of hepatocytes with a classical PKC inhibitor (Go6976) protected against APAP by inhibiting JNK activation. Knockdown of PKC‐α using antisense (ASO) in mice also protected against APAP‐induced liver injury by inhibiting JNK activation. APAP treatment resulted in PKC‐α translocation to mitochondria and phosphorylation of mitochondrial PKC substrates. JNK 1 and 2 silencing in vivo decreased APAP‐induced PKC‐α translocation to mitochondria, suggesting PKC‐α and JNK interplay in a feed‐forward mechanism to mediate APAP‐induced liver injury. Conclusion: PKC‐α and other PKC(s) regulate death (JNK) and survival (AMPK) proteins, to modulate APAP‐induced liver injury. (Hepatology 2014;59:1543‐1554)