Sharon Manley

Differential Roles of Unsaturated and Saturated Fatty Acids on Autophagy and Apoptosis in Hepatocytes

Fatty acid-induced lipotoxicity plays a critical role in the pathogenesis of nonalcoholic liver disease. Saturated fatty acids and unsaturated fatty acids have differential effects on cell death and steatosis, but the mechanisms responsible for these differences are not known. Using cultured HepG2 cells and primary mouse hepatocytes, we found that unsaturated and saturated fatty acids differentially regulate autophagy and apoptosis. The unsaturated fatty acid, oleic acid, promoted the formation of triglyceride-enriched lipid droplets and induced autophagy but had a minimal effect on apoptosis. In contrast, the saturated fatty acid, palmitic acid, was poorly converted into triglyceride-enriched lipid droplets, suppressed autophagy, and significantly induced apoptosis. Subsequent studies revealed that palmitic acid-induced apoptosis suppressed autophagy by inducing caspase-dependent Beclin 1 cleavage, indicating cross-talk between apoptosis and autophagy. Moreover, our data suggest that the formation of triglyceride-enriched lipid droplets and induction of autophagy are protective mechanisms against fatty acid-induced lipotoxicity. In line with our in vitro findings, we found that high-fat diet-induced hepatic steatosis was associated with autophagy in the mouse liver. Potential modulation of autophagy may be a novel approach that has therapeutic benefits for obesity-induced steatosis and liver injury.

Parkin and Mitofusins Reciprocally Regulate Mitophagy and Mitochondrial Spheroid Formation

Background: The mechanistic relationship of Parkin and mitofusins in mitochondria quality control is not known. Results: CCCP-induced mitophagy and mitochondrial spheroid formation differentially require Parkin and mitofusins. Conclusion: Parkin and mitofusins reciprocally regulate mitophagy and mitochondrial spheroid formation. Significance: This study revealed a default response of mitochondria to oxidative stress and a molecular mechanism by which Parkin primes the mitochondria for mitophagy. Mitochondrial homeostasis via mitochondrial dynamics and quality control is crucial to normal cellular functions. Mitophagy (mitochondria removed by autophagy) stimulated by a mitochondrial uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP), requires Parkin, but it is not clear why Parkin is crucial to this process. We found that in the absence of Parkin, carbonyl cyanide m-chlorophenylhydrazone induced the formation of mitochondrial spheroids. Mitochondrial spheroid formation is also induced in vivo in the liver by acetaminophen overdose, a condition causing severe oxidative mitochondrial damages and liver injury. Mitochondrial spheroids could undergo a maturation process by interactions with acidic compartments. The formation of this new structure required reactive oxygen species and mitofusins. Parkin suppressed these mitochondrial dynamics by promoting mitofusin degradation. Consistently, genetic deletion of mitofusins without concomitant expression of Parkin was sufficient to prevent mitochondrial spheroid formation and resumed mitophagy. Mitochondrial spheroid formation and mitophagy could represent different strategies of mitochondrial homeostatic response to oxidative stress and are reciprocally regulated by mitofusins and Parkin.

The Emerging Role of Autophagy in Alcoholic Liver Disease

Autophagy is a highly conserved intracellular catabolic pathway that degrades cellular long-lived proteins and organelles. Autophagy is normally activated in response to nutrient deprivation and other stresses as a cell survival mechanism. Accumulating evidence indicates that autophagy plays a critical role in liver pathophysiology, in addition to maintaining hepatic energy and nutrient balance. Alcohol consumption causes hepatic metabolic changes, oxidative stress, accumulation of lipid droplets and damaged mitochondria; all of these can be regulated by autophagy. This review summarizes the recent findings about the role and mechanisms of autophagy in alcoholic liver disease (ALD), and the possible intervention for treating ALD by modulating autophagy.

Role of p62/SQSTM1 in liver physiology and pathogenesis.

p62/sequestosome-1/A170/ZIP (hereafter referred to as p62) is a scaffold protein that has multiple functions, such as signal transduction, cell proliferation, cell survival, cell death, inflammation, tumourigenesis and oxidative stress response. While p62 is an autophagy substrate and is degraded by autophagy, p62 serves as an autophagy receptor for selective autophagic clearance of protein aggregates and organelles. Moreover, p62 functions as a signalling hub for various signalling pathways, including NF-κB, Nrf2 and mTOR. In this review, we discuss the pathophysiological role of p62 in the liver, including formation of hepatic inclusion bodies, cholestasis, obesity, insulin resistance, liver cell death and tumourigenesis.

New advances in molecular mechanisms and emerging therapeutic targets in alcoholic liver diseases

Alcoholic liver disease is a major health problem in the United States and worldwide. Chronic alcohol consumption can cause steatosis, inflammation, fibrosis, cirrhosis and even liver cancer. Significant progress has been made to understand key events and molecular players for the onset and progression of alcoholic liver disease from both experimental and clinical alcohol studies. No successful treatments are currently available for treating alcoholic liver disease; therefore, development of novel pathophysiological-targeted therapies is urgently needed. This review summarizes the recent progress on animal models used to study alcoholic liver disease and the detrimental factors that contribute to alcoholic liver disease pathogenesis including miRNAs, S-adenosylmethionine, Zinc deficiency, cytosolic lipin-1β, IRF3-mediated apoptosis, RIP3-mediated necrosis and hepcidin. In addition, we summarize emerging adaptive protective effects induced by alcohol to attenuate alcohol-induced liver pathogenesis including FoxO3, IL-22, autophagy and nuclear lipin-1α.

Chronic Deletion and Acute Knockdown of Parkin Have Differential Responses to Acetaminophen-induced Mitophagy and Liver Injury in Mice

Background: Parkin is required for mitophagy in cultured cells, but its role in liver injury is not known. Results: APAP-induced mitophagy was impaired in acute Parkin knockdown mouse livers but not whole body Parkin knock-out mice. Conclusion: Chronic, but not acute, loss of Parkin protects against APAP-induced liver injury by multiple mechanisms independent of mitophagy. Significance: This study revealed a novel role of Parkin in APAP-induced liver injury. We previously demonstrated that pharmacological induction of autophagy protected against acetaminophen (APAP)-induced liver injury in mice by clearing damaged mitochondria. However, the mechanism for removal of mitochondria by autophagy is unknown. Parkin, an E3 ubiquitin ligase, has been shown to be required for mitophagy induction in cultured mammalian cells following mitochondrial depolarization, but its role in vivo is not clear. The purpose of this study was to investigate the role of Parkin-mediated mitophagy in protection against APAP-induced liver injury. We found that Parkin translocated to mitochondria in mouse livers after APAP treatment followed by mitochondrial protein ubiquitination and mitophagy induction. To our surprise, we found that mitophagy still occurred in Parkin knock-out (KO) mice after APAP treatment based on electron microscopy analysis and Western blot analysis for some mitochondrial proteins, and Parkin KO mice were protected against APAP-induced liver injury compared with wild type mice. Mechanistically, we found that Parkin KO mice had decreased activated c-Jun N-terminal kinase (JNK), increased induction of myeloid leukemia cell differentiation protein (Mcl-1) expression, and increased hepatocyte proliferation after APAP treatment in their livers compared with WT mice. In contrast to chronic deletion of Parkin, acute knockdown of Parkin in mouse livers using adenovirus shRNA reduced mitophagy and Mcl-1 expression but increased JNK activation after APAP administration, which exacerbated APAP-induced liver injury. Therefore, chronic deletion (KO) and acute knockdown of Parkin have differential responses to APAP-induced mitophagy and liver injury in mice.

Suppression of Autophagic Flux by Bile Acids in Hepatocytes

Retention of bile acids (BAs) in the liver during cholestasis plays an important role in the development of cholestatic liver injury. Several studies have reported that high concentrations of certain BAs induce cell death and inflammatory response in the liver, and BAs may promote liver tumorigenesis. Macroautophagy (hereafter referred to as autophagy) is a lysosomal degradation process that regulates organelle and protein homeostasis and serves as a cell survival mechanism under a variety of stress conditions. However, it is not known if BAs modulate autophagy in hepatocytes. In the present study, we determined autophagic flux in livers of farnesoid X receptor (FXR) knockout (KO) mice that have increased concentrations of hepatic BAs and in primary cultured mouse hepatocytes treated with BAs. The results showed that autophagic flux was impaired in livers of FXR KO mice and in BA-treated primary mouse hepatocytes. Mechanistically, BAs did not affect the activities of cathepsin or the proteasome, but impaired autophagosomal-lysosomal fusion likely due to reduction of Rab7 protein expression and targeting to autophagosomes. In conclusion, BAs suppress autophagic flux in hepatocytes by impairing autophagosomal-lysosomal fusion, which may be implicated in bile acid-induced liver tumor promotion observed in FXR KO mice.

Farnesoid X receptor regulates forkhead Box O3a activation in ethanol-induced autophagy and hepatotoxicity.

Alcoholic liver disease encompasses a wide spectrum of pathogenesis including steatosis, fibrosis, cirrhosis, and alcoholic steatohepatitis. Autophagy is a lysosomal degradation process that degrades cellular proteins and damaged/excess organelles, and serves as a protective mechanism in response to various stresses. Acute alcohol treatment induces autophagy via FoxO3a-mediated autophagy gene expression and protects against alcohol-induced steatosis and liver injury in mice. Farnesoid X Receptor (FXR) is a nuclear receptor that regulates cellular bile acid homeostasis. In the present study, wild type and FXR knockout (KO) mice were treated with acute ethanol for 16 h. We found that ethanol treated-FXR KO mice had exacerbated hepatotoxicity and steatosis compared to wild type mice. Furthermore, we found that ethanol treatment had decreased expression of various essential autophagy genes and several other FoxO3 target genes in FXR KO mice compared with wild type mice. Mechanistically, we did not find a direct interaction between FXR and FoxO3. Ethanol-treated FXR KO mice had increased Akt activation, increased phosphorylation of FoxO3 resulting in decreased FoxO3a nuclear retention and DNA binding. Furthermore, ethanol treatment induced hepatic mitochondrial spheroid formation in FXR KO mice but not in wild type mice, which may serve as a compensatory alternative pathway to remove ethanol-induced damaged mitochondria in FXR KO mice. These results suggest that lack of FXR impaired FoxO3a-mediated autophagy and in turn exacerbated alcohol-induced liver injury.

Role of Hypoxia Inducing Factor-1β in Alcohol-Induced Autophagy, Steatosis and Liver Injury in Mice

Chronic alcohol causes liver hypoxia and steatosis, which eventually develops into alcoholic liver disease (ALD). While it has been known that alcohol consumption activates hepatic hypoxia inducing factor-1α (HIF-1α), conflicting results regarding the role of HIF-1α in alcohol-induced liver injury and steatosis in mice have been reported. In the present study, we aimed to use hepatocyte-specific HIF-1β knockout mice to eliminate the possible compensatory effects of the single knockout of the 1α subunit of HIF to study the role of HIFs in ALD. C57BL/6 wild type mice were treated with acute ethanol to mimic human binge drinking. Matched wild-type and hepatocyte specific HIF-1β knockout mice were also subjected to a recently established Gao-binge alcohol model to mimic chronic plus binge conditions, which is quite common in human alcoholics. We found that acute alcohol treatment increased BNIP3 and BNIP3L/NIX expression in primary cultured hepatocytes and in mouse livers, suggesting that HIF may be activated in these models. We further found that hepatocyte-specific HIF-1β knockout mice developed less steatosis and liver injury following the Gao-binge model or acute ethanol treatment compared with their matched wild type mice. Mechanistically, protection against Gao-binge treatment-induced steatosis and liver injury was likely associated with increased FoxO3a activation and subsequent induction of autophagy in hepatocyte-specific HIF-1β knockout mice.

Role of farnesoid X receptor and bile acids in alcoholic liver disease

Alcoholic liver disease (ALD) is one of the major causes of liver morbidity and mortality worldwide. Chronic alcohol consumption leads to development of liver pathogenesis encompassing steatosis, inflammation, fibrosis, cirrhosis, and in extreme cases, hepatocellular carcinoma. Moreover, ALD may also associate with cholestasis. Emerging evidence now suggests that farnesoid X receptor (FXR) and bile acids also play important roles in ALD. In this review, we discuss the effects of alcohol consumption on FXR, bile acids and gut microbiome as well as their impacts on ALD. Moreover, we summarize the findings on FXR, FoxO3a (forkhead box-containing protein class O3a) and PPARα (peroxisome proliferator-activated receptor alpha) in regulation of autophagy-related gene transcription program and liver injury in response to alcohol exposure.