Nuclear factor κB up-regulation of CCAAT/enhancer-binding protein ß mediates hepatocyte resistance to tumor necrosis factor α toxicity.

UNLABELLED: The sensitization of hepatocytes to cell death from tumor necrosis factor α (TNFα) underlies many forms of hepatic injury, including that from toxins. Critical for hepatocyte resistance to TNFα toxicity is activation of nuclear factor κB (NF-κB) signaling, which prevents TNFα-induced death by the up-regulation of protective proteins. To further define the mechanisms of hepatocyte sensitization to TNFα killing, immunoblot analysis comparing livers from mice treated with lipopolysaccharide (LPS) alone or LPS together with the hepatotoxin galactosamine (GalN) was performed to identify TNFα-induced protective proteins blocked by GalN. Levels of CCAAT/enhancer-binding protein ß (C/EBPß) were increased after LPS treatment but not GalN/LPS treatment. In a nontransformed rat hepatocyte cell line, TNFα-induced increases in C/EBPß protein levels were dependent on NF-κB-mediated inhibition of proteasomal degradation. Pharmacological inhibition of c-Jun N-terminal kinase (JNK) did not affect C/EBPß degradation, indicating that the process was JNK-independent. C/EBPß functioned to prevent cell death as adenoviral C/EBPß overexpression blocked TNFα-induced apoptosis in cells sensitized to TNFα toxicity by NF-κB inhibition. C/EBPß inhibited TNFα-induced caspase 8 activation and downstream mitochondrial cytochrome c release and caspase 3 and caspase 7 activation. Studies in primary hepatocytes from c/ebpß(-/-) mice confirmed that loss of C/EBPß increased death from TNFα. c/ebpß(-/-) mice were also sensitized to liver injury from a nontoxic dose of LPS or TNFα. The absence of jnk2 failed to reverse the GalN-induced block in C/EBPß induction by LPS, again demonstrating that C/EBPß degradation was JNK-independent. CONCLUSION: C/EBPß is up-regulated by TNFα and mediates hepatocyte resistance to TNFα toxicity by inhibiting caspase-dependent apoptosis. In the absence of NF-κB signaling, proteasomal degradation of C/EBPß is increased by a JNK-independent mechanism and promotes death from TNFα.

Macroautophagy and chaperone-mediated autophagy are required for hepatocyte resistance to oxidant stress.

UNLABELLED: The function of the lysosomal degradative pathway of autophagy in cellular injury is unclear, because findings in nonhepatic cells have implicated autophagy as both a mediator of cell death and as a survival response. Autophagic function is impaired in steatotic and aged hepatocytes, suggesting that in these settings hepatocellular injury may be altered by the decrease in autophagy. To delineate the specific function of autophagy in the hepatocyte injury response, the effects of menadione-induced oxidative stress were examined in the RALA255-10G rat hepatocyte line when macroautophagy was inhibited by a short hairpin RNA (shRNA)-mediated knockdown of the autophagy gene atg5. Loss of macroautophagy sensitized cells to apoptotic and necrotic death from normally nontoxic concentrations of menadione. Loss of macroautophagy led to overactivation of the c-Jun N-terminal kinase (JNK)/c-Jun signaling pathway that induced cell death. Death occurred from activation of the mitochondrial death pathway with cellular adenosine triphosphate (ATP) depletion, mitochondrial cytochrome c release, and caspase activation. Sensitization to death from menadione occurred despite up-regulation of other forms of autophagy in compensation for the loss of macroautophagy. Chaperone-mediated autophagy (CMA) also mediated resistance to menadione. CMA inhibition sensitized cells to death from menadione through a mechanism different from that of a loss of macroautophagy, because death occurred in the absence of JNK/c-Jun overactivation or ATP depletion. CONCLUSION: Hepatocyte resistance to injury from menadione-induced oxidative stress is mediated by distinct functions of both macroautophagy and CMA, indicating that impaired function of either form of autophagy may promote oxidant-induced liver injury.

Autophagy regulates adipose mass and differentiation in mice.

The relative balance between the quantity of white and brown adipose tissue can profoundly affect lipid storage and whole-body energy homeostasis. However, the mechanisms regulating the formation, expansion, and interconversion of these 2 distinct types of fat remain unknown. Recently, the lysosomal degradative pathway of macroautophagy has been identified as a regulator of cellular differentiation, suggesting that autophagy may modulate this process in adipocytes. The function of autophagy in adipose differentiation was therefore examined in the current study by genetic inhibition of the critical macroautophagy gene autophagy-related 7 (Atg7). Knockdown of Atg7 in 3T3-L1 preadipocytes inhibited lipid accumulation and decreased protein levels of adipocyte differentiation factors. Knockdown of Atg5 or pharmacological inhibition of autophagy or lysosome function also had similar effects. An adipocyte-specific mouse knockout of Atg7 generated lean mice with decreased white adipose mass and enhanced insulin sensitivity. White adipose tissue in knockout mice had increased features of brown adipocytes, which, along with an increase in normal brown adipose tissue, led to an elevated rate of fatty acid, beta-oxidation, and a lean body mass. Autophagy therefore functions to regulate body lipid accumulation by controlling adipocyte differentiation and determining the balance between white and brown fat.

Autophagy regulates lipid metabolism.

The intracellular storage and utilization of lipids are critical to maintain cellular energy homeostasis. During nutrient deprivation, cellular lipids stored as triglycerides in lipid droplets are hydrolysed into fatty acids for energy. A second cellular response to starvation is the induction of autophagy, which delivers intracellular proteins and organelles sequestered in double-membrane vesicles (autophagosomes) to lysosomes for degradation and use as an energy source. Lipolysis and autophagy share similarities in regulation and function but are not known to be interrelated. Here we show a previously unknown function for autophagy in regulating intracellular lipid stores (macrolipophagy). Lipid droplets and autophagic components associated during nutrient deprivation, and inhibition of autophagy in cultured hepatocytes and mouse liver increased triglyceride storage in lipid droplets. This study identifies a critical function for autophagy in lipid metabolism that could have important implications for human diseases with lipid over-accumulation such as those that comprise the metabolic syndrome.

Differential effects of JNK1 and JNK2 inhibition on murine steatohepatitis and insulin resistance.

UNLABELLED: Activation of c-Jun N-terminal kinase (JNK) has been implicated as a mechanism in the development of steatohepatitis. This finding, together with the reported role of JNK signaling in the development of obesity and insulin resistance, two components of the metabolic syndrome and predisposing factors for fatty liver disease, suggests that JNK may be a central mediator of the metabolic syndrome and an important therapeutic target in steatohepatitis. To define the isoform-specific functions of JNK in steatohepatitis associated with obesity and insulin resistance, the effects of JNK1 or JNK2 ablation were determined in developing and established steatohepatitis induced by a high-fat diet (HFD). HFD-fed jnk1 null mice failed to develop excessive weight gain, insulin resistance, or steatohepatitis. In contrast, jnk2(-/-) mice fed a HFD were obese and insulin-resistant, similar to wild-type mice, and had increased liver injury. In mice with established steatohepatitis, an antisense oligonucleotide knockdown of jnk1 decreased the amount of steatohepatitis in concert with a normalization of insulin sensitivity. Knockdown of jnk2 improved insulin sensitivity but had no effect on hepatic steatosis and markedly increased liver injury. A jnk2 knockdown increased hepatic expression of the proapoptotic Bcl-2 family members Bim and Bax and the increase in liver injury resulted in part from a Bim-dependent activation of the mitochondrial death pathway. CONCLUSION: JNK1 and JNK2 both mediate insulin resistance in HFD-fed mice, but the JNK isoforms have distinct effects on steatohepatitis, with JNK1 promoting steatosis and hepatitis and JNK2 inhibiting hepatocyte cell death by blocking the mitochondrial death pathway.

Chronic oxidative stress sensitizes hepatocytes to death from 4-hydroxynonenal by JNK/c-Jun overactivation.

Sustained activation of the c-Jun NH(2)-terminal kinase (JNK) signaling pathway mediates the development and progression of experimental diet-induced nonalcoholic fatty liver disease (NAFLD). Delineating the mechanism of JNK overactivation in the setting of a fatty liver is therefore essential to understanding the pathophysiology of NAFLD. Both human and experimental NAFLD are associated with oxidative stress and resultant lipid peroxidation, which have been proposed to mediate the progression of this disease from simple steatosis to steatohepatitis. The ability of oxidants and the lipid peroxidation product 4-hydroxynonenal (HNE) to activate JNK signaling suggested that these two factors may act synergistically to trigger JNK overactivation. The effect of HNE on hepatocyte injury and JNK activation was therefore examined in cells under chronic oxidant stress from overexpression of the prooxidant enzyme cytochrome P450 2E1 (CYP2E1), which occurs in NAFLD. CYP2E1-generated oxidant stress sensitized a rat hepatocyte cell line to death from normally nontoxic concentrations of HNE. CYP2E1-overexpressing cells underwent a more profound depletion of glutathione (GSH) in response to HNE secondary to decreased gamma-glutamylcysteine synthetase activity. GSH depletion led to overactivation of JNK/c-Jun signaling at the level of mitogen-activated protein kinase kinase 4 that induced cell death. Oxidant stress and the lipid peroxidation product HNE cause synergistic overactivation of the JNK/c-Jun signaling pathway in hepatocytes, demonstrating that HNE may not be just a passive biomarker of hepatic oxidant stress but rather an active mediator of hepatocellular injury through effects on JNK signaling.

Loss of macroautophagy promotes or prevents fibroblast apoptosis depending on the death stimulus.

Macroautophagy has been implicated as a mechanism of cell death. However, the relationship between this degradative pathway and cell death is unclear as macroautophagy has been shown recently to protect against apoptosis. To better define the interplay between these two critical cellular processes, we determined whether inhibition of macroautophagy could have both pro-apoptotic and anti-apoptotic effects in the same cell. Embryonic fibroblasts from mice with a knock-out of the essential macroautophagy gene atg5 were treated with activators of the extrinsic and intrinsic death pathways. Loss of macroautophagy sensitized these cells to caspase-dependent apoptosis from the death receptor ligands Fas and tumor necrosis factor-alpha (TNF-alpha). Atg5-/- mouse embryonic fibroblasts had increased activation of the mitochondrial death pathway in response to Fas/TNF-alpha in concert with decreased ATP levels. Fas/TNF-alpha treatment failed to up-regulate macroautophagy, and in fact, decreased activity at late time points. In contrast to their sensitization to Fas/TNF-alpha, Atg5-/- cells were resistant to death from menadione and UV light. In the absence of macroautophagy, an up-regulation of chaperone-mediated autophagy induced resistance to these stressors. These results demonstrate that inhibition of macroautophagy can promote or prevent apoptosis in the same cell and that the response is governed by the nature of the death stimulus and compensatory changes in other forms of autophagy. Experimental findings that an inhibition of macroautophagy blocks apoptosis do not prove that autophagy mediates cell death as this effect may result from the protective up-regulation of other autophagic pathways such as chaperone-mediated autophagy.