Hepatocyte cell death via apoptosis and necrosis are major hallmarks of ethanol-induced liver injury. However, inhibition of apoptosis is not sufficient to prevent ethanol-induced hepatocyte injury or inflammation. Since receptor interacting protein kinase (RIP) 3-mediated necroptosis, a non-apoptotic cell death pathway, is implicated in a variety of pathological conditions, here we tested the hypothesis that ethanol-induced liver injury is RIP3-dependent and RIP1-independent. Increased expression of RIP3 was detected in livers of mice after chronic ethanol feeding, as well as in liver biopsies from patients with alcoholic liver disease. Chronic ethanol feeding failed to induce RIP3 in the livers of cytochrome P450 2E1 (CYP2E1)-deficient mice, indicating CYP2E1-mediated ethanol metabolism is critical for RIP3 expression in response to ethanol feeding. Mice lacking RIP3 were protected from ethanol-induced steatosis, hepatocyte injury and expression of pro-inflammatory cytokines. In contrast, RIP1 expression in mouse liver remained unchanged following ethanol feeding and inhibition of RIP1 kinase by necrostatin-1 did not attenuate ethanol-induced hepatocyte injury. Ethanol-induced apoptosis, assessed by TUNEL-positive nuclei and accumulation of cytokeratin-18 fragments in the liver, was independent of RIP3. Conclusion These data demonstrate that CYP2E1-dependent RIP3 expression induces hepatocyte necroptosis during ethanol feeding. Ethanol-induced hepatocyte injury is RIP3-dependent, but independent of RIP1 kinase activity; intervention of this pathway could be targeted as a potential therapeutic strategy.

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Adiponectin is an adipokine with potent anti-inflammatory properties. However, the mechanisms by which adiponectin suppresses macrophage function are not well understood. Treatment of RAW264.7 macrophages with adiponectin for 18 h decreased lipopolysaccharide (LPS)-stimulated tumor necrosis factor-␣ (TNF-␣) production. Here we demonstrate that globular adiponectin (gAcrp) initially increased TNF-␣ expression in RAW264.7 macrophages; this TNF-␣ then contributed to increased expression of interleukin-10, which in turn was required for the development of tolerance to subsequent LPS exposure. gAcrp-mediated increases in TNF-␣ mRNA accumulation were associated with increased TNF-␣ promoter activity. gAcrp increased the DNA binding activity of both Egr-1 and NFB; mutation of either the Egr-1 or NFB binding sites in the TNF-␣ promoter decreased gAcrp-stimulated promoter activity. Further, co-transfection with either dominant negative Egr-1 or the IB super-repressor prevented gAcrp-stimulated TNF-␣ promoter activity. gAcrp also increased Egr-1 promoter activity, mRNA accumulation, and DNA binding activity. Inhibition of ERK1/2 with U0126 potently suppressed gAcrp-stimulated Egr-1 promoter activity, as well as TNF-␣ promoter activity. In summary, these data demonstrate that adiponectin initially increases TNF-␣ production by macrophages via ERK1/ 23 Egr-1 and NFB-dependent mechanisms; these increases in TNF-␣ in turn lead to increased expression of interleukin-10 and an eventual dampening of LPS-mediated cytokine production in macrophages.

Patients with alcoholic cirrhosis and hepatitis have severe muscle loss. Since ethanol impairs skeletal muscle protein synthesis but does not increase ubiquitin proteasome-mediated proteolysis, we investigated whether alcohol-induced autophagy contributes to muscle loss. Autophagy induction was studied in: A) Human skeletal muscle biopsies from alcoholic cirrhotics and controls, B) Gastrocnemius muscle from ethanol and pair-fed mice, and C) Ethanol-exposed murine C2C12 myotubes, by examining the expression of autophagy markers assessed by immunoblotting and real-time PCR. Expression of autophagy genes and markers were increased in skeletal muscle from humans and ethanol-fed mice, and in myotubes following ethanol exposure. Importantly, pulse-chase experiments showed suppression of myotube proteolysis upon ethanol-treatment with the autophagy inhibitor, 3-methyladenine (3MA) and not by MG132, a proteasome inhibitor. Correspondingly, ethanol-treated C2C12 myotubes stably expressing GFP-LC3B showed increased autophagy flux as measured by accumulation of GFP-LC3B vesicles with confocal microscopy. The ethanol-induced increase in LC3B lipidation was reversed upon knockdown of Atg7, a critical autophagy gene and was associated with reversal of the ethanol-induced decrease in myotube diameter. Consistently, CT image analysis of muscle area in alcoholic cirrhotics was significantly reduced compared with control subjects. In order to determine whether ethanol per se or its metabolic product, acetaldehyde, stimulates autophagy, C2C12 myotubes were treated with ethanol in the presence of the alcohol dehydrogenase inhibitor (4-methylpyrazole) or the acetaldehyde dehydrogenase inhibitor (cyanamide). LC3B lipidation increased with acetaldehyde treatment and increased further with the addition of cyanamide. We conclude that muscle autophagy is increased by ethanol exposure and contributes to sarcopenia.

Chronic ethanol feeding sensitizes Kupffer cells to activation by lipopolysaccharide (LPS), leading to increased production of tumor necrosis factor-α (TNF-α). Adiponectin treatment protects mice from ethanol-induced liver injury. Because adiponectin has anti-inflammatory effects on macrophages, we hypothesized that adiponectin would normalize chronic ethanol-induced sensitization of Kupffer cells to LPS-mediated signals. Serum adiponectin concentrations were decreased by 45% in rats fed an ethanol-containing diet for 4 wk compared with pair-fed rats. Adiponectin dose dependently inhibited LPS-stimulated accumulation of TNF-α mRNA and peptide in Kupffer cells from both pair- and ethanol-fed rats. Kupffer cells from ethanol-fed rats were more sensitive to both globular (gAcrp) and full-length adiponectin (flAcrp) than Kupffer cells from pair-fed controls with suppression at 10 ng/ml adiponectin after chronic ethanol feeding. Kupffer cells expressed both adiponectin receptors 1 and 2; chronic ethanol feeding did not change the expression of adiponectin receptor mRNA or protein. gAcrp suppressed LPS-stimulated ERK1/2 and p38 phosphorylation as well as IκB degradation at 100–1,000 ng/ml in Kupffer cells from both pair- and ethanol-fed rats. However, only LPS-stimulated ERK1/2 phosphorylation was sensitive to 10 ng/ml gAcrp. gAcrp also normalized LPS-stimulated DNA binding activity of early growth response-1 with greater sensitivity in Kupffer cells from rats fed chronic ethanol. In conclusion, these results demonstrate that Kupffer cells from ethanol-fed rats are more sensitive to the anti-inflammatory effects of both gAcrp and flAcrp. Suppression of LPS-stimulated ERK1/2 signaling by low concentrations of gAcrp was associated with normalization of TNF-α production by Kupffer cells after chronic ethanol exposure.

Reactive oxygen species (ROS) contribute to the development of chronic ethanol-induced liver injury. Although ROS modulate the activity of many signal transduction pathways, the molecular targets of ROS during ethanol exposure are not well understood. Here, we investigated whether specific ROS-sensitive signal transduction pathways contribute to increased tumor necrosis factor alpha (TNF-alpha) production by Kupffer cells after chronic ethanol feeding to rats. Lipopolysaccharide (LPS) rapidly increased ROS production, measured by dihydrorhodamine fluorescence, in Kupffer cells from ethanol- and pair-fed rats, and ROS production was 2.5-fold greater in ethanol-fed compared with pair-fed. Pretreatment with diphenyleneiodonium (DPI), which inhibits reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, normalized ROS production in Kupffer cells from ethanol-fed rats. LPS rapidly increased Rac1-guanosinetriphosphatase (GTPase) activity and p67(phox) translocation to the plasma membrane in Kupffer cells from pair-fed rats. After ethanol feeding, Rac1-GTPase activity was already increased over pair-fed at baseline and remained elevated over pair-fed after LPS stimulation. Further, LPS-stimulated p67(phox) translocation to the plasma membrane was enhanced after chronic ethanol feeding. LPS-stimulated extracellular signal-regulated kinase (ERK)1/2 and p38 phosphorylation, two signaling pathways regulated by ROS, were increased twofold in Kupffer cells from ethanol-fed rats compared with pair-fed controls. However, only LPS-stimulated ERK1/2 phosphorylation was inhibited by DPI, which also reduced LPS-stimulated TNF-alpha production in Kupffer cells from pair- and ethanol-fed rats. These results demonstrate that chronic ethanol feeding increases LPS-stimulated NADPH oxidase-dependent production of ROS in Kupffer cells. Further, ERK1/2 is an important target of NADPH oxidase-derived ROS in Kupffer cells, contributing to enhanced LPS-stimulated TNF-alpha production by Kupffer cells after chronic ethanol feeding.

Activation of Kupffer cells by lipopolysaccharide (LPS) is a critical step in the pathogenesis of alcoholic liver disease. Kupffer cells isolated from rats fed ethanol in their diet for 4 wk accumulated 4.3-fold more tumor necrosis factor (TNF)-α in response to LPS compared with pair-fed rats. In contrast, LPS-stimulated interleukin (IL)-1 accumulation was 50% lower after ethanol feeding. LPS-stimulated TNF-α mRNA accumulation was twofold higher after ethanol feeding, whereas IL-1β mRNA accumulation was blunted. To understand the mechanisms for this differential response, we investigated the effects of ethanol on LPS-dependent signal transduction. Chronic ethanol feeding increased LPS-stimulated extracellular receptor-activated kinases 1/2 (ERK1/2) activation. Activation of ERK1/2 was required for maximal increases in TNF-α and IL-1β mRNA and was associated with increased binding of early growth response-1 (Egr-1) to the TNF-α promoter after ethanol feeding. In contrast, ethanol feeding completely abrogated activation of nuclear factor-κB DNA-binding activity by LPS and had no effect on AP-1 binding. Together, these data suggest that enhanced activation of ERK1/2 and Egr-1 contributes to increased TNF-α production after chronic ethanol feeding.

Multiple pathways of programmed cell death are important in liver homeostasis. Hepatocyte death is associated with progression of nonalcoholic fatty liver disease (NAFLD) and inhibition of apoptosis partially protects against liver injury in response to high fat diets (HFD). However, the contribution of necroptosis, a caspase-independent pathway of cell death, to HFD-induced liver injury is not known. Wild-type C57BL/6 and receptor interacting protein (RIP) 3−/− mice were randomized to chow or HFD. HFD-fed C57BL/6 mice increased expression of RIP3, the master regulator of necroptosis, as well as phosphorylated mixed lineage kinase domain-like (pMLKL), an effector of necroptotic cell death, in liver. HFD did not increase pMLKL in RIP3−/− mice. HFD increased fasting insulin and glucose, as well as glucose intolerance, in C57BL/6 mice. RIP3−/− were glucose intolerant even on chow diets; HFD further increased fasting glucose and insulin, but not glucose intolerance. HFD also increased hepatic steatosis, plasma ALT activity, inflammation, oxidative stress and hepatocellular apoptosis in wild-type mice; these responses were exacerbated in RIP3−/− mice. Importantly, increased inflammation and injury was associated with early indicators of fibrosis in RIP3−/− compared to C57BL/6 mice. Culture of AML12 hepatocytes with palmitic acid increased cytotoxicity via apoptosis and necrosis. Inhibition of RIP1 with necrostatin-1 or siRNA knock-down of RIP3 reduced palmitic acid-induced cytotoxicity. Conclusion Absence of RIP3, a key mediator of necroptosis, exacerbated HFD-induced liver injury, associated with increased inflammation and hepatocyte apoptosis, as well as early fibrotic responses. These findings indicate that shifts in the mode of hepatocellular death can influence disease progression and have therapeutic implications because manipulation of hepatocyte cell death pathways is being considered as a target for treatment of NAFLD.