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The pathogenic mechanisms underlying the progression of non-alcoholic fatty liver disease (NAFLD) are not fully understood. In this study, we aimed to assess the relationship between endoplasmic reticulum (ER) stress and autophagy in human and mouse hepatocytes during NAFLD. ER stress and autophagy markers were analyzed in livers from patients with biopsy-proven non-alcoholic steatosis (NAS) or non-alcoholic steatohepatitis (NASH) compared with livers from subjects with histologically normal liver, in livers from mice fed with chow diet (CHD) compared with mice fed with high fat diet (HFD) or methionine-choline-deficient (MCD) diet and in primary and Huh7 human hepatocytes loaded with palmitic acid (PA). In NASH patients, significant increases in hepatic messenger RNA levels of markers of ER stress (activating transcription factor 4 (ATF4), glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP)) and autophagy (BCN1) were found compared with NAS patients. Likewise, protein levels of GRP78, CHOP and p62/SQSTM1 (p62) autophagic substrate were significantly elevated in NASH compared with NAS patients. In livers from mice fed with HFD or MCD, ER stress-mediated signaling was parallel to the blockade of the autophagic flux assessed by increases in p62, microtubule-associated protein 2 light chain 3 (LC3-II)/LC3-I ratio and accumulation of autophagosomes compared with CHD fed mice. In Huh7 hepatic cells, treatment with PA for 8 h triggered activation of both unfolding protein response and the autophagic flux. Conversely, prolonged treatment with PA (24 h) induced ER stress and cell death together with a blockade of the autophagic flux. Under these conditions, cotreatment with rapamycin or CHOP silencing ameliorated these effects and decreased apoptosis. Our results demonstrated that the autophagic flux is impaired in the liver from both NAFLD patients and murine models of NAFLD, as well as in lipid-overloaded human hepatocytes, and it could be due to elevated ER stress leading to apoptosis. Consequently, therapies aimed to restore the autophagic flux might attenuate or prevent the progression of NAFLD.

Macrophage activation in response to proinflammatory cytokines and bacterial cell wall products constitutes a key component of the immune response (23,31,50). Resolution of the process occurs after removal of the proinflammatory stimuli and through the action of negative regulators of the activationsignaling pathways, among them interleukin-10 (IL-10), IL-13, alpha/beta interferons (IFN-␣/␤), and more recently several cyclopentenone prostaglandins (PGs) (8,21,35,36,49). In particular, 15-deoxy-⌬ 12,14 -prostaglandin J 2 (15dPGJ 2 ) has been shown to exert important anti-inflammatory effects on several cell types such as monocytes/macrophages and microglia (4,16,35,36). Controversy exists about the identification of intracellular targets involved in the mechanism of action of cyclopentenone PGs: some of these effects have been explained through the transcriptional inhibition exerted by 15dPGJ 2 -activated peroxisome proliferator receptor gamma (PPAR␥) (12,14,36,39); however, other data suggest a main contribution of PPAR␥-independent mechanisms on the antiinflammatory action of this PG, in view of the lack of effect of synthetic PPAR␥ ligands such as thiazolidinediones (17,35).It has been shown that 15dPGJ 2 inhibits the expression of genes requiring the activation of the transcription factors NF-B, AP-1, and Stat1 (17, 35, 36), which are involved in the induction of several enzymes participating in the development of the inflammatory process, such as type 2 nitric oxide synthase (NOS-2) and cyclooxygenase 2 (COX-2) (7,42,51). In macrophages, activated NF-B complexes are composed mainly of p50 and p65 subunits that translocate to the nucleus in response to cell stimulation with lipopolysaccharide (LPS) and proinflammatory cytokines (13,45,48). This activation of NF-B requires phosphorylation by IB kinase (IKK) of IB proteins in specific serine residues that target these proteins for ubiquitin conjugation and degradation by the 26S proteasome (26, 45). The IKK complex contains two catalytic subunits, IKK1 and IKK2, and a regulatory subunit termed NF-B essential modulator (10,54,56). In turn, activation of IKK is mediated by phosphorylation through NF-B-inducing kinase, which acts preferentially over IKK1, and MEK kinase 1 (MEKK1), which phosphorylates IKK2 (6, 30). Biochemical and genetic data indicate that IKK1 and IKK2, despite the sequence similarity, have different functions (15,55). IKK1 participates in differentiation of various cell types (20), whereas IKK2 is involved in LPS signaling in monocytes/macrophages and in general the response to proinflammatory stimuli (34, 55). IKK2 is rapidly activated after cell challenge with LPS, IL-1␤, or tumor necrosis factor alpha (TNF-␣) and progressively undergoes phosphorylation at multiple serine residues that decreases the kinase activity and therefore contributes to the transient activation of this enzyme (6). In this regard, we have investigated the possibility of early effects of 15dPGJ 2 on LPS and IFN-␥ (collectively termed LPS/IFN-␥) cooperative signaling in R...

Objective While it is accepted that macrophage glycolysis is up-regulated under hypoxic conditions, it is not known whether this is linked to a similar increase in macrophage pro-inflammatory activation and whether specific energy demands regulate cell viability in the atheromatous plaque. Approach and Results We studied the interplay between macrophage energy metabolism, polarization and viability in the context of atherosclerosis. Cultured human and murine macrophages and an in vivo murine model of atherosclerosis were used to evaluate the mechanisms underlying metabolic and inflammatory activity of macrophages in the different atherosclerotic conditions analyzed. We observed that macrophage energetics and inflammatory activation are closely and linearly related, resulting in dynamic calibration of glycolysis to keep pace with inflammatory activity. Additionally, we show that macrophage glycolysis and proinflammatory activation mainly depend on hypoxia-inducible factor (HIF) and on its impact on glucose uptake, and on the expression of hexokinase II and ubiquitous 6-phosphofructo-2-kinase (PFKFB3). As a consequence, hypoxia potentiates inflammation and glycolysis mainly via these pathways. Moreover, when macrophages’ ability to increase glycolysis through PFKFB3 is experimentally attenuated, cell viability is reduced if subjected to proinflammatory and/or hypoxic conditions, but unaffected under control conditions. In addition to this, GM-CSF enhances anaerobic glycolysis while exerting a mild pro-inflammatory activation. Conclusions These findings, in human and murine cells and in an animal model, show that hypoxia potentiates macrophage glycolytic flux in concert with a proportional up-regulation of pro-inflammatory activity, in a manner that is dependent on both HIF-1α and PFKFB3.

Partial hepatectomy (PH) triggers a rapid regenerative response in the remaining tissue to reinstate the organ function and the cell numbers. Among the molecules that change in the course of regeneration is an accumulation of prostaglandin E2 in the sera of rats with PH. Analysis of the cyclooxygenase (COX) isoenzymes in the remnant liver showed the preferential expression of COX-2 in hepatocytes. Cultured regenerating hepatocytes expressed significant levels of COX-2, a process that was not observed in the sham counterparts. Maximal expression of COX-2 was detected 16 h after PH with increased levels present even at 96 h. Pharmacological inhibition of COX-2 activity with NS398 shunted the up-regulation of cell proliferation after PH, which suggests a positive interaction of prostaglandins with the progression of the cell cycle. Similar results were obtained after PH of mice lacking the COX-2 gene. The expression of COX-2 in regenerating liver was concomitant with a decrease in CCAAT-enhancer binding protein (C/EBP-a) level and an increase in the expression of C/EBP-b and C/EBP-d. These results suggest a contribution of the enhanced synthesis of prostaglandins to liver regeneration observed after PH.

Background: Cyclooxygenase 2 (COX-2) and matrix metalloproteinases (MMPs) have been implicated in tissue injury and fibrogenesis in animal models but little is known regarding their role in hepatitis C virus (HCV) related liver disease in humans. Aims: To characterise the intrahepatic expression pattern of COX-2 and MMPs in chronic HCV infection and determine whether HCV core and NS5A proteins could promote their expression in cultured hepatocyte derived cell lines. Patients: Thirty two anti-HCV+ and 10 anti-HCV2 patients were studied. Methods: Western blot, reverse transcription-polymerase chain reaction (RT-PCR), enzyme immunoassay, and immunohistochemistry were used to assess the expression pattern of COX-2 and MMPs in liver biopsy samples from all patients. COX-2 gene expression and MMP-9 protein levels were also determined by immunoblot, RT-PCR, and luciferase assays in core and NS5A transfected hepatocyte derived cells. Results: The intrahepatic expression level of COX-2, MMP-2, and MMP-9 was significantly higher in HCV+ than in HCV2 patients, increasing with the fibrotic stage of liver disease. We further demonstrated that COX-2 mRNA, protein, and activity were induced in resting and activated core and NS5A transfectants. Both viral proteins induced transcriptional activity of the COX-2 gene promoter whereas core, but not NS5A, exerted an inducer effect on MMP-9 protein levels in cultured hepatocyte derived cells. Conclusions: Intrahepatic COX-2, MMP-2, and MMP-9 overexpression is associated with progressive hepatic fibrosis in chronic HCV infection, suggesting their pathogenic role in fibrogenesis. HCV core and NS5A proteins were able to upregulate COX-2 and MMP-9 gene expression in hepatocyte derived cells, providing a potential mechanism for hepatic fibrosis during chronic HCV infection.