Jordi Muntané

Impaired autophagic flux is associated with increased endoplasmic reticulum stress during the development of NAFLD

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 8u2009h triggered activation of both unfolding protein response and the autophagic flux. Conversely, prolonged treatment with PA (24u2009h) 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.

Protein tyrosine phosphatase 1B modulates GSK3β/Nrf2 and IGFIR signaling pathways in acetaminophen-induced hepatotoxicity

Acute hepatic failure secondary to acetaminophen (APAP) poisoning is associated with high mortality. Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of tyrosine kinase growth factor signaling. In the liver, this pathway confers protection against injury. However, the involvement of PTP1B in the intracellular networks activated by APAP is unknown. We have assessed PTP1B expression in APAP-induced liver failure in humans and its role in the molecular mechanisms that regulate the balance between cell death and survival in human and mouse hepatocytes, as well as in a mouse model of APAP-induced hepatotoxicity. PTP1B expression was increased in human liver tissue removed during liver transplant from patients for APAP overdose. PTP1B was upregulated by APAP in primary human and mouse hepatocytes together with the activation of c-jun (NH2) terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), resulting in cell death. Conversely, Akt phosphorylation and the antiapoptotic Bcl2 family members BclxL and Mcl1 were decreased. PTP1B deficiency in mouse protects hepatocytes against APAP-induced cell death, preventing glutathione depletion, reactive oxygen species (ROS) generation and activation of JNK and p38 MAPK. APAP-treated PTP1B−/− hepatocytes showed enhanced antioxidant defense through the glycogen synthase kinase 3 (GSK3)β/Src kinase family (SKF) axis, delaying tyrosine phosphorylation of the transcription factor nuclear factor-erythroid 2-related factor (Nrf2) and its nuclear exclusion, ubiquitination and degradation. Insulin-like growth factor-I receptor-mediated signaling decreased in APAP-treated wild-type hepatocytes, but was maintained in PTP1B−/− cells or in wild-type hepatocytes with reduced PTP1B levels by RNA interference. Likewise, both signaling cascades were modulated in mice, resulting in less severe APAP hepatotoxicity in PTP1B−/− mice. Our results demonstrated that PTP1B is a central player of the mechanisms triggered by APAP in hepatotoxicity, suggesting a novel therapeutic target against APAP-induced liver failure.

Differential activation of the human farnesoid X receptor depends on the pattern of expressed isoforms and the bile acid pool composition

The farnesoid X receptor (FXR) is a key sensor in bile acid homeostasis. Although four human FXR isoforms have been identified, the physiological role of this diversity is poorly understood. Here we investigated their subcellular localization, agonist sensitivity and response of target genes. Measurement of mRNA revealed that liver predominantly expressed FXRα1(+/-), whereas FXRα2(+/-) were the most abundant isoforms in kidney and intestine. In all cases, the proportion of FXRα(1/2)(+) and FXRα(1/2)(-) isoforms, i.e., with and without a 12bp insert, respectively, was approximately 50%. When FXR was expressed in liver and intestinal cells the magnitude of the response to GW4064 and bile acids differs among FXR isoforms. In both cell types the strongest response was that of FXRα1(-). Different efficacy of bile acids species to activate FXR was found. The four FXR isoforms shared the order of sensitivity to bile acids species. When in FXR-deficient cells FXR was transfected, unconjugated, but not taurine- and glycine-amidated bile acids, were able to activate FXR. In contrast, human hepatocytes and cell lines showing an endogenous expression of FXR were sensitive to both unconjugated and conjugated bile acids. This suggests that to activate FXR conjugated, but not unconjugated, bile acids require additional component(s) of the intracellular machinery not related with uptake processes, which are missing in some tumor cells. In conclusion, cell-specific pattern of FXR isoforms determine the overall tissue sensitivity to FXR agonists and may be involved in the differential response of FXR target genes to FXR activation.

Cardiotrophin-1 eliminates hepatic steatosis in obese mice by mechanisms involving AMPK activation

BACKGROUND & AIMSnCardiotrophin-1 (CT-1) is a hepatoprotective cytokine that modulates fat and glucose metabolism in muscle and adipose tissue. Here we analyzed the changes in hepatic fat stores induced by recombinant CT-1 (rCT-1) and its therapeutic potential in non-alcoholic fatty liver disease (NAFLD).nnnMETHODSnrCT-1 was administered to two murine NAFLD models: ob/ob and high fat diet-fed mice. Livers were analyzed for lipid composition and expression of genes involved in fat metabolism. We studied the effects of rCT-1 on lipogenesis and fatty acid (FA) oxidation in liver cells and the ability of dominant negative inhibitor of AMP-activated protein kinase (AMPK) to block these effects.nnnRESULTSnCT-1 was found to be upregulated in human and murine steatotic livers. In two NAFLD mouse models, treatment with rCT-1 for 10days induced a marked decrease in liver triglyceride content with augmented proportion of poly-unsaturated FA and reduction of monounsaturated species. These changes were accompanied by attenuation of inflammation and improved insulin signaling. Chronic administration of rCT-1 caused downregulation of lipogenic genes and genes involved in FA import to hepatocytes together with amelioration of ER stress, elevation of NAD(+)/NADH ratio, phosphorylation of LKB1 and AMPK, increased expression and activity of sirtuin1 (SIRT1) and upregulation of genes mediating FA oxidation. rCT-1 potently inhibited de novo lipogenesis and stimulated FA oxidation in liver cells both in vitro and in vivo. In vitro studies showed that these effects are mediated by activated AMPK.nnnCONCLUSIONSnrCT-1 resolves hepatic steatosis in obese mice by mechanisms involving AMPK activation. rCT-1 deserves consideration as a potential therapy for NAFLD.

Activation of the nuclear receptor FXR enhances hepatocyte chemoprotection and liver tumor chemoresistance against genotoxic compounds.

The success of pharmacological treatments in primary liver cancers is limited by the marked efficacy of mechanisms of chemoresistance already present in hepatocytes. The role of the nuclear receptor FXR is unclear. Although, in non-treated liver tumors, its expression is reduced, the refractoriness to anticancer drugs is high. Moreover, the treatment with cisplatin up-regulates FXR. The aim of this study was to investigate whether FXR is involved in stimulating chemoprotection/chemoresistance in healthy and tumor liver cells. In human hepatocytes, the activation of FXR with the agonist GW4064 resulted in a significant protection against cisplatin-induced toxicity. In human hepatoma Alexander cells, with negligible endogenous expression of FXR, GW4064 also protected against cisplatin-induced toxicity, but only if they were previously transfected with FXR/RXR. Investigation of 109 genes potentially involved in chemoresistance revealed that only ABCB4, TCEA2, CCL14, CCL15 and KRT13 were up-regulated by FXR activation both in human hepatocytes and FXR/RXR-expressing hepatoma cells. In both models, cisplatin, even in the absence of FXR agonists, such as bile acids and GW4064, was able to up-regulate FXR targets genes, which was due to FXR-mediated trans-activation of response elements in the promoter region. FXR-dependent chemoprotection was also efficient against other DNA-damaging compounds, such as doxorubicin, mitomycin C and potassium dichromate, but not against non-genotoxic drugs, such as colchicine, paclitaxel, acetaminophen, artesunate and sorafenib. In conclusion, ligand-dependent and independent activation of FXR stimulates mechanisms able to enhance the chemoprotection of hepatocytes against genotoxic compounds and to reduce the response of liver tumor cells to certain pharmacological treatments.

Modulation of Autophagy by Sorafenib: Effects on Treatment Response

The multikinase inhibitor sorafenib is, at present, the only drug approved for the treatment of hepatocellular carcinoma (HCC), one of the most lethal types of cancer worldwide. However, the increase in the number of sorafenib tumor resistant cells reduces efficiency. A better knowledge of the intracellular mechanism of the drug leading to reduced cell survival could help to improve the benefits of sorafenib therapy. Autophagy is a bulk cellular degradation process activated in a broad range of stress situations, which allows cells to degrade misfolded proteins or dysfunctional organelles. This cellular route can induce survival or death, depending on cell status and media signals. Sorafenib, alone or in combination with other drugs is able to induce autophagy, but cell response to the drug depends on the complex integrative crosstalk of different intracellular signals. In cancerous cells, autophagy can be regulated by different cellular pathways (Akt-related mammalian target of rapamycin (mTOR) inhibition, 5′ AMP-activated protein kinase (AMPK) induction, dissociation of B-cell lymphoma 2 (Bcl-2) family proteins from Beclin-1), or effects of some miRNAs. Inhibition of mTOR signaling by sorafenib and diminished interaction between Beclin-1 and myeloid cell leukemia 1 (Mcl-1) have been related to induction of autophagy in HCC. Furthermore, changes in some miRNAs, such as miR-30α, are able to modulate autophagy and modify sensitivity in sorafenib-resistant cells. However, although AMPK phosphorylation by sorafenib seems to play a role in the antiproliferative action of the drug, it does not relate with modulation of autophagy. In this review, we present an updated overview of the effects of sorafenib on autophagy and its related activation pathways, analyzing in detail the involvement of autophagy on sorafenib sensitivity and resistance.

Essential role of Nrf2 in the protective effect of lipoic acid against lipoapoptosis in hepatocytes.

Excess of saturated free fatty acids, such as palmitic acid (PA), in hepatocytes has been implicated in nonalcoholic fatty liver disease. α-Lipoic acid (LA) is an antioxidant that protects against oxidative stress conditions. We have investigated the effects of LA in the early activation of oxidative and endoplasmic reticulum stress, lipid accumulation, and Nrf2-mediated antioxidant defenses in hepatocytes treated with PA or in rats fed a high-fat diet. In primary human hepatocytes, a lipotoxic concentration of PA triggered endoplasmic reticulum stress, induced the apoptotic transcription factor CHOP, and increased the percentage of apoptotic cells. Cotreatment with LA prevented these effects. Similar results were found in mouse hepatocytes in which LA attenuated PA-mediated activation of caspase 3 and reduced lipid accumulation by decreasing PA uptake and increasing fatty acid oxidation and lipophagy, thereby preventing lipoapoptosis. Moreover, LA augmented the proliferation capacity of hepatocytes after PA challenge. Antioxidant effects of LA ameliorated reactive oxygen species production and endoplasmic reticulum stress and protected against mitochondrial apoptosis in hepatocytes treated with PA. Cotreatment with PA and LA induced an early nuclear translocation of Nrf2 and activated antioxidant enzymes, whereas reduction of Nrf2 by siRNA abolished the benefit of LA on PA-induced lipoapoptosis. Importantly, posttreatment with LA reversed the established damage induced by PA in hepatocytes, as well as preventing obesity-induced oxidative stress and lipoapoptosis in rat liver. In conclusion, our work has revealed that in hepatocytes, Nrf2 is an essential early player in the rescue of oxidative stress by LA leading to protection against PA-mediated lipoapoptosis.

Role of hepatocyte S6K1 in palmitic acid-induced endoplasmic reticulum stress, lipotoxicity, insulin resistance and in oleic acid-induced protection

The excess of saturated free fatty acids, such as palmitic acid, that induces lipotoxicity in hepatocytes, has been implicated in the development of non-alcoholic fatty liver disease also associated with insulin resistance. By contrast, oleic acid, a monounsaturated fatty acid, attenuates the effects of palmitic acid. We evaluated whether palmitic acid is directly associated with both insulin resistance and lipoapoptosis in mouse and human hepatocytes and the impact of oleic acid in the molecular mechanisms that mediate both processes. In human and mouse hepatocytes palmitic acid at a lipotoxic concentration triggered early activation of endoplasmic reticulum (ER) stress-related kinases, induced the apoptotic transcription factor CHOP, activated caspase 3 and increased the percentage of apoptotic cells. These effects concurred with decreased IR/IRS1/Akt insulin pathway. Oleic acid suppressed the toxic effects of palmitic acid on ER stress activation, lipoapoptosis and insulin resistance. Besides, oleic acid suppressed palmitic acid-induced activation of S6K1. This protection was mimicked by pharmacological or genetic inhibition of S6K1 in hepatocytes. In conclusion, this is the first study highlighting the activation of S6K1 by palmitic acid as a common and novel mechanism by which its inhibition by oleic acid prevents ER stress, lipoapoptosis and insulin resistance in hepatocytes.

The protective role of coenzyme Q10 in renal injury associated with extracorporeal shockwave lithotripsy: a randomised, placebo‐controlled clinical trial

To determine the efficacy of coenzyme Q10 (CoQ10) in preventing renal injury in patients with lithiasis undergoing extracorporeal shockwave lithotripsy (ESWL).

Essential Role of Protein-tyrosine Phosphatase 1B in the Modulation of Insulin Signaling by Acetaminophen in Hepatocytes

Background: PTP1B is a negative modulator of insulin receptor-mediated signaling. Results: Acetaminophen increased PTP1B in hepatocytes and impaired insulin signaling. Conclusion: PTP1B plays an essential role in the impairment of insulin signaling mediated by acetaminophen in hepatocytes. Significance: Chronic acetaminophen administration might be associated with insulin resistance in the liver. Many drugs are associated with the development of glucose intolerance or deterioration in glycemic control in patients with pre-existing diabetes. We have evaluated the cross-talk between signaling pathways activated by acetaminophen (APAP) and insulin signaling in hepatocytes with or without expression of the protein-tyrosine phosphatase 1B (PTP1B) and in wild-type and PTP1B-deficient mice chronically treated with APAP. Human primary hepatocytes, Huh7 hepatoma cells with silenced PTP1B, mouse hepatocytes from wild-type and PTP1B-deficient mice, and a mouse model of chronic APAP treatment were used to examine the mechanisms involving PTP1B in the effects of APAP on glucose homeostasis and hepatic insulin signaling. In APAP-treated human hepatocytes at concentrations that did not induce death, phosphorylation of JNK and PTP1B expression and enzymatic activity were increased. APAP pretreatment inhibited activation of the early steps of insulin signaling and decreased Akt phosphorylation. The effects of APAP in insulin signaling were prevented by suramin, a PTP1B inhibitor, or rosiglitazone that decreased PTP1B levels. Likewise, PTP1B deficiency in human or mouse hepatocytes protected against APAP-mediated impairment in insulin signaling. These signaling pathways were modulated in mice with chronic APAP treatment, resulting in protection against APAP-mediated hepatic insulin resistance and alterations in islet alpha/beta cell ratio in PTP1B−/− mice. Our results demonstrate negative cross-talk between signaling pathways triggered by APAP and insulin signaling in hepatocytes, which is in part mediated by PTP1B. Moreover, our in vivo data suggest that chronic use of APAP may be associated with insulin resistance in the liver.