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

This work is licensed under a Creative Commons Attribution-NonCommercialNoDerivs 3.0 Unported License.-- et al.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.We acknowledge the following grant support: SAF2012-33283 (MINECO, Spain), Comunidad de Madrid S2010/BMD-2423, EFSD and Amylin Paul Langerhans Grant and Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM, ISCIII, Barcelona, Spain) to AMV.; SAF2010-16037, SAF2013-43713-R (MINECO) and Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD, ISCIII) to PMS. RD12/0042/0019 (ISCIII) and S2010/BMD-2478 (Comunidad de Madrid) to LB, PI 13/01299 and Fundación Mutua Madrileña 2012 to C G-M and AIRC IG-2012 to GMF.Peer Reviewe

Activation of Pregnane X Receptor by Pregnenolone 16 α-carbonitrile Prevents High-Fat Diet-Induced Obesity in AKR/J Mice

Pregnane X receptor (PXR) is known to function as a xenobiotic sensor to regulate xenobiotic metabolism through selective transcription of genes responsible for maintaining physiological homeostasis. Here we report that the activation of PXR by pregnenolone 16α-carbonitrile (PCN) in AKR/J mice can prevent the development of high-fat diet-induced obesity and insulin resistance. The beneficial effects of PCN treatment are seen with reduced lipogenesis and gluconeogenesis in the liver, and lack of hepatic accumulation of lipid and lipid storage in the adipose tissues. RT-PCR analysis of genes involved in gluconeogenesis, lipid metabolism and energy homeostasis reveal that PCN treatment on high-fat diet-fed mice reduces expression in the liver of G6Pase, Pepck, Cyp7a1, Cd36, L-Fabp, Srebp, and Fas genes and slightly enhances expression of Cyp27a1 and Abca1 genes. RT-PCR analysis of genes involved in adipocyte differentiation and lipid metabolism in white adipose tissue show that PCN treatment reduces expression of Pparγ2, Acc1, Cd36, but increases expression of Cpt1b and Pparα genes in mice fed with high-fat diet. Similarly, PCN treatment of animals on high-fat diet increases expression in brown adipose tissue of Pparα, Hsl, Cpt1b, and Cd36 genes, but reduces expression of Acc1 and Scd-1 genes. PXR activation by PCN in high-fat diet fed mice also increases expression of genes involved in thermogenesis in brown adipose tissue including Dio2, Pgc-1α, Pgc-1β, Cidea, and Ucp-3. These results verify the important function of PXR in lipid and energy metabolism and suggest that PXR represents a novel therapeutic target for prevention and treatment of obesity and insulin resistance

Exploiting antitumor immunity to overcome relapse and improve remission duration

Cancer survivors often relapse due to evolving drug-resistant clones and repopulating tumor stem cells. Our preclinical study demonstrated that terminal cancer patient’s lymphocytes can be converted from tolerant bystanders in vivo into effective cytotoxic T-lymphocytes in vitro killing patient’s own tumor cells containing drug-resistant clones and tumor stem cells. We designed a clinical trial combining peginterferon α-2b with imatinib for treatment of stage III/IV gastrointestinal stromal tumor (GIST) with the rational that peginterferon α-2b serves as danger signals to promote antitumor immunity while imatinib’s effective tumor killing undermines tumor-induced tolerance and supply tumor-specific antigens in vivo without leukopenia, thus allowing for proper dendritic cell and cytotoxic T-lymphocyte differentiation toward Th1 response. Interim analysis of eight patients demonstrated significant induction of IFN-γ-producing-CD8+, -CD4+, -NK cell, and IFN-γ-producing-tumor-infiltrating-lymphocytes, signifying significant Th1 response and NK cell activation. After a median follow-up of 3.6 years, complete response (CR) + partial response (PR) = 100%, overall survival = 100%, one patient died of unrelated illness while in remission, six of seven evaluable patients are either in continuing PR/CR (5 patients) or have progression-free survival (PFS, 1 patient) exceeding the upper limit of the 95% confidence level of the genotype-specific-PFS of the phase III imatinib-monotherapy (CALGB150105/SWOGS0033), demonstrating highly promising clinical outcomes. The current trial is closed in preparation for a larger future trial. We conclude that combination of targeted therapy and immunotherapy is safe and induced significant Th1 response and NK cell activation and demonstrated highly promising clinical efficacy in GIST, thus warranting development in other tumor types

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 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

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 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