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Hepatocellular autophagy modulates the unfolded protein response and fasting-induced steatosis in mice.

ABSTRACT: Autophagy and the unfolded protein response (UPR) are key cellular homeostatic mechanisms and are both involved in liver diseases, including nonalcoholic fatty liver disease (NAFLD). Although increasing but conflicting results link these mechanisms to lipid metabolism, their role and potential cross talk herein have been poorly investigated. Therefore, we assessed the effects of hepatocyte-specific autophagy deficiency on liver parenchyma, the UPR, and lipid metabolism. Adult hepatocellular-specific autophagy-deficient mice (Atg7F/FAlb-Cre+) were compared with their autophagy-competent littermates (Atg7+/+Alb-Cre+). Livers were analyzed by electron microscopy, histology, real-time qPCR, and Western blotting. Atg7F/FAlb-Cre+ mice developed hepatomegaly with significant parenchymal injury, as shown by inflammatory infiltrates, hepatocellular apoptosis, pericellular fibrosis, and a pronounced ductular reaction. Surprisingly, the UPR exhibited a pathway-selective pattern upon autophagy deficiency. The activity of the adaptive activating transcription factor 6 (ATF6) pathway was abolished, whereas the proapoptotic protein kinase RNA-like ER kinase pathway was increased compared with Atg7+/+Alb-Cre+ mice. The inositol-requiring enzyme-1? signal was unaltered. Fasting-induced steatosis was absent in Atg7F/FAlb-Cre+ mice. Remarkably, some isolated islands of fat-containing and autophagy-competent cells were observed in these livers. Hepatocellular autophagy is essential for parenchymal integrity in mice. Moreover, in the case of autophagy deficiency, the three different UPR branches are pathway selectively modulated. Attenuation of the ATF6 pathway might explain the observed impairment of fasting-induced steatosis. Finally, autophagy and lipid droplets are directly linked to each other.

SUBMITTER: Kwanten WJ

PROVIDER: S-EPMC5142201 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Hepatocellular autophagy modulates the unfolded protein response and fasting-induced steatosis in mice.

Kwanten Wilhelmus J WJ Vandewynckel Yves-Paul YP Martinet Wim W De Winter Benedicte Y BY Michielsen Peter P PP Van Hoof Viviane O VO Driessen Ann A Timmermans Jean-Pierre JP Bedossa Pierre P Van Vlierberghe Hans H Francque Sven M SM

American journal of physiology. Gastrointestinal and liver physiology 20160811 4

Autophagy and the unfolded protein response (UPR) are key cellular homeostatic mechanisms and are both involved in liver diseases, including nonalcoholic fatty liver disease (NAFLD). Although increasing but conflicting results link these mechanisms to lipid metabolism, their role and potential cross talk herein have been poorly investigated. Therefore, we assessed the effects of hepatocyte-specific autophagy deficiency on liver parenchyma, the UPR, and lipid metabolism. Adult hepatocellular-spec ...[more]

PMID: 27514483

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Project description:The endoplasmic reticulum (ER) is the cellular organelle responsible for protein folding and assembly, lipid and sterol biosynthesis, and calcium storage. The unfolded protein response (UPR) is an adaptive intracellular stress response to accumulation of unfolded or misfolded proteins in the ER. In this study, we show that the most conserved UPR sensor inositol-requiring enzyme 1 ? (IRE1?), an ER transmembrane protein kinase/endoribonuclease, is required to maintain hepatic lipid homeostasis under ER stress conditions through repressing hepatic lipid accumulation and maintaining lipoprotein secretion. To elucidate physiological roles of IRE1?-mediated signalling in the liver, we generated hepatocyte-specific Ire1?-null mice by utilizing an albumin promoter-controlled Cre recombinase-mediated deletion. Deletion of Ire1? caused defective induction of genes encoding functions in ER-to-Golgi protein transport, oxidative protein folding, and ER-associated degradation (ERAD) of misfolded proteins, and led to selective induction of pro-apoptotic UPR trans-activators. We show that IRE1? is required to maintain the secretion efficiency of selective proteins. In the absence of ER stress, mice with hepatocyte-specific Ire1? deletion displayed modest hepatosteatosis that became profound after induction of ER stress. Further investigation revealed that IRE1? represses expression of key metabolic transcriptional regulators, including CCAAT/enhancer-binding protein (C/EBP) ?, C/EBP?, peroxisome proliferator-activated receptor ? (PPAR?), and enzymes involved in triglyceride biosynthesis. IRE1? was also found to be required for efficient secretion of apolipoproteins upon disruption of ER homeostasis. Consistent with a role for IRE1? in preventing intracellular lipid accumulation, mice with hepatocyte-specific deletion of Ire1? developed severe hepatic steatosis after treatment with an ER stress-inducing anti-cancer drug Bortezomib, upon expression of a misfolding-prone human blood clotting factor VIII, or after partial hepatectomy. The identification of IRE1? as a key regulator to prevent hepatic steatosis provides novel insights into ER stress mechanisms in fatty liver diseases associated with toxic liver injuries.

Project description:BackgroundUnfolded protein response (UPR) is a multifaceted signaling cascade that alleviates protein misfolding. Although well studied in nucleated cells, UPR in absence of transcriptional regulation has not been described. Intricately associated with cardiovascular diseases, platelets, despite being anucleate, respond rapidly to stressors in blood. We investigate the UPR in anucleate platelets and explore its role, if any, on platelet physiology and function.MethodsHuman and mouse platelets were studied using a combination of ex vivo and in vivo experiments. Platelet lineage-specific knockout mice were generated independently for each of the 3 UPR pathways, PERK (protein kinase RNA [PKR]-like endoplasmic reticulum kinase), XBP1 (X-binding protein), and ATF6 (activating transcription factor 6). Diabetes patients were prospectively recruited, and platelets were evaluated for activation of UPR under chronic pathophysiological disease conditions.ResultsTunicamycin induced the IRE1α (inositol-requiring enzyme-1alpha)-XBP1 pathway in human and mouse platelets, while oxidative stress predominantly activated the PERK pathway. PERK deletion significantly increased platelet aggregation and apoptosis and phosphorylation of PLCγ2, PLCβ3, and p38 MAPK. Deficiency of XBP1 increased platelet aggregation, with higher PLCβ3 and PKCδ activation. ATF6 deletion mediated a relatively modest effect on platelet phenotype with increased PKA (protein kinase A). Platelets from diabetes patients exhibited a positive correlation between disease severity, platelet activation, and protein aggregation, with only IRE1α-XBP1 activation. Moreover, IRE1α inhibition increased platelet aggregation, while clinically approved chemical chaperone, sodium 4-phenylbutyrate reduced the platelet hyperactivation.ConclusionsWe show for the first time, that UPR activation occurs in platelets and can be independent of genomic regulation, with selective induction being specific to the source and severity of stress. Each UPR pathway plays a key role and can differentially modulate the platelet activation pathways and phenotype. Targeting the specific arms of UPR may provide a new antiplatelet strategy to mitigate thrombotic risk in diabetes and other cardiovascular diseases.

Project description:Radiotherapy using high linear energy transfer (LET) radiation results in effectively killing tumor cells while minimizing dose (biological effective) to normal tissues to block toxicity. It is well known that high LET radiation leads to lower cell survival per absorbed dose than low LET radiation. High-linear energy transfer (LET) neutron treatment induces autophagy in tumor cells, but its precise mechanisms in osteosarcoma are unknown. Here, we investigated this mechanism and the underlying signaling pathways. Autophagy induction was examined in gamma-ray-treated KHOS/NP and MG63 osteosarcoma cells along with exposure to high-LET neutrons. The relationship between radiosensitivity and autophagy was assessed by plotting the cell surviving fractions against autophagy levels. Neutron treatment increased autophagy rates in irradiated KHOS/NP and MG63 cells; neutrons with high-LETs showed more effective inhibition than those with lower LET gamma-rays. To determine whether the unfolded protein response and Akt-mTOR pathways triggered autophagy, phosphorylated eIF2? and JNK levels, and phospho-Akt, phosphor-mTOR, and phospho-p70S6 levels were, respectively, investigated. High-LET neutron exposure inhibited Akt phosphorylation and increased Beclin 1 expression during the unfolded protein response, thereby enhancing autophagy. The therapeutic efficacy of high-LET neutron radiation was also assessed in vivo using an orthotopic mouse model. Neutron-irradiated mice showed reduced tumor growth without toxicity relative to gamma-ray-treated mice. The effect of high-LET neutron exposure on the expression of signaling proteins LC3, p-elF2a, and p-JNK was investigated by immunohistochemistry. Tumors in high-LET-neutron radiation-treated mice showed higher apoptosis rates, and neutron exposure significantly elevated LC3 expression, and increased p-elF2a and p-JNK expression levels. Overall, these results demonstrate that autophagy is important in radiosensitivity, cell survival, and cellular resistance against high-LET neutron radiation. This correlation between cellular radiosensitivity and autophagy may be used to predict radiosensitivity in osteosarcoma.

Dataset Information

Hepatocellular autophagy modulates the unfolded protein response and fasting-induced steatosis in mice.

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Hepatocellular autophagy modulates the unfolded protein response and fasting-induced steatosis in mice.

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