RESUMO
Hyperinsulinemia is a critical risk factor for the pathogenesis of insulin resistance (IR) in metabolic tissues, including the liver. Ethanolamine phosphate phospholyase (ETNPPL), a newly discovered metabolic enzyme that converts phosphoethanolamine (PEA) to ammonia, inorganic phosphate, and acetaldehyde, is abundantly expressed in liver tissue. Whether it plays a role in the regulation of hyperinsulinemia-induced IR in hepatocytes remains elusive. Here, we established an in vitro hyperinsulinemia-induced IR model in the HepG2 human liver cancer cell line and primary mouse hepatocyte via a high dose of insulin treatment. Next, we overexpressed ETNPPL by using lentivirus-mediated ectopic to investigate the effects of ETNPPL per se on IR without insulin stimulation. To explore the underlying mechanism of ETNPPL mediating hyperinsulinemia-induced IR in HepG2, we performed genome-wide transcriptional analysis using RNA sequencing (RNA-seq) to identify the downstream target gene of ETNPPL. The results showed that ETNPPL expression levels in both mRNA and protein were significantly upregulated in hyperinsulinemia-induced IR in HepG2 and primary mouse hepatocytes. Upon silencing ETNPPL, hyperinsulinemia-induced IR was ameliorated. Under normal conditions without IR in hepatocytes, overexpressing ETNPPL promotes IR, reactive oxygen species (ROS) generation, and AKT inactivation. Transcriptome analysis revealed that salt-inducible kinase 1 (SIK1) is markedly downregulated in the ETNPPL knockdown HepG2 cells. Moreover, disrupting SIK1 prevents ETNPPL-induced ROS accumulation, damage to the PI3K/AKT pathway and IR. Our study reveals that ETNPPL mediates hyperinsulinemia-induced IR through the SIK1/ROS-mediated inactivation of the PI3K/AKT signaling pathway in hepatocyte cells. Targeting ETNPPL may present a potential strategy for hyperinsulinemia-associated metabolic disorders such as type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2 , Hiperinsulinismo , Resistência à Insulina , Animais , Humanos , Camundongos , Diabetes Mellitus Tipo 2/metabolismo , Hepatócitos/metabolismo , Hiperinsulinismo/genética , Hiperinsulinismo/metabolismo , Insulina/metabolismo , Resistência à Insulina/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Background: Hepatocellular carcinoma (HCC) is characterized by high mortality, difficulty in early screening, relapse, and poor prognosis. This study aimed to explore the expression of ethanolamine-phosphate phospho-lyase (ETNPPL) and its clinical significance in HCC. Methods: Differentially expressed mRNAs were screened using microarray analysis. Functional enrichment was performed using GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis. We used qRT-PCR to measure the expression of ETNPPL in HCC tissues and paired paracarcinoma tissues. A receiver operating characteristic (ROC) curve and Kaplan-Meier curve were conducted to assess the diagnostic and prognostic values. Cell behaviors were evaluated using a scratch test and transwell assay. Results: The results showed that numerous mRNAs are abnormally expressed in HCC. ETNPPL was decreased in HCC tissues and cells. The area under curve (AUC) of ETNPPL was 0.9089, demonstrating that ETNPPL had diagnostic value. Low expression of ETNPPL was related to poor prognosis for patients with HCC. Moreover, the over-expression of ETNPPL inhibited HCC cell migration and invasion. Conclusions: In conclusion, downregulated ETNPPL was found in HCC and is related to poor patient prognosis and the promotion of cell metastasis. This suggests that ETNPPL serves both as a promising diagnosis and prognosis biomarker, and a therapy target of HCC.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/diagnóstico , Neoplasias Hepáticas/diagnóstico , Recidiva Local de Neoplasia , Prognóstico , Etanolaminas , FosfatosRESUMO
Excessive free fatty acids (FFAs) accumulation is a leading risk factor for the pathogenesis of insulin resistance (IR) in metabolic tissues, including the liver. Ethanolamine-phosphate phospho-lyase (ETNPPL), a newly identified metabolic enzyme, catalyzes phosphoethanolamine (PEA) to ammonia, inorganic phosphate, and acetaldehyde and is highly expressed in hepatic tissue. Whether it plays a role in regulating FFA-induced IR in hepatocytes has yet to be understood. In this study, we established an in vitro palmitic acid (PA)-induced IR model in human HepG2 cells and mouse AML12 cells with chronic treatment of PA. Next, we overexpressed ETNPPL by using lentivirus-mediated ectopic to investigate the effects of ETNPPL per se on IR without PA stimulation. We show that ETNPPL expression is significantly elevated in PA-induced IR and that silencing ETNPPL ameliorates this IR in hepatocytes. Inversely, overexpressing ETNPPL under normal conditions without PA promotes IR, reactive oxygen species generation, and ARG2 activation in both HepG2 and AML12 cells. Moreover, ETNPPL depletion markedly down-regulates ARG2 expression in hepatocytes. Besides, silencing ARG2 prevents ETNPPL-induced ROS accumulation and inhibition of autophagic flux and IR in hepatocytes. Finally, we found that phytopharmaceutical disruption of ETNPPL by quercetin ameliorates PA-induced IR in hepatocytes. Our study discloses that ETNPPL inhibiting autophagic flux mediates insulin resistance triggered by PA in hepatocytes via ARG2/ROS signaling cascade. Our findings provide novel insights into elucidating the pathogenesis of obesity-associated hepatic IR, suggesting that targeting ETNPPL might represent a potential approach for T2DM therapy.
Assuntos
Resistência à Insulina , Humanos , Camundongos , Animais , Resistência à Insulina/genética , Espécies Reativas de Oxigênio/metabolismo , Ácido Palmítico/toxicidade , Fígado/metabolismo , Hepatócitos/metabolismo , Autofagia/genéticaRESUMO
Astrocytes play diverse roles in the central nervous system (CNS) in both physiological and pathological conditions. Previous studies have identified many markers of astrocytes to analyze their complicated roles. Recently, closure of the critical period by mature astrocytes has been revealed, and the need for finding mature astrocyte-specific markers has been growing. We previously found that Ethanolamine phosphate phospholyase (Etnppl) was almost not expressed in the developing neonatal spinal cord, and its expression level slightly decreased after pyramidotomy in adult mice, which showed weak axonal sprouting, suggesting that its expression level negatively correlates with axonal elongation. Although the expression of Etnppl in astrocytes in adult is known, its utility as an astrocytic marker has not yet been investigated in detail. Here, we showed that Etnppl was selectively expressed in astrocytes in adult. Re-analyses using published RNA-sequencing datasets revealed changes in Etnppl expression in spinal cord injury, stroke, or systemic inflammation models. We produced high-quality monoclonal antibodies against ETNPPL and characterized ETNPPL localization in neonatal and adult mice. Expression of ETNPPL was very weak in neonatal mice, except in the ventricular and subventricular zones, and it was heterogeneously expressed in adult mice, with the highest expression in the cerebellum, olfactory bulb, and hypothalamus and the lowest in white matter. Subcellular localization of ETNPPL was dominant in the nuclei with weak expression in the cytosol in the minor population. Using the antibody, astrocytes in adult were selectively labeled in the cerebral cortex or spinal cord, and changes in astrocytes were detected in the spinal cord after pyramidotomy. ETNPPL is expressed in a subset of Gjb6 + astrocytes in the spinal cord. The monoclonal antibodies we created, as well as fundamental knowledge characterized in this study, will be valuable resources in the scientific community and will expand our understanding of astrocytes and their complicated responses in many pathological conditions in future analyses.