Proteomic identification and characterization of hepatic glyoxalase 1 dysregulation in non-alcoholic fatty liver disease

Christos Spanos,Caroline Evans,Michael H Miller,J Bernadette Moore,Bernard M Corfe,Paul J Thornalley,Alexandra Bermúdez-Fajardo ,Anil Dhawan,Petchpailin Leenutaphong,Mark E Weeks,Elaina M Maldonado,Huan Wang,Naila Rabbani,Alberto Quaglia,John Dillon ,David Windridge,Francisco J Salguero,Ernesto Oviedo‐Orta ,Ciarán Fisher ,Emer Fitzpatrick

doi:10.1186/s12953-018-0131-y

Abstract

BackgroundNon-alcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide. However, its molecular pathogenesis is incompletely characterized and clinical biomarkers remain scarce. The aims of these experiments were to identify and characterize liver protein alterations in an animal model of early, diet-related, liver injury and to assess novel candidate biomarkers in NAFLD patients.MethodsLiver membrane and cytosolic protein fractions from high fat fed apolipoprotein E knockout (ApoE−/−) animals were analyzed by quantitative proteomics, utilizing isobaric tags for relative and absolute quantitation (iTRAQ) combined with nano-liquid chromatography and tandem mass spectrometry (nLC-MS/MS). Differential protein expression was confirmed independently by immunoblotting and immunohistochemistry in both murine tissue and biopsies from paediatric NAFLD patients. Candidate biomarkers were analyzed by enzyme-linked immunosorbent assay in serum from adult NAFLD patients.ResultsThrough proteomic profiling, we identified decreased expression of hepatic glyoxalase 1 (GLO1) in a murine model. GLO1 protein expression was also found altered in tissue biopsies from paediatric NAFLD patients. In vitro experiments demonstrated that, in response to lipid loading in hepatocytes, GLO1 is first hyperacetylated then ubiquitinated and degraded, leading to an increase in reactive methylglyoxal. In a cohort of 59 biopsy-confirmed adult NAFLD patients, increased serum levels of the primary methylglyoxal-derived advanced glycation endproduct, hydroimidazolone (MG-H1) were significantly correlated with body mass index (r = 0.520, p < 0.0001).ConclusionCollectively these results demonstrate the dysregulation of GLO1 in NAFLD and implicate the acetylation-ubquitination degradation pathway as the functional mechanism. Further investigation of the role of GLO1 in the molecular pathogenesis of NAFLD is warranted.

Highlights

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide
Proteome analysis identifies glyoxalase 1 as a candidate biomarker for NAFLD Isobaric tags for relative and absolute quantitation combined with nano-liquid chromatography and tandem mass spectrometry was used to compare expression profiles of cytosolic and membrane proteins extracted from the livers of apolipoprotein E knockout (ApoE−/−) and wild type (WT) mice fed either a normal chow diet (ND) or a high fat diet (HFD) for a 12-week period (n = 3/group)
Gene Ontology (GO) analyses confirmed that cytoskeletal proteins were detected in the cytosolic fractions and membrane, ribosomal and endoplasmic reticulum (ER) proteins were detected in the membrane fraction (Fig. 1b), thereby verifying the enrichment strategy. 91 cytosolic and 81 membranous proteins were identified as significantly differentially expressed (P < 0.05) based on both randomized permutation and Kruskal-Wallis tests with BenjaminiHochberg false discovery rate (FDR) used for multiple testing corrections (Additional file 1: Tables S1 and S2)

Summary

Introduction

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide. Its molecular pathogenesis is incompletely characterized and clinical biomarkers remain scarce The aims of these experiments were to identify and characterize liver protein alterations in an animal model of early, diet-related, liver injury and to assess novel candidate biomarkers in NAFLD patients. Non-alcoholic fatty liver disease (NAFLD) is increasingly the most common chronic liver disease in developed nations, with an estimated global adult prevalence of 25% [1]. Whereas steatosis is considered relatively benign and potentially reversible, NASH can progress to irreversible fibrosis and cirrhosis, leading to an increased risk of mortality from liver-related and cardiovascular-related causes [5]. Due to its increasing burden on health services worldwide, early screening and diagnosis of NAFLD is urgently required

Methods

Results

Discussion

Conclusion