Non-alcoholic fatty liver disease proteomics

Abstract

Purpose: Non-alcoholic fatty liver disease (NAFLD) is an important cause of chronic liver injury that has gained concern in clinical hepatology. The principal aim of this study was to find differences in protein expression between patients with NAFLD and healthy controls.

Experimental design: Changes in protein expression of liver samples from each of the three groups of subjects, controls, non-alcoholic steatosis, and non-alcoholic steatohepatitis (NASH), were analyzed by DIGE combined with MALDI TOF/TOF analysis, a proteomic approach that allows to compare hundreds of proteins simultaneously.

Results: Forty-three proteins exhibiting significant changes (ratio ≥1.5, p<0.05) were characterized, 22 comparing steatosis samples versus control samples and 21 comparing NASH versus control samples. Ten of these proteins were further analyzed by Western blot in tissue samples to confirm the observed changes of protein expression using DIGE. The proteins validated were further tested in serum samples of different cohorts of patients.

Conclusions and clinical relevance: Following this approach we identified two candidate markers, carbamoyl phosphate synthase 1 and 78 kDa glucose-regulated protein, differentially expressed between control and NASH. This proteomics approach demonstrates that DIGE combined with MALDI TOF/TOF and Western blot analysis of tissue and serum samples is a useful approach to identify candidate markers associated with NAFLD, resulting in proteins whose level of expression can be correlated to a disease state.

Figure 1

DIGE 2-DE images of human liver samples. Spots differentially expressed are labeled with numbers. Samples were run in a 3–7 isoelectric point (pI) gradient. A: Steatosis versus control differentially expressed spots. B: NASH versus control differentially expressed spots. NASH and steatosis were differentially labeled with a Cy3 and Cy5. An internal standard composed of disease sample and control sample labeled with Cy2 was added as normalization standard. Gels were further stained with Sypro Ruby for spot picking.

Figure 2

GRP78 and CPS1 were validated by Western blot analysis. GAPDH was used as loading control. A three dimensional view (3D view) of the spots corresponding to the selected proteins is displayed. 20 μg of liver lysate of each sample was incubated against each antibody in order to validate the differences in expression observed by DIGE. GRP78 and CPS1 were down-regulated in NASH samples.

Figure 3

Validation in human serum of two of the NAFLD associated proteins identified in liver samples by 2-DE. Changes in protein expression of CPS1 and GRP78 were validated using 15 serum samples of each of the three groups of subjects, (control, steatosis, and NASH). (A). CPS1 and GRP78 were down-regulated in NASH samples. Quantification of the signal has been obtained by densitometric scanning; data obtained were plotted as intensity values. The horizontal bar represents the media of the intensity values. Three replicas of each Western were performed. Student t-test of the densitometric values were calculated in order to asses the significance of the results The p values obtained for each antibody are as follows: CPS1 (steatosis/control p=4×10⁻²; NASH/control p=7.3×10⁻¹³); GRP78 (steatosis/control p=1×10⁻³; NASH/control p= 1.4×10⁻⁷). (B) Representative immunoblot analysis for the evaluation of six representative patients: C1, C2 (controls) S1, S2 (steatosis), N1, N2 (NASH). The expression levels of CPS1 and GRP78 were analyzed using the antibodies described in Materials and Methods. For evaluation of these proteins, 80 μg of protein was loaded onto a 12% SDS-PAGE. As control loading the PVDF membranes were stained with Ponceau staining (data not shown).