Elsevier

Journal of Proteomics

Volume 221, 15 June 2020, 103683
Journal of Proteomics

Proteomic analysis to identify differentially expressed proteins between subjects with metabolic healthy obesity and non-alcoholic fatty liver disease

https://doi.org/10.1016/j.jprot.2020.103683Get rights and content

Highlights

  • Analysis of differential proteomics between MHO and NAFLD

  • FBLN5 and DHRS2 was selected and validated as candidate to distinguish MHO from NAFLD.

  • MHO has high level of oxidative phosphorylation and NAFLD has high level of ECM organization.

Abstract

Obese subjects with non-alcoholic fatty liver disease (NAFLD) and considered metabolically healthy have not been well differentiated. In this study, obese subjects were divided into metabolic healthy obesity (MHO) and NAFLD groups. Liver tissues were sampled from these two types of subjects undergoing bariatric surgery, and proteins in the liver tissues that expressed differently between the two groups of subjects were identified by Tandem Mass Tags (TMT) assay. Compared with the MHO group, 132 proteins were found to be upregulated and 84 proteins were found to be downregulated (mainly localized in mitochondria) in NAFLD group. The KEGG pathway analysis showed that significantly upregulated metabolic pathways include PPAR signaling, ECM-receptor interaction and oxidative phosphorylation was significantly downregulated. The GO analysis revealed that upregulated proteins were involved in extracellular structure organization, extracellular matrix organization and downregulated proteins took part in the oxidation–reduction process and so on. FBLN5 and DHRS2 were further validated by Western blot, immunohistochemistry and ELISA. All results demonstrate that FBLN5 expression was significantly upregulated but DHRS2 was significantly downregulated.

Significance

The variation between MHO and NAFLD was studied by mass spectroscopy to evaluate the mechanism with which MHO subjects resist the harmful effects induced by obesity.

Introduction

Non-alcoholic fatty liver disease (NAFLD) describes liver steatosis when secondary causes of hepatic fat accumulation do not exist (e.g., alcohol abuse with daily alcohol consumption >20 g/day for women or >30 g/day for men) [1]. NAFLD is one of the leading causes of liver disease in developed countries and its occurrence is also creeping up in developing countries [2]. Currently, it affects about one third of all adults in the US [3,4]. Similarly, 15–30% of the general adult population suffers from NAFLD in Asia [5]. In fact, the severe clinical form of NAFLD, i.e., non-alcoholic steatohepatitis (NASH)-is already one of the top aetiologies to require liver transplantation for adults [6,7]. Hepatic steatosis is regarded as a benign onset of NAFLD because no severe liver injury is observed during this stage, but some patients with hepatic steatosis do eventually progress into NASH, NASH-associated cirrhosis, or even hepatocellular carcinoma (HCC) [8]. Various non-invasive methods have also been used to detect hepatic fatty infiltration, such as hepatic ultrasonography, computed tomography, and magnetic resonance imaging, but liver biopsy is still the golden standard to diagnose NAFLD [[9], [10], [11]]. Searching for new validated biomarker of NAFLD is urgent.

The classical NAFLD patient is an obese/overweight individual exhibiting insulin resistance, type 2 diabetes, cardiovascular disease, and/or hypertension. According to a recent meta-analysis, a patient becomes ~1.2 times more likely to develop NAFLD for every unit increment of body mass index (BMI) [12]. NAFLD was a very important phenotype of metabolic abnormal obesity (MAO). In fact, the incidence of NAFLD is also rising in lean (non-obese) patients, a problem initially observed in the Asian population and gradually recognized as a global health issue [13,14]. On the other hand, research has somewhat neglected obese people without NALFD. Actually, there is a subset of population that exhibit the so-called “metabolically healthy obesity” (MHO) [[15], [16], [17]], a physiological condition free of any metabolic abnormality (e.g., hypertension, dyslipidemia, insulin resistance, unfavorable inflammatory profile, etc.) [[15], [16], [17]], and MHO individuals have a lower risk of mortality despite their apparent obesity. During our studies on bariatric surgery, we found that many obese patients did not have any fatty liver phenotype, and thus speculated that some factors might alter the homeostasis of glycometabolism and lipometabolism in the liver.

In this work, we make use of mass spectrum to identify proteins that are differentially expressed in obese patients with or without NAFLD. Bioinformatics analysis was applied to categorize the proteins involved in the physiological mechanisms of NAFLD. Two significantly altered proteins were selected for validation by western blotting, ELISA, and immunohistochemistry (IHC) analyses. Our findings indicate that MHO people have a low level of extracellular matrix (ECM) organization and are thus less susceptible to hepatic fibrosis, whereas the NAFLD subjects have a low level of oxidative phosphorylation in their mitochondria and the mitochondria dysfunction produces reactive oxygen species (ROS) to aggravate liver injury.

Section snippets

Ethics statement

Patients were selected from obese patients undergoing laparoscopic Roux-en-Y gastric bypass surgery at the Department of Hepatobiliary Surgery of the Affiliated Drum Tower Hospital of the Medical School of Nanjing University. The study was approved by the Ethics Committee of the Affiliated Drum Tower Hospital of the Medical School of Nanjing University, and the ethical permit number was 2017-030-02. The obesity patients with evidence for viral hepatitis, hemochromatosis, or alcohol consumption

Histopathological examinations

A total of 56 obese subjects who participated in the study were divided into the MHO group and the NAFLD group according to the liver pathological assessment. According to the biochemical tests and HE staining results, 12 subjects had abnormal BMI without liver steatosis and the other 44 patients had NAFLD (Fig. 1 and Table 1). The two groups did not have any significant difference in age, BMI, cholesterol, or fasting glucose, whereas significant difference existed between the two groups in the

Discussion

Obesity reduces life expectancy because obese patients are more susceptible to type 2 diabetes, cardiovascular diseases (CVD), and various cancers [22]. The occurrence of obesity is a major public health concern now, and it has been rising over the past 30 years worldwide [22]. Although the prognostic value of MHO is not only controversial but also quite challenging, consensus has been reached on two key characteristics of MHO subjects. That is, compared with patients with metabolic

Conclusion

In summary, we adopted a TMT-based quantitative proteomics approach in this work to screen differentially expressed proteins associated with NAFLD and MHO subjects in their liver tissues. Significantly changes were firstly identified for the FBLN5 and DHRS2 proteins and then confirmed by western blotting, IHC, and ELISA analyses. Both proteins are presumed to play a crucial role in MHO. The results of our study illuminate on the molecular mechanisms of MHO and may help develop novel

Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [30] partner repository with the dataset identifier PXD016405

The following is the supplementary data related to this article.

. Differentially expressed proteins in PNALD compared to control.

Declaration of Competing Interest

All the authors declare that there is no any conflict of interests.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (No. 31371373, 31771572, 31300103), the Nature Science Foundation of Jiangsu Province (BK20191356), the Open Fund of State Key Laboratory of Natural Medicines (No. SKLNMKF201811), Six talent peaks project in Jiangsu Province (yy-014), Research of Institute of Hospital Management Nanjing University (NDYG2017016), the Innovation Capability Development Project of Jiangsu Province (No. BM2015004), the Nanjing Healthy and

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