Endoplasmic Reticulum and Mitochondria Contacts Correlate with the Presence and Severity of NASH in Humans

Abstract

The interaction between the mitochondria and the endoplasmic reticulum (ER) is essential for hepatocyte function. An increase in ER-mitochondria contacts (ERMCs) is associated with various metabolic diseases. Non-alcoholic fatty liver disease (NAFLD) is associated with obesity and type 2 diabetes, and its progressive form non-alcoholic steatohepatitis (NASH) can lead to cirrhosis and hepatocellular carcinoma. However, the role of ERMCs in the progression of NAFL to NASH is still unclear. We assessed whether ERMCs could correlate with NAFLD severity. We used a proximity ligation assay to measure the abundance of ERMCs in liver biopsies from patients with biopsy-proven NAFLD (n = 48) and correlated the results with histological and metabolic syndrome (MetS) features. NAFLD patients were included according to inclusion and exclusion criteria, and then assigned to NAFL (n = 9) and NASH (n = 39) groups. ERMCs density could discriminate NASH from NAFL (sensitivity 61.5%, specificity 100%). ERMCs abundance correlated with hepatocellular ballooning. Moreover, the density of ERMCs increased with an increase in the number of MetS features. In conclusion, ERMCs increased from NAFL to NASH, in parallel with the number of MetS features, supporting a role for this interaction in the pathophysiology of NASH.

Keywords: MAFLD; NAFLD; NASH; endoplasmic reticulum; mitochondria.

Figure A1

The ROC curve for differentiating ERMCs via PLA in NASH and NAFL.

Figure 1

Schematic diagram of the PLA and representative images from NAFL and NASH liver biopsies. (a) Schematic diagram of the PLA; (b) representative images taken from 3dhistech caseviewer under 20× magnification (scale bars: 100 μm).

Figure 2

Comparison of PLA-dots/cell between PLA-dots/cell by NAFL and grades of NASH according to histology. (a) Comparison of PLA-dots/nucleus in NAFL (n = 9) and NASH (n = 39), (b) steatosis (NASH S1, S2, S3: n = 9, 12, 18, respectively), (c) ballooning (NASH B1, B2: n = 31, 8, respectively), (d) lobular inflammation (NASH L1, L2: n = 35, 4, respectively), (e) fibrosis (NASH F0/1/2, F3: n = 26, 13, respectively), and (f) SAF activity (NASH SAF activity score <3, ≥3: n = 30, 9, respectively). Results were analyzed by Student’s t-test or one-way ANOVA with Tukey’s multiple comparison test. A two-tailed p-value < 0.05 was considered significant. All statistical tests were two-sided. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Data are expressed as mean ± SEM.

Figure 3

PLA-dots/cell and metabolic syndrome contributions. (a) ERMC data were grouped and analyzed depending on the number of MetS features that had a contribution. 0MS, n = 5; 1 MS, n = 18; 2 MS, n = 19; 3 MS, n = 6. (b) A more detailed analysis with the histogram reports the mean and SEM of the ERMC in NAFLD patients with different combinations of MetS features, showing an increasing trend from NAFLD with no MetS features to NAFLD with all three MetS features. Abbreviations: Di, diabetes mellitus type 2; Dy, dyslipidemia; MetS, metabolic syndrome; Ob, obesity. Ob(+)Di(+)Dy(+), n = 6; Ob(+)Di(+)Dy(−), n = 4; Di(+)Dy(+)Ob(−), n = 5; Ob(+)Dy(+)Di(−), n = 10; Ob(+)Di(−)Dy(−), n = 8; Dy(+)Ob(−)Di(−), n = 5; Di(+)Ob(−)Dy(−), n = 5; Di(−)Dy(−)Ob(−), n = 5. Results were analyzed by one-way ANOVA with Dunnett’s multiple comparisons test. All data were normalized to the “0 MetS” control group. A two-tailed p-value < 0.05 was considered significant. All statistical tests were two-sided. ** p < 0.01. Data are expressed as mean ± SEM.

Figure 4

Correlation with demographic, metabolic syndrome features, and histopathological features in NAFLD (n = 48). The left panel presents Pearson correlation coefficient r and p-values for each variable. The right panel/vertical histogram shows different r levels for each variable with PLA dots/cell. SAF scores were adopted for histological parameters.