Hepatic Amyloid Beta-42-Metabolizing Proteins in Liver Steatosis and Metabolic Dysfunction-Associated Steatohepatitis

Abstract

Amyloid beta (Aβ) plays a major role in the pathogenesis of Alzheimer's disease and, more recently, has been shown to protect against liver fibrosis. Therefore, we studied Aβ-42 levels and the expression of genes involved in the generation, degradation, and transport of Aβ proteins in liver samples from patients at different stages of metabolic dysfunction-associated liver disease (MASLD) and under steatotic conditions in vitro/in vivo. Amyloid precursor protein (APP), key Aβ-metabolizing proteins, and Aβ-42 were analyzed using RT-PCR, Western blotting, Luminex analysis in steatotic in vitro and fatty liver mouse models, and TaqMan qRT-PCR analysis in hepatic samples from patients with MASLD. Hepatocytes loaded with palmitic acid induced APP, presenilin, and neprilysin (NEP) expression, which was reversed by oleic acid. Increased APP and NEP, decreased BACE1, and unchanged Aβ-42 protein levels were found in the steatotic mouse liver compared to the normal liver. Aβ-42 concentrations were low in MASLD samples of patients with moderate to severe fibrosis compared to the livers of patients with mild or no MASLD. Consistent with the reduced Aβ-42 levels, the mRNA expression of proteins involved in APP degradation (ADAM9/10/17, BACE2) and Aβ-42 cleavage (MMP2/7/9, ACE) was increased. In the steatotic liver, the expression of APP- and Aβ-metabolizing proteins is increased, most likely related to oxidative stress, but does not affect hepatic Aβ-42 levels. Consistent with our previous findings, low Aβ-42 levels in patients with liver fibrosis appear to be caused by the reduced production and enhanced non-amyloidogenic processing of APP.

Keywords: MASH; MASLD; NAFLD; NASH; amyloid; fatty acids; fibrosis; oxidative stress; steatosis.

Figure 5

Expression of APP-processing proteins in relation to liver tissue fibrosis scores. mRNA expression of proteins processing APP and Aβ-42 were plotted regarding their histologically proven fibrosis grade in liver samples from patients with an MAS ≥ 1 (steatosis and MASH). (A) Non-amyloidogenic pathway and (B–D) amyloidogenic pathway of APP and Aβ-42 processing: (A) degradation of APP, (B) degradation of APP followed by processing towards Aβ-42 formation, (C) degradation of Aβ-42 and (D) binding/transport of Aβ-42. mRNA expression was analyzed using qRT-PCR, followed by normalization to three housekeeping genes—GUSB, HPRT1, and TBP (see Table S3). Data are presented as box blots displaying median values, lower and upper quartiles, and the range of the values (whiskers), with outliers shown as circles (values between 1.5 and 3 times the interquartile range). Fibrosis score: 0, n = 27; 1–2, n = 23; 3–4, n = 14. Statistical differences were analyzed using the Kruskal–Wallis test with post hoc Bonferroni correction. * p < 0.05, ** p < 0.01.

Figure 6

Expression of APP-processing proteins in relation to liver tissue steatosis grade. mRNA expression of genes processing APP and Aβ-42 were plotted according to the histologically proven steatosis grade in liver samples from patients without hepatic fibrosis. mRNA expression was analyzed using qRT-PCR, followed by normalization to three housekeeping genes—GUSB, HPRT1, and TBP (see Table S3). Data are presented as box blots displaying median values, lower and upper quartiles, and the range of the values (whiskers), with outliers shown as circles (values between 1.5 and 3 times the interquartile range). Steatosis grade: 0 < 5, n = 26; 5–33, n = 10; >33, n = 17. Statistical differences were analyzed using the Kruskal–Wallis test with post hoc Bonferroni correction. * p < 0.05, ** p < 0.01.

Figure 1

Palmitic acid (PA) induces the mRNA expression of APP and its metabolizing proteins in vitro, which is reduced by mono-unsaturated oleic acid (OA). (A) Huh7 and (B) HepG2 cells were treated without or with indicated concentrations of PA or PA/OA (1/2) for 24 h, and (C) primary human hepatocytes (PHHs) were treated without or with PA for 24 h. (D) Huh7 cells were treated without or with PA or PA/OA (1/2) for 24 h. (E) HepG2 cells were treated with endoplasmic reticulum (ER) stress inducers thapsigargin (Tab, 0.5 µM for 6 h) or tunicamycin (Tun, 10 µg/mL for 16 h). The mRNA levels of genes involved in the amyloidogenic (APP, BACE1, PS1, and NEP) and non-amyloidogenic (ADAM9, ADAM10, and ADAM17) pathways of APP and its metabolizing genes were analyzed using qRT-PCR, and were normalized to HPRT1 (three independent experiments, mean ± SEM). * p < 0.05 differs from untreated control (0, Ctrl); # p < 0.05 differs from 0.4 mM or 0.8 mM PA/OA treatment.

Figure 2

Expression of APP, its metabolizing proteins, and the hepatic levels of Aβ-42 in mice fed a high-fat diet. Male mice were fed a standard diet (SD) or a high-fat diet (HFD) for 14 weeks, resulting in hepatic steatosis in the HFD group. Hepatic liver tissue was analyzed for mRNA expression of (A) APP- and Aβ-42-generating genes, as well as γ-secretase (PS1) substrates NOTCH1/3 and (B) non-amyloidogenic pathway-related genes. The mRNA levels were analyzed using qRT-PCR and were normalized to YWHAZ (n = 5). (C) Total protein extracts were isolated from liver samples and Western blot analysis using specific antibodies was performed with β-actin as loading control. Relative protein abundance was determined using densitometric analysis and was normalized to the loading control. (D) Hepatic Aβ-42 levels in samples from mice fed a standard diet (SD) or a high-fat diet (HFD). Aβ-42 concentrations were determined using multiplex analysis in homogenates from liver tissue. Data are presented either as data points or mean ± SD (n = 5); * p < 0.05 and ** p < 0.01.

Figure 3

Hepatic Aβ-42 levels in samples from patients with MASLD. Aβ-42 concentrations were determined using multiplex analysis in tissue homogenates from patients with MASH (MAS ≥ 5; n = 23; 4.91 ± 2.30), steatosis (MAS 1–4; n = 21; 6.67 ± 2.63), or control liver (MAS 0; n = 12; 7.78 ± 1.35). * p < 0.05 and ** p < 0.01 were considered as significantly different.

Figure 4

Expression of APP- and Aß-42-processing proteins in liver samples from patients with MASLD. The mRNA expression of proteins involved in the processing of APP and Aβ-42 via (A) non-amyloidogenic and (B–D) amyloidogenic pathways was analyzed: (A) degradation of APP, (B) degradation of APP followed by processing towards Aβ-42 formation, (C) degradation of Aβ-42, (D) binding / transport of Aβ-42, and (E) alternative substrate of γ-secretase PS1. mRNA expression was analyzed in hepatic tissue samples from patients with MASH (MAS ≥ 5, n = 36), hepatic steatosis (MAS 1–4, n = 30), and normal liver tissue (MAS 0, n = 26) using qRT-PCR followed by normalization to three housekeeping genes—GUSB, HPRT1, and TBP (see Table S3). Data are presented as box blots displaying median values, lower and upper quartiles, and the range of the values (whiskers), with outliers shown as circles (values between 1.5 and 3 times the interquartile range). Statistical differences were analyzed using the Kruskal–Wallis test with post hoc Bonferroni correction. * p < 0.05, ** p < 0.01.