Fibroblast growth factor 21 is a hepatokine involved in MASLD progression

Abstract

Aim: We aimed to assess the role of FGF21 in metabolic dysfunction-associated steatotic liver disease (MASLD) at a multi-scale level.

Methods: We used human MASLD pathology samples for FGF21 gene expression analyses (qPCR and RNAseq), serum to measure circulating FGF21 levels and DNA for genotyping the FGF21 rs838133 variant in both estimation and validation cohorts. A hepatocyte-derived cell line was exposed to free fatty acids at different timepoints. Finally, C57BL/6J mice were fed a high-fat and choline-deficient diet (CDA-HFD) for 16 weeks to assess hepatic FGF21 protein expression and FGF21 levels by ELISA.

Results: A significant upregulation in FGF21 mRNA expression was observed in the liver analysed by both qPCR (fold change 5.32 ± 5.25 vs. 0.59 ± 0.66; p = 0.017) and RNA-Seq (3.5 fold; FDR: 0.006; p < 0.0001) in MASLD patients vs. controls. Circulating levels of FGF21 were increased in patients with steatohepatitis vs. bland steatosis (386.6 ± 328.9 vs. 297.9 ± 231.5 pg/mL; p = 0.009). Besides, sex, age, A-allele from FGF21, GG genotype from PNPLA3, ALT, type 2 diabetes mellitus and BMI were independently associated with MASH and significant fibrosis in both estimation and validation cohorts. In vitro exposure of Huh7.5 cells to high concentrations of free fatty acids (FFAs) resulted in overexpression of FGF21 (p < 0.001). Finally, Circulating FGF21 levels and hepatic FGF21 expression were found to be significantly increased (p < 0.001) in animals under CDA-HFD.

Conclusions: Hepatic and circulating FGF21 expression was increased in MASH patients, in Huh7.5 cells under FFAs and in CDA-HFD animals. The A-allele from the rs838133 variant was also associated with an increased risk of steatohepatitis and significant and advanced fibrosis in MASLD patients.

Keywords: FGF21; MASH; MASLD; NAFLD; NASH; fibroblast growth factor 21; gene expression; hepatokine; metabolic syndrome; steatohepatitis.

FIGURE 1

FGF21 findings in CDA‐HFD and control mice. (a) Histological findings in CDA‐HFA animals versus controls; (b) Protein expression evaluated in the livers by immunohistochemistry and (c) circulating levels in sera of mice. CDA‐HFD, choline‐deficient and high‐fat diet. ***p < 0.001.

FIGURE 2

FGF21 mRNA expression is found to be increased in Huh7.5 cells treated with free fatty acids at different timepoints (24, 48 and 72 h). ***p < 0.001.

FIGURE 3

(a) Flowchart of clinical studies; (b) FGF21 relative expression in liver biopsies from healthy controls, simple steatosis and MASH patients classified according to SAF Score; (c) Transcriptomic analysis of FGF21 mRNA expression in healthy controls, bland steatosis and steatohepatitis revealed that FGF21 expression correlated with liver damage progression. Data are expressed as mean $\pm$ SEM. FGF21, Fibroblast Growth Factor 21; MASH, metabolic dysfunction associated steatohepatitis; SS, Simple Steatosis. *p $\le$ 0.05; ***p < 0.001.

FIGURE 4

Correlations between hepatic FGF21 mRNA expression and several clinical and histopathological features: (a) BMI; (b) HOMA‐IR; (c) NAS Score; (d) Steatosis degree; (e) ballooning; and (f) fibrosis stage.

FIGURE 5

Serum FGF21 levels are raised in (a) MASLD patients (n = 89) versus healthy controls (n = 28); (b) MASH (n = 44) versus MASLD (n = 44) versus healthy controls (n = 28); (c) according to steatosis degree, 0%–5% steatosis (n = 28), 6%–33% steatosis (n = 44), 34%–66% steatosis(n = 30) and $\ge$ 66% steatosis (n = 15); (d) according to hepatocyte ballooning. ***p < 0.001; **p < 0.01; *p < 0.05. Mann‐Whitney and Kruskal‐Wallis tests corrected for the Bonferroni test have been used for these comparisons.

FIGURE 6

Patients bearing the G_PNPLA3/A_FGF21 haplotype showed an increased susceptibility to both significant and advanced liver fibrosis.