Targeted Analysis of Three Hormonal Systems Identifies Molecules Associated with the Presence and Severity of NAFLD

Abstract

Aims: To investigate circulating levels and liver gene expression of 3 hormonal pathways associated with obesity, insulin resistance, and inflammation to identify leads towards potential diagnostic markers and therapeutic targets in patients with nonalcoholic fatty liver disease (NAFLD).

Methods: We compared circulating levels of (1) proglucagon-derived hormones (glucagon-like peptide [GLP]-1, GLP-2, glicentin, oxyntomodulin, glucagon, major proglucagon fragment [MPGF]), (2) follistatins-activins (follistatin-like [FSTL]3, activin B), (3) IGF axis (insulin-like growth factor [IGF]-1, total and intact IGF binding protein [IGFBP]-3 and IGFBP-4, and pregnancy-associated plasma protein [PAPP]-A) in 2 studies: (1) 18 individuals with early stage NAFLD versus 14 controls (study 1; early NAFLD study) and in (2) 31 individuals with biopsy proven NAFLD (15 with simple steatosis [SS] and 16 with nonalcoholic steatohepatitis [NASH]), vs 50 controls (24 lean and 26 obese) (study 2). Liver gene expression was assessed in 22 subjects (12 controls, 5 NASH, 5 NASH-related cirrhosis).

Results: Patients in early stages of NAFLD demonstrate higher fasting MPGF and lower incremental increase of glicentin during oral glucose tolerance test than controls. In more advanced stages, FSTL3 levels are higher in NASH than simple steatosis and, within NAFLD patients, in those with more severe lobular and portal inflammation. The IGF-1/intact IGFBP-3 ratio is lower in patients with liver fibrosis. Genes encoding follistatin, activin A, activin B, and the IGF-1 receptor are higher in NASH.

Conclusion: MPGF and glicentin may be involved in early stages of NAFLD, whereas FSTL3 and IGF-1/intact IGFBP3 in the progression to NASH and liver fibrosis respectively, suggesting potential as diagnostic markers or therapeutic targets.

Trial registration: ClinicalTrials.gov NCT03986684.

Keywords: GLP-1; NAFLD; NASH; biomarkers; follistatin; liver steatosis.

Figure 1.

Circulating concentrations of proglucagon-derived hormones after oral glucose tolerance tests (OGTT) in groups with (n = 18) and without NAFLD (n = 14); each time point shows the change in the specific parameter in fold compared with baseline (A–F). Mann–Whitney test was performed between the AUCs of NAFLD vs non-NAFLD for GLP-1, GLP-2, glicentin, oxyntomodulin (non-normally distributed), whereas independent t-test was performed for glucagon and MPGF (normally distributed). mRNA expression of genes related to the follistatins/activins and the IGF-hormonal pathway in 12 subjects with normal liver in histology (healthy), 5 subjects with NASH and 5 with NASH-related cirrhosis (G,H). p-AUC: p value for the comparisons of AUCs between NAFLD and the controls (Mann–Whitney test). *P < .05; **P < .001; #P = .060; $P = .086. ANOVA or Kruskal–Wallis test was used (depending on distribution of data) to assess gene expression, and post hoc Dunnett’s or Dunn’s test, respectively, was performed to compare the controls with NASH or with NASH-related cirrhosis (see ref. (37) for absolute values and variability of the baseline.