Association between Serum Atypical Fibroblast Growth Factors 21 and 19 and Pediatric Nonalcoholic Fatty Liver Disease

Abstract

Atypical fibroblast growth factors (FGF) 21 and 19 play a central role in energy metabolism through the mediation of Klotho coreceptor. Contradictory findings are available about the association of FGF21 and FGF19 with nonalcoholic fatty liver disease (NAFLD) in humans. We investigated the association of serum FGF21, FGF19 and liver Klotho coreceptor with non-alcoholic steatohepatitis (NASH) and fibrosis in children with NAFLD. Serum FGF21 and FGF19 were measured in 84 children with biopsy-proven NAFLD and 23 controls (CTRL). The hepatic expression of Klotho coreceptor was measured in 7 CTRL, 9 patients with NASH (NASH+) and 11 patients without NASH (NASH-). FGF21 and FGF19 showed a tendency to decrease from CTRL (median FGF21 = 196 pg/mL; median FGF19 = 201 pg/mL) to NASH- (FGF21 = 89 pg/mL; FGF19 = 81 pg/mL) to NASH+ patients (FGF21 = 54 pg/mL; FGF19 = 41 pg/mL) (p<0.001 for all comparisons) and were inversely associated with the probability of NASH and fibrosis in children with NAFLD. The hepatic expression of Klotho coreceptor was inversely associated with NASH (R(2) = 0.87, p<0.0001) and directly associated with serum FGF21 (R(2) = 0.57, p<0.0001) and FGF19 (R(2) = 0.67, p<0.0001). In conclusion, serum FGF19 and FGF21 and hepatic Klotho expression are inversely associated with hepatic damage in children with NAFLD and these findings may have important implications for understanding the mechanisms of NAFLD progression.

Conflict of interest statement

Competing Interests: Dr. Anna Alisi is a PLOS ONE Editorial Board member, but this does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1. NASH and serum levels of FGF21 and FGF19.

Distribution of serum FGF21 (panel A) and FGF19 (panel B) in controls, NAFLD children without NASH (NASH−) and NASH children with NASH (NASH+). Lines superimposed to dot-plots are medians.

Figure 2. Probability of NASH and fibrosis as a function of serum FGF21 and FGF19.

Probability of NASH and fibrosis of any degree as a function of loge-transformed values of FGF21 and FGF19 (see text for further details on statistical analysis). Abbreviations: NASH = non-alcoholic steatohepatitis; loge = natural logarithm; AIC = Akaike information criterion. Circles are means and bars 95% confidence intervals.

Figure 3. Association between the hepatic expression of Klotho, liver histopathology and serum FGF21 and FGF19.

Association of Klotho with histopathology, FGF21 and FGF19 (see text for further details on statistical analysis). Abbreviations: loge = natural logarithm; RMSE = root mean squared error of the estimate; R2 = coefficient of determination; AIC = Akaike information criterion. Circles are means and bars 95% confidence intervals.

Figure 4. Representative confocal immunofluorescence for expression and intracellular distribution of Klotho co-receptor.

The representative confocal immunofluorescence was performed on liver tissue cryostat sections OCT-embedded. The staining of Klotho co-receptor in the overweight-obese children without NAFLD (A), in the NAFLD overweight-obese children without NASH (B), and in the overweight-obese children with NASH (C) is shown in green. The nuclei are revealed by specific DAPI staining, displayed in blue. The white bar represents a 30 µm length.

Figure 5. Representative confocal immunofluorescence of Klotho co-receptor localization in liver tissues.

The representative confocal immunofluorescence was performed on liver tissue cryostat sections OCT-embedded. (A–C) Co-staining of Klotho co-receptor (green) and CD163 (red) in children without NAFLD (A), with NAFLD NASH− (B) and with NASH+ (C). (D–F) Co-staining of Klotho co-receptor (green) and cytokeratin 8/18 (red) in children without NAFLD (D), with NAFLD NASH− (E) and with NASH+ (F). (G–I) Co-staining of Klotho co-receptor (green) and alpha-SMA (red) in children without NAFLD (G), with NAFLD NASH− (H) and with NASH+ (I). Yellow arrows indicate hepatic stellate cells. The white bar represents a 30 µm length. (J) Histological scores for panels from A to I.