Research ArticlePANCREATITIS

Pancreatitis is an FGF21-deficient state that is corrected by replacement therapy

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Science Translational Medicine  08 Jan 2020:
Vol. 12, Issue 525, eaay5186
DOI: 10.1126/scitranslmed.aay5186
  • Fig. 1 FGF21 replacement ameliorates pancreatitis.

    (A to C) Pancreatic FGF21 mRNA (A) and protein (B), and plasma FGF21 (C) at 4, 8, 12, or 18 hours after the first cerulein injection in CIP. (D) Pancreatic Fgf21 mRNA after 24 hours of AIP and EIP. (E and F) Pancreatic FGF21 mRNA and protein and plasma FGF21 protein in CIP (E) or AIP (F) after a 24-hour treatment regimen of FGF21 (1 mg/kg) (four intraperitoneal injections). (G and H) Plasma amylase activity in CIP (G) and AIP (H). (I and J). Pancreatic MPO activity in CIP (I) and AIP (J). (K and L) Histological grading of mouse pancreata in CIP (K) and AIP (L). (M) FGF21 in plasma (at 6 and 24 hours), and pancreatic FGF21 mRNA and protein (at 24 hours) after inducing EIP with intraductal infusion of contrast agent in the absence or presence of FGF21 (100 μg/ml). (N) Serum amylase activity at 6 and 24 hours from mice in (M). (O and P) Pancreatic MPO activity (O) and histological grading of pancreata (P) of mice in (M). Results are expressed as means ± SEM. n = number of mice per group for all experiments. n = 3 to 4 in (A) to (C); n = 3 for AIP and n = 5 for EIP in (D); n = 6 in (E), (G), (I), and (K); n = 8 in (F), (H), (J), and (L); n = 5 in (M) to (P). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Asterisks indicate statistically different values relative to vehicle. Statistical differences (P > 0.05) are indicated between all values that have different letters.

  • Fig. 2 β-Klotho in acinar cells is required for the therapeutic effects of FGF21 in pancreatitis.

    (A) Pancreatic Klb mRNA in wild-type (Klbfl/fl) and acinar cell–specific β-klotho KO (KlbCela1) mice (n = 3). (B and C) Pancreatic FGF21 mRNA (B) and protein (C) in CIP after a 24-hour treatment regimen of FGF21 (1 mg/kg) (four intraperitoneal injections) in Klbfl/fl and KlbCela1 mice. (D and E) Plasma FGF21 (D) and amylase activity (E) of mice in (B). (F and G) Representative H&E images of stained pancreata (F) and histological grading (G) of mice in (B). Scale bar indicates 50 μm for all images. (H) Pancreatic inflammation (Tnfα and Il6), oxidative stress (Hmox1), and ER stress (Chop) markers of mice in (B). Results are expressed as means ± SEM. n, number of mice per group. n = 3 (vehicle), 4 (CIP), and 5 (CIP + FGF21) in (B) to (G). ****P < 0.0001. Asterisks indicate statistically different values relative to vehicle. Statistical differences (P > 0.05) are indicated between all values that have different letters.

  • Fig. 3 ATF3 is induced in pancreatitis and binds to the Fgf21 promoter.

    (A) Immunoblot of pancreatic ATF4 and ATF3 at 4, 8, 12, or 18 hours after the first cerulein injection in CIP with β-actin as loading control. (B and C) Quantification of ATF4 (B) and ATF3 (C) in (A). (D) Schematic of the mouse −1497/+5 bp region of the Fgf21 promoter with the sequences and positions of the wild-type (WT) and mutant amino acid response elements (AARE). (E to H) ChIP assays quantified by qPCR for ATF4 (E and G) and ATF3 (F and H) binding to the AARE1 + 3 or AARE2 response elements from mouse pancreas in the CIP time-course experiment. IgG, immunoglobulin G. Results are expressed as means ± SEM. n = 3 to 4 mice per group for all experiments. *P < 0.05; **P < 0.01; ****P < 0.0001. Asterisks indicate statistically different values relative to vehicle.

  • Fig. 4 ATF3 competes with ATF4 to repress Fgf21 promoter activity.

    (A) Fgf21 mRNA in 266-6 mouse acinar cells treated with vehicle (black bar) or 100 nM cerulein (white bar) for 12 hours (N = 3). (B and C) Repression of the Fgf21 promoter using a luciferase reporter gene evaluated after Atf3 siRNA knockdown (N = 4) (B) or ATF3 overexpression (N = 4) (C) in 266-6 cells treated as in (A). (D) Wild-type and AARE mutant Fgf21 promoter luciferase (Luc) activity in 266-6 cells overexpressing ATF3 and ATF4 (N = 4). (E) Fgf21 promoter luciferase activity in 266-6 cells expressing increasing amounts (0.01 to 100 ng) of ATF3 expression plasmid and a fixed amount (1 ng) of ATF4 expression plasmid (N = 8). (F) Truncated Fgf21 promoter luciferase assays in 266-6 cells overexpressing ATF3 and ATF4 (N = 6). (G) Wild-type and AARE mutant Fgf21 promoter luciferase activity in 266-6 cells treated as in (A) (N = 4). Results are expressed as means ± SEM. N, number of replicates. ***P < 0.001; ****P < 0.0001. Asterisks indicate statistically different values relative to vehicle. Statistical differences (P > 0.05) are indicated between all values that have different letters.

  • Fig. 5 Pancreatic FGF21, ATF3, and ATF4 concentrations in human patients with pancreatitis.

    (A) H&E staining of pancreata from a representative normal and pancreatitis patient. (B to G) Pancreatic concentrations of FGF21 (B and C), ATF3 (D and E), and ATF4 (F and G) in normal individuals and patients with chronic (CP) and acute (AP) pancreatitis. Representative immunofluorescence images are shown in (B), (D), and (F), and quantification of samples (normal, n = 14; chronic, n = 49; and acute pancreatitis, n = 3) is shown in (C), (E), and (G). In the immunofluorescence images, DAPI staining is blue and FGF21, ATF3, and ATF4 stainings are gray. Scale bar indicates 50 μm for all images. Results in (C), (E), and (G) are expressed as means ± SEM. Statistical differences (P > 0.05) are indicated between all values that have different letters. R.F.U., relative fluorescence units.

  • Fig. 6 Inhibition of PERK ameliorates pancreatitis in an FGF21-dependent manner.

    (A to F) Wild-type (WT) or FGF21 KO mice (FGF21-KO) were treated with FGF21 (1 mg/kg) or GSK2656157 (25 mg/kg) (four intraperitoneal injections) after CIP. (A and B) Pancreatic FGF21 mRNA (A) and protein (B). (C to F) Plasma amylase activity (C), representative H&E images (D), histological grading (E), and pancreatic mRNA markers of inflammation (Tnfα and Il6), oxidative stress (Hmox1), and ER stress (Chop) (F) in mice treated with therapeutic or vehicle control. Scale bar indicates 50 μm for all images. Results are expressed as means ± SEM; n = 6 mice per group. Statistical differences (P > 0.05) are indicated between all values that have different letters.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/525/eaay5186/DC1

    Fig. S1. Experimental design and characterization of acute and chronic pancreatitis mouse models.

    Fig. S2. Experimental design, histology, and gene expression data for FGF21 therapy experiments in three different models of pancreatitis.

    Fig. S3. Atf3 and Atf4 expression in pancreatitis mouse models and ATF3 and ATF4 ChIP experimental controls.

    Fig. S4. ATF3 and ATF4 expression in 266-6 cells.

    Fig. S5. Model of FGF21’s protective role in pancreatitis.

    Table S1. qPCR primer sequences.

    Table S2. Primary antibody list.

    Table S3. ChIP-qPCR primer sequences.

    Data file S1. Human pancreas tissue donor information.

    Data file S2. Raw numbers for each data point in each graph from the study.

  • The PDF file includes:

    • Fig. S1. Experimental design and characterization of acute and chronic pancreatitis mouse models.
    • Fig. S2. Experimental design, histology, and gene expression data for FGF21 therapy experiments in three different models of pancreatitis.
    • Fig. S3. Atf3 and Atf4 expression in pancreatitis mouse models and ATF3 and ATF4 ChIP experimental controls.
    • Fig. S4. ATF3 and ATF4 expression in 266-6 cells.
    • Fig. S5. Model of FGF21’s protective role in pancreatitis.
    • Table S1. qPCR primer sequences.
    • Table S2. Primary antibody list.
    • Table S3. ChIP-qPCR primer sequences.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Human pancreas tissue donor information.
    • Data file S2 (Microsoft Excel format). Raw numbers for each data point in each graph from the study.

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