Volume 137, Issue 5, Pages 1795-1804 (November 2009) Fibroblast Growth Factor 21 Reduces the Severity of Cerulein-Induced Pancreatitis in Mice  Charis L. Johnson, Jacqueline Y. Weston, Sami A. Chadi, Elena N. Fazio, Murray W. Huff, Alexei Kharitonenkov, Anja Köester, Christopher L. Pin  Gastroenterology  Volume 137, Issue 5, Pages 1795-1804 (November 2009) DOI: 10.1053/j.gastro.2009.07.064 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 Increased Fgf21 accumulation is an immediate, cell autonomous response of acinar cells to CCK stimulation. Northern blot (A; n = 3) and qRT-PCR (B; n = 3–6) analyses following cerulein treatment revealed increased Fgf21 expression compared with saline-treated animals (−). 18S rRNA was used as a loading control. (C) Western blot analysis of pancreatic protein showed increased FGF21 during CIP; n = 3. (D and E) In situ hybridization 1 hour after saline (D) or cerulein (E) injection indicates that Fgf21 is limited to acinar cells and absent from pancreatic islets (I). Scale bar, 110 μm. (F) Fgf21 expression was increased following 30 minutes of CCK stimulation of isolated acinar cells compared with PBS treatment as determined by qRT-PCR. *P < .05; n = 5. (B and F) Error bars represent ± SEM. Gastroenterology 2009 137, 1795-1804DOI: (10.1053/j.gastro.2009.07.064) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 Increased Fgf21 accumulation is a response to multiple forms of acinar cell injury. (A) Northern blot analysis demonstrated increased pancreatic Fgf21 expression 4 hours after induction of pancreatitis with L-arginine (+) but not saline (−). (B) QRT-PCR for Fgf21 expression in pancreatic tissue (Panc), immediately following isolation of acinar cells (Fresh) or following overnight culture (O/N). n = 3; *P < .01. Stimulation with thapsigargin (TG) for 1 hour increased Fgf21 expression in isolated acinar cells compared with PBS-treated controls based on (C) Northern blot and (D) qRT-PCR, n = 3; *P < .05. Loading controls were (A) 18S rRNA hybridization or (C) ethidium bromide staining. (B and D) Error bars represent ± SEM. Gastroenterology 2009 137, 1795-1804DOI: (10.1053/j.gastro.2009.07.064) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 Pancreatic acinar cells are responsive to FGF21. (A) RT-PCR for βKlotho, Fgfr1, Fgfr4, and Mrpl1 (control) in whole pancreatic tissue, isolated acinar cells, AR42J, and ARIP cells. Numbers indicate the size (bp) of mouse (m) and rat (r) amplicons. Western blot analysis for active (pERK) and total ERK1/2 in (B) AR42J and ARIP cells 10 minutes after treatment with 10–10,000 ng/mL of FGF21 or over time in (C) AR42J and (D) isolated acinar cells following 1 μg/mL treatment with FGF21. Total ERK1/2 and β-tubulin were used as loading controls. In all cases, n = 3. Gastroenterology 2009 137, 1795-1804DOI: (10.1053/j.gastro.2009.07.064) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 Loss of Fgf21 leads to increased triglyceride accumulation in the exocrine pancreas. (A) QRT-PCR on mRNA extracts from WT, FGF21Tg (21Tg), and Fgf21−/− (21−/−) pancreatic tissue four hours after saline or cerulein injection. Error bars represent ± SEM. Letters indicate statistically different values (n = 3; P < .01). (B) H&E staining in WT (i), FGF21Tg (ii), and Fgf21−/− (iii) pancreatic tissue. Insets depict a single acinus. (C) Trichrome green stain of Fgf21−/− tissue reveals accumulations of adipose tissue (a) and intra-acinar vacuole accumulation (arrow). Scale bar, 35 μm. (D) Quantification of triglyceride levels in pancreatic and liver tissue. Error bars represent SEM. *Indicates P < .05 between triglyceride levels in WT and Fgf21−/− pancreata. Gastroenterology 2009 137, 1795-1804DOI: (10.1053/j.gastro.2009.07.064) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 Alterations in FGF21 expression in mice alter the severity of CIP. H&E (A) and trichrome green (B) histology of pancreatic sections 7 hours after initiating CIP in WT (i), FGF21Tg (ii), and Fgf21−/− mice. Interlobular edema (*), areas of necrosis (arrow), and vascularity (arrowhead) are indicated. Scale bar, 100 μm. (C) Serum amylase analysis 1, 4, and 7 hours (n = 3–12) and (D) acinar cell apoptosis 4 and 7 hours (n = 3–7) after initiating CIP was quantified. *Indicates a significant difference (P < .05). Error bars represent ± SEM. (E) Western blot analysis for phospho-JNK or phospho (p) c-jun 4 hours into CIP. The lower panel is a Coomassie-stained gel to show equivalent loading with amylase indicated (arrowhead). (F) Western blot analysis of protein extracts from WT, FGF21Tg (21Tg), Fgf21−/− (21−/−), and Mist1−/− (M−/−) acini for carboxypeptidase (CPA) or PDGF immediately after isolation or following overnight culture. Active CPA (*) is observed at 37 kilodaltons compared with inactive CPA at 47 kilodaltons. Gastroenterology 2009 137, 1795-1804DOI: (10.1053/j.gastro.2009.07.064) Copyright © 2009 AGA Institute Terms and Conditions

Figure 6 Egr-1 expression is decreased by FGF21. Trichrome green stain in WT (A) and Fgf21−/− (B) reveals increased extracellular matrix (ECM) deposition (arrowhead) in Fgf21−/− mice. (C) Western blot analysis for SMA, PDGF, and β-actin (control) following saline, 4 hour or 7 hour treatment with cerulein. (D) Densitometric analysis for PDGF at the same time points indicates significantly less PDGF expression in saline treated mice (*P < .05). (E) QRT-PCR analysis of pancreatic Egr-1 expression following saline (Sal) or CIP treatment in WT, FGF21Tg, and Fgf21−/− mice. (F) QRT-PCR for Egr-1 expression in isolated acinar cells incubated 30 minutes in PBS or 1 nmol/L CCK following a 30-minute pretreatment with 1 μg/mL FGF21 (+) or PBS (−); n = 4. Error bars represent ± SEM. (E and F) Significantly different values are indicated by a, b, and c, P < .05. Gastroenterology 2009 137, 1795-1804DOI: (10.1053/j.gastro.2009.07.064) Copyright © 2009 AGA Institute Terms and Conditions