Volume 137, Issue 3, Pages 1093-1101.e3 (September 2009) Molecular Mechanisms of Pancreatic Dysfunction Induced by Protein Malnutrition  Stephen J. Crozier, Louis G. D'Alecy, Stephen A. Ernst, Lauren E. Ginsburg, John A. Williams  Gastroenterology  Volume 137, Issue 3, Pages 1093-1101.e3 (September 2009) DOI: 10.1053/j.gastro.2009.04.058 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 Effect of dietary protein on pancreatic growth in mice. Time course of changes in pancreas weight (A) and pancreatic protein content (B) during protein deficiency and subsequent return of dietary protein. NO PRO, 0 g/kg casein chow (dashed line); CON, 200 g/kg casein chow (solid line). Values represent means ± SE; n = 5–11. Statistical significance was assessed using a 1-way ANOVA and Bonferroni multiple comparison posttest. *Significantly different from initial control value; †significantly different from 4-day NO PRO value, P < .05. Gastroenterology 2009 137, 1093-1101.e3DOI: (10.1053/j.gastro.2009.04.058) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 Effect of dietary protein on mTOR pathway activation in the pancreas. Phosphorylation of ribosomal protein S6 (A) and 4E-BP1 (B) as well as the content of 4E-BP1 (C) was assessed by Western blotting in mice fed either 200 g/kg casein chow for 4 days (CON), 0 g/kg casein chow 4 days (NO PRO), CON chow for 1 day following 4 days on NO PRO chow (1D REC), and CON chow for 4 days following 4 days on NO PRO chow (4D REC). Insets: Representative immunoblots. S6(P), S6 phosphorylated on Ser240/244; S6(T), total S6 content; BP1, total 4E-BP1 content; α, α-form of 4E-BP1; β, β-form of 4E-BP1; γ, γ-form of 4E-BP1; actin, β-actin (loading control). Values represent means ± SE; n = 6–8. *Significantly different from control value, P < .05. Gastroenterology 2009 137, 1093-1101.e3DOI: (10.1053/j.gastro.2009.04.058) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 Effect of rapamycin and FK506 on pancreatic growth following protein deficiency. Changes in pancreas weight (A) and protein content (B) in mice fed 200 g/kg casein chow for 4 days (CON), 0 g/kg casein chow for 4 days (NO PRO), NO PRO chow for 4 days and then CON chow for 4 days while administered vehicle daily (REC), NO PRO chow for 4 days and then CON chow for 4 days while administered daily IP injections of rapamycin (RAPA), or NO PRO chow for 4 days and then CON chow for 4 days while administered daily IP injections of FK506 (FK506). *Significantly different from initial control value, P < .05. Gastroenterology 2009 137, 1093-1101.e3DOI: (10.1053/j.gastro.2009.04.058) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 Effect of dietary protein on pancreatic morphology. Electron microscopy of ultrathin sections stained with uranyl acetate and lead citrate. Mice were fed either 200 g/kg casein chow for 4 days (A), 0 g/kg casein chow 4 days (B), 0 g/kg casein chow for 4 days and then 200 g/kg casein chow for 4 days while administered vehicle daily (C), or 0 g/kg casein chow for 4 days and then 200 g/kg casein chow for 4 days while administered daily IP injections of rapamycin (D). Arrows point to acinar lumens; VS indicates vesicular structures. Gastroenterology 2009 137, 1093-1101.e3DOI: (10.1053/j.gastro.2009.04.058) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 Effect of rapamycin on pancreatic secretory capacity following protein deficiency. Total amounts of protein in pancreo-biliary juice from mice under basal conditions and following bethanechol stimulation as determined by protein assay (A). The relative amount of chymotrypsinogen (B) and Coomassie blue stained protein (C) per unit volume of pancreo-biliary juice from mice following bethanechol stimulation. BASAL, unstimulated; STIMULATED, bethanechol stimulated; CON, 200 g/kg casein chow for 4 days; NO PRO, 0 g/kg casein chow 4 days; REC, NO PRO chow for 4 days and then CON chow for 4 days while administered vehicle daily; RAPA, NO PRO chow for 4 days and then CON chow for 4 days while administered daily IP injections of rapamycin. *Significantly different from basal control value, P < .05. Gastroenterology 2009 137, 1093-1101.e3DOI: (10.1053/j.gastro.2009.04.058) Copyright © 2009 AGA Institute Terms and Conditions

Figure 6 Effect of dietary protein on pancreatic growth in CCK-deficient mice. Time course of changes in pancreas weight (A), pancreatic protein content (B), and chymotrypsinogen content (C) during protein deficiency and subsequent return of dietary protein. WT, age-matched C57BL6 mice (black line); CCK−/−, CCK-deficient mice (gray line); CON, 200 g/kg casein chow (solid lines); NO PRO, 0 g/kg casein chow (dashed lines); Letters C, CON chow for 4 days; N, NO PRO chow 4 days; R, NO PRO chow for 4 days and then CON chow for 4 days; Chymo, total chymotrypsinogen content; GAPDH, loading control. Values represent means ± SE; n = 5–11. Statistical significance was assessed using a 1-way ANOVA and Bonferroni multiple comparison posttest. *Significantly different from respective control value; †significantly different from WT control value, P < .05. Gastroenterology 2009 137, 1093-1101.e3DOI: (10.1053/j.gastro.2009.04.058) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 1 Effect of dietary protein on pancreatic growth in mice. Time-course of changes in body weight (A) and pancreatic DNA content (B) during protein deficiency and subsequent return of dietary protein. NO PRO, 0 g/kg casein chow (dashed line); CON, 200 g/kg casein chow (solid line). Values represent means ± SE; n = 5–11. Statistical significance was assessed using a one-way ANOVA and Bonferroni'a Multiple Comparison post-test. *Significantly different from initial control value, †Significantly different from 4 day 0 g/kg casein value, P < .05. Gastroenterology 2009 137, 1093-1101.e3DOI: (10.1053/j.gastro.2009.04.058) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 2 Effect of rapamycin on acinar cell size following protein deficiency. Staining of pancreatic acinar cell nuclei with DAPI (A) and quantitation of nuclei per unit area (B) in mice fed 200 g/kg casein chow for 4 days (CON), 0 g/kg casein chow for 4 days (NO PRO), NO PRO chow for 4 days and then CON chow for 4 days while administered vehicle (REC), or NO PRO chow for 4 days and then CON chow for 4 days while administered daily i.p. injections of rapamycin (RAPA). For quantitation of nuclei, MetaMorph Offline version 6.1 software (Universal Imaging Corporation; Downingtown, PA) was used to count DAPI stained nuclei of a predetermined size limit within a field. Three fields were counted per mouse pancreas. Values represent means ± SE; n = 4. *Significantly different from control value, P < .05. Gastroenterology 2009 137, 1093-1101.e3DOI: (10.1053/j.gastro.2009.04.058) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 3 Effect of dietary protein on organelle structure and the development of cell stress. Immunohistochemistry was performed on pancreatic acinar cells as described previously.17 There was no observable difference in the distribution of a mitochondrial protein, pyruvate dehydrogenase, and an endoplasmic reticulum (ER) protein, calreticulin, between mice fed 200 g/kg casein chow (control) for 4 days and mice fed 0 g/kg casein chow (no protein) for 4 days (A). Immunoblotting showed no change in the relative amounts of calreticulin and pyruvate dehydrogenase in response to protein deficiency (B). Immunoblotting also showed no change in the relative amounts of the ER stress-induced protein BiP and the cell stress-induced protein Hsp70 in response to protein deficiency or rapamycin administration (C). CON, 200 g/kg casein chow for 4 days; NO PRO, 0 g/kg casein chow 4 days; REC, NO PRO chow for 4 days and then CON chow for 4 days while administered vehicle daily; RAPA, NO PRO chow for 4 days and then CON chow for 4 days while administered daily i.p. injections of rapamycin. GAPDH served as a loading control. Gastroenterology 2009 137, 1093-1101.e3DOI: (10.1053/j.gastro.2009.04.058) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 4 Effect of rapamycin on proteolytic pathway activation in the pancreas following protein deficiency. Immunoblotting showed no conversion of LC3 from its cytosolic from (I) to its autophagic vacuole-associated form (II) or in the relative amount of another autophagy-associated protein, beclin 1 (A). Immunoblotting showed an increase in the relative amount of the proteolysis-associated protein FOXO1 during protein deficiency, but not following rapamycin administration (B). CON, 200 g/kg casein chow for 4 days; NO PRO, 0 g/kg casein chow 4 days; REC, NO PRO chow for 4 days and then CON chow for 4 days while administered vehicle daily; RAPA, NO PRO chow for 4 days and then CON chow for 4 days while administered daily i.p. injections of rapamycin. GAPDH served as a loading control. Gastroenterology 2009 137, 1093-1101.e3DOI: (10.1053/j.gastro.2009.04.058) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 5 Effect of rapamycin on pancreatic digestive enzyme content following protein deficiency. The relative amount of amylase (A) and chymotrypsinogen (B) per mg of pancreatic tissue in mice fed 200 g/kg casein chow for 4 days (CON), 0 g/kg casein chow 4 days (NO PRO), NO PRO chow for 4 days and then CON chow for 4 days while administered vehicle daily (REC), NO PRO chow for 4 days and then CON chow for 4 days while administered daily i.p. injections of rapamycin (RAPA). Insets: Representative immunoblots. Amyl, total amylase content; Chymo, total chymotrypsinogen content; GAPDH (loading control). *Significantly different from control value, P < .05. Gastroenterology 2009 137, 1093-1101.e3DOI: (10.1053/j.gastro.2009.04.058) Copyright © 2009 AGA Institute Terms and Conditions