Activation of the mTOR signalling pathway is required for pancreatic growth in protease-inhibitor-fed mice

Abstract

Cholecystokinin (CCK)-induced pancreatic growth in mice involves parallel increases in DNA and protein. The mammalian target of rapamycin (mTOR) signalling pathway regulates mRNA translation and its activation is implicated in growth of various tissues. The aim of this study was to elucidate whether mTOR activation is required for pancreatic growth in a mouse model of increased endogenous CCK release. In mice fed chow containing the synthetic protease inhibitor camostat, protein synthetic rates and phosphorylation of two downstream targets of mTOR, eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and the ribosomal protein S6 (S6), increased in comparison with fasted controls. The camostat-induced increases in protein synthesis and 4E-BP1 and S6 phosphorylation were almost totally abolished by administration of the mTOR inhibitor rapamycin 1 h prior to camostat feeding. In contrast, the phosphorylation of ERK1/2 and JNK and the expression of the early response genes c-jun, c-fos, ATF3 and egr-1 induced by camostat feeding were not affected by rapamycin. In mice fed camostat for 7 days, the ratio of pancreatic to body weight increased by 143%, but when rapamycin was administered daily this was reduced to a 22% increase. Changes in pancreatic mass were paralleled by protein and DNA content following camostat feeding and rapamycin administration. Moreover, while BrdU incorporation, an indicator of DNA synthesis, was increased to 448% of control values after 2 days of camostat feeding, rapamycin administration completely inhibited this increase. We conclude that the mTOR signalling pathway is required for CCK-induced cell division and pancreatic growth.

Figure 1. Effect of rapamycin on the phosphorylation of Akt (A), 4E-BP1 (B) and S6 (C), as biomarkers of mTOR pathway activation

Insets, representative immunoblots. Akt(P), Akt phosphorylated on Ser⁴⁷³; Akt(T), total Akt content; S6(P), S6 phosphorylated on Ser^240/244; S6(T), total S6 content; α, α form of 4E-BP1; β, β form of 4E-BP1; γ, γ form of 4E-BP1. Values represent means ± s.e.m.; n = 6–8. *Significantly different from fasted control value, †significantly different from camostat-fed value, P < 0.05.

Figure 2. Effect of rapamycin on CRHSP-24 (A), p44/p42 MAPK (B) and p54 JNK (C) phosphorylation

The most highly phosphorylated form of CRHSP-24 (arrow) was separated from increasingly dephosphorylated forms by isoelectric focusing and visualized by Western blotting. The amount of phosphorylated and total ERK was determined from the combined value of the p44 and p42 bands, while that of phosphorylated and total JNK was determined from the value of the p54 band only. Insets, representative immunoblots. p44/p42(P), p44/p42 MAPK phosphorylated on Thr²⁰²/Tyr²⁰⁴; p44/p42(T), total p44/p42 MAPK content; p54/p56(P), p54/p56 phosphorylated on Thr¹⁸³/Tyr¹⁸⁵; p54(T), total p54 JNK content. Values represent means ± s.e.m.; n = 6–8. *Significantly different from fasted control value, †significantly different from camostat-fed value, P < 0.05.

Figure 3. Effect of rapamycin on early response gene transcription

Relative mRNA levels of c-jun (A), c-fos (B), egr-1 (C) and ATF-3 (D) in pancreas of fasted and camostat-fed mice administered carboxymethyl cellulose (CMC) or rapamycin as determined by quantitative PCR. Values represent means ± s.e.m.; n = 5–6. *Significantly different from fasted control value, †significantly different from camostat-fed value, P < 0.05.

Figure 4. Effect of rapamycin on pancreatic growth in mice fed camostat for 7 days

Pancreatic weight (A), DNA content (B) and protein content (C) of mice given daily injections of CMC or rapamycin and fed standard chow or camostat-enriched chow for 7 days. Values represent means ± s.e.m.; n = 6–7. *Significantly different from fasted control value, †significantly different from camostat-fed value, P < 0.05.

Figure 5. Effect of rapamycin on pancreatic DNA synthesis

A, immunostaining of BrdU in pancreatic acinar cell nuclei. B, time course of BrdU incorporation in the pancreas of camostat-fed mice. C, BrdU incorporation in pancreas of mice given daily injections of CMC or rapamycin and fed standard chow or camostat-enriched chow for 2 days. Values represent means ± s.e.m.; n = 9. *Significantly different from fasted control value, †significantly different from camostat-fed value, P < 0.05.

Figure 6. Effect of rapamycin on pancreatic protein synthesis

Rates of global pancreatic protein synthesis in fasted and 2 h camostat-fed mice administered CMC or rapamycin as assessed by the incorporation of ³H-Phe into pancreatic protein during a 10 min period. Values represent means ± s.e.m.; n = 5–6. *Significantly different from fasted control value, †significantly different from camostat-fed value, P < 0.05.