Stephen J. Crozier

Immediate Response of Mammalian Target of Rapamycin (mTOR)‐Mediated Signalling Following Acute Resistance Exercise in Rat Skeletal Muscle

The purpose of the present investigation was to determine whether mammalian target of rapamycin (mTOR)‐mediated signalling and some key regulatory proteins of translation initiation are altered in skeletal muscle during the immediate phase of recovery following acute resistance exercise. Rats were operantly conditioned to reach an illuminated bar located high on a Plexiglass cage, such that the animals completed concentric and eccentric contractions involving the hindlimb musculature. Gastrocnemius muscle was extracted immediately after acute exercise and 5, 10, 15, 30 and 60 min of recovery. Phosphorylation of protein kinase B (PKB) on Ser‐473 peaked at 10 min of recovery (282 % of control, P < 0.05) with no significant changes noted for mTOR phosphorylation on Ser‐2448. Eukaryotic initiation factor (eIF) 4E‐binding protein‐1 (4E‐BP1) and S6 kinase‐1 (S6K1), both downstream effectors of mTOR, were altered during recovery as well. 4E‐BP1 phosphorylation was significantly elevated at 10 min (292 %, P < 0.01) of recovery. S6K1 phosphorylation on Thr‐389 demonstrated a trend for peak activation at 10 min following exercise (336 %, P= 0.06) with ribosomal protein S6 phosphorylation being maximally activated at 15 min of recovery (647 %, P < 0.05). Components of the eIF4F complex were enhanced during recovery as eIF4E association with eIF4G peaked at 10 min (292 %, P < 0.05). Events regulating the binding of initiator methionyl‐tRNA to the 40S ribosomal subunit were assessed through eIF2B activity and eIF2α phosphorylation on Ser‐51. No differences were noted with either eIF2B or eIF2α. Collectively, these results provide strong evidence that mTOR‐mediating signalling is transiently upregulated during the immediate period following resistance exercise and this response may constitute the most proximal growth response of the cell.

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

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.

Molecular Mechanisms of Pancreatic Dysfunction Induced by Protein Malnutrition

BACKGROUND & AIMS Dietary protein deficiency results in diminished capacity of the pancreas to secrete enzymes needed for macronutrient digestion. Previous work has suggested that modulation of the mammalian target of rapamycin (mTOR) pathway by the hormone cholecystokinin (CCK) plays an important role in normal digestive enzyme synthesis after feeding. The purpose of this study was to elucidate the role of mTOR in protein deficiency-induced pancreatic dysfunction. METHODS Wild-type and CCK-null mice were fed protein-deficient chow for 4 days and then allowed to recover on control chow in the presence or absence of the mTOR inhibitor rapamycin. RESULTS The size and secretory capacity of the pancreas rapidly decreased after feeding protein-deficient chow. Refeeding protein-replete chow reversed these changes in both wild-type and CCK-null mice. Changes in the size of the pancreas were paralleled by changes in the content and secretion of digestive enzymes, as well as the phosphorylation of downstream targets of mTOR. Administration of the mTOR inhibitor rapamycin decreased regrowth of the pancreas but did not affect digestive enzyme content or secretory capacity. CONCLUSIONS These studies demonstrate that dietary protein modulates pancreatic growth, but not digestive enzyme synthesis, via CCK-independent activation of the mTOR pathway.

CCK-induced pancreatic growth is not limited by mitogenic capacity in mice

In mice fed trypsin inhibitor (camostat) to elevate endogenous CCK, pancreatic growth plateaus by 7 days. It is unknown whether this represents the maximum growth capacity of the pancreas. To test the ability of CCK to drive further growth, mice were fed chow containing camostat (0.1%) for 1 wk, then fed standard chow for 1 wk, and finally returned to the camostat diet for a week. Pancreatic mass increased to 245% of initial value (iv) following 1 wk of camostat feeding, decreased to 147% iv following a 1 wk return to normal chow, and increased to 257% iv with subsequent camostat feeding. Camostat feeding was associated with significant increases in circulating CCK and changes in pancreatic mass were paralleled by changes in protein and DNA content. Moreover, regression of the pancreas following camostat feeding was associated with changes in the expression of the autophagosome marker LC3. Pancreatic protein synthetic rates were 130% of control after 2 days on camostat but were equivalent to control after 7 days. Changes in the phosphorylation of 4E-BP1 and S6, downstream effectors of mammalian target of rapamycin (mTOR), paralleled changes in protein synthetic rates. Cellular content of Akt, an upstream activating kinase of mTOR, decreased after 7 days of camostat feeding whereas expression of the E3 ubiquitin-ligases and the cell cycle inhibitor p21 increased after 2 days. These results indicate that CCK-stimulated growth of the pancreas is not limited by acinar cell mitogenic capacity but is due, at least in part, to inhibition of promitogenic Akt signaling.

CCK-independent mTORC1 activation during dietary protein-induced exocrine pancreas growth

Dietary protein can stimulate pancreatic growth in the absence of CCK release, but there is little data on the regulation of CCK-independent growth. To identify mechanisms whereby protein stimulates pancreatic growth in the absence of CCK release, C57BL/6 control and CCK-null male mice were fed normal-protein (14% casein) or high-protein (75% casein) chow for 7 days. The weight of the pancreas increased by 32% in C57BL/6 mice and 26% in CCK-null mice fed high-protein chow. Changes in pancreatic weight in control mice were due to both cell hypertrophy and hyperplasia since there was an increase in protein-to-DNA ratio, total DNA content, and DNA synthesis. In CCK-null mice pancreatic growth was almost entirely due to hypertrophy with both protein-to-DNA ratio and cell size increasing without significant increases in DNA content or DNA synthesis. ERK, calcineurin, and mammalian target of rapamycin complex 1 (mTORC1) are activated in models of CCK-induced growth, but there were no differences in ERK or calcineurin activation between fasted and fed CCK-null mice. In contrast, mTORC1 activation was increased after feeding and the duration of activation was prolonged in mice fed high-protein chow compared with normal-protein chow. Changes in pancreatic weight and RNA content were completely inhibited, and changes in protein content were partially abated, when the mTORC1 inhibitor rapamycin was administered during high-protein chow feeding. Prolonged mTORC1 activation is thus required for dietary protein-induced pancreatic growth in the absence of CCK.