Dietary Protein and Amino Acid Deficiency Inhibit Pancreatic Digestive Enzyme mRNA Translation by Multiple Mechanisms

Abstract

Background & Aims Chronic amino acid (AA) deficiency, as in kwashiorkor, reduces the size of the pancreas through an effect on mammalian target of rapamycin complex 1 (mTORC1). Because of the physiological importance of AAs and their role as a substrate, a stimulant of mTORC1, and protein synthesis, we studied the effect of acute protein and AA deficiency on the response to feeding. Methods ICR/CD-1 mice were fasted overnight and refed for 2 hours with 4 different isocaloric diets: control (20% Prot); Protein-free (0% Prot); control (AA-based diet), and a leucine-free (No Leu). Protein synthesis, polysomal profiling, and the activation of several protein translation factors were analyzed in pancreas samples. Results All diets stimulated the Protein Kinase-B (Akt)/mTORC1 pathway, increasing the phosphorylation of the kinase Akt, the ribosomal protein S6 (S6) and the formation of the eukaryotic initiation factor 4F (eIF4F) complex. Total protein synthesis and polysome formation were inhibited in the 0% Prot and No Leu groups to a similar extent, compared with the 20% Prot group. The 0% Prot diet partially reduced the Akt/mTORC1 pathway and the activity of the guanine nucleotide exchange factor eIF2B, without affecting eIF2α phosphorylation. The No Leu diet increased the phosphorylation of eIF2α and general control nonderepressible 2, and also inhibited eIF2B activity, without affecting mTORC1. Essential and nonessential AA levels in plasma and pancreas indicated a complex regulation of their cellular transport mechanisms and their specific effect on the synthesis of digestive enzymes. Conclusions These studies show that dietary AAs are important regulators of postprandial digestive enzyme synthesis, and their deficiency could induce pancreatic insufficiency and malnutrition.

Pancreatic Digestive Enzyme Synthesis and its Translational Control

“…Other studies also have shown the role of leucine in the exocrine pancreas of rats (78), pigs (128), and ruminants (23). A short-term study in mice, demonstrated that diets lacking protein or amino acids inhibited pancreatic protein synthesis (measured by the flooding dose technique) (Figure 7) and the amount of ribosomes engaged in translation on a given moment, by polysomal fractionation (Figure 8) (158). Polysomal profiling analysis performed on pancreatic homogenates showed a significant increase in the polysomal fraction (i.e.…”

“…These results confirm that total pancreatic protein synthesis was inhibited in the diets lacking protein or leucine, with a strong reduction of the mRNAs attached to the ribosomes for their translation into protein. This inhibition was accomplished through different intracellular mechanisms: the protein free diet inhibited pancreatic protein synthesis through a partial inhibition of the mTORC1 pathway, and of the eIF2B activity; the leucine free diet inhibition was caused by the inhibition of the eIF2B activity and activation of the stress signals leading to the GCN2 (General Control Nonderepressible 2) kinase activation and eIF2α phosphorylation, induced by the amino acid imbalance in the system and in the pancreas (158). This study shows that dietary AAs are important regulators of postprandial digestive enzyme synthesis, and their deficiency could induce pancreatic insufficiency and malnutrition.…”

“…( In general, the gastrointestinal tract, including the exocrine pancreas, atrophies in the absence of food and protein synthesis that occurs in response to food intake is required to maintain normal function. Individual dietary components, like protein and amino acids (158,162), regulate pancreatic protein synthesis in mice and rats (77,78). In humans, feeding increases both the rate of secretion and synthesis of digestive enzymes, although the rate of zymogen turnover remains fairly constant during feeding and fasting (130).…”

Ribosomal Protein S6

“…All hormones and neurotransmitters that stimulate protein synthesis enhance rpS6 phosphorylation. In the converse, removing protein from the diet acutely partially reduces rpS6 phosphorylation and this affect increases in prolonged protein deprivation (4,28). These studies were largely directed at showing the presence of the Akt -mTOR -S6K pathway in acini and its importance for regulating protein synthesis and cell growth (33); phosphorylation of rpS6 was used as a readout of pathway activation.…”

“…A number of antibodies have been raised against phosphorylated peptide. In our hands the best antibody for Western Blotting is Cell Signaling #2215, a rabbit polyclonal prepared against p-Ser 24/244 which can be used at 1:5000 or higher dilution (5,28,30,31). This gives a bigger response than the Cell Signaling Ab to pSer 235/236 which has a higher basal level of phosphorylation.…”