Intravenous or luminal amino acids are insufficient to maintain pancreatic growth and digestive enzyme expression in the absence of intact dietary protein.

We previously reported that rats receiving total parenteral nutrition (TPN) undergo significant pancreatic atrophy characterized by reduced total protein and digestive enzyme expression due to a lack of intestinal stimulation by nutrients (Baumler MD, Nelson DW, Ney DM, Groblewski GE. Am J Physiol Gastrointest Liver Physiol 292: G857-G866, 2007). Essentially identical results were recently reported in mice fed protein-free diets (Crozier SJ, D'Alecy LG, Ernst SA, Ginsburg LE, Williams JA. Gastroenterology 137: 1093-1101, 2009), provoking the question of whether reductions in pancreatic protein and digestive enzyme expression could be prevented by providing amino acids orally or by intravenous (IV) infusion while maintaining intestinal stimulation with fat and carbohydrate. Controlled studies were conducted in rats with IV catheters including orally fed/saline infusion or TPN-fed control rats compared with rats fed a protein-free diet, oral amino acid, or IV amino acid feeding, all with oral carbohydrate and fat. Interestingly, neither oral nor IV amino acids were sufficient to prevent the pancreatic atrophy seen for TPN controls or protein-free diets. Oral and IV amino acids partially attenuated the 75-90% reductions in pancreatic amylase and trypsinogen expression; however, values remained 50% lower than orally fed control rats. Lipase expression was more modestly reduced by a lack of dietary protein but did respond to IV amino acids. In comparison, chymotrypsinogen expression was induced nearly twofold in TPN animals but was not altered in other experimental groups compared with oral control animals. In contrast to pancreas, protein-free diets had no detectable effects on jejunal mucosal villus height, total mass, protein, DNA, or sucrase activity. These data underscore that, in the rat, intact dietary protein is essential in maintaining pancreatic growth and digestive enzyme adaptation but has surprisingly little effect on small intestinal mucosa.

Total parenteral nutrition attenuates cerulein-induced pancreatitis in rats.

OBJECTIVES: Our aim was to determine if total parenteral nutrition (TPN)-induced pancreatic atrophy and Hsp70 expression attenuates cerulein-induced pancreatitis in rats. METHODS: Rats were randomized to a 7-day course of saline infusion plus a semipurified diet or TPN, with or without an intravenous cerulein injection or vehicle on day 7, and killed 1 or 6 hours after the injection. Based on a pilot study, 1 hour was the primary time point. Pancreatic atrophy was determined by mass, protein, and DNA contents. Pancreatic heat shock protein 70 (Hsp70) expression was measured by Western analysis. Histological examination of the pancreas assessed for edema, inflammation, vacuolization, and apoptosis. Serum amylase activity was measured using the Phadebas assay. Pancreatic trypsinogen activation was measured using a fluorometric substrate assay. RESULTS: The saline-infused rats fed orally gained significantly more weight than TPN rats. The TPN decreased the pancreatic mass and protein content and the protein-DNA ratio and increased the pancreatic DNA content compared with the saline. The TPN increased the pancreatic Hsp70 expression by 91% compared with the saline. The TPN reduced the cerulein-induced pancreatic histological edema, the vacuolization, and the inflammation compared with the saline. The increase in the serum amylase level after cerulein injection was significantly attenuated, and trypsinogen activation was reduced in TPN animals compared with the saline group. CONCLUSIONS: Lack of luminal nutrients with a 7-day course of TPN provides moderate protection against cerulein-induced pancreatitis in rats.

Functional role of J domain of cysteine string protein in Ca2+-dependent secretion from acinar cells.

The heat shock protein 70 family members Hsc70 and Hsp70 are known to play a protective role against the onset of experimental pancreatitis, yet their molecular function in acini is unclear. Cysteine string protein (CSP-alpha) is a zymogen granule (ZG) membrane protein characterized by an NH(2)-terminal "J domain" and a central palmitoylated string of cysteine residues. The J domain functions as a cochaperone by modulating the activity of Hsc70/Hsp70 family members. A role for CSP-alpha in regulating digestive enzyme exocytosis from pancreas was investigated by introducing CSP-alpha truncations into isolated acini following their permeabilization with Perfringolysin O. Incubation of acini with CSP-alpha(1-82), containing the J domain, significantly augmented Ca(2+)-stimulated amylase secretion. Effects of CSP-alpha(1-82) were concentration dependent, with a maximum 80% increase occurring at 200 microg/ml of protein. Although CSP-alpha(1-82) had no effects on basal secretion measured in the presence of < or =10 nM free Ca(2+), it did significantly augment GTP-gammaS-induced secretion under basal Ca(2+) conditions by approximately 25%. Mutation of the J domain to abolish its cochaperone activity failed to augment Ca(2+)-stimulated secretion, implicating the CSP-alpha/Hsc70 cochaperone system as a regulatory component of the secretory pathway. CSP-alpha physically associates with vesicle-associated membrane protein 8 (VAMP 8) on ZGs, and the CSP-alpha-VAMP 8 interaction was dependent on amino acids 83-112 of CSP-alpha. Immunofluorescence analysis of acinar lobules or purified ZGs confirmed the CSP-alpha colocalization with VAMP 8. These data establish a role for CSP-alpha in regulating digestive enzyme secretion and suggest that CSP-alpha and Hsc70 modulate specific soluble N-ethylmaleimide-sensitive attachment receptor interactions necessary for exocytosis.

Loss of exocrine pancreatic stimulation during parenteral feeding suppresses digestive enzyme expression and induces Hsp70 expression.

Luminal nutrients are essential for the growth and maintenance of digestive tissue including the pancreas and small intestinal mucosa. Long-term loss of luminal nutrients such as during animal hibernation has been shown to result in mucosal atrophy and a corresponding stress response characterized by the induction of heat shock protein (Hsp)70 expression. This study was conducted to determine if the loss of luminal nutrients during total parenteral nutrition (TPN) would result in atrophy of the exocrine pancreas and small intestinal mucosa as well as an induction of Hsp70 expression in rats. In experiment 1, the treatment groups included an orally fed control, a saline-infused surgical control, or TPN treatment for 7 days. In experiment 2, the treatment groups included an orally fed control and TPN alone or coinfused with varying doses of glucagon-like peptide (GLP)-2, a mucosal proliferation agent, for 7 days. In experiment 1, TPN resulted in a 40% reduction in pancreatic mass that was associated with a dramatic reduction in digestive enzyme expression, enhanced apoptosis, and a 200% increase in Hsp70 expression. Conversely, heat shock cognate 70, Hsp27, and Hsp60 expression was not changed in the pancreas. In experiment 2, TPN resulted in a 30% reduction in jejunal mucosa mass and a similar induction of Hsp70 expression. The inclusion of GLP-2 during TPN attenuated jejunal mucosal atrophy and inhibited Hsp70 expression, suggesting that Hsp70 induction is sensitive to cell growth. These data indicate that pancreatic and intestinal mucosal atrophy caused by a loss of luminal nutrient stimulation is accompanied by a compensatory response involving Hsp70.