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Glucocorticoids increase gut glutamine synthetase expression at the translational level
ABSTRACTS The Eighth Annual Society for Surgery of the Alimentary Tract Ross Laboratories Residents and Fellows Research Conference Prouts Neck, Maine May 15, 1993 ,
,
Glucocorticoids Increase Gut Glutamine Synthetase Expression at the Translational Level
Plasmid Labeling Confirms Bacterial Translocation in Pancreatitis
P. Sarantos, W. W. Souba Department of Surgery University of Florida Gainesville, FL
G. Kazantsev, R. Rao, D. Hecht, P. Gattuso, I. Fedorak, G. Djuriein, R. Prinz Departments of Surgery, Medicine, and Pathology Loyola University Medical Center Maywood, IL
Glutamine (GLN) is the major fuel for the gut and is essential for mucosal integrity and function. No studies have focused on the ability of the gut mueosa to synthesize GLN de novo during critical illness when G L N availability from the lumen and blood may be reduced. We hypothesized that the glucocortieoid hormones, which are secreted in increased amounts during stress, induce expression of gut glutamine synthetase (GS), the enzyme catalyzing intracellular G L N biosynthesis. MATERIALS AND METHODS: Rats (200 g, n --50, 10 rats/group) received dexamethasone (DEX, 0.5 mg/kg) or saline. At various times after treatment, the specific activity of jejunal mucosal GS was assayed, and m R N A was extracted. GS transcripts were labeled with a 32-P rat GS eDNA probe, quantitated by phosphoimaging, and normalized to ~-actin. A GS antibody was used to quantitate GS protein by Western blotting. The relative amounts of GS translated were measured using a cell-free protein-synthesizing system (retieulocyte lysate assay). Data (mean -4- SEM) analysis was performed using analysis of variance. RESULTS: DEX increased GS activity by 45% at 12 hours (Figure 1). The absence of an associated increase in GS m R N A (Figure 2) indicated post-transcriptional regulation. The quantity of GS translated was increased by 25% after the administration of DEX (Figure 3). Western blot analysis confirmed this DEX-mediated increase in GS. CONCLUSION: Glucocorticoids increase gut GS levels by accelerating protein translation. This adaptive response provides G L N for the gut mucosa during stress states when exogenous GLN supplies may be ratelimiting. h
o I503
3
Figure 1
L ~
Cont4 ,2 24 72 Time~er DEX(hrs) Figure 2
Isolate coH PUC4K Ent. cloacae D Enterococcus Allisolates
~U g~ 751 I::::g:::;t
(J ~ 0 IVll,l.lilil.rll,,I.Vll, t.Vlil.
Cont4 12 24 72 ~ TimeafterDE)((hrs)
MLN Source
E. coil
[,ooo so NNNNN "
Bacterial translocation (BT) is a proposed mechanism of secondary infection in pancreatitis. Specific plasmid labeling (PL) of enteric bacteria was used to determine if the gut is a source of infection in pancreatitis. Oral antibiotics were administered to 16 dogs for 2 days to colonize their guts with a specific strain of Escherichia coli bearing plasmid pUC4K. This plasmid confers kanamycin resistance and can be identified by DNA gel electrophoresis. Colonization was maintained by the administration of oral kanamycin and confirmed throughout the study by surveillance stool cultures. Pancreatitis was induced by extraduodenal sodium taurocholate/trypsin injection in eight dogs (group I). Eight control dogs (group II) underwent laparotomy only. The pancreas, mesenteric lymph nodes (MLN), peritoneal fluid (PF), liver, and spleen were harvested 7 days later and cultured. The pancreas, MLN, small bowel, and cecum were analyzed histologically. DNA gel electrophoresis was used to identify E. coli pUC4K. Histologically; group I had severe pancreatitis and profound isehemic changes in gut mucosa. Group II had no such changes. BT to the pancreas occurred in five of eight dogs, whereas BT to MLN occurred in six of eight dogs with pancreatitis compared with zero of eight dogs in the control group (p <0.05). In addition to E. coli pUC4K, other gram-negative kanamycin-resistant bacteria were isolated. Their enteric origin was confirmed by analyzing plasmid profiles, which showed them to be identical to those of strains obtained by stool cultures.
00, t I i)il
II
4/8 2/8 1/8 2/8 6/8*
0/8 0/8 0/8 0/8 0/8
Pancreas I II 2/8 2/8 3/8 1/8 5/8**
0/8 0/8 0/8 0/8 0/8
PF I
II
1/8 0/8 2/8 0/8 3/8
0/8 0/8 0/8 0/8 0/8
*p <0.02, **p <0.05 by x2test.
CONCLUSIONS: Plasmid labeling confirms BT from the gut in pancreatitis. Intestinal ischemia in pancreatitis Figure 3 may promote BT. Plasmid labeling can be used to identify BT in other conditions. THE AMERICAN JOURNAL OF SURGERY VOLUME 165 JUNE 1993 741
.Sv 03
I
ControlDE)(
,
Glucocorticoids Increase Gut Glutamine Synthetase Expression at the Translational Level
Plasmid Labeling Confirms Bacterial Translocation in Pancreatitis
P. Sarantos, W. W. Souba Department of Surgery University of Florida Gainesville, FL
G. Kazantsev, R. Rao, D. Hecht, P. Gattuso, I. Fedorak, G. Djuriein, R. Prinz Departments of Surgery, Medicine, and Pathology Loyola University Medical Center Maywood, IL
Glutamine (GLN) is the major fuel for the gut and is essential for mucosal integrity and function. No studies have focused on the ability of the gut mueosa to synthesize GLN de novo during critical illness when G L N availability from the lumen and blood may be reduced. We hypothesized that the glucocortieoid hormones, which are secreted in increased amounts during stress, induce expression of gut glutamine synthetase (GS), the enzyme catalyzing intracellular G L N biosynthesis. MATERIALS AND METHODS: Rats (200 g, n --50, 10 rats/group) received dexamethasone (DEX, 0.5 mg/kg) or saline. At various times after treatment, the specific activity of jejunal mucosal GS was assayed, and m R N A was extracted. GS transcripts were labeled with a 32-P rat GS eDNA probe, quantitated by phosphoimaging, and normalized to ~-actin. A GS antibody was used to quantitate GS protein by Western blotting. The relative amounts of GS translated were measured using a cell-free protein-synthesizing system (retieulocyte lysate assay). Data (mean -4- SEM) analysis was performed using analysis of variance. RESULTS: DEX increased GS activity by 45% at 12 hours (Figure 1). The absence of an associated increase in GS m R N A (Figure 2) indicated post-transcriptional regulation. The quantity of GS translated was increased by 25% after the administration of DEX (Figure 3). Western blot analysis confirmed this DEX-mediated increase in GS. CONCLUSION: Glucocorticoids increase gut GS levels by accelerating protein translation. This adaptive response provides G L N for the gut mucosa during stress states when exogenous GLN supplies may be ratelimiting. h
o I503
3
Figure 1
L ~
Cont4 ,2 24 72 Time~er DEX(hrs) Figure 2
Isolate coH PUC4K Ent. cloacae D Enterococcus Allisolates
~U g~ 751 I::::g:::;t
(J ~ 0 IVll,l.lilil.rll,,I.Vll, t.Vlil.
Cont4 12 24 72 ~ TimeafterDE)((hrs)
MLN Source
E. coil
[,ooo so NNNNN "
Bacterial translocation (BT) is a proposed mechanism of secondary infection in pancreatitis. Specific plasmid labeling (PL) of enteric bacteria was used to determine if the gut is a source of infection in pancreatitis. Oral antibiotics were administered to 16 dogs for 2 days to colonize their guts with a specific strain of Escherichia coli bearing plasmid pUC4K. This plasmid confers kanamycin resistance and can be identified by DNA gel electrophoresis. Colonization was maintained by the administration of oral kanamycin and confirmed throughout the study by surveillance stool cultures. Pancreatitis was induced by extraduodenal sodium taurocholate/trypsin injection in eight dogs (group I). Eight control dogs (group II) underwent laparotomy only. The pancreas, mesenteric lymph nodes (MLN), peritoneal fluid (PF), liver, and spleen were harvested 7 days later and cultured. The pancreas, MLN, small bowel, and cecum were analyzed histologically. DNA gel electrophoresis was used to identify E. coli pUC4K. Histologically; group I had severe pancreatitis and profound isehemic changes in gut mucosa. Group II had no such changes. BT to the pancreas occurred in five of eight dogs, whereas BT to MLN occurred in six of eight dogs with pancreatitis compared with zero of eight dogs in the control group (p <0.05). In addition to E. coli pUC4K, other gram-negative kanamycin-resistant bacteria were isolated. Their enteric origin was confirmed by analyzing plasmid profiles, which showed them to be identical to those of strains obtained by stool cultures.
00, t I i)il
II
4/8 2/8 1/8 2/8 6/8*
0/8 0/8 0/8 0/8 0/8
Pancreas I II 2/8 2/8 3/8 1/8 5/8**
0/8 0/8 0/8 0/8 0/8
PF I
II
1/8 0/8 2/8 0/8 3/8
0/8 0/8 0/8 0/8 0/8
*p <0.02, **p <0.05 by x2test.
CONCLUSIONS: Plasmid labeling confirms BT from the gut in pancreatitis. Intestinal ischemia in pancreatitis Figure 3 may promote BT. Plasmid labeling can be used to identify BT in other conditions. THE AMERICAN JOURNAL OF SURGERY VOLUME 165 JUNE 1993 741
.Sv 03
I
ControlDE)(