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Reactive cellularity and bacterial invasion in the stomach of transgenic mice overexpressing TGFα
April 1 9 9 5
ADAPTATION OF GASTRIC EMPTYING TO HIGH AND LOW PROTEIN DIETS IN RATS: IMPLICATION OF CCK: NBentouimou a, G Shia, V Leraya, ~; Brulev des Varannesa, C Cherbutb, C Roz~c, JP Galmiche a. aFonctions Digestives et Nutrition, and blNRA, 44035 Nantes; ClNSERM U410, 75870 Paris - FRANCE. Gastric emptying is modulated by chronic nutrient intake: gastric emptying of peptone meals is faster in rats adapted to a high protein diet than in rats adapted to a low protein diet (Gastroenterology 1994;106:A566). The aim of the present study was to investigate the possible role of CCK in gastric emptying changes induced by chronic diets in rats. M~thods. Seventy-two male Wistar rats (initial weight 130-200g) were fed ad libitum either with a high protein (50% casein and 5.7% crude protein) or with a low protein diet (3.3% casein and 5.7% crude protein) during 3 weeks. At the end of the adaptation period, the effect of increasing doses of intraperitoneally administered CCK-8 (0.4, 1.3, 4.0 I~glkg) on the gastric emptying of a 3 ml 1.5% methylcellulose meal, labeled with phenol red, was determined, Plasma CCK level was determined by bioassay (J Clin Invest 1985;75:1144) 8 min after administration of a 3 ml 40% peptone meal. Duodenal CCK-mRNA levels were analysed by Northern-blot using a specific probe (after 18 h fasting and 6 h after feeding). Normalization of CCK-mRNA values was performed with respect to phosphoglycerate kinase mRNA. Results. CCK-8 dose-dependently delayed the gastric emptying of the liquid meal; no difference was observed between diets (for instance 35 and 37 % respectively emptied after high and low protein diets with 1.3 ~g/kg CCK). After the peptone meal, plasma CCK increased more in rats adapted to the low protein diet (4.4 +_ ,0.9 pmol/I) than in rats adapted to the high protein diet (1.0 + 0.2 pmole/I, P<0.001). Duodenal CCK-mRNA was not significantly different in rats adapted to high and low protein .diets. Conclusions. In animals chronically fed high and low protein diets, CCK-induced delay in gastric emptying is the same. Changes in basal gastric emptying observed after different diets might be related to differences in CCK release. These differences may take place at a post-transcriptional level.
• REACTIVE CELLULARITY AND BACTERIAL INVASION IN THE STOMACH OF TRANSGENIC MICE OVEREXPRESSlNG TGF(x Dale. E. Beckman and Glenn Merlino. Department Of Cellular Biology and AnatOmy, Medical College of Georgia, Augusta, GA, and Laboratory of Molecular Biology, National Cancer Institute, NIH. Bethesda, MD Stomachs of transgenic mica overexpresslng transforming growth factor alpha (TGF~) display changes resembling M6n6trier's disease. The mucosa thickens due primarily to the accumulation of pit (surface mucous) calls. Simultaneously, terminal differentiation of parietal and zymogenic calls is inhibited. Acid secretion and pepsinogen are markedly reduced. Light and electron microscopic comparison of stomachs from transgenics with those from non-transgenic controls reveals significant changes in the interstitium separating gastric glands. Gastric glands in controls directty appose each other. Greatly increased cellularity in the lamina propria in transgenlcs causes gastric glands to separate. Fibrosis is limited. In addition to fibroblasts, monocytes, macrophages, lymphocytes, plasma calls, mast cells, and neutrophils abound in the interstitium. Bacteria are prominent Although they may be numerous in the mucus along the luminal surface, they are not limited to the surface; they may penetrate into enlarged lumina of gastric glands and into the interstitium It is concluded that bacterial overgrowth may occur as a result of lowered acid and enzyme secretion in transgenics. Cells in the interstitium proliferate and accumulate directly or indirectly in response to increased levels of TGF(x and to overgrowth of bacteria. Interaction between the interstitium and the glandular epithelium may be involved in regulating differentiation of the gastric epithelium and therefore may olay a role in the altered morphology and function that is observed in these transgenic mice.
Growth, Development, and Nutrition
A717
IL-11~ ENHANCES PROLIFERATION OF CACO-2 CEI-LS THROUGH ACTIVATION OF THE TGFcz-EGFRECEPTORAUTOCRINEPATHWAY Warren P, Bishop*, James Lin*, and Gary W. Varilek** *Dept. of Pediatrics, University of Iowa, Iowa City, IA and **Dept. of Internal Medicine, University of Kentucky, Lexington, KY Intedeukin-1 beta (IL-I~) is a known mitogen for a variety of epithelial cells, including keratinocytes and thymic epithelium. Previous studies by our laboratories have shown that IL-1I~ enhances proliferationof intestinal epithelial cell lines. We and others have also shown that Caco-2 cells proliferate in response to an autocdne activation of the epidermal growth factor (EGF) receptor by transforming growth factoralpha ('rGF~). The aim of this study was to determinewhether the observed proliferative response to IL-lt3 is mediated by up-regulation of this autocdne pathway. Methods: Caco-2 cells were grown on collagen-coatedTranswells (Costar). For affinity-labeling of the EGF receptor, cells were incubated with 12SI-EGFat 4°C for 4 hr, then crosslinkedwith bis(sulfosuccinimidyl) suberate, subjected to SDS-PAGE, and autoradiographed. For [~H]thymidine uptake, cells were incubated overnight with IL-I~ EGF EGF receptor blocking antibody (mAb 528), or TGF(~ antisense oligonucleotide~followed by a 3 hour exposure to [aH]thymidineand scintillation counting. For determination of TGFc~and EGF receptor mRNA levels, a quantitative reverse-transcdptasePCR was developed. Results: Incubation of Caco-2 cells with 1[.-113enhanced proliferation approximately 2-fold. This effect was not seen in the presence of the EGF receptor blocking antibody. The proliferative effect of IL-113was also blocked by preincubating the cells with a TGFc~antisense oligonucleotide, but not by random oligonucleotide. IL-113was next shown to increase the 'expressionof TGFczmRNA by more than 10-fold. This effect was apparent within two hours of exposure to the cytokine, and persisted for at least 16 hours. In addition, the effect of IL-113on expression of cell-surface EGF receptor and its mRNA was examined; both were significantly increased by IL-I~. Conclusions: I1_-113enhances proliferation of Caco-2 cells through activation of the TGFaJEGF receptor autocrine pathway. This finding may represent a mechanism by which inflammation enhances proliferation of the intestinal mucosa in vivo.
• BENEFICIAL EFFECT OF DIETARY RESTRICTION ON BILE FORMATION IN AGING RATS: EVIDENCE F O R A ROLE OF GLUTATHIONE. G. Bouchard, S. Chevalier, A. Perea, LM. Yousef, B. Tuchwebcr. I)epts of Pharmacology and of Nutrition, Universil~ de Montr6al, M o n t r ~ , Canada. Dietary restriction (DR) is known to prevent the age-related decline in organ function. We have shown that DR exerts a beneficial effect on age-related decrease in bile formation but the mechanism is still unclear. The objective of this study was to identify the changes in the main biliary osmotic factors, e.g. bile salts and reduced glutathione (GSH) in aging rats submitted to DR. Total bile salts and GSH were determined in bile from young (3 too), mature (12 too) and old (23 too) female Sprague-Dawley rats fed ad-libitom (AL) or submitted to a 40%DR enriched (R+) or not ('R) in vitamins and minerals. Bile was collected under pentobarbital anesthesia during 2h, in 15 rain aliquots. Aging resulted in a significant decxe~se in bile flow in AL-rats, but not in DR-rats. Both R and R + groups exhibited significantly higher bile flow compared to age-matched AL-grenps. Increased bile secretion was associated, in both R and R + groups, with marked enhancement (2 fold) in GSH and in increased (not reaching significance) biliary bile salts secretion rate. Estimation of the total osmotic effect of GSH and bile salts showed that, at all ages, DR produced a marked increase in apparent GSH-dependent flow. DR also improved bile salt--dependent flow but its effect contributed less than GSH to total bile flow. Preliminary results show that fiver GSH concentration was also improved by DR at all ages examined. In conclusion, DR, either enriched or not in micronutrients, results in greater generation of bile flow and prevents the age-related deciine in bile formation mainly by stimulation of GSH-bifiary secretion. This research was funded by MRC and NSERC of Canada. GB and SC are recipients of a Canadian Liver Foundation and of a FCAR studentship respectively.
ADAPTATION OF GASTRIC EMPTYING TO HIGH AND LOW PROTEIN DIETS IN RATS: IMPLICATION OF CCK: NBentouimou a, G Shia, V Leraya, ~; Brulev des Varannesa, C Cherbutb, C Roz~c, JP Galmiche a. aFonctions Digestives et Nutrition, and blNRA, 44035 Nantes; ClNSERM U410, 75870 Paris - FRANCE. Gastric emptying is modulated by chronic nutrient intake: gastric emptying of peptone meals is faster in rats adapted to a high protein diet than in rats adapted to a low protein diet (Gastroenterology 1994;106:A566). The aim of the present study was to investigate the possible role of CCK in gastric emptying changes induced by chronic diets in rats. M~thods. Seventy-two male Wistar rats (initial weight 130-200g) were fed ad libitum either with a high protein (50% casein and 5.7% crude protein) or with a low protein diet (3.3% casein and 5.7% crude protein) during 3 weeks. At the end of the adaptation period, the effect of increasing doses of intraperitoneally administered CCK-8 (0.4, 1.3, 4.0 I~glkg) on the gastric emptying of a 3 ml 1.5% methylcellulose meal, labeled with phenol red, was determined, Plasma CCK level was determined by bioassay (J Clin Invest 1985;75:1144) 8 min after administration of a 3 ml 40% peptone meal. Duodenal CCK-mRNA levels were analysed by Northern-blot using a specific probe (after 18 h fasting and 6 h after feeding). Normalization of CCK-mRNA values was performed with respect to phosphoglycerate kinase mRNA. Results. CCK-8 dose-dependently delayed the gastric emptying of the liquid meal; no difference was observed between diets (for instance 35 and 37 % respectively emptied after high and low protein diets with 1.3 ~g/kg CCK). After the peptone meal, plasma CCK increased more in rats adapted to the low protein diet (4.4 +_ ,0.9 pmol/I) than in rats adapted to the high protein diet (1.0 + 0.2 pmole/I, P<0.001). Duodenal CCK-mRNA was not significantly different in rats adapted to high and low protein .diets. Conclusions. In animals chronically fed high and low protein diets, CCK-induced delay in gastric emptying is the same. Changes in basal gastric emptying observed after different diets might be related to differences in CCK release. These differences may take place at a post-transcriptional level.
• REACTIVE CELLULARITY AND BACTERIAL INVASION IN THE STOMACH OF TRANSGENIC MICE OVEREXPRESSlNG TGF(x Dale. E. Beckman and Glenn Merlino. Department Of Cellular Biology and AnatOmy, Medical College of Georgia, Augusta, GA, and Laboratory of Molecular Biology, National Cancer Institute, NIH. Bethesda, MD Stomachs of transgenic mica overexpresslng transforming growth factor alpha (TGF~) display changes resembling M6n6trier's disease. The mucosa thickens due primarily to the accumulation of pit (surface mucous) calls. Simultaneously, terminal differentiation of parietal and zymogenic calls is inhibited. Acid secretion and pepsinogen are markedly reduced. Light and electron microscopic comparison of stomachs from transgenics with those from non-transgenic controls reveals significant changes in the interstitium separating gastric glands. Gastric glands in controls directty appose each other. Greatly increased cellularity in the lamina propria in transgenlcs causes gastric glands to separate. Fibrosis is limited. In addition to fibroblasts, monocytes, macrophages, lymphocytes, plasma calls, mast cells, and neutrophils abound in the interstitium. Bacteria are prominent Although they may be numerous in the mucus along the luminal surface, they are not limited to the surface; they may penetrate into enlarged lumina of gastric glands and into the interstitium It is concluded that bacterial overgrowth may occur as a result of lowered acid and enzyme secretion in transgenics. Cells in the interstitium proliferate and accumulate directly or indirectly in response to increased levels of TGF(x and to overgrowth of bacteria. Interaction between the interstitium and the glandular epithelium may be involved in regulating differentiation of the gastric epithelium and therefore may olay a role in the altered morphology and function that is observed in these transgenic mice.
Growth, Development, and Nutrition
A717
IL-11~ ENHANCES PROLIFERATION OF CACO-2 CEI-LS THROUGH ACTIVATION OF THE TGFcz-EGFRECEPTORAUTOCRINEPATHWAY Warren P, Bishop*, James Lin*, and Gary W. Varilek** *Dept. of Pediatrics, University of Iowa, Iowa City, IA and **Dept. of Internal Medicine, University of Kentucky, Lexington, KY Intedeukin-1 beta (IL-I~) is a known mitogen for a variety of epithelial cells, including keratinocytes and thymic epithelium. Previous studies by our laboratories have shown that IL-1I~ enhances proliferationof intestinal epithelial cell lines. We and others have also shown that Caco-2 cells proliferate in response to an autocdne activation of the epidermal growth factor (EGF) receptor by transforming growth factoralpha ('rGF~). The aim of this study was to determinewhether the observed proliferative response to IL-lt3 is mediated by up-regulation of this autocdne pathway. Methods: Caco-2 cells were grown on collagen-coatedTranswells (Costar). For affinity-labeling of the EGF receptor, cells were incubated with 12SI-EGFat 4°C for 4 hr, then crosslinkedwith bis(sulfosuccinimidyl) suberate, subjected to SDS-PAGE, and autoradiographed. For [~H]thymidine uptake, cells were incubated overnight with IL-I~ EGF EGF receptor blocking antibody (mAb 528), or TGF(~ antisense oligonucleotide~followed by a 3 hour exposure to [aH]thymidineand scintillation counting. For determination of TGFc~and EGF receptor mRNA levels, a quantitative reverse-transcdptasePCR was developed. Results: Incubation of Caco-2 cells with 1[.-113enhanced proliferation approximately 2-fold. This effect was not seen in the presence of the EGF receptor blocking antibody. The proliferative effect of IL-113was also blocked by preincubating the cells with a TGFc~antisense oligonucleotide, but not by random oligonucleotide. IL-113was next shown to increase the 'expressionof TGFczmRNA by more than 10-fold. This effect was apparent within two hours of exposure to the cytokine, and persisted for at least 16 hours. In addition, the effect of IL-113on expression of cell-surface EGF receptor and its mRNA was examined; both were significantly increased by IL-I~. Conclusions: I1_-113enhances proliferation of Caco-2 cells through activation of the TGFaJEGF receptor autocrine pathway. This finding may represent a mechanism by which inflammation enhances proliferation of the intestinal mucosa in vivo.
• BENEFICIAL EFFECT OF DIETARY RESTRICTION ON BILE FORMATION IN AGING RATS: EVIDENCE F O R A ROLE OF GLUTATHIONE. G. Bouchard, S. Chevalier, A. Perea, LM. Yousef, B. Tuchwebcr. I)epts of Pharmacology and of Nutrition, Universil~ de Montr6al, M o n t r ~ , Canada. Dietary restriction (DR) is known to prevent the age-related decline in organ function. We have shown that DR exerts a beneficial effect on age-related decrease in bile formation but the mechanism is still unclear. The objective of this study was to identify the changes in the main biliary osmotic factors, e.g. bile salts and reduced glutathione (GSH) in aging rats submitted to DR. Total bile salts and GSH were determined in bile from young (3 too), mature (12 too) and old (23 too) female Sprague-Dawley rats fed ad-libitom (AL) or submitted to a 40%DR enriched (R+) or not ('R) in vitamins and minerals. Bile was collected under pentobarbital anesthesia during 2h, in 15 rain aliquots. Aging resulted in a significant decxe~se in bile flow in AL-rats, but not in DR-rats. Both R and R + groups exhibited significantly higher bile flow compared to age-matched AL-grenps. Increased bile secretion was associated, in both R and R + groups, with marked enhancement (2 fold) in GSH and in increased (not reaching significance) biliary bile salts secretion rate. Estimation of the total osmotic effect of GSH and bile salts showed that, at all ages, DR produced a marked increase in apparent GSH-dependent flow. DR also improved bile salt--dependent flow but its effect contributed less than GSH to total bile flow. Preliminary results show that fiver GSH concentration was also improved by DR at all ages examined. In conclusion, DR, either enriched or not in micronutrients, results in greater generation of bile flow and prevents the age-related deciine in bile formation mainly by stimulation of GSH-bifiary secretion. This research was funded by MRC and NSERC of Canada. GB and SC are recipients of a Canadian Liver Foundation and of a FCAR studentship respectively.