April 1995 Growth, Development, and Nutrition A725

• BIOSYNTHESIS OF RAT SMALL INTESTINAL BRUSH BORDER MEMBRANE ANGIOTENSIN-CONVERTING ENZYME DURING INDUCTION BY A HIGH PROLINE DIET. R.H. Erickson. B.B. Siddiki, M. Lindstrom, B.C. Yoon, Y.S. Kim. GI Research Lab, VA Med. Ctr. & Dept. of Med., Univ. of CA, San Francisco, CA 94121.

The brush border membrane (BBM) of small intestinal epithelial ceils contains several enzymes such as angiotensin-converting enzyme (ACE) which participate in the digestion of prolyl peptides. Currently very li t t le is known regarding the role of diet and the molecular mechanisms involved in regulating intestinal levels of BBM enzymes. Recently we have observed in rats that after 7 days, intestinal levels of ACE are increased 3-9 fold by high protein diets, particularly those containing high amounts of praline (gelatin)'. These changes occur in the proximal and middle intestinal segments of the small intestine with little change observed distally. Thus ACE is potentially an important model for examining the role of dietary factors in regulating BBM peptidase levels along the longitudinal axis of the small intestine. In this study, early events in the time course of ACE induction were examined by momtonng changes in the biosynthetic rate of the enzyme. Rats were initially maintained for 7 days on a low protein (4% casein) diet. They were then switched to a high protein (50% gelatin) diet and sacrificed at various times (0, 12h, & 1,2,3 days). Explants were prepared from proximal and distal intestinal segments, maintained in organ culture and metabolically labeled for 30 rain with 35S-methionine. ACE was immunoprecipitated from labeled exptants, subjected to SDS-PAGE and radioautography. Densitometry was used to quantify the amount of newly synthesized ACE. The rate of ACE biosynthesis was increased approximately 3 fold at day 1 and maintained at this level in the 2 & 3 day animals. Increased levels of ACE enzyme activity (proximal BBM) were not apparent until 2- 3 days after the diet switch. Steady state levels of ACE mRNA were increased 1.5 fold by day 2. No change in the ACE synthetic rate was observed in distal intestine during this time. Shorter periods of induction were examined by in vivo intestinal perfusion. When solutions containing either gelatin (20 mg/ml), pancreatin treated gelatin (20 mg/ml) or the ACE peptide substrate Bz-Gly-Ala-Pro (10 mM) were perfused through proximal intestinal segments for periods up to 3h, no change in the synthetic rate was observed. The results of this study indicate that: (1) the rate of ACE biosynthesis increases rapidly in response to diet and is limited to specific regions of the small intestine, (2) changes in ACE mRNA do not correspond to increased rates of biosynthesis, (3) post- transcriptional regulatory mechanisms may play a role in the early stages of ACE induction.

ASSOCIATION OF INSULIN RESISTANCE WITH INTRAVENOUS LIPID ADMINISTRATION. A: Farag, S. Bridges, E.C. Opara, O.E. Akwari. Department of Surgery, Duke University Medical Center, Durham, NC

We have previously shown that intravenous administration of 10% intralipid at the rate of 1 ml/hr for 48 hours, in rats fed ad libitum, caused the development of impaired glucose regulation which was abolished by the provision of L- glutamine (GLN) during the infusion. AiM: In the present study; we examined the effect of reducing the dose of the infused lipid on glucose homeostasis. METHOD: Two groups of male Sprague Dawtey rats had their femoral veins cannulated and baseline blood samples were taken, following anesthesia with pentobarbital. All animals were housed individually in a cage and two hours after waking, lipid infusion Was started with free access to food and water. In group A (mean + sem, body weight, 254 + 12 g, n =7) 10% jntralipid was infused at the rate Of 0.5 cc/hr, approximating to 11 kcal/day and each animal in group B, (mean body weight, 239 + 9, n =8) received half of that dose (0.25 cc/hr). Food intake was monitored daily and after 48 hours, all animals were sacrificed and blood samples were taken. RESULTS: Food intake was not significantly different between the two groups (A = 43 -I- 5 vs B = 49 + 3 kcal/day). Compared to baseline, mean plasma glucose levels were increased in both groups, after 48 hours; group A (106 __. 2 vs 143 + 4 mg/dl, p< .005) and group B (108 + 5 vs 140 + 5 mg/dl, p< .001). Although mean plasma glucose levels after lipid infusion were the same for the two groups, it was significantly (p<0.001) different from that previously obtained at intralipid infusion rate of 1 ml/hr( 170 + 5 mg/dl, n=7) in spite of similar baseline levels. In contrast, hyperinsulinemia occurred to the same extent and mean plasma insulin levels obtained before and after infusion were: group A = 203 + 41 vs 1267 + 184, p< .005 and group B = 271 + 52 vs 1303 + 357, p< .005. These changes were associated with depletion of blood glutathione levels, after 48 hours of lipid infusion at each dose, suggestive of oxidative stress. CONCLUSION: Intravenous lipid infusion a t clinically-relevant doses caused impaired glucose regulation associated with oxidative stress. These data support a role for GLN supplementation to prevent deleterious changes in lipid-based TPN.

~Z:FECT OF I/ICREASIII6 DOSES OF L-ABGINIIIE (LA) ON I / ~ D/U~QE ~ NITRIC OXIDE (NO) SYIClIIESIS Ill EgPERII~AL COLITIS. - " - ~ , X Bertr~n, J Marl4, R Bartoli, E Castell~, HA Gassull. Digestive Research Unit, Hospital Germans Trias i Pujol. Bedalona, Spain.

Backg~: LA is the substrate for NO synthesis. Enhanced NO synthesis may promote ~lacosal injury in both experimental colitis and human inflammatory bowel disease (IBD). Enteral diets used in IBD had a variable content in LA (2 to 30 g/lO00 Keal). The influence of LA intake on colonic inflammation has not been studied. Aim: To assess the effect of intragastrie administration of increasing doses of LA on colonic damsge in experimental colitis. Methods: Forty-two Sprague-Dawley rats (200-250 g) with trinitrobenzenesulfonio acid (TNBS; 30 rag, 50%) induced colitis were randomized to receive, from 3 days before to 3 days after TNBS, increasing doses of intragastrie LA (4 groups: 30, i00, 300, 500 rag/d), D-arginine (500 rag/d), or LA (500 rag/d) plus aminoguanidine (2 mg/kg/d). Local eieosenoid release, assessed by intracolonic dialysis (ELISA, pg/ml), and plasma nitrite levels (nM) were measured at the 3rd day after TNBS. Afterwards, colon was removed for macroscopic and histological assessment. Results: There were no differences in macroscopic and histologic scores, and plasma nitrite levels between the different groups. Local thromboxane B2 release was lower in D-arginine and sminoguanidine groups (p=O.O01). Conclusions: In spite of raised intraeolonie TxB2 release as compared to D-arginine, the administration of increasing doses of LA is not related to a worsening of acute colonic damage in TNBS-indueed colitis.

GASTRIC MUCOSAL INJURY STIMULATES bFGF AND ITS RECEPTOR GENE EXPRESSION AND TRIGGERS CUG-INITIATED TRANSLATION OF 20.7 AND 21.7 kDa bFGF ISOFORMS. R.Z. Florkiewicz, A. Santos, K. Tanoue, F.L. Irwin Jr., LJ. Sarfeh and A. Tarnawski. DVA Medical Center, Long Beach, University of California, Irvine; The Scripps Research Institute, La JoUa, California.

Repair of deep alcohol-induced gastric mucosal injury requires not only restitution of the surface epithelium, but also reconstruction of a capillary network by angiogenesis (Gastro 1989,'96"..4505). Exogenous 18kDa basic fibroblast growth factor (bFGF) promotes angiogenesis in injured gastric mucosa (Gastro 1992;102".A176), ostensibly through interactions with its plasma membrane tyrosine kinase receptors (FGFRs). The single copy bFGF gene also encodes multiple gene products that are larger than 18kDa, known as the high molecular weight (HMW) isoforms, We studied whether repair of alcohol-injured gastric mucosa affects the expression of gastric mucosal bFGF and/or FGFR(s) mRNAs and proteins. METHODS: Gastric injury was induced in 52 male Sprague-Dawley rats by administering i.g. Sml/kg of 100% ethanol. Gastric specimens were obtained prior to, and at 8, 24, 48 and 72 hrs after ethanol. STUDIES: A) the expression of bFGF and FGF receptors (FGFR-1 and FGFR-2) mRNAs using RT-PCR; B) assessment of bFGF, FGFR-1 and -2 proteins by Western blot and C) immunostaining of mucosa with specific antibodies against bFGF, FGFR-1 and -2 and quantitation of fluorescent signal with videoimage system. RESULTS: A significant increase in bFGF mRNA was detected at 8 and 24 hrs after injury (724-+26% and 833-+34%, respectively vs normal mucesa; both p<0.001), that was followed, beginning at 24hrs, by an increase in bFGF protein, lasting throughout 72hrs. A similar increases in FGFR-1 and -2 mRNAs and proteins were also detected. As determined by Western blotting, the increase in bFGF protein was specific for the HMW isoforms (20.1 and 21.7kDa), not the t8kDa isoform. Immunostaining of gastric sections indicates that the 240% increase in bFGF signal (from 85+-6 in normal mucosa to 205+-12 units in the mucosa bordering necrosis; p<0.001) was localized to migrating microvascular tubes that will ultimately recenstruet the mucosal capillary network. CONCLUSIONS: 1) Gastric mucosal injury/repair results in a spatially and temporally restricted increase in bFGF and FGFR-1 and -2 mRNAs and proteins. 2) The preferential synthesis of CUG-initiated HMW bFGF translation products is a novel/n vivo regulatory mechanism during repair of gastric injury.