April 1995 Growth, Development, and Nutrition A753

ANTIBODIES TO EPIDERMAL GROWTH FACTOR (EGF) ACCELERATE IN VlVO INTESTINAL PROPULSIVE MOTILITY (IPM) IN SUCKLING RATS. H. Shinohara, C. Williams, O. Koldovsk~,. Depts of Pediatrics and Physiology, University ef Arizona, Tucson, AZ

We have previously reported that EGF delaved IPM in vivo in suckling rats. In our present experiments, we tested the effect of EGF antibodies. Twelve-day-old rats fasted for 15 hours received subcutaneously 50 gl of bovine serum albumin (BSA , 0.1%) either alone or with rabbit antibodies against mouse EGF, in a dose neutralizing 250 ng EGF; [similar dose was used by Berseth (Am. J. Physiol. 253: G662) in other experiments with suckling rats] and orogastrically s~Cr-EDTA (a motility marker). Rats were killed 30 minutes later. Counts were measured separately in the stomach and the small intestine divided into 12 segments. Results are expressed as %o f gastrointestinal total counts present in individual segments (antibody group = open symbols, 22 rats; BSA group = full sy.mbols, 18 rats); no counts were detected beyond segment 8. EGF anhbedies did not affect gastric emptying, but accelerated IPM. (stat.signiL [ANOVA] differences: * = between segments 4, # = between the ratio of segments 5 and 7).

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These studies thus suggest that EGF regulates the motility of the developing small intestine.

DEVELOPMENTAL EXPRESSION OF THE CAUDAL RELATED HOMEODOMAIN TRANSCRIPTION FACTORS CDX-1 AND CDX-2 D.G. Silberg and P.G. Traber Division of Gastroenterology, University of Pennsylvania, Philadelphia, PA

The homeodomain transcription factors have been shown to be of central importance in the development of many organisms. The mouse caudal homeobo_x gene-2 (mCdx-2) can activate transcription of intestine-specific genes, such as sucrase-isomaltase, and is expressed at high levels in small intestine and colon in the adult mouse. Therefore investigation of mCdx-2, and a related intestine- specific family member mCdx-1, may provide information regarding the control of intestinal differentiation and development. These two transcription factors were analyzed to determine their patterns of expression in development. The expression of mCdx-I and mCdx2 mRNAs were analyzed at multiple time points from gastrulation through adulthood. Northern analysis of polyadenylated RNA of whole mouse embryo (d7, d l l , d15 and d17 p.c.) revealed low level expression of mCdx-1 at day 7 p.c. which decreased to undetectable levels by day 11 p.c. and increased again at days 15 and 17 at the expected size of 1.8 k.b. In contrast, hybridization of the mCdx-2 probe revealed a larger transcript of approximately 4.4 kb at day 7 and 11 p.c., with the expected size transcript of 2.4 kb beginning at day 11 p.c which increased in intensity with age. Northern analysis of total RNA from tissue from prenatal and postnatal small intestine and colon showed that a the same sized mRNA was expressed throughout late development and into adulthood. An RNase protection assay was performed to directly compare the expression of mCdx-1 and mCdx-2 throughout development (days 11.5, 12.5, 13.5, 14.5, 15.5, 16.5 p.c. and postnatal days 0, 9 and adult). Expression of mCdx-2 appeared weakly at day 11.5 p.c, three days earlier than mCdx-1. The expression of both mCdx-1 and mCdx-2 increased at day 14.5 and remained at high levels from day 16.5 p.c. to adult in the colon and small intestine. In summary, these developmental studies indicate that there is a difference in the expression of mCdx-1 and mCdx-2 in early embryonic development during gastrulation and coordinated expression in the gut surrounding the endodermal-intestinal epithelial transition which occurs at approximately embryonic day 15. These results suggest that further studies should be performed to determine the importance of these proteins on development of the intestinal epithelium,

PROTEIN DIGESTION AND DISSIMILATORY AMINO ACID METABOLISM IN THE HUMAN COLON: IN VlVO MEASUREMENTS OF BACTERIAL METABOLITES AND IN VITRO FERMENTATION STUDIES. E. A. Smith, J. H. Cummings, G. T. Macfadane. MRC Dunn Clinical Nutrition Centre, Cambridge, U. K.

Although breakdown of carbohydrate by bacteria in the human colon and its physiologic significance to the host are relatively well understood, few studies have been made on protein digestion in this organ. Despite this, it has been known for many years that putrefactive reactions in the colon give rise to a variety of toxic metabolites including, ammonia, sulphide, amines, phenols, indoles and thiols, and that some of these substances are involved in certain disease states. Since putrefactive processes must be controlled to some degree by environmental factors in the colon, our objectives were to investigate protein breakdown in different regions of the large gut, and to determine the effects of carbohydrate (starch) and pH on peptide and amino acid dissimilation using ammonia, branched chain fatty acids (BCFA) and phenols as markers.

Due to inaccessibility of the proximal bowel for routine study, gut contents (caecum, ascending, transverse and descending colons, and sigmoid/rectum (S/R)) were obtained from six subjects (two females, four males) who died suddenly from coronary heart disease or had violent deaths (average age 57 years, range16-89 years). To minimise post-mortem changes in colonic material, samples were taken between 2 and 4 hours after death (mean 3h 20 min). In vitro fermentation experiments were made in stirred vessels to investigate the effects of pH and starch on peptide and amino acid metabolism.

Bacterial cell counts gave values ranging from log 10.2 (SEM 0.2) in the caecum to Iog12.3 (SEM 0.3)in the SIR,Total soluble protein and peptides in caecal contents ranged from 0.1-15.4 g/kg (mean 5.8) and12.4-58.5 g/kg (mean 37.4) respectively. In the S/R, corresponding values were 7.0-22.4 (mean 14.7) and 25.0- 38.0 (31.5). BCFA and total phenols progressively increased from the caecum (0.84 mmol/kg gut contents (SEM 0.57) and 5.2 mmol/kg (SEM 0.6) to the SIR (2.30 (SEM 0.39) and 7.4 (SEM 2.3) respectively. However, the increase in putrefactive products in the distal bowel was not observed with ammonia, pH measurements gave mean values of ca. 5.5 for caecal material and 6.7 for contents in the S/R. Metabolic studies in vitro showed that rates of peptide fermentation by colonic bacteria were 34old higher at pH 6.8 compared to pH 5.5, and that starch (10 mg/ml) reduced rates of ammonia and phenol production by 50%. Free amino acids, which do not accumulate to a significant extent in the colon, were degraded at similar rates to peptides at pH 5.5, but were fermented between 2 and 4 times more slowly at pH 6.8.

Conclusions: Proteins and peptides occur in large amounts throughout the human colon, but dissimilatory metabolism of these substances by intestinal bacteria is reduced in the proximal bowel by fermentable carbohydrates such as starch. They increase bacterial requirements for peptides and amino acids for biosynthetic purposes, as well as exerting an indirect effect by reducing gut pH during their fermentation, thereby changing patterns of bacterial metabolism.

INTESTINAL LIPOPROTEIN (LP) METABOLISM IN DIABETES MELLITUS (DM): DIETARY FAq*I'Y ACID (FA) MODULATES CHYLOMICRON PHOSPHOLIPID (CM.-PL) COMPOSITION. LJ Smith, J Ezekowitz, D Gowda, MT Clandinin, Departments of Pediatrics and Medicine, University of Alberta, Edmonton, Canada. DM is associated with hyperlipidemia leading to an increased risk of atherosclerosis and accelerated cardiovascular disease. The small intestine assumes a greater relative role in lipoprotein metabolism in DM than in normal subjects. The effects of fasting and chronic feeding of physiologically-relevant high-fat diets, with high or low P/S ratio or high in fish oil (FO) lipids on the composition of CM-PL was studied in the streptozotocin (S)-treated rat model of DM. Methods. Male Sprague-Dawley rats were assigned at weaning to control or diabetic (S 65 m~kg ip) groups. Animals were further subdivided into 3 groups receiving high fat (20% w/w) semisynthetic diets with high (2.0), low (0.2) P/S ratio or high FO (25% fish oil concentrate) for 4 weeks. Rats were then subjected to intestinal lymphatic duct and duodenal canmdation, perfused overnight intraduodenally with saline/gincose. Lymph was collected tbr 4h the next day, and the animals then infused for 8h with a fat emulsion closely resembling the diet in FA composition, with lymph collection for the last 4h. LP fractions were separated by ultrascentrifllgation and characterized by TLC and GLC. Results. a) Feeding vs fasting caused a dramatic reduction in arachidonate levels in all dietary groups, especially the diabetic high P/S group (25.6% to 11.5%) and arachidonate levels were lowest in the FO group (9.6% to 5.6%). b) Feeding a high P/S diet resulted in a greater rise in 18:2w6 levels in diabetics of controls (17.6% vs 9.1%). c) Feeding a FO-enriched diet to diabetics resulted in higher levels of 18:2w6 (25.7 vs 17.2%), and 20:5w3 (l 5.4 vs 14.2%) with lower arachidonate levels (5.6%) compared to controls. All p,<0.05. Conclusions. a) Diabetic anirnals respond more favorably to diets rich in polyunsamrates and fish oil than controls in terms of arachidonate and linoleic acid metabolism, b) CM-PL composition is modulated by intestinal desaturase activity more dramatically for diabetics than controls. This study was fllnded by the Muttart Diabetes Foundation.