April 1995 Growth, Development, and Nutrition A765

• EXPRESSION OF THE C-JUN PROTOONCOGENE IS ASSOCIATED WITH ABSORPTIVE CELL DIFFERENTIATION ALONG THE CRYPT-VILLUS AXIS. K. Y. Yeh and M. Yeh. Section of Gastroenterology, Department of Medicine, Lonisiana State University Medical Center, Shreveport, LA

After being produced in the crypt, intestinal absorptive cells leave the crypt and migrate alonl~ the villus and extrude into the lumen. During cell migration toward the villus tip, it expresses enzymes and proteins to perform digestive and absorptive functions. The precise molecular mechanisms that regulate absorptive cell differentiation are unknown. It is known that some of the proteins, such as intestinal alkaline phosphatase (lAP), s u ~ i s o m a l t a s e , and lnctos~phlorizin hydrotase are transcriptionally regulated. For activation of these genes in the villus cell, the expoession of specific transcriptional factors should occur in ImralleL Because the c-]un proto~cogene encodes activation protein ! (AP 1 ) and the IAPgene contains an AP 1 site, we determined c-jun andjun-D mRNA levels, as well as IAP mRNA levels, along the crypt-villus (c-v) axis in the rat duodenum. Six levels of eryostat sections were sequentially collected along the c-v axis from adult rat duodenum for analysis. Total RNA was isolated and afiquot of RNA was size fractionated by denaturing agarose gel eiectrophoresis for quantitafion oftbe 28S rRNA. GAPDH mRNA levels in samples were also measured for normalizing sample

Northern blots showed that c-jun mRNA was barely le in the crypt and steadily increased from the villus base to a

plateau at the 20-40% villus level from the villus tip. Expression ofjun-D mRNA also showed the similar pettem of gradually increasing from the crypt to the villus tip, however, c-jun:jun-D ratios were about 0.5 at the crypt and 3 at the villus tip. The 3.0 and 2.7 kb IAP mRNAS, detected by specific prof. , for each RNA species, were undeteetable in the crypt, appeared at the villus bottom and increased to the plateau at the 50% level of villus. This gradient change in mRNA levels along the c-v axis closely related to lAP activity, which was low at the crypt and peaked at the villus tip. GAPDH mRNA also showed developmental gradient along the bottom haifofthec-v axis. Thus, the c-jun ~ e n e , that has been considered to be the narl~ signal of cell proliferation, is expressed at a high level associating with cell differentiation in villus cells. The detailed relationship between c-jun and lAP gene expression remains to be defined.

• POSTNATAL SHIFTS OF FERRITIN H AND L mRNA LEVELS OCCUR IN RAT DUODENUM AND DICTATE ISOFORM EXPRESSION IN RESPONSE TO DIETARY IRON. K. Y. Yeh, X. Alvarez-Hemandez, J. Glass and M. Yeh. Department of Medicine and Center for Excellence in Cancer Research, Treatment and Education, Louisiana State University Medical Center, Shreveport, LA

Ferritin (Ft) consists of 24 heavy (H) and light (L) subunits in varying proportions in different tissues and plays a significant role in iron metabolism and detoxification. Neither developmental changes nor die~'y iron alteration of intestinal Ft H and L subunit expression have been reported. To determine developmental changes in H and L subunit expression, rats were killed on day 3, 12, 18, 24, 32, and 60. Duodenal Ft H and L mRNA levels were determined by Nortbem blots and subunit contents were analyzed by immunoprecipitation followed by gradient (5- 23%) SDS-PAGE. Duodenal Ft H mRNA levels were the same on day 3 and 12, and increased 2-3 fold on day 18-24 and thereafter. This developmental increase of Ft H mRNA was accompanied by a 60% decrease in Ft L mRNA levels, resulting in a shift of H/L mRNA ratios from about 1 to 7. Duodenal Ft contents, but not mRNA levels, were related to mucosal iron content: decreasing between day 3 and 12 and increasing thereafter. However, the Ft H:L ratios paralleled the H:L mRNA ratios. To determine whether dietary iron had any preferential influence on Ft subunit expression, day 12 rats were fed 0.25 ml Similac low-iron infant formula supplemented with or without 1 mM FeSO4 by intragastric infusion and day 60 rats were fed (after overnight on 5% sucrose solution) 0.25 g chow with or without 8 mg iron. Duodenal expression ofFt H and L was determined 1.5, 3 and 6 h later. Dietary iron did not change duodenal Ft H and L mRNA levels and ratios during the experimental period in both age groups, but increased duodenal Ft contents more than 10-fold at 6 h after iron infusion in day 12 rats and 8-fold at 3 h and 6 h in day 60 rats. Duodenal Ft H:L ratios remained about 1 in day 12 and 7 in day 60 rats after die~a'y iron feeding. We conclude that 1) concomitant increase in Ft H and decrease in Ft L mRNA levels occur in the duodenum during postnatal development; 2) these changes dictate Ft H and L subunit production in response to dietary iron feeding; and 3) the effect of dietary iron on duodenal Ft expression is consistent with well defined translational regulation in other tissues.

EFFECT OF MUSTARD AND TURMERIC ON INTESTINAL PERISTALTIC ACTIVITY. N. Yerra, M.D., C.S. Pitchumoni, M.D., FACP, Division of Gastroenterology Our Lady of Mercy Medical Center, Bronx, New York 10466.

The biologic properties of commonly used spices are not well known. Previously we have shown that the oroceeal transit time (OCTT) is prolonged by red pepper but not by black pepper. In this study, we looked at the effects of Turmeric (Cureuma longa) and mustard (Sinapis alba) on OCTT, using the lactulose breath hydrogen test. After an overnight fast, healthy volunteers ingested 10 gin. of lactulose in an isosmotic solution. Serial end-expiratory breath samples were collected at 15 minute intervals and analyzed for hydrogen content. The time taken for the hydrogen level to rise 10 ppm. above basal values was considered as the baseline OCTT. On different days we measured OCTT after administration of 10 grams of lactulose and 2.0 gin. of turmeric (N= 8) or 1.0 gm. of mustard (N= 6), were given in gelatin capsules. Results are as follows:

Baseline After After I Volunteers I ocTr (rain).

1. 75 2. 90 3. 90 4. 120 5. 75 6. 120 7. 105 8. 90

Spice Mean basal OCTT + SD (Minutes)

Turmeric 95.6+ 16.6 (N=8) Mustard 95.0 + 16.0 (N=6)

Turmeric Mustard 60 45

135 75 75 75

105 45 75 30

105 105 105 Not done 75 Not done

Mean OCTT After Turmeric/Mustard Statistical _+ SD (Minutes) Significance

91.8+23.0 NS

62.5+25.1 S

Conclusion: Mustard significantly reduces OCTT. Turmeric in the dosage used reduces OCq'T minimally with no statistical significance. Projection for the future: The mechanism of reduction and the clinical applications of the observation are to be studied further.

GLUCAGON STIMULATES DNA SYNTHESIS IN SMALL INTESTINAL CELLS. Kristina Zachrisson and Andr6s Uribe. Department of Medicine, Karolinska Institute, Danderyd Hospital, Stockholm, Sweden

Previous studies have shown that the intestinal microflora and

indomethacin trigger atrophic reaction in the intestinal epithelium. The

enhanced DNA sythesis was associated with changes in the tissue

concentration of glucagon, neurotensin and other regulatory peptides. Our

aim was to examine whether glucagon and neurotensin stimulates DNA

synthesis of intestinal cells in vitro to further evaluate a potential role of

these peptides in the trophic reactions described above.

Methods: A small intestinal cell line from germ-free rats (IEC-6 cells)

was allowed to grow for 24 hours in a nutrient-enriched medium.

Thereafter, the cells were incubated during 24 hours with glucagon and

neurotensin at a concentration of I() -6, 10 -7, 10 -8, 10 -9 och 10-10 M.

Epidermal growth factor, EGF, at a concentration of 25, 50, I00, 200 oeh

400 ng/ml was used as a reference substance. The cells were labelled with

3H-methyl-thymidine for 4h and processed for autoradiography. DNA

synthesis was evaluated by the labelling index (LI%) in a light microscope.

Results: Glucagon increased the LI in a dose related manner. The LI was

at least twice as high in cells incubated with glucagon at 10 -6, 10 -7, 10 -8 M

compared to controls (p<0.05). Similarly, EGE at 100, 200 and 400 ng/ml

increased the LI compared to controls (p<0.05). The LI was not

significantly affected by neurotensin.

Conclusion: Glucagon and EGF stimulates DNA synthesis in intestinal

cells. Our results suggests that glucagon may participate in the development

of the trophic reaction elicited by chronic administration of indomethacin

and/or bacterial contamination.