Differential mechanisms of induction of ornithine decarboxylase in rat intestine by L- and D-amino acids.

A single intragastric administration of glycine, L- and D-alanine, and L-and D-serine into rats resulted in a more than 20-fold stimulation of intestinal mucosal ornithine decarboxylase (ODC) within 4 h. The stimulation of ODC activity was accompanied by an increase in the amount of immunoreactive ODC protein. The induction of ODC by D-amino acids was in all likelihood attributable to an enhanced accumulation of ODC-specific mRNA species as revealed by Northern blot and dot-blot hybridization analyses. However, the induction by glycine and L-amino acids was not explainable by changes of mRNA since the changes in mRNA contents were only marginal. Since the turnover rates of L-serine-induced and D-serine-induced intestinal ODC protein were the same as the non-induced control, we concluded that the induction by glycine and L-amino acids was brought about by an increased efficiency of translation of the ODC message.

Circadian rhythm in the ornithine decarboxylase activity of rat small intestine.

Intestinal ornithine decarboxylase [L-ornithine carboxy-lyase, EC 4.1.1.17] activity was found to show a marked circadian rhythm with a peak 4 h after the start of eating in rats on a diet containing protein. In rats with an intestinal blind loop, the enzyme was induced in the portion of the intestine that came in contact with the protein meal, but not in the blind loop. Injection of tetragastrin or CCK-PZ alone had no effect on enzyme induction, but when a protein suspension was introduced into a tied loop of intestine soon after the injection of tetragastrin or CCK-PZ, the enzyme was induced in the segment to almost the same extent as in the intestines of normal rats eating a protein meal. These results suggest that the circadian rhythm in activity of intestinal ornithine decarboxylase is initiated by release of gastrin, or CCK-PZ, or both, and contact of protein with the small intestine after the intake of food containing protein.

Purification and properties of rat small intestinal arginase.

Arginase [L-arginine amidinhydrolase EC 3.5.3.1] from rat small intestine was purified about 2,200-fold and its properties were compared with those of the rat liver and kidney enzymes. Intestinal arginase was extremely labile on storage either at -10 degrees or 4 degrees and lost activity during purification unless 25 mM L-valine was present. The purified enzyme appeared to be homogeneous by disc electrophoresis and its molecular weight was estimated to be 120,000 by Sephadex G-100 filtration...

Induction of intestinal ornithine decarboxylase by single amino acid feeding.

Intestinal and hepatic ornithine decarboxylase (ODC) activities increased to a peak 4 h after administration of a diet containing casein or an amino acid mixture simulating that of casein to rats starved for 12 h. All amino acids except cysteine with a two or three carbon skeleton, including those with a D-configuration, and alpha-amino-isobutyric acid (AIB) strongly induced intestinal ODC when given in the diet or administered intragastrically. Amino acids with a four carbon skeleton were far less effective as inducers and other amino acids did not induce intestinal ODC at all. The amino acids that induced hepatic ODC showed no particular structural characteristics: glycine and cysteine were very effective, threonine, tryptophan, methionine, and phenylalanine were less effective, and serine, valine, isoleucine, and histidine were only slightly effective. Elevation of ODC activity after amino acid administration was not due to stabilization of the enzyme protein with the amino acids. Intestinal ODC was induced by intragastric but not intraperitoneal injection of glycine, although these treatments resulted in similar increases in the tissue concentration of glycine. On the contrary, hepatic ODC was induced by glycine regardless of the administration route. Intestinal ODC was also induced only in the segment of the intestine perfused with a solution of an amino acid with which the activity increased in the feeding experiment. These results suggest that the accumulation of an amino acid per se is not a trigger for induction of intestinal ODC and that an amino acid must act on the mucosal surface to induce the enzyme.

Purification and some properties of rat intestinal ornithine decarboxylase.

Ornithine decarboxylase (ODC) was induced in rat small intestine by treatment with hypotonic solution in vitro and purified by two procedures, a conventional procedure and an immunoaffinity procedure. SDS-polyacrylamide gel electrophoresis showed that the molecular weight of the preparation purified by the immunoaffinity procedure (Mr = 53,000) was slightly larger than that of the preparation obtained by the conventional procedure (Mr = 52,000). Values for the Km for L-ornithine (0.1 mM), the isoelectric point (5.4), and the final specific activity (5.1-5.5 x 10(5) nmol CO2/mg protein/30 min) of the two preparations were similar to those reported for the rat liver ODC. Addition of a protease inhibitor (limabean trypsin inhibitor) to the crude extract prevented the appearance of the smaller enzyme (Mr = 52,000) obtained by the conventional purification procedure. Our result indicates that the large enzyme is native ODC and the smaller one is a partial proteolysis product of native ODC.

Effect of food intake on intestinal absorption and mucosal hydrolases in alloxan diabetic rats.

The relation between food intake and enzyme activity of the small intestine and rate of intestinal absorption were studied in rats 15 days after induction of alloxan diabetes. Diabetic rats were given an ad lib. semisynthetic diet or a restricted diet on the basis of either daily intake or body weight. The rates of absorption of 5 mMD-galactose and L-valine were determined in vitro by the everted sac method. The rates of absorption of the substances, expressed per unit weight or per length of intestine, were higher in diabetic rats than in controls, regardless of the amount of food consumed. Maltase and sucrase activities were significantly increased in diabetic rats, regardless of the amount of food consumed. The activity of intestinal alkaline phosphatase was increased in diabetic rats fed ad lib., but not in those on a restricted diet. These findings suggest that in alloxan diabetic rats the increased disaccharidase activity in the small intestine is due to insulin deficiency, and that the increased activity of alkaline phosphatase is only a secondary effect of insulin deficiency, caused by increased food intake resulting from insulin deficiency.

Changes in the intraluminal protein digestion of pancreatic duct-ligated rats.

After ligature of the pancreatic duct (PDL), the body weight of rats decreased for several days, but began to increase from day 7, returning to that at the time of the operation on day 14. In these PDL animals, the weight gain was not due to improved digestion resulting from duodenal leakage of pancreatic enzymes or a compensatory increase of proteolytic enzyme activities in the intestinal mucosa. There was no significant difference in pepsin activities in the gastric contents and mucosa of control and PDL rats. However, acidic proteolytic activity, with a pH range between 1 and 4 and an optimum at pH 2.8, was found to be extremely high in the intestinal contents of PDL rats. Furthermore, the intraluminal pH of PDL rats was maintained below 4.0, especially in the upper small intestine, because of the absence of pancreatic bicarbonate secretion, suggesting that compensatory digestion by acidic proteolysis accounted in part for the growth of PDL rats. The transit time of orally administered material through the gastrointestinal (GI) tract in PDL rats was longer than that in control rats on days 7 and 14. These results suggest that the weight gain of PDL rats was caused by compensatory digestion by acidic proteolysis in the small intestine and prolongation of the transit time through the GI tract.

A cause of hypercholesterolemia in alloxan diabetic rat.

In studies on the cause of hypercholesterolemia in alloxan diabetic rats, the pool size and basal daily synthesis of conjugated bile salts were measured by the washout method and neutral sterols in the luminal contents were determined using cholestyramine. The results showed that in diabetic rats: 1) the biliary excretions of cholesterol and bile salts were significantly increased; 2) the pool size of conjugated bile salts was increased and its rate of synthesis was higher than in controls; 3) the amount of neutral sterols in the luminal contents doubled when cholesterol absorption was inhibited by administration of cholestyramine; 4) the amount of neutral sterols excreted in the feces was the same as in controls. Thus, it was concluded that hypercholesterolemia may be partly caused by an increased rate of intestinal absorption of cholesterol, derived mainly from sloughed off epithelial cells and bile, and due to facilitated micellar formation by the increased amount of bile salts in the intestine.

Effect of sodium saccharin on rat pancreatic enzyme secretion.

Intragastric administration of casein, oleic acid, or polyvinylpyrrolidone resulted in a marked increase in the pancreatic secretion of enzymes, and the administration of 0.03% sodium saccharin, at the concentration permitted in soft drinks, inhibited this increase with these substances. The stimulation of secretion of pancreatic juice-bile mixture by these substances was also inhibited by administration of sodium saccharin. Intravenous injection of CCK-PZ (cholecystokinin-pancreozymin) during inhibition by sodium saccharin, caused a rapid increase in amylase output, suggesting that sodium saccharin acts on the site(s) of CCK-PZ release to prevent its secretion even in the presence of a stimulant, but does not reduce the sensitivity of the pancreas to CCK-PZ. Sodium saccharin seemed to have an effect on secretin release similar to that on CCK-PZ release, judging from the results for the volume of juice-bile mixture.

Difference in the inductions of duodenal alkaline phosphatase by casein feeding and by starch feeding.

Feeding of starch or casein to rats that had been starved for 4 days resulted in marked increase in duodenal alkaline phosphatase activity in a few hours. In rats with a duodenal blind loop, the enzyme was induced by feeding starch, but not casein. Conditions leading to hyperglycemia, such as an intragastric administration and an intravenous infusion of glucose, induced duodenal alkaline phosphatase to the same extent and in the same period as starch feeding. Subcutaneous injection of insulin alone did not induce the enzyme. In chemically sympathectomized rats, the enzyme was induced by starch feeding. In vagotomized or hexamethonium-treated rats, the enzyme was not induced by starch feeding or intragastric administration of glucose solution. On the contrary, subcutaneous injection of carbachol alone induced the enzyme. These results suggest that stimulation of the parasympathetic nervous system via hyperglycemia is important for induction of duodenal alkaline phosphatase by starch feeding. The mechanism of enzyme induction by casein feeding is still unknown.

Memory of the rhythmic change in activity of duodenal alkaline phosphatase in rats.

Studies were made on memory of the rhythmic change in activity of duodenal alkaline phosphatase in rats. During starvation, the peak enzyme activity decreased gradually disappearing in 4 days. The peak activity of duodenal alkaline phosphatase was retained by feeding starch diet for 4 days instead of starvation for 4 days, but not by feeding casein diet for the same period. Starvation for one day after feeding casein diet for 4 days resulted in disappearance of the peak activity. However, the peak activity was still retained after one day of starvation after 4 days on starch diet. Therefore, starch feeding appears to be important in the memory of the rhythmic change in activity of duodenal alkaline phosphatase.

Changes in brush-border enzyme activities of intestinal epithelial cells isolated from the villus-crypt axis during the early phase of alloxan diabetes in rats.

The sucrase activity in enterocytes isolated from the villus crypt axis was found to increase in all regions of the villus from day 2 after induction of diabetes, and the increase continued until day 4. In contrast, alkaline phosphatase activity increased mainly in the apical one-third of the villus-crypt column, and the increase occurred abruptly on day 4 with increase in food intake.

Diabetes and glucose transporter gene expression in rat small intestine.

The expressions of Na(+)-dependent glucose transporter (SGLT1) and five facilitative glucose transporter genes (GLUT1-5) in the small intestine of streptozotocin (STZ)-induced diabetic rats were examined by RNA blotting analysis. The transcripts of SGLT1 mRNA gave bands of 4.5 Kilobases (Kb) and 2.8Kb (very faint band). The levels of SGLT1 mRNA were significantly increased in 30- and 60-day STZ rats, but not changed in acute diabetic rats (2- to 10- day STZ rats). The GLUT2 mRNA levels changed in parallel with the D-galactose transport activity, being increased about 4-fold in 5-day STZ rats. The transcripts of GLUT5 mRNA gave three bands of 5.1Kb, 2.8Kb and 2.OKb, whose levels were significantly reduced in 30- and 60-day STZ rats. These results suggest that the facilitative glucose transporter (GLUT2), in addition to the Na(+)-dependent glucose transporter (SGLT1), may play an important role in intestinal glucose transport in diabetic rats.

Developmental changes in intestinal glucose transporter mRNA levels.

Developmental changes in glucose transporter mRNA levels in the jejunum of rats of different ages were examined by using slot blot RNA analysis. The level of SGLT1 mRNA did not change significantly through life. The GLUT5 mRNA level was highest in 10-day-old rats and then decreased reaching the adult level by day 20 after birth. The GLUT2 mRNA level was low in rats of 5 and 10 days old, but then increased progressively reaching the adult value by day 25 after birth. These results indicate that the expressions of intestinal facilitative glucose transporter genes change markedly in the third week after birth.

Inhibition of glucose absorption by phlorizin affects intestinal functions in rats.

BACKGROUND: To investigate the mechanism of regulation of intestinal disaccharidase activity and glucose absorption, the effect of dietary intake of phlorizin, a potent and specific inhibitor of intestinal glucose transport, on intestinal disaccharidase activity and Na(+)-dependent glucose transporter was examined in rats. METHODS: Jejunal disaccharidase activity and the number of Na(+)-dependent glucose transporters were determined in rats maintained on a low-starch diet, a high-starch diet, or low-starch diets containing various amounts of phlorizin (0.1%-0.9% wt/wt). RESULTS: Jejunal disaccharidase activity increased in a dose- and time-dependent manner. Stimulation of jejunal disaccharidase activity only occurred when phlorizin was added to starch-containing diets, not when it was added to a carbohydrate-free diet. Addition of the same amount of phloretin and glucose (constituents of phlorizin), to the diet failed to increase disaccharidase activity. The maximum binding of phlorizin to brush border membrane vesicles was increased in the rats fed phlorizin, whereas the dissociation constant remained unchanged, suggesting an increase of glucose transporter expression. CONCLUSIONS: Dietary phlorizin increased the jejunal disaccharidase activity and Na(+)-dependent glucose transporter expression. The trigger for these changes may have been due to an increased luminal glucose content.